Cell growth on metallic glasses: the interaction of amorphous metal alloys with cultured neuronal, osteoblast, endothelial, and fibroblast cells

Ferrous based, corrosion resistant amorphous alloys supported the adhesion and growth of cultured chick neuronal cells, human marrow stromal cells (presumptive osteoblasts), bovine aortal endothelial cells, and hamster kidney fibroblasts. Alloys of compositions Fe60Ni10Cr10P13C7, Fe70Cr10P13C7, and Fe70Cr10P13B7 were found to be suitable. In contrast the crystalline form of these alloys was markedly less effective. Outgrowth of neurites from neuronal cells was promoted by precoating the metal surface with either laminin or neurite promotion factor. The adhesion of osteoblasts and fibroblasts suggests that corrosion resistant metal glasses should be considered as biomaterials useful for orthopedic applications. The adhesion of neuronal cells accompanied by neurite outgrowth indicates that the system might provide a functional interface between the neuromuscular system and an electromagnetic material that could be useful in bionic engineering.     

Adhesion of staphylococcal and Caco-2 cells on diamond-like carbon polymer hybrid coating

Staphylococci cause the majority of the nosocomial implant-related infections initiated by adhesion of planktonic bacteria to the implant surface. It was hypothesized that plasma accelerating filtered pulsed arc discharge method enables combination of the advantageous properties of diamond with the antisoiling properties of polymers. Diamond-like carbon polytetrafluoroethylene hybrid (DLC-PTFE-h) coating was produced. The adhesion of S. aureus ATCC 25923 (10(8) colony-forming units/mL) to surfaces diminished from 2.32%, 2.35%, and 2.57% of high quality DLC, titanium, and oxidized silicon, respectively, to 1.93% of DLC-PTFE-h. For S. epidermidis ATCC 35984 the corresponding figures were 3.90%, 3.32%, 3.47%, and 2.57%. Differences in bacterial adhesion between recombinant DLC-PTFE-h and other materials were statistically significant (p < 0.05). In contrast, human Caco-2 cells adhered as well to DLC-PTFE-h as to DLC, titanium, or silicon, which were all in the MTT test found to be cytocompatible. DLC-PTFE-h coating can be used to modify the surface properties of any surgical implants and is an unfavorable substrate for staphylococcal cells, but compatible with human Caco-2 cells. DLC-PTFE-h coating may help in the combat against Staphylococcus-related implant infections which usually require both antibiotics and surgical removal of the implant for cure.     

Fluoroapatite glass-ceramic coatings on alumina: structural,mechanical and biological characterisation

The aim of this work was to realise bioactive coatings on full density alpha-alumina substrates. An SiO2-CaO-based glass (SC) and an SiO2-Al2O3-P2O5-K2O-CaO-F--based glass-ceramic (SAF) were used for this purpose. Specifically, SAF is a fluoroapatite containing glass-ceramic and previous studies have shown that it is a highly bioactive biomaterial. Furthermore, these fluoroapatite crystals possess a needle-shaped morphology which mimics that of hydroxylapatite found in human hard tissues, particularly in teeth. SAF is a very viscous glass-ceramic and for this reason an intermediate, less viscous, SC layer was interposed in direct contact with alumina aiming to obtain a good coating adhesion. Moreover, this intermediate layer strongly lowers the Al3+ diffusion and thus minimises both compositional changes in the SAF outer layer and the risk of detrimental modifications of the nature of the crystalline phases. A complete characterisation of the coated samples was performed by means of X-ray diffraction, optical and scanning microscopy. Coating adhesion on alumina was tested by comparative shear tests while biocompatibility was investigated on alumina. bulk SAF and on the realised coatings. For this purpose, cytotoxicity, adhesion and proliferation of human osteoblast-like cells were cultured onto the three materials. Results showed that the interposition of the SC layer was successful in allowing a good softening and spreading of the SAF outer layer and in avoiding the crystallisation of undesired crystalline phases maintaining the good bioactive properties of the bulk one. In vitro results on the coatings showed osteoblast-like cell behaviour similar to bulk fluoroapatite glass-ceramic and better respect to alumina substrates, being a promising index of bone material integration and of its in vivo possible applications.     

Nanocrystalline hydroxyapatite/titania coatings on titanium improves osteoblast adhesion

Bulk hydroxyapatite (HA) and titania have been used to improve the osseointegration of orthopedic implants. For this reason, composites of HA and titania have been receiving increased attention in orthopedics as novel coating materials. The objective of this in vitro study was to produce nanophase (i.e., materials with grain size less than 100 nm) HA/titania coatings on titanium. The adhesion of bone forming cells (osteoblasts) on the composite coatings were also assessed and compared with single-phase nanotitania and nano-HA titanium coatings. Nanocrystalline HA powders were synthesized through wet chemistry and hydrothermal treatments at 200 degrees C. Nanocrystalline titania powders obtained commercially were mixed with the nanocrystalline HA powders at various weight ratios. The mixed powders were then deposited on titanium utilizing a room-temperature coating process called IonTite. The results of the present study showed that such coatings maintained the chemistry and crystallite size of the original HA and titania powders. Moreover, osteoblasts adherent on single-phase nanotitania coatings were well-spread whereas they became more round and extended distinct filopodia on the composite and single-phase HA coatings. Interestingly, the number of osteoblasts adherent on the nanotitania/HA composite coatings at weight ratios of 2/1 and 1/2 were significantly greater compared with single-phase nanotitania coatings, currently-used plasma-sprayed HA coatings, and uncoated titanium. These findings suggest that nanotitania/HA coatings on titanium should be further studied for improved orthopedic applications.     

Initial in vitro interaction of osteoblasts with nano-porous alumina

In the present study we have used a characterised primary human cell culture model to investigate cellular interactions with nano-porous alumina. This material, prepared by anodisation, is being developed as a coating on titanium alloy implants. The structure of the alumina, as determined by X-ray diffraction and transmission electron microscopy, was amorphous. When studying cell/material interactions we used both biochemical and morphological parameters. Cell viability, proliferation and phenotype were assessed by measurement of redox reactions in the cells, cellular DNA, tritiated thymidine ([3H]-TdR) incorporation and alkaline phosphatase (ALP) production. Results showed a normal osteoblastic growth pattern with increasing cell numbers during the first 2 weeks. A peak in cell proliferation was seen on day 3, after which cell growth decreased, followed by an increase in ALP production, thus indicating that the osteoblastic phenotype was retained on the alumina. Cell adhesion was observed, the osteoblast-like cells having a flattened morphology with filipodia attached to the pores of the material. SDS-PAGE and western blot measurements showed that the nano-porous alumina was able to adsorb fibronectin. Trace amounts of aluminium ions were measured in the surrounding medium, but no adverse effect on cell activity was observed.     

Evaluation of the TiMo12Zr6Fe2 alloy for orthopaedic implants: in vitro biocompatibility study by using primary human fibroblasts and osteoblasts

To reveal the biocompatibility of TiMo12Zr6Fe2 (TMZF), a new titanium alloy used since 1998 for orthopaedic prosthesis, we compared the behavior of primary human fibroblasts and osteoblasts grown on TMZF discs or on plastic tissue culture dishes, a widely used material specifically treated by the manufacturer to enhance cell growth. Proliferation, differentiation. RNA and collagen type I expression level of human cells were carried out. The analysis were performed over a period of 96 h. Fibroblasts behaved at the same way on the two different supports after 48 h, their number increased after 96 h when cells were grown on the alloy. Osteoblasts adhered and proliferated on the alloy discs as well as on plastic. RNA expression level was not affected. Interestingly, cell number at each time point was higher for fibroblasts than for osteoblasts. The RNA expression level was higher for the osteoblasts. Both cell types cultured on the alloy revealed an increase in the amount of type I collagen and a similar electrophoretic pattern was found for collagen produced by fibroblasts and osteoblasts grown on either supports. These results indicate good biocompatibility of the TMZF alloy, which allowed adhesion and proliferation of both the examined cell types and suggest that TMZF is a promising material for orthopaedic implants.     

