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 Collagen microarchitecture mechanically controls myofibroblast differentiation

2020 ◽  
Vol 117 (21) ◽  
pp. 11387-11398 ◽  
Author(s):  
Bo Ri Seo ◽  
Xingyu Chen ◽  
Lu Ling ◽  
Young Hye Song ◽  
Adrian A. Shimpi ◽  
...  
Keyword(s):  
Collagen Type ◽  
Collagen Type I ◽  
Type I ◽  
Myofibroblast Differentiation ◽  
Dependent Manner ◽  
Fibrillar Collagen ◽  
Collagen Gels ◽  
Gelation Temperature ◽  
Rheological Characterization ◽  
Fiber Thickness

Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness. Collagen gels with controlled fiber thickness and pore size were microfabricated by adjusting the gelation temperature while keeping their concentration constant. Rheological characterization and simulation data indicated that networks with thicker fibers and larger pores exhibited increased strain-stiffening relative to networks with thinner fibers and smaller pores. Accordingly, ASCs cultured in scaffolds with thicker fibers were more contractile, expressed myofibroblast markers, and deposited more extended fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs in scaffolds with thicker fibers exhibited a more proangiogenic phenotype that promoted endothelial sprouting in a contractility-dependent manner. Our findings suggest that changes of collagen microarchitecture regulate myofibroblast differentiation and fibrosis independent of collagen quantity and bulk stiffness by locally modulating cellular mechanosignaling. These findings have implications for regenerative medicine and anticancer treatments.

scholarly journals In vitro production of human dermal equivalent

2010 ◽  
Vol 63 (7-8) ◽  
pp. 459-464 ◽  
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.

Effect of MMP-13 degraded SPARC on type I collagen and its biomarker potential in colorectal cancer.

2018 ◽  
Vol 36 (4_suppl) ◽  
pp. 717-717
Author(s):  
Stephanie Nina Kehlet ◽  
Henrik Harling ◽  
Lars N Jørgensen ◽  
Morten A Karsdal ◽  
Nicholas Willumsen
Keyword(s):  
Colorectal Cancer ◽  
Type I Collagen ◽  
Collagen Degradation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Matricellular Protein ◽  
Elisa Assay ◽  
Type I ◽  
Healthy Controls ◽  
Collagen Deposition

717 Background: Increased collagen deposition and remodeling of the extracellular matrix has been shown to play a role in the pathology of gastrointestinal cancer (GC). The matricellular protein SPARC (secreted proteome acidic and rich in cysteine) binds collagens and hereby regulates collagen fibrillogenesis. Matrix metalloproteinase (MMP) mediated cleavage of SPARC, increases the affinity for collagens up to 20-fold. SPARC has been shown to be overexpressed in GC patients and associated with GC cell invasion and metastasis. Increased expression and cleavage of SPARC might therefore be implicated in GC pathology by increasing collagen deposition. Here, we present the development and validation of a competitive enzyme-linked immunosorbent assay (ELISA) quantifying a specific MMP-13 generated fragment of SPARC - a cleavage site involved in increased collagen affinity. The biomarker potential of this fragment was examined in serum from colorectal cancer (CRC) patients and healthy controls. Moreover, we evaluated the ability of cleaved SPARC to prevent type I collagen degradation in vitro. Methods: A monoclonal antibody was raised against a MMP-13-generated neo-epitope of SPARC and a competitive ELISA assay (SPARC-M) was developed and technically validated. Serum levels were assessed in CRC patients (n=50) and healthy controls (n=30). The ability of cleaved SPARC to prevent collagen degradation was investigated using an ELISA assay measuring type I collagen degradation by MMP-9. Results: SPARC-M was technically robust and specific for SPARC cleaved by MMP-13. The fragment was elevated in CRC patients when compared to healthy controls (p=0.0097). When MMP-13 degraded SPARC was incubated with type I collagen and MMP-9, type I collagen degradation was completely inhibited suggesting that SPARC increases collagen deposition by preventing collagen degradation. Conclusions: SPARC-M was significantly elevated in CRC patients compared to healthy controls suggesting biomarker potential. Biologically, cleaved SPARC may prevent type I collagen degradation hereby leading to a pro-tumorigenic environment. Larger clinical studies are needed to validate the clinical use of this biomarker in GC.

scholarly journals Organotypic Cultures of Intervertebral Disc Cells: Responses to Growth Factors and Signaling Pathways Involved

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Harris Pratsinis ◽  
Dimitris Kletsas
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Intervertebral Disc ◽  
Dna Synthesis ◽  
Signaling Pathways ◽  
Collagen Type I ◽  
Cell Physiology ◽  
Type I ◽  
Collagen Gels

