Nano-structured titanium coating for improving biological performance

Nano-structured titanium coating was obtained by alkali treating the vacuum plasma sprayed samples following hot water immersing for 24 h. The influences of the surface microstructure on the biological performance were studied. A canine model was applied for in vivo evaluation of the bone bonding ability of the coatings. The histological examination results demonstrate that new bone was formed more rapidly on the nano-structured coating implants and grew into the porosity than the as-sprayed one. After 4 weeks implantation, the nano-structured implants were found to appose directly to the surrounding bone while large lacunae could still be observed at the interface between the as-sprayed samples and bone. All these results indicate that a nano-structured surface on the porous titanium coating is favorable for bone bonding.     

Biological behavior of sol-gel coated dental implants

The biocompatibility of dental implants coated with titania/hydroxyapatite (HA) and titania/bioactive glass (BG) composites obtained via sol-gel process was investigated using an in vitro and in vivo model. A device for the in vitro testing of screw-shaped dental implants was developed, in order to well compare the two experimental models studying the behavior of human MG63 osteoblast-like cells seeded onto a particular geometry. The expression of some biochemical parameters of osteoblastic phenotype (alkaline phosphatase specific activity, collagen and osteocalcin production) and some indications on cells morphology obtained by scanning electron microscopy were evaluated. The in vitro and in vivo models were compared after implants insertion in rabbit tibia and femur. The removal torque and histomorphometric parameters (percentage of bone in contact with implant surface and the amount of bone inside the threaded area) were examined. A good agreement was found between the in vitro and in vivo models. These experiments showed better performances of HA and BG sol-gel coated dental implants with respect to uncoated titanium; in particular, it was found that in vitro the HA coating stimulates osteoblastic cells in producing higher level of ALP and collagen, whereas in vivo this surface modification resulted in a higher removal torque and a larger bone-implant contact area. This behavior could be ascribed to the morphology and the chemical composition of the implants with rough and bioactive surfaces.     

Investigation of boundary conditions for biomimetic HA deposition on titanium oxide surfaces

To improve the clinical outcome of metal implants, i.e. earlier loading and reduction of the incidence of revision surgery, better bone bonding ability is wanted. One method to achieve this is to change the surface chemistry to give a surface that facilitates bone bonding in vivo, i.e. a bioactive surface. Crystalline titanium oxide has recently been proven to be bioactive in vitro and is an interesting option to the more common hydroxylapatite (HA) coatings on implants. A materials possible in vitro bioactivity is tested through soaking in simulated body fluid and studies of possible HA formation on the surface. For bioactive materials, the formed HA layer can also be used as a coating. The aim of the current paper is to investigate some boundary conditions for HA formation on crystalline titanium oxide surfaces regarding influence from coating thickness, soaking time and soaking temperature. The influence from soaking time and temperature on the HA growth were investigated on oxidised Ti samples, (24 h at 800°C) resulting in a rutile surface structure. The oxidised samples were tested at three temperatures (4, 37 and 65°C) and four times (1 h, 1 day, 1 week and 4 weeks). The influence from titanium coating thickness on the HA growth was investigated via depositing thin films of crystalline titanium dioxide on Ti plates using a reactive magnetron sputtering process. Four different PVD runs with coating thicknesses between 19 and 74 nm were tested. The soaking temperature had an effect on the HA formation and growth on both rutile surfaces and native oxide on Ti substrates. Higher temperatures lead to easier formation of HA. It was even possible, at 65°C, to grow HA on native titanium oxide from soaking in PBS. The coating quality was better for HA formed at 65°C compared to 37°C. All PVD-coatings showed HA growth after 1 week in PBS at 37°C, thus even very thin coatings of crystalline titanium oxide coatings are bioactive.