Effects of hydroxylapatite coating crystallinity on biosolubility, cell attachment efficiency and proliferation in vitro.

A hydroxylapatite (HA) coating with approximately 97% crystalline HA content (MP-1 treated HA coating, MP-HA) was tested in vitro for its biosolubility and cellular biocompatibility. The MP-HA coating was compared with a standard HA coating with approximately 63% crystallinity (SHA) and an amorphous HA coating with approximately 25% crystallinity (AHA), as well as a titanium (Ti) surface without HA coating as a control. The topographic study with scanning electron microscopy indicated that MP-HA appeared more coarse, with projected nodules which altered the shape of cells attached to the substrate. Biosolubility study indicated that MP-HA had the least effect on the culture medium pH, while AHA (P < 0.01) and SHA (P < 0.05) significantly raised the medium pH up to 8.2 and 7.75, respectively. X-ray diffraction (XRD) analysis showed essentially unchanged levels of the total soluble phases of all coatings after incubation with culture medium, except that the CaO phase was rapidly dissolved from AHA coatings and completely eliminated from SHA coatings. Cultures of human gingival fibroblasts on these HA coatings showed that MP-HA and SHA had about the same cell attachment efficiency which was relatively lower than that of AHA coatings. MP-HA generated significant higher cell proliferation rate relative to AHA (P < 0.01) and SHA (P < 0.05). This study indicated that surface chemistry and topography of lower crystallinity might be favorable to cell attachment, but that elevated medium pH might result in a cytotoxic effect that inhibits the proliferation of attached cells on coating surfaces.     

钛合金表面新型生物玻璃/纳米羟基磷灰石涂层的细胞相容性评价

目的为检验前期制备的新型生物玻璃／纳米羟基磷灰石涂层的细胞相容性。方法在本实验中，采用L929成纤维细胞在涂层浸提液中的培养，检测了涂层的细胞毒性，采用人体骨髓基质干细胞存涂层上的培养，检测了涂层对人体骨髓基质下细胞增殖和代谢的影响。结果低于浓度的涂层浸提液（〈10％）埘L929细胞增殖具有促进作用，而高浓度的涂层浸提液（〉50％）对L929细胞的增殖具有抑制作用，其中100％的涂层浸提液对L929细胞的增殖抑制比例为20.9％，在细胞毒性分级中处于合格范围内。结论在培养早期，人体骨髓基质干细胞在涂层表面的增殖要优于对照组，涂层显示出良好的细胞相容性。由于合格的细胞毒性和良好的细胞相容性，该涂层的钛合金具有作为骨植入物的应用潜力。     

钛植入物促骨整合及抗感染涂层的研究进展

钛及其合金具有稳定的化学性质和良好的人体亲和性,常被作为骨科植入材料,但未经处理的钛植入物易发生骨整合不良和植入物周围感染导致植入失败。近年来,人口老龄化加剧带动植入材料使用量增加,亟需性能更好的钛表面修饰方法以降低植入失败率。理想的骨科植入物应同时具备促成骨和抗菌特性,但目前大量表面改性策略只解决其中一种问题,导致植入物无法达到预期效果。因此,植入材料涂层需满足这两种特性或在两者间寻找平衡点。综述近几年来解决钛植入物无菌性松动或感染问题的最新策略,如表面形貌改善和生物分子涂层等促进成骨,抗菌药物负载和光热治疗等防治感染措施,并通过双因素整合和三因素整合进一步介绍了同时解决这两个问题的研究进展。     

Easy assessment of the biocompatibility of Ni-Ti alloys by in vitro cell culture experiments on a functionally graded Ni-NiTi-Ti material

The biocompatibility of nickel-titanium alloys was investigated by single-culture experiments on functionally graded samples with a stepwise change in composition from pure nickel to pure titanium, including an Ni-Ti shape memory alloy for a 50:50 mixture. This approach permitted a considerable decrease of experimental resources by simultaneously studying a full variation of composition. The results indicate a good biocompatibility for a nickel content up to about 50%. The cells used in the biocompatibility studies comprised osteoblast-like osteosarcoma cells (SAOS-2, MG-63), primary human osteoblasts (HOB), and murine fibroblasts (3T3).     

