Regeneration of articular cartilage--evaluation of osteochondral defect repair in the rabbit using multiphasic implants.

OBJECTIVE: To investigate whether two different multiphasic implants could initiate and sustain repair of osteochondral defects in rabbits. The implants address the malleable properties of cartilage while also addressing the rigid characteristics of subchondral bone. DESIGN: The bone region of both devices consisted of D, D-L, L-polylactic acid invested with hyaluronan (HY). The cartilage region of the first device was a polyelectrolytic complex (PEC) hydrogel of HY and chitosan. In the second device the cartilage region consisted of type I collagen scaffold. Eighteen rabbits were implanted bilaterally with a device, or underwent defect creation with no implant. At 24 weeks, regenerated tissues were evaluated grossly, histologically and via immunostaining for type II collagen. RESULTS: PEC devices induced a significantly better repair than untreated shams. Collagen devices resulted in a quality of repair close to that of the PEC group, although its mean repair score (19.0+/-4.2) did not differ significantly from that of the PEC group (20.4+/-3.7) or the shams (16.5+/-6.3). The percentage of hyaline-appearing cartilage in the repair was highest with collagen implants, while the degree of bonding of repair to the host, structural integrity of the neocartilage, and reconstitution of the subchondral bone was greatest with PEC devices. Cartilage in both device-treated sites stained positive for type II collagen and GAG. CONCLUSIONS: Both implants are capable of maintaining hyaline-appearing tissue at 24 weeks. The physicochemical region between the cartilage and bone compartments makes these devices well suited for delivery of different growth factors or drugs in each compartment, or different doses of the same factor. It also renders these devices excellent vehicles for chondrocyte or stem cell transplantation.

The effect of alendronate (Fosamax) and implant surface on bone integration and remodeling in a canine model.

Patients at high risk for osteoporosis and its associated morbidity, including postmenopausal women, are being pharmacologically managed to stabilize and improve bone mass. Alendronate sodium (Fosamax) is a commonly used antiresorptive agent effective in osteopenic women for reducing bone resorption, increasing bone density, and decreasing fracture incidence. With the increased incidence of alendronate-treated women who are undergoing hip replacement or fracture repair by prosthesis placement, data are needed to predict how alendronate affects host bone integration with uncemented surfaces. The aim of this study was to determine the effect of alendronate on new bone formation and attachment to implant surfaces in a normal and simulated estrogen-deficient, calcium-deficient canine model, using an implantable bone growth chamber. Alendronate did not affect host bone integration to surfaces commonly used in uncemented total joint arthroplasty, but there were significant differences dependent solely on the type of surface.

Transforming growth factor beta superfamily members: role in cartilage modeling.

Normal and abnormal extracellular matrix turnover is thought to result, in part, from the balance in the expression of metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). The clinical manifestations of an imbalance in these relationships are evident in a variety of pathologic states, including osteoarthritis, deficient long-bone growth, rheumatoid arthritis, tumor invasion, and inadequate cartilage repair. Articular cartilage defects commonly heal as fibrocartilage, which is structurally inferior to the normal hyaline architecture of articular cartilage. Transforming growth factor-beta 1 (TGF-beta1), a cytokine central to growth, repair, and inflammation, has been shown to upregulate TIMP-1 expression in human and bovine articular cartilage. Additionally, members of the TGF-beta superfamily are thought to play key roles in chondrocyte growth and differentiation. Bone morphogenetic protein-2 (BMP-2), a member of this superfamily, has been shown to regulate chondrocyte differentiation states and extracellular matrix composition. It was proposed that, by optimizing extracellular matrix composition, BMP-2 would enhance articular cartilage healing. After determining the release kinetics of BMP-2 from a collagen type I implant (Long-Evans male rats; two implants/rat, n = 14), it was found that, in a tissue engineering application, BMP-2 induced a hyaline-like repair of New Zealand White rabbit knee articular cartilage defects (3-mm full-thickness defects in the femoral trochlea; 2 defects/rabbit, n = 36). The quality of cartilage repair with BMP-2 (with or without chondrocytes) was significantly better than defects treated with BMP-2, as assessed by a quantitative scoring scale. Immunohistochemical staining revealed TIMP-1 production in the cartilage defects treated with BMP-2. When studied in vitro, it was found that BMP-2 markedly increased TIMP-1 mRNA by both bovine articular and human rib chondrocytes. Additionally, increased TIMP-1 mRNA was translated into increased TIMP-1 protein production by bovine chondrocytes. Taken together, these data suggest that BMP-2 may be a useful cytokine to improve healing of cartilaginous defects. Furthermore, these data suggest that the beneficial effects of BMP-2 may be, in part, related to alterations in extracellular matrix turnover.

