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

Zn-containing Adhesives Facilitate Collagen Protection and Remineralization at the Resin-Dentin Interface: A Narrative Review

This work was supported by the Ministry of Economy and Competitiveness and the European Regional Development Fund (Project PID2020-114694RB-100 MINECO/AEI/FEDER/UE). M.T.-O. holds a FPU fellowship from the Ministry of Universities [FPU20/00450].This is a narrative review of the literature assessing the potential effectiveness of doping dentin polymeric adhesives with zinc compounds in order to improve bonding efficacy, remineralization and protection against degradation. A literature search was conducted using electronic databases, such as PubMed, MEDLINE, DIMDI andWeb of Science. Through our search, we found literature demonstrating that Zn-doped dentin adhesives promote protection and remineralization of the resin-dentin interfaces. The increased bioactivity has also facilitated dentinal tubules’ occlusion by crystals’ precipitation contributing to improved sealing efficacy of restorations. Loading dentin adhesives with zinc gives rise to an increase of both crystallinity of mineral and crosslinking of collagen. The main role of zinc, in dentin adhesives, is to inhibit collagen proteolysis. We concluded that zinc exerts a protective effect through binding at the collagen-sensitive cleavage sites of matrixmetalloproteinases (MMPs), contributing to dentin matrix stabilization. Zinc may not only act as a MMPs inhibitor, but also influence signaling pathways and stimulate metabolic effects in dentin mineralization and remineralization processes. Zn-doped adhesives increase the longevity of dentin bonding through MMPs inhibition. Zn poses a remineralization strategy in demineralized dentin.Ministry of Economy and CompetitivenessEuropean Commission PID2020-114694RB-100 MINECO/AEI/FEDER/UEMinistry of Universities FPU20/0045

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

Silver improves collagen structure and stability at demineralized dentin: a dynamic-mechanical and Raman analysis.

Objective: This study aimed to evaluate the effect of silver loaded nanoparticles (NPs) application on dentin remineralization. Methods: Polymethylmetacrylate-based NPs and silver loaded NPs (Ag-NPs) were applied on demineralized dentin surfaces. Dentin was characterized morphologically by scanning electron microscopy, mechanically probed by a nanoindenter to test nanohardness and Young modulus, and chemically analyzed by Raman spectroscopy after 24 h and 7 d of storage. Untreated surfaces were used as control. Data were submitted to ANOVA and Student-Newman-Keuls multiple comparisons tests (P<0.05). Results: After Raman analysis, dentin treated with Ag-NPs obtained the lowest mineralization and intensity of stoichiometric hydroxyapatite when compared with specimens treated with undoped-NPs. The lowest relative mineral concentration, expressed as the ratio phosphate or carbonate/phenyl group, and crystallinity was attained by dentin treated with with Ag-NPs, after 7 d. Ag-NPs application generated the highest values of collagen crosslinking (intensity at 1032 cm-1 band). The molecular conformation of the collagen’s polypeptide chains, amide-I and CH2 also attained the highest peaks in dentin treated with Ag-NPs. Staggered and demineralized collagen fibrils were observed covering the dentin surfaces treated with Ag-NPs, at both 24 h and 7 d. Samples treated with Ag-NPs attained the lowest values of nanohardness and Young’s modulus at 7 d of storage. Conclusions: Peritubular and intertubular dentin were remineralized when using undoped-NPs. After 7 d, collagen treated with NPs was remineralized but dentin treated with Ag-NPs attained an improved collagen matrix structure and stability but the lowest mineralization and crystallinity.This work was supported by the Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER). Project MAT2017-85999-P MINECO/AEI/FEDER/UE

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

The Collagen Origin Influences the Degradation Kinetics of Guided Bone Regeneration Membranes

This work was supported by: (1) the Ministry of Economy and Competitiveness and European Regional Development Fund [Project PID2020-114694RB-100 MINECO/AEI/FEDER/UE], (2) University of Granada/Regional Government of Andalusia Research Fund from Spain and European Regional Development Fund (A-BIO-157-UGR-18/FEDER).Collagen membranes are currently the most widely used membranes for guided bone regeneration; however, their rapid degradation kinetics means that the barrier function may not remain for enough time to permit tissue regeneration to happen. The origin of collagen may have an important effect on the resistance to degradation. The aim of this study was to investigate the biodegradation pattern of five collagen membranes from different origins: Biocollagen, Heart, Evolution X-fine, CopiOs and Parasorb Resodont. Membranes samples were submitted to different degradation tests: (1) hydrolytic degradation in phosphate buffer saline solution, (2) bacterial collagenase from Clostridium histolyticum solution, and (3) enzyme resistance using a 0.25% porcine trypsin solution. Immersion periods from 1 up to 50 days were performed. At each time point, thickness and weight measurements were performed with a digital caliper and an analytic microbalance, respectively. ANOVA and Student–Newman–Keuls tests were used for comparisons (p < 0.05). Differences between time-points within the same membranes and solutions were assessed by pair-wise comparisons (p < 0.001). The Evolution X-fine collagen membrane from porcine pericardium attained the highest resistance to all of the degradation tests. Biocollagen and Parasorb Resodont, both from equine origin, experienced the greatest degradation when immersed in PBS, trypsin and C. histolyticum during challenge tests. The bacterial collagenase solution was shown to be the most aggressive testing method.Ministry of Economy and Competitiveness PID2020-114694RB-100European Commission PID2020-114694RB-100 A-BIO-157-UGR-18/FEDERUniversity of Granada/Regional Government of Andalusia Research Fund from Spain A-BIO-157-UGR-18/FEDE

