scholarly journals Development and Validation of Fluorescently Labeled, Functional Type I Collagen Molecules

2021 ◽  
Author(s):  
Seyed Mohammad Siadat ◽  
Monica E Susilo ◽  
Jeffrey A Paten ◽  
Alexandra A Silverman ◽  
Charles A DiMarzio ◽  
...  
Keyword(s):  
Type I Collagen ◽  
De Novo ◽  
Type I ◽  
Growth And Remodeling ◽  
Fibril Structure ◽  
Collagen Molecules ◽  
Vitro Model ◽  
Development And Validation ◽  
Network Morphology

While de novo collagen fibril formation is well-studied, there are few investigations into the growth and remodeling of extant fibrils, where molecular collagen incorporation into and erosion from the fibril surface must delicately balance during fibril growth and remodeling. Observing molecule/fibril interactions is difficult, requiring the tracking of molecular dynamics while, at the same time, minimizing the effect of the observation on fibril structure and assembly. To address the observation-interference problem, exogenous collagen molecules are tagged with small fluorophores and the fibrillogenesis kinetics of labeled collagen molecules as well as the structure and network morphology of assembled fibrils are quantified for the first time. While excessive labeling significantly disturbs fibrillogenesis kinetics and network morphology of assembled fibrils, adding less than ~1.2 labels preserves them. Applications of the functional, labeled collagen probe are demonstrated in both cellular and acellular systems. The functional, labelled collagen associates strongly with native fibrils and when added to an in vitro model of corneal stromal development, is endocytosed rapidly by cells and is translocated into synthesized matrix networks within 24 hours.

scholarly journals Evaluation In Vivo and In Vitro of the Antioxidant, Antinociceptive, and Anti-Inflammatory Activities of Biflavonoids From Ouratea hexasperma and O. ferruginea

2019 ◽  
Vol 14 (6) ◽  
pp. 1934578X1985680 ◽  
Author(s):  
Poliana de Araujo Oliveira ◽  
Queli Cristina Fidelis ◽  
Thayane Ferreira da Costa Fernandes ◽  
Milene Conceição de Souza ◽  
Dayane Magalhães Coutinho ◽  
...  
Keyword(s):  
Type I Collagen ◽  
In Vivo Model ◽  
Type I ◽  
Anti Inflammatory ◽  
Vitro Model ◽  
Factor Α ◽  
Necrosis Factor

Ouratea species are used for the treatment of inflammation-related diseases such as rheumatism and arthritic disorders. The Ouratea genus is a rich source of flavonoids and bioflavonoids and for this reason we evaluated the effects of the biflavonoid fractions from the leaves of O. hexasperma (OHME) and O. ferruginea (OFME) in the in vivo model of complete Freund’s adjuvant (CFA)-induced arthritis and in the in vitro model of oxidative stress and cellular viability. The CFA-induced arthritis model in rats was followed by paw volume, articular incapacitation and Randall-selitto models, as well as quantification of cytokines and serum C-terminal telopeptide of type I collagen levels. OHME and OFME demonstrated antinociceptive and anti-inflammatory activities, as well as improvement in articular incapacity and reduction in levels of interleukin 1β (IL-1β), IL-6, tumor necrosis factor α, and type 1 collagen, and increased cell viability. No adverse effects were observed. The results suggest that OHME and OFME can reduce inflammation and bone resorption besides their antioxidant action.

scholarly journals Identification and characterization of a fibroblast marker: FSP1.

1995 ◽  
Vol 130 (2) ◽  
pp. 393-405 ◽  
Author(s):  
F Strutz ◽  
H Okada ◽  
C W Lo ◽  
T Danoff ◽  
R L Carone ◽  
...  
Keyword(s):  
Cell Fate ◽  
Calcium Binding ◽  
Type I Collagen ◽  
De Novo ◽  
Mesangial Cells ◽  
In Vivo Studies ◽  
Type I ◽  
Tubular Epithelium ◽  
Collagen Gels

