Polysaccharide surface engineering of poly(D, L-lactic acid) via electrostatic self-assembly technique and its effects on osteoblast growth behaviours

The objective of this study was to surface modify the poly (D, L-lactic acid) (PDLLA) films and assess the effects of the modified surfaces on the functions of osteoblasts cultured in vitro. A layer-by-layer (LBL) self assembly technique, was used leading to the formation of multilayers on the PDLLA film surfaces. Chitosan (Chi) and poly (styrene sulfonate, sodium salt) (PSS) were utilized as polycation and polyanion in this study, respectively. The layer structure was investigated by using X-ray photoelectron spectroscopy (XPS) and water contact angle measurement, respectively. XPS analysis displayed the presence of chitosan on PDLLA surface. A full coverage of coating with PSS/Chi layers was achieved on the PDLLA surface only after the deposition layers of PEI/(PSS/Chi)₂. These results showed that PDLLA films could be modified with PSS/Chi pairs which may affect the biocompatibility of the modified PDLLA films. To confirm this hypothesis, cell proliferation, cell viability as well as alkaline phosphtase activity of osteoblasts on layer-by-layer modified PDLLA films as well as control samples were investigated in vitro. The proliferation of osteoblasts on modified PDLLA films was found to be greater than that on control (p < 0.05 and p < 0.01) after 1, 4 and 7 days culture, respectively. Cell viability measurement showed that the PSS/Chi modified PDLLA films have higher cell viability (p < 0.01) than control. Osteoblast differentiation function (ALP) on LBL-modified PDLLA film was found significantly higher (p < 0.01) than that of virgin PDLLA films. These data suggests that PSS/Chi pair was successfully employed to surface modify PDLLA film via a layer-by-layer technique, and enhanced its cell biocompatibility.     

Effect of nanotubular-micro-roughened titanium surface on cell response in vitro and osseointegration in vivo

This study was to evaluate wettability, cell response, and osseointegration of nanotubular titanium (Ti) surface by anodic oxidation. Commercially pure Ti discs were treated by polishing, sandblasting, and anodizing. These surfaces were characterized by scanning electron microscopy and contact angle measurement. MC3T3-E1 osteoblast cell was used to evaluate cell response in vitro. The cell morphology, cell viability, and alkaline phosphatase (ALP) specific activity were assessed. The Ti implants of 2.0 mm diameter and 5.0 mm long treated by anodizing and sandblasting/anodizing were inserted into the tibia of rats. After 3 weeks, the histology of the Ti–bone interface was examined. SEM observations showed that the anodizing and sandblasting/anodizing created the nanotubular surface and graded nanotubular-micro-roughened surfaces, respectively. The anodizing and sandblasting/anodizing significantly improved the hydrophilicity of Ti. The significant greatest cell spreading and ALP specific activity were observed on the graded nanotubular-micro-roughened surfaces treated by sandblasting/anodizing. The in vivo study shows that newly formed bone was intimately in contact with the nanotubular surfaces without adverse immune response. This study has suggested that the graded nanotubular-micro-roughened surface of Ti treated with sandblasting/anodizing is very promising in implantology due to improved hydrophilicity, favorable cell response, and excellent osseointegration.     

In vitro evaluation of osteoconductivity and cellular response of zirconia and alumina based ceramics

Developed ceria/yttria stabilized zirconia and ceria/yttria stabilized zirconia toughened alumina supported formation of apatite layer when immersed in simulated body fluid without any prior surface treatment. The formed mineral layer was confirmed as hydroxyapatite through X-ray diffraction patterns. The calcium/phosphate atomic ratio obtained from energy dispersive X-ray spectroscopy was found to be little less (Ca/P = 1.5) than that of pure hydroxyapatite (Ca/P = 1.7) which indicates the probability of mixed type calcium-phosphate compound formation. The achieved thickness of apatite layer was estimated through a surface profilometer and as high as ~ 17 μm thickness was found after 28 days of soaking. The biocompatibility of the developed materials was ensured through in vitro human osteoblast like cell (MG63) culture on ceramic discs. The morphology of attached cells was characterized through scanning electron microscopy and fluorescent microscopy which show multilayered interconnected cell growth within 8 days of culture period. Moreover, differentiation of MG63 cells was evaluated through MTT assay, total protein content and alkaline phosphatase activity.     

