Isolation, culture and induced differentiation of rabbit mesenchymal stem cells into osteoblasts

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Abstract

Mesenchymal stem cells (MSCs) may be easily isolated from the bone marrow, and possess multi-lineage differentiation potential and various therapeutic applications. The differentiation of MSCs into osteoblasts is a complex process that is regulated by multiple internal and external factors. In the present study, the differentiation of MSCs isolated from rabbit bone marrow into osteoblasts using different osteoblast inductive media in the presence of dexamethasone, bone morphogenetic protein 2 (BMP-2), 1,25-dihydroxyvitamin D3, transforming growth factor β (TGFβ), platelet lysate and cyclooxygenase 2 (COX2), respectively. Alkaline phosphatase (ALP) activity, mineralization, collagen type (Ct) I and osteocalcin activities, and the mRNA and protein expression levels of vascular endothelial growth factor (VEGF), BMP-2 and Ct II were measured during the differentiation process in MSCs treated with different inducers. Rabbit MSCs were successfully isolated and were observed to be predominantly circular in shape after culture for 24 h. Following subculture for 5 days, the cells demonstrated a spindle shape. ALP, Ct I and osteocalcin activities were higher in cells cultured in dexamethasone, BMP-2 and TGFβ compared with the activities in control cells. Following differentiation, the dexamethasone, BMP-2 and TGFβ groups demonstrated significantly enhanced mineralization of MSCs detected by Alizarin Red S staining. The mRNA and protein expression levels of VEGF, BMP-2 and Ct II were significantly increased in the same groups compared with the levels in the control group. In conclusion, rabbit MSCs were successfully isolated from bone marrow and differentiated into osteoblasts indicated by raised ALP, Ct I and osteocalcin activities, mineralization and expression of osteogenesis-inducing genes and proteins. The present study revealed that dexamethasone, BMP-2 and TGFβ have a positive effect on cell differentiation.

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Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors.

Matus Studeny, Frank C. Marini, Richard Champlin … (2002)

Molecules that physiologically control cell proliferation are often produced locally in tissues and are rapidly destroyed when they enter circulation. This allows local effects while avoiding interference with other systems. Unfortunately, it also limits the therapeutic use of these molecules via systemic delivery. We here demonstrate that, for the purpose of anticancer therapy, bone marrow-derived mesenchymal stem cells (MSCs) can produce biological agents locally at tumor sites. We show that the tumor microenvironment preferentially promotes the engraftment of MSCs as compared with other tissues. MSCs with forced expression of IFN-beta inhibited the growth of malignant cells in vivo. Importantly, this effect required the integration of MSCs into the tumors and could not be achieved by systemically delivered IFN-beta or by IFN-beta produced by MSCs at a site distant from the tumors. Our results indicate that MSCs may serve as a platform for delivery of biological agents in tumors.

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Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components.

F Barry, R E Boynton, B. Liu … (2001)

Transforming growth factor (TGF)-beta-induced chondrogenesis of mesenchymal stem cells derived from bone marrow involves the rapid deposition of a cartilage-specific extracellular matrix. The sequential events in this pathway leading from the undifferentiated stem cell to a mature chondrocyte were investigated by analysis of key matrix elements. Differentiation was rapidly induced in cells cultured in the presence of TGF-beta 3 or -beta 2 and was accompanied by the early expression of fibromodulin and cartilage oligomeric matrix protein. An increase in aggrecan and versican core protein synthesis defined an intermediate stage, which also involved the small leucine-rich proteoglycans decorin and biglycan. This was followed by the appearance of type II collagen and chondroadherin. The pathway was also characterized by the appearance of type X collagen, usually associated with hypertrophic cartilage. There was also a change in the pattern of sulfation of chondroitin sulfate, with a progressive increase in the proportion of 6-sulfated species. The major proportion of newly synthesized glycosaminoglycan was part of an aggregating proteoglycan network. These data allow us to define the phenotype of the differentiated cell and to understand in greater detail the sequential process of matrix assembly. Copyright 2001 Academic Press.

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VEGF: an essential mediator of both angiogenesis and endochondral ossification.

J-G Dai, Menan A Rabie (2007)

During bone growth, development, and remodeling, angiogenesis as well as osteogenesis are closely associated processes, sharing some essential mediators. Vascular endothelial growth factor (VEGF) was initially recognized as the best-characterized endothelial-specific growth factor, which increased vascular permeability and angiogenesis, and it is now apparent that this cytokine regulates multiple biological functions in the endochondral ossification of mandibular condylar growth, as well as long bone formation. The complexity of VEGF biology is paralleled by the emerging complexity of interactions between VEGF ligands and their receptors. This narrative review summarizes the family of VEGF-related molecules, including 7 mammalian members, namely, VEGF, placenta growth factor (PLGF), and VEGF-B, -C, -D, -E, and -F. The biological functions of VEGF are mediated by at least 3 corresponding receptors: VEGFR-1/Flt-1, VEGFR-2/Flk-1, VEGFR-3/Flt-4 and 2 co-receptors of neuropilin (NRP) and heparan sulfate proteoglycans (HSPGs). Current findings on endochondral ossification are also discussed, with emphasis on VEGF-A action in osteoblasts, chondroblasts, and chondroclasts/osteoclasts and regulatory mechanisms involving oxygen tension, and some growth factors and hormones. Furthermore, the therapeutic implications of recombinant VEGF-A protein therapy and VEGF-A gene therapy are evaluated. Abbreviations used: VEGF, Vascular endothelial growth factor; PLGF, placenta growth factor; NRP, neuropilin; HSPGs, heparan sulfate proteoglycans; FGF, fibroblast growth factor; TGF, transforming growth factor; HGF, hepatocyte growth factor; TNF, tumor necrosis factor; ECM, extracellular matrix; RTKs, receptor tyrosine kinases; ERK, extracellular signal kinases; HIF, hypoxia-inducible factor.

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Author and article information

Journal

Journal ID (nlm-ta): Exp Ther Med

Journal ID (iso-abbrev): Exp Ther Med

Journal ID (publisher-id): ETM

Title: Experimental and Therapeutic Medicine

Publisher: D.A. Spandidos

ISSN (Print): 1792-0981

ISSN (Electronic): 1792-1015

Publication date (Print): April 2018

Publication date (Electronic): 26 February 2018

Publication date PMC-release: 26 February 2018

Volume: 15

Issue: 4

Pages: 3715-3724

Affiliations

[1 ]Department of Orthopedics, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430014, P.R. China

[2 ]Department of Stomatology, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430014, P.R. China

[3 ]Department of Orthopedics, Wuhan Xinzhou District People's Hospital, Wuhan, Hubei 430400, P.R. China

Author notes

Correspondence to: Dr Hao Liu, Department of Orthopedics, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, 26 ShengLi Road, Wuhan, Hubei 430014, P.R. China, E-mail: haoliu6027@ 123456sina.com

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Publisher ID: ETM-0-0-5894

DOI: 10.3892/etm.2018.5894

PMC ID: 5863588

PubMed ID: 29581732

SO-VID: 3c1a7119-3aa7-497d-bab5-7133176981a9

License:

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Isolation, culture and induced differentiation of rabbit mesenchymal stem cells into osteoblasts

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Most cited references 31

Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors.

Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components.

VEGF: an essential mediator of both angiogenesis and endochondral ossification.

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