The ERα/KDM6B regulatory axis modulates osteogenic differentiation in human mesenchymal stem cells

Osteoporosis is a highly prevalent public health burden associated with an increased risk of bone fracture, particularly in aging women. Estrogen, an important medicinal component for the preventative and therapeutic treatment of postmenopausal osteoporosis, induces osteogenesis by activating the estrogen receptor signaling pathway and upregulating the expression of osteogenic genes, such as bone morphogenetic proteins (BMPs). The epigenetic regulation of estrogen-mediated osteogenesis, however, is still unclear. In this report, we found that estrogen significantly induced the expression of lysine-specific demethylase 6B (KDM6B) and that KDM6B depletion by shRNAs led to a significant reduction in the osteogenic potential of DMSCs. Mechanistically, upon estrogen stimulation, estrogen receptor-α (ERα) was recruited to the KDM6B promoter, directly enhancing KDM6B expression. Subsequently, KDM6B was recruited to the BMP2 and HOXC6 promoters, resulting in the removal of H3K27me3 marks and activating the transcription of BMP2 and HOXC6, the master genes of osteogenic differentiation. Furthermore, we found that estrogen enhanced DMSC osteogenesis during calvarial bone regeneration and that estrogen’s pro-osteogenic effect was dependent on KDM6B in vivo. Taken together, our results demonstrate the vital role of the ERα/KDM6B regulatory axis in the epigenetic regulation of the estrogen-dependent osteogenic response.

Osteogenic Potential of Magnesium (Mg)-Doped Multicomponent Bioactive Glass: In Vitro and In Vivo Animal Studies

The use of bioactive glasses (BGs) has been quite fruitful in hard tissue engineering due to the capability of these materials to bond to living bone. In this work, a melt-derived magnesium (Mg)-doped BG (composition: 45SiO2–3P2O5–26CaO–15Na2O–7MgO–4K2O (mol.%)) was synthesized for being used in bone reconstruction. The prepared BGs were then manufactured as three-dimensional (3D) scaffolds by using the sponge replica approach. The microstructure of the samples was assessed by X-ray diffraction (XRD) and the surface morphology was observed by using scanning electron microscopy (SEM). The in vitro bioactivity and the release of osteo-stimulatory Mg2+ ions from the prepared samples were investigated over 7 days of incubation in simulated body fluids (SBF). In vitro cellular analyses revealed the compatibility of the Mg-doped BGs with human osteosarcoma cells (MG-63 cell line). Moreover, the Mg-doped BGs could induce bone nodule formation in vitro and improve the migratory ability of human umbilical vein endothelial cells (HUVECs). In vivo osteogenic capacity was further evaluated by implanting the BG-derived scaffolds into surgically-created critical-size bone defects in rats. Histological and immunohistological observations revealed an appropriate bone regeneration in the animals receiving the glass-based scaffolds after 12 weeks of surgery. In conclusion, our study indicates the effectiveness of the Mg-doped BGs in stimulating osteogenesis in both in vitro and in vivo conditions.

Graphene–Oxide Porous Biopolymer Hybrids Enhance In Vitro Osteogenic Differentiation and Promote Ectopic Osteogenesis In Vivo

Over the years, natural-based scaffolds have presented impressive results for bone tissue engineering (BTE) application. Further, outstanding interactions have been observed during the interaction of graphene oxide (GO)-reinforced biomaterials with both specific cell cultures and injured bone during in vivo experimental conditions. This research hereby addresses the potential of fish gelatin/chitosan (GCs) hybrids reinforced with GO to support in vitro osteogenic differentiation and, further, to investigate its behavior when implanted ectopically. Standard GCs formulation was referenced against genipin (Gp) crosslinked blend and 0.5 wt.% additivated GO composite (GCsGp/GO 0.5 wt.%). Pre-osteoblasts were put in contact with these composites and induced to differentiate in vitro towards mature osteoblasts for 28 days. Specific bone makers were investigated by qPCR and immunolabeling. Next, CD1 mice models were used to assess de novo osteogenic potential by ectopic implantation in the subcutaneous dorsum pocket of the animals. After 4 weeks, alkaline phosphate (ALP) and calcium deposits together with collagen synthesis were investigated by biochemical analysis and histology, respectively. Further, ex vivo materials were studied after surgery regarding biomineralization and morphological changes by means of qualitative and quantitative methods. Furthermore, X-ray diffraction and Fourier-transform infrared spectroscopy underlined the newly fashioned material structuration by virtue of mineralized extracellular matrix. Specific bone markers determination stressed the osteogenic phenotype of the cells populating the material in vitro and successfully differentiated towards mature bone cells. In vivo results of specific histological staining assays highlighted collagen formation and calcium deposits, which were further validated by micro-CT. It was observed that the addition of 0.5 wt.% GO had an overall significant positive effect on both in vitro differentiation and in vivo bone cell recruitment in the subcutaneous region. These data support the GO bioactivity in osteogenesis mechanisms as being self-sufficient to elevate osteoblast differentiation and bone formation in ectopic sites while lacking the most common osteoinductive agents.

Three-Dimensional Porous Scaffolds Derived from Bovine Cancellous Bone Matrix Promote Osteoinduction, Osteoconduction, and Osteogenesis

The use of three-dimensional porous scaffolds derived from decellularized extracellular matrix (ECM) is increasing for functional repair and regeneration of injured bone tissue. Because these scaffolds retain their native structures and bioactive molecules, in addition to showing low immunogenicity and good biodegradability, they can promote tissue repair and regeneration. Nonetheless, imitating these features in synthetic materials represents a challenging task. Furthermore, due to the complexity of bone tissue, different processes are necessary to maintain these characteristics. We present a novel approach using decellularized ECM material derived from bovine cancellous bone by demineralization, decellularization, and hydrolysis of collagen to obtain a three-dimensional porous scaffold. This study demonstrates that the three-dimensional porous scaffold obtained from bovine bone retained its osteoconductive and osteoinductive properties and presented osteogenic potential when seeded with human Wharton’s jelly mesenchymal stromal cells (hWJ-MSCs). Based on its characteristics, the scaffold described in this work potentially represents a therapeutic strategy for bone repair.

Biocompatibility and Osteogenic Potential of Calcium Silicate-Based Cement Combined with Enamel Matrix Derivative: Effects on Human Bone Marrow-Derived Stem Cells

The characteristics of retrograde filling material are important factors that can affect the long-term success of apical microsurgery. Various calcium silicate-based cements (CSC) were introduced to overcome drawbacks of mineral trioxide aggregate (MTA), while Emdogain is known to be effective in the regeneration of periodontal tissues. The aim of this study is to evaluate the biocompatibility and osteogenic potential of various CSCs combined with Emdogain on human bone marrow-derived mesenchymal stem cells. Experimental groups were classified into eight groups depending on the material and the presence of Emdogain. In the cell-counting kit test, all experimental groups combined with Emdogain showed higher cell viability compared with those without Emdogain at days 1 and 2. In the wound-healing assay, cell migration increased significantly over time, with or without Emdogain. In the alkaline phosphatase assay, all groups treated with Emdogain showed higher activity compared with those without Emdogain at day 3 (p < 0.05). Using alizarin red S staining, all groups treated with Emdogain showed greater calcium nodule formation compared with those without Emdogain at days 7 and 14 (p < 0.05). In conclusion, using CSCs as retrograde filling materials and the application of additional Emdogain will increase bone regeneration and improve the prognosis of apical microsurgery.

Dicalcium silicate-induced mitochondrial dysfunction and autophagy-mediated macrophagic inflammation promotes osteogenic differentiation of BMSCs

