Biphasic Calcium Phosphate Nanoparticles Research Articles

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.

Synthesis, physicochemical characteristics, cytocompatibility, and antibacterial properties of iron-doped biphasic calcium phosphate nanoparticles with incorporation of silver

The application of biphasic calcium phosphate (BCP) in tissue engineering and regenerative medicine has been widely explored due to its extensively documented multi-functionality. The present study attempts to synthesize a new type of BCP nanoparticles, characterised with favourable cytocompatibility and antibacterial properties via modifications in their structure, functionality and assemblage, using dopants. In this regard, this study initially synthesized iron-doped BCP (FB) nanoparticles with silver subsequently incorporated into FB nanoparticles to create a nanostructured composite (FBAg). The FB and FBAg nanoparticles were then characterized using Fourier transform infrared spectroscopy, x-ray diffraction, ultraviolet-visible spectroscopy, and x-ray photoelectron spectroscopy. The results showed that silver was present in the FBAg nanoparticles, with a positive correlation observed between increasing AgNO3 concentrations and increasing shape irregularity and reduced particle size distribution. Additionally, cell culture tests revealed that both FB and FBAg nanoparticles were compatible with bone marrow-derived mesenchymal stem cells (hBMSCs). The antibacterial activity of the FBAg nanoparticles was also tested using Gram-negative E. coli and Gram-positive S. aureus, and was found to be effective against both bacteria. The inhibition rates of FBAg nanoparticles against E. coli and S. aureus were 33.78 ± 1.69–59.03 ± 2.95%, and 68.48 ± 4.11–89.09 ± 5.35%, respectively. These findings suggest that the FBAg nanoparticles have potential use in future biomedical applications.

Dual composition Chondroitin Sulfate and gelatin biomimetic hydrogel based on tyramine crosslinking for tissue regenerative medicine

Hydrogels with bone biomimetic microenvironment are of great importance for the development of bone-graft materials. Herein, a biomimetic hydrogel, which was based on biopolymer as an extracellular matrix (ECM) mimicking components with the incorporation of biphasic calcium phosphate (BCP) nanoparticles, was prepared via the enzymatic crosslinking. Chondroitin sulfate and gelatin were first modified to form tyramine (TA) moieties on the natural polymers, which were ultilized as precursors for in-situ forming hydrogel (CDTA-GTA) under physiological conditions. The amount of CDTA to GTA was optimized for gelation time as well as degraded ability with in collagenase medium. Increasing the CDTA amount delayed the time for crosslinking process, but the water holding capacity was increased. The subjection of BCP into CDTA-GTA hydrogel produced an injectable system with suitable biomineralizated property. This hydrogel formed more rapidly in 90–100 s under 3 mM H2O2 and 0.125 mg/mL HRP enzyme. BCP/CDTA-GTA hydrogel exhibited the porous structure (100–300 µm in diameter) with the homogenous distribution of BCP nanoparticles on the wall of the interconnected networks, suggesting the good environment for bone growth. Further, the degradation process of the obtained hydrogel followed pseudo-first-order kinetics in the collagenase condition. After soaking in SBF for 28 days, SEM following EDS technique confirmed the aggregation of the calcium and phosphorus on the surface of CDTA-GTA hydrogel (Ca/P ratio ∼ 1.34) which improved in case of BCP/ CDTA-GTA hydrogel (Ca/P ratios ∼ 1.77). Through X-ray diffraction, the formation of calcite crystals was only detected with BCP hydrogel. In vitro cytotoxicity assays with hMSC cells exhibited non-toxicity of the biomimetic hydrogel. Interestingly, the remarkably enhancement of the level intracellular calcium deposit as well as the activity of ALP-osteogenic related marker were observed in MSC cells treated with biomimetic hydrogel, showing superior scaffold osteogenic potential. These results indicate that enzymatic cross-linked CDTA-GTA gel with BCP nanoparticles would be a favorable option to accelerate bone repair and thus should potentially be useful for osteochondral tissue engineering.

