scholarly journals EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Junjie Shen ◽  
Yi Sun ◽  
Xuanzhe Liu ◽  
Yu Zhu ◽  
Bingbo Bao ◽  
...  
Keyword(s):  
Bone Formation ◽  
Osteogenic Differentiation ◽  
Distraction Osteogenesis ◽  
Bone Repair ◽  
Angiogenic Factor ◽  
Osteogenic Potential ◽  
Human Umbilical Cord ◽  
Alizarin Red ◽  
Differentiation Potential

Abstract 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 CD31hiEMCNhi 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.

scholarly journals EGFL6 Regulates the Interaction Between Angiogenesis and Osteogenesis via Wnt/β-catenin Signaling in Distraction Osteogenesis

2021 ◽  
Author(s):  
Junjie Shen ◽  
Yi Sun ◽  
Xuanzhe Liu ◽  
Yu Zhu ◽  
Bingbo Bao ◽  
...  
Keyword(s):  
Bone Formation ◽  
Osteogenic Differentiation ◽  
Distraction Osteogenesis ◽  
Bone Repair ◽  
Angiogenic Factor ◽  
Osteogenic Potential ◽  
Human Umbilical Cord ◽  
Alizarin Red ◽  
Differentiation Potential

Abstract 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) and plays a key role in the interaction between osteogenesis 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 CD31hiEMCNhi 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 osteogenic differentiation potential of BMSCs and accelerated bone regeneration by stimulating angiogenesis, thus exerting therapeutic benefit by increasing EGFL6 secretion to promote 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.

scholarly journals Role of Fzd6 in Regulating the Osteogenic Differentiation of Adipose-derived Stem Cells in Osteoporosis

2021 ◽  
Author(s):  
Tianli Wu ◽  
Zhihao Yao ◽  
Gang Tao ◽  
Fangzhi Lou ◽  
Hui Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Wnt Signaling Pathway ◽  
Osteogenic Potential ◽  
Adipose Derived Stem Cells ◽  
Alizarin Red ◽  
Differentiation Potential

Abstract Objective: Although it has been demonstrated that adipose-derived stem cells (ASCs) from osteoporosis mice (OP-ASCs) exhibit impaired osteogenic differentiation potential, the molecular mechanism has not yet been elucidated. We found that Fzd6 was decreased in OP-ASCs compared with ASCs. This study investigates the effects and underlying mechanisms of Fzd6 in the osteogenic potential of OP-ASCs. Methods: Fzd6 expression in ASCs and OP-ASCs was measured by PCR gene chip. Fzd6 overexpression and silencing lentiviruses were used to evaluate the role of Fzd6 in the osteogenic differentiation of OP-ASCs. Real-time PCR (qPCR) and western blotting (WB) was performed to detect the expression of Fzd6 and bone-related molecules, including runt-related transcription factor 2 (Runx2) and osteopontin (Opn). Alizarin red staining and Alkaline phosphatase (ALP) staining was performed following osteogenic induction. Microscopic CT (Micro-CT), hematoxylin and eosin staining (H&E) staining, and Masson staining were used to assess the role of Fzd6 in osteogenic differentiation of osteoporosis (OP) mice in vivo.Results: Expression of Fzd6 was decreased significantly in OP-ASCs. Fzd6 silencing down-regulated the osteogenic ability of OP-ASCs in vitro. Overexpression of Fzd6 rescued the impaired osteogenic capacity in OP-ASCs in vitro. We obtained similar results in vivo.Conclusions: Fzd6 plays an important role in regulating the osteogenic ability of OP-ASCs both in vivo and in vitro. Overexpression of Fzd6 associated with the Wnt signaling pathway promotes the osteogenic ability of OP-ASCs, which provides new insights for the prevention and treatment of OP.

