Mikael Ivarsson

Protein adsorption to surface chemistry and crystal structure modification of titanium surfaces.

ABSTRACT Objectives To observe the early adsorption of extracellular matrix and blood plasma proteins to magnesium-incorporated titanium oxide surfaces, which has shown superior bone response in animal models. Material and Methods Commercially pure titanium discs were blasted with titanium dioxide (TiO2) particles (control), and for the test group, TiO2 blasted discs were further processed with a micro-arc oxidation method (test). Surface morphology was investigated by scanning electron microscopy, surface topography by optic interferometry, characterization by X-ray photoelectron spectroscopy (XPS), and by X-ray diffraction (XRD) analysis. The adsorption of 3 different proteins (fibronectin, albumin, and collagen type I) was investigated by an immunoblotting technique. Results The test surface showed a porous structure, whereas the control surface showed a typical TiO2 blasted structure. XPS data revealed magnesium-incorporation to the anodic oxide film of the surface. There was no difference in surface roughness between the control and test surfaces. For the protein adsorption test, the amount of albumin was significantly higher on the control surface whereas the amount of fibronectin was significantly higher on the test surface. Although there was no significant difference, the test surface had a tendency to adsorb more collagen type I. Conclusions The magnesium-incorporated anodized surface showed significantly higher fibronectin adsorption and lower albumin adsorption than the blasted surface. These results may be one of the reasons for the excellent bone response previously observed in animal studies.

Regulation of fibroblast gene expression by keratinocytes in organotypic skin culture provides possible mechanisms for the antifibrotic effect of reepithelialization

To investigate the mechanisms behind the antifibrotic effect associated with epidermal regeneration, the expression of 12 fibroblast genes important for the modulation of the extracellular matrix (ECM), as well as α‐smooth muscle actin, was studied in a keratinocyte‐fibroblast organotypic skin culture model. The study was performed over time during epidermal generation and in the presence or absence of the profibrotic factor transforming growth factor‐β. the Presence of epidermal differentiation markers in the model was essentially coherent with that of native skin. Fibroblast gene expression was analyzed with real‐time polymerase chain reaction after removal of the epidermal layer. After 2 days of air‐exposed culture, 11 out of the 13 genes studied were significantly regulated by keratinocytes in the absence or presence of transforming growth factor‐β. The regulation of connective tissue growth factor, collagen I and III, fibronectin, plasmin system regulators, matrix metalloproteinases and their inhibitors as well as α‐smooth muscle actin was consistent with a suppression of ECM formation or contraction. Overall, the results support a view that keratinocytes regulate fibroblasts to act catabolically on the ECM in epithelialization processes. This provides possible mechanisms for the clinical observations that reepithelialization and epidermal wound coverage counteract excessive scar formation.

Inhibition of connective tissue growth factor/CCN2 expression in human dermal fibroblasts by interleukin‐1α and β

Connective tissue growth factor (CTGF/CCN2) is a matricellular protein induced by transforming growth factor (TGF)‐β and intimately involved with tissue repair and overexpressed in various fibrotic conditions. We previously showed that keratinocytes in vitro downregulate TGF‐β‐induced expression of CTGF in fibroblasts by an interleukin (IL)‐1 α‐dependent mechanism. Here, we investigated further the mechanisms of this downregulation by both IL‐1α and β. Human dermal fibroblasts and NIH 3T3 cells were treated with IL‐1α or β in presence or absence of TGF‐β1. IL‐1 suppressed basal and TGF‐β‐induced CTGF mRNA and protein expression. IL‐1α and β inhibited TGF‐β‐stimulated CTGF promoter activity, and the activity of a synthetic minimal promoter containing Smad 3‐binding CAGA elements. Furthermore, IL‐1α and β inhibited TGF‐β‐stimulated Smad 3 phosphorylation, possibly linked to an observed increase in Smad 7 mRNA expression. In addition, RNA interference suggested that TGF‐β activated kinase1 (TAK1) is necessary for IL‐1 inhibition of TGF‐β‐stimulated CTGF expression. These results add to the understanding of how the expression of CTGF in human dermal fibroblasts is regulated, which in turn may have implications for the pathogenesis of fibrotic conditions involving the skin. J. Cell. Biochem. 110: 1226–1233, 2010. Published 2010 Wiley‐Liss, Inc.

