1

Title 1

'Biomechanical Properties of Bovine Tendon Xenografts Treated with a Modern 2

Processing Method'. 3

4

Authors & Contributions Statement 5

Mr Henry B Colaço*: study design, data collection/analysis, manuscript preparation 6

Mr Breck R Lord**: data collection, data analysis 7

Ms Diane L Back***: study design, manuscript preparation 8

Mr Andrew J Davies***: study design, manuscript preparation 9

Prof Andrew A Amis**: study design, manuscript preparation, approval 10

Mr Adil Ajuied***: study design, data analysis, manuscript preparation, approval 11

All authors have read and approved the final submitted manuscript. 12

13

*Royal Free London NHS Foundation Trust, London, UK 14

**Imperial College London, London, UK 15

***Guy's and St Thomas' Hospital NHS Foundation Trust, London, UK 16

17

Corresponding Author 18

Mr Henry B Colaço 19

22 Ballingdon Road, London, SW11 6AJ, UK 20

tel: +44(0)7958 567 359 21

e-mail: henry@henrycolaco.com 22

23

Keywords: Xenograft; Tendon; Knee Ligaments; ACL; Sterilization 24

Word Count: 2,359 (Introduction through Discussion) 25

2

Abstract 26

Xenograft tendons have been used in few human studies, with variable results. With 27

the advent of novel tissue processing techniques, which may mitigate against an 28

immune-mediated rejection response without adversely affecting mechanical 29

properties, there may now be a clinical role for xenograft tendons, particularly in knee 30

ligament reconstruction. We hypothesize that ‘BioCleanse®’ processed bovine 31

extensor digitorum medialis (EDM) tendons exhibit favorable time-zero pre-32

implantation biomechanical characteristics when compared to both unprocessed 33

bovine EDM tendons and BioCleanse® processed human cadaveric allograft tibialis 34

anterior tendons. 35

In this in vitro case controlled laboratory study, three groups of tendons underwent a 36

5-stage static loading test protocol: 15 BioCleanse® bovine (BCB), 15 fresh frozen 37

unprocessed bovine (FFB), and 12 BioCleanse® human allograft (BCA) tendons. 38

Cross-sectional area of the grafts was measured using an alginate molding technique, 39

and tendons were mounted within an Instron® 5565 Materials Testing System using 40

cryogenic clamps. 41

BCB tendons displayed a higher ultimate tensile stress (p<0.05), with equivalent 42

ultimate failure load, creep, and modulus of elasticity when compared to the FFB 43

tendons (p>0.05). BCB tendons had an equivalent cross-sectional area to the BCA 44

tendons (p>0.05) whilst exhibiting a greater failure load, ultimate tensile stress, less 45

creep and a higher modulus of elasticity (p<0.05). 46

The BioCleanse® process did not adversely affect the time-zero biomechanical 47

properties of bovine xenograft EDM tendons. BioCleanse® processed bovine 48

xenograft EDM tendons exhibited superior biomechanical characteristics when 49

3

compared with BioCleanse® processed allograft tibialis anterior tendons. These 50

findings support further investigation of xenograft tendons in orthopaedic soft tissue 51

reconstructive surgery. 52

53

Keywords: Knee ligaments; Xenograft; Sterilization; Biomechanics of tendon 54

4

Introduction 55

Current graft options for knee ligament reconstruction include autograft, allograft, and 56

synthetic graft (Strobel et al., 2006; Bonanzinga et al., 2014; Legnani et al., 2010). 57

