聚羟基丁酸及其共聚物在组织工程心脏瓣膜中应用的初步研究

应用组织工程支架，将活细胞种植于可生物降解的瓣膜支架上，制造出一种组织相容性好，不需抗凝，具有修复能力，且支持患终生的理想心脏瓣膜。对照进行聚羟基丁酸（polyhydroxybutyrate,PHB)、聚羟基丁酸／聚羟基戊酸共聚物（Polyhydroxybutyrate/hydroxyvalerate,PHBV)皮下包埋和种植细胞生长研究实验。结果表明PHBV皮下包埋8周仍基本保持膜状结构，10周完全降解，且生物相容性优于PHB。PHBV更适于组织工程心脏瓣膜支架材料的应用。     

电纺丝法制备骨组织工程用聚羟基丁酸酯支架及性能研究

采用电纺丝法成功制备了聚羟基丁酸酯(PHB)电纺丝膜.采用SEM、XRD、力学拉伸等研究方法对其微观形态、力学性能及体外降解行为进行了研究,并讨论了小鼠成纤维细胞(L929)在支架表面的形态,为其在骨组织工程支架上的应用提供理论和实验基础.结果表明纺丝液浓度、注射泵流速以及接收距离等操作参数对PHB纤维的形态有很大的影响.与PHB浇铸膜相比,PHB支架的结晶度及体外降解速率明显大于前者,但拉伸强度和断裂伸长率较小.在支架表面,细胞的形态良好,表明支架具有良好的生物相容性.这种降解性能及生物相容性兼备的PHB骨组织工程支架材料具有潜在的应用前景.     

成纤维细胞在聚羟基丁酸酯表面黏附与种植研究

可降解高分子聚合物广泛应用于组织工程,其表面的黏附特性影响细胞的种植与生长。我们研究了成纤维细胞系NIH3T3在可降解材料聚羟基丁酸酯（PHB）表面的黏附和生长。应用线性变剪切流槽研究NIH3T3细胞在材料表面的黏附力学特性,结果表明,由于在PHB表面的临界脱离剪应力较低,高密度接种时间细胞趋于聚集;在经多聚赖氨酸衣被的表面临界脱离剪应力提高,细胞铺展生长。对三维PHB多孔支架种植细胞实验表明,在PHB材料表面进行多聚赖氨酸衣被,可以有效地提高细胞种植效率。     

无纺网与多孔海绵状材料作为软骨组织工程支架的适用性及体内降解性

背景：聚羟基乙酸无纺网与聚羟基丁酸酯-聚羟基己酸酯共聚物多孔海绵具有良好的塑形适应性、生物降解性与生物相容性。 目的：观察聚羟基乙酸无纺网与聚羟基丁酸酯-聚羟基己酸酯共聚物多孔海绵作为软骨组织工程支架的适用性及体内降解性。 方法：分别制备乳兔软骨细胞-聚羟基乙酸无纺网复合物、乳兔软骨细胞-聚羟基丁酸酯-聚羟基己酸酯共聚物多孔海绵复合物。在实验组成年兔两侧背部皮下分别植入制备的两种复合物,在对照组成年兔两侧背部皮下分别植入聚羟基乙酸无纺网与聚羟基丁酸酯-聚羟基己酸酯共聚物。 结果与结论：组织学观察显示,以聚羟基乙酸无纺网获取的组织工程软骨,植入4 周时软骨细胞较小,软骨内有较多聚羟基乙酸纤维残留,8周时软骨细胞较成熟,包埋在陷窝内,聚羟基乙酸纤维消失,12周时软骨细胞成熟,基质分泌丰富,无聚羟基乙酸存留;以聚羟基丁酸酯-聚羟基己酸酯共聚物多孔海绵获取的组织工程软骨,植入4周时软骨细胞不成熟,软骨基质内似“杂质”样材料残留物较多,8周时软骨细胞较成熟,软骨基质内仍可见材料残留,12周时软骨基质材料残留基本消失。两组组织工程软骨特殊染色与免疫组织化学检测均显示再生软骨胶原与基质黏多糖生成良好,软骨中均检测出Ⅱ型胶原。表明两种材料作为软骨组织工程支架具有良好的适用性,其降解时间均达到组织工程软骨构建的要求。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

聚羟基丁酸酯及其在生物医学领域中的应用

简要介绍了生物可降解聚合物聚羟基丁酸酯（PHB）的合成、提取及改性技术，重点评述了PHB在药物释放和组织工程方面的应用研究进展。     

以羟基丁酸酯与羟基已酸酯共聚物为支架材料构建组织工程喉形态软骨

背景：再生预定形态组织工程软骨的研究为喉软骨病损的修复与重建提供了新思路与新方法。然而,由于喉软骨形态、部位与功能的特殊性,迄今在此领域软骨组织工程研究并未呈现出其应有的优势。目的：探讨带蒂肌筋膜组织瓣构建组织工程喉支架形态软骨方法,为肌筋膜瓣复合组织工程化软骨修复重建喉软骨支架功能提供实验依据。方法：采用溶剂浇铸、模压成形和颗粒滤沥方法制备喉支架形态聚羟基丁酸酯与聚羟基己酸酯共聚物聚羟基丁酸酯与聚羟基己酸酯共聚物聚羟基丁酸酯与聚羟基己酸酯共聚物生物材料塑形物,接种软骨细胞形成细胞-聚羟基丁酸酯与聚羟基己酸酯共聚物复合物,体外共同培养1周后用于体内植入。将新西兰白兔脊背部一侧骶棘肌及其筋膜制备肌筋膜组织瓣,采用筋膜衬里方法充填与包裹软骨细胞聚羟基丁酸酯与聚羟基己酸酯共聚物喉支架形态复合物,原位植入。将单纯聚羟基丁酸酯与聚羟基己酸酯共聚物喉支架体内植入的兔作为对照组。分别于术后6,12和18周取材,行大体形态观察、组织学和免疫组化检测评估喉支架形态组织工程化软骨成形与再生情况。结果与结论：制备的聚羟基丁酸酯与聚羟基己酸酯共聚物多孔生物材料塑形物呈中空半面喇叭状,形似喉支架形态,乙醇静态容积测定孔隙率>90%。筋膜衬里的带蒂肌筋膜组织瓣血运丰富,可有效充填与包裹喉支架形态塑形物。不同时间点均获取形态维持良好的喉支架形态组织工程软骨,组织学和免疫组化检测均证实体内植入6周即可形成软骨组织,12周及18软骨组织进一步成熟,而对照组体内植入未检测到软骨组织。结果证实,带蒂肌筋膜组织瓣可保障血运,采用筋膜衬里的肌筋膜组织瓣充填与包裹方法可构建喉支架形态组织工程软骨。 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

聚羟基丁酸/戊酸酯共聚物膜与人骨髓间充质干细胞的体外生物相容性

背景：聚羟基丁酸/戊酸酯共聚物是近年来受到重视的聚羟基脂肪酸族组织工程支架材料,具有免疫排斥反应低、生物相容性好和降解产物无毒副作用的优点。 目的：观察聚羟基丁酸/戊酸酯共聚物膜材料与人骨髓间充质干细胞的体外生物相容性。 方法：将第3代人骨髓间充质干细胞种植于聚羟基丁酸/戊酸酯共聚物膜上作为实验组,培养板单纯培养细胞作为对照组,计算1,2,4 h两组贴壁细胞的数量,得出细胞贴壁率。MTT比色法观察2,4,6,8 d两组细胞的增殖情况。采用Hoechst33258荧光法,检测3,6,9,12 d两组细胞内的DNA含量。将人骨髓间充质干细胞接种聚羟基丁酸/戊酸酯共聚物膜材料上5 d后,电镜扫描观察细胞在材料上的生长情况。 结果与结论：共培养1 h时,实验组的细胞贴壁率低于对照组;其他时间段两组之间细胞贴壁率差异无显著意义。两组各时点间的细胞增殖差异无显著意义。两组各时间点细胞内DNA含量差异无显著意义。扫描电镜观察人骨髓间充质干细胞在聚羟基丁酸/戊酸酯共聚物膜上生长良好,形态呈梭形,细胞间连接紧密,分泌较多细胞基质。证明聚羟基丁酸/戊酸酯共聚物膜材料与人骨髓间充质干细胞有良好的生物相容性。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

可降解聚羟基丁酸酯-羟基戊酸酯的共聚物膜

背景：聚羟基丁酸/羟基戊酸共聚酯已用于构建心脏生物瓣膜,但不知是否可作为骨再生引导膜？ 目的：观察聚羟基丁酸/羟基戊酸共聚酯膜的生物相容性,并评价其成骨能力。 方法：采用MTT法检测100%,75%,50%,25%聚羟基丁酸/羟基戊酸共聚酯材料浸提液对犬骨髓间充质干细胞相对增殖度的影响,并评价其细胞毒性;在犬胫骨缺损模型左右两侧远心端骨缺损上方覆盖聚羟基丁酸/羟基戊酸共聚酯膜作为实验组,在近心端仅以骨膜瓣复位作为对照组。 结果与结论：100%,75%,50%,25%聚羟基丁酸/羟基戊酸共聚酯膜的细胞毒性分级为0~1级,对骨髓间充质干细胞的生长及增殖无毒性;实验组术后第2周即可见新骨形成,12周时骨缺损区已完全被新骨充填,骨修复质量明显优于对照组。表明聚羟基丁酸/羟基戊酸共聚酯膜具有良好的生物相容性,对骨髓间充质干细胞无明显毒性,引导成骨能力强。     

骨髓间充质干细胞-聚乙醇酸支架复合体种植动物皮下构建组织工程化小口径血管

背景：课题组的前期工作已证实骨髓间充质干细胞可以诱导分化为血管内皮细胞和血管平滑肌细胞,并证实所诱导的细胞和胶原包埋的聚乙醇酸支架具有良好的组织相容性。 目的：探讨利用动物皮下作为生物反应器构建小口径组织工程化血管的可行性。 方法：骨髓间充质干细胞诱导分化为血管平滑肌样细胞和血管内皮样细胞,分层种植于胶原包埋聚乙醇酸支架表面,然后将细胞-支架复合体种植于动物皮下,构建小口径组织工程化血管。 结果与结论：人工血管组织学观察见管壁结构清晰,其大体结构和天然血管相似,可承受26.6 kPa的血管腔内压力不破裂。皮下培养8周免疫荧光观察Brdu标记的部分细胞核呈现明亮的黄绿色荧光。结果说明利用动物的皮下作为生物反应器,采用静态培养的方式构建小口径组织工程化血管是可行的。     

羟基丁酸-羟基辛酸聚合物/胶原软骨组织工程支架的细胞亲和性

背景：已有很多实验证明,单独高分子材料或生物性材料制备的组织工程支架无法满足组织工程研究。 目的：评价羟基丁酸-羟基辛酸聚合物/胶原组织工程支架的生物学特性及细胞亲和性。 方法：以羟基丁酸-羟基辛酸聚合物作为主体材料,按质量分数复合不同比例(2%,4%,6%,8%,10%)的胶原,采用溶剂浇铸-颗粒沥滤法制备组织工程支架。通过扫描电镜观察材料内部结构及孔径大小,液体位移法测定材料孔隙率。将羟基丁酸-羟基辛酸聚合物/胶原支架、羟基丁酸-羟基辛酸聚合物支架分别与兔软骨细胞复合培养,MTT法测定细胞的生长曲线,扫描电镜观察细胞在材料上的生长黏附情况。 结果与结论：羟基丁酸-羟基辛酸聚合物/胶原复合软骨组织工程支架孔径大小200 μm左右,孔隙率为(85±2)%,细胞亲水性随加入胶原比例的增加而升高。与羟基丁酸-羟基辛酸聚合物支架比较,不同比例的羟基丁酸-羟基辛酸聚合物/胶原支架可明显促进软骨细胞的黏附、增殖。证实羟基丁酸-羟基辛酸聚合物/胶原复合支架具备更好的细胞亲和性。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

犬骨髓基质干细胞与聚羟基烷酸酯体外相容性的实验研究

目的：评价聚羟基烷酸酯（PHBV）作为组织工程支架与犬骨髓基质干细胞（BMSCs）的生物相容性。方法:原代培养犬BMSCs，传至3-4代后，接种至PHBV膜和泡沫样三维支架上，以接种至培养板上细胞为对照组，倒置显微镜下观察细胞形态；于培养1、2、3周分别采用4%多聚甲醛固定，常规组织切片，HE染色；在5、10、14 d用Hoechst33258荧光法定量测定细胞内DNA含量，BCA法测定蛋白质含量。结果:倒置显微镜观察PHBV 纤维较粗，且透光性差，在相差显微镜下不易观察。第3-4代BMSCs接种至PHBV膜上2 h后即大部黏附，3 d后伸展良好，呈纺锤形或梭形，在三维支架的孔隙内立体生长，1周开始细胞间连接，3周广泛连接，分泌大量基质；培养接种1周后，取二个膜状PHBV固定，HE染色后，见骨髓基质干细胞增殖。培养接种2周后，骨髓基质干细胞增殖明显，呈梭形密布于膜状PHBV上。培养接种3周后，骨髓基质干细胞增殖较第二周无明显变化。定量测定接种的细胞内DNA含量和蛋白质含量与对照组相比无显著差异。结论:PHBV作为BMSCs的组织工程支架材料，具有良好的生物相容性。     

A novel construct as a cell carrier for tissue engineering

A 3D scaffold, in the form of a foam, with the top surface carrying a micropattern, was constructed from biodegradable polyesters poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) and poly(L-lactide-co-D,L-lactide) (P(L/DL)LA) to serve as a substitute for the extracellular matrix (ECM) of tissues with more than one cell type. The construct was tested in vitro for engineering of such tissues using fibroblasts (3T3) and epithelial cells (retinal pigment epithelial cells, D407). The patterned surface was seeded with D407 cells and the foam was seeded with 3T3 cells to represent a tissue with two different cell types. To improve cell adhesion, the construct was treated with fibronectin. The cells were seeded on the construct in a sequence allowing each type time for adhesion. Cell proliferation, studied by MTS assay, was significantly higher than that of tissue culture polystyrene control by day 14. Scanning electron and fluorescence microscopy showed that the foam side of the construct was highly porous and the pores were interconnected and this allowed cell mobility and proliferation. Immunostaining showed collagen deposition, indicating the secretion of the new ECM by the cells. On the film side of the construct D407 cells formed piles in the grooves and covered the surface completely. It was concluded that the 3D P(L/DL)LA-PHBV construct with one micropatterned surface has a serious potential for use as a tissue engineering carrier in the reconstruction of complex tissues with layered organization and different types of cells in each region.     

Engineering of biologically active living heart valve leaflets using human umbilical cord-derived progenitor cells

This study demonstrates the engineering of biologically active heart valve leaflets using prenatally available human umbilical cord-derived progenitor cells as the only cell source. Wharton's Jelly-derived cells and umbilical cord blood-derived endothelial progenitor cells were subsequently seeded on biodegradable scaffolds and cultured in a biomimetic system under biochemical or mechanical stimulation or both. Depending on the stimulation, leaflets showed mature layered tissue formation with functional endothelia and extracellular matrix production comparable with that of native tissues. This demonstrates the feasibility of heart valve leaflet fabrication from prenatal umbilical cord-derived progenitor cells as a further step in overcoming the lack of living autologous replacements with growth and regeneration potential for the repair of congenital malformation.     

Bone generation on PHBV matrices: an in vitro study

Bone formation was investigated in vitro by culturing rat marrow stromal osteoblasts in biodegradable, macroporous poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) matrices over a period of 60 days. Foams were prepared after solvent evaporation and solute leaching. PHBV solutions with different concentrations were prepared in chloroform: dichloromethane (1:2, v/v). In order to create a matrix with high porosity and uniform pore sizes, sieved sucrose crystals (300-500 microm) were used. PHBV foams were treated with rf-oxygen plasma (100 W 10 min) to modify their surface chemistry and hydrophilicity with the aim of increasing the reattachment of osteoblasts. Osteoblasts were isolated from rat bone marrow and seeded onto PHBV foams. The cell density on and in the foams was determined with MTS assay. MTS results showed that osteoblasts proliferated on PHBV. Twenty-one days after seeding of incubation, growth of osteoblasts on matrices and initiation of mineralization were observed by confocal laser scanning microscopy. Increasing ALP and osteocalcin secretion during 60 days confirmed the osteoblastic phenotype of the derived stromal cells. SEM, histological evaluations and confocal laser scanning microscopy showed that osteoblasts could grow inside the matrices and lead to mineralization. Cells exhibited spindle-like morphology and had a diameter of 10-30 microm. Based on these, it could confidently be stated that PHBV seems to be a promising polymeric matrix material for bone tissue engineering.     

Structural alterations of adhesion mediating components in cells cultured on poly-beta-hydroxy butyric acid

Polymers may serve as a biodegradable material in tissue engineering. To assess the biocompatibility of poly-beta-hydroxy butyric acid (PHB), we studied the structural organization of cellular molecules involved in adhesion using osteoblastic and epithelial cell lines. On PHB, both cell lines revealed a rounded cell shape due to reduced spreading. The filamentous organization of the actin cytoskeleton was impaired. In double immunofluorescence analyses we demostrated that the colocalization of the fibronectin fibrils with the actin filaments was lost in cultures on PHB. Similarly, collagen II distribution was altered, whereas the organization of collagen I was not obviously affected. Further evidence for impaired structural organization was obtained for the beta1-integrin receptor and vinculin which mediate the interaction of the cytoskeleton with the extracellular matrix. In confluent epithelial cells, the tight junction protein ZO-1 showed a larger lateral extension in the cell-cell contacts when cells were grown on PHB. Because structural organization of components which mediate cell-matrix and cell-cell adhesion controls cell physiology these parameters could be a sensitive indicator for the biocompatibility of implant materials.     

热致相分离法制备聚羟基丁酸酯-羟基戊酸酯纳米纤维支架及其表征***

背景：由微生物合成的聚羟基丁酸酯-羟基戊酸酯(polyhydroxybutyrate-hydroxyvalerate,PHBV)是聚羟基脂肪酸酯的一种,具有良好的生物相容性和机械强度。目的：探讨热致相分离法制备PHBV纳米纤维支架的方法及结晶行为。方法：采用扫描电镜、广角X射线衍射、红外光谱和差示扫描量热分析分析基质的结构。结果与结论：凝胶温度对纳米纤维的结晶和热性质有很大的影响。当凝胶温度较高时,PHBV纳米纤维的结晶度和晶粒尺寸随着凝胶温度的降低而减小,而且随着凝胶温度的降低,其结晶的有序性增加。说明温度对PHBV支架形貌和结构的影响可能对PHBV支架的性质-包括生物降解性和对细胞活性的生物应答反应有一定的积极意义。关键词：聚羟基丁酸酯-羟基戊酸酯;纳米纤维;基质;支架;生物相容性doi:10.3969/j.issn.1673-8225.2012.03.002     

Improvement of the cytocompatibility of electrospun poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] mats by Ecoflex

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] (PHBV) is a nature-derived polyester with potential application in tissue engineering scaffolds. However, PHBV is associated with disadvantages including high brittleness, slow degradation, high hydrophobicity, and unsatisfactory biocompatibility. In this study, we sought to improve the properties of PHBV by blending it with Ecoflex, a synthetic biopolyester with a high flexibility, fast degradation, and comparatively higher hydrophilicity. PHBV was codissolved with Ecoflex in dichloromethane at different mass ratios (PHBV/Ecoflex: 100/0, 70/30, 50/50, and 30/70) and electrospun into mats. Compared with the pure PHBV mat, the Ecoflex-containing mats showed decreased contact angles with phosphate-buffered saline (PBS), accelerated weight loss in PBS, and increased strain at break with increasing Ecoflex mass ratios. In vitro cell culture also showed significantly improved adhesion and proliferation of human bone marrow stroma cells with the introduction of Ecoflex. Blending PHBV with Ecoflex is a simple and effective method to improve the chemical, mechanical, and biological properties of PHBV simultaneously and thereby to expedite its application in tissue engineering. To our knowledge, this is the first report showing the biocompatibility of Ecoflex-containing materials with human cells.     

不同类型生物材料的心脏瓣膜应用及安全性评价

背景:组织工程心脏瓣膜是应用工程学和生命科学的原理和方法构建具有生理功能和生物活性的瓣膜替代物,但仍处于动物实验阶段。 目的：总结常用的组织工程心脏瓣膜,对不同类型生物材料的心脏瓣膜应用的安全性进行评价。 方法：以“生物材料,心脏瓣膜,支架材料,综述文献,组织工程”为中文关键词,采用计算机检索2000-01/2010-12相关文章。纳入与生物材料与组织工程心脏瓣膜研究相关的文章;排除重复研究或Meta分析类文章。 结果与结论：共纳入生物材料与组织工程心脏瓣膜研究相关文献20篇。天然支架材料因其优越的生物相容性和三维空间构象,具有其他材料不可比拟的仿生性。合成可降解高分子材料具有良好的可控性和力学性能也备受研究者青睐,而将天然材料和高分子材料融合一体构建的复合支架材料为组织工程心脏瓣膜的研究提供了新的策略和方向,具有广阔的应用前景。     

A novel construct as a cell carrier for tissue engineering

A 3D scaffold, in the form of a foam, with the top surface carrying a micropattern, was constructed from biodegradable polyesters poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) and poly(L-lactide-co-D,L-lactide) (P(L/DL)LA) to serve as a substitute for the extracellular matrix (ECM) of tissues with more than one cell type. The construct was tested in vitro for engineering of such tissues using fibroblasts (3T3) and epithelial cells (retinal pigment epithelial cells, D407). The patterned surface was seeded with D407 cells and the foam was seeded with 3T3 cells to represent a tissue with two different cell types. To improve cell adhesion, the construct was treated with fibronectin. The cells were seeded on the construct in a sequence allowing each type time for adhesion. Cell proliferation, studied by MTS assay, was significantly higher than that of tissue culture polystyrene control by day 14. Scanning electron and fluorescence microscopy showed that the foam side of the construct was highly porous and the pores were interconnected and this allowed cell mobility and proliferation. Immunostaining showed collagen deposition, indicating the secretion of the new ECM by the cells. On the film side of the construct D407 cells formed piles in the grooves and covered the surface completely. It was concluded that the 3D P(L/DL)LA-PHBV construct with one micropatterned surface has a serious potential for use as a tissue engineering carrier in the reconstruction of complex tissues with layered organization and different types of cells in each region.