TGF-β1 diminishes collagen production during long-term cyclic stretching of engineered connective tissue: implication of decreased ERK signaling.
Cyclic stretching and growth factors like TGF-β have been used to enhance extracellular matrix (ECM) production by cells in engineered tissue to achieve requisite mechanical properties. In this study, the effects of TGF-β1 were evaluated during long-term cyclic stretching of fibrin-based tubular constructs seeded with neonatal human dermal fibroblasts. Samples were evaluated at 2, 5, and 7 weeks for tensile mechanical properties and ECM deposition. At 2 weeks, +TGF-β1 samples had 101% higher collagen concentration but no difference in ultimate tensile strength (UTS) or modulus compared to -TGF-β1 samples. However, at weeks 5 and 7, -TGF-β1 samples had higher UTS/modulus and collagen concentration, but lower elastin concentration compared to +TGF-β1 samples. The collagen was better organized in -TGF-β1 samples based on picrosirius red staining. Western blot analysis at weeks 5 and 7 showed increased phosphorylation of ERK in -TGF-β1 samples, which correlated with higher collagen deposition. The TGF-β1 effects were further evaluated by western blot for αSMA and SMAD2/3 expression, which were 16-fold and 10-fold higher in +TGF-β1 samples, respectively. The role of TGF-β1 activated p38 in inhibiting phosphorylation of ERK was evaluated by treating samples with SB203580, an inhibitor of p38 activation. SB203580-treated cells showed increased phosphorylation of ERK after 1 hour of stretching and increased collagen production after 1 week of stretching, demonstrating an inhibitory role of activated p38 via TGF-β1 signaling during cyclic stretching. One advantage of TGF-β1 treatment was the 4-fold higher elastin deposition in samples at 7 weeks. Further cyclic stretching experiments were thus conducted with constructs cultured for 5 weeks without TGF-β1 to obtain improved tensile properties followed by TGF-β1 supplementation for 2 weeks to obtain increased elastin content, which correlated with a reduction in loss of pre-stress during preconditioning for tensile testing, indicating functional elastin. This study shows that a sequential stimulus approach - cyclic stretching with delayed TGF-β1 supplementation - can be used to engineer tissue with desirable tensile and elastic properties.
Department of Chemical Engineering & Materials Science, University of Minnesota, MN 55455, USA.
Zeeshan H
ZH
Syedain
Robert T
RT
Tranquillo
D001391:Azo Compounds / Q000494:pharmacology; D003094:Collagen / Q000737:chemistry; D003238:Connective Tissue / Q000473:pathology*; D004548:Elasticity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D007231:Infant, Newborn; D015398:Signal Transduction; D012867:Skin / Q000473:pathology; D013314:Stress, Mechanical; D013718:Tensile Strength; D023822:Tissue Engineering / Q000379:methods; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D001391:Azo Compounds; D004791:Enzyme Inhibitors; D053773:Transforming Growth Factor beta1; C009798:C.I. direct red 80; D003094:Collagen; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases
Cardiovasc Pathol. 2007 Sep-Oct;16(5):268-76
17868877
Nat Rev Mol Cell Biol. 2000 Dec;1(3):169-78
11252892
Am J Physiol Heart Circ Physiol. 2006 Dec;291(6):H2924-32
16891407
Exp Cell Res. 1996 Feb 25;223(1):127-34
8635484
Biomaterials. 2011 Jan;32(3):714-22
20934214
Am J Physiol. 1999 Nov;277(5):G1074-80
10564114
Am J Pathol. 2003 Feb;162(2):533-46
12547711
Circ Res. 2006 Jan 6;98(1):25-35
16397155
Ann Biomed Eng. 2003 Sep;31(8):937-49
12918909
Matrix Biol. 2003 Jun;22(4):339-50
12935818
Tissue Eng Part A. 2008 Jul;14(7):1161-72
18380592
Anal Biochem. 1976 Aug;74(2):441-7
822746
Clin Chim Acta. 1967 Nov;18(2):267-73
4864804
Am J Physiol Cell Physiol. 2005 Sep;289(3):C766-73
15814588
J Biol Chem. 2007 Apr 6;282(14):10405-13
17317656
Tissue Eng. 2006 Aug;12(8):2367-83
16968176
Histochemistry. 1989;93(1):27-9
2482274
J Biol Chem. 1997 Jan 3;272(1):345-52
8995268
Genes Dev. 2005 Dec 1;19(23):2783-810
16322555
Tissue Eng Part A. 2008 Jun;14(6):1059-66
19230128
J Biomech. 2004 Oct;37(10):1543-50
15336929
Am J Physiol Lung Cell Mol Physiol. 2007 Apr;292(4):L944-52
17209135
Proc Natl Acad Sci U S A. 2008 May 6;105(18):6537-42
18436647
Matrix Biol. 2003 Nov;22(6):477-90
14667840
J Biol Chem. 2002 Feb 22;277(8):6153-61
11748224
Pharm Res. 2008 May;25(5):1212-21
18092140
J Cell Sci. 1995 Mar;108 ( Pt 3):1251-61
7622608
Cardiovasc Res. 2004 Mar 1;61(4):736-44
14985070
Biomaterials. 2009 Sep;30(25):4078-84
19473698
Am J Respir Cell Mol Biol. 2002 Feb;26(2):183-8
11804868
Circulation. 2006 Feb 14;113(6):e85-151
16407573
Tissue Eng. 2007 Jul;13(7):1501-11
17518750
Circ Res. 2004 Aug 6;95(3):253-60
15217906
Tissue Eng Part A. 2008 Jan;14(1):83-95
18333807
Philos Trans R Soc Lond B Biol Sci. 2007 Aug 29;362(1484):1253-5
17569644
J Invest Dermatol. 2002 Apr;118(4):704-11
11918720
J Invest Dermatol. 2008 Aug;128(8):1906-14
18323784
Tissue Eng. 2007 Apr;13(4):799-807
17346099
Biomaterials. 2002 Sep;23(17):3717-31
12109697
Histochemistry. 1982;74(1):153-6
7085347
Osteoarthritis Cartilage. 2007 Sep;15(9):1025-33
17498976