RhBMP-2/ACBM：一种有潜力的组织工程骨支架材料
|
摘要
临床上由于创伤、肿瘤切除、感染以及发育异常等原因而导致的骨缺损需要恢复骨骼的连续性、力学支撑以及再生修复。传统的骨缺损修复主要采用自体骨、同种异体骨移植、骨水泥、陶瓷及金属等。自体骨移植存在来源少,且常造成供骨部位并发症,会给病人带来一定的痛苦;而异体骨移植存在排斥反应问题,上述方法均存在不同程度的缺点。
骨组织病损的修复可由周围健康的成骨前体细胞被趋化或植入缺损局部,通过再生最终实现。基于这种设想,组织工程,即细胞——基质(天然或人工合成材料)复合移植试图利用这一方法构建骨缺损修复的替代物。
由于细胞必须依赖于细胞外基质的存在才能发挥其功能,基质材料必须具备三维结构,并有促进细胞粘附、分化的成分,而且还需要为组织再生提供一定的力学强度。因此,在骨组织工程中,细胞外基质替代物即种植基质材料的选择是一个重要方面。
人工合成的高分子聚合材料因为具有适合成骨作用的前体细胞移动、增殖和分化的网状孔隙结构,常被用作促进骨组织再生的基质材料。但因生物力学特性差、代谢产物对周围组织的毒性作用、应力疲劳及对骨组织的应力遮挡等缺点,使其应用受到限制。其它材料中,天然材料衍生物,如各种方法处理的异体、异种脱钙骨等衍生材料,临床应用表明有促进骨缺损修复的作用。尽管动物实验及临床骨缺损应用表明脱钙骨基质(DBM)具有较好的生物相容性和吸收特性,但由于其力学特性差,而且有一定的抗原性,难以满足骨组织工程材料的要求。因此,研究和开发理想的骨组织工程基质材料有待进一步深入。
研究显示脱细胞基质材料,如脱细胞真皮及血管等保留了良好的力学特性、生物相容性、生物降解特性,粗糙的表面和形状有利于细胞的粘附及生长,经处理后可避免疾病传播和免疫排斥反应。
因此,我们有理由推测:经加工而成的脱细胞骨基质(ACBM)作为支架材料在骨组织工程中的应用前景广阔。本研究首先制备ACBM,并对其生物力学特性,生物相容性,骨诱导和骨传导作用进行评价。在此基础上,用纤维蛋白胶FBG,将具有骨诱导活性的重组人骨形态发生蛋白2(rhBMP-2)与ACBM复合在体外构建组织工程骨缓
释载体,并观察材料的缓释作用。然后,将新西兰大耳白兔的骨髓间充质干细胞(MSC)与材料复合并观察对兔桡骨缺损的修复效果。
主要结果和结论:
本研究从构建组织工程骨的细胞外基质材料入手,将异种骨经系列处理后制备成具有适合管状骨修复的ACBM;体内外实验表明,ACBM具有低抗原性和良好的生物相容性。在此基础上,用人FBG将外源性rhBMP-2与ACBM复合;然后,分离、扩增MSC并在体外诱导分化;将细胞与上述材料复合后,体内植入修复兔桡骨骨缺损效果良好,得出以下主要结论。
1.本实验采用理化联合生物化学的方法,实现了异种骨的脱细胞去抗原处理。体内外实验表明,对植入受体的细胞及体液免疫无明显影响,而且具有良好的生物安全性。
2.优化了制作过程。分析表明,其主要成分胶原羟基磷灰石与正常骨组织相似。组织学观察表明,在制作中其形态结构无破坏,生物力学强度与新鲜骨组织无统计学差异。与现有的骨衍生材料比较,ACBM保留了骨组织的原有结构和生物力学强度,可作为骨缺损,尤其是管状骨缺损的修复材料。
3.超微结构观察显示,ACBM为适合种子细胞生长的三维多孔支架材料,具有天然骨的网孔和孔隙率。体外种植发现,材料有利于种子细胞的粘附及活性表达;体内植入表明,ACBM具有骨传导和骨诱导双重作用。
4.本研究结果表明,具有生物力学强度的块状ACBM材料,在修复骨缺损早期,其成骨量大于无力学强度的骨粒材料。适当的生物力学强度的材料有利于对骨缺损的修复。
5.将FBG引入ACBM,用两步法将rhBMP-2复合进入ACBM,从而制备rhBMP-2的缓释载体。超微结构观察可见,复合材料的网孔结构及网孔率与复合前无明显差别。体外观察证实,缓释载体在4w内能够稳定而有效地释放rhBMP-2,达到缓释目的;体内植入修复骨缺损研究表明,复合rhBMP-2后,能够促进对局部组织中成骨前体细胞的趋化、诱导,加速对骨缺损的修复。
6.本实验分离的MSC,体外具有成骨、成脂肪诱导分化的潜能;体外扩增及成骨诱导分化表明,无论其“质和量”均能够满足构建组织工程骨的需要;rhBMP-2的成骨诱导进一步表明细胞与材料复合的可行性。
7.修复骨缺损的结果表明,4-8w,细胞-材料复合物对兔桡骨节段性骨缺损的修复效果优于无细胞的rhBMP-2/ACBM材料,而后者又较单纯ACBM材料修复为好。
因此,材料-细胞复合物可以加速成骨,更利于骨缺损的修复。8w后,虽然成骨量在前二者无统计学差异,但在新骨的重建和塑型方面仍以细胞-材料复合移植效果为好。
8.荧光小鼠MSC与材料复合移植后的示踪观察表明,12w时,表达绿色荧光蛋白的小鼠MSC不但可以成活,而且能够发挥成骨作用。
Critical size bone defects arising from trauma, tumor resection, infection and skeletal abnormalities require assistance to provide skeletal continuity, mechanical support and eventual regeneration. Traditionally, materials strategies to address bone defects include the use of autogeneous grafts and flaps, allograft bone, non-degradable bone cement, metals and ceramics. All of these options have their associated problems such as the finite amount of tissue that can be harvested for autografts, the difficulty to shape grafts into the desired form, and the risk of immune rejection or disease transfer with allografts.
