Noggin promotes osteogenesis in human adipose-derived mesenchymal stem cells via FGFR2/Src/Akt and ERK signaling pathway.

The balance between Noggin and bone morphogenetic proteins (BMPs) is important during early development and skeletal regenerative therapies. Noggin binds BMPs in the extracellular space, thereby preventing BMP signaling. However, Noggin may affect cell response not necessarily through the modulation of BMP signaling, raising the possibility of direct Noggin signaling through yet unspecified receptors. Here we show that in osteogenic cultures of adipose-derived stem cells (ASCs), Noggin activates fibroblast growth factor receptors (FGFRs), Src/Akt and ERK kinases, and it stabilizes TAZ proteins in the presence of dexamethasone. Overall, this leads ASCs to increased expression of osteogenic markers and robust mineral deposition. Our results also indicate that Noggin can induce osteogenic genes expression in normal human bone marrow stem cells and alkaline phosphatase activity in normal human dental pulp stem cells. Besides, Noggin can specifically activate FGFR2 in osteosarcoma cells. We believe our findings open new research avenues to further explore the involvement of Noggin in cell fate modulation by FGFR2/Src/Akt/ERK signaling and potential applications of Noggin in bone regenerative therapies.

A Biological Study of Composites Based on the Blends of Nanohydroxyapatite, Silk Fibroin and Chitosan.

In this work, the biological properties of three-dimensional scaffolds based on a blend of nanohydroxyapatite (nHA), silk fibroin (SF), and chitosan (CTS), were prepared using a lyophilization technique with various weight ratios: 10:45:45, 15:15:70, 15:70:15, 20:40:40, 40:30:30, and 70:15:15 nHA:SF:CTS, respectively. The basic 3D scaffolds were obtained from 5% (w/w) chitosan and 5% silk fibroin solutions and then nHA was added. The morphology and physicochemical properties of scaffolds were studied and compared. A biological test was performed to study the growth and osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). It was found that the addition of chitosan increases the resistance properties and extends the degradation time of materials. In vitro studies with human mesenchymal stem cells found a high degree of biotolerance for the materials produced, especially for the 20:40:40 and 15:70:15 (nHa:SF:CTS) ratios. The presence of silk fibroin and the elongated shape of the pores positively influenced the differentiation of cells into osteogenic cells. By taking advantage of the differentiation/proliferation cues offered by individual components, the composites based on the nanohydroxyapatite, silk fibroin, and chitosan scaffold may be suitable for bone tissue engineering, and possibly offer an alternative to the widespread use of collagen materials.

Characterization of Collagen/Beta Glucan Hydrogels Crosslinked with Tannic Acid.

Hydrogels based on collagen/ß-glucan crosslinked with tannic acid were obtained by neutralization using dialysis. The presence of tannic acid allowed obtaining stable hydrogel materials with better mechanical properties. Tannic acid was released from matrices gradually and not rapidly. The antioxidant properties of the obtained hydrogels increased over the course of their incubation in culture media and were dependent on the concentration of tannic acid in the matrices. The obtained materials influenced dehydrogenase activity and the ATP level of pathogens. Additionally, the materials' extracts improved the HaCaT cells' viability. Therefore, the obtained hydrogels seem to be promising biocompatible materials which display antimicrobial properties.

The Preparation and Characterization of Chitosan-Based Hydrogels Cross-Linked by Glyoxal.

In this study, hydrogels based on chitosan cross-linked by glyoxal have been investigated for potential medical applications. Hydrogels were loaded with tannic acid at different concentrations. The thermal stability and the polyphenol-releasing rate were determined. For a preliminary assessment of the clinical usefulness of the hydrogels, they were examined for blood compatibility and in the culture of human dental pulp cells (hDPC). The results showed that after immersion in a polyphenol solution, chitosan/glyoxal hydrogels remain nonhemolytic for erythrocytes, and we also did not observe the cytotoxic effect of hydrogels immersed in tannic acid (TA) solutions with different concentration. Tannic acid was successfully released from hydrogels, and its addition improved material thermal stability. Thus, the current findings open the possibility to consider such hydrogels in clinics.

Preparation and Characterization of Fish Skin Collagen Material Modified with ß-Glucan as Potential Wound Dressing.

Collagen possesses unique properties, e.g., biocompatibility, biodegradability, and non-toxicity. However, collagen material degrades too quickly and has low mechanical properties. One of the methods of polymers' modification is mixing them to obtain blends. In this study, the influence of ß-glucan for collagen material was analyzed. The interaction between the functional groups of the polymer was analyzed by ATR-FTIR (attenuated total reflection-fourier transform infrared) spectroscopy. The influence of ß-glucan on mechanical properties was evaluated. The surface properties of materials were assessed using contact angle measurements and the topography of materials was evaluated by AFM (atomic force microscope). The structure of materials was analyzed according to SEM (scanning electron microscopy) pictures. Moreover, the DPPH-free radicals' scavenging ability and biocompatibility against erythrocytes and HaCaT cells were evaluated. Collagen and ß-glucan were bound together by a hydrogen bond. ß-glucan addition increased the roughness of the surface of the film and resulted in a more rigid character of the materials. A small addition of ß-glucan to collagen provided a more hydrophilic character. All the materials could swell in in vitro conditions and showed antioxidant activity. Materials do not cause erythrocyte hemolysis. Finely, our cytotoxicity studies indicated that ß-glucan can be safely added at small (10% or less) quantity to collagen matrix, they sufficiently support cell growth, and the degradation products of such matrices may actually provide some beneficial effects to the surrounding cells/tissues.

How Surface Properties of Silica Nanoparticles Influence Structural, Microstructural and Biological Properties of Polymer Nanocomposites.

The aim of this work was to study effect of the type of silica nanoparticles on the properties of nanocomposites for application in the guided bone regeneration (GBR). Two types of nanometric silica particles with different size, morphology and specific surface area (SSA) i.e., high specific surface silica (hss-SiO2) and low specific surface silica (lss-SiO2), were used as nano-fillers for a resorbable polymer matrix: poly(L-lactide-co-D,L-lactide), called PLDLA. It was shown that higher surface specific area and morphology (including pore size distribution) recorded for hss-SiO2 influences chemical activity of the nanoparticle; in addition, hydroxyl groups appeared on the surface. The nanoparticle with 10 times lower specific surface area (lss-SiO2) characterized lower chemical action. In addition, a lack of hydroxyl groups on the surface obstructed apatite nucleation (reduced zeta potential in comparison to hss-SiO2), where an apatite layer appeared already after 48 h of incubation in the simulated body fluid (SBF), and no significant changes in crystallinity of PLDLA/lss-SiO2 nanocomposite material in comparison to neat PLDLA foil were observed. The presence and type of inorganic particles in the PLDLA matrix influenced various physicochemical properties such as the wettability, and the roughness parameter note for PLDLA/lss-SiO2 increased. The results of biological investigation show that the bioactive nanocomposites with hss-SiO2 may stimulate osteoblast and fibroblast cells'proliferation and secretion of collagen type I. Additionally, both nanocomposites with the nanometric silica inducted differentiation of mesenchymal cells into osteoblasts at a proliferation stage in in vitro conditions. A higher concentration of alkaline phosphatase (ALP) was observed on the material modified with hss-SiO2 silica.

In vitro cytotoxicity of biodegradable Zn-Mg surgical wires in tumor and healthy cells.

In this work, we examined the in vitro cytotoxicity of new biodegradable surgical wires. The wires made of zinc with the addition of a small amount of magnesium (pure zinc, ZnMg 0.0026, ZnMg 0.0068, and ZnMg 0.08) have been investigated. The wires were produced using a technology based on extrusion and subsequent drawing. The resulting wires with a diameter of 0.8-1.0 mm are designed to be used in surgical operations related to bone joints. For cytotoxicity studies, we have selected human dental pulp stem cells (hDPSC) as the cell population representing normal osteoprogenitor cells. Considering that, after bone surgeries, the chance of osteosarcoma increases, we have compared the results obtained in hDPSC to those obtained with Saos-2 human osteosarcoma cell line. Cultured cells were exposed to the extracts obtained from the materials incubated in culture medium for 24 h with and without preincubation. Extracts of different ratios were examined. The results showed that the extracts obtained from wires made of ZnMg 0.0026 alloy exhibit high toxicity to Saos-2 osteosarcoma cells and low toxicity to hDPSC cells. This was in contrast to all reference materials, i.e., commercial surgical sutures made of steel and polymers, that did not display cytotoxicity toward osteosarcoma cells. Thus, the detected phenomenon for the ZnMg 0.0026 alloy can become the basis for creating biodegradable Zn-Mg surgical wires with antitumor activity.

Novel Eco-Friendly Tannic Acid-Enriched Hydrogels-Preparation and Characterization for Biomedical Application.

Sodium alginate and tannic acid are natural compounds that can be mixed with each other. In this study, we propose novel eco-friendly hydrogels for biomedical applications. Thus, we conducted the following assessments including (i) observation of the structure of hydrogels by scanning electron microscope; (ii) bioerosion and the concentration of released tannic acid from subjected material; (iii) dehydrogenase activity assay to determine antibacterial activity of prepared hydrogels; and (iv) blood and cell compatibility. The results showed that hydrogels based on sodium alginate/tannic acid exert a porous structure. The immersion in simulated body fluid (SBF) results in the biomineralization process occurring on their surface while the bioerosion studies revealed that the addition of tannic acid improves hydrogels' stability proportional to its concentration. Besides, tannic acid release concentration depends on the type of hydrogels and the highest amount was noticed for those based on sodium alginate with the content of 30% tannic acid. Antibacterial activity of hydrogels was proven for both Gram-negative and Gram-positive bacteria, the hemolysis rate was below 5% and the viability of the cells was elevated with an increasing amount of tannic acid in hydrogels. Collectively, we assume that obtained materials make the imperative to consider them for biomedical applications.

The role of CaO/SiO2 ratio and P2O5 content in gel-derived bioactive glass-polymer composites in the modulation of their bioactivity and osteoinductivity in human BMSCs.

We obtained a range of PLGA-based composites containing sol-gel bioactive glasses (SBG) from the SiO2-CaO and SiO2-CaO-P2O5 systems. Eight SBGs with different CaO/SiO2 ratios with and without P2O5 were incorporated at 50% w/w to PLGA matrix and structured into thin films suitable for cell culture. The SBG/PLGA composites were examined for their bioactivity in simulated body fluid (SBF), ion release profile in culture media with and without cells, and osteoinductivity in standard human bone marrow stromal cell (hBMSC) cultures without osteogenic growth factors. Our results indicate different surface activity of composites depending on the presence/absence of P2O5 in SBG composition. Furthermore, ion release profile to culture medium differed depending on the presence/absence of cells. Direct culture of hBMSC on the SiO2-CaO/PLGA composite films resulted in elevated Runx-2 mRNA, opposite to low Runx-2 mRNA levels on SiO2-CaO-P2O5/PLGA films. All studied composites increased Osx mRNA levels. Whereas some of SiO2-CaO/PLGA composites did not elevate BMP-2 and -6 proteins in hBMSC cultures, high levels of these BMPs were present in all cultures on SiO2-CaO-P2O5/PLGA composites. All composites induced BMP-related Tak1 signalling, whereas Smad1 signalling was restricted mostly to composites containing three-component SBGs. ALP activity of hBMSC and BMP-related luciferase activity of mouse BRITE cells differed depending on whether the cells were stimulated with culture medium conditioned with SBG/PLGA composites or the cells were directly cultured on the composite surfaces. Altogether, beyond bioactivity and osteoinductivity of SBG/PLGA composites, our studies show key differences in the biological response to both the bioactive material dissolution products and upon direct cell-material contacts.

Antibacterial Activity and Cytocompatibility of Bone Cement Enriched with Antibiotic, Nanosilver, and Nanocopper for Bone Regeneration.

Bacterial infections due to bone replacement surgeries require modifications of bone cement with antibacterial components. This study aimed to investigate whether the incorporation of gentamicin or nanometals into bone cement may reduce and to what extent bacterial growth without the loss of overall cytocompatibility and adverse effects in vitro. The bone cement Cemex was used as the base material, modified either with gentamicin sulfate or nanometals: Silver or copper. The inhibition of bacterial adhesion and growth was examined against five different bacterial strains along with integrity of erythrocytes, viability of blood platelets, and dental pulp stem cells. Bone cement modified with nanoAg or nanoCu revealed greater bactericidal effects and prevented the biofilm formation better compared to antibiotic-loaded bone cement. The cement containing nanoAg displayed good cytocompatibility without noticeable hemolysis of erythrocytes or blood platelet disfunction and good viability of dental pulp stem cells (DPSC). On the contrary, the nanoCu cement enhanced hemolysis of erythrocytes, reduced the platelets aggregation, and decreased DPSC viability. Based on these studies, we suggest the modification of bone cement with nanoAg may be a good strategy to provide improved implant fixative for bone regeneration purposes.

New sol-gel bioactive glass and titania composites with enhanced physico-chemical and biological properties.

We developed TiO2 matrix composites modified by sol-gel bioactive glasses (SBG) of either high CaO content (A2) or high SiO2 content (S2). The latter were mixed with titanium dioxide (TiO2) at 75:25, 50:50, and 25:75 weight ratios and sintered at 1250°C for 2 h. We examined the effects of various types (A2 or S2) and compositional TiO2 :SBG ratios on the mechanical properties of resulting composites, their bioactivity and human bone marrow mesenchymal stem cells (MSC) response. The chemistry of SBGs influenced the phase composition, mechanical and biological properties of the composites. Rutile and titanite prevailed in A2-TiO2 composites, and rutile and crystobalite in S2-TiO2 composites. Compressive strength increased significantly for 25A2-TiO2 composites (140 MPa) compared to matrix TiO2 (58 MPa). Composites containing 50-75 wt % of either SBG displayed bioactive properties as determined by simulated body fluid test. Compared to TiO2, human bone marrow stromal cell (BMSC) viability was enhanced on the composites containing 25 wt % of either SBG, whereas the composites modified by 25 wt % of S2 enhanced alkaline phosphatase activity and mineralization in cultures treated with osteogenic inducers-dexamethasone (Dex) or bone morphogenetic protein. Increasing amounts of A2 in TiO2 matrix decreased cell viability but increased collagen deposition and mineralized matrix production by BMSC. Considering the physico-chemical and biological properties of the presented composites, the modification of TiO2 with SBG may prove useful strategy in several bone tissue related regeneration strategies.

The effect of acrylamide and nitric oxide donors on human mesenchymal progenitor cells.

We have examined the effects of nitric oxide donors and acrylamide on mesenchymal progenitor cell (hMPC) viability, programmed cell death (PCD) and differentiation. Acrylamide was examined at 0.5mM and 1.5mM concentrations, NOC-18 at 10µM and SNP at 100µM. Cell viability was assayed with MTS, PCD was determined by phosphatidylserine, caspase-9 and -3/7 and mitochondrial membrane potential assays, and osteogenic cell differentiation was evaluated by alkaline phosphatase activity (ALP) and mRNA levels for collagen type I, bone sialoprotein, ostepontin and osteocalcin. Serum-free hMPC cultures treated with 1.5mM acrylamide and SNP for 72h demonstrated reduced viability. PCD analyses revealed that SNP stimulated cells to necrosis in reactive species-dependent manner. Acrylamide (1.5mM) led to apoptosis independent of reactive species. Acrylamide and SNP reduced ALP activity and collagen type I mRNA levels but mRNA levels for bone sialoprotein and osteopontin increased in SNP treated cells and remained unchanged in acrylamide. Acrylamide had no effect on guanylate cyclase and cGMP osteogenic signaling pathway. The study suggests that acrylamide might impair bone development and remodeling upon acute or prolonged intoxication with this compound of mesenchymal cells.

Age and skeletal sites affect BMP-2 responsiveness of human bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) contain osteoprogenitors responsive to stimulation by osteogenic growth factors like bone morphogenetic proteins (BMPs). When used as grafts, BMSCs can be harvested from different skeletal sites such as axial, appendicular, and orofacial bones, but the lower therapeutic efficacy of BMPs on BMSCs-responsiveness in humans compared to animal models may be due partly to effects of skeletal site and age of donor. We previously reported superior differentiation capacity and osteogenic properties of orofacial BMSCs relative to iliac crest BMSCs in same individuals. This study tested the hypothesis that recombinant human BMP-2 (rhBMP-2) stimulates human BMSCs differently based on age and skeletal site of harvest. Adult maxilla, mandible, and iliac crest BMSCs from same individuals and pediatric iliac crest BMSCs were comparatively assessed for BMP-2 responsiveness under serum-containing and serum-free insulin-supplemented culture conditions. Adult orofacial BMSCs were more BMP-2-responsive than iliac crest BMSCs based on higher gene transcripts of alkaline phosphatase, osteopontin, and osteogenic transcription factors MSX-2 and Osterix in serum-free insulin-containing medium. Pediatric iliac crest BMSCs were more responsive to rhBMP-2 than adult iliac crest BMSCs based on higher expression of alkaline phosphatase and osteopontin in serum-containing medium. Unlike orofacial BMSCs, MSX-2 and Osterix transcripts were similarly expressed by adult and pediatric iliac crest BMSCs in response to rhBMP-2. These data demonstrate that age and skeletal site-specific differences exist in BMSC osteogenic responsiveness to BMP-2 stimulation and suggest that MSX-2 and Osterix may be potential regulatory transcription factors in BMP-mediated osteogenesis of adult orofacial cells.