Octacalcium Phosphate (OCP) Crystals Induce Synovial Inflammation and Cartilage

Introduction: The presence of intra-articular basic calcium phosphate (BCP) crystals, including OCP, carbonated-apatite, hydroxyapatite and tricalcium phosphate crystals, is associated with severe osteoarthritis and destructive arthropathies such as Milwaukee shoulder. Although BCP crystals displayed, in vitro, mitogenic, anabolic and catabolic responses, their intra-articular effect was never assessed.Objective: To determine the effects of OCP crystals in joints in vivo.Methods: OCP crystals (200 ug in 20 ml PBS) were injected into the right knee joint (the contra-lateral knee joint injected with 20 ul of PBS serving as a control) of wild-type mice treated or not by the IL1R antagonist Anakinra or mice deficient for the inflammasome proteins ASC and NALP3. 4 days and 17 days after crystal injection, mice were sacrificed and knee joints dissected. Histological scoring for synovial inflammation and characterisation of macrophages, neutrophils and T cells were performed. Technetium (Tc) uptake was measured at 6h, 1 and 4 days after OCP injection. Cartilage degradation was evaluated by Safranin O staining and VDIPEN immunohistochemistry. Intra-articular localisation of injected OCP crystals was evidenced by Von Kossa staining.Results: The intra-articular localisation of injected OCP crystals was evidenced by Von Kossa staining performed on non-decalcified samples embedded in methyl-metacrylate. Injection of OCP crystals into knee joints led at day 4 to an inflammatory response with intense macrophage staining and also some neutrophil recruitment in the synovial membrane. This synovitis was not accompanied by increased Tc uptake into the knee joint, Tc uptake being similar in OCP crystal injected knee or control knee at all time points investigated (6h, 1 day, 4 days). The histological modifications persisted over 17 days, with an additional fibrosis evidenced at this later time-point. The OCP crystal-induced synovitis was totally IL-1a and IL-1 independent as shown by the absence of inhibitory effects of anakinra injected into wild-type mice. Accordingly, OCP crystal-induced synovitis was similar in ASC-/- and NALP3-/- mice as no alterations of inflammation were demonstrated between these mice groups. Concerning cartilage matrix degradation, OCP crystals induced a strong breakdown of proteoglycans 4 and 17 days after injection, as measured by loss of red staining from Safranin O-stained sections of cartilage surfaces. In addition, we also measured advanced cartilage matrix destruction mediated by MMPs, as evidenced by VDIPEN staining of cartilage. OCP-mediated cartilage degradation was similar in all experimental conditions tested (WT+Anakinra, or ASC or NALP3 deficient mice).Conclusion: These data indicate in vivo that the intra-articular presence of OCP crystals is associated with cartilage destruction along with synovial inflammation. This is an interesting and new model of destructive arthropathy related to BCP crystals which will allow to assess new therapies in this disease.

Revisiting spatial distribution and biochemical composition of calcium-containing crystals in human osteoarthritic articular cartilage.

INTRODUCTION: Calcium-containing (CaC) crystals, including basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP), are associated with destructive forms of osteoarthritis (OA). We assessed their distribution and biochemical and morphologic features in human knee OA cartilage. METHODS: We prospectively included 20 patients who underwent total knee replacement (TKR) for primary OA. CaC crystal characterization and identification involved Fourier-transform infra-red spectrometry and scanning electron microscopy of 8 to 10 cartilage zones of each knee, including medial and lateral femoral condyles and tibial plateaux and the intercondyle zone. Differential expression of genes involved in the mineralization process between cartilage with and without calcification was assessed in samples from 8 different patients by RT-PCR. Immunohistochemistry and histology studies were performed in 6 different patients. RESULTS: Mean (SEM) age and body mass index of patients at the time of TKR was 74.6 (1.7) years and 28.1 (1.6) kg/m², respectively. Preoperative X-rays showed joint calcifications (chondrocalcinosis) in 4 cases only. The medial femoro-tibial compartment was the most severely affected in all cases, and mean (SEM) Kellgren-Lawrence score was 3.8 (0.1). All 20 OA cartilages showed CaC crystals. The mineral content represented 7.7% (8.1%) of the cartilage weight. All patients showed BCP crystals, which were associated with CPP crystals for 8 joints. CaC crystals were present in all knee joint compartments and in a mean of 4.6 (1.7) of the 8 studied areas. Crystal content was similar between superficial and deep layers and between medial and femoral compartments. BCP samples showed spherical structures, typical of biological apatite, and CPP samples showed rod-shaped or cubic structures. The expression of several genes involved in mineralization, including human homolog of progressive ankylosis, plasma-cell-membrane glycoprotein 1 and tissue-nonspecific alkaline phosphatase, was upregulated in OA chondrocytes isolated from CaC crystal-containing cartilages. CONCLUSIONS: CaC crystal deposition is a widespread phenomenon in human OA articular cartilage involving the entire knee cartilage including macroscopically normal and less weight-bearing zones. Cartilage calcification is associated with altered expression of genes involved in the mineralisation process.

Vitamin D3-induced calcemic and phosphatemic responses in the freshwater mud eel Amphipnous cuchia maintained in different calcium environments

Vitamin D3 (100 ng 100 g body weight-l day-l) was administered intraperitoneally (ip) to the freshwater mud eel Amphipnous cuchia kept in artificial freshwater, calcium-free freshwater, low-calcium freshwater (0.2 mmol/l CaCl2) or calcium-rich freshwater (13.4 mmol/l CaCl2) for 15 days. Analyses of serum calcium and phosphate levels were performed on days 1, 3, 5, 10 and 15 after the beginning of the experiment (six eels from each group at each interval). Administration of vitamin D3 elevated the serum calcium [maximum elevation occurred at day 10 in artificial freshwater (vehicle: 10.55 ± 0.298, vitamin D: 13.90 ± 0.324), low-calcium freshwater (vehicle: 11.17 ± 0.220, vitamin D: 12.98 ± 0.297) and calcium-rich freshwater (vehicle: 11.24 ± 0.373, vitamin D: 14.24 ± 0.208) whereas it occurred at day 5 (vehicle: 8.42 ± 0.253, vitamin D: 11.07 ± 0.328) in calcium-free freshwater] and phosphate levels [maximum elevation at day 15 in artificial freshwater (vehicle: 4.39 ± 0.105, vitamin D: 5.37 ± 0.121), calcium-free freshwater (vehicle: 4.25 ± 0.193, vitamin D: 5.12 ± 0.181), low-calcium freshwater (vehicle: 3.93 ± 0.199, vitamin D: 5.28 ± 0.164) and calcium-rich freshwater (vehicle: 3.77 ± 0.125, vitamin D: 5.46 ± 0.151)] of the fish maintained in the above mentioned environmental media, but the responses were more pronounced in the fish kept in calcium-rich media

Experimental Studies on Non-Metallic Composite Bone Implants With a Special Reference to Staphylococcal Biofilm Infection

Non-metallic implants made of bioresorbable or biostable synthetic polymers are attractive options in many surgical procedures, ranging from bioresorbable suture anchors of arthroscopic surgery to reconstructive skull implants made of biostable fiber-reinforced composites. Among other benefits, non-metallic implants produce less interference in imaging. Bioresorbable polymer implants may be true multifunctional, serving as osteoconductive scaffolds and as matrices for simultaneous delivery of bone enhancement agents. As a major advantage for loading conditions, mechanical properties of biostable fiber-reinforced composites can be matched with those of the bone. Unsolved problems of these biomaterials are related to the risk of staphylococcal biofilm infections and to the low osteoconductivity of contemporary bioresorbable composite implants. This thesis was focused on the research and development of a multifunctional implant model with enhanced osteoconductivity and low susceptibility to infection. In addition, the experimental models for assessment, diagnostics and prophylaxis of biomaterial-related infections were established. The first experiment (Study I) established an in vitro method for simultaneous evaluation of calcium phosphate and biofilm formation on bisphenol-Aglycidyldimethacrylate and triethylenglycoldimethacrylate (BisGMA-TEGDMA) thermosets with different content of bioactive glass 45S5. The second experiment (Study II) showed no significant difference in osteointegration of nanostructured and microsized polylactide-co-glycolide/β-tricalcium phosphate (PLGA /β-TCP) composites in a minipig model. The third experiment (Study III) demonstrated that positron emission tomography (PET) imaging with the novel 68Ga labelled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) CD33 related sialic-acid immunoglobulin like lectins (Siglec-9) tracer was able to detect inflammatory response to S. epidermidis and S. aureus peri-implant infections in an intraosseous polytetrafluoroethylene catheter model. In the fourth experiment (Study IV), BisGMATEGDMA thermosets coated with lactose-modified chitosan (Chitlac) and silver nanoparticles exhibited antibacterial activity against S. aureus and P. aeruginosa strains in an in vitro biofilm model and showed in vivo biocompatibility in a minipig model. In the last experiment (Study V), a selective androgen modulator (SARM) released from a poly(lactide)-co-ε-caprolactone (PLCL) polymer matrix failed to produce a dose-dependent enhancement of peri-implant osteogenesis in a bone marrow ablation model.