Adenovirus-mediated expression of vascular endothelial growth factor-a potentiates bone morphogenetic protein9-induced osteogenic differentiation and bone formation

2016 ◽  
Vol 397 (8) ◽  
pp. 765-775 ◽  
Author(s):  
Chang-jun Pi ◽  
Kai-lu Liang ◽  
Zhen-yong Ke ◽  
Fu Chen ◽  
Yun Cheng ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Bone Formation ◽  
Osteogenic Differentiation ◽  
Bone Repair ◽  
Osteoporotic Fractures ◽  
Angiogenic Factor ◽  
Vascular Endothelial ◽  
Akt Inhibitor ◽  
Endothelial Growth Factor

Abstract Mesenchymal stem cells (MSCs) are suitable seed cells for bone tissue engineering because they can self-renew and undergo differentiation into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Vascular endothelial growth factor-a (VEGF-a), an angiogenic factor, is also involved in osteogenesis and bone repair. However, the effects of VEGF-a on osteogenic MSCs differentiation remain unknown. It was previously reported that bone morphogenetic protein9 (BMP9) is one of the most important osteogenic BMPs. Here, we investigated the effects of VEGF-a on BMP9-induced osteogenesis with mouse embryo fibroblasts (MEFs). We found that endogenous VEGF-a expression was undetectable in MSCs. Adenovirus-mediated expression of VEGF-a in MEFs potentiated BMP9-induced early and late osteogenic markers, including alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). In stem cell implantation assays, VEGF-a augmented BMP9-induced ectopic bone formation. VEGF-a in combination with BMP9 effectively increased the bone volume and osteogenic activity. However, the synergistic effect was efficiently abolished by the phosphoinositide 3-kinase (PI3K)/AKT inhibitor LY294002. These results demonstrated that BMP9 may crosstalk with VEGF-a through the PI3K/AKT signaling pathway to induce osteogenic differentiation in MEFs. Thus, our findings demonstrate the effects of VEGF-a on BMP9-induced bone formation and provide a new potential strategy for treating nonunion fractures, large segmental bony defects, and/or osteoporotic fractures.

scholarly journals ER stress arm XBP1s plays a pivotal role in proteasome inhibition-induced bone formation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Dan Zhang ◽  
Kim De Veirman ◽  
Rong Fan ◽  
Qiang Jian ◽  
Yuchen Zhang ◽  
...  
Keyword(s):  
Bone Formation ◽  
Er Stress ◽  
Osteogenic Differentiation ◽  
Osteoblast Differentiation ◽  
Bone Destruction ◽  
Proteasome Inhibition ◽  
Stress Signaling ◽  
Alizarin Red

Abstract Background Bone destruction is a hallmark of multiple myeloma (MM). It has been reported that proteasome inhibitors (PIs) can reduce bone resorption and increase bone formation in MM patients, but the underlying mechanisms remain unclear. Methods Mesenchymal stem cells (MSCs) were treated with various doses of PIs, and the effects of bortezomib or carfilzomib on endoplasmic reticulum (ER) stress signaling pathways were analyzed by western blotting and real-time PCR. Alizarin red S (ARS) and alkaline phosphatase (ALP) staining were used to determine the osteogenic differentiation in vitro. Specific inhibitors targeting different ER stress signaling and a Tet-on inducible overexpressing system were used to validate the roles of key ER stress components in regulating osteogenic differentiation of MSCs. Chromatin immunoprecipitation (ChIP) assay was used to evaluate transcription factor-promoter interaction. MicroCT was applied to measure the microarchitecture of bone in model mice in vivo. Results We found that both PERK-ATF4 and IRE1α-XBP1s ER stress branches are activated during PI-induced osteogenic differentiation. Inhibition of ATF4 or XBP1s signaling can significantly impair PI-induced osteogenic differentiation. Furthermore, we demonstrated that XBP1s can transcriptionally upregulate ATF4 expression and overexpressing XBP1s can induce the expression of ATF4 and other osteogenic differentiation-related genes and therefore drive osteoblast differentiation. MicroCT analysis further demonstrated that inhibition of XBP1s can strikingly abolish bortezomib-induced bone formation in mouse. Conclusions These results demonstrated that XBP1s is a master regulator of PI-induced osteoblast differentiation. Activation of IRE1α-XBP1s ER stress signaling can promote osteogenesis, thus providing a novel strategy for the treatment of myeloma bone disease.

scholarly journals Identification of WNT16 as a Predictable Biomarker for Accelerated Osteogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells In Vitro

