Design of injectable hydrogels of gelatin and alginate with ferric ions for cell transplantation

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Design of injectable hydrogels of gelatin and alginate with ferric ions for cell transplantation Mina Anamizu , Yasuhiko Tabata PII: DOI: Reference:

S1742-7061(19)30668-3 https://doi.org/10.1016/j.actbio.2019.10.001 ACTBIO 6386

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Acta Biomaterialia

Received date: Revised date: Accepted date:

10 July 2019 30 September 2019 1 October 2019

Please cite this article as: Mina Anamizu , Yasuhiko Tabata , Design of injectable hydrogels of gelatin and alginate with ferric ions for cell transplantation, Acta Biomaterialia (2019), doi: https://doi.org/10.1016/j.actbio.2019.10.001

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Design of injectable hydrogels of gelatin and alginate with ferric ions for cell transplantation

Mina Anamizu and Yasuhiko Tabata*

Laboratory of Biomaterials, Institute for Frontier Life and Medical Sciences, Kyoto University, 53 Kawara-cho Shogoin, Sakyo-ku Kyoto 606-8507, Japan

*Corresponding author. Laboratory of Biomaterials, Department of Regeneration Science and Engineering, Institute for Frontier Life and Medical Sciences, Kyoto University, 53 Kawara-cho Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.

Tel.: +81 75 751 4121 Fax: +81 75 751 4646 E-mail: [email protected]

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Abstract The objective of this study is to design bioabsorbable injectable hydrogels based on the physico-chemical interaction between biocompatible polymers and ferric ions, and evaluate the survival, proliferation, and osteogenic differentiation of cells encapsulated in the hydrogels. The injectable hydrogels were prepared by simply mixing mixed alginate/gelatin solution at various ratios and FeCl3 solution. The hydrogels prepared disappeared within a few days in the phosphate buffered-saline solution (PBS) with containing collagenase although the disappearance rate increased with an increase of the gelatin ratio in the hydrogel. For the hydrogel of alginate/gelatin low ratio, the survival and proliferation of cells in the hydrogel-encapsulated condition were significantly high compared with those of hydrogel at the higher ratios. The cells collected 3 days after cultured in the hydrogel also proliferated to a significantly higher extent than those collected from other hydrogels. The proliferation ability of cells was similar that of cells cultured on the standard tissue culture polystyrene (TCPS) dish. When evaluated to compare with cells cultured on the TCPS dish, the expression of runt-related transcription factor-2 (RUNX2) gene, the alkaline phosphatase (ALP) activity, and the calcium precipitation were significantly high. The cells were encapsulated by the mixed alginate/gelatin and FeCl3 hydrogel and injected in the back subcutis of mice, the percentage of cells retained in the injected site was higher than that of cells injected in the PBS suspension. It is concluded that the injectable hydrogel prepared by simple mixing mixed alginate/gelatin solution and FeCl3 solution is a promising material for the cell transplantation.

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Statement of significance Injectable hydrogels prepared by simple mixing mixed alginate/gelatin solution at various ratios and FeCl3 solution. For the hydrogel of alginate/gelatin low ratio, the survival, the proliferation, and the differentiate properties of cells in the hydrogel-encapsulated condition were similar those of cells cultured on the TCPS dish. When the cells encapsulated hydrogels were injected in the back subcutis of mice, the percentage of cells retained in the injected site was higher than that of cells injected in the PBS suspension. It is concluded that the present injectable hydrogel is a promising material for the cell transplantation.

Keywords gelatin; alginate; physico-chemical interaction; injectable hydrogel

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1. Introduction Cell transplantation is one of the promising therapies in regenerative medicine to induce the regeneration and repairing of damaged tissues and organs [1-3]. Especially, the cell transplantation via injection is attractive as a minimally invasive treatment since surgical operations are not required. Potential cell transplantation is most typically introduced into the tissues of interest directly via the injection of cells, suspended in an appropriate medium [4-6]. However, it is well recognized that the retention of cells transplanted at the injection site is extremely poor by the method [7]. Therefore, it is of prime importance to create a technology and methodology for an enhanced retention of cells transplanted at the injection site. Under these circumstances, significant research efforts have been investigated to design injectable hydrogels that can be injected in a solution form into the body and then rapidly form the hydrogel at the injection site. Injectable hydrogels have many advantages for various biomedical applications, such as their ease of administration, the minimally invasive treatment, and simple cell encapsulation [8]. Various injectable hydrogels have been mainly prepared by chemically or covalently crosslinked of polymers [9-11]. However, the injectable hydrogels of chemical gelation require the introduction of chemically active residues or catalysts to initiate the in situ reaction [12]. Moreover, the cells transplanted with such injectable hydrogels do not always survive nor function efficiently in the body. This is mainly because the injectable hydrogels of chemical crosslinking are not degraded in the body synchronizing with the cell proliferation [13-15]. As one trial to tackle the issue, injectable

