scholarly journals Application of Bone Morphogenetic Protein 7 Enhanced the Osteogenic Differentiation and Mineralization of Bone Marrow-Derived Stem Cells Cultured on Deproteinized Bovine Bone

Coatings ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 642
Author(s):  
Hyunjin Lee ◽  
Sae Kyung Min ◽  
Yoon-Hee Park ◽  
Jun-Beom Park
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Bovine Bone ◽  
Bone Morphogenetic Protein 7 ◽  
Bone Particle ◽  
Morphogenetic Protein ◽  
Cells Cultured ◽  
Deproteinized Bovine Bone

The growth of bone morphogenetic protein 7 (BMP-7) has been applied for tissue regeneration due to its osteoinductive properties. The aim of this research is to analyze the enhancing effects of BMP-7 on the osteogenic differentiation and mineralization of human bone marrow-derived stem cells cultured on the bovine bone particle. After the stem cells were loaded onto the bone graft material, their morphology was observed on day 7. Viability assays based on the application of fluorescent stains were used for qualitative analyses. Alkaline phosphatase activity assays and Alizarin red staining were used for the assessment of osteogenic differentiation on days 7 and 14. Next-generation mRNA sequencing was applied to evaluate global gene expression. Gene ontology and pathway analysis was used to propose the underlying mechanism. Fibroblast-like morphology was attained with the stem cells. The cells were shown to be firmly attached to the bone particle. Most of the stem cells produced an intense green fluorescence. The relative cellular viability assay values for BMP-7 groups at 0, 10, and 100 ng/mL on day 7 were 0.295 ± 0.003, 0.250 ± 0.002, and 0.240 ± 0.003, respectively (p < 0.05). Alkaline phosphatase activity was significantly higher in BMP-7 groups at concentration of 100 ng/mL compared to the control on days 7 and 14 (p < 0.05). The results of the mineralization assay showed significantly higher values for BMP-7 groups at 100 ng/mL concentration when compared with the control (p < 0.05). The expression of RUNX2 was increased with application of BMP-7 and mitogen-activated protein kinase pathway was associated with the target genes. Overall, this study shows that in vitro application of BMP-7 increases alkaline phosphorylase activity and mineralization of stem cells culture on deproteinized bovine bone mineral. The study suggests that combining stem cells with osteoinductive growth factors with scaffolds can have synergy effects on osteogenic differentiation.

scholarly journals The Role of Insulin-Like Growth Factor-2 on the Cellular Viability and Differentiation to the Osteogenic Lineage and Mineralization of Stem Cells Cultured on Deproteinized Bovine Bone Mineral

2020 ◽  
Vol 10 (16) ◽  
pp. 5471
Author(s):  
Hyunjin Lee ◽  
Sae Kyung Min ◽  
Yoon-Hee Park ◽  
Jun-Beom Park
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Stem Cell ◽  
Alkaline Phosphatase Activity ◽  
Bovine Bone ◽  
Cellular Viability ◽  
Bovine Bone Mineral ◽  
Deproteinized Bovine Bone Mineral ◽  
Cells Cultured ◽  
Deproteinized Bovine Bone

Insulin-like growth factors (IGFs) plays various roles, including differentiation and mitogenesis, and IGFs are reported to regulate the bone growth and maintenance. This study was performed to analyze the enhancing effects of IGF-2 on osteogenic differentiation and the mineralization of stem cells cultured on deproteinized bovine bone mineral. Stem cell loaded bone graft material was cultured in the presence of the IGF-2 at final concentrations of 10 and 100 ng/mL and the morphology of the cells was observed on Days 1, 3, and 7. The commercially available, two-color assay based on plasma membrane integrity and esterase activity was also used for qualitative analyses on Days 1, 3, and 7. The level of alkaline phosphatase activity and anthraquinone dye assay were used to evaluate osteogenic differentiation on Days 7 and 14. Real-time polymerase chain reaction was applied in order to identify the mRNA expression of BGLAP, Runx2, and β-catenin. The stem cells were well-attached with fibroblast morphology and most of the stem cells produced a high intensity of green fluorescence, indicating that there were live cells on Day 1. The relative cellular viability assay values for IGF-2 groups at 0, 10, and 100 ng/mL on Day 1 were 0.419 ± 0.015, 0.427 ± 0.013, and 0.500 ± 0.030, respectively (p < 0.05). The absorbance values at 405 nm for alkaline phosphatase activity on Day 7 for IGF-2 at 0, 10, and 100 ng/mL were 2.112 ± 0.152, 1.897 ± 0.144, and 2.067 ± 0.128, respectively (p > 0.05). The mineralization assay results at Day 7 showed significantly higher values for IGF-2 groups at 10 and 100 ng/mL concentration when compared to the control (p < 0.05). The application of IGF-2 groups of 10 and 100 ng/mL produced a significant increase of BGLAP. Conclusively, this study indicates that the use of IGF-2 on stem cell loaded bone graft increased cellular viability, Alizarin red staining, and BGLAP expression of stem cells. This report suggests the combined approach of stem cells and IGF-2 with scaffold may have synergistic effects on osteogenesis.

