NHOst Cells Research Articles

Mechanical properties and osteointegration of the mesh structure of a lumbar fusion cage made by 3D printing

The currently popular 3D printing makes it possible to produce spatial scaffolds, the main purpose of which is to obtain implants that have favourable mechanical properties to promote cell adhesion. This study aims to prove the influence of changes in selected geometrical parameters of scaffolds, used in intervertebral cages, on the mechanical properties obtained and thus on the osteointegration of the studied constructs with osteoblasts and fibroblasts. The stiffness values and maximum failure force of four modifications to geometric dimensions of the meshes were determined from the intendation test. Adhesion assays were conducted (including gentle pendulum motion) for Balb/3T3 fibroblasts and NHOst osteoblasts. The study revealed that an important geometrical parameter affecting the strength of the mesh is the height (h) of the connection point between arms of successive mesh cells. There was no significant effect of the mesh geometry on the abundance and survival of Balb/3T3 and NHOst cells. At the same time, fibroblasts were more likely to form colonies in the area where there is fusion of mesh cells, as opposed to osteoblasts that were more numerous at vertices of the mesh.

Hsa_circ_0006948 regulates the proliferation, migration and invasion in osteosarcoma by regulation of the expression of miR-490-3p target ATG7

Objective: To investigate the effect of hsa_circ_0006948 (circ_0006948) on the proliferation, migration and invasion of osteosarcoma cells and the underlying mechanism. Methods: A total of 120 osteosarcoma tissues and 40 adjacent normal tissue samples were collected from patients admitted to the First People's Hospital of Shangqiu City from 2009 to 2015. Microarray analysis was performed to detect the differential expressions of circRNA in Saos-2 cell. The mRNA expressions of circ_0006948, microRNA (miR)-490-3p and autophagy-related protein 7 (ATG7) in osteosarcoma cells, NHOst cells, osteosarcoma tissues and adjacent tissues were detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Cell clone formation assay was used to detect cell proliferation ability, Transwell assay was used to detect cell invasion ability, and cell scratch assay was used to detect cell migration ability. The interactions between circ_0006948 and miR-490-3p, miR-490-3p and ATG7 were detected by dual luciferase reporter gene assay. The correlation between miR-490-3p and ATG7 was analyzed by TargetScan database, and the expression levels of Bcl-2 and Bax proteins in cells were detected by western blot. Results: The mRNA expression levels of circ_0006948, miR-490-3p and ATG7 in SAOS-2 cells were significantly different from NHOst cells (P<0.01). The mRNA expression levels of circ_0006948, miR-490-3p and ATG7 in osteosarcoma tissues were significantly different from adjacent tissues (P<0.01). The numbers of cell clone, migration and mobility in circ_0006948-siRNA group were (32.78±1.76), (37.58±1.82) and (36.93±1.45)%, respectively, lower than (65.72±1.45), (78.63±1.93) and (65.32±1.74)% in the siRNA NC group (all P<0.01). The numbers of cell clone, migration and mobility in the miR-490-3p mimics group were (20.08±1.54), (30.24±1.78) and (21.15±1.68)%, respectively, lower than (60.36±1.83), (76.93±1.64) and (40.56±1.27)% in the mimics NC group (all P<0.01). The numbers of cell clone, migration and mobility in the miR-490-3p inhibitor+ siRNA NC group were (90.34±1.72), (120.89±2.34) and (70.83±1.93)%, respectively, higher than (61.27±1.73), (75.82±1.82) and (42.38±1.74)% in the inhibitor NC+ siRNA NC group (P<0.01). The numbers of cell clone, migration and mobility in the circ_0006948 siRNA+ miR-490-3p inhibitor group were (58.74±1.98), (73.46±1.04) and (40.35±1.72)%, respectively, lower than (90.34±1.72), (120.89±2.34) and (70.83±1.93)% in the miR-490-3p inhibitor+ siRNA NC group (P<0.01). The numbers of cell clone, migration and mobility in the ATG7 siRNA group were (20.56±1.87), (40.36±1.76) and (20.96±1.73)%, lower than (65.46±1.74), (90.87±2.32) and (40.87±2.03)% in the siRNA NC group (P<0.01). The absorbance of miR-490-3p mimics+ pcDNA-ATG7 group was 0.54±0.11, higher than (0.36±0.08) of miR-490-3p mimics group (P<0.05). The expression levels of Bax and Bcl-2 protein in Saos-2 cells of miR-490-3p mimics group were significantly different from mimics NC group (P<0.01). The protein expression levels of Bax and Bcl-2 in Saos-2 cells of miR-490-3p mimics + pcDNA-ATG7 group were significantly different from miR-490-3p mimics group (P<0.01). Conclusion: Circ_0006948 regulates ATG7 expression through miR-490-3p, therefore regulates the proliferation, migration and invasion of osteosarcoma cells.

