类人胶原蛋白Ⅱ/纳米羟基磷灰石/壳聚糖复合骨修复材料制备关键技术的研究
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摘要
目的:依据结构仿生和过程仿生的思路,采用静电自组装技术制备重组类人胶原蛋白Ⅱ(RHLCⅡ)/纳米羟基磷灰石(nHA)/壳聚糖(CS)复合骨修复材料,解决动物源胶原基骨修复材料存在的病毒隐患等安全问题以及纳米羟基磷灰石陶瓷材料无成骨诱导活性的问题。提高材料的生物相容性、赋予材料成骨活性,探讨材料的优化机制,研究兔源骨髓间充质干细胞种植于材料的诱导成骨情况。为研究新型骨修复和细胞载体材料提供实验数据,获得具有广阔市场前景的骨修复替代材料。
方法:1.纳米羟基磷灰石的制备:依据仿生的思路,实验采用贝壳和无机金属盐,以水热合成法和沉淀法制备羟基磷灰石,通过XRD、SEM以及ZETASIZER粒径测试仪对其结构、晶相、粒径进行分析比较,首先筛选出纳米低结晶羟基磷灰石的制备方法。
2.类人胶原蛋白Ⅱ(RHLCⅡ)/纳米羟基磷灰石(nHA)/壳聚糖(CS)复合骨修复材料的制备:为使材料无机界面与有机界面牢固结合,抗压强度达到骨修复材料标准,本实验采用共混法和原位滴定法制备类人胶原蛋白Ⅱ(RHLCⅡ)/纳米羟基磷灰石(nHA)/壳聚糖(CS)复合骨修复材料。通过机械强度和均匀性的比较,最终筛选出适合材料组分复合的方法。并首次选择天然交联剂京尼平交联复合材料,在增强组分间结合能力的同时避免了使用化学合成交联剂戊二醛等对细胞产生毒性危害的缺陷,制备了新一代RHLCⅡ)/nHA/CS复合骨修复材料。
3.建立模型对制备工艺进行优化:本实验依据单因素实验结果,借助MINITAB统计学软件,采用Plackett-Burman设计和响应面优化法对复合材料成型工艺进行了优化。通过X射线衍射(XRD)、红外光谱(FTIR)、扫描电镜(SEM)、MTT比色法等对材料的微观结构、力学性能和生物相容性进行了表征,同时通过Unico UV对交联反应做了跟踪测定,初步了解了天然化合物京尼平对骨复合材料的交联程度,最终获得制备复合材料的优选方案。
4.成骨诱导性能评价:本实验将骨髓间充质干细胞(BMSCs)种植于骨修复材料上,对BMSCs在复合材料上的成活率、贴附率、ALP活性以及钙结节现象进行了观察分析,评价材料与BMSCs复合的诱导成骨性能。
结果:1.新一代RHLCⅡ/nHA/CS复合骨修复材料的优选制备方案为:(1)原位滴定法按组分配比为nHA/RHLCⅡ=1.53在弱碱性环境下反应制备nHA/RHLCⅡ粉末;(2)加入壳聚糖浓度为2%±1%的粘稠溶液,充分混匀后加入京尼平,使京尼平终浓度达0.61%,倾入不锈钢模具,在4℃下冷藏24h;(3)置于-80℃超低温冰箱预冷冻3 h后真空冷冻干燥48 h。PBS(pH=7.3)缓冲液反复浸洗,二次冻干后置于干燥器中保存。(4)移植前,支架材料经灭菌后与BMSCs细胞在体外共培养,材料具有成骨活性后可直接移入受损部位。
2.制备的骨修复复合材料具有以下特征:无机相为平均大小为30nm的弱结晶纳米羟基磷灰石;材料为三维多孔材料,孔径在75～100μm之间,孔隙率在90%以上;无机相与有机相比为26:74,且相面结合紧密,机械强度可达80.46MPa;细胞毒性评为1级,在诱导培养基条件下,BMSCs细胞能够紧密贴附在材料表面及内部,经14天共培养后无死细胞出现,贴附率接近100%,ALP值是对照空白样的10倍,且7天后就有钙结节产生。
结论:本文首次对RHLCⅡ/nHA/CS骨修复材料的制备关键技术进行了系统研究,通过优化机制制备的RHLCⅡ/nHA/CS多孔支架材料从组成和结构上与自然骨相似,抗压强度可达80.46MPa,与BMSCs细胞复合后表现出良好的成骨活性,是一种功能、结构与自然骨相似且安全性能高的优选骨修复材料。通过控制材料组分的配比可获得不同机械强度需求的骨修复材料,因此有望应用于商品化生产,满足社会对不同机械强度骨修复材料的要求,解决社会骨修复材料供应量不足的问题。
Objective: To prepare the composite of the recombined human-like collagen II (RHLC II)/ nano-hydroxyapatite (nHA)/ chitosan (CS) with a biomimetic method by the electrostatic self-assembly technique, which avoid virus-danger of animal-sourced collagen and osteogenesis-lack of nano-hydroxyapatite based-ceramic materials. Further, study the optimization mechanism of the scaffold molding technology and obtain a novel bone repair material which has a promising broad market. Farther, improve the biocompatibility and inducibility of the scaffold, and study the inducible effect of rabbit sourced BMSCs seeding on scaffold. These were to give preliminary experimental evidence of a novel bone repair materials or cell vehicle materials, and obtain an ideal bone substitution for clinic.
