Response Of Human Osteoblast-like Cells Research Articles

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.

Biological effect of hydrothermally synthesized silica nanoparticles within crystalline hydroxyapatite coatings for titanium implants

Development of functional coatings for artificial bone implants that strengthen the osseointegration and accelerate bone healing processes is urgently needed in the biomedical field. In this study we present biological effect of novel composite coatings with different concentration of silica nanoparticles within crystalline hydroxyapatite matrix (HAp-SiO2) synthesized on titanium under hydrothermal conditions. Samples were analyzed for their elemental composition, structure, bioactivity and in vitro cytotoxicity. The results indicate the formation and homogeneous distribution of silica nanoparticles on the surface of hexagonal hydroxyapatite (HAp) crystals. The coatings show improved bioactivity in comparison with pure HAp after 4 days of immersion in simulated body fluid (SBF). The responses of human osteoblast-like cells (MG-63) cultured onto the synthesized materials provide evidence that HAp-SiO2 composites exhibit good biocompatibility. We propose that this is because HAp-SiO2 composites favor biomineralization process with cell proliferation remaining unaffected, regardless of the amount of silica. Furthermore, SEM and fluorescence measurements demonstrate that HAp-SiO2 had positive effect on cell morphology, favoring cell adhesion.

Cell responses to titanium treated by a sandblast-free method for implant applications

Sandblast and acid-etching (SLA) is the most prevalent treatment to titanium implants, while residual sand particles are inevitably introduced on SLA titanium surfaces. NH4OH and H2O2 mixture was used to etch titanium plates (E) and titanium bars (EB), aiming at substituting sandblast procedure. To study the effects of different scale rough structures on cell response of Human osteoblast-like cells (MG63), traditional H2SO4 and HCl mixture was also used to further etch the titanium plates above (DE). Holes of 10–20μm were obtained on E and DE surfaces, which are very close to the size of osteoblasts. Surfaces with micro/nano and micro/submicro hierarchical structures were obtained on the treated titanium. As-prepared E, DE and EB surfaces are hydrophilic, while only EB stayed hydrophilic after 5days' exposure to air. MG63 cultured on E and EB surfaces showed higher proliferation rate and attachment area than on DE and P surfaces. E and DE showed higher alkaline phosphatases (ALP) activity after 7 and 14days of osteoinduction, while EB showed the highest osteopontin (OPN) and bone sialoprotein (BSP) production after 21days of osteoinduction. These results indicate that E and EB surfaces boost the proliferation and osteogenic differentiation of MG63 without introducing sand particles. This is a promising treatment to titanium implant.

Enhanced Hydrophilicity and Biocompatibility of Dental Zirconia Ceramics by Oxygen Plasma Treatment.

Surface properties play a critical role in influencing cell responses to a biomaterial. The objectives of this study were (1) to characterize changes in surface properties of zirconia (ZrO2) ceramic after oxygen plasma treatment; and (2) to determine the effect of such changes on biological responses of human osteoblast-like cells (MG63). The results indicated that the surface morphology was not changed by oxygen plasma treatment. In contrast, oxygen plasma treatment to ZrO2 not only resulted in an increase in hydrophilicity, but also it retained surface hydrophilicity after 5-min treatment time. More importantly, surface properties of ZrO2 modified by oxygen plasma treatment were beneficial for cell growth, whereas the surface roughness of the materials did not have a significant efficacy. It is concluded that oxygen plasma treatment was certified to be effective in modifying the surface state of ZrO2 and has the potential in the creation and maintenance of hydrophilic surfaces and the enhancement of cell proliferation and differentiation.

