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xperiences with sheep as an animal model for shoulderurgery: Strengths and shortcomings

. Simon Turner, BVSc, MS, Dipl ACVS, Ft. Collins, CO

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heep (and goats) are a convenient large-animalodel for rotator cuff repair because of availability,ase of handling and housing, animal cost, and ac-eptance to society as a research animal. Tenotomy ofhe infraspinatus tendon and subsequent reattachmento the proximal humerus is useful to address the biome-hanical, histologic, and biochemical processes of ro-ator cuff repair. Detaching this tendon and immedi-tely reattaching it does not represent the clinicalicture but serves as a relatively rapid way to screenifferent suture anchors, suture patterns, scaffolds,rowth factors, and other biologics or a combinationf these treatments to enhance the healing process. Toinimize spontaneous reattachment and reproduce ahronic rotator cuff injury, the end of the tendon cane covered and then reattached 4 weeks later if bone-

o-tendon healing is to be evaluated. This chronicodel is useful to understand the biology (degree ofuscle atrophy and fatty infiltration) of rotator cuffuscles as well as innovative methods of repair.lose-stall confinement is required during the convales-ence in acute and chronic studies. Ultrasound in thewake animal can be used to monitor gap formationnd tissue organization. Sheep have also been used

o determine whether capsular healing after plications equivalent to open capsular shift. (J Shoulder Elbowurg 2007;16:158S-163S.)

keletally mature female sheep are rapidly becom-ng a convenient large-animal model for a variety ofrthopedic problems, including rotator cuff repair.18

his is because of availability, ease of handling andousing, animal cost, and acceptance to society as aesearch animal. Although the anatomy of the shoul-er of quadrupeds is quite different than humans,

rom the Department of Clinical Sciences, Veterinary MedicalCenter, Colorado State University.

eprint requests: A. Simon Turner, BVSc, MS, Dipl ACVS, Profes-sor, Department of Clinical Sciences, Veterinary Medical Center,Colorado State University, Ft. Collins, CO 80523 (E-mail:sturner@colostate.edu).opyright © 2007 by Journal of Shoulder and Elbow SurgeryBoard of Trustees.

058-2746/2007/$32.00

ioi:10.1016/j.jse.2007.03.002

58S

enotomy of the infraspinatus of goats17 and sheep,5nd subsequent reattachment to the proximal hu-erus, is useful to address the biomechanical, histo-

ogic, and biochemical processes of rotator cuff re-air.

Sheep have been selected because the similarity ofhe infraspinatus tendon to the human supraspinatusendon.3 Although the sheep infraspinatus tendon isot intraarticular, there is a bursa under the tendon,nd therefore, the repair has some contact with syno-ial fluid that lubricates the bursa. The bursa likelyoes not have the volume of synovial fluid that isontained in the shoulder joint. Therefore, the re-aired tendon has some characteristics of a healing

endon in a milieu of synovial fluid. In most cases,etrieval surgery harvests robust tendon-like scar ma-erial that forms between the retracted tendon stumpnd the bone (gap scar formation). This is not analo-ous to the human condition in which gaping orailure to heal occurs in rotator cuff repair.

This tendon also useful for in vitro studies2,10 be-ause it is relatively easy to prepare for biomechani-al testing owing to its size. Surgery on one shoulders well tolerated in sheep. The Institutional Animalare and Use Committee (IACUC) may only allow the

urgery to be performed on one forelimb, but someACUC’s may allow both. Preemptive and postoper-tive analgesia is essential to all animal studies.

URGICAL APPROACH

Shoulder surgery in sheep is performed under gen-ral anesthesia with the animal in lateral recumbency.n open approach to the shoulder is used becauserthroscopic repair would be impractical. A gentlyurving skin incision is made over the point of thehoulder and then deepened through the subcutane-us tissues. The skin is reflected and the fascia of therachialis muscle is incised. The acromial head of theeltoideus muscle will become visible. A plane ofissection is established along the cranial edge of theeltoideus muscle and the muscle reflected caudad.

Important anatomic structures of the shoulder jointn sheep are displayed in Figure 1. The insertion ofhe infraspinatus tendon is identified, and a curvedorceps placed under the tendon. The tendon is

ncised along its attachment to the greater tuberos-

J Shoulder Elbow Surg Turner 159SVolume 16, Number 5S

Figure 1 A, Intraoperative view of the surgical approach to the right tendinous insertion of the infraspinatus musclein the sheep. The acromial head of the deltoideus muscle is retracted. B, Anatomic dissection of a right shouldershows the location of M. terres minor. C, Further dissection of a right shoulder shows the synovial bursa and the

footprint of M. infraspinatus. D, Deeper dissection of a right shoulder shows the humeral head and joint capsule.E, Bony anatomic landmarks of the right sheep shoulder.

