Neonatal Rat Calvarial Osteoblasts Research Articles

Preparation of composite calcium phosphate cement scaffold loaded with Hedysarum polysaccharides and its efficacy in repairing bone defects

It’s imperative to create a more ideal biological scaffold for bone defect repair. Calcium phosphate bone cements (CPC) could be used as a scaffold. Some ingredients and osteogenic factors could be added to improve its poor mechanical properties and biological activity. As a macromolecule extracted from traditional Chinese medicine, Hedysarum polysaccharides (HPS) would significantly promote the osteogenic activity of bone biomaterials. Zirconium oxide and starch were added to the solid phase and citric acid was added to the liquid phase to optimize CPC. HPS was loaded onto the scaffold as an osteogenic factor, and the prepared CPS + HPS was characterized. Further, the cytocompatibility of CPS + HPS was assessed according to activity, differentiation, and calcification in neonatal rat calvarial osteoblasts, and the biosafety of CPS + HPS was evaluated according to acute toxicity, pyrogen, sensitization, and hemolysis. The success of CPS + HPS in repairing bone defects was evaluated by using a rabbit femur implantation experiment. After optimization, CPS-20-CA-5 containing 10% starch and 5% citric acid displayed the highest mechanical strength of 28.96 ± 0.03 MPa. HPS-50 was demonstrated to exert the best osteogenic effect. The combination of CPS + HPS achieved HPS-loaded CPC. Material characterization, cytocompatibility, biosafety, and femoral implantation experiments indicated that CPS + HPS possessed better pressure resistance and improved osteogenic ability in bone defect repair.CPS + HPS demonstrated effective pressure resistance and superior osteogenic ability, which may be of great significance for bone defects and bone tissue engineering to promote bone regeneration and repair.Graphical The composite CPS loaded with HPS-50 is an excellent scaffold with fine load-bearing ability and sustained release of osteogenic factors to repair bone defects.

Di 2-ethyl hexyl phthalate affects differentiation and matrix mineralization of rat calvarial osteoblasts –in vitro

Di-2-ethyl hexyl phthalate (DEHP), an industrial plasticizer and a ubiquitous environmental contaminant, is an established endocrine disruptor (ED). Increasing evidences indicate that some EDs interfere with osteoblast differentiation and function. In the present study, we investigated the effects of DEHP on the expression of cell cycle proteins, differentiation markers, Runx2 and its co-activator TAZ in osteoblasts derived from neonatal rat calvaria. A significant decrease in protein levels of cyclin D1 and CDK-2 was found at high dosage of DEHP (100μM) after 24h treatment. DEHP treatment caused a significant decrease in ALP mRNA. While DEHP treatment significantly decreased the TAZ at mRNA and protein levels, it decreased only the Runx2protein levels. Histochemical localization of ALP, collagen and mineralized nodules studied from cells treated with DEHP (10 and 100μM) for 21days revealed a drastic decrease in collagen, ALP and mineralization. In conclusion, DEHP affected differentiation of neonatal rat calvarial osteoblasts and mineralization of matrix secreted by these cells.

Effects of exogenous phosphorus and silicon on osteoblast differentiation at the interface with bioactive ceramics

In this study, we have investigated the effects of dissolved phosphorus and silicon on osteoblast differentiation in vitro. Neonatal rat calvarial osteoblasts were seeded on silica-calcium phosphate composites (SCPCS), hydroxyapatite (HA-200), and tissue culture polystyrene (TCPS) and incubated over 4 days in media containing 0 {minimal essential medium [MEM] (-)} or 3 mM β-glycerophosphate [MEM (+)]. Inductively coupled plasma analysis showed that P-content in original MEM (+) was 225% higher than that in MEM (-). Moreover, P-content in MEM (+) significantly increased to 3.4-4.4 mM and 3.6-4.7 mM after 2 and 4 days incubation with SCPC, respectively, owing to material dissolution and exogenous phosphate supplementation. In contrast, P-content in MEM (+) showed no change upon incubation with HA or TCPS. The P-content in MEM (-) incubated with SCPC was considerably lower than that in MEM (+). SCPC exhibited controlled Si-release in cell culture media [MEM (-) or MEM (+)], with Si-rich SCPC showing a significantly greater dissolution than Si-poor SCPC. Moreover, SCPC, unlike HA, demonstrated a cell- and solution-mediated dissolution over 4 days. Quantitative real-time PCR showed that in MEM (-), osteocalcin and osteopontin mRNA expression on Si-rich SCPC was significantly greater than that on HA, suggesting that Si plays an important role in enhancing bone-cell differentiation. However, osteoblast phenotypic expression on SCPC was significantly decreased after 4 days incubation in MEM (+), indicating that sustained exposure to elevated P-levels in the media can downregulate osteoblast function. Our results demonstrate that the controlled dissolution of SCPC provides a natural stimulus for bone-cell differentiation in vitro and could obviate the need of exogenous phosphate supplementation.