Ultrafine-Grained Ti-31Mo-Type Composites with HA and Ag, Ta2O5 or CeO2 Addition for Implant Applications.

Ultrafine-grained Ti31Mo alloy and Ti31Mo5HA, Ti31Mo5HA-Ag (or Ta2O5, CeO2) composites with a grain size of approximately 2 μm were produced by the application of mechanical alloying and powder metallurgy. Additionally, the surface of the Ti31Mo alloy was modified. In the first stage, the specimens were immersed in 5M NaOH for 24 h at 60 °C. In the second stage, hydroxyapatite (HA) was deposited on the sample surface. The cathodic deposition at −5 V vs. open circuit potential (OCP) in the electrolyte containing 0.25M CaNa2-EDTA (di-calcium ethylenediaminetetraacetic acid), 0.25M K2HPO4 in 1M NaOH at 120 °C for 2 h was applied. The bulk Ti31Mo alloy is a single β-type phase. In the alkali-modified surface titanium oxide, Ti3O is formed. After hydrothermal treatment, the surface layer mostly consists of the Ca10(PO4)6(OH)2 (81.23%) with about 19% content of CaHPO4·2H2O. Using optical profiler, roughness 2D surface topography parameters were estimated. The in vitro cytocompatibility of synthesized materials was studied. The cell lines of normal human osteoblasts (NHost) and human periodontal ligament fibroblasts (HPdLF) was conducted in the presence of tested biomaterials. Ultrafine-grained Ti-based composites altered with HA and Ag, Ta2O5 or CeO2 have superior biocompatibility than the microcrystalline Ti metal. NHost and HPdLF cells in the contact with the synthesized biomaterial showed stable proliferation activity. Biocompatibility tests carried out indicate that the ultrafine-grained Ti31Mo5HA composites with Ag, Ta2O5, or CeO2 could be a good candidate for implant applications.

Cell and Tissue Response to Polyethylene Terephthalate Mesh Containing Bone Allograft in Vitro and in Vivo.

Extended polyethylene terephthalate mesh (PET, Dacron) can provide containment of compressed particulate allograft and autograft. This study assessed if PET mesh would interfere with osteoprogenitor cell migration from vertebral plates through particulate graft, and its effect on osteoblast differentiation or the quality of bone forming within fusing vertebra during vertebral interbody fusion. The impact of PET mesh on the biological response of normal human osteoblasts (NHOst cells) and bone marrow stromal cells (MSCs) to particulate bone graft was examined in vitro. Cells were cultured on rat bone particles +/- mesh; proliferation and osteoblast differentiation were assessed. The interface between the vertebral endplate, PET mesh, and newly formed bone within consolidated allograft contained by mesh was examined in a sheep model via microradiographs, histology, and mechanical testing. Growth on bone particles stimulated proliferation and early differentiation of NHOst cells and MSCs, but delayed terminal differentiation. This was not negatively impacted by mesh. New bone formation in vivo was not prevented by use of a PET mesh graft containment device. Fusion was improved in sites containing allograft/demineralized bone matrix (DBM) versus autograft and was further enhanced when stabilized using pedicle screws. Only sites treated with allograft/DBM+screws exhibited greater percent bone ingrowth versus discectomy or autograft. These results were mirrored biomechanically. PET mesh does not negatively impact cell attachment to particulate bone graft, proliferation, or initial osteoblast differentiation. The results demonstrated that bone growth occurs from vertebral endplates into graft material within the PET mesh. This was enhanced by stabilization with pedicle screws leading to greater bone ingrowth and biomechanical stability across the fusion site. The use of extended PET mesh allows containment of bone graft material during vertebral interbody fusion without inhibiting migration of osteoprogenitor cells from vertebral end plates in order to achieve fusion. 5.

