Lower Collagen Production Research Articles

Cell viability and collagen deposition on hydroxyapatite coatings formed on pretreated substrates

A method of thermal deposition that involves localized high peak temperatures at the electrode-electrolyte interface was proposed for hydroxyapatite (HA) coatings deposition. Active surface layers were obtained by HA incorporation from the aqueous electrolyte into the substrate surface due to the decrease of HA solubility with increasing of substrate temperature. Commercially Ti-6Al-4V alloy (grade 4) substrates were pretreated by sandblasting combined with various chemical pre-treatments: 20% NaOH, 20% oxalic acid, and both. After both types of pre-treatment and HA thermal deposition, we can observe uniform coatings with a rough surface. HA presence was confirmed by X-ray Diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR) analysis. According to received data, samples after 6.5 bar, one cycle of sandblasting and etching in 20% oxalic acid 2 h, 20% H3PO4 1 min as well as untreated samples showed the highest wettability. All measured contact angles are lower than 90°, which means that obtained HA surfaces are hydrophilic and indicates that the wetting of the surface is favorable, and the fluid will spread over a large area of the surface.Resazurin reduction assay showed satisfactory U2OS osteoblastic cell adhesion in 1 day of the experiment. There is no significant difference between non-treated polished surfaces and HA-covered with sandblasting pre-treatment. In contrast – the polished surface with HA coating demonstrates significantly less cell adhesion.It was confirmed low collagen production both in untreated and polished-HA surfaces in comparison with the sandblasted-HA ones. The obtained data suggest that roughness plays an important role in cell adhesion and proliferation, but HA coating provides additional stimuli for cell activity (collagen synthesis).

ECM1: A CTGF Binding Protein Inhibiting Ductular Reaction and Liver Fibrosis

IntroductionExtracellular matrix protein 1 (ECM1) has inhibitory roles against transforming growth factor (TGF)β activation and liver fibrosis. We have identified ECM1 as binding protein for connective tissue growth factor (CTGF) in yeast two‐hybrid analysis. This study aims to determine whether ECM1 binding inhibits CTGF‐mediated ductular reaction and liver fibrosis.MethodsMice were fed with 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) to induce ductular reaction and liver fibrosis. ECM1 was overexpressed in adenovirus and intravenously injected into animals before DDC feeding. ECM1 interaction with CTGF, and their effects on liver injury after DDC feeding were analyzed.ResultsECM1 downregulation was correlated with CTGF upregulation during DDC‐induced liver injury. ECM1 overexpression after DDC feeding caused low levels of EpCAM and Sox9 mRNAs, small areas of EpCAM+ cells, and few proliferating ductular epithelial cells in comparison to controls that received GFP adenovirus. Weak fibrogenesis was also observed in the Ecm1 overexpressing livers, as evidenced by low levels of fibrosis‐related genes, small fibrotic areas in staining with α smooth muscle actin antibody and Sirius red as well as low collagen production determined by hepatic hydroxyproline content. Finally, we found that ECM1 could interact with CTGF and integrin αvβ6 leading to low TGFβ activation in vitro and in vivo.ConclusionThese observations indicate that ECM1 is a novel binding protein for CTGF. Its overexpression significantly reduces DDC‐induced ductular reaction and fibrogenic reaction, supporting anti‐fibrotic potentials of this molecule for the treatment of chronic liver disease.Support or Funding InformationThis study is supported by National Institutes of Health NIAAA KO1AA024174 grant and Children Miracle Research Foundation grant awarded to L Pi.

Connective tissue growth factor and integrin αvβ6: a new pair of regulators critical for ductular reaction and biliary fibrosis in mice.