Intervertebral disc (IVD) degeneration is strongly associated with low back pain, a major cause of disability worldwide. An in-depth understanding of IVD cell physiology is required for the design of novel regenerative therapies. Accordingly, aim of this work was the study of IVD cell responses to mitogenic growth factors in a three-dimensional (3D) organotypic milieu, comprising characteristic molecules of IVD’s extracellular matrix. In particular, annulus fibrosus (AF) cells were cultured inside collagen type-I gels, while nucleus pulposus (NP) cells in chondroitin sulfate A (CSA) supplemented collagen gels, and the effects of Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF), and Insulin-Like Growth Factor-I (IGF-I) were assessed. All three growth factors stimulated DNA synthesis in both AF and NP 3D cell cultures, with potencies similar to those observed previously in monolayers. CSA supplementation inhibited basal DNA synthesis rates, without affecting the response to growth factors. ERK and Akt were found to be phosphorylated following growth factor stimulation. Blockade of these two signaling pathways using pharmacologic inhibitors significantly, though not completely, inhibited growth factor-induced DNA synthesis. The proposed culture systems may prove useful for further in vitro studies aiming at future interventions for IVD regeneration.

Bacterial Endotoxin Inhibits Migration, Attachment, and Orientation of Human Gingival Fibroblasts in vitro and Delays Collagen Gel Contraction

1987 ◽  
Vol 66 (9) ◽  
pp. 1449-1455 ◽  
Author(s):  
S. Pitaru ◽  
M. Soldinger ◽  
D. Madgar ◽  
Z. Metzger
Keyword(s):  
Collagen Type ◽  
Cell Attachment ◽  
Collagen Gel ◽  
Collagen Type I ◽  
Human Gingival Fibroblasts ◽  
Bacterial Endotoxin ◽  
Type I ◽  
Gingival Fibroblasts ◽  
Collagen Gels

The purpose of this study was to assess the effect of endotoxin adsorbed to dental surfaces and to collagen type I on the migration, attachment, and orientation of human gingival fibroblasts (HGF). Transversely cut porcine tooth root slices (RS), 200 μm thick, were prepared. Half of the RS obtained were partially demineralized in EDTA. Half of the demineralized and non-demineralized RS were incubated with 400 μg/mL of endotoxin for 24 hr, whereas the other half were maintained in PBS and served as controls. Experimental and control RS were placed on confluent layers of HFG and cultured for six days. Cell migration toward and cell attachment to the periphery of the RS and the formation of oriented cell sheets were assessed by means of photographic techniques. Additionally, six-day-old cultures were fixed and processed for SEM observation. In separate experiments, the effect of endotoxin on cell attachment to collagen type I and on contraction of three-dimensional collagen gels was assessed. It was found that: (i) bacterial endotoxin inhibited migration and attachment of HGF to both demineralized and non-demineralized cementum and interfered with the development of oriented cellular structure ; (ii) the inhibitory effect was significantly more pronounced for non-demineralized than for demineralized cementum; (iii) the morphology of HGF attached to endotoxin-treated dental surfaces was altered compared with that of their controls; and (iv) bacterial endotoxin inhibited cell attachment to collagen type I and delayed the contraction of collagen gel.

scholarly journals Toxicity Effects of Methylene Blue on Rat Intervertebral Disc Annulus Fibrosus Cells

2019 ◽  
Vol 2 (22.2) ◽  
pp. 155-164
Author(s):  
Liang Zhang
Keyword(s):  
Methylene Blue ◽  
Cell Apoptosis ◽  
Annulus Fibrosus ◽  
Caspase 3 ◽  
In Vitro Study ◽  
Collagen Type I ◽  
Type I ◽  
Dependent Manner ◽  
Concentration Dependent Manner

