Oxidized Alginate-Gelatin Hydrogel: A Favorable Matrix for Growth

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Oxidized Alginate-Gelatin Hydrogel: A Favorable Matrix for Growth and Osteogenic Differentiation of Adipose-Derived Stem Cells in 3D Bapi Sarker, Tobias Zehnder, Subha Narayan Rath, Raymund E. Horch, Ulrich Kneser, Rainer Detsch, and Aldo R. Boccaccini ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00188 • Publication Date (Web): 15 Jun 2017 Downloaded from http://pubs.acs.org on June 16, 2017

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ACS Biomaterials Science & Engineering

Oxidized Alginate-Gelatin Hydrogel: A Favorable Matrix for Growth and Osteogenic Differentiation of AdiposeDerived Stem Cells in 3D Bapi Sarker,a,§ Tobias Zehnder,a Subha N. Rath,b,¥ Raymund E. Horch,b Ulrich Kneser,c Rainer Detscha and Aldo R. Boccaccini*,a a

Institute of Biomaterials, Department of Materials Science and Engineering, University of

Erlangen-Nuremberg, 91058 Erlangen, Germany b

Department of Plastic and Hand Surgery, University of Erlangen-Nuremberg, 91054

Erlangen, Germany c

Department of Hand, Plastic, and Reconstructive Surgery- Burn Center, BG Trauma Center

Ludwigshafen and Department of Plastic and Hand Surgery, University of Heidelberg, Germany Present addresses §

Department of Mechanical Engineering & Materials Science, Washington University in St.

Louis, St. Louis, MO 63105, USA ¥

Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, Kandi

502285, Telangana, India *Corresponding author: Email: [email protected]

ACS Paragon Plus Environment


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ABSTRACT Alginate-based hydrogels are extensively used matrices for cell encapsulation, but they need to be modified to recapitulate chemical, microstructural and mechanical properties of the native extracellular matrix. Like other cell types, mesenchymal stem cells exhibit rounded and clustered morphologies when they are embedded in alginate hydrogels. In this study, we use covalently crosslinked oxidized alginate-gelatin hydrogels to encapsulate human adiposederived stem cells in order to investigate cell growth, viability and morphology during osteogenic differentiation taking advantage of the different physicochemical properties of this modified alginate-based hydrogel in comparison to pristine alginate hydrogel. We investigate the effect of hydrogel compositions on stem cell behavior in 3D. Higher viability and spreading morphology of encapsulated cells with interconnected networks were observed in high gelatin containing compositions. More filopodial protrusions from multicellular nodules were noticed during osteogenic differentiation in the hydrogels having a high amount of gelatin, confirming their suitability for cell encapsulation and bone tissue engineering applications. KEYWORDS Alginate, hydrogel, microbead, osteogenic differentiation, stem cell, 3D


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INTRODUCTION Cell encapsulation aims to embed viable cells within a biocompatible matrix in order to provide the entrapped cells with a 3D tissue-like environment, mimicking in vivo conditions. In addition to biocompatibility, a suitable matrix for cell encapsulation must possess semipermeability that allows the exchange of oxygen, nutrients and metabolites and simultaneously protects the encapsulated cells from toxic foreign bodies.1,2 Due to their ability to protect the encapsulated cells from antibodies and the host immune system, this technique has drawn attention in clinical applications as a delivery system enabling the transport of specific cells to the target site in vivo. Allotransplantation and xenotransplantation have been conducted using cell-loaded microcapsules for the treatment of endocrine diseases, such as diabetes,3 anaemia,4 dwarfism,5 haemophilia B,6 kidney7 and liver8 failure, and nervous system insufficiencies.9 In spite of the simplicity of this approach, the progress in this field during the past decades could not meet the high expectations due to issues about biocompatibility, biodegradability and cytocompatibility of the matrices used. Hydrogels are commonly used matrices for cell encapsulation since they exhibit a number of features which are advantageous in terms of providing a biocompatible environment to cells.10 Designing hydrogels for cell microencapsulation techniques is a major challenge. Initially, this approach was proposed as a means to protect encapsulated cells from the external environment, thereby preventing the rejection by the host immune system.11 For applications in tissue engineering, this property is not the only major requirement. Permeability of smaller molecules like oxygen, nutrients, growth factors, and metabolites are equally important for cell survival inside the matrix and these properties play an important role in the success or failure of materials used for tissue engineering applications, especially for cell encapsulation.2 Furthermore equilibrated mass transfer through the whole microbead is very important. If the dimension of the pores and the porosity of the matrix are not



