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Light transmittance of the cDPC and NPC groups when wavelength ranges from 280 nm to 780 nm with a scanning interval of 10 nm.
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This study aims to develop a cross-linked decellularized porcine corneal graft (cDPC) as a substitute for lamellar donor corneas and to evaluate the feasibility of using cDPC to treat fungal keratitis. The cDPC was prepared by decellularization, chemical crosslinking and γ-ray irradiation. Transparency, effectiveness of decellularization and biomec...
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Context 1
... transmittance evaluation of transparency. To obtain a quantitative measure of light transmit- tance (LT), we measure LT over a range from 280 nm to 780 nm using an ultraviolet spectrophotometer. There was no significant difference between the NPC and cDPC groups at any wavelength between these two different wavelengths (n = 10, p > 0.05) (Fig. ...
Similar publications
The cornea is one of the most commonly transplanted tissues worldwide. It is used to restore vision when severe visual impairment or blindness occurs in patients with corneal diseases or after trauma. Due to the global shortage of healthy donor corneas, decellularized corneal tissue has significant potential as an alternative to corneal transplanta...
Citations
... However, decreased mechanical properties and corneal blurring after decellularization remain concerns to peruse. Along with different modifications, such as in vitro corneal crosslinking after decellularization to stabilize the biomechanical properties of the decellularized cornea [12,13], hydrogels of gelatin methacrylate (GelMA) and Collagen I were introduced to modify a decellularized cornea for proper biomechanical and transparency features [14,15]. GelMA has been used for corneal regeneration in different studies via bioprinting [16,17], molding [18,19], or electrospinning [20] techniques, which can be a proper modifier hydrogel for a decellularized cornea. ...
Corneal injury is one of the substantial and challenging global concerns regarding vision loss. Corneal transplantation is the gold standard for restoring vision to a blurred cornea; however, suture-related complications, corneal haziness, and graft failure have led to ongoing research to improve graft efficiency. In this study, a highly suture-able hybrid corneal scaffold composed of decellularized corneas injected with GelMa/PEGDA in situ was fabricated to preserve the structural integrity and transparency of the injured corneas. First, GelMA/PEGDA hydrogels with different ratios (3/7, 1/1, and 7/3) were evaluated in terms of microstructure, degradation rate, cytocompatibility, and equilibrium water content. GelMa/PEGDA 7/3 was chosen based on the cytocompatibility assay, which confirmed the attachment, maintenance, and proliferation of limbal stem cells with an elongated morphology. Next, the explanted rabbit cornea was decellularized successfully to preserve its extracellular components and transparency with less than 1% DNA content. Thereafter, a hybrid cornea was developed by incorporating GelMA/PEGDA hydrogel (7/3) into the decellularized rabbit cornea under ultraviolet light. This hybrid cornea showed improved sutureability and possessed appropriate transparency. In addition, ex vivo examinations of the enucleated eyeballs of rabbits showed that this hybrid cornea effectively adhered to the corneal stromal and surrounding tissues by applying GelMA/PEGDA hydrogel, which is favorable for corneal transplantations. Based on these findings, the introduced hybrid cornea could be a potential candidate for the in situ repair of deep anterior corneal defects.
Graphical abstract
... With no reports of in vivo rejection, xenogeneic scafolds could be used efciently for corneal transplantation with the great advantage of their easy accessibility [5,[20][21][22]. Although sterilisation methods are not within the topic of the current article, it is necessary to mention that they can supplement corneal decellularization methods to ensure maximum efect without rejection after in vivo implantation [23][24][25][26]. ...
... It could also simplify the storage and extend the availability of corneal tissue in developing countries with limited access to eye banking. Efciently decellularized corneal tissue could help treat corneal diseases (keratitis, ectasia, and perforations) or correct hyperopia and presbyopia [26,34,82]. Te main challenge to corneal decellularization is to "sufciently decellularize corneas" while maintaining their transparency, ECM structure, and biocompatibility [2,8,39,58]. ...
