Calcium-deficient Hydroxyapatite Research Articles

Assessment of Streptococcus mutans biofilm formation on calcium phosphate ceramics: The role of crystalline composition and microstructure.

Streptococcus mutans is one of the bacteria that initiates the colonization of the pellicle at the tooth surface. It forms a plaque, together with other bacteria, which gradually dissolves the pellicle and leaves the tooth surface unprotected against the acidic oral environment. Calcium phosphate ceramics are excellent synthetic materials for the study of biofilm formation in dentistry because they are comparable to teeth in chemical composition and structure. Calcium phosphates can be processed to achieve a variety of crystalline compounds with biologically relevant ionic substitutions and structures that allow study of the effect of the surface chemistry and the topography independently. In this article, we describe the preparation and characterization of three types of calcium phosphate-based materials as a suitable surface for the formation of the S. mutans biofilm: beta-tricalcium phosphate (β-TCP); sintered hydroxyapatite (SHA); and calcium-deficient hydroxyapatite (CDHA). The densest biofilms were formed on the surfaces of SHA and CDHA, with no significant differences due to the stoichiometry or microstructure. In contrast, β-TCP showed a lower susceptibility to S. mutans biofilm formation, suggesting that the crystalline structure is the controlling parameter. Subsequently, SHA was selected to develop a dental biofilm model that allowed study of S. mutans biofilm susceptibility to chlorhexidine and ethanol.

Effect of sodium silicate concentration on the physico-chemical properties of dual-setting bone-like apatite cements

Calcium phosphate cements (CPCs) with their biocompatibility, osteoconductivity and injectability plays an important role in bone defects treatment. Dual-setting cement is a promising approach to improve the physicochemical and biological properties of CPCs in which the liquid phase contains dissolved monomers that polymerized during hardening process. In this study, a series of dual-setting cements were prepared by mixing dicalcium phosphate dihydrate (DCPD) as a solid phase and sodium silicate solution (SS) with different concentrations as hardening liquids. The idea was to evaluate the effect of SS concentration on the setting mechanism, physicochemical and mechanical properties of the formulated cements. The setting kinetics was attributed to the DCPD dissolution and calcium deficient hydroxyapatite (CDHA) precipitation in parallel to SS polymerization to form sodium silicate gel. This was responsible for the formation of an interpenetrating composite material in which the formed gel fills the micropores of the CPC cement. The results showed that the setting time can be modulated by varying the SS concentrates in the hardening liquid. It was shown, as the SS content in the cement liquid increased from 10 to 30 wt%, the setting time decreased from 56 to 5 min. Moreover, the DCPD dissolution and CDHA precipitation were accelerated by increasing the SS concentration. The bone-like apatite cement with the highest SS content exhibited an enhanced compressive strength and high resorption rates. Overall, the addition of sodium silicate gel is a promising approach to regulate the different properties of the apatite-forming cement to be used for bone fracture treatment.

Influence of Fish Scale-Based Hydroxyapatite on Forcespun Polycaprolactone Fiber Scaffolds.

Marine waste byproducts, especially fish scales, have proved to be one of the most prominent sources for developing sustainable materials for various applications including biomedical applications. Hydroxyapatite (HAp), being one of such biomaterials that can be synthesized from the massive fish-based waste, has received plentitude of attention due to its excellent ability to promote cell growth and proliferation. However, understanding the influence of HAp on polymer matrices that are tailored for biomedical applications is still a challenge. This study is intended to develop a sophisticated yet inexpensive method to obtain nonwoven polycaprolactone (PCL) nanofibrous scaffolds and analyze the influence of calcium-deficient nanoporous hydroxyapatite (n-HAp) on the thermal, mechanical, and biological properties of these scaffolds. The n-HAp is synthesized using two different types of fish scales, carpa (CA) and pink perch (PP), by calcination followed by nanomilling. The synthesized n-HAp powder is characterized by using X-ray diffraction, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy. The PCL fibrous scaffolds were developed using a novel forcespinning technique with n-HAp as the filler. The morphology of the scaffolds was characterized using SEM and Raman spectroscopy. SEM and TEM results have confirmed the size reduction of the HAp powder after nanomilling. Thermal properties were analyzed using thermogravimetric analysis and differential scanning calorimetry. The major degradation temperature has increased by 3° and was observed to be 398° for 1 wt % filler loading for both carpa and pink perch-derived n-HAp. The increase in filler content has increased the residue left after decomposition and is 4% for 5 wt % filler loading. The crystallinity percent has increased by 7% compared to neat fibers for 1 wt % filler loading. Mechanical properties were tested using tensile tests. The tensile test strength has shown 32% improvement for 1 wt % compared to neat fibers. Cell viability tests were performed using hFOB cells which have shown significant cell growth for a high filler loading of 5 wt %. The results suggest that both CA-n-HAP and PP-n-Hap-incorporated fibrous scaffolds can be used potentially for biomedical applications after careful investigation of the scaffold behavior with longer incubation periods.

Preparation and characterization of injectable gelatin/alginate/chondroitin sulfate/α-calcium sulfate hemihydrate composite paste for bone repair application.

In this study, a group of injectable composite pastes with a novel formulation consisting of two inorganic components: α-calcium sulfate hemihydrate (α-CSH, P/L = 1.8-2.1g/ml) and calcium-deficient hydroxyapatite (CDHA, P/L = 0.1g/ml) nanoparticles; and three biopolymers: gelatin (2, 4wt. %), alginate (1, 1.5wt. %), and chondroitin sulfate (0.5wt. %) were carefully prepared and thoroughly characterized with commensurate characterizations. The composite sample composed of gelatin (2wt. %), alginate (1.5wt. %), chondroitin sulfate (0.5wt. %), and also CDHA nanoparticles and α-CSH with P/L ratios of 0.1 and 2.1g/ml, respectively, exhibited optimal properties in terms of injectability, anti-washout performance, and rheological characteristics. After 14days of immersion of the chosen sample in the simulated body fluid medium, a dense layer of apatite was formed on the surface of the composite paste. The cellular in vitro tests, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT), alkaline phosphatase assay, 4',6-diamidino-2-phenylindole staining, and cellular attachment, revealed the desirable response of MG-63 cells to the composite paste. The chondroitin sulfate significantly improved the injectability, anti-washout performance, and cellular response of the samples. Considering the promising features of the composite paste prepared in this research work, it could be considered as an alternative injectable bioactive material for bone repair applications.[Formula: see text].

Feasibility study on surface morphology regulation of β-tricalcium phosphate bone graft for enhancing cellular response

Beta-tricalcium phosphate (βTCP) granules are commonly used as an artificial bone graft material. Meanwhile, the surface morphology of the bone graft is an important factor for cellular response. In this study, feasibility study on surface morphology regulation of βTCP bone graft for enhancing cellular response were investigated. The regulation was achieved on the basis of a multistep heating process. Briefly, initial heat treatment of βTCP granules at 1300 °C for 12 h resulted in fabrication of alpha-tricalcium phosphate (αTCP) granules with interconnected micropores. In the second heat treatment, exposure of αTCP granules to 100% relative humidity at 100 °C resulted in partial hydrolysis of αTCP, leading to the fabrication of needle-like calcium-deficient hydroxyapatite (cdHAp) crystals on the αTCP surface. After the third heat treatment at 1100 °C, both cdHAp and αTCP converted back to βTCP, and interconnected micropores with a roughened surface structure were formed. In vitro cell evaluation demonstrated that βTCP granules obtained by the series of heat treatments exhibited 24 times higher cell proliferation at day 9, and four times higher alkaline phosphatase activity compared with untreated βTCP granules. Therefore, we concluded that regulation of surface morphology by a series of heat treatments is useful for improving cellular response to βTCP bone grafting materials.

Differentiation of committed osteoblast progenitors by octacalcium phosphate compared to calcium-deficient hydroxyapatite in Lepr-cre/Tomato mouse tibia

This study aimed to investigate the accumulation and differentiation of mesenchymal stem cells (MSCs) around octacalcium phosphate (OCP) compared with those around calcium-deficient hydroxyapatite (CDHA), a material obtained through hydrolysis of the original OCP. Leptin receptor (Lepr)-expressing bone marrow-derived MSCs around the OCP and CDHA were pursued utilizing genetically modified Lepr-cre/Tomato mice. OCP and CDHA granules were implanted into the tibia defect of the mice for 10 weeks and subjected to histomorphometric and immunohistochemical analyses. The structural properties of OCP and CDHA after inoculation into mouse subcutaneous tissue (until 4 weeks) or culture mediums (14 days) were analyzed using physicochemical techniques. In vitro osteoblastic differentiation of primary MSCs was examined with the materials for 14 days. While Lepr-cre/Tomato positive cells (red) accumulated around both OCP and CDHA, Lepr and osteocalcin double-positive osteoblastic cells (yellow) were significantly more abundant around OCP than around CDHA in the early implantation period. OCP enhanced the osteoblastic differentiation of MSCs more than CDHA in vitro. Physicochemical and structual analyses provided evidence that OCP tended to convert to the apatitic phase in the tested physiological environments. The higher osteoconductivity of OCP originated from a capacity-enhancing osteoblastic differentiation of committed osteoblast progenitors in bone marrow accompanied by OCP hydrolysis. Statement of significanceMSCs play a key role in bone regeneration through osteoblastic differentiation. Calcium phosphates have been widely applied as bone substitute materials, and OCP has a better ability to promote osteoblast differentiation of MSCs than that of HA in vitro. However, it is not clear how MSCs accumulate in the bone marrow and differentiate into osteoblasts during bone regeneration in vivo. In this study, we focused on the leptin receptor, a marker of bone marrow-derived MSCs. Using genetically modified mice labeled with the red fluorescent protein Tomato, we observed the accumulation of MSCs around calcium phosphates implanted in tibia bone defects and their differentiation into osteoblasts.

Solvothermal Synthesis of Calcium-Deficient Hydroxyapatite via Hydrolysis of α-Tricalcium Phosphate in Different Aqueous-Organic Media

In the present work, the effects of various organic solvents (solvent nature and fraction within the solution) and solvothermal conditions on the formation of calcium-deficient hydroxyapatite (CDHA) via hydrolysis of α-tricalcium phosphate (α-TCP) are investigated. The wet precipitation method is applied for α-TCP synthesis, and the hydrolysis reaction is performed in solutions with different water-to-organic solvent ratios under solvothermal conditions at 120 °C for 3 h and at 200 °C for 5 h. Ethyl alcohol, isopropyl alcohol, and butyl alcohol did not inhibit the hydrolysis of α-TCP, while methyl alcohol and ethylene glycol have a more prominent inhibitory effect on the hydrolysis, hence the formation of single-phased CDHA. From all the solvents analysed, ethylene glycol has the highest impact on the sample morphology. Under certain water to ethylene glycol ratios and solvothermal conditions, samples containing a significant fraction of rods are obtained. However, samples prepared with ethylene glycol are characterised by a particularly low BET surface area.

Enhanced Mechanical Properties of 3D Printed Nanocomposites Composed of Functionalized Plant-Derived Biopolymers and Calcium-Deficient Hydroxyapatite Nanoparticles

The combination of plant-derived polymer resins and mineral-based nanoparticles into three dimensional (3D) printable, high-performance nanocomposites suggests a means to improve the sustainability profile of rapid prototyping and additive manufacturing. In this work, our previously published nanocomposite biomaterial system of acrylated epoxidized soybean oil (AESO) and polyethylene glycol (PEGDA) diluent composited with calcium-deficient hydroxyapatite (nHA) nanorods was improved by the substitution of AESO with methacrylated AESO (mAESO) and the partial substitution of PEGDA with isosorbide methacrylate (IM). mAESO was used to increase the degree of crosslinking and reduce the ink viscosity. IM was synthesized by reacting the hydroxyl groups on isosorbide with methacrylic anhydride. The effects of partially replacing PEGDA with IM on the rheology and printability of the nanocomposite inks and the mechanical properties of the resulting nanocomposite materials were quantified. These masked stereolithography (mSLA) printed nanocomposites have greatly improved mechanical properties (tensile strength, Young’s modulus, and Mode-I fracture toughness) due to the shift to mAESO and IM. IM greatly improved the tensile fracture strength and Young’s modulus of the nanocomposites by acting as a reactive diluent and as a stiff segment in the polymer system. Dynamic mechanical analysis revealed that the glass transition temperature of the nanocomposite increased due to the addition of IM. However, IM decreased the strain-at–fracture, making the nanocomposites more brittle. This study demonstrates the development of high-performance mAESO-IM-nHA-based novel nanocomposites that can be easily 3D printed using desktop mSLA, suggesting a facile path forward to improved sustainability in rapid prototyping and additive manufacturing using nanocomposites for a broad range of applications.

Hydroxyapatite formation under calcium-deficient concentration conditions modulated by amino acid-capped gold nanoparticles

This study investigated the effects of the surface modification of gold nanoparticles (AuNPs) with negative, positive, and polar uncharged amino acids on hydroxyapatite formation under calcium-deficient conditions. The AuNPs were prepared using a chemical reduction method and capped with eight different amino acids. The different amino acid (AA) side chains had different interactions with the AuNPs, as indicated by the different shifts in the ultraviolet–visible spectra. The calcium and phosphor sources were added at the same concentration to obtain calcium-deficient hydroxyapatite (CDHA). X-ray diffraction revealed the presence of amino acid-capped AuNPs during synthesis resulting in CDHA with low crystallinity. The calculated crystallinity index (CI) indicated that aspartic acid could promote the crystallization of biomimetic hydroxyapatite. The crystallite size decreased by 6%–62%, which is beneficial for osteoinductivity in bone substitute applications. The involvement of AA-AuNPs in the synthesized hydroxyapatite resulted in apatite with a higher Ca/P ratio than that of the control except for cysteine, lysine, and glutamine. The morphology of the samples ranged from quasi-spherical to needle-like, which can potentially be used as an osteoinductive material.

Nanosized calcium deficient hydroxyapatites for tooth enamel protection.

Calcium phosphates (CaP) are extensively studied as additives to dental care products for tooth enamel protection against caries. However, it is not clear yet whether substituted CaP could provide better enamel protection. In this study we produced, characterized and tested in vitro substituted and co‐substituted calcium deficient hydroxyapatite (CDHAp) with Sr2+ and F− ions. X‐ray powder diffractometry, Fourier transformation infrared spectroscopy, scanning electron microscopy, energy‐dispersive X‐ray analysis, Brunauer–Emmett–Teller were used to characterize synthesized powders and also cytotoxicity was evaluated. pH = f(t) test was performed to estimate, weather synthesized CDHAp suspensions are able to increase pH of experimental media after acid addition. Synthesis products were incorporated into paste to perform in vitro remineralization on the bovine enamel. In addition to mentioned instrumental methods, profilometry was used for evaluation of remineralised enamel samples. The obtained results confirmed formation of CDHAp substituted with 1.5–1.6 wt% of fluoride and 7.4–7.8 wt% of strontium. pH = f(t) experiment pointed out that pH increased by approximately 0.3 within 10 min after acid addition for all CDHAp suspensions. A new layer of the corresponding CDHAp was formed on the enamel. Its thickness increased by 0.8 ± 0.1 μm per day and reached up to 5.8 μm after 7 days. Additionally, octa calcium phosphates were detected on the surface of control samples. In conclusion, we can assume that CDHAp substituted with Sr2+ and/or F− could be used as an effective additive to dental care products promoting formation of protecting layer on the enamel, but there was no significant difference among sample groups.

Influence of the Metal Incorporation into Hydroxyapatites on the Deactivation Behavior of the Solids in the Esterification of Glycerol

The effects of the metal incorporation into hydroxyapatites on the deactivation behavior of the solids were examined in the esterification of glycerol (EG) reaction. The introduction of Cu, Co, or Ni ions by ion exchange in calcium-deficient hydroxyapatites resulted in active catalysts for the EG reaction. The metal contents were varied from 2.0 to 17.0%, providing better performances at rather high metal contents. Part of metal species existed in the hydroxyapatite lattice structure and also as isolated Cu2+, Ni2+, and Co2+ entities on the surface, as shown by XPS and EPR. The effects of the reaction temperature, reaction time, and glycerol to acetic acid molar ratios were deeply investigated. The spent solids used in this study were characterized by XRD, FTIR, SEM-EDS, chemical analyses, EPR, and XPS. The Cu2+–OH acid pairs could promote a superior catalytic performance of Cu-containing hydroxyapatites due to the resistance of these solids against leaching of the active species, which is even better than those of Co and Ni-containing counterparts with high metal contents. Cu into hydroxyapatite had a good reusability and long-term utilization for five consecutive cycles of 24 h under a glycerol to acetic acid molar ratio of 0.25 at 80 °C, and longer reaction times provide triacetin formation. This was due to the fact that Cu was stabilized by interacting with Ca, PO4, and OH sites into the hydroxyapatite lattice, being highly active for the EG reaction. The results also revealed that isolated Cu2+ sites played an important role in enhancing the glycerol conversion, intrinsically due to the Cu-containing hydroxyapatites ability to avoid strong adsorption of glycerol oligomers on the catalytic sites.

In vivo effectiveness of carbonated calcium-deficient hydroxyapatite-coated activated carbon fiber cloth on bone regeneration.

We have previously shown that activated carbon fiber cloth (ACC) either uncoated or coated with carbonated calcium-deficient hydroxyapatite (CDA), namely ACC and ACC/CDA, were biocompatible in vitro with human osteoblasts. Here we hypothesized that ACC and ACC/CDA could be used as tissue patches in vivo to accelerate wounded bone healing. In a model of rat femoral defect, we have compared spontaneous cortical bone regeneration with regeneration in the presence of ACC and ACC/CDA patches. At Day 7, 14, and 21, bone formation was evaluated using microcomputed tomography, magnetic resonance imaging, and histological analysis. Our results demonstrate first that these ACC tissues are highly biocompatible in vivo, and second that ACC/CDA patches apposition results in the acceleration of bone reconstruction due to a guiding action of the ACC fibers and an osteogenic effect of the CDA phase. We guess that this approach may represent a valuable strategy to accelerate bone regeneration in human.

Nanocomposites based on apatitic tricalcium phosphate and autofibrin

Nanocomposites based on apatitic tricalcium phosphate in an autofibrin matrix were obtained by precipitation at a Ca/P ratio of 1.50, pH 9 and a maturation time from 30 min to 7–14 days. The resorbability of nanocomposites was determined by the composition of calcium phosphates, which, during long-term maturation, formed as the calcium-deficient hydroxyapatite with a Ca/P ratio of 1.66, whereas biopolymer matrix favored the formation of more soluble calcium phosphates with a Ca/P ratio of 1.53–1.59. It was found that the fibrin clot stabilized, along with apatitic tricalcium phosphate, the phase of amorphous calcium phosphate, which after 800 °C was transformed into resorbable α-tricalcium phosphate. Citrated plasma inhibited the conversion of apatitic tricalcium phosphate into stoichiometric hydroxyapatite, which also facilitated the formation of resorbable β-tricalcium phosphate after 800 °C. The combined effect of the maturation time and the biopolymer matrix determined the composition, physicochemical and morphological properties of nanocomposites and the possibililty to control its extent of resorption

Selective Detection of an Infection Biomarker by an Osteo-Friend Scaffold: Development of a Multifunctional Artificial Bone Substitute.

Developments in three-dimensional (3D) printing technologies have led to many potential applications in various biomedical fields, especially artificial bone substitutes (ABSs). However, due to the characteristics of artificial materials, biocompatibility and infection remain issues. Here, multifunctional ABSs have been designed to overcome these issues by the inclusion of a biochemical modality that allows simultaneous detection of an infection biomarker by osteo-friend 3D scaffolds. The developed multifunctional scaffolds consist of calcium-deficient hydroxyapatite (CDHA), which has a similar geometric structure and chemical composition to human bone, and gold nanoparticles (Au NPs), which assists osteogenesis and modulates the fluorescence of labels in their microenvironment. The Au NPs were subsequently conjugated with fluorescent dye-labeled probe DNA, which allowed selective interaction with a specific target biomarker, and the fluorescent signal of the dye was temporally quenched by the Au NP-derived Förster resonance energy transfer (FRET). When the probe DNA unfolded to bind to the target biomarker, the fluorescence signal was recovered due to the increased distance between the dye and Au NPs. To demonstrate this sensing mechanism, a microbial oligonucleotide was selected as a target biomarker. Consequently, the multifunctional scaffold simultaneously facilitated osteogenic proliferation and the detection of the infection biomarker.

Effect of the liquid-to-powder ratio on physicochemical properties of calcium phosphate cements and of these properties over Biofilm thickness of adhered Staphylococcus Aureus

Tricalcium phosphate (TCP) synthetized by high temperature solid state reaction at 1400°C and three derived calcium phosphate cements (CPCs) prepared at liquid to powder (L/P) ratios of 0.33, 0.44 and 0.55 ml/g, respectively, were physicochemically characterized. Calcium deficient hydroxyapatite crystals were identified by scanning electron microscopy on CPC, and differences in crystal sizes were observed at different L/P ratios. Also, the biofilm thickness of two Staphylococcus Aureus (S.aureus) strains grown for 24 hours on the three CPC are reported. A dependence of the biofilm thickness with the specific surface area (SSA) of CPC was identified. They are directly proportional for non-extracellular polysaccharide substances (EPS) producing S.aureus and inversely proportional for EPS producing S.aureus. Non-proportional behavior between the SSA and mechanical strength of the CPC was observed as L/P ratio increases.

Activated Carbon Fiber Cloth/Biomimetic Apatite: A Dual Drug Delivery System

A biomaterial that is both bioactive and capable of controlled drug release is highly attractive for bone regeneration. In previous works, we demonstrated the possibility of combining activated carbon fiber cloth (ACC) and biomimetic apatite (such as calcium-deficient hydroxyapatite (CDA)) to develop an efficient material for bone regeneration. The aim to use the adsorption properties of an activated carbon/biomimetic apatite composite to synthetize a biomaterial to be used as a controlled drug release system after implantation. The adsorption and desorption of tetracycline and aspirin were first investigated in the ACC and CDA components and then on ACC/CDA composite. The results showed that drug adsorption and release are dependent on the adsorbent material and the drug polarity/hydrophilicity, leading to two distinct modes of drug adsorption and release. Consequently, a double adsorption approach was successfully performed, leading to a multifunctional and innovative ACC-aspirin/CDA-tetracycline implantable biomaterial. In a second step, in vitro tests emphasized a better affinity of the drug (tetracycline or aspirin)-loaded ACC/CDA materials towards human primary osteoblast viability and proliferation. Then, in vivo experiments on a large cortical bone defect in rats was carried out to test biocompatibility and bone regeneration ability. Data clearly highlighted a significant acceleration of bone reconstruction in the presence of the ACC/CDA patch. The ability of the aspirin-loaded ACC/CDA material to release the drug in situ for improving bone healing was also underlined, as a proof of concept. This work highlights the possibility of bone patches with controlled (multi)drug release features being used for bone tissue repair.

Thermodynamic and experimental studies on removal of calcium and sulfate ions from recycling water of complex sulfide flotation operations

A chemical method for removing calcium sulfate saturated solutions (0.016 mol/L CaSO4) using barium chloride (BaCl2·2H2O) and sodium phosphate (Na3PO4) was experimentally studied. The main interest is to remove these ions from the solution through the precipitation of two solid species: sulfate (SO42−) as barite (BaSO4), and calcium (Ca2+) as hydroxyapatite (Ca5(PO4)3OH). Additionally, a solid/liquid separation method (i.e., flotation) was explored, using oleic acid and dodecylamine as collectors. The results show that, the chemical treatment of saturated solutions at 60 °C, pH 11.5 and using 3.9 g/L BaCl2·2H2O and 1.6 g/L Na3PO4, promotes the precipitation of barium sulfate and calcium-deficient hydroxyapatite (Ca10–x(HPO4)x(PO4)6–x(OH)2–x), with residual concentrations of calcium and sulfate below 0.10 and 5 mg/L, respectively. The residual calcium concentration increases to 28 mg/L when using the same amount of reactants, at temperature and pH values below those quoted. The highest flotation recovery of hydroxyapatite with oleic acid at pH 9.5 was about 80%, while that of barite floated with dodecylamine at pH 6.5 was about 90%.

