In Vivo Effects of Biosilica and Spongin-Like Collagen Scaffolds on the Healing Process in Osteoporotic Rats.

Due to bioactive properties, introducing spongin-like collagen (SPG) into the biosilica (BS) extracted from marine sponges would present an enhanced biological material for improving osteoporotic fracture healing by increasing bone formation rate. Our aim was to characterize the morphology of the BS/SPG scaffolds by scanning electron microscopy (SEM), the chemical bonds of the material by Fourier transform infrared spectroscopy (FTIR), and evaluating the orthotopic in vivo response of BS/SPG scaffolds in tibial defects of osteoporotic fractures in rats (histology, histomorphometry, and immunohistochemistry) in two experimental periods (15 and 30 days). SEM showed that scaffolds were porous, showing the spicules of BS and fibrous aspect of SPG. FTIR showed characteristic peaks of BS and SPG. For the in vivo studies, after 30 days, BS and BS/SPG showed a higher amount of newly formed bone compared to the first experimental period, observed both in the periphery and in the central region of the bone defect. For histomorphometry, BS/SPG presented higher %BV/TV compared to the other experimental groups. After 15 days, BS presented higher volumes of collagen type I. After 30 days, all groups demonstrated higher volumes of collagen type III compared to volumes at 15 days. After 30 days, BS/SPG presented higher immunostaining of osteoprotegerin compared to the other experimental groups at the same experimental period. The results showed that BS and BS/SPG scaffolds were able to improve bone healing. Future research should focus on the effects of BS/SPG on longer periods in vivo studies.

Tricalcium phosphate-loaded injectable hydrogel as a promising osteogenic and bactericidal teicoplanin-delivery system for osteomyelitis treatment: An in vitro and in vivo investigation.

Osteomyelitis is an inflammation of bone tissue usually caused by pyogenic bacteria. The most recurrent clinical approach consists of bone debridement followed by parenteral administration of antibiotics. However, systemic antibiotic treatment has limitations regarding absorption rate and bioavailability over time. The main challenge of osteomyelitis treatment consists of coupling the persistent infection treatment with the regeneration of the bone debrided. In this work, we developed an injectable drug delivery system based on poloxamer 407 hydrogel containing undoped Mg, Zn-doped tricalcium phosphate (ß-TCP), and teicoplanin, a broad-spectrum antibiotic. We evaluated how the addition of teicoplanin and ß-TCP affected the micellization, gelation, particle size, and surface charge of the hydrogel. Later, we studied the hydrogel degradation and drug delivery kinetics. Finally, the bactericidal, biocompatibility, and osteogenic properties were evaluated through in vitro studies and confirmed by in vivo Wistar rat models. Teicoplanin was found to be encapsulated in the corona portions of the hydrogel micelles, yielding a bigger hydrodynamics radius. The encapsulated teicoplanin showed a sustained release over the evaluated period, enough to trigger antibacterial properties against Gram-positive bacteria. Besides, the formulations were biocompatible and showed bone healing ability and osteogenic properties. Finally, in vivo studies confirmed that the proposed locally injected formulations yielded osteomyelitis treatment with superior outcomes than parenteral administration while promoting bone regeneration. In conclusion, the presented formulations are promising drug delivery systems for osteomyelitis treatment and deserve further technological improvements.

Amorphous TiO2nano-coating on stainless steel to improve its biological response.

This study delves into the potential of amorphous titanium oxide (aTiO2) nano-coating to enhance various critical aspects of non-Ti-based metallic orthopedic implants. These implants, such as medical-grade stainless steel (SS), are widely used for orthopedic devices that demand high strength and durability. The aTiO2nano-coating, deposited via magnetron sputtering, is a unique attempt to improve the osteogenesis, the inflammatory response, and to reduce bacterial colonization on SS substrates. The study characterized the nanocoated surfaces (SS-a TiO2) in topography, roughness, wettability, and chemical composition. Comparative samples included uncoated SS and sandblasted/acid-etched Ti substrates (Ti). The biological effects were assessed using human mesenchymal stem cells (MSCs) and primary murine macrophages. Bacterial tests were carried out with two aerobic pathogens (S. aureusandS. epidermidis) and an anaerobic bacterial consortium representing an oral dental biofilm. Results from this study provide strong evidence of the positive effects of the aTiO2nano-coating on SS surfaces. The coating enhanced MSC osteoblastic differentiation and exhibited a response similar to that observed on Ti surfaces. Macrophages cultured on aTiO2nano-coating and Ti surfaces showed comparable anti-inflammatory phenotypes. Most significantly, a reduction in bacterial colonization across tested species was observed compared to uncoated SS substrates, further supporting the potential of aTiO2nano-coating in biomedical applications. The findings underscore the potential of magnetron-sputtering deposition of aTiO2nano-coating on non-Ti metallic surfaces such as medical-grade SS as a viable strategy to enhance osteoinductive factors and decrease pathogenic bacterial adhesion. This could significantly improve the performance of metallic-based biomedical devices beyond titanium.

