A comparative study of electrochemical deposition and biomimetic deposition of calcium phosphate on porous titanium

Coating porous titanium with calcium phosphate (Ca-P) is an effective way to enhance titanium's osteoinduction capability. This study investigated the effectiveness of two coating methods: biomimetic deposition (BD) and electrochemical deposition (ED) in aqueous solutions. The titanium surfaces were treated by acidic etching and alkaline before coating. Effects of the pre-coating treatments on Ca-P coating were also investigated. Both deposition methods could produce Ca-P coatings on the inner pore surfaces of the titanium. The BD coatings were thicker and more uniform than were the ED coatings. On the other hand, ED was less sensitive to the condition of the titanium surface, and much faster in the coating deposition. However, ED produces less uniform and thinner coating layers on the inner pore surfaces of the titanium than does BD. The crystal structure of the coating is octacalcium phosphate (OCP) regardless of the deposition method. The morphology of flake-like OCP crystals in the deposition layers is similar for both deposition methods, except that the crystal flakes rupture after ED.     

Bioactive films on metallic surfaces for osteoconduction

A fast and effective electrochemical method was developed to make a dense calcium phosphate films on titanium and stainless steel for hard tissue replacement. The surfaces of titanium and stainless steel were cathodically treated in an electrochemical cell. By controlling the treatment parameters, a film of 100-nm thickness was deposited on the metal surface in several minutes. The thin film was amorphous calcium phosphate containing octacalcium phosphate nuclei, and also dense and ductile. The treated metals were able to induce bioactive calcium phosphate deposition after immersion in simulated body fluid (SBF) for only 1 and 2 days. In vivo study was conducted by implanting the treated specimens of titanium and stainless steel in dog's femur cavity. The treated metallic surfaces showed good ability of osteoconduction. This surface treatment method can be potentially used to enhance bioactivity of any type of metallic surfaces.     

Nano-scale study of the nucleation and growth of calcium phosphate coating on titanium implants

The nucleation and growth of a calcium phosphate (Ca-P) coating deposited on titanium implants from simulated body fluid was investigated by using atomic force microscopy (AFM) and environmental scanning electron microscopy (ESEM). Forty titanium alloy plates were assigned into two groups. One group with a smooth surface having a maximum roughness R(max) < 0.10 microm (s-Ti6Al4V) and a group with a rough surface with an R(max) < 0.25 microm (r-Ti6Al4V) were used. Titanium samples were immersed in SBF concentrated by five (SBF x 5) from 10 min to 5 h and examined by AFM and ESEM. Scattered Ca-P deposits of approximately 15 nm in diameter appeared after only 10 min of immersion in SBF x 5. These Ca-P deposits grew up to 60-100 nm after 4 h on both s- and r-Ti6Al4V substrates. With increasing immersion time, the packing of Ca-P deposits with size of tens of nanometers in diameter formed larger globules and then a continuous Ca-P film on titanium substrates. A direct contact between the Ca-P coating and the Ti6Al4V surface was observed. The Ca-P coating was composed of nanosized deposits and of an interfacial glassy matrix. This interfacial glassy matrix might ensure the adhesion between the Ca-P coating and the Ti6Al4V substrate. In the case of s-Ti6Al4V substrate, failures within this interfacial glassy matrix were observed overtime. Part of the glassy matrix remained on s-Ti6Al4V while part detached with the Ca-P film. The Ca-P coating detached from the smooth substrate, whereas the Ca-P film extended onto the whole rough titanium surface over time. In the case of r-Ti6Al4V, the Ca-P coating covered evenly the substrate after immersion in SBF x 5 for 5 h. The present study suggested that the heterogeneous nucleation of Ca-P on titanium was immediate and did not depend on the Ti6Al4V surface topography. The further growth and mechanical attachment of the final Ca-P coating strongly depended on the surface, for which a rough topography was beneficial.     

Trabecular bone response to surface roughened and calcium phosphate (Ca-P) coated titanium implants.

The influence of calcium phosphate (Ca-P) coating and surface roughness on the trabecular bone response of titanium implants was investigated. Four types of titanium implants, i.e. blasted with titanium powder, sintered with titanium beads, titanium powder blasted and provided with an additional Ca-P coating, and titanium beads with Ca-P coating, were prepared. The Ca-P coating was deposited by ion beam dynamic mixing method. The Ca-P coating was rapid heat-treated with infrared radiation at 700 degrees C. The implants were inserted into the trabecular bone of the left and right femoral condyles of 16 rabbits. After implantation periods of 2, 3, 4 and 12 weeks, the bone-implant interface was evaluated histologically and histomorphometrically. Histological evaluation revealed new bone formation around different implant materials after already 3 weeks of implantation. After 12 weeks, mature trabecular bone surrounded all implants. At 3 and 4 weeks of implantation, no difference existed in bone contact to the various implant materials. On the other hand, after 12 weeks of implantation the highest percentage of bone contact was found around the Ca-P coated beads implants. Supported by the results, we concluded that the combination of surface geometry and Ca-P coating benefits the implant-bone response during the healing phase.     