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hydrogels formed through a physico-chemical interaction force need to be designed. For the hydrogel formed based on the physico-chemical interaction, there are no needs to use the crosslinking agents for in situ reaction. In addition, the survival, proliferation, and biological functions are not suppressed by the in vivo remaining of hydrogels due to the fast degradation [12-15]. Gelatin and alginate are both biocompatible polymers and have been widely used for food, pharmaceutical, and medical applications. The bio-safety also have been proven through their long-term these applications. In addition, ferric ions present in the body and do not show cytotoxicity at the low concentration. It is demonstrated that both the gelatin and alginate physically interact with ferric ions [16, 17]. The properties of hydrogels crosslinked by the physical interaction can be controlled by changing the polymer concentration, molecular weight, and ion concentration [14]. In this study, injectable hydrogels based on the physico-chemical interaction among gelatin, alginate, and ferric ion are designed. The gelatin behavior of hydrogels prepared by changing the mixing ratio of alginate and gelatin in the presence of ferric ions. The viability and proliferation of cells encapsulated in the hydrogels were investigated. As one application of hydrogel, cells are transplanted with the injectable hydrogel for bone regeneration. Therefore, here, the osteogenic differentiation of cells was evaluated as one cell ability. We also evaluate the in vivo cell retention after injection with the hydrogel of alginate/gelatin and ferric ions.

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2. Materials and methods 2.1.Materials Gelatin with an isoelectric point of 5.0 and the weight-averaged molecular weight of 100,000, by bovine bone was kindly supplied from Nitta Gelatin Inc., Osaka, Japan. Sodium alginate with the weight-averaged molecular weight of 2,300,000 was kindly supplied from KIMICA Inc., Tokyo, Japan. Iron chloride (III), glycerol 2-phospate disodium salt hydrate (β-GP) and alizarin red were purchased from Sigma-Aldrich Inc., St. Louis, USA. Hydrochloric acid (HCl), disodium dihydrogen ethylenediaminetetraacetate dihydrate (EDTA-2Na), phenol, sulfuric acid, sodium lauryl sulfate (SDS), dexamethasone, L(+)-ascorbic acid, and ethanol were purchased from Nacalai Tesque. Inc., Kyoto, Japan. Sodium chloride (NaCl), and trisodium citrate dihydrate were purchased from FUJIFILM Wako Pure Chemical Inc., Osaka, Japan. Collagenase D was purchased from Roche Diagnostics, Indianapolis, USA.

2.2.Preparation of hydrogels Hydrogels were prepared by simply mixing the mixed alginate/gelatin solution (A/G solution) and the FeCl3 solution. Alginate and gelatin were dissolved in 10 mM phosphate buffered-saline solution (PBS, pH 7.4) at various mixing ratios at 37 ℃ (Table 1). Then, 100 μl of the FeCl3 solution (20 mM) was added to 100 μl of the A/G solution to give a final polymers and ferric ions concentrations 0.5 wt% and 10 mM, respectively. To evaluate the fluidity of hydrogels prepared the following

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method was performed. Briefly, after mixing of A/G solution and FeCl3 solution for 10 sec, the tube containing the solution was titled. After that, the fluidity of the solution was assessed.

2.3.Degradation test of hydrogels Hydrogels prepared by the A0G10, A2G8, and A3G7 solution were agitated in 1 ml of PBS containing 10 µg/ml collagenase D at 37 ℃. At different time intervals, the PBS supernatant was collected, and the hydrogels were agitated again in the same volume of flesh PBS. The amount of gelatin in the supernatant was determined by Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific Inc., Massachusetts, USA) . The amount of eluted alginate in the supernatant was also determined by phenol sulfuric acid method [18]. The degradation ratio of hydrogels was calculated by dividing the amount of eluted gelatin and alginate by that initially used.

2.4.In vitro cell encapsulation studies MC3T3-E1 cells of a murine bone calvaria pre-osteoblast were cultured in the normal medium (NM) consisting of MEM alpha basic (Gibco Inc., Massachusetts, USA) with 10 vol % fetal calf serum (FCS, HyClone Laboratories, Inc., Logan, USA) and 100 U/ml penicillin G and 100 mg/ml streptomycin (Sigma Aldrich Inc., St. Louis, USA) at 37 ℃ in a humidified atmosphere with 5% CO2 -95% air. To encapsulate cells in the hydrogels, the cells were trypsinized to detach from the tissue culture polystyrene (TCPS) dish, followed by resuspended in the NM. The A/G solution and

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the FeCl3 solution were sterilized by filtration through filters with a pore size of 0.22 µm (Merck Inc., Darmstadt, Germany). Then, the cell suspension in NM (5×106 cells/ml, 60 μl) was added to the A/G solution (240 μl) and then thoroughly mixed by pipetting. Next, the cell suspension in the A/G solution (300 μl) was added to the FeCl3 solution (300 μl) to allow cells to encapsulate in the hydrogels formed. The volumetric ratio of polymer solution, the NM cell suspension, and the FeCl3 solution was 40/10/50. The hydrogels encapsulating cells were cultured in each well of 48 well multi-dish culture plates (Corning Inc., New York, USA) with the NM for 3 days. The medium was changed every day. Every experiments were done independently 3 times for each sample unless otherwise mentioned.

2.5.Evaluation of viability and proliferation of cells after hydrogel encapsulation The viability and proliferation of cells encapsulated in the hydrogels were evaluated using Live/Dead ™

Viability/Cytotoxicity Kit (Invitrogen Inc., Carlsbad, CA) according to the

manufacture’s protocol. After 1 and 3 days culture in the hydrogel-encapsulated condition, the hydrogels were rinsed with PBS, and then incubated in a staining solution containing 500 μl of 2 µM calcein AM and 4 µM ethidium homodimer-1 in PBS for 1 hr at 37℃ in dark. The hydrogels were observed on a fluorescent microscopy BZ-X700 (KEYENCE Co., Ltd., Osaka, Japan). The cell viability was calculated by counting the number of live and dead cells. The cell proliferation ratio of cells encapsulated in the hydrogel was calculated by divided the number of cells cultured for 3 days

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by that for 1 day. The proliferation of cells collected from the hydrogels was also evaluated. Similarly, the cells were encapsulated in the hydrogels and cultured for 3 days in the NM. After that, the hydrogels were placed in the NM containing 100 mM EDTA-2Na for 10 min. Then, the cell suspension was collected by dissolving the hydrogels. After that, the cell suspension was centrifuged at 1000 rpm for 3 min at 4℃. The number of cells collected was set to be 1×104 cells/ml and re-seeded in each well of 96 well multi-dish culture plates (Corning Inc., New York, USA). As a control, cells which had not been encapsulated, were also similarly seeded in each well of 96 well multi-dish culture plates (Corning Inc., New York, USA). The number of cells proliferated was assessed by determining the amount of DNA 1 and 3 days later [19]. Briefly, the cells incubated in 300 µl/cm2 of aqueous solution containing sodium lauryl sulfate (SDS, 0.2 mg/ml), NaCl (3 M), and trisodium citrate dihydrate (0.3 M) for 2 hr at 37 ℃ for cell lysis. Next, 80 µl of the cell lysate was mixed with 80 µl of 1.9 µM bisbenzimide H33258 fluorochrome trihydrochloride DMSO solution (Nacalai Tesque. Inc., Kyoto, Japan), and then the fluorescent intensity was measured at Ex/Em 355/460 nm with Multi-mode Microplate Reader (SpectraMax i3x, Molecular Devices Japan Co., Ltd., Tokyo, Japan) to evaluate the amount of DNA. The proliferation ratio of cells collected from the hydrogels was calculated by divided the number of cells cultured for 3 days by that for 1 day.

2.6.Evaluation of osteogenic differentiation Cells encapsulated in the A2G8 hydrogels were collected from the hydrogels by the same procedure

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described above. Cells cultured in both the non-encapsulated and hydrogel-encapsulated conditions were seeded in each well of 6 well multi-dish culture plates (Corning Inc., New York, USA) at a density of 5×104 cells/well. After 2 days culture in the NM, the medium was replaced by the differentiation medium (DM) of the NM supplemented with 10 nM dexamethasone, 0.2 mM L(+)-ascorbic acid, and 10 mM β-GP. After that, the cells were cultured further for 7 and 14 days while the DM was changed every 3-4 days. As a NM control, the non-encapsulated cells were also cultured in NM in the same condition.

2.7.mRNA expression analysis Runt-related transcription factor-2 (RUNX2) expression of cells was quantified by real-time polymerase chain reaction (PCR) analysis [20]. After culture in the DM for 7 and 14 days, the total RNA was extracted using RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) according to the manufacture’s protocol. Complementary DNA (cDNA) was synthesized using a SuperScript VILO cDNA synthesis kit (Thermo Fisher Scientific Inc., Massachusetts, USA). The cDNA (100 ng, 1 µl), forward and reverse primers (20 µM, each 0.5 µl), and 12.5 µl of Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) were mixed, and real-time PCR was performed on a Prism 7500 real-time PCR thermal cycler (Applied Biosystems, Foster City, CA). The following PCR conditions were used: 95 ℃ for 10 min, followed by 40 cycles of 95 ℃ for 15 s and 60 ℃ for 1 min. β-actin was used as a housekeeping gene, and the expression level was analyzed by

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method comparing with the untreated cells. The primer sequences for RUNX2 and β-actin were as follows, (Forward and Reverse) RUNX2: 5’-AAGTGCGGTGCAAACTTTCT-3’, 5’-TCTCGGTGG CTGGTAGTGA-3’, β-actin: 5’-TCTTGGGTATGGAATCCTGTG-3’, 5’-AGGTCTTTACGGATG TCAACG-3’.

2.8.Alkaline phosphatase and calcium assays Alkaline phosphatase (ALP) enzyme activity was evaluated by LabAssay™ ALP (FUJIFILM Wako Pure Chemical Inc. Osaka, Japan) based on the absorbance measurement of a p-nitrophenol product [21]. Briefly, cells were rinsed twice with PBS, freeze-thawed, and finally incubated in 200 µl/cm2 of aqueous solution containing SDS (0.2 mg/ml), NaCl (3 M), and trisodium citrate dihydrate (0.3 M) for 1 hr at 37 ℃ for cell lysis. Next, 20 µl of the cell lysate was mixed with 100 µl of 6.7 mM p-nitrophenyl phosphate aqueous solution, and then by incubation for 15 min at 37 ℃. After mixing 80 µl of 20 mM NaOH aqueous solution, the absorbance of the solution mixture was measured at 405 nm with Multi-mode Microplate Reader (SpectraMax i3x, Molecular Devices Japan Co., Ltd., Tokyo, Japan) to evaluate ALP activity. Relative ALP activity was calculated by divided the ALP activity of the corresponding cells by that of the cells cultured in NM. Cells were incubated in 2 ml of 1 M HCl solution for 24 hr at 4 ℃. The amount of calcium in the HCl solution was determined with a calcium E-HA kit (FUJIFILM Wako Pure Chemical Inc. Osaka, Japan) according to the manufacture’s protocol [19]. Relative calcium content was calculated at the same procedure

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described above.

