In vitro growth and differentiation of osteoblast-like human bone marrow cells on glass reinforced hydroxyapatite plasma-sprayed coatings

Human osteoblastic bone marrow cells were cultured for periods of up to 28 days in control conditions and on the surface of a glass reinforced hydroxyapatite composite (HA/G1) and commercial hydroxyapatite (HA) plasma-sprayed coatings, in the as-received condition and after immersion treatment in culture medium for 21 days. Cultures were characterized for total protein content and alkaline phosphatase activity. Scanning electron microscope analyses were performed on control cultures, seeded materials and materials incubated in the absence of cells. Culture media were analyzed for total and ionized calcium and phosphorus concentrations throughout the incubation period. Immersion of HA/G1 and HA coatings in culture medium resulted in significant alterations to the levels of calcium and phosphorus in the medium, leading to surface modifications. However, seeded material samples showed significant differences in the pattern of variation of the levels of these species. Cell proliferation was observed in the as-received HA/G1 composite, but cell mediated formation of mineral deposits was not proved. In contrast, as-received HA hardly supported cell growth. Previously immersed material samples showed cell proliferation and evidence of biological formation of mineral deposits. However, the HA/G1 composite presented better surface characteristics for cell growth as the behavior of bone marrow cells was closer to that observed in control cultures. © 1999 Kluwer Academic Publishers     

In vitro testing of plasma-sprayed hydroxyapatite coatings

Hydroxyapatite-coated coupons were tested in various physiological media. Immersion in Ringer's solution showed that heat-treated coatings displayed a weight gain but the assprayed coatings underwent a weight loss. Dissolution of the coating was measured by weighing the specimen before and after ageing. Immersion of hydroxyapatite coatings showed the appearance of small spheres that were identified by X-ray diffraction as hydroxyapatite. Changes in coating morphology were detected and the coating degradation mechanisms are discussed. This paper thus looks at the morphology, composition, crystallinity and dissolution of hydroxyapatite coatings aged in Ringer's solution.     

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.     

In vitro response of osteoblasts to hydroxyapatite-reinforced polyethylene composites

A primary human cell culture model was used to investigate a range of hydroxyapaptite (HA)-reinforced high-density polyethylene (HDPE) composites (HAPEXTM). These materials are being developed as potential bone-substitute materials. When designing and optimizing a second-generation biomaterial, it is important to achieve a balance between mechanical and biological properties without compromising either. Biochemical and histological parameters have been used to compare the biological response of 20% and 40% volume HA in HDPE. Cellular DNA and incorporation of tritiated thymidine was measured to assess cell proliferation. Alkaline phosphatase (ALP) production was used as a marker of osteoblast phenotype expression. In this preliminary study, osteoblasts cultured on the 20% HAPEXTM showed a greater increase in the rate of proliferation and osteoblast expression as indicated by an increase in ALP activity compared to the 40% HAPEXTM over the time period studied. Osteoblast-like cells showed a flattened morphology on both composites and in some cases a greater covering was observed on the 20% HAPEXTM. These results indicate that the composites may not be identical in terms of bioactivity and that further research on surface topography and physico-chemical properties is required to assess fully the biological response of these composites. © 1998 Kluwer Academic Publishers     

Specific response of osteoblast-like cells on hydroxyapatite layer containing serum protein

Accelerations of bone-like apatite deposition and cell growth on an electrically polarized ceramic hydroxyapatite have been reported. A relationship between these phenomena was investigated in a previous report, and then it was suggested that osteoblast-like cell’s (MC3T3-E1) growth had relevance to the mineral growth. The effect of the formed apatite layer especially appeared to be on the cell adhesion. The acceleration of cell proliferation on the polarized HAp has been shown using fibroblastic cell (L929) and nerve cell (SK-N-SH) lines, therefore the effect of the layer on L929 and SK-N-SH was investigated to support the mechanism of acceleration of cell proliferation by polarization of HAp. In this study, the effect of the bone-like apatite layer was not confirmed on L929 cell’s growth. On the other hand, the acceleration of nerve cell’s proliferation was confirmed on the formed apatite layer. However, the remarkable improvement of the cell adhesion of SK-N-SH was not confirmed on the apatite layer. Consequently, it was considered that the bone-like apatite containing serum protein obtained by the coprecipitation of bone-like apatite and serum protein has a pronounced role only in the activity of osteoblast-like cells.     

