Bioactivation of calcium deficient hydroxyapatite with foamed gelatin gel. A new injectable self-setting bone analogue.

An alternative approach to bone repair for less invasive surgical techniques, involves the development of biomaterials directly injectable into the injury sites and able to replicate a spatially organized platform with features of bone tissue. Here, the preparation and characterization of an innovative injectable bone analogue made of calcium deficient hydroxyapatite and foamed gelatin is presented. The biopolymer features and the cement self-setting reaction were investigated by rheological analysis. The porous architecture, the evolution of surface morphology and the grains dimension were analyzed with electron microscopy (SEM/ESEM/TEM). The physico-chemical properties were characterized by X-ray diffraction and FTIR analysis. Moreover, an injection test was carried out to prove the positive effect of gelatin on the flow ensuing that cement is fully injectable. The cement mechanical properties are adequate to function as temporary substrate for bone tissue regeneration. Furthermore, MG63 cells and bone marrow-derived human mesenchymal stem cells (hMSCs) were able to migrate and proliferate inside the pores, and hMSCs differentiated to the osteoblastic phenotype. The results are paving the way for an injectable bone substitute with properties that mimic natural bone tissue allowing the successful use as bone filler for craniofacial and orthopedic reconstructions in regenerative medicine.

Magnesium phosphate cements for endodontic applications with improved long-term sealing ability.

AIM: To characterize three radiopaque Magnesium Phosphate Cements (MPCs) developed for endodontic purposes. METHODOLOGY: Three experimental MPCs containing Bi2 O3 were formulated. The experimental cements, which consisted of mixtures of magnesium oxide with different phosphate salts, were characterized for setting time, injectability, porosity, compressive strength and phase composition. The long-term sealing ability of the experimental MPCs applied in single-rooted teeth as root canal filling material or as sealer in combination with gutta-percha was also assessed using a highly sensitive fluid filtration system. A mineral trioxide aggregate (MTA) cement was used as control. Statistical analysis was performed with two- or three-way analysis of variance (anova) and Tukey's test was used for comparisons. RESULTS: The addition of 10 wt% Bi2 O3 within the composition of the MPCs provided an adequate radiopacity for endodontic applications according to ISO 6876 standard. The reaction products resulting from the MPCs were either struvite (MgNH4 PO4 ·6H2 O) or an amorphous sodium magnesium phosphate. The porosity of the three MPCs ranged between 4% and 11%. The initial setting time of the experimental cements was between 6 and 9 min, attaining high early compressive strength values (17-34 MPa within 2 h). All MPC formulations achieved greater sealing ability than MTA (P < 0.05) after 3 months, which was maintained after 6 months for two of the experimental cements (P < 0.05). CONCLUSIONS: These MPCs had adequate handling and mechanical properties and low degradation rates. Furthermore, a stable sealing ability was demonstrated up to 6 months when using the cement both as root filling material and as sealer in conjunction with gutta-percha.

Influence of nanosecond laser surface patterning on dental 3Y-TZP: Effects on the topography, hydrothermal degradation and cell response.

OBJECTIVES: Laser surface micropatterning of dental-grade zirconia (3Y-TZP) was explored with the objective of providing defined linear patterns capable of guiding bone-cell response. METHODS: A nanosecond (ns-) laser was employed to fabricate microgrooves on the surface of 3Y-TZP discs, yielding three different groove periodicities (i.e., 30, 50 and 100 µm). The resulting topography and surface damage were characterized by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). X-Ray diffraction (XRD) and Raman spectroscopy techniques were employed to assess the hydrothermal degradation resistance of the modified topographies. Preliminary biological studies were conducted to evaluate adhesion (6 h) of human mesenchymal stem cells (hMSC) to the patterns in terms of cell number and morphology. Finally, Staphylococcus aureus adhesion (4 h) to the microgrooves was investigated. RESULTS: The surface analysis showed grooves of approximately 1.8 µm height that exhibited surface damage in the form of pile-up at the edge of the microgrooves, microcracks and cavities. Accelerated aging tests revealed a slight decrease of the hydrothermal degradation resistance after laser patterning, and the Raman mapping showed the presence of monoclinic phase heterogeneously distributed along the patterned surfaces. An increase of the hMSC area was identified on all the microgrooved surfaces, although only the 50 µm periodicity, which is closer to the cell size, significantly favored cell elongation and alignment along the grooves. A decrease in Staphylococcus aureus adhesion was observed on the investigated micropatterns. SIGNIFICANCE: The study suggests that linear microgrooves of 50 µm periodicity may help in promoting hMSC adhesion and alignment, while reducing bacterial cell attachment.