Electrospun chitosan/polyethylene-oxide (PEO)/halloysites (HAL) membranes for bone regeneration applications

Fabricating biomaterials that mimic the extracellular matrix (ECM) environment of human tissue with appropriate functional, biomechanical, and biocompatible properties remains a challenge. This research aims to fabricate chitosan/polyethylene-oxide (PEO) membranes reinforced with halloysite nanotubes (HAL) as potential scaffolds for bone regeneration applications through facile electrospinning method. Morphological study through FESEM and STEM imaging demonstrated the presence of HAL along the chitosan/PEO fiber matrix with average fiber diameter ranged from 72.07 ± 3.30 nm to 154.87 ± 6.77 nm. Furthermore, the membranes incorporated with up to 5 wt% HAL revealed to have a larger pore, which was suitable for bone regeneration applications. Moreover, the mechanical tests showed that the fabricated chitosan/PEO/HAL membranes exhibited appropriate tensile strength properties to support the growth of osteoblast cells. Thermal stability analysis showed an improved behavior with the addition of HAL to the chitosan/PEO membranes. In addition, the surface wettability of the chitosan/PEO membrane reinforced with 0–5 wt% of HAL ranged between 0 to 75o, thus showed a hydrophilic nature appropriate for bone tissue regeneration. Besides, natural human osteoblast cell (NHOst) culturing demonstrated the cell adhesion and cell interaction with chitosan/PEO with 5 wt% HAL membrane through the attachment of matured NHOst and primary osteoblast cells on the membranes. Further in vitro analysis revealed that chitosan/PEO with 5 wt% HAL membranes increased the ALP activity by 2.5 times at the 14 days. In summary, the results obtained from this study demonstrated the potential of chitosan/PEO/HAL membranes for bone regeneration applications.

MiR-33a Controls hMSCS Osteoblast Commitment Modulating the Yap/Taz Expression Through EGFR Signaling Regulation.

Mesenchymal stromal cells (hMSCs) display a pleiotropic function in bone regeneration. The signaling involved in osteoblast commitment is still not completely understood, and that determines the failure of current therapies being used. In our recent studies, we identified two miRNAs as regulators of hMSCs osteoblast differentiation driving hypoxia signaling and cytoskeletal reorganization. Other signalings involved in this process are epithelial to mesenchymal transition (EMT) and epidermal growth factor receptor (EGFR) signalings through the regulation of Yes-associated protein (YAP)/PDZ-binding motif (TAZ) expression. In the current study, we investigated the role of miR-33a family as a (i) modulator of YAP/TAZ expression and (ii) a regulator of EGFR signaling during osteoblast commitments. Starting from the observation on hMSCs and primary osteoblast cell lines (Nh-Ost) in which EMT genes and miR-33a displayed a specific expression, we performed a gain and loss of function study with miR-33a-5p and 3p on hMSCs cells and Nh-Ost. After 24 h of transfections, we evaluated the modulation of EMT and osteoblast genes expression by qRT-PCR, Western blot, and Osteoimage assays. Through bioinformatic analysis, we identified YAP as the putative target of miR-33a-3p. Its role was investigated by gain and loss of function studies with miR-33a-3p on hMSCs; qRT-PCR and Western blot analyses were also carried out. Finally, the possible role of EGFR signaling in YAP/TAZ modulation by miR-33a-3p expression was evaluated. Human MSCs were treated with EGF-2 and EGFR inhibitor for different time points, and qRT-PCR and Western blot analyses were performed. The above-mentioned methods revealed a balance between miR-33a-5p and miR-33a-3p expression during hMSCs osteoblast differentiation. The human MSCs phenotype was maintained by miR-33a-5p, while the maintenance of the osteoblast phenotype in the Nh-Ost cell model was permitted by miR-33a-3p expression, which regulated YAP/TAZ through the modulation of EGFR signaling. The inhibition of EGFR blocked the effects of miR-33a-3p on YAP/TAZ modulation, favoring the maintenance of hMSCs in a committed phenotype. A new possible personalized therapeutic approach to bone regeneration was discussed, which might be mediated by customizing delivery of miR-33a in simultaneously targeting EGFR and YAP signaling with combined use of drugs.

Micromechanical Measurements and Biocompatibility of the High Porosity Ti Scaffold Made with Saccharose as a Space Holder

The paper presents mechanical properties of the porous Ti scaffold made with saccharose as a space holder. The Ti scaffolds had the porosity of 50, 60 and 70% and were made using 100 and 325 mesh Ti as a scaffold material. The pores were established using saccharose spherical space holder particles of 0.7–0.9 mm in diameter. The local mechanical properties of the Ti scaffold have been measured using nanoindenter. Several loading-unloading characteristics of the thin scaffold walls in the microscale have been measured. It has been observed that Ti scaffolds have very high mechanical properties measured in the microscale that are correlated with partial scaffold oxidation and a formation of TiO2, which took place during the production stages. The scaffold is composed mainly of Ti oxide and the structure has been identified by XRD as well as XPS. The highest mechanical properties have scaffolds composed of 100 mesh Ti particles, compared to 325 mesh Ti. The microscale tests measurements are rather slightly affected by the pore presence and are under influence of Ti particles. The MTT test shows slightly higher proliferation of NHost cells on porous Ti scaffold in comparison to the reference Ti sample.

Surface modification of bulk titanium substrates for biomedical applications via low-temperature microwave hydrothermal oxidation.

Micro-to-nanoscale surface topographies of orthopaedic and dental implants can affect fluid wetting and biological response. Nanoscale features can be superimposed on microscale roughness of titanium (Ti) surfaces at high temperatures, resulting in increased osteoblast differentiation. However, high temperatures can compromise mechanical properties of the bulk material. Here, we have developed a novel low-temperature microwave hydrothermal (MWHT) oxidation process for nanomodification of microrough (SLA) Ti surfaces. Nanoscale protuberances (20 -100 nm average diameter) were generated on SLA surfaces via MWHT treatment at 200°C in H2 O, or in aqueous solutions of H2 O2 or NH4 OH, for times ranging from 1 to 40 h. The size, shape, and crystalline content of the nanoprotuberances varied with the solution used and treatment time. The hydrophilicity of all MWHT-modified surfaces was dramatically enhanced. MG63 and normal human osteoblasts (NHOsts) were cultured on MWHT-treated SLA surfaces. While most responses to MWHT-modified surfaces were comparable to those seen on SLA controls, the MWHT-generated nanotopography reduced osteocalcin production by NHOst cells, suggesting that specific nanotopographic characteristics differentially mediate osteoblast phenotypic expression. MWHT processing provides a scalable, low-temperature route for tailoring nanoscale topographies on microroughened titanium implant surfaces with significantly enhanced wetting by water, without degrading the microscale surface structure of such implants. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 782-796, 2018.

