仿去上皮羊膜微纳结构壳聚糖改性胶原—透明质酸复合电纺膜的研制及其性能研究
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摘要
第一部分仿去上皮羊膜微纳结构壳聚糖改性胶原透明质酸复合电纺膜的研制
目的：利用高压静电纺丝与静电吸附表面改性技术探讨制备仿去上皮人羊膜超微结构壳聚糖改性胶原-透明质酸复合电纺膜的可行性,获得胶原以乙酸为溶剂制备纳米纤维以及在聚环氧乙烷为促电纺剂下胶原复合透明质酸制备纳米纤维的最佳浓度配比,在此基础上联合交联和静电吸附表面改性技术优化复合电纺膜的稳定性与表面微纳结构,以期为眼表损伤修复用覆膜的研究提供实验基础,拓宽静电纺丝纳米技术和静电吸附表面改性技术在生物医学领域的应用范围。
方法：首先对人羊膜组织微观形貌进行细致观察分析,在此基础上,采用具有良好生物相容性的天然高分子材料胶原、透明质酸和人工合成高分子材料聚环氧乙烷为主要的研究原料,通过对电纺原材料浓度配比的调整,优化静电纺丝各项参数,制备复合电纺薄膜。通过戊二醛交联和透明质酸与壳聚糖静电吸附作用,改善电纺薄膜的稳定性和微纳结构,肉眼观察评估所制备的复合电纺膜的透明性和可操作性,扫描电镜检测薄膜表面和断面微结构,分析评价壳聚糖改性胶原-透明质酸复合电纺膜与去上皮人羊膜组织在微观形貌上的相似性。
结果：去上皮人羊膜基底膜和基质是由平均直径分别为52.9±17.5nm和41.2±14.7nm的胶原纤维无规则排列组成的片层结构。在25℃,40%湿度环境中,推进速度为0.1ml/h,接收距离为10cm,电压在10-15KV之间时,电纺处于稳定状态。10%w/v胶原以90%v/v乙酸为溶剂电纺可以制备平均直径为79.1±27.8nm的纤维薄膜；10%w/vCOL、0.1%w/vHA和7%w/vPEO以8：1：1体积比电纺可以制备平均直径为106.6±40.9nm,表面光滑,具有良好分散性的纳米纤维薄膜。戊二醛交联不仅使复合电纺膜纳米纤维形态和直径(平均直径为66.3±24.8nm)更接近于羊膜基底膜和基质,而且大大改善了薄膜的透明性和可操作性；在不影响复合膜纤维形态和直径前提下复合膜实现壳聚糖表面改性并与去上皮人羊膜微观形貌更加相近。
结论：本研究首先证实了胶原、透明质酸复合聚环氧乙烷电纺制备具有纳米级直径均一纤维薄膜的可行性；并在此基础上,通过戊二醛交联和静电吸附表面改性方法对复合薄膜透明性和可操作性进行优化,实现了对去上皮人羊膜组织微纳结构的人工模拟,为临床眼表损伤修复用眼表覆膜的研究和应用奠定了可靠的实验基础。
第二部分仿去上皮羊膜微纳结构壳聚糖改性胶原-透明质酸复合电纺膜的物理化学性能研究
目的：对胶原-透明质酸复合电纺膜、交联膜和壳聚糖改性膜的物理化学性能进行表征,探讨交联和表面改性后处理对薄膜理化性能的影响,对比分析壳聚糖改性胶原-透明质酸复合电纺膜与去上皮人羊膜组织相应性能的差异,为进一步优化该类眼表修复覆膜研究奠定实验基础,同时也为临床上眼表组织修复支架的制备提供新的方法和研究思路。
方法：采用紫外-可见光光度仪对不同薄膜的可见光透过率进行检测,傅里叶转换红外线光谱分析不同薄膜的化学组成及变化,万能测试机对薄膜的抗拉强度、杨氏模量和最大伸长率进行测试分析,静态水接触角对不用薄膜的亲疏水性进行分析比较。
结果：干燥状态下,在555nm波长处交联膜和壳聚糖表面改性膜的可见光透过率分别为70%和75%,而羊膜仅为40%；在湿润状态下,在555nm波长处交联膜和壳聚糖表面改性膜的可见光透过率分别增加到88%和89%,羊膜在该处的可见光透过率增加到74%。胶原-透明质酸复合电纺膜、交联膜和壳聚糖改性膜的红外光谱图中均存在胶原、透明质酸特征性吸收峰,但交联膜和壳聚糖改性膜在2000-1000cm-1附近的吸收峰强度降低,位于3314cm-1、1619cm-1和1430cm-1附近的吸收峰信号变弱；壳聚糖改性膜中酰胺A,酰胺Ⅰ,Ⅱ键吸收峰波幅较交联膜增强；在交联膜和壳聚糖改性膜中,均未检测到聚环氧乙烷特征性吸收峰；在壳聚糖改性膜中检测到所有壳聚糖特征性吸收峰；壳聚糖改性膜与去上皮人羊膜相比均具有胶原、透明质酸特征性吸收峰,但壳聚糖改性膜在1750-1000cm-1范围内的吸收峰波幅较去上皮人羊膜者低。壳聚糖改性膜的平均抗拉强度为31.39±6.61Mpa,略低于去上皮人羊膜(39.72±4.43Mpa),而在同等强度外力作用时,形变量为交联膜>壳聚糖改性膜>去上皮人羊膜。交联膜和壳聚糖改性膜的平均水接触角分别为18.6±1.70和23.9±2.60,均小于去上皮人羊膜(63.4±3.30)。
