Skip to main content
SpringerLink
Log in

Engineering Hybrid-Hydrogels Comprised of Healthy or Diseased Decellularized Extracellular Matrix to Study Pulmonary Fibrosis

  • 2022 CMBE Young Innovators
  • Published:
Cellular and Molecular Bioengineering Aims and scope Submit manuscript

Abstract

Idiopathic pulmonary fibrosis is a chronic disease characterized by progressive lung scarring that inhibits gas exchange. Evidence suggests fibroblast-matrix interactions are a prominent driver of disease. However, available preclinical models limit our ability to study these interactions. We present a technique for synthesizing phototunable poly(ethylene glycol) (PEG)-based hybrid-hydrogels comprising healthy or fibrotic decellularized extracellular matrix (dECM) to decouple mechanical properties from composition and elucidate their roles in fibroblast activation. Here, we engineered and characterized phototunable hybrid-hydrogels using molecular techniques such as ninhydrin and Ellman’s assays to assess dECM functionalization, and parallel-plate rheology to measure hydrogel mechanical properties. These biomaterials were employed to investigate the activation of fibroblasts from dual-transgenic Col1a1-GFP and αSMA-RFP reporter mice in response to changes in composition and mechanical properties. We show that reacting functionalized dECM from healthy or bleomycin-injured mouse lungs with PEG alpha-methacrylate (αMA) in an off-stoichiometry Michael-addition reaction created soft hydrogels mimicking a healthy lung elastic modulus (4.99 ± 0.98 kPa). Photoinitiated stiffening increased the material modulus to fibrotic values (11.48 ± 1.80 kPa). Percent activation of primary murine fibroblasts expressing Col1a1 and αSMA increased by approximately 40% following dynamic stiffening of both healthy and bleomycin hybrid-hydrogels. There were no significant differences between fibroblast activation on stiffened healthy versus stiffened bleomycin-injured hybrid-hydrogels. Phototunable hybrid-hydrogels provide an important platform for probing cell-matrix interactions and developing a deeper understanding of fibrotic activation in pulmonary fibrosis. Our results suggest that mechanical properties are a more significant contributor to fibroblast activation than biochemical composition within the scope of the hybrid-hydrogel platform evaluated in this study.

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.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

Data Availability

The raw and processed data required to reproduce these findings are available here: Saleh, Kamiel; Hewawasam, Rukshika; Šerbedžija, Predrag; Blomberg, Rachel; Noreldeen, Saif; Edelman, Benjamin; Smith, Bradford; Riches, David; Magin, Chelsea (2022), “Engineering hybrid-hydrogels comprised of healthy or diseased decellularized extracellular matrix to study pulmonary fibrosis”, Mendeley Data, V1, https://doi.org/10.17632/stxpj6xmgg.1.

References

  1. Arkenberg, M. R., H. D. Nguyen, and C. C. Lin. Recent advances in bio-orthogonal and dynamic crosslinking of biomimetic hydrogels. J. Mater. Chem. B. 8:7835–7855, 2020.

    Article  Google Scholar 

  2. Balestrini, J. L., S. Chaudhry, V. Sarrazy, A. Koehler, and B. Hinz. The mechanical memory of lung myofibroblasts. Integr. Biol. (Camb). 4:410–421, 2012.

    Article  Google Scholar 

  3. Barbarin, V., A. Nihoul, P. Misson, M. Arras, M. Delos, I. Leclercq, D. Lison, and F. Huaux. The role of pro- and anti-inflammatory responses in silica-induced lung fibrosis. Respir. Res. 6:112, 2005.

    Article  Google Scholar 

  4. Bolukbas, D. A., M. M. De Santis, H. N. Alsafadi, A. Doryab, and D. E. Wagner. The preparation of decellularized mouse lung matrix scaffolds for analysis of lung regenerative cell potential. Methods Mol. Biol. 275–295:2019, 1940.

    Google Scholar 

  5. Booth, A. J., R. Hadley, A. M. Cornett, A. A. Dreffs, S. A. Matthes, J. L. Tsui, K. Weiss, J. C. Horowitz, V. F. Fiore, T. H. Barker, B. B. Moore, F. J. Martinez, L. E. Niklason, and E. S. White. Acellular normal and fibrotic human lung matrices as a culture system for in vitro investigation. Am. J. Respir. Crit. Care Med. 186:866–876, 2012.

    Article  Google Scholar 

  6. Christensen, P. J., R. E. Goodman, L. Pastoriza, B. Moore, and G. B. Toews. Induction of lung fibrosis in the mouse by intratracheal instillation of fluorescein isothiocyanate is not T-cell-dependent. Am. J. Pathol. 155:1773–1779, 1999.

    Article  Google Scholar 

  7. Crapo, P. M., T. W. Gilbert, and S. F. Badylak. An overview of tissue and whole organ decellularization processes. Biomaterials. 32:3233–3243, 2011.

    Article  Google Scholar 

  8. de Hilster, R. H. J., P. K. Sharma, M. R. Jonker, E. S. White, E. A. Gercama, M. Roobeek, W. Timens, M. C. Harmsen, M. N. Hylkema, and J. K. Burgess. Human lung extracellular matrix hydrogels resemble the stiffness and viscoelasticity of native lung tissue. Am. J. Physiol. Lung Cell. Mol. Physiol. 318:L698–L704, 2020.

    Article  Google Scholar 

  9. DellaLatta, V., A. Cecchettini, S. DelRy, and M. A. Morales. Bleomycin in the setting of lung fibrosis induction: from biological mechanisms to counteractions. Pharmacol. Res. 97:122–130, 2015.

    Article  Google Scholar 

  10. Dolhnikoff, M., T. Mauad, and M. S. Ludwig. Extracellular matrix and oscillatory mechanics of rat lung parenchyma in bleomycin-induced fibrosis. Am. J. Respir. Crit. Care Med. 160:1750–1757, 1999.

    Article  Google Scholar 

  11. Gillette, B. M., J. A. Jensen, M. Wang, J. Tchao, and S. K. Sia. Dynamic hydrogels: switching of 3D microenvironments using two-component naturally derived extracellular matrices. Adv. Mater. 22:686–691, 2010.

    Article  Google Scholar 

  12. Haak, A. J., Q. Tan, and D. J. Tschumperlin. Matrix biomechanics and dynamics in pulmonary fibrosis. Matrix Biol. 73:64–76, 2018.

    Article  Google Scholar 

  13. Heinzelmann, K., M. Lehmann, M. Gerckens, N. Noskovicova, M. Frankenberger, M. Lindner, R. Hatz, J. Behr, A. Hilgendorff, M. Konigshoff, and O. Eickelberg. Cell-surface phenotyping identifies CD36 and CD97 as novel markers of fibroblast quiescence in lung fibrosis. Am. J. Physiol. Lung Cell. Mol.r Physiol. 315:L682–L696, 2018.

    Article  Google Scholar 

  14. Herrera, J., C. A. Henke, and P. B. Bitterman. Extracellular matrix as a driver of progressive fibrosis. J. Clin. Invest. 128:45–53, 2018.

    Article  Google Scholar 

  15. Jones, M. G., A. Fabre, P. Schneider, F. Cinetto, G. Sgalla, M. Mavrogordato, S. Jogai, A. Alzetani, B. G. Marshall, K. M. O’Reilly, J. A. Warner, P. M. Lackie, D. E. Davies, D. M. Hansell, A. G. Nicholson, I. Sinclair, K. K. Brown, and L. Richeldi. Three-dimensional characterization of fibroblast foci in idiopathic pulmonary fibrosis. JCI Insight. 2016. https://doi.org/10.1172/jci.insight.86375.

    Article  Google Scholar 

  16. Keith, R. C., J. L. Powers, E. F. Redente, A. Sergew, R. J. Martin, A. Gizinski, V. M. Holers, S. Sakaguchi, and D. W. Riches. A novel model of rheumatoid arthritis-associated interstitial lung disease in SKG mice. Exp. Lung Res. 38:55–66, 2012.

    Article  Google Scholar 

  17. Knudsen, L., E. Lopez-Rodriguez, L. Berndt, L. Steffen, C. Ruppert, J. H. T. Bates, M. Ochs, and B. J. Smith. Alveolar micromechanics in bleomycin-induced lung injury. Am. J. Respir. Cell Mol. Biol. 59:757–769, 2018.

    Article  Google Scholar 

  18. Korfei, M., S. Schmitt, C. Ruppert, I. Henneke, P. Markart, B. Loeh, P. Mahavadi, M. Wygrecka, W. Klepetko, L. Fink, P. Bonniaud, K. T. Preissner, G. Lochnit, L. Schaefer, W. Seeger, and A. Guenther. Comparative proteomic analysis of lung tissue from patients with idiopathic pulmonary fibrosis (IPF) and lung transplant donor lungs. J. Proteome Res. 10:2185–2205, 2011.

    Article  Google Scholar 

  19. Liu, F., C. M. Haeger, P. B. Dieffenbach, D. Sicard, I. Chrobak, A. M. F. Coronata, M. M. S. Velandia, S. Vitali, R. A. Colas, P. C. Norris, A. Marinkovic, X. L. Liu, J. Ma, C. D. Rose, S. J. Lee, S. A. A. Comhair, S. C. Erzurum, J. D. McDonald, C. N. Serhan, S. R. Walsh, D. J. Tschumperlin, and L. E. Fredenburgh. Distal vessel stiffening is an early and pivotal mechanobiological regulator of vascular remodeling and pulmonary hypertension. JCI Insight. 2016. https://doi.org/10.1172/jci.insight.86987.

    Article  Google Scholar 

  20. Liu, F., D. Lagares, K. M. Choi, L. Stopfer, A. Marinkovic, V. Vrbanac, C. K. Probst, S. E. Hiemer, T. H. Sisson, J. C. Horowitz, I. O. Rosas, L. E. Fredenburgh, C. Feghali-Bostwick, X. Varelas, A. M. Tager, and D. J. Tschumperlin. Mechanosignaling through YAP and TAZ drives fibroblast activation and fibrosis. Am. J. Physiol. Lung Cell. Mol. Physiol. 308:L344–L357, 2015.

    Article  Google Scholar 

  21. Liu, F., and D. J. Tschumperlin. Micro-mechanical characterization of lung tissue using atomic force microscopy. J. Vis. Exp. 2011. https://doi.org/10.3791/2911.

    Article  Google Scholar 

  22. Liu, S. S., C. Liu, X. X. Lv, B. Cui, J. Yan, Y. X. Li, K. Li, F. Hua, X. W. Zhang, J. J. Yu, J. M. Yu, F. Wang, S. Shang, P. P. Li, Z. G. Zhou, Y. Xiao, and Z. W. Hu. The chemokine CCL1 triggers an AMFR-SPRY1 pathway that promotes differentiation of lung fibroblasts into myofibroblasts and drives pulmonary fibrosis. Immunity. 54:2433–2435, 2021.

    Article  Google Scholar 

  23. Manali, E. D., C. Moschos, C. Triantafillidou, A. Kotanidou, I. Psallidas, S. P. Karabela, C. Roussos, S. Papiris, A. Armaganidis, G. T. Stathopoulos, and N. A. Maniatis. Static and dynamic mechanics of the murine lung after intratracheal bleomycin. BMC Pulm. Med. 11:33, 2011.

    Article  Google Scholar 

  24. Meyvis, T. K. L., S. C. De Smedt, J. Demeester, and W. E. Hennink. Rheological monitoring of long-term degrading polymer hydrogels. J. Rheol. 43:933–950, 1999.

