Full Proceeding Paper INCUBATION OF PLATELET-RICH FIBRIN MATRIX WITH MESENCHYMAL STEM CELLS IMPROVES MATRIX STIFFNESS MIRTA H. REKSODIPUTRO 1 , GITA PRATAMA 2 , BUDI WIWEKO 2,5 , EVANTHI KUSUMAWARDHANI 3 , DENISWARI RAHAYU 3 , RAISA NAULI 3 , VALENCIA JANE MARTIN 3 , NORMALINA SANDORA 4,5* 1 Department of Otorhinolaryngology-Head and Neck, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo Hospital, Jakarta, Indonesia, 2 Department of Obstetrics and Gynaecology, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo Hospital, Jakarta, Indonesia, 3 Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia, 4 Faculty of Medicine, Universitas Riau, Pekanbaru, Indonesia, 5 Indonesian Medical Education and Research Institute, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia Email: normalina.sandora@gmail.com Received: 12 Oct 2019, Revised and Accepted: 17 Feb 2020 ABSTRACT Objective: The platelet-rich fibrin matrix (PRFM) is condensed platelet-rich plasma (PRP) and should possess a comparable biomechanical property to the transplanted sites, for them to be physiologically functional. The aim of this study was to investigate the effect of human bone marrow mesenchymal stem cells (hBM-MSC) or human umbilical cord mesenchymal stem cells (hUC-MSC) on the biomechanical properties of PRFM. Methods: PRFM was prepared by the gelation of PRP using 25 mmol CaCl2. The resulting coin-shaped PRFM pellets, 5 cm in diameter and 300 μm thick, were directly seeded with hUC-MSC or hBM-MSC at 2,000 cells cm -2 , followed by 24 h incubation at 37 °C in 5% (v/v) CO2 in air. The samples were then observed by scanning electron microscopy to determine the morphology of the matrix surface. The PRFM biomechanical properties were determined at a 10 mm. min -1 failure rate using an MCT 2150 universal testing machine (AandD Co. LTD). Results: SEM imaging of the surface of the PRFM seeded with hBM-MSC and hUC-MSC showed a cloudy layer that thickened over time. The elastin slope of the PRFM was significantly improved after seeding with hBM-MSC and hUC-MSC when compared with unseeded PRFM (p<0.002, R 2 =0.983). Both cell types elicited similar biomechanical effects (p=0.99). Conclusion: PRFM seeded with hBM-MSC or hUC-MSC showed significantly increased elasticity. Keywords: Platelet-rich fibrin matrix, Mesenchymal stem cells © 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) DOI: http://dx.doi.org/10.22159/ijap.2020.v12s3.39604 INTRODUCTION Wound healing after open injury or surgical procedures is challenging, and many materials and techniques have been evaluated for their ability to enhance and accelerate complete healing [1]. Normally, fibrin clot is formed in an open wound. It is highly elastic and viscous to stop bleeding and maintains hemostasis by forming a mesh that traps platelets [2]. Platelets are rich in growth factors, including platelet-derived growth factor (PDGF), transforming growth factor (TGF-b), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and insulin-like growth factor (IGF) [3, 4]. In addition, platelets release coagulation factors, serotonin, histamine, endostatin and hydrolytic enzymes [4]. Platelets can also be used to isolate a platelet-rich fibrin matrix (PRFM), a second-generation platelet fraction derived from platelet-rich plasma (PRP) obtained by centrifugation of whole blood. The PRP contains a 6-to 8-fold higher growth factor concentration than whole blood and can form the PRFM fibrin network following the addition of CaCl2 [5]. The growth factors in the PRFM are released slowly between 7 and 14 d. PRFM is used across the globe in various procedures, including orthopedic, cardiothoracic, vascular, general, and plastic surgeries. PRFM serves many different clinical needs due to its favorable promotion of healing, simple processing and ease of suturing to surrounding tissues. The natural fibrin framework in PRFM is thought to protect the growth factors from proteolysis. For clinical use, autologous sources for PRP are favored to eliminate disease transmission from donors. Naturally, the transplanted matrix will be invaded by the surrounding cells. While matrix stiffness is very important for its application that dictates the choice of biomaterial to replace a given tissue, nonetheless, until recently, no study has explored the biomechanical properties of PRFM after infiltrated with cells. Mechanical forces are important drivers of cellular growth and development, but mechanical overload leads to tissue injury [6]. A mechanical force or load that causes a material to change its shape is termed mechanical stress, symbolized by the Greek letter sigma (σ), and is defined as the force per unit area within a material (σ = F/A). Mechanical stress is similar to the concept of pressure and has the same units (N. m -2 ), where one N. m -2 is equal to one Pascal (Pa) of stress or pressure. PRFM, once fully polymerized, is quite stiff, with an elastic modulus of 937.3±314.6 kPa, stress at break value of 1476.0±526.3kPa, and an elongation at break value of 146.3±33.8%, as cited by Lucarelli and colleagues [7]. These values represent a stiffness equivalent to about half that of intact human skin [8]. Human BM-MSC seeded on decellularized tendon had increased matrix ultimate tensile stress after 7 d incubation with 6% strain [9]. The aim of the present study was to investigate the PRFM biomechanics, including the elastic modulus, ultimate tensile stress and ultimate tensile strain, before and after incubation of prepared PRFM with human bone marrow mesenchymal stem cells (hBM-MSC) or human umbilical cord mesenchymal stem cells (hUC-MSC). MATERIALS AND METHODS Upon receipt of ethical approval (no 78/UN2. F1. D/PA/PPM. SIPP.00.02/2018), four blood donors were screened for human immunodeficiency virus [10], hepatitis B, hepatitis C and cytomegalovirus infections. All four donors provided signed informed consent for participation in the study. Peripheral blood samples (8 ml) were withdrawn from each donor for the preparation of PRFM. Matrix preparation Platelet-rich plasma was isolated using RegenKit®-BCT (Regenlab), followed the manufacturer’s instructions. Briefly, the 8 ml of peripheral blood in the collection tube was centrifuged at 1500 g for 10 min, and the resultant plasma supernatant and platelet sediment were inverted and mixed 10 times. A 7 ml volume was removed, International Journal of Applied Pharmaceutics ISSN- 0975-7058 Vol 12, Special Issue 3, 2020