NEUROLOGICAL MECHANISMS UNDERLYING ROTATOR CUFF

NEUROLOGICAL MECHANISMS UNDERLYING ROTATOR CUFF SKELETAL MUSCLE PATHOPHYSIOLOGY AND IMPLICATIONS FOR THERAPY

BY

SANDEEP MANNAVA

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

NEUROSCIENCE

MAY 2012

Winston-Salem, North Carolina

Approved By

Thomas L. Smith, Ph.D., Advisor

Mark E. Van Dyke, Ph.D., Chair

Osvaldo Delbono, M.D., Ph.D.

L. Andrew Koman, M.D.

Zhongyu Li, M.D., Ph.D.

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DEDICATION

I dedicate this dissertation to: my parents, Venkata Rao Mannava, MD and Uma Mannava, MD;

to my wife, Kathleen A. Mannava, MD; and to my sister, Kavitha Mannava,

without your support, love, encouragement, and patience this would not be possible.

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ACKNOWLEDGEMENTS

I would like to sincerely thank my parents: Venkata Rao Mannava, MD and Uma

Mannava, MD. Without your love, support, and guidance, I would not be in the position

I am today. It is my hope that I have been able to take advantage of the many

opportunities that you have given me. I am truly blessed to be your son. I would also like

to thank my beautiful wife Kathleen A. Mannava, MD, who is the love of my life. Thank

you for allowing me to pursue my many interests; your unwavering support and love

keeps me grounded and strong. Finally, I would like to thank my sister Kavitha Mannava.

You have been a friend since you were born. You are the future and have inspired me to

do better…

I would like to thank my mentors Thomas L. Smith, PhD and L. Andrew Koman,

MD. Also, I would like to thank Riyaz H Jinnah, B. Chir, FRCS and Beth P. Smith, PhD.

Your support of my research ideas allowed me to pursue my sincere academic interests

during my time in the laboratory. You all took a chance in giving me this research and

clinical educational opportunity at Wake Forest University. I am grateful for the

opportunity to take part in your innovative program. I have learned a tremendous amount

from all of you, both professionally and personally. Thank you for letting me learn from

mistakes and experience the success of our collective research efforts.

I am grateful to my committee members Osvaldo Delbono, MD, PhD and

Zhongyu Li, MD, PhD. You have both been great role models during my pursuit of the

PhD degree after completing my medical school training. I truly appreciate your

invaluable insights, which have greatly improved the quality of my research endeavors. I

appreciate both of you giving me the most valuable gift of all, your time.

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I want to thank my committee chairman, Mark E. Van Dyke, PhD. Since I arrived

at Wake Forest University, we have met several times a month and I have learned a lot

from your very unique perspective. You have challenged me to improve my research,

have encouraged me after my many grant and manuscript rejections, and have

congratulated me after each success. Thank you for all you do for young orthopaedic

researchers at Wake Forest University and for all you do for our department.

All great research requires a capable and qualified team. Wake Forest University

School of Medicine’s Department of Orthopaedic Surgery has one of the best research

teams in the world. I would like to acknowledge and thank the great faculty for your

insights and help in completing these many projects: Michael F. Callahan, PhD;

Christopher J. Tuohy, MD; Gary G. Poehling, MD; and Walton W. Curl, MD. I would

also like to thank our wonderful orthopaedic residents for their support and help, both

professionally and personally: Thorsten M. Seyler, MD; Johannes F. Plate, MD; Jordan

M. Case, MD; Omar F. Nazir, MD; Maxwell K. Langfitt, MD; Patrick W. Whitlock, MD,

PhD; Jonathan C. Barnwell, MD; Austin V. Stone, MD; and Peter J. Apel, MD, PhD. I

have also been very fortunate to work with some very bright, motivated, and enthusiastic

medical students and graduate students, who I would like to thank: Walter F. Wiggins,

BS; Patrick Haubruck, BS; Simon M. Trach, BS; and Lauren A. Pace, BS. Finally, I

would like to acknowledge and thank the wonderful orthopaedic research staff: Eileen

Martin, Martha Holden, Janet Kelly, Casey Lively Northam, and Deanna Sizemore.

I would like to specifically mention and thank some of our collaborators from

other departments: Katherine R. Saul, PhD; Joel D. Stitzel, PhD; and Josh Tan, MS. I

would also like to thank my neuroscience collaborators and professors Carol Milligan,

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PhD and Ronald W Oppenheim, PhD. I have learned from you both in the classroom and

from our collaborative research efforts. Thank you for making me a better basic scientist

and for supporting my research pursuits.

I am thankful for the research opportunities and mentorship I received earlier in

my educational career. Under the mentorship of Charles Antzelevitch, PhD (Masonic

Medical Research Laboratories, Utica, NY), Robert F Gilmour Jr., PhD (Cornell

University, Ithaca, NY), and Niels F. Otani, PhD (Cornell University, Ithaca, NY), I

developed my scientific curiosity and love of biomedical research. I appreciate the

amount of patience and support you provided me when I was younger, immature, and less

knowledgeable. Thank you for allowing me to learn in your laboratories and for inspiring

me to pursue biomedical research.

On a personal note, I would like to acknowledge the support of my good friends:

Matthew J. Carhart, JD; Robert F. Manfredo, JD; Vikram V. Bellapravalu, MBA;

Andrew Lombardo, MBA; and Philip Lombardo, PhD. We have known each other our

entire lives and have pushed each other to pursue greatness. I am proud of all you have

accomplished. Hopefully we are just getting started...

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TABLE OF CONTENTS

Table of Contents ..................................................................................................................... vi

List of Illustrations and Tables ........................................................................................... ix Chapter 2 Figures and Table .................................................................................................................... ix Chapter 3 Figures and Table .................................................................................................................... ix Chapter 4 Figures and Tables .................................................................................................................. ix Chapter 5 Figures and Table .................................................................................................................... ix Chapter 6 Figure ............................................................................................................................................. x

List Of Abbreviations ............................................................................................................. xi

Abstract .................................................................................................................................... xiv

Chapter 1 ..................................................................................................................................... 1

Introduction ............................................................................................................................... 1 Rotator Cuff Tears: A Description of the Clinical Problem .................................................. 2 Rationale for a Neuroscience Approach to Rotator Cuff Reconstruction Surgery ...... 2 Development and Characterization of a Small Animal Model for the Study of Rotator Cuff Function After Injury ............................................................................................... 5 Neuromodulation of In Vivo Soft-Tissue Biomechanical Properties Following Botulinum Neurotoxin A (BoNT-A) Injection: A Potential Therapeutic Strategy to Improve Operative Outcomes of Rotator Cuff Repair Surgery ........................................... 7 A Novel Paradigm for Rotator Cuff Research: Translating Basic Science Animal Model Studies to Clinical Recommendations Using Simulation Analysis ...................... 9 References .......................................................................................................................................... 10

Chapter 2 ................................................................................................................................... 17

Evaluation of In Vivo Rotator Cuff Muscle Function After Acute and Chronic Detachment of the Supraspinatus Tendon: An Experimental Study in an Animal Model ........................................................................................................................... 17

ADDENDUM ........................................................................................................................................ 17 Abstract ............................................................................................................................................... 18 Introduction ...................................................................................................................................... 20 Materials and Methods: ................................................................................................................. 21

Muscle Force Testing: ................................................................................................................................ 21 Muscle Force Data Standardization ..................................................................................................... 23 EMG Testing ................................................................................................................................................... 24 Histological analysis ................................................................................................................................... 24 Statistics .......................................................................................................................................................... 25 Source of Funding ....................................................................................................................................... 25

Results ................................................................................................................................................. 26 Discussion .......................................................................................................................................... 29 Acknowledgements......................................................................................................................... 34 References .......................................................................................................................................... 35

Chapter 3 ................................................................................................................................... 60

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Chemical Denervation with Botulinum Neurotoxin A Improves the Surgical Manipulation of the Muscle-Tendon Unit: An Experimental Study in an Animal Model .......................................................................................................................................... 60

Abstract ............................................................................................................................................... 61 Introduction ...................................................................................................................................... 63 Materials and Methods .................................................................................................................. 64

Botulinum Toxin Injection ....................................................................................................................... 65 Assessment of Active and Passive Muscle Properties .................................................................. 65 Statistical Analysis ...................................................................................................................................... 67

Results ................................................................................................................................................. 68 Discussion .......................................................................................................................................... 70 Acknowledgements......................................................................................................................... 74 Source of Funding ............................................................................................................................ 75 References .......................................................................................................................................... 76

Chapter 4 ................................................................................................................................... 93

Contributions of Neural Tone to In Vivo Passive Muscle-Tendon Unit Biomechanical Properties in a Rat Rotator Cuff Animal Model ............................. 93

Abstract ............................................................................................................................................... 94 Introduction ...................................................................................................................................... 95 Materials and Methods .................................................................................................................. 97

Botulinum Neurotoxin A and Saline Injection Procedure .......................................................... 97 Surgical Exposure and Experimental Apparatus ........................................................................... 98 Muscle Force Testing .............................................................................................................................. 100 Passive Load-Relaxation Testing ....................................................................................................... 100 Fung’s Quasilinear Viscoelastic Model (QLV) ............................................................................... 101 Statistics ....................................................................................................................................................... 103

Results ............................................................................................................................................... 104 Discussion ........................................................................................................................................ 105 Acknowledgements....................................................................................................................... 112 References ........................................................................................................................................ 113

Chapter 5 ................................................................................................................................ 132

The Science of Rotator Cuff Repairs: Translating Basic Science into Clinical Recommendations .............................................................................................................. 132

Abstract ............................................................................................................................................. 133 Introduction .................................................................................................................................... 135 Rotator Cuff Tears: A Clinical Problem .................................................................................. 135 The Rotator Cuff Muscle Undergoes Changes After Tendon Injury ............................. 137 In Vivo Biomechanics of Rotator Cuff Repair Surgery...................................................... 138 Evaluation of Muscle Function after Rotator Cuff Tears ................................................. 140 Computational Modeling of the Human Upper Extremity and Implications for Orthopaedic Surgery .................................................................................................................... 143 The Effect of Muscle-Tendon Unit Retraction on Passive and Active Tension: A Simulation Analysis of Rotator Cuff Tears ............................................................................ 145 Summary and Conclusions ......................................................................................................... 146 Source of Funding .......................................................................................................................... 147 Acknowledgements....................................................................................................................... 147 References ........................................................................................................................................ 149

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Chapter 6 ................................................................................................................................ 177

Clinical Relevance and Future Directions .................................................................. 177 Summary of Doctoral Thesis ..................................................................................................... 178 Clinical Relevance to Orthopaedic Surgery .......................................................................... 178 Future Direction for Rotator Cuff Surgery Research ........................................................ 179 The Study of Age –Related Changes and the Influence on Rotator Cuff Surgery..... 179 Future Directions of Botulinum Neurotoxin A (BoNT-A) Modulation of Soft-Tissue Biomechanics .................................................................................................................................. 182 A Bioengineering Approach to Rotator Cuff Surgery ........................................................ 184 References ........................................................................................................................................ 185

Curriculum Vitae ................................................................................................................. 191 Education .......................................................................................................................................... 191 Teaching............................................................................................................................................ 191 Professional Societies .................................................................................................................. 191 Research Support .......................................................................................................................... 192 Peer-Reviewed Manuscripts ...................................................................................................... 192 Book Chapters ................................................................................................................................. 194 Thesis Titles .................................................................................................................................... 194 United States Patent ..................................................................................................................... 194 Abstracts and Meeting Presentations .................................................................................... 194 Research Positions and Employment ..................................................................................... 199 Awards and Honors ...................................................................................................................... 200

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LIST OF ILLUSTRATIONS AND TABLES

Chapter 2 Figures and Table

Chapt 2, FIG 1 Pages 45-46 Chapt 2, FIG 2 Pages 47-48 Chapt 2, FIG 3 Pages 49-50 Chapt 2, FIG 4 Pages 51-52 Chapt 2, FIG 5 Pages 53-54 Chapt 2, FIG 6 Pages 55-56 Chapt 2, FIG 7 Pages 57-58 Chapter 2, Table 1 Page 59


Chapt 3, FIG 1 Pages 84-85 Chapt 3, FIG 2 Pages 86-87 Chapt 3, FIG 3 Pages 88-89 Chapt 3, FIG 4 Pages 90-91 Chapt 3, Table 1 2

Chapter 4 Figures and Tables

Chapt 4, FIG 1 Pages 120-121 Chapt 4, FIG 2 Pages 122-123 Chapt 4, FIG 3 Pages 124-125 Chapt 4, FIG 4 Pages 126-127 Chapt 4, FIG 5 Pages 128-129 Chapt 4, Table 1 Page 130 Chapt 4, Table 2 Page 131


Chapt 5, FIG 1 Pages 160-161 Chapt 5, FIG 2 Pages 162-163 Chapt 5, FIG 3 Pages 164-165 Chapt 5, FIG 4 Pages 166-167 Chapt 5, FIG 5 Pages 168-169

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Chapt 5, FIG 6 Pages 170-171 Chapt 5, FIG 7 Pages 172-173 Chapt 5, FIG 8 Pages 174-175 Chapt 5, Table 1 Page 176

Chapter 6 Figure

Chapt 6, FIG 1 Pages 189-190

xi

LIST OF ABBREVIATIONS

α Pennation Angle or Alpha (power analysis) μL Microliters ‘A’ Fung QLV Parameter (linear scaling elastic

component)

Ann of Biomed Eng Annals of Biomedical Engineering ant Anterior AUC Area Under the Curve (for Compound Motor

Action Potential)

‘B’ Fung QLV Parameter (nonlinear scaling elastic component)

BoNT-A Botulinum Neurotoxin A or Botulinum Toxin A ‘C’ Fung QLV Parameter (viscocity) CD1 Cluster-Designation 1 CE Contractile Element (representation of actin-

myosin interaction with a dashpot in the Hill Muscle Model)

CME Continuing Medical Education CMAP Compound Motor Action Potential CST Corticospinal Tract CT-Scan Computer-Tomography Scan ‘D’ Distal EMG Electromyography EPub Electronically Published FT Force Applied to Tendon g Grams

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Hz Hertz J Hand Surg Am Journal of Hand Surgery American Edition JBJS (Am.) The Journal of Bone and Joint Surgery American

Edition

KCl Potassium Chloride kg Kilogram ls

T Tendon Slack Length lo

M Resting Muscle Length lat Lateral ‘M’ Middle mA Milliampere mm Millimeter ms Millisecond N Newton(s) ‘P’ Proximal PCSA Physiological Cross-Sectional Area PMID PubMed Identification Number QLV Quasilinear Viscoelastic Model (described by Fung) R2 Coefficient of Determination RST Rubrospinal Tract s Second(s) Tau1 or τ1 Fung QLV Parameter (fast relaxation component

time constant)

Tau2 or τ2 Fung QLV Parameter (slow relaxation

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component time constant) TST Tectospinal Tract U Units V Volt(s) VST Vestibulospinal Tract

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ABSTRACT

Sandeep Mannava

Introduction: Rotator cuff tears are a common cause of upper-extremity disability. For

chronic, full-thickness, rotator cuff tears, repair surgery can be technically challenging

due to large gap distances and increased stiffness of the muscle-tendon unit. In contrast,

acute rotator cuff tears are associated with lower repair tensions, less fibro-fatty

infiltration of the muscle, and better functional outcomes. Animal models can be used to

understand rotator cuff muscle function and these results can be translated into clinical

recommendations using computational modeling.

Methods: In vivo muscle function, electromyography, and passive muscle-tendon unit

properties were studied before and after supraspinatus tenotomy in a rodent rotator cuff

injury model (acute vs chronic). Chemical denervation and Fung’s QLV model were used

to assess neural contributions to in vivo stress-relaxation biomechanical properties. Then,

a series of simulation experiments were conducted using a validated computational

human musculoskeletal shoulder model to assess both passive and active tension of

rotator cuff repairs based on surgical positioning.

Results: Muscle function was impaired at the tensions required to repair a chronically

torn rotator cuff (45% reduction from maximal twitch amplitude, p<0.05). Dysfunction in

the chronic tear setting was detectable via EMG (p<0.05). Although the chronically

injured muscle-tendon unit becomes more stiff; pharmacological modulation of the

nervous system with BoNT-A improves compliance by approximately 20% (p<0.05). At

adducted postures, computational data from simulated surgical repair of chronically torn

rotator cuff indicated that passive repair tension markedly exceeds the pullout strength of

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fixation techniques typically used in these surgeries and there was approximately a 50%

reduction in moment generating capacity after repair of chronically torn rotator cuff.

Discussion and Conclusion: Rotator cuff surgical outcomes may be improved by earlier

intervention, which results in lower surgical repair tensions and fewer electromyographic

neuromuscular changes. Our data suggest a direct experimental connection between high

repair tensions—chronic tear setting—and impaired contractile force. The

pharmacological modulation of increased muscle-tendon unit stiffness using BoNT-A has

the potential to facilitate the surgical manipulation of the muscle-tendon unit and protect

the repaired tendon. Simulation analysis stresses the importance of proper arm

positioning during intra-operative repair, post-operative healing, and rehabilitation.

1

CHAPTER 1

INTRODUCTION

Sandeep Mannava

2

ROTATOR CUFF TEARS: A DESCRIPTION OF THE CLINICAL PROBLEM

Rotator cuff tears are a common cause of upper extremity pain and disability.

The incidence of rotator cuff tears is approximately 4% in patients under the age of 40

years old, with the incidence increasing to 54% in patients over the age of 60 years old

[1]. About 300,000 rotator cuff surgeries are performed annually in the United States

with an economic burden of $3 billion [2, 3].

In healthy individuals, the four rotator cuff muscles act as dynamic stabilizers,

centering the humeral head on the glenoid in order to optimize upper-extremity function

during motion [4]. Once torn, rotator cuff tendons do not heal spontaneously, which can

lead to decreased range of shoulder motion, joint instability, chondral injury, and

decreased function [5]. Despite considerable surgical advances, these surgeries are still

associated with a high rate (20-90%) of recurrent tearing [5-10]. Factors contributing to

poor operative outcomes include advanced age [2], poor tendon quality [11], extensive

muscle atrophy and fibrofatty infiltration [12], increased time from injury [2, 3, 13], and

large tear size [2, 3, 14, 15]. Currently, there is no “gold-standard” surgical treatment that

successfully restores pre-injury shoulder strength, range of motion, and function [5-9]. A

research approach that utilizes neuroscience techniques may provide the insights needed

to improve functional outcomes after rotator cuff surgery.

RATIONALE FOR A NEUROSCIENCE APPROACH TO ROTATOR CUFF

RECONSTRUCTION SURGERY

Neuroscience is a broad field, encompassing the central nervous system (brain

and spinal cord), the peripheral nervous system, and skeletal muscle. In orthopaedics,

surgeons often deal with the more peripheral manifestations of pathological conditions

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afflicting the central nervous system. For example, orthopaedic surgeons manage and

treat the muscle contractures and joint deformities that are associated with cerebral palsy

[16]. Additionally, orthopaedic surgeons directly operate and clinically manage spine

pathology. Further, the common orthopaedic conditions of carpal tunnel syndrome and

cubital tunnel syndrome are caused by nerve compression.

Insights can be gained from a neuroscience approach to the difficult problem of

fibrofatty degeneration of the rotator cuff muscles after chronic detachment of the tendon.

Many orthopaedic surgeons consider the structural changes that the muscle undergoes

irreversible [12, 17, 18], and little progress has been made since the first modern

description of the condition by E. Armory Codman. In his 1934 book The Shoulder:

Rupture of the Supraspinatus Tendon and Other Lesions in or about the Subacromial

Bursa, Dr Codman stated “the tendon was retracted to such a degree that I could not even

attempt a suture.” Orthopaedic research has focused on the tendon-to-bone interface (i.e.

how to re-attach the tendons to the humerus) [19-26]; however, the rotator cuff muscles

function as dynamic stabilizers and without adequate contractile strength of these

muscles, the repair of a torn tendon may be of little value to the patient. In fact, current

practice guidelines acknowledge the inadequacy of rotator cuff repair surgery in

reversing pathological changes in skeletal muscle structure and function [5-10]. Thus, a

more complete understanding of the pathophysiological mechanisms underlying rotator

cuff skeletal muscle dysfunction may improve clinical care.

The current understanding of rotator cuff muscle pathology is largely based upon

clinical studies, including physical examination and radiological imaging of the disease

state [17, 27, 28]. Some of the pathophysiological changes contributing to the muscle

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dysfunction might be attributable to nerve compression or entrapment. Recently, an

association has been made between large rotator cuff tears and suprascapular neuropathy

in several clinical case series [29-31]. The suprascapular nerve takes a tortuous course

and has the potential to become entrapped or compressed by the atrophied and retracted

muscle-tendon unit in the suprascapular and spinoglenoid notches [32-34]. Unfortunately,

the literature is limited to clinical studies. Basic science data that has directly associated

suprascapular neuropathy to the chronicity of the torn rotator cuff tendon are not

currently available.

Basic science literature on rotator cuff pathology has been limited to structural

gene/protein expression [35, 36], histological examination [37-39], and biomechanical

studies [38, 40, 41]. Previous studies offer a limited physiological profile of rotator cuff

muscle function after injury to the rotator cuff tendon. Specifically, previous studies

examined isometric contractions at repair tensions below those required to surgically

reattach the muscle-tendon unit to the humeral head [42-45]. Many of the previous

studies have been performed in a classical physiology setting of ex vivo explanted whole

muscle or in vitro single muscle fiber studies [42-45]. The in vivo muscle function studies

of pathological rotator cuff muscles have been performed in the sheep model [45].

For advancement of the basic science understanding of how rotator cuff muscles

respond to surgical manipulation and tendinous injury, it is necessary to develop an in

vivo small animal rodent model to test rotator cuff muscle and suprascapular nerve

function. As Codman described in 1934, passive stiffness of the muscle-tendon unit is a

major determinant of surgical success and post-operative functional outcome [38, 40, 41].

Botulinum Neurotoxin A (BoNT-A) is a widely administered pharmacological agent that

5

has been utilized in neuroscience research and clinical management of spasticity [46, 47].

The novel orthopaedic use of this highly potent neurotoxin has the potential to

temporarily and reversibly cause chemical denervation and flaccid paralysis/paresis, thus

modifying passive stiffness and improving the surgical manipulation of otherwise stiff

skeletal muscle-tendon units. In summary, a systematic neuroscience-based approach and

understanding of rotator cuff pathology has the potential to improve orthopaedic clinical

practice for this common cause of upper-extremity pathology.

