4D Printing in Tissue Engineering and Regenerative Medicine: A Transformative Approach to Intelligent Biofabrication
Subject Areas :Azizeh Rahmani Del Bakhshayesh 1 * , Fariba Alizadeh Eghtedar 2 , Mahdiyeh Rahmani Del Bakhshayesh 3
1 - Department of Tissue Engineering, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
2 - . Department of Tissue Engineering, Faculty of Advanced Medical Sciences, Mashhad University of Medical Sciences, Mashhad, Iran
3 - Al-Zahra Technical & Vocational College, Tabriz, Iran
Keywords: 4D Printing, Tissue Engineering, Regenerative Medicine, Stimulus-responsive Materials, Dynamic Scaffolds,
Abstract :
4D printing as an advanced generation of 3D printing has opened new horizons in tissue engineering and regenerative medicine. By integrating additive manufacturing with stimulus-responsive smart materials, this technology introduces time as the fourth dimension, enabling the creation of dynamic and adaptive structures. While conventional 3D printing faces limitations in replicating complex biological architectures, 4D printing overcomes these constraints by utilizing materials responsive to stimuli such as temperature, pH, light, and moisture, allowing controlled shape or functional transformations after fabrication.
In this approach, a 3D-printed scaffold undergoes controlled and programmable transformation when exposed to one or more external stimuli, transitioning into a new, distinct, and stable configuration. This capability allows for a more accurate simulation of the dynamic behavior of living tissues and enhances functional integration within biological environments. By addressing key limitations of traditional biofabrication methods, 4D printing provides unprecedented opportunities for recreating the structural and functional complexity of native tissues.
Applications of 4D printing in tissue engineering and regenerative medicine are rapidly expanding, including the development of dynamic scaffolds and self-assembling structures, engineered vascular networks, shape-morphing implants, adaptive artificial organs, and intelligent drug delivery systems. The technology’s capacity to create responsive and programmable bio-constructs positions it as a promising platform for next-generation regenerative therapies and personalized medicine.
This article reviews the fundamental principles, materials, fabrication strategies, biomedical applications, current challenges, and future perspectives of 4D printing in tissue engineering and regenerative medicine, highlighting its transformative potential in advancing intelligent and functional biofabrication.
1. Del Bakhshayesh AR, Babaie S, Niknafs B, Abedelahi A, Mehdipour A, Ghahremani-Nasab M. High efficiency biomimetic electrospun fibers for use in regenerative medicine and drug delivery: A review. Materials Chemistry and Physics. 2022;279:125785.
2. Rahmani Del Bakhshayesh A, Saghebasl S, Asadi N, Kashani E, Mehdipour A, Nezami Asl A, et al. Recent advances in nano‐scaffolds for tissue engineering applications: toward natural therapeutics. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. 2023;15(6):e1882.
3. Adel IM, ElMeligy MF, Elkasabgy NA. Conventional and recent trends of scaffolds fabrication: a superior mode for tissue engineering. Pharmaceutics. 2022;14(2):306.
4. Rahmani Del Bakhshayesh A, Annabi N, Khalilov R, Akbarzadeh A, Samiei M, Alizadeh E, et al. Recent advances on biomedical applications of scaffolds in wound healing and dermal tissue engineering. Artificial cells, nanomedicine, and biotechnology. 2018;46(4):691-705.
5. Del Bakhshayesh AR, Asadi N, Alihemmati A, Tayefi Nasrabadi H, Montaseri A, Davaran S, et al. An overview of advanced biocompatible and biomimetic materials for creation of replacement structures in the musculoskeletal systems: focusing on cartilage tissue engineering. Journal of biological engineering. 2019;13(1):85.
6. Rahmani Del Bakhshayesh A, Mostafavi E, Alizadeh E, Asadi N, Akbarzadeh A, Davaran S. Fabrication of three-dimensional scaffolds based on nano-biomimetic collagen hybrid constructs for skin tissue engineering. ACS omega. 2018;3(8):8605-11.
7. Saghati S, Akbarzadeh A, Del Bakhshayesh A, Sheervalilou R, Mostafavi E. Electrospinning and 3D printing: prospects for market opportunity. 2018.
