Acute coronavirus infection triggers a TNF-dependent osteoporotic phenotype in mice.

AIMS: Millions of people died during the COVID-19 pandemic, but the vast majority of infected individuals survived. Now, some consequences of the disease, known as long COVID, are been revealed. Although the respiratory system is the target of Sars-CoV-2, COVID-19 can influence other parts of the body, including bone. The aim of this work was to investigate the impact of acute coronavirus infection in bone metabolism. MAIN METHODS: We evaluated RANKL/OPG levels in serum samples of patients with and without acute COVID-19. In vitro, the effects of coronavirus in osteoclasts and osteoblasts were investigated. In vivo, we evaluated the bone phenotype in a BSL2 mouse model of SARS-like disease induced by murine coronavirus (MHV-3). KEY FINDINGS: Patients with acute COVID-19 presented decreased OPG and increased RANKL/OPG ratio in the serum versus healthy individuals. In vitro, MHV-3 infected macrophages and osteoclasts, increasing their differentiation and TNF release. Oppositely, osteoblasts were not infected. In vivo, MHV-3 lung infection triggered bone resorption in the femur of mice, increasing the number of osteoclasts at 3dpi and decreasing at 5dpi. Indeed, apoptotic-caspase-3+ cells have been detected in the femur after infection as well as viral RNA. RANKL/OPG ratio and TNF levels also increased in the femur after infection. Accordingly, the bone phenotype of TNFRp55-/- mice infected with MHV-3 showed no signs of bone resorption or increase in the number of osteoclasts. SIGNIFICANCE: Coronavirus induces an osteoporotic phenotype in mice dependent on TNF and on macrophage/osteoclast infection.

Injectable chitosan/gelatin/bioactive glass nanocomposite hydrogels for potential bone regeneration: In vitro and in vivo analyses.

The present work describes in vitro and in vivo behaviors of thermosensitive composite hydrogels based on polymers/bioactive glass nanoparticles. Assays in SBF (simulated body fluid) solution showed that loss of hydrogel mass in vitro was decreased by 4.3% when bioactive glass nanoparticles (nBG) were incorporated, and confirmed the bioactivity of nBG containing hydrogels. In vitro assays demonstrated the cytocompatibility of the hydrogels with encapsulated rat bone marrow mesenchymal stem cells (BMSC). Crystal violet assays showed a 27% increase in cell viability when these cells were seeded in hydrogels containing nBG. In vivo biocompatibility was examined by injecting hydrogels into the dorsum of Swiss rats. The results indicated that the prepared hydrogels were nontoxic upon subcutaneous injection, and could be candidates for a safe in situ gel-forming system. Injection of the hydrogels into a rat tibial defect allowed preliminary evaluation of the hydrogels' regenerative potential. Micro Computed Tomography analysis suggested that more new tissue was formed in the defects treated with the hydrogels. Taken together, our data suggest that the developed injectable composite hydrogels possess properties which make them suitable candidates for use as temporary injectable matrices for bone regeneration.

Structural analysis of fluorine-containing bioactive glass nanoparticles synthesized by sol-gel route assisted by ultrasound energy.

In the last decades, studies about the specific effects of bioactive glass on remineralization of dentin were the focus of attention, due to their excellent regenerative properties in mineralized tissues. The incorporation of Fluorine in bioactive glass nanoparticles may result in the formation of fluorapatite (FAP), which is chemically more stable than hydroxyapatite or carbonated hydroxyapatite, and therefore is of interest for dental applications. The aim of this study was to synthesize and characterize a new system of Fluorine-containing bioactive glass nanoparticles (BGNPF). A sol-gel route assisted by ultrasound was used for the synthesis of BGNPF. The particles obtained were characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), atomic force microscopy (AFM), X-ray diffraction (XRD), dynamic light scattering (DLS), nitrogen adsorption, and X-ray photoelectron spectroscopy (XPS). SEM micrographs showed that the particles are quite uniform spherical nanostructures, occurring agglomeration or partial sinterization of the particulate system after heat treatment. XRD and XPS analysis results suggest the formation of fluorapatite crystals embedded within the matrix of the bioactive glass nanoparticles. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 360-366, 2018.

