The main focus of WG4 will be evaluating the suitability of the corneal scaffold and its subsequent modifications for corneal regeneration and prepare the techniques and materials (both novel and existing) needed for proceeding to human trials. To achieve this, facilities of different research groups will be combined in which the knowledge on culture conditions and the use of animal models and/or
in-vitro alternatives for animal models will be explored.
This Working Group has the following objectives:
1. To study the scaffold’s cytotoxicity in vitro and in vivo;
2. To assess tissue integration and tissue regeneration in vitro and in vivo of the scaffold;
3. To determine the scaffold’s immunogenicity in vivo.
Research tasks to achieve these objectives are:
• Task 4.1: To study the scaffold’s cytotoxicity in vitro and in vivo. Cytotoxicity and/or proliferation assays with primary human corneal epithelial and stromal cells will be performed to exclude possible toxic effects of the scaffold on these cells. These assays are based on measuring the viability and mitotic activity of the cells. The reaction of the surrounding tissue to the scaffold implanted in animals will be assessed by observing the animals clinically and performing subsequent histology, immunofluorescence and confocal microscopy.
• Task 4.2: To assess tissue integration and tissue regeneration in vitro and in vivo of the scaffold. The scaffolds will be co-cultured with primary human corneal cells, and implanted in animals to analyse and optimize tissue integration and tissue regeneration capabilities. Several culture conditions and several implantation methods and locations will be carried out to find the optimal protocol. Exchange of information on culture conditions is of utter importance.
• Task 4.3: To determine the immunogenicity and biocompatibility in vivo. The immunogenicity of the scaffolds will be observed in vivo upon implantation in animals using surgical microscopy and subsequently by performing immunohistochemistry. In vivo features used to determine an immune reaction are corneal neovascularization and opacity and swelling of the tissue surrounding the implant. The presence of the different infiltrated immune cells will be determined histologically, including the use of markers for neutrophils (myeloperoxidase (MPO)), macrophages (ED1), T-cells (CD3) and antibodies (IgG). Data will be combined in standard operating procedures for testing biocompatibility.