壳聚糖与硫酸软骨素共混膜性质的研究

以壳聚糖和硫酸软骨素按一定比例制备出共混膜，研究了膜片的透光性、含水量、渗透性、力学性质、表面结构、生物降解性、生物相容性等性质。结果表明该共混膜具有较好的透光性、通透性、生物降解性和生物相容性，膜表面较粗糙。以此共混膜为载体培养兔角膜基质细胞，发现细胞在此共混膜上生长良好。制备膜片随着加入CaSO4量的增加，膜的通透性也随之增加。     

兔角膜内皮细胞载体的体外培养及移植的研究

以特定曲率的壳聚糖-硫酸软骨素共混膜为载体，构建兔角膜内皮。研究了共混膜的透光性、体外酶降解性以及兔角膜内皮细胞在载体上的细胞贴附性、细胞形态及膜强度等性质。结果表明，共混膜的透光率达90％以上，生物降解性良好。该共混膜有良好的细胞贴附性及机械强度；兔角膜内皮细胞可在共混膜上长成良好的单层，细胞形态较好，并贴附牢固，振荡后细胞无脱落，膜片保持完整。将培养好的载体植入到去除内皮层的兔角膜中，术眼在56天内基本保持透明，说明体外构建的内皮可执行角膜内皮层的部分功能。     

葡甘聚糖一胶原蛋白一壳聚糖共混膜（I）

用溶液共混法制备了葡甘聚糖一胶原蛋白一壳聚糖（KCCS）共混膜。并用FT—IR，X—RD，SEM及透光率表征了膜的结构，同时测试了膜的抗张强度、断裂伸长率、吸水率、透水汽性、渗透性和吸附性。结果表明：共混膜中葡甘聚糖、胶原蛋白及壳聚糖之间存在着强烈的相互作用和良好的相容性，三者共混明显改善了纯聚合物和二元膜的性能。以共混膜为载体培养内皮细胞，发现共混膜具有良好的细胞相容性，预示着共混膜可作为潜在的组织工程支架材料。     

葡甘聚糖-胶原蛋白-壳聚糖共混膜(I)

用溶液共混法制备了葡甘聚糖-胶原蛋白-壳聚糖(KCCS)共混膜。并用FT-IR,X-RD,SEM及透光率表征了膜的结构,同时测试了膜的抗张强度、断裂伸长率、吸水率、透水汽性、渗透性和吸附性。结果表明:共混膜中葡甘聚糖、胶原蛋白及壳聚糖之间存在着强烈的相互作用和良好的相容性,三者共混明显改善了纯聚合物和二元膜的性能。以共混膜为载体培养内皮细胞,发现共混膜具有良好的细胞相容性,预示着共混膜可作为潜在的组织工程支架材料。     

基于肝素和硫酸软骨素的两种纳米颗粒改性表面对生物相容性影响的研究

本研究合成了基于肝素和硫酸软骨素的两种纳米颗粒,用于316L不锈钢表面的生物功能改性。通过激光粒度分析仪、傅立叶变换红外光谱(FTIR)、原子力显微镜(AFM)、水接触角等对纳米颗粒的性质及颗粒固定前后表面的理化性质进行表征。通过体外血液相容性评价和内皮细胞相容性评价对两种纳米颗粒改性表面的生物相容性进行对比研究。结果表明,两种纳米颗粒均能有效降低材料表面血小板的粘附和聚集行为,但肝素纳米颗粒对内皮细胞的生长增殖表现出抑制作用,而硫酸软骨素纳米颗粒改性表面则具有促进内皮再生的潜能。     

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.     

组织工程骨-软骨复合支架的构建与优化

背景：制备具有细胞识别信号的细胞外基质替代材料及仿生支架是目前组织工程支架材料研究的重点和热点。 目的：制备并筛选出能够满足构建骨-软骨复合组织要求的多孔三维支架,并评价其生物学性能。 方法：制备胶原-壳聚糖、明胶-硫酸软骨素-透明质酸钠、胶原-陶瓷化骨、明胶-陶瓷化骨支架材料,以新鲜关节为对照组。 结果与结论：胶原-壳聚糖支架孔径50-200 μm,孔隙率(90.5±2.1)%;明胶-硫酸软骨素-透明质酸钠支架孔径100- 150 μm,孔隙率(78.0±1.1)%;胶原-陶瓷化骨支架孔径400-500 μm,孔隙率(67.5±2.1)%;明胶-陶瓷化骨支架孔径300-400 μm,孔隙率(65.9±1.2)%。明胶-硫酸软骨素-透明质酸钠与明胶-陶瓷化骨支架基本符合实验要求,其结构与生物化学成分近似于自然细胞外基质,能够模拟细胞外微环境。说明明胶-硫酸软骨素-透明质酸钠与明胶-陶瓷化骨支架可作为复合组织的支架。     