The role of gypsum in the reactions of phosphate with soils

The aim of this study was to examine the mechanisms by which gypsum increases the sorption of fertilizer-P in soils of and and semi-arid regions. Either gypsum or soil (Usher from the UK; pH 7.8, 7% organic matter, 21% CaCO3: Yasouj from Iran; pH 8.2, 1.4% OM, 18% CaCO3: Ghanimeh from Saudi Arabia; pH 7.8, 1% OM, 26% CaCO3, 13% gypsum) was shaken for 24 It with KH2PO4 solutions in 10 mM CaCl2. With gypsum, grinding increased sorption by a factor of about 3, and increase in pH from 5.6 to 7.5 greatly increased sorption. Scanning electron micrographs (SEM) and EDX quantitative analysis showed that small crystals of gypsum disappeared and roughly spherical particles of dicalcium phosphate (DCPD) were formed. Analysis of equilibrium Solutions showed, using GEOCHEM, that octa-calcium phosphate (OCP) coated the DCPD. For the soils, sorption was in the order Ghanimeh > Yasouj > Usher. Removal of gypsum from Ghanimeh reduced sorption, with precipitated gypsum having a greater effect than gypsum mixed physically with the soil. Addition to Usher had no effect. SEM and EDX could not be used in the soil matrix, but solubility analysis again showed that solutions were close to equilibrium with OCP. Usher was unresponsive to added gypsum, presumably because of its small sorption capacity and high organic matter content. In Ghanimeh and Yasouj soils, gypsum increased sorption by being a source of readily available Ca2+ (c) 2005 Elsevier B.V. All rights reserved.

Calcium removal from milk by ion exchange

Calcium removal, using Duolite C433 ion exchange resin, was faster from permeate than from milk. Almost all calcium could be removed, suggesting a fairly rapid conversion from both soluble calcium phosphate and from micellar calcium to ionic calcium. Calcium reduction from milk is accompanied by an increase in pH, a reduction in ionic calcium, an increase in ethanol stability and an increase in the rennet coagulation time. There is a gradual increase in the average casein micelle size with calcium removal, up to a point where the micelle size increases dramatically. Zeta potential becomes more negative with calcium removal. At higher levels of calcium removal, the changes are not reversible, on reducing pH to its original value. For goat's milk, over the range 0-20% total calcium removal, relatively small reductions in total calcium gave rise to proportionally larger reductions in ionic calcium in a ratio of about 1:3.2.

The effect of BMP-2 on the osteoconductive properties of beta-tricalcium phosphate in rat calvaria defects

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Biological Response to Wollastonite Doped alpha-Tricalcium Phosphate Implants in Hard and Soft Tissues in Rats

The biological response following subcutaneous and bone implantation of beta-wollastonite(beta-W)-doped alpha-tricalcium phosphate bioceramics in rats was evaluated. Tested materials were: tricalciurn phosphate (TCP), consisting of a mixture of alpha- and beta-polymorphs; TCP doped with 5 wt. % of beta-W (TCP5W), composed of alpha-TCP as only crystalline phase; and TCP doped with 15 wt. % of beta-W (TCP15), containing crystalline alpha-TCP and beta-W. Cylinders of 2x1 mm were implanted in tibiae and backs of adult male Rattus norvegicus, Holtzman rats. After 7, 30 and 120 days, animals were sacrificed and the tissue blocks containing the implants were excised, fixed and processed for histological examination. TCP, TCP5W and TCP15W implants were biocompatible but neither bioactive nor biodegradable in rat subcutaneous tissue. They were not osteoinductive in connective tissue either. However, in rat bone tissue beta-W-doped alpha-TCP implants (TCP5W and TCP15W) were bioactive, biodegradable and osteoconductive. The rates of biodegradation and new bone formation observed for TCP5W and TCP15W implants in rat bone tissue were greater than for non-doped TCP.

