Role of Iron on Physical and Mechanical Properties of Brushite Cements, and Interaction with Human Dental Pulp Stem Cells.

Improving the physical, mechanical and biological properties of brushite cements (BrC) is of a great interest for using them in bone and dental tissue engineering applications. The objective of this study was to incorporate iron (Fe) at different concentrations (0.25, 0.50, and 1.00 wt.%) to BrC and study the role of Fe on phase composition, setting time, compressive strength, and interaction with human dental pulp stem cells (hDPSCs). Results showed that increase in Fe concentration increases the ß-tricalcium phosphate (ß-TCP)/ dicalcium phosphate dihydrate (DCPD) ratio and prolongs the initial and final setting time due to effective role of Fe on stabilizing the ß-TCP crystal structure and retarding its dissolution kinetic, in a dose dependent manner where the highest setting time was recorded for 1.00 wt.% Fe-BrC sample. Addition of low concentrations of Fe (0.25 and 0.50 wt.%) did not have adverse effect on compressive strength and strength was in the range of 5.7-7.05 (±~1.4) MPa; however, presence of 1.00 wt.% Fe decreases the strength of BrC from 7.05 ± 1.57 MPa to 3.12 ± 1.06 MPa. Interaction between the BrCs and hDPSCs was evaluated by cell proliferation assay, scanning electron microscopy, and live/dead staining. Low concentrations of 0.25, and 0.50 wt.% of Fe did not have any adverse effect on cell attachment and proliferation; while significant decrease in cellular activity was evident in BrC samples doped with 1.00 wt. %. Together, these data show that low concentrations of Fe (equal or less than 0.50 wt. %) can be safely added to BrC without any adverse effect on physical, mechanical and biological properties in presence of hDPSCs.

Antibacterial Composite Material Based on Polyhydroxybutyrate and Zn-Doped Brushite Cement.

A composite material based on electrospinning printed polyhydroxybutyrate fibers impregnated with brushite cement containing Zn substitution was developed for bone implant applications. Powder X-ray Diffraction (PXRD), Fourier Transform Infrared Spectroscopy and Scanning Electron Microscopy were applied for materials characterization. Soaking the composite in Ringer's solution led to the transformation of brushite into apatite phase, accompanied by the morphology changes of the material. The bending strength of the composite material was measured to be 3.1 ± 0.5 MPa. NCTC mouse fibroblast cells were used to demonstrate by means of the MTT test that the developed material was not cytotoxic. The behavior of the human dental pulp stem cells on the surface of the composite material investigated by the direct contact method was similar to the control. It was found that the developed Zn containing composite material possessed antibacterial properties, as testified by microbiology investigations against bacteria strains of Escherichia coli and Staphylococcus aureus. Thus, the developed composite material is promising for the treatment of damaged tissues with bacterial infection complications.

In vivo biocompatibility of SrO and MgO doped brushite cements.

The addition of dopants in biomaterials has emerged as a critical regulator of bone formation and regeneration due to their imminent role in the biological process. The present work evaluated the role of strontium (Sr) and magnesium (Mg) dopants in brushite cement (BrC) on in vivo bone healing performance in a rabbit model. Pure, 1 wt% SrO (Sr-BrC), 1 wt% MgO (Mg-BrC), and a binary composition of 1.0 wt% SrO + 1.0 wt% MgO (Sr + Mg-BrC) BrCs were implanted into critical-sized tibial defects in rabbits for up to 4 months. The in vivo bone healing of three doped and pure BrC samples was examined and compared using sequential radiological examination, histological evaluations, and fluorochrome labeling studies. The results indicated excellent osseous tissue formation for Sr-BrC and Sr + Mg-BrC and moderate bone regeneration for Mg-BrC compared to pure BrC. Our findings indicated that adding small amounts of SrO, MgO, and binary dopants to the BrC can significantly influence new bone formation for bone tissue engineering.

Synergistic Effects of Silicon/Zinc Doped Brushite and Silk Scaffolding in Augmenting the Osteogenic and Angiogenic Potential of Composite Biomimetic Bone Grafts.

