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BioImpacts. 2022;12: 219-231.
doi: 10.34172/bi.2021.43
  Abstract View: 176
  PDF Download: 55

Original Research

Evaluation of in vitro fibroblast migration by electrospun triple-layered PU-CA/gelatin.PRGF/PU-CA scaffold using an AAVS1 targeted EGFP reporter cell line

Forough Shams 1 ORCID logo, Hamideh Moravvej 2 ORCID logo, Simzar Hosseinzadeh 3,4, Bahram Kazemi 1, Masoumrh Rajabibazl 5, Azam Rahimpour 3,4* ORCID logo

1 Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Skin Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3 Medical Nano-Technology and Tissue Engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
5 Department of Clinical Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
*Corresponding Author: Email: rahimpour@sbmu.ac.ir

Abstract

Introduction: Migration of fibroblast cells in wound areas is a critical aspect of the wound healing process. Employment of enhanced green fluorescent protein (EGFP) labeled fibroblast cells facilitates real-time monitoring and functional evaluation of these cells in both in vitro and in vivo settings. Plasma rich in growth factor (PRGF) is a potent accelerator of wound healing; therefore, in this study, a novel method to fabricate an electrospun bioactive scaffold containing PRGF was employed to induce in vitro cell proliferation and migration.
Methods: First, the EGFP reporter gene was integrated into the AAVS1 locus of fibroblast cells using CRISPR/Cas9 system. Then, PRGF was obtained from platelet-rich plasma, and a multi-layered scaffold was fabricated using polyurethane-cellulose acetate (PU-CA) fibers as the outer layers and PRGF-containing gelatin fibers were located in the internal layer like a central strip. Scanning electron microscopy (SEM), tensile, water contact angle, and FTIR tests were performed to assess the characteristics of the scaffolds. The EGFP targeted cells were cultured on scaffolds with or without PRGF to investigate their viability, toxicity, and migration pattern in response to the release profile.
Results: Fluorescence images showed that the number of migrating cells on scaffold containing PRGF was more significant than PU-CA scaffold up to day 6. Increased expression of SGPL1, DDR2, and VEGF genes was also observed on the scaffold containing PRGF compared to PU-CA using real-time polymerase chain reaction (PCR) analysis with around 3-, 2-, and 2-fold enhancement, respectively.
Conclusion: The current scaffold provides the appropriate template for cell attachment and migration. In addition, the present results highlight the potential of reporter gene targeting for the in vitro analysis of biological processes such as migration.
Keywords: Gene targeting, Fibroblast migration, Polyurethane-cellulose acetate, Platelet-rich plasma, AAVS1, Enhanced green fluorescent protein
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Submitted: 01 Jan 2021
Revision: 10 May 2021
Accepted: 22 May 2021
ePublished: 30 Aug 2021
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