与胶原特异性结合的BMP2模拟肽/PLGA 3D打印复合支架的制备及成骨诱导活性

将胶原绑定结构域(CBD)多肽序列与骨形态发生蛋白2模拟肽(BMP2-MP)序列连接制备具有胶原绑定能力的CBD-BMP2-MP, 再将CBD-BMP2-MP与聚丙交酯-乙交酯/胶原(PLGA/COL)3D打印支架相结合, 以支架表面的胶原成分为媒介, 将CBD-BMP2-MP更有效地固定于骨修复材料上, 达到对其进行改性的目的. 利用扫描电子显微镜(SEM)、 电子万能试验机和接触角测量仪对复合支架表面形貌、 力学强度和亲水性等材料学性能进行评价. 用荧光成像法评测 CBD-BMP2-MP及BMP2-MP与支架材料的结合能力. 在各组支架材料表面接种MC3T3-E1细胞进行体外培养, 采用CCK-8、 鬼笔环肽荧光染色、 茜素红染色及qPCR综合评价细胞在材料表面的黏附、 增殖和成骨分化等细胞行为, 研究CBD-BMP2-MP修饰的3D多孔PLGA/COL复合支架的生物学性能. 研究结果表明, 利用3D打印技术制备的多孔支架具有形貌可控的孔隙结构, 为细胞生长创造更有利的细胞微环境, 支架表面胶原成分的加入提高了支架材料的亲水性, 同时对支架材料本身的力学性能无任何影响, 提高了复合支架本身的生物相容性. 与普通BMP2-MP相比, CBD-BMP2-MP具有更好的胶原绑定能力, 与复合支架的结合更稳定, 提高了PLGA/COL复合支架对BMP2-MP的负载能力. 支架表面负载CBD-BMP2-MP后具有极强的促细胞成骨分化能力. MC3T3-E1细胞表现出更高的钙沉积能力, 并且成骨分化相关基因Runx2, ALP, COL-I及OPN等水平也有了明显提升. 表明CBD-BMP2-MP多孔复合支架具有良好的生物相容性和成骨诱导活性, 在骨组织修复领域具有良好的应用前景.     

丝素蛋白修饰改性的聚羟基脂肪酸酯支架上人体平滑肌细胞的生长

聚三羟基丁酸脂和聚三羟基己酸脂的共聚物(PHBHH_x)是一种具有良好强度和韧性的生物可降解高分子材料, 可作为组织工程心脏瓣膜支架的选择材料之一. 但其生物相容性尚不甚理想. 为此, 本工作利用丝素蛋白修饰改性高分子多孔支架, 以提高支架的生物相容性. 并将人体平滑肌细胞接种在该复合支架上进行体外培养, 以证实改性效果. 其中, 用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)方法测试细胞生长, 评估复合支架的细胞相容性. 并用扫描电子显微镜观察细胞在支架上的生长形态. 结果显示, 丝素蛋白修饰改性后的复合支架更有利于细胞的粘附与生长, 平滑肌细胞在支架上表现出良好的生长形态. 这表明, 丝素能够改善多孔支架的生物相容性, 使PHBHH_x/丝素蛋白复合物能更适宜作为组织工程心脏瓣膜的支架材料. 结果对于进一步研究细胞外间质在复合支架上的生长以及体外培养的组织重建有重要的参考意义.     

p(HEMA-MMA)表面固定I-型胶原和碱性成纤维生长因子(bFGF)改善细胞相容性的研究

材料表面固定生物分子一直都被认为是其提高其细胞相容性有效的途径。本文采用羧基二咪唑(CDI)作为缩合剂,使p(HEMA-MMA)表面羟基与胶原中氨基发生缩合反应,从而将I-型胶原接枝在材料表面。然后利用"接枝-吸附"的方法,将I-型胶原和bFGF混合层吸附固定于材料表面,形成稳定的胶原与bFGF涂层。红外光谱(FTIR)和原子力显微镜(AFM)证实了该种固定方法的成功。静态接触角显示材料表面亲水性较改性前略微降低。涂层稳定性实验结果表明在类似人体环境(pH=7.4)下涂层具有良好的稳定性。细胞粘附性实验以及MTT实验一致显示改性后材料的细胞相容性得到明显提高。因此,Col/bFGF-p(HEMA-MMA)有望成为能够被宿主机体所接受的生物材料。     

