Mineralization of calcium phosphate crystals in starch template inducing a brushite kidney stone biomimetic composite

Dicalcium phosphate dihydrate (brushite) and octacalcium phosphate (OCP) crystals are precursors of hydroxyapatite (HAp) for tooth enamel, dentine, and bones formation in living organisms. Here, we introduce a new method for biomimicking brushite and OCP in starch using single and double diffusion techniques. Brushite and OCP crystals were grown by precipitation in starch after gelation. The obtained materials were analyzed by infrared spectroscopy (IR), scanning electron microscopy (SEM), X-ray diffraction (XRD), and confocal laser scanning microscopy (CLSM). IR spectra demonstrate starch inclusion by peak shifts in the 2900–3500 cm–1 region. SEM showed two different morphologies: plate-shaped and needle-like crystals. Calcium phosphate/starch aggregates bear strong resemblance to prismatic brushite kidney stones. This may open up a clue to understand the mechanism of kidney stone formation.

In vitro characterization of a synthetic calcium phosphate bone graft on periodontal ligament cell and osteoblast behavior and its combination with an enamel matrix derivative.

OBJECTIVES Recent studies suggest that a combination of enamel matrix derivative (EMD) with grafting material may improve periodontal wound healing/regeneration. Newly developed calcium phosphate (CaP) ceramics have been demonstrated a viable synthetic replacement option for bone grafting filler materials. AIMS This study aims to test the ability for EMD to adsorb to the surface of CaP particles and to determine the effect of EMD on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. MATERIALS AND METHODS EMD was adsorbed onto CaP particles and analyzed for protein adsorption patterns via scanning electron microscopy and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using CellTiter 96 One Solution Cell Assay (MTS). Cell differentiation was analyzed using real-time PCR for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen1α1, and mineralization was assessed using alizarin red staining. RESULTS Analysis of cell attachment revealed significantly higher number of cells attached to EMD-adsorbed CaP particles when compared to control and blood-adsorbed samples. EMD also significantly increased cell proliferation at 3 and 5 days post-seeding. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers including collagen1α1, alkaline phosphatase, and osteocalcin in osteoblasts and PDL cells cultured on EMD-adsorbed CaP particles at various time points. CONCLUSION The present study suggests that the addition of EMD to CaP grafting particles may influence periodontal regeneration by stimulating PDL cell and osteoblast attachment, proliferation, and differentiation. Future in vivo and clinical studies are required to confirm these findings. CLINICAL RELEVANCE The combination of EMD and CaP may represent an option for regenerative periodontal therapy in advanced intrabony defects.

Poly(ethylene oxide)-b-poly(3-sulfopropyl methacrylate) block copolymers for calcium phosphate mineralization and biofilm inhibition.

Poly(ethylene oxide) (PEO) has long been used as an additive in toothpaste, partly because it reduces biofilm formation on teeth. It does not, however, reduce the formation of dental calculus or support the remineralization of dental enamel or dentine. The present article describes the synthesis of new block copolymers on the basis of PEO and poly(3-sulfopropyl methacrylate) blocks using atom transfer radical polymerization. The polymers have very large molecular weights (over 10(6) g/mol) and are highly water-soluble. They delay the precipitation of calcium phosphate from aqueous solution but, upon precipitation, lead to relatively monodisperse hydroxyapatite (HAP) spheres. Moreover, the polymers inhibit the bacterial colonization of human enamel by Streptococcus gordonii, a pioneer bacterium in oral biofilm formation, in vitro. The formation of well-defined HAP spheres suggests that a polymer-induced liquid precursor phase could be involved in the precipitation process. Moreover, the inhibition of bacterial adhesion suggests that the polymers could be utilized in caries prevention.

Effect of large doses of parenteral vitamin D on glycaemic control and calcium/phosphate metabolism in patients with stable type 2 diabetes mellitus: a randomised, placebo-controlled, prospective pilot study.

OBJECTIVE Vitamin D (D₃) status is reported to correlate negatively with insulin production and insulin sensitivity in patients with type 2 diabetes mellitus (T2DM). However, few placebo-controlled intervention data are available. We aimed to assess the effect of large doses of parenteral D3 on glycosylated haemoglobin (HbA(₁c)) and estimates of insulin action (homeostasis model assessment insulin resistance: HOMA-IR) in patients with stable T2DM. MATERIALS AND METHODS We performed a prospective, randomised, double-blind, placebo-controlled pilot study at a single university care setting in Switzerland. Fifty-five patients of both genders with T2DM of more than 10 years were enrolled and randomised to either 300,000 IU D₃ or placebo, intramuscularly. The primary endpoint was the intergroup difference in HbA(₁c) levels. Secondary endpoints were: changes in insulin sensitivity, albuminuria, calcium/phosphate metabolism, activity of the renin-aldosterone axis and changes in 24-hour ambulatory blood pressure values. RESULTS After 6 months of D₃ supply, there was a significant intergroup difference in the change in HbA(₁c) levels (relative change [mean ± standard deviation] +2.9% ± 1.5% in the D₃ group vs +6.9% ± 2.1% the in placebo group, p = 0.041) as HOMA-IR decreased by 12.8% ± 5.6% in the D₃ group and increased by 10% ± 5.4% in the placebo group (intergroup difference, p = 0.032). Twenty-four-hour urinary albumin excretion decreased in the D₃ group from 200 ± 41 to 126 ± 39, p = 0.021). There was no significant intergroup difference for the other secondary endpoints. CONCLUSIONS D₃ improved insulin sensitivity (based on HOMA-IR) and affected the course of HbA(₁c) positively compared with placebo in patients with T2DM.

Osteoclast-like cells on deproteinized bovine bone mineral and biphasic calcium phosphate: light and transmission electron microscopical observations.

OBJECTIVES The occurrence of multinucleated giant cells (MNGCs) on bone substitute materials has been recognized for a long time. However, there have been no studies linking material characteristics with morphology of the MNGCs. The aim was to analyze the qualitative differences of MNGCs on two commercially available calcium phosphate bone substitute materials retrieved from bone defects. MATERIAL AND METHODS Six defects were prepared bilaterally in the mandibular body of three mini pigs. The defects were randomly grafted with either deproteinized bovine bone mineral (DBBM) or biphasic calcium phosphate (BCP). After a healing period of four weeks, bone blocks were embedded in LR White resin. Three consecutive sections per defect were analyzed as follows: two with light microscopy using toluidine blue and tartrate-resistant acid phosphatase (TRAP) staining and one with transmission electron microscopy. RESULTS Multinucleated giant cells appeared on both biomaterials. On BCP, MNGCs had a flat morphology and were not observed in resorption lacunae. On DBBM, the MNGCs appeared more round and were often found in shallow concavities. MNGCs on both biomaterials demonstrated a varying degree of TRAP staining, with a tendency toward higher staining intensity of MNGCs on BCP. At the ultrastructural level, signs of superficial dissolution of BCP together with phagocytosis of minor fragments were observed. MNGCs on the surface of DBBM demonstrated sealing zones and ruffled borders, both features of mature osteoclasts. CONCLUSION MNGCs demonstrated distinctly different histological features depending on the bone substitute material used. Further research is warranted to understand the clinical implications of these morphological observations.

Bone healing around nanocrystalline hydroxyapatite, deproteinized bovine bone mineral, biphasic calcium phosphate, and autogenous bone in mandibular bone defects.

The individual healing profile of a given bone substitute with respect to osteogenic potential and substitution rate must be considered when selecting adjunctive grafting materials for bone regeneration procedures. In this study, standardized mandibular defects in minipigs were filled with nanocrystalline hydroxyapatite (HA-SiO), deproteinized bovine bone mineral (DBBM), biphasic calcium phosphate (BCP) with a 60/40% HA/β-TCP (BCP 60/40) ratio, or particulate autogenous bone (A) for histological and histomorphometric analysis. At 2 weeks, percent filler amongst the test groups (DBBM (35.65%), HA-SiO (34.47%), followed by BCP 60/40 (23.64%)) was significantly higher than the more rapidly substituted autogenous bone (17.1%). Autogenous bone yielded significantly more new bone (21.81%) over all test groups (4.91%-7.74%) and significantly more osteoid (5.53%) than BCP 60/40 (3%) and DBBM (2.25%). At 8 weeks, percent filler amongst the test groups (DBBM (31.6%), HA-SiO (31.23%), followed by BCP 60/40 (23.65%)) demonstrated a similar pattern and was again significantly higher as compared to autogenous bone (9.29%). Autogenous bone again exhibited statistically significantly greater new bone (55.13%) over HA-SiO (40.62%), BCP 60/40 (40.21%), and DBBM (36.35%). These results suggest that the osteogenic potential of HA-SiO and BCP is inferior when compared to autogenous bone. However, in instances where a low substitution rate is desired to maintain the volume stability of augmented sites, particularly in the esthetic zone, HA-SiO and DBBM may be favored. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 1478-1487, 2015.

Distribution of calcium phosphate in the exoskeleton of larval Exeretonevra angustifrons Hardy (Diptera : Xylophagidae)

Distribution and organisation of the mineral, amorphous calcium phosphate (ACP), has been investigated in the exoskeleton of the xylophagid fly larva Exeretonevra angustifrons Hardy. While head capsule and anal plate are smooth with a thin epicuticle, the epicuticle of the body is thicker and shows unusual micro-architecture comprised of minute hemispherical (dome-shaped) protrusions. Electron microprobe analysis and energy dispersive spectroscopy revealed heterogeneity of mineral elements across body cuticle and a concentration of ACP in the epicuticle, especially associated with the hemispherical structures. Further imaging and analysis showed the bulk of the ACP to be present in nano-sized granules. It is hypothesised that the specific distribution of ACP may enhance cuticular hardness or durability without reducing flexibility. (c) 2004 Elsevier Ltd. All rights reserved.

An X-ray micro-fluorescence study to investigate the distribution of Al, Si, P and Ca ions in the surrounding soft tissue after implantation of a calcium phosphate-mullite ceramic composite in a rabbit animal model

Synthetic calcium phosphates, despite their bioactivity, are brittle. Calcium phosphate-mullite composites have been suggested as potential dental and bone replacement materials which exhibit increased toughness. Aluminium, present in mullite, has however been linked to bone demineralisation and neurotoxicity: it is therefore important to characterise the materials fully in order to understand their in vivo behaviour. The present work reports the compositional mapping of the interfacial region of a calcium phosphate-20 wt% mullite biocomposite/soft tissue interface, obtained from the samples implanted into the long bones of healthy rabbits according to standard protocols (ISO-10993) for up to 12 weeks. X-ray micro-fluorescence was used to map simultaneously the distribution of Al, P, Si and Ca across the ceramic-soft tissue interface. A well defined and sharp interface region was present between the ceramic and the surrounding soft tissue for each time period examined. The concentration of Al in the surrounding tissue was found to fall by two orders of magnitude, to the background level, within similar to 35 mu m of the implanted ceramic.

A study of the formation of amorphous calcium phosphate and hydroxyapatite on melt quenched Bioglass(A (R)) using surface sensitive shallow angle X-ray diffraction

Melt quenched silicate glasses containing calcium, phosphorous and alkali metals have the ability to promote bone regeneration and to fuse to living bone. These glasses, including 45S5 Bioglass(A (R)) [(CaO)(26.9)(Na2O)(24.4)(SiO2)(46.1)(P2O5)(2.6)], are routinely used as clinical implants. Consequently there have been numerous studies on the structure of these glasses using conventional diffraction techniques. These studies have provided important information on the atomic structure of Bioglass(A (R)) but are of course intrinsically limited in the sense that they probe the bulk material and cannot be as sensitive to thin layers of near-surface dissolution/growth. The present study therefore uses surface sensitive shallow angle X-ray diffraction to study the formation of amorphous calcium phosphate and hydroxyapatite on Bioglass(A (R)) samples, pre-reacted in simulated body fluid (SBF). Unreacted Bioglass(A (R)) is dominated by a broad amorphous feature around 2.2 A...(-1) which is characteristic of sodium calcium silicate glass. After reacting Bioglass(A (R)) in SBF a second broad amorphous feature evolves similar to 1.6 A...(-1) which is attributed to amorphous calcium phosphate. This feature is evident for samples after only 4 h reacting in SBF and by 8 h the amorphous feature becomes comparable in magnitude to the background signal of the bulk Bioglass(A (R)). Bragg peaks characteristic of hydroxyapatite form after 1-3 days of reacting in SBF.