An in situ forming gelatin-based hydrogel loaded with soluble amniotic membrane promotes full-thickness wound regeneration in rats

Document Type : Original Article

Authors

1 Trauma Research Center, Aja University of Medical Sciences, Tehran, Iran

2 Department of Tissue Engineering, Faculty of Basic Sciences and Advanced Technologies in Medicine, Royan Institute, ACECR, Tehran, Iran

3 Iranian Tissue Bank and Research Center, Gene, Cell and Tissue Institute, Tehran university of Medical Sciences, Tehran, Iran

Abstract

Objective(s): Early effective treatment and appropriate coverage are vital for full-thickness wounds. Amnion membrane-derived products have recently emerged in tissue engineering. However, the optimal concentration, carrier for controlled release, and handling have remained challenges. This study aims to develop and optimize an in situ forming, amniotic-based hydrogel for wound healing.
Materials and Methods: Here, a composite matrix was fabricated with gelatin hydrogel modified with methacrylate functional group conjugated (GelMA) and keratose (wt.1%), loaded with mesenchymal stem cells (MSCs, 1×105 cell/ml) and optimized soluble amniotic membrane (SAM, 0.5 mg/ml). The physicochemical properties of the final subject were evaluated in vitro and in vivo environments. 
Results: The results of the in vitro assay demonstrated that conjugation of the methacryloyl group with gelatin resulted in the formation of GelMA hydrogel (26.7±1.2 kPa) with higher mechanical stability. Modification of GelMA with a glycosaminoglycan sulfate (Keratose) increased controlled delivery of SAM (47.3% vs. 84.3%).  Metabolic activity (93%) and proliferation (21.2 ± 1.5 µg/ml) of MSCs encapsulated in hydrogel improved by incorporation of SAM (0.5 mg/ml). Furthermore, the migration of fibroblasts was facilitated in the scratched assay by SAM (0.5 mg/ml)/MSCs (1×105 cell/ml) conditioned medium. The GelMA hydrogel groupes revealed regeneration of full-thickness skin defects in rats after 3 weeks due to the high angiogenesis (6.3 ± 0.3), cell migration, and epithelialization. 
Conclusion: The results indicated in situ forming and tunable GelMA hydrogels containing SAM and MSCs could be used as efficient substrates for full-thickness wound regeneration.

Keywords

Main Subjects

References

1. Shukla SK, Sharma AK, Gupta V, Yashavarddhan MH. Pharmacological control of inflammation in wound healing. J Tissue Viability  2019; 28: 218-222.
2.    Geanaliu-Nicolae RE, Andronescu E. Blended natural support materials—collagen based hydrogels used in biomedicine. Mater (Basel) 2020;13:1–31. 
3.    Sorg H, Tilkorn DJ, Hager S, Hauser J, Mirastschijski U. Skin wound healing: An update on the current knowledge and concepts. Eur Surg Res 2017;58:81–94.
4.    Stamm A, Reimers K, Strauß S, Vogt P, Scheper T, Pepelanova I. In vitro wound healing assays–state of the art. BioNanoMat 2016;17:79-87. 
5.    Zheng Y, Zheng S, Fan X, Li L, Xiao Y, Luo P, et al. Amniotic epithelial cells accelerate diabetic wound healing by modulating inflammation and promoting neovascularization. Stem Cells Int 2018; 25: 142-187.
6.    Gushiken LF, Beserra FP, Bastos JK, Jackson CJ, Pellizzon CH. Cutaneous wound healing: An update from physiopathology to current therapies. Life 2021; 11: 665-679.
7.    Wilkinson HN, Hardman MJ. Wound healing: Cellular mechanisms and pathological outcomes. Open Biol 2020; 10: 200223.
8.    Eming SA, Martin P, Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation. Sci Transl Med 2014; 265: p.265sr6-265sr6.
9.    Lipsky BA, Aragón-Sánchez J, Diggle M, Embil J, Kono S, Lavery L, et al. IWGDF guidance on the diagnosis and management of foot infections in persons with diabetes. Diabetes Metab Res Rev 2016; 32: 45–74. 
10.    Boateng J, Catanzano O. Advanced therapeutic dressings for effective wound healing - a review. J Pharm Sci 2015;104: 3653–3680. 
11.    Goula A, Gkioka V, Michalopoulos E, Katsimpoulas M, Noutsias M, Sarri EF, et al. Advanced therapy medicinal products challenges and perspectives in regenerative medicine. J Clin Med Res 2020;12:780–786. 
12.    Balasubramani V, Jeganathan R, Kumar SD. Numerical analysis of porosity effects on mechanical properties for tissue engineering scaffold. Mater Today Proc 2023; 30:124.
13.    Park JW, Hwang SR, Yoon IS. Advanced growth factor delivery systems in wound management and skin regeneration. Molecules 2017; 22.8: 1259
14.    Koizumi N, Inatomi T, Sotozono C, Fullwood NJ, Quantock AJ, Kinoshita S. Growth factor mRNA and protein in preserved human amniotic membrane. Curr Eye Res 2000; 20:173–177. 
15.    Murphy SV, Skardal A, Song L, Sutton K, Haug R, Mack DL, et al. Solubilized amnion membrane hyaluronic acid hydrogel accelerates full-thickness wound healing. Stem Cells Transl Med 2017; 6: 2020–2032. 
16.    Murri MS, Moshirfar M, Birdsong OC, Ronquillo YC, Ding Y, Hoopes PC. Amniotic membrane extract and eye drops: A review of literature and clinical application. Clin Ophthalmol 2018;12:1105–1112. 
17.    Elkhenany H, El-Derby A, Abd Elkodous M, Salah RA, Lotfy A, El-Badri N. Applications of the amniotic membrane in tissue engineering and regeneration: the hundred-year challenge. Stem Cell Res Ther 2022;13:1-9. 
18.    Asl NS, Nejat F, Mohammadi P, Nekoukar A, Hesam S, Ebrahimi M, et al. Amniotic membrane extract eye drop promotes limbal stem cell proliferation and corneal epithelium healing. Cell J 2019; 20: 459–468. 
19.    Momeni, Maryam, et al. In vitro and in vivo investigation of a novel amniotic‐based chitosan dressing for wound healing. Wound Repair Regen 2018; 26: 87–101.
20.    Bhattacharjee M, Escobar Ivirico JL, Kan HM, Shah S, Otsuka T, Bordett R, et al. Injectable amnion hydrogel-mediated delivery of adipose-derived stem cells for osteoarthritis treatment. Proc Natl Acad Sci U S A 2022; 119:e2120968119.
21.    Bayat S, Zabihi AR, Farzad SA, Movaffagh J, Hashemi E, Arabzadeh S, et al. Evaluation of debridement effects of bromelain-loaded sodium alginate nanoparticles incorporated into chitosan hydrogel in animal models. Iran J Basic Med Sci 2021; 24:1404–1412.
