scholarly journals Calcium-Silicate-Incorporated Gellan-Chitosan Induced Osteogenic Differentiation in Mesenchymal Stromal Cells

Polymers ◽  
2021 ◽  
Vol 13 (19) ◽  
pp. 3211
Author(s):  
Krishnamurithy Genasan ◽  
Mohammad Mehrali ◽  
Tarini Veerappan ◽  
Sepehr Talebian ◽  
Murali Malliga Raman ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Calcium Silicate ◽  
Bone Mineralization ◽  
Drying Method ◽  
Alizarin Red ◽  
Osteogenic Gene Expression ◽  
Critical Bone Defects

Gellan-chitosan (GC) incorporated with CS: 0% (GC-0 CS), 10% (GC-10 CS), 20% (GC-20 CS) or 40% (GC-40 CS) w/w was prepared using freeze-drying method to investigate its physicochemical, biocompatible, and osteoinductive properties in human bone-marrow mesenchymal stromal cells (hBMSCs). The composition of different groups was reflected in physicochemical analyses performed using BET, FTIR, and XRD. The SEM micrographs revealed excellent hBMSCs attachment in GC-40 CS. The Alamar Blue assay indicated an increased proliferation and viability of seeded hBMSCs in all groups on day 21 as compared with day 0. The hBMSCs seeded in GC-40 CS indicated osteogenic differentiation based on an amplified alkaline-phosphatase release on day 7 and 14 as compared with day 0. These cells supported bone mineralization on GC-40 CS based on Alizarin-Red assay on day 21 as compared with day 7 and increased their osteogenic gene expression (RUNX2, ALP, BGLAP, BMP, and Osteonectin) on day 21. The GC-40 CS–seeded hBMSCs initiated their osteogenic differentiation on day 7 as compared with counterparts based on an increased expression of type-1 collagen and BMP2 in immunocytochemistry analysis. In conclusion, the incorporation of 40% (w/w) calcium silicate in gellan-chitosan showed osteoinduction potential in hBMSCs, making it a potential biomaterial to treat critical bone defects.

scholarly journals The Role of Pannexin3-Modified Human Dental Pulp-Derived Mesenchymal Stromal Cells in Repairing Rat Cranial Critical-Sized Bone Defects

2017 ◽  
Vol 44 (6) ◽  
pp. 2174-2188 ◽  
Author(s):  
Fangfang Song ◽  
Hualing Sun ◽  
Liyuan Huang ◽  
Dongjie Fu ◽  
Cui Huang
Keyword(s):  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Dental Pulp ◽  
Underlying Mechanism ◽  
Bone Defects ◽  
Dependent Manner ◽  
Alizarin Red ◽  
Human Dental Pulp

Background/Aims: Human dental pulp-derived mesenchymal stromal cells (hDPSCs) are promising seed cells for tissue engineering due to their easy accessibility and multi-lineage differentiation. Pannexin3 (Panx3) plays crucial roles during bone development and differentiation. The aim of the present study was to investigate the effect of Panx3 on osteogenesis of hDPSCs and the underlying mechanism. Methods: Utilizing qRT-PCR, Western blot, and immunohistochemistry, we explored the change of Panx3 during osteogenic differentiation of hDPSCs. Next, hDPSCs with loss (Panx3 knockdown) and gain (Panx3 overexpression) of Panx3 function were developed to investigate the effects of Panx3 on osteogenic differentiation of hDPSC and the underlying mechanism. Finally, a commercial β-TCP scaffold carrying Panx3-modified hDPSCs was utilized to evaluate bone defect repair. Results: Panx3 was upregulated during osteogenic differentiation in a time-dependent manner. Panx3 overexpression promoted osteogenic differentiation of hDPSCs, whereas depletion of Panx3 resulted in a decline of differentiation, evidenced by upregulated expression of mineralization-related markers, increased alkaline phosphatase (ALP) activity, and enhanced ALP and Alizarin red staining. Panx3 was found to interact with the Wnt/β-catenin signaling pathway, forming a negative feedback loop. However, Wnt/β-catenin did not contribute to enhancement of osteogenic differentiation as observed in Panx3 overexpression. Moreover, Panx3 promoted osteogenic differentiation of hDPSCs via increasing ERK signaling pathway. Micro-CT and histological staining results showed that Panx3-modified hDPSCs significantly improved ossification of critical-sized bone defects. Conclusion: These findings suggest that Panx3 is a crucial modulator of hDPSCs differentiation.

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.

scholarly journals Revealing the influence of electron beam melted Ti-6Al-4V scaffolds on osteogenesis of human bone marrow-derived mesenchymal stromal cells

2021 ◽  
Vol 32 (9) ◽  
Author(s):  
Kristin S. Ødegaard ◽  
Lingzi Ouyang ◽  
Qianli Ma ◽  
Glenn Buene ◽  
Di Wan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Electron Beam ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Bone Growth ◽  
Bone Mineralization ◽  
Bone Matrix ◽  
Porous Scaffolds ◽  
Alkaline Phosphatase Staining

AbstractPorous Titanium-6Aluminum-4Vanadium scaffolds made by electron beam-based additive manufacturing (AM) have emerged as state-of-the-art implant devices. However, there is still limited knowledge on how they influence the osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs). In this study, BMSCs are cultured on such porous scaffolds to determine how the scaffolds influence the osteogenic differentiation of the cells. The scaffolds are biocompatible, as revealed by the increasing cell viability. Cells are evenly distributed on the scaffolds after 3 days of culturing followed by an increase in bone matrix development after 21 days of culturing. qPCR analysis provides insight into the cells’ osteogenic differentiation, where RUNX2 expression indicate the onset of differentiation towards osteoblasts. The COL1A1 expression suggests that the differentiated osteoblasts can produce the osteoid. Alkaline phosphatase staining indicates an onset of mineralization at day 7 in OM. The even deposits of calcium at day 21 further supports a successful bone mineralization. This work shines light on the interplay between AM Ti64 scaffolds and bone growth, which may ultimately lead to a new way of creating long lasting bone implants with fast recovery times.

scholarly journals Impact of Vancomycin Treatment on Human Mesenchymal Stromal Cells During Osteogenic Differentiation

2018 ◽  
Vol 3 (3) ◽  
pp. 2473011418S0002
Author(s):  
Jason Bariteau ◽  
Rishin Kadakia ◽  
Brian Traub ◽  
Nick Willett
Keyword(s):  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Bone Healing ◽  
Stromal Cells ◽  
Human Mscs ◽  
Alizarin Red ◽  
Degree Of Mineralization ◽  
Human Mesenchymal Stromal Cells ◽  
Vancomycin Powder ◽  
Vancomycin Treatment

Category: Basic Sciences/Biologics Introduction/Purpose: Vancomycin is often delivered locally for surgical site infection prophylaxis. Recent reports of possible osteotoxicity have led to uncertainty concerning vancomycin’s safety in the setting of arthrodesis and bone healing. Bone formation during arthrodesis takes place as recruited human mesenchymal stromal cells (hMSCs) proliferate and differentiate into mature osteoblasts. The purpose of this research was to determine the impact of vancomycin treatment on hMSCs during osteogenic differentiation. Methods: Human MSCs were cultured in MSC growth media to an appropriate confluence. Cells were cultured for 24 hours to facilitate adherence, after which the media was aspirated and replaced with osteogenic differentiation media (Lonza, Switzerland). Osteogenic differentiation media was supplemented with vancomycin powder to yield solutions with concentrations of 0, 50, 500 & 5000 µg/mL. Fresh vancomycin powder was added with every media change. MSCs viability and proliferation were assessed via live/dead staining with 1 µM calcein-AM and 0.5 µM ethidium homodimer-1 (EthD-1) after 1, 3, and 7 days of differentiation and vancomycin treatment. Mineralization of differentiated cells was assessed via staining with 40 mM alizarin red (ARS; pH 4.1) after 21 days. Semi-quantification of the degree of mineralization was performed by measuring absorbance values at 405 nm using a microplate reader. Microscopy was used for qualitative evaluation. Results: Cell viability decreased with increasing vancomycin concentrations. Impairment of hMSC proliferation was also observed with increasing concentrations of vancomycin. MSCs treated with 5000 µg/mL vancomycin demonstrated significantly less cell growth compared to all other treatment groups (P=0.0001). Absorbance measurements from each well stained with alizarin red was used for semi-quantification of the degree of mineralization. As vancomycin concentrations were increased, absorbance levels decreased (Figure). This reduction in mineralization was also demonstrated qualitatively; with alizarin red less apparent in the wells with increasing vancomycin concentrations (Figure). Conclusion: Local vancomycin is utilized for prevention of infection, often in procedures that necessitate the formation of new bone. Bone healing requires migration, proliferation and differentiation of hMSCs. This work demonstrates impaired viability and function of hMSCs following vancomycin as well as decreased osteoblastic mineralization. Future work will require in vivo studies aimed at determining relative nonunion rates in the setting of vancomycin prophylaxis. Still, the results of this study suggest that vancomycin may be toxic to hMSCs and caution should be exercised by providers when considering vancomycin in foot and ankle patients requiring bony healing following fracture or arthrodesis.

scholarly journals Secretion of niche signal molecules in conditions of osteogenic differentiation of multipotent mesenchymal stromal cells induced by textured calcium phosphate coating

2019 ◽  
Vol 65 (4) ◽  
pp. 339-346
Author(s):  
L.S. Litvinova ◽  
V.V. Shupletsova ◽  
K.A. Yurova ◽  
O.G. Khaziakhmatova ◽  
N.M. Todosenko ◽  
...  
Keyword(s):  
Calcium Phosphate ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Calcium Phosphate Coating ◽  
Signal Molecules ◽  
Humoral Factors ◽  
Alizarin Red

