Chondrocyte-seeded hydroxyapatite for repair of large articular cartilage defects. A pilot study in the goat.

The aim of this study was to evaluate the potential for restoration of a large cartilage defect in the goat knee with hydroxyapatite (HA) loaded with chondrocytes. Isolated chondrocytes were suspended in fibrin glue, seeded on top of the HA, and then the composite graft was implanted in the defect. After transplantation, cell behaviour, newly synthesised matrix and the HA-glue interface were assessed histologically after 2, 4, 12, 26 and 52 weeks. Special attention was paid to the incorporation process of HA in the subchondral bone and interactions between this biomaterial and the fibrin-glue-chondrocyte suspension. Chondrocytes in the glue proved to survive the transplantation procedure and produced new metachromatically stained matrix two weeks after implantation. The glue-cell suspension had penetrated the superficial porous structure of the HA. Four weeks after surgery, islands of hyaline-like cartilage were observed at the HA-glue interface. A layer of fibrous tissue was formed surrounding the HA graft, resulting in a relatively instable fixation of the HA in the defect. This instability of the graft in the defect, possibly together with early weight bearing, resulted in a gradual loss of the newly formed hyaline cartilage-like repair tissue. Progressive resorption of the HA occurred without any sign of active bone remodelling from the host site. One year after surgery part of the defect which extended down to the cancellous bone had been predominantly restored with newly formed lamellar bone. Only small HA remnants were still present at the bottom of the original defect. Resurfacing of the joint had occurred with fibrocartilaginous repair tissue. The absence of adequate fixation capacity of the HA near the joint space resulted in a relative instability of the graft with progressive resorption. Therefore, HA is not a suitable biomaterial to facilitate the repair of large articular cartilage defects.

Chondrocyte behaviour within different types of collagen gel in vitro.

In cartilage repair experiments chondrocytes are transplanted into osteochondral defects. Biological substances are used as cell vehicles and are likely to play an important role in the outcome of these studies. Collagen gel is formed by polymerization of type I collagen and is used in plastic surgery and for three-dimensional culture systems. To test collagen gel as a potential vehicle for transplantation, we evaluated chondrocyte behaviour in vitro in different collagen gels. Collagen type I was extracted and purified from rat tail tendon and fetal calf skin and compared with commercially available collagen type I. After suspension of bovine chondrocytes, five different collagen gels were cultured for 14 days and evaluated by light and electron microscopy. Cells proliferated within all gels and synthesized proteoglycans as assessed by 35S incorporation; 40-90% of cells maintained a chondrocyte-like morphology after 1 week in culture depending on the type of collagen gel. Synthetic and secretory activity was confirmed by electron microscopy. Based on these results, calf skin collagen is recommended for culturing chondrocytes for implantation.

Resurfacing potential of heterologous chondrocytes suspended in fibrin glue in large full-thickness defects of femoral articular cartilage: an experimental study in the goat.

A large full-thickness articular-cartilage defect was created in the medial femoral condyle of 32 adult goats. The defects were xenografted with isolated rabbit chondrocytes suspended in fibrin glue. Sham operated goats, where only a standardized defect was created, were used as controls. Results of cartilage repair were assessed after 3, 8, 13, 26 and 52 weeks. The repair tissue was evaluated macroscopically, histologically and biochemically. Results indicated that xenografted rabbit chondrocytes survived the transplantation and maintained their potential to produce matrix in fibrin glue, particularly if they were located in a non-weight-bearing area. In terms of an immunological reaction to xenografted chondrocytes, only mild signs of synovitis were observed in both groups and rejection of transplanted cells did not occur. From 3 weeks gradually progressive resolvement of the fibrin glue was observed with subsequent replacement by fibrous tissue. Initially xenografted defects histologically showed better tendency for cartilage regeneration, however, 52 weeks after surgery no significant differences could be detected in the repair tissue of both groups macroscopically, histologically and on biochemical scoring. The amount of collagen type II in the newly synthesized matrix was 75% 1 year after surgery. This study shows that isolated heterologous chondrocytes can be used for transplantation in articular cartilage defects, however, fibrin glue does not offer enough biomechanical support to the cells to maintain its function as a three-dimensional scaffold.

The potential of adult human perichondrium to form hyalin cartilage in vitro.

The usefulness of adult human perichondrium for the restoration of articular cartilage defects depends on the potential to form hyalin cartilage. In order to evaluate the capacity of adult human perichondrium to form hyalin cartilage in vitro, perichondrium of the rib of eight adult human beings was cultured in vitro. After removal of residual cartilage, perichondrial explants were cultured for 7 or 10 days. The explants were histologically examined using specific stains to prove the presence of glycosaminoglycans (GAGs) normal for hyalin cartilage. Clear differentiation of perichondrial cells towards chondrocytes was noted. The chondrocytes synthesized new matrix substances normally present in hyalin cartilage. This investigation supports the usefulness of adult human rib perichondrium for the restoration of cartilage defects. Due to the enormous potential of the rib perichondrium to form hyalin cartilage in vitro, even defects in joints with a rather thick cartilage layer might be restored using this biological material.

Perichondral grafting for cartilage lesions of the knee.

Twenty-five patients with 30 chondral lesions of the knee were treated with an autogenous strip of costal perichondrium. The graft was fixed to the subchondral bone with Tissucol (Immuno, Vienna), a human fibrin glue. The leg was then immobilised for two weeks followed by two weeks of continuous passive motion. Weight-bearing was permitted after three months. The mean knee score (Ranawat, Insall and Shine 1976) changed from 73 before operation to 90 one year after; in 14 patients evaluated after two years there was no decrease. In 28 cases the defect was completely filled with tissue resembling articular cartilage. We conclude that in most cases perichondral arthroplasty of cartilage defects of the knee gives excellent results.

