Skip to main content
Log in

Effect of 3D Printing Temperature on Bioactivity of Bone Morphogenetic Protein-2 Released from Polymeric Constructs

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

Abstract

Growth factors such as bone morphogenetic protein-2 (BMP-2) are potent tools for tissue engineering. Three-dimensional (3D) printing offers a potential strategy for delivery of BMP-2 from polymeric constructs; however, these biomolecules are sensitive to inactivation by the elevated temperatures commonly employed during extrusion-based 3D printing. Therefore, we aimed to correlate printing temperature to the bioactivity of BMP-2 released from 3D printed constructs composed of a model polymer, poly(propylene fumarate). Following encapsulation of BMP-2 in poly(dl-lactic-co-glycolic acid) particles, growth factor-loaded fibers were fabricated at three different printing temperatures. Resulting constructs underwent 28 days of aqueous degradation for collection of released BMP-2. Supernatants were then assayed for the presence of bioactive BMP-2 using a cellular assay for alkaline phosphatase activity. Cumulative release profiles indicated that BMP-2 released from constructs that were 3D printed at physiologic and intermediate temperatures exhibited comparable total amounts of bioactive BMP-2 release as those encapsulated in non-printed particulate delivery vehicles. Meanwhile, the elevated printing temperature of 90 °C resulted in a decreased amount of total bioactive BMP-2 release from the fibers. These findings elucidate the effects of elevated printing temperatures on BMP-2 bioactivity during extrusion-based 3D printing, and enlighten polymeric material selection for 3D printing with growth factors.

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

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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

Similar content being viewed by others

References

  1. ASTM F2131: Standard test method for in vitro biological activity of recombinant human bone morphogenetic protein-2 (rh-BMP-2) using the W-20 mouse stromal cell line. 2012.

  2. Bittner, S. M., J. L. Guo, and A. G. Mikos. Spatiotemporal control of growth factors in three-dimensional printed scaffolds. Bioprinting 12:2018, 2018.

    Article  Google Scholar 

  3. Bracaglia, L. G., M. Messina, C. Vantucci, H. B. Baker, A. Pandit, and J. P. Fisher. Controlled delivery of tissue inductive factors in a cardiovascular hybrid biomaterial scaffold. ACS Biomater. Sci. Eng. 3:1350–1358, 2016.

    Article  PubMed  CAS  Google Scholar 

  4. Busatto, C., J. Pesoa, I. Helbling, J. Luna, and D. Estenoz. Heterogeneous hydrolytic degradation of poly(lactic-co-glycolic acid) microspheres: mathematical modeling. J. Appl. Polym. Sci. 134:45464, 2017.

    Article  CAS  Google Scholar 

  5. Caballero Aguilar, L. M., S. M. Silva, and S. E. Moulton. Growth factor delivery: defining the next generation platforms for tissue engineering. J. Control. Release 306:40–58, 2019.

    Article  CAS  PubMed  Google Scholar 

  6. Cai, Z., Y. Wan, M. L. Becker, Y. Z. Long, and D. Dean. Poly(propylene fumarate)-based materials: synthesis, functionalization, properties, device fabrication and biomedical applications. Biomaterials 208:45–71, 2019.

    Article  CAS  PubMed  Google Scholar 

  7. Clark, A., T. A. Milbrandt, J. Z. Hilt, and D. A. Puleo. Retention of insulin-like growth factor I bioactivity during the fabrication of sintered polymeric scaffolds. Biomed. Mater. 9:025015, 2014.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Coleman, J., and A. Lowman. Biodegradable nanoparticles for protein delivery: analysis of preparation conditions on particle morphology and protein loading, activity and sustained release properties. J. Biomater. Sci. Polym. Ed. 23:1129–1151, 2012.

    CAS  PubMed  Google Scholar 

  9. Comminal, R., M. P. Serdeczny, D. B. Pedersen, and J. Spangenberg. Numerical modeling of the strand deposition flow in extrusion-based additive manufacturing. Addit. Manuf. 20:68–76, 2018.

