Metabolic Activity Of Osteoblasts Research Articles

Anti-osteoblastogenic, pro-inflammatory and pro-angiogenic effect of extracellular vesicles isolated from the human osteosarcoma cell line MNNG/HOS.

Extracellular Vesicles (EVs) are becoming increasingly recognized as integral signaling vehicles in several types of cancers, including bone malignancies. However, the specific mechanisms by which EVs influence osteosarcoma progression have not been fully determined. We evaluated the effects of EVs derived from the human osteosarcoma cell line MNNG/HOS (MNNG/HOS-EVs) on bone resident cells. We found that MNNG/HOS-EVs are internalized by osteoblasts and osteoclasts in vitro, with potent inhibitory effects on osteoblast metabolic activity, cell density and alkaline phosphatase activity. Consistently, MNNG/HOS-EVs reduced the expression of cell cycle and pro-osteoblastogenic genes, whilst increasing transcriptional expression and protein release of pro-osteoclastogenic/inflammatory cytokines (RankL, Il1b, Il6 and Lcn2), pro-tumoral cytokines (CCL2,5,6,12 and CXCL1,2,5) and the metalloproteinase MMP3. MNNG/HOS-EVs did not induce osteoclast differentiation, while promoting in vitro and in vivo angiogenesis. Intriguingly, EVs derived from another osteosarcoma cell line (U2OS) reduced ALP activity but had no other effect on osteoblast phenotype. MNNG/HOS-EVs were also found to dramatically increase Serpin b2 expression in osteoblasts. To evaluate the significance of this finding, osteoblasts were forced to overexpress Serpin b2, which however did not affect osteoblast differentiation, while Il6 and Lcn2 mRNAs were up regulated. Overall, we shed light on the interactions of osteosarcoma EVs with the cells of the bone microenvironment, identifying key anti-osteoblastogenic, pro-inflammatory and pro-angiogenic factors that could contribute to osteosarcoma expansion.

Wnt3a and ASCs are capable of restoring mineralization in staph aureus-infected primary murine osteoblasts.

Bone infections are one of the main reasons for impaired bone regeneration and non-union formation. In previous experimental animal studies we could already demonstrate that bone defects due to prior infections showed a markedly reduced healing capacity, which could effectively be enhanced via application of Wnt3a and Adipose-derived stromal cells (ASCs). For a more in-depth analysis, we investigated proliferation and mineralization of cultured osteoblasts infected with staph aureus and sought to investigate effects of Wnt3a and ASCs on infected osteoblasts. Primary murine osteoblasts were isolated from calvariae and infected with staph aureus. Infected osteoblasts received treatment via application of recombinant Wnt3a, ASC conditioned medium and were furthermore cocultured with ASCs. Osteoblasts were evaluated by Alamar blue assay for metabolic activity, TUNEL-assay for apoptosis, ALP and Alizarin Red staining for mineralization. In addition, immunoflourescent staining (IF) and qRT-PCR analyses were performed. Infected osteoblasts showed a markedly reduced ability for mineralization and increased apoptosis, which could be restored to physiological levels by Wnt3a and ASC treatment. Interestingly, metabolic activity of osteoblasts seemed to be unaffected by staph aureus infection. Additional analyses of Wnt-pathway activity revealed effective enhancement of canonical Wnt-pathway activity in Wnt3a-treated osteoblasts. In summary, we gained further osteoblast-related insights into pathomechanisms of reduced bone healing capacity upon infections.

Patient-specific effects of soluble factors from Staphylococcus aureus and Staphylococcus epidermidis biofilms on osteogenic differentiation of primary human osteoblasts

