| Objective①In order to obtain high pure seed cells to establish a simple and effective method of isolation, purification and culture of BMSCs in vitro using Percoll desity gradient centrifugation combined with adherent culture.②To evaluate the biocompatibility of collagen sponge and BMSCs.③To evaluate the osteogenic differentiation of the cells transplanted in the BMSCs-collagen sponge scaffold complex.④To investigate the effectiveness of the BMSCs-collagen sponge scaffold complex for bone formation and lumbar interbody fusion using a rabbit model.Methods⑤BMSCs were isolated from2weeks old rabbit femur bone marrow by Percoll density gradient centrifugation combined with adherent culture methods. and identified by morphological observation, flow cytometry instrument surface phenotype detection and differerntation potential research.②The P3BMSCs and collagen sponge were co-cultured, adhesion rate formula was used to calculate the rate of cell adhesion, the cell proliferation in the scaffold was observe by MTT assay and the proliferation curve was draw. and the situation of cells and the connection between cells and scaffolds was investigated by microscope. HE staining and scanning electron microscope.③The third passage cells were continued to be induced in osteogenic medium for14days, the cells were detected with alkaline phosphatase staining and alizarin red staining for differentiating into osteoblasts.④Forty mature male White Zealand rabbits (weigh,3.5～4.5kg) were randomly allocated to receive one of the following graft materials:porous collagen sponge plus BMSCs (group Ⅰ); porous collagen sponge alone (group Ⅱ); autogenous bone graft (group Ⅲ); and nothing (group Ⅳ). All animals underwent anterior vertebral interbody fusion at the L4/L5level. The lumbar spine was harvested en bloc, and the new bone formation and spinal fusion was evaluated using radiographic analysis, microcomputed tomography, manual palpation test, and histological examination at12weeks after surgery.Results①A form of spindle-shaped uniform adherent cells was isolated with the methods of Percoll density gradient centrifugation and adherent culture, the positive rates of CD44, CD90, CD105, CD34, and CD45in the third passage BMSCs were99.34%,98.12%,96.44%,2.18%and1.92%respectively. The BMSCs, which were continued to be induced in osteogenic medium for14days, differentiated into osteoblasts.②The average adhesion rate was83.13%, the BMSCs were able to proliferate normally in the collagen sponge scaffolds. compared with control group. the platform duration was longer and the number of BMSCs was larger. The number of cells continued to increase with culture time and the cell morphology changed from spindle to polygonal by light microscope. HE staining showed that the scaffold pores filled with BMSCs, and scanning electron microscopy revealed clusters of polygonal cells attached to the scaffold surface and part of them migrated into the scaffold pores.③Alkaline phosphatase staining showed that the cells that were cultured in osteogenic medium after14d were positive. and alizarin red staining revealed there were extracellular calcium deposition and calcium nodules. which indicated that the cells differentiated into osteoblasts.④The weight of all animals were increased two weeks after surgery. By manual palpation test and radiographic analysis. bony fusion was observed in40%(4/10) of rabbits in group I,70%(7/10) of rabbits in group Ⅲ. and in0%(0/10) of rabbits in both group Ⅱ and Ⅳ. There was no statistically significant difference between group I and group Ⅲ. By radiographic analysis.microcomputed tomography and histological analysis, mature bone marrow and trabecular bone were detected at fused segments. New formation bone and the adjacent vertebral segments integrated, fibrous tissue was observed in the defect sites, but no collagen sponge was found in all intervertebral spaces.Conclusion①The combination of Percoll density gradient centrifugation and adherent culture is an ideal method to obtain high purity rabbit BMSCs.②The collagen sponge possesses good biocompatibility and is suitable for scaffolds in tissue engineering.③The BMSCs, which are transplanted in collagen collage, have the ability to differentiate into osteoblasts, and the scaffold dose not affect the differentiation.④The rabbit model can become an optimal small animal experimental one in interbody fusion biological research. The BMSCs-collagen sponge complex in vivo form bone and promote rabbit lumbar interbody fusion. |
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