Cell-laden alginate/polyacrylamide beads as carriers for stem cell delivery: preparation and characterization

Abstract

Stem cell based therapies employ engraftment or systemic administration methods for the delivery of stem cells into the target tissues to enhance their regenerative potential. However, majority of the stem cells were found to migrate away from the target site soon after the transplantation, which directly hinders their clinical efficacy, in particular while treating cartilage defects. Therefore, the present study was designed to explore the feasibility and efficacy of an alginate/polyacrylamide (Algi/PAAm) composite biomaterial in the form of cell-laden hydrogel beads as a suitable carrier system to be able to hold the stem cells at the target site and deliver them efficiently. Human bone marrow-derived mesenchymal stem cells (hBMSCs) have been used as a model cell. The beads prepared at an optimized concentration ratio were characterized to study their physicochemical properties. Furthermore, cell-encapsulated Algi/PAAm beads were evaluated for their biological properties. The result of this study has demonstrated that the Algi/PAAm beads with their optimal composition were able to maintain the viability of the encapsulated cells during the period of study, suggesting the cellular compatibility of the beads. Additionally, the encapsulated cells showed round morphology within the beads, in contrast to the 2D-cultured spindle-like shape of hBMSCs. Based on the experimental data obtained in this study, cell-laden Algi/PAAm beads may serve as a potential carrier system for stem cell delivery.

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1. Introduction

Stem cell therapy holds great promise in the treatment of various diseases as well as in the regeneration of damaged or defective tissues. Stem cells have a great capacity to differentiate into a variety of cell types and therefore, stem cell transplantation has been elucidated as an effective therapeutic strategy for the regeneration of the target tissues. Despite the initial promising results obtained from preclinical/clinical studies, stem cell therapy has not been able to be actualized as a routine therapeutic option in the clinic. The major issues attributed to this, could be the poor stem cell retention, engraftment in the hostile environment of the target tissue and the need for an optimized system for delivering sufficient numbers of stem cells.¹ These issues have been remained to be resolved before the complete translation of stem cell-based therapies. As a matter of fact, for the functional regeneration of the target tissue, sufficient quantity of the stem cells should be re-delivered to the patient. Stem cells can be transplanted directly into the target site and/or administered peripherally via different delivery techniques. However, the transplanted cells experience a harsh environment and in general, more than 95% of cells die shortly after the transplantation,¹ which could be attributed to ischemia due to absence of vascularization and cell attachment sites resulting in cell death (anoikis), host immune response and low cell density.^2,3 Furthermore, the conventional injection methods often result in poor cell survival and low levels of cell integration into the host tissue.⁴

To overcome these limitations, stem cell delivery systems have been developed. Stem cell delivery systems are specially designed to provide the cell attachment sites, protect from host immune cells, and localize the cells to achieve appropriate cell densities. Ideally, the system should be non-immunogenic, biodegradable at appropriate rates and stable at physiological pH and temperature. Several biomaterials-based delivery systems have been used for stem cell delivery in the form of nanofibers, microspheres, beads, hydrogels, etc. Each form of carrier system has its own advantages as well as disadvantages depending on the application. For example, nanofiber system has mostly being used as tissue scaffold but its application in the local delivering of stem cells is limited. Similarly, microspheres cannot be used with higher cell densities of encapsulated cells and, furthermore, there is a chance of migration from the implanted site. Among them, hydrogel provides the hydrated environment to the cultured cells but struggles for its mechanical stability.⁵ Among different types of hydrogels, cell-laden hydrogels have gained much attention for the delivery of stem cells due to their ability to fix stem cells in place while providing a hydrated microenvironment. These systems also provide a means to allow for the factors produced by the encapsulated stem cells to diffuse into the target site.⁴ Therefore, these by enhancing the mechanical stability of them, these cell-laden hydrogels could be used in the development of a clinically translatable delivery system to improve the effectiveness of the stem cell delivery for various tissue engineering and regenerative medicine applications.

