明胶固定化改性壳聚糖膜的制备与性能表征

用碳二亚胺缩合剂将明胶固定于壳聚糖膜表面,采用傅立叶红外光谱、X射线光电子能谱表征改性膜的结构,结果显示明胶成功固定于壳聚糖膜表面.透光率、吸水率和接触角测试表明,改性膜的透光率最高可达98%左右,能满足角膜修复对透光性的要求.改性后膜的吸水率提高到97.6%,接触角下降到73.4°,亲水性提高.以壳聚糖膜和明胶固定化改性壳聚糖膜为载体培养人成纤维细胞,结果显示细胞在明胶固定化改性壳聚糖膜上的生长情况优于壳聚糖膜,改性膜具有良好的细胞亲和性.这种明胶固定化改性壳聚糖膜有望成为一种角膜修复材料.     

PNIPAAm-g-EVA/明胶支架双层人工皮肤的细胞相容性研究

构建了PNIPAAm-g-EVA/明胶支架复合人工皮肤样品,并初步考察了该人工皮肤的细胞相容性。由于明胶支架的生物相容性已经得到充分验证,主要选择成纤维细胞为实验细胞,PNIPAAm-g-EVA膜为研究对象。采用含有胶原酶的消化液消化组织块,在较短时间内可获得足够数量的成纤维细胞。成纤维细胞与PNIPAAm-g-EVA膜共培养结果表明,成纤维细胞在膜表面粘附力强,生长、增殖情况良好。说明与PNIPAAm-g-EVA膜共培养并未影响细胞的正常生理功能,PNIPAAm-g-EVA具有良好的细胞相容性。     

静电自组装表面修饰HA/PA6膜:表征及其细胞相容性研究

通过表面改性引入活性生物分子可以用来增强材料表面的生物相容性。采用静电自组装技术将明胶和聚乙烯亚胺引入羟基磷灰石/聚酰胺6(HA/PA6)材料表面,最终获得大分子修饰的复合材料。利用水接触角测量(WCA)、X射线光电子能谱(XPS)和原子力显微镜(AFM)测试手段对表面改性前后HA/PA6膜的表面化学、亲水性和表面形貌进行表征。并研究了其对细胞活性的影响。结果表明,改性后膜表面的亲水性增强,粗糙度增大。体外细胞实验表明,明胶涂层后的HA/PA6表面细胞更利粘附、铺展和生长。该固定方法模拟细胞外基质组成制备出的生物活性膜能进一步满足生物医学工程需求。     

角膜内皮细胞载体壳聚糖基共混膜的制备及性质研究

为探讨壳聚糖基共混膜作为组织工程化角膜内皮细胞载体的可行性,制备了羟乙基壳聚糖/明胶/硫酸软骨素共混膜,并评价其性质。结果表明该膜片具有良好的透明度,适宜的含水量;细胞毒性0～1级;体外培养的角膜内皮细胞能够很好地贴附和生长于共混膜上;植入大鼠肌肉内,能够稳定地降解,诱发的炎症反应明显低于不可降解手术缝合线,说明该膜片具有良好的组织相容性,有望作为角膜细胞载体构建组织工程化角膜。     

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。     

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。     

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。     

羟基磷灰石/钛合金骨替代材料体外培养成骨细胞的实验研究

实验选用小鼠头盖骨成骨细胞,采用体外细胞培养技术对具有羟基磷灰石涂层的钛合金(HA/Ti)与未经过表面改性的钛合金两种骨替代材料进行细胞相容性评价,动态观察两种骨替代材料对成骨细胞生长、附着的影响。结果表明两种骨替代材料对成骨细胞生长无抑制作用,未发生细胞毒性反应,细胞在两种材料表面均能正常粘附、生长、增殖,均具有良好的细胞附着形态和细胞增殖率,而HA/钛合金材料具有更好的成骨性,是一种骨细胞相容性良好的骨替代材料。     

聚己内酯/明胶电纺纳米纤维支架的制备与细胞相容性研究

构建了聚己内酯/明胶(PCL/GE)电纺纳米纤维支架,考察了该支架在模拟体液中的降解速率及其细胞相容性。结果表明:相比于PCL纤维膜,支架表面具有良好的亲水性,且PCL/GE复合纤维支架降解速率得到极大提高;大鼠脂肪来源间充质干细胞(marrow stromal cells,MSCs)在纤维支架表面粘附力强,生长、增殖情况良好。     

电气石/壳聚糖复合纤维的表征及细胞相容性

以壳聚糖为基体,电气石为分散相,采用溶液纺丝法制备电气石/壳聚糖复合纤维,利用光学显微镜、扫描电镜以及红外光谱仪对材料进行表征。电气石/壳聚糖复合纤维与人骨肉瘤细胞株（MG63）体外共培养,初步评价了材料的细胞相容性。结果显示,电气石颗粒在复合纤维中分散均匀且被壳聚糖包裹,纤维表面无裸露电气石。细胞在电气石/壳聚糖复合纤维表面黏附及生长增殖状况良好,材料对细胞无明显毒性。该材料有望成为一种良好的创伤修复敷料。     

