Composite Bioscaffolds Incorporating Decellularized ECM as a Cell-Instructive Component Within Hydrogels as In Vitro Models and Cell Delivery Systems

  • Arthi Shridhar
  • Elizabeth Gillies
  • Brian G. Amsden
  • Lauren E. Flynn
Part of the Methods in Molecular Biology book series (MIMB, volume 1577)


Decellularized tissues represent promising biomaterials, which harness the innate capacity of the tissue-specific extracellular matrix (ECM) to direct cell functions including stem cell proliferation and lineage-specific differentiation. However, bioscaffolds derived exclusively from decellularized ECM offer limited versatility in terms of tuning biomechanical properties, as well as cell–cell and cell–ECM interactions that are important mediators of the cellular response. As an alternative approach, in the current chapter we describe methods for incorporating cryo-milled decellularized tissues as a cell-instructive component within a hydrogel carrier designed to crosslink under mild conditions. This composite strategy can enable in situ cell encapsulation with high cell viability, allowing efficient seeding with a homogeneous distribution of cells and ECM. Detailed protocols are provided for the effective decellularization of human adipose tissue and porcine auricular cartilage, as well as the cryo-milling process used to generate the ECM particles. Further, we describe methods for synthesizing methacrylated chondroitin sulphate (MCS) and for performing UV-initiated and thermally induced crosslinking to form hydrogel carriers for adipose and cartilage regeneration. The hydrogel composites offer great flexibility, and the hydrogel phase, ECM source, particle size, cell type(s) and seeding density can be tuned to promote the desired cellular response. Overall, these systems represent promising platforms for the development of tissue-specific 3-D in vitro cell culture models and in vivo cell delivery systems.


Cell delivery vehicle Cell therapy Cell-instructive biomaterials Composite scaffolds Decellularization Extracellular matrix (ECM) Hydrogels In vitro In vivo Methacrylated chondroitin sulphate Stem cells 



Scholarship funding to support this work was provided by the NSERC CREATE CONNECT! Program and Western’s Collaborative Graduate Training Program in Musculoskeletal Health Research (CMHR). Operational grant funding support was provided by NSERC. The authors would like to thank Cody Brown for technical assistance with the SEM imaging and the Western Nanofabrication facility for access to their imaging equipment. Finally, we would like to acknowledge Drs. Aaron Grant, Brian Evans and Robert Richards for their clinical collaborations in providing human adipose tissue samples, as well as the Mount Brydges abattoir for providing the porcine auricular cartilage tissue.


  1. 1.
    Frantz C, Stewart KM, Weaver VM (2010) The extracellular matrix at a glance. J Cell Sci 123:4195–4200. doi: 10.1242/jcs.023820 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Jabbari E, Leijten J, Xu Q, Khademhosseini A (2015) The matrix reloaded: the evolution of regenerative hydrogels. Mater Today 19:190–196. doi: 10.1016/j.mattod.2015.10.005 CrossRefGoogle Scholar
  3. 3.
    Knight E, Przyborski S (2015) Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro. J Anat 227:746–756. doi: 10.1111/joa.12257 CrossRefPubMedGoogle Scholar
  4. 4.
    Hinderer S, Layland SL, Schenke-Layland K (2016) ECM and ECM-like materials—biomaterials for applications in regenerative medicine and cancer therapy. Adv Drug Deliv Rev 97:260–269. doi: 10.1016/j.addr.2015.11.019 CrossRefPubMedGoogle Scholar
  5. 5.
    Sart S, Tsai A-C, Li Y, Ma T (2014) Three-dimensional aggregates of mesenchymal stem cells: cellular mechanisms, biological properties, and applications. Tissue Eng Part B Rev 20:365–380. doi: 10.1089/ten.TEB.2013.0537 CrossRefPubMedGoogle Scholar
  6. 6.
    Geckil H, Xu F, Zhang X et al (2010) Engineering hydrogels as extracellular matrix mimics. Nanomedicine 5:469–484. doi: 10.2217/nnm.10.12 CrossRefPubMedGoogle Scholar
  7. 7.