Due to the frequency of biofilm-forming Staphylococcus aureus and Staphylococcus epidermidis in orthopedics, it is crucial to understand the interaction between the soluble factors produced by prokaryotes and their effects on eukaryotes. Our knowledge concerning the effect of soluble biofilm factors (SBF) and their virulence potential on osteogenic differentiation is limited to few studies, particularly when there is no direct contact between prokaryotic and eukaryotic cells. SBF were produced by incubating biofilm from S. aureus and S. epidermidis in osteogenic media. Osteoblasts of seven donors were included in this study. Our results demonstrate that the detrimental effects of these pathogens do not require direct contact between prokaryotic and eukaryotic cells. SBF produced by S. aureus and S. epidermidis affect the metabolic activity of osteoblasts. However, the effect of SBF derived from S. aureus seems to be more pronounced compared to that of S. epidermidis. The influence of SBF of S. aureus and S. epidermidis on gene expression of COL1A1, ALPL, BGLAP, SPP1, RUNX2 is bacteria-, patient-, concentration-, and incubation time dependent. Mineralization was monitored by staining the calcium and phosphate deposition and revealed that the SBF of S. epidermidis markedly inhibits calcium deposition; however, S. aureus shows a less inhibitory effect. Therefore, these new findings support the hypotheses that soluble biofilm factors affect the osteogenic processes substantially, particularly when there is no direct interaction between bacteria and osteoblast.

Coordination Compounds As Multi-Delivery Systems for Osteoporosis.

Osteoporosis therapies leveraging bisphosphonates and mineral components (e.g., magnesium, calcium, and strontium) have been raising attention because of their potential for managing this ever-growing disease. The administration of multicomponent therapeutics (combined therapy) in elderly patients is complex and suffers from low patient adherence. Herein, we report an all-in-one combination of four antiosteoporotic components into a new family of coordination complexes: [M2(H4alen)4(H2O)2]·1.5H2O [where M2+ = Mg2+ (1), (Mg0.535Ca0.465)2+ (2) and (Mg0.505Ca0.450Sr0.045)2+ (3)]. These solid-state complexes were prepared, for the first time, through microwave-assisted synthesis. It is demonstrated that the compounds are capable of releasing their antiosteoporotic components, both in conditions that mimic the path along the gastrointestinal tract and in long periods under physiological conditions (pH ∼7.4). More importantly, when administered in low concentrations, the compounds did not elicit a cytotoxic effect toward liver, kidney, and osteoblast-like cell lines. Besides, it is important to highlight the unique coordination complex with four bone therapeutic components, [(Mg0.505Ca0.450Sr0.045)2(H4alen)4(H2O)2]·1.5H2O (3), which significantly promoted osteoblast metabolic activity up to ca. 1.4-fold versus the control group. These findings bring this type of compounds one-step closer to be considered as an all-in-one and more effective treatment for managing chronic bone diseases, prompting further research on their therapeutic properties.

Fabrication of biocompatible and bioabsorbable polycaprolactone/ magnesium hydroxide 3D printed scaffolds: Degradation and in vitro osteoblasts interactions

Biodegradable polymeric 3D implants are of considerable interest for biomedical applications, however the degradation profile and bioactivity are important considerations for many clinical applications. In this context, bioresorbable magnesium hydroxide (MH) nanoparticles (NPs) (<50 nm) were blended with the degradable polymer poly (ε-caprolactone) (PCL) at concentrations of 5 and 20 wt%, and the composite was manufactured by 3D printing technology. Efficient load transfer was found between the nanofiller and matrix PCL, which was reflected in changes in the tensile properties of the MH-based composite. A statistically significant 44.3% increase in tensile modulus was achieved by the addition of 5 wt% MH, which was in agreement with the Halpin-Tsai theoretical model. The incorporation of MH in the PCL scaffolds accelerated the weight loss of the scaffolds and decreased the molecular weight of PCL over a prolonged soaking period (150 days) in PBS solution (pH 7.37, 37 ± 0.5 °C). The PCL/MH composite scaffolds were shown to be non-cytotoxic in vitro, and ion diffusion into the cell culture media promoted osteoblast metabolic activity, attachment, and proliferation, as compared to PCL-only scaffolds. Moreover, osteoblastic activity, as assessed by the expression of alkaline phosphatase, was significantly higher on the composite PCL/MH scaffold after 14 and 21 days. In summary, the 3D PCL/MH composite scaffolds could enhance osteoblastic activity and demonstrated a moderately accelerated degradation profile, which are characteristics that can be considered favorable for bone regeneration applications.

Evaluation of human osteoblast metabolic activity in modified titanium-conditioned medium.

