Oelschlaeger, C.

Roether, J.

Willenbacher, N.

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Hyaluronic acid cryogels with non-cytotoxic crosslinker genipin

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Materials Letters: X 4 (2019) 100027Contents lists available at ScienceDirectMaterials Letters: Xjournal homepage: www.elsevier .com/locate /mlbluxHyaluronic acid cryogels with non-cytotoxic crosslinker genipinhttps://doi.org/10.1016/j.mlblux.2019.1000272590-1508/ 2019 The Authors. Published by Elsevier B.V.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).⇑ Corresponding author.Johanna Roether ⇑, Claude Oelschlaeger, Norbert WillenbacherKarlsruhe Institute of Technology (KIT), Institute for Mechanical Process Engineering and Mechanics, 76131 Karlsruhe, Germanya r t i c l e i n f o a b s t r a c tArticle history:Received 4 July 2019Accepted 12 October 2019Available online 14 October 2019Keywords:Non-cytotoxic crosslinkingGenipinHyaluronic acid3D scaffoldCryogelationFor the first time, macroporous, elastic, three-dimensional hyaluronic acid cryogels were prepared withgenipin as non-cytotoxic crosslinking agent. These cryogels are characterized by a lamellar porous struc-ture with a homogeneous pore size of ~100 mm, shear elasticity of ~2 kPa and a swelling ratio of 2.5 inwater. Additionally, multiple particle tracking based microrheology measurements reveal the formationof a heterogeneous network. This novel biomaterial owns great potential as non-cytotoxic alternative forapplication in drug delivery, as tissue engineering scaffold or wound healing substrate and can helpreducing toxicity of artificial skin grafts or tissue equivalents. 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).1. Introduction technique. Thus, we compared the swelling capacity, structural,For successful tissue engineering, a scaffold must be biofunc-tional, biodegradable, biocompatible, with a 3D porous architec-ture and high degree of pore interconnectivity. Furthermore, itshould have appropriate mechanical properties to closely mimicmechanical, and ideally also chemical properties of the extracellu-lar matrix. For fabrication of hyaluronic acid (HA) based-scaffolds,phase separation [1], freeze-drying [2] and electrospinning [3]have been established. Therefore, BDDE (1,4-butanediol diglycidylether) [4] and glutaraldehyde [1] were used as crosslinkers. Thefabrication of one-component HA scaffolds via cryogelation [5]facilitates the formation of homogeneous pores at a relativelylow polymer concentration. Crosslinkers that are reactive withHA at low temperatures are ethyleneglycol diglycidylether (EGDE)[6–8] and EDC/NHS (carbodiimide/N-hydroxysuccinimide) [9].Mixed cryogels of HA with collagen, gelatine and chitosan werealso crosslinked by EDC/NHS [10] and glutaraldehyde [11,12]. Mostof the crosslinkers and especially their non-reacting residues arecytotoxic [13,14]. It is consequently desirable to use non-cytotoxic crosslinkers to form stable and biocompatible HA basedhydrogels. Genipin is a natural product extracted from the garde-nia fruit and it was shown that its cytotoxicity is significantlylower than that of common crosslinkers [15–20]. Genipin has beenutilized to crosslink biopolymers, such as chitosan [19] and gela-tine [20] or hybrid systems like chitosan/HA [21] but it has neverbeen used to form pure HA gels before. We now proved the suit-ability of genipin as an alternative crosslinking agent for the fabri-cation of one component HA porous gels using the cryogelationmicro- and macro-viscoelastic properties of HA scaffolds cross-linked with genipin to the corresponding features of such gelscrosslinked with commonly used EGDE.2. Materials and methods2.1. Preparation of cryogels and determination of swelling capacityMacroporous gels were prepared using the cryogelation tech-nique as previously described [8]. Briefly, HA (Mw = 2.0 to 2.2Mio Da, Contipro) was dissolved in 1% NaOH and EGDE was added.For genipin crosslinking, HA was dissolved in PBS and mixed 2:1with genipin in DMSO (20 mg/ml, considering the maximum solu-bility). After mixing for 20 min, solutions were frozen at 20 C for6 days. After thawing, the swelling ratio (SR) was determined bymeasuring the ratio of the mass of the gel equilibrated in waterand un-swollen state.2.2. Rotational rheometryGel bulk linear viscoelastic properties were characterized per-forming oscillatory shear experiments in the linear-viscoelasticregime, using a rotational rheometer Anton Paar MCR 501 (plate/plate, diameter 8 mm, gap 1 mm).2.3. Multiple particle trackingLocal viscoelastic properties of the matrix, namely the porewalls, were investigated using the multiple particle tracking(MPT) technique [22,23]. In MPT experiments, the thermally driven2 J. Roether et al. /Materials Letters: X 4 (2019) 100027motion of inert microspheres that are evenly distributed within asample is monitored. Here, we tracked the Brownian motion ofgreen fluorescent polystyrene microspheres (diameter 0.19 lm).For performing measurements exclusively in the matrix, particleswere added to the polymer solutions before freezing. The displace-ments of particle centers were monitored at a rate of 50 frames/s.Movies of the fluctuating microspheres were analyzed using a cus-tom MPT routine, incorporated into the software Image ProcessingSystem (Visiometrics iPS) and a self-written Matlab program [24],based on the widely used Crocker and Grier algorithm [25].Fig. 2. Dynamic shear moduli G0 (closed symbols) and G00 (open symbols) of 3 wt%HA gels crosslinked with EGDE (blue) and genipin (red). (For interpretation of thereferences to colour in this figure legend, the reader is referred to the web version ofthis article.)Table 1Crosslinker concentrations in a 3 wt% HA hydrogel.Crosslinker Concentration/wt% Reactive groups/mMol/mlSolventEGDE 0.7 40 1% NaOHGenipin 0.5 20 PBS with 30% DMSO3. Results and discussion3.1. Cryogels structural properties and swelling capacityIndependent of the crosslinker used, both types of cryogelsobtained were spongy, elastic with large pores and they swelledinstantaneously when immersed in water. Examples of 3D imagesof the structure of these cryogels in the wet state, obtained withlaser scanning microscopy (LSM 510, Carl Zeiss), are shown inFig. 