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

Doped Electrospinned Material-Guides High Efficiency Regional Bone Regeneration

Grant PID2020-114694RB-I00 funded by MCIN/AEI 10.13039/501100011033. M.T.-O. is Fellow FPU of Ministry of Universities [grant FPU20/0045].The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/polym15071726/s1The main target of bone tissue engineering is 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 (Si-M) were doped with zinc (Zn-Si-M) or doxycycline (Dox-Si-M). Critical bone defects were effectuated on six New Zealand-bred rabbit skulls and then they were covered with the membranes. After six weeks, a histological analysis (toluidine blue technique) was employed to determine bone cell population as osteoblasts, osteoclasts, osteo- cytes, M1 and M2 macrophages and vasculature. Membranes covering the bone defect determined a higher count of bone cells and blood vessels than in the sham group at the top regions of the defect. Pro-inflammatory M1 appeared in a higher number in the top regions than in the bottom regions, when Si-M and Dox-Si-M were used. Samples treated with Dox-Si-M showed a higher amount of anti-inflammatory and pro-regenerative M2 macrophages. The M1/M2 ratio obtained its lowest value in the absence of membranes. On the top regions, osteoblasts were more abundant when using Si-M and Zn-Si-M. Osteoclasts were equally distributed at the central and lateral regions. The sham group and samples treated with Zn-Si-M attained a higher number of osteocytes at the top regions. A preferential osteoconductive, osteoinductive and angiogenic clinical environment was created in the vicinity of the membrane placed on critical bone defects.MCIN/AEIFPU of Ministry of Universities FPU20/004

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

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

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

Antibacterial Effect of Functionalized Polymeric Nanoparticles on Titanium Surfaces Using an In Vitro Subgingival Biofilm Model

This research was funded by the Ministry of Economy and Competitiveness (MINECO) and the European Regional Development Fund (FEDER), grant number (PID2020-114694RB-I00 MINECO/AEI/FEDER/UE). M. Toledano-Osorio holds a FPU fellowship from the Ministry of Universities (FPU20/00450).This investigation aimed to evaluate the antibacterial effect of polymeric nanoparticles (NPs), functionalized with calcium, zinc, or doxycycline, using a subgingival biofilm model of six bacterial species (Streptococcus oralis, Actinomyces naeslundii, Veillonela parvula, Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans) on sandblasted, large grit, acidetched titanium discs (TiDs). Undoped NPs (Un-NPs) or doped NPs with calcium (Ca-NPs), zinc (Zn-NPs), or doxycycline (Dox-NPs) were applied onto the TiD surfaces. Uncovered TiDs were used as negative controls. Discs were incubated under anaerobic conditions for 12, 24, 48, and 72 h. The obtained biofilm structure was studied by scanning electron microscopy (SEM) and its vitality and thickness by confocal laser scanning microscopy (CLSM). Quantitative polymerase chain reaction of samples was used to evaluate the bacterial load. Data were evaluated by analysis of variance (p < 0.05) and post hoc comparisons with Bonferroni adjustments (p < 0.01). As compared with uncovered TiDs, Dox-NPs induced higher biofilm mortality (47.21% and 85.87%, respectively) and reduced the bacterial load of the tested species, after 72 h. With SEM, scarce biofilm formation was observed in Dox-NPs TiDs. In summary, Dox-NPs on TiD reduced biofilm vitality, bacterial load, and altered biofilm formation dynamics.Spanish GovernmentEuropean Commission PID2020-114694RB-I00 MINECO/AEI/FEDER/UEMinistry of Universities FPU20/0045

Evaluating Membranes for Bone Regeneration

This work was supported by: 1)the Ministry of Economy and Competitiveness and European Regional Development Fund [Project MAT2017-85999-P 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).This research is part of M.T-O.’s PhD research study.Objectives:Maxillofacial bone defectsare the main hindering conditions for traditional dental implant strategies. Guided Bone Regeneration (GBR) is used to handle this situation. The principle of GBR is to use a membrane to prevent the colonization of soft tissue cells of the bone defect and favors the migration of osteogenic linages. Currentmembranes do not completely fulfill the requirements that an optimal membrane should have, sometimes resulting in non-predictable results. Thus, the need to develop an ideal membrane to perform this duty is clear. Recent developments in bio-manufacturing are driving innovations in membranes technology permitting the active participation of the membrane in the healing and regenerative process trough native tissue mimicking, drug-delivery and cells interaction, away from being a passive barrier. New membranes features need specific evaluation techniques, beyond the International Standard for membrane materials (last reviewed in 2004), being this the rationale for the present review.Nanotechnology application has completely shifted the way of analyzing structural characterization. New progresses on osteoimmmunomodulation have also switched the understanding of cells-membranes interaction. Data and Sources:To propose an updated protocol for GBR membranes evaluation, critical reading of therelevant published literature was carried out after a MEDLINE/PubMed database search. Conclusions:The main findings are that a potential active membrane should be assessed in its nanostructure, physicochemical and nanomechanical properties,bioactivityand antibacterial,osteoblasts proliferation, differentiation and mineralization. Immunomodulationtesting for macrophages recruitmentand M2 phenotype promotion in osteoblasts co-culture has to beachieved to completely analyze membranes/tissue interactions.Ministry of Economy and Competitiveness and European Regional Development Fund [Project MAT2017-85999-P MINECO/AEI/FEDER/UE]University of Granada/Regional Government of Andalusia Research Fund from Spain and European Regional Development Fund (A-BIO-157-UGR-18/FEDER

Biochemical assessment of nanostructures in human trabecular bone: Proposal of a Raman microspectroscopy based measurements protocol

Background: Improvements to the understating of the compositional contributions of bone mineral and organic components to the competence of trabecular bone are crucial. The purpose of this study was to propose a protocol to study biochemical composition of trabecular bone, based on two combined Raman analysis methodologies. Material and Methods: Both cluster and single point Raman mappings were obtained, in order to assess bone degeneration associated with aging, disease, or injury, and to help in the evaluation and development of successful therapies. In this study, human trabecular bone has been analysed throughout a) Raman cluster analysis: bone mineral content, carbonate-to-phosphate ratio (both from the mineral components), the crosslinking and nature/secondary structure of collagen (both from the organic components); and b) Single point Raman spectra, where Raman points related to the minerals and organic components were also obtained, both techniques were employed in spectra attained at 400 to 1700 cm- 1. Results: Multivariate analysis confirmed: 1) the different spectral composition, 2) the existence of centroids grouped by chemical affinity of the various components of the trabecular bone, and 3) the several traces of centroids and distribution of chemical compositional clusters. Conclusions: This study is important, because it delivers a study protocol that provides molecular variations information in both mineral and collagen structure of trabecular bone tissue. This will enable clinicians to benefit knowing the microstructural differences in the bone subjected to degeneration of their patients.Project MAT2017-85999-P supported by the Ministry of Economy and Competitiveness (MINECO) of Spanish Government and European Regional Development Fund (ERDF)