Journal of Equine Veterinary Science 31 (2011) 147-154

Journal of Equine Veterinary Science

journal homepage: www.j -evs.com

Case Study

Treatment of Bilateral Medial Femoral Condyle Articular CartilageFissures in a Horse Using Bone Marrow-Derived MultipotentMesenchymal Stromal Cells

Leah F. Raheja BS a, Larry D. Galuppo DVMb, Jeanne Bowers-Lepore DVMc,Joseph P. Dowd DVMd, Fern Tablin DVM, PhD a, Clare E. Yellowley PhD a

aDepartment of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, CAbDepartment of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, CAcHarris Farms Horse Division, Coalinga, CAd Equine Medical and Surgical Group, Arcadia, CA

Keywords:StifleCartilageStem cellOCDRepair

Corresponding author at: Larry D. Galuppo, DVSurgical and Radiological Sciences, School of Veterinaryof California, Davis, 2112 Tupper Hall One Shields Ave, D

E-mail address: ldgaluppo@ucdavis.edu (L.D. Galu

0737-0806/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.jevs.2010.12.009

a b s t r a c t

The objective of this study was to describe the use, and outcome, of multipotentmesenchymal stromal cells (MSCs) in the treatment of equine articular cartilage defectsof the medial femoral condyle. A 4-year-old Thoroughbred gelding (n = 1) with bilateralstifle athroscopy was found to have bilateral articular cartilage fissure defects of themedial femoral condyles with concurrent cranial cruciate ligament injury. Bone marrowderived MSCs were isolated, expanded, and suspended in a partially autologous fibringlue. The initial cell/fibrin glue mixture was delivered arthroscopically into the articularcartilage defects 90 days after the initial arthroscopic examination. Follow-up treatmentsincluded two additional injections of MSCs suspended in lactated Ringers solution, 5 and13 months after the initial examination, directly into the joint. Post-treatment outcomewas assessed by arthroscopic examination and by comparison of preinjury and post-treatment performance records. Arthroscopic evaluation 4 months after the initialMSC treatment revealed marked smoothing, reduction in the depth of cartilage defectsand observation of moderate improvement in the cranial cruciate ligament. Approxi-mately 15 months after the initial MSC treatment the horse returned to racing. Analysisof race records demonstrated that the post-treatment (including all three MSC treat-ments) average race earnings (earnings per start) were comparable with those predatingthe initial injury. The favorable clinical response in the face of an unknown, but likely,guarded prognosis suggest that MSC therapy is not deleterious and may augment healingof articular cartilage fissures of the medial femoral condyle. MSCs represent a viable andpromising alternative therapy in the treatment of articular cartilage injuries in perfor-mance horses.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

Joint pathology has been reported to be a major cause oflameness in horses and is estimated to account for 42%-60%[1,2] of lameness cases. The relatively avascular and hypoxic

M, Department ofMedicine, Universityavis, CA 95616.ppo).

ll rights reserved.

nature of cartilage as a tissue, in addition to the non-proliferative characteristics of mature chondrocytes, oftenresults in a poor prognosis for complete recovery. In addi-tion, these cartilage injuries present with highly variableclinical signs and recovery outcomes. Factors that determinethe extent of healing include the depth, location, size, andweight-bearing environment of the lesion(s), in addition topatient age and conformation [3,4]. Current treatments forinjuries related to articular cartilage in horses are ever

Table 1Preinjury performance records

Date of race Distance (furlongs) Placing Winnings ($)

November 25, 2004 6.5 1 13,200February 11, 2005 6 1 21,450March 5, 2005 6.5 1 66,270March 26, 2005 6.5 1 62,415April 22, 2005 5.5 5 2,002May 21, 2005 7 6 0February 19, 2006 6.5 0 0March 23, 2006 6 2 10,000May 20, 2006 7 3 8,580August 12, 2006 6.5 5 880September 16, 2006 6.5 7 400Average 6.41 3 16,836.09Total 70.50 32 185,197.00Starts 11.00