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sufficient for proper diffusion of biomolecules the embedded cells that are far from the surface of the microbead may receive a lower amount of nutrients at a given time.2 Moreover, microbeads must be biodegradable to accommodate cell migration and proliferation. The matrix biodegradability allows for a gradual replacement of the hydrogel material with the encapsulated cells' own deposited extracellular matrix (ECM).12 Another important factor is the mechanical stiffness of the matrix, which should be optimized to protect the encapsulated cells from external stress. Simultaneously, the matrix should not exert a high mechanical barrier to the embedded cells, which could inhibit normal cellular behavior.13 Among the wide ranges of biocompatible hydrogels, alginate (ALG) is widely used for encapsulation of cells due to its excellent ionic gelation properties with divalent cations, which occurs under mild and non-toxic conditions for the encapsulated cells.14 However, it is now well established that encapsulated cells maintain round morphology and form clusters in pristine ALG hydrogel due to the absence of cell adhesion moieties and dense hydrogel structure. Similar morphologic outcomes were observed for MG-63 cells in our previous study15 and for adipose-derived mesenchymal stem cells (ASCs),16 encapsulated in ALG. Inclusion of cell adhesion molecules like gelatin or RGD (Arg-Gly-Asp) to ALG hydrogels did not enhance cell growth, migration and spreading morphologies significantly in 3D,15,17 which proves that the presence of cell adhesion moieties is not the only factor for cell growth in ALG. Other factors, especially matrix porosity and stiffness, are very important for cell migration with spreading morphologies since ionically crosslinked ALG possesses dense hydrogel structure, low degradability (nondegradable in mammals) and high stiffness, which restrict cell migration and spreading.18,19 In our previous studies, covalently crosslinked oxidized alginate (alginate dialdehyde)-gelatin (ADA-GEL) hydrogels were employed successfully for fabrication of microbeads20 and osteoblast-like MG-63 cells were successfully encapsulated within ADA-GEL hydrogel.15,17


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Superior growth, viability, proliferation, migration of MG-63 cells with spreading morphology and high cell-cell interconnections were observed in ADA-GEL microbeads compared to ALG, RGD-modified ALG and GEL-blended ALG microbeads.15 Colony formation has been observed during osteogenic differentiation of ASCs in calcium ALG microbeads at longer cultivation times, where round clusters of cells were formed at initial cultures.16 Monaco et al.21 showed in 2D culture that ASCs exhibited typical elongated fibroblastic morphology initially and formed colonies at longer culture times when they started to differentiate into osteogenic lineage. Since clustering is a common phenomenon of most types of cells in calcium-crosslinked ALG hydrogels, it has remained an open question whether the ASCs will show the same morphology they exhibited in ALG or whether they will show different morphologies when they are encapsulated in ADA-GEL. Finally, it is important to determine the effect of ADA-GEL compositions on morphological behavior and osteogenic differentiation of ASCs in 3D. Therefore, in this study we compared for the first time the growth, morphological behavior and osteogenic differentiation of ASCs in five different compositions of ADA-GEL microbeads.

EXPERIMENTAL SECTION Hydrogel synthesis and analysis of mechanical properties The ADA-GEL hydrogel synthesis process was described in detail in the previous study.20 Briefly, alginate di-aldehyde (ADA) was synthesized from ALG (guluronic acid content 65– 70%, Sigma-Aldrich, Germany) by partial oxidation using sodium metaperiodate (VWR International, Germany). ADA suspension (in ethanol-water mixture) was dialyzed against ultrapure water (Direct-Q®, Merck Millipore, Germany) using a dialysis membrane (MWCO: 6000–8000 Da, Spectrum Lab, USA). The resulted ADA solution was then lyophilized to obtain dry ADA. The lyophilized ADA was dissolved in ultrapure water at a concentration of 2% (w/v) and the resultant solution was sterilized by filtration through a



