Leukocyte-Reduced Platelet-Rich Plasma Normalizes Matrix Metabolism in Torn Human Rotator Cuff Tendons

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1 Leukocyte-Reduced Pltelet-Rich Plsm Normlizes Mtrix Metbolism in Torn Humn Rottor Cuff Tendons Jessic A. Cross,* BS, Brin J. Cole, yz MD, Kyln P. Sptny,* BS, Emily Sundmn,* DVM, Anthony A. Romeo, y MD, Greg P. Nicholson, y MD, Bettin Wgner, DVM, nd Lis A. Fortier,* DVM, PhD Investigtion performed t Cornell University, Ithc, New York, USA Bckground: The optiml pltelet-rich plsm (PRP) for tretment of suprspintus tendinopthy hs not been determined. Purpose: To evlute the effect of low- versus high-leukocyte concentrted PRP products on ctbolic nd nbolic meditors of mtrix metbolism in disesed rottor cuff tendons. Study Design: Controlled lbortory study. Methods: Disesed suprspintus tendons were treted with PRP mde by use of 2 commercil systems: Arthrex Autologous Conditioned Plsm Double Syringe System () nd Biomet GPS III Mini Pltelet Concentrte System (). Tendon explnts were plced in 6-well pltes nd cultured in,, or control medi (Dulbecco s Modified Egle Medium 1 1% fetl bovine serum) for 96 hours. Tendons were processed for hemtoxylin-eosin histologic results nd were scored with the modified Bonr scle. Group 1 tendons were defined s moderte tendinopthy (Bonr score \3); group 2 tendons were ssessed s severely ffected (Bonr score = 3). Trnsforming growth fctor b 1 (TGFb-1), interleukin-1b (IL-1b), interleukin-1 receptor ntgonist (IL-1R), interleukin-6 (IL-6), interleukin-8 (IL-8), nd mtrix metlloproteinse 9 (MMP-9) concentrtions in PRP medi were mesured by use of enzyme-linked immunosorbent ssy fter 96 hours of culture with disesed tendon. Tendon messenger RNA expression of collgen type I (COL1A1), collgen type III (COL3A1), crtilge oligomeric mtrix protein (COMP), MMP-9, MMP-13, nd IL-1b ws mesured with rel-time quntittive polymerse chin rection. Results: Leukocytes nd pltelets were significntly more concentrted in compred with. Incresed IL-1b ws present in fter culture with group 1 tendons. IL-6 ws incresed in fter culture with group 2 tendons. Both TGFb-1 nd MMP-9 were incresed in fter culture with either tendon group. In cultures, IL-1R:IL-1b in PRP used s medi nd COL1A1:COL3A1 gene expression were incresed for group 1 tendon cultures. Gene expression of MMP-9 nd IL-1b ws incresed in group 2 tendons cultured in. There ws no significnt difference in the expression of MMP-13 or COMP in either group of tendons cultured in or. Conclusion: promotes norml collgen mtrix synthesis nd decreses cytokines ssocited with mtrix degrdtion nd inflmmtion to greter extent thn does in modertely degenertive tendons. In severely degenertive tendons, neither PRP preprtion enhnced mtrix synthesis. Clinicl Relevnce: my promote heling in modertely degenertive rottor cuff tendons. Keywords: pltelet-rich plsm; growth fctors; biologicl heling enhncement; shoulder; rottor cuff z Address correspondence to Brin J. Cole, MD, Deprtment of Orthopedics, Rush University Medicl Center, 1653 West Congress Prkwy, Chicgo, IL 6612, USA (emil: cole.reserch@rushortho.com). *Deprtment of Clinicl Sciences, Cornell University, Ithc, New York, USA. y Deprtment of Orthopedics, Rush University Medicl Center, Chicgo, Illinois, USA. Deprtment of Popultion Medicine nd Dignostic Sciences, Cornell University, Ithc, New York, USA. One or more of the uthors hs declred the following potentil conflict of interest or source funding: The study ws funded by Arthrex Inc. B.J.C., A.A.R., nd L.A.F. re pid consultnts for Arthrex. The Americn Journl of Sports Medicine, Vol. 43, No. 12 DOI: / Ó 215 The Author(s) Rottor cuff tendon bnormlities re mong the most frequent cuses of shoulder pin. 8,36 Without intervention, the prognosis for symptomtic ptients with rottor cuff ters remins reltively poor. 8,9 Even with surgicl repir, poor qulity tissue regenertion is ssocited with ntomic nd biologicl filure. This might be ttributed to insufficient gene or protein expression or pucity of undifferentited cells t the heling site. 6,12,14,15 Reltively high filure rtes fter rottor cuff repir nd the chllenges ssocited with compromised tendon structure in chronic rottor cuff conditions remin importnt considertions for the development of techniques tht enhnce the biologicl heling environment. Current techniques include 2898

