Nature-Inspired Effects of Naturally Occurring Trace Element-Doped Hydroxyapatite Combined with Surface Interactions of Mineral-Apatite Single Crystals on Human Fibroblast Behavior

Innovative engineering design for biologically active hydroxyapatites requires enhancing both mechanical and physical properties, along with biocompatibility, by doping with appropriate chemical elements. Herein, the purpose of this investigation was to evaluate and elucidate the model of naturally occurring hydroxyapatite and the effects of doped trace elements on the function of normal human fibroblasts, representing the main cells of connective tissues. The substrates applied (geological apatites with hexagonal prismatic crystal habit originated from Slyudyanka, Lake Baikal, Russia (GAp) and from Imilchil, The Atlas Mountains, Morocco (YAp)) were prepared from mineral natural apatite with a chemical composition consistent with the building blocks of enamel and enriched with a significant F− content. Materials in the form of powders, extracts and single-crystal plates have been investigated. Moreover, the effects on the function of fibroblasts cultured on the analyzed surfaces in the form of changes in metabolic activity, proliferation and cell morphology were evaluated. Apatite plates were also evaluated for cytotoxicity and immune cell activation capacity. The results suggest that a moderate amount of F− has a positive effect on cell proliferation, whereas an inhibitory effect was attributed to the Cl− concentration. It was found that for (100) GAp plate, fibroblast proliferation was significantly increased, whereas for (001) YAp plate, it was significantly reduced, with no cytotoxic effect and no immune response from macrophages exposed to these materials. The study of the interaction of fibroblasts with apatite crystal surfaces provides a characterization relevant to medical applications and may contribute to the design of biomaterials suitable for medical applications and the evaluation of their bioavailability.

Ashing of Bone: Errors Due to Loss of CO2 and Its Correction

Background: Ashing is widely used to determine weight fraction of water-free bone that is mineral, but no standard procedure exists and the range of techniques used spans a range of temperatures and times over which the amount of weight loss is variable. We show that variability is largely due to progressive loss of CO2 from CO3 2- ions in the apatite crystal lattice, beginning at 600⁰C, typically used for ashing. We test the effect of varying temperature, time and weight of sample and develop a reliable method, using small samples.Methods: Replicate samples of bovine cortical bone were tested at 500⁰, 600⁰ and 700⁰C for times ranging up to 24h. We also tested samples of multiple humans at what we concluded to be the optimal conditionsResults: Varying conditions of ashing resulted in variations in apparent ash weight % by up to 7%. Samples between 5 and 20 mg heated to 600⁰C for 1 h gave results agreeing with generally accepted values, but with much smaller variability. Ash wt% values for multiple human bone samples differed by up to 4.8% but replicate data for individuals agree to ± 1 wt%. Conclusions: A satisfactory method is given for ash weight determination using small samples, and yielding highly reproducible data. If accepted widely, ash weight values between laboratories could be used to study variations due to diet, age, drug treatment and disease.

Selective Laser Melting of Hydroxyapatite: Perspectives for 3D Printing of Bioresorbable Ceramic Implants

Hydroxyapatite, being the major mineral component of tooth enamel and natural bones, is a good candidate for bone tissue engineering applications. One of the promising approaches for manufacturing of three-dimensional objects is selective laser sintering/melting which enables the creation of a dense structure directly during 3D printing by adding material layer-by-layer. The effect of laser irradiation with a wavelength of 10.6 μm on the behavior of mechanochemically synthesized hydroxyapatite under different treatment conditions was studied for the first time in this work. It was shown that, in contrast to laser treatment, the congruent melting is impossible under conditions of a relatively slow rate of heating in a furnace. Depending on the mode of laser treatment, hydroxyapatite can be sintered or melted, or partially decomposed into the more resorbable calcium phosphates. It was found that the congruent selective laser melting of hydroxyapatite can be achieved by treating the dense powder layer with a 0.2 mm laser spot at a power of 4 W and at a scanning speed of 700 mm/s. Melting was shown to be accompanied by the crystallization of a dense monolayer of oxyhydroxyapatite while preserving the initial apatite crystal lattice. The thickness of the melted layer, the presence of micron-sized pores, and the phase composition can be controlled by varying the scanning speed and laser power. This set of parameters permits the use of selective laser melting technology for the production of oxyhydroxyapatite biodegradable implants with acceptable properties by 3D printing.

