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http://ajs.sagepub.com/ Medicine The American Journal of Sports http://ajs.sagepub.com/content/41/12/2909 The online version of this article can be found at: DOI: 10.1177/0363546513503810 2013 41: 2909 originally published online September 18, 2013 Am J Sports Med Seok Won Chung, Byung Wook Song, Yeun Ho Kim, Kyoung Un Park and Joo Han Oh Healing in a Rabbit Model Effect of Platelet-Rich Plasma and Porcine Dermal Collagen Graft Augmentation for Rotator Cuff Published by: http://www.sagepublications.com On behalf of: American Orthopaedic Society for Sports Medicine can be found at: The American Journal of Sports Medicine Additional services and information for http://ajs.sagepub.com/cgi/alerts Email Alerts: http://ajs.sagepub.com/subscriptions Subscriptions: http://www.sagepub.com/journalsReprints.nav Reprints: http://www.sagepub.com/journalsPermissions.nav Permissions: What is This? - Sep 18, 2013 OnlineFirst Version of Record - Nov 27, 2013 Version of Record >> by Gutierrez Meneses Arturo on November 28, 2013 ajs.sagepub.com Downloaded from by Gutierrez Meneses Arturo on November 28, 2013 ajs.sagepub.com Downloaded from

The American Journal of Sports Medicinescaffold to facilitate healing. A porcine dermal collagen patch graft is a tissue-engineered xenograft that has been used as a biological scaffold

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http://ajs.sagepub.com/Medicine

The American Journal of Sports

http://ajs.sagepub.com/content/41/12/2909The online version of this article can be found at:

 DOI: 10.1177/0363546513503810

2013 41: 2909 originally published online September 18, 2013Am J Sports MedSeok Won Chung, Byung Wook Song, Yeun Ho Kim, Kyoung Un Park and Joo Han Oh

Healing in a Rabbit ModelEffect of Platelet-Rich Plasma and Porcine Dermal Collagen Graft Augmentation for Rotator Cuff

  

Published by:

http://www.sagepublications.com

On behalf of: 

  American Orthopaedic Society for Sports Medicine

can be found at:The American Journal of Sports MedicineAdditional services and information for    

  http://ajs.sagepub.com/cgi/alertsEmail Alerts:

 

http://ajs.sagepub.com/subscriptionsSubscriptions:  

http://www.sagepub.com/journalsReprints.navReprints:  

http://www.sagepub.com/journalsPermissions.navPermissions:  

What is This? 

- Sep 18, 2013OnlineFirst Version of Record  

- Nov 27, 2013Version of Record >>

by Gutierrez Meneses Arturo on November 28, 2013ajs.sagepub.comDownloaded from by Gutierrez Meneses Arturo on November 28, 2013ajs.sagepub.comDownloaded from

Effect of Platelet-Rich Plasma and PorcineDermal Collagen Graft Augmentation forRotator Cuff Healing in a Rabbit Model

Seok Won Chung,* MD, Byung Wook Song,y MD, Yeun Ho Kim,z MD,Kyoung Un Park,§ MD, PhD, and Joo Han Oh,||{ MD, PhDInvestigation performed at Seoul National University Bundang Hospital, Seongnam, Korea

Background: The rate of healing failure after surgical repair of chronic rotator cuff tears is considerably high.

Purpose: To verify the effect of platelet-rich plasma (PRP) with and without porcine dermal collagen graft augmentation ontendon-to-bone healing, using the rabbit supraspinatus tendon.

Study Design: Controlled laboratory study.

Methods: A total of 80 rabbits were randomly allocated into 4 groups (20 rabbits per group: 12 for histological and 8 for mechan-ical testing): repair (R), repair 1 patch augmentation (RPa), repair 1 PRP (RPr), and repair 1 patch 1 PRP (RPaPr). The rightshoulder was used for experimental interventions, and the left served as a control. Six weeks after the detachment of the supra-spinatus, the torn tendon was repaired in a transosseous manner, simulating double-row repair in all groups. Platelet-rich plasmawas prepared and applied onto the repair site in the RPr and RPaPr groups, and the patch was used to augment the repair in theRPa and RPaPr groups. The mechanical tensile strength test was performed at 8 weeks after repair and the histological evaluationat 4 and 8 weeks.

Results: At 4 weeks, the collagen fibers were poorly organized, and fiber continuity was not established in all groups. However,vascularity and cellularity were higher with granulation tissue formation in the PRP-treated groups (RPr and RPaPr) than the non-treated groups (R and RPa). At 8 weeks, tendon-to-bone integration was much improved with more collagen fibers, and longitu-dinally oriented collagen fibers were visible in all groups. The PRP-treated groups showed better collagen fiber continuity andorientation than the nontreated groups; however, no distinctive difference was found between the patch-augmented groups(RPa and RPaPr) and nonaugmented groups (R and RPr). The mean load-to-failure results were 61.57 6 29.99 N, 76.84 6

16.08 N, 105.35 6 33.82 N, and 117.93 6 12.60 N for the R, RPa, RPr, and RPaPr groups, respectively, and they were significantlydifferent between the R and RPr (P = .018), R and RPaPr (P = .002), and RPa and RPaPr (P = .029) groups.

Conclusion: This animal study showed the enhancement of tendon-to-bone healing after local administration of autologous PRPassessed by histological and biomechanical testing in a rabbit model of chronic rotator cuff tears. However, there was little addi-tive effect of the patch graft.