The regeneration of fractured bone is based on the hypothesis that healthy progenitor cells, either recruited or delivered to an injured site, can eventually replace the lost or damaged bone tissue. Based on this suggestion, tissue engineering approaches attempt to create tissue replacements by culturing cells onto nature or synthetic three-dimensional matrixes. It is difficult to repair tissues by using cultured cells alone. In fact, a biomaterial with appropriate composition and three-dimensional structure to promote cellular adhesion, differentiation and the mechanic strength to provide as a scaffold for tissue regeneration is needed. So, it is very important for materials to be used in bone engineering.
Three-dimensional porous polymeric matrixes are seen as one approach to enhance bone regeneration by creating and maintaining channels that facilitate progenitor cell migration, proliferation, and differentiation. However, their application is limited because of some shortcomings such as poor mechanical properties, toxicity of degradation product to cells and stress shielding to the surrounding bone or fatigue failure of the implants. For other materials to be considered, among which are natural bone derived biomaterials. DBM for example, has been shown to accelerate wound healing, and be used as a biocompatible and absorbable material in both animal and human bone defects. However, these materials can not meet the requirements of bone tissue engineering because of their poor mechanical properties and allergic reactions. Therefore, the research of improving ideal materials in
bone engineering should be continuously carried out.
Acellular matrixes, such as dermis and blood vessels had been shown to have good mechanical properties, biocompatibility, biodegradability. Also, their gross architecture and shape facilitate cells adhesion and proliferation. At the same time, allergic reactions and disease transmission may be avoided after treatment.
Therefore, it is reasonable to predict that ACBM can be used as a prospective candidate to form a structural framework for bone engineering. In this study, ACBM material was prepared and evaluated for mechanical properties, general biocompatibility, effect of osteoinductive and osteoconductive. Subsequently, it was combined with rhBMP-2 by fibrin gelatin to constitute the slowly releasing carrier of tissue engineering bone. The scaffold was seeded with mesenchymal stem cells harvested from bone marrow of NZW rabbits to evaluate their performance as potential bone substitutes in vitro and in vivo.
The main results and conclusions are as follows:
1. United with physical, chemical and biochemical methods, we successfully realized deantigenic treatment of xenogeneic bone in the formulations of ACBM. In vivo and in vitro evaluation of cellular and humoral immunity in recipients proved that ACBM has lower or no antigen with a good reliability.
2. Not only the components and structure of bone are being preserved in ACBM, but also the mechanical properties are similar to that of fresh bone. Compared with natural derived bone matrixs existing now, ACBM reserves the original structure and biomechanics. So, it is a suitable material for the repair of bone defects, especially for segmental bone defects in tubular bone.
