生物活性玻璃组织工程支架的制备及细胞相容性研究
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摘要
通过研制新型的骨组织修复材料，帮助患者修复缺损或缺失的骨组织，更好的恢复人体硬组织功能是医学界和生物医学材料学界多年来一直在探索并试图解决的重要问题。本研究首先采用溶胶-凝胶技术结合模板法，制备出具有卷曲片层结构的生物活性玻璃，然后通过原位凝固成型工艺结合光固化快速成型技术制备出具有较高力学强度和一定孔隙率的具有有序堆砌孔结构的生物活性玻璃陶瓷支架材料，研究了这一支架材料的制备工艺、物理化学性质、体外磷灰石矿化活性和降解性能，并以成骨细胞MG-63为细胞模型研究了生物活性玻璃陶瓷支架的细胞相容性。主要研究工作如下：
首先，通过传统熔融法制备了45S5生物活性玻璃，采用溶胶-凝胶技术结合非离子型嵌段共聚物P123作为模板制备出具有卷曲片层结构的58S生物活性玻璃粉体，然后在模拟体液SBF中浸泡表征其体外矿化性能，发现随浸泡时间的延长，磷灰石晶体在颗粒表面成核生长，并最终覆满整个表面。实验结果表明，本实验制备的45S5和58S生物活性玻璃粉体都具有良好的生物活性和体外矿化性能。
其次，通过调节制备工艺参数来改善生物活性玻璃浆料的流动性，制备出了流动性好，性能稳定的生物活性玻璃浆料，采用原位凝固成型工艺结合光固化快速成型技术制备出具有较高力学强度和一定孔隙率的具有有序堆砌孔结构的生物活性玻璃陶瓷支架材料。结果表明，我们制备的这一支架材料是一种部分结晶的生物活性玻璃陶瓷材料，结晶部分为针状Na_4Ca_4(Si_6O_(18))晶体；μCT结果表明支架材料的孔隙率约为61%，基本满足松质骨孔隙率要求；万能力学电子试验机测试结果表明该支架材料的抗压强度约在12.37±1.25MPa，基本满足松质骨力学强度要求。
再次，我们将制备的生物活性玻璃陶瓷支架材料浸泡于SBF溶液中，通过观察其表面磷灰石的生成速率来评价其生物活性和矿化能力。实验结果如下：支架材料在浸泡过程中离子溶出的同时伴随着碳酸磷灰石HCA矿化层的生成，磷灰石晶体起初在支架表面成核生长，14天后覆满了整个表面，说明我们制备的支架材料具有良好的体外矿化能力；通过检测浸泡支架后SBF溶液的变化，发现pH值在第1天呈现一个快速增长趋势，然后增长速率有所减缓，3天后达到一个相对平稳的状态，最终稳定在7.65左右。我们同时对浸泡后支架的重量变化做了记录，发现浸泡1天后支架的重量损失约在8-9%，随后保持稳定，7天后又开始下降，21天后重量损失达到20.7%左右。我们认为重量损失的原因可能是由于支架材料在SBF溶液浸泡过程中的离子溶出导致的，在1-7天浸泡时间段出现的动态平衡，可能是由于支架材料表面的磷灰石矿化生成速率与离子溶出速率一致引起的。我们还通过ICP技术测试了浸泡支架后SBF溶液中离子浓度的变化，发现在24h内溶液中Si离子浓度呈快速增加，24h后仍持续增加但增速变缓，这说明浸泡初期Si离子释放主要由扩散作用控制，24h后还受到磷灰石矿化层的抑制而有所减缓。浸泡24h后，溶液中的Ca~(2+)离子浓度趋向于一个稳定值，可能是因为此时磷灰石的生成速率和支架材料中的离子溶出速率达到了一个动态平衡。溶液中P离子浓度随时间增加呈逐渐降低的趋势，是因为P是生成磷灰石的必须元素，材料中溶解出的P不足也抑制了磷灰石的生长。最后，我们对支架浸泡过程中Si离子的释放动力学进行了研究，发现支架材料中Si元素在0-24小时时间段内的释放基本遵循一级动力学释放模型，此过程属于扩散-溶解控制。支架开始溶解时，材料本身的Si含量较大，而溶液中几乎为0，所以在水分子的扩散-释放机制作用下，Si的释放速率在浸泡初始阶段较大，而后趋向于平缓。
最后，我们还探讨了规则孔隙结构与不规则孔隙结构的支架材料对成骨细胞粘附、增殖的影响。实验采用成骨细胞MG-63为细胞模型，经过一天的共培养，扫描电镜观察发现大量细胞已经伸出伪足并紧紧粘附在支架材料上，并通过MTT法检测细胞的增殖情况，结果发现规则孔隙结构支架材料上的细胞活性明显高于不规则孔隙结构支架材料上的细胞活性，并且两者具有统计学显著性差异。
To develop a new type of bone tissue repair material to help the patients repair the defector missing bone tissue and restoration of human hard tissue, the medical academia and thebiomedical materials academia have been trying to solve this problem over the years. In thisstudy, first, the bioactive glasses with curled lamellar structure were prepared by sol-gelmethods, and then the bioactive glass-ceramic scaffolds were produced by in-situsolidification molding process with rapid prototyping techniques. As a result, the bioactiveglass-ceramic scaffolds with regular pore structure have a high mechanical strength andcertain porosity; we also studied the preparation process, the physical and chemical properties,the apatite biomineralization activity and the cytocompatibility between the scaffold andMG-63osteoblasts. The main results as follows:
First, the45S5bioactive glass were prepared by conventional melting method, the58Sbioactive glass powders with curled lamellar structure were prepared by sol-gel technologyusing non-ionic block copolymer P123as the template. After immersion in SBF, we foundthat HCA crystals gradually nucleation and growth on the surface，finally covered the wholesurface with the immersion time increased. This indicated that the45S5and58S bioactiveglass have a good bioactivity and in vitro biomineralization.
