Supercritical Fluid Technology as a Tool to Prepare Gradient Multifunctional Architectures Towards Regeneration of Osteochondral Injuries

  • Ana Rita C. DuarteEmail author
  • Vitor E. Santo
  • Manuela E. Gomes
  • Rui L. Reis
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1058)


Platelet lysates (PLs) are a natural source of growth factors (GFs) known for its stimulatory role on stem cells which can be obtained after activation of platelets from blood plasma. The possibility to use PLs as growth factor source for tissue healing and regeneration has been pursued following different strategies. Platelet lysates are an enriched pool of growth factors which can be used as either a GFs source or as a three-dimensional (3D) hydrogel. However, most of current PLs-based hydrogels lack stability, exhibiting significant shrinking behavior. This chapter focuses on the application of supercritical fluid technology to develop three-dimensional architectures of PL constructs, crosslinked with genipin. The proposed technology allows in a single step operation the development of mechanically stable porous structures, through chemical crosslinking of the growth factors present in the PL pool, followed by supercritical drying of the samples. Furthermore gradient structures of PL-based structures with bioactive glass are also presented and are described as an interesting approach to the treatment of osteochondral defects.


Supercritical fluid technology Platelet lysate Genipin Polymerization Gradient structures 



The research leading to these results has received funding from the project “Accelerating tissue engineering and personalized medicine discoveries by the integration of key enabling nanotechnologies, marine-derived biomaterials and stem cells,” supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).


