Treatment of Bilateral MeFissures in a Horse UsingMesenchymal Stromal Ce
Leah F. Raheja BS a, Larry D. GaluJoseph P. Dowd DVMd, Fern TabaDepartment of Anatomy, Physiology and Cell Biology, SbDepartment of Surgical and Radiological Sciences, SchcHarris Farms Horse Division, Coalinga, CAd Equine Medical and Surgical Group, Arcadia, CA
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
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 to
Journal of Equine Veterinary ScCorresponding author at: Larry D. Galuppo, DVM, Department ofcartilage 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 ssures 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.Keywords:StieCartilageStem cellOCDRepairSurgical and Radiological Sciences, School of Veterinaryof California, Davis, 2112 Tupper Hall One Shields Ave, D
E-mail address: firstname.lastname@example.org (L.D. Galu
0737-0806/$ - see front matter 2011 Elsevier Inc. Adoi:10.1016/j.jevs.2010.12.009dial Femoral Condyle Articular CartilageBone Marrow-Derived Multipotentlls
ppo DVMb, Jeanne Bowers-Lepore DVMc,lin DVM, PhD a, Clare E. Yellowley PhD a
chool of Veterinary Medicine, University of California, Davis, CAool of Veterinary Medicine, University of California, Davis, CA
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 bilateralstie athroscopy was found to have bilateral articular cartilage ssure defects of themedial femoral condyles with concurrent cranial cruciate ligament injury. Bone marrowderived MSCs were isolated, expanded, and suspended in a partially autologous bringlue. The initial cell/brin glue mixture was delivered arthroscopically into the articularCase StudyJournal of Equine
journal homepMedicine, Universityavis, CA 95616.ppo).
ll rights reserved.eterinary Science
ience 31 (2011) 147-154patient age and conformation [3,4]. Current treatments forinjuries related to articular cartilage in horses are ever
different between individuals receiving MSC-laden brin
2.1. Clinical Findings
A 4-year-old Thoroughbred gelding was presented forbilateral stie 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 stieradiographic examination were inconclusive. However,there was slight attening 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 stie problem wassuspected. Screening for equine protozoal myeloencephalitis
e Veteevolving. A diagnosis is generally conrmed througharthroscopic examination and treatments include partialmeniscectomy, curettage or drilling into the subchondralbone, cartilage ap/fragment xation, 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 . 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 . 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 . 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 efcacy 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 ap. The efcacy of such therapy, asmeasured by a combination of function and pain , hasbeen reported to be >80% in human beings , withcomparable results in horses using a similar technique forthe repair of manufactured full-thickness defects in minorload-bearing areas of the tibiotarsal joint . However,induction of defects to harvest chondrocytes is a concernwith this type of cell therapy and comparable clinicalresults have been obtained with microfracture . 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 inammatory 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 . With therelative abundance and accessibility of MSCs for clinicalharvest, many clinicians and researchers have begun look-ing at the use of MSCs for cartilage , tendon [23,24],
L.F. Raheja et al. / Journal of Equin148ligament [23,24], and bone repair . Recently, there wasa report of the use of autologous MSCs in a brin glueenhancing the early repair of manufactured full-thicknessarticular cartilage/subchondral bone defect in the equineglue or control (MSC-free brin glue), the study did notcompare an untreated control, thus it is difcult to deter-mine whether the brin glue alone may have played a rolein healing. There is evidence in the published data thatbrin may serve as a scaffold for tissue repair, includingthat of cartilage, thereby contributing to the healingprocess [27,28]. The apparent benets 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 efcacy ofsuch treatment in naturally occurring supercial 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 brin glue to treatbilateral diffuse ssure fractures of the medial femoralcondyles of a horse.
2. Materials and Methodsfemoropatellar joint . Although the report noted thatlong-term healing at 8 months was not signicantly
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
rinary Science 31 (2011) 147-154(indirect uorescent 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
e Veteintra-articular blocks and previous radiographic examina-tion, the horse was referred for diagnostic arthroscopy ofboth sties.
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 exion bilaterally. On the basis of the referralworkup, bilateral diagnostic stie arthroscopy was per-formed. Both sties were approached in a similar mannerusing standard arthroscopic approaches  to the femo-ropatellar, medial (cranial and caudal compartments), andlateral femorotibial joints. The most marked abnormalnding was moderate to severe, diffuse ssures 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, andbrillation of the adjacent axial aspect of the medialfemoral condyle of the left leg (Fig. 2A-C). The right legshowed signs of moderate ssure 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-
Fig. 1. Initial cranial to caudal radiographic projections of the left and rightsties demonstrating mild attening of the medial femoral condyles bilat-erally. The radiographic examination was performed 4 months beforediagnostic arthroscopy.L.F. Raheja et al. / Journal of Equinville, 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 brin glue in an attempt toll the ssures 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 saltsolution. 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 asksand growth media was added to a nal volume of 50 mL perask. Growth media was then changed every 2 days. Afterapproximately 1 week, numerous large patches of adherent,linear cells with a broblastic-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% conuency, cells were sub-cultured. Briey, 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 quantied 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 certied) 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. Afte...