Connective tissue growth factor (CTGF) is a matricellular protein that mediates cell-matrix interaction through various subtypes of integrin receptors. This study investigated the role of CTGF and integrin αvβ6 in hepatic progenitor/oval cell activation, which often occurs in the form of ductular reactions (DRs) when hepatocyte proliferation is inhibited during severe liver injury. CTGF and integrin αvβ6 proteins were highly expressed in DRs of human cirrhotic livers and cholangiocarcinoma. Confocal microscopy analysis of livers from Ctgf promoter-driven green fluorescent protein reporter mice suggested that oval cells and cholangiocytes were the main sources of CTGF and integrin αvβ6 during liver injury induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Deletion of exon 4 of the Ctgf gene using tamoxifen-inducible Cre-loxP system down-regulated integrin αvβ6 in DDC-damaged livers of knockout mice. Ctgf deficiency or inhibition of integrin αvβ6, by administrating the neutralizing antibody, 6.3G9 (10 mg/kg body weight), caused low levels of epithelial cell adhesion molecule and cytokeratin 19 gene messenger RNAs. Also, there were smaller oval cell areas, fewer proliferating ductular epithelial cells, and lower cholestasis serum markers within 2 weeks after DDC treatment. Associated fibrosis was attenuated, as indicated by reduced expression of fibrosis-related genes, smaller areas of alpha-smooth muscle actin staining, and low collagen production based on hydroxyproline content and Sirius Red staining. Finally, integrin αvβ6 could bind to CTGF mediating oval cell adhesion to CTGF and fibronection substrata and promoting transforming growth factor (TGF)-β1 activation in vitro. CTGF and integrin αvβ6 regulate oval cell activation and fibrosis, probably through interacting with their common matrix and signal partners, fibronectin and TGF-β1. CTGF and integrin αvβ6 are potential therapeutic targets to control DRs and fibrosis in related liver disease.

Angiotensinase C mRNA and Protein Downregulations Are Involved in Ethanol-Deteriorated Left Ventricular Systolic Dysfunction in Spontaneously Hypertensive Rats.

The influences of angiotensinase C on ethanol-induced left ventricular (LV) systolic function were assessed in spontaneously hypertensive rats (SHRs). SHRs were fed by a liquid diet with or without ethanol for 49 days. The normotensive Wistar Kyoto rats (WKY) were fed by the liquid diet without ethanol and used as control. We evaluated LV systolic function, angiotensinase C mRNA and protein expressions, activation of the renin-angiotensin system (RAS), and the gene expressions of LV collagen (Col) III a1 and matrix metalloproteinases- (MMP-) 9. Compared to the WKY, LV systolic dysfunction (expressed by decreased fractional shortening and ejection fraction) was observed in the SHRs before ethanol treatment and further deteriorated by ethanol treatment. In the ethanol-treated SHRs, the following were observed: downregulations of angiotensinase C mRNA and protein, increased RAS activity with low collagen production as evidenced by angiotensin II and angiotensin type 1 receptor (AT1R) protein upregulation, AT1aR mRNA downregulation, and an MMP-9 mRNA expression upregulation trend with the downregulation of Col III a1 mRNA expression in LV. We conclude that chronic ethanol regimen is sufficient to promote the enhanced RAS activity-induced decrease in the production of cardiac collagen via downregulated angiotensinase C, leading to the further deterioration of LV systolic dysfunction in SHRs.

Time and dose-dependent effects of chondroitinase ABC on growth of engineered cartilage

Tissue engineering techniques have been effective in developing cartilage-like tissues in vitro. However, many scaffold-based approaches to cultivating engineered cartilage have been limited by low collagen production, an impediment for attaining native functional load-bearing tensile mechanical properties. Enzymatic digestion of glycosaminoglycans (GAG) with chondroitinase ABC (chABC) temporarily suppresses the construct's GAG content and compressive modulus and increases collagen content. Based on the promising results of these early studies, the aim of this study was to further promote collagen deposition through more frequent chABC treatments. Weekly dosing of chABC at a concentration of 0.15U/mL resulted in a significant cell death, which impacted the ability of the engineered cartilage to fully recover GAG and compressive mechanical properties. In light of these findings, the influence of lower chABC dosage on engineered tissue (0.004 and 0.015U/mL) over a longer duration (one week) was investigated. Treatment with 0.004U/mL reduced cell death, decreased the recovery time needed to achieve native compressive mechanical properties and GAG content, and resulted in a collagen content that was 65% greater than the control. In conclusion, the results of this study demonstrate that longer chABC treatment (one week) at low concentrations can be used to improve collagen content in developing engineered cartilage more expediently than standard chABC treatments of higher chABC doses administered over brief durations.