结论：胶原-透明质酸交联膜和壳聚糖改性膜具有优于去上皮人羊膜组织的良好透明性；经电纺、交联和壳聚糖表面改性三步法处理后胶原和透明质酸仍保持原有活性不变；壳聚糖改性膜具有与去上皮人羊膜相似的化学组成；虽然复合膜的抗拉强度稍逊于去上皮人羊膜组织,但其更好的弹性和更高的亲水性对于我们后续细胞和组织相容性研究奠定了有利的理论基础。
第三部分仿去上皮羊膜微纳结构壳聚糖改性胶原-透明质酸复合电纺膜生物相容性研究
目的：分析评价壳聚糖改性胶原-透明质酸复合电纺膜的生物相容性,为仿羊膜微纳结构眼表覆膜的临床应用和产业化提供理论依据和实验基础,从根本上推进眼表修复用仿生人工薄膜材料的研制。
方法：以去上皮人羊膜为对照,采用体外细胞培养法将人角膜上皮、结膜上皮细胞接种于壳聚糖改性膜上,扫描电镜观察细胞的粘附形态,活死细胞染色检测薄膜对细胞的毒性作用,CCK-8法比较细胞在薄膜表面的增殖能力；构建大鼠角膜急性碱烧伤模型,荧光素钠和苏木精-伊红染色观察壳聚糖改性膜移植后角膜上皮再生水平,肉眼观察基质水肿和炎症反应情况,评价薄膜的可操作性和在体生物相容性。
结果：角膜上皮细胞在壳聚糖改性膜上具有类似于正常角膜上皮的无规则扁平样外观,结膜上皮细胞在该膜上表现出细胞与材料相互融合现象；接种24h后在壳聚糖改性膜上角膜上皮细胞可检测到极少量的细胞死亡而结膜上皮细胞未检测到细胞死亡；细胞的增殖能力随培养时间的延长而增强,差异有统计学意义(p<0.05),而同种细胞在不同薄膜上的增殖能力无明显差异(p=1.12)；壳聚糖改性膜在大鼠急性碱烧伤角膜表面保持良好的透明性,与基质面贴合紧密；薄膜下可见角膜上皮再生并与其下基质粘连紧密,角膜基质水肿和炎症反应均减轻。
结论：壳聚糖改性胶原-透明质酸复合电纺膜具有良好的细胞相容性,在动物角膜急性碱烧伤模型中表现出良好的可操作性和透明性,具有与去上皮人羊膜组织相似的促上皮化、抑制基质水肿和炎症反应特性,本研究结果为探寻仿羊膜结构与功能材料的研制进而为眼表修复用仿生薄膜材料的研究奠定了扎实的实验基础,为临床上眼表疾病的治疗提供了一条新的途径。
Part I Development of chitosan modified collagen-hyaluronic acid compositeelectrospinning film with biomimetic micro-nano structure ofdenuded amniotic membrane
Objective
The purpose of this study was to investigate the feasibility of chitosan modified collagen-hyaluronic acid composite film with biomimetic micro-nano structure of denuded amniotic membrane by using electrospinning and electrostatic adsorption surface modification technology. Electrospinning agent concentration ratio between collagen and its solvent acetic acid as well as with the use of poly (ethylene oxide), collagen composite hyaluronic acid electrospinning film were facilitated. On the basis of the above results, by using chemical cross-linking and electrostatic adsorption surface modification technology to optimize the stability and surface micro-nano structure of the composite electrospinning film, so as to provide an experimental basis on the studies of the ocular surface damage repair film and to broaden the range of applications of electrospinning nanotechnology and electrostatic adsorption surface modification technology in the biomedical field.
Methods
On the basis of careful observation and analysis of the morphology of human amniotic membrane, we used natural polymer materials of collagen, hyaluronic acid and a synthetic polymer material polyethylene oxide as the main raw materials to prepare the composite electrospinning film. The ratio of the concentration of electrospinning raw materials and the parameters of the electrospinning were optimized. Glutaraldehyde cross-linking and electrostatic adsorption between hyaluronic acid and chitosan were used to improve the the stability and micro-and nano-structure of the electrospinning film. Transparency and operability of the composite electrospun fiber films were observed. Surface and cross-section microstructure of the films were detected by scanning electron microscopy. The similarity of the microscopic morphology between chitosan modified collagen-hyaluronic acid composite electro spinning film and denuded amniotic membrane was also evaluated.
Results
The basement membrane and matrix of denuded human amniotic membrane were composed of lamellar structure consisting of irregular collagen fibers with the average diameter was52.9±17.5nm and41.2±14.7nm, respectively. Under25°C and40%humidity, stable electrospinning state could be achieved with advancing speed for0.1ml/h,10cm receiver distanc and10-15kV voltage.10%w/v collagen with90%v/v acetic acid as solvent could be electrospun successfully, the average fiber diameter was79.1±27.8nm.10%w/v COL,0.1%w/v HA and7%w/v PEO with8:1:1volume ratio could prepare film with the average diameter was106.6±40.9nm. Smooth surface and a good dispersion of the nano-fiber diameter could be observed. Glutaraldehyde cross-linking not only rendered the morphology and nano-fiber diameters of composite electrospinning film similar to the amniotic membrane (average diameter of66.3±24.8nm), but also greatly improved the transparency and operability of the film. Without affecting fiber morphology and diameter the composite film was modified by chitosan,which made the composite membrane surface much similar to denuded human amniotic membrane.
Conclusion
This study was first confirmed that collagen, hyaluronic acid and polyethylene oxide could be prepared by electrospinning to fabricate uniform nanoscale fiber diameter. glutaraldehyde crosslinking and electrostatic adsorption surface-modification could optimize the transparency and operability of the composite film. Artificial simulation of the micro-nano structure of denuded human amniotic membrane has been achieve. This will lay a reliable experimental basis for clinical ocular surface damage repair.
Part II Physical and chemical properties of chitosan modified collagen-hyaluronic acid composite electrospinning film with biomimeticmicro-nano structure of denuded amniotic membrane
Objective
The purpose of this study was to investigate the physical and chemical properties of the collagen-hyaluronic acid composite electrospun film, post-crosslinking film and chitosan modified film. To explore the influence of crosslinking and surface modification on the physical and chemical properties of the films. Comparatively analyze the differences of corresponding performance between chitosan-modified collagen-hyaluronic acid composite electrospinning film and denuded human amniotic membrane. To lay the experimental foundation of further optimization of this kind of ocular surface repair film. Also to prepare new methods and research ideas to the fabrication of tissue repair scaffolds in clinic.
Methods
UV-visible spectrophotometer was used to detect the visible light transmittance of the films. Fourier transform infrared spectral analysis was used to analyze the chemical composition and changes in the films. The tensile strength, Young's modulus and the maximum elongation of the film were detected by the universal testing machine. Static water contact angle was tested to analyzed the film's hydrophobicity and hydrophilicity.
Results
In dry state the visible light transmittance at the wavelength of555nm were70%and75%in the crosslinked film and chitosan modified membrane, respectively. While that of amniotic membrane was only40%. In the wet state, at the wavelength of555nm, the visible light transmittance of the crosslinked film and chitosan surface modified membrane was increased to88%and89%, respectively, that of the amniotic membrane was increased to74%. In the infrared spectra, characteristic absorption peak of Collagen and hyaluronic acid could be observed on the composite electrospun film, crosslinked film and chitosan modified film. At2000-1000cm-1the absorption peaks had lower intensity. At3314cm-1,1619cm-1and1430cm-1the absorption peaks had weaker signals. In chitosan modified film, the amide A, amide I and II chemical bond had high absorption peak than the crosslinked film. There was no absorption peak of polyethylene was detected on crosslinked film and chitosanmodified film. The characteristic absorption peak of chitosan were all detected on the chitosan modified membrane. In comparation with denuded human amniotic membrane, the chitosan modified film had similar absorption peaks of collagen and hyaluronic acid. While the absorption peak in the range of1750-1000cm-1was lower than that of denuded human amniotic membrane. The average tensile strength of the chitosan modified membrane was31.39±6.61MPa, which slightly lower than that of denuded human amniotic membrane (39.72±4.43MPa). Under the equal strength of external force, the deformation level was cross-linked film> chitosan modified film> denuded human amniotic membrane. The average water contact angle of the crosslinked film and the chitosan modified film were18.6±1.7°and23.9±2.6°, which were less than that of the denuded human amnion (63.4±3.3°).
Conclusion
Collagen-hyaluronic acid crosslinked film and chitosan modified membrane had better transparency than that of denuded human amniotic membrane. The three-step treatment of electrospinning. crosslinking and chitosan surface modification did not change the original activity of collagen and hyaluronic acid. Chitosan modified film had almost the same chemical compositon of denuded human amniotic membrane. Although the tensile strength of the composite membrane was slightly inferior to denuded human amniotic membrane, but its better flexibility and higher hydrophilicity laid a favorable theoretical basis to our subsequent cellular and tissue compatibility.
Part IH Biocompatibilities of chitosan modified collagen-hyaluronic acidcomposite electrospinning film with biomimetic micro-nano structureof denuded amniotic membrane
Objective
The purpose of this study was to analyze and evaluate the biocompatibility of chitosan modified collagen-hyaluronic acid composite electrospun membrane. Lay a theoretical and experimental basis to the clinical application and industrialization of the biomimetic amniotic membrane in the ocular surface reconstruction. To fundamentally promote the development bionic artificial film materials in ocular surface repair.
Methods
Human corneal epithelial cells and conjunctival epithelial cells were seeded on chitosan modified film. Scanning electron microscopy was used to observed the morphology of cells, live/dead assay was used to detect toxic effects of the film to the cells. CCK-8method was used to compare the proliferation of cells on the surface of film, denuded human amniotic membrane was used as control. The rat acute corneal alkali burn model was established, sodium fluorescein and hematoxylin-eosin staining were use to detect the level of corneal epithelial regeneration. The situation of stromal edema and inflammation were visually observed, the operability and in vivo biocompatibility of the film were evaluated, denuded human amniotic membrane was used as control.
Results
Corneal epithelial cells in chitosan modified collagen-hyaluronic acid composite electrospun membrane showd a flat-like appearance which was similar to that of normal corneal epithelium. Conjunctival epithelial cells in the film performed a cell-material merging phenomenon. Live/dead assay showed that24hours after incubation a small amount of corneal epithelial cell death was detected on the composite electro-spun membrane as well as denuded amniotic membrane,while no conjunctival epithelial cell death was detected on both two kind of membrane. Cell proliferation was enhanced with prolonged incubation time, the difference was statistically significant (p<0.05). The same kind of cell proliferation on different films was not significant different (p=1.12). Chitosan modified membrane transplanted on the surface of the acute alkali burned cornea maintained good transparency, and tightly bonded with the substrate surface. Promotion of corneal epithelial regeneration, close adhesion between re-newed corneal epithelial cells and matrix and reduction of stromal edema and inflammation could be observed on the chitosan modified membrane transplanted eye, which was similar to that of denuded human amniotic membrane.
Conclusion
Chitosan modified collagen-hyaluronic acid composite electrospinning film has good cell compatibility. On animal acute corneal alkali burn model it shows good operability and transparency. Its properties of epithelium promotion, stromal edema inhibition as well as inflammatory response suppression lay a solid experimental basis for clinical explore of the materials research imitating the structure and function of amniotic membrane in ocular surface repair. Also this provides a new way in the treatment of ocular surface disease.
目的：利用高压静电纺丝与静电吸附表面改性技术探讨制备仿去上皮人羊膜超微结构壳聚糖改性胶原-透明质酸复合电纺膜的可行性,获得胶原以乙酸为溶剂制备纳米纤维以及在聚环氧乙烷为促电纺剂下胶原复合透明质酸制备纳米纤维的最佳浓度配比,在此基础上联合交联和静电吸附表面改性技术优化复合电纺膜的稳定性与表面微纳结构,以期为眼表损伤修复用覆膜的研究提供实验基础,拓宽静电纺丝纳米技术和静电吸附表面改性技术在生物医学领域的应用范围。
方法：首先对人羊膜组织微观形貌进行细致观察分析,在此基础上,采用具有良好生物相容性的天然高分子材料胶原、透明质酸和人工合成高分子材料聚环氧乙烷为主要的研究原料,通过对电纺原材料浓度配比的调整,优化静电纺丝各项参数,制备复合电纺薄膜。通过戊二醛交联和透明质酸与壳聚糖静电吸附作用,改善电纺薄膜的稳定性和微纳结构,肉眼观察评估所制备的复合电纺膜的透明性和可操作性,扫描电镜检测薄膜表面和断面微结构,分析评价壳聚糖改性胶原-透明质酸复合电纺膜与去上皮人羊膜组织在微观形貌上的相似性。
结果：去上皮人羊膜基底膜和基质是由平均直径分别为52.9±17.5nm和41.2±14.7nm的胶原纤维无规则排列组成的片层结构。在25℃,40%湿度环境中,推进速度为0.1ml/h,接收距离为10cm,电压在10-15KV之间时,电纺处于稳定状态。10%w/v胶原以90%v/v乙酸为溶剂电纺可以制备平均直径为79.1±27.8nm的纤维薄膜；10%w/vCOL、0.1%w/vHA和7%w/vPEO以8：1：1体积比电纺可以制备平均直径为106.6±40.9nm,表面光滑,具有良好分散性的纳米纤维薄膜。戊二醛交联不仅使复合电纺膜纳米纤维形态和直径(平均直径为66.3±24.8nm)更接近于羊膜基底膜和基质,而且大大改善了薄膜的透明性和可操作性；在不影响复合膜纤维形态和直径前提下复合膜实现壳聚糖表面改性并与去上皮人羊膜微观形貌更加相近。
结论：本研究首先证实了胶原、透明质酸复合聚环氧乙烷电纺制备具有纳米级直径均一纤维薄膜的可行性；并在此基础上,通过戊二醛交联和静电吸附表面改性方法对复合薄膜透明性和可操作性进行优化,实现了对去上皮人羊膜组织微纳结构的人工模拟,为临床眼表损伤修复用眼表覆膜的研究和应用奠定了可靠的实验基础。
第二部分仿去上皮羊膜微纳结构壳聚糖改性胶原-透明质酸复合电纺膜的物理化学性能研究
目的：对胶原-透明质酸复合电纺膜、交联膜和壳聚糖改性膜的物理化学性能进行表征,探讨交联和表面改性后处理对薄膜理化性能的影响,对比分析壳聚糖改性胶原-透明质酸复合电纺膜与去上皮人羊膜组织相应性能的差异,为进一步优化该类眼表修复覆膜研究奠定实验基础,同时也为临床上眼表组织修复支架的制备提供新的方法和研究思路。
方法：采用紫外-可见光光度仪对不同薄膜的可见光透过率进行检测,傅里叶转换红外线光谱分析不同薄膜的化学组成及变化,万能测试机对薄膜的抗拉强度、杨氏模量和最大伸长率进行测试分析,静态水接触角对不用薄膜的亲疏水性进行分析比较。
结果：干燥状态下,在555nm波长处交联膜和壳聚糖表面改性膜的可见光透过率分别为70%和75%,而羊膜仅为40%；在湿润状态下,在555nm波长处交联膜和壳聚糖表面改性膜的可见光透过率分别增加到88%和89%,羊膜在该处的可见光透过率增加到74%。胶原-透明质酸复合电纺膜、交联膜和壳聚糖改性膜的红外光谱图中均存在胶原、透明质酸特征性吸收峰,但交联膜和壳聚糖改性膜在2000-1000cm-1附近的吸收峰强度降低,位于3314cm-1、1619cm-1和1430cm-1附近的吸收峰信号变弱；壳聚糖改性膜中酰胺A,酰胺Ⅰ,Ⅱ键吸收峰波幅较交联膜增强；在交联膜和壳聚糖改性膜中,均未检测到聚环氧乙烷特征性吸收峰；在壳聚糖改性膜中检测到所有壳聚糖特征性吸收峰；壳聚糖改性膜与去上皮人羊膜相比均具有胶原、透明质酸特征性吸收峰,但壳聚糖改性膜在1750-1000cm-1范围内的吸收峰波幅较去上皮人羊膜者低。壳聚糖改性膜的平均抗拉强度为31.39±6.61Mpa,略低于去上皮人羊膜(39.72±4.43Mpa),而在同等强度外力作用时,形变量为交联膜>壳聚糖改性膜>去上皮人羊膜。交联膜和壳聚糖改性膜的平均水接触角分别为18.6±1.70和23.9±2.60,均小于去上皮人羊膜(63.4±3.30)。
结论：胶原-透明质酸交联膜和壳聚糖改性膜具有优于去上皮人羊膜组织的良好透明性；经电纺、交联和壳聚糖表面改性三步法处理后胶原和透明质酸仍保持原有活性不变；壳聚糖改性膜具有与去上皮人羊膜相似的化学组成；虽然复合膜的抗拉强度稍逊于去上皮人羊膜组织,但其更好的弹性和更高的亲水性对于我们后续细胞和组织相容性研究奠定了有利的理论基础。