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ty of the humerus (Figure 1, A and B). The footprintf the tendon is approximately 10 mm � 20 mmFigure 1, C). Immediately under the tendon is aynovial bursa that does not communicate with thehoulder joint (Figure 1, C). The shoulder joint is notntered in this approach (Figure 1, D).

At this point, a Hall air drill and burr are used toreate a bed of bleeding bone, and if a bone troughs to be used, it is prepared at this stage. Orthopedistsew to this model will be surprised and pleased atow dense sheep bone is compared with adult orlderly human bone. Predrilling of the bone is neces-ary for suture anchor insertion. Acute and chronictudies of infraspinatus reattachment will be dis-ussed.

CUTE STUDIES

Clearly, detaching the ovine (or caprine) infraspi-atus tendon and immediately reattaching it does notepresent the clinical picture. It does, however, serves a relatively rapid way to screen different suturenchors, suture patterns, scaffolds, growth factors,nd other biologics, or a combination of these treat-ents, to enhance the healing process (Figure 2). Sur-ival times frequently used are 6 and 12 weeks.roup sizes are typically 12 animals if mechanical

esting and histology are end points. Within eachroup, 9 animals are allocated to mechanical testingnd 3 for histology.

Some of the earlier studies using sheep (and goats)ave evaluated different methods of tendon reattach-ent. St. Pierre el al,17 used goats to evaluate tendonealing to cortical bone compared with a cancellousrough. They evaluated the repairs histologically andiomechanically and found the tendon-to-bone heal-

igure 2 Reattachment of the infraspinatus tendon of the sheepsing modified Mason-Allen suture pattern and a bone trough.

ng process quite similar. c

Other acute studies using sheep have looked atifferent suture anchors (Figure 3),6 a mixture of boneorphogenetic proteins,15 suture patterns,5,9 low-

ntense pulsed ultrasound (Figure 4)12,13 and swinemall intestinal submucosa.16

HRONIC STUDIES

The repair of a chronic, massive, rotator cuff injurys a challenge to the shoulder surgeon. This hasrompted the search for a clinically relevant animalodel to evaluate methods to enhance repair andnderstand the biology behind the chronic tear. Ini-ially, delayed repair of the released infraspinatusendon was not recommended because of the diffi-

igure 3 Deployment of suture anchors in the dense bone of theateral epicondyle of the humerus on sheep.

igure 4 Low-intensity pulsed ultrasound treatment after immedi-te reattachment of the infraspinatus.12,13 Other large animalodels (including goats) compared with sheep, would not tolerate

he harness shown.

ulty in distinguishing scar tissue from normal tendon

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J Shoulder Elbow Surg Turner 161SVolume 16, Number 5S

t the time of reattachment.5 As a rule, animals arenown to heal rapidly, with abundant neovascular-zation and fibrous tissue ingrowth; sheep are noxception.

In later studies, methods to cover the end of thevine infraspinatus and minimize spontaneous reat-

achment were refined. To reproduce a chronic rota-or cuff injury, Coleman et al1 covered the end of thenfraspinatus with Gore-Tex (PRECLUDE; W. L. Gore

Associates, Flagstaff, AZ; Figure 5). The sheepere reoperated on 6 weeks later. The PRECLUDE-overed tendon was located, isolated, and reat-ached to the lateral tuberosity of the humerus. Theendon had retracted an average of 2.6 cm (range,.9-2.9). In that study, some sheep were reoperatedn at 18 weeks. These animals had irreparable ten-ons because of excessive retraction, which wereecured using a synthetic scaffold as a bridge.

To aid in identification of the detached infraspina-us tendon after long periods of detachment, Gerbert al4 performed an osteotomy of the greater tuberos-ty of the humerus. To prevent spontaneous healing,he end of the tendon was covered with a 5-cm-longilicone rubber tube. They also found fatty infiltrationnd muscle atrophy in proportion to the amount ofusculotendinous retraction. Therefore, the chronicetachment model in sheep is also useful to under-tand the biology (degree of muscle atrophy and fattynfiltration) of rotator cuff muscles as well as innova-ive methods of repair. The chronic model gives theurgeon a better understanding of the timing of theepair and the temporal aspects of healing.1,4,11

These long-standing chronic models (detached foreriods greater than 8 weeks) are more useful to

igure 5 Identification of the infraspinatus tendon covered withore-Tex (PRECLUDE, W. L. Gore & Associates, Flagstaff, AZ) in a

hronic model.1

valuate bioimplants (eg, collagen scaffolds) rather n

han attempted reattachment to the bone. In sheep, weurrently recommend infraspinatus detachment and cov-ring, and then reattachment, as soon as 4 weeks ifone-to-tendon healing is to be evaluated. If the abilityf a bioimplant, scaffold, autologous platelet-rich fibrinatrix, a growth factor or a combination of these toridge a large gap is to be evaluated, the reattach-ent surgery can be scheduled 8 weeks after theetachment and covering surgery.