    Article  Google Scholar 

  25. Moore, B. B., and C. M. Hogaboam. Murine models of pulmonary fibrosis. Am. J. Physiol. Lung Cell. Mol. Physiol. 294:L152-160, 2008.

    Article  Google Scholar 

  26. National Research Council (U.S.). Committee for the update of the guide for the care and use of laboratory animals, Institute for Laboratory Animal Research (U.S.) and National Academies Press (U.S.). Guide for the care and use of laboratory animals. Washington, D.C.: National Academies Press, 2011, p. xxv, 220 p.

  27. Nizamoglu, M., R. H. J. de Hilster, F. Zhao, P. K. Sharma, T. Borghuis, O. M. C. Harmsen, and J. K. Burgess. An in vitro model of fibrosis using crosslinked native extracellular matrix-derived hydrogels to modulate biomechanics without changing composition. bioRxiv. 2022. https://doi.org/10.1101/2022.02.02.478812.

    Article  Google Scholar 

  28. Parker, M. W., D. Rossi, M. Peterson, K. Smith, K. Sikstrom, E. S. White, J. E. Connett, C. A. Henke, O. Larsson, and P. B. Bitterman. Fibrotic extracellular matrix activates a profibrotic positive feedback loop. J. Clin. Investig. 124:1622–1635, 2014.

    Article  Google Scholar 

  29. Petrou, C. L., T. J. D’Ovidio, D. A. Bolukbas, S. Tas, R. D. Brown, A. Allawzi, S. Lindstedt, E. Nozik-Grayck, K. R. Stenmark, D. E. Wagner, and C. M. Magin. Clickable decellularized extracellular matrix as a new tool for building hybrid-hydrogels to model chronic fibrotic diseases in vitro. J. Mater. Chem. B. 8:6814–6826, 2020.

    Article  Google Scholar 

  30. Pouliot, R. A., P. A. Link, N. S. Mikhaiel, M. B. Schneck, M. S. Valentine, F. J. Kamga Gninzeko, J. A. Herbert, M. Sakagami, and R. L. Heise. Development and characterization of a naturally derived lung extracellular matrix hydrogel. J. Biomed. Mater. Res. A. 104:1922–1935, 2016.

    Article  Google Scholar 

  31. Redente, E. F., B. P. Black, D. S. Backos, A. N. Bahadur, S. M. Humphries, D. A. Lynch, R. M. Tuder, R. L. Zemans, and D. W. H. Riches. Persistent, progressive pulmonary fibrosis and epithelial remodeling in mice. Am. J. Respir. Cell. Mol. Biol. 64:669–676, 2021.

    Article  Google Scholar 

  32. Richeldi, L., H. R. Collard, and M. G. Jones. Idiopathic pulmonary fibrosis. Lancet. 389:1941–1952, 2017.

    Article  Google Scholar 

  33. Rosales, A. M., S. L. Vega, F. W. DelRio, J. A. Burdick, and K. S. Anseth. Hydrogels with reversible mechanics to probe dynamic cell microenvironments. Angew. Chem. Int. Ed. Engl. 56:12132–12136, 2017.

    Article  Google Scholar 

  34. Rube, C. E., D. Uthe, K. W. Schmid, K. D. Richter, J. Wessel, A. Schuck, N. Willich, and C. Rube. Dose-dependent induction of transforming growth factor beta (TGF-beta) in the lung tissue of fibrosis-prone mice after thoracic irradiation. Int. J. Radiat. Oncol. Biol. Phys. 47:1033–1042, 2000.

    Article  Google Scholar 

  35. Saldin, L. T., M. C. Cramer, S. S. Velankar, L. J. White, and S. F. Badylak. Extracellular matrix hydrogels from decellularized tissues: structure and function. Acta Biomater. 49:1–15, 2017.

    Article  Google Scholar 

  36. Sava, P., A. Ramanathan, A. Dobronyi, X. Peng, H. Sun, A. Ledesma-Mendoza, E. L. Herzog, and A. L. Gonzalez. Human pericytes adopt myofibroblast properties in the microenvironment of the IPF lung. JCI Insight. 2:e96352, 2017.

    Article  Google Scholar 

  37. Schiller, H. B., I. E. Fernandez, G. Burgstaller, C. Schaab, R. A. Scheltema, T. Schwarzmayr, T. M. Strom, O. Eickelberg, and M. Mann. Time- and compartment-resolved proteome profiling of the extracellular niche in lung injury and repair. Mol. Syst. Biol. 11:819, 2015.

    Article  Google Scholar 

  38. Shi, X., Y. Chen, Q. Liu, X. Mei, J. Liu, Y. Tang, R. Luo, D. Sun, Y. Ma, W. Wu, W. Tu, Y. Zhao, W. Xu, Y. Ke, S. Jiang, Y. Huang, R. Zhang, L. Wang, Y. Chen, J. Xia, W. Pu, H. Zhu, X. Zuo, Y. Li, J. Xu, F. Gao, D. Wei, J. Chen, W. Yin, Q. Wang, H. Dai, L. Yang, G. Guo, J. Cui, N. Song, H. Zou, S. Zhao, J. H. W. Distler, L. Jin, and J. Wang. LDLR dysfunction induces LDL accumulation and promotes pulmonary fibrosis. Clin. Transl. Med.12:e711, 2022.

    Article  Google Scholar 

  39. Silver, F. H., and D. E. Birk. Molecular-structure of collagen in solution—comparison of type-I, type-Ii, type-Iii and type-V. Int. J. Biol. Macromol. 6:125–132, 1984.

    Article  Google Scholar 

  40. Smith, B. J., G. S. Roy, A. Cleveland, C. Mattson, K. Okamura, C. M. Charlebois, K. L. Hamlington, M. V. Novotny, L. Knudsen, M. Ochs, R. D. Hite, and J. H. T. Bates. Three alveolar phenotypes govern lung function in murine ventilator-induced lung injury. Front. Physiol. 11:660, 2020.

    Article  Google Scholar 

  41. Tsukui, T., K. H. Sun, J. B. Wetter, J. R. Wilson-Kanamori, L. A. Hazelwood, N. C. Henderson, T. S. Adams, J. C. Schupp, S. D. Poli, I. O. Rosas, N. Kaminski, M. A. Matthay, P. J. Wolters, and D. Sheppard. Collagen-producing lung cell atlas identifies multiple subsets with distinct localization and relevance to fibrosis. Nat. Commun. 11:1920, 2020.

    Article  Google Scholar 

  42. Wagner, D. E., N. R. Bonenfant, D. Sokocevic, M. J. DeSarno, Z. D. Borg, C. S. Parsons, E. M. Brooks, J. J. Platz, Z. I. Khalpey, D. M. Hoganson, B. Deng, Y. W. Lam, R. A. Oldinski, T. Ashikaga, and D. J. Weiss. Three-dimensional scaffolds of acellular human and porcine lungs for high throughput studies of lung disease and regeneration. Biomaterials. 35:2664–2679, 2014.

    Article  Google Scholar 

  43. Wang, Y., L. Zhang, T. Huang, G. R. Wu, Q. Zhou, F. X. Wang, L. M. Chen, F. Sun, Y. Lv, F. Xiong, S. Zhang, Q. Yu, P. Yang, W. Gu, Y. Xu, J. Zhao, H. Zhang, W. Xiong, and C. Y. Wang. The methyl-CpG-binding domain 2 facilitates pulmonary fibrosis by orchestrating fibroblast to myofibroblast differentiation. Eur. Respir. J. 2022. https://doi.org/10.1183/13993003.03697-2020.

    Article  Google Scholar 

  44. Wolters, P. J., H. R. Collard, and K. D. Jones. Pathogenesis of idiopathic pulmonary fibrosis. Annu. Rev. Pathol. 9:157–179, 2014.

    Article  Google Scholar 

  45. Wu, S. M., J. J. Tsai, H. C. Pan, J. L. Arbiser, L. Elia, and M. L. Sheu. Aggravation of pulmonary fibrosis after knocking down the aryl hydrocarbon receptor in the insulin-like growth factor 1 receptor pathway. Br. J. Pharmacol. 179:3430–3451, 2022.

    Article  Google Scholar 

  46. Zhang, Y., M. Jiang, M. Nouraie, M. G. Roth, T. Tabib, S. Winters, X. Chen, J. Sembrat, Y. Chu, N. Cardenes, R. M. Tuder, E. L. Herzog, C. Ryu, M. Rojas, R. Lafyatis, K. F. Gibson, J. F. McDyer, D. J. Kass, and J. K. Alder. GDF15 is an epithelial-derived biomarker of idiopathic pulmonary fibrosis. Am. J. Physiol. Lung Cell. Mol. Physiol. 317:L510–L521, 2019.

    Article  Google Scholar 

Download references

Acknowledgments

This work was supported by funding from the National Heart, Lung, and Blood Institute of the National Institutes of Health (NIH) under Awards R01 HL080396 (CMM), R01 HL153096 (CMM, KSS, PS, and DWHR), R01HL151630 (BJS), and T32 HL 07085 (RB); the National Cancer Institute of the NIH under Award R21 CA252172 (CMM and RB); the National Science Foundation under Award 1941401 (CMM and RH); the Department of the Army under Award W81XWH-20-1-0037 (CMM).

Author information

Author notes

  1. Kamiel S. Saleh and Rukshika Hewawasam have contributed equally to this work.

Authors and Affiliations

  1. Department of Bioengineering, University of Colorado, Denver | Anschutz Medical Campus, 2115 N Scranton Street, Suite 3010, Aurora, CO, 80045, USA

    Kamiel S. Saleh, Rukshika Hewawasam, Predrag Šerbedžija, Rachel Blomberg, Saif E. Noreldeen, Bradford J. Smith & Chelsea M. Magin

  2. Program in Cell Biology, Department of Pediatrics, National Jewish Health, Denver, CO, USA

    Benjamin Edelman & David W. H. Riches

  3. Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA

    David W. H. Riches & Chelsea M. Magin

  4. Department of Research, Veterans Affairs Eastern Colorado Health Care System, Aurora, CO, USA

    David W. H. Riches

  5. Department of Immunology and Microbiology, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA

    David W. H. Riches

  6. Department of Pediatrics, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA

    Bradford J. Smith & Chelsea M. Magin

Authors
  1. Kamiel S. Saleh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rukshika Hewawasam
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Predrag Šerbedžija
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rachel Blomberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Saif E. Noreldeen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Benjamin Edelman
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Bradford J. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. David W. H. Riches
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Chelsea M. Magin
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

KSS, RH, PS, RB, BJS, DWHR, and CMM conceived the research plan. KSS, RH, PS, RB, BJS, SEN, and BE carried out experiments. All authors wrote, reviewed, and edited the manuscript.

Corresponding author

Correspondence to Chelsea M. Magin.

Ethics declarations

Conflict of interest

Dr. Magin is an inventor on a pending patent related to the technology described in this manuscript. All remaining authors (KSS, RH, PS, RB, BJS, SEN, BE, and DWHR) have no conflicts of interest to disclose.

Human Studies

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

Animal Studies

All institutional and national guidelines for the care and use of laboratory animals were followed and approved by the appropriate institutional committees.

Additional information

Associate Editor Cheng Dong oversaw the review of this article.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Saleh, K.S., Hewawasam, R., Šerbedžija, P. et al. Engineering Hybrid-Hydrogels Comprised of Healthy or Diseased Decellularized Extracellular Matrix to Study Pulmonary Fibrosis. Cel. Mol. Bioeng. 15, 505–519 (2022). https://doi.org/10.1007/s12195-022-00726-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12195-022-00726-y

Keywords

Search

Navigation