Combined Porogen Leaching and Emulsion Templating to produce Bone Tissue Engineering Scaffolds

Authors

  • Robert Owen Department of Materials Science and Engineering, INSIGNEO Institute for in silico Medicine, University of Sheffield, UK；Department of Materials Science and Engineering, The Kroto Research Institute, University of Sheffield, UK；Regenerative Medicine and Cellular Therapies, School of Pharmacy, University of Nottingham Biodiscovery Institute,University Park, UK
  • Colin Sherborne Department of Materials Science and Engineering, The Kroto Research Institute, University of Sheffield, UK
  • Richard Evans Bioengineering, Interdisciplinary Programmes Engineering, University of Sheffield, UK
  • Gwendolen C. Reilly Department of Materials Science and Engineering, INSIGNEO Institute for in silico Medicine, University of Sheffield, UK；Department of Materials Science and Engineering, The Kroto Research Institute, University of Sheffield, UK
  • Frederik Claeyssens Department of Materials Science and Engineering, INSIGNEO Institute for in silico Medicine, University of Sheffield, UK；Department of Materials Science and Engineering, The Kroto Research Institute, University of Sheffield, UK

DOI:

https://doi.org/10.18063/ijb.v6i2.265

Keywords:

Polymerized high internal phase emulsions, Emulsion templating, Alginate, Multiscale porosity, Bone tissue engineering

Abstract

Bone has a hierarchy of porosity that is often overlooked when creating tissue engineering scaffolds where pore sizes are typically confined to a single order of magnitude. High internal phase emulsion (HIPE) templating produces polymerized HIPEs (polyHIPEs): highly interconnected porous polymers which have two length scales of porosity covering the 1–100 µm range. However, additional larger scales of porosity cannot be introduced in the standard emulsion formulation. Researchers have previously overcome this by additively manufacturing emulsions; fabricating highly microporous struts into complex macroporous geometries. This is time consuming and expensive; therefore, here we assessed the feasibility of combining porogen leaching with emulsion templating to introduce additional macroporosity. Alginate beads between 275 and 780 µm were incorporated into the emulsion at 0, 50, and 100 wt%. Once polymerized, alginate was dissolved leaving highly porous polyHIPE scaffolds with added macroporosity. The compressive modulus of the scaffolds decreased as alginate porogen content increased. Cellular performance was assessed using MLO-A5 post-osteoblasts. Seeding efficiency was significantly higher and mineralized matrix deposition was more uniformly deposited throughout porogen leached scaffolds compared to plain polyHIPEs. Deep cell infiltration only occurred in porogen leached scaffolds as detected by histology and lightsheet microscopy. This study reveals a quick, low cost and simple method of producing multiscale porosity scaffolds for tissue engineering.

References

Wu S, Liu X, Yeung KW, et al., 2014, Biomimetic Porous Scaffolds for Bone Tissue Engineering. Materials Sci Eng R Rep, 80:1–36.

Gupta D, Singh AK, Dravid A, et al., 2019, Multiscale Porosity in Compressible Cryogenically 3D Printed Gels for Bone Tissue Engineering. ACS Appl Mater Interfaces, 11:20437–52. DOI: 10.1021/acsami.9b05460

Rustom LE, Boudou T, Nemke BW, et al., 2017, Multiscale Porosity Directs Bone Regeneration in Biphasic Calcium Phosphate Scaffolds. ACS Biomater Sci Eng, 3:2768–78. DOI: 10.1021/acsbiomaterials.6b00632

Woodard JR, Hilldore AJ, Lan SK, et al., 2007, The Mechanical Properties and Osteo conductivity of Hydroxyapatite Bone Scaffolds with Multi-scale Porosity. Biomaterials, 28:45–54. DOI: 10.1016/j.biomaterials.2006.08.021

Roosa SM, Kemppainen JM, Moffitt EN, et al., 2010, The Pore Size of Polycaprolactone Scaffolds has Limited Influence on Bone Regeneration in an in vivo Model. J Biomed Mater Res A, 92:359–68. DOI: 10.1002/jbm.a.32381

Vand K, Kaplan D, 2005, Porosity of 3D Biomaterial Scaffolds and Osteogenesis. Biomaterials, 26:5474–91. DOI:10.1016/j.biomaterials.2005.02.002

