Age-dependent responsiveness of rabbit and human cartilage cells to sex steroids in vitro

Rabbit epiphyseal carilage tissue has been shown to convert testosterone (T) to dihydrotestosterone (DHT). In this report, the metabolic conversion of T into DHT is shown to be age-dependent, being most active in cartilage from animal at the age of gonadal maturation. Human cartilage from newborn and prepubertal children is also shown to convert T into DHT and—to a lesser extent—to estradiol.

Low concentrations of DHT and 17β-estradiol (E₂) (10⁻¹¹−10⁻⁹ M) were also shown to stimulate in vitro cartilage cells from boys and girls respectively. As previously shown for cultured rabbit chondrocytes, the stimulating effects of both hormones on human chondrocytes was age-dependent. Cartilage cells derived from children up to one year old did not respond, while cells from boys and girls in the early phase of puberty responded best.

These data indicate that human cartilage tissue in vivo, contains both 5-reductase and aromatase activities during post-natal skeletal growth. Androgens may act on cartilage after their metabolic conversion to estrogens. The mechanism of age-dependency of both cartilage androgen enzymatic activities and chondrocyte responsiveness to sex steroids in vitro remains to be explained.     

Regulation of articular chondrocyte catabolic genes by growth factor interaction

Osteoarthritis is characterized by a loss of articular cartilage homeostasis in which degradation exceeds formation. Several growth factors have been shown to promote cartilage formation by augmenting articular chondrocyte anabolic activity. This study tests the hypothesis that such growth factors also play an anticatabolic role. We transferred individual or combinations of the genes encoding insulin-like growth factor-I, bone morphogenetic protein-2, bone morphogenetic protein-7, transforming growth factor-β1, and fibroblast growth factor-2, into adult bovine articular chondrocytes and measured the expression of catabolic marker genes encoding A disintegrin and metalloproteinase with thrombospondin motifs-4 and -5, matrix metalloproteinases-3 and -13, and interleukin-6. When delivered individually, or in combination, these growth factor transgenes differentially regulated the direction, magnitude, and time course of expression of the catabolic marker genes. In concert, the growth factor transgenes regulated the marker genes in an interactive fashion that ranged from synergistic inhibition to synergistic stimulation. Synergistic stimulation prevailed over synergistic inhibition, reaching maxima of 15.2- and 2.7-fold, respectively. Neither the magnitude nor the time course of the effect of the transgene combinations could be predicted on the basis of the individual transgene effects. With few exceptions, the data contradict our hypothesis. The results demonstrate that growth factors that are traditionally viewed as chondrogenic tend also to promote catabolic gene expression. The competing actions of these potential therapeutic agents add an additional level of complexity to the selection of regulatory factors for restoring articular cartilage homeostasis or promoting repair.     

Perlecan: an important component of the cartilage pericellular matrix

Perlecan (Pln) is a large proteoglycan that can bear HS (heparan sulfate) and chondroitin sulfate glycosaminoglycans. Previous studies have demonstrated that Pln can interact with growth factors and cell surfaces either via its constituent glycosaminoglycan chains or core protein. Herein, we summarize studies demonstrating spatially and temporally regulated expression of Pln mRNA and protein in developing and mature cartilage. Mutations either in the Pln gene or in genes involved in glycosaminoglycan assembly result in severe cartilage phenotypes seen in both human syndromes and mouse model systems. In vitro studies demonstrate that Pln can trigger chondrogenic differentiation of multipotential mouse CH310T1/2 stem cells as well as maintain the phenotype of adult human chondrocytes. Structural mapping indicates that these activities lie entirely within domain I, a region unique to Pln, and that they require glycosaminoglycans. We also discuss data indicating that Pln cooperates with the key chondrogenic growth factor, BMP-2, to promote expression of hypertrophic chondrocyte markers. Collectively, these studies indicate that Pln is an important component of human cartilage and may have useful applications in tissue engineering and cartilage-directed therapeutics.     

Optimal combination of soluble factors for tissue engineering of permanent cartilage from cultured human chondrocytes

Since permanent cartilage has poor self-regenerative capacity, its regeneration from autologous human chondrocytes using a tissue engineering technique may greatly benefit the treatment of various skeletal disorders. However, the conventional autologous chondrocyte implantation is insufficient both in quantity and in quality due to two major limitations: dedifferentiation during a long term culture for multiplication and hypertrophic differentiation by stimulation for the redifferentiation. To overcome the limitations, this study attempted to determine the optimal combination in primary human chondrocyte cultures under a serum-free condition, from among 12 putative chondrocyte regulators. From the exhaustive 2(12) = 4,096 combinations, 256 were selected by fractional factorial design, and bone morphogenetic protein-2 and insulin (BI) were statistically determined to be the most effective combination causing redifferentiation of the dedifferentiated cells after repeated passaging. We further found that the addition of triiodothyronine (T3) prevented the BI-induced hypertrophic differentiation of redifferentiated chondrocytes via the suppression of Akt signaling. The implant formed by the human chondrocytes cultured in atelocollagen and poly(l-latic acid) scaffold under the BI + T3 stimulation consisted of sufficient hyaline cartilage with mechanical properties comparable with native cartilage after transplantation in nude mice, indicating that BI + T3 is the optimal combination to regenerate a clinically practical permanent cartilage from autologous chondrocytes.     

Human polymer-based cartilage grafts for the regeneration of articular cartilage defects

The use of autologous chondrocyte implantation (ACI) and its further development combining autologous chondrocytes with bioresorbable matrices may represent a promising new technology for cartilage regeneration in orthopaedic research. Aim of our study was to evaluate the applicability of a resorbable three-dimensional polymer of pure polyglycolic acid (PGA) for the use in human cartilage tissue engineering under autologous conditions. Adult human chondrocytes were expanded in vitro using human serum and were rearranged three-dimensionally in human fibrin and PGA. The capacity of dedifferentiated chondrocytes to re-differentiate was evaluated after two weeks of tissue culture in vitro and after subcutaneous transplantation into nude mice by propidium iodide/fluorescein diacetate (PI/FDA) staining, scanning electron microscopy (SEM), gene expression analysis of typical chondrocyte marker genes and histological staining of proteoglycans and type II collagen. PI/FDA staining and SEM documented that vital human chondrocytes are evenly distributed within the polymer-based cartilage tissue engineering graft. The induction of the typical chondrocyte marker genes including cartilage oligomeric matrix protein (COMP) and cartilage link protein after two weeks of tissue culture indicates the initiation of chondrocyte re-differentiation by three-dimensional assembly in fibrin and PGA. Histological analysis of human cartilage tissue engineering grafts after 6 weeks of subcutaneous transplantation demonstrates the development of the graft towards hyaline cartilage with formation of a cartilaginous matrix comprising type II collagen and proteoglycan. These results suggest that human polymer-based cartilage tissue engineering grafts made of human chondrocytes, human fibrin and PGA are clinically suited for the regeneration of articular cartilage defects.