Surfactant as a Critical Factor When Tuning the Hydrophilicity in Three-Dimensional Polyester-Based Scaffolds: Impact of Hydrophilicity on Their Mechanical Properties and the Cellular Response of Human Osteoblast-Like Cells

In tissue engineering, the hydrophilicity of porous scaffolds is essential and influences protein and cell adhesion as well as nutrient diffusion into the scaffold. The relative low hydrophilicity of degradable polyesters, which limits diffusion of nutrients, is a major drawback in large porous scaffolds of these materials when used for bone tissue engineering and repair of critical size defects. Designing porous biodegradable polymer scaffolds with improved hydrophilicity, while maintaining their mechanical, thermal, and degradation properties is therefore of clinical interest. Here, surfactants were used to tune the hydrophilicity and material properties. A total of 3-20% (w/w) of surfactant, polysorbate 80 (Tween 80), was used as an additive in poly(l-lactide-co-1,5-dioxepan-2-one) [poly(LLA-co-DXO)] and poly(l-lactide)-co-(ε-caprolactone) [poly(LLA-co-CL)] scaffolds. A significantly decreased water contact angle was recorded for all the blends and the crystallinity, glass transition temperature and crystallization temperature were reduced with increased amounts of surfactant. Copolymers with the addition of 3% Tween 80 had comparable mechanical properties as the pristine copolymers. However, the E-modulus and tensile stress of copolymers decreased significantly with the addition of 10 and 20% Tween 80. Initial cell response of the material was evaluated by seeding human osteoblast-like cells (HOB) on the scaffolds. The addition of 3% Tween 80 did not significantly influence cell attachment or proliferation, while 20% Tween 80 significantly inhibited osteoblast proliferation. RT-PCR results showed that 3% Tween 80 stimulated mRNA expression of alkaline phosphatase (ALP), osteoprotegerin (OPG), and bone morphogenetic protein-2 (BMP-2).

Effects of density of anisotropic microstamped silica thin films on guided bone tissue regeneration—In vitrostudy

The growing demand for better implant aesthetics has led to increased research on the development of all-ceramic dental implants. The use of microtextured coatings with enhanced properties has been presented as a viable way to improve tissue integrability of all-ceramic implants. The aim of this study was to evaluate the effects of different densities of anisotropic microtextured silica thin films, which served as a model coating, on the behavior of human osteoblast-like cells. The differential responses of human osteoblast-like cells to anisotropic silica microtextures with varying densities, produced via a combination of sol-gel and soft lithography processing, were evaluated in terms of alignment, elongation (using fluorescence microscopy), overall cellular activity, and the expression/activity levels of alkaline phosphatase (ALP). Statistical analysis was conducted using one-way ANOVA/Tukey HSD post hoc test. The thin films were thoroughly characterized via scanning electron microscopy/energy dispersive spectroscopy, Fourier transform infrared, and contact angle measurements. Thin film characterization revealed increased nanoscale roughness and reduced wettability on the micropatterned surfaces. Cell culture experiments indicated that the microtextures induced cell alignment, elongation, and guided colonization on the surface. Cells cultured on denser micropatterns exhibited increased metabolic activity (t = 14-21 days). The early expression/activity levels of ALP released into the medium were found to be significantly higher only on the least dense micropattern. These results suggest the possibility that microstructured silica thin films could be used to guide and enhance peri-implant cell/tissue responses, potentially improving tissue integration for metallic and all-ceramic dental implants.

An in vitro evaluation of the responses of human osteoblast-like SaOs-2 cells to SLA titanium surfaces irradiated by erbium:yttrium–aluminum–garnet (Er:YAG) lasers

