Icariin Protects against Glucocorticoid-Induced Osteonecrosis of the Femoral Head in Rats

Background/Aims: Glucocorticoid (GC)-related osteonecrosis of the femoral head (ONFH) is a common complication following administration of steroids to treat many diseases. Our previous study demonstrated that icariin (ICA) might have a beneficial effect on the bone marrow mesenchymal stem cells (BMSCs) of patients with steroid-associated osteonecrosis. In this study, we investigated the underlying mechanisms of ICA associated with the potential enhancement of osteogenesis and anti-adipogenesis in GC-related ONFH. Methods: In vitro cell proliferation was evaluated by CCK-8 assay. Alizarin red S and alkaline phosphatase (ALP) activity were used to measure osteogenic differentiation, while adipogenic differentiation was revealed by oil red O staining and TG content assay. The expression level of osteogenesis-associated genes and PPARγ was evaluated by RT-qPCR, western blotting and immunofluorescence. A total of 30 female SD rats were randomly separated into three groups: a control group, a methylprednisolone (MPS) group and a MPS + ICA group. Serum ALP and TG (triglyceride), micro-CT scanning, histological and immunohistochemical analyses were performed in the animal model. Results: In the in vitro study, ICA promoted proliferation, improved osteogenic differentiation and suppressed adipogenic differentiation of BMSCs treated with MPS. The group treated with MPS and 10-6 M ICA expressed higher levels of Runx2, ALP, bone morphogenetic protein (BMP) 2, and OC and lower expression of PPARγ than the MPS group. In the in vivo study, ICA prevented bone loss in a rat model of GC-related ONFH as shown by micro-CT scanning, histological and immunohistochemical analyses. Conclusions: ICA is an effective compound for promoting bone repair and preventing or delaying the progression of GC-associated ONFH in rats. This effect can be explained by its ability to improve the balance between adipogenesis and osteogenesis, indicating that ICA is an effective candidate for management of GC-associated ONFH.

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The Mechanism, Regulatory Effect of Wnt5α/Ca2+ Signaling Pathway on hMSCs Osteogenic Differentiation Under Infection Conditions

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2019.2191 ◽

2019 ◽

Vol 9 (12) ◽

pp. 1706-1711

Author(s):

Yilizati Yilihamu ◽

Liu Yansong ◽

Xu Mingliang ◽

Cao Tianyong ◽

Shi Rongjian

Keyword(s):

Osteogenic Differentiation ◽

Signaling Pathway ◽

Calcium Concentration ◽

Wnt Signaling Pathway ◽

Quantitative Detection ◽

Control Group ◽

Specific Gene ◽

Beta Catenin ◽

Alizarin Red

Background: The paper aimed to reveal the role and possible regulatory mechanisms of Wnt5 gene and its non-canonical Beta-catenin signaling pathway in osteogenic differentiation and bone repairment of hMSCs under infection environment. Material and Methods : CCK-8 method was employed to detect the effect of SPA on the proliferation of different hMSCs cells. The histological staining of alizarin red was used to observe the differentiation of cells into osteoblasts. The semi-quantitative detection of osteoblast alkaline phosphatase (ALP) was performed. Fluo-2 assay was employed to measure the intracellular calcium concentration of different hMSCs before and after osteogenic differentiation stimulated by SPA. RT-PCR was applied to detect the specific gene fluorescence of osteoblasts differentiation. Western Blotting was employed to determine the differentiation of hMSCs into osteoblasts. Results: Different hMSCs were able to proliferate in vitro under the stimulation of 100 ng/mL SPA. CCK-8 kit was used to detect lentivirus-infected hMSCs and non-transduced hMSCs in vitro. The results showed that all the cells gradually proliferate with time and an increasing rate. The hMSCs treated with different Wnt5 genes were stained with alizarin red after osteogenic differentiation, and the activities of AMSCs ALP increased. The intracellular free calcium concentration increased with the induction time and peaked in the Wnt5 overexpression group. PCR results showed that ALP, Runx-2, Collagen I, OC, Osterix and BSP genes were all increased in contrast with the non-induction Control group. Conclusion: Under the inflammatory condition of SPA virulence factors, non-canonical Wnt signaling pathway is associated with the classical Wnt/ β-catenin signaling pathway in the process of osteogenic differentiation of hMSCs in vitro, which has a certain synergistic effect.