Effect of calcium phosphate coating crystallinity and implant surface roughness on differentiation of rat bone marrow cells

In this study, we examined the effect of calcium phosphate (Ca-P) coating crystallinity and of surface roughness on growth and differentiation of osteogenic cells. Grit-blasted titanium substrates were provided with Ca-P coatings of different crystallinities. Rat bone marrow (RBM) cells were cultured on these substrates and on noncoated rough and smooth titanium substrates. After specific culture times, expression of osteogenic markers by the cells was studied. Cells cultured on crystalline coatings and on titanium substrates proliferate, express alkaline phosphatase, osteocalcin (OC), and show mineralization of the extracellular matrix. Rough titanium substrates only express low OC levels. Significantly higher OC levels were expressed on smooth titanium, and even higher levels on the crystalline Ca-P coating. No difference was found in calcification between smooth and rough titanium. The crystalline coating showed more calcification than the titanium substrates. When substrates without cells were incubated in medium, precipitation of calcium was found. On the titanium substrates, this precipitate disappeared after prolonged incubation. The precipitate on the crystalline coating was stable and increased with longer incubation times. On the amorphous coatings, no proliferation and differentiation of RBM cells were found. After longer culture periods, substrates showed extensive dissolution. Cells on the amorphous coatings did express high levels of prostaglandin E2. In contrast, prostaglandin E2 expression was low for the other substrates. We conclude that crystalline Ca-P coatings stimulate differentiation of RBM cells, to a higher extent than titanium substrates. Surface roughness only has a limited effect on phenotype expression of the cells. In contrast, thin amorphous coatings show negative effects on the growth and differentiation of cultured RBM cells.     

A novel nano-porous alumina biomaterial with potential for loading with bioactive materials

Nano-porous alumina, with the potential for being loaded with bioactive materials, has been proposed as a novel material for coating implants. In this study, the shear strength of the interface between such nano-porous anodic aluminium oxide (AAO) coatings and titanium substrates, their biocompatibility, and their potential for pore loading have been investigated. An interface shear strength in excess of 29 MPa was obtained which is comparable with that of conventional plasma sprayed hydroxyapatite implant coatings. The viability and differentiation of MG63 osteoblastic cells co-cultured on the coating was found to be broadly comparable to that of similar cells co-cultured on conventional bioinert implant materials such as titanium and fully dense alumina. Extensive pore loading with silica nano-particles of different sizes and in different combinations was demonstrated throughout the thickness of AAO layers 1 microm and 60 microm thick. This work has demonstrated, that with suitable choice of pore filling materials, this novel coating might simultaneously combat infection, encourage bone regeneration, and secure fixation of the implant to bone.     

Induction plasma sprayed Sr and Mg doped nano hydroxyapatite coatings on Ti for bone implant

In this study, we report fabrication of strontium (Sr) and magnesium (Mg) doped hydroxyapatite (HA) coating on commercially pure titanium (Cp-Ti) substrates using inductively coupled radio frequency (RF) plasma spray. HA powder was doped with 1 wt % Sr (Sr-HA) and 1 wt % Mg (Mg-HA), heat treated at 800°C for 6 h and then used for plasma spray coating. X-ray diffraction (XRD) and Fourier transformed infrared spectroscopic (FTIR) analysis indicated that the coatings were primarily composed of phase pure crystalline HA. When compared to undoped HA coating, physical properties such as microstructure, grain size, and adhesive bond strength of the doped HA coatings did not change significantly. Microstructure of the coatings showed coherency in the structure with an average grain size of 200-280 μm HA particles, where each of the HA grains consisted of 20-30 nm sized particles. An average adhesive bond strength of 17 MPa ensured sufficient mechanical strength of the coatings. A chemistry dependent improvement in bone cell-coating interaction was noticed for doped coatings although it had minimal effect on physical properties of the coatings. In vitro cell-materials interactions using human fetal osteoblasts (hFOB) showed better cell attachment and proliferation on Sr-HA coatings compared to HA or Mg-HA coatings. Presence of Sr in the coating also stimulated hFOB cell differentiation and alkaline phosphatase (ALP) expression. Improvement in bioactivity of Sr doped HA coatings on Ti without compromising its mechanical properties makes it an excellent material of choice for coated implant.     

