In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors

Lima S.A.F., Wodewotzky T.I., Lima-Neto J.F., Beltrao-Braga P.C.B. & Alvarenga F.C.L. 2012. [In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors.] Diferenciacao in vitro de celulas-tronco mesenquimais da medula ossea de caes em precursores osteogenicos. Pesquisa Veterinaria Brasileira 32(5):463-469. Departamento de Reproducao Animal e Radiologia Veterinaria, Faculdade de Medicina Veterinaria e Zootecnia, Universidade Estadual Paulista, Campus de Botucatu, Distrito de Rubiao Junior s/n, Botucatu, SP 18618-970, Brazil. E-mail: silviavet@usp.br The aim of our research was to evaluate the potential for osteogenic differentiation of mesenchimal stem cells (MSC) obtained from dog bone marrow. The MSC were separated using the Ficoll method and cultured under two different conditions: DMEM low glucose or DMEM/F12, both containing L-glutamine, 20% of FBS and antibiotics. MSC markers were tested, confirming CD44+ and CD34- cells with flow cytometry. For osteogenic differentiation, cells were submitted to four different conditions: Group 1, same conditions used for primary cell culture with DMEM supplemented media; Group 2, same conditions of Group 1 plus differentiation inductors Dexametazone, ascorbic acid and beta-glicerolphosphate. Group 3, Cells cultured with supplemented DMEM/F12 media, and Group 4, same conditions as in Group 3 plus differentiation inductors Dexametazone, ascorbic acid and beta-glicerolphosphate. The cellular differentiation was confirmed using alizarin red and imunostaining with SP7/Osterix antibody. We observed by alizarin staining that calcium deposit was more evident in cells cultivated in DMEM/F12. Furthermore, by SP/7Osterix antibody immunostaining we obtained 1:6 positive cells when using DMEM/F12 compared with 1:12 for low-glucose DMEM. Based on our results, we conclude that the medium DMEM/F12 is more efficient for induction of differentiation of mesenchymal stem cells in canine osteogenic progenitors. This effect is probably due to the greater amount of glucose in the medium and the presence of various amino acids.

Ciprofloxacin has antifibrotic effects in scleroderma fibroblasts via downregulation of Dnmt1 and upregulation of Fli1

Systemic sclerosis (SSc) is characterized by fibrosis of the skin and internal organs. The present study was undertaken to examine the effects of ciprofloxacin, a fluoroquinolone antibiotic implicated in matrix remodeling, on dermal and lung fibroblasts obtained from SSc patients. Dermal and lung fibroblasts from SSc patients and healthy subjects were treated with ciprofloxacin. Western blotting was used to analyze protein levels and RT-PCR was used to measure in RNA expression. The pharmacologic inhibitor UO126 was used to block Erk1/2 signaling. SSc dermal fibroblasts demonstrated a significant decrease in collagen type I mRNA and protein levels after antibiotic treatment, while healthy dermal fibroblasts were less sensitive to ciprofloxacin, downregulating collagen only at the protein levels. Connective tissue growth factor (CCN2) gene expression was significantly reduced and matrix metalloproteinase (MMPI) levels were enhanced after ciprofloxacin treatment to a similar extent in healthy and SSc fibroblasts. Ciprofloxacin induced Erk1/2 phosphorylation, and Erk1/2 blockade completely prevented MMP1 upregulation. However. Smad1 and Smad3 activation in response to TGF beta was not affected. The expression of friend leukemia integration factor 1 (Fli1). a transcriptional repressor of collagen, was increased after treatment with ciprofloxacin only in SSc fibroblasts, and this was accompanied by a decrease in the levels of DNA methyltransferase 1 (Dnmt1). Similar effects were observed in SSc-interstitial lung disease (ILD) lung fibroblasts. In summary, our study demonstrates that ciprofloxacin has antifibrotic actions in SSc dermal and lung fibroblasts via the downregulation of Dnmt1, the upregulation of Fli1 and induction of MMPI gene expression via an Erk1/2-dependent mechanism. Thus, our data suggest that ciprofloxacin may he an attractive therapy for SSc skin and lung fibrosis.

