大鼠脂肪干细胞在纳米羟基磷灰石-壳聚糖-果胶/壳聚糖-明胶支架的成骨向分化研究

目的 探索纳米羟基磷灰石-壳聚糖-果胶/壳聚糖-明胶（nHCP/CG）支架对大鼠脂肪干细胞（r ADSC）定向成骨分化的影响，为其临床应用提供理论支持。方法 选择SD成年大鼠5只，体质量60～80 g，雌雄不限。从SD成年大鼠两侧腹股沟脂肪组织原代分离、培养并鉴定r ADSC，然后将其接种在nHCP/CG支架材料上，并以壳聚糖-明胶（CG）支架为对照。通过四甲基偶氮唑盐（MTT）法、Live/Dead及苏木精-伊红（HE）染色法评价支架的细胞相容性；在添加和不添加成骨诱导剂培养条件下，以碱性磷酸酶（ALP）活性及多种免疫组织化学染色测定r ADSC在nHCP/CG支架中的成骨分化行为。结果 r ADSC具有间充质干细胞的特性，其在nHCP/CG复合支架材料上活性较好。nHCP/CG支架较CG支架更利于r ADSC的黏附和增殖；r ADSC在nHCP/CG支架中培养1 d、3 d、7 d时细胞数量明显高于CG支架中的细胞数量（0.432±0.028 vs 0.174±0.004、0.532±0.017 vs 0.291±0.023、0.595±0.014 vs 0.754±0.038）且...     

壳聚糖-透明质酸共混膜对兔角膜基质细胞生长的影响

观察各种壳聚糖-透明质酸共混膜对角膜基质细胞生长的影响作用,研究透明质酸混入比例对共混膜与细胞相容性的影响.以共混膜为载体培养兔角膜基质细胞,通过光学和电子显微镜,观察细胞在膜上的生长情况;通过MTT法,检测细胞在共混膜上的贴附率和生长活性;通过检测培养基中乳酸脱氢酶的活性,预示壳聚糖-透明质酸共混膜与角膜基质细胞的相容性.以低于1:0.1的比例混入透明质酸.可以提高细胞在共混膜上的贴附率、生长速度,细胞在共混膜上的生长状态好于在壳聚糖膜上的生长状态.结果提示,以低于1:0.1的比例混入透明质酸,可以提高壳聚糖膜与角膜基质细胞的相容性,促进细胞生长;而以高于1:0.1的比例混入透明质酸,则不利于细胞在共混膜上的生长,降低了壳聚糖膜与角膜基质细胞的相容性.     

构建缓慢释放碱性成纤维细胞生长因子的脱细胞羊膜人工活性真皮**☆

背景：目前人工皮肤替代品的种类较多,各有优缺点,仍然没有一种理想的产品应用于临床。 目的：探讨构建一种可以缓慢释放碱性成纤维细胞生长因子的新型人工活性真皮的可行性。 方法：组织块法培养幼儿包皮成纤维细胞;采用酶-去垢剂法制备人脱细胞羊膜;双相法制备碱性成纤维细胞生长因子-明胶-壳聚糖缓释微球;缓释微球黏附于脱细胞羊膜上;三四代成纤维细胞培养于负载缓释微球的脱细胞羊膜上。 结果与结论：制备的脱细胞羊膜为白色半透明状薄膜有较高的孔隙率,空隙不规则,孔径大小为10~100 nm,无细胞毒性;碱性成纤维细胞生长因子-明胶-壳聚糖缓释微球分散较均匀,呈球形,粒径均匀,球体表面比较光滑,载药率为20 ng/g,包封率为80.5%,体外药物缓释曲线显示药物控释效果良好;成纤维细胞在支架表面爬行生长良好,层粘连蛋白表达较对照组高。表明将成纤维细胞种植于负载碱性成纤维细胞生长因子-明胶-壳聚糖缓释微球的脱细胞羊膜上,缓释微球能较好地黏附于脱细胞羊膜表面。     

壳聚糖-明胶-透明质酸薄膜的制备及其性能的研究

本实验采用表面修饰法和共混法两种不同的方法将透明质酸引入壳聚糖-明胶体系，制备了壳聚糖-明胶-透明质酸复合薄膜，考察了薄膜的表面形貌、亲水性和力学性能等理化性能以及生物相容性。实验结果表明，壳聚糖-明胶-透明质酸薄膜具有良好的亲水性，而共混法和表面修饰法制备的薄膜在力学性能上各有所长。体外细胞培养结果表明，成纤维细胞在共混法制备的薄膜上的黏附和生长情况要明显好于表面修饰法制备的薄膜。     

