Osteoblast Lineage Research Articles

Decreasing miR-433-3p activity in the osteoblast lineage blunts glucocorticoid-mediated bone loss.

Glucocorticoid excess causes bone loss due to decreased bone formation and increased bone resorption; miR-433-3p is a miRNA that negatively regulates bone formation in male mice by targeting Runx2 as well as RNAs involved in Wnt, protein kinase A and endogenous glucocorticoid signaling. To examine the impact of miR-433-3p on glucocorticoid-mediated bone loss, transgenic mice expressing a miR-433-3p tough decoy inhibitor in the osteoblast lineage were administered prednisolone via slow-release pellets. Bone loss was greater in control mice treated with prednisolone compared with miR-433-3p tough decoy mice due to higher osteoclast activity in the controls. In whole femurs, Rankl was significantly higher in prednisolone-treated controls compared with miR-433-3p tough decoy mice. Surprisingly, negative regulators of Wnt signaling Sost and Dkk1 were higher in miR-433-3p tough decoy mice and were unaffected by prednisolone. Luciferase-3' UTR reporter assays demonstrated that Sost is a novel miR-433-3p target, whereas Dkk1 is a previously validated miR-433-3p target. miR-433-3p levels are lower in matrix synthesizing osteoblasts than in more osteocytic cells, thus the impact of miR-433-3p on the osteoblast lineage maybe dependent on cell context: it is a negative regulator in matrix-depositing osteoblasts by targeting RNAs important for differentiation and function, but a positive regulator in osteocytes, due to its ability to target prominently expressed negative regulators of Wnt signaling, Sost and Dkk1. The mechanisms by which miR-433-3p indirectly regulates glucocorticoid-mediated osteoclastogenesis remain unknown. However, we speculate that this regulation may be mediated miR-433-3p activity in osteocytes, which play an important role in controlling osteoclastogenesis.

Mapping the spatial atlas of the human bone tissue integrating spatial and single-cell transcriptomics.

Bone is a multifaceted tissue requiring orchestrated interplays of diverse cells within specialized microenvironments. Although significant progress has been made in understanding cellular and molecular mechanisms of component cells of bone, revealing their spatial organization and interactions in native bone tissue microenvironment is crucial for advancing precision medicine, as they govern fundamental signaling pathways and functional dependencies among various bone cells. In this study, we present the first integrative high-resolution map of human bone and bone marrow, using spatial and single-cell transcriptomics profiling from femoral tissue. This multi-modal approach discovered a novel bone formation-specialized niche enriched with osteoblastic lineage cells and fibroblasts and unveiled critical cell-cell communications and co-localization patterns between osteoblastic lineage cells and other cells. Furthermore, we discovered a novel spatial gradient of cellular composition, gene expression and signaling pathway activities radiating from the trabecular bone. This comprehensive atlas delineates the intricate bone cellular architecture and illuminates key molecular processes and dependencies among cells that coordinate bone metabolism. In sum, our study provides an essential reference for the field of bone biology and lays the foundation for advanced mechanistic studies and precision medicine approaches in bone-related disorders.

Atlas of Fshr expression from novel reporter mice.

The FSH-FSHR pathway has been considered an essential regulator in reproductive development and fertility. But there has been emerging evidence of FSHR expression in extragonadal organs. This poses new questions and long-term debates regarding the physiological role of the FSH-FSHR, and underscores the need for reliable, in vivo analysis of FSHR expression in animal models. However, conventional methods have proven insufficient for examining FSHR expression due to several limitations. To address this challenge, we developed Fshr-ZsGreen reporter mice under the control of Fshr endogenous promoter using CRISPR-Cas9. With this novel genetic tool, we provide a reliable readout of Fshr expression at single-cell resolution level in vivo and in real time. Reporter animals were also subjected to additional analyses,to define the accurate expression profile of FSHR in gonadal and extragonadal organs/tissues. Our compelling results not only demonstrated Fshr expression in intragonadal tissues but also, strikingly, unveiled notably increased expression in Leydig cells, osteoblast lineage cells, endothelial cells in vascular structures, and epithelial cells in bronchi of the lung and renal tubes. The genetic decoding of the widespread pattern of Fshr expression highlights its physiological relevance beyond reproduction and fertility, and opens new avenues for therapeutic options for age-related disorders of the bones, lungs, kidneys, and hearts, among other tissues. Exploiting the power of the Fshr knockin reporter animals, this report provides the first comprehensive genetic record of the spatial distribution of FSHR expression, correcting a long-term misconception about Fshr expression and offering prospects for extensive exploration of FSH-FSHR biology.

