Microscopic Structure of Swollen Hydrogels by Scanning Electron and Light Microscopies: Artifacts and Reality

. 2020 Mar 05 ; 12 (3) : . [epub] 20200305

Status PubMed-not-MEDLINE Jazyk angličtina Země Švýcarsko Médium electronic

Typ dokumentu časopisecké články

Perzistentní odkaz   https://www.medvik.cz/link/pmid32150859

Grantová podpora
17-08531S Grantová Agentura České Republiky
TE01020118 Technologická Agentura České Republiky
TN01000008 Technologická Agentura České Republiky

The exact knowledge of hydrogel microstructure, mainly its pore topology, is a key issue in hydrogel engineering. For visualization of the swollen hydrogels, the cryogenic or high vacuum scanning electron microscopies (cryo-SEM or HVSEM) are frequently used while the possibility of artifact-biased images is frequently underestimated. The major cause of artifacts is the formation of ice crystals upon freezing of the hydrated gel. Some porous hydrogels can be visualized with SEM without the danger of artifacts because the growing crystals are accommodated within already existing primary pores of the gel. In some non-porous hydrogels the secondary pores will also not be formed due to rigid network structure of gels that counteracts the crystal nucleation and growth. We have tested the limits of true reproduction of the hydrogel morphology imposed by the swelling degree and mechanical strength of gels by investigating a series of methacrylate hydrogels made by crosslinking polymerization of glycerol monomethacrylate and 2-hydroxyethyl methacrylate including their interpenetrating networks. The hydrogel morphology was studied using cryo-SEM, HVSEM, environmental scanning electron microscopy (ESEM), laser scanning confocal microscopy (LSCM) and classical wide-field light microscopy (LM). The cryo-SEM and HVSEM yielded artifact-free micrographs for limited range of non-porous hydrogels and for macroporous gels. A true non-porous structure was observed free of artifacts only for hydrogels exhibiting relatively low swelling and high elastic modulus above 0.5 MPa, whereas for highly swollen and/or mechanically weak hydrogels the cryo-SEM/HVSEM experiments resulted in secondary porosity. In this contribution we present several cases of severe artifact formation in PHEMA and PGMA hydrogels during their visualization by cryo-SEM and HVSEM. We also put forward empirical correlation between hydrogel morphological and mechanical parameters and the occurrence and intensity of artifacts.

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Hoffman A.S. Hydrogels for biomedical applications. Adv. Drug Deliv. Rev. 2002;54:3–12. doi: 10.1016/S0169-409X(01)00239-3. PubMed DOI

Macková H., Plichta Z., Hlídková H., Sedláček O., Konefal R., Sadakbayeva Z., Dušková-Smrčková M., Horák D., Kubinová Š. Reductively Degradable Poly(2-hydroxyethyl methacrylate) Hydrogels with Oriented Porosity for Tissue Engineering Applications. ACS Appl. Mater. Interf. 2017;9:10544–10553. doi: 10.1021/acsami.7b01513. PubMed DOI

Šprincl L., Kopeček J., Lím D. Effect of porosity of heterogeneous poly(glycol monomethacrylate) gels on the healing-in of test implants. J. Biomed. Mater. Res. 1971;4:447–458. doi: 10.1002/jbm.820050503. PubMed DOI

Kopeček J., Yang J. Hydrogels as smart biomaterials. Polym. Int. 2007;56:1078–1098. doi: 10.1002/pi.2253. DOI

Savina I.N., Cnudde V., D’Hollander S., van Hoorebeke L., Mattiasson B., Galaev I.Y., Du Prez F. Cryogels from poly(2-hydroxyethyl methacrylate): Macroporous, interconnected materials with potential as cell scaffolds. Soft Matter. 2007;3:1176–1184. doi: 10.1039/b706654f. PubMed DOI

Allen P.E.M., Bennett D.J., Williams D.R.G. Water in methacrylates-I. Sorption and desorption properties of poly(2-hydroxyethyl methacrylate-co-glycol dimethacrylate) networks. Eur. Polym. J. 1992;28:347–352. doi: 10.1016/0014-3057(92)90252-W. DOI

Orakdogen N., Okay O. Influence of the initiator system on the spatial inhomogeneity in acrylamide-based hydrogels. J. Appl. Polym. Sci. 2007;103:3228–3237. doi: 10.1002/app.24977. DOI

Shibayama M. Universality and specificity of polymer gels viewed by scattering methods. Bull. Chem. Soc. Jpn. 2006;79:1799–1819. doi: 10.1246/bcsj.79.1799. DOI

Annabi N., Mithieux S.M., Weiss A.S., Dehghani F. The fabrication of elastin-based hydrogels using high pressure CO2. Biomaterials. 2009;30:1–7. doi: 10.1016/j.biomaterials.2008.09.031. PubMed DOI

