牙种植体表面生物化修饰及构建组织工程支架的实验研究
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摘要
钛及钛合金具有优良的机械性能和生物相容性,成为口腔医学及矫形外科领域广泛应用的种植体材料。但不足之处是,它们属于生物惰性材料,在临床应用中所需愈合时间较长,且植入体内后组织反应表现为非特异性和随机性。对钛种植材料的表面进行改性可以有效地改善其各种性能,获得生物相容性更好、愈合更快、具有生物活性的新型种植体。传统的牙种植体表面改性的方法如化学、物理学及形态学等方法仅在一定程度上改善了其生物相容性和骨结合能力。组织工程渗透到牙种植相关领域,给牙种植体的表面改性及界面愈合方式的研究开辟了一条新路。本研究旨在探讨一种牙种植体表面生物化修饰及组织工程支架构建的研究方法。在对钛种植体表面进行生物化改性的基础上,运用层层自组装技术构建了一种聚电解质多层膜组织工程支架结构,并将大鼠骨髓基质细胞分层包裹在细胞支架内进行培养,以期为促进临床上牙种植体-骨组织界面的组织愈合以及种植体周牙周组织再生研究提供实验依据。研究由以下三个系列实验所组成。
1.钛种植体表面磷酸化及仿生矿化。应用磷酸对钛种植体表面进行磷酸化处理,模拟体液仿生矿化以及大鼠骨髓基质细胞的增殖分化实验鉴定体外矿化诱导性能和磷酸化及矿化材料表面的细胞亲和性。结果发现,磷酸化的钛基材表面沉积了高结晶度的磷酸钛聚合物层,不同晶相的聚合物皆能成功诱导磷灰石晶体的生成,仿生矿化的表层可以诱导骨髓基质细胞的成骨向分化。本实验为钛种植体表面改性提供了一种新的实验方法,并为进一步的生物化改性确立实验基础。
2.研究生物化钛种植体表面对骨形态发生蛋白生长因子的缓释效应。通过层层自组装技术在磷酸化钛种植体表面构建以壳聚糖/海藻酸钠为组分的聚电解质多层膜结构,同时复合骨形态发生蛋白生长因子BMP-2,应用ELISA方法测定BMP-2的体外缓释效果。结果发现,在磷酸化的钛种植体表面构建的PEC多层膜结构能够复合骨生长因子BMP-2并在体外对其进行缓释。细胞学研究证实,体外缓释的BMP-2保持其活性并能发挥骨诱导的生物学效应。本研究建立了一种牙种植体表面构建PEC多层膜结构并实现骨生长因子缓释的技术方法,为牙种植体表面组织工程支架的构建提供了实验依据。
3.在上述实验结果的基础上,在生物化钛种植体表面构建以壳聚糖/海藻酸钠为组分的聚电解质多层膜组织工程支架结构,细胞增殖分化实验鉴定其细胞相容性,并应用层层自组装技术将大鼠骨髓基质细胞分层包裹在细胞支架内进行培养。结果发现,多层膜组织工程支架能够明显促进骨髓基质细胞的增殖和分化,该支架结构能够在种植体表面对细胞进行分层包裹培养。本实验在种植体表面成功构建了组织工程支架,并为牙种植体周牙周组织再生重建研究提供了新的思路和实验依据。
本研究通过系列实验,成功实现对牙种植体表面的生物化改性,在此基础上应用层层自组装技术构建以壳聚糖和海藻酸钠为组分的聚电解质多层膜组织工程支架结构,首次成功实现在牙种植体表面对目的细胞的分层包裹培养,为牙种植体表面组织工程化牙周组织再生研究提供了实验基础。
Titanium and its alloys have been extensively used in dental and orthopedic fields for implants due to their excellent mechanical properties and biocompatibility. However, these implant materials display non-specificity and randomness after implantation into the living body and will spend a long time for clinical healing result from their bioinert properties. The strategy of titanium surface modification can effectively improve the performance of these materials involve preferably biocompatibility and bioactive. Several surface modifications, including physico-chemical and morphologic ones, have been investigated to improve the biocompatibility and bone-bonding ability of Ti implants in a certain extent. One approach, biomimetic surface engineering, develop a new strategy of the study on the surface modification and the bone-interface healing. The purpose of this study is to investigate the research strategy of the biolization modification and the scaffold constructed on the surface of titanium implants. Based on the biolization modification of the titanium surface, the polyelectrolyte complex (PEC) hydrogel composited of polycationic chitosan (CS) and polyanionic alginate(ALG) was self-organized by the Layer-by-Layer self-assemble technique on the surface of titanium, and the PEC hydrogel was used as the scaffold to encapsulate the rat bone marrow stroma cells(BMSCs) delamination for co-culture. The result of the study is the foundation of the experimental research to improve the bone-implant interface healing and the regeneration of the periodontal ligament. The research consist of three serial experiments.
