Annals of Biomedical Engineering

, Volume 43, Issue 11, pp 2618–2629 | Cite as

Reinforcement of Mono- and Bi-layer Poly(Ethylene Glycol) Hydrogels with a Fibrous Collagen Scaffold

  • K. R. C. Kinneberg
  • A. Nelson
  • M. E. Stender
  • A. H. Aziz
  • L. C. Mozdzen
  • B. A. C. Harley
  • S. J. Bryant
  • V. L. FergusonEmail author


Biomaterial-based tissue engineering strategies hold great promise for osteochondral tissue repair. Yet significant challenges remain in joining highly dissimilar materials to achieve a biomimetic, mechanically robust design for repairing interfaces between soft tissue and bone. This study sought to improve interfacial properties and function in a bi-layer hydrogel interpenetrated with a fibrous collagen scaffold. ‘Soft’ 10% (w/w) and ‘stiff’ 30% (w/w) PEGDM was formed into mono- or bi-layer hydrogels possessing a sharp diffusional interface. Hydrogels were evaluated as single-(hydrogel only) or multi-phase (hydrogel + fibrous scaffold penetrating throughout the stiff layer and extending >500 μm into the soft layer). Including a fibrous scaffold into both soft and stiff mono-layer hydrogels significantly increased tangent modulus and toughness and decreased lateral expansion under compressive loading. Finite element simulations predicted substantially reduced stress and strain gradients across the soft—stiff hydrogel interface in multi-phase, bi-layer hydrogels. When combining two low moduli constituent materials, composites theory poorly predicts the observed, large modulus increases. These results suggest material structure associated with the fibrous scaffold penetrating within the PEG hydrogel as the major contributor to improved properties and function—the hydrogel bore compressive loads and the 3D fibrous scaffold was loaded in tension thus resisting lateral expansion.


Mechanical properties Tissue engineering Osteochondral Interface Hydrogel Scaffold Multi-phase 



Research reported in this publication was partially supported by the University of Colorado Innovative Seed Grant Program and NSF CAREER Award CBET #1055989 (K.R.C.K., A.N., M.S., V.L.F.); NSF CAREER Award DMR #0847390 (A.H.A., S.J.B.), NIH R21 AR063331 (L.C.M., B.A.C.H), and a NIH Pharmaceutical Biotechnology Training fellowship to A.H.A. Imaging experiments were performed in the University of Colorado Anschutz Medical Campus Advanced Light Microscopy Core supported in part by NIH/NCATS Colorado CTSI Grant #UL1 TR001082. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or NSF. The authors also thank Dr. Justine J. Roberts for assistance related to hydrogel synthesis and Rachael C. Paietta for contributions to mechanical testing methods and analysis.