Second, to improve its fluidity and stability, a certain dispersing agent were added into the45S5bioactive glass slurry, then the bioactive glass-ceramic scaffolds were prepared byin-situ solidification molding process with a high mechanical strength and certain porosity,partially crystallized with Na_4Ca_4(Si_6O_(18)) crystals. The μCT results showed that the porosityis about61%which meet to the basic requirements of cancellous bone porosity; thecompressive strength of the scaffold is12.37±1.25MPa also meet to the mechanical strengthof cancellous bone requirements.
Third, to test its bioactivity and biomineralization, the bioactive glass scaffolds weresoaked into SBF and observed apatite generated. The results showed that the HCA crystalnucleation and growth on the surface of the scaffold gradually with the immersion time increased, and covered with the whole surface after immersion for14days, it indicated thatthe scaffold had a good biomineralization. The pH value of the SBF were recordedsimultaneously after immersion, it presented a fast-growing trend at first day, then growthslowed, three days later, it reached a relatively stable state at around7.65. The weight loss ofthe scaffold after immersion was tested; it underwent an immediate loss of8-9%during thefirst day, and held this value until7days, then decreased to20.7%on21days. A dynamicequilibrium appeared and thus the scaffold weight was stabilized during1-7days immersion,the reason maybe the scaffold dissolution consistent to the formation of HCA. The ionconcentration of the SBF after the scaffold immersion was also tested; Si ion concentrationshowed a rapid increase and continued to increase slowly after24h of immersion, Si ionrelease mainly controlled by diffusion at early age and slowing down for the formation of theHCA mineralization layer. Ca~(2+)ion concentration tends to a stable value after immersion for24h; this maybe due to the formation of apatite and ion dissolution reached a dynamicequilibrium. The concentration of P was gradually decreased with the time increased, thatbecause P was an essential element to generate apatite but it was dissolved from the scaffoldinsufficiently. Finally, we studied the kinetics of Si ion release, found that the release of Si in0-24h period followed to the release of first-order kinetics model and belonged todiffusion-dissolved control process. This Si release process is started due to the scaffold, theSi concentration in solution is almost0at this time, according to the diffusion releasemechanism, the release rate of Si is rapidly at the initial stage, and then tend to be gentlerelease stage.
