scholarly journals Optimizing Osteogenic Differentiation of Ovine Adipose-Derived Stem Cells by Osteogenic Induction Medium and FGFb, BMP2, or NELL1 In Vitro

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Emil Østergaard Nielsen ◽  
Li Chen ◽  
Jonas Overgaard Hansen ◽  
Matilda Degn ◽  
Søren Overgaard ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Stromal Cells ◽  
Basic Medium ◽  
Induction Medium ◽  
Control Analysis ◽  
Alizarin Red ◽  
Osteogenic Induction Medium ◽  
Osteogenic Induction ◽  
Fibroblast Growth Factor Basic

Although adipose-derived stromal cells (ADSCs) have been a major focus as an alternative to autologous bone graft in orthopedic surgery, bone formation potential of ADSCs is not well known and cytokines as osteogenic inducers on ADSCs are being investigated. This study aimed at isolating ADSCs from ovine adipose tissue (AT) and optimizing osteogenic differentiation of ovine ADSCs (oADSC) by culture medium and growth factors. Four AT samples were harvested from two female ovine (Texel/Gotland breed), and oADSCs were isolated and analyzed by flow cytometry for surface markers CD29, CD44, CD31, and CD45. Osteogenic differentiation was made in vitro by seeding oADSCs in osteogenic induction medium (OIM) containing fibroblast growth factor basic (FGFb), bone morphogenetic protein 2 (BMP2), or NEL-like molecule 1 (NELL1) in 4 different dosages (1, 10, 50, and 100 ng/ml, respectively). Basic medium (DMEM) was used as control. Analysis was made after 14 days by Alizarin red staining (ARS) and quantification. This study successfully harvested AT from ovine and verified isolated cells for minimal criteria for adipose stromal cells which suggests a feasible method for isolation of oADSCs. OIM showed significantly higher ARS to basic medium, and FGFb 10 ng/ml revealed significantly higher ARS to OIM alone after 14 days.

scholarly journals MEK1/2 Inhibitor (GDC0623) Promotes Osteogenic Differentiation of Primary Osteoblasts Inhibited by IL-1β through the MEK-Erk1/2 and Jak/Stat3 Pathways

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Zeng-Qiao Zhang ◽  
Xiao-Shen Hu ◽  
Ye-Chen Lu ◽  
Jun-Peng Zhang ◽  
Wen-Yao Li ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Western Blotting ◽  
Cell Counting ◽  
Nuclear Antigen ◽  
Control Group ◽  
Induction Medium ◽  
Alizarin Red ◽  
Real Time Quantitative Pcr ◽  
Osteogenic Induction Medium ◽  
Osteogenic Induction

Objective. We evaluated the effects and mechanisms of GDC0623 on osteogenic differentiation of osteoblasts induced by IL-1β. Methodology. Osteoblasts were treated with 20 ng/ml IL-1β and 0.1 µM GDC0623. Cell proliferation levels were evaluated by the cell counting kit 8 (CCK8), EdU assay, and western blotting [proliferating cell nuclear antigen (PCNA) and Cyclin D1]. Osteoblasts were cultured in an osteogenic induction medium for 1–3 weeks after which their differentiations were assessed by alkaline phosphatase (ALP) staining, Alizarin Red staining, calcium concentration, immunocytochemistry staining, real-time quantitative PCR (RT-qPCR), and immunofluorescence staining. The osteogenesis-associated mechanisms were further evaluated by western blotting using appropriate antibodies. Results. Relative to the control group, IL-1β induced the rapid proliferation of osteoblasts and suppressed their osteogenic differentiations by upregulating the activities of MEK-Erk1/2 as well as Jak-Stat3 pathways and by elevating MMP13 and MMP9 levels. However, blocking of the MEK-Erk1/2 signaling pathway by GDC0623 treatment reversed these effects. Conclusion. Inhibition of Jak-Stat3 pathway by C188-9 downregulated the expression levels of MMP9 and MMP13, activated MEK-Erk1/2 pathway, and inhibited osteogenic differentiation.

scholarly journals Mechanical Sensing Element PDLIM5 Promotes Osteogenesis of Human Fibroblasts by Affecting the Activity of Microfilaments

