Novel Ultrashort Self-Assembling Peptide Bioinks for 3D Culture of Muscle Myoblast Cells

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Wafaa Arab, Sakandar Rauf, Ohoud Al-Harbi, Charlotte Hauser


The ability of skeletal muscle to self-repair after a traumatic injury, tumor ablation, or muscular disease is slow and limited, and the capacity of skeletal muscle to self-regenerate declines steeply with age. Tissue engineering of functional skeletal muscle using 3D bioprinting technology is promising for creating tissue constructs that repair and promote regeneration of damaged tissue. Hydrogel scaffolds used as biomaterials for skeletal muscle tissue engineering can provide chemical, physical and mechanical cues to the cells in three dimensions thus promoting regeneration. Herein, we have developed two synthetically designed novel tetramer peptide biomaterials. These peptides are self-assembling into a nanofibrous 3D network, entrapping 99.9% water and mimicking the native collagen of an extracellular matrix. Different biocompatibility assays including MTT, 3D cell viability assay, cytotoxicity assay and live-dead assay confirm the biocompatibility of these peptide hydrogels for mouse myoblast cells (C2C12). Immunofluorescence analysis of cell-laden hydrogels revealed that the proliferation of C2C12 cells was well-aligned in the peptide hydrogels compared to the alginate-gelatin control. These results indicate that these peptide hydrogels are suitable for skeletal muscle tissue engineering. Finally, we tested the printability of the peptide bioinks using a commercially available 3D bioprinter. The ability to print these hydrogels will enable future development of 3D bioprinted scaffolds containing skeletal muscle myoblasts for tissue engineering applications.


Biomaterials; Bioinks; 3D cell culture; 3D Scaffold; Tissue Engineering; Skeletal Muscle Cells.

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Stilhano R S, Madrigal J L, Wong K, et al., 2016, Injectable alginate hydrogel for enhanced spatiotemporal control of lentivector delivery in murine skeletal muscle. J Control Release, 237: 42–49.

Chaturvedi V, Dye D E, Kinnear B F, et al., 2015, Interactions between skeletal muscle myoblasts and their extracellular matrix revealed by a serum free culture system. PLO S, 10(6): 1–27.

Järvinen T A H, Järvinen T L N, Kääriäinen M, et al., 2007, Muscle injuries: Optimising recovery. Best Pract Res Clin Rheumatol, 2(2): 317–331.

Manring H, Abreu E, Brotto N, et al., 2014, Novel excitation-contraction coupling related genes reveal aspects of muscle weakness beyond atrophy: New hopes for treatment of musculoskeletal diseases. Front Physiol, 5: 1–12. http://dx.doi:10.3389/fphys.2014.00037

Grasman J M, Zayas M J, Page R L, et al., 2015, Biomimetic scaffolds for regeneration of volumetric muscle loss in skeletal muscle injuries. Acta Biomater, 25: 2–15.

Zorlutuna P, Annabi N, Camci-Unal G, et al., 2012, Microfabricated biomaterials for engineering 3D tissues. Adv Mater, 24(14): 1782–1804. https://dx.doi:10.1002/adma.201104631

Sato M, Ito A, Kawabe Y, et al., 2011, Enhanced contractile force generation by artificial skeletal muscle tissues using IGF-I gene-engineered myoblast cells. J Biosci Bioeng, 112(3): 273–278.

Lepper C, Partridge T A, Fan C M, 2011, An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration. Development, 138(17): 3639–3646.

Kuraitis D, Giordano C, Ruel M, et al., 2012, Exploiting extracellular matrix-stem cell interactions: A review of natural materials for therapeutic muscle regeneration. Biomaterials, 33(2): 428–443.

Atala A, Bauer S B, Soker S, et al., 2006, Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet, 367(9518): 1241–1246.http://dx.doi:10.1016/S0140-6736(06)68438-9

Carsin H, Ainaud P, Le Bever H, et al., 2000, Cultured epithelial autografts in extensive burn coverage of severely traumatized patients: A five year single center experience with 30 patients. Burns, 26(4): 379–387.

Raya-Rivera A, Esquiliano D R, Yoo J J, et al., 2011, Tissue-engineered autologous urethras for patients who need reconstruction: An observational study. Lancet, 377(9772): 1175–1182.

Warnke P H, Springer I N, Wiltfang J, et al., 2004, Growth and transplantation of a custom vascularised bone graft in a man. Lancet, 364(9436): 766–770.

Atala A, Kasper F K, Mikos A G, 2012, Engineering complex tissues. Sci Transl Med, 4(160): 160 rv12.

Murphy S V, Atala A, 2014, 3D bioprinting of tissues and organs. Nat Biotechnol, 32(8): 773–785.

Derby B, 2012, Printing, and prototyping of tissues and scaffolds. Science, 338(6109): 921–926.

Sundaramurthi D, Rauf S, Hauser C A, 2016, 3D bioprinting technology for regenerative medicine applications. Int J Bioprint, 2(2): 117–135.

Hauser C A, Zhang, 2010, Designer self-assembling peptide nanofiber biological materials, 2010, Chem Soc Rev, 39(8): 2780–2790.