k-Casein upregulates osteogenic differentiation on bone marrow mesenchymal stem cells cultured on agarose microcarriers

2019 ◽  
Vol 69 (6) ◽  
pp. 373-380 ◽  
Author(s):  
Hammed Tanimowo Aiyelabegan ◽  
Malihe Ebadi ◽  
Gholam Ali Kardar ◽  
Nasrin Lotfibakhshaiesh ◽  
Farid Abedin Dorkoosh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Marrow Mesenchymal Stem Cells ◽  
Cells Cultured

Osteogenic Differentiation of Bone-Marrow-Derived Stem Cells Cultured with Mixed Gelatin and Chitooligosaccharide Scaffolds

2011 ◽  
Vol 22 (8) ◽  
pp. 1083-1098 ◽  
Author(s):  
Juthamas Ratanavaraporn ◽  
Siriporn Damrongsakkul ◽  
Sorada Kanokpanont ◽  
Masaya Yamamoto ◽  
Yasuhiko Tabata
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Cells Cultured

286 OSTEOGENIC DIFFERENTIATION IN VITRO OF PORCINE ADULT MESENCHYMAL STEM CELLS

2008 ◽  
Vol 20 (1) ◽  
pp. 223
Author(s):  
A. Lima ◽  
E. Monaco ◽  
S. Wilson ◽  
D. Kim ◽  
C. Feltrin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Adipose Tissue ◽  
Osteogenic Differentiation ◽  
Subcutaneous Adipose Tissue ◽  
Cell Types ◽  
Alizarin Red ◽  
Subcutaneous Adipose

The quantity and accessibility of subcutaneous adipose tissue in humans make it an attractive alternative to bone marrow as a source of adult stem cells for therapeutic purposes. However, before such a cell source substitution can be proposed, the properties of stem cells derived from adipose (ADSCs) and bone marrow (MSCs) and their differentiated progeny must be compared in an animal model that adequately simulates the structure and physiology of humans. The objective of this work was to induce adult porcine stem cells isolated from subcutaneous adipose tissue and bone marrow to differentiate in vitro along the osteoblastic lineage and to compare their morphological, phenotypic, and genotypic properties. MSCs and ADSCs were isolated respectively from femurs and subcutaneous adipose tissue of adult pigs and cultured in vitro using DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin G-streptomycin, and 5.6 mg L–1 amphotericin B. After 3 passages, cells were differentiated along the osteogenic lineage using lineage-specific inducing medium. Osteogenic medium contained 100 nm dexamethasone, 10 mm β-glycerophosphate, and 0.005 mm ascorbic acid-2-phosphate. Osteogenic cultures were incubated for 4 weeks in 95% air and 5% CO2 at 39�C. Spent medium was replaced with fresh medium every 3 days. Histological staining with alkaline phosphatase, Von Kossa, and alizarin red S were performed at 0, 2, 4, 7, 14, 21, and 28 days of differentiation (dd). At the same time points, RNA was extracted. qPCR was performed on COL1A1, BGLAP, SPARC, and SPP1. As internal control, the geometrical mean of GTF2H, NUBP, and PPP2C was used. Relative mRNA abundance between cell types was calculated using 1/efficiencydCT. The osteogenic differentiation of both MSCs and ADScs was confirmed by the organization of the cells in nodules and by alkaline phosphatase-, Von Kossa-, and alizarin red S-positive staining. The percent relative abundance of the 4 genes in both cell types was COL1A1 (ca. 50) > SPARC (ca. 45) > SPP1 (ca. 5) > BGLAP ( < 0.1). Cell types showed similar mRNA abundance for COL1A1 and SPARC while SPP1 and BGLAP were, respectively, 10- and 19-fold higher in MSCs than in ADSCs. All of the genes had the same pattern among tissues during differentiation except for SPP1, which showed a >10-fold increase at 14 v. 0 dd only for MSCs. Adipose-derived stem cells demonstrated a clear osteogenic differentiation and similar expression and pattern of the two osteogenic genes most abundant in MSCs (COL1A1 and SPARC). However, the higher abundance of SPP1 and BGLAP and the different behavior of SPP1 in MSCs suggest a different transcription profile between the two cell types. From these preliminary results, adipose tissue can be a practical alternative source for stem cells in future human clinical applications.

scholarly journals Impact of Age on Human Adipose Stem Cells for Bone Tissue Engineering

2017 ◽  
Vol 26 (9) ◽  
pp. 1496-1504 ◽  
Author(s):  
Denis Dufrane
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Bone Tissue Engineering ◽  
Osteogenic Differentiation ◽  
Bone Tissue ◽  
Pathological Condition ◽  
Growth Potential ◽  
Matrix Mineralization

