Conclusions
The method of MC culture is feasible under in vitro free-swelling and dynamic loading conditions, simulating in vivo posttransplantation. Mechanical stimulation significantly provokes cellular outgrowth and long-term chondrogenic maturation at the mRNA level, whereas histology depicts immature neotissue where typical cartilage matrix is expected.
Methods
Minced, fibrin-associated, bovine articular cartilage fragments were cultured in vitro within polyurethane scaffold rings. Constructs were maintained either free swelling for two or four weeks (control), underwent direct mechanical knee-joint-specific bioreactor-induced dynamic compression and shear, or they were maintained free swelling for two weeks followed by two weeks of bioreactor stimulation. Samples were collected for glycosaminoglycan (GAG)/DNA quantification; collagen type I, collagen type II, aggrecan, cartilage oligomeric matrix protein (COMP), proteoglycan-4 (PRG-4) messenger RNA (mRNA) analysis; histology and immunohistochemistry.
Purpose
Our aim was to explore the effect of varying in vitro culture conditions on general chondrogenesis of minced cartilage (MC) fragments.
Results
Cellular outgrowth and neomatrix formation was successfully accomplished among all groups. GAG/DNA and collagen type I mRNA were not different between groups; chondrogenic genes collagen type II, aggrecan and COMP revealed a significant downregulation among free-swelling constructs over time (week two through week four). Mechanical loading was able to maintain chondrogenic expression with significantly stronger expression at long-term time points (four weeks) in comparison with four-week control. Histology and immunohistochemistry revealed that bioreactor culture induced stronger cellular outgrowth than free-swelling constructs. However, weaker collagen type II and aggrecan expression with an increased collagen type I expression was noted among this outgrowth neotissue. Conclusions: The method of MC culture is feasible under in vitro free-swelling and dynamic loading conditions, simulating in vivo posttransplantation. Mechanical stimulation significantly provokes cellular outgrowth and long-term chondrogenic maturation at the mRNA level, whereas histology depicts immature neotissue where typical cartilage matrix is expected.