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evolving. A diagnosis is generally confirmed througharthroscopic examination and treatments include partialmeniscectomy, curettage or drilling into the subchondralbone, cartilage flap/fragment fixation, and surgicaldebridement and microfracture [5-12]. In horses withlameness localized to the medial femorotibial joint havingsubtle radiographic changes of the medial femoral condyles,it was found that only two of six horses with generalizedcartilage lesions were reported as being sound and withoutany evidence of joint effusion after arthroscopic-guidedabrasion arthroplasty and microfracture [12]. Prognosisdepends on the severity and location of the lesion(s),among other factors, and it is generally accepted thatthe percentage of patients making a complete recoverydecreases with increasing severity of injury [13]. In anotherseries, 86% of horses with focal lesions of themedial femoralcondyle treated with arthroscopic curettage and debride-ment returned to normal function, whereas none of thehorses that presented with extensive and diffuse damage, inwhich arthroscopic treatmentwas not performed, recoveredcompletely [6]. Both studies highlight a guarded prognosisfor horses with moderate to extensive damage to the carti-lage of the medial femoral condyle.

With limited options available for articular cartilagerepair and the efficacy of such options being variable,especially for extensive injuries, clinicians and researchersin both human and veterinary medicine have, over the pastdecade, looked into the potential of progenitor or stem cellsto aid in tissue regeneration and repair. Currently, only onecell-based therapy has been approved by the Food andDrug Administration for use in human beings (Carticel,Genzyme). This uses autologous chondrocytes harvestedfrom a non- or low load-bearing location that are thenexpanded in vitro, implanted into articular defects, andcovered by a periosteal flap. The efficacy of such therapy, asmeasured by a combination of function and pain [14], hasbeen reported to be >80% in human beings [15], withcomparable results in horses using a similar technique forthe repair of manufactured full-thickness defects in minorload-bearing areas of the tibiotarsal joint [16]. However,induction of defects to harvest chondrocytes is a concernwith this type of cell therapy and comparable clinicalresults have been obtained with microfracture [17]. Thus,other tissue sources for cellular therapy as well asimplantation methods are being considered.

Mesenchymal stromal cells (MSCs) are a multipotentadult stem cell population capable of differentiating intotissues of the mesenchymal lineages including bone,cartilage, and fat, and may also serve as trophic mediatorsaiding in attenuating the inflammatory response [18-20].These cells have been referred to in the published data andby industry by a variety of names including mesenchymalstem cells and bone marrow stromal cells; we have chosento use the namemultipotentMSCs, as recommended by theInternational Society for Cellular Therapy [21]. With therelative abundance and accessibility of MSCs for clinicalharvest, many clinicians and researchers have begun look-ing at the use of MSCs for cartilage [22], tendon [23,24],ligament [23,24], and bone repair [25]. Recently, there wasa report of the use of autologous MSCs in a fibrin glueenhancing the early repair of manufactured full-thicknessarticular cartilage/subchondral bone defect in the equine

femoropatellar joint [26]. Although the report noted thatlong-term healing at 8 months was not significantlydifferent between individuals receiving MSC-laden fibringlue or control (MSC-free fibrin glue), the study did notcompare an untreated control, thus it is difficult to deter-mine whether the fibrin glue alone may have played a rolein healing. There is evidence in the published data thatfibrin may serve as a scaffold for tissue repair, includingthat of cartilage, thereby contributing to the healingprocess [27,28]. The apparent benefits of MSCs on earlyhealing should not be disregarded and may providea foundation for additional intra-articular cellular therapyto improve overall joint health. Although experimentalstudies are limited, there is less evidence for the efficacy ofsuch treatment in naturally occurring superficial lesions inwhich the subchondral bone is intact. In this case report,we describe the use of arthroscopically delivered autolo-gous MSCs in a partially autologous fibrin glue to treatbilateral diffuse fissure fractures of the medial femoralcondyles of a horse.

