Enrichment of chitosan hydrogels with perfluorodecalin promotesgelation and stem cell vitality

Timothy E.L. Douglas a,n, Maciej Pilarek b, Ilona Kalaszczyńska c, Ilona Senderek b,Agata Skwarczyńska d, Vincent M.J.I. Cuijpers e, Zofia Modrzejewska d,Małgorzata Lewandowska-Szumieł c, Peter Dubruel a

a Polymer Chemistry and Biomaterials (PBM) Group, Department of Organic Chemistry, University of Ghent, Krijgslaan 281 S4, 9000 Gent, Belgiumb Biotechnology and Bioprocess Engineering Division, Faculty of Chemical and Process Engineering, Warsaw University of Technology, Waryńskiego 1,00-645 Warsaw, Polandc Tissue Engineering Laboratory, Department of Histology and Embryology, Medical University of Warsaw, Chałubińskiego 5, 02-004 Warsaw, Polandd Department of Environmental Systems Engineering, Faculty of Process and Environmental Engineering, Technical University of Łódź, ul. Wólczańska 213,90-924, Łódź, Polande Department of Biomaterials, Radboud University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands

a r t i c l e i n f o

Article history:Received 23 September 2013Accepted 29 March 2014Available online 5 April 2014

Keywords:BiomaterialsSol–gel preparationComposite materialsLiquid perfluorochemical (fluorocarbon)

a b s t r a c t

Thermosensitive injectable hydrogels for bone regeneration consisting of chitosan, sodium beta-glycerophosphate (Na-β-GP) and alkaline phosphatase (ALP) were enriched with oxygenated perfluor-odecalin (PFD), a liquid hydrophobic perfluorochemical with high oxygen affinity, in order to improvecell growth on the hydrogels. Furthermore, influence of PFD concentration on hydrogel physicochemicalproperties relevant for bone regeneration, namely gelation speed, radiopacity and homogenicity, wasinvestigated. Addtionally, ALP-mediated and non-ALP-mediated mineralization were evaluated byincubation in 0.1 M calcium glycerophosphate and simulated body fluid. 2% (w/v) chitosan hydrogelscontaining 2.5 mg/ml ALP were enriched with PFD at five concentrations, namely 0 (control), 0.069,0.138, 0.207 and 0.276 ml/ml hydrogel, denoted A, B, C, D and E, respectively. Rheometrical investigationsrevealed that gelation speed increased with increasing PFD concentration. Micro-CT analysis revealedhomogenicity of all sample groups except E and that radiopacity increased in the orderB4C4A4D4E. ALP-mediated and non-ALP-mediated mineralization were not affected adversely byPFD. Growth of human adipose tissue-derived mesenchymal stem cells (ADSC) encapsulated inhydrogels was markedly higher in sample groups containing PFD, i.e. B–E. Hence, incorporation ofoxygenated PFD can improve the suitability of hydrogels as bone regeneration materials.

& 2014 Elsevier B.V. All rights reserved.

1. Introduction

Chitosan, a deacetylated derivative of chitin, is a popularpolysaccharide biomaterial due to its biocompatibility, degradabil-ity, non-mammalian origin, antibacterial properties [1–3], andsolubility in acidic solutions, permitting formation of thermosen-sitive injectable chitosan hydrogels by neutralization with sodiumbeta-glycerophosphate (Na-β-GP) coupled with increasing tem-perature to body temperature [4,5]. Na-β-GP reduces electrostaticrepulsion between positively charged chitosan chains by neutra-lization. It also promotes, in combination with temperatureincrease, proton transfer from chitosan to its phosphate part andchitosan chain dehydration, enhanced by its glycerol part, in turn

promoting interchain hydrogen bonding as well as hydrophobicinteractions [6,7].

When hydrogels are applied as bone regeneration materials,mineralization with a ceramic phase based on calcium phosphate(CaP) is considered advantageous [8]. Previously, addition of theenzyme alkaline phosphatase (ALP) to chitosan/Na-β-GP hydrogelsnot only induced mineralization after incubation in a solution ofcalcium glycerophosphate (Ca–GP), which served as a source ofcalcium ions and substrate for ALP, but also accelerated gelation [9].

Advantages of chitosan hydrogels as bone regeneration materi-als include the ease of encapsulation of growth factors andmesenchymal stem cells (MSC) [10,11], which can differentiateinto bone-forming cells, supporting new bone formation [12].However, hypoxia threatens to compromise the survival, prolifera-tion and differentiation of MSC seeded on biomaterials [13,14].One strategy to improve oxygen delivery to such cells is incorpora-tion of oxygenated liquid perfluorochemicals (PFC) within the

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Materials Letters

http://dx.doi.org/10.1016/j.matlet.2014.03.1730167-577X/& 2014 Elsevier B.V. All rights reserved.

n Corresponding author. Tel.: þ32 9 264 4508; fax: þ32 9 264 4972.E-mail address: Timothy.Douglas@UGent.be (T.E.L. Douglas).

Materials Letters 128 (2014) 79–84

biomaterial. PFC are derivatives of hydrocarbons in which hydro-gen atoms have been substituted by fluorine atoms. PFC have ahigh affinity for oxygen and thus have been used as tissueoxygenating fluids in biomedical applications and as oxygencarriers in biotechnological applications to promote growth ofplant and mammalian cells and microbial cultures (for reviews,see [15–18]). PFC covalently grafted onto photocrosslinked chit-osan hydrogels have increased O2-binding capacity and, afteroxygenation, promoted fibroblast growth [19]. The growth ofhepatocytes encapsulated in alginate beads has been promotedby the addition of droplets of perfluorooctyl bromide by emulsi-fication [20,21]. One commonly applied PFC is perfluorodecalin(PFD), which has improved growth of microbial cultures bypromoting O2 transfer [22–26] and can be easily recycledand reoxygenated due to its hydrophobicity and immiscibilitywith aqueous solutions. PFD is cytocompatible for mammaliancells [27], and has increased radiopacity of bone cement [28].

In this study, oxygenated PFD was incorporated into chitosan/Na-β-GP/ALP hydrogels in order to improve viability of embeddedMSC, in this case human adipose tissue-derived mesenchymalstem cells (ADSC). A second aim was to investigate PFD's influenceon hydrogel physicochemical properties relevant for bone regen-eration, namely gelation speed, radiopacity, homogeneity andmineralizability. These were evaluated by rheometry, micro-computer topography (Micro-CT) and incubation in mineralizationsolutions and assessment of dry mass percentage, i.e. the hydrogelweight percentage attributable to mineral and polymer (i.e. not towater), which served as a measure of the extent of mineralformation. Influence of PFD on both ALP-mediated and non-ALP-mediated mineralizability was studied by incubation in Ca–GPsolution or simulated body fluid (SBF), respectively.

2. Materials and methods

Production of chitosan/Na-β-GP/ALP hydrogels and PFD incor-poration: Chitosan from shrimp shells, Na-β-GP, ALP and Ca–GP(Product nos. 50494, 50020, P7640 and 50043, respectively), wereobtained from Sigma-Aldrich (BE). PFD was obtained from ABCRGmbH (D). Chitosan and Na-β-GP powder were sterilized byethylene oxide in a Steri Vac 5XL device (3 M), at room tempera-ture, exposure time 3 h, post-treatment aeration time 48 h.

Chitosan hydrogels were produced similarly to the methoddescribed previously [9]. Briefly, solutions of chitosan (25 mg/ml0.1 M HCl), Na-β-GP (1 g/ml Milli-Q ultra pure water) and ALP(25 mg/ml Milli-Q water), PFD and Milli-Q water were pre-cooledto 4 1C and mixed vigorously on ice to form an emulsion. Theproportions of the aforementioned components in five differentsample groups, denoted as A, B C, D and E and differing in volumeof Milli-Q water and PFD added, are given in Table 1.

Rheometry: Hydrogel components with a total volume of 0.5 mlwere mixed on ice at 4 1C and analyzed by rheometry using aPhysica, MCR 301 rheometer (Anton Paar, B) (rotating head

diameter 25 mm, sample gap 0.7 mm). Storage modulus (G0) wasrecorded for 3000 s at 37 1C and 1.6 Hz. Measurements wereperformed in triplicate.

Micro-CT analysis: Hydrogels of volume 0.5 ml were preparedin Eppendorf microreaction tubes for Micro-CT examination usinga Skyscan 1072 desktop X-ray micro-CT system (Skyscan, B) withscanning settings: exposure time 1.9 s, no aluminum filter, 20�magnification¼14,16 pix size, rotation step 0.91, range of rotation1801. Cone beam reconstruction was performed using NReconv1.4.4 (Skyscan) software with standardized settings: (600 images,step size: 1): beam hardening correction 4%, ring artefact reduc-tion on, change dynamic image range: 0.002–0.094 (in attenuationcoefficient). A standard volume of interest (VOI) of diameter 6 mmand 10 mm in height was examined. CT-An V1.13 (Skyscan) waschosen for image quantification. Data were expressed as gray scale

Table 1Composition of hydrogel types A–E used in this study.

Samplegroup

Volume of individual component added per ml of hydrogel (ml)

25 mg/mlchitosan

1 g/ml NaGP 25 mg/ml ALP Milli-Q PFD

A 0.554 0.069 0.100 0.276 0B 0.554 0.069 0.100 0.207 0.069C 0.554 0.069 0.100 0.138 0.138D 0.554 0.069 0.100 0.069 0.207E 0.554 0.069 0.100 0 0.276

Fig. 1. Gelation kinetics of chitosan hydrogel sample groups A–E containing2.5 mg/ml ALP at 37 1C. Mean values (n¼3) of groups A–E (a), groups A and D(b) and groups A and E (c). Error bars show standard deviation.

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values on an index from 0 to 255. Values in the VOI from 53 up to255, corresponding to the hydrogel, were analyzed.

Mineralization studies: Hydrogels of volume 2 ml, produced bygelation at 37 1C overnight, were incubated for 7 days in 0.1 MCa–GP or SBF, rinsed three times with Milli-Q water and incubatedin Milli-Q water for 24 h. Ca–GP serves as a source of calcium ionsand substrate for ALP, while SBF contains no substrate for ALP. Thedry mass percentage was calculated as: (weight after incubationand subsequent freeze-drying for 24 h/weight before freeze-dry-ing)n100.

Adipose tissue-derived mesenchymal stem cells (ADSC) encapsulatedin hydrogels: ADSC were obtained from two patients during cosmeticliposuction (local ethics committee approval KB/85/A/2012). Written

consent was obtained. ADSC were cultured in DMEM supplementedwith 10% (v/v) fetal calf serum, 0.05 U/ml penicillin, 0.05 U/mlstreptomycin at 37 1C (Invitrogen). 270,000 ADSC at passage 3 wereencapsulated in hydrogels of volume 0.5 ml (sample groups A–E,Table 1). Prior to cell culture studies, PFD was sterilized andoxygenated to its maximum capacity as described previously[26,29]. Hydrogels were cast in wells of 24-well plates in duplicateand covered with 1 ml of culture medium. Medium was changedevery 2 days. Four plates were prepared for analysis at four timepoints, namely day 1, 2, 4 and 7. At each time point, ADSC viabilitywas investigated by fluorescence microscopy after staining withfluorescein diacetate (FDA), propidium iodide (PI) and the ThiazolylBlue Tetrazolium Blue (MTT) assay to detect living cells, dead cells

Fig. 2. Micro-CT analysis of chitosan hydrogel sample groups A–E. (i) Image of Eppendorf microreaction tube containing hydrogel. Hydrogel cross-section chosen for analysisis indicated. (A–E) Micro-CT images displaying gray scale values for sample groups A–E, respectively. Black areas represent air, green areas represent Eppendorf tube, and redareas represent hydrogel. Lighter shades of red indicate higher gray scale values and hence radiopacity. (For interpretation of the references to color in this figure caption, thereader is referred to the web version of this paper.)

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and cell metabolic activity, respectively. Phase contrast microscopywas also performed. All reagents were from Sigma-Aldrich.

3. Results and discussion

Effect of PFD on gelation kinetics: Gelation curves (Fig. 1a)revealed that gelation speed increased with increasing PFD con-tent. The influence of PFD was most apparent for groups D and E

(Fig. 1b and c). Gelation in groups B–E appeared to have beencompleted after 3000 s, at which point similar storage modulus(G0) values had been reached. Group A appeared not to havecompleted gelation after 3000 s and its G0 value was markelylower. PFD is well known for its hydrophobicity and immiscibilitywith aqueous solutions [26,29,30]. As the volume of PFD contentwas raised, the aqueous content of the gel decreased by the samevolume (Table 1). Hence, with increasing PFD content, the con-centration of chitosan in the aqueous phase would be expected torise, promoting interchain interactions and thus gelation.An alternative and parallel explanation could be that hydrophobicregions on chitosan chains interact with the surface of PFDdroplets dispersed within the hydrogel, i.e. that the dropletsthemselves act as a crosslinker for the chitosan chains and thuspromote hydrogel formation.

Radiopacity and homogenicity of PFD-loaded hydrogels: Micro-CTanalysis yielded the following mean gray scale values for thesample groups A, B, C, D and E: 74.5, 71.3, 72.8, 76.9, and 80.2,respectively. Hence, radiopacity increased in the order BoCoAoDoE and thus rose with increasing PFD content with theexception of the PFD-free sample group A. Radiopacity would beexpected to increase with increasing PFD content due to PFD0shigher radiopacity compared to water. However, the radiopacity ofthe PFD-free group A was higher than that of the groups B and C.Only groups D and E, which had higher PFD contents, displayed ahigher radiopacity than group A. It is not inconceivable that thelower overall radiopacity in groups B and C is diminished byrestructuring of the chitosan hydrogel caused by PFD addition,however such discussion of possible underlying mechanisms mustremain speculative and is beyond the scope of this manuscript.Micro-CT images (Fig. 2) demonstrated that sample groups A—Dwere homogeneous with regard to gray scale values and thusradiopacity, suggesting good dispersion of PFD droplets andstability of the emulsion of PFD droplets in these groups. However,the sample group with the highest PFD content, namely E, wasdistinctly more inhomogeneous.

Influence of PFD on ALP-mediated and non-ALP-mediated miner-alization: Regarding mineralizability, only group C displayed ahigher dry mass percentage after mineralization in 0.1 M Ca–GP(Fig. 3a). In contrast, only group E displayed a lower dry masspercentage after mineralization in SBF (Fig. 3b). PFD can be

Fig. 3. Dry mass percentage of chitosan hydrogel sample groups A–E containing2.5 mg/ml ALP incubated for 7 days in (a) 0.1 M Ca–GP and (b) SBF. Error bars showstandard deviation.

Group A

Group E

Phase contrast Live Dead

Fig. 4. Analysis of ADSC viability in hydrogels without PFD (group A) and containing PFD (group E). Pictures from each group were taken from the same location. Cells werecultured in hydrogels for 7 days. Left column: phase contrast images; middle column: fluorescein diacetate staining of live cells (green); right column: propidium iodidestaining of dead cells (red). (For interpretation of the references to color in this figure caption, the reader is referred to the web version of this paper.)

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expected to influence dry mass percentage in two ways. Firstly,due to PFD's hydrophobicity and immiscibility with water, increas-ing PFD content hampers diffusion of ions, ALP and ALP substrate(Ca–GP) and thus influences mineral formation. Secondly, PFD0shigh density (1.92 g/ml) compared to water [28] favors a decreasein dry mass percentage with increasing PFD content. Dry masspercentages did not increase with increasing PFD content aftermineralization in SBF, with the exception of group E (Fig. 3b),suggesting that PFD does not hinder diffusion of ions from SBF intothe hydrogel network. When hydrogels were incubated in Ca–GPsolution, dry mass percentage did not decrease with increasingPFD content and was higher for group C (Fig. 3b), suggesting thatthere is an optimal PFD content to promote mineralization.ALP release from chitosan hydrogels has been demonstratedpreviously [9] and increasing PFD content may hinder not onlyALP release, promoting mineralization, but also Ca–GP diffusioninto the hydrogel network, reducing mineral formation.

Influence of PFD on ADSC viability: Fluorescence microscopyimages (Fig. 4) showed that viability of ADSC encapsulated insample groups containing PFD (B–E) was markedly higher thanthat of the cells in sample group A, which contained no PFD.A markedly higher number of dead cells were observed inhydrogels without PFD (A) in contrast to sample groups containingPFD (B–E) (Fig. 4). Moreover, a subtle increase in the cell numberof sample group E in comparison to groups B, C and D could beobserved (Supplementary Fig. S1).

From day 2 of analysis, cells in direct contact with PFD dropletsstarted to adhere and spread on droplet surfaces. This was evenmore pronounced on day 7 (Fig. 5). ADSC surrounding PFDdroplets were metabolically active, as shown by the formation offormazan needles. The effect of higher survival of cells in hydro-gels containing PFD was confirmed on ADSC derived from adiposetissue of two donors.

4. Conclusions and outlook

PFD was incorporated homogeneously into chitosan/Na-β-GP/ALP hydrogels in groups B–E. Speed of gelation increases with

increasing PFD content. PFD content did not negatively influenceALP-mediated and non-ALP-mediated mineralization. OxygenatedPFD markedly improved ADSC viability and reduced cell death.

PFD-mediated acceleration of gelation and promotion of MSCgrowth can increase hydrogels0 suitability for bone regenerationapplications. Future tasks include optimization of PFD droplet size,which has been reported to be the critical factor affecting O2

transfer [27]. The effect of increasing PFD concentration on MSCneeds to be invesitgated quantitatively, as does the effect of O2

delivery via PFD on osteogenic differentiation.

Acknowledgment

T.E.L.D. acknowledges FWO, Belgium, for a postdoctoral fellowship.

Appendix A. Supplementary material

Supplementary data associated with this paper can be found inthe online version at http://dx.doi.org/10.1016/j.matlet.2014.03.173.

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