Osteoblast differentiation of human bone marrow stromal cells (hBMSC) according to age for bone tissue engineering

Gin-Ah Song; Hyun-Mo Ryoo; Jin-Young Choi

doi:10.5125/jkaoms.2010.36.4.243

Journal List > J Korean Assoc Oral Maxillofac Surg > v.36(4) > 1032395

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Song, Ryoo, and Choi: Osteoblast differentiation of human bone marrow stromal cells (hBMSC) according to age for bone tissue engineering

Original Article

J Korean Assoc Oral Maxillofac Surg 2010;36(4):243-249.

Published online: 10 January 2010

DOI: https://doi.org/10.5125/jkaoms.2010.36.4.243

Osteoblast differentiation of human bone marrow stromal cells (hBMSC) according to age for bone tissue engineering

Gin-Ah Song^1,², Hyun-Mo Ryoo¹, Jin-Young Choi²

¹Department of Molecular Genetics, School of Dentistry and Dental Research Institute, BK21 Program, Seoul, Korea

²Department of Oral and Maxillofacial Surgery, School of Dentistry, Seoul National University, Seoul, Korea

Jin-Young Choi Department of Oral and Maxillofacial Surgery, School of Dentistry, Seoul National University 28, Yeongeon-dong, Jongno-gu, Seoul, 110-749, Korea Tel: +82-2-2072-3992 Fax: +82-2-766-4948 E-mail: jinychoi@plaza.snu.ac.kr

Received 12 April 2010 Revised 10 June 2010 Accepted 23 June 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Tissue engineered bone (TEB) can replace an autogenous bone graft requiring an secondary operation site as well as avoid complications like inflammation or infection from xenogenic or synthetic bone graft. Adult mesenchymal stem cells (MSC) for TEB are considered to have various ranges of differentiation capacity or multipotency by the donor site and age. This study examined the effect of age on proliferation capacity, differentiation capacity and bone morphogenetic protein-2 (BMP-2) responsiveness of human bone marrow stromal cells (hBMSC) according to the age. In addition, to evaluate the effect on enhancement for osteoblast differentiation, the hBMSC were treated with Trichostatin A (TSA) and 5-Azacitidine (5-AZC) which was HDAC inhibitors and methyltransferase inhibitors respectively affecting chromatin remodeling temporarily and reversibly. The young and old group of hBMSC obtained from the iliac crest from total 9 healthy patients, showed similar proliferation capacity. Cell surface markers such as CD34, CD45, CD90 and CD105 showed uniform expression regardless of age. However, the young group showed more prominent transdifferentiation capacity with adipogenic differentiation. The osteoblast differentiation capacity or BMP responsiveness was low and similar between young and old group. TSA and 5-AZC showed potential for enhancing the BMP effect on osteoblast differentiation by increasing the expression level of osteogenic master gene, such as DLX5, ALP. More study will be needed to determine the positive effect of the reversible function of HDAC inhibitors or methyltransferase inhibitors on enhancing the low osteoblast differentiation capacity of hBMSC.

Keywords: Mesenchymal stem cells, Osteoblast differentiation, Histone deacetylase inhibitors, DNA methyltransferase inhibitors, Aging

References

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Fig. 1.

Proliferation assay of hBMSC. A. Total cell numbers of harvested hBMSC after P0 culture was manually counted by hemocytometer between culture day D13 and D17. B. CCK-8 assay was performed after 12, 36, 60, 120 hours culture with P3–5 cells of respective hBMSC. C. Population doubling time (PDT) was analyzed. (CCK: cell counting kit)

Fig. 2.

Surface marker expression of hBMSC between young and old aged group. FACS of the immunophenotypic surface profile for CD34, CD45, CD90 and CD105 from isolated hBMSC was analyzed. Black line empty histograms represent the fluorescence from negative-control cells incubated without antibody: blue colored histograms represent the counts of singals incubated with the relevant cell surface antibody. (FACS: fluorescence-activated cell sorting)

Fig. 3.

Multipotency of hBMSC. A. Oil-red O staining after adipogenic differentiation for 2 weeks in adipogenic medium. B. Alizarin-Red S staining after osteogenic differentiation for 2 weeks in osteogenic medium.

Fig. 4.

Low rhBMP-2 responsiveness of hBMSC. (rhBMP-2: recombinant human bone morphogenetic protein-2)

Fig. 5.

Osteogenic marker gene expression of hBMSC after TSA, 5-AZC pretreatment by semiquantitive RT-PCR. (TSA: trichostatin A, 5-AZC: 5-azacytidine, DLX5: distal-less homeobox 5, ALP: Alkaline phosphatase, GAPDH: glyceraldehyde-3-phosphate dehydrogenase)

Table 1.

Characteristics of hBMSC donors from iliac crest

Subject	Age/Sex
Y1	21/M
Y2	25/F
Y3	26/F
M1	39/F
M2	39/F
M3	33/F
E1	54/F
E2	61/F
E3	70/M

(hBMSC: human bone marrow stromal cell, M: male, F: female)

Table 2.

Primer sets for RT-PCR

Symbol	Name	GenBank No.	Primer
DLX5	Distal-less homeobox 5	NM_005221.5	5′-AGCTCCTACCACCAGTACGG-3′
DLX5	Distal-less homeobox 5	NM_005221.5	5′-GTTTGCCATTCACCATTCTCAC-3′
ALP	Alkaline phosphatase, liver/bone/kidney	NM_000478.3	5′-ATGGGATGGGTGTCTCCACA-3′
ALP	Alkaline phosphatase, liver/bone/kidney	NM_000478.3	5′-CCACGAAGGGGAACTTGTC-3′
GAPDH	Glyceraldehyde-3-phosphate dehydrogenase	NM_002046.3	5′-CATGAGAAGTATGACAACAGCCT-3′
GAPDH	Glyceraldehyde-3-phosphate dehydrogenase	NM_002046.3	5′-AGTCCTTCCACGATACCAAAGT-3′

(RT-PCR: reverse transcription polymerase chain reaction)

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