The cornea is one of the most commonly transplanted tissues worldwide. It is used to restore vision when severe visual impairment or blindness occurs in patients with corneal diseases or after trauma. Due to the global shortage of healthy donor corneas, decellularized corneal tissue has significant potential as an alternative to corneal transplantation. It preserves the native and biological ultrastructure of the cornea and, therefore, represents the most promising scaffold. This article discusses different methods of corneal decellularization based on the current literature. We searched PubMed.gov for articles from January 2009 to December 2023 using the following keywords: corneal decellularization, decellularization methods, and corneal transplantation. Although several methods of decellularization of corneal tissue have been reported, a universal standardised protocol of corneal decellularization has not yet been introduced. In general, a combination of decellularization methods has been used for efficient decellularization while preserving the optimal properties of the corneal tissue.
... 39 Therefore, EDC-crosslinked matrices exhibit very low cytotoxicity, which has already been demonstrated in vitro and in animal models. 40 immune response, and anti-calcification ability compared with glutaraldehyde treatment. 44,45 The reason for anti-calcification may be that the EDC treatment does not contain aldehyde groups, which can protect elastin. ...
Tissue‐engineered blood vessel substitutes have been developed due to the lack of suitable small‐diameter vascular grafts. Xenogeneic extracellular matrix (ECM) scaffolds have the potential to provide an ideal source for off‐the‐shelf vascular grafts. In this study, porcine carotid arteries were used to develop ECM scaffolds by decellularization and coating with heparin and hepatocyte growth factor (HGF). After decellularization, cellular and nucleic materials were successfully removed with preservation of the main compositions (collagen, elastin, and basement membrane) of the native ECM. The ultimate tensile strength, suture strength, and burst pressure were significantly increased after cross‐linking. Pore size distribution analysis revealed a porous structure within ECM scaffolds with a high distribution of pores larger than 10 μm. Heparinized scaffolds exhibited sustained release of heparin in vitro and showed potent anticoagulant activity by prolonging activated partial thromboplastin time. The scaffolds showed an enhanced HGF binding capacity as well as a constant release of HGF as a result of heparin modification. When implanted subcutaneously in rats, the modified scaffolds revealed good biocompatibility with enzyme degradation resistance, mitigated immune response, and anti‐calcification. In conclusion, heparinized and HGF‐coated acellular porcine carotid arteries may be a promising biological scaffold for tissue‐engineered vascular grafts.
... Therefore, the crosslinking of collagen is necessary to improve its physicochemical properties [16]. Previous studies have reported that the preparation of crosslinked membranes using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-Hydroxy succinimide (NHS) improves optical and mechanical performance [17]. However, the long-term stability of implants stabilized by EDC/NHS is limited, and these membranes are not suitable for the interrupted suture method commonly used in clinical procedures [18]. ...
Corneal disease has become the second leading cause of blindness in the world. Corneal transplantation is currently considered to be one of the common treatments for vision loss. This paper presents a novel approach utilizing dual-crosslinked membranes composed of polyrotaxane multiple aldehydes (PRAs), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS) in the development process. Collagen was crosslinked, respectively, by EDC/NHS and PRAs to form stable amide bonds and imine groups. Through the formation of a double interpenetrating network, dual-crosslinked (Col-EDC-PRA) membranes exhibited enhanced resistance to collagenase degradation and superior mechanical properties compared to membranes crosslinked with a single crosslinker. Furthermore, Col-EDC-PRA membranes display favorable light transmittance and water content characteristics. Cell experiments showed that Col-EDC-PRA membranes were noncytotoxic and were not significantly different from other membranes. In a rabbit keratoplasty model, corneal stromal repair occurred at 5 months, evidenced by the presence of stromal cells and neo-stroma, as depicted in hematoxylin–eosin-stained histologic sections and optical coherence tomography images of the anterior segment. Moreover, there was no inflammation and corneal neovascularization, as well as no corneal rejection reaction in the surgical area. Overall, the results demonstrated that the dual-crosslinked membranes served effectively for corneal tissue regeneration after corneal defect.