Dicalcium silicate (Ca2SiO4, C2S) has osteogenic potential but induces macrophagic inflammation. Mitochondrial function plays a vital role in macrophage polarization and macrophagic inflammation. The mitochondrial function of C2S-treated macrophages is still unclear. This study hypothesized: (1) the C2S modulates mitochondrial function and autophagy in macrophages to regulate macrophagic inflammation, and (2) C2S-induced macrophagic inflammation regulates osteogenesis. We used RAW264.7 cells as a model of macrophage. The C2S (75-150 μg/mL) extract was used to analyze the macrophagic mitochondrial function and macrophage-mediated effect on osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs). The results showed that C2S extract (150 μg/mL) induced TNF-α, IL-1β, and IL-6 production in macrophages. C2S extract (150 μg/mL) enhanced reactive oxygen species (ROS) level and intracellular calcium level but reduced mitochondrial membrane potential (MtMP) and ATP production. TEM images showed reduced mitochondrial abundance and altered the mitochondrial morphology in C2S (150 μg/mL)-treated macrophages. Protein level expression of PINK1, Parkin, Beclin1, and LC3 was upregulated but TOMM20 was downregulated. mRNA sequencing and KEGG analysis showed that C2S-induced differentially expressed mRNAs in macrophages were mainly distributed in the essential signaling pathways involved in mitochondrial function and autophagy. The conditioned medium from C2S-treated macrophage (C2S-CM) robustly promoted osteogenic differentiation in BMSCs. In conclusion, our results indicate mitochondrial dysfunction and autophagy as the possible mechanism of C2S-induced macrophagic inflammation. The promotion of osteogenic differentiation of BMSCs by the C2S-induced macrophagic inflammation suggests the potential application of C2S in developing immunomodulatory bone grafts.

Comparative analysis of local stimulation methods of reparative osteogenesis

Objective To determine the ratio of the main growth factors when using various methods of local stimulation of reparative osteogenesis.Material and methods The study consisted of two parts: in the first part a comparative analysis of the content of growth factors by ELISA was carried out (PDGF – platelet derived growth factor, TGF – transforming growth factor, VEGF – vascular endothelial growth factor, IGF – insulin-like growth factor, BMP6 and BMP7 – morphogenetic proteins 6 and 7), capable of stimulating reparative osteogenesis in blood plasma, plateletrich plasma, red bone marrow and bone autoregenerate. The second part presented the results of approbation of the autoregenerate obtained according to the original method in the framework of an acute experiment on animals.Results The most important cytokines affecting the process of reparative osteogenesis are fibroblast growth factor – FGF1 and bone morphogenetic protein 7 – BMP7. Based on the results of a comparative enzymelinked immunosorbent assay, it has been established that the autoregenerate, obtained by the original method, and a bone marrow aspirate concentrate have the highest osteogenic potential.Conclusion Autoregenerate is an effective and promising means of local stimulation of reparative osteogenesis, and its transplantation is a simple and highly effective procedure.

COMPARISON OF BONE PARAMETERS BY BODY REGION IN UNIVERSITY ATHLETES: SYSTEMATIC REVIEW

ABSTRACT Introduction: Bone mineral density (BMD) and bone mineral content (BMC) vary depending on the type of sport practiced and the body region, and their measurement can be an effective way to predict health risks throughout an athlete’s life. Objective: To describe the methodological aspects (measurement of bone parameters, body regions, precision errors and covariates) and to compare BMD and BMC by body region (total body, upper limbs, lower limbs and trunk) among university athletes practicing different sports. Methods: A search was performed on the databases PubMed, Web of Science, Scopus, ScienceDirect, EBSCOhost, SportDiscus, LILACS and SciELO. Studies were selected that: (1) compared BMD and BMC of athletes practicing at least two different sports (2) used dual-energy X-ray absorptiometry (DXA) to assess bone parameters (3) focused on university athletes. The extracted data were: place of study, participant selection, participants’ sex, sport practiced, type of study, bone parameters, DXA model, software used, scan and body regions, precision error, precision protocol, covariates and comparison of bone parameters between different sports by body region. Results: The main results were: 1) BMD is the most investigated bone parameter; 2) total body, lumbar spine and proximal femur (mainly femoral neck) are the most studied body regions; 3) although not recommended, the coefficient of variation is the main indicator of precision error; 4) total body mass and height are the most commonly used covariates; 5) swimmers and runners have lower BMD and BMC values; and 6) it is speculated that basketball players and gymnasts have greater osteogenic potential. Conclusions: Swimmers and runners should include weight-bearing exercises in their training routines. In addition to body mass and height, other covariates are important. The results of this review can help guide intervention strategies focused on preventing diseases and health problems during and after the athletic career. Level of evidence II; Systematic Review.