8. Kalogeropoulou M, Díaz-Payno PJ, Mirzaali MJ, van Osch GJ, Fratila-Apachitei LE, Zadpoor AA. 4D printed shape-shifting biomaterials for tissue engineering and regenerative medicine applications. Biofabrication. 2024;16(2):022002.
9. Gueche YA, Sanchez-Ballester NM, Cailleaux S, Bataille B, Soulairol I. Selective laser sintering (SLS), a new chapter in the production of solid oral forms (SOFs) by 3D printing. Pharmaceutics. 2021;13(8):1212.
10. Panda BK, Sahoo S. Thermo-mechanical modeling and validation of stress field during laser powder bed fusion of AlSi10Mg built part. Results in Physics. 2019;12:1372-81.
11. An J, Leong KF. Multi-material and multi-dimensional 3D printing for biomedical materials and devices. Biomedical Materials & Devices. 2023;1(1):38-48.
12. Kong B, Zhao Y. 3D bioprinting for biomedical applications. BME frontiers. 2023;4:0010.
13. Vanaei S, Parizi M, Salemizadehparizi F, Vanaei H. An overview on materials and techniques in 3D bioprinting toward biomedical application. Engineered Regeneration. 2021;2:1-18.
14. Tyagi N, Bhardwaj V, Sharma D, Tomar R, Chaudhary V, Khanuja M, et al. 3D printing technology in the pharmaceutical and biomedical applications: a critical review. Biomedical Materials & Devices. 2024;2(1):178-90.
15. Dong Y, Wang S, Ke Y, Ding L, Zeng X, Magdassi S, et al. 4D printed hydrogels: fabrication, materials, and applications. Advanced Materials Technologies. 2020;5(6):2000034.
16. Del Bakhshayesh AR, Ghahremani-nasab M, Babaie S, Del Bakhshayesh MR. Revolutionizing Wound Healing: Integrating 4D Bioprinting with Adaptive Bioactive Coatings for Dynamic Tissue Regeneration. 2025.
17. Li Y, Zhang F, Liu Y, Leng J. 4D printed shape memory polymers and their structures for biomedical applications. Science China Technological Sciences. 2020;63(4):545-60.
18. Abolhassani S, Fattahi R, Safshekan F, Saremi J, Hasanzadeh E. Advances in 4D bioprinting: The next frontier in regenerative medicine and tissue engineering applications. Advanced Healthcare Materials. 2025;14(4):2403065.
19. Zeenat L, Zolfagharian A, Sriya Y, Sasikumar S, Bodaghi M, Pati F. 4D printing for vascular tissue engineering: progress and challenges. Advanced Materials Technologies. 2023;8(23):2300200.
20. Cui C, Kim D-O, Pack MY, Han B, Han L, Sun Y, et al. 4D printing of self-folding and cell-encapsulating 3D microstructures as scaffolds for tissue-engineering applications. Biofabrication. 2020;12(4):045018.
21. Kuang X, Roach DJ, Wu J, Hamel CM, Ding Z, Wang T, et al. Advances in 4D printing: materials and applications. Advanced Functional Materials. 2019;29(2):1805290.
22. Sossou G, Demoly F, Belkebir H, Qi HJ, Gomes S, Montavon G. Design for 4D printing: Modeling and computation of smart materials distributions. Materials & Design. 2019;181:108074.
23. Pugliese R, Regondi S. Artificial intelligence-empowered 3D and 4D printing technologies toward smarter biomedical materials and approaches. Polymers. 2022;14(14):2794.
24. Cosola A, Roppolo I, Frascella F, Napione L, Barrera G, Tiberto P, et al. 4D Printing of multifunctional devices induced by synergistic role of magnetite and silver nanoparticles in polymeric nanocomposites. Advanced Functional Materials. 2024;34(41):2406226.
25. Zhao W, Yan Y, Chen X, Wang T. Combining printing and nanoparticle assembly: Methodology and application of nanoparticle patterning. The Innovation. 2022;3(4).
26. Guo J, Zhang R, Zhang L, Cao X. 4D printing of robust hydrogels consisted of agarose nanofibers and polyacrylamide. ACS Macro Letters. 2018;7(4):442-6.
27. Tiwari M, Mishra D. Applications of 3D-and 4D-Printed Polymer Nanocomposites in the Medical and Biomedical Field. Polymer Nanocomposites for 3D, 4D and 5D Printing: Fundamental to Applications: Springer; 2025. p. 293-320.
