The effects of nanoscale pits on primary human osteoblast adhesion formation and cellular spreading

Current understanding of the mechanisms involved in ossesoinegration following implantation of a biomaterial has led to an emphasis being placed on the modification of material topography to control interface reactions. Recent studies have inferred nanoscale topography as an important mediator of cell adhesion and differentiation. Biomimetic strategies in orthopaedic research aim to exploit these influences to regulate cellular adhesion and subsequent bony tissue formation. Here experimental topographies of nanoscale pits demonstrating varying order have been fabricated by electron-beam lithography in (poly)carbonate. Osteoblast adhesion to these nanotopographies was ascertained by quantification of the relation between adhesion complex formation and total cell area. This study is specifically concerned with the effects these nanotopographies have on adhesion formation in S-phase osteoblasts as identified by BrdU incorporation. Nanopits were found to reduce cellular spreading and adhesion formation.     

Formation and structure of zinc-substituted calcium hydroxyapatite

Zn-substituted Ca hydroxyapatites were synthesized by precipitation method under the specific conditions (pH 8, 90 °C) and their structural properties were investigated. The substituting limit of Zn was estimated at about 15 mol%. The lattice parameter a decreased up to 5 mol% Zn, and started to increase over 5 mol% Zn. The lattice parameter c monotonously decreased with increase in Zn fraction. The increase in lattice parameter a for higher Zn fraction was ascribed to increasing amount of lattice H₂O which substituted for OH sites in the apatite structure. The lattice H₂O in the Zn-substituted apatites was lost by the heat treatment at 400 °C. As a result, both the lattice parameters a and c of the heat-treated apatites decreased with increasing Zn fraction. Only the difference in ionic radius between Zn²⁺ (0.074 nm) and Ca²⁺ (0.099 nm) was decisive to the change in both lattice parameters after the heat treatment at 400 °C.     

The role of surface charge on the uptake and biocompatibility of hydroxyapatite nanoparticles with osteoblast cells

The objective of this study is to evaluate the effect of hydroxyapatite (HAP) nanoparticles with different surface charges on the cellular uptake behavior and in vitro cell viability and proliferation of MC3T3-E1 cell lines (osteoblast). The nanoparticles' surface charge was varied by surface modification with two carboxylic acids: 12-aminododecanoic acid (positive) and dodecanedioic acid (negative). The untreated HAP nanoparticles and dodecanoic acid modified HAP nanoparticles (neutral) were used as the control. X-ray diffraction (XRD) revealed that surface modifications by the three carboxylic acids did not change the crystal structure of HAP nanoparticles; Fourier transform infrared spectroscopy (FT-IR) confirmed the adsorption and binding of the carboxylic acids on the HAP nanoparticles' surfaces; and zeta potential measurement confirmed that the chemicals successfully modified the surface charge of HAP nanoparticles in water based solution. Transmission electron microscopy (TEM) images showed that positively charged, negatively charged and untreated HAP nanoparticles, with similar size and shape, all penetrated into the cells and cells had more uptake of HAP nanoparticles with positive charge compared to those with negative charge, which might be attributed to the attractive or repulsive interaction between the negatively charged cell membrane and positively/negatively charged HAP nanoparticles. The neutral HAP nanoparticles could not penetrate the cell membrane due to their larger size. MTT assay and LDH assay results indicated that as compared with the polystyrene control, greater cell viability and cell proliferation were measured on MC3T3-E1 cells treated with the three kinds of HAP nanoparticles (neutral, positive, and untreated), among which positively charged HAP nanoparticles showed the strongest improvement for cell viability and cell proliferation. In summary, the surface charge of HAP nanoparticles can be modified to influence the cellular uptake of HAP nanoparticles and the different uptake also influences the behavior of cells. These in vitro results may also provide useful information for investigations of HAP nanoparticle applications in gene delivery and intracellular drug delivery.     