Background: There is an increasing local application of methylene blue (MB) in the treatment of discogenic low back pain (LBP) and percutaneous transforaminal endoscopic discectomy (PTED) procedures. MB could generate DNA damage and induce apoptosis in different cell types; however, the effects of MB on intervertebral disc (IVD) annulus fibrosus (AF) cells are not clearly understood. Objective: The objective of this study was to investigate the effects of different concentrations of MB on rat AF cells in vitro. Study Design: This study used an experimental design. Setting: This research was conducted at the Orthopaedic Institute of the Clinical Medical College of Yangzhou University. Methods: AF cells were isolated and cultured with different concentrations of MB (0, 2, 20, and 200 μg/mL) and assessed to determine the possible cytotoxic effects of MB. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. The inverted phase-contrast microscopy was used to perform morphological observation of apoptotic cells, and flow cytometry was used to measure the incidence of cell apoptosis. The mRNA and protein expression levels of apoptosis-associated genes (caspase-3, Bcl-2, and Bax) and other related genes (collagen type I, transforming growth factor β1 [TGF-β1], fibroblast growth factor [bFGF], and tissue inhibitor of metalloproteinase-1 [TIMP-1]) were analyzed by quantitative real-time PCR (RT-PCR) and Western blotting. Results: Our results indicated that MB reduced cell viability in a concentration- and timedependent manner. MB also induced marked AF cell apoptosis in a concentration-dependent manner observed by inverted phase-contrast microscopy, flow cytometry, and indicated by the increased expression of caspase-3. Both RT-PCR and Western blotting revealed significant upregulation of Bax and caspase-3 expression levels accompanied by decreased expression of Bcl2 in a concentration-dependent manner. Moreover, collagen type I, TGF-β1, bFGF, and TIMP-1 mRNA and protein levels were also found to be decreased by MB in a concentration-dependent manner. Limitations: Limitations of this study were the in vitro study design and lack of in vivo validation of the observed effects of MB on human IVD cells. Conclusions: Our results indicate that a high concentration of MB can not only inhibit proliferation and paracrine function of AF cells, but can also induce cell apoptosis in a concentration-dependent manner, suggesting that it is necessary to choose low concentrations of MB in practical application and limit the use of MB in the treatment of discogenic LBP to research protocols. Key words: Methylene blue, annulus fibrosus cell, proliferation, apoptosis, paracrine

Bradykinin augments fibroblast-mediated contraction of released collagen gels

2001 ◽  
Vol 281 (1) ◽  
pp. L164-L171 ◽  
Author(s):  
Tadashi Mio ◽  
Xiangde Liu ◽  
Myron L. Toews ◽  
Yuichi Adachi ◽  
Debra J. Romberger ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Type I Collagen ◽  
Inflammatory Diseases ◽  
Fetal Lung ◽  
Lung Fibroblasts ◽  
Type I ◽  
Dependent Manner ◽  
Collagen Gels ◽  
Protein Kinase C Inhibitors

Bradykinin is a multifunctional mediator of inflammation believed to have a role in asthma, a disorder associated with remodeling of extracellular connective tissue. Using contraction of collagen gels as an in vitro model of wound contraction, we assessed the effects of bradykinin tissue on remodeling. Human fetal lung fibroblasts were embedded in type I collagen gels and cultured for 5 days. After release, the floating gels were cultured in the presence of bradykinin. Bradykinin significantly stimulated contraction in a concentration- and time-dependent manner. Coincubation with phosphoramidon augmented the effect of 10−9 and 10−8 M bradykinin. A B2 receptor antagonist attenuated the effect of bradykinin, whereas a B1 receptor antagonist had no effect, suggesting that the effect is mediated by the B2 receptor. An inhibitor of intracellular Ca2+mobilization abolished the response; addition of EGTA to the culture medium attenuated the contraction of control gels but did not modulate the response to bradykinin. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase C inhibitors staurosporine and GF-109203X attenuated the responses. These data suggest that by augmenting the contractility of fibroblasts, bradykinin may have an important role in remodeling of extracellular matrix that may result in tissue dysfunction in chronic inflammatory diseases, such as asthma.

scholarly journals RNA-Containing Immune Complexes Formed by Anti-Melanoma Differentiation Associated Gene 5 Autoantibody Are Potent Inducers of IFN-α

2021 ◽  
Vol 12 ◽  
Author(s):  
Kaiwen Wang ◽  
Jiangfeng Zhao ◽  
Wanlong Wu ◽  
Wenwen Xu ◽  
Shuhui Sun ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Dependent Manner ◽  
Type I Ifn ◽  
Cytoplasmic Rna ◽  
Healthy Donors ◽  
Rna Immunoprecipitation ◽  
High Level