Osteoblastic cell behaviour on modified titanium surfaces

IntroductionThe surfaces of endoosseous dental implants have been subjected to numerous modifications in order to create a surface which can provide rapid bone healing and fast implant loading. Each modification has involved changes to the chemical composition and topography of the surfaces which have resulted in various biological reactions to the implanted material. AimThe aim of this study was to evaluate the surface topography and chemistry of various modified titanium surfaces: (1) machined surface (MA), (2) alumina-blasted (Al2O3), (3) alumina-blasted and acid-etched (Al2O3 DE), (4) hydroxyapatite/tricalcium phosphate grit-blasted (HA/TCP) and (5) hydroxyapatite/tricalcium phosphate grit-blasted and acid-etched (HA/TCP DE) and to analyse the effects of surface roughness, and chemical composition on human osteoblast vitality, differentiation, morphology and orientation. Materials and methodsThe modified surfaces were subjected to topographic analysis using Scanning Electron Microscopy (SEM), optical profilometry, roughness analysis and chemical composition evaluation using Energy Dispersion Spectroscopy (EDS) analysis. The biological effects of the titanium modifications was analysed using human osteoblasts cell culture where the cell morphology, vitality (MTS assay) and differentiation (ALP activity) was analysed. ResultsThe machined surfaces were classified as anisotropic, smooth and composed of titanium and oxygen.The blasted surface samples along with the blasted and etched samples were found to be isotropic and rough. The grit-blasting procedure resulted in the incorporation of components from the blasting material. In the case of the blasted and etched samples, etching decreased the surface development as indicated by the Sdr and also reduced the amount of chemical compounds incorporated into the surfaces during the blasting procedure.The attached NHOst cells, proliferated the surfaces. With regard to the MA samples, the cells spread close to the titanium surface, with expanded cytoplasmic extensions and lamelipodia and were oriented in line with the groves left after machining. On the rough substrates, cells were less dispersed and exhibited numerous cytoplasmic extensions, filopodia and interconnections, they were not oriented with respect to the surfaces features. The cell viability of all samples except for Al2O3 decreased after the first day of culture. For all Al2O3, Al2O3 DE and HA samples the viability increased with culture time after an initial reduction.At the end of the culture period the ALP activity was slightly greater on Al2O3 and HA samples compared to the control with the HA DE sample having the same activity as the control. The Al2O3, HA and HA DE ALP samples showed comparable activity and were statistically different from MA and Al2O3 DE samples. ConclusionsIn this study, variously treated titanium surfaces were correlated with osteoblastic cell viability, morphology and differentiation in comparison with the plastic and smooth titanium. All examined surfaces were found to be biocompatible. Favourable cell reactions were observed for Al2O3 and HA blasted surfaces. The surface roughness patterns influenced the growth orientation while the surface topography influenced osteoblast morphology. Further animal studies are necessary to compare the in-vivo effect on osseointegration of these modified titanium surfaces.

Morphological and Mechanical Properties of Osteosarcoma Microenvironment Cells Explored by Atomic Force Microscopy.

Cell mechanical properties that depend on cytoskeleton architecture are critical to the mechanotransduction process, and have great potential for cancer diagnosis and therapy. In this study, the morphological and mechanical properties of typical osteosarcoma microenvironment cells, including mesenchymal stem cells (MSC), normal human osteoblast cells (NHOst) and osteosarcoma cells (MG-63), were compared using atomic force microscopy (AFM). The MG-63 cells were smaller and thicker than the MSC and NHOst cells. The membrane roughness of MG-63 cells was higher than that of MSC and NHOst cells. The MG-63 cells had lower stiffness than their normal counterparts due to their reduced organization of the cytoskeleton structure. The cell stiffness influenced the mechanotransduction. The MG-63 cells had a lower percentage of nuclear YAP/TAZ compared with the MSC and NHOst cells. The F-actin assembly was disrupted by the cytochalasin D (cyto D) treatment used to investigate its influence on mechanotransduction. Disruption of the cytoskeleton leaded to a decrease of the cell stiffness, and reduced the nuclear YAP/TAZ percentage, indicating its inhibition in the cell mechanotransduction process. This study would shed light on the development of a novel cancer diagnosis strategy and would contribute to reveal the relationship between the cytoskeleton structure and the cell mechanical properties.

Osteogenic response of human MSCs and osteoblasts to hydrophilic and hydrophobic nanostructured titanium implant surfaces.

Microstructured implant surfaces created by grit blasting and acid etching titanium (Ti) support osseointegration. This effect is further enhanced by storing in aqueous solution to retain hydrophilicity, but this also leads to surface nanostructure formation. The purpose of this study was to assess the contributions of nanostructures on the improved osteogenic response of osteoblast lineage cells to hydrophilic microstructured Ti. Human mesenchymal stem cells (MSCs) and normal human osteoblasts (NHOsts) were cultured separately on non-nanostructured/hydrophobic (SLA), nanostructured/hydrophilic (modSLA), or nanostructured/hydrophobic (SLAnano) Ti surfaces. XPS showed elevated carbon levels on SLA and SLAnano compared to modSLA. Contact angle measurements indicated only modSLA was hydrophilic. Confocal laser microscopy revealed minor differences in mean surface roughness. SEM showed the presence of nanostructures on modSLA and SLAnano. MSCs and NHOst cells exhibited similar morphology on the substrates and osteoblastic differentiation and maturation were greatest on modSLA. These results suggest that when the appropriate microstructure is present, hydrophilicity may play a greater role in stimulating MSC and NHOst osteoblastic differentiation and maturation than the presence of nanostructures generated during storage in an aqueous environment. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3137-3148, 2016.