Thermogelling chitosan-collagen-bioactive glass nanoparticle hybrids as potential injectable systems for tissue engineering.

Recently, stimuli-responsive nanocomposite-derived hydrogels have gained prominence in tissue engineering because they can be applied as injectable scaffolds in bone and cartilage repair. Due to the great potential of these systems, this study aimed to synthesize and characterize novel thermosensitive chitosan-based composites, chemically modified with collagen and reinforced by bioactive glass nanoparticles (BG) on the development of injectable nanohybrids for regenerative medicine applications. Thus, the composite hydrogels were extensively characterized by structural, morphological, rheological, and biological testing. The composites showed thermosensitive response with the gelation temperature at approximately 37 °C, which is compatible with the human body temperature. In addition, scanning electron microscopy (SEM) analysis indicated that the chitosan hydrogels exhibited 3D-porous structures, and the incorporation of collagen in the system caused increase on the average pore size. Fourier transform infrared spectroscopy (FTIR) analysis indicated the main functional groups of each component of the composite system and their chemical interactions forming the scaffold. Moreover, rheological measurements were employed to assess the viscoelastic behavior of the hydrogels as a function of the temperature. The results demonstrated that the addition of collagen and bioactive glass increases the mechanical properties after the gelation process. The addition of 2 wt.% of BG nanoparticles caused an increase of approximately 39% on stiffness compared to pure chitosan and the addition of 30 wt.% collagen caused a further increase on the stiffness by 95%. The cytotoxicity and cell viability of the hydrogels were assessed by MTT and LIVE/DEAD® assays, where the results demonstrated no toxic effect of the composites on the human osteosarcoma cell culture (SAOS) and kidney cells line of human embryo (HEK 293 T). Hence, it can be stated that innovative composites were successfully designed and synthesized in this research with promising potential to be used as thermoresponsive biomaterials for bone-tissue bioapplications.

Chitosan and carboxymethyl-chitosan capping ligands: Effects on the nucleation and growth of hydroxyapatite nanoparticles for producing biocomposite membranes.

Synthetic biomaterials based on calcium phosphates (CaP) have been widely studied for bone tissue reconstruction therapies, but no definitive solution that fulfills all of the required properties has been identified. Thus, this study reports the synthesis of composite membranes based on nanohydroxyapatite particles (nHA) embedded in chitosan (CHI) and O-carboxymethyl chitosan (CMC) matrices produced using a one-step co-precipitation method in water media. Biopolymers were used as capping ligands for simultaneously controlling the nucleation and growth of the nHA particles during the precipitation process and also to form the polymeric network of the biocomposites. The bionanocomposites were extensively characterized using light microscopy (LM), scanning and transmission electron microscopy (SEM/TEM), energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), atomic force microscopy (AFM), X-ray micro-CT analysis (µCT), andMTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide) cell proliferation assays for cell cytotoxicity. The results demonstrated that the ligands used during the synthesis highly affected the composites produced, primarily due the changes in the mechanisms and kinetics of nucleation and growth of the HA particles at the nanoscale level. The SEMimages revealed that the use of carboxyl-functionalized chitosan (CMC) ligands significantly reduced the average size of theHA nanoparticles and caused the formation of a narrower size distribution (90±20nm) compared to theHAnanoparticles producedwith chitosan ligands (220±50nm). The same trend was verified by the AFM analysis,where the nHA particles were formed evenly dispersed in the polymer matrix. However, the CMC-based composites were more homogeneously distributed, which was endorsed by the images collected via X-ray micro-CT. The FTIR spectra and the XRD analysis indicated that nanosized hydroxyapatite was the predominant calcium phosphate phase produced during the co-precipitation aqueous process for both the chitosan and CMC biocomposites. These novel hybrid systems based on chitosan and chitosan-derivatives with nHA composites were non-cytotoxic to a human osteoblast-like model cell line (SAOS) according to MTT in vitro assays. Moreover, the CMC-nHA biocomposites revealed a striking improvement in the cell viability response compared to the CHI-nHA biocomposite, which was attributed to the much higher surface area caused by the refinement of the nanoparticles size. Thus, the results of this study demonstrate that these novel bionanocomposite membranes offer promising perspectives as biomaterials for potential repair and replacement of cartilage and bone tissues.

