Comparison Of Airdried And Lyophilized Acellular Porcine Cornea As Scaffold For Corneal Tissue Engineering

BackgroundDiseases affecting the cornea are a major cause of blindness worldwide and the vast majority of affected patients would benefit from a suitable corneal replacement . The severe shortage of donor corneas and graft failures resulting from immunological rejection have prompted various efforts to develop corneal substitutes, with the dual aims of providing a plentiful supply of graft material and reducing the rate of rejection. Various kinds of corneal substitutes have been developed, including the nondegradable synthetic corneal replacement, keratoprosthesis (KPros), and tissue engineered corneas. Although KPros can maintain the integrity of the globe and permits light transmission, the elaborate surgical techniques required and the high complication rate limit its clinical applicability .Many natural or synthetic materials, such as amniotic membrane, crosslinked collagens, and crosslinked collagen–chitosan hydrogels, have been used as scaffolds for corneal tissue engineering . Despite their ability to restore the integrity of globe, many problems remain, such as the lack of the natural corneal conformation, low clarity, vulnerability, intolerance of sutures, and complications, which have restricted their clinical applications. It seems that the most critical barrier to successful tissue engineering of the cornea lies in the construction of a suitably strong, clear stromal matrix. The strength and clarity of the cornea is attributed to its exquisite arrangement of collagen fibrils, which is difficult to imitate using synthetic methods . The method of decellularization has been widely used in tissue engineering, because of its advantages in preserving the natural extracellular matrix (ECM) and removing cell antigenicity . Therefore, we postulated that it should be feasible to use decellularization to fabricate a scaffold that could retain the natural corneal ECM. Based on the concept of using porous three-dimensional(3D) scaffolds to provide physical support and a local environment for cells, lyophilization is a popular method used in tissue engineering to dry materials and to generate pores in scaffolds . We used this method in our previous study to consider the feasibility of seeding keratocytes on the scaffold in vitro . Although the porous lyophilized porcine acellular corneal stroma (l-PACS) was amenable to cell seeding in vitro, the generation of pores by lyophilization was accompanied by a disturbance of the natural regular collagen arrangement, which is directly responsible for corneal transparency. However, unlike other tissues, the corneal stroma in adults is a relatively acellular tissue, with only 3%–10% quiescent keratocytes . Therefore, in engineering such a specific tissue, its transparency, the basis of the visual pathway, might be more important than cell seeding in vitro. Furthermore, as the biomaterial is degraded, the ingrowth of host cells might be achieved in vivo.Air drying has a long history as a traditional method of storage in many disciplines. It is a relatively moderate method of gradually evaporating water from a material without disrupting its inner conformation. We hypothesized that air drying might be the best alternative for corneal tissue engineering, to evaporate any residual water without destroying the delicate natural collagen arrangement, thus preserving the material.Objectives, Methods and ResultsObjective: The objectives of this study were to fabricate a clear and strong corneal substitute with a natural conformation using the methods of decellularization and air drying, to test its properties, and to evaluate its potential for clinical applications.Methods: Although various corneal substitutes have been fabricated, none seems to simultaneously provide good transparency, suitable tensile strength, and a natural structure. In this study, an air-dried porcine acellular corneal stroma (a-PACS), with good transparency and mechanical properties, was prepared from the anterior lamella of porcine cornea by decellularization and air drying, with the Bowman's layer well preserved. We evaluated its physical and biological properties, comparing it with lyophilized porcine acellular corneal stroma (l-PACS) as the control.Results: An in vitro study showed that the a-PACS was clearly superior to the l-PACS in transparency, mechanical properties, collagen arrangement, surface morphology of the Bowman's layer, support for epithelial growth, resistance to collagenase, and equilibrium water content. An MTT assay showed that the conditioned culture media prepared from both substitutes promoted the proliferation of L929 cells. After both substitutes were subcutaneously implanted in rats, a more regular collagen arrangement and more uniform cell ingrowth were seen in the a-PACS substitute, in which the rate of biodegradation was slower but which seemed more appropriate for cell migration and proliferation than the l-PACS. Both a-PACS and l-PACS restored the lamellar corneal defect in a New Zealand rabbit model. The transparency at each time point was better in the a-PACS group, with fewer new vessels formed, than in the l-PACS group, which makes a-PACS the much more attractive option.Conclusion: With all these advantages, a-PACS is considered a promising corneal substitute.