多孔丝素蛋白材料的血管化及其组织再生性能的研究
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摘要
近几十年来,生物材料的研究从最初的惰性材料到生物活性材料,进而转向开发可原位诱导组织再生、器官形成的组织诱导性生物材料。丝素蛋白由于其良好的组织相容性、易加工性和可降解性等,作为组织诱导性生物医用材料的研究日益受到关注。
对于机体的绝大多数组织而言,血管新生是损伤修复过程中必不可少的环节。因此,利用生物材料辅助修复缺损组织时,必然要求生物材料能够快速有效地血管化。尽管目前已经有很多研究关注改善生物材料的血管化问题,比如,设计和加工具有恰当孔径、孔隙率和连通性的多孔材料,向材料中添加血管生长因子、接种细胞及人造血管等等。但是,生物材料血管化的详细过程及其与组织再生之间的相互关系,生物材料性能与血管化及组织再生的相互关系,体内环境对生物材料的修饰作用和该作用对血管化的影响以及材料降解性能与组织再生之间的相互关系等方面,尚无系统的研究。故目前关于材料血管化方面的研究,在应用水平上仍然没有取得实际意义的成功,生物材料的血管化问题仍然是制约生物材料研究与应用的主要因素。
本文以丝素蛋白基医用生物材料研发中的关键问题——材料的血管化及其组织再生性能为目标进行研究和探讨。通过对多孔丝素蛋白材料和聚乙烯醇(PVA)海绵在不同组织中的血管化过程的仔细观察以及对生物材料血管化与组织再生之间的关系的探讨,从生物学和材料科学的角度分析与讨论了生物材料血管化及组织再生的过程与实质。
进一步,通过结合体内外方法考察了体内环境对植入材料的修饰作用及其对细胞增殖、伤口愈合的影响。体内环境对生物材料的修饰作用,主要包括蛋白吸附及细胞外基质在材料中的组装。经过体内环境的修饰作用,生物材料即被活化,为细胞生长提供适宜的微环境。
本文最后提出一种评价材料降解与组织再生之间的协调性的方法,半定量的考察材料的降解性能及其对组织再生的影响,进一步揭示材料降解与组织再生之间的关系。
本研究将形态结构相似的多孔丝素蛋白材料和聚乙烯醇(PVA)海绵分别植入大鼠背部真皮组织和股部肌肉组织,通过组织学切片详细观察其血管化过程和组织再生情况。结果表明,多孔丝素蛋白材料和PVA海绵,无论植入真皮组织还是肌肉组织,其血管化过程具有共同的步骤和特征。这个过程包括4个依次连续发生的阶段:吸附阶段,细胞外基质组装阶段,组织及血管快速增生阶段及重塑阶段。据此推测,此模式为多孔生物材料在体内血管化所共有。这为开发多孔医用生物材料以促进生物材料快速有效的血管化提供了实验基础。
通过考察多孔丝素蛋白材料和PVA海绵中的血管化和组织再生之间的关系,结果表明,植入早期细胞外基质在生物材料中的组装及细胞增殖首先为材料的血管化提供了良好的条件,材料良好的血管化又为新生组织的进一步生长提供保证。通过比较,多孔丝素蛋白材料的血管化情况优于PVA海绵,其组织再生的速度快于PVA海绵,新生组织的质量优于PVA海绵。由此可知,生物材料的化学组成对材料的血管化及其组织再生重要影响。
此外,结合体内外方法,进一步考察了体内环境对生物材料的修饰作用及其对细胞增殖和伤口愈合的影响,考察了材料性能对这种修饰作用的影响。结果表明,生物材料植入体内以后,体内环境会立即对材料进行修饰,这种修饰作用对细胞增殖活性及伤口愈合时间及愈合质量有重要影响。多孔丝素蛋白材料和PVA海绵的比较表明,材料的表面化学组成及表面形态结构不同,体内环境的修饰效果也就不同。
最后,通过定义残余材料面积百分比、降解材料面积百分比和组织再生面积百分比来半定量描述生物材料的降解程度和组织再生情况,通过生物材料降解曲线与组织再生曲线来比较材料降解速率与组织再生速率之间的协调性。从而提出了一种判定生物材料降解速率与组织再生速率之间协调程度的半定量方法。经过初步的实验验证,这种方法的评价结果与形态学观察结果一致,说明该方法简便、快速、可行。
总之,本研究通过观察多孔生物材料血管化过程,进一步分析了生物材料血管化与组织再生之间的关系。通过体内外方法对生物材料的体内修饰作用及其对细胞增殖和伤口愈合的作用进行了考察,通过比较多孔丝素蛋白材料和PVA海绵不同的修饰结果,表明生物材料的化学组成及表面形态结构对体内修饰作用的效果有重要影响。最后,本文提出了一种评价材料降解速率与组织再生速率协调性的半定量方法,考察了生物材料降解与组织再生之间的关系。期望本文研究结果能够为生物材料的血管化和组织再生提供实验依据,为血管新生、伤口愈合等基础理论研究提供参考,为进一步建立生物材料与组织再生之间的量化模型提供基础。
Within the past decades, biomaterials have been evolved from inert materials to bioactive materials and tissue inducing biomaterials which may induce tissue regeneration in situ. Because of nice biocompatibility, processible attributes and biodegradability of silk fibroin, study on the biomaterials based on silk fibroin has attracted more and more intrest.