3. Scanning with SEM, it proved that the three-dimensional porous ACBM maintain channels that facilitate progenitor cell migration, proliferati
骨组织病损的修复可由周围健康的成骨前体细胞被趋化或植入缺损局部,通过再生最终实现。基于这种设想,组织工程,即细胞——基质(天然或人工合成材料)复合移植试图利用这一方法构建骨缺损修复的替代物。
由于细胞必须依赖于细胞外基质的存在才能发挥其功能,基质材料必须具备三维结构,并有促进细胞粘附、分化的成分,而且还需要为组织再生提供一定的力学强度。因此,在骨组织工程中,细胞外基质替代物即种植基质材料的选择是一个重要方面。
人工合成的高分子聚合材料因为具有适合成骨作用的前体细胞移动、增殖和分化的网状孔隙结构,常被用作促进骨组织再生的基质材料。但因生物力学特性差、代谢产物对周围组织的毒性作用、应力疲劳及对骨组织的应力遮挡等缺点,使其应用受到限制。其它材料中,天然材料衍生物,如各种方法处理的异体、异种脱钙骨等衍生材料,临床应用表明有促进骨缺损修复的作用。尽管动物实验及临床骨缺损应用表明脱钙骨基质(DBM)具有较好的生物相容性和吸收特性,但由于其力学特性差,而且有一定的抗原性,难以满足骨组织工程材料的要求。因此,研究和开发理想的骨组织工程基质材料有待进一步深入。
研究显示脱细胞基质材料,如脱细胞真皮及血管等保留了良好的力学特性、生物相容性、生物降解特性,粗糙的表面和形状有利于细胞的粘附及生长,经处理后可避免疾病传播和免疫排斥反应。
因此,我们有理由推测:经加工而成的脱细胞骨基质(ACBM)作为支架材料在骨组织工程中的应用前景广阔。本研究首先制备ACBM,并对其生物力学特性,生物相容性,骨诱导和骨传导作用进行评价。在此基础上,用纤维蛋白胶FBG,将具有骨诱导活性的重组人骨形态发生蛋白2(rhBMP-2)与ACBM复合在体外构建组织工程骨缓
释载体,并观察材料的缓释作用。然后,将新西兰大耳白兔的骨髓间充质干细胞(MSC)与材料复合并观察对兔桡骨缺损的修复效果。
主要结果和结论:
本研究从构建组织工程骨的细胞外基质材料入手,将异种骨经系列处理后制备成具有适合管状骨修复的ACBM;体内外实验表明,ACBM具有低抗原性和良好的生物相容性。在此基础上,用人FBG将外源性rhBMP-2与ACBM复合;然后,分离、扩增MSC并在体外诱导分化;将细胞与上述材料复合后,体内植入修复兔桡骨骨缺损效果良好,得出以下主要结论。
1.本实验采用理化联合生物化学的方法,实现了异种骨的脱细胞去抗原处理。体内外实验表明,对植入受体的细胞及体液免疫无明显影响,而且具有良好的生物安全性。
2.优化了制作过程。分析表明,其主要成分胶原羟基磷灰石与正常骨组织相似。组织学观察表明,在制作中其形态结构无破坏,生物力学强度与新鲜骨组织无统计学差异。与现有的骨衍生材料比较,ACBM保留了骨组织的原有结构和生物力学强度,可作为骨缺损,尤其是管状骨缺损的修复材料。
3.超微结构观察显示,ACBM为适合种子细胞生长的三维多孔支架材料,具有天然骨的网孔和孔隙率。体外种植发现,材料有利于种子细胞的粘附及活性表达;体内植入表明,ACBM具有骨传导和骨诱导双重作用。
4.本研究结果表明,具有生物力学强度的块状ACBM材料,在修复骨缺损早期,其成骨量大于无力学强度的骨粒材料。适当的生物力学强度的材料有利于对骨缺损的修复。
5.将FBG引入ACBM,用两步法将rhBMP-2复合进入ACBM,从而制备rhBMP-2的缓释载体。超微结构观察可见,复合材料的网孔结构及网孔率与复合前无明显差别。体外观察证实,缓释载体在4w内能够稳定而有效地释放rhBMP-2,达到缓释目的;体内植入修复骨缺损研究表明,复合rhBMP-2后,能够促进对局部组织中成骨前体细胞的趋化、诱导,加速对骨缺损的修复。
6.本实验分离的MSC,体外具有成骨、成脂肪诱导分化的潜能;体外扩增及成骨诱导分化表明,无论其“质和量”均能够满足构建组织工程骨的需要;rhBMP-2的成骨诱导进一步表明细胞与材料复合的可行性。
7.修复骨缺损的结果表明,4-8w,细胞-材料复合物对兔桡骨节段性骨缺损的修复效果优于无细胞的rhBMP-2/ACBM材料,而后者又较单纯ACBM材料修复为好。
因此,材料-细胞复合物可以加速成骨,更利于骨缺损的修复。8w后,虽然成骨量在前二者无统计学差异,但在新骨的重建和塑型方面仍以细胞-材料复合移植效果为好。
8.荧光小鼠MSC与材料复合移植后的示踪观察表明,12w时,表达绿色荧光蛋白的小鼠MSC不但可以成活,而且能够发挥成骨作用。
Critical size bone defects arising from trauma, tumor resection, infection and skeletal abnormalities require assistance to provide skeletal continuity, mechanical support and eventual regeneration. Traditionally, materials strategies to address bone defects include the use of autogeneous grafts and flaps, allograft bone, non-degradable bone cement, metals and ceramics. All of these options have their associated problems such as the finite amount of tissue that can be harvested for autografts, the difficulty to shape grafts into the desired form, and the risk of immune rejection or disease transfer with allografts.
