journal article·2026-02Open access

Hybrid Bioprinting for functionally graded tissue engineering constructs with patterned and localized biochemical signals

Jiannan Li, Carolyn Kim, Hossein V. Alizadeh, Shreya Garg, Arnaud Bruyas, Peng Zhao, Isadora S. D. Passos, Chi-Chun Pan, Andrea S. Flores Pérez, Mark A. Skylar-Scott, Sungwoo Kim, Yunzhi P. Yang

Abstract

Abstract Engineering native-mimetic tissue constructs is challenging due to their intricate biological and structural gradients. To address this, Hybprinter-SAM was developed by integrating three bioprinting technologies: syringe extrusion (SE), acoustic droplet ejection (ADE) and molten material extrusion (MME). This system not only enables the creation of mechanical gradients by integrating soft and rigid materials spanning 7 order magnitude of stiffness but also facilitates precise patterning and controlled localization of biochemical signals within printed scaffolds. This capability is beneficial in replicating the complexity of native tissues to enhance functionality. Both the printing process and biomaterials were optimized to balance printability, mechanical integrity, and biocompatibility. As a proof of concept, Hybprinter-SAM was used in a bone-tendon regeneration study to engineer a multi-material construct with patterned fibroblast growth factor 2 (FGF-2), resulting in markers indicative of fibrocartilage development. These findings highlight the potential of Hybprinter-SAM as a versatile platform for diverse tissue engineering applications that require complex, functionally graded tissue constructs.
Source: crossref · Quality: baseline

Research context

OpenAlex topics: 3D Printing in Biomedical Research · Nanomaterials and Printing Technologies · Electrospun Nanofibers in Biomedical Applications

SciVX discovery tags are terminology-based. OpenAlex topics are source-provided classifications.

References (80)

Show source-provided references
1. Khademhosseini A, Langer R (2016) A decade of progress in tissue engineering. Nat Protoc 11:1775–1781. https://doi.org/10.1038/nprot.2016.12310.1038/nprot.2016.123 ↗
2. Shafiee A, Atala A, Caskey C (2017) Tissue engineering: toward a new era of medicine. Annual Review of Medicine, Vol 68 68:29–40. https://doi.org/10.1146/annurev-med-102715-09233110.1146/annurev-med-102715-092331 ↗
3. Yen H, Tseng C, Hsu S, Tsai C (2009) Evaluation of chondrocyte growth in the highly porous scaffolds made by fused deposition manufacturing (FDM) filled with type II collagen. Biomed Microdevices 11:615–624. https://doi.org/10.1007/s10544-008-9271-710.1007/s10544-008-9271-7 ↗
4. Ceretti E et al (2017) Multi-layered scaffolds production via fused deposition modeling (FDM) using an open source 3D printer: process parameters optimization for dimensional accuracy and design reproducibility. 3rd Cirp Conf Biomanufacturing 65:13–18. https://doi.org/10.1016/j.procir.2017.04.04210.1016/j.procir.2017.04.042 ↗
5. Masood S, Singh J, Morsi Y (2005) The design and manufacturing of porous scaffolds for tissue engineering using rapid prototyping. Int J Adv Manuf Technol 27:415–420. https://doi.org/10.1007/s00170-004-2187-310.1007/s00170-004-2187-3 ↗
6. Hoque ME, Chuan YL, Pashby I (2012) Extrusion based rapid prototyping technique: an advanced platform for tissue engineering scaffold fabrication. Biopolymers 97:83–93. https://doi.org/10.1002/bip.2170110.1002/bip.21701 ↗
