Preparation Of Corneal Scaffold By Bovine Collagen And Its Biocompatibility Evaluation With Human Corneal Stromal Cells

The cornea is a transparent tissue, locating in the front of eyeballs, plays an important role in protecting eyes, providing refractive power and guaranteeing visual function. Corneal diseases which are caused by thermal burn, chemical burn, mechanical damage and infection are the second major cause of blindness (the first is cataract). At present, transplantation with human donor corneal tissue is the only widely accepted treatment. However, there is an increasing need for the donor corneal tissues and a severe shortage of suitable cornea donors in many countries. Fortunately, with development of tissue engineering research, reconstruction of tissue-engineered cornea in vitro which has a similar structure and function to the normal cornea makes it possible, thus overcoming corneal shortage. Scaffold, an important element in reconstruction of tissue-engineered cornea, should have a good biocompatibility and tissue structure characteristics. It is known that materials for the reconstruction of tissue-engineered cornea mainly include collagen, chitosan, heterologous corneal stroma and other natural materials, besides some synthetic materials and composite materials. Comparing with other materials, collagen has obvious advantages on degradation, biological safety, and immunogenicity. There are limitations to the scaffolds prepared by using the recombinant human collagen because of poor mechanical strength and high level of consumption. While bovine collagen as material to the reconstruction of tissue-engineered cornea, shows good prospect, such as its rich sources and the simple methods of purification. So far, there is no report about the reconstruction of tissue-engineered cornea using bovine collagen and its biocompatibility.Bovine collagen (130 mg/mL) is used to prepare scaffolds by lyophilization. EDC/NHS are used to crosslink the bovine collagen scaffolds and then the physicochemical properties and structure are detected. The bovine collagen scaffolds had flat surface and uniform thickness. The scaffolds exhibited transmittance between 41.5%-89.6%(water as contrast, WL=300-800 nm), and the moisture content was 83.66% ± 1.75%. The bovine collagen scaffolds formed porous lamellar structures. These results showed that the scaffold was similar to the natural cornea, no matter the transparency, water content, or organizational structure.The human corneal stromal cells (HCSCs, the sixty-eighth generation) were conducted to evaluate the biocompatibility of the bovine collagen scaffolds. The results show that HCSCs appeared as a typical fiber-like characteristic, strong proliferation, and the population doubling time of the cells was 39.03 h. The cells positively expressed the marker protein (vimentin) and the function proteins (integrin ?1 and connexin 43, Aldehyde Dehydrogenase 3A1). The real-time quantitative PCR results showed that the HCSCs can normally express growth related factors (Kras, mTOR and STAT3), proliferation related factors (p27 and CDK2) and differentiation related factor (vimentin). These results suggested that the HCSCs still had normal phenotypes and potential to form normal corneal stroma.The biocompatibility of bovine collagen scaffold was evaluated using the HCSCs. The results showed that the scaffolds had no cytotoxicity to the HCSCs. After the HCSCs were inoculated on the collagen scaffolds for 5 days, it showed a good state, and some of HCSCs moved into the scaffold. The marker protein (vimentin) and the function proteins (integrin ?1 and connexin-43, Aldehyde Dehydrogenase 3A1) could be expressed by the HCSCs on/in the scaffold. The results of real-time quantitative PCR showed that the relative expression of growth related factors (Kras, mTOR and STAT3), proliferation related factors (p27 and CDK2) and differentiation related factor (vimentin) in these cells were similar to the normal HCSCs. These results suggested that the collagen scaffold had ideal biocompatibility.In conclusion, bovine collagen is used to prepare corneal scaffolds in this study. The scaffolds have desired physical and chemical properties, and ideal biocompatibility. These works lay the foundation for the reconstruction of tissue-engineered cornea in vitro.