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Silica (SiO2)/chitosan (CS) composite aerogels are bioactive when they are submerged in simulated body fluid (SBF), causing the formation of bone-like hydroxyapatite (HAp) layer. Silica-based hybrid aerogels improve the elastic behavior, and the combined CS modifies the network entanglement as a crosslinking biopolymer. Tetraethoxysilane (TEOS)/CS is used as network precursors by employing a sol-gel method assisted with high power ultrasound (600 W). Upon gelation and aging, gels are dried in supercritical CO2 to obtain monoliths. Thermograms provide information about the condensation of the remaining hydroxyl groups (400-700 degrees C). This step permits the evaluation of the hydroxyl group's content of 2 to 5 OH nm(-2). The formed Si-OH groups act as the inductor of apatite crystal nucleation in SBF. The N-2 physisorption isotherms show a hysteresis loop of type H3, characteristic to good interconnected porosity, which facilitates both the bioactivity and the adhesion of osteoblasts cells. After two weeks of immersion in SBF, a layer of HAp microcrystals develops on the surface with a stoichiometric Ca/P molar ratio of 1.67 with spherulite morphology and uniform sizes of 6 mu m. This fact asserts the bioactive behavior of these hybrid aerogels. Osteoblasts are cultured on the selected samples and immunolabeled for cytoskeletal and focal adhesion expression related to scaffold nanostructure and composition. The initial osteoconductive response observes points to a great potential of tissue engineering for the designed composite aerogels., This research was 80% supported by Andalucia FEDER/ITI 2014-2020 Grant for PI 013/017 and Junta de Andalucia TEP115 and CTS 253 PAIDI research groups (Spain). The work has also been co-financed by the 2014-2020 ERDF Operational Programme and by the Department of Economy, Knowledge, Business and University of the Regional Government of Andalusia. Project reference: FEDER-UCA18_106598.

The authors of this manuscript were partially supported by Research Groups #CTS-138 and #CTS-1028 (Junta de Andalucía, Spain). This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors., All experiments were performed after the approval of the Committee on Animal Research of the University of Granada (CEEA 2014/357) and under the European Union and Spanish regulations for ethics in animal research (EU Directive 63/2010 and Spanish RD 53/2013) and reported following the ARRIVE guidelines., The data that support the findings of this study are available from the corresponding author upon reasonable request., The authors would especially like to acknowledge the contribution to the histochemical and immunohistochemical studies of Serafin Vélez García and Carmen Ruíz Guzmán, technicians in the Department of Surgery, and María Dolores Rodriguez, in the Department of Pathology and IBIMER of the University of Granada and Justin G. Davis for assistance with the writing style., Musashi-1 (MSI1) is an RNA-binding protein that regulates progenitor cells in adult and developing organisms to maintain self-renewal capacities. The role of musashi-1 in the bone healing environment and its relation with other osteogenic factors is unknown. In the current study, we analyze the expression of MSI1 in an experimental model of rat femoral bone fractures. We also analyze the relation between MSI1 expression and the expression of two osteogenic markers: periostin (POSTN) and runt-related transcription factor 2 (RUNX2). We use histological, immunohistochemical, and qPCR techniques to evaluate bone healing and the expression of MSI1, POSTN, and RUNX2 over time (4, 7, and 14 days). We compare our findings with non-fractured controls. We find that in bone calluses, the number of cells expressing MSI1 and RUNX2 increase over time and the intensity of POSTN expression decreases over time. Within bone calluses, we find the presence of MSI1 expression in mesenchymal stromal cells, osteoblasts, and osteocytes but not in hypertrophic chondrocytes. After 14 days, the expression of MSI1, POSTN, and RUNX2 was significantly correlated. Thus, we conclude that musashi-1 potentially serves in the osteogenic differentiation of mesenchymal stromal cells and bone healing. Therefore, further studies are needed to determine the possibility of musashi-1 ' s role as a clinical biomarker of bone healing and therapeutic agent for bone regeneration., Junta de Andalucia European Commission CTS-138 CTS-1028

Silica (SiO2)/chitosan (CS) composite aerogels are bioactive when they are submerged in simulated body fluid (SBF), causing the formation of bone-like hydroxyapatite (HAp) layer. Silica-based hybrid aerogels improve the elastic behavior, and the combined CS modifies the network entanglement as a crosslinking biopolymer. Tetraethoxysilane (TEOS)/CS is used as network precursors by employing a sol-gel method assisted with high power ultrasound (600 W). Upon gelation and aging, gels are dried in supercritical CO2 to obtain monoliths. Thermograms provide information about the condensation of the remaining hydroxyl groups (400&ndash, 700 &deg, C). This step permits the evaluation of the hydroxyl group&rsquo, s content of 2 to 5 OH nm&minus, 2. The formed Si-OH groups act as the inductor of apatite crystal nucleation in SBF. The N2 physisorption isotherms show a hysteresis loop of type H3, characteristic to good interconnected porosity, which facilitates both the bioactivity and the adhesion of osteoblasts cells. After two weeks of immersion in SBF, a layer of HAp microcrystals develops on the surface with a stoichiometric Ca/P molar ratio of 1.67 with spherulite morphology and uniform sizes of 6 &mu, m. This fact asserts the bioactive behavior of these hybrid aerogels. Osteoblasts are cultured on the selected samples and immunolabeled for cytoskeletal and focal adhesion expression related to scaffold nanostructure and composition. The initial osteoconductive response observes points to a great potential of tissue engineering for the designed composite aerogels.