2 AJSM Vol. 43, No. 12, 215 PRP Enhnces Rottor Cuff Tendon Heling 2899 extrcellulr mtrices, llogenic or utogenic tendon trnsplnts, synthetic mesh, or the ddition of growth fctors to the surgicl site. 1 The use of extrcellulr or synthetic mtrices for shoulder repir hs been ssocited with poor results in some studies. 17,32 However, introduction of growth fctors fter soft tissue trumtic event cn stimulte enhnced repir nd ngiogenesis. 27 Pltelet-rich plsm (PRP) contins growth fctors of interest for tendon regenertion, mking it ttrctive for use in rottor cuff repir ugmenttion. Concurrent with the growth in PRP technologies is the increse in the diversity of preprtion systems offering differing cellulr concentrtions in the resultnt finl PRP product delivered to the ptient. In ddition to pltelet content vribility, lrge differences in leukocyte concentrtion exist between PRP systems. Whether leukocytes hve positive or negtive influence on heling tissues remins controversil issue nd subject of investigtion. 3 Multiple studies demonstrte how reduced inflmmtion promotes norml collgen genertion nd my reduce the degree of tendon dmge during the heling process by limiting ctbolic ctivity, such s extrcellulr mtrix ctbolism. 24,25,35,37 Additionl properties of PRP tht re thought to be beneficil for the tretment of tendon injuries include provision of fibrin-mtrix scffold rich in growth fctors tht promotes collgen orgniztion, fiber pttern lignment, tenocyte prolifertion, nd collgen type I synthesis. 23 The objective of this study ws to compre PRP generted from Arthrex Autologous Conditioned Plsm Double Syringe System (; Arthrex Inc) nd Biomet GPS III Mini Pltelet Concentrte Kit (; Biomet Inc) nd their effects on disesed rottor cuff tendon metbolism. This is in contrst to number of studies investigting the effects of PRP on helthy tendon or in niml models tht do not reflect clinicl relity. 7,24,31 With the knowledge tht the system concentrtes pltelets nd reduces leukocytes nd tht the system concentrtes both pltelets nd leukocytes compred with whole blood, we hypothesized tht incresed leukocyte concentrtion would result in incresed delivery of ctbolic cytokines nd mtrix metlloproteinses (MMPs) with resultnt incresed tendon mtrix degrdtion. METHODS All procedures were pproved by the pproprite regultory bodies t Cornell University nd Rush University Medicl Center. All investigtions were conducted in conformity with ethicl principles of reserch, nd informed consent for prticiption in the study ws obtined from ll prticipnts. Pilot Study to Determine the Effects of Acid Citrte Dextrose in PRP on Tendon Metbolism The use of cid citrte dextrose (ACD) nticogulnt in 1 of the 2 PRP systems (Arthrex Double Syringe ACP System; ) is optionl ccording to the mnufcturer s directions if the PRP is used within 4 hours of initil blood collection. The second system (Biomet GPS III Mini Pltelet Concentrte Seprtion Kit; ) requires 1% finl volume ACD. The exclusion of ACD, which hs ph of 4.98, hs the dvntges of mintining physiologic ph, minimizing pin fter injection of PRP, 26 nd simplifying the steps nd regents needed to generte PRP. A pilot study ws performed to determine the effect of inclusion or exclusion of ACD in on tendon metbolism. Humn biceps tendons (n = 6) were procured from cdveric donors nd dissected into mm explnts. Venous blood ws obtined with nd without ACD from helthy humn volunteers to generte. Explnts were cultured in PRP s medi for 96 hours with PRP from 1 donor being used on tendon explnts from 1 cdver. Medi ph ws mesured by use of