多巴胺表面修饰胶原膜促进细胞粘附和增殖的研究

多巴胺已经被广泛地用于材料的表面修饰改性,能够提高材料的生物相容性,赋予材料新的反应活性。为了考察多巴胺表面修饰胶原膜对其机械强度、湿热稳定性、亲水性和生物相容性的影响,对多巴胺自组装表面修饰胶原膜不同时间形成的膜材料进行研究,结果发现,经过多巴胺自组装表面修饰后,胶原保持完整的三股螺旋结构,膜材料的机械强度、湿热稳定性和亲水性均得到提高,而且成纤维细胞更易于在膜上粘附和增殖。     

成纤维细胞和血小板在多壁碳纳米管上粘附性

使用化学气相沉积(CVD)的方法在碳纸基底上制备了多壁碳纳米管(MWCNTs),并研究了L929小鼠成纤维细胞在多壁碳纳米管和碳纸对照组上的粘附及增殖等生长行为,以及各种血液蛋白吸附于这两种材料表面后对人皮肤成纤维细胞粘附产生的影响,同时对比了这两种材料的血小板粘附情况.结果表明:种植在多壁碳纳米管上的成纤维细胞生长明显比碳纸上的旺盛,细胞浓度从第1天的12.5×10~5/mL明显增加到第7天的4.1×10~5/mL,多壁碳纳米管对细胞无毒性反应.预吸附白蛋白、纤维蛋白原、免疫球蛋白对细胞粘附量有促进作用,并且多壁碳纳米管的血小板粘附率低于碳纸.这些结果证明多壁碳纳米管具有良好的组织相容性和一定的血液相容性.     

多巴胺表面修饰胶原膜促进细胞粘附和增殖的研究

多巴胺已经被广泛地用于材料的表面修饰改性,能够提高材料的生物相容性,赋予材料新的反应活性.为了考察多巴胺表面修饰胶原膜对其机械强度、湿热稳定性、亲水性和生物相容性的影响,对多巴胺自组装表面修饰胶原膜不同时间形成的膜材料进行研究,结果发现,经过多巴胺自组装表面修饰后,胶原保持完整的三股螺旋结构,膜材料的机械强度、湿热稳定性和亲水性均得到提高,而且成纤维细胞更易于在膜上粘附和增殖.     

胶原/海藻酸钙互穿网络水凝胶的构建及其对细胞行为的影响

利用溶液共混法制备了胶原/海藻酸钠混合液,建立了制备胶原/海藻酸钙互穿网络水凝胶的方法,并证实互穿网络凝胶中,胶原与海藻酸钙二者共存且相互独立,胶原结构保持完好。与传统海藻酸钙水凝胶相比,本文建立的胶原/海藻酸钙互穿网络水凝胶表面疏水性增强且凝胶结构更加疏松,更利于细胞粘附与物质传递。以人源成纤维细胞为模型,通过考察细胞在凝胶表面与凝胶内部的形态,进一步证实胶原分子在胶原/海藻酸钙互穿网络水凝胶的表面与内部均有分布。细胞迁移实验结果也表明该胶原/海藻酸钙互穿网络水凝胶不仅能有效保持细胞活性,且为细胞迁移行为的研究提供了一种新的研究模型。     

Ⅰ胶原/岩藻聚糖硫酸酯复合支架的制备及性能

以I型胶原、岩藻聚糖硫酸酯为主要原料,采用0.04~0.06M醋酸溶解的Ⅰ胶原、岩藻聚糖硫酸酯的水溶液通过共混的方法制备I型胶原、岩藻聚糖硫酸酯混合溶液,经过冷冻干燥制备复合支架材料。用SEM对复合支架进行性能表征,测定了复合支架的结构、吸水率、体外降解性能,还做了复合支架材料的细胞相容性实验,主要是细胞毒性检测,以及与成纤维细胞复合共培养,检测细胞在材料上的生长情况。结果表明:岩藻聚糖硫酸酯-胶原共混支架材料具有一定孔径的网状结构,有良好的生物相容性,细胞能粘附在材料上生长。它作为一种潜在的生物材料可望在生物医学领域得到应用。     

