[An in vitro study on three-dimensional cultivation with dynamic compressive stimulation for cartilage tissue engineering]

Abstract

Objective: To investigate the effect of three-dimensional cultivation with dynamic compressive stimulation on promotion of cartilage growth in vitro, by constructing tissue engineered cartilage with three-dimensional porous articular cartilage extracellular matrix (ECM) scaffolds laden with rabbit chondrocytes and performing mechanical stimulation by compressive stress in bioreactor.

Methods: Chondrocytes of healthy adult New Zealand rabbits were isolated, and passage 2 chondrocytes were seeded onto three-dimensional porous articular cartilage ECM scaffolds for 5 days pre-cultivation, and then were divided into 2 groups: Group A continued static culture as control; group B (dynamic culture condition) underwent dynamic compressive strain stimulation (compressive strain of 15%, frequence of 1 Hz) in a bioreactor. Cell viability and distribution in scaffolds were observed; the glycosaminoglycan (GAG) content, collagen content, and total DNA content were measured after 3 weeks of culturing; and elastic modulus was evaluated by mechanical test.

Results: Laser scanning confocal microscopy indicated that cells grew well and evenly distributed in the scaffold of group B, while poor cells growth and loss of staining in the central region of the scaffolds were observed in group A. Scanning electron microscopy showed that chondrocytes possessed good adhesion, proliferation, and growth on the scaffolds of group B; while the number of chondrocytes was significantly reduced, and cells scattered in group A. Biochemical composition analysis showed that collagen, GAG, and DNA contents of cell-scaffold constructs were (675.85 ± 27.93) μg/mg, (621.72 ± 26.75) μg/mg, and (16.98 ± 3.23) μg/sample in group B, and were (438.72 ± 6.35) μg/mg, (301.63 ± 30.51) μg/mg, and (10.18 ± 4.39) μg/sample in group A respectively, which were significantly higher in group B than in group A (t = -18.512, P = 0.000; t = 17.640, P = 0.000; t = 2.790, P = 0.024). Mechanical testing indicated that the elastic modulus of group B [(0.67 ± 0.09 ) MPa] was significantly higher than that of group A [(0.49 ± 0.16) MPa] and cell-free scaffolds [(0.43 ± 0.12) MPa] (P < 0.05).

Conclusion: Mimetic compressive stress with three-dimensional dynamic conditions created in the bioreactor is superior to the ordinary static three-dimensional cultivation, it can provide the optimal environment for chondrocytes on the ECM scaffolds, which may be a good way to construct tissue engineered cartilage in vitro.