28. Quan H, Pirosa A, Yang W, Ritchie RO, Meyers MA. Hydration-induced reversible deformation of the pine cone. Acta biomaterialia. 2021;128:370-83.
29. Taghipour YD, Hokmabad VR, Del Bakhshayesh AR, Asadi N, Salehi R, Nasrabadi HT. The application of hydrogels based on natural polymers for tissue engineering. Current medicinal chemistry. 2020;27(16):2658-80.
30. Díaz‐Payno PJ, Kalogeropoulou M, Muntz I, Kingma E, Kops N, D'Este M, et al. Swelling‐dependent shape‐based transformation of a human mesenchymal stromal cells‐laden 4D bioprinted construct for cartilage tissue engineering. Advanced healthcare materials. 2023;12(2):2201891.
31. Lv C, Sun X-C, Xia H, Yu Y-H, Wang G, Cao X-W, et al. Humidity-responsive actuation of programmable hydrogel microstructures based on 3D printing. Sensors and Actuators B: Chemical. 2018;259:736-44.
32. Yang GH, Kim W, Kim J, Kim G. A skeleton muscle model using GelMA-based cell-aligned bioink processed with an electric-field assisted 3D/4D bioprinting. Theranostics. 2021;11(1):48.
33. Kim SH, Seo YB, Yeon YK, Lee YJ, Park HS, Sultan MT, et al. 4D-bioprinted silk hydrogels for tissue engineering. Biomaterials. 2020;260:120281.
34. Kirillova A, Maxson R, Stoychev G, Gomillion CT, Ionov L. 4D biofabrication using shape‐morphing hydrogels. Advanced Materials. 2017;29(46):1703443.
35. Constante G, Apsite I, Alkhamis H, Dulle M, Schwarzer M, Caspari A, et al. 4D biofabrication using a combination of 3D printing and melt-electrowriting of shape-morphing polymers. ACS Applied Materials & Interfaces. 2021;13(11):12767-76.
36. Liang Z, Liu Y, Zhang F, Ai Y, Liang Q. Dehydration-triggered shape morphing based on asymmetric bubble hydrogel microfibers. Soft Matter. 2018;14(32):6623-6.
37. Cao J, Zhou C, Su G, Zhang X, Zhou T, Zhou Z, et al. Arbitrarily 3D configurable hygroscopic robots with a covalent–noncovalent interpenetrating network and self‐healing ability. Advanced Materials. 2019;31(18):1900042.
38. Mulakkal MC, Trask RS, Ting VP, Seddon AM. Responsive cellulose-hydrogel composite ink for 4D printing. Materials & Design. 2018;160:108-18.
39. Ji Z, Yan C, Yu B, Zhang X, Cai M, Jia X, et al. 3D printing of hydrogel architectures with complex and controllable shape deformation. Advanced Materials Technologies. 2019;4(4):1800713.
40. Sydney Gladman A, Matsumoto EA, Nuzzo RG, Mahadevan L, Lewis JA. Biomimetic 4D printing. Nature materials. 2016;15(4):413-8.
41. Apsite I, Uribe JM, Posada AF, Rosenfeldt S, Salehi S, Ionov L. 4D biofabrication of skeletal muscle microtissues. Biofabrication. 2020;12(1):015016.
42. Ding A, Lee SJ, Ayyagari S, Tang R, Huynh CT, Alsberg E. 4D biofabrication via instantly generated graded hydrogel scaffolds. Bioactive materials. 2022;7:324-32.
43. Naficy S, Gately R, Gorkin III R, Xin H, Spinks GM. 4D printing of reversible shape morphing hydrogel structures. Macromolecular Materials and Engineering. 2017;302(1):1600212.
44. Taylor MJ, Tomlins P, Sahota TS. Thermoresponsive gels. Gels. 2017;3(1):4.
45. Solis DM, Czekanski A. The effect of the printing temperature on 4D DLP printed pNIPAM hydrogels. Soft Matter. 2022;18(17):3422-9.
46. Chen G, Zhou Y, Dai J, Yan S, Miao W, Ren L. Calcium alginate/PNIPAAm hydrogel with body temperature response and great biocompatibility: Application as burn wound dressing. International Journal of Biological Macromolecules. 2022;216:686-97.