Erbium:yttrium-aluminum-garnet (Er:YAG) laser treatment is an effective option for the removal of bacterial plaques. Many studies have shown that Er:YAG lasers cannot re-establish the biocompatibility of titanium surfaces. The aim of this study was to evaluate the responses of the human osteoblast-like cell line, SaOs-2, to sand-blasted and acid-etched (SLA) titanium surface irradiation using different energy settings of an Er:YAG laser by examining cell viability and morphology. Forty SLA titanium disks were irradiated with an Er:YAG laser at a pulse energy of either 60 or 100 mJ with a pulse frequency of 10 Hz under water irrigation and placed in a 24-well plate. Human osteoblast-like SaOs-2 cells were seeded onto the disks in culture media. Cells were then kept in an incubator with 5% carbon dioxide at 37 °C. Each experimental group was divided into two smaller groups to evaluate cell morphology by scanning electron microscope and cell viability using 3-4,5-dimethylthiazol 2,5-diphenyltetrazolium bromide test. In both the 60 and the 100 mJ experimental groups, spreading morphologies, with numerous cytoplasmic extensions, were observed prominently. Similarly, a majority of cells in the control group exhibited spreading morphologies with abundant cytoplasmic extensions. There were no significant differences among the laser and control groups. The highest cell viability rate was observed in the 100 mJ laser group. No significant differences were observed between the cell viability rates of the two experimental groups (p = 1.00). In contrast, the control group was characterized by a significantly lower cell viability rate (p < 0.001). Treatments with an Er:YAG laser at a pulse energy of either 60 or 100 mJ do not reduce the biocompatibility of SLA titanium surfaces. In fact, modifying SLA surfaces with Er:YAG lasers improved the biocompatibility of these surfaces.

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).

Sphene ceramics for orthopedic coating applications: An in vitro and in vivo study

The host response to titanium alloy (Ti–6Al–4V) is not always favorable as a fibrous layer may form at the skeletal tissue–device interface, causing aseptic loosening. Recently, sphene (CaTiSiO 5) ceramics were developed by incorporating Ti in the Ca–Si system, and found to exhibit improved chemical stability. The aim of this study is to evaluate the in vitro response of human osteoblast-like cells, human osteoclasts and human microvascular endothelial cells to sphene ceramics and determine whether coating Ti–6Al–4V implants with sphene enhances anchorage to surrounding bone. The study showed that sphene ceramics support human osteoblast-like cell attachment with organized cytoskeleton structure and express increased mRNA levels of osteoblast-related genes. Sphene ceramics were able to induce the differentiation of monocytes to form functional osteoclasts with the characteristic features of f-actin and α vβ 3 integrin, and express osteoclast-related genes. Human endothelial cells were also able to attach and express the endothelial cell markers ZO-1 and VE-Cadherin when cultured on sphene ceramics. Histological staining, enzyme histochemistry and immunolabelling were used for identification of mineralized bone and bone remodelling around the coated implants. Ti–6Al–4V implants coated with sphene showed new bone formation and filled the gap between the implants and existing bone in a manner comparable to that of the hydroxyapatite coatings used as control. The new bone was in direct contact with the implants, whereas fibrous tissue formed between the bone and implant with uncoated Ti–6Al–4V. The in vivo assessment of sphene-coated implants supports our in vitro observation and suggests that they have the ability to recruit osteogenic cells, and thus support bone formation around the implants and enhance osseointegration.

Biological response of human bone cells to zinc-modified Ca–Si-based ceramics

Calcium silicate (CaSiO 3) ceramics have received considerable attention in recent years due to their excellent bioactivity and degradability. However, their poor chemical stability limits their biological applications. Hardystonite (Ca 2ZnSi 2O 7) ceramics are Ca–Si-based materials developed by incorporating zinc into the Ca–Si system to improve their chemical stability. However, the biological responses of Ca 2ZnSi 2O 7 to bone cells are unknown. The objective of this study is to investigate and compare the in vitro responses of human osteoblast-like cells (HOBs) and osteoclasts when cultured on Ca 2ZnSi 2O 7 and CaSiO 3 ceramic disks. The ability of Ca 2ZnSi 2O 7 ceramics to support HOB attachment, cytoskeleton organization, proliferation and differentiation was assessed by scanning electron microscopy, confocal microscopy, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, alkaline phosphatase activity and quantitative real-time polymerase chain reaction. Our results show that Ca 2ZnSi 2O 7 supported HOB attachment with a well-organized cytoskeleton structure, and significantly increased cellular proliferation and differentiation compared to CaSiO 3. In addition, Ca 2ZnSi 2O 7 showed increased expression levels of osteoblast-related mRNAs (alkaline phosphatase, collagen type I, osteocalcin, receptor activator of NF κB ligand and osteoprotegerin) compared to CaSiO 3. Ca 2ZnSi 2O 7 ceramic supported the formation of mature and functional osteoclasts and formed resorption imprints. On CaSiO 3 ceramics, the cells failed to differentiate from the monocytes into osteoclasts. Taken together, these results indicate that Hardystonite ceramics are conducive to both types of bone cells, osteoblast-like cells and osteoclasts, suggesting their potential use for skeletal tissue regeneration and as coatings onto currently available orthopedic and dental implants.