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Laser surface modification of titanium substrate for pulsed laser deposition of highly adherent hydroxyapatite

Biomedical implant devices made out of titanium and its alloys are benefited by a modified surface or a bioactive coating to enhance bone bonding ability and to function effectively in vivo for the intended period of time. In this respect hydroxyapatite coating developed through pulsed laser deposition is a promising approach. Since the success of the bioactive ceramic coated implant depends mainly on the substrate-coating strength; an attempt has been made to produce micro patterned surface structure on titanium substrate for adherent hydroxyapatite coating. A pulsed Nd-YAG laser beam (355 nm) with 10 Hz repetition rate was used for surface treatment of titanium as well as hydroxyapatite deposition. The unfocussed laser beam was used to modify the substrate surface with 500–18,000 laser pulses while keeping the polished substrate in water. Hydroxyapatite deposition was done in a vacuum deposition chamber at 400°C with the focused laser beam under 1 × 10⁻³ mbar oxygen pressure. Deposits were analyzed to understand the physico-chemical, morphological and mechanical characteristics. The obtained substrate and coating surface morphology indicates that laser treatment method can provide controlled micro-topography. Scratch test analysis and microindentation hardness values of coating on laser treated substrate indicate higher mechanical adhesion with respect to coatings on untreated substrates.     

Correlation of in vitro and in vivo results of vacuum plasma sprayed titanium implants with different surface topography

Research has proven that rough surfaces improve both biologic and biomechanical responses to titanium (Ti) implants. The purpose of this study was to evaluate the correlation between the expression of bone cell-associated proteins to Vacuum Plasma-Sprayed Titanium implants (VPS-Ti) with different surface textures in vitro and the bone integration in vivo. The biological performances of the surfaces were evaluated over a period of 8 weeks using human bone marrow cell cultures and Göttinger mini pigs. Cells were cultured on VPS-Ti with two respectively different surface-roughnesses (Ra). The level of Osteoprotegerin (OPG), Osteocalcin (OC) and alkaline phosphatase activity (ALP) were evaluated. The bone integration in vivo was evaluated by histomorphological analyses. A cancellous structured titanium (CS-Ti) construct was used as reference material in both study designs. Comparison of data was conducted using the Scheffé tests and the paired t-test with Bonferroni’s correction. A comparative analysis was done to measure the degree of association between the in vitro and in vivo data. A total amount of OC was significantly increased for VPS-Ti for cells cultured on both VPS-Ti and CS-Ti, while OPG was only detectable after 8 weeks without any significant differences. The ALP activity on all surfaces was not statistically increased. For VPS-Ti with Ra ranging from 0.025 mm up to 0.059 mm, bone integration response was increased, but there was no statistical difference between the VPS-Ti. Expression of OPG, OC and ALP correlated with the histomorphological data over the 8-week period. The in vitro data suggest the superiority of VPS-Ti over CS-Ti, but more importantly, the biocompatibility of testing an in vitro model to predict the outcome and possible integration of implants in vivo.     

Surface characterization and biological response of carbon-coated oxygen-diffused titanium having different topographical surfaces

The materials (C-ODTi) with different topographical surfaces that possess interstitial oxygen atoms into the host titanium lattice and an upper nanometric surface layer of anatase-TiO₂ covered by a carbon thin layer were fabricated in this study. The carbon thin layer on the surface of C-ODTi was composed of amorphous carbon and nano-graphite crystals. In vitro tests, using human bone marrow-derived mesenchymal cells (hBMCs), were performed to check cytotoxicity, examining in particular cell morphology, cell proliferation, cell differentiation, and mineralization capability. After 10 days of culture a higher degree of cell viability was observed on the surface of C-ODTi with an abraded surface. We also observed that hBMCs cultured in direct contact with C-ODTi maintained their capability to express alkaline phosphatase activity (ALP) and formed mineralized nodules similar to the control cultures. Our results demonstrate that the carbon layer coating on the surface of C-ODTi possess better biological response than commercially pure titanium (cp Ti), which was evidenced by the higher proliferation rates of osteoblasts, higher osteo-differentiation and a higher mineralization capability.     

经碱处理的等离子喷涂钛涂层的成骨性能研究

用碱处理方法对等离子喷涂的钛涂层进行了表面改性.涂层的骨结合能力通过植入到狗股骨中进行探查.涂层与骨组织之间的结合强度用推出实验进行测量.获得的结果表明改性的涂层不但能直接与骨组织结合,还促进了骨组织的长入.植入一个月后,改性的涂层展示了较好的成骨性能,取得了较高的结合强度.对碱处理改善钛涂层成骨性能的机理作了进一步的分析.这主要是由于碱处理后涂层表面的微观结构以及化学组成的改变所引起的.     

