Promotion of osteogenic differentiation of stem cells and increase of bone-bonding ability in vivo using urease-treated titanium coated with calcium phosphate and gelatin

Abstract

Because of its excellent biocompatibility and low allergenicity, titanium has been widely used for bone replacement and tissue engineering. To produce a desirable composite with enhanced bone response and mechanical strength, in this study bioactive calcium phosphate (CaP) and gelatin composites were coated onto titanium (Ti) via a novel urease technique. The cellular responses to the CaP/gelatin/Ti (CaP/gel/Ti) and bone bonding ability were evaluated with proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) on CaP/gel/Ti and CaP/Ti in vitro. The results showed that the optical density values, alkaline phosphatase expression and genes expression of MSCs on CaP/gel/Ti were similar to those on CaP/Ti, yet significantly higher than those on pure Ti (p < 0.05). CaP/gel/Ti and CaP/Ti rods (2 mm in diameter, 10 mm in length) were also implanted into femoral shaft of rabbits and pure Ti rods served as control (n = 10). Histological examination, scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) measurements were performed at 4 and 8 weeks after the operation. The histological and SEM observations demonstrated clearly that more new bone formed on the surface of CaP/gel/Ti than in the other two groups at each time point. The CaP/gel/Ti bonded to the surrounding bone directly with no intervening soft tissue layer. An interfacial layer, containing Ti, Ca and P, was found to form at the interface between bone and the implant on all three groups by EDS analysis. However, the content of Ca, P in the surface of CaP/gel/Ti implants was more than in the other two groups at each time point. The CaP/gel/Ti modified by the urease method was not only beneficial for MSCs proliferation and osteogenic differentiation, but also favorable for bone bonding ability on Ti implants in vivo, suggesting that Ti functionalized with CaP and gelatin might have a great potential in clinical joint replacement or dental implants.     

In vivo synthesis of calcium oxalate whiskers on CoCrMo alloy surfaces via biomineralization

Surface treatments using bio-technology are valuable and fascinating in the sense that such treatments are natural and yield good biocompatibility. Calcium oxalate whiskers for biomedical applications were successfully synthesized on the CoCrMo alloy surfaces implanted in Aloe leaves which consist of many active bio-chemical elements. The effect of surface wettability and surface morphology on the formation of whiskers was investigated using four differently treated CoCrMo surfaces: (i) smoothly polished surface, (ii) electrochemical etched surface, (ii) textured surface with dimples, and (iv) parallel orientated-grooved surface. Results showed that the formed whiskers had a length ranging between 100 μm and 600 μm, and a diameter in the range of 2 μm to 5 μm. Electrochemically etched surfaces had better wettability and were favorably for growing whiskers. Surface morphology with (i) dimple textures or (ii) parallel grooves facilitated the effective control of the size and amount of the grown whiskers.     

In vivo release of rhBMP-2 loaded porous calcium phosphate cement pretreated with albumin

In this study, the release of rhBMP-2 loaded porous Ca-P cement was studied in vitro and in vivo. We hypothesized that adsorption sites of Ca-P ceramic with high affinity for rhBMP-2 can be blocked by pretreatment of the ceramic with albumin prior to rhBMP-2 loading, which would result in weaker rhBMP-2 binding and enhanced release of rhBMP-2. Preset porous Ca-P cement discs with a diameter of 6.35 mm (volume: 75 mm³) were pretreated by incubation in a solution of 10% rat serum albumin for 24 h or in ddH₂O (control group) prior to administration of 5 μg radiolabeled ¹³¹I-rhBMP-2. Release was assessed in vitro in phosphate buffered saline (PBS) and fetal calf serum and in vivo by longitudinal scintigraphic imaging of radiolabeled ¹³¹I-rhBMP-2 and gamma counting of dissected implants. In vitro release from pretreated discs was higher during the first day. For both formulations, release in PBS was limited compared to release in serum. In vivo release considerably exceeded in vitro release. In vivo release kinetics showed no significant difference of half-lives between pretreated and control discs. Both formulations showed sustained release during at least 4 weeks. Ex vivo gamma counting of retrieved samples confirmed scintigraphic results and showed that the capsule and surrounding tissues only contained a minor fraction rhBMP-2. We conclude that 1. scintigraphy of ¹³¹I-labeled rhBMP-2 provides a reliable method for longitudinal measurement of rhBMP-2 release kinetics in vivo. 2. albumin pretreatment of porous Ca-P cement does not results in relevant increase of initial release of rhBMP-2 in vivo, and 3. preset rhBMP-2 loaded porous Ca-P cement discs exhibit one phase exponential release kinetics in the rat ectopic model, characterized by a retention of 20–30% after 4 weeks.     