血管组织工程支架材料的细胞相容性

背景：胶原与透明质酸均有利于组织培养中细胞的黏附、增殖和分化。 目的：观察血管内皮细胞、平滑肌细胞与胶原/透明质酸膜、明胶海绵的细胞相容性,并筛选最佳种植方法。 方法：将第3-5代兔血管平滑肌细胞种植在胶原/透明质酸膜(或明胶海绵)材料上,连续培养2周后将兔内皮细胞接种在平滑肌细胞-胶原/透明质酸膜(或明胶海绵)复合体上,并设置单纯平滑肌细胞与内皮细胞共同接种组。 结果与结论：①光镜和扫描电镜观察：细胞在两种材料上均随着培养时间,接种次数增加而生长加快,其中在胶原/透明质酸膜上的细胞生长更好,细胞连接更致密。②WST-1法检测：胶原/透明质酸膜组平滑肌细胞的黏附率及增殖率均高于明胶海绵组(P < 0.05),且细胞在材料上的生长随着接种次数的增加有不同程度提高。③³H-TDR掺入法检测DNA合成率：在胶原/透明质酸膜上的细胞DNA合成最高,明胶海绵上的较差。表明胶原/透明质酸膜具有较理想的细胞相容性,采用适当间隔、反复接种的方法可提高细胞的黏附和增殖。     

不同接种方式对组织工程牙龈种子细胞黏附效果的影响

背景：如何在体外将支架材料和种子细胞高效地复合以构建组织工程牙周组织是目前牙周病治疗及牙周缺损修复研究的重要方向。 目的：比较传统沉淀接种法和胶原包裹接种法的细胞黏附状况,优化细胞接种方式。 方法：将一定浓度的犬牙龈成纤维细胞,分别采用传统沉淀接种法和胶原包裹接种法接种到聚乳酸-壳聚糖-明胶梯度孔径和均匀孔径支架上,通过细胞计数测定支架上贴附的细胞数量,计算其接种率,并进行对比分析。 结果与结论：采用胶原凝胶包裹接种法将细胞接种至均匀孔径支架和梯度孔径支架上,其接种率均明显高于传统的沉淀接种法(P < 0.01)。用胶原凝胶包裹种子细胞行细胞接种可以有效提高种子细胞的接种率,增加支架上的细胞初始浓度,可以选用胶原凝胶包裹细胞接种方式用于牙龈工程组织构建。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：     

以壳聚糖-胶原共混膜为三维支架同种异体工程化软骨的构建

目的探讨以壳聚糖-胶原共混膜为三维支架材料的同种异体软骨细胞构建组织工程化软骨的能力。方法将分离、培养、扩传兔软骨细胞，接种在壳聚糖-胶原共混膜上，倒置显微镜下观察细胞在共混膜上的生长情况。体外培养7d后，将细胞-材料复合物种植在新西兰兔皮下，6周取材，对获得的同种异体工程化软骨进行组织学评价。结果兔软骨细胞接种于壳聚糖-胶原共混膜上4h后有贴壁现象出现，细胞呈梭形。培养48h后，软骨细胞分裂增殖越来越多并向周围延伸，培养第7天取材，HE染色示细胞生长良好，呈梭形。体内培养6周取材，HE染色、Masson染色为均一的成熟软骨组织，且共混膜已降解。结论以壳聚糖-胶原共混膜为支架材料同种异体软骨细胞在有免疫力的动物体内可形成工程化软骨。     

角膜修复用改性明胶交联膜的生物相容性评价

本文对自制的改性壳聚糖-明胶交联膜(MC-Gel)与壳聚糖-明胶交联膜(CS-Gel)的理化性能、生物相容性进行测定与评价,探讨其用于角膜组织修复与重建的可行性。采用紫外分光光度计测定了交联膜的透光率及渗透性,结果显示两组交联膜的透光率超过90%,且对葡萄糖、色氨酸与NaCl的渗透率均接近或优于人角膜的渗透率,可满足角膜修复对透光率及营养物质透过的要求。同时,采用MTT法测定了人角膜上皮细胞(HCEC)在两组膜上的生长活性,并评价了两组交联膜在兔眼角膜内的组织相容性。实验结果表明,MC-Gel能较好地支持HCEC在其上增殖,且植入兔眼角膜后无明显炎症反应,术后4个月材料全部降解。因此,该交联膜具有良好的理化性能与眼组织相容性,有望成为一种良好的角膜修复材料。     