To evaluate human osteoblast metabolic activity cultured in medium conditioned with commercially pure titanium after surface treatments with alumina or ceramic grit-blasting followed by acid etching. Commercially available, pure Grade 4 titanium disks were used and subjected to seven different surface modifications: (1) machined (MA)-used as the control group; (2) blasted with Al2O3 (Al2O3); (3) blasted with sintered ceramic (HAS); (4) blasted with non-sintered ceramics (HA); (5) blasted with Al2O3 and etched with HCl/H2SO4 (Al2O3 DE); (6) blasted with sintered ceramic and etched with HCl/H2SO4 (HAS DE), and (7) blasted with non-sintered ceramic and etched with HCl/H2SO4 (HA DE). A samples roughness evaluation test was carried out with an interference microscope, and energy-dispersive X-ray spectroscopy was performed to evaluate the presence of aluminum, phosphorus, and calcium deposited during the titanium surface treatment along with carbon contaminants acquired by the surface during processing. A culture medium conditioned with the respective samples was prepared in five dilutions, and its effect on human osteoblast cell viability was evaluated using the relative viability of cells. Human osteoblast metabolic activity was found to be the most intensive for the Al2O3 DE sample. The lowest activity was observed for the HAS DE. The material's cytocompatibility depended on both the surface roughness and its chemical composition. Etching had a dual effect on cell activity, depending on the chemical composition of the titanium surface after blasting.

Cross talk between redox signalling and metabolic activity of osteoblasts and fibroblasts in the presence of hydroxyapatite-based biomaterials influences bone regeneration.

Regeneration of bone tissue defects that result from metabolic disorders, including periodontal diseases, can be supported by biomaterials based on hydroxyapatite. Despite of good biocompatibility of biomaterials they can cause oxidative stress and inflammatory processes as a result of mechanical interaction with surrounding tissues. Because osteoblasts are responsible for bone regeneration process in which gingival fibroblasts may also participate, the aim of the work was to investigate the influence of hydroxyapatite-based biomaterials (allogeneic and xenogeneic) and biomaterials combined with enamel matrix derivative (Emdogain) on osteoblast and fibroblast redox balance in the context of osteoblast proliferation and differentiation. The results showed that examined substitutes were not cytotoxic in vitro, but affected redox balance of osteoblasts and fibroblasts (ROS level increase and GSH level decrease) which led to oxidative stress (MDA and protein carbonyl groups level increase) resulting in an increase of the Nrf2 and NFκB expression. The consequence of these changes was partial inhibition of proliferation and osteoblast differentiation. Emdogain alone and combined with biomaterials decreased ROS generation and increased GSH level in both osteoblasts and fibroblasts leading to reduction of transcription factors expression especially proinflammatory NFκB, which promoted osteoblast differentiation and mineralization process.

Bacteria Death and Osteoblast Metabolic Activity Correlated to Hydrothermally Synthesised TiO₂ Surface Properties.

Orthopaedic surgery comes with an inherent risk of bacterial infection, prolonged antibiotic therapy and revision surgery. Recent research has focused on nanostructured surfaces to improve the bactericidal and osseointegrational properties of implants. However, an understanding of the mechanical properties of bactericidal materials is lacking. In this work, the surface properties of hydrothermal TiO2 nanostructured surfaces are investigated for their effect on bactericidal efficiency and cellular metabolic activity of human osteoblast cells. TiO2 nanostructures, approximately 307 nm in height and 14 GPa stiffness, were the most effective structures against both gram-positive (Staphylococcus aureus) and gram-negative (Pseudomonas aeruginosa) bacteria. Statistical analysis significantly correlated structure height to the death of both bacteria strains. In addition, the surface contact angle and Young’s modulus were correlated to osteoblast metabolic activity. Hydrophilic surfaces with a contact angle between 35 and 50° produced the highest cellular metabolic activity rates after 24 h of incubation. The mechanical tests showed that nanostructures retain their mechanical stability and integrity over a long time-period, reaffirming the surfaces’ applicability for implants. This work provides a thorough examination of the surface, mechanical and wettability properties of multifunctional hydrothermally synthesised nanostructured materials, capable of killing bacteria whilst improving osteoblast metabolic rates, leading to improved osseointegration and antibacterial properties of orthopaedic implants.