Effects of baicalin-modified poly(D,L-lactic acid) surface on the behavior of osteoblasts

In the present study, the functions of rat calvaria osteoblasts on baicalin-modified poly(D,L-lactic acid) (PDLLA) films were investigated in vitro. The surface characteristics of surfaces (both modified and control) were investigated by water contact angle measurement and electron spectroscopy for chemical analysis (ESCA). Cell morphologies on these surfaces were examined by scanning electron microscopy (SEM). Cell adhesion and proliferation were used to assess cell growth on the modified and control surfaces. The MTT assay was used to determine cell viability and alkaline phosphatase (ALP) activity was performed to evaluate differentiated cell function. Compared to control films, cell attachment of osteoblasts on baicalin-modified PDLLA film was significantly higher (P<0.05 and P<0.01) after 6 h and 8 h culture, and cell proliferation was also significantly greater (P<0.05 and P<0.01) at the end of 4th and 7th day, respectively. The MTT assay suggested that the cell viability of osteoblasts cultured on baicalin-modified PDLLA film was significantly higher (P<0.05) than that seeded on the control. Meanwhile, the ALP activity of osteoblasts cultured on modified films was also considerably enhanced (P<0.01) compared to that found on control. These results revealed that the biocompatibility PDLLA could be improved by surface modification with baicalin.     

Biocompatibility and bioactivity of PDLLA/TiO2 and PDLLA/TiO2/Bioglass® nanocomposites

Biocompatibility and bioactivity of polymer matrix composites containing titanium dioxide (TiO₂) nanoparticles were investigated. The solvent casting method was used to prepare poly (d,l-lactic acid) (PDLLA) films with 0 and 20 wt.% TiO₂ nanoparticles and with 20 wt.% TiO₂ mixed with 5 wt.% micrometre-sized (< 5 μm) Bioglass® particles. The samples were characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD) and energy Dispersive X-ray (EDX) analyses. A Zygo® light interferometer was used to examine the surface roughness of the samples. The bioactivity and the surface reactivity of the materials were determined by investigating the formation of hydroxyapatite (HA) on the surface of samples upon immersion in simulated body fluid (SBF) for up to 28 days. Heterogeneous distributed HA crystals were found on composite films containing TiO₂ after 21 days exposure to SBF. Cell cytotoxicity and viability were determined by using live/dead and MTS assay on osteoblast-like MG-63 cells. The PDLLA films containing different concentrations of TiO₂ and Bioglass® particulate inclusions showed no effect on cell viability in live/dead assay after incubation period of 7 days. All three groups of samples demonstrated significant increase in relative metabolic activity in MTS assay after 7 days incubation (while a slower proliferation rate was obtained for cells on the PDLLA film containing both TiO₂ and Bioglass® compared to the Thermanox® control). The bioactive behaviour of the nanocomposites may make them attractive materials for fabrication of tissue engineering scaffolds.     

Effect of grain size on mechanical,surface and biological properties of microwave sintered hydroxyapatite

Hydroxyapatite (HA) compacts having average grain sizes of 168 ± 0.086 nm, 1.48 ± 0.627 μm and 5.01 ± 1.02 μm are processed from synthesized HA powder by microwave sintering at varying sintering temperature for different times. Superior mechanical and biological properties are shown by nano-grain HA compacts as compared to their micron grained counterparts. Compressive strength, indentation hardness, and indentation fracture toughness are increased with the decrease in HA grain size. The highest surface energy and maximum wettability are exhibited by nano-grain HA. HA compacts are assessed for cell–material interaction by SEM, MTT and immunochemistry assays using human osteoblast cell line for 1, 5 and 11 days. MTT assays showed higher number of living cells and faster proliferation on nano-grain HA surface. Osteoblast cells on nano-grain HA surface expressed significantly higher amount of vinculin and alkaline phosphatase (ALP) protein markers for cell adhesion and differentiation respectively. This study shows the effect of grain size on physical, mechanical and in vitro biological properties of microwave sintered HA compacts.     

