Characterization of regenerated neo-corneas. (a) H&E sections through a healthy, unoperated cornea, and regenerated neo-corneas at 12 months after implantation with control RHCIII-MPC and CLP-PEG scaffolds. Epithelial hyperplasia was noted in the implanted corneas, which is normal in post-grafting tissues (b). Scale bars, 100 mm. The regenerated corneal epithelium shows staining with cytokeratin 3/12, a marker of differentiated cells in all three samples. Stromal collagens types III (c) and V (d) are present in the implanted as well as unoperated corneas, showing in particular that remodeling and extracellular matrix production is occurring in CLP-PEG implants that contained no collagen. Sub-epithelial nerve plexus stained with b-tubulin (e) showing nerve regeneration in CLP-PEG hydrogels as in control RHCIII-MPC and unoperated contralateral eyes. Scale bars, 100 mm. At the ultrastructural level, TEM micrographs of all three samples show an epithelium with distinct layers of elongated, cuboidal and flattened cells, characteristic of healthy corneas (f). Scale bar, 50 mm. TEM images show a regular, lamellar arrangement in both unoperated and regenerated stromas (g). Scale bar, 20 mm. The CLP-PEG neo-corneas have slightly less regular stromas as seen in 3D reconstructed SBF-SEM images (epithelium rendered in blue) (h) suggesting an on-going process. Both neo-corneas contained collagen fibrils decorated with proteoglycans (i), similar to the matrix of the healthy, unoperated cornea. Scale bar, 200 nm. 

Characterization of regenerated neo-corneas. (a) H&E sections through a healthy, unoperated cornea, and regenerated neo-corneas at 12 months after implantation with control RHCIII-MPC and CLP-PEG scaffolds. Epithelial hyperplasia was noted in the implanted corneas, which is normal in post-grafting tissues (b). Scale bars, 100 mm. The regenerated corneal epithelium shows staining with cytokeratin 3/12, a marker of differentiated cells in all three samples. Stromal collagens types III (c) and V (d) are present in the implanted as well as unoperated corneas, showing in particular that remodeling and extracellular matrix production is occurring in CLP-PEG implants that contained no collagen. Sub-epithelial nerve plexus stained with b-tubulin (e) showing nerve regeneration in CLP-PEG hydrogels as in control RHCIII-MPC and unoperated contralateral eyes. Scale bars, 100 mm. At the ultrastructural level, TEM micrographs of all three samples show an epithelium with distinct layers of elongated, cuboidal and flattened cells, characteristic of healthy corneas (f). Scale bar, 50 mm. TEM images show a regular, lamellar arrangement in both unoperated and regenerated stromas (g). Scale bar, 20 mm. The CLP-PEG neo-corneas have slightly less regular stromas as seen in 3D reconstructed SBF-SEM images (epithelium rendered in blue) (h) suggesting an on-going process. Both neo-corneas contained collagen fibrils decorated with proteoglycans (i), similar to the matrix of the healthy, unoperated cornea. Scale bar, 200 nm. 

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Statement of significance: Although biomaterials comprising full-length recombinant human collagen and extracted animal collagen have been evaluated and used clinically, these macromolecules provide only a limited number of functional groups amenable to chemical modification or crosslinking and are demanding to process. Synthetic, customizable ana...

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... corneas performed in compliance with OECD GLP principles by a blinded vet patholo- gist (BioVet AB, Sollentuna, Sweden) concluded that there was no difference between CLP-PEG and RHCIII-MPC implanted corneas. Epithelial hyperplasia was noted in all regenerated neo-corneas but otherwise, they resembled unoperated, healthy mini-pig cor- neas (Fig. ...
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... CLP-PEG and RHCIII-MPC regenerated neo-corneas had cytokeratin 3-positive, fully differentiated epithelia, like un- operated controls (Fig. 4b). The regenerated neo-cornea stromas were positively stained for collagen type III (Fig. 4c) which co- exists with type I within the stromal ECM [26], and type V (Fig. 4d), which makes up 10-20% of corneal collagen [27]. No blood ves- sels (no CD31 staining) or lymphatic vessels (no LYVE1 staining) were found within the regenerated ...