Bone Mineral Density and Molecular Genetic Markers of Bone Remodeling in Blood of Cosmonauts after Long-term Missions on Board the International Space Station

The results of the investigation of the bone system of 24 Russian cosmonauts after long-term (124–199 days) missions on board the International space station (ISS) are presented. Functional adaptation of the bone system involves some complex changes in the metabolic activity of osteoblasts and osteoclasts, such as alterations of the serum concentrations of osteocalcin, tartrate-resistant acid phosphatase (TRAP), osteoprotegerin, and the activator ligand of the receptor of nuclear factor kappa-B (RANKL); in addition, in peripheral blood leucocytes, there are changes in the expression of genes regulating the development of skeletal system and bone mineral metabolism. Significant variability in the mineral density of femoral neck and molecular genetic markers studied after long-term space flights indicates individual variability of the balance of the processes of bone remodeling, bone formation and resorption. Significant bone mass losses in the femoral bone of cosmonauts are associated with more pronounced changes in the markers of metabolic activity of osteoclasts.

The Biocompatibility and Occlusion Ability of a Zein-Based Biomaterial for Bone Surgery

During surgical procedures on bone, a common method of producing haemostasis at bleeding cancellous bone is the occlusion of blood vessels. This is often achieved with bone wax, which is not bioresorbable, unlike the zein-based biomaterial investigated in the present research. Zein is a prolamin derived from corn, and has been gaining importance as a bio-medical material. Taking advantage of its solubility in ethanol-water solvents but insolubility in water, a zein-based viscoelastic solid can be produced which effectively occludes the flow of fluids through a porous surface modelling cancellous bone. Zein powder was dissolved into a 70% ethanol-in-water solution, and the ethanol was later leached out through exposure to an alcohol-free media. The insoluble zein ‘resin’ produced could occlude water flow through a porous surface. Experiments were conducted to determine the optimum composition of the precursor zein solution, varying the proportion of zein dissolved in the ethanol-water solvent. A 0.7 w/v composition was selected as the preferred ratio. A cell viability test using the resazurin assay showed that unleached ethanol in the zein-based biomaterial does not pose a threat, as the metabolic activity of osteoblasts on zein resin outperformed that on bone wax after 24 hours of incubation. Subsequent characterisation of the zein resin was performed with a rheometer: results showed that the 0.7 w/v composition had a higher storage modulus and loss modulus for the range of frequencies tested.

Resistin promotes the abnormal Type I collagen phenotype of subchondral bone in obese patients with end stage hip osteoarthritis

The purpose of this study was to determine the effect of adiposity on the architecture and composition of hip OA subchondral bone, and to examine the pathological role of adipokines. Femoral heads were collected from normal-weight or over-weight/obese patients with hip OA. Structural parameters of subchondral bone were determined by MicroCT and type I collagen α1/α2 ratio was determined by SDS PAGE and by qRT-PCR in ex-vivo bone explants. The serum concentration of adipokines was determined by Luminex. The effect of resistin on primary OA osteoblasts was determined by analysis of Wnt pathway signal transduction, bone nodule formation, and osteoblast metabolic activity. Subchondral bone from over-weight/obese hip OA patients exhibited reduced trabecular thickness, increased bone surface/bone volume ratio, and an increase in the Type I collagen α1/α2, compared to normal-weight hip OA patients. The serum concentration of resistin was higher in overweight/obese OA patients, compared to normal-weight OA patients. Stimulation of normal-weight bone explant with recombinant resistin increased the Type I collagen α1/α2 ratio. Stimulation of primary OA osteoblasts with recombinant resistin increased Wnt signalling activation, osteoblast metabolic activity, and bone nodule formation. Increased adiposity in hip OA patients is associated with altered subchondral bone architecture and type I collagen composition.

Combining 3D human in vitro methods for a 3Rs evaluation of novel titanium surfaces in orthopaedic applications.

