Biphasic Calcium Phosphate Ceramics Research Articles

Study of biphasic calcium phosphate (BCP) ceramics of tilapia fish bones by age

Biphasic calcium phosphate (BCP), consisting of bioceramics such as HAp + β-TCP and Ca10(PO4)6(OH)2 + Ca3(PO4)2, is a popular choice for optimizing performance due to its superior biological reabsorption and osseointegration. In this study, BCP was produced by calcining the bones of tilapia fish (Oreochromis niloticus) reared in net cages and slaughtered at an age ranging from 15 to 420 days. The bones were cleaned and dried, calcined at 900 °C for 8 h, and then subjected to high-energy grinding for 3 h to produce BCP powders. After the calcination process, the crystalline phase’s hydroxyapatite (HAp) and/or beta-tricalcium phosphate (β-TCP) were present in the composition of the bioceramic. The age-dependent variation in phase composition was confirmed by complementary vibrational spectroscopy techniques, revealing characteristic peaks and bands of the bioceramic. This variation was marked by an increase in HAp phase and a decrease in β-TCP phase. Thermogravimetric Analysis (TGA) and Differential Thermal Analysis (DTA) from 25 to 1400 °C showed the characteristic mass losses of the material, with a greater loss observed for younger fish, indicating the complete removal of organic components at temperatures above 600 °C. Comparison of the results obtained by X-Ray Diffraction (XRD) and Rietveld refinement with Raman spectroscopy showed excellent agreement. These results showed that with temperature and environment control and adequate fish feeding, it is possible to achieve the desired amounts of each phase by choosing the ideal age of the fish. This bioceramic enables precise measurement of HAp and β-TCP concentrations and Ca/P molar ratio, suitable for medical orthopedics and dentistry.

Calcium phosphate ceramic-induced osteoimmunomodulation: Submicron-surface-treated macrophage-derived exosomes driving osteogenesis

The development of osteoimmunology has highlighted the crucial role of biomaterial surface topography in modulating macrophage responses, subsequently affecting bone formation. However, the underlying mechanism remains largely unexplored. This study introduced two biphasic calcium phosphate (BCP) ceramics with different surface architecture, finding that compared to micron-scale counterparts (BCP2, ∼3.07 μm), BCP with submicron-scale structure (BCP1, ∼0.66 μm) enhanced ectopic bone formation and facilitated M2 macrophage polarization in vivo. Previous studies have focused predominantly on soluble cytokines secreted by macrophages, the significance of exosomes in intercellular communication has been overlooked. This study isolated exosomes from macrophages treated with either submicron- or micron-scale surfaces, designated as B1-Exos and B2-Exos, respectively. Notably, B1-Exos could significantly improve osteogenic differentiation of mesenchymal stem cells (MSCs), potentially attributable to miRNA cargos within these exosomes. SmallRNA sequencing, western blotting, qRT-PCR, and bioinformatics analyses indicated that submicron-sized BCP1 ceramic markedly elevated miR-142a-5p levels in macrophage-derived exosomes, activating the PTEN/AKT signaling pathway, and consequently guiding the differentiation of MSCs towards osteoblast lineage. These findings not only deepen our understanding of the biomaterial-mediated osteoinduction mechanism, but also inform the design of bone repair materials with specialized surface design for proper immunomodulation to initiate osteogenesis.

