Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A 3D bioprinting system to produce human-scale tissue constructs with structural integrity

Abstract

A challenge for tissue engineering is producing three-dimensional (3D), vascularized cellular constructs of clinically relevant size, shape and structural integrity. We present an integrated tissue–organ printer (ITOP) that can fabricate stable, human-scale tissue constructs of any shape. Mechanical stability is achieved by printing cell-laden hydrogels together with biodegradable polymers in integrated patterns and anchored on sacrificial hydrogels. The correct shape of the tissue construct is achieved by representing clinical imaging data as a computer model of the anatomical defect and translating the model into a program that controls the motions of the printer nozzles, which dispense cells to discrete locations. The incorporation of microchannels into the tissue constructs facilitates diffusion of nutrients to printed cells, thereby overcoming the diffusion limit of 100–200 μm for cell survival in engineered tissues. We demonstrate capabilities of the ITOP by fabricating mandible and calvarial bone, cartilage and skeletal muscle. Future development of the ITOP is being directed to the production of tissues for human applications and to the building of more complex tissues and solid organs.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: ITOP system.
Figure 2: 2D/3D patterning using the ITOP system.
Figure 3: Mandible bone reconstruction.
Figure 4: Calvarial bone reconstruction.
Figure 5: Ear cartilage reconstruction.
Figure 6: Skeletal muscle reconstruction.

References

  1. Mikos, A.G. et al. Engineering complex tissues. Tissue Eng. 12, 3307–3339 (2006).

    CAS  Article  Google Scholar 

  2. Atala, A., Kasper, F.K. & Mikos, A.G. Engineering complex tissues. Sci. Transl. Med. 4, 160rv12 (2012).

    Article  Google Scholar 

  3. Murphy, S.V. & Atala, A. 3D bioprinting of tissues and organs. Nat. Biotechnol. 32, 773–785 (2014).

    CAS  Article  Google Scholar 

  4. Mironov, V. et al. Organ printing: tissue spheroids as building blocks. Biomaterials 30, 2164–2174 (2009).

    CAS  Article  Google Scholar 

  5. Jones, N. Science in three dimensions: the print revolution. Nature 487, 22–23 (2012).

    CAS  Article  Google Scholar 

  6. Ferris, C.J., Gilmore, K.G., Wallace, G.G. & In het Panhuis, M. Biofabrication: an overview of the approaches used for printing of living cells. Appl. Microbiol. Biotechnol. 97, 4243–4258 (2013).

    CAS  Article  Google Scholar 

  7. Xu, T. et al. Complex heterogeneous tissue constructs containing multiple cell types prepared by inkjet printing technology. Biomaterials 34, 130–139 (2013).

    Article  Google Scholar 

  8. Durmus, N.G., Tasoglu, S. & Demirci, U. Bioprinting: Functional droplet networks. Nat. Mater. 12, 478–479 (2013).

    CAS  Article  Google Scholar 

  9. Cooper, G.M. et al. Inkjet-based biopatterning of bone morphogenetic protein-2 to spatially control calvarial bone formation. Tissue Eng. Part A 16, 1749–1759 (2010).

    CAS  Article  Google Scholar 

  10. Costa, K.D., Lee, E.J. & Holmes, J.W. Creating alignment and anisotropy in engineered heart tissue: role of boundary conditions in a model three-dimensional culture system. Tissue Eng. 9, 567–577 (2003).

    Article  Google Scholar 

  11. Cui, X., Breitenkamp, K., Finn, M.G., Lotz, M. & D'Lima, D.D. Direct human cartilage repair using three-dimensional bioprinting technology. Tissue Eng. Part A 18, 1304–1312 (2012).

    CAS  Article  Google Scholar 

  12. da Graca, B. & Filardo, G. Vascular bioprinting. Am. J. Cardiol. 107, 141–142 (2011).

