scholarly journals Gene targeting of the transcription factor Mohawk in rats causes heterotopic ossification of Achilles tendon via failed tenogenesis

2016 ◽  
Vol 113 (28) ◽  
pp. 7840-7845 ◽  
Author(s):  
Hidetsugu Suzuki ◽  
Yoshiaki Ito ◽  
Masahiro Shinohara ◽  
Satoshi Yamashita ◽  
Shizuko Ichinose ◽  
...  
Keyword(s):  
Achilles Tendon ◽  
Osteogenic Differentiation ◽  
Heterotopic Ossification ◽  
Chondrogenic Differentiation ◽  
Molecular Mechanisms ◽  
Mechanical Load ◽  
Adipogenic Differentiation ◽  
Tendon Repair ◽  
Mechanical Stretch ◽  
Tendon Cells

Cell-based or pharmacological approaches for promoting tendon repair are currently not available because the molecular mechanisms of tendon development and healing are not well understood. Although analysis of knockout mice provides many critical insights, small animals such as mice have some limitations. In particular, precise physiological examination for mechanical load and the ability to obtain a sufficient number of primary tendon cells for molecular biology studies are challenging using mice. Here, we generated Mohawk (Mkx)−/− rats by using CRISPR/Cas9, which showed not only systemic hypoplasia of tendons similar to Mkx−/− mice, but also earlier heterotopic ossification of the Achilles tendon compared with Mkx−/− mice. Analysis of tendon-derived cells (TDCs) revealed that Mkx deficiency accelerated chondrogenic and osteogenic differentiation, whereas Mkx overexpression suppressed chondrogenic, osteogenic, and adipogenic differentiation. Furthermore, mechanical stretch stimulation of Mkx−/− TDCs led to chondrogenic differentiation, whereas the same stimulation in Mkx+/+ TDCs led to formation of tenocytes. ChIP-seq of Mkx overexpressing TDCs revealed significant peaks in tenogenic-related genes, such as collagen type (Col)1a1 and Col3a1, and chondrogenic differentiation-related genes, such as SRY-box (Sox)5, Sox6, and Sox9. Our results demonstrate that Mkx has a dual role, including accelerating tendon differentiation and preventing chondrogenic/osteogenic differentiation. This molecular network of Mkx provides a basis for tendon physiology and tissue engineering.

scholarly journals Macrophages in heterotopic ossification: from mechanisms to therapy

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Yifei Huang ◽  
Xinyi Wang ◽  
Daixuan Zhou ◽  
Wenwen Zhou ◽  
Fengyi Dai ◽  
...  
Keyword(s):  
Growth Factor ◽  
Osteogenic Differentiation ◽  
Heterotopic Ossification ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
Oncostatin M ◽  
Sequence Of Events ◽  
Mediators Of Inflammation

AbstractHeterotopic ossification (HO) is the formation of extraskeletal bone in non-osseous tissues. It is caused by an injury that stimulates abnormal tissue healing and regeneration, and inflammation is involved in this process. It is worth noting that macrophages are crucial mediators of inflammation. In this regard, abundant macrophages are recruited to the HO site and contribute to HO progression. Macrophages can acquire different functional phenotypes and promote mesenchymal stem cell (MSC) osteogenic differentiation, chondrogenic differentiation, and angiogenesis by expressing cytokines and other factors such as the transforming growth factor-β1 (TGF-β1), bone morphogenetic protein (BMP), activin A (Act A), oncostatin M (OSM), substance P (SP), neurotrophin-3 (NT-3), and vascular endothelial growth factor (VEGF). In addition, macrophages significantly contribute to the hypoxic microenvironment, which primarily drives HO progression. Thus, these have led to an interest in the role of macrophages in HO by exploring whether HO is a “butterfly effect” event. Heterogeneous macrophages are regarded as the “butterflies” that drive a sequence of events and ultimately promote HO. In this review, we discuss how the recruitment of macrophages contributes to HO progression. In particular, we review the molecular mechanisms through which macrophages participate in MSC osteogenic differentiation, angiogenesis, and the hypoxic microenvironment. Understanding the diverse role of macrophages may unveil potential targets for the prevention and treatment of HO.