  1. 1.
    Broom, N. D., and C. A. Poole. A functional morphological-study of the tidemark region of articular-cartilage maintained in a non-viable physiological condition. J. Anat. 135:65–82, 1982.PubMedCentralPubMedGoogle Scholar
  2. 2.
    Bryant, S. J., R. J. Bender, K. L. Durand, and K. S. Anseth. Encapsulating Chondrocytes in degrading PEG hydrogels with high modulus: Engineering gel structural changes to facilitate cartilaginous tissue production. Biotechnol. Bioeng. 86:747–755, 2004.CrossRefPubMedGoogle Scholar
  3. 3.
    Bullough, P., and J. Goodfellow. The significance of the fine structure of articular cartilage. J. Bone Joint Surg. Br. 50:852–857, 1968.PubMedGoogle Scholar
  4. 4.
    Burdick, J. A., and K. S. Anseth. Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering. Biomaterials 23:4315–4323, 2002.CrossRefPubMedGoogle Scholar
  5. 5.
    Caliari, S. R., and B. A. Harley. Collagen-GAG scaffold biophysical properties bias MSC lineage choice in the presence of mixed soluble signals. Tissue Eng. Part A 20:2463–2472, 2014.PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Caliari, S. R., and B. A. Harley. Structural and biochemical modification of a collagen scaffold to selectively enhance MSC tenogenic, chondrogenic, and osteogenic differentiation. Adv. Healthc. Mater. 3:1086–1096, 2014.PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Caliari, S. R., D. W. Weisgerber, M. A. Ramirez, D. O. Kelkhoff, and B. A. Harley. The influence of collagen-glycosaminoglycan scaffold relative density and microstructural anisotropy on tenocyte bioactivity and transcriptomic stability. J. Mech. Behav. Biomed. Mater. 11:27–40, 2012.PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Caliari, S. R., L. C. Mozdzen, O. Armitage, M. L. Oyen, and B. A. Harley. Periodically perforated core-shell collagen biomaterials balance cell infiltration, bioactivity, and mechanical properties. J. Biomed. Mater. Res. A 102:917–927, 2014.PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Caliari, S. R., D. W. Weisgerber, W. K. Grier, Z. Mahmassani, M. D. Boppart, and B. A. Harley. Collagen scaffolds incorporating coincident gradations of instructive structural and biochemical cues for osteotendinous junction engineering. Adv. Healthc. Mater. 4:831–837, 2015.Google Scholar
  10. 10.
    Campbell, S. E., V. L. Ferguson, and D. C. Hurley. Nanomechanical mapping of the osteochondral interface with contact resonance force microscopy and nanoindentation. Acta Biomater. 8:4389–4396, 2012.CrossRefPubMedGoogle Scholar
  11. 11.
    Carter, D. R., G. S. Beaupré, M. Wong, R. L. Smith, T. P. Andriacchi, and D. J. Schurman. The mechanobiology of articular cartilage development and degeneration. Clin. Orthop. Relat. Res.® 427:S69–S77, 2004.CrossRefGoogle Scholar
  12. 12.
    Chawla, K. K. Composite Materials Science and Engineering. New York: Springer, 2012.Google Scholar
  13. 13.
    Coburn, J., M. Gibson, P. A. Bandalini, C. Laird, H. Q. Mao, L. Moroni, D. Seliktar, and J. Elisseeff. Biomimetics of the extracellular matrix: an integrated three-dimensional fiber-hydrogel composite for cartilage tissue engineering. Smart Struct. Syst. 7:213–222, 2011.PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Coburn, J. M., M. Gibson, S. Monagle, Z. Patterson, and J. H. Elisseeff. Bioinspired nanofibers support chondrogenesis for articular cartilage repair. Proc. Natl. Acad. Sci. USA 109:10012–10017, 2012.PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Cui, W., Q. Wang, G. Chen, S. Zhou, Q. Chang, Q. Zuo, K. Ren, and W. Fan. Repair of articular cartilage defects with tissue-engineered osteochondral composites in pigs. J. Biosci. Bioeng. 111:493–500, 2011.CrossRefPubMedGoogle Scholar
  16. 16.
    Duan, P., Z. Pan, L. Cao, Y. He, H. Wang, Z. Qu, J. Dong, and J. Ding. The effects of pore size in bilayered poly(lactide-co-glycolide) scaffolds on restoring osteochondral defects in rabbits. J. Biomed. Mater. Res. A 102:180–192, 2013.CrossRefPubMedGoogle Scholar
  17. 17.
