Dexamethasone and Doxycycline Doped Nanoparticles Increase the Differentiation Potential of Human Bone Marrow Stem Cells

Non-resorbable polymeric nanoparticles (NPs) are proposed as an adjunctive treatment for bone regenerative strategies. The present in vitro investigation aimed to evaluate the effect of the different prototypes of bioactive NPs loaded with zinc (Zn-NPs), doxycycline (Dox-NPs) or dexamethasone (Dex-NPs) on the viability, morphology, migration, adhesion, osteoblastic differentiation, and mineralization potential of human bone marrow stem cells (hBMMSCs). Cell viability, proliferation, and differentiation were assessed using a resaruzin-based assay, cell cycle analysis, cell migration evaluation, cell cytoskeleton staining analysis, Alizarin Red S staining, and expression of the osteogenic-related genes by a real-time quantitative polymerase chain reaction (RT-qPCR). One-Way ANOVA and Tukey’s test were employed. The resazurin assay showed adequate cell viability considering all concentrations and types of NPs at 24, 48, and 72 h of culture. The cell cycle analysis revealed a regular cell cycle profile at 0.1, 1, and 10 g/mL, whereas 100 g/mL produced an arrest of cells in the S phase. Cells cultured with 0.1 and 1 g/mL NP concentrations showed a similar migration capacity to the untreated group. After 21 days, mineralization was increased by all the NPs prototypes. Dox-NPs and Dex-NPs produced a generalized up-regulation of the osteogenic-related genes. Dex-NPs and Dox-NPs exhibited excellent osteogenic potential and promoted hBMMSC differentiation. Future investigations, both in vitro and in vivo, are required to confirm the suitability of these NPs for their clinical application.Ministry of Economy and CompetitivenessEuropean Commission PID2020-114694RB-I00 PID2020-115887GB-I00Ministry of Universities FPU20/00450European Social Fund (ESF)Center for Forestry Research & Experimentation (CIEF)European Commissio

Biomimetic Remineralization of an Extracellular Matrix Collagen Membrane for Bone Regeneration

Natural extracellular matrix (ECM) collagen membranes are frequently used for bone regeneration procedures. Some disadvantages, such as rapid degradation and questionable mechanical properties, limit their clinical use. These membranes have a heterologous origin and may proceed from different tissues. Biomineralization is a process in which hydroxyapatite deposits mainly in collagen fibrils of the matrices. However, when this deposition occurs on the ECM, its mechanical properties are increased, facilitating bone regeneration. The objective of the present research is to ascertain if different membranes from distinct origins may undergo biomineralization. Nanomechanical properties, scanning electron (SEM) and multiphoton (MP) microscopy imaging were performed in three commercially available ECMs before and after immersion in simulated body fluid solution for 7 and 21 d. The matrices coming from porcine dermis increased their nanomechanical properties and they showed considerable mineralization after 21 d, as observed in structural changes detected through SEM and MP microscopy. It is hypothesized that the more abundant crosslinking andMinistry of Economy and CompetitivenessEuropean Commission PID2020-114694RB-I00 PID2020-113919RB-I00Ministry of Universities FPU20/0045

Novel Pastes Containing Polymeric Nanoparticles for Dentin Hypersensitivity Treatment: An In Vitro Study

Tubule occlusion and remineralization are considered the two main goals of dentin hypersensitivity treatment. The objective is to assess the ability of dentifrices containing zinc-doped polymeric nanoparticles (NPs) to enduringly occlude the dentinal tubules, reinforcing dentin’s mechanical properties. Fifteen dentin surfaces were acid-treated for dentinal tubule exposure and brushed with (1) distilled water, or with experimental pastes containing (2) 1% of zinc-doped NPs, (3) 5% of zinc-doped NPs, (4) 10% of zinc-doped NPs or (5) Sensodyne®. Topographical and nanomechanical analyses were performed on treated dentin surfaces and after a citric acid challenge. ANOVA and Student–Newman–Keuls tests were used (p < 0.05). The main results indicate that all pastes produced tubule occlusion (100%) and reinforced mechanical properties of intertubular dentin (complex modulus was above 75 GPa). After the citric acid challenge, only those pastes containing zinc-doped NPs were able to maintain tubular occlusion, as specimens treated with Sensodyne® have around 30% of tubules opened. Mechanical properties were maintained for dentin treated with Zn-doped NPs, but in the case of specimens treated with Sensodyne®, complex modulus values were reduced below 50 GPa. It may be concluded that zinc-doped NPs at the lowest tested concentration produced acid-resistant tubular occlusion and increased the mechanical properties of dentin.Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER), grant number PID2020-114694RB-I00 MINECO/AEI/ FEDER/UELaboratorios KIN® fabricated and provided tested experimental pastes loaded with Zn-doped NP

Assessing bone quality through mechanical properties in postmenopausal trabecular bone.

