Regulation of gene expression in tissue engineering, differentiation and 
bone regeneration of ossifying stem cells

Abstract Background The repair of critical-sized bone defects is always a challenging problem. Electromagnetic fields (EMFs), used as a physiotherapy for bone defects, have been suspected to cause potential hazards to human health due to the long-term exposure. To optimize the application of EMF while avoiding its adverse effects, a combination of EMF and tissue engineering techniques is critical. Furthermore, a deeper understanding of the mechanism of action of EMF will lead to better applications in the future. Methods In this research, bone marrow mesenchymal stem cells (BMSCs) seeded on 3D-printed scaffolds were treated with sinusoidal EMFs in vitro. Then, 5.5 mm critical-sized calvarial defects were created in rats, and the cell scaffolds were implanted into the defects. In addition, the molecular and cellular mechanisms by which EMFs regulate BMSCs were explored with various approaches to gain deeper insight into the effects of EMFs. Results The cell scaffolds treated with EMF successfully accelerated the repair of critical-sized calvarial defects. Further studies revealed that EMF could not directly induce the differentiation of BMSCs but improved the sensitivity of BMSCs to BMP signals by upregulating the quantity of specific BMP (bone morphogenetic protein) receptors. Once these receptors receive BMP signals from the surrounding milieu, a cascade of reactions is initiated to promote osteogenic differentiation via the BMP/Smad signalling pathway. Moreover, the cytokines secreted by BMSCs treated with EMF can better facilitate angiogenesis and osteoimmunomodulation which play fundamental roles in bone regeneration. Conclusion In summary, EMF can promote the osteogenic potential of BMSCs and enhance the paracrine function of BMSCs to facilitate bone regeneration. These findings highlight the profound impact of EMF on tissue engineering and provide a new strategy for the clinical treatment of bone defects.

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Micro-Computed Tomography Analysis on Administration of Mesenchymal Stem Cells - Bovine Teeth Scaffold Composites for Alveolar Bone Tissue Engineering

Journal of Biomimetics Biomaterials and Biomedical Engineering ◽

10.4028/www.scientific.net/jbbbe.52.86 ◽

2021 ◽

Vol 52 ◽

pp. 86-96

Author(s):

Desi Sandra Sari ◽

Fourier Dzar Eljabbar Latief ◽

Ferdiansyah ◽

Ketut Sudiana ◽

Fedik Abdul Rantam

Keyword(s):

Computed Tomography ◽

Tissue Engineering ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Regeneration ◽

Bone Growth ◽

Alveolar Bone ◽

Micro Computed Tomography ◽

Freeze Dried ◽

Significant Difference

The tissue engineering approach for periodontal tissue regeneration using a combination of stem cells and scaffold has been vastly developed. Mesenchymal Stem Cells (MSCs) seeded with Bovine Teeth Scaffold (BTSc) can repair alveolar bone damage in periodontitis cases. The alveolar bone regeneration process was analyzed by micro-computed tomography (µ-CT) to observe the structure of bone growth and to visualize the scaffold in 3-Dimensional (3D). The purpose of this study is to analyze alveolar bone regeneration by µ-CT following the combination of MSCs and bovine teeth scaffold (MSCs-BTSc) implantation in the Wistar rat periodontitis model. Methods. MSCs were cultured from adipose-derived mesenchymal stem cells of rats. BTSc was taken from bovine teeth and freeze-dried with a particle size of 150-355 µm. MSCs were seeded on BTSc for 24 hours and transplanted in a rat model of periodontitis. Thirty-five Wistar rats were made as periodontitis models with LPS induction from P. gingivalis injected to the buccal section of interproximal gingiva between the first and the second mandibular right-molar teeth for six weeks. There were seven groups (control group, BTSc group on day 7, BTSc group on day 14, BTSc group on day 28, MSCs-BTSc group on day 7, MSCs-BTSc group on day 14, MSCs-BTSc group on day 28). The mandibular alveolar bone was analyzed and visualized in 3D with µ-CT to observe any new bone growth. Statistical Analysis. Group data were subjected to the Kruskal Wallis test followed by the Mann-Whitney (p <0.05). The µ-CT qualitative analysis shows a fibrous structure, which indicates the existence of new bone regeneration. Quantitative analysis of the periodontitis model showed a significant difference between the control model and the model with the alveolar bone resorption (p <0.05). The bone volume and density measurements revealed that the MSCs-BTSc group on day 28 formed new bone compared to other groups (p <0.05). Administration of MSCs-BTSc combination has the potential to form new alveolar bone.