The availability of large quantities of mesenchymal stem cells (MSCs), multilineage differentiation potential, readily propagation in culture and no requirement for adjunctive immunosuppressive therapies,⁶ has made MSCs the most relevant progenitor cell source for tissue engineering applications, in particular cartilage tissue engineering. MSCs have the ability to migrate and engraft onto sites of injury and undergo site-specific differentiation. Therefore, MSCs have been used readily in direct injection for target tissue repair (for example, cartilage). Although the results of this clinical approach were reassuring due to its minimal invasiveness, but it has been observed that the direct injection of MSCs often results in poor cell viability. This could be attributed to the direct exposure of injected stem cells to the damaging mechanical forces that can injure the cells, thereby emphasizing on the need of an optimized delivery system.¹ Additionally, a majority of the MSCs were found to migrate away from the target site soon after the transplantation, which being a crucial process further hinders its clinical efficacy.⁷ The method and site of delivery most likely affects the trafficking of MSCs to target organs. Therefore, encapsulation system based on stem-cell laden hydrogels (Algi/PAAm, for example) could be used as a suitable system for maintaining the natural cell phenotypes, and for delivering MSCs in situ at the target site. With three-dimensional (3D) scaffolds, cell survival and graft integration can be augmented during cell transplantation.⁸ Furthermore, 3D scaffolds are required to maintain proper growth, preservation of the morphology and production of natural chondrocyte matrix components.^8–11

In view of this, immobilization of MSCs in alginate gels is a well-known technology used for various tissue engineering applications. MSCs immobilized in alginate gels maintain good viability during long-term culture due to the mild environment of the gel network. These alginate gels may function as a protective barrier towards physical stress and to avoid immunological reactions with the host.¹² For most of the stem cell delivery applications hydrogel beads have been used to allow diffusion of essential nutrients.¹³ Although the cell-crosslinked alginate hydrogel beads show excellent bioactivities, the network exhibits low strength and toughness, which may limit its practical applications.¹² These limitations of the alginate-based scaffolds could be overcome by the use of the combination of tough polymers such as polyacrylamide (PAAm) in the form of composite cell-laden hydrogel beads which can show potent mechanical and physical stability properties closer to natural human cartilage tissue.¹⁴ as well as could prove suitable for the controlled delivery of MSCs in situ.¹⁵ PAAm is a biocompatible synthetic polymer and has been clinically proved to be non-toxic, non-immunogenic and stable in human body.¹⁶ PAAm is an ingredient in a variety of cosmetic and beauty products, and has been approved by the U.S, Food and Drug Administration (FDA) with limitations in quantity, to be used in food-packaging adhesives, paper and paperboards, to wash or peel fruits and vegetables and in imprinting soft-shell gelatin capsules.¹⁷ Keeping the above points in view, in this study, authors report the preparation and characterization of cell-laden Algi/PAAm beads suitable for the delivery of stem cells, in particular hBMSCs, for tissue engineering applications such as cartilage tissue regeneration.

2. Experimental

Materials

For material preparation, acrylamide (Cat. no. 164855000), N,N′-methylenebisacrylamide (Cat. no. 163805000) and N,N′,N′,N′-tetramethylethylenediamine (TEMED) (Cat. no. 138455000) were purchased from Acros Organics, Belgium. Ammonium persulfate (APS) (Cat. no. 15055) and calcium chloride (Cat no. 10043524) was bought from Qualigen India. Sodium alginate (Cat. no. W201502) was obtained from Sigma-Aldrich USA and used as received. For cell culture experiments, MEM alpha modification (1×) medium (Cat. no. SH30265.01) was purchased from HyClone. Fetal bovine serum, (Cat. no. 12003C-500ML) from SAFC Biosciences, and penicillin/streptomycin (Cat. no. 15140-122) and L-glutamine 200 mM (100×) (Cat. no. 25030-081) were obtained from Life Technologies. Dulbecco's Phosphate Buffer Saline (PBS) (Cat. no. D8537) purchased from Sigma-Aldrich, USA. Ficoll-Paque (Cat. no. 17-1440-02) from GE Healthcare, 0.25% trypsin–EDTA solution 1× (Cat. no. TCL014-6X500ML) from Himedia and CryoSure-DMSO (Cat. no. 0482) from WAK-Chemie Medical GmbH received, respectively. For cell staining experiments the live/dead kit (Cat. no. L3224) was purchased from Life Technologies and cell counting kit (CCK-8) (Cat. no. 96992) from Sigma (USA).

