Giuseppe Cama

Can the Hydrophilicity of Functional Monomers Affect Chemical Interaction

The number of carbon atoms and/or ester/polyether groups in spacer chains may influence the interaction of functional monomers with calcium and dentin. The present study assessed the chemical interaction and bond strength of 5 standard-synthesized phosphoric-acid ester functional monomers with different spacer chain characteristics, by atomic absorption spectroscopy (AAS), ATR-FTIR, thin-film x-ray diffraction (TF-XRD), scanning electron microscopy (SEM), and microtensile bond strength (μTBS). The tested functional monomers were 2-MEP (two-carbon spacer chain), 10-MDP (10-carbon), 12-MDDP (12-carbon), MTEP (more hydrophilic polyether spacer chain), and CAP-P (intermediate hydrophilicity ester spacer). The intensity of monomer-calcium salt formation measured by AAS differed in the order of 12-MDDP=10-MDP>CAP-P>MTEP>2-MEP. FTIR and SEM analyses of monomer-treated dentin surfaces showed resistance to rinsing for all monomer-dentin bonds, except with 2-MEP. TF-XRD confirmed the weaker interaction of 2-MEP. Highest µTBS was observed for 12-MDDP and 10-MDP. A shorter spacer chain (2-MEP) of phosphate functional monomers induced formation of unstable monomer-calcium salts, and lower chemical interaction and dentin bond strength. The presence of ester or ether groups within longer spacer carbon chains (CAP-P and MTEP) may affect the hydrophilicity, μTBS, and also the formation of monomer-calcium salts.

Accuracy of CBCT for volumetric measurement of simulated periapical lesions.

AIMnTo compare the accuracy of cone beam computed tomography (CBCT) and micro-computed tomography (μCT) when measuring the volume of bone cavities.nnnMETHODOLOGYnTen irregular-shaped cavities of varying dimensions were created in bovine bone specimens using a rotary diamond bur. The samples were then scanned using the Accuitomo 3D CBCT scanner. The scanned information was converted to the Digital Imaging and Communication in Medicine (DICOM) format ready for analysis. Once formatted, 10 trained and calibrated examiners segmented the scans and measured the volumes of the lesions. Intra/interexaminer agreement was assessed by each examiner re-segmenting each scan after a 2-week interval. Micro-CT scans were analysed by a single examiner. To achieve a physical reading of the artificially created cavities, replicas were created using dimensionally stable silicone impression material. After measuring the mass of each impression sample, the volume was calculated by dividing the mass of each sample by the density of the set impression material. Further corroboration of these measurements was obtained by employing Archimedes principle to measure the volume of each impression sample. Intraclass correlation was used to assess agreement.nnnRESULTSnBoth CBCT (mean volume: 175.9 mm3) and μCT (mean volume: 163.1 mm3) showed a high degree of agreement (intraclass correlation coefficient >0.9) when compared to both weighed and Archimedes principle measurements (mean volume: 177.7 and 182.6 mm3, respectively).nnnCONCLUSIONnCone beam computed tomography is an accurate means of measuring volume of artificially created bone cavities in an ex vivo model. This may provide a valuable tool for monitoring the healing rate of apical periodontitis; further investigations are warranted.

A novel method of forming micro- and macroporous monetite cements

Second to autologous bone grafts are the calcium phosphate cements (CPCs) used as synthetic bone substitutes due to their chemical similarity to the mineral component of bone. Their ability to conform to complex bone defects and excellent osteoconductivity also render them excellent scaffolds for bone tissue engineering, although they do have their own limitations. Calcium phosphates can be divided into two main categories, namely apatite and brushite. Apatites exhibit low solubility, whereas, calcium phosphates that set to form brushite, are metastable, which degrade rapidly, but do subsequently form hydroxyapatite that retards the rate. In contrast dicalcium phosphate anhydrous (monetite) has a higher solubility than octacalcium phosphate and does not transform to an apatite; thus, it is able to continue to degrade with time. Herein, a new method was used via the addition of sodium chloride to β-tricalcium phosphate and monocalcium phosphate monohydrate to form micro- and macroporous monetite (DCPA). The X-ray diffraction and FTIR spectra confirmed the formation of monetite in the presence of both, 6.2 M NaCl solution or 60% of solid sodium chloride. The maximum compressive strength (σc = 12.3 ± 1.8 MPa) and the Youngs modulus (E = 1.0 ± 0.1 GPa) of the monetite cements obtained were comparable to the upper limits of the values reported for cancellous bone and also higher than that reported by other routes used to form monetite. The porous cements analysed by microCT revealed an interconnected porosity with the preliminary in vitro biological evaluation indicating favourable osteoblast cell attachment and growth.

Preparation of multicompartment sub-micron particles using a triple-needle electrohydrodynamic device

Control over the size and morphology of polymeric carriers for drug delivery systems is essential to optimize their functionality. In the current study, we demonstrate the feasibility of using an electrohydrodynamic process with a triple-needle device to prepare nearly mono-dispersed, spherical, tri-layered sub-micron particles. Three biocompatible polymer solutions of poly (lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL) and polymethylsilsesquioxane (PMSQ) were used to prepare particles with three distinct layers. Optimized particles were shown to be spherical with an average size ranging from 320 nm (±80 nm) to 220 (±8 nm), which varied with a change in the working distance in the electrohydrodynamic processing. The surface and internal structure and morphology were studied using confocal, transmission and scanning electron microscopy combined with focused ion beam sectioning. Cytotoxicity was shown to be negligible in an in vitro assay. The ability to fabricate such multilayered particles in a single step, under ambient conditions has considerable potential for a range of applications in particular controlled release drug delivery system.

In vitro osteoinductive potential of porous monetite for bone tissue engineering

Tissue engineering–based bone grafts are emerging as a viable alternative treatment modality to repair and regenerate tissues damaged as a result of disease or injury. The choice of the biomaterial component is a critical determinant of the success of the graft or scaffold; essentially, it must induce and allow native tissue integration, and most importantly mimic the hierarchical structure of the native bone. Calcium phosphate bioceramics are widely used in orthopaedics and dentistry applications due to their similarity to bone mineral and their ability to induce a favourable biological response. One such material is monetite, which is biocompatible, osteoconductive and has the ability to be resorbed under physiological conditions. The osteoinductive properties of monetite in vivo are known; however, little is known of the direct effect on osteoinduction of human mesenchymal stem cells in vitro. In this study, we evaluated the potential of monetite to induce and sustain human mesenchymal stem cells towards osteogenic differentiation. Human mesenchymal stem cells were seeded on the monetite scaffold in the absence of differentiating factors for up to 28 days. The gene expression profile of bone-specific markers in cells on monetite scaffold was compared to the control material hydroxyapatite. At day 14, we observed a marked increase in alkaline phosphatase, osteocalcin and osteonectin expressions. This study provides evidence of a suitable material that has potential properties to be used as a tissue engineering scaffold.

Gene Expression Responses to Mechanical Stimulation of Mesenchymal Stem Cells Seeded on Calcium Phosphate Cement

INTRODUCTIONnThe aim of the study reported here was to investigate the molecular responses of human mesenchymal stem cells (MSC) to loading with a model that attempts to closely mimic the physiological mechanical loading of bone, using monetite calcium phosphate (CaP) scaffolds to mimic the biomechanical properties of bone and a bioreactor to induce appropriate load and strain.nnnMETHODSnHuman MSCs were seeded onto CaP scaffolds and subjected to a pulsating compressive force of 5.5±4.5 N at a frequency of 0.1 Hz. Early molecular responses to mechanical loading were assessed by microarray and quantitative reverse transcription-polymerase chain reaction and activation of signal transduction cascades was evaluated by western blotting analysis.nnnRESULTSnThe maximum mechanical strain on cell/scaffolds was calculated at around 0.4%. After 2 h of loading, a total of 100 genes were differentially expressed. The largest cluster of genes activated with 2 h stimulation was the regulator of transcription, and it included FOSB. There were also changes in genes involved in cell cycle and regulation of protein kinase cascades. When cells were rested for 6 h after mechanical stimulation, gene expression returned to normal. Further resting for a total of 22 h induced upregulation of 63 totally distinct genes that were mainly involved in cell surface receptor signal transduction and regulation of metabolic and cell division processes. In addition, the osteogenic transcription factor RUNX-2 was upregulated. Twenty-four hours of persistent loading also markedly induced osterix expression. Mechanical loading resulted in upregulation of Erk1/2 phosphorylation and the gene expression study identified a number of possible genes (SPRY2, RIPK1, SPRED2, SERTAD1, TRIB1, and RAPGEF2) that may regulate this process.nnnCONCLUSIONnThe results suggest that mechanical loading activates a small number of immediate-early response genes that are mainly associated with transcriptional regulation, which subsequently results in activation of a wider group of genes including those associated with osteoblast proliferation and differentiation. The results provide a valuable insight into molecular events and signal transduction pathways involved in the regulation of MSC osteogenic differentiation in response to a physiological level of mechanical stimulation.

Structural changes and biological responsiveness of an injectable and mouldable monetite bone graft generated by a facile synthetic method

Brushite (dicalcium phosphate dihydrate) and monetite (dicalcium phosphate anhydrous) are of considerable interest in bone augmentation owing to their metastable nature in physiological fluids. The anhydrous form of brushite, namely monetite, has a finer microstructure with higher surface area, strength and bioresorbability, which does not transform to the poorly resorbable hydroxyapatite, thus making it a viable alternative for use as a scaffold for engineering of bone tissue. We recently reported the formation of monetite cements by a simple processing route without the need of hydrothermal treatment by using a high concentration of sodium chloride in the reaction mix of β-tricalcium phosphate and monocalcium phosphate monohydrate. In this paper, we report the biological responsiveness of monetite formed by this method. The in vitro behaviour of monetite after interaction and ageing both in an acellular and cellular environment showed that the crystalline phase of monetite was retained over three weeks as evidenced from X-ray diffraction measurements. The crystal size and morphology also remained unaltered after ageing in different media. Human osteoblast cells seeded on monetite showed the ability of the cells to proliferate and express genes associated with osteoblast maturation and mineralization. Furthermore, the results showed that monetite could stimulate osteoblasts to undergo osteogenesis and accelerate osteoblast maturation earlier than cells cultured on hydroxyapatite scaffolds of similar porosity. Osteoblasts cultured on monetite cement also showed higher expression of osteocalcin, which is an indicator of the maturation stages of osteoblastogenesis and is associated with matrix mineralization and bone forming activity of osteoblasts. Thus, this new method of fabricating porous monetite can be safely used for generating three-dimensional bone graft constructs.

Di-Calcium Phosphate and Phytosphingosine as an Innovative Acid-Resistant Treatment to Occlude Dentine Tubules.

The present investigation evaluated the ability of an experimental di-calcium phosphate (DCP) desensitising agent used alone or combined with phytosphingosine (PHS) to occlude dentine tubules and resist a citric acid (CA) or artificial saliva (AS) challenge. Three groups of human dentine specimens (DS) were treated with the following: (1) PHS alone, (2) DCP or (3) a combination of PHS and DCP. Dentine hydraulic conductance of DS was evaluated using a digital flow sensor at 6.9 kPa. The average fluid volume for each of the treated DS was used to calculate the total dentine permeability reduction (%P) prior to and following CA immersion for 1 min or AS immersion for 4 weeks. The treated DS were subjected to both scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy analysis. Statistically significant differences (%P) were identified between the groups by ANOVA and Fishers multiple comparison test (p < 0.05), respectively. Interestingly, both PHS and DCP appeared to work synergistically. DS treated with DCP or PHS/DCP demonstrated a significant reduction (%P) prior to and following CA or AS challenge (p < 0.05). Both the SEM and FTIR analyses showed consistent brushite crystals occluding the dentine tubules. Conversely, the application of PHS alone failed to demonstrate any significant reduction of dentine permeability (p > 0.05) or show any evidence of occlusion of the dentine tubules. DCP can be used alone or combined with PHS to decrease the dentine permeability as well as to resist a CA and AS challenge. These results would, therefore, suggest that DCP may be a suitable treatment option for dentine hypersensitivity.

The role of new zinc incorporated monetite cements on osteogenic differentiation of human mesenchymal stem cells

β-Tricalcium phosphate particles were sintered in the presence of different amounts (0-0.72mol) of zinc oxide (ZnO) to prepare zinc doped β-TCP (Znβ-TCP) particles for further use in novel monetite (DCPA: CaHPO4) zinc incorporated bone cements with osteogenic differentiation potential towards human mesenchymal stem cells (hMSCs). XRD analysis of zinc incorporated cements prepared with β-TCP reagent particles doped with different amount of ZnO (i.e. 0.03, 0.09 and 0.18mol ZnO) revealed the presence of unreacted Znβ-TCP and monetite. Furthermore, it was shown that zinc ions preferentially occupied the β-TCP crystal lattice rather than the monetite one. Release experiments indicated a burst release of ions from the different fabricated cements during the first 24h of immersion with zinc concentrations ranging between 85 and 100% of the total concentration released over a period of 21days. Cell proliferation significantly increased (P<0.05) on zinc incorporated monetite respect to control samples (Zinc-free cement) at 7 and 14days post seeding. The expression of Runx-2 was significantly up regulated (P<0.05) in the case of cells seeded on monetite prepared with β-TCP doped with 0.03 moles of ZnO. On the other hand, the cell mineralization as well as the expression of osteogenic marker genes ALP and OSC decreased significantly (P<0.05) at 14days post cell seeding. In conclusion, these results suggest that the zinc ions released from the cements during the first 24h of culture played a critical role in regulating the osteogenic differentiation of hMSCs.

Sprouty 2, an Early Response Gene Regulator of FosB and Mesenchymal Stem Cell Proliferation During Mechanical Loading and Osteogenic Differentiation

Sprouty 2 (Spry2), an inhibitor of MAP kinase signaling was previously shown by our group to be induced during mechanical loading of mesenchymal stem cells (MSCs). Here, we studied the implication of Spry2 activation during mechanical loading and chemically induced MSC differentiation. Spry 2 expression showed an immediate early response during mechanical loading and chemical induction of osteogenic differentiation and followed the same pattern as osteogenic associated gene FosB and was necessary for the induction of FosB, as Spry 2 knock down also abrogated the upregulation of FosB expression. Spry 2 knock down was, also associated with an early response of the osteogenic genes Runx‐2 and ALP. Neither the knock‐down of Spry 2 nor the subsequent reduction in FosB had any effect on mid‐late osteogenesis or mineralization but was associated with a significant increase in proliferation of MSC. These effects were possibly governed by negative regulation of MEK/Erk signaling as Spry 2 knock down resulted in an increase in phosphorylation of Erk1/2. In summary, our results shows the involvement of Spry2 in regulation of FosB and Runx2 genes, MAPK signaling and proliferation of MSC. Taken together these results suggest a possible role for Spry2 in regulation of MSC functions in response to mechanical loading and osteogenic differentiation. J. Cell. Biochem. 118: 2606–2614, 2017.