Porous Biphasic Calcium Phosphate Granules from Oyster Shell Promote the Differentiation of Induced Pluripotent Stem Cells.

Oyster shells are rich in calcium, and thus, the potential use of waste shells is in the production of calcium phosphate (CaP) minerals for osteopathic biomedical applications, such as scaffolds for bone regeneration. Implanted scaffolds should stimulate the differentiation of induced pluripotent stem cells (iPSCs) into osteoblasts. In this study, oyster shells were used to produce nano-grade hydroxyapatite (HA) powder by the liquid-phase precipitation. Then, biphasic CaP (BCP) bioceramics with two different phase ratios were obtained by the foaming of HA nanopowders and sintering by two different two-stage heat treatment processes. The different sintering conditions yielded differences in structure and morphology of the BCPs, as determined by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Brunauer-Emmett-Teller (BET) surface area analysis. We then set out to determine which of these materials were most biocompatible, by co-culturing with iPSCs and examining the gene expression in molecular pathways involved in self-renewal and differentiation of iPSCs. We found that sintering for a shorter time at higher temperatures gave higher expression levels of markers for proliferation and (early) differentiation of the osteoblast. The differences in biocompatibility may be related to a more hierarchical pore structure (micropores within macropores) obtained with briefer, high-temperature sintering.

Pulsed laser deposition temperature effects on strontium-substituted hydroxyapatite thin films for biomedical implants.

Substituting small molecule drugs with abundant and easily affordable ions may have positive effects on the way countless disease treatments are approached. The interest in strontium cation in bone therapies soared in the wake of the success of strontium ranelate in the treatment of osteoporosis. A new method for producing thin strontium-containing hydroxyapatite (Sr-HA, Ca9Sr(PO4)6(OH)2) films as coatings that render bioinert titanium implant bioactive is reported here. The method is based on the combination of a mechanochemical synthesis of Sr-HA targets and their deposition in form of thin films on top of titanium with the use of laser ablation at low pressure. The films were 1-2 µm in thickness and their formation was studied at different temperatures, including 25, 300, and 500 °C. Highly crystalline Sr-HA target transformed during pulsed laser deposition to a fully amorphous film, whose degree of long-range order recovered with temperature. Particle edges became somewhat sharper and surface roughness moderately increased with temperature, but the (Ca+Sr)/P atomic ratio, which increased 1.5 times during the film formation, remained approximately constant at different temperatures. Despite the mostly amorphous structure of the coatings, their affinity for capturing atmospheric carbon dioxide and accommodating it as carbonate ions that replace both phosphates and hydroxyls of HA was confirmed in an X-ray photoelectron spectroscopic analysis. As the film deposition temperature increased, the lattice voids got reduced in concentration and the structure gradually "closed," becoming more compact and entailing a linear increase in microhardness with temperature, by 0.03 GPa/°C for the entire 25-500 °C range. Biocompatibility and bioactivity of Sr-HA thin films deposited on titanium were confirmed in an interaction with dental pulp stem cells, suggesting that these coatings, regardless of the processing temperature, may be viable candidates for the surface components of metallic bone implants.

In vivo evaluation of interactions between biphasic calcium phosphate (BCP)-niobium pentoxide (Nb2O5) nanocomposite and tissues using a rat critical-size calvarial defect model.

Natural or synthetic biomaterials are increasingly being used to support bone tissue repair or substitution. The combination of natural calcium phosphates with biocompatible alloys is an important route towards the development of new biomaterials with bioperformance and mechanical responses to mimic those of human bones. This article evaluated the structural, physical, mechanical and biological properties of a new mechanical improved nanocomposite elaborated by association of fish biphasic calcium phosphate (BCP) and niobium pentoxide (Nb2O5). The nanocomposite (Nb-BCP) and the pure BCP, used as a positive control, were obtained by powder metallurgy. The density, porosity and microhardness were measured. The structural analysis was determined by X-ray diffraction (XRD) and the biological properties were studied in histological sections of critical size calvaria defects in rats, 7, 15, 30, 45 and 60 days after implantation of disks of both materials. Morphological description was made after scanning electron microscopy (SEM) and optical microscopy analysis. After sintering, the Nb-BCP nanocomposite presented four crystalline phases: 34.36% calcium niobate (CaNb2O6), 21.68% phosphorus niobium oxide (PNb9O25), 42.55% ß-tricalcium phosphate (Ca3(PO4)2) and 1.31% of niobium pentoxide (Nb2O5) and exhibited increases of 17% in density, 66% in Vickers microhardness and 180% in compressive strength compared to pure BCP. In vivo study, showed biocompatibility, bioactivity and osteoconductivity similar to pure BCP. SEM showed the formation of globular accretions over the implanted nanocomposites, representing one of the stages of bone mineralization. In conclusion, the BCP and Nb2O5 formed a nanocomposite exhibiting characteristics that are desirable for a biomaterial, such as bioperformance, higher ß-TCP percentage and improved physical and mechanical properties compared to pure BCP. These characteristics demonstrate the promise of this material for supporting bone regeneration.

Effects on growth and osteogenic differentiation of mesenchymal stem cells by the strontium-added sol-gel hydroxyapatite gel materials.

In the present study, strontium-modified hydroxyapatite gels (Sr-HA) at different concentrations were prepared using sol-gel approach and their effect on human-bone-marrow-derived mesenchymal stem cells, were evaluated. The effect of Strontium on physico-chemical and morphological properties of hydroxyapatite gel were evaluated. Morphological analyses (SEM and TEM) demonstrate that an increasing in the amount of Sr ions doped into HA made the agglomerated particles smaller. The substitution of large Sr2+ for small Ca2+ lead to denser atomic packing of the system causing retardation of crystals growth. The biological results demonstrated that hydroxyapatite gel containing from 0 to 20 mol% of Sr presented no cytotoxicity and promote the expression of osteogenesis related genes including an early marker for osteogenic differentiation ALP; a non-collagen protein OPN and a late marker for osteogenic differentiation OCN. Finally, the Sr-HA gels could have a great potential application as filler in bone repair and regeneration and used in especially in the osteoporotic disease.

Novel mineralized heparin-gelatin nanoparticles for potential application in tissue engineering of bone.

Nanoparticles (NPs) were prepared from succinylated gelatin (s-GL) cross-linked with aldehyde heparin (a-HEP) and used subsequently as a nano-template for the mineralization of hydroxyapatite (HAP). Gelatin was functionalized with succinyl groups that made it soluble at room temperature. Heparin was oxidized to generate aldehyde groups and then used as a cross-linker that can react with s-GL to form NPs via Schiff's base linkage. The polymer concentrations, feed molar ratios and pH conditions were varied to fabricate NPs suspension. NPs were obtained with a spheroid shape of an average size of 196 nm at pH 2.5 and 202 nm at pH 7.4. These NPs had a positive zeta potential of 7.3 ± 3.0 mV and a narrow distribution with PDI 0.123 at pH 2.5, while they had a negative zeta potential of -2.6 ± 0.3 mV and formed aggregates (PDI 0.257) at pH 7.4. The NPs prepared at pH 2.5 with a mean particle size of 196 nm were further used for mineralization studies. The mineralization process was mediated by solution without calcination at 37 °C. The HAP formed on NPs was analyzed by Fourier transform infrared spectroscopy and X-ray diffraction. HAP coated s-GL/a-HEP NPs developed in this study may be used in future as osteoinductive fillers enhancing the mechanical properties of injectable hydrogel or use as potential multifunctional device for nanotherapeutic approaches.

Preparation, evaluation, and first clinical use of 177Lu-labeled hydroxyapatite (HA) particles in the treatment of rheumatoid arthritis: utility of cold kits for convenient dose formulation at hospital radiopharmacy.

While radiation synovectomy (RSV) constitutes a successful paradigm for the treatment of arthritis, a major cornerstone of its success resides in the selection of appropriate radiolabeled agent. Among the radionuclide used for RSV, the scope of using (177)Lu [T1/2 = 6.65 d, Eß(max) = 497 keV, Eγ = 113 KeV (6.4%), 208 KeV (11%)] seemed to be attractive owing to its suitable decay characteristics, easy availability, and cost-effective production route. The present article describes a formulation of (177)Lu-labeled hydroxyapatite (HA) using ready-to-use kits of HA particles of 1-10 µm size range. The developed kits enable convenient one-step preparation of (177)Lu-HA (400 ± 30 MBq doses) in high radiochemical purity (>99%) and stability at hospital radiopharmacy. The preparation showed promising results in pre-clinical studies carried out in Wistar rats bearing arthritis in knee joints. In preliminary clinical investigation, significant improvement in the disease conditions was reported in 10 patients with rheumatoid arthritis of knee joints treated with 333 ± 46 MBq doses of (177)Lu-HA. The studies reveal that while (177)Lu labeled HA particles holds considerable promise as a cost-effective agent for RSV, the adopted strategy of using HA kits could be a potential step toward wider clinical utilization of radiolanthanide-labeled HA particles.

[Preparation of alpha-tricalcium phosphate/HA whisker/carboxymethyl chitosan-gelatin composite porous bone cement].

In order to investigate the effects of HA whisker and carboxymethyl chitosan-gelatin(CMC-Gel) on the mechanical properties of porous calcium phosphate cement, a series of alpha-tricalcium phosphate (alpha-TCP), HA whisker and L-sodium glutamate porogen with different mass fractions were mixed, and setting liquid was added to them to prepare alpha-TCP/HA whisker composite porous bone cement. Then, the cement was immersed in a series of CMC-Gel solutions which had different weight ratios of CMC to Gel to prepare alpha-TCP/HA whisker/CMC-Gel composite porous bone cement. The compressive strengths and microstructure of cement were characterized by mechanical testing machine and SEM. The results showed that when the mass fraction of HA whisker is 4%, the compressive strength of alpha-TCP/HA whisker composite porous bone cement reaches 2.57MPa, which is 1.81 times that of alpha-TCP bone cement. When the weight ratio of CMC to Gel is 50:50, the compressive strength of alpha-TCP/HA whisker/CMC-Gel composite porous bone cement is 3. 34MPa, which is 2.35 times that of alpha-TCP bone cement, and the toughness of the composite cement is greatly improved as well.

Facile synthesis of a three-dimensional hydroxyapatite monolith for protein adsorption.

Hydroxyapatite (HA) shows promising applications in the clinical treatment of bone defects owing to its excellent physicochemical properties, such as biocompatibility, bioactivity, and osteoconductivity. However, it is difficult to maintain a porous structure in HA materials because of processing difficulties. In this study, a hard template method was developed to prepare a porous HA monolith with a hierarchical pore structure and high porosity. The cellulose monolith template was prepared from cellulose acetate using a thermally induced phase separation method. The cellulose monoliths were then immersed into the HA slurry to form a cellulose_HA composite monolith, which was converted to an HA monolith by burning in air to remove the cellulose monolith. Owing to the hierarchically porous structure of the cellulose monolith template, the obtained HA monolith demonstrated a hierarchically porous structure. Furthermore, the HA monolith was explored to study the adsorption and release properties of bovine serum albumin (BSA), which indicated that the HA monolith had a high adsorption capacity (388.6 mg g-1) and sustained release from the BSA-loaded HA monolith. Thus, HA monoliths have potential applications in the field of protein purification and biomaterials.

Rapid regeneration of enamel-like-oriented inorganic crystals by using rotary evaporation.

Enamel, the hardest tissue in the human body, has excellent mechanical properties, mainly due to its highly ordered spatial structure. Fabricating enamel-like structure is still a challenge today. In this work, a simple and highly efficient method was introduced, using the silk fibroin as a template to regulate calcium- and phosphate- supersaturated solution to regenerate enamel-like hydroxyapatite crystals on various substrates (enamel, dentin, titanium, and polyethylene) under rotary evaporation. The enamel-like zinc oxide nanorod array structure was also successfully synthesized using the aforementioned method. This strategy provides a new approach to design and fabricate mineral crystals with particular orientation coatings for materials.

Structural modification of nanohydroxyapatite Ca10(PO4)6(OH)2 related to Eu3+ and Sr2+ ions doping and its spectroscopic and antimicrobial properties.

The Eu3+ and Sr2+ ions co-doped hydroxyapatite nanopowders (Ca10(PO4)6(OH)2) were synthesized via a precipitation method and post heat-treated at 500 °C. The concentration of Eu3+ ions was established in the range of 0.5-5 mol% to investigate the site occupancy preference. The concentration of Sr2+ ions was set at 5 mol%. The structural and morphological properties of the obtained materials were studied by an X-ray powder diffraction, a transmission electron microscopy techniques and infrared spectroscopy. As synthesized nanoparticles were in the range of 11-17 nm and annealed particles were in the range of 20-26 nm. The luminescence properties in dependence of the dopant concentration and applied temperature were investigated. The 5D0 → 7F0 transition shown the abnormally strong intensity for annealed materials connected with the increase of covalency character of Eu3+-O2- bond, which arise as an effect of charge compensation mechanism. The Eu3+ ions occupied three possible crystallographic sites in these materials revealed in emission spectra: one Ca(1) site with C3 symmetry and two Ca(2) sites with Cs symmetry arranged as cis and trans symmetry. The antibacterial properties of Eu3+ and Sr2+ ions doped and co-doped hydroxyapatite nanopowders were also determined against Gram-negative pathogens such as Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli. Obtained results suggest that both europium and strontium ions may implement antibacterial properties for hydroxyapatites. In the most cases, better antibacterial effect we noticed for dopants at 5 mol% ratio. However, the effect is strongly species- and strain-dependent feature.

Dentinal tubules driven wetting of dentin: Cassie-Baxter modelling.

We investigate the wetting properties of dentin surfaces submitted to a phosphoric acid etching followed by an air drying procedure, as in clinical situations of adhesive dentistry. The surface topography of the etched surfaces was characterized by AFM, and the wetting properties of water on these rough and heterogeneous surfaces were studied, by contact angle measurements. We showed that the contact angle increases with the acid exposure time and consequently with both surface roughness and the organicmineral ratio of the dentin components. From the whole results, obtained on dentin and also on synthesized hydroxyapatites samples, we inferred a water contact angle of ~133° on the dentinal tubule. These experimental results may be described by the Cassie-Baxter approach, and it is suggested that small air pockets could be formed inside the dentinal tubules.

Template-oriented synthesis of hydroxyapatite nanoplates for 3D bone printing.

The design of hydroxyapatite (HA) nanoarchitecture is critical for fabricating artificial bone tissues as it dictates the biochemical and the mechanical properties of the final product. Herein, we incorporated a simple hard-template approach to synthesise single crystal nanoplates of HA. We used the 2D graphitic nitride (g-C3N4) material to prepare an HA sol-gel under hydrothermal conditions. A new HA nanostructure was then formed during the calcination and removal of g-C3N4 at a higher temperature, which finally led to the production of nanoplates (thickness of ∼100 nm) while in lateral dimension the average size was in the micrometre scale. We characterised the synthesised HA nanoplates with XRD, TEM, and HRTEM. The theoretically predicted nanostructure construction based on Wulff's method is in full agreement with the experimental observations. We then prepared different weight ratios of HA and polylactic acid (PLA) composites for artificial 3D bone fabrication. The strong interaction between PLA and HA's (110) facet, which was the second most prevalent, resulted in the composite's mechanical robustness. After mechanical testing, an optimum ratio was selected for biological studies and 3D printing. Biological experiments demonstrated that the synthesised composite had excellent viability in vitro.

Corrosion resistance and drug release profile of gentamicin-loaded polyelectrolyte multilayers on magnesium alloys: Effects of heat treatment.

Magnesium (Mg) alloys have received considerable attentions as the emerging biodegradable implant materials in orthopedic surgery applications. However, the rapid corrosion rate and the susceptibility to bacterial infection have prevented their wide spread applications to date. In this work, the gentamicin-loaded multilayers have been constructed on Mg alloys through spin-assisted layer-by-layer (SLbL) assembly. Heat treatment is applied for improving the corrosion resistance and prolonging the drug release profile. In addition, the treated multilayer can promote the formation of hydroxyapatite (HA) during the long-time immersion in Hank's balanced salt solution (HBSS).

Osteoblast interactions with various hydroxyapatite based biomaterials consolidated using a spark plasma sintering technique.

This study investigated the osteoblast behaviors on various hydroxyapatite based biomaterials that were consolidated at 1100 degrees C for 3 min by a spark plasma sintering technique. The osteoblasts from human fetal osteoblast cell line were cultured in the medium on the various biomaterials surfaces (HA, RF21, 1SiHA, and 5SiHA) to assess the cell morphology and proliferation as well as cell differentiation (alkaline phosphatase activity). Moreover, the bone gamma-carboxyglutamic protein or osteocalcin in the medium were determined at different periods of culture. The present results indicated that the amount of osteocalcin in the medium decreased during the periods of culture. The highest osteocalcin production obtained from the biomaterial 5SiHA after cell culture for 2 days demonstrated that the presence of silica in the biomaterials enhanced the cell differentiation by the rapid release of silicate and calcium ions.

Preparation and evaluation of a new radiopharmaceutical for radiosynovectomy, 111Ag-labelled hydroxyapatite (HA) particles.

Many radionuclides, namely, 166Ho, 90Y, 165Dy, 32P, 198Au, 186Re, etc. have been used for radio-synovectomy. Silver-111 (T 1/2 7.45 d) can be produced in a nuclear reactor and is a potential therapeutic radionuclide decaying by beta(-) emission (92% E beta max=1.037MeV and 8% by beta(-) decay associated with emission of gamma-rays (E gamma=245.4keV, I gamma=1.33%; E gamma=342.1keV, I gamma=6.7%)). Because of the production feasibility and favourable nuclear properties, 111Ag may find use as a suitable radionuclide for radio-synovectomy. Hydroxyapatite (HA), Ca10(PO4)6(OH)2 is one of the preferred particulates for this application. In this work, [111Ag]Ag-HA particulates were successfully prepared with high-labelling yield ( approximately 97%) at various pH values. The radiochemical purity of the [111Ag]Ag-HA particles was 99.9%. Stability studies for 7 days showed that the [111Ag]Ag-HA particles retained their stability. gamma camera images at 15 min, 24h and 5 d after injection of the particles into rabbits revealed the retention of the activity in the synovial joints of the knee, thereby indicating excellent in vivo stability of [111Ag]Ag-HA particles. Therefore, [111Ag]Ag-HA particles would be a potential therapeutic agent in the management of arthritis.

Effects of liquid phase on basic properties of alpha-tricalcium phosphate-based apatite cement.

Effects of liquid phase on the basic properties of alpha-tricalcuim phosphate (alpha-TCP)-based cement, BIOPEX, were investigated by employing three liquid phases: distilled water, neutral sodium hydrogen phosphate solution, and succinic acid disodium salt solution containing sodium salt of chondroitin sulfate. When mixed with neutral sodium hydrogen phosphate or succinic acid disodium salt solution, the initial setting times of the cement were 19.4 +/- 0.55 and 11.8 +/- 0.45 minutes respectively. These setting times were much shorter than that of distilled water, 88.4 +/- 0.55 minutes. Formation of needle-like crystals typical of apatite was much faster when neutral sodium hydrogen phosphate solution was used, as compared to distilled water or succinic acid disodium salt solution. Moreover, at 24 hours after mixing, the largest amount of apatite was formed when neutral sodium hydrogen phosphate solution was used, whereas use of succinic acid resulted in the least. On the final mechanical strength of the cement, that yielded with neutral sodium hydrogen phosphate solution was the highest. In contrast, lower mechanical strength was observed--especially at the initial stage--when succinic acid sodium salt was used. It was thus concluded that alpha-TCP-based cement allowed accelerated transformation to apatite, and that higher mechanical strength since the initial stage was achieved when neutral sodium hydrogen phosphate solution was used as the liquid phase.

Premixed calcium phosphate cements: synthesis, physical properties, and cell cytotoxicity.

OBJECTIVES: Calcium phosphate cement (CPC) is a promising material for dental, periodontal, and craniofacial repairs. However, its use requires on-site powder-liquid mixing that increases the surgical placement time and raises concerns of insufficient and inhomogeneous mixing. The objective of this study was to determine a formulation of premixed CPC (PCPC) with rapid setting, high strength, and good in vitro cell viability. METHODS: PCPCs were formulated from CPC powder+non-aqueous liquid+gelling agent+hardening accelerator. Five PCPCs were thus developed: PCPC-Tartaric, PCPC-Malonic, PCPC-Citric, PCPC-Glycolic, and PCPC-Malic. Formulations and controls were compared for setting time, diametral tensile strength, and osteoblast cell compatibility. RESULTS: Setting time (mean+/-S.D.; n=4) for PCPC-Tartaric was 8.2+/-0.8 min, significantly less than the 61.7+/-1.5 min for the Premixed Control developed previously (p<0.001). On 7th day immersion, the diametral tensile strength of PCPC-Tartaric reached 6.5+/-0.8 MPa, higher than 4.5+/-0.8 MPa of Premixed Control (p=0.036). Osteoblast cells displayed a polygonal morphology and attached to the nano-hydroxyapatite crystals in the PCPCs. All cements had similar live cell density values (p=0.126), indicating that the new PCPCs were as cell compatible as a non-premixed CPC control known to be biocompatible. Each of the new PCPCs had a cell viability that was not significantly different (p>0.1) from that of the non-premixed CPC control. SIGNIFICANCE: PCPCs will eliminate the powder-liquid mixing during surgery and may also improve the cement performance. The new PCPCs supported cell attachment and yielded a high cell density and viability. Their mechanical strengths approached the reported strengths of sintered porous hydroxyapatite implants and cancellous bone. These nano-crystalline hydroxyapatite cements may be useful in dental, periodontal, and craniofacial repairs.

Fabrication of porous bioactive structures using the selective laser sintering technique.

Hydroxyapatite, a ceramic with which natural bone inherently bonds, has been incorporated into a polymer matrix to enhance the bioactivity of implant materials. In order to manufacture custom-made bioactive implants rapidly, selective laser sintering has been investigated to fabricate hydroxyapatite and polyamide composites and their properties investigated. One objective of this research was to identify the maximum hydroxyapatite content that could be incorporated into the matrix, which was sintered at various parameters. The study focused on investigating the control of porosity and pore size of the matrix by manipulating the selective laser sintering parameters of the laser power and laser scan speed. The interception method was used to analyse the internal porous morphology of the matrices which were cross-sectioned through the vertical plane. Most notably, all structures built demonstrated interconnection and penetration throughout the matrix. Liquid displacement was also used to analyse the porosity of the matrices. The laser power showed a negative relationship between porosity and variation in parameter values until a critical power value was reached. However, the same relationship for laser scan speed matrices was inconsistent. The effects of the laser power and laser scanning speed on the features of porous structures that could influence cell spreading, proliferation, and bone regeneration are presented.

A perfusion bioreactor system capable of producing clinically relevant volumes of tissue-engineered bone: in vivo bone formation showing proof of concept.

In an effort to produce clinically relevant volumes of tissue-engineered bone products, we report a direct perfusion bioreactor system. Goat bone marrow stromal cells (GBMSCs) were dynamically seeded and proliferated in this system in relevant volumes (10 cc) of small sized macroporous biphasic calcium phosphate scaffolds (BCP, 2-6 mm). Cell load and cell distribution were shown using methylene blue block staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining was used to demonstrate viability of the present cells. After 19 days of cultivation, the scaffolds were covered with a viable, homogeneous cell layer. The hybrid structures became interconnected and a dense layer of extracellular matrix was present as visualized by environmental scanning electron microscopy (ESEM). ESEM images showed within the extracellular matrix sphere like structures which were identified as calcium phosphate nodules by energy dispersive X-ray analysis (EDX). On line oxygen measurements during cultivation were correlated with proliferating GBMSCs. It was shown that the oxygen consumption can be used to estimate GBMSC population doubling times during growth in this bioreactor system. Implantation of hybrid constructs, which were proliferated dynamically, showed bone formation in nude mice after 6 weeks of implantation. On the basis of our results we conclude that a direct perfusion bioreactor system is capable of producing clinically relevant volumes of tissue-engineered bone in a bioreactor system which can be monitored on line during cultivation and show bone formation after implantation in nude mice.

Novel synthesis and structural characterization of fluorine and chlorine co-substituted hydroxyapatites.

Fluorine and chlorine co-substituted hydroxyapatites (HAP) were prepared through aqueous precipitation method. Characterization results from X-ray diffraction (XRD) revealed that co-substitution of these anions resulted in the formation of pure HAP phase except for the small observed difference in the lattice parameters of the resultant apatites. The elemental analysis and FT-IR results have also confirmed the incorporation of substituted anions in the apatite structure. The calculated crystallite size for all anionic substituted apatite fall well below the range of 50 nm size that tend to coincide with the value of bone mineral crystallite size.