Exploring the osteogenic potential of semisynthetic triterpenes from Combretum leprosum: An in vitro and in silico study.

Combretum leprosum Mart. is a plant of the Combretaceae family, widely distributed in the Northeast region of Brazil, popularly used as an anti-inflammatory agent, and rich in triterpenes. This study evaluated in vitro and in silico potential osteogenic of two semisynthetic triterpenes (CL-P2 and CL-P2A) obtained from the pentacyclic triterpene 3ß,6ß,16ß-trihydroxylup-20(29)-ene (CL-1) isolated from C. leprosum. Assays were carried out in cultured murine osteoblasts (OFCOL II), first investigating the possible toxicity of the compounds on these cells through viability assays (MTT). Cell proliferation and activation were investigated by immunohistochemical evaluation of Ki-67, bone alkaline phosphatase (ALP) activity, and mineralization test by Von Kossa. Molecular docking analysis was performed to predict the binding affinity of CL-P2 and CL-P2A to target proteins involved in the regulation of osteogenesis, including: bone morphogenetic protein 2 (BMP-2), proteins related to Wingless-related integration (WNT) pathway (Low-density lipoprotein receptor-related protein 6-LRP6 and sclerostin-SOST), and receptor activator of nuclear factor (NF)-kB-ligand (RANK-L). Next, Western Blot and immunofluorescence investigated BMP-2, WNT, RANK-L, and OPG protein expressions in cultured murine osteoblasts (OFCOL II). None of the CL-P2 and CL-P2A concentrations were toxic to osteoblasts. Increased cell proliferation, ALP activity, and bone mineralization were observed. Molecular docking assays demonstrated interactions with BMP-2, LRP6, SOST, and RANK-L/OPG. There was observed increased expression of BMP-2, WNT, and RANK-L/OPG proteins. These results suggest, for the first time, the osteogenic potential of CL-P2 and CL-P2A.

In vitro effects and mechanisms of Humulus lupulus extract on bone marrow progenitor cells and endothelial cells.

Osteoporosis is the most common metabolic bone disorder and is associated with a high incidence of fractures. Angiogenesis and adequate blood flow are important during bone repair and maintenance. Estrogens play a key role in bone formation, in the prevention of bone resorption and vasculature maintenance. Hormone replacement therapy (HRT) has been used with great benefits for bone fracture prevention but has been linked to the development of serious important side effects, including cancer and stroke. Phytoestrogens are an attractive alternative to HRT because their chemical structure is similar to estradiol but, they could behave as selective modulators: acting as antagonists of estrogen receptors in the breast and endometrium and as agonists in the vascular endothelium and bone. Hops contain a wide variety of phytoestrogens that have individually been shown to possess estrogenic activity by either blocking or mimicking. In this study we have to evaluate the in vitro effects and mechanisms of action of hops extracts on the osteogenic and adipogenic capacity of bone marrow progenitor cells (BMPCs), and the angiogenic potential of EA.hy926 endothelial cells. We show that hops extracts increase the proliferative capacity of BMPCs and promote their osteogenic differentiation while decreasing their pro-osteoclastogenic capacity; and that these effects are mediated by the MAPK pathway. Additionally, hops extracts prevent the adipogenic differentiation of BMPCs and promote endothelial cell activity, by mechanisms also partially mediated by MAPK.

Biological evaluation of critical bone defect regeneration using hydroxyapatite/ alginate composite granules.