[Release of the lateral retinaculum of the knee in retropatellar chondropathy]. / Klieven van het laterale retinaculum van de knie bij retropatellaire chondropathie.

Retropatellar chondropathy is a frequent cause of pain in the knee; it usually runs a favourable natural course. When the symptoms interfere with work or sports, reduction of pain can be achieved by cleaving the lateral retinaculum (lateral release). To assess the results, a questionnaire was sent to 96 persons; 80 questionnaires could be evaluated. Only patients who had minor or no visible damage of the patellar cartilage were included in this study. Fifty-three patients reported improvement, in spite of which 34 had persistent mild symptoms which forced 12 of them to change the nature of their work. Thirty-seven patients had to change their sports activities in spite of the operation.

Delayed intraosseous venous blood flow does not correlate with patellar cartilage changes in anterior knee pain.

In 67 patients with longstanding anterior knee pain intra-osseous phlebography of the patella and arthroscopy of the knee were performed. No statistical correlation could be demonstrated between the delayed drainage of the contrast medium from the patellar veins and the degree of damage to the patellar cartilage.

Long-term inversion stability of the ankle after rupture of the lateral ligaments.

The late results of surgical and nonsurgical treatment of recent ruptures of the lateral ligaments of the ankle are compared in two groups of 52 patients. By means of a questionnaire, physical examination and inversion stress roentgenography (at an average of three years after the injury), the functional and mechanical stability of the ankle were determined. After both surgical repair and treatment with plaster cast alone, more than 90% of patients achieved inversion stability of the ankle.

Repair of sheep articular cartilage defects with a rabbit costal perichondrial graft.

A xenograft of costal rabbit perichondrium was transplanted to an articular cartilage defect in a sheep knee. After 12 weeks, cartilage was formed with increased calcification of the basal layer and a mean of 74 percent collagen type II. The synovium did not show any infiltration, indicating the absence of any immunologic reaction.

Repair of articular defects by perichondrial grafts. Experiments in the rabbit.

A defect was created in the articular cartilage of the rabbit knee leaving the subchondral bone intact. The lesion was repaired by an autologous graft of costal perichondrium and fixed with fibrin glue. The result was compared with a nontreated defect in the contralateral knee. In 26 out of 30 knees, graft fixation proved to be adequate. In the grafted group the perichondrium developed macroscopically and histologically into normal hyaline cartilage. The nongrafted defects showed only limited repair.

Chondrocyte behavior in fibrin glue in vitro.

To test fibrin glue as a vehicle in chondrocyte transplantation, chondrocytes, isolated from articular cartilage of young rabbits, were mixed with Tissucol, a highly concentrated fibrin glue, and cultured for 7 days. Histology, autoradiography (35-S) and electron microscopy were used to study chondrocyte behavior and phenotypic expression. Chondrocytes multiplied, retained their morphology, and produced matrix in fibrin glue as long as the cells were surrounded by the glue. Glue disintegration started after 3 days and was accelerated by higher cell concentrations.

Optimization of cryopreservative procedures for human articular cartilage chondrocytes.

Procedures are being developed which use isolated articular cartilage (AC) chondrocytes to restore damaged articular surfaces. The availability of isolated human chondrocytes for transplantation may be increased by low-temperature storage (banking). At present, no single method of freezing chondrocytes has been proven to be optimal. In this project, two different freezing protocols, I and II, were compared. Protocol I used freezing rates of -1 degrees C/min down to a temperature of -40 degrees C. Protocol II used a freezing rate of -1 degree C/min down to -10 degrees C and faster rates thereafter. Cells were stored for 2 weeks at -196 degrees C. Survival and function of the cells after thawing were evaluated by histological examination and determination of 35S- and 3H-thymidine incorporation after 1 and 2 weeks of high-density monolayer culture. Cells frozen with protocol I showed better function and survival (99.75%) than cells frozen with protocol II (85%). Both groups showed slowing of metabolism and replication after freezing when compared with controls. We conclude that controlled freezing of adult human chondrocytes at rates of -1 degrees C/min improves survival. Banking of human AC chondrocytes may be feasible using protocol I, although some questions regarding the long-term behaviour of human AC chondrocytes after cryopreservation remain to be answered.

Culture of chondrocytes in alginate and collagen carrier gels.

In this in vitro study, we compared the potential of collagen and alginate gels as carriers for chondrocyte transplantation and we studied the influence of demineralized bone matrix (DBM) on chondrocytes in the gels. Chondrocytes were assessed for cell viability, phenotype (histology), proliferation rate and sulfate incorporation. Collagen gels showed a significant increase in cell numbers, but the chondrocytes dedifferentiated into fibroblast-like cells from day 6 onwards. In alginate gels, initial cell loss was found, but the cells maintained their typical chondrocyte phenotype. Although the total quantity of proteoglycans initially synthesized per cell in collagen gel was significantly higher, expressed per cell, the quantity in alginate gel eventually surpassed collagen. No effects of culturing chondrocytes in combination with DBM could be demonstrated on cell proliferation and sulfate incorporation. The collagen and alginate gels have different advantages as carriers for chondrocyte transplantation. The high proliferation rate of chondrocytes in collagen gel may be an advantage, but the preservation of the chondrocyte phenotype and the gradually increasing proteoglycan synthesis in alginate gel is a promising method for creating a hyaline cartilage implant in vitro.