Hyaluronic acid (HA)-based hydrogels have been widely used as cell culture substrates, adhesives and fillers for clinical therapeutics, and drug delivery vehicles, with promising results. Due to the limited mechanical properties, degradation rates, and biological activity of HA, it is important that HA be chemically modified, cross-linked or reinforced with specific components to confer a versatile range of viscoelastic and biophysical cues. Herein, we present a toolbox comprising of a class of in situ cross-linkable hydrogels encompassing a blend of three components: HA, cellulose nanocrystals (CNCs) and tropoelastin (TROPO). The resulting hydrogels confer tenable mechanical and biological properties which benefit from the merits of the individual components. Incorporation of CNCs enhanced the hydrogel’s stability against hydrolytic and enzymatic degradation, due to the chemical integration of the nanoparticles within the HA mesh. Additionally, the incorporation of TROPO enhanced elasticity and resilience. Finally, the developed hydrogels demonstrated a positive in vitro biological response as they were able to support and promote the adhesion, viability and growth of the seeded fibroblasts. These results demonstrate a feasible approach for the production of hydrogels exhibiting key structural stability, biological response and improved elastic resilience that can be advantageously explored in acellular and cellular tissue engineering strategies for the regeneration of various tissues, such as the vocal fold.
TibbittMWAnsethKS.Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol Bioeng2009;
103: 655–663.
2.
GeckilHZhangXMoonS, et al.
Engineering hydrogels as extracellular matrix mimics. Nanomedicine (Lond)2010;
5: 469–484.
3.
GrahamNB.Hydrogels: their future, Part I. Med Device Technol1998;
9: 18–22.
4.
JeongBBaeYHLeeDS, et al.
Biodegradable block copolymers as injectable drug-delivery systems. Nature1997;
388: 860–862.
5.
CheniteAChaputCWangD, et al.
Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials2000;
21: 2155–2161.
6.
TemenoJSMikosAG.Injectable biodegradable materials for orthopedic tissue engineering. Biomaterials2000;
21: 2405–2412.
7.
DominguesRMASilvaMGershovichP, et al.
Development of injectable hyaluronic acid/cellulose nanocrystals bionanocomposite hydrogels for tissue engineering applications. Bioconjugate Chem2015;
26: 1571–1581.
8.
ThibeaultSLKlemukSAChenX, et al.
In vivo engineering of the vocal fold ECM with injectable HA hydrogels — late effects on tissue repair and biomechanics in a rabbit model. J Voice2011;
25: 249–253.
9.
GuptaDTatorCHShoichetMS.Fast-gelling injectable blend of hyaluronan and methylcellulose for intrathecal, localized delivery to the injured spinal cord. Biomaterials2006;
27: 2370–2379.
10.
PriceRDMyersSLeighIM, et al.
The role of hyaluronic acid in wound healing – assessment of clinical evidence. Am J Clin Dermatol2005;
6: 393–402.
11.
BishopPN.Structural macromolecules and supramolecular organisation of the vitreous gel. Prog Retin Eye Res2000;
19: 323–344.
12.
HuberMTrattnigSLintnerF.Anatomy, biochemistry, and physiology of articular cartilage. Invest Radiol2000;
35: 573–580.
13.
ButlerJEHammondTHGraySD.Gender-related differences of hyaluronic acid distribution in the human vocal fold. Laryngoscope2001;
111: 907–911.
TooleBP.Hyaluronan: from extracellular glue to pericellular cue. Nat Rev Cancer2004;
4: 528–539.
16.
ChenWYJAbatangeloG.Functions of hyaluronan in wound repair. Wound Repair Regen1999;
7: 79–89.
17.
YangXBakaicEHoareT, et al.
Injectable polysaccharide hydrogels reinforced with cellulose nanocrystals: morphology, rheology, degradation, and cytotoxicity. Biomacromolecules2013;
14: 4447–4455.
18.
DashRFostonMRagauskasAJ.Improving the mechanical and thermal properties of gelatin hydrogels cross-linked by cellulose nanowhiskers. Carbohydr Polym2013;
91: 638–645.
19.
YangJHanCXuF, et al.
