【作者】 黄沙；

【导师】 金岩；

【作者基本信息】 第四军医大学 ， 口腔基础医学， 2008， 博士

【摘要】 在组织工程众多研究领域中,具有多种生物学性能的生长因子等生物活性物质是一个不可或缺的重要环节。生物活性物质是细胞间传递信息并对细胞生长具有调节功能的一些多肽类物质,它们能够在细胞增殖与分化的不同阶段促进或者抑制细胞的增殖、分化、迁移和基因的表达。近年来生长因子在组织工程技术中有着大量的应用,为引导修复重建和促进组织再生创造了有利条件。由于蛋白本身的特性,它们又极易受到外界条件的影响,例如温度、pH值及有机试剂等。在体内半衰期极短,进入体内后迅速扩散,变性和酶解,因此,如何有效地保护和释放生长因子是组织工程需要解决的问题之一。近年来迅速发展的药物控释技术正好为发挥生长因子的最佳效能提供了合适的保障。考虑到组织工程产品的特殊性,控释系统应该达到以下几条标准:生物相容性、载体本身的降解产物或其本身可被生物体吸收、易于使用、安全无毒、半透、可以抑制病理过程、有利于愈合与再生且可以作为新组织生成时的结构支撑。本研究在组织工程和控制释放技术的基础上,设计研发既可控释生长因子又符合组织工程特殊需要的可植入功能型生物载体,并对这类产品进行相关性能研究,使其具备组织工程领域的应用价值和开发前景。实验分为三个部分:1.复合生长因子的控释微球载体的研制及其相关性能研究在改良的乳化冷凝法基础上,采用三种不同固化方法(化学交联、光交联和热交联)交联制备复合成纤维细胞生长因子(bFGF)的明胶微球载体,测定微球形态、粒径、降解率、载药率和包裹率以及bFGF的体外释放效能;检测微球的生物相容性和对组织工程种子细胞的作用,以证明其作为体内植入物的安全性和对生长因子的生物活性保存能力。结论:①不同交联方法制备的复合bFGF明胶控释微球工艺简便,性能优良,各有优势;在较长时间内能持续释放活性bFGF,具有作为载体构建功能型组织工程皮肤替代物的可行性。②控释微球抗原性低、相容性好,能明显促进成纤维细胞的增殖,并呈现时间依赖性,可以作为构建功能型组织工程产品的载体作进一步研究。2.复合控释系统的双层组织工程皮肤替代物的研制及其性能研究采用天然可吸收的明胶海绵和控释微球经物理交联方法复合作为内层,进一步通过粘着聚氨酯膜为外层,模拟皮肤结构构建复合控释系统的双层结构的新型皮肤替代物。观察内部结构和控释性能;并评价其细胞和组织象容性;将复合控释系统的双层组织工程皮肤替代物移植全层皮肤缺损创面模型,检测创面愈合的效果。结论:①制备的双层组织工程皮肤替代物为双层复合结构,复合材料一侧为致密状结构的聚氨酯膜,提供强度和控制体液平衡,另一侧为疏松多孔结构的明胶海绵层,微球牢固镶嵌其中,对孔隙无明显影响;②细胞在皮肤替代物上能粘附存活,增殖良好,无明显炎性细胞,皮肤替代物能诱导血管和成纤维细胞长入;③皮肤替代物可有效促进皮肤全层缺损创面的愈合。3.功能型微粒皮肤替代物的研制及其性能研究在明胶控释微球的制备基础上,采用热交联固化方法和梯度冷冻干燥方法制备复合bFGF的大粒径型微粒载体,测定微球形态、粒径、降解率、载药率和包裹率和bFGF的体外释放效能;利用人成纤维细胞和大粒径型微粒载体(GM-bFGF)复合构建功能型微粒皮肤替代物——即同时具备细胞培养底物、生长因子控释载体和可植入性皮肤替代物的三种功能,探讨其对皮肤创面损伤的促愈合作用。结论:①所得大粒径型微粒载体依实验条件不同呈现三种不同表面性状:平滑型、脑回型和多孔型,作为载体构建功能型微粒皮肤替代物各有优势;②利用成纤维细胞和大粒径型微粒载体可以复合构建具有活性的功能型微粒皮肤替代物(SGM-bFGF、GGM-bFGF和PGM-bFGF),PGM-bFGF的综合性能最佳;③三种类型皮肤替代物均可有效促进皮肤创面损伤的愈合,是具有开发和应用前景的新型皮肤替代物。综上所述,本课题选用具有良好生物学性能的控释系统单独或者复合作为载体,研制一系列具有控释功能的组织工程皮肤替代物,控释系统可有效延长生长因子应有有效性、靶向输送并且可以作为承载细胞的支架。通过对控制释放载体构建的功能型组织工程皮肤替代物的形态学、功能特性、对组织工程种子细胞的促增殖作用和体内植入安全性以及体内疗效等相关性能研究,本课题成功构建的复合生长因子的控释微球载体、复合控释系统的双层组织工程皮肤替代物以及控释型微粒皮肤替代物为功能型组织工程产品的完善和发展探索了一条切实可行的新途径。更多还原

【Abstract】 Bioactive compounds, especially the growth factors with multiple biological efficacies, play the vital role among the numerous research fields of tissue engineering. Bioactive compounds are polypeptide materials, which take the part of both informational deliverers between cells and growth regulator on cells. They can improve or suppress the cell proliferation, differentiation, immigration and gene expression during the different stages of cell growth. The considerable application of growth factors theses days makes for renovation and regeneration of tissues. However, due to the property of being protein, growth factors are subjected to external condition (such as temperature, pH and organic reagents); and the half-life period of growth factors is so short, that they diffused, denaturized and degraded quickly. Therefore, effective conservation and delivery of growth factors is urgent needs for the development of tissue engineering. The drug delivery system (DDS), which is an advanced technology today, provides the suitable vehicle of releasing the growth factors. Considering the particularity of tissue engineering products, the DDS in this study should be accord with the standards below: biocompatibility, absorbability of vehicle or degradation product, safety and asepsis, half- penetrated, suppression of pathological period, improved tissue renovation and regeneration and being the scaffolds of renewal tissue. The objective of this study is to design and construct the functional products based on tissue engineering and controlled release technology, which play the dual role of growth factor delivery vehicle and implantable scaffold, and to survey the characteristics, thus giving an indication of possible application as a promising candidate in tissue engineering. The study was composed of threesections as follows:1. Preparation and related characteristics survey of controlled release microspheres incorporated growth factors: Three kinds of solidification (chemical crosslinking, photo-crosslinking and thermo-crosslinking) were applied with improved emulsifying condense method to prepare the controlled release microspheres incorporated basic fibroblast growth factor (bFGF), the characteristics including morphology, diameter, degradation, drug content, encapsulation and bFGF releasing efficacy were surveyed, and the biocompatibility and their effects on fibroblasts were investigated to testify the security and bioactivity of growth factors. Conclusion:①The microspheres incorporated bFGF, which were all rapid and accurate constructed by different methods, have excellent function and respective advantages, and they could control release bFGF through a long period.②The microspheres incorporated bFGF with low antigenicity and well biocompatibility can promote the proliferation of fibroblasts with time obviously. Due to the unique beneficial effects above, it could be hypothesized that the functional implantable microspheres incorporated bFGF might substantially be fundament for future study.2. Preparation and related characteristics survey of bilayer wound dressings containing bFGF- incorporated microspheres: The purpose of this study was to synthesize a novel wound dressing containing bFGF-loaded microspheres to provide an optimum healing milieu for promoting healing and tissue regeneration. For this purpose, a natural, nontoxic and biocompatible material, gelatin, was chosen as the underlying layer and the porous matrices in sponge form were prepared from gelatin by physical crosslinking technique. As the external layer, elastomeric polyurethane membranes were adhered. The composite dressings were characterized for structure, in vitro protein release and compatibility, and further tested via in vivo experiments on full-thickness skin defects created on york pigs. Conclusion:①The full structure of a bilayer wound dressing is composed of a thin polyurethane film over which the gelatin sponge containing bFGF-loaded microspheres was attached. The two layers adhered firmly to each other; the gelatin sponges show the porous and inter-connected network structures, and the microspheres did not interfere with the pore structure of the sponge.②Fibroblasts grew and proliferated well on wound dressings and there were no immune rejection in the subdermal implantation. The wound dressings could induce the infiltration of host cells and the vascularization.③The application of these novel bilayer wound dressing containing bFGF-loaded microspheres provided an optimum healing milieu for regenerating tissues in pig’s skin defect models.3. Preparation and related characteristics survey of multifunctional particle skin equivalents: The gelatin particles encapsulating bFGF based on thermo-crosslinking microspheres were prepared and modified by cryogenic freeze-drying treatment to develop the surface with three kinds of features on morphology during the post-preparation. The particles were characterized and their influence on fibroblasts has been assessed, and in vivo examinations have been made to observe guided dermal tissue regeneration after implantable particles transplantation. The multifunctional implantable particles might play a triple role as a culture substrate, protein transplantation vehicle, and biodegradable implant in this study. Conclusion:①The features on morphology were smooth, gyrus-patterned and porous according to different experiment condition, and they expressed respective advantages;②The according results we present in the text indicate that the feasibility of multifunctional implantable particles(SGM-bFGF, GGM-bFGF, PGM-bFGF),especially PGM-bFGF as an excellent skin equivalent;③They could deliver cultured cells and bioactive molecules to correct dermal defects, thus giving an indication of a promising and novel candidate of skin equivalents.In summary, we present a series of functional tissue engineering products constructed with unique controlled release vehicle. As a beneficial drug delivery system, growth factors can be introduced to be responsible for cell growth onto polymer surfaces in cell culture and induce cell ingrowth and vascularization at the initial stages of wound healing. Specifically, basic fibroblast growth factor (bFGF) is a multifunctional protein that promotes angiogenesis and regulates many aspects of cellular activity, including cell proliferation, migration, and metabolism in a concentration-dependent manner, and the best way to retain the biological activity and enhance the efficacy of bFGF is to achieve sustained release and to maintain suitable concentration over an extended time period by controlled release vehicle. In the present study, we performed a overall evaluation of controlled release microspheres incorporated growth factors, bilayer wound dressings containing bFGF- incorporated microspheres, multifunctional particle skin equivalents. The according results we present in the text indicate that the feasibility of these products for skin regeneration, thus giving an indication of possible application as a promising candidate in tissue engineering.更多还原