DEVELOPMENT AND CHARACTERIZATION OF A SMALL

ANIMAL MODEL FOR THE STUDY OF ROTATOR CUFF

FUNCTION AFTER INJURY

This doctoral thesis is divided into several chapters based upon the development

and characterization of a small animal model system for the study of rotator cuff

neuromuscular function after injury. To the best of our knowledge, there are no published

studies that evaluate in vivo rodent rotator cuff muscle function after rotator cuff tear. The

lack of these studies is probably due to the technical limitations of performing the

surgical dissection of the suprascapular nerve and isolation of the single rotator cuff

tendon in the rat model. Supraspinatus muscle atrophy following chronic rotator cuff

tears may place excessive traction on the suprascapular nerve, contributing to functional

decline. Electromyography (EMG) is an inexpensive, minimally invasive test for rapid

electrophysiological assessment of patients during normal and athletic upper extremity

motion [48-50]. After establishing an in vivo rat shoulder model, the utility of EMG in

diagnosing rotator cuff neuromuscular dysfunction was examined.

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In Chapter 2, we describe the development of a functional rodent rotator cuff

animal model and studied the natural history of rotator cuff dysfunction after injury. The

rat shoulder is an established animal model for the study of human rotator cuff pathology

based on similarities in bony architecture of the shoulder region in these two species.

Unfortunately, most of the current rotator cuff literature has not addressed muscle and

nerve function after rotator cuff tears. The first goal of the doctoral thesis was to develop

a rat shoulder animal model to test in vivo muscle force and electromyography (EMG).

Muscle force testing was used to evaluate in vivo rat rotator cuff stimulation frequency-

dependent muscle recruitment, muscle-tendon unit displacement, single-twitch muscle

contraction, tetanic contraction, and fatigue in animals with acutely and chronically torn

rotator cuffs. Then EMG was performed in acute and chronic in vivo rat rotator cuff tear

model to study compound motor action potential (CMAP) amplitude and area under the

curve, as well as CMAP regional location heterogeneity. In Chapter 2, we were also able

to study passive changes to in vivo soft tissue biomechanical properties of the muscle-

tendon unit that occur after acute and chronic rotator cuff tears. We hypothesized that a

chronically torn rotator cuff tendon results in increased passive muscle-tendon unit

stiffness and reduced in vivo skeletal muscle contractile strength. Further, we

hypothesized that neuromuscular dysfunction can be detected by electromyography

(EMG) in the chronic tendon tear setting.

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NEUROMODULATION OF IN VIVO SOFT-TISSUE BIOMECHANICAL

PROPERTIES FOLLOWING BOTULINUM NEUROTOXIN A (BONT-A)

INJECTION: A POTENTIAL THERAPEUTIC STRATEGY TO IMPROVE

OPERATIVE OUTCOMES OF ROTATOR CUFF REPAIR SURGERY

Based on the findings from Chapter 2, we identified excessive repair tension as a

major determinant of reduced muscle function following rotator cuff tears. Surgical repair

of a large chronic rotator cuff tear requires manipulation of a muscle-tendon unit that is

scarred, retracted, and stiffer than normal secondary to dis-use muscle atrophy [38, 40,

41]. Altering the passive properties of skeletal muscle by chemical denervation has the

potential to improve the surgical manipulation of the muscle-tendon unit by reducing

muscle stiffness and may ultimately improve surgical outcomes. Surgical and

rehabilitation experience with patients who have sustained neurological insults such as

cerebral palsy demonstrates significant influence of the nervous system over the passive

biomechanical properties of muscle. Passive visco-elastic properties of skeletal muscle-

tendon units are key determinants of surgical and post-operative functional success [38,

40, 41]. Most tendon repair studies have attempted to overcome increased muscle-tendon

unit stiffness and improve healing by focusing on the tendon-to-bone interface through

improvement of repair site strength by varying surgical techniques and suture materials

[19-26]. Injection of BoNT-A into the skeletal muscle shifts attention for repair to the

proximal portion of the muscle-tendon unit. Altering the passive properties of skeletal

muscle by chemical denervation has the potential to improve the surgical manipulation of

the muscle-tendon unit and ultimately improve surgical outcomes. Further, this strategy

has the added benefit of “bioprotecting” the tendon repair site. Presumably, by causing

8

temporary chemical paresis, the muscle will not be able to generate enough active force

to rupture the surgically repaired tendon [51, 52].

In Chapter 3, we first tested the hypothesis that Botulinum neurotoxin A (BoNT-

A) injection temporarily and reversibly changes active and passive skeletal muscle

properties. Specifically, the chemical denervation effected by the toxin alters the visco-

elastic properties of the muscle when compared to control, saline injected skeletal

muscle. Chapter 3 is our first published study demonstrating that the soft-tissue passive

properties of the muscle-tendon unit can be altered via chemical denervation in a mouse

gastrocnemius animal model. We postulated and then proved that BoNT-A injection can

improve the surgical manipulation of the muscle-tendon unit.

In Chapter 4, we utilized the rat rotator cuff animal model to study the influence

and contribution of neuronal tone on passive skeletal muscle-tendon unit visco-elastic

properties after chemical denervation using BoNT-A. Fung’s quasi-linear visco-elastic

mathematical model was used to study the effects of chemical denervation on passive

muscle properties. A standard muscle force testing experimental apparatus was used to

record and analyze the load-relaxation characteristics of skeletal muscle after chemical

denervation with BoNT-A. In Chapter 4, we report on our mathematical and experimental

determination of the relative contribution of the nervous system to soft-tissue

biomechanical properties.

9

A NOVEL PARADIGM FOR ROTATOR CUFF RESEARCH: TRANSLATING

BASIC SCIENCE ANIMAL MODEL STUDIES TO CLINICAL

RECOMMENDATIONS USING SIMULATION ANALYSIS

In conclusion, Chapter 5 summarizes our basic science animal data and discusses

the current literature on rotator cuff injuries and repairs. We further attempt to translate

our animal studies into clinical recommendations using a computational human upper-

extremity movement biomechanics model to perform a simulation analysis. In Chapter 5,

we present data that explores passive and active muscle properties in the context of acute

and chronic rotator cuff tears. For chronic, full-thickness, rotator cuff tears, repair surgery

can be technically challenging due to large gap distances and increased stiffness of the

muscle-tendon unit. In contrast, acute rotator cuff tears are associated with lower repair

tensions, less fibro-fatty infiltration of the muscle, and better functional outcomes.

Chapter 5 demonstrates how animal models can be used to understand rotator cuff muscle

function in the acute and chronic tear setting and how these results can be translated into

clinical recommendations using computational modeling. Chapter 6 briefly discusses the

clinical relevance of our basic science findings presented in this thesis and outlines future

experiments that we would like to pursue, related to rotator cuff dysfunction and repair

surgery.

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17

CHAPTER 2

EVALUATION OF IN VIVO ROTATOR CUFF MUSCLE

FUNCTION AFTER ACUTE AND CHRONIC DETACHMENT OF

THE SUPRASPINATUS TENDON: AN EXPERIMENTAL STUDY IN

AN ANIMAL MODEL

Sandeep Mannava, Johannes F. Plate, Patrick W. Whitlock, Michael F. Callahan,

Thorsten M. Seyler, L. Andrew Koman, Thomas L. Smith, Christopher J. Tuohy

The following manuscript was published in JBJS (Am.). 2011 Sept 21. PMID: 21938374.

This chapter is reprinted with permission. Stylistic variations are due to the requirements

of the journal. Mannava performed the experiments, prepared the manuscript, and served

as corresponding author. T.L. Smith acted in an advisory and editorial capacity.

ADDENDUM: During committee review of the dissertation, it was determined that the

photomicrographs that comprise chapter 2, figure 2 may have been taken on two separate microscopes with

different focal lengths, resulting in images that have different magnification. The figure has been published

after peer-review. The study of muscle atrophy following tenotomy in the rat supraspinatus was extensively

studied by Barton et al (J Orthop Res. 2005 Mar; 23(2):259-65) who performed quantitative histological

analysis. Barton et al concluded that tendon detachment results in decreased muscle mass, decreased

muscle fiber size, and increased collagen content/fibrosis—these findings were significant when compared

to controls. For further information regarding histology of the rat rotator cuff after injury, please refer to the

manuscript Barton ER, Gimbel JA, Williams GR, Soslowsky LJ. Rat supraspinatus muscle atrophy after

tendon detachment. J Orthop Res. 2005 Mar; 23(2):259-65. PMID 15734235.

http://www.ncbi.nlm.nih.gov/pubmed/15734235�

18

ABSTRACT

Background: Surgical repair of large chronic rotator cuff tears can be technically

demanding because it requires manipulation of a muscle-tendon unit that is scarred,

retracted, and stiffer than normal, which contribute to increased repair tensions. This

study characterizes in vivo rotator cuff muscle-tendon unit function after acute and

chronic injury at surgically relevant preload tensions.

Methods: Sixty-two Sprague-Dawley rats—healthy uninjured controls (n=22), acute

injury group (n=20), and chronic injury group (n=20)—underwent in vivo muscle force

testing and electromyography (EMG) of their supraspinatus muscle-tendon unit at various

preload tensions.

Results: Preload tension affected the maximal supraspinatus muscle contractile force in

all experimental groups (p<0.05). At the peak tension required to repair an acute tear,

there was between a 28%-30% reduction in maximal tetanic contraction amplitude in all

experimental groups (p<0.05). At the peak tension required to repair a chronic tear, there

was between a 40%-53% reduction in maximal tetanic contraction amplitude in all

experimental groups (p<0.05). The uninjured control group showed increased muscle

endurance (p<0.05) compared to the acute injury and chronic injury experimental groups

at all preload tensions. The chronically injured group showed reduced compound motor

action potential (CMAP) amplitude (p<0.05).

Conclusions: Both acute and chronic injury experimental groups demonstrated a

functional impairment related to increasing preload tensions, with the only difference

being the amount of tension required for surgical repair. The present study also associates

19

increased time from rotator cuff tendon injury to impaired in vivo rotator cuff muscle

EMG findings.

Clinical Relevance: The findings suggest that earlier surgical intervention, requiring

lower repair tension, may lead to improved functional outcomes.

20

INTRODUCTION

With the increasing age and activity level of the population, there has been an

increase in both prevalence and operative interventions for symptomatic rotator cuff tears

[2, 3]. Often, surgical intervention for rotator cuff tears is delayed until after conservative

treatment has failed [3, 45]. Surgical repair of a large chronic rotator cuff tear may

require the manipulation of a muscle-tendon unit that is scarred, retracted, and stiffer than

normal [38, 40, 41]. These physical changes contribute to the technical challenge of

rotator cuff surgery.

Changes in the structure of the muscle-tendon unit after rotator cuff injury

contribute to the increased tension required to attach the torn rotator cuff tendon back to

its anatomic humeral head footprint [38, 45, 53-55]. The increased repair tension may

impair healing at the tendon-bone interface, contributing to recurrent tearing after surgery

[53]. Additionally, the function of chronically torn rotator cuff muscle-tendon units is

also impaired after injury [42-45, 56]. With current repair techniques, reduced muscle

function is sometimes irreversible, even after a relatively short period of time from injury

[55].

The present study characterizes rotator cuff muscle-tendon unit function under

acute and chronic injury conditions at surgically relevant preload tensions. The surgically

relevant preload tensions were chosen to recreate the stress placed upon the muscle-

tendon unit during rotator cuff repair surgery. These preload tensions were determined

based on previously reported repair tensions in this animal model [53]. Our study used a

rat rotator cuff model to study in vivo muscle force and electromyography (EMG) of the

rotator cuff after acute and chronic detachment of the supraspinatus tendon. The study

21

hypothesized that observed muscle function would be impaired at tensions required to

perform rotator cuff repair surgery and that electrophysiological EMG assessment of the

muscle would be impaired in the chronic tendon tear setting.

MATERIALS AND METHODS:

Muscle function in 26 male Sprague-Dawley rats weighing 400-450 g (Charles

River, Wilmington, MA) were studied. Six un-injured rats served as healthy controls; the

remaining 20 rats had unilateral supraspinatus tendon detachment by methods previously

described [53]. These animals were divided into an acute injury group (n=10) with

muscle function tested four weeks after supraspinatus tenotomy and a chronic injury

group (n=10) with muscle function tested twelve weeks after supraspinatus tenotomy.

Additionally, EMG was studied in a total of 36 male Sprague-Dawely rats. Sixteen un-

injured rats served as healthy controls. The remaining 20 rats had their supraspinatus

tendon sharply detached and were divided into an acute injury group (n=10) and a

chronic injury group (n=10). Animals were sacrificed immediately after in vivo muscle

force and EMG testing. All experimentation was conducted after approval from the

Institutional Animal Care and Use Committee.

Muscle Force Testing:

The surgical exposure and in vivo physiological testing of the rat supraspinatus

muscle-tendon unit were conducted based upon previously described experimental

methods [57], using a physiological testing apparatus (Fig. 1) [57, 58]. The muscle-

tendon unit then was pretensioned to tensions ranging between 0.05N and 4N. Gimbel et

al. determined that these preload tensions are clinically relevant for this animal model

[53, 54], approximately 1.7N of peak repair tension was required at four weeks post-

22

tenotomy, and approximately 2.5N of peak repair tension was required at twelve weeks

post-tenotomy [53, 54]. A maximal preload tension of 4N was used to attain a full

preload tension versus maximum amplitude curve without causing gross injury.

Suprascapular nerve stimulation protocols were executed at these varying preload

tensions. The suprascapular nerve was supra-maximally stimulated with 1.5V during

muscle force recordings. To ensure that 1.5V was a supra-maximal stimulation, a range

of stimuli were tested between 0.6V to 2.0V, and in all cases, 1.5V produced maximal

single twitch muscle contraction.

To test frequency-dependent muscle fiber recruitment, the suprascapular nerve

was stimulated at frequencies of 1Hz, 60Hz, 80Hz, 100Hz, 150Hz, and 200Hz for one

second, and the maximal amplitude of the force of contraction was recorded. A recovery

time of 60 seconds was allowed between stimuli. This recovery time allowed for full

recovery of maximal contractile force. For the frequency-dependant recruitment protocol,

nerve stimulation was performed at a preload tension of 0.4N, the tension required to

repair the supraspinatus tendon immediately after tenotomy [53, 54].

The compliance and stiffness of the muscle was assessed by measuring the

displacement of the muscle-tendon unit (from the starting preload tension of 0.05N,

which was set as 0mm displacement) required to attain the various preload tensions. After

the muscle-tendon unit was immobilized to the linearly translating stage (Fig. 1A), the

stage was displaced away from the force transducer, to preload tensions ranging from

0.05N to 4N, and the amount of displacement required to attain these tension was

recorded. To test tension versus maximal single twitch and tetany force of contraction,

the muscle-tendon unit was ranged from 0.05N to 4N of preload tension, and the nerve

23

was stimulated at 1Hz and 150Hz for five seconds with a recovery time of 60 seconds

between stimuli.

For the fatigue protocol, the nerve was stimulated at 100Hz for 150 seconds at

preload tensions of 0.4N, 1.3N, and 2N with 300 seconds between stimuli to allow for

complete recovery. The amplitude of contraction was continually measured and recorded

throughout the entire 150 seconds of nerve stimulation. These data were expressed as

change from maximum standardized amplitude as a function of time at one second (max

amplitude), 30 seconds, 60 seconds, 90 seconds, 120 seconds, and 150 seconds.

Muscle Force Data Standardization

All muscle force measurements during testing were expressed as percentages of

the maximal contractile force generated by the tested muscle in order to normalize for

animal variability. Physiological testing in the in vivo setting results in data that is

variable due to experimental preparation and other environmental differences beyond the

control of the investigator. Increasing the number of animals studied could have reduced

the variability of the muscle force measurements; but in the present study this was not

feasible due to the large number of animals that would have been required. Therefore, in

order to compare the animals and groups, standardization was necessary. Previously

published reports used similar percentages in expressing their data [42-44], normalizing

against either the same muscle or the contralateral muscle in order to facilitate the

comparison of contractile amplitudes between animals and groups. After consultation

with a statistician, use of the contralateral muscle for standardization was determined to

be inappropriate because it was possible that muscle function of the contralateral

uninjured limb could be affected by injuring the opposite supraspinatus tendon.

24

EMG Testing

EMG testing was performed using a Sierra Wave (Caldwell Laboratories,

Kennewick, WA) electrodiagnostic system. EMG testing consisted of supramaximal

stimulation of the suprascapular nerve for a duration of 0.1ms with a range of constant

currents (0.5mA to 2mA) to ensure that maximal compound motor action potential

amplitude was attained. Maximal compound motor action potential amplitude and area

under the curve (AUC) were recorded using a needle electrode placed at different

locations (proximal, middle, distal) on the supraspinatus muscle belly. The testing

protocol was repeated three times at each location, and the single result with the highest

amplitude and appropriate compound motor action potential waveform morphometry was

recorded.

Histological analysis

A total of 18 samples were collected from the uninjured control (n = 6), four

weeks post-tenotomy (n = 6), and twelve weeks post-tenotomy (n = 6) groups, after

muscle force testing. The mid-substance portion of the supraspinatus muscles were

processed for histology, embedded in paraffin, and sectioned with a microtome to obtain

5.0 μm thick cross-sections. The sections were mounted on slides and stained using

hemotoxylin and eosin (H & E, Sigma, St. Louis, MO), Masson’s Trichrome (Sigma, St.

Louis, MO), and 4,6-diamidino-2-phenylindole (DAPI, Vector, Burlingame, CA).

Representative light (H&E and Masson’s Trichrome) and fluorescence (DAPI)

micrographs were taken at 200x magnification. For DAPI stained sections, three high-

powered 200x, representative photomicrographs were taken per sample and then a

modified counting procedure [59] was used to quantify the fluorescence using ImageJ

25

(NIH, Bethesda, MD). Several of the samples were counted manually to verify the

accuracy of the automated quantification method. The quantified DAPI fluorescence was

averaged for the sample, and the average of the three images was used for further

statistical comparisons between groups. This method of analysis was employed to help

reduce sampling error.

Statistics

For a desired power of 0.8 and α = 0.05, a sample size of n = 5 for each group was

calculated. This calculation was consistent with historical physiological data in a rat

skeletal muscle model system [51, 57]. Muscle force measurements were converted to

percentages, and the data were expressed as averages ± standard error of measurement

(SEM) for each group. All other measurements were expressed as averages ± standard

error of the mean (SEM) for each group. For comparisons between and within groups, a

Two-Way Repeated Measures ANOVA followed by a Tukey post hoc test was

performed. An ANOVA followed by a Tukey post hoc test was used for comparisons of

DAPI quantification, maximal compound motor action potential amplitude, and

compound motor action potential area under the curve between groups. Significance was

defined as p<0.05. A linear regression was generated using the tension applied versus

displacement of the muscle-tendon unit data. Stiffness/compliance was calculated based

on regression analysis.

Source of Funding

All studies were completed with intra-departmental physician-scientist start-up funds. No

external funding source was utilized and no external funding source played a role in this

investigation.

26

RESULTS

Histological analysis revealed an increase in inter-fascicular area in the post-

injury groups compared to the uninjured control group (Figs. 2A-F). There was evidence

of inter-fascicular fibrosis in the Masson’s Trichrome stain for the injured groups (Figs. 2

E-F). The uninjured control group appeared to have more highly organized and densely

packed muscle fibers when compared to the muscles that had undergone tenotomy (Figs.

2A-F). There also appeared to be less inter-fascicular space in the twelve-weeks post-

tenotomy group when compared to the four-weeks tenotomy group (Figs. 2B-C and E-F).

However, the muscle fibers in the twelve-weeks post-tenotomy group were not as densely

packed as the uninjured control group (Figs. 2A and C, D and F). DAPI stain indicated

that there was a decrease in nuclear material in the tenotomized groups when compared to

uninjured control muscles (Figs. 2G-J). The reduction in DAPI fluoresence was

statistically significant between the uninjured control group and the tenotomy groups

(p<0.05, Fig. 2J). Significant differences were not detected between the two tenotomy

groups.

As preload tension increased, the displacement of the muscle-tendon unit also

increased in all three groups (Fig. 3). There was a statistically significant interaction

between preload tension and time from injury (p<0.05, Fig 3 & Table 1). At 1.3N of

preload tension, post-hoc testing revealed there was a difference between the control

group and the four-weeks post-tenotomy group (p<0.05) as well as between the control

group and the twelve-weeks post-tenotomy group (p<0.05). At 2.1N of preload tension,

there was a difference between the control versus four-weeks post-tenotomy group

(p<0.05), control versus twelve-weeks post-tenotomy group (p<0.05), and four-weeks

27

post-tenotomy versus twelve-weeks post-tenotomy groups (p<0.05). No differences in

compliance/stiffness were detected between groups below 1.3N (Fig. 3). Linear

regression analysis was used to calculate both the stiffness and compliance of the

supraspinatus muscle-tendon units in vivo (Table 1). The data indicate that both the four-

weeks post-tenotomy and twelve-week post-tenotomy groups had reduced compliance or

increased stiffness when compared to the control group (p<0.05, Table 1).

Increasing the frequency of stimulation resulted in an increased force of

contraction (Fig. 4). There was no significant difference between groups at each of the

stimulation frequencies, but there was a significant effect of frequency of stimulation on

standardized maximum amplitude (p<0.05).

The effect of preload tension on maximal supraspinatus muscle single twitch

contraction amplitude was statistically significant (p<0.05, Fig. 5A). The effect of the

time from injury on single twitch was not statistically significant. Post-hoc Tukey test

showed a significant decrease in the maximum single twitch force generated at 1.7N and

2.5N of preload tension from maximum twitch generated in all of the groups (p<0.05).

For all groups, there was between a 22%-45% reduction in maximal single twitch

amplitude at acute repair tensions and between a 45%-56% reduction in maximal single

twitch amplitude at chronic repair tensions. The preload tensions that produced the

maximal standardized amplitude for supraspinatus single twitch ranged between 0.2N to

1N for all experimental groups.

The effect of preload tension on maximal supraspinatus muscle tetanic contraction

amplitude was statistically significant (p<0.05, Fig. 5B). The effect of the time from

injury (uninjured control, four weeks post-tenotomy, and twelve weeks post-tenotomy)

28

on standardized tetanic contraction amplitude was not statistically significant. Post-hoc

Tukey test showed a significant (p<0.05) decrease in the maximum tetanic force

generated at 1.7N and 2.5N of preload tension from maximum tetanic contraction

generated in all groups. This reduction in force was greater than 28% maximal tetanic

contraction at acute repair tensions, and between 40%-53% at chronic repair tensions.

