| Biological material as the scaffold for bone regeneration combining with the seed cells formed cell-scaffold hybrid materials. The hybrid materials were transplanted into bone injury site to regenerate via immune system function, cell proliferation and growth factors secretion. The hybrid materials were able to be completely degraded and seed cells were able to differentiate into osteocytes in vivo, which achieved the regeneration of bone.Choosing seed cell and scaffolds are the hot and difficult topics for Bone regeneration. Owing to biological characteristics of stem cells, they are to be seen as important means to treat pathological and physiological diseases such as bone defect, systemic lupus erythematosus and hypohemia. However, the sources of stem cells are limited. Scaffolds for bone regeneration must be non-poisonous, and have feasible mechanical performance, stable and continuous degradation and good biocompatibility. Currently known single material is difficult to meet the requirements. Some materials including KGM, HA, HAP, β-TCP and CS are frequently used to prepare scaffolds. But they are far from to the salvation of the Bone regeneration ScaffoldsIn order to solve the two hot topic above, the thesis were written into three parts:The first part dealt with the KGM/HA and HAP/β-TCP/CS preparation according to the physicochemical properties and biological characteristics of above materials in order to overcome the difficult of single material. The second part concentrated with reprogram of somatic cells into induced multipotent stem cells (iMS) via fish oocyte extracts. The third part contributed to regeneration of bone defects via animal model with the aid of iMS cells as seed cells combining with scaffolds. The three parts and a short summary were given below.1. Preparation of KGM/HA and HAP/β-TCP/CS scaffolds:(1) Preparation of KGM/HA scaffoldsKGM/HA composite scaffolds were produced following the basic procedures below. KGM/HA mixture were prepared by sol-gel method with ammonia solution served to adjust pH value at room temperature. The mixture formed the KGM/HA composite scaffolds by bath curing, freezing and freeze drying in turn. The structure of KGM/HA were analyzed by SEM and optical microscope methods. In addition, the porosity, pore diameter, compressive strength, water absorption, degradation rate and compatibility with stem cells of KGM/HA scaffold were studied.The results showed that:KGM/HA composite scaffolds displayed three-dimensional meshed structure, porosity of70～90%. About80to90%of all pore diameters were between100～250μm and the compressive strength were about0.4MP. KGM/HA composie scaffolds formed three-dementional meshed structure. HA has a good compatibility with KGM. HA and KGM formed three-dementional porous scaffolds by hydrogen bond. The water absorption of scaffolds was up to1168%and gradually rised with increasing KGM in composition scaffolds. The scaffolds showed certain degradation performance and about40%could be degradated within12weeks. The degradation ration decreased with increasing the ratio of KGM. The scaffolds had a good compatibility by culturing iMS experiment. Cell number decreased with increasing HA.(2) Preparation of HAP/β-TCP/CS scaffoldsThe HAP powders were synthesized by wet chemical precipitation. HAP/β-TCP scaffolds were prepared via foam dipping method. The HAP/β-TCP/CS composite scaffolds were fabricated through soaking of chitosan acetic solution into the HAP/β-TCP scaffolds.The soaking solution was produced after certain concentration of chitosan acetic acid solution was vacuumed. Then, the scaffolds with chitosan formed after few minutes vacuum of soaking scaffold into solution. The composite scaffolds were obtained after drying. The acetic acid was removed with an equal volume of sodium carbonate solution. The residual salt was removed by dipping the scaffolds into the distilled water at2centigrade. HAP/β-TCP/CS composite scaffolds were produced after drying at37centigrade.HAP/β-TCP/CS composite bone scaffolds were three-dimension cross-connection porous structure, and average pore rate of HAP/β-TCP was90%. After attached to CS, forming network structure closed the scaffolds’ pore structure, which decreased the porosity of locality. Average pore rate of HAP/β-TCP/CS was83%. Apertures were mainly distributed in the range of50～400μm, and compressive strength was about1.38MPa. The in vitro degradation process of HAP/β-TCP/CS mainly included two stages. The first stage was that attached CS in the outside and β-TCP were completely degraded within nine weeks. The second stage was that mainly HAP was degraded after nine weeks. The degradation speed of HAP was very slow, and HAP couldn’t be completely degraded at12th week yet. However, CS and β-TCP could be completely degraded within nine weeks.2. Reprogram of somatic cells into induced multipotent stem cells (iMS cells)The oocyte extract were used as revulsant and fibroblasts as seed cells. Fibroblasts were reprogrammed into induced multipotent stem cells by certain exposure time of revulsive. Cell surface antigens were detected by immunohistochemistry and flow cytometry. The gene expression was detected by PCR. The differentiation potentials of iMS cells in vitro and in vivo were studied by directed induction and forming subcutaneous teratoma of nude mouse, respectively. The epigenetic mechanisms of differentiating somatic cells into stem cells were studied by DNA methylation and phosphorylation analysis.The results showed that:the cell morphology constantly changed along with the increasing concentration and exposure time of revulsive. The cell shape presented more irregular and bigger than control. The expression of stem cell markers Oct3/4, Sox2, Nanog and c-Myc showed positive. In vitro directional induction and teratoma tissue slice results showed that the induced multipotent stem cells were able to differentiate into osteoblast-like cells, neurocyte-like cells, adipocyte-like cells and blood vessel, nerve, muscle and adipose tissue. The results of RT-PCR and real-time PCR showed that the expression of pluripotent marker genes Oct3/4and Nanog were strongest by exposure of72hours. The extent of DNA methylation of the four regions of promoters of the genes Oct3/4and Nanog were lower than that of the control group. The phosphorylation level of these proteins inculuding p38, AS160, P-catenin in three pathways became higher than that of control group. Three pathways were up-regulated or opened.3. The biocompatibility of cells and composition scaffoldsInduced multipotent stem cells and bone marrow mesenchymal stem cells as seed cells were respectively co-cultured with KGM/HA and HAP/β-TCP/CS as support carriers. We detected the cell proliferation in the composite scaffolds by MTT method. SEM was used to test cell adhesion ability. The biocompatibility of iMS cells and composite scaffolds were studied with the methods of in vitro co-culture, and the in vivo subcutaneous embedding. The cells differentiating into osteocytes and the degradation of scaffolds were studied by bone defect repairing of model animal, X-ray, tissue slice and SEM.The results showed that:The co-cultured iMS cells with composite scaffolds KGM/HA and BMMSCs with HAP/β-TCP/CS grew well and grew into the hole. The composition scaffolds implanted cells were transplanted into subcutaneous tissue of Kunming mouse, and didn’t cause inflammation. The BMMSCs-HAP/β-TCP/CS composite materials were transplanted into dog bone defect site. After6months, new bone tissue generated at the bone defect site which indicated BMMSCs combined with HAP/β-TCP/CS were able to repair and treat bone defect. The iMS cells-KGM/HA composite materials were transplanted into rabbit bone defect site. After8weeks, the defect was partly repaired. However, the difference of repair effects of8and12weeks was not significant which indicated KGM/HA had the function of Osteoconduction, but no Osteo-induction.In summary, the composite scaffolds HAP/p-TCP/CS and KGM/HA were produced using polymeric materials and ceramics materials. Both composite scaffolds had higher porosity, pore size distribution and good biocompatibility. Reprogram of fibroblasts into iMS cells by exposure of fish oocyte extracts were archived. This reprogramming method provided a new source for stem cells which can be served to repair bone tissue defect. Animal models of repairing bone damage with iMS cells combining with composition scaffolds were preliminarily established that provided the important scientific and theoretical data for clinical application research.
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