Construção de substituto da pele composto por matriz de colágeno porcino povoada por fibroblastos dérmicos e queratinócitos humanos: avaliação histológica

INTRODUÇÃO: O uso de enxertos autólogos é limitado pela extensão da área doadora e pelo estado clínico dos pacientes, no caso de lesões extensas. Alotransplantes coletados de cadáveres ou voluntários são rejeitados após uma ou duas semanas, servindo apenas como cobertura temporária para essas lesões. O tratamento de grandes lesões cutâneas com tegumento autólogo reconstruído constitui alternativa atraente, já que, a partir de um pequeno fragmento de pele do paciente, pode-se obter culturas de células que se multiplicam rapidamente e podem ser criopreservadas, permitindo, assim, sua utilização em novos tratamentos por tempo indeterminado. Este estudo pretendeu avaliar o comportamento histológico de queratinócitos e fibroblastos humanos cultivados sobre uma matriz de colágeno porcino derivada da submucosa intestinal. MÉTODO: Células da epiderme e derme humana foram cultivadas separadamente e semeadas sobre matriz de colágeno porcino, onde permaneceram em ambiente controlado por 21 dias, antes de serem submetidas a análise histológica. RESULTADOS: Observou-se que os fibroblastos invadem e colonizam a matriz de colágeno, enquanto os queratinócitos se organizam de forma laminar e estratificada sobre a superfície em que foram semeados. CONCLUSÕES: A utilização da matriz de colágeno porcino como carreador de células da pele humana é possível e a organização dessas células se assemelha à arquitetura da pele humana.

Influence of a hypercholesterolemic diet on the collagen composition of the bladder wall extracellular matrix in rats

Purpose: To investigate the effects of hypercholesterolemic diet on the collagen composition of urinary bladder wall. Materials and methods: Forty-five female 4-week-old Wistar rats were divided into three groups: 1) control group fed a normal diet (ND); 2) model of bladder outlet obstruction (BOO) group fed a ND; and 3) group fed a HCD (1.25% cholesterol). Total serum cholesterol, LDL cholesterol and body weight were assessed at baseline. Four weeks later, group 2 underwent a surgical procedure resulting in a partial BOO, while groups 1 and 3 underwent a sham similar surgical procedure. Six weeks later, all animals had their bladders removed; serum cholesterol and LDL cholesterol levels and body weights were measured. Morphological and morphometric analysis was performed by Picrosirius staining and collagen types I and III were identified by immunofluorescence. Statistical analysis was completed and significance was considered when p<0.05. Results: Rats fed an HCD exhibited a significant increase in LDL cholesterol levels (p<0.001) and body weight (p=0.017), when compared to the groups fed a ND during the ten-week study period. Moreover, the HCD induced morphological alterations of the bladder wall collagen, regarding thin collagen fibers and the amounts of type III collagen when compared to the control group (p=0.002 and p=0.016, respectively), resembling the process promoted in the BOO model. Conclusions: A hyper-cholesterolemic diet in Wistar rats promoted morphological changes of the bladder types of collagen, as well as increases in body weight and LDL cholesterol.

Effect of dental tissue conditioners and matrix metalloproteinase inhibitors on type I collagen microstructure analyzed by Fourier transform infrared spectroscopy

This study aimed to evaluate the chemical interaction of collagen with some substances usually applied in dental treatments to increase the durability of adhesive restorations to dentin. Initially, the similarity between human dentin collagen and type I collagen obtained from commercial bovine membranes of Achilles deep tendon was compared by the Attenuated Total Reflectance technique of Fourier Transform Infrared (ATR-FTIR) spectroscopy. Finally, the effects of application of 35% phosphoric acid, 0.1M ethylenediaminetetraacetic acid (EDTA), 2% chlorhexidine, and 6.5% proanthocyanidin solution on microstructure of collagen and in the integrity of its triple helix were also evaluated by ATR-FTIR. It was observed that the commercial type I collagen can be used as an efficient substitute for demineralized human dentin in studies that use spectroscopy analysis. The 35% phosphoric acid significantly altered the organic content of amides, proline and hydroxyproline of type I collagen. The surface treatment with 0.1M EDTA, 2% chlorhexidine, or 6.5% proanthocyanidin did not promote deleterious structural changes to the collagen triple helix. The application of 6.5% proanthocyanidin on collagen promoted hydrogen bond formation. (c) 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.

Extracellular Matrix Composition of the Cricopharyngeus Muscle

The aim of this study was to analyze the presence and distribution of total collagen, type I and type III collagen, elastic fibers, fibronectin, and versican in the endomysium of cricopharyngeus muscles from adults of various ages. The study was a cross-sectional analysis of human cricopharyngeus muscles. Twenty-seven muscles obtained from autopsies of men and women ranging in age from 28 to 92 years were analyzed with the Picrosirius method, oxidized Weigert resorcin-fuchsin, immunohistochemistry, and image analysis. Collagen had the highest density among the analyzed components. Elastic fibers surrounded each muscle cell; they were aligned longitudinally by their long axis and associated with traversing fibers, thereby forming a fiber network with embedded muscle cells. The fibronectin and versican contents varied widely among the specimens. We found no statistically significant differences between the proportion of extracellular matrix (ECM) components and factors such as gender and race. We conclude that the higher proportion of type I and type III collagen is compatible with the cricopharyngeus muscle's sphincteric behavior, and the arrangement of the elastic fibers may also contribute to the muscle's elasticity. We found no statistically significant correlation between the ECM components and age.