Effects of void defects on the mechanical properties of biphasic calcium phosphate nanoparticles: A molecular dynamics investigation

Porous biphasic calcium phosphate (BCP) ceramics are widely used in bone tissue engineering, and the mechanical properties of BCP implants must be reliable. However, the effects of pore structure (e.g., shape and size) on the mechanical properties are not well understood. In this study, we used molecular dynamics simulations to investigate the influence of pore shape and size on the mechanical behavior of BCP nanoparticles. BCP void models with cylindrical and cuboid pores ranging from 2 to 16 nm in diameter were constructed, and the elastic moduli were calculated. In addition, uniaxial tensile and compressive tests were performed on the models. We found that the pore size had a more significant impact on the mechanical properties of BCP than pore shape. Further, the elastic moduli decreased nonlinearly with increasing pore size. In addition, the tensile and compressive strength also decreased with the increase in pore size, but the ductility improved. Furthermore, deformation and fracture were more likely to occur near the pores and at the phase interfaces as a result of high atomic local strain in the calcium-deficient hydroxyapatite area. The results of this work reveal the effects of pore parameters on the mechanical properties of porous BCP at the nanometer level, which may aid the design of improved porous and multiphase CaP-based biomaterials for bone regeneration.

The role of integrin αvβ3 in biphasic calcium phosphate ceramics mediated M2 Macrophage polarization and the resultant osteoinduction

Calcium phosphate ceramics-based biomaterials were reported to have good biocompatibility and osteoinductivity and have been widely applied for bone defect repair and regeneration. However, the mechanism of their osteoinductivity is still unclear. In our study, we established an ectopic bone formation in vivo model and an in vitro macrophage cell co-culture system with calcium phosphate ceramics to investigate the effect of biphasic calcium phosphate on osteogenesis via regulating macrophage M1/M2 polarization. Our micro-CT data suggested that biphasic calcium phosphate had significant osteoinductivity, and the fluorescence co-localization detection found increased F4/80+/integrin αvβ3+ macrophages surrounding the biphasic calcium phosphate scaffolds. Besides, our study also revealed that biphasic calcium phosphate promoted M2 polarization of macrophages via upregulating integrin αvβ3 expression compared to tricalcium phosphate, and the increased M2 macrophages could subsequently augment the osteogenic differentiation of MSCs in a TGFβ mediated manner. In conclusion, we demonstrated that macrophages subjected to biphasic calcium phosphate could polarize toward M2 phenotype via triggering integrin αvβ3 and secrete TGFβ to increase the osteogenesis of MSCs, which subsequently enhances bone regeneration.

One-step co-doping of ZnO and Zn2+ in osteoinductive calcium phosphate ceramics with synergistic antibacterial activity for regenerative repair of infected bone defect

How to endow bone grafts with long-term antibacterial activity and good bone regenerative ability to achieve the regenerative repair of infected bone defects has been the focus of the clinical treatment of osteomyelitis. The present study introduced a novel one-step route to realizing the co-doping of zinc oxide (ZnO) and zinc ion (Zn2+) in biphasic calcium phosphate (BCP) ceramics to utilize their synergistic antibacterial. Compared with the conventional BCP ceramics (BCP-Ca), the ZnO/Zn2+ co-doping ones (BCP-Zn) possessed strong antibacterial ability on E. coli and S. aureus as well as stimulated the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) effectively. The synergistic antibacterial mechanism of ZnO and Zn2+ was also investigated. BCP-Zn showed excellent osteoinductivity and angiogenesis at three months postoperatively in the canine intramuscular implantation model. Moreover, BCP-Zn exhibited excellent anti-infective ability and bone regenerative repair compared to BCP-Ca and control groups in the infected bone defect model of rat femur. Collectively, these findings suggest that the simultaneous introduction of ZnO/Zn2+ could have immense potential to expand the application of osteoinductive BCP ceramics in the regenerative repair of infected bone defects.

Nanowhiskers Orchestrate Bone Formation and Bone Defect Repair by Modulating Immune Cell Behavior.

Immunomodulatory biomaterials have emerged as promising treatment agents for bone defects. However, it is unclear how such biomaterials control immune cell behaviors to facilitate large-segment bone defect repair. Herein, we fabricated biphasic calcium phosphate ceramics with nanowhisker structures to explore the immunoregulation features and influence on large-segment bone defect repair. We found that the nanowhisker structures markedly facilitated large-segment bone defect repair by promoting bone regeneration and scaffold resorption. Our in vitro experiment and transcriptomic analysis showed that mechanical stress derived from nanowhisker structures may activate the transcription of Egr-1 to induce early switch of macrophage phenotype to M2, which could not only facilitate osteogenic differentiation of BMSCs but also enhance the expression of osteoclast differentiation-regulating genes of M2 macrophage. In vivo study showed that the nanowhisker structures relieved local inflammatory responses by inducing early switch of macrophage phenotype from M1 to M2, which resulted in accelerated osteoclastogenesis for biomaterial resorption and osteogenesis for ectopic bone formation. Hence, we presume that nanowhisker structures may orchestrate bone formation and material resorption coupling to facilitate large-segment bone defect repair by controlling the switch of macrophage phenotype. This study provides new insight into the designing of immunomodulatory tissue engineering biomaterials for treating large-segment bone defects.

Molecular Dynamics Simulation of Biomimetic Biphasic Calcium Phosphate Nanoparticles.

Biphasic calcium phosphate (BCP) is used as a bone substitute and bone tissue repair material due to its better control over bioactivity and biodegradability. It is crucial to stabilize the implanted biomaterial while promoting bone ingrowth. However, a lack of standard experimental and theoretical protocols to characterize the physicochemical properties of BCP limits the optimization of its composition and properties. Computational simulations can help us better to learn BCP at a nanoscale level. Here, the Voronoi tessellation method was combined with simulated annealing molecular dynamics to construct BCP nanoparticle models of different sizes, which were used to understand the physicochemical properties of BCP (e.g., melting point, infrared spectrum, and mechanical properties). We observed a ∼20 to 30 Å layer of calcium-deficient hydroxyapatite at the HAP/β-TCP interface due to particle migration, which may contribute to BCP stability. The BCP model may stimulate further research into BCP ceramics and multiphasic ceramics. Moreover, our study may facilitate the optimization of compositions of BCP-based biomaterials.

Enhanced reparative dentinogenesis of biphasic calcium phosphate ceramics containing calcium-deficient hydroxyapatite (CDHA) and strontium-incorporated CDHA in direct pulp capping

Despite that dental materials and therapeutic strategies have made progress, reserving dental pulp vitality remains a great challenge during clinical practices of dentistry. Biphasic calcium phosphate (BCP) ceramic is a promising biomaterial for bone regeneration and dentin restoration, whose biological activities need to be further improved to meet the needs of regenerative medicine. Calcium-deficient hydroxyapatite (CDHA), which is more similar in composition to natural bone and tooth minerals, imparts superior bioactivity to BCP ceramics. Besides, owing to the positive roles of strontium in stimulating bone formation, the strontium-incorporated CDHA has excellent biocompatibility and further promotes bone regeneration. However, the effect of CDHA and strontium-incorporated CDHA in dental pulp capping has been rarely investigated. Herein, we presented an effective method to integrate CDHA and strontium-incorporated CDHA into BCP ceramics and investigated their biological properties as a potential strategy for dental pulp-capping and dentin regeneration. The characterization of phase composition and crystal structure confirmed that novel BCP ceramics with high CDHA content (BCP-A) and strontium-incorporated CDHA (BCP-A/Sr) have been successfully synthesized. In vitro, BCP-C (conventional BCP), BCP-A, and BCP-A/Sr provided an appropriate microenvironment and significantly promoted the proliferation of human dental pulp cells (hDPCs). BCP-A did not affect the migration of hDPCs, yet BCP-C and BCP-A/Sr displayed a suppressing trend. In vivo, BCP-A significantly reduced the inflammation response and improved the formation of reparative dentin bridges. Compared with mineral trioxide aggregate (MTA), a clinical direct pulp capping material, the texture of reparative dentin bridges in BCP-A showed more uniformity. Moreover, RNA-seq analysis further clarified that direct co-culture of hDPCs with BCP-A mainly up-regulated the cell cycle signaling pathway and CDC20 protein. These findings indicate that the novel BCP-A is a promising biomaterial for direct dental pulp capping due to its unique intrinsic biological activities, which warrants further investigation.

A Novel Hollow‐Tube‐Biphasic‐Whisker‐Modified Calcium Phosphate Ceramics with Simultaneously Enhanced Mechanical Strength and Osteogenic Activity

AbstractDue to the inherent brittleness and low mechanical strength, it is still a challenge for calcium phosphate (Ca‐P) ceramics to be used in load‐bearing bone defect repair. To achieve a good balance between mechanical strength and osteogenic activity, hollow‐tube‐whisker‐modified biphasic calcium phosphate (BCP) ceramics (BCP‐HW) are successfully fabricated by an in situ growth process in the present study. Compared to the initial BCP ceramics (BCP‐C) and those with solid whiskers, BCP‐HW exhibits larger specific surface area (3.9 times vs BCP‐C) and higher mechanical strength (3.4 times vs BCP‐C), endowing it with stronger stimulation on adhesion, proliferation, and osteogenic differentiation of bone marrow mesenchymal stem cells. In an intramuscular implantation model of canine, BCP‐HW shows excellent osteoinductivity and promotes the maturation of new bone, and the resultant compressive strength of the implant increases to ≈12 MPa at 3 months postoperatively. In another critical‐sized segmental bone defect model of rabbit femur, BCP‐HW has the best repairing effect. After implantation for 6 months, much more new bone ingrowth and higher bending load are observed in BCP‐HW than BCP‐C. Collectively, these findings suggest that the in situ hollow‐tube whisker construction possesses immense potential in expanding the applications of Ca‐P ceramics to load‐bearing bone defect repair.

HA/β-TCP Biphasic Calcium Phosphate Ceramics Derived From Butterfish Bones Loaded With Bone Marrow Mesenchymal Stem Cells Promote Osteogenesis

Biphasic calcium phosphate (BCP) containing hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) is considered the gold standard for bone repair. However, the complex synthesis of BCP restricts the wide application of BCP. In the preliminary work, we proved that the HA/β-TCP BCP ceramic material extracted from fishery waste had a good prospect as a bone replacement graft material for filling bone defects. This study aimed to explore the osteogenesis effect of BCP material derived from butterfish bones in vivo and in vitro. After loading human bone marrow mesenchymal stem cells (hBMSCs) with materials, we used scanning electron microscopy to observe cell adhesion and survival. Western blot analysis was used to detect osteogenic expression in vitro. Micro-computed tomography and hematoxylin–eosin staining were used to detect bone regeneration and material degradation rate in the rat femoral defect model. The results showed that hBMSCs grew well and adhered closely to the material. In vitro, the expression levels of bone formation–related markers ALP and Runx-2 of butterfish bones calcined at 900°C were generally higher than those in the other groups. Notably, in vivo, the osteogenesis ability of butterfish bones calcined at 900°C was almost comparable to that of the most commonly used Bio-Oss in clinical practice and was significantly better than that in the other groups. In summary, the BCP ceramic material derived from butterfish bones had good biocompatibility, osteoconductivity, and osteoinductivity, and had a good application prospect in the field of bone graft substitutes.

Digital light processing (DLP) of nano biphasic calcium phosphate bioceramic for making bone tissue engineering scaffolds

Nano-sized ceramic particles are beneficial for the properties of 3D printed structures via digital light processing (DLP). However, achieving optimal conditions for the preparation and processing of ceramic nanoparticle-based slurry is a significant challenge. In this work, a stable and well dispersed slurry with nano-sized biphasic calcium phosphate (BCP) powders was developed and porous BCP bioceramic scaffolds with good bioactivity were accurately fabricated via the DLP additive manufacturing technology. Effects of dispersant, photoinitiator and solid loading on rheological properties and curing abilities of BCP slurries were systematically studied. The slurry having 65 wt % of nano-sized BCP powders showed a relatively low viscosity of 400 mPa s at the 50 s−1 shear rate. It was found that the optimal photoinitiator concentration was tightly associated with the energy dosage in DLP. Furthermore, the effects of sintering temperature (1100 °C, 1200 °C and 1300 °C) on the surface morphology, shrinkage, phase constitution, hardness, compressive properties, and in vitro biological performance of DLP-formed bioceramics were comprehensively investigated. The results showed that DLP-formed BCP ceramics sintered at 1300 °C had the best mechanical properties, in vitro cytocompatibility, and homogeneous bone-like apatite formation ability. Finally, DLP-formed 3D scaffolds with different pore sizes ranging from 300 μm to 1 mm were successfully fabricated, which exhibited high fidelity and accuracy. The current study has demonstrated the great potential of using DLP technology to construct functional complex BCP bioceramic scaffolds for bone tissue regeneration.

Dynamics of the natural genesis of β-TCP/HAp phases in postnatal fishbones towards gold standard biocomposites for bone regeneration

The search for gold-standard materials for bone regeneration is still a challenge in reconstruction surgery. The ratio between hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP) in biphasic calcium phosphate ceramics (BCPs) is one of the most important factors in osteoinduction promotion and controlled biodegradability, configurating what is currently considered as a possible gold standard material for bone substitution in reconstructive surgery. Exploring the natural genesis of the HAp and β-TCP phases in fishbones during their postnatal growth, this study developed a biphasic bioceramic obtained from the calcination of Nile tilapia (Oreochromis niloticus) bones as a function of their ages. The natural genesis dynamics of the structural evolution of the β-TCP and HAp phases were characterized by physicochemical methods, taking into account of the age of the fish and the material processing conditions. Thermal analysis (TGA / DTA) showed complete removal of the organic matter and transitions associated with the transformation of carbonated hydroxyapatite (CDHA) to HAp and β-TCP phases. After calcination at 900 °C, the material was characterized by: X-ray diffraction (XRD) and refinement by the Rietveld method; Fourier Transform Infrared Spectroscopy with Attenuated Total Reflection (FTIR-ATR); Raman spectroscopy; Scanning Electron Microscopy (SEM) and Flame Atomic Absorption Spectroscopy (FAAS). The analysis allowed identification and quantitative estimate of the variations of the HAp and β-TCP phases in the formation of the BCPs. The results showed that the decrease in β-TCP against the increase in the HAp phases is symmetrical to the dynamics of the natural genesis of these phases, surprisingly maintaining the balanced phase proportion even when bones of young fishes were used. The microstructure analysis confirms the observed transformation. In addition, in vivo tests demonstrated the osteoinductive potential of BCP scaffolds implanted in an ectopic site, and their remarkable regenerative functionality, as bone graft, was demonstrated in alveolar bone after tooth extraction. MTT cytotoxicity assay for BCP samples for MC3T3-E1 pre-osteoblasts and L929 fibroblasts cells showed viability equal or higher than 100%. A logistic empirical model is presented to explain the three stages of HAp natural formation with fish age and it is also compared to the fish size evolution.

Reinforcing the function of bone graft via the Ca-P ceramics dynamic behavior-enhanced osteogenic microenvironment for optimal bone regeneration and reconstruction

Bone tissue engineering is one of the most potential strategies in treating bone defect. However, implantation failure caused by the loss of biological activity has always been a difficulty in clinical transformation, resulting from the numerous cells death after implantation. Therefore, it is a burdensome challenge to design bone grafts with biological function adaptability. In this paper, we report a means to construct dynamically functional bone grafts (DFBGs) with Ca/P ceramics based on mechanical field drive-coupling and mechanobiology theory. In this study, the osteogenic microenvironment and cell functional activity are optimized by micro-vibration (MV) external field drive-coupled with biphasic calcium phosphate (BCP) ceramic, so that the DFBGs are endowed with optimal bone regeneration ability. The test results show that DFBGs have higher osteogenic differentiation ability and mechanical adaptability. The calcium release of BCP ceramics and the activation of calcium channels of cells are enhanced under MV field, thus the osteogenic potential of DFBGs is further up-regulated. The application results show that DFBGs system has excellent efficacy in guiding bone regeneration and the amount of new bone formation is about 35% higher than that of bone grafts (BGs) under static environment after implanted for 1 week, indicating expectable application prospect in treating bone defects.

Study on the difference of osteogenesis and Notch signaling pathway expression in biphasic calcium-phosphorus ceramic granule materials with different microstructure.

Different microstructures including micropore diameter, micropore volume, and micropore area of biphasic calcium phosphate (BCP, hydroxyapatite: β-tricalcium phosphate=8:2) ceramics granules were obtained by varying their sintering temperatures. Sprague-Dawley rat bone marrow-derived stem cells (BMSCs) were co-cultured with BCPs in vitro study and the BMSCs showed different degrees of proliferative activity under the influence of three materials. Cell proliferation and vitality were assessed. Three kinds of BCPs were implanted in the dorsal muscle of beagle dogs. At 1, 2, and 3 months, histological analyses were conducted to estimate the rate of osteogenesis. Expression of Notch pathway genes and osteogenic-related genes were detected by quantitative real-time polymerase chain reaction (q-rtPCR). The proportion of osteogenesis area increased to:48.75 ± 4.20%, 29.48 ± 1.55%, and 26.58 ± 3.86% at 3 months after the implantation (1050, 1150, 1250). Significant differences were observed in the upregulation of Notch pathway genes among different BCPs. BCPs with different micropore diameters have different ectopic osteogenesis effects and led to up-regulation of the Notch signaling pathway genes to different extents.

Pathway to tailor the phase composition, microstructure and mechanical properties of pulsed laser deposited cobalt-substituted calcium phosphate coatings on titanium

Dense calcium phosphate-based ceramics were fabricated to be used as targets for pulsed laser deposition (PLD). Nanostructured cobalt-substituted hydroxyapatite (Co:HAP) was used as a starting powder. To vary phase composition and microstructure of targets, two sintering approaches were applied, conventional (CS) and two-step sintering (TSS). The obtained results show that in both cases biphasic calcium phosphate (BCP) ceramics (targets) were prepared, with slightly different HAP-to-β–TCP amount ratio and a significantly different microstructure. While the CS method yielded fully dense ceramics with an average grain size of 1.3 μm, the ceramics prepared by TSS had a density of 98.5%, with a predominant grain size below 100 nm. (Ca + Co)P coatings were prepared by PLD of (Ca + Co)P targets. The temperature of the Ti substrate was adjusted to be 25 and 500 °C. The results show that the phase composition of (Ca + Co)P coatings depended on the phase composition of targets as well as on the temperature of the Ti substrate. The coating prepared at 25 °C using CS target consisted of three calcium phosphate phases, HAP, β–TCP and α–TCP; when the TSS target was used, the coating was biphasic, containing HAP and β–TCP. When the substrate was heated to 500 °C, regardless of whether the CS or the TSS target was used, the deposited coatings were composed of HAP and α–TCP. Due to different phase compositions, the (Ca + Co)P coatings deposited at 25 °C showed an improved hardness compared to those deposited at 500 °C. The obtained results confirmed that the phase composition, morphology and mechanical properties of 0.3 μm thick (Ca + Co)P coatings on a Ti substrate can be tailored by employing (Ca + Co)P targets with different microstructures, and also by varying the temperature of the Ti substrate during deposition experiments.

Biomimetic Ceramic Composite: Characterization, Cell Response, and In Vivo Biocompatibility.

