Strontium-releasing fluorapatite glass-ceramic scaffolds: Structural characterization and in vivo performance.

There is increasing interest in biodegradable ceramic scaffolds for bone tissue engineering capable of in situ delivery of ionic species favoring bone formation. Strontium has been shown to be osteogenic, but strontium-containing drugs such as strontium ranelate, used in Europe for the treatment of osteoporosis, are now restricted due to clinical evidence of systemic effects. By doping fluorapatite-based glasses with strontium, we developed ceramic scaffolds with fully interconnected macroporosity and cell size similar to that of cancellous bone, that are also capable of releasing strontium. The crystallization behavior, investigated by XRD and SEM, revealed the formation of akermanite and fluorapatite at the surface of strontium-free glass-ceramic scaffolds, and strontium-substituted fluorapatite at the surface of the strontium-doped scaffolds. At 8 weeks after implantation in a rat calvarial critical size defect, scaffolds doped with the highest amount of strontium led to the highest mineral apposition rate. A significantly higher amount of newly-formed bone was found with the strontium-free glass-ceramic scaffold, and possibly linked to the presence of akermanite at the scaffold surface. We demonstrate by energy dispersive XRF analyses of skull sections that strontium was present in newly formed bone with the strontium-doped scaffolds, while a significant amount of fluorine was incorporated in newly formed bone, regardless of composition or crystallization state. STATEMENT OF SIGNIFICANCE: The present work demonstrates the in vivo action of strontium-containing glass-ceramic scaffolds. These bone graft substitutes are targeted at non load-bearing bone defects. Results show that strontium is successfully incorporated in newly formed bone. This is associated with a significantly higher Mineral Apposition Rate. The benefits of in situ release of strontium are demonstrated. The broader scientific impact of this works builds on the concept of resorbable ceramic scaffolds as reservoirs of ionic species capable of enhancing bone regeneration.

Influences of doping mesoporous magnesium silicate on water absorption, drug release, degradability, apatite-mineralization and primary cells responses to calcium sulfate based bone cements.

In this study, composite cements containing mesoporous magnesium silicate (m-MS) and calcium sulfate (CS) were fabricated. The results revealed that the setting time of the m-MS/CS composite cements (m-MSC) slightly prolonged with the increase of m-MS content while the compressive strength suffered a little loss. The doping of m-MS improved the water absorption, drug release (vancomycin) and degradability of the m-MSC in Tris-HCl solution (pH=7.4). In addition, addition of m-MS facilitated the apatite-mineralization of m-MSC in simulated body fluid (SBF), indicating good bioactivity. For cell cultural experiments, the results revealed that the m-MSC promoted the cells adhesion and proliferation, and improved the alkaline phosphatase (ALP) activity of MC3T3-E1 cells, revealing good cytocompatibility. It could be suggested that the m-MSC might be promising cements biomaterials for bone tissue regeneration.

Effect of Mg content on the bioactivity and biocompatibility of Mg-substituted fluorapatite nanopowders fabricated via mechanical activation.

The aim of this work was preparation, characterization, bioactivity and biocompatibility evaluation of Mg-substituted fluorapatite (Mg-FA) nanopowders. Mg-FA nanopowders with a chemical composition of Ca10-xMgx(PO4)6F2, with x=0, 0.5, 1, and 2 were prepared by mechanically activated method. The in vitro bioactivity was investigated by soaking the powders in simulated body fluid (SBF) for various time periods to analyze the nucleation and growth of bone-like apatite on the surface of the samples. Cell viability and cell attachment were studied by MTT assay. Results indicated that the bioactivity of all of samples with different Mg content was improved compared with the pure FA. However, the mechanism of bioactivity is different and depends on the amount of Mg substitution. Finally, cell culture suggested that the addition of Mg(2+) has no adverse effect and Mg-FA samples have good biocompatibility. The Mg-FA material shows potential in satisfying the requirements of biomedical applications.

Injectability, microstructure and release properties of sodium fusidate-loaded apatitic cement as a local drug-delivery system.