Angiogenesis is a key step during wound healing in most tissues, so biomaterials aided wound healing requires rapid and effective neovascularization of biomaterials after implantation. Althogh lots of researches focus on the problem such as design and preparation of porous biomaterials, application of growth factors and (stem) cells and artificial blood vessels, little real success has been abtained and bad neovascularization of biomaterials has been the main obstacle in terms of clinical application.
In addition, little knowledge of detailed neovascularization of biomaterials, effects of in vivo circumstance on biomaterials implanted, and how to evaluate the degradation rate of biomaterials and tissue regeneration rate is known.
Thus, the purpose of this study is to explore and analyze the essence of neovascularization and inducibility in biomaterials through observation on the neovascularization process occurred in porous silk fibroin materials and polyvinyl alcohol (PVA) sponges.
In this study, porous silk fibroin materials and PVA sponges were implanted in muscles of hind limb and back skin in rats, and the neovascularization process in both materials has been observed in detail via tissue sections. In addition, the biocompatibility of porous silk fibroin materials and PVA sponges has been semi-quantitively compared through histological scoring method. The results showed that identical neovascularization process occurred in both materials either implanted in the muscles or the back skin. The process mainly included four sequential steps as follows: adsorptioin, organization of extracellular matrix (ECM), rapid proliferation of tissues and remodeling of the tissues and blood vessels networks. This might be the implication of general mode of neovascularization process in porous biomaterials. Furthermore, the results showed that porous silk fibroin materials have better biocompatibility than PVA sponges.
The in vivo decoration of porous silk fibroin materials and tissue regenration in porous silk fibroin materials have been further examined by combination with in vivo implantation and in vitro cell culture. The definition of in vivo decoration mainly indicates the protein adsorption and ECM organization in the materials in vivo. As for the results that porous silk fibroin materials have been more advantageous than PVA sponges to neovascularization and tissue regeneration, there might be 4 reasons as follows: (1) silk fibroin, as a natural protein, has better biocompatibility than polymers; (2) the surface texture of porous silk fibroin materials might be suitable for protein adsorption and ECM organization; (3) the porous structure of porous silk fibroin materials facilitates the ingrowth of endothelial cells and fibroblasts; (4) the biodegradation rate of porous silk fibroin materials accords with the tissue regeneration rate.
In the last chapter, the degradation of biomaterials and tissue regeneration were quantified by definition of area percentage of residual biomaterials (Rirs(%)=Sis/S1s×100%) and area percentage of newly formed tissues (Rint(%)=Sit/Sg×100%) in histological sections. Besides, biodegradation rate of biomaterials and tissue regeneration rate were compared by comparison of growth time of newly formed tissues (tr) and biodegradation time (tm) of biomaterials. In brief, a semi-quantitive method has been established in this paper to evaluate the synchronization extent between the degradation rate of biomaterials and regeneration rate of tissues.
Where Sis is the area of residual biomaterials i days after implantation, S1s is the area of biomaterials 1 day after implantation, Sit is the area of newly formed tissues i days after implantation, Sg is the area of full recovery tissues. And tr indicates the time (day) when a certain amount of tissues have been regenerated and thus they could continue growing without any stent. tm indicates the time (day) when a certain amount of biomaterials have been biodegraded and thus they lose their whole support.
Through this method, the accordance between tissue regeneration and biodegradation of biomaterials could be evaluated semi-quantitatively. Moreover, the feasibility of this method was testified through in vivo experiments, and the results suggested that this method is rapid, feasible and easy to operate. This work might provide solid basis for further quantifying attributes of biomaterials.
对于机体的绝大多数组织而言,血管新生是损伤修复过程中必不可少的环节。因此,利用生物材料辅助修复缺损组织时,必然要求生物材料能够快速有效地血管化。尽管目前已经有很多研究关注改善生物材料的血管化问题,比如,设计和加工具有恰当孔径、孔隙率和连通性的多孔材料,向材料中添加血管生长因子、接种细胞及人造血管等等。但是,生物材料血管化的详细过程及其与组织再生之间的相互关系,生物材料性能与血管化及组织再生的相互关系,体内环境对生物材料的修饰作用和该作用对血管化的影响以及材料降解性能与组织再生之间的相互关系等方面,尚无系统的研究。故目前关于材料血管化方面的研究,在应用水平上仍然没有取得实际意义的成功,生物材料的血管化问题仍然是制约生物材料研究与应用的主要因素。
本文以丝素蛋白基医用生物材料研发中的关键问题——材料的血管化及其组织再生性能为目标进行研究和探讨。通过对多孔丝素蛋白材料和聚乙烯醇(PVA)海绵在不同组织中的血管化过程的仔细观察以及对生物材料血管化与组织再生之间的关系的探讨,从生物学和材料科学的角度分析与讨论了生物材料血管化及组织再生的过程与实质。
进一步,通过结合体内外方法考察了体内环境对植入材料的修饰作用及其对细胞增殖、伤口愈合的影响。体内环境对生物材料的修饰作用,主要包括蛋白吸附及细胞外基质在材料中的组装。经过体内环境的修饰作用,生物材料即被活化,为细胞生长提供适宜的微环境。
本文最后提出一种评价材料降解与组织再生之间的协调性的方法,半定量的考察材料的降解性能及其对组织再生的影响,进一步揭示材料降解与组织再生之间的关系。
本研究将形态结构相似的多孔丝素蛋白材料和聚乙烯醇(PVA)海绵分别植入大鼠背部真皮组织和股部肌肉组织,通过组织学切片详细观察其血管化过程和组织再生情况。结果表明,多孔丝素蛋白材料和PVA海绵,无论植入真皮组织还是肌肉组织,其血管化过程具有共同的步骤和特征。这个过程包括4个依次连续发生的阶段:吸附阶段,细胞外基质组装阶段,组织及血管快速增生阶段及重塑阶段。据此推测,此模式为多孔生物材料在体内血管化所共有。这为开发多孔医用生物材料以促进生物材料快速有效的血管化提供了实验基础。
通过考察多孔丝素蛋白材料和PVA海绵中的血管化和组织再生之间的关系,结果表明,植入早期细胞外基质在生物材料中的组装及细胞增殖首先为材料的血管化提供了良好的条件,材料良好的血管化又为新生组织的进一步生长提供保证。通过比较,多孔丝素蛋白材料的血管化情况优于PVA海绵,其组织再生的速度快于PVA海绵,新生组织的质量优于PVA海绵。由此可知,生物材料的化学组成对材料的血管化及其组织再生重要影响。
此外,结合体内外方法,进一步考察了体内环境对生物材料的修饰作用及其对细胞增殖和伤口愈合的影响,考察了材料性能对这种修饰作用的影响。结果表明,生物材料植入体内以后,体内环境会立即对材料进行修饰,这种修饰作用对细胞增殖活性及伤口愈合时间及愈合质量有重要影响。多孔丝素蛋白材料和PVA海绵的比较表明,材料的表面化学组成及表面形态结构不同,体内环境的修饰效果也就不同。
最后,通过定义残余材料面积百分比、降解材料面积百分比和组织再生面积百分比来半定量描述生物材料的降解程度和组织再生情况,通过生物材料降解曲线与组织再生曲线来比较材料降解速率与组织再生速率之间的协调性。从而提出了一种判定生物材料降解速率与组织再生速率之间协调程度的半定量方法。经过初步的实验验证,这种方法的评价结果与形态学观察结果一致,说明该方法简便、快速、可行。
总之,本研究通过观察多孔生物材料血管化过程,进一步分析了生物材料血管化与组织再生之间的关系。通过体内外方法对生物材料的体内修饰作用及其对细胞增殖和伤口愈合的作用进行了考察,通过比较多孔丝素蛋白材料和PVA海绵不同的修饰结果,表明生物材料的化学组成及表面形态结构对体内修饰作用的效果有重要影响。最后,本文提出了一种评价材料降解速率与组织再生速率协调性的半定量方法,考察了生物材料降解与组织再生之间的关系。期望本文研究结果能够为生物材料的血管化和组织再生提供实验依据,为血管新生、伤口愈合等基础理论研究提供参考,为进一步建立生物材料与组织再生之间的量化模型提供基础。
Within the past decades, biomaterials have been evolved from inert materials to bioactive materials and tissue inducing biomaterials which may induce tissue regeneration in situ. Because of nice biocompatibility, processible attributes and biodegradability of silk fibroin, study on the biomaterials based on silk fibroin has attracted more and more intrest.
