Bacterial nanocellulose-reinforced gelatin methacryloyl hydrogel enhances biomechanical property and glycosaminoglycan content of 3D-bioprinted cartilage
Tissue-engineered ear cartilage scaffold based on three-dimensional (3D) bioprinting technology presents a new strategy for ear reconstruction in individuals with microtia. Natural hydrogel is a promising material due to its excellent biocompatibility and low immunogenicity. However, insufficient mechanical property required for cartilage is one of the major issues pending to be solved. In this study, the gelatin methacryloyl (GelMA) hydrogel reinforced with bacterial nanocellulose (BNC) was developed to enhance the biomechanical properties and printability of the hydrogel. The results revealed that the addition of 0.375% BNC significantly increased the mechanical properties of the hydrogel and promoted cell migration in the BNC-reinforced hydrogel. Constructs bioprinted with chondrocyte-laden BNC/GelMA hydrogel bio-ink formed mature cartilage in nude mice with higher Young’s modulus and glycosaminoglycan content. Finally, an auricle equivalent with a precise shape, high mechanics, and abundant cartilage-specific matrix was developed in vivo. In this study, we developed a potentially useful hydrogel for the manufacture of auricular cartilage grafts for microtia patients.
Tanzer RC, 1961, Total reconstruction of the external auricle. Arch Otolaryngol, 73: 64–68. https://doi.org/10.1001/archotol.1961.00740020068008
Brent B, 1974, Ear reconstruction with an expansile framework of autogenous rib cartilage. Plast Reconstr Surg, 53: 619–628. https://doi.org/10.1097/00006534-197406000-00001
Conway H, Neumann CG, Gelb J, et al., 1948, Reconstruction of the external ear. Ann Surg, 128: 226–239. https://doi.org/10.1097/00000658-194808000-00005
Brent B, Tanzer RC, Rueckert F, et al., 1992, Auricular repair with autogenous rib cartilage grafts. Plast Reconstr Surg, 90: 375–376. https://doi.org/10.1097/00006534-199209000-00002
Nagata S, 1993, A new method of total reconstruction of the auricle for microtia. Plast Reconstr Surg, 92: 187–201. https://doi.org/10.1097/00006534-199308000-00001
Brent B, 1999, Technical advances in ear reconstruction with autogenous rib cartilage grafts: Personal experience with 1200 cases. Plast Reconstr Surg, 104: 319–334. https://doi.org/10.1097/00006534-199908000-00001
Zhang Q, Zhang R, Xu F, et al., 2009, Auricular reconstruction for microtia: personal 6-year experience based on 350 microtia ear reconstructions in china. Plast Reconstr Surg, 123: 849–858. https://doi.org/10.1097/PRS.0b013e318199f057
Cao Y, Vacanti JP, Paige KT, et al., 1997, Transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue-engineered cartilage in the shape of a human ear. Plast Reconstr Surg, 100: 297–302. https://doi.org/10.1097/00006534-199708000-00001
Zhou G, Jiang H, Yin Z, et al., 2018, In vitro regeneration of patient-specific ear-shaped cartilage and its first clinical application for auricular reconstruction. EBioMedicine, 28:287–302. https://doi.org/10.1016/j.ebiom.2018.01.011
Sterodimas A, de Faria J, Correa WE, et al., 2009, Tissue engineering and auricular reconstruction: A Review. J Plast Reconstr Aesthet Surg, 62: 447–452. https://doi.org/10.1016/j.bjps.2008.11.046
Jia L, Zhang Y, Yao L, et al., 2020, Regeneration of human-ear-shaped cartilage with acellular cartilage matrix-based biomimetic scaffolds. Appl Mater Today, 20: 100639. https://doi.org/10.1016/j.apmt.2020.100639
Ning L, Mehta R, Cao C, et al., 2020, Embedded 3D bioprinting of gelatin methacryloyl-based constructs with highly tunable structural fidelity. ACS Appl Mater Interfaces, 12: 44563–44577. https://doi.org/10.1021/acsami.0c15078
