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Roles of SATB2 in osteogenic differentiation and bone regeneration.

ABSTRACT: Expressed in branchial arches and osteoblast-lineage cells, special AT-rich sequence-binding protein (SATB2) is responsible for preventing craniofacial abnormalities and defects in osteoblast function. In this study, we transduced SATB2 into murine adult stem cells, and found that SATB2 significantly increased expression levels of bone matrix proteins, osteogenic transcription factors, and a potent angiogenic factor, vascular endothelial growth factor. Using an osterix (Osx) promoter-luciferase construct and calvarial cells isolated from runt-related transcription factor 2 (Runx2)-deficient mice, we found that SATB2 upregulates Osx expression independent of Runx2, but synergistically enhances the regulatory effect of Runx2 on Osx promoter. We then transplanted SATB2-overexpressing adult stem cells genetically double-labeled with bone sialoprotein (BSP) promoter-driven luciferase and ?-actin promoter-driven enhanced green fluorescent protein into mandibular bone defects. We identified increased luciferase-positive cells in SATB2-overexpressing groups, indicating more transplanted cells undergoing osteogenic differentiation. New bone formation was consequently accelerated in SATB2 groups. In conclusion, SATB2 acts as a potent transcription factor to enhance osteoblastogenesis and promote bone regeneration. The application of SATB2 in bone tissue engineering gives rise to a higher bone forming capacity as a result of multiple-level amplification of regulatory activity.

SUBMITTER: Zhang J

PROVIDER: S-EPMC3118613 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Roles of SATB2 in osteogenic differentiation and bone regeneration.

Zhang Jin J Tu Qisheng Q Grosschedl Rudolf R Kim Min Seok MS Griffin Terrence T Drissi Hicham H Yang Pishan P Chen Jake J

Tissue engineering. Part A 20110421 13-14

Expressed in branchial arches and osteoblast-lineage cells, special AT-rich sequence-binding protein (SATB2) is responsible for preventing craniofacial abnormalities and defects in osteoblast function. In this study, we transduced SATB2 into murine adult stem cells, and found that SATB2 significantly increased expression levels of bone matrix proteins, osteogenic transcription factors, and a potent angiogenic factor, vascular endothelial growth factor. Using an osterix (Osx) promoter-luciferase ...[more]

PMID: 21385070

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Project description:BACKGROUND:The proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMScs) are modulated by a variety of microRNAs (miRNAs). SATB homeobox 2 (SATB2) is a critical transcription factor that contributes to maintain the balance of bone metabolism. However, it remains unclear how the regulatory relationship between miR-103 and SATB2 on HBMScs proliferation and osteogenic differentiation. METHODS:HBMScs were obtained from Cyagen Biosciences and successful induced osteogenic differentiation. The proliferation abilities of HBMScs after treatment with agomiR-103 and antagomiR-103 were assessed using a cell counting Kit-8 (CCK-8) assay, and osteogenic differentiation was determined using alizarin red S staining and alkaline phosphatase (ALP) activity assay. The expression levels of miR-103, SATB2, and associated osteogenic differentiation biomarkers, including RUNX family transcription factor 2 (RUNX2), bone gamma-carboxyglutamate protein (BGLAP), and secreted phosphoprotein 1 (SPP1), were evaluated using real-time qPCR and Western blot. The regulatory sites of miR-103 on SATB2 were predicted using bioinformatics software and validated using a dual luciferase reporter assay. The underlying mechanism of miR-103 on SATB2-medicated HBMScs proliferation and osteogenic differentiation were confirmed by co-transfection of antagomiR-103 and SATB2 siRNA. RESULTS:The expression of miR-103 in HBMScs after induction of osteogenic differentiation was reduced in a time-dependent way. Overexpression of miR-103 by transfection of agomiR-103 suppressed HBMScs proliferation and osteogenic differentiation, while silencing of miR-103 by antagomiR-103 abolished these inhibitory effects. Consistently, RUNX2, BGLAP and SPP1 mRNA and protein expression were decreased in agomiR-103 treated HBMScs compared with those in agomiR-NC group. Meanwhile, antagomiR-103 upregulated the mRNA and protein expression levels of RUNX2, BGLAP and SPP1 in HBMScs. Further studies revealed that SATB2 was a direct target gene of miR-103. BMSCs transfected with agomiR-103 exhibited significantly downregulated protein expression level of SATB2, whereas knockdown of miR-103 promoted it. Additionally, rescue assays confirmed that silencing of SATB2 partially reversed the effects of antagomiR-103 induced HBMScs proliferation and osteogenic differentiation. CONCLUSIONS:The present results suggested that miR-103 negatively regulates SATB2 to serve an inhibitory role in the proliferation and osteogenic differentiation of HBMScs, which sheds light upon a potential therapeutic target for treating bone-related diseases.