Angiogenesis is a key step during wound healing in most tissues, so biomaterials aided wound healing requires rapid and effective neovascularization of biomaterials after implantation. Althogh lots of researches focus on the problem such as design and preparation of porous biomaterials, application of growth factors and (stem) cells and artificial blood vessels, little real success has been abtained and bad neovascularization of biomaterials has been the main obstacle in terms of clinical application.
In addition, little knowledge of detailed neovascularization of biomaterials, effects of in vivo circumstance on biomaterials implanted, and how to evaluate the degradation rate of biomaterials and tissue regeneration rate is known.
Thus, the purpose of this study is to explore and analyze the essence of neovascularization and inducibility in biomaterials through observation on the neovascularization process occurred in porous silk fibroin materials and polyvinyl alcohol (PVA) sponges.
In this study, porous silk fibroin materials and PVA sponges were implanted in muscles of hind limb and back skin in rats, and the neovascularization process in both materials has been observed in detail via tissue sections. In addition, the biocompatibility of porous silk fibroin materials and PVA sponges has been semi-quantitively compared through histological scoring method. The results showed that identical neovascularization process occurred in both materials either implanted in the muscles or the back skin. The process mainly included four sequential steps as follows: adsorptioin, organization of extracellular matrix (ECM), rapid proliferation of tissues and remodeling of the tissues and blood vessels networks. This might be the implication of general mode of neovascularization process in porous biomaterials. Furthermore, the results showed that porous silk fibroin materials have better biocompatibility than PVA sponges.
The in vivo decoration of porous silk fibroin materials and tissue regenration in porous silk fibroin materials have been further examined by combination with in vivo implantation and in vitro cell culture. The definition of in vivo decoration mainly indicates the protein adsorption and ECM organization in the materials in vivo. As for the results that porous silk fibroin materials have been more advantageous than PVA sponges to neovascularization and tissue regeneration, there might be 4 reasons as follows: (1) silk fibroin, as a natural protein, has better biocompatibility than polymers; (2) the surface texture of porous silk fibroin materials might be suitable for protein adsorption and ECM organization; (3) the porous structure of porous silk fibroin materials facilitates the ingrowth of endothelial cells and fibroblasts; (4) the biodegradation rate of porous silk fibroin materials accords with the tissue regeneration rate.
In the last chapter, the degradation of biomaterials and tissue regeneration were quantified by definition of area percentage of residual biomaterials (Rirs(%)=Sis/S1s×100%) and area percentage of newly formed tissues (Rint(%)=Sit/Sg×100%) in histological sections. Besides, biodegradation rate of biomaterials and tissue regeneration rate were compared by comparison of growth time of newly formed tissues (tr) and biodegradation time (tm) of biomaterials. In brief, a semi-quantitive method has been established in this paper to evaluate the synchronization extent between the degradation rate of biomaterials and regeneration rate of tissues.
Where Sis is the area of residual biomaterials i days after implantation, S1s is the area of biomaterials 1 day after implantation, Sit is the area of newly formed tissues i days after implantation, Sg is the area of full recovery tissues. And tr indicates the time (day) when a certain amount of tissues have been regenerated and thus they could continue growing without any stent. tm indicates the time (day) when a certain amount of biomaterials have been biodegraded and thus they lose their whole support.
Through this method, the accordance between tissue regeneration and biodegradation of biomaterials could be evaluated semi-quantitatively. Moreover, the feasibility of this method was testified through in vivo experiments, and the results suggested that this method is rapid, feasible and easy to operate. This work might provide solid basis for further quantifying attributes of biomaterials.
引文
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