PURPOSE: to evaluate biocompatibility and osteogenic potential of hydroxyapatite/alginate composite after its implantation on rat calvarian critical bone defect. METHODS: thirty adults male Wistar rats were randomly distributed into two groups: GHA - critical bone defect filled with hydroxyapatite/alginate composite granules (HA/Alg) and CG - critical bone defect without biomaterial; evaluated at biological points of 15, 45 and 120 days. RESULTS: the histomorphometrically analyses for GHA showed osteoid matrix deposition (OM) among the granules and towards the center of the defect in centripetal direction throughout the study, with evident new bone formation at 120 days, resulting in filling 4/5 of the initial bone defect. For CG, this finding was restricted to the edges of the bone margins and formation of connective tissue on the residual area was found in all biological points. Inflammatory response on GHA was chronic granulomatous type, discrete and regressive for all biological points. Throughout the study, the CG presented mononuclear inflammatory infiltrate diffuse and regressive. Histomorphometry analyses showed that OM percentage was evident for GHA group when compared to CG group in all analyzed periods (p > 0.05). CONCLUSIONS: the biomaterial evaluated at this study showed to be biocompatible, bioactive, osteoconductive and biodegradable synchronously with bone formation.

Modulation of osteoclastogenesis by macrogeometrically designed hydrophilic dual acid-etched titanium surfaces.

The aim of this study was to evaluate the influence of implant macrodesign and surface hydrophilicity on osteoclast (OC) differentiation, activation, and survival in vitro. Titanium disks were produced with a sandblasted, dual acid-etched surface, with or without additional chemical modification for increasing hydrophilicity (SAE-HD and SAE, respectively) and different macrodesign comprising trapezoidal (HLX) or triangular threads (TMX). This study evaluated 7 groups in total, 4 of which were experimental: HLX/SAE-HD, HLX-SAE, TMX/SAE-HD, and TMX/SAE; and 3 control groups comprising OC differentiated on polystyrene plates (CCPC): a positive CCPC (+), a negative CCPC (-), and a lipopolysaccharide-stimulated assay positive control group, CCPC-LPS. Murine macrophage RAW264.7 cells were seeded on the disks, differentiated to OC (RAW-OC) by receptor activator of nuclear factor-κB ligand (RANKL) treatment and cultured for 5 days. Osteoclast differentiation and cell viability were respectively assessed by specific enzymatic Tartrate-Resistant Acid Phosphatase (TRAP) activity and MTT assays. Expression levels of various OC-related genes were measured at the mRNA level by quantitative polymerase chain reaction (qPCR). HLX/SAE-HD, TMX/SAE-HD, and HLX/SAE significantly suppressed OC differentiation when compared to CCPC (+). Cell viability was significantly increased in TMX/SAE and reduced in HLX/SAE-HD. In addition, the expression of Interleukin (IL)-6 and Tumour Necrosis Factor (TNF)-α was upregulated in TMX/SAE-HD compared to CCPC (+). Hydrophilic surfaces negatively modulate macrophage/osteoclast viability. Specifically, SAE-HD with double triangular threads increases the cellular pro-inflammatory status, while surface hydrophilicity and macrodesign do not seem to have a distinct impact on osteoclast differentiation, activation, or survival.

Additive manufacturing of bioactive and biodegradable poly (lactic acid)-tricalcium phosphate scaffolds modified with zinc oxide for guided bone tissue repair.

Bioactive and biodegradable scaffolds that mimic the natural extracellular matrix of bone serve as temporary structures to guide new bone tissue growth. In this study, 3D-printed scaffolds composed of poly (lactic acid) (PLA)-tricalcium phosphate (TCP) (90-10 wt.%) were modified with 1%, 5%, and 10 wt.% of ZnO to enhance bone tissue regeneration. A commercial chain extender named Joncryl was incorporated alongside ZnO to ensure the printability of the composites. Filaments were manufactured using a twin-screw extruder and subsequently used to print 3D scaffolds via fused filament fabrication (FFF). The scaffolds exhibited a homogeneous distribution of ZnO and TCP particles, a reproducible structure with 300 µm pores, and mechanical properties suitable for bone tissue engineering, with an elastic modulus around 100 MPa. The addition of ZnO resulted in enhanced surface roughness on the scaffolds, particularly for ZnO microparticles, achieving values up to 241 nm. This rougher topography was responsible for enhancing protein adsorption on the scaffolds, with an increase of up to 85% compared to the PLA-TCP matrix. Biological analyses demonstrated that the presence of ZnO promotes mesenchymal stem cell (MSC) proliferation and differentiation into osteoblasts. Alkaline phosphatase (ALP) activity, an important indicator of early osteogenic differentiation, increased up to 29%. The PLA-TCP composite containing 5% ZnO microparticles exhibited an optimized degradation rate and enhanced bioactivity, indicating its promising potential for bone repair applications.

Amorphous titanium oxide (aTiO2) thin films biofunctionalized with CAP-p15 induce mineralized-like differentiation of human oral mucosal stem cells (hOMSCs).

Insufficient osseointegration of titanium-based implants is a factor conditioning their long-term success. Therefore, different surface modifications, such as multifunctional oxide coatings, calcium phosphates, and the addition of molecules such as peptides, have been developed to improve the bioactivity of titanium-based biomaterials. In this work, we investigate the behavior of human oral mucosal stem cells (hOMSCs) cultured on amorphous titanium oxide (aTiO2), surfaces designed to simulate titanium (Ti) surfaces, biofunctionalized with a novel sequence derived from cementum attachment protein (CAP-p15), exploring its impact on guiding hOMSCs towards an osteogenic phenotype. We carried out cell attachment and viability assays. Next, hOMSCs differentiation was assessed by red alizarin stain, ALP activity, and western blot analysis by evaluating the expression of RUNX2, BSP, BMP2, and OCN at the protein level. Our results showed that functionalized surfaces with CAP-p15 (1 µg ml-1) displayed a synergistic effect increasing cell proliferation and cell attachment, ALP activity, and expression of osteogenic-related markers. These data demonstrate that CAP-p15 and its interaction with aTiO2surfaces promote osteoblastic differentiation and enhanced mineralization of hOMSCs when compared to pristine samples. Therefore, CAP-p15 shows the potential to be used as a therapeutical molecule capable of inducing mineralized tissue regeneration onto titanium-based implants.

Inhibition of lipopolysaccharide-induced NF-κB maintains osteogenesis of dental pulp and periodontal ligament stem cells.

Dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) can differentiate into osteoblasts, indicating that both are potential candidates for bone tissue engineering. Osteogenesis is influenced by many environmental factors, one of which is lipopolysaccharide (LPS). LPS-induced NF-κB activity affects the osteogenic potencies of different types of MSCs differently. This study evaluated the effect of LPS-induced NF-κB activity and its inhibition in DPSCs and PDLSCs. DPSCs and PDLSCs were cultured in an osteogenic medium, pretreated with/without NF-κB inhibitor Bay 11-7082, and treated with/without LPS. Alizarin red staining was performed to assess bone nodule formation, which was observed under an inverted light microscope. NF-κB and alkaline phosphatase (ALP) activities were measured to examine the effect of Bay 11-7082 pretreatment and LPS supplementation on osteogenic differentiation of DPSCs and PDLSCs. LPS significantly induced NF-κB activity (p = 0.000) and reduced ALP activity (p = 0.000), which inhibited bone nodule formation in DPSCs and PDLSCs. Bay 11-7082 inhibited LPS-induced NF-κB activity, and partially maintained ALP activity and osteogenic potency of LPS-supplemented DPSCs and PDLSCs. Thus, inhibition of LPS-induced NF-κB activity can maintain the osteogenic potency of DPSCs and PDLSCs.

Skeletal Effects of a Prolonged Oral Metformin Treatment in Adult Wistar Rats.

INTRODUCTION: We previously showed that a 3-week oral metformin (MET) treatment enhances the osteogenic potential of bone marrow stromal cells (BMSCs) and improves several bone histomorphometric parameters in Wistar rats with metabolic syndrome (MetS). However, the skeletal effects of extended periods of MET need to be completely elucidated. Hence, in this study, the impact of a prolonged (3-month) MET treatment was investigated on bone architecture, histomorphometric and biomechanics variables, and osteogenic potential of BMSCs in Wistar rats with or without MetS. MATERIALS AND METHODS: Young male Wistar rats (n=36) were randomized into four groups (n=9) that received either 20% fructose (F), MET (MET), F plus MET treatments (FMET), or drinking water alone (Veh). Rats were euthanized, blood was collected, and bones were dissected and processed for peripheral quantitative computed tomography (pQCT) analysis, static and dynamic histomorphometry, and bone biomechanics. In addition, BMSCs were isolated to determine their osteogenic potential. RESULTS: MET affected trabecular and cortical bone, altering bone architecture and biomechanics. Furthermore, MET increased the pro-resorptive profile of BMSCs. In addition, fructose-induced MetS practically did not affect the the structural or mechanical variables of the skeleton. CONCLUSION: A 3-month treatment with MET (with or without MetS) affects bone architecture and biomechanical variables in Wistar rats.

Coenzyme Q10 prevents RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways.

Coenzyme Q10 (CoQ10) is a potent antioxidant that is implicated in the inhibition of osteoclastogenesis, but the underlying mechanism has not been determined. We explored the underlying molecular mechanisms involved in this process. RAW264.7 cells received receptor activator of NF-κB ligand (RANKL) and CoQ10, after which the differentiation and viability of osteoclasts were assessed. After the cells were treated with CoQ10 and/or H2O2 and RANKL, the levels of reactive oxygen species (ROS) and proteins involved in the PI3K/AKT/mTOR and MAPK pathways and autophagy were tested. Moreover, after the cells were pretreated with or without inhibitors of the two pathways or with the mitophagy agonist, the levels of autophagy-related proteins and osteoclast markers were measured. CoQ10 significantly decreased the number of TRAP-positive cells and the level of ROS but had no significant impact on cell viability. The relative phosphorylation levels of PI3K, AKT, mTOR, ERK, and p38 were significantly reduced, but the levels of FOXO3/LC3/Beclin1 were significantly augmented. Moreover, the levels of FOXO3/LC3/Beclin1 were significantly increased by the inhibitors and mitophagy agonist, while the levels of osteoclast markers showed the opposite results. Our data showed that CoQ10 prevented RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways in RAW264.7 cells.

A new hydroxyapatite-alginate-gelatin biocomposite favor bone regeneration in a critical-sized calvarial defect model.

This study aimed to evaluate the osteogenic potential of hydroxyapatite (HA), Alginate (Alg), and Gelatine (Gel) composite in a critical-size defect model in rats. Twenty-four male rats were divided into three groups: a negative control with no treatment (Control group), a positive control treated with deproteinized bovine bone mineral (DBBM group), and the experimental group treated with the new HA-Alg-Gel composite (HA-Alg-Gel group). A critical size defect (8.5mm) was made in the rat's calvaria, and the bone formation was evaluated by in vivo microcomputed tomography analysis (µCT) after 1, 15, 45, and 90 days. After 90 days, the animals were euthanized and histological and histomorphometric analyses were performed. A higher proportion of mineralized tissue/biomaterial was observed in the DBBM group when compared to the HA-Alg-Gel and Control groups in the µCT analysis during all analysis periods. However, no differences were observed in the mineralized tissue/biomaterial proportion observed on day 1 (immediate postoperative) in comparison to later periods of analysis in all groups. In the histomorphometric analysis, the HA-Alg-Gel and Control groups showed higher bone formation than the DBBM group. Moreover, in histological analysis, five samples of the HA-Alg-Gal group exhibited formed bone spicules adjacent to the graft granules against only two of eight samples in the DBBM group. Both graft materials ensured the maintenance of defect bone thickness, while a tissue thickness reduction was observed in the control group. In conclusion, this study demonstrated the osteoconductive potential of HA-Alg-Gel bone graft by supporting new bone formation around its particles.

Novel Organic-Inorganic Nanocomposite Hybrids Based on Bioactive Glass Nanoparticles and Their Enhanced Osteoinductive Properties.

Inorganic-organic hybrid biomaterials have been proposed for bone tissue repair, with improved mechanical flexibility compared with scaffolds fabricated from bioceramics. However, obtaining hybrids with osteoinductive properties equivalent to those of bioceramics is still a challenge. In this work, we present for the first time the synthesis of a class II hybrid modified with bioactive glass nanoparticles (nBGs) with osteoinductive properties. The nanocomposite hybrids were produced by incorporating nBGs in situ into a polytetrahydrofuran (PTHF) and silica (SiO2) hybrid synthesis mixture using a combined sol-gel and cationic polymerization method. nBGs ~80 nm in size were synthesized using the sol-gel technique. The structure, composition, morphology, and mechanical properties of the resulting materials were characterized using ATR-FTIR, 29Si MAS NMR, SEM-EDX, AFM, TGA, DSC, mechanical, and DMA testing. The in vitro bioactivity and degradability of the hybrids were assessed in simulated body fluid (SBF) and PBS, respectively. Cytocompatibility with mesenchymal stem cells was assessed using MTS and cell adhesion assays. Osteogenic differentiation was determined using the alkaline phosphatase activity (ALP), as well as the gene expression of Runx2 and Osterix markers. Hybrids loaded with 5, 10, and 15% of nBGs retained the mechanical flexibility of the PTHF-SiO2 matrix and improved its ability to promote the formation of bone-like apatite in SBF. The nBGs did not impair cell viability, increased the ALP activity, and upregulated the expression of Runx2 and Osterix. These results demonstrate that nBGs are an effective osteoinductive nanoadditive for the production of class II hybrid materials with enhanced properties for bone tissue regeneration.

Novel Fe3O4 Nanoparticles with Bioactive Glass-Naproxen Coating: Synthesis, Characterization, and In Vitro Evaluation of Bioactivity.

Immune response to biomaterials, which is intimately related to their surface properties, can produce chronic inflammation and fibrosis, leading to implant failure. This study investigated the development of magnetic nanoparticles coated with silica and incorporating the anti-inflammatory drug naproxen, aimed at multifunctional biomedical applications. The synthesized nanoparticles were characterized using various techniques that confirmed the presence of magnetite and the formation of a silica-rich bioactive glass (BG) layer. In vitro studies demonstrated that the nanoparticles exhibited bioactive properties, forming an apatite surface layer when immersed in simulated body fluid, and biocompatibility with bone cells, with good viability and alkaline phosphatase activity. Naproxen, either free or encapsulated, reduced nitric oxide production, an inflammatory marker, while the BG coating alone did not show anti-inflammatory effects in this study. Overall, the magnetic nanoparticles coated with BG and naproxen showed promise for biomedical applications, especially anti-inflammatory activity in macrophages and in the bone field, due to their biocompatibility, bioactivity, and osteogenic potential.

Mineral matrix deposition of MC3T3-E1 pre-osteoblastic cells exposed to silicocarnotite and nagelschmidtite bioceramics: In vitro comparison to hydroxyapatite.

This work presents the effect of the silicocarnotite (SC) and nagelschmidtite (Nagel) phases on in vitro osteogenesis. The known hydroxyapatite of biological origin (BHAp) was used as a standard of osteoconductive characteristics. The evaluation was carried out in conventional and osteogenic media for comparative purposes to assess the osteogenic ability of the bioceramics. First, the effect of the material on cell viability at 24 h, 7 and 14 days of incubation was evaluated. In addition, cell morphology and attachment on dense bioceramic surfaces were observed by fluorescence microscopy. Specifically, alkaline phosphatase (ALP) activity was evaluated as an osteogenic marker of the early stages of bone cell differentiation. Mineralized extracellular matrix was observed by calcium phosphate deposits and extracellular vesicle formation. Furthermore, cell phenotype determination was confirmed by scanning electron microscope. The results provided relevant information on the cell attachment, proliferation, and osteogenic differentiation processes after 7 and 14 days of incubation. Finally, it was demonstrated that SC and Nagel phases promote cell proliferation and differentiation, while the Nagel phase exhibited a superior osteoconductive behavior and could promote MC3T3-E1 cell differentiation to a higher extent than SC and BHAp, which was reflected in a higher number of deposits in a shorter period for both conventional and osteogenic media.

Resveratrol Alleviates Osteoporosis by Promoting Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells via SITR1/PI3K/AKT Pathway / El Resveratrol Alivia la Osteoporosis al Promover la Diferenciación Osteogénica de las Células Madre Mesenquimales de la Médula Ósea a Través de la Vía SITR1/PI3K/AKT

SUMMARY: Senile osteoporosis is mainly caused by reduced osteoblast differentiation and has become the leading cause of fractures in the elderly worldwide. Natural organics are emerging as a potential option for the prevention and treatment of osteoporosis. This study was designed to study the effect of resveratrol on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in osteoporosis mice. A mouse model of osteoporosis was established by subcutaneous injection of dexamethasone and treated with resveratrol administered by gavage. In vivo and in vitro, we used western blot to detect protein expression, and evaluated osteogenic differentiation of BMSCs by detecting the expression of osteogenic differentiation related proteins, calcium deposition, ALP activity and osteocalcin content. Resveratrol treatment significantly increased the body weight of mice, the level of serum Ca2+, 25(OH)D and osteocalcin, ration of bone weight, bone volume/total volume, trabecular thickness, trabecular number, trabecular spacing and cortical thickness in osteoporosis mice. In BMSCs of osteoporosis mice, resveratrol treatment significantly increased the expression of Runx2, osterix (OSX) and osteocalcin (OCN) protein, the level of calcium deposition, ALP activity and osteocalcin content. In addition, resveratrol treatment also significantly increased the expression of SIRT1, p-PI3K / PI3K and p-AKT / AKT in BMSCs of osteoporosis mice. In vitro, resveratrol increased the expression of SIRT1, p-PI3K / PI3K and p-AKT / AKT, Runx2, OSX and OCN protein, the level of calcium deposition, ALP activity and osteocalcin content in BMSCs in a concentration-dependent manner, while SIRT1 knockdown significantly reversed the effect of resveratrol. Resveratrol can attenuate osteoporosis by promoting osteogenic differentiation of bone marrow mesenchymal stem cells, and the mechanism may be related to the regulation of SIRT1/PI3K/AKT pathway.

La osteoporosis senil es causada principalmente por una diferenciación reducida de osteoblastos y se ha convertido en la principal causa de fracturas en las personas mayores en todo el mundo. Los productos orgánicos naturales están surgiendo como una opción potencial para la prevención y el tratamiento de la osteoporosis. Este estudio fue diseñado para estudiar el efecto del resveratrol en la diferenciación osteogénica de las células madre mesenquimales de la médula ósea (BMSC) en ratones con osteoporosis. Se estableció un modelo de osteoporosis en ratones mediante inyección subcutánea de dexametasona y se trató con resveratrol administrado por sonda. In vivo e in vitro, utilizamos Western blot para detectar la expresión de proteínas y evaluamos la diferenciación osteogénica de BMSC detectando la expresión de proteínas relacionadas con la diferenciación osteogénica, la deposición de calcio, la actividad de ALP y el contenido de osteocalcina. El tratamiento con resveratrol aumentó significativamente el peso corporal de los ratones, el nivel sérico de Ca2+, 25(OH)D y osteocalcina, la proporción de peso óseo, el volumen óseo/ volumen total, el espesor trabecular, el número trabecular, el espaciado trabecular y el espesor cortical en ratones con osteoporosis. En BMSC de ratones con osteoporosis, el tratamiento con resveratrol aumentó significativamente la expresión de las proteínas Runx2, osterix (OSX) y osteocalcina (OCN), el nivel de deposición de calcio, la actividad de ALP y el contenido de osteocalcina. Además, el tratamiento con resveratrol también aumentó significativamente la expresión de SIRT1, p-PI3K/PI3K y p-AKT/AKT en BMSC de ratones con osteoporosis. In vitro, el resveratrol aumentó la expresión de las proteínas SIRT1, p-PI3K/PI3K y p- AKT/AKT, Runx2, OSX y OCN, el nivel de deposición de calcio, la actividad de ALP y el contenido de osteocalcina en BMSC de manera dependiente de la concentración, mientras que La caída de SIRT1 revirtió significativamente el efecto del resveratrol. El resveratrol puede atenuar la osteoporosis al promover la diferenciación osteogénica de las células madre mesenquimales de la médula ósea, y el mecanismo puede estar relacionado con la regulación de la vía SIRT1/PI3K/AKT.

Kisspeptin (Kp-10) inhibits in vitro osteogenic differentiation of multipotent mesenchymal stromal cells extracted from the bone marrow of adult rats.

Kisspeptin (Kp-10) is a neuropeptide that binds to GPR54 receptors, exerting several functions mainly in the nervous and reproductive systems of the body. However, its effects and mechanisms of action on the skeletal system remain poorly understood. This study evaluated the effects of different concentrations of Kp-10 on in vitro osteogenic differentiation of multipotent mesenchymal stromal cells (MSCs) extracted from the bone marrow (BM) of adult Wistar rats. Two-month-old female rats were euthanized to extract BM from long bones to obtain MSCs. Four experimental groups were established in vitro: a control and Kp-10 at concentrations of 0.01, 0.05 and, 0.1 µg/mL. After induction of osteogenic differentiation, cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay, alkaline phosphatase activity, collagen synthesis, percentage of area covered by MSCs/field and mineralized nodules/field, and immunocytochemistry of the GPR54 receptor tests. Furthermore, evaluation of gene transcripts for type I collagen, Runx-2, Bmp-2, bone sialoprotein, osteocalcin and osteopontin was performed using real-time RT-qPCR. It was observed that MSCs expressed GPR54 receptor to which Kp-10 binds during osteogenic differentiation, promoting a negative effect on osteogenic differentiation. This effect was observed at all the Kp-10 concentrations in a concentration-dependent manner, characterized by a decrease in the activity of alkaline phosphatase, collagen synthesis, mineralized nodules, and decreased expression of gene transcripts for type I collagen, osteocalcin, osteopontin, and Runx-2. Thus, Kp-10 inhibits in vitro osteogenic differentiation of MSCs extracted from the BM of adult Wistar rats.

PLA/Hydroxyapatite scaffolds exhibit in vitro immunological inertness and promote robust osteogenic differentiation of human mesenchymal stem cells without osteogenic stimuli.

