The influence of target stoichiometry on early cell adhesion of co-sputtered calcium–phosphate surfaces

The nature of the initial interaction between calcium phosphate (Ca–P) thin films and osteoblasts can be influenced by a number of different properties including the phase, crystallinity, stoichiometry and composition of the surface. There is still a strong interest in developing and studying Ca–P surfaces that have the ability to accurately control the osteoblast response. Radio frequency (RF) magnetron sputtering is a technique that allows for accurate control of the properties of deposited Ca–P coatings and has been studied extensively because of this fact. In this work, Ca–P coatings were co-deposited using RF magnetron sputtering in order to study the effect of changing the target stoichiometry on the initial in vitro behavior of MG63 osteoblast-like cells. The samples produced were analysed both as-deposited and after thermal annealing to 500 °C. After annealing XPS analyses of the samples co-deposited using tricalcium phosphate (TCP) materials gave a Ca/P ratio of 1.71 ± 0.01, as compared to those co-deposited from hydroxyapatite (HA) materials, with a Ca/P of 1.82 ± 0.06. In addition to this, the curve fitted XPS data indicated the presence of low levels of carbonate in the coatings. Despite this the XRD results for all of the annealed coatings were shown to be characteristic of pure HA with a preferred 002 orientation. The atomic force microscopy results also highlighted that both types of coatings had surface features of a similar size (200–220 nm). Both surfaces exhibited a degree of surface degradation, even after 1 h of cell culture. However, the TCP derived surfaces showed an enhanced osteoblastic cell response in terms of cell adhesion and cell proliferation in the earlier stages of cell culture than the surfaces deposited from HA. An improvement in the initial cell attachment and a potential for increased cell proliferation rates is viewed as a highly advantageous result in relation to controlling the osteoblast response on these surfaces.     

Preparation of hydroxyapatite-carbon nanotube composite nanopowders

Here we report a simple preparation of composite nanopowders made of hydroxyapatite (HA) and carbon nanotube (CNT). In particular, CNTs were ionically modified to dissolve homogeneously in a series of organic solvents, including tetrahydrofuran and ethanol. The addition of HA nanopowders within the CNTs solution resulted in rapid precipitation of the composite HA-CNTs nanopowders. High resolution electron image revealed individual CNTs were evenly distributed within the cluster of HA nanoparticulates. A maximal concentration of CNTs to be organized within the HA nanopowder was highly dependent on the physicochemical characteristics of HA powders. A pilot biological assessment of the composite powders demonstrated favorable adhesion and growth of tissue cells. The developed HA-CNTs composite nanopowders may be potentially useful as an initial powder source for HA-CNT nanocomposites or coatings in biomedical applications.     

Enhanced in vivo responses of osteoblasts in electrostatically activated zones by hydroxyapatite electrets

Matured osteoblasts were proved to be located in the bone formation accelerated by induced large surface charges on the electrically polarized hydroxyapatite (HA) ceramics regardless of the charge polarities, whereas the spatial cell distribution patterns were different. Polarized HA ceramic plates with an average electric charge of 3.9 μCcm⁻² were implanted in widely spaced defects of canine femora for 3 and 7 days. The osteoblasts were identified by immunochemical detections of osteocalcin and osteopontin. Expressions of osteocalcin and osteopontin were detected throughout the gaps between the implanted HA plates and the cut cortical bone surfaces, especially in the vicinities of the cut cortical bone surfaces and the osteoids regardless of the polarity of the induced charges. Additionally, the newly formed bone tissue that directly bonded to the negatively charged HA surfaces was lined by an osteoblast layer. As soon as 7 days after the implantation, the presence of well-developed osteoblasts suggested that the electrostatic force of the HA ceramics had conditioned the field in the biointerface zone of the polarized HA surfaces.     