ObjectiveAnti-melanoma differentiation-associated gene 5 (MDA5) autoantibody is a distinctive serology hallmark of dermatomyositis (DM). As an autoantigen, MDA5 is a cytoplasmic RNA recognition receptor. The aim of this study was to address the question of whether the RNA-containing immune complex (IC) formed by MDA5 and anti-MDA5 could activate type I interferon (IFN) response.MethodPatients with anti-MDA5+ DM (n = 217), anti-MDA5− DM (n = 68), anti-synthase syndrome (ASyS, n = 57), systemic lupus erythematosus (SLE, n = 245), rheumatoid arthritis (RA, n = 89), and systemic sclerosis (SSc, n = 30) and healthy donors (HD, n = 94) were enrolled in our studies. Anti-MDA5 antibody was detected by line blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and Western blotting. Cytokine profiling was determined by multiplex flow cytometry, and IFN-α was further measured by ELISA. Type I IFN-inducible genes were detected by quantitative PCR (qPCR). RNA–IC binding was analyzed by RNA immunoprecipitation. Plasmacytoid dendritic cells (pDCs) derived from healthy donors were cultivated and stimulated with MDA5 ICs with or without RNase and Toll-like receptor 7 (TLR-7) agonist. The interaction between MDA5 ICs and TLR7 was evaluated by immunoprecipitation and confocal microscopy.ResultsAccording to our in-house ELISA, the presence of anti-MDA5 antibody in 76.1% of DM patients, along with 14.3% of SLE patients who had a lower titer yet positive anti-MDA5 antibody, was related to the high level of peripheral IFN-α. ICs formed by MDA5 and anti-MDA5 were potent inducers of IFN-α via TLR-7 in an RNA-dependent manner in vitro.ConclusionOur data provided evidence of the mechanistic relevance between the anti-MDA5 antibody and type I IFN pathway.

scholarly journals Effects of Adiponectin on Diastolic Function in Mice Underwent Transverse Aorta Constriction

2019 ◽  
Vol 13 (2) ◽  
pp. 225-237 ◽  
Author(s):  
Xueting Han ◽  
Yanyan Wang ◽  
Mingqiang Fu ◽  
Yu Song ◽  
Jingfeng Wang ◽  
...  
Keyword(s):  
Diastolic Dysfunction ◽  
Collagen Type ◽  
Lysyl Oxidase ◽  
Pressure Overload ◽  
Collagen Type I ◽  
Myocardial Tissue ◽  
Type I ◽  
Compound C ◽  
Adult Cardiomyocytes

Abstract Diastolic dysfunction is common in various cardiovascular diseases, which could be affected by adiponectin (APN). Nevertheless, the effects of APN on diastolic dysfunction in pressure overload model induced by transverse aorta constriction (TAC) remain to be further elucidated. Here, we demonstrated that treatment of APN attenuated diastolic dysfunction and cardiac hypertrophy in TAC mice. Notably, APN also improved active relaxation of adult cardiomyocytes, increased N2BA/N2B ratios of titin isoform, and reduced collagen type I to type III ratio and lysyl oxidase (Lox) expressions in the myocardial tissue. Moreover, APN supplementation suppressed TAC-induced oxidative stress. In vitro, inhibition of AMPK by compound C (Cpc) abrogated the effect of APN on modulation of titin isoform shift and the anti-hypertrophic effect of APN on cardiomyocytes induced by AngII. In summary, our findings indicate that APN could attenuate diastolic dysfunction in TAC mice, which are at least partially mediated by AMPK pathway.

In Vitro Osteogenic, Angiogenic, and Inflammatory Effects of Copper in β-Tricalcium Phosphate

MRS Advances ◽  
2019 ◽  
Vol 4 (21) ◽  
pp. 1253-1259
Author(s):  
Weiguo Han ◽  
Haley Cummings ◽  
Murali Krishna Duvuuru ◽  
Sarah Fleck ◽  
Sahar Vahabzadeh ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Tricalcium Phosphate ◽  
Physical And Mechanical Properties ◽  
Early Time ◽  
Collagen Type I ◽  
Type I ◽  
Dependent Manner ◽  
Dental Tissue ◽  
Engineering Applications

AbstractTricalcium phosphate (TCP) is a promising candidate in bone and dental tissue engineering applications. Though osteoconductive, its low osteoinductivity is a major concern. Trace elements addition at low concentrations are known for their impact on not only the osteoinductivity, but also physical and mechanical properties of TCP. Copper (Cu) is known for its role in vascularization and angiogenesis in biological systems. Here, we studied the effects of Cu addition on phase composition, porosity, microstructure and in vitro interaction with osteoblast (OB) cells. Our results showed that Cu stabilized the TCP structure, while no significant effect of microstructure and porosity was found. Cu at concentrations less than 1 wt.% did not have any cytotoxic effect while decreased proliferation of OBs were observed at 1 wt.% Cu doped TCP. Addition of Cu upregulated collagen type I and vascular endothelial growth factor expression in a dose dependent manner at early time-point. Furthermore, Cu reduced inflammatory gene expression by human osteoblasts. These findings show that addition of Cu to TCP may provide a therapeutic strategy that can be applied in bone tissue engineering applications.

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