角膜组织工程支架壳聚糖基共混膜的制备及性能评价

以脱乙酰度50%左右的水溶性壳聚糖(Cts)为主要成分,添加不同比例的明胶(gel)、硫酸软骨素(CS)共混制备出3组共混膜,测定了共混膜的透光率、含水量、细胞毒性及兔角膜基质细胞在膜上的生长活性,并评价了筛选出的共混膜在大鼠股部肌肉和兔眼前房内的组织相容性。实验结果表明,V(Cts)∶V(gel)∶V(CS)=9∶1∶0.1的共混膜具有较好的透光率,合适的含水量,细胞毒性反应为0级,而且适合兔角膜基质细胞生长贴附;植入大鼠股部肌肉和兔眼前房后炎症反应较轻。该种膜片有望作为载体支架用于构建组织工程角膜。     

壳聚糖-硫酸软骨素共混膜性质的研究

以溶液共沸法制成不同共混比例的壳聚糖－硫酸软骨素共混膜,通过观察共混膜的表面形态结构、结晶度、红外吸收及透光率,发现壳聚糖和硫酸软骨素两种分子具有较好的相容性,分子间具有较强的相互作用,所形成的共混膜表面结构均匀单一。通过研究共混膜的各种性质发现硫酸软骨素的混入可以改善膜的力学特性,提高膜的透光性及渗透性,降低膜的吸水性及对蛋白的吸附性。以共混膜为载体培养兔角膜内皮细胞,发现硫酸软骨素的引入可明显提高膜和细胞的相容性,兔角膜内皮细胞可在膜上长期生长,结果提示此共混膜可作为细胞培养的良好载体,用于器官损伤修复及细胞移植。     

防粘连壳聚糖衍生物共混膜的生物学性质研究

将甲壳素、羧甲基壳聚糖和羟丙基壳聚糖按一定比例共混交联,流延成膜法制备CM-C-HP共混膜.利用细胞培养、扫描电镜、HE染色等方法对共混膜的吸水率、溶胀比、细胞相容性、细胞贴附性、皮内刺激反应、急性全身毒性、体内外降解性和生物相容性进行了评价.结果表明,该共混膜具有一定吸水率,溶胀比小,无皮内刺激反应和急性全身毒性,具有良好的细胞相容性,生物可降解性和生物相容性,对成纤维细胞的生长有较强的抑制作用,是一种极具潜力的具有良好生物安全性的可吸收防粘连膜片.     

壳聚糖-透明质酸共混膜性质的研究

以溶液共混法制成不同比例的壳聚糖一透明质酸共混膜,通过观察各种共混膜的表面形态结构、结晶度、透光率等,发现在以较低比例混入透明质酸所形成的共混膜中两种高分子的相容性较好,分子间存在较强的相互作用力,形成的共混膜表面结构均匀单一。通过对共混膜理化性质的研究,发现透明质酸的混入可以有效的改变壳聚糖膜的力学特性、吸水性、吸附性以及对小分子物质的渗透性。以共混膜和壳聚糖膜为载体培养兔角膜细胞,结果发现较低比例的透明质酸可以显著提高壳聚糖膜与角膜细胞的相容性,能够有效的支持细胞在膜上生长,结果提示以一定共混比例制成的壳聚糖一透明质酸共混膜可以作为细胞体外培养的良好载体,可用于器官损伤修复以及细胞移植。     

壳聚糖-硫酸软骨素共混膜与兔角膜基质细胞相容性的研究

为了探讨硫酸软骨素对壳聚糖膜与兔角膜基质细胞相容性的影响,在制备了不同壳聚糖-硫酸软骨素共混膜的基础上,以壳聚糖-硫酸软骨素共混膜作为载体培养兔角膜基质细胞,研究兔角膜基质细胞在共混膜上贴附、生长和代谢的情况,并观察了细胞的形态结构.结果发现,硫酸软骨素的混入可以有效地提高兔角膜基质细胞在膜上的贴附和生长速度,促进蛋白质的代谢,降低共混膜对细胞的损伤,有利于细胞在膜上长成密集单层,预示了以一定比例混入硫酸软骨素可以显著提高壳聚糖膜与兔角膜基质细胞的相容性,壳聚糖-硫酸软骨素共混膜具有作为兔角膜基质细胞培养和移植载体的潜能.     

