控制释放载体的研制及其构建功能型组织工程皮肤的实验研究
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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。
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 three
sections 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.
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。
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 three
sections 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.
引文
1. 付小兵,王德文主编.现代创伤修复学.北京:人民军医出版社,1999.2-3.
2 周廷冲主编.多肽生长因子基础与临床.北京:中国科学技术出版社,1992.42-46.
3 Pierec GF,Mustor TA. Pharmacologic enhancement of wound healing.Annu Rev Med,1995,46:467-469.
4 Lieberman JR,Le LQ,Wu L.Regional geng therapy with a BMP-2-producing murine stromal cell line induces heterotopic and orthotopic bone formation in rodents.J Orthop Res,1998,16(3):330.
5 Frenkel S,Cesare P.Degradation and repair of articular cartilage.Front Biosci,1999,4:671-685.
6 Ripamonti U,Reddi AH.Tissue engineering,morphogenesis,and regeneration of the periodontal tissues by bone morphogenetic proteins.Crit Rev Oral Biol Med,1997,8(2):154-163.
7 Lu L,Yaszemski MJ,Mikos AG.TGF-betal release from biodegradable polymer microparticles:its effects on marrow stromal osteoblast function.J Bone Joint Surg Am,2001,83(Pt2):S82-91.
8 Mann BK , Schmedlen RH , west JL . Tethered—TGF-beta increases extracellular matrix production of vascular smooth muscle cells.Biomaterials,2001,22(5):439-444.
9 Lockin RM,Williamson MC,Beresford JN,et a1.In vitro effects of growth factors and dexamethasone on rat marrow stromal cells.Clin Orthop,1995,313(1):27-35.
10 Lu W W, Ip W W. Biomechanical properties of thin skin flap after basicfibroblast growth factor (bFGF) administration. British Journal of Plastic Surgery ,2000, 53:225.
11 Bennett NT.Schultz GS.Growth factors and wound healing(partⅡ):role in normal and chronic wound healing.Ann J Surg,1993,166:74-78.
12 A ntoniades HN ,Theofanis NG. Expression of growth factor and receptor mRNAs in skin epithelial cells following acute cutaneous injury.Am J Pathol,1993,142:1101-1107.
13 姚敏,许伟石,史济湘.大鼠浅度烫伤创面愈合中 PDGF 及其受体基因的表达.中华创伤杂志,1999,15:55-56.
14 韦多,葛绳德.小面积Ⅱ度烫伤创面愈合过程中内源性 TGF-α 的动态表达.中华整形烧伤外科杂志,1997,5:323-325.
15 方培耀,程枫.不同深度的Ⅱ度烧伤创面中单核巨噬细胞生长因子的 mRNA表达.中华创伤杂志,1998,6:353-355.
16 Armelin.HA.Pituitary extracts and steroid hormobnes in the control of 3t3 cell growth. Proc Natl Acad Sci U SA,1973,70:2702-2706.
17 冯江,杜文华,王劲.多种生长因子促糖尿病患者难愈合性创面的临床研究.中国修复与重建外科杂志,l999,l3:278-279.
18 Willinger EB. The role of growth factors in wound healing. Skin Pharmacol, 1991, 4:175. L9 付小兵,王亚平.碱性成纤维细胞生长因子加速猪背部创伤修复的实验研究.中华创伤杂志,1995,3:131-133.
20 付小兵.生长因子促(抑)创伤修复的临床应用与展望.中华创伤杂志,1998,6:365-366.
21 RalphN,Martins J,Chleboun0,eta1.The role of PDGF-BB on the development of the collateral circulation after acute arterial occlusion.Growth factors,1994,10:299-306.
22 合廷敏,牛星筹.创面愈合过程中内源性 EGF 变化的实验研究.中华整形烧伤外科杂志,1997,2:153-152.
23 Broadley KN ,Aquino AM ,W oodward SC,et a1. M onospecific anti—bodies im plicate bFGF in norm al wound repair.Lab Invest,l989,6l:57l-575.
24 Walgenbach KJ,Bruenaged G, Lovett JE,et a1. Therapeutic angiogenesis in wounds:the influence of growth factors at a muscle flap—ischemic tissue interface.Biol Matrices Tissue Reconstr,l998,57:279-282.
25 曹卫红,柴家科,杨志祥,等.大鼠急性放射性皮肤溃疡愈合过程中PDGF-β 及其受体的表达.感染、炎症、修复,2005,6(4):210-2l3.
26 卞徽宁,陈华德,郑少逸,等.外源性 bFGF 对创面愈合病理变化的影响.感染、炎症、修复,2006,7(4):206-209.
27 熊美华,程飚,刘宏伟,等.rhPDGF 对糖尿病全层皮肤缺损创面微血管形成的影响和意义.感染、炎症、修复,2006,7(4):210-212.
28 Chapekar MS. Tissue engineering: challenges and opportunities.J Biomech Mater Res (Appl Biomater),2000,53:617–620.
29 Hacker MC, Mikos AG. Trends in tissue engineering research. Tissue Eng,2006,12(8):2049–2057.
30 Chen VJ, Ma PX. Nano-fibrous poly(L-lactic acid) scaffolds with interconnected spherical macro-pores. Biomaterials,1996,25:2065–2073.
31 Nair LS, Laurencin CT. Polymers as biomaterials for tissue engineering and controlled drug delivery. Adv Biochem Eng Biotechnol,2006,102:47–90.
32 Kleinman HK, Philip D, Hoffman MP. The role of the extracellular matrix in morphogenesis. Curr Opin Biotechnol,2003,14:526–532.
33 Hosokawa R,Kikuzaki K,Kimoto T,et a1.Controlled local application of basic fibroblast growth factor(bFGF)accelerates the healing of GBR : Anexperimental study in beagle dogs.J Clin Oral Implans Res,2000,l1(4):345-357.
34 Maeda M,Kadota K,Kajihara M,et a1.Sustained release of human growth hormone (hGH) from collagen film and evaluation of effect on wound healing in db/db mice.J Control Release,2001,77(3):261-272.
35 Elissef J,McIntosh W .Fu K,et at.Controlled-release of IGF-I and TGF-beta l in a photo polymerizing hydrogel for cartilage tissue engineering.J Orthop Res,2001,19:1098-1104.
36 Rosner BI,Siegel RA,Grosberg A,et a1.Rational design of contact guiding,neurotrophic matrices for peripheral nerve regeneration.Ann Biomed Eng,2003,31:1383-1401.
37 Wang S,Wan ACA.Xu X,et a1.A new nerve guide conduit material composed of a biodegradable poly(phosphoester).Biomaterials,2001.22:1157-1169.
38 Saito N, Takaoka K. New synthetic biodegradable polymers as BMP carriers for bone tissue engineering.Biomaterials,2003,24:2287-2293.
39 M. Yamamoto, Y. Tabata, L. Hong, S. Miyamoto, N. Hashimoto and Y. Ikada, Bone regeneration by transforming growth factor beta1 released from a biodegradable hydrogel, J. Controlled Release,2000,64:133–142.
40 K.Y. Lee, M.C. Peters and D.J. Mooney, Comparison of vascular endothelial growth factor and basic fibroblast growth factor on angiogenesis in SCID mice, J. Controlled Release,2003,87:49–56.
41 Holland TA, Mikos AG. Biodegradable polymeric scaffolds: improvements in bone tissue engineering through controlled drug delivery. Adv Biochem Eng Biotechnol,2006(102):161–85.
42 颜文龙,孙恩杰,郭海英,等.组织工程支架材料.上海生物医学工程,2004,25(1):51-54.
43 Lee M, Chen TT, Iruela-Arispe ML, Wu BM, Dunn JC. Modulation of protein delivery from modular polymer scaffolds. Biomaterials , 2007 , 28(10) :1862–1870.
44 Nillesen ST, Geutjes PJ, Wismans R, Schalkwijk J, Daamen WF, van Kuppevelt TH. Increased angiogenesis and blood vessel maturation in acellular collagen-heparin scaffolds containing both FGF2 and VEGF. Biomaterials,2007,28(6):1123–1131.
45 Ozeki M, Ishii T, Hirano Y, Tabata Y. Controlled release of hepatocyte growth factor from gelatin hydrogels based on hydrogel degradation. J Drug Target,2001,9(6):461–471.
46 周文常,但卫华,廖隆理,等.胶原蛋白及其与高聚物形成的复合材料在医学中的应用.皮革科学与工程,2004,14(2):30-33.
47 刘刚,胡蕴玉,颜永年,等.I 型胶原修饰的多孔材料聚乙醇酸-乳酸共聚物对兔骨髓间充质干细胞粘附和增殖及成骨细胞基因表达的影响.中华医学杂志.甲,2003,83(7):580-583.
48 尹玉姬,姚康德。刘义广,等. 组织工程相关壳聚糖-明胶基生物材料.应用化学,2004,21(3):217-222.
49 Park YJ, Lee YM, Park SN, Sheen SY, Chung CP, Lee SJ. Platelet derived growth factor releasing chitosan sponge for periodontal bone regeneration. Biomaterials,2000,21(2):153–159.
50 Lu Y, Chen SC. Micro and nano-fabrication of biodegradable polymers for drug delivery. Adv Drug Deliv Rev,2004,56:1621–1633.
51 N Adhirajan , N Shanmugasundaram , Mary Babu. Gelatin microspheres cross-linked with EDC as a drug delivery system for doxycyline: Development and characterization. J Microencapsul,2007,24 (7):659-671.
53 Lee KY, Peters MC, Anderson KW, Mooney DJ. Controlled growth factor release from synthetic extracellular matrices. Nature,2000,408(6815):998–1000.
54 A.C. Rapraeger, et al., Regulation by heparan sulfate in fibroblast growth factor signaling, Methods Enzymol. 1994,245:219–240.
55 Heller J, Barr J, Ng SY, Abdellauoi KS, Gurny R.Poly(ortho esters): synthesis, characterization, properties and uses. Adv Drug Deliv Rev,2002,54:1015–1039.
56 Heller J, Barr J. Poly(ortho esters)——from concept to reality. Biomacromolecules,2004,5:1625–1632.
57 Heller J. Ocular delivery using poly(ortho esters). Adv Drug Deliv Rev,2005,57:2053–2062.
58 Chen RR, Silva EA, Yuen WW,Mooney DJ. Spatio-temporal VEGF and PDGF delivery patterns blood vessel formation and maturation.Pharmaceut Res, 2007,24(2):258–264.
59 Wang YU. Computer modeling and simulation of solid-state sintering: a phase field approach. Acta Mater,2006,54:953–961.
60 Richardson TP, Peters MC, Ennett AB, Mooney DJ. Polymeric system for dual growth factor delivery. Nat Biotechnol,2001,19(11):1029–1034.
61 Fu K, Harrell R, Zinski K, Um C, Jaklenec A, Frazier J, et al. A potential approach for decreasing the burst effect of protein from PLGA microspheres. J Pharm Sci 2003,92(8):1582–1591.
62 Ageorges C, Ye L, Hou M. Advances in fusion bonding techniques for joining thermoplastic matrix composites: a review. Composites: Part A: Appl Sci Manuf 2001,32:839–857.
63 Levenberg S, Huang NF, Lavik E, Rogers AB, Itskovitz-Eldor J, Langer R. Differentiation of human embryonic stem cells on threedimensional polymerscaffolds. Proc Natl Acad Sci USA,2003,100(22):12741–12746.
64 Langer R, Tirrell DA. Designing materials for biology and medicine. Nature,2004,428(6982):487–492.
65 Weigel T, Schinkel G, Lendlein A. Design and preparation of polymeric scaffolds for tissue engineering. Expert Rev Med Devices,2006,3(6):835–851.
66 Tsang VL, Bhatia SN. Fabrication of three-dimensional tissues. Adv Biochem Eng Biotechnol,2007,103:189–205.
67 Boccaccini AR, Blaker JJ. Bioactive composite materials for tissue engineering scaffolds. Expert Rev Med Devices,2005,2(3):303–317.
68 Rosso F, Marino G, Giordano A, Barbaris M, Parmeggiani D,Barbarisi A. Smart materials as scaffolds for tissue engineering. J Cell Physiol,2005,203(3):465–470.
69 Lockin RM,Williamson MC,Beresford JN,et a1.In vitro effects of growth factors and dexamethasone on rat marrow stromal cells.Clin Orthop,1995,313(1):27-35.
70 Arevalo-Silva CA,Cao Y,Weng Y,et a1.The effect of fibroblast growth factor and transforming growth factor-beta on porcine chondrocytes and tissue-engineered autologous elastic cartilage.Tissue Eng,2001,7(1):81-88.
71 Tabata Y,Miyao M,Inamoto T,et a1.De novo formation of adipose tissue by controlled release of basic fibroblast growth factor.Tissue Eng,2000,6(3):279-289.
72 Soker S,Machado M,Atala A.Systems for therapeutic angiogenesis in tissue engineering.World J Urol,2000,18(1):10-18.
73 Tanaka H,Quarto R,Williams S,et a1.In vivo and in vitro effects of insulin-like growth factor-1(IGF-1)on femoral mRNA expression in old rats.Bone,1994,15(6):647-653.
74 Tabata Y,Morimoto K,Katsumata H,et a1.Surfactant-free preparation of biodegradable hydrogel microspheres for protein release.J Bioact Compat Polym,1999,14:371-373.
75 Yu K,Makoto 0,Takashi I,et a1.Time course of de novo adiposeness in Matrigel by gelatin microspheres incorporating basic fibroblast growth factor.Tissue Eng,2002,4:609-613.
76 Orban JM,Marra KG,HollingerJO.Composition options for tissue en gineered bone.Tissue Eng,2002,8:529-539.
77 T.P. Richardson, M.C. Peters, A.B. Ennett, D.J. Mooney, Polymeric system for dual growth factor delivery, Nat. Biotechnol,2001,19:1029–1034.
78 S. Young, M. Wong, Y. Tabata, A.G. Mikos, Gelatin as a delivery vehicle for the controlled release of bioactive molecules, J Control Release,2005,109:256–274.
79 C. Yan, X. Li, X. Chen, D. Wang, D. Zhong, T. Tan, H. Kitano, Anticancer gelatin microspheres with multiple function, Biomaterials,1991,12:640–644.
80 P.T. Prisell, O. Camber, J. Hiselius, G. Norstedt, Evaluation of hyaluronan as a vehicle for peptide growth factors, Int. J. Pharm,1992,85:51–56.
81 CaoT,Ho KH,Teoh SW.Scafold design and in vitro study ofoseochondral coculture in a three—dimensional porous polycaprolactone scaffold fabricated by fused deposition modeling.Tissue Eng,2003,9:103-112.
82 Y. Tabata, Tissue regeneration based on growth factor release, Tissue Eng,2003,9:S5–S15.
83 Yamamoto M,Ikada Y,Tabata Y.Controlled release of growth factors based on biodegradation of gelatin hydrogel:J .J Biomer Sci Polym Ed,2001,12:77-88
84 Tabata Y,Ikada Y. Vascularization effect of basic fibroblast growth factorreleased from gelatin hydrogels with different biodegradabilities. Biomaterials,1999,20:2169-2175.
85 Tabata Y,Yamada K,Miyamot S.et a1.Bone regeneration by basic fibroblast growth factor complexed with biodegradable hydrogels Biomaterials,1998,19:807-815.
86 Kawai K,Suzuki S,Tab ata Y,et a1.Accelerated tissue regeneration through incorporation of basic fibroblast growth factor—impregnated gelatin microspheres into artificial dermis,Biomaterials,2000,21:489-499.
87 Yang CF , Yasukawa T . Kimura H , et al Experimental corueal neovaseularization by basic fibroblast growt h factor incorporated into gelatin bydrogel,Ophthalmic Res,2000,32:19-24.
88 Han g L,Mivamoto S,Yamada K,et a1.Enhanced formation of fibrosis in a rabbit aneurysm by gelatin hydrogel incorporating basic fibroblast growth faetor,Neurosurgery,2001,49:954-960.
89 Masanori F,Masayuki I,Masafumi S,et a1.Vascularization in vivo caused by the controlled release of fibroblast growth factor-2 from an injectable chitosan/non-anticoagulant heparin hydrogel.Biomaterials,2004.25:699-706.
90 J.H. Distler, A. Hirth, M. Kurowska-Stolarska, R.E. Gay, S. Gay, O. Distler, Angiogenic and angiostatic factors in the molecular control of angiogenesis, Q. J. Nucl. Med,2003,47:149-161.
91 D.W. Losordo, S. Dimmeler, Therapeutic angiogenesis and vasculogenesis for ischemic disease. Part I: angiogenic cytokines, Circulation,2004,109:2487–2491.
92 唐群委,林建明,吴季怀,等.功能材料, 2006,9(37): 1510-1513.
93 Sung HW,Chang Y,Chin CT,et a1.Crosslinking characteristics and mechanical properties od a bovine pericardium fixed with a naturally occurringcrosslinking agent.J Biomed Mater Res,1999,47(2): l16-126.
94 韩津生.戊二醛检测研究进展.环境与健康杂志,2005,22(4):319-320.
95 崔玉明,胡蕴玉,吕昌伟,等.骨髓基质细胞与两种不同载体材料生物相容性的实验研究.中国矫形外科杂志,2003,11(16):1113-1116.
96 Oreffo RO,Driessens FC,P1anell JA,et a1.Growth and diferentiation of human bone marrow osteoprogenitors on novel calcium phosphate cements.Biomaterials,1998,19(20):1845-1854.
97 Hansbrough JF,Cooper ML,Cohen R,et al. Evaluation of a biodegradable matrix containing cultured human fibroblasts as a dermal replacement beneath meshed skin grafts on athymic mice. Surgery,1992,111(4):438-446.
98 中华人民共和国国家标准GB/T 16886.12 1996,医疗器械生物学评价第12部分:样品制备与参照样品.
99 Nimni ME. Polypeptide growth factors:targeted delivery systems. Biomaterials ,1997,18:1201-25
100 Coulomb B,Friteau L,Barueh J,eta1.Advantage of the presence of living derm al fibroblasts within in vitro reconstructed skin for grafting in humans.Hast Reconstr Surg,1998,101(7):1891-1903.
101 杨志明.组织工程基础与临床.成都:四川科学技术出版社,2000.233-237.
102 肖仕初,夏照帆.真皮替代物.国外医学,生物医学工程分册,2001,24(1):19.
103 徐成晦.真空冷冻干燥技术.真空与低温,1994,13 (2):95-99.
104 华泽钊,任禾盛著. 低温生物医学技术,北京:科学出版社.1994
105 张洁,华泽钊. 模拟生物组织冻结过程. 实验与分析工程热物理学报.2000,3:350-353.
106 Steven T Boyce.Fabrication,quality assurance,and assessment of culturedskin substitutes for treatment of skin wounds.Biochemical Engineering Journal,2004,20:107-l12.
107 Mi Fwu—Long,Wu Yu-Bey,Shyu Shin-Shing,et a1.Asymmetric chitosan membranes prepared by dry/wet phase separation;a new type of wound dressing for controlled antibacterial release.Journal of Membrane Science,2003,212:237-254.
108 Masashi Nomi, Anthony Atala, Paolo De Coppi, Shay Soker. Principals of neovascularization for tissue engineering. Molecular Aspects of Medicine,2002,23:463–483.
109 Ulubayram K,Cakar AN,Korkusuz P,et a1.EGF contmning gelatin based wound dressings. Biomaterials,2001,22:1345-1356.
110 Hang L,Mivamoto S,Yamada K,et a1.Enhanced formation of fibrosis in a rabbit aneurysm by gelatin hydrogel incorporating basic fibroblast growth faetor. Neurosurgery,2001,49:954-960.
111 Paul. Willi,Chandra P. Sharma. Chitosan and Alginate Wound Dressings: A Short Review. Trends Biomater. Artif.Organs,2004,18(1):18-23.
112 Lin FH,Chen TM,Chen KS,et a1.An animal study of a novel trilayer wound dressing materials non·-woven fabric grafted with N-isopropyl acrylamide and gelatin,et al. Mater Chem Phys,2000,64:189-195.
113 Lee CH,Singla A,Lee Y. Biomedical applications of collagen.Int J Pharm,2001,221(1-2):1-22
114 Baker PG, Haig G. Metronidazole in the treatment of chronic pressure sores and ulcers: a comparison with standart treatments in general practice. Practitioner 1981;225:569-73.
115 Kaya AZ, Turani N, Akyuz M. The effectiveness of a hydrogel dressing compared with standard management of pressure ulcers. J Wound Care. 2005,14(1):42-4.
116 Bruin P, Jonkman MF, Meijer HJ, Pennings AJ. A new porous polyetherurethane wound covering. J Biomed Mater Res 1990;24:217-26.
117 Lynch SE, Colvin RB, Antoniades HN. Growth factors in wound healing- single and synergistic effects on partial thickness porcine skin wounds. J Clin Invest 1989;84:640-6.
118 Ko IK, Iwata H. An approach to constructing three-dimensional tissue. Ann N Y Acad Sci. 2001;944:443–55.
119 T. Kushibiki, H. Matsuoka and Y. Tabata, Synthesis and physical characterization of poly(ethylene glycol)-gelatin conjugates, Biomacromolecules 2004 (5):202–208.
120 A. Muvaffak, I. Gurhan and N. Hasirci, Prolonged cytotoxic effect of colchicine released from biodegradable microspheres, J. Biomed. Mater. Res. 2004 (71B):295–304.
121 A.J. Kuijpers, G.H. Engbers, P.B. van Wachem, J. Krijgsveld, S.A. Zaat, J. Dankert and J. Feijen, Controlled delivery of antibacterial proteins from biodegradable matrices, J. Controlled Release 1998 (53):235–247.
122 L.D.Silvio and W. Bonfield, Biodegradable drug delivery system for the treatment of bone infection and repair, J. Mater. Sci., Mater. Med. 1999 (10):653–658.
123 Malda J, Kreijveld E, Temenoff JS, et al. Expansion of human nasal chondrocytes on macroporous microcarriers enhances redifferentiation. Biomaterials 2003; 24:5153-61.
124 詹国平,韩彦红,谢恩伟.阿霉素明胶微球的制备及体外释药特性.中国医学工程 [J], 2005,13(5): 483-488
125 Liu JY, Hafner J, Dragieva G, et al. Autologous cultured keratinocytes onporcine gelatine microbeads effectively heal chronic venous leg ulcers. Wound Repair Regen 2004;12:148-56.
126 Baker, T.L., and Goodwin, T.J. Three-dimensional culture of bovine chondrocytes in rotating-wall vessels. In Vitro Cell. Dev. Biol Anim. 1997, 33, 358.
127 Himes VB, Hu WS.Attachment and growth of mammalian cells on microcarriers with different ion exchange capacities. Biotechnol Bioeng , 1987,24:1155–63.
128 Reuveny S, Mizrahi A, Kotler M, Freeman A. Factors effecting cell attachment, spreading, and growth on derivatized microcarriers: II introduction of hydrophobicelements. Biotechnol Bioeng . 1983,5:2969–80.
129 Kiremitci M, Piskin E. Cell adhesion to the surfaces of polymeric beads. Biomaterials: 1990, Art Cells Art Org; 18:599–603.
130 Chun, K.W. Biodegradable PLGA microcarriers for injectable delivery of chondrocytes: effect of surface modification on cell attachment and function. Biotechnol. Prog. 2004,20, 1797–1801
131 Brittberg M, Lindahl A, Nilsson A, et al. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 1994,331:889–95.
132 Drury JL, Mooney DJ. Hydrogels for tissue engineering: Scaffold design variables and applications. Biomaterials 2003,24:4337–51.
133 Christiansen, J., Ek, L., and Tegner, E. Pinch grafting of leg ulcers. A retrospective study of 412 treated ulcers in 146 patients. Acta Derm. Venereol. 1997,77: 471.
134 Lin, T.-W., and Horng, S.-Y. A new method of microskin mincing. Burns1994,20: 526.
135 Dai NT, Williamson MR, Khammo N, Adams EF, Coombes AG. Composite cell support membranes based on collagen and polycaprolactone for tissue engineering of skin. Biomaterials. 2004,25:4263–71.
136 Griffith LG,Naughton G. Tissue engineering——current challenges and expanding opportunities. Science,2002,295:1009-1012.
2 周廷冲主编.多肽生长因子基础与临床.北京:中国科学技术出版社,1992.42-46.
3 Pierec GF,Mustor TA. Pharmacologic enhancement of wound healing.Annu Rev Med,1995,46:467-469.
4 Lieberman JR,Le LQ,Wu L.Regional geng therapy with a BMP-2-producing murine stromal cell line induces heterotopic and orthotopic bone formation in rodents.J Orthop Res,1998,16(3):330.
5 Frenkel S,Cesare P.Degradation and repair of articular cartilage.Front Biosci,1999,4:671-685.
6 Ripamonti U,Reddi AH.Tissue engineering,morphogenesis,and regeneration of the periodontal tissues by bone morphogenetic proteins.Crit Rev Oral Biol Med,1997,8(2):154-163.
7 Lu L,Yaszemski MJ,Mikos AG.TGF-betal release from biodegradable polymer microparticles:its effects on marrow stromal osteoblast function.J Bone Joint Surg Am,2001,83(Pt2):S82-91.
8 Mann BK , Schmedlen RH , west JL . Tethered—TGF-beta increases extracellular matrix production of vascular smooth muscle cells.Biomaterials,2001,22(5):439-444.
9 Lockin RM,Williamson MC,Beresford JN,et a1.In vitro effects of growth factors and dexamethasone on rat marrow stromal cells.Clin Orthop,1995,313(1):27-35.
10 Lu W W, Ip W W. Biomechanical properties of thin skin flap after basicfibroblast growth factor (bFGF) administration. British Journal of Plastic Surgery ,2000, 53:225.
11 Bennett NT.Schultz GS.Growth factors and wound healing(partⅡ):role in normal and chronic wound healing.Ann J Surg,1993,166:74-78.
12 A ntoniades HN ,Theofanis NG. Expression of growth factor and receptor mRNAs in skin epithelial cells following acute cutaneous injury.Am J Pathol,1993,142:1101-1107.
13 姚敏,许伟石,史济湘.大鼠浅度烫伤创面愈合中 PDGF 及其受体基因的表达.中华创伤杂志,1999,15:55-56.
14 韦多,葛绳德.小面积Ⅱ度烫伤创面愈合过程中内源性 TGF-α 的动态表达.中华整形烧伤外科杂志,1997,5:323-325.
15 方培耀,程枫.不同深度的Ⅱ度烧伤创面中单核巨噬细胞生长因子的 mRNA表达.中华创伤杂志,1998,6:353-355.
16 Armelin.HA.Pituitary extracts and steroid hormobnes in the control of 3t3 cell growth. Proc Natl Acad Sci U SA,1973,70:2702-2706.
17 冯江,杜文华,王劲.多种生长因子促糖尿病患者难愈合性创面的临床研究.中国修复与重建外科杂志,l999,l3:278-279.
18 Willinger EB. The role of growth factors in wound healing. Skin Pharmacol, 1991, 4:175. L9 付小兵,王亚平.碱性成纤维细胞生长因子加速猪背部创伤修复的实验研究.中华创伤杂志,1995,3:131-133.
20 付小兵.生长因子促(抑)创伤修复的临床应用与展望.中华创伤杂志,1998,6:365-366.
21 RalphN,Martins J,Chleboun0,eta1.The role of PDGF-BB on the development of the collateral circulation after acute arterial occlusion.Growth factors,1994,10:299-306.
22 合廷敏,牛星筹.创面愈合过程中内源性 EGF 变化的实验研究.中华整形烧伤外科杂志,1997,2:153-152.
23 Broadley KN ,Aquino AM ,W oodward SC,et a1. M onospecific anti—bodies im plicate bFGF in norm al wound repair.Lab Invest,l989,6l:57l-575.
24 Walgenbach KJ,Bruenaged G, Lovett JE,et a1. Therapeutic angiogenesis in wounds:the influence of growth factors at a muscle flap—ischemic tissue interface.Biol Matrices Tissue Reconstr,l998,57:279-282.
25 曹卫红,柴家科,杨志祥,等.大鼠急性放射性皮肤溃疡愈合过程中PDGF-β 及其受体的表达.感染、炎症、修复,2005,6(4):210-2l3.
26 卞徽宁,陈华德,郑少逸,等.外源性 bFGF 对创面愈合病理变化的影响.感染、炎症、修复,2006,7(4):206-209.
27 熊美华,程飚,刘宏伟,等.rhPDGF 对糖尿病全层皮肤缺损创面微血管形成的影响和意义.感染、炎症、修复,2006,7(4):210-212.
28 Chapekar MS. Tissue engineering: challenges and opportunities.J Biomech Mater Res (Appl Biomater),2000,53:617–620.
29 Hacker MC, Mikos AG. Trends in tissue engineering research. Tissue Eng,2006,12(8):2049–2057.
30 Chen VJ, Ma PX. Nano-fibrous poly(L-lactic acid) scaffolds with interconnected spherical macro-pores. Biomaterials,1996,25:2065–2073.
31 Nair LS, Laurencin CT. Polymers as biomaterials for tissue engineering and controlled drug delivery. Adv Biochem Eng Biotechnol,2006,102:47–90.
32 Kleinman HK, Philip D, Hoffman MP. The role of the extracellular matrix in morphogenesis. Curr Opin Biotechnol,2003,14:526–532.
33 Hosokawa R,Kikuzaki K,Kimoto T,et a1.Controlled local application of basic fibroblast growth factor(bFGF)accelerates the healing of GBR : Anexperimental study in beagle dogs.J Clin Oral Implans Res,2000,l1(4):345-357.
34 Maeda M,Kadota K,Kajihara M,et a1.Sustained release of human growth hormone (hGH) from collagen film and evaluation of effect on wound healing in db/db mice.J Control Release,2001,77(3):261-272.
35 Elissef J,McIntosh W .Fu K,et at.Controlled-release of IGF-I and TGF-beta l in a photo polymerizing hydrogel for cartilage tissue engineering.J Orthop Res,2001,19:1098-1104.
36 Rosner BI,Siegel RA,Grosberg A,et a1.Rational design of contact guiding,neurotrophic matrices for peripheral nerve regeneration.Ann Biomed Eng,2003,31:1383-1401.
37 Wang S,Wan ACA.Xu X,et a1.A new nerve guide conduit material composed of a biodegradable poly(phosphoester).Biomaterials,2001.22:1157-1169.
38 Saito N, Takaoka K. New synthetic biodegradable polymers as BMP carriers for bone tissue engineering.Biomaterials,2003,24:2287-2293.
39 M. Yamamoto, Y. Tabata, L. Hong, S. Miyamoto, N. Hashimoto and Y. Ikada, Bone regeneration by transforming growth factor beta1 released from a biodegradable hydrogel, J. Controlled Release,2000,64:133–142.
40 K.Y. Lee, M.C. Peters and D.J. Mooney, Comparison of vascular endothelial growth factor and basic fibroblast growth factor on angiogenesis in SCID mice, J. Controlled Release,2003,87:49–56.
41 Holland TA, Mikos AG. Biodegradable polymeric scaffolds: improvements in bone tissue engineering through controlled drug delivery. Adv Biochem Eng Biotechnol,2006(102):161–85.
42 颜文龙,孙恩杰,郭海英,等.组织工程支架材料.上海生物医学工程,2004,25(1):51-54.
43 Lee M, Chen TT, Iruela-Arispe ML, Wu BM, Dunn JC. Modulation of protein delivery from modular polymer scaffolds. Biomaterials , 2007 , 28(10) :1862–1870.
44 Nillesen ST, Geutjes PJ, Wismans R, Schalkwijk J, Daamen WF, van Kuppevelt TH. Increased angiogenesis and blood vessel maturation in acellular collagen-heparin scaffolds containing both FGF2 and VEGF. Biomaterials,2007,28(6):1123–1131.
45 Ozeki M, Ishii T, Hirano Y, Tabata Y. Controlled release of hepatocyte growth factor from gelatin hydrogels based on hydrogel degradation. J Drug Target,2001,9(6):461–471.
46 周文常,但卫华,廖隆理,等.胶原蛋白及其与高聚物形成的复合材料在医学中的应用.皮革科学与工程,2004,14(2):30-33.
47 刘刚,胡蕴玉,颜永年,等.I 型胶原修饰的多孔材料聚乙醇酸-乳酸共聚物对兔骨髓间充质干细胞粘附和增殖及成骨细胞基因表达的影响.中华医学杂志.甲,2003,83(7):580-583.
48 尹玉姬,姚康德。刘义广,等. 组织工程相关壳聚糖-明胶基生物材料.应用化学,2004,21(3):217-222.
49 Park YJ, Lee YM, Park SN, Sheen SY, Chung CP, Lee SJ. Platelet derived growth factor releasing chitosan sponge for periodontal bone regeneration. Biomaterials,2000,21(2):153–159.
50 Lu Y, Chen SC. Micro and nano-fabrication of biodegradable polymers for drug delivery. Adv Drug Deliv Rev,2004,56:1621–1633.
51 N Adhirajan , N Shanmugasundaram , Mary Babu. Gelatin microspheres cross-linked with EDC as a drug delivery system for doxycyline: Development and characterization. J Microencapsul,2007,24 (7):659-671.
53 Lee KY, Peters MC, Anderson KW, Mooney DJ. Controlled growth factor release from synthetic extracellular matrices. Nature,2000,408(6815):998–1000.
54 A.C. Rapraeger, et al., Regulation by heparan sulfate in fibroblast growth factor signaling, Methods Enzymol. 1994,245:219–240.
55 Heller J, Barr J, Ng SY, Abdellauoi KS, Gurny R.Poly(ortho esters): synthesis, characterization, properties and uses. Adv Drug Deliv Rev,2002,54:1015–1039.
56 Heller J, Barr J. Poly(ortho esters)——from concept to reality. Biomacromolecules,2004,5:1625–1632.
57 Heller J. Ocular delivery using poly(ortho esters). Adv Drug Deliv Rev,2005,57:2053–2062.
58 Chen RR, Silva EA, Yuen WW,Mooney DJ. Spatio-temporal VEGF and PDGF delivery patterns blood vessel formation and maturation.Pharmaceut Res, 2007,24(2):258–264.
59 Wang YU. Computer modeling and simulation of solid-state sintering: a phase field approach. Acta Mater,2006,54:953–961.
60 Richardson TP, Peters MC, Ennett AB, Mooney DJ. Polymeric system for dual growth factor delivery. Nat Biotechnol,2001,19(11):1029–1034.
61 Fu K, Harrell R, Zinski K, Um C, Jaklenec A, Frazier J, et al. A potential approach for decreasing the burst effect of protein from PLGA microspheres. J Pharm Sci 2003,92(8):1582–1591.
62 Ageorges C, Ye L, Hou M. Advances in fusion bonding techniques for joining thermoplastic matrix composites: a review. Composites: Part A: Appl Sci Manuf 2001,32:839–857.
63 Levenberg S, Huang NF, Lavik E, Rogers AB, Itskovitz-Eldor J, Langer R. Differentiation of human embryonic stem cells on threedimensional polymerscaffolds. Proc Natl Acad Sci USA,2003,100(22):12741–12746.
64 Langer R, Tirrell DA. Designing materials for biology and medicine. Nature,2004,428(6982):487–492.
65 Weigel T, Schinkel G, Lendlein A. Design and preparation of polymeric scaffolds for tissue engineering. Expert Rev Med Devices,2006,3(6):835–851.
66 Tsang VL, Bhatia SN. Fabrication of three-dimensional tissues. Adv Biochem Eng Biotechnol,2007,103:189–205.
67 Boccaccini AR, Blaker JJ. Bioactive composite materials for tissue engineering scaffolds. Expert Rev Med Devices,2005,2(3):303–317.
68 Rosso F, Marino G, Giordano A, Barbaris M, Parmeggiani D,Barbarisi A. Smart materials as scaffolds for tissue engineering. J Cell Physiol,2005,203(3):465–470.
69 Lockin RM,Williamson MC,Beresford JN,et a1.In vitro effects of growth factors and dexamethasone on rat marrow stromal cells.Clin Orthop,1995,313(1):27-35.
70 Arevalo-Silva CA,Cao Y,Weng Y,et a1.The effect of fibroblast growth factor and transforming growth factor-beta on porcine chondrocytes and tissue-engineered autologous elastic cartilage.Tissue Eng,2001,7(1):81-88.
71 Tabata Y,Miyao M,Inamoto T,et a1.De novo formation of adipose tissue by controlled release of basic fibroblast growth factor.Tissue Eng,2000,6(3):279-289.
72 Soker S,Machado M,Atala A.Systems for therapeutic angiogenesis in tissue engineering.World J Urol,2000,18(1):10-18.
73 Tanaka H,Quarto R,Williams S,et a1.In vivo and in vitro effects of insulin-like growth factor-1(IGF-1)on femoral mRNA expression in old rats.Bone,1994,15(6):647-653.
74 Tabata Y,Morimoto K,Katsumata H,et a1.Surfactant-free preparation of biodegradable hydrogel microspheres for protein release.J Bioact Compat Polym,1999,14:371-373.
75 Yu K,Makoto 0,Takashi I,et a1.Time course of de novo adiposeness in Matrigel by gelatin microspheres incorporating basic fibroblast growth factor.Tissue Eng,2002,4:609-613.
76 Orban JM,Marra KG,HollingerJO.Composition options for tissue en gineered bone.Tissue Eng,2002,8:529-539.
77 T.P. Richardson, M.C. Peters, A.B. Ennett, D.J. Mooney, Polymeric system for dual growth factor delivery, Nat. Biotechnol,2001,19:1029–1034.
78 S. Young, M. Wong, Y. Tabata, A.G. Mikos, Gelatin as a delivery vehicle for the controlled release of bioactive molecules, J Control Release,2005,109:256–274.
79 C. Yan, X. Li, X. Chen, D. Wang, D. Zhong, T. Tan, H. Kitano, Anticancer gelatin microspheres with multiple function, Biomaterials,1991,12:640–644.
80 P.T. Prisell, O. Camber, J. Hiselius, G. Norstedt, Evaluation of hyaluronan as a vehicle for peptide growth factors, Int. J. Pharm,1992,85:51–56.
81 CaoT,Ho KH,Teoh SW.Scafold design and in vitro study ofoseochondral coculture in a three—dimensional porous polycaprolactone scaffold fabricated by fused deposition modeling.Tissue Eng,2003,9:103-112.
82 Y. Tabata, Tissue regeneration based on growth factor release, Tissue Eng,2003,9:S5–S15.
83 Yamamoto M,Ikada Y,Tabata Y.Controlled release of growth factors based on biodegradation of gelatin hydrogel:J .J Biomer Sci Polym Ed,2001,12:77-88
84 Tabata Y,Ikada Y. Vascularization effect of basic fibroblast growth factorreleased from gelatin hydrogels with different biodegradabilities. Biomaterials,1999,20:2169-2175.
85 Tabata Y,Yamada K,Miyamot S.et a1.Bone regeneration by basic fibroblast growth factor complexed with biodegradable hydrogels Biomaterials,1998,19:807-815.
86 Kawai K,Suzuki S,Tab ata Y,et a1.Accelerated tissue regeneration through incorporation of basic fibroblast growth factor—impregnated gelatin microspheres into artificial dermis,Biomaterials,2000,21:489-499.
87 Yang CF , Yasukawa T . Kimura H , et al Experimental corueal neovaseularization by basic fibroblast growt h factor incorporated into gelatin bydrogel,Ophthalmic Res,2000,32:19-24.
88 Han g L,Mivamoto S,Yamada K,et a1.Enhanced formation of fibrosis in a rabbit aneurysm by gelatin hydrogel incorporating basic fibroblast growth faetor,Neurosurgery,2001,49:954-960.
89 Masanori F,Masayuki I,Masafumi S,et a1.Vascularization in vivo caused by the controlled release of fibroblast growth factor-2 from an injectable chitosan/non-anticoagulant heparin hydrogel.Biomaterials,2004.25:699-706.
90 J.H. Distler, A. Hirth, M. Kurowska-Stolarska, R.E. Gay, S. Gay, O. Distler, Angiogenic and angiostatic factors in the molecular control of angiogenesis, Q. J. Nucl. Med,2003,47:149-161.
91 D.W. Losordo, S. Dimmeler, Therapeutic angiogenesis and vasculogenesis for ischemic disease. Part I: angiogenic cytokines, Circulation,2004,109:2487–2491.
92 唐群委,林建明,吴季怀,等.功能材料, 2006,9(37): 1510-1513.
93 Sung HW,Chang Y,Chin CT,et a1.Crosslinking characteristics and mechanical properties od a bovine pericardium fixed with a naturally occurringcrosslinking agent.J Biomed Mater Res,1999,47(2): l16-126.
94 韩津生.戊二醛检测研究进展.环境与健康杂志,2005,22(4):319-320.
95 崔玉明,胡蕴玉,吕昌伟,等.骨髓基质细胞与两种不同载体材料生物相容性的实验研究.中国矫形外科杂志,2003,11(16):1113-1116.
96 Oreffo RO,Driessens FC,P1anell JA,et a1.Growth and diferentiation of human bone marrow osteoprogenitors on novel calcium phosphate cements.Biomaterials,1998,19(20):1845-1854.
97 Hansbrough JF,Cooper ML,Cohen R,et al. Evaluation of a biodegradable matrix containing cultured human fibroblasts as a dermal replacement beneath meshed skin grafts on athymic mice. Surgery,1992,111(4):438-446.
98 中华人民共和国国家标准GB/T 16886.12 1996,医疗器械生物学评价第12部分:样品制备与参照样品.
99 Nimni ME. Polypeptide growth factors:targeted delivery systems. Biomaterials ,1997,18:1201-25
100 Coulomb B,Friteau L,Barueh J,eta1.Advantage of the presence of living derm al fibroblasts within in vitro reconstructed skin for grafting in humans.Hast Reconstr Surg,1998,101(7):1891-1903.
101 杨志明.组织工程基础与临床.成都:四川科学技术出版社,2000.233-237.
102 肖仕初,夏照帆.真皮替代物.国外医学,生物医学工程分册,2001,24(1):19.
103 徐成晦.真空冷冻干燥技术.真空与低温,1994,13 (2):95-99.
104 华泽钊,任禾盛著. 低温生物医学技术,北京:科学出版社.1994
105 张洁,华泽钊. 模拟生物组织冻结过程. 实验与分析工程热物理学报.2000,3:350-353.
106 Steven T Boyce.Fabrication,quality assurance,and assessment of culturedskin substitutes for treatment of skin wounds.Biochemical Engineering Journal,2004,20:107-l12.
107 Mi Fwu—Long,Wu Yu-Bey,Shyu Shin-Shing,et a1.Asymmetric chitosan membranes prepared by dry/wet phase separation;a new type of wound dressing for controlled antibacterial release.Journal of Membrane Science,2003,212:237-254.
108 Masashi Nomi, Anthony Atala, Paolo De Coppi, Shay Soker. Principals of neovascularization for tissue engineering. Molecular Aspects of Medicine,2002,23:463–483.
109 Ulubayram K,Cakar AN,Korkusuz P,et a1.EGF contmning gelatin based wound dressings. Biomaterials,2001,22:1345-1356.
110 Hang L,Mivamoto S,Yamada K,et a1.Enhanced formation of fibrosis in a rabbit aneurysm by gelatin hydrogel incorporating basic fibroblast growth faetor. Neurosurgery,2001,49:954-960.
111 Paul. Willi,Chandra P. Sharma. Chitosan and Alginate Wound Dressings: A Short Review. Trends Biomater. Artif.Organs,2004,18(1):18-23.
112 Lin FH,Chen TM,Chen KS,et a1.An animal study of a novel trilayer wound dressing materials non·-woven fabric grafted with N-isopropyl acrylamide and gelatin,et al. Mater Chem Phys,2000,64:189-195.
113 Lee CH,Singla A,Lee Y. Biomedical applications of collagen.Int J Pharm,2001,221(1-2):1-22
114 Baker PG, Haig G. Metronidazole in the treatment of chronic pressure sores and ulcers: a comparison with standart treatments in general practice. Practitioner 1981;225:569-73.
115 Kaya AZ, Turani N, Akyuz M. The effectiveness of a hydrogel dressing compared with standard management of pressure ulcers. J Wound Care. 2005,14(1):42-4.
116 Bruin P, Jonkman MF, Meijer HJ, Pennings AJ. A new porous polyetherurethane wound covering. J Biomed Mater Res 1990;24:217-26.
117 Lynch SE, Colvin RB, Antoniades HN. Growth factors in wound healing- single and synergistic effects on partial thickness porcine skin wounds. J Clin Invest 1989;84:640-6.
118 Ko IK, Iwata H. An approach to constructing three-dimensional tissue. Ann N Y Acad Sci. 2001;944:443–55.
119 T. Kushibiki, H. Matsuoka and Y. Tabata, Synthesis and physical characterization of poly(ethylene glycol)-gelatin conjugates, Biomacromolecules 2004 (5):202–208.
120 A. Muvaffak, I. Gurhan and N. Hasirci, Prolonged cytotoxic effect of colchicine released from biodegradable microspheres, J. Biomed. Mater. Res. 2004 (71B):295–304.
121 A.J. Kuijpers, G.H. Engbers, P.B. van Wachem, J. Krijgsveld, S.A. Zaat, J. Dankert and J. Feijen, Controlled delivery of antibacterial proteins from biodegradable matrices, J. Controlled Release 1998 (53):235–247.
122 L.D.Silvio and W. Bonfield, Biodegradable drug delivery system for the treatment of bone infection and repair, J. Mater. Sci., Mater. Med. 1999 (10):653–658.
123 Malda J, Kreijveld E, Temenoff JS, et al. Expansion of human nasal chondrocytes on macroporous microcarriers enhances redifferentiation. Biomaterials 2003; 24:5153-61.
124 詹国平,韩彦红,谢恩伟.阿霉素明胶微球的制备及体外释药特性.中国医学工程 [J], 2005,13(5): 483-488
125 Liu JY, Hafner J, Dragieva G, et al. Autologous cultured keratinocytes onporcine gelatine microbeads effectively heal chronic venous leg ulcers. Wound Repair Regen 2004;12:148-56.
126 Baker, T.L., and Goodwin, T.J. Three-dimensional culture of bovine chondrocytes in rotating-wall vessels. In Vitro Cell. Dev. Biol Anim. 1997, 33, 358.
127 Himes VB, Hu WS.Attachment and growth of mammalian cells on microcarriers with different ion exchange capacities. Biotechnol Bioeng , 1987,24:1155–63.
128 Reuveny S, Mizrahi A, Kotler M, Freeman A. Factors effecting cell attachment, spreading, and growth on derivatized microcarriers: II introduction of hydrophobicelements. Biotechnol Bioeng . 1983,5:2969–80.
129 Kiremitci M, Piskin E. Cell adhesion to the surfaces of polymeric beads. Biomaterials: 1990, Art Cells Art Org; 18:599–603.
130 Chun, K.W. Biodegradable PLGA microcarriers for injectable delivery of chondrocytes: effect of surface modification on cell attachment and function. Biotechnol. Prog. 2004,20, 1797–1801
131 Brittberg M, Lindahl A, Nilsson A, et al. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 1994,331:889–95.
132 Drury JL, Mooney DJ. Hydrogels for tissue engineering: Scaffold design variables and applications. Biomaterials 2003,24:4337–51.
133 Christiansen, J., Ek, L., and Tegner, E. Pinch grafting of leg ulcers. A retrospective study of 412 treated ulcers in 146 patients. Acta Derm. Venereol. 1997,77: 471.
134 Lin, T.-W., and Horng, S.-Y. A new method of microskin mincing. Burns1994,20: 526.
135 Dai NT, Williamson MR, Khammo N, Adams EF, Coombes AG. Composite cell support membranes based on collagen and polycaprolactone for tissue engineering of skin. Biomaterials. 2004,25:4263–71.
136 Griffith LG,Naughton G. Tissue engineering——current challenges and expanding opportunities. Science,2002,295:1009-1012.