Straining Mode–Dependent Collagen Remodeling in Engineered Cardiovascular Tissue

Similar to native cardiovascular tissues, the mechanical properties of engineered cardiovascular constructs depend on the composition and quality of the extracellular matrix, which is a net result of matrix remodeling processes within the tissue. To improve tissue remodeling, and hence tissue mechanical properties, various mechanical conditioning protocols, such as strain-based or flow-based conditioning, have been applied to engineered cardiovascular constructs with promising results. We hypothesize that tissue remodeling is dependent on the mode of straining. Therefore, the effects of two modes of straining, being either static or dynamic, were quantified on several indices of tissue remodeling. Differences in matrix composition (collagen and glycosaminoglycans [GAGs]) and maturity (collagen cross-links) were quantified with time on gene expression and protein levels. In addition, the secretion of specific collagen remodeling markers (matrix metalloproteinase-1), collagen synthesis marker (procollagen type I carboxy-terminal propeptide, PIP), and collagen degradation marker (carboxyterminal telopeptide of type I, ICTP) was investigated with time. Static strain stimulated collagen gene expression and production with time. Dynamic straining resulted in (1) lower collagen gene expression and production, but (2) enhanced collagen cross-link expression and density, and GAG production, and (3) stimulated collagen remodeling processes, as expressed by enhanced production of remodeling markers. Thus, despite a lower collagen production, the quality of the neotissue was enhanced by a dynamic straining component. These straining mode-dependent remodeling responses allow us for the first time to balance collagen and cross-link production and, thus, to fine tune tissue mechanical properties via mechanical conditioning protocols. This is of utmost importance for cardiovascular tissue engineering, where insufficient mechanical properties are currently a main limiting factor for present in vivo application.

Effect of TGF-?/Smad signaling pathway on lung myofibroblast differentiation

Myofibroblasts play important roles in the pathogenesis of lung fibrosis. Transforming growth factor (TGF)-beta 1 has been widely recognized as a key fibrogenic cytokine. The major signaling pathway of (TGF)-beta(1) is through cytoplasmic Smad proteins. Our study investigated the role of individual (TGF)-beta(1)/Smad signal proteins in mediating alpha-smooth muscle actin (alpha-SMA) gene expression, which is a well-known key marker of myofibroblast differentiation. We transiently cotransfected alpha-SMA promoter-luciferase fusion plasmid (p895-Luc) and Smad expression plasmids and measured Luc activity in (TGF)-beta(1)-treated human fetal lung fibroblasts. We induced Smad3 knockout mice lung fibrosis by bleomycin. alpha-SMA protein expression was assessed by Western blotting. Collagen protein was analyzed by measuring hydroxyprolin. Myofibroblast morphology was assessed by immunohistochemistry. We found that the overexpression of Smad3, not Smad2 markedly increased (TGF)-beta(1)-induced alpha-SMA promoter activity and alpha-SMA protein expression in vitro, whereas the overexpression of dominant negative mutant Smad3 and Smad7 repressed (TGF)-beta(1)-induced alpha-SMA gene expression. Compared to wild-type mice, Smad3 knockout mice showed attenuated lung fibrosis after bleomycin treatment, manifested by lower collagen production and myofibroblast differentiation. Our study suggested (TGF)-beta(1)/Smad3 is a major pathway which regulated the myofibroblast differentiation. This result indicates a potential significance for future attempts of attenuating the progression of human lung fibrosis by the inhibition of the Smad3 cascade.

Differential and stage dependent effects of retinoic acid on chondrogenesis and synthesis of extracellular matrix macromolecules in chick craniofacial mesenchyme in vitro

Abstract Retinoic acid (RA) is well known to be a potent teratogen and induces a variety of facial defects in vivo, but at concentration levels lower than those that cause facial defects, RA seems to play an important role in normal facial development. In a previous study, we demonstrated the ability of RA to stimulate chondrogenesis in vitro in HH stage 23/24 chick mandibular (MND) but not frontonasal (FNP) mesenchyme cultured in a serum-free medium. The present study furthers these results by examining the effects of RA on chondrogenesis of chick facial mesenchyme at earlier embryonic stages and the effects on cell proliferation and synthesis of specific extracellular matrix macromolecules at stage 23/24. MND and FNP cells were cultured as micromasses for 4 days in defined media. As described previously, chondrogenesis in stage 23/24 MND cells was significantly enhanced by concentrations of RA of 0.1–1 ng/ml; however, at all earlier stages examined (18 to 22) RA at these concentrations had no significant effect. Higher concentrations of the retinoid inhibited chondrogenesis in MND cultures from all stages tested. Cells of the FNP from all stages displayed no significant change in chondrogenesis below 1 ng/ml RA and a dose dependent inhibition at higher concentrations. Thus RA's promotional effects in the face are not only tissue specific (MND), but also stage-dependent (HH 23/24). The specific effects of RA on matrix production and cell proliferation of stage 23/24 MND and FNP cells was examined by analysis of 35S sulfate, 3H thymidine and 3H proline incorporation. Analysis of 35S sulfate incorporation into sulfated proteoglycans confirmed that concentrations of RA of 0.1–1 ng/ml stimulated cartilage matrix production in MND but not FNP cultures. Above this level of RA, 35S sulfate incorporation was reduced in both. Likewise, 3H proline incorporation into collagenous protein, and to a lesser extent non-collagenous proteins, was stimulated by low levels of RA in MND, but not FNP cultures. Higher concentrations of the retinoid in either MND or FNP cultures did not lower collagen production, undoubtedly due to stimulation of non-chondrogenic cells within the population. This indicates that levels of RA as high as 100 ng/ml cause phenotypic change rather than cell death. This last point is corroborated by the analysis of 3H thymidine uptake in the cultures which was only transiently modified in most. The data indicate that cell proliferation occurred even in the presence of high RA levels.