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Effects of Concentrated Growth Factor on the Proliferation, Migration, and Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells: An in vitro study

10.21203/rs.3.rs-29138/v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Xiang Yu ◽

Hui Ren ◽

Qi Shang ◽

Gengyang Shen ◽

Kai Tang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Osteogenic Differentiation ◽

Control Group ◽

Elisa Assay ◽

Alizarin Red ◽

Expression Levels ◽

Marrow Mesenchymal Stem Cells ◽

Mrna Expression Levels

Abstract Background Concentrated growth factor (CGF) has been reported to be effective in bone formation or soft/hard tissue healing in recent years. Despite a few studies regarding the effects of CGF on the proliferation, migration, and osteogenic differentiation of BMSCs, their underlying mechanisms are not fully understood. The purpose of this study is to investigate the effects and possible mechanisms of CGF on the proliferation, migration, and osteogenic differentiation of rat-derived bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods CGF was extracted from the Sprague Dawley (SD) rats by venipuncture of the abdominal aortic vein, and scanning electron microscopy (SEM) was used for the structural characterization. The release of bone morphogenetic protein 2 (BMP-2) from CGF was measured over the periods of 1 ~ 14 days, using the enzyme-linked immunosorbent (Elisa) assay. Cell Counting Kit-8 (CCK-8) assay was used to measure cell proliferation. Migration capacity was analyzed using the transwell assay. The osteogenic differentiation and mineralization ability were determined by Alkaline phosphatase activity (ALP) staining and Alizarin Red staining respectively. Quantitative real-time PCR (RT-qPCR), was used to evaluate the mRNA expression levels of Runx2, Ocn, Smad1, and Smad5 after culture for 14 days. Further, the protein expression of BMP-2, phosphorylated-Smad1/5 (p-Smad1/5), and Smad1/5/8 was determined by Western blot after a 14-day cell culture. Results The SEM analysis showed a porous and dense three-dimensional fibrin network in CGF. The Elisa assay showed that BMP-2 was released from CGF extract for more than 14d, and it reached a peak at the time point of 5d. The cell densities of the CGF group at the different concentrations (5%, 10%, and 20%) were significantly higher than that of the control group at the periods of day 1 to day 5 (p < 0.05). Moreover, the number of migratory cells of the CGF group was greater than that of the control group at 24 h. ALP activity analysis and Alizarin Red staining results demonstrated that CGF may successfully induce osteogenic differentiation of BMSCs. Moreover, the RT-qPCR results showed that CGF extracts dramatically enhanced the mRNA expression levels of Runx2, Ocn, Smad1, and Smad5 in BMSCs at days 14 (p < 0.05). Furthermore, Western blot results showed that CGF extracts markedly up-regulated the protein expression levels of BMP-2, p-Smad1/5, and Smad1/5/8. Conclusions CGF can promote the proliferation, migration, and promote the osteogenic differentiation potential of BMSCs in vitro. The BMP-2/Smad signaling pathway was involved in the osteogenic differentiation and mineralization of BMSCs induced by CGF. Therefore, CGF has good application potential in tissue engineering for bone regeneration and repair.

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Calcium Polyphosphate Nanoparticles Act as an Effective Inorganic Phosphate Source during Osteogenic Differentiation of Human Mesenchymal Stem Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20225801 ◽

2019 ◽

Vol 20 (22) ◽

pp. 5801 ◽

Cited By ~ 4

Author(s):

Luan Phelipe Hatt ◽

Keith Thompson ◽

Werner E. G. Müller ◽

Martin James Stoddart ◽

Angela Rita Armiento

Keyword(s):