Response of human osteoblast-like cells to fluid flow shear: A potential role for the microtubule network and primary ciliums

Introduction: A limited understanding of the cellular mechanisms governing bone mechanotransduction has inhibited the development of clinical treatments for a variety of bone disorders, including osteoporosis, osteoarthritis and microgravity-associated bone atrophy. The cytoskeleton is thought to play a role in cellular mechanotransduction, however the exact mechanism in bone cells has not yet been clearly elucidated. Studies involving cytoskeletal inhibitors have not generally considered secondary effects on cellular organelles such as the primary cilia. These cellular projections could account for the disparity between shear stresses predicted to occur in vivo and the minimum threshold of membrane deformation required to elicit a cellular response in vitro.&#x0D; Methods: MG-63 (human osteoblast-like) cells were cultured in vitro. Cultures were exposed to intermittent cyclic fluid flow shear stress (1 Pa amplitude), for 8 or 12 hrs. Some cultures were loaded in the presence of nocodazole (a microtubule inhibitor) or cytochalasin D (an actin filament inhibitor). The cellular response was analyzed through RT-PCR assessment of messenger RNA levels for specific molecules related to matrix metabolism. The effects of drug treatments on cytoskeletal disorganization and the primary cilia were assessed with immunocytochemistry and electron microscopy.&#x0D; Results: In untreated cultures, shear stress was associated with significant increases in mRNA levels for collagen I and matrix metalloproteinases 1 and 3, for both time points assessed. These increases were maintained in cultures loaded in the presence of cytochalasin D, but were almost completely abrogated in nocodazole-treated cultures. Cytoskeletal inhibitors exerted some dose-dependent effects on length and structure of primary cilia in MG-63 cells.&#x0D; Conclusions: The microtubule network appears to be necessary for some shear-induced responses of osteoblast-like cells. MG-63 cells possess primary cilia, organelles that could amplify fluid flow shear, accounting for some apparent contradictions between studies related to osteoblast mechanosensitivity. Since these structures are composed of microtubules, the observation that microtubule disruptors inhibit the shear response of osteoblast-like cells suggests the primary cilium may have a role in osteoblast mechanotransduction. The effects of cytoskeletal inhibitors on cilium structure may explain the conflicting results of earlier mechanotransduction studies.

Ti-6Al-7Nb promotes cell spreading and fibronectin and osteopontin synthesis in osteoblast-like cells

The purpose of this study was to compare the early response of human osteoblast-like cells (SaOS-2) on commercially pure titanium (cpTi) and titanium-6-aluminium-7-niobium (Ti-6Al-7Nb) using glass slide as a control. In terms of cell attachment, no significance was observed when cells were seeded on the materials. However, morphological analysis by scanning electron microscope revealed that cells on Ti-6Al-7Nb showed better spreading after 4 hrs. After 48 hrs, both Western analysis and reverse transcription polymerase chain reaction analyses showed that cells cultured on Ti-6Al-7Nb synthesized a higher amount of fibronectin and osteopontin as compared to cells seeded on cpTi or on glass slide. These results suggest that Ti-6Al-7Nb possess a good potential to support SaOS-2 cells on spreading and fibronectin and osteopontin synthesis, therefore, this material may be one of a candidate material used in implant dentistry.

Influence of surface pretreatment of titanium- and cobalt-based biomaterials on covalent immobilization of fibrillar collagen

Collagen type-I is a major component of the extracellular matrix of most tissues and it is increasingly utilized for surface engineering of biomaterials to accelerate receptor-mediated cell adhesion. In the present study, coatings with layers of fibrillar type-I collagen were prepared on titanium, titanium alloy, and cobalt alloy to improve initial osteoblast adhesion and implant–tissue integration. To suppress the quick in vivo degradation rate of collagen the deposited layers were covalently immobilized at the metal surfaces as well as chemically cross-linked. The application of different oxidation techniques to the metallic substrates resulted in surfaces with varying hydroxyl group contents, which directly influenced the amount of immobilized silane coupling agents. It was found that a high density of surface-bound coupling agents increased the stability of the covalently linked collagen layers. After coating of metallic biomaterials with a cross-linked collagen layer, an improved cellular response of human osteoblast-like cells (MG-63) in vitro could be recognized.

Compositional effects on the formation of a calcium phosphate layer and the response of osteoblast-like cells on polymer-bioactive glass composites

Biodegradable polymer-ceramic composites are attractive systems for bone tissue engineering applications. These composites have the combined advantages of the component phases, as well as the inherent ease in optimization where desired material properties can be tailored in a well-controlled manner. This study focuses on the optimization of a polylactide- co-glycolide (PLAGA) and 45S5 bioactive glass (BG) composite for bone tissue engineering. The first objective is to examine the effects of composition or overall BG content on the formation of a Ca–P layer on the PLAGA–BG composite. It is expected that with increasing BG content (0%, 10%, 25%, 50% by weight), the required incubation time in a simulated body fluid (SBF) for the composite to form a detectable surface Ca–P layer will decrease. Both the kinetics and the chemistry will be determined using SEM+EDAX, FTIR, and μ -CT methods. Solution phosphorous and calcium concentrations will also be measured. The second objective of the study is to determine the effects of BG content on the maturation of osteoblast-like cells on the PLAGA–BG composite. It is hypothesized that mineralization will increase with increasing BG content, and the composite will support the proliferation and differentiation of osteoblasts. Specifically, cell proliferation, alkaline phosphatase activity and mineralization will be monitored as a function of BG content (0%, 10%, 50% by weight) and culturing time. It was found that the kinetics of Ca–P layer formation and the resulting Ca–P chemistry were dependent on BG content. The response of human osteoblast-like cells to the PLAGA–BG composite was also a function of BG content. The 10% and 25% BG composite supported greater osteoblast growth and differentiation compared to the 50% BG group. The results of this study suggest that there is a threshold BG content which is optimal for osteoblast growth, and the interactions between PLAGA and BG may modulate the kinetics of Ca–P formation and the overall cellular response.

Response of Human Osteoblast-Like Cells and Fibroblasts to Titanium Alloy Nitrided under Glow Discharge Conditions

Cell adhesion to biomaterials and cell proliferation are affected, among others, by the chemical composition and surface microtopography of the biomaterial. These are, in turn, influenced by sterilisation processes. The aim of this study was to evaluate the sensitivity of osteoblasts and fibroblasts to TiN surfaces deprived of an oxide film or with an enhanced oxide film due to sterilisation in plasma or in an autoclave, respectively. Type TiN+T2N+aTi(N) nitrided surface layers were produced under glow discharge conditions and sterilized in plasma Sterrad 100 and in a steam autoclave. The results provide evidence that nitrided surface layers improved wearand corrosion resistance of the titanium alloy and that these features were not influenced by sterilisation procedures that resulted in various oxide films. In vitro study of human fibroblast and osteoblast-like cells cultured on TiN surfaces modified by sterilisation processes showed that cells respond to the presence of an enhanced oxide film by aggregating, exhibiting lower viability and increased apoptosis, while to a surface deprived of oxides, by regular spreading and high viability. It can be concluded that differences in surface composition and topography of the nitrided surface layers due to various types of sterilisation elicit specific cell responses, and plasma sterilisation seems to be optimal for bone implants with this surface layer.

Modulation of the responses of human osteoblast-like cells to physiologic mechanical strains by biomaterial surfaces

In a previous study we demonstrated that MG-63 cells cultured on Ti-6Al-4V discs covered by alumina ceramic and submitted to intermittent mechanical strain (IMS) presented morphological alteration associated with enhanced differentiation. Here we examine how the mechanical response of osteoblasts can be modulated by the nature of the substrate. MG-63 cells were cultured on four materials: polystyrene and Ti-6Al-4V (average roughness =0.48 μm) as smooth substrates; Ti-6Al-4V (average roughness =5.76 μm) and Ti-6Al-4V covered with alumina (average roughness =5.21 μm) as rough substrates. Mechanical strains were applied for 15 min, three times a day for 1–5 days with a 600 μstrains magnitude and a 0.25 Hz frequency. IMS stimulated alkaline phosphatase activity by 25–35% on all substrates and had no effect on cell growth on either substrate. Fibronectin (FN) was chosen as representative of cell-matrix interaction. FN production was increased by 60% after 1 day of stretching only on alumina-coated discs. FN organization examined on smooth substrates was affected by 5 days of IMS, showing a thickening of the fibres. The same modifications induced by IMS were previously observed on alumina-covered discs. Vinculin expression was not affected by IMS whatever the substrate. Cell–cell interactions were determined by N-cadherin immunoblotting. N-cadherin expression was increased by IMS specifically on rough substrates. Our results suggest that the nature of the surface did not influence the up-regulation of alkaline phosphatase activity induced by IMS, but modulates specifically cell–substrate as well as cell–cell interactions in response to IMS.

Three-dimensional, bioactive, biodegradable, polymer-bioactive glass composite scaffolds with improved mechanical properties support collagen synthesis and mineralization of human osteoblast-like cells in vitro.

In the past decade, tissue engineering-based bone grafting has emerged as a viable alternative to biological and synthetic grafts. The biomaterial component is a critical determinant of the ultimate success of the tissue-engineered graft. Because no single existing material possesses all the necessary properties required in an ideal bone graft, our approach has been to develop a three dimensional (3-D), porous composite of polylactide-co-glycolide (PLAGA) and 45S5 bioactive glass (BG) that is biodegradable, bioactive, and suitable as a scaffold for bone tissue engineering (PLAGA-BG composite). The objectives of this study were to examine the mechanical properties of a PLAGA-BG matrix, to evaluate the response of human osteoblast-like cells to the PLAGA-BG composite, and to evaluate the ability of the composite to form a surface calcium phosphate layer in vitro. Structural and mechanical properties of PLAGA-BG were measured, and the formation of a surface calcium phosphate layer was evaluated by surface analysis methods. The growth and differentiation of human osteoblast-like cells on PLAGA-BG were also examined. A hypothesis was that the combination of PLAGA with BG would result in a biocompatible and bioactive composite, capable of supporting osteoblast adhesion, growth and differentiation, with mechanical properties superior to PLAGA alone. The addition of bioactive glass granules to the PLAGA matrix resulted in a structure with higher compressive modulus than PLAGA alone. Moreover, the PLAGA-BA composite was found to be a bioactive material, as it formed surface calcium phosphate deposits in a simulated body fluid (SBF), and in the presence of cells and serum proteins. The composite supported osteoblast-like morphology, stained positively for alkaline phosphatase, and supported higher levels of Type I collagen synthesis than tissue culture polystyrene controls. We have successfully developed a degradable, porous, polymer bioactive glass composite possessing improved mechanical properties and osteointegrative potential compared to degradable polymers of poly(lactic acid-glycolic acid) alone. Future work will focus on the optimization of the composite scaffold for bone tissue-engineering applications and the evaluation of the 3-D composite in an in vivo model.