In vitro dissolution and corrosion study of calcium phosphate coatings elaborated by pulsed electrodeposition current on Ti6Al4V substrate

Calcium-deficient hydroxyapatite (Ca-def-HAP) coatings on titanium alloy (Ti6Al4V) substrates are elaborated by pulsed electrodeposition. In vitro dissolution/precipitation process is investigated by immersion of the coated substrate into Dulbecco’s Modified Eagle Medium (DMEM) from 1 h to 28 days. Calcium and phosphorus concentrations evolution in the biological liquid are determined by Induced Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) for each immersion time. Physical and chemical characterizations of the coating are performed by scanning electron microscopy (SEM) associated to Energy Dispersive X-ray Spectroscopy (EDXS) for X-ray microanalysis. Surface modifications are investigated by an original method based on the three-dimensional reconstruction of SEM images (3D-SEM). Moreover, corrosion measurements are carried out by potentiodynamic polarization experiments. The results show that the precipitation rate of the Ca-def HAP coating is more pronounced in comparison with that of stoichiometric hydroxyapatite (HAP) used as reference. The precipitated bone-like apatite coating is thick, homogenous and exhibits an improved link to the substrate. Consequently, the corrosion behaviour of the elaborated prosthetic material is improved.     

In situ study on the curing process of calcium phosphate bone cement

The aim of this study was to follow the entire curing process of modified alpha-TCP cement, and to explore how the liquid phase affects the curing reaction. Two calcium phosphate bone cements (CPCs) with a variety of aqueous solution were studied for comparison. In situ X-ray diffraction analysis and pH testing were employed to follow the chemical reaction, while quantitative ultrasonic measurement (QUS) was carried out to monitor the physical change. Results showed that CPC powders were completely consumed after 72 h. Two steps were presented in apatite formation. The first step was the precipitation of carbonated hydroxyapatite (CHA), and in the second step, conversion of calcium deficient hydroxyapatite (CDHA) was the dominant reaction. Finally, CPCs were fully converted to apatite except the cement with NaH2PO4 as liquid phase, because acidic environment inhibited the conversion of apatite. The pH increased linearly after mixing, when supersaturation was reached, it decreased to pH approximately 6.0 gradually. Ultrasound measurement indicated that the variation of speed of sound (SOS) was related to both apatite formation and microstructural evolution. Ultrasonic attenuation coefficient (UAC) was able to quantitatively describe the curing process from viscous paste to elastic solid as a function of curing time. Moreover, the curing reaction conformed to classical dissolution-precipitation mechanism.     

Biocompatibility of calcium phosphate bone cement with optimised mechanical properties: an in vivo study

This work establishes the in vivo performance of modified calcium phosphate bone cements for vertebroplasty of spinal fractures using a lapine model. A non-modified calcium phosphate bone cement and collagen-calcium phosphate bone cements composites with enhanced mechanical properties, utilising either bovine collagen or collagen from a marine sponge, were compared to a commercial poly(methyl methacrylate) cement. Conical cement samples (8?mm height?×?4?mm base diameter) were press-fit into distal femoral condyle defects in New Zealand White rabbits and assessed after 5 and 10 weeks. Bone apposition and tartrate-resistant acid phosphatase activity around cements were assessed. All implants were well tolerated, but bone apposition was higher on calcium phosphate bone cements than on poly(methyl methacrylate) cement. Incorporation of collagen showed no evidence of inflammatory or immune reactions. Presence of positive tartrate-resistant acid phosphatase staining within cracks formed in calcium phosphate bone cements suggested active osteoclasts were present within the implants and were actively remodelling within the cements. Bone growth was also observed within these cracks. These findings confirm the biological advantages of calcium phosphate bone cements over poly(methyl methacrylate) and, coupled with previous work on enhancement of mechanical properties through collagen incorporation, suggest collagen-calcium phosphate bone cement composite may offer an alternative to calcium phosphate bone cements in applications where low setting times and higher mechanical stability are important.     

