scholarly journals Arginine Supplementation Promotes Extracellular Matrix and Metabolic Changes in Keratoconus

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2076
Author(s):  
Tina B. McKay ◽  
Shrestha Priyadarsini ◽  
Tyler Rowsey ◽  
Dimitrios Karamichos
Keyword(s):  
Corneal Stroma ◽  
Biomechanical Properties ◽  
Collagen Type I ◽  
Type I ◽  
Collagen Deposition ◽  
Corneal Fibroblasts ◽  
Steady State Levels ◽  
Vitro Model ◽  
Arginine Supplementation

Keratoconus (KC) is a common corneal ectatic disease that affects 1:500–1:2000 people worldwide and is associated with a progressive thinning of the corneal stroma that may lead to severe astigmatism and visual deficits. Riboflavin-mediated collagen crosslinking currently remains the only approved treatment to halt progressive corneal thinning associated with KC by improving the biomechanical properties of the stroma. Treatments designed to increase collagen deposition by resident corneal stromal keratocytes remain elusive. In this study, we evaluated the effects of arginine supplementation on steady-state levels of arginine and arginine-related metabolites (e.g., ornithine, proline, hydroxyproline, spermidine, and putrescine) and collagen protein expression by primary human corneal fibroblasts isolated from KC and non-KC (healthy) corneas and cultured in an established 3D in vitro model. We identified lower cytoplasmic arginine and spermidine levels in KC-derived constructs compared to healthy controls, which corresponded with overall higher gene expression of arginase. Arginine supplementation led to a robust increase in cytoplasmic arginine, ornithine, and spermidine levels in controls only and a significant increase in collagen type I secretion in KC-derived constructs. Further studies evaluating safety and efficacy of arginine supplementation are required to elucidate the potential therapeutic applications of modulating collagen deposition in the context of KC.

scholarly journals Relative rates of biosynthesis of collagen type I, type V and type VI in calf cornea

1991 ◽  
Vol 274 (2) ◽  
pp. 615-617 ◽  
Author(s):  
P Kern ◽  
M Menasche ◽  
L Robert
Keyword(s):  
Type I Collagen ◽  
Collagen Type ◽  
Corneal Stroma ◽  
Collagen Type I ◽  
Type I ◽  
Type Vi Collagen ◽  
Sds Page ◽  
Proline Incorporation ◽  
Type V

The biosynthesis of type I, type V and type VI collagens was studied by incubation of calf corneas in vitro with [3H]proline as a marker. Pepsin-solubilized collagen types were isolated by salt fractionation and quantified by SDS/PAGE. Expressed as proportions of the total hydroxyproline solubilized, corneal stroma comprised 75% type I, 8% type V and 17% type VI collagen. The rates of [3H]proline incorporation, linear up to 24 h for each collagen type, were highest for type VI collagen and lowest for type I collagen. From pulse-chase experiments, the calculated apparent half-lives for types I, V and VI collagens were 36 h, 10 h and 6 h respectively.

scholarly journals A Mouse Model of Brittle Cornea Syndrome caused by mutation in Zfp469

2021 ◽  
Author(s):  
Chloe M. Stanton ◽  
Amy S. Findlay ◽  
Camilla Drake ◽  
Mohammad Z. Mustafa ◽  
Philippe Gautier ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Corneal Stroma ◽  
Therapeutic Benefit ◽  
Type I ◽  
Loss Of Function ◽  
Brittle Cornea Syndrome ◽  
Vitro Model ◽  
Biomechanical Strength ◽  
Corneal Thinning

Brittle Cornea Syndrome (BCS) is a rare recessive condition characterised by extreme thinning of the cornea and sclera. BCS results from loss-of-function mutations in the poorly understood genes ZNF469 or PRDM5. In order to determine the function of ZNF469 and to elucidate pathogenic mechanisms, we used genome editing to recapitulate a human ZNF469 BCS mutation in the orthologous mouse gene, Zfp469. Ophthalmic phenotyping showed that homozygous Zfp469 mutation causes significant central and peripheral corneal thinning arising from reduced stromal thickness. Expression of key components of the corneal stroma in primary keratocytes from Zfp469BCS/BCS mice is affected, including decreased Col1a1 and Col1a2 expression. This alters the type I:type V collagen ratio and results in collagen fibrils with smaller diameter and increased fibril density in homozygous mutant corneas, correlating with decreased biomechanical strength in the cornea. Cell-derived matrices generated by primary keratocytes show reduced deposition of type I collagen offering an in vitro model for stromal dysfunction. Work remains to determine whether modulating ZNF469 activity will have therapeutic benefit in BCS or in conditions such as keratoconus where the cornea thins progressively.