Nitric oxide modulates metalloproteinase-2, collagen deposition and adhesion rate after polypropylene mesh implantation in the intra-abdominal wall

Prosthetic meshes are commonly used to correct abdominal wall defects. However, the inflammatory reaction induced by these devices in the peritoneum is not completely understood. We hypothesized that nitric oxide (NO), produced by nitric oxide synthase 2 (NOS2) may modulate the response induced by mesh implants in the abdominal wall and, consequently, affect the outcome of the surgical procedure. Polypropylene meshes were implanted in the peritoneal side of the abdominal wall in wild-type and NOS2-deficient (NOS2(-/-)) mice. After 15 days tissues around the mesh implant were collected, and inflammatory markers (the cytokine interleukin 1 beta (IL-1 beta) and NO) and tissue remodeling (collagen and metalloproteinases (MMP) 2 and 9) were analyzed. The lack of NOS2-derived NO induced a higher incidence of visceral adhesions at the mesh implantation site compared with wild-type mice that underwent the same procedure (P < 0.05). Additionally, higher levels of IL-1 beta were present in the mesh-implanted NOS2(-/-) animals compared with control and wild-type mice. Mesh implantation induced collagen I and III deposition, but in smaller amounts in NOS2(-/-) mice. MMP-9 activity after the surgical procedure was similarly increased in both groups. Conversely, MMP-2 activity was unchanged in mesh-implanted wild-type mice, but was significantly increased in NOS2(-/-) mice (P < 0.01), due to decreased S-nitrosylation of the enzyme in these animals. We conclude that NOS2-derived NO is crucial for an adequate response to and integration of polypropylene mesh implants in the peritoneum. NO deficiency results in a prolonged inflammatory reaction to the mesh implant, and reduced collagen deposition may contribute to an increased incidence of visceral adhesions. (C) 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

QUANTIFICATION OF LEFT VENTRICULAR MYOCARDIAL COLLAGEN SYSTEM IN CHILDREN, YOUNG ADULTS, AND THE ELDERLY

Studies on the collagen system of the human myocardium are still limited compared to those on small laboratory animals. The aim of this work was to observe the collagen tissue of the myocardium of the human heart as a function of age. The types of collagen, as well as the density of collagen tissue and the diameter of collagen fibrils, were examined. Fragments of the left ventricular wall from 15 hearts, 5 from children, 5 from young adults, and 5 from elderly individuals, were analyzed by using the Picrosirius-polarization method and by transmission electron microscopy (TEM). The results showed the presence of collagen type III and collagen type I, both in the endomysium and perimysium of the 3 groups studied. Measurements of collagen content in myocardial tissue displayed that both endomysial and perimysial collagen increase in number and thickness in the adult and elderly. These histochemical results coincided with the observations obtained with the electron microscope in showing an increase in the number of collagen fibrils with a large diameter in the adult and elderly hearts. The present results on cardiac collagen may be important for assessing the pathogenesis of several cardiopathies in the hearts of children, young adults, and the elderly.

Extracellular matrix in airway smooth muscle is associated with dynamics of airway function in asthma

Background: Altered deposition of extracellular matrix (ECM) in the airway smooth muscle (ASM) layer as observed in asthma may influence ASM mechanical properties. We hypothesized that ECM in ASM is associated with airway function in asthma. First, we investigated the difference in ECM expression in ASM between asthma and controls. Second, we examined whether ECM expression is associated with bronchoconstriction and bronchodilation in vivo. Methods: Our cross-sectional study comprised 19 atopic mild asthma patients, 15 atopic and 12 nonatopic healthy subjects. Spirometry, methacholine responsiveness, deep-breath-induced bronchodilation (Delta R-rs) and bronchoscopy with endobronchial biopsies were performed. Positive staining of elastin, collagen I, III and IV, decorin, versican, fibronectin, laminin and tenascin in ASM was quantified as fractional area and mean density. Data were analysed using Pearson's or Spearman's correlation coefficient. Results: Extracellular matrix expression in ASM was not different between asthma and controls. In asthmatics, fractional area and mean density of collagen I and III were correlated with methacholine dose-response slope and DRrs, respectively (r = 0.71, P < 0.01; r = 0.60, P = 0.02). Furthermore, ASM collagen III and laminin in asthma were correlated with FEV1 reversibility (r = -0.65, P = 0.01; r = -0.54, P = 0.04). Conclusion: In asthma, ECM in ASM is related to the dynamics of airway function in the absence of differences in ECM expression between asthma and controls. This indicates that the ASM layer in its full composition is a major structural component in determining variable airways obstruction in asthma.