Effect of the ionic product of bioglass 60s on osteoblastic activity in canines.

BACKGROUND: The objective of the present study was to evaluate the effect of the ionic product (IP) of BG60S on osteoblastic activity. The following media groups were created: DMEM, which is formed by osteoblasts in basal medium; IP DMEM, which is formed by osteoblasts in IP with basal medium; OST, which is formed by osteoblasts in osteogenic medium; and IP OST, which is formed by osteoblasts in IP with osteogenic medium. The osteoblasts were cultivated in an incubator at 37 °C and 5 % CO2 for 7, 14 and 21 days. After each period, the alkaline phosphatase (AP) activity, mineralised area per field and expression of osterix (OSX), bone sialoprotein (BSP), osteonectin (ON) and osteocalcin (OC) were evaluated by reverse transcription (RT)-PCR. RESULTS: The IP significantly increased the AP activity in the IP DMEM group at 7 and 14 days and reduced the AP activity in the IP OST group at 14 and 21 days relative to their respective controls (DMEM and OST). The groups that received the IP displayed a significant increase in the percentage of mineralised area per field and more advance maturation of the extracellular matrix relative to those that did not receive IP. The IP significantly increased the expression of OSX, BSP and ON in osteoblast cultures maintained in IP DMEM compared with the control (DMEM) for the majority of studied periods. In osteogenic medium, IP also significantly increased OSX, BSP, ON and OC expression compared with the control (OST) for the majority of studied periods. CONCLUSIONS: The IP of BG60S alters the gene expression of canine osteoblasts, favouring the synthesis and mineralisation of the extracellular matrix.

Hybrid matrix grafts to favor tissue regeneration in rabbit femur bone lesions.

At present, typical approaches employed to repair fractures and other bone lesions tend to use matrix grafts to promote tissue regeneration. These grafts act as templates, which promote cellular adhesion, growth and proliferation, osteoconduction, and even osteoinduction, which commonly results in de novo osteogenesis. The present work aimed to study the bone-repairing ability of hybrid matrixes (HM) prepared with polyvinyl alcohol (PVA) and bioactive glass in an experimental rabbit model. The HM were prepared by combining 30% bioactive glass (nominal composition of 58% SiO2 -33 % CaO - 9% P2O5) and 70% PVA. New Zealand rabbits were randomly divided into the control group (C group) and two groups with bone lesions, in which one received a matrix implant HM (Implant group), while the other did not (no Implant group). Clinical monitoring showed no altered parameters from either the Implant or the no Implant groups as compared to the control group, for the variables of diet grades, day and night temperatures and hemograms. In the Implant group, radiologic and tomographic studies showed implanted areas with clean edges in femoral non-articular direction, and radio-dense images that suggest incipient integration. Minimum signs of phlogosis could be observed, whereas no signs of rejection at this imaging level could be identified. Histological analysis showed evidence of osteo-integration, with the formation of a trabecular bone within the implant. Together, these results show that implants of hybrid matrixes of bioactive glass are capable of promoting bone regeneration.

Characterization and induction of cementoblast cell proliferation by bioactive glass nanoparticles.

