Precise immunomodulation of the M1 to M2 macrophage transition enhances mesenchymal stem cell osteogenesis and differs by sex

Objectives Up to 10% of fractures result in undesirable outcomes, for which female sex is a risk factor. Cellular sex differences have been implicated in these different healing processes. Better understanding of the mechanisms underlying bone healing and sex differences in this process is key to improved clinical outcomes. This study utilized a macrophage–mesenchymal stem cell (MSC) coculture system to determine: 1) the precise timing of proinflammatory (M1) to anti-inflammatory (M2) macrophage transition for optimal bone formation; and 2) how such immunomodulation was affected by male versus female cocultures. Methods A primary murine macrophage-MSC coculture system was used to demonstrate the optimal transition time from M1 to M2 (polarized from M1 with interleukin (IL)-4) macrophages to maximize matrix mineralization in male and female MSCs. Outcome variables included Alizarin Red staining, alkaline phosphatase (ALP) activity, and osteocalcin protein secretion. Results We found that 96 hours of M1 phenotype in male cocultures allowed for maximum matrix mineralization versus 72 hours in female cocultures. ALP activity and osteocalcin secretion were also enhanced with the addition of IL-4 later in male versus female groups. The sex of the cells had a statistically significant effect on the optimal IL-4 addition time to maximize osteogenesis. Conclusion These results suggest that: 1) a 72- to 96-hour proinflammatory environment is critical for optimal matrix mineralization; and 2) there are immunological differences in this coculture environment due to sex. Optimizing immunomodulation during fracture healing may enhance and expedite the bone regeneration response. These findings provide insight into precise immunomodulation for enhanced bone healing that is sex-specific. Cite this article: K. Nathan, L. Y. Lu, T. Lin, J. Pajarinen, E. Jämsen, J-F. Huang, M. Romero-Lopez, M. Maruyama, Y. Kohno, Z. Yao, S. B. Goodman. Precise immunomodulation of the M1 to M2 macrophage transition enhances mesenchymal stem cell osteogenesis and differs by sex. Bone Joint Res 2019;8:481–488. DOI: 10.1302/2046-3758.810.BJR-2018-0231.R2.

Download Full-text

Melatonin promotes bone marrow mesenchymal stem cell osteogenic differentiation and prevents osteoporosis development through modulating circ_0003865 that sponges miR-3653-3p

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02224-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xudong Wang ◽

Taiqiu Chen ◽

Zhihuai Deng ◽

Wenjie Gao ◽

Tongzhou Liang ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Osteogenic Differentiation ◽

Luciferase Reporter ◽

Specific Gene ◽

Biological Processes ◽

Alizarin Red ◽

Qrt Pcr ◽

Dual Luciferase Reporter Assay

Abstract Background Little is known about the implications of circRNAs in the effects of melatonin (MEL) on bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation and osteoporosis (OP) progression. The aim of our study was to investigate circRNAs in MEL-regulated BMSC differentiation and OP progression. Methods BMSC osteogenic differentiation was measured by qRT-PCR, western blot (WB), Alizarin Red, and alkaline phosphatase (ALP) staining. Differential circRNA and mRNA profiles of BMSCs treated by MEL were characterized by deep sequencing, followed by validation using RT-PCR, Sanger sequencing, and qRT-PCR. Silencing and overexpression of circ_0003865 were conducted for functional investigations. The sponged microRNAs and targeted mRNAs were predicted by bioinformatics and validated by qRT-PCR, RNA pull-down, and dual-luciferase reporter assay. The function of miR-3653-3p and circ_0003865/miR-3653-3p/growth arrest-specific gene 1 (GAS1) cascade was validated for the osteogenic differentiation of BMSCs by CCK-8, qRT-PCR, WB, Alizarin Red, and ALP staining. The effects of circ_0003865 on OP development were tested in murine OP model. Results MEL promoted osteogenic differentiation of BMSCs. RNA sequencing revealed significant alterations in circRNA and mRNA profiles associated with multiple biological processes and signaling pathways. Circ_0003865 expression in BMSCs was significantly decreased by MEL treatment. Silencing of circ_0003865 had no effect on proliferation while promoted osteogenic differentiation of BMSCs. Overexpression of circ_0003865 abrogated the promotion of BMSC osteogenic differentiation induced by MEL, but proliferation of BMSCs induced by MEL had no change whether circ_0003865 was overexpression or not. Furthermore, circ_0003865 sponged miR-3653-3p to promote GAS1 expression in BMSCs. BMSC osteogenic differentiation was enhanced by miR-3653-3p overexpression while BMSC proliferation was not affected. By contrast, miR-3653-3p silencing mitigated the promoted BMSC osteogenic differentiation caused by circ_0003865 silencing, but had no effect on proliferation. Finally, circ_0003865 silencing repressed OP development in mouse model. Conclusion MEL promotes BMSC osteogenic differentiation and inhibits OP pathogenesis by suppressing the expression of circ_0003865, which regulates GAS1 gene expression via sponging miR-3653-3p.