    Benders KEM, van Weeren PR, Badylak SF et al (2013) Extracellular matrix scaffolds for cartilage and bone regeneration. Trends Biotechnol 31:169–176. doi: 10.1016/j.tibtech.2012.12.004 CrossRefGoogle Scholar
  8. 8.
    Hoshiba T, Lu H, Kawazoe N, Chen G (2010) Decellularized matrices for tissue engineering. Expert Opin Biol Ther 10:1717–1728. doi: 10.1517/14712598.2010.534079 CrossRefPubMedGoogle Scholar
  9. 9.
    Crapo PM, Gilbert TW, Badylak SF (2011) An overview of tissue and whole organ decellularization processes. Biomaterials 32:3233–3243. doi: 10.1016/j.biomaterials.2011.01.057 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Flynn LE (2010) The use of decellularized adipose tissue to provide an inductive microenvironment for the adipogenic differentiation of human adipose-derived stem cells. Biomaterials 31:4715–4724. doi: 10.1016/j.biomaterials.2010.02.046 CrossRefPubMedGoogle Scholar
  11. 11.
    Huang S-P, Hsu C-C, Chang S-C et al (2012) Adipose-derived stem cells seeded on acellular dermal matrix grafts enhance wound healing in a murine model of a full-thickness defect. Ann Plast Surg 69:656–662. doi: 10.1097/SAP.0b013e318273f909 CrossRefPubMedGoogle Scholar
  12. 12.
    Dainese L, Guarino A, Burba I et al (2012) Heart valve engineering: decellularized aortic homograft seeded with human cardiac stromal cells. J Heart Valve Dis 21:125–134PubMedGoogle Scholar
  13. 13.
    Woods T, Gratzer PF (2005) Effectiveness of three extraction techniques in the development of a decellularized bone–anterior cruciate ligament–bone graft. Biomaterials 26:7339–7349. doi: 10.1016/j.biomaterials.2005.05.066 CrossRefPubMedGoogle Scholar
  14. 14.
    Han TTY, Toutounji S, Amsden BG, Flynn LE (2015) Adipose-derived stromal cells mediate in vivo adipogenesis, angiogenesis and inflammation in decellularized adipose tissue bioscaffolds. Biomaterials 72:125–137. doi: 10.1016/j.biomaterials.2015.08.053 CrossRefPubMedGoogle Scholar
  15. 15.
    Turner AEB, Yu C, Bianco J et al (2012) The performance of decellularized adipose tissue microcarriers as an inductive substrate for human adipose-derived stem cells. Biomaterials 33:4490–4499. doi: 10.1016/j.biomaterials.2012.03.026 CrossRefPubMedGoogle Scholar
  16. 16.
    Yu C, Bianco J, Brown C et al (2013) Porous decellularized adipose tissue foams for soft tissue regeneration. Biomaterials 34:3290–3302. doi: 10.1016/j.biomaterials.2013.01.056 CrossRefPubMedGoogle Scholar
  17. 17.
    Ki H, Tian T, Han Y et al (2014) Biomaterials composite hydrogel scaffolds incorporating decellularized adipose tissue for soft tissue engineering with adipose-derived stem cells. Biomaterials 35:1914–1923. doi: 10.1016/j.biomaterials.2013.11.067 CrossRefGoogle Scholar
  18. 18.
    Brown CFC, Yan J, Han TTY et al (2015) Effect of decellularized adipose tissue particle size and cell density on adipose-derived stem cell proliferation and adipogenic differentiation in composite methacrylated chondroitin sulphate hydrogels. Biomed Mater 10:45010. doi: 10.1088/1748-6041/10/4/045010 CrossRefGoogle Scholar
  19. 19.
    Heinbuch D (2016) Adipose-derived stem cell differentiation in cell aggregates supplemented with micronized, decellularized extracellular matrix. University of Western Ontario, LondonGoogle Scholar
  20. 20.
    Agmon G, Christman KL (2016) Controlling stem cell behavior with decellularized extracellular matrix scaffolds. Curr Opin Solid State Mater Sci 20:193–201. doi: 10.1016/j.cossms.2016.02.001 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Kang H, Peng J, Lu S et al (2014) In vivo cartilage repair using adipose-derived stem cell-loaded decellularized cartilage ECM scaffolds. J Tissue Eng Regen Med 8:442–453. doi: 10.1002/term.1538 CrossRefPubMedGoogle Scholar
  22. 22.
    Quint C, Kondo Y, Manson RJ et al (2011) Decellularized tissue-engineered blood vessel as an arterial conduit. Proc Natl Acad Sci U S A 108:9214–9219. doi: 10.1073/pnas.1019506108 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Badylak SF, Freytes DO, Gilbert TW (2009) Extracellular matrix as a biological scaffold material: structure and function. Acta Biomater 5:1–13. doi: 10.1016/j.actbio.2008.09.013 CrossRefGoogle Scholar
  24. 24.
    Tsai C-H, Chou M-Y, Jonas M et al (2002) A composite graft material containing bone particles and collagen in osteoinduction in mouse. J Biomed Mater Res 63:65–70CrossRefGoogle Scholar
  25. 25.
    Mazzitelli S, Luca G, Mancuso F et al (2011) Production and characterization of engineered alginate-based microparticles containing ECM powder for cell/tissue engineering applications. Acta Biomater 7:1050–1062. doi: 10.1016/j.actbio.2010.10.005 CrossRefPubMedGoogle Scholar
  26. 26.
    Chang C-H, Chen C-C, Liao C-H et al (2014) Human acellular cartilage matrix powders as a biological scaffold for cartilage tissue engineering with synovium-derived mesenchymal stem cells. J Biomed Mater Res Part A 102:2248–2257. doi: 10.1002/jbm.a.34897 CrossRefGoogle Scholar
  27. 27.
    Beck EC, Barragan M, Libeer TB et al (2016) Chondroinduction from naturally derived cartilage matrix: a comparison between devitalized and decellularized cartilage encapsulated in hydrogel pastes. Tissue Eng Part A 22(7–8):665–679. doi: 10.1089/ten.tea.2015.0546 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Drury JL, Mooney DJ (2003) Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials 24:4337–4351. doi: 10.1016/S0142-9612(03)00340-5 CrossRefPubMedGoogle Scholar
  29. 29.
    Cai S, Liu Y, Zheng Shu X, Prestwich GD (2005) Injectable glycosaminoglycan hydrogels for controlled release of human basic fibroblast growth factor. Biomaterials 26:6054–6067. doi: 10.1016/j.biomaterials.2005.03.012 CrossRefPubMedGoogle Scholar
  30. 30.
    Lim HL, Hwang Y, Kar M, Varghese S (2014) Smart hydrogels as functional biomimetic systems. Biomater Sci 2:603. doi: 10.1039/c3bm60288e CrossRefGoogle Scholar
  31. 31.
    Spiller KL, Maher SA, Lowman AM (2011) Hydrogels for the repair of articular cartilage defects. Tissue Eng Part B Rev 17:281–299. doi: 10.1089/ten.teb.2011.0077 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Hayami JWS, Waldman SD, Amsden BG (2015) Photo-cross-linked methacrylated polysaccharide solution blends with high chondrocyte viability, minimal swelling, and moduli similar to load bearing soft tissues. Eur Polym J 72:1–11. doi: 10.1016/j.eurpolymj.2015.01.038 CrossRefGoogle Scholar
  33. 33.
    Flynn LE, Prestwich GD, Semple JL, Woodhouse KA (2008) Proliferation and differentiation of adipose-derived stem cells on naturally derived scaffolds. Biomaterials 29:1862–1871. doi: 10.1016/j.biomaterials.2007.12.028 CrossRefPubMedGoogle Scholar
  34. 34.
    Kong HJ, Smith MK, Mooney DJ (2003) Designing alginate hydrogels to maintain viability of immobilized cells. Biomaterials 24:4023–4029. doi: 10.1016/S0142-9612(03)00295-3 CrossRefPubMedGoogle Scholar
  35. 35.