    Google Scholar 

  10. Emmermacher, J., D. Spura, J. Cziommer, D. Kilian, T. Wollborn, U. Fritsching, J. Steingroewer, T. Walther, M. Gelinsky, and A. Lode. Engineering considerations on extrusion-based bioprinting: interactions of material behavior, mechanical forces and cells in the printing needle. Biofabrication 12:025022, 2020.

    Article  CAS  PubMed  Google Scholar 

  11. England, J. L., and G. Haran. Role of solvation effects in protein denaturation: from thermodynamics to single molecules and back. Annu. Rev. Phys. Chem. 62:257–277, 2011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Farshid, B., G. Lalwani, M. ShirMohammadi, J. Simonsen, and B. Sitharaman. Boron nitride nanotubes and nanoplatelets as reinforcing agents of polymeric matrices for bone tissue engineering. J. Biomed. Mater. Res. B 105:406–419, 2017.

    Article  CAS  Google Scholar 

  13. Fox, N., and I. Streinu. Redundant and critical noncovalent interactions in protein rigid cluster analysis. In: Discrete and Topological Models in Molecular Biology, edited by N. Jonoska, and M. Saito. Berlin: Springer, 2014, pp. 167–196.

    Chapter  Google Scholar 

  14. Freeman, F. E., P. Pitacco, L. H. A. van Dommelen, J. Nulty, D. C. Browe, J.-Y. Shin, E. Alsberg, and D. J. Kelly. 3D bioprinting spatiotemporally defined patterns of growth factors to tightly control tissue regeneration. Sci. Adv. 6:5093, 2020.

    Article  CAS  Google Scholar 

  15. Galarraga, J. H., M. Y. Kwon, and J. A. Burdick. 3D bioprinting via an in situ crosslinking technique towards engineering cartilage tissue. Sci. Rep. 9:19987, 2019.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Holland, T. A., Y. Tabata, and A. G. Mikos. In vitro release of transforming growth factor-β1 from gelatin microparticles encapsulated in biodegradable, injectable oligo(poly(ethylene glycol) fumarate) hydrogels. J. Control. Release 91:299–313, 2003.

    Article  CAS  PubMed  Google Scholar 

  17. Jain, S., T. Fuoco, M. A. Yassin, K. Mustafa, and A. Finne-Wistrand. Printability and critical insight into polymer properties during direct-extrusion based 3D printing of medical grade polylactide and copolyesters. Biomacromolecules 21:388–396, 2020.

    Article  CAS  PubMed  Google Scholar 

  18. Ji, S., K. Dube, J. P. Chesterman, S. L. Fung, C. Y. Liaw, J. Kohn, and M. Guvendiren. Polyester-based ink platform with tunable bioactivity for 3D printing of tissue engineering scaffolds. Biomater. Sci. 7:560–570, 2019.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kasper, F. K., K. Tanahashi, J. P. Fisher, and A. G. Mikos. Synthesis of poly(propylene fumarate). Nat. Protoc. 4:518–525, 2009.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kempen, D. H., L. Lu, T. E. Hefferan, L. B. Creemers, A. Maran, K. L. Classic, W. J. Dhert, and M. J. Yaszemski. Retention of in vitro and in vivo BMP-2 bioactivities in sustained delivery vehicles for bone tissue engineering. Biomaterials 29:3245–3252, 2008.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kempen, D. H., L. Lu, C. Kim, X. Zhu, W. J. Dhert, B. L. Currier, and M. J. Yaszemski. Controlled drug release from a novel injectable biodegradable microsphere/scaffold composite based on poly(propylene fumarate). J. Biomed. Mater. Res. A 77:103–111, 2006.