In Vitro Biodegradation Pattern of Collagen Matrices for Soft Tissue Augmentation

This work was supported by: (1) the Ministry of Economy and Competitiveness and the European Regional Development Fund (Project PID2020-114694RB-100 MINECO/AEI/FEDER/UE), (2) the University of Granada/Regional Government of Andalusia Research Fund from Spain, and the European Regional Development Fund (A-BIO-157-UGR-18/FEDER). This research is part of C.V.'s PhD research study.Collagen matrices have become a great alternative to the use of connective tissue grafts for soft tissue augmentation procedures. One of the main problems with these matrices is their volume instability and rapid degradation. This study has been designed with the objective of examining the degradation of three matrices over time. For this purpose, pieces of 10 10 mm2 of Fibro-Gide, Mucograft and Mucoderm were submitted to three different degradation tests— (1) hydrolytic degradation in phosphate buffer solution (PBS); (2) enzyme resistance, using a 0.25% porcine trypsin solution; and (3) bacterial collagenase resistance (Clostridium histolyticum)—over different immersion periods of up to 50 days. Weight measurements were performed with an analytic microbalance. Thickness was measured with a digital caliper. A stereomicroscope was used to obtain the matrices’ images. ANOVA and Student–Newman–Keuls tests were used for mean comparisons (p < 0.05), except when analyzing differences between time-points within the same matrix and solution, where pair-wise comparisons were applied (p < 0.001). Fibro-Gide attained the highest resistance to all degradation challenges. The bacterial collagenase solution was shown to constitute the most aggressive test as all matrices presented 100% degradation before 14 days of storage.Ministry of Economy and CompetitivenessEuropean Commission PID2020-114694RB-100 A-BIO-157-UGR-18/FEDERUniversity of Granada/Regional Government of Andalusia Research Fund from Spai

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)

Nanoscopic dynamic mechanical analysis of resin-infiltrated dentine, under in vitro chewing and bruxism events

The aim of this study was to evaluate the induced changes in mechanical behavior and bonding capability of resin–infiltrated dentine interfaces, after application of mechanical stimuli. Dentine surfaces were subjected to partial demineralization through 37% phosphoric acid etching followed by the application of an etch-and-rinse dentine adhesive, Single Bond (3M/ESPE). Bonded interfaces were stored in simulated body fluid during 24 h, and then tested or submitted to the mechanical loading challenge. Different loading waveforms were applied: No cycling (I), 24 h cycled in sine (II) or square (III) waves, sustained loading held for 24 h (IV) or sustained loading held for 72 h (V). Microtensile bond strength (MTBS) was assessed for the different groups. Debonded dentine surfaces were studied by field emission scanning electron microscopy (FESEM). At the resin–dentine interface, both the hybrid layer (HL) and the bottom of the hybrid layer (BHL), and both peritubular and intertubular were evaluated using a nanoindenter in scanning mode. The load and displacement responses were used to perform the nano-Dynamic Mechanical analysis and to estimate the complex and storage modulus. Dye assisted Confocal Microscopy Evaluation was used to assess sealing ability. Load cycling increased the percentage of adhesive failures in all groups. Specimens load cycled in held 24 h attained the highest complex and storage moduli at HL and BHL. The storage modulus was maximum in specimens load cycled in held 24 h at peritubular dentine, and the lowest values were attained at intertubular dentine. The storage modulus increased in all mechanical tests, at peritubular dentine. An absence of micropermeability and nanoleakage after loading in sine and square waveforms were encountered. Porosity of the resin–dentine interface was observed when specimens were load cycled in held 72 h. Areas of combined sealing and permeability were discovered at the interface of specimens load cycled in held 24 h. Crack-bridging images appeared in samples load cycled with sine waveform, after FESEM examination.This work was supported by grants MINECO/FEDER MAT2014-52036-P and FIS2013-41821-R