Osteogenic Differentiation ◽

Bone Repair ◽

Human Mesenchymal Stem Cells ◽

Bone Diseases ◽

Alizarin Red ◽

Alp Activity ◽

Calcium Polyphosphate ◽

Phosphate Source ◽

Matrix Mineralisation

The ability of bone-marrow-derived mesenchymal stem/stromal cells (BM-MSCs) to differentiate into osteoblasts makes them the ideal candidate for cell-based therapies targeting bone-diseases. Polyphosphate (polyP) is increasingly being studied as a potential inorganic source of phosphate for extracellular matrix mineralisation. The aim of this study is to investigate whether polyP can effectively be used as a phosphate source during the in vitro osteogenic differentiation of human BM-MSCs. Human BM-MSCs are cultivated under osteogenic conditions for 28 days with phosphate provided in the form of organic β-glycerolphosphate (BGP) or calcium-polyP nanoparticles (polyP-NP). Mineralisation is demonstrated using Alizarin red staining, cellular ATP content, and free phosphate levels are measured in both the cells and the medium. The effects of BGP or polyP-NP on alkaline phosphatase (ALP) activity and gene expression of a range of osteogenic-related markers are also assessed. PolyP-NP supplementation displays comparable effects to the classical BGP-containing osteogenic media in terms of mineralisation, ALP activity and expression of osteogenesis-associated genes. This study shows that polyP-NP act as an effective source of phosphate during mineralisation of BM-MSC. These results open new possibilities with BM-MSC-based approaches for bone repair to be achieved through doping of conventional biomaterials with polyP-NP.

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EGFL6 regulates angiogenesis and osteogenesis in distraction osteogenesis via Wnt/β-catenin signaling

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02487-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Junjie Shen ◽

Yi Sun ◽

Xuanzhe Liu ◽

Yu Zhu ◽

Bingbo Bao ◽

...

Keyword(s):

Bone Formation ◽

Osteogenic Differentiation ◽

Distraction Osteogenesis ◽

Bone Repair ◽

Angiogenic Factor ◽

Osteogenic Potential ◽

Human Umbilical Cord ◽

Alizarin Red ◽

Differentiation Potential

Abstract Background Osteogenesis is tightly coupled with angiogenesis during bone repair and regeneration. However, the underlying mechanisms linking these processes remain largely undefined. The present study aimed to test the hypothesis that epidermal growth factor-like domain-containing protein 6 (EGFL6), an angiogenic factor, also functions in bone marrow mesenchymal stem cells (BMSCs), playing a key role in the interaction between osteogenesis and angiogenesis. Methods We evaluated how EGFL6 affects angiogenic activity of human umbilical cord vein endothelial cells (HUVECs) via proliferation, transwell migration, wound healing, and tube-formation assays. Alkaline phosphatase (ALP) and Alizarin Red S (AR-S) were used to assay the osteogenic potential of BMSCs. qRT-PCR, western blotting, and immunocytochemistry were used to evaluate angio- and osteo-specific markers and pathway-related genes and proteins. In order to determine how EGFL6 affects angiogenesis and osteogenesis in vivo, EGFL6 was injected into fracture gaps in a rat tibia distraction osteogenesis (DO) model. Radiography, histology, and histomorphometry were used to quantitatively evaluate angiogenesis and osteogenesis. Results EGFL6 stimulated both angiogenesis and osteogenic differentiation through Wnt/β-catenin signaling in vitro. Administration of EGFL6 in the rat DO model promoted CD31hiEMCNhi type H-positive capillary formation associated with enhanced bone formation. Type H vessels were the referred subtype involved during DO stimulated by EGFL6. Conclusion EGFL6 enhanced the osteogenic differentiation potential of BMSCs and accelerated bone regeneration by stimulating angiogenesis. Thus, increasing EGFL6 secretion appeared to underpin the therapeutic benefit by promoting angiogenesis-coupled bone formation. These results imply that boosting local concentrations of EGFL6 may represent a new strategy for the treatment of compromised fracture healing and bone defect restoration.

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EGFL6 Regulates the Interaction Between Angiogenesis and Osteogenesis via Wnt/β-catenin Signaling in Distraction Osteogenesis

10.21203/rs.3.rs-251914/v1 ◽

2021 ◽

Author(s):

Junjie Shen ◽

Yi Sun ◽

Xuanzhe Liu ◽

Yu Zhu ◽

Bingbo Bao ◽

...