    Farrell, E., F. J. O’Brien, P. Doyle, J. Fischer, I. Yannas, B. A. Harley, B. O’Connell, P. J. Prendergast, and V. A. Campbell. A collagen-glycosaminoglycan scaffold supports adult rat mesenchymal stem cell differentiation along osteogenic and chondrogenic routes. Tissue Eng. 12:459–468, 2006.CrossRefPubMedGoogle Scholar
  18. 18.
    Ferguson, V. L., A. J. Bushby, and A. Boyde. Nanomechanical properties and mineral concentration in articular calcified cartilage and subchondral bone. J. Anat. 203:191–202, 2003.PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Galperin, A., R. A. Oldinski, S. J. Florczyk, J. D. Bryers, M. Q. Zhang, and B. D. Ratner. Integrated bi-layered scaffold for osteochondral tissue engineering. Adv. Healthc. Mater. 2:872–883, 2013.PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Gotterbarm, T., W. Richter, M. Jung, S. Berardi Vilei, P. Mainil-Varlet, T. Yamashita, and S. J. Breusch. An in vivo study of a growth-factor enhanced, cell free, two-layered collagen-tricalcium phosphate in deep osteochondral defects. Biomaterials 27:3387–3395, 2006.CrossRefPubMedGoogle Scholar
  21. 21.
    Guo, X., J. Liao, H. Park, A. Saraf, R. M. Raphael, Y. Tabata, F. K. Kasper, and A. G. Mikos. Effects of TGF-beta 3 and preculture period of osteogenic cells on the chondrogenic differentiation of rabbit marrow mesenchymal stem cells encapsulated in a bilayered hydrogel composite. Acta Biomater. 6:2920–2931, 2010.PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Halpin Affdl, J. C., and J. L. Kardos. The Halpin-Tsai equations. Polymer Sci. Eng. 16:344–352, 1976.CrossRefGoogle Scholar
  23. 23.
    Harley, B. A., J. H. Leung, E. Silva, and L. J. Gibson. Mechanical characterization of collagen-glycosaminoglycan scaffolds. Acta Biomater. 3:463–474, 2007.CrossRefPubMedGoogle Scholar
  24. 24.
    Harley, B. A., A. K. Lynn, Z. Wissner-Gross, W. Bonfield, I. V. Yannas, and L. J. Gibson. Design of a multiphase osteochondral scaffold III: Fabrication of layered scaffolds with continuous interfaces. J. Biomed. Mater. Res. Part A 92A:1078–1093, 2010.Google Scholar
  25. 25.
    Hashin, Z., and S. Shtrikman. A variational approach to the theory of the elastic behaviour of multiphase materials. J. Mech. Phys. Solids 11:127–140, 1963.CrossRefGoogle Scholar
  26. 26.
    Hortensius, R. A., and B. A. Harley. The use of bioinspired alterations in the glycosaminoglycan content of collagen-GAG scaffolds to regulate cell activity. Biomaterials 34:7645–7652, 2013.PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Im, G. I., J. H. Ahn, S. Y. Kim, B. S. Choi, and S. W. Lee. A hyaluronate-atelocollagen/beta-tricalcium phosphate-hydroxyapatite biphasic scaffold for the repair of osteochondral defects: a porcine study. Tissue Eng. Part A 16:1189–1200, 2010.CrossRefPubMedGoogle Scholar
  28. 28.
    Jiang, C. C., H. Chiang, C. J. Liao, Y. J. Lin, T. F. Kuo, C. S. Shieh, Y. Y. Huang, and R. S. Tuan. Repair of porcine articular cartilage defect with a biphasic osteochondral composite. J. Orthop. Res. 25:1277–1290, 2007.CrossRefPubMedGoogle Scholar
  29. 29.
    Jin, G. Z., J. J. Kim, J. H. Park, S. J. Seo, J. H. Kim, E. J. Lee, and H. W. Kim. Biphasic nanofibrous constructs with seeded cell layers for osteochondral repair. Tissue Eng. Part C Methods 20:895–904, 2014.CrossRefPubMedGoogle Scholar
  30. 30.
    Kandel, R. A., M. Grynpas, R. Pilliar, J. Lee, J. Wang, S. Waldman, P. Zalzal, M. Hurtig, and C. B. S. T. Team. Repair of osteochondral defects with biphasic cartilage-calcium polyphosphate constructs in a Sheep model. Biomaterials 27:4120–4131, 2006.CrossRefPubMedGoogle Scholar
  31. 31.
    Khanarian, N. T., N. M. Haney, R. A. Burga, and H. H. Lu. A functional agarose-hydroxyapatite scaffold for osteochondral interface regeneration. Biomaterials 33:5247–5258, 2012.PubMedCentralCrossRefPubMedGoogle Scholar
  32. 32.
    Khanarian, N. T., J. Jiang, L. Q. Wan, V. C. Mow, and H. H. Lu. A hydrogel-mineral composite scaffold for osteochondral interface tissue engineering. Tissue Eng. Part A 18:533–545, 2012.PubMedCentralCrossRefPubMedGoogle Scholar
