仿生合成丝素蛋白/羟基磷灰石类骨质复合生物材料的研究
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摘要
仿生合成技术模仿了自然界生物体合成自身矿物的过程，即无机成分在有机模板的精确调控下合成有机／无机复合体。在合成过程中先形成有机自组装体(如：蛋白质、多糖、核酸、生物膜、脂质体等)，使无机成分在有机自组装聚集体和溶液的相界面发生化学反应，在自组装体模板的精确调控下，形成有机／无机复合体。
本论文研究主要以丝素蛋白为模板，使钙、磷溶液在一定的PH值及一定的温度下，通过分子键的作用，在丝素蛋白表面自组装沉积、成核形成类骨羟基磷灰石，并研究不同温度对磷灰石晶体在丝素蛋白表面取向的影响，制备丝素蛋白／羟基磷灰石类骨质复合生物材料，用于骨缺损的修复。
首先，仿生合成丝素蛋白／羟基磷灰石类骨质复合生物材料的制备。以蚕丝丝素蛋白为模板，按照有机／无机为3／7的比例将蚕丝丝素蛋白和磷酸的混合溶液逐步滴定到氢氧化钙溶液中(Ca∶P=1.67)，同时用氢氧化钠调节PH值为9～10，强力搅拌，沉化48小时，形成丝素蛋白／羟基磷灰石复合生物材料。采用梯度温度(25℃、37℃、50℃、60℃、70℃)制备不同的样本，通过FTIR、XRD、SEM、TEM对样本进行表征，筛选出最佳类骨质结构的复合生物材料制备的条件。结果显示：通过对不同温度下制备的SF／HA复合材料进行表征显示，FTIR、XRD显示所制备的复合生物材料中，磷灰石晶体为含有碳酸根的低结晶度纳米羟基磷灰石，与天然骨组织中的磷灰石晶体相似；同时XRD显示磷灰石晶体尺寸为15.6～22.8nm，扫描电镜图片Smile View软件测量显示SF／HA复合材料晶体颗粒为0.229～0.409μm，表明HA晶体以及SF／HA复合材料晶体颗粒均晶体随温度的增加而增大；SF／HA复合材料晶体颗粒XRD图谱显示，随着温度的增加晶体沿c轴择优取向生长，同时TEM电子衍射图显示，在60℃、70℃水浴时，晶体明显沿c轴方向择优生长，生长方向与纤维长轴平行，并且水浴70℃时晶体取向稍优于60℃时复合材料的晶体：这与天然骨组织中，羟基磷灰石晶体在胶原纤维表面生长与排列方向相同。因此，所制备的丝素蛋白／羟基磷灰石复合生物材料为类骨质结构。
论文的第二部分研究了所制备的丝素蛋白／羟基磷灰石类骨质复合生物材料的生物学特性。首先将制备好的材料粉体利用压片机在20MPa的机械力作用下，压制成直径约1.2mm的圆盘型，并利用纯羟基磷灰石材料作为对照培养MG63细胞，观察细胞的增殖，测试其生物学特性。通过检测MTT，ALP，Ca及OC值，利用SEM观察不同时间细胞的粘附和生长，显示所制备的丝素蛋白／羟基磷灰石类骨质复合生物材料有利于细胞的粘附、增殖和生长，可以促进细胞ALP活性表达，提高细胞基质钙离子的释放，并且可以诱导细胞OC分泌增加，促进细胞成骨性能的表达，是一种理想的复合生物材料。
综上所述，本论文以蚕丝丝素蛋白纤维为模板，钙磷在丝素蛋白模板的调控下，在纤维表面沉积、成核形成类骨羟基磷灰石晶体，在丝素蛋白模板精确的调控下，磷灰石晶体在纤维表面沿c轴择优取向生长，晶体生长方向与纤维长轴平行，所制备的类骨质复合生物材料与天然骨具有组成和结构上的统一；并对其生物学特性进行了评价，有利于细胞的粘附，增殖和生长以及成骨活性的表达，是一种理想的复合生物材料。
Multiplicity mineralized composite procedures in Nature provide a rich foundation for biomineralization and novel materials designs. i.e. sea hells, bone and other hard tissue. The procedure is the Natural inorganic mineral formatted by the organic template regulation. The formation of Natural inorganic-organic composites is a complex process, including the assembly of the organic template (i.e. protein, polysaccharides, nucleinic acid, biological membrane and liposome) subsequent inorganic mineral deposition, nucleation and growth on the organic template surface by the the organic template regulation.