Simple approach to reinforce hydrogels with cellulose nanocrystals. Nanoscale; 2014; 6: 5934–5943.
BondesonDMathewAOksmanK.Optimization of the isolation of nanocrystals from microcrystalline cellulose by acid hydrolysis. Cellulose2006;
13: 171–180.
22.
DominguesRMAGomesMEReisRL.The potential of cellulose nanocrystals in tissue engineering strategies. Biomacromolecules2014;
15: 2327–2346.
23.
WiseSGWeissAS.Tropoelastin. Int J Biochem Cell Biol2009;
41: 494–497.
24.
YeoGCBaldockCWiseSG, et al.
Targeted modulation of tropoelastin structure and assembly. ACS Biomater Sci Eng2017;
3: 2832–2844.
25.
MithieuxSMWiseSGWeissAS.Tropoelastin – a multifaceted naturally smart material. Adv Drug Deliv Rev2013;
65: 421–428.
26.
BroekelmannTJKozelBAIshibashiH, et al.
Tropoelastin interacts with cell-surface glycosaminoglycans via its COOH-terminal domain. J Biol Chem2005;
280: 40939–40947.
27.
BaldockCOberhauserAFMaL, et al.
Shape of tropoelastin, the highly extensible protein that controls human tissue elasticity. Proc Natl Acad Sci2011;
108: 4322–4327.
28.
SilvaCRBaboPSGulinoM, et al.
Injectable and tunable hyaluronic acid hydrogels releasing chemotactic and angiogenic growth factors for endodontic regeneration. Acta Biomater2018;
77: 155–171.
29.
VercruysseKPMarecakDMMarecekJF, et al.
Synthesis and in vitro degradation of new polyvalent hydrazide cross-linked hydrogels of hyaluronic acid. Bioconjugate Chem. 1997;
8: 686–694.
30.
YeoYHighleyCBBellasE, et al.
In situ cross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesions in a rabbit model. Biomaterials2006;
27: 4698–4705.
31.
DahlmannJKrauseAMöllerL, et al.
Fully defined in situ cross-linkable alginate and hyaluronic acid hydrogels for myocardial tissue engineering. Biomaterials2013;
34: 940–951.
32.
TanHChuCRPayneKA, et al.
Injectable in situ forming biodegradable chitosan – hyaluronic acid based hydrogels for cartilage tissue engineering. Biomaterials2009;
30: 2499–2506.
33.
BaboPSantoVEDuarteARC, et al.
Platelet lysate membranes as new autologous templates for tissue engineering applications. Inflamm Regen2014;
34: 033–044.
34.
LüSLiuMNiB.An injectable oxidized carboxymethylcellulose/N-succinyl-chitosan hydrogel system for protein delivery. Chem Eng J2010;
160: 779–787.
35.
LiuCXiaZCzernuszkaJT.Design and development of three-dimensional scaffolds for tissue engineering. Chem Eng Res Des2001;
85: 1051–1064.
36.
CollinsMNBirkinshawC.Hyaluronic acid based scaffolds for tissue engineering – a review. Carbohydr Polym2013;
92: 1262–1279.
TathamASShewryPR.Comparative structures and properties of elastic proteins. Philos Trans R Soc Lond, B, Biol Sci2002;
357: 229–234.
39.
UrryDWHugelTSeitzM, et al.
Elastin: a representative ideal protein elastomer. Philos Trans R Soc Lond, B, Biol Sci2002;
357: 169–184.
40.
OssipovDAPiskounovaSVargheseOP, et al.
Functionalization of hyaluronic acid with chemoselective groups via a disulfide-based protection strategy for in situ formation of mechanically stable hydrogels. Biomacromolecules2010;
11: 2247–2254.
41.
ItoTYeoYHighleyCB, et al.
The prevention of peritoneal adhesions by in situ cross-linking hydrogels of hyaluronic acid and cellulose derivatives. Biomaterials2007;
28: 975–983.
42.
GheduzziDGuerraDBochicchioB, et al.
Heparan sulphate interacts with tropoelastin, with some tropoelastin peptides and is present in human dermis elastic fibers. Matrix Biol2005;
24: 15–25.
43.