    Article  PubMed  CAS  Google Scholar 

  22. Kim, K., D. Dean, J. Wallace, R. Breithaupt, A. G. Mikos, and J. P. Fisher. The influence of stereolithographic scaffold architecture and composition on osteogenic signal expression with rat bone marrow stromal cells. Biomaterials 32:3750–3763, 2011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kim, S., Y. Kang, C. A. Krueger, M. Sen, J. B. Holcomb, D. Chen, J. C. Wenke, and Y. Yang. Sequential delivery of BMP-2 and IGF-1 using a chitosan gel with gelatin microspheres enhances early osteoblastic differentiation. Acta Biomater. 8:1768–1777, 2012.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kim, K., J. Lam, S. Lu, P. P. Spicer, A. Lueckgen, Y. Tabata, M. E. Wong, J. A. Jansen, A. G. Mikos, and F. K. Kasper. Osteochondral tissue regeneration using a bilayered composite hydrogel with modulating dual growth factor release kinetics in a rabbit model. J. Control. Release 168:166–178, 2013.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Koons, G. L., M. Diba, and A. G. Mikos. Materials design for bone-tissue engineering. Nat. Rev. Mater. 5:584–603, 2020.

    Article  CAS  Google Scholar 

  26. Koons, G. L., and A. G. Mikos. Progress in three-dimensional printing with growth factors. J. Control. Release 295:50–59, 2019.

    Article  CAS  PubMed  Google Scholar 

  27. Kyle, S., Z. M. Jessop, A. Al-Sabah, and I. S. Whitaker. ‘Printability’ of candidate biomaterials for extrusion based 3D printing: state-of-the-art. Adv. Healthcare Mater. 6:1700264, 2017.

    Article  CAS  Google Scholar 

  28. Lakshmanan, R., P. Kumaraswamy, U. M. Krishnan, and S. Sethuraman. Engineering a growth factor embedded nanofiber matrix niche to promote vascularization for functional cardiac regeneration. Biomaterials 97:176–195, 2016.

    Article  CAS  PubMed  Google Scholar 

  29. Lee, C.-U., J. Vandenbrande, A. E. Goetz, M. A. Ganter, D. W. Storti, and A. J. Boydston. Room temperature extrusion 3D printing of polyether ether ketone using a stimuli-responsive binder. Addit. Manuf. 28:430–438, 2019.

    CAS  Google Scholar 

  30. Lee, J., J. Walker, K. S. Kang, S. H. Lee, J. Y. Kim, B. K. Lee, and D. W. Cho. Bone regeneration using a microstereolithography-produced customized poly(propylene fumarate)/diethyl fumarate photopolymer 3D scaffold incorporating BMP-2 loaded PLGA microspheres. Biomaterials 32:744–752, 2011.

    Article  CAS  PubMed  Google Scholar 

  31. Lee, J., J. Walker, S. Natarajan, and S. Yi. Prediction of geometric characteristics in polycaprolactone (PCL) scaffolds produced by extrusion-based additive manufacturing technique for tissue engineering. Rapid Prototyp. J. 26:238–248, 2020.

    Article  Google Scholar 

  32. Luca, L., A.-L. Rougemont, B. H. Waploth, R. Gurny, and O. Jordan. The effects of carrier nature and pH on rhBMP-2-induced ectopic bone formation. J. Control. Release 147:38–44, 2010.

    Article  CAS  PubMed  Google Scholar 

  33. Luo, Y., G. Le Fer, D. Dean, and M. L. Becker. 3D printing of poly(propylene fumarate) oligomers: evaluation of resin viscosity, printing characteristics and mechanical properties. Biomacromolecules 20:1699–1708, 2019.

    Article  CAS  PubMed  Google Scholar 

  34. Mistry, A. S., A. G. Mikos, and J. A. Jansen. Degradation and biocompatibility of a poly(propylene fumarate)-based/alumoxane nanocomposite for bone tissue engineering. J. Biomed. Mater. Res. A 83:940–953, 2007.

    Article  CAS  PubMed  Google Scholar 

  35. Nyberg, E., C. Holmes, T. Witham, and W. L. Grayson. Growth factor-eluting technologies for bone tissue engineering. Drug Deliv. Transl. Res. 6:184–194, 2016.

    Article  CAS  PubMed  Google Scholar 

  36. Ohta, H., S. Wakitani, K. Tensho, H. Horiuchi, S. Wakabayashi, N. Saito, Y. Nakamura, K. Nozaki, Y. Imai, and K. Takaoka. The effects of heat on the biological activity of recombinant human bone morphogenetic protein-2. J. Bone Miner. Metab. 23:420–425, 2005.