scholarly journals Increased Collagen Crosslinking in Stiff Clubfoot Tissue: Implications for the Improvement of Therapeutic Strategies

2021 ◽  
Vol 22 (21) ◽  
pp. 11903
Author(s):  
Jarmila Knitlova ◽  
Martina Doubkova ◽  
Adam Eckhardt ◽  
Martin Ostadal ◽  
Jana Musilkova ◽  
...  
Keyword(s):  
Lysyl Oxidase ◽  
Second Harmonic ◽  
Collagen Type I ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Dependent Manner ◽  
Collagen Deposition ◽  
Collagen Gels ◽  
Second Harmonic Generation Imaging

Congenital clubfoot is a complex musculoskeletal deformity, in which a stiff, contracted tissue forms in the medial part of the foot. Fibrotic changes are associated with increased collagen deposition and lysyl oxidase (LOX)‑mediated crosslinking, which impair collagen degradation and increase the tissue stiffness. First, we studied collagen deposition, as well as the expression of collagen and the amount of pyridinoline and deoxypyridinoline crosslinks in the tissue of relapsed clubfoot by immunohistochemistry, real-time PCR, and enzyme-linked immunosorbent assay (ELISA). We then isolated fibroblast‑like cells from the contracted tissue to study the potential inhibition of these processes in vitro. We assessed the effects of a LOX inhibitor, β‑aminopropionitrile (BAPN), on the cells by a hydroxyproline assay, ELISA, and Second Harmonic Generation imaging. We also evaluated the cell-mediated contraction of extracellular matrix in 3D cell‑populated collagen gels. For the first time, we have confirmed significantly increased crosslinking and excessive collagen type I deposition in the clubfoot-contracted tissue. We successfully reduced these processes in vitro in a dose-dependent manner with 10–40 µg/mL of BAPN, and we observed an increasing trend in the inhibition of the cell‑mediated contraction of collagen gels. The in vitro inhibitory effects indicate that BAPN has good potential for the treatment of relapsed and resistant clubfeet.

scholarly journals A Mouse Model of Brittle Cornea Syndrome caused by mutation in Zfp469.

2021 ◽  
Author(s):  
Chloe M. Stanton ◽  
Amy S. Findlay ◽  
Camilla Drake ◽  
Mohammad Z. Mustafa ◽  
Philippe Gautier ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Corneal Stroma ◽  
Therapeutic Benefit ◽  
Type I ◽  
Loss Of Function ◽  
Brittle Cornea Syndrome ◽  
Vitro Model ◽  
Biomechanical Strength ◽  
Corneal Thinning

Brittle Cornea Syndrome (BCS) is a rare recessive condition characterised by extreme thinning of the cornea and sclera. BCS results from loss-of-function mutations in the poorly understood genes ZNF469 or PRDM5. In order to determine the function of ZNF469 and to elucidate pathogenic mechanisms, we used genome editing to recapitulate a human ZNF469 BCS mutation in the orthologous mouse gene, Zfp469. Ophthalmic phenotyping showed that homozygous Zfp469 mutation causes significant central and peripheral corneal thinning arising from reduced stromal thickness. Expression of key components of the corneal stroma in primary keratocytes from Zfp469BCS/BCS mice is affected, including decreased Col1a1 and Col1a2 expression. This alters the type I:type V collagen ratio and results in collagen fibrils with smaller diameter and increased fibril density in homozygous mutant corneas, correlating with decreased biomechanical strength in the cornea. Cell-derived matrices generated by primary keratocytes show reduced deposition of type I collagen offering an in vitro model for stromal dysfunction. Work remains to determine whether modulating ZNF469 activity will have therapeutic benefit in BCS or in conditions such as keratoconus where the cornea thins progressively.

scholarly journals Integration of Biomechanical and Biological Characterization in the Development of Porous Poly(caprolactone)-Based Membranes for Abdominal Wall Hernia Treatment