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 759
Author(s):  
Xiaolan Huang ◽  
Rongmei Qu ◽  
Yan Peng ◽  
Yuchao Yang ◽  
Tingyu Fan ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Morphological Changes ◽  
Human Fibroblasts ◽  
Inhibitory Effect ◽  
Induction Medium ◽  
Sensing Element ◽  
Differentiation Potential ◽  
Osteogenic Induction Medium ◽  
Osteogenic Induction ◽  
Related Proteins

Human skin fibroblasts (HSFs) approximate the multidirectional differentiation potential of mesenchymal stem cells, so they are often used in differentiation, cell cultures, and injury repair. They are an important seed source in the field of bone tissue engineering. However, there are a few studies describing the mechanism of osteogenic differentiation of HSFs. Here, osteogenic induction medium was used to induce fibroblasts to differentiate into osteoblasts, and the role of the mechanical sensitive element PDLIM5 in microfilament-mediated osteogenic differentiation of human fibroblasts was evaluated. The depolymerization of microfilaments inhibited the expression of osteogenesis-related proteins and alkaline phosphatase activity of HSFs, while the polymerization of microfilaments enhanced the osteogenic differentiation of HSFs. The evaluation of potential protein molecules affecting changes in microfilaments showed that during the osteogenic differentiation of HSFs, the expression of PDLIM5 increased with increasing induction time, and decreased under the state of microfilament depolymerization. Lentivirus-mediated PDLIM5 knockdown by shRNA weakened the osteogenic differentiation ability of HSFs and inhibited the expression and morphological changes of microfilament protein. The inhibitory effect of knocking down PDLIM5 on HSF osteogenic differentiation was reversed by a microfilament stabilizer. Taken together, these data suggest that PDLIM5 can mediate the osteogenic differentiation of fibroblasts by affecting the formation and polymerization of microfilaments.

scholarly journals In Vitro Fabrication of Hybrid Bone/Cartilage Complex Using Mouse Induced Pluripotent Stem Cells

2020 ◽  
Vol 21 (2) ◽  
pp. 581 ◽  
Author(s):  
Phoonsuk Limraksasin ◽  
Takeru Kondo ◽  
Maolin Zhang ◽  
Hiroko Okawa ◽  
Thanaphum Osathanon ◽  
...  
Keyword(s):  
Cartilage Tissue ◽  
Marker Genes ◽  
Induction Medium ◽  
Mechanical Stimuli ◽  
Tissue Formation ◽  
Osteogenic Induction Medium ◽  
Osteogenic Induction ◽  
Chondrogenic Marker ◽  
Induced Pluripotent

Cell condensation and mechanical stimuli play roles in osteogenesis and chondrogenesis; thus, they are promising for facilitating self-organizing bone/cartilage tissue formation in vitro from induced pluripotent stem cells (iPSCs). Here, single mouse iPSCs were first seeded in micro-space culture plates to form 3-dimensional spheres. At day 12, iPSC spheres were subjected to shaking culture and maintained in osteogenic induction medium for 31 days (Os induction). In another condition, the osteogenic induction medium was replaced by chondrogenic induction medium at day 22 and maintained for a further 21 days (Os-Chon induction). Os induction produced robust mineralization and some cartilage-like tissue, which promoted expression of osteogenic and chondrogenic marker genes. In contrast, Os-Chon induction resulted in partial mineralization and a large area of cartilage tissue, with greatly increased expression of chondrogenic marker genes along with osterix and collagen 1a1. Os-Chon induction enhanced mesodermal lineage commitment with brachyury expression followed by high expression of lateral plate and paraxial mesoderm marker genes. These results suggest that combined use of micro-space culture and mechanical stimuli facilitates hybrid bone/cartilage tissue formation from iPSCs, and that the bone/cartilage tissue ratio in iPSC constructs could be manipulated through the induction protocol.