Carbon nanotube-based coatings on titanium

This paper reports results of the modification of titanium surface with multiwalled carbon nanotubes (CNTs). The Ti samples were covered with CNTs via electrophoretic deposition (EPD) process. Prior to EPD process, CNTs were functionalized by chemical treatment. Mechanical, electrochemical and biological properties of CNT-covered Ti samples were studied and compared to those obtained for unmodified titanium surface. Atomic force microscopy was used to investigate the surface topography. To determine micromechanical characteristics of CNT-covered metallic samples indentation tests were conducted. Throughout electrochemical studies were performed in order to characterize the impact of the coating on the corrosion of titanium substrate. In vitro experiments were conducted using the human osteoblast NHOst cell line. CNT layers shielded titanium from corrosion gave the surface-enhanced biointegrative properties. Cells proliferated better on the modified surface in comparison to unmodified titanium. The deposited layer enhanced cell adhesion and spreading as compared to titanium sample.

Translationally controlled tumor protein supplemented chitosan modified glass ionomer cement promotes osteoblast proliferation and function.

The objective of this study was to evaluate the effect of translationally controlled tumor protein (TCTP) supplemented in a novel glass ionomer cement (BIO-GIC) on normal human osteoblasts (NHost cells). BIO-GIC was a glass ionomer cement (GIC) modified by adding chitosan and albumin to promote the release of TCTP. NHost cells were seeded on specimens of GIC, GIC+TCTP, BIO-GIC and BIO-GIC+TCTP. Cell proliferation was determined by BrdU assay. It was found that BIO-GIC+TCTP had significantly higher proliferation of cells than other specimens. Bone morphogenetic protein-2 (BMP-2) and osteopontin (OPN) gene expressions assessed by quantitative real time PCR and alkaline phosphatase (ALP) activity were used to determine cell differentiation. Bone cell function was investigated by calcium deposition using alizarin assay. Both BMP-2 and OPN gene expressions of cells cultured on specimens with added TCTP increased gradually up-regulation after day 1 and reached the highest on day 3 then down-regulation on day 7. The ALP activity of cells cultured on BIO-GIC+TCTP for 7 days and calcium content after 14 days were significantly higher than other groups. BIO-GIC+TCTP can promote osteoblast cells proliferation, differentiation and function.

Cytotoxicity evaluation of biodegradable Zn-3Mg alloy toward normal human osteoblast cells.

The recent proposal of using Zn-based alloys for biodegradable implants was not supported with sufficient toxicity data. This work, for the first time, presents a thorough cytotoxicity evaluation of Zn-3Mg alloy for biodegradable bone implants. Normal human osteoblast cells were exposed to the alloy's extract and three main cell-material interaction parameters: cell health, functionality and inflammatory response, were evaluated. Results showed that at the concentration of 0.75mg/ml alloy extract, cell viability was reduced by ~50% through an induction of apoptosis at day 1; however, cells were able to recover at days 3 and 7. Cytoskeletal changes were observed but without any significant DNA damage. The downregulation of alkaline phosphatase protein levels did not significantly affect the mineralization process of the cells. Significant differences of cyclooxygenase-2 and prostaglandin E2 inflammatory biomarkers were noticed, but not interleukin 1-beta, indicating that the cells underwent a healing process after exposure to the alloy. Detailed analysis on the cell-material interaction is further discussed in this paper.