Method:1.The preparation of nano-hydroxyapatite: HA were prepared from oyster shell or inorganic metallic salt by hydrothermal synthesis or coprecipitation method. The structure of HA was evaluated by the X-ray diffraction (XRD), scanning electron microscope (SEM) and ZETASIZER; Finally some useful strategies were chosed to prepare nature bone-like hydroxyapatite.
2.The preparation of RHLC II/ nHA/ CS composites: In order to meet the needs of repair bone materials with high compressive strength, i.e. the binding between inorganic and organic phase is tight or compact, in this research, RHLC II/ nHA/ CS composites were prepared by mixing method and in situ synthesis at moderate temperature. After analyzing their mechanic properties and homogeneity, a better way was selected to prepare them. Moreover, to avoid the toxic danger of chemical crosslink agent such as glutaraldehyde and strengthen the composite, the natural crosslink agent-genipin was firstly chosed to fabricate the scaffold and a novel RHLC II/ nHA/ CS composite with good compatibility was obtained.
3. Establishing a model for optimizing the preparation: It is a wise way to reduce the process cost and tailor the needs of board market. In this step, an optimized modeling technique of composite was obtained with response surface methodology (RSM) by Minitab soft. The micro-structure, mechanical properties and the biocompatible of the composite were studied by the X-ray diffraction (XRD), flourier transformation infrared spectra (FTIR), scanning electron microscope (SEM) and MTT colorimetric assay. At the same time, the degree of the cross-linking of the genipin and the biomimetic bone composite was determined by the Unico UV. In addition, we acquire stiffer scaffold by new crosslink method.
4. The Evaluation of bone induction activity: Seeded the rabbit bone marrow stem cells on scaffold, then observed the percent of live cells、cell attachment、ALP activity and the appearance of mineralized nodules, we finally acquired a materials with bone induction activity.
Result: 1. A novel preparation of RHLC II/nHA/CS bone repair materials was:
(1)n-HA/ RHLC II powder was prepared with nHA/ RHLC II =1.53 by in situ synthesis at alkalescency environment;
(2)2wt% chitosan was uniform mixed with n-HA/ RHLC II powder, then added genipin at 0.61% final concentration and in poured the stainless steel tooting .Cold preservation at 4℃for 24 h;
(3)Precooled the scaffold in the -80℃ultra low temperature freezer for 3h and formed the shaping in vacuum freeze-drying for 48h.The scaffold was preserve at desiccator after soaking in PBS (pH=7.3) for many times.
(4)The scaffold and BMSCs was cultured in vitro for several days until the scaffold showing the induction bone activity .Then this material could be a substitution for bone repair.
2. The composite bone repair material of RHLC II/nHA/CS had much similarity in the compositions and the structure of the nature bone, which was prepared from the weak crystalline nHA deposited in the recombined human-like collagen and the polysaccharides. Three dimensional porous scaffolds was formed by the interaction among the aforementioned three and the strongly combination of the organic and inorganic surfaces(26∶74). Finally a composite with 75～100μm of pore diameter, above 90% of the porosity, 80.46 MPa of maximum compressive strength, 1 degree of the cytotoxicity and with strong bone induction activity (100% of attachment, higher ALP activity and the appearance of mineralized nodules et al. in induced media) was successfully synthesized, which was an excellent biomimetic bone scaffold in composition, structure, function and close to the nature bone in mechanical strength.
