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New Horizons in Articular Repair

Alan J. Nixon, BVSc, MS, Diplomate ACVS, and Lisa A. Fortier, DVM, PhD, Diplomate ACVS

1. Introduction (full or partial thickness) is a critical determinant in Articular cartilage rarely reforms a functional hya- healing, the size of the defect, its location in relation line surface after injury. Most simple cartilage lac- to weight-bearing or non-weight-bearing areas, and erative injuries reach a benign nonhealing phase, the age of the animal influence the repair rate and which remains unchanged over time.1,2 Deeper resiliency of new cartilage surfaces. Convery et al. cartilage lesions, which violate the tide-mark and showed that lesions in the equine femorotibial joint extend into the subchondral plate, result in an that were less than 3 mm in diameter healed with improved healing response.3 This is largely be- little residual deformity.6 More recently, Hurtig et cause of the proliferation of undifferentiated mesen- al. determined that lesions larger than 15 mm2 in chymal cells from the deeper tissues. In horses, the surface area tend to show reasonably good repair at progression in cartilage defects from granulation to 5 months but degenerate with increasing time.7 fibrous tissue and, finally, fibrocartilage is slower Given these and other studies, repair of full-thick- than it is in rodents; the healing of defects in dogs ness articular cartilage defects in the horse may not 4 tends to be somewhere between these extremes. be as satisfactory or as complete as that documented The fibrous tissue undergoes progressive chondrifi- in smaller animals. Metaplasia of fibrous tissue to cation to form a fibrocartilaginous mass that is fibrocartilage is not always evident and, depending loosely attached to the original cartilage edges. on the time of examination, degeneration to fibrous The subchondral bony plate occasionally reforms to tissue and later mechanical erosion of the repair the same approximate level as the adjacent undam- tissue can occur. Repair tissue is biomechanically aged bone. Immediately above the reformed sub- inferior to normal articular cartilage, even though chondral plate, areas of cartilage proliferation the histological appearance is often fibrocartilage or predominate. The deeper cartilage layers and sur- 8 face fibrous tissue generally follow a pattern of de- even hyaline-like tissue. Repair tissue generally creasing cellularity as the defect matures. The has significantly less and, to some ex- phenomenon of matrix flow, an intrinsic repair tent, type II than does normal cartilage. mechanism, may also contribute to the healing of Additionally, the development of subchondral archi- equine articular cartilage defects by forming over- tecture and re-establishment of a tide-mark is often hanging lips of cartilage on the perimeter of the irregular and inconsistent. This creates suscepti- lesion that tend to migrate in a centripetal manner.5 bility to cartilage deterioration with normal joint In small defects, this can result in significant reduc- activity. Poor-quality, relatively short-lived repair tion in lesion size. In larger defects, matrix flow cartilage has led to the development of pharmaco- plays an insignificant role, compared with mesen- logic and surgical methods to improve the repair chymal cell proliferation. Although depth of injury process.

