J Korean Assoc Oral Maxillofac Surg.  2010 Aug;36(4):243-249. 10.5125/jkaoms.2010.36.4.243.

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

• Song GA
• Ryoo HM
• Choi JY

• KMID: 1457774
• DOI: http://doi.org/10.5125/jkaoms.2010.36.4.243

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.

Keyword

Mesenchymal stem cells; Osteoblast differentiation; Histone deacetylase inhibitors; DNA methyltransferase inhibitors; Aging

MeSH Terms

Adult
Aging
Bone and Bones
Bone Marrow
Chromatin Assembly and Disassembly
Durapatite
Histone Deacetylase Inhibitors
Humans
Hydroxamic Acids
Inflammation
Mesenchymal Stromal Cells
Osteoblasts
Tissue Donors
Transplants
Durapatite
Histone Deacetylase Inhibitors
Hydroxamic Acids

Figure

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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)

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  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)

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  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.

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  Fig. 4. Low rhBMP-2 responsiveness of hBMSC. (rhBMP-2: recombinant human bone morphogenetic protein-2)

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  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)

Reference

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