Anodic plasma-chemical treatment of CP titanium surfaces for biomedical applications

The anodic plasma-chemical (APC) process was used to modify CP titanium surfaces for biomedical applications. This technique allows for the combined chemical and morphological modification of titanium surfaces in a single process step. The resulting conversion coatings, typically several micrometer thick, consist mainly of titanium oxide and significant amounts of electrolyte constituents. In this study, a new electrolyte was developed containing both calcium-stabilized by complexation with EDTA-and phosphate ions at pH 14. The presence of the Ca-EDTA complex, negatively charged at high pH, favors incorporation of high amounts of calcium into the APC coatings during the anodic (positive) polarization. The coating properties were evaluated as a function of the process variables by XPS, GD-OES, Raman spectroscopy, SEM and tensile testing, and compared to those of calcium-free APC coatings and uncoated CP titanium surfaces. The maximal Ca/P atomic ratio in the coating produced with the new APC electrolyte was approximately 1.3, with higher Ca concentrations than reported in conventional APC coatings. The dissolution behavior of the incorporated, amorphous CaP phases was investigated by exposure to a diluted EDTA solution. The coatings produced in the new electrolyte system exhibit favorable mechanical stability. The new APC technology is believed to be a versatile and cost-effective coating technique to render titanium implant surfaces bioactive.     

细胞培养法评价含硅羟基磷酸钙涂层镁合金的生物相容性

目的 采用细胞培养法在体外评估镁合金表面不同硅含量的羟基磷酸钙涂层对成骨细胞的毒性反应.方法 采用经鉴定的SD大鼠成骨细胞接种于三种含有不同浓度硅含量的羟基磷酸钙涂层的镁合金及对照组裸镁合金,MTT法检测细胞活力并榆测成骨细胞的碱性磷酸酶表达;扫描电镜观察细胞生长情况.结果 细胞活力检测和碱性磷酸酶活性分析均表明,硅含...     

Transmission electron microscopy of coatings formed by plasma electrolytic oxidation of titanium

Transmission electron microscopy and supporting film analyses are used to investigate the changes in composition, morphology and structure of coatings formed on titanium during DC plasma electrolytic oxidation in a calcium- and phosphorus-containing electrolyte. The coatings are of potential interest as bioactive surfaces. The initial barrier film, of mixed amorphous and nanocrystalline structure, formed below the sparking voltage of 180 V, incorporates small amounts of phosphorus and calcium species, with phosphorus confined to the outer ～63% of the coating thickness. On commencement of sparking, calcium- and phosphorus-rich amorphous material forms at the coating surface, with local heating promoting crystallization in underlying and adjacent anodic titania. The amorphous material thickens with increased treatment time, comprising almost the whole of the ～5.7-μm-thick coating formed at 340 V. At this stage, the coating is ～4.4 times thicker than the oxidized titanium, with a near-surface composition of about 12 at.% Ti, 58 at.% O, 19 at.% P and 11 at.% Ca. Further, the amount of titanium consumed in forming the coating is similar to that calculated from the anodizing charge, although there may be non-Faradaic contributions to the coating growth.     

