Authors: Yajing Fu, Dan Ma, Fengyan Fan...

PURPOSE

Stem cells from the apical papilla (SCAPs) are promising seed cells for tissue regeneration medicine and possess the osteogenic differentiation potential. Wnt5a, a typical ligand of the noncanonical Wnt pathway, exhibits diverse roles in the regulation of osteogenesis. The transcriptional co-activator with PDZ-binding motif (TAZ, WWTR1) is a core regulator in the Hippo pathway and regulates stem behavior including osteogenic differentiation. This study aims to examine how Wnt5a regulates SCAPs osteogenesis and explore the precise mechanistic relationship between Wnt5a and TAZ.

METHODS

SCAPs were isolated from developing apical papilla tissue of extracted human immature third molars in vitro. ALP staining, ALP activity and Alizarin red staining were used to evaluate osteogenic capacity. Osteogenic-related factors were assessed by qRT-PCR or Western blotting. Additionally, the receptor tyrosine kinase-like orphan receptor 2 (ROR2) was detected by immunocytofluorescence staining and silenced by small interfering RNA to verify the function of Wnt5a/ROR2 in TAZ-mediated osteogenesis. And we constructed TAZ-overexpression and β-catenin-overexpression SCAPs generated by lentivirus to explore the precise mechanistic relationship between Wnt5a and TAZ.

RESULTS

Wnt5a (100ng/mL) significantly suppressed ALP activity, mineralization nodules formation, expression of osteogenic-related factors. Meanwhile, it decreased the expression of TAZ mRNA and protein. TAZ overexpression promoted osteogenesis of SCAPs while Wnt5a could block TAZ-mediated osteogenesis. Furthermore, ROR2 siRNA (siROR2) was found to upregulate TAZ and canonical Wnt pathway signaling related molecules such as β-catenin, GSK3β and p-GSK3β. The suppression of Wnt5a/ROR2 on osteogenesis was significantly reversed by β-catenin overexpression through Wnt5a/ROR2/β-catenin/TAZ pathway.

CONCLUSION

Taken together, the present study demonstrates that Wnt5a suppresses TAZ-mediated osteogenesis of SCAPs and there may be a Wnt5a/ROR2/β-catenin/TAZ pathway regulating osteogenesis of SCAPs. Moreover, Wnt5a could be a candidate for regulators in tissue regeneration.

Topics: Cell Differentiation; Cells, Cultured; Hippo Signaling Pathway; Humans; Osteogenesis; Stem Cells; Transcriptional Coactivator with PDZ-Binding Motif Proteins; Wnt Signaling Pathway; Wnt-5a Protein

PubMed: 35237028
DOI: 10.2147/DDDT.S350698

Authors: Junjie Shen, Yi Sun, Xuanzhe Liu...

BACKGROUND

Osteogenesis is tightly coupled with angiogenesis during bone repair and regeneration. However, the underlying mechanisms linking these processes remain largely undefined. The present study aimed to test the hypothesis that epidermal growth factor-like domain-containing protein 6 (EGFL6), an angiogenic factor, also functions in bone marrow mesenchymal stem cells (BMSCs), playing a key role in the interaction between osteogenesis and angiogenesis.

METHODS

We evaluated how EGFL6 affects angiogenic activity of human umbilical cord vein endothelial cells (HUVECs) via proliferation, transwell migration, wound healing, and tube-formation assays. Alkaline phosphatase (ALP) and Alizarin Red S (AR-S) were used to assay the osteogenic potential of BMSCs. qRT-PCR, western blotting, and immunocytochemistry were used to evaluate angio- and osteo-specific markers and pathway-related genes and proteins. In order to determine how EGFL6 affects angiogenesis and osteogenesis in vivo, EGFL6 was injected into fracture gaps in a rat tibia distraction osteogenesis (DO) model. Radiography, histology, and histomorphometry were used to quantitatively evaluate angiogenesis and osteogenesis.