The preload tensions that produced the maximal standardized amplitude for supraspinatus

tetanic contraction ranged between 0.4N to 0.8N for all experimental groups.

The uninjured control group showed significantly increased muscle endurance, or

functional capacity (p<0.05) compared to the four-weeks post-tenotomy and twelve-

weeks post-tenotomy groups at all preload tensions (Fig. 6). There also was significantly

increased muscle endurance or functional capacity (p<0.05) for the twelve-weeks post-

tenotomy group when compared to the four-weeks post-tenotomy group at the lower

preload tension of 0.4N. This relationship was not significantly different between four-

weeks post-tenotomy and twelve-weeks post-tenotomy groups in the higher preload

tensions of 1.3N and 2N.

The maximal compound motor action potential amplitude of the supraspinatus

muscle was influenced by the time from the initial supraspinatus tendon injury (Figs. 7A

and C). There was a statistically significant difference between the maximal compound

motor action potential amplitudes between the groups (p<0.05). Tukey post-hoc testing

showed a statistically significant difference between the control and twelve-weeks post-

tenotomy groups (p<0.05), the four-weeks post-tenotomy and twelve-weeks post-

tenotomy groups (p<0.05), but no difference was detected between the control group and

29

the four-weeks post-tenotomy group. No significant difference was detected for the area

under the curve between the experimental groups (Fig. 7B).

The heterogeneity of compound motor action potential amplitude was related to

the location of the recording needle in the supraspinatus muscle (Fig. 7C). There was a

significant difference between the compound motor action potential amplitude between

groups (p<0.05, Figs. 7A and C). There was also a significant difference in maximal

compound motor action potential amplitude based on the location of the recording

electrode on the supraspinatus muscle within the experimental groups (p<0.05). The

Tukey test post-hoc comparison indicated a significant decline in compound motor action

potential amplitude between the proximal and distal locations (Fig. 7C, p<0.05) and

between the proximal and middle locations (Fig. 7C, p<0.05) in the control and four-

weeks post-tenotomy groups. No significant difference was detected in the maximal

compound motor action potential amplitudes between any of the recording locations in

the twelve-weeks post-tenotomy group.

DISCUSSION

Muscle function is significantly impaired at the tensions required to perform

rotator cuff reconstruction surgery. Changes in the muscle-tendon unit after rotator cuff

injury include decreased muscle volume, increased fat content of the muscle, altered

muscle fiber composition, increased fibrotic infiltration of the muscle [36-41, 60],

alterations in the muscle architecture [35, 36], and changes in the molecular composition

of the muscle [35, 36]. These structural changes result in a rotator cuff muscle-tendon

unit that is difficult to manipulate during surgery [38, 40, 41]. Our data demonstrate that

30

the preload tensions applied to the acutely and chronically torn rotator cuff muscle-

tendon units during repair result in significantly decreased function.

The frequency dependent recruitment of supraspinatus muscle fibers was similar

regardless of injury group (Fig. 4). Even after tenotomy, the supraspinatus muscle is still

able to produce force when stimulated under certain supra-physiological conditions.

However, current single-stage repair methods using high repair tensions and

physiological nerve activation below the frequencies tested in this experiment may result

in decreased muscle function.

Similar to previous reports, muscle-tendon unit stiffness increased with time from

injury (Fig. 3, Table 1) [38, 40, 41]. There are several possible molecular mechanisms,

including change in sarcomere length and histological changes in muscle structure, which

might contribute to increased stiffness of the muscle-tendon unit after injury [35, 36].

Changes in muscle structure observed histologically (Fig. 2) might be partially

responsible for changes in passive biomechanical properties of the muscle-tendon unit

(Fig 3, Table 1).

Our study demonstrates, that the chronically injured group’s muscle-tendon unit

might gain some compliance (inversely related to stiffness, Table 1). An explanation for

this trend is that secondary attachment due to scarring of the detached tendon to

surrounding tissue results in the re-establishment of load during muscle contraction [42].

This attachment might result in partial reversal of the atrophic changes (Fig. 2) that occur

in the muscle after tendon injury, resulting in improved passive properties (Fig. 3, Table

1). The muscle does not recover fully after the repair of a chronic tear because the tendon

does not heal in a location where the muscle is ideally preloaded or stretched.

31

Muscle function testing demonstrated that there was a significant decline in the

force of muscle contraction at the peak tensions required to repair acutely injured and

chronically injured rotator cuff tears (Figs. 5A and B) [53, 54]. After internally

standardizing the amplitude of supraspinatus contraction, all of the groups responded to

increased preload tension similarly. This finding may indicate that an increased emphasis

needs to be placed on the tensions at which tendons are repaired, as muscle function was

significantly impaired at high repair tensions.

The exercise capacity of supraspinatus muscles associated with completely torn

tendons declines (Fig. 6). The difference between the chronically injured and acutely

injured group might be partially explained by the re-establishment of contraction after

secondary attachment of the torn tendon to surrounding tissue [42]. Our data showed that

as preload tension increases from 0.4N to 1.3N and 2N, the difference between the

acutely injured and chronically injured group was not significant. The greater strain

placed on these muscles associated with the injured tendons resulted in the impaired

ability of the muscle to sustain maximal contraction for an extended period of time. In the

setting of acute and chronic rotator cuff tears, the peak surgical repair tensions would be

sufficient to result in a significantly reduced ability for the muscle to sustain contraction.

This finding has implications for recommendations made for post-operative rehabilitation

protocols and exercise capacity [61-63].

Other investigators have demonstrated that rotator cuff muscles, associated with

torn rotator cuff tendons, fatigue faster and have a lower maximum force of contraction

after injury [42-45, 56, 64, 65]. Our study represents a more complete characterization of

muscle function at surgically relevant preload tensions that simulate the stress placed

32

upon the muscle-tendon unit during and following rotator cuff reconstruction surgery. It

is unclear how accounting for the increased tension will influence outcomes after repair

of torn rotator cuffs. Since a broad range for ideal repair tension was observed in our

study, future studies are needed to investigate, determine, and optimize the ideal repair

tension when repairs are performed.

Neuromuscular function of the supraspinatus muscle as detected by EMG is

significantly impaired after chronic injury to the supraspinatus tendon (Figs. 7 A and C).

Electrophysiological impairment of the supraspinatus muscle following tenotomy may

indicate that suprascapular nerve dysfunction contributes to decreased function following

rotator cuff tears (CME). However, it is possible that these observed compound motor

action potential changes are due to muscle architecture changes described previously in

the literature [36-41, 60] and observed in our histological analysis (Fig. 2). Alternatively,

the decreased compound motor action potential amplitude may result from a combination

of both nerve traction and muscle atrophy. Recently, nerve injury has been implicated as

possibly inciting some of the structural muscular changes that occur after rotator cuff

tendon detachment [29, 66].

Several case series have described an association between rotator cuff tears and

suprascapular neuropathy [30, 67]; however, the present study makes a direct connection

between impaired EMG findings and chronicity of the rotator cuff tear (CME). Future

studies are needed to characterize the mechanisms underlying the electrophysiological

changes observed in the present study, such as nerve tension after tenotomy or specific

histological markers for neuromuscular changes. Ideally nerve conduction velocity would

also be studied during experimentation. However, technical and anatomical limitations of

33

the in vivo model did not allow for consistent placement of the stimulator and the

recording electrode at a fixed distance to allow for measurement of the distance between

the stimulating electrode and the recording electrode.

Limitation of the physiological testing related to replication of joint-biomechanics

and the use of general anesthesia when using the rat rotator cuff animal model system are

previously noted in the literature [57]. The testing of more animals would have reduced

variability and increased the power of the study, perhaps revealing a significant

experimental group effect on preload tension versus maximal standardized muscle force

generated. Based upon the confidence intervals (Fig. 5), we conclude that a statistically

significant difference would probably not represent a physiological or clinically relevant

difference in this animal system. The experimental groups responded similarly to preload

tension, with increasing preload tensions reducing the force of muscle contraction

(p<0.05, Fig. 5).

The four-weeks and twelve-weeks post-tenotomy time-points were selected to

represent an acute and chronic rotator cuff tear, as the repair tension essentially plateaus

at twelve weeks post-tenotomy [53, 54]. In this rat rotator cuff model, spontaneous

healing and formation of a pseudo-tendon probably results in a reversal of atrophy, which

is not consistent with the human condition. Further, this animal model does not undergo

the robust fibro-fatty infiltration (Fig. 2) that is observed in chronic human rotator cuff

tears and large animal rotator cuff chronic injury models [37-39, 68, 69].

Based on the findings of our study, it is likely that impaired rotator cuff muscle

function at the peak repair tensions required to perform rotator cuff repair surgery will

disrupt the balance of gleno-humeral joint forces, thus affecting post-operative shoulder

34

biomechanics. Both acute and chronic injury experimental groups demonstrated a

functional impairment related to increasing preload tensions, with the only difference

being the amount of tension required at the time of surgical repair. Similarly, Davidson et

al demonstrated that excessive repair tension (in excess of eight pounds force) is

associated with worse clinical outcomes, when compared to patients whose rotator cuffs

required lower tension for repair (approximately two pounds force) [70]. Further,

impaired neuromuscular activity associated with the chronically torn rotator cuff may

contribute to impaired function following injury. Our study directly associates impaired

EMG findings to time from injury of the rotator cuff tendon. The findings suggest that

functional outcomes may be improved by earlier intervention at lower repair tensions;

however, future studies are needed to test this hypothesis.

ACKNOWLEDGEMENTS

We would like to thank Dr. Beth P. Smith, Dr. Riyaz H. Jinnah, Mrs. Eileen

Martin, and Mrs. Casey Northam for their support in the planning and execution of the

experiments, as well as useful suggestions in the preparation of this manuscript.

This project is taken in part from a dissertation submitted to the Neuroscience

Program, Wake Forest University Graduate School of Arts and Sciences, in partial

fulfillment of the requirements for the degree of Doctor of Philosophy.

35

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FIG 1: Experimental Apparatus. The experimental apparatus and key components are

labeled in these schematic diagrams. Panel A: A view of the entire physiological

apparatus is depicted. Panel B: The image focuses on the rat supraspinatus muscle-tendon

unit during force recording.

46

Chapter 2, Fig. 1

47

FIG 2: Histological Analysis of Mid-Substance Supraspinatus Muscle After Tenotomy.

After tenotomy, there is an increase in inter-fascicular space, fibrotic infiltration, and

muscle atrophy when compared to the highly order, densely packed, uninjured control

skeletal muscle (H & E, Panels A-C; Masson’s Trichrome, Panels D-F). The post-

tenotomy muscle also displayed a significant decline in nuclear material, as quantified by

measurement of DAPI fluorescence (Panels G-J; p<0.05 compared to uninjured controls,

denoted by *). Data are presented as the mean and standard error of the mean.

Photomicrographs taken at 200x and scale bars represent 20µm.

48

Chapter 2, Fig. 2

49

FIG 3: The Effect of Preload Tension on Displacement of the Supraspinatus Tendon. The

displacement of the supraspinatus muscle-tendon unit increases as preload tension

increases. A significant difference exists between the muscle-tendon unit displacement of

the control group and the 4 weeks post-tenotomy group at repair tensions above 1.3N

(p<0.05, denoted by *). Significant differences between the muscle-tendon unit

displacement of the control group and the 12 weeks post-tenotomy group (p<0.05), the

control group and the 4 weeks post-tenotomy group (p<0.05), and finally the 4 weeks and

12 weeks post-tenotomy groups (p<0.05) were detected at preload tensions above 2.1N

(denoted by ψψ). Data are presented as the mean and standard error of the mean.

50

Chapter 2, Fig. 3

51

FIG 4: Frequency Dependent Recruitment of Supraspinatus Muscle Fibers. Increasing

the frequency of stimulation resulted in an increased force of contraction in all

experimental groups. There is no significant difference between uninjured control, 4

weeks post-tenotomy, and 12 weeks post-tenotomy experimental groups at each of the

stimulation frequencies. Data are presented as the mean and standard error of the mean.

52

Chapter 2, Fig. 4

53

FIG 5: The Effect of Preload Tension on Supraspinatus Muscle Contraction. Peak

tensions required to repair a 4 weeks post-tenotomy and 12 weeks post-tenotomy rat

supraspinatus tendon (as determined by Gimbel et al. [21,22]) are denoted by the dashed-

dotted line. Panel A: The amount of preload tension significantly affected the maximal

supraspinatus muscle single twitch contraction in all experimental groups (p<0.05). For

all groups, there was between a 22%-45% reduction in maximal single twitch amplitude

at 4 week post-tenotomy repair tensions and between a 45%-56% reduction in maximal

single twitch amplitude at 12 week post-tenotomy repair tensions. Both of these

reductions in contractile force were significant (p<0.05). Panel B: The amount of preload

tension significantly affected the maximal supraspinatus muscle tetanic contraction

amplitude in all experimental groups (p<0.05). At the peak tension required to repair a

rotator cuff at 4 weeks post-tenotomy, there was between a 28%-30% reduction in

maximal tetanic contraction amplitude in all experimental groups (p<0.05). At the peak

tension required to repair a rotator cuff at 12 weeks post-tenotomy, there was between a

40%-53% reduction in maximal tetanic contraction amplitude in all experimental groups

(p<0.05). Data are presented as mean and standard error of the mean.

54

Chapter 2, Fig. 5

55

FIG 6: The Effect Of Time From Injury And Preload Tension On Supraspinatus Muscle

Endurance. The uninjured control group showed significantly greater muscle endurance

(p<0.05) compared to the 4 weeks post supraspinatus tenotomy and 12 weeks post

supraspinatus tenotomy experimental groups at all repair tensions. The 12 weeks post-

tenotomy group had significantly increased muscle endurance when compared to the 4

weeks post-tenotomy group at 0.4N repair tension (p<0.05), but this difference between

experimental groups was not significant at the higher preload tensions of 1.3N and 2.0N.

The data are expressed as change from maximal amplitude over time. The greater the

change from maximal amplitude indicates an inability to sustain maximal contraction.

Data are presented as mean and standard error of the mean.

56

Chapter 2, Fig. 6

57

FIG 7: The Effect Of Time From Injury On Supraspinatus Electromyography (EMG)

With Suprascapular Nerve Stimulation. Panel A: The chronic injury group has

significantly lower maximal compound motor action potential (CMAP) amplitude when

compared to both the uninjured control (p<0.05, denoted by *) and acutely injured groups

(p<0.05, denoted by *). Panel B: No significant differences were detected between the

experimental groups when evaluating the area under the curve (AUC) of the compound

motor action potential. Panel C: The heterogeneity of compound motor action potential

amplitude was related to the location of the recording needle in the supraspinatus muscle.

The regional heterogeneity in compound motor action potential amplitude exists in the

control group between the proximal and distal (p<0.05, denoted by *) and proximal and

middle locations (p<0.05, denoted by *). Similarly, a significant difference existed in the

4 weeks post-tenotomy group between the proximal and distal locations (p<0.05, denoted

by ψ) and between the proximal and middle locations (p<0.05, denoted by ψ). This

decline in maximal compound motor action potential amplitude, detected in the control

and 4 weeks post-tenotomy groups, was not detected in the 12 weeks post-tenotomy

group. There was not a statistically significant difference in the maximal compound

motor action potential amplitudes between any of recording locations in the 12 weeks

post-tenotomy group. Panel D: The experimental apparatus and key components are

labeled in this schematic diagram. The image focuses on the rat supraspinatus muscle-

tendon unit during EMG recording. The recording ELECTRODE locations are labeled

‘D’ for distal, ‘M’ for middle, and ‘P’ for proximal. All data are presented as mean and

standard error of the mean.

58

Chapter 2, Fig.7

59

Chapter 2, Table 1

Group

Slope Compliance

(mm/N) R2 P

Inverse of Slope Stiffness

(N/mm)

Control (n=5) 3.255 ± 0.214 0.974 <0.05 0.307 ± 0.021

Acute Injury (n=10) 1.7918 ± 0.094 * 0.929 <0.05 0.558 ± 0.029 *

Chronic Injury (n=7) 2.396 ± 0.092 *, ‡ 0.966 <0.05 0.417 ± 0.016 *

Table 1. Mean Linear Regression Analysis for Compliance/Stiffness Data * Signficant difference (p<0.05) when compared to the control group, Tukey post-hoc

‡ Signficant difference (p<0.05) when compared to the acute group, Tukey post-hoc

Average compliance and stiffness data expressed as MEAN ± SEM

60

CHAPTER 3

CHEMICAL DENERVATION WITH BOTULINUM NEUROTOXIN

A IMPROVES THE SURGICAL MANIPULATION OF THE

MUSCLE-TENDON UNIT: AN EXPERIMENTAL STUDY IN AN

ANIMAL MODEL

Sandeep Mannava, Michael F. Callahan, Simon M. Trach, Walter F. Wiggins, Beth P.

Smith, L. Andrew Koman, Thomas L. Smith, Christopher J. Tuohy

The following manuscript was published in the J Hand Surg Am. 2011

Feb;36(2):222-31. PMID: 21276885. This chapter is reprinted with permission.

Stylistic variations are due to the requirements of the journal. S. Mannava performed

the experiments, prepared the manuscript, and served as corresponding author. T.L.

Smith acted in an advisory and editorial capacity.

61

ABSTRACT

Purpose: The chemical denervation that results from botulinum neurotoxin A (BoNT-A)

causes a temporary reversible paresis that may result in easier surgical manipulation of

the muscle-tendon unit in the context of tendon rupture and repair. The purpose of the

study was to determine if BoNT-A injections can be utilized to temporarily and

reversibly modulate active and passive skeletal muscle properties.

Methods: Male CD1 mice weighing 40-50g were divided into a 1 week post-injection

group (n=13: n=5 saline and n=8 BoNT-A) and a 2 week post-injection group (n=17: n=7

saline and n=10 BoNT-A). The animals underwent in vivo muscle force testing and in

vivo biomechanical evaluation.

Results: There was a significant decline in the maximal single twitch amplitude (p<0.05)

and tetanic amplitude (p<0.05) at 1 week and at 2 weeks after BoNT-A injection, when

compared to saline injected controls. BoNT-A injection significantly reduced the peak

passive properties of the muscle-tendon unit as a function of displacement at 1 week

(p<0.05). Specifically, the stiffness of the BoNT-A injected muscle-tendon unit was

0.417 N/mm compared to the control saline injected group which was 0.634 N/mm, a

35% reduction in stiffness (p<0.05).

Conclusions: Pre-surgical treatment with BoNT-A may improve the surgical

manipulation of the muscle-tendon unit, thus improving operative outcomes. The results

implicate neural tone as a substantial contributor to the passive repair tension of the

muscle-tendon unit. The modulation of neural tone through temporary reversible paresis

is a novel approach that may improve intra-operative and post-operative passive muscle

properties, allowing for progressive rehabilitation while protecting the surgical repair site.

62

Type of Study: Basic Science

Key Words: Botulinum Neurotoxin Type A (BoNT-A); Chemical Denervation; Skeletal

Muscle; Tendon Repair Surgery;

63

INTRODUCTION

Tendon injuries commonly cause disability and place a high cost burden on the

United States economy [1]. Surgical repair of torn muscle-tendon units can be technically

demanding. In certain settings, these injuries have been associated with high rates of re-

tearing and continued diminished functional capacity after surgery [2-6]. In particular,

hand zone 2 flexor tendon repairs have been associated with re-rupture rates ranging from

0% to 16.6% [6-9]. Factors associated with poor post-operative outcomes following

muscle-tendon unit repair have been studied extensively in torn rotator cuff and include

advanced age [2], diminished tendon quality [10], muscle atrophy [11], and prolonged

time from injury [2, 12, 13].

After tendon rupture, the muscle undergoes many structural changes as it

atrophies including fibro-fatty infiltration of the muscle belly [14-18], decreased capillary

density [19], and reduced muscle volume [15, 16, 20]. These structural changes result in a

stiff and retracted muscle-tendon unit that is difficult to manipulate surgically [15, 21-

23]. After undergoing these changes, the repair tension required to re-attach the ruptured

muscle-tendon unit to its anatomical insertion progressively increases from the time of

injury. This increased repair tension results in impaired healing biomechanics [24, 25].

Historically, most tendon repair studies have attempted to overcome increased muscle-

tendon unit stiffness and improve healing by focusing on the tendon-to-tendon and the

tendon-to-bone interfaces. These studies documented improved repair site strength using

varying surgical techniques and suture materials [26-33]. More recently, there have been

attempts to completely eliminate repair tension through muscle paralysis by chemical

denervation using botulinum neurotoxin A (BoNT-A) and cast immobilization [34].

64

BoNT-A is a widely used pharmacological agent that, when injected

intramuscularly, can modulate skeletal muscle contractility by inhibiting presynaptic

release of acetylcholine [35, 36]. The chemical denervation that results from BoNT-A

causes a temporary reversible paresis that may result in easier surgical manipulation of

the muscle-tendon unit. These results have implications for potentially improving upper

extremity tendon repair surgery for ruptured zone II flexor tendons, biceps tendons,

triceps tendons, and rotator cuff. To date, most studies have focused on the active

isometric contractile properties of skeletal muscle after BoNT-A injection at repair

tensions below what is required to successfully repair the muscle-tendon unit to its

anatomical insertion [37-39]. The purpose of the present study was to characterize the

active contractile properties of the muscle-tendon unit over a wide range of surgically

relevant preload tensions after chemical denervation and recovery using a well-

characterized murine model. Further, the current study characterizes the neuronal

contribution to passive skeletal muscle tone. The study hypothesis was that BoNT-A

injections can be utilized to temporarily and reversibly alter active and passive skeletal

muscle properties at surgically relevant preload tensions and displacement distances.

MATERIALS AND METHODS

A total of 30 male CD1 mice weighing 40-50g (Charles River, Wilmington, PA)

were studied after approval from the Institutional Animal Care and Use Committee in

accordance with standards set forth by the National Institutes of Health and any national

law on the care and use of laboratory animals. While housed in the controlled

temperature vivarium with 12 hour light-dark cycles, chow and water were provided to

the mice ad libitum. The animals were divided into a 1 week post-injection group (n=13:

65

n=5 saline and n=8 BoNT-A) and a 2 week post-injection group (n=17: n=7 saline and

n=10 BoNT-A). All animals were immediately injected with a lethal dose of KCl under

general anesthesia after in vivo muscle function testing. All experimentation was triple-

blinded in that one scientist injected the animal, a second scientist performed all surgery,

and a separate third scientist performed the in vivo muscle function testing. The second

and third scientists were not aware of the injection status (saline versus BoNT-A) until

after experimentation and analysis was completed.

