钛基种植体表面仿细胞外基质活性涂层的设计、构建及其生物学评价
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摘要
缩短种植体骨整合时间,达到种植体即刻负载或早期负载是种植体表面改性研究的主要目标。本研究从仿生的视角,采用层层自组装的方法(LbL technique),在纯钛片表面以设计、构建仿细胞外基质活性涂层为目标,进行了方法学探讨。在此基础上,对各种涂层进行了详细的生物学评价。
首先,纯钛片表面经打磨抛光及氧化性混酸溶液处理,得到我们需要的基底面。研究表明这是一个富含羟基且具有良好生物活性的基底面,由于羟基与富含氨基的聚电解质生物大分子之间的氢键作用,为我们后续的组装提供了可能。接着选择了4种具有良好骨诱导性的生物大分子(Ⅰ型胶原、壳聚糖、透明质酸和多聚谷氨酸),根据它们等电点的不同配成带电荷的聚电解质溶液(Ⅰ型胶原和壳聚糖带正电荷,透明质酸和多聚谷氨酸负电荷),通过层层自组装技术将上述聚电解质两两配比组装到纯钛基底面上,共得到4种不同的涂层并进行表征。扫描电镜观察发现与纯钛基底面相比,涂层的表面形貌发生了改变,也是布满了各种不同大小的纳米级别的颗粒；AFM检测结果也证实了相同的变化趋势。X射线光电子能谱仪分析发现涂层表面元素成分氮的含量随着组装层数的增加而不断升高；而石英晶体微量天平检测的涂层厚度也在随着组装层数的增加而不断增厚；最后,接触角实验显示涂层表面的可湿性随着钛片表面聚电解质组装层数的变化而不断变化。以上结果证明我们所选择的有机质大分子被成功的组装到了纯钛表面。接着,在各种涂层上体外培养成骨前体细胞(MC3T3-E1),观察细胞的粘附、增殖和分化情况。结果我们发现以Ⅰ型胶原和透明质酸为骨架的涂层最有利于成骨前体细胞的生长。
接下来,我们设计合成了GRGDSPC多肽序列,并将该多肽序列引入到透明质酸结构中,然后利用自组装的方法构建了胶原／透明质酸聚电解质复合层。由于该多肽序列中端基为半胱氨酸残基,该残基中有活泼的巯基,而巯基之间可以在温和的条件下进行交联形成双硫键,从而使得最后的胶原／透明质酸聚电解质复合膜稳定。这样,我们在引入RGD序列的同时,亦实现了聚电复合膜的交联。体外降解研究显示,该交联的胶原／透明质酸复合膜稳定性显著提高；不仅如此,该涂层对谷胱甘肽具有降解响应性,在谷胱甘肽模拟体液中的降解速度大大提高,暗示这种涂层可以在体内通过谷胱甘肽而加速降解。体外生物学评价显示：与胶原／透明质酸涂层相比,引入RGD的涂层显著不仅促进了成骨前体细胞的粘附,而且也促进了成骨前体细胞的增殖、分化和钙化。
在此基础上,我们计划在种植体表面的涂层中引入活性生长因子。BMP-2分子由于具有强烈的骨诱导性而备受关注,本实验也选择BMP-2作为研究对象。首先将BMP-2溶入胶原溶液而带上正电荷,接着通过LBL技术将BMP-2引入到涂层中,接下来同样将涂层交联。体外降解实验表明,该涂层在增加稳定性的同时也实现了对BMP-2分子的缓释作用,而释放出来的BMP-2分子也没有丧失原有的活性。生物学评价结果也表明,与RGD功能化的胶原／透明质酸复合膜相比,该活性涂层显著促进了细胞的分化和功能表达。与细胞外基质的结构功能类似,该涂层不仅仅是作为成骨细胞的支架让其黏附生长,而且涂层中的RGD序列与缓释出来的生长因子能够通过与整合素或其它特定的细胞表面受体与成骨细胞直接或间接的作用,从而促进成骨细胞的生长、迁移和分化,因而具有明显的仿生特征。
不仅如此,当以同样的策略在已经引入RGD的胶原／透明质酸复合膜中组装bFGF分子(最近的研究发现bFGF不仅能刺激血管的再生,也能明显促进成骨),也取得了成功,同样使涂层稳定性大大提高,也具有对bFGF分子的缓释功能。同样较RGD功能化的胶原／透明质酸复合膜促进了成骨细胞的分化和钙化,该活性涂层也具有仿生的特点。
最后,我们在涂层中同时组装上BMP-2和bFGF分子,降解结果显示该涂层实现了对BMP-2和bFGF分子的缓释作用,而且BMP-2和bFGF它们之间能独立释放,互不干扰。生物学评价结果显示,组装了多个生长因子的活性涂层与分别组装BMP-2或bFGF或RGD功能化的涂层相比较,该涂层显著促进了成骨细胞的黏附增殖和分化,而且BMP-2和bFGF分子之间具有明显的协同作用。这种用有机质来改性的新型的仿生的种植体表面能显著促进MC3T3-E1细胞的黏附、增殖和分化,有助于种植体的早期骨整合。
显然,以上研究显示可以通过自组装的方法进行仿细胞外基质活性涂层的构建,而且该表面可以既可以极大促进成骨前体细胞的黏附,又显著促进了成骨前体细胞的增殖和分化；显而易见,如果用于种植体的表面,该表面有望促进新骨的早期形成,并增强种植体-骨组织之间的结合强度。
The main purpose of surface modification of dental implant is to shorten the time of osseointegration, and as to reach immediately or early loading. From the aspect of biomimetics, this investigation focused on the detailed methodology research of designing and constructing a biomimetic ECM bioactive coating on pure commercial titanium implant surface. Based on this, biological evaluation of different coatings was undertaken.