The present study aimed to synthesize biphasic calcium phosphate ceramics (CaPs) composed of β-tricalcium phosphate (β-TCP) and hydroxyapatite (HAp) from the propagated Scleractinian coral and dicalcium phosphate anhydrous using a solid-state reaction followed by heat treatment at a temperature of 1100 °C for 1 h to 7 days. The as-prepared coral and coral-derived biphasic CaPs samples were characterized through scanning electron microscopy, X-ray diffractometry, Fourier transform infrared spectroscopy, and Raman spectroscopy. The cell response of the biphasic CaPs was evaluated by in vitro cytotoxicity assessment using mouse fibroblast (L929) cells. The bilateral femoral defect rabbit model was used to assess the early local reaction of the coral-derived biphasic CaPs bone graft on tissue. The results confirmed that the co-existence of β-TCP and HAp was formed at 1100 °C for 1 h. The ratio of HA/β-TCP increased as the heat-treatment time increased. The coral-derived biphasic CaPs comprising 61% HAp and 39% β-TCP (defined as HT-3) were not cytotoxic. Furthermore, no significant differences in local tissue reaction were observed between the HT-3 sample and autogenous bone. Therefore, the synthesized coral-derived biphasic CaPs is a candidate for bone grafting due to its good biocompatibility.

Break monopoly of polarization: CD301b+ macrophages play positive roles in osteoinduction of calcium phosphate ceramics

The pro-inflammatory M1 and anti-inflammatory M2 polarization macrophages were considered crucial during the immune regulation of bone regeneration. However, M2 polarization macrophages cannot strictly represent regenerative macrophage phenotypes. There is an urgent need to classify a reliable regenerative macrophage to guide the development of osteoimmune modulation and bone regeneration biomaterials. Herein, we report a new CD301b+ macrophage phenotype in the osteoinduction process of calcium phosphate ceramics. There were far more CD301b+ macrophages around biphasic calcium phosphate ceramics than those around β-tricalcium phosphate ceramics. Additionally, CD301b-deficient mice failed to form new bone around biphasic calcium phosphate ceramics. Moreover, macrophages stimulated by biphasic calcium phosphate ceramics were beneficial to osteogenic differentiation of mesenchymal stem cells. CD301b+ macrophages can activate the IGF1R/Akt/mTOR pathway of mesenchymal stem cells by IGF1 expression, thereby promoting osteogenic differentiation. Furthermore, transplanting the sorted CD301b+ macrophages could further facilitate the osteoinduction of biphasic calcium phosphate ceramics. These data highlighted the critical role of CD301b+ macrophages in the osteoinduction of calcium phosphate ceramics. It may provide a novel target for potential osteoimmune modulation in bone regeneration.

Sustained calcium ion release from bioceramics promotes CaSR-mediated M2 macrophage polarization for osteoinduction.

Innate immune cells, especially macrophages, play a dual role in tissue repair and the defense against foreign bodies. Although biphasic calcium phosphate (BCP) ceramics have been confirmed as an excellent osteoimmunoregulatory biomaterial, it is unclear whether the ions release of BCP directly affects macrophage polarization and the mechanism by which the ions release is involved in osteoimmunomodulation. Herein, we verified the superior osteoinductive capacity of BCP in wild-type mice and showed its inability to promote this process in macrophage-deficient (LysM-/- ) mice. Moreover, scanning electron microscopy, ion release curve, and calcein AM-staining results confirmed that BCP-released Ca2+ in a sustained manner, thereby maintaining the long-term induction of M2 macrophage polarization and promoting the differentiation of mesenchymal stem cells into osteoblasts during osteogenesis. Furthermore, Ca2+ targeted the Wnt/β-catenin signaling pathway and activated Arg1 and IL-10 (M2 marker genes) transcription through the calcium-sensing receptor (CaSR) in macrophages. Under treatment with a CaSR antagonist, macrophages cultured with the BCP fluid extract exhibited lower Ca2+ intake and weaker M2 macrophage polarization. These findings underscore the critical role of macrophages in bone regeneration and clarify the molecular mechanisms of Ca2+ -mediated osteoinduction by biomaterials, which is of great significance for the future design of biomaterial-oriented tissue regeneration engineering.