Background: The inner structure of trabecular bone is a result of structural optimisation provided by remodeling processes. Changes in hormonal status related to menopause cause bone tissue loss and micro-architectural deterioration with a consequent susceptibility to fracture. Accumulation of micro-damage in bone, as a function of the rate of production and rate of repair, underlies the development of stress fractures, increasing fragility associated to age and osteoporosis, especially in transmenopausal women. Patients and Methods: Quasi-static and nano-dynamic mechanical characterisation were undertaken in trabecular bone from femoral neck biopsies of postmenopausal women. AFM complementary studies were performed to determine nano-roughness (SRa) and the fibrils width of collagen. Nanoindentations were used to quantify transmenopausal changes in intrinsic mechanical properties of trabecular bone: hardness (Hi), modulus of Young (Ei), complex modulus (E*), tan delta (δ), storage modulus (E') and loss modulus (E"). Results: As result of the quasi-static measurements, 0.149 (0.036) GPa and 2.95 (0.73) GPa of Hi and Ei were obtained, respectively. As result of the nano-dynamic measurements, 17.94 (3.15), 0.62 (0.10), 13.79 (3.21 and 6.39 (1.28) GPa of E*, tan (δ), E' and E" were achieved, respectively. 101.07 SRa and 831.28 nm of fibrils width were additionally obtained. Conclusion: This study poses a first approach to the measurement of bone quality in postmenopausal trabecular bone by combining quasistatic, nano-DMA analysis and tribology of dentin surface through AFM characterizationProject MAT2017-85999-P supported by the Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER)

SEM and AFM characterization of surface of two RMGICs for degradation before and after modification with bioactive glass ceramic

“This is an Accepted Manuscript of an article published by Taylor & Francis Group in Journal of Adhesion Science and Technology on 09/12/2015, available online: http://www.tandfonline.com/10.1080/01694243.2015.1115603."Objectives: The aim of this study was to evaluate the effect of bioactive glass–ceramic particles (Biosilicate®) addition on surface nanoroughness and topography of Resin-modified glass ionomer cements (RMGICs).Methods: Experimental materials were made by incorporating 2 wt% of Biosilicate® into Fuji II LC® (FL) and Vitremer® (VT) powders. Disks of RMGICs (with and without Biosilicate®) measuring 0.5 cm (diameter) × 0.5 mm (thickness) were fabricated and polished. Samples were stored at 37 °C in dry or immersed in distilled water for 30 days. Digital images (20 × 20 μm) from the surfaces were obtained by means of an atomic force microscopy. Three images were acquired for each sample, and four nanoroughness measurements were performed in each image. Nanoroughness (Ra, nm) was assessed by Nanoscope Software V7. Data were analyzed with ANOVA and Student–Newman–Keuls multiple comparisons (p < 0.05). SEM images were obtained for surface topography analysis.Results: FL was significantly rougher than VT (p < 0.05) in wet and dry conditions. The addition of Biosilicate® increased the surface roughness in VT and decreased in FL, regardless of the storage media (p ≤ 0.05). No differences existed between materials and storage conditions after Biosilicate® addition. Significance: The Biosilicate® particles addition produced changes on the surface nanoroughness of the RMGICs. These changes depended on the particles size of the original cements in dry conditions. In water storage, dissolution of the Biosilicate® particles, a silica-rich gel formation, and a hydroxyl carbonate apatite precipitation on the surface of the materials changed the nanoroughness surface. FL was the roughest in both conditions.Significance: The Biosilicate® particles addition produced changes on the surface nanoroughness of the RMGICs. These changes depended on the particles size of the original cements in dry conditions. In water storage, dissolution of the Biosilicate® particles, a silica-rich gel formation, and a hydroxyl carbonate apatite precipitation on the surface of the materials changed the nanoroughness surface. FL was the roughest in both conditions.MINECO/FEDERMAT2014-52036-P

In vitro mechanical stimulation facilitates stress dissipation and sealing ability at the conventional glass ionomer cement-dentin interface.