Quantification of carbon nanotube induced adhesion of osteoblast on hydroxyapatite using nano-scratch technique

This paper explores the nano-scratch technique for measuring the adhesion strength of a single osteoblast cell on a hydroxyapatite (HA) surface reinforced with carbon nanotubes (CNTs). This technique efficiently separates out the contribution of the environment (culture medium and substrate) from the measured adhesion force of the cell, which is a major limitation of the existing techniques. Nano-scratches were performed on plasma sprayed hydroxyapatite (HA) and HA-CNT coatings to quantify the adhesion of the osteoblast. The presence of CNTs in HA coating promotes an increase in the adhesion of osteoblasts. The adhesion force and energy of an osteoblast on a HA-CNT surface are 17 ± 2 μN/cell and 78 ± 14 pJ/cell respectively, as compared to 11 ± 2 μN/cell and 45 ± 10 pJ/cell on a HA surface after 1 day of incubation. The adhesion force and energy of the osteoblasts increase on both the surfaces with culture periods of up to 5 days. This increase is more pronounced for osteoblasts cultured on HA-CNT. Staining of actin filaments revealed a higher spreading and attachment of osteoblasts on a surface containing CNTs. The affinity of CNTs to conjugate with integrin and other proteins is responsible for the enhanced attachment of osteoblasts. Our results suggest that the addition of CNTs to surfaces used in medical applications may be beneficial when stronger adhesion of osteoblasts is desired.     

Statistical demonstration of the relative effect of surface chemistry and roughness on human osteoblast short-term adhesion

The effects of material composition, surface chemistry or surface topography on cell attachment (short-term adhesion) have been largely studied on bone-derived cells. However, no statistical demonstration of these effects has been performed until now. With this objective, we quantified the attachment after 24 hours of human osteoblasts on pure titanium, titanium alloy and stainless steel substrates presenting 6 different surface morphologies and 2 different roughness amplitude obtained by sand-blasting, electro-erosion, acid etching, polishing and machine-tooling. The coating by a gold-palladium layer of these surfaces allowed determining the relative effect of the surface roughness and of the surface chemistry. By multiple analysis of variance, we demonstrated that neither material composition nor surface roughness amplitude influenced cell attachment except on sandblasted pure titanium substrates. On the contrary, a high significant influence of the process used to produce the surface was observed meaning that the main influent factor on cell attachment could be either the surface morphology or the surface chemistry induced by the process. As the coating of surfaces by a gold-palladium layer decreased significantly the attachment of cells on the majority of substrates, we concluded that attachment is rather influenced by surface chemistry than by surface topography.     

The inhibition of lamellar hydroxyapatite and lamellar magnetic hydroxyapatite on the migration and adhesion of breast cancer cells

Hydroxyapatite nanoparticles have been reported to exhibit potent anti-tumor effects in some cancer cells. In our previous study, we have successfully synthesized two types of hydroxyapatite nanoparticles, laminated hydroxyapatite (L-HAp) and laminated magnetic hydroxyapatite (LM-HAp). In this study, we wanted to investigate the effects of L-HAp and LM-HAp with various concentrations on human breast cancer MDA-MB-231 cells. Cell proliferation was assessed with a MTT colorimetric assay. Scratch and adhesion assays were used to detect the effects of these two materials on migration and adhesion. The expressions of integrin β1 and Akt were measured by Western blotting. Our results showed that L-HAp and LM-HAp had little cell cytotoxicity and significantly reduced cell mobility and adhesion. LM-HAp showed greater inhibitor ability on migration and adhesion of MDA-MB-231 cells. Moreover, results from western blotting showed that L-HAp and LM-HAp impacted the phosphorylation of integrin β1, but showed no regular impact on Akt. This study suggests that L-HAp and LM-HAp may be potential anti-tumor and delivery system for breast cancer therapy.     

RGD peptide immobilized on TiO2 nanotubes for increased bone marrow stromal cells adhesion and osteogenic gene expression

Recently, TiO₂ nanotube layers are widely used in orthopedics and dental applications because of their good promotion effect on bone cells. Furthermore, peptide sequences such as arginine–glycine–aspartic acid are used to modify Ti implant for binding to cell surface integrins through motif. In this study, a cellular adhesive peptide of arginine–glycine–aspartic acid–cysteine (RGDC) was immobilized onto anodized TiO₂ nanotubes on Ti to examine its in vitro responses on rat bone marrow stromal cells (BMSCs). Materials were characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy techniques. High-resolution C1s scans suggested the presence of RGDC on the surface and SEM images confirmed the nanotubes were not destroyed after modification. BMSCs adhesion and osteogenic gene expression were detected in TiO₂ nanotube layers with and without RGDC modification by fluorescence microscopy, confocal laser scanning microscopy, SEM, and realtime polymerase chain reaction (Real-time PCR). Results showed that the TiO₂ nanotube layers immobilized with RGDC increased BMSCs adhesion compared to nonfunctionalized nanotubes after 4 h of cultivation. Furthermore, the osteogenic gene expression of BMSCs was dramatically enhanced on the TiO₂ nanotube layers immobilized with RGDC (10 mM) compared to the TiO₂ nanotube layers immobilized with RGDC (1 mM) and non-functionalized anodized Ti. Our results from in vitro study provided evidence that Ti anodized to possess nanotubes and then further functionalized with RGDC should be further studied for the design of better biomedical implant surfaces.     

