Preparation Of Composite Collagen Scaffold And Its Biocompatibility With Corneal Stromal Cells

The number of blindness caused by corneal disease is second to cataract. Cornealdisease results when opacity occurs due to infection, trauma and diseases of theimmune system. The corneal disease occurred in the early time can be cured throughpositive and effective treatment. If the corneal disease not treated timely wouldeventually lead to blindness. There are more than40million corneal blindnessindividuals worldwide. At present, the only widely accepted treatment istransplantation with human donor corneal tissue. However there is an increasing needfor human donor corneal tissue and a severe shortage of suitable cornea donors inmany countries. Recently, corneal tissue engineering opens a new path for in vitroreconstruction of tissue-engineered cornea and tissue-engineered corneal stroma andtheir clinical application. One of the most important steps is material in thepreparation of the tissue engineered cornea. Now, natural materials such as collagen,chitosan and heterologous corneal stroma are the main materials, while other syntheticmaterials and composite materials are used to build the tissue engineered cornea.Collagen is the main component of connective tissue, its chemical and biologicalcharacteristics of different animal origin are very similar. Compared to other tissueengineering corneal materials, collagen has low immunogenicity, low toxicity, lowantigenicity, and have has good biocompatibility and biodegradability. In this paper,human type I, type V, fibronectin and chondroitin are the principal raw materials ofthe composite collagen scaffold. The preparation process of compound collagen gelscaffold is optimized by adjustment of type I collagen concentration. The cornealstromal cell line cells established in our laboratory are inoculated in the compositecollagen scaffold to detect its biocompatibility.Human type I, type V, fibronectin and chondroitin were used to build compositecollagen scaffold. As the crosslinker of crosslinking reaction, EDC/NHS （1-Ethyl- 3-（3-dim-ethyllaminopropyl） carbodiimide hydrochloride/N-Hydroxysuccinimide）were used to enhance the mechanical strength and toughness of the compositecollagen scaffold. Performances of transparency, water content and structure weredetermined of the composite collagen scaffolds whose concentration of human type Iwere9mg/mL,10mg/mL and11mg/mL. The results of Light transmittance andwater content showed that: With the increase of concentration of collagen, thetransparency of the composite collagen scaffold increased, and the water contentdeclined slightly. The results of HE and TEM showed that: the apertures’ continuity ofthe composite collagen scaffold（Human type I,9mg/mL） was good, but the internalstructure organization was too loose. The internal structure organization of thecomposite collagen scaffold（Human type I,11mg/mL） was compact, but thecontinuity of its apertures was not well. The structure organization and its apertures’continuity of the composite collagen scaffold（Human type I,10mg/mL） were suitablefor cells to move in. The results of SEM, the combination of these composite collagenscaffolds’ collagen fibers was well-organized. So, the corneal stromal cell line cellswere inoculated in the composite collagen scaffold（Human type I,10mg/mL） todetect its biocompatibility.To ensure the features of the human corneal stromal cell line, the human cornealstromal cell line（passage20） was conducted. According to the results of opticalmicroscope observation, the human corneal stromal cells that cultured in vitro grew ingood condition and kept strong ability to cleavage. The immunofluorescence showedthat human corneal stromal cell lines expressed the marker protein-vimentin, theconnection protein-integrin β1and connexin-43, the function protein-Aldehydedehydrogenase3A1. The real time quantitative PCR showed that human cornealstromal cell lines expressed growth factors （Kras, mTOR, Stat3and RRN3）,multiplication factors （CDK2and p27） and differentiation factor （Vimentin）. Theseresults suggested that the human corneal stromal cells still had normal phenotypes andthe potential to form normal human corneal stroma.Biological compatibility of composite collagen scaffolds was detected by usingpassage20human corneal stromal cells as seeder cells and the composite collagenscaffold（Human type I,10mg/mL） as scaffold carries, which was produced in DMEM/F12（1:1） medium containing15%fetal bovine serum at37°C with5%CO₂.The results of frozen section DiI and HE, human cornea stromal cells moved into thecomposite collagen scaffold and distributed evenly. The results of TEM and SEM,certain connections formed among cells and between cells and scaffold. The results ofimmunohistochemical staining, human cornea stromal cells inoculated in thecomposite collagen scaffold still expressed the marker protein-vimentin, theconnection protein-integrin β1and connexin-43, the function protein-Aldehydedehydrogenase3A1. The results of real time quantitative PCR showed that therelative expression of human corneal stromal cells’ growth factors （Kras, mTOR,Stat3and RRN3）, multiplication factors （CDK2and p27） and differentiation factor（Vimentin） were similar to the normal cells. These results suggested that thebiocompatibility of the composite collagen scaffold is good.In conclusion, identification results of water content, transparency and structureshowed that the composite collagen scaffold（Human type I,10mg/mL） had idealperformances compared with the other two scaffolds. The results of biocompatibilityshowed that it had fine biological compatibility with human corneal stromal cells.This study is the base to the preparation both of the tissue-engineered human cornealstroma and the tissue-engineered human cornea.