Modulation of Macrophage Phenotype, Maturation, and Graft Integration through Chondroitin Sulfate Cross-Linking to Decellularized Cornea

Abstract

Decellularized corneas obtained from other species have gained intense popularity in the field of tissue engineering due to its role to serve as an alternative to the limited availability of high-quality donor tissues. However, the decellularized cornea is found to evoke an immune response inspite of the removal of the cellular contents and antigens due to the distortion of the collagen fibrils that exposes certain antigenic sites, which often lead to graft rejection. Therefore, in this study we tested the hypothesis that cross-linking the decellularized corneas with chondroitin sulfate may help in restoring the distorted conformationation changes of fibrous matrix and thus help in reducing the occurrence of graft rejection. Cross-linking of the decellularized cornea with oxidized chondroitin sulfate was validated by ATR-FTIR analysis. An in vitro immune response study involving healthy monocytes and differentiated macrophages with their surface marker analysis by pHrodo red, Lysotracker red, ER tracker, and CD63, LAMP-2 antibodies confirmed that the cross-linked decellularized matrices elicited the least immune response compared to the decellularized ones. We implanted three sets of corneal scaffolds obtained from goat, i.e., native, decellularized, and decellularized corneas conjugated with chondroitin sulfate into the rabbit stroma. Histology analysis, three months after implantation into the rabbit corneal stromal region, confirmed the restoration of the collagen fibril conformation and the migration of cells to the implanted constructs, affirming proper graft integration. Hence we conclude that the chondroitin sulfate cross-linked decellularized corneal matrix may serve as an efficient alternative to the allograft and human cadaveric corneas.

Host Response to Biomaterials for Cartilage Tissue Engineering: Key to Remodeling

“…Similar to the natural matrix environment, acellular ECM can regulate the biological function of resident cells and multifunctional stem cells and promote the recovery of the structure and function of the damaged tissue ( Agrawal et al, 2010 ; Li et al, 2018 ). In addition to its application in the repair of articular cartilage, acellular ECM has been applied in various tissues (or organs) such as bone ( Huber et al, 2017 ), tendon ( Zhang S. et al, 2018 ), nerve ( Chen et al, 2019 ), blood vessels ( Gong et al, 2016 ), cornea ( Chakraborty et al, 2019 ), and skin ( Milan et al, 2019 ), and some success in achieving repair.…”

Generation and Application of a Reporter Cell Line for the Quantitative Screen of Extracellular Vesicle Release

“…To test EV induction we used PMA, a protein kinase C activator, which increases intracellular Ca 2+ levels in THP-1 and other cell types (Reisine and Guild, 1987;Gonczi et al, 2002;Yu et al, 2003). PMA stimulates increased expression levels of CD63 compared to vehicle-treated THP-1 cells (Chakraborty et al, 2019) and the increase in intracellular Ca 2+ generates exosome secretion (Savina et al, 2003;Messenger et al, 2018). These reports are consistent with our finding that PMA enhanced CD63 expression in both parent THP-1 and the reporter cells and led to the choice of PMA as a positive control for the HTS.…”

Corneal endothelium tissue engineering: An evolution of signaling molecules, cells, and scaffolds toward 3D bioprinting and cell sheets

“…Different decellularization methods survive different surface ligands or recognition signals which are essential for binding to growth factors, cytokines, and target cells (Wilson et al, 2016). Decellularization removes the native cells, immunogenic compounds, and infectious agents from the scaffold but preserves the structural and functional proteins of the ECM (J. Chakraborty et al, 2018; Lynch & Ahearne, 2013). Different decellularized corneal scaffolds are used for human application, including decellularized porcine corneas (Ju, Gao, et al, 2012; Yoeruek et al, 2012), decellularized porcine cornea lamellae (C. Zhang et al, 2017), decellularized human corneal stromal lamellae (He, Forest, Bernard, et al, 2016), decellularized posterior lamellae of the bovine cornea (Bayyoud et al, 2012), and decellularized ostrich corneal stroma (X.‐N.…”

“…Decellularization methods are not able to completely remove the cellular components from the porcine cornea; therefore, the acellular porcine cornea scaffolds are transparent only for two months in humans (Shi et al, 2017). Human nonspecific inflammatory reactions initiate the slow immune rejection process by IL‐2 and TNF‐α secretion from the surrounding transplanted cells (J. Chakraborty et al, 2018; Wilson et al, 2016). The complete removal of xenoantigen from the xenograft tissues reduces the immune rejection rate.…”

“…The complete removal of xenoantigen from the xenograft tissues reduces the immune rejection rate. Modified decellularization methods and CRISPR-Cas9 technology have been introduced for this purpose (H. J.. As an example, chondroitin sulfate crosslinking with the decellularized xenograft scaffold is one of the modulations that reduces the immune rejection by masking the antigen presentation to the immune system (J Chakraborty et al, 2018)…”