Skip to main content
SpringerLink
Log in

Scaffold-Free Tissue Engineering: Organization of the Tissue Cytoskeleton and Its Effects on Tissue Shape

  • Published:
Annals of Biomedical Engineering Aims and scope Submit manuscript

Abstract

Work described herein characterizes tissues formed using scaffold-free, non-adherent systems and investigates their utility in modular approaches to tissue engineering. Immunofluorescence analysis revealed that all tissues formed using scaffold-free, non-adherent systems organize tissue cortical cytoskeletons that appear to be under tension. Tension in these tissues was also evident when modules (spheroids) were used to generate larger tissues. Real-time analysis of spheroid fusion in unconstrained systems illustrated modular motion that is compatible with alterations in tensions, due to the process of disassembly/reassembly of the cortical cytoskeletons required for module fusion. Additionally, tissues generated from modules placed within constrained linear molds, which restrict modular motion, deformed upon release from molds. That tissue deformation is due in full or in part to imbalanced cortical actin cytoskeleton tensions resulting from the constraints imposed by mold systems is suggested from our finding that treatment of forming tissues with Y-27632, a selective inhibitor of ROCK phosphorylation, reduced tissue deformation. Our studies suggest that the deformation of scaffold-free tissues due to tensions mediated via the tissue cortical cytoskeleton represents a major and underappreciated challenge to modular tissue engineering.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11

Similar content being viewed by others

References

  1. Amack, J. D., and M. L. Manning. Knowing the boundaries: extending the differential adhesion hypothesis in embryonic cell sorting. Science 338(6104):212–215, 2012.

    Article  PubMed  CAS  Google Scholar 

  2. Dean, D. M., and J. R. Morgan. Cytoskeletal-mediated tension modulates the directed self-assembly of microtissues. Tissue Eng. Part A 14(12):1989–1997, 2008.

    Article  PubMed  CAS  Google Scholar 

  3. Dean, D. M., A. P. Napolitano, J. Youssef, and J. R. Morgan. Rods, tori, and honeycombs: the directed self-assembly of microtissues with prescribed microscale geometries. FASEB J. 21(14):4005–4012, 2007.

    Article  PubMed  CAS  Google Scholar 

  4. Fleming, P. A., W. S. Argraves, C. Gentile, A. Neagu, G. Forgacs, and C. J. Drake. Fusion of uniluminal vascular spheroids: a model for assembly of blood vessels. Dev. Dyn. 239(2):398–406, 2010.

    Article  PubMed Central  PubMed  Google Scholar 

  5. Foty, R. A., C. M. Pfleger, G. Forgacs, and M. S. Steinberg. Surface tensions of embryonic tissues predict their mutual envelopment behavior. Development 122(5):1611–1620, 1996.

    PubMed  CAS  Google Scholar 

  6. Foty, R. A., and M. S. Steinberg. The differential adhesion hypothesis: a direct evaluation. Dev. Biol. 278(1):255–263, 2005.

    Article  PubMed  CAS  Google Scholar 

  7. Fung, Y. C. What are the residual stresses doing in our blood vessels? Ann. Biomed. Eng. 19(3):237–249, 1991.

    Article  PubMed  CAS  Google Scholar 

  8. Gentile, C., P. A. Fleming, V. Mironov, K. M. Argraves, W. S. Argraves, and C. J. Drake. VEGF-mediated fusion in the generation of uniluminal vascular spheroids. Dev. Dyn. 237(10):2918–2925, 2008.

    Article  PubMed Central  PubMed  Google Scholar 

  9. Han, H. C., S. Marita, and D. N. Ku. Changes of opening angle in hypertensive and hypotensive arteries in 3-day organ culture. J. Biomech. 39(13):2410–2418, 2006.

    Article  PubMed  Google Scholar 

  10. Heidemann, S. R., S. Kaech, R. E. Buxbaum, and A. Matus. Direct observations of the mechanical behaviors of the cytoskeleton in living fibroblasts. J. Cell Biol. 145(1):109–122, 1999.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  11. Holtfreter, J. Gewebeaffinitat, ein mittel der embryonal formbildung. Arch. Exptl. Zellforsch. Gewebezucht 23:169–209, 1939.

    Google Scholar 

  12. Ingber, D. E. Tensegrity: the architectural basis of cellular mechanotransduction. Annu. Rev. Physiol. 59:575–599, 1997.

    Article  PubMed  CAS  Google Scholar 

  13. Ingber, D. E. Integrins, tensegrity, and mechanotransduction. Gravit. Space Biol. Bull. 10(2):49–55, 1997.

    PubMed  CAS  Google Scholar 

  14. Katoh, K., Y. Kano, M. Amano, K. Kaibuchi, and K. Fujiwara. Stress fiber organization regulated by MLCK and rho-kinase in cultured human fibroblasts. Am. J. Physiol. Cell Physiol. 280(6):C1669–C1679, 2001.

    PubMed  CAS  Google Scholar 

  15. Katoh, K., Y. Kano, M. Amano, H. Onishi, K. Kaibuchi, and K. Fujiwara. Rho-kinase-mediated contraction of isolated stress fibers. J. Cell Biol. 153(3):569–584, 2001.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  16. Kelm, J. M., V. Lorber, J. G. Snedeker, D. Schmidt, A. Broggini-Tenzer, M. Weisstanner, et al. A novel concept for scaffold-free vessel tissue engineering: self-assembly of microtissue building blocks. J. Biotechnol. 148(1):46–55, 2010.

    Article  PubMed  CAS  Google Scholar 

  17. Korff, T., and H. G. Augustin. Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation. J. Cell Biol. 143(5):1341–1352, 1998.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  18. Korff, T., S. Kimmina, G. Martiny-Baron, and H. G. Augustin. Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness. FASEB J. 15(2):447–457, 2001.

    Article  PubMed  CAS  Google Scholar 

  19. Lei, S., Y. P. Tian, W. D. Xiao, S. Li, X. C. Rao, J. L. Zhang, et al. ROCK is involved in vimentin phosphorylation and rearrangement induced by dengue virus. Cell Biochem. Biophys. 67(3):1333–1342, 2013.

    Google Scholar 

  20. Liu, S. Q., and Y. C. Fung. Zero-stress states of arteries. J. Biomech. Eng. 110(1):82–84, 1988.

    Article  PubMed  CAS  Google Scholar 

  21. Lyle, K. S., J. A. Corleto, and T. Wittmann. Microtubule dynamics regulation contributes to endothelial morphogenesis. Bioarchitecture 2(6):220–227, 2012.

    Article  PubMed Central  PubMed  Google Scholar 

  22. Manning, M. L., R. A. Foty, M. S. Steinberg, and E. M. Schoetz. Coaction of intercellular adhesion and cortical tension specifies tissue surface tension. Proc. Natl Acad. Sci. USA. 107(28):12517–12522, 2010.

    Article  PubMed Central  PubMed  Google Scholar 

  23. Mironov, V., V. Kasyanov, C. Drake, and R. R. Markwald. Organ printing: promises and challenges. Regen. Med. 3(1):93–103, 2008.

    Article  PubMed  CAS  Google Scholar 

  24. Moscana, A., and H. Moscana. The dissociation and aggregation of cells from organ rudiments of the early chick embryo. J. Anat. 86(3):287–301, 1952.

    Google Scholar 

  25. Nicol, A., and D. R. Garrod. The sorting out of embryonic cells in monolayer, the differential adhesion hypothesis and the non-specificity of cell adhesion. J. Cell Sci. 38:249–266, 1979.

