| Objectives: 1. To extract rabbit's platelet-rich plasma using two steps centrifuging in laboratory and count it's quantity of platelet. To study the influence of PRP with different concentration on the proliferation of bone marrow stromal cells (BMSCs). 2. To construct an injectable tissue-engineered bone graft with fibrin glue (FG),autologous PRP and BMSCs using tissue-engineered technique. Observing the growth condition of seed cells in scaffold materials and studying the micro-constitution of injectable tissue-engineered bone.3. To construct a compound injectable bone graft with medical fibrin glue and autologous PRP ,using this bone graft only or compounded with BMSCs to repair the segmental defect of long bone .To discuss the feasibility of using autologous PRP repairing bone defect and probe into a new path-way of repairing bone defect with multiplicate autologous growth factors. The final pouporse of this study is to develop a new -style compound injectable tissue-engineered bone graft which involved multiplicate bio-active growth factors and BMSCs ,then using this tissue-engineered bone to repairing bone defect.Methods:The first part The extract of rabbit' PRP and it's influence on BMSCs: The blood sample was gained from rabbits' central artery in the back of ear. Sodium citrate was used as anticoagulant. In order to get PRP, we take of two steps centrifuging. The quantity of platelet in PRP or whole blood was calculated respectively from the observation of microscope.Bone marrow stromal cells which were isolated from rabbits' iliac marrow were culture-expanded in vitro using "whole marrow cultured method". Five different concentration of PRP(1%n 5%> 10%n 20%% 30%)were take as experimental groups ,the group without PRP as control group . The MTT method was utilized to detect the influence of PRP on BMSCs.The second part The construction of an injectable tissue-engineered bone with autologous PRP and observeing it's ultramicro-structure. Bone marrow stromal cells which were isolated from rabbits' iliac marrow were culture-expanded in vitro using "whole marrow cultured method". In order to induce BMSCs' polarization, the conditional medium was utilized ,which involved 10% fetal bovine serum (FBS), 50ug/ml ascorbic acid, 10~8mol/L dexamethasone, 103mol/L beta Sodium Glycerophosphate.The injectable tissue-engineered bone constructed from autologous PRP, FQ and BMSCs was cultured in vitro. Inverted microscope was used to observe the growth of cells. After the tissue-engineered bone was cultured a week , some samples were took out to make a histological section and have HE stain .Some samples were sent to have a scanning electron microscope observation. And the other tissue-engineered bone (that was cultured a week in vitro) was cut into little chips , digested with 0.25% trypsin and cultured again, observing the alive rate of the seed cells.The third part 36 New-Zealand rabbits were divided into 3groups randomly and equally,12 in each group . A 1.5cm segmental defect in right radius was made ineach animal and three different bone grafts were implanted into it respectively. Group A: FG+PRP+MSCs; Group B:FG+PRP; Group C: FGo About a month before the operation ,MSCs aspirated from cilium of rabbits were cultured and multiplied in vitro .PRP was extracted from autologous artery blood and stored in a refrigeratory (-70 ° C ).In addition , another 4 rabbits which the same defects in radius left empty were regarded as the blank group.At the time-points of 4n 8, 12 weeks after implanting operation ,anteroposterior radiographs were taken on the right radius in all animals , and two rabbits were sacrificed and take specimens from the middle of bone defect to have a histological observation.After 12 weeks of the implanting all animals were sacrificed and the radius were take to analysis the biomechanics strength .The reparative effects of bone defects were evaluated by radiographic analysis > histological observation and biomechanics analysis .Results: l.The average platelet in PRP was more than four times than that in whole blood . 2.The outcome shows that all experimental groups have accelerated BMSCs' proliferation in the experiment of MTT .(compared with the control group ,using analysis of variance, P<0.05). PRP's effect of accelerated cells proliferation rely on it's dosage. At the range from 1% to 20% concentrateion of PRP, this effect enhances with the increase of PRP(P<0.05). However, compared with the 20% group, the 30% group shows a weaken effect on cells' proliferation. (P<0.05) and there are not significance difference between the30% group and 10% group. ( P>0.05) .2. In this experiment,we construct a kind of injectable tissue-engineered bone,which begin to form a kind of gel between20 to 30 seconds after the mixture of solutions A and solutions B. In the invert microscope , it can be seen a lot of cells which have all kinds of shape such as:globular shape> multiangel shape and fusiform cell. After cultured one week, the gelatine begin to degrade, fusiform cells could beseen adhered to the bottom of the Petri dish.Histological analysis exhibited so much globular and olivary cells distribute in the gel extensively. Using scanning electron microscopy, it can be found that globular and olivary cells embed in the fibrin glue ,around of the cells ,there are a lot of little particales,some particales condense in to a mass and some adhere on the surface of cell membrane . The tissue-engineered bone that was cultured a week in vitro)was cut into little chips , digested with 0.25% trypsin and cultured again, Three days later, many of the productive multi-angel shape and fusiform cell adhered on the bottom of the petri dish and lived prosperously.3. Roentgenographic scores and callus density at bone defect in group A and B were better than those in group C at postoperative 4^ 8^ 12weeks.There were no significant statistical differences between group A and group B in the Roentgenographic scores. But the callus density in group A was better than that in group B .The bone defects in blank group were the same size after 16 weeks of the operation. Histological analysis exhibited that the new bone formation in group A and group B are superior to what in group C at each point of time .In group A and B,most of the FG was absorbed and substituted by new formed cortical bone 8 weeks postoperatively. After 12 weeks of the operation ,a matured and plenty of bone matrix new bone filled in the bone defect and there formed a medullary cavity in group A. There were no significant statistical difference between the biomechanical strength of radius in group A and that in normal group(P>0.05) .But what in group B was significant lower than that in group A (P<0.05).Conclusion : PRP have an effect of acceleration the proliferateion of BMSCs cultured in vitro . this effect rely on the dosage of PRP in a some degree .the construction of a injectable tissue-engineered bone with FG > BMSCs and autologous PRP is a simplified metho. Injectable tissue-engineered bone involved more livelyseed cells. Since PRP can promote healing of bone defect s,the injectable bone grafts compound with autologous PRP , with or without BMSCs ,are both effective on repairing segmental bone defect.
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