Osteogenic differentiation of equine cord blood multipotent mesenchymal stromal cells within coralline hydroxyapatite scaffolds in vitro

2011 ◽  
Vol 24 (05) ◽  
pp. 354-362 ◽  
Author(s):  
R. J. Figueroa ◽  
T. G. Koch ◽  
D. H. Betts
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Viability ◽  
Osteogenic Differentiation ◽  
Alkaline Phosphatase Activity ◽  
Stromal Cells ◽  
Culture Medium ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Coralline Hydroxyapatite ◽  
Osteogenic Induction

SummaryObjective: To investigate the osteogenic differentiation potential of equine umbilical cord blood-derived multipotent mesenchymal stromal cells (CB-MSC) within coralline hydro-xyapatite scaffolds cultured in osteogenic induction culture medium.Methods: Scaffolds seeded with equine CBMSC were cultured in cell expansion culture medium (control) or osteogenic induction medium (treatment). Cell viability and distribution were confirmed by the MTT cell viability assay and DAPI nuclear fluorescence staining, respectively. Osteogenic differentiation was evaluated after 10 days using reverse transcription polymerase chain reaction, alkaline phosphatase activity, and secreted osteocalcin concentration. Cell morphology and matrix deposition were assessed by scanning electron microscopy (SEM) after 14 days in culture.Results: Cells showed viability and adequate distribution within the scaffold. Successful osteogenic differentiation within the scaffolds was demonstrated by the increased expression of osteogenic markers such as Runx2, osteopontin, osteonectin, collagen IA increased levels of alkaline phosphatase activity increased osteocalcin protein secretion and bone-like matrix presence in the scaffold pores upon SEM evaluation.Clinical significance: These results demonstrate that equine CB-MSC maintain viability and exhibit osteogenic potential in coralline hydroxyapatite scaffolds when induced in vitro. Equine CB-MSC scaffold constructs deserve further investigation for their potential role as biologically active fillers to enhance bone-gap repair in the horse.

scholarly journals Effect on Osteogenic Differentiation of Genetically Modified IL4 or PDGF-BB Over-Expressing and IL4-PDGF-BB Co-Over-Expressing Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro

2021 ◽  
Vol 8 (11) ◽  
pp. 165
Author(s):  
Masanori Tsubosaka ◽  
Masahiro Maruyama ◽  
Elijah Ejun Huang ◽  
Ning Zhang ◽  
Takeshi Utsunomiya ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Early Stage ◽  
Genetically Modified ◽  
Interleukin 4 ◽  
Alizarin Red ◽  
Cell Based Therapy

The use of genetically modified (GM) mesenchymal stromal cells (MSCs) and preconditioned MSCs (pMSCs) may provide further opportunities to improve the outcome of core decompression (CD) for the treatment of early-stage osteonecrosis of the femoral head (ONFH). GM interleukin-4 (IL4) over-expressing MSCs (IL4-MSCs), platelet-derived growth factor (PDGF)-BB over-expressing MSCs (PDGF-BB-MSCs), and IL4-PDGF-BB co-over-expressing MSCs (IL4-PDGF-BB-MSCs) and their respective pMSCs were used in this in vitro study and compared with respect to cell proliferation and osteogenic differentiation. IL4-MSCs, PDGF-BB-MSCs, IL4-PDGF-BB-MSCs, and each pMSC treatment significantly increased cell proliferation compared to the MSC group alone. The percentage of Alizarin red-stained area in the IL4-MSC and IL4-pMSC groups was significantly lower than in the MSC group. However, the percentage of Alizarin red-stained area in the PDGF-BB-MSC group was significantly higher than in the MSC and PDGF-BB-pMSC groups. The percentage of Alizarin red-stained area in the IL4-PDGF-BB-pMSC was significantly higher than in the IL4-PDGF-BB-MSC group. There were no significant differences in the percentage of Alizarin red-stained area between the MSC and IL4-PDGF-BB-pMSC groups. The use of PDGF-BB-MSCs or IL4-PDGF-BB-pMSCs increased cell proliferation. Furthermore, PDGF-BB-MSCs promoted osteogenic differentiation. The addition of GM MSCs may provide a useful supplementary cell-based therapy to CD for treatment of ONFH.

miR-217 Suppresses Osteogenic Differentiation of Human Dental Pulp Stem Cells by Down-Regulating Sirtuin 1