The introduction of an antibiotic, sodium fusidate (SF), into the liquid phase of calcium carbonate-calcium phosphate (CaCO3-CaP) bone cement was evaluated, considering the effect of the liquid to powder ratio (L/P) on the composition and microstructure of the set cement and the injectability of the paste. In all cases, we obtained set cements composed mainly of biomimetic carbonated apatite analogous to bone mineral. With this study, we evi-denced a synergistic effect of the L/P ratio and SF presence on the injectability (i.e., the filter-pressing pheno-menon was suppressed) and the setting time of the SF-loaded cement paste compared to reference cement (without SF). In addition, the in vitro study of SF release, according to the European Pharmacopoeia recommendations, showed that, regardless of the L/P ratio, the cement allowed a sustained release of the antibiotic over 1month in sodium chloride isotonic solution at 37°C and pH7.4; this release is discussed considering the microstructure characteristics of SF-loaded cements (i.e., porosity, pore-size distribution) before and after the release test. Finally, modelling antibiotic release kinetics with several models indicated that the SF release was controlled by a diffusion mechanism.

Innovative delivery of siRNA to solid tumors by super carbonate apatite.

RNA interference (RNAi) technology is currently being tested in clinical trials for a limited number of diseases. However, systemic delivery of small interfering RNA (siRNA) to solid tumors has not yet been achieved in clinics. Here, we introduce an in vivo pH-sensitive delivery system for siRNA using super carbonate apatite (sCA) nanoparticles, which is the smallest class of nanocarrier. These carriers consist simply of inorganic ions and accumulate specifically in tumors, yet they cause no serious adverse events in mice and monkeys. Intravenously administered sCA-siRNA abundantly accumulated in the cytoplasm of tumor cells at 4 h, indicating quick achievement of endosomal escape. sCA-survivin-siRNA induced apoptosis in HT29 tumors and significantly inhibited in vivo tumor growth of HCT116, to a greater extent than two other in vivo delivery reagents. With innovative in vivo delivery efficiency, sCA could be a useful nanoparticle for the therapy of solid tumors.

Bone mineral crystallisation kinetics.

The kinetics of bone apatite crystallisation are examined using a novel approach to obtain quantitative, direction dependence features such as growth rate and activation energy. X-ray diffraction was employed for analysis of bovine, porcine and 'anorganic' bone specimens. Apatite coherence length was utilised as the independent variable within a Johnson-Mehl-Avrami (JMA) model. A direction averaged crystallisation activation energy of 183 ± 8 kJ mol(-1) was observed for the three bone groups. The Johnson-Mehl-Avrami 'n' exponent decreased with increasing temperature for all bone groups, indicating that apatite crystallisation changes to a diffusion limited process at higher temperatures. The results revealed little evidence to support any organic component 'protective' effect, and, on the contrary indicated that the organic matrix promotes apatite crystallisation.

Reversing multidrug resistance in breast cancer cells by silencing ABC transporter genes with nanoparticle-facilitated delivery of target siRNAs.

BACKGROUND: Multidrug resistance, a major impediment to successful cancer chemotherapy, is the result of overexpression of ATP-binding cassette (ABC) transporters extruding internalized drugs. Silencing of ABC transporter gene expression with small interfering RNA (siRNA) could be an attractive approach to overcome multidrug resistance of cancer, although delivery of siRNA remains a major hurdle to fully exploit the potential of siRNA-based therapeutics. Recently, we have developed pH-sensitive carbonate apatite nanoparticles to efficiently carry and transport siRNA across the cell membrane, enabling knockdown of the cyclin B1 gene and consequential induction of apoptosis in synergy with anti-cancer drugs. METHODS AND RESULTS: We report that carbonate apatite-mediated delivery of the siRNAs targeting ABCG2 and ABCB1 gene transcripts in human breast cancer cells which constitutively express both of the transporter genes dose-dependently enhanced chemosensitivity to doxorubicin, paclitaxel and cisplatin, the traditionally used chemotherapeutic agents. Moreover, codelivery of two specific siRNAs targeting ABCB1 and ABCG2 transcripts resulted in a more robust increase of chemosensitivity in the cancer cells, indicating the reversal of ABC transporter-mediated multidrug resistance. CONCLUSION: The delivery concept of multiple siRNAs against ABC transporter genes is highly promising for preclinical and clinical investigation in reversing the multidrug resistance phenotype of breast cancer.