2. Materials and Methods

2.1. Clinical Findings

A 4-year-old Thoroughbred gelding was presented forbilateral stifle arthroscopy. The horse began racing inNovember 2004 as a 2-year-old and raced successfullythrough September 2006 with 11 starts (Table 1). Althoughthere was a clinical indication of subtle hindlimbperformance-limiting lameness in July 2006, physicalexamination, nuclear scintigraphy, and associated stifleradiographic examination were inconclusive. However,there was slight flattening noted on the medial femoralcondyles bilaterally (Fig. 1). In October 2006, the horse wasremoved from race training because of its inability toperform. There was no reported evidence of lameness at thetrot, however, the horse was unwilling to canter or gallop.On the basis of clinical assessment, a stifle problem wassuspected. Screening for equine protozoal myeloencephalitis(indirect fluorescent antibody test [IFA] for Sarcocystis neu-rona andNeospora hughesi) was negative. Bilateral diagnosticintra-articular anesthesia resulted in markedly improvedwillingness to canter. On the basis of the response to the

Fig. 1. Initial cranial to caudal radiographic projections of the left and rightstifles demonstrating mild flattening of the medial femoral condyles bilat-erally. The radiographic examination was performed 4 months beforediagnostic arthroscopy.

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intra-articular blocks and previous radiographic examina-tion, the horse was referred for diagnostic arthroscopy ofboth stifles.

On presentation in October 2006, physical examinationrevealed mild right tibiotarsal effusion, mild right tarsalsheath effusion, and mild bilateral hindlimb fetlock effu-sion. All other physical parameters were within normallimits. The horse was sound at the trot and mildly positiveto hindlimb flexion bilaterally. On the basis of the referralworkup, bilateral diagnostic stifle arthroscopy was per-formed. Both stifles were approached in a similar mannerusing standard arthroscopic approaches [29] to the femo-ropatellar, medial (cranial and caudal compartments), andlateral femorotibial joints. The most marked abnormalfinding was moderate to severe, diffuse fissures of thearticular cartilage on the central portion of the medialfemoral condyle, mild fraying of the cranial aspect of themedial meniscus with associated cartilage cavitations,moderate fraying of the cranial cruciate ligament, andfibrillation of the adjacent axial aspect of the medialfemoral condyle of the left leg (Fig. 2A-C). The right legshowed signs of moderate fissure fractures of the articularcartilage on the central portion of the medial femoralcondyle without evidence of additional ligament ormeniscal damage (Fig. 3). The widely distributed surfacearea of the articular cartilage damage of the medial femoralcondyle precluded the possibility of surgical debridement.All joint compartments were thoroughly lavaged and skinincisions were closed with 2-0 prolene (Ethicon, Somer-ville, NJ) in an interrupted suture pattern. The horserecovered from anesthesia without complications.

Given the guarded prognosis for a successful return toracing, a decision was made to use bone marrow-derivedMSCs in a partially autologous fibrin glue in an attempt tofill the fissures and thus augment the repair process.

2.2. Bone Marrow Aspiration and Stem Cell Expansion

Two days after surgery, bone marrow aspirate wascollected aseptically from the sternum using a 13-gauge,2.5-inch (6.4-cm) bone marrow aspiration needle. Justbefore aspiration, 3 mL of 500 U/mL preservative freeheparin was injected into the sternum and a total volume of35 mL of bone marrow was collected. Aspirate was injectedinto a sterile polypropylene tube and inverted to mix thor-oughly. In a sterile biosafety cabinet, the aspirate wasdivided evenly and diluted 2:1 with Hanks balanced salt