  33. 33.
    Lee, J. C., C. Pereira, X. Ren, W. Huang, D. W. Weisgerber, D. T. Yamaguchi, B. A. Harley, and T. A. Miller. Optimizing collagen scaffolds for bone engineering: effects of crosslinking and mineral content on structural contraction and osteogenesis. J. Craniofac. Sur. 2015.
  34. 34.
    Lin, D. C., D. I. Shreiber, E. K. Dimitriadis, and F. Horkay. Spherical indentation of soft matter beyond the Hertzian regime: numerical and experimental validation of hyperelastic models. Biomech. Model. Mechanobiol. 8:345–358, 2009.PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Lin-Gibson, S., S. Bencherif, J. A. Cooper, S. J. Wetzel, J. M. Antonucci, B. M. Vogel, F. Horkay, and N. R. Washburn. Synthesis and characterization of PEG dimethacrylates and their hydrogels. Biomacromolecules 5:1280–1287, 2004.CrossRefPubMedGoogle Scholar
  36. 36.
    Lopa, S., and H. Madry. Bioinspired Scaffolds for osteochondral regeneration. Tissue Eng. Part A 20:2052–2076, 2014.CrossRefPubMedGoogle Scholar
  37. 37.
    Lu, S., J. Lam, J. E. Trachtenberg, E. J. Lee, H. Seyednejad, J. J. den van Beucken, Y. Tabata, M. E. Wong, J. A. Jansen, A. G. Mikos, and F. K. Kasper. Dual growth factor delivery from bilayered, biodegradable hydrogel composites for spatially-guided osteochondral tissue repair. Biomaterials 35:8829–8839, 2014.PubMedCentralCrossRefPubMedGoogle Scholar
  38. 38.
    Lynn, A. K., S. M. Best, R. E. Cameron, B. A. Harley, I. V. Yannas, L. J. Gibson, and W. Bonfield. Design of a multiphase osteochondral scaffold. I. Control of chemical composition. J. Biomed. Mater. Res. Part A 92A:1057, 2010.Google Scholar
  39. 39.
    Mente, P. L., and J. L. Lewis. Elastic-modulus of calcified cartilage is an order of magnitude less-than that of subchondral bone. J. Orthop. Res. 12:637–647, 1994.CrossRefPubMedGoogle Scholar
  40. 40.
    Mohan, N., V. Gupta, B. Sridharan, A. Sutherland, and M. S. Detamore. The potential of encapsulating “raw materials” in 3D osteochondral gradient scaffolds. Biotechnol. Bioeng. 111:829–841, 2014.PubMedCentralCrossRefPubMedGoogle Scholar
  41. 41.
    Moutos, F. T., and F. Guilak. Composite scaffolds for cartilage tissue engineering. Biorheology 45:501–512, 2008.PubMedCentralPubMedGoogle Scholar
  42. 42.
    Nicodemus, G. D., S. C. Skaalure, and S. J. Bryant. Gel structure impacts pericellular and extracellular matrix deposition which subsequently alters metabolic activities in chondrocyte-laden PEG hydrogels. Acta Biomater. 7:492–504, 2011.PubMedCentralCrossRefPubMedGoogle Scholar
  43. 43.
    O’Brien, F. J., B. A. Harley, I. V. Yannas, and L. Gibson. Influence of freezing rate on pore structure in freeze-dried collagen-GAG scaffolds. Biomaterials 25:1077–1086, 2004.CrossRefPubMedGoogle Scholar
  44. 44.
    O’Brien, F. J., B. A. Harley, I. V. Yannas, and L. J. Gibson. The effect of pore size on cell adhesion in collagen-GAG scaffolds. Biomaterials 26:433–441, 2005.CrossRefPubMedGoogle Scholar
  45. 45.
    Roberts, J. J., A. Earnshaw, V. L. Ferguson, and S. J. Bryant. Comparative study of the viscoelastic mechanical behavior of agarose and poly(ethylene glycol) hydrogels. J. Biomed. Mater. Res. B Appl. Biomater. 99:158–169, 2011.CrossRefPubMedGoogle Scholar
  46. 46.
    Roberts, J. J., G. D. Nicodemus, E. C. Greenwald, and S. J. Bryant. Degradation improves tissue formation in (Un)Loaded chondrocyte-laden hydrogels. Clin. Orthop. Relat. Res. 469:2725–2734, 2011.PubMedCentralCrossRefPubMedGoogle Scholar
  47. 47.
    Sharma, B., C. G. Williams, M. Khan, P. Manson, and J. H. Elisseeff. In vivo chondrogenesis of mesenchymal stem cells in a photopolymerized hydrogel. Plast. Reconstr. Surg. 119:112–120, 2007.CrossRefPubMedGoogle Scholar
  48. 48.
    Sherwood, J. K., S. L. Riley, R. Palazzolo, S. C. Brown, D. C. Monkhouse, M. Coates, L. G. Griffith, L. K. Landeen, and A. Ratcliffe. A three-dimensional osteochondral composite scaffold for articular cartilage repair. Biomaterials 23:4739–4751, 2002.CrossRefPubMedGoogle Scholar
  49. 49.