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Yu-Hee Kim ◽  
Kyung-Ah Cho ◽  
Hyun-Ji Lee ◽  
Minhwa Park ◽  
Han Su Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Selection Criterion ◽  
Osteogenic Potential ◽  
Differentiation Potential ◽  
Real Time Quantitative Pcr ◽  
Single Cell Cloning

The application of mesenchymal stem cells (MSCs) for treating bone-related diseases shows promising outcomes in preclinical studies. However, cells that are isolated and defined as MSCs comprise a heterogeneous population of progenitors. This heterogeneity can produce variations in the performance of MSCs, especially in applications that require differentiation potential in vivo, such as the treatment of osteoporosis. Here, we aimed to identify genetic markers in tonsil-derived MSCs (T-MSCs) that can predict osteogenic potential. Using a single-cell cloning method, we isolated and established several lines of nondifferentiating (ND) or osteoblast-prone (OP) clones. Next, we performed transcriptome sequencing of three ND and three OP clones that maintained the characteristics of MSCs and determined the top six genes that were upregulated in OP clones. Upregulation of WNT16 and DCLK1 expression was confirmed by real-time quantitative PCR, but only WNT16 expression was correlated with the osteogenic differentiation of T-MSCs from 10 different donors. Collectively, our findings suggest that WNT16 is a putative genetic marker that predicts the osteogenic potential of T-MSCs. Thus, examination of WNT16 expression as a selection criterion prior to the clinical application of MSCs may enhance the therapeutic efficacy of stem cell therapy for bone-related complications, including osteoporosis.

Oestrogen receptors are involved in the osteogenic differentiation of periodontal ligament stem cells

2010 ◽  
Vol 31 (2) ◽  
pp. 117-124 ◽  
Author(s):  
Feng Pan ◽  
Rui Zhang ◽  
Guang Wang ◽  
Yin Ding
Keyword(s):  
Stem Cells ◽  
Bone Formation ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Oestrogen Receptors ◽  
Periodontal Ligament Stem Cells ◽  
Differentiation Potential ◽  
Reverse Transcription Pcr ◽  
Pdl Cells

The existence of PDLSCs [PDL (periodontal ligament) stem cells] in PDL has been identified and such cells may function in periodontal reconstruction, including bone formation. Oestrogens/ERs (oestrogen receptors; ERα and ERβ) exert important effects in bone formation, however, the relationship between ERs and PDLSCs has not been established. In the present study, PDLSCs were isolated and assays for detecting stem-cell biomarkers and multipotential differentiation potential confirmed the validity of human PDLSCs. The results of RT–PCR (reverse transcription–PCR) and Western blotting showed that ERα and ERβ were expressed at higher levels in PDLSCs as compared with PDLCs (PDL cells), and 17β-oestradiol obviously induced the osteogenic differentiation of PDLSCs in vitro. Furthermore, a pan-ER inhibitor or lentivirus-mediated siRNA (small interfering RNA) targeting ERα or ERβ blocked the oestrogen-induced osteogenic differentiation of PDLSCs. The results indicate that both ERα and ERβ were involved in the process of osteogenic differentiation of PDLSCs.

scholarly journals Icariin Protects against Glucocorticoid-Induced Osteonecrosis of the Femoral Head in Rats

2018 ◽  
Vol 47 (2) ◽  
pp. 694-706 ◽  
Author(s):  
Zengfa Huang ◽  
Cheng Cheng ◽  
Beibei Cao ◽  
Jing Wang ◽  
Hui Wei ◽  
...  
Keyword(s):  
Femoral Head ◽  
Osteogenic Differentiation ◽  
Bone Repair ◽  
Adipogenic Differentiation ◽  
Ct Scanning ◽  
Control Group ◽  
Micro Ct ◽  
Alizarin Red ◽  
Immunohistochemical Analyses