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0.22 µm filter (Carl Roth GmbH + Co. KG, Germany). The sterilized ADA solution was lyophilized under sterile conditions and 5% (w/v) solution was prepared by dissolving the required amount of dry ADA in sterile phosphate buffered saline (PBS, Gibco, Thermo Fisher Scientific, Germany). Gelatin (GEL, Bloom 300, Type A, from porcine skin, SigmaAldrich, Germany) was dissolved in ultrapure water at 37 °C to get 5% (w/v) solution, which was then sterilized by filtration through a 0.22 µm filter. The GEL solution was added slowly into the ADA solution under continuous stirring to synthesize ADA-GEL hydrogels with five compositions abbreviated as ADA70-GEL30, ADA60-GEL40, ADA50-GEL50, ADA40GEL60 and AD30-GEL70 to denote respective weight ratios of ADA and GEL. For performing the nanoindentation study, the mixed solution of ADA and GEL was poured into a 35 mm polystyrene Petri dish prior to gelation. After gelation further ionic crosslinking was performed with 0.1 M calcium chloride solution (calcium chloride dihydrate salt, VWR International, Germany) for 10 minutes. The ~2 mm thick hydrogel was washed with Hank´s Balanced

Salt

Solution

(HBSS,

Sigma-Aldrich,

Germany)

prior

to

performing

nanoindentation. Then the sample was submerged in HBSS at room temperature with the nanoindenter tip below the surface of HBSS all the time during the experiment in order to avoid any error occurred due to the adhesive force at the interface between water and air.22 The study was performed using a ferruled optical fiber-based nanoindenter (PIUMA, Optics11, Amsterdam; The Netherlands) at room temperature. A probe with a tip diameter of 96 µm was used in this study. Measurements were performed at different positions of each hydrogel to receive an average value. The loading period, holding time and unloading time were set to 2 s, 1 s and 2 s, respectively. The reduced Young’s modulus was calculated based on the load-displacement curve obtained from each experiment. Isolation of hASCs


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Primary human adipose-derived mesenchymal stem cells (hASCs) were used to investigate in vitro cytocompatibility of the ADA-GEL hydrogels in three-dimensional (3D) environments. Cells were isolated from the lipoaspirate of a healthy adult donor while undergoing abdominal fat reduction surgery. The procedure was approved by the Ethics Committee of the University Hospital Erlangen and informed consent was obtained. The lipoaspirate was washed with phosphate buffered saline (PBS) (Gibco, Thermo Fisher Scientific, Germany). Digestion was performed with 0.3 U/ml of collagenase NB 4G (SERVA GmbH, Germany) for 30 min at 37 °C with gentle and continuous shaking. CD-271+ hASCs were positively selected by magnetic-activated cell sorting using CD-271 microbead kit (MACS, Miltenyi Biotec GmbH, Germany) as recommended by the manufacturer. The CD 271+ cells were then cultured in basal culture media composed of DMEM/Ham’s F-12 (1:1) supplemented with 10% FCS, 2 mg/L of L-glutamine (Biochrom AG, Germany), and cytomix-MSC (Miltenyi Biotec GmbH, Germany). After achieving passage 5, the cells were trypsinized and used for the experiment. Tri-lineage (osteogenic, adipogenic and chondrogenic) differentiation of the isolated hASCs was demonstrated according to a standard protocol to confirm their multilineage differentiation ability.23 Cell encapsulation and in vitro culture ADA-GEL hydrogels of the five various compositions were used for cell encapsulation. During the hydrogel synthesis process, specifically after 2 minutes of adding GEL to ADA, cells were added to the hydrogel precursor mixture at a density of 2×106 cells/ml of the hydrogel. The resulted mixture was subsequently transferred into an extrusion cartridge (Nordson EFD, USA), which was connected to a high precision fluid dispenser (Ultimus V, Nordson EFD, USA). Cell embedded microbeads were fabricated from ADA-GEL of various compositions according to the procedure described in our previous study,20 where cell-free microbeads were fabricated. Microbeads were generated by applying different air pressure



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(0.55 to 2.5 bars) and collected in a beaker containing 0.1 M calcium chloride solution and kept for 10 minutes to allow complete ionic gelation. The fabricated microbeads were then sieved using cell strainer with 100 µm sized pores (BD Bioscience, Germany) and washed three times with serum-free Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Thermo Fisher Scientific, Germany) to remove the residual calcium chloride solution from the surface of the microbeads. The cell embedded microbeads were cultured in basal culture media for 2 days, followed by adding osteoinductive medium, which was composed of basal culture medium supplemented with 0.1 µM dexamethasone, 50 µg/ml of ascorbic acid, 10 mM βglycerophosphate (all supplements were purchased from Sigma-Aldrich GmbH, Germany). All experiments were conducted with three sets of samples (triplicate) of each hydrogel and each set contained approximate 30 microbeads. Phase-contrast microscopy Distribution and morphology of hASCs in the ADA-GEL microbeads after cell encapsulation and after specific culturing periods (day 3, 7, 14 and 28) were assessed by phase-contrast microscopy (Primovert, Carl Zeiss Microimaging GmbH, Germany). Moreover, the size of the microbeads was also assessed using the phase-contrast microscopy. The diameter of the beads produced from all hydrogels was found in the range of 600-900 µm. Immunofluorescence staining and microscopy Cytocompatibility was assessed by live-dead fluorescence staining using Calcein AM (InvitrogenTM, Molecular probes® by Life technologiesTM, USA) and Propidium iodide (PI) (InvitrogenTM, Molecular probes® by Life technologiesTM, USA). To visualize the live and dead cells in the microbeads after 4 weeks of cultivation, a mixture of calcein AM and PI (4 µL calcein and 5 µL PI per 1 mL Hank's balanced salt solution (HBSS, Gibco, Thermo Fisher Scientific, Germany) was added to the microbeads and incubated for 25 minutes at 37 °C. After incubation, the encapsulated cells were fixed using 4% paraformaldehyde


