{
  "abstract": "Purpose Descemet’s membrane (DM) is the specialized extracellular matrix (ECM) located between the posterior corneal stroma and the endothelium. Thanks to its composition and mechanical properties, this basement membrane provides the biochemical cues that sustain endothelium viability and function, making it an optimal cell niche. Several decellularization techniques have been developed to isolate the ECM removing all cellular components from a variety of tissues. Once decellularized, these tissues can be employed in all those pathological conditions involving closure defects or tissue damage, which can be improved providing an exogenous healthy ECM that drives cell migration and repair of the damaged area. An example of these conditions is full-thickness macular hole, for which various scaffolds have been used to induce hole closure, considering also extraocular derivatives like amniotic membrane. The present work describes the tailored-made decellularization protocol to obtain an acellular scaffold from DM, suitable for regenerative medicine purposes in ocular surgery.Methods For this study, twelve DMs were mechanically stripped from donor corneas. To induce cell lysis and eliminate all cytoplasmic and nuclear material, isolated DMs were subjected to osmotic (hypotonic) shock for two hours under constant agitation. The efficiency of the decellularization process was evaluated in terms of nuclear material removal, and therefore total residual DNA was quantified and analyzed by gel electrophoresis to exclude the presence of remaining high molecular weight DNA fragments. Furthermore, nuclear removal and ECM integrity after treatment were verified with immunofluorescence analysis. In particular, the maintenance of collagen (type VIII, IV and I), glycoproteins (fibronectin and laminin), proteoglycans (HSPG2) and elastin was analyzed in decellularized DMs compared to untreated controls.Results DNA quantification showed a 99% reduction of total DNA amount in decellularized DMs compared to control ones, with only 4 ng DNA/mg wet tissue residual. Furthermore, the agarose gel electrophoresis confirmed the absence of DNA fragments bigger than 200 base pairs. Concerning the immunofluorescence staining, no DAPI-positive nucleus was visible after decellularization, confirming the efficiency of the process. Moreover, ECM components staining maintained the same pattern and intensity as the fresh untreated control, with the exception of the signal localized in the cytoplasm, absent in decellularized samples, thus indicating the depletion of cytoplasmic content.Conclusions The results collected in this study show that this decellularization protocol is effective to obtain a decellularized DM. In particular, the product is depleted of the donor DNA, thus avoiding the risk of unwanted immunogenicity when used as heterologous implant. Furthermore, the treatment preserves tissue collagen and elastin, which are both responsible for the maintenance of tissue biomechanical properties. Glycoproteins and proteoglycans, important mediators of cell adhesion and cell-matrix crosstalk, are also maintained. Thanks to these features, this tissue could find application in surgery for conditions like full-thickness macular hole. DM is already widely employed in intraocular surgery and is currently prestripped and provided by eye banks, and therefore a decellularized DM could represent a valid candidate as a scaffold for macular hole treatment.",
  "authors": [
    {
      "affiliations": [
        "Veneto Eye Bank Foundation, Venice, Italy"
      ],
      "name": "Stefania D’Agostino"
    },
    {
      "affiliations": [
        "Veneto Eye Bank Foundation, Venice, Italy"
      ],
      "name": "Elena Daniele"
    },
    {
      "affiliations": [
        "Veneto Eye Bank Foundation, Venice, Italy"
      ],
      "name": "Stefano Ferrari"
    },
    {
      "affiliations": [
        "Veneto Eye Bank Foundation, Venice, Italy"
      ],
      "name": "Diego Ponzin"
    }
  ],
  "title": "3.5. Decellularized descemet’s membrane: a new scaffold on sight for full-thickness macular hole",
  "uid": "bd3348a5-8d88-5609-bf01-fe6d109cfeb2"
}