Preparation and biological evaluation of hydroxyapatite-coated nickel-free high-nitrogen stainless steel

Abstract

Calcium phosphate was formed on nickel-free high-nitrogen stainless steel (HNS) by chemical solution deposition. The calcium phosphate deposition was enhanced by glutamic acid covalently immobilized on the surface of HNS with trisuccinimidyl citrate as a linker. X-ray diffraction patterns and Fourier transform infrared spectra showed that the material deposited on glutamic acid-immobilized HNS within 24 h was low-crystallinity calcium-deficient carbonate-containing hydroxyapatite (HAp). The biological activity of the resulting HAp-coated HNS was investigated by using a human osteoblast-like MG-63 cell culture. The HAp-coated HNS stimulated the alkaline-phosphate activity of the MG-63 culture after 7 days. Therefore, HAp-coated HNS is suitable for orthopedic devices and soft tissue adhesion materials.     

Cytocompatibility of High Nitrogen Nickel-Free Stainless Steel for Orthopedic Implants

Cytocompatibility of high nitrogen nickel-free stainless steel(HNS) with different nitrogen content was evaluated and compared with a conventional austenitic stainless steel 317L.The MTT assay(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed on MG63 osteoblasts to assess the cytotoxicity.The expression of selected marker typical of differentiated osteoblasts,such as alkaline phosphatase activity(AKP),was also monitored in MG63 cells cultured on the tested materials.As a result,HNS had higher cell growth than 317L;meanwhile the cytocompatibility was increased with increasing nitrogen content.Furthermore,HNS enhanced osteoblasts differentiation,as confirmed by AKP activity.Overall these facts indicated that HNS had higher cytocompatibility than 317L and the nitrogen content contributed to the higher cytocompatibility of HNS.The influence of nitrogen on surface energy further explained the cytocompatibility of HNS.     

In vitro testing of Nd:YAG laser processed calcium phosphate coatings

Nd:YAG laser cladding is a new method for deposition of a calcium phosphate onto metallic surfaces of interest in implantology. The aim of this study was to compare the biologic response of MG-63 human osteoblast-like cells grown on Ti-6Al-4V substrates coated with a calcium phosphate layer applied using different methods: plasma spraying as reference material and Nd:YAG laser cladding as test material. Tissue culture polystyrene was used as negative control. The Nd:YAG laser clad material showed a behaviour similar to the reference material, plasma spray, respective to cell morphology (SEM observations), cell proliferation (AlamarBlue assay) and cytotoxicity of extracts (MTT assay). Proliferation, as measured by the AlamarBlue assay, showed little difference in the metabolic activity of the cells on the materials over an 18 day culture period. There were no significant differences in the cellular growth response on the test material when compared to the ones exhibited by the reference material. In the solvent extraction test all the extracts had some detrimental effect on cellular activity at 100% concentration, although cells incubated in the test material extract showed a proliferation rate similar to that of the reference material. To better understand the scope of these results it should be taken into account that the Nd:YAG clad coating has recently been developed. The fact that its in vitro performance is comparable to that produced by plasma spray, a material commercially available for more than ten years, indicates that this new laser based method could be of commercial interest in the near future.     

Silicate and borate glasses as composite fillers: a bioactivity and biocompatibility study

Composites filled with a silicate glass (CSi) and a new borate glass (CB) were developed and compared in terms of their in vitro behaviour both in acellular and cellular media. Acellular tests were carried out in SBF and the composites were characterized by SEM-EDS, XRD and ICP. Biocompatibility studies were investigated by in vitro cell culture with MG-63 osteoblast-like and human bone marrow cells. The growth of spherical calcium phosphate aggregates was observed in acellular medium on all composite surfaces indicating that these materials became potentially bioactive. The biological assessment resulted in a dissimilar behavior of the composites. The CSi demonstrated an inductive effect on the proliferation of cells. The cells showed a normal morphology and high growth rate when compared to standard culture plates. Contrarily, inhibition of cell proliferation occurred in the CB probably due to its high degradation rate, leading to high B and Mg ionic concentration in the cell culture medium.     