Copper-Doped Biphasic Calcium Phosphate Powders: Dopant Release, Cytotoxicity and Antibacterial Properties.

Cytotoxicity and antibacterial properties associated with the dopant release of Cu-doped Biphasic Calcium Phosphate (BCP) powders, mainly composed of hydroxyapatite mixed with β-tricalcium phosphate powders, were investigated. Twelve BCP ceramics were synthesized at three different sintering temperatures (600 °C, 900 °C and 1200 °C) and four copper doping rates (x = 0.0, 0.05, 0.10 and 0.20, corresponding to the stoichiometric amount of copper in Ca10Cux(PO4)6(OH)2-2xO2x). Cytotoxicity assessments of Cu-doped BCP powders, using MTT assay with human-Mesenchymal Stem Cells (h-MSCs), indicated no cytotoxicity and the release of less than 12 ppm of copper into the biological medium. The antibacterial activity of the powders was determined against both Gram-positive (methicillin-sensitive (MS) and methicillin resistant (MR) Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. The Cu-doped biomaterials exhibited a strong antibacterial activity against MSSA, MRSA and E. coli, releasing approximatively 2.5 ppm after 24 h, whereas 10 ppm were required to induce an antibacterial effect against P. aeruginosa. This study also demonstrated that the culture medium used during experiments can directly impact the antibacterial effect observed; only 4 ppm of Cu2+ were effective for killing all the bacteria in a 1:500 diluted TS medium, whereas 20 ppm were necessary to achieve the same result in a rich, non-diluted standard marrow cell culture medium.

Proteomic analysis identified LBP and CD14 as key proteins in blood/biphasic calcium phosphate microparticle interactions

Immediately upon implantation, scaffolds for bone repair are exposed to the patient's blood. Blood proteins adhere to the biomaterial surface and the protein layer affects both blood cell functions and biomaterial bioactivity. Previously, we reported that 80-200 µm biphasic calcium phosphate (BCP) microparticles embedded in a blood clot, induce ectopic woven bone formation in mice, when 200-500 µm BCP particles induce mainly fibrous tissue. Here, in a LC-MS/MS proteomic study we compared the differentially expressed blood proteins (plasma and blood cell proteins) and the deregulated signaling pathways of these osteogenic and fibrogenic blood composites. We showed that blood/BCP-induced osteogenesis is associated with a higher expression of fibrinogen (FGN) and an upregulation of the Myd88- and NF-κB-dependent TLR4 signaling cascade. We also highlighted the key role of the LBP/CD14 proteins in the TLR4 activation of blood cells by BCP particles. As FGN is an endogenous ligand of TLR4, able to modulate blood composite stiffness, we propose that different FGN concentrations modify the blood clot mechanical properties, which in turn modulate BCP/blood composite osteoactivity through TLR4 signaling. The present findings provide an insight at the protein level, into the mechanisms leading to an efficient bone reconstruction by blood/BCP composites. Statement of significanceUpon implantation, scaffolds for bone repair are exposed to the patient's blood. Blood proteins adhere to bone substitute surface and this protein layer affects both biomaterial bioactivity and bone healing. Therefore, for the best outcome for patients, it is crucial to understand the molecular interactions between blood and bone scaffolds. Biphasic calcium phosphate (BCP) ceramics are considered as the gold standard in bone reconstruction surgery. Here, using proteomic analyses we showed that the osteogenic properties of 80-200 µm BCP particles embedded in a blood clot is associated with a higher expression of fibrinogen. Fibrinogen upregulates the Myd88- and NF-κB-dependent TLR4 pathway in blood cells and, BCP-induced TLR4 activation is mediated by the LBP and CD14 proteins.