    Article  CAS  PubMed  Google Scholar 

  37. Olthof, M. G. L., D. H. R. Kempen, J. L. Herrick, M. J. Yaszemski, W. J. A. Dhert, and L. Lu. Effect of different sustained bone morphogenetic protein-2 release kinetics on bone formation in poly(propylene fumarate) scaffolds. J. Biomed. Mater. Res. B 106:477–487, 2018.

    Article  CAS  Google Scholar 

  38. Placone, J. K., and A. J. Engler. Recent advances in extrusion-based 3D printing for biomedical applications. Adv. Healthcare Mater. 7:e1701161, 2018.

    Article  CAS  Google Scholar 

  39. Ramburrun, P., P. Kumar, Y. E. Choonara, D. Bijukumar, L. C. du Toit, and V. Pillay. A review of bioactive release from nerve conduits as a neurotherapeutic strategy for neuronal growth in peripheral nerve injury. Biomed. Res. Int. 2014:132350, 2014.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Roberts, J. J., P. Naudiyal, L. Jugé, L. E. Bilston, A. M. Granville, and P. J. Martens. Tailoring stimuli responsiveness using dynamic covalent cross-linking of poly(vinyl alcohol)-heparin hydrogels for controlled cell and growth factor delivery. ACS Biomater. Sci. Eng. 1:1267–1277, 2015.

    Article  CAS  PubMed  Google Scholar 

  41. Seto, S. P., T. Miller, and J. S. Temenoff. Effect of selective heparin desulfation on preservation of bone morphogenetic protein-2 bioactivity after thermal stress. Bioconjug. Chem. 26:286–293, 2015.

    Article  CAS  PubMed  Google Scholar 

  42. Sevim, K., and J. Pan. A mechanistic model for acidic drug release using microspheres made of PLGA 50:50. Mol. Pharm. 13:2729–2735, 2016.

    Article  CAS  PubMed  Google Scholar 

  43. Shah, S. R., A. M. Henslee, P. P. Spicer, S. Yokota, S. Petrichenko, S. Allahabadi, G. N. Bennett, M. E. Wong, F. K. Kasper, and A. G. Mikos. Effects of antibiotic physicochemical properties on their release kinetics from biodegradable polymer microparticles. Pharm. Res. 31:3379–3389, 2014.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Smith, B. T., S. M. Bittner, E. Watson, M. M. Smoak, L. Diaz-Gomez, E. R. Molina, Y. S. Kim, C. D. Hudgins, A. J. Melchiorri, D. W. Scott, K. J. Grande-Allen, J. J. Yoo, A. Atala, J. P. Fisher, and A. G. Mikos. Multi-material dual gradient 3D printing for osteogenic differentiation and spatial segregation. Tissue Eng. Part A 26:239–252, 2020.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Suntornnond, R., E. Y. S. Tan, J. An, and C. K. Chua. A highly printable and biocompatible hydrogel composite for direct printing of soft and perfusable vasculature-like structures. Sci. Rep. 7:16902, 2017.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Tarafder, S., A. Koch, Y. Jun, C. Chou, M. R. Awadallah, and C. H. Lee. Micro-precise spatiotemporal delivery system embedded in 3D printing for complex tissue regeneration. Biofabrication 8:025003, 2016.

    Article  PubMed  CAS  Google Scholar 

  47. Thibault, R. A., L. S. Baggett, A. G. Mikos, and F. K. Kasper. Osteogenic differentiation of mesenchymal stem cells on pregenerated extracellular matrix scaffolds in the absence of osteogenic cell culture supplements. Tissue. Eng. Part A 16:431–440, 2010.

    Article  CAS  PubMed  Google Scholar 

  48. Timmer, M. D., H. Shin, R. A. Horch, C. G. Ambrose, and A. G. Mikos. In vitro cytotoxicity of injectable and biodegradable poly(propylene fumarate)-based networks: unreacted macromers, cross-linked networks, and degradation products. Biomacromolecules 4:1026–1033, 2003.