Clinical Relevance: The use of PRP might be a biological supplement to increase the rotator cuff healing rate, which still remainslow even after successful cuff repair, but this result should be interpreted with caution regarding clinical applications.

Keywords: shoulder; rotator cuff; rabbit model; growth factors/healing enhancement; platelet-rich plasma; porcine dermal col-lagen graft

A rotator cuff tear is a common condition that causes painand dysfunction.21,25 In spite of the rapid development ofthe techniques and instruments used for surgical repairof torn rotator cuffs, failure of rotator cuff healing aftersurgical repair remains one of the most common andwell-known complications. Recent studies have reportedthat approximately 50% of surgically repaired rotator cuffsdo not heal properly, independent of the surgical

procedures utilized.8,12,14,38 Consequently, many efforts tosupport the repair construct mechanically and enhancetendon-to-bone healing biologically have been undertakento decrease the failure rate after rotator cuff repair.

Platelet-rich plasma (PRP) is blood plasma that isenriched with platelets by centrifugation, and it containsnumerous growth factors such as platelet-derived growthfactor (PDGF), epidermal growth factor (EGF), transform-ing growth factor–b 1 (TGF-b1), insulin-like growthfactor-1 (IGF-1), vascular endothelial growth factor(VEGF), and basic fibroblast growth factor (bFGF), whichare also known to be involved in tendon healing.27,39

Recently, PRP has gained widespread use clinically in the

The American Journal of Sports Medicine, Vol. 41, No. 12DOI: 10.1177/0363546513503810� 2013 The Author(s)

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orthopaedic field, and much attention has been paid toenhance the healing process by providing endogenousgrowth factors as a biological approach. However, the clini-cal results are somewhat controversial, and basic experi-mental studies are still lacking, especially on tendon-to-bone healing after rotator cuff repair.2,13,41

In addition, many attempts have been made to augmentthe repair with a patch graft as a mechanical and biologicalscaffold to facilitate healing. A porcine dermal collagenpatch graft is a tissue-engineered xenograft that hasbeen used as a biological scaffold to provide a strongerrepair.5 It is commercially made of a chemically cross-linked, acellular collagen/elastin material derived fromthe porcine dermis; thus, it is resistant to enzymatic degra-dation and minimizes potential cell-mediated responses.17

The use of patch grafts for rotator cuff repair may act asa mechanical augmentation and improve the rate andquality of biological healing by providing a chemicallyand structurally beneficial environment for host cells.6

Therefore, the purpose of this study was to verify theeffect of PRP with or without porcine dermal collagen graftaugmentation on tendon-to-bone healing in a chronic rota-tor cuff tear model, using the rabbit supraspinatus tendon.We hypothesized that locally administered PRP and por-cine dermal collagen graft augmentation could enhancetendon-to-bone healing.

MATERIALS AND METHODS

All experimental procedures were approved by the Experi-mental Animal Committee of the Clinical Research Instituteof the authors’ institution (IACUC No. BA1104-081/027-01).

Allocation of Rabbits

Power analysis indicated that a required sample size wouldbe 8 for biomechanical tests to detect a significant differencein ultimate load to failure (mean difference, 90 6 40 N; a

error = .05; b error = .2; dropout rate = 25%), based on pre-vious studies.32,46 For the histological evaluation, weassigned 6 rabbits per group at 4 weeks and 8 weeks(Figure 1). Thus, 80 mature New Zealand White male rab-bits (weight, 3.5-4.0 kg) were randomly allocated into 4groups (20 rabbits per group: 12 for histological and 8 formechanical testing): the repair group (R), repair 1 patchaugmentation group (RPa), repair 1 PRP group (RPr),and repair 1 patch 1 PRP group (RPaPr). All experimentalprocedures were performed in the right shoulder, and shamoperations were performed in the left shoulder (control).

Chronic Rotator Cuff Tear Model

Under anesthesia and sterile conditions, a 3-cm skin inci-sion was made between just proximal to the acromion pro-cess and greater tuberosity, and then the supraspinatustendon was exposed after incising and retracting the del-toid muscle. For all rabbits, a chronic tear was created inthe right shoulder by completely severing the supraspina-tus tendon at the insertion site to the greater tuberosityand wrapping the torn tendon with a 10 mm–long siliconePenrose drain (8 mm in outer diameter, Yushin Corp,Bucheon, Korea) to inhibit adhesion to the surroundingsoft tissue, and it was left alone for 6 weeks (Figure 2).For the left shoulder, a sham operation was performedthat omitted the critical steps of tear or repair, includingPRP application and patch augmentation.

Week 0

Group RRepair

Week 6

Supraspinatus tear (4 groups, 20 rabbits per each group)Right: supraspinatus tear, Left: control

Group RPaRepair + Patch

Group RPrRepair + PRP

Group RPaPrRepair + PRP

+ Patch

Week 10 Histological evaluation (6 per group): H&E stain,Masson’s Trichrome stain

Week 14 Histological evaluation (6 per group): H&E stain, Masson’s Trichrome stainMechanical evaluation (8 per group): Mode of failure, Load to failure

Figure 1. Flowchart showing the allocation of rabbits in the study.

{Address correspondence to Joo Han Oh, MD, PhD, Seoul National University College of Medicine, Seoul National University Bundang Hospital, 82Gumi-ro 173 beon-gil, Bundang-gu, Seongnam 463-707, Korea (e-mail: [email protected] / [email protected] / [email protected]).

*Department of Orthopaedic Surgery, Konkuk University School of Medicine, Konkuk University Medical Center, Seoul, Korea.yDepartment of Orthopaedic Surgery, Nalgae Hospital, Seoul, Korea.zDepartment of Orthopaedic Surgery, Anjung Seoul Jeil Hospital, Pyeongtaek, Korea.§Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seoul, Korea.||Department of Orthopaedic Surgery, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seoul, Korea.

One or more of the authors has declared the following potential conflict of interest or source of funding: This research was supported by the SeoulNational University Bundang Hospital’s research fund (No. 03-2011-013) and by the Department of Laboratory Medicine of Seoul National University Bun-dang Hospital and Anatomic Pathology of Seoul National University Hospital.

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Repair of Rotator Cuff Tendon

After 6 weeks, we repaired the torn supraspinatus tendonwith No. 2-0 Ticron (Tyco, Waltham, Massachusetts) ina transosseous manner after creating a bleeding bed at thefootprint of the greater tuberosity. Repair procedures wereas follows: under the same anesthesia and with the sameapproach, we removed the Penrose drain wrapped aroundthe supraspinatus tendon and then gently roughened theexposed greater tuberosity with a scalpel blade. Four bonetunnels were created at the anterior and posterior aspectsof the footprint and mediolaterally at the articular surfaceand 5 mm from it to simulate a double-row repair. The sutureends grasping the tendon at the medial and lateral aspects ofthe tendon were then passed through the bone tunnels (Fig-ure 3) and tied over the lateral humeral cortex, reattachingthe supraspinatus tendon to the footprint.

PRP Preparation

To prepare PRP, 10 mL of autologous blood was acquiredfrom the femoral vein of each RPr and RPaPr specimen ina sterile manner under anesthesia before surgical proceduresof the shoulder. Blood sampling was performed after expos-ing the femoral vein by dissection to acquire blood quicklyand safely to prevent blood clotting. Then, 10 mL of autolo-gous blood drawn from each rabbit was combined with 1.1mL of anticoagulant citrate dextrose phosphate (ACD-A) toprevent coagulation. The PRP preparation was based onthe method described in a previous study.1 Each blood sam-ple was first centrifuged at 210g for 10 minutes to separatethe plasma containing the platelets from the red cells inthe inferior layer. The plasma was drawn off the top andwas centrifuged for another 10 minutes at 860g to obtaina 2-part plasma (platelet-poor plasma constituting the upper

part and PRP constituting the lower part). After the removalof platelet-poor plasma, PRP was gently aspirated witha pipette and placed in a sterile tube. The amount of PRPwas approximately 1 mL and was used within minutes toadd to the repair site after activation by 0.1 mL of 10% cal-cium chloride. The ratio of PRP to the calcium chloride acti-vator was approximately 10:1. Platelets and leukocytes inwhole blood and PRP were counted on each sample. Themean number of platelets in whole blood was 259.06 6

88.04 3 103/mL and that in PRP was 924.77 6 330.86 3

103/mL. In addition, the mean number of leukocytes in wholeblood was 6.18 6 2.01 3 103/mL and that in PRP was muchdecreased with 0.06 6 0.09 3 103/mL. The numbers of plate-lets and leukocytes in PRP were not statistically differentbetween the RPr and RPaPr groups (P = .625 and .450 forplatelets and leukocytes, respectively). The preparation ofPRP and counting of platelets and leukocytes were performedby a specialist in the Department of Laboratory Medicine(K.U.P.), who was unaware of the present study.

PRP Application and/or Patch Augmentation

The prepared activated PRP (type 4B according to the clas-sification by Mishra et al35), which was changed intoa gelatinous substance by adding calcium chloride (PRPgel), was immediately administered on the repaired tendonof each specimen in the RPr group. That is, the PRP gelwith adhesive properties was applied onto the site of therepaired tendon-to-bone approximation, as Beck et al7 did(Figure 4). The 1 mm–thick porcine dermal collagen graft(Permacol, Tissue Science Laboratories Plc, Aldershot,United Kingdom) was prepared as a 1 3 1–cm2 patch.The collagen patch was not soaked in saline before usage,as no hydration or other preparatory procedures wererequired according to manufacturer instructions.

The collagen patch was used to augment the repair site bystitching the proximal portion to the supraspinatus tendonand the distal portion to the soft tissue at the lateral portionof the proximal humerus for the RPa group (Figure 5). Wedid not mix or soak the patch with PRP to minimize the pos-sible loss of PRP. For the RPaPr group, the patch was appliedover PRP after it had been added to the repair site.

Histological Evaluation

At 4 and 8 weeks after repair, the rabbits (6 per group foreach period) were fully anesthetized and euthanized withcarbon dioxide, and the greater tubercle of the humeralhead with the attached supraspinatus tendon of bothshoulders of each rabbit was harvested. Specimens werefixed in neutral buffered 10% formalin (pH 7.4) and decal-cified, and paraffin blocks were made in the repair siteincluding the supraspinatus tendon and greater tuberos-ity. Sections that were 5 mm thick were cut in the coronalplane and stained with hematoxylin and eosin (H&E) andMasson trichrome. We assessed vascularity, cellularity,collagen fiber continuity, and proportion of fibers orientedparallel at the tendon-to-bone interface, and we also evalu-ated the inflammation rate and the absorption rate of thegraft at the tendon-to-graft interface. For each of these

Figure 2. Creation of a chronic rotator cuff tear. The supraspi-natus tendon was completely severed at the insertion site to thegreater tuberosity, and the torn end was wrapped with a siliconePenrose drain to inhibit adhesion to the surrounding tissue.

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items, the histological findings were graded semiquantita-tively into 4 stages (grades 0, 1, 2, and 3). Vascularity, cel-lularity, inflammation rate around the graft, andabsorption rate around the graft were graded as absentor minimally present (grade 0), mildly present (grade 1),moderately present (grade 2), and severe or markedly pres-ent (grade 3). In addition, for the items of collagen fibercontinuity and collagen fibers oriented parallel, we dividedtheir stages by percentage: present with \25% of propor-tion (grade 0), 25%-50% of proportion (grade 1), 50%-75%of proportion (grade 2), and .75% of proportion (grade3). All examinations were performed in a randomized andblinded fashion to eliminate observer bias by a musculo-skeletal specialized pathologist with 10 years of training,who was not involved in the study. The cross-sectionswere examined under a microscope (DP 70, Olympus,Tokyo, Japan) and analyzed using an image software

system (Image-Pro Plus, Media Cybernetics Inc, Bethesda,Maryland).

Mechanical Evaluation

In addition, 8 weeks after repair, the entire supraspinatustendon of both shoulders along with the humeral head ofeach rabbit was harvested after being euthanized (8 pergroup). We then evaluated the mode of tear and the loadto failure at a rate of 1 mm/s with a preload of 5 N after5 consecutive preconditioning loads (5-50 N at a loadingrate of 15 N/s), using a custom fixture clamping systemand an Instron materials testing machine (5565A, Instron,Norwood, Massachusetts) (Figure 6). We designed and

Figure 4. The PRP gel was administered on the repair site.Figure 5. The porcine dermal collagen graft was used to aug-ment the repair site by stitching the proximal portion of thepatch to the supraspinatus tendon and the distal portion tothe soft tissue at the lateral portion of the proximal humerus.

Figure 3. Repair procedures of the torn supraspinatus tendon (SST) to the greater tuberosity (GT) in a transosseous manner, sim-ulating a double-row repair. (A) Two suture ends for the medial row and lateral row were passed through the 4 bone tunnels. (B)Each suture end was then tied over the lateral humeral cortex, reattaching the supraspinatus tendon to the footprint. Inf, inferior;Lat, lateral; Med, medial; Sup, superior.

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manufactured a custom fixture clamping system for tensiletesting of the supraspinatus tendon that consisted of 2 sepa-rate fixtures: a humeral head fixation unit, which rigidlyfixed the humeral head and permitted the supraspinatus ten-don and muscle attached to the humeral head to come outthrough a hole, and a cryogenic tendon fixation unit, whichsecured the myotendinous junction of the tendon usinga sandpaper-attached clamp and liquid nitrogen to preventslippage. The supraspinatus tendon was fixed to this systemalong its anatomic direction to allow tensile loading and thetendon-to-bone interface to form a right angle. The tensileload-to-failure data were automatically collected with a per-sonal computer–based data acquisition system.

Statistical Analysis

Statistical analysis was performed using SPSS software(version 15.0, SPSS Inc, Chicago, Illinois). Comparisonsbetween the groups were performed using a 1-way analysisof variance, followed by Bonferroni post hoc analysis withthe significance level set at P \ .05. Before the analysis ofvariance test, the Kolmogorov-Smirnov test for normalityand the Levene test for homogeneity of variances were per-formed, and they fulfilled the assumption of normality andhomogeneity of variances with all P values ..05.

RESULTS

Four specimens (1 RPa, 1 RPr, and 2 RPaPr) showeda deep wound infection at the time of harvest, and another4 rabbits died either during the operation (2 rabbits: 1 RPrand 1 RPaPr) or within a few days after the operation (2rabbits: 1 R and 1 RPa) of unknown causes. These eventswere distributed relatively evenly among the groups, andwe do not believe that the added procedures of blood collec-tion for PRP or patch augmentation affected these events.In addition, a wide dehiscence of the supraspinatus tendonrepair was noted in 3 rabbits (1 R, 1 RPa, and 1 RPr) atharvest. These 11 rabbits were excluded from furtherassessment; therefore, 69 rabbits were used in the finalanalysis (4-week histological testing: n = 6, 5, 5, and 5; 8-week histological testing: n = 5, 5, 5, and 5; 8-weekmechanical testing: n = 7, 7, 7, and 7 for R, RPa, RPr,and RPaPr groups, respectively), as shown in Table 1.

Histological Evaluation

The results of histological grading are described in Table2. At 4 weeks, the collagen fibers were poorly organized,and fiber continuity with bone had not yet been estab-lished in all 4 groups. Evidence of vascularization wasnoted by the presence of blood vessels, and cells werepresent in the irregularly arranged fibrovascular inter-face tissues in all 4 groups. However, vascularity and cel-lularity (Figure 7, A and B) were higher with granulationtissue formation in the PRP-treated groups (RPr andRPaPr) than in the nontreated groups (R and RPa). Inall 4 groups, the histological findings of the 4-week speci-mens were clearly different from those of the controlside, which showed dense regular collagen fibers withcomplete continuity to the bone. At this period, most ofthe graft tissue was not integrated into the surroundingtissue, mostly keeping its shape, which was covered withfibrous tissue and had many inflammatory cells aroundit (Figure 8A).