2020 ◽  
Vol 10 (7) ◽  
pp. 978-986
Author(s):  
Haiquan Yue ◽  
Yidan Guo ◽  
Juan Song ◽  
Ruimin Liu
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Dental Pulp ◽  
Dental Pulp Stem Cells ◽  
Sirtuin 1 ◽  
Luciferase Reporter ◽  
Induction Medium ◽  
Alizarin Red ◽  
Human Dental Pulp ◽  
Osteogenic Induction

The paper is committed to uncovering the effect of miR-217 on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and its mechanism. hDPSCs were separated from human dental pulp tissues for measurement of stemness. The osteogenic differentiation of hDPSCs was induced in an osteogenic induction medium. The hDPSCs were transfected with miR-217 mimic, miR-217 inhibitor and/or sh-SIRT1 accordingly. The expressions of miR-217 and SIRT1 were detected in hDPSCs after cell transfection and osteogenic differentiation. Calcium nodules were showed by alizarin red staining. Moreover, the expressions of osteogenic differentiation-related genes were also assessed. The binding of miR-217 to SIRT1 was predicted on starBase and further determined by dual-luciferase reporter assay. Down-regulated miR-217 and up-regulated SIRT1 were found during osteogenic differentiation of hDPSCs. The osteogenic differentiation of hDPSCs was suppressed after transfection of miR-217 mimic or sh-SIRT1 while promoted by miR-217 inhibition. Taken together, miR-217 can suppress osteogenic differentiation of hDPSCs by negatively regulating SIRT1.

scholarly journals Insulin Enhances the In Vitro Osteogenic Capacity of Flexor Tendon-Derived Progenitor Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Sushmitha S. Durgam ◽  
Nadine N. Altmann ◽  
Haley E. Coughlin ◽  
Audrey Rollins ◽  
Laura D. Hostnik
Keyword(s):  
Alkaline Phosphatase ◽  
Monolayer Culture ◽  
Tendon Healing ◽  
Cell Aggregation ◽  
Induction Medium ◽  
Dependent Manner ◽  
Alizarin Red ◽  
Igf I ◽  
Osteogenic Induction

There is increased incidence of tendon disorders and decreased tendon healing capacity in people with diabetes mellitus (DM). Recent studies have also suggested pathological ossification in repair tendon of people with DM. Therefore, the objective of this study is to investigate the effects of insulin supplementation, an important pathophysiologic stimulus of DM, on tendon progenitor cell (TPC) proliferation and in vitro osteogenic capacity. Passage 3 TPCs were isolated from collagenase-digested, equine superficial digital flexor tendons. TPC proliferation was measured via MTT assay after 3 days of monolayer culture in medium supplemented with 0, 0.007, 0.07, and 0.7 nM insulin. In vitro osteogenic capacity of TPCs (Alizarin Red staining, osteogenic mRNA expression, and alkaline phosphatase bioactivity) was assessed with 0, 0.07, and 0.7 nM insulin-supplemented osteogenic induction medium. Insulin (0.7 nM) significantly increased TPC proliferation after 3 days of monolayer culture. Alizarin Red staining of insulin-treated TPC osteogenic cultures demonstrated robust cell aggregation and mineralized matrix secretion, in a dose-dependent manner. Runx2, alkaline phosphatase, and Osteonectin mRNA and alkaline phosphatase bioactivity of insulin-treated TPC cultures were significantly higher at day 14 of osteogenesis compared to untreated controls. Addition of picropodophyllin (PPP), a selective IGF-I receptor inhibitor, mitigated the increased osteogenic capacity of TPCs, indicating that IGF-I signaling plays an important role. Our findings indicate that hyperinsulinemia may alter TPC phenotype and subsequently impact the quality of repair tendon tissue.

scholarly journals TMT-Based Proteomic Explores the Influence of DHEA on the Osteogenic Differentiation of hBMSCs

