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4-Axis printing microfibrous tubular scaffold and tracheal cartilage application

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  • ReceivedJun 23, 2019
  • AcceptedJul 26, 2019
  • PublishedSep 6, 2019

Abstract

Long-segment defects remain a major problem in clinical treatment of tubular tissue reconstruction. The design of tubular scaffold with desired structure and functional properties suitable for tubular tissue regeneration remains a great challenge in regenerative medicine. Here, we present a reliable method to rapidly fabricate tissue-engineered tubular scaffold with hierarchical structure via 4-axis printing system. The fabrication process can be adapted to various biomaterials including hydrogels, thermoplastic materials and thermosetting materials. Using polycaprolactone (PCL) as an example, we successfully fabricated the scaffolds with tunable tubular architecture, controllable mesh structure, radial elasticity, good flexibility, and luminal patency. As a preliminary demonstration of the applications of this technology, we prepared a hybrid tubular scaffold via the combination of the 4-axis printed elastic poly(glycerol sebacate) (PGS) bio-spring and electrospun gelatin nanofibers. The scaffolds seeded with chondrocytes formed tubular mature cartilage-like tissue both via in vitro culture and subcutaneous implantation in the nude mouse, which showed great potential for tracheal cartilage reconstruction.


Funded by

the National Key Research and Development Program of China(2018YFB1105602,2017YFC1103900)

the National Natural Science Foundation of China(21574019,81320108010,81571823,81871502)

the Natural Science Foundation of Shanghai(18ZR1401900)

the Fundamental Research Funds for the Central Universities

DHU Distinguished Young Professor Program(LZA2019001)

the Science and Technology Commission of Shanghai(17DZ2260100,15DZ1941600)

the Program for Shanghai Outstanding Medical Academic Leader

and the Program of Shanghai Technology Research Leader.


Acknowledgment

This work was supported by the National Key Research and Development Program of China (2018YFB1105602 and 2017YFC1103900), the National Natural Science Foundation of China (21574019, 81320108010, 81571823 and 81871502), the Natural Science Foundation of Shanghai (18ZR1401900), the Fundamental Research Funds for the Central Universities, DHU Distinguished Young Professor Program (LZA2019001), the Science and Technology Commission of Shanghai (17DZ2260100 and 15DZ1941600), the Program for Shanghai Outstanding Medical Academic Leader, and the Program of Shanghai Technology Research Leader.


Interest statement

The authors declare no conflict of interest.


Contributions statement

Lei D designed and performed all experiments, analyzed the data and prepared the manuscript. Xu Y performed the chondrocytes culture and trachea reconstruction. Luo B and Shen A contributed to 3D printing. Guo Y performed SEM characterization and statistic analysis. Liu Z and Wang S performed PGS polymer synthesis. Wang D and Yang H performed the manufacture of receivers. Xuan H contributed to electrospinning. Zhang Y contributed to mechanical testing. He C and Qing FL contributed to the discussion and analysis of the experimental result. Zhou G discussed the concept and contributed to the writing of the manuscript. You Z supervised the whole work and wrote the manuscript.


Author information

Dong Lei is a PhD student at the State Key Laboratory for Modification of Chemical Fibers and Polymer Materials at Donghua University. From 2013 to now, he has been conducting his master and doctoral research at Donghua University. His current research involves biomaterials, 3D printing and tissue regeneration.


Yong Xu received his MCs degree from the Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine in 2018. Now he is continuing to study for a PhD degree in the Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine. His research interests focus on cartilage regenerative biomaterials and functional tracheal reconstruction.


Guangdong Zhou is a professor of Medical College of Shanghai Jiao Tong University, Executive Vice-Director of National Center for Tissue Engineering Research, Former Secretary-General and Executive Director of Tissue Engineering and Regenerative Medicine Branch of Chinese Society of Biomedical Engineering. His research interests focus on the functional cartilage regeneration and clinical application transformation, construction of cartilage in vitro and precise regulation of its three-dimensional morphology (auricle, trachea, etc.), and repair of various cartilage defects in large animals.


Zhengwei You is a professor and the chair of Department of Composite Materials at Donghua University. He received his BSc degree from Shanghai Jiao Tong University and PhD degree from Shanghai Institute of Organic Chemistry. He conducted his postdoctoral research at Georgia Institute of Technology and University of Pittsburgh. Prior to joining Donghua University, he was an innovation manager in Bayer Material Science. His current research involves smart polymers, biomaterials, 3D printing, and stretchable electronics.