Initial biocompatibility and enhanced osteoblast response of Si doping in a porous BCP bone graft substitute

Granular shape biphasic calcium phosphate (BCP) bone grafts with and without doping of silicon cations were evaluated in regards to biocompatibility and MG-63 cellular response. To do this we studied Cellular cytotoxicity, cellular adhesion and spreading behavior and cellular differentiation with alizarin red S staining. Gene expression in MG-63 cells on the implanted bone substitutes was also examined at different time points using RT-PCR. In comparison, the Si-doped BCP granule showed more cellular viability than the BCP granule without doping in MTT assay. Moreover, cell proliferation was much higher when Si doping was employed. The cells grown on the silicon-doped BCP substitutes had more active filopodial growth with cytoplasmic webbing that proceeded to the flattening stage, which was indicative of well cellular adhesion. When these cells were exposed to Si-doped BCP granules for 14 days, well differentiated MG-63 cells were observed. Osteonectin and osteopontin genes were highly expressed in the late stage of differentiation (14 days), whereas collagen type I mRNA were found to be highly expressed during the early stage (day 3). These combined results of this study demonstrate that silicon-doped BCP enhanced osteoblast attachment/spreading, proliferation, differentiation and gene expression.     

Micromorphological effect of calcium phosphate coating on compatibility of magnesium alloy with osteoblast

Abstract

Octacalcium phosphate (OCP) and hydroxyapatite (HAp) coatings were developed to control the degradation speed and to improve the biocompatibility of biodegradable magnesium alloys. Osteoblast MG-63 was cultured directly on OCP- and HAp-coated Mg-3Al-1Zn (wt%, AZ31) alloy (OCP- and HAp-AZ31) to evaluate cell compatibility. Cell proliferation was remarkably improved with OCP and HAp coatings which reduced the corrosion and prevented the H₂O₂ generation on Mg alloy substrate. OCP-AZ31 showed sparse distribution of living cell colonies and dead cells. HAp-AZ31 showed dense and homogeneous distribution of living cells, with dead cells localized over and around corrosion pits, some of which were formed underneath the coating. These results demonstrated that cells were dead due to changes in the local environment, and it is necessary to evaluate the local biocompatibility of magnesium alloys. Cell density on HAp-AZ31 was higher than that on OCP-AZ31 although there was not a significant difference in the amount of Mg ions released in medium between OCP- and HAp-AZ31. The outer layer of OCP and HAp coatings consisted of plate-like crystal with a thickness of around 0.1 μm and rod-like crystals with a diameter of around 0.1 μm, respectively, which grew from a continuous inner layer. Osteoblasts formed focal contacts on the tips of plate-like OCP and rod-like HAp crystals, with heights of 2–5 μm. The spacing between OCP tips of 0.8–1.1 μm was wider than that between HAp tips of 0.2–0.3 μm. These results demonstrated that cell proliferation depended on the micromorphology of the coatings which governed spacing of focal contacts. Consequently, HAp coating is suitable for improving cell compatibility and bone-forming ability of the Mg alloy.     

Promotion of initial cell adhesion on trisuccinimidyl citrate-modified nickel-free high-nitrogen stainless steel

The surface of nickel-free high-nitrogen stainless steel (HNS) was modified with a citric acid-based cross-linker, trisuccinimidyl citrate (TSC), to promote initial cell adhesion in external skeletal fixation pins. The remaining active ester groups on TSC-immobilized HNS reacted with the amino groups of serum proteins. The immobilized serum proteins formed cell recognition sites to promote the initial cell adhesion immediately after cell seeding. The amount of fibronectin, which is a typical cell adhesion protein, immobilized on the TSC-immobilized HNS surface was threefold greater than on the original HNS after only 15 min. The fibroblastic cell culture experiments showed that the initial cell adhesion was significantly enhanced on the TSC-immobilized HNS compared with the original HNS at 3 h. Furthermore, the cell adhesion activity of the TSC-immobilized HNS continued to promote cell proliferation even at 7 days. Therefore, TSC-immobilized HNS may enable the rapid integration of soft tissues through its reaction with the patient’s serum proteins and extracellular proteins around the surgical site.     