2018 ◽  
Vol 2018 ◽  
pp. 1-15
Author(s):  
Federico Vozzi ◽  
Tiziana Nardo ◽  
Ilenia Guerrazzi ◽  
Claudio Domenici ◽  
Silvia Rocchiccioli ◽  
...  
Keyword(s):  
Abdominal Wall ◽  
Abdominal Wall Hernia ◽  
Biomechanical Properties ◽  
Collagen Type I ◽  
Porous Membrane ◽  
Biological Properties ◽  
Type I ◽  
Molecular Weights ◽  
Intermediate Surface

Aims. Synthetic meshes are the long-standing choice for the clinical treatment of abdominal wall hernias: the associated long-term complications have stimulated the development of a new generation of bioresorbable prostheses. In this work, polycaprolactone (PCL) porous membranes prepared by solvent casting/porogen leaching of PCL/poly(ethylene glycol) (PEG) blends with different compositions (different PCL/PEG weight ratios and PEG molecular weights) were investigated to be applied in the field. An optimal porous membrane structure was selected based on the evaluation of physicochemical, biomechanical, and in vitro biological properties, compared to a reference commercially available hernia mesh (CMC). Findings. Selected PCL7-2i membranes, derived from PCL/PEG 70/30 (PCL: Mw 70,000-90,000 Da; PEG: 35,000 Da), showed suitable pore size for the application, intermediate surface hydrophilicity, and biomimetic mechanical properties. In vitro cell tests performed on PCL7-2i membranes showed their cytocompatibility, high cell growth during 21 days, a reduced production of proinflammatory IL-6 with respect to CMC, and a significant secretion of collagen type I. Conclusions. PCL7-2i membranes showed biomimetic biomechanical properties and in vitro biological properties similar to or even better than - in the case of anti-inflammatory behavior and collagen production - CMC, a commercially available product, suggesting potentially improved integration in the host tissue.

scholarly journals The Senescence Difference Between the Central and Peripheral Cornea Induced by Sutures

2021 ◽  
Author(s):  
Suxia Li ◽  
Qiaoqiao Dong ◽  
Muchen Dong ◽  
Mingli Qu ◽  
Yao Wang ◽  
...  
Keyword(s):  
Mouse Model ◽  
In Vitro Model ◽  
Corneal Stroma ◽  
Corneal Neovascularization ◽  
Tissue Fibrosis ◽  
Corneal Fibroblasts ◽  
Vitro Model ◽  
Peripheral Cornea

Abstract Introduction: Cell senescence plays a regulatory role in tissue fibrosis, and corneal scarring is usually more severe in the central cornea based on clinical observation. We attempted to explore the senescence difference between the central and peripheral cornea in an in vivo mouse model of suture-induced senescence and an in vitro model of senescence with hydrogen peroxide (H2O2)-induced rabbit corneal fibroblasts. Methods: Male Balb/c mice (6-8 weeks) received sutures in the central, superior, inferior, nasal, and temporal cornea, and the sutures were removed on day 14. Corneal neovascularization (CNV) was observed under a slit lamp microscope with a digital camera. Fibroblasts isolated from central and peripheral rabbit corneas were induced with H2O2 to establish an in vitro model of senescence. The senescence was evaluated with SA-β-gal staining and gene expression analysis of p21, p27, and p53. Results: Senescent cells accumulated in the corneal stroma from day 3 to day 14 after the operation and peaked on day 14, later than CNV. More senescent keratocytes were observed in the peripheral cornea in the mouse model. The peripheral corneal fibroblasts were more likely to become senescent related to H2O2 in vitro. The PCR results revealed that the senescence-related genes, p21, p27, and p53, were all highly expressed in the peripheral corneal fibroblasts. Conclusions: As senescent fibroblasts can limit tissue fibrosis, the senescence difference between the central and peripheral cornea may contribute to the difference in scarring.