solution. Samples were then centrifuged at 300 � g for 15minutes at room temperature. Supernatants were removedand pellets were combined and mixed thoroughly.Combined pellets were divided and diluted 2:1 in Hanksbalanced salt solution. Samples were then centrifuged1,000 � g for 5 minutes at room temperature. Supernatantswere removed and each pellet was diluted 2:1 and resus-pended in growth media consisting of RPMI 1640 (Invi-trogen, Carlsbad, CA), supplemented with 10% fetal bovineserum and 1% antibioticeantimycotic (A/A). Each samplewas divided evenly between four 75-cm2 cell culture flasksand growth media was added to a final volume of 50 mL perflask. Growth media was then changed every 2 days. Afterapproximately 1 week, numerous large patches of adherent,linear cells with a fibroblastic-like morphology wereapparent. On the basis of several in vitro experiments in ourlaboratory and by others, these cells have been found todisplay characteristics indicative of MSCs including theability to differentiate down the osteogenic and chondro-genic lineages given the appropriate stimuli [30,31]. Afterreaching approximately 80% confluency, cells were sub-cultured. Briefly, growth media was aspirated and disheswere washed using phosphate-buffered saline. Cells werethen collected using 0.05% trypsin and centrifuged at 500 �g for 5 minutes at room temperature. After the supernatantwas removed, the cell pellet was resuspended, cells werethen quantified using a hemocytometer and an invertedlightmicroscope and then replated at 5,000 cells/cm2 on cellculture dishes. Growthmediawas changed every 2 days. Forcryopreservation of MSCs, cells were centrifuged at 500 � gfor 5 minutes and reconstituted in 5% dimethyl sulphoxide(biotechnology performance certified) in growth media andfrozen at a concentration of 2 � 106 cells/mL in liquidnitrogen. For cell resurrection after cryopreservation, cellswere reconstituted in growth media and centrifuged at500 � g for 5 minutes. Supernatant was removed and theremaining cell pellet was reconstituted in growth media.Cells were then plated at 5,000 cells/cm2 and cultured usingthe methods described earlier in the text. For MSC injection,growth media was aspirated and dishes were washed usingphosphate-buffered saline. Cells were then collected using0.05% trypsin, suspended in lactated Ringers solution (LRS),and centrifuged at 500 � g for 5 minutes at room temper-ature. After the supernatant was removed, the cell pelletwas resuspended in LRS, and cells were then quantifiedusing a hemocytometer and an inverted light microscope.

2.3. Thrombin Preparation

Because of the lack of availability of equine thrombin,commercially available bovine thrombin(Calbiochem/EMDBiosciences, Gibbstown, NJ) was reconstituted using 10% (g/mL) calcium chloride solution in nanopure water to a finalconcentration of 0.5 mg/mL and filter sterilized using 0.22-mmvacuum filters. Samples were then aliquoted and storedat �20�C until use.

2.4. Cryoprecipitation of Autologous Fibrinogen

Two days before the jointMSC augmentation procedure,whole blood (approximately 250 mL) was collected in anACD bag and incubated at 4�C for approximately 2.5 hours

Fig. 2. Diagnostic arthroscopic images of the left medial femorotibial joint demonstrating cartilage fissures (arrows) of the femoral condyle (A), fraying of thecranial medial meniscus (white arrow) and abnormal cartilage folding (black arrow) (B), and fibrillation of the cartilage of the axial aspect of the medial femoralcondyle (black arrow). The cranial cruciate ligament appeared mildly abnormal (white arrow) (C). MFC, medial femoral condyle; CM, cranial aspect of medialmeniscus; CCr, cranial cruciate ligament; ICE, intercondylar eminence.

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to allow erythrocytes to separate from plasma [32]. Ina sterile biosafety cabinet, erythrocytes were drained usinga 14-gauge needle, and plasma was collected into poly-propylene conical tubes, then later centrifuged at 1,000 � gfor 7 minutes at room temperature. Supernatants (plasma)were removed, divided into clean polypropylene conicaltubes, and frozen at �80�C for a minimum of 12 hours.Samples were then thawed slowly at 4�C in styrofoam rackand then centrifuged at 450 � g in a swinging bucketcentrifuge at 4�C for 15 minutes. Supernatants wereremoved and the remaining pellet (fibrinogen) was resus-pended in 5 mL of supernatant. Fibrinogen samples werealiquoted and stored at �20�C.

Fig. 3. Diagnostic arthroscopic image of the right medial femorotibial jointdemonstrating cartilage fissures (arrow) similar to those found in the leftmedial femorotibial joint.