Project description:BackgroundThe selection of appropriate scaffold plays an important role in ensuring the success of bone regeneration. The use of scaffolds with different materials and their effect on the osteogenic performance of cells is not well studied and this can affect the selection of suitable scaffolds for transplantation. Hence, this study aimed to investigate the comparative ability of two different synthetic scaffolds, mainly hydroxyapatite (HA) and polycaprolactone (PCL) scaffolds in promoting in vitro and in vivo bone regeneration.MethodIn vitro cell viability, morphology, and alkaline phosphatase (ALP) activity of MC3T3-E1 cells on HA and PCL scaffolds were determined in comparison to the accepted model outlined for two-dimensional systems. An in vivo study involving the transplantation of MC3T3-E1 cells with scaffolds into an artificial bone defect of 4 mm length and 1.5 mm depth in the rat's left maxilla was conducted. Three-dimensional analysis using micro-computed tomography (micro-CT), hematoxylin and eosin (H&E), and immunohistochemistry analyses evaluation were performed after six weeks of transplantation.ResultsMC3T3-E1 cells on the HA scaffold showed the highest cell viability. The cell viability on both scaffolds decreased after 14 days of culture, which reflects the dominant occurrence of osteoblast differentiation. An early sign of osteoblast differentiation can be detected on the PCL scaffold. However, cells on the HA scaffold showed more prominent results with intense mineralized nodules and significantly (p < 0.05) high levels of ALP activity with prolonged osteoblast induction. Micro-CT and H&E analyses confirmed the in vitro results with bone formation were significantly (p < 0.05) greater in HA scaffold and was supported by IHC analysis which confirmed stronger expression of osteogenic markers ALP and osteocalcin.ConclusionDifferent scaffold materials of HA and PCL might have influenced the bone regeneration ability of MC3T3-E1. Regardless, in vitro and in vivo bone regeneration was better in the HA scaffold which indicates its great potential for application in bone regeneration.

Project description:BackgroundThere is growing evidence that extracellular vesicles (EVs) play a crucial role in the paracrine mechanisms of transplanted human mesenchymal stem cells (hMSCs). Little is known, however, about the influence of microenvironmental stimuli on the osteogenic effects of EVs. This study aimed to investigate the properties and functions of EVs derived from undifferentiated hMSC (Naïve-EVs) and hMSC during the early stage of osteogenesis (Osteo-EVs). A further aim was to assess the osteoinductive potential of Osteo-EVs for bone regeneration in rat calvarial defects.MethodsEVs from both groups were isolated using size-exclusion chromatography and characterized by size distribution, morphology, flow cytometry analysis and proteome profiling. The effects of EVs (10 µg/ml) on the proliferation, migration, and osteogenic differentiation of cultured hMSC were evaluated. Osteo-EVs (50 µg) or serum-free medium (SFM, control) were combined with collagen membrane scaffold (MEM) to repair critical-sized calvarial bone defects in male Lewis rats and the efficacy was assessed using µCT, histology and histomorphometry.ResultsAlthough Osteo- and Naïve-EVs have similar characteristics, proteomic analysis revealed an enrichment of bone-related proteins in Osteo-EVs. Both groups enhance cultured hMSC proliferation and migration, but Osteo-EVs demonstrate greater efficacy in promoting in vitro osteogenic differentiation, as evidenced by increased expression of osteogenesis-related genes, and higher calcium deposition. In rat calvarial defects, MEM with Osteo-EVs led to greater and more consistent bone regeneration than MEM loaded with SFM.ConclusionsThis study discloses differences in the protein profile and functional effects of EVs obtained from naïve hMSC and hMSC during the early stage of osteogenesis, using different methods. The significant protein profile and cellular function of EVs derived from hMSC during the early stage of osteogenesis were further verified by a calvarial bone defect model, emphasizing the importance of using differentiated MSC to produce EVs for bone therapeutics.