1.EGDE cryogels show interconnected round pores of size~100 lm and thin matrix wall of ~5–20 lm [8]. For genipin cryo-gels, the images suggest the formation of a more lamellar porousstructure with a pore size almost similar to that of EGDE gels. Toour knowledge, this is the first time that pure HA hydrogels havebeen fabricated with non-cytotoxic crosslinker genipin. Differentcrosslinking mechanisms are the origin of the formation of thesetwo morphologically different gels. In HA/EGDE gels, epoxy groupsof the EGDE are covalently bond to the HA hydroxyl groups underalkaline conditions [6]. Genipin generally reacts with primaryamino groups of biopolymers but HA does not have such groups.However, HA has multiple highly reactive hydroxy groups thatare able to form e.g. glycosidic bonds, presumably with genipin[26]. The latter are known to be stable as seen in other polycarbo-hydrates, such as starch. Other gels, based on non-covalent bonds,are known to be stable in water, too. [27]. Although the crosslink-ing mechanism cannot be fully unravelled here, cryogelation of HAand genipin leads to stable intermolecular bonds, that are strongenough to allow for the swelling of HA/genipin gels in water with-out dissolving. Without genipin, no stable structures can beobtained.Despite the higher molar ratio of crosslinker to polymer,HA/EGDE gels exhibit a higher degree of swelling with SR  7.5compared to HA/genipin gels with SR  2.5. This may be due to aheterogeneous gel structure with percolating domains of highFig. 1. LSM images of gels structures, crosslinked with EGDE and genipin, vicrosslink density that limits the swellability of genipin gels. Afterimmersion in water, both compositions show high long-term sta-bility (several months) and shape fidelity, which can be consideredmandatory in tissue engineering applications.3.2. Mechanical propertiesIn bulk oscillatory shear measurements, gels of both composi-tions show frequency independent elastic moduli and G0 domi-nates over G00 in the frequency range from 0.1 to 10 rad/s (Fig. 2).sualized by fluorescence of tracer particles embedded in the pore walls.Fig. 3. MSDs of PS particles (d = 0.19 lm) dispersed in the matrix of HA 3 wt% gels crosslinked with EGDE (A) and genipin (B). Blue and red curves are the ensemble-averagedMSD.J. Roether et al. /Materials Letters: X 4 (2019) 100027 3This is considered as a typical gel-like behavior. Correspondingshear elastic plateau modulus data G0 (average of G0 valuesobtained in the probed frequency range) show that the less swollengel made of genipin provides a higher elastic modulus valueG0 ~ 2000 Pa compared to the highly swollen EGDE gel whereG0 ~ 200 Pa. This is direct evidence of the different crosslink den-sity of the swollen gels. The three-times lower water uptake ofthe genipin gel outweighs the two-times lower molar ratio ofcrosslinker to polymer compared to the EGDE gel (see Table 1and see 3.1).In order to characterize the local elasticity of the pore walls,microstructural and local viscoelastic properties of the gels wereinvestigated by means of MPT microrheology. Fig. 3 shows thevariation of mean square displacements (MSDs) as a function oflag time s for tracer particles with diameter 0.19 mm dispersed inthe gel network. In both cases, almost no time dependence of theindividual MSDs is found and this result indicates that particlesare highly constrained by the surrounding fluid which is consistentwith an elastic trapping of particles in a gel-like network.Additionally, for gels crosslinked with genipin (Fig. 3B), therange of displacements at a given lag time is much broader thanfor the gel crosslinked with EGDE (Fig. 3A). At s = 0.1 s, MSDs varyabout two orders of magnitude, from ~3.104 to 102 lm2 for gen-ipin gels compared to only one order of magnitude from ~5.105 to7.104 lm2 for EGDE gels.This indicates a more heterogeneous structure of theHA/genipin network with a non-Gaussian parameter [28] a = 5.5,compared to the HA/EGDE gel where a = 1.4. As already mentionedabove, this might be the reason for the reduced swelling capacity ofgenipin gels (see 3.1). The higher absolute value of the averageMSD for genipin gels indicates that particles explore a softer envi-ronment than in EGDE gels. The discrepancy between micro- andmacrorheology which is more pronounced for genipin than forEGDE hydrogels, is presumably due to densely crosslinked regionsin the more heterogeneous genipin hydrogels, that are inaccessiblefor tracer particles but seem to contribute to the overall mechani-cal strength of the constructs.4. ConclusionGenipin can be used as crosslinking agent for producing non-cytotoxic macroporous hyaluronic acid cryogels. Bulk elasticity ofgenipin and conventional EDGE gels are in the same range,whereas the local mechanical properties of genipin gels are moreheterogeneous. Both gels show similar pore sizes in a well-suitable range for cell culture applications. 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Hyaluronic acid cryogels with non-cytotoxic crosslinker genipin

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