  1. 1.
    Yang PJ, Temenoff JS (2009) Engineering orthopedic tissue interfaces. Tissue Eng. Part B Rev. 15:127–141CrossRefGoogle Scholar
  2. 2.
    Ahmed TAE, Hincke MT (2010) Strategies for articular cartilage lesion repair and functional restoration. Tissue Eng. Part B. Rev. 16:305–329CrossRefGoogle Scholar
  3. 3.
    Lefebvre V, Smits P (2005) Transcriptional control of chondrocyte fate and differentiation. Birth Defects Res Part C Embryo Today Rev 75:200–212CrossRefGoogle Scholar
  4. 4.
    Malafaya PB, Silva GA, Reis RL (2007) Natural-origin polymers as carriers and scaffolds for biomolecules and cell delivery in tissue engineering applications. Adv Drug Deliv Rev 59:207–233CrossRefGoogle Scholar
  5. 5.
    O’Shea TM, Miao X (2008) Bilayered scaffolds for osteochondral tissue engineering. Tissue Eng. Part B. Rev. 14:447–464CrossRefGoogle Scholar
  6. 6.
    Chen J et al (2011) Simultaneous regeneration of articular cartilage and subchondral bone in vivo using MSCs induced by a spatially controlled gene delivery system in bilayered integrated scaffolds. Biomaterials 32:4793–4805CrossRefGoogle Scholar
  7. 7.
    Mano JF, Reis RL (2007) Osteochondral defects: present situation and tissue engineering approaches. J. Tissue Eng. Regen. Med. 1:261–273CrossRefGoogle Scholar
  8. 8.
    Chen FM, Zhang M, Wu ZF (2010) Toward delivery of multiple growth factors in tissue engineering. Biomaterials 31:6279–6308CrossRefGoogle Scholar
  9. 9.
    Kon E, Mutini A, Arcangeli E, Delcogliano M, Filardo G, Nicoli Aldini N, Pressato D, Quarto R, Zaffagnini S, Marcacci M (2008) Novel nanostructured scaffold for osteochondral regeneration: pilot study in horses. J. Tissue Eng. Regen. Med. 2:408–417CrossRefGoogle Scholar
  10. 10.
    Lu HH, Subramony SD, Boushell MK, Zhang X (2010) Tissue engineering strategies for the regeneration of orthopedic interfaces. Ann. Biomed. Eng. 38:2142–2154CrossRefGoogle Scholar
  11. 11.
    Anitua E et al (2006) New insights into and novel applications for platelet-rich fibrin therapies. Trends Biotechnol 24:227–234CrossRefGoogle Scholar
  12. 12.
    Haberhauer M et al (2008) Cartilage tissue engineering in plasma and whole blood scaffolds. Adv. Mater. 20:2061–2067CrossRefGoogle Scholar
  13. 13.
    Santo VE et al (2012) Enhancement of osteogenic differentiation of human adipose derived stem cells by the controlled release of platelet lysates from hybrid scaffolds produced by supercritical fluid foaming. J. Control. Release 162:19–27CrossRefGoogle Scholar
  14. 14.
    Duarte a RC, Mano JF, Reis RL (2009) Perspectives on: supercritical fluid technology for 3D tissue engineering scaffold applications. J. Bioact. Compat. Polym. 24:385–400CrossRefGoogle Scholar
  15. 15.
    Duarte ARC et al (2013) Unleashing the potential of supercritical fluids for polymer processing in tissue engineering and regenerative medicine. J. Supercrit. Fluids 79:177–185CrossRefGoogle Scholar
  16. 16.
    Santo VE, Duarte ARC, Gomes ME, Mano JF, Reis RL (2010) Hybrid 3D structure of poly(d,l-lactic acid) loaded with chitosan/chondroitin sulfate nanoparticles to be used as carriers for biomacromolecules in tissue engineering. J Supercrit Fluids 54:320–327CrossRefGoogle Scholar
  17. 17.
    van de Witte P, Dijkstra PJJ, van den Berg JWA, Feijen J (1996) Phase separation processes in polymer solutions in relation to membrane formation. J. Memb. Sci. 117:1–31CrossRefGoogle Scholar
  18. 18.
    Eckert CA, Knutson BL, Debenedetti PG (1996) Supercritical fluids as solvents for chemical and materials processing. Nature 383:313–318CrossRefGoogle Scholar
  19. 19.
    Temtem M et al (2009) Supercritical CO2 generating chitosan devices with controlled morphology. Potential application for drug delivery and mesenchymal stem cell culture. J. Supercrit. Fluids 48:269–277CrossRefGoogle Scholar
  20. 20.
    Keeney M, Pandit A (2009) The osteochondral junction and its repair via bi-phasic tissue engineering scaffolds. Tissue Eng. Part B. Rev. 15:55–73CrossRefGoogle Scholar
  21. 21.
    Gadjanski I, Vunjak-Novakovic G (2015) Challenges in engineering osteochondral tissue grafts with hierarchical structures. Expert Opin. Biol. Ther. 2598:1–17Google Scholar
  22. 22.
    Nukavarapu SP, Dorcemus DL (2012) Osteochondral tissue engineering: current strategies and challenges. Biotechnol Adv.