Bone nonunion is a pathological condition in which all bone healing processes have stopped, resulting in abnormal mobility between 2 bone segments. The incidence of bone-related injuries will increase in an aging population, leading to such injuries reaching epidemic proportions. Tissue engineering and cell therapy using mesenchymal stem cells (MSCs) have raised the possibility of implanting living tissue for bone reconstruction. Bone marrow was first proposed as the source of stem cells for bone regeneration. However, as the quantity of MSCs in the bone marrow decreases, the capacity of osteogenic differentiation of bone marrow stem cells is also impaired by the donor’s age in terms of reduced MSC replicative capacity; an increased number of apoptotic cells; formation of colonies positive for alkaline phosphatase; and decreases in the availability, growth potential, and temporal mobilization of MSCs for bone formation in case of fracture. Adipose-derived stem cells (ASCs) demonstrate several advantages over those from bone marrow, including a less invasive harvesting procedure, a higher number of stem cell progenitors from an equivalent amount of tissue harvested, increased proliferation and differentiation capacities, and better angiogenic and osteogenic properties in vivo. Subcutaneous native adipose tissue was not affected by the donor’s age in terms of cellular senescence and yield of ASC isolation. In addition, a constant mRNA level of osteocalcin and alkaline phosphatase with a similar level of matrix mineralization of ASCs remained unaffected by donor age after osteogenic differentiation. The secretome of ASCs was also unaffected by age when aiming to promote angiogenesis by vascular endothelial growth factor (VEGF) release in hypoxic conditions. Therefore, the use of adipose cells for bone tissue engineering is not limited by the donor’s age from the isolation of stem cells up to the manufacturing of a complex osteogenic graft.

scholarly journals MicroRNA-320a Regulates the Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells by Targeting HOXA10

2016 ◽  
Vol 38 (1) ◽  
pp. 40-48 ◽  
Author(s):  
Jianhou Huang ◽  
Yake Meng ◽  
Yan Liu ◽  
Yu Chen ◽  
Haisong Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Ectopic Expression ◽  
In Silico Analysis ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Matrix Mineralization ◽  
Morphogenetic Protein

Background/Aims: Human bone marrow-derived mesenchymal stem cells (hMSCs) are a promising cell source for bone engineering owing to their high potential to differentiate into osteoblasts. The bone morphogenetic protein-inducible gene homeobox a10 (HOXA10) is a critical regulator of osteogenesis. The objective of the present study was to identify microR-NAs (miRNAs) targeting HOXA10 and examine the effects on the osteogenic differentiation of hMSCs. Methods: Based on in silico analysis, HOXA10-targeting miRNAs were selected and their regulatory roles in osteoblast differentiation were investigated. Results: Six HOXA10-targeting miRNAs were identifIed by computational analysis, of which miR-320a was selected for further analysis because it was downregulated during osteogenic induction. Overexpression of miR-320a downregulated HOXA10 and significantly inhibited osteogenesis in hMSCs, as determined by the downregulation of the osteogenic markers Runx2, ALP, and OC and the inhibition of ALP activity and matrix mineralization, whereas miR-320a inhibition had the opposite effects. Furthermore, ectopic expression of HOXA10 (not including 3′-UTR) rescued the effects of miR-320a on osteogenic differentiation. Conclusion: These results suggest that miR-320a acts as a critical regulator of osteogenic differentiation of hMSCs by repressing its target HOXA10.

scholarly journals Human Amnion-Derived Mesenchymal Stem Cells Promote Osteogenic Differentiation in Human Bone Marrow Mesenchymal Stem Cells by Influencing the ERK1/2 Signaling Pathway

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Yuli Wang ◽  
Fei Jiang ◽  
Yi Liang ◽  
Ming Shen ◽  
Ning Chen
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Osteoblastic Differentiation ◽  
Human Amnion ◽  
Bone Deficiency ◽  
Marrow Mesenchymal Stem Cells

Human amnion-derived mesenchymal stem cells (HAMSCs) are considered to be an important resource in the field of tissue engineering because of their anti-inflammatory properties and fewer ethical issues associated with their use compared with other sources of stem cells. HAMSCs can be obtained from human amniotic membranes, a readily available and abundant tissue. However, the potential of HAMSCs as seed cells for treating bone deficiency is unknown. In this study, HAMSCs were used to promote proliferation and osteoblastic differentiation in human bone marrow mesenchymal stem cells (HBMSCs) in a Transwell coculture system. Proliferation levels were investigated by flow cytometry and immunofluorescence staining of 5-ethynyl-2′-deoxyuridine (EdU). Osteoblastic differentiation and mineralization were evaluated in chromogenic alkaline phosphatase (ALP) activity substrate assays, Alizarin red S staining, and RT-PCR analysis of early HBMSCs osteogenic marker expression. We demonstrated that HAMSCs stimulated increased alkaline phosphatase (ALP) activity, mRNA expression of osteogenic marker genes, and mineralized matrix deposition. Moreover, the effect of HAMSCs was significantly inhibited by U0126, a highly selective inhibitor of extracellular signaling-regulated kinase 1/2 (ERK1/2) signaling. We demonstrate that HAMSCs promote osteogenic differentiation in HBMSCs by influencing the ERK1/2 signaling pathway. These observations confirm the potential of HAMSCs as a seed cell for the treatment of bone deficiency.

Sign in / Sign up

Export Citation Format

Share Document