Nano rough micron patterned titanium for directing osteoblast morphology and adhesion

Previous studies have demonstrated greater functions ofosteoblasts (bone-forming cells) on nanophase compared with conventional metals. Nanophase metals possess a biologically inspired nanostructured surface that mimics the dimensions of constituent components in bone, including collagen and hydroxyapatite. Not only do these components possess dimensions on the nanoscale, they are aligned in a parallel manner creating a defined orientation in bone. To date, research has yet to evaluate the effect that organized nanosurface features can have on the interaction of osteoblasts with material surfaces. Therefore, to determine if surface orientation of features can mediate osteoblast adhesion and morphology, this study investigated osteoblast function on patterned titanium substrates containing alternating regions of micron rough and nano rough surfaces prepared by novel electron beam evaporation techniques. This study was also interested in determining whether or not the size of the patterned regions had an effect on osteoblast behavior and alignment. Results indicated early controlled osteoblast alignment on these patterned materials as well as greater osteoblast adhesion on the nano rough regions of these patterned substrates. Interestingly, decreasing the width of the nano rough regions (from 80 microm to 22 microm) on these patterned substrates resulted in a decreased number of osteoblasts adhering to these areas. Changes in the width of the nano rough regions also resulted in changes in osteoblast morphology, thus, suggesting there is an optimal pattern dimension that osteoblasts prefer. In summary, results of this study provided evidence that aligned nanophase metal features on the surface of titanium improved early osteoblast functions (morphology and adhesion) promising for their long term functions, criteria necessary to improve orthopedic implant efficacy.     

Less harmful acidic degradation of poly(lacticco-glycolic acid) bone tissue engineering scaffolds through titania nanoparticle addition

In the last 10 years, biodegradable aliphatic polyesters, such as poly(lactic-co-glycolic acid) (PLGA), have attracted increasing attention for their use as scaffold materials in bone tissue engineering because their degradation products can be removed by natural metabolic pathways. However, one main concern with the use of these specific polymers is that their degradation products reduce local pH, which in turn induces an inflammatory reaction and damages bone cell health at the implant site. Thus, the objective of the present in vitro study was to investigate the degradation behavior of PLGA when added with dispersed titania nanoparticles. The results of this study provided the first evidence that the increased dispersion of nanophase titania in PLGA decreased the harmful change in pH normal for PLGA degradation. Moreover, previous studies have demonstrated that the increased dispersion of titania nanoparticles into PLGA significantly improved osteoblast (bone-forming cell) functions (such as adhesion, collagen synthesis, alkaline phosphatase activity, and calcium-containing minerals deposition). In this manner, nanophase titania-PLGA composites may be promising scaffold materials for more effective orthopedic tissue engineering applications.     

Charge and potential of a dust grain versus the intergrain distance and establishment of the latter in a low-pressure plasma

Results from experimental studies of ordered dust structures in plasma are reviewed. The experimental conditions and the data on the grain size and intergrain distance in plasma dust crystals are analyzed. It is shown that intergrain distance is a function of the grain size. The range of the ratio of the dust grain size to the Debye radius within which plasma dust crystals can form is determined. A volume cell surrounding a dust grain in plasma is considered. It is found that the potential and charge of the grain depend substantially on the intergrain distance. The charge, potential, and potential energy of a dust grain in a plasma dust crystal, as well as the electrostatic force exerted by the plasma field on the grain, are calculated by the method of molecular dynamics as functions of the intergrain distance. The corresponding analytic approximations and the criterion for the establishment of a steady-state intergrain distance are proposed.     

Selective adhesion and mineral deposition by osteoblasts on carbon nanofiber patterns

In an effort to develop better orthopedic implants, osteoblast (bone-forming cells) adhesion was determined on microscale patterns (30 microm lines) of carbon nanofibers placed on polymer substrates. Patterns of carbon nanofibers (CNFs) on a model polymer (polycarbonate urethane [PCU]) were developed using an imprinting method that placed CNFs in selected regions. Results showed the selective adhesion and alignment of osteoblasts on CNF patterns placed on PCU. Results also showed greater attraction forces between fibronectin and CNF (compared with PCU) patterns using atomic force microscope force-displacement curves. Because fibronectin is a protein that mediates osteoblast adhesion, these results provide a mechanism of why osteoblast adhesion was directed towards CNF patterns. Lastly, this study showed that the directed osteoblast adhesion on CNF patterns translated to enhanced calcium phosphate mineral deposition along linear patterns of CNFs on PCU. Since CNFs are conductive materials, this study formulated substrates that through electrical stimulation could be used in future investigations to further promote osteoblasts to deposit anisotropic patterns of calcium-containing mineral similar to that observed in long bones.     