scholarly journals Mechanical stimulation regulates differentiation of bone marrow stem cells by modulating miR-140-5p via TGFβ1/Smad2 signaling pathway

2020 ◽  
Author(s):  
Yuepeng Qian ◽  
Guozheng Zhu ◽  
Zelin Ye ◽  
Canjun Zeng ◽  
Runguang Li
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Mechanical Stimulation ◽  
Adipogenic Differentiation ◽  
Mechanical Stretch ◽  
Culture Conditions ◽  
Sprague Dawley ◽  
Effective Strategies ◽  
Patient Health

Abstract Background: Osteoporosis is a common progressive bone disease that drastically impairs patient health, independent mobility, and quality of life, and there is an urgent need for improved preventive and therapeutic strategies. A shift in bone marrow mesenchymal stem cell (BMSC) differentiation from osteogenic to adipogenic may contribute to disease pathogenesis. Mechanical stress on BMSCs is reported to promote osteogenesis, so we examined the effects of mechanical stimulation on BMSC differentiation and associated signaling pathways.Methods: A sinusoidal tensile stress loading device was developed and examined effects of mechanical (stretch) stimulation on cultured BMSC (isolated from Sprague-Dawley rats) phenotype under osteogenic and adipogenic culture conditions. Osteogenic differentiation of BMSCs was assessed by alkaline phosphatase (ALP) staining and expression of Runx2 and BMP2, while adipogenic differentiation was evaluated by oil red O staining and expression of PPARγ and C/EBPα.Results: It demonstrated that appropriate mechanical stimulation could promote osteogenic differentiation of BMSCs and inhibit differentiation into adipocytes. The mechanic stimuli could inhibit the expression of miR-140-5P in BMSCs, and the overexpression of miR-140-5P inhibited the osteogenic differentiation, whereas the inhibition of miR-140-5P promoted the osteogenic differentiation. Further, both mechanical stimulation and miR-140-5p knockdown promoted TGFβ1/Smad2 signaling, while miR-140-5p overexpression downregulated TGFβ1/ Smad2 signaling.Conclusions: Appropriate mechanical stimulation promoted osteogenic differentiation and inhibited the adipogenic differentiation of BMSCs by lowering miR-140-5p expression, which in turn upregulates the TGFβ1/Smad2 signaling pathway. Our results provide a foundation for the development of effective strategies to promote bone remodeling, thereby lowering the burden of osteoporosis.

scholarly journals Zonal Characterization and Differential Trilineage Potentials of Equine Intrasynovial Deep Digital Flexor Tendon-Derived Cells

2020 ◽  
Author(s):  
Vivian G Quam ◽  
Nadine N Altmann ◽  
Matthew T Brokken ◽  
Sushmitha Durgam
Keyword(s):  
Chondrogenic Differentiation ◽  
Flexor Tendon ◽  
Adipogenic Differentiation ◽  
Tendon Repair ◽  
Cell Surface Antigens ◽  
In Vitro Proliferation ◽  
Repair Capacity ◽  
Tendon Regeneration ◽  
Poor Outcomes

Abstract Background: Intrasynovial deep digital flexor tendon (DDFT) injury is a common cause for forelimb lameness in horses and are typically associated with poor outcomes. Intrasynovial tendon contains a superficial fibrocartilage in response to compressive forces from an opposing boney prominence, and is critical for tendon gliding function. Characterization of tendon-derived cells (TDC) from intrasynovial tendon fibrocartilage has not been conducted as for prototypical extrasynovial tendon and is necessary for developing improved therapeutic strategies. Results: In this study, we successfully isolated homogenous TDC from the fibrocartilaginous (fTDC) and tendinous (tTDC) zones of equine forelimb intrasynovial DDFT via low-density plating method. During monolayer passage, both TDC subpopulations exhibited clonogenicity, high in vitro proliferation rate, and fibroblast-like morphology. Third passage fTDC and tTDC were positive for cell surface antigens CD90 and CD29 and negative for CD44 and CD45. There were no significant differences in the basal tenogenic, osteogenic and chondrogenic marker expressions of fTDC and tTDC. Trilineage differentiation demonstrated that fTDC were largely restricted to chondrogenic differentiation; whereas, those from the tendinous zone underwent osteogenic and chondrogenic differentiation. Both TDC subpopulations displayed weak adipogenic differentiation potentials. Conclusions: These results provide a foundation for studies exploring cell-based therapies for intrasynovial tendon repair as these TDC are potential targets to enhance intrinsic repair capacity. Pending further investigation, promoting chondrogenic properties in cells administered exogenously into the intrasynovial space may be beneficial for intrasynovial tendon regeneration.