Botulinum Toxin Injection

Botulinum neurotoxin A (Allergan, Irvine, CA) was reconstituted as previously

described [37-39]. Briefly, a 100U vial of lyophylized BoNT-A was reconstituted with

normal saline. The toxin was injected into the gastrocnemius muscle at a dosage of 3

units/kg body weight in an injection volume of 10 µL with a Hamilton syringe (Fischer

Scientific, Pittsburgh, PA) equipped with a 30 gauge needle. For the control animals,

normal saline (0.9%) was injected in a similar manner. The dose and volume were chosen

based on previous experiments using this animal model with this dosing schedule, which

produced temporary reversible paresis in a relatively short period of time (i.e. 2 weeks)

[37]. All injections were performed percutaneously. Each head of the left gastrocnemius,

medial and lateral aspects, was injected with equal volumes (5 µL) and units of toxin

while the animals were anesthetized with isoflurane (Webster Veterinary, Patterson

Companies Inc., Sterling, MA).

Assessment of Active and Passive Muscle Properties

Control saline injected (n=12) and BoNT-A injected mice (n=18) were

anesthetized using isoflurane (1.0 – 1.5 vol % in oxygen) and remained under inhalation

66

anesthesia for the duration of the experiment. The surgical approach and isolation of the

gastrocnemius muscle and the sciatic nerve has been described previously [37-39]. After

the muscle and nerve were isolated with the aid of a dissection microscope, the mouse

was moved and strapped to a linearly translating stage. The lower limb of the animal was

pinned at the ankle joint and the femur with Kirschner wires to isolate gross movement.

In all cases, the strap and pins ensured that all contractile and passive force produced by

the gastrocnemius muscle-tendon unit translated perpendicular and “in line” with the

force transducer. After the mouse was sufficiently immobilized, the calcaneus was

disarticulated with the Achilles tendon still attached. The muscle-tendon-bone were then

attached to a force transducer (model FORT100; World Precision Instruments Inc.,

Sarasota, FL) with wire suture (Figure 1). The transducer was connected to an amplifier

(model 13-G4615-50; Gould, Cleveland, OH) through an interface controller (EMKA

Technologies Inc., Falls Church, VA) and an analog-to-digital converter card

(model:PCI-6023E, National Instruments, Austin, TX) connected to a personal computer.

The sciatic nerve then was stimulated using a bipolar hook electrode (FHC,

Bowdoinham, ME) and a pulse generator (model Tenma TGP110, BioSurplus Inc., San

Diego, CA). To electrically isolate the simulating hook electrode and sciatic nerve from

the surrounding tissue, a piece of Silastic® sheeting (Dow Corning Co., Midland, MI)

was wrapped around the exterior of the nerve and electrode. The stimulation protocol, the

force of muscle contraction, and the passive muscle-tendon unit properties were

controlled and recorded by IOX software, Slow Wave Analyzer (EMKA Technologies

Inc., Falls Church, VA). The body temperature of the mice was maintained during

experimentation with an external heat lamp.

67

Active muscle force testing consisted of supramaximal sciatic nerve stimulation to

produce a maximal single twitch contraction. A range of stimuli were tested between

0.6V and 2.0V, and maximal single twitch was recorded. In all cases, 1.5V was sufficient

to produce maximal single twitch contraction; thus, this voltage was used throughout

experimentation. The muscle-tendon unit then was pretensioned to preload tensions

ranging between 0.05N to 1N, the maximal preload tension of 1N corresponded to

approximately twice the average mouse body weight. The maximal preload tension of 1N

reduced the maximal amplitude of contraction without causing gross injury to the muscle-

tendon unit. To test preload tension versus maximal single twitch and tetany force of

contraction, the muscle-tendon unit was ranged from 0.05N to 1N of preload tension, and

the nerve was stimulated at 1Hz and 150Hz for 5 seconds with a recovery time of 60

seconds between stimuli. This rest period allowed for full recovery of maximal

contractile force.

Peak passive muscle-tendon unit properties were assessed after muscle force

testing was completed. The linear translating stage, to which the mouse was securely

attached, was displaced 1mm, 2mm, 3mm, 4mm, and 5mm. The peak passive force

produced by displacing the muscle-tendon unit from 0mm to each of the displacements

(1mm-5mm) was recorded by the force transducer. Between each of the displacement

experiments, the muscle-tendon unit was returned to 0mm displacement (set to 0.05N

preload tension) and was allowed to rest for 5 minutes.

Statistical Analysis

A power analysis was conducted prior to experimentation to determine the sample

size needed in each experimental group to detect statistically significant differences. For

68

a desired power of 0.8 and α = 0.05, a sample size of n = 5 for each group was calculated.

This calculation was consistent with a previous botulinum neurotoxin A report in this

animal model [37]. To ensure adequate statistical power, a minimum of n = 6 mice in

each experimental group was tested.

All values were expressed as the mean ± standard error of the mean. All statistical

analysis was performed using SigmaStat (Systat Software, Inc., San Jose, CA). T-test or

Mann-Whitney U Non-Parametric Test was used for comparison of maximal single

twitch and maximal tetanic contraction force at 1 week and 2 weeks post-injection

between groups (saline versus BoNT-A). For comparisons of the preload tension versus

maximal contraction (single and tetanic) between and within groups, a two-way repeated

measures ANOVA followed by Tukey post hoc test was performed. Additionally, two-

way repeated measures ANOVA followed by Tukey post hoc test and a linear regression

analysis assessed peak passive tension versus displacement of the muscle-tendon unit

data. A significance level of p ≤ 0.05 was used for all tests.

RESULTS

The injection of BoNT-A resulted in a significant decline (p<0.05) in both

maximal single twitch and tetanic contraction at both 1 and 2 weeks post-injection when

compaired to saline injected gastrocnemius muscles (Figure 2A-D). The BoNT-A group

had an average maximal single-twitch contractile force of 0.236 N ± 0.0199 N (saline

injected group had a single twitch contraction of 0.812 N ± 0.123 N) at 1 week post-

injection. There was a partial single twitch recovery of active force to 0.543 N ± 0.121 N

(saline injected group had a single twitch contraction of 0.972 N ± 0.0981N) at 2 weeks

post-BoNT-A injection. A similar active force recovery was observed in the maximal

69

tetanic contraction, with the BoNT-A group having an average maximal tetanic

contraction of 0.85 N ± 0.13 N (saline injected group had a tetanic contraction of 3.33 N

± 0.04 N) at 1 week post-BoNT-A injection. There was a partial tetanic recovery of

active force to 1.57 N ± 0.28 N (saline injected group had a tetanic contraction of 2.92 N

± 0.12 N) at 2 weeks post-BoNT-A injection.

Preload tension significantly affected the maximal single twitch and tetanic

contraction amplitude in both saline and BoNT-A injected groups at 1 and 2 weeks post-

injection (p<0.05, Figure 3A-D). At 1 week after BoNT-A injection, there was a

significant difference between the preload tension versus maximal single twitch and

tetanic amplitude curves when compared to saline injected controls (p<0.05, Figure 3A

and B). Although the injection of BoNT-A decreased the amplitude of maximal single

twitch and tetanic contraction (Figure 2C and D), differences between BoNT-A and

saline injected preload tension versus maximal muscle force curves were not statistically

significant at 2 weeks post-injection (p=0.082 for Figure 3C single twitch and p=0.053

for Figure 3D tetanic contraction). The curve depicting the relationship between preload

tension and maximal contractile force appeared to flatten at 1 week post BoNT-A

injection, but the shape of the curve appeared to become more parabolic at 2 weeks after

injection (Figure 3A-D).

BoNT-A injection significantly reduced the peak passive properties of the

muscle-tendon unit as a function of displacement at 1 week (p<0.05, Table 1 and Figure

4A). Specifically, the stiffness of the BoNT-A injected muscle-tendon unit was 0.417

N/mm compared to the control saline injected group, which was 0.634 N/mm, a 35%

reduction in stiffness (p<0.05, Table 1). Post-hoc Tukey pairwise comparison testing

70

revealed that starting at 3mm displacement, there was a statistically significant difference

between the control saline injected group and the BoNT-A injected group (p<0.05) at 1

week post injection (as denoted by † on Figure 4A). This change in the muscle-tendon

unit stiffness (slope of the lines in Figure 4, calculated and expressed in Table 1), was not

detectable at 2 weeks after injection, as there was no significant difference between the

BoNT-A injected and saline-injected groups (Table 1 and Figure 4B). There was a

statistically significant interaction (p<0.05) between displacement and peak passive

properties detected in both the saline injected and BoNT-A injected groups at 1 and 2

weeks after injection. As the displacement of the muscle-tendon unit increased, the

passive peak tension increased linearly (Table 1).

DISCUSSION

Pre-surgical treatment with BoNT-A modulates neural tone by chemical

denervation [35, 36], thus facilitating the surgical manipulation of the muscle-tendon

unit. Our study, which focuses on passive muscle-tendon unit properties after chemical

denervation, complements previous reports that have focused on active muscle properties

and their relationship to surgical outcomes of tendon repair [4, 38].

The use of BoNT-A in the pre-surgical treatment of skeletal muscle results in a

significant reduction in contractile force for both single twitch and tetanic contraction

(Figure 2). This reduction in force occurs over a wide range of preload tensions (Figure

3). In both instances, the data demonstrate that the reduced force of contraction is

temporary and reversible (Figures 2 and 3). Active contraction, resulting in mechanical

strain, is critical for tendon-to-bone healing [34, 40-43], and early motion results in

improved clinical outcomes [9, 44-46]. However, the benefits of mechanical strain and

71

early motion must be weighed against failure at the surgical repair site [26-33]. Thus,

titrating a pre-operative dose of BoNT-A to cause temporary reversible paresis is a novel

strategy by which the surgeon can improve surgical outcomes. The pharmacokinetics of

the contractile paresis and reversal of impairment ideally would correspond to the

progressive post-operative strengthening and rehabilitation protocol, ensuring optimal

mechanical strain and motion, even in the non-compliant patient. This strategy has been

termed “bioprotection” [38] and “chemoprotection” [4] in the literature, and the data

from this present study further expand upon this concept by providing a more

comprehensive, surgically relevant, preload tension versus maximal single and tetanic

contraction profile and recovery (Figure 3).

Often, the surgical manipulation of the muscle-tendon unit can be technically

difficult following a chronic tear of the tendon because of structural changes that increase

its stiffness [14-24]. Pre-surgical treatment of the muscle-tendon unit with BoNT-A can

be used to modulate stiffness and improve surgical manipulation (Figure 4 and Table 1).

After 1 week of chemical denervation, the average stiffness of the BoNT-A injected

group was 0.417 N/mm compared to the control saline injected group which was 0.634

N/mm, a 35% significant reduction in stiffness (Table 1). Our data demonstrate that

temporary and reversible chemical denervation of the muscle-tendon unit with BoNT-A

results in a reversible reduction in the stiffness, and thus, improved surgical manipulation

(Figure 4 and Table 1). In particular, reduced stiffness allows for greater excursion of the

muscle-tendon unit during surgery and reduced tension at the tendon-to-bone repair site.

Muscle physiology literature attributes most of the passive properties of the muscle-

tendon unit to three factors: 1.) the contractile component (actin-myosin cross-bridging

72

interactions); 2.) the intra-myofibril series elastic component (titin and desmin); and 3.)

the parallel elastic components (connective tissue of the epimysium, perimysium, and the

endomysium) [47]. Often, the neural component of tone has been de-emphasized as a

significant contributor to passive biomechanical muscle-tendon unit properties [47]. By

chemically denervating the muscle with BoNT-A, our data demonstrate that neural tone

is an important contributor to muscle tone, and the flaccid paresis that follows pre-

operative BoNT-A injection may facilitate tendon repair surgery.

Although our study suggests that pre-surgical BoNT-A injection can improve

surgical manipulation of the muscle-tendon unit, future studies are needed to optimize the

presurgical dose of BoNT-A, and pharmacokinetic response studies are needed to

determine if outcomes will improve with bioprotection/chemoprotection. A limitation of

the present study is that longer period of time was not studied to ensure that the primary

end-point of muscle-tendon unit stiffness returned to “normal.” There are some recent

basic science reports and clinical reports that indicate that botulinum toxin A injection

may result in persistent structural changes in injected skeletal muscle [48], which can be

detected by needle EMG months after injection [49]. However, the majority of clinical

studies indicate that the orthopaedic applications of botulinum neurotoxin A injection are

both safe and effective, even after repeat injections, for treatment and management of

spasticity associated with cerebral palsy [35, 50, 51] and flexion contractures following

total knee arthroplasty [52]. Some initial studies in the literature suggest that BoNT-A

pretreatment will improve outcomes in both a rat animal model and a clinical hand zone 2

flexor tendon injury study [4, 38]. Accordingly, the notable findings presented in this

study warrant further investigation. The present study has the limitation of being

73

performed in a mouse gastrocnemius model. Despite the similarities that exist between

the muscle-tendon unit of a mouse and human, there are differences in size, volume, and

muscle fiber composition between the animal model and humans. Further, all

experimentation was performed in healthy muscle-tendon units that had not undergone

tenotomy or injury. Therefore, future studies are needed to study the effects of BoNT-A

in the post-tenotomy setting. All experimentation for this study was performed in vivo.

The tendon, attached to the calcaneus, was disarticulated and linearized with the force

transducer to ensure accurate force recordings from the muscle. Our in vivo

experimentation has the limitation that it does not replicate the exact biomechanics of

normal muscle-tendon-bone action. Further, it is possible that general anesthesia

influences muscle tone and function. However, muscle-tendon unit testing in both BoNT-

A and saline-injected groups were performed under general anesthesia in a similar

manner, with the same anesthetic agent and dose.

Focusing on the proximal muscle portion of the muscle-tendon unit has been

described as a “paradigm shift” [4] from historical attempts at tendon repair [4, 38]. In a

rat Achilles’ tendon animal model, temporary, chemical, partial paresis results in

“bioprotection” of the tendon repair, resulting in improved structural outcomes by

reducing the active muscle contractile force [38]. Similar favorable outcomes were

observed clinically in a study of hand zone 2 flexor tendon repairs [4]. However,

complete absence of tension has been demonstrated to impair the biomechanics of tendon

healing [34], as some mechanical strain provided by muscle contraction is required to

improve molecular remodeling and osteointegration [40-43]. Early motion is beneficial

because it reduces stiffness and increases range of motion, but overly strenuous early

74

rehabilitation may lead to gap formation at the repair site or tendon rupture [9, 44-46].

Clinically, the surgeon must balance post-operative rehabilitation and repair site integrity.

The study results suggest that pre-surgical treatment with BoNT-A will facilitate

the surgical manipulation of the muscle-tendon unit, thus potentially improving operative

outcomes. The results implicate neural tone as a considerable contributor to the passive

repair tension of the muscle-tendon unit. Our study suggests that modulation of neural

tone by causing temporary reversible paresis is a novel approach that may improve intra-

operative and post-operative passive muscle properties, allowing for progressive

rehabilitation while protecting the surgical repair site; however, future studies will be

necessary to confirm this hypothesis. The results of our study also have implications for

orthopaedic surgical repair of ruptured tendons, such as hand zone 2 flexor tendons,

biceps tendon, triceps tendon, rotator cuff, the Achilles tendon, and the quadriceps

tendon.

ACKNOWLEDGEMENTS

We would like to thank Dr Thorsten M. Seyler, Mrs Eileen Martin, and Mrs

Casey Northam for their support in the planning and execution of the experiments, as

well as useful suggestions in the preparation of this manuscript.




75

SOURCE OF FUNDING



investigation.

76

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labeled in this schematic diagram.

85

Chapter 3, Fig. 1

86

FIG 2: The Effect of BoNT-A on Gastrocnemius Muscle Contraction. There is a

significant reduction in both maximal single twitch and tetanic force of muscle

contraction at 1 week and 2 weeks after injection of BoNT-A when compared to saline

injected controls (p<0.05, denoted by *). Panels A-B represent maximal force of single

twitch and tetanic contraction one-week post-injection. Panels C-D represent maximal

force of single twitch and tetanic contraction two-weeks post injection. Data are

presented as the mean and standard error of the mean.

87

Chapter 3, Fig. 2

88

FIG 3: The Effect of Preload Tension on Gastrocnemius Muscle Contraction. Panels A-B

represent the relationship between preload tension and maximal single-twitch and tetanic

contraction 1 week post BoNT-A injection. Panels C-D represent the relationship

between preload tension and maximal single-twitch and tetanic contraction 2 weeks post-

BoNT-A injection. The amount of preload tension applied to the muscle tendon unit

significantly affected the maximal muscle single twitch and tetanic contractile force for

the saline injected controls (p<0.05, Panels A-D). Further, there was a significant

reduction in the muscle contractile force when the BoNT-A group was compared to the

saline injected controls at 1 week post-injection (Panels A-B). Data are presented as mean

and standard error of the mean.

89

Chapter 3, Fig. 3

90

FIG 4: The Effect of Displacement of the Gastrocnemius-Muscle-Achilles-Tendon-Unit

on Passive Peak Tension. The passive peak tension of the muscle-tendon unit increases as

displacement increases. A significant difference exists between the muscle-tendon unit

peak passive tension BoNT-A injected experimental group compared to the saline

injected controls at 1 week post-injection starting above 3 mm displacement (p<0.05,

denoted by †, Panel A). Significant differences were not detected between the

experimental groups 2 weeks post-injection (Panel B). Data are presented as the mean

and standard error of the mean.

91

Chapter 3, Fig. 4

92

Chapter 3, Table 1

Table 1. Linear Regression Analysis for Muscle-Tendon Unit Displacement v. Peak Tension Curves 1 Week Post-Injection:

Subject Slope (N/mm) R2 P

Average Saline Injected Controls (n=6) 0.634 ± 0.0502 0.985 <0.001

Average Botulinum Toxin A Injected

Group (n=6) 0.417 ± 0.0449 * 0.996 <0.001

2 Weeks Post-Injection:

Subject Slope (N/mm) R2 P

Average Saline Injected Controls (n=7) 0.679 ± 0.0459 0.984 <0.001

Average Botulinum Toxin A Injected

Group (n=10) 0.600 ± 0.0306 0.984 <0.001

Average slope data expressed as MEAN ± SEM * T-test reveals significant difference (p<0.05)

93

CHAPTER 4

CONTRIBUTIONS OF NEURAL TONE TO IN VIVO PASSIVE

MUSCLE-TENDON UNIT BIOMECHANICAL PROPERTIES IN A

RAT ROTATOR CUFF ANIMAL MODEL

Sandeep Mannava, Walter F. Wiggins, Katherine R. Saul, Joel D. Stitzel, Beth P. Smith,

L. Andrew Koman, Thomas L. Smith, Christopher J. Tuohy

The following manuscript was published in the Ann of Biomed Eng. 2011 Mar 29. EPub.

PMID: 21445691. This chapter is reprinted with permission. Stylistic variations are due

to the requirements of the journal. S. Mannava performed the experiments, prepared the

manuscript, and served as corresponding author. T.L. Smith acted in an advisory and

editorial capacity.

94

ABSTRACT

Passive viscoelastic properties of muscle-tendon units are key determinants of

intra- and post-operative success. Atrophied, retracted, and stiff muscle-tendon units are

technically challenging to manipulate and perform poorly after surgical repair. This study

employs botulinum neurotoxin A (BoNT-A)-mediated inhibition of presynaptic

acetylcholine release to examine in vivo neural contributions to soft tissue biomechanical

properties. In vivo load-relaxation and active muscle contractile force testing protocols

were performed in the rat rotator cuff model. The passive properties were assessed using

linear regression analysis and Fung’s quasi-linear viscoelastic (QLV) model. BoNT-A

injected muscle-tendon units had a significant reduction in force of contraction

(p=0.001). When compared to saline-injected controls, the BoNT-A significantly

decreased parameter ‘A’ of the QLV model, which represents the linear elastic response

(p=0.032). The viscous properties in the BoNT-A treatment group were not significantly

different from saline-injected controls, as determined by comparison of QLV model

parameters ‘C,’ ‘τ1,’ and ‘τ2.’ In conclusion, neural tone contributes significantly to

muscle-tendon unit passive biomechanical properties. Pre-surgical treatment with BoNT-

A may improve the rehabilitation of muscle by altering its passive elastic properties.

Accordingly, pharmacological modulation of skeletal muscle stiffness with BoNT-A

increases flexibility, potentially improving function. Chemical denervation with BoNT-A

may also improve the manipulation of stiff and difficult to mobilize muscles during

surgical procedures.

95

INTRODUCTION

Approximately 300,000 rotator cuff repair surgeries are performed annually in the

United States, as rotator cuff tears are a common cause of upper-extremity pain and

disability [1]. Chronically torn rotator cuffs can pose an operative challenge because the

scarred and retracted muscle-tendon units are difficult to manipulate surgically [2-4].

Studies in rat [5, 6], sheep [7, 8], dog [2], and human rotator cuff [9] have demonstrated

that the muscle-tendon unit becomes retracted and stiffer after injury, which is attributed

to changes in the muscle’s structure [2, 5-8]. Specifically, as the muscle atrophies

following tendon injury, it undergoes sarcopenia [10], decreased muscle volume [2, 11,

12], fibrofatty infiltration [2, 11-13], decreased capillary density [2, 7, 8, 14], and muscle

fiber-type switching [12].

In some instances, full excursion of the injured muscle-tendon unit back to its

anatomical footprint on the humeral head is not possible during surgical repair of torn

rotator cuffs [6, 9]. Even when operative repair is possible, the repair tension might be

excessive, leading to early repair site failure or re-tears after surgery [6, 15, 16]. Other

investigators have demonstrated that repair tension, which is determined by the muscle-

tendon unit biomechanical properties, is an important factor that influences tendon-to-

bone healing in the rotator cuff [6]. In particular, excessive repair tension has been

associated with impaired tendon-to-bone healing [6] and worse patient-perceived

functional outcomes after rotator cuff repair surgery [9].