We performed subtractive and differential hybridization for transcript comparison between murine fibroblasts and isogenic epithelium, and observed only a few novel intracellular genes which were relatively specific for fibroblasts. One such gene encodes a filament-associated, calcium-binding protein, fibroblast-specific protein 1 (FSP1). The promoter/enhancer region driving this gene is active in fibroblasts but not in epithelium, mesangial cells or embryonic endoderm. During development, FSP1 is first detected by in situ hybridization after day 8.5 as a postgastrulation event, and is associated with cells of mesenchymal origin or of fibroblastic phenotype. Polyclonal antiserum raised to recombinant FSP1 protein stained the cytoplasm of fibroblasts, but not epithelium. Only occasional cells stain with specific anti-FSP1 antibodies in normal parenchymal tissue. However, in kidneys fibrosing from persistent inflammation, many fibroblasts could be identified in interstitial sites of collagen deposition and also in tubular epithelium adjacent to the inflammatory process. This pattern of anti-FSP1 staining during tissue fibrosis suggests, as a hypothesis, that fibroblasts in some cases arise, as needed, from the local conversion of epithelium. Consistent with this notion that FSP1 may be involved in the transition from epithelium to fibroblasts are experiments in which the in vitro overexpression of FSP1 cDNA in tubular epithelium is accompanied by conversion to a mesenchymal phenotype, as characterized by a more stellate and elongated fibroblast-like appearance, a reduction in cytokeratin, and new expression of vimentin. Similarly, tubular epithelium submerged in type I collagen gels exhibited the conversion to a fibroblast phenotype which includes de novo expression of FSP1 and vimentin. Use of the FSP1 marker, therefore, should further facilitate both the in vivo studies of fibrogenesis and the mapping of cell fate among fibroblasts.

Effect of Adhesion or Collagen Molecules on Cell Attachment, Insulin Secretion, and Glucose Responsiveness in the Cultured Adult Porcine Endocrine Pancreas: A Preliminary Study

2003 ◽  
Vol 12 (4) ◽  
pp. 439-446 ◽  
Author(s):  
Kazuya Edamura ◽  
Koko Nasu ◽  
Yukiko Iwami ◽  
Hiroyuki Ogawa ◽  
Nobuo Sasaki ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Type I Collagen ◽  
Cell Function ◽  
Cell Attachment ◽  
Control Group ◽  
Type I ◽  
Collagen Group ◽  
Collagen Molecules ◽  
Glucose Responsiveness

The effect of either adhesion or collagen molecules on cell attachment, insulin secretion, and glucose responsiveness was investigated in adult porcine pancreatic endocrine (PE) cells that were cultured for a longer term in vitro. Six different types of molecules—laminin, fibronectin, poly-L-lysine (PLL), type I collagen, gelatin, and Matrigel—were used. Approximately 2.0 × 105 cells per dish of each molecule type were cultured for 4 weeks. In the laminin group, the insulin accumulation was maintained at a significantly higher level than in the control group at 4 weeks of culture, and glucose-stimulated insulin secretion and the insulin-positive rate were also higher than in the control group. In the Matrigel group, islet-like cell clusters were formed, but insulin accumulation rapidly decreased at 3–4 weeks of culture. A large number of PE cells attached tightly and spread in the fibronectin group until the fourth week of culture, but their function was not better than those in the control group. In the PLL and gelatin groups, the PE cell function was not significantly different from that of the control group. In the type I collagen group, insulin secretion was inferior to that of the other groups. The results of this study suggest that laminin is the most suitable extracellular matrix for the long-term culture preservation of PE cells.

scholarly journals Anti-Photoaging Effect of Plant Extract Fermented with Lactobacillus buchneri on CCD-986sk Fibroblasts and HaCaT Keratinocytes

2020 ◽  
Vol 11 (1) ◽  
pp. 3 ◽  
Author(s):  
Yun-Mi Kang ◽  
Chul-Hee Hong ◽  
Sa-Haeng Kang ◽  
Dong-Seok Seo ◽  
Seong-Oh Kim ◽  
...  
Keyword(s):  
In Vitro Model ◽  
Type I Collagen ◽  
Dermal Fibroblasts ◽  
Epidermal Keratinocytes ◽  
Type I ◽  
Mrna Levels ◽  
Protective Effects ◽  
Lactobacillus Buchneri ◽  
Vitro Model

