Cultures Of Human Osteoblast-like Cells Research Articles

Osteoblast-Derived Matrix Vesicles Exhibit Exosomal Traits and a Unique Subset of microRNA: Their Caveolae-Dependent Endocytosis Results in Reduced Osteogenic Differentiation.

Matrix vesicles (MVs) are nano-sized extracellular vesicles that are anchored in the extracellular matrix (ECM). In addition to playing a role in biomineralization, osteoblast-derived MVs were recently suggested to have regulatory duties. The aims of this study were to establish the characteristics of osteoblast-derived MVs in the context of extracellular vesicles like exosomes, assess their role in modulating osteoblast differentiation, and examine their mechanism of uptake. MVs were isolated from the ECM of MG63 human osteoblast-like cell cultures and characterized via enzyme activity, transmission electron microscopy, nanoparticle tracking analysis, Western blot, and small RNA sequencing. Osteoblasts were treated with MVs from two different culture conditions (growth media [GM]; osteogenic media [OM]) to evaluate their effects on the differentiation and production of inflammatory markers and on macrophage polarization. MV endocytosis was assessed using a lipophilic, fluorescent dye and confocal microscopy with the role of caveolae determined using methyl-β-cyclodextrin. MVs exhibited a four-fold enrichment in alkaline phosphatase specific activity compared to plasma membranes; were 50-150 nm in diameter; possessed exosomal markers CD63, CD81, and CD9 and endosomal markers ALIX, TSG101, and HSP70; and were selectively enriched in microRNA linked to an anti-osteogenic effect and to M2 macrophage polarization. Treatment with GM or OM MVs decreased osteoblast differentiation. Osteoblasts endocytosed MVs using a mechanism that involves caveolae. These results support the hypothesis that osteoblasts produce MVs that participate in the regulation of osteogenesis.

Magnetic 3D scaffolds for tissue engineering applications: bioactive glass (45S5) coated with iron-loaded hydroxyapatite nanoparticles

Magnetic 45S5 bioactive glass (BG) based scaffolds covered with iron-loaded hydroxyapatite (Fe-HA-BG) nanoparticles were obtained and its cytotoxicity investigated. Fe-HA nanoparticles were synthesized by a wet chemical method involving the simultaneous addition of Fe2+/Fe3+ ions. BG based scaffolds were prepared by the foam replica procedure and covered with Fe-HA by dip-coating. Fe-HA-BG magnetic saturation values of 0.049 emu g−1 and a very low remanent magnetization of 0.01 emu g−1 were observed. The mineralization assay in simulated body fluid following Kokubo’s protocol indicated that Fe-HA-BG scaffolds exhibited improved hydroxyapatite formation in comparison to uncoated scaffolds at shorter immersion times. The biocompatibility of the material in vitro was assessed using human osteoblast-like MG-63 cell cultures and mouse bone marrow-derived stroma cell line ST-2. Overall, the results herein discussed suggest that magnetic Fe-HA coatings seem to enhance the biological performance of 45S5 BG based scaffolds. Thus, this magnetic Fe-HA coated scaffold is an interesting system for bone tissue engineering applications and warrant further investigation.

3D Bioprinted Bacteriostatic Hyperelastic Bone Scaffold for Damage-Specific Bone Regeneration.

Current strategies for regeneration of large bone fractures yield limited clinical success mainly due to poor integration and healing. Multidisciplinary approaches in design and development of functional tissue engineered scaffolds are required to overcome these translational challenges. Here, a new generation of hyperelastic bone (HB) implants, loaded with superparamagnetic iron oxide nanoparticles (SPIONs), are 3D bioprinted and their regenerative effect on large non-healing bone fractures is studied. Scaffolds are bioprinted with the geometry that closely correspond to that of the bone defect, using an osteoconductive, highly elastic, surgically friendly bioink mainly composed of hydroxyapatite. Incorporation of SPIONs into HB bioink results in enhanced bacteriostatic properties of bone grafts while exhibiting no cytotoxicity. In vitro culture of mouse embryonic cells and human osteoblast-like cells remain viable and functional up to 14 days on printed HB scaffolds. Implantation of damage-specific bioprinted constructs into a rat model of femoral bone defect demonstrates significant regenerative effect over the 2-week time course. While no infection, immune rejection, or fibrotic encapsulation is observed, HB grafts show rapid integration with host tissue, ossification, and growth of new bone. These results suggest a great translational potential for 3D bioprinted HB scaffolds, laden with functional nanoparticles, for hard tissue engineering applications.

Photobiomodulation of human osteoblast-like cells invitro by low-intensity-pulsed LED light.

Visible light irradiation is an emerging area in regenerative medicine research. We hypothesized that low‐intensity‐pulsed LED light irradiance may exert photobiomodulatory effects on cultured osteoblast‐like cells. To test this hypothesis, we investigated cell proliferation and markers of cell maturation and metabolic activity following pulsed LED irradiance. Monolayer explant cultures of human osteoblast‐like cells were exposed four times in 24‐h intervals to 2 min of pulsed white LED irradiance of 2.4–2.5 mW·cm−2 and its different spectra of 0.2–0.5 mW·cm−2 (frequency range of 10–40 Hz). Cell proliferation was estimated from microscopic cell counting and cell death by lactate dehydrogenase activity in culture media (measured by a colorimetric method). The early markers of osteoblast maturation and metabolic activity, that is, cellular alkaline phosphatase activity and osteocalcin content, were measured using a colorimetric method and ELISA, respectively. Irradiance of 40 Hz caused the highest increase in cell number (P < 0.01). Osteocalcin content in cells decreased following 40 Hz and 10 Hz irradiance (P < 0.05). The 40 Hz blue range irradiance (diffuse transmittance 420–580 nm, maximal cell irradiance 0.5 mW·cm−2) caused a decrease in alkaline phosphatase cellular activity (P < 0.001) and an increase in media osteocalcin content (P < 0.05). The 40 Hz green range (diffuse transmittance 560–650 nm, maximal cell irradiance 0.4 mW·cm−2) irradiance caused an increase in the number of cells and in cell death. In summary, pulsed (40 Hz) white light irradiance has photomodulatory effects, with its green range spectrum affecting cell proliferation and cell death, and its blue range spectrum affecting cellular maturation and metabolism. The results indicate a low‐intensity threshold of photobiomodulation of osteoblast‐like cells in vitro.

Baghdadite/Polycaprolactone nanocomposite scaffolds: preparation, characterisation, and in vitro biological responses of human osteoblast-like cells (Saos-2 cell line)

ABSTRACT In the present research, porous composite scaffolds consisting of baghdadite scaffolds with polycaprolactone (PCL) coating, were fabricated via combining polyurethane sponge replication method and dip-coating processes and characterised by x-ray diffraction (XRD), scanning electron microscope (SEM), and porosity and compressive strength measurements. MTT assay and cell attachment were carried out via human osteoblast-like cell culture on the scaffolds. Results showed that the scaffolds possessed porous structures with porosity about 70-83%, and compressive strengths 0.18–1.31 MPa. Polycaprolactone can affect a significant role in improving the mechanical properties of baghdadite scaffold. It could enhance the compressive strength of baghdadite scaffold from 0.18 to 0.41 MPa while its porous structure is maintained. The evaluation of cell viability and attachment, verified no cytotoxicity, and good attachment and proliferation of cells on the scaffolds. To summarise, baghdadite–PCL composite scaffold with a porous structure, great mechanical properties and excellent biological responses is a promising candidate for bone tissue engineering.

Bioactive glass (45S5)-based 3D scaffolds coated with magnesium and zinc-loaded hydroxyapatite nanoparticles for tissue engineering applications

Bioactive glass (BG)-based scaffolds of 45S5 composition covered with hydroxyapatite nanoparticles loaded with Mg2+, Zn2+ and, both Mg2+ and Zn2+ ions, were developed and tested as materials for tissue engineering applications. The scaffolds were prepared by the foam replica technique and mono- and bi-metal loaded and unloaded hydroxyapatite nanoparticles (HA, Zn-HA, Mg-HA and Mg-Zn-HA) were obtained by an adaptation of the wet chemical deposition method. Coating of BG with these nanoparticles was performed by dip-coating to obtain HA-BG, Zn-HA-BG, Mg-HA-BG and Mg-Zn-HA-BG scaffolds.As predictor of the bone bonding ability of the produced scaffolds, in this study we investigated the formation of an apatite layer on the scaffold surfaces in the presence of simulated body fluid. The cytotoxicity and osteogenic properties of the materials in vitro was evaluated using human osteoblast-like MG-63 cell cultures. The mineralization assay following Kokubo’s protocol indicated that bi-metal loaded Mg-Zn-HA-BG scaffolds exhibited higher/faster bioactivity than mono-metal loaded scaffolds while mineralization of HA-BG, Zn-HA-BG and Mg-HA-BG was similar to that of uncoated scaffolds. Moreover, an increase of proliferation of MG-63 cells after 48 h and 7 days was measured by BrdU assays for Mg-Zn-HA-BG scaffolds. In agreement with these results, SEM images confirmed increased interaction between these scaffolds and cells, in comparison to that observed for mono-metal-loaded HA-coated scaffolds. Altogether, the obtained results suggest that nanocrystalline Mg-Zn-HA coatings enhance the biological performance of standard scaffolds of 45S5 BG composition. Thus these novel ion doped HA coated scaffolds are attractive systems for bone tissue engineering.

Development of hydroxyapatite from eggshell waste and a chitosan-based composite: In vitro behavior of human osteoblast-like cell (Saos-2) cultures

Hydroxyapatite was successfully synthesized using eggshell waste as a raw material. Eggshell waste and orthophosphoric acid were co-precipitated for 2 h at an ambient temperature. The pH of the solution was adjusted to 10 using ammonium hydroxide. Then, 10–30 wt% of hydroxyapatite was loaded into the chitosan film. Scanning electron microscopy and energy dispersive analyses confirmed the morphological properties and dispersion. A thermogravimetric analysis showed a significant enhancement in the thermal stability of the composite. The existence of hydroxyapatite resulted in a higher thermal stability. Furthermore, atomic force microscopy was used to investigate the roughness of the surface. With the addition of hydroxyapatite, the roughness significantly increased. The swelling behavior of the composite was observed in phosphate buffer saline solution. The hydroxyapatite offered the inferiority on the swelling behavior. A preliminary investigation on the in vitro behavior of Saos-2 was also performed. The composite presented good cytotoxicity, and thus, excellent properties as a tissue engineering material.

Bone Environment Influences Irreversible Adhesion of a Methicillin-Susceptible Staphylococcus aureus Strain.

Prosthesis and joint infections are an important threat in public health, especially due to the development of bacterial biofilms and their high resistance to antimicrobials. Biofilm-associated infections increase mortality and morbidity rates as well as hospitalization costs. Prevention is the best strategy for this serious issue, so there is an urgent need to understand the signals that could induce irreversible bacterial adhesion on a prosthesis. In this context, we investigated the influence of the bone environment on surface adhesion by a methicillin-susceptible Staphylococcus aureus strain. Using static and dynamic biofilm models, we tested various bone environment factors and showed that the presence of Mg2+, lack of oxygen, and starvation each increased bacterial adhesion. It was observed that human osteoblast-like cell culture supernatants, which contain secreted components that would be found in the bone environment, increased bacterial adhesion capacity by 2-fold (p = 0.015) compared to the medium control. Moreover, supernatants from osteoblast-like cells stimulated with TNF-α to mimic inflammatory conditions increased bacterial adhesion by almost 5-fold (p = 0.003) without impacting on the overall biomass. Interestingly, the effect of osteoblast-like cell supernatants on bacterial adhesion could be counteracted by the activity of synthetic antibiofilm peptides. Overall, the results of this study demonstrate that factors within the bone environment and products of osteoblast-like cells directly influence S. aureus adhesion and could contribute to biofilm initiation on bone and/or prosthetics implants.

Influence of high glucose and advanced glycation end-products (ages) levels in human osteoblast-like cells gene expression.

BackgroundType 2 diabetes mellitus (T2DM) is associated with an increased risk of osteoporotic fracture. Several factors have been identified as being potentially responsible for this risk, such as alterations in bone remodelling that may have been induced by changes in circulating glucose or/and by the presence of non-oxidative end products of glycosylation (AGEs). The aim of this study is to assess whether such variations generate a change in the gene expression related to the differentiation and osteoblast activity (OPG, RANKL, RUNX2, OSTERIX, and AGE receptor) in primary cultures of human osteoblast-like cells (hOB).MethodsWe recruited 32 patients; 10 patients had osteoporotic hip fractures (OP group), 12 patients had osteoporotic hip fractures with T2DM (T2DM group), and 10 patients had hip osteoarthritis (OA group) with no osteoporotic fractures and no T2DM. The gene expression was analyzed in hOB cultures treated with physiological glucose concentration (4.5 mM) as control, high glucose (25 mM), and high glucose plus AGEs (2 μg/ml) for 24 h.ResultsThe hOB cultures from patients with hip fractures presented slower proliferation. Additionally, the hOB cultures from the T2DM group were the most negatively affected with respect to RUNX2 and OSX gene expression when treated solely with high glucose or with high glucose plus AGEs. Moreover, high levels of glucose induced a major decrease in the RANKL/OPG ratio when comparing the OP and the T2DM groups to the OA group.ConclusionsOur data indicates an altered bone remodelling rate in the T2DM group, which may, at least partially, explain the reduced bone strength and increased incidence of non-traumatic fractures in diabetic patients.

Biological Evaluation of Polydimethylsiloxane Modified by Calcium Phosphate Nanoparticles for Potential Application in Spine Surgery

The biological properties of a composite made of polydimethylsiloxane matrix and two kinds of calcium phosphate nanoparticles (hydroxyapatite, beta tricalcium phosphate) were tested by means of in vitro and in vivo investigations. Chemical and phase analyses of the nanoparticles were performed. The effects of different volume fractions of nanoparticles were investigated. Human osteoblast-like cell cultures (MG 63) demonstrated that the addition of 5–15 vol.% of nanoparticles had a significantly positive effect on osteoblast viability, and that high concentration did not support cell proliferation. The highest rates of osteointegration in pig bone were attained for implants modified by 15 vol.% hydroxyapatite and more intensive formation of new bone tissue was observed. Our study indicates that hydroxyapatite probably better imitates the composition and structure of the mineral phase of real bone.

Preparation and biological evaluation of hydroxyapatite-coated nickel-free high-nitrogen stainless steel

Calcium phosphate was formed on nickel-free high-nitrogen stainless steel (HNS) by chemical solution deposition. The calcium phosphate deposition was enhanced by glutamic acid covalently immobilized on the surface of HNS with trisuccinimidyl citrate as a linker. X-ray diffraction patterns and Fourier transform infrared spectra showed that the material deposited on glutamic acid-immobilized HNS within 24 h was low-crystallinity calcium-deficient carbonate-containing hydroxyapatite (HAp). The biological activity of the resulting HAp-coated HNS was investigated by using a human osteoblast-like MG-63 cell culture. The HAp-coated HNS stimulated the alkaline-phosphate activity of the MG-63 culture after 7 days. Therefore, HAp-coated HNS is suitable for orthopedic devices and soft tissue adhesion materials.

Pulsed Low Intensity Electromagnetic Field (PEMF) Affects Cell Cycle of Human Osteoblast-like Cells &lt;i&gt;in &lt;/i&gt;&lt;i&gt;Vi&lt;/i&gt;&lt;i&gt;tro&lt;/i&gt;

High frequency electro magnetic field in the range of 4 - 33 kHz given in low frequency bursts of 7-15 Hz (PEMF) is efficient in stimulating osteoblast proliferation and differentiation in vitro. We hypothesize that osteoblast cell cycle changes following this stimulatory PEMF. In order to explore this effect we used primary explant cultures of human osteoblast-like cells. These cells were subjected to low frequency of 20-30 Hz, h igher frequency of 5-15 kHz electro magnetic fields and to PEM F (5-15 kHz basic frequency in pulses of 20-30 Hz) with maximal magnetic flu x of 10-12 Gauss. The maturation state of the cells was estimated by the measurement of cellular alkaline phosphatase activity and the overall cell death by the LDH activity in culture media. The cell cycle was analyzed by cytometry. The PEMF stimulation significantly increased the proportion of cells in the G1 phase (p<.001) and decreased the proportion of apoptotic and necrotic cells (p<.001), with parallel decrease in cellular alkaline phosphatase and media LDH act ivities. Pure 20-30 Hz or 5-15 kHz electro magnetic stimu lations had no effect on these parameters. Therefore we show that PEM F with a basic frequency in the kHz range provided in pulses in the infrasonic range alters human osteoblast cell cycle by reducing cell death and cellular maturation.

Mineral Trioxide Aggregate–based Endodontic Sealer Stimulates Hydroxyapatite Nucleation in Human Osteoblast-like Cell Culture

IntroductionThe main purpose of this study was to evaluate the biocompatibility and bioactivity of a new mineral trioxide aggregate (MTA)-based endodontic sealer, MTA Fillapex (MTA-F; Angelus, Londrina, Brazil), in human cell culture. MethodsHuman osteoblast-like cells (Saos-2) were exposed for 1, 2, 3, and 7 days to MTA-F, Epiphany SE (EP-SE; SybronEndo, Orange, CA), and zinc oxide–eugenol sealer (ZOE). Unexposed cultures were the control group (CT). The viability of the cells was assessed by MTT assay and the morphology by scanning electron microscopy (SEM). The bioactivity of MTA-F was evaluated by alkaline phosphatase activity (ALP) and the detection of calcium deposits in the culture with alizarin red stain (ARS). Energy-dispersive X-ray spectroscopy (EDS) was used to chemically characterize the hydroxyapatite crystallites (HAP). Saos-2 cells were cultured for 21 days for ARS and SEM/EDS. ARS results were expressed as the number of stained nodules per area. Statistical analysis was performed with analysis of variance and Bonferroni tests (P < .01). ResultsMTA-F exposure for 1, 2, and 3 days resulted in increased cytotoxicity. In contrast, viability increased after 7 days of exposure to MTA-F. Exposure to EP-SE and ZOE was cytotoxic at all time points. At day 7, ALP activity increase was significant in the MTA-F group. MTA-F presented the highest percentage of ARS-stained nodules (MTA-F > CT > EP-SE > ZOE). SEM/EDS analysis showed hydroxyapatite crystals only in the MTA-F and CT groups. In the MTA-F group, crystallite morphology and chemical composition were different from CT. ConclusionsAfter setting, the cytotoxicity of MTA-F decreases and the sealer presents suitable bioactivity to stimulate HAP crystal nucleation.

Synthesis of strontium substituted hydroxyapatite whiskers used as bioactive and mechanical reinforcement material

The strontium substituted hydroxyapatite (SrHA) whiskers with 6.6mol% of Ca replaced by Sr were successfully synthesized via homogeneous precipitation method. The product had a width of 0.2–8μm and length up to 155μm. The human osteoblast-like cell (MG-63) culture showed that Sr substitution could stimulate the proliferation of MG-63 at certain extracted concentrations. Furthermore, the addition of SrHA whiskers could significantly improve the mechanical properties of calcium phosphate cement (CPC). The 5wt.% SrHA whisker reinforced CPC revealed the highest compressive strength of 2.92MPa, which was almost 2-times higher than that of the pure CPC. Our study indicated that SrHA whisker might be a potential candidate as a new bioactive and mechanical reinforcement material for hard tissue regeneration applications.

Human osteoblast-like cell spreading and proliferation on Ti-6Al-7Nb surfaces of varying roughness

There have been a number of attempts to modify the surface of dental implants to improve osseointegration. These modifications include alterations of surface chemistry and roughness. The purpose of this study was to examine the early response of human osteoblast-like cells placed on Ti-6Al-7Nb disks that had a similar grooved surface topography and three different levels of surface roughness (Ra = 0.0374, 0.0911, and 0.2435 µm). Cultures of human osteoblast-like cells revealed no significant difference in initial cell attachment among the various surfaces; however, cell spread was greater on rough surfaces than on glass slides and smoother surfaces. In addition, cell proliferation and Ki-67 expression were increased when cells were cultured on rough surfaces. These results suggest that a greater Ti-6Al-7Nb surface roughness support the initial spread and proliferation of human osteoblast-like cells. Thus, modification of the surface roughness of dental implants might hasten osseointegration. (J Oral Sci 53, 23-30, 2011).

Cytocompatibility of Medical Biomaterials Containing Nickel by Osteoblasts: a Systematic Literature Review

The present review is based on a survey of 21 studies on the cytocompatibility of medical biomaterials containing nickel, as assessed by cell culture of human and animal osteoblasts or osteoblast-like cells. Among the biomaterials evaluated were stainless steel, NiTi alloys, pure Ni, Ti, and other pure metals. The materials were either commercially available, prepared by the authors, or implanted by various techniques to generate a protective layer of oxides, nitrides, acetylides. The observation that the layers significantly reduced the initial release of metal ions and increased cytocompatibility was confirmed in cell culture experiments. Physical and chemical characterization of the materials was performed. This included, e.g., surface characterization (roughness, wettability, corrosion behavior, quantity of released ions, microhardness, and characterization of passivation layer). Cytocompatibility tests of the materials were conducted in the cultures of human or animal osteoblasts and osteoblast-like cells. The following assays were carried out: cell proliferation and viability test, adhesion test, morphology (by fluorescent microscopy or SEM). Also phenotypic and genotypic markers were investigated. In the majority of works, it was found that the most cytocompatible materials were stainless steel and NiTi alloy. Pure Ni was rendered and less cytocompatible. All the papers confirmed that the consequence of the formation of protective layers was in significant increase of cytocompatibility of the materials. This indicates the possible further modifications of the manufacturing process (formation of the passivation layer).

The Role of Activin A in The Human Osteoblast Cell Cycle: A Preliminary Experimental in Vitro Study

Animal studies have previously shown that activin A enhances osteoblast proliferation IN VITRO and increases bone formation and bone mechanical strength IN VIVO. For the further understanding of its action in human osteoblast, we studied the pattern of a cell cycle response to the treatment with activin A. We hypothesize that activin A alters the cell cycle pattern of human osteoblast. Primary cultures of human osteoblast-like cells were treated by activin A in a biologically effective concentration (100 ng/mL). The cells in cultured samples were counted, assayed for cellular alkaline phosphatase activity and calcitonin expression, LDH activity in the medium, cellular BrdU incorporation, cell cycle cytometry and compared to untreated controls. The treated by activin A cells responded by a significant shift toward the G1 phase of the cell cycle with parallel decrease in cell death rate (lower LDH activity and less necrotic cells in cytometric analysis). The treated cells also showed a lower alkaline phosphatase activity and calcitonin expression, indicating their undifferentiated state, and didn't change their proliferation rate. The number of cells in culture increased following treatment with activin A. We show that activin A increases the net osteoblast number in culture by reducing the cell death rate without affecting the cell proliferation. These findings should be part of cellular pathways that are involved in the initial stages of bone tissue generation.

Molecular and mechano-biology of collagen gel contraction mediated by human MG-63 cells: involvement of specific intracellular signaling pathways and the cytoskeleton

Culture of human osteoblast-like MG-63 cells within collagen gels results in the generation of intrinsic stress. Release of such collagen gels from attachment results in gel contraction and enhanced MMP-1, MMP-3, and alpha2 integrin mRNA levels. To understand the potential role of microtubules and signaling pathways involved in MG-63 cell-mediated contraction and gene expression, cells were cultured in collagen gels. After 24 h collagen gels were released, then immediately treated with nocodazole or specific protein kinase inhibitors. Contraction was assessed, RNA isolated, and real-time PCR analysis performed. Treatment with high concentrations of a microtubule depolymerization agent, nocodazole, enhanced early contraction and led to elevated mRNA levels for MMP-3, whereas low concentrations inhibited contraction at later time points and did not affect mRNA levels. ROCK inhibitor treatment (Y27632) inhibited collagen gel contraction and led to depressed mRNA levels. The ERK1/2 inhibitor U0126 did not affect contraction, but treatment led to depressed MMP-1, MMP-3, and alpha2 mRNA levels. The p38MAPK inhibitor SB203580 modestly affected contraction, but did not affect mRNA levels. These results suggest the potential role of cytoskeletal integrity and multiple kinase signaling pathways in specific bone-remodeling events.

The use of confluence stages does not decrease the overall variability in primary human osteoblasts but can give additional information on differentiation in vitro.

Primary cultures of human osteoblast-like cells are frequently used to study osteoblast function. Due to inhomogeneous growth of primary osteoblasts in culture, it is of interest if the use of confluence stages for analysis would reduce the overall variability. Consequently, we have tested the influence of degree of confluence and passage number on the growth and differentiation of human primary osteoblast-like cells. Phenotypic features of primary human osteoblast-like cells were compared at four successive cell densities defined as stage of confluence I (50%), stage II (75%), stage III (100%) and stage IV (5 days post confluence). The stability of the system was also tested by comparing these observations obtained using cells from the 2nd and 4th passages. As a sign for further differentiation, the number of AP-positive cells increased with a decrease in proliferation. The secretion of procollagen-I decreased to 50% during culture while procollagen I mRNA doubled from proliferation to confluence. A constant activity of alkaline phosphatase, procollagen-I secretion and procollagen I gene expression over passages was seen together with a decrease in growth. The paper introduces a potential model of osteoblastic differentiation in vitro for human primary osteoblast-like cells. We were able to show an increasing differentiation with a decrease in proliferation and the stability of this differentiation behaviour over cell passages in this model, but we were not able to reduce the overall variability.

The effect of dual frequency cyclic compression on matrix deposition by osteoblast-like cells grown in 3D scaffolds and on modulation of VEGF variant expression

As a strategy to optimise osteointegration of biomaterials by inducing proper extracellular matrix synthesis, and specifically angiogenic growth factor production and storage, we tested the effects of cyclic mechanical compression on 3D cultures of human osteoblast-like cells. MG-63 cells were seeded into 3D porous hydroxyapatite ceramics under vacuum to enable a homogenous cellular distribution. A four-day culture period allowed cell proliferation throughout the scaffolds. Low amplitude cyclic compressions were then applied to the scaffolds for 15 min with different regimens generated by the ZetOS™ system. A 3 Hz sinusoidal (sine) signal increased slightly collagen and fibronectin expression. When 50 Hz or 100 Hz vibrations were superimposed to the 3 Hz signal, matrix protein expression was down-regulated. In contrast, adding a 25 Hz vibration up-regulated significantly collagen and fibronectin. Moreover, expression of a matrix-bound variant of vascular endothelial growth factor-A (VEGF-A) was specifically stimulated compared to control or 3 Hz sine, and non-soluble VEGF protein was increased. Our study enabled us to identify low-amplitude, high-frequency strain regimen able to increase major matrix proteins of bone tissue and to regulate the expression of VEGF variants, showing that an appropriate combined loading has the potential to functionalise cellularized bone-like constructs.