Inhibition of Rac and ROCK Signalling Influence Osteoblast Adhesion,Differentiation and Mineralization on Titanium Topographies

Reducing the time required for initial integration of bone-contacting implants with host tissues would be of great clinical significance. Changes in osteoblast adhesion formation and reorganization of the F-actin cytoskeleton in response to altered topography are known to be upstream of osteoblast differentiation, and these processes are regulated by the Rho GTPases. Rac and RhoA (through Rho Kinase (ROCK)). Using pharmacological inhibitors, we tested how inhibition of Rac and ROCK influenced osteoblast adhesion, differentiation and mineralization on PT (Pre-treated) and SLA (sandblasted large grit, acid etched) topographies. Inhibition of ROCK, but not Rac, significantly reduced adhesion number and size on PT, with adhesion size consistent with focal complexes. After 1 day, ROCK, but not Rac inhibition increased osteocalcin mRNA levels on SLA and PT, with levels further increasing at 7 days post seeding. ROCK inhibition also significantly increased bone sialoprotein expression at 7 days, but not BMP-2 levels. Rac inhibition significantly reduced BMP-2 mRNA levels. ROCK inhibition increased nuclear translocation of Runx2 independent of surface roughness. Mineralization of osteoblast cultures was greater on SLA than on PT, but was increased by ROCK inhibition and attenuated by Rac inhibition on both topographies. In conclusion, inhibition of ROCK signalling significantly increases osteoblast differentiation and biomineralization in a topographic dependent manner, and its pharmacological inhibition could represent a new therapeutic to speed bone formation around implanted metals and in regenerative medicine applications.     

Influence of nanophase titania topography on bacterial attachment and metabolism

Surfaces with nanophase compared to conventional (or nanometer smooth) topographies are known to have different properties of area, charge, and reactivity. Previously published research indicates that the attachment of certain bacteria (such as Pseudomonas fluorescens 5RL) is higher on surfaces with nanophase compared to conventional topographies, however, their effect on bacterial metabolism is unclear. Results presented here show that the adhesion of Pseudomonas fluorescens 5RL and Pseudomonas putida TVA8 was higher on nanophase than conventional titania. Importantly, in terms of metabolism, bacteria attached to the nanophase surfaces had higher bioluminescence rates than on the conventional surfaces under all nutrient conditions. Thus, the results from this study show greater select bacterial metabolism on nanometer than conventional topographies, critical results with strong consequences for the design of improved biosensors for bacteria detection.     

Ti2448合金种植体表面不同纳米管径生物活性膜对成骨细胞早期黏附的影响

观察Ti2448合金表面不同纳米管径生物活性膜对成骨细胞早期黏附的影响,筛选出能够抵抗细菌在种植体表面黏附和定植的最适管径,为研制开发具有抗菌性能的种植体提供实验基础。采用阳极氧化法,设定不同氧化电压,在Ti2448合金表面生成具有不同纳米管径的生物活性膜。通过MTT实验检测不同管径纳米管表面在不同时间点黏附的MG-63细胞数量差异;通过免疫荧光法观察接种24h后不同管径纳米管表面黏附细胞形态学差异。结果显示,细胞接种3h和24h后,30nm组的OD值显著高于对照组及其他实验组(P0.05);随着纳米管径的增加,细胞骨架蛋白的铺展范围逐渐减小,细胞由多角形渐变为梭形、椭圆形、圆形。研究结果表明,30nm管径生物活性膜更有利于成骨细胞的早期黏附,可能更能抵抗细菌在钛种植体上的黏附与定植。     

Mechanics and electrostatics of the interactions between osteoblasts and titanium surface

