Anabolic effects of acellular bone marrow, platelet rich plasma, and serum on equine suspensory ligament fibroblasts in vitro

Summary The purpose of this study was to investigate the response of suspensory ligament fibroblasts (SLF) to in vitro stimulation using acellular bone marrow (ABM), platelet rich plasma (in vitro PRP), and serum as potential treatment modalities for suspensory desmitis. Blood, bone marrow, and suspensory ligaments were collected from five horses. SLF were harvested, grown until confluent, and stimulated with various concentrations of ABM, PRP, equine serum, foetal bovine serum, and medium (control). The responses to the treatments were assessed using a combination of radiolabeling for total protein synthesis and an ELISA for quantification of Cartilage Oligomeric Matrix Protein (COMP) production. Addition of all of the samples resulted in significant increases in COMP and total protein synthesis over controls (P<0.001). ABM caused the greatest increase in both COMP and total protein synthesis by the SLF. Equine ABM, PRP, and serum contain anabolic factors that promote matrix synthesis by SLF in vitro, with ABM having the greatest effect. Application of bone marrow to injured ligaments may enhance healing by providing anabolic factors, other than or in addition to mesenchymal stem cells, which stimulate matrix production.


Introduction
Suspensory ligamentdesmitis is acommon injuryi na thletic horses. Normal healing requiresap rolongedc onvalescent period, ranging from threetonine months (10).The repair process results in alteredfibrealignment andamechanically inferior structure making the damagedregionsprone to re-injury. In horses with severe lesions,t he injuries can become career limiting.
Herthel (13) haspreviously reportedthe enhanced healing of suspensory ligament injuriestreated with intra-lesionalinjection of autologous bone marrow,a nd proposed thatt he beneficial effectso ft he treatment were due to the provision of pluripotential mesenchymal stem cellsand/or growth factors. However, this treatment hasremained controversial due to our lackoftrueunderstanding of its mechanismo fa ction,t he smalln umber of mesenchymal stem cells (MSC)o btainedi natypical bone marrow aspirated(1M SC/100,000nucleated cells) (18), andthe lackofscientific investigations to elucidate whetherthe MSC or the growth factors withint he bone marrow( acellular bone marrow) have the capacity to improve the healing response. Furthermore,'growth factors' have not yetbeen readilyidentified in equine bone marrow.
'Plateletrichplasma'is consideredarich sourceo fm ultipleg rowthf actors andh as been used to acceleratee xisting wound healing pathways ( 18). Processing of plateletrichplasmainvolves sequestration and concentration of platelets in ordert oa mplifythe delivery of the growth factors they contain to injuredtissues. Unlikeindividual recombinantg rowthf actors, platelet rich plasma can modulate and'up-regulate'one growth factor'sf unction in the presence of additional growth factors.

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Approaches to studying the efficacyo f treatments on soft tissue healing have employedo ur knowledge of the structural propertiesa nd composition of normal tendons andl igaments,a nd have included measurements of cellulardivision or proliferation,aswellasextracellularmatrix synthesis (proteoglycan,collagen,total protein, Cartilage Oligomeric Matrix Protein [COMP])and organization (22). COMP is a non-collagenous glycoproteinthatisfound predominantlyi nt issues thatr esist load, such as tendon,ligament, cartilage,meniscus, andi ntervertebrald isc. High levels have been found in equine tendon, whereas lowl evelsh aveb een found in fibrous scar tissue (21). This proteinmay playasubstantial role in tendon development andm aturation and, because it is largelyabsentfrom fibrous scar tissue, it mayserve as amarker of appropriatet endon matrix( re)modeling (22). Tendon fibroblasts, in culture, were shownt os ynthesize COMP slowly in the absence of growth factors, butadramatic rise in COMPs ynthesis waso bservedf ollowing the addition of various growth factors, especially transforming growth factor beta one (TGF-β 1) (22).

Sample collection
Blood andt issues amples were harvested aseptically from five mixed age( range sevento18years),breed,and genderhorses at an abattoir immediately post-mortem.
Serum ands odium citrate vacutainert ubes were used to collect blood intendedfor use as equine serum andplateletrichplasma, respectively.Bone marrowaspirateswerecollected aseptically from the sternum of the horsesusing an 11 gauge bone marrowaspiraten eedle a ands terile syringe andt hen placed in sodium citrate vacutainert ubes. Distal forelimbs were obtainedfrom horses without grosssignsofsuspensorydesmitis. Alls amples were kept on ice untilt ransported to the lab for further processing.

Statistical analysis
Theresults were analyzed with astatistical softwareprogramme l using analysis of variance (ANOVA )inamixed model approach. When appropriate, ap ost-hoc Tukey'st est wasu sed for 'pairwise' comparisons. Differences between the meanswereconsidered significant when P<0.05.