NOTES

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Proceedings of the Annual Convention of the AAEP 2001 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: CURRENT CONCEPTS IN EQUINE OSTEOARTHRITIS down to actively bleeding bone, which provides a source of pluripotential cells. The subsequent fi- brous or fibrocartilaginous response is determined primarily by lesion location and size, degree of weight-bearing, and age of the animal. The carti- lage resection perimeter should be vertical rather than beveled because this results in better attach- ment of tissue regrowth to original cartilage. Forage Drilling the subchondral bone in addition to debrid- ing the overlying cartilage opens up wider channels into the subchondral marrow spaces. Vachon et al. have shown that repair tissue is superior following Fig. 1. Histologic appearance of articular cartilage from imma- subchondral drilling after cartilage debridement of 10 ture (6-month-old, left) and mature (3-year-old, right) horses the third carpal bone. Forage is still occasionally shows the markedly reduced cell density, lack of vascular access performed following debridement of subchondral to the attached subchondral bone, and well-established tide mark cystic lesions, to perforate the dense sclerotic perim- of adult cartilage. Cartilage healing is enhanced in young eter that surrounds most mature lesions. How- horses through these and other features. ever, none of the drilling experiments performed in horses resulted in the reported to have been seen in rabbit drilling experiments. Cyst expansion following forage of the cyst perime- 2. Cartilage Repair ter has also been described, resulting in continued 11 Generally, the healing of chondral and osteochon- lameness. Moreover, micropicking has largely dral defects is more complete in young animals replaced drilling techniques. because of the increased mitotic capacity of chondro- Microfracture/Micropick cytes, more active matrix synthesis, and closer prox- imity to the vascular supply in the depths of the Perforation of the subchondral bone after full-thick- articular–epiphyseal complex (Fig. 1).9 Examples ness cartilage debridement can more precisely be of improved repair capacity are easily seen in the accomplished with micropick awls (Fig. 2). The ta- resurfacing potential following OCD flap removal pered awl can be driven 2–3 mm into the subchon- when compared with the debridement of articular dral plate to open a channel for vascular ingress, erosions in adults. Facilitation of cartilage repair pluripotent delivery, and associated fluxes, all of which coalesce in the falls into one of two categories: repair by stimula- 12 tion of pluripotential cells from the subarticular so-called “super-clot.” The simplicity of the tech- level or by transplantation of cartilage, osteochon- nique is appealing but experimental evidence of dral grafts, or from remote regions. superior healing in horses is limited to an in- crease in tissue volume in full-thickness defects and Local Cartilage Repair minor improvement in type II collagen content.13 Cartilage Debridement Methods to stimulate cartilage repair from mesen- chymal cells in the subchondral marrow spaces rely on full-thickness cartilage debridement to open a communication to the subchondral region. These methods occasionally are supplemented by subchon- dral bone drilling (forage) or spongialization (sau- cerization). Partial-thickness cartilage resection reduces the dissipation of cartilage breakdown prod- ucts into the synovial environment, thus reducing irritation to the synovial membrane and subsequent production of . In general, partial-thick- ness chondrectomy, which is usually performed with mechanical abraders, is a smoothing procedure. It does not attenuate or terminate continued fibril- lation. Full-thickness cartilage debridement is the procedure of choice for deeply fibrillated cartilage with areas of exposed subchondral bone. Cartilage Fig. 2. Micropick procedure being used after removal of a basal debridement with hand tools or mechanical debrid- fracture of the sesamoid. The residual fibrillated cartilage sur- ers allows removal of the dense subchondral plate face (left) has been debrided and is being micropicked (right).

