Dynamic interaction between the growing Ca–P minerals and bacterial cellulose nanofibers during early biomineralization process

Bone is a composite of organic phase (collagen nanofibers) and Ca–P minerals (hydroxylapatite) and an important biological structure in the field of biomineralization, but the interaction between organic matrixes and inorganic minerals is still too ambiguous. In order to investigate the interaction between the growing Ca–P minerals and organic nanofibers during early biomineralization process, bacterial cellulose (BC) nanofibers were used as templates to mimic collagen nanofibers for Ca–P minerals deposition via biomineralization for periods from as short as 4–72 h. Our findings pointed out that the resultant Ca–P minerals formed on BC nanofibers were platelet-like calcium-deficient HAp which was analogous to those in natural bone tissue. Strikingly, we found that the growth of Ca–P minerals had influence on the structure and properties of BC nano-templates during biomineralization process.     

Surface modification of P(EMA-co-HEA)/SiO2 nanohybrids for faster hydroxyapatite deposition in simulated body fluid?

P(EMA-co-HEA)/SiO₂ nanocomposites with 0, 15 and 30 wt% of silica were obtained by copolymerization of ethyl methacrylate, EMA, and hydroxyethyl acrylate, HEA, during the simultaneous acid-catalyzed sol–gel polymerization of tetraethoxysilane, TEOS. A surface modification treatment was applied in order to reduce the induction time for hydroxyapatite (HAp) nucleation, combining a previous NaOH attack to increase the number of surface nucleating sites, and an alternate soaking process in Ca and P solutions to form apatite precursors, prior to the immersion in a simulated body fluid (SBF). The NaOH treatment was not effective by itself in shortening the HAp induction time. It introduced sodium carboxylates in the copolymer but hydrolyzed the silica network excessively, thus reducing the surface nucleating potential of its boundary silanols. Therefore, bioactivity was only due to the surface carboxylate groups of the organic phase. Maybe a controlled dissolution extent of the silica network so as to improve bioactivity could be attained by reducing the duration of the NaOH-treatment. This would be interesting in the hybrid with 30 wt% of silica, because its dense silica network is not able to hydrolyze in SBF without any previous treatment, whereas the silica network in the hybrid with 15 wt% of silica hydrolyzes at the surface promoting the deposition of HAp. The CaP treatment was able to coat the surfaces of the samples with a calcium phosphate layer within minutes. This amorphous calcium phosphate acted as HAp precursor, skipping the induction period in SBF.     

Fabrication and properties of mineralized collagen‐chitosan/hydroxyapatite scaffolds

A hydroxyapatite (HAp)/biopolymer composite scaffold was fabricated by mineralizing a crosslinked collagen/chitosan, which was pre‐mineralized with Ca²⁺ and phosphate salts, in simulated body fluid (SBF) for only 24 hr. A self‐organized structure similar to bone is expected. Microstructures of the crosslinked collagen/chitosan scaffold, the pre‐mineralized collagen–chitosan scaffold (CCS), and the mineralized collagen‐chitosan/HAp scaffolds (MCCHS) were characterized by scanning electron microscopy (SEM), revealing non‐alteration of the porous structure and formation of the HAp particles. X‐ray diffractometer (XRD) confirmed the crystalline structure of the HAp. Thermal gravimetric analysis found that more HAp particles were formed when the CCSs were pre‐mineralized in a higher concentration of Ca²⁺. Water‐uptake ratio of the crosslinked CCS was ～160, decreased to ～120 after incubating in Ca²⁺ solution, and further decreased to ～20 after mineralization. Mechanical strength of the CCS was improved significantly after the in situ mineralization too. The method introduced here may be potentially applied to obtain other biopolymer/HAp composite in a short period. Copyright © 2008 John Wiley & Sons, Ltd.     

