- Email: [email protected]
Thermo-reversible injectable hydrogel composing of pluronic F127 and carboxymethyl hexanoyl chitosan for cell-encapsulation
Colloids and Surfaces B: Biointerfaces 185 (2020) 110606
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Thermo-reversible injectable hydrogel composing of pluronic F127 and carboxymethyl hexanoyl chitosan for cell-encapsulation
T
Lie-Sian Yap, Ming-Chien Yang* Department of Materials Science and engineering, National Taiwan University of Science and Technology, Taipei, 10607, Taiwan
A R T I C LE I N FO
A B S T R A C T
Keywords: Injectable hydrogel Thermo-reversibility F127 MG-63 Cell encapsulation
This study demonstrated a novel injectable-thermoreversible hydrogel scaffold composing of PLuronic F127, carboxymethyl hexanoyl chitosan (CA) and glyoxal (Gx) for encapsulating human osteosarcoma MG-63 cells. The hydrogel was prepared by simply mixing CA, F127 and Gx. In so doing, this system exhibited short gelation time and higher gelation temperature. In addition, this hydrogel exhibited thermo-reversibility, that is, the hydrogel can liquefy at room temperature and revert to gel state at body temperature. The encapsulated cells in this hydrogel proliferated more than 400% in the 5-day incubation. Based on these results, these F127/CA/Gx hydrogels can be used to encapsulate cells for tissue engineering applications.
1. Introduction
In our previous work, we developed a thermosensitive hydrogel system comprising of F127, CA and GA [13]. However, F127/CA/GA lacks thermal reversibility. That is, the F127/CA/GA hydrogel would not liquefy at lower temperature. Other researchers also found similar phenomenon in the poloxamer-chitosan-glutaraldehyde system [14]. This makes the stability and storage time of hydrogel shorter. To overcome this problem, we tried to design a thermo-reversible hydrogel with longer storage time and stability. In this work, we developed a thermo-reversible injectable hydrogel composing of F127 and CA using Gx as the crosslinker for encapsulating MG-63 cells. The functional groups before and after chemical crosslinking were examined. Eagle’s minimum essential medium Furthermore, the biocompatibility of the hydrogel and morphology of encapsulated-cell after injection were investigated.
Injectable scaffold has received a lot of attention in the last decades. It offers benefits such as less discomfort, minimally invasive manner, easy administration, and cost effectiveness. The main purpose of injectable scaffold is to overcome the needs of operation. By injecting hydrogel solution to the defect area, the body environment will make the solution gelled in a short period into hydrogel and filled any size or shape of the defect [1,2]. Pluronic F127 (F127) is common thermo-responsive polymers for clinical applications [3–5]. At temperature above the lower critical solution temperature (LCST) and above the critical micelle concentration, F127 forms micelles and the solution becomes hydrogel [4] However, F127 hydrogel are easy to dissolve in the aqueous environment. To improve the integrity of F127, Carboxymethyl hexanoyl chitosan (CA) and glyoxal (Gx) were used to modify F127 hydrogel in this study. CA, commercially known as chitosonic acid®, is a chitosan derivative soluble in aqueous solution at neutral pH. CA exhibits antimicrobial activity, biocompatibility, water-solubility, good hydration activity for absorbing, stable, and has functional groups that can be used to crosslink in hydrogel formation [6]. Gx is the smallest dialdehyde with the chemical formula OHCCHO. Some researchers state that Gx has lower toxicity than glutaraldehyde (GA) [7–10]. Gx as a crosslinker exhibited no toxicity to the cell [7,11], increased the viscoelasticity of the biomaterials [8], and increased thermal stability [12].
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2. Materials and methods 2.1. Materials CA (Advanced Delivery Technology, Taiwan), F127 (Sigma, USA), and Gx (Nippon Shinyaku, Japan) were used for preparing injectable hydrogels. Eagle’s minimum essential medium (MEM), sodium bicarbonate, sodium pyruvate, 3-(4,5-dimethyllthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma, USA. Penicillin and streptomycin, fetal bovine serum (FBS), trypsin, and phosphate buffered saline (PBS) were purchased from Caisson Laboratories Inc., USA. Ethanol (95%) (Acros
Corresponding Author. E-mail address: [email protected] (M.-C. Yang).
https://doi.org/10.1016/j.colsurfb.2019.110606 Received 24 April 2019; Received in revised form 22 September 2019; Accepted 20 October 2019 Available online 23 October 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
Colloids and Surfaces B: Biointerfaces 185 (2020) 110606
L.-S. Yap and M.-C. Yang
Table 1 Effect of CA and Gx on the gelation characteristics of F127 hydrogel. Hydrogel composition
Tgel (°C)
F127-20 F127-20/CA-0.5/Gx F127-20/CA-1.0/Gx F127-20/CA-1.5/Gx
24.2 32.2 30.7 29.3
± ± ± ±
tgel (s) 0.4 1.6 0.1 0.6
72.2 72.0 70.1 64.0
± ± ± ±
4.0 5.5 0.7 3.1
G’ at 37 °C (kPa)
G” at 37 °C (kPa)
Dissolution time in medium
7.2 4.7 4.4 5.2
2.1 2.7 2.6 2.9
< > > >
Fig. 1. The effect of temperature on the damping factor for F127-20/CA-1.5/ Gx.
± ± ± ±
0.4 0.4 0.2 0.4
± ± ± ±
0.2 0.3 0.2 0.2
1h 5d 5d 5d
Fig. 2. Viability of encapsulated MG-63 cells.
2.5. MG-63 cells culture
Organics, USA), calcein AM (eBioscience, USA) and trypan blue 0.4% (American Bioinovations, USA) were used for cell staining. All of these materials were used directly without any further purification.
MG-63 cells were cultured in MEM containing 10% FBS, 0.1% penicillin, 0.15% sodium bicarbonate in 75 cm2 cell culture flasks at 37 °C under 5% CO2. The MG-63 cells were harvested every 3 days by using trypsin-EDTA.
2.2. Preparation of injectable hydrogels Firstly, 0.25 to 0.75 g of CA were dissolved in 50 ml of distilled water. After stirring, 10 g of F127 were then added to the CA solution before stirring for 30 min at 4 °C. Thereafter, 10 μl of Gx were added and mixed for 10 min at 4 °C.
2.6. Viability of encapsulated cells
The gelation temperature, storage modulus and loss modulus were determined using a rotational rheometer (MCR 102, Anton Paar, Austria) with a stainless steel cone and plate geometry (25 mm diameter and a gap of 0.45 mm between the cone and plate) under temperature ramp step oscillation. Hydrogel solution was heated from 15 to 45 °C, at a heating rate of 0.02 °C/s. The dynamic mechanical properties of hydrogels were carried out under a constant stress (10 Pa) at a frequency of 0.1 Hz and maximum strain amplitude of 1% at 37 °C.
The F127/CA/Gx solution was sterilized with UV light (UV-C at 253.7 nm) for 1 h in cold environment. Then 60 μl of the hydrogel solution were mixed with 40 μl of MEM containing 2.5 × 103 MG-63 cells in each well of a 24-well plate. After standing 2 min for gelation, 900 μl of MEM were added into each well. The encapsulated cells were incubated for 1, 3, 5 days at 37 °C. The medium was changed every 2 days. The growth of MG-63 cell was determined with MTT assay and analyzed using an ELISA plate reader at 570 nm. The viability of the cell was calculated as the absorbance of the sample divided by the initial absorbance. At least four replicates were analyzed for each data point. For determining the cytotoxicity, the positive control was un-encapsulated cells cultured in PBS without nutrition, while the negative control is un-encapsulated cells cultured in MEM.
2.4. Gelation time
2.7. Survivability of encapsulated cells after injection
The gelation time of hydrogel was measured in a transparent vial. A magnetic stirrer was put into the hydrogel solution, then the vial was placed in a water bath at 37 °C and stirred at 200 rpm. The gelation time was registered when the magnetic bar stopped due to the gelation [15,16].
After sterilization, 60 μl of F127/CA/Gx and 40 μl of MEM containing 2500 cells were mixed in the syringe, and the mixture was injected into the 4-chamber slide through a needle of 19 G × 1½ in. Afterward, 900 μl of medium were added into each chamber. The encapsulated cells were stained with calcein AM (8 μM) for 30 min at 37 °C. Calcein AM is known as live-cell staining in the
2.3. Rheological measurements
2
Colloids and Surfaces B: Biointerfaces 185 (2020) 110606
L.-S. Yap and M.-C. Yang
Fig. 3. Staining of MG-63 cells.
fluorescent techniques. The live cells would appear green under the light emission of the blue laser (488 nm, BX-51, Olympus, Japan).
The addition of CA and Gx can keep the gel from dissolution in the medium. The pristine 20 wt% F127 dissolved within 1 h in the medium, while F127/CA/Gx remained intact for at least 5 days. This would prevent the release of encapsulated cells from the scaffold after injection. Among these three ingredients, F127 conferred the thermo-responsivity of the hydrogel, CA increased the gelation temperature, and Gx enhanced the integrity of the hydrogel without dissolving in the medium. The micellization of F127 would be interrupted by the presence of CA, thus the gelation temperature of the F127/CA/Gx would be higher than pure F127 (24.2 ± 0.4 °C). In the literature, the presence of chitosan would interfere the gelation of Pluronic [19]. In this work, CA was playing the role as chitosan to interrupt the gelation of F127. Lastly, Gx would crosslink with the amino groups of CA to form a network which would keep the hydrogel intact in the aqueous environment [21]. Indeed, without Gx, the mixture of F127 and CA dissolved rapidly in the medium.
3. Results and discussion 3.1. Gelation of F127/CA/Gx Table 1 summarizes the gelation behaviors of F127/CA/Gx hydrogels. The gelation time of original F127 hydrogel was 72 s. The addition of CA and Gx was able to reduce the gelation time and increase the gelation temperature. Gelation temperature of F127/CA/Gx was determined from the intersection of storage modulus [G’] and loss modulus [G”] [17–19]. The best temperature range for the injectable hydrogel is 25–37 °C [20]. Once the hydrogel solution was injected to the defect area in the body, the solution will gel at the body temperature. As shown in Table 1, the gelation temperature of modified hydrogel was around 29–32 °C. 3
Colloids and Surfaces B: Biointerfaces 185 (2020) 110606
L.-S. Yap and M.-C. Yang
allowing them to stretch into spindle-like shape. Park et al. reported that encapsulated chondrocytes in chitosan-Pluronic hydrogel also adopted spherical morphology [24]. Encapsulated human bone stromal cells (hBMSC) also retained a rounded morphology and showed high viability [7]. Other encapsulated mesenchymal stem cells in injectable chitosan/collagen microbeads showed high viability after injection process. The viability was not significantly affected by injection through needle [9].
3.2. Thermo-reversibility of F127/CA/Gx In this system, F127/CA/Gx hydrogel was designed to be thermoreversible. For injectable scaffold, the hydrogel can remain in gel state and become liquid for encapsulating cells. The thermo-reversibility of the hydrogel was evaluated based on the damping factor (tanδ), which is the ratio of G” to G’. Hydrogel solution was first heated to 40 °C, then cooled down to 14 °C, then heated again to 40 °C. The hydrogel appeared as a gel when the tanδ was less than 1. Fig. 1 shows that the first heating cycle and second heating cycle exhibited the same gelation temperature for F127/CA/Gx hydrogel. Furthermore, both the tanδ curves of first heating cycle and second heating cycle were mostly overlapped, indicating that this hydrogel exhibited thermo-reversibility. The tanδ curve of the first cooling cycle showed that the hydrogel liquefied around 24 °C, while the gelation in the first and second heating cycles occurred around 28.4 °C. The second heating curve was slightly lower than the first heating curve due to water loss. The result showed that this hydrogel was thermo-reversible. This phenomenon is possibly because that GX is shorter than GA, the crosslinked CA chains would form a more compact and hydrophobic network, causing less interruption to the micelles of F127 than in the case of GA [22]. This allows F127 to keep its thermo-reversibility.
4. Conclusion This study showed that CA and Gx were able to retard the dissolution of F127, increase Tgel to above 25 °C, and keep gelation time within around 70 s. The encapsulated cells after injection survived and proliferated. These results demonstrated that F127/CA/Gx hydrogels were able to serve as injectable scaffolds in the tissue engineering applications. Declaration of competing interest None. Appendix A. Supplementary data
3.3. Viability of encapsulated MG-63 in F127/CA/Gx
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110606.
The viability increased day by day except for the positive control (Fig. 2. Viability of encapsulated MG-63 cells.). On the first day, the viability of ecapsulated-MG-63 was below 100%. This is because in the early incubation stage, encapsulated cells were in the adaptation period. On the third day, the viability increased by more than 200% of that of the first-day incubation. On the fifth day of incubation, the increase of viability was not so fast because cells almost reached confluency. Positive control always dropped their viability along with the incubation time. The increased viability of encapsulated-cells indicated that the hydrogel exhibited no cytotoxicity to MG-63 and the encapsulated cells can grow safely. Fig. 2 also reveals that the viability of the encapsulated cells was lower than the negative control (un-encapsulated cells in MEM) because these cells were trapped in a 3D hydrogel matrix, limiting their nutrients and oxygen supply, whereas the negative control was cultured in a 2D environment, allowing the cells to easily access the nutrients and oxygen supply. In the literature, the viability of encapsulated human articular chondrocytes (HACs) and human bone marrow-derived mesenchymal stromal cells (MSCs) in gelatin-methacryloyl hydrogel showed an increase in cell viability over time. Along with the incubation time, the viability of both HACs and MSCs increased from 60% to more than 80% in a 3-week culture period [23]. This behavior was similar to the encapsulated MG-63 cells in our experiments. Thermosensitive chitosan-Pluronic hydrogel also showed increasing number of chondrocytes in the duration from 14 to 28 days of incubation [24]. Other chitosan/glycerol phosphate/hydroxyl ethyl cellulose/glyoxal hydrogel can also successfully encapsulate human embryonic kidney cells (HEK293). The presence of Gx (0.1–0.15 mM) was able to promote cell proliferation [10].
References [1] B. Chang, N. Ahuja, C. Ma, X. Liu, Injectable scaffolds: preparation and application in dental and craniofacial regeneration, Mater. Sci. Eng. R Rep. 111 (2017) 1–26. [2] Q. Hou, P.A. De Bank, K.M. Shakesheff, Injectable scaffolds for tissue regeneration, J. Mater. Chem. 14 (2004) 1915–1923. [3] R. Basak, R. Bandyopadhyay, Encapsulation of hydrophobic drugs in Pluronic F127 micelles: effects of drug hydrophobicity, solution temperature, and pH, Langmuir 29 (2013) 4350–4356. [4] J.J. Escobar-Chavez, M. Lopez-Cervantes, A. Naik, Y.N. Kalia, D. QuintanarGuerrero, A. Ganem-Quintanar, Applications of thermo-reversible pluronic F-127 gels in pharmaceutical formulations, J. Pharm. Pharm. Sci. 9 (2006) 339–358. [5] D.R. Devi, P. Sandhya, B.N.V. Hari, Poloxamer a novel functional molecule for drug delivery and gene Therapy.pDf, J. Pharm. Sci. Res. 5 (2013) 159–165. [6] S.-M. Lee, K.-H. Liu, Y.-Y. Liu, Y.-P. Chang, C.-C. Lin, Y.-S. Chen, Chitosonic® acid as a novel cosmetic ingredient: evaluation of its antimicrobial, antioxidant and hydration activities, Materials 6 (2013) 1391–1402. [7] L. Wang, J.P. Stegemann, Glyoxal crosslinking of cell-seeded chitosan/collagen hydrogels for bone regeneration, Acta Biomater. 7 (2011) 2410–2417. [8] H.K. Heris, N. Latifi, H. Vali, N. Li, L. Mongeau, Investigation of Chitosan-glycol/ glyoxal as an injectable biomaterial for vocal fold tissue engineering, Procedia Eng. 110 (2015) 143–150. [9] L. Wang, R.R. Rao, J.P. Stegemann, Delivery of mesenchymal stem cells in Chitosan/Collagen microbeads for orthopedic tissue repair, Cells Tissues Organs 197 (2013) 333–343. [10] C.D. Hoemann, A. Chenite, J. Sun, M. Hurtig, A. Serreqi, Z. Lu, E. Rossomacha, M.D. Buschmann, Cytocompatible gel formation of chitosan-glycerol phosphate solutions supplemented with hydroxyl ethyl cellulose is due to the presence of glyoxal, J. Biomed. Mater. Res. A. 83A (2007) 521–529. [11] M. Dessi, A. Borzacchiello, T.H. Mohamed, W.I. Abdel-Fattah, L. Ambrosio, Novel biomimetic thermosensitive beta-tricalcium phosphate/chitosan-based hydrogels for bone tissue engineering, J. Biomed. Mater. Res. A 101 (2013) 2984–2993. [12] S. Kim, Y. Kang, C.A. Krueger, M. Sen, J.B. Holcomb, D. Chen, J.C. Wenke, Y. Yang, Sequential delivery of BMP-2 and IGF-1 using a chitosan gel with gelatin microspheres enhances early osteoblastic differentiation, Acta Biomater. 8 (2012) 1768–1777. [13] L.-S. Yap, M.-C. Yang, Evaluation of hydrogel composing of Pluronic F127 and carboxymethyl hexanoyl chitosan as injectable scaffold for tissue engineering applications, Colloids Surf. B Biointerfaces 146 (2016) 204–211. [14] T.-W. Chung, S.-Y. Lin, D.-Z. Liu, Y.-C. Tyan, J.-S. Yang, Sustained release of 5-FU from Poloxamer gels interpenetrated by crosslinking chitosan network, Int. J. Pharm. 382 (2009) 39–44. [15] E. Khodaverdi, M. Tafaghodi, F. Ganji, K. Abnoos, H. Naghizadeh, In vitro insulin release from thermosensitive chitosan hydrogel, AAPS PharmSciTech 13 (2012) 460–466. [16] C. Li, S. Ren, Y. Dai, F. Tian, X. Wang, S. Zhou, S. Deng, Q. Liu, J. Zhao, X. Chen, Efficacy, pharmacokinetics, and biodistribution of Thermosensitive Chitosan/βGlycerophosphate hydrogel loaded with docetaxel, AAPS PharmSciTech 15 (2014) 417–424. [17] G. Dumortier, J.L. Grossiord, M. Zuber, G. Couarraze, J.C. Chaumeil, Rheological
3.4. Survivability of encapsulated MG-63 The main purpose of this work was to develop a hydrogel that can be injected into the defect site. The influence of needle of injection was one of the important issues in this study. Fig. 3 shows that cells survived after encapsulation and injection. The number of cells increased with the incubation time. Encapsulated cells assumed spherical morphology while the un-encapsulated cells appear spindle-like. This is reasonable because encapsulated cells were surrounded by the hydrogel matrix, whereas un-encapsulated cells were attached to the bottom of the flask, 4
Colloids and Surfaces B: Biointerfaces 185 (2020) 110606
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[18]
[19]
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interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications, Eur. J. Pharm. Biopharm. 57 (2004) 19–34. [22] K.C. Gupta, F.H. Jabrail, Glutaraldehyde and glyoxal cross-linked chitosan microspheres for controlled delivery of centchroman, Carbohydr. Res. 341 (2006) 744–756. [23] K.S. Lim, B.S. Schon, N.V. Mekhileri, G.C.J. Brown, C.M. Chia, S. Prabakar, G.J. Hooper, T.B.F. Woodfield, New visible-light photoinitiating system for improved print fidelity in gelatin-based bioinks, ACS Biomater. Sci. Eng. 2 (2016) 1752–1762. [24] K.M. Park, S.Y. Lee, Y.K. Joung, J.S. Na, M.C. Lee, K.D. Park, Thermosensitive chitosan–Pluronic hydrogel as an injectable cell delivery carrier for cartilage regeneration, Acta Biomater. 5 (2009) 1956–1965.
study of a thermoreversible morphine gel, Drug Dev. Ind. Pharm. 17 (1991) 1255–1265. L. Mayol, F. Quaglia, A. Borzacchiello, L. Ambrosio, M.I. La Rotonda, A novel poloxamers/hyaluronic acid in situ forming hydrogel for drug delivery: rheological, mucoadhesive and in vitro release properties, Eur. J. Pharm. Biopharm. 70 (2008) 199–206. T. Gratieri, G.M. Gelfuso, E.M. Rocha, V.H. Sarmento, O. de Freitas, R.F. Lopez, A poloxamer/chitosan in situ forming gel with prolonged retention time for ocular delivery, Eur. J. Pharm. Biopharm. 75 (2010) 186–193. D.S. Shaker, M.K. Ghorab, A. Klingner, M.S. Teiama, In-situ injectable thermosensitive gel based on poloxamer as a new carrier for tamoxifen citrate, Int. J. Pharm. Pharm. Sci. 5 (2013) 429–437. J. Berger, M. Reist, J.M. Mayer, O. Felt, N.A. Peppas, R. Gurny, Structure and
5
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Thermo-reversible injectable hydrogel composing of pluronic F127 and carboxymethyl hexanoyl chitosan for cell-encapsulation
T
Lie-Sian Yap, Ming-Chien Yang* Department of Materials Science and engineering, National Taiwan University of Science and Technology, Taipei, 10607, Taiwan
A R T I C LE I N FO
A B S T R A C T
Keywords: Injectable hydrogel Thermo-reversibility F127 MG-63 Cell encapsulation
This study demonstrated a novel injectable-thermoreversible hydrogel scaffold composing of PLuronic F127, carboxymethyl hexanoyl chitosan (CA) and glyoxal (Gx) for encapsulating human osteosarcoma MG-63 cells. The hydrogel was prepared by simply mixing CA, F127 and Gx. In so doing, this system exhibited short gelation time and higher gelation temperature. In addition, this hydrogel exhibited thermo-reversibility, that is, the hydrogel can liquefy at room temperature and revert to gel state at body temperature. The encapsulated cells in this hydrogel proliferated more than 400% in the 5-day incubation. Based on these results, these F127/CA/Gx hydrogels can be used to encapsulate cells for tissue engineering applications.
1. Introduction
In our previous work, we developed a thermosensitive hydrogel system comprising of F127, CA and GA [13]. However, F127/CA/GA lacks thermal reversibility. That is, the F127/CA/GA hydrogel would not liquefy at lower temperature. Other researchers also found similar phenomenon in the poloxamer-chitosan-glutaraldehyde system [14]. This makes the stability and storage time of hydrogel shorter. To overcome this problem, we tried to design a thermo-reversible hydrogel with longer storage time and stability. In this work, we developed a thermo-reversible injectable hydrogel composing of F127 and CA using Gx as the crosslinker for encapsulating MG-63 cells. The functional groups before and after chemical crosslinking were examined. Eagle’s minimum essential medium Furthermore, the biocompatibility of the hydrogel and morphology of encapsulated-cell after injection were investigated.
Injectable scaffold has received a lot of attention in the last decades. It offers benefits such as less discomfort, minimally invasive manner, easy administration, and cost effectiveness. The main purpose of injectable scaffold is to overcome the needs of operation. By injecting hydrogel solution to the defect area, the body environment will make the solution gelled in a short period into hydrogel and filled any size or shape of the defect [1,2]. Pluronic F127 (F127) is common thermo-responsive polymers for clinical applications [3–5]. At temperature above the lower critical solution temperature (LCST) and above the critical micelle concentration, F127 forms micelles and the solution becomes hydrogel [4] However, F127 hydrogel are easy to dissolve in the aqueous environment. To improve the integrity of F127, Carboxymethyl hexanoyl chitosan (CA) and glyoxal (Gx) were used to modify F127 hydrogel in this study. CA, commercially known as chitosonic acid®, is a chitosan derivative soluble in aqueous solution at neutral pH. CA exhibits antimicrobial activity, biocompatibility, water-solubility, good hydration activity for absorbing, stable, and has functional groups that can be used to crosslink in hydrogel formation [6]. Gx is the smallest dialdehyde with the chemical formula OHCCHO. Some researchers state that Gx has lower toxicity than glutaraldehyde (GA) [7–10]. Gx as a crosslinker exhibited no toxicity to the cell [7,11], increased the viscoelasticity of the biomaterials [8], and increased thermal stability [12].
⁎
2. Materials and methods 2.1. Materials CA (Advanced Delivery Technology, Taiwan), F127 (Sigma, USA), and Gx (Nippon Shinyaku, Japan) were used for preparing injectable hydrogels. Eagle’s minimum essential medium (MEM), sodium bicarbonate, sodium pyruvate, 3-(4,5-dimethyllthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma, USA. Penicillin and streptomycin, fetal bovine serum (FBS), trypsin, and phosphate buffered saline (PBS) were purchased from Caisson Laboratories Inc., USA. Ethanol (95%) (Acros
Corresponding Author. E-mail address: [email protected] (M.-C. Yang).
https://doi.org/10.1016/j.colsurfb.2019.110606 Received 24 April 2019; Received in revised form 22 September 2019; Accepted 20 October 2019 Available online 23 October 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
Colloids and Surfaces B: Biointerfaces 185 (2020) 110606
L.-S. Yap and M.-C. Yang
Table 1 Effect of CA and Gx on the gelation characteristics of F127 hydrogel. Hydrogel composition
Tgel (°C)
F127-20 F127-20/CA-0.5/Gx F127-20/CA-1.0/Gx F127-20/CA-1.5/Gx
24.2 32.2 30.7 29.3
± ± ± ±
tgel (s) 0.4 1.6 0.1 0.6
72.2 72.0 70.1 64.0
± ± ± ±
4.0 5.5 0.7 3.1
G’ at 37 °C (kPa)
G” at 37 °C (kPa)
Dissolution time in medium
7.2 4.7 4.4 5.2
2.1 2.7 2.6 2.9
< > > >
Fig. 1. The effect of temperature on the damping factor for F127-20/CA-1.5/ Gx.
± ± ± ±
0.4 0.4 0.2 0.4
± ± ± ±
0.2 0.3 0.2 0.2
1h 5d 5d 5d
Fig. 2. Viability of encapsulated MG-63 cells.
2.5. MG-63 cells culture
Organics, USA), calcein AM (eBioscience, USA) and trypan blue 0.4% (American Bioinovations, USA) were used for cell staining. All of these materials were used directly without any further purification.
MG-63 cells were cultured in MEM containing 10% FBS, 0.1% penicillin, 0.15% sodium bicarbonate in 75 cm2 cell culture flasks at 37 °C under 5% CO2. The MG-63 cells were harvested every 3 days by using trypsin-EDTA.
2.2. Preparation of injectable hydrogels Firstly, 0.25 to 0.75 g of CA were dissolved in 50 ml of distilled water. After stirring, 10 g of F127 were then added to the CA solution before stirring for 30 min at 4 °C. Thereafter, 10 μl of Gx were added and mixed for 10 min at 4 °C.
2.6. Viability of encapsulated cells
The gelation temperature, storage modulus and loss modulus were determined using a rotational rheometer (MCR 102, Anton Paar, Austria) with a stainless steel cone and plate geometry (25 mm diameter and a gap of 0.45 mm between the cone and plate) under temperature ramp step oscillation. Hydrogel solution was heated from 15 to 45 °C, at a heating rate of 0.02 °C/s. The dynamic mechanical properties of hydrogels were carried out under a constant stress (10 Pa) at a frequency of 0.1 Hz and maximum strain amplitude of 1% at 37 °C.
The F127/CA/Gx solution was sterilized with UV light (UV-C at 253.7 nm) for 1 h in cold environment. Then 60 μl of the hydrogel solution were mixed with 40 μl of MEM containing 2.5 × 103 MG-63 cells in each well of a 24-well plate. After standing 2 min for gelation, 900 μl of MEM were added into each well. The encapsulated cells were incubated for 1, 3, 5 days at 37 °C. The medium was changed every 2 days. The growth of MG-63 cell was determined with MTT assay and analyzed using an ELISA plate reader at 570 nm. The viability of the cell was calculated as the absorbance of the sample divided by the initial absorbance. At least four replicates were analyzed for each data point. For determining the cytotoxicity, the positive control was un-encapsulated cells cultured in PBS without nutrition, while the negative control is un-encapsulated cells cultured in MEM.
2.4. Gelation time
2.7. Survivability of encapsulated cells after injection
The gelation time of hydrogel was measured in a transparent vial. A magnetic stirrer was put into the hydrogel solution, then the vial was placed in a water bath at 37 °C and stirred at 200 rpm. The gelation time was registered when the magnetic bar stopped due to the gelation [15,16].
After sterilization, 60 μl of F127/CA/Gx and 40 μl of MEM containing 2500 cells were mixed in the syringe, and the mixture was injected into the 4-chamber slide through a needle of 19 G × 1½ in. Afterward, 900 μl of medium were added into each chamber. The encapsulated cells were stained with calcein AM (8 μM) for 30 min at 37 °C. Calcein AM is known as live-cell staining in the
2.3. Rheological measurements
2
Colloids and Surfaces B: Biointerfaces 185 (2020) 110606
L.-S. Yap and M.-C. Yang
Fig. 3. Staining of MG-63 cells.
fluorescent techniques. The live cells would appear green under the light emission of the blue laser (488 nm, BX-51, Olympus, Japan).
The addition of CA and Gx can keep the gel from dissolution in the medium. The pristine 20 wt% F127 dissolved within 1 h in the medium, while F127/CA/Gx remained intact for at least 5 days. This would prevent the release of encapsulated cells from the scaffold after injection. Among these three ingredients, F127 conferred the thermo-responsivity of the hydrogel, CA increased the gelation temperature, and Gx enhanced the integrity of the hydrogel without dissolving in the medium. The micellization of F127 would be interrupted by the presence of CA, thus the gelation temperature of the F127/CA/Gx would be higher than pure F127 (24.2 ± 0.4 °C). In the literature, the presence of chitosan would interfere the gelation of Pluronic [19]. In this work, CA was playing the role as chitosan to interrupt the gelation of F127. Lastly, Gx would crosslink with the amino groups of CA to form a network which would keep the hydrogel intact in the aqueous environment [21]. Indeed, without Gx, the mixture of F127 and CA dissolved rapidly in the medium.
3. Results and discussion 3.1. Gelation of F127/CA/Gx Table 1 summarizes the gelation behaviors of F127/CA/Gx hydrogels. The gelation time of original F127 hydrogel was 72 s. The addition of CA and Gx was able to reduce the gelation time and increase the gelation temperature. Gelation temperature of F127/CA/Gx was determined from the intersection of storage modulus [G’] and loss modulus [G”] [17–19]. The best temperature range for the injectable hydrogel is 25–37 °C [20]. Once the hydrogel solution was injected to the defect area in the body, the solution will gel at the body temperature. As shown in Table 1, the gelation temperature of modified hydrogel was around 29–32 °C. 3
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allowing them to stretch into spindle-like shape. Park et al. reported that encapsulated chondrocytes in chitosan-Pluronic hydrogel also adopted spherical morphology [24]. Encapsulated human bone stromal cells (hBMSC) also retained a rounded morphology and showed high viability [7]. Other encapsulated mesenchymal stem cells in injectable chitosan/collagen microbeads showed high viability after injection process. The viability was not significantly affected by injection through needle [9].
3.2. Thermo-reversibility of F127/CA/Gx In this system, F127/CA/Gx hydrogel was designed to be thermoreversible. For injectable scaffold, the hydrogel can remain in gel state and become liquid for encapsulating cells. The thermo-reversibility of the hydrogel was evaluated based on the damping factor (tanδ), which is the ratio of G” to G’. Hydrogel solution was first heated to 40 °C, then cooled down to 14 °C, then heated again to 40 °C. The hydrogel appeared as a gel when the tanδ was less than 1. Fig. 1 shows that the first heating cycle and second heating cycle exhibited the same gelation temperature for F127/CA/Gx hydrogel. Furthermore, both the tanδ curves of first heating cycle and second heating cycle were mostly overlapped, indicating that this hydrogel exhibited thermo-reversibility. The tanδ curve of the first cooling cycle showed that the hydrogel liquefied around 24 °C, while the gelation in the first and second heating cycles occurred around 28.4 °C. The second heating curve was slightly lower than the first heating curve due to water loss. The result showed that this hydrogel was thermo-reversible. This phenomenon is possibly because that GX is shorter than GA, the crosslinked CA chains would form a more compact and hydrophobic network, causing less interruption to the micelles of F127 than in the case of GA [22]. This allows F127 to keep its thermo-reversibility.
4. Conclusion This study showed that CA and Gx were able to retard the dissolution of F127, increase Tgel to above 25 °C, and keep gelation time within around 70 s. The encapsulated cells after injection survived and proliferated. These results demonstrated that F127/CA/Gx hydrogels were able to serve as injectable scaffolds in the tissue engineering applications. Declaration of competing interest None. Appendix A. Supplementary data
3.3. Viability of encapsulated MG-63 in F127/CA/Gx
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110606.
The viability increased day by day except for the positive control (Fig. 2. Viability of encapsulated MG-63 cells.). On the first day, the viability of ecapsulated-MG-63 was below 100%. This is because in the early incubation stage, encapsulated cells were in the adaptation period. On the third day, the viability increased by more than 200% of that of the first-day incubation. On the fifth day of incubation, the increase of viability was not so fast because cells almost reached confluency. Positive control always dropped their viability along with the incubation time. The increased viability of encapsulated-cells indicated that the hydrogel exhibited no cytotoxicity to MG-63 and the encapsulated cells can grow safely. Fig. 2 also reveals that the viability of the encapsulated cells was lower than the negative control (un-encapsulated cells in MEM) because these cells were trapped in a 3D hydrogel matrix, limiting their nutrients and oxygen supply, whereas the negative control was cultured in a 2D environment, allowing the cells to easily access the nutrients and oxygen supply. In the literature, the viability of encapsulated human articular chondrocytes (HACs) and human bone marrow-derived mesenchymal stromal cells (MSCs) in gelatin-methacryloyl hydrogel showed an increase in cell viability over time. Along with the incubation time, the viability of both HACs and MSCs increased from 60% to more than 80% in a 3-week culture period [23]. This behavior was similar to the encapsulated MG-63 cells in our experiments. Thermosensitive chitosan-Pluronic hydrogel also showed increasing number of chondrocytes in the duration from 14 to 28 days of incubation [24]. Other chitosan/glycerol phosphate/hydroxyl ethyl cellulose/glyoxal hydrogel can also successfully encapsulate human embryonic kidney cells (HEK293). The presence of Gx (0.1–0.15 mM) was able to promote cell proliferation [10].
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