RESULTS

EGFL6 stimulated both angiogenesis and osteogenic differentiation through Wnt/β-catenin signaling in vitro. Administration of EGFL6 in the rat DO model promoted CD31EMCN type H-positive capillary formation associated with enhanced bone formation. Type H vessels were the referred subtype involved during DO stimulated by EGFL6.

CONCLUSION

EGFL6 enhanced the osteogenic differentiation potential of BMSCs and accelerated bone regeneration by stimulating angiogenesis. Thus, increasing EGFL6 secretion appeared to underpin the therapeutic benefit by promoting angiogenesis-coupled bone formation. These results imply that boosting local concentrations of EGFL6 may represent a new strategy for the treatment of compromised fracture healing and bone defect restoration.

Topics: Animals; Cell Differentiation; Cells, Cultured; Fracture Healing; Human Umbilical Vein Endothelial Cells; Humans; Osteogenesis; Osteogenesis, Distraction; Rats; Wnt Signaling Pathway; beta Catenin

PubMed: 34294121
DOI: 10.1186/s13287-021-02487-3

Authors: Feng Wang, Huijuan Qian, Lingchi Kong...

Both osteoblasts and preosteoclasts contribute to the coupling of osteogenesis and angiogenesis, regulating bone regeneration. Astragaloside IV (AS-IV), a glycoside of cycloartane-type triterpene derived from the Chinese herb , exhibits various biological activities, including stimulating angiogenesis and attenuating ischemic-hypoxic injury. However, the effects and underlying mechanisms of AS-IV in osteogenesis, osteoclastogenesis, and bone regeneration remain poorly understood. In the present study, we found that AS-IV treatment inhibited osteoclastogenesis, preserved preosteoclasts, and enhanced platelet-derived growth factor-BB (PDGF-BB)-induced angiogenesis. Additionally, AS-IV promoted cell viability, osteogenic differentiation, and angiogenic gene expression in bone marrow mesenchymal stem cells (BMSCs). The activation of AKT/GSK-3β/β-catenin signaling was found to contribute to the effects of AS-IV on osteoclastogenesis and osteogenesis. Furthermore, AS-IV accelerated bone regeneration during distraction osteogenesis (DO), as evidenced from the improved radiological and histological manifestations and biomechanical parameters, accompanied by enhanced angiogenesis within the distraction zone. In summary, AS-IV accelerates bone regeneration during DO, by enhancing osteogenesis and preosteoclast-induced angiogenesis simultaneously, partially through AKT/GSK-3β/β-catenin signaling. These findings reveal that AS-IV may serve as a potential bioactive molecule for promoting the coupling of osteogenesis and angiogenesis, and imply that AKT/GSK-3β/β-catenin signaling may be a promising therapeutic target for patients during DO treatment.

Topics: Animals; Bone Marrow; Bone Regeneration; Cell Proliferation; Cells, Cultured; Drugs, Chinese Herbal; Male; Models, Animal; Neovascularization, Physiologic; Osteoblasts; Osteogenesis; Rats; Rats, Sprague-Dawley; Saponins; Triterpenes

PubMed: 33994865
DOI: 10.7150/ijbs.57681

Authors: Yuan Xue, Sihan Hu, Chichi Chen...

Irisin is well-known to contribute to bone homeostasis due to its bidirectional regulation on osteogenesis and osteoclastogenesis. However, the mechanisms of irisin involved in mesenchymal stem/stromal cells (MSCs)-derived osteogenesis are still under investigated. Fibronectin type III domain-containing protein 5 (FNDC5) is the precursor protein of irisin, compare with wild type (WT) littermates, FNDC5 mice lost bone mass significantly, collectively evidenced by the decrease of bone mineral density (BMD), impaired bone formation and reduced N-terminal propertied of type I procollagen (P1NP) in sera. Meanwhile, the bone resorbing of FNDC5 mice has enhanced accompanied by increased tartrate phosphatase (TRAP) staining cells morphologically and cross-Linked C-telopeptide of type 1 collagen (CTX) level in sera. study showed that lack of irisin impeded the MSC-derived osteogenesis of FNDC5 mice. The addition of irisin promote the osteogenesis of WT and irisin-deficient MSCs, by activating αV integrin-induced ERK/STAT pathway subsequently enhancing bone morphogenetic protein 2 (BMP2) expression and BMP/SMAD signaling activation. Taken together, these findings further indicate that irisin regulates bone homeostasis. Moreover, irisin promotes MSC-derived osteogenesis by binding to αV integrin and activating BMP/SMAD signaling consequently. Thus, irisin may be a promising therapeutic target for osteoporosis and bone defects.

