ABSTRACT
OBJECTIVE: To date numerous attempts have been undertaken to conquer the challenging problem of reconstructing long segmental tracheal defects, as yet without lasting success. Recently, employing concepts of tissue engineering in animals, cartilage-like constructs were transplanted in vivo. However, both the feasibility of fabricating tracheal replacements and the use of human tracheal chondrocytes (HTC) for tissue engineering are still under investigation. In this study, we optimized isolation and cultivation techniques for human tracheal cartilage, assessing the feasibility of seeding these cells onto a novel, three-dimensional (3-D) polyester-urethane polymer (DegraPol). METHODS: Human tracheal cartilage was harvested from the trachea of lung donors, digested in 0.3% collagenase II, and the condrocytes serially passaged every 7-9 days. Cells were also cultivated over agar plate during the total 6-8 weeks expansion phase. Thereafter, chondrocytes were seeded onto DegraPol (pore sizes 150-200 microm) with a seeding density of 2.4 x 10(7)/ml, and chondrocyte-polymer constructs maintained during in vitro static culture. RESULTS: HTC displayed stable proliferation kinetics in monolayer culture with positive expression of collagen type II. Following polymer seeding, both cellular proliferation and extracellular matrix (ECM) production, as measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and glycosaminoglycan assays, continued over extended culture. Active growth of HTC on DegraPol was further demonstrated by Alcian blue staining, with the histomorphological appearance of the construct resembling that of native cartilage. Scanning electron microscopy showed chondrocyte growth and ECM synthesis both on the surface and inside the porous scaffold, with a dense cell layer on the surface of the scaffold and a lower cell distribution in the scaffold's interior. CONCLUSIONS: The harvested chondrocytes from human trachea cartilage expand well in vitro and possess the ability to form new cartilage-like tissue when seeded onto DegraPol matrix. However, improved culture conditions are needed to permit cellular growth throughout cell-polymer constructs.