Long non-coding RNA Malat1 fine-tunes bone homeostasis and repair by orchestrating cellular crosstalk and β-catenin-OPG/Jagged1 pathway.

The IncRNA Malat1 was initially believed to be dispensable for physiology due to the lack of observable phenotypes in Malat1 knockout (KO) mice. However, our study challenges this conclusion. We found that both Malat1 KO and conditional KO mice in the osteoblast lineage exhibit significant osteoporosis. Mechanistically, Malat1 acts as an intrinsic regulator in osteoblasts to promote osteogenesis. Interestingly, Malat1 does not directly affect osteoclastogenesis but inhibits osteoclastogenesis in a non-autonomous manner in vivo via integrating crosstalk between multiple cell types, including osteoblasts, osteoclasts, and chondrocytes. Our findings substantiate the existence of a novel remodeling network in which Malat1 serves as a central regulator by binding to β-catenin and functioning through the β-catenin-OPG/Jagged1 pathway in osteoblasts and chondrocytes. In pathological conditions, Malat1 significantly promotes bone regeneration in fracture healing. Bone homeostasis and regeneration are crucial to well-being. Our discoveries establish a previous unrecognized paradigm model of Malat1 function in the skeletal system, providing novel mechanistic insights into how a lncRNA integrates cellular crosstalk and molecular networks to fine tune tissue homeostasis, remodeling and repair.

Integrated single-cell analysis reveals heterogeneity and therapeutic insights in osteosarcoma

Osteosarcoma (OSA) is a primary bone malignancy characterized by its aggressive nature and high propensity for metastasis. Despite advancements in multimodal therapies, the clinical outcomes for OSA patients remain suboptimal, necessitating deeper molecular insights for improved therapeutic strategies. Here, we employed single-cell RNA sequencing (scRNA-seq) to elucidate the cellular heterogeneity and transcriptional dynamics of OSA tumors. Our study identified eleven distinct tumor cell subpopulations, including osteoblastic, chondroblastic, and myeloid lineages, each exhibiting unique transcriptional profiles associated with disease progression and metastasis. Epithelial-mesenchymal transition (EMT) emerged as a critical process driving aggressive phenotypes, supported by gene set enrichment analyses (GSVA) and transcription factor regulatory network analyses. Integration of copy number variation (CNV) data highlighted genomic alterations in osteoblastic and chondroblastic cells, implicating potential therapeutic targets. Furthermore, immune cell infiltration analyses revealed distinct immune profiles across OSA subtypes, correlating with tumor mutational burden (TMB) and clinical outcomes. Our findings underscore the complexity of OSA biology and provide a foundation for developing personalized treatment strategies targeting tumor heterogeneity and immune interactions.

Dual scalable osteogenic microtissue engineering via GelMA microsphere-inspired mechanical training and autonomous assembling of dental pulp stem cell

Large bone tissue defects present a significant clinical challenge due to the lack of stem cells and an osteogenic microenvironment, leading to fibrotic healing and impaired bone regeneration. Microsphere-based cell-on three-dimensional (3D) culture systems show great promise for constructing osteogenic microtissues. However, the underlying mechanisms require further investigation. In this study, we propose a simple, scalable framework for highly efficient osteogenic microtissue construction, utilizing gelatin methacryloyl (GelMA) microspheres and dental pulp stem cells (DPSCs). The GelMA microspheres provide an extensive, scalable 3D framework for the autonomous adhesion, migration, and proliferation of DPSCs. Within the enormous 3D space created by the microspheres, DPSCs anchor to the microspheres and neighboring cells, inducing intrinsic tensile stress and simulating a mechanical force akin to “rock climbing training”. Transcriptomic sequencing results reveal that the 3D spatial and mechanical microenvironment modulates biological processes involved in cell adhesion, extracellular matrix organization, and the positive regulation of cell migration. Further investigations demonstrate that triggering the FAK/YAP pathway mediate mechanical driven differentiation of DPSCs into the osteoblastic lineage in the excellent osteogenic microtissues. Moreover, this simple scalable 3D framework strategy is expected to enable the efficient and large-scale preparation of stem cell-based microtissues.