2.5. Delivery of MSCs in Fibrin Glue Mixture

Approximately 90 days after the initial arthroscopicexamination and bone marrow aspiration, the patientreturned to the clinic for a procedure using autologousMSCs. Two days before surgery, cell culture supernatantswere submitted to clinical microbiology laboratory foraerobic bacteria and mycoplasma testing. Cell cultureswere confirmed as negative before injection. Arthroscopicexamination of both medial femorotibial joints wasrepeated. After observing and verifying the previouslyidentified cartilage fissures (Fig. 4), several 18-gauge nee-dles were placed into each medial femorotibial joint andinserted into the crevices of the fissures (Fig. 5) in the leftand right femoral condyles. In all, 1 � 107 MSCs/mL werereconstituted in fibrinogen and equal volumes of thrombinand the MSC fibrinogen mixture were drawn into separate1-mL syringes. The syringes were attached to a sterile Ysyringe connector and the MSC/fibrinogen mixture and thethrombin were slowly and evenly dispensed into thecrevices, polymerizing as they combined.

The patient had a prolonged (approximately 1.5 hours)recovery from anesthesia but eventually did recoverwithout incident. To document any potential osseouschanges, bilateral baseline post-treatment stifle radio-graphs were taken. These radiographs revealed similarfindings to the previous radiographs, including slight

flattening of the medial condyle of the stifles. The horsewas transported to the farm 3 days after surgery and wasconfined to a stall for 4 weeks. After the initial 2 weeks,hand walking for 10-15 minutes twice daily was instituted.For the next 4 weeks, the patient was housed in a stall withpaddock and then turned out into a small paddock or corralfor 8 additional weeks.

3. Results

Four months after the initial stem cell treatment,arthroscopic examination of both stifleswas repeated usingthe previously described procedure. Exploration of the leftand right medial femorotibial joints showed persistentfissures of the central articular cartilage of the medialcondyles. However, these fissures were more blunted and

Fig. 4. Arthroscopic images of the left (L) and right (R) medial femorotibial joints using CO2 gas for joint distension demonstrating the surgical access for applicationof mesenchymal stromal cells (MSCs) in partially autologous fibrin glue. The cartilage fissures appeared more pronounced using gas arthroscopy.

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shallow than in previous exploration (Fig. 6). The areaof the suspected cranial cruciate injury appeared to bemoderately improved, as determined by observation,which revealed smoothing of the surface irregularities, andby palpation, where the ligament felt to be more firm.Although intra-articular biopsies would have been helpfulin assessing articular cartilage quality and possible contri-bution made by injected cells, the potential detrimentaleffects to the already compromised articular surface werefelt too great. The joint was thoroughly lavaged and nodebridement was performed. The horse recovered withoutincident.

On the basis of the persistent fissures, a plan was madeto thaw additional MSCs for intra-articular therapy. Fivemonths after initial stem cell treatment (30 days after thelast diagnostic arthroscopy), the patient was presented fora second stem cell injection. On physical examination, thepatient was found to be bright and alert. There was mild

Fig. 5. An arthroscopic image demonstrating application of the mesen-chymal stromal cells (MSCs) in a partially autologous fibrin glue with an 18-guage needle. The needle was replaced several times to obtain the appro-priate plane that would allow the fissures to be filled with the MSCs andfibrin glue.

effusion of the right femorotibial joint. A brief lamenessexamination showed no obvious gait abnormalities. A totalvolume of 10 mL of LRS containing 7.5 � 106/cells/mL wasinjected into each medial femorotibial joint. There were noadverse effects noted after injecting both stifles and noother medications were administered. The patient wasconfined to a stall for 30 days during which he was handwalked for 10 minutes twice daily. After 30 days, he wasmoved to a small paddock with the same walk regimen for30 additional days. The horse was to resume training at 60days postinjection only if there were no signs of lamenessat the trot and he was willing to canter.

