Injectable melatonin-loaded carboxymethyl chitosan (CMCS)-based hydrogel accelerates wound healing by reducing inflammation and promoting angiogenesis and collagen deposition

doi:10.1016/j.jmst.2020.06.001

Abstract

Abstract:

Carboxymethyl chitosan (CMCS)-based hydrogels have antibacterial activity, and have shown the abilities of preventing wound infection, promoting cell proliferation, accelerating collagen deposition, and stimulating hyaluronic acid formation during wound healing. As a hormone produced by the pineal gland in humans and animals, melatonin promotes skin wound healing by regulating the release of inflammatory mediators and accelerating the proliferation and migration of cells, angiogenesis, and collagen deposition. However, the combined effects of CMCS and melatonin on wound healing remain unclear. Injectable CMCS-based hydrogels containing melatonin were prepared, and their healing effects were evaluated using a full-thickness cutaneous wound model in rats. Compared with the control and the hydrogel with no melatonin groups, the melatonin-loaded hydrogel significantly increased the percentage of wound closure, promoted the proliferation of granulation tissue and re-epithelialization, and accelerated collagen deposition. Additionally, the melatonin-loaded hydrogel promoted angiogenesis and vascular endothelial growth factor receptor protein expression and increased the expression of cyclooxygenase-2 and inducible nitric oxide synthase. The melatonin-loaded hydrogel also markedly increased the expression of collagen III, α-smooth muscle actin, and transforming growth factor-β1 proteins and reduced collagen I expression. These results suggest that the melatonin-loaded hydrogel promoted granulation tissue formation and accelerated wound healing by reducing inflammation and promoting angiogenesis and collagen deposition.

Key words: Carboxymethyl chitosan, Injectable melatonin-containing hydrogel, Wound healing, Antiinflammatory, Angiogenesis, Collagen deposition

Kai Chen, Changci Tong, Jinge Yang, Peifang Cong, Ying Liu, Xiuyun Shi, Xu Liu, Jun Zhang, Rufei Zou, Keshen Xiao, Yuyang Ni, Lei Xu, Mingxiao Hou, Hongxu Jin, Yunen Liu. Injectable melatonin-loaded carboxymethyl chitosan (CMCS)-based hydrogel accelerates wound healing by reducing inflammation and promoting angiogenesis and collagen deposition[J]. J. Mater. Sci. Technol., 2021, 63: 236-245.

Figures/Tables 7

Fig. 1. Storage modulus (G’) and loss modulus (G’’)-time dependence of hydrogel with and without melatonin.

Fig. 2. SEM images of hydrogels without (A) and with (B) melatonin. The morphology of CMCS/alginate hydrogels with and without melatonin was observed by scanning electron microscopy (SEM, inspect F50, FEI).

Fig. 3. Injectable melatonin-loaded hydrogel significantly increased the wound healing rate. (A) Body weight change during the treatment period. (B) Healing rate of full-thickness wounds in each treatment group. *p < 0.05, vs. control and hydrogel groups. (C) Photographs of wound closure in the control, hydrogel, and melatonin-loaded hydrogel groups from 0 to 12 days after surgery.

Fig. 4. Injectable melatonin-loaded hydrogel accelerated wound healing in rats. (A) Representative histopathological images in each group (HE staining). Scale bar =50 μm. (B) ImageJ software was used to measure the levels of re-epithelialization of full-thickness wounds in each group. *p < 0.05, vs. control and hydrogel groups.

Fig. 5. Injectable melatonin-loaded hydrogel accelerated collagen deposition during wound healing in rats. (A) Representative histopathological images in each group (modified Masson staining). Scale bar =50 μm. (B) ImageJ software was used to measure collagen deposition in full-thickness wounds in each group. *p < 0.05, vs. control and hydrogel groups.

Fig. 6. Injectable melatonin-loaded hydrogel reduced inflammation and accelerated angiogenesis and collagen deposition. (A) Representative Western blot images of VEGFR, COX-2, iNOS, α-SMA, collagen III, collagen I, TGF-β1, and GAPDH. (B-H) Quantitative analysis of VEGFR, COX-2, iNOS, α-SMA, collagen III, collagen I, and TGF-β1 expression relative to GAPDH. All of the experiments were repeated at least three times. The results are expressed as mean ± SEM. *p < 0.05, vs. control and hydrogel groups.