No evidence to indicate topographic dependency on bone formation around cp titanium implants under masticatory loading

In vitro studies have proved the topographic dependency upon osteogenesis on titanium plate by investigating the cell-adhesion, -shape, -proliferation, -differentiation, ALP activity and osteocalcin production of osteogenic stem cells, MG36, MC3T3-E1 and wild strains of bone formative cells from animal and human. However, this in vivo study on bone growth around cp titanium dental implants under masticatory loading did not demonstrate significant difference among the different surface roughness in the range of Ra 0.4–1.9 μm, Rz 2.8–11.2 μm, Rmax 3.6–28.1 μm and Sm 2.9–41.0 μm, which was estimated by measuring the bone contacts, bone occupancies and bone bonding strengths at the implant/bone marrow interface. It is revealed that the topographic dependency on the osteogenetic activity is apt to be covered with wide variation in bone healing potential under the clinical condition with functional biting load.     

Appearance of cell-adhesion factor in osteoblast proliferation and differentiation of apatite coating titanium by blast coating method

We have already reported that the apatite coating of titanium by the blast coating (BC) method could show a higher rate of bone contact from the early stages in vivo, when compared to the pure titanium (Ti) and the apatite coating of titanium by the flame spraying (FS) method. However, the detailed mechanism by which BC resulted in satisfactory bone contact is still unknown. In the present study, we investigated the importance of various factors including cell adhesion factor in osteoblast proliferation and differentiation that could affect the osteoconductivity of the BC disks. Cell proliferation assay revealed that Saos-2 could grow fastest on BC disks, and that a spectrophotometric method using a LabAssay^TM ALP kit showed that ALP activity was increased in cells on BC disks compared to Ti disks and FS disks. In addition, higher expression of E-cadherin and Fibronectin was observed in cells on BC disks than Ti disks and FS disks by relative qPCR as well as Western blotting. These results suggested that the expression of cell-adhesion factors, proliferation and differentiation of osteoblast might be enhanced on BC disks, which might result higher osteoconductivity.     

不同表面特征的多孔钛与皮下软组织间生物力学性质的研究

为解决经皮器械长期生物密封问题, 选择具有三维支架结构和合适的表面微孔多孔钛作为其皮下固定材料, 表面预沉积一层类骨磷灰石, 空白样品作为对照, 皮下植入兔子的背部. 术后40天和70天对样品与周围软组织间的结合强度进行测定, 并对断口处组织与材料间的结合形貌和区域成分进行分析. 结果表明, 随着时间的延长, 不同表面特征的材料与组织的结合强度都不断增大; 表面具有类骨磷灰石层的样品无论是结合强度还是结合形态都优于空白样品, 涂层样品40天时与组织的结合强度几乎与对照样品70天时与组织的结合强度相当. 说明类骨磷灰石层在体内有利于较早形成活跃的细胞材料表面, 有助于皮下结缔组织的长入以及与材料表面的牢固结合.     

Incorporation of bovine serum albumin into biomimetic coatings on titanium with high loading efficacy and its release behavior

Bovine serum albumin (BSA) was employed as a model protein to study its loading efficiency into a calcium phosphate (CaP) coating on titanium substrates. It is found that the protein loading efficiency can be adjusted by varying the specific configurations of the coating system such as simulated body fluid (SBF) volume, solution height and container selection for the SBF. A BSA loading efficiency as high as 90% was achieved when the ratio of the substrate surface area to modified SBF (m-SBF) volume was as high as 0.072. The release of BSA from the biomimetic coatings was also investigated in vitro. A sustained release was achieved although a large quantity of BSA was still trapped in the coating after 15 days of immersion in a phosphate buffer solution. A much faster release rate would be expected when the coating is implanted in vivo due to the active involvement of osteoclast cells and enzymes.     