神经干细胞在PLGA、壳聚糖和明胶膜表面的生长规律

目的探索神经干细胞在PLGA、壳聚糖和明胶膜表面的生长规律,寻找适合中枢神经再生的支架材料.方法大鼠胚胎神经干细胞以105/mL密度分别种植于PLGA、壳聚糖和明胶膜表面,观察神经干细胞的生长、分化情况.结果接种后12h神经干细胞在壳聚糖膜表面呈团簇状贴附,在明胶膜表面24h贴附牢固;第2d起明胶膜表面贴附的神经干细胞周围见有突起的分化细胞;第3d壳聚糖膜表面贴附的神经干细胞周围也见有突起的分化细胞,壳聚糖膜部分溶解,明胶膜完全溶解,第5d分化细胞长满膜表面;观察期内PLGA膜表面无神经干细胞贴附,PLGA膜保持完整.结论神经干细胞不能在PLGA膜表面贴附和生长,在壳聚糖和明胶膜表面贴附和分化良好,壳聚糖比明胶更利于神经干细胞贴附,明胶比壳聚糖更利于神经干细胞分化.PLGA、壳聚糖和明胶都不宜单独作为生物支架材料和神经干细胞共同构建中枢神经工程化组织.     

表面修饰对羟基磷灰石修复骨缺损的影响

精氨酸-甘氨酸-天冬氨酸（Arg-Gly-Asp,RGD）多肽是介导种子细胞与支架材料黏附的多肽链．RGD序列可被固有黏附蛋白受体特异性结合．在生物材料表面自发形成一分子层．为与受体介导的种子细胞提供特异性位点而促进细胞的黏附和分化。笔者旨在应用RGD多肽对羟基磷灰石（hydroxyapatite．HA）材料进行表面修饰处理．以促进骨髓基质干细胞（marrow stromal cells．MSC）在其表面的黏附和生长．为骨组织工程提供一种支架材料的表面修饰手段。     

Modulation of mesenchymal stem cells behaviors by chitosan/gelatin/pectin network films

In this article, the chitosan/gelatin/pectin (CGP) network films were prepared to build appropriate physicochemical and mechanical microenvironment for attachment, proliferation, and differentiation of mesenchymal stem cells (MSCs). Results suggested that the hydrophilicity and mechanical character of CGP composites films could be modulated via adjusting the pectin content in the composites. The investigations of attachment and proliferation behaviors of mesenchymal stem cells (MSCs) on the CGP films were carried out. The morphology of cells was observed with hematoxylin/eosin staining (HE) and scanning electron microscope (SEM). The osteogenic differentiation of MSCs was investigated via ALP and polymerase chain reaction (PCR). Results suggested that the CGP films have excellent biocompatibility. MSCs seeded on CGP (0.1) film show higher proliferation capacity compared with other samples. Moreover, osteogenic differentiation of MSCs also depends on the properties of the substrate. The MSCs seeded on CGP (0.5) expressed the highest ALP activity, osteogenic gene expression and mineral formation capacity. These results suggest that the composition of the CGP network films could effectively modulate their physicochemical and mechanical properties and further regulate the cell behaviors of MSCs.     

Improved osteogenic differentiation of human marrow stromal cells cultured on ion-induced chemically structured poly-epsilon-caprolactone

The ability to control cell proliferation/differentiation, using material surface, is a main goal in tissue engineering. The objective of this study was to evaluate the attachment, proliferation and differentiation to the osteoblastic phenotype of human marrow stromal cells (MSC) when seeded on poly-epsilon-caprolactone (PCL) thin films before and after irradiation with 10 keV He+. The polymeric surface was characterized as surface chemical structure and composition, roughness and morphology on the micro- and nano-scale, wettability and surface free energy parameters. MSC were obtained from patients undergoing routine hip replacement surgery, expanded in vitro and cultured on untreated PCL and He+ irradiated PCL films for up to 4-5 weeks in osteogenic medium. He+-irradiation led to slight smoothening of the surface and different nanoscale surface chemical structure, while surface free energy resulted unchanged in comparison to untreated PCL. The results from biological testing demonstrated that early attachment and further proliferation, as well as osteoblastic markers, were higher for MSC on He+-irradiated PCL. In conclusion, the change of PCL surface properties induced by ion beam irradiation is confirmed to enhance the adhesion of MSC and support their differentiation.     