Fig. 7. Effects of injectable melatonin-loaded hydrogel on VEGFR, collagen III, and α-SMA expression. (A, C, E) Representative immunofluorescent staining images of VEGFR, collagen III, and α-SMA. (B, D, F) Semi-quantitative analysis of VEGFR, collagen III, and α-SMA. All of the experiments were repeated at least three times. The results are expressed as mean ± SEM. *p < 0.05, vs. control and hydrogel groups.

References 39

[1]	J. Skiveren, S. Bermark, K. LeBlanc, S. Baranoski, J. Wound Care 24 (2015) 388-392. DOI URL PMID
[2]	R. Jayakumar, M. Prabaharan, S.V. Nair, S. Tokura, H. Tamura, N. Selvamurugan, Prog. Mater. Sci. 55 (2010) 675-709. DOI URL
[3]	L. Upadhyaya, J. Singh, V. Agarwal, R.P. Tewari, Carbohydr. Polym. 91 (2013) 452-466. DOI URL PMID
[4]	X. Li, X. Kong, Z. Zhang, Int. J. Mol. Sci. 50 (2012) 1299-1305.
[5]	N. Joondan, S. Jhaumeer-Laulloo, P. Caumul, Microbiol. Res. 169 (2014) 675-685. DOI URL
[6]	T. Minagawa, Y. Okamura, Y. Shigemasa, S. Minami, Y. Okamoto, Carbohydr. Polym. 67 (2007) 640-644. DOI URL
[7]	X. Huang, Y. Zhang, X. Zhang, L. Xu, X. Chen, S. Wei, Mater. Sci. Eng. C 33 (2013) 4816-4824. DOI URL
[8]	X. Lv, Y. Liu, S. Song, C.C. Tong, X.Y. Shi, Y. Zhao, J. Zhang, M. Hou, Carbohydr. Polym. 205 (2019) 312-321. DOI URL PMID
[9]	R. Hardeland, Int. J. Mol. Sci. 20 (2019) 1223-1256. DOI URL
[10]	N.J. Prado, L. Ferder, W. Manucha, E.R. Diez, Curr. Hypertens. Rep. 20 (2018) 45-56. DOI URL PMID
[11]	P. Maria, L.P. Mónica, G.C. Antonio, M. Fernando, Int. J. Mol. Sci. 18 (2017) 865-878. DOI URL
[12]	T. Farias, R.I.D. Paixao, M.M. Cruz, R. deSa, J.J. Simao, V.J. Antraco, M.I.C. Alonso-Vale, Cells 8 (2019) E1041. DOI URL PMID
[13]	K. Kleszczy´nski, S. Zwicker, S. Tukaj, M. Kasperkiewicz, D. Zillikens, R. Wolf, T.W. Fischer, J. Pineal Res. 58 (2015) 117-126. DOI URL PMID
[14]	S. Goswami, C. Haldar, Br. J. Dermatol. 171 (2014) 1147-1155. DOI URL PMID
[15]	T. Tunali, G. Sener, A. Yarat, N. Emekli, Life Sci. 76 (2005) 1259-1265. DOI URL PMID
[16]	G. Soybir, C. Topuzlu, O. Odabas, K. Dolay, A. Bilir, F. Koksoy, Surg. Today 33 (2003) 896-901. DOI URL
[17]	K. Pugazhenthi, M. Kapoor, A.N. Clarkson, I. Hall, I. Appleton, J. Pineal. Res. 44 (2008) 387-396. DOI URL PMID
[18]	R. Song, L. Ren, H. Ma, R. Hu, J. Liu, Am. J. Transl. Res. 8 (2016) 4682-4693. URL PMID
[19]	F. Blažević, T. Milekić, M. Duvnjak Romić, M. Juretić, I. Pepić, J. Filipović-Grčić, J. Lovrić, A. Hafner, Carbohydr. Polym. 146 (2016) 445-454. DOI URL PMID
[20]	R. Fernanda, S.P.C.-F. de Abreu, Polímeros: Ciência e Tecnologia 15 (2005) 79-83. DOI URL
[21]	X. Lv, W. Zhang, Y. Liu, Y. Zhao, J. Zhang, M. Hou, Carbohydr. Polym. 198 (2018) 86-93. DOI URL PMID
[22]	X. Zhao, H. Wu, B. Guo, R. Dong, Y. Qiu, P.X. Ma, Biomaterials 122 (2017) 34-47. DOI URL PMID
[23]	C.C. Tong, Y. Liu, Y.B. Zhang, P.F. Cong, X.Y. Shi, Y. Liu, L. Shi, H.X. Jin, M.X. Hou, Exp. Biol. Med. (Maywood) 243 (2018) 934-944. DOI URL
[24]	Z. Atoufi, S.K. Kamrava, S.M. Davachi, M. Hassanabadi, S. Saeedi Garakani, R. Alizadeh, M. Farhadi, S. Tavakol, Z. Bagher, G. Hashemi Motlagh, Int. J. Mol. Sci. 139 (2019) 1168-1181.
[25]	Z. Naghizadeh, A. Karkhaneh, A. Khojasteh, J. Biomed. Mater. Res. A 106 (2018) 1932-1940. DOI URL PMID
[26]	M.D. Romic, M.S. Klaric, J. Lovric, I. Pepic, B. Cetina-Cizmek, J. Filipovic-Grcic, A. Hafner, Eur. J. Pharm. Biopharm. 107 (2016) 67-79. DOI URL PMID
[27]	Y. Hu, Z. Zhang, Y. Li, X. Ding, D. Li, C. Shen, F.J. Xu, Macromol. Rapid Commun. 39 (2018), e1800069. DOI URL PMID
[28]	W.G. Deng, S.T. Tang, H.P. Tseng, K.K. Wu, Blood 108 (2006) 518-524. DOI URL PMID
[29]	R. Fardid, A. Salajegheh, M.A. Mosleh-Shirazi, S. Sharifzadeh, M.A. Okhovat, M. Najafi, A. Rezaeyan, A. Abaszadeh, Cells 19 (2017) 324-331.
[30]	M. P. Ramírez-Fernández, J.L. Calvo-Guirado, Clin. Oral. Invesig. 17 (2013) 147-158.
[31]	Z. Jiang, B. Han, H. Li, Y. Yang, W. Liu, Carbohydr. Polym. 129 (2015) 1-8. DOI URL PMID
[32]	J. Chi, Z. Jiang, J. Qiao, Y. Peng, W. Liu, B. Han, Carbohydr. Polym. 15 (2019) 80-89.
[33]	N. Bulbuller, O. Dogru, H. Yekeler, Z. Cetinkaya, N. Ilhan, C. Kirkil, J. Surg. Res. 123 (2005) 3-7. DOI URL PMID
[34]	C. Akyuz, N.F. Yasar, O. Uzun, K.D. Peker, O. Sunamak, M. Duman, A.O. Sehirli, S. Yol, Singapore Med. J. 59 (2018) 545-549. DOI URL PMID
[35]	H. Jin, Z. Zhang, C. Wang, Q. Tang, J. Wang, X. Bai, Q. Wang, M. Nisar, N. Tian, Q. Wang, C. Mao, X. Zhang, X. Wang, Exp. Mol. Med. 50 (2018) 154-160.
[36]	K. Pugazhenthi, M. Kapoor, A.N. Clarkson, I. Hall, I. Appleton, J. Pineal Res. 44 (2008) 387-396. DOI URL PMID
[37]	E.A. Vorotelyak, L.A. Malchenko, O.S. Rogovaya, D.S. Lazarev, N.N. Butorina, V.Y. Brodsky, Bull. Exp. Biol.Med. 168 (2019) 242-246.
[38]	S. Bona, G. Rodrigues, A.J. Moreira, F. C. Di Naso, A.S. Dias, T. R. Da Silveira, C.A. Marroni, N.P. Marroni, JGH Open 2 (2018) 117-123. DOI URL PMID
[39]	I. Crespo, B.S. Miguel, A. Fernández, J.O. Urbina, J. González-Gallego, M.J. Tũnón, Transl. Res. 165 (2015) 346-357. DOI URL PMID