Electrospun gelatin/poly(ε-caprolactone) fibrous scaffold modified with calcium phosphate for bone tissue engineering

In this study gelatin (Gel) modified with calcium phosphate nanoparticles (SG5) and polycaprolactone (PCL) were used to prepare a 3D bi-layer scaffold by collecting electrospun PCL and gelatin/SG5 fibers separately in the same collector. The objective of this study was to combine the desired properties of PCL and Gel/SG5 in the same scaffold in order to enhance mineralization, thus improving the ability of the scaffold to bond to the bone tissue. The scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and the wide angle X-ray diffraction (WAXD) measurements confirmed that SG5 nanoparticles were successfully incorporated into the fibrous gelatin matrix. The composite Gel/SG5/PCL scaffold exhibited more enhanced mechanical properties than individual Gel and Gel/SG5 scaffolds. The presence of SG5 nanoparticles accelerated the nucleation and growth of apatite crystals on the surface of the composite Gel/SG5/PCL scaffold in simulated body fluid (SBF). The osteoblast response in vitro to developed electrospun scaffolds (PCL and Gel/SG5/PCL) was investigated by using normal human primary NHOst cell lines. NHOst cell culture studies showed that higher alkaline phosphatase (ALP) activity and better mineralization were obtained in the case of composite materials than in pure PCL scaffolds. The mechanically strong PCL scaffold served as a skeleton, while the Gel/SG5 fibers facilitated cell spreading and mineralization of the scaffold.

Short-term moderate hypothermia stimulates alkaline phosphatase activity and osteocalcin expression in osteoblasts by upregulating Runx2 and osterix in vitro

Exposure of Normal Human Osteoblast cells (NHOst) to a period of hypothermia may interrupt their cellular functions, lead to changes in bone matrix and disrupt the balance between bone formation and resorption, resulting in bone loss or delayed fracture healing. To investigate this possibility, we exposed NHOst cells to moderate (35°C) and severe (27°C) hypothermia for 1, 12, 24 and 72h. The effects of hypothermia with respect to cell cytoskeleton organization, metabolic activity and the expression of cold shock chaperone proteins, osteoblast transcription factors and functional markers, were examined. Our findings showed that prolonged moderate hypothermia retained the polymerization of the cytoskeletal components. NHOst cell metabolism was affected differently according to hypothermia severity. The osteoblast transcription factors Runx2 and osterix were necessary for the transcription and translation of bone matrix proteins, where alkaline phosphatase (Alp) activity and osteocalcin (OCN) bone protein were over expressed under hypothermic conditions. Consequently, bone mineralization was stimulated after exposure to moderate hypothermia for 1 week, indicating bone function was not impaired. The cold shock chaperone protein Rbm3 was significantly upregulated (p<0.001) during the cellular stress adaption under hypothermic conditions. We suggest that Rbm3 has a dual function: one as a chaperone protein that stabilizes mRNA transcripts and a second one in enhancing the transcription of Alp and Ocn genes. Our studies demonstrated that hypothermia permitted the in vitro maturation of NHOst cells probably through an osterix-dependent pathway. For that reason, we suggest that moderate hypothermia can be clinically applied to counteract heat production at the fracture site that delays fracture healing.

Resorbable Polymer Membranes for Medical Applications

A bioresorbable polymer poly-ε-caprolactone (PCL) was tested in order to obtain porous materials suitable for membranes. The commercial PCL with various molecular weights (2kDa, 60kDa, 80 kDa) but similar polydispersity has been chosen. The membranes were produced by the casting method and the membrane materials underwent microstructure investigation (SEM) to assess the size of pores and an average porosity of the membranes. The membranes permeability was established by means of ultrafiltration. Also wettabilility and basic mechanical properties (such as: tensile strength Rm, Youngs modulus, E) were established. The membranes durability was tested in in vitro conditions (PBS/37°C) by monitoring of changes by means of ion conductivity measurement and changes in the molecular weight (the Ubbelohde method). The porous materials were tested towards biocompatibility, i.e. the membrane was contacted with the osteoblast line of NHOst cells (viability test, cells morphology). Non-perforated PCL foil was used as a reference material. The best physicochemical, mechanical and biological properties of the membranes were observed in case of application of PCL with molecular weight of 60 kDa.

A polysaccharide from Agaricus blazei inhibits proliferation and promotes apoptosis of osteosarcoma cells

Many reports have proved that traditional Chinese herbal medicines (TCM) have become popular used in disease prevention and as alternatives to cancer chemotherapy. In this study, we purified a polysaccharide (ABP-Ia) from the fruiting bodies of Agaricus blazei and identified its molecular weight to be 4.2×105Da. ABP-Ia was a heteropolysaccharide fraction consisting of glucose, mannose, and galactose in a molar ratio of 1:1:1, along with trace of rhamnose. The effect of ABP-Ia at three concentrations of 100, 200 and 400μg/mL on the cell growth and apoptosis was evaluated in osteosarcoma cell lines HOS and a normal human osteoblast cell line NHOst. ABP-Ia had a significant inhibitory effect against the growth of HOS cells, whereas a mild cytotoxicity to the HOS cells mediated by ABP-Ia was observed, which was in accordance with the results that ABP-Ia substantially induced apoptosis in a dose-dependent fashion in the HOS cells. However ABP-Ia had no or minor inhibitory and cytotoxic effects on the viability of NHOst cells even at the high concentration of 400μg/mL. Base on all the observations, we could conclude that ABP-Ia had an evident inhibitory effect on the growth of HOS cells mainly through induction of apoptosis, with a minor toxicity to normal human osteoblast cell.

Wnt Inhibitory Factor 1 Decreases Tumorigenesis and Metastasis in Osteosarcoma

It has been reported that the progression of osteosarcoma was closely associated with the aberrant activation of canonical Wnt signaling. Wnt inhibitory factor-1 (WIF-1) is a secreted Wnt inhibitor whose role in human osteosarcoma remains unknown. In this study, WIF-1 expression in NHOst and osteosarcoma cell lines was determined by real-time reverse transcription-PCR, methylation-specific PCR, and Western blotting analysis. In addition, tissue array from patient samples was examined for WIF-1 expression by immunohistochemistry. Compared with normal human osteoblasts, WIF-1 mRNA and protein levels were significantly downregulated in several osteosarcoma cell lines. The downregulation of WIF-1 mRNA expression is associated with its promoter hypermethylation in these tested cell lines. Importantly, WIF-1 expression was also downregulated in 76% of examined osteosarcoma cases. These results suggest that the downregulation of WIF-1 expression plays a role in osteosarcoma progression. To further study the potential tumor suppressor function of WIF-1 in osteosarcoma, we established stable 143B cell lines overexpressing WIF-1. WIF-1 overexpression significantly decreased tumor growth rate in nude mice as examined by the s.c. injection of 143B cells stably transfected with WIF-1 and vector control. WIF-1 overexpression also markedly reduced the number of lung metastasis in vivo in an orthotopic mouse model of osteosarcoma. Together, these data suggest that WIF-1 exerts potent antiosteosarcoma effect in vivo in mouse models. Therefore, the reexpression of WIF-1 in WIF-1-deficient osteosarcoma represents a potential novel treatment and preventive strategy.

Osteoprotegerin (OPG) Production by Cells in the Osteoblast Lineage is Regulated by Pulsed Electromagnetic Fields in Cultures Grown on Calcium Phosphate Substrates

Pulsed electromagnetic fields (PEMF) used clinically to stimulate bone formation enhance the osteogenic effects of BMP-2 on human mesenchymal stem cells (MSCs) if the MSCs are grown in osteogenic medium and are cultured on calcium phosphate (CaP) surfaces rather than tissue culture polystyrene plastic (TCPS). This study tested if PEMF's effects on cells in the osteoblast lineage are substrate dependent and if factors produced by osteoblasts that regulate osteoclastic bone resorption, might also be regulated by PEMF. Human MSCs treated with BMP-2 and human osteoblast-like cells (normal human osteoblasts [NHOst cells], MG63 cells, SaOS-2 cells) were cultured on CaP or TCPS and their response to PEMF (4.5 ms bursts of 20 pulses repeating at 15 Hz for 8 h/day) determined as a function of decoy receptor osteoprotegerin (OPG) and RANK ligand (RANKL) production, both of which are associated with regulation of osteoclast differentiation. The results showed that when osteoblast-like cells were cultured on CaP, PEMF decreased cell number and increased production of paracrine factors associated with reduced bone resorption like OPG. RANKL was unaffected, indicating that the OPG/RANKL ratio was increased, further supporting a surface-dependent osteogenic effect of PEMF. Moreover, effects of estrogen were surface dependent and enhanced by PEMF, demonstrating that PEMF can modulate osteogenic responses to anabolic regulators of osteoblast function. These effects of PEMF would not be evident in models examining cells in traditional culture on plastic.