In vitro assessment of the biological response to nano-sized hydroxyapatite

Nano-sized, rod-like hydroxyapatite (nHA) crystals were produced and shown to be phasepure by X-ray diffraction analysis, as no secondary phases were observed. The nHA suspension was electrosprayed onto glass substrates using a novel processing routine to maintain nanocrystals of hydroxyapatite. The biocompatibility of nHAwas determined using human monocyte-derived macrophages and human osteoblast-like (HOB) cell models. The release of lactate dehydrogenase (LDH) from human monocyte-derived macrophages was measured as an indicator of cytotoxicity. The release of the inflammatory cytokine, tumour necrosis factor alpha (TNF-alpha) from cells in the presence of nHA crystallites was used as a measure of the inflammatory response. Although there was some evidence of LDH release from human monocyte-derived macrophages when in contact with high concentrations of nHA crystals, there was no significant release of TNF-alpha. Moreover, nHA-sprayed substrates were able to support the attachment and the growth of HOB cells. These results indicate that nHA crystals may be suitable for intraosseous implantation and offers the potential to formulate enhanced composites for biomedical applications.     

Effect of hydroxyapatite porosity on growth and differentiation of human osteoblast-like cells

To study whether hydroxyapatite (HA) porosity can influence its osteoconductive properties, cell adhesion, proliferation and differentiation were compared in human osteoblast-like cells grown on HA disks of different porosity (A = 20%, B = 40%, C = 60%). Human osteoblast-like cells were isolated and characterized. Proliferation rate and alkaline phosphatase (ALP) activity were assessed at 3, 7, 15, 21, and 28 days. Type I collagen and osteonectin production were demonstrated with fluorescence microscopy and osteoblast adhesion studied at 7 and 21 days by scanning electron microscopic analysis. Cell growth on HA was three- to six-fold lower than on polystyrene control disks. At 28 days, 2141 (±350) cells/well grew on the most porous disks (Group C), with highly significant differences from controls (p < 0.005). The ALP production was 2–3 fold lower on HA than on plastic. In the Group C the mean ALP activity was of 2.95 (±0.07) UI/well after 28 days, higher than in the other two groups. At 21 and 28 days, proliferation rate and ALP activity on the three HA cultures were significantly different (p < 0.05). A decrease in cell population and increased ALP activity were observed on the most porous material, and high proliferation and poor differentiation rates on the less porous disks.     

Antibacterial hydroxyapatite/chitosan complex coatings with superior osteoblastic cell response

Chitosan was widely used as an antibacterial component. While most antibacterial materials also possess cytotoxicities, we hypothesize that selectively destruction of bacterial cells can be achieved by controlling the material parameters of chitosan, due to its intrinsic antibacterial mechanism. In this study, porous hydroxyapatite coatings prepared by the liquid precursor plasma spraying process were used for loading the chitosan with different concentrations: 10, 20, 50, and 100 g/L, respectively. The antibacterial properties and osteoblastic cell response of the hydroxyapatite/chitosan complex coatings were studied as a function of chitosan concentration. The results indicated that the antimicrobial activity was directly proportional to the chitosan concentration, while loading of chitosan with lower concentrations (10 and 20 g/L) was even beneficial to the proliferation of osteoblastic cells. Overall, our study demonstrated that combined antibacterial activity and superior osteoblast cell response can be achieved by using hydroxyapatite/chitosan complex coatings, which have great potential in bone replacement and regeneration applications.     

In vitro biological,chemical and electrochemical evaluation of titania reinforced hydroxyapatite sol–gel coatings on surgical grade 316L SS

Development and characterization of titania (TiO₂)/Hydroxyapatite (HAP) reinforced coating derived by sol–gel dip coating process was studied. Structural, electrochemical and biological behavior of the reinforced coatings were conducted and elaborated in this paper. The bonding between the HAP matrix and TiO₂ reinforcement might have been achieved predominantly through a chemical bond that resulted from the mutual chemical reaction among the components. In vitro electrochemical studies of the reinforced coatings shows good corrosion resistance and minimum leach out of metal ions in the simulated body fluid (Hank's balanced salt solution) environment. The cell culture cytotoxicity studies were also carried out using L929 cell line. Cell culture studies showed that the reinforced coatings were non-cytotoxic to mouse fibroblast cell line. On the basis of these observations, it's concluded that the titania reinforced materials are biocompatible and corrosion resistant and hence are a candidate material for biomedical applications.     