scholarly journals Physical Exercise Modulates miR-21-5p, miR-129-5p, miR-378-5p, and miR-188-5p Expression in Progenitor Cells Promoting Osteogenesis

Cells ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 742 ◽  
Author(s):  
Maria Teresa Valenti ◽  
Michela Deiana ◽  
Samuele Cheri ◽  
Monica Dotta ◽  
Francesco Zamboni ◽  
...  
Keyword(s):  
Physical Exercise ◽  
Osteogenic Differentiation ◽  
Progenitor Cells ◽  
Molecular Mechanisms ◽  
Adipogenic Differentiation ◽  
Marathon Runners ◽  
Beneficial Effects ◽  
Protein Levels ◽  
Smad4 Protein ◽  
Smad7 Expression

Physical exercise is known to promote beneficial effects on overall health, counteracting risks related to degenerative diseases. MicroRNAs (miRNAs), short non-coding RNAs affecting the expression of a cell’s transcriptome, can be modulated by different stimuli. Yet, the molecular effects on osteogenic differentiation triggered by miRNAs upon physical exercise are not completely understood. In this study, we recruited 20 male amateur runners participating in a half marathon. Runners’ sera, collected before (PRE RUN) and after (POST RUN) the run, were added to cultured human mesenchymal stromal cells. We then investigated their effects on the modulation of selected miRNAs and the consequential effects on osteogenic differentiation. Our results showed an increased expression of miRNAs promoting osteogenic differentiation (miR-21-5p, miR-129-5p, and miR-378-5p) and a reduced expression of miRNAs involved in the adipogenic differentiation of progenitor cells (miR-188-5p). In addition, we observed the downregulation of PTEN and SMAD7 expression along with increased AKT/pAKT and SMAD4 protein levels in MSCs treated with POST RUN sera. The consequent upregulation of RUNX2 expression was also proven, highlighting the molecular mechanisms by which miR-21-5p promotes osteogenic differentiation. In conclusion, our work proposes novel data, which demonstrate how miRNAs may regulate the osteogenic commitment of progenitor cells in response to physical exercise.

scholarly journals New Insights into Osteogenic and Chondrogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells and Their Potential Clinical Applications for Bone Regeneration in Pediatric Orthopaedics

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Nicola Giuliani ◽  
Gina Lisignoli ◽  
Marina Magnani ◽  
Costantina Racano ◽  
Marina Bolzoni ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Transcription Factor ◽  
Bone Regeneration ◽  
Osteogenic Differentiation ◽  
Chondrogenic Differentiation ◽  
Adult Stem Cells ◽  
Molecular Mechanisms ◽  
Human Mesenchymal Stem Cells ◽  
Signal Pathways

Human mesenchymal stem cells (hMSCs) are pluripotent adult stem cells capable of being differentiated into osteoblasts, adipocytes, and chondrocytes. The osteogenic differentiation of hMSCs is regulated either by systemic hormones or by local growth factors able to induce specific intracellular signal pathways that modify the expression and activity of several transcription factors. Runt-related transcription factor 2 (Runx2) and Wnt signaling-related molecules are the major factors critically involved in the osteogenic differentiation process by hMSCs, and SRY-related high-mobility-group (HMG) box transcription factor 9 (SOX9) is involved in the chondrogenic one. hMSCs have generated a great interest in the field of regenerative medicine, particularly in bone regeneration. In this paper, we focused our attention on the molecular mechanisms involved in osteogenic and chondrogenic differentiation of hMSC, and the potential clinical use of hMSCs in osteoarticular pediatric disease characterized by fracture nonunion and pseudarthrosis.

scholarly journals Expression Profile of New Marker Genes Involved in Differentiation of Canine Adipose-Derived Stem Cells into Osteoblasts