等离子体处理偶联胶原提高聚乳酸材料的细胞相容性

本研究通过结合氨气等离子体处理以及胶原改性的方法来改善聚乳酸材料的细胞相容性。水接触角分析表明氨气等离子体处理可以改善聚乳酸材料的亲水性,促进细胞的粘附。光学显微镜以及扫描电镜的分析结果表明,氨气等离子体处理后通过胶原改性可以进一步促进细胞在聚乳酸材料表面的生长。并且,与涂覆胶原相比,偶联胶原可以更好地使胶原蛋白固定在聚乳酸材料表面,从而表现出更好的细胞相容性。     

胶原分子酰化修饰对其结构与性能的影响

在碱性条件下,采用琥珀酸酐(SA)、马来酸酐(MA)、邻苯二甲酸酐(PA)对提取的酶溶性胶原进行分子修饰,并通过总碳含量、等电点、红外、X射线衍射、圆二色谱、超灵敏差示扫描量热以及体外成纤等方法对酰化前后胶原的结构与性能进行了表征。结果表明,酸酐改性后的胶原侧链羧基含量明显增加,等电点显著下降,且酰化后的胶原仍保有特殊的三股螺旋构象;不同修饰程度的琥珀酰化胶原的红外、圆二色谱表明,胶原的酰化程度越高,其三股螺旋构象的完整性越差。与PA改性胶原相比,SA、MA酰化胶原有着更完整的三股螺旋构象和更高的热变性温度。体外成纤实验显示,酰化胶原不具备生理条件下的成纤能力,表明三股螺旋结构是胶原成纤的必要不充分条件。     

胶原、壳聚糖及其共混膜对红细胞的影响

利用原子力显微镜高分辨、低损伤的成像能力,研究比较了壳聚糖膜(CSF)、胶原膜(COF)、胶原/壳聚糖共混膜(COF/CSF)对红细胞的影响。结果显示,壳聚糖膜对红细胞的粘附、聚集作用明显,导致红细胞形态异常甚至碎裂。胶原膜上的红细胞外形不规则,但细胞无碎裂。共混膜对红细胞有一定的粘附作用,对细胞形态的影响程度界于两种单组分膜之间。     

海藻酸钠水凝胶中粘附位点及空间构建促进MG-63细胞铺展及成骨分化

细胞粘附与铺展是三维水凝胶基质中贴壁依赖型细胞存活所必须的两个条件,将细胞粘附位点的引入和凝胶中细胞铺展空间的构建相结合,提出了同时含有RGD多肽和明胶微球的粘附型大孔水凝胶模型,以促进细胞在其中的铺展与分化。该模型采用光交联海藻酸钠水凝胶为基础,同时引入RGD多肽和明胶微球,通过RGD多肽的共价接枝为细胞粘附提供前提,利用明胶微球在37℃下的快速降解性,为细胞的进一步增殖和铺展以及分化提供所需空间。结果显示,明胶微球的加入提高了凝胶的力学性能,同时降低了凝胶的溶胀率。RGD和明胶微球的引入能够很好地支持MG-63细胞在其中的增殖、粘附与铺展,并显著提高其碱性磷酸酶活性,上调成骨相关基因(BMP-2,COL-I和OCN)的表达。而在不含微球的RGD-ALG和ALG凝胶中,细胞铺展及成骨分化均受到很大抑制。     

热变性对鱼胶原海绵材料性能的影响

以草鱼皮为原料提取天然胶原蛋白,经DSC、圆二色谱和SDS-PAGE分析证实为具有完整三螺旋分子结构的Ⅰ型胶原。在不同条件下对该胶原进行热处理后分别获得了低变性、高变性和完全变性的胶原样品,热变性焓测定结果表明热变性程度分别为16.9%,57.9%和100%。在此基础上,重点研究了热变性处理后胶原结构和胶原海绵材料性能的变化。结果表明,草鱼皮胶原变性的起始温度(To,33℃)是其分子结构和胶原海绵材料性能改变的阀值温度。高于该温度条件下的短暂受热历史即可导致胶原三螺旋结构的部分或完全解离。同时,胶原变性程度的增加,材料结构的有序性、材料力学性能和耐酶解能力也随之降低,但是促细胞增殖能力有所提升。可见,低于胶原热变性的起始温度是天然胶原提取和胶原材料应用的安全温度条件。     

Cells behaviors and genotoxicity on topological surface

To investigate different cells behaviors and genotoxicity, which were driven by specific microenvironments, three patterned surfaces (pillars, wide grooves and narrow grooves) and one smooth surface were prepared by template-based technique. Vinculin is a membrane-cytoskeletal protein in focal adhesion plaques and associates with cell–cell and cell–matrix junctions, which can promote cell adhesion and spreading. The immunofluorescence staining of vinculin revealed that the narrow grooves patterned substrate was favorable for L929 cell adhesion. For cell multiplication, the narrow grooves surface was fitted for the proliferation of L929, L02 and MSC cells, the pillars surface was only in favor of L929 cells to proliferate during 7 days of cell cultivation. Cell genetic toxicity was evaluated by cellular micronuclei test (MNT). The results indicated that topological surfaces were more suitable for L929 cells to proliferate and maintain the stability of genome. On the contrary, the narrow grooves surface induced higher micronuclei ratio of L02 and MSC cells than other surfaces. With the comprehensive results of cell multiplication and MNT, it was concluded that the wide grooves surface was best fitted for L02 cells to proliferate and have less DNA damages, and the smooth surface was optimum for the research of MSC cells in vitro.     

Comparison of physical, chemical and cellular responses to nano- and micro-sized calcium silicate/poly(epsilon-caprolactone) bioactive composites.

In this study, we fabricated nano-sized calcium silicate/poly(epsilon-caprolactone) composite (n-CPC) and micro-sized calcium silicate/poly(epsilon-caprolactone) composite (m-CPC). The composition, mechanical properties, hydrophilicity and degradability of both n-CPC and m-CPC were determined, and in vitro bioactivity was evaluated by investigating apatite forming on their surfaces in simulated body fluid (SBF). In addition, cell responses to the two kinds of composites were comparably investigated. The results indicated that n-CPC has superior hydrophilicity, compressive strength and elastic modulus properties compared with m-CPC. Both n-CPC and m-CPC exhibited good in vitro bioactivity, with different morphologies of apatite formation on their surfaces. The apatite layer on n-CPC was more homogeneous and compact than on m-CPC, due to the elevated levels of calcium and silicon concentrations in SBF from n-CPC throughout the 14-day soaking period. Significantly higher levels of attachment and proliferation of MG63 cells were observed on n-CPC than on m-CPC, and significantly higher levels of alkaline phosphatase activity were observed in human mesenchymal stem cells (hMSCs) on n-CPC than on m-CPC after 7 days. Scanning electron microscopy observations revealed that hMSCs were in intimate contact with both n-CPC and m-CPC surfaces, and significantly cell adhesion, spread and growth were observed on n-CPC and m-CPC. These results indicated that both n-CPC and m-CPC have the ability to support cell attachment, growth, proliferation and differentiation, and also yield good bioactivity and biocompatibility.     

Specific response of osteoblast-like cells on hydroxyapatite layer containing serum protein

Accelerations of bone-like apatite deposition and cell growth on an electrically polarized ceramic hydroxyapatite have been reported. A relationship between these phenomena was investigated in a previous report, and then it was suggested that osteoblast-like cell’s (MC3T3-E1) growth had relevance to the mineral growth. The effect of the formed apatite layer especially appeared to be on the cell adhesion. The acceleration of cell proliferation on the polarized HAp has been shown using fibroblastic cell (L929) and nerve cell (SK-N-SH) lines, therefore the effect of the layer on L929 and SK-N-SH was investigated to support the mechanism of acceleration of cell proliferation by polarization of HAp. In this study, the effect of the bone-like apatite layer was not confirmed on L929 cell’s growth. On the other hand, the acceleration of nerve cell’s proliferation was confirmed on the formed apatite layer. However, the remarkable improvement of the cell adhesion of SK-N-SH was not confirmed on the apatite layer. Consequently, it was considered that the bone-like apatite containing serum protein obtained by the coprecipitation of bone-like apatite and serum protein has a pronounced role only in the activity of osteoblast-like cells.     

Polymer films with surfaces unmodified and modified by non-thermal plasma as new substrates for cell adhesion

The surface properties of biomaterials, such as wettability, polar group distribution, and topography, play important roles in the behavior of cell adhesion and proliferation. Gaseous plasma discharges are among the most common means to modify the surface of a polymer without affecting its properties. Herein, we describe the surface modification of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) films using atmospheric pressure plasma processing through exposure to a dielectric barrier discharge (DBD). After treatment the film surface showed significant changes from hydrophobic to hydrophilic as the water contact angle decreasing from 95° to 37°. All plasma-treated films developed more hydrophilic surfaces compared to untreated films, although the reasons for the change in the surface properties of PS and PMMA differed, that is, the PS showed chemical changes and in the case of PMMA they were topographical. Excellent adhesion and cell proliferation were observed in all films. In vitro studies employing flow cytometry showed that the proliferation of L929 cells was higher in the film formed by a 1:1 mixture of PS/PMMA, which is consistent with the results of a previous study. These findings suggest better adhesion of L929 onto the 1:1 PS/PMMA modified film, indicating that this system is a new candidate biomaterial for tissue engineering.     

明胶固定化壳聚糖膜的细胞相容性评价

用碳二亚胺将明胶固定于壳聚糖膜表面,以改善壳聚糖的细胞相容性.采用MTT法评价膜材料对L929细胞的毒性.将壳聚糖膜(CS)、明胶固定化壳聚糖膜(CS-GEL)与人角膜上皮细胞(HCEC)体外复合培养,观察细胞的形态及生长情况,考察材料的细胞粘附率、材料对细胞活性与增殖的影响,并通过流式细胞术对细胞周期和凋亡进行分析.结果表明,CS-GEL具有良好的细胞粘附性,HCEC在CS-GEL上的生长和增殖情况优于CS,与CS相比,CS-GEL上HCEC的G1期比例下降,S期和G2期增加.明胶的引入缩短了细胞在膜表面的适应时间,使其尽快进入正常的细胞周期;同时表面固定的明胶可抑制细胞凋亡.因此,明胶固定化壳聚糖膜具有良好的体外细胞相容性,有望成为一种较好的角膜修复材料.     

Fabrication and characterization of bioactive silk fibroin/wollastonite composite scaffolds

Composite scaffolds of silk fibroin (SF) with bioactive wollastonite were prepared by freeze-drying. X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy analysis showed that random coil and β-sheet structure co-existed in the SF scaffold. The mechanical performance, surface hydrophilicity and water-uptake capacity of the composite scaffolds were improved compared with those of pure SF scaffold. The bioactivity of the composite scaffold was evaluated by soaking in a simulated body fluid (SBF), and formation of a hydroxycarbonate apatite (HCA) layer was determined by FT-IR and XRD. The results showed that the SF/wollastonite composite scaffold was bioactive as it induced the formation of HCA on the surface of the composite scaffold after soaking in SBF for 5 days. In vitro cell attachment and proliferation tests showed that the composite scaffold was a good matrix for the growth of L929 mouse fibroblast cells. Consequently, the incorporation of wollastonite into the SF scaffold can enhance both the mechanical strength and bioactivity of the scaffold, which suggests that the SF/wollastonite composite scaffold may be a potential biomaterial for tissue engineering.     