Ultraviolet (UV) exposure triggers the abnormal production of reactive oxygen (ROS) species and the expression of matrix metalloproteinases (MMPs) that are responsible for photoaging. Probiotics are widely used in healthcare and for immune enhancement. One probiotic, Lactobacillus buchneri is found in Kimchi. This study was aimed at assessing the anti-photoaging effect of plant extracts fermented with L. buchneri (PELB) to develop functional cosmetics. We investigated the anti-photoaging effect of PELB in a UVB-induced photoaging in vitro model and selected effective extracts using the elastase inhibition assay, ELISA for Type I procollagen and collagenase-1, and quantitative real time PCR. Normal human dermal fibroblasts and epidermal keratinocytes were pre-treated with PELB and exposed to UVB. We found that PELB decreased elastase activity and increased type I collagen expression in a UVB-induced photoaging in vitro model. In addition, PELB greatly reduced collagenase activity and MMP mRNA levels in a UVB-induced photoaging in vitro model. Furthermore, PELB promoted the expression of moisture factor and anti-oxidant enzymes in a UVB-induced photoaging in vitro model. These results indicated that the PELB could be potential candidates for the protective effects against UVB-induced photoaging. Overall, these results suggest that PELB might be useful natural components of cosmetic products.

scholarly journals Organic / inorganic bioactive materials Part I: Synthesis, structure and in vitro assessment of collagen/silicocarnotite biocoatings

2009 ◽  
Vol 7 (4) ◽  
pp. 702-710 ◽  
Author(s):  
Vladimir Hristov ◽  
Lachezar Radev ◽  
Bisserka Samuneva ◽  
Georgi Apostolov
Keyword(s):  
Type I Collagen ◽  
Sol Gel ◽  
Weight Ratio ◽  
Type I ◽  
Bioactive Materials ◽  
Organic Part ◽  
Carboxylate Groups ◽  
Cross Linkage ◽  
Collagen Molecules

AbstractThe silicocarnotite, as an inorganic part of the coatings, has been synthesized using a polystep sol-gel method. The chemical composition of the prepared silicocarnotite sol is described as 58.12 CaO, 29.42 P2O5, 12.45 SiO2 (wt%), where Ca/P+Si = 1,67. The acid soluble type I collagen, as an organic part of the obtained coatings, was mixed with silicocarnotite powder in a weight ratio of 25:75 and 75:25 weight ratio without cross-linkage. The acidity of the obtained mixture was readjust with 25% NH4OH to pH = 9.0. The mixture was then dried at 37°C for 12 h.The growth of B-type carbonate containing hydroxyapatite (B-type CO3HA) in which CO3 2+→PO4 3− on the surface of collagen/silicocarnotite coatings soaked in 1.5 simulated body fluid (1.5 SBF) was observed. The nucleation of B-type CO3HA was estimated on the obtained coatings after 3 days immersion in 1.5 SBF. The negatively charged carboxylate groups from the collagen surface may be responsible for the HA deposition. This was confirmed by the “red shift” of carboxylate groups of collagen molecules in the FTIR spectra. After soaking in 1.5 SBF, the morphology of prepared coatings and HA formation was observed by SEM.

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.

A serum-free in vitro model of renal microvessel development

1998 ◽  
Vol 274 (6) ◽  
pp. F1150-F1160 ◽  
Author(s):  
Lisa M. Antes ◽  
Monica M. Villar ◽  
Sylvia Decker ◽  
Roberto F. Nicosia ◽  
Dean A. Kujubu
Keyword(s):  
Growth Factors ◽  
Endothelial Cell ◽  
In Vitro Model ◽  
Type I Collagen ◽  
Type I ◽  
Renal Vasculature ◽  
Serum Free ◽  
Polypeptide Growth Factors ◽  
Vitro Model