At 8 weeks, the tendon-to-bone interface was composedof less cellular and less vascular fibrous tissues in all 4groups. Instead, the number of collagen fibers in the inter-face was greatly increased, and collagen organization wasobviously improved. In addition, more collagen fibersbridged the interface, showing good tendon-to-bone inte-gration, and longitudinally oriented collagen fibers werevisible in all 4 groups. However, the PRP-treated groups(RPr and RPaPr) showed better collagen fiber continuityand more regularly arranged collagen fibers than the non-treated groups (R and RPa) (Figure 7, C and D). The colla-gen fibers in the interface were more perpendicular fromthe bone in the PRP-treated groups. However, no distinc-tive difference was found in tendon-to-bone healingbetween the patch-augmented groups and nonaugmentedgroups (RPa vs R and RPaPr vs RPr). Even though thehealing process was much progressed up to 8 weeks, itdid not reach the level of the control side in terms of theregularity of newly formed collagen fibers and continuityto the bone. Most of the patch graft was integrated intothe surrounding tissue at this period (Figure 8B).

Figure 6. (A) Materials testing machine with a custom fixtureclamping system. (B) Tensile testing of the repaired supraspi-natus tendon using the materials testing machine.

TABLE 1Complications and Number of Rabbits

Included in Each Analysisa

RGroup

RPaGroup

RPrGroup

RPaPrGroup

Deep infection 0 1 1 2No recovery from anesthesia 0 0 1 1Death from unknown causes 1 1 0 0Wide dehiscence of repaired

supraspinatus tendon1 1 1 0

4-week histological evaluation 6 5 5 58-week histological evaluation 5 5 5 58-week mechanical evaluation 7 7 7 7

aR, repair; RPa, repair 1 patch augmentation; RPaPr, repair 1

patch 1 platelet-rich plasma; RPr, repair 1 platelet-rich plasma.

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Mechanical Evaluation

In the failure mode analysis, the ratio of the insertionaltear to the midsubstance tear was 5:2 in the R, 4:3 in theRPa, 3:4 in the RPr, and 3:4 in the RPaPr groups (Table3); the midsubstance tear suggests strong tendon-to-bonehealing, while the insertional tear suggests relativelyweak tendon-to-bone healing.45 In this study, midsub-stance tears were more prevalent in the RPr (57.1%)than the R (28.6%) group and in the RPaPr (57.1%) thanthe RPa (42.8%) group. The load-to-failure values of theoperated side (right side) were significantly lower thanthose of the control side (left side) in all groups (P \ .05for all cases). Mean load-to-failure values for the R, RPa,RPr, and RPaPr groups were 61.57 6 29.99 N, 76.84 6

16.08 N, 105.35 6 33.82 N, and 117.93 6 12.60 N, respec-tively (P = .001). The Bonferroni post hoc analysis (Table 4)revealed significant differences in load to failure betweenthe R and RPr (P = .018), R and RPaPr (P = .002), andRPa and RPaPr (P = .029) groups. However, there was

no difference between the R and RPa (P = .662) and RPrand RPaPr (P = .779) groups.

DISCUSSION

This study demonstrated that the local administration ofPRP on the repaired supraspinatus tendon facilitated bio-logical tendon-to-bone healing and increased the load tofailure of the repaired rotator cuff; however, these biologi-cal and mechanical properties were not enhanced by por-cine dermal collagen graft augmentation.

Platelet-rich plasma is an autologous platelet concen-trate that contains various growth factors that can stimu-late tendon healing. The platelets collected in PRP by thecentrifugation process are activated by the addition ofthrombin or calcium chloride, which induces the releaseof these growth factors from a granules.9 Platelet-richplasma usually represents a 3- to 5-fold increase in plateletcount compared with whole blood. Even though there is

TABLE 2Results of Histological Testing (Operated Side)a

R Group RPa Group RPr Group RPaPr Group

G0 G1 G2 G3 G0 G1 G2 G3 G0 G1 G2 G3 G0 G1 G2 G3

4-week evaluation, nVascularity 0 3 2 1 0 2 2 1 0 0 4 1 0 0 3 2Cellularity 0 3 2 1 0 2 3 0 0 1 3 1 0 1 2 2Collagen fiber continuity 3 3 0 0 1 4 0 0 3 2 0 0 4 1 0 0Collagen fibers oriented parallel 3 3 0 0 3 2 0 0 1 4 0 0 3 2 0 0Inflammation rate around graft 0 1 2 2 0 0 2 3Absorption rate around graft 5 0 0 0 5 0 0 0

8-week evaluation, nVascularity 2 3 0 0 2 3 0 0 2 3 0 0 0 5 0 0Cellularity 2 2 1 0 1 2 2 0 2 3 0 0 1 3 1 0Collagen fiber continuity 0 3 2 0 0 3 2 0 0 1 3 1 0 1 3 1Collagen fibers oriented parallel 0 3 2 0 0 4 1 0 0 2 3 0 0 2 3 0Inflammation rate around graft 0 2 3 0 0 3 2 0Absorption rate around graft 0 1 1 3 0 1 0 4

aGrades were as follows: G0 = absent or minimal or \25% of proportion; G1 = mild degree or 25%-50%; G2 = moderate degree or 50%-75%;and G3 = severe (marked) degree or .75%. R, repair; RPa, repair 1 patch augmentation; RPaPr, repair 1 patch 1 platelet-rich plasma; RPr,repair 1 platelet-rich plasma.

TABLE 3Results of Mechanical Testinga

R Group (n = 7) RPa Group (n = 7) RPr Group (n = 7) RPaPr Group (n = 7)

Load to failure–operated side, N 61.57 6 29.99 76.84 6 16.08 105.35 6 33.82 117.93 6 12.60Load to failure–control side, N 161.59 6 19.02 153.42 6 35.63 159.64 6 28.54 157.35 6 32.55P value \.001 \.001 .007 .018Insertional tear–operated side, n 5 4 3 3Midsubstance tear–operated side, n 2 3 4 4

aValues are expressed as mean 6 standard deviation unless otherwise indicated. R, repair; RPa, repair 1 patch augmentation; RPaPr,repair 1 patch 1 platelet-rich plasma; RPr, repair 1 platelet-rich plasma.

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some controversy, the 3- to 5-fold concentrate seems to besuggested as a therapeutic PRP concentrate for tissuehealing and regeneration.33 In the current study, we dem-onstrated a consistent result in platelet count in PRP,which increased approximately 4-fold compared withwhole blood (mean, 924.77 6 330.86 3 103/mL vs 259.066 88.04 3 103/mL, respectively). Thus, we believe thatthe PRP preparation process was well performed andthat the level of platelets was within the therapeuticrange. In addition, the reduction of leukocytes in PRPmay enhance healing by decreasing the recruitment ofleukocyte-induced catabolic signaling molecules such asinterleukin-1 b, tumor necrosis factor a, and matrixmetalloproteinase-9.34,44 As it is known that the presenceof leukocytes can result in proinflammatory cellular signal-ing and local tissue catabolism, a higher concentration ofleukocytes is likely undesirable for musculoskeletal appli-cations associated with tendon healing.11,28 We believethat even though prepared PRP contains a mixture of ana-bolic and catabolic mediators, the net effect of PRP wasanabolic, thus facilitating tendon-to-bone healing, as weused leukocyte-reduced PRP in this study. In addition,we used calcium chloride alone to activate PRP and to pro-duce an autologous PRP gel. Calcium chloride withoutthrombin may allow platelets to be functionally viableover a 7-day time and also a slower release of growth fac-tors enhancing cell migration and healing.18 Moreover,the formation of fibrin glue (PRP gel) may provide an

adhesive support, which can confine the secretion ofgrowth factors to a chosen site, thus resulting in enhancedactivity.3

By applying this PRP gel onto the repair site of torn rabbitsupraspinatus tendons, we were able to verify the facilitationof biological tendon-to-bone healing and improvement in themechanical load to failure. This positive effect of PRP on rota-tor cuff healing may be caused by the growth factors secretedfrom the a granules of platelets. Growth factors releasedfrom platelets include PDGF, TGF-b1, IGF-1, VEGF, EGF,platelet-derived angiogenesis factor, and bFGF, which arealso known to be upregulated or involved during tendon heal-ing.36 These growth factors contribute to stem cell recruit-ment, mitogenesis, angiogenesis, collagen synthesis,extracellular matrix production, and remodeling.3,24 Severalin vitro studies have demonstrated that PRP may possiblycontain and release these growth factors abundantly andstimulate the proliferation of fibroblasts, tendon cells, osteo-blasts, and mesenchymal stem cells.15,30 In addition, hepato-cyte growth factor, which is a plasmatic factor, released fromPRP could induce cell proliferation and promote the synthe-sis of angiogenic factors during the healing process.3,47 Theactions of these growth factors released from the plateletsin PRP may have helped tendon-to-bone healing of the rabbitsupraspinatus tendon in this study. However, this should beinterpreted with caution because the dose-response relation-ship between platelet count and growth factor level in PRPwas previously demonstrated only in TGF-b1,48 and we did

Figure 7. At 4 weeks, the collagen fibers were poorly orga-nized, and fiber continuity with bone had not yet been estab-lished in all groups. Vascularity and cellularity were higherwith granulation tissue formation in the PRP-treated groups(A) than the nontreated groups (B) (H&E stain, 1003 magnifi-cation). At 8 weeks, the tendon-to-bone interface was lesscellular, and more collagen fibers bridged the interface,showing good tendon-to-bone integration, and longitudinallyoriented collagen fibers were also visible. The PRP-treatedgroups (C) showed better collagen fiber continuity andmore regularly arranged collagen fibers than the nontreatedgroups (D) (Masson trichrome stain, 1003 magnification).

Figure 8. (A) At 4 weeks, most of the graft tissue remained inthe specimen (white arrow), which was covered with fibroustissue and had many inflammatory cells around it; (B) how-ever, at 8 weeks, most of the patch graft was integratedinto the surrounding tissue (black arrow) (H&E stain, 403

magnification).

