Impaired Osteogenic Differentiation Of Mesenchymal Stem Cells Derived From Bone Marrow Of Patients With Low-Risk Myelodysplastic Syndromes

Abstract Background The pathogenesis of MDS has not been completely understood, and insufficiency of the hematopoietic microenvironment can be an important factor. MSCs and osteoblasts are key components of the hematopoietic microenvironment. Studying osteoblastic differentiation of MSCs quantitatively may help to understand the pathogenesis of MDS. Methods 38 patients with MDS and 15 normal donors were investigated in this study. Osteoblastic differentiation assays were performed in 16 MDS cases and 8 controls. The expression of osteogenic differentiation markers were measured by real-time PCR. Alkaline phosphatase staining was performed with Alkaline Phosphatase staining kit after 3,7,14 days of incubation. ALP activity was assessed at 3, 7, and 10 days after osteogenic differentiation. Mineralization analysis was performed at 7, 14 and 21 days of osteogenic induction. The areas of mineralization were measured by Image-Pro Plus 6.0 software. Results Both MDS-MSCs and normal cells displayed same fibroblast-like morphology and similar antigen expression. The expression level of RUNX2 was significantly decreased in MSCs from MDS, compaired with normal controls, especially in lower-risk MDS. After osteogenic induction, lower-risk MDS showed lower alkaline phosphatase activity, less intense alizarin red S staining, and lower gene expression of osteogenic differentiation markers, however, higher-risk MDS was normal. Conclusions We concluded that impaired osteogenic differentiation of MSCs was seen mainly in patients with lower-risk MDS. It may contribute to the ineffective hamatopoiesis of MDS. Disclosures: No relevant conflicts of interest to declare.

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microRNA-214-3p Suppresses Ankylosing Spondylitis Fibroblast Osteogenesis via BMP–TGFβ Axis and BMP2

Frontiers in Endocrinology ◽

10.3389/fendo.2020.609753 ◽

2021 ◽

Vol 11 ◽

Author(s):

Lixiang Ding ◽

Yukun Yin ◽

Yu Hou ◽

Haoran Jiang ◽

Ji Zhang ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Ankylosing Spondylitis ◽

Osteogenic Differentiation ◽

Target Gene ◽

Loss Of Function ◽

Alizarin Red ◽

Neck Fracture ◽

Osteogenic Induction ◽

Related Proteins ◽

Bone Morphogenic Protein 2

Recent investigations suggest microRNAs (miRs) exert functions in fibroblast osteogenesis in ankylosing spondylitis (AS), an inflammatory rheumatic disease. But the mechanism of miR-214-3p in osteogenic differentiation in AS is not clearly understood yet. In this study, fibroblasts were obtained from the capsular ligament of patients with AS and femoral neck fracture and cultured for osteogenic induction and identified. The roles of miR-214-3p and bone morphogenic protein 2 (BMP2) in AS fibroblast osteogenesis were assessed via gain- and loss-of-function, alizarin red S staining, and alkaline phosphatase (ALP) detection. Levels of miR-214-3p, BMP2, osteogenic differentiation-related proteins, and BMP–TGFβ axis-related proteins were further measured. Consequently, miR-214-3p was downregulated in AS fibroblasts, with enhanced ALP activity and calcium nodules, which were reversed by miR-214-3p overexpression. BMP2 was a target gene of miR-214-3p and promoted AS fibroblast osteogenesis by activating BMP–TGFβ axis, while miR-214-3p inhibited AS fibroblast osteogenesis by targeting BMP2. Together, miR-214-3p could prevent AS fibroblast osteogenic differentiation by targeting BMP2 and blocking BMP–TGFβ axis. This study may offer a novel insight for AS treatment.