    Rouillard AD, Berglund CM, Lee JY et al (2011) Methods for photocrosslinking alginate hydrogel scaffolds with high cell viability. Tissue Eng Part C Methods 17:173–179. doi: 10.1089/ten.tec.2009.0582 CrossRefPubMedGoogle Scholar
  36. 36.
    Koh W, Revzin A, Pishko MV (2002) Poly (ethylene glycol) hydrogel microstructures encapsulating living cells. Langmuir 18:2459–2462. doi: 10.1021/la0115740 CrossRefGoogle Scholar
  37. 37.
    Thanh T, Thi H, Lee JS et al (2016) Enhanced cellular activity in gelatin-poly (ethylene glycol) hydrogels without compromising gel stiffness 16(3): 334–340Google Scholar
  38. 38.
    Wang D-A, Varghese S, Sharma B et al (2007) Multifunctional chondroitin sulphate for cartilage tissue-biomaterial integration. Nat Mater 6:385–392. doi: 10.1038/nmat1890 CrossRefPubMedGoogle Scholar
  39. 39.
    Li Q, Williams CG, Sun DDN et al (2004) Photocrosslinkable polysaccharides based on chondroitin sulfate. J Biomed Mater Res 68A:28–33. doi: 10.1002/jbm.a.20007 CrossRefGoogle Scholar
  40. 40.
    Goude MC, McDevitt TC, Temenoff JS (2014) Chondroitin sulfate microparticles modulate transforming growth factor-β1-induced chondrogenesis of human mesenchymal stem cell spheroids. Cells Tissues Organs 199:117–130. doi: 10.1159/000365966 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Varghese S, Hwang NS, Canver AC et al (2008) Chondroitin sulfate based niches for chondrogenic differentiation of mesenchymal stem cells. Matrix Biol 27:12–21. doi: 10.1016/j.matbio.2007.07.002 CrossRefPubMedGoogle Scholar
  42. 42.
    Ravanat J-L, Douki T, Cadet J (2001) Direct and indirect effects of UV radiation on DNA and its components. J Photochem Photobiol B 63:88–102. doi: 10.1016/S1011-1344(01)00206-8 CrossRefPubMedGoogle Scholar
  43. 43.
    Park H, Temenoff JS, Holland TA et al (2005) Delivery of TGF-β1 and chondrocytes via injectable, biodegradable hydrogels for cartilage tissue engineering applications. Biomaterials 26:7095–7103. doi: 10.1016/j.biomaterials.2005.05.083 CrossRefPubMedGoogle Scholar
  44. 44.
    Park H, Temenoff JS, Tabata Y et al (2007) Injectable biodegradable hydrogel composites for rabbit marrow mesenchymal stem cell and growth factor delivery for cartilage tissue engineering. Biomaterials 28:3217–3227. doi: 10.1016/j.biomaterials.2007.03.030 CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Temenoff JS, Park H, Jabbari E et al (2004) Thermally cross-linked oligo(poly(ethylene glycol) fumarate) hydrogels support osteogenic differentiation of encapsulated marrow stromal cells in vitro. Biomacromolecules 5:5–10. doi: 10.1021/bm030067p CrossRefPubMedGoogle Scholar
  46. 46.
    Gilbert TW, Sellaro TL, Badylak SF (2006) Decellularization of tissues and organs. Biomaterials 27:3675–3683. doi: 10.1016/j.biomaterials.2006.02.014 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Arthi Shridhar
    • 1
  • Elizabeth Gillies
    • 1
    • 2
  • Brian G. Amsden
    • 3
  • Lauren E. Flynn
    • 1
    • 4
  1. 1.Department of Chemical and Biochemical EngineeringThe University of Western OntarioLondonCanada
  2. 2.Department of ChemistryThe University of Western OntarioLondonCanada
  3. 3.Department of Chemical EngineeringQueen’s UniversityKingstonCanada
  4. 4.Department of Anatomy and Cell Biology, Schulich School of Medicine and DentistryThe University of Western OntarioLondonCanada

Personalised recommendations