Osteoblast attachment to a textured surface in the absence of exogenous adhesion proteins

The present study investigated whether osteoblasts could attach to a culture substratum through a surface texture-dependent mechanism. Four test groups were used: (A) untextured, and three texture groups with maximum feature sizes of (B) <0.5 /spl mu/m, (C) 2 /spl mu/m, and (D) 4 /spl mu/m, respectively. All surfaces were coated with the nonadhesive protein bovine serum albumin (BSA). Osteoblasts were allowed to adhere in serum-free medium for either 1 or 4 h, at which time nonadherent cells were removed. At 4 h, untextured surface A exhibited no cell attachment, while textured surfaces B, C, and D exhibited 9%, 32%, and 16% cell adhesion, respectively. At 16 h of incubation, adherent osteoblasts on textured surface C exhibited focal adhesion contacts and microfilament stress-fiber bundles. These results indicate that microtextured surfaces in the absence of exogenous adhesive proteins can facilitate osteoblast adhesion.     

The cell attachment and morphology of neonatal rat calvarial osteoblasts on the surface of Ti-6Al-4V and plasma-sprayed HA coating: Effect of surface roughness and serum contents

The biocompatibility of material plays an important role in the bone–implant interface for the prosthetic implant fixation. The biocompatibility of implants is associated with the chemical composition, surface topography, surface energy and surface roughness of biomaterials. The effects of two factors, surface roughness and serum contents, on osteoblast behavior at the surface of Ti-6Al-4V and plasma sprayed HA coating were investigated in the experiment. The osteoblasts derived from neonatal rat calvarial were cultured in Dulbeccos modified Eagle medium (DMEM) with fetal bovine serum (FBS) on the surface of polished Ti-6Al-4V (Ti-p), grit-blasted Ti-6Al-4V (Ti-b), polished HA coating (HAC-p), and as-sprayed HA coating (HAC). Under culture medium containing 4% FBS, the level of cell attachment to the polished surface is significantly higher than the rough surface of the same experimental materials during all culture periods. Increasing the contents of FBS up to 10%, the difference of osteoblast attachment is not found between Ti-p and Ti-b. Under 4% serum condition, the cell morphology attached to smooth surfaces (Ti-p and HAC-p) is spread faster and are more flattened than the one to rough surface of the same experimental materials by SEM. After 24 h culture, the corroded cracks are easily observed at the surface of polished HA coatings, and the cell morphology on HAC-p coatings are elongated and less flattened compared with Ti-p. The result is consistent with statistical difference of cell attachment between Ti-p and HAC-p under 4% serum condition.     

Influences of magnetized hydroxyapatite on the growth behaviors of osteoblasts and the mechanism from molecular dynamics simulation

To investigate the influence of magnetized hydroxyapatite on the growth and differentiation of osteoblasts, hydroxyapatite (HA) and magnetized hydroxyapatite (mHA) were synthesized and characterized. The cell viability, differentiation, and morphologies of osteoblasts were investigated in vitro, respectively. The results showed that compared to HA, cells cultured with mHA had better cell viability, and both HA and mHA were beneficial to the early differentiation of osteoblasts. Furthermore, the interaction mechanism between mHA and osteoblasts was elucidated using a molecular dynamics simulation. The simulation results indicated that when cultured with osteoblasts, HA adsorbed bovine serum protein onto its surface from the medium immediately, which was beneficial to the adhesion and proliferation of osteoblasts. The main driving force for the adsorption of bovine serum was the electronic properties of HA crystal faces. The (211) crystal face of HA had the highest electron density among its all crystal faces, thus mainly contributing to the protein adsorption of HA. Nevertheless, the (211) crystal face of mHA still had a relatively higher electron density than that of HA, thus possessing better protein adsorption than that of HA, and in turn promoting the biological functions of osteoblasts.     

