{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,25]],"date-time":"2025-11-25T17:42:48Z","timestamp":1764092568376,"version":"3.45.0"},"reference-count":35,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,11,24]],"date-time":"2025-11-24T00:00:00Z","timestamp":1763942400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ongoing Research Funding Program","award":["ORF-2025-1467"],"award-info":[{"award-number":["ORF-2025-1467"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Gels"],"abstract":"<jats:p>Gelatin methacryloyl (GelMA) hydrogels are widely used in tissue engineering because of their tunable mechanical and biological properties. However, many studies have arbitrarily selected key synthesis parameters, such as methacrylic anhydride (MA) concentration, (lithium phenyl-2 4 6-trimethyl-benzoyl phosphinate) LAP concentration, GelMA content, UV exposure time, and reaction duration, without clear justification. This study aimed to systematically optimize GelMA hydrogel fabrication and evaluate the mechanical and biological performances of the resulting hydrogels for craniofacial muscle tissue engineering. Hydrogels were synthesized following a standardized protocol, and the reaction progress was confirmed via proton nuclear magnetic resonance (1H-NMR). The swelling ratio, degradation behavior, compressive strength, and metabolic activity (AlamarBlue assay using C2C12 myoblasts) were assessed. Statistical analysis was performed using independent t-tests and one-way ANOVA with Tukey\u2019s post hoc test (p &lt; 0.05). The results showed that small variations in MA concentration and reaction time significantly affected the hydrogel properties. Higher GelMA concentrations (10\u201320%) enhanced the mechanical strength but reduced the biological activity. LAP \u2265 0.5% and prolonged UV exposure lowered metabolic activity, whereas 0.1% LAP with 1\u20132 min of UV exposure provided an optimal balance. These findings provide a reproducible framework for GelMA fabrication and establish a foundation for developing tailored biomaterials for muscle tissue engineering.<\/jats:p>","DOI":"10.3390\/gels11120945","type":"journal-article","created":{"date-parts":[[2025,11,25]],"date-time":"2025-11-25T17:31:34Z","timestamp":1764091894000},"page":"945","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Optimizing Gelatin Methacryloyl for Craniofacial Muscle Regeneration: Material Design and Application"],"prefix":"10.3390","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8575-7330","authenticated-orcid":false,"given":"Mohammad B.","family":"Aljaber","sequence":"first","affiliation":[{"name":"Department of Dental Health, College of Applied Medical Sciences, King Saud University, P.O. Box 10219, Riyadh 12372, Saudi Arabia"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5569-1668","authenticated-orcid":false,"given":"Omar","family":"Alageel","sequence":"additional","affiliation":[{"name":"Department of Dental Health, College of Applied Medical Sciences, King Saud University, P.O. Box 10219, Riyadh 12372, Saudi Arabia"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9200-6749","authenticated-orcid":false,"given":"David Y. S.","family":"Chau","sequence":"additional","affiliation":[{"name":"Division of Biomaterials and Tissue Engineering, Eastman Dental Institute, University College London, Royal Free Hospital Campus, Rowland Hill Street, London NW3 2PF, UK"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3917-3446","authenticated-orcid":false,"given":"Jonathan C.","family":"Knowles","sequence":"additional","affiliation":[{"name":"Division of Biomaterials and Tissue Engineering, Eastman Dental Institute, University College London, Royal Free Hospital Campus, Rowland Hill Street, London NW3 2PF, UK"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2142","DOI":"10.1039\/C7SM02187A","article-title":"Tailoring the mechanical properties of gelatin methacryloyl hydrogels through manipulation of the photocrosslinking conditions","volume":"14","author":"Zhang","year":"2018","journal-title":"Soft Matter"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"130","DOI":"10.18063\/IJB.2017.02.003","article-title":"A dual crosslinking strategy to tailor rheological properties of gelatin methacryloyl","volume":"3","author":"Zhou","year":"2017","journal-title":"Int. J. Bioprint."