Bone defects stand out as one of the greatest challenges of reconstructive surgery. Fused deposition modelling (FDM) allows for the printing of 3D scaffolds tailored to the morphology and size of bone damage in a patient-specific and high-precision manner. However, FDM still suffers from the lack of materials capable of efficiently supporting osteogenesis. In this study, we developed 3D-printed porous scaffolds composed of polylactic acid/hydroxyapatite (PLA/HA) composites with high ceramic contents (above 20%, w/w) by FDM. The mechanical properties of the PLA/HA scaffolds were compatible with those of trabecular bone. In vitro degradation tests revealed that HA can neutralize the acidification effect caused by PLA degradation, while simultaneously releasing calcium and phosphate ions. Importantly, 3D-printed PLA/HA did not induce the upregulation of activation markers nor the expression of inflammatory cytokines in dendritic cells thus exhibiting no immune-stimulatory properties in vitro. Evaluations using human mesenchymal stem cells (MSC) showed that pure PLA scaffolds exerted an osteoconductive effect, whereas PLA/HA scaffolds efficiently induced osteogenic differentiation of MSC even in the absence of any classical osteogenic stimuli. Our findings indicate that 3D-printed PLA scaffolds loaded with high concentrations of HA are most suitable for future applications in bone tissue engineering.

Investigation of teratogenic effects of letrozole on fetal bone development / Investigación de los efectos teratogénicos de letrozol en el desarrollo óseo fetal

SUMMARY: Letrozole is mainly used for the treatment of unexplained infertility, breast cancer and polycystic ovarian syndrome, with secondary use in ovarian stimulation. In cases of unexpected or unknown pregnancy during the use of letrozole, letrozole may cause a teratogenic effect on the fetus. In this reason, in this study, we aimed to determine the effect of letrozole on fetal bone development. In this study, 32 pregnant Wistar albino rats were used. The rats were divided into four groups: Control (saline) and high; 0.3 mg/kg, medium; 0.03 mg/kg, low; 0.003 mg/ kg letrozole. Saline and letrozole were administered in 100 mL solutions by intraperitonaly from day 11 to day 15 of pregnancy. The skeletal system development of fetuses was examined with double skeletal staining, immunohistochemical staining methods and mineral density scanning electron microscopy. A total of 100 fetuses from female rats, 25 in each group, were included in the study. As a result of that, ossification rates were observed to decrease depending on the dose of letrozole in the forelimb limb (scapula, humerus, radius, ulna) and hindlimb (femur, tibia, fibula) limb bones. As a result of the statistical analysis, a statistically significant decrease was found in the ossification rates of all bones between the control group and low, medium, high letrozole groups (p<0.001). Exposure to letrozole during pregnancy adversely affected ossification and bone growth. However, the teratogenic effects of letrozole are unclear. Therefore, it needs to be investigated more extensively.

RESUMEN: Letrozol se usa principalmente para el tratamiento de la infertilidad inexplicable, el cáncer de mama y el síndrome de ovario poliquístico, con estimulación ovárica de uso secundario. En casos de embarazo inesperado o desconocido durante el uso de letrozol, puede causar un efecto teratogénico en el feto. Por esta razón, en este estudio, nuestro objetivo fue determinar el efecto de letrozol en el desarrollo óseo fetal. Se utilizaron 32 ratas albinas Wistar preñadas las cuales se distribuyeron en cuatro grupos: Control (solución salina) y alta; 0,3 mg/kg, medio; 0,03 mg/kg, bajo; 0,003 mg/kg de letrozol. Se administró solución salina y letrozol en soluciones de 100 mL por vía intraperitoneal desde el día 11 hasta el día 15 de la preñez. El desarrollo del sistema esquelético de los fetos se examinó con tinción esquelética doble, métodos de tinción inmunohistoquímica y microscopía electrónica de barrido de densidad mineral. Se incluyeron en el estudio un total de 100 fetos de ratas hembra, 25 en cada grupo. Como resultado, se observó que las tasas de osificación disminuían dependiendo de la dosis de letrozol en los huesos de los miembros torácicos (escápula, húmero, radio, ulna) y de las miembros pélvicos (fémur, tibia, fíbula). Se encontró una disminución estadísticamente significativa en las tasas de osificación de todos los huesos entre el grupo control y los grupos de letrozol bajo, medio y alto (p<0,001). La exposición a letrozol durante la preñez afectó negativamente la osificación y el crecimiento óseo. Sin embargo, los efectos teratogénicos del letrozol no están claros por lo que debe ser investigado más extensamente.