TABLE 4Results of Bonferroni Post Hoc Analysis for Load to Failure

of Operated Side for Each Groupa

R Group RPa Group RPr Group RPaPr Group

R group .662 .018b .002b

RPa group .662 .166 .029b

RPr group .018b .166 .779RPaPr group .002b .029b .779

aR, repair; RPa, repair 1 patch augmentation; RPaPr, repair 1

patch 1 platelet-rich plasma; RPr, repair 1 platelet-rich plasma.bStatistically significant: P \ .05.

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not evaluate whether a high amount of the growth factorswas actually released and worked on tendon-to-bone healingon a molecular level.

There have been several studies that investigated theeffect of PRP on tendon healing, and most of the basicresearch using animal models has suggested that PRPmay improve tendon healing.4,10,31 However, all these stud-ies investigated the effect of PRP on tendon-to-tendon heal-ing and not on tendon-to-bone healing. To the best of ourknowledge, there has only been 1 animal study, by Becket al,7 which investigated the effect of PRP on tendon-to-bone healing of the rotator cuff. Beck et al7 showed thatthe application of PRP during rotator cuff repair in ratsrather decreased tissue strain because of the cellular infil-tration, increased areas of fibrinoid necrosis, and the lackof collagen orientation at the 7-day point and did not affectthe load to failure of the repair. However, in their study, theinitial histological changes at the 7-day time point were nor-malized at the 14-day point, and, compared with the controlgroup, the collagen fibers of the PRP group were more orga-nized in a more linear fashion, directed toward the nativetendon footprint. Moreover, they only evaluated the effectof PRP at an early time point up to 21 days. We believethat there can be a positive effect of PRP at a later timepoint when the maturation of collagen is more progressed.Recently, several authors performed prospective random-ized clinical trials and tested the ability of PRP to enhancetendon-to-bone healing of the rotator cuff and functionaloutcome; however, there seems to be a lack of consensus.Randelli et al40 and, more recently, Gumina et al23 reportedthat the use of PRP in the treatment of rotator cuff tearsimproved repair integrity. Conversely, Castricini et al13

and Rodeo et al42 revealed that PRP application at thetime of rotator cuff repair had no effect on healing or func-tional scores. We believe that these conflicting results maycome from the variability of growth factor content betweenthe donors and methods of PRP preparation.49 The methodsand length of time involved in blood collection, centrifuga-tion, platelet activation, and delivery to the target sitealso affect the final concentration and composition of growthfactors and may alter the effectiveness of PRP.29 In addi-tion, the presence or absence of leukocytes in preparationscould affect the effectiveness of PRP, as a higher concentra-tion of leukocytes is likely undesirable for tendon healingbecause of their inflammatory and catabolic effect on localtissue.11,28 Moreover, the effect of PRP may not reach thelevel at which the retear rate shows a significant differenceor at which patients could detect a subjective difference offunction in clinical studies, considering that the clinicalstudies dealt not with massive tears but with mostly small-to large-sized tears, which already are expected to havegood anatomic and functional results.13,42 In this study,we collected the blood quickly and safely from the femoralvein under anesthesia to prevent blood clotting and destruc-tion of platelets, and all procedures for PRP extraction wereconsistently performed by 1 senior specialist in laboratorymedicine (K.U.P.). In addition, the administration ofPRP on the repaired rotator cuff was performed immedi-ately, within minutes after extracting and activating PRP.We believe that these efforts to reduce the variability

resulting from the PRP preparation may contribute inenhancing the effect of PRP and further rotator cuff healingin this study.

Regarding the delivery method, there could have been dif-ferent results if we had infiltrated PRP into the tendon orbone. We applied the PRP gel onto the repaired tendonbecause the infiltration of whole PRP into a chronicallytorn small rabbit tendon will cause a lot of resistance, accom-panied by much leakage outside the tendon to an unwantedspace. This loss of PRP may influence the results. The place-ment of PRP between the tendon and bone may be anotherpossible method. However, this method would also causemuch loss of PRP by the squeezing effect during the repairprocess. On the other hand, the infiltration of PRP intobone may stimulate mesenchymal stem cells, thus enhancingtissue healing. Moreover, much of the leukocytes were sepa-rated from PRP by the centrifugation protocol of this study,and the final product contained a very small amount of leu-kocytes (0.06 6 0.09 3 103/mL in PRP). This reduction of leu-kocytes in PRP may be another factor to enhance the effect ofPRP on tissue healing by decreasing the recruitment ofleukocyte-induced catabolic signaling molecules.34

As a secondary objective, we evaluated the effect of thepatch graft on rabbit rotator cuff healing by using a porcinedermal collagen graft. However, the result was less satisfac-tory, showing no significant effect on rotator cuff healingboth mechanically and biologically, even with the applica-tion of PRP. Several authors have investigated the effectof the patch graft on rotator cuff healing in animal modelsand showed improved biological and mechanical propertiesby the use of a porcine small intestinal submucosapatch.16,50,51 However, all these studies used the patch asan interpositional graft, which was different from the pres-ent study, where it was used as an augmentation graft inrotator cuff repair. In one animal study, Schlegel et al43

used the patch to augment sheep infraspinatus tendonrepair and showed that the load to failure and histologicalfindings did not indicate a significant difference betweenthe augmented and nonaugmented groups at 12 weeks.They demonstrated that patch augmentation was insuffi-cient to prevent the anatomic failure of rotator cuff repair.Their result was quite similar to that of the present study.