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Effect of miR-192-5p on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Idiopathic Scoliosis by Regulating Wnt/β-catenin Signaling Pathway via RSPO1

10.21203/rs.3.rs-961628/v1 ◽

2021 ◽

Author(s):

Yifan Yang ◽

Jing Xu ◽

Qingxin Su ◽

Yiran Wu ◽

Qizheng Li ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Alkaline Phosphatase ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Signaling Pathway ◽

Alizarin Red ◽

Alkaline Phosphatase Staining ◽

Marrow Mesenchymal Stem Cells ◽

Related Proteins

Abstract BackgroundIdiopathic scoliosis (IS) is the most common structural scoliosis, which seriously affects not only patient’s physical and mental health but also quality of patient’s life. Abnormal osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is one of the causes of IS. However, the regulation mechanism of osteogenic differentiation of BMSCs in patients with IS remains to be further studied.MethodsSerum samples of 135 patients with IS were collected, and the expression of miRNA were detected by RT-qPCR. BMSCs from patients with IS were collected and the expression of miR-192-5p in BMSCs from IS patients and normal BMSCs was detected by RT-qPCR. Double luciferase reporter genes assay was used to verify the targeting relationship between miR-192-5p and RSPO1. The levels of RSPO1, osteogenic related proteins (OC, OPN and RUNX2) and Wnt/β-catenin signaling pathway related proteins (WNT3A and β-catenin) were detected by Western blotting. Alkaline phosphatase staining and alizarin red staining were used to evaluate the osteogenesis of BMSCs.ResultsmiR-192-5p was significantly up-regulated in serum and BMSCs of patients with IS. Alkaline phosphatase staining and alizarin red staining showed that miR-192-5p inhibitor promoted the osteogenic differentiation of BMSCs from IS patients. miR-192-5p targeted down-regulated the expression of RSPO1 in BMSCs from IS patients. In addition, overexpression of RSPO1 activated Wnt/β-catenin signaling pathway in BMSCs from IS patients. Furthermore, miR-192-5p/RSPO1 axis regulated levels of osteogenic related proteins (OC, OPN and RUNX2) in BMSCs from IS patients through Wnt/β-catenin signaling pathway, and affected the osteogenic differentiation of BMSCs.ConclusionmiR-192-5p, which was highly expressed in patients with IS, inhibited Wnt/β-catenin signaling pathway by down-regulating RSPO1 protein and then reduced the osteogenic differentiation ability of BMSCs.

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Inhibition of the Mammalian Target of Rapamycin Pathway Stimulates Osteoblast Proliferation and Differentiation Through Autophagy in MC3T3-E1 Cells

10.21203/rs.3.rs-36977/v1 ◽

2020 ◽

Author(s):

Xining Li ◽

Xi-Ning Li ◽

Yu Zhao ◽

Zhi-Gang Zhou ◽

Hong-Chang Zhou ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Mammalian Target Of Rapamycin ◽

Mtor Pathway ◽

Target Of Rapamycin ◽

Differentiation Markers ◽

Alizarin Red ◽

Sequestosome 1 ◽

Alkaline Phosphatase Staining ◽

Proliferation And Differentiation ◽

Ribosomal S6 Kinase

Abstract Background: OP(Osteoporosis) is a common bone metabolic disorder in the elderly characterized by loss of bone mass and a tendency to fracture. The mammalian target of rapamycin (mTOR) pathway in autophagy plays an indispensable role in maintaining the stability of the intracellular environment and ensuring the normal physiological functions of cells. Methods: In this study, different concentrations(20, 40, 60, 80, 100, 120, 140, 160, 180 and 200nM) of rapamycin were used to act on MC3T3-E1 osteoblasts for different time lengths(6, 12, 24, 36 and 48 hours). CCK8 was used to detect the proliferative activity of cells and screen suitable rapamycin concentration for subsequent experiments. Western blot and real-time quantitative PCR were used to detect the expression changes of phosphorylated mTOR, upstream and downstream mTOR pathway, autophagy and osteogenic differentiation markers. The expression of LC3 was observed by immunofluorescence. The differentiation ability of osteoblasts was observed by alizarin red and alkaline phosphatase staining.Results: The results showed that the induction of proliferation activity of osteoblasts from 20 nM to 200 nM presented a parabolic feature. After the action time of 50 μM rapamycin exceeded 12 hours, the proportion of S stage cells was significantly increased. The results of gene and protein analysis showed that rapamycin significantly inhibited the phosphorylation of mTOR, and the phosphorylation of the downstream factors of mTOR, 4E-BP1(eIF4E-binding protein 1) and S6K1(p70 ribosomal S6 kinase 1) also decreased. Rapamycin significantly increased the expression of LC3 II (microtubule associated protein 1 light chain 3-α), significantly increased the ratio of LC3II/LC3I, and significantly decreased the expression of p62(sequestosome-1). Rapamycin significantly induced the expression of ALP(Alkaline phosphatase), Runx2(Runt-related transcription factor 2) and osterix. Conclusions: This study confirmed that rapamycin stimulates the autophagy of osteoblasts by inhibiting mTOR and promotes their proliferation and differentiation, suggesting that mTOR may be a potential therapeutic target for osteoporosis.