Attachment and proliferation of human osteoblast-like cells (MG-63) on laser-ablated titanium implant material

Demand is increasing for shortening the long (3–6 months) osseointegration period to rehabilitate patients' damaged chewing apparatus in as short a time as possible. For dental implants, as for biomaterials in general, the bio- and osseointegration processes can be controlled at molecular and cellular levels by modification of the implant surface. One of the most promising of such surface modifications is laser ablation, as demonstrated by our previous results [46]. Commercially pure (CP4) sand-blasted, acid-etched titanium disks (Denti® System Ltd., Hungary) were irradiated with a KrF excimer laser (248 nm, fluence 0.4 J/cm², FWHM 18 ns, 2000 pulses), or with a Nd:YAG laser (532 nm, 1.3 J/cm², 10 ns, 200 pulses) then examined by SEM, AFM, and XPS. In vitro attachment (24 h) and proliferation (72 h) of MG-63 osteoblast cells were investigated via dimethylthiazol-diphenyl tetrazolium bromide (MTT), alamarBlue (AB) assays alkaline phosphatase quantification (ALP) and SEM. SEM and AFM revealed significant changes in morphology and roughness. XPS confirmed the presence of TiO₂ on each sample; after Nd:YAG treatment a reduced state of Ti (Ti^3 +) was also observed. MTT, AB and ALP measurements detected an increase in the number of cells between the 24- and 72 hour observations; however, laser treatment did not affect cell attachment and proliferation significantly.     

Study on the genotoxicity of HA/ZrO2 composite particles in vitro

To evaluate the genotoxicity of the HA/ZrO₂ composite particles by using the micronucleus test (MNT) in vitro. HA/ZrO₂ composite particles prepared by sintering at high temperature and pressure, that used powder of HA and ZrO₂ of different proportions, were compared with pure HA particles and pure ZrO₂ particles. The effect of the composite particles on cell proliferation of rabbit mesenchymal stem cells, and its the genotoxicity to rabbit mesenchymal stem cells were detected by MNT method. The MTT test showed that both pure HA particles and composite particles which contained HA promoted cell proliferation of rabbit mesenchymal stem cells, while pure ZrO₂ particles did not, and there was a significant difference (P < 0.05). The MNT test showed no significant difference between the HA group and the negative control group (P > 0.05), but a significant difference between the HA group and the positive control group (P < 0.05). The difference between the ZrO₂ group and the negative control group was significant (P < 0.01), while the difference between the ZrO₂ group and the positive control group was insignificant (P > 0.05). The genotoxicity of the HA/ZrO₂ composite particle increased with a higher proportion of ZrO₂ and an increase in the concentration of the composite, and the 30 wt.% HA/70% ZrO₂ composite with 200 μg/mL concentration showed significant genotoxicity (P < 0.01).     

High-speed deposition of dense,dendritic and porous SiO2 films by Nd: YAG laser chemical vapor deposition

Dense, dendritic and porous SiO₂ films were prepared by laser chemical vapor deposition (LCVD) using a high-power continuous-wave mode Nd: YAG laser (206 W) and a TEOS (tetraethyl orthosilicate) precursor. The effects of laser power (P_L) and total chamber pressure (P_tot) on the microstructure and deposition rate (R_dep) were investigated. Amorphous SiO₂ films were obtained independent of P_L and P_tot. Flame formation was observed between the nozzle and the substrate at P_L > 160 W and P_tot > 15 kPa. At P_L = 206 W, dense, dendritic and porous SiO₂ films were obtained at P_tot < 20 kPa, P_tot = 23 kPa and P_tot > 25 kPa, respectively. The R_dep increased thousands of times under flame formation conditions, the highest R_dep being reached at 1200 μm h^?1, 22,000 μm h^?1 and 28,000 μm h^?1 for the dense, dendritic and porous SiO₂ films, respectively.     