    Article  Google Scholar 

  13. Duan, B., Hockaday, L.A., Kang, K.H. & Butcher, J.T. III. Bioprinting of heterogeneous aortic valve conduits with alginate/gelatin hydrogels. J. Biomed. Mater. Res. A 101, 1255–1264 (2013).

    Article  Google Scholar 

  14. Ilkhanizadeh, S., Teixeira, A.I. & Hermanson, O. Inkjet printing of macromolecules on hydrogels to steer neural stem cell differentiation. Biomaterials 28, 3936–3943 (2007).

    CAS  Article  Google Scholar 

  15. Norotte, C., Marga, F.S., Niklason, L.E. & Forgacs, G. Scaffold-free vascular tissue engineering using bioprinting. Biomaterials 30, 5910–5917 (2009).

    CAS  Article  Google Scholar 

  16. Skardal, A., Zhang, J. & Prestwich, G.D. Bioprinting vessel-like constructs using hyaluronan hydrogels crosslinked with tetrahedral polyethylene glycol tetracrylates. Biomaterials 31, 6173–6181 (2010).

    CAS  Article  Google Scholar 

  17. Xu, T. et al. Hybrid printing of mechanically and biologically improved constructs for cartilage tissue engineering applications. Biofabrication 5, 015001 (2013).

    Article  Google Scholar 

  18. Derby, B. Printing and prototyping of tissues and scaffolds. Science 338, 921–926 (2012).

    CAS  Google Scholar 

  19. Mironov, V., Boland, T., Trusk, T., Forgacs, G. & Markwald, R.R. Organ printing: computer-aided jet-based 3D tissue engineering. Trends Biotechnol. 21, 157–161 (2003).

    CAS  Article  Google Scholar 

  20. Cui, X. & Boland, T. Human microvasculature fabrication using thermal inkjet printing technology. Biomaterials 30, 6221–6227 (2009).

    CAS  Article  Google Scholar 

  21. Khalil, S. & Sun, W. Bioprinting endothelial cells with alginate for 3D tissue constructs. J. Biomech. Eng. 131, 111002 (2009).

    Article  Google Scholar 

  22. Lee, W. et al. Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication. Biomaterials 30, 1587–1595 (2009).

    CAS  Article  Google Scholar 

  23. Xu, T., Baicu, C., Aho, M., Zile, M. & Boland, T. Fabrication and characterization of bio-engineered cardiac pseudo tissues. Biofabrication 1, 035001 (2009).

    Article  Google Scholar 

  24. Chang, C.C., Boland, E.D., Williams, S.K. & Hoying, J.B. Direct-write bioprinting three-dimensional biohybrid systems for future regenerative therapies. J. Biomed. Mater. Res. B Appl. Biomater. 98, 160–170 (2011).

    Article  Google Scholar 

  25. Fedorovich, N.E., De Wijn, J.R., Verbout, A.J., Alblas, J. & Dhert, W.J. Three-dimensional fiber deposition of cell-laden, viable, patterned constructs for bone tissue printing. Tissue Eng. Part A 14, 127–133 (2008).

    CAS  Article  Google Scholar 

  26. Jakab, K., Neagu, A., Mironov, V., Markwald, R.R. & Forgacs, G. Engineering biological structures of prescribed shape using self-assembling multicellular systems. Proc. Natl. Acad. Sci. USA 101, 2864–2869 (2004).

    CAS  Article  Google Scholar 

  27. Landers, R., Hübner, U., Schmelzeisen, R. & Mülhaupt, R. Rapid prototyping of scaffolds derived from thermoreversible hydrogels and tailored for applications in tissue engineering. Biomaterials 23, 4437–4447 (2002).

    CAS  Article  Google Scholar 

  28. Hoffman, A.S. Hydrogels for biomedical applications. Adv. Drug Deliv. Rev. 54, 3–12 (2002).

    CAS  Article  Google Scholar 

  29. Nicodemus, G.D. & Bryant, S.J. Cell encapsulation in biodegradable hydrogels for tissue engineering applications. Tissue Eng. Part B Rev. 14, 149–165 (2008).