We produced a basal surface by the following treatments:first grinding and polishing, then acid-etching with a mixture of H2SO4/H2O2. Previous studies indicated it is a basal surface with plenty of hydroxy group and owns quite good biological activity. The interaction of hydrogen bonds, between hydroxy group and polyelectrolyte with lots of amino group, provides the feasibility of the following self-assembly. Then four biomacromolecules with good osteoinductive, collagen typeⅠ, chitosan, hyaluronic acid and polyglutamic acid were choosed. and four polyelectrolyte solutions with disparity charge were prepared according to their different isoelectric point. Four different coatings were prepared on pure titanium basal surface with layer by layer technique and were sent for characterization. SEM analysis demonstrated that surface morphology of the coatings was changed compared with that of basal surface, and were also covered with different granules in nano size. The results of AFM examination also proved almost the same tendancy. XPS analysis showed that the element nitrogen content of the coating surface was dramatically increased with deposition number. On the other hand, the results of quartz crystal microbalance measurement indicated that the thickness of coating was also increased with approximately linearly with the deposition number. Finally, contact angle measurement showed that surface wettability of the coating was dramatically altered along the deposition number. Taken together, we can draw a conclusion that organic biomacromolecules we applied have been successfully constructed on the titanium surface. After that, we cultured pre-osteoblasts (MC3T3-E1) on different coatings in order to evaluate cell attachment, cell proliferation and cell differentiation. The biological evaluation results suggested that the coating based on collagen type I and hyaluronic acid promote cell growth at mostly.
Next, GRGDSPC polypeptide sequence was designed and synthesized, and was then tointroduced to hyaluronic acid. Finally, collagen/hyaluronic acid PEM was constructed on titanium surface by LBL technique. Owing to the high activity of sulfhydryl group from aminothiopropionic acid reside of the polypeptide; the sulfhydryl group can change into disulfide bond with cross linkage reaction under moderate condition, which makes the collagen/hyaluronic acid PEM more stable. Thus, the RGD sequence was introduced to the PEM and the PEM was cross linked at the same time. The in vitro degradation experiment showed, the stability of collagen/hyaluronic acid PEM, which was cross linked, was significantly promoted. Furthermore, the degradation rate of this coating was largely elevated in GSH fluid, which suggested the degradation rate of this coating could be accelerated with the action of in vivo glutathion. In vitro biological evaluation demonstrated, compared with collagen/hyaluronic acid PEM, the RGD functionalized PEM significantly promoted the attachmen, proliferation, differentiation and calcification of preosteoblasts.
