Three-Dimensional Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells Promotes Matrix Metallopeptidase 13 (MMP13) Expression in Type I Collagen Hydrogels

Autologous bone transplantation is the principal method for reconstruction of large bone defects. This technique has limitations, such as donor site availability, amount of bone needed and morbidity. An alternative to this technique is tissue engineering with bone marrow-derived mesenchymal stem cells (BMSCs). In this study, our aim was to elucidate the benefits of culturing BMSCs in 3D compared with the traditional 2D culture. In an initial screening, we combined BMSCs with four different biogels: unmodified type I collagen (Col I), type I collagen methacrylate (ColMa), an alginate and cellulose-based bioink (CELLINK) and a gelatin-based bioink containing xanthan gum (GelXA-bone). Col I was the best for structural integrity and maintenance of cell morphology. Osteogenic, adipogenic, and chondrogenic differentiations of the BMSCs in 2D versus 3D type I collagen gels were investigated. While the traditional pellet culture for chondrogenesis was superior to our tested 3D culture, Col I hydrogels (i.e., 3D) favored adipogenic and osteogenic differentiation. Further focus of this study on osteogenesis were conducted by comparing 2D and 3D differentiated BMSCs with Osteoimage® (stains hydroxyapatite), von Kossa (stains anionic portion of phosphates, carbonates, and other salts) and Alizarin Red (stains Ca2+ deposits). Multivariate gene analysis with various covariates showed low variability among donors, successful osteogenic differentiation, and the identification of one gene (matrix metallopeptidase 13, MMP13) significantly differentially expressed in 2D vs. 3D cultures. MMP13 protein expression was confirmed with immunohistochemistry. In conclusion, this study shows evidence for the suitability of type I collagen gels for 3D osteogenic differentiation of BMSCs, which might improve the production of tissue-engineered constructs for treatment of bone defects.

Download Full-text

Effect of Hepatic Kinase B1 (LKB1) on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells During Senescence

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2020.2247 ◽

2020 ◽

Vol 10 (2) ◽

pp. 246-251

Author(s):

Wenxiao Jiang ◽

Yijun Zhang ◽

Ye Huang ◽

Yunfeng Cheng ◽

Zhigang Liu

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Type I Collagen ◽

Mtor Pathway ◽

Nodule Formation ◽

Type I ◽

Alp Activity ◽

Marrow Mesenchymal Stem Cells

Hepatic kinase B1 (LKB1) is a tumor suppressor and regulates cell proliferation and apoptosis. However, whether LKB1 affects bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation of during aging remains unclear. Two BMSCs derived from Zempster24−/− (aging) and Zempster24+/+ (normal) mice were cultured in vitro followed by measurement of LKB1 expression by real-time quantitative PCR and Western blot. LKB1 siRNA was transfected into Zempster24−/−BMSCs and LKB1 expression was measured. 14 days after osteogenic induction, mineralized nodule formation was evaluated by alizarin red staining, expression of Calcin, type I collagen, RUNX2 and OPN mRNA expression was measured, together with alkaline phosphatase (ALP) activity and the PI3K/mTOR pathway activity. Compared with normal BMSCs, LKB1 expression was significantly increased, calcified nodules were decreased, with reduced expression of osteocalcin, type I collagen, RUNX2 and OPN mRNA as well as decreased ALP activity and PI3K/mTOR signaling protein expression (P < 0.05). LKB1 siRNA transfection into senescent BMSCs down-regulated LKB1 expression, increased calcification nodule formation, expression of osteocalcin, type I collagen, RUNX2 and OPN mRNA, as well as increased ALP activity and PI3K/mTOR pathway protein expression (P < 0.05). Aging can promote the increase of LKB1 expression and inhibit the osteogenic differentiation of BMSCs. Down-regulation of LKB1 expression in BMSCs during senescence can promote osteogenic differentiation through regulating PI3K/mTOR pathway.