Effect of acidulated phosphate fluoride gel application time on enamel demineralization of deciduous and permanent teeth.

Although the effect of acidulated phosphate fluoride gel (APF gel) on caries reduction in permanent teeth is based on evidence, the relevance of the clinical application time is still under debate. Also, the effect of 4- versus 1-min application has not been evaluated in deciduous enamel. In a blind, crossover, in situ study of 14 days, 16 adult volunteers wore palatal appliances containing slabs of human permanent and deciduous enamel. At the beginning of each phase, the slabs were submitted to one of the following treatments: no APF application (negative control); APF gel (1.23% F) application for 1 or 4 min. Biofilm accumulation on the slab surface was allowed and the slabs were subjected eight times a day to 20% sucrose, simulating a high cariogenic challenge condition. On the 15th day of each phase, fluoride retained as CaF(2) and fluorapatite (FAp) was determined on the enamel of the slabs and demineralization was assessed by cross-sectional microhardness. Fluoride as CaF(2) and FAp, formed by APF gel application on the enamel slabs not subjected to the cariogenic challenge, was also determined. APF gel reduced demineralization in both enamel types (p < 0.05), but the difference between 1 and 4 min was not statistically significant (p > 0.05). CaF(2) and FAp formed and retained on deciduous and permanent enamel was significantly higher in APF gel groups (p < 0.05), but no significant difference was found between 1 and 4 min (p > 0.05). The findings suggest that 1 min of APF gel application provides a similar effect on inhibition of demineralization as 4 min, for both permanent and deciduous enamel.

Effect of fluoride, lesion baseline severity and mineral distribution on lesion progression.

The present study investigated the effects of fluoride (F) concentration, lesion baseline severity (ΔZ(base)) and mineral distribution on lesion progression. Artificial caries lesions were created using three protocols [methylcellulose acid gel (MeC), hydroxyethylcellulose acid gel (HEC), carboxymethylcellulose acid solution (CMC)] and with low and high ΔZ(base) groups by varying demineralization times within protocols. Subsequently, lesions were immersed in a demineralizing solution for 24 h in the presence of 0, 1, 2 or 5 ppm F. Changes in mineral distribution characteristics of caries lesions were studied using transverse microradiography. At baseline, the protocols yielded lesions with three distinctly different mineral distributions. Secondary demineralization revealed differences in F response between and within lesion types. In general, lowΔZ lesions were more responsive to F than highΔZ lesions. LowΔZ MeC lesions showed the greatest range of response among all lesions, whereas highΔZ HEC lesions were almost unaffected by F. Laminations were observed in the presence of F in all but highΔZ HEC and CMC lesions. Changes in mineral distribution effected by F were most pronounced in MeC lesions, with remineralization/mineral redeposition in the original lesion body at the expense of sound enamel beyond the original lesion in a dose-response manner. Both ΔZ(base) and lesion mineral distribution directly impact the F response and the extent of secondary demineralization of caries lesions. Further studies - in situ and on natural white spot lesions - are required to better mimic in vivo caries under laboratory conditions.

Hybrid structure in PCL-HAp scaffold resulting from biomimetic apatite growth.

Polymer-ceramic composites are favourite candidates when aiming to replace bone tissue. We present here scaffolds made of polycaprolactone-hydroxyapatite (PCL-HAp) composites, and investigate in vitro mineralisation of the scaffolds in SBF after or without a nucleation treatment. In vitro bioactivity is enhanced by HAp incorporation as well as by nucleation treatment, as demonstrated by simulated body fluid (SBF) mineralization. Surprisingly, we obtained a hybrid interconnected organic-inorganic structure, as a result of micropore invasion by biomimetic apatite, which results in a mechanical strengthening of the material after two weeks of immersion in SBF92. The presented scaffolds, due to their multiple qualities, are expected to be valuable supports for bone tissue engineering.

Comparison of carbonate apatite and beta-tricalcium phosphate (resorbable calcium phosphates) implanted subcutaneously into the back of rats.