47. Ansari MJ, Rajendran RR, Mohanto S, Agarwal U, Panda K, Dhotre K, et al. Poly (N-isopropylacrylamide)-based hydrogels for biomedical applications: A review of the state-of-the-art. Gels. 2022;8(7):454.
48. Ding X, Wang M, Wei L, Wang L, Yuan M, Yu F, et al. Mechanical properties of three N-isopropylacrylamide-based semi-interpenetrating network bilayer hydrogels and their effects on multi-stimuli responsiveness. Iranian Polymer Journal. 2024;33(3):273-87.
49. Yue C, Zhai H, Zhang H, Wang T, Li Z, Ma L, et al. Synthesis of a PNIPAM-based composite hydrogel and its multipurpose applications in piezoresistive and temperature sensing. ACS Applied Electronic Materials. 2024;6(5):3216-26.
50. Gheysoori P, Paydayesh A, Jafari M, Peidayesh H. Thermoresponsive nanocomposite hydrogels based on Gelatin/poly (N–isopropylacrylamide)(PNIPAM) for controlled drug delivery. European Polymer Journal. 2023;186:111846.
51. Huang Y-C, Cheng Q-P, Jeng U-S, Hsu S-h. A biomimetic bilayer hydrogel actuator based on thermoresponsive gelatin methacryloyl–poly (N-isopropylacrylamide) hydrogel with three-dimensional printability. ACS Applied Materials & Interfaces. 2023;15(4):5798-810.
52. Bakarich SE, Gorkin R, Spinks GM. 4D Printing with Mechanically Robust, Thermally Actuating Hydrogels. Macromolecular rapid communications. 2015;36(12).
53. Halperin A, Kröger M, Winnik FM. Poly (N‐isopropylacrylamide) phase diagrams: fifty years of research. Angewandte Chemie International Edition. 2015;54(51):15342-67.
54. Nasef SM, Khozemy EE, Mahmoud GA. pH-responsive chitosan/acrylamide/gold/nanocomposite supported with silver nanoparticles for controlled release of anticancer drug. Scientific Reports. 2023;13(1):7818.
55. Pourmadadi M, Darvishan S, Abdouss M, Yazdian F, Rahdar A, Díez-Pascual AM. pH-responsive polyacrylic acid (PAA)-carboxymethyl cellulose (CMC) hydrogel incorporating halloysite nanotubes (HNT) for controlled curcumin delivery. Industrial Crops and Products. 2023;197:116654.
56. Rizwan M, Yahya R, Hassan A, Yar M, Azzahari AD, Selvanathan V, et al. pH sensitive hydrogels in drug delivery: Brief history, properties, swelling, and release mechanism, material selection and applications. Polymers. 2017;9(4):137.
57. Hu Y, Wang Z, Jin D, Zhang C, Sun R, Li Z, et al. Botanical‐inspired 4D printing of hydrogel at the microscale. Advanced Functional Materials. 2020;30(4):1907377.
58. Dai W, Guo H, Gao B, Ruan M, Xu L, Wu J, et al. Double network shape memory hydrogels activated by near-infrared with high mechanical toughness, nontoxicity, and 3D printability. Chemical Engineering Journal. 2019;356:934-49.
59. Huang J, Zhao L, Wang T, Sun W, Tong Z. NIR-triggered rapid shape memory PAM–GO–gelatin hydrogels with high mechanical strength. ACS applied materials & interfaces. 2016;8(19):12384-92.
60. Regato-Herbella M, Mantione D, Blachman A, Gallastegui A, Calabrese GC, Moya SE, et al. Multiresponsive 4d printable hydrogels with anti-inflammatory properties. ACS Macro Letters. 2024;13(9):1119-26.
61. Morrison RJ, Hollister SJ, Niedner MF, Mahani MG, Park AH, Mehta DK, et al. Mitigation of tracheobronchomalacia with 3D-printed personalized medical devices in pediatric patients. Science translational medicine. 2015;7(285):285ra64-ra64.
62. Liu B, Li H, Meng F, Xu Z, Hao L, Yao Y, et al. 4D printed hydrogel scaffold with swelling-stiffening properties and programmable deformation for minimally invasive implantation. Nature Communications. 2024;15(1):1587.