indictor pper (Whtmn) to ssess exhustion of culture medi nutrients. At the time of culture termintion, tendons were pulverized in freezer-mill, nd totl RNA ws extrcted by use of the Qigen RNesy Kit (RNesy Mini Hndbook; Qigen). Rel-time quntittive polymerse chin rection (RTqPCR) ws performed to mesure nbolic mrkers collgen type I (COL1A1), collgen type III (COL3A1), crtilge oligomeric mtrix protein (COMP), ctbolic mrkers including MMP-9 nd MMP-13, tumor necrosis fctor (TNF-), nd interleukin-1b (IL-1b) by use of ABIPRISM 79HT Sequence-Detection with Tqmn Gene Expression Assys-Inventoried (Applied Biosystems). Expression results were normlized to 18S RNA expression by use of the 2 DDC T method. A Shpiro-Wilks test indicted tht the dt were normlly distributed. Gene expression nd medi ph were compred by use of 2-smple t tests. The ph of PRP without ACD (ph, 8.) ws significntly higher compred with tht of PRP contining ACD (ph, 7.; P \.1). There were no differences in expression genes between the PRP groups with or without ACD. Expression of TNF- ws undetectble. Results of the pilot study indicted tht ACD t finl volume of 1% does not ffect norml tendon metbolism nd should not be fctor in outcome ssessment when compring PRPs with or without ACD. The following principl study ws therefore performed using without ACD nd with ACD to reflect common clinicl prctice. Rottor Cuff Tendon Acquisition nd Preprtion Tendon biopsy specimens were tken from the lterl spect of chroniclly torn suprspintus tendons fter routine exposure of the glenohumerl joint from 2 ptients between the ges of 6 nd 8 yers who were undergoing reverse shoulder rthroplsty for rottor cuff rthropthy. Tendons were dissected into mm explnt pieces nd rinsed with Hnk s Blnced Slt Solution (HBSS), nd 5 explnts were plced per well of 6-well plte (Figure 1). Pltelet-Rich Plsm Venous blood ws collected from helthy humn volunteer popultion distinct from the rottor cuff donor ptients. Blood ws used to generte (Arthrex

3 29 Cross et l The Americn Journl of Sports Medicine Tendons: n = 2 6 ml blood in ACD Blood donors: n = 2 15 ml blood no ACD 5 explnts into ech of 3 wells. Explnt for histology. Centrifuge Histology: H&E stining 3.5 ml into well 1A 3.5 ml into well 2A 3.5 ml medi into well 3A Tendon explnts RT PCR COL1A1 COL3A1 COMP IL-1β MMP-9 MMP-13 Culture for 96 hours Culture medi ELISA IL-1β IL-1R IL-6 IL-8 MMP-9 TGFβ-1 Figure 1. Schemtic of study design nd method. ACD, cid citrte dextrose; COL1A1, collgen type I; COL3A1, collgen type III; COMP, crtilge oligomeric mtrix protein; ELISA, enzyme-linked immunosorbent ssy; H&E, hemtoxylin nd eosin; IL-1b, interleukin-1b; IL-1R, interleukin-1 receptor ntgonist; IL-6, interleukin-6; IL-8, interleukin-8;, leukocyte-high plteletrich plsm;, leukocyte-low pltelet-rich plsm; MMP-9, mtrix metlloproteinse 9; MMP-13, mtrix metlloproteinse 13; RT-PCR, rel-time polymerse chin rection; TGFb-1, trnsforming growth fctor b 1. ACP without ACD) nd (Biomet PRP with ACD) ccording to mnufcturer directions. One blood donor ws used per tendon, nd blood from tht donor ws used to mke both nd. Whole blood, pltelet-poor plsm,, nd smples were submitted for complete blood counts. Immeditely fter PRP preprtion, tendon explnts from single ptient were incubted in, from single donor, or control medi (Dulbecco s Modified Egle s Medium [DMEM] 1 1% fetl bovine serum [FBS]) for 96 hours. At conclusion of the incubtion period, ph vlues for ech smple were recorded to verify there were no lrge decreses in ph indicting exhustion of nutrients. Tendon explnts were wshed with phosphte-buffered sline (PBS), snp frozen, nd stored t 8 C for RNA isoltion. Medi were stored t 8 C in independent liquots for ech of the enzyme-linked immunosorbent ssys (ELI- SAs) to void repeted freeze/thw cycles. RNA Purifiction nd qpcr Gene expression of COL1A1, COL3A1, COMP, MMP-13, MMP-9, nd IL-1b ws quntified s described for the pilot study. Growth Fctor nd Cytokine Quntifiction