Still on this base, we plan to introduce growth factors to this coating. BMP-2 has been extensively explored because of its strong osteoinductivity, for the same reason BMP-2 was choosed in this experiment. So BMP-2 was dissolved in collagen solution and thus positively charged, and was introduced to the coating by LBL technique, then cross linkage reaction was also carried out. Degradation test manifestated BMP-2 bioactive molecules could be slowly released from the treated coating, which also shared a higher stability. Biological evaluation results suggested that the released BMP-2 molecules do not loss any activity. Compared with RGD functionalized COL/HA PEM, this bioactive coating significantly improved the differentiation and function expression of preosteoblasts. Similar to the unique structure and function of ECM, that coating serves not only as a passive scaffold for preosteoblasts, but RGD sequence and several growth factors slowly released from this coating can interact with presosteoblasts via integrins or other specific cell surface receptors, thus directly and indirectly promoting growth, migration and differentiation of these cells.
Moreover, bFGF can also be incorporated into the RGD functionalized COL/HA PEM successfully with the same strategy. However, recently reseach has verified that bFGF can not only stimulate vascularization, but also promote osteogenesis. The constructed coating also shares several advantages such as stability and drug delivery. This bioactive coating also shares some character of biomimetics, and significantly increased the differentiation and calcification of preosteoblasts than RGD functionalized COL/HA PEM.
Finally, we try to introduce both BMP-2 and bFGF to the coating at the same time, and in vitro degradation test also indicated that BMP-2 and bFGF could be slowly released from the coating respectively, without any intervention. Compared with all the coatings mentioned above, this special coating which incorporated with both BMP-2 and bFGF could significantly promote cell attachment, cell proliferation and cell differentiation, however, there exists conspicuous synergistic effect between BMP-2 and bFGF. This newly biomimetic ECM bioactive coating on dental surface, which was modified with organic macromolecules, could encourage attachment, proliferation and differentiation of MC3T3-E1 cells, and might facilitate early bone integration to dental implant surface.
Obviously, from this investigation, we can get a conclution that biomimetic ECM bioactive coating could be constructed on dental implant surface successfully by LBL technique, and this coating remarkably enhance preosteoblasts attachment, proliferation and differentiation, and might facilitate better integration with bone.
首先,纯钛片表面经打磨抛光及氧化性混酸溶液处理,得到我们需要的基底面。研究表明这是一个富含羟基且具有良好生物活性的基底面,由于羟基与富含氨基的聚电解质生物大分子之间的氢键作用,为我们后续的组装提供了可能。接着选择了4种具有良好骨诱导性的生物大分子(Ⅰ型胶原、壳聚糖、透明质酸和多聚谷氨酸),根据它们等电点的不同配成带电荷的聚电解质溶液(Ⅰ型胶原和壳聚糖带正电荷,透明质酸和多聚谷氨酸负电荷),通过层层自组装技术将上述聚电解质两两配比组装到纯钛基底面上,共得到4种不同的涂层并进行表征。扫描电镜观察发现与纯钛基底面相比,涂层的表面形貌发生了改变,也是布满了各种不同大小的纳米级别的颗粒；AFM检测结果也证实了相同的变化趋势。X射线光电子能谱仪分析发现涂层表面元素成分氮的含量随着组装层数的增加而不断升高；而石英晶体微量天平检测的涂层厚度也在随着组装层数的增加而不断增厚；最后,接触角实验显示涂层表面的可湿性随着钛片表面聚电解质组装层数的变化而不断变化。以上结果证明我们所选择的有机质大分子被成功的组装到了纯钛表面。接着,在各种涂层上体外培养成骨前体细胞(MC3T3-E1),观察细胞的粘附、增殖和分化情况。结果我们发现以Ⅰ型胶原和透明质酸为骨架的涂层最有利于成骨前体细胞的生长。
接下来,我们设计合成了GRGDSPC多肽序列,并将该多肽序列引入到透明质酸结构中,然后利用自组装的方法构建了胶原／透明质酸聚电解质复合层。由于该多肽序列中端基为半胱氨酸残基,该残基中有活泼的巯基,而巯基之间可以在温和的条件下进行交联形成双硫键,从而使得最后的胶原／透明质酸聚电解质复合膜稳定。这样,我们在引入RGD序列的同时,亦实现了聚电复合膜的交联。体外降解研究显示,该交联的胶原／透明质酸复合膜稳定性显著提高；不仅如此,该涂层对谷胱甘肽具有降解响应性,在谷胱甘肽模拟体液中的降解速度大大提高,暗示这种涂层可以在体内通过谷胱甘肽而加速降解。体外生物学评价显示：与胶原／透明质酸涂层相比,引入RGD的涂层显著不仅促进了成骨前体细胞的粘附,而且也促进了成骨前体细胞的增殖、分化和钙化。
在此基础上,我们计划在种植体表面的涂层中引入活性生长因子。BMP-2分子由于具有强烈的骨诱导性而备受关注,本实验也选择BMP-2作为研究对象。首先将BMP-2溶入胶原溶液而带上正电荷,接着通过LBL技术将BMP-2引入到涂层中,接下来同样将涂层交联。体外降解实验表明,该涂层在增加稳定性的同时也实现了对BMP-2分子的缓释作用,而释放出来的BMP-2分子也没有丧失原有的活性。生物学评价结果也表明,与RGD功能化的胶原／透明质酸复合膜相比,该活性涂层显著促进了细胞的分化和功能表达。与细胞外基质的结构功能类似,该涂层不仅仅是作为成骨细胞的支架让其黏附生长,而且涂层中的RGD序列与缓释出来的生长因子能够通过与整合素或其它特定的细胞表面受体与成骨细胞直接或间接的作用,从而促进成骨细胞的生长、迁移和分化,因而具有明显的仿生特征。
不仅如此,当以同样的策略在已经引入RGD的胶原／透明质酸复合膜中组装bFGF分子(最近的研究发现bFGF不仅能刺激血管的再生,也能明显促进成骨),也取得了成功,同样使涂层稳定性大大提高,也具有对bFGF分子的缓释功能。同样较RGD功能化的胶原／透明质酸复合膜促进了成骨细胞的分化和钙化,该活性涂层也具有仿生的特点。
最后,我们在涂层中同时组装上BMP-2和bFGF分子,降解结果显示该涂层实现了对BMP-2和bFGF分子的缓释作用,而且BMP-2和bFGF它们之间能独立释放,互不干扰。生物学评价结果显示,组装了多个生长因子的活性涂层与分别组装BMP-2或bFGF或RGD功能化的涂层相比较,该涂层显著促进了成骨细胞的黏附增殖和分化,而且BMP-2和bFGF分子之间具有明显的协同作用。这种用有机质来改性的新型的仿生的种植体表面能显著促进MC3T3-E1细胞的黏附、增殖和分化,有助于种植体的早期骨整合。
显然,以上研究显示可以通过自组装的方法进行仿细胞外基质活性涂层的构建,而且该表面可以既可以极大促进成骨前体细胞的黏附,又显著促进了成骨前体细胞的增殖和分化；显而易见,如果用于种植体的表面,该表面有望促进新骨的早期形成,并增强种植体-骨组织之间的结合强度。
The main purpose of surface modification of dental implant is to shorten the time of osseointegration, and as to reach immediately or early loading. From the aspect of biomimetics, this investigation focused on the detailed methodology research of designing and constructing a biomimetic ECM bioactive coating on pure commercial titanium implant surface. Based on this, biological evaluation of different coatings was undertaken.
We produced a basal surface by the following treatments:first grinding and polishing, then acid-etching with a mixture of H2SO4/H2O2. Previous studies indicated it is a basal surface with plenty of hydroxy group and owns quite good biological activity. The interaction of hydrogen bonds, between hydroxy group and polyelectrolyte with lots of amino group, provides the feasibility of the following self-assembly. Then four biomacromolecules with good osteoinductive, collagen typeⅠ, chitosan, hyaluronic acid and polyglutamic acid were choosed. and four polyelectrolyte solutions with disparity charge were prepared according to their different isoelectric point. Four different coatings were prepared on pure titanium basal surface with layer by layer technique and were sent for characterization. SEM analysis demonstrated that surface morphology of the coatings was changed compared with that of basal surface, and were also covered with different granules in nano size. The results of AFM examination also proved almost the same tendancy. XPS analysis showed that the element nitrogen content of the coating surface was dramatically increased with deposition number. On the other hand, the results of quartz crystal microbalance measurement indicated that the thickness of coating was also increased with approximately linearly with the deposition number. Finally, contact angle measurement showed that surface wettability of the coating was dramatically altered along the deposition number. Taken together, we can draw a conclusion that organic biomacromolecules we applied have been successfully constructed on the titanium surface. After that, we cultured pre-osteoblasts (MC3T3-E1) on different coatings in order to evaluate cell attachment, cell proliferation and cell differentiation. The biological evaluation results suggested that the coating based on collagen type I and hyaluronic acid promote cell growth at mostly.
Next, GRGDSPC polypeptide sequence was designed and synthesized, and was then tointroduced to hyaluronic acid. Finally, collagen/hyaluronic acid PEM was constructed on titanium surface by LBL technique. Owing to the high activity of sulfhydryl group from aminothiopropionic acid reside of the polypeptide; the sulfhydryl group can change into disulfide bond with cross linkage reaction under moderate condition, which makes the collagen/hyaluronic acid PEM more stable. Thus, the RGD sequence was introduced to the PEM and the PEM was cross linked at the same time. The in vitro degradation experiment showed, the stability of collagen/hyaluronic acid PEM, which was cross linked, was significantly promoted. Furthermore, the degradation rate of this coating was largely elevated in GSH fluid, which suggested the degradation rate of this coating could be accelerated with the action of in vivo glutathion. In vitro biological evaluation demonstrated, compared with collagen/hyaluronic acid PEM, the RGD functionalized PEM significantly promoted the attachmen, proliferation, differentiation and calcification of preosteoblasts.
Still on this base, we plan to introduce growth factors to this coating. BMP-2 has been extensively explored because of its strong osteoinductivity, for the same reason BMP-2 was choosed in this experiment. So BMP-2 was dissolved in collagen solution and thus positively charged, and was introduced to the coating by LBL technique, then cross linkage reaction was also carried out. Degradation test manifestated BMP-2 bioactive molecules could be slowly released from the treated coating, which also shared a higher stability. Biological evaluation results suggested that the released BMP-2 molecules do not loss any activity. Compared with RGD functionalized COL/HA PEM, this bioactive coating significantly improved the differentiation and function expression of preosteoblasts. Similar to the unique structure and function of ECM, that coating serves not only as a passive scaffold for preosteoblasts, but RGD sequence and several growth factors slowly released from this coating can interact with presosteoblasts via integrins or other specific cell surface receptors, thus directly and indirectly promoting growth, migration and differentiation of these cells.
Moreover, bFGF can also be incorporated into the RGD functionalized COL/HA PEM successfully with the same strategy. However, recently reseach has verified that bFGF can not only stimulate vascularization, but also promote osteogenesis. The constructed coating also shares several advantages such as stability and drug delivery. This bioactive coating also shares some character of biomimetics, and significantly increased the differentiation and calcification of preosteoblasts than RGD functionalized COL/HA PEM.
Finally, we try to introduce both BMP-2 and bFGF to the coating at the same time, and in vitro degradation test also indicated that BMP-2 and bFGF could be slowly released from the coating respectively, without any intervention. Compared with all the coatings mentioned above, this special coating which incorporated with both BMP-2 and bFGF could significantly promote cell attachment, cell proliferation and cell differentiation, however, there exists conspicuous synergistic effect between BMP-2 and bFGF. This newly biomimetic ECM bioactive coating on dental surface, which was modified with organic macromolecules, could encourage attachment, proliferation and differentiation of MC3T3-E1 cells, and might facilitate early bone integration to dental implant surface.
Obviously, from this investigation, we can get a conclution that biomimetic ECM bioactive coating could be constructed on dental implant surface successfully by LBL technique, and this coating remarkably enhance preosteoblasts attachment, proliferation and differentiation, and might facilitate better integration with bone.
引文
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