U-Pb geochronology of apatite crystallized within a terrestrial impact melt sheet: Manicouagan as a geochronometer test site

ABSTRACT The Manicouagan impact event has been the subject of multiple age determinations over the past ~50 yr, providing an ideal test site for evaluating the viability of different geochronometers. This study highlights the suitability of Manicouagan’s essentially pristine impact melt body as a medium for providing insight into the U-Pb isotope systematics of geochronometers in the absence of shock-related overprinting. We performed in situ laser-ablation–inductively coupled plasma–mass spectrometry (LA-ICP-MS) U-Pb geochronology on apatite and zircon, both of which crystallized as primary phases. This study is the first application of U-Pb geochronology to apatite crystallized within a terrestrial impact melt sheet. U-Pb analyses were obtained from 200 melt-grown apatite grains (n = 222 spots), with a data subset providing a lower-intercept age of 212.5 ± 8.0 Ma. For melt-grown zircon, a total of 30 analyses from 28 grains were obtained, with a subset of the data yielding a lower-intercept age of ± 1.6 Ma. The lower precision (±8.0 Ma; ±3%) obtained from apatite is a consequence of low U and a high and variable common-Pb composition. This resulted from localized Pb*/PbC heterogeneity within the impact melt sheet that was incorporated into the apatite crystal structure during crystallization (where Pb*/PbC is the ratio of radiogenic Pb to common Pb). While considered a limitation to the precision obtainable from melt-grown apatite, its ability to record local-scale isotopic variations highlights an advantage of U-Pb studies on melt-grown apatite. The best-estimate ages from zircon and apatite overlap within error and correlate with previously determined ages for the Manicouagan impact event. An average formation age from the new determinations, combined with previous age constraints, yields a weighted mean age of 214.96 ± 0.30 Ma for the Manicouagan impact structure.

Review Article Recent Advances in Preventing Dental Erosion

Dental erosion is defined as an irreversible loss of dental hard tissue due to exposure to chelating agents or non-bacterial acids. The occurrence of this condition was noted and the incidence and prevalence of dental erosion has been increasingly documented.The Ant erosive agents such as Anacardic acid in which the key component is the cashewnut shell liquid is phenolic lipids. It is a mixture of molecules which are saturated and unsaturated. It is also considered to have an anti-microbial effect and has been studied for the treatment of cancer, oxidative damage, inflammation and obesity disorders. Other anti-erosive agent like Fluoride helps in tooth remineralization. Fluorapatite, rather than hydroxyapatite, forms during the process of remineralization when fluoride is found in oral fluids. In apatite crystal lattice formation, fluoride ions replace hydroxy ions. Fluorapatite, even under acidic conditions, is less soluble than hydroxyapatite, which helps to regenerate tooth enamel. Fluoride is therefore a stronger anti-erosive agent. Various Recent advances in anti-erosive agents are Calcium and phosphate, Casein phosphopeptide amorphous calcium phosphate (CPP-ACP), Protease inhibitors, Oils, Chitosan chitosan and Multivalent metal ions Various techniques to evaluate dental erosion are in vitro techniques and in vivo techniques. In vitro techniques are Scanning electron microscope, Surface Profilometry, Polarized Light Microscopyand Non-Contact Confocal Laser Scanning Microscopy (CLSM). And iv vivo techniques are Photographs Clinical review and indices. The most important point of treatment is identifying and removing the erosion factor, above all current materials and methods. Therefore, early identification of the lesions, evaluation and removal of the etiological variables are relevant topics.

A time-course Raman spectroscopic analysis of spontaneous in vitro microcalcifications in a breast cancer cell line

Microcalcifications are early markers of breast cancer and can provide valuable prognostic information to support clinical decision-making. Current detection of calcifications in breast tissue is based on X-ray mammography, which involves the use of ionizing radiation with potentially detrimental effects, or MRI scans, which have limited spatial resolution. Additionally, these techniques are not capable of discriminating between microcalcifications from benign and malignant lesions. Several studies show that vibrational spectroscopic techniques are capable of discriminating and classifying breast lesions, with a pathology grade based on the chemical composition of the microcalcifications. However, the occurrence of microcalcifications in the breast and the underlying mineralization process are still not fully understood. Using a previously established model of in vitro mineralization, the MDA-MB-231 human breast cancer cell line was induced using two osteogenic agents, inorganic phosphate (Pi) and β-glycerophosphate (βG), and direct monitoring of the mineralization process was conducted using Raman micro-spectroscopy. MDA-MB-231 cells cultured in a medium supplemented with Pi presented more rapid mineralization (by day 3) than cells exposed to βG (by day 11). A redshift of the phosphate stretching peak for cells supplemented with βG revealed the presence of different precursor phases (octacalcium phosphate) during apatite crystal formation. These results demonstrate that Raman micro-spectroscopy is a powerful tool for nondestructive analysis of mineral species and can provide valuable information for evaluating mineralization dynamics and any associated breast cancer progression, if utilized in pathological samples.

Hydroxyl Groups Induce Bioactivity in Silica/Chitosan Aerogels Designed for Bone Tissue Engineering. In Vitro Model for the Assessment of Osteoblasts Behavior

Silica (SiO2)/chitosan (CS) composite aerogels are bioactive when they are submerged in simulated body fluid (SBF), causing the formation of bone-like hydroxyapatite (HAp) layer. Silica-based hybrid aerogels improve the elastic behavior, and the combined CS modifies the network entanglement as a crosslinking biopolymer. Tetraethoxysilane (TEOS)/CS is used as network precursors by employing a sol-gel method assisted with high power ultrasound (600 W). Upon gelation and aging, gels are dried in supercritical CO2 to obtain monoliths. Thermograms provide information about the condensation of the remaining hydroxyl groups (400–700 °C). This step permits the evaluation of the hydroxyl group’s content of 2 to 5 OH nm−2. The formed Si-OH groups act as the inductor of apatite crystal nucleation in SBF. The N2 physisorption isotherms show a hysteresis loop of type H3, characteristic to good interconnected porosity, which facilitates both the bioactivity and the adhesion of osteoblasts cells. After two weeks of immersion in SBF, a layer of HAp microcrystals develops on the surface with a stoichiometric Ca/P molar ratio of 1.67 with spherulite morphology and uniform sizes of 6 μm. This fact asserts the bioactive behavior of these hybrid aerogels. Osteoblasts are cultured on the selected samples and immunolabeled for cytoskeletal and focal adhesion expression related to scaffold nanostructure and composition. The initial osteoconductive response observes points to a great potential of tissue engineering for the designed composite aerogels.

Osteopontin regulates biomimetic calcium phosphate crystallization from disordered mineral layers covering apatite crystallites

Details of apatite formation and development in bone below the nanometer scale remain enigmatic. Regulation of mineralization was shown to be governed by the activity of non-collagenous proteins with many bone diseases stemming from improper activity of these proteins. Apatite crystal growth inhibition or enhancement is thought to involve direct interaction of these proteins with exposed faces of apatite crystals. However, experimental evidence of the molecular binding events that occur and that allow these proteins to exert their functions are lacking. Moreover, recent high-resolution measurements of apatite crystallites in bone have shown that individual crystallites are covered by a persistent layer of amorphous calcium phosphate. It is therefore unclear whether non-collagenous proteins can interact with the faces of the mineral crystallites directly and what are the consequences of the presence of a disordered mineral layer to their functionality. In this work, the regulatory effect of recombinant osteopontin on biomimetic apatite is shown to produce platelet-shaped apatite crystallites with disordered layers coating them. The protein is also shown to regulate the content and properties of the disordered mineral phase (and sublayers within it). Through solid-state NMR atomic carbon-phosphorous distance measurements, the protein is shown to be located in the disordered phases, reaching out to interact with the surfaces of the crystals only through very few sidechains. These observations suggest that non-phosphorylated osteopontin acts as regulator of the coating mineral layers and exerts its effect on apatite crystal growth processes mostly from afar with a limited number of contact points with the crystal.

Preparation of Hydroxyapatite/Silk Fibroin Composite and Its Adoption in Posterolateral Spinal Fusion of Rats

Hydroxyapatite/silk fibroin (HAp/SF) composite was prepared and applied to the posterolateral spinal fusion model in rats to observe the effect of bone fusion. Method: Calcium chloride, diammonium phosphate, SF, and polyvinyl alcohol were used as raw materials, HAp/SF composites were prepared by chemical precipitation. The microstructure of the composite, crystal phase composition, and chemical structure were analyzed by the scanning electron microscope (SEM) and X-ray diffraction (XRD), and fourier transform infrared spectrometer (FTIR Spectrometer). Through the cultivation of osteoblasts MC3T3-E1 in vitro, the adhesion and proliferation (A&P) of cells on the face of materials were investigated. Thereby, the biocompatibility of the material was characterized. HAp/SF material was applied to the rat posterolateral spinal fusion model. The osteogenesis and spinal fusion were evaluated by the imaging observation, histological observation and manual palpation. The results showed that the rod-shaped HAp with uniform size and high purity was obtained, with a diameter of 20∼40 nm and a length of 200∼500 nm, similar to the apatite crystal in natural bone tissue (BT). In composite materials, a spatial network structure was formed by the interweaving of the SF fibers, and HAp was deposited on the face of the SF or in the middle of its network structure. In the obtained HAp/SF materials, the calcium ions of HAp and the carbonyl groups of SF were used to form thermally stable complexes through strong chemical bonds. Besides, SF was a template for the directional induction of HAp crystal growth, and the growth of HAp crystal along the C axis was regulated by SF. The growth direction was parallel to the long axis of SF fibers, and was consistent with the structure of apatite crystals deposited on the face of collagen fibers in natural BT. The results of cell culture in vitro showed that: after comparison with the control group (CG) with pure Hap, the adhesion ability of cells to HAp/SF material was significantly improved. The proliferation capacity of bone artificial bone (BAM) material and HAp/SF material was also significantly improved. The nuclear and skeletal staining results of MC3T3-E1 cells on the face of three groups of materials (HAp, BAM and HAp/SF) were combined, and the results also indicated that BAM and HAp/SF materials had good ability to promote cell A&P. The results of posterolateral spinal fusion in rats showed that HAp/SF materials group palpated the posterolateral spine for fusion. The formation of new BT on the posterolateral side of the spine was revealed by the Micro-computed tomography (Micro-CT) examination. In conclusion, HAp/SF composite had good osteoblastic compatibility and can achieve good spinal fusion effect.

Preparation and Histological Study of Multi-Walled Carbon Nanotubes Bone Graft in Management of Class II Furcation Defects in Dogs

BACKGROUND: The main target of periodontal disease and alveolar bone defeat treatment is the regeneration of the lost structures. AIM: This work deals with the evaluation of the effect of functionalised multi-walled carbon nanotubes (MWCNTs), as grafting material in the management of furcation defects created in dogs. MATERIAL AND METHODS: Potential cytotoxicity of the grafting material was assessed. Scanning electron microscope (SEM) and energy dispersive x-ray (EDX) analysis after incubation of the grafting material in simulated body fluid (SBF) at pH 7.4 and 37°C for one week was done. In six healthy mongrel dogs' full-thickness mucoperiosteal flaps were raised on the buccal aspects to create two walls intrabony defects at the furcation areas. The mandibular premolar area received the grafting material. Histological evaluation was carried out at 1, 2- and 3-months’ period. RESULTS: Cytotoxicity results proved the safety of grafting material application. The prepared material exhibited good Ca-apatite crystal patterns at the surface revealed by SEM and high calcium content showed by EDX results. Good bone formation ability was also apparent histologically. CONCLUSION: The prepared grafting material (MWCNTs) can serve as a delivery vehicle for osteogenic cells and osteogenic growth factor proteins in the bone development process.