The major of thesis focused on the study of the like-bone Silk fibroin (SF)/ hydroxyapatite(HA) biocomposite. First prepare the SF organic template, secondly under controlled PH and temperature calcium and phosphorus begin deposition and nucleation and growth on the organic template surface by the the organic template precise regulation and form like-bone silk fibroin /hydroxyapatite biocomposite. at the same time, research the effect of temperature on the hydroxyapatite crystal direction on the silk fibroin template surface.
Firstly the preparation of like-bone Silk fibroin (SF)/ hydroxyapatite (HA) biocomposite by biomineralization. Ca(OH)_2, H_3PO_4 and SF were used as starting materials, and their composites were synthesized by a biomineralization. The starting materials were prepared for the composition of the SF/HA composite to be 30/70 in weight ratio. add SF template into the H3PO4 solution with vigorous stirring until uniform mixing, the prepare of the composite was conducted by adding the H_3PO_4 solution with the SF slowly into the Ca(OH)_2 solution with vigorous stirring and adjust PH to 9～10 With NaOH at grade temperature (25℃、37℃、50℃、60℃、70℃). Checking the biomaterial prepared at grade temperature, chose the better reparation condition. The results from the tests of FTIR、XRD show that the hydroxyapatite is low crystallinity nano-carbonated apatite [CO_3-substituted Ca_(10)(PO_4)_6(OH)_2].it is consistent with the hydroxyapatite crystals in bone. the size of hydroxyapatite crystals is 15.6～22.8nm showed by XRD, and the crystals size of composite is 0.229~0.409um.they indicate that the crystals size increase with the rising of temperature. The results from the tests of SEM,TEM and electron diffraction show that the needle-like hydroxyapatite crystal grows along the c-axes of hydroxyapatite crystal and The crystallographic c-axes of hydroxyapatite were aligned along the long axes of the fibers, consistent with observations of collagen fibrils and hydroxyapatite crystals in bone at the 60℃、70℃.the like-bone structure is better at the aqueous bath of 70℃than 60℃.
Secondly, in vitro experiments were designed and carried out to evaluate the biological reaction of the biomaterial prepared as described in the first part. Compress the SF/HA powder into disk shape with 1.2mm diameter by tablet machine. and MG63, which is human osteoblast cell phenotypes was adopted to valuate the biological reaction. controlled by HA. Morphology observation ,SEM and MTT results show that the like-bone SF/HA is better benefit to MG63 that HA, it is benefit to MG63 adhesion differentiation and proliferation. At the same time the results of ALP, Ca~(2+) concentration and OC indicate that The SF/HA can promote the ALP express, hasten the delivery of cell matrix Ca~(2+) and induce the OC perspire controlled with HA. So the like-bone SF/HA biomaterial is a better biomaterial.
Based on the evidences from the experiments. calcium and phosphorus begin deposition , nucleation and growth on the organic template surface by the organic template precise regulation and form like-bone silk fibroin/hydroxyapatite biocomposite. The hydroxyapatite is low crystallinity nano-carbonated apatite [CO_3-substituted Ca_(10)(PO_4)_6(OH)_2]. It is consistent with the hydroxyapatite crystals in bone. and the needle-like hydroxyapatite crystal grows along the c-axes of hydroxyapatite crystal and The crystallographic c-axes of hydroxyapatite were aligned along the long axes of the fibers, consistent with observations of collagen fibrils and hydroxyapatite crystals in bone. At the same time, evaluate the biological reaction, the like-bone SF/HA can promote MG63 adhesion differentiation and proliferation and ossify activity.