Passive skeletal muscle-tendon unit load-relaxation properties are key

determinants of operative success following tendon repair surgery [6, 17]. Most

biomechanical studies occur in the ex vivo or in vitro setting and focus on structural

96

components of the muscle, such as actin, myosin, titin, and desmin [17]. However, these

studies fail to evaluate the influence of the nervous system on skeletal muscle tone [17].

Surgical and rehabilitation experience with patients who have sustained neurological

insults, such as cerebral palsy or cerebrovascular accidents, demonstrates that the nervous

system influences the passive biomechanical properties of muscle [18-21].

The purpose of the current study is to use botulinum neurotoxin A (BoNT-A) to

examine and quantify in vivo neurological contributions to skeletal muscle tone, which

consists of elasticity and viscosity. BoNT-A is a potent, naturally occurring toxin that

results in a temporary and reversible muscle paresis through pre-synaptic neurological

blockade of acetylcholine release [18, 19, 22]. The present study employs Fung’s quasi-

linear visco-elastic (QLV) model to characterize the load-relaxation behavior of muscle-

tendon units in an in vivo animal model system. The use of QLV modeling to help

characterize a multi-component, muscle-tendon system in vivo was chosen because of the

clinical relevance for tendon repair surgery. Since the tendon of the supraspinatus is short

relative to the length of the muscle, in vivo biomechanical testing followed by QLV

analysis can be used to characterize neuronal influences on the muscle, as the tendon

contributions to passive biomechanical properties will likely be negligible in this

particular anatomic location [23]. The study hypothesis is that the nervous system

contributes to in vivo soft-tissue biomechanical properties; therefore, muscle-tendon unit

tone, specifically stiffness and elasticity, can be pharmacologically modulated with

BoNT-A.

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MATERIALS AND METHODS

The rat shoulder model has been established in previous experiments as a suitable

model for the study of human rotator cuff pathology [24, 25]. A total of 12 male Sprague-

Dawley rats weighing 400-450g (Charles River, Wilmington, MA) were studied. While

housed in the controlled temperature vivarium with 12 hour light-dark cycles, chow and

water were provided to the rats ad libitum. The animals were divided into two

experimental groups based upon injection type. Six rats were injected with normal saline

and served as controls; the remaining six rats were injected with equal volumes of BoNT-

A. The injection protocol is described in greater detail below. At 1-week post-injection,

in vivo muscle function and biomechanical load-relaxation testing was performed. The

animals were sacrificed immediately after in vivo testing. Experimentation was blinded in

that one scientist injected the animal, and a second scientist performed all surgery and in

vivo testing. The second scientist was not made aware of the group designation of the

animals until after all data was collected and analyzed. All experimentation was

conducted after approval from the Institutional Animal Care and Use Committee.

Botulinum Neurotoxin A and Saline Injection Procedure

Botulinum neurotoxin A (Allergan, Irvine, CA) was reconstituted as previously

described.[26-28] Briefly, a 100U vial of lyophylized BoNT-A was reconstituted with

normal saline. The toxin was injected into the supraspinatus muscle at a dosage of 9

units/kg body weight in an injection volume of 10 µL with a Hamilton syringe (Fischer

Scientific, Pittsburg, PA) equipped with a 30 gauge needle. The dosage of BoNT-A was

determined based on previously published reports in this animal model system and

98

muscle [29, 30]. Control animals received an equal volume injection of normal saline

(0.9%) in a similar manner.

All injections were performed percutaneously. Animals were anesthetized with

isoflurane (Webster Veterinary, Patterson Companies Inc., Sterling, MA) and placed in

the prone position. The scapula and shoulder were shaved and prepped with alcohol. The

scapular spine was palpated, and the muscle was injected superior and caudal to that bony

landmark. The needle was advanced to the bone (suprascapular fossa) and then

withdrawn slightly. The injection volume was distributed over the volume of the muscle.

Surgical Exposure and Experimental Apparatus

Saline injected control animals (n=6) and BoNT-A animals (n=6) were

anesthetized with isoflurane 1-week post-injection. The time period of 1 week was

chosen because the period of time from injection was thought to be sufficient to cause

maximal skeletal muscle paresis, which is defined as a reduction in active contractile

force. Similarly, we hypothesized that passive biomechanical properties would also

change during this initial period of time following injection. Further, we felt that there

would be minimal changes in the muscle’s structure in the 1 week following BoNT-A

injection, making passive biomechanical comparisons of the load-displacement data

between experimental groups more likely to be attributable to the neural contributions

and less likely to be attributable to structural changes (i.e. atrophy). The injected upper-

limbs were shaved and prepped with the animal in the prone position. A skin incision was

made over the scapula, extending towards the humerus. The overlying musculature

(deltoid and trapezius) were reflected, and the acromioclavicular ligament was cut,

exposing the injected supraspinatus muscle-tendon unit. A custom titanium plate (6.5 mm

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x 3 mm) with roughened contact surfaces was affixed to the freed supraspinatus tendon.

Clamp slippage was not observed during the experimental protocol. A rigidly affixed

custom clamp was then securely fastened to a linearly translating experimental table. This

custom clamp was attached to the inferior aspect of the scapula, below the scapular spine.

The clamp prevented the freely-gliding scapula from moving during experimentation.

The scapula was gently retracted from the thorax and a brachial plexus dissection was

performed to isolate the suprascapular nerve. A bipolar hook electrode was placed on the

suprascapular nerve. The nerve plexus and surrounding tissue was electrically isolated by

wrapping a piece of Silastic sheeting (Dow Corning Co, Midland, MI) around the exterior

of the suprascapular nerve and bipolar electrode. The rat’s upper extremity was

immobilized with straps and pins on a platform that translated linearly. A single body

strap and pins through the wrist and elbow were used to isolate other directional force

vectors, ensuring that contractile force and passive force produced by the supraspinatus

muscle-tendon unit translated perpendicular and ‘in line’ with the force transducer in all

experimental cases (Fig 1). If force was not directed perpendicular/‘in line’ with the force

transducer, it is possible that the recorded values of tension would be lower than what is

actually produced during active force of contraction and passive stretching protocols.

A steel wire was used to connect the custom titanium plate holding the

supraspinatus tendon to a force transducer (model FORT100; World Precision

Instruments Inc., Sarasota, FL). The transducer was connected to an amplifier (model 13-

G4615-50; Gould, Cleveland, OH) that was interfaced with a personal computer through

an interface controller (EMKA TECHNOLOGIES Inc., Falls Church, VA) and an

analog-to-digital converter card (model: PCI-6023E, National Instruments, Austin, TX).

100

A pulse generator (model Tenma TGP110, BioSurplus Inc., San Diego, CA) was used to

stimulate the suprascapular nerve. The stimulation protocol and the force recordings were

controlled and recorded by IOX software, Slow Wave Analyzer (EMKA Technologies

Inc., Falls Church, VA). During all experimental recordings, the body temperature of the

animals was monitored and maintained with an external heat lamp.

Muscle Force Testing

Supramaximal stimulation of the suprascapular nerve was produced with 1.5V. A

range of 0.6V to 2.0V was tested to ensure that maximal single twitch was attained at

1.5V of stimulation, and in all cases, this stimulation voltage produced maximal single

twitch contraction. The muscle-tendon unit was then pretensioned to 0.1N of preload

tension by moving the platform, based on previously published reports in this animal

model system [5, 6], and the suprascapular nerve was stimulated at a frequency of 100Hz,

150Hz, and 200Hz for 1 second, and maximal amplitude of the force of contraction was

recorded. A recovery time of 60 seconds was allowed between stimuli. This recovery

time allowed for full recovery of maximal contractile force. A total of three trials were

performed at each stimulation frequency, and the largest tetanic contractile amplitude was

recorded for each frequency. The stimulation protocol was performed to record the

maximal tetanic contractile force for saline injected controls and BoNT-A injected

animals 1-week after injection.

Passive Load-Relaxation Testing

A starting preload tension of 0.05N was set as 0mm displacement. Prior to the

passive load-relaxation testing, a preconditioning protocol of stretching the muscle-

tendon unit from 0mm to 1mm was executed and held for a duration of 10 seconds, then

101

relaxed to 0 mm for 30 seconds and this was repeated 10 times, based on a previously

published report of in vivo biomechanical testing in this rat rotator cuff model system [5].

The supraspinatus muscle-tendon unit remained connected to the experimental apparatus

after muscle force testing. The stage was then displaced away from the force transducer

to specific displacement increments (between 0 to 8 mm). The passive tension of the

muscle-tendon unit was assessed in vivo by measuring the passive tension produced by

the muscle-tendon unit while the muscle-tendon unit was displaced from 0mm to 8mm of

displacement in 2mm increments. Between load-relaxation cycles, the muscle-tendon unit

was returned to 0mm displacement and allowed to rest for 10 minutes. The passive

tension was continuously recorded for a duration of 210 seconds after displacement,

throughout the experimental protocol.

Fung’s Quasilinear Viscoelastic Model (QLV)

Fung’s quasilinear viscoelastic model (QLV) [31] has been used by others to

provide a mathematical description of the passive biomechanical behavior of the entire,

intact, in vivo muscle-tendon unit during loading and relaxation [5, 29]. Specifically, the

QLV model allows for the extraction of elastic and viscous parameters from the load-

relaxation experiments.

Fung [31] proposed that the uniaxial deformation, ε(t), of a viscoelastic tissue and

the resultant uniaxial stress, σ(t), exhibited the following relationship:

(1)

where G(t) is the reduced relaxation function proposed by Fung,

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(2)

where E1 denotes the Euler integral E1(y) = ; σe(εA) is the elastic stress

response, and εA is the applied step increase in strain. In the special nonlinear elastic

case,

(3)

where: ‘A’ represents the linear component of the elastic response and ‘B’ represents the

nonlinear component of the elastic response. In our analysis, we assumed the idealized

case of the relaxation experiment where an immediate and constant deformation is

applied to the tissue such that ε(t) = a. Thus, (1) reduces to

(4)

as demonstrated by Nigul and Nigul,[32] where ‘C’ describes the scaling of the viscous

properties during load-relaxation; ‘τ1’ describes the early or fast component of

relaxation—on the order of seconds; and ‘τ 2’ describes the late or slow component of

relaxation—on the order of minutes to hours. Equation (4) was employed to fit the

experimental data using the nlinfit nonlinear least squares regression function intrinsic to

MATLAB (MathWorks, Natick, MA). Additionally, the goodness of fit was quantified

for elastic and viscous responses of the QLV model. A coefficient of determination ‘R2’

was calculated and used to quantify goodness-of-fit for the nonlinear least-squares

103

regression. Others have established this technique as an appropriate and validated method

in the rat rotator cuff animal model [5, 29].

When the QLV parameters A and B are varied, the passive tension versus time

curve undergoes a shift and there is a corresponding change in the peak and equilibrium

passive tensions (Fig 2A). Varying parameter ‘A’ results in a change in the passive

tension versus time graph which scales “linearly” (Fig 2A); whereas varying parameter

‘B’ results in a change in the passive tension versus time graph which scales nonlinearly

(Fig 2A). Varying the QLV parameters ‘C,’ ‘τ1,’ and ‘τ 2,’ affects the rate of load-

relaxation and may influence the equilibrium passive tension (Fig 2B). Examples of the

time histories for passive tension at varying displacements are shown (Fig 3). These

curves (Fig 3) are parameterized by the Fung’s QLV method described previously.

Statistics

All values were expressed as the mean ± standard error of the mean. Statistical

analysis was performed using SigmaStat (Systat Software Inc., San Jose, CA). A mixed

model ANOVA followed by a Tukey post hoc test was performed for between and within

group comparison of muscle force measurements stimulated at various frequencies (Fig.

4) and passive tension versus muscle-tendon unit displacement data (Fig 5). T-tests were

performed for comparison of peak stiffness, equilibrium stiffness, and QLV parameters

between BoNT-A experimental and saline-injected control groups. In vivo stiffness of the

muscle-tendon unit was determined based on the linear regression analysis (slope of the

line) for the tension (peak and equilibrium) versus displacement data (Fig 5). Linear

regression analysis was performed on the data recorded between 2 mm to 8 mm on the

passive tension versus displacement data (Fig 5). At 2mm of displacement, the preload

104

tension has been documented in the literature [5] to be on the linear portion of the load-

displacement curve. Therefore, regression analysis on this portion of the data would

presumably avoid the non-linear toe-region.

RESULTS

One week after the injection of BoNT-A, a significant decline (p=0.001) in

maximal tetanic contraction force was documented at all frequencies of stimulation when

compared to saline injected controls (Fig 4). At 1-week after the injection, the BoNT-A

injected group had an average maximal tetanic contractile force of 0.24 N ± 0.05 N, while

the saline injected group had an average maximal tetanic contractile force of 5.06 N ±

0.05 N.

Sample time histories for the control versus BoNT-A passive force development

at various displacements are shown (Fig. 3). Botulinum neurotoxin A significantly

reduced the peak passive tension of the muscle-tendon unit as a function of displacement

one week after injection (p=0.022, Fig 5 A). Post-hoc analysis revealed that there were

significant differences in peak passive tension between experimental groups at 6mm

displacement (p=0.002, Fig 5A) and at 8 mm displacement (p=0.012, Fig 5A). Similarly,

BoNT-A also reduced the equilibrium passive tension of the muscle-tendon unit as a

function of displacement one week after injection (p=0.043, Fig 5B). Post-hoc analysis

revealed that there were significant differences in equilibrium passive tension between

experimental groups at 6mm displacement (p=0.013, Fig 5B) and at 8 mm displacement

(p=0.006, Fig 5B).

As the displacement of the muscle-tendon unit increased, the passive tensions

(both peak and equilibrium) increased linearly (Fig 5 and Table 1). The slopes of the lines

105

representing in vivo stiffness of the muscle-tendon unit (Fig 5) were significantly

different between the BoNT-A group and the saline injected group for peak tension

(p=0.043, Table 1), as there was a 16.32% reduction in the in vivo stiffness of the

muscle-tendon unit for the BoNT-A injected group (Table 1). However, a significant

difference was not detected for the equilibrium tension (p=0.067, Table 1), as in vivo

stiffness was calculated from the displacement versus equilibrium tension data (Fig 5B),

which demonstrated a 22.7% reduction in the stiffness for the chemically denervated

experimental group.

QLV parameter A (representing linear scaling of the elastic response) was

significantly influenced by chemical denervation with BoNT-A one week post-injection

(Table 2). No significant differences in the elastic parameter B and the viscous

parameters C, τ1, and τ 2 were detected between experimental groups (Table 2). A

nonlinear least-squares regression (R2) was calculated to quantify the goodness-of-fit for

all of the time-history recordings to the QLV model: average Saline R2elastic = 0.9704

(Saline R2elastic range: 0.8769 – 0.9997); average Saline R2

viscous = 0.9968 (Saline R2viscous

range: 0.9888 – 0.9998); BoNT-A R2elastic = 0.9889 (BoNT-A R2

elastic range: 0.9757 –

0.9951); and BoNT-A R2viscous = 0.9990 (BoNT-A R2

viscous range: 0.9966 – 0.9997).

DISCUSSION

Neural tone significantly contributes to the passive, in vivo, soft-tissue, load-

relaxation biomechanical properties of the muscle-tendon unit. Chemical denervation

with BoNT-A influences both the active contractile force and passive viscoelastic

properties of the muscle-tendon unit. More specifically, the nervous system influences

the elastic component of the load-relaxation properties. These findings help to quantify

106

the neural contribution to in vivo muscle tone, which is experienced clinically during

physical therapy and rehabilitation of patients with insults to their nervous system (i.e.

when stretching a spastic muscle of a patient with cerebral palsy, the therapist often

experiences a stiff muscle that resists manipulation) [18-21]. Further, pre-operative

injection of BoNT-A facilitates the intra-operative surgical manipulation of the muscle-

tendon unit by decreasing stiffness (Table 1) and the elastic component ‘A ‘of Fung’s

QLV (Table 2). Pre-surgical chemical denervation is a novel approach to modulating the

in vivo soft-tissue biomechanics of the muscle-tendon unit and may facilitate the

mobilization of scarred, retracted, shortened, and atrophied muscle-tendon units during

surgery.

Resting muscle tone is attributed to a neural reflex loop, in which afferent sensory

nerves detect the contractile state of the muscle and then send a signal to efferent motor

neurons, via a synapse in the spinal cord. These efferent motor neurons provide

stimulation or inhibition of the muscle, resulting in appropriate contraction or relaxation.

In addition to the structural elements (i.e. actin, myosin, titin) of the musculoskeletal

system that contribute to passive muscle-tendon unit properties, this reflex arc helps to

maintain a basal level of contractile tone. The reflex loop is modulated by central nervous

system input via the corticospinal and reticulospinal tracts, both of which provide

inhibitory modulation of this reflex. BoNT-A blocks the transmission of the efferent

nerve input to the skeletal muscle, and thus disrupts the reflex loop [33]. It is likely that

the BoNT-A influences elasticity (or QLV parameter ‘A’) by disrupting this neural reflex

loop, blocking neural input to the skeletal muscle, and reducing stiffness.

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Based upon our clinical experience, the effects of a one-time dose of BoNT-A on

inhibition of active contractile force are reversed by 4-6 months [18]. Yet in animal

studies, modulation of the passive stiffness of the muscle-tendon unit, by a one-time dose

of BoNT-A, seem to reverse two weeks after injection [34]. Our data demonstrate that

BoNT-A significantly reduces supramaximal contractile force (Fig 4). Recently, BoNT-A

chemical denervation was employed in the perioperative setting, in an animal model, to

protect a surgically repaired tendon injury. In this study, the chemically denervated

muscles could not generate enough strength to produce suture rupture [27]. Similar

favorable outcomes were demonstrated in a clinical tendon repair study [35]. In the non-

compliant patient, who attempts excessive activity before healing is allowed to occur

following surgery, pre-surgical BoNT-A injection provides “bio-protection” or “chemo-

protection” of the repair site because the chemically denervated muscle cannot generate

the necessary force to exceed the suture fixation techniques used to secure the torn

muscle-tendon unit to the bone [27, 35].

In addition to the proposed “chemo-protection” or “bio-protection” mechanisms,

it is likely that repair tension was also influenced by the use of BoNT-A. The healing of a

muscle-tendon unit to bone is based on many factors; however, the role of repair tension

has recently been appreciated. In the clinically relevant rat rotator cuff animal model,

repair tension increased with time after injury [5]. The higher tensioned repairs

demonstrated impaired healing at the tendon-bone interface as assessed by biomechanical

testing, histology, and molecular assays [6].

Another rat rotator cuff animal study employed BoNT-A in combination with

cast-immobilization of the rodent limb in order to reduce repair tension, with the goal of

108

chemically protecting the surgical repair. In this study by Galatz et al., the reduced repair

tension coupled with a complete absence of mechanical stimulation during healing led to

impaired histological and biomechanical tendon-to-bone healing [29]. Hettrich et al. also

reported mixed biomechanical and histological results of tendon healing after rotator cuff

injury and repair in the rat rotator cuff animal model [36]. However, these studies focused

on tendon-to-bone healing and did not quantify the passive biomechanical properties of

the muscle-tendon unit. Both studies conclude that further investigation is required at

earlier and later time points to determine the efficacy and influence of chemodenervation

on tendon-to-bone healing [29, 36].

Based upon previous reports in the literature regarding the relationship between

repair tension and tendon-to-bone healing, some investigators have attempted to optimize

the mechanical loading of the muscle-tendon unit after surgery. These investigators

repaired rotator cuff tendons in the rodent model and then studied various rehabilitation

protocols after surgery that employed treadmill running and continuous passive motion

[37, 38]. One study demonstrated that healing was not impaired with motion as assessed

biomechanically and histologically; however, the passive range of joint motion might be

adversely affected [37]. The other study demonstrated increased scar formation with

excessive early motion, which adversely affected tendon healing [38]. Biologically, some

motion or tension at the repair sight is necessary to improve healing; however, repair

tensions that are too high result in impaired healing [6, 29, 37-40]. Ideally, the BoNT-A

dose would be optimized for both pharmaco-kinetics and pharmaco-dynamics in the pre-

surgery setting to correspond with the patient’s rehabilitation protocol after tendon repair

surgery. In this way, pre-surgical injection of BoNT-A has the potential to allow for the

109

pharmacological control of repair site tension throughout the healing process, further

improving the mechano-biology of healing during rehabilitation following surgery [6, 29,

37-40].

Stress and strain is typically used for Fung’s QLV analysis of biological tissue,

such as tendon, ligament, or muscle. However, QLV parameters in the present study were

determined based upon loads and displacements because cross-sectional area and length

of the muscle-tendon unit are not known during in vivo experimentation. This method has

the limitation of being highly sensitive to differences in length and size of the samples

used. In the animal model system used, there was enough consistency between the

experimental subjects to detect significant differences. Additionally, other investigators

have successfully employed the method of parameterization of load-displacement data

using an in vivo rat rotator cuff animal model system [5]. It is possible that after 1 week

of BoNT-A injection, the muscles could have undergone an adaptive response, such as

atrophy, which might explain the differences in passive properties observed. A limitation

of this study is that muscle volume or physiological cross-sectional area (PCSA) was not

measured after experimentation to ensure that these structural changes did not occur.

Galatz et al. reported minimal structural changes at 14 days post-injection in the muscle’s

architecture, as determined by histological examination of rat supraspinatus injected with

9 units/kg of BoNT-A, the same dose used in our present study. However, Galatz et al.

did report some structural changes during their analysis [29]. Further, Hettrich et al.

reported a significant reduction in rat supraspinatus weight at 4 and 8 weeks after

injection [36]. Analysis of stress-strain data, as opposed to load-relaxation data, would

account for these potential differences in muscle structure after BoNT-A injection.

110

However, at 1 week after BoNT-A injection, we expect minimal atrophic changes to the

muscle’s structure.

A limitation of the present study is that only a single dose-volume combination of

BoNT-A was examined. Future studies are needed to elucidate the time-course of BoNT-

A’s modulation on the passive, in vivo, biomechanical properties of the muscle-tendon

unit. In addition, studying the effects of BoNT-A over a longer period of time would

determine if the stiffness and QLV parameters return to baseline. However, it is possible

that the passive properties might not return to the pre-injection state. A report in the

literature concluded that repeated BoNT-A injections may result in persistent structural

changes in skeletal muscle [41]. However, experience with orthopaedic surgical

applications has demonstrated that BoNT-A injections are both safe and reversible [18,

21, 42].

The present study also has the limitation of being performed in an animal model.