新型壳聚糖基仿生网络复合膜诱导骨髓间充质干细胞定向成骨分化

目的 探讨新型壳聚糖基网络复合膜诱导骨髓间充质干细胞（MSCs）定向成骨分化的可行性.方法 采用仿生学方法,壳聚糖、明胶、果胶按照一定比例制作成新型壳聚糖基仿生网络复合膜.设计4个组：实验组1（复合膜＋常规培养基）,对照组1（常规培养基）,实验组2（复合膜＋成骨诱导（OS）培养基）,对照组2（OS培养基）.通过倒置相差显微镜、四甲基偶氮唑盐（MTT）法、扫描电镜（SEM）检测细胞在复合膜上的生长和增殖情况;通过测定碱性磷酸酶（ALP）活性来评价MSCs在复合膜上的成骨分化能力;通过特殊染色和能谱分析（EDX）来评定钙盐的沉积.结果 MSCs在网络复合膜上贴附、生长良好且增殖旺盛.MTr法检测细胞活力显示实验组吸光度（OD）值与对照组比较无统计学意义（P＞0.05）.SEM观察到细胞在支架材料表面呈聚集生长,分泌大量的细胞外基质,可见散在的结节形成.实验组1的ALP活性明显增高,与实验组2、对照组比较有统计学意义（P＜0.01）.茜素红和Von Kossa染色可见实验组细胞分化后形成的钙化结节;ALP染色可见胞浆内蓝染颗粒;EDX检测到Ca、P沉积.结论 新型壳聚糖基网络复合材料具有良好的生物相容性,在不添加诱导剂的条件下,可以诱导MSCs定向成骨分化.     

Stem cell attachment to layer-by-layer assembled TiO2 nanoparticle thin films

Surface topography is one of the most important factors influencing the attachment and spreading of cells. In the present study, layer-by-layer assembled titanium dioxide (TiO2) nanoparticle thin films were chosen for attachment, proliferation and spreading studies on mouse mesenchymal stem cells (MSC). Increasing surface roughness was observed with increasing number of layer-by-layer assembled TiO2 thin films. Four layer TiO2 thin film showed higher number of attached cells than a one layer thin film and control surfaces. MSCs experienced no cytotoxic effects after culture on the TiO2 coated substrates as observed from the cytotoxicity tests. Cell spreading, visualized with scanning electron microscopy, showed a faster rate of spreading on a rougher surface. Cells on a four-layer substrate, at 12 h showed complete spreading, where as most of the cells on a control surface and a one-layer surface, at 24 h, retained a rounded morphology. In conclusion, TiO2 nanoparticle thin films were successfully assembled in alternation with polyelectrolytes and in-vitro studies with MSC showed an increase in the attachment and faster spreading of cells on rougher surfaces.     

Osteogenic differentiation of mesenchymal stem cells in self-assembled peptide-amphiphile nanofibers

The proliferation and differentiation of mesenchymal stem cells (MSC) was investigated in a three dimensional (3-D) network of nanofibers formed by self-assembly of peptide-amphiphile (PA) molecules. PA was synthesized by standard solid phase chemistry that ends with the alkylation of the NH(2) terminus of the peptide. The sequence of arginine-glycine-aspartic acid (RGD) was included in peptide design as well. A 3-D network of nanofibers was formed by mixing cell suspensions in media with dilute aqueous solution of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. When rat MSC were seeded into the PA nanofibers with or without RGD, larger number of cells attached was observed in the PA nanofibers including RGD. When measured to evaluate the osteogenic differentiation of MSC, the alkaline phosphatase (ALP) activity and osteocalcin content became maximum for the PA nanofibers including RGD compared with those without RGD, although both the values were significantly higher compared with those in the static tissue culture plate (2-D culture). We concluded that the attachment, proliferation, and osteogenic differentiation of MSC were influenced by PA nanofibers as the cell scaffold.     

Attachment, spreading and short-term proliferation of human osteoblastic cells cultured on chitosan films with different degrees of acetylation

Chitosan (Ch) is being actively investigated as a non-protein template for the growth of an increasing number of anchorage-dependent cells, including chondrocytes and bone cells. In the present work, Ch films with degrees of N-acetylation (DAs) in the range of 4 to 49% were evaluated with respect to the attachment, spreading and short-term proliferation of osteoblasts, using human osteoblastic MG-63 cells. The films were characterized in terms of surface morphology and surface charge by atomic force microscopy and streaming potential measurements, respectively. Cell attachment was assessed after 3 and 24 h of cell culture. After 24 h of incubation, cell attachment was found to be dependent on the DA, lower DAs favouring cell adhesion. With time, cell spreading and cytoskeleton organization were only attained for DAs 相似文献   

Attachment,spreading and short-term proliferation of human osteoblastic cells cultured on chitosan films with different degrees of acetylation