Collagen fibrillogenesis in vitro: interaction of types I and V collagen

1986 ◽  
Vol 44 ◽  
pp. 210-211
Author(s):  
Arthur J. Wasserman ◽  
Kathy C. Kloos ◽  
David E. Birk
Keyword(s):  
Type I Collagen ◽  
Corneal Stroma ◽  
Minor Component ◽  
Homogeneous Distribution ◽  
Type I ◽  
Type V Collagen ◽  
Fibril Morphology ◽  
Type V ◽  
In Vitro Interaction

Type I collagen is the predominant collagen in the cornea with type V collagen being a quantitatively minor component. However, the content of type V collagen (10-20%) in the cornea is high when compared to other tissues containing predominantly type I collagen. The corneal stroma has a homogeneous distribution of these two collagens, however, immunochemical localization of type V collagen requires the disruption of type I collagen structure. This indicates that these collagens may be arranged as heterpolymeric fibrils. This arrangement may be responsible for the control of fibril diameter necessary for corneal transparency. The purpose of this work is to study the in vitro assembly of collagen type V and to determine whether the interactions of these collagens influence fibril morphology.

scholarly journals Collagen-Based Electrospun Materials for Tissue Engineering: A Systematic Review

2021 ◽  
Vol 8 (3) ◽  
pp. 39
Author(s):  
Britani N. Blackstone ◽  
Summer C. Gallentine ◽  
Heather M. Powell
Keyword(s):  
Systematic Review ◽  
Tissue Engineering ◽  
Collagen Type I ◽  
The Body ◽  
Pool Data ◽  
Type I ◽  
High Concentrations ◽  
Increased Strength

Collagen is a key component of the extracellular matrix (ECM) in organs and tissues throughout the body and is used for many tissue engineering applications. Electrospinning of collagen can produce scaffolds in a wide variety of shapes, fiber diameters and porosities to match that of the native ECM. This systematic review aims to pool data from available manuscripts on electrospun collagen and tissue engineering to provide insight into the connection between source material, solvent, crosslinking method and functional outcomes. D-banding was most often observed in electrospun collagen formed using collagen type I isolated from calfskin, often isolated within the laboratory, with short solution solubilization times. All physical and chemical methods of crosslinking utilized imparted resistance to degradation and increased strength. Cytotoxicity was observed at high concentrations of crosslinking agents and when abbreviated rinsing protocols were utilized. Collagen and collagen-based scaffolds were capable of forming engineered tissues in vitro and in vivo with high similarity to the native structures.

scholarly journals Mechanical and Degradation Properties of Hybrid Scaffolds for Tissue Engineered Heart Valve (TEHV)

2021 ◽  
Vol 12 (1) ◽  
pp. 20
Author(s):  
Rabia Nazir ◽  
Arne Bruyneel ◽  
Carolyn Carr ◽  
Jan Czernuszka
Keyword(s):  
Heart Valve ◽  
Crosslinking Density ◽  
Biomechanical Properties ◽  
Collagen Type I ◽  
Type I ◽  
Aortic Heart Valve ◽  
Tissue Engineered Heart Valve ◽  
Human Aortic Valve ◽  
Degradation Properties ◽  
Hybrid Scaffolds

In addition to biocompatibility, an ideal scaffold for the regeneration of valvular tissue should also replicate the natural heart valve extracellular matrix (ECM) in terms of biomechanical properties and structural stability. In our previous paper, we demonstrated the development of collagen type I and hyaluronic acid (HA)-based scaffolds with interlaced microstructure. Such hybrid scaffolds were found to be compatible with cardiosphere-derived cells (CDCs) to potentially regenerate the diseased aortic heart valve. This paper focused on the quantification of the effect of crosslinking density on the mechanical properties under dry and wet conditions as well as degradation resistance. Elastic moduli increased with increasing crosslinking densities, in the dry and wet state, for parent networks, whereas those of interlaced scaffolds were higher than either network alone. Compressive and storage moduli ranged from 35 ± 5 to 95 ± 5 kPa and 16 ± 2 kPa to 113 ± 6 kPa, respectively, in the dry state. Storage moduli, in the dry state, matched and exceeded those of human aortic valve leaflets (HAVL). Similarly, degradation resistance increased with increasing the crosslinking densities for collagen-only and HA-only scaffolds. Interlaced scaffolds showed partial degradation in the presence of either collagenase or hyaluronidase as compared to when exposed to both enzymes together. These results agree with our previous findings that interlaced scaffolds were composed of independent collagen and HA networks without crosslinking between them. Thus, collagen/HA interlaced scaffolds have the potential to fill in the niche for designing an ideal tissue engineered heart valve (TEHV).

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.

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