Despite the similarities between the rat and human shoulder, anatomical, structural (i.e.

muscle fiber composition, muscle size, muscle volume, etc.), and functional differences

(i.e. quadra-pedal versus bi-pedal) exist between the rodent model and humans. Despite

the advantages of in vivo testing in the rat model, the present experimental apparatus

does not allow for replication of the exact joint biomechanics of normal muscle-tendon-

bone action because the rotator cuff muscle-tendon unit was configured linearly with the

force-transducer to ensure accurate recording. A final limitation was that the in vivo

biomechanical testing was performed under general anesthesia, which may influence

resting muscle tone and function. However, identical muscle-tendon unit testing was

111

performed for both experimental groups, BoNT-A injected and saline injected, under the

same anesthetic conditions, with the same anesthetic agent and dose.

Recently, there has been an increased interest in understanding the nervous

system’s role during the pathogenesis and healing of rotator cuff tears [43-46]. Since the

shoulder is a dynamic joint with a complex range of motion, it requires the simultaneous

activation and coordination of many nerves, muscles, tendons, and bones to perform a

movement. Our data demonstrate that even at “rest,” the nervous system has a significant

role in passive skeletal muscle tone, which ultimately influences the motion and function

of the joint. The findings of the present study demonstrate that the nervous system

influences elasticity of the muscle-tendon unit, as QLV model parameter ‘A’ is

significantly changed after BoNT-A injection. These findings have implications for

mathematical modeling of the musculoskeletal system. Based on our QLV analysis, it is

likely that varying elasticity of the tissue in a model system would simulate neural

influences. More specifically, our data demonstrate that linear scaling of the elastic tissue

response would be most appropriate for investigation of neuronal influences using

computational techniques. However, these findings should be interpreted with caution, as

further investigation using computational analysis techniques and verification with in

vivo experimentation is necessary to validate this conclusion.

In conclusion, neural tone significantly contributes to muscle-tendon unit passive

biomechanical properties, and pre-surgical treatment with BoNT-A may improve the

rehabilitation of muscle by altering the passive elastic properties. Accordingly,

pharmacological modulation of skeletal muscle stiffness with BoNT-A increases

flexibility and may improve function. Chemical denervation with BoNT-A may also

112

improve the manipulation of stiff and difficult to mobilize muscles during surgical

procedures. Besides the rotator cuff, these findings have implications for the operative

repair and rehabilitation of many muscle-tendon units, such as biceps tendon, triceps

tendon, quadriceps tendon, Achilles tendon, and hand flexor tendons.

ACKNOWLEDGEMENTS

We would like to thank Mrs. Eileen Martin, Mr. Patrick Haubruck, and Dr.

Michael F. Callahan for their support in the planning and execution of the experiments.

No external financial support or potential conflicts were identified related to the subject

of this study. This project is taken in part from a dissertation submitted to the

Neuroscience Program, Wake Forest University Graduate School of Arts and Sciences, in

partial fulfillment of the requirements for the degree of Doctor of Philosophy.

113

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Chapter 4 Figures and Tables


shown in this diagram. Panel A represents 0 mm of muscle-tendon unit displacement and

Panel B represents 8 mm of muscle-tendon unit displacement (achieved by moving the

linearly translating stage away from the force-transducer).

121

Chapter 4, Fig. 1

122

FIG 2: The Effect of Varying QLV Model Parameters on a Sample Time History of Data

for a 4mm Displacement of the Rat Supraspintus Muscle-Tendon Unit. Panel A

demonstrates how doubling parameters ‘A’ and ‘B’ influence the passive tension versus

time sample data. Panel B demonstrates how doubling parameters ‘C,’ ‘τ1,’ and ‘τ 2,’

influence the passive tension versus time sample data.

123

Chapter 4, Fig. 2

124

FIG 3: Sample Time Histories of BoNT-A versus Saline Injected Supraspinatus Muscle-

Tendon Units after Various Displacements. The BoNT-A demonstrated lower passive

tension when compared to saline-injected controls throughout the time history.

Goodness-of-fit of the time-histories to the QLV model were assessed by nonlinear least-

squares regression and reported in the results.

125

Chapter 4, Fig. 3

126

FIG 4: The Effect of BoNT-A on Rat Supraspinatus Muscle Contraction. There is a

significant reduction in the maximal tetanic muscle contractile force at all frequencies of

stimulation studied at one week after injection of BoNT-A when compared to saline

injected controls (p=0.001). Data are presented as the mean ± standard error of the mean.

127

Chapter 4, Fig. 4

128

FIG 5: The Effect of Displacement of the Rat Supraspinatus Muscle-Tendon Unit on

Passive Peak and Equilibrium Tension. The passive peak and equilibrium tension of the

muscle-tendon unit increased as displacement increased. There was a significant

difference between the muscle-tendon unit peak passive tension in the BoNT-A injected

experimental group compared to the saline injected controls at one week post-injection

(p=0.022, Panel A). Significant differences in passive equilibrium tension were also

detected between the experimental groups at 1-week post-injection (p=0.043, Panel B).

The linear regression trend lines are shown for all experimental groups and the data for

this analysis are presented in Table 1. Data are presented as the mean ± standard error of

the mean.

129

Chapter 4, Fig. 5

130

Chapter 4, Table 1

Table 1. Linear Regression Analysis for rat supraspinatus muscle tendon unit before and after botulinum neurotoxin A injection Displacement versus Peak Tension (Figure 2A)

Subject n

Slope Stiffness (N/mm) R2 P

Average Control 6 0.98 ± 0.074 0.952 0.005

Average

Botulinum Toxin A

6 0.82 ± 0.146 * 0.934 0.007

Displacement versus Equilibrium Tension (Figure 2B)

Subject

Slope Stiffness (N/mm) R2 P

Average Control 6 0.651 ±

0.0779 0.947 0.005

Average

Botulinum Toxin A

6 0.503 ± 0.0751 0.94 0.006

Average slope data expressed as MEAN ± SEM * T-test reveals significant difference (p=0.040)

131

Chapter 4, Table 2

Table 2. QLV parameters for rat supraspinatus muscle tendon unit after botulinum neurotoxin A injection

Subject n A (N) B (mm-1) C τ1 (s) τ2 (s) Average Control 6 2.008 ± 0.996 0.282 ± 0.0573 0.210 ± 0.0382 1.140 ± 0.239 35336.2 ± 4934.438

Average Botulinum Toxin A

6 0.398 ± 0.124 * 0.405 ± 0.0366 0.182 ± 0.0285 1.257 ± 0.251 49718.2 ± 12944.25

Parameters obtained from load-relaxation behavior; All data expressed as MEAN ± SEM * T-test reveals significant difference (p=0.032)

132

CHAPTER 5

THE SCIENCE OF ROTATOR CUFF REPAIRS: TRANSLATING

BASIC SCIENCE INTO CLINICAL RECOMMENDATIONS

Sandeep Mannava, Christopher J. Tuohy, Thorsten M. Seyler, Johannes F. Plate,

L. Andrew Koman, Patrick W. Whitlock, Thomas L. Smith, Katherine R. Saul

S. Mannava performed the experiments, prepared the manuscript that was presented at

the 2011 AAOS Annual Meeting in San Diego, CA, and served as corresponding author.

T.L. Smith acted in an advisory and editorial capacity. K.R. Saul served in an advisory

role and helped complete the simulation analysis.

133

ABSTRACT

Introduction: For chronic, full-thickness, rotator cuff tears, repair surgery can be

technically challenging due to large gap distances and increased stiffness of the muscle-

tendon unit. In contrast, acute rotator cuff tears are associated with lower repair tensions,

less fibro-fatty infiltration of the muscle, and better functional outcomes. This study

demonstrates how animal models can be used to understand rotator cuff muscle function

in the acute and chronic tear setting and how these results can be translated into clinical

recommendations using computational modeling.

Methods: In vivo muscle function, electromyography (EMG), and passive muscle-tendon

unit properties were studied before and after supraspinatus tenotomy in a rodent rotator

cuff injury model (acute vs chronic). Then, a series of simulation experiments were

conducted using a validated computational human musculoskeletal shoulder model to

assess both passive and active tension of rotator cuff repairs based on surgical

positioning.

Results: In vivo muscle function was impaired at the tensions required to repair a

chronically torn rotator cuff (approximately a 50% reduction from maximal twitch

amplitude, p<0.05). Dysfunction in the chronic tear setting was detectable via EMG

(p<0.05). At adducted postures, computational data from simulated surgical repair of

chronically torn rotator cuff indicated that passive repair tension markedly exceeds the

pullout strength of suture and anchor techniques typically used in these surgeries. Further,

simulation analysis indicated there was approximately a 50% reduction in moment

generating capacity after repair of chronically torn rotator cuff.

134

Conclusions: The rat data suggest a direct experimental connection between high repair

tensions—chronic tear setting—and impaired contractile force. Simulation analysis

confirmed that functional outcomes are compromised when excessively retracted rotator

cuff muscle-tendon units are repaired in a single-stage procedure.

Clinical Relevance: Functional outcomes following surgical repair of rotator cuffs may

be improved by earlier intervention because the acute tear setting requires lower surgical

repair tension and is associated with fewer neuromuscular changes.

135

INTRODUCTION

Rotator cuff tears are a common cause of upper-extremity disability, particularly

in elderly patients over 60 years of age [55, 71, 72]. For chronic, full-thickness, rotator

cuff tears, repair surgery can be technically challenging due to large gap distances and

increased stiffness of the muscle-tendon unit. In contrast, acute rotator cuff tears are

associated with lower repair tensions, less fibro-fatty infiltration of the muscle, and better

functional outcomes. This study demonstrates how animal models can be used to

understand rotator cuff muscle function in the acute and chronic tear setting and how

these results can be translated into clinical recommendations using computational

modeling.

ROTATOR CUFF TEARS: A CLINICAL PROBLEM

Rotator cuff tears are a common cause of upper extremity pain and disability that

results in nearly 4.5 million physician visits [55] and more than 75,000 operative repairs

annually [68], in the United States. Approximately 20% of all adults have a torn rotator

cuff [72] and the distribution of tears is skewed towards the older patient population [55,

71, 72]. In one study of shoulder magnetic resonance imaging, approximately 4% of

patient’s under the age of 40 were diagnosed with a rotator cuff tear; this number rose to

approximately 54% of patients over the age of 60 [62].

Disability from rotator cuff pathology affects approximately 20% of older adults,

who struggle to maintain their independence because of reduced upper-limb function

secondary to pain and weakness [1, 56]. As the average age of the United States’

population increases and older patients live a more active lifestyle, the number of rotator

cuff repair surgeries performed annually continues to rise [54, 55]. Despite many

136

technological advances, recent studies have reported variable clinical operative results,

with some studies reporting a high rotator cuff repair failure rate [21, 24] and others

reporting more favorable outcomes [52].

The rotator cuff functions as a dynamic stabilizer, acting to optimize the position

of the humeral head on the articular surface of glenoid during motion and to provide

initiation of arm forward flexion and abduction. Rotator cuff failure can lead to abnormal

and unbalanced gleno-humeral joint forces, which can ultimately lead to pain during

movement of the affected limb [19, 37, 48, 69]. Current treatment recommendations for

rotator cuff pathology often delay surgical intervention until conservative management

options are exhausted [7, 55]. Delayed surgical repair may result in disease progression

and changes to the muscle-tendon unit [2, 16, 30, 31, 39, 51, 60], which some consider

irreversible with current single-staged repair surgery [7, 49, 59, 67].

Since the rotator cuff muscles require complex neuro-physiological coordination

to perform their function, many research efforts have focused on studying rotator cuff

pathology using animal models. These studies have employed rat [28, 29], rabbit [49, 58,

59, 67], dog [15], sheep [7, 26], and non-human primates [63] to study the natural history

of the disease process and test innovative operative solutions. However, findings in

animal models can be difficult to interpret and translate into clinical practice for patient

benefit. The present study will demonstrate how animal studies are used to gain insights

regarding rotator cuff pathologies. In particular, we will present data related to muscle

function after acute and chronic rotator cuff tears in a rat rotator cuff animal model. We

then performed a simulation analysis using a computational model of human upper-

extremity motion in order to “scale” the small animal findings to the human condition.

137

This research paradigm of using advanced computational modeling to help interpret

animal studies will improve the pace of basic-science innovation. Further, this process

may help facilitate the translation of research findings into clinical solutions that improve

the care of patients, resulting in an expedited return to work or recreation following

rotator cuff surgery.

THE ROTATOR CUFF MUSCLE UNDERGOES CHANGES AFTER TENDON

INJURY

Numerous animal studies have examined the changes that occur in the skeletal

muscle associated with the torn rotator cuff tendon. Previous experimental investigations

have established the rat shoulder model as suitable for the study of human rotator cuff

pathology because of upper-extremity anatomy similarities between the two species (Fig.

1) [64, 66]. Barton et. al reported that the rat supraspinatus undergoes a rapid dis-use

atrophy, with muscle fiber type switching to fast-muscle fibers, decreased muscle mass,

and increased inter-fasicular fibrosis [2]. Findings from our laboratory in the rat model

demonstrate similar changes (Fig. 2) [45]. Interestingly, after isolated rotator cuff

tenotomy, the rat supraspinatus muscle does not undergo the anticipated fibro-fatty

infiltration of the muscle-belly, which has been observed in some instances of clinical

rotator cuff tears [22, 32, 40].

Studies that utilize the rabbit animal model to study muscle structure after

tenotomy have demonstrated similar atrophic changes described in the rat literature, with

the added benefit of recapitulating the fatty degeneration seen clinically [49, 58, 59, 67].

Utilizing the rabbit rotator cuff model, various authors suggested that rotator cuff tendon

tears have neuromuscular implications [38, 57]. In particular, the fatty infiltration that is

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seen after rotator cuff tears may result from a nerve-injury component [33, 57] or a more

complex neuromuscular connectivity dysfunction [38]. These basic science studies are

consistent with recent case series that demonstrated electromyographic (EMG)

dysfunction after rotator cuff tears, which were reversible upon rotator cuff repair. These

studies concluded that “reducing” the atrophied and retracted rotator cuffs during repair

surgery resulted in decreased tension placed upon the suprascapular nerve as it coursed

through the suprascapular or spinoglenoid notch [9, 43]. Similarly, our group recently

reported EMG changes in compound motor action potential amplitude in chronically

injured rat rotator cuffs, directly relating electrophysiological rotator cuff dysfunction to

time from tendon injury (Fig. 3) [45]. These studies are among the first published reports

that relate rotator cuff tendon tears to a complex neuromuscular system, requiring

activation and coordination of many nerves and muscles in concert in order to produce

purposeful upper-extremity movement.

Skeletal muscle changes following rotator cuff tears have also been described in

larger animal model systems, such as sheep [7, 26] and dog [60]. These studies confirmed

that the muscle-tendon unit changes after rotator cuff tears. In particular, the rotator cuff

muscle atrophies, resulting in less mass and higher fat content [7, 26, 60].

IN VIVO BIOMECHANICS OF ROTATOR CUFF REPAIR SURGERY

Numerous animal studies have studied the natural history of rotator cuff injuries.

In particular, these studies quantified the passive tension required to “reduce” the injured

tendon back to its humeral insertion [26, 28, 29, 60]. The muscle-tendon unit structural

changes described previously [2, 7, 26, 38, 57, 60] result in a stiff an retracted rotator cuff

that is difficult to manipulate during surgery. In the rat rotator cuff animal model, Gimbel

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et al. reported that the repair tension required to re-appose the supraspinatus tendon to the

humeral head rose rapidly and progressively from the time of injury [28]. Further, they

demonstrated that higher repair tensions were associated with a reduced quality of

tendon-to-bone healing, as demonstrated biomechanically [29]. Similar changes to the

passive biomechanics of the muscle-tendon unit were demonstrated in the canine model

[60] and sheep model [2, 26]. These studies reported that passive stiffness of the rotator

cuff was increased in the chronically torn setting, presumably because of the progressive

changes in the skeletal muscle and rotator cuff tendon structure [7, 26, 60]. A cadaveric

study performed by Halder et al. supports the animal studies and predicts that retraction

following rotator cuff tears contributes to decreased shoulder function, particularly

decreased torque and strength [34]. A clinical study by Davidson and Rivenburgh

measured intraoperative rotator cuff repair tensions in 67 consecutive patients and

correlated these tensions to clinical outcomes, as determined by strength measurements

and validated clinical metrics (Constant Score, American Shoulder and Elbow Surgeons’

functional criteria, and visual analog scale score). The study concluded that high repair

tensions were correlated with worse subjective and objective outcome measures [10].

Recently, our group published two reports which explored a novel mechanism by

which surgeons can chemically denervate a muscle-tendon unit pre-operatively in order

to improve the manipulation of the muscle-tendon unit during surgery [44, 46]. Our

initial animal study was performed in a mouse gastrocnemius model and demonstrated

that the stiffness of the muscle-tendon unit can be reduced by approximately 30% one-

week following an intramuscular botulinum neurotoxin A injection [44]. In a follow-up

report using the rat rotator cuff model, we demonstrated that injection of botulinum

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neurotoxin A could pharmacologically modulate and reduce the elasticity of the in vivo

viscoelastic muscle-tendon unit. Based upon our animal data, the nervous system

contributes to the in vivo passive biomechanical properties of repair tension experienced

during rotator cuff repair [46]. Treatment of the rotator cuff with botulinum neurotoxin A

prior to surgery, may improve the surgical manipulation of the stiff and retracted tendon

[44, 46], with the additional benefit of “bioprotecting” the tendon-to-bone repair. By

temporarily and reversibly weakening the muscle, the active contractile force cannot

generate enough force to cause rupture of the repair site [42]. Recent reports in the rat

rotator cuff model, in which “bioprotection” of the repair site was employed,

demonstrated either equivocal or reduced healing biomechanics at the tendon-to-bone

junction [25, 35]. However, further studies are needed to investigate the clinical benefit

of botulinum neurotoxin A modulation of biomechanical stiffness and the potential for

“bioprotection” of the repair site. Our department is currently conducting a prospective,

randomized, double-blinded study exploring the benefit of adjunct botulinum neurotoxin

A injection for rotator cuff repair.

EVALUATION OF MUSCLE FUNCTION AFTER ROTATOR CUFF TEARS

The shoulder has an extensive range of motion that requires the simultaneous

neuromuscular coordination of numerous muscle-tendon units to produce purposeful

movement. Rotator cuff investigators have largely focused their basic science efforts on

tendon-to-bone fixation, where surgical interventions occur during operative repair [3-5,

23, 47]. The structural changes in the muscle-tendon unit that result in increased repair

tension have also been the subject of many investigations [7, 26, 28, 60]. Since the rotator

cuff acts as a dynamic stabilizer during upper extremity motion, the consequences of

141

structural muscle changes (i.e. atrophy, fibro-fatty infiltration, fiber-type switching, etc)

on contractile force possibly influences post-operative functional outcomes.

Unfortunately, limited literature exists on the function of the rotator cuff muscles after

tendon tears [6, 8, 10, 17, 18, 27, 45, 50].

Several studies have examined exercise capacity in the rabbit shoulder model,

correlating reduced muscle force with histological and radiographic changes that occur

with dis-use atrophy following rotator cuff tendon tears [6, 17, 18]. Similarly, Meyer et

al. demonstrated that chronic tendon tears in the sheep model were associated with

atrophy, retraction, and fatty infiltration, which resulted in reduced amplitude of muscle

contractile force in this chronically injured experimental group. The authors concluded

that computer tomography of the rotator cuff and the associated radiographic changes

could only indirectly and approximately predict the reduced function [50]. Coleman et al.

demonstrated that muscle function was likely to improve in the sheep model when repair

was conducted in the acute tear setting. The authors suggested that when repair is

undertaken in the chronic tear setting, the muscle might not recover full function.

Essentially, as conservative management is pursued and surgery is delayed, their data

suggest that a “point of no return” exists where full function will not be achieved with

current single-stage rotator cuff repair [7].

In a study conducted by Gerber et al., thirteen patients were evaluated by

magnetic resonance imaging, and fibrofatty infiltration of their rotator cuff muscles was

assessed using Goutallier staging [27]. Intraoperative muscle contractile amplitude was

assessed after electrical nerve stimulation and was correlated to the anatomic cross-

sectional area and the amount of fibrofatty infiltration. The authors concluded that higher

142

Goutallier staging resulted in impaired muscle function [27]. Davidson and Rivenburgh

demonstrated reduced isokinetic strength in their patients, when the rotator cuff was

repaired under excessive tension (i.e. repair tension exceeding 8 lbs-force) [10].

Recently, we published a report that examined the functional consequences of

rotator cuff tears in a rat model. There were three experimental groups: 1.) uninjured

controls; 2.) acute injury (muscular function was tested 4-weeks after supraspinatus

tenotomy); and 3.) chronic injury (muscular function was tested 12-weeks after

supraspinatus tenotomy). Histological changes (Fig. 2) in the muscle architecture were

identified that were consistent with increased in vivo stiffness of the muscle-tendon unit

in the acute and chronic tear setting [45]. Our findings were similar to previous published

reports in this animal model [2, 28, 29]. Then in vivo muscle-function testing of the

supraspinatus muscle-tendon unit was performed for a wide range of repair tensions,

creating a comprehensive contractile amplitude versus preload/repair tension profile of

rotator cuff function after injury (Fig. 4). Our data demonstrates that regardless of

experimental group, muscle function is exquisitely sensitive to preload tension [45].

Specifically, we demonstrated that there was a 50% reduction in maximal contractile

amplitude (Fig. 4, p<0.05) [45] at preload tensions equivalent to the chronic repair

tension required to reappose the stiff, retracted supraspinatus muscle-tendon unit to the

humeral head in this rat model [28, 29]. The findings indicate that there is a broad range

of repair tensions in which the supraspinatus muscle is able to generate maximal force;

however, current single-stage repair techniques, which occasionally require excessive

repair tensions, reduce functional capacity significantly. These data provide evidence that

link histological changes after rotator cuff tears to increased stiffness of the muscle

143

tendon unit to reduced muscle contractile amplitude. In conclusion, the study suggests

that improved functional outcomes would be achieved by earlier surgical interventions

for rotator cuff repair [45], thereby avoiding what some investigators consider

irreversible structural and biomechanical changes to the muscle-tendon unit.