Bioresorption and biocompatibility of carbonate apatites, both sintered and non-sintered (S-CAP and N-CAP), and of sintered beta-tricalcium phosphate (beta-TCP) were compared by implanting particles of these materials into the back of adult rats. Bioresorption--when evaluated non-destructively with non-decalcified tissues using microfocus X-ray tomography--was essentially the same for N-CAP and beta-TCP, while S-CAP exhibited statistically lower bioresorption at 2, 4, and 12 weeks postoperatively. Biocompatibility--when evaluated by ED1 immunostaining--was in the order of beta-TCP > N-CAP > S-CAP. The intensity of ED1 immunostaining decreased with time, but persisted longer in beta-TCP than in S-CAP and N-CAP, indicating that beta-TCP produced the strongest and most enduring stimulation of macrophages. Although no statistical differences were found in tartrate-resistant acid phosphatase (TRAP) staining among the materials at each implantation period, the degree of TRAP staining for S-CAP was statistically greater at 12 weeks than at 2 and 4 weeks, indicating that osteoclast-like cells were in part responsible for the resorption of the carbonate apatite.

Interactions of cisplatin with calcium phosphate nanoparticles: in vitro controlled adsorption and release.

A new system for the local delivery of chemotherapy to malignant solid tumors has been developed based on calcium phosphate (CaP) nanoparticles. The adsorption of the anti-neoplastic drug cis-diamminedichloroplatinum (cisplatin) was characterized on three types of apatitic CaP (poorly and well crystallized hydroxyapatite, and carbonated apatite). Adsorption isotherms obtained in chloride-free phosphate solutions at pH = 7.4 (24 and 37 degrees C) indicate that cisplatin adsorption increases with temperature and increases with decreasing crystallinity. Release studies in phosphate buffer saline (containing the chloride ion essential for release) showed that while the cumulative amount of released drug was the same for all apatites at 20 days (approximately 70% of the total bound), the least crystalline material released the drug more slowly. The drug release rate increased slightly with temperature. Cytotoxicity testing was conducted in a K8 clonal murine osteosarcoma cell line to verify that drug activity was retained after adsorption onto the apatite crystals. K8 cells were plated onto dried films of the apatite/cisplatin conjugates and after 24 h, viability was measured with tritiated uridine. The apatite/cisplatin formulations exhibited cytotoxic effects with a dose dependent diminishment of cell viability.

[Investigation of biocompatibility of the apatite cement]. / Badanie biozgodnosci cementu apatytowego.

The results of experimental studies of biocompatibility of the apatite cement, implanted in the muscular tissue of rats and bone tissue of rabbits were presented in this study. Investigations of the local reactions of the muscular tissue were carried out on 18 rats of Wistar type by implanting samples from the apatite cement in the dorsal muscles. Macroscopic and microscopic investigations were carried out 7, 14, 30, 180, 270 days after the implantation. Investigations of local reactions of bone tissue were carried out on 12 rabbits of New Zealand breed. By implanting the tested material into the femoral bone in the region of trochanter. Sections of the animals were made 1, 3, 6, and 9 months after the surgery. In the early period active inflammatory process was seen in the muscles in the direct vicinity of the implant and then it disappeared leading to formation of a thin fibrous connective tissue capsule. Histological investigations made of bone tissue later showed, formation of young tissue with small focuses of fibrous connective tissue. In the carried out investigations it was shown that the apatite cement is characterized with a high degree of biocompatibility and osteoinductive activity.

Mechanical properties and biochemical activity of remineralizing resin-based Ca-PO4 cements.