组织工程甲壳素载体支架对角膜上皮损伤修复作用的研究

探讨甲壳素载体支架培养角膜上皮细胞的可行性以及对角膜上皮的损伤修复作用。以自制的甲壳素膜片为材料,测定其厚度、含水量、透光率、机械强度、生物相容性和降解性,在甲壳素膜片上培养兔角膜上皮细胞观察其细胞相容性,构建组织工程角膜上皮,将其移植到受损的角膜上皮层,观察其修复作用。结果甲壳素膜厚度为0.20mm,含水量为81.59%,不同波长下的透光率均大于80%,干态和湿态下的载荷强度分别为6.56和29.85N,植入肌肉及皮下后炎症反应较轻,有利于细胞贴附,并能促进角膜上皮的损伤修复。该材料基本符合组织工程角膜支架材料的要求,为组织工程角膜支架的研究指明了方向。     

柞蚕丝素/壳聚糖共混膜的结构及细胞相容性

用碳化二亚胺(EDC)作为交联剂,流延法制备柞蚕丝素(ASF)/壳聚糖(CS)共混膜。用扫描电镜(SEM)、红外光谱(FT-IR)、热重分析(TG)和四甲基偶氮唑盐比色法(MTT)对膜的结构及细胞相容性进行了研究。结果显示,柞蚕丝素和壳聚糖具有较好的相容性和较强的相互作用,壳聚糖能阻碍共混膜内的丝素蛋白形成β-折叠构象。EDC能分别与柞蚕丝素及壳聚糖反应,从而对共混膜进行有效的交联,并使膜的热稳定性提高。与ASF膜或CS膜相比,共混比为80/20和40/60的ASF/CS共混膜更有利于细胞生长和增殖,作为一种新型的生物材料具有良好的研究和开发应用前景。     

淀粉/PVA膜在血浆模拟液及唾液模拟液中的体外降解行为及相关体外生物相容性评价

通过熔融浇注法制备出一系列厚度在0.05 mm～0.10 mm的淀粉/聚乙烯醇（PVA）（SP）薄膜。研究了薄膜在血浆（SBF）及唾液模拟液（SSF）中的降解行为,分析了降解过程中力学性能、失重率、溶胀度、热性能以及表面形态的变化。研究结果表明,膜在30天的降解过程中能够维持良好的尺寸稳定性和一定的力学强度。通过细胞毒性、细胞贴壁及溶血试验表征了膜的体外生物相容性。结果表明,SP膜具有良好的细胞和血液相容性。所有测试结果证明,SP薄膜是一种应用于诱导组织再生薄膜的潜在材料。      

魔芋葡甘聚糖/羧甲基纤维素钠共混膜的制备及其性能表征

以魔芋葡甘聚糖和羧甲基纤维素钠为主要原料,添加丙三醇为增塑剂,乳酸为改性剂,结合微波处理工艺,采用流延成型的方法制备出魔芋葡甘聚糖/羧甲基纤维素钠共混膜。通过FT-IR、X-射线衍射对共混膜进行性能表征,同时测定了共混膜的吸水率、力学性能和降解率。结果表明:膜有较好的降解性能和细胞相容性,作为一种潜在的术后防粘连材料将具有良好的应用前景。     

再生丝素蛋白肽/聚乙烯醇膜材料性能的实验研究

采用不同配比的再生丝素蛋白肽(SFP)与聚乙烯醇(PVA)水溶液共混的方法制备了再生丝素蛋白肽/聚乙烯醇(SFP/PVA)膜材料,对SFP/PVA膜材料进行了红外表征。通过电子拉力试验机进行了不同配比SFP/PVA膜材料的力学性能试验,结果显示配比为30/70的膜最大承受力为10.57N,拉伸强度为35.2MPa,力学性能最佳。膜材料降解性实验在人工体液中进行,结果显示其降解从SFP开始,随后与SFP结合部分的PVA在接触到水介质后逐步溶失。MTT法对HepG2细胞的促生长试验结果表明,各配比SFP/PVA膜材料均具有良好的促HepG2细胞快速黏附和生长性。综合各性能指标,配比为30/70的SFP/PVA膜具有作为组织工程支架载体的应用前景。     

Preparation and properties of a chitosan-based carrier of corneal endothelial cells

A novel chitosan-based membrane that was made of hydroxypropyl chitosan, gelatin and chondroitin sulfate was used as a carrier of corneal endothelial cells. The characteristics of the blend membrane, such as transparency, equilibrium water content, permeability, mechanical properties, protein absorption ability, hydrophilicity and surface morphology, were determined. To study the effects of the membrane on cell attachment and growth, rabbit corneal endothelial cells were cultured on this artificial membrane. The biodegradability and biocompatibility of the blend membrane were in vivo evaluated by its implantation into the muscle of the rats. Glucose permeation results demonstrated that the blend membrane had higher glucose permeability than natural human cornea. Scanning electron microscopy (SEM) analysis of the membranes demonstrated that no fibrils were observed. As a result, the optical transparency of the membrane was as good as the natural human cornea. The average value of tensile strength of the membrane was 13.71 MPa for dry membrane and 1.48 MPa for wet membrane. The value of elongation at break of the wet was 45.64%. The cultured rabbit corneal endothelial cells formed a monolayer on the blend membrane which demonstrated that the membrane was suitable for corneal endothelial cells to attach and grow. In addition, the membranes in vivo showed a good bioabsorption property. The mild symptoms of inflammation at sites of treatment could be resolved as the implant was absorbed by the host. The results of this study demonstrated that the hydroxypropyl chitosan-chondroitin sulfate-gelatin blend membrane can potentially be used as a carrier for corneal endothelial cell transplantation.     

Fabrication and characters of a corneal endothelial cells scaffold based on chitosan

A novel chitosan-based membrane that made of hydroxyethyl chitosan, gelatin and chondroitin sulfate was used as a carrier of corneal endothelial cells. The characteristics of the blend membrane including transparency, equilibrium water content, ion and glucose permeability were determined. The results showed that the optical transparency of the membrane was as good as the natural human cornea. The water content of this scaffold was 81.32% which was remarkably close to the native cornea. The membrane had a good ion permeability and its glucose permeability was even higher than natural human cornea. The cultured rabbit corneal endothelial cells formed a monolayer on the membrane. The results demonstrated that the membrane was suitable for corneal endothelial cells to attach and grow on it. In addition, the membranes in vivo could be degraded steadily with less inflammation and showed a good histocompatibility. These results demonstrated that the hydroxyethyl chitosan-chondroitin sulfate-gelatin blend membrane can potentially be used as a carrier for corneal endothelial cell transplantation.     

Tissue-engineered membrane based on chitosan for repair of mechanically damaged corneal epithelium

In this study, hydroxyethyl chitosan (HECTS), a water-soluble derivative of chitosan, was used to create a blend membrane and its function and application as a scaffold in repair of mechanically damaged corneal epethelia were examined. The results showed that HECTS significantly promoted growth of corneal epithelial cells (CEpCs) in vitro and that CEpCs grew well on the HECTS-based blend membrane. Fluorescent imaging showed that CEpCs were interconnected and formed layers on the membrane. After transplanting the CEpCs-seeded membranes onto the damaged corneal epithelium, we found that the epithelium was repaired faster compared with control. The repaired corneal epithelium with the membrane had a more compact structure and a smoother surface than control when observed by histology and scanning electron microscope. These results demonstrate an ability of the tissue-engineered scaffold to speed up the repair of mechanically damaged corneal epithelium.     

Hydrogel membranes based on genipin-cross-linked chitosan blends for corneal epithelium tissue engineering

Novel polymeric hydrogel scaffolds for corneal epithelium cell culturing based on blends of chitosan with some other biopolymers such as hydroxypropylcellulose, collagen and elastin crosslinked with genipin, a natural substance, were prepared. Physicochemical and biomechanical properties of these materials were determined. The in vitro cell culture experiments with corneal epithelium cells have indicated that a membrane prepared from chitosan-collagen blend (Ch-Col) provided the regular stratified growth of the epithelium cells, good surface covering and increased number of the cell layers. Ch-Col membranes are therefore the most promising material among those studied. The performance of Ch-Col membranes is comparable with that of the amniotic membrane which is currently recommended for clinical applications.     

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)—the effect of biomolecular ligands to balance cell adhesion and stimulated detachment

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na⁺/K⁺-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.     

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)—the effect of biomolecular ligands to balance cell adhesion and stimulated detachment

Abstract

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na⁺/K⁺-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.     

An attempt to construct the stroma of cornea using primary cultured corneal cells

The number of patients currently awaiting corneal transplantation has resulted in the need to develop an artificial corneal replacement. In this study, we aimed to construct the corneal stroma using non-transformed corneal cells and a perfusion cell culture method. Corneal cells isolated from chicken embryos or rabbit and were embedded in the alkaline solubilized collagen gels crosslinked by TSG (Pentaerythritol polyethyleneglycol ether tetrasuccinimidyl glutarate). During culture, the majority of cells migrated from inside of the gel. The chicken and rabbit cells changed their morphology and stratified structures were constructed within the gels. These microstructures were similar to the natural corneal tissue. TEM analysis was performed to confirm the nano-microstructure of the constructs. Contrary to expectation, the cornea-like nanostructure of collagen fibrils was not observed within the gels. Further study including for example, such as the addition of dynamic stress or co-culture with endothelial cells, are therefore required in order to produce artificial constructs with the same superstructure as natural corneal tissue.     

Smartphone-based imaging of the corneal endothelium at sub-cellular resolution

This aim of this study was to test the feasibility of smartphone-based specular microscopy of the corneal endothelium at a sub-cellular resolution. Quantitative examination of endothelial cells is essential for evaluating corneal disease such as determining a diagnosis, monitoring progression and assessing treatment. Smartphone-based technology promises a new opportunity to develop affordable devices to foster quantitative examination of endothelial cells in rural and underserved areas. In our study, we incorporated an iPhone 6 and a slit lamp to demonstrate the feasibility of smartphone-based microscopy of the corneal endothelium at a sub-cellular resolution. The sub-cellular resolution images allowed quantitative calculation of the endothelial cell density. Comparative measurements revealed a normal endothelial cell density of 2978 cells/mm² in the healthy cornea, and a significantly reduced cell density of 1466 cells/mm² in the diseased cornea with Fuchs’ dystrophy. Our ultimate goal is to develop a smartphone-based telemedicine device for low-cost examination of the corneal endothelium, which can benefit patients in rural areas and underdeveloped countries to reduce health care disparities.