Supplement

Supplementary information

Supporting data are available in the online version of the paper.


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  • Figure 1

    Schematic illustration of the 4-axis printing system to prepare tubular scaffolds from various materials.

  • Figure 2

    Printed PCL tubular scaffolds with diverse geometries. Tubular scaffolds with different diameters (a, b); SEMs of circular tubular scaffold in top view (c) and section view (d); triangular prism receiver (e) and corresponding tubular scaffold (f); SEMs of triangular tubular scaffold in top view (g) and section view (h); hexagonal prism shaped receiver (i) and corresponding tubular scaffold (j); SEMs of hexagonal tubular scaffold in top view (k) and section view (l).

  • Figure 3

    Microstructures and mechanical properties of PCL tubular scaffolds. Schematic models of printing process (a) and interweaved fibrous-network structure (b); photographs of tubular scaffolds with various microstructures (c); SEM images of crisscrossed fibers with different densities (d–g); cyclic compressive (h) and tensile (i) tests for 10 cycles of 3D printed PCL tubular scaffolds.

  • Figure 4

    Predictable and reproducible printing with controllable structural parameters. The relation curves of screw pitch (a), fiber spacing (b), diameters (c) and fabric angles (d) with rotational speed.

  • Figure 5

    Versatility of the 4-axis printing strategy to fabricate hydrogel scaffold and porous thermoset bio-spring. Photographs of alginate/polyacrylamide tubular hydrogel scaffold ((a) top view; (b) visualized with red dye; (c) hydrogel scaffold filled with water); printing and crosslinking of PGS bio-spring (d); flexible and elastic PGS bio-spring (e); SEMs of PGS bio-spring with hierarchical structure in section view ((f) tube architecture; (g) woven fibers; (h) micropore structure).

  • Figure 6

    Upgradeable bio-spring for hybrid tubular tissue engineered scaffold. Schematic of hybrid scaffold with exterior nanofibrous structure (a) and interior PGS biospring (b); optical images of hybrid PGS/gelatin tubular scaffold (c); SEM images of scaffold with tubular structure (d), bilayer structure (e), and nanofibrous external surface (f).

  • Figure 7

    Biocompatibility of the hybrid scaffold. Engineered tubular cartilage constructs (a, b); no obvious difference in cell proliferation was found between the hybrid scaffold and the control group (culture medium only) (c); after cell seeding, live & dead staining shows that cells proliferated from 1 d (d) to 4 d (e) of in vitro culture; almost no apoptosis was observed from 1 d (f) to 4 d (g) of in vitro culture. Nuclear was stained by blue color, while apoptosis cell was stained by green color.

  • Figure 8

    In vitro engineered tubular cartilage. After cell seeding, samples at 8 weeks retain their original tubular shape and form cartilage-like tissues with a matured cartilage appearance (a, b); as shown in the histological analysis of HE (c), safranin-O (d), Masson’s trichrome (e) and type II collagen (f), the engineered cartilage presented typical lacunae structures and cartilage-specific ECM deposition accompanied by gradual degradation of the hybrid scaffolds. Yellow arrows indicate residual scaffold; black arrows indicate mature cartilage.

  • Figure 9

    In vivo tubular cartilage regeneration. After 2 weeks of cultivation and 12 weeks of in vivo implantation, samples successfully regenerate relatively homogeneous mature tubular cartilage with typical lacunae structures (a, b) and cartilage-specific ECM deposition ((c) HE; (d) safranin-O; (e) Masson’s trichrome; (f) type II collagen); the quantitative analyses showed that the hybrid scaffold with chondrocyte group (scaffold & cells) achieved the highest indexes of wet weight (g), thickness (h), and DNA content (j) with significant differences among groups; as for the quantitative indexes of Young’s modulus (i), GAG content (k), and total collagen content (l), the scaffold & cells group are still significantly higher compared with scaffold only group (without cells), but slightly lower than fresh normal trachea cartilage group. Green arrow indicates normal connective tissue; black arrow indicates neocartilage; yellow arrow indicates residual scaffold. Statistically significance: **p<0.01.

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