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Proceedings of the Annual Convention of the AAEP 2001 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: CURRENT CONCEPTS IN EQUINE OSTEOARTHRITIS However, in many situations this may prolong ac- simultaneous multiple anchoring is being explored. tive exercise. Resolution of joint effusion and radiographically ob- vious subchondral lysis, as well as reformation of the Abrasion Chondroplasty subchondral contour, are better than that which In human knee arthroplasty, performing uniform follows cartilage flap removal. debridement with a burr, to remove a layer of dense sclerotic eburnated bone following full thickness car- Transplantation Resurfacing: Tissues tilage loss, has been shown to cause tufts of fibro- Different tissues and methods have been used ex- cartilage to occur at sites of vessel protrusion perimentally for cartilage resurfacing. Five types through the subchondral plate.14 This technique of donor tissue have been investigated: periosteal remains poorly developed in equine arthroscopic and perichondrial autografts, osteochondral au- surgery. This is compounded by the fact that most tografts or allografts, chondral autografts, isolated end-stage joints are poor candidates for surgical autografts or allografts, and stromal therapy because the aim usually is a return to some (mesenchymal) stem cell autotransplants. actively functional state rather than simple attenu- ation of joint pain. Abrasion chondroplasty has, Periosteum/Perichondrium however, been used quite successfully in improving Several investigators have examined periosteal and the status of eburnated regions in the trochlear perichondrial grafts for cartilage resurfacing in ridges of the hock and may have potential in ebur- horses. Although O’Driscoll et al and Rubak both nated areas of other rotatory action joints. found hyaline cartilage production following perios- teal transfer to cartilage defects in experimental Cartilage Flap Reattachment small animals, these results have not been dupli- Under defined conditions where an OCD cartilage cated in studies of horses by Vachon et al.15–17 flap has not detached along its entire perimeter, local debridement of the necrotic cartilage and mar- Osteochondral Grafts row fibrosis can be accomplished arthroscopically Osteochondral grafting using autogenous sternal or and the partially attached flap can be replaced and carpal donor fragments has been generally unsuc- secured with PDS pins (OrthoSorb) or PLLA tacks cessful.18 Although cartilage incorporation has (chondral darts, Arthrex). This has worked satis- been satisfactory, the attached subchondral bone factorily on large flaps in the fetlock, hock, and stifle has not incorporated well.19 Mosaicplasty and sim- (Fig. 3). Criteria for use include a large flap only ilar osteochondral dowel systems are technically dif- partially detached with a relatively normal-appear- ficult to harvest and transplant arthroscopically; ing surface structure. The fibrosis intervening be- results in the carpus and fetlock of horses suggest tween cartilage and bony defect must be removed that the cartilage is susceptible to degeneration.20 if the procedure is to work. Several diverging A suitable donor site is also a significant issue. pins are placed with the kit provided. Use of the multishot chondral dart system (Arthrex) for Transplantation Resurfacing: Isolated Cells Chondrocyte Transplantation Chondrocyte grafting of articular defects has re- cently entered clinical trials in man.21 However, the experimental study of chondrocyte transplantation has expanded over the past 30 years. The potential benefits of transplanting chondrocytes without the surrounding cartilage matrix include the transfer of a pool of metabolically active cells that will fill in- congruities in the articular surface without the problems of articular and subchondral fit or the problems of secure attachment of solid tissue trans- plants such as periosteum, cartilage, or osteochon- dral grafts. Difficulties in achieving incorporation of osteochondral grafts, as well as failures in the horse of many other solid tissue transfers, has en- couraged the study of free-cell transplants. Chondrocyte transfer to full-thickness cartilage defects has been extensively studied in experimental animals. Many studies simply implanted chondro- cytes of articular or physeal origin into cartilage 22–26 Fig. 3. Stifle OCD flap lesion after placing PDS pins along the defects. Although the results were often posi- length of the lateral trochlear ridge flap (arrowheads). An addi- tive, the articular defects and the experimental an- tional OCD of the patella (arrow) was debrided. imals used were small. Nevertheless, the use of