We do not know the exact reason why there was noeffect of patch augmentation in this study; however, webelieve that the evaluation time point may be a reason.Even though Zheng et al51 suggested that the graftedpatch was totally replaced by dense irregular collagenfibers at 8 weeks in a rabbit supraspinatus tendon model,on the other hand, Funakoshi et al19 showed that the scaf-fold materials still remained in the regenerated tissues at12 weeks and that the tensile strength in the regeneratedtissue after patch grafts was significantly improved from 4to 12 weeks postoperatively in a rabbit rotator cuff model.Thus, 8 weeks may not be enough for the full integration ofpatch grafts with the surrounding tendon tissue and formature tendon-to-bone healing. Further study witha long-term evaluation may be necessary. However, an 8-week period is sufficient for the evaluation of early to mid-term tendon healing. Thus, we believe that the results ofthis study, which inform us that patch augmentation was

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not effective on tendon-to-bone healing at an early to midtermperiod, are still meaningful, even though we do not know thechanges at a late period. In addition, if the tension for therepaired supraspinatus tendon to be pulled back to the foot-print was high, such as the clinical situation of chronicretracted rotator cuff tears, the result could be differentbecause of the load distribution effect of patch augmentationat an early to midterm period. However, all supraspinatustendons of each rabbit were repaired onto the greater tuber-osity with almost no tension in this study. That is, if the pos-sibility of good healing already exists in a situation of very lowtension at the repair site, patch augmentation at the repairsite may not have the ability to show improvement. Moreover,the different kinds of patch grafts used may affect the result.The porcine dermal collagen patch graft used in this studyhas a long residence time after implantation,37 which mayresult in late integration with the surrounding tendon tissueand a late effect on tendon-to-bone healing, which we werenot able to check in this study.

To our knowledge, this is the first study evaluating theeffect of PRP on rotator cuff repair combined with patch aug-mentation in a rabbit model using a relatively large numberof animals. In this animal model, we used only 1 shoulder forthe repair procedure with and without PRP or patch augmen-tation and the other shoulder as a control. The use of bilateralshoulders for the experimental procedures in each rabbitcould reduce the number of animals needed. However, webelieve that it may cause too much harm or sometimes belethal to the animals because of the long operation time andmuch blood loss, especially for the animals for which bloodwas collected for the PRP preparation. In addition, to useonly 1 shoulder for the experimental intervention may enablethe reduced use of that shoulder during the initial phase ofhealing, which may decrease the initial detachment and pos-sibly aid in enhancing tendon-to-bone healing.22 Another con-sideration is that the healing pathway is fundamentallydifferent between an acute injury and chronic injury. Differ-ent from acute injuries, chronic injuries presumably havea terminated inflammatory phase, with a paucity of plateletsand a decrease in healing potential because of poor-quality tissues.20,39 In this situation, the increased growthfactors produced by PRP may stimulate the tissue and restartthe inflammatory and stalled healing process, thereby facili-tating healing. Considering that rotator cuff tears usuallydevelop over a long period of time as a result of intrinsic orextrinsic factors, and that most rotator cuff surgeries are per-formed on chronic tears, the use of a chronic tear model, likein this study, is essential for this research; still, most animalmodels evaluate acute injuries and repair conditions.

Nevertheless, the current study has several limitations.First, this study was an animal study, which limits thegeneralization of the results. The differences in the anat-omy of the shoulder, the intrinsic healing potential, andthe platelet function between rabbits and humans preventthe clinical application of the findings of this preliminarystudy without further investigation. Second, we studiedthe status of tendon-to-bone healing only at an early tomidterm period until 8 weeks after repair with and with-out PRP and patch augmentation and had no informationon further changes at a late postoperative period. Even

though we selected 8 weeks based on previous studies,26,46

there certainly could be more mechanical and biologicalchanges after 8 weeks. Further study with a longer periodmay be needed. Third, we did not measure the repair ten-sion or tendon quality. The different tension and tendonqualities may affect the result of tendon-to-bone healingprofoundly. Finally, we could not confirm that PRP stayedin the desired site after closure nor determine how thegrowth factors secreted from PRP acted in the enhance-ment of tendon-to-bone healing. However, as the PRP-treated groups actually showed a significant effect ontendon-to-bone healing in this study, we believe that PRPwas present at the repair site in sufficient quantities to cre-ate an effect, and the effect might come from the growthfactors or cytokines released from PRP. Further studies,such as an immunohistochemical assay, to detect theexpression of PRP-related growth factors or cytokines,using different application methods of direct infiltrationof PRP into the tendon or bone, may be needed as a nextstep and to confirm the results of this study.

In conclusion, this controlled animal study showed theenhancement of tendon-to-bone healing after local admin-istration of autologous PRP assessed by histological andbiomechanical testing in a chronic rotator cuff tear modelin rabbits. However, there was little additive effect of theporcine dermal collagen graft as a biological augmenterof PRP. This finding suggests that PRP might be used asa biological supplement to increase the rotator cuff healingrate, which still remains low even after successful rotatorcuff repair. However, because of the limitations of this ani-mal study, our results should be interpreted with cautionregarding clinical application.

ACKNOWLEDGMENT

The authors especially thank Kyong Bun Lee (SeoulNational University Hospital) for the professional evalua-tion of histological grading of the specimens.

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