Download Full-text

99mTc–HDP Quantification of Extracellular Matrix Mineralization in 3d-Human Mesenchymal Stem Cell Culturesy

Journal of Stem Cell Research & Therapy ◽

10.4172/2157-7633.1000374 ◽

2017 ◽

Vol 07 (01) ◽

Author(s):

Tobias Großner ◽

Uwe Haberkorn ◽

Tobias Gotterbarm

Keyword(s):

Extracellular Matrix ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Human Mesenchymal Stem Cell ◽

Matrix Mineralization

Download Full-text

Temporal Changes in PEG Hydrogel Structure Influence Human Mesenchymal Stem Cell Proliferation and Matrix Mineralization

Advances in Experimental Medicine and Biology - Tissue Engineering ◽

10.1007/978-0-387-34133-0_10 ◽

2007 ◽

pp. 135-149 ◽

Cited By ~ 10

Author(s):

Charles R Nuttelman ◽

April M Kloxin ◽

Kristi S Anseth

Keyword(s):

Cell Proliferation ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Human Mesenchymal Stem Cell ◽

Temporal Changes ◽

Matrix Mineralization ◽

Stem Cell Proliferation

Download Full-text

Inhibition of GDF11 could promote bone healing in the tooth extraction socket and facilitate mesenchymal stem cell osteogenic differentiation in T2DM pigs

Journal of Periodontology ◽

10.1002/jper.20-0011 ◽

2020 ◽

Vol 91 (12) ◽

pp. 1645-1652

Author(s):

Shuyan Wang ◽

Lei Wang ◽

Shaojie Shi ◽

Xingxing Wang ◽

Chunxia He ◽

...

Keyword(s):

Stem Cell ◽

Mesenchymal Stem Cell ◽

Osteogenic Differentiation ◽

Bone Healing ◽

Tooth Extraction ◽

Extraction Socket

Download Full-text

Role of Mesenchymal Stem Cell-Conditioned Medium (MSC-CM) in the Bone Regeneration: A Systematic Review from 2007-2018

Annual Research & Review in Biology ◽

10.9734/arrb/2019/v31i230045 ◽

2019 ◽

pp. 1-16

Author(s):

Ismail Hadisoebroto Dilogo ◽

Jessica Fiolin

Keyword(s):

Stem Cell ◽

Mesenchymal Stem Cell ◽

Bone Regeneration ◽

Conditioned Medium ◽

Bone Healing ◽

In Vitro Studies ◽

In Vivo Studies

Background: The therapeutic value of mesenchymal stem cells (MSCs) in tissue engineering and regenerative medicine is attributable in part to paracrine pathways triggered by several secreted factors secreted into culture media. The secreted factor here is known as the conditioned medium (CM) or secretome. Objectives: This review is aimed to investigate and summarise the in-vitro, pre-clinical in-vivo studies regarding the role of CM-MSC in bone regeneration from 2007 until 2018 Data Sources: A systematic literature search on PubMed, MEDLINE, OVID, Scopus and Cochrane library was carried out by using search terms: Secretome, conditioned medium, mesenchymal stem cell, bone healing, osteogenic, osteogenesis. Methods: A total of 611 articles were reviewed. Ten articles were identified as relevant for this systematic literature review. Results: Three tables of studies were constructed for in vitro studies and in-vivo studies. Conclusion: All of the included in-vitro studies and in-vivo studies have shown a promoting effect of bone regeneration at various stages. Although there are no clinical studies regarding the use of CM-MSC in the human bone regeneration that have been conducted, transplantation of secretome has shown a promising result in the acceleration of bone healing process.