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280 MULTILINEAGE POTENTIAL OF PORCINE BONE MARROW AND ADIPOSE-DERIVED MESENCHYMAL STEM CELLS IN 3-D ALGINATE HYDROGELS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab280 ◽

2009 ◽

Vol 21 (1) ◽

pp. 237 ◽

Cited By ~ 1

Author(s):

D. Kim ◽

A. J. Maki ◽

H.-J. Kong ◽

E. Monaco ◽

M. Bionaz ◽

...

Keyword(s):

Gene Expression ◽

Tissue Engineering ◽

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Adipogenic Differentiation ◽

Cell Types ◽

Scaffold Material ◽

Over Time

Adipose tissue presents an appealing alternative to bone marrow as a source of mesenchymal stem cells (MSC). However, in order to enhance cell proliferation and differentiation, 3-dimensional (3-D) culture may be required. A 3-D culture has benefits due to its more in vivo-like environment. Further, to form a functional tissue, a scaffold material is required to ensure proper shape and allow for efficient delivery of nutrients and growth factors. Alginate, a resorbable hydrogel, is a potential injectable scaffold for fat and bone tissue engineering due to its high biocompatibility, gelation with calcium and slow dissolution in a physiologic environment. In the present study, we examined the viability, gene expression and morphology of MSC, isolated from porcine adipose (ADSC) and bone marrow (BMSC), during osteogenic and adipogenic differentiation in a 3D alginate hydrogel environment for 0, 7 and 14 days (d). ADSC and BMSC were infused into alginate hydrogels, which polymerized upon the addition of Ca+2 ions. Both stem cell types were differentiated into osteoblasts using 0.1 μm dexamethasone, 10 mm beta glycerophosphate and 50 μm ascorbic acid, whereas adipocytes were differentiated using 10 μm insulin, 1 μm dexamethasone, and 0.5 mm IBMX. Osteogenic differentiation was confirmed using alkaline phosphatase, Von Kossa, and alizarin red S staining and adipogenic differentiation was confirmed using Oil Red O. Cell viability and proliferation was quantified using the MTT assay. Gene expression was measured using qPCR. The morphology of ADSC and BMSC differentiated toward osteogenic lineages changed with both cell types forming osteogenic nodules over time. The nodules formed by ADSC were larger in diameter than those formed by BMSC. Unlike the osteogenic cells that formed nodules, the ADSC and BMSC differentiated into adipogenic cells showed no significant changes in cell size or aggregation. Gene expression results indicated increased PPARG expression in BMSC with time whereas ADSC showed a peak of expression on day 7 and then decreased. ADSC showed increased (14-fold) PPRG expression when compared with BMSC. ADSC had 160-fold less expression of ALP than BMSC. BMSC showed a 16-fold higher expression level of BGLAP than ADSC. ADSC showed a 15.8% higher expression than BMSC for COL1a1. Both ADSC and BMSC showed similar trends SPARC expression, but BMSC had a 12-fold higher expression of SPP1 than ADSC. In summary, both types of mesenchymal stem cells successfully differentiated into both lineages and maintained viability in the hydrogel over time. In conclusion, alginate is a viable scaffold material for the differentiation of mesenchymal stem cells for tissue engineering applications. These results allow for future studies using the pig as an in vivo fat and bone tissue engineering model. This research was supported by the Illinois Regenerative Medicine Institute.

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The TAZ2 domain of CBP/p300 directs acetylation towards H3K27 within chromatin

10.1101/2020.07.21.214338 ◽

2020 ◽

Author(s):

Thomas W. Sheahan ◽

Viktoria Major ◽

Kimberly M. Webb ◽

Elana Bryan ◽

Philipp Voigt

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Enzymatic Activity ◽

Crucial Role ◽

Regulation Of Gene Expression ◽

Histone H3 ◽

Embryonic Stem ◽

Site Specific ◽

Lysine Residues

AbstractThe closely related acetyltransferases CBP and p300 are key regulators of gene expression in metazoans. CBP/p300 acetylate several specific lysine residues within nucleosomes, including histone H3 lysine 27 (H3K27), a hallmark of active enhancers and promoters. However, it has remained largely unclear how specificity of CBP/p300 towards H3K27 is achieved. Here we show that the TAZ2 domain of CBP is required for efficient acetylation of H3K27, while curbing activity towards other lysine residues within nucleosomes. We find that TAZ2 is a sequence-independent DNA binding module, promoting interaction between CBP and nucleosomes, thereby enhancing enzymatic activity and regulating substrate specificity of CBP. TAZ2 is further required to stabilize CBP binding to chromatin in mouse embryonic stem cells, facilitating specificity towards H3K27 and modulating gene expression. These findings reveal a crucial role of TAZ2 in regulating H3K27ac, while highlighting the importance of correct site-specific acetylation for proper regulation of gene expression.