Study on the synthesis and ion-exchange properties of layered titanate Na2Ti3O7 powders with different sizes

Layered titanate Na₂Ti₃O₇ powders with varying sizes were prepared by solid-state reaction of Na₂CO₃ and TiO₂ with different average particle sizes. The structures of the titanates and the products which had undergone H⁺ and Ag⁺ exchange were investigated by XRD, TEM and BET analysis. The influence of the particle size of starting material TiO₂ on the reaction rate, the particle size and ion-exchange property of the resulting products was studied. It is found that nanometer sized TiO₂ facilitates the solid-state reaction and leads to the formation of ultrafine titanate. The H⁺-exchange property is improved by decreasing the particle size of Na₂Ti₃O₇ and the small sized layered titanate can be exfoliated easily by AgNO₃ solution.     

In vitro biocompatibility study of calcium phosphate glass ceramic scaffolds with different trace element doping

Porous calcium phosphate based glass ceramics (CaO-P₂O₅-Na₂O) containing different trace elements (2.0 mol% Mg, Sr and Zn respectively) were prepared by coating polyurethane foams with sol-gel derived glass slurry. After heat treatment at suitable temperatures, main phase catena hexaphosphate (Ca₄P₆O₁₉) and minor phase calcium pyrophosphate (β-Ca₂P₂O₇) crystallized from the glass matrix. These scaffolds were soaked in simulated body fluid (SBF) to determine the solubility and apatite formation, and mouse MC3T3-E1 cells were used to investigate the bioactivity and biocompatibility. The Sr doped scaffold showed a higher degradability than those samples containing Zn or Mg, inducing the formation of an apatite layer with a high (Sr + Ca)/P molar ratio of 1.64, whereas only some discontinuous CaP layers and spare apatite agglomerates were found on the scaffolds doped with Mg ((Mg + Ca)/P = 1.12) and Zn ((Zn + Ca)/P = 1.55) respectively. In vitro cell culture, a high degree of cell adhesion and spreading was achieved on the samples containing Sr or Zn, while only a few cells adhered to the Mg doped sample. These results implied that the bioactivity and biocompatibility of the scaffolds were not only strongly associated with the apatite forming ability, but also related with the Ca/P molar ratios of the deposits.     

In vivo absorption study of ritodrine hydrochloride in the buccal administration to rats

Background: Although ritodrine (RD)-hydrochloride (HCl), named RD-HCl, is widely used in the treatment of premature labor by intravenous prolonged infusion or frequent oral dosing of tablets, those administrations often lower patients’ quality of life (QOL) or cause undesirable side effects, such as tachycardia; therefore, in this study, the potential usefulness of buccal administration as a novel administration method was examined in vivo.

Method: First, the HPLC method was assessed for the determination of plasma RD concentration. Then, after RD-HCl solution in saline was administered intravenously (1?mg/kg), intragastrically (10?mg/kg) or buccally (10?mg/kg) in rats, the plasma concentration–time profiles were investigated, and the absorption extent and rate compared.

Results: The present modified determination method by HPLC with fluorescence detection (Ex. 278?nm, Em. 306?nm) was suitable to analyze the plasma level at 8–200?ng/mL. Buccal administration gave the best plasma concentration–time profile for maintenance of an effective plasma level and fewer side effects. Absorption rates calculated by deconvolution also supported better sustained absorption in buccal dosing.

Conclusion: Buccal application of RD-HCl was demonstrated to be a potentially useful dosing method in the treatment of premature labor with RD-HCl.     

A study of the chemical and morphological alterations of PS and PC surfaces induced by excimer laser treatments

The effect of laser irradiation on polystyrene (PS) and bisphenol-A polycarbonate (PC) has been studied by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), optical microscopy and surface profilometry. The dependence of the surface chemical and morphological properties on wavelength and fluence has been investigated.On both materials no apparent chemical modification was induced by irradiation at 350 nm, there is no evidence of non-linear effects. Morphological alterations were induced in the PS subsurface, due to thermal shock effects. The irradiation of both polymers at 248 and 193 nm did not result in photoassisted oxidation. In the case of PS, etching is evident at 193 nm. Irradiation of PC at 248 nm induces oxygen depletion and photo-Fries rearrangements, whereas at 193 nm oxygen depletion and etching are predominant. In both polymers etching yields peculiar surface morphologies, namely the formation of debris and cone-like structures.     

Effect of hot water and heat treatment on the apatite-forming ability of titania films formed on titanium metal via anodic oxidation in acetic acid solutions

Titanium and its alloys have been widely used for orthopedic implants because of their good biocompatibility. We have previously shown that the crystalline titania layers formed on the surface of titanium metal via anodic oxidation can induce apatite formation in simulated body fluid, whereas amorphous titania layers do not possess apatite-forming ability. In this study, hot water and heat treatments were applied to transform the titania layers from an amorphous structure into a crystalline structure after titanium metal had been anodized in acetic acid solution. The apatite-forming ability of titania layers subjected to the above treatments in simulated body fluid was investigated. The XRD and SEM results indicated hot water and/or heat treatment could greatly transform the crystal structure of titania layers from an amorphous structure into anatase, or a mixture of anatase and rutile. The abundance of Ti–OH groups formed by hot water treatment could contribute to apatite formation on the surface of titanium metals, and subsequent heat treatment would enhance the bond strength between the apatite layers and the titanium substrates. Thus, bioactive titanium metals could be prepared via anodic oxidation and subsequent hot water and heat treatment that would be suitable for applications under load-bearing conditions.     

In vitro study investigating the mechanical properties of acrylic bone cement containing calcium carbonate nanoparticles

A successful total hip replacement has an expected service life of 10-20 years with over 75% of failures due to aseptic loosening which is directly related to cement mantle failure. The aim of the present study was to investigate the addition of nanoparticles of calcium carbonate to acrylic bone cement. It was anticipated that an improvement in mechanical performance of the resultant nanocomposite bone cement would be achieved. A design of experiment approach was adopted to maximise the mechanical properties of the bone cement containing nanoparticles of calcium carbonate and to determine the constituents and preparation methods for which these occur. The selected conditions provided improvements of 21% in energy to maximum load, 10% in elastic modulus, 7% in bending strength and 8% in bending modulus when compared with bone cement without nanoparticles. Although cement containing nanoCaCO(3) coated in sodium citrate also enhanced the energy to maximum load by 28% and the elastic modulus by 14% when compared with control cement, it is not recommended as a factor in the production of nanocomposite bone cement due to reduction in the bending properties of the final bone cement.     

Improving the thromboresistivity of chemical sensors via nitric oxide release: fabrication and in vivo evaluation of NO-releasing oxygen-sensing catheters

The development and in vivo analytical performance of a nitric oxide (NO)-releasing amperometric oxygen sensor with greatly enhanced thromboresistivity are reported. Gas permeable coatings formulated with cross-linked silicone rubber (SR) containing NO-generating compounds (diazeniumdiolates) are shown to release NO for extended periods of time (> 20 h) while reducing platelet adhesion and activation. Oxygen-sensing catheters prepared by dip-coating the NO-releasing films over the outer SR tubes of the implantable devices display similar analytical response properties in vitro (sensitivity, selectivity, response times) when compared to analogous sensors prepared without the NO release coatings. Superior analytical accuracy (relative to blood PO2 values measured in vitro) and greatly reduced thrombus formation on the outer surface of the sensors are observed in vivo (in canine model) with the NO release PO2 sensors compared to control sensors (without NO release) implanted simultaneously within the same animals. Based on these preliminary studies, the use of NO release polymers to fabricate catheter-style chemical sensors may be a potential solution to lingering biocompatibility and concomitant performance problems encountered when attempting to employ such devices for continuous intravascular measurements of blood gases and electrolytes.     

In vitro and in vivo evaluation of the biocompatibility of a calcium phosphate/poly(lactic-co-glycolic acid) composite

This study assess the effects of bioceramic and poly(lactic-co-glycolic acid) composite (BCP/PLGA) on the viability of cultured macrophages and human dental pulp fibroblasts, and we sought to elucidate the temporal profile of the reaction of pulp capping with a composite of bioceramic of calcium phosphate and biodegradable polymer in the progression of delayed dentine bridge after (30 and 60 days) in vivo. Histological evaluation of inflammatory infiltrate and dentin bridge formation were performed after 30 and 60 days. There was similar progressive fibroblast growth in all groups and the macrophages showed viability. The in vivo study showed that of the three experimental groups: BCP/PLGA composite, BCP and calcium hydroxide (Ca(OH)₂) dentin bridging was the most prevalent (90 %) in the BCP/PLGA composite after 30 days, mild to moderate inflammatory response was present throughout the pulp after 30 days. After 60 days was observed dentine bridging in 60 % and necrosis in 40 %, in both groups. The results indicate that understanding BCP/PLGA composite is biocompatible and by the best tissue response as compared to calcium hydroxide in direct pulp capping may be important in the mechanism of delayed dentine bridge after 30 and 60 days.     