2021 ◽  
Vol 22 (13) ◽  
pp. 6663
Author(s):  
Maurycy Jankowski ◽  
Mariusz Kaczmarek ◽  
Grzegorz Wąsiatycz ◽  
Claudia Dompe ◽  
Paul Mozdziak ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Osteogenic Differentiation ◽  
Molecular Mechanisms ◽  
Marker Genes ◽  
P Value ◽  
Illumina Platform

Next-generation sequencing (RNAseq) analysis of gene expression changes during the long-term in vitro culture and osteogenic differentiation of ASCs remains to be important, as the analysis provides important clues toward employing stem cells as a therapeutic intervention. In this study, the cells were isolated from adipose tissue obtained during routine surgical procedures and subjected to 14-day in vitro culture and differentiation. The mRNA transcript levels were evaluated using the Illumina platform, resulting in the detection of 19,856 gene transcripts. The most differentially expressed genes (fold change >|2|, adjusted p value < 0.05), between day 1, day 14 and differentiated cell cultures were extracted and subjected to bioinformatical analysis based on the R programming language. The results of this study provide molecular insight into the processes that occur during long-term in vitro culture and osteogenic differentiation of ASCs, allowing the re-evaluation of the roles of some genes in MSC progression towards a range of lineages. The results improve the knowledge of the molecular mechanisms associated with long-term in vitro culture and differentiation of ASCs, as well as providing a point of reference for potential in vivo and clinical studies regarding these cells’ application in regenerative medicine.

scholarly journals Circular RNA AFF4 modulates osteogenic differentiation in BM-MSCs by activating SMAD1/5 pathway through miR-135a-5p/FNDC5/Irisin axis

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Chao Liu ◽  
An-Song Liu ◽  
Da Zhong ◽  
Cheng-Gong Wang ◽  
Mi Yu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Formation ◽  
Osteogenic Differentiation ◽  
Molecular Mechanisms ◽  
Expression Patterns ◽  
Regulatory System ◽  
Circular Rna ◽  
Luciferase Reporter ◽  
Integrin Αv

AbstractBone marrow-derived mesenchymal stem cells (BM-MSCs), the common progenitor cells of adipocytes and osteoblasts, have been recognized as the key mediator during bone formation. Herein, our study aim to investigate molecular mechanisms underlying circular RNA (circRNA) AFF4 (circ_AFF4)-regulated BM-MSCs osteogenesis. BM-MSCs were characterized by FACS, ARS, and ALP staining. Expression patterns of circ_AFF4, miR-135a-5p, FNDC5/Irisin, SMAD1/5, and osteogenesis markers, including ALP, BMP4, RUNX2, Spp1, and Colla1 were detected by qRT-PCR, western blot, or immunofluorescence staining, respectively. Interactions between circ_AFF4 and miR-135a-5p, FNDC5, and miR-135a-5p were analyzed using web tools including TargetScan, miRanda, and miRDB, and further confirmed by luciferase reporter assay and RNA pull-down. Complex formation between Irisin and Integrin αV was verified by Co-immunoprecipitation. To further verify the functional role of circ_AFF4 in vivo during bone formation, we conducted animal experiments harboring circ_AFF4 knockdown, and born samples were evaluated by immunohistochemistry, hematoxylin and eosin, and Masson staining. Circ_AFF4 was upregulated upon osteogenic differentiation induction in BM-MSCs, and miR-135a-5p expression declined as differentiation proceeds. Circ_AFF4 knockdown significantly inhibited osteogenesis potential in BM-MSCs. Circ_AFF4 stimulated FNDC5/Irisin expression through complementary binding to its downstream target molecule miR-135a-5p. Irisin formed an intermolecular complex with Integrin αV and activated the SMAD1/5 pathway during osteogenic differentiation. Our work revealed that circ_AFF4, acting as a sponge of miR-135a-5p, triggers the promotion of FNDC5/Irisin via activating the SMAD1/5 pathway to induce osteogenic differentiation in BM-MSCs. These findings gained a deeper insight into the circRNA-miRNA regulatory system in the bone marrow microenvironment and may improve our understanding of bone formation-related diseases at physiological and pathological levels.

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