Time course involvement of matrix metalloproteinases in the vascular alterations of renovascular hypertension

Increased vascular matrix metalloproteinases (MMPs) levels play a role in late phases of hypertensive vascular remodeling. However, no previous study has examined the time course of MMPs in the various phases of two-kidney, one-clip hypertension (2K1C). We examined structural vascular changes, collagen and elastin content, vascular oxidative stress, and MMPs levels/activities during the development of 2K1C hypertension. Plasma angiotensin converting enzyme (ACE) activity was measured to assess renin-angiotensin system activation. Sham or 2K1C hypertensive rats were studied after 2, 4, 6, and 10 weeks of hypertension. Systolic blood pressure (SBP) was monitored weekly. Morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin, orcein and picrosirius red sections. Aortic NADPH activity and superoxide production was evaluated. Aortic gelatinolytic activity was determined by in situ zymography, and MMP-2, MMP-14, and tissue inhibitor of MMPs (TIMP)-2 levels were determined by gelatin zymography, immunofluorescence and immunohistochemistry. 2K1C hypertension was associated with increased ACE activity, which decreased to normal after 10 weeks. We found increased aortic collagen and elastin content in the early phase of hypertension, which were associated with vascular hypertrophy, increased vascular MMP-2 and MMP-14 (but not TIMP-2) levels, and increased gelatinolytic activity, possibly as a result of increased vascular NADPH oxidase activity and oxidative stress. These results indicate that vascular remodeling of renovascular hypertension is an early process associated with early increases in MMPs activities, enhanced matrix deposition and oxidative stress. Using antioxidants or MMPs inhibitors in the early phase of hypertension may prevent the vascular alterations of hypertension. (C) 2012 Elsevier B.V. All rights reserved.

Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation

Possa SS, Charafeddine HT, Righetti RF, da Silva PA, Almeida-Reis R, Saraiva-Romanholo BM, Perini A, Prado CM, Leick-Maldonado EA, Martins MA, Tiberio ID. Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation. Am J Physiol Lung Cell Mol Physiol 303: L939-L952, 2012. First published September 21, 2012; doi:10.1152/ajplung.00034.2012.-Several studies have demonstrated the importance of Rho-kinase in the modulation of smooth muscle contraction, airway hyperresponsiveness, and inflammation. However, the effects of repeated treatment with a specific inhibitor of this pathway have not been previously investigated. We evaluated the effects of repeated treatment with Y-27632, a highly selective Rho-kinase inhibitor, on airway hyperresponsiveness, oxidative stress activation, extracellular matrix remodeling, eosinophilic inflammation, and cytokine expression in an animal model of chronic airway inflammation. Guinea pigs were subjected to seven ovalbumin or saline exposures. The treatment with Y-27632 (1 mM) started at the fifth inhalation. Seventy-two hours after the seventh inhalation, the animals' pulmonary mechanics were evaluated, and exhaled nitric oxide (E-NO) was collected. The lungs were removed, and histological analysis was performed using morphometry. Treatment with Y-27632 in sensitized animals reduced E-NO concentrations, maximal responses of resistance, elastance of the respiratory system, eosinophil counts, collagen and elastic fiber contents, the numbers of cells positive for IL-2, IL-4, IL-5, IL-13, inducible nitric oxide synthase, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, transforming growth factor-beta, NF-kappa B, IFN-gamma, and 8-iso-prostaglandin F2 alpha contents compared with the untreated group (P < 0.05). We observed positive correlations among the functional responses and inflammation, remodeling, and oxidative stress pathway activation markers evaluated. In conclusion, Rho-kinase pathway activation contributes to the potentiation of the hyperresponsiveness, inflammation, the extracellular matrix remodeling process, and oxidative stress activation. These results suggest that Rho-kinase inhibitors represent potential pharmacological tools for the control of asthma.

Immunophenotyping and extracellular matrix remodeling in pulmonary and extrapulmonary sarcoidosis

Objective: To investigate the significance of cellular immune markers, as well as that of collagen and elastic components of the extracellular matrix, within granulomatous structures in biopsies of patients with pulmonary or extrapulmonary sarcoidosis. Methods: We carried out qualitative and quantitative evaluations of inflammatory cells, collagen fibers, and elastic fibers in granulomatous structures in surgical biopsies of 40 patients with pulmonary and extrapulmonary sarcoidosis using histomorphometry, immunohistochemistry, picrosirius red staining, and Weigert's resorcin-fuchsin staining. Results: The extrapulmonary tissue biopsies presented significantly higher densities of lymphocytes, macrophages, and neutrophils than did the lung tissue biopsies. Pulmonary granulomas showed a significantly higher number of collagen fibers and a lower density of elastic fibers than did extrapulmonary granulomas. The amount of macrophages in the lung samples correlated with FVC (p < 0.05), whereas the amount of CD3+, CD4+, and CD8+ lymphocytes correlated with the FEV1/FVC ratio and VC. There were inverse correlations between TLC and the CD1a+ cell count (p < 0.05), as well as between DLCO and collagen/elastic fiber density (r = -0.90; p = 0.04). Conclusions: Immunophenotyping and remodeling both showed differences between pulmonary and extrapulmonary sarcoidosis in terms of the characteristics of the biopsy samples. These differences correlated with the clinical and spirometric data obtained for the patients, suggesting that two different pathways are involved in the mechanism of antigen clearance, which was more effective in the lungs and lymph nodes.

A bayesian network meta-analysis on comparisons of enamel matrix derivatives, guided tissue regeneration and their combination therapies

Aims: Guided tissue regeneration (GTR) and enamel matrix derivatives (EMD) are two popular regenerative treatments for periodontal infrabony lesions. Both have been used in conjunction with other regenerative materials. We conducted a Bayesian network meta-analysis of randomized controlled trials on treatment effects of GTR, EMD and their combination therapies. Material and Methods: A systematic literature search was conducted using the Medline, EMBASE, LILACS and CENTRAL databases up to and including June 2011. Treatment outcomes were changes in probing pocket depth (PPD), clinical attachment level (CAL) and infrabony defect depth. Different types of bone grafts were treated as one group and so were barrier membranes. Results: A total of 53 studies were included in this review, and we found small differences between regenerative therapies which were non-significant statistically and clinically. GTR and GTR-related combination therapies achieved greater PPD reduction than EMD and EMD-related combination therapies. Combination therapies achieved slightly greater CAL gain than the use of EMD or GTR alone. GTR with BG achieved greatest defect fill. Conclusion: Combination therapies performed better than single therapies, but the additional benefits were small. Bayesian network meta-analysis is a promising technique to compare multiple treatments. Further analysis of methodological characteristics will be required prior to clinical recommendations.

Hybrid Scaffolds Built From PET and Collagen as a Model For Vascular Graft Architecture

A hybrid material with excellent mechanical and biological properties is produced by electrospinning a co-solution of PET and collagen. The fibers are mapped using SEM, confocal Raman microscopy and collagenase digestion assays. Fibers of different compositions and morphologies are intermingled within the same membrane, resulting in a heterogeneous scaffold. The collagen distribution and exposure are found to depend on the PET/collagen ratio. The materials are chemically and mechanically characterized and biologically tested with fibroblasts (3T3-L1) and a HUVEC culture in vitro. All of the hybrid scaffolds show better cell attachment and proliferation than PET. These materials are potential candidates to be used as vascular grafts.

Calcium Binding to Leptospira Outer Membrane Antigen LipL32 Is Not Necessary for Its Interaction with Plasma Fibronectin, Collagen Type IV, and Plasminogen

LipL32 is the most abundant outer membrane protein from pathogenic Leptospira and has been shown to bind extracellular matrix (ECM) proteins as well as Ca2+. Recent crystal structures have been obtained for the protein in the apo-and Ca2+-bound forms. In this work, we produced three LipL32 mutants (D163-168A, Q67A, and S247A) and evaluated their ability to interact with Ca2+ and with ECM glycoproteins and human plasminogen. The D163-168A mutant modifies aspartate residues involved in Ca2+ binding, whereas the other two modify residues in a cavity on the other side of the protein structure. Loss of calcium binding in the D163-D168A mutant was confirmed using intrinsic tryptophan fluorescence, circular dichroism, and thermal denaturation whereas the Q67A and S247A mutants presented the same Ca2+ affinity as the wild-type protein. We then evaluated if Ca2+ binding to LipL32 would be crucial for its interaction with collagen type IV and plasma proteins fibronectin and plasminogen. Surprisingly, the wild-type protein and all three mutants, including the D163-168A variant, bound to these ECM proteins with very similar affinities, both in the presence and absence of Ca2+ ions. In conclusion, calcium binding to LipL32 may be important to stabilize the protein, but is not necessary to mediate interaction with host extracellular matrix proteins.