Addition of Ca-compounds nanoparticles in melt-textured Bi : 2212

Superconducting BSCCO samples made by melt-texturing process were prepared with the addition of calcium zirconate and calcium silicate nanoparticles. Bi:2212 melt-textured composites prepared with I wt.% of either addition showed different behavior for the critical current density as a function of the applied field, indicating that for each additional compound the improvement can be associated to different enhancement mechanisms, such as the creation of pinning centers and the increase on the connectivity of the grains. The estimated pinning forces indicated higher values for the calcium compound containing samples. (c) 2007 Elsevier B.V. All rights reserved.

Factors determining rock phosphate solubilization by microorganisms isolated from soil

Forty two soil isolates (31 bacteria and 11 fungi) were studied for their ability to solubilize rock phosphate and calcium phosphate in culture medium. Eight bacteria and 8 fungi possessed solubilizing ability. Pseudomonas cepacia and Penicillium purpurogenum showed the highest activity. There was a correlation between final pH value and titratable acidity (r = - 0.29 to -0.87) and between titratable acidity and soluble phosphate (r = 0.22 to 0.99). Correlation values were functions of insoluble phosphate and of the group of microorganisms considered. A high correlation was observed between final pH and soluble phosphate only for the rock phosphates inoculated with the highest concentration of solubilizing bacteria (r = -0.73 to -0.98).

Biological response to wollastonite doped α-tricalcium phosphate implants in hard and soft tissues in rats

The biological response following subcutaneous and bone implantation of β-wollastonite(β-W)-doped α-tricalcium phosphate bioceramics in rats was evaluated. Tested materials were: tricalcium phosphate (TCP), consisting of a mixture of α- and β-polymorphs; TCP doped with 5 wt. % of β-W (TCP5W), composed of α-TCP as only crystalline phase; and TCP doped with 15 wt. % of β-W (TCP 15), containing crystalline α-TCP and β-W. Cylinders of 2×1 mm were implanted in tibiae and backs of adult male Rattus norvegicus, Holtzman rats. After 7, 30 and 120 days, animals were sacrificed and the tissue blocks containing the implants were excised, fixed and processed for histological examination. TCP, TCP5W and TCP15W implants were biocompatible but neither bioactive nor biodegradable in rat subcutaneous tissue. They were not osteoinductive in connective tissue either. However, in rat bone tissue β-W-doped α-TCP implants (TCP5W and TCP 15W) were bioactive, biodegradable and osteoconductive. The rates of biodegradation and new bone formation observed for TCP5W and TCP15W implants in rat bone tissue were greater than for non-doped TCP.

Effect of low-fluoride dentifrices supplemented with calcium glycerophosphate on enamel demineralization in situ

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vitro effect of calcium-containing prescription-strength fluoride toothpastes on bovine enamel erosion under hyposalivation-simulating conditions

Purpose: To evaluate the ability of calcium-containing prescription-strength fluoride (F) toothpastes in preventing enamel erosion under low salivary flow simulating conditions. Methods: Enamel and dentin bovine specimens were assigned to the following groups: A - placebo; B - 1,100 ppm F/NaF (Aquafresh Advanced); C - 5,000 ppm F/NaF (Prevident 5000 Booster); D - 5000 ppm F/NaF+calcium sodium phosphosilicate (Topex Renew); and E - 5,000 ppm F/NaF+tri-calcium phosphate (Clinpro 5000). Specimens were positioned in custom-made devices, creating a sealed chamber on the surface, connected to peristaltic pumps. Citric acid was injected into the chamber for 2 minutes, followed by artificial saliva (0.05 ml/minute), for 60 minutes, 4x/day, for 3 days. Aquafresh was also tested under normal salivary flow (0.5 ml/minute), as reference (Group F). Specimens were exposed to the toothpastes for 2 minutes, 2x/day. After cycling, surface loss (SL) and concentration of loosely- and firmly-bound F were determined. Data were analyzed by ANOVA. Results: Group A (placebo) presented highest surface loss (SL), while Group F had the lowest, for both substrates. For enamel, none of the dentifrices differed from Group B or among each other. For dentin, none of the dentifrices differed from Group B, but Group E showed greater protection than Group C. Group E presented the highest F concentrations for both substrates, only matched by Group D for firmly-bound fluoride on enamel. All fluoridated dentifrices tested reduced SL, with no additional benefit from higher F concentrations. Some formulations, especially Clinpro 5000, increased F availability on the dental substrates, but no further erosion protection was observed.

C-Phycocyanin protects SH-SY5Y cells from oxidative injury, rat retina from transient ischemia and rat brain mitochondria from Ca2+/phosphate-induced impairment

Oxidative stress and mitochondrial impairment are essential in the ischemic stroke cascade and eventually lead to tissue injury. C-Phycocyanin (C-PC) has previously been shown to have strong antioxidant and neuroprotective actions. In the present study, we assessed the effects of C-PC on oxidative injury induced by tert-butylhydroperoxide (t-BOOH) in SH-SY5Y neuronal cells, on transient ischemia in rat retinas, and in the calcium/phosphate-induced impairment of isolated rat brain mitochondria (RBM). In SH-SY5Y cells, t-BOOH induced a significant reduction of cell viability as assessed by an MTT assay, and the reduction was effectively prevented by treatment with C-PC in the low micromolar concentration range. Transient ischemia in rat retinas was induced by increasing the intraocular pressure to 120 mmHg for 45 min, which was followed by 15 min of reperfusion. This event resulted in a cell density reduction to lower than 50% in the inner nuclear layer (INL), which was significantly prevented by the intraocular pre-treatment with C-PC for 15 min. In the RBM exposed to 3 mM phosphate and/or 100 mu M Ca2+, C-PC prevented in the low micromolar concentration range, the mitochondrial permeability transition as assessed by mitochondrial swelling, the membrane potential dissipation, the increase of reactive oxygen species levels and the release of the pro-apoptotic cytochrome c. In addition, C-PC displayed a strong inhibitory effect against an electrochemically-generated Fenton reaction. Therefore, C-PC is a potential neuroprotective agent against ischemic stroke, resulting in reduced neuronal oxidative injury and the protection of mitochondria from impairment. (C) 2012 Elsevier Inc. All rights reserved.

Impact of experimental Nano-HAP pastes on bovine enamel and dentin submitted to a pH cycling model

This in vitro study evaluated the preventive potential of experimental pastes containing 10% and 20% hydroxyapatite nanoparticles (Nano-HAP), with or without fluoride, on dental demineralization. Bovine enamel (n=15) and root dentin (n=15) specimens were divided into 9 groups according to their surface hardness: control (without treatment), 20 Nanop paste (20% HAP), 20 Nanop paste plus (20% HAP + 0.2% NaF), 10 Nanop paste (10% HAP), 10 Nanop paste plus (10% HAP + 0.2% NaF), placebo paste (without fluoride and HAP), fluoride paste (0.2% NaF), MI paste (CPP-ACP, casein phosphopeptide-amorphous calcium phosphate), and MI paste plus (CPP-ACP + 0.2% NaF). Both MI pastes were included as commercial control products containing calcium phosphate. The specimens were treated with the pastes twice a day (1 min), before and after demineralization. The specimens were subjected to a pH-cycling model (demineralization–6-8 h/ remineralization-16-18 h a day) for 7 days. The dental subsurface demineralization was analyzed using cross-sectional hardness (kgf/mm 2 , depth 10-220 µm). Data were tested using repeated-measures two-way ANOVA and Bonferroni's test (p<0.05). The only treatment able to reduce the loss of enamel and dentin subsurface hardness was fluoride paste (0.2% NaF), which differed significantly from the control at 30- and 50-µm depth (p<0.0001). The other treatments were not different from each other or compared with the control. The experimental Nanop pastes, regardless of the addition of fluoride, were unable to reduce dental demineralization in vitro.