Duin S, Schutz K, Ahlfeld T, et al., 2019, 3D Bioprinting of functional islets of langerhans in an alginate/methylcellulose hydrogel blend. Adv Healthc Mater, 8: e1801631. https://doi.org/10.1002/adhm.201801631
Rakin RH, Kumar H, Rajeev A, et al., 2021, Tunable metacrylated hyaluronic acid-based hybrid bioinks for stereolithography 3D bioprinting. Biofabrication, 13: 044109. https://doi.org/10.1088/1758-5090/ac25cb
Rastogi P, Kandasubramanian B, 2019, Review of alginate-based hydrogel bioprinting for application in tissue engineering. Biofabrication, 11: 042001. https://doi.org/10.1088/1758-5090/ab331e
Levett PA, Melchels FP, Schrobback K, et al., 2014, A biomimetic extracellular matrix for cartilage tissue engineering centered on photocurable gelatin, hyaluronic acid and chondroitin sulfate. Acta Biomater, 10: 214–223. https://doi.org/10.1016/j.actbio.2013.10.005
Gungor-Ozkerim PS, Inci I, Zhang YS, et al., 2018, Bioinks for 3D bioprinting: An overview. Biomater Sci, 6: 915–946. https://doi.org/10.1039/c7bm00765e
Huang J, Xiong J, Wang D, et al., 2021, 3D bioprinting of hydrogels for cartilage tissue engineering. Gels, 7: 144. https://doi.org/10.3390/gels7030144
Kang HW, Lee SJ, Ko IK, et al., 2016, A 3D bioprinting system to produce human-scale tissue constructs with structural integrity. Nat Biotechnol, 34: 312–319. https://doi.org/10.1038/nbt.3413
Sun Y, You Y, Jiang W, et al., 2019, 3D-bioprinting a genetically inspired cartilage scaffold with GDF5-conjugated bmsc-laden hydrogel and polymer for cartilage repair. Theranostics, 9: 6949–6961. https://doi.org/10.7150/thno.38061
Matai I, Kaur G, Seyedsalehi A, et al., 2020, Progress in 3D bioprinting technology for tissue/organ regenerative engineering. Biomaterials, 226: 119536. https://doi.org/10.1016/j.biomaterials.2019.119536
Klotz BJ, Gawlitta D, Rosenberg A, et al., 2016, Gelatin-methacryloyl hydrogels: Towards biofabrication-based tissue repair. Trends Biotechnol, 34: 394–407. https://doi.org/10.1016/j.tibtech.2016.01.002
Yang R, Chen F, Guo J, et al., 2020, Recent advances in polymeric biomaterials-based gene delivery for cartilage repair. Bioact Mater, 5: 990–1003. https://doi.org/doi.org/10.1016/j.bioactmat.2020.06.004
Yue K, Trujillo-de Santiago G, Alvarez MM, et al., 2015, Synthesis, properties, and biomedical applications of gelatin methacryloyl (gelma) hydrogels. Biomaterials, 73: 254–271. https://doi.org/10.1016/j.biomaterials.2015.08.045
Bhamare N, Tardalkar K, Parulekar P, et al., 2021, 3D printing of human ear pinna using cartilage specific ink. Biomed Mater, 16: 055008. https://doi.org/10.1088/1748-605X/ac15b0
Taghipour YD, Hokmabad VR, Del Bakhshayesh AR, et al., 2020, The application of hydrogels based on natural polymers for tissue engineering. Curr Med Chem, 27: 2658–2680. https://doi.org/10.2174/0929867326666190711103956
Pertile RA, Moreira S, Gil da Costa RM, et al., 2012, Bacterial cellulose: Long-term biocompatibility studies. J Biomater Sci Polym Ed, 23: 1339–1354. https://doi.org/10.1163/092050611X581516
McKenna BA, Mikkelsen D, Wehr JB, et al., 2009, Mechanical and structural properties of native and alkali-treated bacterial cellulose produced by gluconacetobacter xylinus strain ATCC 53524. Cellulose, 16: 1047–1055. https://doi.org/10.1007/s10570-009-9340-y
Athukoralalage SS, Balu R, Dutta NK, et al., 2019, 3D bioprinted nanocellulose-based hydrogels for tissue engineering applications: A brief review. Polymers (Basel), 11: 898. https://doi.org/10.3390/polym11050898