    PubMed  CAS  Google Scholar 

  26. Omelchenko, T., E. Fetisova, O. Ivanova, E. M. Bonder, H. Feder, J. M. Vasiliev, et al. Contact interactions between epitheliocytes and fibroblasts: formation of heterotypic cadherin-containing adhesion sites is accompanied by local cytoskeletal reorganization. Proc. Natl Acad. Sci. USA. 98(15):8632–8637, 2001.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  27. Parsons, J. T., A. R. Horwitz, and M. A. Schwartz. Cell adhesion: integrating cytoskeletal dynamics and cellular tension. Nat. Rev. Mol. Cell Biol. 11(9):633–643, 2010.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  28. Phillips, H. M., and M. S. Steinberg. Embryonic tissues as elasticoviscous liquids. I. rapid and slow shape changes in centrifuged cell aggregates. J. Cell Sci. 30:1–20, 1978.

    PubMed  CAS  Google Scholar 

  29. Pollard, T. D., and J. A. Cooper. Actin, a central player in cell shape and movement. Science 326(5957):1208–1212, 2009.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  30. Rachev, A., and S. E. Greenwald. Residual strains in conduit arteries. J. Biomech. 36(5):661–670, 2003.

    Article  PubMed  CAS  Google Scholar 

  31. Shutova, M., C. Yang, J. M. Vasiliev, and T. Svitkina. Functions of nonmuscle myosin II in assembly of the cellular contractile system. PLoS One 7(7):e40814, 2012.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  32. Sokabe, M., K. Naruse, S. Sai, T. Yamada, K. Kawakami, M. Inoue, et al. Mechanotransduction and intracellular signaling mechanisms of stretch-induced remodeling in endothelial cells. Heart Vessels Suppl 12:191–193, 1997.

    PubMed  CAS  Google Scholar 

  33. Stamenovic, D. Rheological behavior of mammalian cells. Cell Mol. Life Sci. 65(22):3592–3605, 2008.

    Article  PubMed  CAS  Google Scholar 

  34. Steinberg, M. S. Differential adhesion in morphogenesis: a modern view. Curr. Opin. Genet. Dev. 17(4):281–286, 2007.

    Article  PubMed  CAS  Google Scholar 

  35. Steinberg, M. S., and M. Takeichi. Experimental specification of cell sorting, tissue spreading, and specific spatial patterning by quantitative differences in cadherin expression. Proc. Natl Acad. Sci. USA. 91(1):206–209, 1994.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  36. Stirbat, T. V., A. Mgharbel, S. Bodennec, K. Ferri, H. C. Mertani, J. P. Rieu, et al. Fine tuning of tissues’ viscosity and surface tension through contractility suggests a new role for alpha-catenin. PLoS One 8(2):e52554, 2013.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  37. Wang, N., J. P. Butler, and D. E. Ingber. Mechanotransduction across the cell surface and through the cytoskeleton. Science 260(5111):1124–1127, 1993.

    Article  PubMed  CAS  Google Scholar 

  38. Wang, N., K. Naruse, D. Stamenovic, J. J. Fredberg, S. M. Mijailovich, I. M. Tolic-Norrelykke, et al. Mechanical behavior in living cells consistent with the tensegrity model. Proc. Natl Acad. Sci. USA. 98(14):7765–7770, 2001.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  39. Youssef, J., A. K. Nurse, L. B. Freund, and J. R. Morgan. Quantification of the forces driving self-assembly of three-dimensional microtissues. Proc. Natl Acad. Sci. USA. 108(17):6993–6998, 2011.

    Article  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank Dr. Susan M. Lessner, Ph.D. for her critical reading of this manuscript. This work was supported by NIH R01 DE019355 (MJY), NIH R01 HL080168 (CJD), NSF EPSCoR RII EPS-0903795 (CJD), and by NIH predoctoral fellowship T32 HL007260 (supporting CAC, awarded to Dr. Donald R. Menick, MUSC, PI). This study used the services of the Morphology, Imaging and Instrumentation Core, which is supported by NIH-NIGMS P30 GM103342 to the South Carolina COBRE for Developmentally Based Cardiovascular Diseases.

Author information

Authors and Affiliations

  1. Regenerative Medicine and Cell Biology, Medical University of South Carolina, 173 Ashley Avenue, BSB 626, MSC 508, Charleston, SC, 29425, USA

    Caitlin A. Czajka, Agnes Nagy Mehesz, Thomas C. Trusk, Michael J. Yost & Christopher J. Drake

  2. Department of Surgery, Medical University of South Carolina, 173 Ashley Avenue, BSB 601, MSC 508, Charleston, SC, 29425, USA

    Michael J. Yost

Authors
  1. Caitlin A. Czajka
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Agnes Nagy Mehesz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thomas C. Trusk
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael J. Yost
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christopher J. Drake
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christopher J. Drake.

Additional information

Associate Editor Scott I. Simon oversaw the review of this article.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Fig. 1. Diagram depicting toroid loading onto Bose ElectroForce 3100 uniaxial testing apparatus and initial dimensions of toroid. A1: Toroid is removed from agarose mold using forceps and placed around two horizontal cantilevers; A2-3: top cantilever is translocated 1.5 mm vertically to establish initial state, in which toroidal internal diameter matches that of the agarose mold central post (2.5 mm). Initial force mounted toroid exerts on cantilevers (avg: 0.09 ± 0.017 g) is set to zero at this time. B: Enlarged front-view of mounted toroid. Initial toroid length Lo was set as 2.5 mm, corresponding to the internal diameter at initial state. Cross-sectional area was obtained from measuring the cross-sectional diameters of intact, paraformaldehyde-fixed toroids and by imaging the fixed cross-sections and measuring cross-sectional diameters on the images. The average value 0.566 mm2 (n = 7) was used for all calculations. C: Example of stress–strain plot denoting locations from which values for ultimate tensile strength, Young’s modulus, and displacement at failure calculations were obtained.

Supplementary Fig. 2. Stress–strain plots obtained from load–displacement data acquired during uniaxial tensile testing of day 3 toroids. Day 3 Y-27632-treated toroids (bottom plot) showed elongated toe and linear elastic regions as compared to day-matched control samples (top plot).

Supplementary Table 1. Summary of results of Student’s t-test analysis of toroid ring opening experiments corresponding to Fig. 6 (p < 0.05). Confidence Intervals are expressed as absolute values (±). Based on F test, assumption of equal variance among samples was valid for all except T2. Significance level p < 0.05.

Supplementary Table 2. Summary of data used to calculate two-way ANOVA corresponding to biomechanical characterization of toroids in Figs. 8 and 9. As listed from top to bottom, each cell contains mean, variance, standard deviation, and standard error. Units for mean Young’s modulus and ultimate tensile strength are kPa; mean displacement at failure is reported in mm.

Supplementary Table 3. Summary of results of two-way ANOVA (two factors, two levels each: Y-27632 treatment vs. control and 3 vs. 6 days in culture) corresponding to biomechanical characterization of toroids in Figs. 8 and 9. SS—sum of squares; df—degrees of freedom; MS—mean square. Significance level p < 0.05.

Supplementary Table 4. Summary of results of post-test calculations with Bonferroni correction corresponding to biomechanical characterization of toroids in Figs. 8 and 9. ‘3’ or ‘6’ indicates culture duration (i.e., day 3 or day 6, respectively); ‘C’ or ‘I’ indicates treatment (i.e., control or Y-27632-treated, respectively). Significance level p < 0.05.

Supplementary material 1 (PDF 1655 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Czajka, C.A., Mehesz, A.N., Trusk, T.C. et al. Scaffold-Free Tissue Engineering: Organization of the Tissue Cytoskeleton and Its Effects on Tissue Shape. Ann Biomed Eng 42, 1049–1061 (2014). https://doi.org/10.1007/s10439-014-0986-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10439-014-0986-8

Keywords

Search

Navigation