In vitro behavior of fluoridated hydroxyapatite coatings in organic-containing simulated body fluid

A dense and pure hydroxyapatite [HA, Ca₁₀(PO₄)₆(OH)₂] coating and a fluoridated HA [Ca₁₀(PO₄)₆(OH)_0.67F_1.33] are deposited on Ti6Al4V substrates by sol-gel dip coating method. Glucose and bovine serum albumin have been added in standard simulated body fluid (SBF) to form organic-containing SBF in simulation of the physiological blood plasma. The HA and the fluoridated HA coatings are immersed in the standard and modified SBF for time periods of 2, 4, 7, 14 and 28 days at 37 ± 0.1°C. After soaking, the coating surface is examined for nucleation and growth of apatite using SEM morphological observation. The post-soaking SBF solutions are analyzed via Inductively Coupled Plasma spectroscopy for calcium ion concentration. The results show that at concentration of 40 g/L, bovine serum albumin has significant retardation effect on apatite precipitation from SBF onto pure or fluoridated HA coatings; Fluorine-incorporation in HA has positive bio-activation effect in both standard SBF and organic-containing SBF. However, glucose addition in SBF does not generate significant influence on the bioactivity of HA and fluoridated HA.     

Nanocrystalline spherical hydroxyapatite granules for bone repair: in vitro evaluation with osteoblast-like cells and osteoclasts

Conventionally sintered hydroxyapatite-based materials for bone repair show poor resorbability due to the loss of nanocrystallinity. The present study describes a method to establish nanocrystalline hydroxyapatite granules. The material was prepared by ionotropic gelation of an alginate sol containing hydroxyapatite (HA) powder. Subsequent thermal elimination of alginate at 650 °C yielded non-sintered, but unexpectedly stable hydroxyapatite granules. By adding stearic acid as an organic filler to the alginate/HA suspension, the granules exhibited macropores after thermal treatment. A third type of material was achieved by additional coating of the granules with silica particles. Microstructure and specific surface area of the different materials were characterized in comparison to the already established granular calcium phosphate material Cerasorb M^®. Cytocompatibility and potential for bone regeneration of the materials was evaluated by in vitro examinations with osteosarcoma cells and osteoclasts. Osteoblast-like SaOS-2 cells proliferated on all examined materials and showed the typical increase of alkaline phosphatase (ALP) activity during cultivation. Expression of bone-related genes coding for ALP, osteonectin, osteopontin, osteocalcin and bone sialoprotein II on the materials was proven by RT-PCR. Human monocytes were seeded onto the different granules and osteoclastogenesis was examined by activity measurement of tartrate-specific acid phosphatase (TRAP). Gene expression analysis after 23 days of cultivation revealed an increased expression of osteoclast-related genes TRAP, vitronectin receptor and cathepsin K, which was on the same level for all examined materials. These results indicate, that the nanocrystalline granular materials are of clinical interest, especially for bone regeneration.     

The interactions of bisphosphonates in solution and as coatings on hydroxyapatite with osteoblasts

Aseptic loosening is one of the major causes of failure of artificial hip joints, and it can occur for several reasons, including osteolysis of the bone tissue in response to stress shielding or cellular reactions to wear debris. Any treatment of the prosthesis which could minimize the osteolytic response of bone tissue may be able to extend the life-time of the implant. Bisphosphonates are potent inhibitors of osteoclastic bone resorption, and they bind avidly to hydroxyapatite (HA). Coating the prostheses with bisphosphonates may therefore inhibit osteolysis. We have investigated the potential for this approach by determining whether bisphosphonates interact with osteoblasts in vitro. The effects of pamidronate (P), clodronate (C), and etidronate (E) in solution and when coated onto HA were investigated. P inhibited protein and collagen syntheses potently when in solution, but not after being bound to HA. When bound to HA, both P and C increased DNA, protein and collagen syntheses of osteoblasts and may encourage the osseointegration of implants. The pharmacological effects of the bisphosphonates studied altered dramatically after binding to HA. This must be fully investigated before this approach to prolonging prostheses stability can be evaluated.     

Mineralization of osteoblasts with electrospun collagen/hydroxyapatite nanofibers