Effect of preparative conditions on crystallinity of apatite particles obtained from simulated body fluids

Preparation of colloidal hydroxyapatite (HAp) particles under body fluid conditions was investigated with focusing on the effect of preparative conditions on crystallinity of the resulting particles. Tris(hydroxymethyl)aminomethane was added to 1.5SBF (a solution having 1.5 times higher ion concentrations than those of a simulated body fluid, SBF) to increase the solution pH, which resulted in induction of homogeneous nucleation of HAp in the solution. Colloidal HAp particles having diameters about 300 nm were obtained. When the reaction was proceeded at 70°C and the sample was dried by heating, it was effective to obtain HAp particles having high crystallinity. Experimental results support that remaining water in the sample contributed to increase HAp crystallinity.     

Biomimetic approach towards the preparation of hydroxyapatite and hydroxyapatite/chitosan/β-cyclodextrin nanoparticles: application to controlled drug release

Hydroxyapatite (HAp) and hydroxyapatite/chitosan/β-cyclodextrin (HAp/CS/β-CD) nanoparticles were successfully prepared in the modified simulated body fluid (SBF) solution at the physiological conditions (pH 7.4, temperature?=?37 °C). CS/β-CD nanoparticles acted as templates for the synthesis of HAp/CS/β-CD nanoparticles to improve the nanoarchitecture of HAp and its crystallinity.The nanoparticles were characterized by FT-IR spectroscopy, scanning electron microscopy (SEM), and X-ray diffraction (XRD). Kneading and coprecipitation methods were applied to prepare the inclusion complex involving β-CD and p-THPP (5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin), a photosensitizer for anti-cancer drugs. The 1:1 stoichiometric ratio of the formed inclusion complex was characterized by a formation constant of 7.216?×?10² mol^?1 dm³ and analyzed by ¹H NMR, FTIR, and UV–Vis. The p-THPP delivery release in vitro was in this order: HAp/CS/β-CD?<?CS/β-CD?<?<?HAp/β-CD?<?β-CD, hinting at a better controlled release by HAp/CS/β-CD nanoparticles.     

Enhancing the stiffness of electrospun nanofiber scaffolds with a controlled surface coating and mineralization

A new method was developed to coat hydroxyapatite (HAp) onto electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers for tendon-to-bone insertion site repair applications. Prior to mineralization, chitosan and heparin were covalently immobilized onto the surface of the fibers to accelerate the nucleation of bone-like HAp crystals. Uniform coatings of HAp were obtained by immersing the nanofiber scaffolds into a modified, 10-fold-concentrated simulated body fluid (m10SBF) for different periods of time. The new method resulted in thicker and denser coatings of mineral on the fibers compared to those produced by previously reported methods. Scanning electron microscopy measurements confirmed the formation of nanoscale HAp particles on the fibers. A mechanical property assessment demonstrated a higher stiffness with respect to previous coating methods. A combination of the nanoscale fibrous structure and bonelike mineral coating could mimic the structure, composition, and function of mineralized tissues.     

Organic / inorganic bioactive materials Part I: Synthesis, structure and in vitro assessment of collagen/silicocarnotite biocoatings

The silicocarnotite, as an inorganic part of the coatings, has been synthesized using a polystep sol-gel method. The chemical composition of the prepared silicocarnotite sol is described as 58.12 CaO, 29.42 P₂O₅, 12.45 SiO₂ (wt%), where Ca/P+Si = 1,67. The acid soluble type I collagen, as an organic part of the obtained coatings, was mixed with silicocarnotite powder in a weight ratio of 25:75 and 75:25 weight ratio without cross-linkage. The acidity of the obtained mixture was readjust with 25% NH₄OH to pH = 9.0. The mixture was then dried at 37°C for 12 h. The growth of B-type carbonate containing hydroxyapatite (B-type CO₃HA) in which CO₃ ²⁺→PO₄ ³⁻ on the surface of collagen/silicocarnotite coatings soaked in 1.5 simulated body fluid (1.5 SBF) was observed. The nucleation of B-type CO₃HA was estimated on the obtained coatings after 3 days immersion in 1.5 SBF. The negatively charged carboxylate groups from the collagen surface may be responsible for the HA deposition. This was confirmed by the “red shift” of carboxylate groups of collagen molecules in the FTIR spectra. After soaking in 1.5 SBF, the morphology of prepared coatings and HA formation was observed by SEM.     