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Stretch-Induced Lncrna SNHG8 Inhibits Osteogenic Differentiation by Regulating EZH2 in Hpdlscs

10.21203/rs.3.rs-362066/v1 ◽

2021 ◽

Author(s):

Zijie Zhang ◽

Qin He ◽

Xiaolu Zhao ◽

Xiaoyu Li ◽

Fulan Wei

Keyword(s):

Alkaline Phosphatase ◽

Western Blot ◽

Osteogenic Differentiation ◽

High Throughput ◽

High Throughput Sequencing ◽

Tooth Movement ◽

Alizarin Red ◽

Qrt Pcr ◽

Osteogenic Induction

Abstract Background: Periodontal ligament stem cells (PDLSCs) are important for the remodeling of the alveolar bone while tooth moving. However, the effect of long non-coding RNA (lncRNA) on osteogenic differentiation of PDLSCs under mechanical force remains unclear.Methods: In this study, we compared stretched and non-stretched PDLSCs by high-throughput sequencing. The verification and selection of lncRNAs were achieved by quantitative reverse transcription polymerase chain reaction (qRT-PCR). PDLSCs osteogenic differentiation potentials were assessed by alkaline phosphatase (ALP) staining, Alizarin Red staining, qRT-PCR, and western blot. The application of mechanical force used Flexcell-FX-6000-Tension System in vitro, and constructing rats’ tooth movement model in vivo. To verify the osteogenic regulation ability of small nucleolar RNA host gene 8 (SNHG8), PDLSCs were stretched or applied osteogenic induction after been infected by lentivirus. RNA fluorescence in situ hybridization, isolation of nuclear and cytoplasmic RNA, qRT-PCR and western blot were performed to locate SNHG8. Western blot and qRT-PCR to find the relationship between enhancer of zeste homolog 2 (EZH2) and SNHG8.Results: Our results demonstrated that among lncRNAs altered screened by high-throughput sequencing, the expression level of SNHG8 steadily decreased after being stretched. Analysis of mRNA expression and protein levels revealed an upregulation of ALP and RUNX2, ALP and Alizarin Red staining showed more obvious alkaline phosphatase and more mineralized nodules in SNHG8 knockdown PDLSCs. In vivo experiments showed lower expression of the homologous gene of SNHG8 after tooth movement, and better ability of ectopic osteogenesis after knockdown SNHG8. The verification of SNHG8’s nuclear location led us to infer that SNHG8 may interact with EZH2. The qRT-PCR and western blot results disclosed EZH2 expression reduced along with the knockdown of SNHG8. Furthermore, knockdown of EZH2 lead to PDLSCs’ osteogenic differentiation ability increasing under osteogenic induction according to the mRNA level of ALP and RUNX2 accompanied by ALP and Alizarin Red staining results.Conclusion: In general, our study confirmed that mechanically sensitive lncRNA SNHG8 can influence the osteogenic differentiation of PDLSCs through epigenetic pathways without directly encoding protein, which provides solid evidence for the regulation by non-coding genes.

