Application of benzonase in preparation of decellularized lamellar porcine corneal stroma for lamellar keratoplasty

Abstract

This study was to develop anovel and efficient method using endonuclease (benzonase) to preparedecellularized lamellar porcine corneal stroma (DLPCS). The DLPCS was preparedfrom native lamellar porcine corneal stroma (NLPCS) and was treated with 1000 U/ml benzonase for 5hours. We conducted the following measurements and animal transplantation tocompare DLPCS and NLPCS. The residual DNA was decreased significantly from 367.13 ± 19.96 ng/mg to 15.41 ± 0.65 ng/mg after treatment of benzonase by the detection of fluorescentnucleic acid stain. The residual benzonase was also less than detection limit.There was no significant difference in light transmittance of DLPCS comparedwith NLPCS. The extracts of DLPCS did not inhibit cell proliferation of human cornealepithelial cells, mouse fibroblast (L‐929) and African green monkey kidney cell(Vero cell). The DLPCS was transplanted into the corneas of rabbit by lamellarkeratoplasty. There was no corneal melting and graft rejection been observedwithin 12 months. The images demonstrated that the repairment of corneal nervesand keratocytes of DLPCS were in indentical shape and reflection compared withnormal cornea, and no obvious inflammatory cells were observed postoperation, byin vivo confocal microscopy. We provided novel evidence that the application ofbenzonase may improve the quality of DLPCS.

Tissue-Specific Decellularization Methods: Rationale and Strategies to Achieve Regenerative Compounds

“…Benzonase endonuclease is often used as the main decellularizing agent in a detergent-free approach, based on its ability to quickly infiltrate the corneal stroma, combined with its easy removal by repeated washes. This approach minimizes the destruction of the ECM, with minimal loss of optical transparency and proper results in animal transplantation assays [186].…”

Decellularization for the retention of tissue niches

“… 39 Benzonase, while also unable to act as a principal decellularizing agent, is compatible for use with several detergent-free decellularization protocols. 173 – 175 DNase can be used as a replacement for TNBP, which similarly works to reduce negative impacts caused by chemical agents, as DNase achieves similar results with shorter protocols and higher mechanical stability and GAG content. 39 ECM treated with DNase or RNase must be put through numerous rinse cycles, as they are immunogenic compounds that can hinder recellularization attempts.…”

Assessment and validation of enrichment and target capture approaches to improveMycobacterium tuberculosisWGS from direct patient samples

“…Benzonase breaks down both ssDNA, dsDNA, RNA and DNA:RNA hybrids, while DNase targets primarily dsDNA with reduced specificity for ssDNA and DNA:RNA hybrids. Benzonase is thus an ideal candidate for extracellular DNA depletion that may provide a more efficient solution than the more frequently used DNase (Sutton et al ., 1997; Liu et al ., 2019; Amar et al ., 2021).…”

“…This is congruent with previous studies where the application of a saline wash or treatment with the MolYsis kit (Molzym, Germany), reduced contaminating DNA(Votintseva et al, 2015;Votintseva et al, 2017).The benzonase treatment protocol demonstrated a significant enrichment effect. We initially hypothesized that benzonase was superior to a simple wash or DNase treatment because it more effectively removes extracellular contaminating ssDNA in addition to dsDNA, RNA and DNA:RNA hybrids, while DNase targets only dsDNA(Sutton et al, 1997;Liu et al, 2019;Amar et al, 2021). However, there was only a small difference in total DNA by the Qubit ssDNA between the DNase and benzonase treated samples (0,55 ± 0,302 ng/µl and 0,51 ± 0,213 ng/µl respectively), arguing that more effective ssDNA degradation is not the only reason for the benzonase effect.…”