Sains Malaysiana 49(8)(2020): 1865-1874

http://dx.doi.org/10.17576/jsm-2020-4908-09

Effect of FGF-2 and PDGF-BB on a Co-Culture of Human Gingival Fibroblasts and Umbilical Vein Endothelial Cells

(Kesan FGF-2 dan PDGF-BB ke atas Ko-Kultur Sel Fibroblas Gingiva dan Sel Endotelial Vena Umbilikal Manusia)

NASAR UM MIN ALLAH¹, ZURAIRAH BERAHIM¹*, AZLINA AHMAD¹ & KANNAN THIRUMULU PONNURAJ^1,2

¹School of Dental Sciences, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan Darul Naim, Malaysia

²Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan Darul Naim, Malaysia

Received: 2 October 2019/Accepted: 27 March 2020

ABSTRACT

Gingival recession can be treated by root coverage procedure with tissue graft. The ideal gingiva graft should mimic the properties of the native gingiva. Gingival fibroblasts are main cells that reside in human gingiva, while the endothelial cells are the basis for blood vessel formation. The co-culture of these cells, will help in better understanding of gingival tissue regeneration. This study was aimed to determine the effects of fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-BB (PDGF-BB) on a co-culture of human gingival fibroblasts (HGFs) and human umbilical vein endothelial cells (HUVECs). In this in vitro experimental study, the medium for the establishment of monolayer and co-culture of these cells were first optimised. Then, the optimal concentrations of these growth factors were determined by assessing the cell viability using MTT assay. Next, to study the stimulatory effect of these growth factors, both HGF and HUVECs were co-cultured and gene expression analysis for fibroblast and angiogenic biomarkers was assessed using Real-Time RT-PCR. Cell viability assay showed that the effect of FGF-2 on HGF was dose-dependent and was optimum at a concentration of 5 ng mL^-1, while that of PDGF-BB on HUVEC was optimum at a concentration of 20 ng mL^-1. The stimulatory effect of FGF-2 and PDGF-BB was further supported by the Real-Time PCR results which showed that there is a significant expression of VIM, COL1A1, FN, CD31, VE-Cadherin, and vWFin the treatment group of both cells after three days of co-culture experiment, compared to control group. This study indicates a possible synergistic effect of FGF-2 and PDGF-BB growth factors in a co-culture of HGF and HUVEC leading to proangiogenic activity.

Keywords: Co-culture; FGF-2; HUVEC; PDGF-BB; tissue engineering

ABSTRAK

Penyusutan gingiva boleh dirawat menggunakan prosedur penutupan akar bersama geraf tisu. Geraf tisu gingiva yang unggul mestilah hampir menyerupai tisu gingiva yang asli. Fibroblas gingiva adalah sel utama yang terdapat dalam gingiva manusia, manakala sel endotelium adalah asas untuk pembentukan salur darah. Ko-kultur sel-sel ini akan membantu dalam pemahaman yang lebih baik mengenai pertumbuhan semula tisu gingiva. Kajian ini bertujuan untuk menentukan kesan faktor pertumbuhan fibroblas (FGF-2) dan faktor terbitan platlet (PDGF-BB) ke atas ko-kultur sel fibroblas gingiva (HGFs)  dan  sel endotelium vena umbilikus manusia (HUVECs). Di dalam uji kaji in vitro ini, media untuk memantapkan kultur sel satu lapisan dan ko-kultur kedua-dua jenis sel tersebut dioptimumkan terlebih dahulu. Berikutan itu kepekatan yang terbaik bagi kedua-dua faktor pertumbuhan ditentukan dengan menilai kebolehidupan sel menggunakan ujian MTT. Seterusnya, untuk mengkaji kesan perangsangan faktor pertumbuhan ini, kedua-dua HGFs dan HUVECs telah menjalani ko-kultur dan analisis pengekspresan gen untuk biopenanda fibroblas dan angiogenik telah dilakukan dengan masa nyata RT-PCR. Asai kebolehidupan sel menunjukkan bahawa kesan FGF-2 ke atas HGF adalah mengikut dos dan optimum pada kepekatan 5 ng mL^-1, manakala kesan PDGF-BB ke atas HUVEC adalah optimum pada kepekatan 20 ng mL^-1. Kesan rangsangan FGF-2 dan PDGF-BB turut disokong oleh keputusan masa nyata PCR yang menunjukkan pengekspresan VIM, COL1A1, FN, CD31, VE-Cadherin dan vWF yang signifikan  antara kumpulan yang dirawat untuk kedua-dua jenis sel selepas 3 hari menjalani ko-kultur berbanding kumpulan kawalan. Kajian ini menunjukkan bahawa terdapat kemungkinan kesan sinergistik antara faktor pertumbuhan  FGF-2 dan PDGF-BB di dalam ko-kultur sel HGF dan HUVEC yang mengarah kepada aktiviti proangiogenik.

Kata kunci: FGF-2; HUVEC; kejuruteraan tisu; ko-kultur; PDGF-BB

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*Corresponding author; email: zurairah@usm.my