Land LQ, Choong C, 2013, Three-dimensional Scaffolds for Tissue Engineering Applications: Role of Porosity and Pore Size. Tissue Eng Part B Rev, 19:485-502. DOI: 10.1089/ten.teb.2012.0437

Owen R, Sherborne C, Paterson T, et al., 2016, Emulsion Templated Scaffolds with Tunable Mechanical Properties for Bone Tissue Engineering. J Mech Behav Biomed Mater, 54:159–72. DOI: 10.1016/j.jmbbm.2015.09.019

Sherborne C, Owen R, Reilly GC, et al., 2018, Light-based Additive Manufacturing of PolyHIPEs: Controlling the Surface Porosity for 3D Cell Culture Applications. Mater Des, 156:494–503. DOI: 10.1016/j.matdes.2018.06.061

Malayeri A, Sherborne C, Paterson T, et al., 2016, Osteosarcoma growth on trabecular bone mimicking structures manufactured via laser direct write. Int J Bioprint, 2:67–72. DOI: 10.18063/ijb.2016.02.005

Owen R, Sherborne C, Reilly GC, et al., 2015, Data for the Analysis of PolyHIPE Scaffolds with Tunable Mechanical Properties for Bone Tissue Engineering. Data Brief, 5:616–20. DOI: 10.1016/j.dib.2015.09.051

Paterson TE, Gigliobianco G, Sherborne C, et al., 2018, Porous Microspheres Support Mesenchymal Progenitor Cell Ingrowth and Stimulate Angiogenesis. APL Bioeng, 2:026103. DOI: 10.1063/1.5008556

Wang AJ, Paterson T, Owen R, et al., 2016, Photocurable High Internal Phase Emulsions (HIPEs) Containing Hydroxyapatite for Additive Manufacture of Tissue Engineering Scaffolds with Multi-scale Porosity. Mater Sci Eng C, 67:51–8. DOI: 10.1016/j.msec.2016.04.087

Whitely M, Rodriguez-Rivera G, Waldron C, et al., 2019, Porous PolyHIPE Microspheres for Protein Delivery from an Injectable Bone Graft. Acta Biomat, 93:169–79. DOI: 10.1016/j.actbio.2019.01.044

Lee A, Langford CR, Rodriguez-Lorenzo LM, et al., 2017, Bioceramic Nanocomposite Thiol-acrylate polyHIPE Scaffolds for Enhanced Osteoblastic Cell Culture in 3D. Biomat Sci, 5:2035–47. DOI: 10.1039/c7bm00292k

Dikici BA, Reilly GC, Claeyssens F, 2020, Boosting the Osteogenic and Angiogenic Performance of Multiscale Porous Polycaprolactone Scaffolds by in vitro Generated Extracellular Matrix Decoration. ACS Appl Mater Interfaces, 12:12510–24. DOI: 10.1021/acsami.9b23100

Dikici BA, Sherborne C, Reilly GC, et al., 2019, Emulsion Templated Scaffolds Manufactured from Photocurable Polycaprolactone. Polymer, 175:243–54. DOI: 10.1016/j.polymer.2019.05.023

Dikici BA, Dikici S, Reilly GC, et al., 2019, A Novel Bilayer Polycaprolactone Membrane for Guided Bone Regeneration: Combining Electrospinning and Emulsion Templating. Materials (Basel), 12:12162643. DOI: 10.3390/ma12162643

Cameron NR, 2005, High Internal Phase Emulsion Templating as a Route to Well-defined Porous Polymers. Polymer, 46:1439–49. DOI: 10.1016/j.polymer.2004.11.097

Krajnc PH, 2014, PolyHIPEs from Methyl Methacrylate: Hierarchically Structured Microcellular Polymers with Exceptional Mechanical Properties. Polymer, 55:4420–4. DOI: 10.1016/j.polymer.2014.07.007

Iand G, Silverstein MS, 2010, Polymerized pickering HIPEs: Effects of synthesis parameters on porous structure. J Polym Sci Part A Polym Chem, 48:1516–25. DOI: 10.1002/pola.23911

Bokhari M, Carnachan RJ, Przyborski SA, et al., 2007, Emulsion-templated Porous Polymers as Scaffolds for Three Dimensional Cell Culture: Effect of Synthesis Parameters on Scaffold Formation and Homogeneity. J Mater Chem, 17:4088–94. DOI: 10.1039/b707499a

Carnachan RJ, Bokhari M, Przyborski SA, et al., 2006, Tailoring the Morphology of Emulsion-templated Porous Polymers. Soft Matter, 2:608–16. DOI: 10.1039/b603211g

Richez A, Deleuze H, Vedrenne P, et al., 2005, Preparation of Ultra-low-density Microcellular Materials. J Appl Polym Sci, 96:2053–63. DOI: 10.1002/app.21668

Xu H, Zheng X, Huang Y, et al., 2016, Interconnected Porous Polymers with Tunable Pore Throat Size Prepared via Pickering High Internal Phase Emulsions. Langmuir, 32:38–45. DOI: 10.1021/acs.langmuir.5b03037

Williams JM, Gray AJ, Wilkerson MH, 1990, Emulsion Stability and Rigid Foams from Styrene or Divinylbenzene Water-in-oil Emulsions. Langmuir, 6:437–44. DOI: 10.1021/la00092a026

Williams JM, Wrobleski DA, 1988, Spatial Distribution of the Phases in Water-in-oil Emulsions. Open and Closed Microcellular Foams from Cross-linked Polystyrene. Langmuir, 4:656–62. DOI: 10.1021/la00081a027

Robinson JL, Moglia RS, Stuebben MC, et al., 2013, Achieving Interconnected Pore Architecture in Injectable PolyHIPEs for Bone Tissue Engineering. Tissue Eng Part A, 20:1103–12. DOI: 10.1089/ten.tea.2013.0319

Knight E, Murray B, Carnachan R, et al., 2011, Alvetex®: Polystyrene Scaffold Technology for Routine Three Dimensional Cell Culture. In: Haycock JW, editor. 3D Cell Culture: Methods and Protocols. Humana Press, Totowa, NJ.pp. 323–40. DOI: 10.1007/978-1-60761-984-0_20

Viswanathan P, Johnson DW, Hurley C, et al., 2014, 3D Surface Functionalization of Emulsion-Templated Polymeric Foams. Macromolecules, 47:7091–8. DOI: 10.1021/ma500968q

Cameron NR, Sherrington DC, Albiston L, et al., 1996, Study of the Formation of the Open-cellular Morphology of Poly(styrene/divinylbenzene) polyHIPE Materials by cryo-SEM. Colloid Polym Sci, 274:592–5. DOI: 10.1007/bf00655236

Stevens M Mand George J H, 2005, Exploring and Engineering the Cell Surface Interface. Science, 310:1135–8.

Sears NA, Dhavalikar PS, Cosgriff-Hernandez EM, 2016, Emulsion Inks for 3D Printing of High Porosity Materials. Macromol Rapid Commun, 37:1369–74. DOI: 10.1002/marc.201600236

Sears N, Dhavalikar P, Whitely M, et al., 2017, Fabrication of Biomimetic Bone Grafts with Multi-material 3D Printing. Biofabrication, 9:025020. DOI: 10.1088/1758-5090/aa7077

Susec M, Ligon SC, Stampfl J, et al., 2013, Hierarchically Porous Materials from Layer-by-layer Photopolymerization of High Internal Phase Emulsions. Macromol Rapid Commun, 34:938–43. DOI: 10.1002/marc.201300016

Johnson DW, Sherborne C, Didsbury MP, et al., 2013, Macrostructuring of Emulsion-templated Porous Polymers by 3D Laser Patterning. Adv Mater, 25:3178–81. DOI: 10.1002/adma.201300552

Lee JM, Ng WL, Yeong WY, 2019, Resolution and Shape in Bioprinting: Strategizing Towards Complex Tissue and Organ Printing. Appl Phys Rev, 6:011307. DOI: 10.1063/1.5053909

Ng WL, Lee JM, Zhou M, et al., 2020, Vat Polymerizationbased Bioprinting Process, Materials, Applications and Regulatory Challenges. Biofabrication, 12:022001. DOI: 10.1088/1758-5090/ab6034

Zhang J, Hu Q, Wang S, et al., 2020, Digital Light Processing Based Three-dimensional Printing for Medical Applications. Int J Bioprint, 6:1–10.