The regeneration of fractured bone is based on the hypothesis that healthy progenitor cells, either recruited or delivered to an injured site, can eventually replace the lost or damaged bone tissue. Based on this suggestion, tissue engineering approaches attempt to create tissue replacements by culturing cells onto nature or synthetic three-dimensional matrixes. It is difficult to repair tissues by using cultured cells alone. In fact, a biomaterial with appropriate composition and three-dimensional structure to promote cellular adhesion, differentiation and the mechanic strength to provide as a scaffold for tissue regeneration is needed. So, it is very important for materials to be used in bone engineering.
Three-dimensional porous polymeric matrixes are seen as one approach to enhance bone regeneration by creating and maintaining channels that facilitate progenitor cell migration, proliferation, and differentiation. However, their application is limited because of some shortcomings such as poor mechanical properties, toxicity of degradation product to cells and stress shielding to the surrounding bone or fatigue failure of the implants. For other materials to be considered, among which are natural bone derived biomaterials. DBM for example, has been shown to accelerate wound healing, and be used as a biocompatible and absorbable material in both animal and human bone defects. However, these materials can not meet the requirements of bone tissue engineering because of their poor mechanical properties and allergic reactions. Therefore, the research of improving ideal materials in
bone engineering should be continuously carried out.
Acellular matrixes, such as dermis and blood vessels had been shown to have good mechanical properties, biocompatibility, biodegradability. Also, their gross architecture and shape facilitate cells adhesion and proliferation. At the same time, allergic reactions and disease transmission may be avoided after treatment.
Therefore, it is reasonable to predict that ACBM can be used as a prospective candidate to form a structural framework for bone engineering. In this study, ACBM material was prepared and evaluated for mechanical properties, general biocompatibility, effect of osteoinductive and osteoconductive. Subsequently, it was combined with rhBMP-2 by fibrin gelatin to constitute the slowly releasing carrier of tissue engineering bone. The scaffold was seeded with mesenchymal stem cells harvested from bone marrow of NZW rabbits to evaluate their performance as potential bone substitutes in vitro and in vivo.
The main results and conclusions are as follows:
1. United with physical, chemical and biochemical methods, we successfully realized deantigenic treatment of xenogeneic bone in the formulations of ACBM. In vivo and in vitro evaluation of cellular and humoral immunity in recipients proved that ACBM has lower or no antigen with a good reliability.
2. Not only the components and structure of bone are being preserved in ACBM, but also the mechanical properties are similar to that of fresh bone. Compared with natural derived bone matrixs existing now, ACBM reserves the original structure and biomechanics. So, it is a suitable material for the repair of bone defects, especially for segmental bone defects in tubular bone.
3. Scanning with SEM, it proved that the three-dimensional porous ACBM maintain channels that facilitate progenitor cell migration, proliferati
引文
Freed CE, Vunjak-novakovic G, Iron KJ. Biodegradable polymer scaffolds for tissue engineering. J Biotechnology NY, 1994;12(7):689-693
Reddi AH. Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat Biotechnol, 1998;16:247–252
Kim BS, Mooney DJ. Devlopment of biocompatible synthetic extracellular matrices for tissue engineering. J Trends Biotechnol,1998;16(5):224-230
Agrawal C, Achanasion KA. Technique to control pH in vicinity of biodegrading PLA- PGA implants. J Biomed Mater Res, 1997;38(2):105-114
Sims CD, Butler PE, Cao YL ,et al. Tissue engineered neocartilage using plasma derived polymersubstrates and chondrocytes. Plast Reconstr Surg, 1998;101 (6): 1580- 1585
颜晓慧,郑磊,洪华容,等. 骨组织工程应用可降解聚合物的可能性探讨.国外医学生物医学工程分册,2000;23(1):11-17
Devin JE, Attawia MA, Laurencin CT,et al. Three dimensional degradable porous polymer ceramic matrices for use in bone repair. J Biomater Sci Polymer,1996; 7(8): 661-669
刘彦春,王炜,曹谊林,等. 卵磷脂、多聚赖氨酸和 PL A包埋 PGA与软骨细胞体外培养的实验研究. 实用美容整形外科杂志,1997;8(5):225-227
Lin FH, Liao CJ, Chen KS, et al. Preparation of a biphasic porous bioceramic by heating bovine cancellous bone with Na4P207·10H2O addition. J Biomaterials, 1999; 20(5): 475- 484.
