RESUMO
Objective To construct genipin-crosslinked rat acellular spinal cord scaffolds and evaluate their enzymatic degradation rate,biomechanical properties and cytotoxicity.Methods Rat spinal cord scaffolds were decellularized by chemical extraction and chemically crosslinked with 5 g/L genipin solution.Micro-structure of the uncrosslinked and genipin-crosslinked acellular spinal cord scaffolds were observed by HE staining and scanning electron microscopy and properties of pore size,porosity,water ratio,and degradation rate in 2.5 g/L trypsin enzyme solution were examined.Ultimate tensile strength and elastic modulus of normal rat thoracic spinal cord,uncrosslinked and genipin-crosslinked acellular spinal cord scaffolds were determined on Instron mechanical testing instrument.Rat bone marrow mesenchymal stem cells were cultured in lixivium of uncrosslinked and genipin-crosslinked acellular spinal cord scaffolds and MTT assay for relative cell growth rate was test to evaluate the cytotoxicity of scaffolds.Results The uncrosslinked and the genipin-crosslinked acellular spinal cord scaffolds possessed a similar three-dimensional mesh-porous structure with a mean pore diameter about 30 μm and a porosity over 80％,but there was a statistical difference between the two groups(P ＞ 0.05).Water ratio of genipincrosslinked scaffolds was (229.7 ± 12.5) ％,far lower than (283.4 ± 11.2) ％ of uncrosslinked scaffolds (P ＜ O.05) ; genipin-crosslinked acellular spinal cord scaffolds had lower weight loss at each time point than the uncrosslinked acellular spinal cord scaffolds (P ＜ 0.05),but the stability in trypsin,ultimate tensile strength and elastic modulus of acellular spinal cord scaffolds were significantly enhanced by genipin-crosslinking (P ＜ 0.05).Furthermore,no obvious cytotoxicity was observed in the uncroslinked and genipin-crosslinked scaffolds.Conclusions Rat acellular spinal cord scaffolds present no obvious change in structure after genipin-crosslinking,but there is significant improvement in the biomechanical properties and ability against enzymatic degradation and no marked cytotoxicity.Hence,the genipincrosslinked scaffolds are promising in tissue engineering for spinal injury.
RESUMO
Objective To observe three-dimensional structure and biological features of rat acellular spinal cord scaffold prepared by sonic oscillation and chemical extraction in order to offer an ideal scaffold for spinal cord tissue engineering research.Methods Rat spinal cord underwent acellular treatment with sonic oscillation and chemical extraction (Triton X-100 at volume fracture of 2％ and sodium deoxycholate at volume fracture of 2％) (acellular spinal cord group).In contrast with spinal cord tissue of normal rats (control group),general morphology,histology and ultramicro three-dimensional structure of acellular spinal cord scaffold were observed and aperture size,factor of porosity,water ratio,enzymolysis ratio and stability in water solution of the scaffold were also detected.Results Acellular spinal cord group showed effective removal of original cell components with factor of porosity for (94.57 ±3.45) ％ and water content for (88.62 ± 1.0) ％,and satisfactory three-dimensional structure with average aperture of 46 μm.Scaffold showed gradual degradation in enzymolysis solution and enzymolysis rate reached (69.03 ± 2.19)％ at 20 hours.Besides,scaffold showed stepwise disintegration in double distilled water and hydrolysis rate was (62.55 ± 1.70) ％ at 8 days.While,normal spinal cord showed close structure,generous neurons and myelin sheath with factor of porosity for (0.04 ± 0.02) ％ and water content for (62.4 ± 1.5) ％,and unobvious pore structure under scanning electron microscope.Normal spinal cord were degraded gradually in enzymolysis solution and enzymolysis rate was (37.62 ± 0.9)％ at 20hours.In the meantime,normal spinal cord were disintegrated gradually in double distilled water and hydrolysis rate was (40.97 ± 0.81) ％ at 8 days.Conclusions Acellular spinal cord scaffold prepared by sonic oscillation plus chemical extraction achieves complete removal of cell components,intact extracellular matrix,and satisfactory results in three-dimensional network structures,factor of porosity and water content.Also,the scaffold meets theoretical demands of tissue-engineered spinal cord scaffold and is an ideal alterative for tissue-engineered spinal cord scaffold.