Background/Aims: Glucocorticoid (GC)-related osteonecrosis of the femoral head (ONFH) is a common complication following administration of steroids to treat many diseases. Our previous study demonstrated that icariin (ICA) might have a beneficial effect on the bone marrow mesenchymal stem cells (BMSCs) of patients with steroid-associated osteonecrosis. In this study, we investigated the underlying mechanisms of ICA associated with the potential enhancement of osteogenesis and anti-adipogenesis in GC-related ONFH. Methods: In vitro cell proliferation was evaluated by CCK-8 assay. Alizarin red S and alkaline phosphatase (ALP) activity were used to measure osteogenic differentiation, while adipogenic differentiation was revealed by oil red O staining and TG content assay. The expression level of osteogenesis-associated genes and PPARγ was evaluated by RT-qPCR, western blotting and immunofluorescence. A total of 30 female SD rats were randomly separated into three groups: a control group, a methylprednisolone (MPS) group and a MPS + ICA group. Serum ALP and TG (triglyceride), micro-CT scanning, histological and immunohistochemical analyses were performed in the animal model. Results: In the in vitro study, ICA promoted proliferation, improved osteogenic differentiation and suppressed adipogenic differentiation of BMSCs treated with MPS. The group treated with MPS and 10-6 M ICA expressed higher levels of Runx2, ALP, bone morphogenetic protein (BMP) 2, and OC and lower expression of PPARγ than the MPS group. In the in vivo study, ICA prevented bone loss in a rat model of GC-related ONFH as shown by micro-CT scanning, histological and immunohistochemical analyses. Conclusions: ICA is an effective compound for promoting bone repair and preventing or delaying the progression of GC-associated ONFH in rats. This effect can be explained by its ability to improve the balance between adipogenesis and osteogenesis, indicating that ICA is an effective candidate for management of GC-associated ONFH.

scholarly journals Effects of strontium ions with potential antibacterial activity on in vivo bone regeneration

2020 ◽  
Author(s):  
Nafiseh Baheiraei ◽  
Hossein Eyni ◽  
Bita bakhshi ◽  
Raziyeh Najafloo
Keyword(s):  
Antibacterial Activity ◽  
Bone Formation ◽  
Bone Regeneration ◽  
Bone Repair ◽  
Early Stage ◽  
Antibacterial Activities ◽  
Calvarial Bone ◽  
Alizarin Red

Abstract Background: Bioactive glasses (BGs) have attracted added attention in the structure of the scaffolds for bone repair applications. Different metal ions could be doped in BGs to induce specific biological responses. Among these ions, strontium (Sr) is considered as an effective and safe doping element with promising effects on bone formation and regeneration.Methods: In this experiment, we evaluated the antibacterial activities of the gelatin-BG (Gel-BG) and Gel-BG/Sr scaffolds in vitro. The osteogenic properties of the prepared scaffolds were also assessed in rabbit calvarial bone defects for 12 weeks. Alizarin Red, Hematoxylin & Eosin (H&E) and Masson’s Trichrome staining were performed to assess bone regeneration and the obtained results were compared with those without Sr. Also, histomorphometric data were obtained to evaluate the new bone, residual graft, and connective tissue.Results: Both scaffolds showed in vivo bone formation during 12 weeks with the newly formed bone area in Gel-BG/Sr scaffold was higher than that in Gel-BG scaffolds after the whole period. Based on the histological results, Gel-BG/Sr exhibited acceleration of early-stage bone formation in vivo. The results of antibacterial investigation showed that although both Gel-BG/Sr and Gel-BG effectively inhibited the growth of Escherichia coli (E. coli) but, only Gel-BG/Sr structure could lead to a 3 log reduction in Staphylococcus aureus (S. aureus). Conclusions: Our results confirmed that Sr doped BG is a favorable candidate for bone tissue engineering with superior antibacterial activity and bone regeneration capacity compared with similar counterparts having no Sr ion.

scholarly journals Nanog/NFATc1/Osterix signaling pathway-mediated promotion of bone formation at the tendon-bone interface after ACL reconstruction with De-BMSCs transplantation

2021 ◽  
Author(s):  
Kai Tie ◽  
Jinghang Cai ◽  
Jun Qin ◽  
Hao Xiao ◽  
Yangfan Shangguan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Formation ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Molecular Mechanism ◽  
Bone Healing ◽  
Osteogenic Potential ◽  
Differentiation Potential ◽  
Bone Interface ◽  
Biomechanical Strength