63. Mirani B, Pagan E, Currie B, Siddiqui MA, Hosseinzadeh R, Mostafalu P, et al. An advanced multifunctional hydrogel‐based dressing for wound monitoring and drug delivery. Advanced healthcare materials. 2017;6(19):1700718.
64. Kuribayashi-Shigetomi K, Onoe H, Takeuchi S. Cell origami: self-folding of three-dimensional cell-laden microstructures driven by cell traction force. PloS one. 2012;7(12):e51085.
65. Saska S, Pilatti L, Blay A, Shibli JA. Bioresorbable polymers: advanced materials and 4D printing for tissue engineering. Polymers. 2021;13(4):563.
66. Norotte C, Marga FS, Niklason LE, Forgacs G. Scaffold-free vascular tissue engineering using bioprinting. Biomaterials. 2009;30(30):5910-7.
67. Song KH, Highley CB, Rouff A, Burdick JA. Complex 3D‐printed microchannels within cell‐degradable hydrogels. Advanced Functional Materials. 2018;28(31):1801331.
68. Ding A, Lee SJ, Tang R, Gasvoda KL, He F, Alsberg E. 4D cell‐condensate bioprinting. Small. 2022;18(36):2202196.
69. Kitana W, Apsite I, Hazur J, Boccaccini AR, Ionov L. 4D biofabrication of T‐shaped vascular bifurcation. Advanced Materials Technologies. 2023;8(1):2200429.
70. Hwangbo H, Lee H, Roh EJ, Kim W, Joshi HP, Kwon SY, et al. Bone tissue engineering via application of a collagen/hydroxyapatite 4D-printed biomimetic scaffold for spinal fusion. Applied Physics Reviews. 2021;8(2):021403.
71. Lin C, Liu L, Liu Y, Leng J. 4D printing of bioinspired absorbable left atrial appendage occluders: a proof-of-concept study. ACS Applied Materials & Interfaces. 2021;13(11):12668-78.
72. Zarek M, Mansour N, Shapira S, Cohn D. 4D printing of shape memory‐based personalized endoluminal medical devices. Macromolecular rapid communications. 2017;38(2):1600628.
73. Sahafnejad-Mohammadi I, Karamimoghadam M, Zolfagharian A, Akrami M, Bodaghi M. 4D printing technology in medical engineering: a narrative review. Journal of the Brazilian Society of Mechanical Sciences and Engineering. 2022;44(6):233.
74. Shin SR, Farzad MR, Tamayol A, Manoharan MV, Mostafalu P, Zhang YS, et al. A bioactive carbon nanotube-based ink for printing 2D and 3D flexible electronics. Advanced materials (Deerfield Beach, Fla). 2016;28(17):3280.
75. Grinberg D, Siddique S, Le MQ, Liang R, Capsal JF, Cottinet PJ. 4D Printing based piezoelectric composite for medical applications. Journal of Polymer Science Part B: Polymer Physics. 2019;57(2):109-15.
76. Mahmoud DB, Schulz‐Siegmund M. Utilizing 4D printing to design smart gastroretentive, esophageal, and intravesical drug delivery systems. Advanced healthcare materials. 2023;12(10):2202631.
77. Kirtane AR, Abouzid O, Minahan D, Bensel T, Hill AL, Selinger C, et al. Development of an oral once-weekly drug delivery system for HIV antiretroviral therapy. Nature communications. 2018;9(1):2.
78. Malachowski K, Breger J, Kwag HR, Wang MO, Fisher JP, Selaru FM, et al. Stimuli‐responsive theragrippers for chemomechanical controlled release. Angewandte Chemie International Edition. 2014;53(31):8045-9.
79. Lin M, Firoozi N, Tsai C-T, Wallace MB, Kang Y. 3D-printed flexible polymer stents for potential applications in inoperable esophageal malignancies. Acta biomaterialia. 2019;83:119-29.
80. Melocchi A, Inverardi N, Uboldi M, Baldi F, Maroni A, Pandini S, et al. Retentive device for intravesical drug delivery based on water-induced shape memory response of poly (vinyl alcohol): design concept and 4D printing feasibility. International journal of pharmaceutics. 2019;559:299-311.
81. Pedron S, Van Lierop S, Horstman P, Penterman R, Broer D, Peeters E. Stimuli responsive delivery vehicles for cardiac microtissue transplantation. Advanced Functional Materials. 2011;21(9):1624-30.