Keyword(s):

Bone Formation ◽

Osteogenic Differentiation ◽

Distraction Osteogenesis ◽

Bone Repair ◽

Angiogenic Factor ◽

Osteogenic Potential ◽

Human Umbilical Cord ◽

Alizarin Red ◽

Differentiation Potential

Abstract Background: Osteogenesis is tightly coupled with angiogenesis during bone repair and regeneration. However, the underlying mechanisms linking these processes remain largely undefined. The present study aimed to test the hypothesis that epidermal growth factor-like domain-containing protein 6 (EGFL6), an angiogenic factor, also functions in bone marrow mesenchymal stem cells (BMSCs) and plays a key role in the interaction between osteogenesis angiogenesis.Methods: We evaluated how EGFL6 affects angiogenic activity of human umbilical cord vein endothelial cells (HUVECs) via proliferation, transwell migration, wound healing, and tube-formation assays. Alkaline phosphatase (ALP) and Alizarin Red S (AR-S) were used to assay the osteogenic potential of BMSCs. qRT-PCR, western blotting, and immunocytochemistry were used to evaluate angio- and osteo-specific markers and pathway-related genes and proteins. In order to determine how EGFL6 affects angiogenesis and osteogenesis in vivo, EGFL6 was injected into fracture gaps in a rat tibia distraction osteogenesis (DO) model. Radiography, histology, and histomorphometry were used to quantitatively evaluate angiogenesis and osteogenesis. Results: EGFL6 stimulated both angiogenesis and osteogenic differentiation through Wnt/β-catenin signaling in vitro. Administration of EGFL6 in the rat DO model promoted CD31hiEMCNhi type H-positive capillary formation associated with enhanced bone formation. Type H vessels were the referred subtype involved during DO stimulated by EGFL6.Conclusion: EGFL6 enhanced osteogenic differentiation potential of BMSCs and accelerated bone regeneration by stimulating angiogenesis, thus exerting therapeutic benefit by increasing EGFL6 secretion to promote angiogenesis-coupled bone formation. These results imply that boosting local concentrations of EGFL6 may represent a new strategy for the treatment of compromised fracture healing and bone defect restoration.

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Glucocorticoids induce osteonecrosis of the femoral head through the Hippo signaling pathway

Open Life Sciences ◽

10.1515/biol-2021-0102 ◽

2021 ◽

Vol 16 (1) ◽

pp. 1130-1140

Author(s):

Yugang Li ◽

Zechuan Xu ◽

Shan Chang

Keyword(s):

Femoral Head ◽

Signaling Pathway ◽

Adipogenic Differentiation ◽

Hippo Pathway ◽

Control Group ◽

Hippo Signaling ◽

Dependent Manner ◽

Alizarin Red ◽

Hippo Signaling Pathway ◽

Normal Differentiation

Abstract Osteonecrosis of the femoral head (ONFH) induced by glucocorticoids (GCs) has been considered to be associated with the dysfunction of bone marrow mesenchymal stem cells (BMSCs). Studies have reported that GCs can regulate the normal differentiation of BMSCs. However, the exact mechanism of this regulation remains unclear. In this study, we used methylprednisolone (MPS) to induce BMSCs, and then found that the Hippo signaling pathway was upregulated in a dose-dependent manner compared to that in the control group. In addition, the osteogenic ability of BMSCs was decreased, as evaluated by Alizarin Red S staining analysis and alkaline phosphatase activity assays, accompanied by the downregulated expression of Runx2, osteopontin, and osteocalcin. Additionally, the adipogenic capacity of BMSCs under the MPS conditions was increased, as identified by Oil Red O staining with upregulated triglyceride and PPARγ expression. Moreover, suppression by knockdown of MST1 was found to attenuate the Hippo signaling pathway and adipogenic differentiation, while enhancing osteogenic differentiation. In conclusion, our findings revealed that the Hippo signaling pathway was involved in GC-ONFH by affecting the osteogenic and adipogenic differentiation capacities of BMSCs. Our study could provide a basis for further investigation of the specific function of the Hippo pathway in ONFH.

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186 The effects of different concentrations of MgSO4 in osteogenic differentiation

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab186 ◽

2019 ◽

Vol 31 (1) ◽

pp. 217

Author(s):

L. R. Padoveze ◽

M. Rubessa ◽

C. E. Ambrosio ◽

M. B. Wheeler

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

In Vitro Study ◽

Control Group ◽

Nodule Formation ◽

Osteogenic Medium ◽

Alizarin Red ◽

Physiological Processes

Tissue engineering offers a viable alternative to bone grafts in repairing large bone defects. Magnesium-based materials are biocompatible in vivo, and it is possible to determine the degradation period according to the necessities (Farraro et al. 2014 J. Biomech. 47, 1979-1986). Magnesium (Mg) is part of many physiological processes, and it promotes the osteogenesis of mesenchymal stem cells (Díaz-Tocados et al. 2017 Sci. Rep. 7, 7839.). Moreover, Mg up-regulates important genes associated with the osteogenic differentiation (Yoshizawa et al. 2014 Acta Biomater. 10, 2834-2842). The aim of this study was to evaluate the effect of different Mg concentrations in the osteogenic medium on the number of nodules of bone. Swine adipose stem cells (ASC) were previously isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20-33). In this in vitro study, ASC were cultured during 4 weeks in osteogenic medium with addition of 0.1, 0.2, 1, 2, 10, or 20mM MgSO4. The medium was changed twice a week. Alizarin Red and Von Kossa staining were performed to evaluate the formation of nodules by mineralization of extracellular matrix (ECM), evidenced by dark red nodules and calcium deposit. The experiment was replicated 3 times in triplicate. Data were analysed using the generalized linear model (GLM) procedure, and Bonferroni’s post hoc test was used to perform statistical multiple comparison (SPSS Inc./IBM Corp., Chicago, IL, USA). The results showed enhanced nodule formation with 2mM Mg in the osteogenic medium (35.6v. 15.3, respectively for 2mM and Control). This result confirms the ability of magnesium to act in bone formation. There was no statistical difference among the different groups when we evaluated the Von Kossa staining results, indicating that the quality of the new formations was comparable to that of the control group even in an elevated nodule formation. In conclusion, a higher concentration of magnesium can improve nodule formation into osteogenic differentiation in vitro; the 2mM concentration showed the best nodule formation compared with the other groups. These results showed the value of magnesium in bone physiology.

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Synthesis and characterization of melatonin-loaded chitosan microparticles promote differentiation and mineralization in pre-osteoblastic cells

Journal of Oral Implantology ◽

10.1563/aaid-joi-d-19-00208 ◽

2020 ◽

Author(s):

Ren-Yeong Huang ◽

Po-Yan Hsiao ◽

Lian-Ping Mau ◽

Yi-Wen Cathy Tsai ◽

David L. Cochran ◽

...

Keyword(s):

Sustained Release ◽

Osteogenic Differentiation ◽

Control Group ◽

Alizarin Red ◽

Positive Effects ◽

Related Transcription Factor ◽

Release Assay ◽

Investigation Period ◽

Novel Scaffold

In terms of a novel scaffold with well osteoinductive and osteoconductive capacity, melatonin (Mel) possesses positive effects on chemical linkage in scaffold structures, which may allow osteogenic differentiation. The aim of this study is to fabricate Mel-loaded chitosan (CS) microparticles (MPs) as a novel bone substitute through generating a Mel sustained release system from Mel-loaded CS MPs, and evaluating its effect on the osteogenic capacity of MC3T3-E1 in vitro. The physical-chemical characteristics of the prepared CS MPs were examined by both Fourier transform infrared spectroscopy and scanning electron microscopy. The released profile and kinetics of Mel from MPs were quantified, and the bioactivity of the released Mel on pre-osteoblasts MC3T3-E1 cells was characterized in vitro. In vitro drug release assay has shown high encapsulation efficiency and sustained release of Mel over the investigation period. In an osteogenesis assay, Mel-loaded CS MPs have significantly enhanced Alkaline phosphatase (Alp) mRNA expression and ALP activity compared to the control group. Meanwhile, the osteoblast specific differentiation genes, including runt related transcription factor 2 (Runx2), bone morphogentic protein-2 (Bmp2), and collagen I (Col I) and osteocalcin (Ocn) were also significantly up-regulated. Furthermore, quantificational alizarin red-based assay demonstrated that Mel-loaded CS MPs notably enhanced the calcium deposit of MC3T3-E1 compared to the controls. In essence, Mel-loaded CS MPs can control the release of Mel for a period of time to accelerate osteogenic differentiation of pre-osteoblast cells in vitro.

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Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses

Stem Cell Research & Therapy ◽

10.1186/s13287-020-02085-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ping Zhou ◽

Jia-Min Shi ◽

Jing-E Song ◽

Yu Han ◽

Hong-Jiao Li ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Telomerase Activity ◽

Human Pluripotent Stem Cells ◽

Cell Counting ◽

Cell Type ◽

Alizarin Red

Abstract Background Derivation of osteoblast-like cells from human pluripotent stem cells (hPSCs) is a popular topic in bone tissue engineering. Although many improvements have been achieved, the low induction efficiency because of spontaneous differentiation hampers their applications. To solve this problem, a detailed understanding of the osteogenic differentiation process of hPSCs is urgently needed. Methods Monolayer cultured human embryonic stem cells and human-induced pluripotent stem cells were differentiated in commonly applied serum-containing osteogenic medium for 35 days. In addition to traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and alizarin red staining, we also applied studies of cell counting, cell telomerase activity, and flow cytometry as essential indicators to analyse the cell type changes in each week. Results The population of differentiated cells was quite heterogeneous throughout the 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type and tumourigenicity of the obtained cells. Finally, a dynamic map was made to integrate the analysis of these results during osteogenic differentiation of hPSCs, and the cell types at defined stages were concluded. Conclusions Our results lay the foundation to improve the in vitro osteogenic differentiation efficiency of hPSCs by supplementing with functional compounds at the desired stage, and then establishing a stepwise induction system in the future.

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Effect of dexamethasone as osteogenic supplementation in in vitro osteogenic differentiation of stem cells from human exfoliated deciduous teeth

Journal of Materials Science Materials in Medicine ◽

10.1007/s10856-020-06475-6 ◽

2021 ◽

Vol 32 (1) ◽

Author(s):

Mariane Beatriz Sordi ◽

Raissa Borges Curtarelli ◽

Izabella Thaís da Silva ◽

Gislaine Fongaro ◽

Cesar Augusto Magalhães Benfatti ◽

...

Keyword(s):

Stem Cells ◽

Extracellular Matrix ◽

Osteogenic Differentiation ◽

Culture Media ◽

Deciduous Teeth ◽

Free Calcium ◽

Alizarin Red ◽

Matrix Mineralization ◽

Media Supplementation

AbstractIn in vitro culture systems, dexamethasone (DEX) has been applied with ascorbic acid (ASC) and β-glycerophosphate (βGLY) as culture media supplementation to induce osteogenic differentiation of mesenchymal stem cells. However, there are some inconsistencies regarding the role of DEX as osteogenic media supplementation. Therefore, this study verified the influence of DEX culture media supplementation on the osteogenic differentiation, especially the capacity to mineralize the extracellular matrix of stem cells from human exfoliated deciduous teeth (SHED). Five groups were established: G1—SHED + Dulbecco’s Modified Eagles’ Medium (DMEM) + fetal bovine serum (FBS); G2—SHED + DMEM + FBS + DEX; G3—SHED + DMEM + FBS + ASC + βGLY; G4—SHED + DMEM + FBS + ASC + βGLY + DEX; G5—MC3T3-E1 + α Minimal Essential Medium (MEM) + FBS + ASC + βGLY. DNA content, alkaline phosphatase (ALP) activity, free calcium quantification in the extracellular medium, and extracellular matrix mineralization quantification through staining with von Kossa, alizarin red, and tetracycline were performed on days 7 and 21. Osteogenic media supplemented with ASC and β-GLY demonstrated similar effects on SHED in the presence or absence of DEX for DNA content (day 21) and capacity to mineralize the extracellular matrix according to alizarin red and tetracycline quantifications (day 21). In addition, the presence of DEX in the osteogenic medium promoted less ALP activity (day 7) and extracellular matrix mineralization according to the von Kossa assay (day 21), and more free calcium quantification at extracellular medium (day 21). In summary, the presence of DEX in the osteogenic media supplementation did not interfere with SHED commitment into mineral matrix depositor cells. We suggest that DEX may be omitted from culture media supplementation for SHED osteogenic differentiation in vitro studies.

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