Injectable multifunctional hydrogel containing Sphingosine 1-phosphate and human acellular amniotic membrane for skin wound healing

Document Type : Original Article


1 School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran

2 Tissue Engineering and Stem Cells Research Center, Shahroud University of Medical Sciences, Shahroud, Iran

3 Department of Tissue Engineering, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran

4 Department of Hematology, School of Allied Medical Sciences, Shahroud University of Medical Sciences, Shahroud, Iran

5 Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

6 Department of Mechanical Engineering, New Jersey Institute of Technology, New Jersey, United States of America (USA)

7 Health Technology Incubator Center, Shahroud University of Medical Sciences, Shahroud, Iran

8 Sexual Health and Fertility Research Center, Shahroud University of Medical Sciences, Shahroud, Iran



Objective(s): The skin serves as the main defense barrier, protecting against injuries, and preventing infection and water loss. Consequently, wound healing and skin regeneration are crucial aspects of wound management. A novel hydrogel scaffold was developed by incorporating carboxymethyl cellulose (CMC) and gelatin (Gel) hydrogels cross-linked with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) containing Sphingosine 1-phosphate (S1P). This hydrogel is applied topically to treat acute wounds and is covered with a human acellular amniotic membrane (hAAM) as a secondary dressing. 
Materials and Methods: The scaffold was subjected to in vitro cell viability, red blood cell hemolysis, blood clotting index, and in vivo assays. Real-time PCR was implemented to verify the expression of genes involved in skin wounds. The physical and chemical properties of the scaffolds were also tested using weight loss, swelling ratio, scanning electron microscopy (SEM), Fourier transform infrared (FTIR), and mechanical tensile analysis. 
Results: The synthetic scaffold is biocompatible as evidenced by the high percentage of 3T3 cell viability (127%) after 72 hr. Additionally, excellent hemocompatibility with a low hemolytic effect (2.26%) was observed. Our in vivo wound healing assay demonstrated that CMC/Gel/S1P/hAAM wound dressing led to faster wound healing in treated rats compared to the control group over 14.
Also, the mechanical tests showed that the amniotic membrane and the hAAM had very different Young’s modulus and elongation at break values.
Conclusion: This study demonstrates the effectiveness of the CMC/Gel/EDC hydrogel with S1P as a wound dressing. Additionally, hAAM exhibits excellent characteristics as a protective layer for the treatment of acute wounds.


Main Subjects

1. Zhou H, Wang L, Zhang C, Hu J, Chen J, Du W, et al. Feasibility of repairing full-thickness skin defects by iPSC-derived epithelial stem cells seeded on a human acellular amniotic membrane. 
Stem Cell Res Ther 2019;10:1-13.
2. Yu R, Zhang H, Guo B. Conductive biomaterials as bioactive wound dressing for wound healing and skin tissue engineering. Nanomicro Lett 2022;14:1-46.
3. Brumberg V, Astrelina T, Malivanova T, Samoilov A. Modern wound dressings: Hydrogel dressings. Biomedicines 2021;9:1-15.
4. RHEINIWALD J. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975;6:331-343.
5. Zhang J, Liu Z, Li Y, You Q, Yang J, Jin Y, et al. FGF-2-induced human amniotic mesenchymal stem cells seeded on a human acellular amniotic membrane scaffold accelerated tendon-to-bone healing in a rabbit extra-articular model. Stem Cells Int 2020;2020:1-14.
6. Hartmeier PR, Pham NB, Velankar KY, Issa F, Giannoukakis N, Meng WS. Hydrogel dressings for chronic wound healing in diabetes: beyond hydration. J Pharm Drug Deliv Res 2021;10:1-21.
7. Khan S, Anwar N. Gelatin/carboxymethyl cellulose based stimuli-responsive hydrogels for controlled delivery of 5-fluorouracil, development, in vitro characterization, in vivo safety and bioavailability evaluation. Carbohydr Polym 2021;257:1-13.
8. Azarifar M, Ghanbarzadeh B, Khiabani MS, Basti AA, Abdulkhani A, Noshirvani N, et al. The optimization of gelatin-CMC based active films containing chitin nanofiber and Trachyspermum ammi essential oil by response surface methodology. Carbohydr Polym 2019;208:457-468.
9. Samadian H, Zamiri S, Ehterami A, Farzamfar S, Vaez A, Khastar H, et al. Electrospun cellulose acetate/gelatin nanofibrous wound dressing containing berberine for diabetic foot ulcer healing: In vitro and in vivo studies. Sci Rep 2020;10:1-12.
10. Camci-Unal G, Cuttica D, Annabi N, Demarchi D, Khademhosseini A. Synthesis and characterization of hybrid hyaluronic acid-gelatin hydrogels. Biomacromolecules 2013;14:1085-1092.
11. Kanikireddy V, Varaprasad K, Jayaramudu T, Karthikeyan C, Sadiku R. Carboxymethyl cellulose-based materials for infection control and wound healing: A review. Int J Biol Macromol 2020;164:963-975.
12. Lai J-Y, Li Y-T. Functional assessment of cross-linked porous gelatin hydrogels for bioengineered cell sheet carriers. Biomacromolecules 2010;11:1387-1397.
13. Nazarnezhada S, Abbaszadeh-Goudarzi G, Samadian H, Khaksari M, Ghatar JM, Khastar H, et al. Alginate hydrogel containing hydrogen sulfide as the functional wound dressing material: In vitro and in vivo study. Int J Biol Macromol 2020;164:3323-3331.
14. Hla T, Lee M-J, Ancellin N, Liu CH, Thangada S, Thompson BD, et al. Sphingosine-1-phosphate: extracellular mediator or intracellular second messenger? Biochem Pharmacol 1999;58:201-207.
15. Vogler R, Sauer B, Kim D-S, Schäfer-Korting M, Kleuser B. Sphingosine-1-phosphate and its potentially paradoxical effects on critical parameters of cutaneous wound healing. J Invest Dermatol 2003;120:693-700.
16. Kawanabe T, Kawakami T, Yatomi Y, Shimada S, Soma Y. Sphingosine 1-phosphate accelerates wound healing in diabetic mice. 
J Dermatol Sci 2007;48:53-60.
17. Lee H, Goetzl EJ, An S. Lysophosphatidic acid and sphingosine 1-phosphate stimulate endothelial cell wound healing. 
Am J Physiol Cell Physiol 2000;278:612-618.
18. Aoki M, Aoki H, Mukhopadhyay P, Tsuge T, Yamamoto H, Matsumoto NM, et al. Sphingosine-1-phosphate facilitates skin wound healing by increasing angiogenesis and inflammatory cell recruitment with less scar formation. 
Int J Mol Sci  2019;20:3381:1-16.
19. Wacker BK, Alford SK, Scott EA, Thakur MD, Longmore GD, Elbert DL. Endothelial cell migration on RGD-peptide-containing PEG hydrogels in the presence of sphingosine 1-phosphate. Biophys J 2008;94:273-285.
20. Murakami M, Saito T, Tabata Y. Controlled release of sphingosine-1-phosphate agonist with gelatin hydrogels for macrophage recruitment. Acta Biomater 2014;10:4723-4729.
21. Kim YH, Tabata Y. Recruitment of mesenchymal stem cells and macrophages by dual release of stromal cell‐derived factor‐1 and a macrophage recruitment agent enhances wound closure. 
J Biomed Mater Res A 2016;104:942-956.
22. Philippe A. Les pansements secondaires. Soins 2016;802:51-53.
23. Loeffelbein DJ, Rohleder NH, Eddicks M, Baumann CM, Stoeckelhuber M, Wolff K-D, et al. Evaluation of human amniotic membrane as a wound dressing for split-thickness skin-graft donor sites. Biomed Res Int 2014;2014:1-12.
24. Moravvej H, Memariani H, Memariani M, Kabir-Salmani M, Shoae-Hassani A, Abdollahimajd F. Evaluation of fibroblast viability seeded on acellular human amniotic membrane. Biomed Res Int 2021; 2021:1-6.
25. Sabapathy V, Sundaram B, VM S, Mankuzhy P, Kumar S. Human Wharton’s jelly mesenchymal stem cells plasticity augments scar-free skin wound healing with hair growth. PLoS One 2014;9:1-10.
26. Xiao S, Xiao C, Miao Y, Wang J, Chen R, Fan Z, et al. Human acellular amniotic membrane incorporating exosomes from adipose-derived mesenchymal stem cells promotes diabetic wound healing. Stem Cell Res Ther 2021;12:1-16.
27. Xue S-L, Liu K, Parolini O, Wang Y, Deng L, Huang Y-C. Human acellular amniotic membrane implantation for lower third nasal reconstruction: a promising therapy to promote wound healing. Int J Burns Trauma 2018;6:1-8.
28. Hashemi SS, Mohammadi AA, Moshirabadi K, Zardosht M. Effect of dermal fibroblasts and mesenchymal stem cells seeded on an amniotic membrane scaffold in skin regeneration: A case series. 
J Cosmet Dermatol 2021;20:4040-4047.
29. Wilshaw S-P, Kearney JN, Fisher J, Ingham E. Production of an acellular amniotic membrane matrix for use in tissue engineering. Tissue Eng 2006;12:2117-2129.
30. Tang K, Wu J, Xiong Z, Ji Y, Sun T, Guo X. Human acellular amniotic membrane: a potential osteoinductive biomaterial for bone regeneration. J Biomater Appl 2018;32:754-764.
31. Jahit I, Nazmi N, Isa M, Sarbon N. Preparation and physical
properties of gelatin/CMC/chitosan composite films as affected
by drying temperature. Int Food Res J  2015;23:1068-1074.
32. Nazmi N, Isa M, Sarbon N. Preparation and characterization of chicken skin gelatin/CMC composite film as compared to bovine gelatin film. Food Biosci 2017;19:149-155.
33. Sweah ZJ, editor A swelling study in different PH and mechanical properties of biodegradable films based on Pluronic F-127/Poly-Vinyl Alcohol. Mater Sci Forum 2020;1002:389-398.
34. Sze Huei GO, Muniyandy S, Sathasivam T, Veeramachineni AK, Janarthanan P. Iron cross-linked carboxymethyl cellulose–gelatin complex coacervate beads for sustained drug delivery. Chem pap 2016;70:243-252.
35. Singh D, Rai V, Agrawal DK. Regulation of collagen I and collagen III in tissue injury and regeneration. Cardiol Cardiovasc Med 2023;7:1-18.
36. Roberts RE, Cavalcante-Silva J, Kineman RD, Koh TJ. Liver is a primary source of insulin-like growth factor-1 in skin wound healing. J Endocrinol 2022;252:59-70.
37. Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic‐Canic M. Growth factors and cytokines in wound healing. Wound Repair Regen 2008;16:585-601.
38. Gaudry M, Brégerie O, Andrieu Vr, El Benna J, Pocidalo M-A, Hakim J. Intracellular pool of vascular endothelial growth factor in human neutrophils. Blood 1997;90:4153-4161.
39. Islam S, Chuensirikulchai K, Khummuang S, Keratibumrungpong T, Kongtawelert P, Kasinrerk W, et al. Accumulation of versican facilitates wound healing: implication of its initial ADAMTS-cleavage site. Matrix Biol 2020;87:77-93.
40. Garoufalia Z, Papadopetraki A, Karatza E, Vardakostas D, Philippou A, Kouraklis G, et al. Insulin-like growth factor-I and wound healing, a potential answer to non-healing wounds: A systematic review of the literature and future perspectives. Biomed Rep 2021;15:1-5.
41. Ramirez H, Patel SB, Pastar I. The role of TGFβ signaling in wound epithelialization. Adv Wound Care 2014;3:482-491.
42. Johnson KE, Wilgus TA. Vascular endothelial growth factor and angiogenesis in the regulation of cutaneous wound repair. Adv Wound Care 2014;3:647-661.
43. Zhao X, Wu H, Guo B, Dong R, Qiu Y, Ma PX. Antibacterial anti-oxidant electroactive injectable hydrogel as self-healing wound dressing with hemostasis and adhesiveness for cutaneous wound healing. Biomaterials 2017;122:34-47.
44. Lim NSJ, Sham A, Chee SML, Chan C, Raghunath M. Combination of ciclopirox olamine and sphingosine‐1‐phosphate as granulation enhancer in diabetic wounds. 
Wound Repair Regen 2016;24:795-809.
45. Sanluis-Verdes A, Yebra-Pimentel Vilar MT, García-Barreiro JJ, García-Camba M, Ibáñez JS, Doménech N, et al. Production of an acellular matrix from amniotic membrane for the synthesis of a human skin equivalent. Cell Tissue Bank 2015;16:411-423.
46. Taghiabadi E, Beiki B, Aghdami N, Bajouri A. Amniotic membrane seeded fetal fibroblasts as skin substitute for wound regeneration. Methods Mol Biol 2019;1879:211-219.
47. Jantrawut P, Bunrueangtha J, Suerthong J, Kantrong N. Fabrication and characterization of low methoxyl pectin/gelatin/carboxymethyl cellulose absorbent hydrogel film for wound dressing applications. Materials 2019;12:1-11.
48. Ohta S, Mitsuhashi K, Chandel AKS, Qi P, Nakamura N, Nakamichi A, et al. Silver-loaded carboxymethyl cellulose nonwoven sheet with controlled counterions for infected wound healing. Carbohydr Polym 2022;286:1-29.