第三部分仿去上皮羊膜微纳结构壳聚糖改性胶原-透明质酸复合电纺膜生物相容性研究
目的：分析评价壳聚糖改性胶原-透明质酸复合电纺膜的生物相容性,为仿羊膜微纳结构眼表覆膜的临床应用和产业化提供理论依据和实验基础,从根本上推进眼表修复用仿生人工薄膜材料的研制。
方法：以去上皮人羊膜为对照,采用体外细胞培养法将人角膜上皮、结膜上皮细胞接种于壳聚糖改性膜上,扫描电镜观察细胞的粘附形态,活死细胞染色检测薄膜对细胞的毒性作用,CCK-8法比较细胞在薄膜表面的增殖能力；构建大鼠角膜急性碱烧伤模型,荧光素钠和苏木精-伊红染色观察壳聚糖改性膜移植后角膜上皮再生水平,肉眼观察基质水肿和炎症反应情况,评价薄膜的可操作性和在体生物相容性。
结果：角膜上皮细胞在壳聚糖改性膜上具有类似于正常角膜上皮的无规则扁平样外观,结膜上皮细胞在该膜上表现出细胞与材料相互融合现象；接种24h后在壳聚糖改性膜上角膜上皮细胞可检测到极少量的细胞死亡而结膜上皮细胞未检测到细胞死亡；细胞的增殖能力随培养时间的延长而增强,差异有统计学意义(p<0.05),而同种细胞在不同薄膜上的增殖能力无明显差异(p=1.12)；壳聚糖改性膜在大鼠急性碱烧伤角膜表面保持良好的透明性,与基质面贴合紧密；薄膜下可见角膜上皮再生并与其下基质粘连紧密,角膜基质水肿和炎症反应均减轻。
结论：壳聚糖改性胶原-透明质酸复合电纺膜具有良好的细胞相容性,在动物角膜急性碱烧伤模型中表现出良好的可操作性和透明性,具有与去上皮人羊膜组织相似的促上皮化、抑制基质水肿和炎症反应特性,本研究结果为探寻仿羊膜结构与功能材料的研制进而为眼表修复用仿生薄膜材料的研究奠定了扎实的实验基础,为临床上眼表疾病的治疗提供了一条新的途径。
Part I Development of chitosan modified collagen-hyaluronic acid compositeelectrospinning film with biomimetic micro-nano structure ofdenuded amniotic membrane
Objective
The purpose of this study was to investigate the feasibility of chitosan modified collagen-hyaluronic acid composite film with biomimetic micro-nano structure of denuded amniotic membrane by using electrospinning and electrostatic adsorption surface modification technology. Electrospinning agent concentration ratio between collagen and its solvent acetic acid as well as with the use of poly (ethylene oxide), collagen composite hyaluronic acid electrospinning film were facilitated. On the basis of the above results, by using chemical cross-linking and electrostatic adsorption surface modification technology to optimize the stability and surface micro-nano structure of the composite electrospinning film, so as to provide an experimental basis on the studies of the ocular surface damage repair film and to broaden the range of applications of electrospinning nanotechnology and electrostatic adsorption surface modification technology in the biomedical field.
