INTRODUCTION 14 15 25 48 l 5 38 5 13 9 in vivo in vivo 39 In vitro 13 27 24 32 3 17 21 47 22 35 26 46 31 The definition of the optimal condition of culture, such as coating of the substrate, synergistic effect of the substrate and growth factors are required for ligament tissue engineering. in vitro MATERIALS AND METHODS Cells and Culture Conditions 2 l l 3 2 Substrate Surface Treatment 4 2 Biochemical Stimuli 37 42 −1 −1 −1 −1 Fibroblast Proliferation Experiments MTT Assay μ 570 nm 700 nm Cell Cycle Analysis g 4 5 RNA Isolation and RT-PCR g g 260 nm 280 nm 1 TABLE 1. Prime sequence for RT-PCR. MRNA Primer sequence Product size (bp) Type I collagen 5′-TCC CCA GCC ACA AAG AGT CTA CA-3′ 155 5′-GTG ATT GGT GGG ATG TCT TCG TC-3′ Type III collagen 5′-CTG CCA TCC TGA ACT CAA GAG TGG-3′ 447 5′-CCA TCC TCC AGA ACT GTG TAG G-3′ Β-actin 5′-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-3′ 838 5′-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3′ Immuonofluorescence Labelling ex em para Statistical Analysis ® p RESULTS All the works were realized on three types of substrates (silicon sheet, silicon sheet with 1% gelatin coating as testing surfaces and glass surface used as a control). Effect of PDGF-AB on Cell Proliferation We studied the effect of PDGF-AB on fibroblast proliferation according to substrates and PDGF concentrations by two different and complementary methods. MTT Assays 1 −1 p −1 p −1 FIGURE 1. p 0.05 p 0.05 p 0.05 n  −1 Cell Cycle Analysis 2 3 FIGURE 2. −1 n  FIGURE 3. −1 p 0.05 n  3 −1 p Effect of TGF-β1 on the Expression of Collagens Effect of TGF-β1 on Type I and Type III Collagens mRNA Expressions 4 5 FIGURE 4. −1  FIGURE 5. p 0.05 p 0.05 p 0.05 n  5 −1 5 −1 −1 p −1 p −1 Qualitative Expression of Type I and Type III Collagens 6 FIGURE 6. μ −1 β −1 β  DISCUSSION in vitro 45 6 30 8 36 33 27 46 18 et al. 41 et al. 18 −1 −1 −1 −1 21 36 in vivo in vitro 2 12 19 43 20 23 11 16 40 44 Therefore, in the second step of our works, we study the effect of TGF-β1 on the ECM synthesis. Our data showed that the exogenous TGF-β1 influenced the expression of type I and type III collagen in a substrate- dependant manner. 1 10 49 However, on glass, we observed the highest expression of type III collagen mRNA in the control group. In the present work, we used culture medium containing 10% FBS. Although, we did not add any TGF-β1 in the medium, it seemed that this increase was due to the presence of growth factors in the serum and which normally stimulate protein synthesis and other different biological processes. −1 −1 21 4 7 28 29 32 34 in vitro Our results implied that it is better to use protein-coating substrate to improve the proliferation and the synthesis of collagen for later mechanical stretching study. Further studies are needed to determine the combination of these two growth factors and their effect on the biological processes.