不同分子量壳聚糖膜性质的研究

分别以分子量为130，000、220，000、300，000、550，000道尔顿的壳聚糖制备壳聚糖膜，并研究了各膜的表面结构、结晶性、力学特性、渗透性、透光透气性、吸附性、生物降解性等。结晶表明壳聚糖膜的各种特性和壳聚糖的分子量相关，高分子量的壳聚糖膜表面较为光滑，透光性较好，透气性、渗透性和生物降解性较差；低分子量的壳聚糖膜表面较为粗糙，透气性、渗透性和生物降解性较好，但透光性较差。经分析认为膜的结晶性和超微结构决定了不同分子量壳聚糖膜具有不同的性质。     

硫酸长春新碱缓释药膜的制备及稳定性初步研究

目的制备出载硫酸长春新碱微球的胶原-壳聚糖缓释药膜,并考察该制剂的稳定性。方法采用W/O/O溶剂挥发法制备载硫酸长春新碱的聚乳酸/聚羟基乙酸共聚物(PLGA)微球,后把微球与壳聚糖、胶原溶液共混及二次冻干,制备出载硫酸长春新碱微球的胶原-壳聚糖药膜。对微球和药膜表面形态进行了电镜观察,测定了微球和药膜的包封率、载药量及药物释放,药物含量采用高效液相法检测。此外,还初步考察了缓释药膜的稳定性。结果制备的微球包封率达到79.0%±1.0%,微球药物的突释为27.2%±1.2%,制备成药膜后降低到18.0%±1.1%,采用该工艺流程制备出来的缓释药膜,药物突释明显减少。稳定性实验显示,该药膜在40℃条件下放置3个月药物含量下降到97.9%±0.1%,而高湿度或光照环境下放置10 d药物含量下降到91.4%±0.3%和91.2%±0.4%。结论药物包囊制成微球后与胶原、壳聚糖共混制备出的缓释药膜具有较好的释放特性和稳定性,有望成为一种实用的新型缓释抗肿瘤制剂。     

不同脱乙酰度对壳聚糖膜与角膜基质细胞相容性的影响

以分子量为30万．脱乙酰度分别为63．3％、73．7％、83％和97％的壳聚糖制备不同的壳聚糖膜，在不同脱乙酰度的壳聚糖膜上培养兔角膜基质细胞．通过观察角膜基质细胞在不同壳聚糖膜上的生长状态、贴附情况、生长曲线以及乳酸脱氢酶的活性，研究壳聚糖分子脱乙酰度对壳聚糖膜与角膜基质细胞生物相容性的影响。实验结果表明壳聚糖脱乙酰度越高。壳聚糖膜对细胞的损伤越小。越有利于细胞在膜上的生长和贴附，反之．低脱乙酰度的壳聚糖膜与角膜细胞的相容性较差。     

Structural Characteristics and Properties of Silk Fibroin/Poly(lactic acid) Blend Films

This paper describes the preparation and characterization of blend films composed of regenerated silk fibroin (SF) and poly(lactic acid) (PLA). FT-IR and XRD of the SF/PLA blend films with different ratios indicated that the secondary structural transition of SF from Silk I to Silk II was induced upon blending with PLA. The effects of SF/PLA blend ratios on the mechanical and physical properties of the blend films were investigated. Compared to pure SF film, the mechanical and thermal properties of the blend films were improved, and surface hydrophilicity and swelling capacity decreased due to the secondary structural transition of SF to Silk II. Among the blend films with different ratios, the SF/PLA blend film with 7 wt% PLA content showed excellent mechanical properties. Meanwhile, the BSA adsorption amount on the blend film increased with the increase of PLA content. In vitro cell adhesion test showed that the blend film was a good matrix for the growth of L929 mouse fibroblast cells. Consequently, controlling the PLA content in the SF film can improve the mechanical and physical properties of the SF film and provide a promising opportunity to widen potential application of SF in the biomaterials field.     

Porous Silk Fibroin Film as a Transparent Carrier for Cultivated Corneal Epithelial Sheets

Biological carriers, such as the amniotic membrane and serum-derived fibrin, are currently used to deliver cultivated corneal epithelial sheets to the ocular surface. Such carriers require being transparent and allowing the diffusion of metabolites in order to maintain a healthy ocular surface. However, safety issues concerning biological agents encouraged the development of safer, biocompatible materials as cell carriers. We examined the application of porous silk fibroin films with high molecular permeability prepared by mixing silk fibroin and poly(ethylene glycol) (PEG), and then removal of PEG from the silk-PEG films. Molecular permeability of porous silk fibroin film is higher than untreated silk fibroin film. Epithelial cells were isolated from rabbit limbal epithelium, and seeded onto silk fibroin coated wells and co-cultured with mitomycin C-treated 3T3 fibroblasts. Stratified epithelial sheets successfully engineered on porous silk fibroin film expressed the cornea-specific cytokeratins K3 and K12, as well as the corneal epithelial marker pax6. Basement membrane components such as type-IV collagen and integrin β1 were expressed in the stratified epithelial sheets. Further more, colony-forming efficiency of dissociated cells was similar to primary corneal epithelial cells showing that progenitor cells were preserved. The biocompatibility of fibroin films was confirmed in rabbit corneas for up to 6 months. Porous silk fibroin film is a highly transparent, biocompatible material that may be useful as a carrier of cultivated epithelial sheets in the regeneration of corneal epithelium.     

