Preliminary Study Of Effect Of Percutaneous CPC/FG/OM Composite Material Filling Combined With Translation Of HBMSCs On Treatment Of Femoral Head Osteonecrosis

Chapter I Evaluation of feasibility of human bone marrow mesenchymal stem cell cultured by autologous serumObjective:Translations of human bone marrow mesenchymal stem cell cultured by fetal bovine serum have various risks which limit its clinical application. The experiment was to investigate the feasibility of establishing a suitable hBMSCs lines for clinical application cultured by autologous serum instead of fetal bovine serum in vitro.Methods:The hBMSCs was isolated using whole bone marrow adherent way. The passage3of hBMSCs was devided as two groups according the different culturing serum:group of fetal bovine serum and group of autologous serum. The growth of cells were observed by using inverted phase contrast microscope; the MTT method was used to measured the proliferation of passage3and5, then the results were turned to the cell proliferation curve drawing; the passage3of two groups were taken to differentiating into cartilage cells (toluidine blue staining and collagen Ⅱ staining), osteoblasts cells (alkaline phosphate staining and VonKossa staining) and fat cells (oil red O staining) respectively, the positive rates of cell differentiation of two group were calculated; the passage5of autologous serum group was identified CD34,C44,CD45and CD90antigen expression by flow cytometry.Results:The hBMSCs purified by whole bone marrow adherent way was observed taking a elongated spindle presentation under inverted phase contrast microscope. Cells of two groups showed a spiral-like matter of growth in near fusion. The results of MTT show that the passage3and5of two groups possessed a good potential for growth and proliferation. The period of logarithmic growth phase was3-6d. There is no statistic difference between the two groups. hBMSCs of two groups can be differentiated into cartilage cells(stained purple by toluidine blue and yellow by collagen II), osteoblasts(stained ALP positive and lack in cell nuclear by VonKossa) and fat cells(stained red lipid droplets by oil red O), the positive rate of cell differentiation show no significant statistic difference between two groups; the passage5cultured by autologous serum was identified the CD44and CD90antigen expression by flow cytometry without CD34and C45expression. Conclusions:Whole bone marrow adherent culture way is simple and has high purification rate of hBMSCs. The hBMSCs cultured by autologous serum maintained the typical growth characteristics and potentials of proliferation and differentiation of stem cells. The hBMSCs of autologous serum group did not express hematopoietic stem cell surface antigen but BMSCs surface antigen which fit the BMSCs characteristics. Chapter II Preparation of CPC/FG/OM composite material and evaluation of its physical propertiesObjective:The clinical treatment of femoral head osteonecrosis remains a tough issue. Researches have shown that supporting the subchondral bone of femoral head is a key factor for treatment. Calcium phosphate cement (CPC) is a bone tissue material combined repairing and building, which have properties of self-setting and bone conduction. However, due to defects of CPC materials, CPC has some disadvantages such as low strength and lack of bone inducing activity which limit its clinical application especially in weight-bearing area of human body. Fibrin glue(FG) is widely used in clinical as a excellent adhesives, which can increase compressive strength of the CPC by using its viscosity. Osteogenesis medium(OM) has been proved having capability to differentiate stem cells into osteoblasts. The experiment is to evaluate physical property of a novel material, CPC/FG/OM composite material, for treatment of femoral head osteonecrosis. The novel material was expected to remain properties of self-setting and bone conduction of CPC, along with increased compressive strength by adding FG to fulfill the requirement for support the subchondral bone of femoral head and property of bone induction by adding OM to fulfill the requirement for improving the osteogenic activity in femoral head osteonecrosis.Methods:OM was added into FG to making FG/OM solution. According to a3:1power/liquid ratio, CPC powder was mixed with FG/OM solution to making CPC/FG/OM composite materials. CPC/FG material and CPC material was made as the same powder/liquid ratio and regarded as two control groups. Evaluation the physical properties include scanning electron microscopy observation; XRD analysis; setting time (Gilmore needle method); compressive strength and elastic modulus(MTS S10mechanical properties testing machine) and porosity(Archimedes method).Results:Uneven pore distribution and large size pore were observed in both CPC/FG and CPC/FG/OM group comparing with smooth distribution and small size pore in CPC group(SEM). The result of XRD analysis find that the three group has similar crystal phase to hydroxyapatite; the initial setting time of CPC/FG and CPC/FG/OM [(6.8±0.5)min,(7.28±0.6)min]were significantly longer than the CPC group[(3.38±0.4) min](there is significant statistic differences, P<0.05); the final setting time of three group was similar, there is no significant statistic difference among them(P>0.05); the compressive strength and elastic modulus of CPC/FG and CPC/FG/OM [(8.94±0.5)MPa,(9.1±0.4) MPa;(2.73±0.07)GPa,(2.846±0.1)GPa] were significantly higher than the CPC group[(5.08±0.5)MPa,(1.064±0.14)GPa](there is significant statistic differences,P＜0.05); the porosity