Methods
On the basis of careful observation and analysis of the morphology of human amniotic membrane, we used natural polymer materials of collagen, hyaluronic acid and a synthetic polymer material polyethylene oxide as the main raw materials to prepare the composite electrospinning film. The ratio of the concentration of electrospinning raw materials and the parameters of the electrospinning were optimized. Glutaraldehyde cross-linking and electrostatic adsorption between hyaluronic acid and chitosan were used to improve the the stability and micro-and nano-structure of the electrospinning film. Transparency and operability of the composite electrospun fiber films were observed. Surface and cross-section microstructure of the films were detected by scanning electron microscopy. The similarity of the microscopic morphology between chitosan modified collagen-hyaluronic acid composite electro spinning film and denuded amniotic membrane was also evaluated.
Results
The basement membrane and matrix of denuded human amniotic membrane were composed of lamellar structure consisting of irregular collagen fibers with the average diameter was52.9±17.5nm and41.2±14.7nm, respectively. Under25°C and40%humidity, stable electrospinning state could be achieved with advancing speed for0.1ml/h,10cm receiver distanc and10-15kV voltage.10%w/v collagen with90%v/v acetic acid as solvent could be electrospun successfully, the average fiber diameter was79.1±27.8nm.10%w/v COL,0.1%w/v HA and7%w/v PEO with8:1:1volume ratio could prepare film with the average diameter was106.6±40.9nm. Smooth surface and a good dispersion of the nano-fiber diameter could be observed. Glutaraldehyde cross-linking not only rendered the morphology and nano-fiber diameters of composite electrospinning film similar to the amniotic membrane (average diameter of66.3±24.8nm), but also greatly improved the transparency and operability of the film. Without affecting fiber morphology and diameter the composite film was modified by chitosan,which made the composite membrane surface much similar to denuded human amniotic membrane.
Conclusion
This study was first confirmed that collagen, hyaluronic acid and polyethylene oxide could be prepared by electrospinning to fabricate uniform nanoscale fiber diameter. glutaraldehyde crosslinking and electrostatic adsorption surface-modification could optimize the transparency and operability of the composite film. Artificial simulation of the micro-nano structure of denuded human amniotic membrane has been achieve. This will lay a reliable experimental basis for clinical ocular surface damage repair.
Part II Physical and chemical properties of chitosan modified collagen-hyaluronic acid composite electrospinning film with biomimeticmicro-nano structure of denuded amniotic membrane
Objective
The purpose of this study was to investigate the physical and chemical properties of the collagen-hyaluronic acid composite electrospun film, post-crosslinking film and chitosan modified film. To explore the influence of crosslinking and surface modification on the physical and chemical properties of the films. Comparatively analyze the differences of corresponding performance between chitosan-modified collagen-hyaluronic acid composite electrospinning film and denuded human amniotic membrane. To lay the experimental foundation of further optimization of this kind of ocular surface repair film. Also to prepare new methods and research ideas to the fabrication of tissue repair scaffolds in clinic.
Methods
UV-visible spectrophotometer was used to detect the visible light transmittance of the films. Fourier transform infrared spectral analysis was used to analyze the chemical composition and changes in the films. The tensile strength, Young's modulus and the maximum elongation of the film were detected by the universal testing machine. Static water contact angle was tested to analyzed the film's hydrophobicity and hydrophilicity.
