M.M. Beloti

Hedgehog signaling and osteoblast gene expression are regulated by purmorphamine in human mesenchymal stem cells

Several biological events are controlled by Hedgehog (Hh) signaling, including osteoblast phenotype development. This study aimed at evaluating the gene expression profile of human mesenchymal stem cells (hMSCs) treated with the Hh agonist, purmorphamine, focusing on Hh signaling and osteoblast differentiation. hMSCs from bone marrow were cultured in non‐osteogenic medium with or without purmorphamine (2u2009µM) for periods of up to 14 days. Purmorphamine up‐regulated gene expression of the mediators of Hh pathway, SMO, PTCH1, GLI1, and GLI2. The activation of Hh pathway by purmorphamine increased the expression of several genes (e.g., RUNX2 and BMPs) related to osteogenesis. Our results indicated that purmorphamine triggers Hh signaling pathway in hMSCs, inducing an increase in the expression of a set of genes involved in the osteoblast differentiation program. Thus, we conclude that Hh is a crucial pathway in the commitment of undifferentiated cells to the osteoblast lineage. J. Cell. Biochem. 113: 204–208, 2012.

Nanotopography drives stem cell fate toward osteoblast differentiation through α1β1 integrin signaling pathway.

The aim of our study was to investigate the osteoinductive potential of a titanium (Ti) surface with nanotopography, using mesenchymal stem cells (MSCs) and the mechanism involved in this phenomenon. Polished Ti discs were chemically treated with H2SO4/H2O2 to yield nanotopography and rat MSCs were cultured under osteogenic and non‐osteogenic conditions on both nanotopography and untreated polished (control) Ti surfaces. The nanotopography increased cell proliferation and alkaline phosphatase (Alp) activity and upregulated the gene expression of key bone markers of cells grown under both osteogenic and non‐osteogenic conditions. Additionally, the gene expression of α1 and β1 integrins was higher in cells grown on Ti with nanotopography under non‐osteogeneic condition compared with control Ti surface. The higher gene expression of bone markers and Alp activity induced by Ti with nanotopography was reduced by obtustatin, an α1β1 integrin inhibitor. These results indicate that α1β1 integrin signaling pathway determines the osteoinductive effect of nanotopography on MSCs. This finding highlights a novel mechanism involved in nanosurface‐mediated MSCs fate and may contribute to the development of new surface modifications aiming to accelerate and/or enhance the process of osseointegration. J. Cell. Biochem. 115: 540–548, 2014.

Effect of microcapsules containing TAK-778 on bone formation around osseointegrated implants: histomorphometric analysis in dogs.

Purpose:The aim of this study was to evaluate the in vivo effect of TAK-778 on osseointegration of titanium implants. Materials and Methods:Mandibular premolars were extracted from 8 dogs. After 3 months, 2 titanium implants were bilaterally placed, and each implantation site randomly received 1 of the following treatments: sustained-release microcapsules of TAK-778, placebo microcapsules, or no treatment. At 8 and 12 weeks after implantation, the hemi-mandibles containing the implants were removed, and processed for morphologic and histomorphometric analysis. Data were submitted to 2-way analysis of variance. Results:The histologic sections of the 3 experimental groups at 8 and 12 weeks did not show morphologic differences related to applied treatment. The percentage of bone-implant contact, mineralized bone matrix between implant threads, and mineralized bone matrix within mirror area were not affected either by treatments or evaluated periods. Conclusions:No effect of TAK-778 was observed on osseointegration of titanium implants, which most likely occurred because microcapsules may not be retained and, therefore, available at the implant sites. An alternative is the manufacture of a release system, which can be immobilized on implant surface, ensuring the drug permanence in the implant site at least at the initial periods of bone formation.

In vitro biocompatibility of poly(vinylidene fluoride–trifluoroethylene)/barium titanate composite using cultures of human periodontal ligament fibroblasts and keratinocytes