Preparation of flexible bone tissue scaffold utilizing sea urchin test and collagen

Gonads of sea urchin are consumed in Japan and some countries as food and most parts including its tests are discarded as marine wastes. Therefore, utilization of them as functional materials would reduce the waste as well as encourage Japanese fishery. In this study, magnesium containing calcite granules collected from sea urchin tests were hydrothermally phosphatized and the obtained granules were identified as approximately 82% in mass of magnesium containing β-tricalcium phosphate and 18% in mass of nonstoichiometric hydroxyapatite, i.e., a biphasic calcium phosphate, maintaining the original porous network. Shape-controlled scaffolds were fabricated with the obtained biphasic calcium phosphate granules and collagen. The scaffolds showed good open porosity (83.84%) and adequate mechanical properties for handling during cell culture and subsequent operations. The MG-63 cells showed higher proliferation and osteogenic differentiation in comparison to a control material, the collagen sponge with the same size. Furthermore, cell viability assay proved that the scaffolds were not cytotoxic. These results suggest that scaffold prepared using sea urchin test derived calcium phosphate and collagen could be a potential candidate of bone void fillers for non-load bearing defects in bone reconstruction as well as scaffolds for bone tissue engineering.     

A new method for the preparation of bioactive calcium phosphate films hybridized with 1α,25-dihydroxyvitamin D3

The primary goal of this investigation was to develop a calcium phosphate film hybridized with 1α,25-dihydroxyvitamin D₃ for the improvement of osteoconductivity of bone substitutes. The hybrid films (hCaP) were prepared at the different concentrations of 1 × 10⁻¹⁰, 1 × 10⁻⁸, and 1 × 10⁻⁶ M designated as hCaPL, hCaPM, and hCaPH, respectively. The change of the hormone concentration during the preparation of the hybrid films did not cause significant variations on the physical properties of hCaPs, i.e. surface morphology and roughness. On the other hand, X-ray photon spectroscope (XPS) measurements revealed that the concentration change affected the chemical composition of the hybrid films. Recruitment of osteoblast-like MG-63 cells was considerably improved on hCaPs compared to tissue culture plate (TCP). However, cell proliferation on hCaPs was substantially suppressed and inversely proportional to the hormone concentration used. It was observed that bone-like nodules which consisted of bead-like components and well-developed matrix were rapidly formed on hCaPs. Masson’s trichrome and safranin-O stainings elucidated that the bead-like components were MG-63 cells. Safranin-O staining showed that proteoglycan was produced actively. These results indicate that the cells cultured on hCaPs were strongly stimulated by the hormone to produce proteoglycan which can be considered as an induction of premature bone formation. The number of the nodules was increased with hormone concentration and most pronounced at the hCaPH. Gene expression patterns of alkaline phosphatase (ALP), transforming growth factor-β (TGF-β), and osteopontin (OPN) were strongly modulated by hybridized the hormone. For ALP and OPN, gene expressions were activated earlier on hCaPs than untreated calcium phosphate (CaP) confirming the effect of the hybridization was substantial. The TGF-β gene expression was immediately activated after seeding but difference between samples was not significant suggesting that the gene expression was modulated not by the hormone hybridization but by CaP itself. As a result, hybridization of 1,25(OH)₂D₃ with CaP can be a potentially strong candidate to promote osteoconductivity of implant materials.     