Project description:Deficient bone regeneration causes bone defects or nonunion in a substantial proportion of trauma patients that urges for novel therapies. To develop a reliable therapy, we investigated the effect of negative pressure wound therapy (NPWT) on bone regeneration in vivo in a rat calvarial defect model. Negative pressure (NP) treatment in vitro was mimicked to test its effect on osteoblast differentiation in rat mesenchymal stem cells (MSCs) and MC3T3-E1 cells. Transcriptomic analyses, pharmaceutical interventions, and shRNA knockdowns were conducted to explore the underlying mechanism and their clinical relevance was investigated in samples from patients with nonunion. The potential application of a combined therapy of MSCs in hydrogels with negative pressure was tested in the rat critical-size calvarial defect model. We found that NPWT promoted bone regeneration in vivo and NP treatment induced osteoblast differentiation in vitro. NP induced osteogenesis via activating macroautophagy/autophagy by AMPK-ULK1 signaling that was impaired in clinical samples from patients with nonunion. More importantly, the combined therapy involving MSCs in hydrogels with negative pressure significantly improved bone regeneration in rat critical-size calvarial defect model. Thus, our study identifies a novel AMPK-ULK1-autophagy axis by which negative pressure promotes osteoblast differentiation of MSCs and bone regeneration. NPWT treatment can potentially be adopted for therapy of bone defects.Abbreviations: ADP, adenosine diphosphate; AICAR/Aic, acadesine; ALP, alkaline phosphatase; ALPL, alkaline phosphatase, biomineralization associated; AMP, adenosine monophosphate; AMPK, AMP-activated protein kinase; ARS, alizarin red S staining; ATG7, autophagy related 7; ATP, adenosine triphosphate; BA1, bafilomycin A1; BGLAP/OCN, bone gamma-carboxyglutamate protein; BL, BL-918; BS, bone surface; BS/TV, bone surface per tissue volume; BV/TV, bone volume per tissue volume; C.C, compound C; CCN1, cellular communication network factor 1; COL1A1, collagen type I alpha 1 chain; COL4A3, collagen type IV alpha 3 chain; COL4A4, collagen type IV alpha 4 chain; COL18A1, collagen type XVIII alpha 1 chain; CQ, chloroquine; GelMA, gelatin methacryloyl hydrogel; GO, Gene Ontology; GSEA, gene set enrichment analysis; HIF1A, hypoxia inducible factor 1 subunit alpha; HPLC, high-performance liquid chromatography; ITGAM/CD11B, integrin subunit alpha M; ITGAX/CD11C, integrin subunit alpha X; ITGB1/CdD9, integrin subunit beta 1; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; micro-CT, microcomputed tomography; MSCs, mesenchymal stem cells; MTOR, mechanistic target of rapamycin kinase; NP, negative pressure; NPWT, negative pressure wound therapy; PRKAA1/AMPKα1, protein kinase AMP-activated catalytic subunit alpha 1; PRKAA2, protein kinase AMP-activated catalytic subunit alpha 2; PTPRC/CD45, protein tyrosine phosphatase receptor type C; ROS, reactive oxygen species; RUNX2, RUNX family transcription factor 2; SBI, SBI-0206965; SPP1/OPN, secreted phosphoprotein 1; THY1/CD90, Thy-1 cell surface antigen; SQSTM1, sequestosome 1; TGFB3, transforming growth factor beta 3; ULK1/Atg1, unc-51 like autophagy activating kinase 1.

Project description:BackgroundMesenchymal stem cells (MSCs) are known to have different differentiation potential depending on the tissue of origin. Dedifferentiated fat cells (DFATs) are MSC-like multipotent cells that can be prepared from mature adipocytes by ceiling culture method. It is still unknown whether DFATs derived from adipocytes in different tissue showed different phenotype and functional properties. In the present study, we prepared bone marrow (BM)-derived DFATs (BM-DFATs), BM-MSCs, subcutaneous (SC) adipose tissue-derived DFATs (SC-DFATs), and adipose tissue-derived stem cells (ASCs) from donor-matched tissue samples. Then, we compared their phenotypes and multilineage differentiation potential in vitro. We also evaluated in vivo bone regeneration ability of these cells using a mouse femoral fracture model.MethodsBM-DFATs, SC-DFATs, BM-MSCs, and ASCs were prepared from tissue samples of knee osteoarthritis patients who received total knee arthroplasty. Cell surface antigens, gene expression profile, and in vitro differentiation capacity of these cells were determined. In vivo bone regenerative ability of these cells was evaluated by micro-computed tomography imaging at 28 days after local injection of the cells with peptide hydrogel (PHG) in the femoral fracture model in severe combined immunodeficiency mice.ResultsBM-DFATs were successfully generated at similar efficiency as SC-DFATs. Cell surface antigen and gene expression profiles of BM-DFATs were similar to those of BM-MSCs, whereas these profiles of SC-DFATs were similar to those of ASCs. In vitro differentiation analysis revealed that BM-DFATs and BM-MSCs had higher differentiation tendency toward osteoblasts and lower differentiation tendency toward adipocytes compared to SC-DFATs and ASCs. Transplantation of BM-DFATs and BM-MSCs with PHG enhanced bone mineral density at the injection sites compared to PHG alone in the mouse femoral fracture model.ConclusionsWe showed that phenotypic characteristics of BM-DFATs were similar to those of BM-MSCs. BM-DFATs exhibited higher osteogenic differentiation potential and bone regenerative ability compared to SC-DFATs and ASCs. These results suggest that BM-DFATs may be suitable sources of cell-based therapies for patients with nonunion bone fracture.

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Roles of SATB2 in osteogenic differentiation and bone regeneration.

Publications

Roles of SATB2 in osteogenic differentiation and bone regeneration.

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