2.9.Alizarin red staining After 7 and 14 days culture in the DM, the cells were stained with alizarin red to observe the calcium precipitation [20]. The cells were washed with PBS three times and fixed in 75% ethanol for 30 min at 4 ℃. Next, the cells were washed with double-distilled water (DDW) three times and stained in 1% alizarin red solution for 30 min at 37 ℃. Unbound dye was removed by washing three times with DDW.

2.10. In vivo cell transplantation Cells suspension in PBS were injected and the cells encapsulated A2G8 hydrogel were implanted into the back subcutis of C57BL/6n mouse (2 points for every mouse). First, the cells were stained with PKH26 Red Fluorescent Cell Linker Mini Kit for Cell Membrane Labeling for Phanos Technologies (Sigma Aldrich Inc., St. Louis, USA) according to the manufacturer’s protocol, followed by the preparation of cells suspension in PBS 100 μl and cells encapsulated in the A2G8 hydrogel at a final density of 1×107 cells/ml at the same procedure described above. Then, the cell suspension (100 μl/head) was subcutaneously injected into the back of mice via a 1 ml syringe with 22 G needle while the cell encapsulated hydrogel was transplanted by a spatula. Mice was sacrificed and 1 cm of the skin around the transplanted area was collected 0, 1 and 3 days after treatment. Then,

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the skins collected were treated with 10 ml of 0.4 wt% collagenase PBS solution and 10 ml of 100 mM EDTA-2Na PBS solution for 2 hr at 37 ℃. Following the collection of cells by filtration through a cell strainer with a mesh size of 40 µm, the cells were re-seeded in each well of 96 well-multi dish culture plates. Next, the cells were observed by the fluorescent microscopy BZ-X700 (KEYENCE Co., Ltd., Osaka, Japan). Three fluorescence of images were taken at random and the number of PKH stained cells were counted. The cell retention was calculated by divided the number of cells transplanted after 1 or 3 days by that for 0 day.

2.11. Statistical analysis All the statistical data were expressed as the mean ± standard deviations. The data were analyzed by t-test and the statistical significance was accepted at p < 0.05.

3. Results 3.1.Hydrogel preparation Figure 1 shows the hydrogel formation at the various mixing conditions of A/G solution. For the A10G0, A8G2, A7G3, A5G5, A3G7, and A2G8 mixed solutions, the hydrogel formation was observed, and the solution fluidity disappeared. On the other hand, the solution fluidity did not change for the gelatin solution without alginate (A0G10). Figure 2 shows the in vitro disappearance

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profiles of hydrogels. The A3G7, A2G8, and A0G10 hydrogels disappeared within 96, 72, and 2 hr, respectively. The hydrogels disappeared fast with a decrease in the alginate/gelatin ratio.

3.2.Viability and proliferation of cells encapsulated in the hydrogels The viability and proliferation of cells encapsulated in different types of hydrogels were evaluated (Figure 3). The percentage of survived cells increased with a decrease of the alginate/gelatin in the hydrogel. For the A2G8 hydrogel, the percent survival was significantly higher than that of other types of hydrogels 1 and 3 days after encapsulation (Figure 3B). The cell proliferation in the hydrogels increased when the ratio of alginate/gelatin in the hydrogel decreased. A significant cell proliferation was observed for the A3G7 and A2G8 hydrogels compared with other hydrogels. In addition, there was no significant difference in the proliferation between the cells encapsulated in the A3G7 or A2G8 hydrogel and these on the TCPS (Figure 3C). Figure 3D shows the viability and proliferation of cells encapsulated in the hydrogels. Cells collected from the hydrogels still showed a proliferation ability. For the A3G7 and A2G8 hydrogels, the proliferation of cells collected was in a similar level to that of cells cultured on the TCPS dish.

3.3.Osteogenic differentiation of cells encapsulated in the hydrogels Figure 4 shows the osteogenic differentiation behavior of cells encapsulated in the hydrogels. The level of RUNX2 expression was high on 7 days after the osteogenic differentiation culture compared

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with that on 14 days after the culture. However, there was not significant difference in the RUNX2 expression level between cells in the non-encapsulated and encapsulated in the A2G8 hydrogel in the DM (Figure 4A). The ALP activity of cells differentiation cultured for 14 days was significantly higher than that of cells differentiation cultured for 7 days. The ALP activity of cells encapsulated in the A2G8 hydrogel was similar that of cells non-encapsulated at the corresponding day (Figure 4B). The calcium precipitation of cells differentiation cultured for 14 days significantly increased compared with that of cells differentiation cultured for 7 days. The calcium precipitation of cells encapsulated in the A2G8 hydrogel was similar to that of cells non-encapsulated at the corresponding day (Figure 4C). No calcium precipitation of cells encapsulated in the hydrogel was observed after cultured in the non-differentiation medium (data not shown). Figure 4D shows the staining images by alizarin red. The NM control showed no red staining on 7 and 14 days after culture. However, the cells cultured in both the non-encapsulated and encapsulated condition were stained in red.

3.4.In vivo cell transplantation Figure 5 shows the percentage of cell retention at the transplanted area. The percentages of cells retained after the PBS injection were 3.27 and 0.18, 1 and 3 days after cell injection, respectively. On the contrary, for the cells encapsulated in the A2G8 hydrogel 1 and 3 days after cell transplantation, the cell retention percentages were 82.5 and 32.8%, respectively.

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4. Discussion The present study demonstrates that the injectable hydrogel formed based on the physico-chemical interaction of biocompatible polymers and ferric ions is promising for cell transplantation in terms of cell survival, proliferation, and a differentiation property. The hydrogels with different degradabilities were prepared by changing the mixing ratio of alginate to gelatin in the same ferric ion concentration. The cells encapsulated in the hydrogels prepared at a low ratio of alginate/gelatin showed a superior cell survival, proliferation, and osteogenic differentiate properties which are similar to those of cells cultured on the 2D standard TCPS dish, the hydrogels increased the cell retention at the transplantation to a significantly high extent compared with that of cell suspension injection. The mixing ratio of alginate and gelatin had an influence on the gelation of mixed solution (Figure 1). It is reported that gelatin interacts with ferric ions [17]. However, the gelation was not observed for the A0G10 solution of pure gelatin without alginate. The ferric ion concentration would be too low to set a hydrogel. The hydrogel formation by mixing the gelatin and the FeCl 3 solution was observed at the high concentrations. However, the high concentration of ferric ions showed cytotoxicity. In this study, as one trial to reduce the ferric ion concentration, the alginate which also can interact, was mixed with the gelatin. It is considered that the gelation by mixing mixed

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alginate/gelatin solution and FeCl3 solution is due to the ionic coordination between COO - of alginate and gelatin, and ferric ions, which is proven by other researches [17, 22]. Since the COO- of alginate and gelatin fast interact with ferric ions, it is likely that cells can be encapsulated in the hydrogel fast and easily. Based on this concept, ferric ions were selected as the ions. The hydrogels fast disappeared as a decrease of the alginate/gelatin ratio (Figure 2). There are two possible reasons to explain this phenomenon. One of the reasons is the gelatin degradation by collagenase [23-25]. The other reason is that the interaction between the alginate and ferric ions is stronger than that between the gelatin and ferric ions. Therefore, ferric ions will be diffused readily away from the hydrogel at the low ratio of alginate/gelatin. The A2G8 and A3G7 hydrogels disappeared within 72 and 96 hr, respectively (Figure 2). Most researches have reported that the injectable hydrogels disappear within about 3 weeks at the earliest or remains in the body [26-28]. The present hydrogel is faster than the conventional injectable hydrogels. This faster degradation can be explained in terms of crosslinking style. It is likely that the salt crosslinking of the present hydrogels is readily dissociated in the body due to several components to chelate ferric ions. Consequently, the hydrogel would be solubilized in water to disappear faster than that chemically crosslinked. The fast disappearance of hydrogels in a few days will be suitable from the viewpoint of survival and the consequent function retention of cells injected. The viability and proliferation of cells encapsulated increased when the alginate/gelatin ratio in the hydrogel became low (Figure 3). This can be explained by the cytocompatibility of gelatin itself. It

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is likely that the presence of gelatin gives cells an environment suitable for their attachment and proliferation [29, 30]. On the contrary, it is demonstrated that alginate is not the material of cell adhesion [31, 32]. In addition, there are some reports that cells survive better when adhered in a cell friendly environment [33, 34]. The viability of cells encapsulated is not always high in the hydrogels of alginate. However, the introduction of Arg-Gly-Asp (RGD) of a cell adhesion motif allowed alginate to show better cell adhesion [35, 36]. It is highly conceivable that there is a stronger interaction of the alginate functional groups with ferric ions, as compared to gelatin ones. Therefore, the higher content of alginate, the greater the number of attached ferric ions. From this viewpoint, we have to also think that the cytotoxicity of ferric ions that affects the behavior of cells. The balance of gelatin cytocompatibility and ferric ions cytotoxicity would be a better cell viability and proliferation. We can say with certainty that a surrounding environment to allow cells to adhere is essential to improve the survival and proliferation of cells encapsulated in the hydrogels. In this study, in addition to the proliferation of cells in the hydrogels, cells collected from the hydrogels after 3 days incubation also proliferated in the similar level to those cultured on the standard TCPS dish (Figures 5B, and 5C). Although many researches on injectable hydrogels have been reported for cell transplantation, there are few reports to experimentally confirm whether or not the encapsulated cells proliferate in the hydrogels. The effect of alginate/gelatin mixing ratio on the proliferation and osteogenic differentiation abilities of cells encapsulated has been hardly investigated. This study indicates that there exists a mixing ratio suitable to increase the ability of cells encapsulated. For the

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A2G8 hydrogel, the cells encapsulated survived and proliferated, which implies the promising hydrogel for cell encapsulation. In addition, the cells collected from the A2G8 hydrogels showed an ability of osteogenic differentiation. It is apparent from Figures 4A-4C that the level of the relative RUNX2 expression, ALP activity, and calcium precipitation of cells encapsulated in the A2G8 hydrogel was similar to that of cells cultured on the TCPS dish. In the experiment of cell transplantation with the A2G8 hydrogel, the percent cell retention was 82.5 and 32.8% after 1 and 3 days implantation, respectively (Figure 5). In the research of conventional injectable hydrogels, the cell retention was as high as 20% [37-39]. This indicates that from the viewpoint of cell retention, the A2G8 hydrogel is superior to the conventional hydrogels previously reported. To apply this hydrogel in the future, it is necessary to evaluate the in vivo degradability of the hydrogel or the viability, the proliferation, and the functions of cells implanted in vivo. As technologies and methodologies to improve the in vivo survival and functions of cells transplanted, there have been reported on some approaches to increase the efficacy of cell therapy by combining drugs and growth factors with cells [40-42]. We have explored the materials and techniques to encapsulate drugs in gelatin hydrogels, films, particles, and micelles for a good controlled release of drugs [43-45]. In future by combining the techniques with the injectable hydrogel of this study, it will be possible to promote the therapeutic efficacy of cell transplantation at the targeted treatment

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site.

Conclusions The injectable hydrogels were prepared by simple mixing mixed alginate/gelatin solution at various mixing ratios and FeCl3 solution. The hydrogels prepared fast disappeared within a few days in the PBS with containing collagenase. For the hydrogel of alginate/gelatin low ratio, the survival, the proliferation, and the differentiate properties of cells in the hydrogel-encapsulated condition were similar those of cells cultured on the standard TCPS dish. The cells were encapsulated by the hydrogel and injected in the back subcutis of mice, the percentage of cells retained in the injected site was higher than that of cells injected in the PBS suspension.

References [1] S. Bajada, I. Mazakova, J.B. Richardson, N. Ashammakhi, Updates on stem cells and their applications in regenerative medicine, J Tissue Eng Regen Med 2(4) (2008) 169-83. [2] S.P. Liu, R.H. Fu, S.J. Huang, Y.C. Huang, S.Y. Chen, C.H. Chang, C.H. Liu, C.H. Tsai, W.C. Shyu, S.Z. Lin, Stem cell applications in regenerative medicine for neurological disorders, Cell Transplant 22(4) (2013) 631-7. [3] R.S. Mahla, Stem Cells Applications in Regenerative Medicine and Disease Therapeutics, Int J Cell Biol 2016 (2016) 6940283. [4] S.M. Evans, C. Mummery, P.A. Doevendans, Progenitor cells for cardiac repair, Semin Cell Dev Biol 18(1) (2007) 153-60. [5] S. Porat, Y. Dor, New sources of pancreatic beta cells, Curr Diab Rep 7(4) (2007) 304-8. [6] D. Skuk, M. Paradis, M. Goulet, J.P. Tremblay, Ischemic central necrosis in pockets of transplanted

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myoblasts in nonhuman primates: implications for cell-transplantation strategies, Transplantation 84(10) (2007) 1307-15. [7] S.A. Young, S.E. Sherman, T.T. Cooper, C. Brown, F. Anjum, D.A. Hess, L.E. Flynn, B.G. Amsden, Mechanically resilient injectable scaffolds for intramuscular stem cell delivery and cytokine release, Biomaterials 159 (2018) 146-160. [8] D.Y. Ko, U.P. Shinde, B. Yeon, B. Jeong, Recent progress of in situ formed gels for biomedical applications, Prog Polym Sci 38(3-4) (2013) 672-701. [9] Q. Xu, S. A, Y. Gao, L. Guo, J. Creagh-Flynn, D. Zhou, U. Greiser, Y. Dong, F. Wang, H. Tai, W. Liu, W. Wang, W. Wang, A hybrid injectable hydrogel from hyperbranched PEG macromer as a stem cell delivery and retention platform for diabetic wound healing, Acta Biomater 75 (2018) 63-74. [10] H.Y. Wang, D.Q. Zhu, A. Paul, L. Cai, A. Enejder, F. Yang, S.C. Heilshorn, Covalently Adaptable Elastin-Like Protein-Hyaluronic Acid (ELP-HA) Hybrid Hydrogels with Secondary Thermoresponsive Crosslinking for Injectable Stem Cell Delivery, Adv Funct Mater 27(28) (2017). [11] S.H. Park, J.S. Kwon, B.S. Lee, J.H. Park, B.K. Lee, J.H. Yun, B.Y. Lee, J.H. Kim, B.H. Min, T.H. Yoo, M.S. Kim, BMP2-modified injectable hydrogel for osteogenic differentiation of human periodontal ligament stem cells, Sci Rep 7(1) (2017) 6603. [12] X. Ding, Y. Wang, Weak Bond-Based Injectable and Stimuli Responsive Hydrogels for Biomedical Applications, J Mater Chem B 5(5) (2017) 887-906. [13] Y.L. Li, J. Rodrigues, H. Tomas, Injectable and biodegradable hydrogels: gelation, biodegradation and biomedical applications, Chem Soc Rev 41(6) (2012) 2193-2221. [14] J.A. Yang, J. Yeom, B.W. Hwang, A.S. Hoffman, S.K. Hahn, In situ-forming injectable hydrogels for regenerative medicine, Prog Polym Sci 39(12) (2014) 1973-1986. [15] Q. Feng, K. Wei, S. Lin, Z. Xu, Y. Sun, P. Shi, G. Li, L. Bian, Mechanically resilient, injectable, and bioadhesive supramolecular gelatin hydrogels crosslinked by weak host-guest interactions assist cell infiltration and in situ tissue regeneration, Biomaterials 101 (2016) 217-28. [16] V. Privman, S. Domanskyi, R.A. Luz, N. Guz, M.L. Glasser, E. Katz, Diffusion of Oligonucleotides from within Iron-Cross-Linked, Polyelectrolyte-Modified Alginate Beads: A Model System for Drug Release, Chemphyschem 17(7) (2016) 976-84. [17] G. Zhang, L. Lv, Y. Deng, C. Wang, Self-Healing Gelatin Hydrogels Cross-Linked by Combining Multiple Hydrogen Bonding and Ionic Coordination, Macromol Rapid Commun 38(12) (2017). [18] H.D. Graham, Determination of alginate in dairy products, J Dairy Sci 52(4) (1969) 443-8. [19] S. Tajima, Y. Tabata, Preparation of cell aggregates incorporating gelatin hydrogel microspheres containing bone morphogenic protein-2 with different degradabilities, J Biomater Sci Polym Ed 29(7-9) (2018) 775-792. [20] Y. Feng, L. Su, X. Zhong, W. Guohong, H. Xiao, Y. Li, L. Xiu, Exendin-4 promotes proliferation and differentiation of MC3T3-E1 osteoblasts by MAPKs activation, J Mol Endocrinol 56(3) (2016) 189-99. [21] Y. Fukunishi, Y. Tabata, Osteogenic differentiation enhances the MC3T3-E1 secretion of glycosaminoglycans with an affinity for basic fibroblast growth factor and bone morphogenetic protein-2,