Premixed injectable calcium phosphate cement with excellent suspension stability

Premixed injectable calcium phosphate cement (p-ICPC) pastes have advantages over aqueous injectable calcium phosphate cement (a-ICPC) because p-ICPC remain stable during storage and harden only after placement into the defect. This paper focused on the suspension stability of p-ICPC paste by using fumed silica as a stabilizing agent and propylene glycol (PEG) as a continuous phase. Multiple light scanning techniques were first applied to evaluate the suspension stability. The results indicated that fumed silica effectively enhanced the suspension stability of p-ICPC pastes. The stabilizing effect of fumed silica results from the network structure formed in PEG because of its thixotropy. The p-ICPC could be eventually hydrated to form hydroxyapatite under aqueous circumstances by the unique replacement between water and PEG. p-ICPC (1) not only possesses proper thixotropy and compressive strength but has good injectability as well. p-ICPC (1) was cytocompatible and had no adverse effect on the attachment and proliferation of MG-63 cells in vitro. These observations may have applicability to the development of other nonaqueous injectable biomaterials for non-immediate filling and long-term storage.     

Carbon plasma immersion ion implantation and DLC deposition on Ni−Ti alloy

Carbon plasma immersion ion implantation (PIII-C) has been performed on Ni?Ti alloy surface using methane as a precursor gas at low temperature and it has been followed by deposition of diamond-like carbon (DLC) coating. Untreated and coated alloys are characterized with field emission scanning electron microscopy, atomic force microscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy. Electrochemical impedance spectroscopy, and corrosion testing in Hanks’ solution which is simulated body fluid show that corrosion resistance has been enhanced in PIII-C?+?DLC-coated alloy compared to untreated alloy. The in vitro studies of untreated and PIII-C?+?DLC-coated alloys have been evaluated using osteoblast-like cells (MG-63). Cellular behavior in terms of cell morphology along with the viability and cell spreading has been evaluated using scanning electron microscopy and in vitro cell culture assay, respectively. In comparison to Ni–Ti alloy, the coated alloy exhibits better cell viability indicating their biocompatibility.     

羟基磷灰石表面形貌对人成骨肉瘤细胞生物学性能的影响

本文研究了羟基磷灰石(HA)表面形貌对人成骨肉瘤细胞(MG-63)生物学性能的影响。通过单轴压片技术与粒子占位法相结合控制陶瓷表面孔尺度、形态及分布, 从而获得具有不同表面孔结构的HA陶瓷材料。将材料与MG-63共培养, 通过扫描电子显微镜(SEM), MTT检测法表征材料表面形貌对细胞的黏附和增殖影响, 并通过碱性磷酸酶活性(ALP)检测和实时荧光定量(RT-PCR)技术探讨了HA陶瓷材料的表面结构对MG-63成骨分化的诱导作用。结果表明, 大孔结构(孔径大于200 μm)更有利于细胞的黏附和增殖, 而小孔结构(孔径小于100 μm)能促进细胞的成骨分化。孔形貌和孔分布也能影响细胞的生物功能, 相同尺度的孔径, 不规则蜂窝状的多级微孔结构比光滑孔壁的浅孔结构更能诱导细胞的成骨分化。     

Initial in vitro biocompatibility of a bone cement composite containing a poly-ε-caprolactone microspheres

The biocompatibility of a reinforced calcium phosphate injectable bone substitute (CPC-IBS) containing 30% poly-ε-caprolactone (PCL) microspheres was evaluated. The IBS consisted of a solution of chitosan and citric acid as the liquid phase and tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) powder as the solid phase with 30% PCL microspheres. The surface of the CPC-IBS was observed by SEM, and analyzed by EDX profiles. The initial setting of the sample was lower in the IBS containing 0% citric acid than in the IBS containing 10 or 20% citric acid. The compressive strength of the PCL-incorporated CPC-IBS was measured using a Universal Testing Machine. The 20% citric acid samples had the highest mechanical strength at day 12, which was dependent on both time and the citric acid concentration. The in vitro bioactivity experiments with simulated body fluid (SBF) confirmed the formation of apatite on the sample surfaces after 2, 7, and 14 days of incubation in SBF. Ca and P ion release profile by ICP method also confirmed apatite nucleation on the CPC-IBS surfaces. The in vitro biocompatibility of the CPC-IBS was evaluated by using MTT, cellular adhesion, and spreading studies. In vitro cytotoxicity tests by MTT assay showed that the 0 and 10% CPC-IBS was cytocompatible for fibroblast L-929 cells. The SEM micrograph confirmed that MG-63 cells maintained their phenotype on all of the CPC-IBS surfaces although cellular attachment was better in 0 and 10% CPC-IBS than 20% samples.     