    Shimomura, K., Y. Moriguchi, C. D. Murawski, H. Yoshikawa, and N. Nakamura. Osteochondral tissue engineering with biphasic scaffold: current strategies and techniques. Tissue Eng. Part B Rev. 20:463–476, 2014.CrossRefGoogle Scholar
  50. 50.
    Steinmetz, N. J., E. A. Aisenbrey, K. K. Westbrook, H. J. Qi, and S. J. Bryant. Mechanical loading regulates human MSC differentiation in a multi-layer hydrogel for osteochondral tissue engineering. Acta Biomater. 2015. doi: 10.1016/j.actbio.2015.04.015.
  51. 51.
    Vickers, S. M., L. S. Squitieri, and M. Spector. Effects of cross-linking type II collagen-GAG scaffolds on chondrogenesis in vitro: Dynamic pore reduction promotes cartilage formation. Tissue Eng. 12:1345–1355, 2006.CrossRefPubMedGoogle Scholar
  52. 52.
    Villanueva, I., D. S. Hauschulz, D. Mejic, and S. J. Bryant. Static and dynamic compressive strains influence nitric oxide production and chondrocyte bioactivity when encapsulated in PEG hydrogels of different crosslinking densities. Osteoarthr. Cartil. 16:909–918, 2008.PubMedCentralCrossRefPubMedGoogle Scholar
  53. 53.
    Wang, D. A., C. G. Williams, F. Yang, N. Cher, H. Lee, and J. H. Elisseeff. Bioresponsive phosphoester hydrogels for bone tissue engineering. Tissue Eng. 11:201–213, 2005.CrossRefPubMedGoogle Scholar
  54. 54.
    Wang, X., E. Wenk, X. Zhang, L. Meinel, G. Vunjak-Novakovic, and D. L. Kaplan. Growth factor gradients via microsphere delivery in biopolymer scaffolds for osteochondral tissue engineering. J. Control Release 134:81–90, 2009.PubMedCentralCrossRefPubMedGoogle Scholar
  55. 55.
    Wang, Y., H. Meng, X. Yuan, J. Peng, Q. Guo, S. Lu, and A. Wang. Fabrication and in vitro evaluation of an articular cartilage extracellular matrix-hydroxyapatite bilayered scaffold with low permeability for interface tissue engineering. Biomed. Eng. Online 13:80, 2014.PubMedCentralCrossRefPubMedGoogle Scholar
  56. 56.
    Weisgerber, D. W., D. O. Kelkhoff, S. R. Caliari, and B. A. Harley. The impact of discrete compartments of a multi-compartment collagen-GAG scaffold on overall construct biophysical properties. J. Mech. Behav. Biomed. Mater. 28:26–36, 2013.PubMedCentralCrossRefPubMedGoogle Scholar
  57. 57.
    Wong, M., and D. R. Carter. Articular cartilage functional histomorphology and mechanobiology: a research perspective. Bone 33:1–13, 2003.CrossRefPubMedGoogle Scholar
  58. 58.
    Yannas, I. V., E. Lee, D. P. Orgill, E. M. Skrabut, and G. F. Murphy. Synthesis and characterization of a model extracellular matrix that induces partial regeneration of adult mammalian skin. Proc. Natl. Acad. Sci. USA 86:933–937, 1989.PubMedCentralCrossRefPubMedGoogle Scholar
  59. 59.
    Yannas, I. V., D. S. Tzeranis, B. A. Harley, and P. T. So. Biologically active collagen-based scaffolds: advances in processing and characterization. Philos. Trans. A Math. Phys. Eng. Sci. 368:2123–2139, 2010.PubMedCentralCrossRefPubMedGoogle Scholar
  60. 60.
    Yodmuang, S., S. L. McNamara, A. B. Nover, B. B. Mandal, M. Agarwal, T. A. Kelly, P. H. Chao, C. Hung, D. L. Kaplan, and G. Vunjak-Novakovic. Silk microfiber-reinforced silk hydrogel composites for functional cartilage tissue repair. Acta Biomater. 11:27–36, 2015.CrossRefPubMedGoogle Scholar

Copyright information

© Biomedical Engineering Society 2015

Authors and Affiliations

  • K. R. C. Kinneberg
    • 1
  • A. Nelson
    • 2
  • M. E. Stender
    • 1
  • A. H. Aziz
    • 2
    • 3
  • L. C. Mozdzen
    • 4
  • B. A. C. Harley
    • 4
  • S. J. Bryant
    • 2
    • 3
    • 5
  • V. L. Ferguson
    • 1
    • 3
    • 5
    Email author
  1. 1.Department of Mechanical EngineeringUniversity of ColoradoBoulderUSA
  2. 2.Department of Chemical and Biological EngineeringUniversity of ColoradoBoulderUSA
  3. 3.BioFrontiers InstituteUniversity of ColoradoBoulderUSA
  4. 4.Department of Chemical and Biomolecular EngineeringUniversity of Illinois at Urbana-ChampaignChampaignUSA
  5. 5.Material Science & Engineering ProgramUniversity of ColoradoBoulderUSA

Personalised recommendations