Abstract Background: Bone formation plays an important role in early tendon-bone healing after anterior cruciate ligament reconstruction (ACLR). Dedifferentiated osteogenic bone marrow mesenchymal stem cells (De-BMSCs) have enhanced osteogenic potential. This study aimed to investigate the effect of De-BMSCs transplantation on the promotion of bone formation at the tendon-bone interface after ACLR and to further explore the molecular mechanism of the enhanced osteogenic potential of De-BMSCs.Methods: BMSCs from the femurs and tibias of New Zealand White rabbits were subjected to osteogenic induction and then cultured in medium without osteogenic factors; the obtained cell population was termed De-BMSCs. De-BMSCs were induced to undergo osteo-, chondro- and adipo-differentiation in vitro to examine the characteristics of primitive stem cells. An ACLR model with a semitendinosus tendon was established in rabbits, and the animals were divided into a control group, BMSCs group and De-BMSCs group. At 12 weeks after surgery, the rabbits in each group were sacrificed to evaluate tendon-bone healing by histologic staining, micro–computed tomography (micro-CT) examination, and biomechanical testing. During osteogenic differentiation of De-BMSCs, an siRNA targeting nuclear factor of activated T cells 1 (NFATc1) was used to verify the molecular mechanism of the enhanced osteogenic potential of De-BMSCs.Results: De-BMSCs exhibited some properties similar to BMSCs, including multiple differentiation potential and cell surface markers. Bone formation at the tendon-bone interface in the De-BMSCs group was significantly increased, and biomechanical strength was significantly improved. During the osteogenic differentiation of De-BMSCs, the expression of Nanog and NFATc1 was synergistically increased, which promoted the interaction of NFATc1 and Osterix, resulting in increased expression of osteoblast marker genes such as COL1A, OCN, and OPN.Conclusions: De-BMSCs transplantation could promote bone formation at the tendon-bone interface after ACLR and improve the biomechanical strength of the reconstruction. The Nanog/NFATc1/Osterix signaling pathway mediated the enhanced osteogenic differentiation efficiency of De-BMSCs.

Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells on Biodegradable Calcium-deficient Hydroxyapatite Tubular Bacterial Cellulose Composites

2014 ◽  
Vol 1621 ◽  
pp. 71-79 ◽  
Author(s):  
Pelagie Favi ◽  
Madhu Dhar ◽  
Nancy Neilsen ◽  
Roberto Benson
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Bacterial Cellulose ◽  
Periodate Oxidation ◽  
Osteogenic Potential ◽  
Alizarin Red ◽  
Native Bone ◽  
Cellulose Composites

ABSTRACTAdvanced biomaterials that mimic the structure and function of native tissues and permit stem cells to adhere and differentiate is of paramount importance in the development of stem cell therapies for bone defects. Successful bone repair approaches may include an osteoconductive scaffold that permits excellent cell adhesion and proliferation, and cells with an osteogenic potential. The objective of this study was to evaluate the cell proliferation, viability and osteocyte differentiation of equine-derived bone marrow mesenchymal stem cells (EqMSCs) when seeded onto biocompatible and biodegradable calcium-deficient hydroxyapatite (CdHA) tubular-shaped bacterial cellulose scaffolds (BC-TS) of various sizes. The biocompatible gel-like BC-TS was synthesized using the bacterium Gluconacetobacter sucrofermentans under static culture in oxygen-permeable silicone tubes. The BC-TS scaffolds were modified using a periodate oxidation to yield biodegradable scaffolds. Additionally, CdHA was deposited in the scaffolds to mimic native bone tissues. The morphological properties of the resulting BC-TS and its composites were characterized using scanning electron microscopy. The ability of the BC-TS and its composites to support and maintain EqMSCs growth, proliferation and osteogenic differentiation in vitro was also assessed. BC-TS and its composites exhibited aligned nanofibril structures. MTS assay demonstrated increasing proliferation and viability with time (days 1, 2 and 3). Cell-scaffold constructs were cultured for 8 days under osteogenic conditions and the resulting osteocytes were positive for alizarin red. In summary, biocompatible and biodegradable CdHA BC-TS composites support the proliferation, viability and osteogenic differentiation of EqMSCs cultured onto its surface in vitro, allowing for future potential use for tissue engineering therapies.

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