Objective: The aim of this study was to evaluate the induced changes in the chemical and mechanical performance at the glass-ionomer cement-dentin interface after mechanical load application. Methods: A conventional glass-ionomer cement (GIC) (Ketac Bond), and a resin-modified glass-ionomer cement (RMGIC) (Vitrebond Plus) were used. Bonded interfaces were stored in simulated body fluid, and then tested or submitted to the mechanical loading challenge. Different loading waveforms were applied: No cycling, 24 h cycled in sine or loaded in sustained hold waveforms. The cement-dentin interface was evaluated using a nano-dynamic mechanical analysis, estimating the complex modulus and tan δ. Atomic Force Microscopy (AFM) imaging, Raman analysis and dye assisted confocal microscopy evaluation (CLSM) were also performed. Results: The complex modulus was lower and tan delta was higher at interfaces promoted with the GIC if compared to the RMGIC unloaded. The conventional GIC attained evident reduction of nanoleakage. Mechanical loading favored remineralization and promoted higher complex modulus and lower tan delta values at interfaces with RMGIC, where porosity, micropermeability and nanoleakage were more abundant. Conclusions: Mechanical stimuli diminished the resistance to deformation and increased the stored energy at the GIC-dentin interface. The conventional GIC induced less porosity and nanoleakage than RMGIC. The RMGIC increased nanoleakage at the porous interface, and dye sorption appeared within the cement. Both cements created amorphous and crystalline apatites at the interface depending on the type of mechanical loading.This work was supported by the Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER) [Project MAT2017-85999-P]

Doxycycline and Zinc Loaded Silica-Nanofibrous Polymers as Biomaterials for Bone Regeneration

The authors are grateful to Antonio L. Medina-Castillo (NanoMyP-University of Granada Spin-Off enterprise) for the scientific and technical support at experimental membranes’ designing and fabrication and to Gertrudis Gomez Villaescusa (University of Granada) for technical assistance at the laboratory.Themain target of bone tissue engineeringis to design biomaterials that support bone regeneration and vascularization. Nanostructured membranes of (MMA)1-co-(HEMA)1/(MA)3-co-(HEA)2 loaded with 5% wt of SiO2-nanoparticles (HOOC-Si-Membrane) were doped with zinc (Zn-HOOC-Si-Membrane) or doxycycline (Dox-HOOC-Si-Membrane). Critical bone defects were effectuated on six New Zealand-bred rabbit skulls and covered with the membranes. After six weeks, the bone architecture was evaluated with micro computed tomography. Three histological analyses were utilized to analyse bone regeneration, including von Kossa silver nitrate, toluidine blue and fluorescence. All membrane-treated defects exhibited higher number of osteocytes and bone perimeter than the control group without the membrane. Zn-HOOC-Si-Membranes induced higher new bone and osteoid area than those treated with HOOC-Si-Membranes, and control group, respectively. Zn-HOOC-Si-Membranes and Dox-HOOC-Si-Membranes attained the lowest ratio M1 macrophages/M2 macrophages. Dox-HOOC-Si-Membranes caused the lowest number of osteoclasts, and bone density. At the trabecular new bone, Zn-HOOC-Si-Membranes produced the highest angiogenesis, bone thickness, connectivity, junctions and branches. Zn-HOOC-Si-Membranes enhanced biological activity, attained a balanced remodeling, and achieved the greatest regenerative efficiency after osteogenesis and angiogenesis assessments. The bone-integrated Zn-HOOC-Si-Membranes can be considered as bioactive modulators provoking a M2 macrophages (pro-healing cells) increase, being a potential biomaterial for promoting bone repair.Ministry of Economy and CompetitivenessEuropean Union (EU) MINECO/FEDER MAT2017-85999

Ex vivo detection and characterization of remineralized carious dentin, by nanoindentation and single point Raman spectroscopy, after amalgam restoration