1. Biomineralization and phosphorylation performed on the Ti implant surfaces. The titanium surfaces was treated in the phosphoric acid, and then the phosphorylation specimens were soaked in the simulated body fluid (SBF) to investigate the function of biomineralization. To investigate the biocompatibility of the altered biomimetic surface, the rat bone marrow stroma cells (BMSCs) were cultured on these treated sample surfaces. The results of the study showed that a layer of titanium dihydrogen orthophosphate (Ti(H2PO4)3) have been formed on the native oxide surfaces on the phosphorylation titanium, which have the ability to induce formation of bone-like apatite during immersing in SBF and thus considered bioactive. The biomineralization surfaces can induce the osteogenesis differentiation of the BMSCs. The study provide a new experimental strategy of the dental implants surface modification. The result of this study is the foundation of the experiment of the Ti surface biolization modification.
2. To investigate the effects of delayed release of BMP-2 adsorbed in the biolization Ti surface, the PEC hydrogel composited of CS and ALG was self-organized on the surfaces of titanium. Subsequently, the effects of delayed release of BMP-2 encapsulated in the PEC multilayer films was evaluated by enzyme linked immunosorbent assay (ELISA). The results showed that BMP-2 can be loaded in the PEC multilayer films and controlled release in vitro. The BMP-2 encapsulated in the PEC can be kept activity and develop its osteoinduction biological effect. The study provided a new experimental strategy of constructing the PEC multilayer films on titanium surfaces. Moreover, the results also offered a foundation of the experiment of the scaffold constructed on titanium surfaces.
3. Based on the above-mentioned study results, the PEC of CS and ALG was self-organized on the surface of titanium, which was used as the scaffold to encapsulate the BMSCs delamination for co-culture. The results showed that the PEC multilayer scaffolds can obviously enhance the proliferation and differentiation of rat BMSCs and can encapsulate experimental cells delamination for co-culture. The engineering scaffolds were constructed on titanium surfaces successfully in this study, which offered a new research approach and experimental foundation of the regeneration of the periodontal ligament.
In this paper, based on a serial of experiments, the biolization modification on the surface of titanium implant was performed successfully and the PEC of CS and ALG was self-organized on the surface of titanium, which was used as the scaffold to encapsulate the BMSCs delamination for co-culture. The results of the study offered a new research approach and experimental foundation of the regeneration of the periodontal ligament.
1.钛种植体表面磷酸化及仿生矿化。应用磷酸对钛种植体表面进行磷酸化处理,模拟体液仿生矿化以及大鼠骨髓基质细胞的增殖分化实验鉴定体外矿化诱导性能和磷酸化及矿化材料表面的细胞亲和性。结果发现,磷酸化的钛基材表面沉积了高结晶度的磷酸钛聚合物层,不同晶相的聚合物皆能成功诱导磷灰石晶体的生成,仿生矿化的表层可以诱导骨髓基质细胞的成骨向分化。本实验为钛种植体表面改性提供了一种新的实验方法,并为进一步的生物化改性确立实验基础。
2.研究生物化钛种植体表面对骨形态发生蛋白生长因子的缓释效应。通过层层自组装技术在磷酸化钛种植体表面构建以壳聚糖/海藻酸钠为组分的聚电解质多层膜结构,同时复合骨形态发生蛋白生长因子BMP-2,应用ELISA方法测定BMP-2的体外缓释效果。结果发现,在磷酸化的钛种植体表面构建的PEC多层膜结构能够复合骨生长因子BMP-2并在体外对其进行缓释。细胞学研究证实,体外缓释的BMP-2保持其活性并能发挥骨诱导的生物学效应。本研究建立了一种牙种植体表面构建PEC多层膜结构并实现骨生长因子缓释的技术方法,为牙种植体表面组织工程支架的构建提供了实验依据。
3.在上述实验结果的基础上,在生物化钛种植体表面构建以壳聚糖/海藻酸钠为组分的聚电解质多层膜组织工程支架结构,细胞增殖分化实验鉴定其细胞相容性,并应用层层自组装技术将大鼠骨髓基质细胞分层包裹在细胞支架内进行培养。结果发现,多层膜组织工程支架能够明显促进骨髓基质细胞的增殖和分化,该支架结构能够在种植体表面对细胞进行分层包裹培养。本实验在种植体表面成功构建了组织工程支架,并为牙种植体周牙周组织再生重建研究提供了新的思路和实验依据。
本研究通过系列实验,成功实现对牙种植体表面的生物化改性,在此基础上应用层层自组装技术构建以壳聚糖和海藻酸钠为组分的聚电解质多层膜组织工程支架结构,首次成功实现在牙种植体表面对目的细胞的分层包裹培养,为牙种植体表面组织工程化牙周组织再生研究提供了实验基础。