7. Elomaa L et al (2015) Three-dimensional fabrication of cell-laden biodegradable poly(ethylene glycol-. J Mater Chem B 3:8348–8358. https://doi.org/10.1039/c5tb01468a10.1039/c5tb01468a ↗
8. Mondschein RJ, Kanitkar A, Williams CB, Verbridge SS, Long TE (2017) Polymer structure-property requirements for stereolithographic 3D printing of soft tissue engineering scaffolds. Biomaterials 140:170–188. https://doi.org/10.1016/j.biomaterials.2017.06.00510.1016/j.biomaterials.2017.06.005 ↗
9. Li JN et al (2018) A plug-and-play, drug-on-pillar platform for combination drug screening implemented by microfluidic adaptive printing. Anal Chem 90:13969–13977. https://doi.org/10.1021/acs.analchem.8b0345610.1021/acs.analchem.8b03456 ↗
10. Li JN et al (2018) Microfluidic print-to-synthesis platform for efficient preparation and screening of combinatorial peptide microarrays. Anal Chem 90:5833–5840. https://doi.org/10.1021/acs.analchem.8b0037110.1021/acs.analchem.8b00371 ↗
11. Ding YZ et al (2015) Microfluidic-enabled print-to-screen platform for high-throughput screening of combinatorial chemotherapy. Anal Chem 87:10166–10171. https://doi.org/10.1021/acs.analchem.5b0082610.1021/acs.analchem.5b00826 ↗
12. Bettinger CJ (2018) Recent advances in materials and flexible electronics for peripheral nerve interfaces. Bioelectron Med 4:6. https://doi.org/10.1186/s42234-018-0007-610.1186/s42234-018-0007-6 ↗
13. Schuurman W et al (2011) Bioprinting of hybrid tissue constructs with tailorable mechanical properties. Biofabrication. https://doi.org/10.1088/1758-5082/3/2/02100110.1088/1758-5082/3/2/021001 ↗
14. Shim J, Kim J, Park M, Park J, Cho D (2011) Development of a hybrid scaffold with synthetic biomaterials and hydrogel using solid freeform fabrication technology. Biofabrication. https://doi.org/10.1088/1758-5082/3/3/03410210.1088/1758-5082/3/3/034102 ↗
15. Kang H et al (2016) A 3D bioprinting system to produce human-scale tissue constructs with structural integrity. Nat Biotechnol 34:312. https://doi.org/10.1038/nbt.341310.1038/nbt.3413 ↗
16. Lee H-R et al (2021) 3D microextrusion-inkjet hybrid printing of structured human skin equivalents. Bioprinting 22:e00143. https://doi.org/10.1016/j.bprint.2021.e0014310.1016/j.bprint.2021.e00143 ↗
17. Peng X et al (2022) 4D printing of freestanding liquid crystal elastomers via hybrid additive manufacturing. Adv Mater. https://doi.org/10.1002/adma.20220489010.1002/adma.202204890 ↗
18. Peng X et al (2021) Integrating digital light processing with direct ink writing for hybrid 3D printing of functional structures and devices. Addit Manuf. https://doi.org/10.1016/j.addma.2021.10191110.1016/j.addma.2021.101911 ↗
19. Axolotl (ed) <https://www.axolotlbio.com/axo-a6 (
20. RegenHu https://www.regenhu.com/ (
21. CelLink BIO X6, https://www.cellink.com/bioprinting/bio-x6-3d-bioprinter/ (
22. Li J et al (2022) Hybprinting for musculoskeletal tissue engineering. iScience 25:104229. https://doi.org/10.1016/j.isci.2022.10422910.1016/j.isci.2022.104229 ↗
23. Shanjani Y, Pan C, Elomaa L, Yang Y (2015) A novel bioprinting method and system for forming hybrid tissue engineering constructs. Biofabrication. https://doi.org/10.1088/1758-5090/7/4/04500810.1088/1758-5090/7/4/045008 ↗
24. Guo Q et al (2021) A review on acoustic droplet ejection technology and system. Soft Matter 17:3010–3021. https://doi.org/10.1039/d0sm02193h10.1039/d0sm02193h ↗