Loo Y, Zhang S, Hauser C A, 2012, From short peptides to nanofibers to macromolecular assemblies in biomedicine. Biotechnol Advs, 30(3): 593–603.

Wu E C, Zhang S G, Hauser C A E, 2012, Self-assembling peptides as cell-interactive scaffolds. Adv Funct Mater, 22(3): 456–468.

Hauser C A, Deng R, Mishra A, et al., 2011, Natural tri- to hexapeptides self-assemble in water to amyloid β-type fiber aggregates by unexpected α-helical intermediate structures. Proceed Natl Acad Sci, 108(4): 1361–1366.

Mishra A, Loo Y, Deng R, et al., 2011, Ultrasmall natural peptides self-assemble to strong temperature-resistant helical fibers in scaffolds suitable for tissue engineering. Nano Today, 6: 232–239.

Reithofer M R, Chan K H, Lakshmanan A, et al., 2014, Ligation of anti-cancer drugs to selfassembling ultrashort peptides by click chemistry for localized therapy. Chem Sci, 5: 625–630.

Loo Y, Wong Y C, Cai E Z, et al., 2014, Ultrashort peptide nanofibrous hydrogels for the acceleration of healing of burn wounds. Biomaterials, 35(17): 4805–4814. 047

Kroehne V, Heschel I, Schügner F, et al., 2008, Use of a novel collagen matrix with oriented pore structure for muscle cell differentiation in cell culture and in grafts. J Cell Mol Med, 12(5a): 1640–1648.

Kang H W, Lee S J, Ko I K, et al., 2016, A 3D bioprinting system to produce human-scale tissue constructs with structural integrity. Nat Biotechnol, 34(3): 312–319.

Chen S, Nakamoto T, Kawazoe N, et al., 2015, Engineering multi-layered skeletal muscle tissue by using 3D micro­grooved collagen scaffolds. Biomaterials, 73: 23–31.

Jana S, Cooper A, Zhang M, 2013, Chitosan scaffolds with unidirectional microtubular pores for large skeletal myotube generation. Adv Healthc Mater, 2(4): 557–561.

Jana S, Levengood S K L, Zhang M, 2016, Anisotropic materials for skeletal-muscle-tissue engineering. Adv Mater, 28(48): 10588–10612. 02/adma.201600240

Koning M, Harmsen M C, Van Luyn M J A, et al., 2009, Current opportunities and challenges in skeletal muscle tissue engineering. J Tissue Eng Regen Med, 3(6): 407–415.

Bian W, Bursac N, 2008, Tissue engineering of functional skeletal muscle: Challenges and recent advances. IEEE Eng Med Biol Mag, 27(5): 109–113.

Pollot B E, Rathbone C R, Wenke J C, et al., 2017, Natural polymeric hydrogel evaluation for skeletal muscle tissue engineering. J Biomed Mater Res B Appl Biomater.

Loo Y, Lakshmanan A, Ni M, et al., 2015, Peptide bioink: Self-assembling nanofibrous scaffolds for three-dimensional organotypic cultures. Nano Lett, 15(10): 6919–6925.

Taylor S E, Cao T, Talauliker P M, et al., 2013, Objective morphological quantification of microscopic images using a fast fourier transform (FFT) analysis. Curr Protoc Essent Lab Tech, 7(1):9.5.1–9.5.12.

Bajaj P, Reddy B Jr, Millet L, et al., 2011, Patterning the differentiation of C2C12 skeletal myoblasts. RSC, 3(9):897–909.

Matthew D S, Ronald T R, 2010, Collagen structure and stability. Annu Rev Biochem, 78: 929–958.

Shadrin I Y, Khodabukus A, Bursac N, 2016, Striated muscle function, regeneration, and repair cell. Mol Life Sci, 73(22): 4175–4202.

Fuoco C, Petrilli L, Cannata S, et al., 2016, Matrix scaffolding for stem cell guidance toward skeletal muscle tissue engineering, J Orthop Surg Res, 11: 86.

Fuoco C, Cannata S, Gargioli C, 2016, Could a functional artificial skeletal muscle be useful in muscle wasting? Curr Opin Clin Nutr Metab Care, 19(3): 182– 187. 271

Mironov V, Kasyanov V, Drake C, et al., 2008, Organ printing: Promises and challenges. Regen Med, 3(1): 93–103.

Choi Y J, Kim T G, Jeong J, et al., 2016, 3D cell printing of functional skeletal muscle constructs using skeletal muscle derived bioink. Adv Healthc Mater, 5(20): 2636–2645. 201600483

Fedorovich N E, De Wijn J R, Verbout A J, et al., 2008, Three-dimensional fiber deposition of cell-laden, viable, patterned constructs for bone tissue printing. Tissue Eng Part A, 14(1): 127–133.

Aviss K J, Gough J E, Downes S, 2010, Aligned electrospun polymer fibres for skeletal muscle regeneration. Eur Cell Mater, 19(1): 193–204.