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Downregulation of the LncRNA MEG3 Promotes Osteogenic Differentiation of BMSCs and Bone Repairing by Activating Wnt/β-Catenin Signaling Pathway

Journal of Clinical Medicine ◽

10.3390/jcm11020395 ◽

2022 ◽

Vol 11 (2) ◽

pp. 395

Author(s):

Juan Liu ◽

Xin Qi ◽

Xiao-Hong Wang ◽

Hong-Sheng Miao ◽

Zi-Chao Xue ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Osteogenic Differentiation ◽

Signaling Pathway ◽

Rat Model ◽

Cell Counting ◽

Alizarin Red ◽

Skull Defects ◽

Endogenous Expression ◽

Alkaline Phosphatase Staining ◽

Bone Repairing

Background: Previous studies have demonstrated that long non-coding RNA maternally expressed gene 3 (MEG3) emerged as a key regulator in development and tumorigenesis. This study aims to investigate the function and mechanism of MEG3 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and explores the use of MEG3 in skull defects bone repairing. Methods: Endogenous expression of MEG3 during BMSCs osteogenic differentiation was detected by quantitative real-time polymerase chain reaction (qPCR). MEG3 was knockdown in BMSCs by lentiviral transduction. The proliferation, osteogenic-related genes and proteins expression of MEG3 knockdown BMSCs were assessed by Cell Counting Kit-8 (CCK-8) assay, qPCR, alizarin red and alkaline phosphatase staining. Western blot was used to detect β-catenin expression in MEG3 knockdown BMSCs. Dickkopf 1 (DKK1) was used to block wnt/β-catenin pathway. The osteogenic-related genes and proteins expression of MEG3 knockdown BMSCs after wnt/β-catenin inhibition were assessed by qPCR, alizarin red and alkaline phosphatase staining. MEG3 knockdown BMSCs scaffold with PHMG were implanted in a critical-sized skull defects of rat model. Micro-computed tomography(micro-CT), hematoxylin and eosin staining and immunohistochemistry were performed to evaluate the bone repairing. Results: Endogenous expression of MEG3 was increased during osteogenic differentiation of BMSCs. Downregulation of MEG3 could promote osteogenic differentiation of BMSCs in vitro. Notably, a further mechanism study revealed that MEG3 knockdown could activate Wnt/β-catenin signaling pathway in BMSCs. Wnt/β-catenin inhibition would impair MEG3-induced osteogenic differentiation of BMSCs. By using poly (3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHHx)-mesoporous bioactive glass (PHMG) scaffold with MEG3 knockdown BMSCs, we found that downregulation of MEG3 in BMSCs could accelerate bone repairing in a critical-sized skull defects rat model. Conclusions: Our study reveals the important role of MEG3 during osteogenic differentiation and bone regeneration. Thus, MEG3 engineered BMSCs may be effective potential therapeutic targets for skull defects.

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Effects of rutin on osteoblast MC3T3-E1 differentiation, ALP activity and Runx2 protein expression

European Journal of Histochemistry ◽

10.4081/ejh.2021.3195 ◽

2021 ◽

Vol 65 (1) ◽

Author(s):

Xin-Wei Liu ◽

Bin Ma ◽

Ying Zi ◽

Liang-Bi Xiang ◽

Tian-Yu Han

Keyword(s):

Western Blot ◽

Osteogenic Differentiation ◽

Real Time ◽

Real Time Pcr ◽

Osteoblastic Differentiation ◽

Control Group ◽

Alizarin Red ◽

Wide Range ◽

Osteogenic Induction ◽

Orthopedic Diseases

As a flavonoid, rutin has been found to have a wide range of biological functions, such as resisting inflammation and oxidation, and preventing cerebral hemorrhage and hypertension. It has been found to play an important role in osteoporosis and other orthopedic diseases in recent years. MC3T3-E1 cells were randomly divided into a control group, a rutin-1 group (0.01 mmol/L), a rutin-2 group (0.05 mmol/L) and a rutin-3 group (0.1 mmol/L). Osteogenic differentiation of cells was induced by osteogenic induction fluid. The control group was treated with the maximum dose of drug solvent. 2~3 days later, the solvent was replaced with fresh osteogenic induction fluid containing rutin. After a certain period of routine culture, the cells were collected for subsequent experiments. The expression of Runx2 gene in cells in all groups was detected by Real-time PCR; the expression of Runx2 protein was detected by Western blot and immunocytochemistry; the activity of ALP was detected by reagent kit method; osteogenic differentiation was analyzed by alizarin red staining. The results of Real-time PCR showed that, compared with the control group, the treatment of cells with rutin can significantly increase the expression of Runx2 gene (p<0.05); the higher the concentration, the higher the expression of Runx2 gene, and significant differences were found among groups in which different concentrations were used (p<0.05); the results of Western blot and IHC showed that the expression trend of Runx2 protein in each group was consistent with PCR results. In drug treatment groups, the activity of ALP was significantly higher than that in the control group (p<0.05); there were significant differences among groups in which different concentrations were used (p<0.05). The results of alizarin red staining showed that calcified nodules were formed in all groups and that the area of calcified nodules formed in groups treated with rutin was greater than that in the control group; the greater the concentration, the larger the area. Rutin can promote osteoblastic differentiation; and the greater the concentration, the more effective it is.