Long non-coding RNA Malat1 fine-tunes bone homeostasis and repair by orchestrating cellular crosstalk and the β-catenin-OPG/Jagged1 pathway.

The IncRNA Malat1 was initially believed to be dispensable for physiology due to the lack of observable phenotypes in Malat1 knockout (KO) mice. However, our study challenges this conclusion. We found that both Malat1 KO and conditional KO mice in the osteoblast lineage exhibit significant osteoporosis. Mechanistically, Malat1 acts as an intrinsic regulator in osteoblasts to promote osteogenesis. Interestingly, Malat1 does not directly affect osteoclastogenesis but inhibits osteoclastogenesis in a non-autonomous manner in vivo via integrating crosstalk between multiple cell types, including osteoblasts, osteoclasts and chondrocytes. Our findings substantiate the existence of a novel remodeling network in which Malat1 serves as a central regulator by binding to β-catenin and functioning through the β-catenin-OPG/Jagged1 pathway in osteoblasts and chondrocytes. In pathological conditions, Malat1 significantly promotes bone regeneration in fracture healing. Bone homeostasis and regeneration are crucial to well-being. Our discoveries establish a previous unrecognized paradigm model of Malat1 function in the skeletal system, providing novel mechanistic insights into how a lncRNA integrates cellular crosstalk and molecular networks to fine tune tissue homeostasis, remodeling and repair.

Exposure effects of synthetic glucocorticoid drugs on skeletal developmental and immune cell function in zebrafish

Synthetic glucocorticoids (GCs) are used to treat a wide range of human health conditions and as such are frequently detected in the aquatic environment. This, together with the highly conserved nature of the glucocorticoid system across vertebrates means that the potential for biological effects of GCs in fish is relatively high. Here, we found that exposure of zebrafish (Danio rerio) to environmentally relevant concentrations of 4 of the most widely used synthetic GCs (beclomethasone dipropionate, budesonide, fluticasone propionate, and prednisolone), from 0 to 4 days post fertilisation (dpf), resulted in no effects on embryo-larval development or bone and cartilage formation. However, after exposure to equivalents of human therapeutic plasma levels, developmental abnormalities were observed that included pericardial oedema, blood pooling and alterations in jaw cartilage. Furthermore, using a double transgenic zebrafish osteoblast and chondrocyte reporter line, exposure up to 10 dpf resulted in alterations to lower jaw cartilage and bone development for all compounds at, and above, human therapeutic plasma concentrations. In the case of beclomethasone dipropionate, a reduction in lower jaw intercranial distance was observed at the environmentally relevant concentration of 0.1 μg/L. Using further transgenic reporter lines with fluorescently tagged neutrophils and macrophages, we also show exposure of embryo-larvae (0–4 dpf) to the GCs tested resulted in altered immune cell migration, but only at relatively high exposure concentrations. Collectively, our findings show GC exposure impacts embryo-larval zebrafish development, immune function, and skeletal formation, but predominantly at concentrations greater than those currently reported for the aquatic environment. Despite this, however, it is suggested that studies with longer exposure times, and to mixtures of multiple GCs (many GCs act via the same mechanism of action) are warranted before we can confidently assert that these commonly detected contaminants do not pose a risk to fish in the wild.

The Regulatory Role of miRNAs in Zebrafish Fin Regeneration

Since Teleostei fins have a strong regenerative capacity, further research was conducted on the regulation of gene expression during fin regeneration. This research focuses on miRNA, which is a key post-transcriptional regulatory molecule. In this study, a miRNA library for the fin regeneration of zebrafish was constructed to reveal the differential expression of miRNA during fin regeneration and to explore the regulatory pathway for fin regeneration. Following the injection of miRNA agomir into zebrafish, the proliferation of blastema cells and the overall fin regeneration area were significantly reduced. It was observed that the miRNAs impaired blastocyte formation by affecting fin regeneration through the inhibition of the expressions of genes and proteins associated with blastocyte formation (including yap1 and Smad1/5/9), which is an effect associated with the Hippo pathway. Furthermore, it has been demonstrated that miRNAs can impair the patterns and mineralization of newly formed fin rays. The miRNAs influenced fin regeneration by inhibiting the expression of a range of bone-related genes and proteins in osteoblast lineages, including sp7, runx2a, and runx2b. This study provides a valuable reference for the further exploration of morphological bone reconstruction in aquatic vertebrates.

CRISPR activation of Tfeb , a master regulator of autophagy and lysosomal biogenesis, in osteoblast lineage cells increases bone mass and strength.

Autophagy is a recycling pathway in which damaged or dysfunctional proteins, protein aggregates, and organelles are delivered to lysosomes for degradation. Insufficiency of autophagy is thought to contribute to several age-related diseases including osteoporosis. Consistent with this, elimination of autophagy from the osteoblast lineage reduces bone formation and causes low bone mass. However, whether increasing autophagy would benefit bone health is unknown. Here, we increased expression of the endogenous Transcription Factor EB gene ( Tfeb ) in osteoblast lineage cells in vivo via CRISPR activation. Tfeb overexpression stimulated autophagy and lysosomal biogenesis in osteoblasts. Tfeb overexpressing male mice displayed a robust increase in femoral and vertebral cortical thickness at 4.5 months of age. Histomorphometric analysis revealed that the increase in femoral cortical thickness was due to increased bone formation at the periosteal surface. Tfeb overexpression also increased femoral trabecular bone volume. Consistent with these results, bone strength was increased in Tfeb overexpressing mice. Female Tfeb overexpressing mice also displayed a progressive increase in bone mass over time and at 12 months of age had high cortical thickness and trabecular bone volume. This increase in vertebral trabecular bone volume was due to elevated bone formation. Osteoblastic cultures showed that Tfeb overexpression increased proliferation and osteoblast formation. Overall, these results demonstrate that stimulation of autophagy in osteoblast lineage cells promotes bone formation and strength and may represent an effective approach to combat osteoporosis.

3D-printed injectable nanocomposite cryogel scaffolds for bone tissue regeneration

Cryogels are known for their high water content and interconnected macroporosity, two relevant features in tissue engineering approaches. The cryogel structure can support tissue growth as it allows nutrient and oxygen diffusion, removal of waste products, as well as an enhancement of cell infiltration and proliferation. Bioactive glass nanoparticles are biocompatible and clinically approved bioactive materials widely used as implants in the human body to repair or replace diseased or damaged bones. They are known to facilitate bone binding while stimulating bone growth. Indeed, the combination of cryogels with bioactive nanoparticles has already demonstrated promising results for bone regeneration. Although the developed biomaterials succeed in bone regeneration, they lack suitability for minimal invasive procedures or patient-specificity. Here, we demonstrate a freeform 3D printed nanocomposite cryogel, resorting to an ink composed of functionalized gelatin and bioactive glass nanoparticles with methacrylate groups. Complex structures with multiple layers were 3D printed in a xanthan gum supporting bath. The developed 3D printed nanocomposite cryogels demonstrate the ability to recover their shape without any permanent damage, withstanding up to 65 % compression upon injection. Additionally, they stimulate the differentiation of human adipose-derived stem cells into the osteoblast lineage, therefore promoting bone tissue growth. We further demonstrated their suitability for minimal invasive therapeutics by filling a reproduction of a maxillofacial defect. The developed 3D-printed nanocomposite cryogels offer robust shape-recovery properties, easy injectability, tailored geometry into patient-specific injuries, and high osteogenic bioactivity, showcasing its versatility for bone regeneration purposes.

Regulation of Skeletal Development and Maintenance by Runx2 and Sp7.