Degradation and repair of articular cartilage.

Approximately 95,000 total knee replacements and 41,000 other surgical procedures to repair cartilaginous defects of the knee are performed annually in the United States (1). The response of normal articular cartilage to injury or arthritic degeneration is often a sub-optimal repair; the biochemical and mechanical properties of the new tissue differ from the native cartilage, resulting in inadequate or altered function. It is believed that the chondrocytes from the surrounding areas, although perhaps capable of some limited migration at the damaged site, are not able to proliferate and produce the macromolecules necessary to create an organized matrix characteristic of normal articular cartilage (2,3). Current therapeutic options for articular cartilage injuries and degeneration have resulted in repair tissue which may be hyaline-like, but does not approximate the durability and function of the normal articular surface. Numerous studies have been performed to increase our understanding of the normal repair process of articular cartilage and its limitations, and to devise methods and materials to regenerate the joint surface.

Repair of articular cartilage defects: part II. Treatment options.

Articular cartilage injuries result in numerous clinical symptoms, such as pain and decreased functional levels. Current therapeutic options being used include articular surface debridement, such as chondral shaving, abrasion chondroplasty, and subchondral perforation; soft-tissue arthroplasties, such as perichondrial and periosteal grafts; and osteochondral transplantation. None of these therapies, however, has resulted in the successful regeneration of a hyaline-like tissue that withstands normal joint loading and activity over prolonged periods. As a result, research is also being conducted on alternative therapeutic procedures to enhance the repair process and to stimulate the regeneration of a repair tissue with hyaline-like structural and biologic properties. Part I of this paper, which was published in January, discussed the basic science of cartilage healing. Part II presents the treatment options.

Repair of articular cartilage defects: part I. Basic Science of cartilage healing.

Articular cartilage injuries result in numerous clinical symptoms, such as pain and decreased functional levels. The limited reparative capabilities of hyaline cartilage results in the generation of repair tissue that lacks the structure and biomechanical properties of normal cartilage. Chondrocytes are unable to adequately proliferate, migrate, and synthesize high-quality repair tissue in response to blunt, superficial, or deep penetrating trauma. Extensive research has been conducted to understand the healing process and devise techniques that would enhance this response. Part I of this paper will discuss the basic science of cartilage repair. Part II, which will be published in the February issue, will present the treatment options.

Development of a novel osteochondral graft for cartilage repair.

This study reports the development of a novel osteochondral graft for cartilage repair. A technique of proteoglycan extraction via timed enzymatic digestion with hyaluronidase and trypsin and subsequent processing with a chloroform-methanol solution to remove cellular debris from a fresh-frozen bovine osteochondral sample is a method described to prepare a stable biological carrier of low immunogenicity. Lyophilization of the carrier followed by rehydration in a suspension of lapine chondrocytes produced a chimeric xenograft that succeeded in vivo in enhancing cartilage repair. In a pilot study, full-thickness articular cartilage defects treated with these xenografts demonstrated improved healing compared to untreated defects or defects treated with unseeded grafts at 2, 6, and 12 weeks postimplantation. The xenograft provoked a mild inflammatory response; however this did not impede the repair process. Further investigation of this novel chimeric xenograft eventually may yield a method of cartilage repair superior to current methods of treatment.

Chondrocyte transplantation using a collagen bilayer matrix for cartilage repair.

We have developed a novel, two-layered, collagen matrix seeded with chondrocytes for repair of articular cartilage. It consists of a dense collagen layer which is in contact with bone and a porous matrix to support the seeded chondrocytes. The matrices were implanted in rabbit femoral trochleas for up to 24 weeks. The control groups received either a matrix without cells or no implant. The best histological repair was seen with cell-seeded implants. The permeability and glycosaminoglycan content of both implant groups were nearly normal, but were significantly less in tissue from empty defects. The type-II collagen content of the seeded implants was normal. For unseeded implants it was 74.3% of the normal and for empty defects only 20%. The current treatments for articular injury often result in a fibrous repair which deteriorates with time. This bilayer implant allowed sustained hyaline-like repair of articular defects during the entire six-month period of observation.

Transplantation of adenovirally transduced allogeneic chondrocytes into articular cartilage defects in vivo.

Gene transfer to chondrocytes followed by intra-articular transplantation may allow for functional modulation of chondrocyte biology and enhanced repair of damaged articular cartilage. We chose to examine the loss of chondrocytes transduced with a recombinant adenovirus containing the gene for Escherichia coli beta-galactosidase (Ad.RSVntlacZ), followed by transplantation into deep and shallow articular cartilage defects using New Zealand White rabbits as an animal model. A type I collagen matrix was used as a carrier for the growth of the transduced chondrocytes and to retain the cells within the surgically created articular defects. Histochemical analysis of matrices recovered from the animals 1, 3 and 10 days after implantation showed the continued loss of lacZ positive chondrocytes. The number of cells recovered from the matrices was also compared with the initial innoculum of transduced cells present within the matrices at the time of implantation. The greatest loss of transduced cells was observed in the first 24 h after implantation. The numbers of transduced cells present within the matrices were relatively constant between 1 and 3 days postimplantation, but had progressively declined by 10 days postimplantation. These results suggest that transduction of chondrocytes followed by intra-articular transplantation in this rabbit model may enable us to examine the biological effects of focal transgenic overexpression of proteins involved in cartilage homeostasis and repair.

Chondrocyte transplantation and experimental treatment options for articular cartilage defects.

Current treatment options for injured articular cartilage have resulted in temporary improvements in clinical symptoms and functional levels. None of these modalities, however, has resulted in restoration of an articular surface that is able to withstand long-term joint loading and function. As a result, numerous investigators have attempted to devise alternative therapies. The limited regnerative potential of articular cartilage has led investigators to attempt using cells with the potential for differentiation and proliferation to repair chondral defects. Chondrocyte transplantation, both allogeneic and autogenous, has shown early promising results in regenrating hyaline-like tissue in both animals and humans. Encouraging results in animals have also been demonstrated with alternative sources of osteoprogenitor cells as grafts, as well as with natural/synthetic implants and the use of growth factors and cytokines. However, despite encouraging short-term results, long-term data concerning the regenerate tissue are still needed. As more research is being conducted to understand the processes of cartilage maintenance and healing, there is hope that cartilage regeneration and neochondrogenesis will be possible in the future.

Effects of nitric oxide on chondrocyte migration, adhesion, and cytoskeletal assembly.

OBJECTIVE: The migration of cells of chondrocyte lineage is believed to play a role in cartilage growth and repair. The present study examined 1) whether chondrocytes are capable of migration in vitro; and 2) the effects of nitric oxide (NO) on chondrocyte migration, adhesion, and cytoskeletal assembly. METHODS: Chondrocyte migration was evaluated by 2 assays: 1) "centrifugal" migration within a 3-dimensional collagen matrix (dot culture); and 2) directed migration under agarose in response to bone morphogenetic protein. To assess the effects of NO, chondrocytes were treated with either exogenous NO (S-nitrosoglutathione [SNO-GSH]) or a mixture of cytokines known to induce endogenous NO production. The effects of NO on chondrocyte adhesion to fibronectin-coated surfaces, as well as on actin polymerization (determined by indirect immunofluorescence), were also examined. RESULTS: The capacity of chondrocytes to migrate was demonstrated both by the dot culture and by agarose methods. Both SNO-GSH and endogenous NO induced by cytokines inhibited this migration. Exposure to NO also inhibited attachment of chondrocytes to fibronectin and disrupted assembly of actin filaments. These effects of SNO-GSH and cytokine-induced NO production were reversed in the presence of hemoglobin and the NO synthase inhibitor NG-monomethyl arginine, respectively. CONCLUSION: NO interferes with chondrocyte migration and attachment to fibronectin, an extracellular matrix protein, probably via effects on the actin cytoskeleton. These effects of NO may result in impairment of cartilage repair, by interfering with the extracellular matrix regulation of chondrocyte function.

A comparison of abrasion burr arthroplasty and subchondral drilling in the treatment of full-thickness cartilage lesions in the rabbit.

The purpose of this study was to observe the difference in healing of full-thickness articular cartilage defects treated with burr arthroplasty versus subchondral drilling. Cartilage was shaved off the medial femoral condyles of 39 rabbits without penetrating the subchondral plate. In left knees, two 2.0-mm holes were drilled into the condyle until bleeding was obtained. Right knees underwent a burr arthroplasty until punctate bleeding was observed. Animals were sacrificed at 6, 12, and 24 weeks postoperatively. Joint resurfacing and degenerative changes were evaluated grossly and histologically. Degenerative changes in the cartilage surface were observed with both treatments. Rabbits undergoing subchondral drilling had increased fibrocartilaginous healing with time, with a slight increase in degenerative changes. With burr arthroplasty, there was significant decrease in cartilaginous coverage of the exposed surface as well as progressive increase in degenerative changes. Although both techniques were suboptimal, histological evidence at 6 months suggests that subchondral drilling may result in a longer-lived repair than abrasion arthroplasty in the treatment of full-thickness lesions.

Effects of growth-factor-enhanced culture on a chondrocyte-collagen implant for cartilage repair.

The effects of incubation and addition of growth factors to a chondrocyte-seeded collagen implant for cartilage repair were studied. Type I collagen matrices seeded with lapine articular chondrocytes and unseeded controls cultured in the presence and absence of fibroblast growth factor and insulin for 2, 6, and 9 weeks were subjected to biomechanical, biochemical, and histological analysis. Aggregate modulus of elasticity of seeded implants decreased by half at 6 weeks, then rose by a factor of 10 above initial values. Permeability of seeded implants and their controls decreased steadily. Glycosaminoglycan content peaked at 6 weeks, coinciding with the greatest number of chondrocytes and mitotic activity in seeded implants. Chondrocytes remained phenotypically stable and metabolically active; they incorporated glycosaminoglycan into the extracellular matrix, and formed an organized pericellular environment despite the predicted resorption of the collagen matrix. Adding fibroblast growth factor and insulin tripled the rate of cell turnover and doubled the glycosaminoglycan content of seeded implants, but had no effect on their material properties. In vitro incubation for 6 weeks in the presence of fibroblast growth factor and insulin creates a metabolically and mitotically active chondrocyte-collagen composite for implantation into articular cartilage defects.

Demineralized bone matrix. Enhancement of spinal fusion.

A study was conducted to determine the ability of demineralized bone matrix gel to act as an osteoconductive/osteoinductive material to enhance canine spinal fusion. Seven dogs underwent posterior spinal fusion. Four-level fusions were performed with one of four procedures at each level: decortication alone, with gel added, with autograft, or with both gel and autograft. Dogs were killed at 6 weeks and early histologic response was studied. At untreated control sites, little bone formation was evident. Gel-filled sites showed abundant osteoid, with 60% of demineralized particles fused to or surrounded by new bone. Sites filled with autograft had more new bone, but there was more osteoid at gel-treated sites. Autograft augmented with gel showed the most vigorous response, with extensive bridging between demineralized particles, host bone, autograft, and new bone. Significantly less autograft was needed to induce a similar amount of new bone formation when gel was added. Use of the gel as an autograft extender may improve the chance for successful spinal fusion.

Fibroblast growth factor: effects on osteogenesis and chondrogenesis in the chick embryo.

In vivo effects of fibroblast growth factor (FGF) on osteogenesis were evaluated in the chick embryo. Autoradiographic studies of 3H-proline labeling over bone matrix indicated that 24 h after treatment on day 11, FGF stimulated osteogenic cell proliferation, while inhibiting the production of bone matrix collagen. However, 4 days after multiple doses of FGF, the large pool of newly formed osteogenic and chondrogenic cells expressed their function with the increased formation of matrix. The data provide in vivo evidence of the effects of exogenous FGF on osteogenesis and also point to a possible role for FGF both in embryonic osteogenesis and in fracture repair.

Fibroblast growth factor in chick osteogenesis.

In vivo effects of FGF on osteogenesis in the chick embryo were evaluated. Day-11 embryos were injected with FGF followed by radiotracers. 3H-thymidine labelling of osteogenic cells was significantly higher following FGF administration; 3H-proline labelling over bone matrix was greater in the controls. Cartilage cells and matrix were sparsely labelled indicating a low level of metabolic activity. These data provide the first in vivo evidence that FGF stimulates osteogenic cell proliferation, while inhibiting production of bone matrix collagen. A role for FGF in embryonic osteogenesis and in fracture repair is suggested.

Nerve growth factor in skeletal tissues of the embryonic chick.

This study demonstrates, via immunohistochemistry and bioassay, the presence of NGF in embryonic bone and cartilage of the chick. Embryos were killed on days 6-9 of incubation at 12 h intervals, and on days 10-18 at 24 h intervals. Paraffin-embedded sections of hind limbs or buds were immunostained with a polyclonal antibody against NGF and the biotin-avidin-horseradish peroxidase technique. Immunostaining was positive in both bone and cartilage, with cartilage staining more intensely. For bioassay, bones from the hind limbs of 9- and 12-day embryos were fast-frozen, lyophilized, and homogenized with Medium 199 (M199). Dorsal root ganglia from 8-day embryos were cultured for 24-36 h with rooster plasma, M199, and varying concentrations of bone homogenate. Significant neurite outgrowth was seen, with the greatest response elicited by 12-day bone homogenate. Addition of anti-NGF to the cultures abolished neurite outgrowth. The results indicate that NGF is present in cartilage and bone of the chick embryo; it may determine the density of sympathetic innervation to the developing skeletal tissues.