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solution and the images were taken with a fluorescence microscope (Axio Scope A.1, Carl Zeiss Microimaging GmbH, Germany). Fluorescent labeling was also performed to analyze encapsulated cell morphology after 4 weeks. The encapsulated cells were fixed with 4% paraformaldehyde solution for 12 minutes and permeabilized with Triton-X 100 containing permeabilization buffer for 12 minutes. Subsequently, F-actin staining was performed with rhodamine phalloidin (InvitrogenTM, molecular probes® by Life technologiesTM, USA), which selectively stains F-actin and nuclei were visualized with the green fluorescent stain, SYTOX® (InvitrogenTM, molecular probes® by life technologiesTM, USA). Afterward, the microbeads were washed three times with HBSS and the images were taken by fluorescence microscopy. AlamarBlue assay The influence of the various compositions of ADA-GEL on the metabolic activity of encapsulated cells was measured by the reduction of resazurin to resorufin by the alamarBlue assay. For investigation of metabolic activity, the microbeads were cultivated inside a cell strainer, which was placed in 6 tissue culture well-plates in order to prevent the influence of cells that migrated out of the hydrogels and adhered to the bottom of the well. After 4 weeks of incubation, the cell strainers with microbeads were placed in new wells of a 6 well-plate and subsequently freshly prepared cell culture medium containing 10 v% alamarBlue® reagent (Invitrogen, Germany) was added prior to the incubation for 4 hours. After incubation, 100 µL of the medium from each sample was transferred into a well of a new 96 tissue culture well-plate and the absorbance was measured photometrically at 570 and 600 nm (SpectraMax 190 and SoftMax Pro v5.4.1, Molecular Devices GmbH, Germany). Osteogenic differentiation assays The osteogenic differentiation of the encapsulated hASCs was assessed by light microscopy of alkaline phosphatase (ALP)-stained encapsulated cells and the specific ALP activity was



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measured photometrically by analyzing ALP and total protein concentration in the samples. ALP staining of encapsulated hASCs was performed after 4 weeks of cultivation in the osteoinductive medium by using an ALP staining kit (Sigma-Aldrich, Germany), which was added to the fixed encapsulated cells and incubated for 20 minutes. Afterward, the microbeads were washed with HBSS and images were taken with a light microscope (Primovert, Carl Zeiss Microimaging GmbH, Germany). ALP production was measured using an assay, based on the change in absorbance of p-nitrophenol as it is enzymatically cleaved by ALP. For quantifying the ALP in the encapsulated cells, these were lysed using lysis-buffer (pH 7.5, 10 mm Tris–HCl, 1 mm MgCl2, and 0.05% Triton X-100) after 2 and 4 weeks of cultivation. Then the supernatants were vortexed thoroughly and subsequently centrifuged for 5 min at 2000 rpm. 100 µL 9 mM Para-nitrophenylphosphate (pNPP) solution was added to the 250 µl supernatant and incubated at 37°C for 60 minutes. The reaction was stopped by adding 650 µL 1 M NaOH solution and the absorbance was measured at 405 and 690 nm by UV-Vis spectrophotometer (Specord 40, AnalyticJena AG, Germany). The ALP activity was normalized to the total protein content. Bradford test was used to determine the total protein content of the samples. 975 µL Bradford protein assay kit (AppliChem GmbH, Germany) was added to 25 µL supernatant (the same supernatant that was used for ALP study). After 10 minutes incubation in the dark, the absorbance was measured at 595 nm by UV-Vis spectrophotometer. The specific ALP activity was calculated from the ALP measurement and the Bradford test, which was presented as the amount of nano molar paraNitrophenylphosphate (pNPP) that was converted to para-Nitrophenol (pNP) by the ALP, produced by encapsulated cells per minute and mg of total protein. Statistical analysis


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Unless specified otherwise, data are reported as mean ± standard deviation. Statistical differences among the experimental groups (various compositions of ADA-GEL hydrogels) were performed with a one-way ANOVA followed by Bonferroni’s post-hoc test.