Osteoblast-like cellular response to dynamic changes in the ionic extracellular environment produced by calcium-deficient hydroxyapatite.

Solution-mediated reactions due to ionic substitutions are increasingly explored as a strategy to improve the biological performance of calcium phosphate-based materials. Yet, cellular response to well-defined dynamic changes of the ionic extracellular environment has so far not been carefully studied in a biomaterials context. In this work, we present kinetic data on how osteoblast-like SAOS-2 cellular activity and calcium-deficient hydroxyapatite (CDHA) influenced extracellular pH as well as extracellular concentrations of calcium and phosphate in standard in vitro conditions. Since cells were grown on membranes permeable to ions and proteins, they could share the same aqueous environment with CDHA, but still be physically separated from the material. In such culture conditions, it was observed that gradual material-induced adsorption of calcium and phosphate from the medium had only minor influence on cellular proliferation and alkaline phosphatase activity, but that competition for calcium and phosphate between cells and the biomaterial delayed and reduced significantly the cellular capacity to deposit calcium in the extracellular matrix. The presented work thus gives insights into how and to what extent solution-mediated reactions can influence cellular response, and this will be necessary to take into account when interpreting CDHA performance both in vitro and in vivo.

Cell response to collagen-calcium phosphate cement scaffolds investigated for nonviral gene delivery.

Collagen-hydroxyapatite (HA) scaffolds for the non-viral delivery of a plasmid encoding the osteoinductive protein bone morphogenetic protein (BMP)-7 were developed. The collagen-HA was obtained by the combination of calcium phosphate cement in a collagen template. The effect on cell behavior of increasing amounts of HA in the scaffolds was evaluated. Collagen-HA scaffolds containing 13, 23 or 83 wt% HA were prepared. Cell proliferation was reduced in the 83% HA scaffold after 1 day compared to 13 and 23% HA, but by 14 days the number of cells in 83% HA considerably increased. Alkaline phosphatase (ALP) activity was 8 times higher for the 83% HA scaffolds. BMP-7 plasmid was incorporated into the 83% HA scaffold. The transfection was low, although significant levels of BMP7 were expressed, associated with an increase in cell proliferation.

Reduction of Ni release and improvement of the friction behaviour of NiTi orthodontic archwires by oxidation treatments.

This work studies NiTi orthodontic archwires that have been treated using a new oxidation treatment for obtaining Ni-free surfaces. The titanium oxide on the surface significantly improves corrosion resistance and decreases nickel ion release, while barely affecting transformation temperatures. This oxidation treatment avoids the allergic reactions or toxicity in the surrounding tissues produced by the chemical degradation of the NiTi. In the other hand, the lack of low friction coefficient for the NiTi superelastic archwires makes difficult the optimal use of these materials in Orthodontic applications. In this study, the decrease of this friction coefficient has been achieved by means of oxidation treatment. Transformation temperatures, friction coefficient and ion release have been determined.

Fluoride-containing nanoporous calcium-silicate MTA cements for endodontics and oral surgery: early fluorapatite formation in a phosphate-containing solution.

AIM: To test the chemical-physical properties and apatite-forming ability of experimental fluoride-doped calcium silicate cements designed to create novel bioactive materials for use in endodontics and oral surgery. METHODOLOGY: A thermally treated calcium silicate cement (wTC) containing CaCl(2) 5%wt was modified by adding NaF 1%wt (FTC) or 10%wt (F10TC). Cements were analysed by environmental scanning electron microscopy with energy-dispersive X-ray analysis, IR and micro-Raman spectroscopy in wet conditions immediately after preparation or after ageing in a phosphate-containing solution (Dulbecco's phosphate-buffered saline). Calcium and fluoride release and pH of the storage solution were measured. The results obtained were analysed statistically (Tukey's HSD test and two-way anova). RESULTS: The formation of calcium phosphate precipitates (spherulites) was observed on the surface of 24 h-aged cements and the formation of a thick bone-like B-type carbonated apatite layer (biocoating) on 28 day-aged cements. The rate of apatite formation was FTC>F10TC>wTC. Fluorapatite was detected on FTC and F10TC after 1 day of ageing, with a higher fluoride content on F10TC. All the cements released calcium ions. At 5 and 24 h, the wTC had the significantly highest calcium release (P<0.001) that decreased significantly over the storage time. At 3-28 days, FTC and F10TC had significantly higher calcium release than wTC (P<0.05). The F10TC had the significantly highest fluoride release at all times (P<0.01) that decreased significantly over storage time. No significant differences were observed between FTC and wTC. All the cements had a strong alkalinizing activity (OH(-) release) that remained after 28 days of storage. CONCLUSIONS: The addition of sodium fluoride accelerated apatite formation on calcium silicate cements. Fluoride-doped calcium silicate cements had higher bioactivity and earlier formation of fluorapatite. Sodium fluoride may be introduced in the formulation of mineral trioxide aggregate cements to enhance their biological behaviour. F-doped calcium silicate cements are promising bone cements for clinical endodontic use.