25. Ning J, Lei Y, Hu H, Gai C (2023) A comprehensive review of surface acoustic wave-enabled acoustic droplet ejection technology and its applications. Micromachines. https://doi.org/10.3390/mi1408154310.3390/mi14081543 ↗
26. Grant R et al (2009) Achieving accurate compound concentration in cell-based screening: validation of acoustic droplet ejection technology. J Biomol Screen 14:452–459. https://doi.org/10.1177/108705710933658810.1177/1087057109336588 ↗
27. Roberts K et al (2016) Implementation and challenges of direct acoustic dosing into cell-based assays. SLAS Technol 21:76–89. https://doi.org/10.1177/221106821559521210.1177/2211068215595212 ↗
28. Mackenzie T et al (2024) Acoustic droplet ejection facilitates cell-based high-throughput screenings using natural products. SLAS Technol. https://doi.org/10.1016/j.slast.2023.10.00310.1016/j.slast.2023.10.003 ↗
29. Shanjani Y, Siebert S, Ker D, Mercado-Pagan A, Yang Y (2020) Acoustic patterning of growth factor for three-dimensional tissue engineering. Tissue Eng Part A. https://doi.org/10.1089/ten.tea.2019.027110.1089/ten.tea.2019.0271 ↗
30. Sahoo S, Toh S, Goh J (2010) A bFGF-releasing silk/PLGA-based biohybrid scaffold for ligament/tendon tissue engineering using mesenchymal progenitor cells. Biomaterials 31:2990–2998. https://doi.org/10.1016/j.biomaterials.2010.01.00410.1016/j.biomaterials.2010.01.004 ↗
31. Peterson D et al (2015) Evaluation of a collagen-coated, resorbable fiber scaffold loaded with a peptide basic fibroblast growth factor mimetic in a sheep model of rotator cuff repair. J Shoulder Elbow Surg 24:1764–1773. https://doi.org/10.1016/j.jse.2015.06.00910.1016/j.jse.2015.06.009 ↗
32. Zhao S et al (2014) Biological augmentation of rotator cuff repair using bFGF-loaded electrospun poly(lactide-co-glycolide) fibrous membranes. Int J Nanomed 9:2373–2385. https://doi.org/10.2147/IJN.S5953610.2147/IJN.S59536 ↗
33. Sugimoto Y et al (2013) Scx(+)/Sox9(+) progenitors contribute to the establishment of the junction between cartilage and tendon/ligament. Development 140:2280–2288. https://doi.org/10.1242/dev.09635410.1242/dev.096354 ↗
34. Murchison N et al (2007) Regulation of tendon differentiation by scleraxis distinguishes force-transmitting tendons from muscle-anchoring tendons. Development 134:2697–2708. https://doi.org/10.1242/dev.00193310.1242/dev.001933 ↗
35. Blitz E et al (2009) Bone ridge patterning during musculoskeletal assembly is mediated through SCX regulation of Bmp4 at the tendon-skeleton junction. Dev Cell 17:861–873. https://doi.org/10.1016/j.devcel.2009.10.01010.1016/j.devcel.2009.10.010 ↗
36. Kolesky D, Homan K, Skylar-Scott M, Lewis J (2016) Three-dimensional bioprinting of thick vascularized tissues. Proc Natl Acad Sci USA 113:3179–3184. https://doi.org/10.1073/pnas.152134211310.1073/pnas.1521342113 ↗
37. Li J et al (2023) Development and systematic characterization of GelMA/alginate/PEGDMA/xanthan gum hydrogel bioink system for extrusion bioprinting. Biomaterials 293:121969. https://doi.org/10.1016/j.biomaterials.2022.12196910.1016/j.biomaterials.2022.121969 ↗
38. Bruyas A et al (2018) Systematic characterization of 3D-printed PCL/β-TCP scaffolds for biomedical devices and bone tissue engineering: Influence of composition and porosity. J Mater Res 33:1948–1959. https://doi.org/10.1557/jmr.2018.11210.1557/jmr.2018.112 ↗