Isolation and culture of human bone marrow derived mesenchymal stem cells (hBMSCs)

Human bone marrow sample was obtained from donors with their written consent in Christian Medical College and Hospital, India. The following experimental protocol was used for isolation of hBMSCs. Briefly, about 5 ml of bone marrow suspension was harvested from posterior iliac crest of donor and mixed with same volume of heparinized (10 U ml⁻¹) PBS to prevent clotting. The mixture was kept at 4 °C prior to further processing in the laboratory. Small aliquot of 100 μl volume were taken for the monocyte counting. Mononuclear cells (MNCs) were isolated as previously described protocol with some modifications.¹⁸ Briefly, bone marrow sample was subsequently loaded on the top with Ficoll-Paque solution in a 2 : 1 ratio (bone marrow to Ficoll-Paque ratio) preloaded in a centrifuge tube to form a density gradient, followed by centrifugation at 400g for 30 min at 22 °C. After centrifugation, the yellow plasma layer obtained from the bone marrow sample was discarded and thin MNCs layer was carefully collected and washed twice with 12 ml of PBS, 700 g for 6 min. After washing it twice, the cell pellet was dispersed into 2 ml of α-MEM (10% FBS) media for further seeding and cell counting. The isolated cells were suspended in α-MEM of 440 ml mixed with 5 ml of penicillin/streptomycin (1%), 5 ml of glutamine (1%) and 50 ml of FBS (10%) and seeded into 25 cm² flasks. Cells were cultured by incubating at 37 °C, 5% CO₂. After 3 days, non-adherent cells were removed by washing twice with PBS. Media was changed once in every 3 days. The cell confluence and morphology was monitored under an inverted microscope (CKX41, Olympus). Individual fibroblast-like cells and small colonies of these cells can be seen by inverted microscope after 3 to 4 days. As the cells proliferate the colonies increase in size and become more distinct by 7 days of culture. After 10–14 days of culture the seeded cells can be seen to acquire spindle morphology that seemingly confirms the presence of MSCs. When these primary cultured cells reached 60–70% confluence, they were harvested using 0.25% trypsin/EDTA and sub cultured. The third passage cells were used for subsequent studies. Cells isolated from bone marrow samples at passage 3 were characterized for identification of the phenotypes using flow cytometry and morphology using inverted microscope.

Synthesis of Algi/PAAm beads

The experimental protocol for synthesis of Algi/PAAm beads is given below. In brief, a master solution of 40% acrylamide and 2% N,N′-methylenebisacrylamide monomers was prepared in deionized (DI) water. Solution was filtered using 0.45 micron filtration flask and kept in dark for storage until further use. 1.5% (w/v) alginate solution was prepared separately in DI water with magnetic stirring. Pre-polymer solutions i.e., acrylamide master solution and alginate solution, were mixed together in 1 : 5 ratios, respectively, under constant magnetic stirring of 200 rpm at room temperature (RT), making final volume of 5 ml. This solution was transferred to a 5 ml syringe with a built-in 24 G needle. This pre-polymer solution was subsequently dispensed from the syringe drop by drop into the gel-forming solution i.e., 16 ml of CaCl₂ – 10² mM solution mixed with 4 ml of (20% v/v) TEMED with moderate magnetic movement, allowing alginate and acrylamide monomers to completely polymerize (∼10 min) until the beads were completely crosslinked.

For comparative analysis, PAAm and alginate beads were also synthesized separately. Briefly, PAAm hydrogels were synthesized by mixing the appropriate volume of the master solution of acrylamide and bisacrylamide with ammonium phosphate (APS) (20 wt%) and TEMED (20% v/v) in the total volume ratio of 1/100 and 1/1000 respectively, under constant stirring. Alginate beads were prepared with the same protocol except for the addition of the acrylamide master solution and TEMED.

Encapsulation of hBMSCs in Algi/PAAm beads

The hBMSCs were encapsulated in Algi/PAAm beads by using the following method. The cells were trypsinized from tissue culture flask, followed by washing in PBS and then isolation of the cell pellet. The isolated hBMSCs at passage 3 were suspended at a seeding density of 1000 cells per cm² in pre-polymer solution. The cell suspension mixed with pre-polymer solution, was slowly dispensed through a 24 G needle and dropped into the gel-forming solution. The beads with approximately 500 cells per bead (diameter 3 mm) were allowed to polymerize for 10 min and washed twice with DPBS (Fig. 1). The beads were then transferred into 96-well plate with 4 bead in each well filled with medium (α-MEM supplemented with 10% FBS, 1% penicillin/streptomycin and 1% L-glutamine). The beads were cultured at 37 °C in a humidified atmosphere of 5% CO₂ for up to 2 day. No dissolution of the beads was observed during the culture period.

Fig. 1 Schematic representation for the synthesis of hBMSCs encapsulated Algi/PAAm beads. Abbreviations: hBMSCs – human bone marrow-derived mesenchymal stem cells; Am – acrylamide; BIS – N-methylenebisacrylamide; CaCl₂ – calcium chloride; TEMED – tetramethylethylenediamine.