2021 ◽  
Vol 9 ◽  
Author(s):  
Xiaonan Liang ◽  
Mingwei He ◽  
Bo Zhu ◽  
Yongjia Zhu ◽  
Xixi He ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Metabolic Pathways ◽  
Osteoblast Differentiation ◽  
Bioinformatic Analysis ◽  
Induction Medium ◽  
Complete Medium ◽  
Tandem Mass ◽  
Osteogenic Induction Medium ◽  
Osteogenic Induction ◽  
Underlying Mechanisms

Dehydroepiandrosterone (DHEA) has been revealed to implicate in facilitating osteoblast differentiation of human bone marrow mesenchymal stem cells (hBMSCs) and inhibiting osteoporosis (OP). However, the underlying molecular mechanism remains largely unknown. Here, we induced osteogenic differentiation of hBMSCs derived from elders using an osteogenic induction medium with or without DHEA. The results showed that osteogenic induction medium (OIM) with DHEA could significantly promote the proliferation and osteogenic differentiation of hBMSCs than OIM alone. By using a Tandem Mass Tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology, we screened out 604 differentially expressed proteins (DEPs) with at least one unique peptide were identified [524: OIM vs. complete medium (CM), and 547: OIM+DHEA vs. CM], among these proteins, 467 DEPs were shared in these two different comparative groups. Bioinformatic analysis revealed these DEPs are mainly enriched in metabolic pathways. Interestingly, the expression levels of the DEPs in the metabolic pathways showed a more noticeable change in the OIM+DHEA vs. CM group than OIM vs. CM group. Moreover, the protein-protein interaction (PPI) network analysis revealed that three potential proteins, ATP5B, MT-CYB, and MT-ATP6, involved in energy metabolism, might play a key role in osteogenic differentiation induced by OIM+DHEA. These findings offer a valuable clue for us to better understand the underlying mechanisms involved in osteoblast differentiation of hBMSCs caused by DHEA and assist in applying DHEA in hBMSCs-based therapy for osteogenic regeneration.

scholarly journals Surface Modification of Biodegradable Mg-Based Scaffolds for Human Mesenchymal Stem Cell Proliferation and Osteogenic Differentiation

Materials ◽  
2021 ◽  
Vol 14 (2) ◽  
pp. 441
Author(s):  
Si-Han Wang ◽  
Shiao-Pieng Lee ◽  
Chung-Wei Yang ◽  
Chun-Min Lo
Keyword(s):  
Mechanical Properties ◽  
Surface Modification ◽  
Osteogenic Differentiation ◽  
Magnesium Alloys ◽  
Human Mesenchymal Stem Cells ◽  
Human Mesenchymal Stem Cell ◽  
Induction Medium ◽  
Culture Dish ◽  
Osteogenic Induction Medium ◽  
Osteogenic Induction

Magnesium alloys with coatings have the potential to be used for bone substitute alternatives since their mechanical properties are close to those of human bone. However, the surface modification of magnesium alloys to increase the surface biocompatibility and reduce the degradation rate remains a challenge. Here, FHA-Mg scaffolds were made of magnesium alloys and coated with fluorohydroxyapatite (FHA). Human mesenchymal stem cells (hMSCs) were cultured on FHA-Mg scaffolds and cell viability, proliferation, and osteogenic differentiation were investigated. The results showed that FHA-Mg scaffolds display a nano-scaled needle-like structure of aggregated crystallites on their surface. The average Mg2+ concentration in the conditioned media collected from FHA-Mg scaffolds (5.8–7.6 mM) is much lower than those collected from uncoated, Mg(OH)2-coated, and hydroxyapatite (HA)-coated samples (32.1, 17.7, and 21.1 mM, respectively). In addition, compared with hMSCs cultured on a culture dish, cells cultured on FHA-Mg scaffolds demonstrated better proliferation and comparable osteogenic differentiation. To eliminate the effect of osteogenic induction medium, hMSCs were cultured on FHA-Mg scaffolds in culture medium and an approximate 66% increase in osteogenic differentiation was observed three weeks later, indicating a significant effect of the nanostructured surface of FHA-Mg scaffolds on hMSC behaviors. With controllable Mg2+ release and favorable mechanical properties, porous FHA-Mg scaffolds have a great potential in cell-based bone regeneration.

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