聚己内酯/卵磷脂的纺丝支架构建组织工程血管

合成的聚己内酯(PCL)经天然的卵磷脂(Phosphatidylcholine,PC)填充改性后,通过电纺丝技术加工得到三维多孔的纤维支架。卵磷脂含有的两性离子基团,可以显著改善PCL支架材料的亲水性,进而提高支架材料的细胞相容性。体外细胞增殖实验表明,骨髓间充质干细胞(MSCs)在含有5wt%卵磷脂的改性支架表面生长得最好。作为种子细胞的MSCs在流动培养下,通过力学刺激在管状支架内壁形成了多层细胞。通过对样品染色切片和荧光照片的观察,种子细胞MSCs与对照组的血管平滑肌细胞(SMCs)一样,有向改性支架内部生长的趋势。本文以这种PC填充改性PCL材料的纤维支架作为组织工程血管,对其进行了初步的探索。     

ATRP法合成弹性蛋白接枝共聚物及其性能研究

弹性蛋白经α-溴异丁酰溴化制备了大分子ATRP引发剂溴化弹性蛋白(E-Br), 再以E-Br作为引发剂, 在CuCl/2,2-联吡啶催化体系下, 用原子转移自由基聚合方法合成了弹性蛋白-g-聚甲基丙烯酸-β-羟乙酯接枝聚合物. 用红外光谱(FTIR)、X射线光电子能谱(XPS)、热重分析(TGA)、扫描电镜(SEM)、离子色谱和动态接触角对接枝聚合物进行了表征. 结果表明, PHEMA键接到了弹性蛋白表面; SEM显示接枝改性后弹性蛋白的表面比未改性前光滑, 但改性后样品的热性能均比未改性样品的低, 起始热分解温度由改性前的307 ℃变为265 ℃; 动态接触角实验结果表明, 接枝改性后的样品具有良好的亲水性, 反应72 h后, 其前进角由接枝前的130.45°下降到29.80°.     

聚乳酸接枝改性纳米生物玻璃/PLGA复合材料的制备、表面性质及生物活性

以丙交酯开环聚合原位接枝改性的纳米生物玻璃(PLLA-g-BG)与聚丙交酯-乙交酯(PLGA)复合材料为研究对象, 采用TGA, ESEM和EDX分析其接枝率, 粒子分散性和表面元素分布, 通过将兔成骨细胞种植于材料膜表面进行体外培养, 采用荧光染色法、NIH Image J图像分析软件、MTT法和流式细胞术等手段检测细胞在材料表面的平均黏附数量、扩展面积比、增殖能力和细胞周期的变化, 综合评价新型改性纳米复合材料的生物相容性和生物活性. 结果表明, 聚乳酸表面接枝改性可明显改善纳米生物玻璃粒子的团聚; PLGA中掺入一定比例的改性PLLA-g-BG可明显促进兔成骨细胞的黏附、扩展与增殖; 改性纳米生物玻璃的应用可提高生物可降解聚酯材料的生物相容性和生物活性.     

超支化聚胺-酯改性聚二甲基硅氧烷微流控芯片的紫外检测应用

采用超支化聚胺-酯对经过氧气氛处理的PDMS微流控芯片表面进行改性.成功地将超支化聚胺-酯涂覆到PDMS表面,使其表面的接触角由108°±1°降到32°±20°,改善了其亲水性;改性过后通道内的电渗流得到了有效抑制,远低于未改性通道内的电渗流.同时,将芯片通过专门设计的通道与毛细管连接在一起,在紫外检测波长214nm,...     