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1021\/bm990017d","article-title":"Structural and rheological properties of methacrylamide modified gelatin hydrogels","volume":"1","author":"Bogdanov","year":"2000","journal-title":"Biomacromolecules"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Muthuramalingam, K., and Lee, H.J. (2023). Effect of GelMA hydrogel properties on long-term encapsulation and myogenic differentiation of C2C12 spheroids. Gels, 9.","DOI":"10.3390\/gels9120925"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Zhu, M., Wang, Y., Ferracci, G., Zheng, J., Cho, N.J., and Lee, B.H. (2019). Gelatin methacryloyl and its hydrogels with an exceptional degree of controllability and batch-to-batch consistency. Sci. Rep., 9.","DOI":"10.1038\/s41598-019-42186-x"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Ahadian, S., Ram\u00f3n-Azc\u00f3n, J., Estili, M., Liang, X., Ostrovidov, S., Shiku, H., Ramalingam, M., Nakajima, K., Sakka, Y., and Bae, H. (2014). Hybrid hydrogels containing vertically aligned carbon nanotubes with anisotropic electrical conductivity for muscle myofiber fabrication. Sci. Rep., 4.","DOI":"10.1038\/srep04271"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1016\/j.biomaterials.2016.12.026","article-title":"Sequentially-crosslinked bioactive hydrogels as nano-patterned substrates with customizable stiffness and degradation for corneal tissue engineering applications","volume":"120","author":"Rizwan","year":"2017","journal-title":"Biomaterials"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Basara, G., Yue, X., and Zorlutuna, P. (2019). Dual crosslinked gelatin methacryloyl hydrogels for photolithography and 3D printing. Gels, 5.","DOI":"10.3390\/gels5030034"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"14887","DOI":"10.1038\/s41467-020-14887-9","article-title":"Fiber reinforced GelMA hydrogel to induce the regeneration of corneal stroma","volume":"11","author":"Kong","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"582","DOI":"10.1002\/term.1956","article-title":"Three-dimensional co-culture of C2C12\/PC12 cells improves skeletal muscle tissue formation and function","volume":"11","author":"Ostrovidov","year":"2017","journal-title":"J. Tissue Eng. Regen. Med."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"110578","DOI":"10.1016\/j.msec.2019.110578","article-title":"Multi-material 3D bioprinting of porous constructs for cartilage regeneration","volume":"109","author":"Gleadall","year":"2020","journal-title":"Mater. Sci. Eng. C"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Stratesteffen, H., K\u00f6pf, M., Kreimendahl, F., Blaeser, A., Jockenh\u00f6vel, S., and Fischer, H. (2017). GelMA-collagen blends enable drop-on-demand 3D printability and promote angiogenesis. Biofabrication, 9.","DOI":"10.1088\/1758-5090\/aa857c"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1800266","DOI":"10.1002\/macp.201800266","article-title":"Hydrolytic stability of methacrylamide and methacrylate in gelatin methacryloyl and decoupling of gelatin methacrylamide from gelatin methacryloyl through hydrolysis","volume":"219","author":"Zheng","year":"2018","journal-title":"Macromol. Chem. Phys."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Seyedmahmoud, R., \u00c7elebi-Saltik, B., Barros, N., Nasiri, R., Banton, E., Shamloo, A., Ashammakhi, N., Dokmeci, M., and Ahadian, S. (2019). Three-dimensional bioprinting of functional skeletal muscle tissue using gelatin methacryloyl-alginate bioinks. Micromachines, 10.","DOI":"10.3390\/mi10100679"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"5536","DOI":"10.1016\/j.biomaterials.2010.03.064","article-title":"Cell-laden microengineered gelatin methacrylate hydrogels","volume":"31","author":"Nichol","year":"2010","journal-title":"Biomaterials"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"551","DOI":"10.1002\/mabi.201200471","article-title":"Gelatin-methacrylamide hydrogels as potential biomaterials for fabrication of tissue-engineered cartilage constructs","volume":"13","author":"Schuurman","year":"2013","journal-title":"Macromol. Biosci."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Aldana, A.A., Malatto, L., Ur Rehman, M.A., Boccaccini, A.R., and Abraham, G.A. (2019). Fabrication of gelatin methacrylate (Gel-MA) scaffolds with nano- and micro-topographical and morphological features. Nanomaterials, 9.","DOI":"10.3390\/nano9010120"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1039\/C9BM01236B","article-title":"Cell-loaded 3D bioprinted GelMA hydrogels for corneal stroma engineering","volume":"8","author":"Hasirci","year":"2020","journal-title":"Biomater. Sci."