Cementum is a mineralized tissue that lines the surface of the tooth root enabling attachment of the periodontal ligament to the root and surrounding alveolar bone. Studies examining the mechanisms involved in the formation of root cementum have been hindered by an inability to isolate and culture the cells required for cementum production (cementoblasts). This study isolated and characterized cementoblast cells derived from rat molar periodontal ligament. It was observed that the isolated cells expressed F-Spondin, a cementoblast marker, while F-Spondin expression was not observed in the cells of other tissues such as gingival fibroblasts and osteoblasts. As expected, the isolated cementoblast cells also expressed osteocalcin (OC), bone sialoprotein (BSP), alkaline phosphatase (ALP), and type I collagen, demonstrating the presence of mineralized tissues genes in cementoblast cells. These cells showed high ALP activity and calcified nodule formation in vitro. Since cementogenesis could be a critical event for regeneration of periodontal tissues, this study investigated whether bioactive glass particles could affect the proliferation of cementoblasts since they are known to enhance osteoblast proliferation. It was found that the ionic products from bioactive glass nanoparticles increased cementoblast viability, mitochondrial activity, and induced cell proliferation. Together, these results show the characterization of cementoblast cells from rat molar periodontal ligament. Additionally, it was shown that bioactive glass nanoparticles induced cementoblast to proliferate, indicating that they could be a potential material for use in cement regeneration through tissue engineering.

In vitro and in vivo osteogenic potential of bioactive glass-PVA hybrid scaffolds colonized by mesenchymal stem cells.

Bioactive glass/polymer composites are promising materials for bone tissue engineering. The present research group has developed porous hybrid scaffolds comprised of 50% polyvinyl alcohol/50% bioactive glass with a 70%SiO(2)-30%CaO composition. Prior studies have also shown the adequate structural and mechanical behavior of these scaffolds. As such, the present study investigates the in vitro and in vivo osteogenic potential of the scaffold, using mesenchymal stem cells (MSC) from the bone marrow of female rats. MTT, alkaline phosphatase activity, collagen secretion and Von Kossa staining were conducted to evaluate the differentiation ability of MSC in an osteogenic medium. The in vitro results indicate an increase in both cell proliferation and osteogenic differentiation when the hybrid material is present. Von Kossa staining showed a progressive increase in mineralization nodules, coupled with time differentiation. For the in vivo evaluation, three groups were studied: (1) group implanted with the hybrid scaffold, (2) group implanted with scaffold colonized by non-differentiated MSC and (3) group implanted with scaffold colonized by differentiated MSC. The scaffolds were subcutaneously implanted on the back of Wistar rats for 1-8 weeks, and histological and histomorphometric analyses were performed. The tissue ingrowth proved to be higher in the groups colonized by MSC in the first week. In the second week, only the hybrid colonized by differentiated MSC presented a larger percentage of connective tissue. In the third, fourth and eighth weeks, all groups presented 70% of the hybrid scaffold filled with tissue. However, only the group with differentiated MSC presented some form of osteoid tissue, indicating that the hybrid scaffold with differentiated MSC does indeed present osteogenic potential.

Synthesis and characterization of chitosan-polyvinyl alcohol-bioactive glass hybrid membranes.

The tissue engineering strategy is a new approach for the regeneration of cementum, which is essential for the regeneration of the periodontal tissue. This strategy involves the cell cultures present in this tissue, called cementoblasts, and located on an appropriate substrate for posterior implantation in the regeneration site. Prior studies from our research group have shown that the proliferation and viability of cementoblasts increase in the presence of the ionic dissolution products of bioactive glass particles. Therefore, one possible approach to obtaining adequate substrates for cementoblast cultures is the development of composite membranes containing bioactive glass. In the present study, composite films of chitosan-polyvinyl alcohol-bioactive glass containing different glass contents were developed. Glutaraldehyde was also added to allow for the formation of cross-links and changes in the degradation rate. The glass phase was introduced in the material by a sol-gel route, leading to an organic-inorganic hybrid. The films were characterized by Fourier-transformed infrared spectroscopy (FTIR), scanning electron microscopy (SEM) with electron dispersive spectroscopy (EDS) and X-ray diffraction (XRD) analysis. Bioactivity tests were also conducted by immersion of the films in simulated body fluid (SBF). Films containing up to 30% glass phase could be obtained. The formation of calcium phosphate was observed after the immersion of the films. A calcium phosphate layer formed more quickly on materials containing higher bioactive glass contents. In the hybrid containing 23% bioactive glass, a complete layer was formed after 24 h immersion, showing the high bioactivity of this material. However, despite the higher in vitro bioactivity, the film with 23% glass showed lower mechanical properties compared with films containing up to 17% glass.