Secretion of 21 cytokines, chemokines and growth factors (LIF, SCF, SDF-1a, SCGF-b, M-CSF, MCP-3, MIF, MIG, TRAIL, GRO-a; IL-1a, IL-2ra, IL-3, IL-12(p40), IL-16, IL-18, HGF, TNF-b, b-NGF, IFN-a2, CTACK) has been studied in vitro in the culture of human adipose-derived multipotent mesenchymal stromal cells (hAMMSCs) in conditions of its osteogenic differentiation caused by 14-day contact with calcium phosphate (CP) surface with different roughness. Bilateral X-ray amorphous CP coatings were prepared on the samples of commercially pure titanium in the anodal regime using a micro-arc method. An aqueous solution prepared from 20 wt% phosphoric acid, 6 wt% dissolved hydrohyapatite nanopowder (particle diameter 10-30 nm with single agglomerates up to 100 nm), and 9 wt% dissolved calcium carbonate was used to obtain CP coating. hAMMSCs isolated from lipoaspirate were co-cultured after 4 passages with the CP-coated samples at final concentration of 1.5´105 viable karyocytes per 1.5 mL of standard nutrition medium (without osteogenic stimulators) for 14 days (a determination of [CD45,34,14,20], CD73, CD90 и CD105 cell immunophenotype; an analysis of secretory activity) and 21 days (alizarin red S staining of culture) with medium replacement every 3-4 days. Under conditions of in vitro contact with rough CP coating hAMMSCs differentiated into osteoblasts synthesizing the mineralized bone matrix; this was accompanied by 2-3-fold increasing ratio of [CD45,34,14,20]+ hemopoietic cells. The following humoral factors of hemopoietic niches acted as the signal molecules escalating in vitro the hemopoietic base in 14 days of differentiating three-dimensional culture of hAMMSCs: either leukemia inhibitory factor (LIF) and stem cell factor (SCF) cytokines under mean index of CP roughness Ra=2.4-2.6 mm or stromal derived factor-1 (SDF-1a, CXCL12 chemokine) under Ra=3.1-4.4 mm.

Effects of hypoxia on osteogenic differentiation of mesenchymal stromal cells used as a cell therapy for avascular necrosis of the femoral head

Cytotherapy ◽  
2016 ◽  
Vol 18 (9) ◽  
pp. 1087-1099 ◽  
Author(s):  
Gabriela Ciapetti ◽  
Donatella Granchi ◽  
Caterina Fotia ◽  
Lucia Savarino ◽  
Dante Dallari ◽  
...  
Keyword(s):  
Femoral Head ◽  
Cell Therapy ◽  
Osteogenic Differentiation ◽  
Avascular Necrosis ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
A Cell

scholarly journals The oncometabolite R-2-hydroxyglutarate dysregulates the differentiation of human mesenchymal stromal cells via inducing DNA hypermethylation

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Lizhen Liu ◽  
Kaimin Hu ◽  
Jingjing Feng ◽  
Huafang Wang ◽  
Shan Fu ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Chondrogenic Differentiation ◽  
Cell Counting ◽  
Human Mscs ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Dna Hypermethylation ◽  
Cartilaginous Tumors

Abstract Background Isocitrate dehydrogenase (IDH1/2) gene mutations are the most frequently observed mutations in cartilaginous tumors. The mutant IDH causes elevation in the levels of R-enantiomer of 2-hydroxylglutarate (R-2HG). Mesenchymal stromal cells (MSCs) are reasonable precursor cell candidates of cartilaginous tumors. This study aimed to investigate the effect of oncometabolite R-2HG on MSCs. Methods Human bone marrow MSCs treated with or without R-2HG at concentrations 0.1 to 1.5 mM were used for experiments. Cell Counting Kit-8 was used to detect the proliferation of MSCs. To determine the effects of R-2HG on MSC differentiation, cells were cultured in osteogenic, chondrogenic and adipogenic medium. Specific staining approaches were performed and differentiation-related genes were quantified. Furthermore, DNA methylation status was explored by Illumina array-based arrays. Real-time PCR was applied to examine the signaling component mRNAs involved in. Results R-2HG showed no influence on the proliferation of human MSCs. R-2HG blocked osteogenic differentiation, whereas promoted adipogenic differentiation of MSCs in a dose-dependent manner. R-2HG inhibited chondrogenic differentiation of MSCs, but increased the expression of genes related to chondrocyte hypertrophy in a lower concentration (1.0 mM). Moreover, R-2HG induced a pronounced DNA hypermethylation state of MSC. R-2HG also improved promotor methylation of lineage-specific genes during osteogenic and chondrogenic differentiation. In addition, R-2HG induced hypermethylation and decreased the mRNA levels of SHH, GLI1and GLI2, indicating Sonic Hedgehog (Shh) signaling inhibition. Conclusions The oncometabolite R-2HG dysregulated the chondrogenic and osteogenic differentiation of MSCs possibly via induction of DNA hypermethylation, improving the role of R-2HG in cartilaginous tumor development.

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