Biomimetic nanocrystalline apatites analogous to bone mineral can be prepared using soft chemistry. Due to their high similarity to bone apatite, as opposed to stoichiometric hydroxyapatite for example, they now represent an appealing class of compounds to produce bioactive ceramics for which drug delivery and ion exchange abilities have been described extensively. However, immersion in aqueous media of dried non-carbonated biomimetic apatite crystals may generate an acidification event, which is often disregarded and not been clarified to-date. Yet, this acidification process could limit their further development if it is not understood and overcome if necessary. This may, for example, alter biological test outcomes, during their evaluation as bone repair materials, due to potentially deleterious effects of the acidic environment on cells, especially in in vitro static conditions. In this study, we explore the origins of this acidification phenomenon based on complementary experimental data and we point out the central role of the hydrated ionic layer present on apatite nanocrystals. We then propose a practical strategy to circumvent this acidification effect using an adequate post-precipitation equilibration step that was optimized. Using this enutralization protocol, we then showed the possibility of performing (micro)biological assessments on such compounds and provide an illustration with the examples of post-equilibrated Cu²⁺- and Ag⁺-doped nanocrystalline apatites. We demonstrate their non-cytotoxicity to osteoblast cells and their antibacterial features as tested versus five major pathogens involved in bone infections, therefore pointing to their relevance in the field of antibacterial bone substitutes. The preliminary in vivo implantation of a relevant sample in a rat's calvarial defect confirmed its biocompatibility and the absence of adverse reaction. Understanding and eliminating this technical barrier should help promoting biomimetic apatites as a genuine new class of biomaterial-producing compounds for bone regeneration applications, e.g., with antibacterial features, far from being solely considered as "laboratory curiosities".

Multi-substituted hydroxyapatites (ms-HAPs) are currently gaining more consideration owing to their multifunctional properties and biomimetic structure, owning thus an enhanced biological potential in orthopaedic and dental applications. In this study, nano-hydroxyapatite (HAP) substituted with multiple cations (Sr2+, Mg2+ and Zn2+) for Ca2+ and anion ( Si O 4 4 - ) for P O 4 3 - and OH-, specifically HAPc-5%Sr and HAPc-10%Sr (where HAPc is HAP-1.5%Mg-0.2%Zn-0.2%Si), both lyophilized non-calcined and lyophilized calcined, were evaluated for their in vitro ions release. These nanomaterials were characterized by scanning electron microscopy, field emission-scanning electron microscopy and energy-dispersive X-ray, as well as by atomic force microscope images and by surface specific areas and porosity. Further, the release of cations and of phosphate anions were assessed from nano-HAP and ms-HAPs, both in water and in simulated body fluid, in static and simulated dynamic conditions, using inductively coupled plasma optical emission spectrometry. The release profiles were analysed and the influence of experimental conditions was determined for each of the six nanomaterials and for various periods of time. The pH of the samples soaked in the immersion liquids was also measured. The ion release mechanism was theoretically investigated using the Korsmeyer-Peppas model. The results indicated a mechanism principally based on diffusion and dissolution, with possible contribution of ion exchange. The surface of ms-HAP nanoparticles is more susceptible to dissolution into immersion liquids owing to the lattice strain provoked by simultaneous multi-substitution in HAP structure. According to the findings, it is rational to suggest that both materials HAPc-5%Sr and HAPc-10%Sr are bioactive and can be potential candidates in bone tissue regeneration.

Vertebrates are practically unique among the Metazoa in their possession of a skeleton made from calcium phosphate rather than calcium carbonate. Interpretation of the origin of a phosphatic skeleton in early vertebrates has previously centered primarily on systemic requirements for phosphate and/or calcium storage or excretion. These interpretations afford no anatomical or physiological advantage(s) that would not have been equally valuable to many invertebrates. We suggest the calcium phosphate skeleton is distinctly advantageous to vertebrates because of their relatively unusual and ancient pattern of activity metabolism: intense bursts of activity supported primarily by rapid intramuscular formation of lactic acid. Bursts of intense activity by vertebrates are followed by often protracted periods of marked systemic acidosis. This postactive acidosis apparently generates slight skeletal dissolution, associated with simultaneous vascular hypercalcemia. A variety of apparently unrelated histological features of the skeleton in a number of vertebrates may minimize this postactive hypercalcemia. We present new data that suggest that postactive skeletal dissolution would be significantly exacerbated if bone were composed of calcium carbonate rather than calcium phosphate. The former is far less stable both in vivo and in vitro than is calcium hydroxyapatite, under both resting and postactive physiological conditions.

Eight dissolution models of calcium apatites (both fluorapatite and hydroxyapatite) in acids were drawn from the published literature, analyzed and discussed. Major limitations and drawbacks of the models were conversed in details. The models were shown to deal with different aspects of apatite dissolution phenomenon and none of them was able to describe the dissolution process in general. Therefore, an attempt to combine the findings obtained by different researchers was performed which resulted in creation of the general description of apatite dissolution in acids. For this purpose, eight dissolution models were assumed to complement each other and provide the correct description of the specific aspects of apatite dissolution. The general description considers all possible dissolution stages involved and points out to some missing and unclear phenomena to be experimentally studied and verified in future. This creates a new methodological approach to investigate reaction mechanisms based on sets of affine data, obtained by various research groups under dissimilar experimental conditions.

The constant composition (CC) method has been used to study the dissolution kinetics of whole powdered human dentin as a function of calcium phosphate concentration at relative undersaturations with respect to hydroxyapatite (sigma HAP), ranging from +0.8 to -2.8, ionic strength from 0.05 to 0.30 mol/L-1 in sodium chloride or potassium nitrate, pH 4.00 to 5.50, and molar calcium/phosphate ratio in the reaction solutions from 0.05 to 11.1. The results suggest that human dentin behaves as a mixture of at least two calcium phosphate phases, HAP-like and octacalcium phosphate-like, OCP-like. Significant dissolution took place in solutions that were even supersaturated with respect to HAP, and the rates exhibited a striking insensitivity to relative undersaturation, while influenced by ionic strength, pH, and molar calcium/phosphate ratio in the reaction solutions. Although the dissolution was retarded in the presence of magnesium ion, the reaction rate showed the same insensitivity to undersaturation with respect to calcium phosphate.

Equilibrium experiments with bone powder, at pH values ranging from 6.3 to 3.5, show a linear relation between log([Ca2+]/[Ca2+]0) (where [Ca2+]0 = 1 M-Ca2+) and pH, indicating that [Ca2+] could reach levels of 25 mM at pH 5 and 90 mM at pH 4. These elevated Ca2+ concentrations stimulated the lysis of insoluble bone collagen in vitro by purified lysosomes and by mouse bone collagenase, whose activities were additive at acid pH. At neutral pH, the addition of 10-100 mM-CaCl2 did not influence the susceptibility of acid-soluble skin collagen in solution towards bone collagenase, but increased it markedly towards collagen in the fibrillar form. Increasing the [Ca2+] did not influence the susceptibility of collagen to trypsin. Elevated [Ca2+] and a co-operation between lysosomal cysteine proteinases and matrix collagenase could thus participate in the osteoclastic breakdown of bone collagen.

Cadmium ions adsorb onto calcium hydroxyapatite crystals (HA) and are as effective as inorganic pyrophosphate and aluminum ions in retarding the rate of in vitro dissolution of HA. In contrast, cadmium ions have no important retarding effect on the growth of HA, but are built into the crystals, thus making them very resistant to subsequent dissolution. These effects could interfere with bone remodeling, with cadmium protecting normal sites of resorption and thus causing resorption at pathological sites.

ATP is as effective as inorganic pyrophosphate in retarding the rate of dissolution of calcium hydroxyapatite at pH 7, ADP is less effective, and AMP is without effect. The inhibition is due to the adsorption of ATP and ADP units on the crystal surface.

The relationship between changes in medium phosphate concentration and three indices of cell-mediated resorption in fetal rat bone cultures--calcium release, the activity of the lysosomal enzyme beta-glucuronidase in the medium, and the morphology of osteoclasts--has been investigated. Bones treated with either 1 mM or 4 mM phosphate, with or without parathyroid hormone, were examined. After 2 h of culture we found the predominant effect of changes in medium phosphate to be on non-cell-mediated resorption. However, after 24 h changes in medium phosphate affected both cell-mediated and non-cell-mediated resorptive mechanisms. The 24 h effects of phosphate were not associated with either a change in the activity of beta-glucuronidase in the medium or in the area of the ruffled border of osteoclasts, but 4 mM phosphate did prevent parathyroid hormone from increasing the area of the clear zone of osteoclasts. These results imply that changes in medium phosphate alter cell-mediated resorption by affecting mechanisms that are independent of increases in beta-glucuronidase activity or changes in the ruffled border of osteoclasts but that may involve effects on the clear zone of osteoclasts.