of CPC/FG and CPC/FG/OM [(38.64±1.5)%,(37.9±1.4)%]were significantly lower than the CPC group[(48.82±8.2)%](there is significant statistic differences, P<0.05).Conclusions:The three groups have similar crystal phase to hydroxyapatite which means the degraded products in vivo is the same as inorganic constitutes of human bone. Adding OM did not influence the physical property of CPC/FG material:setting time reach the property of injectability when applying percutaneous clinical operation; compressive strength and elastic modulus reach the requirement of support the subchondral bone of femoral head; despite of lower porosity, the surface of CPC/FG/OM have large size pore which can easier for early attachment of seed cells. ChapterⅢ Evaluation of autologous serum cultured hBMSCs seeding on CPC/FG/OM composite materialObjective:There are two key elements need to be concerned in treatment of femoral head osteonecrosis:rebuilding both the biomechanical strength (supporting the subchondral bone) and biological strength (autologous repairing) of femoral head. The development in both bone tissue engineering and stem cell engineering make it a new direction for treatment of femoral head osteonecrosis-translation of stem cells. Study in chapter II has proved that CPC/FG/OM composite material can be an ideal material to rebuild biomechanical strength of femoral head immediately. This chapter aims to evaluate the biocompatibility and property of bone induction of CPC/FG/OM composite material by culturing autologous serum-cultured hBMSCs with material.Methods:Preparations of materials were according to same methods described in chapter Ⅱ. Autologous serum-cultured hBMSCs was prepared in ways described in chapter I. Three materials were put into three separated24-well plate with hBMSCs seeding. SEM (scanning electron microscope) was used to observe the growth of cells in three materials. The proliferation of cell were measured by MTT method in the period of1to7-day respectively; mRNA expression of ALP, COL-I and OCN were measured by RT-PCR method; and activity of ALP(pNPP kit) and content of OCN(human OCN ELISA kit) were measured in the period of3-day,7-day and14-day respectively.Results:hBMSCs were observed in good shape and lots of cell-cell junction in both CPC/FG and CPC/FG/OM group with less junction in CPC group(SEM observation). The proliferations of hBMSCs were significant increased in both CPC/FG and CPC/FG/OM group. The expression of mRNA of ALP, COL-I and OCN was highest in CPC/FG/OM group with CPC/FG lower and CPC lowest. The acticities of ALP were highest in CPC/FG/OM group with CPC/FG lower and CPC lowest. The content of OCN was positive in CPC/FG/OM group after3-day culturation; however it is positive until post-14-day culturation in CPC and CPC/FG group.Conclusions:CPC/FG/OM composite material has excellent biocompatibility and strong property of bone induction comparing with CPC/FG group and CPC group. Chapter IV Animal studies on percutaneous CPC/FG/OM composite material filling combined with translation of hBMSCs in treatment of femoral head osteonecrosisObjective:Evaluating the effect of percutaneous CPC/FG/OM composite material filling combined with translation of hBMSCs on treatment of femoral head osteonecrosis through animal studies.Methods:The hormonal combined endotoxin injection method was chosen to establish New Zealand white rabbit model of femoral head osteonecrosis.(Ear vein injection of Escherichia coli endotoxin (lOug/kg) combined with intramuscular injection methyl-prednisolone (40mg/kg in New Zealand white rabbits). After the animal model have been successfully established,24rabbits were randomly divided into four groups:group A: percutaneous group(simple decompression group); group B:percutaneous hBMSCs translation group; group C:pertaneous CPC/FG/OM filling group and group D:percutaneous CPC/FG/OM filling combined with translation of hBMSCs group. Every group has six rabbits. One side was operated and the contralateral side was regarded as the control group with no operation taken. Radiology and histology evaluation were made every4w,8w and12w.Results:There are no statistic difference among group A,B and control group, radiology exam show that cystic area in subchondral area increased in early stage with narrowing joint space of operation side. The gross view of femoral head show many pit in the femoral head which turn to be a white color in both group, HE staining show that rare intramedullary osteoblasts in the subchondral bone of femoral head and few bone matrix formation, there are no statistic difference about cell count of intramedullary osteoblasts and percentage of bone resorption area among this three group in12w; group C and D have a good effect treatment. Radiology exam show the round shape of femoral head remained in12w and found no cystic lesion in subchondral area of femoral head. Group C has a clearer image of material than group D which has new bone formation image around the material. Gross view of femoral head show white color in group C comparing with the normal color in group D. HE staining show more osteoblasts and new bone matrix formation in group D. More cell count of osteoblasts and less percentage of bone resorption in subchondral area was observed in group D comparing with group C in12w.Conclusions:Results of the animal study show that percutaneous CPC/FG/OM composite materail filling combined with translation of hBMSCs has a significant effect on dealing with femoral head osteonecrosis, compared with core decompression, hBMSCs translation and CPC/FG/OM filling method.