OBJECTIVE: This study examined strength and bioactive (remineralizing) properties of a powder/liquid formulation (Cement I) and a more practical two-paste formulation (Cement II) of a fluoride-releasing resin-based Ca-PO(4) cement. METHODS: For the remineralization potential, the dissolution of calcium, total ionic phosphate and fluoride from set cement specimens were determined in buffered saline and saliva-like solution (SLS). Artificial caries lesions were produced on coronal dentin of extracted human molars. On each tooth, two defined areas were coated, one with a composite resin (calcium-free control), the other with either Cement I or II. After incubation in SLS, the mineral loss under the cement was analyzed by digitized microradiography and compared to that under the control. RESULTS: The diametral tensile strength of Cement II of approximately 30 MPa after 24 h and 23 MPa after 4 weeks was 2 to 3 times higher than that of Cement I (ANOVA, t-test, p<0.05). Calculations of the ion-activity products and Gibb's free energy from solution ion concentrations indicated a significant potential for the formation of fluor- and/or hydroxyapatite. Concurrently, both cements caused increases of 47% (Cement I) and 38% (Cement II) in the lesion mineral content over that underneath the corresponding controls. SIGNIFICANCE: These in vitro results suggest that the stronger Cement II could serve as a restoration-supporting lining material and could remineralize dentin in areas where complete removal of carious tissue is contra-indicated.

Formation of apatite layers on modified canasite glass-ceramics in simulated body fluid.

Canasite glass-ceramics were modified by either increasing the concentration of calcium in the glass, or by the addition of P2O5. Samples of these novel materials were placed in simulated body fluid (SBF), along with a control material (commercial canasite), for periods ranging from 12 h to 28 days. After immersion, surface analysis was performed using thin film X-ray diffraction, Fourier transform infrared reflection spectroscopy, and scanning electron microscopy equipped with energy dispersive X-ray detectors. The concentrations of sodium, potassium, calcium, silicon, and phosphorus in the SBF solution were measured using inductively coupled plasma emission spectroscopy. No apatite was detected on the surface of commercial canasite, even after 28 days of immersion in SBF. A crystalline apatite layer was formed on the surface of a P2O5-containing canasite after 5 days, and after 3 days for calcium-enriched canasite. Ion release data suggested that the mechanism for apatite deposition was different for P2O5 and non-P2O5-containing glass-ceramics.

Synthesis of functionally graded CO3 apatite as surface biodegradable crystals.

Apatite, which will have a degradable surface and a relatively stable core, was synthesized at 80+/-1 degrees C and pH 7.4+/-0.2 using a specially generated gradient carbonate supply system. X-ray diffraction analysis showed that the (300) reflection peak shifted to the higher angle direction due to the substitution of CO3(2-) ions into the PO4(3-) positions and was broader than that of homogeneous hydroxyapatite. Scanning electron microscopy indicated that the apatite was composed of small coagulated ellipsoidal crystals differing from needle-like crystals with hexagonal section of hydroxyapatite. Electron spectroscopy for chemical analysis showed a negative gradient of carbonate concentration with depth in the crystals. The apparent solubility in 0.5 moll(-1) acetate buffer solution (37 degrees C, pH 4.0) was much higher than that of homogeneous hydroxyapatite. The residual sample showed slim needle-like crystals. These results suggested that graded carbonate-containing apatite, namely CO3 apatite with solubility gradient in its crystal structure, was formed by this process and may be useful from the view point of surface biodegradable materials as bone representatives.

In vitro influence of apatite-granule-specific area on human growth hormone loading and release.

Although calcium phosphate biomaterials often are used as drug delivery systems (DDS) at bone sites, the conditions affecting the loading of the therapeutic agent (TA) have not been well documented. A human growth hormone (hGH) adsorption method was used in this study to investigate the influence of the formulated apatite (AP)-specific area on loading and release. AP powders were formulated with a 200-500 microm granulometry and various specific areas. Two milligrams of hGH in solution were deposited for 24 h at 37 degrees C on 100 mg of AP with different specific areas. The amount of hGH loaded was determined by immunoradiometric assay (IRMA) and eluted stain bioassay (ESTA) using Nb2 lymphoma rat cells. Although loading was not greatly influenced by a specific area between 3 and 25 m2/g, dependency was noted for higher specific areas. Human GH release was measured by IRMA and ESTA over a 33-day period, with half-time release between 25 and 79 h. Comparison of IRMA and ESTA measurements for the hGH amounts loaded showed that hGH biologic activity was conserved. Results indicate that it is feasible to control the quantity of TA loading on AP by modifying specific areas for in vivo applications.