The aim of this study was to assess the mechanical and chemical performance of sound and caries-affected dentin (CAD), after Zn-free vs containing amalgam restorations placement and thermocycling. Dentin surfaces were studied by Atomic Force Microscopy (AFM) analysis for surface morphological characterization (including fibril diameter assessment), nano-indentation (to measure nano-hardness-Hi and modulus of Young-Ei), and single point Raman spectroscopy for chemical analysis. Measurements were performed before amalgam placement, after amalgam removal, and after 3 months of thermocycling (100,000cy/5 ºC and 55 ºC). Restorations increased both Hi and Ei at intertubular dentin of CAD. The highest values of Hi were achieved at intertubular dentin after restoring with Zn-containing amalgams. Remineralization of dentin was attributed to the increase of both amorphous and crystalline new mineral, as lower degrees of crystal imperfections in junction with crystal disorders, and improvement in structural stability of collagen were found. Higher presence of minerals were also confirmed after the decrease of fluoridated apatite and the increase of the total water content. Proteoglycans, lipids, and proteins, augmented in both sound and CAD, providing support for the mineral growing. The increase of bands assigned to vibration of carbonate calcium phosphate contributed to a decrease of crystallinity.Project MAT2014-52036-P supported by the Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER)

Self-etching Zinc-doped adhesives improve the potential of caries-affected dentin to be functionally remineralized

The aim of this study was to evaluate if mechanical cycling influences bioactivity at the resin-carious dentin interface after bonding with Zn-doped self-etching adhesives. Caries-affected dentin (CAD) surfaces were bonded with: Clearfil SE Bond (SEB), and 10 wt% ZnO nanoparticles or 2 wt% ZnCl2 were added into the SEB primer or bonding components. Bonded interfaces were stored during 24 h, and then tested or submitted to mechanical loading. Microtensile bond strength (MTBS) was assessed. Debonded dentin surfaces were studied by field emission scanning electron microscopy (FESEM). Remineralization of the bonded interfaces was evaluated through nanohardness (Hi) and Young’s modulus (Ei), Raman spectroscopy/cluster analysis, and Masson's trichrome staining technique. Load cycling increased the percentage of adhesive failures. New precipitation of minerals composed of zinc-base salts and multiple Zn-rich phosphate deposits were observed in samples infiltrated with the Zn-doped adhesives. At the hybrid layer, specimens treated with ZnO incorporated in the primer (SEB·P-ZnO), after load cycling, attained the highest Ei and Hi. Load cycling increased Ei at the bottom of the hybrid layer when both, SEB un-doped and SEB with ZnCl2 included in the bonding (SEB·Bd-ZnCl2), were used. ZnO incorporated in the primer promoted an increase in height of the phosphate and carbonate peaks, crystallinity, relative mineral concentration, and lower collagen crosslinking. ZnCl2 included in the bonding attained similar results, but relative mineral concentration decreased, associated to higher crosslinking and restricted collagen maturation. Staining techniques permitted to observe no signs of exposed protein at the resin-dentin interface after using SEB·PZnO.This work was supported by grant MINECO/FEDERMAT2014-52036-P

Advanced zinc-doped adhesives for high performance at the resin-carious dentin interface

The purpose of this study was to evaluate the remineralization ability of an etch-and-rinse Zn-doped resin applied on caries-affected dentin (CAD). CAD surfaces were subjected to: (i) 37% phosphoric acid (PA) or (ii) 0.5M ethylenediaminetetraacetic acid (EDTA). 10wt% ZnO nanoparticles or 2wt% ZnCl2 were added into the adhesive Single Bond (SB), to create the following groups: PA+SB, PA+SB-ZnO, PA+SB-ZnCl2, EDTA+SB, EDTA+SB-ZnO, EDTA+SB-ZnCl2. Bonded interfaces were submitted to mechanical loading or stored during 24h. Remineralization of the bonded interfaces was studied by AFM nano-indentation (hardness and Young׳s modulus), Raman spectroscopy [mapping with principal component analysis (PCA), and hierarchical cluster analysis (HCA)] and Masson׳s trichrome staining technique. Dentin samples treated with PA+SB-ZnO attained the highest values of nano-mechanical properties. Load cycling increased both mineralization and crystallographic maturity at the interface; this effect was specially noticed when using ZnCl2-doped resin in EDTA-treated carious dentin. Crosslinking attained higher frequencies indicating better conformation and organization of collagen in specimens treated with PA+SB-ZnO, after load cycling. Trichrome staining technique depicted a deeper demineralized dentin fringe that became reduced after loading, and it was not observable in EDTA+SB groups. Multivariate analysis confirmed de homogenizing effect of load cycling in the percentage of variances, traces of centroids and distribution of clusters, especially in specimens treated with EDTA+SB-ZnCl2.Project MAT2014-52036-P supported by the Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER)