scholarly journals Plant Recombinant Human Collagen Type I Hydrogels for Corneal Regeneration

2021 ◽  
Author(s):  
Michel Haagdorens ◽  
Elle Edin ◽  
Per Fagerholm ◽  
Marc Groleau ◽  
Zvi Shtein ◽  
...  
Keyword(s):  
Collagen Type ◽  
Collagen Type I ◽  
Collagen Fibrils ◽  
Type I ◽  
Safe Alternative ◽  
Human Donor ◽  
Human Collagen ◽  
Donor Corneas

Abstract Purpose To determine feasibility of plant-derived recombinant human collagen type I (RHCI) for use in corneal regenerative implants Methods RHCI was crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form hydrogels. Application of shear force to liquid crystalline RHCI aligned the collagen fibrils. Both aligned and random hydrogels were evaluated for mechanical and optical properties, as well as in vitro biocompatibility. Further evaluation was performed in vivo by subcutaneous implantation in rats and corneal implantation in Göttingen minipigs. Results Spontaneous crosslinking of randomly aligned RHCI (rRHCI) formed robust, transparent hydrogels that were sufficient for implantation. Aligning the RHCI (aRHCI) resulted in thicker collagen fibrils forming an opaque hydrogel with insufficient transverse mechanical strength for surgical manipulation. rRHCI showed minimal inflammation when implanted subcutaneously in rats. The corneal implants in minipigs showed that rRHCI hydrogels promoted regeneration of corneal epithelium, stroma, and nerves; some myofibroblasts were seen in the regenerated neo-corneas. Conclusion Plant-derived RHCI was used to fabricate a hydrogel that is transparent, mechanically stable, and biocompatible when grafted as corneal implants in minipigs. Plant-derived collagen is determined to be a safe alternative to allografts, animal collagens, or yeast-derived recombinant human collagen for tissue engineering applications. The main advantage is that unlike donor corneas or yeast-produced collagen, the RHCI supply is potentially unlimited due to the high yields of this production method. Lay Summary A severe shortage of human-donor corneas for transplantation has led scientists to develop synthetic alternatives. Here, recombinant human collagen type I made of tobacco plants through genetic engineering was tested for use in making corneal implants. We made strong, transparent hydrogels that were tested by implanting subcutaneously in rats and in the corneas of minipigs. We showed that the plant collagen was biocompatible and was able to stably regenerate the corneas of minipigs comparable to yeast-produced recombinant collagen that we previously tested in clinical trials. The advantage of the plant collagen is that the supply is potentially limitless.

scholarly journals WNT-5A regulates TGF-β-related activities in liver fibrosis

2017 ◽  
Vol 312 (3) ◽  
pp. G219-G227 ◽  
Author(s):  
Leonie Beljaars ◽  
Sara Daliri ◽  
Christa Dijkhuizen ◽  
Klaas Poelstra ◽  
Reinoud Gosens
Keyword(s):  
Liver Fibrosis ◽  
Functional Role ◽  
Collagen Type ◽  
Collagen Type I ◽  
Matrix Proteins ◽  
Type I ◽  
Human Livers

WNT-5A is a secreted growth factor that belongs to the noncanonical members of the Wingless-related MMTV-integration family. Previous studies pointed to a connection between WNT-5A and the fibrogenic factor TGF-β warranting further studies into the functional role of WNT-5A in liver fibrosis. Therefore, we studied WNT-5A expressions in mouse and human fibrotic livers and examined the relation between WNT-5A and various fibrosis-associated growth factors, cytokines, and extracellular matrix proteins. WNT-5A gene and protein expressions were significantly increased in fibrotic mouse and human livers compared with healthy livers. Regression or therapeutic intervention in mice resulted in decreased hepatic WNT-5A levels paralleled by lower collagen levels. Immunohistochemical analysis showed WNT-5A staining in fibrotic septa colocalizing with desmin staining indicating WNT-5A expression in myofibroblasts. In vitro studies confirmed WNT-5A expression in this cell type and showed that TGF-β significantly enhanced WNT-5A expression in contrast to PDGF-BB and proinflammatory cytokines IL-1β and TNF-α. Additionally, TGF-β induces the expression of the WNT receptors FZD2 and FZD8. After silencing of WNT-5A, reduced levels of collagen type I, vimentin, and fibronectin in TGF-β-stimulated myofibroblasts were measured compared with nonsilencing siRNA-treated controls. Interestingly, the antifibrotic cytokine IFNγ suppressed WNT-5A in vitro and in vivo. IFNγ-treated fibrotic mice showed significantly less WNT-5A expression compared with untreated fibrotic mice. In conclusion, WNT-5A paralleled collagen I levels in fibrotic mouse and human livers. WNT-5A expression in myofibroblasts is induced by the profibrotic factor TGF-β and plays an important role in TGF-β-induced regulation of fibrotic matrix proteins, whereas its expression can be reversed upon treatment, both in vitro and in vivo. NEW & NOTEWORTHY This study describes the localization and functional role of WNT-5A in human and mouse fibrotic livers. Hepatic WNT-5A expression parallels collagen type I expression. In vivo and in vitro, the myofibroblasts were identified as the key hepatic cells producing WNT-5A. WNT-5A is under control of TGF-β and its activities are primarily profibrotic.

Electrospun poly(vinyl) alcohol/collagen nanofibrous scaffold hybridized by graphene oxide for accelerated wound healing

2018 ◽  
Vol 41 (8) ◽  
pp. 467-473 ◽  
Author(s):  
Rethinam Senthil ◽  
Robert Berly ◽  
Thimmiah Bhargavi Ram ◽  
Nallathambi Gobi
Keyword(s):  
Wound Healing ◽  
Graphene Oxide ◽  
Vinyl Alcohol ◽  
Collagen Type ◽  
Collagen Type I ◽  
Fish Bone ◽  
Poly Vinyl Alcohol ◽  
Type I

Purpose: In this study, a blend of synthetic polymer (poly(vinyl) alcohol), natural polymer (collagen type I from fish bone), and graphene oxide nanoparticles is used to fabricate a composite nanofibrous scaffold, by electrospinning, for their potential application in accelerated wound healing. Methods: The scaffold was characterized for its physicochemical and mechanical properties. In vitro studies were carried out using human keratinocyte cell line (HaCaT) which proved the biocompatibility of the scaffold. In vivo study using mice model was carried out and the healing pattern was evaluated using histopathological studies. Results: Scaffold prepared from poly(vinyl) alcohol, collagen type I from fish bone, and graphene oxide possessed better physicochemical and mechanical properties. In addition, in vivo and in vitro studies showed its accelerated wound healing properties. Conclusion: The scaffold with required strength and biocompatibility may be tried as a wound dressing material in large animals after getting necessary approval.

A novel nano-structured porous polycaprolactone scaffold improves hyaline cartilage repair in a rabbit model compared to a collagen type I/III scaffold: in vitro and in vivo studies

2011 ◽  
Vol 20 (6) ◽  
pp. 1192-1204 ◽  
Author(s):  
Bjørn Borsøe Christensen ◽  
Casper Bindzus Foldager ◽  
Ole Møller Hansen ◽  
Asger Albæk Kristiansen ◽  
Dang Quang Svend Le ◽  
...  
Keyword(s):  
Cartilage Repair ◽  
Collagen Type ◽  
Hyaline Cartilage ◽  
Rabbit Model ◽  
Collagen Type I ◽  
In Vivo Studies ◽  
Type I

scholarly journals Hydrogel-hydroxyapatite-monomeric collagen type-I scaffold with low-frequency electromagnetic field treatment enhances osteochondral repair in rabbits

