Hierarchical polymeric scaffolds support the growth of MC3T3-E1 cells


Tissue engineering makes use of the principles of biology and engineering to sustain 3D cell growth and promote tissue repair and/or regeneration. In this study, macro/microporous scaffold architectures have been developed using a hybrid solid freeform fabrication/thermally induced phase separation (TIPS) technique. Poly(lactic-co-glycolic acid) (PLGA) dissolved in 1,4-dioxane was used to generate a microporous matrix by the TIPS method. The 3D-bioplotting technique was used to fabricate 3D macroporous constructs made of polyethylene glycol (PEG). Embedding the PEG constructs inside the PLGA solution prior to the TIPS process and subsequent extraction of PEG following solvent removal (1,4-dioaxane) resulted in a macro/microporous structure. These hierarchical scaffolds with a bimodal pore size distribution (<50 and >300 μm) contained orthogonally interconnected macro-channels generated by the extracted PEG. The diameter of the macro-channels was varied by tuning the dispensing parameters of the 3D bioplotter. The in vitro cell culture using murine MC3T3-E1 cell line for 21 days demonstrated that these scaffolds could provide a favorable environment to support cell adhesion and growth.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8


  1. 1.

    Swieszkowski W, Tuan BHS, Kurzydlowski KJ, Hutmacher DW. Repair and regeneration of osteochondral defects in the articular joints. Biomol Eng. 2007;24:489–95.

    Article  Google Scholar 

  2. 2.

    Bhumiratana S, Vunjak-Novakovic G. Concise review: personalized human bone grafts for reconstructing head and face. Stem Cells Trans Med. 2012;1:64–9.

    Article  Google Scholar 

  3. 3.

    Saito E, Kang H, Taboas JM, Diggs A, Flanagan CL, Hollister SJ. Experimental and computational characterization of designed and fabricated 50:50 PLGA porous scaffolds for human trabecular bone applications, Journal of materials science. Mater Med. 2010;21(8):2371–83.

    Article  Google Scholar 

  4. 4.

    Yoo D. New paradigms in hierarchical porous scaffold design for tissue engineering. Mater Sci Eng C. 2013;33(3):1759–72.

    Article  Google Scholar 

  5. 5.

    Sachot N, Castaño O, Mateos-Timoneda MA, Engel E, Planell JA. Hierarchically engineered fibrous scaffolds for bone regeneration. J R Soc Interface. 2013;10(88):20130684.

    Article  Google Scholar 

  6. 6.

    Zhang J, Zhou H, Yang K, Yuan Y, Liu C. RhBMP-2-loaded calcium silicate/calcium phosphate cement scaffold with hierarchically porous structure for enhanced bone tissue regeneration. Biomaterials. 2013;34(37):9381–92.

    Article  Google Scholar 

  7. 7.

    Lee K-W, Wang S, Fox BC, Ritman EL, Yaszemski MJ, Lu L. Poly(propylene fumarate) bone tissue engineering scaffold fabrication using stereolithography: effects of resin formulations and laser parameters. Biomacromolecules. 2007;8(4):1077–84.

    Article  Google Scholar 

  8. 8.

    Talib M. Fabrication and characterization of 3 dimensional scaffold for tissue engineering application via microstereolithography technique, PhD Thesis, University of Warwick; 2012.

  9. 9.

    Williams JM, Adewunmi A, Schek RM, Flanagan CL, Krebsbach PH, Feinberg SE, et al. Bone tissue engineering using polycaprolactone scaffolds fabricated via selective laser sintering. Biomaterials. 2005;26(23):4817–27.

    Article  Google Scholar 

  10. 10.

    Liao HT, Lee MY, Tsai WW, Wang HC, Lu WC. Osteogenesis of adipose-derived stem cells on polycaprolactone–β-tricalcium phosphate scaffold fabricated via selective laser sintering and surface coating with collagen type I. J Tissue Eng Regen Med. 2013. doi:10.1002/term.1811.

    Google Scholar 

  11. 11.