Improvement of <Emphasis Type="Italic">in vitro</Emphasis> behavior of an Mg alloy using a nanostructured composite bioceramic coating

Magnesium (Mg) alloys as a new group of biodegradable metal implants are being extensively investigated as a promising selection for biomaterials applications due to their apt mechanical and biological performance. However, as a foremost drawback of Mg alloys, the high degradation in body fluid prevents its clinical applications. In this work, a bioceramic composite coating is developed composed of diopside, bredigite, and fluoridated hydroxyapatite on the AZ91 Mg alloy in order to moderate the degradation rate, while improving its bioactivity, cell compatibility, and mechanical integrity. Microstructural studies were performed using a transmission electron microscope (TEM), scanning electron microscope (SEM), X-ray diffraction (XRD) analysis, and energy dispersive spectroscopy (EDS). The degradation properties of samples were carried out under two steps, including electrochemical corrosion test and immersion test in simulated body fluid (SBF). Additionally, compression test was performed to evaluate the mechanical integrity of the specimens. L-929 fibroblast cells were cultured on the samples to determine the cell compatibility of the samples, including the cell viability and attachment. The degradation results suggest that the composite coating decreases the degradation and improves the bioactivity of AZ91 Mg alloy substrate. No considerable deterioration in the compression strength was observed for the coated samples compared to the uncoated sample after 4 weeks immersion. Cytotoxicity test indicated that the coatings improve the cell compatibility of AZ91 alloy for L-929 cells.     

Cellular Interactions and Modulated Osteoblasts Functions Mediated by Protein Adsorption

The rapid adsorption of proteins is one of the first few events that occur when a biomedical device is implanted and strongly affects attachment and proliferation of cells on the material surface. We describe here for the first time the favorable modulation of osteoblasts functions and cell–substrate interactions induced by protein adsorption (bovine serum albumin) on silicone elastomers. It was intriguing that the cell–substrate interactions on protein‐adsorbed silicone elastomer were significantly different from those observed in stand alone silicone. The origin and differences in cell–substrate interactions in terms of cell attachment, viability, and proliferation and assessment of proteins actin, vinculin, and fibronectin are addressed. Cell attachment and proliferation of cells were significantly enhanced on protein‐adsorbed surface in comparison to the bare surface. Similarly, the expression level of fibronectin, actin, and vinculin was appreciably greater on the protein‐adsorbed surface. The study underscores that protein adsorption on artificial biomedical devices can be envisaged as one of the methods to enhance the bioactivity on the surface and regulate biological functions.     

In vitro behavior of human osteoblast-like cells (SaOS2) cultured on surface modified titanium and titanium–zirconium alloy

In this study, titanium (Ti) and titanium–zirconium (TiZr) alloy samples fabricated through powder metallurgy were surface modified by alkali-heat treatment and calcium (Ca)-ion-deposition. The alteration of the surface morphology and the chemistry of the Ti and TiZr after surface modification were examined. The bioactivity of the Ti and TiZr alloys after the surface modification was demonstrated. Subsequently, the cytocompatibility of the surface modified Ti and TiZr was evaluated via in vitro cell culture using human osteoblast-like cells (SaOS2). The cellular attachment, adhesion and proliferation after cell culture for 14 days were characterized by scanning electron microscopy (SEM) and MTT assay. The relationship between surface morphology and chemical composition of the surface modified Ti and TiZr and cellular responses was investigated. Results indicated that the surface-modified Ti and TiZr alloys exhibited excellent in vitro cytocompatibility together with satisfactory bioactivity. Since osteoblast adhesion and proliferation are essential prerequisites for a successful implant in vivo, these results provide evidence that Ti and TiZr alloys after appropriate surface modification are promising biomaterials for hard tissue replacement.