The effect of fluorhydroxyapatite-derived fluoride on acid production by streptococci.

The effect of fluoride derived from fluorhydroxyapatite (FHAp) minerals on bacterial glycolysis under aerobic and strictly anaerobic conditions was studied to validate the claims that this mineral could be used as a reservoir of fluoride in plaque. To isolate the direct effect of fluoride on bacterial glycolysis from that of an indirect pH-buffering effect of hydroxyl or phosphate ions which are also dissolved from the mineral, we equalized the pH-fall time course of reactions by manually adding KOH or HCl. This ensured that pH effects on glycolysis were minimized. Under controlled pH-fall and strictly anaerobic conditions, fluoride derived from the dissolution of FHAp containing more than 30,100 ppm fluoride (i.e., when the substitution of OH by F in the mineral was greater than 80%) had a direct inhibitory effect on lactic acid production in Streptococcus mutans. Under free pH-fall and strictly anaerobic conditions, increasing amounts of fluoride in FHAp (starting as low as 2000 ppm fluoride), appeared to have a pronounced indirect inhibitory effect on lactic acid production. This was probably mediated through a reducing pH buffer effect of the mineral. Even in the presence of high-fluoride FHAp, only 0.01 to 0.025 mmol/L fluoride was found in the reaction mixtures, a probable result of non-stoichiometric dissolution of FHAp. In spite of such low levels of fluoride, marked inhibitory effects on bacterial glycolysis were demonstrated. The results of this study suggest that high-fluoride FHAp may serve as a reservoir of fluoride for the inhibition of anaerobic acid production by S. mutans.

Solid-state phosphorus-31 nuclear magnetic resonance differentiation of bone mineral and synthetic apatite used to fill bone defects.

Bioabsorption of synthetic apatite compounds used to promote bone healing and remodeling has been difficult to evaluate. In this study, solid-state phosphorus-31 nuclear magnetic resonance (NMR) has been used to characterize and quantitate bone mineral and a synthetic apatite in order to establish a model for bioabsorption studies. Pulverized solid samples of cortical rabbit bone and a synthetic fluoridated apatite were examined in vitro at variable degrees of hydration. A 9.4 T superconducting spectrometer was used to obtain 31P magic angle spinning NMR spectra and T1 relaxation times. Quantitation was attempted in mixed samples using T1 recovery data. Bone mineral and synthetic apatite could be distinguished by chemical shift and T1 relaxation time in variable hydration states, and were readily differentiated in mixtures by their T1 relaxation time. NMR estimates of relative proportions of components in mixed samples were accurate within 2% of evaluations based on weight. Solid-state 31P NMR therefore provides a suitable method for monitoring the bioabsorption of synthetic apatites.

Solution-mediated transformation of octacalcium phosphate (OCP) to apatite.

OCP crystals were hydrolyzed in solutions containing Ca2+, Mg2+, HPO4(2-), CO3(2-), F-, citrate or P2O7 ions. Products of hydrolysis were analyzed using scanning (SEM) and transmission (TEM) electron microscopy, infrared spectroscopy and x-ray diffraction. Results demonstrated that the OCP to Apatite (AP) transformation is influenced by: (1) types of ions in solution: inhibited by Mg2+, citrate or P2O7(4-); facilitated by F-, CO3(2-), HPO4(2-) or Ca2+ ions; (2) ionic concentrations; (3) solution pH; (4) OCP crystal size. SEM showed needle-like micro-crystals on the surfaces and ends of OCP macrocrystals. TEM showed side-to-side and end-to-end arrangements and presence of central defects in the apatite crystals. IR spectra showed the incorporation of CO3, or HPO4, the HPO4 incorporation being least from F-containing solutions. These results suggest that OCP to AP transformation occurred by the process of dissolution of OCP and subsequent precipitation of Ca-deficient apatites, incorporating CO3(2-), HPO4(2-) or F- present in solution. These results indicate that the observed stability of OCP in pathological calcifications may be due to the presence of Mg2+, citrate and/or P2O7(4-) and/or low levels of CO3(2-), F-, Ca2+, HPO4(2-) ions in the biological fluids.