Porous biodegradable polyurethane nanocomposites: preparation, characterization, and biocompatibility tests

A porous biodegradable polyurethane nanocomposite based on poly(caprolactone) (PCL) and nanocomponents derived from montmorillonite (Cloisite®30B) was synthesized and tested to produce information regarding its potential use as a scaffold for tissue engineering. Structural and morphological characteristics of this nanocomposite were studied by infrared spectroscopy (FTIR), X-ray diffraction (XRD), small angle X-ray scattering (SAXS) and scanning electron microscopy (SEM). The reaction between polyurethane oligomers with isocyanate endcapped chains and water led to the evolution of CO2, which was responsible for building interconnected pores with sizes ranging from 184 to 387 μm. An in vitro cell-nanocomposite interaction study was carried out using neonatal rat calvarial osteoblasts. The ability of cells to proliferate and produce an extracellular matrix in contact with the synthesized material was assessed by an MTT assay, a collagen synthesis analysis, and the expression of alkaline phosphatase. In vivo experiments were performed by subcutaneously implanting samples in the dorsum of rats. The implants were removed after 14, 21, and 29 days, and were analyzed by SEM and optical microscopy after tissue processing. Histology crosssections and SEM analyses showed that the cells were able to penetrate into the material and to attach to many location throughout the pore structure. In vitro and in vivo tests demonstrated the feasibility for polyurethane nanocomposites to be used as artificial extracellular matrices onto which cells can attach, grow, and form new tissues.

Enhancement of osteoblast gene expression by mechanically compatible porous Si‐rich nanocomposite

Synthesis of a porous bioactive ceramic implant for load bearing applications is a challenging task in maxillofacial and orthopedic surgeries. A novel bioactive resorbable silica-calcium phosphate nanocomposite (SCPC) has recently been introduced as a potential bone graft. In the present study, we employed SCPC to develop a resorbable porous scaffold and analyzed the effects of composition and porosity on the mechanical properties. The ranges of compressive strength and modulus of elasticity of SCPC containing 32-56% porosity were 1.5-50 MPa and 0.14-2.1 GPa, respectively, which matched the corresponding values for trabecular bone. The compressive strength of dense SCPC was dependent on the Si content and acquired values (93-285 MPa) comparable to that of cortical bone. The superior mechanical properties of SCPC are attributed to the intricate interactions at the boundaries of the nanograins and to the homogenous distribution of hierarchical pore-structure throughout the material volume. X-ray computed tomography and mercury porosimetry analyses revealed high interconnectivity of the pores in the size range 3 nm to 650 microm. Quantitative real-time PCR analyses showed that neonatal rat calvarial osteoblasts attached to Si-rich SCPC expressed 5- and 26-fold higher osteocalcin mRNA levels compared to cells attached to ProOsteon hydroxyapatite disks and tissue culture polystyrene plates respectively, after four days in culture. Results of the present study strongly suggest that porous, bioactive resorbable SCPCs can serve as tissue engineering scaffolds for cell delivery to treat load-bearing bone defects in orthopedic and maxillofacial surgeries.