This research was funded 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 Manuel Toledano-Osorio’s PhD research study., Polymeric membranes are employed in guided bone regeneration (GBR) as physical barriers to facilitate bone in-growth. A bioactive and biomimetic membrane with the ability to participate in the healing and regeneration of the bone is necessary. The aim of the present study was to analyze how novel silicon dioxide composite membranes functionalized with zinc or doxycycline can modulate the osteoblasts' proliferation, differentiation, and expression of selected antigenic markers related to immunomodulation. Nanostructured acrylate-based membranes were developed, blended with silica, and functionalized with zinc or doxycycline. They were subjected to MG63 osteoblast-like cells culturing. Proliferation was assessed by MTT-assay, differentiation by evaluating the alkaline phosphatase activity by a spectrophotometric method and antigenic phenotype was assessed by flow cytometry for selected markers. Mean comparisons were conducted by one-way ANOVA and Tukey tests (p < 0.05). The blending of silica nanoparticles in the tested non-resorbable polymeric scaffold improved the proliferation and differentiation of osteoblasts, but doxycycline doped scaffolds attained the best results. Osteoblasts cultured on doxycycline functionalized membranes presented higher expression of CD54, CD80, CD86, and HLA-DR, indicating a beneficial immunomodulation activity. Doxycycline doped membranes may be a potential candidate for use in GBR procedures in several challenging pathologies, including periodontal disease., European Commission MAT2017-85999-P A-BIO-157-UGR-18/FEDER, Ministry of Economy and Competitiveness MAT2017-85999-P, University of Granada/Regional Government of Andalusia Research Fund from Spain A-BIO-157-UGR-18/FEDER

Biogenesis and function of microRNAs can be influenced by genetic variants in the pri-miRNA sequences leading to phenotypic variability. This study aims to identify single nucleotide polymorphisms (SNPs) affecting the expression levels of bone-related mature microRNAs and thus, triggering an osteoporotic phenotype. An association analysis of SNPs located in pri-miRNA sequences with bone mineral density (BMD) was performed in the OSTEOMED2 cohort (n = 2183). Functional studies were performed for assessing the role of BMD-associated miRNAs in bone cells. Two SNPs, rs6430498 in the miR-3679 and rs12512664 in the miR-4274, were significantly associated with femoral neck BMD. Further, we measured these BMD-associated microRNAs in trabecular bone from osteoporotic hip fractures comparing to non-osteoporotic bone by qPCR. Both microRNAs were found overexpressed in fractured bone. Increased matrix mineralization was observed after miR-3679-3p inhibition in human osteoblastic cells. Finally, genotypes of rs6430498 and rs12512664 were correlated with expression levels of miR-3679 and miR-4274, respectively, in osteoblasts. In both cases, the allele that generated higher microRNA expression levels was associated with lower BMD values. In conclusion, two osteoblast-expressed microRNAs, miR-3679 and miR-4274, were associated with BMD; their overexpression could contribute to the osteoporotic phenotype. These findings open new areas for the study of bone disorders.

Background Type 2 diabetes mellitus (T2DM) is associated with an increased risk of osteoporotic fracture. Several factors have been identified as being potentially responsible for this risk, such as alterations in bone remodelling that may have been induced by changes in circulating glucose or/and by the presence of non-oxidative end products of glycosylation (AGEs). The aim of this study is to assess whether such variations generate a change in the gene expression related to the differentiation and osteoblast activity (OPG, RANKL, RUNX2, OSTERIX, and AGE receptor) in primary cultures of human osteoblast-like cells (hOB). Methods We recruited 32 patients; 10 patients had osteoporotic hip fractures (OP group), 12 patients had osteoporotic hip fractures with T2DM (T2DM group), and 10 patients had hip osteoarthritis (OA group) with no osteoporotic fractures and no T2DM. The gene expression was analyzed in hOB cultures treated with physiological glucose concentration (4.5 mM) as control, high glucose (25 mM), and high glucose plus AGEs (2 μg/ml) for 24 h. Results The hOB cultures from patients with hip fractures presented slower proliferation. Additionally, the hOB cultures from the T2DM group were the most negatively affected with respect to RUNX2 and OSX gene expression when treated solely with high glucose or with high glucose plus AGEs. Moreover, high levels of glucose induced a major decrease in the RANKL/OPG ratio when comparing the OP and the T2DM groups to the OA group. Conclusions Our data indicates an altered bone remodelling rate in the T2DM group, which may, at least partially, explain the reduced bone strength and increased incidence of non-traumatic fractures in diabetic patients.