Preparation of chitosan films using different neutralizing solutions to improve endothelial cell compatibility

The development of chitosan-based constructs for application in large-size defects or highly vascularized tissues is still a challenging issue. The poor endothelial cell compatibility of chitosan hinders the colonization of vascular endothelial cells in the chitosan-based constructs, and retards the establishment of a functional microvascular network following implantation. The aim of the present study is to prepare chitosan films with different neutralization methods to improve their endothelial cell compatibility. Chitosan salt films were neutralized with either sodium hydroxide (NaOH) aqueous solution, NaOH ethanol solution, or ethanol solution without NaOH. The physicochemical properties and endothelial cell compatibility of the chitosan films were investigated. Results indicated that neutralization with different solutions affected the surface chemistry, swelling ratio, crystalline conformation, nanotopography, and mechanical properties of the chitosan films. The NaOH ethanol solution-neutralized chitosan film (Chi-NaOH/EtOH film) displayed a nanofiber-dominant surface, while the NaOH aqueous solution-neutralized film (Chi-NaOH/H₂O film) and the ethanol solution-neutralized film (Chi-EtOH film) displayed nanoparticle-dominant surfaces. Moreover, the Chi-NaOH/EtOH films exhibited a higher stiffness as compared to the Chi-NaOH/H₂O and Chi-EtOH films. Endothelial cell compatibility of the chitosan films was evaluated with a human microvascular endothelial cell line, HMEC-1. Compared with the Chi-NaOH/H₂O and Chi-EtOH films, HMECs cultured on the Chi-NaOH/EtOH films fully spread and exhibited significantly higher levels of adhesion and proliferation, with retention of the endothelial phenotype and function. Our findings suggest that the surface nanotopography and mechanical properties contribute to determining the endothelial cell compatibility of chitosan films. The nature of the neutralizing solutions can affect the physicochemical properties and endothelial cell compatibility of chitosan films. Therefore, selection of suitable neutralization methods is highly important for the application of chitosan in tissue engineering.     

Fabrication and characterization of chemically crosslinked keratin films

A film was prepared by casting the reduced keratin solution after mixing with chemical crosslinkers such as ethylene glycol diglycidyl ether (EGDE) and glycerol diglycidyl ether (GDE). Although the keratin without an addition of a plasticizer gave rise to a fragile film, the keratin treated with those crosslinkers gave a tenacious and flexible film similarly to the mixing with chitosan, which we previously reported [Biomaterials 23 (2002) 817]. Chemically crosslinked keratin films stretched longer than keratin–chitosan composite film and showed much improved waterproof characteristics, that is, no swelling was observed under acidic and neutral aqueous conditions and they swelled to a lesser extent under basic condition, although keratin–chitosan composite film significantly swelled in acidic and neutral solution. Chemically crosslinked keratin film as well as keratin–chitosan composite film maintained their mechanical properties upon re-drying after swelling.When mouse fibroblast cells are cultured on crosslinked keratin-coated surface, the attachment of cells is a little delayed compared with the cells on keratin, keratin–chitosan composite and chitosan films. However, the cells once attached to the crosslinked keratin surface well proliferated, suggesting the biocompatibility of chemically crosslinked keratin film.     

Rapid adhesion and proliferation of keratinocytes on the gold colloid/chitosan film scaffold

The gold colloid/chitosan film scaffold, which could enhance the attached ratio and accelerate proliferation of newborn mice keratinocytes, was fabricated by nanotechnology and self-assembly technology. This nanometer scaffold was characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The keratinocytes were cultured and observed on three different extracellular matrices (ECM): gold colloid/chitosan film scaffold, chitosan film and cell culture plastic (control groups). 6 h, 12 h, 24 h after inoculation, the cell attached ratios were calculated respectively. In comparison to control groups, this scaffold could significantly (P < 0.01) increase the attached ratio of keratinocytes and promote their growth. Meanwhile, there were not any fusiform fibroblasts growing on this scaffold. The rapidly proliferating keratinocytes were indentified and characterized by immunohistochemistry and transmissive electron microscope (TEM), which showed the cells maintain their biological activity well. The results indicated that gold colloid/chitosan film scaffold was nontoxic to keratinocytes, and was a good candidate for wound dressing in skin tissue engineering.     

新型壳聚糖/PBLG-co-PGA复合膜的研究

采用壳聚糖和聚谷氨酸苄酯-co-聚谷氨酸(PBLG-co-PGA)通过1-乙基-3-(3-二甲基氨基丙基)碳化二亚胺(EDC)与N-羟基琥珀酰亚胺(NHS)交联得到复合膜.采用FTIR和XRD对复合膜的结构进行了表征,同时对复合膜的吸水率进行了研究.研究结果表明,复合膜中壳聚糖和PBLG-co-PGA分子之间成功地发生了交联反应,二者之间的交联与氢键作用改变了二者原有的结晶结构,这对复合膜的性能有着重要的影响.以复合膜为载体培养软骨细胞发现,复合膜与壳聚糖膜相比,具有更高的细胞粘附率,预示着该复合膜是一种具有良好应用前景的生物医用材料.     

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.     

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.     

表面活性剂对壳聚糖成膜体系物理特性的影响

目的 为研究不同表面活性剂对壳聚糖成膜体系物理与力学性能的影响,以期制备性能优良的壳聚糖膜。方法 文中配制质量分数为1%的壳聚糖膜液,以壳聚糖质量分数为30%的甘油为增塑剂,分别以质量分数为0.05%的吐温20、40、80,司盘20、40、80为表面活性剂,采用流延法制备可食膜。考察膜液流变性质、表面张力、Zeta电位,成膜力学性能、水分敏感性（水蒸气透过率、溶胀性、溶解性）、透光性,并利用AHP–CRITIC联合评价法对膜性能进行综合评价。结果 在质量分数为1%时,壳聚糖膜液表现为弱胀塑性;表面活性剂的加入能够有效降低膜液表面张力,使其保持稳定状态（Zeta电位>30 mV）;成膜均为淡黄色半透明状,透光范围为45%～77%;对同一系列表面活性剂,随着亲水亲油平衡值（HLB）的增加,成膜力学性能下降,但阻水能力增强,同时,吐温添加组膜的力学性能均优于司盘添加组,而阻水性能则相反。结论 综合评价膜体系性能发现,加入司盘20的壳聚糖膜得分最高,吐温系列最高分为吐温20。研究结果为成膜体系中表面活性剂的选择提供了理论依据。     

Bovine pericardium coated with biopolymeric films as an alternative to prevent calcification: In vitro calcification and cytotoxicity results

Bovine pericardium, for cardiac valve fabrication, was coated with either chitosan or silk fibroin film. In vitro calcification tests of coated and non coated bovine pericardium were performed in simulated body fluid solution in order to investigate potential alternatives to minimize calcification on implanted heart valves. Complementary, morphology was assessed by scanning electron microscopy — SEM; X-ray diffraction (XRD) and infrared spectroscopy (FTIR-ATR) were performed for structural characterization of coatings and biocompatibility of chitosan. Silk fibroin films were assayed by in vitro cytotoxicity and endothelial cell growth tests. Bovine pericardium coated with silk fibroin or chitosan did not present calcification during in vitro calcification tests, indicating that these biopolymeric coatings do not induce bovine pericardium calcification. Chitosan and silk fibroin films were characterized as non cytotoxic and silk fibroin films presented high affinity to endothelial cells. The results indicate that bovine pericardium coated with silk fibroin is a potential candidate for cardiac valve fabrication, since the affinity of silk fibroin to endothelial cells can be explored to induce the tissue endothelization and therefore, increase valve durability by increasing their mechanical resistance and protecting them against calcification.     

Cell supports of chitosan/hyaluronic acid and chondroitin sulphate systems. Morphology and biological behaviour

Films and sponges of chitosan (CHI), chitosan/hyaluronic acid (CHI–HA) and chitosan/chondroitin sulphate (CHI–CHOS), were prepared by film deposition or lyophilization (sponges), avoiding the formation of interpolyelectrolyte complexes. The biological behaviour of the systems was analysed by studying the cell behaviour using a fibroblast cell line and standard biological MTT and Alamar Blue tests. The morphology of films, sponges and cell seeded samples was analysed by ESEM. The results obtained indicate that all the systems can be considered as good supports for cell adhesion and proliferation, but there is specific activation of the proliferative process in the presence of hyaluronic acid and chondroitin sulphate.     

Structure,morphology and cell affinity of poly(l-lactide) films surface-functionalized with chitosan nanofibers via a solid–liquid phase separation technique

Poly(l-lactide) films with a nano-structured surface by immobilizing chitosan nanofibers (CSNFs) for improving the cell affinity were fabricated via a solid-liquid phase separation technique. The successful grafting of CSNFs on the surface of poly(l-lactide) films was confirmed by the binding energy of N1s at 398.0 eV in the X-ray photoelectron spectroscopy and the amide I and II bands of chitosan at 1650 and 1568 cm^? 1 in the Fourier transform infrared spectroscopy. Compared with the poly(l-lactide) film, the hydrophilicity was improved with a lower water contact angle of 83.3 ± 1.9° and 75.3 ± 2.5° for the CSNFs-grafted and CSNFs-grafted/anchored poly(l-lactide) films respectively. The scanning electron microscopy and atomic force microscopy analyses showed that the grafted CSNFs with 50–500 nm in diameter were randomly arranged on the film surface and entangled with the anchored CSNFs on the outermost layer. The 3T3 fibroblasts culture indicated cells tended to attach and stretch along the CSNFs on the film surface. The cell viability measurement revealed that among all the samples, the film with both grafted and anchored CSNFs exhibited the highest cell proliferation rate that was twice as much of the poly(l-lactide) film at 7 d. Herein, engineering a nano-structured surface by solid–liquid phase separation will be a promising tool for surface modification of biomaterials.