Surface energy of hydroxyapatite and β-tricalcium phosphate ceramics driving serum protein adsorption and osteoblast adhesion

The main objective of this work was to evaluate the specific role of calcium phosphates surface energy on serum protein adsorption and human osteoblast adhesion, by isolating chemical effects from those caused by topography. Highly dense phosphate ceramics (single-phase hydroxyapatite HA and β-tricalcium phosphates β-TCP) presenting two distinct nano roughnesses were produced. Some samples were gold-sputter coated in order to conveniently mask the surface chemical effects (without modification of the original roughness) and to study the isolated effect of surface topography on cellular behavior. The results indicated that the nano topography of calcium phosphates strongly affected the protein adsorption process, being more important than surface chemistry. The seeding efficacy of osteoblasts was not affected nor by the topography neither by the calcium phosphate chemistries but the β-TCP chemistry negatively influenced cell spreading. We observed that surface hydrophobicity is another way to change protein adsorption on surfaces. The decrease of the polar component of surface energy on gold-coated samples leaded to a decreased albumin and fibronectin adsorption but to an increased cell adhesion. Overall, this work contributes to better understand the role of topography and surface chemistry of calcium phosphates in serum protein adsorption and osteoblast adhesion.     

Effects of nano-surface properties on initial osteoblast adhesion and Ca/P adsorption ability for titanium alloys

Titanium alloys (Ti6Al4V), while subjected to high temperature surface treatment, experience altered nano-surface characteristics. The effects of such surface treatments are examined, including the initial adhesion force experienced by osteoblasts, the Ca/P adsorption capability, and the nano-surface properties, including the amounts of amphoteric Ti-OH groups, surface topography, and surface roughness. The initial adhesion force is considered a quantitative indicator of cyto-compatibility in?vitro. Previously, a cyto-detacher was applied in a pioneer attempt measuring the initial adhesion force of fibroblasts on a metal surface. Presently, the cyto-detacher is further applied to evaluate the initial adhesion force of osteoblasts. Results reveal that (1)?titanium alloys subjected to heat treatment could promote the adsorption capability of Ca and P; (2)?titanium alloys subjected to heat treatment could have higher initial osteoblast adhesion forces; (3)?the adhesion strength of osteoblasts, ranging from 38.5 to 58.9?nN (nanonewtons), appears stronger for rougher surfaces. It is concluded that the heat treatment could have impacted the biocompatibility in terms of the initial osteoblast adhesion force and Ca/P adsorption capability.     

Bioactive evaluation of 45S5 bioactive glass fibres and preliminary study of human osteoblast attachment

Bioactive glass fibres can be used as tissue engineering scaffolds. In this investigation, the bioactive response of 45S5 glass fibres was assessed in simulated body fluid (SBF). Preliminary attachment of osteoblasts to the fibre surface was assessed, as were the fibre tensile strength and fracture toughness. Fourier transform infrared spectroscopy (FTIR) analysis revealed that hydroxyapatite (HA) was formed on the fibres' surface after 2-4 days in SBF. Raman micro-spectroscopic analysis was used to monitor development of the HA layer during immersion. A correlation was found between increase in intensity of the PO4(3-) peak near 964cm(-1) and appearance of crystalline HA (P-O bending peaks) using FTIR. Such results are encouraging for in situ bioactivity monitoring, as Raman spectra are insensitive to the presence of water, unlike FTIR. Average tensile strength of 45S5 fibres (79 microm diameter) was 340+/-140 MPa. Fracture toughness, determined using fracture surface analysis, was 0.7+/-0.1 MPa m1/2. Confocal microscopy revealed osteoblasts attached and spread along the fibres after 15-90 min culture. Scanning electron microscopy analysis showed that cells with filopodia and dorsal ruffles remained attached after 14 days in culture. These results are encouraging, as cell adhesion is an important first step prior to proliferation and differentiation.     

Preparation of HA/chitosan composite coatings on alkali treated titanium surfaces through sol-gel techniques

In this paper we demonstrated for the first time the feasibility to generate well crystallized hydroxyapatite/chitosan (HA/CS) composite coatings on alkali treated titanium surfaces through combining the in situ hydrothermal precipitation and sol-gel dip coating technique without introducing any other coupling agent. Alkali treatment converting hydrophobic Ti surfaces into super-hydrophilic surfaces was the pre-requirement of uniform coatings. Preliminary cell culturing results revealed that the coatings were biocompatible and supported osteoblasts attachment.     