The influence of novel bioactive glasses on in vitro osteoblast behavior

Implant success requires a direct bond between bone and implant surface. Bioinert implants, such as titanium alloys, are commonly plasma-spray-coated with a bone-bonding, bioactive material such as hydroxyapatite. Such coatings tend to be chemically and topographically inhomogeneous without reproducible properties. A family of bioactive glasses that can be enameled and reliably adheres to titanium alloy has been developed. In this study the cytocompatibility of two of these glass compositions was tested in the as-cast condition. The effects of these glasses on the early and late events of osseous tissue formation in vitro were determined with MC3T3-E1.4 mouse osteoblast-like cells. MC3T3-E1.4 cells were cultured on glasses containing 55 and 50 wt % SiO(2), with titanium alloy (Ti6Al4V) and tissue culture polystyrene as controls. Cellular adhesion and proliferation, and alkaline phosphatase activity were studied over 5 to 15 days in culture. Qualitative and quantitative assays of mineralization were conducted. The osteoblast-like cells showed increased proliferation when grown on a bioactive glass containing 50 wt % silica. However, the adhesion, differentiation and mineralization behavior were similar on both glass compositions used in this study. These bioactive glasses proved to be cytocompatible substrata for osteoblast-like cell culture, and yielded higher cellular proliferation than titanium alloy.     

Behavior in simulated body fluid of calcium phosphate coatings obtained by laser ablation

Three types of calcium phosphate coatings onto titanium alloy substrates, deposited by the laser ablation technique, were immersed in a simulated body fluid in order to determine their behavior in conditions similar to the human blood plasma. Neither the hydroxyapatite coating nor the amorphous calcium phosphate coating do dissolve and the alpha-tricalcium phosphate phase of the coating of beta-tricalcium phosphate with minor alpha phase slightly dissolves. Precipitation of an apatitic phase is favored onto the hydroxyapatite coating and onto the coating of beta-tricalcium phosphate with minor alpha phase. Onto the titanium alloy substrate reference there is also precipitation but at larger induction times. However, onto the amorphous calcium phosphate coating no precipitate is formed.     

壳聚糖-酪蛋白磷酸肽修饰钛合金表面的成骨细胞黏附与增殖

背景：钛合金表面只有吸附某些有机分子才能使生长因子活化,促进细胞增殖,从而激活周围的骨细胞发挥作用。 目的：在钛合金种植体表面制备生物活性涂层,寻找一种有效促进骨整合的方法。 方法：采用多步组装方法在硅烷偶联剂修饰的钛合金表面依次接枝壳聚糖与0,5,10 g/L的酪蛋白磷酸肽,形成生物活性复合涂层,以未修饰的钛合金为对照。将经过不同处理的钛合金与成骨细胞共培养,利用细胞计数法、荧光染色法、MTT比色法评价涂层的生物学性能。 结果与结论：与未修饰的钛合金相比,在壳聚糖上接枝酪蛋白磷酸肽后,钛合金表面成骨细胞的早期黏附与增殖明显提高  (P < 0.05),并且随酪蛋白磷酸肽质量浓度的增大促进作用显著增强(P < 0.05)。说明制备的壳聚糖-酪蛋白磷酸肽复合涂层可以显著提高成骨细胞的功能,增强钛合金的生物学性能,有望成为有效促进骨整合的方法。     

Nanocrystalline hydroxyapatite coatings from ultrasonated electrolyte: preparation, characterization, and osteoblast responses

An electrochemical method of producing nanograined hydroxyapatite coatings on titanium surface is reported in this article. The electrolyte contained Ca(NO(3))(2) and NH(4)H(2)PO(4) in the molar ratio of 1.67:1. The electrolyte had physiological pH and was ultrasonically agitated throughout the time of electrolysis. Coatings were deposited for 30 minutes at 10 and 15 mA/cm(2) and contained monohydroxyapatite phase whose grain sizes were 18 and 25 nm, respectively. These sizes are comparable with the grain size of bone. Small globules of hydroxyapatite covered the coating surface completely. Cell viability and total protein assay studies were carried out using SaOS-2 human osteoblast-like cell line. Of the two, the coating produced at 10 mA/cm(2) showed higher viability and protein activity and seems to be a promising material for osseointegration.