Due to oxidation and adsorption of chloride and hydroxyl anions, the surface of titanium (Ti) implants is negatively charged. A possible mechanism of the attractive interaction between the negatively charged Ti surface and the negatively charged osteoblasts is described theoretically. It is shown that adhesion of positively charged proteins with internal charge distribution may give rise to attractive interaction between the Ti surface and the osteoblast membrane. A dynamic model of the osteoblast attachment is presented in order to study the impact of geometrically structured Ti surfaces on the osteoblasts attachment. It is indicated that membrane-bound protein complexes (PCs) may increase the membrane protrusion growth between the osteoblast and the grooves on titanium (Ti) surface and thereby facilitate the adhesion of osteoblasts to the Ti surface. On the other hand, strong local adhesion due to electrostatic forces may locally trap the osteoblast membrane and hinder the further spreading of osteointegration boundary. We suggest that the synergy between these two processes is responsible for successful osteointegration along the titanium surface implant.     

MC3T3-E1 osteoblast attachment and proliferation on porous hydroxyapatite scaffolds fabricated with nanophase powder

Porous bone tissue engineering scaffolds were fabricated using both nano hydroxyapatite (nano HA) powder (20 nm average particle size) and micro HA powder (10 microm average particle size), resulting in sintered scaffolds of 59 vol% porosity and 8.6 +/- 1.9 microm average grain size and 72 vol% porosity and 588 +/- 55nm average grain size, respectively. Scanning electron microscopy was used to measure both the grain size and pore size. MC3T3-E1 osteoblast (OB) attachment and proliferation on both nano HA and micro HA porous scaffolds were quantified. As expected, OB cell number was greater on nano HA scaffolds compared with similarly processed micro HA scaffolds 5 days after seeding, while OB attachment did not appear greater on the nano HA scaffolds (p < 0.05).     

Mediation of Rac1 activation by kindlin-2: an essential function in osteoblast adhesion, spreading, and proliferation

Kindlins are focal adhesion proteins that regulate integrin signaling. Although integrin activation is critical for bone development, little is known about the expression and role of kindlins in osteoblasts. We therefore investigated the function of kindlin-2 in osteoblast adhesion, spreading, and proliferation using small interfering RNA. In MC3T3-E1 cells, only kindlin-2 is highly expressed and localizes to focal adhesion. We found that kindlin-2 was involved in integrin activation in MC3T3-E1 cells and that kindlin-2 knockdown osteoblasts resulted in diminished cell adhesion, spreading, and proliferation. In this process, kindlin-2 knockdown impaired transient Rac1 activation, influencing Akt activation and AP-1 activity. In agreement with these data, pharmacological inhibition of Rac1 reduced MC3T3-E1 cell adhesion, spreading, and proliferation. Overall, these findings demonstrated that kindlin-2 governs Rac1 activation, which controls osteoblast function. Our findings provide the first insights concerning the function of kindlin-2 in osteoblast, and suggest that kindlin-2 is a critical mediator for osteoblast physiology.     

Study of semiconducting nanomaterials under pressure

The pressure induced structural and mechanical properties of nanocrystalline ZnO, ZnS, ZnSe, GaN, CoO, CdSe, CeO(2), SnO(2), SiC, c-BC(2)N, and β-Ga(2)O(3) with different grain sizes have been analyzed under high pressures. The molecular dynamics simulation model has been used to compute isothermal equation of state, volume collapse and bulk modulus of these materials in nano and bulk phases at ambient and high pressures and compared with the experimental data. It is evident from these calculations that the change in particle size affects directly the phase transition pressure and bulk modulus. The values of phase transition pressure and bulk modulus increase with decrease in grain size of the material. The equilibrium cell volume and volume collapse in parent phase is directly proportional to the grain size of the materials. Present results are in good agreement with experimental data. The model is able to explain these thermodynamic properties at varying temperatures and pressures successfully.     