Proliferation of human-derived osteoblast-like cells depends on the cycle number and frequency of uniaxial strain

We tested the hypothesis whether the number of applied load cycles and the frequency of uniaxial strain have an effect on proliferation of human bone derived osteoblast-like cells. A new approach was developed in order to differentiate between the effects of frequency and the effects of cycle number and strain duration. Monolayers of subconfluently grown cells were stretched in rectangular silicone dishes with cyclic predominantly uniaxial movement along there longitudinal axes. Strain was applied over 2 days varying the number of applied load cycles (4–3600) at a constant frequency (1 Hz) or varying the frequency (0.1–30 Hz) at a constant number of applied cycles (1800) or at a constant strain duration (5 min). At a constant frequency, proliferative response increases (103%) with the number of applied cycles until a cycle number maximum (1800 cycles) was reached. 3600 cycles reduced cell number (43%) in contrast to the maximum. The variation of the frequency of applied strain tended to result in slight differences with regard to cell proliferation when cycle number was left constant. However, combined with an appropriate number of cycles there was an optimal frequency (1 Hz) as stimulus for bone cell proliferation (84%). A higher frequency (30 Hz) in combination with a high cycle number (9000) reduced cell number to control level (4%). This study demonstrates a frequency and cycle number dependent proliferative response of human osteoblast-like cells. It could be shown that effects of the frequency should not be considered separately from the effects of the cycle number.

Glass reinforced hydroxyapatite for hard tissue surgery—Part II: in vitro evaluation of bone cell growth and function

Hydroxyapatite (HA)-based materials are considered to be potentially useful as bone implant materials, particularly those reinforced with glass to improve mechanical strength. However, the precise effects of glass-reinforced HA on the growth and functions of bone cells are still unclear. The present study has therefore examined the response of human osteoblast-like cells to HA and HA reinforced with two different proportions of glass, namely 2.5% and 5%. All materials enabled the cells to attach and proliferate during 7 days in culture and, although the growth was less than on control plastic surfaces, there was no deleterious effect of the 5% glass composite compared with HA alone. Flow cytometry analysis showed that there was no effect on cell size and granularity, but there were marked and highly selective changes in the expression of certain connective tissue proteins. Thus, while bone sialoprotein and osteonectin were down-regulated on HA alone, the expression of these antigens was relatively enhanced on the composite materials, and collagen type I was also up-regulated on the glass-reinforced HA. Thus, modulation of the glass composition of HA materials could be used to produce not only improved mechanical strength, but also enhanced biocompatibility.

Designing biomaterials to direct biological responses.

We have set forth a design strategy for creating biomimetic materials that direct the formation of tissue surrounding implants or regeneration within porous scaffolds. Our studies have established that heterogeneous mimetic peptide surfaces (MPS) containing both the -RGD- (cell-binding) and-FHRRIKA- (putative heparin-binding) peptides, unique to BSP, in the ratio of 75:25 (MPS II) or 50:50 (MPS III) proved to be more biologically relevant and specific for RCO cell function. The initial response of human osteoblast-like cells to these surfaces was mediated by the collagen (alpha 2 beta 1) and vitronectin receptors (alpha v beta 3), whereas the vitronectin receptor alone dominated longer-term events (> 30 min). MPS II and III surfaces enhanced cell spreading and long-term events such as mineralization of the extracellular matrix compared to homogenous peptide surfaces and controls. Furthermore, extensive mineralization of the ECM deposited by RCOs occurred when the peptide was coupled to an interfacial interpenetrating polymer network (IPN) that resisted protein deposition (i.e., non-specific adsorption) and fouling. Work on thermo-reversible P(NIPAAm-co-AAc) hydrogels demonstrated the ability to create materials that can be delivered to the body in a minimally invasive manner and support tissue regeneration. These hydrogels can be modified to incorporate biofunctional components such as the biomimetic peptides, theoretically enhancing their ability to foster tissue regeneration. These results suggest that biomaterials can be engineered to mimic ECM components of bone (e.g., various organs) by grafting peptides in the appropriate ratios of the cell and heparin-binding domains, and ultimately modulate the expression of the osteoblast cell phenotype. Approaches similar to the one presented in this work can be used to design materials for hybrid artificial organs and other tissues.