Experimental Study On Tissue-engineered Bone Constructed By Goat Osteoinduced ADSCs With Surface-modified Spinal Cage

Objective:Nowadays, the incidence of spinal degenerative diseases keeps rising. The main treatment method includes spinal fusion and non-fusion procedures. In this study,nano-scaled beta-tricalcium phosphate/chitosan/polycarpolactone(β-TCP/CS/PCL)and titanium cages were used to construct different tissue-engineered bone with goat osteoinduced adipose derived stromal cells(ADSCs). Its compatibility and osteogenic potential were evaluated, and its feasibility was also detected.Methods:1.Goat ADSCs’ aspiration, isolation : goat fat tissue was collected, digested with0.1% collagenase type I. The third passage of cells was prepared to be used.2.Goat ADSCs’ osteoindution and grouping: The third passage of cells were divided into experimental and control groups. The experimental group was cultured with complete medium containing osteogenic reagents. The control group was cultured with complete medium. Alkaline phosphatase staining, Alizarin red staining and osteocalcin expression were detected to verify osteoinduced potential.3.Preparation and surface-modification of nano-scaled β-TCP/CS/PCL cage and Ti cage: Nano-scaled β-TCP/CS/PCL cages were divided into two groups.Group A was modified with RGD polypeptide, and group B was not modified. Ti cages were divided into two groups. Group C was surface-modified with femtosecond laser, and group D was not surface-modified.4.Four groups were planted with goat osteoinduced ADSCs and photoed by scanning electron microscopy on day 1,7,14 and 21. MTT detection and ALP quantitative detection were examined on day 1,4,7,10,13,16,19 and 22.Results:1.Primary ADSCs have settled down and adhered after 24 h. Primary ADSCs were irregular, mostly circular or multi-angel. ADSCs have gradually extended to the spindle and parts of them had coarse protrusions after 72 h. The third passage of ADSCs were regular and spindle with active proliferation. Osteoinduced ADSCs’ shaped into multi-angel after inducing. ALP staining shows that the majority of nucleus and cytoplasm stained positive. Alizarin red staining shows that red or orangecalcium deposited between cells. Osteocalcin expression of experimental group was significantly increased than the control group, the difference was significant.2.SEM image of cages which were cultured with cells shows: a large number of cells extended fully and adhered to the cages in group A, B. Cell growth of group A was better than group B. The number of cell adhered to Group C was less. The amount of cells of group D was less than group C. Cell proliferation activity of group A and B was better than group C.3.In the previous 14 days, MTT results and ALP activity of Group A, B, C increased as time continued while the rate of cell proliferation slowed down in the later period. OD values and ALP activity of Group A, B were higher than group C, D for each time point, the difference was significant(P<0.05). And Group A were higher than group B(P<0.05). Group C were higher than group D(P<0.05).Conclusion:1.We can separate goat ADSCs using collagenase digestion and adherence separation. Osteogenic potential were evaluated according to test results. Goat ADSCs can be used to build spinal tissue engineering models as seed cells.2.The cells grow well and secrete large amounts of extracellular matrix after ADSCs were cultured with nano-scaledβ-TCP/CS/PCL cage. It indicates that tissue engineered bone was constructed successfully in vitro. RGD polypeptide helps seed cell adhesion and promotes cell proliferation.3.The cells grow in the surface and secretion of extracellular matrix was not obvious after ADSCs were cultured with Ti cage. The amount of cells shedding was large in the surface of non-surface-modified cages. It showed that femtosecond helps seed cell adhesion and promotes cell proliferation.4.The biodegradable cage which has been chemically modified by RGD exists porosity and appropriate cell growth environment. However, The laser physics modified cells which were inoculated in the titanium cage can only stay on the surface, not grow deep inside. So the biodegradable cage has more significant biological advantages and application potential than the titanium cage.