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Proceedings of the Annual Convention of the AAEP 2001 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: CURRENT CONCEPTS IN EQUINE OSTEOARTHRITIS allograft chondrocytes was established and, despite brin cultures in vitro indicate that fibrin is a tena- the known immune response of host tissues to these cious that supports chondrocyte phenotypic chondrocytes,27–31 the rapid proliferation of matrix expression and matrix elaboration.44 Autogenous led to an apparent “sequestration” of the trans- fibrinogen also has anabolic effects on chondrocyte planted chondrocytes, which provided a barrier to function that are not evident in commercially pre- 32–34 immune recognition. Assessment of free allo- pared fibrinogen (Fig. 4).45 graft or autograft chondrocytes in larger, more clin- The ability to inject chondrocyte-laden fibrinogen ically relevant lesions has not been done. Given and activated thrombin through a needle provides the larger size of the defects and the complete lack of site-specific deposition of self-adhering graft mate- adherent properties of the grafted cells, there would rial that can be placed arthroscopically. Research be little reason to expect the chondrocytes to remain studies of the equine femoropatellar joint indicated in the target defect for more than a few minutes that fibrin–chondrocyte grafts improved the mor- during postoperative recovery. Several studies phologic and biochemical properties of healing tis- suggest that isolated cultured chondrocytes may sue in 12-mm, full-thickness cartilage defects.46 have a role in the reconstruction of physeal injuries Classic markers of hyaline cartilage such as collagen where the grafts can be secured by overlying soft 35,36 type II were significantly elevated in chondrocyte- tissues. grafted stifles (61% compared with 25% in control Transplanting chondrocytes in a vehicle or adher- ungrafted lesions at 8 months). The mechanics of ent matrix composite provides better assurance that stifle arthroscopy and chondrocyte–fibrin grafting the transplanted cells will remain in position for have been adapted to routine surgical procedures. periods long enough to synthesize a new pericellular Currently, autogenous fibrinogen is harvested the matrix and to establish a bond to the subchondral day before surgery, 30 ϫ 106 allograft chondrocytes bone. Additionally, the new matrix assumes the are thawed and cultured for a minimum of 24 hours, immune isolating effect initially provided by the and both are mixed with calcium-activated bovine transplant vehicle. Various biologic and synthetic thrombin as they enter a needle, which is inserted materials have been described for this purpose. percutaneously so that the tip is placed in the car- Biologically derived materials have been studied tilage defect. The only modification to routine ar- most extensively. Cultures of allograft and au- throscopy is temporary insufflation of the joint with tograft chondrocytes transplanted in fibrin, collagen helium or carbon dioxide to allow fibrin polymeriza- , and hyaluronate products have been shown to tion. Clearly, access to a lab capable of harvesting stimulate hyaline cartilage repair in various exper- 37–43 and storing the cells is a prerequisite for chondro- imental models. The use of fibrin-based vehi- 47 cles is appealing because of the ready source of cyte-based resurfacing programs. However, such autogenous fibrinogen from plasma, its application facilities are becoming more frequently associated as a self-polymerizing liquid, and the inherent with referral institutions and this trend will proba- “glue”-like properties of thrombin-activated fibrino- bly continue. gen. Experimental studies evaluating the survival Long-term studies of equine clinical cases follow- and metabolic activity of equine chondrocytes in fi- ing chondrocyte grafting are not available. Initial results in horses with subchondral cysts and related defects of the femoral or metacarpal condyles indi- cates a more rapid resolution of lameness and effu- sion and an accelerated bony fill of the defects on follow-up radiographs. Articular defects that re- main after lag-screw repair of fractures of the meta- carpal condyles (2 horses) and third carpal bone (4 horses) have also been grafted with chondrocyte– fibrin transplants inserted following arthroscopic debridement of the cartilage edges. Although these grafts have had no apparent impact on bony union of the fracture, clinical symptoms have resolved and the third carpal fracture cases have re-entered training. Although these procedures show prom- ise, the techniques involved are still experimental and combining chondrocytes with growth factors seems to be more appropriate.48 Other matrix vehicles have been evaluated for use in the horse. Although maintaining arthroscopi- Fig. 4. Collagen type II gene expression is enhanced in fibrin cally applied techniques is an important criterion for vehicles derived from autogenous fibrinogen from plasma (left) application in the horse, other products and meth- compared with equine fibrinogen purchased from commercial ods to secure the grafts in extensive cartilage defects vendors (right). have also been developed. Collagen mesh and lat-

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Proceedings of the Annual Convention of the AAEP 2001 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: CURRENT CONCEPTS IN EQUINE OSTEOARTHRITIS tice materials containing cultured chondrocytes Chondrocyte-based studies and clinical applica- meet these surgical criteria and also provide some tion in horses utilize allograft chondrocytes har- strength to the implant by virtue of the collagen vested from foals and weanlings. Although the network. Chondrocytes readily populate these metabolic and mitogenic capacities of fetal chondro- absorbable scaffolds and actively synthesize a new cytes are somewhat better than those of weanlings, cartilage matrix (Fig. 5).49 However, long-term ex- they do not maintain this advantage after being perimental trials in horses with 15-mm, full-thick- cryopreserved for several months.b Additionally, ness defects in the stifle have shown only moderate viability declines to unsatisfactory levels (Ͻ50%) af- improvement in cartilage healing.50,51 The concept ter 3 months at Ϫ196°C.47 Chondrocytes derived of chondrocytes in a pre-existing collagen meshwork from mature horses survive cryopreservation for ex- forming “artificial cartilage” has not met with the tended periods but their metabolic capacity is dimin- success seen in small-animal research models.40,41,52 ished. As a result, the ideal donor age is considered Hyaluronate vehicles for chondrocyte transfer to be 4–6 months.47 Preparing chondrocytes in have special appeal because of their similarity to the monolayer for several days prior to transplantation hyaluronate-rich cartilage matrix, the apparent has also been shown to provide a metabolic stimu- beneficial effects of hyaluronate-containing synovial lus, which allows partially cryodamaged cells to re- fluid on cartilage metabolism, and previous research cover or disintegrate and be discarded at the initial showing upregulation of chondrocyte metabolism media exchange.44 under the influence of exogenous hyaluronate.53–55 The advantages of autogenous chondrocyte trans- Studies of chondrocyte–hyaluronate grafts in small plantation are tempered by a lack of a suitable donor defects were quite positive.42,43 However, hyaluro- joint, the cost of preliminary surgery for cartilage nate gels that dissolve rapidly present a handling harvest, and the time delay between harvest and problem for implantation and have no adherent ca- reimplantation. Although these factors may be pabilities in cartilage defects.a satisfactory in man,21 they are generally unsatisfac- Synthetic matrix have the advantage of tory for treating acute injury in horses. Moreover, being biologically inert. They also hydrolyze at a the minimum number of chondrocytes required for specific rate, which can be altered in the production substantive contribution to cartilage repair is 10 phase to tailor to specific grafting applications. million cells per ml of injected fibrin.44,62 Average Several studies have defined useful polymer con- yields of chondrocytes from cartilage derived from structs for chondrocyte seeding and subsequent ar- 3–6-year-old horses are only 12–15 million cells per ticular implantation.56–61 The results of in-vivo gm of tissue.47 To obtain 3 gm of cartilage for re- studies are satisfactory but not superior to other surfacing purposes would denude an area approxi- biological methods. Integrating tissue-engineered mately one-third to one-half the size of the composites to surrounding normal cartilage has femoropatellar joint in a mature horse. Clearly, been unsatisfactory. The real advantage of using other sites for chondrocyte harvest are required; polymers as vehicles for transplantation may be in studies of chondrocytes derived from sternal carti- a dual role: as carriers for growth factors and to lage provide a possible solution. The intuitive ad- slowly facilitate the metabolism of transplanted vantages of autogenous chondrocyte transplants chondrocytes. have not been experimentally ratified. The most recent study compared articular healing at 26 and 52 weeks after autogenous or allogenous chondro- cyte grafting and found no difference in the success rates of each.63 More attention is now being fo- cused on the use of undifferentiated mesenchymal cells as transplant donor cells. Mesenchymal Cell Transplantation Harnessing the pluripotent undifferentiated stem cell, or mesenchymal stem cell as it has become known, to bolster the local pool of stem cells in an articular defect has distinct advantages. Mesen- chymal stem cells are a ready source of autogenous graft cells and there are several regions of the body where stem cells can be harvested for in vitro proliferation and differentiation prior to reimplan- tation in articular defects. An obvious example is the use of the cambial layers of the periosteum.64,65 Fig. 5. Tissue engineered collagen matrix, which has been However, other pools of stem cells, such as bone 66 seeded with chondrocytes and cultured for 21 days, shows active marrow, are more readily available and it is these cartilage matrix deposition between the collagen network of the pools that show considerable promise in cartilage composite, which is ready for transfer to the joint. resurfacing.67 Under appropriate conditions, such

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Proceedings of the Annual Convention of the AAEP 2001 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: CURRENT CONCEPTS IN EQUINE OSTEOARTHRITIS as lowered oxygen tension, high cell density, dexa- of the experiment and that the levels were similar to methasone exposure, and fetal calf serum–enriched the levels evident in parallel chondrocyte cultures. media, mesenchymal stem cells can be directed to The effect of various growth factors on the differen- differentiate toward chondrocytes.64,66,68 The de- tiation and synthetic activity of these cells is also of velopment of chondrocytes rather than osteoblasts is significance and may eventually be used in conjunc- largely dependent on vascular access and the result- tion with mesenchymal cell grafts for cartilage re- ing oxygen tension.66 For full-thickness articular surfacing (Fig. 6).71,72 Use of gene-enhanced MSC defects, components of both tissue types are re- chondrogenesis to derive chondrocytes for trans- quired for reconstructing the subchondral plate and plant to cartilage surfaces offers real potential for the overlying hyaline cartilage surface. Transplan- autogenous chondrocyte grafting in horses.73 tation of mesenchymal cells into collagen gels has been used to successfully resurface 3 ϫ 6 mm defects Growth-Factor–Enhanced Cartilage Repair in the rabbit femoral condyle.67 Hyaline cartilage Several naturally occurring polypeptide growth fac- and reformed subchondral tissues were apparent at tors play an important role in cartilage homeosta- 6 months. Mechanical testing of these new sur- sis.74 The differentiating and matrix anabolic faces indicated that they were softer than normal activity of insulin-like growth factor-I (IGF-I) and cartilage but quite improved over ungrafted control transforming growth factor-␤ (TGF-␤) are particu- defects. Longer-term studies are needed because larly important in counteracting the degradatory mechanical test data are critical determinants of the and catabolic activities of cytokines, serine pro- likely durability of such tissue. teases, and neutral metalloproteases. The manip- In horses, the sternum provides a better source of ulation of this balance in disease conditions such as mesenchymal cells than does the tuber coxae. arthritis and acute cartilage injury may be possible Bone marrow can be harvested standing and is rich by exogenous administration of IGF-I and TGF- in undifferentiated stem cells.69 In-vitro culture ␤.75–80 The effects of these and other growth fac- results indicate a similar progression of differentia- tors have been studied extensively in culture tion in high-density monolayer cultures. Markers systems of many types. Most data have been gen- of cartilage phenotype such as proteoglycan and erated from chondrocyte monolayer and cartilage type II collagen were evident by day 7 of culture. explant cultures, where IGF-I and TGF-␤ generally The behavior of mesenchymal stem cells in three- result in elevated matrix molecule elaboration, con- dimensional culture is of greater importance be- current with minor to moderate mitogenic ef- cause this mimics the cartilage environment more fects.81–86 Similar results were evident in closely.70 Mesenchymal cells assumed a rounded monolayer cultures of equine chondrocytes, where appearance with active proteoglycan production in a dose-dependent stimulation of proteoglycan produc- parallel study of chondrocytes and mesenchymal tion occurred in serum-free and serum-supple- cells in fibrin three-dimensional cultures. The mented cultures.87 Three-dimensional culture comparative and temporal aspects of the study indi- assessment of the effect of these same growth factors cated that the mesenchymal cells continued to syn- on equine chondrocyte metabolism have also been thesize cartilage matrix components over the course performed with fibrin gels, which provide a stable suspension culture resembling the cartilage matrix environment.88,89 The dose-response to growth fac- tors was largely influenced by the presence of fetal calf serum in the media. When cultured without serum supplements, these two growth factors stim- ulated matrix component elaboration in a dose-de- pendent manner; the most profound effects occurred at the highest concentrations of IGF-I and TGF-␤. Serum-free cultures are not representative of the in-vivo environment experienced by cartilage, par- ticularly in immature horses; therefore, inclusion of serum or defined media-derived supplements are important components of culture experiments. Enhanced proteoglycan and collagen synthesis, as well as stimulated cellular replication, were evident only at lower dose rates of IGF-I (10 ng/ml) and TGF-␤ (1 ng/ml) in serum-supplemented cultures. Increased concentrations of IGF-I (50 and 100 ng/ ml) resulted in minimal further improvements. ␤ Fig. 6. Equine bone marrow–derived mesenchymal stem cells in Increased levels of TGF- (5 and 10 ng/ml) eventu- culture 2 days after addition of transforming growth factor-␤1(5 ally suppressed matrix synthesis and cell divi- 88,89 ng/ml). Assay for collagen type II showed enhanced chondrogen- sion. Other studies in the horse largely focused esis. on IGF-I because it was not detrimental to chondro-

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Proceedings of the Annual Convention of the AAEP 2001 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: CURRENT CONCEPTS IN EQUINE OSTEOARTHRITIS cyte metabolism when present in excess concentra- tions.90 Several cartilage explant studies that used both normal and interleukin-1–depleted cartilage also revealed that IGF-I had a positive effect on equine cartilage homeostasis.91 Given these re- sults, IGF-I was selected as a suitable growth factor for in-vivo studies in the horse. Other investiga- tors evaluated articular repair following TGF-␤ administration.2,92 However, synovitis and osteo- phyte development have been alarming features of TGF-␤ use in these animal studies.93,94 Slow-release delivery of IGF-I within a cartilage defect, to facilitate matrix production in local and transplanted chondrocytes, provides a mechanism Fig. 8. Effect of bone morphogenetic protein-7 (BMP-7) gene- for enhanced cartilage repair. Elution studies us- enhanced chondrocyte function 4 weeks after implantation to the ing IGF-I–laden equine fibrin indicate that maxi- stifle of horses. A null gene (AdCD) was used to transduce mally stimulatory levels of IGF-I (Ͼ50 ng/ml) chondrocytes used as controls. remain for a minimum of 3 weeks following an ini- tial loading dose of 20 ␮g (Fig. 7).95 Although the dissolution rate of fibrin in the synovial environ- combination with chondrocyte or mesenchymal stem ment is not known, it is not expected to vary consid- cell grafts, where more complete cartilage repair erably from the buffered polyionic saline used in the develops.48 Healing evaluation at 8 months, fol- in-vitro elution studies. In-vivo evaluation of a lowing implantation of a mixture of chondrocytes self-polymerizing fibrin vehicle that is devoid of cells and 25 ␮g IGF-I in stifle defects of 8 horses, showed but is loaded with 25 ␮g IGF-I and injected into a considerably improved joint surface. There was cartilage lesions in the femoropatellar joints showed 58% type II collagen and better neocartilage integra- improved cell population with more cartilage-like tion at the defect edges. Studies of IGF-I and architecture after 6 months.96 However, markers BMP-7 gene-enhanced chondrocyte function in sim- of hyaline cartilage such as type II collagen in- ilar transplant models suggest that both may stim- creased to 47%, far short of the 90% minimum evi- ulate healing beyond that seen in unstimulated dent in normal articular cartilage. Nevertheless, chondrocyte-grafted cases (Fig. 8).98–100 simple fibrin vehicle grafts used in control stifles did Clinical resurfacing trials in horses have used a not significantly enhance healing. There was a regimen of autogenous fibrin laden with 50 ␮g IGF-I mean collagen type II content of 39%, which is sim- and 30 million chondrocytes per milliliter of fibrin. ilar to healing in empty full-thickness defects.46 The chondrocytes were mixed with fibrinogen and Other studies using injected combinations of IGF-I IGF-I with activated thrombin to provide a 2-com- and pentosan polysulfate showed attenuation of the ponent system for immediate injection (Fig. 9). symptoms of synovitis in OA models in sheep.97 The polymerization process developed immediately In general, IGF-I seems to have better application in upon injection into the articular defect. Currently,

Fig. 9. Chondrocyte–IGF-I grafting of subchondral cyst of the femoral condyle. Debridement is followed by cancellous bone Fig. 7. Elution profile of IGF-I over time from polymerized fibrin grafting of the deeper portion of the cyst and injection of the after loading IGF-I (25 ␮g) to each 1 ml aliquot and assaying thrombin-activated mixture of fibrinogen, chondrocytes, and IGF-I in medium above the fibrin composite. IGF-I.