Transforming growth factor-β modulates proliferation and differentiation of mouse clonal osteoblastic MC3T3-E1 cells depending on their maturation stages

The effect of transforming growth factor-β (TGF-β) on proliferation and differentiation of mouse clonal osteoblastic cells (MC3T3-E1) was examined in vitro in three different stages of their differentiation. Stage I (1–3 days after plating) was characterized by rapid cell growth, negligible alkaline phosphatase (ALP) activity and high proteoglycan synthesis, but low collagen production. In stage II (3–5 days after plating), proteoglycan synthesis sharply decreased and ALP activity and collagen synthesis began to increase. Stage III (7–9 days after plating) was characterized by maximal osteoblastic phenotypes. Treating MC3T3-E1 cells with 1 ng/ml of TGF-β greatly inhibited DNA synthesis in stage I but not in stage II. In contrast, TGF-β dose-dependently stimulated the synthesis of collagenase digestible proteins (CDP), noncollagenous proteins (NCP) and proteoglycan, especially in stage II. The minimum effective dose of TGF-β in this stage was as low as 0.04–0.2 ng/ml. In stages I and III, the MC3T3-E1 cells were rather insensitive to TGF-β in increasing three osteoblastic phenotypes. The increase in ALP activity in stages II and III was inhibited by 1 ng/ml of TGF-β. These results indicate that the response to TGF-β of mouse clonal osteoblastic MC3T3-E1 cells changes depending on their maturation stages.

Metabolism of collagen in aspartylglycosaminuria: Urinary excretion of hydroxyproline

Aspartylglycosaminuria (AGU) is a lysosomal storage disorder of glycoprotein degradation characterized by severe mental retardation and connective tissue alterations. We have previously described low collagen production in skin fibroblast cultures from AGU patients. In the present work we showed that the urinary excretion of hydroxyproline (total, non-dialysable and free hydroxyproline as indicators of collagen metabolism) was reduced in young AGU patients in comparison with age-matched controls. In adult patients no significant difference was detected. The results support the view that reduced collagen production is associated with the connective tissue abnormalities in this disorder.

Morphological, autoradiographic, immunochemical and cytochemical investigation of a cell strain with trisomy 7 from a spontaneous abortus.

A complex investigation of the phenotype of a stable strain of LHC—162 cells derived from a spontaneous abortus was carried out. The karyotype, of the strain was 47,XY,+C. The extra chromosome was identified by Giemsa staining as a No. 7. The strain was subjected to cytomorphological, autoradiographic, immunochemical and virological examination in comparison with diploid cell strains. The cells of the LHC—162 strain had no oncogenic activity. Their susceptibility to the three types of poliomyelitis virus did not differ from those of diploid strains. The growth of the LHC—162 cells was poorly organized, and they had a reduced capacity for the formation of histotypical structures. There was low collagen production, poor accumulation of lipid granules, high acid phosphatase, activity, and high glycogen content. Radioautographic investigation of cell cycle revealed lengthening of the mitotic cycle in the G2, period which was twice as long as in diploid strains. Immunochemical investigation showed that fibroblasts of both trisomic and diploid strains synthesized proteins in α1 and α2 globulin zones. However, the synthesis of a protein in α1 zone was considerably more intensive and in α2 zone less intensive in the LHC—162 cells than in the diploid strain.