39. Kang J et al (2020) The influence of electron beam sterilization on In Vivo degradation of β-TCP/PCL of different composite ratios for bone tissue engineering. Micromachines. https://doi.org/10.3390/mi1103027310.3390/mi11030273 ↗
40. Bruyas A, Moeinzadeh S, Kim S, Lowenberg D, Yang Y (2019) Effect of electron beam sterilization on Three-Dimensional-Printed Polycaprolactone/Beta-Tricalcium phosphate scaffolds for bone tissue engineering. Tissue Eng Part A 25:248–256. https://doi.org/10.1089/ten.tea.2018.013010.1089/ten.tea.2018.0130 ↗
41. Mercado-Pagan AE, Stahl AM, Ramseier ML, Behn AW, Yang Y (2016) Synthesis and characterization of Polycaprolactone urethane Hollow fiber membranes as small diameter vascular grafts. Mater Sci Eng C Mater Biol Appl 64:61–73. https://doi.org/10.1016/j.msec.2016.03.06810.1016/j.msec.2016.03.068 ↗
42. Skylar-Scott M et al (2022) Orthogonally induced differentiation of stem cells for the programmatic patterning of vascularized organoids and bioprinted tissues. Nat Biomed Eng 6:449. https://doi.org/10.1038/s41551-022-00856-810.1038/s41551-022-00856-8 ↗
43. McAndrews KM, Kim MJ, Lam TY, McGrail DJ, Dawson MR (2014) Architectural and mechanical cues direct mesenchymal stem cell interactions with crosslinked gelatin scaffolds. Tissue Eng Part A 20:3252–3260. https://doi.org/10.1089/ten.TEA.2013.075310.1089/ten.TEA.2013.0753 ↗
44. Schultz KM, Kyburz KA, Anseth KS (2015) Measuring dynamic cell-material interactions and remodeling during 3D human mesenchymal stem cell migration in hydrogels. Proc Natl Acad Sci U S A 112:E3757–3764. https://doi.org/10.1073/pnas.151130411210.1073/pnas.1511304112 ↗
45. Horton ER et al (2020) Extracellular matrix production by mesenchymal stromal cells in hydrogels facilitates cell spreading and is inhibited by FGF-2. Adv Healthc Mater 9:e1901669. https://doi.org/10.1002/adhm.20190166910.1002/adhm.201901669 ↗
46. Shanmugasundaram S, Logan-Mauney S, Burgos K, Nurminskaya M (2012) Tissue transglutaminase regulates chondrogenesis in mesenchymal stem cells on collagen type XI matrices. Amino Acids 42:1045–1053. https://doi.org/10.1007/s00726-011-1019-710.1007/s00726-011-1019-7 ↗
47. Zhang Y et al (2023) Mechanisms and therapeutic prospects of mesenchymal stem cells-derived exosomes for tendinopathy. Stem Cell Res Ther 14:307. https://doi.org/10.1186/s13287-023-03431-310.1186/s13287-023-03431-3 ↗
48. Wechsler ME, Rao VV, Borelli AN, Anseth KS (2021) Engineering the MSC secretome: a hydrogel focused approach. Adv Healthc Mater 10:e2001948. https://doi.org/10.1002/adhm.20200194810.1002/adhm.202001948 ↗
49. Naddaf A, Tsibranska I, Bart H (2010) Kinetics of BSA release from poly(N-isopropylacrylamide) hydrogels. Chem Eng Process Process Intensif 49:581–588. https://doi.org/10.1016/j.cep.2010.05.00810.1016/j.cep.2010.05.008 ↗
50. Waheed A, Mazumder MAJ, Al-Ahmed A, Roy P, Ullah N (2019) Functional biopolymers (eds Mohammad Abu Jafar Mazumder, Heather Sheardown, & Amir Al-Ahmed). Springer International Publishing, pp 377–42710.1007/978-3-319-95990-0_4 ↗
51. Deo K, Singh K, Peak C, Alge D, Gaharwar A (2020) Bioprinting 101: design, fabrication, and evaluation of cell-laden 3D bioprinted scaffolds. Tissue Eng Part A 26:318–338. https://doi.org/10.1089/ten.tea.2019.029810.1089/ten.tea.2019.0298 ↗
52. Unagolla J, Jayasuriya A (2020) Hydrogel-based 3D bioprinting: a comprehensive review on cell-laden hydrogels, bioink formulations, and future perspectives. Appl Mater Today. https://doi.org/10.1016/j.apmt.2019.10047910.1016/j.apmt.2019.100479 ↗
53. Jeon O, Ryu S, Chung J, Kim B (2005) Control of basic fibroblast growth factor release from fibrin gel with heparin and concentrations of fibrinogen and thrombin. J Control Release 105:249–259. https://doi.org/10.1016/j.jconrel.2005.03.02310.1016/j.jconrel.2005.03.023 ↗
54. Campbell P, Miller E, Fisher G, Walker L, Weiss L (2005) Engineered spatial patterns of FGF-2 immobilized on fibrin direct cell organization. Biomaterials 26:6762–6770. https://doi.org/10.1016/j.biomaterials.2005.04.03210.1016/j.biomaterials.2005.04.032 ↗
55. Miller E, Fisher G, Weiss L, Walker L, Campbell P (2006) Dose-dependent cell growth in response to concentration modulated patterns of FGF-2 printed on fibrin. Biomaterials 27:2213–2221. https://doi.org/10.1016/j.biomaterials.2005.10.02110.1016/j.biomaterials.2005.10.021 ↗
56. Antonov YA, Zhuravleva IL (2012) Macromolecular complexes of BSA with gelatin. Int J Biol Macromol 51:319–328. https://doi.org/10.1016/j.ijbiomac.2012.05.01210.1016/j.ijbiomac.2012.05.012 ↗
57. Antonov YA, Zhuravleva IL (2013) Effect of protein thermo aggregation on the binding of BSA to gelatin type A. Int J Biol Macromol 53:160–167. https://doi.org/10.1016/j.ijbiomac.2012.11.01610.1016/j.ijbiomac.2012.11.016 ↗
58. Shkilnyy A, Proulx P, Sharp J, Lepage M, Vermette P (2012) Diffusion of Rhodamine B and bovine serum albumin in fibrin gels seeded with primary endothelial cells. Colloids Surf B Biointerfaces 93:202–207. https://doi.org/10.1016/j.colsurfb.2012.01.00510.1016/j.colsurfb.2012.01.005 ↗
59. Tellado SF, Balmayor ER, Van Griensven M (2015) Strategies to engineer tendon/ligament-to-bone interface: biomaterials, cells and growth factors. Adv Drug Deliv Rev 94:126–14010.1016/j.addr.2015.03.004 ↗
60. Hernigou P et al (2014) Biologic augmentation of rotator cuff repair with mesenchymal stem cells during arthroscopy improves healing and prevents further tears: a case-controlled study. Int Orthop 38:1811–1818. https://doi.org/10.1007/s00264-014-2391-110.1007/s00264-014-2391-1 ↗
61. Claudepierre P, Voisin MC (2005) The entheses: histology, pathology, and pathophysiology. Joint Bone Spine 72:32–37. https://doi.org/10.1016/j.jbspin.2004.02.01010.1016/j.jbspin.2004.02.010 ↗
62. Ralphs J et al (1998) Regional differences in cell shape and gap junction expression in rat Achilles tendon: relation to fibrocartilage differentiation. J Anat 193:215–222. https://doi.org/10.1046/j.1469-7580.1998.19320215.x10.1046/j.1469-7580.1998.19320215.x ↗
63. Rossetti L et al (2017) The microstructure and micromechanics of the tendon-bone insertion. Nat Mater. https://doi.org/10.1038/nmat486310.1038/nmat4863 ↗
64. Lei T et al (2021) Biomimetic strategies for tendon/ligament-to-bone interface regeneration. Bioact Mater 6:2491–2510. https://doi.org/10.1016/j.bioactmat.2021.01.02210.1016/j.bioactmat.2021.01.022 ↗
65. Calejo I, Costa-Almeida R, Gomes ME (2019) Cellular complexity at the interface: challenges in enthesis tissue engineering. Adv Exp Med Biol 1144:71–90. https://doi.org/10.1007/5584_2018_30710.1007/5584_2018_307 ↗