陈希哲,杨连甲. 生物陶瓷与骨组织工程. 国外医学生物医学工程分册,2000;23(1):6-10
杨维东,毛天球. 口腔颌面部骨替代材料与骨组织工程. 实用口腔医学杂志,1999;15(6):469-471
陈际达,王远亮,蔡绍皙,等. 仿真骨科材料制备的新方法. 高技术通讯, 2001;8:22-25
Wainwright DJ. Use of an acellular allograft dermal matrix (Alloderm) in the management of full thickness burns. Burns, 1995; 21:243–248
Chaplin JM, Costantino PD, Wolpoe ME, et al. Use of an acellular dermal allograft for
dural replacement: an experimental study. Neurosurgery, 1999;45:320–327
Sclafani AP, Jacono AA, McCormick S, et al. Evaluation of acellular dermal graft in sheet (Alloderm) and injectable (micronized Alloderm) forms for soft tissue augmentation. Arch Facial Plast Surg, 2000;2:130–136
Srivastava EZ, DeSagun LJ, Jennings S, et al. Use of porcine acellular dermal matrix as a dermal substitute in rates. Ann Surg, 2000;233:400–408
刘流,梁德江,申鹏飞,等. 脱细胞膀胱细胞外基质作为膀胱替代材料的实验研究. 中华实验外科杂志, 1999;16:181-183
Lee Y, Chen MY, Kuo CY, et al. Preparation of porcine dermal matrix graft. Global Chinese Symposium on Biomaterials and Controlled Release, 1999:584–587
Badylak SF, Matheny R, Obermiller J, et al. ECM scaffold for myocardial reconstruction. Symposium on Tissue Engineering Science. Aegean Conferences Series, 2002;4:65-69
Badylak SF, Ling F, Record R, et al. Vascularization of 3-dimensional scaffolds. Symposium on Tissue Engineering Science. Aegean Conferences Series, 2002;4:63-65
Adams PW, Lee HS, Waldmam WJ, et al. Alloantigenity of human endothelial cells:Frequency and phenotype of human T helper lymphocytes that can react with allogeneic endothelial cells. J Immunol, 1992;148:3753-3759
刘玮,陆裕朴,胡蕴玉,等. 异种植骨抗原性的免疫组织化学实验研究. 中华骨科杂志,1989;9:53-55
李彦林,杨志明,解琪慧,等. 生物衍生组织工程骨支架材料的制备及理化特性.生物医学工程学杂志,2002;19(1):10-12
郑磊,王前. 骨组织工程基质材料的现状及展望. 生物医学工程学杂志,2001;18(3)∶ 470-472
Becker W, Urist MR, Tucker LM, et al. Human demineralized freeze-dried bone: inadequate induced bone formation in athymic mice. A preliminary report. J. Periodontol, 1995;669:822–828
Reynolds MA, Bowers GM. Fate of demineralized freeze-dried bone allografts in human intrabony defects. J Periodontol, 1996;672:150–157
Urist MR, Wallace TH ,Adams T, et al. The function of fibrocartilaginous fracture callus. JBJS, 1965; 47B:304–318
Urist MR, Silverman BF, Buring K, et al. The bone induction principle. Clin Orthop Rel Res, 1967;53:243–283
Strates BS ,Urist MR. Origin of the indutive signal in implants of normal and lathyritic bone matrix. Clin Orthop, 1969; 66:226–240.
