Fred Harwood

Rib perichondrial grafts for the repair of full-thickness articular-cartilage defects. A morphological and biochemical study in rabbits.

The purpose of this study was to investigate the use of perichondrial grafts in articular cartilage defects and to characterize the newly formed cartilage. In a rabbit model, rib perichondrium was used to repair full-thickness defects in the femoral condyle. The quality of repair was then evaluated histologically and biochemically at six and twelve weeks after grafting. Unacceptable results were obtained in 50 per cent of the rabbits. These failures were due to condylar fracture in 20 per cent, failure of graft attachment in 20 per cent, and infection in 10 per cent. The technique of grafting must be improved to increase the percentage of successful grafts in which neocartilage with a relatively normal chemical composition fills the articular cartilage defect. Successful grafts proliferate to fill the full-thickness defect with neocartilage, which has biochemical characteristics that are similar to those of hyaline cartilage.

A histological and biochemical assessment of the cartilage matrix obtained from in vitro storage of osteochondral allografts.

Fresh osteochondral allografts were stored at 4 degrees C in tissue culture media at variable time periods (3, 7, 14 and 28 days). Sterilely dissected tibial plateaus with a standardized 1/2 cm subchondral bone shell were obtained from canines 1-3 hrs post mortem. X-rays were taken to determine maturity of the animals. Only mature animals (closed epiphyses) were considered for the study. Histologically, safranin 0 (metachromatic stain for glycosaminoglycans) was observed in all experimental specimens. H&E stained sections showed at all time periods of 3, 7, 14 and 28 days that the cell morphology and arrangements were similar in the superficial and deep areas of the cartilage obtained from the stored osteochondral allograft when compared to the control articular cartilage. The cells were in lacunae and arranged in clusters. Biochemically, glycosaminoglycans and collagen content showed no difference at the 95% level of confidence during the duration of the study (28 days) when compared to the 0 day control cartilage. Collagen typing, based on the assessment by HPLC of the CNBr peptides showed the major presence of type II collagen (no evidence of dedifferentiation was observed). No type I was found to be present. Some apparent variations in the proportions of minor collagen components were noted--e.g. at 14 days the cartilage appeared to contain increased amounts of type XI but little or no type IX collagen (HMW, LMW) when compared to the day 0 control. At 28 days a shift to a larger amount of type IX collagen occurs, especially in the LMW component, with a small amount of type XI collagen when compared to normal day 0 articular cartilage. Cell viability, i.e., the ability of the allograft tissue to incorporate 35SO4 in the synthesis of glycosaminoglycans, was intact up to 28 days of storage.

Value of hyaluronic acid in the prevention of contracture formation.

Joint stiffness secondary to immobilization was inhibited by intra-articular hyaluronic acid injection in an experimental joint contracture in rabbits. Biochemical and biomechanical parameters were used to evaluate the joint stiffness after nine weeks of immobilization. In all treatments, hyaluronic acid reduced the measured stiffness in the contracture by approximately 50% as compared to the contractures of the untreated rabbits. In addition, hyaluronic acid prevented the loss of glycosaminoglycans (GAGs) (as measured by hexosamine), which normally occurs in untreated contractures. The results are related to a working hypothesis that intra-articular injections of drugs such as hyaluronic acid (Healon-R) will stimulate hyaluronic acid synthesis within the matrices of periarticular connective tissue (PCT). If the spacing and lubricating properties of the glycosaminoglycans could be maintained in the stress-deprived state, the centripetal collapse of the fibrillar matrix could be avoided, anomalous cross-links could be minimized, and more normal joint mechanics could be retained.

Nutrition of cruciate ligament reconstruction by diffusion: Collagen synthesis studied in rabbits

The reconstructed anterior cruciate ligament was studied in the rabbit using the medial third of the patellar tendon. Tritiated proline, 100 microCi/kg body weight, was injected intra-articularly to insure detection of the metabolic conversion product 3H-hydroxyproline in the avascular graft. During the immediate postoperative period, nutrients were found to derive from the synovial fluid through a process of diffusion, demonstrating that synovial nutrition occurs prior to revascularization of the graft.

Collagen Types in Neocartilage Tissue Resulting from Rib Perichondrial Graft in an Articular Defect — a Rapid Semi-Quantitative Methodology

A method for estimating type II to type I collagen ratios in small tissue samples has been developed. The cyanogen bromide peptides of the tissue collagens were analyzed by SDS-gel electrophoresis. Marker peptides representative of each collagen type were established and their relative amounts determined by integration of the stained peptide bands following gel scans. Marker peptide ratios were then computed for each of several standard type II/type I mixtures and these peptide ratios were mathematically correlated with the corresponding type II/type I collagen ratios. A linear relationship between marker peptide ratio and collagen type ratio was established. This relationship was applied to the analyses of type II/type I ratios in samples of rib perichondrium and neocartilage derived from perichondrial graft repairs of full thickness femoral condyle defects. The results indicated that perichondrial grafts synthesize both types II and I collagens and that the proportion of type II increases with increasing post-transplant time.

Effect of Low Dosage Schedule of D-Penicillamine on Collagen Cross-Linking in a Nine Week Immobilized Rabbit Knee

Rabbits immobilized with one leg pinned in a fully flexed position, were treated with 60 mg of D-penicillamine/kg three times a week for 9 weeks. After this period of immobilization the animals were sacrificed and the periarticular connective tissue collagen reduced with [3H] NaBH4 for determination of collagen cross-links. There was a significant decrease in the formation of cross-links in the control and immobilized knee in the treated animals as compared to the untreated animals. Penicillamine also blocked the increase in DHLNL, HLNL and HHMD previously demonstrated in immobilized rabbit knees. This is the first demonstration that in vivo administration of penicillamine directly inhibits cross-linking reactions in collagen.

Architectural remodeling of fresh meniscal allograft transplantation in a rabbit joint : Its long-term protective effect on articular cartilage

This study assessed a fresh medial meniscal allograft transplantation model in the rabbit knee. Biological characterization included assessment of collagen remodeling of the allograft and the potential protection against cartilage degeneration. Allograft transplantation was performed on the left knee, and total meniscectomy on the right knee. Forty-seven rabbits were operated on and assessed at 9, 12, and 26 weeks. Fresh medial meniscal allografts showed collagen remodeling that paralleled the revascularization and cellular proliferation of the allografts. Revascularization was shown as early as 9 weeks from the periphery, extending to the inner one-third of the allograft by 26 weeks. Viability assessment of the meniscal allograft cells showed live cells at the periphery of the allograft at 9 and 12 weeks. At time 0, i.e., the time of the transplant, few viable cells were observed within the donor tissues. Biochemically, collagen remodeling, in terms of increased reducible collagen crosslinks, i.e., dihydroxylysinonorleucine, and the percentage of collagen present, was seen throughout the 26-week observation period. At 26 weeks, the meniscal allografts inhibited degenerative changes of the femoral and tibial cartialage compared to results with total meniscectomy.