强韧支架用丝素蛋白基生物墨水及其3D打印支架模拟软件的开发

基于丝素蛋白（SF）和有限元分析方法，开发了一种强韧支架用丝素蛋白基生物墨水及其3D打印支架压缩性能模拟软件。表征了墨水的可打印性及对应水凝胶和3D打印支架的力学性能，评估了相关打印支架的细胞相容性。基于所述丝素蛋白基生物墨水，设计并制备了不同高度和孔隙率的3D打印支架，利用所开发软件和电子万能材料试验机对打印支架的压缩性能进行了模拟预测和实测对比。结果表明：该墨水可打印性佳，对应支架强度高韧性好、细胞相容性好。所开发软件操作简便，具有3D打印支架建模、模型压缩力学性能预测以及指导3D打印支架快速制备等功能。     

硫代-iniferters法改性弹性蛋白的研究

弹性蛋白经对氯甲基苯甲酰氯氯化、二乙基二硫代氨基甲酸钠(NaSD)硫化制备了大分子iniferter剂(E-S), 再以E-S为引发剂, 在紫外光(UV)照射下引发甲基丙烯酸-β-羟乙酯(HEMA)聚合, 合成了聚甲基丙烯酸-β-羟乙酯(E-PHEMA)改性的弹性蛋白聚合物. 用红外(FTIR)和X射线光电子能谱(XPS)、热重分析(TGA)、扫描电镜(SEM)和动态接触角对改性弹性蛋白进行了表征. 结果表明: PHEMA键接到了弹性蛋白上; SEM显示改性后弹性蛋白的表面比未改性前变得光滑, 但改性后样品的热性能均低于未改性样品, 起始热分解温度由改性前的307.0 ℃变为260.2 ℃, 最大失重速率温度由347 ℃降到316.3 ℃; 动态接触角实验表明改性后样品具有良好的亲水性, 反应72 h后前进角由改性前的130.45°下降到35.40°, 接触角滞后由70.42°变为35.40°.     

羟基磷灰石/聚乳酸微粒复合生物支架的体外细胞相容性评价

利用溶液共混法以及溶剂挥发法制备了羟基磷灰石(Nano-HA)/聚乳酸(PLA)微粒,再粘结微粒加工成三维多孔Nano-HA/PLA微粒复合生物支架。借助相差显微镜、扫描电子显微镜和MTT法检测了鼠骨髓基质细胞(BMSCs)在该支架材料上的生长情况,通过细胞形态学观察和细胞增殖情况评价了该复合生物支架材料的生物相容性。结果表明,SEM观察到支架材料上培养细胞4d后,细胞主要附着、铺展在支架的低洼处或孔洞处表面,并向孔洞深部沿壁生长;在支架材料上培养细胞8d后,细胞多为梭形形态,并有许多生长角,直接贴附于支架的微粒表面,开始连片生长,有明显的增值,各组没有变形、坏死现象。支架材料上培养细胞2,3,4,5,6和8d的MTT检测表明,各实验组RGR均达到100%以上,细胞毒性为0级;细胞在支架材料上的生长曲线显示,实验组细胞活力比对照组高26%。因此,该Nano-HA/PLA微粒复合生物支架没有细胞毒性,并对细胞有良好的粘附和增殖能力,为较具潜力的骨修复材料。     

Degradation of electrospun SF/P(LLA-CL) blended nanofibrous scaffolds in vitro

Nanofibrous scaffolds of silk fibroin (SF) and poly(l-lactic acid-co-?-caprolactone) (P(LLA-CL)) blends fabricated via electrospinning possessed good mechanical property and biocompatibility, as demonstrated by a previous study in vitro. However, the degradation behavior of the scaffolds, which may significantly influence tissue repair and regeneration, needs further exploration. In this study, in vitro degradation of pure SF, P(LLA-CL) and SF/P(LLA-CL) blended nanofibrous scaffolds were performed in phosphate-buffered saline (PBS, pH 7.4 ± 0.1) at 37 °C for 6 months. A series of analyses and characterizations (including morphologic changes, loss weight, pH changes of PBS solutions, DSC, XRD and FTIR-ATR) were conducted to the nanofibrous scaffolds after degradation and the results showed that the pure SF nanofibrous scaffolds were not completely degradable in PBS while pure P(LLA-CL) nanofibrous scaffolds had the fastest degradation rate. Moreover, the addition of SF reduced the degradation rate of P(LLA-CL) in SF/P(LLA-CL) blended nanofibrous scaffolds. This was probably caused by the intermolecular interactions between SF and P(LLA-CL), which hindered the movement of P(LLA-CL) molecular chains.     