Macchiarini P, Jungebluth P, Go T, et al., 2008, Clinical transplantation of a tissue-engineered airway. Lancet, 372(9655): 2023–2030.

Martinello T, Bronzini I, Volpin A, et al., 2012, Successful recellularization of human tendon scaffolds using adipose-derived mesenchymal stem cells and collagen gel. J Tissue Eng Regen Med, 8(8): 612–619.

Badylak S F, 2004, Xenogeneic extracellular matrix as a scaffold for tissue reconstruction. Transpl Immunol, 12(3–4): 367–377.

Jia J, Richards D J, Pollard S, et al., 2014, Engineering alginate as bioink for bioprinting. Acta Biomater, 10(10): 4323–4331.

Pataky K, Braschler T, Negro A, et al., 2012 Microdrop printing of hydrogel bioinks into 3D tissue like geometries. Adv Mater, 24(3): 391–396.

Huijun L, Tan Y J, Leong K F, et al., 2017, 3D bioprinting of highly thixotropic alginate/methylcellulose hydrogel with strong interface bonding. ACS Appl Mater Interfaces, 9(23): 20086–20097.

Li H, Liu S, Li L, 2016, Rheological study on 3D printability of alginate hydrogel and effect of graphene oxide. Int J Bioprint, 2(2): 54–66.

Luo N C and Grover L M, 2010, Cell encapsulation using biopolymer gels for regenerative medicine. Biotechnol Lett, 32(6): 733−742.

Luo K, Yang Y, Shao Z, 2016, Physically crosslinked biocompatible silk-fibroin-based hydrogels with high mechanical performance. Adv Funct Mater, 26(6): 872−880.

Kuen Y L, David J M, 2012, Alginate: Properties and biomedical applications, Prog Polym Sci, 37(1): 106–126. 06.003

Dreesmann L, Ahlers M, Schlosshauer B L, 2007, The pro-angiogenic characteristics of a cross-linked gelatin matrix. Biomaterials, 28(36): 5536–5543.

Sandrasegaran K, Lall C, Rajesh A, et al., 2005, Distinguishing gelatin bioabsorbable sponge and postoperative abdominal abscess on. Am J Roentgenol, 184(2): 475–480.

Balakrishnan B, Mohanty M, Umashankar P R, et al., 2005, Evaluation of an in situ forming hydrogel wound dressing based on oxidized alginate and gelatin, Biomaterials, 26(32), 6335–6342.

Rosellini E, Cristallini C, Barbani N, et al., 2009, Pre­paration and characterization of alginate-gelatin blend films for cardiac tissue engineering. J Biomed Mater Res A, 91(2): 447–453.

Dong Z, Wang Q, Du Y, 2006, Blend films and their properties for drug controlled release. J Memb Sci, 280(1–2): 37–44. 2006.01.002

Fan L, Du L, Huang R, et al., 2005, Preparation and characterization of alginate-gelatin blend fibers. J Appl Polym Sci, 96(5):1625–1629.

Li S, Yan Y, Xiong Z, et al., 2009, Gradient hydrogel construct based on an improved cell assembling system. J Bioact Compat Polym, 24(1): 84–99.

Yan Y, Wang X, Xiong Z, 2005, Direct construction of a three-dimensional structure with cells and hydrogel. J Bioact Compat Polym, 20(3): 259–269.

Li S, Yan Y, Xiong Z, et al., 2009, Gradient hydrogel construct based on an improved cell assembling system. J Bioact Compat Polym, 24(1): 84–99.

Roberto D, Kenneth C H, 2011, Actin structure and function. Annu Rev Biophys, 40: 169–186. http://dx.doi. org/10.1146/annurev-biophys-042910-155359

Dado D, Levenberg S, 2009, Cell-scaffold mechanical interplay within engineered tissue. Semin Cell Dev Biol, 20(6): 656–664. http:// dx. doi. org/ 10. 1016/ j. semcdb. 2009.02.001

Phillips J, Bunting S, Hall S, et al., 2005, Neural tissue engineering: A self-organizing collagen guidance conduit. Tissue Eng, 11(9–10): 1611–1617. 1089/ten.2005.11.1611

Chung C, Bien H, Entcheva E, 2007, The role of cardiac tissue alignment in modulating electrical function. J Cardiovasc Electrophysiol, 18(12): 1323– 1329. /j. 15408167. 2007. 009 59.x

Zhao Y, Zeng H, Nam J, et al., 2009, Fabrication of skeletal muscle constructs by topographic activation of cell alignment. Biotechnol Bioeng, 102(2): 624–631.

Crabb R A, Chau E P, Evans M C, et al., 2006, Biomechanical and Microstructural Characteristics of a Collagen Film-Based Corneal Stroma Equivalent, Tissue Eng, 12(6): 1565–1575.

Zhu Y, Cao Y, Pan J, et al., 2010, Macro-alignment of electrospun fibers for vascular tissue engineering. J Biomed Mater Res B, 92(2): 508–516.

Bajaj P, Khang D and Webster T J, 2006, Control of spatial cell attachment on carbon nanofiber patterns on polycarbonate urethane. Int J Nanomed, 1(3): 361–365.



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