Topics: Animals; Apoptosis Regulatory Proteins; Bone Density; Bone Morphogenetic Protein 2; Cell Differentiation; Cells, Cultured; Collagen Type I; Fibronectins; Integrin alphaV; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Mitochondrial Proteins; Osteogenesis; Peptides; Signal Transduction

PubMed: 35002510
DOI: 10.7150/ijbs.63505

Authors: Kyung-Ran Park, Ji Eun Park, Bomi Kim...

Stem cells have received attention in various diseases, such as inflammatory, cancer, and bone diseases. Mesenchymal stem cells (MSCs) are multipotent stem cells that are critical for forming and repairing bone tissues. Herein, we isolated calycosin-7-O-β-glucoside (Caly) from the roots of , which is one of the most famous medicinal herbs, and investigated the osteogenic activities of Caly in MSCs. Caly did not affect cytotoxicity against MSCs, whereas Caly enhanced cell migration during the osteogenesis of MSCs. Caly increased the expression and enzymatic activities of ALP and the formation of mineralized nodules during the osteogenesis of MSCs. The osteogenesis and bone-forming activities of Caly are mediated by bone morphogenetic protein 2 (BMP2), phospho-Smad1/5/8, Wnt3a, phospho-GSK3β, and phospho-AKT, inducing the expression of runt-related transcription factor 2 (RUNX2). In addition, Caly-mediated osteogenesis and RUNX2 expression were attenuated by noggin and wortmannin. Moreover, the effects were validated in pre-osteoblasts committed to the osteoblast lineages from MSCs. Overall, our results provide novel evidence that Caly stimulates osteoblast lineage commitment of MSCs by triggering RUNX2 expression, suggesting Caly as a potential anabolic drug to prevent bone diseases.

Topics: Animals; Astragalus propinquus; Bone Marrow Cells; Bone Morphogenetic Protein 2; Calcification, Physiologic; Cell Differentiation; Core Binding Factor Alpha 1 Subunit; Glucosides; Humans; Isoflavones; Mesenchymal Stem Cells; Mice; NIH 3T3 Cells; Osteoblasts; Osteogenesis; Plant Extracts

PubMed: 34768792
DOI: 10.3390/ijms222111362

Authors: Yi He, Guanhui Chen, Ye Li...

The biological behaviors of magnetic graphene oxide (MGO) in a static magnetic field (SMF) are unknown. The current study is to investigate the cellular behaviors, osteogenesis and the mechanism in BMSCs treated with MGO combined with an SMF. Results showed that the synthetic MGO particles were bio-compatible and could significantly improve the osteogenesis of BMSCs under SMFs, as verified by elevated alkaline phosphatase activity, mineralized nodule formation, and expressions of mRNA and protein levels. Under SMF at the same intensity, the addition of graphene oxide to FeO could increase the osteogenic ability of BMSCs. The Wnt/β-catenin pathway was indicated to be related to the MGO-driven osteogenic behavior of the BMSCs under SMF. Taken together, our findings suggested that MGO under an SMF could promote osteogenesis in BMSCs through the Wnt/β-catenin pathway and hence should attract more attention for practical applications in bone tissue regeneration.

