Although the low power of our study prevents definitive conclusions, ACI and mosaicplasty are cartilage repair techniques that are clinically equivalent and similar in performance. The high percentage of spontaneous improvement ((1/3) of the patients) observed after simple debridement calls into question the need for prompt surgical treatment of patients with lesions similar to those included in this clinical trial. Moreover, this finding warrants further investigation, ideally through randomized clinical trials in which patients subjected to debridement alone are compared with patients undergoing reconstructive surgery.

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Human articular cartilage heals poorly in adults and current surgical procedures do not provide long-term repair. Cell therapy and tissue engineering could become the treatment of choice, but suffer a major limitation as chondrocytes in vitro lose the differentiated phenotype. In vivo, the chondrogenic lineage is specified by transcription factor Sox9. Thus, cell-based therapy could be successful if Sox9 expression and chondrogenic commitment of the expanded cells were preserved. To achieve this goal, we developed a serum-free medium that supports cell proliferation and preserves the differentiation potential. Indeed, expression of Sox9 is maintained when the conventionally used serum is substituted for by this defined supplement. Spontaneous cartilage formation after expansion in serum-free medium is obtained in vitro in a high-density pellet culture and confirmed in vivo in a functional assay in immunodeficient mice. By contrast, cells grown in serum lose the expression of Sox9 and fail to reform cartilage both in vitro and in vivo unless they are rescued by chondrogenic inducers such as transforming growth factor beta(1) and dexamethasone. Our data emphasize the importance of the microenvironment in modulating commitment, plasticity, and phenotype of chondrocytes, and provide an experimental system to study their physiological or pathological metabolism in a controlled context.

Abstract. Differentiation of hypertrophic chondrocytes toward an osteoblast-like phenotype occurs in vitro when cells are transferred to anchorage-dependent culture conditions in the presence of ascorbic acid (Descalzi Cancedda, E, C. Gentili, P. Manduca, and R. Cancedda. 1992. J. Cell Biol. 117:427-435). This process is enhanced by retinoic acid addition to the culture medium. Here we compare the growth of hypertrophic chondrocytes undergoing this differentiation process to the growth of hypertrophic chondrocytes maintained in suspension culture as such. The proliferation rate is significantly higher in the adherent hypertrophic chondrocytes differentiating to osteoblastlike cells. In cultures supplemented with retinoic acid the proliferation rate is further increased. In both cases cells stop proliferating when mineralization of the extracellular matrix begins. We also report on the ultrastructural organization of the osteoblast-like Cell cultures and we show virtual identity with cultures of osteoblasts grown from bone chips. Cells are embedded in a dense meshwork of type I collagen fibers and mineral is observed in the extracellular matrix associated with collagen fibrils. Differentiating hypertrophic chondrocytes secrete large amounts of an 82-kD glycoprotein. The protein has been purified from conditioned medium and identified as ovotransferrin. It is transiently expressed during the in vitro differentiation of hypertrophic chondrocytes into osteoblast-like cells. In cultured hypertrophic chondrocytes treated with 500 nM retinoic acid, ovotransferrin is maximally expressed 3 d after retinoic acid addition, when the cartilage-bone-specific collagen shift occurs, and decays between the 5th and the 10th day, when cells have fully acquired the osteoblast-like phenotype. Similar results were obtained when retinoic acid was added to the culture at the 50 nM "physiological" concentration. Cells expressing ovotransferrin also coexpress ovotransferrin receptors. This suggests an autocrine mechanism in the control of chondrocyte differentiation to osteoblast-like cells.T H~ chick epiphyseal growth plate is a unique site to investigate ontogenic processes leading to the formation of endochondral bone. The sequence of events includes the formation of hypertrophic cartilage, its invasion by blood vessels from the perichondrium, the erosion of the calcified cartilage, and its replacement by bone tissue. It is generally accepted that hypertrophic chondrocytes in the growth plate degenerate at the site of the transition region from cartilage to osteoid and ultimately die. Several authors have reported that in organ cultures hypertrophic chondrocytes start expressing bone markers and they have proposed that hypertrophic chondrocytes may contribute to the formation of a bone matrix (37,41,43). We have recently shown that in vitro chick hypertrophic chondrocytes, obtained as single isolated cells after 3 wk in suspension culture, further differentiate to osteoblast-like ceils when transferred to anchorage-dependent culture c...

Autologous chondrocyte implantation is currently applied in clinics as an innovative tool for articular cartilage repair. Animal models have been and still are being used to validate and further improve the technique. However, in various species, the outcome varies from hyaline-like cartilage to fibrocartilage. This may be due partly to the spontaneous dedifferentiation of chondrocytes once cultured in vitro. Here we assessed whether the extent of dedifferentiation varies between species and we hypothesized that the level of chondrocyte phenotype stability during expansion may contribute to the maintenance of their chondrogenic commitment and redifferentiation potential. Condyle chondrocytes were harvested from sheep, dog, and human, and expanded for 1, 6, or 12 cell duplications. At each interval, cell phenotype was monitored (morphology and biosynthesis of cartilage markers) and redifferentiation was assessed by an in vitro assay of chondrogenesis in micromass pellet and an in vivo assay of ectopic cartilage formation in immunodeficient mice. Results indicate that, during culture, the sheep chondrocyte phenotype is maintained better than that of human chondrocytes, which in turn dedifferentiate to a lesser extent than dog chondrocytes Accordingly, after expansion, sheep chondrocytes spontaneously reform hyaline-like cartilage; human chondrocytes redifferentiate only under stimulation with chondrogenic inducers whereas, after a few passages, dog chondrocytes lose any capacity to redifferentiate regardless of the presence of inducers. Thus, conditions allowing cartilage formation in one species are not necessarily transposable to other species. Therefore, results with animal models should be cautiously applied to humans. In addition, for tissue-engineering purposes, the number of cell duplications must be, for each species, carefully monitored to remain in the range of amplification allowing redifferentiation and chondrogenesis.