Singhsa P, Narain R, Manuspiya H, 2017, Bacterial cellulose nanocrystals (BCNC) preparation and characterization from three bacterial cellulose sources and development of functionalized bcncs as nucleic acid delivery systems. ACS Appl Nano Mater, 1:209–221. https://doi.org/10.1021/acsanm.7b00105
Duchi S, Onofrillo C, O’Connell CD, et al., 2017, Handheld co-axial bioprinting: application to in situ surgical cartilage repair. Sci Rep, 7: 5837. https://doi.org/10.1038/s41598-017-05699-x
Jia L, Hua Y, Zeng J, et al., 2022, Bioprinting and regeneration of auricular cartilage using a bioactive bioink based on microporous photocrosslinkable acellular cartilage matrix. Bioact Mater, 16: 66–81. https://doi.org/10.1016/j.bioactmat.2022.02.032
Xu Y, Zhou J, Liu C, et al., 2021, Understanding the role of tissue-specific decellularized spinal cord matrix hydrogel for neural stem/progenitor cell microenvironment reconstruction and spinal cord injury. Biomaterials, 268: 120596. https://doi.org/10.1016/j.biomaterials.2020.120596
Hua Y, Xia H, Jia L, et al., 2021, Ultrafast, tough, and adhesive hydrogel based on hybrid photocrosslinking for articular cartilage repair in water-filled arthroscopy. Sci Adv, 7: eabg0628. https://doi.org/10.1126/sciadv.abg0628
Mendoza L, Batchelor W, Tabor RF, et al., 2018, Gelation mechanism of cellulose nanofibre gels: A colloids and interfacial perspective. J Colloid Interface Sci, 509: 39–46. https://doi.org/10.1016/j.jcis.2017.08.101
Fourati Y, Tarres Q, Delgado-Aguilar M, et al., 2021, Cellulose nanofibrils reinforced PBAT/TPS blends: Mechanical and rheological properties. Int J Biol Macromol, 183: 267–275. https://doi.org/10.1016/j.ijbiomac.2021.04.102
Fan Y, Yue Z, Lucarelli E, et al., 2020, Hybrid printing using cellulose nanocrystals reinforced GelMA/HAMA hydrogels for improved structural integration. Adv Healthc Mater, 9: e2001410. https://doi.org/10.1002/adhm.202001410
Rathan S, Dejob L, Schipani R, et al., 2019, Fiber reinforced cartilage ECM functionalized bioinks for functional cartilage tissue engineering. Adv Healthc Mater, 8: e1801501. https://doi.org/10.1002/adhm.201801501
Loh E, Fauzi MB, Ng MH, et al., 2018, Cellular and molecular interaction of human dermal fibroblasts with bacterial nanocellulose composite hydrogel for tissue regeneration. ACS Appl Mater Interfaces, 10: 39532–39543. https://doi.org/10.1021/acsami.8b16645
Zhang P, Chen L, Zhang Q, et al., 2016, Using in situ nanocellulose-coating technology based on dynamic bacterial cultures for upgrading conventional biomedical materials and reinforcing nanocellulose hydrogels. Biotechnol Prog, 32:1077–1084. https://doi.org/10.1002/btpr.2280
Griffin MF, Premakumar Y, Seifalian AM, et al., 2016, Biomechanical characterisation of the human auricular cartilages; implications for tissue engineering. Ann Biomed Eng, 44:3460–3467. https://doi.org/10.1007/s10439-016-1688-1
Qasim M, Chae DS, Lee NY, 2019, Advancements and frontiers in nano-based 3D and 4D scaffolds for bone and cartilage tissue engineering. Int J Nanomed, 14:4333–4351. https://doi.org/10.2147/IJN.S209431
Zhao X, Lang Q, Yildirimer L, et al., 2016, Photocrosslinkable gelatin hydrogel for epidermal tissue engineering. Adv Healthc Mater, 5:108–118. https://doi.org/10.1002/adhm.201500005
Markstedt K, Mantas A, Tournier I, et al., 2015, 3D bioprinting human chondrocytes with nanocellulose-alginate bioink for cartilage tissue engineering applications. Biomacromolecules, 16: 1489–1496. https://doi.org/10.1021/acs.biomac.5b00188
Ávila HM, Schwarz S, Rotter N, et al., 2016, 3D bioprinting of human chondrocyte-laden nanocellulose hydrogels for patient-specific auricular cartilage regeneration. Bioprinting, 1–2: 22–35. https://doi.org/10.1016/j.bprint.2016.08.003