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Downregulation of the long non-coding RNA MEG3 promotes osteogenic differentiation of BMSCs and bone repairing by activating Wnt/β-catenin signaling pathway

10.21203/rs.3.rs-413260/v1 ◽

2021 ◽

Author(s):

Juan Liu ◽

Xin Qi ◽

Hong-Sheng Miao ◽

Zi-Chao Xue ◽

San-Hu Zhao ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Rat Model ◽

Alizarin Red ◽

Skull Defects ◽

Alkaline Phosphatase Staining ◽

Non Coding Rna ◽

Bone Repairing ◽

Long Non Coding Rna

Abstract Background: Previous studies have demonstrated long non-coding RNA maternally expressed gene 3 (MEG3) emerged as a key regulator in development and tumorigenesis. However, whether MEG3 participate in osteogenic differentiation and bone regeneration remains unclear. This study aims to investigate the function and mechanism of MEG3 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), and explores the use of MEG3 in skull defects bone repairing. Methods: Endogenous expression of MEG3 during BMSCs osteogenic differentiation were detected by qPCR. MEG3 was knockdown in BMSCs by lentivirus. The proliferation, osteogenic-related genes and proteins expression were assessed by the CCK-8, PCR, alizarin red and alkaline phosphatase staining in MEG3 knockdown BMSCs. Western blot was used to detected β-catenin expression in MEG3 knockdown BMSCs. DKK1 was used to block wnt/β-catenin pathway, the osteogenic-related genes and proteins expression were assessed by PCR, alizarin red and alkaline phosphatase staining in MEG3 knockdown BMSCs. MEG3 knockdown BMSCs scaffold with PHMG were implanted in a critical-sized skull defects of rat model, micro-CT, hematoxylin and eosin staining, and immunohistochemistry were performed to evaluate the bone repairing. Results: MEG3 was increased during osteogenic differentiation of BMSCs. Downregulation of MEG3 could promote osteogenic differentiation of BMSCs in vitro. Notably, a further mechanism study revealed MEG3 knockdown could activate Wnt/β-catenin signaling pathway in BMSCs. Wnt/β-catenin inhibition would impair MEG3-induced osteogenic differentiation of BMSCs. By using PHMG scaffold with MEG3 knockdown BMSCs, we found that downregulation of MEG3 in BMSCs could accelerated bone repairing in a critical-sized skull defects rat model. Conclusions: Our study reveals the important role of MEG3 during osteogenic differentiation and bone regeneration. Thus, MEG3 engineered BMSCs may be effective potential therapeutic targets for skull defects.

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The inhibitory effects of vancomycin on rat bone marrow–derived mesenchymal stem cell differentiation

Journal of Neurosurgery Spine ◽

10.3171/2020.10.spine201511 ◽

2021 ◽

pp. 1-5

Author(s):

Kari Hanson ◽

Carly Isder ◽

Kristen Shogren ◽

Anthony L. Mikula ◽

Lichun Lu ◽

...