Mineralization of Collagen‐Coated Electrospun Poly(lactide‐co‐glycolide) Nanofibrous Mesh to Enhance Growth and Differentiation of Osteoblasts and Bone Marrow Mesenchymal Stem Cells

To obtain the biomimetic scaffolding materials for bone tissue engineering, poly(lactide‐co‐glycolide) (PLGA) nanofibrous mesh (NFM) was mineralized in a 5× simulated body fluid (SBF) for different time after it was treated by air plasma for 15 min and subsequent collagen coating. The apatite particles were nucleated on the surface of individual nanofibers, gradually grew up, and finally covered the whole NFM surface. The mineral aggregates were mainly composed of tiny hydroxyapatite (HA) nanoparticles, whose content reached a constant value of 54 µg · cm^?2 after 9 days. The collagen coating and apatite deposition enhanced the NFM strength pronouncedly too. In vitro cell culture demonstrated that the non‐ or less mineralized NFMs were more beneficial of cell spreading and proliferation than those highly mineralized NFMs, but the latter ones could strongly promote secretion of alkaline phosphatase (ALP) by osteoblasts after cultured for 14 days. Moreover, the highly mineralized NFMs also could significantly up‐regulated ALP activity and calcium synthesis of bone marrow mesenchymal stem cells (BMSCs), demonstrating that these NFMs are more favorable of the osteoblast phenotype expression and osteogenic induction. Therefore, the biomimetic apatite deposited PLGA/collagen NFM could be a promising candidate scaffold for bone tissue engineering.     

Effect of surface structure on biomechanical properties and osseointegration

The purpose of this study is to improve the bone-bonding ability between titanium implants and living bone through the control of geometric design and chemical compositions of an implant surface. We compared the tissue healing response and resulting implant stability for three surface designs by characterizing the histological and mechanical properties of the healing tissue around smooth-surfaced Ti–6Al–4V (SS), CP-Ti plasma-spray-coated (PSC), alkali- and heat-treated (AHT) implants. The implants were transversely inserted into a dog thighbone and evaluated at 4, 8, and 12 weeks. Histological examination indicated that initial matrix mineralization leading to osseointegration occurred more rapidly with the AHT implant. During the 4, 8, and 12 week healing periods, new bone on the surface of AHT implant showed denser growth than that on the SS and PSC implants. The more extensive tissue integration and more rapid matrix mineralization with the AHT implant were reflected in the mechanical test data, which demonstrated superior attachment strength and interfacial stiffness for the AHT implant after healing for 4, 8 and 12 weeks of healing because of the mechanical interlocking in the micrometer sized rough surface and the large bonding area between bone and implant caused by the nanosized porous surface structure. Histological and mechanical data demonstrate that with the appropriate surface design selection, bone bone-bonding ability can be improved and can induce acceleration of the healing response, thereby improving the potential for implant osseointegration.     

Hydroxyapatite coating on magnesium with MgF<Subscript>2</Subscript> interlayer for enhanced corrosion resistance and biocompatibility

Hydroxyapatite (HA) was coated onto pure magnesium (Mg) with an MgF₂ interlayer in order to reduce the surface corrosion rate and enhance the biocompatibility. Both MgF₂ and HA were successfully coated in sequence with good adhesion properties using the fluoride conversion coating and aerosol deposition techniques, respectively. In a simulated body fluid (SBF), the double layer coating remarkably enhanced the corrosion resistance of the coated Mg specimen. The in vitro cellular responses of the MC3T3-E1 pre-osteoblasts were examined using a cell proliferation assay and an alkaline phosphatase (ALP) assay, and these results demonstrated that the double coating layer also enhanced cell proliferation and differentiation levels. In the in vivo study, the HA/MgF₂ coated Mg corroded less than the bare Mg and had a higher bone-to-implant contact (BIC) ratio in the cortical bone area of the rabbit femora 4 weeks after implantation. These in vitro and in vivo results suggested that the HA coated Mg with the MgF₂ interlayer could be used as a potential candidate for biodegradable implant materials.     