Nitric oxide protects osteoblasts from oxidative stress‐induced apoptotic insults via a mitochondria‐dependent mechanism

Nitric oxide (NO) contributes to the regulation of osteoblast activities. In this study, we evaluated the protective effects of NO pretreatment on oxidative stress-induced osteoblast apoptosis and its possible mechanism using neonatal rat calvarial osteoblasts as the experimental model. Exposure of osteoblasts to sodium nitroprusside (SNP) at a low concentration of 0.3 mM significantly increased cellular NO levels without affecting cell viability. However, when the concentration reached a high concentration of 2 mM, SNP increased the levels of intracellular reactive oxygen species and induced osteoblast injuries. Thus, administration of 0.3 and 2 mM SNP in osteoblasts were respectively used as sources of NO and oxidative stress. Pretreatment with NO for 24 h significantly ameliorated the oxidative stress-caused morphological alterations and decreases in alkaline phosphatase activity, and reduced cell death. Oxidative stress induced osteoblast death via an apoptotic mechanism, but NO pretreatment protected osteoblasts against the toxic effects. The mitochondrial membrane potential was significantly reduced following exposure to the oxidative stress. However, pretreatment with NO significantly lowered the suppressive effects. Oxidative stress increased cellular Bax protein production and cytochrome c release from mitochondria. Pretreatment with NO significantly decreased oxidative stress-caused augmentation of Bax and cytochrome c protein levels. In parallel with cytochrome c release, oxidative stress induced caspase-3 activation and DNA fragmentation. Pretreatment with NO significantly reduced the oxidative stress-enhanced caspase-3 activation and DNA damage. Results of this study show that NO pretreatment can protect osteoblasts from oxidative stress-induced apoptotic insults. The protective action involves a mitochondria-dependent mechanism.

2,6‐Diisopropylphenol Protects Osteoblasts from Oxidative Stress‐Induced Apoptosis through Suppression of Caspase‐3 Activation

2,6-Diisopropylphenol is an intravenous anesthetic agent used for induction and maintenance of anesthesia. Since it is similar to alpha-tocopherol, 2,6-diisopropylphenol may have antioxidant effects. Osteoblasts play important roles in bone remodeling. In this study, we attempted to evaluate the protective effects of 2,6-diisopropylphenol on oxidative stress-induced osteoblast insults and their possible mechanisms, using neonatal rat calvarial osteoblasts as the experimental model. Clinically relevant concentrations of 2,6-diisopropylphenol (3 and 30 microM) had no effect on osteoblast viability. However, 2,6-diisopropylphenol at 300 microM time-dependently caused osteoblast death. Exposure to sodium nitroprusside (SNP), a nitric oxide donor, increased amounts of nitrite in osteoblasts. 2,6-Diisopropylphenol did not scavenge basal or SNP-releasing nitric oxide. Hydrogen peroxide (HP) enhanced levels of intracellular reactive oxygen species in osteoblasts. 2,6-Diisopropylphenol significantly reduced HP-induced oxidative stress. Exposure of osteoblasts to SNP and HP decreased cell viability time-dependently. 2,6-Diisopropylphenol protected osteoblasts from SNP- and HP-induced cell damage. Analysis by a flow cytometric method revealed that SNP and HP induced osteoblast apoptosis. 2,6-Diisopropylphenol significantly blocked SNP- and HP-induced osteoblast apoptosis. Administration of SNP and HP increased caspase-3 activities. However, 2,6-diisopropylphenol significantly decreased SNP- and HP-enhanced caspase-3 activities. This study shows that a therapeutic concentration of 2,6-diisopropylphenol can protect osteoblasts from SNP- and HP-induced cell insults, possibly via suppression of caspase-3 activities.

Nitric Oxide Induces Osteoblast Apoptosis through a Mitochondria‐Dependent Pathway

Osteoblasts contribute to bone remodeling. Nitric oxide can regulate osteoblast activities. In this study, we attempted to evaluate the pathophysiological effects of nitric oxide on osteoblasts and its possible mechanism using neonatal rat calvarial osteoblasts as the experimental model. Exposure of osteoblasts to sodium nitroprusside, a nitric oxide donor, decreased alkaline phosphatase activities and cell viability in a concentration- and time-dependent manner. Apoptotic analysis revealed that sodium nitroprusside time-dependently increased the percentages of osteoblasts undergoing apoptosis. Administration of sodium nitroprusside reduced the mitochondrial membrane potential of osteoblasts. In parallel with the mitochondrial dysfunction, levels of intracellular reactive oxygen species and cytochrome c were significantly elevated following sodium nitroprusside administration. Exposure of osteoblasts to sodium nitroprusside significantly increased caspase-3 activity. Results of this study show that nitric oxide, decomposed from sodium nitroprusside, can induce osteoblast apoptosis through a mitochondrion-dependent cascade that causes mitochondrial dysfunction, release of intracellular reactive oxygen species and cytochrome c from mitochondria to cytoplasm, and activation of caspase-3.