Finally，the effect on cell adhesion and proliferation were discussed between the regularand irregular pore structure using MG-63cells as a model. After one day co-culture, MG-63cells have been found in a large number of pseudopodia and adhered to the scaffold surface bySEM. MTT methods were used to test the proliferation of the cells on the bioactive glassceramic scaffold, and found that the regular porous structure was significantly higher cellactivity than the irregular porous structure, and the difference between two groups isstatistically significant.
首先，通过传统熔融法制备了45S5生物活性玻璃，采用溶胶-凝胶技术结合非离子型嵌段共聚物P123作为模板制备出具有卷曲片层结构的58S生物活性玻璃粉体，然后在模拟体液SBF中浸泡表征其体外矿化性能，发现随浸泡时间的延长，磷灰石晶体在颗粒表面成核生长，并最终覆满整个表面。实验结果表明，本实验制备的45S5和58S生物活性玻璃粉体都具有良好的生物活性和体外矿化性能。
其次，通过调节制备工艺参数来改善生物活性玻璃浆料的流动性，制备出了流动性好，性能稳定的生物活性玻璃浆料，采用原位凝固成型工艺结合光固化快速成型技术制备出具有较高力学强度和一定孔隙率的具有有序堆砌孔结构的生物活性玻璃陶瓷支架材料。结果表明，我们制备的这一支架材料是一种部分结晶的生物活性玻璃陶瓷材料，结晶部分为针状Na_4Ca_4(Si_6O_(18))晶体；μCT结果表明支架材料的孔隙率约为61%，基本满足松质骨孔隙率要求；万能力学电子试验机测试结果表明该支架材料的抗压强度约在12.37±1.25MPa，基本满足松质骨力学强度要求。
再次，我们将制备的生物活性玻璃陶瓷支架材料浸泡于SBF溶液中，通过观察其表面磷灰石的生成速率来评价其生物活性和矿化能力。实验结果如下：支架材料在浸泡过程中离子溶出的同时伴随着碳酸磷灰石HCA矿化层的生成，磷灰石晶体起初在支架表面成核生长，14天后覆满了整个表面，说明我们制备的支架材料具有良好的体外矿化能力；通过检测浸泡支架后SBF溶液的变化，发现pH值在第1天呈现一个快速增长趋势，然后增长速率有所减缓，3天后达到一个相对平稳的状态，最终稳定在7.65左右。我们同时对浸泡后支架的重量变化做了记录，发现浸泡1天后支架的重量损失约在8-9%，随后保持稳定，7天后又开始下降，21天后重量损失达到20.7%左右。我们认为重量损失的原因可能是由于支架材料在SBF溶液浸泡过程中的离子溶出导致的，在1-7天浸泡时间段出现的动态平衡，可能是由于支架材料表面的磷灰石矿化生成速率与离子溶出速率一致引起的。我们还通过ICP技术测试了浸泡支架后SBF溶液中离子浓度的变化，发现在24h内溶液中Si离子浓度呈快速增加，24h后仍持续增加但增速变缓，这说明浸泡初期Si离子释放主要由扩散作用控制，24h后还受到磷灰石矿化层的抑制而有所减缓。浸泡24h后，溶液中的Ca~(2+)离子浓度趋向于一个稳定值，可能是因为此时磷灰石的生成速率和支架材料中的离子溶出速率达到了一个动态平衡。溶液中P离子浓度随时间增加呈逐渐降低的趋势，是因为P是生成磷灰石的必须元素，材料中溶解出的P不足也抑制了磷灰石的生长。最后，我们对支架浸泡过程中Si离子的释放动力学进行了研究，发现支架材料中Si元素在0-24小时时间段内的释放基本遵循一级动力学释放模型，此过程属于扩散-溶解控制。支架开始溶解时，材料本身的Si含量较大，而溶液中几乎为0，所以在水分子的扩散-释放机制作用下，Si的释放速率在浸泡初始阶段较大，而后趋向于平缓。
最后，我们还探讨了规则孔隙结构与不规则孔隙结构的支架材料对成骨细胞粘附、增殖的影响。实验采用成骨细胞MG-63为细胞模型，经过一天的共培养，扫描电镜观察发现大量细胞已经伸出伪足并紧紧粘附在支架材料上，并通过MTT法检测细胞的增殖情况，结果发现规则孔隙结构支架材料上的细胞活性明显高于不规则孔隙结构支架材料上的细胞活性，并且两者具有统计学显著性差异。
To develop a new type of bone tissue repair material to help the patients repair the defector missing bone tissue and restoration of human hard tissue, the medical academia and thebiomedical materials academia have been trying to solve this problem over the years. In thisstudy, first, the bioactive glasses with curled lamellar structure were prepared by sol-gelmethods, and then the bioactive glass-ceramic scaffolds were produced by in-situsolidification molding process with rapid prototyping techniques. As a result, the bioactiveglass-ceramic scaffolds with regular pore structure have a high mechanical strength andcertain porosity; we also studied the preparation process, the physical and chemical properties,the apatite biomineralization activity and the cytocompatibility between the scaffold andMG-63osteoblasts. The main results as follows:
First, the45S5bioactive glass were prepared by conventional melting method, the58Sbioactive glass powders with curled lamellar structure were prepared by sol-gel technologyusing non-ionic block copolymer P123as the template. After immersion in SBF, we foundthat HCA crystals gradually nucleation and growth on the surface，finally covered the wholesurface with the immersion time increased. This indicated that the45S5and58S bioactiveglass have a good bioactivity and in vitro biomineralization.