本论文研究主要以丝素蛋白为模板，使钙、磷溶液在一定的PH值及一定的温度下，通过分子键的作用，在丝素蛋白表面自组装沉积、成核形成类骨羟基磷灰石，并研究不同温度对磷灰石晶体在丝素蛋白表面取向的影响，制备丝素蛋白／羟基磷灰石类骨质复合生物材料，用于骨缺损的修复。
首先，仿生合成丝素蛋白／羟基磷灰石类骨质复合生物材料的制备。以蚕丝丝素蛋白为模板，按照有机／无机为3／7的比例将蚕丝丝素蛋白和磷酸的混合溶液逐步滴定到氢氧化钙溶液中(Ca∶P=1.67)，同时用氢氧化钠调节PH值为9～10，强力搅拌，沉化48小时，形成丝素蛋白／羟基磷灰石复合生物材料。采用梯度温度(25℃、37℃、50℃、60℃、70℃)制备不同的样本，通过FTIR、XRD、SEM、TEM对样本进行表征，筛选出最佳类骨质结构的复合生物材料制备的条件。结果显示：通过对不同温度下制备的SF／HA复合材料进行表征显示，FTIR、XRD显示所制备的复合生物材料中，磷灰石晶体为含有碳酸根的低结晶度纳米羟基磷灰石，与天然骨组织中的磷灰石晶体相似；同时XRD显示磷灰石晶体尺寸为15.6～22.8nm，扫描电镜图片Smile View软件测量显示SF／HA复合材料晶体颗粒为0.229～0.409μm，表明HA晶体以及SF／HA复合材料晶体颗粒均晶体随温度的增加而增大；SF／HA复合材料晶体颗粒XRD图谱显示，随着温度的增加晶体沿c轴择优取向生长，同时TEM电子衍射图显示，在60℃、70℃水浴时，晶体明显沿c轴方向择优生长，生长方向与纤维长轴平行，并且水浴70℃时晶体取向稍优于60℃时复合材料的晶体：这与天然骨组织中，羟基磷灰石晶体在胶原纤维表面生长与排列方向相同。因此，所制备的丝素蛋白／羟基磷灰石复合生物材料为类骨质结构。
论文的第二部分研究了所制备的丝素蛋白／羟基磷灰石类骨质复合生物材料的生物学特性。首先将制备好的材料粉体利用压片机在20MPa的机械力作用下，压制成直径约1.2mm的圆盘型，并利用纯羟基磷灰石材料作为对照培养MG63细胞，观察细胞的增殖，测试其生物学特性。通过检测MTT，ALP，Ca及OC值，利用SEM观察不同时间细胞的粘附和生长，显示所制备的丝素蛋白／羟基磷灰石类骨质复合生物材料有利于细胞的粘附、增殖和生长，可以促进细胞ALP活性表达，提高细胞基质钙离子的释放，并且可以诱导细胞OC分泌增加，促进细胞成骨性能的表达，是一种理想的复合生物材料。
综上所述，本论文以蚕丝丝素蛋白纤维为模板，钙磷在丝素蛋白模板的调控下，在纤维表面沉积、成核形成类骨羟基磷灰石晶体，在丝素蛋白模板精确的调控下，磷灰石晶体在纤维表面沿c轴择优取向生长，晶体生长方向与纤维长轴平行，所制备的类骨质复合生物材料与天然骨具有组成和结构上的统一；并对其生物学特性进行了评价，有利于细胞的粘附，增殖和生长以及成骨活性的表达，是一种理想的复合生物材料。
Multiplicity mineralized composite procedures in Nature provide a rich foundation for biomineralization and novel materials designs. i.e. sea hells, bone and other hard tissue. The procedure is the Natural inorganic mineral formatted by the organic template regulation. The formation of Natural inorganic-organic composites is a complex process, including the assembly of the organic template (i.e. protein, polysaccharides, nucleinic acid, biological membrane and liposome) subsequent inorganic mineral deposition, nucleation and growth on the organic template surface by the the organic template regulation.