Role of N-cadherin in bone formation

Cell-cell adhesion mediated by cadherins is essential for the function of bone forming cells during osteogenesis. Here, the evidence that N-cadherin is an important regulator of osteoblast differentiation and osteogenesis is reviewed. Osteoblasts express a limited number of cadherins, including the classic N-cadherin. The expression profile of N-cadherin in osteoblasts during bone formation in vivo and in vitro suggests a role of this molecule in osteogenesis. Functional studies using neutralizing antibodies or antisense oligonucleotides indicate that N-cadherin is involved in the control the expression of osteoblast marker gene expression and differentiation. Cleavage of N-cadherin during osteoblast apoptosis also suggests a role of N-cadherin-mediated-cell-cell adhesion in osteoblast survival. Hormonal and local factors that regulate osteoblast function also regulate N-cadherin expression and subsequent cell-cell adhesion associated with osteoblast differentiation or survival. Signaling mechanisms involved in N-cadherin-mediated cell-cell adhesion and osteoblast gene expression have also been identified. Alterations of N-cadherin expression are associated with abnormal osteoblast differentiation and osteogenesis in pathological conditions. These findings indicate that N-cadherin plays a role in normal and pathological bone formation and provide some insight into the process involved in N-cadherin-mediated cell-cell adhesion and differentiation in osteoblasts.     

Nanostructured metal coatings on polymers increase osteoblast attachment

Bioactive coatings are in high demand to increase the functions of cells for numerous medical devices. The objective of this in vitro study was to characterize osteoblast (bone-forming cell) adhesion on several potential orthopedic polymeric materials (specifically, polyetheretherketone, ultra-high molecular weight polyethylene, and polytetrafluoroethylene) coated with either titanium or gold using a novel Ionic Plasma Deposition process which creates a surface-engineered nanostructure (with features below 100 nm). Results demonstrated that compared to currently-used titanium and uncoated polymers, polymers coated with either titanium or gold using Ionic Plasma Deposition significantly increased osteoblast adhesion. Qualitative cell morphology results supported quantitative adhesion results as increased osteoblast cell spreading was observed on coated polymers compared to uncoated polymers. In this manner, this in vitro study strongly suggests that Ionic Plasma Deposition should be further studied for creating nanometer surface features on a wide variety of materials to enhance osteoblast functions necessary for orthopedic applications.     

Decreased fibroblast and increased osteoblast adhesion on nanostructured NaOH-etched PLGA scaffolds

To facilitate locomotion and support the body, the skeleton relies on the transmission of forces between muscles and bones through complex junctions called entheses. The varying mechanical and biological properties of the enthesis make healing this avascular tissue difficult; hence the need for an engineered alternative. Cells in situ interact with their environment on the nano-scale which suggests that engineered approaches to enthesis regeneration should include such biologically-inspired nano-scale surface features. The present in vitro study investigated the effects of etching poly-lactic-co-glycolic acid (PLGA) scaffolds to produce nano-topography on the adhesion of fibroblasts and osteoblasts, two integral enthesis cell types. Nano-topography was produced on PLGA by etching the scaffolds in NaOH. Results showed that etching PLGA with NaOH to create nano-scale surface features decreased fibroblast adhesion while it increased osteoblast adhesion; criteria critical for the spatial control of osteoblast and fibroblast adhesion for a successful enthesis tissue engineering material. Thus, the results of this study showed for the first time collective evidence that PLGA can be either treated with NaOH or not on ends of an enthesis tissue engineering construct to spatially increase osteoblast and fibroblast adhesion, respectively.     

Mechanical and In Vitro Biological Performance of Graphene Nanoplatelets Reinforced Calcium Silicate Composite

Calcium silicate (CaSiO₃, CS) ceramic composites reinforced with graphene nanoplatelets (GNP) were prepared using hot isostatic pressing (HIP) at 1150°C. Quantitative microstructural analysis suggests that GNP play a role in grain size and is responsible for the improved densification. Raman spectroscopy and scanning electron microscopy showed that GNP survived the harsh processing conditions of the selected HIP processing parameters. The uniform distribution of 1 wt.% GNP in the CS matrix, high densification and fine CS grain size help to improve the fracture toughness by ∼130%, hardness by ∼30% and brittleness index by ∼40% as compared to the CS matrix without GNP. The toughening mechanisms, such as crack bridging, pull-out, branching and deflection induced by GNP are observed and discussed. The GNP/CS composites exhibit good apatite-forming ability in the simulated body fluid (SBF). Our results indicate that the addition of GNP decreased pH value in SBF. Effect of addition of GNP on early adhesion and proliferation of human osteoblast cells (hFOB) was measured in vitro. The GNP/CS composites showed good biocompatibility and promoted cell viability and cell proliferation. The results indicated that the cell viability and proliferation are affected by time and concentration of GNP in the CS matrix.     