Download Full-text

Distal Low Homeobox 3 Promotes Bone Marrow Mesenchymal Stem Cells Osteogenic Differentiation and Bone Synthesis and Ameliorates Osteoporosis by Activating Typical Wnt Signaling

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2019.2209 ◽

2019 ◽

Vol 9 (12) ◽

pp. 1776-1782

Author(s):

Yongyi Xu ◽

Lei Chen

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Bone Density ◽

Osteogenic Differentiation ◽

Caspase 3 ◽

Type I Collagen ◽

Type I ◽

Alp Activity ◽

Marrow Mesenchymal Stem Cells

The distal low homeobox 3 (DLX3) regulates the bone marrow mesenchymal stem cells (BMSC) osteogenic differentiation. However, whether DLX3 affects osteoporosis (OP) remains unclear. An OVX-induced OP rat model was constructed and DLX3 plasmid was injected followed by analysis of bone mineral density and ALP activity. Rat BMSCs were isolated and divided into control group, OP group and DLX3 group (transfected with DLX3 plasmid) followed by analysis of chondrocytes survival rate by MTT assay, Caspase 3 activity, type I collagen and Osterix expression by Real time PCR and -catenin level by Western blot. DLX3 expression was significantly down-regulated in OP rats with deceased bone density and ALP activity compared to sham group (P < 0 05). When DLX3 was transfected into OP rats, DLX3 expression was significantly up-regulated with increased bone density and ALP activity compared with OP group (P < 0 05). BMSCs survival was significantly decreased in OP group and Caspase 3 activity was significantly increased with downregulated type I collagen, Osterix and -catenin (P < 0 05). However, transfection of DLX3 plasmid into OP group BMSCs cells can significantly reverse the above changes, compared to OP group (P < 0 05). DLX3 expression is reduced in osteoporosis. Up-regulation of DLX3 can promote osteogenic differentiation of BMSCs by regulating typical Wnt signaling, promote differentiation into osteoblasts, increase bone density increase, and then ameliorate osteoporosis.

Download Full-text

miRNA-126 Inhibits Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells (BMSCs)

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2022.2943 ◽

2022 ◽

Vol 12 (4) ◽

pp. 794-799

Author(s):

Le Chang ◽

Wei Duan ◽

Chuang Wang ◽

Jian Zhang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Mrna Level ◽

Alizarin Red ◽

Runx2 Expression ◽

Alp Activity ◽

Smad4 Protein ◽

Marrow Mesenchymal Stem Cells

This study was to determine whether microRNA (miRNA)-126 regulates osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Rat BMSCs were extracted and stimulated for osteogenic differentiation. Functional experiments were conducted to assess miR-126’s impact on BMSCs differentiation. Western blot and RT-qPCR determined miR-126 expression. ALP activity detection and alizarin red staining detection were also performed. After osteogenic differentiation of BMSCs, miR-126 expression was gradually decreased over time. Overexpression of miR-26 decreased ALP activity, Notch signaling activity as well as declined Runx2 expression and calcium Salt nodules after treatment. Importantly, we found that Smad4 serves as a target of miR-126 while upregulation of the miRNA was accompanied with the decreased Smad4 protein expression without affecting the Smad4 mRNA level. In conclusion, miR-126 restrains osteogenic differentiation through inhibition of SMAD4 signaling, providing a novel insight into the mechanism.

Download Full-text

APPLICATION OF DENDRIMER/PLASMID hBMP-2 COMPLEXES LOADED INTO β-TCP/COLLAGEN SCAFFOLD IN THE TREATMENT OF FEMORAL DEFECTS IN RATS

Biomedical Engineering Applications Basis and Communications ◽

10.4015/s1016237214500057 ◽

2014 ◽

Vol 26 (01) ◽

pp. 1450005 ◽

Cited By ~ 1

Author(s):

Tingwei Bao ◽

Huiming Wang ◽

Wentao Zhang ◽

Xuefeng Xia ◽

Jiabei Zhou ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Type I Collagen ◽

Cell Attachment ◽

Collagen Scaffold ◽

Bone Defects ◽

Bone Morphogenetic Protein 2 ◽

Porous Scaffolds ◽

Type I

Purpose: Plasmid loading into scaffolds to enhance sustained release of growth factors is an important focus of regenerative medicine. The aim of this study was to build gene-activated matrices (GAMs) and examine the bone augmentation properties. Methods: Generation 5 polyamidoamine dendrimers (G5 dPAMAM)/plasmid recombinant human bone morphogenetic protein-2 (rhBMP-2) complexes were immobilized into beta-tricalcium phosphate (β-TCP)/type I collagen porous scaffolds. After cultured with rat mesenchymal stem cells (rMSCs), transfection efficiencies were examined. The secretion of rhBMP-2 and alkaline phosphatase (ALP) were detected to evaluate the osteogenic properties. Scanning electron microscopy (SEM) was used to observe attachment and proliferation. Moreover, we applied these GAMs directly into freshly created segmental bone defects in rat femurs, and their osteogenic efficiencies were evaluated. Results: Released plasmid complexes were transfected into stem cells and were expressed, which caused osteogenic differentiations of rat mesenchymal stem cells (rMSCs). SEM analysis showed excellent cell attachment. Bioactivity of plasmid rhBMP-2 was maintained in vivo, and the X-ray observation, histological analysis and immunohistochemistry (IHC) of bone tissue demonstrated that the bone healing in segmental femoral defects was enhanced by implantation of GAMs. Conclusions: Such biomaterials offer therapeutic opportunities in critical-sized bone defects.

Download Full-text

Osteogenic differentiation potential and marker gene expression of different porcine bone marrow mesenchymal stem cell subpopulations selected in different basal media

10.1101/2020.04.27.063230 ◽

2020 ◽

Author(s):

Sangeetha Kannan ◽

Jyotirmoy Ghosh ◽

Sujoy K. Dhara

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Marker Gene ◽

Microscopical Examination ◽

Alizarin Red ◽

Differentiation Potential ◽

Basal Media ◽

Selection Of

AbstractMultipotent porcine mesenchymal stem cells (pMSC) are indispensable for research and therapeutic use. Derivation and culture media might affect the selection of MSC subpopulation and thus the differentiation potential of cells. In this study we evaluated the effects of αMEM, aDMEM, M199, αMEM/M199, aDMEM/M199 and αMEM/aDMEM media on porcine bone marrow MSC derivation; pre-differentiation expression of ALP, COL1A1, SPP1 and BGLAP osteogenic marker genes at passage 5 and 10 pMSC; and differentiation potential of passage 5 pMSC. Morphological changes and matrix formation in osteogenic cells were evaluated by microscopical examination and calcium deposit in osteocytes was confirmed by Alizarin Red S staining. Results indicated media independent selection of different bone marrow MSC subpopulations with different surface marker gene expressions. Many pMSC subpopulations in different media had CD14+ expressing cells. We also observed basal media dependent changes in osteogenic markers expression and differentiation potential of pMSC. The αMEM/aDMEM media grown pMSC showed best osteogenic differentiation potential. We thus recommended the testing of αMEM/aDMEM mixed media in other species for pre-differentiation MSC culture that are intended for better osteogenic differentiation.SummaryPre-differentiation basal media influence osteogenic differentiation potential of mesenchymal stem cells (MSC). Among the tested media, αMEM/aDMEM was the best for pre-differentiation porcine MSC culture intending to use in osteogenesis.

