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Development of injectable Hyaluronic Acid / Cellulose nanocrystals bionanocomposite hydrogels for tissue engineering applications Rui M. A. Domingues, Marta Silva, Pavel Gershovich, Stefano Betta, Pedro Babo, Sofia G Caridade, João F. Mano, Antonella Motta, Rui L. Reis, and Manuela E. Gomes Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00209 • Publication Date (Web): 24 Jun 2015 Downloaded from http://pubs.acs.org on July 2, 2015
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cellulose nanocrystals (CNCs) were employed as nanofillers in a fully biobased strategy for the production of reinforced HA nanocomposite hydrogels. Herein we report the development of a new class of injectable hydrogels composed of adipic acid dihydrazide-modified HA (ADH-HA) and aldehyde-modified HA (a-HA) reinforced with varying contents of aldehyde-modified CNCs (a-CNCs). The obtained hydrogels were characterized in terms of internal morphology, mechanical properties, swelling and degradation behaviour in the presence of hyaluronidase. Our findings suggest that the incorporation of a-CNCs in the hydrogel resulted in a more organized and compact network structure and led to stiffer hydrogels (maximum storage modulus, E’, of 152.4 kPa for 0.25 wt.% a-CNCs content) with improvements of E’ up to 135% comparatively to unfilled hydrogels. In general, increased amounts of a-CNCs led to lower equilibrium swelling ratios and higher resistance to degradation. The biological performance of the developed nanocomposites was assessed towards human adipose derived stem cells (hASCs). HA-CNCs nanocomposite hydrogels exhibited preferential cell supportive properties in in vitro culture conditions due to higher structural integrity and potential interaction of microenvironmental cues with CNC’s sulphate groups. hASCs encapsulated in HA-CNCs hydrogels demonstrated ability to spread within the volume of gels and exhibited pronounced proliferative activity. Together, these results demonstrate that the proposed strategy is a valuable toolbox for fine tuning the structural, biomechanical and biochemical properties of injectable HA hydrogels, expanding their potential range of application in the biomedical field.
Keywords Hyaluronic acid, cellulose nanocrystals, hydrogels, injectable, tissue engineering
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Introduction Hyaluronic acid (HA) is a high molecular mass linear polysaccharide of alternating Dglucuronic acid and N-acetyl-D-glucosamine, ubiquitously found in tissues and body fluids of vertebrates and some bacteria
1-3
. It is a crucial component of the natural
extracellular matrix having an important role in several cellular and tissue functions 4. HA presents unique properties such as good biocompatibility, gel-forming properties and ability to be easily modified through both its carboxyl and hydroxyl groups, which potentiate its use in tissue engineering (TE) and regenerative medicine as a starting material for the production of hydrogels
5, 6
. Moreover, it is naturally degraded by
hyaluronidases (HAses), which hydrolyze the hexosaminidic β(1→4) linkages between N-acetyl d-glucosamine and d-glucuronic acid residues. Since the degradation rate of HA, mediated by HAses, can be increased in case of injury, HA-based materials have the advantage to be degraded and release their content in a controlled manner
2, 7
.
Recent studies have been focusing on the development of HA-based injectable hydrogels, which can be formed in situ after being injected into the body 6, 8, 9. The main advantages of this approach rely on the ability of injectable hydrogels to adapt to the defect shape and to be easily laden with cells and/or drugs. Additionally, its delivery is performed through minimally invasive surgical procedures, resulting in a faster recovery, reduced scaring and less pain for patients
10
. Chemically crosslinked HA-
based hydrogels can be obtained through a variety of chemistries, such as Schiff’s base, enzyme-mediated, photopolymerization, Michael-type addition, disulfide formation and several click chemistry reactions 6, 11-13. Specifically, the hydrazone bond click reaction between hydrazide and aldehyde groups, has been widely used for the preparation of natural polymer based hydrogels
14-16
. Moreover, it has been previously demonstrated
for several polymeric systems that hydrazone cross-links are formed when the
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functionalized hydrogel components come into contact under normal physiological conditions and does not pose considerable cytotoxicity issues
17-19
. Despite the above
mentioned features that makes the HA an appealing precursor for the development of biomaterials for tissue engineering applications, HA as well as other natural polymerbased hydrogels have limited mechanical properties and degradation rates, restraining their clinical applications
12, 20
. Biomaterials used in tissue engineering applications,
besides providing specific biological cues, should create an adequate mechanical environment to support cell migration, proliferation and differentiation aiming the regeneration of functional tissues
10, 21
. Moreover, they must maintain suitable
mechanical features during new tissue formation without deformation or collapse 21, 22. In order to improve the mechanical properties and control the degradability of HAbased hydrogels, HA can be chemically modified or physically crosslinked
15, 23
.
Additional strategies consist in the reinforcement of hydrogels with nanoparticles, such as natural based cellulose nanocrystals (CNCs), resulting in a composite material with improved mechanical properties 19. CNCs can be extracted from a wide range of highly available cellulose sources following well established chemical routes
24
. Along with
the general good biocompatibility, CNCs present high strength (elastic modulus ranging 25-29
from 110 to 220 GPa and tensile strength is in the range of 7.5 - 7.7 GPa
), low
density, high aspect ratio and surface area that greatly contribute to their potential use in regenerative medicine and TE applications
30
. Moreover, its surface chemistry enables
different specific surface modifications, such as oxidation, esterification, etherification, silylation, or polymer grafting, which allow the effective incorporation of CNCs into the hydrogels matrices, where it can act as both filler and cross-linker
30, 31
. Another
interesting aspect of CNCs obtained from the typical sulfuric acid hydrolysis for potential TE applications is the presence of abundant surface SO3- half-ester groups.
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This feature may also confer additional interesting functional properties to the hydrogels, as this functionality is also characteristic of the ECM sulfated glycosaminoglycans which are known to induce and control specific cell functions on the cellular microenvironment 32. In this work we developed injectable hydrogels based on the hydrazone covalent crosslinking reaction, composed of adipic acid dihydrazide-modified HA (ADH-HA) and aldehyde-modified HA (a-HA) reinforced with varying contents of aldehydemodified CNCs (a-CNCs) (Figure 1). The hydrogel precursors and cross-linked hydrogels were thoroughly characterised regarding their chemical, morphological, microstructural and mechanical properties, as well as their swelling and degradation profiles.
Figure 1. Schematic representation of the proposed injectable hyaluronic acid-based hydrogels (HAX) reinforced with CNCs, prepared using a double barrel syringe. a) Hydrazide-functionalized hyaluronic acid (ADH-HA), and b) aldehyde functionalized hyaluronic acid (a-HA), with or without aldehyde-modified CNCs (a−CNCs), will be co-injected and mixed. Stable hydrogels will be formed upon crosslink reaction between the hydrazide and carbonyl moieties of precursor components.
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Results and Discussion Chemical and morphological characterization of HAX and HAX-CNC Considering their unique mechanical properties, CNCs have been incorporated as fillers in several polymeric hydrogel matrices, such as for example to improve the thermal and mechanical properties of gelatin based hydrogels mechanical properties of poly(vinyl alcohol)
33
or to improve the stability and the
34, 35
or α-cyclodextrin
36
hydrogels.
However, only a few studies explored the possibility of using CNCs not only as fillers (i.e., with no covalent attachment to the hydrogel matrix) but also as crosslinker agents, a strategy that usually results in enhanced mechanical properties when compared with the mere physical entrapment of the CNCs within the hydrogel matrix 19, 33. To be used as crosslinkers, chemical functionalization of their surfaces is usually required. In this work, CNCs were functionalized following the well-described
33, 37
sodium periodate
oxidation route that induces the cleavage of the C2—C3 glycol bond of glucose residues, leading to the formation of dialdehyde groups at the respective carbon atoms along the cellulose chains. The reactive aldehyde groups are particularly appealing for a wide range of biomedical applications due to their ability to spontaneously react with amine groups in a Schiff base reaction or condensate with hydrazide groups to form covalent hydrazone linkages. The later chemical route was used to synthesize injectable covalently cross-linked hydrogels by in situ hydrazone formation between adipic acid dihydrazide modified hyaluronic acid (ADH-HA) and aldehyde modified HA (a-HA). Thus, aldehyde modified CNCs (a-CNCs, Figure S2) are expected to participate in the cross-linked polymeric network resulting in HAX-CNC nanocomposite hydrogel with enhanced mechanical and structural performance.
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Injectable and covalently cross-linked hyaluronic acid hydrogels (HAX) reinforced with a-CNCs were produced by the co-injection of the precursor components using a commercial double barrel syringe (Figure 1). In general, all the hydrogel formulations formed quickly upon contact of the two HA derivatives and the a-CNCs (see Movie S3 in supplementary information), a characteristic of several hydrazone cross-linked HA hydrogels which present typical gelation times within 3 to 30 s
19, 38, 39
. The
incorporation of a-CNC didn’t have a significant impact on the apparent gelation time of the hydrogels which were in the range of 15±0.3 to 17±1 s. Successful crosslinking of the hydrazide-functionalized HA with the aldehyde moieties of the a-HA or a-CNC was confirmed by FTIR. Overall the spectra of the hydrogel precursor components and the HAX-CNC showed similar patterns (Figure 2). In the spectrum of HA-ADH, the main differences compared to that of HA are the appearance of the characteristic peaks of the ADH-modification at 1710cm-1 (only seen as a slight slop), 1550cm-1 and 1375cm-1 (stronger than in the non-modified HA), while in the spectrum of a-HA is possible to observe the carbonyl stretch band at around 1735 cm-1, similarly to a-CNC. In the HAX-CNC hydrogels, the band of the carbonyls that are consumed in the covalent hydrazone cross-linking reaction disappears, confirming the covalent crosslinking between the hydrogel components.
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Figure 2. FTIR transmittance spectra of Hyaluronic acid (HA), hydrazide modified hyaluronic acid (ADH-HA), aldehyde functionalized hyaluronic acid (a-HA) and HA hydrogel containing 0.5 wt.% of a-CNCs (HAX-CNC 0.5%). At dashed lines are signalized the peaks corresponding to the CH2 deformation, vibrational bending at ~1375cm-1; amide II (R-C=O-NH-R’) NH bending at ~1569cm-1, and carbonyl stretch at ~1710cm-1; and anhydrous aldehyde (R-C=O) stretch at ~1735cm-1.
The morphology of the HAX hydrogels and the influence of a-CNCs incorporation on their microstructure were investigated by scanning electron microscopy (SEM). Crosssections of the HAX hydrogel formulations were produced by fracturing freeze-dried hydrogels after immersion in liquid nitrogen. Representative images of the crosssections analyzed by SEM are depicted in Figure 3.
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Figure 3. A) cross-sectional SEM pictures of the hydrogel formulations a) HAX-CNC 0%; b) HAX-CNC 0.125%; c) HAX-CNC 0.25%; d) HAX-CNC 0.375%; e) HAX-CNC 0.5%. Magnification 300X (scale bar 50 µm). B) Box-plot representation of the pore size distribution in the formulations HAX, HAX-CNC 0.125%, HAX-CNC 0.25%; HAX-CNC 0.375% and HAX-CNC 0.5%. N>50; three symbols p