Enhanced osteoblast and osteoclast responses to a thin film sputtered hydroxyapatite coating

A sputtering technique followed by a low temperature hydrothermal treatment has been demonstrated to produce a dense-and-bioactive hydroxyapatite thin film coating. The purpose of the present study was to investigate osteoblast and osteoclast responses to the hydroxyapatite coated plates and titanium plates with similar roughness. Rat bone marrow stromal cells were cultured on these plates to induce osteoblasts. The cells showed a significantly enhanced proliferation on the hydroxyapatite surface, accompanied by increase of osteoblastic phenotypes. The co-cultured osteoclasts exhibited the significantly different cell number and morphology between the hydroxyapatite and the titanium surfaces. A series of osteoclast marker genes were more stimulated on the hydroxyapatite and thirty two percent of the hydroxyapatite surface area could be resorbed by osteoclasts. The thin film sputtered hydroxyapatite could provide a favorable surface for both osteoblast and osteoclast formation and their function, indicating its good osteoconductivity and biodegradability.     

The impact of the RGD peptide on osteoblast adhesion and spreading on zinc-substituted hydroxyapatite surface

The incorporation of zinc into the hydroxyapatite structure (ZnHA) has been proposed to stimulate osteoblast proliferation and differentiation. Another approach to improve cell adhesion and hydroxyapatite (HA) performance is coating HA with adhesive proteins or peptides such as RGD (arginine–glycine–aspartic acid). The present study investigated the adhesion of murine osteoblastic cells to non-sintered zinc-substituted HA disks before and after the adsorption of RGD. The incorporation of zinc into the HA structure simultaneously changed the topography of disk’s surface on the nanoscale and the disk’s surface chemistry. Fluorescence microscopy analyses using RGD conjugated to a fluorescein derivative demonstrated that ZnHA adsorbed higher amounts of RGD than non-substituted HA. Zinc incorporation into HA promoted cell adhesion and spreading, but no differences in the cell density, adhesion and spreading were detected when RGD was adsorbed onto ZnHA. The pre-treatment of disks with fetal bovine serum (FBS) greatly increased the cell density and cell surface area for all RGD-free groups, overcoming the positive contribution of zinc to cell adhesion. The presence of RGD on the ZnHA surface impaired the effects of FBS pre-treatment possibly due to competition between FBS proteins and RGD for surface binding sites.     

Biomaterial surface properties modulate in vitro rat calvaria osteoblasts response: Roughness and or chemistry?

The aim of this study was a better understanding of the regulation mechanisms of in vitro osteoblast activity on biomaterials. Rat osteoblast behaviour on different surfaces was studied. Surfaces with different roughness (and a similar surface chemistry) or with different surface chemistry (and a similar roughness) were compared. Cellular morphology was observed by scanning electron microscopy and cell adhesion was quantified using an image analysis system. Osteoblast proliferation was quantified by a MTT test and total protein content and alkaline phosphatase (ALP) activity were evaluated by spectrophotometry. Data were compared by statistical analysis.

Results showed that NiTi surface roughness did not influence osteoblasts morphology, adhesion, total protein content and ALP activity whereas it modulated cell proliferation. Roughness was shown to stimulate cell proliferation. For smooth surfaces exhibiting two different chemical compositions, adhesion rate was found to be higher on Thermanox® than on NiTi whereas proliferation was shown to be smaller. ALP activity was also modulated by surface chemistry. Thus, cell adhesion and ALP activity were found to be more governed by surface chemistry than by roughness whereas cell proliferation was shown to be modulated by roughness (this effect increasing during cell culture) and by chemistry (this effect remaining stable in time) together. Total protein content and cell morphology were found to be independent of both parameters (roughness and chemistry). Effects of surface chemistry were discussed in terms of wettability and electron acceptor/donor properties of the surfaces of interest. Immunofluorescence images of adhesion proteins could not demonstrate differences between the three surfaces.     