Biomimetic versus sintered macroporous calcium phosphate scaffolds enhanced bone regeneration and human mesenchymal stromal cell engraftment in calvarial defects

In contrast to sintered calcium phosphates (CaPs) commonly employed as scaffolds to deliver mesenchymal stromal cells (MSCs) targeting bone repair, low temperature setting conditions of calcium deficient hydroxyapatite (CDHA) yield biomimetic topology with high specific surface area. In this study, the healing capacity of CDHA administering MSCs to bone defects is evaluated for the first time and compared with sintered beta-tricalcium phosphate (β-TCP) constructs sharing the same interconnected macroporosity. Xeno-free expanded human bone marrow MSCs attached to the surface of the hydrophobic β-TCP constructs, while infiltrating the pores of the hydrophilic CDHA. Implantation of MSCs on CaPs for 8 weeks in calvaria defects of nude mice exhibited complete healing, with bone formation aligned along the periphery of β-TCP, and conversely distributed within the pores of CDHA. Human monocyte-osteoclast differentiation was inhibited in vitro by direct culture on CDHA compared to β-TCP biomaterials and indirectly by administration of MSC-conditioned media generated on CDHA, while MSCs increased osteoclastogenesis in both CaPs in vivo. MSC engraftment was significantly higher in CDHA constructs, and also correlated positively with bone in-growth in scaffolds. These findings demonstrate that biomimetic CDHA are favorable carriers for MSC therapies and should be explored further towards clinical bone regeneration strategies. Statement of significanceDelivery of mesenchymal stromal cells (MSCs) on calcium phosphate (CaP) biomaterials enhances reconstruction of bone defects. Traditional CaPs are produced at high temperature, but calcium deficient hydroxyapatite (CDHA) prepared at room temperature yields a surface structure more similar to native bone mineral. The objective of this study was to compare the capacity of biomimetic CDHA scaffolds with sintered β-TCP scaffolds for bone repair mediated by MSCs for the first time. In vitro, greater cell infiltration occurred in CDHA scaffolds and following 8 weeks in vivo, MSC engraftment was higher in CDHA compared to β-TCP, as was bone in-growth. These findings demonstrate the impact of material features such as surface structure, and highlight that CDHA should be explored towards clinical bone regeneration strategies.

Hydrothermal processing of 3D-printed calcium phosphate scaffolds enhances bone formation in vivo: a comparison with biomimetic treatment

Hydrothermal (H) processes accelerate the hydrolysis reaction of α-tricalcium phosphate (α-TCP) compared to the long-established biomimetic (B) treatments. They are of special interest for patient-specific 3D-printed bone graft substitutes, where the manufacturing time represents a critical constraint. Altering the reaction conditions has implications for the physicochemical properties of the reaction product. However, the impact of the changes produced by the hydrothermal reaction on the in vivo performance was hitherto unknown. The present study compares the bone regeneration potential of 3D-printed α-TCP scaffolds hardened using these two treatments in rabbit condyle monocortical defects. Although both consolidation processes resulted in biocompatible scaffolds with osseointegrative and osteoconductive properties, the amount of newly formed bone increased by one third in the hydrothermal vs the biomimetic samples. B and H scaffolds consisted mostly of high specific surface area calcium-deficient hydroxyapatite (38 and 27 m2 g-1, respectively), with H samples containing also 10 wt.% β-tricalcium phosphate (β-TCP). The shrinkage produced during the consolidation process was shown to be very small in both cases, below 3%, and smaller for H than for B samples. The differences in the in vivo performance were mainly attributed to the distinct crystallisation nanostructures, which proved to have a major impact on permeability and protein adsorption capacity, using BSA as a model protein, with B samples being highly impermeable. Given the crucial role that soluble proteins play in osteogenesis, this is proposed to be a relevant factor behind the distinct in vivo performances observed for the two materials. Statement of significanceThe possibility to accelerate the consolidation of self-setting calcium phosphate inks through hydrothermal treatments has aroused great interest due to the associated advantages for the development of 3D-printed personalised bone scaffolds. Understanding the implications of this approach on the in vivo performance of the scaffolds is of paramount importance. This study compares, for the first time, this treatment to the long-established biomimetic setting strategy in terms of osteogenic potential in vivo in a rabbit model, and relates the results obtained to the physicochemical properties of the 3D-printed scaffolds (composition, crystallinity, nanostructure, nanoporosity) and their interaction with soluble proteins.

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.