Suchý T., Šupová M., Bartoš M., Sedláček R., Piola M., Soncini M., Fiore G.B., Sauerová P., Kalbáčová M.H. Dry versus hydrated collagen scaffolds: Are dry states representative of hydrated states? J. Mater. Sci. Mater. Med. 2018;29 doi: 10.1007/s10856-017-6024-2. PubMed DOI

Dahl R., Staehelin L.A. High-pressure freezing for the preservation of biological structure: Theory and practice. J. Electron. Micros. Tech. 1989;12:165–174. doi: 10.1002/jemt.1060130305. PubMed DOI

Paterson S.M., Casadio Y.S., Brown D.H., Shaw J.A., Chirila T.V., Baker M.V. Laser scanning confocal microscopy versus scanning electron microscopy for characterization of polymer morphology: Sample preparation drastically distorts morphologies of poly(2-hydroxyethyl methacrylate)-based hydrogels. J. Appl. Polym. Sci. 2013;127:4296–4304. doi: 10.1002/app.38034. DOI

Trieu H.H., Qutubuddin S. Polyvinyl alcohol hydrogels I. Microscopic structure by freeze-etching and critical point drying techniques. Colloid Polym. Sci. 1994;3:301–309. doi: 10.1007/BF00655501. DOI

Miller D.R., Peppas N.A. Bulk characterization and scanning electron microscopy of hydrogels of P(VA-co-NVP) Biomaterials. 1986;7:329–339. doi: 10.1016/0142-9612(86)90003-7. PubMed DOI

Aston R., Sewell K., Klein T., Lawrie G., Grøndahl L. Evaluation of the impact of freezing preparation techniques on the characterisation of alginate hydrogels by cryo-SEM. Eur. Polym. J. 2016;82:1–15. doi: 10.1016/j.eurpolymj.2016.06.025. DOI

Šlouf M., Vacková T., Lednický F., Wandrol P. Polymer surface morphology: Characterization by electron microscopies. In: Sabbatini L., editor. Polymer Surface Characterization. DE GRUYTER; Berlin, Germany: Boston, MA, USA: 2014. Chapter 6.

Přádný M., Dušková-Smrčková M., Dušek K., Janoušková O., Sadakbayeva Z., Šlouf M., Michálek M. Macroporous 2-hydroxyethyl methacrylate hydrogels of dual porosity for cell cultivation: Morphology, swelling, permeability, and mechanical behavior. J. Polym. Res. 2014;21:579. doi: 10.1007/s10965-014-0579-0. DOI

Kim J., Yaszemski M.J., Lu L. Three-Dimensional Porous Biodegradable Polymeric Scaffolds Fabricated with Biodegradable Hydrogel Porogens. Tissue Eng. Part. C Methods. 2009;15:583–594. doi: 10.1089/ten.tec.2008.0642. PubMed DOI PMC

Dušek K., Sedláček B. Phase separation in poly(2-hydroxyethyl methacrylate) gels in the presence of water. Eur. Polym. J. 1971;7:1275–1285. doi: 10.1016/0014-3057(71)90118-2. DOI

Karpushkin E., Dušková-Smrčková M., Šlouf M., Dušek K. Rheology and porosity control of poly(2-hydroxyethyl methacrylate) hydrogels. Polymer. 2013;54:661–672. doi: 10.1016/j.polymer.2012.11.055. DOI

Lozinsky V.I. Cryogels on the basis of natural and synthetic polymers: Preparation, properties and application. Russ. Chem. Rev. 2002;71:489–511. doi: 10.1070/RC2002v071n06ABEH000720. DOI

Hanson Shepherd J.N., Parker S.T., Shepherd R.F., Gillette M.U., Lewis J.A., Nuzzo R.G. 3D microperiodic hydrogel scaffolds for robust neuronal cultures. Adv. Funct. Mater. 2011;21:47–54. doi: 10.1002/adfm.201001746. PubMed DOI PMC

Woerly S., Marchand R. Interactions of copolymeric poly( glyceryl methacrylate)-collagen hydrogels with neural tissue: Effects of structure and polar groups. Biomaterials. 1991;12:197–203. doi: 10.1016/0142-9612(91)90200-T. PubMed DOI

Dušková-Smrčková M., Sadakbayeva Z., Steinhart M., Dušek K. The Manifold Varieties of Poly(2-Hydroxyethyl Methacrylate) Hydrogels−IPNs. Macromol. Symp. 2017;372:28–42. doi: 10.1002/masy.201700018. DOI

Karpushkin E., Dušková-Smrčková M., Remmler T., Lapčíková M., Dušek K. Rheological properties of homogeneous and heterogeneous poly(2-hydroxyethyl methacrylate) hydrogels. Polym. Int. 2012;61:328–336. doi: 10.1002/pi.3194. DOI

Refojo M.F. Glyceryl methacrylate hydrogels. J. Appl. Polym. Sci. 1965;9:3161–3170. doi: 10.1002/app.1965.070090920. DOI

