力学环境下天然骨组织体外三维培养的研究
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摘要
研究背景和目的：
用骨组织工程技术构建的人工骨在临床治疗中能对骨折和骨损伤病人能够起到修复和置换的作用，使其在体内能够部分或短期地代替患者原有骨的功能、维持基本的骨形态，然而，这种有生命的人工骨的生物活性、与机体天然骨的相容性、排异反应等诸多问题一直都没有解决，影响着人工骨研究的发展进程。因此，有必要首先对机体内的天然骨的结构、组成及其生长环境进行充分研究，利用天然骨的构成及生长的相关研究成果来开展骨组织工程的研究将会更有目的性和导向性。目前国内外就此方面的研究一致认为，骨是一种有生命的生物材料，又是能进行新陈代谢且具有重建行为的生物组织。这种活性组织在体内的生长和构建受力学因素作用明显。早在19世纪，Julis wolff就提出外部应力能影响骨的形状与结构；越来越多的研究也显示，骨组织的形状、骨量及其内部结构的变化取决于骨所处的应力环境的改变，在外来应力刺激下，其能自动地进行骨组织的聚集与吸收，形成完美的自控反馈系统，而其他的非生物力学因素则起辅助调控作用。然而，在机体内部对骨的力学环境研究受其它因素影响较多，因此，若能在体外制备骨生长的模型，并建立有效的力学培养环境，将有利于开展骨的生物力学研究；但至今，这方面的研究工作并不多见，而且难度也非常大。本项目以来自兔股骨头松质骨制备的骨组织体外模型进行体外力学刺激实验，目的是探讨在体外培养环境下，力学刺激对三维培养的骨组织及其内部相关骨细胞生长的影响，以便在组织层面对应力环境与骨生长的关系进行研究。
研究方法：
1．摘取3月龄新西兰大白兔的股骨，无菌处理后将股骨头的松质骨部分制备成直径为8mm，厚度为3mm的质骨体外培养模型；
2．将制备好的松质骨体外培养模型放入含15%胎牛血清的DMEM培养基中，用本室设计的力学负载和循环灌流生物反应器系统进行培养，分别在培养3d、5d和7d后对其进行HE染色和电镜扫描，并和未培养的骨组织模型进行比较，评价模型在体外生长情况；
3．利用Micro-CT对骨组织模型进行扫描及用Mimics、Gemagic等软件进行模型的3D重建，并用有限元分析软件分析表观应力1000με、2000με和3000με对模型内部的影响作用；然后结合骨组织在体内的生理应力作用情况及本课题组的前期研究结果，用动态负载和灌流生物反应器系统对体外培养的松质骨模型实施了1000με、2000με、3000με和4000με，频率为1Hz的表观应力刺激；
4．分别在表观应力刺激5d和14d后，检测不同强度刺激组的碱性磷酸酶(AKP)的活性，并分析不同强度的表观应力刺激对松质骨内成骨细胞生长的影响作用；
5．根据成骨细胞内AKP活性表达情况，分别设定1000με和2000με表观应力为力学负载组进行松质骨模型体外生长试验，然后用Instron5865力学性能测试机对其进行力学性能检测；用Micro-CT扫描对其进行骨密度（TMD）检测；依据Von-kossa染色试验和四环素钙黄绿素双标记试验评价力学刺激对新骨形成的影响；用ELISA、Western blot和qRT-PCR分别检测Collagen-I、OPG和BMP-2蛋白和基因在不同刺激组的表达变化；
6．检测不同强度力学刺激对骨组织模型破骨细胞内抗酒石酸酸性磷酸酶（TRAP）活性表达的影响；分别在3000με和4000με，频率为1Hz的表观应力刺激下，用DNALadder试验检测骨组织模型细胞内DNA的变化，用Caspase活性试剂盒分别检测Caspase-3/8/9的活性。
结果：
1．制备好的松质骨体外培养模型经过修剪、去脂肪组织后，直径为8mm，厚度为3mm，大小均匀，表面平整，适合在动态力学负载系统上进行力学环境下培养；
2．用力学负载和循环灌流生物反应器系统培养制备好的松质骨体外培养模型能够保证培养体上下各个面接受到均匀的培养基营养供应，分别培养3d、5d和7d后经过HE染色和扫描电镜检测显示，模型内活细胞形态明显，且分布密集，在细胞分布和形态上与未培养组无明显区别；
3．松质骨培养模型经Micro-CT扫描后可以看见清晰的组织结构，完整密集，而通过对重建的3D模型有限元分析发现，当模型受到3000με的表观应力作用时，模型内超过4000με的节点数明显增加（1600个），而低于2000με的表观应力作用时，其内部多数节点受力保持在3000με以下；
4．经过5d和14d的表观应力刺激培养，骨组织模型的AKP活性表达分别为27.350±0.071（控制组）、26.309±0.034U/gprot、28.121±0.212U/gprot、13.365±0.105U/gprot、10.161±0.121U/gprot和26.126±0.013U/gpro（t控制组）、29.181±0.041U/gprot、33.218±0.034U/gprot、11.151±0.108U/gprot、10.603±0.010U/gprot；分析显示，AKP活性在表观应力为1000με和2000με间呈递增趋势，而在3000με和4000με间呈递减趋势；
5．在初步检测骨组织模型AKP活性基础之上，分别在培养14d和21d后，表观应力为1000με、2000με及未刺激组的其它检测结果是：弹性模量和最大受力负荷呈刺激强度和刺激时间递增，并且与未刺激组相比，2000με能显著增加模型的弹性模量和最大受力负荷，而1000με刺激时也已使模型的最大负荷发生显著变化；骨密度检测结果为模型培养21d后，刺激组的TMD都发生了显著增加，而培养14d则不能明显改善其骨密度；Von-kossa染色和钙黄绿素荧光双标记试验结果显示，力学负载刺激能促进新的类骨质形成，并随着负载强度和培养时间的增加，新生类骨质的生成也呈显著性增加，但Von-kossa染色和钙黄绿素荧光双标记的试验结果并未呈等量增加，显示出两种方法间又有一定的区别；分别对Collagen-I、OPG和BMP-2蛋白和基因表达的检测结果显示，三者都随刺激强度和时间增加呈依赖性，但在蛋白和基因层次的表达规律又不完全一致;
6．分别经1000με、2000με、3000με和4000με表观应力刺激5d后，骨组织模型破骨细胞内TRAP活性变化结果分别为19.261±0.103（控制组）、18.911±0.081、18.126±0.134、15.961±0.089和16.023±0.101；而在3000με和4000με刺激下，DNA Ladder结果显示，细胞内DNA表达为明显的梯形条带，呈180-200bp大小；Caspase-3/8/9活性检测结果发现，3000με和4000με表观应力都能显著增强三者的表达活性。
结论：
1.在无菌条件下，采用三月龄新西兰大白兔股骨头松质骨制备的骨组织体外培养模型，结构和大小能够保持一致，适于用力学负载和循环灌流生物反应器系统进行培养；
2.通过Micro-CT对松质骨体外培养模型的扫描可以对其进行3D模型的重建及有限元分析，后者从理论上分析了模型在不同力学强度刺激作用下，其内部的应力分布及结构变化，从而为探讨该骨组织模型实体在体外合适力学环境的培养奠定了理论基础；
3.通过对不同表观应力（1000με、2000με、3000με和4000με）刺激作用下骨组织体外培养模型内AKP和TRAP的检测分析，以及一系列的力学性能、TMD、新骨形成、分子表达和细胞凋亡实验评价，初步认为体外培养该模型时，低强度（1000με和2000με）力学刺激可能会促进其内部的骨组织生长，并呈剂量和时间的依赖性；相反，高强度（3000με和4000με）的力学刺激则抑制了其内部细胞（Osteoblasts和Osteoclasts）的增殖和分化，这可能与高强度的力学刺激引起其内部细胞凋亡有关。
4.该骨组织模型的成功制备及体外力学环境的培养是骨的体外细胞培养研究与体内整体研究的有效桥梁，对深入研究骨生长及发育与力学环境之间的关系具有基本的平台作用。
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.
用骨组织工程技术构建的人工骨在临床治疗中能对骨折和骨损伤病人能够起到修复和置换的作用，使其在体内能够部分或短期地代替患者原有骨的功能、维持基本的骨形态，然而，这种有生命的人工骨的生物活性、与机体天然骨的相容性、排异反应等诸多问题一直都没有解决，影响着人工骨研究的发展进程。因此，有必要首先对机体内的天然骨的结构、组成及其生长环境进行充分研究，利用天然骨的构成及生长的相关研究成果来开展骨组织工程的研究将会更有目的性和导向性。目前国内外就此方面的研究一致认为，骨是一种有生命的生物材料，又是能进行新陈代谢且具有重建行为的生物组织。这种活性组织在体内的生长和构建受力学因素作用明显。早在19世纪，Julis wolff就提出外部应力能影响骨的形状与结构；越来越多的研究也显示，骨组织的形状、骨量及其内部结构的变化取决于骨所处的应力环境的改变，在外来应力刺激下，其能自动地进行骨组织的聚集与吸收，形成完美的自控反馈系统，而其他的非生物力学因素则起辅助调控作用。然而，在机体内部对骨的力学环境研究受其它因素影响较多，因此，若能在体外制备骨生长的模型，并建立有效的力学培养环境，将有利于开展骨的生物力学研究；但至今，这方面的研究工作并不多见，而且难度也非常大。本项目以来自兔股骨头松质骨制备的骨组织体外模型进行体外力学刺激实验，目的是探讨在体外培养环境下，力学刺激对三维培养的骨组织及其内部相关骨细胞生长的影响，以便在组织层面对应力环境与骨生长的关系进行研究。
研究方法：
1．摘取3月龄新西兰大白兔的股骨，无菌处理后将股骨头的松质骨部分制备成直径为8mm，厚度为3mm的质骨体外培养模型；
2．将制备好的松质骨体外培养模型放入含15%胎牛血清的DMEM培养基中，用本室设计的力学负载和循环灌流生物反应器系统进行培养，分别在培养3d、5d和7d后对其进行HE染色和电镜扫描，并和未培养的骨组织模型进行比较，评价模型在体外生长情况；
3．利用Micro-CT对骨组织模型进行扫描及用Mimics、Gemagic等软件进行模型的3D重建，并用有限元分析软件分析表观应力1000με、2000με和3000με对模型内部的影响作用；然后结合骨组织在体内的生理应力作用情况及本课题组的前期研究结果，用动态负载和灌流生物反应器系统对体外培养的松质骨模型实施了1000με、2000με、3000με和4000με，频率为1Hz的表观应力刺激；
4．分别在表观应力刺激5d和14d后，检测不同强度刺激组的碱性磷酸酶(AKP)的活性，并分析不同强度的表观应力刺激对松质骨内成骨细胞生长的影响作用；
5．根据成骨细胞内AKP活性表达情况，分别设定1000με和2000με表观应力为力学负载组进行松质骨模型体外生长试验，然后用Instron5865力学性能测试机对其进行力学性能检测；用Micro-CT扫描对其进行骨密度（TMD）检测；依据Von-kossa染色试验和四环素钙黄绿素双标记试验评价力学刺激对新骨形成的影响；用ELISA、Western blot和qRT-PCR分别检测Collagen-I、OPG和BMP-2蛋白和基因在不同刺激组的表达变化；
6．检测不同强度力学刺激对骨组织模型破骨细胞内抗酒石酸酸性磷酸酶（TRAP）活性表达的影响；分别在3000με和4000με，频率为1Hz的表观应力刺激下，用DNALadder试验检测骨组织模型细胞内DNA的变化，用Caspase活性试剂盒分别检测Caspase-3/8/9的活性。
结果：
1．制备好的松质骨体外培养模型经过修剪、去脂肪组织后，直径为8mm，厚度为3mm，大小均匀，表面平整，适合在动态力学负载系统上进行力学环境下培养；
2．用力学负载和循环灌流生物反应器系统培养制备好的松质骨体外培养模型能够保证培养体上下各个面接受到均匀的培养基营养供应，分别培养3d、5d和7d后经过HE染色和扫描电镜检测显示，模型内活细胞形态明显，且分布密集，在细胞分布和形态上与未培养组无明显区别；
3．松质骨培养模型经Micro-CT扫描后可以看见清晰的组织结构，完整密集，而通过对重建的3D模型有限元分析发现，当模型受到3000με的表观应力作用时，模型内超过4000με的节点数明显增加（1600个），而低于2000με的表观应力作用时，其内部多数节点受力保持在3000με以下；
4．经过5d和14d的表观应力刺激培养，骨组织模型的AKP活性表达分别为27.350±0.071（控制组）、26.309±0.034U/gprot、28.121±0.212U/gprot、13.365±0.105U/gprot、10.161±0.121U/gprot和26.126±0.013U/gpro（t控制组）、29.181±0.041U/gprot、33.218±0.034U/gprot、11.151±0.108U/gprot、10.603±0.010U/gprot；分析显示，AKP活性在表观应力为1000με和2000με间呈递增趋势，而在3000με和4000με间呈递减趋势；
5．在初步检测骨组织模型AKP活性基础之上，分别在培养14d和21d后，表观应力为1000με、2000με及未刺激组的其它检测结果是：弹性模量和最大受力负荷呈刺激强度和刺激时间递增，并且与未刺激组相比，2000με能显著增加模型的弹性模量和最大受力负荷，而1000με刺激时也已使模型的最大负荷发生显著变化；骨密度检测结果为模型培养21d后，刺激组的TMD都发生了显著增加，而培养14d则不能明显改善其骨密度；Von-kossa染色和钙黄绿素荧光双标记试验结果显示，力学负载刺激能促进新的类骨质形成，并随着负载强度和培养时间的增加，新生类骨质的生成也呈显著性增加，但Von-kossa染色和钙黄绿素荧光双标记的试验结果并未呈等量增加，显示出两种方法间又有一定的区别；分别对Collagen-I、OPG和BMP-2蛋白和基因表达的检测结果显示，三者都随刺激强度和时间增加呈依赖性，但在蛋白和基因层次的表达规律又不完全一致;
6．分别经1000με、2000με、3000με和4000με表观应力刺激5d后，骨组织模型破骨细胞内TRAP活性变化结果分别为19.261±0.103（控制组）、18.911±0.081、18.126±0.134、15.961±0.089和16.023±0.101；而在3000με和4000με刺激下，DNA Ladder结果显示，细胞内DNA表达为明显的梯形条带，呈180-200bp大小；Caspase-3/8/9活性检测结果发现，3000με和4000με表观应力都能显著增强三者的表达活性。
结论：
1.在无菌条件下，采用三月龄新西兰大白兔股骨头松质骨制备的骨组织体外培养模型，结构和大小能够保持一致，适于用力学负载和循环灌流生物反应器系统进行培养；
2.通过Micro-CT对松质骨体外培养模型的扫描可以对其进行3D模型的重建及有限元分析，后者从理论上分析了模型在不同力学强度刺激作用下，其内部的应力分布及结构变化，从而为探讨该骨组织模型实体在体外合适力学环境的培养奠定了理论基础；
3.通过对不同表观应力（1000με、2000με、3000με和4000με）刺激作用下骨组织体外培养模型内AKP和TRAP的检测分析，以及一系列的力学性能、TMD、新骨形成、分子表达和细胞凋亡实验评价，初步认为体外培养该模型时，低强度（1000με和2000με）力学刺激可能会促进其内部的骨组织生长，并呈剂量和时间的依赖性；相反，高强度（3000με和4000με）的力学刺激则抑制了其内部细胞（Osteoblasts和Osteoclasts）的增殖和分化，这可能与高强度的力学刺激引起其内部细胞凋亡有关。
4.该骨组织模型的成功制备及体外力学环境的培养是骨的体外细胞培养研究与体内整体研究的有效桥梁，对深入研究骨生长及发育与力学环境之间的关系具有基本的平台作用。
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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