Download Full-text

Differential Expression of MicroRNA-3148 in Patients with Osteoporosis and Its Impacts on the Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2021.2635 ◽

2021 ◽

Vol 11 (5) ◽

pp. 957-962

Author(s):

Ainiwaerjiang Damaola ◽

Maerdan Aierken ◽

Mieralimu Muertizha ◽

Abudouaini Abudoureheman ◽

Haishan Lin ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Serum Samples ◽

Alizarin Red ◽

Differentiation Potential ◽

Qrt Pcr ◽

Marrow Mesenchymal Stem Cells ◽

Rat Bone

We aimed to explore the effects of rat bone marrow mesenchymal stem cells (BMSCs) on osteogenic differentiation via analyzing miR-3148 expression in patients with osteoporosis. Realtime quantitative PCR was conducted for assessing microRNA-3148 expression. BMSCs from SD rats were transfected with microRNA-3148 mimics and microRNA-3148 inhibitor via liposomal trans-fection method utilizing Lipo2000, followed by analysis of microRNA-3148 level. After 10-days of osteogenic differentiation induction, alkaline phosphatase (ALP) staining and alizarin red (ARS) staining were done to investigate the osteogenic differentiation potential. Simultaneously, qRT-PCR measured the expression of osteogenesis marker genes (BMP and Runx2) in each group. qRT-PCR analysis revealed a high expression of miR-3148 in the bone tissue and the serum samples from patients with osteoporosis in comparison with healthy individuals. In addition, miRNA-3148 mimics could retard the osteogenic differentiation of BMSCs, while microRNA-3148 inhibitor could prompt the procedure. MicroRNA-3148 was highly expressed in the skeletal tissues and the serum samples from patients with osteoporosis and it could restrain the differentiation of BMSCs into osteoblasts, suggesting that it might be a novel therapeutic target for treating osteoporosis.

Download Full-text

Osteogenic differentiation potential of porcine bone marrow mesenchymal stem cell subpopulations selected in different basal media

Biology Open ◽

10.1242/bio.053280 ◽

2020 ◽

Vol 9 (10) ◽

pp. bio053280

Author(s):

Sangeetha Kannan ◽

Jyotirmoy Ghosh ◽

Sujoy K. Dhara

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Alizarin Red ◽

Differentiation Potential ◽

Osteogenic Lineage ◽

Osteogenic Markers ◽

Basal Media ◽

Selection Of

ABSTRACTMultipotent porcine mesenchymal stem cells (pMSC) are invaluable for research and therapeutic use in regenerative medicine. Media used for derivation and expansion of pMSC may play an important role for the selection of MSC subpopulation at an early stage and thereby, the specific basal medium may also affect differentiation potential of these cells. The present study was undertaken to evaluate the effects of αMEM, aDMEM, M199, αMEM/M199, aDMEM/M199 and αMEM/aDMEM media on (1) porcine bone marrow MSC derivation; (2) expression of number of osteogenic markers (ALP, COL1A1, SPP1 and BGLAP) at 5th and 10th passage in pMSC before differentiation; and (3) differentiation of pMSC (at 5th passage) to osteogenic lineage. Morphological changes and matrix formation in osteogenic cells were evaluated by microscopic examination. Calcium deposits in osteocytes were confirmed by Alizarin Red S staining. Based on expression of different markers, it was evident that selection of bone marrow pMSC subpopulations was independent of basal media used. However, the differentiation of those pMSCs, specifically to osteogenic lineage, was dependent on the medium used for expansion of pMSC at the pre-differentiation stage. We demonstrated here that the pMSC grown in combined αMEM/aDMEM (1:1) medium expressed number of osteogenic markers and these pMSC underwent osteogenic differentiation most efficiently, in comparison to porcine mesenchymal stem cells grown in other media. In conclusion, osteogenic differentiation potential of pMSC maintained in αMEM/aDMEM medium was observed significantly higher compared to cells cultivated in other media and therefore, the combined medium αMEM/aDMEM (1:1) may preferentially be used for expansion of pMSC, if needed for osteogenic differentiation.