Topics: Animals; Cell Differentiation; Cell Proliferation; Gene Expression Regulation, Developmental; Graphite; Humans; Magnetic Fields; Magnetite Nanoparticles; Mesenchymal Stem Cells; Osteogenesis; Rats; Wnt Signaling Pathway

PubMed: 34186257
DOI: 10.1016/j.nano.2021.102435

Authors: Tao Zhang, Weidong Jiang, Fengchun Liao...

BACKGROUND

Distraction osteogenesis (DO), a kind of bone regenerative process, is not only extremely effective, but the osteogenesis rate is far beyond ordinary bone fracture (BF) healing. Exosomes (Exo) are thought to play a part in bone regeneration and healing as key players in cell-to-cell contact. The object of this work was to determine whether exosomes derived from DO and BF serum could stimulate the Osteogenic Differentiation in these two processes, and if so, which genes could be involved.

METHODS

The osteogenesis in DO-gap or BF-gap was evaluated using radiographic analysis and histological analysis. On the 14th postoperative day, DO-Exos and BF-Exos were isolated and cocultured with the jaw of bone marrow mesenchymal stem cells (JBMMSCs). Proliferation, migration and osteogenic differentiation of JBMMSCs were ascertained, after which exosomes RNA-seq was performed to identify the relevant gene.

RESULTS

Radiographic and histological analyses manifested that osteogenesis was remarkably accelerated in DO-gap in comparison with BF-gap. Both of the two types of Exos were taken up by JBMMSCs, and their migration and osteogenic differentiation were also seen to improve. However, the proliferation showed no significant difference. Finally, exosome RNA-seq revealed that the lncRNA MSTRG.532277.1 and the mRNA F-box and leucine-rich repeat protein 14(FBXL14) may play a key role in DO.

CONCLUSIONS

Our findings suggest that exosomes from serum exert a critical effect on the rapid osteogenesis in DO. This promoting effect might have relevance with the co-expression of MSTRG.532277.1 and FBXL14. On the whole, these findings provide new insights into bone regeneration, thereby outlining possible therapeutic targets for clinical intervention.

Topics: Mesenchymal Stem Cells; Osteogenesis; Osteogenesis, Distraction; RNA, Long Noncoding; RNA, Messenger

PubMed: 35643547
DOI: 10.1186/s13018-022-03163-9

Authors: Ziyi Li, Yafei Wang, Shilun Li...

Bone regeneration is a complex process that requires the coordination of osteogenesis and osteoclastogenesis. The balance between osteogenesis and adipogenesis of bone marrow mesenchymal stem cells (BMSCs) plays a major role in the process of bone formation. Recently, intercellular communication between bone cells and surrounding cells has been gradually recognized, and macrophages on the surface of bone have been proven to regulate bone metabolism. However, the underlying mechanisms have not been fully elucidated. Recent studies have indicated that exosomes are vital messengers for cell-cell communication in various biological processes. In this experiment, we found that exosomes derived from M2 macrophages (M2D-Exos) could inhibit adipogenesis and promote osteogenesis of BMSCs. M2D-Exo intervention increased the expression of miR-690, IRS-1, and TAZ in BMSCs. Additionally, miR-690 knockdown in M2 macrophages with a miR-690 inhibitor partially counteracted the effect of M2D-Exos on BMSC differentiation and the upregulation of IRS-1 and TAZ expression. Taken together, the results of our study indicate that exosomes isolated from M2 macrophages could facilitate osteogenesis and reduce adipogenesis through the miR-690/IRS-1/TAZ axis and might be a therapeutic tool for bone loss diseases.

Topics: Adipogenesis; Animals; Exosomes; Interleukin-4; Macrophages; Mesenchymal Stem Cells; Mice; Osteogenesis

PubMed: 34295306
DOI: 10.3389/fendo.2021.680328

Authors: Chenglong Li, Lu Yang, Xiaohua Ren...

