title: Three-dimensional culture environment increases the efficacy of platelet rich plasma releasate in prompting skin fibroblast differentiation and extracellular matrix formation.
abstract: Platelet rich plasma clot- releasate (PRCR) shows significant influence on tissue regeneration in clinical trials. Although, the mechanism of PRCR effect on fibroblast differentiation has been studied on 2D culture system, a detailed investigation is needed to establish the role of PRCR in cell seeded in 3D scaffolds. Therefore, a study was conducted to evaluate the influence of PRCR in fibroblasts (DFB) differentiation and extracellular matrix formation on both 3D and 2D culture systems. Cell viability was measured using MTT assay and DFB differentiation was evaluated by determining the expression levels of nucleostamin and alpha smooth muscle actin (α-SMA), using indirect immunostaining and Western blotting. The expression levels of extracellular matrix genes (collagen-I, collagen-III, fibronectin and laminin) and focal adhesion formation gene (integrin beta-1) were measured using Real-time PCR. The PRCR at 10% showed significant effect on cells viability compared with 5% and 20% in both culture environments. The decrease in the expression levels of nucleostamin and the increase in α-SMA signify the DFB differentiation to myofibroblast-like cells that was prominently greater in 3D compared to 2D culture. In 3D culture systems, the total collage production, expression levels of the extracellular matrix gene and the focal adhesion gene were increased significantly compared to 2D culture. In conclusion, 3D culture environments enhances the proliferative and differentiation effects of PRCR on DFB, thereby potentially increases the efficacy of DFB for future tissue engineering clinical application.
authors.affiliation: 1. Department of Molecular Medicine, Faculty of Medicine, University of Malaya. 50603 Kuala Lumpur, Malaysia.
authors.forename: Hussin A
authors.initials: HA
authors.lastname: Rothan
authors.affiliation: 2. Department of Biomedical Engineering, Faculty of Engineering, University of Malaya. 50603 Kuala Lumpur, Malaysia.
authors.forename: Ivan
authors.initials: I
authors.lastname: Djordjevic
authors.affiliation: 1. Department of Molecular Medicine, Faculty of Medicine, University of Malaya. 50603 Kuala Lumpur, Malaysia.
authors.forename: Hirbod
authors.initials: H
authors.lastname: Bahrani
authors.affiliation: 3. Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Mohammadjavad
authors.initials: M
authors.lastname: Paydar
authors.affiliation: 2. Department of Biomedical Engineering, Faculty of Engineering, University of Malaya. 50603 Kuala Lumpur, Malaysia.
authors.forename: Fatimah
authors.initials: F
authors.lastname: Ibrahim
authors.affiliation: 4. Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Noorsaadah
authors.initials: N
authors.lastname: Abd Rahmanh
authors.affiliation: 1. Department of Molecular Medicine, Faculty of Medicine, University of Malaya. 50603 Kuala Lumpur, Malaysia.
authors.forename: Rohana
authors.initials: R
authors.lastname: Yusof
mesh_terms: D015153:Blotting, Western; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D008855:Microscopy, Electron, Scanning; D053657:Platelet-Rich Plasma / Q000166:cytology* / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D012867:Skin
keywords: PCL-CA scaffold.; Platelet rich plasma; growth factors; human skin fibroblast