[논문]조직공학 재생골을 위한 연구에서 사람 골수 기원 간엽줄기세포의 나이에 따른 조골세포 분화능에 관한 연구

송진아; 류현모; 최진영

doi:10.5125/jkaoms.2010.36.4.243

[국내논문] 조직공학 재생골을 위한 연구에서 사람 골수 기원 간엽줄기세포의 나이에 따른 조골세포 분화능에 관한 연구
Osteoblast differentiation of human bone marrow stromal cells (hBMSC) according to age for bone tissue engineering 원문보기

大韓口腔顎顔面外科學會誌 = Journal of the Korean Association of Oral and Maxillofacial Surgeons, v.36 no.4, 2010년, pp.243 - 249

Abstract ▼ AI-Helper

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.

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문제 정의

This study has showed that hBMSC from old donors have identical proliferation and cell surface marker character with young donors BMSC. And TSA, 5-azacitidine showed the possibility of increase of the BMP effect on osteoblast differentiations.

가설 설정

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)

제안 방법

Next, FACS analysis of the immunophenotypic surface profile for CD34, CD45, CD90 and CD105 of isolated hBMSC was performed to find the discrepancy of the cell surface marker expression between young and old donors.(Fig.
Chromatin state of histone acetylation and demethylation of highly methylated chromatin part such as CpG islands can cause easily gene expression to permit an osteogenesis. To test this possibility of HDAC inhibitor and methyltransferase inhibitor, we used the pretreatment with representative drug, TSA and 5-azacytidine for 24 hours before BMP-2 treatment.(Fig.
In this study, we investigated the effects of donor age on hBMSC proliferation and differentiation potentials towards adipogenic and osteogenic lineages. Although there were many reports about murine BMSC data, the controversy on several points was still remained in hBMSC works.
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.

대상 데이터

After washing with PBS, the cell pellets (1×10⁵ cells per antibody) were resuspended in 100 μL PBS, 1 μL primary antibody was added and incubated for 30 min. Monoclonal primary antibodies (fluorescein isothiocyanate-conjugated or Alexa Fluor 488-conjugated mouse anti-human) recognizing surface markers CD34, CD45, CD90 (Becton Dickinson, San Jose, USA) and CD105 (Invitrogen, Carlsbad, CA, USA) were used. After three washing steps with PBS, the cells were re-suspended in 400 μL PBS and analyzed using BD FACSCalibur and BD FACSCalibur software (Becton Dickinson, San Jose, USA).

성능/효과

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.
Also our data showed old donor’s BMSC showed more declines in differentiation capacity by donor age’s increasing despite of the maintained multipotency.
And 5-azacitidine, methyltransferase inhibitor, has been used optionally to differentiate BMSC to adipocyte or myoblast up to now. Our results showed more highly expressed osteogenic marker gene such as DLX5, ALP after TSA, 5-azacitine pretreatment.

후속연구

Based on the results of this study and many other previous studies, it appears that the osteoblast differentiation of hBMSC is considered to be less successful than animal cells. Future studies should be necessary for positive effect of the reversible inhibitory function of the HDAC inhibitors or methyltransferase inhibitors on enhancing the low capacity of osteoblast differentiation of hBMSC.

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