Some of the earlier problems using the sheepodel of rotator cuff repair were related to suture

upture caused by failure to protect the repair from fulleight bearing. Because slinging was poorly toler-ted in sheep and not permitted by our IACUC, aubber ball was placed under the hoof of the involvedimb to see if this restricted limb movement, and thisas removed at 5 weeks.8Lewis et al8 looked at the effect of immobilization

n infraspinatus healing using a modified Mason-llen suture. They found that that there was no differ-nce between the treatment groups for load-to-failurend stiffness, so this cannot be recommended as aethod of immobilization. Rather, we have reverted

o close-stall confinement during the convalescence.In summary, it is virtually impossible to control the

ostoperative loads on the repaired tendon, andhere are no data to suggest that the ball on the hoofctually leads to diminished weight bearing. Further-ore, other researchers with experience with thisodel suggest that the sheep fires the muscles as it

esists and “fights” the ball on the hoof. Therefore, thiss not a method to “immobilize” the repaired shoul-er. Because sheep and goats are large, heavy ani-als, they likely fire the shoulder girdle muscles im-ediately on standing up after surgery.The high loads imposed by weight-bearing result in

ome separation of the repair in this model. The gapetween the distal end of the transected tendon and

he proximal humerus can be identified on the ultra-ound images in the awake but restrained sheep,ithout costly general anesthesia, to monitor progres-

ion or lack of healing (Figure 6).Although many of these repairs fail to heal by

endon-to-bone healing, the model is still useful totudy the effect of chronic tendon detachment onuscle atrophy and fatty infiltration. The model canlso be used to study the effect of various devices andactors on decreasing the rate of healing of primaryepairs for them to remain intact or the biologic effectsn the formation and maturation of tendon-like scarormation in a preclinical model using a rotator cuffurgical site.

Other issues with this model that should be men-ioned are that the forelimb is weight bearing andhere is no clavicle, a less-developed acromion, and

o coracoacromial arch. These differences between

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he sheep (goat) and humans are probably not asmportant as the issue mentioned above.

An important factor related in this, and likely otherarge-animal models, is that all of the repairs detacho some degree. The intervening gap fills in withbrous scar tissue, unlike humans where a gap typi-ally forms, which may be due to the extraarticularature of the repair. As a result, this is a model ofissue formation in a gap. As such, the model mayave real advantages as a “tissue engineering” ap-roach to tissue formation, but likely it does notvaluate direct healing between tendon and bone.

One distinct disadvantage of the model is a lack ofpecies-specific probes and reagents for sheep (eg,ntibodies), which limits the ability to conduct de-

ailed histologic analyses such as immunohistochem-stry and in-situ hybridization.

LENOHUMERAL INSTABILITY ASSESSMENT

The shoulder of the sheep can be used to evaluatehe response of the glenohumeral joint capsule. Obr-ut et al14 used explanted sheep shoulders to evalu-te the effect of radiofrequency energy on the lengthnd temperature properties of the capsule. Sheepave been used to determine whether capsular heal-ng after plication is equivalent to open capsular shiftFigure 7).7 As determined from histologic appear-nce, there was no difference in the 2 techniques.

UMMARY

Research facilities around the world are continuing

Figure 6 Monitoring healing with ultrasound imagesof the infraspinatus tendon with gap formation (left) an

o gain more experience with sheep as a practical

nd economic large-animal model for various thera-ies of the shoulder joint, particularly the enhance-ent of bone-to-tendon healing. In addition, research-rs are continuing to refine the model and learn morebout its limitations. Studies of growth factors at dif-erent doses and stem-cell therapy, in combinationith different scaffold materials and their configura-

ions, are likely to dominate this exciting field ofrthopedic research.

EFERENCES

1. Coleman SH, Fealy S, Ehteshami JR, Macgillivary JD, AltcheckDW, Warren RF, et al. Chronic rotator cuff injury and repair

right shoulder in the awake sheep after reattachmentganized tissue (right).

