Study Of Bone Marrow-derived Mesenchymal Stem Cells Combined With Silk Fibroin/Nano-hydroxyapatite In Constructing Tissue-engineering Bone In Vitro

ObjectivesTo investigate the degree of binding silk fibroin (SF)/nano-hydroxyapatite (nHA) composite to rabbit bone marrow mesenchymal stem cells (BMSC) culture in vitro as well as the feasibility of constructing tissue-engineered bone.MethodsStep one:Obtain rabbit bone marrow mesenchymal stem cells (BMSC) through separation and purification in vitro with whole marrow method followed by subculture, drawing growth curve with cell number as ordinate axis and time as the abscissa axis.Step two:Judge whether cultured cells are rabbit BMSC. Perform ossific inducing differentiation to the generation of BMSC with strongest proliferation capability according to the growth curve, observing the changes in cell morphology and follow by alkaline phosphatase staining test. Determinate whether it is BMSC based on combination of the two.Step three:Co-culture rabbit BMSC and SF/nHA in vitro constructing tissue-engineered bone model; observe the degree of binding cells to composites by microscope and scanning electron microscope (SEM); construct the control group (rabbit BMSC and bone induced artificial bone), observing the difference in the degree of binding at several different time points (2d,5d,2w). Step four:Inoculate and culture the cells eluted from two materials. Observe cell morphology and growth with normal cells cultured on the same condition as control group and evaluate the biocompatibility of composite.Results1. We obtained rabbit BMSC through separation and purification in vitro with whole marrow method followed by subculture and performed osteoblast differentiation. The cell morphology presented polygon and cone under microscope. Then we performed alkaline phosphatase staining test to cells. We could prove the cultured cells in the study were BMSC based on cell morphology, osteogenic induction and alkaline phosphatase staining.2. The cells adherence rate in SF/nHA group and the cell adherence rate of artificial bone were31.8%and17.6%respectively. There was a significant difference between them (P<0.05).3. Inoculated and cultured the cells eluted from the two materials. There was no significant difference in cell morphology compared with normal cells (inoculated in the same cell density and cultured conventionally).4. The adhesion, quantity and its distributed surface of BMSC on the two materials observed by SEM as well as SF/nHA were superior to artificial bone. Conclusions1. The tissue-engineering bone constructed by SF/nHA and BMSC in vitro is ideal.2. SF/nHA can better promote the adhesion, proliferation and differentiation of bone cells than artificial bone.3. SF/nHA has satisfactory biocompatibility.4. That rabbit BMSC successfully separated and cultured can differentiate into the osteoblast after directional induction and culture, which are seed cells for bone-tissue engineering.