66. Calejo I, Costa-Almeida R, Reis RL, Gomes ME (2019) Enthesis tissue engineering: biological requirements meet at the interface. Tissue Eng Part B Rev 25:330–356. https://doi.org/10.1089/ten.TEB.2018.038310.1089/ten.TEB.2018.0383 ↗
67. Derwin KA, Galatz LM, Ratcliffe A, Thomopoulos S (2018) Enthesis repair: challenges and opportunities for effective tendon-to-bone healing. J Bone Joint Surg Am 100:e109. https://doi.org/10.2106/JBJS.18.0020010.2106/JBJS.18.00200 ↗
68. Akyol E, Hindocha S, Khan W (2015) Use of stem cells and growth factors in rotator cuff tendon repair. Curr Stem Cell Res Ther 10:5–1010.2174/1574888X09666140710101001 ↗
69. Dyment N et al (2014) Lineage tracing of resident tendon progenitor cells during growth and natural healing. PLoS One. https://doi.org/10.1371/journal.pone.009611310.1371/journal.pone.0096113 ↗
70. Hurley-Novatny A et al (2020) Concurrent multi-lineage differentiation of mesenchymal stem cells through spatial presentation of growth factors. Biomed Mater. https://doi.org/10.1088/1748-605X/ab9bb010.1088/1748-605X/ab9bb0 ↗
71. Murakami S, Kan M, McKeehan W, de Crombrugghe B (2000) Up-regulation of the chondrogenic < i > Sox9 gene by fibroblast growth factors is mediated by the mitogen-activated protein kinase pathway. Proc Natl Acad Sci U S A 97:1113–1118. https://doi.org/10.1073/pnas.97.3.111310.1073/pnas.97.3.1113 ↗
72. Chen Z et al (2014) FGF signaling activates a Sox9-Sox10 pathway for the formation and branching morphogenesis of mouse ocular glands. Development 141:2691–2701. https://doi.org/10.1242/dev.10894410.1242/dev.108944 ↗
73. Jensen PT, Lambertsen KL, Frich LH (2018) Assembly, maturation, and degradation of the supraspinatus enthesis. J Shoulder Elbow Surg 27:739–750. https://doi.org/10.1016/j.jse.2017.10.03010.1016/j.jse.2017.10.030 ↗
74. Killian ML, Thomopoulos S (2016) Scleraxis is required for the development of a functional tendon enthesis. FASEB J 30:301–311. https://doi.org/10.1096/fj.14-25823610.1096/fj.14-258236 ↗
75. Li Y et al (2022) Transforming growth factor-β signalling pathway in tendon healing. Growth Factors 40:98–107. https://doi.org/10.1080/08977194.2022.208229410.1080/08977194.2022.2082294 ↗
76. Benjamin M et al (2006) Where tendons and ligaments meet bone: attachment sites ('entheses’) in relation to exercise and/or mechanical load. J Anat 208:471–490. https://doi.org/10.1111/j.1469-7580.2006.00540.x10.1111/j.1469-7580.2006.00540.x ↗
77. Locke R et al (2017) Strain distribution of intact rat rotator cuff Tendon-to-Bone attachments and attachments with defects. J Biomech Engineering-Transactions Asme 139. https://doi.org/10.1115/1.403811110.1115/1.4038111 ↗
78. Brown M, Puetzer J (2023) Enthesis maturation in engineered ligaments is differentially driven by loads that mimic slow growth elongation and rapid cyclic muscle movement. Acta Biomater 172:106–122. https://doi.org/10.1016/j.actbio.2023.10.01210.1016/j.actbio.2023.10.012 ↗
79. Wang T, Lai J, Yang F (2016) Effects of hydrogel stiffness and extracellular compositions on modulating cartilage regeneration by mixed populations of stem cells and chondrocytes < i > in vivo. Tissue Eng Part A 22:1348–1356. https://doi.org/10.1089/ten.tea.2016.030610.1089/ten.tea.2016.0306 ↗
80. Zhao P et al (2023) Scar-degrading endothelial cells as a treatment for advanced liver fibrosis. Adv Sci. https://doi.org/10.1002/advs.20220331510.1002/advs.202203315 ↗