Download Full-text

Laser-Sintered Constructs with Bio-inspired Porosity and Surface Micro/Nano-Roughness Enhance Mesenchymal Stem Cell Differentiation and Matrix Mineralization In Vitro

Calcified Tissue International ◽

10.1007/s00223-016-0184-9 ◽

2016 ◽

Vol 99 (6) ◽

pp. 625-637 ◽

Cited By ~ 14

Author(s):

Alice Cheng ◽

David J. Cohen ◽

Barbara D. Boyan ◽

Zvi Schwartz

Keyword(s):

Stem Cell ◽

Cell Differentiation ◽

Mesenchymal Stem Cell ◽

Stem Cell Differentiation ◽

Matrix Mineralization ◽

Mesenchymal Stem Cell Differentiation

Download Full-text

Melatonin Promotes Bone Marrow Mesenchymal Stem Cell Osteogenic Differentiation and Prevents Osteoporosis Development Through Modulating circ_0003865 that Sponges miR-3653-3p

10.21203/rs.3.rs-50665/v1 ◽

2020 ◽

Author(s):

Xudong Wang ◽

Taiqiu Chen ◽

Zhihuai Deng ◽

Wenjie Gao ◽

Tongzhou Liang ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Osteogenic Differentiation ◽

Luciferase Reporter ◽

Specific Gene ◽

Biological Processes ◽

Alizarin Red ◽

Qrt Pcr ◽

Dual Luciferase Reporter Assay

Abstract Background: To investigate circRNAs in Melatonin (MEL)-regulated bone marrow mesenchymal stem cell (BMSC) differentiation and osteoporosis.Methods: BMSC osteogenic differentiation was measured by qRT-PCR, western blot (WB), Alizarin Red and alkaline phosphatase (ALP) staining. Differential circRNA and mRNA profiles of BMSCs treated by MEL were characterized by deep sequencing, followed by validation using RT-PCR, Sanger sequencing, and qRT-PCR. Silencing and overexpression of circ_0003865 were conducted for functional investigations. The sponged microRNAs and targeted mRNAs were predicted by bioinformatics and validated by qRT-PCR, RNA pull-down, and dual-luciferase reporter assay. The function of miR-3653-3p and circ_0003865/miR-3653-3p/growth arrest-specific gene 1 (GAS1) cascade were validated for the osteogenic differentiation of BMSCs by qRT-PCR, WB, Alizarin Red, and ALP staining. The effects of circ_0003865 on osteoporosis (OP) development was tested in murine osteoporosis model.Results: MEL promoted osteogenic differentiation of BMSCs. RNA sequencing revealed significant alterations in circRNA and mRNA profiles associated with multiple biological processes and signaling pathways. Circ_0003865 expression in BMSCs was significantly decreased by MEL treatment. Silencing of circ_0003865 promoted osteogenic differentiation of BMSCs. Overexpression of circ_0003865 abrogated the promotion of BMSCs osteogenic differentiation induced by MEL. Furthermore, circ_0003865 sponged miR-3653-3p to promote GAS1 expression in BMSCs. BMSC osteogenic differentiation was enhanced by miR-3653-3p overexpression. By contrast, miR-3653-3p silencing mitigated the promoted BMSC osteogenic differentiation caused by circ_0003865 silencing. Finally, circ_0003865 silencing repressed OP development in mouse model.Conclusion: MEL promotes BMSC osteogenic differentiation and inhibits osteoporosis pathogenesis by suppressing the expression of circ_0003865, which regulates GAS1 gene expression via sponging miR-3653-3p.