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Regen Ther 8 (2018) 58-62. [22] M. Gamella, M. Privman, S. Bakshi, A. Melman, E. Katz, DNA Release from Fe(3+) -Cross-Linked Alginate Films Triggered by Logically Processed Biomolecular Signals: Integration of Biomolecular Computing and Actuation, Chemphyschem 18(13) (2017) 1811-1821. [23] M. Matsui, Y. Tabata, Enhanced angiogenesis by multiple release of platelet-rich plasma contents and basic fibroblast growth factor from gelatin hydrogels, Acta Biomater 8(5) (2012) 1792-801. [24] T. Saito, Y. Tabata, Hypoxia-induced angiogenesis is increased by the controlled release of deferoxiamine from gelatin hydrogels, Acta Biomaterialia 10(8) (2014) 3641-3649. [25] Y. Murata, J.I. Jo, Y. Tabata, Intracellular Controlled Release of Molecular Beacon Prolongs the Time Period of mRNA Visualization, Tissue Eng Part A (2019). [26] A.C. Grayson, G. Voskerician, A. Lynn, J.M. Anderson, M.J. Cima, R. Langer, Differential degradation rates in vivo and in vitro of biocompatible poly(lactic acid) and poly(glycolic acid) homo- and co-polymers for a polymeric drug-delivery microchip, J Biomater Sci Polym Ed 15(10) (2004) 1281-304. [27] N. Landa, L. Miller, M.S. Feinberg, R. Holbova, M. Shachar, I. Freeman, S. Cohen, J. Leor, Effect of injectable alginate implant on cardiac remodeling and function after recent and old infarcts in rat, Circulation 117(11) (2008) 1388-96. [28] S. Dobner, D. Bezuidenhout, P. Govender, P. Zilla, N. Davies, A synthetic non-degradable polyethylene glycol hydrogel retards adverse post-infarct left ventricular remodeling, J Card Fail 15(7) (2009) 629-36. [29] K.Y. Lee, D.J. Mooney, Alginate: properties and biomedical applications, Prog Polym Sci 37(1) (2012) 106-126. [30] S. Lu, E.J. Lee, J. Lam, Y. Tabata, A.G. Mikos, Evaluation of Gelatin Microparticles as Adherent-Substrates for Mesenchymal Stem Cells in a Hydrogel Composite, Ann Biomed Eng 44(6) (2016) 1894-907. [31] J.A. Rowley, G. Madlambayan, D.J. Mooney, Alginate hydrogels as synthetic extracellular matrix materials, Biomaterials 20(1) (1999) 45-53. [32] B. Sarker, J. Rompf, R. Silva, N. Lang, R. Detsch, J. Kaschta, B. Fabry, A.R. Boccaccini, Alginate-based hydrogels with improved adhesive properties for cell encapsulation, Int J Biol Macromol 78 (2015) 72-8. [33] J.A. Burdick, K.S. Anseth, Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering, Biomaterials 23(22) (2002) 4315-23. [34] F. Chen, Y. Ni, B. Liu, T. Zhou, C. Yu, Y. Su, X. Zhu, X. Yu, Y. Zhou, Self-crosslinking and injectable hyaluronic acid/RGD-functionalized pectin hydrogel for cartilage tissue engineering, Carbohydr Polym 166 (2017) 31-44. [35] J. Yu, Y. Gu, K.T. Du, S. Mihardja, R.E. Sievers, R.J. Lee, The effect of injected RGD modified alginate on angiogenesis and left ventricular function in a chronic rat infarct model, Biomaterials 30(5) (2009) 751-6. [36] S.J. Bidarra, C.C. Barrias, K.B. Fonseca, M.A. Barbosa, R.A. Soares, P.L. Granja, Injectable in situ crosslinkable RGD-modified alginate matrix for endothelial cells delivery, Biomaterials 32(31) (2011)

22

7897-904. [37] M.E. Danoviz, J.S. Nakamuta, F.L. Marques, L. dos Santos, E.C. Alvarenga, A.A. dos Santos, E.L. Antonio, I.T. Schettert, P.J. Tucci, J.E. Krieger, Rat adipose tissue-derived stem cells transplantation attenuates cardiac dysfunction post infarction and biopolymers enhance cell retention, PLoS One 5(8) (2010) e12077. [38] A.A. Foster, R.E. Dewi, L. Cai, L. Hou, Z. Strassberg, C.A. Alcazar, S.C. Heilshorn, N.F. Huang, Protein-engineered hydrogels enhance the survival of induced pluripotent stem cell-derived endothelial cells for treatment of peripheral arterial disease, Biomater Sci 6(3) (2018) 614-622. [39] H. Niu, X. Li, H. Li, Z. Fan, J. Ma, J. Guan, Thermosensitive, fast gelling, photoluminescent, highly flexible, and degradable hydrogels for stem cell delivery, Acta Biomater 83 (2019) 96-108. [40] H. Park, J.S. Temenoff, Y. Tabata, A.I. Caplan, A.G. Mikos, Injectable biodegradable hydrogel composites for rabbit marrow mesenchymal stem cell and growth factor delivery for cartilage tissue engineering, Biomaterials 28(21) (2007) 3217-27. [41] K.H. Park, K. Na, Effect of growth factors on chondrogenic differentiation of rabbit mesenchymal cells embedded in injectable hydrogels, J Biosci Bioeng 106(1) (2008) 74-9. [42] H. Park, J.S. Temenoff, Y. Tabata, A.I. Caplan, R.M. Raphael, J.A. Jansen, A.G. Mikos, Effect of dual growth factor delivery on chondrogenic differentiation of rabbit marrow mesenchymal stem cells encapsulated in injectable hydrogel composites, J Biomed Mater Res A 88(4) (2009) 889-97. [43] T. Tanigo, R. Takaoka, Y. Tabata, Sustained release of water-insoluble simvastatin from biodegradable hydrogel augments bone regeneration, J Control Release 143(2) (2010) 201-6. [44] Y.H. Kim, Y. Tabata, Enhancement of wound closure by modifying dual release patterns of stromal-derived cell factor-1 and a macrophage recruitment agent from gelatin hydrogels, J Tissue Eng Regen Med 11(11) (2017) 2999-3013. [45] Y. Murata, J.I. Jo, Y. Tabata, Preparation of cationized gelatin nanospheres incorporating molecular beacon to visualize cell apoptosis, Sci Rep 8(1) (2018) 14839.

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Figure 1. Photographs of solution fluidity after mixing of alginate/gelatin and FeCl3 solutions at the various alginate/gelatin ratios. Scale bar is 1 cm.

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Figure 2. In vitro disappearance profiles of A3G7 (◯), A2G8 (△), and A0G10 hydrogels (☐).

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Figure 3. The viability and proliferation of cells encapsulated in the hydrogels. (A) Live/Dead staining images of cells 1 and 3 days after culture in the hydrogel-encapsulated condition. Green: Live cells. Red: Dead cells. Scale bar is 100 μm. (B) The viability of cells 1 (☐) and 3 days after culture in the hydrogel-encapsulated condition (■). *, p < 0.05; significant difference against the viability of cells encapsulated in other hydrogels at the corresponding day. (C) The proliferation of cells 3 days after culture in the hydrogel-encapsulated condition. The percent proliferation is calculated by divided the number of cells cultured for 3 days by that for 1 day. *, p < 0.05; significant difference against the proliferation of cells encapsulated in other hydrogels at the corresponding day. (D) The proliferation of cells collected from the hydrogels for 3 days. The cells were encapsulated in the hydrogels for 3 days. The percent proliferation is calculated by divided the number of cells cultured for 3 days by that for 1 day. *, p < 0.05; significant difference against the value of TCPS control.

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Figure 4. Osteogenic differentiation assay of cells encapsulated in the hydrogels. (A) Relative RUNX2 expression, (B) relative ALP activity, and (D) relative calcium content of cells 7 and 14 days after differentiation culture in the non-encapsulated (☐) and encapsulated in the A2G8 hydrogel (■). *, p < 0.05; significant difference against the value 7 days after differentiation culture at the corresponding cells. (C) Mineralization of staining by alizarin red in the cells after 7 and 14 days after culture in the NM and the DM. Scale bar is 1 cm.

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Figure 5. In vivo cell retention 1 (☐) and 3 days after cell transplantation in the A2G8 hydrogel-encapsulated or PBS suspension condition (■). *, p<0.05; significant difference against the percent cell retention after the PBS injection of cell suspension at the corresponding day.

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Table 1. Preparation condition of mixed alginate/gelatin hydrogels. Code

Alginate (wt%)

Gelatin (wt%)

A10G00

1.00

0.00

A08G02

0.80

0.20

A07G03

0.70

0.30

A05G05

0.50

0.50

A03G07

0.30

0.70

A02G08

0.20

0.80

A00G10

0.00

1.00

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Graphical Abstract

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