| Background and objective:In orthopedic therapy, the artificial bone produced by bone tissue engineering technology isgenerally used to repair bone defects, which can replace original bone in function and appearacepartially or short-time. However, advancement of this artificial bone is limited due to its someissues,such as its bioactive in body, compatibility with the natural bone, rejection by the body,and so on. Therefore, it is necessary first of all to adequately study the structure, compositionand growth environment of natural bone in vivo and vitro, then basing on these research ofnatural bone, the related research in bone tissue engineering will be more purposeful andoriented.At present, studies indicated that bone is a biological material with bioactive, and hasthe characteristics of metabolism and reconstruction. In body, growth and remodeling of thesebioactive tissue could be apparently affected by mechanics, and in1900s, a researcher, Juliswolff, suggested that stress load toward bone could lead its development of appearance andstructure, other studies also agreed that the development of bone in appearance, bone mass andits structure would be affected by stress environment. In reponse to the external mechanicsstimulus, there is a self-control feedback system in bone which can antomatically regulate thefunction of bone formation or bone absorption,but non-biomechanical factors play only asupporting role in this self-feedback system. However,growth environment of bone in body iscomplex which is not towardly for research on effects of stress load to bone, so it is a good ideafor research of mechanics stimulus on bone with bone tissue vitro cultivation model. In thisstudy, we established cancellous bone explant models made from rabbit femur head, and bycultivation of models in a new stress load and circulatory perfusion biological reactor, the effectsof stress load stimulus on bone tissue were studied.Methods:1. The femur were extracted from3-month-old New Zealand white rabbit, then after beingsteriled with75%alcohol,the femur heads were made into cancellous bone explant models withdiameter of8mm and3mm thickness.2. The bone explant models were cultured used a new stress load and circulatory perfusionbiological reactor made by ourself in DMEM medium containing15%fetal bovine serum(FBS).Then, in3d,5d and7d of cultivation respectively, the effects of these models cultured in vitro were evaluated and comparing to the uncultured models by HE staining and scanning electronmicroscope.3. The bone tissue models were scaned by Micro-CT, then the computer3D model was rebuiltand analyzed by Mimics snd finite elememt(FEM) analysis. Considering the effects of FEManalysis and bone physiological stress in vivo,these bone tissue explant models were cultured inthe new stress load and circulatory perfusion biological reactor with1000με,2000με,3000μεand4000με in frequency of1Hz.4. After stress load stimuls for5d and4d, activity of AKP in these bone tissue models weretested, by activity of AKP, it would be understanded whether stress load could affectdifferentiation and growth of osteoblasts in these bone tissue models.5. Based on the expression of AKP activity in osteoblasts,bone explant models were designed incultivation with unstress load(control),1000με,2000με,3000με and4000με respectively infrequency of1Hz. Then,the bone tissue models cultured with1000με and2000με for14d and21d were evaluated by the following assay:mechanical propterties by Instron5865, bone mineraldensity(BMD) by Micro-CT, fresh bone formation by Von-kossa staining and tetracyclinecalcein double labeling assay, and expressing of Collagen-I, OPG and BMP-2in protein andgene detected by ELISA assay, Western-blot assay and Quantitative real time PCR assay.6. To bone explant models with3000με and4000με, the apoptosis occuring in these modelswere detected by activity of tartrate resistant acid phosphatase(TRAP), DNA expressing byDNA Ladder assay and caspase-3/8/9activity in5d.Results:1. The bone explant models which have been prepared from rabbit femur head have an uniformsize(diameter of8mm, thickness of3mm) and a smooth surface, it is suitable for cultivation inthe new stress load and circulatory perfusion biological reactor.2. With cultivation in the new stress load and circulatory perfusion biological reactor, these boneexplant models received an uniform nutrient supply,by the detection of HE staining andscanning electron microscopy in cultivation of3d,5d and7d, it indicated that there were livingcells which were intensive distribution in the models, but between unculture models and culturemodels, there were no significant about distribution and morphology of cells in it.3. By Micro-CT scaning, the clear tissue architecture could be found in the bone tissue models,and from the FEM analysis, we knew that there were majority of node in the3D models with4000με stress load when the3D model were stimulated with3000με, in turn, there weremajority of node with lower2000με stress load when the3D model were stimulated with lower2000με. 4. By cultivation of5d and14d with mechanical load, AKP activity in bone tissue models were27.350±0.071U/gprot(unload),26.309±0.034U/gprot(1000με),28.121±0.212U/gprot(2000με),13.365±0.105U/gprot(3000με),10.161±0.121U/gprot(4000με) and26.126±0.013U/gprot,29.181±0.041U/gprot,33.218±0.034U/gprot,11.151±0.108U/gprot,10.603±0.010U/gprot; andit showed an increasing trend in the AKP activity of bone tissue models stimulated betweent1000με and2000με, in turn, it showed a decreasing trend between3000με and4000με.5. Many effects of mechanical load(1000με and2000με) stimulus on bone tissue vitrocultivation models in14d and21d were found as following:the elastic modulus and themaximum stress load were increasing with stimulus intensity increasing,and compared with thecontrol groups, there had been significant improvement in maximum stress load to1000μεstimulus; To BMD,there were all significant in1000με and2000με with cultivation of21d, butwith cultivation of14d, it wasn t;In Von-kossa staining and tetracycline calcein double labelingassay, there were all fresh osteoid to be found,and it is particularly obvious and widely incultivation for21d with2000με; To protein and gene expression of Collagen-I, OPG and BMP-2,it showed a positive correlation between protein expression or gene expression and stimuluslevel, but between ptotein expression and gene expression, it not appeard the same pattern.6. In the cultivation with3000με and4000με for5d, TRAP activity was obviously inhibitedcomparing to the control group; DNA ladder bands could also observate after agarose gelelectrophoresis, it appeared more DNA fragmentation in180-200bp; Compared with the control,Caspase-3/8/9activity were apparently improved by the mechanical stimulus.Conclusion:1. The bone tissue explant models which were prepared from the3month-old New Land rabbitfemur head under sterile conditions were consistent in structure and size, and suitable forcultivation in vitro with a new stress load and circulatory perfusion bioreactor.2. The3D models were rebuilt from the bone tissue explant models and treated by FEMananlysis after Micro-CT scanning. The FEM analysis revealed in theory the effect of stress loadto bone tissue explant models.So we consider that it will help to culture the bone tissue explantmodels in vitro by the above work.3. These bone tissue explant models were tested in activity of AKP and TRAP after stressstimulus of1000με,2000με,3000με and4000με, and evaluated by some other assays, includingmechanical propties, BMD, fresh bone formation, molecular expression and apoptosis. From it,we knew that it could promote the growth of bone tissue explant models under low mechanicallevels such as1000με and2000με in a dose-and time-dependent manners. On the contrary, thedifferentiation and proliferation of cells(osteoblasts and osteoclasts) in the bone tissue explant models could be inhibited under high mechanical levels such as3000με and4000με,which maybe related with apoptosis caused by mechanical stimulus.4. It was a effective bridge between research of bone cells cultured in vitro and bone study inbody to bone explant models successfully prepared in vitro and cultured in mechanicalenvironment which plays an important role in the association research between bone growth andmechanical environment. |
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