In December 2007, the patient returned to racing (Table2). After an unsuccessful second start, MSCs were shippedto the racetrack and the patient was treated with an addi-tional MSC injection (5 mL LRS with 2.0� 106 cells/mL) intothe left and right medial femorotibial joints. The horse hada marked inflammatory reaction resulting in excessivepericapsular swelling and lameness approximately 5 hoursafter the injection. Treatment with nonsteroidal anti-inflammatory medications (phenylbutazone, 2.2 mg/kgintravenous [IV], twice per day [BID] and flunixin meglu-mine, 0.5 mg/kg, IV, BID) and antibiotics (gentamicinsulfate solution, 6.6 mg/kg IV, once per day [SID]) andpenicillin (20,000 IU/kg, intramuscular [IM], BID) wascontinued for 5 days. The swelling and lameness wassubstantially improved by 12 hours and the horse hadcomplete resolution of clinical signs after 3 days. The horsewas given an additional 3 weeks of rest after the resolution

Table 2Postinjury performance records

Date of race Distance (furlongs) Placing Winnings ($)

December 21, 2007 6 1 39,000January 26, 2008 6 10 0April 4, 2008 6.5 2 27,100May 10, 2008 6 3 13,260June 13, 2008 6.5 1 49,080Average 6.20 3.4 25,688.00Total 31 17 128,440.00Starts 5

Fig. 6. Diagnostic arthroscopic examination of the left (A) and right (B) medial femorotibial joints taken 4 months after the application of mesenchymal stromalcells (MSCs) in fibrin glue. The cartilage fissures were still present but appeared blunted and less distinct.

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of clinical signs and then resumed training. He stillcontinues to race to date with three additional starts. Of hisfive postoperative starts, he finished in the top three onfour occasions, winning twice (Table 2). Average raceearnings (earnings per start) were comparable with thosepredating the initial injury.

4. Discussion

This case demonstrates the use of biologics such asautologous MSCs and partially autologous fibrin glue for thetreatmentof bilateral articular cartilagefissures on themedialfemoral condyles of a horse. Autologous chondrocyte trans-plants, as well asMSCs, have been used to successfully repairartificially created or enhanced full-thickness lesions thattraverse the articular cartilage and/or subchondral boneboundary in both human and veterinarymedicine [15,16,26].It is hypothesized that full-thickness cartilage lesions repairmore successfully than partial thickness defects as a result ofgrowth factors and nutrient supply from the blood vessels inthe subchondral bone and thepotential for infiltrationof localMSCs or other chondroprogenitor cells [33]. The potential useofMSCsorprogenitor cells harvested froma site distant to theinjury and implantation directly into partial thickness defectsmay circumvent the lack of subchondral bone contact andmayavoidpotentialdeleteriouseffectsof subchondraldrillingor cartilage harvest. In addition, although full-thicknessdefects heal, the quality, functionality, and integration of thenew tissue is often inferior to that of the original architecture[34]. Interestingly, a recent study compared the efficacy ofa variety of cell types including chondrocytes and MSCs torepair lesions in the femorotibial joint of rabbits [35]. Theydemonstrated that lesions treated with either chondrocytesor MSCs resulted in hyaline-like cartilage production,whereas those treated with MSCs resulted in better cellularorganization and integration into surrounding cartilage.Ultimately, integration of new tissue and restoration of theoriginal architecture are vital to the success of articularcartilage repair and ultimate return to function [36].

The MSC therapy instituted at each stage of manage-ment was based largely on the initial response to treat-ment. The horse seemed to respond favorably to the initial

surgical procedure and postoperative rehabilitationprogram, as was reflected in the blunting and filling of theinitial crevices. The second surgery was performed toevaluate healing and to help predict the appropriate timefor return to training. On the basis of clinical signs, a secondMSC implantation procedure was not planned. However, inretrospect, it would have been more appropriate to havethe horse’s cells thawed, expanded, and prepared fora second augmentation procedure. Although we did notbelieve it was the ideal time or route of administration, thefirst postoperative intra-articular treatment was performedin an attempt to further support cartilage repair as well asto potentially enhance the healing of the cranial cruciateand meniscal injuries diagnosed during the originalprocedure.