The differentiation and organization of the embryonic renal vasculature is a crucial event in renal development. To study this process, we developed a serum-free in vitro model of renal microvessel development. Mouse embryonic kidney explants, when embedded specifically in type I collagen, demonstrate outgrowth of microvascular structures when stimulated by the phorbol ester 12- O-tetradecanoylphorbol 13-acetate (TPA, 10–50 ng/ml). Other polypeptide growth factors stimulated little, if any, microvessel outgrowth from the explants. Similar outgrowths were not observed when other embryonic tissue explants were used. The number of microvessels observed depended on the gestational age of the explants. We hypothesize that TPA induces the in situ differentiation of metanephric mesenchymal cells into endothelial cell precursors and that specific matrix proteins and cell-matrix interactions are necessary for the organization of these precursors into microvessels. Our model will allow us to examine in detail the responsiveness of metanephric kidney cells to both growth factors and extracellular matrix molecules and to understand how they influence renal endothelial cell differentiation.

scholarly journals In Vitro Model of Bartonella henselae-Induced Angiogenesis

2004 ◽  
Vol 72 (12) ◽  
pp. 7315-7317 ◽  
Author(s):  
James E. Kirby
Keyword(s):  
In Vitro Model ◽  
Type I Collagen ◽  
Bartonella Henselae ◽  
Collagen Matrix ◽  
In Vitro Models ◽  
Type I ◽  
Blood Vessel Formation ◽  
Vitro Model

ABSTRACT Bartonella henselae is a gram-negative pathogen that causes angiogenesis. Here, I establish in vitro models to study Bartonella-induced blood vessel formation. I found that B. henselae induces long-term endothelial survival and tubular differentiation within type I collagen matrix.

scholarly journals Pseudopterosin and O-Methyltylophorinidine Suppress Cell Growth in a 3D Spheroid Co-Culture Model of Pancreatic Ductal Adenocarcinoma

2020 ◽  
Vol 7 (2) ◽  
pp. 57
Author(s):  
Bailu Xie ◽  
Jan Hänsel ◽  
Vanessa Mundorf ◽  
Janina Betz ◽  
Irene Reimche ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
In Vitro Model ◽  
Type I Collagen ◽  
Ductal Adenocarcinoma ◽  
Pancreatic Tumors ◽  
Type I ◽  
Culture In Vitro ◽  
Drug Candidates ◽  
Vitro Model

Current therapies for treating pancreatic ductal adenocarcinoma (PDAC) are largely ineffective, with the desmoplastic environment established within these tumors being considered a central issue. We established a 3D spheroid co-culture in vitro model using a PDAC cell line (either PANC-1 or Capan-2), combined with stellate cells freshly isolated from pancreatic tumors (PSC) or hepatic lesions (HSC), and human type I collagen to analyze the efficiency of the chemotherapeutic gemcitabine (GEM) as well as two novel drug candidates derived from natural products: pseudopterosin (PsA-D) and O-methyltylophorinidine (TYLO). Traditional 2D in vitro testing of these agents for cytotoxicity on PANC-1 demonstrated IC50 values of 4.6 (±0.47) nM, 34.02 (±1.35) µM, and 1.99 (±0.13) µM for Tylo, PsA-D, and GEM, respectively; these values were comparable for Capan-2: 5.58 (±1.74) nM, 33.94 (±1.02) µM, and 0.41 (±0.06) µM for Tylo, PsA-D, and GEM, respectively. Importantly, by assessing the extent of viable cells within 3D co-culture spheroids of PANC-1 with PSC or HSC, we could demonstrate a significant lack of efficacy for GEM, while TYLO remained active and PsA-D showed slightly reduced efficacy: GEM in PANC-1/PSC (IC50 = >100 µM) or PANC-1/HSC (IC50 = >100 µM) spheroids, TYLO in PANC-1/PSC (IC50 = 3.57 ± 1.30 nM) or PANC-1/HSC (IC50 = 6.39 ± 2.28 nM) spheroids, and to PsA-D in PANC-1/PSC (IC50 = 54.42 ± 12.79 µM) or PANC-1/HSC (IC50 = 51.75 ± 0.60 µM). Microscopic 3D rendering supported these cytotoxicity outcomes, showing little or no morphological spheroid structure change during this period of rapid cell death. Our results support the use of this 3D spheroid co-culture in vitro model having a desmoplastic microenvironment for the identification of possible novel chemotherapeutic drug candidates for PDAC, such as TYLO and PsA-D.

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.

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