Characterization

Flow cytometry analysis. Flow cytometry analysis was performed for analyzing phenotypes of isolated cells (prior to their encapsulation within the beads) to make sure that the cells pertain to the family of hBMSCs. Cells were harvested by trypsin, and centrifuged. The cell pellet obtained was then mixed with PBS and following anti-human antibodies, which were conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE) and peridinin-chlorophyll proteins (PerCP): IgG FITC/PE/PerCP, CD45-FITC, CD34-PerCP, CD14-PerCP, CD73-PE, CD90-PE and CD105-PE (Becton Dickinson, USA). Surface markers such as CD34, CD14, CD45, CD73, CD90, and CD105 are the hematopoietic lineage marker, monocyte differentiation antigen 14, monocyte differentiation antigen, ecto-5′-nucleotidase, human Ig superfamily glycoprotein Thy-1 and endoglin, respectively. Approximately 1 × 10⁵ cells were analyzed using a flow cytometer (FACSCalibur, Becton Dickinson, USA) and the data were interpreted. Replicate experiments were performed twice using the same donor cells sample.

Microscopic analysis. For morphological analysis of hBMSCs isolated from human bone marrow sample (prior to their encapsulation within the beads), the cells were observed under inverted microscope (Leica inverted microscope imaging system, USA) in phase-contrast mode and images were taken after cells reached to 60–70% confluence state.

Fourier transforms infrared spectroscopy. The physiochemical properties, such as chemical group functionality of Algi/PAAm beads (without cells) were analyzed using a Fourier transform infrared (FTIR) spectrometer (Nicolet is10, Thermo Scientific, USA) using attenuated total reflectance (ATR) mode. The spectra were recorded over the wave number range of 4000–400 cm⁻¹. For FTIR analysis, Algi/PAAm beads and alginate beads of 0.5 mm diameter were prepared followed by washing with DI water and one hour desiccation, prior to measurement. For PAAm sample measurement, hydrogel was prepared freshly followed by 24 h desiccation.

Thermogravimetric analysis. The thermogravimetric thermograms of PAAm, alginate bead (without cells) and Algi/PAAm beads were obtained using a simultaneous DSC-TGA analyzer (SDT Q600, TA Instruments Inc., USA) at a heating rate of 20 °C min⁻¹ in nitrogen atmosphere. Here, DSC stands for differential scanning calorimetry and TGA stands for thermogravimetric analysis. The TGA thermograms were recorded over a temperature range from ambient to 500 °C. The alumina crucible was used with the capacity of 90 μl. The samples were washed twice with DI water and then lyophilized for 24 h before pre-freezing at −80 °C for overnight. The weight of the samples used for each analysis was 5–10 mg.

Differential scanning calorimetric analysis. The differential scanning calorimetry (DSC) thermograms were recorded using simultaneous thermal analyzer (SDT Q600, TA Instruments Inc., USA). PAAm, alginate bead and Algi/PAAm bead (without cells) were tested in alumina crucibles at a heating rate of 20 °C min⁻¹ under nitrogen gas (100 ml min⁻¹) over a temperature range from 30 °C to 500 °C. Melting temperature was taken as the peak of the melting endotherm. The error in each measurement was estimated to be ±0.5 °C. The sample preparation was similar to TGA samples.

Scanning electron microscope. Morphological analysis of the Algi/PAAm beads (without cells) was studied using a scanning electron microscope (SEM) (Model: FEI Quantan FEG 200, USA). The lyophilized samples were cut to expose the cross-section, coated with gold for 120 seconds using a gold sputter coater (Edwards S150B, Perkin-Elmer, USA) and then analyzed under SEM at an accelerating voltage of 10 kV.

Live/dead assay. Qualitative analysis of the cell viability was performed by using live/dead staining kit (Life Technologies, USA) for the Algi/PAAm bead encapsulated hBMSCs at day 2. For this assay, supernatant of the samples were aspirated and supplemented with 200 μl of staining solution (composition: 2.5 μl of calcein AM and 2.5 μl of ethidium bromide added to 5 ml of culture medium). The staining solution kept protected from light by covering the plate with aluminum foil followed by 10 min incubation. After incubation, the stained samples were observed under fluorescent microscope (Leica DMI6000, USA) to study the cell viability of the hBMSCs encapsulated Algi/PAAm beads. The hBMSCs culture in well plates was taken as a control 2D culture samples with seeding density of 1000 cells per cm².

Cell counting kit-8 (CCK-8) assay. The quantitative analysis of the cell viability was performed using CCK-8 assay. CCK-8 allows sensitive colorimetric assays for the determination of the number of viable cells. For this assay, supernatant were aspirated from the sample wells and hBMSC encapsulated Algi/PAAm bead samples were gently crushed with a sterilized micropestle. Subsequently, all sample wells were supplemented with CCK-8 solution (i.e., 20 μl of CCK-8 and 180 μl of media for each well) in 96-well plate followed by 4 h incubation. For control 2D samples, CCK-8 solution was added over the wells followed by 4 h incubation and then analyzed. The absorbance of the solution was measured at 450 nm using ELISA plate reader (Original Multiskan EX, Thermo Scientific, USA), which is the direct measure of cell viability in the samples. The readings were obtained at day 2 of culture (n = 4) and then plotted for comparison between 2D monolayer culture and cells encapsulated 3D bead culture.

3. Results and discussion

This study demonstrates the synthesis and analysis of the cell-laden Algi/PAAm hydrogel beads in an optimal composition, which could be useful as hBMSCs delivery system for tissue regenerative applications, in particular, cartilage repair. The hypothesis has been supported through the following experimental data.

Culture and characterization of hBMSCs

Flow cytometry analysis. As a first step, the hBMSCs were isolated and characterized for their phenotypic confirmation, prior to their encapsulation. Flow cytometry was used to characterize the cells using surface markers. Surface markers CD34, CD14 and CD45 were found to be negligibly expressed, while hBMSCs markers including CD73, CD90 and CD105 were detected positively (Fig. 2), confirming that the cells isolated from human bone marrow belongs to mesenchymal stem cell lineage.¹⁹ The isolated cells i.e. hBMSCs were further used for encapsulation studies into Algi/PAAm bead system.

Fig. 2 Flow cytometric analysis for the typical surface markers expressed in hBMSCs. The hBMSCs were stained with phycoerythrin (PE), peridinin chlorophyll (PerCP) or fluorescein isothiocyanate (FITC) conjugated antibodies against the indicated surface markers: CD73-PE; CD105-PE; CD90-PE; CD34-PE; CD14-PerCP; CD45-FITC. The blue shaded region shows signals from hBMSCs surface marker antibodies and the red color region represent the isotype controls for background fluorescence.

Microscopic analysis. The morphology of hBMSCs cultured in a tissue culture flask under defined culture medium is studied using inverted microscope. Fig. 3 shows phase contrast micrographs of hBMSCs. The results showed that the cells are confluent and has spindle-shaped morphology similar to mesenchymal lineage originated cells, which further confirming that the isolated cells are MSCs.²⁰ The hBMSCs proliferated at a faster rate after primary culture and attained confluence after 5 days of passage 1. After first passage, about 1 × 10⁶ live cells have been detached for further culturing. Trypan blue staining confirmed almost 100% vitality.

Fig. 3 Phase contrast micrographs of confluent hBMSCs (passage 3) cultured in a tissue culture flask under defined culture medium. (A) 10× magnified view- and (B) 20× magnified view of the cultured cells exhibiting spindle-morphology of hBMSCs and confluence. Marked area has been magnified by 20× as shown in panel B.

Synthesis and characterization of Algi/PAAm beads

The general gelation mechanism involved in the preparation of cell-laden alginate beads through the ionotropic gelation technique involves extrusion of cell-alginate mixture into CaCl₂ solution. By this method, the alginate in the outer layer of the drop which is in immediate contact with calcium ions becomes insoluble and after some time of curing in CaCl₂ solution the alginate present inside the droplet also gets converted into calcium alginate due to the diffusion of calcium ions from the bulk external phase.¹² Preparation of Algi/PAAm beads involves the dropping of the acrylamide/alginate mixture through a syringe needle into a CaCl₂/TEMED solution with continuous stirring at room temperature. Since five parts of alginate is mixed with one part of acrylamide solution, the immediate visible gelation of the bead can be attributed to the ionotropic gelation of the alginate.²¹ The outer layer of the bead becomes solidified immediately due to the formation of calcium alginate entrapping the acrylamide solution. Meanwhile, it can be proposed that the presence of TEMED in the curing solution could also contribute to the polymerization of the acrylamide, leading to an interpenetrating polymeric network of alginate and polyacrylamide within the beads. The composite system is a novel, biocompatible and fast in situ forming gel bead system in a unique composition ratio of alginate and polyacrylamide. The hydroxyl groups in alginate crosslinks with free amide groups on acrylamide for formulating nontoxic gel beads with highly porous internal structures. The intermolecular hydrogen bonding provides an additional mechanical strength to the cross-linked polymeric gel network. Alginate to PAAm concentration has been optimized so as to minimally affect the cellular compatibilities and also the mechanical stability of the system. The photographic images of prepared Algi/PAAm hydrogel beads are shown in Fig. 4. It can be seen from the figure that the shape of the beads is round and size is ∼3 mm. However, by manipulating the experimental conditions, the size of the beads could be modulated.

Fig. 4 Photographic images of Algi/PAAm hydrogel beads. (A & C) shows the dimensions and shape of the hydrogel bead sample used for cell culture experiment. (B) shows the change in dimensions in complete dry and swollen state.

Fourier transform infrared spectroscopy. FTIR analysis of alginate, PAAm and Algi/PAAm beads (without cells) were carried out to determine the chemical functional groups of the beads. The changes observed in peak or peak shifting could reveal bonding or interactions between the functional groups of the polymers. Fig. 5 shows the FTIR spectra of alginate, PAAm and Algi/PAAm beads. The spectral analysis of polyacrylamide hydrogel showed the presence of band at 3354.60 cm⁻¹ which can be attributed to symmetrical and asymmetrical stretching of N–H group. The characteristic C=O stretching vibration bands of amide and acid groups have been observed at 1703.76 cm⁻¹ and the peak for CH=CH₂ group was observed at 1620.25 cm⁻¹. The FTIR spectra of alginate bead exhibited characteristic broad band at 3362.32 cm⁻¹ corresponding to O–H stretching vibrations of alginate. A sharp peak observed at 1620.90 cm⁻¹ corresponds to carbonyl group of –COO⁻ moiety present in Ca²⁺ crosslinked alginate polymers. A characteristic carboxylate group peak was also observed at 1463.85 cm⁻¹. In the spectrum of Algi/PAAm beads, the shoulder peaks appeared at 3417.65 cm⁻¹ and a sharp peak at 1673.69 cm⁻¹ corresponding to the N–H stretching and C=O stretching vibrations, respectively, which confirms the crosslinking between the two groups. The presence of a peak at 1434 cm⁻¹ in the Algi/PAAm beads corresponds to the C–N bending vibration, which further supports the crosslinking reaction. The sharp hydroxyl peak of alginate bead at 1620.90 cm⁻¹ was observed to be split and shifted to a higher value of 1673.69 cm⁻¹ and 1625.55 cm⁻¹. The shift in the wavenumber of hydroxyl group of alginate bead may be due to an effect on bond formation involving adjacent hydroxyl groups in alginate and the CONH₂ groups in PAAm as a result of the conformational changes in the crosslinked alginate and PAAm. A shift in the sharp peak at 649.07 cm⁻¹ in PAAm to 612.51 cm⁻¹ in Algi/PAAm bead was observed which could be attributed to NH₂ bond wagging and bending vibration for the out of the plane carbon. The intermolecular hydrogen bonding provides an additional mechanical strength to the cross-linked polymeric gel network. These observations supported the successful crosslinking of PAAm polymeric chains with the alginate macromolecules in the given ratio.

Fig. 5 FTIR spectra of (A) PAAm, (B) alginate bead and (C) Algi/PAAm beads.

Thermogravimetric analysis. During the process of crosslinking, initiator generates free radical sites on sodium alginate and acrylamide. These reactive sites were crosslinked together due to presence of multifunctional property of N,N′-methylenebisacrylamide. Thus, crosslinkage forms 3D networks of the Algi/PAAm gel at its phase-transition temperature. The TGA profile of PAAm, alginate bead and Algi/PAAm beads (without cells) are shown in Fig. 6. Alginate bead thermogram shows two pyrolysis stages, the first thermal degradation process occurred in the temperature range 200–300 °C with an average weight loss of 37.5% and the second degradation occurred in the range of 330 to 490 °C with 12.73% weight loss. Alginate is a polysaccharide and contains sugar units, galactose and mannose. The first degradation loss could be attributed to the degradation of the carboxyl group; CO₂ is released followed by the second degradation process mainly due to loss of volatile components, rupture of chain, and fragmentation of alginate into monomers with final conversion into carbonate.^21,22 The moisture loss occurred below 100 °C in all the samples. The thermogram profile of PAAm shows three characteristic degradations in the range of 220 to 330 °C and 350 to 460 °C with 12.41% weight loss. In the temperature range 220–330 °C, one ammonia molecule is liberated for every two amide groups, resulting in the formation of imide.²³ Subsequently, thermal degradation of imides and depolymerization of the polymer backbone occurs in the later stage. The degradation profile of Algi/PAAm bead clearly shows distinct decomposition pattern from the individual alginate and PAAm. The Algi/PAAm bead shows weight loss at between 50 to 200 °C and 250 to 350 °C was 60.42% due to the moisture loss, dissociation of chemical crosslinkage, and bonding between PAAm with alginate. On comparison of results obtained with reported thermograms of alginate and PAAm, peak shifts were observed indicating transformation which further provide thermal stability to the gel network in comparison to the PAAm and alginate macromolecules. Hydroxyl groups of alginate could form hydrogen bonding with PAAm. It has been observed that the mechanisms of decomposition in all three samples were quite different and the different decomposition trend and shifted peaks in thermogram qualitatively support the formation of new covalent bonds between alginate and PAAm.

Fig. 6 TGA thermograms of (A) Algi/PAAm bead, (B) alginate bead and (C) PAAm.

Differential scanning calorimetric analysis. The DSC thermogram profile of PAAm, alginate bead and Algi/PAAm beads (without cells) is shown in Fig. 7. The graph of alginate bead exhibits two melting endotherms at 250.73 and 347.74 °C respectively, which is in accordance to the TGA peaks discussed above. Similarly, PAAm thermogram showed a sharp melting endotherm at 121.36 and 305.98 °C, respectively. Whereas Algi/PAAm bead thermogram showed multiple endotherm peaks at 91.69 °C, 179.09 °C and 395.21 °C. In Algi/PAAm bead, a shift in the peak of alginate bead from 347.74C to 395.21 °C and considerable thermal stability of Algi/PAAm beads was observed. This shifting could be attributed to the addition of the bulky groups as side chain and presence of amide bonds on alginate of Algi/PAAm bead, which imparts rigidity to the Algi/PAAm bead network.²³ Thus, DSC supports the crosslinking of PAAm with alginate. The crosslinked form of Algi/PAAm could provide sufficient mechanical strength to the polymer network.

Fig. 7 DSC thermograms of (A) alginate bead, (B) Algi/PAAm bead and (C) PAAm.

Scanning electron microscope. SEM was employed to characterize the surface topography of the lyophilized Algi/PAAm beads (without cells). A representative SEM micrograph of lyophilized Algi/PAAm bead is shown in Fig. 8. The analyzed Algi/PAAm beads show coarse surface morphology with noticeable porosity. The cross-sectional view of the bead shows interconnecting porous 3D network with an average pore size of 59.02 μm diameter. It is anticipated that the interconnected porosity and porous internal 3D network of the beads could enhance the mass transportation properties within the scaffold, support cell infiltration and tissue in-growth in 3D space.

Fig. 8 Scanning electron microscopic images of as lyophilized Algi/PAAm beads. Magnifications for (A), (B) and (C) are 70×, 500× and 3000× respectively.

Evaluation of hBMSCs encapsulated Algi/PAAm beads

Live/dead assay. Cell viability analysis was performed in order to visualize the spatial distribution of live and dead cells throughout the 3D Algi/PAAm bead network as well as in 2D tissue culture (TC) flasks system at day 2. Also the live/dead assay was used to characterize the biocompatibility of the in situ gelling system for hBMSCs, due to their capacity to differentiate into cartilage tissues for example. The qualitative viability analysis of the hBMSCs encapsulated in the bead system is studied by live/dead staining kit. The hBMSCs cultured in 96-well tissue culture plate served as the positive control. It was observed that the hBMSCs were readily encapsulated within the bead system in all samples with roughly same affinity following initial seeding. As demonstrated in Fig. 9, most of the cells were viable in Algi/PAAm bead system at day 2 of culture. Bright green fluorescence throughout the hBMSCs clusters indicates that the cells were mostly viable and homogenously distributed within the bead matrix. However, few dead cells (marked red) were also could be seen in some parts of the bead matrix. In both the culture systems (2D monolayer and 3D bead system) cells were observed to be significantly viable, migrate and form interconnected networks with neighboring cells (in 2D system). These observations clearly demonstrate the cellular compatibility of the Algi/PAAm bead systems for hBMSCs and also confirm that the polymerization conditions employed in the experiments did not adversely affect the cell viability.

Fig. 9 Live/dead micrographs of hBMSCs encapsulated into the Algi/PAAm beads in a 3D culture system stained with calcein AM and ethidium homodimer on day 1. Bright green spots shows the viable cells stained with calcein and red spots shows dead cells. Scale bar is 100 μm. (A–C) shows phase contrast images of hBMSCs cultured on TC (tissue culture) flasks (2D system) and hBMSCs encapsulated in beads whereas (3D system) (D–F) shows stained cells cultured on TC flasks and hBMSCs encapsulated in beads. (E) shows boundary between two beads and homogeneous distribution of cells throughout the bead.

Nevertheless, enhancing the ability of cells to elongate, migrate and connect with neighboring cells within 3D scaffold format is vital to recreate native tissue morphology and function in engineered tissues.²⁴ Therefore, there is an urgent need for engineered tissues to mimic the native tissues, recreate the complex 3D cellular distribution and organization found in vivo, while maintaining the cell viability and function of the emulated tissues.²⁵ In this study, morphologies of hBMSCs seeded onto 2D substrates were also compared to those cultured within the 3D bead matrix. While hBMSCs shows even spreading over the 2D surfaces, the cells encapsulated in the PAAm/alginate bead displays well-spread 3D interactions within the polymer network resulting into round shape morphology. 3D round morphology is the native cell morphology found in biological tissues.^26,27 It is attributed as an advantage of the cell-laden bead matrix that can resemble the tissue-like microenvironments for their application in tissue engineering and regenerative medicine.^28,29

CCK-8 assay. The CCK-8 assay was implemented to quantitatively investigate the cell viability of hBMSCs in the Algi/PAAm bead system on day 2. Fig. 10 shows quantitative viability profile of the hBMSCs cultured in bead system as well as in 2D TC flasks monolayer system on day 2. Results from the CCK-8 assay showed an average OD value for Algi/PAAm bead 3D systems and 2D monolayer culture of 1.15 and 2.69 respectively. This significant amount of cell viability within the 3D bead matrix supports the material's biocompatibility and its ability to provide required physical and biochemical cues for the adhesion and proliferation of the cells. However, the low OD value for bead system resulting in lower viability could be attributed to the encapsulation stress, nutrient limitations or stress due to transient swelling after placement in media. Cells presented in the 3D format within the polymeric network might cause slow rate of migration and proliferation, but the material shows biocompatible behavior because of cell viability and supporting cells for the adhesion. It is worth to pointing out that the biomaterials are commonly used in stem cell delivery and regenerative medicine in a 3D format rather than the 2D and thus the developed 3D Algi/PAAm gel system might be useful for these applications. However, further studies in terms of effect of photoinitiator concentrations with respective cell seeding density and culture conditions are required which are under progress.

Fig. 10 Day 2 proliferation study of hBMSCs in Algi/PAAm beads using CCK-8 assay: the absorbance was read at 450 nm. Cells cultured in TC flasks as monolayer were treated as 2D controls. The error bar represents the standard deviation (n = 4).

4. Conclusion

To best of our knowledge, this is the first report that demonstrates the development of cell-laden Algi/PAAm gels for hBMSCs delivery. Algi/PAAm gels were prepared and their physicochemical and biological properties were systematically characterized in the context of stem cell delivery. The in situ gelation approach has been followed for the encapsulation of hBMSCs within a selected ratio of the two polymers so as to minimize the detrimental effect of the polymers and their gelation mechanisms. The physicochemical properties of these quickly in situ gel-forming beads were investigated in terms of chemical group functionality (FTIR data), thermal degradation profiling (TGA and DSC data), internal porosity and interconnected pore morphology (SEM data). Stem cell encapsulation method within the bead matrix has been optimized and used to evaluate the cellular compatibility of the bead system using hBMSCs as model stem cells. The results confirmed the cellular viability as well as homogenous distribution of the cells within the bead matrix during the period of study (2 days). However, the reduced initial cell viability may be attributed to the encapsulation stress, nutrient limitations or stress due to transient swelling after placement in media. Additionally, it was observed that the hBMSCs cultured over 2D surfaces (culture plates) showed even spreading, whereas the cells encapsulated within the Algi/PAAm beads displayed well-spread 3D interactions within the polymer network resulting into round shape morphology. Other advantages of the bead system includes tunable system properties depending on cell type used as well as the application, easy preparation method (even possible in surgical theatre) and cost-effectiveness. The multi advantageous features make this gel bead system as an attractive stem cell carrier in general and hBMSCs in particular for stem cell delivery and scaffold-based tissue engineering application such as cartilage tissue regeneration. The results show that in situ forming beads from alginate and PAAm are suitable scaffolds to support the survival of hBMSCs with their potential in cartilage repair as well as their suitability as a novel stem cell delivery system.

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgements

This work was supported by CSCR. The author, Deepti Rana, would like to thank CSCR for the award of junior research fellowship.

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