An electron microscopical study of the influence of different glycosaminoglycans on the fibrillogenesis of collagen type I and II in vitro

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.

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Plant Recombinant Human Collagen Type I Hydrogels for Corneal Regeneration

Regenerative Engineering and Translational Medicine ◽

10.1007/s40883-021-00220-3 ◽

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

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00160.2016 ◽

2017 ◽

Vol 312 (3) ◽

pp. G219-G227 ◽

Cited By ~ 21

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.

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Investigation of the effects of prostaglandin E2 on equine superficial digital flexor tendon fibroblasts in vitro

Veterinary and Comparative Orthopaedics and Traumatology ◽

10.3415/vcot-10-03-0044 ◽

2010 ◽

Vol 23 (06) ◽

pp. 417-423 ◽

Cited By ~ 4

Author(s):

J. M. Cissell ◽

S. C. Milton ◽

L. A. Dahlgren

Keyword(s):

Gene Expression ◽

Matrix Protein ◽

Collagen Type ◽

Flexor Tendon ◽

Cell Metabolism ◽

Collagen Type I ◽

Type I ◽

Matrix Metalloproteinase 1 ◽

Superficial Digital Flexor Tendon

Summary Objectives: To evaluate the effects of pros-taglandin E2 (PGE2) treatment on the metabolism of equine tendon fibroblasts in vitro to aid in investigating the response of tendon fibroblasts to injury and novel therapeutics. Methods: Superficial digital flexor tendon fibroblasts isolated via collagenase digestion from six young adult horses were grown in monolayer in four concentrations of PGE2 (0, 10, 50, 100 ng/ml) for 48 hours. Cells and medium were harvested for gene expression (collagen types I and III, cartilage oligomeric matrix protein [COMP], decorin, and matrix metalloproteinase-1, –3, and –13), biochemical analysis (glycosaminoglycan, DNA, and collagen content), and cytological staining. Results: Gene expression for collagen type I was significantly increased at 100 ng/ml PGE2 compared to 10 and 50 ng/ml. There were not any significant differences detected for gene expression of collagen type III, COMP or dec-orin or for biochemical content and cell morphology. Clinical significance: Under the conditions investigated, exogenous treatment of equine tendon fibroblasts with PGE2 failed to alter cell metabolism in a manner useful as a model of tendon injury. A model that applies cyclic strain to a three dimensional construct seeded with tendon fibroblasts may prove to be a more useful model and merits further investigation for this purpose. The ability to assess cellular responses in an environment where the cells are supported within the extracellular matrix may prove beneficial.

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Mysterious path of Borrelia spielmanii: spreading without morphological alteration of collagen type I and IV

Future Microbiology ◽

10.2217/fmb-2019-0217 ◽

2019 ◽

Vol 14 (17) ◽

pp. 1469-1475

Author(s):

Pavle Banović ◽

Ivan Čapo ◽

Dejan Ogorelica ◽

Nenad Vranješ ◽

Verica Simin ◽

...

Keyword(s):

Collagen Type ◽

Erythema Migrans ◽

Collagen Type I ◽

Punch Biopsy ◽

Morphological Alteration ◽

Type I ◽

Contributing Factors ◽

Metalloproteinase 9 ◽

Serology Testing

The majority of suggested mechanisms of Borrelia spreading inside erythema migrans (EM) are developed from in vitro studies and animal models. This report is the first to describe pathomorphological substrate of EM caused by Borrelia spielmanii in humans, addressing the hypothesis of enhanced Borrelia penetration through extracellular matrix. In the process of ruling out of atypical Masters’ disease, we conducted a punch biopsy of suspected EM and a two-tier serology testing for Lyme borreliosis, where we registered antibodies against B. spielmanii. Skin biopsy showed CD4+ and CD8+ lymphocyte involvement and high activity of matrix metalloproteinase 9. No alterations were detected in distribution and morphology of collagen type I and IV. Therefore, it is suggested that other mechanisms should be considered as major contributing factors to local spreading of B. spielmanii.

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In vitro production of human dermal equivalent

Medicinski pregled ◽

10.2298/mpns1008459m ◽

2010 ◽

Vol 63 (7-8) ◽

pp. 459-464 ◽

Cited By ~ 1

Author(s):

Zoran Milosavljevic ◽

Biljana Ljujic

Keyword(s):