Characterization of rhenium nitride films produced by reactive pulsed laser deposition

Rhenium nitride (ReN_x) films were grown on (100)-Si substrates by the reactive pulsed laser deposition (PLD) method using a high purity Re rod in an environment of molecular nitrogen. The resulting films are characterized by several techniques, which include in situ Auger electron spectroscopy, X-ray photoelectron spectroscopy and ex situ X-ray diffraction, scanning electron and atomic force microscopy. Additionally, the four-probe method is used to determine the sheet resistance of deposited layers. Results show that films with N/Re ratios (x) lower than 1.3 are very good conductors. In fact, the resistivity of ReN films for 0.2 < x < 1.3 is of the order of 5% of that of Re films, while at x = 1.3 there is an abrupt increment in resistivity, resulting in dielectric films for 1.3 < x < 1.35. These results differ from the prior understanding that in transition metals, resistivity should increase with nitrogen incorporation.     

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.     

Texture and magnetic properties of electrodeposited FePd films

FePd films were deposited on brass substrates through electrodeposition using an alkaline electrolyte, which were annealed at 500, 600, and 700 °C for 1 h to form the ordered γ₁-FePd fct phase. After annealing, the γ₁-FePd fct phase is observed at 500, 600 and 700 °C. FePd films show the normal direction close to <101>, <111> and <421> directions after annealing 500, 600 and 700 °C, respectively. After annealing 500, 600 and 700 °C the average area grain size is 0.28 μm, 0.37 μm and 1.22 μm, respectively. The maximum coercivity H_c of 309 Oe observed at 500 °C can be explained with both effects of the <001> easy axis and the small grain size.     

Improvement in the morphology of micro-arc oxidised titanium surfaces: A new process to increase osteoblast response

This study describes a method for combining sandblast-acid etching and micro-arc oxidation to optimise titanium implant surfaces, and examines the effects of these surfaces on osteoblast response. Titanium discs were grouped as: micro-arc oxidised (MAO), sandblast-acid etched and micro-arc oxidised (MAO-SA), micro-arc oxidised and heated (MAO-HT), and untreated smooth surface. The combination of sandblast-acid etching and micro-arc oxidation in the MAO-SA group created an average surface roughness of 2.02 ± 0.15 μm compared to the untreated machined surface of 0.31 ± 0.06 μm. Scanning electron microscopy observations of the surface structures showed that the irregularly ordered valleys created by sandblast-acid etching remained after micro-arc oxidation and that micropores had also formed. These microstructures provided a better place for osteoblasts to spread compared with the other surfaces. In addition, our results indicated that adherent osteoblasts expressed greater alkaline phosphatase (ALP) activity and osteocalcin (OC) production on MAO-SA surfaces compared with MAO, MAO-HT, and smooth surfaces. The overall results clearly indicate that combining sandblast-acid etching and micro-arc oxidation techniques improves the titanium surface morphology and increases the roughness, which provides an optimal surface for cell differentiation and osseointegration.     

Bacterial adhesion studies on titanium,titanium nitride and modified hydroxyapatite thin films

A qualitative study on adhesion of the oral bacteria Porphyromonas gingivalis on titanium (Ti), titanium nitride (TiN), fluorine modified hydroxyapatite (FHA) and zinc modified FHA (Zn-FHA) thin films is investigated. Ti and TiN thin films were deposited by DC magnetron sputtering and hydroxyapatite-based films were prepared by solgel method. The crystalline structure, optical characteristics, chemical composition and surface topography of the films were studied by XRD, optical transmission, XPS, EDAX and AFM measurements. The predominant crystallite orientation in the Ti and TiN films was along (002) and (111) of hcp and cubic structures, respectively. The Ti : O : N composition ratio in the surface of the Ti and TiN films was found to be 7 : 21 : 1 and 3 : 8 : 2, respectively. The atomic concentration ratio (Zn + Ca) / P in Zn-FHA film was found to be 1.74 whereby the Zn replaced 3.2% of Ca. The rough surface feature in modified HA films was clearly observed in the SEM images and the surface roughness (rms) of Ti and TiN films was 2.49 and 3.5 nm, respectively, as observed using AFM. The film samples were sterilized, treated in the bacteria culture medium, processed and analyzed using SEM. Surface roughness of the films was found to have least influence on the bacterial adhesion. More bacteria were observed on the TiN film with oxide nitride surface layer and less number of adhered bacteria was noticed on the Ti film with native surface oxide layer and on Zn-FHA film.     