Woerly S., Maghami G., Duncan R. Poly(Glyceryl Methacrylate) Hydrogels—Effect of Composition and Crosslinking Density on Structure and Release of Dextran as a Model Macromolecule. J. Bioact. Compat. Polym. 1992;7:305–323. doi: 10.1177/088391159200700401. DOI

Yu B., Wang C., Ju Y.M., West L., Harmon J., Moussy Y., Moussy F. Use of hydrogel coating to improve the performance of implanted glucose sensors. Biosens. Bioelectron. 2008;23:1278–1284. doi: 10.1016/j.bios.2007.11.010. PubMed DOI

González-Méijome J.M., López-Alemany A., Almeida J.B., Parafita M.A., Refojo M.F. Microscopic observations of superficial ultrastructure of unworn siloxane-hydrogel contact lenses by cryo-scanning electron microscopy. J. Biomed. Mater. Res. Part. B Appl. Biomater. 2006;76:419–423. doi: 10.1002/jbm.b.30386. PubMed DOI

Kuhn W., Peterli E., Majer H. Freezing point depression of gels produced by high polymer network. J. Polym. Sci. 1955;16:539–548. doi: 10.1002/pol.1955.120168238. DOI

Serp D., Mueller M., von Stockar U., Marison I.W. Low-temperature electron microscopy for the study of polysaccharide ultrastructures in hydrogels. I. Theoretical and technical considerations, Biotechnol. Bioeng. 2002;79:243–252. doi: 10.1002/bit.10286. PubMed DOI

Shotton D.M. A practical introduction to rapid freezing techniques. In: Severs N.J., Shotton D.M., editors. Rapid Freezing, Freeze Fracture, and Deep Etching. 1st ed. Wiley-Liss; New York, NY, USA: 1995. pp. 31–49.

Harmon M.E., Schrof W., Frank C.W. Fast-responsive semi-interpenetrating hydrogel networks imaged with confocal fluorescence microscopy. Polymer. 2003;44:6927–6936. doi: 10.1016/j.polymer.2003.07.006. DOI

Moor H. Theory and Practice of High Pressure Freezing. In: Steinbrecht R.A., Zierold K., editors. Cryotechniques in Biological Electron Microscopy. Springer; Berlin Heidelberg, Germany: 1987. pp. 175–191. DOI

Apkarian R.P., Wright E.R. Cryo and Cryo-Etch Methods for Quality Preservation of Hydrogels Imaged at High Magnification by Low Temperature SEM. Microsc. Microanal. 2005;11:1088–1089. doi: 10.1017/S1431927605500163. DOI

Liu Q., Hedberg E.L., Liu Z., Bahulekar R., Meszlenyi R.K., Mikos A.G. Preparation of macroporous poly(2-hydroxyethyl methacrylate) hydrogels by enhanced phase separation. Biomaterials. 2000;21:2163–2169. doi: 10.1016/S0142-9612(00)00137-X. PubMed DOI

Baker M.V., Brown D.H., Casadio Y.S., Chirila T.V. The preparation of poly(2-hydroxyethyl methacrylate) and poly{(2-hydroxyethyl methacrylate)-co-[poly(ethylene glycol) methyl ether methacrylate]} by photoinitiated polymerisation-induced phase separation in water. Polymer. 2009;50:5918–5927. doi: 10.1016/j.polymer.2009.10.047. DOI

Dušková-Smrčková M., Valentová H., Ďuračková A., Dušek K. Diluent Induced Cyclization and Phase Separation in Polymer Networks. Macromol. Symp. 2011;306:67–76. doi: 10.1002/masy.201000133. DOI

Sadakbayeva Z., Dušková-Smrčková M., Šturcová A., Pfleger J., Dušek K. Microstructured poly(2-hydroxyethyl methacrylate)/poly(glycerol monomethacrylate) interpenetrating network hydrogels: UV-scattering induced accelerated formation and tensile behavior. Eur. Polym. J. 2018;101:304–313. doi: 10.1016/j.eurpolymj.2018.02.035. DOI

Turner J.S., Cheng Y.-L. Preparation of PDMS-PMAA Interpenetrating Polymer Network Membranes Using the Monomer Immersion Method. Macromolecules. 2000;33:3714–3718. doi: 10.1021/ma991873k. DOI

Yin L., Fei L., Cui F., Tang C., Yin C. Superporous hydrogels containing poly(acrylic acid-co-acrylamide)/O-carboxymethyl chitosan interpenetrating polymer networks. Biomaterials. 2007;28:1258–1266. doi: 10.1016/j.biomaterials.2006.11.008. PubMed DOI

Lee F., Kurisawa M. Formation and stability of interpenetrating polymer network hydrogels consisting of fibrin and hyaluronic acid for tissue engineering. Acta Biomater. 2013;9:5143–5152. doi: 10.1016/j.actbio.2012.08.036. PubMed DOI

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