Hydroxyapatite scaffolds (HASs) are widely studied as suitable materials for bone replacement scaffolds due to their chemical similarities to organic materials. In our previous study, a novel HAS with a 25‑30‑µm groove structure (HAS‑G) exhibited enhanced osteogenesis of bone mesenchymal stromal cells (BMSCs) compared with HAS, potentially by modulating the macrophage‑induced immune microenvironment. However, the exact effects of different surface patterns on the physiological processes of attached cells is not known. The present study aimed to determine the effects of HAS‑G on the osteogenesis and physiological processes in BMSCs. Cell counting kit‑8 assays and propidium iodide staining followed by flow cytometry were performed, and the results demonstrated that both in normal medium and differentiating medium, HAS‑G promoted cell proliferation by decreasing the proportion of G1/G0 cells and decreased reactive oxygen species (ROS) accumulation in BMSCs compared with HAS. Detection markers of osteogenesis revealed that compared with HAS, HAS‑G increased runt‑related transcription factor 2, osteocalcin and osteopontin protein levels and promoted osteogenesis, which was further confirmed by Alizarin Red S staining. Following JC‑1 staining, it was observed that HAS‑G maintained the mitochondrial membrane potential, similar to that achieved by N‑acetylcysteine pretreatment. In addition, compared with those of HAS, HAS‑G decreased mitochondrial ROS levels, which potentially contributed to the promotion of osteogenesis. The results also demonstrated that HAS‑G inhibited mitophagy induced by ROS accumulation and ATP synthesis compared with HAS. In conclusion, HAS‑G decreased ROS accumulation and mitophagy and thus promoted osteogenesis of BMSCs, indicating that ROS modulation of HAS‑G may serve a key role in osteogenesis.

Topics: Animals; Bone Marrow; Cell Cycle; Cell Proliferation; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Durapatite; Female; Homeostasis; Membrane Potential, Mitochondrial; Mesenchymal Stem Cells; Microscopy, Electron, Scanning; Mitochondria; Mitophagy; Osteocalcin; Osteogenesis; Osteopontin; Porosity; Rats, Sprague-Dawley; Reactive Oxygen Species; Tissue Scaffolds

PubMed: 32700750
DOI: 10.3892/mmr.2020.11352

Authors: Joohyun Lim, Caressa Lietman, Matthew W Grol...

Osteogenesis imperfecta (OI) is a genetic disorder that features wide-ranging defects in both skeletal and nonskeletal tissues. Previously, we and others reported that loss-of-function mutations in FK506 Binding Protein 10 () lead to skeletal deformities in conjunction with joint contractures. However, the pathogenic mechanisms underlying joint dysfunction in OI are poorly understood. In this study, we have generated a mouse model in which is conditionally deleted in tendons and ligaments. removal substantially reduced telopeptide lysyl hydroxylation of type I procollagen and collagen cross-linking in tendons. These biochemical alterations resulting from ablation were associated with a site-specific induction of fibrosis, inflammation, and ectopic chondrogenesis followed by joint deformities in postnatal mice. We found that the ectopic chondrogenesis coincided with enhanced Gli1 expression, indicating dysregulated Hedgehog (Hh) signaling. Importantly, genetic inhibition of the Hh pathway attenuated ectopic chondrogenesis and joint deformities in mutants. Furthermore, Hh inhibition restored alterations in gait parameters caused by loss. Taken together, we identified a previously unappreciated role of in tendons and ligaments and pathogenic mechanisms driving OI joint dysfunction.

Topics: Animals; Animals, Newborn; Chondrocytes; Chondrogenesis; Collagen Type I; Disease Models, Animal; Fibrosis; Gait; Gene Deletion; Gene Expression Regulation; Hedgehog Proteins; Hydroxylation; Inflammation; Joints; Ligaments; Lysine; Mice; Models, Biological; Motor Activity; Ossification, Heterotopic; Osteogenesis; Osteogenesis Imperfecta; Peptides; Sequence Analysis, RNA; Signal Transduction; Tacrolimus Binding Proteins; Tendons

PubMed: 34161280
DOI: 10.1073/pnas.2100690118