Electrospray deposition of nanohydroxyapatite coatings: A strategy to mimic bone apatite mineral

The biological performance of orthopaedic and oral metallic implants can be enhanced significantly by the application of bioactive coatings. In this work, a cost-efficient alternative to the traditional technique to produce a hydroxyapatite (HA) coating with a nanostructured feature onto a metallic implant surface at room temperature via electrospray deposition, is presented. To evaluate the bioactive capacity of these nanoHA (nHA) coatings in vitro, an acellular simulated body fluid soaking experiment and a human osteoblast (HOB) cell culture work were conducted. Under these physiological conditions, the accelerated apatite precipitation process occurred on the nHA-coated titanium surfaces as compared to the uncoated titanium surfaces. HOB cells developed mature cytoskeletons with distinct evidence of actin stress fibres and vinculin adhesion plaques, on these nHA coatings. Hence, this deposition technique holds great potential in producing high quality nHA coatings for biomedical applications.     

碳纳米管/羟基磷灰石复合材料的制备及表征

以含钴介孔分子筛为催化剂、乙醇为碳源, 采用CVD法制备碳纳米管(CNTs)。通过原位合成法制备一系列不同碳纳米管含量的碳纳米管/羟基磷灰石(CNTs/HA)复合材料。分别采用XRD、FTIR、TEM、N₂吸附-脱附和Raman光谱等分析手段, 对所合成CNTs/HA复合材料的晶相、结构、形貌和比表面积等进行了表征。同时研究了碳纳米管的添加量对所合成CNTs/HA复合材料形貌的影响。XRD与Raman结果表明, 所得CNTs/HA复合粉体中仅有CNTs与HA两种物相, 纯度较高, 结晶度较好; TEM结果显示, CNTs/HA复合材料中CNTs表面均匀包裹着一层纳米级的针状HA晶粒, 两者形成了较强的界面结合, 且当CNTs与HA的质量比为3:17时, CNTs与HA形成最佳结合状态; N₂吸附-脱附表征结果表明, 与HA的比表面积相比, CNTs/HA复合材料具有较高比表面积。     

Specific proliferation rates of human osteoblasts on calcium phosphate surfaces with variable concentrations of α-TCP

Ideally, ceramics used in the repair of bone defects need to be resorbed and replaced by newly formed bone in vivo. Tricalcium phosphate (TCP) has been widely used in association with hydroxyapatite (HA) due to its higher resorption kinetics when compared with HA alone. The aim of our study was to quantitatively investigate the effect of α-tricalcium phosphate (α-TCP) on human osteoblasts' adhesion and proliferation. Ceramic samples with variable concentrations of α-TCP and HA were produced by the calcination of calcium-deficient and stoichiometric HA. Human osteoblasts were cultured on the materials in three distinct experiments with different concentrations of cells. Numerical evaluation of cellular growth along time in culture was performed for each condition. The quantity of cells seeded onto the ceramics seems to influence the osteoblast behavior once proliferation was lower when more cells were seeded onto the samples. However, a smaller content of α-TCP in relation to that of HA did not significantly modify the specific proliferation rates of the osteoblasts. Only after a long time in culture, the increasing of the α-TCP content seems to change the cells' behavior.     

In vitro cellular response to titanium electrochemically coated with hydroxyapatite compared to titanium with three different levels of surface roughness

The in vitro response of primary human osteoblast-like (HOB) cells to a novel hydroxyapatite (HA) coated titanium substrate, produced by a low temperature electrochemical method, was compared to three different titanium surfaces: as-machined, Al₂O₃-blasted, plasma-sprayed with titanium particles. HOB cells were cultured on different surfaces for 3, 7 and 14 days at 37 °C. The cell morphology was assessed using scanning electron microscopy (SEM). Cell growth and proliferation were assessed by the measurement of total cellular DNA and tritiated thymidine incorporation. Measurement of alkaline phosphatase (ALP) production was used as an indicator of the phenotype of the cultured HOB cells. After three days incubation, the electrochemically coated HA surface produced the highest level of cell proliferation, and the Al₂O₃-blasted surface the lowest. Interestingly, as the incubation time was increased to 7 days all surfaces produced a large drop in tritiated thymidine incorporation apart from the Al₂O₃-blasted surface, which showed a small increase. Cells cultured on all four surfaces showed an increased expression of ALP with increased incubation time, although there was not a statistically significant difference between surfaces at each time point. Typical osteoblast morphology was observed for cells cultured on all samples. The HA coated sample showed evidence of a deposited phase after three days of incubation, which was not observed on any other surface. Cells incubated on the HA coated substrate appeared to exhibit the highest number of cell processes attaching to the surface, which was indicative of optimal cell attachment. The crystalline HA coating, produced by a low temperature route, appeared to result in a more bioactive surface on the c.p. Ti substrate than was observed for the other three different Ti surfaces.     

