41 research outputs found

    Bioactive glass/polymer composite scaffolds mimicking bone tissue

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    The aim of this work was the preparation and characterization of scaffolds with mechanical and functional properties able to regenerate bone. Porous scaffolds made of chitosan/gelatin (POL) blends containing different amounts of a bioactive glass (CEL2), as inorganic material stimulating biomineralization, were fabricated by freeze-drying. Foams with different compositions (CEL2/POL 0/100; 40/60; 70/30 wt %/wt) were prepared. Samples were crosslinked using genipin (GP) to improve mechanical strength and thermal stability. The scaffolds were characterized in terms of their stability in water, chemical structure, morphology, bioactivity, and mechanical behavior. Moreover, MG63 osteoblast-like cells and periostealderived stem cells were used to assess their biocompatibility. CEL2/POL samples showed interconnected pores having an average diameter ranging from 179 6 5 lm for CEL2/POL 0/100 to 136 6 5 lm for CEL2/POL 70/30. GP-crosslinking and the increase of CEL2 amount stabilized the composites to water solution (shown by swelling tests). In addition, the SBF soaking experiment showed a good bioactivity of the scaffold with 30 and 70 wt % CEL2. The compressive modulus increased by increasing CEL2 amount up to 2.1 6 0.1 MPa for CEL2/POL 70/30. Dynamical mechanical analysis has evidenced that composite scaffolds at low requencies showed an increase of storage and loss modulus with increasing frequency; furthermore, a drop of E0 and E00 at 1 Hz was observed, and for higher frequencies both moduli increased again. Cells displayed a good ability to interact with the different tested scaffolds which did not modify cell metabolic activity at the analyzed points. MTT test proved only a slight difference between the two cytotypes analyze

    Myoconductive and osteoinductive free-standing polysaccharide membranes

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    Free-standing (FS) membranes have increasing applications in the biomedical field as drug delivery systems for wound healing and tissue engineering. Here, we studied the potential of free-standing membranes made by the layer-by-layer assembly of chitosan and alginate to be used as a simple biomimetic system of the periosteum. The design of a periosteum-like membrane implies the elaboration of a thick membrane suitable for both muscle and bone formation. Our aim was to produce well-defined ∼50 μm thick polysaccharide membranes that could be easily manipulated, were mechanically resistant, and would enable both myogenesis and osteogenesis in vitro and in vivo. The membranes were chemically crosslinked to improve their mechanical properties. Crosslinking chemistry was followed via Fourier transform infrared spectroscopy and the mechanical properties of the membranes were assessed using dynamic mechanical analysis. The loading and release of the potent osteoinductive growth factor bone morphogenetic protein 2 (BMP-2) inside and outside of the FS membrane was followed by fluorescence spectroscopy in a physiological buffer over 1 month. The myogenic and osteogenic potentials of the membranes in vitro were assessed using BMP-2-responsive skeletal myoblasts. Finally, their osteoinductive properties in vivo were studied in a preliminary experiment using a mouse ectopic model. Our results showed that the more crosslinked FS membranes enabled a more efficient myoblast differentiation in myotubes. In addition, we showed that a tunable amount of BMP-2 can be loaded into and subsequently released from the membranes, depending on the crosslinking degree and the initial BMP-2 concentration in solution. Only the more crosslinked membranes were found to be osteoinductive in vivo. These polysaccharide-based membranes have strong potential as a periosteum-mimetic scaffold for bone tissue regeneration.This work was financially supported by the Foundation for Science and Technology (FCT) through the scholarship SFRH/BPD/96797/2013, Fundo Social Europeu (FSE), and Programa Diferencial de Potencial Human (POPH) granted to Sofia G. Caridade. C.M. is indebted to the Association Francaise contre les Myopathies for financial support via a post-doctoral fellowship (AFM project 16673). J.A. acknowledges the Whitaker International Fellows and Scholars Program for support via a post-doctoral fellowship. This work was supported by the European Commission (FP7 program) via a European Research Council starting grant (BIOMIM, GA 259370 to C.P.) and by the AFM (grant Microtiss, 16530). We thank Isabelle Paintrand for her technical help with the confocal apparatus