... This phenomenon was not observed when lenticules were crosslinked without decellularization in our preliminary study (data not shown). Moreover, Lin Y et al. reported a high transparency of EDC/NHS crosslinked acellular porcine corneas for fungal keratitis therapy [42] . The effect of EDC/NHS crosslinking could be influenced by different solvents and working temperatures [43] . ...
Substantial evidence has demonstrated the application of fresh and decellularized human corneal lenticules from increasing myopic surgeries. Further preservation of decellularized corneal lenticules would extend its clinical application. However, whether fresh and preserved decellularized lenticules have the same effects in vivo, including refractive correction, remains unclear. Here, we made comprehensive comparisons between fresh human lenticules (FHLs) and preserved decellularized human lenticules (DHLs). Another group of decellularized lenticules was combined with crosslinking for potential keratoconus therapy. Optical transparency, biomechanical properties, and fibrillar ultrastructure were analyzed to evaluate the DHLs and crosslinked DHLs (cDHLs) in vitro. The DHLs retained high transparency and regular ultrastructure, with genetic materials mostly being eliminated. The strength of lenticules in the cDHL group was markedly increased by crosslinking. Moreover, after storage in glycerol for 3 months, the lenticules were reimplanted into rabbit corneal lamellar pockets assisted by a femtosecond laser. The rabbits were followed for another 3 months. There were no obvious rejective complications in any of the three groups. From 1 week to 3 months postoperatively, the host corneas of the FHL group remained highly transparent, while slight hazes were observed in the DHL group. However, the corneas of the cDHL group displayed opacity throughout the 3-month postoperative period. Furthermore, all the lenticules could effectively induce corneal steepening and refractive changes. Taken together, our data indicated that FHLs are ideal inlay products, whereas preserved DHLs could be an alternative for intrastromal lamellar keratoplasty. Our study provides new insights into the clinical application of human lenticule recycling.
Statement of significance
: Currently, substantial evidence has demonstrated the application of fresh and decellularized human corneal lenticules from increasing myopic surgeries. Further preservation of decellularized lenticules would extend its clinical application. However, whether fresh and preserved decellularized lenticules have the same effects in vivo, including refractive correction, remains unclear. Herein, we decellularized human lenticules with or without mechanically strengthened crosslinking. After storage in glycerol for 3 months, the lenticules were reimplanted into rabbit corneas. Comprehensive comparisons were performed among fresh human lenticules (FHLs), decellularized human lenticules (DHLs) and crosslinked DHLs. Our study indicated that FHLs are ideal inlay products, whereas preserved DHLs could be an alternative for intrastromal lamellar keratoplasty. Our study provides new insights into the clinical application of human lenticule recycling.
... However, they can crosslink and precipitate collagens, altering the structural integrity of the 3D proteomic network. 17 Alcohols such as isopropanol, 77,79 glycerol, 80 ethanol, [81][82][83] and methanol 21 have all proven to be effective for removal of lipid content, though some reports indicate that isopropanol may be a superior agent for this purpose. 84 Like alcohols, acetone removes cellular content from ECM by acting on lipids. ...
... The ECM of the cornea is composed of water, inorganic salts, proteoglycans, glycoproteins, and several types of 80 indicates that decellularization of cornea can be performed using glycerol with chemical crosslinking (Figure 2). The application of glycerol to the tissue through a pressure based osmotic system followed by gamma-ray irradiation preserved the fibrous collagen morphology and GAG content. ...
... Moreover, it provided a fully transparent and non-immunogenic decellularized graft with long-term stability. 80 ...