RESULTS AND DISCUSSION Mechanical properties of ADA-GEL hydrogels can be tailored by changing the compositions The mechanical properties of the ADA-GEL hydrogels of five compositions having various ratios of ADA to GEL were investigated using a nanoindenter. The results of the calculation of the reduced Young´s modulus (rYm) from the load-displacement curves are shown in Figure 1. The hydrogels containing higher amount of ADA exhibited higher mechanical properties compared to the compositions with lower ADA content. rYm of ADA70-GEL30 and ADA30-GEL70 were found to be 9.2 ± 4.2 kPa and 3 ± 1.3 kPa, respectively. Moreover, the compositions with higher ADA content showed similar outcomes and the other two compositions with lower ADA content also showed similar values. The significant loss of mechanical properties for the lower ADA composition hydrogels compared to the other three compositions can be attributed to the low crosslinking degree due to the lack of reactive aldehyde groups. Thus a significant number of ɛ-amino groups of gelatin remained uncrosslinked.20 Moreover, due to the low ADA content less ionic gelation occurred by calcium ions, which could also be the cause of low mechanical stiffness of the hydrogels with high GEL content. Though ADA70-GEL30, ADA60-GEL40, and ADA50-GEL50 hydrogels have a different amount of ADA, their mechanical properties appeared to be similar. Due to the presence of an equal amount of ADA and GEL in the composition, ADA50-GEL50, most of the ADA is probably crosslinked with GEL. Therefore, it shows the high mechanical property. In the other two compositions (ADA70-GEL30 and ADA60-GEL40), due to having a higher amount of ADA



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compared to GEL, some ADA might remain uncrosslinked. Therefore, the excess amount of ADA in ADA70-GEL30 and ADA60-GEL40 hydrogels did not contribute to the mechanical properties.

Figure 1. Mechanical properties of ADA-GEL hydrogels with varying ratios of ADA and GEL. Box-and-whisker plot showing the mechanical properties of the hydrogels, measured as reduced Young`s modulus (rYm) using a nanoindenter (n=10). The box-and-whisker plot indicates median (middle line), mean (small square), 25% and 75% of values (box lower and upper bounds, respectively), and minimum and maximum values (whiskers). Asterisks denote the significant difference of pairwise comparison, where *p< 0.05, **p< 0.01 and ***p< 0.001.

Viability and metabolic activity of encapsulated hASCs enhanced in high GEL containing compositions The viability of encapsulated hASCs in ADA-GEL of various compositions was assessed by live-dead staining after 4 weeks of cultivation in the osteoinductive medium. Fluorescence microscopy images of live-dead stained encapsulated cells are shown in Figure 2A. Viable cells were stained with calcein AM, which was cleaved by the naturally occurring intracellular esterases in the viable cells, resulting in a fluorescent green dye trapped in the


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cell which is impermeable to the cell membrane.24 On the other hand, propidium iodide preferentially stained the diploid DNA of apoptotic nuclei and appeared in red.25 After 4 weeks of cultivation, it is clearly observed that the encapsulated cells in all compositions of ADA-GEL were mostly alive, which are shown in green, with relatively few dead cells shown in red. However, the dead cells are comparatively high in number in the microbeads of low GEL containing ADA-GEL compositions (ADA70-GEL30 and ADA60GEL40). This result can be ascribed to the comparatively less availability of cell adhesion ligands due to having a low amount of GEL. On the other hand, high GEL containing ADAGEL microbeads provided more adhesion ligands to the cells. Moreover, as discussed later, the ADA-GEL hydrogels with high GEL content exhibit comparatively high degradation behavior that might reduce the steric hindrance on the encapsulated cells, enabling them to grow and proliferate.