Concentrtion of trnsforming growth fctor b 1 (TGFb-1) ws determined by use of the E mx ImmunoAssy System (Promeg Corp), nd MMP-9 ws mesured with the Biotrk Activity Assy (GE Helthcre Biosciences) by use of multiple-detection plte reder (Tecn Sfire). IL-1b, IL-1 receptor ntgonist protein (IL-1RA), IL-6, nd IL-8 were mesured with Fluorokine MAP Humn Elis Kits on Fluorokine MAP Humn Bse Pnel (R&D Systems). Histologic Testing Tendon smples tht were not subjected to culture were histologiclly prepred nd scored ccording to chnges in tenocyte morphologic chrcteristics, collgen bundle chrcteristics, nd vritions in vsculrity. Briefly, tendon explnts were fixed in 4% prformldehyde, dehydrted in grde lcohol, clered in xylene, nd embedded in prffin. Sequentil 4-mm sections were cut, stined with hemtoxylin-eosin, nd then exmined under light microscopy. Sections were independently scored by 2 reders (J.A.C., L.A.F.) using the modified 4-point Bonr scle (-3, with being norml). 11 The Bonr scle scores were

4 AJSM Vol. 43, No. 12, 215 PRP Enhnces Rottor Cuff Tendon Heling 291 plotted, nd there ws distinct seprtion into 2 equl groups of n = 7. For further nlysis, tendons were seprted into group 1 tendons, defined s those demonstrting moderte tendinopthy (Bonr score \3), nd group 2 tendons, defined s those with severe tendinopthy (Bonr score = 3). Five ptients did not hve enough smple for histologic exmintion, nd 1 explnt hd no histologic evidence of tendon tissue. These 6 ptients were removed from the study becuse they could not be histologiclly clssified ccording to disese sttus. Sttisticl Anlyses A Shpiro-Wilks test indicted norml distribution of cell counts in blood nd PRP (pltelet, hemtocrit, neutrophil, lymphocyte, nd monocyte concentrtions), so 2-smple t test ws used to determine differences between tretments. ELISA nd gene expression dt were not normlly distributed (Shpiro-Wilks test). A Kruskl-Wllis 1-wy nlysis of vrince (ANOVA) ws used to determine significnt differences in gene expression nd ELISA dt with tendons ctegorized s group 1 or group 2. A P vlue less thn.5 ws considered significnt. Sttistix 9 softwre (Anlyticl Softwre) ws used to perform the nlyses. RESULTS PRP Composition Both systems successfully generted PRP. Neutrophil, lymphocyte, nd monocyte fold chnges (PRP/whole blood vlue) were significntly greter in compred with (Figure 2). Pltelet nd RBC concentrtions were significntly greter in compred with L lo PRP. Pltelets were concentrted pproximtely 23 over venous blood in nd 43 in. Histologic Results Retrieved tendons hd histologic evidence of thinning, seprtion, nd disorgniztion of collgen fibers. Tendon fibroblsts were often round in shpe with evidence of hyperplsi (Figure 3). There were scttered res of mononucler cell infiltrtion, vsculr prolifertion, ftty infiltrtion, nd lipoid nd myxoid degenertion. Ctbolic Cytokines (IL-1, IL-1RA, IL-6, IL-8, MMP-9) in PRP After Culture With Disesed Tendon In modertely degenertive group 1 tendon cultures, L lo PRP hd significntly lower IL-1b concentrtion (Figure 4A), no difference in IL-1RA (Figure 4A), nd incresed IL-1RA:IL-1b rtio (Figure 4B) compred with. In severely degenertive group 2 tendon cultures, there were no differences in IL-1b between nd L hi PRP (Figure 4A), significnt increse in IL-1RA in L hi Fold chnge (PRP/blood) P <.1 P <.1 P <.1 P <.1 Hemtocrit Neutrophils Lymphocytes Monocytes Pltelets PRP (Figure 4A), but no resultnt chnge in the IL- 1RA:IL-1b rtio (Figure 4B). No significnt difference ws found in IL-6 concentrtion between nd cultured with group 1 tendons (Figure 5). There ws significntly greter IL-6 in group 2 tendon cultures treted with (Figure 5). No significnt difference ws noted in IL-8 concentrtion fter PRP tretment in either group 1 or group 2 cultures (Figure 5). MMP-9 ws significntly incresed in L hi PRP when cultured with either group 1 or 2 tendons compred with treted tendons (Figure 6). Growth Fctor (TGFb-1) in PRP After Culture