Molecular mechanism of nitric oxide-induced osteoblast apoptosis

Nitric oxide (NO) can regulate osteoblast activities. Our previous study showed that NO induced osteoblast apoptosis [Chen RM, Liu HC, Lin YL, Jean WC, Chen JS, Wang JH. Nitric oxide induces osteoblast apoptosis through the de novo synthesis of Bax protein. J Orthop Res 2002;20:295–302]. This study was further aimed to evaluate the mechanism of NO-induced osteoblast apoptosis from the viewpoints of mitochondrial functions, intracellular oxidative stress, and the anti-apoptotic Bcl-2 protein using neonatal rat calvarial osteoblasts as the experimental model. Exposure of osteoblasts to sodium nitroprusside (SNP), an NO donor, significantly increased amounts of lactate dehydrogenase in the culture medium, and decreased cell viability in concentration- and time-dependent manners. Administration of SNP in osteoblasts time-dependently led to DNA fragmentation. The mitochondrial membrane potential was significantly reduced following SNP administration. SNP decreased complex I NADH dehydrogenase activity in a time-dependent manner. Levels of cellular adenosine triphosphate (ATP) were suppressed by SNP. In parallel with the mitochondrial dysfunction, SNP time-dependently increased levels of intracellular reactive oxygen species. Immunoblotting analysis revealed that SNP reduced Bcl-2 protein levels. Exposure to lipopolysaccharide (LPS) and IFN-γ significant increased endogenous nitrite production. In parallel with the increase in endogenous NO, administration of LPS and IFN-γ suppressed cell viability, mitochondrial membrane potential, and ATP synthesis. Results of this study show that NO released from SNP can induce osteoblast insults and apoptosis, and the mechanism may involve the modulation of mitochondrial functions, intracellular reactive oxygen species, and Bcl-2 protein.

Attachment and proliferation of neonatal rat calvarial osteoblasts on Ti6Al4V: effect of surface chemistries of the alloy

This study examined the cell attachment and proliferation of neonatal rat calvarial osteoblasts on Ti6Al4V alloy as affected by the surface modifications. The modifications could alter simultaneously the surface chemistries of the alloy (elemental difference of Ti, Al, V, Cu and Ni about 300–600 μm thick examined by EDS) as well as the XPS nano-surface characteristics of oxides on the metal surface (chemistries of oxides, amphoteric OH group adsorbed on oxides, and oxide thickness). Three materials including two from modifications and a control were examined. It is argued that a slight change of the nano-surface characteristics of oxides as a result of the modifications neither alters the in vitro capability of Ca and P ion adsorption nor affects the metal ion dissolution behavior of the alloy. This implies that any influence on the cytocompatibility of the materials should only be correlated to the effect of surface chemistries of the alloy and the associated metal ion dissolution behavior of the alloy. The experimental results suggest that the cell response of neonatal rat calvarial osteoblasts on the Ti6Al4V alloy should neither be affected by the variation of surface chemistries of the alloy in a range studied.

Factors in the fracture microenvironment induce primary osteoblast angiogenic cytokine production.