    CAS  Article  Google Scholar 

  30. Wang, X., Yan, Y. & Zhang, R. Recent trends and challenges in complex organ manufacturing. Tissue Eng. Part B Rev. 16, 189–197 (2010).

    Article  Google Scholar 

  31. Guillotin, B. & Guillemot, F. Cell patterning technologies for organotypic tissue fabrication. Trends Biotechnol. 29, 183–190 (2011).

    CAS  Article  Google Scholar 

  32. De Coppi, P. et al. Isolation of amniotic stem cell lines with potential for therapy. Nat. Biotechnol. 25, 100–106 (2007).

    CAS  Article  Google Scholar 

  33. Kim, J. et al. In vitro osteogenic differentiation of human amniotic fluid-derived stem cells on a poly(lactide-co-glycolide) (PLGA)-bladder submucosa matrix (BSM) composite scaffold for bone tissue engineering. Biomed. Mater. 8, 014107 (2013).

    Article  Google Scholar 

  34. Bian, W., Juhas, M., Pfeiler, T.W. & Bursac, N. Local tissue geometry determines contractile force generation of engineered muscle networks. Tissue Eng. Part A 18, 957–967 (2012).

    CAS  Article  Google Scholar 

  35. Binder, K.W. et al. In situ bioprinting of the skin for burns. J. Am. Coll. Surg. 211, 7 (2010).

    Article  Google Scholar 

  36. Marga, F. et al. Toward engineering functional organ modules by additive manufacturing. Biofabrication 4, 022001 (2012).

    Article  Google Scholar 

  37. Schuurman, W. et al. Bioprinting of hybrid tissue constructs with tailorable mechanical properties. Biofabrication 3, 021001 (2011).

    CAS  Article  Google Scholar 

  38. Shim, J.-H., Lee, J.-S., Kim, J.Y. & Cho, D.-W. Bioprinting of a mechanically enhanced three-dimensional dual cell-laden construct for osteochondral tissue engineering using a multi-head tissue/organ building system. J. Micromech. Microeng. 22, 085014 (2012).

    Article  Google Scholar 

  39. Serrano, M.C. et al. In vitro biocompatibility assessment of poly(epsilon-caprolactone) films using L929 mouse fibroblasts. Biomaterials 25, 5603–5611 (2004).

    CAS  Article  Google Scholar 

  40. Sun, H., Mei, L., Song, C., Cui, X. & Wang, P. The in vivo degradation, absorption and excretion of PCL-based implant. Biomaterials 27, 1735–1740 (2006).

    CAS  Article  Google Scholar 

  41. Ignatius, A.A. & Claes, L.E. In vitro biocompatibility of bioresorbable polymers: poly(L, DL-lactide) and poly(L-lactide-co-glycolide). Biomaterials 17, 831–839 (1996).

    CAS  Article  Google Scholar 

  42. Zignani, M. et al. Improved biocompatibility of a viscous bioerodible poly(ortho ester) by controlling the environmental pH during degradation. Biomaterials 21, 1773–1778 (2000).

    CAS  Article  Google Scholar 

  43. Lippens, E. et al. Cell survival and proliferation after encapsulation in a chemically modified Pluronic(R) F127 hydrogel. J. Biomater. Appl. 27, 828–839 (2013).

    Article  Google Scholar 

  44. Amini, A.R., Laurencin, C.T. & Nukavarapu, S.P. Bone tissue engineering: recent advances and challenges. Crit. Rev. Biomed. Eng. 40, 363–408 (2012).

    Article  Google Scholar 

  45. Bichara, D.A. et al. The tissue-engineered auricle: past, present, and future. Tissue Eng. Part B Rev. 18, 51–61 (2012).

    CAS  Article  Google Scholar 

  46. Ostrovidov, S. et al. Skeletal muscle tissue engineering: methods to form skeletal myotubes and their applications. Tissue Eng. Part B Rev. 20, 403–436 (2014).

    Article  Google Scholar 

  47. Ker, E.D. et al. Bioprinting of growth factors onto aligned sub-micron fibrous scaffolds for simultaneous control of cell differentiation and alignment. Biomaterials 32, 8097–8107 (2011).

    CAS  Article  Google Scholar 

  48. Phillippi, J.A. et al. Microenvironments engineered by inkjet bioprinting spatially direct adult stem cells toward muscle- and bone-like subpopulations. Stem Cells 26, 127–134 (2008).

    CAS  Article  Google Scholar 

  49. Choi, J.S., Lee, S.J., Christ, G.J., Atala, A. & Yoo, J.J. The influence of electrospun aligned poly(epsilon-caprolactone)/collagen nanofiber meshes on the formation of self-aligned skeletal muscle myotubes. Biomaterials 29, 2899–2906 (2008).

    CAS  Article  Google Scholar 

  50. Wakelam, M.J. The fusion of myoblasts. Biochem. J. 228, 1–12 (1985).

    CAS  Article  Google Scholar 

  51. Jain, R.K., Au, P., Tam, J., Duda, D.G. & Fukumura, D. Engineering vascularized tissue. Nat. Biotechnol. 23, 821–823 (2005).

    CAS  Article  Google Scholar 

  52. Boland, T., Xu, T., Damon, B. & Cui, X. Application of inkjet printing to tissue engineering. Biotechnol. J. 1, 910–917 (2006).

    CAS  Article  Google Scholar 

  53. Lee, Y.-B. et al. Bio-printing of collagen and VEGF-releasing fibrin gel scaffolds for neural stem cell culture. Exp. Neurol. 223, 645–652 (2010).

    CAS  Article  Google Scholar 

  54. Cabodi, M. et al. A microfluidic biomaterial. J. Am. Chem. Soc. 127, 13788–13789 (2005).

    CAS  Article  Google Scholar 

  55. Ling, Y. et al. A cell-laden microfluidic hydrogel. Lab Chip 7, 756–762 (2007).

    CAS  Article  Google Scholar 

  56. Stachowiak, A.N., Bershteyn, A., Tzatzalos, E. & Irvine, D.J. Bioactive hydrogels with an ordered cellular structure combine interconnected macroporosity and robust mechanical properties. Adv. Mater. 17, 399–403 (2005).

    CAS  Article  Google Scholar 

  57. Atala, A., Bauer, S.B., Soker, S., Yoo, J.J. & Retik, A.B. Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet 367, 1241–1246 (2006).

    Article  Google Scholar 

  58. Raya-Rivera, A. et al. Tissue-engineered autologous urethras for patients who need reconstruction: an observational study. Lancet 377, 1175–1182 (2011).

    Article  Google Scholar 

  59. Raya-Rivera, A.M. et al. Tissue-engineered autologous vaginal organs in patients: a pilot cohort study. Lancet 384, 329–336 (2014).

    Article  Google Scholar 

  60. Liao, Y.S. & Chiu, Y.Y. A new slicing procedure for rapid prototyping systems. Int. J. Adv. Manuf. Technol. 18, 579–585 (2001).

    Article  Google Scholar 

  61. Park, S.C. & Choi, B.K. Tool-path planning for direction-parallel area milling. Comput. Aided Des. 32, 17–25 (2000).

    Article  Google Scholar 

  62. Lee, S.J., Broda, C., Atala, A. & Yoo, J.J. Engineered cartilage covered ear implants for auricular cartilage reconstruction. Biomacromolecules 12, 306–313 (2011).

    CAS  Article  Google Scholar 

  63. Roy, R. et al. Analysis of bending behavior of native and engineered auricular and costal cartilage. J. Biomed. Mater. Res. A 68, 597–602 (2004).