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... CLP-PEG and RHCIII-MPC regenerated neo-corneas had cytokeratin 3-positive, fully differentiated epithelia, like un- operated controls (Fig. 4b). The regenerated neo-cornea stromas were positively stained for collagen type III (Fig. 4c) which co- exists with type I within the stromal ECM [26], and type V (Fig. 4d), which makes up 10-20% of corneal collagen [27]. No blood ves- sels (no CD31 staining) or lymphatic vessels (no LYVE1 staining) were found within the regenerated neo-corneas or healthy controls (Fig. S4). b-tubulin staining of whole mounts confirmed ...
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... CLP-PEG and RHCIII-MPC regenerated neo-corneas had cytokeratin 3-positive, fully differentiated epithelia, like un- operated controls (Fig. 4b). The regenerated neo-cornea stromas were positively stained for collagen type III (Fig. 4c) which co- exists with type I within the stromal ECM [26], and type V (Fig. 4d), which makes up 10-20% of corneal collagen [27]. No blood ves- sels (no CD31 staining) or lymphatic vessels (no LYVE1 staining) were found within the regenerated neo-corneas or healthy controls (Fig. S4). b-tubulin staining of whole mounts confirmed restoration of parallel nerve fibres of the sub-epithelial nerve plexus in both implant ...
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... neo-cornea stromas were positively stained for collagen type III (Fig. 4c) which co- exists with type I within the stromal ECM [26], and type V (Fig. 4d), which makes up 10-20% of corneal collagen [27]. No blood ves- sels (no CD31 staining) or lymphatic vessels (no LYVE1 staining) were found within the regenerated neo-corneas or healthy controls (Fig. S4). b-tubulin staining of whole mounts confirmed restoration of parallel nerve fibres of the sub-epithelial nerve plexus in both implant groups that resembled the unoperated controls (Fig. ...
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... [27]. No blood ves- sels (no CD31 staining) or lymphatic vessels (no LYVE1 staining) were found within the regenerated neo-corneas or healthy controls (Fig. S4). b-tubulin staining of whole mounts confirmed restoration of parallel nerve fibres of the sub-epithelial nerve plexus in both implant groups that resembled the unoperated controls (Fig. ...
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... confirmed the similarity of epithelial cells of CLP-PEG and RHCIII-MPC regenerated neo-corneas to those of the unoperated corneas but with hyperplasia (Fig. 4f). The underlying regenerated stromas contained cells arranged in lamellae (Fig. 4 g). However, the lamellae were less structured in the neo-corneas compared to controls, and particularly in the CLP-PEG implanted corneas as seen by serial block face-SEM (SBF-SEM) 3D reconstruction ( Fig. 4h; Videos S1-3). Staining for proteoglycans below ...
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... confirmed the similarity of epithelial cells of CLP-PEG and RHCIII-MPC regenerated neo-corneas to those of the unoperated corneas but with hyperplasia (Fig. 4f). The underlying regenerated stromas contained cells arranged in lamellae (Fig. 4 g). However, the lamellae were less structured in the neo-corneas compared to controls, and particularly in the CLP-PEG implanted corneas as seen by serial block face-SEM (SBF-SEM) 3D reconstruction ( Fig. 4h; Videos S1-3). Staining for proteoglycans below the newly regenerated epithelium as visualised by TEM showed that their arrangement ...
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... to those of the unoperated corneas but with hyperplasia (Fig. 4f). The underlying regenerated stromas contained cells arranged in lamellae (Fig. 4 g). However, the lamellae were less structured in the neo-corneas compared to controls, and particularly in the CLP-PEG implanted corneas as seen by serial block face-SEM (SBF-SEM) 3D reconstruction ( Fig. 4h; Videos S1-3). Staining for proteoglycans below the newly regenerated epithelium as visualised by TEM showed that their arrangement within RHCIII-MPC and CLP-PEG was similar to the arrangement in an unoperated mini pig cornea (Fig. ...
Context 10
... and particularly in the CLP-PEG implanted corneas as seen by serial block face-SEM (SBF-SEM) 3D reconstruction ( Fig. 4h; Videos S1-3). Staining for proteoglycans below the newly regenerated epithelium as visualised by TEM showed that their arrangement within RHCIII-MPC and CLP-PEG was similar to the arrangement in an unoperated mini pig cornea (Fig. ...

