CONTINUOUS TREATMENT OF TGF-β IS NEEDED TO INDUCE CHONDROGENESIS FROM MESENCHYMAL STEM CELLS

*Kim, H J; **Koo, H J; *Tae, S K; *Jung N H, +*Im, G I

+Dongguk University International Hospital, Goyang, Korea gunil@duih.org

Introduction

Transforming growth factor (TGF-β) treatment is essential for the chondrogenic differentiation of MSCs [1-4]. Because growth factors are expensive, a precise knowledge in employing them in a proper and economic way would help to minimize the cost of cartilage tissue engineering from mesenchymal stem cells. There is a controversy about on how long TGF-β should be given to induce effective chondrogenesis from mesenchymal stem cells. To answer the question, we investigated the response of mesenchymal stem cells to various treatments of TGF-β. The hypothesis of this study was that effective chondrogenesis can be also achieved with brief administration of TGF-β from human MSCs comparable to continuous treatment.

Materials and methods MSCs were isolated from human bone marrow aspirates, and

expanded to passage 3, then further cultured for chondrogenesis in DMEM/ F-12 supplemented with 1% ITS, 10-7 M dexamethasone, 50uM ascorbate-2-phosphate, 50uM L-proline, and 1mM sodium pyruvate. Four sets of growth factor treatments were tried: #1 Monolayer culture for 3 days followed by pellet culture for 4 weeks. No growth factors were given in any period and this set was used as the control; #2 Monolayer culture in 5ng/ml of TGF-β2 for 3 days followed by pellet culture without treatment of growth factors; #3 Monolayer culture in 5ng/ml of TGF-β2 for 3 days followed by a pellet culture for 4 weeks during which 5 ng/ml of TGF-β2 was treated for first 3 days; #4 Monolayer culture in 5ng/ml of TGF-β2 for 3 days followed pellet culture for 4 weeks under continuous treatment of 5 ng/ml of TGF-β2. Then, the amount of DNA and glycosaminoglycan in the pellet was analyzed. RT-PCR was done for the mRNAs of type I collagen, type II collagen, COMP and GAPDH. Pellets were also fixed for Safranin-O staining and immunohistochemical staining for type II collagen.

Results The amount of DNA significantly increased compared with the control (no growth factors) only when TGF-β2 was continuously given (Fig.1), GAG to DNA ratio also significantly increased only when continuously treated with TGF-β2 (Fig.2). RT-PCR showed remarkable increase in the expression in type II collagen and COMP only with continuous exposure to TGF-β2 (Fig.3). Safranin-O and immunohistochemical staining showed gradual increase in the production of proteoglycan and type II collagen with the longer duration of exposure to TGF-β2 (Fig.4).

Discussion

The over all results of this study demonstrates that continuous administration of TGF-β is necessary for an effective chondrogenesis in mesenchymal stem cells from bone marrow.

Fig.1 The amount of DNA per pellet with various conditions of TGF-β2 treatment

Fig.2 GAG to DNA ratio from each pellet

Fig.3 RT-PCR from each pellet

Fig.4 Safranin-O staining from each pellet (x40) References

1. Johnstone B et al. Exp Cell Res 238:265(1998). 2. Fortier LA et al. Am J Vet Res 59:1182(1998). 3. Mackay AM et al. Tissue Eng 4:418(1998) 4. Im GI et al. Tissue Eng 12:527(2006). Acknowledgement

This study was supported by a grant (A060215) of the Korea Health 21 R & D Project, Ministry of Health & Welfare, Repubric of Korea.

(Mean+ S.E., n=5, p<0.05) *

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53rd Annual Meeting of the Orthopaedic Research Society

Poster No: 0471