Neoangiogenesis is essential for successful wound repair. Platelets are among the earliest cells recruited to a site of skeletal injury and are thought to provide numerous factors critical to successful repair. The release of platelet-derived growth factor (PDGF) after skeletal injury increases osteoblast proliferation, chemotaxis, and collagen synthesis; however, its angiogenic effect on osteoblast biology remains unknown. The purpose of this study was to investigate the effect of recombinant human (rh)PDGF-BB on the synthesis of vascular endothelial growth factor (VEGF) by primary neonatal rat calvarial osteoblasts. Furthermore, the authors investigated whether PDGF works in concert with hypoxia, another component of the fracture microenvironment, to additively or synergistically induce VEGF production. Osteoblast cultures were stimulated with varying concentrations of rhPDGF-BB (1, 10, 50, and 100 ng/ml) in normoxic and hypoxic (<1% oxygen) conditions for 0, 3, 6, 12, and 24 hours, and VEGF gene expression was analyzed by Northern blot analysis. To determine whether rhPDGF-BB-induced VEGF messenger RNA (mRNA) expression was transcriptionally mediated or required de novo protein synthesis, transcription, and translation, studies were performed using actinomycin D and cycloheximide, respectively. Treatment with 50 ng/ml rhPDGF-BB resulted in a 2.4-fold increase in VEGF mRNA expression after 3 hours. Interestingly, rhPDGF-BB and hypoxia seemed to have an additive effect, resulting in a 3.7-fold increase in VEGF mRNA expression after 6 hours in primary neonatal rat calvarial osteoblasts. Furthermore, by using actinomycin D and cycloheximide, the authors demonstrated that the rhPDGF-BB-induced VEGF mRNA expression was transcriptionally mediate and not dependent on de novo protein synthesis. These data demonstrate that rhPDGF-BB transcriptionally increases osteoblasts VEGF mRNA expression in vitro. Furthermore, the semiquantitative results suggest that rhPDGF-BB and hypoxia act additively to increase VEGF mRNA expression. It is postulated that similar mechanisms may occur in vivo, at a site of skeletal injury, to induce neoangiogenesis and promote fracture repair.

Nitric oxide induces osteoblast apoptosis through the de novo synthesis of Bax protein

Nitric oxide (NO) plays a crucial role in the physiological and pathophysiological regulations of osteoblast functions. This study is designed to evaluate the toxic effects of NO released by sodium nitroprusside (SNP), an NO donor, on neonatal Wistar rat calvarial osteoblasts from the analyses of cell viability, alkaline phosphatase (ALP) activity, cell morphology, apoptotic cells, terminal deoxynucleotidyl transferase-mediated dUTP nick end-label (TUNEL) assay, DNA ladder, and immunocytochemistry and Western blot for proapoptotic Bax protein. SNP increased the levels of nitrite, an oxidative product of NO, in the culture medium of osteoblasts in concentration- and time-dependent manners, and altered cell morphologies to round and shrinkage shapes. Administration of osteoblasts with SNP resulted in concentration- and time-dependent decreases of cell viability and ALP activity. Analysis of apoptotic cells revealed that SNP increased the percentages of osteoblasts processing apoptosis. Analyses of TUNEL and DNA ladder showed that SNP caused DNA fragmentation. Pretreatment with cycloheximide, an inhibitor of protein synthesis, partially blocked SNP-induced osteoblast apoptosis. Imunocytochemical and immunoblotting analyses revealed that SNP increased Bax protein in osteoblasts. This study suggests that SNP could increase the levels of NO in osteoblasts, and cause osteoblast apoptosis possibly through the de novo synthesis of proapoptotic Bax protein.

Biological responses of neonatal rat calvarial osteoblasts on plasma-sprayed HA/ZrO2 composite coating.

Plasma-sprayed hydroxyapatite (HA) coating, applied to metal substrates, can induce a direct chemical bond with bone and hence achieve a biological fixation of the implant. However, the poor bonding strength between the HA coating and the substrate has been a concern for the orthopedists. In a previous study, the zirconia-reinforced hydroxyapatite composite coatings (HA/ZrO(2)) could significantly improve the mechanical strength before and after soaking in simulated body fluid. This study aims to investigate the biological responses of osteoblasts on plasma-sprayed HA/ZrO(2) coating. The osteoblasts derived from neonatal rat calvarial were cultured in Dulbecco's modified Eagle medium (DMEM) with fetal bovine serum (FBS) on the surface of plasma-sprayed HA coating, HA/ZrO(2) coating, and ZrO(2) coating, respectively. The biological responses were investigated by the cell growth (1, 3, 5, and 10 days) and the cell morphology under scanning electron microscopy (SEM) (3, 6, 12, 24 and 48 h). Examination by SEM revealed that osteoblasts on HA coatings exhibit less spreading during the medium phase (6 and 12 h), while, better morphologies were observed at the latter phases (24 and 48 h). This should be derived by the dissolution of HA coating in the culture medium. On HA/ZrO(2) coating, the cells showed the poor morphologies at the latter phases (24 and 48 h). This could be explained by the no apatite formed at the surface HA/ZrO(2) coating after soaking in simulated body fluid. The lower contents of ZrO(2) coating in HA coating and the addition of other solid solution (ZrO(2)-MgO, CaO-ZrO(2), ZrO(2)-CeO(2)) in HA coating are the two possible methods to improve the cytocompatibility of HA/ZrO(2) coating.