Download Full-text

Bone marrow mesenchymal stem cell-derived exosomes promote rotator cuff tendon-bone healing by promoting angiogenesis and regulating M1 macrophages in rats

10.21203/rs.3.rs-52310/v2 ◽

2020 ◽

Author(s):

Yao Huang ◽

Bing He ◽

Lei Wang ◽

Bin Yuan ◽

Hao Shu ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Rotator Cuff ◽

Mesenchymal Stem Cell ◽

Bone Healing ◽

Umbilical Vein ◽

Rotator Cuff Tears ◽

M1 Macrophages ◽

Rotator Cuff Tendon ◽

Bone Interface

Abstract Background: Rotator cuff tears (RCTs) often require reconstructive surgery. Tendon-bone healing is critical for the outcome of rotator cuff reconstruction, but the process of tendon-bone healing is complex and difficult. Mesenchymal stem cells (MSCs) are considered to be an effective method to promote tendon-bone healing. MSCs have strong paracrine, anti-inflammatory, immunoregulatory, and angiogenic potential. Recent studies have shown that MSCs achieve many regulatory functions through exosomes. The purpose of this study was to explore the role of bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) in tendon-bone healing. Methods: Our study found that BMSC-Exos promote the proliferation, migration, and angiogenic tube formation of Human Umbilical Vein Endothelial Cells (HUVECs). The mechanism by which BMSC-Exos achieve this may be through the regulation of the angiogenic signaling pathway. In addition, BMSC-Exos can inhibit the polarization of M1 macrophages and inhibit the secretion of proinflammatory factors by M1 macrophages. After rotator cuff reconstruction in rats, BMSC-Exos were injected into the tail vein to analyze their effect on the rotator cuff tendon-bone interface healing. Results: It was confirmed that BMSC-Exos increased the breaking load and stiffness of the rotator cuff after reconstruction in rats, induced angiogenesis around the rotator cuff endpoint, and promoted growth of the tendon-bone interface. Conclusion: BMSC-Exos promote tendon-bone healing after rotator cuff reconstruction in rats by promoting angiogenesis and inhibiting inflammation.

Download Full-text

Quercetin promotes bone marrow mesenchymal stem cell proliferation and osteogenic differentiation through the H19/miR-625-5p axis to activate the Wnt/β-catenin pathway

BMC Complementary Medicine and Therapies ◽

10.1186/s12906-021-03418-8 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Wei Bian ◽

Shunqiang Xiao ◽

Lei Yang ◽

Jun Chen ◽

Shifang Deng

Keyword(s):

Bone Marrow ◽

Osteogenic Differentiation ◽

Protein Level ◽

Cell Counting ◽

Alizarin Red ◽

Matrix Mineralization ◽

Stem Cell Proliferation ◽

Alp Activity ◽

Marrow Mesenchymal Stem Cells ◽

Quercetin Treatment

Abstract Background Quercetin and H19 can promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). However, whether quercetin regulates H19 expression to promote osteogenic differentiation of BMSCs is unclear. Methods BMSC proliferation, matrix mineralization, and alkaline phosphatase (ALP) activity were assessed using the Cell Counting Kit-8, ALP assay kit, and alizarin red staining kit, respectively. Expression of H19, miR-625-5p, BMP-2, osteocalcin, and RUNX2 were measured by qRT-PCR; β-catenin protein level was measured by western blotting. Results Quercetin promoted BMSC proliferation, enhanced ALP activity, and upregulated the expression of BMP-2, osteocalcin, and RUNX2 mRNAs, suggesting that it promoted osteogenic differentiation of BMSCs. Moreover, quercetin increased H19 expression, while the effect of quercetin on BMSCs was reversed by silencing H19 expression. Additionally, miR-625-5p, interacted with H19, was downregulated during quercetin-induced BMSC osteogenic differentiation, which negatively correlated with H19 expression. Silencing miR-625-5p expression promoted BMSC proliferation and osteogenic differentiation, whereas miR-625-5p overexpression weakened the effect of quercetin on BMSCs. Finally, quercetin treatment or downregulation of miR-625-5p expression increased β-catenin protein level in BMSCs. Upregulation or downregulation of miR-625-5p or H19 expression, respectively, inhibited β-catenin protein level in quercetin treated-BMSCs. Conclusion H19 promotes, while miR-625-5p inhibits BMSC osteogenic differentiation. Quercetin activates the Wnt/β-catenin pathway and promotes BMSC osteogenic differentiation via the H19/miR-625-5p axis.

Download Full-text