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Figure 2. Viability and metabolic activity of encapsulated hASCs in ADA-GEL hydrogels with varying compositions. (A) Fluorescence microscopy images of viable (green, calcein AM) and dead (red, propidium iodide) hASCs encapsulated in ADA-GEL hydrogels of various compositions after 4 weeks of culture in the osteoinductive cell-culture medium. The majority of the encapsulated cells are viable in all hydrogels compositions. Comparatively more dead cells are found in low GEL containing ADA-GEL microbeads. Scale bar: 500 µm. (B) Metabolic activity of encapsulated hASCs in ADA-GEL of various compositions after 4 weeks of culture presented as % reduction of alamarBlue. Asterisks denote significant difference, *p< 0.05 and ***p< 0.001 (mean ± SD of n=5 measurements).


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The metabolic activity of encapsulated hASCs in ADA-GEL hydrogels of various compositions was analyzed by alamarBlue reduction assay, presented in Figure 2B. The metabolic activity of encapsulated hASCs increased in microbeads of ADA-GEL hydrogels having a high amount of GEL after 4 weeks of cultivation, a result that is in agreement with the observed live-dead staining assay results shown in Figure 2A. The reduction of cell activity for the ADA30-GEL70 microbeads might be the result of the high degradability26 that facilitates migration of the encapsulated cells.15 Therefore, a higher amount of cells migrated out of the microbeads of ADA30-GEL70 compared to other compositions and grew on tissue culture plate. This migration phenomenon was found to decrease for the compositions with increasing ADA content. The encapsulated cells in ADA40-GEL60 exhibited significantly higher metabolic activity compared to other compositions, which proves that this is the optimal composition for better growth and viability of hASCs in 3D. The 3D microstructure of the ADA40-GEL60 hydrogel and the availability of cell adhesion ligands are probably optimized due to hydrogel degradation over the cultivation period that contributes to transport of matrix molecules leading to the development of the pericellular and extracellular matrices. Stiffness and degradability of the hydrogels are the key factors for encapsulated cell morphology Cell morphology is correlated with important biochemical functions of cells such as adhesion, proliferation and migration.27 The morphology of encapsulated hASCs in ADAGEL hydrogels of various compositions was investigated using phase-contrast and fluorescence microscopies over the cultivation period. The phase-contrast microscopy images were taken directly after the encapsulation (Figure 3), showing that encapsulating cells display rounded morphology, as expected, proving that the encapsulation technique has no negative effect on cell morphology. After 4 weeks of cultivation the encapsulated hASCs in



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high GEL containing ADA-GEL microbeads appeared elongated and exhibited spindle-like fibroblastic morphology, establishing their filopodia and intercellular connections to create a 3D cellular network (Figure 3). On the other hand, cells encapsulated in low GEL containing ADA-GEL compositions (ADA70-GEL30 and ADA60-GEL40) exhibited pronounced rounded morphology with some filopodial protrusions. In order to investigate the changes in the morphology of cells encapsulated in low and high GEL containing ADA-GEL hydrogels over the culturing periods, phase-contrast microscopy images of cells embedded in ADA60GEL40 and ADA40-GEL60 were taken at specific intervals over the culturing period. As shown in Figure 4, after 3 days of encapsulation, cells embedded in both compositions exhibited rounded morphology with some filopodial protrusions. Over the longer culturing times cells in ADA60-GEL40 retained the same morphology, whereas cells in ADA40GEL60 elongated and exhibited spindle-like morphology. Low GEL containing ADA-GEL hydrogels showed lower degradation behavior compared to higher GEL containing compositions, which was reported in our previous study.26 In addition, the hydrogels composed of high ADA exhibited higher stiffness compared to lower ADA-containing compositions (Figure 1). Due to the high amount of polysaccharide (ADA), which results in higher stiffness and low degradability, the embedded cells in ADA70-GEL30 and ADA60GEL40 experienced high mechanical stress and their motility was impaired by steric hindrance. Moreover, cell adhesion was not facilitated due to low protein content in the hydrogels that retained the embedded cells in rounded morphology.28,29 On the other hand, cells in high GEL containing ADA-GEL hydrogels were elongated and adopted a strong cellmatrix adhesion-guided morphology due to high adhesive protein content, low stiffness and high degradation of the surrounding matrix. Moreover, ADA-GEL hydrogel exhibits highly porous structure30 and the porous structure might be increased in the high GEL containing compositions due to the release and degradation of the high amount of GEL. Therefore, the


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embedded cells might experience low steric hindrance. The matrices in low ADA-containing hydrogels could be easily deformed by cell-induced stresses, which could facilitate cell spreading and migration due to low degree of crosslinking of the hydrogels compared to high ADA-containing hydrogels.20

Figure 3. Morphological analysis of encapsulated hASCs in ADA-GEL hydrogels. Phase contrast microscopy images of hASCs encapsulated in ADA-GEL microbeads of various compositions immediately after encapsulation and after 4 weeks culture. Images in the left



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panel show homogeneous distribution of cells in the microbeads of all ADA-GEL compositions. At higher magnification (middle panel) the majority of the encapsulated cells appeared in single round shape, confirming the cell encapsulation process did not cause detrimental effects on cellular integrity. After 4 weeks of culture, encapsulated cells exhibited more elongated morphologies with interconnected structures in high GEL composing ADAGEL microbeads. Scale bar: 200 µm (left panel), 100 µm (middle and right panels).