Bone response to calcium phosphate-coated and bisphosphonate-immobilized titanium implants

Thin calcium phosphate (Ca-P) coatings have been introduced to overcome the shortcomings of plasma-sprayed Ca-P coatings. In our previous experiments, thin Ca-P coatings also enabled the immobilization of bisphosphonate, which is a drug used to treat osteoporosis. The present study was designed to evaluate the bone response to titanium implants treated with a thin Ca-P coating and bisphosphonate. Forty cylindrical commercially pure titanium implants with a length of 7 mm and a diameter of 3 mm were used as test implant fixtures. Three groups of surface-treated implants were prepared: (1) blasted with titanium powder and etched with a solution of 10% HF + 5% HNO3 (control); (2) modified with 0.5-microm thick Ca-P coatings and rapid heat-treating, and (3) immobilized with bisphosphonate by immersion in pamidronate disodium solution (10(-2) M) for 24 h at 37 degrees C. These surface-treated implants were inserted into edentulous areas in the mandibular molar region of five beagle dogs. After implantation periods of 4 and 12 weeks, the bone implant interface was evaluated histologically and histomorphometrically. All measurements were statistically evaluated using a one-way ANOVA and Fisher PLSD test for multiple comparisons among the means. Four weeks after the implantation, higher percentage of bone contact was found around the thin Ca-P-coated implants compared to that of the control group. The highest percentage of bone contact was found around the bisphosphonate-immobilized implants after 12 weeks of implantation. These data suggest that a thin coating of calcium phosphate followed by bisphosphonate-immobilization is effective in the promotion of osteogenesis on surfaces of dental implants.     

The role of calcium gluconate in electrochemical activation of titanium for biomimetic coating of calcium phosphate

Supersaturation of calcium and phosphate in the bath solution and activation of the metal substrate is essential for effective biomimetic growth of apatite on orthopedic implants. In this work, bioactivation of titanium surface was achieved by electrodeposition of a thin layer of calcium phosphate followed by an alkaline treatment to obtain pure hydroxyapatite crystals. The influence of calcium gluconate in the electrolyte solution was evaluated and optimized. Adhesive strength, thickness, structural, and surface characteristics were evaluated. A highly adhesive and uniform layer of hydroxyapatite was formed on titanium surface when the electrodeposition was carried out with an electrolyte solution-containing calcium gluconate. The electrodeposited hydroxyapatite coatings were subjected for biomimetic growth in Kokubo's simulated body fluid (SBF) and Kokubo's modified SBF containing 1.5 times higher concentration of Ca. Biomimetic growth was also improved by the addition of calcium gluconate in the SBF solution.     

多孔纯钛种植体表面快速沉积钙磷涂层的研究

商业纯钛片经磨平、喷砂、清洗、酸洗后分成A、B两组。A组不作任何处理,B组用H2SO4和HCl混合液酸蚀,然后两组钛片同时置于37℃的仿生液SBF-A中1d及SBF-B中2d。结果两组钛片表面均可沉积磷灰石涂层,涂层为多孔片状结构,X射线衍射分析证实其为碳酸化羟基磷灰石和磷酸八钙的混合物,钙磷原子量比约1.51。这说明在多孔纯钛表面可以快速仿生沉积碳酸化磷灰石涂层。     

Effect of water vapor treatment on apatite formation on precalcified titanium and bond strength of coatings to substrates.

In previous investigations, a simple method, precalcification, was developed for bioactivating titanium. After a titanium sample was precalcified in a boiling saturated Ca(OH)(2) solution and then immersed in a calcium phosphate supersaturated solution, an apatite coating rapidly precipitated onto its surface. In the present study, heat-treatment in water vapor was carried out prior to precalcification. Heat-treatment in water vapor stimulated the chemical reaction between titanium, calcium, and phosphate. Coating properties were improved, and the bond strength of the coating to substrate was enhanced.     

纯钛表面微弧氧化法制备含羟基磷灰石氧化膜的实验研究

应用微弧氧化技术在纯钛表面制备含羟基磷灰石的氧化物膜.在氧化过程中,将钛试件放入含Ca、P电解液中,用直流脉冲电源处理.用扫描电镜(SEM)观察试件的表面和横断面形貌,X射线能谱(EDS)及X射线衍射(XRD)分析其元素成分和晶相结构.结果表明微弧氧化处理后,纯钛表面生成微孔结构的氧化膜,膜层厚度约20μm,由O、Ti、Ca和P四种元素组成.膜层表面有分布不均匀的火山丘状的微孔分布,直径小于5μm.膜层表面的钙磷原子比为1.63,界面处的钙磷原子比为0.51.膜层由金红石型和锐钛矿型二氧化钛及少量结晶相羟基磷灰石组成.微弧氧化技术在纯钛表面生成了内层致密外层多孔的晶相二氧化钛膜,同时含有少量羟基磷灰石,表明该技术在人工种植牙和人工骨关节等领域具有良好的医学应用前景.     

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.     

The biocompatibility of nanostructured calcium phosphate coated on micro-arc oxidized titanium

To achieve improved osseointegration, there have been many efforts to modify the surface composition and topography of dental implants. Recently, the anodic oxidation treatment of titanium (Ti) has attracted a great deal of attention. Meanwhile, calcium phosphate is commonly applied to metallic implants as a coating material for fast fixation and firm implant-bone attachment on the account of its demonstrated bioactive and osteoconductive properties. In the present study, anodized surface and calcium phosphate deposition by electron beam evaporation were combined. Nanostructured calcium phosphate film was deposited on the micro-arc oxidized Ti. New apatite layer formed easily on the coated film when incubating in DPBS solution at 37 degrees C. By adding basic fibroblast growth factor (bFGF) in the DPBS solution, the bFGF could be immobilized in the newly formed apatite layer. The coated film enhanced osseointegration of Ti implants in vivo.     

Formation and growth of calcium phosphate on the surface of oxidized Ti-29Nb-13Ta-4.6Zr alloy

The bioconductivity of a new biomedical titanium alloy Ti-29Nb-13Ta-4.6Zr achieved by a combination of surface oxidation and alkali treatment is reported in this paper. Oxidation treatment at 400 degrees C for 24 h was found to result in the formation of a hard layer on the surface of the alloy. Immersion in a protein-free simulated body fluid and fast calcification solution led to the growth of calcium phosphate (Ca-P) phase on the oxidized and alkali-treated alloy, and the new bioconductive surface was still harder than the substrate. The surface processes during various treatment and immersion processes were investigated in detail, and the morphology of the calcium phosphate crystals was shown to be determined by the concentrations of Ca and P in the solution.     

Osteogenecity of octacalcium phosphate coatings applied on porous metal implants

The biomimetic route allows the homogeneous deposition of calcium phosphate (Ca-P) coatings on porous implants by immersion in simulated physiologic solution. In addition, various Ca-P phases, such as octacalcium phosphate (OCP) or bone-like carbonated apatite (BCA), which are stable only at low temperatures, can be deposited. In this pilot study, experiments were designed with a twofold-purpose: (1) to investigate the osteoinduction of OCP-coated and noncoated porous tantalum cylinders and of dense titanium alloy cylinders (5 mm in diameter and 10 mm in length) in the back muscle of goats at 12 and 24 weeks (n = 4); and (2) to compare the osteogenic potentials of BCA-coated, OCP-coated, and bare porous tantalum cylinders in a gap of 1 mm created in the femoral condyle of a goat at 12 weeks (n = 2). In the goat muscle, after 12 weeks the OCP-coated porous cylinder had induced ectopic bone as well as bone within the cavity of the OCP-coated dense titanium cylinder. In the femoral condyle, bone did not fill the gap in any of the porous implants. In contrast with the two other groups, OCP-coated porous cylinders exhibited bone formation in the center of the implant. The nature of the Ca-P coating, via its microstructure, its dissolution rate, and its specific interactions with body fluids, may influence the osteogenecity of the Ca-P biomaterial.     