Titanium and its alloys have been extensively used in dental and orthopedic fields for implants due to their excellent mechanical properties and biocompatibility. However, these implant materials display non-specificity and randomness after implantation into the living body and will spend a long time for clinical healing result from their bioinert properties. The strategy of titanium surface modification can effectively improve the performance of these materials involve preferably biocompatibility and bioactive. Several surface modifications, including physico-chemical and morphologic ones, have been investigated to improve the biocompatibility and bone-bonding ability of Ti implants in a certain extent. One approach, biomimetic surface engineering, develop a new strategy of the study on the surface modification and the bone-interface healing. The purpose of this study is to investigate the research strategy of the biolization modification and the scaffold constructed on the surface of titanium implants. Based on the biolization modification of the titanium surface, the polyelectrolyte complex (PEC) hydrogel composited of polycationic chitosan (CS) and polyanionic alginate(ALG) was self-organized by the Layer-by-Layer self-assemble technique on the surface of titanium, and the PEC hydrogel was used as the scaffold to encapsulate the rat bone marrow stroma cells(BMSCs) delamination for co-culture. The result of the study is the foundation of the experimental research to improve the bone-implant interface healing and the regeneration of the periodontal ligament. The research consist of three serial experiments.
1. Biomineralization and phosphorylation performed on the Ti implant surfaces. The titanium surfaces was treated in the phosphoric acid, and then the phosphorylation specimens were soaked in the simulated body fluid (SBF) to investigate the function of biomineralization. To investigate the biocompatibility of the altered biomimetic surface, the rat bone marrow stroma cells (BMSCs) were cultured on these treated sample surfaces. The results of the study showed that a layer of titanium dihydrogen orthophosphate (Ti(H2PO4)3) have been formed on the native oxide surfaces on the phosphorylation titanium, which have the ability to induce formation of bone-like apatite during immersing in SBF and thus considered bioactive. The biomineralization surfaces can induce the osteogenesis differentiation of the BMSCs. The study provide a new experimental strategy of the dental implants surface modification. The result of this study is the foundation of the experiment of the Ti surface biolization modification.
2. To investigate the effects of delayed release of BMP-2 adsorbed in the biolization Ti surface, the PEC hydrogel composited of CS and ALG was self-organized on the surfaces of titanium. Subsequently, the effects of delayed release of BMP-2 encapsulated in the PEC multilayer films was evaluated by enzyme linked immunosorbent assay (ELISA). The results showed that BMP-2 can be loaded in the PEC multilayer films and controlled release in vitro. The BMP-2 encapsulated in the PEC can be kept activity and develop its osteoinduction biological effect. The study provided a new experimental strategy of constructing the PEC multilayer films on titanium surfaces. Moreover, the results also offered a foundation of the experiment of the scaffold constructed on titanium surfaces.
3. Based on the above-mentioned study results, the PEC of CS and ALG was self-organized on the surface of titanium, which was used as the scaffold to encapsulate the BMSCs delamination for co-culture. The results showed that the PEC multilayer scaffolds can obviously enhance the proliferation and differentiation of rat BMSCs and can encapsulate experimental cells delamination for co-culture. The engineering scaffolds were constructed on titanium surfaces successfully in this study, which offered a new research approach and experimental foundation of the regeneration of the periodontal ligament.
In this paper, based on a serial of experiments, the biolization modification on the surface of titanium implant was performed successfully and the PEC of CS and ALG was self-organized on the surface of titanium, which was used as the scaffold to encapsulate the BMSCs delamination for co-culture. The results of the study offered a new research approach and experimental foundation of the regeneration of the periodontal ligament.
引文
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