The aim of this work was to evaluate the biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane to be used in guided tissue regeneration (GTR). Fibroblasts from human periodontal ligament (hPDLF) and keratinocytes (SCC9) were plated on P(VDF-TrFE)/BT and polytetrafluorethylene membranes at a cell density of 20,000 cells well(-1) and cultured for up to 21 days. Cell morphology, adhesion and proliferation were evaluated in hPDLF and keratinocytes, while total protein content and alkaline phosphatase (ALP) activity were assayed only for hPDLF. Using a higher cell density, real-time polymerase chain reaction (PCR) was performed to assess the expression of typical genes of hPDLF, such as periostin, PDLs17, S100A4 and fibromodulin, and key phenotypic markers of keratinocytes, including involucrin, keratins 1, 10 and 14. Expression of the apoptotic genes bax, bcl-2 and survivin was evaluated for both cultures. hPDLF adhered and spread more on P(VDF-TrFE)/BT, whereas keratinocytes showed a round shape on both membranes. hPDLF adhesion was greater on P(VDF-TrFE)/BT at 2 and 4h, while keratinocyte adhesion was similar for both membranes. Whereas proliferation was significantly higher for hPDLF on P(VDF-TrFE)/BT at days 1 and 7, no signs of keratinocyte proliferation could be noticed for both membranes. Total protein content was greater on P(VDF-TrFE)/BT at 7, 14 and 21 days, and higher levels of ALP activity were observed on P(VDF-TrFE)/BT at 21 days. Real-time PCR revealed higher expression of phenotypic markers of hPDLF and keratinocytes as well as greater expression of apoptotic genes in cultures grown on P(VDF-TrFE)/BT. These results indicate that, by favoring hPDLF adhesion, spreading, proliferation and typical mRNA expression, P(VDF-TrFE)/BT membrane should be considered an advantageous alternative for GTR.

Mandibular symphysis and ramus as sources of osteoblastic cells for bone tissue engineering

OBJECTIVESnAutografts from mandibular symphysis and ramus are often used for bone reconstruction. Based on this, we hypothesized that these sites could be useful cell sources for bone tissue engineering approaches. Thus, our study aimed at evaluating the proliferation and osteoblast phenotype development of cells derived from mandibular symphysis and ramus.nnnMATERIALS AND METHODSnCells were isolated from bone fragments of four patients by enzymatic digestion and cultured under osteogenic condition for up to 17xa0days. Cultures were assayed for cell proliferation, gene expression of key bone markers runt-related transcription factor 2 (Runx2), distal-less homeobox 5 (DLX5), SATB homeobox 2 (SATB2), Osterix (OSX), family with sequence similarity 20, member C (FAM20C), bone sialoprotein (BSP), osteopontin (OPN) and osteocalcin (OC), alkaline phosphatase (ALP) expression and activity, and extracellular matrix mineralization. Data were compared by two-way ANOVA or t-test for independent samples when appropriate.nnnRESULTSnCells derived from ramus displayed lower proliferative activity and higher gene expression of Runx2, DLX5, SATB2, OSX, FAM20C, BSP, OPN and OC, ALP protein expression and activity and extracellular matrix mineralization compared with symphysis-derived cells.nnnCONCLUSIONnSymphysis and ramus may be considered as cell sources for bone tissue engineering approaches but due to the higher osteogenic potential, ramus-derived cells are more appealing for constructing cell-based biomaterials.

Effect of autogenous and fresh-frozen bone grafts on osteoblast differentiation

OBJECTIVEnFresh-frozen bone allograft (FFBA) is an alternative to autogenous bone (AB) for reconstructing maxillary bone. Despite the promising clinical results, cell responses to FFBA and AB were not evaluated. Thus, our aim was to compare cells harvested from maxillary reconstructed sites with either AB or FFBA in terms of osteoblast differentiation and to evaluate the effect of culturing cells in contact with FFBA.nnnMETHODSnCells harvested from three patients submitted to bilateral maxillary reconstruction with AB and FFBA were cultured to evaluate: proliferation, alkaline phosphatase activity, extracellular matrix mineralization and gene expression of osteoblastic markers. The effect of FFBA on osteoblast differentiation was studied by culturing cells harvested from AB in contact with FFBA and evaluating the same parameters. Data were compared using either two-way ANOVA followed by Tukey-b test or Students t test (p≤0.05).nnnRESULTSnCell proliferation was higher in cultures from AB grafted sites and extracellular matrix mineralization was higher in cultures derived from FFBA grafted sites. The gene expression of alkaline phosphatase, RUNX2, bone sialoprotein and osteocalcin was higher in cells derived from FFBA compared with cells from AB grafted sites. However, the exposure of cells derived from AB to FFBA particles did not have any remarkable effect on osteoblast differentiation.nnnCONCLUSIONSnThese results indicate the higher osteogenic activity of cells derived from FFBA compared with AB reconstructed sites, offering an explanation at cellular level of why FFBA could be a suitable alternative to AB for reconstructing maxillary bone defects.