The second postoperative intra-articular MSC treatmentwas timed after the horse performed poorly in his secondevent after returning to race. Although other intra-articulartreatment modalities could have been instituted, a decisionwas made to use the horse’s MSCs because of the previouspositive treatment response. The reaction associated withthis treatment cannot be completely explained. However,because the cells were negative for bacterial culture,appropriate aseptic technique was used for intra-articularinjection and the total cell number was lower than previ-ously used, it was assumed to be associated with cell deathduring transportation of the cells over a 36-hour period. Ifthis was the cause, this complicationmight be prevented byusing fresh cells and/or reducing the time between pro-cessing and injection. Another potential cause for a reactioncould be foreign proteins associated with cell cultureand expansion. Although extreme care is taken to washcells thoroughly, there could be a potential for residualbound proteins. Further research will be necessary todetermine the appropriate mechanisms to ensure cellviability during transportation as well as optimum cultureand processing techniques for cellular therapy. Practi-tioners should use cautionwhen administering MSCs intra-articularly until the events underlying the flairs arecompletely elucidated. The administration of systemicnonsteroidal anti-inflammatory drugs (NSAIDS) such asflunixin meglumine to patients before stem cell injection

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has been suggested to dampen similar inflammatoryresponses. If such preloading is considered, practitionersmust weigh the potential negative effects on injected stemcells. There is mounting evidence in the previously pub-lished data to suggest that there may be negative effects ofNSAIDS on the proliferation and differentiation of MSCs[37,38].

Although intra-articular therapy with MSCs is consid-ered controversial, its use can be supported by stem cellresearch performed in a caprine model of osteoarthritis[39]. It was demonstrated that joints treated with bonemarrow-derived MSCs had evidence of marked regenera-tion in an experimentally created injury that involvedresection of the anterior cruciate ligament and completeremoval of the medial meniscus. In addition, MSC-treatedjoints demonstrated reduced degeneration of articularcartilage, osteophytic remodeling, and subchondral bonesclerosis as compared with control joints. It was concludedthat local delivery of adult MSCs stimulated regeneration ofmeniscal tissue and helped reduce the progression of OAthat was seen in control joints.

Although it is difficult to draw a direct relationshipbetween the successful recovery of this horse and thetreatment outlined in this report, on the basis of previousstudies [6,12], it is likely that the prognosis for a completerecovery of this patient, given the nature of the articularcartilage lesions, was guarded at best. It must be noted thatbecause of the recovery time, other possibilities exist,including healing by natural methods alone, which couldexplain the outcome in this patient. However, it does seemto be promising that a horse with this degree of damage tothe cartilage surface could recover sufficiently to becompetitive at a similar level than before injury. Althoughcellular and other biological therapies do not seem to bedetrimental and may provide therapeutic benefit in thetreatment of a unique joint injury of the equine stifle, thereis a need for further research into the efficacy of suchtechniques. More specifically, it is necessary to determinethe most appropriate dosages, substrates, and timing inwhich to administer such therapies.

Acknowledgments

The authors thank Jonathan Hirsch, Bill Symm, LisaFortier. Location of work: The initial lameness examinationand associated diagnostics (nuclear scintigraphy, and stifleradiographs) were performed at the Southern CaliforniaEquine Foundation Hospital at the Santa Anita Racetrack,Arcadia, CA, whereas follow-up lameness examination,(CBC and chemistry panel screening, equine protozoalmyeloencephalitis testing, and diagnostic intra-articularanesthesia) were performed at the Harris Farms HorseDivision facility in Coalinga, CA. Preoperative examinations,arthroscopy, initial postoperative care, bone marrowcollections, and stem cell injections were performed at theUC Davis VeterinaryMedical Teaching Hospital in Davis, CA.Stromal cell isolation and expansion, as well as autologousfibrinogen collection, were performed at the Department ofAnatomy, Physiology, and Cell Biology in the UC DavisSchool of Veterinary Medicine.

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