Conclusion: We first time systematic study on the key technology of Preparation of recombined human-like collagen II/nano-hydroxyapatite/chitosan composite used for bone repair. After optimize the preparation, we acquired a novel composite, whose structure and function are close to the nature bone. Finally we obtained an ideal bone repair material with bone induction activity, which was seeded BMSCs in induced media. The compressive strength of these porous scaffolds could be up to 80.46 MPa and by adjusting the ratio of the compositions, scaffolds with different compressive strength could be composed. Thus, this kind of composite could satisfy the needs for the bone tissue repairing or substitute and be the promising material applied in clinic. It will tailor the mechanic properties to different social needs and solve shortage of bone repair materials.
方法:1.纳米羟基磷灰石的制备:依据仿生的思路,实验采用贝壳和无机金属盐,以水热合成法和沉淀法制备羟基磷灰石,通过XRD、SEM以及ZETASIZER粒径测试仪对其结构、晶相、粒径进行分析比较,首先筛选出纳米低结晶羟基磷灰石的制备方法。
2.类人胶原蛋白Ⅱ(RHLCⅡ)/纳米羟基磷灰石(nHA)/壳聚糖(CS)复合骨修复材料的制备:为使材料无机界面与有机界面牢固结合,抗压强度达到骨修复材料标准,本实验采用共混法和原位滴定法制备类人胶原蛋白Ⅱ(RHLCⅡ)/纳米羟基磷灰石(nHA)/壳聚糖(CS)复合骨修复材料。通过机械强度和均匀性的比较,最终筛选出适合材料组分复合的方法。并首次选择天然交联剂京尼平交联复合材料,在增强组分间结合能力的同时避免了使用化学合成交联剂戊二醛等对细胞产生毒性危害的缺陷,制备了新一代RHLCⅡ)/nHA/CS复合骨修复材料。
3.建立模型对制备工艺进行优化:本实验依据单因素实验结果,借助MINITAB统计学软件,采用Plackett-Burman设计和响应面优化法对复合材料成型工艺进行了优化。通过X射线衍射(XRD)、红外光谱(FTIR)、扫描电镜(SEM)、MTT比色法等对材料的微观结构、力学性能和生物相容性进行了表征,同时通过Unico UV对交联反应做了跟踪测定,初步了解了天然化合物京尼平对骨复合材料的交联程度,最终获得制备复合材料的优选方案。
4.成骨诱导性能评价:本实验将骨髓间充质干细胞(BMSCs)种植于骨修复材料上,对BMSCs在复合材料上的成活率、贴附率、ALP活性以及钙结节现象进行了观察分析,评价材料与BMSCs复合的诱导成骨性能。
结果:1.新一代RHLCⅡ/nHA/CS复合骨修复材料的优选制备方案为:(1)原位滴定法按组分配比为nHA/RHLCⅡ=1.53在弱碱性环境下反应制备nHA/RHLCⅡ粉末;(2)加入壳聚糖浓度为2%±1%的粘稠溶液,充分混匀后加入京尼平,使京尼平终浓度达0.61%,倾入不锈钢模具,在4℃下冷藏24h;(3)置于-80℃超低温冰箱预冷冻3 h后真空冷冻干燥48 h。PBS(pH=7.3)缓冲液反复浸洗,二次冻干后置于干燥器中保存。(4)移植前,支架材料经灭菌后与BMSCs细胞在体外共培养,材料具有成骨活性后可直接移入受损部位。
2.制备的骨修复复合材料具有以下特征:无机相为平均大小为30nm的弱结晶纳米羟基磷灰石;材料为三维多孔材料,孔径在75～100μm之间,孔隙率在90%以上;无机相与有机相比为26:74,且相面结合紧密,机械强度可达80.46MPa;细胞毒性评为1级,在诱导培养基条件下,BMSCs细胞能够紧密贴附在材料表面及内部,经14天共培养后无死细胞出现,贴附率接近100%,ALP值是对照空白样的10倍,且7天后就有钙结节产生。
结论:本文首次对RHLCⅡ/nHA/CS骨修复材料的制备关键技术进行了系统研究,通过优化机制制备的RHLCⅡ/nHA/CS多孔支架材料从组成和结构上与自然骨相似,抗压强度可达80.46MPa,与BMSCs细胞复合后表现出良好的成骨活性,是一种功能、结构与自然骨相似且安全性能高的优选骨修复材料。通过控制材料组分的配比可获得不同机械强度需求的骨修复材料,因此有望应用于商品化生产,满足社会对不同机械强度骨修复材料的要求,解决社会骨修复材料供应量不足的问题。
Objective: To prepare the composite of the recombined human-like collagen II (RHLC II)/ nano-hydroxyapatite (nHA)/ chitosan (CS) with a biomimetic method by the electrostatic self-assembly technique, which avoid virus-danger of animal-sourced collagen and osteogenesis-lack of nano-hydroxyapatite based-ceramic materials. Further, study the optimization mechanism of the scaffold molding technology and obtain a novel bone repair material which has a promising broad market. Farther, improve the biocompatibility and inducibility of the scaffold, and study the inducible effect of rabbit sourced BMSCs seeding on scaffold. These were to give preliminary experimental evidence of a novel bone repair materials or cell vehicle materials, and obtain an ideal bone substitution for clinic.