Figure 4. Encapsulated cell morphology in high and low gelatin containing ADA-GEL hydrogels over the cultivation time. Phase contrast microscopy images of hASCs


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encapsulated in ADA-GEL microbeads of two compositions with low and high GEL content at different cultivation periods in osteoinductive cell-culture medium, showing the morphologies of the cells over time. More elongated cells at longer cultivation periods were observed in high GEL containing hydrogel microbeads. Scale bar: 20 µm.

Figure 5. Distribution of F-actin of encapsulated hASCs in ADA-GEL hydrogels with varying compositions. Fluorescence microscopy images of hASCs encapsulated in ADAGEL hydrogels of various compositions after 4 weeks of cultivation in osteoinductive cellculture medium, showing the distribution of the cytoskeleton of cells in the ADA-GEL



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microbeads of various compositions. The cells were stained for F-actin (red) and nuclei (green). Scale bar: 200 µm (left panel) and 50 µm (right panel).

A similar morphology of encapsulated hASCs in all compositions of ADA-GEL microbeads cultivated for 4 weeks in osteoinductive cell culture medium was observed using fluorescence microscopy (Figure 5). F-actin of encapsulated hASCs was observed to be elongated and spread through the whole microbeads of ADA-GEL with high GEL content. Similar to the outcomes of phase-contrast microscopy investigation, the most spread and elongated morphology of the encapsulated hASCs was observed in the ADA40-GEL60 composition. Nonlinear relationship between osteogenic differentiation and ADA-GEL compositions Although many studies have been performed to evaluate the osteogenic differentiation capability of ASCs in which the cells are grown and differentiated in classical 2D culture, very few reports are available on the growth and differentiations of these stem cells in 3D environments.16,31 The osteogenic differentiation potential of hASCs in a 3D environment is very important in tissue engineering since it mimics the real tissue-like environment. Encapsulated porcine ASCs in ALG microbeads have been successfully differentiated into osteogenic lineage in the study of Kim et al.16 It has been observed that encapsulated cells formed aggregates in early stage and formed colonies when the cells were positive for ALP staining. Though the nodule or colony formation is a characteristic phenomenon for osteogenic differentiation of stem cells, the aggregation of encapsulated cells is a common behavior not only for stem cells but also for other cell types in 3D conditions when cells are embedded in ALG matrices.17,32,33 Interestingly, in this study the encapsulated hASCs spread, migrated and exhibited elongated morphology with cell-cell interaction in ADA-GEL microbeads while keeping their osteogenic differentiation potentiality. The osteogenic differentiation behavior was proven by positive staining for ALP of encapsulated hASCs in


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all ADA-GEL compositions after 4 weeks of cultivation in osteoinductive cell culture medium (Figure 6A). In addition, multicellular nodules were formed in all compositions of ADA-GEL microbeads. It is important to note that only cell nodules were positive for ALP staining, proving that the cells, forming nodules were differentiated to the osteogenic lineage, which is in accordance with outcomes observed for porcine ASCs embedded in pristine ALG hydrogel.16 The characteristic features of ASCs that have never been observed in ALG hydrogels, like filopodial protrusions, linkages between the multicellular nodules and intercellular networks, were observed in ADA-GEL hydrogels while the cells maintained their osteogenic differentiation. These characteristics were found to be enhanced in ADAGEL compositions with relatively high amount of GEL. It must be noted that some multicellular nodules remained unstained for ALP, an effect which may be attributed to the poor and slow diffusion of differentiation factors (dexamethasone and β-glycerol phosphate) through the hydrogels or the insufficiency of staining reagents inside the hydrogel due to poor diffusion.16 More ALP-stained cells were observed in ADA-GEL microbeads having higher amount of GEL and that might be attributed to the combined effect of higher diffusion of differentiation factors and staining reagents due to the highly porous structure of the hydrogels and the presence of higher amount of GEL. As stated in the previous sections, ADA-GEL hydrogels having higher amount of GEL exhibit higher degradation behavior compared to the compositions with lower GEL content, which might create more porous structures.26,30 The expression of typical in vitro osteogenic differentiation marker, the ALP expression of encapsulated hASCs for all five compositions of ADA-GEL, was analyzed after 2 and 4 weeks of cultivation. As shown in Figure 6B, the encapsulated cells for all compositions expressed the osteogenic specific marker after both time periods of cultivation. The ALP expression of the encapsulated hASCs was found to be lower in the composition having the same amount of ADA and GEL after both cultivation periods. However, no