Thadavirul N, Pavasant P, Supaphol P, 2014, Development of Polycaprolactone Porous Scaffolds by Combining Solvent Casting, Particulate Leaching, and Polymer Leaching Techniques for Bone Tissue Engineering. J Biomed Mater Res Part A, 102:3379–92. DOI: 10.1002/jbm.a.35010

Kim TG, Chung HJ, Park TG, 2008, Macroporous and Nanofibrous Hyaluronic Acid/collagen Hybrid Scaffold Fabricated by Concurrent Electrospinning and Deposition/leaching of Salt Particles. Acta Biomater, 4:1611–9. DOI: 10.1016/j.actbio.2008.06.008

Sin D, Miao X, Liu G, et al., 2010, Polyurethane (PU) Scaffolds Prepared by Solvent Casting/particulate Leaching (SCPL) Combined with Centrifugation. Mater Sci Eng C, 30:78–85. DOI: 10.1016/j.msec.2009.09.002

Bencherif SA, Braschler TM, Renaud P, 2013, Advances in the Design of Macroporous Polymer Scaffolds for Potential Applications in Dentistry. J Periodontal Implant Sci, 43:251–61. DOI: 10.5051/jpis.2013.43.6.251

Hutmacher DW, 2001, Scaffold Design and Fabrication Technologies for Engineering Tissues State of the Art and Future Perspectives. J Biomater Sci Polym Ed, 12:107–24.

Venkatesan J, Bhatnagar I, Manivasagan P, et al., 2015, Alginate Composites for Bone Tissue Engineering: A Review. Int J Biol Macromol, 72:269–81.

Schindelin J, Arganda-Carreras I, Frise E, et al., 2012, Fiji: An Open-source Platform for Biological-image Analysis. Nat Methods, 9:676–82. DOI: 10.1038/nmeth.2019

Schneider CA, Rasband WS, Eliceiri KW, 2012, NIH Image to Image J: 25 Years of Image Analysis. Nat Methods, 9:671–5. DOI: 10.1038/nmeth.2089

Owen R, Bahmaee H, Claeyssens F, et al., 2020, Comparison of the Anabolic Effects of Reported Osteogenic Compounds on Human Mesenchymal Progenitor-derived Osteoblasts. Bioengineering (Basel), 7:7010012. DOI: 10.3390/bioengineering7010012

Linkert M, Rueden CT, Allan C, et al., 2010, Metadata Matters: Access to Image Data in the Real World. J Cell Biol, 189:777–82.

Paljevac M, Gradišnik L, Lipovšek S, et al., 2018, MultipleLevel Porous Polymer Monoliths with Interconnected Cellular Topology Prepared by Combining Hard Sphere and Emulsion Templating for Use in Bone Tissue Engineering. Macromol Biosci, 18:1700306. DOI: 10.1002/mabi.201700306

Hayward AS, Eissa AM, Maltman DJ, et al., 2013, Galactosefunctionalized PolyHIPE Scaffolds for Use in Routine Three Dimensional Culture of Mammalian Hepatocytes. Biomacromolecules, 14:4271–7. DOI: 10.1021/bm401145x

Akay G, Birch MA, Bokhari MA, 2004, Microcellular polyhipe Polymer Supports Osteoblast Growth and Bone Formation in vitro. Biomaterials, 25:3991–4000. DOI: 10.1016/j.biomaterials.2003.10.086

Workman VL, Dunnett SB, Kille P, et al., 2008, On-Chip Alginate Microencapsulation of Functional Cells. Macromol Rapid Commun, 29:165–70. DOI: 10.1002/marc.200700641

Beck GR Jr., Zerler B, Moran E, 2001, Gene Array Analysis of Osteoblast Differentiation. Cell Growth Differ, 12:61–83.

Downloads

Published

2020-04-30

Issue

Vol. 6 No. 2 (2020): International Journal of Bioprinting

Section

Research Article

License

Copyright (c) 2024 Robert Owen, Colin Sherborne, Richard Evans, Gwendolen C. Reilly, Frederik Claeyssens

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.