纳米羟基磷灰石/丝素蛋白复合支架材料的降解特性及生物相容性研究

应用共混法制备了纳米羟基磷灰石/丝素蛋白复合支架材料, 通过体外降解和细胞培养实验研究了复合支架材料的降解特性和生物相容性. 体外降解实验结果显示, 复合支架材料具有稳定的降解能力; 在降解过程中, 羟基磷灰石由于与降解液发生钙、磷等离子的交换, 使其结晶得到了进一步生长和完善. 利用细胞计数法、四甲基偶氮唑盐(MTT)比色法和碱性磷酸酶(ALP)活性测定等分析了复合支架材料的生物相容性, 结果表明, MG63细胞在复合支架材料上具有良好的粘附、增殖能力, 并可引起早期的骨分化. 因此, 纳米羟基磷灰石/丝素蛋白复合支架作为骨组织工程的支架材料具有良好的应用前景.     

Influence of Oxygen Plasma Modification on Surface Free Energy of PMMA Films and Cell Attachment

For any biomaterial placed into a biological medium, the surface properties of the material, such as porosity, crystallinity, presence and distribution of electrical charge and functional groups are very critical parameters that determine the acceptance or rejection of the material. Applications, especially tissue engineering require some surface modifications at the molecular level without disturbing the bulk properties of the implants in order to enhance the cell attachment on the material. An appropriate technique is the application of glow discharge plasma which employs no solvents, takes place at ambient temperatures, and alterations take place only at the surface by changing the surface chemistry along with surface free energy (SFE) and efficiency for cell-material interaction. In this study, poly(methyl methacrylate) (PMMA) film surfaces were modified with oxygen plasma. SFE and its dispersive and polar (acidic-basic) components of the modified surfaces were calculated by means of several theoretical approaches including geometric mean, harmonic mean and acid-base equations. The relation between SFE and its dispersive and polar components and cell attachment on surfaces were studied. The highest 3T3 cell attachment was obtained for the surface with the total SFE of 61.77 mJ/m² and polar component of 50.91 mJ/m² according to Geometric mean. The total SFE of this surface was calculated to be 61.06 mJ/m² and the polar component as 40.96 mJ/m² using the Harmonic mean method.     

Improved mechanical property and biocompatibility of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) for blood vessel tissue engineering by blending with poly(propylene carbonate)

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and poly(propylene carbonate) (PPC) were blended by solvent casting method into films at various weight ratios in order to obtain materials with properties more suitable for blood vessel tissue engineering than pure PHBHHx alone. FTIR and XRD analysis indicated the crystal structure of PHBHHx was not altered but the crystallinity was reduced by the interfusion of PPC. Mechanical properties of the films were improved significantly by blending with PPC. A lower elastic modulus and a higher elongation at break were obtained with the increase of PPC content. Wettability, fibronectin adsorption and adhesion of rabbit aorta smooth muscle cells (RaSMCs) on blend films were similar to or better than that on PHBHHx film. All these results showed promises of PHBHHx/PPC blended materials as scaffold material for blood vessel tissue engineering.     

Crosslinked Silk Fibroin/Gelatin/Hyaluronan Blends as Scaffolds for Cell-Based Tissue Engineering

3D porous scaffolds fabricated from binary and ternary blends of silk fibroin (SF), gelatin (G), and hyaluronan (HA) and crosslinked by the carbodiimide coupling reaction were developed. Water-stable scaffolds can be obtained after crosslinking, and the SFG and SFGHA samples were stable in cell culture medium up to 10 days. The presence of HA in the scaffolds with appropriate crosslinking conditions greatly enhanced the swellability. The microarchitecture of the freeze-dried scaffolds showed high porosity and interconnectivity. In particular, the pore size was significantly larger with an addition of HA. Biological activities of NIH/3T3 fibroblasts seeded on SFG and SFGHA scaffolds revealed that both scaffolds were able to support cell adhesion and proliferation of a 7-day culture. Furthermore, cell penetration into the scaffolds can be observed due to the interconnected porous structure of the scaffolds and the presence of bioactive materials which could attract the cells and support cell functions. The higher cell number was noticed in the SFGHA samples, possibly due to the HA component and the larger pore size which could improve the microenvironment for fibroblast adhesion, proliferation, and motility. The developed scaffolds from ternary blends showed potential in their application as 3D cell culture substrates in fibroblast-based tissue engineering.     