Magnesium-doped biphasic calcium phosphate nanoparticles with incorporation of silver: Synthesis, cytotoxic and antibacterial properties

The development of new calcium phosphate nanoparticles with excellent cytocompatibility and antibacterial properties is generating substantial interest in the biomedical field. In this regard, the present study demonstrated the synthesis of magnesium-doped biphasic calcium phosphate nanoparticles containing silver (AgMgB-NPs) via the employment of the chemical wet-precipitation method. Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and ultraviolet–visible spectroscopy (UV–Vis) methods were used to confirm the successful synthesis of AgMgB-NPs. X-ray photoelectron spectroscopy (XPS) and Raman spectra indicated that Mg2+ was doped at the Ca2+ position. The excellent cytocompatibility of AgMgB-NPs was confirmed using the cell counting kit-8 (CCK-8) analysis which employed a culture of human bone marrow-derived mesenchymal stem cells (hBMSCs). Additionally, the antibacterial activity of AgMgB-NPs was evaluated using Gram-negative E. coli and Gram-positive S. aureus micro-organisms. The present study therefore developed novel calcium phosphate nanoparticles that were demonstrated to have the potential for biomedical applications.

Nanostructured selenium-doped biphasic calcium phosphate with in situ incorporation of silver for antibacterial applications

Selenium-doped nanostructure has been considered as an attractive approach to enhance the antibacterial activity of calcium phosphate (CaP) materials in diverse medical applications. In this study, the selenium-doped biphasic calcium phosphate nanoparticles (SeB-NPs) were first synthesized. Then, silver was in situ incorporated into SeB-NPs to obtain nanostructured composite nanoparticles (AgSeB-NPs). Both SeB-NPs and AgSeB-NPs were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), ultraviolet–visible spectroscopy (UV–Vis), X-ray photoelectron spectroscopy (XPS), and Raman spectra. The results confirmed that the SeO32− was doped at the PO43− position and silver nanoparticles were deposited on the surface of SeB-NPs. Next, Transmission Electron Microscopy (TEM) analysis displayed that the prepared AgSeB-NPs had a needle-cluster-like morphology. CCK-8 analysis revealed SeB-NPs and AgSeB-NPs had good cytocompatibility with osteoblasts. The antibacterial activity of the prepared AgSeB-NPs was confirmed by using Gram-negative E. coli and Gram-positive S. aureus. The above results manifested the significance of the final AgSeB-NPs for biomedical applications.

Investigation of biphasic calcium phosphate (BCp)/polyvinylpyrrolidone (PVp) /graphene oxide (GO) composite for biomedical implants

The potential advantages of using 2D graphene oxide (GO)-based composites stimulating cellular differentiation as well as improve the compressive strength of the scaffolds. The present study investigated the biphasic calcium phosphate (BCp)/polyvinylpyrrolidone (PVp)/Graphene oxide (GO) composite with different GO (w/w) content of 1 wt%, 3 wt%, and 5 wt%. When increasing GO content, the compressive strength of scaffolds increased by 366 ± 37, 387 ± 28, and 558 ± 14 for 1 wt%, 3 wt%, and 5 wt% respectively.The TEM images showed the presence of a homogeneous distribution of BCp nanoparticles onto the graphene oxide surface; also Raman and XRD results confirm the formation of composite scaffolds. Furthermore, in vitro cytocompatibility of the BCp-PVp/GO 5 wt% composites were evaluated by cell adhesion and live/dead analysis using MG-63 cells. The obtained results showed that no adverse effect was witnessed, which evidences the superior bio compatibility nature of the composite scaffold. These results suggested that the BCp-PVp/GO composites could be a promising material for tissue engineering application.

In vitro biomineralization on poly(vinyl alcohol)/biphasic calcium phosphate hydrogels

Biomineralized tissue is considered the final product of successful cell culture in bone tissue engineering. Dulbecco’s modified Eagle’s medium (DMEM) with fetal bovine serum (FBS) not only is used as a common culture medium but also provides a natural biomineralization environment, due to having similar ionic concentrations as blood plasma. Here, poly(vinyl alcohol) hydrogel with incorporated biphasic calcium phosphate nanoparticles was immersed in a DMEM–FBS cell culture medium, and then biomineralization occurred on the nanocomposite surface, which was characterized by Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy. Such formed bone-like apatite on the surface facilitated the proliferation of osteoblasts, identified by Cell Counting Kit-8 analysis and fluorescent microscopy. This study verified the spontaneous biomineralization on the surface of a calcium phosphate-based nanocomposite by using a simple DMEM–FBS immersion strategy, which was promising for biomodification of bone substitutes.

Development of chitosan/gelatin hydrogels incorporation of biphasic calcium phosphate nanoparticles for bone tissue engineering

The chitosan/gelatin hydrogel incorporated with biphasic calcium phosphate nanoparticles (BCP-NPs) as scaffold (CGB) for bone tissue engineering was reported in this article. Such nanocomposite hydrogels were fabricated by using cycled freeze-thawing method, of which physicochemical and biological properties were regulated by adjusting the weight ratio of chitosan/gelatin/BCP-NPs. The needle-like BCP-NPs were dispersed into composites uniformly, and physically cross-linked with chitosan and gelatin, which were identified via Scanning Electron Microscope (SEM) images and Fourier Transform Infrared Spectroscopy (FT-IR) analysis. The porosity, equilibrium swelling ratio, and compressive strength of CGB scaffolds were mainly influenced by the BCP-NPs concentration. In vitro degradation analysis in simulated body fluids (SBF) displayed that CGB scaffolds were degraded up to at least 30 wt% in one month. Also, CCK-8 analysis confirmed that the prepared scaffolds had a good cytocompatibility through in culturing with bone marrow mesenchymal stem cells (BMSCs). Finally, In vivo animal experiments revealed that new bone tissue was observed inside the scaffolds, and gradually increased with increasing months, when implanted CGB scaffolds into large necrotic lesions of rabbit femoral head. The above results suggested that prepared CGB nanocomposites had the potential to be applied in bone tissue engineering.

In situ Fabrication of Biological Chitosan and Gelatin-Based Hydrogels Loading Biphasic Calcium Phosphate Nanoparticles for Bone Tissue Regeneration

Adjustably biodegradable materials have gained much attention in biomedical applications. Among of them, various hydrogel-based scaffolds have applied for regenerating soft and hard tissues. In this study, according to differently biological properties of gelatin or chitosan as well as biphasic calcium phosphate nanoparticles (BCPNPs), several injectable nanocomposite hydrogels (INgel) were enzymatically fabricated from a phenolic chitosan derivative (PCD), phenolic gelatin derivative (PGD) and BCPNPs. According the change of H2O2 concentration with follow-up the time, the in situ formation of INgel was varied from 35 to 80 s. The degradation rate of the nanocomposite materials significantly related to in presence of collagenase that expended from 3 days to over one month depending on amount of the formulated PCD. The BCPNPs-encapsulated PCD-PGD INgel enhanced mineralization in the simulated biofluid. Fluorescent cytotoxicity assay indicated that the INgel was fabricated from a higher amount of the PGD resulting in a significant proliferation of bone marrow mesenchymal stem cells. These preliminary results exhibited a great potential of the INgel for bone regeneration.

ENZYMATIC PREPARATION OF MODULATED–BIODEGRADABLE HYDROGEL NANOCOMPOSITES BASED CHITOSAN/GELATIN AND BIPHASIC CALCIUM PHOSPHATE NANOPARTICLES

In the study, injectable chitosan–4 hydroxyphenylacectamide acid (CHPA) and gelatin–tyramine (GTA)–based hydrogels were enzymatically prepared, in which could encapsulate biphasic calcium phosphate nanoparticles (BCP NPs) for enhancing bone regeneration. The in situ formation of hydrogel composite was varied from 35 to 80 seconds depending on concentration of H2O2. Collagenase–mediated biodegradation of the hydrogel composite could be modulated from 3 days to over one month depending on amount of the formulated CHPA. Live/dead cell viability assay indicated that the hydrogel composite enhanced bone marrow mesenchymal stem cells (MSCs). The obtained results show a great potential of the hydrogel composites for bone regeneration due to its adjustable biodegradation, biocompatibility and enhancement in new bone formation.

Preparation of dexamethasone-loaded biphasic calcium phosphate nanoparticles/collagen porous composite scaffolds for bone tissue engineering.

Scaffolds play a crucial role for regeneration of large bone defects. Biomimetic scaffolds having the same composition of natural bone and a controlled release of osteoinductive factors are desirable for promotion of bone regeneration. In this study, composite scaffolds of collagen and biphasic CaP nanoparticles (BCP NPs) with a controlled release nature of dexamethasone (DEX) were prepared and their porous structures were controlled by using ice particulates. In vitro cell culture and in vivo implantation experiments demonstrated the composite scaffolds exerted synergistic effects on the osteogenic differentiation of hMSCs and bone regeneration. The composite scaffolds also showed promotive effect on the formation of capillary blood vessels in the regenerated bone. This study is the first research to prepare DEX-loaded BCP NPs/collagen porous composite scaffolds. The superior performance of the composite scaffolds indicates the composite scaffolds should be useful for bone tissue engineering.

Biphasic Calcium Phosphate (BCP)-Immobilized Porous Poly (d,l-Lactic-co-Glycolic Acid) Microspheres Enhance Osteogenic Activities of Osteoblasts.

The purpose of this study was to evaluate the potential of porous poly (d,l-lactic-co-glycolic acid) (PLGA) microspheres (PMSs) immobilized on biphasic calcium phosphate nanoparticles (BCP NPs) (BCP-IM-PMSs) to enhance osteogenic activity. PMSs were fabricated using a fluidic device, and their surfaces were modified with l-lysine (aminated-PMSs), whereas the BCP NPs were modified with heparin–dopamine (Hep-DOPA) to obtain heparinized–BCP (Hep-BCP) NPs. BCP-IM-PMSs were fabricated via electrostatic interactions between the Hep-BCP NPs and aminated-PMSs. The fabricated BCP-IM-PMSs showed an interconnected pore structure. In vitro studies showed that MG-63 cells cultured on BCP-IM-PMSs had increased alkaline phosphatase activity, calcium content, and mRNA expression of osteocalcin (OCN) and osteopontin (OPN) compared with cells cultured on PMSs. These data suggest that BCP NP-immobilized PMSs have the potential to enhance osteogenic activity.

Surface immobilization of biphasic calcium phosphate nanoparticles on 3D printed poly(caprolactone) scaffolds enhances osteogenesis and bone tissue regeneration

Abstract We fabricated biphasic calcium phosphate nanoparticles (BCP NPs)-immobilized on the surface of 3D printed PCL (BCP-IM-PCL) scaffolds, and evaluated in vitro osteogenesis and in vivo new bone formation in rat tibial defect model. In vitro and in vivo studies showed that BCP-IM-PCL significantly enhanced osteogenic markers ( i.e. , ALP activity, calcium deposition, and the expression of osteocalcin and osteopontin) and markedly increased new bone formation and mineralized bone tissues in tibial defect area, compared to unmodified PCL and BCP-mixed PCL scaffolds. This study demonstrated that BCP NPs-immobilized on the surface of PCL scaffolds are promising templates for bone tissue regeneration.

Preparation of dexamethasone-loaded calcium phosphate nanoparticles for the osteogenic differentiation of human mesenchymal stem cells.

