Effects of Intermittent and Incremental Cyclic Stretch on ERK Signaling and Collagen Production in Engineered Tissue

Abstract

Intermittent cyclic stretching and incrementally increasing strain amplitude cyclic stretching were explored to overcome the reported adaptation of fibroblasts in response to constant amplitude cyclic stretching, with the goals of accelerating collagen production and understanding the underlying cell signaling. The effects of constant amplitude, intermittent, and incremental cyclic stretching regimens were investigated for dermal fibroblasts entrapped in a fibrin gel by monitoring the extracellular signal-regulated kinase (ERK1/2) and p38 pathways, collagen transcription, and finally the deposited collagen protein. Activation of ERK1/2, which has been shown to be necessary for stretch-induced collagen transcription, was maximal at 15 min and decayed by 1 h. ERK1/2 was reactivated by an additional onset of stretching or by an increment in the strain amplitude 6 h after the initial stimulus, which was approximately the lifetime of activated p38, a known ERK1/2 inhibitor. While both intermittent and incremental regimens reactivated ERK1/2, only incremental stretching increased collagen production compared to samples stretched with constant amplitude, resulting in a 37% increase in collagen per cell after 2 weeks. This suggests that a regimen with small, frequent increments in strain amplitude is optimal for this system and should be used in bioreactors for engineered tissues requiring high collagen content.

This is a preview of subscription content, log in to check access.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Abbreviations

ANOVA:

Analysis of variance

COL1A1/COL3A1:

Collagen Iα1/collagen IIIα1

DMSO:

Dimethylsulfoxide

ERK1/2:

Extracellular signal-regulated kinase 1/2

MAPK:

Mitogen-activated protein kinase

PBS:

Phosphate buffered saline

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TBS:

Tris-buffered saline

References

  1. 1.

    Albro, M. B., N. O. Chahine, R. Li, K. Yeager, C. T. Hung, and G. A. Ateshian. Dynamic loading of deformable porous media can induce active solute transport. J. Biomech. 41:3152–3157, 2008.

    Article  Google Scholar 

  2. 2.

    Berry, C. C., J. C. Shelton, D. L. Bader, and D. A. Lee. Influence of external uniaxial cyclic strain on oriented fibroblast-seeded collagen gels. Tissue Eng. 9:613–624, 2003.

    Article  Google Scholar 

  3. 3.

    Canty, E. G., and K. E. Kadler. Procollagen trafficking, processing and fibrillogenesis. J. Cell Sci. 118:1341–1353, 2005.

    Article  Google Scholar 

  4. 4.

    Chahine, N. O., M. B. Albro, E. G. Lima, V. I. Wei, C. R. Dubois, C. T. Hung, and G. A. Ateshian. Effect of dynamic loading on the transport of solutes into agarose hydrogels. Biophys. J. 97:968–975, 2009.

    Article  Google Scholar 

  5. 5.

    Flanagan, T. C., C. Cornelissen, S. Koch, B. Tschoeke, J. S. Sachweh, T. Schmitz-Rode, and S. Jockenhoevel. The in vitro development of autologous fibrin-based tissue-engineered heart valves through optimised dynamic conditioning. Biomaterials 28:3388–3397, 2007.

    Article  Google Scholar 

  6. 6.

    Grassl, E. D., T. R. Oegema, and R. T. Tranquillo. A fibrin-based arterial media equivalent. J. Biomed. Mater. Res. 66A:550–561, 2003.

    Article  Google Scholar 

  7. 7.

    Jansen, J. H., F. A. Weyts, I. Westbroek, H. Jahr, H. Chiba, H. A. Pols, J. A. Verhaar, J. P. van Leeuwen, and H. Weinans. Stretch-induced phosphorylation of ERK1/2 depends on differentiation stage of osteoblasts. J. Cell. Biochem. 93:542–551, 2004.

    Article  Google Scholar 

  8. 8.

    Kim, S. G., T. Akaike, T. Sasagaw, Y. Atomi, and H. Kurosawa. Gene expression of type I and type III collagen by mechanical stretch in anterior cruciate ligament cells. Cell Struct. Funct. 27:139–144, 2002.

    Article  Google Scholar 

  9. 9.

    Mol, A., N. J. B. Driessen, M. C. M. Rutten, S. P. Hoerstrup, C. V. C. Bouten, and F. P. T. Baaijens. Tissue engineering of human heart valve leaflets: a novel bioreactor for a strain-based conditioning approach. Ann. Biomed. Eng. 33:1778–1788, 2005.

    Article  Google Scholar 

  10. 10.

    Neidert, M. R., E. S. Lee, T. R. Oegema, and R. T. Tranquillo. Enhanced fibrin remodeling in vitro with TGF-beta 1, insulin and plasmin for improved tissue-equivalents. Biomaterials 23:3717–3731, 2002.

    Article  Google Scholar 

  11. 11.

    Nishimura, K., P. Blume, S. Ohgi, and B. E. Sumpio. Effect of different frequencies of tensile strain on human dermal fibroblast proliferation and survival. Wound Repair. Regen. 15:646–656, 2007.

    Article  Google Scholar 

  12. 12.

    O’Neill, R. A., A. Bhamidipati, X. Bi, D. Deb-Basu, L. Cahill, J. Ferrante, E. Gentalen, M. Glazer, J. Gossett, K. Hacker, C. Kirby, J. Knittle, R. Loder, C. Mastroieni, M. Maclaren, T. Mills, U. Nguyen, N. Parker, A. Rice, D. Roach, D. Suich, D. Voehringer, K. Voss, J. Yang, T. Yang, and P. B. Vander. Horn. Isoelectric focusing technology quantifies protein signaling in 25 cells. Proc. Natl. Acad. Sci. USA 103:16153–16158, 2006.

    Article  Google Scholar 

  13. 13.

    Papakrivopoulou, J., G. E. Lindahl, J. E. Bishop, and G. J. Laurent. Differential roles of extracellular signal-regulated kinase 1/2 and p38MAPK in mechanical load-induced procollagen alpha1(I) gene expression in cardiac fibroblasts. Cardiovasc. Res. 61:736–744, 2004.

    Article  Google Scholar 

  14. 14.

    Paxton, J. Z., P. Hagerty, J. J. Andrick, and K. Baar. Optimizing an intermittent stretch paradigm using ERK1/2 phosphorylation results in increased collagen synthesis in engineered ligaments. Tissue Eng. Part A 18:277–284, 2012.

    Article  Google Scholar 

  15. 15.

    Raghupathy, R., C. Witzenburg, S. P. Lake, E. A. Sander, and V. H. Barocas. Identification of regional mechanical anisotropy in soft tissue analogs. J. Biomech. Eng. 133:091011, 2011.

    Article  Google Scholar 

  16. 16.

    Rubbens, M. P., A. Mol, R. A. Boerboom, R. A. Bank, F. P. Baaijens, and C. V. Bouten. Intermittent straining accelerates the development of tissue properties in engineered heart valve tissue. Tissue Eng. Part A 15:999–1008, 2009.

    Article  Google Scholar 

  17. 17.

    Ruberti, J. W., and N. J. Hallab. Strain-controlled enzymatic cleavage of collagen in loaded matrix. Biochem. Biophys. Res. Commun. 336:483–489, 2005.

    Article  Google Scholar 

  18. 18.

    Rubin, J., T. C. Murphy, X. Fan, M. Goldschmidt, and W. R. Taylor. Activation of extracellular signal-regulated kinase is involved in mechanical strain inhibition of RANKL expression in bone stromal cells. J. Bone Miner. Res. 17:1452–1460, 2002.

    Article  Google Scholar 

  19. 19.

    Solan, A., S. L. Dahl, and L. E. Niklason. Effects of mechanical stretch on collagen and cross-linking in engineered blood vessels. Cell Transplant. 18:915–921, 2009.

    Article  Google Scholar 

  20. 20.

    Stegemann, H., and K. Stalder. Determination of hydroxyproline. Clin. Chim. Acta 18:267–273, 1967.

    Article  Google Scholar 

  21. 21.

    Stekelenburg, M., M. C. Rutten, L. H. Snoeckx, and F. P. Baaijens. Dynamic straining combined with fibrin gel cell seeding improves strength of tissue-engineered small-diameter vascular grafts. Tissue Eng. Part A 15:1081–1089, 2009.

    Article  Google Scholar 

  22. 22.

    Syedain, Z. H., L. A. Meier, J. W. Bjork, A. Lee, and R. T. Tranquillo. Implantable arterial grafts from human fibroblasts and fibrin using a multi-graft pulsed flow-stretch bioreactor with noninvasive strength monitoring. Biomaterials 32:714–722, 2011.

    Article  Google Scholar 

  23. 23.

    Syedain, Z., L. Meier, J. Reimer, and R. Tranquillo. Tubular heart valves from decellularized engineered tissue. Ann. Biomed. Eng. 41:2645–2654, 2013.

    Article  Google Scholar 

  24. 24.

    Syedain, Z. H., and R. T. Tranquillo. Controlled cyclic stretch bioreactor for tissue-engineered heart valves. Biomaterials 30:4078–4084, 2009.

    Article  Google Scholar 

  25. 25.

    Syedain, Z. H., J. S. Weinberg, and R. T. Tranquillo. Cyclic distension of fibrin-based tissue constructs: evidence of adaptation during growth of engineered connective tissue. Proc. Natl. Acad. Sci. USA 105:6537–6542, 2008.

    Article  Google Scholar 

  26. 26.

    Van Geemen, D., A. Driessen-Mol, F. P. T. Baaigens, and C. Bouten. Understanding strain-induced collagen matrix development in engineered cardiovascular tissues from gene expression profiles. Cell Tissue Res. 352:727–737, 2013.

    Article  Google Scholar 

  27. 27.

    Weinbaum, J., J. Schmidt, and R. Tranquillo. Combating adaptation to cyclic stretching by prolonging activation of extracellular signal-regulated kinase. Cell. Mol. Bioeng. 6:279–286, 2013.

    Article  Google Scholar 

  28. 28.

    Williams, C., S. L. Johnson, P. S. Robinson, and R. T. Tranquillo. Cell sourcing and culture conditions for fibrin-based valve constructs. Tissue Eng. 12:1489–1502, 2006.

    Article  Google Scholar 

Download references

Acknowledgments

The authors thank Naomi Ferguson, Sandra Johnson, Jay Reimer, Dr. Colleen Witzenburg, and Dr. M. Cristine Charlesworth for technical assistance and Kiley Schmidt for providing illustrations. Nanoimmunoassay was performed in the Mayo Clinic Proteomics Core. This study was funded by a National Science Foundation Graduate Research Fellowship (to J.B.S) and National Institutes of Health/National Heart, Lung, and Blood Institute award HL107572 (to R.T.T.).

Conflict of interest

Jillian B. Schmidt, Kelley Chen, and Robert T. Tranquillo declare that they have no conflicts of interest.

Ethical Standards

No human or animal studies were carried out by the authors for this article.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Robert T. Tranquillo.

Additional information

Associate Editor Michael R. King oversaw the review of this article.

Electronic Supplementary Material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Schmidt, J.B., Chen, K. & Tranquillo, R.T. Effects of Intermittent and Incremental Cyclic Stretch on ERK Signaling and Collagen Production in Engineered Tissue. Cel. Mol. Bioeng. 9, 55–64 (2016). https://doi.org/10.1007/s12195-015-0415-6

Download citation

Keywords

  • Fibrin
  • Fibroblast
  • Mitogen activated protein kinase
  • p38
  • Mechanical conditioning