    Hutmacher DW, Schantz T, Zein I, Ng KW, Teoh SH, Tan KC. Mechanical properties and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused deposition modeling. J Biomed Mater Res. 2001;55(2):203–16.

    Article  Google Scholar 

  12. 12.

    Hsu S, Yen H, Tseng C, Cheng C, Tsai C. Evaluation of the growth of chondrocytes and osteoblasts seeded into precision scaffolds fabricated by fused deposition manufacturing. J Biomed Mater Res. 2007;80(2):519–27.

    Article  Google Scholar 

  13. 13.

    Sherwood JK, Riley SL, Palazzolo R, Brown SC, Monkhouse DC, Coates M, et al. A three-dimensional osteochondral composite scaffold for articular cartilage repair. Biomaterials. 2002;23(24):4739–51.

    Article  Google Scholar 

  14. 14.

    Shanjani Y, De Croos JNA, Pilliar RM, Kandel RA, Toyserkani E. Solid freeform fabrication and characterization of porous calcium polyphosphate structures for tissue engineering purposes. J Biomed Mater Res B Appl Biomater. 2010;93(2):510–9.

    Article  Google Scholar 

  15. 15.

    Stosich MS, Moioli EK, Wu JK, Lee CH, Rohde C, Yoursef AM, et al. Bioengineering strategies to generate vascularized soft tissue grafts with sustained shape. Methods. 2009;47(2):116–21.

    Article  Google Scholar 

  16. 16.

    El-Ayoubi R, DeGrandpré C, DiRaddo R, Yousefi A-M, Lavigne P. Design and dynamic culture of 3D-scaffolds for cartilage tissue engineering. J Biomater Appl. 2011;25(5):429–44.

    Article  Google Scholar 

  17. 17.

    Huang YX, Ren J, Chen C, Ren TB, Zhou XY. Preparation and properties of poly(lactide-co-glycolide) (PLGA)/nano-hydroxyapatite (NHA) scaffolds by thermally induced phase separation and rabbit MSCs culture on scaffolds. J Biomater Appl. 2008;22(5):409–32.

    Article  Google Scholar 

  18. 18.

    Blaker JJ, Knowles JC, Day RM. Novel fabrication techniques to produce microspheres by thermally induced phase separation for tissue engineering and drug delivery. Acta Biomater. 2008;4(2):264–72.

    Article  Google Scholar 

  19. 19.

    Gong Y, Ma Z, Gao C, Wang W, Shen J. Specially elaborated thermally induced phase separation to fabricate poly(L-lactic acid) scaffolds with ultra large pores and good interconnectivity. J Appl Polym Sci. 2006;101(5):3336–42.

    Article  Google Scholar 

  20. 20.

    Shao J, Chen C, Wang Y, Chen X, Du C. Early stage structural evolution of PLLA porous scaffolds in thermally induced phase separation process and the corresponding biodegradability and biological property. Polym Degrad Stab. 2012;97(6):955–63.

    Article  Google Scholar 

  21. 21.

    Sabir MI, Xu X, Li L. A review on biodegradable polymeric materials for bone tissue engineering applications. J Mater Sci. 2009;44(21):5713–24.

    Article  Google Scholar 

  22. 22.

    Mehrabanian M, Nasr-Esfahani M. HA/nylon 6,6 porous scaffolds fabricated by salt-leaching/solvent casting technique: effect of nano-sized filler content on scaffold properties. Int J Nanomed. 2011;6:1651–9.

    Google Scholar 

  23. 23.

    Eshraghi S, Das S. Mechanical and microstructural properties of polycaprolactone scaffolds with one-dimensional, two-dimensional, and three-dimensional orthogonally oriented porous architectures produced by selective laser sintering. Acta Biomater. 2010;6(7):2467–76.

    Article  Google Scholar 

  24. 24.

    Ji C, Annabi N, Hosseinkhani M, Sivaloganathan S, Dehghani F. Fabrication of poly-DLlactide/polyethylene glycol scaffolds using the gas foaming technique. Acta Biomater. 2012;8(2):570–8.

    Article  Google Scholar 

  25. 25.

    Singh M, Sandhu B, Scurto A, Berkland C, Detamore MS. Microsphere-based scaffolds for cartilage tissue engineering: using subcritical CO(2) as a sintering agent. Acta Biomater. 2010;6(1):137–43.

    Article  Google Scholar 

  26. 26.

    Dehghani F, Annabi N. Engineering porous scaffolds using gas-based techniques. Current opinion in biotechnology. Elsevier Ltd; 2011;22(5):661–666.

  27. 27.

    Whang K, Thomas CH, Healy KE, Nuber G. A novel method to fabricate bioabsorbable scaffolds. Polymer. 1995;36(4):837–842.

  28. 28.

    Whang K, Tsai DC, Nam EK, Aitken M, Sprague SM, Patel PK, et al. Ectopic bone formation via rhBMP-2 delivery from porous bioabsorbable polymer scaffolds. J Biomed Mater Res. 1998;42(4):491–9.

    Article  Google Scholar 

  29. 29.

    Cui Y, Liu Y, Cui Y, Jing X, Zhang P, Chen X. The nanocomposite scaffold of poly(lactide-co-glycolide) and hydroxyapatite surface-grafted with L-lactic acid oligomer for bone repair. Acta Biomater. 2009;5(7):2680–92.

    Article  Google Scholar 

  30. 30.

    Oh SH, Park SC, Kim HK, Koh YJ, Lee J-H, Lee MC, et al. Degradation behavior of 3D porous polydioxanone-b-polycaprolactone scaffolds fabricated using the melt-molding particulate-leaching method. J Biomater Sci Polym Ed. 2010;22:225–37.

    Article  Google Scholar 

  31. 31.

    Yoshimoto H, Shin YM, Terai H, Vacanti JP. A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering. Biomaterials. 2003;24(12):2077–82.

    Article  Google Scholar 

  32. 32.

    Zhang H, Chen Z. Fabrication and characterization of electrospun PLGA/MWNTs/hydroxyapatite biocomposite scaffolds for bone tissue engineering. J Bioact Compat Polym. 2010;25(3):241–59.

    Article  Google Scholar 

  33. 33.

    Bhardwaj N, Kundu SC. Electrospinning: a fascinating fiber fabrication technique. Biotechnol Adv. 2010;28(3):325–47.

    Article  Google Scholar 

  34. 34.

    Yang S, Leong KF, Du Z, Chua CK. The design of scaffolds for use in tissue engineering. Part II. Rapid prototyping techniques. Tissue Eng. 2002;8(1):1–11.

  35. 35.

    Jones AC, Milthorpe B, Averdunk H, Limaye A, Senden TJ, Sakellariou A, et al. Analysis of 3D bone ingrowth into polymer scaffolds via micro-computed tomography imaging. Biomaterials. 2004;25(20):4947–54.

    Article  Google Scholar 

  36. 36.

    Lee CH, Cook JL, Mendelson A, Moioli EK, Yao H, Mao JJ. Regeneration of the articular surface of the rabbit synovial joint by cell homing: a proof of concept study. Lancet. 2010;376:440–8.

    Article  Google Scholar 

  37. 37.

    Hollister SJ, Lin CY, Saito E, Schek RD, Taboas JM, Williams JM, et al. Engineering craniofacial scaffolds. Orthod Craniofac Res. 2005;8(3):162–73.

    Article  Google Scholar 

  38. 38.

    Park SA, Lee SH, Kim WD. Fabrication of porous polycaprolactone/hydroxyapatite (PCL/HA) blend scaffolds using a 3D plotting system for bone tissue engineering. Bioprocess Biosyst Eng. 2011;34(4):505–13.

    Article  Google Scholar 

  39. 39.

    Taboas JM, Maddox RD, Krebsbach PH, Hollister SJ. Indirect solid free form fabrication of local and global porous, biomimetic and composite 3D polymer-ceramic scaffolds. Biomaterials. 2003;24(1):181–94.

    Article  Google Scholar 

  40. 40.

    Pielichowska K. Polyoxymethylene-homopolymer/hydroxyapatite nanocomposites for biomedical applications. J Appl Polym Sci. 2012;123(4):2234–43.

    Article  Google Scholar 

  41. 41.

    Zhang R, Ma PX. Poly(alpha-hydroxyl acids)/hydroxyapatite porous composites for bone-tissue engineering. I. Preparation and morphology. J Biomed Mater Res. 1999;44:446–55.

    Article  Google Scholar 

  42. 42.

    Nam YS, Park TG. Biodegradable polymeric microcellular foams by modified thermally induced phase separation method. Biomaterials. 1999;20:1783–90.

    Article  Google Scholar 

  43. 43.

    Nam YS, Park TG. Porous biodegradable polymeric scaffolds prepared by thermally induced phase separation. J Biomed Mater Res. 1999;47(1):8–17.

    Article  Google Scholar 

  44. 44.

    Martínez-Pérez CA, Olivas-Armendariz I, Castro-Carmona JS, García-Casillas PE. 2011, Scaffolds for Tissue Engineering Via Thermally Induced Phase Separation. Advances in regenerative medicine, Wislet-Gendebien S (Ed.), ISBN: 978-953-307-732-1, 2011; InTech. doi:10.5772/25476.

  45. 45.

    Smith LA, Liu X, Hu J, Ma PX. The influence of three-dimensional nanofibrous scaffolds on the osteogenic differentiation of embryonic stem cells. Biomaterials. 2009;30:2516–22.

    Article  Google Scholar 

  46. 46.

    Li S, Chen X, Li M. Effect of some factors on fabrication of poly(L-lactic acid) microporous foams by thermally induced phase separation using N,N-dimethylacetamide as solvent. Prep Biochem Biotechnol. 2011;41:53–72.

    Article  Google Scholar 

  47. 47.

    Muschler GE, Nakamoto C, Griffith LG. Engineering principles of clinical cell-based tissue engineering. J Bone Joint Surg Am. 2004;86A:1541–58.

    Google Scholar 

  48. 48.

    Kim J, Magno MH, Waters H, Doll BA, McBride S, Alvarez P, et al. Bone regeneration in a rabbit critical-sized calvarial model using tyrosine-derived polycarbonate scaffolds. Tissue Eng Part A. 2012;18:1132–9.

    Article  Google Scholar 

  49. 49.

    Liang Y, Zheng X, Zhai W, Sun T. 3D PLLA/nano-hydroxyapatite scaffolds with hierarchical porous structure fabricated by low-temperature deposition manufacturing. J Wuhan Univ Technol Mater Sci. 2012;27(2):265–9.

    Article  Google Scholar 

  50. 50.

    Flemming RG, Murphy CJ, Abrams GA, Goodman SL, Nealey PF. Effects of synthetic micro- and nano-structured surfaces on cell behavior. Biomaterials. 1999;20(6):573–88.

    Article  Google Scholar 

  51. 51.

    Wu YC, Shaw SY, Lin HR, Lee TM, Yang CY. Bone tissue engineering evaluation based on rat calvaria stromal cells cultured on modified PLGA scaffolds. Biomaterials. 2006;27:896–904.

    Article  Google Scholar 

  52. 52.

    Liu L, Xiong Z, Yan Y, Zhang R, Wang X, Jin L. Multinozzle low-temperature deposition system for construction of gradient tissue engineering scaffolds. J Biomed Mater Res B Appl Biomater. 2009;88(1):254–63.

    Article  Google Scholar 

  53. 53.

    Saito E, Liao EE, Hu WW, Krebsbach PH, Hollister SJ. Effects of designed PLLA and 50:50 PLGA scaffold architectures on bone formation in vivo. J Tissue Eng Regen Med. 2013;7(2):99–111.

    Article  Google Scholar 

  54. 54.

    Hollister SJ. Porous scaffold design for tissue engineering. Nat Mater. 2005;4(7):518–24.

    Article  Google Scholar 

  55. 55.

    Akbarzadeh R, Yousefi AM. Effects of processing parameters in thermally induced phase separation technique on porous architecture of scaffolds for bone tissue engineering. J Biomed Mater Res B-Appl Biomater; 2014. doi:10.1002/jbm.b.33101.

  56. 56.

    Yousefi AM, Gauvin C, Sun L, DiRaddo RW, Fernandes J. Design and fabrication of 3d-plotted polymeric scaffolds in functional tissue engineering. Polym Eng Sci. 2007;47:608–18.

    Article  Google Scholar 

  57. 57.

    St-Pierre J-P, Gauthier M, Lefebvre L-P, Tabrizian M. Three-dimensional growth of differentiating MC3T3-E1 pre-osteoblasts on porous titanium scaffolds. Biomaterials. 2005;26(35):7319–28.

    Article  Google Scholar 

  58. 58.

    Landers R, Hübner U, Schmelzeisen R, Mülhaupt R. Rapid prototyping of scaffolds derived from thermoreversible hydrogels and tailored for applications in tissue engineering. Biomaterials. 2002;23(23):4437–47.

    Article  Google Scholar 

  59. 59.

    Moroni L, Schotel R, Sohier J, de Wijn JR, van Blitterswijk CA. Polymer hollow fiber three-dimensional matrices with controllable cavity and shell thickness. Biomaterials. 2006;27(35):5918–26.

    Article  Google Scholar 

  60. 60.

    Schek RM, Wilke EN, Hollister SJ, Krebsbach PH. Combined use of designed scaffolds and adenoviral gene therapy for skeletal tissue engineering. Biomaterials. 2006;27:1160–6.

    Article  Google Scholar 

  61. 61.

    Li JP, Habibovic P, van den Doel M, et al. Bone ingrowth in porous titanium implants produced by 3D fiber deposition. Biomaterials. 2007;28:2810–20.

    Article  Google Scholar 

  62. 62.

    Karageorgiou V, Kaplan D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials. 2005;26(27):5474–91.

  63. 63.

    Singhvi R, Kumar A, Lopez GP, Stephanopoulos GN, Wang DI, Whitesides GM, Ingber DE. Engineering cell shape and function. Science. 1994;264(5159):696–8.

    Article  Google Scholar 

  64. 64.

    Zeltinger J, Sherwood JK, Graham DA, Müeller R, Griffith LG. Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition. Tissue Eng. 2001;7(5):557–72.

    Article  Google Scholar 

  65. 65.

    Grayson WL, Bhumiratana S, Cannizzaro C, Vunjak-Novakovic G. Bioreactor cultivation of functional bone grafts. In: Mesenchymal stem cell assays and applications, methods in molecular biology; Mohan C. Vemuri et al (eds.); 2011, vol 698, p. 231–241.

  66. 66.

    Budyanto L, Ooi CP, Goh YQ. Fabrication and characterization of porous poly (L-lactide) (PLLA) scaffolds using liquid–liquid phase separation. In: 4th Kuala Lumpur international conference on biomedical engineering 2008; 2008, vol 21, p. 322–325.

  67. 67.

    Sun L, Parker ST, Syoji D, Wang X, Lewis JA, Kaplan DL. Direct-write assembly of 3D silk/hydroxyapatite scaffolds for bone co-cultures. Adv Healthc Mater. 2012;1:729–35.

    Article  Google Scholar 

  68. 68.

    Joly P, Duda GN, Schöne M, Welzel PB, Freudenberg U, Werner C, Petersen A. Geometry-driven cell organization determines tissue growths in scaffold pores: consequences for fibronectin organization. PLoS ONE. 2013;8:e73545.

    Article  Google Scholar 

  69. 69.

    Rumpler M, Woesz A, Dunlop JW, van Dongen JT, Fratzl P. The effect of geometry on three-dimensional tissue growth. J R Soc Interface. 2008;5:1173–80.

    Article  Google Scholar 

  70. 70.

    Billiet T, Vandenhaute M, Schelfhout J, Van Vlierberghe S, Dubruel P. A review of trends and limitations in hydrogel-rapid prototyping for tissue engineering. Biomaterials. 2012;33(26):6020–41.

    Article  Google Scholar 

  71. 71.

    Stevens MM, George JH. Exploring and engineering the cell surface interface. Science. 2005;310(5751):1135–8.

    Article  Google Scholar 

  72. 72.

    Ng R, Zang R, Yang KK, Liu N, Yang S-T. Three-dimensional fibrous scaffolds with microstructures and nanotextures for tissue engineering. RSC Adv. 2014;2:10110–24.

    Article  Google Scholar 

  73. 73.

    Mikael PE, Amini AR, Basu J, Josefina Arellano-Jimenez M, Laurencin CT, Sanders MM, Barry Carter C, Nukavarapu SP. Functionalized carbon nanotube reinforced scaffolds for bone regenerative engineering: fabrication, in vitro and in vivo evaluation. Biomed Mater. 2014;9(3):035001.

    Article  Google Scholar 

  74. 74.

    Kim Y, Kim G. Collagen/alginate scaffolds comprising core (PCL)–shell (collagen/alginate) struts for hard tissue regeneration: fabrication, characterisation, and cellular activities. J. Mater Chem B. 2013;1:3185–94.

    Article  Google Scholar 

  75. 75.

    He C, Zhang F, Cao L, Feng W, Qiu K, Zhang Y, Wang J. Rapid mineralization of porous gelatin scaffolds by electrodeposition for bone tissue engineering. J Mater Chem. 2012;22:2111–9.

    Article  Google Scholar 

  76. 76.

    Feng P, Niu M, Gao C, Peng S, Shuai C. A novel two-step sintering for nano-hydroxyapatite scaffolds for bone tissue engineering. Sci Rep. 2014;4(5599):1–10.

    Google Scholar 

  77. 77.

    El-Ayoubi R, Eliopoulos N, DiRaddo R, Galipeau J, Yousefi AM. Design and fabrication of 3D porous scaffolds to promote cell-based gene therapy. Tissue Eng. 2008;14:1037–48.

    Article  Google Scholar 

  78. 78.

    Coutu DL, Cuerquis J, El-Ayoubi R, Forner KA, Roy R, Francois M, Hyatt A, Griffith M, Yousefi AM, Blostein MD, Galipeau J. Engineering optimized 3D biocompatible porous scaffolds for mesenchymal stromal cell-based gene therapy of hemophilia B. Biomaterials. 2011;32:295–305.

    Article  Google Scholar 

  79. 79.

    Liao SS, Cui FZ. In vitro and in vivo degradation of mineralized collagen-based composite scaffold: nanohydroxyapatite/collagen/poly(L-lactide). Tissue Eng. 2004;10:73–80.

    Article  Google Scholar 

  80. 80.

    Kim K, Dean D, Lu A, Mikos AG, Fisher JP. Early osteogenic signal expression of rat bone marrow stromal cells is influenced by both hydroxyapatite nanoparticle content and initial cell seeding density in biodegradable nanocomposite scaffolds. Acta Biomater. 2011;7:1249–64.

    Article  Google Scholar 

Download references


This work was supported by the Ohio Board of Regents, the Ohio Third Frontier Program grant entitled: “Ohio Research Scholars in Layered Sensing”, IDCAST, Miami University’s Office for the Advancement of Research, the College of Engineering and Computing, and the Department of Biology. The authors wish to thank Dr. Gilbert Pacey, Dr. James Oris, Dr. Marek Dollár and Dr. Shashi Lalvani for contributing funds to this study. The authors also acknowledge the technical assistance of Dr. Richard Edelmann, Matt Duley, Doug Hart, Carlie Focke, Bill Lack, Barry Landrum and Jayson Alexander, and the administrative assistance of Laurie Edwards.

Author information



Corresponding author

Correspondence to Azizeh-Mitra Yousefi.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Akbarzadeh, R., Minton, J.A., Janney, C.S. et al. Hierarchical polymeric scaffolds support the growth of MC3T3-E1 cells. J Mater Sci: Mater Med 26, 116 (2015). https://doi.org/10.1007/s10856-015-5453-z

Download citation


  • Bone Tissue Engineering
  • Induce Phase Separation
  • Solid Freeform Fabrication
  • Scaffold Fabrication
  • Bimodal Pore Size Distribution