Effect of a three-dimensional chitosan porous scaffold on the differentiation of mesenchymal stem cells into chondrocytes.

Cartilage tissue has a poor capacity for self-repair, especially in the case of severe cartilage damage due to trauma or age-related degeneration. Cell-based tissue engineering using scaffolds has provided an option for the repair of cartilage tissue. The present work demonstrates that a three-dimensional (3D) chitosan scaffold increases the efficiency of the adhesion and differentiation of mesenchymal stem cells (MSCs) after the addition of a chondrogenic medium. These culture conditions promoted MSC differentiation into chondrocytes during the first 9 weeks of monolayer or 3D culture in a scaffold composed of chitosan or chitosan/gelatin. The results demonstrated that a chitosan scaffold caused a reduction in alkaline phosphatase production and an increase in the collagen concentration indicating phenotypic changes in the cells. In support of these results, the production of collagen type II by the MSCs cultured in the chitosan scaffold increased after 3 weeks of culture, indicating the beginning of differentiation. However, the addition of gelatin to the chitosan scaffold did not improve on the results obtained with chitosan alone. These results suggest that this 3D chitosan scaffold is a promising candidate for biomaterial implants designed to promote MSC colonization and has applications in regenerative medicine.

Synthesis, neutralization and blocking procedures of organic/inorganic hybrid scaffolds for bone tissue engineering applications.

Bioactive glasses (BaG) can bind to human bone tissues and have been used in many biomedical applications for the last 30 years. However they usually are weak and brittle. On the other hand, composites that combine polymers and BaG are of particular interest, since they often show an excellent balance between stiffness and toughness. Bioactive glass-poly(vinyl alcohol) foams to be used in tissue engineering applications were previously developed by our group, using the sol-gel route. Since bioactive glass-polymer composite derived from the sol-gel process cannot be submitted to thermal treatments at high temperatures (above 400 degrees C), they usually have unreacted species that can cause cytotoxicity. This work reports a technique for stabilizing the sol-gel derived bioactive glass/poly(vinyl alcohol) hybrids by using glutaraldehyde (GA), NH(4)OH solutions and a blocking solution containing bovine serum albumin. PVA/BaG/GA hybrids were characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM/EDX) analyses. Moreover, MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) biocompatibility and cytotoxicity assays were also conducted. The hybrids exhibited pore size varying from 80 to 820 mum. After treatments, no major changes in the pore structure were observed and high levels of cell viability were obtained.

Properties and biocompatibility of chitosan films modified by blending with PVA and chemically crosslinked.

In the present work we report the synthesis, characterization, and preliminary biocompatibility of polymer blends based on Chitosan and poly(vinyl alcohol) (PVA) with low degree of hydrolysis and chemically crosslinked by glutaraldehyde for potential application on skin tissue repairing. The microstructure and morphology of the blended hydrogels were characterized through Fourier Transform Infrared spectroscopy (FTIR) and Scanning electron microscopy (SEM/EDX) analysis. Hydrogels were investigated by swelling as preliminary in vitro test using simulated body fluid. In addition, biocompatibility, cytotoxicity, and cell viability were assessed via MTT assay with VERO cell culture and cell spreading-adhesion analysis. It was found that by increasing the chitosan to PVA ratio, simulated body fluid uptake of the material was significantly altered. All the tested hydrogels have clearly presented adequate cell viability, non-toxicity, and suitable properties which can be tailored for prospective use in skin tissue engineering.

In vivo evaluation of bioactive glass foams associated with platelet-rich plasma in bone defects.

