Temporal matrix synthesis and histologic features of a chondrocyte-laden porous collagen cartilage analogue
Nixon, A.J.; Sams, A.E.; Lust, G.; Grande, D.; Mohammed, H.O.
American Journal of Veterinary Research 54(2): 349-356
1993
ISSN/ISBN: 0002-9645 PMID: 8430945 Document Number: 417750
Cartilage resurfacing by chondrocyte transplantation, using porous collagen matrices as a vehicle to secure the cells in cartilage defects, has been used experimentally in animals. This in vitro study evaluated the temporal morphologic features and proteoglycan synthesis of chondrocyte-laden collagen matrices. Forty-two porous collagen disks were implanted with a minimum of 6 times 10-6 viable chondrocytes, covered by a polymerized collagen gel layer, and 6 disks were harvested after 0, 3, 7, 10, 14, 18, or 22 days of incubation in supplemented Ham's F12 medium at 37 C and 5% CO-2. Histologic and histochemical evaluation of formalin-fixed segments of the cultured disks indicated that the chondrocytes proliferated in the implant, producing small groups and linear segments of cells by day 14. The collagen framework remained intact over the course of the study with thick areas attributable to depositions of matrix material after day 10. Alcain blue-stained matrix was evaluated in the pericellular region of chondrocytes in sections of disks harvested on days 14, 18, and 22. Glycosaminoglycan (GAG) assay by dimethyl-methylene blue dye binding after papain digestion of the disk segments revealed negligible amounts of GAG at day 0. Significant (P ltoreq 0.0001) increase in total GAG content was observed by day 3 (0.329 mu-g/mg of disk) and further increases were observed until a plateau in GAG quantity was seen on day 14. Mean peak GAG content was 0.553 +- 0.062 mu-g/mg. Secondary treatment of the papain-digested implants with keratanase and chondroitinase ABC yielded similar trends in chondroitin sulfate (CS) and keratan sulfate (KS) concentrations. The CS content significatnly (P = 0.0002) increased for the first 14 days of incubation, then a plateau was observed for the remainder of the study. Peak CS content was 0.354 +- 0.037 mu-g/mg. Concentration of KS reached a pleateu earlier than did CS content, with peak amount of 0.193 +- 0.027 mu-g/mg on day 10. Fluctuations in KS content were not significant until an increase on day 22. Chondrocytes actively populated the collagen implants, increasing in number and synthesizing matrix GAG epitopes over the 22 days of incubation. These results indicate that chondrocyte-laden porous collagen matrices may be suitable cartilage analogue materials and the optimal metabolic time for transfer to cartilage defects is 10 to 14 days.