Vitamin C ◽

Collagen Type ◽

Collagen Type I ◽

Type I ◽

Collagen Gels ◽

In Vitro Production ◽

Dermal Equivalent ◽

Enzymatic Dissociation ◽

Vitro Production

Introduction. Human dermal tissue is composed of loose and dense connective tissue. Main cell populations are fibroblasts and the dominant fibers are built from collagen type I. The aim of our study was to determine the precise method and time frame for the in vitro production of human dermal equivalent and to investigate the effects of ratio of structural elements and vitamin C on characteristics of the engineered tissue. Material and methods. Primary isolation of the foreskin fibroblasts was performed by explant method and enzymatic dissociation. Various collagen gels were obtained by mixing cells (from 25x103 to 200x103/ml) and neutralized collagen type I (from 2 to 4 mg/ml), with or without vitamin C. The routine histological and morphometrical examination was performed. Results. Enzymatic dissociation of the foreskin proved to be a faster method for production of desired number of fibroblasts (7.5x105 for 4 days). The contraction of collagen-gels started from day one through day seven and was dependent on cell and collagen concentration with higher density gels being contracted to a greater extent, except for the lowest/highest values. The best result was achieved with 100x103 cells and 2 mg/ml collagen. Vitamin C at 50 ?g/ml had no effect on speed of tissue formation. Conclusion. A precise approach that mimic the in vivo conditions is needed for the in vitro production of the dermal equivalent suitable for the possible treatment of tissue defects. Nearly ten days are necessary from the donor tissue dissociation to the final product.

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Collagen Type I Containing Hybrid Hydrogel Enhances Cardiomyocyte Maturation in a 3D Cardiac Model

Polymers ◽

10.3390/polym11040687 ◽

2019 ◽

Vol 11 (4) ◽

pp. 687 ◽

Cited By ~ 4

Author(s):

Sam G. Edalat ◽

Yongjun Jang ◽

Jongseong Kim ◽

Yongdoo Park

Keyword(s):

Growth Factor ◽

Collagen Type ◽

Transforming Growth Factor ◽

Embryonic Stem ◽

Collagen Type I ◽

Transforming Growth Factor Β1 ◽

Type I ◽

Hybrid Hydrogel ◽

Cardiac Model

In vitro maturation of cardiomyocytes in 3D is essential for the development of viable cardiac models for therapeutic and developmental studies. The method by which cardiomyocytes undergoes maturation has significant implications for understanding cardiomyocytes biology. The regulation of the extracellular matrix (ECM) by changing the composition and stiffness is quintessential for engineering a suitable environment for cardiomyocytes maturation. In this paper, we demonstrate that collagen type I, a component of the ECM, plays a crucial role in the maturation of cardiomyocytes. To this end, embryonic stem-cell derived cardiomyocytes were incorporated into Matrigel-based hydrogels with varying collagen type I concentrations of 0 mg, 3 mg, and 6 mg. Each hydrogel was analyzed by measuring the degree of stiffness, the expression levels of MLC2v, TBX18, and pre-miR-21, and the size of the hydrogels. It was shown that among the hydrogel variants, the Matrigel-based hydrogel with 3 mg of collagen type I facilitates cardiomyocyte maturation by increasing MLC2v expression. The treatment of transforming growth factor β1 (TGF-β1) or fibroblast growth factor 4 (FGF-4) on the hydrogels further enhanced the MLC2v expression and thereby cardiomyocyte maturation.

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Preliminary Study of In Vitro Three-Dimensional Skin Model Using an Ovine Collagen Type I Sponge Seeded with Co-Culture Skin Cells: Submerged versus Air-Liquid Interface Conditions

Polymers ◽

10.3390/polym12122784 ◽

2020 ◽

Vol 12 (12) ◽

pp. 2784

Author(s):

Mh Busra Fauzi ◽

Zahra Rashidbenam ◽

Aminuddin Bin Saim ◽

Ruszymah Binti Hj Idrus

Keyword(s):

Collagen Type ◽

Three Dimensional ◽

Collagen Type I ◽

Liquid Interface ◽

Type I ◽

Skin Model ◽

Skin Cells ◽

Air Liquid Interface ◽

In Vitro Skin Model

Three-dimensional (3D) in vitro skin models have been widely used for cosmeceutical and pharmaceutical applications aiming to reduce animal use in experiment. This study investigate capability of ovine tendon collagen type I (OTC-I) sponge suitable platform for a 3D in vitro skin model using co-cultured skin cells (CC) containing human epidermal keratinocytes (HEK) and human dermal fibroblasts (HDF) under submerged (SM) and air-liquid interface (ALI) conditions. Briefly, the extracted OTC-I was freeze-dried and crosslinked with genipin (OTC-I_GNP) and carbodiimide (OTC-I_EDC). The gross appearance, physico-chemical characteristics, biocompatibility and growth profile of seeded skin cells were assessed. The light brown and white appearance for the OTC-I_GNP scaffold and other groups were observed, respectively. The OTC-I_GNP scaffold demonstrated the highest swelling ratio (~1885%) and water uptake (94.96 ± 0.14%). The Fourier transformation infrared demonstrated amide A, B and I, II and III which represent collagen type I. The microstructure of all fabricated sponges presented a similar surface roughness with the presence of visible collagen fibers and a heterogenous porous structure. The OTC-I_EDC scaffold was more toxic and showed the lowest cell attachment and proliferation as compared to other groups. The micrographic evaluation revealed that CC potentially formed the epidermal- and dermal-like layers in both SM and ALI that prominently observed with OTC-I_GNP compared to others. In conclusion, these results suggest that OTC_GNP could be used as a 3D in vitro skin model under ALI microenvironment.

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