Development And Performance Studies Of Chitosan Modified Collagen-hyaluronic Acid Composite Electrospinning Film With Biomimetic Micro-nano Structure Of Denuded Amniotic Membrane

Part I Development of chitosan modified collagen-hyaluronic acid compositeelectrospinning film with biomimetic micro-nano structure ofdenuded amniotic membraneObjectiveThe 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.MethodsOn 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.ResultsThe 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.ConclusionThis 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 membraneObjectiveThe 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.MethodsUV-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.ResultsIn 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°).ConclusionCollagen-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 membraneObjectiveThe 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.MethodsHuman 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.ResultsCorneal 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.ConclusionChitosan 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.