The objective of this study was to evaluate the use of bioactive glass foams produced by the sol-gel process, associated or not with platelet-rich plasma (PRP), in the regeneration of bone defects. Mongrel dogs (n = 14) were divided into two groups after having their superior first premolar removed. A small piece of vestibular bone from the alveolus was intentionally removed. The area was filled with bioactive glass foam produced by the sol-gel method. Two groups were tested: group A was the glass foam; group B was the same material associated with PRP, prepared from each animal. The other side of alveolar bone was used as a control group, in which the bone defect did not receive any biomaterial. The thickness of the bone area was measured before and after the intervention. After a period of 60 days implantation, the right and left bones were measured again, and a bone biopsy on both regions was conducted for histological analysis. The findings show an increase of bone thickness for both materials implanted compared to the control group. Group B, implanted with bioactive glass foam associated with PRP, showed a thicker bone area compared to Group A. Histological results indicate bone formation for both materials used. However, the bioactive glass associated with PRP gave rise to a more mature bone formation. These results show that bioactive glass foams processed by a sol-gel method is effective in maintaining the thickness of the alveolar ridge, and the use of PRP associated with the foams improve bone formation.

Apatite formation on poly(2-hydroxyethyl methacrylate)-silica hybrids prepared by sol-gel process.

Hybrids of poly(2-hydroxyethyl methacrylate) (PHEMA), a polymer that has been employed in a wide variety of biomedical applications, and silica-gel, which exhibits a well-known bioactivity, were produced. The obtained hybrids were characterized and their in vitro ability to induce the formation of a calcium phosphate layer on the surface was evaluated. The surface area of hybrids decreased with increasing amounts of PHEMA so that hybrids with more than approximately 40% PHEMA are virtually non-porous. All hybrids induced the formation of a calcium phosphate layer on their surfaces when soaked into simulated body fluid. The induction time and the morphology of the apatite layer varied according to the polymer content.

The effect of ionic products from bioactive glass dissolution on osteoblast proliferation and collagen production.

Bioactive ceramics developed during the past few decades have interesting properties from the biological standpoint, but their effects on cellular events remain partially unknown. In the current work, we investigated cellular viability, proliferation, morphology changes and metabolic activity of rat primary culture osteoblasts in contact with the ionic products from the dissolution of a bioactive glass with 60% of silica (BG60S) and a biphasic calcium phosphate (BCP). We observed that although osteoblasts cultured with BG60S showed vacuole formation, cell viability was increased when compared to BCP and control. The vacuole formation was not due to the presence of high calcium concentration in the ionic products from the dissolution of BG60S and was not related to nitric oxide production from the osteoblasts. We did find that high silicon concentration could induce cellular vacuole formation. Additionally, energy dispersive spectroscopy analysis indicated that vacuole contained 75% more silicon than other regions in the cell outside the vacuole. We further found that collagen production was higher in osteoblast cultured in the presence of BG60S compared to BCP and control, while alkaline phosphatase production was similar among cells incubated with BG60S, BCP and control. Together, our results indicate that osteoblast vacuole formation was due to high silicon contents in the dissolution of BG60S and we can suggest that despite the vacuole formation, there is no significant alteration in the bioceramic cell interaction.

Structure and dosimetric analysis of biodegradable glasses for prostate cancer treatment.

This work analyzes SiO2 and SiO2-CaO glasses incorporated with samarium atoms produced by sol-gel synthesis. The goal is to provide biocompatible and biodegradable radioactive seeds as an alternative to be used in brachytherapy for the treatment of prostate cancer. The chemical and physical characteristics of the obtained glasses were analyzed by energy dispersive x-ray spectroscopy, x-ray diffraction, He picnometry, and nitrogen adsorption analysis. A theoretical analysis of the process of neutron activation of the samples was also conducted through the calculation of the activity of the seeds and the beta- and gamma-ray doses emitted by the seeds. The results demonstrate the incorporation of samarium atoms in the glass matrix. The experimental data coupled with the theoretical studies in neutron activation suggest that it is possible to obtain radioactive seeds with activities equivalent to 125I seeds used in prostatic brachytherapy.