The cell attachment and morphology of neonatal rat calvarial osteoblasts on the surface of Ti-6Al-4V and plasma-sprayed HA coating: effect of surface roughness and serum contents.

The biocompatibility of material plays an important role in the bone-implant interface for the prosthetic implant fixation. The biocompatibility of implants is associated with the chemical composition, surface topography, surface energy and surface roughness of biomaterials. The effects of two factors, surface roughness and serum contents, on osteoblast behavior at the surface of Ti-6Al-4V and plasma sprayed HA coating were investigated in the experiment. The osteoblasts derived from neonatal rat calvarial were cultured in Dulbecco's modified Eagle medium (DMEM) with fetal bovine serum (FBS) on the surface of polished Ti-6Al-4V (Ti-p), grit-blasted Ti-6Al-4V (Ti-b), polished HA coating (HAC-p), and as-sprayed HA coating (HAC). Under culture medium containing 4% FBS, the level of cell attachment to the polished surface is significantly higher than the rough surface of the same experimental materials during all culture periods. Increasing the contents of FBS up to 10%, the difference of osteoblast attachment is not found between Ti-p and Ti-b. Under 4% serum condition, the cell morphology attached to smooth surfaces (Ti-p and HAC-p) is spread faster and are more flattened than the one to rough surface of the same experimental materials by SEM. After 24 h culture, the corroded cracks are easily observed at the surface of polished HA coatings, and the cell morphology on HAC-p coatings are elongated and less flattened compared with Ti-p. The result is consistent with statistical difference of cell attachment between Ti-p and HAC-p under 4% serum condition.

Hypoxia Regulates Osteoblast Gene Expression

Vascular disruption secondary to fracture creates a hypoxic gradient of injury wherein the oxygen tension at the center of the wound is very low. In vivo this hypoxic microenvironment stimulates the expression of a variety of cytokines from inflammatory cells, fibroblasts, endothelial cells, and osteoblasts. In order to begin to dissect this complex system, we have examined the effects of hypoxia on isolated osteoblast gene expression in vitro. Understanding gene expression in this system may facilitate the development of targeted therapeutic modalities designed to accelerate fracture repair and reduce complications. Using an established model of in vitro hypoxia, we have analyzed the expression of genes involved in bone matrix production and turnover. Subconfluent neonatal rat calvarial osteoblasts were exposed to hypoxia (pO2 = 35–40 mm Hg) and total cellular RNA was collected at 0, 3, 6, 24, and 48 h. Northern analysis was used to analyze the expression patterns of (1) transforming growth factors (TGFs)-β1, -β2, and -β3 and their type I receptor; (2) collagens I and III; and (3) tissue inhibitor of metalloproteinase-1. We have demonstrated a marked elevation of TGF-β1 gene expression within 3 h of hypoxia. Although neither TGF-β2 nor TGF-β3 expression was affected by hypoxia, the TGF-β type I receptor was substantially upregulated within 6 h. In addition, extracellular matrix scaffolding molecules (collagens I and III) were markedly, but differentially, upregulated. Finally, we have demonstrated that the expression of an inhibitor of extracellular matrix turnover, the tissue inhibitor of metalloproteinase-1, was strikingly decreased in response to hypoxia. These results imply that hypoxia can affect osseous healing by altering the expression of cytokines, bone-specific extracellular matrix molecules, and their regulators.

Titanium particles inhibit osteoblast adhesion to fibronectin‐coated substrates

To illuminate the effect of titanium particles on osteoblast function, we compared the adhesion force of neonatal rat calvarial osteoblasts on fibronectin-coated glass after incubation with titanium particles (80% had diameters of less than 5 microm). The cells were incubated with the particles for 1.5-72 hours. Using a micropipette single-cell manipulation system, we showed that the adhesion force of the osteoblasts to fibronectin-coated glass (1.0 microg/ml) was significantly affected by the presence of particulate debris. The adhesion force of the cells incubated with titanium particles for less than 4 hours was not significantly affected by exposure to the particles; after 4 hours, however, it was significantly reduced relative to that of controls. Aspiration of particle-challenged osteoblasts into the micropipette demonstrated that the particles were not stripped from the cell surface and therefore confirmed that the osteoblasts had ingested them. During aspiration, the particles traveled through the cytoplasm rather than on the cell surface. When the osteoblasts were exposed to the particles and cytochalasin D, they exhibited much lower adhesion forces than did the controls or the cells exposed to titanium particles only; this indicates an important role of actin filaments in the osteoblastic response to particles. Staining for F-actin also indicated an influence of internalized titanium particulate on cytoskeletal arrangement and cell spreading. Furthermore, with standard Northern blotting techniques, levels of mRNA for collagen type I and fibronectin were significantly reduced as early as 4 hours after exposure to particles compared with levels in controls, and this effect continued to 72 hours. These data indicate that direct exposure of osteoblasts to titanium particles, which we propose to be ingested by the osteoblasts, can significantly decrease osteoblast adhesion force; this may lead to decreased cellular activity and gene expression of fibronectin and collagen type I in the presence of titanium wear debris.

Synthesis, analytical characterization, and osteoblast adhesion properties on RGD-grafted polypyrrole coatings on titanium substrates

The covalent attachment of an Arg-Gly-Asp (RGD) containing peptide to polypyrrole(PPy)-coated titanium substrates has been investigated in order to develop a bioactive material of potential use in orthopedic fields. Polypyrrole has been employed as the coating polymer because of its suitability to be electrochemically grown directly onto metallic substrates of different shapes, leading to remarkably adherent films. The synthetic peptide Cys-Gly-(Arg-Gly-Asp)-Ser-Pro-Lys, containing the cell-adhesive region of fibronectin(RGD), has been grafted to the polymer substrate via the cysteine residue using a procedure recently developed in the authors laboratory. The effectiveness of grafting was monitored by X-ray photoelectron spectroscopy (XPS), which assessed the presence of the peptide grafted onto the polymer surface exploiting the cysteine sulfur as target element. Neonatal rat calvarial osteoblasts were attached to RGD-modified PPy-coated Ti substrates at levels significantly greater than on unmodified PPy-coated Ti and glass coverslip substrates.

Differential regulation of syndecan expression by osteosarcoma cell lines in response to cytokines but not osteotropic hormones

Bone cells are regulated by interactions with both growth factors and components of the extracellular matrix (ECM). Syndecans are cell-surface heparan sulfate proteoglycans known to play a role in cell adhesion and migration, and binding of growth factors. This study was performed to investigate the expression of syndecans by osteoblasts. Reverse transcription-linked polymerase chain reaction (RT-PCR) and Northern analysis detected syndecan transcripts in the human osteosarcoma cell lines MG-63, TE-85, SaOS-2, and U2OS; human osteoblast-like cells; rat calvarial osteoblasts; and in human bone. Western blot analysis of proteoglycans from MG-63 and TE-85 cells detected multiple heparan sulfate proteoglycan core proteins consistent with syndecan expression. Regulation of syndecan-1, -2, and -4 expression was investigated in TE-85, MG-63, and SaOS-2 cells, in response to interleukin (IL)-1β, and IL-6, parathyroid hormone [PTH(1-34)], and 1,25(OH) 2-vitamin D 3. Northern analysis demonstrated that in the osteosarcoma cell lines there was no regulation of syndecan transcript levels in response to PTH(1-34) or 1,25(OH) 2-vitamin D 3 for 24 or 48 h. In contrast, when MG-63 and SaOS-2 cells were incubated with IL-1β (0.01–10 ng/mL) and IL-6 (0.1–50 ng/mL) there was a dose-dependent decrease in mRNA levels for syndecan-1 and -2 at 24 and 48 h, but in response to IL-1β upregulation in the levels of syndecan-4 transcripts. In addition, Northern analysis was performed on RNA isolated from neonatal rat calvarial osteoblasts cultured under conditions that promote osteogenesis for 0, 5, 13, 21, and 35 days. Syndecan-1 expression was observed to decrease during the culture period, syndecan-2 transcript levels increased, and there appeared to be no overall change in syndecan-4 levels. Controlled expression of syndecans by cells of the osteoblast lineage may be important in the regulation of osteoblastic proliferation and differentiation.

Development and analytical characterization of cysteine-grafted polypyrrole films electrosynthesized on Ptand Ti-substrates as precursors of bioactive interfaces

The grafting of cysteine to polypyrrole(PPY)-coated platinum and titanium substrates has been investigated with the aim of developing innovative bioactive materials of interest for bone implants. Polypyrrole has been chosen as the coating polymer because of its suitability to be electrochemically grown directly onto metallic substrates, of any shape and dimension, leading to remarkably adherent overlayers. The effectiveness of grafting was monitored by X-ray photoelectron spectroscopy (XPS) which showed the presence of aminoacid residues onto the polymer surface. Information obtained by an accurate curve fitting of significant regions in the spectra (Cls, Nls, and Ols signals) and by a cross-check of peak area ratios, before and after the grafting process, gave evidence that cysteine forms covalent bonds to pyrrole rings, preferentially in β-positions, via the sulfydryl group, leaving both amino and carboxylic functionalities available for further chemistry. The surface density of cysteine residues was evaluated by microgravimetric measurements performed by the electrochemical quartz crystal microbalance and was found suitable for the exploitation of these modified surfaces as bioactive systems. Some preliminary results are reported on the adhesion of neonatal rat calvarial osteoblasts onto titanium substrates after coating by a PPY film modified by a polypeptide having cysteine as a terminal residue and containing the Arg-Gly-Asp aminoacid adhesive sequence.

Three-dimensional culture of rat calvarial osteoblasts in porous biodegradable polymers

Neonatal rat calvarial osteoblasts were cultured in 90% porous, 75 : 25 poly( dl-lactic- co-glycolic acid) (PLGA) foam scaffolds for up to 56 days to examine the effects of the cell seeding density, scaffold pore size, and foam thickness on the proliferation and function of the cells in this three-dimensional environment. Osteoblasts were seeded at either 11.1×10 5 or 22.1×10 5 cells per cm 2 onto PLGA scaffolds having pore sizes in the range of 150–300 or 500–710 μm with a thickness of either 1.9 or 3.2 mm. After 1 day in culture, 75.6 and 68.6% of the seeded cells attached and proliferated on the 1.9 mm thick scaffolds of 150–300 μm pore size for the low and high seeding densities, respectively. The number of osteoblasts continued to increase throughout the study and eventually leveled off near 56 days, as indicated by a quantitative DNA assay. Osteoblast/foam constructs with a low cell seeding density achieved comparable DNA content and alkaline phosphatase (ALPase) activity after 14 days, and mineralization results after 56 days to those with a high cell seeding density. A maximum penetration depth of osseous tissue of 220±40 μm was reached after 56 days in the osteoblast/foam constructs of 150–300 μm pore size initially seeded with a high cell density. For constructs of 500–710 μm pore size, the penetration depth was 190±40 μm under the same conditions. Scaffold pore size and thickness did not significantly affect the proliferation or function of osteoblasts as demonstrated by DNA content, ALPase activity, and mineralized tissue formation. These data show that comparable bone-like tissues can be engineered in vitro over a 56 day period using different rat calvarial osteoblast seeding densities onto biodegradable polymer scaffolds with pore sizes in the range of 150–710 μm. When compared with the results of a previous study where similar polymer scaffolds were seeded and cultured with marrow stromal cells, this study demonstrates that PLGA foams are suitable substrates for osteoblast growth and differentiated function independent of cell source.