22.    Soheilifar MH, Masoudi-Khoram N. Wound dressings incorporating microRNAs: Innovative therapy for diabetic wound treatment. Iran J Basic Med Sci 2022; 26:1042–1044.
23.    Samadian H, Vahidi R, Salehi M, Hosseini-Nave H, Shahabi A, Zanganeh S, et al. Hydrogel nanocomposite based on alginate/zeolite for burn wound healing: In vitro and in vivo study. Iran J Basic Med Sci 2023; 26:708–716. 
24.    Shin H, Olsen BD, Khademhosseini A. The mechanical properties and cytotoxicity of cell-laden double-network hydrogels based on photocrosslinkable gelatin and gellan gum biomacromolecules. Biomater 2012; 33:3143–3152. 
25.    Xiao S, Zhao T, Wang J, Wang C, Du J, Ying L, et al. Gelatin methacrylate (GelMA)-based hydrogels for cell transplantation: an effective strategy for tissue engineering. Stem Cell Rev Rep 2019;15: 664–679. 
26.    Nguyen AH, McKinney J, Miller T, Bongiorno T, McDevitt TC. Gelatin methacrylate microspheres for controlled growth factor release. Acta Biomater 2015; 13:101–110.
27.    Öztürk E, Arlov Ø, Aksel S, Li L, Ornitz DM, Skjåk‐Bræk G, Zenobi‐Wong M. Sulfated hydrogel matrices direct mitogenicity and maintenance of chondrocyte phenotype through activation of FGF signaling. Adv Funct Mater. 2016; 26: 3649-3662.
28.    Zhang Q, Chang C, Qian C, Xiao W, Zhu H, Guo J, et al. Photo-crosslinkable amniotic membrane hydrogel for skin defect healing. Acta Biomater 2021; 125:197–207. 
29.    Ledford B, Barron C, Van Dyke M, He JQ. Keratose hydrogel for tissue regeneration and drug delivery. Semin Cell Dev Biol 2022; 128:145–153. 
30.    Song Y, Nagai N, Saijo S, Kaji H, Nishizawa M, Abe T. In situ formation of injectable chitosan-gelatin hydrogels through double crosslinking for sustained intraocular drug delivery. Mater Sci Eng C Mater Biol Appl 2018; 88:1–12. 
31.    Keriquel V, Guillemot F, Arnault I, Guillotin B, Miraux S, Amédée J, et al. In vivo bioprinting for computer-and robotic-assisted medical intervention: preliminary study in mice. Biofabrication 2010; 2:014101.
32.    Hong IS. Enhancing stem cell-based therapeutic potential by combining various bioengineering technologies. Front Cell Dev Biol 2022; 10: 901661.
33.    Luo G, Cheng W, He W, Wang X, Tan J, Fitzgerald M, et al. Promotion of cutaneous wound healing by local application of mesenchymal stem cells derived from human umbilical cord blood. Wound Repair Regen 2010; 18: 506–513. 
34.    Zhao X, Lang Q, Yildirimer L, Lin ZY, Cui W, Annabi N, et al. Photocrosslinkable gelatin hydrogel for epidermal tissue engineering. Adv Healthc Mater 2016; 5: 108–118. 
35.    Brinkman WT, Nagapudi K, Thomas BS, Chaikof EL. Photo-cross-linking of type I collagen gels in the presence of smooth muscle cells: Mechanical properties, cell viability, and function. Int J Biol Macromol 2003; 4: 890–895. 
36.    Ågren MS, Taplin CJ, Woessner JF, Eaglstein WH, Mertz PM. Collagenase in wound healing: Effect of wound age and type. J Invest Dermatol 1992; 99: 709–714.
37.    Zhou X, Zhu W, Nowicki M, Miao S, Cui H, Holmes B, et al. 3D bioprinting a cell-laden bone matrix for breast cancer metastasis study. ACS Appl Mater Interfaces 2016; 8:30017–30026.
38.    Fonseca DFS, Costa PC, Almeida IF, Dias-Pereira P, Correia-Sá I, Bastos V, et al. Swellable gelatin methacryloyl microneedles for extraction of interstitial skin fluid toward minimally invasive monitoring of urea. Macromol Biosci 2020; 20:2000195.
39.    Baei P, Daemi H, Mostafaei F, Azam Sayahpour F, Baharvand H, Baghaban Eslaminejad M. A tough polysaccharide-based cell-laden double-network hydrogel promotes articular cartilage tissue regeneration in rabbits. Chem Eng J 2021; 418:129277.
40.    Kim MH, Wu WH, Choi JH, Kim JH, Hong SH, Jun JH, et al. Conditioned medium from the three-dimensional culture of human umbilical cord perivascular cells accelerate the migration and proliferation of human keratinocyte and fibroblast. J Biomater Sci Polym Ed 2018; 29:1066–1080. 
41.    Hur W, Lee HY, Min HS, Wufuer M, Lee C won, Hur JA, et al. Regeneration of full-thickness skin defects by differentiated adipose-derived stem cells into fibroblast-like cells by fibroblast-conditioned medium. Stem Cell Res Ther 2017; 8:1-3.
42.    Maarof M, Mh Busra MF, Lokanathan Y, Bt Hj Idrus R, Rajab NF, Chowdhury SR. Safety and efficacy of dermal fibroblast conditioned medium (DFCM) fortified collagen hydrogel as acellular 3D skin patch. Drug Deliv Transl Res 2019; 9: 144–161. 
43.    Armstrong DG. The efficacy of Apligraf in the treatment of diabetic foot ulcers. Plast Reconstr Surg 2006;117:152S-157S. 
44.    Marston WA. Dermagraft®, a bioengineered human dermal equivalent for the treatment of chronic nonhealing diabetic foot ulcer. Expert Rev Med Devices 2004; 1:21–31. 
45.    Alamouty MA, Asl NS, Safari A, Ebrahimi M, Daemi H. Fabrication of cell-laden AME-loaded collagen-based hydrogel promotes fibroblast proliferation and wound healing in vitro. Cell J 2023; 25:255–263. 
46.    Tehrani FA, Ahmadiani A, Niknejad H. The effects of preservation procedures on antibacterial property of amniotic membrane. Cryobiology 2013; 67:293–298. 
47.    Haugh AM, Witt JG, Hauch A, Darden M, Parker G, Ellsworth WA, et al. Amnion membrane in diabetic foot wounds: A meta-analysis. Plast Reconstr Surg - Glob Open 2017; 5:e1302. 
48.    Chopra, Aditi, and Betsy S. Thomas. Amniotic membrane: A novel material for regeneration and repair. J Biomim Biomater Tissue Eng 18.1: 2013; 1-8.
49.    Taghiabadi E, Nasri S, Shafieyan S, Firoozinezhad SJ, Aghdami N. Fabrication and characterization of spongy denuded amniotic membrane based scaffold for tissue engineering. Cell J 2015;16:476–487.
50.    Heydary HA, Karamian E, Poorazizi E, Khandan A, Heydaripour J. A novel nano-fiber of iranian gum tragacanth-polyvinyl alcohol/nanoclay composite for wound healing applications. Procedia Mater Sci 2015; 11:176–182. 
51.    Doozandeh Z, Saber-Samandari S, Khandan A. Preparation of novel arabic gum-c6h9no biopolymer as a bedsore for wound care application. Acta Med Iran 2020; 58:520–530. 
52.    Karimi M, Asefnejad A, Aflaki D, Surendar A, Baharifar H, Saber-Samandari S, et al. Fabrication of shapeless scaffolds reinforced with baghdadite-magnetite nanoparticles using a 3D printer and freeze-drying technique. J Mater Res Technol 2021;14: 3070–3079. 
53.    Stanton AE, Tong X, Yang F. Extracellular matrix type modulates mechanotransduction of stem cells. Acta Biomater 2019; 96: 310–320. 
54.    de Guzman RC, Saul JM, Ellenburg MD, Merrill MR, Coan HB, Smith TL, et al. Bone regeneration with BMP-2 delivered from keratose scaffolds. Biomaterials 2013; 34:1644–1656.
55.    Vojdani Z, Babaei A, Vasaghi A, Habibagahi M, Talaei-Khozani T. The effect of amniotic membrane extract on umbilical cord blood mesenchymal stem cell expansion: Is there any need to save the amniotic membrane besides the umbilical cord blood? Iran J Basic Med Sci 2016; 19: 89–96.
56.    Arno AI, Amini-Nik S, Blit PH, Al-Shehab M, Belo C, Herer E, Tien CH, et al. Human Wharton’s jelly mesenchymal stem cells promote skin wound healing through paracrine signaling. Stem Cell Res Ther 2014; 5: 1–13.
57.    Dinh T, Braunagel S, Rosenblum BI. Growth Factors in Wound Healing: The present and the future?.” Clin Podiatr Med Surg  2015; 32.1: 109-119.
58.    Rustad KC, Wong VW, Sorkin M, Glotzbach JP, Major MR, Rajadas J, et al. Enhancement of mesenchymal stem cell angiogenic capacity and stemness by a biomimetic hydrogel scaffold. Biomaterials 2012; 33:80–90. 
59.    Yu A, Matsuda Y, Takeda A, Uchinuma E, Kuroyanagi Y. Effect of EGF and bFGF on fibroblast proliferation and angiogenic cytokine production from cultured dermal substitutes. J Biomater Sci Polym Ed 2012; 23: 1315–1324. 
Iranian Journal of Basic Medical Sciences
Volume 27, Issue 8
August 2024
Pages 1005-1014

Files

Share

How to cite

Statistics