Growth of calcium hydroxyapatite (Ca-HAp) on cholesterol and cholestanol crystals from a simulated body fluid: A possible insight into the pathological calcifications associated with atherosclerosis

An experimental study into calcium phosphate (CP) nucleation and growth on cholesterol and cholestanol surfaces from a supersaturated simulated body fluid (SBF) is presented with the overall aim of gaining some fundamental insights into the pathological calcifications associated with atherosclerosis. Soaking of pressed cholesterol disks at physiological temperature in SBF solutions was found to lead to CP nucleation and growth if the disks were surface roughened and if an SBF with concentrations of the calcium and hydrogen phosphate ions at 2.25x physiological concentrations was used. The CP phase deposited was shown via SEM micrographs to possess a florette type morphology akin to that observed in earlier reported studies. The use of recrystallised cholesterol and cholestanol microcrystals as substrates for soaking in SBF facilitated the observation of CP deposition. In general, cholesterol recrystallised from polar solvents like 95% ethanol as a cholesterol monohydrate phase which was a better substrate for CP growth than cholesterol recrystallised from more non-polar solvents (e.g., benzene) which produced anhydrous cholesterol phases. CP was also observed to form on recrystallised cholestanol microcrystals, a molecule closely related to cholesterol. Inductively coupled plasma optical emission spectrometry (ICP-OES) data gave confirmation that Ca:P mole ratios of the grown CP were 1.3-1.5 suggesting a mixed phase of octacalcium phosphate (OCP) and Ca-deficient HAp and that the CP coating grows (with time of soaking) on the substrates after nucleation in the SBF growth medium. Infrared (IR) spectra of the extracted coatings from the cholesterol substrates confirmed that the CP phase deposited is a semi crystalline HAp with either carbonate substituted into its structure or else co-deposited as calcium carbonate. Soaking experiments involving modified cholesterol substrates in which the OH group in the molecule was replaced with the oleiyl or phosphonate group showed no CP nucleation and growth. This observation illustrates the importance of the known epitaxial relationship between cholesterol and HAp (which theoretically predicts favourable deposition of one phase upon the other) and the consequences of its destruction (by chemical modification of the cholesterol). In the case of the phosphorylated cholesterol, failure of this substrate to nucleate CP phases may have also been caused by the reduction in concentration of free solution Ca2+ in the SBF medium by complexation with the phosphonate groups on the phosphorylated cholesterol. This would have reduced the ion product of Ca2+ and inorganic phosphate and lowered the degree of supersaturation in the SBF medium.     

Controlling apatite microparticles formation by calcining electrospun sol–gel derived ultrafine silica fibers

Electrospun ultrafine silica fibers were calcined at 150–800 °C. The relation of calcination temperature to the ability to form biomimetic apatite in a simulated body fluid solution (SBF) was evaluated. The largest apatite particles, formed on non-calcined fibers after 1 week of soaking in SBF, were 10 μm in diameter, had a narrow size distribution (coefficient of variation 9%), and were similar to pearls on string. The particles size decreased with increasing calcination temperature below 250 °C and the particles formed on the fibers calcined at 250 °C were 4.5 μm in diameter. No particles were found on those calcined above 500 °C. By using a concentrated SBF at 1.5-times higher ionic concentrations than SBF, the size of apatite microparticles increased about 50%. The fibrous substrate covered with apatite particles was effective for osteoblastic differentiation of pre-osteoblastic cells.     

Micro-PIXE analysis of bioconductive hydroxyapatite coatings on titanium alloy

Bioconductive materials and in particular implants using Ti alloy (Ti6–Al4–V) coated with hydroxyapatite (HAp) have proved to be a suitable surgical procedure. However, experience has shown that these implants not always have the required reliability to guarantee their expected life-span of approximate 15 years. In this research, experimental Ti alloy-implants coated with HAp and incubated in a simulated body fluid (r-SBF) under controlled physiological conditions were studied by nuclear microprobe (NMP). Selected HAp coatings, were analysed by micro-PIXE using protons of 1.5 MeV at the iThemba LABS NMP facility. Major elements (Ti, Al, V, Ca and P) as well as trace elements (Si, K, Fe, Zn and Sr) were determined. The effect of longer incubation time was of particular interest. Results confirmed that secondary Ca-deficient defect hydroxyapatite precipitated from the simulated body solution onto the HAp coating surface after prolonged incubation. This newly formed layer is thought to be of vital importance for bonding of implants with living bone tissue.     

含骨脱细胞基质电纺纤维的成骨性能

通过脱细胞技术制备了猪骨脱细胞基质(DBM), 用胃蛋白酶消化DBM使其变为可溶形式, 采用静电纺丝技术制备了含有DBM的左旋聚乳酸(PLLA)电纺纤维(PLLA/DBM), 并对PLLA/DBM的形貌、 亲水性、 细胞相容性、 成骨性能和体外矿化能力进行评价. 研究结果表明, 脱细胞处理能够有效去除骨组织中的细胞成分, 使DNA含量显著下降. DBM经胃蛋白酶处理后溶于六氟异丙醇(HFIP), 可进行静电纺丝, 制备的PLLA/DBM[m(PLLA)∶m(DBM)=10∶0, 9∶1, 7∶3, 5∶5]电纺纤维具有良好的亲水性, 且无细胞毒性, 对骨髓间充质干细胞的黏附及成骨分化有明显的诱导促进作用, 体外生物矿化效果优良.     

Preparation and applications of novel fluoroalkyl end‐capped vinyltrimethoxysilane oligomer/hydroxyapatite nanocomposites

Novel fluoroalkyl end‐capped vinyltrimethoxysilane oligomer/hydroxyapatite (HAp) nanocomposites were prepared by the reaction of calcium nitrate tetrahydrate and phosphoric acid in the presence of the corresponding oligomer. These fluorinated oligomer/HAp composites thus obtained are nanometer size‐controlled fine particles (83–173 nm), and were found to exhibit good dispersibility in methanol, ethanol, and isopropyl alcohol. These fluorinated HAp nanocomposites were applied to the surface modification of glass and poly(methyl methacrylate) (PMMA) to exhibit good hydro‐ and oleophobic characteristics imparted by fluorine on their surface. In addition, the surface structural changes of the modified polyethylene terephtalate and PMMA films treated with these fluorinated nanocomposites before and after soaking in a simulated body fluid (SBF) were analyzed by using SEM, XRD, and EDX to observe the formation of spherical HAp deposits on the surface. Copyright © 2009 John Wiley & Sons, Ltd.     

Surface characterization and dissolution study of biodegradable calcium metaphosphate coated by sol–gel method

Coatings of biomedical implant surfaces by a bioactive calcium phosphate film render bioactivity to the implant surface and shorten the healing time. In this present study, calcium metaphosphate (CMP) sol was synthesized by sol–gel method and coated onto the titanium alloys (Ti-6Al-4V). CMP sol was first synthesized by reacting Ca(NO₃)₂4H₂O (Sigma–Aldrich 99%, USA) with (OC₂H₅)₃P (Fluka 97%, Japan) in methyl alcohol. A stoichiometric Ca/P ratio of 0.5 was obtained by varying the amounts of the reactants. Sol was then coated on Ti-6Al-4V substrates by spin coating. The coated-specimens were then dried at 70 °C for 24 h, followed by a heat treatment at 650 °C for 1 h. Structural and chemical properties of the coatings were evaluated using XRD, SEM, and EPMA. The dissolution property of the coated-CMP layer was investigated by immersing the samples in the simulated body fluid (SBF) for 1, 3, 7 and 21 days. The concentration of Ca²⁺ released was measured using ICP. After heat treatment, SEM indicated a smooth and uniform CMP layer, with CMP grains of approximately 100 nm. The CMP phase was identified with δ-CMP (JCPDS #9-363). After immersion in SBF, coatings were observed to be roughened and porous. The concentration of Ca²⁺ in SBF was observed to increase over time, indicating continuous dissolution. The presence of titanium oxide phosphate compounds were also observed on CMP surfaces after immersion. It was thus concluded that the ability to control coating properties as well as the need for low heat treatment temperature offers advance for the use of CMP coating by sol–gel process on Ti-6Al-4V implant surfaces.     

Synthesis and In Vitro Bioactivity of TiO2 Nanorods

Titanium dioxide (TiO₂) powders were synthesized by the hydrothermal method. The TiO₂ powders were composed of nanorods with dimensions of 10–18 nm and 60–180 nm in diameter and length, respectively. The in vitro bioactivity of the TiO₂ powders was examined by evaluation of hydroxyapatite (HAp) formation ability in simulated body fluid (SBF). The results showed that TiO₂ nanorods induced the formation of nanocrystalline HAp after soaking in SBF after 1 day rapidly. Our study indicates that TiO₂ nanorods are bioactive and might be used for preparation of new biomaterials.     

Self-assembled structures of hydroxyapatite in the biomimetic coating on a bioinert ceramic substrate

The tribological properties of alumina ceramic are excellent due in part to a high wettability because of the hydrophilic surface and fluid film lubrication that minimizes the adhesive wear. Such surfaces are further modified with bioactive glass/ceramic coating to promote direct bone apposition in orthopedic applications. The present communication reports the biomimetic coating of calcium hydroxyapatite (HAp) on dense (2-3% porosity) alumina (alpha-Al(2)O(3)) substrate (1cmx1cmx0.5cm), at 37 degrees C. After a total period of 6 days immersion in simulated body fluid (SBF), at 37 degrees C, linear self-assembled porous (pore size: approximately 0.2mum) structures (length: approximately 375.39mum and width: 5-6mum) of HAp were obtained. The phenomenon has been demonstrated by self-assembly and diffusion-limited aggregation (DLA) principles. Structural and compositional characterization of the coating was carried out using SEM with EDX facility, XRD and FT-IR data.     

Surface Properties of Graphene Functionalized TiO2/nHA Hybrid Coatings Made on Ti6Al7Nb Alloys via Plasma Electrolytic Oxidation (PEO)

Nano-hydroxyapatite (nHA)-matrix coatings containing graphene nanosheets (GNS)-nHA were coated on Ti6Al7Nb alloys by plasma electrolytic oxidation (PEO) treatment for the improvement of their surface properties. Crystallographic properties, functional groups, and elemental analysis of coatings were characterized by XRD, ATR–FTIR, and EDS analysis. Surface morphological changes of the coated surfaces were investigated by AFM and SEM. The electrochemical corrosion behavior of the coatings was examined by using the potentiodynamic scanning (PDS) tests under in-vitro conditions in simulated body fluid (SBF). The results showed that the GNS was successfully deposited in ceramic matrix coatings on Ti6Al7Nb alloys. Also, the microstructural observations revealed that the coatings have a porous and rough structure. The XRD and ATR–FTIR quantitative analysis have proved the appearance of HA and GNS in the coating layers. An increase in the coating thickness, surface hardness, and anatase/rutile transformation rate was determined, while the GNS ratio in the coating layers was increased. The microhardness of the nHA coating reinforced with 1.5 wt% GNS was measured at 862 HV, which was significantly higher than that of GNS-free (only nHA) coating (584 HV). The best in-vitro resistance to corrosion in SBF was observed in the nHA/1.5GNS wt% coating.     

造孔剂对电泳沉积制备多孔HA涂层及其生物活性的影响

采用水热法制得的羟基磷灰石(HA)纳米粉体,分别与造孔剂葡萄糖(Glu)、壳聚糖(CS)、炭粉(C)3种微粒(＜38.5 μm)配置成质量比1∶1的悬浮液,电泳沉积 烧结制备钛基多孔HA涂层,并对制得的3种多孔HA涂层在模拟体液浸泡前后的表面形貌、化学组成及物相变化进行表征。 结果表明,经700 ℃烧结处理后制得的3种多孔HA涂层在1.5倍人体模拟体液中浸泡5 d后,多孔HA涂层表面均被层状生长的碳磷灰石颗粒完全覆盖,颗粒直径在5~25 μm,说明这些多孔HA涂层均具有良好的生物活性。 其中以CS为造孔剂制得的多孔HA涂层结合强度最高,达19.5 MPa,有望开发成为新型的人骨植入生物陶瓷材料。     

Effect of buffer in simulated body fluid on morphology and crystallinity of hydroxyapatite precipitated on 45S5 bioactive glass-derived glass–ceramic scaffolds: comparison of Good’s buffer systems and TRIS

In vitro tests that verify the ability of a material to form bone-like apatite precipitated (HAp) use a solution that imitates the inorganic part of blood plasma, simulated body fluid (SBF). During such tests, it is necessary to maintain a neutral pH, for which purpose the International Standards Organization recommends the TRIS (tris(hydroxymethyl)aminomethane) buffer (ISO 23,317:2014). To do this, TRIS buffer must remain inert, but, as we have previously reported, TRIS interacts with highly bioreactive materials (such as 45S5 Bioglass-derived scaffolds), thereby accelerating scaffold dissolution. In the search for an alternative to TRIS, we have also recently published results for the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 3-(N-morpholino) propanesulfonic acid (MOPS) buffers, which were also unable to maintain a neutral pH in SBF. Thus, we here continue our search for a more suitable Good’s buffer by comparing BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid) and TES (2-[(2-hydroxy-1,1-bis(hydroxymethyl) ethyl) amino] ethanesulfonic acid) with TRIS. 45S5-derived glass–ceramic scaffolds were tested under static–dynamic conditions for bone-like apatite formation using SBF + BES and SBF + TES solutions. The pH measurements, leachate analysis [atomic absorption spectrophotometry (AAS) (Ca²⁺, Si^IV), UltraViolet-Visible (UV-VIS) spectrophotometry (PO₄)^3?], and scaffold analysis [X-Ray powder diffraction analysis (XRD), X-ray fluorescent analysis (XRF), scanning electron microscopy (SEM)/energy dispersive spectroscopy (EDS), Brunauer.Emmett.Teller specific surface area analysis (BET)] all showed that both BES and TES quickly interacted with the tested material. Moreover, the kinetics of the glass–ceramic dissolution affected the crystallinity and morphology of the precipitated HAp. Based on our previous results and the present data, it appears that Good’s buffers are not suitable for the in vitro testing of bioactivity of highly bioreactive materials.     

In vitro and in vivo degradation of an injectable bone repair composite

In vitro and in vivo degradation behaviors of an injectable bone regeneration composite (IBRC) which comprised of nano-hydroxyapatite/collagen (nHAC) particles in alginate hydrogel carrier were investigated. In vitro degradation quantitative testing indicated that the alginate had a faster degradation rate in simulated body fluid (SBF) than in deionized water at 37 °C. Similarly, IBRC also had a higher degradation rate in SBF than in deionized water at 37 °C, which was evaluated by alginate molecular weight measurement, mechanical properties test and degradation kinetics evaluation. But molecular weight of alginate degraded slower in IBRC than that in aqueous solution. In vitro results showed that degradation medium SBF had influence on degradation of alginate molecules. In the in vivo degradation study, surprisingly, there was no obvious decreasing of molecular weight of alginate from 0 to 8 weeks. IBRC degraded mostly after 24 weeks implantation and was replaced by connective tissue. No fibrous capsule and acute inflammatory reaction were found during the observed 24 weeks after IBRC implantation. There is only a mild short-term inflammatory response in rat dorsum muscle. These results indicated that IBRC had a controllable degradability and biocompatibility. Therefore, IBRC may be a promising degradable material for bone repair and bone tissue engineering.     

Synthesis of PDMS-Based Porous Materials for Biomedical Applications

Polydimethylsiloxane (PDMS) and tetraethoxysilane (TEOS)-based porous organically modified silicates (ORMOSILs) for biomedical applications were synthesized through a sol-gel process, using sucrose particles as templates. These materials were characterized by ²⁹Si CP-MAS NMR spectroscopy, thin film X-ray diffraction, and scanning electron microscopy. Their bioactivity was evaluated using a simulated body fluid (SBF) of Kokubo recipe. These materials had a bimodal porous structure with pores of 300–500 m and 10–50 m in diameter. NMR showed that the silanol groups of the PDMS chain cross-linked to silica derived from the hydrolysis and condensation of TEOS. The samples containing Ca(II) exhibited apatite deposition on the pore walls within 3 days in SBF.