As an earliest known and readily available osteogenic inducer for stem cells, dexamethasone (DEX) plays a key role in affecting cell functions and cellular processes, especially for cell proliferation and differentiation. However, the clinical application of DEX has been limited because of its uncontrolled release. An ideal carrier is desired to control the DEX release for the osteogenic differentiation of stem cells and bone tissue engineering. Biphasic calcium phosphate nanoparticles (BCP-NPs) should be potential carriers for DEX due to their osteoconductive properties and good biocompatibility as a bone graft biomaterial. In this study, DEX-loaded BCP-NPs were prepared by two methods: (1) immersion of BCP-NPs in a DEX solution (denoted DEX/BCP-NPs), (2) DEX incorporation during BCP-NP formation in a calcifying solution (denoted DEX@BCP-NPs). The DEX@BCP-NPs showed a higher DEX loading amount and more sustainable DEX release than did the DEX/BCP-NPs. The DEX@BCP-NPs were used for the culture of human bone marrow-derived mesenchymal stem cells (hMSCs) and showed a promotive effect on the proliferation of hMSCs. Furthermore, the DEX@BCP-NPs significantly increased the alkaline phosphatase (ALP) activity, calcium deposition and gene expressions of the osteogenic markers of hMSCs when compared to BCP-NPs without DEX loading. The results demonstrated BCP-NPs were good carriers for DEX loading and the DEX@BCP-NPs should be useful for bone tissue engineering.

Preparation and Biomineralization of Injectable Hydrogel Composite Based Chitosan-tetronic and BCP Nanoparticles

In this study, a hydrogel composite was enzymatically prepared with rapid gelation time from tyramine-tetronic-conjugated chitosan and biphasic calcium phosphate (BCP) nanoparticles. Compressive stress of the hydrogel composite was reached at 591±20 KPa with 10 wt% of loaded BCP. Degradation study of the material showed 20% of weight loss after 4 weeks. In vitro study with MG 63 osteoblast cell evidenced that the cells were well-attached on hydrogel composite surfaces. Biomineralization on the hydrogel composites surfaces was well-observed after soaking for 14 days in simulated body fluid (SBF) solution. The obtained results indicated that the hydrogel composite could be an injectable potential material for bone regeneration.

Nanoparticle biphasic calcium phosphate loading on gelatin-pectin scaffold for improved bone regeneration.

Biphasic calcium phosphate (BCP) nanoparticles were loaded with porous gelatin-pectin (GE-P) scaffolds. The biodegradable gelatin-pectin-BCP scaffolds were produced as miscible mixtures with well-defined interconnected pores to facilitate osteoconductivity and enhance bone formation. It was observed that the compressive strength increased with the loading of BCP nanoparticles. From in vitro results, cell adhesion, viability, and proliferation were found in the GE-P-10 scaffolds in comparison with those without BCP, resulting in high alkaline phosphate (ALP), and osteopontin (OPN) expression at 21 days. Micro-computed tomography data, hematoxylin and eosin staining, and immunohistochemistry (OPN, OCN, COL I, and COL II) confirmed rapid new bone formation in rabbit models. Our results provide a novel and simple method to provide an adequate scaffold, and thus GE-P-BCP porous scaffolds may be appropriate candidates for bone tissue engineering.

In vitro and in vivo studies of BMP-2-loaded PCL-gelatin-BCP electrospun scaffolds.

To confirm the effect of recombinant human bone morphogenetic protein-2 (BMP-2) for bone regeneration, BMP-2-loaded polycaprolactone (PCL)-gelatin (Gel)-biphasic calcium phosphate (BCP) fibrous scaffolds were fabricated using the electrospinning method. The electrospinning process to incorporate BCP nanoparticles into the PCL-Gel scaffolds yielded an extracellular matrix-like microstructure that was a hybrid system composed of nano- and micro-sized fibers. BMP-2 was homogeneously loaded on the PCL-Gel-BCP scaffolds for enhanced induction of bone growth. BMP-2 was initially released at high levels, and then showed sustained release behavior for 31 days. Compared with the PCL-Gel-BCP scaffold, the BMP-2-loaded PCL-Gel-BCP scaffold showed improved cell proliferation and cell adhesion behavior. Both scaffold types were implanted in rat skull defects for 4 and 8 weeks to evaluate the biological response under physiological conditions. Remarkable bone regeneration was observed in the BMP-2/PCL-Gel-BCP group. These results suggest that BMP-2-loaded PCL-Gel-BCP scaffolds should be considered for potential bone tissue engineering applications.

