scholarly journals Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT-Raman spectroscopy

2023 ◽  
Vol 83 ◽  
Author(s):  
V. A. Nascimento ◽  
S. M. Malmonge ◽  
A. R. Santos Jr.
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Raman Spectroscopy ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Functional Groups ◽  
Culture Media ◽  
Cell Types ◽  
Ft Raman Spectroscopy ◽  
Ft Raman

Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

scholarly journals Fluid Flow Mechanical Stimulation-Assisted Cartridge Device for the Osteogenic Differentiation of Human Mesenchymal Stem Cells

Micromachines ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 927
Author(s):  
Ki-Taek Lim ◽  
Dinesh-K. Patel ◽  
Sayan-Deb Dutta ◽  
Keya Ganguly
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Fluid Flow ◽  
Osteogenic Differentiation ◽  
Flow Condition ◽  
Cultured Cells ◽  
Human Mesenchymal Stem Cells ◽  
Proliferation And Differentiation ◽  
Static Conditions

Human mesenchymal stem cells (hMSCs) have the potential to differentiate into different types of mesodermal tissues. In vitro proliferation and differentiation of hMSCs are necessary for bone regeneration in tissue engineering. The present study aimed to design and develop a fluid flow mechanically-assisted cartridge device to enhance the osteogenic differentiation of hMSCs. We used the fluorescence-activated cell-sorting method to analyze the multipotent properties of hMSCs and found that the cultured cells retained their stemness potential. We also evaluated the cell viabilities of the cultured cells via water-soluble tetrazolium salt 1 (WST-1) assay under different rates of flow (0.035, 0.21, and 0.35 mL/min) and static conditions and found that the cell growth rate was approximately 12% higher in the 0.035 mL/min flow condition than the other conditions. Moreover, the cultured cells were healthy and adhered properly to the culture substrate. Enhanced mineralization and alkaline phosphatase activity were also observed under different perfusion conditions compared to the static conditions, indicating that the applied conditions play important roles in the proliferation and differentiation of hMSCs. Furthermore, we determined the expression levels of osteogenesis-related genes, including the runt-related protein 2 (Runx2), collagen type I (Col1), osteopontin (OPN), and osteocalcin (OCN), under various perfusion vis-à-vis static conditions and found that they were significantly affected by the applied conditions. Furthermore, the fluorescence intensities of OCN and OPN osteogenic gene markers were found to be enhanced in the 0.035 mL/min flow condition compared to the control, indicating that it was a suitable condition for osteogenic differentiation. Taken together, the findings of this study reveal that the developed cartridge device promotes the proliferation and differentiation of hMSCs and can potentially be used in the field of tissue engineering.

scholarly journals 14-3-3ε protein-loaded 3D hydrogels favor osteogenesis

2020 ◽  
Vol 31 (11) ◽  
Author(s):  
Ana A. Aldana ◽  
Marina Uhart ◽  
Gustavo A. Abraham ◽  
Diego M. Bustos ◽  
Aldo R. Boccaccini
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
3D Printing ◽  
Osteogenic Differentiation ◽  
Alginate Hydrogel ◽  
Hydrogel Scaffolds ◽  
Future Developments ◽  
Positive Effect ◽  
Cell Adhesion And Proliferation

Abstract3D printing has emerged as vanguard technique of biofabrication to assemble cells, biomaterials and biomolecules in a spatially controlled manner to reproduce native tissues. In this work, gelatin methacrylate (GelMA)/alginate hydrogel scaffolds were obtained by 3D printing and 14-3-3ε protein was encapsulated in the hydrogel to induce osteogenic differentiation of human adipose-derived mesenchymal stem cells (hASC). GelMA/alginate-based grid-like structures were printed and remained stable upon photo-crosslinking. The viscosity of alginate allowed to control the pore size and strand width. A higher viscosity of hydrogel ink enhanced the printing accuracy. Protein-loaded GelMA/alginate-based hydrogel showed a clear induction of the osteogenic differentiation of hASC cells. The results are relevant for future developments of GelMA/alginate for bone tissue engineering given the positive effect of 14-3-3ε protein on both cell adhesion and proliferation.

Dextran-coated fluorapatite nanorods doped with lanthanides in labelling and directing osteogenic differentiation of bone marrow mesenchymal stem cells

2014 ◽  
Vol 2 (23) ◽  
pp. 3609-3617 ◽  
Author(s):  
Haifeng Zeng ◽  
Xiyu Li ◽  
Fang Xie ◽  
Li Teng ◽  
Haifeng Chen
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Bone Tissue Engineering ◽  
Osteogenic Differentiation ◽  
Bone Tissue ◽  
Novel Approach ◽  
Marrow Mesenchymal Stem Cells

A novel approach for labelling and tracking BMSCs in bone tissue engineering by using dextran-coated fluorapatite nanorods doped with lanthanides.

scholarly journals Ca(2+) Oscillations and Its Transporters in Mesenchymal Stem Cells

2010 ◽  
pp. 323-329 ◽  
Author(s):  
B Ye
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Replacement Therapy ◽  
Cell Types ◽  
Cell Replacement Therapy ◽  
Cell Replacement ◽  
Cell Functions ◽  
The Past ◽  
Intracellular Ca

Intracellular free Ca(2+) is one of important biological signals regulating a number of cell functions. It has been discussed widely and extensively in several cell types during the past two decades. Attention has been paid to the Ca2+ transportation in mesenchymal stem cells in recent years as mesenchymal stem cells have gained considerable interest due to their potential for cell replacement therapy and tissue engineering. In this paper, roles of intracellular Ca(2+) oscillations and its transporters in mesenchymal stem cells have been reviewed.

scholarly journals MicroRNA-214 Suppresses Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Targeting ATF4

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Siqi Yao ◽  
Wei Zhao ◽  
Qianmin Ou ◽  
Lanchen Liang ◽  
Xuefeng Lin ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Target Prediction ◽  
Cell Types ◽  
Luciferase Reporter ◽  
Periodontal Ligament Stem Cells ◽  
Activating Transcription Factor 4 ◽  
Activating Transcription Factor

Periodontitis is the main cause of adult tooth loss. Stem cell-based tissue engineering has become a promising therapy for periodontitis treatment. To date, human periodontal ligament stem cells (hPDLSCs) have been shown to be a favorable source for tissue engineering, but modulatory mechanisms of hPDLSCs remain unclear. Approximately 60% of mammalian genes are the targets of over 2000 miRNAs in multiple human cell types, and miRNAs are able to influence various biological processes in the human body, including bone formation. In this study, we found that after osteogenic induction, miR-214 was significantly decreased in hPDLSCs; therefore, we examined the effects of miR-214 on osteogenic differentiation. Computational miRNA target prediction analyses and luciferase reporter assays revealed that activating transcription factor 4 (ATF4) is a direct target of miR-214. We prepared cells overexpressing miR-214 and found that miR-214 negatively regulates osteogenic differentiation of hPDLSCs. For the target of miR-214, ATF4 protein expression level was decreased after induction. In conclusion, we found that miR-214-ATF4 axis is a novel pathway for regulating hPDLSC osteogenic differentiation.

280 MULTILINEAGE POTENTIAL OF PORCINE BONE MARROW AND ADIPOSE-DERIVED MESENCHYMAL STEM CELLS IN 3-D ALGINATE HYDROGELS

2009 ◽  
Vol 21 (1) ◽  
pp. 237 ◽  
Author(s):  
D. Kim ◽  
A. J. Maki ◽  
H.-J. Kong ◽  
E. Monaco ◽  
M. Bionaz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tissue Engineering ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Adipogenic Differentiation ◽  
Cell Types ◽  
Scaffold Material ◽  
Over Time

Adipose tissue presents an appealing alternative to bone marrow as a source of mesenchymal stem cells (MSC). However, in order to enhance cell proliferation and differentiation, 3-dimensional (3-D) culture may be required. A 3-D culture has benefits due to its more in vivo-like environment. Further, to form a functional tissue, a scaffold material is required to ensure proper shape and allow for efficient delivery of nutrients and growth factors. Alginate, a resorbable hydrogel, is a potential injectable scaffold for fat and bone tissue engineering due to its high biocompatibility, gelation with calcium and slow dissolution in a physiologic environment. In the present study, we examined the viability, gene expression and morphology of MSC, isolated from porcine adipose (ADSC) and bone marrow (BMSC), during osteogenic and adipogenic differentiation in a 3D alginate hydrogel environment for 0, 7 and 14 days (d). ADSC and BMSC were infused into alginate hydrogels, which polymerized upon the addition of Ca+2 ions. Both stem cell types were differentiated into osteoblasts using 0.1 μm dexamethasone, 10 mm beta glycerophosphate and 50 μm ascorbic acid, whereas adipocytes were differentiated using 10 μm insulin, 1 μm dexamethasone, and 0.5 mm IBMX. Osteogenic differentiation was confirmed using alkaline phosphatase, Von Kossa, and alizarin red S staining and adipogenic differentiation was confirmed using Oil Red O. Cell viability and proliferation was quantified using the MTT assay. Gene expression was measured using qPCR. The morphology of ADSC and BMSC differentiated toward osteogenic lineages changed with both cell types forming osteogenic nodules over time. The nodules formed by ADSC were larger in diameter than those formed by BMSC. Unlike the osteogenic cells that formed nodules, the ADSC and BMSC differentiated into adipogenic cells showed no significant changes in cell size or aggregation. Gene expression results indicated increased PPARG expression in BMSC with time whereas ADSC showed a peak of expression on day 7 and then decreased. ADSC showed increased (14-fold) PPRG expression when compared with BMSC. ADSC had 160-fold less expression of ALP than BMSC. BMSC showed a 16-fold higher expression level of BGLAP than ADSC. ADSC showed a 15.8% higher expression than BMSC for COL1a1. Both ADSC and BMSC showed similar trends SPARC expression, but BMSC had a 12-fold higher expression of SPP1 than ADSC. In summary, both types of mesenchymal stem cells successfully differentiated into both lineages and maintained viability in the hydrogel over time. In conclusion, alginate is a viable scaffold material for the differentiation of mesenchymal stem cells for tissue engineering applications. These results allow for future studies using the pig as an in vivo fat and bone tissue engineering model. This research was supported by the Illinois Regenerative Medicine Institute.

scholarly journals Dose-Response Tendon-Specific Markers Induction by Growth Differentiation Factor-5 in Human Bone Marrow and Umbilical Cord Mesenchymal Stem Cells

2020 ◽  
Vol 21 (16) ◽  
pp. 5905
Author(s):  
Maria Camilla Ciardulli ◽  
Luigi Marino ◽  
Erwin Pavel Lamparelli ◽  
Maurizio Guida ◽  
Nicholas Robert Forsyth ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cell Types ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Differentiation Factor ◽  
Anti Inflammatory ◽  
Growth Differentiation Factor 5

Mesenchymal stem cells derived from human bone marrow (hBM-MSCs) are utilized in tendon tissue-engineering protocols while extra-embryonic cord-derived, including from Wharton’s Jelly (hWJ-MSCs), are emerging as useful alternatives. To explore the tenogenic responsiveness of hBM-MSCs and hWJ-MSCs to human Growth Differentiation Factor 5 (hGDF-5) we supplemented each at doses of 1, 10, and 100 ng/mL of hGDF-5 and determined proliferation, morphology and time-dependent expression of tenogenic markers. We evaluated the expression of collagen types 1 (COL1A1) and 3 (COL3A1), Decorin (DCN), Scleraxis-A (SCX-A), Tenascin-C (TNC) and Tenomodulin (TNMD) noting the earliest and largest increase with 100 ng/mL. With 100 ng/mL, hBM-MSCs showed up-regulation of SCX-A (1.7-fold) at Day 1, TNC (1.3-fold) and TNMD (12-fold) at Day 8. hWJ-MSCs, at the same dose, showed up-regulation of COL1A1 (3-fold), DCN (2.7-fold), SCX-A (3.8-fold) and TNC (2.3-fold) after three days of culture. hWJ-MSCs also showed larger proliferation rate and marked aggregation into a tubular-shaped system at Day 7 (with 100 ng/mL of hGDF-5). Simultaneous to this, we explored the expression of pro-inflammatory (IL-6, TNF, IL-12A, IL-1β) and anti-inflammatory (IL-10, TGF-β1) cytokines across for both cell types. hBM-MSCs exhibited a better balance of pro-inflammatory and anti-inflammatory cytokines up-regulating IL-1β (11-fold) and IL-10 (10-fold) at Day 8; hWJ-MSCs, had a slight expression of IL-12A (1.5-fold), but a greater up-regulation of IL-10 (2.5-fold). Type 1 collagen and tenomodulin proteins, detected by immunofluorescence, confirming the greater protein expression when 100 ng/mL were supplemented. In the same conditions, both cell types showed specific alignment and shape modification with a length/width ratio increase, suggesting their response in activating tenogenic commitment events, and they both potential use in 3D in vitro tissue-engineering protocols.

scholarly journals Prospect of Stem Cells in Bone Tissue Engineering: A Review

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Azizeh-Mitra Yousefi ◽  
Paul F. James ◽  
Rosa Akbarzadeh ◽  
Aswati Subramanian ◽  
Conor Flavin ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Bone Tissue Engineering ◽  
Bone Tissue ◽  
Cell Types ◽  
3D Scaffold ◽  
Stem Cell Source ◽  
Induced Pluripotent

Mesenchymal stem cells (MSCs) have been the subject of many studies in recent years, ranging from basic science that looks into MSCs properties to studies that aim for developing bioengineered tissues and organs. Adult bone marrow-derived mesenchymal stem cells (BM-MSCs) have been the focus of most studies due to the inherent potential of these cells to differentiate into various cell types. Although, the discovery of induced pluripotent stem cells (iPSCs) represents a paradigm shift in our understanding of cellular differentiation. These cells are another attractive stem cell source because of their ability to be reprogramed, allowing the generation of multiple cell types from a single cell. This paper briefly covers various types of stem cell sources that have been used for tissue engineering applications, with a focus on bone regeneration. Then, an overview of some recent studies making use of MSC-seeded 3D scaffold systems for bone tissue engineering has been presented. The emphasis has been placed on the reported scaffold properties that tend to improve MSCs adhesion, proliferation, and osteogenic differentiation outcomes.

scholarly journals Growth on Metallo-Supramolecular Coordination Polyelectrolyte (MEPE) Stimulates Osteogenic Differentiation of Human Osteosarcoma Cells (MG63) and Human Bone Marrow Derived Mesenchymal Stem Cells

Polymers ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 1090 ◽  
Author(s):  
Janina Belka ◽  
Joachim Nickel ◽  
Dirk G. Kurth
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Differentiation ◽  
Osteogenic Differentiation ◽  
Three Dimensional ◽  
Cell Types ◽  
Growth Conditions ◽  
Cellular Environment ◽  
Human Osteosarcoma Cells ◽  
Supramolecular Coordination

Background: Culturing of cells is typically performed on standard tissue culture plates generating growth conditions, which in general do not reflect the native three-dimensional cellular environment. Recent investigations provide insights in parameters, which strongly affect the general cellular behavior triggering essential processes such as cell differentiation. The physical properties of the used material, such as stiffness, roughness, or topology, as well as the chemical composition of the cell-surface interface are shown to play a key role in the initiation of particular cellular responses. Methods: We extended our previous research, which identified thin films of metallo-supramolecular coordination polyelectrolytes (MEPEs) as substrate to trigger the differentiation of muscular precursor cells. Results: Here, we show that the same MEPEs similarly stimulate the osteogenic differentiation of pre-osteoblasts. Remarkably, MEPE modified surfaces also trigger the differentiation of primary bone derived mesenchymal stem cells (BMSCs) towards the osteogenic lineage. Conclusion: This result leads to the conclusion that these surfaces individually support the specification of cell differentiation toward lineages that correspond to the natural commitment of the particular cell types. We, therefore, propose that Fe-MEPEs may be used as scaffold for the treatment of defects at least in muscular or bone tissue.

178 Differential Behavior of Porcine Mesenchymal Stem Cells from Bone Marrow and Adipose During Osteogenic Differentiation on a Glycosaminoglycan Hydrogel Scaffold for Bone and Cartilage Tissue Engineering

2018 ◽  
Vol 30 (1) ◽  
pp. 229 ◽  
Author(s):  
S. A. Womack ◽  
D. J. Milner ◽  
D. W. Weisgerber ◽  
B. A. Harley ◽  
M. B. Wheeler
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Cartilage Tissue Engineering ◽  
Cartilage Tissue ◽  
Scaffold Material ◽  
Alizarin Red ◽  
And Cartilage

The pig is an ideal species for use in tissue engineering studies targeted towards repair of bone and cartilage defects. Novel collagen-glycosaminoglycan hydrogel (CG) scaffolds have shown promise for supporting bone and cartilage growth from mesenchymal stem cells. In order to determine the suitability of these scaffolds for use in porcine model systems for bone and cartilage tissue engineering, we have begun to investigate the behaviour of porcine mesenchymal stem cells on this material. The purpose of this study was to determine whether mesenchymal stem cells from adipose (ASC) and bone marrow (BMSC) form bone on a CG scaffold material. Primary BMSC and ASC from 6-month-old Yorkshire pigs were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum. The ASC and BMSC were then trypsinized at passage 4 or 5 and used to seed ~4-mm-diameter CG scaffolds with 2 million cells/scaffold. Scaffolds were seeded by suspending the cells in medium that had been equilibrated for 30 min, and then placing the CG scaffold into the medium. This method of seeding was determined to be most effective in previous experiments. Scaffolds were then cultured for 7 days in DMEM followed by 21 days in osteogenic media. At the conclusion of the incubation period, the diameter of the scaffolds was measured, and they were fixed with 4% paraformaldehyde and cryosectioned. Then, 10-µm sections were stained with Alizarin Red to assay for mineralization, a hallmark of osteogenic differentiation. Both ASC- and BMSC-loaded scaffolds showed Alizarin Red staining throughout the section after incubation, demonstrating that both undergo osteogenesis on the scaffold material (n = 4). During osteogenic differentiation, scaffolds seeded with both ASC and BMSC showed a decrease in diameter. Unseeded scaffolds showed no decrease in size when in media. The BMSC scaffolds demonstrated a more extensive decrease in size than ASC. The average diameter of ASC loaded scaffolds after differentiation was 2.49 ± 0.39 mm, and that of BMSC-loaded scaffolds was 1.47 ± 0 0.18 mm (n = 3, P < 0.05, Student’s t-test). This suggests a differential ability of ASC and BMSC to break down and metabolize the scaffold matrix, and may indicate that one cell type may be preferable to the other for repairing osteogenic defects using these scaffolds. Current experiments underway will analyse expression of matrix-degrading enzymes to determine the source of the difference between cell types in scaffold shrinkage during differentiation. We will also quantify mineralization in ASC- v. BMSC-loaded scaffolds and assay gene expression of osteogenic markers to determine if there is a difference in osteogenic potential between sources of mesenchymal stem cells on these scaffolds.

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