IN VITRO NEOCARTILAGE PRODUCTION USING 3D CHONDROCYTE CULTURES IN COLLAGEN GELS

Abstract

Purpose: Damaged cartilage has very limited potential for self-repair. Tissue bioengineering offers an interesting alternative for repair of cartilage injury caused by joint trauma or osteochondritis dessicans. The purpose of this work was to use primary chondrocytes cultivated in vitro on collagen gel to produce a neocartilage which can be reimplanted.

Material and methods: Chondrocytes were extracted by enzymatic digestion from calf feet harvested from animals aged less than six months. Two million cells were seeded on collagen gels in multiple-well plates and covered with culture medium (1 ml). Type I collagen was acquired from ground calf skin used at a concentration of 1.25 mg/ml. The culture medium was a v/v mixture of RPMI 1640 and NCTC 109. This mixture was supplemented with 10% foetal calf serum, 100 U/ml penicillin, and 250 ng/ml amphotericin B. Cell proliferation was assess fluorometrically and synthesis of glycosaminoglycans (sGAG) by colorimetric assay. Histological study (safranine O) and immunohistochemistry tests (type I and II collagen) were performed to monitor synthesis of matrix components. Expression of genes coding for certain matrix proteins (collagen Ia 2 and 1, II, X, agrecan and MMP13) was studied using RT-PCR.

Results: The chondrocyte phenotype was preserved. Type II collagen as well as agrecan was expressed and expression of type I collagen did not increase during the culture. Progressive synthesis of sGAG was observed as was moderate cell proliferation. Cell distribution within the gel was apparently homogeneous. The chondrocytes retained their round shape throughout the study. Type II collagen deposits were visible on day 9 in peripheral cells in areas of high-cell density, then progressed with time.

Discussion: Our in vitro results show that three-dimensional cultures of chondrocytes using a collagen gel can produce construction of an extracellular matrix with preservation of chondrocyte phenotype during the culture period.

Conclusion: The collagen matrix offers an environment favouring the formation of a functional artificial cartilage by chondrocytes and opens promising perspectives for repairing damaged cartilage.