    Article  Google Scholar 

  64. Ko, I.K. et al. The effect of in vitro formation of acetylcholine receptor (AChR) clusters in engineered muscle fibers on subsequent innervation of constructs in vivo. Biomaterials 34, 3246–3255 (2013).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported, in part, by grants from the Armed Forces Institute of Regenerative Medicine (W81XWH-08-2-0032), the Telemedicine and Advanced Technology Research Center at the US Army Medical Research and Material Command (W81XWH-07-1-0718) and the Defense Threat Reduction Agency (N66001-13-C-2027). We would like to thank D.M. Eckman for editorial comments on this manuscript, and H.S. Kim and G.V. Kulkarni for technical assistance.

Author information

Authors and Affiliations

Authors

Contributions

H.-W.K., S.J.L., J.J.Y. and A.A. developed the concept of the integration tissue and organ printing (ITOP) system and designed all experiments. H.-W.K. performed in vitro experiments and composite hydrogel development, analyzed data and wrote the manuscript. C.K. performed in vivo experiments of the printed cartilage and bone constructs and analyzed data. I.K.K. performed in vivo experiments of the printed skeletal muscle construct and analyzed data. S.J.L., J.J.Y. and A.A. analyzed data and wrote the manuscript. A.A. provided direction and supervised the project. S.J.L., J.J.Y. and A.A. edited the manuscript.

Corresponding author

Correspondence to Anthony Atala.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Integrated tissue and organ printing (ITOP) system

The ITOP system consists of three major units; 1) 3-axis stage/controller, 2) dispensing module including multi-cartridge and pneumatic pressure controller, and 3) a closed acrylic chamber with temperature controller and humidifier.

Supplementary Figure 2 Optimization of composite hydrogel system for 3D bioprinting

Dispensing rates (dispensed volume per unit time) with different concentrations of (a) gelatin (n=30) and (b) HA (n=30). The dispensing rate could by increased or decreased by decreasing or increasing the concentration of gelatin, respectively. Irregularities in dispensing rates to varying gelatin concentrations were directly related to the coefficient of variation (COV); where a COV of greater than 30% was associated with uneven dispensing of the hydrogel. However, introduction of HA to gelatin significantly improved uniformity of dispensing rate with low COV values. *Coefficient of variation (COV) was calculated by dividing average with standard deviation.

Supplementary Figure 3 Optimization of PCL and TCP ratio

(a) Compression modulus and (b) water uptake ability of the printed PCL/TCP constructs with different ratios (*P<0.05, n = 3). (c) Quantification of calcium content showing the osteogenic differentiation of hAFSCs seeded on the PCL/TCP constructs with different ratios (no significance, n = 3).

Supplementary Figure 4 Vascularization of 3D printed ear

The engineered cartilage tissues showed vascularization of the implanted constructs in the periphery region at 1 and 2 months after implantation, as confirmed by vWF immunostaining, but similar to normal cartilage tissue, no vascularization was noted in the central region.

Supplementary Figure 5 Immunofluorescent images of 3D printed muscle organization

3D printed muscle structures were cultured in the growth medium up to 3 days, and then induced myotube formation in the differentiation medium for 7 days. Live/dead staining of the printed muscle organization at (a) 1 day and (b) 3 days. (c) Immunofluorescent staining for MHC of the printed muscle organization at 7 days after cell differentiation. The myotubes formed in the printed constructs showed unidirectionally organized myotubes that are consistently aligned along the longitudinal axis of the printed organization.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 533 kb)

Supplementary Table 1

Supplementary Table 1 (PDF 68 kb)

Supplementary Source Code (PDF 9448 kb)

Supplementary Movie 1

Supplementary Movie 1 (MOV 16386 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kang, HW., Lee, S., Ko, I. et al. A 3D bioprinting system to produce human-scale tissue constructs with structural integrity. Nat Biotechnol 34, 312–319 (2016). https://doi.org/10.1038/nbt.3413

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.3413

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing