Osteogenic potential of gingival stromal progenitor cells cultured in platelet rich fibrin is predicted by core-binding factor subunit-α1/Sox9 expression ratio (in vitro)

Abstract

Background: Alveolar bone defect regeneration has long been problematic in the field of dentistry. Gingival stromal progenitor cells (GSPCs) offer a promising solution for alveolar bone regeneration. In order to optimally differentiate and proliferate progenitor cells, growth factors (GFs) are required. Platelet rich fibrin (PRF) has many GFs and can be easily manufactured. Core-binding factor subunit-α1 (CBF-α1) constitutes a well-known osteogenic differentiation transcription factor in SPCs. Sox9, as a chondrogenic transcription factor, interacts and inhibits CBF-α1, but its precise role in direct in vitro osteogenesis remains unknown. GSPCs cultured in vitro in PRF to optimally stimulate osteogenic differentiation has been largely overlooked. The aim of this study was to analyze GSPCs cultured in PRF osteogenic differentiation predicted by CBF-α1/Sox9. Methods: This study used a true experimental with post-test only control group design and random sampling. GPSCs isolated from the lower gingiva of four healthy, 250-gram, 1-month old, male Wistar rats ( Rattus Novergicus) were cultured for two weeks, passaged every 4-5 days. GSPCs in passage 3-5 were cultured in five M24 plates (N=108; n=6/group) for Day 7, Day 14, and Day 21 in three different mediums (control negative group: αModified Eagle Medium; control positive group: High Glucose-Dulbecco’s Modified Eagle Medium (DMEM-HG) + osteogenic medium; Treatment group: DMEM-HG + osteogenic medium + PRF). CBF-α1 and Sox9 were examined with ICC monoclonal antibody. A one-way ANOVA continued with Tukey HSD test (p<0.05) based on Kolmogorov–Smirnov and Levene's tests (p>0.05) was performed. Results: The treatment group showed the highest CBF-α1/Sox9 ratio (16.00±3.000/14.33±2.517) on Day 7, while the lowest CBF-α1/Sox9 ratio (3.33±1.528/3.67±1.155) occurred in the control negative group on Day 21, with significant difference between the groups (p<0.05). Conclusion: GSPCs cultured in PRF had potential osteogenic differentiation ability predicted by the CBF-α1/sox9 ratio.

<i>In vitro</i> bone sialoprotein-I expression in combined gingival stromal cells and platelet rich fibrin during osteogenic differentiation

“…Decreased expression of BSP-I will cause osteoporosis. BSP-I is also expressed in osteoclasts and osteoblasts responsible for bone remodeling in bone homeostasis [19,22,24].…”

Erken Osteojenik Farklılaşma (In Vitro) Süresince Wistar Sıçanlarda (Rattus novergicus) Gingival Medicinal Signaling Hücrelere Post Trombositten Zengin Fibrin Uygulamasının Agrekan Ekspresyonuna Etkisi

“…In minimizing the suffering of animal model used rodent anesthesia with an intramuscular (IM) injection at the dosage of 0.05-0.1 mL/10 g body weight, they were ketamine, xylazine, acepromazine, and a sterile isotonic saline from Sigma Aldrich, USA. It followed the method of Nugraha et al [5] , GMSCs was passaged every 4-5 days also based on the culture method of Nugraha et al [5] in Gingival Mesenchymal Stem Cells (MSCs) [6] . The GMSCs in passage 3-5 were cultured in five M24 plates from Sigma-Aldrich with (N=54) and (n=6) per group until 7 th , 14 th , an 21 st day in three different culture mediums, which were control negative group, control positive group and treatment group (see below for details) [5,6] .…”

“…Next, the whole blood (6 mL) was aspirated using a 10 mL disposable syringe and inserted in a non-coagulant vacutainer tube then centrifuged at 3000 rpm/min for 10 min (Kubota, Tokyo, Japan). Thus, PRF obtained mince and it was inserted into each culture plate of the treatment group [5,6] .…”

“…PRF increases the early indicator of osteogenic differentiation such as Bone Alkaline Phosphatase (BALP) and Runt-related Transcription Factor 2 (RUNX2) /Core-Binding Factor Subunit Alpha-1 (CBF-alpha-1) in 7 th day and late marker of osteogenic differentiation such as Osteocalcin in 21 st day [5,6] . Our previous study showed that PRF administration in GMSCs' osteogenic culture medium decreases Sox9 expression which is the master gene of chondrogenic differentiation [6] . During the early stage of osteogenic differentiation and pre-osteoblast, some chondrogenic differentiation markers play have important role.…”

“…Even though the osteogenic and chondrogenic differentiation are considered as two separate processes during endochondral bone formation. The previous study mentioned that there is a correlation between them as a continuous developmental lineage which defines the biological process [6,7] . Furthermore, Aggrecan as the early chondrogenic differentiation marker also has an important role during the osteogenic differentiation for osteoblast formation.…”