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Biomimetic Synthesis of Nanocrystalline Hydroxyapatite Composites: Therapeutic Potential and Effects on Bone Regeneration

International Journal of Molecular Sciences ◽

10.3390/ijms20236002 ◽

2019 ◽

Vol 20 (23) ◽

pp. 6002 ◽

Cited By ~ 6

Author(s):

Chih-Hsiang Fang ◽

Yi-Wen Lin ◽

Feng-Huei Lin ◽

Jui-Sheng Sun ◽

Yuan-Hung Chao ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Bone Regeneration ◽

Stromal Cell ◽

Alveolar Bone ◽

Therapeutic Potential ◽

Bone Defects ◽

Stromal Cell Derived Factor ◽

Biomimetic Hydroxyapatite

The development of a novel alloplastic graft with both osteoinductive and osteoconductive properties is still necessary. In this study, we tried to synthesize a biomimetic hydroxyapatite microspheres (gelatin/nano-hydroxyapatite microsphere embedded with stromal cell-derived factor-1: GHM-S) from nanocrystalline hydroxyapatites and to investigate their therapeutic potential and effects on bone regeneration. In this study, hydroxyapatite was synthesized by co-precipitation of calcium hydroxide and orthophosphoric acid to gelatin solution. The microbial transglutaminase was used as the agent to crosslink the microspheres. The morphology, characterization, and thermal gravimetric analysis of microspheres were performed. SDF-1 release profile and in vitro biocompatibility and relative osteogenic gene expression were analyzed, followed by in vivo micro-computed tomography study and histological analysis. The synthesized hydroxyapatite was found to be similar to hydroxyapatite of natural bone tissue. The stromal cell-derived factor-1 was embedded into gelatin/hydroxyapatite microsphere to form the biomimetic hydroxyapatite microsphere. The stromal cell-derived factor-1 protein could be released in a controlled manner from the biomimetic hydroxyapatite microsphere and form a concentration gradient in the culture environment to attract the migration of stem cells. Gene expression and protein expression indicated that stem cells could differentiate or develop into pre-osteoblasts. The effect of bone formation by the biomimetic hydroxyapatite microsphere was assessed by an in vivo rats’ alveolar bone defects model and confirmed by micro-CT imaging and histological examination. Our findings demonstrated that the biomimetic hydroxyapatite microsphere can enhance the alveolar bone regeneration. This design has potential be applied to other bone defects.

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Effect of epigenetic modulation on interleukin-6 (IL-6) gene expression with adjuvant selenomethionine (Se-Met) with imatinib mesylate (IM) in remission of chronic myelogenous leukemia (CML-cp) patients.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.e17022 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. e17022-e17022

Author(s):

Eugene McPherson

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Histone Deacetylase ◽

Histone Deacetylase Inhibitors ◽

Kinase Inhibitors ◽

Regulation Of Gene Expression ◽

Myelogenous Leukemia ◽

Abl Tyrosine Kinase ◽

Nfkb Activation ◽

Epigenetic Modulation

e17022 Background: Histone deacetylase (HDAC) are enzymes involved remodeling chromatin and epigenetic regulation of gene expression. Histone deacetylase inhibitors (HDACI) can reverse aberrant epigenetic changes in CML stem cells. BCR-abl tyrosine kinase inhibitors IM is effective in inducing remission in CML-cp patients but residual leukemia stem cells (LSC) may give rise to resistance and relapse. Pan-HDACI may arrest this with downregulation of IL-6, and NFkb activation when adjuvant Se-Met is combined with IM. Methods: CML-cp , CML-bc cases were presented with evidence of overexpression of BCR-abl fusion protein reported previously in elderly patients. A 91 YOF with CML-cp , subclinical hypothyroidism with elevated IL-6, CRP, sIL-2R, VEGF, NFkb, and IGF-1 was treated for seven years with adjuvant Se-Met, IM and levothyroxine ith normalization, minmal toxicity and extended trending down of IL-6, CRP,sIL-2R. Results: After seven years of adjuvant therapy with Se-Met, IM, and Levothyroxine a significant reduction of IL-6, CRP of 9.8075 down from 30.783 mg/L. Subsequuent normalization of the other pro-inflammatory cytokines were normalized. Conclusions: HDACI activity expressed in synergism of Se-Met with IM and Levothyroxine may lower the apoptotic threshold in CML-cp stem cells decreasing BCR-abl expression and rescuing quiescent progenitor cells that may prolong remission and decrease disease relapse.

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Essential Cytoplasmic Role of the Histone Lysine Methyltransferase Setdb1 in Post-Transcriptional Regulation in Embryonic Stem Cells

10.21203/rs.3.rs-145869/v1 ◽

2021 ◽

Author(s):

Roberta Rapone ◽

Laurence Del Maestro ◽

Costas Bouyioukos ◽

Sonia Albini ◽

Paola Cruz-Tapias ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Transcriptional Regulation ◽

Mrna Stability ◽

Regulation Of Gene Expression ◽

Embryonic Stem ◽

Histone Lysine ◽

Histone Lysine Methyltransferase ◽

Lysine Methyltransferase ◽

Post Transcriptional Regulation

Abstract Embryonic stem cells (ESCs) fate is regulated both at transcriptional and post-transcriptional levels. Indeed, several studies showed that, in addition to gene transcription, mRNA stability and protein synthesis are finely tuned and strongly control the ESCs pluripotency and fate changes. An increasing number of RNA-binding proteins (RBPs) involved in post-transcriptional and translational regulation of gene expression has been identified as regulators of ESC identity. The major lysine methyltransferase Setdb1 is essential for the self-renewal and viability of ESCs. Setdb1 was primarily known to methylate the lysine 9 of histone 3 (H3K9) in the nucleus, where it regulates chromatin functions. However, Setdb1 is also massively localized in the cytoplasm, including in mouse ESCs, where its role remains unknown. Here we show that the cytoplasmic Setdb1 (cSetdb1) is essential for the survival of mESCs. Functional assays further demonstrate that cSetdb1 regulates gene expression post-transcriptionally, affecting the abundance of mRNAs and the rate of newly synthetized proteins. A yeast-two-hybrid assay shows that cSetdb1 interacts with several regulators of mRNA stability and protein translation machinery, such as the ESCs-specific E3 ubiquitin ligase and mRNA silencer Trim71/Lin41. Finally, proteomic analyses reveal that cSetdb1 is required for the integrity of Trim71 complexes involved in mRNA metabolism and translation. Altogether, our data uncover the essential cytoplasmic function of a firstly supposed nuclear “histone” lysine methyltransferase, Setdb1, and provide new insights into the cytoplasmic/post-transcriptional regulation of gene expression mediated by a key epigenetic regulator.

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Comparison of PLGA/Fibrin and PLGA/Hyaluronic Acid Scaffolds for Chondrogenesis of Human Adipose-Derived Stem Cells

International Journal of Medical Laboratory ◽

10.18502/ijml.v7i2.2920 ◽

2020 ◽

Author(s):

Zeinolabedin Sharifian ◽

Batool Hashemibeni ◽

Majid Pourentezari ◽

Ali Valiani ◽

Mohammad Mardani ◽

...

Keyword(s):

Gene Expression ◽

Tissue Engineering ◽

Stem Cells ◽

Hyaluronic Acid ◽

Real Time ◽

Cartilage Regeneration ◽

Type Ii Collagen ◽

Control Group ◽

Adipose Derived Stem Cells ◽

X Gene

Background and Aims: Tissue engineering is a relatively novel field that has been intensely developing during recent years and has shown to be excessively promising when used for cartilage regeneration. Scaffolds represent important components for tissue engineering. Materials and Methods: The Poly Lactic-Co-Glycolic Acid (PLGA) impregnated with fibrin and hyaluronic acid (HA) produce hybrid scaffolds. human adipose-derived stem cells (hADSCs) were seeded in scaffolds and cultured in chondrogenic media. The viability of cells in different groups was assessed by MTT. The expression of chondrogenic related genes [Sox9, type II collagen (Col II), Aggrecan(AGG)] and type X collagen (Col X) was quantified by real-time polymerase chain reaction. Results: The results of the real-time PCR showed SOX9, AGG and Col X gene expression in the control groups being significantly lower than the other groups (p≤0.05). It also demonstrated Col II gene expression in the control group being significantly lower than the PLGA/Fibrin and PLGA/Fibrin/HA groups (p≤0.05). The Col X gene expression of cells in PLGA/HA and PLGA/Fibrin/HA groups significantly decreased in comparison with the PLGA/Fibrin group (p≤0.05). Conclusions: These conclusions indicate that administration of PLGA/ Fibrin and PLGA/HA scaffolds, particularly PLGA/Fibrin/ HA, motivates chondrogenesis in hADSCs. This can be diminished by decreasing hypertrophic markers and increasing characteristic markers of hyaline cartilage.