Results
In dry state the visible light transmittance at the wavelength of555nm were70%and75%in the crosslinked film and chitosan modified membrane, respectively. While that of amniotic membrane was only40%. In the wet state, at the wavelength of555nm, the visible light transmittance of the crosslinked film and chitosan surface modified membrane was increased to88%and89%, respectively, that of the amniotic membrane was increased to74%. In the infrared spectra, characteristic absorption peak of Collagen and hyaluronic acid could be observed on the composite electrospun film, crosslinked film and chitosan modified film. At2000-1000cm-1the absorption peaks had lower intensity. At3314cm-1,1619cm-1and1430cm-1the absorption peaks had weaker signals. In chitosan modified film, the amide A, amide I and II chemical bond had high absorption peak than the crosslinked film. There was no absorption peak of polyethylene was detected on crosslinked film and chitosanmodified film. The characteristic absorption peak of chitosan were all detected on the chitosan modified membrane. In comparation with denuded human amniotic membrane, the chitosan modified film had similar absorption peaks of collagen and hyaluronic acid. While the absorption peak in the range of1750-1000cm-1was lower than that of denuded human amniotic membrane. The average tensile strength of the chitosan modified membrane was31.39±6.61MPa, which slightly lower than that of denuded human amniotic membrane (39.72±4.43MPa). Under the equal strength of external force, the deformation level was cross-linked film> chitosan modified film> denuded human amniotic membrane. The average water contact angle of the crosslinked film and the chitosan modified film were18.6±1.7°and23.9±2.6°, which were less than that of the denuded human amnion (63.4±3.3°).
Conclusion
Collagen-hyaluronic acid crosslinked film and chitosan modified membrane had better transparency than that of denuded human amniotic membrane. The three-step treatment of electrospinning. crosslinking and chitosan surface modification did not change the original activity of collagen and hyaluronic acid. Chitosan modified film had almost the same chemical compositon of denuded human amniotic membrane. Although the tensile strength of the composite membrane was slightly inferior to denuded human amniotic membrane, but its better flexibility and higher hydrophilicity laid a favorable theoretical basis to our subsequent cellular and tissue compatibility.
Part IH Biocompatibilities of chitosan modified collagen-hyaluronic acidcomposite electrospinning film with biomimetic micro-nano structureof denuded amniotic membrane
Objective
The purpose of this study was to analyze and evaluate the biocompatibility of chitosan modified collagen-hyaluronic acid composite electrospun membrane. Lay a theoretical and experimental basis to the clinical application and industrialization of the biomimetic amniotic membrane in the ocular surface reconstruction. To fundamentally promote the development bionic artificial film materials in ocular surface repair.
Methods
Human corneal epithelial cells and conjunctival epithelial cells were seeded on chitosan modified film. Scanning electron microscopy was used to observed the morphology of cells, live/dead assay was used to detect toxic effects of the film to the cells. CCK-8method was used to compare the proliferation of cells on the surface of film, denuded human amniotic membrane was used as control. The rat acute corneal alkali burn model was established, sodium fluorescein and hematoxylin-eosin staining were use to detect the level of corneal epithelial regeneration. The situation of stromal edema and inflammation were visually observed, the operability and in vivo biocompatibility of the film were evaluated, denuded human amniotic membrane was used as control.
Results
Corneal epithelial cells in chitosan modified collagen-hyaluronic acid composite electrospun membrane showd a flat-like appearance which was similar to that of normal corneal epithelium. Conjunctival epithelial cells in the film performed a cell-material merging phenomenon. Live/dead assay showed that24hours after incubation a small amount of corneal epithelial cell death was detected on the composite electro-spun membrane as well as denuded amniotic membrane,while no conjunctival epithelial cell death was detected on both two kind of membrane. Cell proliferation was enhanced with prolonged incubation time, the difference was statistically significant (p<0.05). The same kind of cell proliferation on different films was not significant different (p=1.12). Chitosan modified membrane transplanted on the surface of the acute alkali burned cornea maintained good transparency, and tightly bonded with the substrate surface. Promotion of corneal epithelial regeneration, close adhesion between re-newed corneal epithelial cells and matrix and reduction of stromal edema and inflammation could be observed on the chitosan modified membrane transplanted eye, which was similar to that of denuded human amniotic membrane.
Conclusion
Chitosan modified collagen-hyaluronic acid composite electrospinning film has good cell compatibility. On animal acute corneal alkali burn model it shows good operability and transparency. Its properties of epithelium promotion, stromal edema inhibition as well as inflammatory response suppression lay a solid experimental basis for clinical explore of the materials research imitating the structure and function of amniotic membrane in ocular surface repair. Also this provides a new way in the treatment of ocular surface disease.
引文
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