IL‐1α Counteract TGF‐β Regulated Genes and Pathways in Human Fibroblasts

Dysregulated wound healing is commonly associated with excessive fibrosis. Connective tissue growth factor (CTGF/CCN2) is characteristically overexpressed in fibrotic diseases and stimulated by transforming growth factor‐β (TGF‐β) in dermal fibroblasts. We previously showed that interleukin‐1 (IL‐1α) counteracts TGF‐β‐stimulated CTGF mRNA and protein expression in these cells. The aim of this study was to explore the effects of IL‐1α on further genes and pathways in TGF‐β regulated fibroblasts. Transcriptional microarray and multiple comparison analysis showed that the antagonizing effects of IL‐1α was much more prominent than the synergistic effects, both with respect to number of genes and extent of changes in gene expression. Moreover, comparing canonical pathways by gene set enrichment analysis and the Ingenuity Pathway Analysis tool revealed that IL‐1α counteracted TGF‐β in the top six most confident pathways regulated by both cytokines. Interferon and IL‐1 signaling, as well as two pathways involved in apoptosis signaling were suppressed by TGF‐β and activated by IL‐1α. Pathways involving actin remodeling and focal adhesion dynamics were activated by TGF‐β and suppressed by IL‐1α. Analyzing upstream regulators in part corroborate the comparison of canonical pathways and added cell cycle regulators as another functional group regulated by IL‐1α. Finally, gene set enrichment analysis of fibrosis‐related genes indicated that IL‐1 moderately counteracts the collective effect of TGF‐β on these genes. Microarray results were validated by qPCR. Taken together, the results indicate prominent antagonistic effects of IL‐1α on TGF‐β regulated interferon signaling, as well as on a wide variety of other genes and pathways in fibroblasts. J. Cell. Biochem. 117: 1622–1632, 2016.

Staphylococcus epidermidis and Staphylococcus aureus trigger different interleukin-8 and intercellular adhesion molecule-1 in lung cells: implications for inflammatory complications following neonatal sepsis

Staphylococci are a major contribution for neonatal sepsis, which is the main risk factor for bronchopulmonary dysplasia. This study investigated the expression of pro‐inflammatory mediators in endothelial and respiratory cells from newborns exposed to staphylococci.

Effects of synovial fluid from aseptic prosthesis loosening on collagen production in osteoblasts.

Synovial fluid from a loose prosthesis may act as a vehicle for factors that regulate bone turnover. The effect of such synovial fluid on osteoblasts has been studied. Synovial fluid obtained from patients who underwent revision hip arthroplasty because of aseptic prosthesis loosening was studied regarding the effect on protein synthesis, procollagen I mRNA expression, the secretion of procollagen I carboxyterminal propeptide (PICP) and osteocalcin in MG63 osteoblasts. Protein synthesis was increased and procollagen I mRNA expression was decreased by synovial fluid from patients with prosthesis loosening. Synovial fluid stimulated the total PICP in cell medium, but there was no change after correction for cell protein content in the cells. Synovial fluid in patients with prosthesis loosening has a general stimulatory effect on collagen formation and osteoblast proliferation because of a stimulatory effect on cell growth. Aseptic prosthesis loosening may be associated with an increase in bone formation.RésuméLe liquide synovial provenant de prothèses descellées peut être considéré comme le support de facteurs de la régulation du turn over osseux. Pour cela ont été étudiés les effets de ce liquide sur les ostéoblastes. Le liquide synovial obtenu de patients ayant nécessité une révision de prothèse de hanche descellée pour descellement aseptique a été utilisé afin d’évaluer les effets de ce liquide sur la synthèse des protéines, pro collagène, I mRNA expression sur la sécrétion du pro collagène I carboxyterminale propeptide et sur l’ostéocalcine dans les ostéoblastes MG63. Cette synthèse protéinique est augmentée et l’expression du pro collagène I mRNA diminue lorsque le liquide synovial de ces patients est adjoint. Le liquide synovial stimule le PICP mais il n’y a pas de modification des protéines cellulaires contenues dans les ostéoblastes. Conclusion, le fluide synovial chez ces patients présentant un descellement prothétique a un effet stimulant sur la formation du collagène et la prolifération des ostéoblastes. Le descellement aseptique des prothèses peut être associé avec une augmentation de la formation osseuse.

Interleukin-1-mediated effects of normal oral keratinocytes and head and neck squamous carcinoma cells on extracellular matrix related gene expression in fibroblasts

OBJECTIVES The composition of tumor stroma and the activity of tumor associated fibroblasts are important for tumor growth. Interactions between carcinoma cells and fibroblasts regulate the turnover of extracellular matrix (ECM). Here, the in vitro effects of oral squamous cell carcinoma (SCC) cells (UT-SCC-30 and UT-SCC-87) on fibroblast expression of genes for ECM components and connective tissue growth factor (CTGF/CCN2), were compared to those of normal oral keratinocytes (NOK). MATERIALS AND METHODS Cocultures with fibroblasts in collagen gels and keratinocytes with the two cell types separated by a semi permeable membrane were used, and relative gene expression was measured with real-time PCR. RESULTS All investigated genes were regulated by NOK and the SCCs. The downregulation of pro-collagens α1(I) and α1(III) was more pronounced in cocultures with NOK, while the expression of CCN2 and fibronectin was downregulated by both NOK and the SCCs to a similar extent. UT-SCC-87, but not UT-SCC-30, secreted significantly more IL-1α than NOK. A recombinant interleukin-1 receptor antagonist reversed many of the observed effects on fibroblast gene expression suggesting involvement of IL-1 in cocultures with NOK as well as with SCCs. CONCLUSION The observed differential effects on fibroblast gene expression suggest that NOK are more antifibrotic compared to UT-SCC-30 and UT-SCC-87. These findings may contribute to a better understanding of the mechanisms behind ECM turnover in tumors.