  23. 23.
    Canadas RF, Marques AP, Reis RL, Oliveira JM (2017) In: Oliveira JM, Reis RL (eds) Regenerative strategies for the treatment of knee joint disabilities. Springer International, Berlin, pp 213–233. CrossRefGoogle Scholar
  24. 24.
    Yan LP et al (2015) Bilayered silk/silk-nanoCaP scaffolds for osteochondral tissue engineering: In vitro and in vivo assessment of biological performance. Acta Biomater. 12:227–241CrossRefGoogle Scholar
  25. 25.
    Yan LP, Oliveira JM, Oliveira AL, Reis RL (2013) Silk fibroin/nano-CaP bilayered scaffolds for osteochondral tissue engineering. Key Eng. Mater. 587:245–248CrossRefGoogle Scholar
  26. 26.
    Zaky SH, Ottonello A, Strada P, Cancedda R, Mastrogiacomo M (2008) Platelet lysate favours in vitro expansion of human bone marrow stromal cells for bone and cartilage engineering. J. Tissue Eng. Regen. Med. 2:472–481CrossRefGoogle Scholar
  27. 27.
    Santo VE et al (2016) Engineering enriched microenvironments with gradients of platelet lysate in hydrogel fibers. Biomacromolecules 17:1985–1997CrossRefGoogle Scholar
  28. 28.
    Babo PS et al (2016) Assessment of bone healing ability of calcium phosphate cements loaded with platelet lysate in rat calvarial defects. J. Biomater. Appl. 31:637–649CrossRefGoogle Scholar
  29. 29.
    Yan L, Oliveira JM, Oliveira AL, Reis RL (2015) Current concepts and challenges in osteochondral tissue engineering and regenerative medicine. ACS Biomater. Sci. Eng. 1(4):150220124046001. CrossRefGoogle Scholar
  30. 30.
    Ribeiro V, Pina S, Oliveira JM, Reis RL (2017) In: Oliveira JM, Reis RL (eds) Regenerative strategies for the treatment of knee joint disabilities. Springer International, Berlin, pp 129–146. CrossRefGoogle Scholar
  31. 31.
    Cengiz IF, Oliveira JM, Reis RL (2014) In: Magnenat-Thalmann N, Ratib O, Choi HF (eds) 3D multiscale physiological human. Springer, London, pp 25–47. CrossRefGoogle Scholar
  32. 32.
    Wang C, Lau TT, Loh WL, Su K, Wang D (2011) Cytocompatibility study of a natural biomaterial crosslinker—Genipin with therapeutic model cells. J Biomed Mater Res B Appl Biomater 97:58–65. CrossRefPubMedGoogle Scholar
  33. 33.
    Muzzarelli RAA (2009) Genipin-crosslinked chitosan hydrogels as biomedical and pharmaceutical aids. Carbohydr. Polym. 77:1–9CrossRefGoogle Scholar
  34. 34.
    Butler MF, Ng Y, Pudney PDA (2003) Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups and Genipin. J Polym Sci Part A Polym Chem 41:3941–3953CrossRefGoogle Scholar
  35. 35.
    Mu C, Zhang K, Lin W, Li D (2012) Ring-opening polymerization of genipin and its long-range crosslinking effect on collagen hydrogel. J Biomed Mater Res A 101:385–393. CrossRefPubMedGoogle Scholar
  36. 36.
    Chuang M, Johannsen M (2009) Characterization of pH in aqueous CO2—systems. Polym Degrad Stab 97(6):839–848CrossRefGoogle Scholar
  37. 37.
    Babo P et al (2014) Platelet lysate membranes as new autologous templates for tissue engineering applications. Inflamm. Regen. 34:033–044CrossRefGoogle Scholar
  38. 38.
    Santo VE, Popa EG, Mano JF, Gomes ME, Reis RL (2015) Natural assembly of platelet lysate-loaded nanocarriers into enriched 3D hydrogels for cartilage regeneration. Acta Biomater. 19:56–65CrossRefGoogle Scholar
  39. 39.
    Duarte ARC, Caridade SG, Mano JF, Reis RL (2009) Processing of novel bioactive polymeric matrixes for tissue engineering using supercritical fluid technology. Mater. Sci. Eng. C 29:2110–2115CrossRefGoogle Scholar
  40. 40.
    Rezwan K, Chen QZ, Blaker JJ, Boccaccini AR (2006) Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials 27:3413–3431CrossRefGoogle Scholar
  41. 41.
    Rey C, Combes C (2016) Biomineralization and biomaterials. pp 95–127. CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Ana Rita C. Duarte
    • 1
    • 2
    Email author
  • Vitor E. Santo
    • 1
    • 2
  • Manuela E. Gomes
    • 1
    • 2
  • Rui L. Reis
    • 1
    • 2
  1. 1.3B’s Research Group—Biomaterials, Biodegradables and Biomimetics, European Institute of Excellence on Tissue Engineering and Regenerative Medicine, University of MinhoBarco/GuimarãesPortugal
  2. 2.ICVS/3B’s—PT Government Associate LaboratoryBraga/GuimarãesPortugal

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