- Top of page
- Materials and Methods
Purpose: To evaluate the potential use of decellularized porcine corneas (DPCs) as a carrier matrix for cultivating human corneal cells in tissue engineering.
Methods: Corneal cells were isolated from human corneoscleral rims. Porcine corneas were decellularized using hypotonic tris buffer, ethylene diamine tetra-acetic acid (EDTA, 0.1%), aprotinin (10 KIU/ml) and 0.3% sodium dodecyl sulphate. Haematoxylin–eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining were performed to confirm removal of the corneal cells. Quantitative analysis was performed to determine levels of desoxyribonucleic acid (DNA) using DNA Purification Kit (Fermentas, St. Leon-Rot, Germany). Alcian blue staining was carried out to analyse the structure of the extracellular matrix (ECM). Corneal stromal cells were injected into the DPCs; limbal corneal epithelial cells and corneal endothelial cells were seeded onto the anterior and posterior surfaces of the DPCs, respectively. Evaluation was undertaken at days 14 and 30. The phenotypical properties of the cultivated corneal cells were investigated using Immunolocalization of type I collagen, keratocan, lumican, cytokeratin 3 (AE5) and type VIII collagen.
Results: Haematoxylin–eosin and DAPI staining showed efficient elimination of porcine corneal cells, whereas alcian blue confirmed gross preservation of the ECM. The quantitative analysis of the DNA content showed a significant reduction (mean before decellularization: 75.45 ± 13.71 ng/mg; mean after decellularization: 9.87 ± 2.04 ng/mg, p < 0.001). All three types of corneal cells were efficiently cultured and expanded on the DPCs.
Conclusions: Decellularized porcine corneas might serve as a potential scaffold for tissue engineering of the cornea, possibly providing xenogenic substrate for corneal transplantation.
- Top of page
- Materials and Methods
However, many difficulties still exist for tissue engineering of the cornea and its clinical use. In general, the properties of native soft tissues cannot easily be duplicated by synthetic materials or biomaterials (Porter et al. 1998; Tegtmeyer et al. 2001). The most appropriate matrix for a tissue-engineered cornea is a cornea itself. While human material is limited, xenogenic corneal material is abundant.
Porcine material especially has been investigated extensively as a candidate for xenografting. Previously, it has been shown that the α-gal epitope, which induces hyperacute rejection, is almost absent in the porcine cornea except for several keratocytes in the most anterior part, suggesting that the porcine cornea has clinical potential. Although this may limit the risk of graft rejection, cross-species transmission of porcine pathogens is still a major concern (Amano et al. 2003).
Our results confirm that the decellularization method used for other organs may also be suitable for the porcine cornea. The three different corneal cell types were successfully incorporated into the decellularized scaffold. Although an incomplete reduction of porcine DNA with 0.3% SDS was noted. A threshold for an immunogenic reaction needs still to be determined. Moreover, SDS seems to lower the corneal transparency most evidently immediately after its application. An acceptable transparency level was restored by 100% glycerol for 6 h. Alcian blue staining of the ECM confirmed a partial disorganization of the fibres possibly explaining the reduced transparency. Still, no gross histological disruption was observed. The intensity of the immunohistochemical images was lower in the porcine tissue compared with the human controls. An explanation of this phenomenon could have been the usage of passage 1 of cultivated cells or an altered surface of the DPCs.
Gonzalez-Andrades et al. compared two decellularization processes. It was shown that both NaCl and SDS achieved a sufficient decellularization level for all three corneal cell types (Gonzalez-Andrades et al. 2011). The authors concluded that the application of a 1.5 m NaCl solution on porcine corneas produces an acellular corneal stroma with adequate histological and optical properties. Human keratocytes were able to penetrate, spread and differentiate within this scaffold. In conclusion, the authors suggest that the decellularization of animal corneas with 1.5 m NaCl represents a useful method for the development of human bioengineered corneas with therapeutic potential. In comparison with our study, the authors used 0.1% SDS and no endothelial or epithelial cells were investigated. However, Du et al. optimised a protocol to produce an acellular porcine corneal scaffold and investigated its mechanical integrity and biocompatibility. They concluded that only SDS resulted in an almost complete decellularization after 24 h. Histological analysis of the acellular matrix showed that the CSCs had been removed substantially, collagen fibres were kept arranged in an orderly fashion, and Bowman’s layer and Descemet’s membrane were both still intact (Du et al. 2011). Sasaki et al. evaluated stability and biocompatibility of an artificial corneal stroma which was decellularized by ultra-high, hydrostatic pressure. They concluded that completely decellularized porcine corneal stroma has an extremely high biocompatibility and has the potential to serve as a scaffold for an artificial cornea (Sasaki et al. 2009).
Zhou et al. supported the results that SDS is able to produce a porcine corneal acellular matrix. Although water absorption and light transmission of the acellular matrix decreased, it showed similar biocompatibility and biomechanical properties as the natural equivalent. After xenotransplantation into rabbit corneal stromal layers, and observation for 4 weeks, complete transparency was noted. For almost 1 year postoperatively, the corneas remained transparent with regular stromal structures and appeared stable in situ without an immunological rejection. They concluded that these acellular matrices, similar to natural corneas in structure, strength, and transparency, have tremendous potential for tissue engineering and corneal transplantation (Zhou et al. 2011).
Fu et al. used Triton X-100 (1%) plus a freeze-drying process for decellularization of porcine corneas and measured the in vivo biocompatibility in a rabbit model with a follow-up of 3 months. The authors observed no sign of rejection and that the acellular matrix gradually integrated into the rabbit cornea, indicating a good biocompatibility. They concluded that the corneal acellular matrix can be used as a scaffold for a tissue-engineered cornea and that a biological corneal equivalent can be reconstructed in a dynamic culturing system (Fu et al. 2010).
Further in vivo studies are necessary to determine whether the xenocorneas can maintain stromal dehydration, long-term transparency and in-vivo biocompatibility. Also, it has to be clarified whether a significant reduction of immunogenic epitopes was achieved to guarantee a long-term immunological tolerance. Although promising studies on other decellularized organs give a genuine hope for decellularized xenocorneas, the above stated questions have to be meticulously addressed before defining its future potential as a realistic treatment alternative. In conclusion, our results demonstrate that by decellularization of porcine corneas and recellularization with human corneal cells, it is possible to obtain viable xenografts for corneal transplantation. In principle, this technique could establish a new dimension of tissue engineering of the cornea and has the theoretical potential to overcome the immanent problem of corneal transplant shortage.