Cell instructive scaffolding platforms displaying synergistic effects by virtue of their chemical and physical cues have tremendous scope in modulating cell phenotype and thus improving the success of any graft. In this regard, we report here the development of Si- and Zn-doped brushite cement composited with silk scaffolding that hierarchically emulated the cancellous bone. The composite scaffolds fabricated exhibited an open porous network capable of enhanced osteoblast survival as attested by increased alkaline phosphatase activity and also sustaining osteoclast activity affirmed by tartrate resistant acid phosphatase staining. Moreover, the chemical cues presented by dissolutions products from the composite scaffold enabled the osteoblasts to secrete proangiogenic factors which favored better endothelial cell survival, confirmed through in vitro experiments. Moreover, the efficacy of these composite biomimetic scaffolds was validated in vivo in volumetric femur defects in rabbits, which revealed that these matrices influenced vascular cell infiltration and favored the formation of matured bony plate. Fluorochrome labeling studies and microtomography analysis revealed that at the end of three months, the implanted composite scaffolds had completely resorbed, leaving behind neo-osseous tissue and vouching for clinical translation of these composite matrices as viable and affordable bone-graft substitutes.

Effects of platelet-rich plasma on biological activity and bone regeneration of brushite-based calcium phosphate cement.

In this study, we focused on identifying the effects of platelet-rich plasma (PRP) on bioactivity and bone ingrowths incorporated into brushite cement. We introduced PRP as a series of substitutions with an aqueous citrate-ion solution, and the optimized cement with PRP showed no disintegration of the paste consistency. Incorporating PRP showed that the setting time decreased with the increasing of PRP ratios, although the compressive strength was not significantly changed. We evaluated in vitro degradation and bioactivity with the simulated body fluid test, and the result showed that adding PRP accelerated the carbonated apatite nucleation and markedly improved the surface reactivity of the cement. The in vitro studies demonstrated that incorporating PRP into the brushite cement improved cell adhesion and proliferation. The in vivo effects of PRP were faster degradation, improved tissue response in the early stage, and bone ingrowths. We demonstrated based on our results of this study, incorporation of PRP into brushite cement could be helpful for improving biological activity of the cement as well as bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2316-2326, 2018.

The in vitro evolution of resorbable brushite cements: A physico-chemical, micro-structural and mechanical study.

The mechanisms by which calcium phosphate bone substitutes evolve and are resorbed in vivo are not yet fully known. In particular, the formation of intermediate phases during resorption and evolution of the mechanical properties may be of crucial interest for their clinical efficiency. The in vitro tests proposed here are the first steps toward understanding these phenomena. Microporous Dicalcium Phosphate Dihydrate (DCPD) samples were immersed in tris(hydroxymethyl)aminomethane (TRIS) and Phosphate Buffered Saline (PBS) solutions, with or without daily refresh of the medium, for time-points up to 14days. Before and after immersion, samples were extensively characterised in terms of morphology, chemistry (XRD coupled with Rietveld analysis), microstructure (X-ray tomography, SEM observations) and local mechanical properties (instrumented micro-indentation). The composition of the immersion solutions was monitored in parallel (pH, elemental analysis). The results show the influence and importance of the experimental set-up and protocol on the formation of apatite and octacalcium phosphate concurrently to DCPD dissolution; moreover, strong inter-correlations between physico-chemistry, microstructure and mechanics are demonstrated. STATEMENT OF SIGNIFICANCE: Ideally, the resorption kinetics of biodegradable bone substitutes should be controlled to favor the healing processes of bone. Although biodegradable bone grafts are already used in surgeries, their resorption process is still partially unknown. The present work studies these resorption phenomena, their kinetics and mechanisms and their consequences on the properties of a calcium phosphate resorbable material. The original in vitro approach developed in this work couples for the first time physico-chemical, micro-structural and mechanical assessments. The dissolution of the CaP phase in body fluids and the reprecipitation of more stable phases are studied on a local scale, which has permitted to evidence and monitor the development of a gradient of properties between the surface and the core of the samples.

In Situ Synchrotron X-ray Diffraction Analysis of the Setting Process of Brushite Cement: Reaction and Crystal Growth.

Brushite cements are fast self-setting materials that can be used as bone substitute materials. Although tracing their fast setting process is a challenge, it is important for the understanding of the same, which in turn is important for the material's further development and use in the clinics. In this study, the setting rate, phase formation, and crystal growth of brushite cements were quantitatively studied by in situ synchrotron powder X-ray diffraction (SXRD) on a time scale of seconds. The influence of reactant ratios and a retardant (citric acid) on the setting reaction were analyzed. To complement the in situ investigations, scanning electron microscopy was carried out for ex situ morphological evolution of crystals. The initial reaction followed a four-step process, including a fast nucleation induction period, nucleation, crystal growth, and completion of the setting. The brushite crystal size grew up to the micro scale within 1 min, and the brushite content increased linearly after the nucleation until all monocalcium phosphate monohydrate (MCPM; Ca(H2PO4)2·H2O) had dissolved within minutes, followed by a slow increase until the end of the monitoring. By adjusting the MCPM to the ß-tricalcium phosphate (ß-TCP, ß-Ca3(PO4)2) ratio in the starting powders, the brushite/monetite ratio in the cements could be modified. In the presence of citric acid, the formation of brushite nuclei was not significantly retarded, whereas the increase in brushite content and the growth of crystal size were effectively hindered. The amount of monetite also increased by adding citric acid. This is the first time that the brushite setting process has been characterized in the first seconds and minutes of the reaction by SXRD.

Scavenging effect of Trolox released from brushite cements.

In this study a brushite cement was doped with the chain-breaking antioxidant Trolox. The effect of the antioxidant on the physical properties of the cement was evaluated and the release of Trolox was monitored by UV spectroscopy. The ability of the Trolox set free to scavenge reactive oxygen species (ROS) released by macrophages was determined in vitro using a luminol-amplified chemiluminescence assay. Trolox did not modify the crystalline phases of the set cement, which mainly formed crystalline brushite after 7 days in humid conditions. The setting time, compressive strength and morphology of the cement also remained unaltered after the addition of the antioxidant. Trolox was slowly released from the cement following a non-Fickian transport mechanism and nearly 64% of the total amount was released after 3 days. Moreover, the capacity of Trolox to scavenge the ROS released by macrophages increased in a dose-dependent manner. Trolox-loaded cements are expected to reduce some of the first harmful effects of acute inflammation and can thus potentially protect the surrounding tissue during implantation of these as well as other materials used in conjunction.

Effects of Silicon on Osteoclast Cell Mediated Degradation, In Vivo Osteogenesis and Vasculogenesis of Brushite Cement.

Calcium phosphate cements (CPCs) are being widely used for treating small scale bone defects. Among the various CPCs, brushite (dicalcium phosphate dihydrate, DCPD) cement is widely used due to its superior solubility and ability to form new bone. In the present study, we have studied the physical, mechanical, osteoclast-like-cells differentiation and in vivo osteogenic and vasculogenic properties of silicon (Si) doped brushite cements. Addition of Si did not alter the phase composition of final product and regardless of Si level, all samples included ß-tricalcium phosphate (ß-TCP) and DCPD. 1.1 wt. % Si addition increased the compressive strength of undoped brushite cement from 4.78±0.21 MPa to 5.53±0.53 MPa, significantly. Cellular activity was studied using receptor activator of nuclear factor κß ligand (RANKL) supplemented osteoclast-like-cells precursor RAW 264.7 cell. Phenotypic expressions of the cells confirmed successful differentiation of RAW264.7 monocytes to osteoclast-like-cells on undoped and doped brushite cements. An increased activity of osteoclast-like cells was noticed due to Si doping in the brushite cement. An excellent new bone formation was found in all cement compositions, with significant increase in Si doped brushite samples as early as 4 weeks post implantation in rat femoral model. After 4 weeks of implantation, no significant difference was found in blood vessel formation between the undoped and doped cements, however, a significant increase in vasculgenesis was found in 0.8 and 1.1 wt. % Si doped brushite cements after 8 weeks. These results show the influence of Si dopant on physical, mechanical, in vitro osteoclastogenesis and in vivo osteogenic and vasculogenic properties of brushite cements.