Decellularization of natural tissues to produce extracellular matrix is a promising method for three-dimensional scaffolding and for understanding microenvironment of the tissue of interest. Due to the lack of a universal standard protocol for tissue decellularization, recent investigations seek to develop novel methods for whole or partial organ decellularization capable of supporting cell differentiation and implantation towards appropriate tissue regeneration. This review provides a comprehensive and updated perspective on the most recent advances in decellularization strategies for a variety of organs and tissues, highlighting techniques of chemical, physical, biological, enzymatic, or combinative-based methods to remove cellular contents from tissues. In addition, the review presents modernized approaches for improving standard decellularization protocols for numerous organ types.
... The origin of the cells used for recellularization is an important issue to consider. Non-human animal cells, immortalized cell lines, and primary human corneal cells have been used in most studies involving repopulation of DCs [17,19,23,53,69,70]. Although primary human cells can bypass the safety concerns related to immortalized cell lines and animal-derived cells, their limited proliferation potential has hampered their clinical use. ...
Up to 40% of donor corneas are deemed unsuitable for transplantation, aggravating the shortage of graft tissue. In most cases, the corneal extracellular matrix is intact. Therefore, their decellularization followed by repopulation with autologous cells may constitute an efficient alternative to reduce the amount of discarded tissue and the risk of immune rejection after transplantation. Although induced pluripotent (hiPSCs) and orbital fat-derived stem cells (OFSCs) hold great promise for corneal epithelial (CE) reconstruction, no study to date has evaluated the capacity of decellularized corneas (DCs) to support the attachment and differentiation of these cells into CE-like cells. Here, we recellularize DCs with hiPSCs and OFSCs and evaluate their differentiation potential into CE-like cells using animal serum-free culture conditions. Cell viability and adhesion on DCs were assessed by calcein-AM staining and scanning electron microscopy. Cell differentiation was evaluated by RT-qPCR and immunofluorescence analyses. DCs successfully supported the adhesion and survival of hiPSCs and OFSCs. The OFSCs cultured under differentiation conditions could not express the CE markers, TP63, KRT3, PAX6, and KRT12, while the hiPSCs gave rise to cells expressing high levels of these markers. RT-qPCR data suggested that the DCs provided an inductive environment for CE differentiation of hiPSCs, supporting the expression of PAX6 and KRT12 without the need for any soluble induction factors. Our results open the avenue for future studies regarding the in vivo effects of DCs as carriers for autologous cell transplantation for ocular surface reconstruction.
... Light transmittance across the visible spectrum from 350 to 700 nm wavelength was quantified using a spectrophotometer (BioTek Synergy HTX, ThermoFisher) with the scaffold immersed in ultrapure water. Glycerol has been shown to re-establish the thickness and curvature of a swollen cornea [21]. For this reason, the scaffold transmittance was then assessed after immersion in glycerol for 2 hours. ...
Decellularized porcine corneal scaffolds are a potential alternative to human cornea for keratoplasty. Although clinical trials have reported promising results, there can be corneal haze or scar tissue. Here, we examined if recellularizing the scaffolds with human keratocytes would result in a better outcome. Scaffolds were prepared that retained little DNA (14.89 ± 5.56 ng/mg) and demonstrated a lack of cytotoxicity by in vitro . The scaffolds were recellularized using human corneal stromal cells and cultured for between 14 in serum-supplemented media followed by a further 14 days in either serum free or serum-supplemented media. All groups showed full-depth cell penetration after 14 days. When serum was present, staining for ALDH3A1 remained weak but after serum-free culture, staining was brighter and the keratocytes adopted a native dendritic morphology with an increase (p < 0.05) of keratocan, decorin, lumican and CD34 gene expression. A rabbit anterior lamellar keratoplasty model was used to compare implanting a 250 μm thick decellularized lenticule against one that had been recellularized with human stromal cells after serum-free culture. In both groups, host rabbit epithelium covered the implants, but transparency was not restored after 3 months. Post-mortem histology showed under the epithelium, a less-compact collagen layer, which appeared to be a regenerating zone with some α-SMA staining, indicating fibrotic cells. In the posterior scaffold, ALDH1A1 staining was present in all the acellular scaffold, but in only one of the recellularized lenticules. Since there was little difference between acellular and cell-seeded scaffolds in our in vivo study, future scaffold development should use acellular controls to determine if cells are necessary.