Maturation of biomimetic hydroxyapatite in physiological fluids: a physicochemical and proteomic study.

Biomimetic calcium-deficient hydroxyapatite (CDHA) as a bioactive material exhibits exceptional intrinsic osteoinductive and osteogenic properties because of its nanostructure and composition, which promote a favorable microenvironment. Its high reactivity has been hypothesized to play a relevant role in the in vivo performance, mediated by the interaction with the biological fluids, which is amplified by its high specific surface area. Paradoxically, this high reactivity is also behind the in vitro cytotoxicity of this material, especially pronounced in static conditions. The present work explores the structural and physicochemical changes that CDHA undergoes in contact with physiological fluids and to investigate its interaction with proteins. Calcium-deficient hydroxyapatite discs with different micro/nanostructures, coarse (C) and fine (F), were exposed to cell-free complete culture medium over extended periods of time: 1, 7, 14, 21, 28, and 50 days. Precipitate formation was not observed in any of the materials in contact with the physiological fluid, which would indicate that the ionic exchanges were linked to incorporation into the crystal structure of CDHA or in the hydrated layer. In fact, CDHA experienced a maturation process, with a progressive increase in crystallinity and the Ca/P ratio, accompanied by an uptake of Mg and a B-type carbonation process, with a gradual propagation into the core of the samples. However, the reactivity of biomimetic hydroxyapatite was highly dependent on the specific surface area and was amplified in nanosized needle-like crystal structures (F), whereas in coarse specimens the ionic exchanges were restricted to the surface, with low penetration in the material bulk. In addition to showing a higher protein adsorption on F substrates, the proteomics study revealed the existence of protein selectivity toward F or C microstructures, as well as the capability of CDHA, and more remarkably of F-CDHA, to concentrate specific proteins from the culture medium. Finally, a substantial improvement in the material's ability to support cell proliferation was observed after the CDHA maturation process.

Peptidic biofunctionalization of laser patterned dental zirconia: A biochemical-topographical approach.

A dual approach employing peptidic biofunctionalization and laser micro-patterns on dental zirconia was explored, with the aim of providing a flexible tool to improve tissue integration of restorations. Direct laser interference patterning with a femtosecond Ti:Sapphire laser was employed, and two periodic grooved patterns were produced with a periodicity of 3 and 10 µm. A platform containing the cell-adhesive RGD and the osteogenic DWIVA peptides was used to functionalize the grooved surfaces. Topography and surface damage were characterized by confocal laser scanning (CLSM), scanning electron and scanning transmission electron microscopy techniques. The surface patterns exhibited a high homogeneity and subsurface damage was found in the form of nano-cracks and nano-pores, at the bottom of the valleys. Accelerated tests in water steam were carried out to assess hydrothermal degradation resistance, which slightly decreased after the laser treatment. Interestingly, the detrimental effects of the laser modification were reverted by a post-laser thermal treatment. The attachment of the molecule was verified trough fluorescence CLSM and X-ray photoelectron spectroscopy. Finally, the biological properties of the surfaces were studied in human mesenchymal stem cells. Cell adhesion, morphology, migration and differentiation were investigated. Cells on grooved surfaces displayed an elongated morphology and aligned along the patterns. On these surfaces, migration was greatly enhanced along the grooves, but also highly restricted in the perpendicular direction as compared to flat specimens. After biofunctionalization, cell number and cell area increased and well-developed cell cytoskeletons were observed. However, no effects on cell migration were found for the peptidic platform. Although some osteogenic potential was found in specimens grooved with a periodicity of 10 µm, the largest effects were observed from the biomolecule, which favored upregulation of several genes related to osteoblastic differentiation in all the surfaces.

In vivo efficiency of antimicrobial inorganic bone grafts in osteomyelitis treatments.

The purpose of the present work was to evaluate in vivo different antimicrobial therapies to eradicate osteomyelitis created in the femoral head of New Zealand rabbits. Five phosphate-based cements were evaluated: calcium phosphate cements (CPC) and calcium phosphate foams (CPF), both in their pristine form and loaded with doxycycline hyclate, and an intrinsic antimicrobial magnesium phosphate cement (MPC; not loaded with an antibiotic). The cements were implanted in a bone previously infected with Staphylococcus aureus to discern the effects of the type of antibiotic administration (systemic vs. local), porosity (microporosity, i.e. <5 µm vs. macroporosity, i.e. >5 µm) and type of antimicrobial mechanism (release of antibiotic vs. intrinsic antimicrobial activity) on the improvement of the health state of the infected animals. A new method was developed, with a more comprehensive composite score that integrates 5 parameters of bone infection, 4 parameters of bone structural integrity and 4 parameters of bone regeneration. This method was used to evaluate the health state of the infected animals, both before and after osteomyelitis treatment. The results showed that the composite score allows to discern statistically significant differences between treatments that individual evaluations were not able to identify. Despite none of the therapies completely eradicated the infection, it was observed that macroporous materials (CPF and CPFd, the latter loaded with doxycycline hyclate) and intrinsic antimicrobial MPC allowed a better containment of the osteomyelitis. This study provides novel insights to understand the effect of different antimicrobial therapies in vivo, and a promising comprehensive methodology to evaluate the health state of the animals was developed. We expect that the implementation of such methodology could improve the criteria to select a proper antimicrobial therapy.

Control of stem cell response and bone growth on biomaterials by fully non-peptidic integrin selective ligands.

In this communication we report that anchoring αvß3 or α5ß1 integrin-selective RGD peptidomimetics to titanium efficiently tunes mesenchymal stem cell response in vitro and bone growth in rat calvarial defects. Our results demonstrate that this molecular chemistry-derived approach could be successful to engineer instructive coatings for orthopedic applications.

Wet or dry mechanochemical synthesis of calcium phosphates? Influence of the water content on DCPD-CaO reaction kinetics.

Mechanosynthesis of calcium phosphates can be performed under wet or dry conditions. In most papers and patents, grinding under wet conditions was selected. So far, only a few papers were devoted to dry mechanosynthesis of calcium phosphates. To understand why wet mechanosynthesis was preferred, the influence of water addition on the kinetics of the mechanochemical reaction of dicalcium phosphate dihydrate with calcium oxide was investigated. The DCPD disappearance rate constant k and the final reaction time t(f) were determined in each case and correlated with the water content present in the slurry. Results showed that the addition water (i) slowed down the reaction rate and (ii) increased the powder contamination by mill material (hard porcelain) due to ball and vial erosion; and that (iii) wet milling did not generate the expected products, in contrast to dry grinding, because porcelain induced hydroxyapatite decomposition with the formation of beta-tricalcium phosphate and silicon-stabilized tricalcium phosphate. Consequently, dry mechanosynthesis appears preferable to wet milling in the preparation of calcium phosphates of biological interest.

In vitro degradation of calcium phosphates: Effect of multiscale porosity, textural properties and composition.

The capacity of calcium phosphates to be replaced by bone is tightly linked to their resorbability. However, the relative importance of some textural parameters on their degradation behavior is still unclear. The present study aims to quantify the effect of composition, specific surface area (SSA), and porosity at various length scales (nano-, micro- and macroporosity) on the in vitro degradation of different calcium phosphates. Degradation studies were performed in an acidic medium to mimic the osteoclastic environment. Small degradations were found in samples with interconnected nano- and micropores with sizes below 3µm although they were highly porous (35-65%), with maximum weight loss of 8wt%. Biomimetic calcium deficient hydroxyapatite, with high SSA and low crystallinity, presented the highest degradation rates exceeding even the more soluble ß-TCP. A dependence of degradation on SSA was indisputable when porosity and pore sizes were increased. The introduction of additional macroporosity with pore interconnections above 20µm significantly impacted degradation, more markedly in the substrates with high SSA (>15m2/g), whereas in sintered substrates with low SSA (<1m2/g) it resulted just in a linear increase of degradation. Up to 30 % of degradation was registered in biomimetic substrates, compared to 15 % in ß-TCP or 8 % in sintered hydroxyapatite. The incorporation of carbonate in calcium deficient hydroxyapatite did not increase its degradation rate. Overall, the study highlights the importance of textural properties, which can modulate or even outweigh the effect of other features such as the solubility of the compounds. STATEMENT OF SIGNIFICANCE: The physicochemical features of calcium phosphates are crucial to tune biological events like resorption during bone remodeling. Understanding in vitro resorption can help to predict the in vivo behavior. Besides chemical composition, other parameters such as porosity and specific surface area have a strong influence on resorption. The complexity of isolating the contribution of each parameter lies in the close interrelation between them. In this work, a multiscale study was proposed to discern the extent to which each parameter influences degradation in a variety of calcium phosphates, using an acidic medium to resemble the osteoclastic environment. The results emphasize the importance of textural properties, which can modulate or even outweigh the effect of the intrinsic solubility of the compounds.

Evaluation of bone formation in calcium phosphate scaffolds with µCT-method validation using SEM.

There is a plethora of calcium phosphate (CaP) scaffolds used as synthetic substitutes to bone grafts. The scaffold performance is often evaluated from the quantity of bone formed within or in direct contact with the scaffold. Micro-computed tomography (µCT) allows three-dimensional evaluation of bone formation inside scaffolds. However, the almost identical x-ray attenuation of CaP and bone obtrude the separation of these phases in µCT images. Commonly, segmentation of bone in µCT images is based on gray scale intensity, with manually determined global thresholds. However, image analysis methods, and methods for manual thresholding in particular, lack standardization and may consequently suffer from subjectivity. The aim of the present study was to provide a methodological framework for addressing these issues. Bone formation in two types of CaP scaffold architectures (foamed and robocast), obtained from a larger animal study (a 12 week canine animal model) was evaluated by µCT. In addition, cross-sectional scanning electron microscopy (SEM) images were acquired as references to determine thresholds and to validate the result. µCT datasets were registered to the corresponding SEM reference. Global thresholds were then determined by quantitatively correlating the different area fractions in the µCT image, towards the area fractions in the corresponding SEM image. For comparison, area fractions were also quantified using global thresholds determined manually by two different approaches. In the validation the manually determined thresholds resulted in large average errors in area fraction (up to 17%), whereas for the evaluation using SEM references, the errors were estimated to be less than 3%. Furthermore, it was found that basing the thresholds on one single SEM reference gave lower errors than determining them manually. This study provides an objective, robust and less error prone method to determine global thresholds for the evaluation of bone formation in CaP scaffolds.

Critical review: Injectability of calcium phosphate pastes and cements.

Calcium phosphate cements (CPC) have seen clinical success in many dental and orthopaedic applications in recent years. The properties of CPC essential for clinical success are reviewed in this article, which includes properties of the set cement (e.g. bioresorbability, biocompatibility, porosity and mechanical properties) and unset cement (e.g. setting time, cohesion, flow properties and ease of delivery to the surgical site). Emphasis is on the delivery of calcium phosphate (CaP) pastes and CPC, in particular the occurrence of separation of the liquid and solid components of the pastes and cements during injection; and established methods to reduce this phase separation. In addition a review of phase separation mechanisms observed during the extrusion of other biphasic paste systems and the theoretical models used to describe these mechanisms are discussed. STATEMENT OF SIGNIFICANCE: Occurrence of phase separation of calcium phosphate pastes and cements during injection limits their full exploitation as a bone substitute in minimally invasive surgical applications. Due to lack of theoretical understanding of the phase separation mechanism(s), optimisation of an injectable CPC that satisfies clinical requirements has proven difficult. However, phase separation of pastes during delivery has been the focus across several research fields. Therefore in addition to a review of methods to reduce phase separation of CPC and the associated constraints, a review of phase separation mechanisms observed during extrusion of other pastes and the theoretical models used to describe these mechanisms is presented. It is anticipated this review will benefit future attempts to develop injectable calcium phosphate based systems.

Calcium phosphate cements as bone drug delivery systems: a review.

Since calcium phosphate cements were proposed, several formulations have been developed, some of them commercialised, and they have proven to be very efficient bone substitutes in different applications. Some of their properties, such as the injectability, or the low-temperature setting, which allows the incorporation of different drugs, make them very attractive candidates as drug carriers. In this article, the performance of calcium phosphate cements as carriers of different types of drugs, such as antibiotics, analgesics, anticancer, anti-inflammatory, as well as growth factors is reviewed.

Regulating the antibiotic drug release from ß-tricalcium phosphate ceramics by atmospheric plasma surface engineering.

Calcium phosphate (CaP) ceramics are of interest in bone substitution due to their good biocompatibility and bioresorbability. Currently certain CaPs in the market are loaded with antibiotics in order to prevent infections but further control is needed over antibiotic release patterns. Cold plasmas have emerged as a useful means of modifying the interactions with drugs through surface modification of polymer materials. In this work we explore the possibility of using atmospheric pressure plasmas as a tool for the surface modification of these CaP materials with newly populated bonds and charges, with views on enabling higher loading and controlled drug release. Herein the surface modification of ß-tricalcium phosphate ceramics is investigated using an atmospheric pressure helium plasma jet as a tool for tuning the controlled release of the antibiotic doxycycline hyclate, employed as a drug model. The surface chemistry is tailored mainly by plasma jet surface interaction with an increasing O/C ratio without changes in the topography as well as by build-up of surface charges. With this surface tailoring it is demonstrated that the atmospheric plasma jet is a new promising tool that leads to the design of a control for drug release from bioceramic matrices.

Ion-doping as a strategy to modulate hydroxyapatite nanoparticle internalization.

Although it is widely acknowledged that ionic substitutions on bulk hydroxyapatite substrates have a strong impact on their biological performance, little is known of their effect on nanoparticles (NPs) especially when used for gene transfection or drug delivery. The fact that NPs would be internalized poses many questions but also opens up many new possibilities. The objective of the present work is to synthesize and assess the effect of a series of hydroxyapatite-like (HA) NPs doped with various ions on cell behavior, i.e. carbonate, magnesium and co-addition. We synthesized NPs under similar conditions to allow comparison of results and different aspects in addition to assessing the effect of the doping ion(s) were investigated: (1) the effect of performing the cell culture study on citrate-dispersed NPs and on agglomerated NPs, (2) the effect of adding/excluding 10% of foetal bovine serum (FBS) in the cell culture media and (3) the type of cell, i.e. MG-63 versus rat mesenchymal stem cells (rMSCs). The results clearly demonstrated that Mg-doping had a major effect on MG-63 cells with high cytotoxicity but not to rMSCs. This was a very important finding because it proved that doping could be a tool to modify NP internalization. The results also suggest that NP surface charge had a large impact on MG-63 cells and prevents their internalization if it is too negative-this effect was less critical for rMSCs.

In vitro degradation behavior of a novel bioresorbable composite material based on PLA and a soluble CaP glass.

Poly(alpha-hydroxy acids), and in particular polylactic acid (PLA), are nowadays amongst the most used bioabsorbable materials. However, this polymer may not meet some application requirements due to inadequate mechanical properties and or its degradation characteristics. A possible strategy to tackle this problem is the incorporation of an inorganic phase into the polymeric matrix. In this work a new fully biodegradable composite material made with PLA and calcium phosphate soluble glass particles has been developed. The behaviour of the PLA/glass composite has been analysed during its degradation in simulated physiological conditions by means of weight loss, molecular weight and thermal properties analysis and electron microscopy observation. The results showed that the incorporation of phosphate glass particles into the polymer significantly accelerated the degradation of the PLA and induced the formation of calcium phosphate precipitates at the composite surface.

Effect of the particle size on the micro and nanostructural features of a calcium phosphate cement: a kinetic analysis.

The aim of this work is to investigate the possibility of controlling the final micro and nanostructural features of a calcium phosphate cement by modifying the particle size of the starting powder, and to study the effect of this parameter on the kinetics of the setting reaction. The development of calcium phosphate materials with tailored structures at the micro and nanoscale levels could allow the modulation of some specific responses in biologic phenomena such as protein adsorption and cell adhesion, which strongly depend on the nano-sized roughness of the interface. It is shown that the higher specific surface, produced by the reduction of the particle size of the powder, strongly accelerates the hydrolysis of the alpha-TCP into calcium-deficient hydroxyapatite. The higher degree of supersaturation attained in the solution favours the nucleation of smaller crystals. Thus, by increasing the specific surface of the starting powder in a factor of 5, the size of the precipitated crystals is strongly reduced, and the specific surface of the set cement increases by a factor of 2. The reduction of the particle size produces a substantial decrease of the setting time and accelerates the hardening of the cement without significantly affecting the final strength attained. The mechanical strength achieved by the cement cannot be univocally related to the degree of reaction, without considering the microstructural features.