Mesenchymal Stem Cells Embedded In An Injectable Atelocollagen Scaffold For Disc Regeneration In A Rabbit Model

Part1Rabbit mesenchymal stem cells co-culture with an injectableatelocollagen scaffold: an in vitro studyObjective: To investigate the biological characteristics of an injectable cell-scaffoldcomposite with rabbit mesenchymal stem cells (MSCs) embedded in the atelocollagenscaffold in vitro.Methods: The rabbit tibia bone was punctured and2mL bone marrow was extracted.The MSCs were processed by culture, proliferation and passage. After identify the cellsurface markers (i.e., CD34, CD44and CD90) with a flow cytometry machine, the MSCswere co-cultured with an injectable atelocollagen scaffold. An inversion microscope wasused to observe the growth characteristics and FDA-PI staining was employed to clarifythe growth and survival rate of MSCs in the scaffold.Results: The MSCs expressed CD44and CD90, however, did not express CD34. Inaddition, the MSCs embedded in the atelocollagen grew well,3days after the embedment,the cells with typical fibroblast-shaped morphology could be observed and after14days,the edge of the hydrogel was degrade and the cells fell off and adhered to the culturetemplate. The FDA-PI staining showed none dead cells were detected after14days culture.Conclusions: The cultured cells have the essential phenotypes and characteristics ofthe MSCs, and seeding cells embedded in atelocollagen scaffold can grow and proliferatewell, atelocollagen is an ideal injectable scaffold for embedding MSCs for culture. Part2Mesenchymal stem cells embedded in an injectable atelocollagenscaffold for disc regeneration in a rabbit modelObjective: To explore the feasibility of mesenchymal stem cells embedded inatelocollagen scaffold for intervertebral disc degeneration.Methods:20white New Zealand rabbits are randomly divided into three groups:1)normal control group (n=4);2) degeneration model group (n=8); and3) transplantationgroup (n=8). In degeneration model group, induce and establish the degeneration merelyby means of puncturing the annulus fibrosus with needles of defined gauges and aspiratingsome nucleus pulposus tissue from the intervertebral discs; in transplantation group,2weeks after inducing degeneration model, the cell-scaffold (MSCs co-culture withatelocollagen) composite was transplanted. The X-ray, MRI, histological and RT-PCR wereapplied to clarify the regeneration effects in vivo.Results: the disc height index (DHI) obviously decreases in the degeneration modelgroup and moderate decreases in transplantation group (P＜0.05). MRI results show anincreasingly aggravated degeneration in the degeneration model group while thedegeneration in the transplantation group is not obvious (P＜0.05). Macroscopicevaluation showed significant disc degeneration in degeneration model group, however, inthe transplantation group, the basic structure of annulus fibrosus was normal, the discsdemonstrated nucleus pulposus regeneration, but the structure of nucleus pulposus wasincomplete. Histological analysis demonstrated that degeneration model group showedsignificantly disorder, and the transplantation group showed slightly disordered annulusfibrosus with nucleus pulposus regeneration. RT-PCR confirmed that there werestatistically significant differences in expression of the II collagen and aggrecan betweentransplantation and degeneration model group (P＜0.05).Conclusion: the cell-scaffold composite could restore the disc height and increase theII collagen and aggrecan content in the degenerated discs, which illustrated that the composite can prohibit or delay intervertebral disc degeneration.