ReinbothBHanssenEClearyEG, et al.
Molecular interactions of biglycan and decorin with elastic fiber components. J Biol Chem2002;
277: 3950–3957.
44.
KozelBARongishBJCzirokA, et al.
Elastic fiber formation: a dynamic view of extracellular matrix assembly using timer reporters. J Cell Physiol2006;
207: 87–96.
45.
TuYWeissAS.Glycosaminoglycan-mediated coacervation of tropoelastin abolishes the critical concentration, accelerates coacervate formation, and facilitates spherule fusion: implications for tropoelastin microassembly. Biomacromolecules2008;
9: 1739–1744.
46.
KucurMKaradagBIsmanFK, et al.
Plasma hyaluronidase activity as an indicator of atherosclerosis in patients with coronary artery disease. Bratislava Med J2009;
110: 21–26.
47.
TungJ-SMarkGEHollisGF.A microplate assay for hyaluronidase and hyaluronidase inhibitors. Anal Biochem1994;
223: 149–152.
48.
ZhouCWuQYueY, et al.
Application of rod-shaped cellulose nanocrystals in polyacrylamide hydrogels. J Colloid Interface Sci2011;
353: 116–123.
49.
AnnabiNFathiAMithieuxSM, et al.
The effect of elastin on chondrocyte adhesion and proliferation on poly (ɛ-caprolactone)-elastin composites. Biomaterials2011;
32: 1517–1525.
50.
WangKNuneKCMisraR.The functional response of alginate-gelatin-nanocrystalline cellulose injectable hydrogels toward delivery of cells and bioactive molecules. Acta Biomater2016;
36: 143–151.
51.
ChanRWTitzeIR.Hyaluronic acid (with fibronectin) as a bioimplant for the vocal fold mucosa. Laryngoscope1999;
109: 1142–1149.
52.
KlemukSATitzeIR.Viscoelastic properties of three vocal-fold injectable biomaterials at low audio frequencies. Laryngoscope2004;
114: 1597–1603.
53.
BrienFJOHarleyBAYannasIV, et al.
The effect of pore size on cell adhesion in collagen-GAG scaffolds. Biomaterials2005;
26: 433–441.
54.
ZeltingerJPhDSherwoodJK, et al.
Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition. Tissue Eng2001;
7: 557–572.
55.
PricolaKLKuhnNZHaleem-SmithH, et al.
Interleukin-6 maintains bone marrow-derived mesenchymal stem cell stemness by an ERK1/2-dependent mechanism. J Cell Biochem2009;
108: 577–588.
56.
BolandGMPerkinsGHallDJ, et al.
Wnt 3a promotes proliferation and suppresses osteogenic differentiation of adult human mesenchymal stem cells. J Cell Biochem2004;
93: 1210–1230.
57.
AnnabiNMithieuxSMBoughtonEA, et al.
Synthesis of highly porous crosslinked elastin hydrogels and their interaction with fibroblasts in vitro. Biomaterials2009;
30: 4550–4557.
58.
YeungTGeorgesPCFlanaganLA, et al.
Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil Cytoskeleton2005;
60: 24–34.
59.
ZamanMHTrapaniLMSieminskiA, et al.
Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis. Proc Natl Acad Sci USA2006;
103: 10889–10894.
60.
SchwartzMA.Integrins and Extracellular Matrix in Mechanotransduction. Cold Spring Harb Perspect Biol2010;
2: a005066
61.
DeForestCAAnsethKS.Advances in bioactive hydrogels to probe and direct cell fate. Annu Rev Chem Biomol Eng2012;
3: 421–444.
62.
SalbachJRachnerTDRaunerM, et al.
Regenerative potential of glycosaminoglycans for skin and bone. J Mol Med2012;
90: 625–635.
63.
HortensiusRAHarleyB.The use of bioinspired alterations in the glycosaminoglycan content of collagen-GAG scaffolds to regulate cell activity. Biomaterials2013;
34: 7645–7652.
64.
EhmannHMAMohanTKoshanskayaM, et al.
Design of anticoagulant surfaces based on cellulose nanocrystals. Chem Commun (Camb)2014;
50: 13070–13072.