ABSTRACTIn this study, we report on a group of complementary human osteoblast in vitro test methods for the preclinical evaluation of 3D porous titanium surfaces. The surfaces were prepared by additive manufacturing (electron beam melting [EBM]) and plasma spraying, allowing the creation of complex lattice surface geometries. Physical properties of the surfaces were characterized by SEM and profilometry and 3D in vitro cell culture using human osteoblasts. Primary human osteoblast cells were found to elicit greater differences between titanium sample surfaces than an MG63 osteoblast‐like cell line, particularly in terms of cell survival. Surface morphology was associated with higher osteoblast metabolic activity and mineralization on rougher titanium plasma spray coated surfaces than smoother surfaces. Differences in osteoblast survival and metabolic activity on titanium lattice structures were also found, despite analogous surface morphology at the cellular level. 3D confocal microscopy identified osteoblast organization within complex titanium surface geometries, adhesion, spreading, and alignment to the biomaterial strut geometries. Mineralized nodule formation throughout the lattice structures was also observed, and indicative of early markers of bone in‐growth on such materials. Testing methods such as those presented are not traditionally considered by medical device manufacturers, but we suggest have value as an increasingly vital tool in efficiently translating pre‐clinical studies, especially in balance with current regulatory practice, commercial demands, the 3Rs, and the relative merits of in vitro and in vivo studies. Biotechnol. Bioeng. 2016;113: 1586–1599. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Development of a Multifunctional Platform Based on Strong, Intrinsically Photoluminescent and Antimicrobial Silica‐Poly(citrates)‐Based Hybrid Biodegradable Elastomers for Bone Regeneration

Biodegradable biomaterials with intrinsically multifunctional properties such as high strength, photoluminescent ability (bioimaging monitoring), and antimicrobial activity (anti‐infection), as well as high osteoblastic differentiation ability, play a critical role in successful bone tissue regeneration. However, fabricating a biomaterial containing all these functions is still a challenge. Here, urethane cross‐linked intrinsically multifunctional silica‐poly(citrate) (CMSPC)‐based hybrid elastomers are developed by first one‐step polymerization and further chemical crosslinked using isocyanate. CMSPC hybrid elastomers demonstrate a high modulus of 976 ± 15 MPa, which is superior compared with most conventional poly(citrate)‐based elastomers. Hybrid elastomers show strong and stable intrinsic photoluminescent ability (emission 400–600 nm) due to the incorporation of silica phase. All elastomers exhibit high inherent antibacterial properties against Staphylococcus aureus. In addition, CMSPC hybrid elastomers significantly enhance the proliferation and metabolic activity of osteoblasts (MC3T3‐E1). CMSPC hybrid elastomers significantly promote the osteogenic differentiation of MC3T3‐E1 by improving alkaline phosphatase activity and calcium biomineralization deposits, as well as expressions of osteoblastic genes. These hybrid elastomers also show a minimal inflammatory response indicated by subcutaneous transplantation in vivo. These optimized structure and multifunctional properties make this hybrid elastomer highly promising for bone tissue regeneration and antiinfection and bioimaging applications.

Combined effects of direct current stimulation and immobilized BMP‐2 for enhancement of osteogenesis

Direct current (DC) stimulation has been used to promote bone repair and osteogenesis, but problems associated with the implanted metal electrodes may limit its application and compromise the therapeutic results. The replacement of the metal electrodes with a biodegradable conductive polymer film can potentially overcome these problems. In our work, polypyrrole/chitosan films comprising polypyrrole nanoparticles dispersed in a chitosan matrix were prepared. The polypyrrole/chitosan film meets the requirements for DC delivery, as indicated by its electrical conductivity, biodegradability, and mechanical properties. The film supports osteoblast growth to the same degree as dentine discs (a bone-like mineralized substrate), confirming that it is non-cytotoxic. Our results showed that optimal DC stimulation was achieved with 200 µA for 4 h per day, and under this condition, osteoblast metabolic activity on Day 7 increased by 1.8-fold over that without DC stimulation. To further improve osteogenesis on the polypyrrole/chitosan film, bone morphogenetic protein-2 (BMP-2) was covalently immobilized on the film surface. Osteoblasts cultured on the BMP-2-functionalized polypyrrole/chitosan film and subjected to the optimal DC stimulation exhibited a significant increase in cellular metabolic activity (2.3-fold on Day 7), ALP activity (1.7-fold on Day 21) and mineralization (twofold on Day 21) over those cultured on polypyrrole/chitosan film without DC stimulation. Osteogenic gene expression results showed that BMP-2 and DC stimulation by itself enhanced osteoblast differentiation, and a combination of these two factors resulted in synergistic effects on osteoblast differentiation and maturation.

Substance P and norepinephrine modulate murine chondrocyte proliferation and apoptosis

Substance P (SP) and norepinephrine (NE) containing sensory and sympathetic nerve fibers innervate bone and fracture callus. They are involved in controlling vascularization and matrix differentiation during skeletal growth. Both types of nerve fibers are known to modulate growth and metabolic activity of osteoblasts and osteoclasts. The aim of this study was to understand the roles of SP and NE in chondrocyte metabolism and their impact on chondrocyte proliferation, apoptosis, and cell adhesion. Primary costal chondrocytes were isolated from newborn mice. Micromass and monolayer cell culture regimens were used to analyze the effects of SP and NE on matrix formation, as determined by quantitative polymerase chain reaction and immunohistochemistry. The effects of SP and NE on proliferation, adhesion, and apoptosis of chondrocytes were determined by enzyme-linked immunosorbent assay, bromodeoxyuridine, TUNEL, and morphometric analyses. SP, neurokinin type 1 (NK-1) receptor, α-adrenergic receptor (α-AR), and β-AR were abundantly expressed in primary costal chondrocytes. Stimulation with SP or NE did not affect extracellular matrix formation with respect to types I, II, and IX collagen and aggrecan in micromass pellets. SP dose-dependently increased the rate of proliferation of chondrocytes via the NK-1 receptor, whereas NE decreased the apoptosis rate of chondrocytes by stimulating β-AR. Both neurotransmitters induced the formation of focal adhesion contacts. Transmitters of sympathetic and sensory nerve fibers modulate the metabolic activity of chondrocytes. Endogenous SP, NK-1 receptor, and adrenoceptor expression in chondrocytes implicates as-yet-unknown, presumably trophic, functions of neurotransmitter for skeletal growth and might be of interest for use in cartilage regenerative medicine.

Poly(Ethylene Glycol) and Hydroxy Functionalized Alkane Phosphate Self-Assembled Monolayers Reduce Bacterial Adhesion and Support Osteoblast Proliferation

Presently there is interest today in designing improved titanium surfaces capable of high bioactivity in order to promote strong anchorage of the bone surrounding implants while at the same time discouraging bioadhesion. Poly(ethylene glycol)-modified (PEG) alkane phosphate and OH-terminated alkane phosphates have been demonstrated to be spontaneously adsorbed onto titanium oxide surfaces and produce surfaces with different protein resistance in relation to the PEG surface density. This study aims to evaluate caries-associated Streptococcus mutans (S. mutans) adhesion and osteoblast proliferation while varying the PEG surface density of titanium surfaces. Bacterial adhesion was quantified by fluorescence microscopy and SAOS-2 human osteoblast proliferation was evaluated up to 7 days of culture in vitro. Metabolic activity of osteoblasts was measured by MTT test and the secretion of extracellular matrix proteins (osteopontin, osteocalcin and type I collagen) in culture medium was determined by immunoenzymatic assays. As the PEG surface density increased, the bacterial adhesion considerably decreased when compared to uncoated titanium surfaces. The monomolecular coatings proved to be capable of supporting osteoblast proliferation with the greatest levels of metabolic activity at the highest PEG surface concentrations. These results are extremely promising for potential clinical application in implant uses where both reduction of bacteria adhesion and stimulation of bone formation are highly desirable.

The effect of electrochemically simulated titanium cathodic corrosion products on ROS production and metabolic activity of osteoblasts and monocytes/macrophages

Nowadays aseptic loosening is the most common cause of orthopaedic implant failure. Some of its reasons have already been described up to now; however, others remain still hypothetical. Besides the inflammatory response to wear particles originating at different sources, the role of reactive oxygen species as products of cellular reactions and/or as a result of the process of corrosion of an implant leading to implant failure has recently been discussed too. In the present study, we used a galvanostatic polarization to simulate the cathodic partial reaction of the corrosion process at a titanium alloy surface. With respect to cells occurring at the interface of a metal implant, the behaviour of osteoblasts and monocytes/macrophages was investigated. It has been found that cathodic polarization of Ti6Al4V induces an increase in the level of intracellular reactive oxygen species as well as suppressing the metabolic activity of cells in a dose-dependent manner. This is in agreement with the results obtained with cells after external addition of hydrogen peroxide as another kind of oxidative stress. In both approaches, monocytes/macrophages show a higher tolerance to oxidative stress than osteoblasts. It could be concluded that the electrochemical setup developed induced intracellular changes occurring during oxidative stress and it could be used for future detailed analysis of the consequences of corrosion processes for cellular reactions.

O‐phospho‐L‐serine modified calcium phosphate cements – material properties, in vitro and in vivo investigations

AbstractOne of the most important requirements for calcium phosphate bone cements (CPBC) is remodelling in vivo. Degradation of the artificial material and the formation of native new bone matrix have to interdigitate to avoid instability. The remodelling process should not last longer than one year. In this study a CPBC was modified with O‐phospho‐L‐serine (phosphoserine) to improve the material properties in order to speed up remodelling in vivo. The results showed that the modified cements exhibit a nano‐crystalline microstructure with a high specific surface area and increased compressive strength (about 50 %). Metabolic activity of osteoblasts was improved on the modified material. Monocytes were found to be highly activated on the cements containing phosphoserine. In addition the formation of multinucleated giant cells (osteoclast‐like cells) was not impaired on phosphoserine modified cement composites. In vivo experiments in Wistar rats and mini pigs clearly revealed that phosphoserine modified cements showed a higher capability of remodelling compared to the cements without phosphoserine.

The role of the cytoskeleton in mechanotransduction in human osteoblast-like cells.

The regulation of osteoblast proliferation is a key factor in maintaining bone mass. The enhancement of this process can be achieved by stimulating the proliferation of these cells. Mechanical stimulation is one of the important enhancing factors, but the exact cellular mechanisms of mechanical stimulation, i.e., mechanotransduction, are unknown. In order to investigate the role of the cytoskeleton components in mechanotransduction for cell proliferation, I compared the total DNA content in cultured replicates of osteoblast-like cells derived from three human donors following their exposure to enhancing mechanical stimulation, with and without added specific microtubular and microfilament polymerization blockers (Colchicin and Cytochalasin D, respectively). The results revealed the essential and unique role of the microtubular component of the cytoskeleton in mechanotransduction for proliferation by showing that Colchicin blocked the expected increase in the DNA content after mechanical stimulation of the cultured replicates without altering the total DNA content in replicates at static conditions. Conversely, a specific blockage of the microfilament polymerization presented uniform cytotoxic effect in both static and biomechanically active environments. Since previous reports indicated the essential role of microfilament polymerization for the osteoblast metabolic activity, the results of this study further support the hypothesis that the mechanotransduction mechanisms for proliferation and metabolic activity are mediated by different intracellular pathways.

Cytokine-stimulated expression of inducible nitric oxide synthase by mouse, rat, and human osteoblast-like cells and its functional role in osteoblast metabolic activity.

Recent evidence suggests that the production of nitric oxide (NO) may have important roles in the regulation of osteoblast and osteoclast metabolism. The present study was performed to investigate the effects of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) on the expression of inducible NO-synthase (iNOS) and to measure high-output production of NO by primary rat osteoblasts and osteoblastic cell lines ROS 17/2.8, MC3T3-E1 and MG-63. In addition, we have investigated if NO may mediate some of the effects of these cytokines on osteoblast metabolism. Northern blots and immunocytochemistry revealed time-dependent iNOS messenger RNA and protein expression in primary rat osteoblasts in response to cytokine treatment. Reverse transcription polymerase chain reaction amplified an 807-base pair (bp) product from ROS 17/2.8 cells, which had a size and restriction enzyme-cut pattern identical to that predicted for authentic rat iNOS. Nitrite accumulation in culture medium was induced by IFN-gamma in a time- and dose-dependent manner and inhibited by cotreatment with inhibitors of NOS activity and by dexamethasone. IL-1 beta, TNF-alpha, and bacterial lipopolysaccharide were found to have weak stimulatory effects on nitrite production on their own. However, IL-1 beta and TNF-alpha showed strong synergy with IFN-gamma, but, surprisingly, lipopolysaccharide was found to exert potent inhibitory effects on IFN-gamma-induced nitrite synthesis. Basal production of nitrite and induction of its synthesis was similarly observed with primary rat osteoblasts as well as ROS 17/2.8, MC3T3-E1, and MG-63 cell lines. Cytokine-induced NO production significantly reduced osteoblast activity, as was evidenced by inhibition of DNA synthesis, cell proliferation, alkaline phosphatase activity, and osteocalcin production. The results provide evidence for a basal expression of iNOS activity and show that the iNOS messenger RNA, protein, and enzyme activity are all induced by cytokines across the species. The data further suggest that osteoblast-derived NO may have an important role in mediation of localized bone destruction associated with inflammatory bone diseases such as rheumatoid arthritis.