Fabrication, characterization, and biological assessment of multilayered DNA-coatings for biomaterial purposes

This study describes the fabrication of two types of multilayered coatings onto titanium by electrostatic self-assembly (ESA), using deoxyribosenucleic acid (DNA) as the anionic polyelectrolyte and poly-d-lysine (PDL) or poly(allylamine hydrochloride) (PAH) as the cationic polyelectrolyte. Both coatings were characterized using UV-vis spectrophotometry, atomic force microscopy (AFM), X-ray photospectroscopy (XPS), contact angle measurements, Fourier transform infrared spectroscopy (FTIR), and for the amount of DNA immobilized. The mutagenicity of the constituents of the coatings was assessed. Titanium substrates with or without multilayered DNA-coatings were used in cell culture experiments to study cell proliferation, viability, and morphology. Results of UV-vis spectrophotometry, AFM, and contact angle measurements clearly indicated the progressive build-up of the multilayered coatings. Furthermore, AFM and XPS data showed a more uniform build-up and morphology of [PDL/DNA]-coatings compared to [PAH/DNA]-coatings. DNA-immobilization into both coatings was linear, and approximated 3microg/cm(2) into each double-layer. The surface morphology of both types of multilayered DNA-coatings showed elevations in the nanoscale range. No mutagenic effects of DNA, PDL, or PAH were detected, and cell viability and morphology were not affected by the presence of either type of multilayered DNA-coating. Still, the results of the proliferation assay revealed an increased proliferation of primary rat dermal fibroblasts on both types of multilayered DNA-coatings compared to non-coated controls. The biocompatibility and functionalization of the coatings produced here, will be assessed in subsequent cell culture and animal-implantation studies.     

钛表面纳米仿生磷灰石涂层对成骨样细胞活性的生物增强效应

目的：观察钛表面纳米仿生磷灰石涂层对成骨样细胞行为的影响，为骨科常用钛植入体的表面改性及其生物效应提供实验依据。方法: 商业用纯钛经过物理、化学和生物处理，表面生成均匀薄层仿生的纳米磷灰石涂层，将仿生涂层的钛金属板与成骨样细胞复合培养，以纯钛和只经磨砂、酸蚀处理的钛板作为对照，采用MTT法检测细胞活力和增殖变化、扫描电镜和激光共聚焦荧光显微镜观察细胞形态、RT-PCR检测碱性磷酸酶基因表达。结果: 纳米仿生磷灰石涂层比非涂层钛金属表面细胞的增殖数量明显增高，细胞的形态和分布也优于对照组；培养12 d，涂层对细胞ALP基因表达的量明显高于对照组。结论: 钛金属表面纳米仿生磷灰石涂层可以增强细胞的生物效应，提高钛植入体的骨界面早期结合，具有很好的应用前景。     

Immobilization of hyaluronic acid on plasma-sprayed porous titanium coatings for improving biological properties

In the present study, hyaluronic acid (HyA) was covalently immobilized onto titanium coatings to improve their biological properties. Diffuse reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy were employed to characterize the HyA-modified titanium coating. HyA-modified titanium coatings possess better cell-material interaction, and human mesenchymal stem cells present good adhesive morphologies on the surface of TC-AAH. The results of subsequent cellular evaluation showed that the immobilization of HyA on titanium coatings could improve hMSC attachment, proliferation, and differentiation. In vivo evaluation of implants in rabbit femur condyle defect model showed improvements of early osseointegration and bone-to-implant contact of TC-AAH. In conclusion, immobilization of HyA could improve biological properties of titanium coatings.     

Fabrication and characterization of functionally graded nano-micro porous titanium surface by anodizing

The purpose of this study was to fabricate and characterize nanotubular structure on machined titanium (MA) and resorbable blast media (RBM) treated titanium by anodizing. The anodized MA and RBM were characterized with scanning electron microscopy, transmission electron microscopy, X-ray diffraction, energy disperse spectra, X-ray photoelectron spectra, and nano-indentation and scratch test. Highly ordered nanotubular layers of individually 100 nm in diameter and 500 nm in length approximately were formed regardless of the substrates. The nanotubular layers consisted mainly of amorphous TiO(2) with trace fluorine. The nanotubular surfaces on both the substrates significantly reduced water contact angles and elastic modulus compared with those prior to anodizing. The anodizing treatment significantly increased the surface roughness of the smooth MA, but significantly decreased the surface roughness of the roughened RBM. The critical delamination forces of the nanotubular layer were not obtained due to the limitation of surface roughness. The anodized RBM consisted of a nano-micro porous graded structure, or a nanotubular amorphous fluoride containing TiO(2) layer on top of micro-roughened titanium surface, which is expected to significantly improve the surface area that can be used to deliver drugs and growth factors and alleviate the interfacial elastic modulus mismatch as to enhance osseointegration when compared with conventional dental and orthopedic implant devices with smooth or acid etched surface.     

Preparation and characterization of thin film surface coatings for biological environments

An approach to the problem of selecting synthetic materials for use in biological media is presented. Firstly, a surface energetic criterion of biocompatibility of foreign surfaces is suggested. This criterion, which is based on an analysis of the surface interactions between a typical biological fluid (i.e. blood) and synthetic surfaces, is founded on the premise that a sufficiently low (but not very low) solid-bioloical fluid interfacial free energy of the order of 1–3 dyne/cm, is necessary in order to fulfil the dual requirements of maintaining a low thermodynamic driving force for the adsorption of fluid components on the solid surface as well as a mechanically stable solid-fluid interface.

In the second part of this investigation, an experimental approach involving the radio frequency (rf) sputter deposition of thin solid films of tightly adhering polymeric compounds on materials with the desired bulk characteristics, is shown to he a promising method of tailoring the surface properties of many types of synthetic materials for use in biological environments. The preparation and surface characterization of thin, solid films of oxidized fluorocarbon coatings (from a Teflon FEP target) by rf sputtering is illustrated. The deposited polymer films were characterized for their surface morphology, thickness, elemental surface chemical composition and their wetting properties in a biological environment, by the techniques of scanning electron microscopy (SEM), ellipsometry, electron spectroscopy for chemical analysis (ESCA) and contact angle measurements, respectively. Based on the ESCA and contact angle results, it emerges that the surfaces of such polymeric coatings possess sufficient mobility to considerably alter their structures between different environments (such as air and water) and thereby present different wetting characters to these environments. The contact angle procedure developed in this investigation permitted the estimation of the relevant wetting properties of such mobile surfaces in an aqueous environment (which is the environment encountered in most biological fluids).

In the final part of this investigation, the possibility of effecting a drastic reduction in the solid-water interfacial free energy of the sputtered polymer surfaces by physical and or chemical modification of their surfaces and thereby improving their biocompatibility is illustrated.     

钛合金表面介孔二氧化钛涂层的制备及表征**◆

背景：介孔结构的二氧化钛涂层除了具有一般介孔材料的优点外,还具有很好的生物相容性和独特的抗菌性,并且在钛及钛合金表面很易形成。 目的：对介孔诱导型二氧化钛涂层和普通的二氧化钛涂层进行形貌结构分析和比较,为钛合金表面介孔结构二氧化钛涂层在生物医学领域的应用提供实验和理论基础。 方法：在Ti-6Al-4V合金表面采用模板法和非模板法制备介孔诱导型二氧化钛涂层和普通二氧化钛涂层,使用场发射扫描电子显微镜、快速比表面积/孔隙分析仪和X射线衍射仪对两种二氧化钛涂层的表面形貌结构进行比较分析。 结果与结论：在Ti-6Al-4V合金表面制备的稳定介孔二氧化钛涂层,其平均介孔孔径、比表面积和孔容分别为6.668 0 nm、124.190 6 m2/g和0.256 470 cm3/g,具有介孔结构大比表面积和孔容的特点,适合对医用钛及钛合金改性。     

Synthesis,analytical characterization,and osteoblast adhesion properties on RGD-grafted polypyrrole coatings on titanium substrates

The covalent attachment of an Arg-Gly-Asp (RGD) containing peptide to polypyrrole(PPy)-coated titanium substrates has been investigated in order to develop a bioactive material of potential use in orthopedic fields. Polypyrrole has been employed as the coating polymer because of its suitability to be electrochemically grown directly onto metallic substrates of different shapes, leading to remarkably adherent films. The synthetic peptide Cys-Gly-(Arg-Gly-Asp)-Ser-Pro-Lys, containing the cell-adhesive region of fibronectin(RGD), has been grafted to the polymer substrate via the cysteine residue using a procedure recently developed in the authors laboratory. The effectiveness of grafting was monitored by X-ray photoelectron spectroscopy (XPS), which assessed the presence of the peptide grafted onto the polymer surface exploiting the cysteine sulfur as target element. Neonatal rat calvarial osteoblasts were attached to RGD-modified PPy-coated Ti substrates at levels significantly greater than on unmodified PPy-coated Ti and glass coverslip substrates.     

Synthesis, analytical characterization, and osteoblast adhesion properties on RGD-grafted polypyrrole coatings on titanium substrates

The covalent attachment of an Arg-Gly-Asp (RGD) containing peptide to polypyrrole(PPy)-coated titanium substrates has been investigated in order to develop a bioactive material of potential use in orthopedic fields. Polypyrrole has been employed as the coating polymer because of its suitability to be electrochemically grown directly onto metallic substrates of different shapes, leading to remarkably adherent films. The synthetic peptide Cys-Gly-(Arg-Gly-Asp)-Ser-Pro-Lys, containing the cell-adhesive region of fibronectin (RGD), has been grafted to the polymer substrate via the cysteine residue using a procedure recently developed in the authors laboratory. The effectiveness of grafting was monitored by X-ray photoelectron spectroscopy (XPS), which assessed the presence of the peptide grafted onto the polymer surface exploiting the cysteine sulfur as target element. Neonatal rat calvarial osteoblasts were attached to RGD-modified PPy-coated Ti substrates at levels significantly greater than on unmodified PPy-coated Ti and glass coverslip substrates.     

Fabrication and characterization of oxygen-diffused titanium for biomedical applications

Although titanium has been successful as an orthopedic or dental implant material, performance problems still persist concerning implant–bone interfacial bonding strength. In this study a novel oxygen-diffused titanium (ODTi), fabricated by introducing oxygen into the titanium crystal lattice by thermal treatment, was investigated. The fabricated material is the result of a surface modification made on commercially pure titanium (cp Ti) previously coated with poly(vinyl alcohol) (PVA) by means of a thermal treatment performed at 700 °C in an ultra-pure argon atmosphere. The thermal treatment at 700 °C led to the formation of an anatase TiO₂ film on the cp Ti surface and a concentration gradient of oxygen into titanium. The surface of the fabricated ODTi consisted of an outer nanometric layer of anatase TiO₂ and an inner nanometric layer of Ti₂O_x (x < 1) in which the oxygen is in solid solution with the titanium metal. It was found that ODTi possesses in vitro apatite formation ability after being soaked into simulated body fluid (SBF) solution. This apatite formation ability is attributed to the presence of the anatase TiO₂ outermost surface layer and to abundant hydroxyl groups (–OH) formed on the ODTi surface after immersion in SBF.     

Roughness induced dynamic changes of wettability of acid etched titanium implant modifications

Dynamic contact angle analysis (DCA) was used to investigate time-dependent wettability changes of sandblasted and acid-etched commercially pure (cp) titanium (Ti) implant modifications during their initial contact with aqueous systems compared to a macrostructured reference surface. Surface topography was analyzed by scanning electron microscopy and by contact stylus profilometry. The microstructured Ti surfaces were found to be initially extremely hydrophobic. This hydrophobic configuration can shift to a completely wettable surface behavior with water contact angles of 0 degrees after the first emersion loop during DCA experiments. It is suggested that a hierarchically structured surface topography could be responsible for this unexpected wetting phenomenon. Roughness spatial and hybrid parameters could describe topographical features interfering with dynamic wettability significantly better than roughness height parameters. The Ti modifications which shift very sudden from a hydrophobic to a hydrophilic state adsorbed the highest amount of immunologically assayed fibronectin. The results suggest that microstructuring greatly influences both the dynamic wettability of Ti implant surfaces during the initial host contact and the initial biological response of plasma protein adsorption. The microstructured surfaces, once in the totally wettable configuration, may improve the initial contact with host tissue after implantation, due to the drastically increased hydrophilicity.     

Biological properties of acid etched titanium implants: effect of sandblasting on bone anchorage

The SLA (sandblasted with large grit and acid etched) surface is a textured surface that has been documented to lead to a rapid and strong implant fixation. The purpose of the present study was to determine the contribution of sandblasting in addition to etching to implant anchorage. It was also aimed to determine if the pits carved during etching alone have a bone-interlocking capacity that leads to microanchorage between the implant and bone. SLA implants and machined-and-acid-etched (MA) implants were placed in the maxilla of Land Race pigs. After 10 weeks of healing, they were reverse torqued. The reverse torque of the SLA and MA implants was 157.29 +/- 38.04 N cm and 105.33 +/- 25.12 N cm, respectively. Sandblasting increased bone anchorage by 49.3%; the difference was statistically significant (p =.028). Bone was found attached to both surfaces; bone ingrowth was found in the pits of both surfaces. It is suggested that the two surfaces are able to generate bone interlocking and mechanical coupling at the interface. When finite-element modeling is performed with these surfaces, it is suggested that the bound mode be used instead of the slip mode.     

Bone growth enhancement in vivo on press-fit titanium alloy implants with acid etched microtexture

Early bone ongrowth secures long-term fixation of primary implants inserted without cement. Implant surfaces roughened with a texture on the micrometer scale are known to be osseoconductive. The aim of this study was to evaluate the bone formation at the surface of acid etched implants modified on the micro-scale. We compared implants with a nonparticulate texture made by chemical milling (hydrofluoric acid, nitric acid) (control) with implants that had a dual acid etched (hydrofluoric acid, hydrochloric acid) microtexture surface superimposed on the primary chemically milled texture. We used an experimental joint replacement model with cylindrical titanium implants (Ti-6Al-4V) inserted paired and press-fit in cancellous tibia metaphyseal bone of eight canines for 4 weeks and evaluated by histomorphometric quantification. A significant twofold median increase was seen for bone ongrowth on the acid etched surface [median, 36.1% (interquartile range, 24.3-44.6%)] compared to the control [18.4% (15.6-20.4%)]. The percentage of fibrous tissue at the implant surface and adjacent bone was significantly less for dual acid textured implants compared with control implants. These results show that secondary roughening of titanium alloy implant surface by dual acid etching increases bone formation at the implant bone interface.     

Surface and biological evaluation of hydroxyapatite-based coatings on titanium deposited by different techniques.

Hydroxyapatite coatings have been deposited on titanium cp by plasma spray, sol-gel, and sputtering techniques for dental implant applications. The latter two techniques are of current interest, as they allow coatings of micrometer dimensions to be deposited. Coating morphology, composition, and structure have been investigated by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). All coatings were homogeneous and exhibited a rough morphology suitable for implant applications. The sputtered (after annealing), plasma spray, and sol-gel coatings all showed diffraction peaks corresponding to hydroxyapatite. The surface contaminants were observed to be different for the different coating types. The sputtered coatings were found to have a composition most similar to hydroxyapatite; the sol-gel deposits also showed a high concentration of hydroxyl ions. A discrepancy in the Ca/P ratio was observed for the plasma spray coatings, and a small concentration of carbonate ions was found in the sputter-deposited coatings. The in vitro cell-culture studies using MG63 osteoblast-like cells demonstrated the ability of cells to proliferate on the materials tested. The sol-gel coating promotes higher cell growth, greater alkaline phosphatase activity, and greater osteocalcin production compared to the sputtered and plasma-sprayed coatings.     

Characterization and biocompatibility of a titanium dental implant with a laser irradiated and dual-acid etched surface

The biological properties of commercial pure titanium (cp-Ti) dental implants can be improved by surface treatment. In this study, the cp-Ti surfaces were prepared to enable machined surfaces (TM) to be compared to the machined, sandblasted, laser irradiated and dual-acid etched surfaces (TA). The surface elements and roughness were characterized. The biocompatibility was evaluated by cell and organ culture in vitro. The removal torque was measured in rabbit implantation. Surface characterization revealed that TA surface was more oxidized than TM surface. The TA surface had micrometric, beehive-like coarse concaves. The average roughness (2.28 mum) was larger than that typical of acid-etched surfaces. Extracts of both materials were not cytotoxic to bone cells. The morphology of cells attached on the TA surface was superior to that on the TM surface. TA promoted cell migration and repaired damaged bones more effectively in organ culture. The formation of bone-like nodules on TA disk exceeded that on TM disk. Rabbit tibia implantation also proved that TA implant had greater removal torque value. These results suggested that TA had good osteoconductivity and was a potential material for dental implantation.     

Chemical-physical characterization and in vitro preliminary biological assessment of hyaluronic acid benzyl ester-hydroxyapatite composite

HYAFF11 is a biocompatible, biodegradable benzyl ester of hyaluronic acid. However, in order to use it for orthopedic application, its mechanical performance needs to be improved. In this study, a novel composite based on HYAFF11 polymer matrix reinforced with hydroxylapatite (HA) has been developed. Its advantage is having a similar component of the mineral phase of bone resulting in favorable osteoconductive properties. The present study has examined the compressive mechanical and surface chemical-physical properties of the novel HYAFF11-HA composite. Preliminary biological investigations, including pH and cytotoxicity studies of the material extracts, have also been performed using an in vitro primary human osteoblast-like cell model. Moreover, protein, especially fibronectin adsorption has been investigated following incubation in culture medium and human plasma. The results show a grainy surface topography composed mainly of C, P, and Ca, with a Ca/P atomic ratio indicating HA on the composite surface. Mechanical analysis shows an improvement of the compressive properties of HYAFF11 matrix, both in the dry and swollen states, with values in the range of that of spongy bone. No cytotoxic effects and no inhibition of cell proliferation have been observed in the presence of the material extracts with pH values within acceptable ranges for cell vitality. Protein studies reveal a similar pattern, but a higher amount of fibronectin following incubation in human plasma when compared with culture medium. The results show that the novel HYAFF11-HA composite shows a great potential for application in orthopedic fields, especially as vertebral trabecular bone substitute.     

钛表面梯度生物活性涂层材料在颅骨修补中的生物性能

BACKGROUND: Titanium mesh has good clinical effect in repairing skull defects, but due to the lack of bone induction ability, the titanium mesh has a poor integration with the bone tissue. OBJECTIVE: To observe the biological properties of the gradient bioactive coating materials on the titanium surface in the skull repair. METHODS: Osteoblasts were co-cultured with the titanium mesh with or without gradient bioactive coatings for 14 days, and then cell proliferation was detected using MTT method. Seventy-one patients with skull defects were enrolled, including 43 males and 28 females, aged 15-60 years, and were subjected to skull repair using the titanium mesh with (observation group, n=3) or without (control group, n=38) gradient bioactive coatings. During the postoperative follow-up of 12 months, the repairing effects and adverse reactions were observed in the two groups. RESULTS AND CONCLUSION: (1) In vitro cell culture: the cell proliferative ability was increased significantly in the observation group as compared with the control group at 8, 10, 12 and 14 days after cell culture. (2) In vivo repair: the hospital stay and wound healing time in the observation group were significantly lower than those in the control group (P < 0.05), and at the final follow-up, the postoperative recovery effect was significantly higher in the observation group than the control group (P < 0.05). The titanium meshes were fixed firmly in the two groups, with no floating, infection and exposure. These results show that the titanium mesh with gradient bioactive coating has good biocompatibility and osteoinduction capacity.      

Influence of multilayer rhBMP-2 DNA coating on the proliferation and differentiation of MC3T3-E1 cells seeded on roughed titanium surface

For bone morphogenetic protein (BMP) gene therapy to be a viable approach for enhancing implant osseointegration clinically, requires the development of efficient nonviral delivery vectors that can coat the implant. This study evaluated a multilayer cationic liposome-DNA complex (LDc) coating as a delivery vehicle for recombinant human BMP-2 (rhBMP-2). Multilayered coatings, comprising hyaluronic acid (HA) and LDc, were fabricated onto titanium using a layer-by-layer (LBL) assembly technique. Preosteoblastic MC3T3-E1 cells were cultured on the roughened titanium surfaces coated with multilayers of HA/LDc, or on uncoated or HA/liposome only surfaces as controls. The amount of rhBMP-2 secreted by the MC3T3-E1 cells and the effect of the various surfaces on cell viability, proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) secretion, and calcium deposition were evaluated. Messenger RNA levels of OC, ALP, Runx2, and Osx were also investigated. The results demonstrated that rhBMP-2 protein secreted into culture medium at 3 days was significantly higher than control groups. MC3T3-E1 cells cultured on the HA/LDc coating displayed significantly higher ALP activity and OC secretion at 7 days and 14 days culture, respectively. MC3T3-E1 cells cultured on HA/LDc upregulated expression of the osteoblast differentiation markers, especially on days 12 for OC and on days 6 and 12 for ALP and Osx. In conclusion, MC3T3-E1 cell cultured on the multilayer HA/LDc coating surface can secret rhBMP-2 protein and the protein levels were effective in inducing early osteogenic differentiation. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A: 2766-2774, 2012.     

Immobilization and characterization of gamma-aminobutyric acid on gold surface

gamma-Aminobutyric acid (GABA) is one of two main inhibitory neurotransmitters in the central nervous system that plays an important role in neuronal function and dysfunction. Immobilization of GABA molecules on a rigid surface in an ordered fashion will provide an opportunity to understand some of the fundamental properties related to its structure and function. In this study, we report a novel strategy for immobilization of bioactive GABA on gold substrate. GABA was immobilized in three consecutive steps, namely gold substrate amination, dextran covalent attachment, and GABA immobilization. Surface chemistry was verified at each step using XPS and FTIR. Bioactivity of GABA immobilized on the gold surface was studied using atomic force microscopy to reveal antigen-antibody binding. Nonspecific protein adsorption on the bioactive surface was analyzed quantitatively using anti-GABA antibody and an enzyme linked nonspecific anti-immunoglobulin-G antibody in an ELISA assay. GABA functionalized surface has high affinity for anti-GABA, while showing significantly low affinity for nonspecific anti-IgG antibody. All these data support the presence of a bio-functional immobilized GABA on the gold surface. In conclusion, we report a novel technique for immobilizing bioactive GABA molecules in an orderly fashion on gold substrates.     

Physical, chemical, and biological characterization of pulsed laser deposited and plasma sputtered hydroxyapatite thin films on titanium alloy

The physical, chemical, and biological properties of pulsed laser deposited (PLD) and plasma sputtered (PS) hydroxyapatite (HA) coatings were compared. Human osteoblast-like cell responses to these coatings in vitro were assayed for proliferation and phenotypic expression. PS coatings formed smooth and continuous thin films that followed the contours of the substrate surface. PLD coatings consisted of numerous spheroidal micro- and macroparticles. The crystallinity of all coatings was quantified by comparison with the HA target used for both the PS and PLD processes. The XRD and FTIR results indicated that unannealed PLD coatings deposited at room temperature had X-ray spectra consistent with an amorphous structure and were found to dissolve after only a few hours in saline solution. Annealing at 400 degrees C increased the crystallinity (87-98%), which resulted in improved stability and cell activity. The PS coatings showed greater chemical stability than the unannealed PLD coatings and contained an approximate 15% crystalline phase, increasing to 65% postannealing. Cell proliferation and alkaline phosphatase production were significantly higher on unannealed PS specimens than the other coating treatments. There may be benefits in engineering the presence of a minor percentage of a microcrystalline phase in an amorphous or nanometer scale polycrystalline HA structure.     

Structural analysis of hydroxyapatite coatings on titanium

Hydroxyapatite from two sources was electrophoretically deposited onto flat titanium plate material. Depending upon the deposition conditions various changes in the structure of the ceramic were identified. A well-adhering Ti-P compound was present at the interface. Hydroxyapatite oxygenated to various degrees and tetracalcium phosphate were reproducibly formed in the coating.