With Disesed Tendon P =.2 Figure 2. Distribution of hemtocrit, neutrophil, lymphocyte, monocyte, nd pltelet concentrtions in leukocyte-low pltelet-rich plsm () or leukocyte-high PRP (L hi PRP) divided by corresponding venous blood vlues to generte fold chnge. Brs represent men (n = 2) 6 stndrd error. A 2-smple t test ws performed to compre vlues between nd. The concentrtion of TGFb-1 ws significntly greter in used to tret both group 1 nd group 2 tendons compred with treted groups (Figure 7). Mtrix Gene Expression (COL1A1:COL3A1, COMP) in Tendons Cultured in PRP In modertely degenertive group 1 tendons, COL1A1:- COL3A1 gene expression ws significntly incresed in tendons cultured in either or, with expression gretest in tendons cultured in (P =.4) (Figure 8A). In severely degenertive group 2 tendons, there ws no chnge in expression of COL1A1:COL3A1 between tendons cultured in,, or control (P =.29). There ws no significnt difference in the expression of COMP fter PRP tretment for either group 1 (P =.73) or group 2 (P =.36) tendons.

5 292 Cross et l The Americn Journl of Sports Medicine Figure 3. (A) Norml suprspintus tendon demonstrting closely rrnged, prllel fibers; sprse distribution of elongted fibroblsts; nd vsculr, lymph, nd nerve bundles contined within the epitenon nd running prllel with collgen fibers. (B-O) disesed suprspintus tendon from ptients who underwent reverse shoulder rthroplsty demonstrting thinning, seprted, nd disorientted collgen fibers (double rrows); rounded fibroblsts (rrows); fibroblstic hyperplsi; infiltrtion of mononucler cells; rndom vsculr prolifertion (white rrows); ftty infiltrtion or lipoid degenertion (rrowheds); nd myxoid degenertion. (B-H) Group 1 tendons clssified s mild tendinopthy. (I-O) Group 2 tendons clssified s severe tendinopthy. Hemtoxylineosin stin, scle br = 1 mm. Ctbolic Gene Expression (MMP-9, MMP-13, IL-1b) in Tendons Cultured in PRP Expression of MMP-9 ws similr in control tendons but ws stimulted to greter extent in group 2 tendons cultured in either Llo PRP or Lhi PRP compred with group 1 tendons, with Llo PRP being greter thn Lhi PRP (P \.1) (Figure 8B). No significnt chnge in MMP-9 expression ws noted fter PRP tretment in group 1 tendons (P =.9). There ws no significnt difference in the expression of MMP-13 fter Llo PRP or Lhi PRP tretment for either group 1 (P =.99) or group 2 (P =.12) tendons. Expression of IL-1b in group 2 tendons hd significnt response to PRP (P =.3), but group 1 tendons did not (P =.17) (Figure 8C). In group 2 tendons, both Llo PRP nd Lhi PRP tretments resulted in significntly incresed IL1-b expression, with Llo PRP being greter thn Lhi PRP. DISCUSSION The primry objective of this study ws to evlute the effect of low versus high leukocyte nd pltelet concentrtion PRP products on ctbolic nd nbolic meditors of disesed tendon mtrix metbolism. The findings of this study support our hypothesis tht Llo PRP contins less ctbolic cytokines thn Lhi PRP, resulting in blnce towrd tendon mtrix regenertion in mild to modertely ffected tendons. However, the results observed in severely degenertive tendons were dissimilr, with neither PRP cpble of enhncing mtrix synthesis. This could be due to the infiltrtion of mononucler cells nd neovsculriztion in severely ffected tendons, which ws visible on the histologic imges. This ssumption is supported by the observtion tht even though the sme Llo PRP nd Lhi PRP were used s culture medi for group 1 nd group 2 tendons, group 2 severely degenertive tendons responded