钛表面微弧氧化涂层的细胞生物活性

背景：微弧氧化技术可改善钛或钛合金的表面特征。 目的：研究纯钛表面微弧氧化涂层的表面性能及其对MC3T3-E1细胞早期黏附、增殖及成骨能力的影响。 方法：将46个直径10 mm、厚度2 mm圆盘状纯钛试件分为实验组和对照组。实验组置于含0.02 mol/Lβ-甘油磷酸二钠盐及0.2 mol/L乙酸钙的电解液中进行微弧氧化处理,对照组对试件进行机械抛光。扫描电子显微镜观察试件表面形貌,X射线能谱分析检测涂层表面钙磷比,X射线衍射分析检测涂层晶相构成。将MC3T3-E1细胞接种在两组试件表面,1,2,4 h电镜下观察细胞形态,在2,4,7 d通过CCK-8方法检测细胞增殖,并于7,14 d检测碱性磷酸酶活性。 结果与结论：经微弧氧化处理后,钛表面形成粗糙多孔的钙磷涂层,微弧氧化涂层主要元素为Ca、P、O及Ti,微弧氧化膜层主要由氧化钛、钛酸钙、磷酸钙及偏磷酸钙构成。电镜观察显示1 h 微弧氧化涂层表面细胞已伸出伪足,4 h呈现较典型的细胞形态。细胞在微弧氧化处理钛表面4,7 d的细胞增殖和7,14 d的碱性磷酸酶活性高于对照组。表明微弧氧化技术生成的粗糙多孔钙磷涂层能显著促进MC3T3-E1细胞的早期黏附、增殖及成骨活性。     

Incorporation of tobramycin into biomimetic hydroxyapatite coating on titanium

Calcium phosphate coatings containing an antibiotic were produced on titanium alloy (Ti6Al4V) implants using a biomimetic approach. Thin, amorphous calcium phosphate (ACP) coatings were first deposited onto Ti6Al4V plates by immersion in 5 times concentrated simulated body fluid (SBF), for 24h at 37 degrees C. The ACP-coated implants were then immersed in a supersaturated calcium phosphate (SCP) solution containing 0, 100, 200, 400, 600 or 800 mg/l of tobramycin for 48 h at 37 degrees C. A carbonated hydroxyapatite (CHA) layer, approximately 40 microm thick, was formed. Approximately 3 microg/mg of tobramycin was co-precipitated with the CHA crystals onto titanium alloy plates, using 800mg/l tobramycin in the coating solution. For comparison, plasma-sprayed calcium phosphate coatings were also immersed in solutions containing 100, 200, 400 or 1,000 mg/l of tobramycin for 10, 40 min, or 48 h. A maximum of about 0.3 microg/mg could be adsorbed onto the plasma-sprayed calcium phosphate coating with the comparable concentration of 800 mg/l in solution. The dissolution of coating and release of tobramycin were also measured in vitro using saline solution buffered at pH 5.0 or 7.3 at 37 degrees C. The release rate of tobramycin was faster at pH 7.3 than at pH 5, with 50 and 4 microg/ml/min, respectively. Tobramycin released from the biomimetic-coated plates could inhibit growth of Staphylococcus aureus bacteria. The result of this study, therefore, indicates that the biomimetic CHA coatings containing antibiotics could be used to prevent post-surgical infections in orthopaedic or trauma.     

Carbonate apatite coating on titanium induced rapidly by precalcification.

Chemical treatments have been thought to be promised methods for improving bioactivity of titanium. In this work, the effect of precalcification with boiling saturated Ca(OH)2 solution on bioactivation of titanium was investigated. After precalcification and soaking in supersaturated Ca-P solution (SCP), calcium phosphate rapidly precipitated onto the surfaces of titanium, and after only three days an uniform apatite layer was found up to thickness of a few micrometers. The observation using scanning electron microscopy (SEM) showed that the coating was composed of a number of small crystal grains. The investigation by X-ray energy dispersion spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) indicated that the coating was Ca-deficient carbonate apatite. Based on the analyses for the surfaces and SCP, a mechanism of precipitation of apatite was proposed in thermal dynamics and kinetics.     

Porous calcium phosphate coating over phosphorylated chitosan film by a biomimetic method

A porous calcium phosphate coating deposited on chitosan films was studied using scanning electron microscopy, energy-dispersive X-ray analysis, micro-Fourier transform infrared spectroscopy (micro-FTIR) and thin-film X-ray diffractometry (XRD). Chitosan films were first prepared by dissolving chitosan powder in dilute acetic acid and drying in a flat petri dish. The films were phosphorylated using urea and H3PO4 with the P content being 0.1-0.2 wt%. Phosphorylated films soaked in saturated Ca(OH)2 solution for 8 days led to the formation of a calcium phosphate precursor phase over the entire surface. This precursor phase stimulated the growth of a porous coating of calcium-deficient hydroxy apatite when immersed in 1.5 x SBF for more than 20 days. Phosphorylated films not treated with Ca(OH)2 did not show any calcium phosphate growth upon immersion in SBF solution. The precursor phase is thought to be octacalcium phosphate, which nucleates a HAP phase during SBF treatment. Initially, this treatment in SBF results in the formation of a single-layer calcium phosphate particles over the film surface. As immersion time in SBF increases, further nucleation and growth produce a porous HAP coating. The Ca/P ratio of the HAP coating is a function of SBF immersion time.     

Mussel-inspired bioceramics with self-assembled Ca-P/polydopamine composite nanolayer: Preparation,formation mechanism,improved cellular bioactivity and osteogenic differentiation of bone marrow stromal cells

The nanostructured surface of biomaterials plays an important role in improving their in vitro cellular bioactivity as well as stimulating in vivo tissue regeneration. Inspired by the mussel’s adhesive versatility, which is thought to be due to the plaque–substrate interface being rich in 3,4-dihydroxy-l-phenylalamine (DOPA) and lysine amino acids, in this study we developed a self-assembly method to prepare a uniform calcium phosphate (Ca-P)/polydopamine composite nanolayer on the surface of β-tricalcium phosphate (β-TCP) bioceramics by soaking β-TCP bioceramics in Tris–dopamine solution. It was found that the addition of dopamine, reaction temperature and reaction time are three key factors inducing the formation of a uniform Ca-P/polydopamine composite nanolayer. The formation mechanism of a Ca-P/polydopamine composite nanolayer involved two important steps: (i) the addition of dopamine to Tris–HCl solution decreases the pH value and accelerates Ca and P ionic dissolution from the crystal boundaries of β-TCP ceramics; (ii) dopamine is polymerized to form self-assembled polydopamine film and, at the same time, nanosized Ca-P particles are mineralized with the assistance of polydopamine, in which the formation of polydopamine occurs simultaneously with Ca-P mineralization (formation of nanosized microparticles composed of calcium phosphate-based materials), and finally a self-assembled Ca-P/polydopamine composite nanolayer forms on the surface of the β-TCP ceramics. Furthermore, the formed self-assembled Ca-P/polydopamine composite nanolayer significantly enhances the surface roughness and hydrophilicity of β-TCP ceramics, and stimulates the attachment, proliferation, alkaline phosphate (ALP) activity and bone-related gene expression (ALP, OCN, COL1 and Runx2) of human bone marrow stromal cells. Our results suggest that the preparation of self-assembled Ca-P/polydopamine composite nanolayers is a viable method to modify the surface of biomaterials by significantly improving their surface physicochemical properties and cellular bioactivity for bone regeneration application.     

Surface treatments of titanium in aqueous solutions containing calcium and phosphate ions.

Immersion treatment of titanium in aqueous solutions containing various kinds of ion concentrations of calcium and phosphate (pH 5.8, 7.0, and 8.0) were attempted to accelerate calcium phosphate precipitation on titanium in body fluid. The performance was confirmed using scanning electron microscopy, X-ray diffractometry, and Fourier transformed infrared absorption spectrometry with a reflection absorption spectrometer of the specimen immersed in Hanks' solution. Calcium phosphate precipitation on titanium in Hanks' solution is accelerated by the immersion treatment in aqueous solutions containing calcium and phosphate ions. The amount, composition, and shape of calcium phosphate precipitate vary according to the pH and ion concentrations of the solutions in which titanium is immersed. This method is effective for the surface treatment of inside pore narrow space of titanium materials.     

Early apatite deposition and osteoblast growth on plasma-sprayed dicalcium silicate coating

Dicalcium silicate coating was deposited onto a Ti-6Al-4V substrate using plasma-spraying technology. The coating was immersed in simulated body fluid (SBF) for 1, 3, 6, 12, 24, and 48 h to investigate early apatite formation on the coating. Osteoblasts were also seeded onto the surface of the dicalcium silicate coating to evaluate its biocompatibility. Cold field-emission scanning electron microscopy and energy-dispersive X-ray spectrometry were used to evaluate the morphologies and determine the chemical composition of the coatings. The surface structural changes caused by immersion in SBF were analyzed using thin-film X-ray diffraction. After the dicalcium silicate coating was soaked in SBF solution 1-6 h, two types of particles containing calcium and phosphorus were formed on the surface. One type consisted of relatively larger particles (P1) precipitated on the surface of the coating from the precursor cluster formed in the SBF solution. The second type was composed of particles (P2) nucleated on the surface of the coating. With increasing immersion time, the particles coalesced to form a surface Ca-P layer. The Ca-P layer was composed of amorphous calcium phosphate that was not transformed to crystalline apatite until the immersion time in SBF exceeded 24 h. The formation mechanism of the Ca-P layer and apatite on the surface of the coating is believed to be involved in the formation of the Si 3-ring active surface site with negative charge. The cell-seeding test revealed that osteoblasts grew and proliferated very well on the surface of the dicalcium silicate coating.