Comparison of mesenchymal stem cell and osteosarcoma cell adhesion to hydroxyapatite

Immortalized cells are often used to model the behavior of osteogenic cells on orthopaedic and dental biomaterials. In the current study we compared the adhesive behavior of two osteosarcoma cell lines, MG-63 and Saos-2, with that of mesenchymal stem cells (MSCs) on hydroxyapatite (HA). It was found that osteosarcoma cells demonstrated maximal binding to fibronectin-coated HA, while MSCs alternately preferred HA coated with collagen-I. Interesting, the binding of MG-63 and Saos-2 cells to fibronectin was mediated by both α5 and αv-containing integrin heterodimers, whereas only αv integrins were used by MSCs. Cell spreading was also markedly different for the three cell types. Osteosarcoma cells exhibited optimal spreading on fibronectin, but poor spreading on HA disks coated with fetal bovine serum. In contrast, MSCs spread very well on serum-coated surfaces, but less extensively on fibronectin. Finally, we evaluated integrin expression and found that MSCs have higher levels of α2 integrin subunits relative to MG-63 or Saos-2 cells, which may explain the enhanced adhesion of MSCs on collagen-coated HA. Collectively our results suggest that osteosarcoma cells utilize different mechanisms than MSCs during initial attachment to protein-coated HA, thereby calling into question the suitability of these cell lines as in vitro models for cell/biomaterial interactions.     

Novel 3D scaffold with enhanced physical and cell response properties for bone tissue regeneration,fabricated by patterned electrospinning/electrospraying

Developing three dimensional scaffolds mimicking the nanoscale structure of native extracellular matrix is a key parameter in tissue regeneration. In this study, we aimed to introduce a novel 3D structures composed of nanofibers (NF) and micro particles (MP) and compare their efficiency with 2D nanofibrous scaffold. The conventional nanofibrous PCL scaffolds are 2D mats fabricated by the electrospinning technique, whereas the NF/MP and patterned NF/MP PCL scaffolds are three dimensional structures fabricated by a modified electrospinning/electrospraying technique. The mentioned method was carried out by varying the electrospinning solution parameters and use of a metal mesh as the collector. Detailed fabrication process and morphological properties of the fabricated structures is discussed and porosity, pore size and PBS solution absorption value of the prepared structures are reported. Compared with the 2D structure, 3D scaffolds possessed enhanced porosity and pore size which led to the significant increase in their water uptake capacity. In vitro cell experiments were carried out on the prepared structures by the use of MG-63 osteosarcoma cell line. The fabricated 3D structures offered significantly increased cell attachment, spread and diffusion which were confirmed by SEM analysis. In vitro cytocompatibility assessed by MTT colorimetric assay indicated a continuous cell proliferation over 21 days on the innovative 3D structure, while on 2D mat cell proliferation stopped at early time points. Enhanced osteogenic differentiation of the seeded MG-63 cells on 3D scaffold was confirmed by the remarkable ALP activity together with increased and accelerated calcium deposition on this structure compared to 2D mat. Massive and well distributed bone minerals formed on patterned 3D structure were shown by EDX analysis. In comparison between NF/MP quasi-3D and Patterned NF/MP 3D scaffolds, patterned structures proceeded in all of the above properties. As such, the innovative Patterned NF/MP 3D scaffold could be considered as a proper bone graft substitute for bone tissue regeneration.     