    Epigenetic regulation during fetal femur development: DNA methylation matters

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    Epigenetic modifications are heritable changes in gene expression without changes in DNA sequence. DNA methylation has been implicated in the control of several cellular processes including differentiation, gene regulation, development, genomic imprinting and X-chromosome inactivation. Methylated cytosine residues at CpG dinucleotides are commonly associated with gene repression; conversely, strategic loss of methylation during development could lead to activation of lineage-specific genes. Evidence is emerging that bone development and growth are programmed; although, interestingly, bone is constantly remodelled throughout life. Using human embryonic stem cells, human fetal bone cells (HFBCs), adult chondrocytes and STRO-1+ marrow stromal cells from human bone marrow, we have examined a spectrum of developmental stages of femur development and the role of DNA methylation therein. Using pyrosequencing methodology we analysed the status of methylation of genes implicated in bone biology; furthermore, we correlated these methylation levels with gene expression levels using qRT-PCR and protein distribution during fetal development evaluated using immunohistochemistry. We found that during fetal femur development DNA methylation inversely correlates with expression of genes including iNOS (NOS2) and COL9A1, but not catabolic genes including MMP13 and IL1B. Furthermore, significant demethylation was evident in the osteocalcin promoter between the fetal and adult developmental stages. Increased TET1 expression and decreased expression of DNA (cytosine-5-)-methyltransferase 1 (DNMT1) in adult chondrocytes compared to HFBCs could contribute to the loss of methylation observed during fetal development. HFBC multipotency confirms these cells to be an ideal developmental system for investigation of DNA methylation regulation. In conclusion, these findings demonstrate the role of epigenetic regulation, specifically DNA methylation, in bone development, informing and opening new possibilities in development of strategies for bone repair/tissue engineering.<br/

    Polymer-mineral scaffold augments in vivo equine multipotent stromal cell osteogenesis

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    Abstract Background Use of bioscaffolds to direct osteogenic differentiation of adult multipotent stromal cells (MSCs) without exogenous proteins is a contemporary approach to bone regeneration. Identification of in vivo osteogenic contributions of exogenous MSCs on bioscaffolds after long-term implantation is vital to understanding cell persistence and effect duration. Methods This study was designed to quantify in vivo equine MSC osteogenesis on synthetic polymer scaffolds with distinct mineral combinations 9 weeks after implantation in a murine model. Cryopreserved, passage (P)1, equine bone marrow-derived MSCs (BMSC) and adipose tissue-derived MSCs (ASC) were culture expanded to P3 and immunophenotyped with flow cytometry. They were then loaded by spinner flask on to scaffolds composed of tricalcium phosphate (TCP)/hydroxyapatite (HA) (40:60; HT), polyethylene glycol (PEG)/poly-l-lactic acid (PLLA) (60:40; GA), or PEG/PLLA/TCP/HA (36:24:24:16; GT). Scaffolds with and without cells were maintained in static culture for up to 21 days or implanted subcutaneously in athymic mice that were radiographed every 3 weeks up to 9 weeks. In vitro cell viability and proliferation were determined. Explant composition (double-stranded (ds)DNA, collagen, sulfated glycosaminoglycan (sGAG), protein), equine and murine osteogenic target gene expression, microcomputed tomography (μCT) mineralization, and light microscopic structure were assessed. Results The ASC and BMSC number increased significantly in HT constructs between 7 and 21 days of culture, and BMSCs increased similarly in GT constructs. Radiographic opacity increased with time in GT-BMSC constructs. Extracellular matrix (ECM) components and dsDNA increased significantly in GT compared to HT constructs. Equine and murine osteogenic gene expression was highest in BMSC constructs with mineral-containing scaffolds. The HT constructs with either cell type had the highest mineral deposition based on μCT. Regardless of composition, scaffolds with cells had more ECM than those without, and osteoid was apparent in all BMSC constructs. Conclusions In this study, both exogenous and host MSCs appear to contribute to in vivo osteogenesis. Addition of mineral to polymer scaffolds enhances equine MSC osteogenesis over polymer alone, but pure mineral scaffold provides superior osteogenic support. These results emphasize the need for bioscaffolds that provide customized osteogenic direction of both exo- and endogenous MSCs for the best regenerative potential
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