Method:1.The preparation of nano-hydroxyapatite: HA were prepared from oyster shell or inorganic metallic salt by hydrothermal synthesis or coprecipitation method. The structure of HA was evaluated by the X-ray diffraction (XRD), scanning electron microscope (SEM) and ZETASIZER; Finally some useful strategies were chosed to prepare nature bone-like hydroxyapatite.
2.The preparation of RHLC II/ nHA/ CS composites: In order to meet the needs of repair bone materials with high compressive strength, i.e. the binding between inorganic and organic phase is tight or compact, in this research, RHLC II/ nHA/ CS composites were prepared by mixing method and in situ synthesis at moderate temperature. After analyzing their mechanic properties and homogeneity, a better way was selected to prepare them. Moreover, to avoid the toxic danger of chemical crosslink agent such as glutaraldehyde and strengthen the composite, the natural crosslink agent-genipin was firstly chosed to fabricate the scaffold and a novel RHLC II/ nHA/ CS composite with good compatibility was obtained.
3. Establishing a model for optimizing the preparation: It is a wise way to reduce the process cost and tailor the needs of board market. In this step, an optimized modeling technique of composite was obtained with response surface methodology (RSM) by Minitab soft. The micro-structure, mechanical properties and the biocompatible of the composite were studied by the X-ray diffraction (XRD), flourier transformation infrared spectra (FTIR), scanning electron microscope (SEM) and MTT colorimetric assay. At the same time, the degree of the cross-linking of the genipin and the biomimetic bone composite was determined by the Unico UV. In addition, we acquire stiffer scaffold by new crosslink method.
4. The Evaluation of bone induction activity: Seeded the rabbit bone marrow stem cells on scaffold, then observed the percent of live cells、cell attachment、ALP activity and the appearance of mineralized nodules, we finally acquired a materials with bone induction activity.
Result: 1. A novel preparation of RHLC II/nHA/CS bone repair materials was:
(1)n-HA/ RHLC II powder was prepared with nHA/ RHLC II =1.53 by in situ synthesis at alkalescency environment;
(2)2wt% chitosan was uniform mixed with n-HA/ RHLC II powder, then added genipin at 0.61% final concentration and in poured the stainless steel tooting .Cold preservation at 4℃for 24 h;
(3)Precooled the scaffold in the -80℃ultra low temperature freezer for 3h and formed the shaping in vacuum freeze-drying for 48h.The scaffold was preserve at desiccator after soaking in PBS (pH=7.3) for many times.
(4)The scaffold and BMSCs was cultured in vitro for several days until the scaffold showing the induction bone activity .Then this material could be a substitution for bone repair.
2. The composite bone repair material of RHLC II/nHA/CS had much similarity in the compositions and the structure of the nature bone, which was prepared from the weak crystalline nHA deposited in the recombined human-like collagen and the polysaccharides. Three dimensional porous scaffolds was formed by the interaction among the aforementioned three and the strongly combination of the organic and inorganic surfaces(26∶74). Finally a composite with 75～100μm of pore diameter, above 90% of the porosity, 80.46 MPa of maximum compressive strength, 1 degree of the cytotoxicity and with strong bone induction activity (100% of attachment, higher ALP activity and the appearance of mineralized nodules et al. in induced media) was successfully synthesized, which was an excellent biomimetic bone scaffold in composition, structure, function and close to the nature bone in mechanical strength.
Conclusion: We first time systematic study on the key technology of Preparation of recombined human-like collagen II/nano-hydroxyapatite/chitosan composite used for bone repair. After optimize the preparation, we acquired a novel composite, whose structure and function are close to the nature bone. Finally we obtained an ideal bone repair material with bone induction activity, which was seeded BMSCs in induced media. The compressive strength of these porous scaffolds could be up to 80.46 MPa and by adjusting the ratio of the compositions, scaffolds with different compressive strength could be composed. Thus, this kind of composite could satisfy the needs for the bone tissue repairing or substitute and be the promising material applied in clinic. It will tailor the mechanic properties to different social needs and solve shortage of bone repair materials.
引文
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