6 AJSM Vol. 43, No. 12, 215 PRP Enhnces Rottor Cuff Tendon Heling 293 ng IL-6 / ml PRP P =.48 P =.4 P =.55 P = ng IL-8 / ml PRP Figure 5. Interleukin-6 (IL-6) nd IL-8 concentrtions in leukocyte-low pltelet-rich plsm () or leukocytehigh PRP () used s culture medi for 96 hours for modertely degenertive (group 1) or severely degenertive (group 2) suprspintus tendons. Brs represent men (n = 7) 6 stndrd error. Significnce ws determined by use of Kruskl-Wllis 1-wy nlysis of vrince. Figure 4. (A) Concentrtion of interleukin-1b (IL-1b), interleukin-1 receptor ntgonist protein (IL-1R), nd (B) the resultnt IL-R:IL-1b rtio in leukocyte-low pltelet-rich plsm () or leukocyte-high PRP () fter 96 hours of incubtion with modertely degenertive (group 1) or severely degenertive (group 2) suprspintus tendons. Brs represent men (n = 7) 6 stndrd error. Significnce ws determined by use of Kruskl-Wllis 1-wy nlysis of vrince. ng MMP-9 / ml PRP P =.1 P = to greter extent by nerly n order of mgnitude in mtrix gene expression of IL-1b nd MMP-9. This would suggest tht there is not just single PRP preprtion for ll situtions, nd determining the optiml preprtion for ech tissue should be pursued in clinicl studies. The results of this study cn be further extrpolted to ptients with rottor cuff disese. Improved methods to clssify tendon disese such tht moderte versus severely degenertive rottor cuff tendinopthy cn be identified preopertively would provide informtion to design tretment pproch tilored to the extent of the disese process. This ex vivo study hs some inherent limittions. First, in nturlly occurring tendinopthy, numerous nbolic nd ctbolic meditors re lredy present tht could lter the effect of PRP on tissues, nd those confounding fctors would be excluded in n ex vivo study. Second, since this ws retrievl study, we did not know how long ptients were ffected by rottor cuff ters. Histologic ssessment nd scoring of the smples llowed for grouping the tendons bsed on severity of degenertion to ccommodte for the vrying levels of degenertion of tendon Figure 6. Mtrix metlloproteinse 9 (MMP-9) concentrtion in leukocyte-low pltelet-rich plsm () or leukocyte-high PRP () used s culture medi for 96 hours for modertely degenertive (group 1) or severely degenertive (group 2) suprspintus tendons. Brs represent men (n = 7) 6 stndrd error. Significnce ws determined by use of Kruskl-Wllis 1-wy nlysis of vrince. between ptients. In ddition, dose-response study could not be performed given the smll quntity of tissue retrieved from these ptients. Finlly, the pilot study investigting the effects of ACD ws completed on norml tendon, nd thus it is possible tht the conclusions from this portion of the study my hve differed if the presence or bsence of ACD hd been investigted on disesed tendon. Leukocytes nd the proinflmmtory cytokine IL-1b were significntly incresed in. IL-1b is produced by ctivted mcrophges, blood neutrophils, B-lymphocytes, nd endothelil cells. Incresed expression of IL-1b in the

7 294 Cross et l The Americn Journl of Sports Medicine A Control b COL1A1:COL3A1 c Figure 7. Concentrtion of trnsforming growth fctor b 1 (TGFb-1) in leukocyte-low pltelet-rich plsm () or leukocyte-high PRP () fter 96 hours of culture with modertely degenertive (group 1) or severely degenertive (group 2) suprspintus tendons. Brs represent men (n = 7) 6 stndrd error. Significnce ws determined by use of Kruskl-Wllis 1-wy nlysis of vrince. subcromil burs of disesed rottor cuffs hs been correlted with incresed pin in ptients due to its cpcity to incite inflmmtion nd hyperesthesi. 37 An incresed rtio of IL-1R:IL-1b suggests greter cpcity to competitively inhibit binding of IL-1b to type I nd II cell surfce receptors, thereby decresing the ctbolic effects of IL-1b. 33 In group 1 tendons, but not in group 2 tendons, the rtio of IL-1R:IL-1b ws incresed in, but gene expression of IL-1b ws not significntly ffected in these tendons by either PRP. The results in group 2 tendons were dissimilr, with no chnge in the rtio of IL-1R:IL-1b but significnt increses in IL-1b gene expression in tendons cultured in either PRP. Regultion of the IL-1 pthwy in tendinopthies is not well understood, but these dt suggest tht the entire pthwy should be considered, prticulrly in cses of severe tendinopthy where gene expression levels in the tendon tissue suggest the presence of potentilly proinflmmtory environment. In this study, hd significntly greter concentrtion of IL-6 compred with for group 2 tendons, nd there ws no significnt difference between the biologics for either group for IL-8 concentrtions. Limited dt re vilble detiling the interction between IL-6 nd IL-8 in rottor cuff tendon tissue, but recent studies involving ptients with cute Achilles tendon rupture showed tht IL-6 nd IL-8 concentrtions were significntly incresed during the heling phse, while proinflmmtory cytokines TNF- nd IL-1b were below detectble levels. It ws lso observed tht infusion of IL-6 into peritendinous tissue round the humn Achilles tendon incresed the synthesis of collgen type I. 1,2 These studies suggest tht IL-6 nd IL-8 my be ssocited with nti-inflmmtory nd regenertive effects in heling tendon. Incresed expression of MMPs hs been shown to be ssocited with complete rottor cuff ters nd tendinopthy. 22 B C Control Control 1 MMP IL-1β 1 2 These zinc-dependent MMPs re cpble of degrding intct nd degenertive tendon extrcellulr mtrix. More 2 b b c c Figure 8. Gene expression in modertely degenertive (group 1) or severely degenertive (group 2) tendons fter culture for 96 hours in leukocyte-low pltelet-rich plsm () or leukocyte-high PRP (). (A) Rtio of collgen type I (COL1A1) to collgen type 3 (COL3A1) genes ssocited with tendon mtrix synthesis. (B) Mtrix metlloproteinse 9 (MMP-9) nd (C) interleukin-1b (IL-1b) gene expression ssocited with tendon ctbolism. Brs represent men (n = 7) 6 stndrd error. Superscript letters indicte significnce difference between groups by use of Kruskl-Wllis 1-wy nlysis of vrince. 5

8 AJSM Vol. 43, No. 12, 215 PRP Enhnces Rottor Cuff Tendon Heling 295 specificlly, MMP-13 cn degrde ll subtypes of collgen, including those tht provide mechnicl strength to tendons such s collgen type I. 13 Smller collgen frgments re primrily the trget of the geltinse MMP-9. In this study, L hi PRP hd significntly lrger concentrtion of MMP-9 thn did in both groups. This is expected becuse hd more neutrophils nd pltelets thn did. MMP-9 is stored in circulting neutrophils nd pltelets nd is relesed upon their ctivtion. 16 However, severely degenertive group 2 tendons cultured in hd significntly lrger MMP-9 gene expression compred with, nd both were incresed compred with control tendon cultures. Much of the informtion regrding regultion of MMP-9 in tendinopthy is unknown, but it is thought tht the ction of MMP-9 to cleve dentured collgen nd collgen type III in degenertive tissue llows for formtion of structurl collgens, such s collgen type I, in the remodeling process. 28 Pltelets re nturl reservoir of TGFb-1, which is key growth fctor for stimultion of collgen synthesis, cell prolifertion, recruitment, nd migrtion nd is ssocited with reduced scr formtion in heling tendons. 3,5,18 In rottor cuff repir procedures, TGFb-1 improves heling nd mechnicl strength t the tendon-bone interfce. 4,19,2,29,34 The incresed concentrtion of TGFb-1 in compred with in the present study is not surprising since pltelet concentrtion ws lso greter in. Although the quntity of TGFb-1 ws 3 to 4 times greter in thn in, norml mtrix synthesis, s mesured by the rtio of COL1A1:COL3A1 gene expression in tendons, ws incresed only in group 1 modertely degenertive tendons, nd within tht group, stimulted greter synthesis thn did. Norml rottor cuff tendon is predominntly composed of type I collgen (.95%) nd smll mounts of type III, IV, nd V collgen (\5%). 38 Type III collgen is normlly restricted to the endotenon surrounding fiber bundles. It is incresed fter injury nd is bundnt in wound bed grnultion tissue. In the nturl heling of torn rottor cuff tendons, successful remodeling is typified by replcement of type III collgen with type I collgen. 21 The dt in this study suggest tht in modertely disesed tendon, influences norml tendon mtrix metbolism butisunbletoelicitspecificmetbolicresponseinmore degenertive cses of rottor cuff tendinopthy. 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