In vitro and in vivo antimicrobial properties of silver-containing hydroxyapatite prepared via ultrasonic spray pyrolysis route

Hydroxyapatite (HAp), with its high biocompatibility and osteoconductivity, readily absorbs proteins, amino acids and other substances, which in turn favor the adsorption and colonization of bacteria. To prevent bacterial growth and biofilm formation on HAp discs, silver-containing (1–20 mol%) HAp (Ag-HAp) powders were synthesized using an ultrasonic spray pyrolysis (USSP) technique. The X-ray diffraction (XRD) peaks were very broad, indicating low crystallinity, and this induced the release of Ag⁺ ions from Ag-HAp powders. In addition, a gradual increase in Ca^2 + ion release was observed. These results suggest that dissolution of Ca^2 + ion in Ag-HAp triggered the release of Ag⁺ ions.The antimicrobial efficacy of Ag-HAp disc was tested against Staphylococcus aureus. Samples with Ag contents of more than 5 mol% were found to be highly effective against bacterial colonization and biofilm formation in vitro. In vivo antibacterial tests using bioluminescent strains also showed reductions in the viability of bacteria with Ag-HAp (5 mol%) discs. Biocompatibility tests using a modified Transwell® insert method showed that Ag-HAp (5 mol%) discs have negative effects on osteoblast proliferation. These results indicate that Ag-HAp (5 mol%) has effective antibacterial activity and good biocompatibility both in vitro and in vivo together with good biocompatibility, thus confirming its utility as a bactericidal material.     

Preparation and in vitro evaluation of mesoporous hydroxyapatite coated β-TCP porous scaffolds

A mesoporous hydroxyapatite (HA) coating was prepared on a β-tricalcium phosphate (β-TCP) porous scaffold by a sol-gel dip-coating method using the block copolymer Pluronic F127 (EO₁₀₆PO₇₀EO₁₀₆) as the template. For application as a bone graft, in vitro cell response and bone-related protein expression of mesoporous HA coated β-TCP scaffold were investigated, using the non-mesoporous HA coated scaffold as the control group, to evaluate the influence of the mesoporous structure on the biological properties of HA coating. It was found that the increased surface area of the mesoporous HA coating greatly affected the response of MC3T3-E1 osteoblasts and the expression of proteins. An enzyme-linked immunosorbent assay recorded a significantly higher expression of alkaline phosphatase (ALP) and bone sialoprotein (BSP) in the mesoporous group than those in the control group (*p < 0.05) after different incubation periods. The introduction of mesopores enhanced the expression of ALP and BSP in the cells grown on the mesoporous HA coatings, on the premise of maintaining the protein expression in a sequence to ensure the correct temporo-spatial expression in osteogenesis. These results indicated that the mesoporous HA coating would provide a good environment for cell growth, suggesting that it could be used as the coating material for the surface modification of the tissue engineering scaffolds.     

Synthesis and characterization of calcium phosphate/collagen biocomposites doped with Zn2+

Composites were developed using calcium phosphate (CaP)/collagen (COL) doped with Zn⁺² to attempt the materials association with adequate properties for biological applications in the recovery of the bone tissue by trauma or pathogenies. Hydroxyapatite (HAP) and hydroxyapatite-βtricalcium phosphate (HAPβTCP) were synthesized and doped with zinc nitrate. High purity grade type I collagen was extracted and purified from bovine pericardium. CaP doped and undoped with Zn⁺² were produced with COL and the composites were developed using a simple mixture process. All samples were characterized by Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR) and X-ray diffraction analysis (XRD. In addition, biocompatibility and cell viability were assessed by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) using osteoblast cell culture. The results have indicated that both morphological and structural features and chemical composition of the composites were very similar to their precursors, collagen and calcium phosphate components. Also, the biocomposites presented a homogeneous aspect with the calcium phosphate particles aggregated to the collagen fibers. The biological evaluation of the composites in vitro showed cellular viability, presenting proliferation of the osteoblasts compared to the control cells (P < 0.05). The composites showed appropriate physical and biological properties creating more biologically active scaffolds that may support bone growth. Therefore, the novel developed biocomposites have high potential to be used for rebuilding small lesions in bone tissue engineering.     

Osteoblast behavior on TiO2 microgrooves prepared by soft-lithography and sol–gel methods

This study focused on the effects of microgrooved TiO₂ surfaces on osteoblast behavior. Microgrooved TiO₂ surfaces with different widths (12 μm and 40 μm) and flat surfaces were fabricated on glass substrates based on the combination of a sol–gel technique and soft-lithography. Osteoblasts (MC3T3-E1) were cultured on the as-prepared microgrooved and flat TiO₂ surfaces. Optical microscopy and scanning electron microscopy were used to analyze the adherent cell behavior by examining the cell morphology. Orientation angle analysis indicated that the cells tended to align along the microgrooves. This tendency was stronger on the microgrooves with smaller widths and became weak with increasing width. Alamar Blue assay indicated that the microgrooves restricted cell proliferation and the alkaline phosphatase assay revealed that the microgrooves limited the differentiation rate. This restriction increased with decreasing microgroove width. The surface energy of the TiO₂ surfaces was size-dependent and followed the order γ _12 μm < γ _40 μm < γ _{flat surfaces}. Osteoblast proliferation and differentiation on the surface with high surface energy exhibited high proliferation and differentiation rates. These results indicated that surface energy appeared to be a dominant factor for cell activity. Thus, surface energy would be a valuable index for the cell compatibility of a micropatterned surface.     

Bioactivity and mineralization of hydroxyapatite with bioglass as sintering aid and bioceramics with Na3Ca6(PO4)5 and Ca5(PO4)2SiO4 in a silicate matrix

Hydroxyapatite and Bioglass^®-45S5 were sintered together creating new ceramic compositions that yielded increased apatite deposition and osteoblast differentiation and proliferation in vitro compared to hydroxyapatite. The sintered products characterized by X-ray diffraction, revealed hydroxyapatite as the main phase when small quantities (1, 2.5 and 5 wt.%) of bioglass was added. Bioglass behaved as a sintering aid with β-TCP (Ca₃(PO₄)₂) being the minor phase. The amount of β-TCP increased with the amount of bioglass added. In compositions with larger additions of bioglass (10 and 25 wt.%), new phases with compositions of calcium phosphate silicate (Ca₅(PO₄)₂SiO₄) and sodium calcium phosphate (Na₃Ca₆(PO₄)₅) were formed respectively within amorphous silicate matrices. In vitro cell culture studies of the ceramic compositions were examined using bone marrow stromal cell (BMSC). Cell proliferation and differentiation of bone marrow stromal cells into osteoblasts were determined by Pico Green DNA assays and alkaline phosphatase (ALP) activity, respectively. All hydroxyapatite–bioglass co-sintered ceramics exhibited larger cell proliferation compared to pure hydroxyapatite samples. After 6 days in cell culture, the ceramic with Ca₅(PO₄)₃SiO₄ in a silicate matrix formed by reacting hydroxyapatite with 10 wt.% bioglass exhibited the maximum proliferation of the BMSC's. The ALP activity was found to be largest in the ceramic with Na₃Ca₆(PO₄)₅ embedded in a silicate matrix synthesized by reacting hydroxyapatite with 25 wt.% bioglass.