Local delivery of alendronate eluting chitosan scaffold can effectively increase osteoblast functions and inhibit osteoclast differentiation

The aim of this study was to investigate the effect of alendronate released from chitosan scaffolds on enhancement of osteoblast functions and inhibition of osteoclast differentiation in vitro. The surface and cell morphologies of chitosan scaffolds and alendronate-loaded chitosan scaffolds were characterized by variable pressure field emission scanning electron microscope (VP-FE-SEM). Alendronate was released in a sustained manner. For evaluating osteoblast functions in MG-63 cells, we investigated cell proliferation, alkaline phosphatase (ALP) activity, and calcium deposition. Furthermore, for evaluating inhibition of osteoclast differentiation in RAW 264.7 cells, we investigated tartrate-resistant acid phosphatase (TRAP) activity, TRAP staining, and gene expressions. The in vitro studies revealed that osteoblasts grown on alendronate-loaded chitosan scaffold showed a significant increment in cell proliferation, ALP activity, and calcium deposition as compared to those grown on chitosan scaffolds. In addition, the in vitro study showed that osteoclast differentiation in RAW 264.7 cells cultured on alendronate-loaded chitosan scaffolds was greatly inhibited as compared to those cultured on chitosan scaffolds by the results of TRAP activity, TRAP staining, and gene expressions. Taken together, alendronate-loaded chitosan scaffolds could achieve the dual functions of improvement in osteoblast functions and inhibition of osteoclast differentiation. Thus, alendronate-eluting chitosan substrates are promising materials for enhancing osteoblast functions and inhibiting osteoclast differentiation in orthopedic and dental fields.     

接枝RGD多肽磷灰石-硅灰石材料的矿化和生物学特性

应用等离子辅助化学接枝方法在磷灰石-硅灰石(AW)生物活性玻璃的表面接枝精氨酸-甘氨酸-天冬氨酸(RGD)多肽。采用模拟体液(SBF)浸泡方法研究了AW表面接枝RGD基团对材料体外矿化特性的影响。SEM和EDS检测结果表明,RGD多肽的引入有利于羟基磷灰石(HA)的沉积,能够增强RGD-AW复合材料的体外矿化能力,HA形貌为蠕虫状。材料MG-63细胞共培养实验以及材料新西兰成年大白兔体内植入实验的结果表明,表面化学接枝RGD多肽的RGD-AW复合材料能够显著地促进类成骨细胞的黏附和铺展,并且在2周、4周和8周时均能够加速新骨的生成及骨组织结构和功能的重建。     

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.     

Hydrodechlorination of 4-chlorophenol in water with formic acid using a Pd/activated carbon catalyst

The ultra-fine HNS (2,2',4,4',6,6'-hexanitrostilbene) with desired properties is needed for military and civilian applications because of its reliable threshold energy to short impulse shock waves and its excellent thermal and shock stability. This paper reports on prefilming twin-fluid nozzle assisted precipitation (PTFN-P) to obtain ultra-fine HNS explosive with high specific surface area (SSA), high purity, and narrow particle size distribution. The properties of ultra-fine HNS have been confirmed by SEM, BET, HPLC, XRD, DSC and TGA-SDTA. SEM photograph revealed that the PTFN-P process offers ellipsoid crystalline morphology with particle size of 90-150 nm. The BET and Langmuir SSA of nanocrystalline HNS with purity of 99.44 wt.% were determined to be 19.28 m(2)/g and 29.26 m(2)/g, respectively. The XRD peaks of nanocrystalline HNS seemed to have similar diffraction angles as those of synthesized HNS, and the weakening of peak strength was observed apparently. DSC results of the nanocrystalline HNS showed that the exothermic decomposing at the temperature range of 323-398 degrees C. Furthermore, HNS samples were submitted to impact and small scale gap test and the results indicated that nanocrystalline HNS is less sensitive than synthesized HNS (50 microm) to impact and shock stimuli.