New Horizons in Articular Cartilage Repair - IVIS

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IN DEPTH: CURRENT CONCEPTS IN EQUINE OSTEOARTHRITIS

New Horizons in Articular Cartilage Repair Alan J. Nixon, BVSc, MS, Diplomate ACVS, and Lisa A. Fortier, DVM, PhD, Diplomate ACVS

1.

Introduction

Articular cartilage rarely reforms a functional hyaline surface after injury. Most simple cartilage lacerative injuries reach a benign nonhealing phase, which remains unchanged over time.1,2 Deeper cartilage lesions, which violate the tide-mark and extend into the subchondral bone plate, result in an improved healing response.3 This is largely because of the proliferation of undifferentiated mesenchymal cells from the deeper tissues. In horses, the progression in cartilage defects from granulation to fibrous tissue and, finally, fibrocartilage is slower than it is in rodents; the healing of defects in dogs tends to be somewhere between these extremes.4 The fibrous tissue undergoes progressive chondrification to form a fibrocartilaginous mass that is loosely attached to the original cartilage edges. The subchondral bony plate occasionally reforms to the same approximate level as the adjacent undamaged bone. Immediately above the reformed subchondral plate, areas of cartilage proliferation predominate. The deeper cartilage layers and surface fibrous tissue generally follow a pattern of decreasing cellularity as the defect matures. The phenomenon of matrix flow, an intrinsic repair mechanism, may also contribute to the healing of equine articular cartilage defects by forming overhanging lips of cartilage on the perimeter of the lesion that tend to migrate in a centripetal manner.5 In small defects, this can result in significant reduction in lesion size. In larger defects, matrix flow plays an insignificant role, compared with mesenchymal cell proliferation. Although depth of injury

(full or partial thickness) is a critical determinant in healing, the size of the defect, its location in relation to weight-bearing or non-weight-bearing areas, and the age of the animal influence the repair rate and resiliency of new cartilage surfaces. Convery et al. showed that lesions in the equine femorotibial joint that were less than 3 mm in diameter healed with little residual deformity.6 More recently, Hurtig et al. determined that lesions larger than 15 mm2 in surface area tend to show reasonably good repair at 5 months but degenerate with increasing time.7 Given these and other studies, repair of full-thickness articular cartilage defects in the horse may not be as satisfactory or as complete as that documented in smaller animals. Metaplasia of fibrous tissue to fibrocartilage is not always evident and, depending on the time of examination, degeneration to fibrous tissue and later mechanical erosion of the repair tissue can occur. Repair tissue is biomechanically inferior to normal articular cartilage, even though the histological appearance is often fibrocartilage or even hyaline-like tissue.8 Repair tissue generally has significantly less proteoglycan and, to some extent, type II collagen than does normal cartilage. Additionally, the development of subchondral architecture and re-establishment of a tide-mark is often irregular and inconsistent. This creates susceptibility to cartilage deterioration with normal joint activity. Poor-quality, relatively short-lived repair cartilage has led to the development of pharmacologic and surgical methods to improve the repair process.

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IN DEPTH: CURRENT CONCEPTS IN EQUINE OSTEOARTHRITIS down to actively bleeding bone, which provides a source of pluripotential cells. The subsequent fibrous or fibrocartilaginous response is determined primarily by lesion location and size, degree of weight-bearing, and age of the animal. The cartilage resection perimeter should be vertical rather than beveled because this results in better attachment of tissue regrowth to original cartilage.

Fig. 1. Histologic appearance of articular cartilage from immature (6-month-old, left) and mature (3-year-old, right) horses shows the markedly reduced cell density, lack of vascular access to the attached subchondral bone, and well-established tide mark of adult cartilage. Cartilage healing is enhanced in young horses through these and other features.

2.

Cartilage Repair

Generally, the healing of chondral and osteochondral defects is more complete in young animals because of the increased mitotic capacity of chondrocytes, more active matrix synthesis, and closer proximity to the vascular supply in the depths of the articular– epiphyseal complex (Fig. 1).9 Examples of improved repair capacity are easily seen in the resurfacing potential following OCD flap removal when compared with the debridement of articular erosions in adults. Facilitation of cartilage repair falls into one of two categories: repair by stimulation of pluripotential cells from the subarticular level or by transplantation of cartilage, osteochondral grafts, or chondrocytes from remote regions.

Forage Drilling the subchondral bone in addition to debriding the overlying cartilage opens up wider channels into the subchondral marrow spaces. Vachon et al. have shown that repair tissue is superior following subchondral drilling after cartilage debridement of the third carpal bone.10 Forage is still occasionally performed following debridement of subchondral cystic lesions, to perforate the dense sclerotic perimeter that surrounds most mature lesions. However, none of the drilling experiments performed in horses resulted in the hyaline cartilage reported to have been seen in rabbit drilling experiments. Cyst expansion following forage of the cyst perimeter has also been described, resulting in continued lameness.11 Moreover, micropicking has largely replaced drilling techniques.

Local Cartilage Repair

Microfracture/Micropick Perforation of the subchondral bone after full-thickness cartilage debridement can more precisely be accomplished with micropick awls (Fig. 2). The tapered awl can be driven 2–3 mm into the subchondral plate to open a channel for vascular ingress, pluripotent stem cell delivery, and associated growth factor fluxes, all of which coalesce in the so-called “super-clot.”12 The simplicity of the technique is appealing but experimental evidence of superior healing in horses is limited to an increase in tissue volume in full-thickness defects and minor improvement in type II collagen content.13

Cartilage Debridement Methods to stimulate cartilage repair from mesenchymal 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 subchondral bone drilling (forage) or spongialization (saucerization). Partial-thickness cartilage resection reduces the dissipation of cartilage breakdown products into the synovial environment, thus reducing irritation to the synovial membrane and subsequent production of cytokines. In general, partial-thickness chondrectomy, which is usually performed with mechanical abraders, is a smoothing procedure. It does not attenuate or terminate continued fibrillation. Full-thickness cartilage debridement is the procedure of choice for deeply fibrillated cartilage with areas of exposed subchondral bone. Cartilage debridement with hand tools or mechanical debriders allows removal of the dense subchondral plate

Fig. 2. Micropick procedure being used after removal of a basal fracture of the sesamoid. The residual fibrillated cartilage surface (left) has been debrided and is being micropicked (right).

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IN DEPTH: CURRENT CONCEPTS IN EQUINE OSTEOARTHRITIS However, in many situations this may prolong active exercise. Abrasion Chondroplasty In human knee arthroplasty, performing uniform debridement with a burr, to remove a layer of dense sclerotic eburnated bone following full thickness cartilage loss, has been shown to cause tufts of fibrocartilage to occur at sites of vessel protrusion through the subchondral plate.14 This technique remains poorly developed in equine arthroscopic surgery. This is compounded by the fact that most end-stage joints are poor candidates for surgical therapy because the aim usually is a return to some actively functional state rather than simple attenuation of joint pain. Abrasion chondroplasty has, however, been used quite successfully in improving the status of eburnated regions in the trochlear ridges of the hock and may have potential in eburnated areas of other rotatory action joints. Cartilage Flap Reattachment Under defined conditions where an OCD cartilage flap has not detached along its entire perimeter, local debridement of the necrotic cartilage and marrow fibrosis can be accomplished arthroscopically and the partially attached flap can be replaced and secured with PDS pins (OrthoSorb) or PLLA tacks (chondral darts, Arthrex). This has worked satisfactorily on large flaps in the fetlock, hock, and stifle (Fig. 3). Criteria for use include a large flap only partially detached with a relatively normal-appearing surface structure. The fibrosis intervening between cartilage and bony defect must be removed if the procedure is to work. Several diverging pins are placed with the kit provided. Use of the multishot chondral dart system (Arthrex) for

Fig. 3. Stifle OCD flap lesion after placing PDS pins along the length of the lateral trochlear ridge flap (arrowheads). An additional OCD of the patella (arrow) was debrided.

simultaneous multiple anchoring is being explored. Resolution of joint effusion and radiographically obvious subchondral lysis, as well as reformation of the subchondral contour, are better than that which follows cartilage flap removal. Transplantation Resurfacing: Tissues

Different tissues and methods have been used experimentally for cartilage resurfacing. Five types of donor tissue have been investigated: periosteal and perichondrial autografts, osteochondral autografts or allografts, chondral autografts, isolated chondrocyte autografts or allografts, and stromal (mesenchymal) stem cell autotransplants. Periosteum/Perichondrium Several investigators have examined periosteal and perichondrial grafts for cartilage resurfacing in horses. Although O’Driscoll et al and Rubak both found hyaline cartilage production following periosteal transfer to cartilage defects in experimental small animals, these results have not been duplicated in studies of horses by Vachon et al.15–17 Osteochondral Grafts Osteochondral grafting using autogenous sternal or carpal donor fragments has been generally unsuccessful.18 Although cartilage incorporation has been satisfactory, the attached subchondral bone has not incorporated well.19 Mosaicplasty and similar osteochondral dowel systems are technically difficult to harvest and transplant arthroscopically; results in the carpus and fetlock of horses suggest that the cartilage is susceptible to degeneration.20 A suitable donor site is also a significant issue. Transplantation Resurfacing: Isolated Cells

Chondrocyte Transplantation Chondrocyte grafting of articular defects has recently 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 incongruities in the articular surface without the problems of articular and subchondral fit or the problems of secure attachment of solid tissue transplants such as periosteum, cartilage, or osteochondral grafts. Difficulties in achieving incorporation of osteochondral grafts, as well as failures in the horse of many other solid tissue transfers, has encouraged the study of free-cell transplants. Chondrocyte transfer to full-thickness cartilage defects has been extensively studied in experimental animals. Many studies simply implanted chondrocytes of articular or physeal origin into cartilage defects.22–26 Although the results were often positive, the articular defects and the experimental animals used were small. Nevertheless, the use of AAEP PROCEEDINGS Ⲑ Vol. 47 Ⲑ 2001


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IN DEPTH: CURRENT CONCEPTS IN EQUINE OSTEOARTHRITIS allograft chondrocytes was established and, despite the known immune response of host tissues to these chondrocytes,27–31 the rapid proliferation of matrix led to an apparent “sequestration” of the transplanted chondrocytes, which provided a barrier to immune recognition.32–34 Assessment of free allograft or autograft chondrocytes in larger, more clinically relevant lesions has not been done. Given the larger size of the defects and the complete lack of adherent properties of the grafted cells, there would be little reason to expect the chondrocytes to remain in the target defect for more than a few minutes during postoperative recovery. Several studies suggest that isolated cultured chondrocytes may have a role in the reconstruction of physeal injuries where the grafts can be secured by overlying soft tissues.35,36 Transplanting chondrocytes in a vehicle or adherent matrix composite provides better assurance that the transplanted cells will remain in position for periods long enough to synthesize a new pericellular matrix and to establish a bond to the subchondral bone. Additionally, the new matrix assumes the immune isolating effect initially provided by the transplant vehicle. Various biologic and synthetic materials have been described for this purpose. Biologically derived materials have been studied most extensively. Cultures of allograft and autograft chondrocytes transplanted in fibrin, collagen gels, and hyaluronate products have been shown to stimulate hyaline cartilage repair in various experimental models.37– 43 The use of fibrin-based vehicles is appealing because of the ready source of autogenous fibrinogen from plasma, its application as a self-polymerizing liquid, and the inherent “glue”-like properties of thrombin-activated fibrinogen. Experimental studies evaluating the survival and metabolic activity of equine chondrocytes in fi-

Fig. 4. Collagen type II gene expression is enhanced in fibrin vehicles derived from autogenous fibrinogen from plasma (left) compared with equine fibrinogen purchased from commercial vendors (right). 220

brin cultures in vitro indicate that fibrin is a tenacious polymer that supports chondrocyte phenotypic expression and matrix elaboration.44 Autogenous fibrinogen also has anabolic effects on chondrocyte function that are not evident in commercially prepared fibrinogen (Fig. 4).45 The ability to inject chondrocyte-laden fibrinogen and activated thrombin through a needle provides site-specific deposition of self-adhering graft material that can be placed arthroscopically. Research studies of the equine femoropatellar joint indicated that fibrin– chondrocyte grafts improved the morphologic and biochemical properties of healing tissue in 12-mm, full-thickness cartilage defects.46 Classic markers of hyaline cartilage such as collagen type II were significantly elevated in chondrocytegrafted stifles (61% compared with 25% in control ungrafted lesions at 8 months). The mechanics of stifle arthroscopy and chondrocyte–fibrin grafting have been adapted to routine surgical procedures. Currently, autogenous fibrinogen is harvested the day before surgery, 30 ⫻ 106 allograft chondrocytes are thawed and cultured for a minimum of 24 hours, and both are mixed with calcium-activated bovine thrombin as they enter a needle, which is inserted percutaneously so that the tip is placed in the cartilage defect. The only modification to routine arthroscopy is temporary insufflation of the joint with helium or carbon dioxide to allow fibrin polymerization. Clearly, access to a lab capable of harvesting and storing the cells is a prerequisite for chondrocyte-based resurfacing programs.47 However, such facilities are becoming more frequently associated with referral institutions and this trend will probably continue. Long-term studies of equine clinical cases following chondrocyte grafting are not available. Initial results in horses with subchondral cysts and related defects of the femoral or metacarpal condyles indicates a more rapid resolution of lameness and effusion and an accelerated bony fill of the defects on follow-up radiographs. Articular defects that remain after lag-screw repair of fractures of the metacarpal 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 promise, 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 arthroscopically applied techniques is an important criterion for application in the horse, other products and methods to secure the grafts in extensive cartilage defects have also been developed. Collagen mesh and lat-





IN DEPTH: CURRENT CONCEPTS IN EQUINE OSTEOARTHRITIS tice materials containing cultured chondrocytes meet these surgical criteria and also provide some strength to the implant by virtue of the collagen network. Chondrocytes readily populate these absorbable scaffolds and actively synthesize a new cartilage matrix (Fig. 5).49 However, long-term experimental trials in horses with 15-mm, full-thickness defects in the stifle have shown only moderate improvement in cartilage healing.50,51 The concept of chondrocytes in a pre-existing collagen meshwork forming “artificial cartilage” has not met with the success seen in small-animal research models.40,41,52 Hyaluronate vehicles for chondrocyte transfer have special appeal because of their similarity to the hyaluronate-rich cartilage matrix, the apparent beneficial effects of hyaluronate-containing synovial fluid on cartilage metabolism, and previous research showing upregulation of chondrocyte metabolism under the influence of exogenous hyaluronate.53–55 Studies of chondrocyte– hyaluronate grafts in small defects were quite positive.42,43 However, hyaluronate gels that dissolve rapidly present a handling problem for implantation and have no adherent capabilities in cartilage defects.a Synthetic matrix polymers have the advantage of being biologically inert. They also hydrolyze at a specific rate, which can be altered in the production phase to tailor to specific grafting applications. Several studies have defined useful polymer constructs for chondrocyte seeding and subsequent articular implantation.56 – 61 The results of in-vivo studies are satisfactory but not superior to other biological methods. Integrating tissue-engineered composites to surrounding normal cartilage has been unsatisfactory. The real advantage of using polymers as vehicles for transplantation may be in a dual role: as carriers for growth factors and to slowly facilitate the metabolism of transplanted chondrocytes.

Fig. 5. Tissue engineered collagen matrix, which has been seeded with chondrocytes and cultured for 21 days, shows active cartilage matrix deposition between the collagen network of the composite, which is ready for transfer to the joint.

Chondrocyte-based studies and clinical application in horses utilize allograft chondrocytes harvested from foals and weanlings. Although the metabolic and mitogenic capacities of fetal chondrocytes are somewhat better than those of weanlings, they do not maintain this advantage after being cryopreserved for several months.b Additionally, viability declines to unsatisfactory levels (⬍50%) after 3 months at ⫺196°C.47 Chondrocytes derived from mature horses survive cryopreservation for extended periods but their metabolic capacity is diminished. As a result, the ideal donor age is considered to be 4 – 6 months.47 Preparing chondrocytes in monolayer for several days prior to transplantation has also been shown to provide a metabolic stimulus, which allows partially cryodamaged cells to recover or disintegrate and be discarded at the initial media exchange.44 The advantages of autogenous chondrocyte transplantation are tempered by a lack of a suitable donor joint, the cost of preliminary surgery for cartilage harvest, and the time delay between harvest and reimplantation. Although these factors may be satisfactory in man,21 they are generally unsatisfactory for treating acute injury in horses. Moreover, the minimum number of chondrocytes required for substantive contribution to cartilage repair is 10 million cells per ml of injected fibrin.44,62 Average yields of chondrocytes from cartilage derived from 3– 6-year-old horses are only 12–15 million cells per gm of tissue.47 To obtain 3 gm of cartilage for resurfacing purposes would denude an area approximately one-third to one-half the size of the femoropatellar joint in a mature horse. Clearly, other sites for chondrocyte harvest are required; studies of chondrocytes derived from sternal cartilage provide a possible solution. The intuitive advantages of autogenous chondrocyte transplants have not been experimentally ratified. The most recent study compared articular healing at 26 and 52 weeks after autogenous or allogenous chondrocyte grafting and found no difference in the success rates of each.63 More attention is now being focused 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. Mesenchymal 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 reimplantation in articular defects. An obvious example is the use of the cambial layers of the periosteum.64,65 However, other pools of stem cells, such as bone marrow, are more readily available66 and it is these pools that show considerable promise in cartilage resurfacing.67 Under appropriate conditions, such AAEP PROCEEDINGS Ⲑ Vol. 47 Ⲑ 2001


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IN DEPTH: CURRENT CONCEPTS IN EQUINE OSTEOARTHRITIS as lowered oxygen tension, high cell density, dexamethasone exposure, and fetal calf serum– enriched media, mesenchymal stem cells can be directed to differentiate toward chondrocytes.64,66,68 The development of chondrocytes rather than osteoblasts is largely dependent on vascular access and the resulting oxygen tension.66 For full-thickness articular defects, components of both tissue types are required for reconstructing the subchondral plate and the overlying hyaline cartilage surface. Transplantation of mesenchymal cells into collagen gels has been used to successfully resurface 3 ⫻ 6 mm defects in the rabbit femoral condyle.67 Hyaline cartilage and reformed subchondral tissues were apparent at 6 months. Mechanical testing of these new surfaces indicated that they were softer than normal cartilage but quite improved over ungrafted control defects. Longer-term studies are needed because mechanical test data are critical determinants of the likely durability of such tissue. In horses, the sternum provides a better source of mesenchymal cells than does the tuber coxae. Bone marrow can be harvested standing and is rich in undifferentiated stem cells.69 In-vitro culture results indicate a similar progression of differentiation in high-density monolayer cultures. Markers of cartilage phenotype such as proteoglycan and type II collagen were evident by day 7 of culture. The behavior of mesenchymal stem cells in threedimensional culture is of greater importance because this mimics the cartilage environment more closely.70 Mesenchymal cells assumed a rounded appearance with active proteoglycan production in a parallel study of chondrocytes and mesenchymal cells in fibrin three-dimensional cultures. The comparative and temporal aspects of the study indicated that the mesenchymal cells continued to synthesize cartilage matrix components over the course

Fig. 6. Equine bone marrow– derived mesenchymal stem cells in culture 2 days after addition of transforming growth factor-␤1 (5 ng/ml). Assay for collagen type II showed enhanced chondrogenesis. 222

of the experiment and that the levels were similar to the levels evident in parallel chondrocyte cultures. The effect of various growth factors on the differentiation and synthetic activity of these cells is also of significance and may eventually be used in conjunction with mesenchymal cell grafts for cartilage resurfacing (Fig. 6).71,72 Use of gene-enhanced MSC chondrogenesis to derive chondrocytes for transplant to cartilage surfaces offers real potential for autogenous chondrocyte grafting in horses.73 Growth-Factor–Enhanced Cartilage Repair

Several naturally occurring polypeptide growth factors play an important role in cartilage homeostasis.74 The differentiating and matrix anabolic activity of insulin-like growth factor-I (IGF-I) and transforming growth factor-␤ (TGF-␤) are particularly important in counteracting the degradatory and catabolic activities of cytokines, serine proteases, and neutral metalloproteases. The manipulation of this balance in disease conditions such as arthritis and acute cartilage injury may be possible by exogenous administration of IGF-I and TGF␤.75– 80 The effects of these and other growth factors have been studied extensively in culture systems of many types. Most data have been generated from chondrocyte monolayer and cartilage explant cultures, where IGF-I and TGF-␤ generally result in elevated matrix molecule elaboration, concurrent with minor to moderate mitogenic effects.81– 86 Similar results were evident in monolayer cultures of equine chondrocytes, where dose-dependent stimulation of proteoglycan production occurred in serum-free and serum-supplemented cultures.87 Three-dimensional culture assessment of the effect of these same growth factors on equine chondrocyte metabolism have also been performed with fibrin gels, which provide a stable suspension culture resembling the cartilage matrix environment.88,89 The dose-response to growth factors was largely influenced by the presence of fetal calf serum in the media. When cultured without serum supplements, these two growth factors stimulated matrix component elaboration in a dose-dependent 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, particularly 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. Increased levels of TGF-␤ (5 and 10 ng/ml) eventually suppressed matrix synthesis and cell division.88,89 Other studies in the horse largely focused on IGF-I because it was not detrimental to chondro-





IN DEPTH: CURRENT CONCEPTS IN EQUINE OSTEOARTHRITIS cyte metabolism when present in excess concentrations.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 results, IGF-I was selected as a suitable growth factor for in-vivo studies in the horse. Other investigators evaluated articular repair following TGF-␤ administration.2,92 However, synovitis and osteophyte 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 for enhanced cartilage repair. Elution studies using IGF-I–laden equine fibrin indicate that maximally stimulatory levels of IGF-I (⬎50 ng/ml) remain for a minimum of 3 weeks following an initial loading dose of 20 ␮g (Fig. 7).95 Although the dissolution rate of fibrin in the synovial environment is not known, it is not expected to vary considerably from the buffered polyionic saline used in the in-vitro elution studies. In-vivo evaluation of a self-polymerizing fibrin vehicle that is devoid of cells but is loaded with 25 ␮g IGF-I and injected into cartilage lesions in the femoropatellar joints showed improved cell population with more cartilage-like architecture after 6 months.96 However, markers of hyaline cartilage such as type II collagen increased to 47%, far short of the 90% minimum evident in normal articular cartilage. Nevertheless, simple fibrin vehicle grafts used in control stifles did not significantly enhance healing. There was a mean collagen type II content of 39%, which is similar to healing in empty full-thickness defects.46 Other studies using injected combinations of IGF-I and pentosan polysulfate showed attenuation of the symptoms of synovitis in OA models in sheep.97 In general, IGF-I seems to have better application in

combination with chondrocyte or mesenchymal stem cell grafts, where more complete cartilage repair develops.48 Healing evaluation at 8 months, following implantation of a mixture of chondrocytes and 25 ␮g IGF-I in stifle defects of 8 horses, showed a considerably improved joint surface. There was 58% type II collagen and better neocartilage integration at the defect edges. Studies of IGF-I and BMP-7 gene-enhanced chondrocyte function in similar transplant models suggest that both may stimulate healing beyond that seen in unstimulated chondrocyte-grafted cases (Fig. 8).98 –100 Clinical resurfacing trials in horses have used a regimen of autogenous fibrin laden with 50 ␮g IGF-I and 30 million chondrocytes per milliliter of fibrin. The chondrocytes were mixed with fibrinogen and IGF-I with activated thrombin to provide a 2-component system for immediate injection (Fig. 9). The polymerization process developed immediately upon injection into the articular defect. Currently,

Fig. 7. Elution profile of IGF-I over time from polymerized fibrin after loading IGF-I (25 ␮g) to each 1 ml aliquot and assaying IGF-I in medium above the fibrin composite.

Fig. 9. Chondrocyte–IGF-I grafting of subchondral cyst of the femoral condyle. Debridement is followed by cancellous bone grafting of the deeper portion of the cyst and injection of the thrombin-activated mixture of fibrinogen, chondrocytes, and IGF-I.

Fig. 8. Effect of bone morphogenetic protein-7 (BMP-7) geneenhanced chondrocyte function 4 weeks after implantation to the stifle of horses. A null gene (AdCD) was used to transduce chondrocytes used as controls.

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IN DEPTH: CURRENT CONCEPTS IN EQUINE OSTEOARTHRITIS the predominant application has been OCD and subchondral cystic defects of the fetlock (8 horses) and stifle (43 horses), although several shoulder and carpal articular lesions have been treated. The fibrin polymer and growth factor have been well tolerated and have effected resolution of effusion and lameness. Assessment of stifle OC cyst cases 1–5 years after implant indicates that the horses respond by increased bone deposition in the subchondral plate and then generally throughout the cyst over the ensuing first year. Soundness has taken as long as 1 year to develop, but 22 of 30 horses beyond the first post-operative year have remained in active work without lameness. Six of 8 horses with grafted fetlock subchondral cysts have been evaluated beyond 1 year and all returned to racing or nonracing athletic work. Several members of the bone-morphogenetic protein (BMP) family also have considerable benefits in cartilage healing. Of these, BMP2 and BMP7 show the most promise. In-vitro studies show that BMP2 has matrix stimulatory effects similar to IGF-I. Long-term in-vivo studies show enhanced cartilage repair in rabbits.101,102 References and Footnotes 1. Mankin HJ. The response of articular cartilage to mechanical injury. J Bone Joint Surg 1982;64-A:460 – 466. 2. Hunziker EB, Rosenberg LC. Repair of partial-thickness defects in articular cartilage: cell recruitment from the synovial membrane. J Bone Joint Surg 1996;78-A:721– 733. 3. Campbell CJ. The healing of cartilage defects. Clin Orthop 1969;64:45– 63. 4. Riddle WE. Healing of articular cartilage in the horse. J Am Vet Med Assoc 1970;157:1471–1479. 5. Ghadially JA, Ghadially FN. Evidence of cartilage flow in deep defects in articular cartilage. Arch B Cell Path 1975; 18:193–204. 6. Convery FR, Akeson WH, Keown GH. The repair of large osteochondral defects. Clin Orthop 1972;82:253–262. 7. Hurtig MB, Fretz PB, Doige CE, et al. Effects of lesion size and location on equine articular cartilage repair. Can J Vet Res 1988;52:137–146. 8. Ahsan T, Sah RL. Biomechanics of integrative cartilage repair. Osteoarthritis Cartilage 1999;7:29 – 40. 9. Madsen K, Moskalewski S, von der Mark K, et al. Synthesis of proteoglycans, collagen, and elastin by cultures of rabbit auricular chondrocytes—relation to age of the donor. Dev Biol 1983;96:63–73. 10. Vachon A, Bramlage LR, Gabel AA, et al. Evaluation of the repair process of cartilage defects of the equine third carpal bone with and without subchondral bone perforation. Am J Vet Res 1986;47:2637–2645. 11. Howard RD, McIlwraith CW, Trotter GW. Arthroscopic surgery for subchondral cystic lesions of the medial femoral condyle in horses: 41 cases (1988 –1991). J Am Vet Med Assoc 1995;206:842– 850. 12. Rodrigo JJ, Steadman JR, Silliman JF, et al. Improvement of full-thickness chondral defect healing in the human knee after debridement and microfracture using continuous passive motion. Am J Knee Surg 1994;7:109 –116. 13. Frisbie DD, Trotter GW, Powers BE, et al. Arthroscopic subchondral bone plate microfracture technique augments healing of large chondral defects in the radial carpal bone and medial femoral condyle of horses. Vet Surg 1999;28: 242–255. 224

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a

Nixon, AJ and Foley, RL. Unpublished observation. Nixon. Unpublished observations.

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