Regeneration of fractured or diseased bones is the challenge faced by current technologies in tissue engineering. The major solid components of human bone consist of collagen and hydroxyapatite. Collagen (Col) and hydroxyapatite (HA) have potential in mimicking natural extracellular matrix and replacing diseased skeletal bones. More attention has been focused on HA because of its crystallographic structure similar to inorganic compound found in natural bone and extensively investigated due to its excellent biocompatibility, bioactivity and osteoconductivity properties. In the present study, electrospun nanofibrous scaffolds are fabricated with collagen (80 mg/ml) and Col/HA (1:1). The diameter of the collagen nanofibers is around 265 ± 0.64 nm and Col/HA nanofibers are 293 ± 1.45 nm. The crystalline HA (29 ± 7.5 nm) loaded into the collagen nanofibers are embedded within nanofibrous matrix of the scaffolds. Osteoblasts cultured on both scaffolds and show insignificant level of proliferation but mineralization was significantly (p < 0.001) increased to 56% in Col/HA nanofibrous scaffolds compared to collagen. Energy dispersive X-ray analysis (EDX) spectroscopy results proved the presence of higher level of calcium and phosphorous in Col/HA nanocomposites than collagen nanofibrous scaffolds grown osteoblasts. The results of the present study suggested that the designed electrospun nanofibrous scaffold (Col/HA) have potential biomaterial for bone tissue engineering.     

In vitro cytocompatibility and corrosion resistance of zinc-doped hydroxyapatite coatings on a titanium substrate

To improve biocompatibility and corrosion resistance during the initial implantation stage, zinc-substituted hydroxyapatite (ZnHAp) coating was fabricated on pure titanium by the electrolytic deposition method. The morphology, microstructure and chemical composition of the coating were investigated by X-ray diffraction, scanning electron microscopy, energy-dispersive X-ray analysis and Fourier transform infrared spectroscopy. The prepared ZnHAp crystals were calcium deficient and were carbonated owing to the incorporation of some Zn²⁺. This incorporation of Zn²⁺ into the HAp significantly reduced porosity and caused the coating to become noticeably denser. In addition, the Zn²⁺ ions were homogeneously distributed in the coating. The potentiodynamic polarisation test revealed that the ZnHAp-coated surface showed superior corrosion resistance over that of the HAp-coated surface and bare Ti. The in vitro bioactivity was evaluated in a simulated body fluid, which revealed that the ZnHAp coating can rapidly induce bone-like apatite formation of nuclear and growth features. In addition, the cell response tests showed that the MC3T3-E1 cells on the ZnHAp coating clearly enhanced the in vitro cytocompatibility of Ti compared with the same cells on HAp coating. ZnHAp coating was thus beneficial for improving biocompatibility.     

Preparation and characterization of a novel hydroxyapatite/carbon nanotubes composite and its interaction with osteoblast-like cells

Hydroxyapatite (HA) and its based biomaterials could chemically bond directly to bone when implanted, resulting in the formation of a strong bone-implant interface. Carbon nanotubes (CNT) are believed to be very promising in the enhancement ceramic matrix and played an important role as reinforcement for imparting strength and toughness to brittle HA bioceramic. Here we demonstrate the potential use in reinforcing biomaterials through an attempt to use CNT strengthen brittle HA bioceramic. This work aims to study the optimum sintering conditions of CNT modified HA to get CNT–HA composite with improved mechanical properties using a rapid spark plasma sintering system. The microstructure, phases, chemical compositions and mechanical properties of the ceramic samples were characterized by various advanced methods. Though no obvious chemical reaction between CNT and HA has been detected, the physical crosslink caused by the presence of CNT resulted in that a CNT–HA composite with a relatively high value of modulus (~ 131.1 GPa) and hardness (~ 6.86 GPa) achieved at the sintering temperature of 1100 °C. In vitro cellular responses to the composites were assessed to investigate the proliferation and morphology of a human osteoblast cell line cultured on the various composites.     

Adhesion formation of primary human osteoblasts and the functional response of mesenchymal stem cells to 330nm deep microgrooves.

The surface microtexture of an orthopaedic device can regulate cellular adhesion, a process fundamental in the initiation of osteoinduction and osteogenesis. Advances in fabrication techniques have evolved to include the field of surface modification; in particular, nanotechnology has allowed for the development of experimental nanoscale substrates for investigation into cell nanofeature interactions. Here primary human osteoblasts (HOBs) were cultured on ordered nanoscale groove/ridge arrays fabricated by photolithography. Grooves were 330nm deep and either 10, 25 or 100mum in width. Adhesion subtypes in HOBs were quantified by immunofluorescent microscopy and cell-substrate interactions were investigated via immunocytochemistry with scanning electron microscopy. To further investigate the effects of these substrates on cellular function, 1.7K gene microarray analysis was used to establish gene regulation profiles of mesenchymal stem cells cultured on these nanotopographies. Nanotopographies significantly affected the formation of focal complexes (FXs), focal adhesions (FAs) and supermature adhesions (SMAs). Planar control substrates induced widespread adhesion formation; 100mum wide groove/ridge arrays did not significantly affect adhesion formation yet induced upregulation of genes involved in skeletal development and increased osteospecific function; 25mum wide groove/ridge arrays were associated with a reduction in SMA and an increase in FX formation; and 10mum wide groove/ridge arrays significantly reduced osteoblast adhesion and induced an interplay of up- and downregulation of gene expression. This study indicates that groove/ridge topographies are important modulators of both cellular adhesion and osteospecific function and, critically, that groove/ridge width is important in determining cellular response.     