Enzyme-mediated in situ preparation of biocompatible hydrogel composites from chitosan derivative and biphasic calcium phosphate nanoparticles for bone regeneration

Injectable chitosan-based hydrogels have been widely studied toward biomedical applications because of their potential performance in drug/cell delivery and tissue regeneration. In this study we introduce tetronic–grafted chitosan containing tyramine moieties which have been utilized for in situ enzyme-mediated hydrogel preparation. The hydrogel can be used to load nanoparticles (NPs) of biphasic calcium phosphate (BCP), mixture of hydroxyapatite (HAp) and tricalcium phosphate (TCP), forming injectable biocomposites. The grafted copolymers were well-characterized by 1H NMR. BCP nanoparticles were prepared by precipitation method under ultrasonic irradiation and then characterized by using x-ray powder diffraction (XRD) and scanning electron microscopy (SEM). The suspension of the copolymer and BCP nanoparticles rapidly formed hydrogel biocomposite within a few seconds of the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The compressive stress failure of the wet hydrogel was at 591 ± 20 KPa with the composite 10 wt% BCP loading. In vitro study using mesenchymal stem cells showed that the composites were biocompatible and cells are well-attached on the surfaces.

Gelation behavior of in situ forming gels based on HPMC and biphasic calcium phosphate nanoparticles

In this study, in situ forming gels are prepared using biphasic calcium phosphate (BCP) as filler and hydroxypropyl methylcellulose (HPMC) as a matrix exhibiting temperature-sensitive behavior. BCP was composed of β-tricalcium phosphate (β-TCP) with plate-like morphology and nano-sized hyadroxyapatite (HAp). Gel permeation chromatography (GPC) and rheological results showed that low molecular weight HPMC had lower gelation temperature. Effects of BCP content and HAp/β-TCP ratio on rheological behavior of the gels were investigated. According to the results, all samples showed a pseudoplastic behavior and their viscosity increased with increasing mineral phase, especially β-tricalcium phosphate. In order to investigate interaction mechanisms between the mineral phase and polymer and also the effects of ion release, particle size, hydrophobisity, and hydrophilisity, hydrophobic and hydrophilic silica with different particle sizes were also utilized. Results showed that factors affecting the hydrophobisity and hydrophilisity of solution may influence the rheological properties.

Functional nanofiber mat of polyvinyl alcohol/gelatin containing nanoparticles of biphasic calcium phosphate for bone regeneration in rat calvaria defects

New biodegradable mats was successfully obtained by functional polyvinyl alcohol (PVA)/Gelatin (GE) blend fiber mats containing different BCP amounts (20, 40, and 50 w/v%) of biphasic calcium phosphate (BCP) nanoparticles for bone regeneration. BCP nanoparticles were loaded and dispersed successfully in the PVA/GE fibrous matrix. The addition of BCP was found to have increased fiber diameter, tensile strength, osteoblast cell adhesion, proliferation, and protein expression. Compared to the others, the 50% BCP-loaded electrospun PVA/GE fibers had the most favorable mechanical properties, cell attachment and growth, and protein expression. In vivo bone formation was examined using rat models, and increased bone formation was observed for the 50% BCP-loaded electrospun PVA/GE blends within 2 and 4 weeks. This result suggests that the 50% BCP-PVA/GE composite nanofiber mat has high potential for use in the field of bone regeneration and tissue engineering.