Keyword(s):

Bone Marrow ◽

Alkaline Phosphatase ◽

Osteogenic Differentiation ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

High Dose ◽

Inhibitory Effects ◽

Alizarin Red ◽

Osteogenic Factors

OBJECTIVE The use of intrawound vancomycin powder in spine surgery has been shown to decrease the rate of surgical site infections; however, the optimal dose is unknown. High-dose vancomycin inhibits osteoblast proliferation in vitro and may decrease the rate of solid arthrodesis. Bone marrow–derived mesenchymal stem cells (BMSCs) are multipotent cells that are a source of osteogenesis in spine fusions. The purpose of this study was to determine the effects of vancomycin on rat BMSC viability and differentiation in vitro. METHODS BMSCs were isolated from the femurs of immature female rats, cultured, and then split into two equal groups; half were treated to stimulate osteoblastic differentiation and half were not. Osteogenesis was stimulated by the addition of 50 µg/mL l-ascorbic acid, 10 mM β-glycerol phosphate, and 0.1 µM dexamethasone. Vancomycin was added to cell culture medium at concentrations of 0, 0.04, 0.4, or 4 mg/mL. Early differentiation was determined by alkaline phosphatase activity (4 days posttreatment) and late differentiation by alizarin red staining for mineralization (9 days posttreatment). Cell viability was determined at both the early and late time points by measurement of formazan colorimetric product. RESULTS Viability within the first 4 days decreased with high-dose vancomycin treatment, with cells receiving 4 mg/mL vancomycin having 40%–60% viability compared to the control. A gradual decrease in alizarin red staining and nodule formation was observed with increasing vancomycin doses. In the presence of the osteogenic factors, vancomycin did not have deleterious effects on alkaline phosphatase activity, whereas a trend toward reduced activity was seen in the absence of osteogenic factors when compared to osteogenically treated cells. CONCLUSIONS Vancomycin reduced BMSC viability and impaired late osteogenic differentiation with high-dose treatment. Therefore, the inhibitory effects of high-dose vancomycin on spinal fusion may result from both reduced BMSC viability and some impairment of osteogenic differentiation.

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Alpha-Lipoic Acid Alleviates High-Glucose Suppressed Osteogenic Differentiation of MC3T3-E1 Cells via Antioxidant Effect and PI3K/Akt Signaling Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000479605 ◽

2017 ◽

Vol 42 (5) ◽

pp. 1897-1906 ◽

Cited By ~ 12

Author(s):

Kai Dong ◽

Pengjie Hao ◽

Sheng Xu ◽

Shutai Liu ◽

Wenjuan Zhou ◽

...

Keyword(s):

Oxidative Damage ◽

Osteogenic Differentiation ◽

Lipoic Acid ◽

High Glucose ◽

Osteoblastic Differentiation ◽

Antioxidant Effect ◽

Akt Pathway ◽

Alpha Lipoic Acid ◽

Western Blots ◽

Alizarin Red

Background/Aims: Patients with diabetes mellitus have a higher risk of dental implant failure. One major cause is high-glucose induced oxidative stress. Alpha-lipoic acid (ALA), a naturally occurring compound and dietary supplement, has been established as a potent antioxidant that is a strong scavenger of free radicals. However, few studies have yet investigated the effect of ALA on osteogenic differentiation of osteoblasts cultured with high glucose medium. The aim of this study is to investigate the effects of ALA on the osteoblastic differentiation in MC3T3-E1 cells under high glucose condition. Methods: MC3T3-E1 cells were divided into 4 groups including normal glucose (5.5 mM) group (control), high glucose (25.5 mM) group, high glucose + 0.1 mM ALA group, and high glucose + 0.2 mM ALA group. The proliferation, osteogenic differentiation and mineralization of cells were evaluated by MTT assay, alkaline phosphatase (ALP) activity assay, alizarin red staining and real time-polymerase chain reaction. High-glucose induced oxidative damage was also assessed by the production of reactive oxygen species (ROS) and superoxide dismutase (SOD). Western blots were performed to examine the role of PI3K/Akt pathway. Results: The proliferation, osteogenic differentiation and mineralization of MC3T3-E1 cells were significantly decreased by the ROS induced by high-glucose. All observed oxidative damage and osteogenic dysfunction induced were inhibited by ALA. Moreover, the PI3K/Akt pathway was activated by ALA. Conclusions: We demonstrate that ALA may attenuate high-glucose mediated MC3T3-E1 cells dysfunction through antioxidant effect and modulation of PI3K/Akt pathway.

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

Stem Cells International ◽

10.1155/2018/9781393 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 5

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.

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