COMPUTATIONAL MODELING OF THE HUMAN UPPER EXTREMITY AND

IMPLICATIONS FOR ORTHOPAEDIC SURGERY

In the chronic rotator cuff tear setting, retraction and excessive repair tension

were identified as key contributors to reduced functional capacity in a rat rotator cuff

model. Despite anatomical similarities between rat and human shoulders (Fig. 1), the

mass of the rotator cuff muscles between the two differs by orders of magnitude. Many

surgeons are hesitant to change their management of patients based upon animal data. To

aid in the interpretation of the functional rat data, retraction of chronically torn rotator

cuff muscle-tendon units was examined in a computational human upper-extremity

model.

Computational musculoskeletal models are libraries of data derived from

anatomical (i.e. muscle architecture, anatomical paths) and functional experiments (i.e.

joint moments, muscle force) compiled into an accessible and predictive mathematical

framework (Fig. 5). The force that a muscle-tendon unit generates in these model systems

is dependent on several factors, such as the architecture of the muscle-tendon unit (fiber

length, muscle cross-sectional area, pennation, tendon length) and the stretch in the

muscle and tendon. Hill-type muscle models are mathematical representations of the

mechanical properties of muscle that use non-linear springs (length-dependent elements)

and dashpots (velocity-dependent elements) to capture the force-generating behavior of

144

muscle (Fig. 6) [73]. Graphical representations of mathematical models are useful

clinically because they permit surgeons to interact intuitively with the models. These

graphical models facilitate research because parameters can be easily altered to examine

different clinical conditions (Fig. 6).

We have developed a musculoskeletal model of the upper limb representing the

anthropometry and muscle parameters of a 50th percentile male (Fig. 6) [36]. The

shoulder portion of the musculoskeletal model has three degrees of freedom represented

with definitions recommended by the International Society of Biomechanics [70].

Scapular and clavicular movements representing a normal shoulder rhythm are

incorporated into the model [11]. The upper-extremity musculoskeletal model was

implemented in OpenSim, an open-source software platform [12]. The model

incorporates:

• Skeletal representations of the torso, clavicle, scapula, humerus, radius,

ulna, wrist, and hand

• 15 degrees of freedom including glenohumeral joint, elbow, forearm,

wrist, index finger, and thumb

• 50 Hill-type muscle-tendon actuators representing the muscles and muscle

compartments crossing the shoulder, elbow, forearm, and wrist based on

architectural parameters measured from cadaveric experiments

• Moment arm and maximum isometric joint moment profiles consistent

with experimental values.

The upper limb model used in our study has been used previously to examine

multiple clinically relevant scenarios, including the consequences of Steindler

145

flexorplasty [61], brachioradialis to flexor pollicis longus transfer after spinal cord injury

[53], and latissimus dorsi transfer [20]. Computational models permit us to alter

parameters to represent clinical conditions or interventions in order to understand the

consequences in a way that would be impractical or impossible to explore in vivo (Table

1). Through computational modeling, researchers and surgeons can work together to

identify approaches to improve upper limb function.

THE EFFECT OF MUSCLE-TENDON UNIT RETRACTION ON PASSIVE AND

ACTIVE TENSION: A SIMULATION ANALYSIS OF ROTATOR CUFF TEARS

Numerous animal studies and our own data suggest that the tension required to

repair torn rotator cuff tendons to the humeral head influences surgical outcomes and

may be influenced by gap length and posture. A more detailed understanding of the

forces produced in the human shoulder throughout a full range shoulder postures for a

variety of clinically relevant gap distances may allow surgeons to predict surgical

outcomes, determine surgical candidacy, and to make evidence-based recommendations

for post-operative immobilization and rehabilitation that protect the repair site. The

purpose of our simulation analysis was to characterize the dependence of passive force

and active moment-generating capacity on shoulder posture and tear size in the

supraspinatus following surgical repair of uninjured, acute, and chronic retracted gap

tendon lengths. To represent a surgically repaired supraspinatus muscle-tendon unit, the

tendon slack length was shortened by a gap distance, while the anatomical path of the

muscle was maintained. Tendon slack length was shortened by 1.5cm to represent the

acute tear setting and 3cm to represent the chronic tear setting. Passive forces were

compared to previously reported values for suture pull out strength and anchoring

146

techniques typically used in these repairs (191-287N) [13, 41]. The calculated outcome

measures included passive muscle-tendon force throughout various ranges of motion

(thoracohumeral angle, plane of elevation, and axial rotation) and active moment-

generating capacity throughout abduction (Fig. 7).

Larger gap distances, simulating a chronically torn rotator cuff tendon, and

smaller thoracohumeral angles are associated with increased passive forces (Fig. 7). In

certain postures, the passive tension of chronically torn supraspinatus muscle-tendon

units exceeds anchor fixation methods. Further, larger gap distances are associated with

reduced peak isometric moment-generating capacity, with reductions up to 53% for

simulated chronic rotator cuff tears (Fig. 8). This study elucidates the biomechanical

consequences of repairs of large gap lengths in the rotator cuff and provides insight as to

why repairs in the chronic tear setting are associated with poorer clinical outcomes.

Increased abduction of the arm used to reduce the supraspinatus tendon intraoperatively

for the repair of larger gaps may ultimately lead to increased failure postoperatively

during shoulder adduction. For larger tendon defects, the loss of strength in the

supraspinatus may be substantial following repair, even if retearing is prevented.

Methods for reducing gap lengths, such as tendon grafts or graft substitutes, may limit the

negative biomechanical consequences of the surgical repair of excessively retracted

rotator cuff muscle-tendon units.

SUMMARY AND CONCLUSIONS

Chronically torn rotator cuff muscle-tendon units may be characterized by

increased fibro-fatty infiltration, resulting in a reduced ability to tolerate high repair

tensions. Outcomes of rotator cuff repair may be improved by earlier surgical

147

intervention, which results in lower surgical repair tensions and fewer electromyographic

neuromuscular changes. Our data suggest a direct experimental connection between high

repair tensions in the chronic tear setting and impaired contractile force. There is a

similar reduction of muscle contractile force by approximately 50% in the chronic tear

setting based on our research using both rat (Fig. 4) and simulation analysis (Fig. 8).

Therefore, we are confident our clinical recommendations are translatable from animals

to humans. Simulation analysis stresses the importance of proper arm positioning during

intra-operative repair, post-operative healing, and rehabilitation. Modifications in patient

instructions regarding post-operative arm positioning and range of motion guidelines will

reduce repair site tension. Current research efforts in our laboratory are focused on

reducing repair tension by using tissue-engineered constructs to bridge the large gap

distances that are common after chronic tears in order to improve patient outcomes.

SOURCE OF FUNDING



investigation.

ACKNOWLEDGEMENTS

The three-dimensional CT-scans shown in Figure 1 were produced in

collaboration with the Center for Biomolecular Imaging of Wake Forest School of

Medicine. Part of this study was performed in collaboration with the Wake Forest

University Graduate School of Arts and Sciences Summer Research Opportunities

Program. We would like to thank Dr. Beth P. Smith, Dr. Riyaz H. Jinnah, Mrs. Eileen

148

Martin, and Mr Anthony Santago for their support in the planning and execution of the

experiments, as well as useful suggestions in the preparation of this manuscript.




149

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biomechanics, 44(4): 669-75, 2011.

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55. Oh, L. S.; Wolf, B. R.; Hall, M. P.; Levy, B. A.; and Marx, R. G.: Indications for

rotator cuff repair: a systematic review. Clin Orthop Relat Res, 455: 52-63, 2007.

56. Rejeski, W. J.; Ip, E. H.; Marsh, A. P.; Miller, M. E.; and Farmer, D. F.:

Measuring disability in older adults: the International Classification System of

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supraspinatus tendon in an animal model: A histologic and biomechanical study. Journal

of Shoulder and Elbow Surgery, 9(2): 79-84.

67. Uhthoff, H. K.; Matsumoto, F.; Trudel, G.; and Himori, K.: Early reattachment

does not reverse atrophy and fat accumulation of the supraspinatus--an experimental

study in rabbits. Journal of orthopaedic research : official publication of the Orthopaedic

Research Society, 21(3): 386-92, 2003.

68. Vitale, M. A.; Vitale, M. G.; Zivin, J. G.; Braman, J. P.; Bigliani, L. U.; and

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comparison of asymptomatic and symptomatic shoulders. The Journal of bone and joint

surgery. American volume, 88(8): 1699-704, 2006.

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160


FIG 1: Anatomical Similarities Between the Rat and Human Shoulder. Three-

dimensional reconstructions of computer-tomography radiographic studies for rat

shoulder (Panel A) and for human shoulder (Panel B) are depicted. The supraspinatus

tendon in both species pass beneath an enclosed arch, as described by Soslowsky et al.

[65].

161

Chapter 5, Fig. 1

162

FIG 2: Histological Analysis of Supraspinatus Muscle After Rotator Cuff Tears.

Following rotator cuff tears, the rat supraspinatus undergoes atrophy (Panel B & C).

Histological analysis demonstrates increased inter-fascicular space and fibrotic

infiltration of the supraspinatus following Acute Injury (Panel B) and Chronic Injury

(Panel C), compared to the densely packed and ordered Uninjured Control skeletal

muscle (Panel A). Photomicrographs taken at 200x and scale bars represent 20µm.

Masson’s Trichrome Stain, 5µm sections of mid-substance supraspinatus muscle after

tenotomy of the supraspinatus tendon. Acute Injury: Analysis Conducted 4-weeks post-

tenotomy; Chronic Injury: Analysis Conducted 12-weeks post-tenotomy. (Figure adapted

from Mannava et al.[45] and reproduced with permission from The Journal of Bone and

Joint Surgery.)

163

Chapter 5, Fig. 2

164

FIG 3: Chronicity of Rotator Cuff Injury Influences Supraspinatus Electromyography

(EMG). Compound motor action potential (CMAP) and area under the curve (AUC) were

recorded by electromyography (EMG) from the supraspinatus muscle, following

suprascapular nerve stimulation. Panel A: After the rat rotator cuff was chronically

injured, there was lower maximal compound motor action potential (CMAP) amplitude

when compared to both the uninjured control (p<0.05, denoted by *) and acutely injured

groups (p<0.05, denoted by *). Panel B: Significant differences in AUC of the CMAP

were not detected between the experimental groups. All data are presented as mean and

standard error of the mean. Acute Injury: EMG Tested 4-weeks post-tenotomy; Chronic

Injury: EMG Tested 12-weeks post-tenotomy. (Figure adapted from Mannava et al.[45]

and reproduced with permission from The Journal of Bone and Joint Surgery.)

165

Chapter 5, Fig. 3

166

FIG 4: Repair Tension Influences Supraspinatus Muscle Function. The peak tension

required to repair an acutely torn rat rotator cuff is approximately 1.7N of preload

tension.[28, 29] The peak tension required to repair a chronically torn rat rotator cuff is

approximately 2.5N.[28, 29] Preload or repair tension affected the maximal supraspinatus

muscle single twitch contraction in all experimental groups (p<0.05). For all groups,

there was between a 22%-45% reduction in maximal single twitch amplitude at acute

repair tensions and between a 45%-56% reduction in maximal single twitch amplitude at

chronic repair tensions. Both of these reductions in contractile force were significant

(p<0.05). Data are presented as mean and standard error of the mean. (Figure adapted

from Mannava et al. [45] and reproduced with permission from The Journal of Bone and

Joint Surgery.)

167

Chapter 5, Fig. 4

168

FIG 5. Schematic diagram depicting the elements required to create an orthopaedic movement biomechanics model.

169

Chapter 5, Fig. 5

170

FIG 6. The Shoulder Portion of the Musculoskeletal Model. A musculoskeletal model

was developed for of the upper limb representing the anthropometry and muscle

parameters of a 50th percentile male [36]. Some of the parameters used for the

development of this model are shown in schematic form.

171

Chapter 5, Fig. 6

172

FIG 7. Passive Muscle-Tendon Unit Tension After Simulated Acute and Chronic Rotator

Cuff Tears. Simulation analysis was performed to study the passive tension during

abduction developed by the muscle-tendon unit after rotator cuff tears for 1.) Uninjured

control; 2.) Acute Tear (simulated by 1.5cm retraction); and 3.) Chronic Tear (simulated

by 3cm retraction). A representative pull-out strength of 215N is indicated with a gray

line and represents the passive force required for failure of suture and anchoring

techniques used during rotator cuff repair surgery [14]. The chronic tear group is

associated with increased passive forces, especially when the arm is adducted (smaller

thoracohumeral angle). Passive forces predicted by the simulation analysis at 60 degrees

abduction are consistent with observed intraoperative passive forces (0-45N) [10].

173

Chapter 5, Fig. 7

174

FIG 8. . Total (Active & Passive) Isometric Moment-Generating Capacity After

Simulated Acute and Chronic Rotator Cuff Tears. Function of the rotator cuff muscle-

tendon unit is assessed after simulated repair. Throughout the abduction range of motion,

simulation analysis was performed to study the active moment generating capacity

developed by the muscle-tendon unit after rotator cuff tears for 1.) Uninjured control; 2.)

Acute Tear (simulated by 1.5cm retraction); and 3.) Chronic Tear (simulated by 3cm

retraction). Chronic tears, which are simulated by larger gap lengths, are associated with

reduced peak isometric moment-generating capacity, with reductions up to 53%. When

repairing a chronically torn and retracted rotator cuff, total moment is affected by two

mechanisms : 1.) lengthening of the muscle results in less optimal actin-myosin cross

bridging and 2.) the interaction between the force and moment arm profiles are altered.

175

Chapter 5, Fig. 8

176

Chapter 5, Table 1

Table 1. Applications for Musculoskeletal Modeling Model Parameters Clinical Conditions Interventions

join kinematics arthritis, bony deformities joint replacement, arthrodesis, osteotomy

muscle architecture atrophy, muscle contracture strength training, tendon repair, tendon lengthening

muscle paths scarring tendon transfer

muscle activations spinal cord injury, altered neural control

functional electrical stimulation, botulinum neurotoxin A injection

177

CHAPTER 6

CLINICAL RELEVANCE AND FUTURE DIRECTIONS

Sandeep Mannava

178

SUMMARY OF DOCTORAL THESIS

In summary, the data presented in this thesis suggest that rotator cuff surgical

outcomes may be improved by earlier intervention, which results in lower surgical repair

tensions and fewer electromyographic neuromuscular changes. Our data suggest a direct

experimental connection between high repair tensions—chronic tear setting—and

impaired contractile force. The pharmacological modulation of increased muscle-tendon

unit stiffness using BoNT-A has the potential to reduce the requirement for high-repair

tensions, facilitating the surgical manipulation of the muscle-tendon unit and protecting

the repaired tendon. Simulation analysis stresses the importance of proper arm

positioning during intra-operative repair, post-operative healing, and rehabilitation. Thus,

changing patient instructions on post-operative arm positioning and ranges of motion will

reduce repair site tension.

CLINICAL RELEVANCE TO ORTHOPAEDIC SURGERY

The universal goals of rotator cuff surgery are to restore normal joint motion,

return the patient to full upper-extremity function, and to prevent further degenerative

disease. Despite many recent surgical advances, favorable outcomes after rotator cuff

repair surgery remain a challenge for the orthopedic surgeon. Thus, surgeons must

continue to unravel the biology that underlies rotator cuff pathophysiology in an attempt

to improve surgical intervention. By unraveling the biological mechanisms underlying

rotator cuff pathology in this clinically-relevant animal model system, the results of this

thesis may have the potential to improve the understanding of neuromuscular function

after rotator cuff tears, which may eventually lead to patient-specific interventions and

management for rotator cuff tears. Ultimately, an improved biological understanding of

179

rotator cuff pathophysiology allows for evidence-based guidelines in the treatment and

management of rotator cuff dysfunction. Improved surgical techniques will eventually

lead to an expedited return to independent living and recreational activities for patients

who have undergone rotator cuff surgery.

FUTURE DIRECTION FOR ROTATOR CUFF SURGERY RESEARCH

Our research efforts are centered on addressing clinical problems, and ultimately,

to improve patient care by translating this basic-science knowledge into clinical practice.

The experiments in this thesis allowed us to test the feasibility of utilizing an established

and well-characterized animal model system in order to improve our understanding of

rotator cuff neuromuscular function after injury in a small animal model. This proposal

represents a critical step in building a portfolio of functional rotator cuff data. However,

future studies will need to study the cellular and molecular basis of the

pathophysiological phenomena observed after rotator cuff injury in a small animal model,

large animal models [1-3] and, ultimately, clinical rotator cuff reconstruction studies.

Further, future molecular studies of in vivo osteointegration, tendon remodeling, and

muscle healing are needed to better elucidate the molecular mechanisms, cell signaling

pathways, and growth factor expression underlying rotator cuff healing. These future

studies will improve our molecular understanding of rotator cuff healing, which may

ultimately lead to improved surgical outcomes following tendon repair surgery.

THE STUDY OF AGE –RELATED CHANGES AND THE INFLUENCE ON

ROTATOR CUFF SURGERY

With increasing age and activity level of the population, there has been a similar

increase in both the prevalence and the number of operative interventions for

180

symptomatic rotator cuff tears [4-6]. Rotator cuff tears are a common cause of upper

extremity pain and disability in older adults. It is estimated that 20% of adults experience

a rotator cuff tear, with prevalence increasing to near 50% in adults over the age of 70

[7]. Chronic disability from rotator cuff tendinopathy affects approximately 20% of older

adults who struggle to maintain independence while faced with reduced mobility and

subsequent difficulty performing activities of daily living (ADLs) [8]. Difficulty

performing upper limb tasks such as lifting is one of the first indicators of disability [9].

Advanced age has been associated with decreased muscle function [10-14] and

healing potential after injury [13, 14], possibly due to molecular and structural changes in

the muscle occurring throughout the normal aging process [10, 11]. Some of the

detrimental changes associated with aging include loss of muscle mass [15, 16], a decline

in growth factor or trophic support of the muscle tissue (i.e. decreased IGF-1) [17, 18],

excitation-contraction uncoupling, impaired intracellular calcium handling [19, 20], age-

related denervation [21], and altered molecular expression of proteins essential for force

production [19, 20]. Myogenic regulatory factors, such as MyoD and Myf5, play an

important role during skeletal muscle development and its response to injury following

rotator cuff tears [22].

Despite the clinical knowledge that advanced patient age is a risk factor for sub-

optimal rotator cuff repair outcomes [13, 14, 23-25], there is limited understanding of

why senescent patients perform poorly following surgery. The rat rotator cuff is an

established animal model suitable for the study of human rotator cuff pathology [26, 27].

However, many basic science rodent studies have not addressed the influence of age on

181

rotator cuff pathology because the National Institutes of Health has limited access to the

Aged Rodent Colony to National Institutes of Aging funded researchers.

We are amongst the first in orthopaedic surgery to utilize the National Institutes

of Aging in vivo Aged Rodent Colony animal model to study the effects of age on

supraspinatus muscle dysfunction. We are actively funded through the Orthopaedic

Research and Education Foundation to characterize how the normal aging process

influences rotator cuff muscle-tendon unit structure and function. Due to the high

prevalence of rotator cuff tears in the elderly patient population and the heterogeneity of

patient activity level, it is difficult to study how tendon injury and age-related changes

influence rotator cuff function in the clinical setting. The N.I.A. animal model utilized in

this study allows us to explore the natural history of age-related rotator cuff changes in

isolation from rotator cuff injury. We hypothesized that aged muscle undergoes structural

and molecular changes that reduce its functional capacity. We are also actively funded to

study the influence of age on tendon-to-bone healing, through the use of biomechanical

and histological techniques.

Additionally, we have recently begun a pilot project studying the natural history

of aging on rotator cuff muscle-tendon unit properties in a characterized aged vervet

monkey model. Current research efforts are focused on studying age related histological

changes to the skeletal muscle structure, biomechanical characterization of the upper

extremity musculature (using laser diffraction, advanced radiological imaging, and

careful dissection), and tendon-to-bone biomechanical properties. We are hoping to use

these studies as pilot data to obtain funding to study the natural history of rotator cuff

182

changes after tendon tear and attempt experimental interventions in this large animal

model.

FUTURE DIRECTIONS OF BOTULINUM NEUROTOXIN A (BONT-A)

MODULATION OF SOFT-TISSUE BIOMECHANICS

We are currently conducting a series of murine studies with BoNT-A to better

understand the mechanism by which chemical denervation improves the surgical

manipulation of the muscle-tendon unit. In particular, we have designed a series of

experiments to study the myotatic reflex (FIG. 1), whose component parts of the reflex

loop consist of the Golgi tendon organ, the intrafusal muscle fibers, afferent / efferent

neural connections to the α- and γ-motoneurons, as well as inter-neurons. In this series of

experiments, we explore the role of BoNT-A on inhibiting the normal recovery of the

resting tone after stretching. We propose and will study the three possible mechanisms by

which the BoNT-A might influence passive properties:

1. Muscle response to passive stretch is partially controlled by the efferent arm

of the γ-motoneuron pathway. The γ-activation of the intrafusal muscle fibers

utilizes the neurotransmitter (acetylcholine); therefore, γ-motoneurons are

susceptible to the inhibiting effect of BoNT-A [28]. Pharmacological

inhibition with BoNT-A may prevent the intrafusal muscle fibers from

adapting to length changes; therefore, the length sensor for the muscle might

become dysfunctional. This proposed mechanism prevents the whole muscle

from contracting to its normal length after stretching, thus, maintenance of

resting tone may be impaired after BoNT-A injection.

183

2. Stretching of the muscle-tendon unit is sensed by the Golgi tendon organs,

which excite the α-motoneurons to activate muscle contraction. This reflex

loop protects the muscle from damage due to excessive strain. The efferent

arm of the α-motorneurons is inhibited by the injection of the BoNT-A.

Therefore, a percentage of tension initially produced at resting length is

decreased in chemically denervated muscle due to pharmacological, pre-

synaptic, neuromuscular, blockade of transmission that ultimately results in

decreased muscle contractility.

3. Titin has the ability to adapt its stiffness based upon the concentration of

intramuscular calcium [29]. One study reported that calcium induced titin

confirmation changes resulted in a 25% increase in total muscle stiffness [30].

BoNT-A injection may modulate passive in vivo muscle-tendon unit

properties through its indirect interaction with the titin protein [31]. After

BoNT-A injection, the denervated muscle contracts less frequently and each

contraction produces less force. The concentration of intracellular calcium in

BoNT-A denervated muscle is reduced, presumably resulting in a less stiff

titin configuration, contributing to decreased passive tension.

We are also conducting a series of experiments in the mouse gastrocnemius muscle-

tendon unit model that studies structural changes in the skeletal muscle after BoNT-A

injection. Specifically, we will attempt to calculate an in vivo modulus for the muscle-

tendon unit, before and after chemical denervation. This series of experiments will allow

us to characterize the contributions of structural alterations to improved muscle

manipulation versus the neural contributions to improved muscle manipulation.

184

Ultimately, we would like to study the effect of pre-surgical BoNT-A injection on

clinical outcomes following rotator cuff repair surgery. Our department is currently

conducting a double-blinded, placebo-controlled, clinical study testing the role of BoNT-

A on improved operative rotator cuff outcomes. Based on this initial study, we might

propose expanding the use of BoNT-A in future rotator cuff studies.

A BIOENGINEERING APPROACH TO ROTATOR CUFF SURGERY

We are also actively collaborating with our colleagues in biomedical engineering

using our basic-science mathematical findings to drive movement biomechanical

computational analysis of upper extremity motion. This collaboration represents another

source of translational research funding and support. Through this collaborative effort,

we are attempting to develop a laser-diffraction device for the intra-operative evaluation

of z-band distance. With powerful mathematical techniques, we can “scale” our murine

and rodent findings to a human. Further, upper-extremity movement biomechanics

analysis and radiographic imaging in human subjects allows us to test our hypotheses in

our patient populations.

185

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Shoulder Elbow Surg. 19(3): p. 467-76.

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general population. J Shoulder Elbow Surg, 2010. 19(1): p. 116-20.

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and the International Arthroscopy Association, 2009. 25(1): p. 30-9.

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receptor signaling impairment in aging rat skeletal muscle. Biochemical and biophysical

research communications, 1997. 235(3): p. 784-9.

18. Owino, V., et al., Age-related loss of skeletal muscle function and the inability to

express the autocrine form of insulin-like growth factor-1 (MGF) in response to

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19. Payne, A.M., et al., Neurogenesis of excitation-contraction uncoupling in aging

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low sprouting competence in motoneuron diseases. The Journal of neuroscience : the

official journal of the Society for Neuroscience, 2000. 20(7): p. 2534-42.

22. Frey, E., et al., Adipogenic and myogenic gene expression in rotator cuff muscle

of the sheep after tendon tear. Journal of orthopaedic research : official publication of the

Orthopaedic Research Society, 2009. 27(4): p. 504-9.

23. Verma, N.N., et al., Outcomes of arthroscopic rotator cuff repair in patients aged

70 years or older. Arthroscopy : the journal of arthroscopic & related surgery : official

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24. Romeo, A.A., et al., Repair of full thickness rotator cuff tears. Gender, age, and

other factors affecting outcome. Clinical orthopaedics and related research, 1999(367): p.

243-55.

25. Yamaguchi, K., et al., The demographic and morphological features of rotator

cuff disease. A comparison of asymptomatic and symptomatic shoulders. The Journal of

bone and joint surgery. American volume, 2006. 88(8): p. 1699-704.

26. Soslowsky, L.J., et al., Development and use of an animal model for

investigations on rotator cuff disease. J Shoulder Elbow Surg, 1996. 5(5): p. 383-92.

188

27. Soslowsky, L.J., et al., Neer award 1999 Overuse activity injures the

supraspinatus tendon in an animal model: A histologic and biomechanical study. Journal

of Shoulder and Elbow Surgery. 9(2): p. 79-84.

28. Stampacchia, G., et al., Change of stretch reflex threshold in spasticity: effect of

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189

Chapter 6 Figure

Figure 1: Schematic diagram depicting the myotatic reflex arc. The myotatic or stretch

reflex functions as a regulatory sensor for changes in muscle length and contributes to the

resting tone of skeletal muscle. This reflex loop is modulated by the central nervous

system through long-tract pathways:

Voluntary: corticospinal tract (CST): anterior (ant) and lateral (lat)

Postural: Vestibulaspinal Tract (VST), Tectospinal Tract (TST). Rubrospinal Tract (RST)

190

Chapter 6, Fig. 1

191

CURRICULUM VITAE

SANDEEP MANNAVA, MD Resident, Physician Scientist Training Program

Department of Orthopaedic Surgery Wake Forest University School of Medicine

EDUCATION

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES Doctor of Philosophy (Ph.D.), The Neuroscience Program Winston-Salem, North Carolina May 2012 STATE UNIVERSITY OF NEW YORK AT SYRACUSE—UPSTATE MEDICAL UNIVERSITY Doctor of Medicine (M.D.) Syracuse, New York May 2009 CORNELL UNIVERSITY Bachelor of Science (B.S), Biological Sciences (concentration: Animal Physiology) Cum Laude, High Honors in Biology, Distinction in Research Ithaca, New York May 2005

TEACHING

Clinical Volunteer Faculty Facilitator at the Wake Forest University School of Medicine: • Case Centered Learning Course (1st year medical student curriculum 2009-2010) • Standardized Patient Assessment (2nd year medical student curriculum 2011) • Standardized Patient Assessment (1st year medical student curriculum 2011-2012)

PROFESSIONAL SOCIETIES

Orthopaedic Research Society—Associate Member American Academy of Orthopaedic Surgery—Resident Member Society for Neuroscience (SfN)—Student Member Biomedical Engineering Society (BMES)—Student Member

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RESEARCH SUPPORT

Principal Investigator: Orthopaedic Research and Education Foundation Resident Clinician Scientist Training Grant ($20,000) Title: The Effect of Age on Tendon-to-Bone Healing: A Biomechanical and Histological Assessment of Surgically Repaired Rotator Cuffs PI: Sandeep Mannava Co-I: Thomas L. Smith (mentor/sponsor), Joel D Stitzel, Christopher J Tuohy Co-Investigator: Wake Forest University Intramural Research Grant Re-Submission ($20,000) Title: Optimization of bioreactor preconditioning protocols for the maturation of seeded, naturally-derived scaffolds PI: Christopher J Tuohy Co-I: Sandeep Mannava, Thorsten M Seyler, Thomas L Smith, Patrick W Whitlock

PEER-REVIEWED MANUSCRIPTS

1. Plate JF, Haubruck P, Walters J, Mannava S, Smith BP, Smith TL, Tuohy CJ. Rotator Cuff Injuries in Professional and Recreational Athletes. Accepted for publication in J Surg Orthop Adv, March 6, 2012. IN PRESS

2. Lang JE, Mannava S, Floyd AJ, Goddard MS, Smith BP, Modifi A, Jinnah RH. Robotic Systems in Orthopaedic Surgery. JBJS (Br.). 2011 Oct;93(10):1296-9.

3. Mannava S, Plate JF, Stone AV, Smith TL, Smith BP, Koman LA, Tuohy CJ. Recent advances for the management of Raynaud phenomenon using botulinum neurotoxin A. J Hand Surg (Am.). 2011 Oct;36(10):1708-10. PMID 21903346.

4. Mannava S. Plate, JF, Seyler, TM, Whitlock, PW, Callahan, MF, Koman, LA, Smith, TL, Tuohy, CJ. Evaluation of in vivo rotator cuff muscle function after acute and chronic detachment of the supraspinatus tendon: an experimental study in an animal model. JBJS (Am.). 2011 Sept 21;93(18):1702-11. PMID: 21938374

5. Saul KR, Hayon S, Smith TL, Tuohy CJ, Mannava S. Postural dependence of

passive tension in the supraspinatus following rotator cuff repair: a simulation analysis. J Clin Biomech. 2011 Oct;26(8):804-10. PMID: 21596463

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6. Mannava S, Wiggins WF, Saul KS, Stitzel JD, Smith BP, Koman LA, Smith TL, Tuohy CJ. Contributions of Neural Tone to In Vivo Passive Muscle-Tendon Unit Biomechanical Properties in a Rat Rotator Cuff Animal Model. Ann of Biomed Eng. 2011 Mar 29. PMID: 21445691

7. Mannava S, Callahan MF, Trach SM, Wiggins WF, Smith BP, Koman LA,

Smith TL, Tuohy CJ. Chemical Denervation with Botulinum Neurotoxin A Improves the Surgical Manipulation of the Muscle-Tendon Unit: An Experimental Study in an Animal Model. J Hand Surg Am. 2011 Feb;36(2):222-31. PMID: 21276885

8. Otani NF, Mo A, Mannava S, Fenton FH, Cherry EM, Luther S, Gilmour RF Jr.

Characterization of multiple spiral wave dynamics as a stochastic predator-prey system. Phys Rev E Stat Nonlin Soft Matter Phys. 2008 Aug; 78(2 Pt 1):021913. PMID: 18850871

9. Burashnikov A, Mannava S, Antzelevitch C. Transmembrane action potential

heterogeneity in the canine isolated arterially perfused right atrium: effect of IKr and IKur/Ito block. Am J Physiol Heart Circ Physiol. 2004 Jun;286(6):H2393-400. PMID: 15148061

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BOOK CHAPTERS

1. Brown PJ, Plate JF, Mannava S, Stitzel JD. Injury Mechanisms of Five Common Sports Related Orthopaedic Injuries. Orthopaedic Biomechanics. Beth Winklestein, editor. Submitted July 8, 2011 to CRC/Taylor and Francis Publishing Company. IN PRESS.

2. Whitlock PW, Seyler TM, Mannava S, Poehling GG. A tissue-engineered approach to ligament and tendon reconstruction. Sports Injuries: Prevention, Diagnosis, Treatment, Rehabilitation. Editors: Mahmut Doral et al. Springer. 1st Edition., 2011, XII, 1020 p. 706 illus., 481 in color., Hardcover SBN: 978-3-642-15629-8. DOI: 10.1007/978-3-642-15630-4_157, © Springer-Verlag Berlin Heidelberg 2011

THESIS TITLES

1. Mannava S. (2012). The Neurological Mechanisms Underlying Rotator Cuff Skeletal Muscle Pathophysiology and Implications for Therapy. Wake Forest University Graduate School of Arts and Sciences. Doctoral Thesis (The Neuroscience Program)

2. Mannava S, Otani NF, Gilmour RF. (2005). The Predator-Prey Relationship in

2D Spiral Wave Simulations. Cornell University Undergraduate Honors Thesis.

UNITED STATES PATENT

Mannava S, Tanaka MF, Tuohy CJ. Tissue tensioning devices and related methods. Utility Patent Application No. 12/942,311 filed November 9, 2010 in United States Patent and Trademark Office MULTIMEDIA/VIDEO: • Case JM, Mannava S, Cheetham S, Long BJ, Stubbs AJ. Physical Examination of the

Shoulder. [MULTIMEDIA/VIDEO]. AAOS 2012 Annual Meeting. San Francisco, CA. February 7, 2012. Selected as a FEATURED PRESENTATION

ABSTRACTS AND MEETING PRESENTATIONS

1. Mannava S, Plate JF, Seyler TM, Whitlock PW, Callahan MF, Smith TL, Tuohy CJ. Muscle function after rotator cuff injuries in an animal model. [ABSTRACT].

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Surgical Sciences Research Day. Wake Forest University School of Medicine. Winston-Salem, NC. November 19, 2009.

2. Mannava S, Plate JF, Seyler TM, Tuohy CJ, Callahan MF, Koman LA, Smith

TL. Evaluation of Rotator Cuff Muscle Dysfunction. [ABSTRACT]. 10th Annual Graduate Student Research Day, Spring 2010. Wake Forest University Graduate School of Arts and Sciences. Winston-Salem, NC.

3. Mannava S, Plate JF, Seyler TM, Whitlock PW, Callahan MF, Smith TL, Tuohy

CJ. The pathophysiology of rotator cuff dysfunction and implications for therapy: A series of related in vivo animal studies. [ABSTRACT]. Resident Research Day. Wake Forest University School of Medicine. Winston-Salem, NC. April 16, 2010.

4. Mannava S, Plate JF, Seyler TM, Callahan MF, Smith TL, Koman LA, Tuohy

CJ. Novel Use of Electromyography (EMG) to Evaluate In Vivo Rotator Cuff Muscle Function After Acute and Chronic Detachment of the Supraspinatus Tendon: An Experimental Study in an Animal Model. [ABSTRACT]. Poster Presentation at the Southern Orthopaedic Association 2010 Annual Meeting, Fajardo, Puerto Rico.

5. Mannava S, Plate, JF, Seyler, TM, Callahan MF, Smith TL, Koman LA, Tuohy

CJ. Evaluation of In Vivo Rotator Cuff Muscle Function After Acute and Chronic Detachment of the Supraspinatus Tendon: An Experimental Study in an Animal Model. [ABSTRACT]. Podium Presentation at the Southern Orthopaedic Association 2010 Annual Meeting, Fajardo, Puerto Rico.

6. Mannava S, Wiggins WF, Callahan MF, Smith TL, Koman LA, Tuohy CJ.

Innovations in the management of complex and chronic tendon injuries. [ABSTRACT] Podium Presentation at the North Carolina Orthopaedic Association 2010 Annual Meeting. Asheville, NC.

7. Tuohy CJ, Plate JF, Callahan MF, Koman LA, Smith TL, Mannava S.

Pathophysiologic Mechanisms of Rotator Cuff Dysfunction and Implications for Therapy. [ABSTRACT] Podium Presentation at the North Carolina Orthopaedic Association 2010 Annual Meeting. Asheville, NC.

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8. Mannava S, Plate JF, Callahan MF, Smith TL, Tuohy CJ, Koman LA. Evaluation of In Vivo Muscle Function After Rotator Cuff Tears Using Electromyography in an Animal Model. [ABSTRACT] Podium Presentation at the Eastern Orthopaedic Association 2010 Annual Meeting. Naples, FL. TRAVEL AWARD WINNING PAPER.

9. Mannava S, Wiggins WF, Stitzel J, Callahan MF, Koman, LA, Smith TL, Tuohy

CJ. Contributions of neural tone to muscle-tendon unit stress-relaxation biomechanical properties. [ABSTRACT] Podium Presentation at the Biomedical Engineering Society (BMES) 2010 Annual Meeting. Austin, TX. TRAVEL AWARD WINNING PAPER.

10. Holzbaur KS, Hayon S, Smith T, Tuohy C, Mannava S. Passive tension in

supraspinatus following rotator cuff repair: a simulation analysis. [ABSTRACT] Podium Presentation at the Biomedical Engineering Society (BMES) 2010 Annual Meeting. Austin, TX. TRAVEL AWARD WINNING PAPER.

11. Mannava S, Wiggins WF, Callahan MF, Stitzel J, Koman, LA, Tuohy CJ, Smith

TL. Neurological influences and modulation of skeletal muscle tone. [ABSTRACT] Poster Presentation at the Society for Neuroscience (SfN) 2010 Annual Meeting. San Diego, CA.

12. Mannava S, Tuohy CJ, Seyler TM, Stitzel J, Hayon S, Whitlock PW, Smith TL,

Saul KR. The Science of Rotator Cuff Repairs: Translating Basic Science into Clinical Recommendations. [Scientific Exhibit ABSTRACT] Presentation at the AAOS 2011 Annual Meeting. San Diego, CA. SELECTED AS A TOP 20 EXHIBIT.

13. Mannava S, Seyler TM, Pace LA, Whitlock PW, Smith BP, Koman LA, Smith

TL, Tuohy CJ. Age-Related Changes Affect Supraspinatus Muscle Function: An Experimental Study in an Animal Model. [ABSTRACT] Presentation at the ORS 2011 Annual Meeting. San Diego, CA.

14. Sikes CV, Brown PJ, Danelson KA, Seyler TM, Mannava S, Jinnah RH, Stitzel

JD, Lang JE. Biomechanical Analysis of Hip Core Decompression Techniques and Fracture Risk. [ABSTRACT] Presentation at the ORS 2011 Annual Meeting. San Diego, CA.

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15. Mannava S, Saul KR, Seyler TM, Koman LA, Smith TL, Tuohy CJ. Rotator Cuff

Dysfunction After Acute and Chronic Tears: Translating Animal Studies to Clinical Recommendations Using Simulation Analysis. [ABSTRACT] Presentation at the 2010 OREF / ORS Atlantic Coast Resident Research Symposium—Duke University. Durham, NC.

16. Mannava S, Saul KR, Plate JF, Seyler TM, Koman LA, Tuohy CJ, Smith TL.

Rotator Cuff Dysfunction After Acute and Chronic Tears: Translating Animal Studies to Clinical Recommendations Using Simulation Analysis. [ABSTRACT] Surgical Sciences Research Day. Wake Forest University School of Medicine. Winston-Salem, NC. November 11, 2010. GOLD MEDAL WINNER OF BASIC SCIENCE AWARD

17. Mannava S, Wiggins WF, Saul KR, Stitzel J, Koman, LA, Tuohy CJ, Smith TL.

Chemical Denervation with Botulinum Neurotoxin A Modulates Skeletal Muscle Tone: Assessment of Neural Contributions to In Vivo Soft-Tissue Biomechanics. [ABSTRACT] Poster Presentation at the Western North Carolina Society for Neuroscience (SfN).Wake Forest University School of Medicine. Winston-Salem, NC. November 11, 2010.

18. Pace LA, Hill P, Garrett J, Ma J, Apel P, Mannava S, Barnwell J, Smith B, Li Z,

Koman LA, Smith T, Van Dyke M. Clinical Translation of a Keratin Biomaterial Hydrogel for Nerve Repair. [ABSTRACT] Podium Presentation at NCTERM 2010.

19. Pace LA and Mannava S. Clinical Translation of a Keratin Biomaterial Hydrogel

for Nerve Repair: From Mice to Men. [Invited Lecture]. Wake Forest University School of Medicine, Regenerative Medicine Interest Group. Winston-Salem, NC. January 21, 2011.

20. Mannava S, Koman LA, Seyler TM, Plate JF, Tuohy CJ, Saul KR, Smith TL.

Translating Animal Studies to Clinical Recommendations with Computational Analysis for the Study of Rotator Cuff Dysfunction After Acute and Chronic Tears. [ABSTRACT] 11th Annual WFU Post-Doctoral Research Day. March 2011. Winston-Salem, NC. AWARD WINNER (runner-up): INTEGRATIVE SCIENCES CATEGORY.

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21. Mannava S, Plate JF, Seyler TM, Whitlock PW, Saul KR, Smith TL, Tuohy CJ.

The Science of Rotator Cuff Repairs: Translating Basic Science into Clinical Recommendations. [ABSTRACT]. Resident Research Day. Wake Forest University School of Medicine. Winston-Salem, NC. April 1, 2011.

22. Schweppe ML, Goddard MS, Mannava S, Plate JF, Seyler TM, Lang JA.

Accuracy of Component Positioning and the Associated Learning Curve of the Direct Anterior Approach in Total Hip Arthroplasty. [ABSTRACT]. Resident Research Day. Wake Forest University School of Medicine. Winston-Salem, NC. April 1, 2011.

23. Tuohy CJ, Plate JF, Pace LA, Saul KR, Koman LA, Smith TL, Mannava S. Age-

related changes affect rotator cuff muscle function. [ABSTRACT]. Eastern Orthopaedic Association Annual Meeting. Williamsburg, VA. October 19, 2011.

24. Koman LA, Plate JF, Mannava S, Stone AV, Jinnah RH, Smith TL. Plate JF,

Pace LA, Saul KR, Addressing The Shortage of Academic Orthopaedic Surgeons: Evaluation of an Innovative Seven-Year Physician Scientist Residency Training Program. [ABSTRACT]. Eastern Orthopaedic Association Annual Meeting. Williamsburg, VA. October 19, 2011.

25. Smith TL, Haubruck P, Saul KR, Wiggins WF, Stitzel JD, Smith BP, Tuohy CJ, Mannava S. In vivo biomechanics of neural mechanisms influencing stretching of the muscle-tendon unit. [ABSTRACT]. Biomedical Engineering Society Annual Meeting. Hartford, CT. October 12, 2011.

26. Plate JF, Mannava S, Seyler TM, Saul KR, Pace LA, Smith TL, Tuohy CJ. Age Influences Healing and Function of Rotator Cuff Muscles. [ABSTRACT]. North Carolina Orthopaedic Association. Kiawah Island, SC. October 7, 2011.

27. Haubruck P, Mannava S, Schmidmaier G, Smith BP, Saul KR, Koman LA, Tuohy CJ, Smith TL. Bioprotection of the muscle-tendon unit improves surgical manipulation in an animal model. [ABSTRACT]. European Orthopaedic Research Society 2011 Annual Meeting. Vienna, Austria.

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RESEARCH POSITIONS AND EMPLOYMENT

2009-present Physician Scientist/Resident, Department of Orthopaedic Surgery Wake Forest University School of Medicine Post-Doctoral Research Fellow Wake Forest University Institute For Regenerative Medicine (Mentors: Thomas L. Smith, PhD and L. Andrew Koman, MD)

2006-2007 Graduate Research Assistant, State University of New York at Syracuse -

Upstate Medical University, Department of Pharmacology, Patch Clamp Analysis of Ion Channel Dynamics in Heart Failure and Fibrillation (Ik1 and Gap Junctions) (Mentor: Justus MB Anumonwo, PhD)

2004-2005 Research Assistant, Cornell University

Department of Biomedical Sciences Mathematical Modeling Of Ventricular Fibrillation Using Small-World Network Theory, Scale Free Network Theory, and The Predator-Prey Differential Equation Model (Mentor: Robert F. Gilmour Jr, PhD)

2003 Urban Semester Student, Weill Cornell Medical School

Department of Physiology and Biophysics Three Dimensional Characterizations of Ventricular Fibrillation and Real Time Fibrillation Control (Mentor: David J Christini, PhD)

2002-2003 Summer Research Fellow, Masonic Medical Research Laboratory,

Department of Experimental Cardiology Studied Electrical Conduction Heterogeneity During Atrial Fibrillation (Mentor: Charles Antzelevitch, PhD)

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AWARDS AND HONORS

• Wake Forest University 11th Annual Graduate Student and Post-Doc Research Day, March 22, 2011. Integrative Sciences Category (RUNNER-UP Paper)

• Wake Forest University School of Medicine Surgical Science Basic Science Award, First Place (2010)

• Wake Forest University Graduate School of Arts and Sciences, Alumni Student Award (2010)

• Eastern Orthopaedic Association Travel Award (2010)

• Howard Hughes Research Scholarship (2004)

• American Heart Association Award (2004)

• Masonic Medical Research Laboratory Summer Fellowship (2002 and 2003)

NEUROLOGICAL MECHANISMS UNDERLYING ROTATOR CUFF

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