Human-osteoblast proliferation and differentiation on grit-blasted and bioactive titanium for dental applications

Physico-chemical and topographical surface quality of commercially pure titanium (c.p. Ti) dental implants is one of the most influencing factors in the improvement of their osseointegration. In this sense, previously, a two-step method (2S) for obtaining bioactive blasted-rough titanium surfaces was developed for improving short-term (due to its bioactivity) and long-term (due to its roughness) osseointegration. This 2S-method consists of: (1) Grit blasting on titanium surface in order to roughen it, and (2) thermo-chemical (TCh) treatment in order to obtain a bioactive surface with bone-bonding ability. The aim of the present work is to evaluate the in vitro human-osteoblast response (proliferation, differentiation – ALP activity- and cell morphology-studied by environmental scanning electron microscopy) of rough c.p. Ti (grit blasted), bioactive c.p. Ti (thermo-chemically treated) and rough-bioactive c.p. Ti (2S-treated). Different grit materials (Al₂O₃ and SiC) have been used in order to investigate their influence. The results showed that cell adhesion was statistically higher for the rough and bioactive surfaces, whatever the grit used. Cells proliferated very well on all the c.p. Ti surfaces. If comparing groups with and without TCh (all other treatments being equal) the ALP was always higher in the groups with TCh, indicating stimulation of osteoblast differentiation because of TCh, more significantlly in the groups that were first blasted. Those ALP results were accompanied by a decrease in the value of proliferation, which shows the good behavior of the cells. This results suggest that a rough and bioactive-titanium surface obtained by 2S-treatment enhances adhesion and differentiation activity of human osteoblasts cells.     

Modell zur Analyse des knöchernen Anwachsverhaltens auf bioaktiv beschichteten Implantatoberflächen

Model to analyse the bone on‐growth on bioactive coated implant surfaces Especially on the field of bone regeneration, transient and permanent implants are an important method of therapy in the Orthopaedic Surgery. In this context, bioactive surfaces on metallic implants provide an improved contact to the surrounding bone. The goal of our study was to establish an in‐vitro test system to evaluate the on‐growth of bone‐derived cells on different surface coatings. Therefore, we invented a special kind of clamps made of commercially‐pure (c‐p) titanium and blasted with hydroxyapatite particles followed by electrochemically coating with calcium phosphate (BONIT®‐HA, BONIT®). Definite pieces of human cancellous bone were attached to these clamps, inserted onto tissue culture plates and cultivated in DMEM for ten days. Finally, the contact area between human cancellous bone and the implant surface was analyzed and the spreading of osteoblast‐like cells evaluated by scanning electron microscopy (SEM). A well‐spread morphology of bone cells was observed on the implant surfaces coated with calcium phosphate (CaP). In comparison the clamps without CaP coatings showed only a marginal growth of bone cells on the clamp surface. The presented newly in‐vitro test setup using titanium clamps coated with bioactive layers attached to human cancellous bone represents a well‐functioning model for qualitative evaluation of bone on‐growth.     

Mg ion implantation on SLA-treated titanium surface and its effects on the behavior of mesenchymal stem cell

Magnesium (Mg) is one of the most important ions associated with bone osseointegration. The aim of this study was to evaluate the cellular effects of Mg implantation in titanium (Ti) surfaces treated with sand blast using large grit and acid etching (SLA). Mg ions were implanted into the surface via vacuum arc source ion implantation. The surface morphology, chemical properties, and the amount of Mg ion release were evaluated by scanning electron microscopy (SEM), Auger electron spectroscopy (AES), Rutherford backscattering spectroscopy (RBS), and inductively coupled plasma-optical emission spectrometer (ICP-OES). Human mesenchymal stem cells (hMSCs) were used to evaluate cellular parameters such as proliferation, cytotoxicity, and adhesion morphology by MTS assay, live/dead assay, and SEM. Furthermore, osteoblast differentiation was determined on the basis of alkaline phosphatase (ALP) activity and the degree of calcium accumulation. In the Mg ion-implanted disk, 2.3 × 10¹⁶ ions/cm² was retained. However, after Mg ion implantation, the surface morphology did not change. Implanted Mg ions were rapidly released during the first 7 days in vitro. The MTS assay, live/dead assay, and SEM demonstrated increased cell attachment and growth on the Mg ion-implanted surface. In particular, Mg ion implantation increased the initial cell adhesion, and in an osteoblast differentiation assay, ALP activity and calcium accumulation. These findings suggest that Mg ion implantation using the plasma source ion implantation (PSII) technique may be useful for SLA-treated Ti dental implants to improve their osseointegration capacity.     

Osteoblast proliferation and maturation on bioactive fiber-reinforced composite surface

The objective of this study was to evaluate the proliferation and osteogenic potential of bone-marrow derived osteoblast-like cells on fiber-reinforced composite (FRC) substrates with and without bioactive glass surface modification. Three FRC materials were fabricated for the study: (a) grit-blasted FRC, (b) grit-blasted FRC with bidirectional net reinforcement and (c) FRC with bioactive glass (BAG) coating. Rat bone-marrow derived osteoblast-like cells were harvested and cultured on experimental material plates and on cp. titanium plates (control) for 21 days. The materials' surfaces were characterized by roughness testing and scanning electron microscopy. Cell growth and differentiation kinetics were subsequently investigated by evaluating proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) and bone sialoprotein (BSP) production. On day 14, the cell proliferation was significantly lower (P < 0.05) on FRC-BAG than on titanium and FRC. The proliferation on the other three materials was equal throughout the experiment. The maximal ALP activities on FRC, FRC-Net, and titanium were observed on day 21, whereas FRC-BAG had already reached the maximal level on day 14. Expression of osteoblastic markers (OC, BSP) indicates that the fastest osteogenic differentiation takes place on FRC after 7 days. In contrast, a slower differentiation process was observed on titanium than on any other tested material (P < 0.015) at 21 days, as was confirmed by increased mRNA expression of OC and BSP. It can be concluded that the proliferation and maturation of osteoblast-like cells on FRC appears to be comparable to titanium. Presence of BAG enhances cell maturation.     

A novel snail-inspired bionic design of titanium with strontium-substituted hydroxyapatite coating for promoting osseointegration

As the population ages,more and more people are suffering from osteoarthritis (OA),resulting in an increasing requirement for joint implants.Surface modification to improve the topology and compo-sition of the implants has been proved to be an effective way to improve the primary stability and long-term success rate of joint implants.In this work,a bionic micro/nano-structure accompanied with a strontium-substituted hydroxyapatite (SrHA) coating was fabricated on titanium (Ti) surface via elec-trochemical corrosion,ultrasonic treatment,and hydrothermal deposition methods.The in vitro study demonstrated that the bionic structure and the bioactive apatite could synergistically increase the expres-sions of integrin-related gene (ITG α5β1) and osteoblastic genes (Col-I and OCN),and thus promote osteoblast growth.In addition,owing to the anti-bone resorption property of Sr2+,the coating could effectively inhibit osteoclast differentiation and proliferation.In a word,the prepared samples not only promoted osteogenesis but also inhibited osteoclastogenesis.The in vivo experiment via a rabbit model found that the bionic structured surface provided the pore for new bone ingrowth,which was beneficial to the mechanical interlocking between the implant and bone.Moreover,the bionic structure and bioactive SrHA coating had a synergistic effect on promoting bone formation,osseointegration,and bone-implant bonding strength.This study therefore presented a new strategy to fabricate bio-functionalized Ti-based implants for potential application in orthopedics field.     

Adhesion study of pulsed laser deposited hydroxyapatite coating on laser surface nitrided titanium

Hydroxyapatite (HA) coatings were fabricated by pulsed laser deposition (PLD) on commercially pure titanium which had been subjected to different types of pre-treatment. These include: (i) 60-grit SiC grinding, (ii) 320-grit SiC grinding, (iii) 1-µm diamond paste mirror-finishing, (iv) etching with Knoll solution, and (v) laser surface nitriding followed by selective etching. The HA coatings were pulsed laser deposited at different water-vapor pressures to determine the optimal processing conditions. The nitrided-etched specimen exhibits a three dimensional TiN dendritic network which promotes the adhesion between HA coating and titanium substrate. Among the specimens with different pre-treatments, the adhesion strength of HA is the highest for the nitrided-etched specimen, reaching about twice that for the mirror-finished specimen. Thin-film X-ray diffraction shows a high degree of crystallinity for the PLD deposited HA. According to energy-dispersive X-ray analysis, the Ca/P ratio of the deposited HA reaches an approximate value of 1.7, similar to that of the HA target. Scanning-electron microscopy reveals that the deposited HA is about 4 μm in thickness. Growth of apatite was rapidly induced on the HA coated specimens when immersed in Hanks' solution for 4 days, indicating that the PLD HA coating is highly bone bioactive. This could be partly due to the high wettability of the PLD HA surface.