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Proceedings of the Annual Convention of the AAEP 2001 Reprinted in the IVIS website with the permission of AAEP Close window to return to IVIS IN DEPTH: CURRENT CONCEPTS IN EQUINE OSTEOARTHRITIS the predominant application has been OCD and sub- 14. Johnson LL. Characteristics of the immediate postarthro- chondral cystic defects of the fetlock (8 horses) and scopic blood clot formation in the knee joint. Arthroscopy 1991;7:14–23. stifle (43 horses), although several shoulder and car- 15. O’Driscoll SW, Keeley FW, Salter RB. The chondrogenic pal articular lesions have been treated. The fibrin potential of free autogenous periosteal grafts for biological polymer and growth factor have been well tolerated resurfacing of major full-thickness defects in joint surfaces and have effected resolution of effusion and lame- under the influence of continuous passive motion. J Bone ness. Assessment of stifle OC cyst cases 1–5 years Joint Surg 1986;68-A:1017–1035. 16. Rubak JM. Reconstruction of articular cartilage defects after implant indicates that the horses respond by with free periosteal grafts. An experimental study. Acta increased bone deposition in the subchondral plate Ortho Scand 1982;53:175–180. and then generally throughout the cyst over the 17. Vachon AM, McIlwraith CW, Trotter GW, et al. Morpho- ensuing first year. Soundness has taken as long as logic study of induced osteochondral defects of the distal 1 year to develop, but 22 of 30 horses beyond the first portion of the radial carpal bone in horses by use of glued periosteal autografts. Am J Vet Res 1991;52:317–327. post-operative year have remained in active work 18. Howard RD, McIlwraith CW, Trotter GW, et al. Long-term without lameness. Six of 8 horses with grafted fet- fate and effects of exercise on sternal cartilage autografts lock subchondral cysts have been evaluated beyond used for repair of large osteochondral defects in horses. 1 year and all returned to racing or nonracing ath- Am J Vet Res 1994;55:1158–1168. letic work. 19. Stover SM, Moritz AF, Benton HP, et al. Autologous artic- ular osteochondral grafts for resurfacing joint defects (ab- Several members of the bone-morphogenetic pro- stract). Vet Surg 1993;22:400. tein (BMP) family also have considerable benefits in 20. Hurtig MB. Experimental use of small osteochondral cartilage healing. Of these, BMP2 and BMP7 show grafts for resurfacing the equine third carpal bone. Equine the most promise. In-vitro studies show that Vet J 1988;(suppl 6):23–27. 21. Brittberg M, Lindahl A, Nilsson A, et al. Treatment of deep BMP2 has matrix stimulatory effects similar to cartilage defects in the knee with autologous chondrocyte IGF-I. Long-term in-vivo studies show enhanced transplantation. New Eng J Med 1994;331:889–941. 101,102 cartilage repair in rabbits. 22. Chesterman PJ, Smith AU. Homotransplantation of artic- ular cartilage and isolated chondrocytes. An experimental study in rabbits. J Bone Joint Surg 1968;50-B:184–197. References and Footnotes 23. Green WT. Articular cartilage repair. Behavior of rabbit 1. Mankin HJ. The response of articular cartilage to mechan- chondrocytes during tissue culture and subsequent al- ical injury. J Bone Joint Surg 1982;64-A:460–466. lografting. Clin Orthop 1977;124:237–250. 2. Hunziker EB, Rosenberg LC. Repair of partial-thickness 24. Bentley G, Smith AU, Mukerjhee R. Isolated epiphyseal defects in articular cartilage: cell recruitment from the chondrocyte allografts into joint surfaces. Ann Rheum Dis synovial membrane. J Bone Joint Surg 1996;78-A:721– 1978;37:449–458. 733. 25. Bentley G, Greer RB. Homotransplantation of isolated 3. Campbell CJ. 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