Figure 7 Plication of the shoulder joint capsule in the sheep.7

of the

model in sheep. J Bone Joint Surg Am 2003;85:2391-402.

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2. Cummins CA, Appleyard RC, Strickland S, Haen PS, Chen S,Murrell GA. Rotator cuff repair: an ex-vivo analysis of sutureanchor repair techniques on initial load to failure Arthroscopy2005;21:1236-41.

3. Gerber C, Schneeberger AG, Beck M, Schlegel U. Mechanicalstrength of repairs of the rotator cuff. J Bone Joint Surg Br 1994;76:371-80.

4. Gerber C, Meyer DC, Schneeberger AG, Hoppeler H, vonRechenberg B. Effect of tendon release and delayed repair on thestructure of the muscles of the rotator cuff: an experimental study insheep. J Bone Joint Surg Am 2004;86:1973-82.

5. Gerber C, Schneeberger AG, Perren SM, Nyffeler RW. Experi-mental rotator cuff repair. A preliminary study. J Bone Joint SurgAm 1999;81:1281-90.

6. Harrison JA, Wallace D, Van Sickle D, Martin T, Sonnabend DH,Walsh WR. A novel suture anchor of high-density collagencompared with a metallic anchor. Results of a 12-week study insheep. Am J Sports Med 2000;28:883-7.

7. Kelly BT, Turner AS, Bansal1 M, et al. In-vivo healing responseafter capsular plication in an ovine shoulder model. Proceedingsof the 50th Annual Meeting of the Orthopaedic Research Society,San Francisco, CA; 2004. p. 209.

8. Lewis CW, Schlegel TF, Hawkins RJ, James SP, Turner AS. Compar-ison of tunnel suture and suture anchor methods as a function of timein a sheep model. Biomed Sci Instrum 1999;35:403-8.

9. Lewis CW, Schlegel TF, Hawkins RJ, James SP, Turner AS. Theeffect of immobilization on rotator cuff healing using modifiedMason-Allen: a biomechanical study in sheep Biomed Sci Instrum2001;37:263-8.

0. Ma CB, MacGillivray JD, Clabeaux J, Lee S, Otis JC. Biome-chanical evaluation of arthroscopic rotator cuff stitches. J BoneJoint Surg Am 2004;86:1211-6.

1. Meyer DC, Hoppeler H, von Rechenberg B, Gerber C. Apathomechanical concept explains muscle loss and fatty muscular

changes following surgical tendon release. J Orthop Res2004;22:1004-7.

2. Motta T, Tokish JM, Schlegel TF, Hawkins RJ, Trumble TN,MacLeay JM, et al. Enhancement of infraspinatus tendon healingwith pulsed low intensity ultrasound in an ovine model: biome-chanical evaluation [abstract]. Proceedings of the 51st AnnualMeeting of the Orthopaedic Research Society, Washington, DC;2005. p. 725.

3. Motta T, Tokish JM, Schlegel TF, Hawkins RJ, Trumble TN,MacLeay JM, et al. Histological response to low intensity pulsedultrasound on rotator cuff repair healing in an ovine model[abstract]. Proceedings of the 51st Annual Meeting of the Ortho-paedic Research Society, Washington, DC; 2005. p. 632.

4. Obrzut SL, Hecht P, Hayashi K, Fanton GS, Thabit G 3rd, MarkelMD. The effect of radiofrequency energy on the length andtemperature properties of the glenohumeral joint capsule. Arthro-scopy 1998;14:395-400.

5. Rodeo S, Atkinson B, Kim HJ, Campbell D, Turner AS, Potter H.Augmentation of rotator cuff tendon-to-bone repair using a mixtureof bone morphogenetic proteins in an ovine model [abstract].Proceedings of the 48th Annual Meeting of the OrthopaedicResearch Society, Dallas, TX; 2002. p. 145.

6. Schlegel TF, Hawkins RJ, Lewis CW, Motta T, Turner AS. Theeffects of augmentation with swine small intestine submucosa ontendon healing under tension: histologic and mechanical evalu-ations in sheep. Am J Sports Med 2006;34:275-80.

7. St Pierre P, Olson EJ, Elliott JJ, O’Hair KC, McKinney LA, Ryan J.Tendon-healing to cortical bone compared with healing to acancellous trough a biomechanical and histological evaluation ingoats. J Bone Joint Surg Am 1995;77:1858-66.

8. Turner AS. Research in orthopaedic surgery. In: Souba W,Wilmore D, editors. Surgical research. San Diego, CA: Aca-

demic Press; 2001. p. 80-1; 80-64.