胡运生,范清宇,周勇,等. 异体脱钙骨基质明胶骨粒/骨水泥复合材料的生物力学性能研究. 中国修复重建外科杂志,2002;16(2):97-99
马贵骧,韩巽,王艺明,等. 天然型无机骨的制备及其成骨效应的实验研究. 中国修复重建外科杂志,1996;10(1):35-38
Livesey SA, Herndon DN, Hollyoak MA, et al. Transplanted acellular allograft dermal matrix. Potential for the reconstruction of viable dermis. Transplantation, 1995;60:1–9
Takami Y, Matsuda T, Yoshitake M, et al. Dispase/detergent treated dermal matrix as a dermal substitute. Burns, 1996;22:182–190
韩雪峰,杨大平,郭铁芳. 曲拉通X-100对制备脱细胞血管基质影响的实验研究.中华外科杂志, 2002;40(1):27-29
Abraham GA, Murray J, Billiar K ,et al. Evaluation of the porcine intestinal collagen layer as a biomaterial. J Biomed Mater Res, 2000;51:442–452
Walter RJ, Matsuda T, Reyes HM, et al. Characterization of acellular dermal matrices (ADMs) prepared by two different methods. Burns, 1998;24: 104–113
夏良树,聂长明. 洗涤剂用复合酶的配伍性能研究. 日用化学工业,2001;5(31):58-61
Ole K, Torben VB,Claus CF. Industrial enzyme applications. Biotechnology, 2002; 13:345–351
Schmidt CE ,Baier JM. Acellular vascular tissues: natural biomaterials for tissue repair and tissue engineering. Biomaterials, 2000;21:2215–2231
龚志锦,朱明华,郑建明,等. 显示粘蛋白、弹力蛋白和胶原蛋白的复合染色法及其应用. 中国组织化学与细胞化学杂志,2002;11(4)503-504
Jin QM, Takita H, Kohgo T, et al. Effects of geometry of hydroxyapatite as a cell substratum in BMP-induced ectopic bone formation in PPHAP and PBHAP while only endochondral ossification took place in HCHAP. Interestingly, cartilage in the central .J Biomed Mater Res, 2000;51:491–499
Young C, Sandstedt P, Skoglund A. A comparative study of anicxenogenic bone and
autogenous bone implants for bone regeneration in rabbits. Int Joral Maxillofac Implants, 1999;14:72~76
Roberta H, Raeder, Stephen F, et al,Natural anti-galactose1,3 galactose antibodies delay, but do not prevent the acceptance of extracellular matrix xenografts, Transplant Immunology, 2002;10:15~24
李彦林,杨志明,解慧琪,等. 生物衍生骨支架材料的体外细胞相容性实验研究.中国修复重建外科杂志,2001;15(4):227-231
Oliveira JG, Ramos JP, Xarier S, et al, Analysis of fine needl as piration biopsies by flow cytometry in kidney transplant patients. Transplantation, 1997;64:97-102
汪子伟. 流式细胞术在移植医学中的应用. 中华器官移植杂志,2000;21(1):62-63
Shishido S, Naziruddin B, Xu XC, et al. Indirect recognition of porcine xenoantigens by human CD4+T cell clones. Transplation, 1998;65:706-711
沈佰华,谢晋,李宁丽,等. 人特异性T细胞系间接识别猪抗原. 上海免疫学杂志,2001;21(4)210-212
丁勇,范清宇,张怡,等. 活性煅烧骨复合骨水泥修复骨缺损:修复后的血管及结构变化. 中国临床康复,2002:2(6):190-191
钟红兴,苏泽轩,邓文锋,等. 肾移植术后外周血CD3+细胞CD69、CD25及CD71的表达及意义. 中华器官移植杂志,2000;21(4):201-203
马晶波,李锋,冯树芳. 系统红斑狼疮患者外周血CD4+、CD8+和CD22+淋巴细胞活化分子的表达. 中华风湿病学杂志,2003;2(7):71-74
Masaomi Y, Yoshifumi S, Itaru N, et al. Humoral immune responses during acute rejection in rat lung transplantation. Transplant Immunology, 2003;11:31-37
Crespo M, Lozano M, Sole M, et al. Diagnosis and treatment of acute humoral rejection after kidney transplantation: preliminary experience. Transplantation Proceedings, 2003; 35: 1677-1678
医疗器械生物学标准汇编(第1部分-第12部分),中国标准出版社第一编辑室.北京.中国标准出版社,2003
ISO10993 1:1992 Biological evaluation of medical device, Part1-12,1996
高景恒. 关于医用生物材料 (植入体 )的安全性评价与评价标准. 实用美容整形外科杂志,2000;4(11):216-218
Chapekar MS. Regulatory concerns in the development of biologic–biomaterial
combinations. J Biomed Mater Res, 1996;33:199–203
Wilke A, Orth J, Lomb M, et al. Biocompatibility analysis of different biomaterials in human bone marrow cell cultures. J Biomed Mater Res, 1998;40 (2) :301-307
Kue R, Sohrabi A, Nagle D, et al. Enhanced proliferation and osteocalcin production by human osteoblast like MG6 3 cells on silicon nitride ceramic discs. Biomaterials, 1999;20 (13):1195-1202
周馨,郑昌琼,王方瑚,等. 一种新型磷酸钙骨水泥的制备、基本性能的研究及生物安全性评价. 生物医学工程学杂志,1999:16(增刊 ):56-59
Reddi AH. Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat Biotechnol, 1998;16:247–252
Kim BS, Mooney DJ. Devlopment of biocompatible synthetic extracellular matrices for tissue engineering. J Trends Biotechnol,1998;16(5):224-230
Agrawal C, Achanasion KA. Technique to control pH in vicinity of biodegrading PLA- PGA implants. J Biomed Mater Res, 1997;38(2):105-114
Sims CD, Butler PE, Cao YL ,et al. Tissue engineered neocartilage using plasma derived polymersubstrates and chondrocytes. Plast Reconstr Surg, 1998;101 (6): 1580- 1585
颜晓慧,郑磊,洪华容,等. 骨组织工程应用可降解聚合物的可能性探讨.国外医学生物医学工程分册,2000;23(1):11-17
Devin JE, Attawia MA, Laurencin CT,et al. Three dimensional degradable porous polymer ceramic matrices for use in bone repair. J Biomater Sci Polymer,1996; 7(8): 661-669
刘彦春,王炜,曹谊林,等. 卵磷脂、多聚赖氨酸和 PL A包埋 PGA与软骨细胞体外培养的实验研究. 实用美容整形外科杂志,1997;8(5):225-227
Lin FH, Liao CJ, Chen KS, et al. Preparation of a biphasic porous bioceramic by heating bovine cancellous bone with Na4P207·10H2O addition. J Biomaterials, 1999; 20(5): 475- 484.
陈希哲,杨连甲. 生物陶瓷与骨组织工程. 国外医学生物医学工程分册,2000;23(1):6-10
杨维东,毛天球. 口腔颌面部骨替代材料与骨组织工程. 实用口腔医学杂志,1999;15(6):469-471
陈际达,王远亮,蔡绍皙,等. 仿真骨科材料制备的新方法. 高技术通讯, 2001;8:22-25
Wainwright DJ. Use of an acellular allograft dermal matrix (Alloderm) in the management of full thickness burns. Burns, 1995; 21:243–248
Chaplin JM, Costantino PD, Wolpoe ME, et al. Use of an acellular dermal allograft for
dural replacement: an experimental study. Neurosurgery, 1999;45:320–327
Sclafani AP, Jacono AA, McCormick S, et al. Evaluation of acellular dermal graft in sheet (Alloderm) and injectable (micronized Alloderm) forms for soft tissue augmentation. Arch Facial Plast Surg, 2000;2:130–136
Srivastava EZ, DeSagun LJ, Jennings S, et al. Use of porcine acellular dermal matrix as a dermal substitute in rates. Ann Surg, 2000;233:400–408
刘流,梁德江,申鹏飞,等. 脱细胞膀胱细胞外基质作为膀胱替代材料的实验研究. 中华实验外科杂志, 1999;16:181-183
Lee Y, Chen MY, Kuo CY, et al. Preparation of porcine dermal matrix graft. Global Chinese Symposium on Biomaterials and Controlled Release, 1999:584–587
Badylak SF, Matheny R, Obermiller J, et al. ECM scaffold for myocardial reconstruction. Symposium on Tissue Engineering Science. Aegean Conferences Series, 2002;4:65-69
Badylak SF, Ling F, Record R, et al. Vascularization of 3-dimensional scaffolds. Symposium on Tissue Engineering Science. Aegean Conferences Series, 2002;4:63-65
Adams PW, Lee HS, Waldmam WJ, et al. Alloantigenity of human endothelial cells:Frequency and phenotype of human T helper lymphocytes that can react with allogeneic endothelial cells. J Immunol, 1992;148:3753-3759
刘玮,陆裕朴,胡蕴玉,等. 异种植骨抗原性的免疫组织化学实验研究. 中华骨科杂志,1989;9:53-55
李彦林,杨志明,解琪慧,等. 生物衍生组织工程骨支架材料的制备及理化特性.生物医学工程学杂志,2002;19(1):10-12
郑磊,王前. 骨组织工程基质材料的现状及展望. 生物医学工程学杂志,2001;18(3)∶ 470-472
Becker W, Urist MR, Tucker LM, et al. Human demineralized freeze-dried bone: inadequate induced bone formation in athymic mice. A preliminary report. J. Periodontol, 1995;669:822–828
Reynolds MA, Bowers GM. Fate of demineralized freeze-dried bone allografts in human intrabony defects. J Periodontol, 1996;672:150–157
Urist MR, Wallace TH ,Adams T, et al. The function of fibrocartilaginous fracture callus. JBJS, 1965; 47B:304–318
Urist MR, Silverman BF, Buring K, et al. The bone induction principle. Clin Orthop Rel Res, 1967;53:243–283
Strates BS ,Urist MR. Origin of the indutive signal in implants of normal and lathyritic bone matrix. Clin Orthop, 1969; 66:226–240.
胡运生,范清宇,周勇,等. 异体脱钙骨基质明胶骨粒/骨水泥复合材料的生物力学性能研究. 中国修复重建外科杂志,2002;16(2):97-99
马贵骧,韩巽,王艺明,等. 天然型无机骨的制备及其成骨效应的实验研究. 中国修复重建外科杂志,1996;10(1):35-38
Livesey SA, Herndon DN, Hollyoak MA, et al. Transplanted acellular allograft dermal matrix. Potential for the reconstruction of viable dermis. Transplantation, 1995;60:1–9
Takami Y, Matsuda T, Yoshitake M, et al. Dispase/detergent treated dermal matrix as a dermal substitute. Burns, 1996;22:182–190
韩雪峰,杨大平,郭铁芳. 曲拉通X-100对制备脱细胞血管基质影响的实验研究.中华外科杂志, 2002;40(1):27-29
Abraham GA, Murray J, Billiar K ,et al. Evaluation of the porcine intestinal collagen layer as a biomaterial. J Biomed Mater Res, 2000;51:442–452
Walter RJ, Matsuda T, Reyes HM, et al. Characterization of acellular dermal matrices (ADMs) prepared by two different methods. Burns, 1998;24: 104–113
夏良树,聂长明. 洗涤剂用复合酶的配伍性能研究. 日用化学工业,2001;5(31):58-61
Ole K, Torben VB,Claus CF. Industrial enzyme applications. Biotechnology, 2002; 13:345–351
Schmidt CE ,Baier JM. Acellular vascular tissues: natural biomaterials for tissue repair and tissue engineering. Biomaterials, 2000;21:2215–2231
龚志锦,朱明华,郑建明,等. 显示粘蛋白、弹力蛋白和胶原蛋白的复合染色法及其应用. 中国组织化学与细胞化学杂志,2002;11(4)503-504
Jin QM, Takita H, Kohgo T, et al. Effects of geometry of hydroxyapatite as a cell substratum in BMP-induced ectopic bone formation in PPHAP and PBHAP while only endochondral ossification took place in HCHAP. Interestingly, cartilage in the central .J Biomed Mater Res, 2000;51:491–499
Young C, Sandstedt P, Skoglund A. A comparative study of anicxenogenic bone and
autogenous bone implants for bone regeneration in rabbits. Int Joral Maxillofac Implants, 1999;14:72~76
Roberta H, Raeder, Stephen F, et al,Natural anti-galactose1,3 galactose antibodies delay, but do not prevent the acceptance of extracellular matrix xenografts, Transplant Immunology, 2002;10:15~24
李彦林,杨志明,解慧琪,等. 生物衍生骨支架材料的体外细胞相容性实验研究.中国修复重建外科杂志,2001;15(4):227-231
Oliveira JG, Ramos JP, Xarier S, et al, Analysis of fine needl as piration biopsies by flow cytometry in kidney transplant patients. Transplantation, 1997;64:97-102
汪子伟. 流式细胞术在移植医学中的应用. 中华器官移植杂志,2000;21(1):62-63
Shishido S, Naziruddin B, Xu XC, et al. Indirect recognition of porcine xenoantigens by human CD4+T cell clones. Transplation, 1998;65:706-711
沈佰华,谢晋,李宁丽,等. 人特异性T细胞系间接识别猪抗原. 上海免疫学杂志,2001;21(4)210-212
丁勇,范清宇,张怡,等. 活性煅烧骨复合骨水泥修复骨缺损:修复后的血管及结构变化. 中国临床康复,2002:2(6):190-191
钟红兴,苏泽轩,邓文锋,等. 肾移植术后外周血CD3+细胞CD69、CD25及CD71的表达及意义. 中华器官移植杂志,2000;21(4):201-203
马晶波,李锋,冯树芳. 系统红斑狼疮患者外周血CD4+、CD8+和CD22+淋巴细胞活化分子的表达. 中华风湿病学杂志,2003;2(7):71-74
Masaomi Y, Yoshifumi S, Itaru N, et al. Humoral immune responses during acute rejection in rat lung transplantation. Transplant Immunology, 2003;11:31-37
Crespo M, Lozano M, Sole M, et al. Diagnosis and treatment of acute humoral rejection after kidney transplantation: preliminary experience. Transplantation Proceedings, 2003; 35: 1677-1678
医疗器械生物学标准汇编(第1部分-第12部分),中国标准出版社第一编辑室.北京.中国标准出版社,2003
ISO10993 1:1992 Biological evaluation of medical device, Part1-12,1996
高景恒. 关于医用生物材料 (植入体 )的安全性评价与评价标准. 实用美容整形外科杂志,2000;4(11):216-218
Chapekar MS. Regulatory concerns in the development of biologic–biomaterial
combinations. J Biomed Mater Res, 1996;33:199–203
Wilke A, Orth J, Lomb M, et al. Biocompatibility analysis of different biomaterials in human bone marrow cell cultures. J Biomed Mater Res, 1998;40 (2) :301-307
Kue R, Sohrabi A, Nagle D, et al. Enhanced proliferation and osteocalcin production by human osteoblast like MG6 3 cells on silicon nitride ceramic discs. Biomaterials, 1999;20 (13):1195-1202
周馨,郑昌琼,王方瑚,等. 一种新型磷酸钙骨水泥的制备、基本性能的研究及生物安全性评价. 生物医学工程学杂志,1999:16(增刊 ):56-59