Second, to improve its fluidity and stability, a certain dispersing agent were added into the45S5bioactive glass slurry, then the bioactive glass-ceramic scaffolds were prepared byin-situ solidification molding process with a high mechanical strength and certain porosity,partially crystallized with Na_4Ca_4(Si_6O_(18)) crystals. The μCT results showed that the porosityis about61%which meet to the basic requirements of cancellous bone porosity; thecompressive strength of the scaffold is12.37±1.25MPa also meet to the mechanical strengthof cancellous bone requirements.
Third, to test its bioactivity and biomineralization, the bioactive glass scaffolds weresoaked into SBF and observed apatite generated. The results showed that the HCA crystalnucleation and growth on the surface of the scaffold gradually with the immersion time increased, and covered with the whole surface after immersion for14days, it indicated thatthe scaffold had a good biomineralization. The pH value of the SBF were recordedsimultaneously after immersion, it presented a fast-growing trend at first day, then growthslowed, three days later, it reached a relatively stable state at around7.65. The weight loss ofthe scaffold after immersion was tested; it underwent an immediate loss of8-9%during thefirst day, and held this value until7days, then decreased to20.7%on21days. A dynamicequilibrium appeared and thus the scaffold weight was stabilized during1-7days immersion,the reason maybe the scaffold dissolution consistent to the formation of HCA. The ionconcentration of the SBF after the scaffold immersion was also tested; Si ion concentrationshowed a rapid increase and continued to increase slowly after24h of immersion, Si ionrelease mainly controlled by diffusion at early age and slowing down for the formation of theHCA mineralization layer. Ca~(2+)ion concentration tends to a stable value after immersion for24h; this maybe due to the formation of apatite and ion dissolution reached a dynamicequilibrium. The concentration of P was gradually decreased with the time increased, thatbecause P was an essential element to generate apatite but it was dissolved from the scaffoldinsufficiently. Finally, we studied the kinetics of Si ion release, found that the release of Si in0-24h period followed to the release of first-order kinetics model and belonged todiffusion-dissolved control process. This Si release process is started due to the scaffold, theSi concentration in solution is almost0at this time, according to the diffusion releasemechanism, the release rate of Si is rapidly at the initial stage, and then tend to be gentlerelease stage.
Finally，the effect on cell adhesion and proliferation were discussed between the regularand irregular pore structure using MG-63cells as a model. After one day co-culture, MG-63cells have been found in a large number of pseudopodia and adhered to the scaffold surface bySEM. MTT methods were used to test the proliferation of the cells on the bioactive glassceramic scaffold, and found that the regular porous structure was significantly higher cellactivity than the irregular porous structure, and the difference between two groups isstatistically significant.
引文
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