Discussion
The results of this study supportour hypothesis andthe concept that bone marrowaspirates contain factors that maye nhancel igamenth ealing as has been previouslyd escribed by Herthel (13).D ue to thes mall number of mesenchymal stem cells obtained in at ypicalb one marrowa spirate ( 18),the beneficial effects of the treatment mayb e due to factors other than,o ri na dditiont o, MSC's contained within thebone marrow.
Quantitative analysis of individual equine growth factors is complicated by the lack of equine specific commercialassays, as well as the presence of activeand latent forms of growth factors andg rowthf actor inhibitors. Hence, we tookanalternativeapproach using an activity assayt om easure the effectsofthe various samples on the suspensoryl igamentf ibroblasts. Ab eneficial mix of growth factors for ligament repair mustb ea blet oi nducea ppropriatep rotein synthesis andl igamentm atrix formation. The effectsofABM, PRP, ES,and FBSon the suspensory ligamentf ibroblastsw ere therefore assessed by measuring overall proteinsynthesis andCOMP production in vitro by the suspensory ligamentf ibroblasts.
COMP wasc hosen because of previous work suggesting this protein mayp laya n importantr ole in tendon development and maturation (21).A lthoughC OMP levels have not previouslyb een measuredi n equine suspensory ligaments,t endons and ligaments ares imilarc onnectivet issues containingfibroblastic cells thathavesimilarhealing characteristics. While its precise role remains unclear at present,t here is some evidencetosuggest thatCOMP levels duringt endon maturation correlatew ith tendon strength (23). COMP maybeagood marker of connectivet issuem atrix formation sinceitispresent in lowlevelsin tendinous scar tissue.
Acquisition of acellularbone marrowisa minimallyi nvasive procedure that requires minimalp rocessing of the samples.I nt his study,A BM caused as ignificantd ose-de-pendent responseb yt he suspensory ligament fibroblasts. The10% ABMresultedin the highest COMP and totalp rotein synthesis overall, with an early eight-foldi ncrease in mean COMPp roduction anda four-fold increase in mean totalprotein synthesis by the fibroblasts when comparedto controls. Perhaps ABM containsh igher concentrationso fg rowthf actors or hasa morea ppropriatep hysiologic distribution of growth factors than the other biological fluids used in this study.
Platelet richp lasmaa nd equine serum areconsideredtoberichsources of various growth factors (18), areless invasive to obtain thanabone marrowa spirate,a nd requireprocessing that is similartoacellular bone marrow. In this study,PRP andESalso demonstrateds ignificanta nabolice ffects on suspensory ligamentf ibroblasts. However,neitherPRP nor ES were as effectiveas ABM. Serum, thrombin, ando therp latelet activators were not addedt ot he PRP samples to encourage clot formation in our study.Whilst activation of platelets triggers the release of the growth factors ando ther contents of the alpha granules withint he platelets,s pontaneous clot formation was observedw ithin the wells following addition of the PRPs amples,a nd therefore further activation wasdeemed unnecessary. Additionally,higher levels of TGF-B1 were quantifiedwithin PRP, as comparedtoplateletp oor plasma, without prior activation of the samples with thrombininaprevious study (3). It wasalsosuggested thatthrombin influences wound healing (3). Therefore,t he addition of thrombint ot he PRP samples might have interferedwith ourability to accurately assesst he responseo ft he suspensory fibroblasts to PRPalone.
Foetal bovineserum is available commerciallyand wasshown to be themost potent stimulatoro fc ell proliferation and protein synthesis in previous studies usingr abbit flexortendonswhen compared to individual growth factors (14). However, in thes tudy presented here, FBSdemonstrated minimal anabolic effects on suspensoryligamentfibroblasts in vitro ,indicatingthat even adult, species specific serum is more active than foetal serum fromanother species.
In summary,a ni ncrease in COMPa nd total proteins ynthesis by the suspensory

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ligamentf ibroblastsw as observedf ollowing stimulation with the various biological fluids.ABM, PRP, ES,and FBSall resulted in significant increases in COMPsynthesis over controls while 10%A BM resultedi n the greatest synthesis of COMP and total proteinc omparedt oo thert reatments.This suggeststhatABM maycontain appropriate combinationsofgrowthfactors that act synergistically to enhanceh ealing,a nd which mayb eb eneficialf or the successful managemento fs uspensoryd esmitis.F uture work should be directed at evaluating the in vivo responseo fs uspensoryl igaments to various biological fluids,d eterminingt he optimal dosages andt iming of administration of the fluids,a nd potentiallyu sing theseb iological fluids as vehiclesf or growth factor and/or mesenchymal stem cell delivery to injuredsoft tissues.