    Article  CAS  PubMed  Google Scholar 

  49. Tousif Ayyub, K., K. Moravkar, M. Maniruzzaman, and P. Amin. Effect of melt extrudability and melt binding efficiency of polyvinyl caprolactam polyvinyl acetate polyethylene glycol graft copolymer (Soluplus®) on release pattern of hydrophilic and high dose drugs. Mater. Sci. Eng. C 99:563–574, 2019.

    Article  CAS  Google Scholar 

  50. Trachtenberg, J. E., J. K. Placone, B. T. Smith, C. M. Piard, M. Santoro, D. W. Scott, J. P. Fisher, and A. G. Mikos. Extrusion-based 3D printing of poly(propylene fumarate) in a full-factorial design. ACS Biomater. Sci. Eng. 2:1771–1780, 2016.

    Article  CAS  PubMed  Google Scholar 

  51. Turner, B. N., R. Strong, and S. A. Gold. A review of melt extrusion additive manufacturing processes: I. Process design and modeling. Rapid Prototyping J. 20:192–204, 2014.

    Article  Google Scholar 

  52. Wang, M. O., J. M. Etheridge, J. A. Thompson, C. E. Vorwald, D. Dean, and J. P. Fisher. Evaluation of the in vitro cytotoxicity of cross-linked biomaterials. Biomacromolecules 14:1321–1329, 2013.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Wang, M. O., C. M. Piard, A. Melchiorri, M. L. Dreher, and J. P. Fisher. Evaluating changes in structure and cytotoxicity during in vitro degradation of three-dimensional printed scaffolds. Tissue Eng. Part A 21:1642–1653, 2015.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Wei, C., N. G. Solanki, J. M. Vasoya, A. V. Shah, and A. T. M. Serajuddin. Development of 3D printed tablets by fused deposition modeling using polyvinyl alcohol as polymeric matrix for rapid drug release. J. Pharm. Sci. 109:1558–1572, 2020.

    Article  CAS  PubMed  Google Scholar 

  55. Xiao, J., Y. Wang, S. Bellusci, and X. Li. Pharmacological application of growth factors: basic and clinical. Biomed. Res. Int. 2015:141794, 2015.

    PubMed  PubMed Central  Google Scholar 

  56. Xu, Y., C.-S. Kim, D. M. Saylor and D. Koo. Polymer degradation and drug delivery in PLGA-based drug–polymer applications: a review of experiments and theories. J. Biomed. Mater. Res. B Appl. Biomater. 105:1692–1716, 2017.

    Article  CAS  PubMed  Google Scholar 

  57. Yamakawa, S. and K. Hayashida. Advances in surgical applications of growth factors for wound healing. Burns Trauma 7:10, 2019.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Yao, B., R. Wang, Y. Wang, Y. Zhang, T. Hu, W. Song, Z. Li, S. Huang and X. Fu. Biochemical and structural cues of 3D-printed matrix synergistically direct MSC differentiation for functional sweat gland regeneration. Sci. Adv. 6:eaaz1094, 2020.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Institutes of Health (Grant P41 EB023833). G.L.K. is supported by the Robert and Janice McNair Foundation MD/PhD Student Scholar Program. M.D. is supported by a Rubicon postdoctoral fellowship from the Dutch Research Council (NWO; Project No. 019.182EN.004). The authors gratefully acknowledge Anthony J. Melchiorri and Yu Seon Kim for their guidance on polymer synthesis, and Luis Diaz-Gomez for his input on 3D printing.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Antonios G. Mikos.

Additional information

Associate Editor Stefan M Duma 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

Below is the link to the Supplementary Information.

Supplementary Information 1 (DOCX 703 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Koons, G.L., Kontoyiannis, P.D., Diba, M. et al. Effect of 3D Printing Temperature on Bioactivity of Bone Morphogenetic Protein-2 Released from Polymeric Constructs. Ann Biomed Eng 49, 2114–2125 (2021). https://doi.org/10.1007/s10439-021-02736-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10439-021-02736-9

Keywords

Navigation