Porous composite scaffolds of hydroxyapatite/silk fibroin via two‐step method

A two‐step method was used to fabricate the hydroxyapatite (HAP)/silk fibroin (SF) scaffolds, i.e. the nano‐sized HAP/SF composite powders were prepared by co‐precipitation, which were then blended with SF solution to fabricate the HAP/SF composite scaffolds. The obtained scaffolds showed a 3D porous structure. The porosity was higher than 90% with the average macropore size of 214.2 µm. Moreover, the nano‐sized HAP/SF composite powders were uniformly dispersed in the silk fibroin matrix, which provided the scaffolds enhanced compressive properties. The cell culture assay showed that the scaffolds fabricated by the two‐step method could improve the cell proliferation and osteogenic differentiation when compared with those prepared by the conventional one‐step blending method. The results suggested that the two‐step method could promote the uniform dispersion of HAP in the SF matrix and efficient combination between the HAP and the matrix, which may provide a potential application in the composite scaffold preparation for tissue engineering. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of poly(hydroxybutyrate-co-hydroxyhexanoate) microparticles on growth of murine fibroblast L929 cells

Poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) microparticles were successfully prepared and their proliferative effects on cultured fibroblasts were studied. PHBHHx microparticles (0.005-0.1 g/L) promoted cell proliferation in murine fibroblast L929 and elevated intracellular calcium concentrations ([Ca²⁺]_i). EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid) inhibited PHBHHx microparticle-induced cell proliferation by chelating the extracellular Ca²⁺ and blocking the PHBHHx particle-induced [Ca²⁺]_i increase. Transwell experiments demonstrated that PHBHHx microparticles stimulated fibroblast proliferation when separated from cells by a 0.4 μm filter as effectively as when applied directly to cells. Since PHBHHx microparticles had a diameter of 75 μm, the stimulatory effect of PHBHHx particles on cell growth was attributed to degradation products smaller than 0.4 μm in diameter. The trophic effect of these microparticles is consistent with our previous reports demonstrating good biocompatibility for PHBHHx.     

The Hydrophobicity Study of Silica and Glass Surfaces Modified with Polyalkylhydrosiloxanes

A procedure based on the analysis of the shape of a sessile drop was used to measure the contact angles of water in air, _a = 93°–97°, for quartz and glass plates modified with polyalkylhydrosiloxanes. The specific heat of wetting, q = 36 mJ/m², and the surface pressure of adsorbed water film, = 48 mJ/m², were determined from adsorption-calorimetric experiment on macroporous disperse silica modified with polymethylhydrosiloxane GKZh-94M. The contact angle in saturated water vapors, _v = 84°, was calculated from these values. It was shown that the liquid-phase modification of silica and glass with polyalkylhydrosiloxanes leads to distinct hydrophobization of the studied surfaces.     

Preparation and characterization of new materials based on silk fibroin,chitosan and nanohydroxyapatite

Abstract

In this paper, a series of porous nanohydroxyapatite/silk fibroin/chitosan (nHA/SF/CTS) scaffolds were successfully prepared using the freeze-drying method. The biomaterials were characterized by attenuated total reflection Fourier transform infrared spectroscopy, and mechanical testing and thermogravimetric analysis. Moreover, studies of porosity, pore size, swelling properties and in vitro degradation test were performed. Research has proved that micro-structure, porosity, water adsorption and compressive strength were greatly affected by the components’ concentration, in particular the content of silk fibroin. SEM observations showed that the scaffolds of nHA/SF/CTS are highly porous, with pore size in wide range from 25 to 300?µm which is suitable for cell growth. nHA/SF/CTS scaffolds have sufficient mechanical integrity to resist handling during implantation and in vivo loading. Both, the compressive modulus and compressive strength of the scaffold, decrease with the increase in silk fibroin content.