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Human acellular amniotic membrane: A potential osteoinductive biomaterial for bone regeneration

Journal of Biomaterials Applications ◽

10.1177/0885328217739753 ◽

2017 ◽

Vol 32 (6) ◽

pp. 754-764 ◽

Cited By ~ 12

Author(s):

Kai Tang ◽

Jiayi Wu ◽

Zekang Xiong ◽

Yanhui Ji ◽

Tingfang Sun ◽

...

Keyword(s):

Gene Expression ◽

Tissue Engineering ◽

Bone Marrow ◽

Bone Regeneration ◽

Tissue Regeneration ◽

Amniotic Membrane ◽

Matrix Material ◽

Defect Model ◽

Femoral Defect ◽

Marrow Mesenchymal Stem Cells

Human acellular amniotic membrane is an acellular, naturally extracellular matrix material with various bioactive factors, which applied in tissue engineering in clinic. Several studies have applied human acellular amniotic membrane in skin and ocular surface tissue engineering to enhance tissue regeneration. However, the application of human acellular amniotic membrane in bone tissue engineering was rarely investigated. The aim of the current study was to investigate the osteoinductivity, angiogenesis and the early molecular changes of human acellular amniotic membrane to bone regeneration. Our results showed that human acellular amniotic membrane with excellent biocompatibility was beneficial for bone marrow mesenchymal stem cells proliferation and osteogenic differentiation. In rat femoral defect model, the existence of human acellular amniotic membrane significantly improved bone regeneration in the defects. The gene expression of CXCR-4, MCP-1, OC and CatK which were connected with cells recruitment and bone remodeling, was enhanced in the defects implanted with human acellular amniotic membrane. The results of this study suggest that human acellular amniotic membrane is an osteoinductive biomaterial for bone regeneration.

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Low level laser therapy promotes bone regeneration by coupling angiogenesis and osteogenesis

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02493-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jie Bai ◽

Lijun Li ◽

Ni Kou ◽

Yuwen Bai ◽

Yaoyang Zhang ◽

...

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Bone Tissue Engineering ◽

Bone Regeneration ◽

Bone Tissue ◽

Low Level Laser Therapy ◽

Level Laser ◽

Vascularized Bone

Abstract Background Bone tissue engineering is a new concept bringing hope for the repair of large bone defects, which remains a major clinical challenge. The formation of vascularized bone is key for bone tissue engineering. Growth of specialized blood vessels termed type H is associated with bone formation. In vivo and in vitro studies have shown that low level laser therapy (LLLT) promotes angiogenesis, fracture healing, and osteogenic differentiation of stem cells by increasing reactive oxygen species (ROS). However, whether LLLT can couple angiogenesis and osteogenesis, and the underlying mechanisms during bone formation, remains largely unknown. Methods Mouse bone marrow mesenchymal stem cells (BMSCs) combined with biphasic calcium phosphate (BCP) grafts were implanted into C57BL/6 mice to evaluate the effects of LLLT on the specialized vessel subtypes and bone regeneration in vivo. Furthermore, human BMSCs and human umbilical vein endothelial cells (HUVECs) were co-cultured in vitro. The effects of LLLT on cell proliferation, angiogenesis, and osteogenesis were assessed. Results LLLT promoted the formation of blood vessels, collagen fibers, and bone tissue and also increased CD31hiEMCNhi-expressing type H vessels in mBMSC/BCP grafts implanted in mice. LLLT significantly increased both osteogenesis and angiogenesis, as well as related gene expression (HIF-1α, VEGF, TGF-β) of grafts in vivo and of co-cultured BMSCs/HUVECs in vitro. An increase or decrease of ROS induced by H2O2 or Vitamin C, respectively, resulted in an increase or decrease of HIF-1α, and a subsequent increase and decrease of VEGF and TGF-β in the co-culture system. The ROS accumulation induced by LLLT in the co-culture system was significantly decreased when HIF-1α was inhibited with DMBPA and was followed by decreased expression of VEGF and TGF-β. Conclusions LLLT enhanced vascularized bone regeneration by coupling angiogenesis and osteogenesis. ROS/HIF-1α was necessary for these effects of LLLT. LLLT triggered a ROS-dependent increase of HIF-1α, VEGF, and TGF-β and resulted in subsequent formation of type H vessels and osteogenic differentiation of mesenchymal stem cells. As ROS also was a target of HIF-1α, there may be a positive feedback loop between ROS and HIF-1α, which further amplified HIF-1α induction via the LLLT-mediated ROS increase. This study provided new insight into the effects of LLLT on vascularization and bone regeneration in bone tissue engineering.

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