Chapter 2 Materials and Methods
2.6 Statistic Analysis
The data was presented as means ± standard error of the mean (SEM). The statistical analyses between different groups were determined with Student- Newman-Keuls Multiple Comparisons Test. A value of p ≤ 0.05 was considered statistically significant difference.
Figure 2-1 Synthesis of phenol groups modified glycol chitosan via EDC & NHS method.
Figure 2-2 Fabrication of HPP-GC hydrogel.
Figure 2-3 Mechanism of photochemical crosslinking [35].
Figure 2-4 (A) Digital force gauge. (B) Procedure of testing the detachment stress of hydrogel by using egg membrane.
Egg membrane
HPP-GC hydrogel
(A)
(B)
Chapter 3
Results and Discussion
3.1 Synthesis and Characterization of HPP-GC
HPP-GC was synthesized by the reaction of glycol chitosan (GC) with 3-(4-Hydroxyphenyl) propionic acid (HPP). The successful derivatization was verified by
1H NMR (Figure 3-1). Quantitative Uv-visible analysis (Figure 3-2) was used to
determine the degree of substitution (DS) of phenol groups in HPP-GC samples.
The successful synthesis was verified by 1H NMR. Figure 3-1 shows 1H NMR spectra of glycol chitosan and HPP-GC. By comparison with the spectrum of glycol chitosan, new signals were observed at δ = 6.8 ppm and δ = 7.2 ppm in the spectrum of HPP-GC. The signals of new peaks corresponding to aromatic protons confirmed the successful graft of phenol groups in the HPP-GC polymer. The reduction of the signal of hydrogen on amino groups at δ = 2.7 ppm after the reaction confirmed that the reaction completed correctly.
The degree of substitution (DS) was determined by Uv-visible spectrophotometer.
According to the Figure 3-2, the phenol groups on HPP-GC could be detected at 276 nm wavelength. Among all the samples produced, the DS arranged from 0.200 to 0.600 μmoles of phenol groups per mg of HPP-GC. The DS could be controlled by the amount of HPP. After the actual DS was determined, the following experiments of different
characteristics could be analyzed and explained.
3.2 Fabrication of Photocrosslinked HPP-GC Hydrogel
After mixed with light-activated metal catalyst RuII(bpy)3Cl2 and SPS, the solution become orange ready to be crosslinked. This injectable solution could be crosslinked whenever the user wants and could be made into any shape (Figure 3-3). In the photo crosslinking process, the solution could be crosslinked rapidly within 5 seconds, yet waiting for 30 seconds to make the hydrogel crosslinked completely in our procedure.
These advantages make applying hydrogel to irregular wound possible and the moment for crosslinking could be controlled easily. Compared with other crosslinking methods, widely used enzymatically crosslinked hydrogels always need special device to fabricate hydrogel such as double syringes [30, 34], that is an additional expense and hard to operate. Besides, after mixing with HRP and H2O2, the gelation will occur immediately and the moment is hard to control that would definitely cause inconvenience in certain condition. Moreover, some hybrid crosslinking methods are under restraint like temperature and pH of environment [33]. This photochemically crosslinked hydrogel can conquer those disadvantages with the merits mentioned above.
As the Figure 3-4 shown, different DS have significant influence on the mechanical properties, structure and appearance of hydrogels. High DS (> 0.500 µmole/mg
HPP-GC) would make the hydrogel shrink, and the high crosslinking density would squeeze out the water of hydrogels. Sometimes the hydrogel may be stiff and crack after
crosslinked.
3.3 Strong Tissue Adhesion
Strong tissue adhesion is the main advantage of this photochemically crosslinked HPP-GC hydrogel. In previous study, it puts forth the fact that by irradiation of blue light, the phenol groups can not only form intermolecular crosslinking but bind the amino and thiol groups on animals’ tissue as well [35, 41]. Tissue adhesion could be measured by utilizing digital force gauge to measure the detachment stress at the break point of hydrogels when they were pulled apart. The results indicates that different DS would critically affect the tissue adhesion of hydrogels. Figure 3-5 shows that the detachment stress of the least DS (around 0.250 µmole/mg HPP-GC) is 15.99 ± 9.29 kpa, the moderate DS (around 0.370 µmole/mg HPP-GC) is 77.96 ± 12.10 kpa, the higher DS (larger than 0.534 µmole/mg HPP-GC) is lower than 10.40 ± 7.90 kpa. This photochemically crosslinked HPP-GC hydrogel possess approximately same or larger adhesive strength than other crosslinking method or polymer [31, 33, 64]. Especially, compared with the commercial fibrin sealant fabricated by same photochemical method [46], the adhesive strength of this HPP-GC hydrogel is much larger.
From Figure 3-5, we could also find out that low DS (< 0.300 µmole/mg HPP-GC)
would not provide enough phenol groups to crosslink that the hydrogel would not have adequate crosslinking density; therefore, it would lose the cohesive ability to become hydrogel and could not form enough bonds with animal tissue for adhesion as well. As for hydrogels with high DS, they tend to have too high crosslinking density and that would cause the hydrogel have less elasticity, shrink and sometimes crack into pieces [41]. This tendency would severely destroy the structure of hydrogel and finally fail to bind animal tissue. After finding out the proper DS of hydrogels, which possess the strongest tissue adhesion, this most promising hydrogel would serve as the major object of this research.
In addition, as Figure 3-6 shown, different concentration also have crucial effect on tissue adhesion. Higher concentration (3%) possessed better tissue adhesion and moderate concentration (2%) just slightly lower than the higher one (3%); nevertheless, low concentration (1%) could not become strong hydrogel and it still had a lot of water which had not been crosslinked by irradiation of blue light. Therefore, it is ideal to
fabricate hydrogel between 2% to 3%.
3.4 Rheological Analysis and Mechanical Property
The rheological analysis can characterize the elastic and viscous property of this photocrosslinked HPP-GC hydrogel. As Figure 3-7 shown, the storage modulus of 3%
HPP-GC hydrogel is approximately 6663 ± 65 Pa, which indicates the fact that this
hydrogel is highly elastic and it has great mechanical strength [65]. Previous studies have shown that photo cross-linking 2% glycol chitosan solution has storage modulus of 1800 Pa [66]. Similarly, crosslinking 1.5% glycol chitosan solution with various concentrations of benzaldehyde functionalized PEG analogues has storage modulus ranging from 210~1400 Pa [30]. Hence, we can say that this ruthenium-based photochemical crosslinking process make HPP-GC hydrogel have much higher elastic and mechanical strength. The high storage modulus can also verify the fact that this photochemical crosslinking method can make hydrogel form strong intermolecular covalent bond between polymers. The outstanding elastic and viscosity of HPP-GC hydrogels is significant for tissue adhesion, which can better bear load and dissipate
elastic energy, and can be applicable to flexible soft tissue [67].
3.5 Hydration of Different DS HPP-GC Hydrogels
Swelling and hydration ability of taking up liquid play important part in wound dressing.
Stable swelling and hydration can provide proper absorbency for wound dressing to treat wound exudate. The values of hydration of different DS of hydrogel were measured. As Figure 3-8 shows that the dry hydrogels took up water fast in the first 5 hours. The hydration equilibrium was reached at approximately 24 hours, and the values of hydration of least DS hydrogels (0.250 µmole/mg), moderate DS hydrogels and high DS hydrogels are 2222% ± 33%, 1610% ± 314% and 1288% ± 261%
respectively. It is obvious that different DS of hydrogel can affect crosslinking density of hydrogel, and that definitely change the morphology and porosity of hydrogel [68].
The higher the crosslinking density is, the smaller the porosity is. Therefore, smaller porosity absorbs less liquid. On the contrary, if the crosslinking density is lower, the higher porosity can absorb liquid more and faster. The appropriate hydration can be controlled by the degree of substitution of phenol groups. Since the hydrogels can also serve as hemostatic material, the hydration of taking up blood is significant as well
[69].
3.6 Degradation and Stability of HPP-GC Hydrogel
Degradation is an important factor for the application of this hydrogel. The hydrogel should sustain for sufficient time and have adequate strength for sealing the wound and the curation [69]. Moreover, for the using purpose of being drug delivery system and wound dressing, the hydrogel must have ideal rate of degradation to control the drug release or the degradation at expected time point [58]. According to the Figure 3-9, the hydrogel initially degrade 20% of weight since the hydrogel would shrink and squeeze out some water, and the weight of hydrogel would lose along with the renewal of PBS.
The following 13 days, the hydrogel only lost 20% of mass, which indicates that it cannot only sustain for adequate time but degrade with proper rate. In order to observe the tendency of degradation after the initial loss of water squeezed out, another
experiment was done as Figure 3-10. The low DS cause hydrogels have more loose structure; thus, they would degrade faster than moderate ones. Nevertheless, high DS often results in crack of hydrogel make them lose their weight even faster than low DS
hydrogel.
3.7 Cell Viability and Cytotoxicity
In this study, this photocrosslinked hydrogels were designed to be applied and crosslinked directly on the site of wound. Thus, the cytotoxicity of the HPP-GC and this photochemically crosslinking processes were evaluated by cultivating L929 cells with extraction of hydrogel and then quantify via MTS method. By immersing hydrogel into serum-free medium to get extraction, it could be regarded as the condition that when hydrogel adhere to human tissue, how body fluid would change and whether it would cause cytotoxicity. Different concentration of extraction were utilized to cultivate L929 for 1 day. As shown in Figure 3-11, the 100%, 50% and 10% have 53%
± 7%, 77% ± 2% and 97% ± 4% of cell viability respectively. The result demonstrates that most of the toxicity of SPS was consumed after the rapid photocrosslinking process, which has been studied in previous study [39]; hence, the hydrogels have low toxicity
at most of concentration except the highest concentration.
3.8 In Vitro Drug Release
Hydrogels are frequently used as drug delivery system and have promising controlled
release by modifying their structure and porosity [58, 60]. The Figure 3-12 presents an initial fast release, which may be attributed to the diffusion caused by swelling and the water squeezed out toward the surface of hydrogel after crosslinking. Since this photocrosslinked hydrogel expected to serve as wound dressing and sealant of surgical procedure such as sealing incision of gastrointestinal tract surgery, this fast release of amoxicillin is a desirable feature in an acidic condition of the stomach to overcome the limitations of gastric emptying times [59, 70]. In previous study of hydration, the porous structure of hydrogel also strongly correlate with the rate of drug release. For different using purpose, user can design different degree of substation of phenol groups
to modify porous structure.
3.9 Measurement of Antibacterial Ability
In this in-vitro antibacterial experiment, E coli. and S. epidermidis were used to test the antimicrobial ability of hydrogel, which entrapped gentamycin by photocrosslinking.
Different concentration of gentamycin (1000, 200, 50 μg/ml) were tested in order to observer the efficacy and then user could select proper concentration of antibiotics to use in the future. As Figure 3-13 shown, both antibiotic presented evident inhibition zone, it demonstrates the fact that gentamycin can against wide range of bacteria including gram-positive and gram-negative ones. By comparing with commercial gauze, there is only small inhibition zone around gauze, and it shows that this hydrogels are
more efficient drug delivery and better release system. Although antibiotics can prevent bacterial infection, user must choose appropriate concentration of antibiotics for not causing damage to cells. Therefore, another groups of different concentration of samples were tested, and the Figure 3-14 shows that different concentrations of gentamycin (200, 100, 50 μg/ml) would affect the inhibition zone. As Table 3-1 shown, when using E coli. as the object of the experiment, 200, 100 and 50 μg/ml of gentamycin-entrapped hydrogel had 306% ± 41%, 304% ± 38% and 249% ± 6% of inhibition respectively. When using S. epidermidis as the object of the experiment, 200, 100 and 50 μg/ml of gentamycin-entrapped hydrogel had 305% ± 42%, 271% ± 32%
and 192% ± 22% of inhibition respectively. The result demonstrates that 200 and 100 μg/ml of gentamycin-entrapped hydrogel could efficiently treat against two kinds of bacterial. Nonetheless, 50 μg/ml of gentamycin-entrapped hydrogel had relatively small inhibition zone. As a result, 200 and 100 μg/ml of gentamycin-entrapped hydrogel can
serve as promising antibacterial material.
3.10 In Vivo Hemostatic Ability
This rapidly crosslinked hydrogel can utilized as hemostatic material because of outstanding adhesive property and fast gelation. This injectable hydrogel is able to be applied onto the hemorrhaging site and adhere to tissue around directly by irradiation of blue light and then become a hemostatic barrier. Moreover, glycol chitosan, which is
a kind of derivative of chitosan, have some hemostatic ability. The hemostatic ability was measured by determine the quantity of bleeding on mice liver (Figure 3-15). As shown in Figure 3-16, for the control group, the mice live was bleeding without applying any material and the blood loss was 179.2 ± 95.5 mg. The other group was applying HPP-GC solution and then crosslink it by blue light, the blood loss was 73.0
± 15.9 mg. According to the observation, the bleeding site on mice wound could be covered and stopped it from bleeding by this hydrogel within 10s, and significantly reduce the blood loss of the wound. Likewise, the filter papers could also indicates that there was obvious different amount of blood was absorbed (Figure 3-17). As a result, with the adhesive and in situ crosslinked ability, this HPP-GC hydrogel can serve as antibleeding barrier to deal with irregular wound and bleeding of wound. Additionally, by these results, the applicability of this hydrogel to animal tissue is verified.
Figure 3-1 1H NMR analysis of glycol chitosan and HPP-GC.
Deacetylate
monomer Acetylate
monomer
Glycol Chitosan
HPP-GC
1 2
3-6
7
Phenol Conjugation
Figure 3-2 Uv-visible analysis of glycol chitosan, HPP and HPP-GC.
0 1 2 3 4 5
200 220 240 260 280 300 320 340
Absobance
Wavelength (nm) 276 nm
1
2
3
1. HPP-100 μg/ml 2. HPP-GC
3. Glycol Chitosan
Figure 3-3 Gelation of HPP-GC hydrogel.
Figure 3-4 Appearance of hydrogel with different DS. (A) low DS (~0.250 µmole/mg HPP-GC) (B) moderate DS (~ 0.370 µmole/mg HPP-GC) (C) high DS (> 0.534
µmole/mg HPP-GC)
(A) (B) (C)
Figure 3-5 Detachment stress of hydrogel with different DS. (n = 5, * and***
represent p < 0.05 and < 0.001 in comparison with sample of 0.378 DS) 0
0.270 0.347 0.368 0.378 0.400 0.534 0.623 0.794
Detachment Stress (kpa)
DS (μmole/mg HPP-GC)
*** ***
*** ***
*
Figure 3-6 Detachment stress of hydrogel with different concentration. (DS of sample:
0.368 μmole/mg HPP-GC) (n = 5, * represents p < 0.05 in comparison with 3%) 0
10 20 30 40 50 60 70 80 90 100
3% 2% 1%
Detachment Stress (kpa)
Concentration (wt%)
*
Figure 3-7 Storage modulus (G’) and viscous modulus (G’’) of HPP-GC hydrogel as a function of frequency (Hz) at 37 ℃.
1 10 100 1000 10000
0 0.2 0.4 0.6 0.8 1 1.2
Modulus G', G'' (Pa)
Frequency (Hz)
G' G''
Figure 3-8 Hydration of dry hydrogel with different DS. (n = 4, * represents p < 0.05 in comparison with sample of moderate DS)
0%
500%
1000%
1500%
2000%
2500%
0 5 10 15 20 25 30
Hydration (%)
Hours
Low DS Moderate DS Excessive DS
*
Figure 3-9 Degradation of hydrogels. (n = 5) 0%
20%
40%
60%
80%
100%
0 2 4 6 8 10 12 14 16
Mass Remains (%)
Days
Figure 3-10 Degradation of hydrogels after removing the water on surface. (n = 4) 40%
50%
60%
70%
80%
90%
100%
0 1 2 3 4 5 6 7
Mass Remains (%)
Weeks
Low DS Moderate DS Excessive DS
Figure 3-11 Cell viability of different concentration of extraction form hydrogel. (n = 5)
0%
20%
40%
60%
80%
100%
120%
100% 50% 10% blank control
Cell Viability (%)
Exraction concentration & Control
Figure 3-12 Amoxicillin released from hydrogel. (n = 4) 0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 50 100 150 200 250 300 350
Release Ratio (%)
Minutes
Figure 3-13 (A) E coli. and (B) S. epidermidis were the used. (1) 1000, (2) 200 and (3) 50 μg/ml of gentamycin were entrapped in hydrogel. (4) Commercial gauzes were immersed in 1000 μg/ml gentamycin and (5) 10 µl 1000 μg/ml gentamycin as control.
Figure 3-14 (A) E coli. and (B) S. epidermidis were the used. (1) 200, (2) 100 and (3) 50 μg/ml of gentamycin were entrapped in hydrogel. (4) HPP-GC hydrogel without
gentamycin (5) 10 µl 200 μg/ml gentamycin as control.
(1)
(2) (3)
(4) (5)
(1)
(2) (3)
(4) (5)
(A) (B)
Figure 3-15 Procedure of in vivo hemostatic experiment.
Figure 3-16 Blood loss from wound. (n = 3) 0.0
50.0 100.0 150.0 200.0 250.0 300.0
Control Applying HPP-GC hydrogel
Blood (mg)
Figure 3-17 Filter paper, which absorbed the blood from wound (A) Control (B) Applying HPP-GC without crosslinking (C) Applying hydrogel.
(A) (B) (C)
Table 3-1 Inhibition of zone of different concentration of gentamycin against E coli.
and S. epidermidis. (n = 4)
200 (µg/ml) 100µg/ml 50µg/ml E coli. 306% ± 41% 304% ± 38% 249% ± 6%
S. epidermidis 305% ± 42% 271% ± 32% 192% ± 22%
Chapter 4 Conclusion
In this study, an injectable phenol conjugated glycol chitosan hydrogel were successfully fabricated by rapid photochemical crosslinking. This hydrogel shows outstanding tissue adhesion and its ability of flexible application to irregular wound for its in-situ gelation. Because of these advantages and stable characteristics, this hydrogel is able to seal wound and sustain for adequate time that make it be a great sealant and wound dressing. With antibiotics, this hydrogel cannot only serve as ideal drug release system but also perform desirable antibacterial ability. In vivo hemostatic experiment, photocrosslinked HPP-GC hydrogels were successfully crosslink on bleeding site of mice liver, adhere to the tissue around wound, and arrested blood within 10 seconds that showed excellent hemostatic ability. This result demonstrates that the hydrogel indeed have ability to be utilized on animal tissue. All the merits mentioned above prove that photochemically crosslinked HPP-GC hydrogels are promising tissue adhesives, drug release system, hemostatic and antibacterial materials.
References
[1] N. Annabi, K. Yue, A. Tamayol, A. Khademhosseini, Elastic sealants for surgical applications, Eur J Pharm Biopharm 95(Pt A) (2015) 27-39.
[2] W.D. Spotnitz, S. Burks, Hemostats, sealants, and adhesives: components of the surgical toolbox, Transfusion 48(7) (2008) 1502-16.
[3] B. Mizrahi, C. Weldon, D.S. Kohane, Tissue Adhesives as Active Implants, 8 (2010) 39-56.
[4] J.C. Wheat, J.S. Wolf, Jr., Advances in bioadhesives, tissue sealants, and hemostatic agents, Urol Clin North Am 36(2) (2009) 265-75, x.
[5] T.B. Reece, T.S. Maxey, I.L. Kron, A prospectus on tissue adhesives, Am J Surg 182(2) (2001) 40s-44s.
[6] S. Ozawa, Patient blood management: use of topical hemostatic and sealant agents, AORN J 98(5) (2013) 461-78.
[7] P.T. Kumar, V.K. Lakshmanan, T.V. Anilkumar, C. Ramya, P. Reshmi, A.G.
Unnikrishnan, S.V. Nair, R. Jayakumar, Flexible and microporous chitosan hydrogel/nano ZnO composite bandages for wound dressing: in vitro and in vivo evaluation, ACS Appl Mater Interfaces 4(5) (2012) 2618-29.
[8] M. Kozicki, M. Kolodziejczyk, M. Szynkowska, A. Pawlaczyk, E. Lesniewska, A.
Matusiak, A. Adamus, A. Karolczak, Hydrogels made from chitosan and silver nitrate,
Carbohydr Polym 140 (2016) 74-87.
[9] M. Perez-Diaz, E. Alvarado-Gomez, M. Magana-Aquino, R. Sanchez-Sanchez, C.
Velasquillo, C. Gonzalez, A. Ganem-Rondero, G. Martinez-Castanon, N. Zavala-Alonso, F. Martinez-Gutierrez, Anti-biofilm activity of chitosan gels formulated with silver nanoparticles and their cytotoxic effect on human fibroblasts, Mater Sci Eng C Mater Biol Appl 60 (2016) 317-23.
[10] F. Wahid, J.J. Yin, D.D. Xue, H. Xue, Y.S. Lu, C. Zhong, L.Q. Chu, Synthesis and characterization of antibacterial carboxymethyl Chitosan/ZnO nanocomposite hydrogels, Int J Biol Macromol 88 (2016) 273-9.
[11] H.A.G. M. Radosevich, T. Burnoufa, Fibrin Sealant Scientific Rationale,Production M ethods, Properties, andCurrent Clinical Use, Vox Sang 72 (1997) 133-143.
[12] D.J. Meng-G Martin Lee, Applications of Fibrin Sealant in Surgery, Surgical Innovation 12 (2005) 203–213.
[13] G. Hidas, A. Kastin, M. Mullerad, J. Shental, B. Moskovitz, O. Nativ, Sutureless nephron-sparing surgery: use of albumin glutaraldehyde tissue adhesive (BioGlue), Urology 67(4) (2006) 697-700; discussion 700.
[14] M. Ryou, C.C. Thompson, Tissue Adhesives: A Review, Techniques in Gastrointestinal Endoscopy 8(1) (2006) 33-37.
[15] R. Cirocchi, E. Farinella, F. La Mura, L. Cattorini, B. Rossetti, D. Milani, P. Ricci, P. Covarelli, M. Coccetta, G. Noya, F. Sciannameo, Fibrin glue in the treatment of anal fistula: a systematic review, Ann Surg Innov Res 3 (2009) 12.
[16] J.A. Saldana-Cortes, F. Larios-Arceo, E. Prieto-Diaz-Chavez, E.P. De Buen, S.
Gonzalez-Mercado, A.S. Alvarez-Villasenor, M.R. Prieto-Aldape, C. Fuentes-Orozco, A. Gonzalez-Ojeda, Role of fibrin glue in the prevention of cervical leakage and strictures after esophageal reconstruction of caustic injury, World J Surg 33(5) (2009) 986-93.
[17] A. Lauto, D. Mawad, L.J.R. Foster, Adhesive biomaterials for tissue reconstruction, Journal of Chemical Technology & Biotechnology 83(4) (2008) 464-472.
[18] B.J. Kober, A.M. Scheule, V. Voth, N. Deschner, E. Schmid, G. Ziemer, Anaphylactic reaction after systemic application of aprotinin triggered by aprotinin-containing fibrin sealant, Anesth Analg 107(2) (2008) 406-9.
[19] S. Ghosh, J.D. Cabral, L.R. Hanton, S.C. Moratti, Strong poly(ethylene oxide) based gel adhesives via oxime cross-linking, Acta Biomater 29 (2016) 206-14.
[20] M.D. Thomas E. MacGillivray, Fibrin Sealants and Glues, J Card Surg 18 (2003) 480-485.
[21] L. Durham, D. Willatt, M. Yung, I. Jones, P. Stevenson, M. Ramadan, A method for preparation of fibrin glue, The Journal of Laryngology & Otology 101(11) (1987)
1182-1186.
[22] A.E. Ardis, Preparation of monomeric alkyl alpha-cyano-acrylates, Google Patents, 1949.
[23] H. Coover, F. Joyner, N. Shearer, T. Wicker, Chemistry and performance of cyanoacrylate adhesives, J Soc Plast Eng 15 (1959) 413-417.
[24] W. Furst, A. Banerjee, Release of glutaraldehyde from an albumin-glutaraldehyde tissue adhesive causes significant in vitro and in vivo toxicity, Ann Thorac Surg 79(5) (2005) 1522-8; discussion 1529.
[25] P.A. Leggat, D.R. Smith, U. Kedjarune, Surgical applications of cyanoacrylate adhesives: a review of toxicity, ANZ J Surg 77(4) (2007) 209-13.
[26] J.B.H. Pt. Stephen C. Woodward, John L. Cameron, , George Brandes, Edwin J.
Pulaski, , Fred Leonard, Histotoxicity of Cyanoacrylate Tissue Adhesive, Annals of Surgery 162 (1964) 114-122.
[27] B.J. Vote, M.J. Elder, Cyanoacrylate glue for corneal perforations: a description of a surgical technique and a review of the literature, Clinical & experimental ophthalmology 28(6) (2000) 437-442.
[28] K. Kimura, K. Sugiura, Adhesive composition, Google Patents, 1982.
[29] J.H. Ryu, S. Hong, H. Lee, Bio-inspired adhesive catechol-conjugated chitosan for biomedical applications: A mini review, Acta Biomater 27 (2015) 101-15.
[30] S.V. Gohil, S.B. Brittain, H.-M. Kan, H. Drissi, D.W. Rowe, L.S. Nair, Evaluation of enzymatically crosslinked injectable glycol chitosan hydrogel, J. Mater. Chem. B 3(27) (2015) 5511-5522.
[31] E. Lih, J.S. Lee, K.M. Park, K.D. Park, Rapidly curable chitosan-PEG hydrogels as tissue adhesives for hemostasis and wound healing, Acta Biomater 8(9) (2012) 3261-9.
[32] J.H. Ryu, Y. Lee, M.J. Do, S.D. Jo, J.S. Kim, B.S. Kim, G.I. Im, T.G. Park, H. Lee, Chitosan-g-hematin: enzyme-mimicking polymeric catalyst for adhesive hydrogels, Acta Biomater 10(1) (2014) 224-33.
[33] J.H. Ryu, Y. Lee, W.H. Kong, T.G. Kim, T.G. Park, H. Lee, Catechol-functionalized chitosan/pluronic hydrogels for tissue adhesives and hemostatic
[33] J.H. Ryu, Y. Lee, W.H. Kong, T.G. Kim, T.G. Park, H. Lee, Catechol-functionalized chitosan/pluronic hydrogels for tissue adhesives and hemostatic