These exhibit different biomechanical properties, complication profiles, handling 58

characteristics, availability, host integration, cost, and the ability to replicate native 59

anatomy (Barrera Oro et al., 2011). The limit of available autograft represents a 60

significant challenge in the context of revision soft tissue reconstructions and the 61

multi-ligament knee reconstruction (Levy et al., 2009; Lind et al., 2009). One possible 62

solution would be to implant tendon grafts of animal origin (xenograft) (Laurencin 63

and El-Amin, 2008). This is an established concept in soft tissue reconstruction, 64

which has been applied with varying degrees of success over the last 60 years, 65

although there remains a paucity of research in this field (Allen et al., 1987, Dahlstedt 66

et al., 1989, Stone et al., 2007a). 67

The potential benefits of xenografts include avoidance of donor site morbidity, a 68

reduction of surgical anaesthetic time, a potentially lower cost profile, elimination of 69

the risk of human blood-borne diseases (e.g. HIV, Hep C), and an abundance of 70

source material resulting in a more robust supply chain (Cooper et al., 2010, Nagda et 71

al., 2010, Simonds et al., 1992, Asselmeier et al., 1993, Patel and Trampuz, 2004, 72

Kainer et al., 2004). Commercially available xenograft products for orthopaedic 73

surgery range from osteoconductive hydroxyapatite and collagen-based bone graft 74

substitutes, to soft tissue extracellular matrix grafts from dermis and small intestine 75

submucosa (Mucalo, 2007; Neen et al., 2006, Soler et al., 2007, Iannotti et al., 2006). 76

At present, there are no widely available commercial xenogeneic tendons or ligaments 77

suitable for implantation in humans. 78

5

If unprocessed bovine or porcine xenografts, which express α-gal and other epitopes 79

(e.g. Neu-Gc) known collectively as ‘non-gal epitopes’, are implanted into humans or 80

non-human primates, the graft will trigger an acute immune mediated rejection 81

response and result in catastrophic failure (Stone et al., 2007b, Galili, 1993; Galili, 82

2013; Galili, 2012). Xenograft tissue also carries with it the inherent risks of immune 83

mediated rejection and transmission of zoonoses (e.g. Bovine Spongiform 84

Encephalopathy (BSE) (Laurencin and El-Amin, 2007). For bovine tissue, the 85

foremost risk can be mitigated by sourcing all cattle from BSE-free countries, as 86

recognized by the World Organization for Animal Health (OIE). The challenge lies in 87

developing processing techniques that effectively sterilise the tissue and overcome 88

immune-mediated rejection, whilst maintaining favourable biomechanical properties 89

and graft-host integration. 90

Previous methods of terminal sterilization such as ethylene oxide, gamma irradiation, 91

and glutaraldehyde have been shown to adversely affect the handling and 92

biomechanical properties of graft tissue (Allen et al., 1987; Jackson et al., 1990; 93

Gibbons et al., 1991; Rasmussen et al., 1994). Lyophilization can have similar 94

adverse effects and does not guarantee removal of pathogens (Kamiński et al., 2009; 95

Galili, 2013; Crawford et al., 2004). Although a large cohort study recently identified 96

a number of patient and graft-related factors, including processing method, that were 97

associated with revision risk after allograft ACL reconstruction, the use of allograft 98

for ACL and multiligament knee reconstruction remains standard practice (Tejwani et 99

al). 100

Several novel proprietary methods have been developed for treating allograft, which 101

involve physical and chemical processes to achieve decontamination and sterilization 102

6

(Vangsness et al., 2003; Vangsness and Dellamaggiora, 2009; McAllister et al., 103

2007). These processes have the potential to play a role in processing xenograft tissue 104

for implantation in humans if the biomechanical properties of the tissue are not 105

adversely affected, and if antigen expression is reduced below a threshold where no 106

significant immune response is triggered (Stone et al., 1997; Stone et al., 2007a). 107

The purpose of this study was to compare the pre-implantation tensile biomechanical 108

properties of chemically processed bovine extensor digitorum medialis (EDM) 109

tendons against unprocessed fresh-frozen bovine tendons, and chemically processed 110

tibialis anterior allograft tendons. It was hypothesized that chemically processed 111

bovine extensor tendons would have similar biomechanical properties to unprocessed 112

bovine and chemically processed human allograft tendons. The clinical relevance of 113

the study is that if the bovine xenograft tendons exhibit similar or superior mechanical 114

properties to human allograft tendons, and the BioCleanse® process does not 115

adversely affect the time zero biomechanical properties, we will have identified a 116

potential tendon graft source for further study in animal and human trials. 117

7

Methods 118

Following institutional approval, bovine extensor digitorum medialis (EDM) tendons 119

were harvested aseptically from certified BSE-free cattle with a minimum weight of 120

295kg and age under 30 months, confirmed by dentition (Maverick Biosciences Pty 121

Ltd., Dubbo, East NSW, Australia). Fifteen fresh frozen bovine (FFB) tendons 122

underwent no further processing, and were stored at -30°C. A further 15 bovine EDM 123

tendons (BCB: BioCleanse bovine) and 12 human tibialis anterior allograft (BCA: 124

BioCleanse allograft) tendons were processed using the proprietary BioCleanse® 125

process (RTI, Alachua, Florida, USA). All tendons were imported frozen on dry ice 126

and stored at -30°C until testing, at which time the grafts were placed in a 23°C water 127

bath to achieve controlled thawing in watertight sealed packs. 128

Tendon specimens were prepared to 90mm length, allowing secure cryogenic clamp 129

fixation of a 30mm segment at each end, leaving the central 30mm section, similar to 130

the length of the native anterior cruciate ligament (ACL), to undergo mechanical 131

tensile testing. The mean cross sectional area (CSA) of each tendon was calculated 132

prior to testing using a ‘replica molding’ technique (Goodship and Birch, 2005). An 133

alginate dental impression material (Blueprint cremix, Dentsply DeTrey, Germany) 134

was used to form a cast and axial digital photographs taken of 3 equally spaced 135

sections using a mounted Canon EOS600D digital SLR with an 18-megapixel sensor 136

and fixed focal length Canon EF 50mm f/1.8 II standard lens. The mean CSA was 137

calculated using image analysis software (Image J 1.48, National Institute of Health, 138

Bethesda, Maryland, US). This method is accurate to within 0.8%, and reproducible 139

(coefficient of variation = 1.42%), allowing calculation of ultimate tensile stress and 140

Young’s modulus of elasticity. 141

8

The custom soft-tissue cryogenic clamps (Riemersma and Schamhardt, 1982), used 142

corrugated plates and injection of liquid carbon dioxide (CO2) to freeze the tissue and 143

prevent slippage. The rounded edges of the clamps reduced the stress riser at the 144

interface between the clamp and the central 30mm length of tendon to be tested. An 145

initial clamp temperature of approximately -30°C was reached by expansion of liquid 146

CO2. At this temperature, tissue thermal transition is acceptable, even at a distance of 147

5 mm from the device, which minimizes alteration of viscoelastic properties (Rincón 148

et al., 2001; Lam et al., 1990; Woo et al., 1987). 149

Tensile mechanical testing was performed using a single-axis materials testing 150

machine (Model 5565, Instron, High Wycombe, UK) accurate to +/- 0.02 mm. The 151

upper cryogenic clamp was attached to a 5kN load cell (accuracy < 0.025 %) mounted 152

to the mobile crosshead, with the lower clamp secured to a static base plate. The 153

automated test protocol was set using Instron® Bluehill® 2 modular applications 154

software with data capture every 100ms. Testing was conducted at room temperature 155

(23°C). Each tendon was secured within the clamps using moderate compression 156

before increasing the tension manually using fine position control to achieve a starting 157

load of 0 to 0.005 N. 158

The test protocol was divided into five phases (Table 1): each tendon underwent an 159

initial preload to allow uncrimping of collagen fibers (phases 1&2), followed by a 160

steady ramp to 500 N (phase 3), which exceeded the maximum load experienced in 161

ACL graft during accelerated rehabilitation (Noyes and Grood, 1976; Woo et al., 162

1991). The load was maintained at 500N for two minutes (phase 4) to allow for 163

measurement of creep (Jaglowski et al., 2014). Mean creep strain rate (S s-1) was 164

calculated for each tendon by dividing change in strain (S), where S = creep 165

9

elongation/original length, over the 120 second period. Finally the specimen was 166

loaded to failure (phase 5). The ultimate tensile stress (UTS) was calculated for each 167

tendon, by dividing the ultimate failure load (UFL, N) by CSA (mm2); so UTS = UFL 168

/ CSA (MPa). Young’s modulus of elasticity (E) was calculated for each tendon from 169

the linear, elastic portion of the force versus extension curve during phase 5 ramp to 170

failure loading, which was converted to a stress versus strain curve by dividing the 171

force by the CSA and the extension by the original gauge length of the specimen. 172

Statistical analysis was performed using IBM® SPSS® Statistics v22.0.0.0 (IBM, 173

Armonk, NY, USA). One-way analysis of variance (ANOVA) test was applied, and 174

post hoc analysis was performed using the Tukey Honestly Significantly Different 175

(HSD) test to identify significant differences between pairs of compared groups. A 176

significance level of P<0.05 was applied throughout. 177

As the effect of the BioCleanse® process on bovine tendons was not yet known, an 178

accurate a priori power calculation could not be made with certainty. The number of 179

tendons required to adequately power the study ( = 0.8) was therefore estimated 180

from a review of similar published data (Schimizzi et al., 2007; Reid et al., 2010; 181

Conrad et al., 2013). A post-hoc power analysis confirmed the assumption that the 182

study was adequately powered (>0.8) to reach significance. 183

10

Results 184

The cross-sectional areas (CSA) of both BioCleanse® groups were smaller than the 185

unprocessed bovine group (p< 0.01), but a difference was not found between BCB 186

and BCA (p>0.05) (Table 2). Levene’s test for equality of variances was >0.05, which 187

means that we could assume homogeneity of variances, or an equal variance within 188

each group when compared with the other two groups. 189

The ultimate failure load (UFL) was higher in both bovine groups compared with 190

allograft (p< 0.05 and p< 0.01 for BCB and FFB, respectively), but a difference was 191

not found between BCB and FFB (p >0.05) (Table 2). The ultimate tensile stress 192

(UTS) was higher in the processed bovine group than both other groups (p<0.05 and 193

p<0.01 for FFB and BCA, respectively). , but a difference was not found between 194

FFB and BCA (p >0.05) (Table 2). 195

The allograft tendons exhibited more creep and a lower Young’s modulus of elasticity 196

(E) than both bovine groups. Analysis found that the BCA tendons crept faster than 197

BCB (p< 0.05) and FFB (p< 0.01), but did not find a difference in creep between 198

BCB and FFB (p >0.05), and that while E was significantly lower in BCA than BCB 199

(p< 0.01) and FFB tendons (p< 0.05), a difference was not found between BCB and 200

FFB (p >0.05) (Table 2). 201

Interestingly, the BioCleanse® treated bovine tendons exhibited a closer correlation 202

between CSA and ultimate failure load (r2 = 0.745) (Figure 1), when compared with 203

fresh frozen bovine (r2 = 0.28735), and BioCleanse® treated human tendons (r2 = 204

0.220) (Table 2, Figure 1). A post-hoc data analysis revealed the power to be 0.86 205

(>0.8), which confirmed pre-test estimates that the sample size would be sufficient to 206

adequately power the study. 207

11

Discussion 208

The most important finding of this study was that the chemically treated tendons had 209

similar or superior mechanical properties to their fresh-frozen controls. The processed 210

bovine tendons (BCB) had greater ultimate tensile stress, with equivalent ultimate 211

failure load, creep, and modulus of elasticity when compared with the fresh frozen 212

bovine tendons (FFB). The processed bovine (BCB) tendons had similar cross-213

sectional area, higher ultimate tensile stress, failure load, and modulus of elasticity, 214

with less creep, when compared with the processed human allograft (BCA) tibialis 215

anterior tendons. Thus, the ‘time zero’ mechanical properties of the treated tendons 216

are suitable for a ligament graft. 217

The effects of the BioCleanse process on the time-zero biomechanical properties of 218

human allograft have been investigated previously. Schimizzi et al. (2007) compared 219

BioCleanse treated allograft human tibialis anterior tendons against gamma-irradiated, 220

and fresh-frozen tendons, and found no significant difference in failure load and 221

failure stress. 222

Jones et al. (2007) compared BioCleanse treated human bone-patellar tendon-bone 223

(BTB) grafts against fresh frozen BTB grafts, again using a cyclic testing protocol 224

followed by ramp to failure. They found no difference in ultimate failure load, 225

ultimate stress, cyclic creep, or stiffness. 226

Reid et al. (2010) compared three groups of human bone-patellar tendon-bone 227

allografts: BioCleanse, low-dose irradiated (50kGy) fresh frozen, and Clearant 228

(Clearant Inc., Los Angeles, California, USA) treated specimens, half of which were 229

terminally irradiated with 50 kGy gamma radiation. They found no significant 230

differences between the groups for stiffness under cyclic loading, or ultimate stress. 231

12

Conrad et al. (2013) compared BioCleanse treated human allograft Achilles tendons 232

against tendons treated with 1.5-2.5 MRads gamma radiation, and fresh-frozen 233

tendons. The BioCleanse group exhibited similar ultimate failure load and stiffness, 234

but a lower Young’s modulus and ultimate stress. 235

This study compared chemically processed tendon with frozen human allograft 236

tendons and not with fresh specimens. That was because it is not possible to source, 237

transport and store them prior to use without freezing. Freezing of fresh tendons is a 238

standard method of tissue preparation and storage for allograft (Schimizzi et al., 239

2007; Reid et al., 2010; Conrad et al., 2013). Studies have found minimal effects of 240

deep-freezing and thawing on the biomechanical characteristics of human and animal 241

tendons, including multiple freeze-thaw cycles (Huang et al, 2011; Moon et al, 2006; 242

Jung et al, 2011; Lee et al, 2009). 243

One limitation of our study is that although the temperature of the cryogenic clamps 244

was known, the temperature at the mid-substance of the tendon was not measured 245

during testing. However, although tissue temperature can alter biomechanical 246

properties, the thermal transition profile was acceptable and unlikely to affect results 247

properties (Rincón et al., 2001; Lam et al., 1990; Woo et al., 1987). 248

Although this study has demonstrated acceptable mechanical properties of the 249

chemically treated xenografts when compared to untreated xenografts and human 250

tibialis anterior allografts, this does not mean that they are suitable for clinical use. It 251

is well-known that ligament constructs other than autografts suffer from differing 252

tissue reactions and changes of strength post-implantation, and may also provoke 253

unwanted host reactions (Wong and Griffiths, 2014); those aspects require testing in 254

animals, ideally non-human primates, prior to human trials. 255

13

Conclusion 256

This study has established that the time-zero biomechanical characteristics of 257

BioCleanse® processed bovine tendons are suitable for a ligament graft. This 258

supports further investigation of the application of xenograft tendon in orthopaedic 259

soft tissue reconstructive surgery prior to clinical use. 260

261

Acknowledgements 262

Funding was received in the form of donated tendons from Regeneration 263

Technologies Inc., Alachua, FL. 264

The Instron machine was donated by the Arthritis Research UK charity. 265

No authors have any relevant financial disclosures or conflicts of interest do declare. 266

14

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Phase Start load (N) End load (N) Time (s) Load rate

1 Ramp to preload 0 10 10 1 N/s

2 Preload 10 10 60 0

3 Ramp to 500N 10 500 10 49 N/s

4 Creep 500 500 120 0

5 Ramp to failure 500 (failure) (variable) 10 mm/s

427

Table 1. Tensile testing load protocol. 428

429

430

BCB FFB BCA

CSA (mm2) 24 ± 6 33* ± 5 24 ± 4

UFL (N) 2,132 ± 473 2,286 ± 573 1,559* ± 176

UTS (MPa) 88.1* ± 10.2 70.2 ± 13.2 68.9 ± 9.3

Creep (S s-1) 0.035 ± 0.011 0.028 ± 0.010 0.046* ± 0.020

Young’s modulus (E; MPa) 725.5 ± 139.8 642.8 ± 182.4 484.2* ± 99.1

431

Table 2. Results of tensile testing. * denotes significant difference (p<0.05) 432

433

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434

Fig 1. 435