Citations

... Several in vitro and in vivo studies have reported that short ECM-mimicking peptides can stimulate regeneration in a range of organ systems. As an alternative to fulllength collagen, short collagen-like peptides (CLPs) have been used to fabricate soft hydrogels [154] or have been conjugated to synthetic polymer for better mechanical strength as implants [164,165]. ...
... One synthetic polymer that is commonly used to conjugate CLP is polyethylene glycol (PEG) which is chemically inert [164,166,167]. CLP-PEG implants can be enhanced with 2-methacryloyloxyethyl phosphorylcholine (MPC), an artificial lipid that suppress the inflammation. ...
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The cornea and the skin are two organs that form the outer barrier of the human body. When either is injured (e.g., from surgery, physical trauma, or chemical burns), wound healing is initiated to restore integrity. Many cells are activated during wound healing. In particular, fibroblasts that are stimulated often transition into repair fibroblasts or myofibroblasts that synthesize extracellular matrix (ECM) components into the wound area. Control of wound ECM deposition is critical, as a disorganized ECM can block restoration of function. One of the most abundant structural proteins in the mammalian ECM is collagen. Collagen type I is the main component in connective tissues. It can be readily obtained and purified, and short analogs have also been developed for tissue engineering applications, including modulating the wound healing response. This review discusses the effect of several current collagen implants on the stimulation of corneal and skin wound healing. These range from collagen sponges and hydrogels to films and membranes.
... Despite the cornea have the relative immune privilege, allogeneic rejection is the most common reason of corneal transplantation failure [51]. A relative study has proposed that collagen V was secreted by the corneal epithelium into the corneal stroma by EVs pathway, which meant that EV secretion is involved in the biomaterialsinduced corneal regeneration [52]. Furthermore, MSC-Exos with co-delivery of siRNA against Fas receptor and miR-375 inhibitor successfully improved islet transplantation [53], making exosomes possible to promote immune tolerance of corneal grafts. ...
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Background Ocular surface and retinal diseases are widespread problems that cannot be ignored in today’s society. However, existing prevention and treatment still have many shortcomings and limitations, and fail to effectively hinder the occurrence and development of them. Main body The purpose of this review is to give a detailed description of the potential mechanism of exosomes and autophagy. The eukaryotic endomembrane system refers to a range of membrane-bound organelles in the cytoplasm that are interconnected structurally and functionally, which regionalize and functionalize the cytoplasm to meet the needs of cells under different conditions. Exosomal biogenesis and autophagy are two important components of this system and are connected by lysosomal pathways. Exosomes are extracellular vesicles that contain multiple signaling molecules produced by multivesicular bodies derived from endosomes. Autophagy includes lysosome-dependent degradation and recycling pathways of cells or organelles. Recent studies have revealed that there is a common molecular mechanism between exosomes and autophagy, which have been, respectively, confirmed to involve in ocular surface and retinal diseases. Conclusion The relationship between exosomes and autophagy and is mostly focused on fundus diseases, while a deeper understanding of them will provide new directions for the pathological mechanism, diagnosis, and treatment of ocular surface and retinal diseases.
... -In vitro disease modelling in support of the 3Rs; -COVID- 19 and in vitro disease modelling; ...
... Dr Reddy also highlighted how the ECM can be modulated through the mechanical tuning of its components, in order to fabricate cancer organoid cultures for personalised therapy. [19][20][21] NeuroSAFE -A Next Gen cruelty-free solution for testing vaccine safety for human application Dr Subhadra Dravida (Founder, Transcell Biologics, India) and Saikat Biswas (Global Head, Life Sciences Digital Operations Wipro Ltd, India) jointly presented a non-animal cruelty-free workstation strategy for the safety-related risk assessment of vaccines in production, with respect to neurovirulence in clinics. The testing of vaccines for neurovirulence risk has always been a requirement for many regulatory authorities globally. ...
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The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models. Oncoseek Bio-Acasta Health, which is an innovative biotechnology start-up company based in Hyderabad and Vishakhapatnam, India, organises an annual International Conference on 3Rs Research and Progress. In 2021, this conference was on ‘Advances in Research Animal Models and Cutting-Edge Research in Alternatives’. This annual conference is a platform that brings together eminent scientists and researchers from various parts of the world, to share recent advances from their research in the field of alternatives to animals including new approach methodologies, and to promote practices to help refine animal experiments where alternatives are not available. This report presents the proceedings of the conference, which was held in hybrid mode (i.e. virtual and in-person) in November 2021.