Characterization of surface modified glycerol/silk fibroin film for application to corneal endothelial cell regeneration

Corneal endothelial cells (CEnCs) play a fundamental role in maintaining the transparency of the cornea. CEnCs lose their full proliferating capacity when tissue damages occur. The loss in proliferation rate is associated with corneal edema and decrease in visual acuity, leading in severe cases, to blindness. In these situations, a corneal transplant is usually needed to restore the original tissue functions. Tissue engineering is an efficient alternative for the production of implantable films, which can regenerate the tissue functions regulating at the same time the immune-response.

In this study, we proposed a stable and transparent film, composed of silk fibroin modified with glycerol (G/SF), as a potential substrate for corneal endothelial cells regeneration. Our results confirmed that G/SF films have a uniform structure, rougher surface and lower thickness respect to the SF film. In vitro tests show that G/SF films can induce a slight increase in CEnCs initial adhesion and proliferation rate if compared with the SF film. Morphology and gene expression evaluations demonstrated that the bioactive effects of silk fibroin were not affected by the presence of glycerol. For this reason, the G/SF films are suitable as CEnCs carrier and promising for the corneal damages treatments.     

胶原-葡甘聚糖-硫酸软骨素复合膜对全层皮肤损伤修复的实验研究

用小猪皮提取胶原,与葡甘聚糖和硫酸软骨素复合改性制成复合膜,研究用于修复全层皮肤缺损。选用新西兰大白兔14只,1.5~2.0kg,在胸腰脊柱两侧切割建立2cm×4cm全层皮肤缺损模型,左侧为试验组,敷以复合膜,右侧不经任何处理,作为空白对照组。观察创面的变化,定期取材,作组织学检查。复合膜有防止创口出血和感染的作用,实验组创伤愈合形成矩性痂,空白对照组形成线性痂。无明显的免疫排斥反应,有促进皮肤结构再生的能力。胶原-葡甘聚糖-硫酸软骨素复合膜对创伤的修复有促进作用。     

Experimental approaches to generate compositional gradients in the fully biodegradable polymer blend system based on poly(3‐hydroxybutyric acid)

The fully biodegradable polymer blend films with compositional gradients from the surface to the inside of the films were prepared by the solution‐diffuse technique. A solution of bacterial poly(3‐hydroxybutyric acid) (PHB) in hexafluoro‐2‐propanol (HFIP), which is also a good solvent for poly(vinyl alcohol) (PVA), was cast on a PVA film and the solvent HFIP was evaporated. By controlling the solvent evaporation rate, the PHB/PVA blend film with a compositional gradient was obtained. The phase structure and biodegradation profile of the PHB/PVA blend film were found to be different from those of the corresponding blend film prepared by the conventional solution‐cast method.     

Ocular cell monolayers cultured on biodegradable substrates.

The aim of this study was to culture retinal pigment epithelial (RPE) and corneal endothelial cells on biodegradable substrates for future use in monolayer transplantation in the eye. The biodegradable polymers, poly-l-lactic (PLLA) and poly-dl-lactic-co-glycolic acid (85:15) (PLGA) (both of molecular weight 105 kd) were the biomaterials used. All materials were seeded with either pig/human retinal pigment epithelial cells or rabbit corneal endothelial cells and were maintained in tissue culture conditions. Upon confluency, the cell density was calculated and cell viability determined. All monolayers were stained with phalloidin-rhodamine for F-actin and antibodies to the tight junction (zonula occludens) protein, ZO1, to demonstrate the presence of tight junctions. The final cell density of human RPE monolayers on PLLA films was 2950 cells/mm(2) (+/-185). The final cell density of pig RPE on PLLA and PLGA film was 2350 cells/mm(2) (+/-152 and 178, respectively). Rabbit corneal endothelial cells had a final cell density of 2650 cells/mm(2) (+/-164). F-actin staining revealed a circumferential ring of actin filaments in all of the cells grown on substrates. ZO(1) immunohistochemistry demonstrated staining along the lateral cell borders of all cell types. The successful culture of retinal pigment epithelial and corneal endothelial monolayers on these substrates may have potential for transplanting cell monolayers in the eye to improve vision.