Effects of chondrogenic microenvironment on construction of cartilage tissues using marrow stromal cells in vitro.

OBJECTIVE: To investigate whether it is feasible to use the chondrogenic microenvironment provided by cartilage cells to construct cartilage tissues in vitro with bone marrow stromal cells (BMSC).

MATERIALS AND METHODS: We isolated and cultured BMSC and cartilage cells from Sprague Dawley rats (SD rats). The supernatant of cartilage culture was used as inducing solution to cause differentiation of BMSC from the second generation of cells cultured in vitro. Cells were examined seven days later, using immunohistochemistry to determine the expression of collagen specific to type II cartilage. RT-PCR was used to detect the expression of type II collagen and aggrecan mRNA. BMSC and cartilage cells were isolated from SD rats and cultured in vitro. The BMSC and cartilage cells in culture were mixed evenly in an 8:2 ratio and inoculated into a polyglycolic acid/polylactic acid (PGA/PLA) scaffold to a final concentration of 5.0x10(7) cells/ml. PGA/PLA preparations with pure cartilage cells or pure BMSC served as the positive and negative controls, respectively. The control group of low-concentration cartilage cells consisted of PGA/PLA preparations containing cartilage cells at 20% of the above mentioned concentration (1.0x10(7) cells/ml). Samples were collected eight weeks later, at which time general observations, wet weight, and glycosaminoglycan (GAG) levels were determined, and histological and immunohistochemical examinations were performed.

RESULTS: Immunohistochemistry showed the induction of BMSC type II collagen, and RT-PCR indicated the expression of type II collagen and aggrecan mRNA. In the mixed-cell group and the positive control group, pure mature cartilage cells were produced after eight weeks of culture in vitro, and the size and shape of the scaffold were maintained throughout the culture period. The two groups gave rise to newly generated cartilage cells essentially identical in appearance and histological properties. The immunohistochemical results showed that the cartilage cells of both groups expressed abundant cartilage-specific type II collagen. The average wet weight and GAG content were more than 70% of the values in the positive control group. Only an extremely small amount of immature cartilage tissue formed in local regions in the BMSC-only sample, and the scaffold was obviously shrunken and deformed. Although the wet weight of newly generated cartilage tissue in the low-concentration cartilage cell sample reached 30% of the value of the positive control group, the scaffold was obviously shrunken and deformed. Only regional and discontinuous cartilage tissues were formed, and the amount of newly generated cartilage was obviously less than in the co-culture and positive control groups.

CONCLUSIONS: Cartilage cells can provide a microenvironment for cartilage formation to some extent, and also effectively induce BMSC to differentiate into cartilage cells and form tissue-engineered cartilage in vitro.