Runx2 (runt related transcription factor 2) and Sp7 (Sp7 transcription factor 7) are crucial transcription factors for bone development. The cotranscription factor Cbfb (core binding factor beta), which enhances the DNA-binding capacity of Runx2 and stabilizes the Runx2 protein, is necessary for bone development. Runx2 is essential for chondrocyte maturation, and Sp7 is partly involved. Runx2 induces the commitment of multipotent mesenchymal cells to osteoblast lineage cells and enhances the proliferation of osteoprogenitors. Reciprocal regulation between Runx2 and the Hedgehog, fibroblast growth factor (Fgf), Wnt, and parathyroid hormone-like hormone (Pthlh) signaling pathways and Dlx5 (distal-less homeobox 5) plays an important role in these processes. The induction of Fgfr2 (Fgf receptor 2) and Fgfr3 expression by Runx2 is important for the proliferation of osteoblast lineage cells. Runx2 induces Sp7 expression, and Runx2+ osteoprogenitors become Runx2+Sp7+ preosteoblasts. Sp7 induces the differentiation of preosteoblasts into osteoblasts without enhancing their proliferation. In osteoblasts, Runx2 is required for bone formation by inducing the expression of major bone matrix protein genes, including Col1a1 (collagen type I alpha 1), Col1a2, Spp1 (secreted phosphoprotein 1), Ibsp (integrin binding sialoprotein), and Bglap (bone gamma carboxyglutamate protein)/Bglap2. Bglap/Bglap2 (osteocalcin) regulates the alignment of apatite crystals parallel to collagen fibrils but does not function as a hormone that regulates glucose metabolism, testosterone synthesis, and muscle mass. Sp7 is also involved in Co1a1 expression and regulates osteoblast/osteocyte process formation, which is necessary for the survival of osteocytes and the prevention of cortical porosity. SP7 mutations cause osteogenesis imperfecta in rare cases. Runx2 is an important pathogenic factor, while Runx1, Runx3, and Cbfb are protective factors in osteoarthritis development.

Gradient Boosting Prediction of Overlapping Genes From Weighted Co-expression and Differential Gene Expression Analysis of Wnt Pathway: An Artificial Intelligence-Based Bioinformatics Study.

Introduction The Wnt (wingless-related integration site) signalling pathway is crucial for bone formation and remodelling, regulating the commitment of mesenchymal stem cells (MSCs) to the osteoblastic lineage. It triggers the transcriptional activation of Wnt target genes and promotes osteoblast proliferation and survival. Weighted co-expression network analysis (WGCNA) and differential gene expression analysis help researchers understand gene roles. Gradient boosting, a machine learning technique, enhances understanding of genetic and molecular mechanisms contributing to overlap genes, improving gene regulation and functional genomics. The aim is to predict overlapping genes in the Wnt signalling pathway. Methods Differential gene expression analysis was performed using the National Center for Biotechnology Information (NCBI) geo dataset-GSE251951, focusing on the effect of Wnt signaling on treatment. The WGCNA module was analyzed using the iDEP tool to identify interconnected gene clusters. Hub genes were identified by calculating module eigengenes, correlated with external traits, and ranked based on module membership values. The study utilized gradient boosting, an ensemble learning method, to predict models, evaluate their performance using metrics like accuracy, precision, recall, and F1 score, and adjust predictions based on gradient and learning rate. Results The dendrogram uses the "Dynamic TreeCut" algorithm to analyze gene clusters, aiding researchers in understanding gene modules and biological processes, identifying co-expressed genes, and discovering new pathways. The confusion matrix displays 88 actual and predicted cases. The gradient boosting model achieves 78.9% accuracy in predicting Wnt pathway overlapping genes, with a respectable area under the curve (AUC) and classification accuracy values. It accurately predicts 73.9% of samples, with a high precision ratio and low recall. Conclusion Future research should enhance differential expression analysis and WGCNA to identify key Wnt pathway genes, improve sensitivity, specificity, hyperparameter tuning, and validation experiments, and use larger datasets.

An Exploration of the Role of Osteoclast Lineage Cells in Bone Tissue Engineering.

Bone defects because of age, trauma, and surgery, which are exacerbated by medication side effects and common diseases such as osteoporosis, diabetes, and rheumatoid arthritis, are a problem of epidemic scale. The present clinical standard for treating these defects includes autografts and allografts. Although both treatments can promote robust regenerative outcomes, they fail to strike a desirable balance of availability, side effect profile, consistent regenerative efficacy, and affordability. This difficulty has contributed to the rise of bone tissue engineering (BTE) as a potential avenue through which enhanced bone regeneration could be delivered. BTE is founded upon a paradigm of using biomaterials, bioactive factors, osteoblast lineage cells (ObLCs), and vascularization to cue deficient bone tissue into a state of regeneration. Despite promising preclinical results, BTE has had modest success in being translated into the clinical setting. One barrier has been the simplicity of its paradigm relative to the complexity of biological bone. Therefore, this paradigm must be critically examined and expanded to better account for this complexity. One potential avenue for this is a more detailed consideration of osteoclast lineage cells (OcLCs). Although these cells ostensibly oppose ObLCs and bone regeneration through their resorptive functions, a myriad of investigations have shed light on their potential to influence bone equilibrium in more complex ways through their interactions with both ObLCs and bone matrix. Most BTE research has not systematically evaluated their influence. Yet contrary to expectations associated with the paradigm, a selection of BTE investigations has demonstrated that this influence can enhance bone regeneration in certain contexts. In addition, much work has elucidated the role of many controllable scaffold parameters in both inhibiting and stimulating the activity of OcLCs in parallel to bone regeneration. Therefore, this review aims to detail and explore the implications of OcLCs in BTE and how they can be leveraged to improve upon the existing BTE paradigm.

NOR1 promotes the osteoblastic differentiation of human periodontal ligament stem cells via TGF-β signaling pathway

Alveolar bone loss is a main manifestation of periodontitis. Human periodontal ligament stem cells (PDLSCs) are considered as optimal seed cells for alveolar bone regeneration due to its mesenchymal stem cell like properties. Osteogenic potential is the premise for PDLSCs to repair alveolar bone loss. However, the mechanism regulating osteogenic differentiation of PDLSCs remain elusive. In this study, we identified Neuron-derived orphan receptor 1 (NOR1), was particularly expressed in PDL tissue in vivo and gradually increased during osteogenic differentiation of PDLSCs in vitro. Knockdown of NOR1 in hPDLSCs inhibited their osteogenic potential while NOR1 overexpression reversed this effect. In order to elucidate the downstream regulatory network of NOR1, RNA-sequencing was used. We found that downregulated genes were mainly enriched in TGF-β, Hippo, Wnt signaling pathway. Further, by western blot analysis, we verified that the expression level of phosphorylated-SMAD2/3 and phosphorylated-SMAD4 were all decreased after NOR1 knockdown. Additionally, ChIP-qPCR and dual luciferase reporter assay indicated that NOR1 could bind to the promoter of TGFBR1 and regulate its activity. Moreover, overexpression of TGFBR1 in PDLSCs could rescue the damaged osteogenic potential after NOR1 knockdown. Taken together, our results demonstrated that NOR1 could activate TGF-β/SMAD signaling pathway and positively regulates the commitment of osteoblast lineages of PDLSCs by targeting TGFBR1 directly.

Strategies for the Codelivery of Osteoclasts and Mesenchymal Stem Cells in 3D-Printable Osteochondral Scaffolds.

Osteochondral defects, characterized by structural compromises to articular cartilage and subchondral bone, can cause pain and lead to progressive cartilage damage and eventual osteoarthritis. Unfortunately, repairing these defects remains difficult because of the poor regenerative properties of cartilage and complex mechanical demands of the joint. As such, the field of tissue engineering aims to develop multiphasic implants that replace pathological cartilage and bone tissue and restore mechanical functionality to the joint. Recent bone physiology investigations have demonstrated that osteoclast (OC) lineage cells are inextricably involved in osteoblastic bone formation through an extensive network of anabolic signaling pathways, and so the codelivery OC and osteoblast (OB) lineage cells within scaffolds is being actively explored for bone tissue engineering purposes. However, it remains unclear how these cells can be incorporated into the design of multiphasic osteochondral scaffolds to potentially enhance subchondral bone formation and subsequent implant osseointegration. To explore this question, we examined direct surface seeding and hydrogel encapsulation as potential scaffold cellularization strategies. First, we examined how OC precursor cells and peripheral blood monocytes (PBMCs) influence early-stage bone matrix development and osteogenesis in 2D coculture. Then, we evaluated the osteogenic potential of mesenchymal stem cells (MSCs) and PBMCs cocultures encapsulated within a gelatin methacrylate (GelMA) hydrogel system. Our findings demonstrate that coculturing PBMCs with MSCs in 2D cultures significantly enhanced cell proliferation, early bone matrix deposition, and the formation of cell clusters by Day 28. However, we observed no significant difference in type I collagen deposition between GelMA hydrogel scaffolds cultured in basal and OC conditions during the same period. In addition, we found that the GelMA hydrogel system with MSC/PBMC cocultures in OC conditions exhibited decreased osteogenic activity by Day 28. Collectively, our findings support the osteogenic potential of OC-lineage cells in 2D culture conditions, and the potential benefits of surface-seeding for the codelivery of OC-lineage cells and MSCs in osteo-scaffolds for enhanced osteochondral regeneration and broader bone tissue engineering purposes.

Enhanced osteogenic differentiation for osteoporosis treatment through controlled icariin release in the bone cavity via extracorporeal shock wave

Controlling drug release from the bone cavity through physical stimulation remains a challenge because the unique shielding properties of the bone structure make it difficult for many physical stimuli to be effective in skeletal disorders. Herein, we designed a type of high-loading nanocomposites self-assembled from Icariin (ICA), Ca2+, and Zoledronic acid (ZOL), which could respond to extracorporeal shock wave (ESW) to control drug release in the bone cavity and govern osteoblast-adipocyte lineage commitment to prevent osteoporotic fracture. The nanocomposites contain more than 70 % of the payloads and exhibit excellent function in bone marrow targeting. When shocked with ESW, the payloads including ICA, Ca2+, and ZOL, can be released in the bone marrow. ICA can inhibit the formation of adipocyte lineages and promote the formation of osteoblast lineages by inhibiting the transcription of peroxisome proliferators-activated receptor γ (PPARγ) and fatty acid-binding protein 4 (FABP4), ESW, and Ca2+ further increase osteoblast activity meanwhile, and ZOL acts as an inhibitor of osteoclasts, leading to an overall anabolism. In vivo, the treatment contributed to a significant increase in bone anabolism, including bone-related parameters, bone strength, and bone resilience, ultimately greatly reducing the risk of osteoporotic fracture. This study demonstrated the high feasibility of combining the bone-penetrating ESW-responsive drug-controlled release system with the regulation of osteoblast-adipocyte lineage commitment for osteoporotic fracture prevention.

Deletion of the transcription factor EBF1 in perivascular stroma disrupts skeletal homeostasis and precipitates premature aging of the marrow microenvironment

Early B cell factor 1 (EBF1) is a transcription factor expressed by multiple lineages of stromal cells within the bone marrow. While cultures of Ebf1-deficient cells have been demonstrated to have impaired differentiation into either the osteoblast or adipogenic lineage in vitro by several groups, in vivo there has been a nominal consequence of the loss of EBF1 on skeletal development. In this study we used Prx-cre driven deletion of Ebf1 to eliminate EBF1 from the entire mesenchymal lineage of the skeleton and resolve this discrepancy. We report here that EBF1 is expressed primarily in the Mesenchymal Stem and Progenitor Cell (MSPC)-Adipo, MSPC-Osteo, and the Early Mesenchymal Progenitors, and that loss of EBF1 has a plethora of consequences to maintenance of the skeleton throughout adulthood. Stroma from the Prx-cre;Ebf1fl/fl bones had impaired osteogenic differentiation, an age-dependent loss of CFU-F, and elevated senescence accompanying Ebf1-deletion. New bone formation was reduced after 3 months, and resulted in a quiescent bone environment with fewer osteoblasts and an accompanied reduction in osteoclast-mediated remodeling. Consequently, bones were less ductile at a younger age, and deletion of EBF1 dramatically impaired fracture repair. Disruption of EBF1 in perivascular populations also rearranged the vascular network within these bones and disrupted cytokine signaling from key hematopoietic niches resulting in anemia, reductions in B cells, and myeloid skewing of marrow hematopoietic lineages. Mechanistically we observed disrupted BMP signaling within Ebf1-deficient progenitors with reduced SMAD1-phosphorylation, and elevated secretion of the soluble BMP-inhibitor Gremlin from the MSPC-Adipo cells. Ebf1-deficient progenitors also exhibited posttranslational suppression of glucocorticoid receptor expression. Together, these results suggest that EBF1 signaling is required for mesenchymal progenitor mobilization to maintain the adult skeleton, and that the primary action of EBF1 in the early mesenchymal lineage is to promote proliferation, and differentiation of these perivascular cells to sustain a healthy tissue.

The Involvement of microRNAs in Bone Remodeling Signaling Pathways and Their Role in the Development of Osteoporosis.

Bone remodeling, crucial for maintaining the balance between bone resorption and formation, relies on the coordinated activity of osteoclasts and osteoblasts. During osteoclastogenesis, hematopoietic stem cells (HSCs) differentiate into the osteoclast lineage through the signaling pathways OPG/RANK/RANKL. On the other hand, during osteoblastogenesis, mesenchymal stem cells (MSCs) differentiate into the osteoblast lineage through activation of the signaling pathways TGF-β/BMP/Wnt. Recent studies have shown that bone remodeling is regulated by post-transcriptional mechanisms including microRNAs (miRNAs). miRNAs are small, single-stranded, noncoding RNAs approximately 22 nucleotides in length. miRNAs can regulate virtually all cellular processes through binding to miRNA-response elements (MRE) at the 3' untranslated region (3'UTR) of the target mRNA. miRNAs are involved in controlling gene expression during osteogenic differentiation through the regulation of key signaling cascades during bone formation and resorption. Alterations of miRNA expression could favor the development of bone disorders, including osteoporosis. This review provides a general description of the miRNAs involved in bone remodeling and their significance in osteoporosis development.

Direct activation of PI3K in osteoblasts and osteocytes strengthens murine bone through sex-specific actions on cortical surfaces.

Intracellular phosphoinositide 3-kinase (PI3K) signaling is activated by multiple bone-active receptors. Genetic mutations activating PI3K signaling are associated with clinical syndromes of tissue overgrowth in multiple organs, often including the skeleton. While one formation is increased by removing the PI3K inhibitor (phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)), the effect of direct PI3K activation in the osteoblast lineage has not been reported. We introduced a known gain-of-function mutation in Pik3ca, the gene encoding the p110α catalytic subunit of PI3K, in osteocytes and late osteoblasts using the dentin matrix protein-1 Cre (Dmp1Cre) mouse and assessed the skeletal phenotype. Femur shape was grossly normal, but cortical thickness was significantly greater in both male and female Dmp1Cre.Pik3caH1047R mice, leading to almost doubled bone strength at 12wk of age. Both sexes had smaller marrow areas from 6wk of age. Female mice also exhibited greater cross-sectional area, which continued to increase until 24wk of age, resulting in a further increase in bone strength. Although both male and female mice had increased endocortical mineralizing surface, only female mice had increased periosteal mineralizing surface. The bone formed in the Dmp1Cre.Pik3caH1047R mice showed no increase in intracortical remodeling nor any defect in cortical bone consolidation. In contrast, on both endocortical and periosteal surfaces, there was more lamellar bone formation, including highly organized osteocyte networks extending along the entire surface at a greater thickness than in control mice. In conclusion, direct activation of PI3Kα in cells targeted by Dmp1Cre leads to high cortical bone mass and strength with abundant lamellar cortical bone in female and male mice with no increase in intracortical remodeling. This differs from the effect of PTEN deletion in the same cells, suggesting that activating PI3Kα in osteoblasts and osteocytes may be a more suitable target to promote formation of lamellar bone.