Promotion of adhesion and proliferation of endothelial progenitor cells on decellularized valves by covalent incorporation of RGD peptide and VEGF

Tissue engineered heart valve is a promising alternative to current heart valve surgery, for its capability of growth, repair, and remodeling. However, extensive development is needed to ensure tissue compatibility, durability and antithrombotic potential. This study aims to investigate the biological effects of multi-signal composite material of polyethyl glycol-cross-linked decellularized valve on adhesion and proliferation of endothelial progenitor cells. Group A to E was decellularized valve leaflets, composite material of polyethyl glycol-cross-linked decellularized valves leaflets, vascular endothelial growth factor-composite materials, Arg-Gly-Asp peptide-composite materials and multi-signal modified materials of polyethyl glycol-cross-linked decellularized valve leaflets, respectively. The endothelial progenitor cells were seeded for each group, cell adhesion and proliferation were detected and neo-endothelium antithrombotic function of the multi-signal composite materials was evaluated. At 2, 4, and 8?h after the seeding, the cell numbers and 3H-TdR incorporation in group D were the highest. At 2, 4, and 8 days after the seeding, the cell numbers and 3H-TdR incorporation were significantly higher in groups C, D, and E compared with groups A and B (P?<?0.05) and cell numbers and the expression of t-PA and eons in the neo-endothelium were quite similar to those in the human umbilical vein endothelial cells at 2, 4, and 8 days after the seeding. The Arg-Gly-Asp- peptides (a sequential peptide composed of arginine (Arg), glycine (Gly) and aspartic acid (Asp)) and VEGF-conjugated onto the composite material of PEG-crosslinked decellularized valve leaflets synergistically promoted the adhesion and proliferation of endothelial progenitor cells on the composite material, which may help in tissue engineering of heart valves.     

The effect of surface treatments on the adhesion of electrochemically deposited hydroxyapatite coating to titanium and on its interaction with cells and bacteria

The effect of different mechanical and chemical pre-treatments on the adhesion strength of hydroxyapatite (HAp) coating on a commercially pure titanium (CP-Ti) substrate was studied by means of a standard tensile test followed by microscopic and chemical analysis to determine the locus of fracture. In addition, the effects of either these pre-treatments or post-treatment by low-energy electron irradiation, which allowed tuning the wettability of the surface, on both osteoblast progenitor attachment and S. aureus bacteria attachment were investigated. A dedicated program was developed for unambiguous identification and count of stained cells. A single-phase HAp coating was formed by electrodeposition. A series of surface pre-treatments consisted of grinding down to P1000, etching in HNO₃/HF solution, grit blast, soaking in NaOH and subsequent heat treatment provided the highest adhesion strength to the HAp coating. Osteoblast progenitors derived from rats may be attached preferentially to a hydrophilic surface (post-treatment to θ = 30°), while the bacteria seemed to be less attached to hydrophobic surfaces (post-treatment to θ = 105°). However, the results were not statistically different. The bacteria seemed to be less attached to the smoother, uncoated surfaces.     

Sr-containing hydroxyapatite: morphologies of HA crystals and bioactivity on osteoblast cells

A series of Sr-substituted hydroxyapatites (HA), of general formula Ca_(10 ? x)Sr_x(PO₄)₆(OH)₂, where x = 2 and 4, were synthesized by solid state methods and characterized extensively. The reactivity of these materials in cell culture medium was evaluated, and the behavior towards MG-63 osteoblast cells (in terms of cytotoxicity and proliferation assays) was studied. Future in vivo studies will give further insights into the behavior of the materials.A paper by Lagergren et al. (1975), concerning Sr-substituted HA prepared by a solid state method, reports that the presence of Sr in the apatite composition strongly influences the apatite diffraction patterns. Zeglinsky et al. (2012) investigated Sr-substituted HA by ab initio methods and Rietveld analyses and reported changes in the HA unit cell volume and shape due to the Sr addition.To further clarify the role played by the addition of Sr on the physico-chemical properties of these materials we prepared Sr-substituted HA compositions by a solid state method, using different reagents, thermal treatments and a multi-technique approach. Our results indicated that the introduction of Sr at the levels considered here does influence the structure of HA. There is also evidence of a decrease in the crystallinity degree of the materials upon Sr addition. The introduction of increasing amounts of Sr into the HA composition causes a decrease in the specific surface area and an enrichment of Sr-apatite phase at the surface of the samples. Bioactivity tests show that the presence of Sr causes changes in particle size and/or morphology during soaking in MEM solution; on the contrary the morphology of pure HA does not change after 14 days of reaction. The presence of Sr, as Sr-substituted HA and SrCl_2, in cultures of human MG-63 osteoblasts did not produce any cytotoxic effect. In fact, Sr-substituted HA increased the proliferation of osteoblast cells and enhanced cell differentiation: Sr in HA has a positive effect on MG-63 cells. In contrast, Sr ions alone, at the concentrations released by Sr-HA (1.21–3.24 ppm), influenced neither cell proliferation nor differentiation. Thus the positive effects of Sr in Sr-HA materials are probably due to the co-action of other ions such as Ca and P.     

Effect of filler surface morphology on the impact behaviour of hydroxyapatite reinforced high density polyethylene composites

Instrumented falling weight impact tests have been carried out to characterize the impact behaviour of hydroxyapatite reinforced high-density polyethylene composite (HA-HDPE) in order to use this biomaterial in skull implants. The effects of HA filler surface morphology and volume fraction on the fracture toughness were studied, and fracture mechanism investigated. Impact resistance was found to be markedly improved by using a sintered grade HA filler with smooth particle surface instead of spray dried grade HA with rough surface. SEM examination of impacted fracture surfaces revealed that the improvement of impact resistance was due to the stronger interfacial bonding between smooth HA particles and HDPE polymer matrix compared with that between rough HA and HDPE, which results in more energy absorption during impact and hence better fracture resistance.     

Human osteoblast adhesion on titanium alloy, stainless steel, glass and plastic substrates with same surface topography

Osteoblast adhesion on materials will depend on the surface aspects of materials which may be described according to their surface chemistry, surface topography or surface energy. To separate the effects of roughness and composition of materials on osteoblast response, we chose to compare substrates with various surface composition but with the same smooth surface. Ti6Al4V alloy, stainless steel, glass and standard tissue culture polystyrene were tested. Adhesion was evaluated using specific antibodies against adhesion proteins and by a quantitative cell detachment assay. After 1, 7 and 14 days, cells expressed extracellularly fibronectin fibers, and intracellularly type I collagen and osteopontin. Vinculin-labeled focal contacts were visible on all materials but were more frequent on glass and stainless steel surfaces. ₁-integrin subunit-labeled patches were visible on all surfaces at each delay. The quantitative cell detachment assay showed few differences between materials. Adhesion was higher on metallic substrates although cell proliferation was higher on glass and stainless steel compared to tissue culture polystyrene and Ti6Al4V alloy. Substrates with various surface composition but with the same surface topography did not induce significant differences of adhesion although cell proliferation was variable.     

Mesenchymal stem cell adhesion and spreading on nanostructured biomaterials

Bone marrow-derived human mesenchymal stem cells were seeded in serum-free media onto ion beam-deposited nanostructured metalloceramic (Ti-Cr-N) films and plasma-nitrided titanium disks, which were left uncoated as well as precoated with fetal bovine serum. Precoating the disks with serum appears to stimulate cell spreading on both the titanium nitride and metalloceramic materials for as little as 1 hour incubation time. The implication is that both of these materials can adsorb serum proteins in amounts sufficient to influence cell adhesion and spreading for potentially improved in vivo response of orthopedic and dental implants. The materials in this study may prove to exhibit enhanced biological and mechanical properties when compared to conventional micron-scale implant materials such as titanium or cobalt-chrome alloys.     

RGD修饰钛表面对人牙龈成纤维细胞初期黏附和铺展的影响

用羰基二咪唑(1,1'-carbonyldiimidazole,CDI)将含RGD的短肽共价连接到纯钛表面,研究接枝后的钛表面对原代培养的人牙龈成纤维细胞(human gingival fibroblasts,HGF)初期黏附和铺展的影响.结果表明,RGD修饰的纯钛表面粘附的细胞数比未修饰钛表面多,细胞铺展面积比钛表面的大,应力纤维的形成比钛表面早.RGD接枝钛表面更有利于人牙龈成纤维细胞的粘附,改善了纯钛的生物相容性.     

Molecular dynamics simulation of RGD peptide adsorption on titanium oxide surfaces

Peptide Arg-Gly-Asp (RGD) sequence is a ubiquitous adhesive motif found in various bone extracellular matrix proteins and is crucial in the biomaterial surface/interface reaction. This study analyzed the adsorption of RGD on different titanium oxide surfaces with molecular dynamics simulation. The simulation results indicate that the RGD peptide binds strongly with anatase (001) and rutile (010). RGD conformation changes due to the variation of the backbone torsion angle in the middle of the RGD chain. Pair correlation function analysis indicates that the interaction of the RGD peptide and the titanium oxide results from hydrogen bonding and the groups in RGD play different roles during the adsorption process. This study provides useful information on how to design titanium surfaces in order to modulate peptide or protein adsorption.     

The effects of micro arc oxidation of gamma titanium aluminide surfaces on osteoblast adhesion and differentiation

The adhesion and proliferation of human fetal osteoblasts, hFOB 1.19, on micro arc oxidized (MAO) gamma titanium aluminide (γTiAl) surfaces were examined in vitro. Cells were seeded on MAO treated γTiAl disks and incubated for 3 days at 33.5 °C and subsequently for 7 days at 39.5 °C. Samples were then analyzed by scanning electron microscopy (SEM) and alkaline phosphatase assay (ALP) to evaluate cell adhesion and differentiation, respectively. Similar Ti-6Al-4V alloy samples were used for comparison. Untreated γTiAl and Ti-6Al-4V disks to study the effect of micro arc oxidation and glass coverslips as cell growth controls were also incubated concurrently. The ALP Assay results, at 10 days post seeding, showed significant differences in cell differentiation, with P values <0.05 between MAO γTiAl and MAO Ti-6Al-4V with respect to the corresponding untreated alloys. While SEM images showed that hFOB 1.19 cells adhered and proliferated on all MAO and untreated surfaces, as well as on glass coverslips at 10 days post seeding, cell differentiation, determined by the ALP assay, was significantly higher for the MAO alloys.     

The influence of surface chemistry and topography on the contact guidance of MG63 osteoblast cells

The purpose of the present study was to determine in vitro the effects of different surface topographies and chemistries of commercially pure titanium (cpTi) and diamond-like carbon (DLC) surfaces on osteoblast growth and attachment. Microgrooves (widths of 2, 4, 8 and 10 μm and a depth of 1.5–2 μm) were patterned onto silicon (Si) substrates using microlithography and reactive ion etching. The Si substrates were subsequently vapor coated with either cpTi or DLC coatings. All surfaces were characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. Using the MG63 Osteoblast-Like cell line, we determined cell viability, adhesion, and morphology on different substrates over a 3 day culture period. The results showed cpTi surfaces to be significantly more hydrophilic than DLC for groove sizes larger than 2 μm. Cell contact guidance was observed for all grooved samples in comparison to the unpatterned controls. The cell viability tests indicated a significantly greater cell number for 8 and 10 μm grooves on cpTi surfaces compared to other groove sizes. The cell adhesion study showed that the smaller groove sizes, as well as the unpatterned control groups, displayed better cell adhesion to the substrate.     

Immobilization of RGD peptide on HA coating through a chemical bonding approach

In this work, Arg-Gly-Asp (RGD) sequence containing peptide was immobilized on hydroxyapatite (HA) coatings through a chemical bonding approach in two steps, surface modification with 3-aminopropyltriethoxysilane (APTES) and RGD immobilization. The results indicate that RGD has been successfully immobilized on HA coatings. Comparing with physical adsorption coatings, the chemically bonded RGD on the coatings shows much better anti-wash-out ability. Since RGD is able to recognize cell-membrane integrins on biointerfaces, the present method will be an effective way to favor interaction of cells with HA coatings.     

Multilayer coatings on biomaterials for control of MG-63 osteoblast adhesion and growth

Here, the layer-by-layer technique (LbL) was used to modify glass as model biomaterial with multilayers of chitosan and heparin to control the interaction with MG-63 osteoblast-like cells. Different pH values during multilayer formation were applied to control their physico-chemical properties. In the absence of adhesive proteins like plasma fibronectin (pFN) both plain layers were rather cytophobic. Hence, the preadsorption of pFN was used to enhance cell adhesion which was strongly dependent on pH. Comparing the adhesion promoting effects of pFN with an engineered repeat of the FN III fragment and collagen I which both lack a heparin binding domain it was found that multilayers could bind pFN specifically because only this protein was capable of promoting cell adhesion. Multilayer surfaces that inhibited MG-63 adhesion did also cause a decreased cell growth in the presence of serum, while an enhanced adhesion of cells was connected to an improved cell growth.