Comparative Evaluation on the In Vitro Biological Performance of Ti45Al8.5Nb Intermetallic with Ti6Al4V and Ti6Al7Nb Alloys

The corrosion resistance of Ti45Al8.5Nb intermetallic alloy in artificial saliva and its cytocompatibility was studied via electrochemical tests, scanning electron microscopy, ion release measurement, and MTT assay, with contemporary biomedical Ti6Al4V and Ti6Al7Nb alloys as comparison. The results demonstrate that the corrosion potential (E_corr) and the corrosion current density (i_corr) of the three experimental alloy samples are similar and there is no statistically significant difference among them (p > 0.05). The Al³⁺ ion releasing concentration for Ti45Al8.5Nb intermetallic and Ti6Al7Nb alloy after anodic polarization are close. The relative cell proliferation rates of the three experimental alloy extract groups are all over 90% at various cultivation periods (1, 3, and 5 d), and there is no obvious difference for the MG63 cell morphologies comparing with that of the negative group, reaching confluence after 5 d culture and showing well stretched, which indicates that Ti45Al8.5Nb intermetallic alloy has a good cytocompatibility with the Grade 1 RGR value (no toxicity) according to ISO 10993‐5: 1999.     

Temperature dependence of the segregation of calcium titanate from solid solutions of (Ba,Ca) (Ti,Zr)O3 and its effect on the dielectric properties

The maximum solubility of calcium titanate in the ferroelectric solid solution Ba_1?xCa_x (Ti_1?yZr_y)O₃ depends greatly on the temperature and on the rate of cooling. According to the amount of segregated calcium titanate the permittivity of ceramic samples is reduced appreciably. The relative lowering of ? with rising amounts of the second phase, calcium titanate, is given with adequate accuracy by the Lichtenecker rule. The absolute deviations can be explained by characteristic changes of the microstructure.     

Temperature programmed desorption study of the oxide layer formed by the interaction of polycrystalline nickel with oxygen

In a 100 l exposure of oxygen, an oxide layer was formed on a nickel surface, this surface was heated and maintained at 623 K to dissociate the oxide, then by exposure to 5 l oxygen, a small amount of oxygen was adsorbed again. From the Temperature Programmed Desorption spectrum of this O₂/Ni surface it can be concluded that the original oxide layer on the nickel surface was not a stoichiometric nickel (II) oxide, but may be a compound of a variety of oxides such as nickel (II) oxide, nickel (III) species and excess oxygen.     

In vitro study in stimulating the secretion of angiogenic growth factors of strontium-doped calcium polyphosphate for bone tissue engineering

Angiogenesis of tissue-engineered bone remains a limited factor for the engineering of larger bone tissue constructs. Attempts to stimulate angiogenesis, using recombinant protein or gene transfer of angiogenic growth factors, have been proposed; however, these approaches have been associated with some problems regarding such as complex technique, expensive prices as well as safety problems and short half-life of angiogenic growth factors. This study was performed to determine the ability of strontium-doped calcium polyphosphate (SCPP) to induce angiogenesis via researching its effect on the mRNA expressions and protein secretion of VEGF and bFGF in/from cultured osteoblasts (ROS17/2.8 cells). We cultured osteoblasts with SCPP scaffolds containing various doses of strontium as well as calcium polyphosphate (CPP) scaffold. Through the detection of MTT and SEM, we have found that SCPP could promote cell proliferation and maintain their morphology. The results of RT–PCR and ELISA indicated that, compared with those in CPP group, the mRNA expression as well as protein levels of VEGF and bFGF in/from cultured osteoblasts were dose-dependent increasing in response to increasing strontium before reaching the peak in SCPP groups, and 8% SCPP showed the optimal promoting role. Therefore, SCPP containing proper dose of strontium could be served as a potential biomaterial with stimulating angiogenesis in bone tissue engineering and bone repair.