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jiyuan Yan ◽  
Chaoxu Liu ◽  
Chang Tu ◽  
Ruizhuo Zhang ◽  
Xiangyu Tang ◽  
...  
Keyword(s):  
Chondrogenic Differentiation ◽  
Collagen Type ◽  
Composite Scaffold ◽  
Low Frequency ◽  
Collagen Type I ◽  
Type I ◽  
Osteochondral Defects ◽  
Osteochondral Repair

Abstract Background Cartilage damage is a common medical issue in clinical practice. Complete cartilage repair remains a significant challenge owing to the inferior quality of regenerative tissue. Safe and non-invasive magnetic therapy combined with tissue engineering to repair cartilage may be a promising breakthrough. Methods In this study, a composite scaffold made of Hydroxyapatite-Collagen type-I (HAC) and PLGA-PEG-PLGA thermogel was produced to match the cartilage and subchondral layers in osteochondral defects, respectively. Bone marrow mesenchymal stem cells (BMSC) encapsulated in the thermogel were stimulated by an electromagnetic field (EMF). Effect of EMF on the proliferation and chondrogenic differentiation potential was evaluated in vitro. 4 mm femoral condyle defect was constructed in rabbits. The scaffolds loaded with BMSCs were implanted into the defects with or without EMF treatment. Effects of the combination treatment of the EMF and composite scaffold on rabbit osteochondral defect was detected in vivo. Results In vitro experiments showed that EMF could promote proliferation and chondrogenic differentiation of BMSCs partly by activating the PI3K/AKT/mTOR and Wnt1/LRP6/β-catenin signaling pathway. In vivo results further confirmed that the scaffold with EMF enhances the repair of osteochondral defects in rabbits, and, in particular, cartilage repair. Conclusion Hydrogel-Hydroxyapatite-Monomeric Collagen type-I scaffold with low-frequency EMF treatment has the potential to enhance osteochondral repair.

Comprehensive In Vitro and In Vivo Characterization of Loss and Gain-of-Function Von Willebrand Factor Collagen Binding Variants Using a Mouse Model System,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3266-3266
Author(s):  
Yasuaki Shida ◽  
Christine Brown ◽  
Jeff Mewburn ◽  
Kate Sponagle ◽  
Ozge Danisment ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
Platelet Adhesion ◽  
Collagen Type ◽  
Collagen Type I ◽  
Type I ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Collagen Binding

Abstract Abstract 3266 Von Willebrand Factor (VWF) is a large multimeric glycoprotein that mediates platelet adhesion to the damaged blood vessel wall and subsequent platelet aggregation at the site of injury. Rare mutations in the VWF A3 domain, that disrupt collagen binding, have been found in patients with a mild bleeding phenotype. However, the analysis of these aberrant VWF-collagen interactions has been relatively limited. Thus, in this study, we have developed mouse models of collagen binding mutants and analyzed the function of the A3 and A1 domains using comprehensive in vitro and in vivo approaches. All of the collagen binding variant AAs are conserved in mice. 6 loss-of-function (S1731T, W1745C, S1783A, H1786D, A1 deletion, A3 deletion) and 1 gain-of-function (L1757A) variant was generated in the context of the mouse VWF cDNA. The 4 loss-of-function missense mutants have all been described in patients with mild bleeding phenotypes. The recombinant mouse VWFs (rmVWF) were synthesized in HEK293T cells and analyzed for type I and III collagen binding in both a static assay (CBA) and a flow-based assay at 2,500s−1 in which VWF is bound to collagen on a surface, and labeled platelet adhesion is quantified. The multimer profile of all the rmVWFs was normal. The expression level of the rmVWF derived from HEK293T cells was quantified. W1745C and the A3 deletion showed significantly lower levels of expression and the A1 deletion mutant showed strong intracellular retention. In the static collagen binding assay, S1731T showed almost normal binding to collagen type I and a 50% reduction in binding to collagen type III. The other 3 missense variants, W1745C, S1783A and H1786D, showed reduced binding to both collagens I and III, and the A3 deletion mutant showed absent binding. In the in vitro flow assay, the sensitivity to detect defects in collagen binding was superior to the static assay, although the patterns of binding defects were similar. W1745C showed similar low levels of platelet adhesion to both types of collagen, while S1783A and H1786D showed a lack of platelet binding on the collagen III surface similar to the A3 deletion mutant, and a reduced binding to collagen type I similar to W1745C. The gain-of-function mutant showed consistent enhanced collagen binding and platelet adhesion in the static and flow assays, respectively. In vivo studies delivered the mVWF cDNAs with a strong liver specific promoter by hydrodynamic injection. At 7 days post-delivery, the VWF:Ag levels in the WT and collagen binding variant mice were similar, apart from the W1745C mutant, that showed 14.6% levels compared to WT. Platelet counts and multimer patterns were normal with the collagen binding variants. In vivo intravital microscopy studies were performed using the cremaster arteriolar model when VWF levels were in a physiological range. Thrombosis was induced by 10%FeCl3 applied for 3 mins. Platelets were labeled in vivo by Rhodamine 6G and the thrombus development was analyzed by spinning disc confocal microscopy. Loss-of-function mutants showed transient platelet adhesion at the site of injury, however the adhesion was unstable and vessel occlusion was not observed. Using three complementary experimental systems we have been able to confirm the collagen binding defects in this group of variant VWFs. There is a differential sensitivity to the two forms of collagen and of the three experimental systems. The A3 deletion mutant consistently resulted in the most severe phenotype while the missense mutants showed variable degrees of functional deficit. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Model for human skin reconstructed in vitro composed of associated dermis and epidermis

2006 ◽  
Vol 124 (2) ◽  
pp. 71-76 ◽  
Author(s):  
Luís Ricardo Martinhão Souto ◽  
Jussara Rehder ◽  
José Vassallo ◽  
Maria Letícia Cintra ◽  
Maria Helena Stangler Kraemer ◽  
...  
Keyword(s):  
Human Skin ◽  
Collagen Type ◽  
Human Fibroblasts ◽  
Skin Grafting ◽  
Human Keratinocytes ◽  
Collagen Type I ◽  
Type I ◽  
Bovine Collagen

CONTEXT AND OBJECTIVE: The technique of obtaining human skin with dermis and epidermis reconstructed from cells isolated from patients can enable autologous skin grafting on patients with few donor sites. It also enables in vitro trials on chemicals and drugs. The objective of this work was to demonstrate a method for obtaining human skin composed of associated dermis and epidermis, reconstructed in vitro. DESIGN AND SETTING: Experimental laboratory study, in the Skin Cell Culture Laboratory of Faculdade de Ciências Médicas, Universidade Estadual de Campinas. METHODS: Cells from human fibroblast cultures are injected into bovine collagen type I matrix and kept immersed in specific culturing medium for fibroblasts. This enables human dermis reconstruction in vitro. On this, by culturing human keratinocytes and melanocytes, differentiated epidermis is formed, leading to the creation of human skin composed of associated dermis and epidermis, reconstructed in vitro. RESULTS: We showed that human skin composed of associated dermis and epidermis can be successfully reconstructed in vitro. It is histologically formed in the same way as human skin in vivo. Collagen tissue can be identified in the dermis, with cells and extracellular matrix organized in parallel to multilayer epidermis. CONCLUSIONS: It is possible to obtain completely differentiated human skin composed of associated dermis and epidermis, reconstructed in vitro, from injection of human fibroblasts into bovine collagen type I matrix and culturing of human keratinocytes and melanocytes on this matrix.

scholarly journals Non-cross-linked collagen type I/III materials enhance cell proliferation: in vitro and in vivo evidence

2014 ◽  
Vol 22 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Ines WILLERSHAUSEN ◽  
Mike BARBECK ◽  
Nicole BOEHM ◽  
Robert SADER ◽  
Brita WILLERSHAUSEN ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Collagen Type ◽  
Collagen Type I ◽  
Type I ◽  
Proliferation In Vitro
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