Selectively Promoting or Preventing Osteoblast Growth on Titanium Functionalized with Polyelectrolyte Multilayers

Titanium plays an important role in medical applications, such as hip joint implants or fixation plates. These implants must perform differently depending on their clinical application. In particular, the osseointegrative properties required of the implant vary with clinical application. The present work is aimed at the functionalization of titanium surface using polyelectrolyte multilayers consisting of natural biopolymers and testing their cell adhesive properties with respect to the osseointegration capacity. Multilayered coatings were created from chitosan (Chi), hyaluronic acid (HA), and gelatine (Gel) through layer‐by‐layer deposition. Cell adhesion, proliferation, and viability were tested in vitro with the human osteoblast cell line CAL‐72 at timescales up to 7 d. Two multilayer coatings consisting of alternated chitosan/gelatin or chitosan/hyaluronic acid layers with the outmost layer of gelatin (Chi/Gel) or hyaluronic acid (Chi/HA), respectively, were tested. The experimental results showed that surfaces functionalized with Chi/Gel and Chi/HA multilayers demonstrated a good initial adhesion of osteoblasts. After 4 d culturing, osteoblast cells were almost completely detached from the substrates functionalized with Chi/HA multilayers. In contrast to Chi/HA, the proliferation of osteoblasts on substrates with Chi/Gel multilayer coatings was statistically significantly higher compared to the control titanium. We have shown that the growth of osteoblasts can be enhanced or completely prevented on a titanium surface functionalized with polyelectrolyte multilayers consisting of natural biopolymers, as desired. Both multilayer coatings, Chi/Gel and Chi/HA, have potential for applications in the field of titanium implants, where rapid osseointegration is essential, and/or where no ingrowth of the implant is desired, respectively.     

羟基磷灰石/钛合金骨替代材料体外培养成骨细胞的实验研究

实验选用小鼠头盖骨成骨细胞,采用体外细胞培养技术对具有羟基磷灰石涂层的钛合金(HA/Ti)与未经过表面改性的钛合金两种骨替代材料进行细胞相容性评价,动态观察两种骨替代材料对成骨细胞生长、附着的影响。结果表明两种骨替代材料对成骨细胞生长无抑制作用,未发生细胞毒性反应,细胞在两种材料表面均能正常粘附、生长、增殖,均具有良好的细胞附着形态和细胞增殖率,而HA/钛合金材料具有更好的成骨性,是一种骨细胞相容性良好的骨替代材料。     

Effects of silicate and carbonate substitution on the properties of hydroxyapatite prepared by aqueous co-precipitation method

This research was conducted to investigate the effects of silicate and carbonate substitutions in hydroxyapatite (HA) on phase retention, physical properties, and in vitro biological response. A wet chemical method was employed to synthesize silicate-substituted HA (Si–HA) and carbonate-substituted HA (CHA). It was shown that the presence of silicate and carbonate ions in the HA lattice increases the specific surface area and Ca/P molar ratio. In addition, the substitution also resulted in a reduction in the crystallinity of the HA powder and promoted better bioactivity of the sintered body. This was confirmed through in vitro cell culture experiment which revealed that the osteoblast cells adhered well on the surface of the ceramic and that ionic-substituted HA exhibited better cell performance than the control HA.