Phosphoproteome reveals an atlas of protein signaling networks during osteoblast adhesion

Cell adhesion on surfaces is a fundamental process in the emerging biomaterials field and developmental events as well. However, the mechanisms regulating this biological process in osteoblasts are not fully understood. Reversible phosphorylation catalyzed by kinases is probably the most important regulatory mechanism in eukaryotes. Therefore, the goal of this study is to assess osteoblast adhesion through a molecular prism under a peptide array technology, revealing essential signaling proteins governing adhesion‐related events. First, we showed that there are main morphological changes on osteoblast shape during adhesion up to 3 h. Second, besides classical proteins activated upon integrin activation, our results showed a novel network involving signaling proteins such as Rap1A, PKA, PKC, and GSK3β during osteoblast adhesion on polystyrene. Third, these proteins were grouped in different signaling cascades including focal adhesion establishment, cytoskeleton rearrangement, and cell‐cycle arrest. We have thus provided evidence that a global phosphorylation screening is able to yield a systems‐oriented look at osteoblast adhesion, providing new insights for understanding of bone formation and improvement of cell–substratum interactions. Altogether, these statements are necessary means for further intervention and development of new approaches for the progress of tissue engineering. J. Cell. Biochem. 109: 957–966, 2010. © 2010 Wiley‐Liss, Inc.     

Coagulation of dust grains in the plasma of an RF discharge in argon

Results are presented from experimental studies of coagulation of dust grains of different sizes injected into a low-temperature plasma of an RF discharge in argon. A theoretical model describing the formation of dust clusters in a low-temperature plasma is developed and applied to interpret the results of experiments on the coagulation of dust grains having large negative charges. The grain size at which coagulation under the given plasma conditions is possible is estimated using the developed theory. The theoretical results are compared with the experimental data.     

Modulatory effects of silibinin in cell behavior during osteogenic phenotype

Natural molecules, such as flavonoid, are very welcome strategies to modulate bone turnover. This prompted us to comprehend better the effect of silibinin on osteoblast metabolism, mainly considering intracellular pathways able to drive cell adhesion to differentiation. By exploring in vitro approaches, our data show a modulatory effect of the silibinin (200 μg/mL) on the osteoblast intracellular signaling, contributing with decisive pathways governing cell adhesion, differentiation, and further mineralization, recapitulating important stages of osteogenesis. Within the first 24 hours of adhesion (acute stage), osteoblasts respond to silibinin by rearranging their cytoskeleton and start mechanisms responsible to extracellular matrix (ECM) remodeling, which reach intense profile at 28 days of treatment (chronic stage) by favoring matrix metalloproteinases (MMPs-2, and -9) activities, concomitant to mineralizing phenotype. Importantly, silibinin seems to reprogram genes related to inflammatory landscape and significantly upmodulating osteoprotegerin (>25 fold-changes), signaling molecule involved with osteoclastogenesis. Altogether, our results show for the first time that silibinin drives in vitro osteoblast differentiation by requiring specific intracellular signaling. In conjunction, this molecular landscape contributes to understand the effect of silibinin on osteoblasts performance and open novel therapeutic possibilities to silibinin in bone disorders, such as osteoporosis.     

Modelling and simulation of substrate elasticity sensing in stem cells

Recently, we have developed a multiscale soft matter cell model aiming at improving the understanding of mechanotransduction mechanism of stem cells, which is responsible for information exchange between cells and their extracellular environment. In this paper, we report the preliminary results of our research on multiscale modelling and simulation of soft contact and adhesion of stem cells. The proposed multiscale soft matter cell model may be used to model soft contact and adhesion between cells and their extracellular substrates. To the authors' best knowledge, this may be the first time that a soft matter model has been developed for cell contact and adhesion. Moreover, we have developed and implemented a Lagrange-type meshfree Galerkin formulation and related computational algorithms for the proposed cell model. Comparison study with experimental data has been conducted to validate the parameters of the cell model. By using the soft matter cell model, we have simulated the soft adhesive contact process between cells and extracellular substrates. The simulation shows that the cell can sense substrate elasticity by responding it in different manners from cell spreading to cell contact configuration and molecular conformation changes.