[Fabrication of a novel cartilage acellular matrix scaffold for cartilage tissue engineering]

Abstract

Objective: To develop a novel cartilage acellular matrix (CACM) scaffold and to investigate its performance for cartilage tissue engineering.

Methods: Human cartilage microfilaments about 100 nm-5 microm were prepared after pulverization and gradient centrifugation and made into 3% suspension after acellularization treatment. After placing the suspension into moulds, 3-D porous CACM scaffolds were fabricated using a simple freeze-drying method. The scaffolds were cross-linked by exposure to ultraviolet radiation and immersion in a carbodiimide solution 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysucinimide. The scaffolds were investigated by histological staining, SEM observation and porosity measurement, water absorptiofl rate analysis. MTT test was also done to assess cytotoxicity of the scaffolds. After induced by conditioned medium including TGF-beta1, canine BMSCs were seeded into the scaffold. Cell proliferation and differentiation were analyzed using inverted microscope and SEM.

Results: The histological staining showed that there are no chondrocyte fragments in the scaffolds and that toluidine blue, safranin O and anti-collagen II immunohistochemistry staining were positive. The novel 3-D porous CACM scaffold had good pore interconnectivity with pore diameter (155 +/- 34) microm, 91.3% +/- 2.0% porosity and 2451% +/- 155% water absorption rate. The intrinsic cytotoxicity assessment of novel scaffolds using MTT test showed that the scaffolds had no cytotoxic effect on BMSCs. Inverted microscope showed that most of the cells attached to the scaffold. SEM micrographs indicated that cells covered the scaffolds uniformly and majority of the cells showed the round or elliptic morphology with much matrix secretion.

Conclusion: The 3-D porous CACM scaffold reserved most of extracellular matrix after thoroughly decellularization, has good pore diameter and porosity, non-toxicity and good biocompatibility, which make it a suitable candidate as an alternative cell-carrier for cartilage tissue engineering.