... The cells acquired potency to proliferate and covered vascular luminal surface. The existence of specific molecules mainly glycosaminoglycans is conceived as one of the most important factors of tissue rejection after transplantation [22]. Although, these factors could facilitate cell attachment and activate specific signaling pathways correlated with cell growth and survival [23]. ...
Objective: The current experiment aimed to assess the impact of detergents
such as 3% Triton X-100, 1% peracetic acid, 1% Tween-20, and 1% SDS in
combination with Trypsin-EDTA on acellularization of ovine aortae after 7 days.
Results: Hematoxylin-Eosin staining showed an appropriate acellularization
rate in ovine aortae, indicated by a lack of cell nuclei in the tunica media layer. DAPI
staining confirmed the lack of nuclei in the vascular wall after being exposed to the
combination of chemical and enzymatic solutions. Verhoeff-Van Gieson staining
showed that elastin fibers were diminished in acellular samples compared to the
control group while collagen stands were unchanged. CCK-8 survival assay showed
enhanced viability in human umbilical vein endothelial cells 5 days after being cultured
on decellularized samples compared to the cells cultured on a plastic surface (p<0.05).
SEM imaging showed flattening of endothelial cells on the acellular surface.
... Also, the Boston Kpro requires the use of a corneal graft, thus not reducing the impact of lack of donor tissues in the treatment of corneal blindness. Besides, we will not consider methods using extracellular matrix deposition by cells (Nishida et al., 2004;Kobayashi et al., 2013) and methods using decellularized materials (Lin et al., 2017;Hashimoto et al., 2010). Thus, we will present the works using either collagen (type I or type III) or other biopolymers to produce a mimetic scaffold. ...
Bio-engineering technologies are currently used to produce biomimetic artificial corneas that should present structural, chemical, optical, and biomechanical properties close to the native tissue. These properties are mainly supported by the corneal stroma which accounts for 90% of corneal thickness and is mainly made of collagen type I. The stromal collagen fibrils are arranged in lamellae that have a plywood-like organization. The fibril diameter is between 25 and 35 nm and the interfibrillar space about 57 nm. The number of lamellae in the central stroma is estimated to be 300. In the anterior part, their size is 10-40 μm. They appear to be larger in the posterior part of the stroma with a size of 60-120 μm. Their thicknesses also vary from 0.2 to 2.5 μm. During development, the acellular corneal stroma, which features a complex pattern of organization, serves as a scaffold for mesenchymal cells that invade and further produce the cellular stroma. Several pathways including Bmp4, Wnt/β-catenin, Notch, retinoic acid, and TGF-β, in addition to EFTFs including the mastering gene Pax-6, are involved in corneal development. Besides, retinoic acid and TGF- β seem to have a crucial role in the neural crest cell migration in the stroma. Several technologies can be used to produce artificial stroma. Taking advantage of the liquid-crystal properties of acid-soluble collagen, it is possible to produce transparent stroma-like matrices with native-like collagen I fibrils and plywood-like organization, where epithelial cells can adhere and proliferate. Other approaches include the use of recombinant collagen, cross-linkers, vitrification, plastically compressed collagen or magnetically aligned collagen, providing interesting optical and mechanical properties. These technologies can be classified according to collagen type and origin, presence of telopeptides and native-like fibrils, structure, and transparency. Collagen matrices feature transparency >80% for the appropriate 500-μm thickness. Non-collagenous matrices made of biopolymers including gelatin, silk, or fish scale have been developed which feature interesting properties but are less biomimetic. These bioengineered matrices still need to be colonized by stromal cells to fully reproduce the native stroma.