... Gonzalez-Nolasco et al. have recently reviewed the role of exosomes in recognition, rejection, and tolerance of allografts (Gonzalez-Nolasco et al., 2018). Another study shows that short collagen-like peptides conjugated to polyethylene glycol (CLP-PEG) exert proregenerating effects through stimulation of EVs production by host cells (Jangamreddy et al., 2018). Additionally, epithelial-derived exosomes are reported to have a potential role in corneal wound healing and neovascularization. ...
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Exosomes are a subset of extracellular vesicles (EVs) that are secreted by most cell types. They are nanosized EVs ranging from 30 to 150 nm. The membrane-enclosed bodies originate by the process of endocytosis and mainly comprise DNA, RNA, protein, and lipids. Exosomes not only act as cell-to-cell communication signaling mediators but also have the potential to act as biomarkers for clinical application and as a promising carrier for drug delivery. Unfortunately, the purification methods for exosomes remain an obstacle. While most of the exosome researches are mainly focused on cancer, there are limited studies highlighting the importance of exosomes in ocular biology, specifically cornea-associated pathologies. Here, we summarize a brief description of exosome biogenesis, roles of exosomes and exosome-based therapies in corneal pathologies, and exosome bioengineering for tissue-specific therapy.
... Samples from implanted and control corneas were processed for TEM as described in McTiernan et al. [14,21,22]. In brief, samples were prepared for TEM by fixing a quarter of each cornea in 2.5% glutaraldehyde solution in 0.1 M sodium cacodylate buffer (pH 7.4). ...
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... Вторым по популярности природным биоматериалом является желатин, который получают из коллагена и используют в качестве гелеобразующего вещества в производстве средств личной гигиены, косметики, продуктов питания, пищевых добавок, напитков, фармацевтической продукции и т. д. [46][47][48]. ...
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... 27 When conjugated to a multiarm polyethylene glycol (PEG) through a short peptide and thiolmaleimide, the resulting CLP-PEG hydrogel could be fabricated into corneal implants that promoted regeneration in the corneas of mini-pigs. 28,29 However, the N-(3-dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride (EDC) used for stabilizing the hydrogels was possibly pro-inflammatory. 20,30 As mentioned above there are currently no or limited treatment options for patients awaiting corneal transplantation that are at high-risk of graft rejection. ...
... We showed that CLP-PEG-MPC implants, like previously described CLP-PEG ones, were readily and reliably mouldable. 29 Both implants had refractive indices of 1.34, in keeping with their high water content. However, while both implants were highly transparent in visible light, CLP-PEG-MPC but not CLP-PEG implants filtered up to 60% of potentially damaging UV-A, which is essential for lens and retina protection (from cataract and macular degeneration). ...
... 18,19 Here, as in Fagerholm et al., 19 the original implant was remodeled during corneal regeneration. Furthermore, Jangamreddy et al. 29 showed that the weaker, cell-free CLP-PEG hydrogels implanted into rabbit corneas transformed into regenerated neo-corneas with mechanical properties approximating those of healthy, unoperated corneas, the desired end-points. Here, the objective was to synthesize a decreased-cost, peptide-based implant with the immunosuppressive qualities of MPC. ...
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The long-term survival of biomaterial implants is often hampered by surgery-induced inflammation that can lead to graft failure. Considering that most corneas receiving grafts are either pathological or inflamed before implantation, the risk of rejection is heightened. Here, we show that bioengineered, fully synthetic, and robust corneal implants can be manufactured from a collagen analog (collagen-like peptide-polyethylene glycol hybrid, CLP-PEG) and inflammation-suppressing polymeric 2-methacryloyloxyethyl phosphorylcholine (MPC) when stabilized with the triazine-based crosslinker 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. The resulting CLP-PEG-MPC implants led to reduced corneal swelling, haze, and neovascularization in comparison to CLP-PEG only implants when grafted into a mini-pig cornea alkali burn model of inflammation over 12 months. Implants incorporating MPC allowed for faster nerve regeneration and recovery of corneal sensation. CLP-PEG-MPC implants appear to be at a more advanced stage of regeneration than the CLP-PEG only implants, as evidenced by the presence of higher amounts of cornea-specific type V collagen, and a corresponding decrease in the presence of extracellular vesicles and exosomes in the corneal stroma, in keeping with the amounts present in healthy, unoperated corneas.
... Besides, these peptide analogs showed pro-regeneration effects via stimulating the production of the extracellular vesicle by the host cells. In addition, researchers declared that the CLP-PEG hydrogel implants can be applied for the regeneration of corneal epithelial tissue [63,64] . ...
... No Pig Suitable integration, reepithelialization, no redness & inflammation, neovascularization or lymphatic vessels [63,64] Collagen vitrigel. ...
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Blindness of the cornea is the major cause of blindness globally. Many approaches and strategies have been developed to replace or regenerate damaged cornea. Different biomaterials like biopolymeric hydrogels with excellent mechanical & physical properties have been used for the regeneration of corneal tissue. In this review, we focused on using the different types of hydrogels throughout in-vivo studies for the regeneration of corneal epithelial tissue.
... So far, there has been considerable diversity in the scaffolds for corneal treatment using both synthetic and natural materials [19,20]. These scaffolds have indicated different degrees of success once they were implanted into an in vivo cellular microenvironment [10,[21][22][23][24][25][26][27][28][29]. Most of the natural materials do not have an unexpected inflammatory response during degradation, resulting in biocompatibility and mostly bioactivity that incorporate them into surrounding native tissues [30][31][32][33]. ...
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A hybrid scaffold of gelatin-glycosaminoglycan matrix and fibrin (FGG) has been synthesized to improve the mechanical properties, degradation time and cell response of fibrin-like scaffolds. The FGG scaffold was fabricated by optimizing some properties of fibrin-only gel and gelatin-glycosaminoglycan (GG) scaffolds. Mechanical analysis of optimized fibrin-only gel showed the Young module and tensile strength of up to 72 and 121 KPa, respectively. Significantly, the nine-fold increase in the Young modulus and a seven-fold increase in tensile strength was observed when fibrin reinforced with GG scaffold. Additionally, the results demonstrated that the degradation time of fibrin was enhanced successfully up to 7 days which was much longer time compared to fibrin-only gel with 38 h of degradation time. More than 45% of FGG initial mass was preserved on day 7 in the presence of aprotinin. Human corneal fibroblast cells (HCFCs) were seeded on the FGG, fibrin-only gel and GG scaffolds for 5 days. The FGG scaffold showed excellent cell viability over 5 days, and the proliferation of HCFCs also increased significantly in comparison with fibrin-only gel and GG scaffolds. The FGG scaffold illustrates the great potential to use in which appropriate stability and mechanical properties are essential to tissue functionality.
... ALK was used to implant the hydrogels into minipig eyes and the transplanted corneas were followed up for 12 months. Implanted corneas remained clear and both epithelium and stroma regenerated successfully [204]. ...
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Full-text available
Background: Since the cornea is responsible for transmitting and focusing light into the eye, injury or pathology affecting any layer of the cornea can cause a detrimental effect on visual acuity. Aging is also a reason for corneal degeneration. Depending on the level of the injury, conservative therapies and donor tissue transplantation are the most common treatments for corneal diseases. Not only is there a lack of donor tissue and risk of infection/rejection, but the inherent ability of corneal cells and layers to regenerate has led to research in regenerative approaches and treatments. Methods: In this review, we first discussed the anatomy of the cornea and the required properties for reconstructing layers of the cornea. Regenerative approaches are divided into two main categories; using direct cell/growth factor delivery or using scaffold-based cell delivery. It is expected delivered cells migrate and integrate into the host tissue and restore its structure and function to restore vision. Growth factor delivery also has shown promising results for corneal surface regeneration. Scaffold-based approaches are categorized based on the type of scaffold, since it has a significant impact on the efficiency of regeneration, into the hydrogel and non-hydrogel based scaffolds. Various types of cells, biomaterials, and techniques are well covered. Results: The most important characteristics to be considered for biomaterials in corneal regeneration are suitable mechanical properties, biocompatibility, biodegradability, and transparency. Moreover, a curved shape structure and spatial arrangement of the fibrils have been shown to mimic the corneal extracellular matrix for cells and enhance cell differentiation. Conclusion: Tissue engineering and regenerative medicine approaches showed to have promising outcomes for corneal regeneration. However, besides proper mechanical and optical properties, other factors such as appropriate sterilization method, storage, shelf life and etc. should be taken into account in order to develop an engineered cornea for clinical trials.