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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.

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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.

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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.

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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.

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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.

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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

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