Osteogenic differentiation of equine adipose tissue derived mesenchymal stem cells using CaCl2.
Abstract: Adipose tissue derived mesenchymal stem cells (ASCs) may be used to cure bone defects after osteogenic differentiation. In this study we tried to optimize osteogenic differentiation for equine ASCs using various concentrations of CaCl in comparison to the standard osteogenic protocol. ASCs were isolated from subcutaneous adipose tissue from mixed breed horses. The osteogenic induction protocols were (1) the standard osteogenic medium (OM) composed of dexamethasone, ascorbic acid and β-glycerol phosphate; (2) CaCl based protocol composed of 3, 5 and 7.5mM CaCl. Differentiation and proliferation were evaluated at 7, 10, 14 and 21days post-differentiation induction using the alizarin red staining (ARS) detecting matrix calcification. Semi-quantification of cell protein content, ARS and alkaline phosphatase activity (ALP) were performed using an ELISA reader. Quantification of the transcription level for the common osteogenic markers alkaline phosphatase (ALP) and Osteopontin (OP) was performed using RT-qPCR. In the presence of CaCl, a concentration dependent effect on the osteogenic differentiation capacity was evident by the ARS evaluation and OP gene expression. We provide evidence that 5 and 7mM CaCl enhance the osteogenic differentiation compared to the OM protocol. Although, there was a clear commitment of ASCs to the osteogenic fate in the presence of 5 and 7mM CaCl, cell proliferation was increased compared to OM. We report that an optimized CaCl protocol reliably influences ASCs osteogenesis while conserving the proliferation capacity. Thus, using these protocols provide a platform for using ASCs as a cell source in bone tissue engineering.
Copyright © 2017 Elsevier Ltd. All rights reserved.
Publication Date: 2017-11-21 PubMed ID: 29175012DOI: 10.1016/j.rvsc.2017.11.010Google Scholar: Lookup
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Summary
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This research studies the use of equine adipose tissue-derived mesenchymal stem cells (ASCs), focusing on improving their osteogenic differentiation using various concentrations of CaCl2, compared to traditional osteogenic protocols. The optimized CaCl2 protocol was found to enhance osteogenesis while preserving the cells’ ability to proliferate, making them a potential resource for bone tissue engineering.
Osteogenic Differentiation of ASCs
- The focus of this study is adipose tissue-derived mesenchymal stem cells, which can be tailored to cure bone defects after inducing osteogenic differentiation.
- ASCs were obtained from the subcutaneous adipose tissue of mixed breed horses.
- The research aimed at enhancing the osteogenic differentiation of ASCs using various concentrations of calcium chloride (CaCl2), and comparing the results with those from conventional osteogenic methodologies.
Study Design and Methodology
- Two main osteogenic induction protocols were utilized: the standard osteogenic medium (OM), comprising of dexamethasone, ascorbic acid, and β-glycerol phosphate, and a protocol based on CaCl2, using 3, 5 and 7.5mM concentrations.
- Following the differentiation induction, differentiation and proliferation were examined at four predetermined time-points: 7, 10, 14, and 21 days.
- The assessments involved the use of alizarin red staining (ARS) for detecting matrix calcification, semi-quantification of cell protein content, and the activity level of alkaline phosphatase (ALP), a common osteogenic marker, undertaken using an ELISA reader.
- Transcription levels of ALP and Osteopontin (OP), another marker for osteogenesis, were quantified using RT-qPCR.
Results and Findings
- The presence of CaCl2 showed a concentration-dependent impact on the osteogenic differentiation capacity, as evidenced by the ARS assessment and OP gene expression.
- Notably, the use of CaCl2 at concentrations of 5 and 7mM significantly enhanced osteogenic differentiation compared to the standard OM protocol.
- The commitment of ASCs to the osteogenic fate with 5 and 7mM concentrations of CaCl2 was evident, showing increased cell proliferation compared to the OM protocol.
- The study concludes that the optimized protocol using specific concentrations of CaCl2 greatly influences ASC osteogenesis while conserving their proliferative capacity.
Significance of the Study
- Through the successful optimization of the CaCl2 protocol, this study contributes to creating a platform for harnessing ASCs as a viable cell source in bone tissue engineering.
- The findings potentially open up new avenues in regenerative medicine, particularly in the treatment of bone defects.
Cite This Article
APA
Elashry MI, Baulig N, Heimann M, Bernhardt C, Wenisch S, Arnhold S.
(2017).
Osteogenic differentiation of equine adipose tissue derived mesenchymal stem cells using CaCl2.
Res Vet Sci, 117, 45-53.
https://doi.org/10.1016/j.rvsc.2017.11.010 Publication
Researcher Affiliations
- Dept. of Veterinary -Anatomy, -Histology and -Embryology, University of Giessen, 35392 Giessen, Germany; Anatomy and Embryology Department, Faculty of Veterinary Medicine, University of Mansoura, 35516, Egypt.
- Dept. of Veterinary -Anatomy, -Histology and -Embryology, University of Giessen, 35392 Giessen, Germany.
- Dept. of Veterinary -Anatomy, -Histology and -Embryology, University of Giessen, 35392 Giessen, Germany.
- Dept. of Veterinary -Anatomy, -Histology and -Embryology, University of Giessen, 35392 Giessen, Germany.
- Dept. of Veterinary Clinical Sciences, Small Animal Clinic c/o Institute of Veterinary Anatomy, Histology and -Embryology, University of Giessen, Frankfurter Str. 98, 35392 Giessen, Germany.
- Dept. of Veterinary -Anatomy, -Histology and -Embryology, University of Giessen, 35392 Giessen, Germany. Electronic address: Stefan.arnhold@vetmed.uni-giessen.de.
MeSH Terms
- Adipose Tissue / cytology
- Alkaline Phosphatase
- Animals
- Calcium Chloride / pharmacology
- Cell Differentiation / drug effects
- Cell Proliferation
- Glycerophosphates
- Horses
- Humans
- Mesenchymal Stem Cells / physiology
- Osteogenesis / physiology
- Tissue Engineering / methods
Citations
This article has been cited 4 times.- Wolint P, Näf L, Schibler D, Hild N, Stark WJ, Giovanoli P, Calcagni M, Buschmann J. Suspension of Amorphous Calcium Phosphate Nanoparticles Impact Commitment of Human Adipose-Derived Stem Cells In Vitro. Biology (Basel) 2021 Jul 16;10(7).
- Elashry MI, Baulig N, Wagner AS, Klymiuk MC, Kruppke B, Hanke T, Wenisch S, Arnhold S. Combined macromolecule biomaterials together with fluid shear stress promote the osteogenic differentiation capacity of equine adipose-derived mesenchymal stem cells. Stem Cell Res Ther 2021 Feb 12;12(1):116.
- Li H, Zhang R. The role of calcium ions and the transient receptor potential vanilloid (TRPV) channel in bone remodelling and orthodontic tooth movement. Mol Biol Rep 2025 Mar 10;52(1):297.
- Zhai ZH, Li J, You Z, Cai Y, Yang J, An J, Zhao DP, Wang HJ, Dou MM, Du R, Qin J. Feline umbilical cord-derived mesenchymal stem cells: isolation, identification, and antioxidative stress role through NF-κB signaling pathway. Front Vet Sci 2023;10:1203012.
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