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Pepelanova, I., Kruppa, K., Scheper, T., and Lavrentieva, A. (2018). Gelatin-methacryloyl (GelMA) hydrogels with defined degree of functionalization as a versatile toolkit for 3D cell culture and extrusion bioprinting. Bioengineering, 5.","DOI":"10.3390\/bioengineering5030055"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2212","DOI":"10.1002\/jbm.a.37206","article-title":"Utilization of GelMA with phosphate glass fibers for glial cell alignment","volume":"109","author":"Patel","year":"2021","journal-title":"J. Biomed. Mater. Res. A"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Costantini, M., Testa, S., Fornetti, E., Barbetta, A., Trombetta, M., Cannata, S.M., Gargioli, C., and Rainer, A. (2017). Engineering muscle networks in 3D gelatin methacryloyl hydrogels: Influence of mechanical stiffness and geometrical confinement. Front. Bioeng. Biotechnol., 5.","DOI":"10.3389\/fbioe.2017.00022"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1713","DOI":"10.1089\/ten.tea.2010.0666","article-title":"Synthesis and characterization of tunable poly(ethylene glycol): Gelatin methacrylate composite hydrogels","volume":"17","author":"Hutson","year":"2011","journal-title":"Tissue Eng. Part A"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"7891","DOI":"10.2147\/IJN.S175619","article-title":"Electrospun nanofiber blend with improved mechanical and biological performance","volume":"13","author":"Lobo","year":"2018","journal-title":"Int. J. Nanomed."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2093","DOI":"10.1039\/C7BM00110J","article-title":"In vitro and in vivo analysis of visible light crosslinkable gelatin methacryloyl (GelMA) hydrogels","volume":"5","author":"Noshadi","year":"2017","journal-title":"Biomater. Sci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1305","DOI":"10.1016\/j.jbiomech.2016.03.014","article-title":"Change in viability of C2C12 myoblasts under compression, shear and oxidative challenges","volume":"49","author":"Hong","year":"2016","journal-title":"J. Biomech."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1845","DOI":"10.1016\/j.biomaterials.2013.11.009","article-title":"Interpenetrating networks based on gelatin methacrylamide and PEG formed using concurrent thiol click chemistries for hydrogel tissue engineering scaffolds","volume":"35","author":"Daniele","year":"2014","journal-title":"Biomaterials"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Young, A.T., White, O.C., and Daniele, M.A. (2020). Rheological properties of coordinated physical gelation and chemical cross-linking in gelatin methacryloyl (GelMA) hydrogels. Macromol. Biosci., 20.","DOI":"10.1002\/mabi.202000183"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1021\/acs.biomac.7b01221","article-title":"Quantification of substitution of gelatin methacryloyl: Best practice and current pitfalls","volume":"19","author":"Truffault","year":"2018","journal-title":"Biomacromolecules"},{"key":"ref_29","first-page":"797","article-title":"Nanoengineered myogenic scaffolds for skeletal muscle tissue engineering","volume":"3","author":"Quint","year":"2014","journal-title":"Nanoscale"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"254","DOI":"10.1016\/j.biomaterials.2015.08.045","article-title":"Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels","volume":"73","author":"Yue","year":"2015","journal-title":"Biomaterials"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1002\/jbm.a.36226","article-title":"Gelatin methacrylate scaffold for bone tissue engineering: The influence of polymer concentration","volume":"106","author":"Celikkin","year":"2018","journal-title":"J. Biomed. Mater. Res. A"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Li, X., Chen, S., Li, J., Wang, X., Zhang, J., Kawazoe, N., and Chen, G. (2016). 3D culture of chondrocytes in gelatin hydrogels with different stiffness. Polymers, 8.","DOI":"10.3390\/polym8080269"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Aljaber, M.B., Verisqa, F., Keskin-Erdogan, Z., Patel, K.D., Chau, D.Y.S., and Knowles, J.C. (2023). Influence of gelatin source and Bloom number on gelatin methacryloyl hydrogels mechanical and biological properties for muscle regeneration. Biomolecules, 13.","DOI":"10.3390\/biom13050811"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1038\/nprot.2016.037","article-title":"Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue culture platforms","volume":"11","author":"Loessner","year":"2016","journal-title":"Nat. Protoc."},{"key":"ref_35","unstructured":"Aljaber, M.B.S. (2024). The Utilisation of GelMA-Based Surface-Patterned Scaffolds for Craniofacial Muscle Regeneration Applications, University College London."}],"container-title":["Gels"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2310-2861\/11\/12\/945\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,25]],"date-time":"2025-11-25T17:40:10Z","timestamp":1764092410000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2310-2861\/11\/12\/945"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,24]]},"references-count":35,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["gels11120945"],"URL":"https:\/\/doi.org\/10.3390\/gels11120945","relation":{},"ISSN":["2310-2861"],"issn-type":[{"value":"2310-2861","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,24]]}}}