The response of primary rat and human osteoblasts and an immortalized rat osteoblast cell line to orthopaedic materials: comparative sensitivity of several toxicity indices

When studying the biocompatibility of orthopaedic biomaterials it isoften necessary to discriminate between responses which show mild cytotoxicity.It is therefore essential to use a very sensitive index of toxicity. We havecompared the sensitivity of four well-established indices of toxicity: totalcell protein content, leakage of lactate dehydrogenase (LDH), reducedglutathione content and the MTT assay, with that of a novel index, alkalinephosphatase (ALP) activity. Comparisons were made by detecting nickel chloridetoxicity in osteoblasts. ALP activity, the novel method, proved the mostsensitive index of toxicity and it provides a convenient automated assay forassessing the interactions of materials with osteoblasts. The responses tonickel chloride and to aqueous extracts prepared from carbon fibre reinforcedepoxy and polyetheretherketone (peek), two candidate materials for orthopaedicimplants, were compared in primary and immortalized rat osteoblasts, and !in primary human osteoblasts. Although the immortalized rat osteoblast cell line,FFC, was consistently the most sensitive cell type, the responses of the humancells and the FFC cell line were similar in terms of ALP activity throughout therange of nickel concentrations studied. Neither peek nor epoxy material extractsshowed a significant decrease in the MTT or ALP responses in any of the threecell types. Our data suggest that immortalized rat osteoblasts may provide anin vitro model system for screening the biocompatibility of orthopaedicpolymers.     

The effect of varying percentage hydroxyapatite in poly(ethylmethacrylate) bone cement on human osteoblast-like cells

Poly(ethylmethacrylate) (PEMA) bone cement has been developed, and the cements mechanical properties are improved by the incorporation of particulate fillers, such as hydroxyapatite (HA). In this in vitro study, human osteoblast-like (HOB) cells were used to examine the effect on cellular behavior of the addition of HA to PEMA using a plain PEMA control. Thymidine uptake (³H-TdR) and total DNA were used to assess cell growth and proliferation. Confocal laser scanning microscopy (CLSM) was used to study focal contacts and actin cytoskeletal organisation. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to assess cell morphology and cellular ultrastucture. The early time points showed preferential anchorage to the HA exposed on the cement surface, but no difference in adhesion or proliferation. These results have been attributed to increases in residual monomer with HA incorporation, as shown by proton nuclear magnetic resonance (H¹-NMR) spectra.     

Electrostatic ligand coatings of nanoparticles enable ligand-specific gene delivery to human primary cells

A general method of coating polymer/DNA nanoparticles was developed. Peptide coated nanoparticles were found to have favorable biophysical characteristics including small particle size, near-neutral zeta potential, and stability in serum. At appropriate formulation conditions including near-neutral charge ratio, the coated nanoparticles enabled effective ligand-specific gene delivery to human primary endothelial cells in serum-containing media. As this nanoparticulate drug delivery system has high efficacy, ligand-based specificity, biodegradability, and low cytotoxicity, it may be potentially useful in several clinical applications.     

Behavior of osteoblast-like cells on calcium-deficient hydroxyapatite ceramics composed of particles with different shapes and sizes

In designing the biomaterials, it is important to control their surface morphologies, because they affect the interactions between the materials and cells. We previously reported that porous calcium-deficient hydroxyapatite (HA) ceramics composed of rod-like particles had advantages over sintered porous HA ceramics; however, the effects of the surface morphology of calcium-deficient HA ceramics on cell behavior have remained unclear. Using a hydrothermal process, we successfully prepared porous calcium-deficient HA ceramics with different surface morphologies, composed of plate-like particles of 200–300, 500–800 nm, or 2–3 μm in width and rod-like particles of 1 or 3–5 μm in width, respectively. The effects of these surface morphologies on the behavior of osteoblast-like cells were examined. Although the numbers of cells adhered to the ceramic specimens did not differ significantly among the specimens, the proliferation rates of cells on the ceramics decreased with decreasing particle size. Our results reveal that controlling the surface morphology that is governed by particle shape and size is important for designing porous calcium-deficient HA ceramics.