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significant difference was observed among the compositions at both time points. Moreover, no linear relation could be confirmed between the hydrogel compositions and the ALPexpression of encapsulated cells.

Figure 6. Osteogenic differentiation of encapsulated hASCs in ADA-GEL hydrogels. (A) Light microscopy images of ALP-stained hASCs encapsulated in ADA-GEL of various compositions after 4 weeks of cultivation in osteoinductive cell-culture media. The cells that are positively stained for ALP are in reddish purple color. Scale bar: 200 µm. (B) Specific ALP activity of encapsulated hASCs in ADA-GEL of various compositions after 2 and 4


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weeks of cultivation. Asterisks denote significant difference, *p< 0.05 (mean ± SD of n=5 measurements).

It is important to note that the encapsulated hASCs for all compositions expressed higher ALP after 4 weeks compared to that after 2 weeks. However, the significant difference of the expression was observed only for the compositions having high GEL content (ADA40GEL60 and ADA30-GEL70). Therefore it can be hypothesized that the high amount of GEL and related higher degradation of the hydrogel are the major influencing factors stimulating of ALP expression of encapsulated hASCs. Several studies illustrate that the incorporation of cell binding peptides, specifically RGD, into hydrogels improves the osteogenic differentiation of progenitor or mesenchymal stem cells.34–37 Since GEL possesses the RGD sequence of collagen,26,38 the presence of the RGD peptide in the hydrogel matrices likely stimulates the activity of cell integrin receptors that may play a role in osteogenic differentiation. As shown in the previous work,30 ADA-GEL hydrogel exhibits high porosity due to the cleavage of polysaccharide ring of alginate during synthesis of ADA that facilitates hydrolytic degradation. Due to exhibiting porous structure, exogenous osteogenic supplements could easily transport to the encapsulated cells in the ADA-GEL microbeads. Moreover, since GEL is bound to ADA due to the crosslinking between ɛ-amino groups of GEL and aldehyde groups of ADA,20 higher amount of GEL retains in the ADA-GEL compared to the ALG-GEL blended hydrogels.15 The presence of high amount of GEL in the ADA-GEL microbeads could enhance encapsulated cell viability and osteogenic differentiation as discussed earlier.

CONCLUSIONS ADA-GEL hydrogels of various compositions supported osteogenic differentiation of encapsulated adipose-derived stem cells while exhibiting filopodial protrusions of



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multicellular nodules with interconnected cellular networks in the microbeads, specific characteristics that have never been observed in 3D ALG-based hydrogels. Though all ADAGEL compositions supported osteogenic differentiation, the hydrogels with a high amount of GEL promoted the growth and viability of encapsulated hASCs with high interconnected morphology and more filopodial protrusions due to the availability of cell adhesive moieties and low steric hindrance. No linear correlation was observed between the elongated, interconnected cell morphology and osteogenic differentiation of hASCs. However, the investigation of ALP expression is only a preliminary osteogenic differentiation study. A complete study including other assays, e.g. type I collagen, osteocalcin, bone sialo protein, is required. Nevertheless, the present results demonstrate that cell encapsulation with ADAGEL hydrogels is a viable strategy to investigate cell growth, morphological behavior and differentiation in 3D. The outcomes of this study unravel that the morphology of adiposederived stem cells or cell nodules during osteogenic differentiation is highly dependent on the microstructure, physico-mechanical properties and composition of the surrounding matrix.

ACKNOWLEDGEMENTS This work was supported by the Emerging Fields Initiative (EFI) of the University of Erlangen-Nuremberg, Germany (project TOPbiomat). B. Sarker acknowledges the German Academic Exchange Service (DAAD) for financial support. The authors thank Dr. Andreas Arkudas (Department of Plastic and Hand Surgery, University of Erlangen-Nuremberg) for helpful discussions.

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Oxidized Alginate-Gelatin Hydrogel: A Favorable Matrix for Growth

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