Chitosan (Ch) is being actively investigated as a non-protein template for the growth of an increasing number of anchorage-dependent cells, including chondrocytes and bone cells. In the present work, Ch films with degrees of N-acetylation (DAs) in the range of 4 to 49% were evaluated with respect to the attachment, spreading and short-term proliferation of osteoblasts, using human osteoblastic MG-63 cells. The films were characterized in terms of surface morphology and surface charge by atomic force microscopy and streaming potential measurements, respectively. Cell attachment was assessed after 3 and 24 h of cell culture. After 24 h of incubation, cell attachment was found to be dependent on the DA, lower DAs favouring cell adhesion. With time, cell spreading and cytoskeleton organization were only attained for DAs ≤ 13%. Regarding cell proliferation, cells grown on films with the lowest DA (4%) revealed a higher specific growth rate as compared to those grown on films with higher DAs. Films with a DA of 49% failed to sustain cell proliferation. In addition, a longer lag-phase was observed on Ch, as compared to TCPS, in accordance to an observed delay of cell spreading. The present findings revealed that differences in the DA as small as 9% may be critical in terms of the osteoblast response to two-dimensional Ch-based matrices.     

微弧氧化陶瓷膜表面骨髓间充质干细胞生物相容性实验

目的观察微弧氧化（micro-arc,MAO）陶瓷膜表面骨髓间充质干细胞（mesenchymal stem cell,MSC）的黏附和增殖生长情况,通过MSC在三组不同表面处理材料表面的黏附和增殖情况评价微弧氧化陶瓷膜表面的生物相容性。方法贴壁法培养大鼠骨髓间充质干细胞,骨诱导培养后,做碱性磷酸酶染色和茜素红钙结节染色,鉴定其成骨潜能。取生长良好的第三代MSC,调整细胞密度为5×10^4/mL,接种到三组钛片表面。在接种细胞后第1、3、5、7天每组分别取出5枚钛片,一个做电镜扫描,另外4个细胞计数。观察MSC在不同材料表面的黏附增殖情况。电镜扫描MAO陶瓷膜表面形貌特征,EDX分析其表面主要元素含量。结果MSC具有良好的成骨特性,在MAO陶瓷膜表面的增殖优于光滑组和喷沙组。电镜下微弧氧化陶瓷膜表面有无数2～10m的微孔,并含有钙、磷成分。结论MAO陶瓷膜具有良好的生物相容性,大鼠骨髓间充质干细胞在其多孔,含有钙、磷元素表面的黏附及增殖均优于其它组。     

High-efficiency cell seeding method by relatively hydrophobic culture strategy

Cell adhesion efficiency is one of the key factors affecting the results of manufacturing tissue engineering constructs. High efficiency is required for seeding low proliferation cells onto scaffolds. In this study, we designed a strategy to improve the efficiency of cell adhesion using hydrophobic cell culture environment to enhance cells adhering to a scaffold. Cells have lower affinity to the surface of polydimethylsiloxane (PDMS) than tissue culture polystyrene (TCPS) plates. When cells were cultured with gelatin microspheres or chitosan films in a PDMS-coated plate instead of a normal TCPS plate, there was a significant increase in cell attachment efficiency. Cells cultured in the PDMS-coated system tended to selectively attach onto the gelatin microspheres or chitosan films, which are relatively more hydrophilic than the PDMS surface. However, minimal cell attachment on gelatin microspheres or chitosan films was observed when gelatin microspheres or chitosan films were placed in normal TCPS plate. Cell counting experiments with gelatin microspheres in the PDMS-coated system resulted in a cell attachment efficiency of 89.8% after 1 day of cultivation, whereas the cell attachment efficiency was less than 1% in normal TCPS plate. The results demonstrate that the method is easy to use and could be useful for fast cultivation of cell-scaffold constructs.     

骨组织工程支架材料表面修饰的试验研究

观察骨髓基质细胞 (BMSC)在吸附了多聚赖氨酸 (Poly -L -Lysine,PLL)的羟基磷灰石 (HA)上的生长情况。探讨改进材料表面活性 ,促进种子细胞黏附和生长的方法。利用组织工程学的方法 ,将体外培养的BMSC种植于吸附了多聚赖氨酸的HA上 ,立体培养一周 ,用环境扫描电镜和倒置相差显微镜观察细胞的生长情况。结果表明 ,细胞在体外可以立体培养成活 ,吸附了多聚赖氨酸的HA上细胞较多。多聚赖氨酸可以改善HA的表面活性 ,促进细胞的黏附和生长。