Download Full-text

MicroRNA-346-5p Regulates Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells by Inhibiting Transmembrane Protein 9

BioMed Research International ◽

10.1155/2020/8822232 ◽

2020 ◽

Vol 2020 ◽

pp. 1-7

Author(s):

Yicai Zhang ◽

Yi Sun ◽

Jinlong Liu ◽

Yu Han ◽

Jinglong Yan

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Protein Level ◽

Molecular Mechanisms ◽

Transmembrane Protein ◽

Luciferase Reporter ◽

Alizarin Red ◽

Protein Levels

The molecular mechanisms how bone marrow-derived mesenchymal stem cells (BMSCs) differentiate into osteoblast need to be investigated. MicroRNAs (miRNAs) contribute to the osteogenic differentiation of BMSCs. However, the effect of miR-346-5p on osteogenic differentiation of BMSCs is not clear. This study is aimed at elucidating the underlying mechanism by which miR-346-5p regulates osteogenic differentiation of human BMSCs. Results of alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining indicated that upregulation of miR-346-5p suppressed osteogenic differentiation of BMSCs, whereas downregulation of miR-346-5p enhanced this process. The protein levels of the osteoblastic markers Osterix and Runt-related transcription factor 2 (Runx2) were decreased in cells treated with miR-346-5p mimic at day 7 and day 14 after being differentiated. By contrast, downregulation of miR-346-5p elevated the protein levels of Osterix and Runx2. Moreover, a dual-luciferase reporter assay revealed that Transmembrane Protein 9 (TMEM9) was a target of miR-346-5p. In addition, the Western Blot results demonstrated that the TMEM9 protein level was significantly reduced by the miR-346-5p mimic whereas downregulation of miR-346-5p improved the protein level of TMEM9. These results together demonstrated that miR-346-5p served a key role in BMSC osteogenic differentiation of through targeting TMEM9, which may provide a novel target for clinical treatments of bone injury.

Download Full-text

Biological Characteristics and Osteogenic Differentiation of Ovine Bone Marrow Derived Mesenchymal Stem Cells Stimulated with FGF-2 and BMP-2

International Journal of Molecular Sciences ◽

10.3390/ijms21249726 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9726

Author(s):

Sandra Gromolak ◽

Agnieszka Krawczenko ◽

Agnieszka Antończyk ◽

Krzysztof Buczak ◽

Zdzisław Kiełbowicz ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Collagen Type ◽

Collagen Type I ◽

Biological Characteristics ◽

Immunofluorescent Staining ◽

Type I ◽

Differentiation Potential

Cell-based therapies using mesenchymal stem cells (MSCs) are a promising tool in bone tissue engineering. Bone regeneration with MSCs involves a series of molecular processes leading to the activation of the osteoinductive cascade supported by bioactive factors, including fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2). In this study, we examined the biological characteristics and osteogenic differentiation potential of sheep bone marrow MSCs (BM-MSCs) treated with 20 ng/mL of FGF-2 and 100 ng/mL BMP-2 in vitro. The biological properties of osteogenic-induced BM-MSCs were investigated by assessing their morphology, proliferation, phenotype, and cytokine secretory profile. The osteogenic differentiation was characterized by Alizarin Red S staining, immunofluorescent staining of osteocalcin and collagen type I, and expression levels of genetic markers of osteogenesis. The results demonstrated that BM-MSCs treated with FGF-2 and BMP-2 maintained their primary MSC properties and improved their osteogenic differentiation capacity, as confirmed by increased expression of osteocalcin and collagen type I and upregulation of osteogenic-related gene markers BMP-2, Runx2, osterix, collagen type I, osteocalcin, and osteopontin. Furthermore, sheep BM-MSCs produced a variety of bioactive factors involved in osteogenesis, and supplementation of the culture medium with FGF-2 and BMP-2 affected the secretome profile of the cells. The results suggest that sheep osteogenic-induced BM-MSCs may be used as a cellular therapy to study bone repair in the preclinical large animal model.

Download Full-text