Protein fibrils as scaffold material for cartilage tissue engineering: effects on cell viability and proliferation

Abstract

preparation of scaffold-free cartilage-like cell-sheets derived from hBMSCs before, that the sheets were shrunk under the serum-free chondrogenic medium, but not under the serum mixed chondrogenic medium. Moreover, other groups have shown the addition of basic fibroblast growth factor (bFGF) during expansion phase enhanced not only their proliferation, but also their chondrogenic capacity of hBMSCs with chondrogenic pellet culture system after their expansion. This modification during expansion phase might be applied to the cell-sheets. The aim of this study is to investigate better condition to provide stably and securely the scaffold-free cartilage-like cell-sheets from hBMSCs. Methods: The hBMSCs were harvested from bone marrow obtained by routin iliac crest aspiration from 3 young male volunteers (21yo, 27yo, and 31yo) with their agreement before lower extremity joint surgeries. The proliferative and chodrogenic capacity were assessed at P1 stage. hBMSCs were expanded with DMEM-LG containing 1% Penicillin/ Streptomycin, condition (1)5%FBS, condition (2)10%FBS, or condition (3) 5%FBS with 10ng/ml of bFGF, in monolayer culture. The proliferation rate was determined from hemocytometer counts. Then, the cells harvested from the cultivation under (1), (2), and (3) were applied to vshaped non-adherent 96-well plate at 250,000 cells per well to perform chondrogenic pellet culture system for 21 days. Chondrogenic differentiation medium consisting of DMEM-HG with 1% ITS-premix, 10 nM Dexamethasone, 130uM ascorbate-2-phosphate, 1% sodium pyruvate,1% Penicillin/Streptomycin and 10ng/ml TGFb1 was employed for the pellet, and changed freshly every other day. The glycosaminoglycan (GAG) contents of each pellet were measured and normalized with DNA (GAG/ DNA) to evaluate their chondrogenic differentiation capacity. Moreover, the BMSCs from one donor (21yo) were expanded under the condition (3)5%FBS with 10ng/ml of bFGF, until P3 stage to fabricate scaffold-free cartilage-like cell-sheets with cell culture inserts (CCIs: 0.3 cm^2, thickness, 20-25mm, polyethylene terephthalate). The sheets were generated with condition (A) serum-free chodrogenic differentiation medium alone or condition (B) equal mixture of serum-free chondrogenic medium and growth medium with 10% FBS, condition(2), for 21 days. The pellets and cell-sheets were fixed with 10% formalin and embedded in paraffin and they were sectioned and stained with Safranin O/Fast Green. Results: The proliferation rates of condition(2) were 2 times higher than (1) (p < 0.05) and (3) were 4.5 times higher than (1) (p < 0.001). The difference between GAG/DNA of (1) and (2) was not significant, but that of (3) was 3 times higher than others (p < 0.05). Safranin-O staining (3) was stained well compared (1), and (2). The cell-sheets, neither under (A) and (B) were not shrunk nor detached from the inside wall of CCIs, but once taken from CCIs, the sheets under (A) looked shrunk and the edge was curling (Figure). The intensity of the Safranin-O staining showed no difference betwee...