The major of thesis focused on the study of the like-bone Silk fibroin (SF)/ hydroxyapatite(HA) biocomposite. First prepare the SF organic template, secondly under controlled PH and temperature calcium and phosphorus begin deposition and nucleation and growth on the organic template surface by the the organic template precise regulation and form like-bone silk fibroin /hydroxyapatite biocomposite. at the same time, research the effect of temperature on the hydroxyapatite crystal direction on the silk fibroin template surface.
Firstly the preparation of like-bone Silk fibroin (SF)/ hydroxyapatite (HA) biocomposite by biomineralization. Ca(OH)_2, H_3PO_4 and SF were used as starting materials, and their composites were synthesized by a biomineralization. The starting materials were prepared for the composition of the SF/HA composite to be 30/70 in weight ratio. add SF template into the H3PO4 solution with vigorous stirring until uniform mixing, the prepare of the composite was conducted by adding the H_3PO_4 solution with the SF slowly into the Ca(OH)_2 solution with vigorous stirring and adjust PH to 9～10 With NaOH at grade temperature (25℃、37℃、50℃、60℃、70℃). Checking the biomaterial prepared at grade temperature, chose the better reparation condition. The results from the tests of FTIR、XRD show that the hydroxyapatite is low crystallinity nano-carbonated apatite [CO_3-substituted Ca_(10)(PO_4)_6(OH)_2].it is consistent with the hydroxyapatite crystals in bone. the size of hydroxyapatite crystals is 15.6～22.8nm showed by XRD, and the crystals size of composite is 0.229~0.409um.they indicate that the crystals size increase with the rising of temperature. The results from the tests of SEM,TEM and electron diffraction show that the needle-like hydroxyapatite crystal grows along the c-axes of hydroxyapatite crystal and The crystallographic c-axes of hydroxyapatite were aligned along the long axes of the fibers, consistent with observations of collagen fibrils and hydroxyapatite crystals in bone at the 60℃、70℃.the like-bone structure is better at the aqueous bath of 70℃than 60℃.
Secondly, in vitro experiments were designed and carried out to evaluate the biological reaction of the biomaterial prepared as described in the first part. Compress the SF/HA powder into disk shape with 1.2mm diameter by tablet machine. and MG63, which is human osteoblast cell phenotypes was adopted to valuate the biological reaction. controlled by HA. Morphology observation ,SEM and MTT results show that the like-bone SF/HA is better benefit to MG63 that HA, it is benefit to MG63 adhesion differentiation and proliferation. At the same time the results of ALP, Ca~(2+) concentration and OC indicate that The SF/HA can promote the ALP express, hasten the delivery of cell matrix Ca~(2+) and induce the OC perspire controlled with HA. So the like-bone SF/HA biomaterial is a better biomaterial.
Based on the evidences from the experiments. calcium and phosphorus begin deposition , nucleation and growth on the organic template surface by the organic template precise regulation and form like-bone silk fibroin/hydroxyapatite biocomposite. The hydroxyapatite is low crystallinity nano-carbonated apatite [CO_3-substituted Ca_(10)(PO_4)_6(OH)_2]. It is consistent with the hydroxyapatite crystals in bone. and the needle-like hydroxyapatite crystal grows along the c-axes of hydroxyapatite crystal and The crystallographic c-axes of hydroxyapatite were aligned along the long axes of the fibers, consistent with observations of collagen fibrils and hydroxyapatite crystals in bone. At the same time, evaluate the biological reaction, the like-bone SF/HA can promote MG63 adhesion differentiation and proliferation and ossify activity.
引文
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