Bone alkaline phosphatase and osteocalcin expression of rat's Gingival mesenchymal stem cells cultured in platelet-rich fibrin for bone remodeling (in vitro study)

Abstract

Objective:The aim of this study was to analyze the osteogenic differentiation of rat GMSCs cultured in PRF for bone remodeling.Materials and Methods:GMSCs were isolated from the lower gingival tissue of four healthy, 250 g, 1-month old, male rats (Rattus norvegicus) cut into small fragments, cultured for 2 weeks, and subsequently passaged every 4–5 days. GMSCs isolated in passage 3 were characterized by CD34, CD45, CD44, CD73, CD90, and CD105 using fluorescein isothiocyanate immunocytochemistry (ICC) examination. GMSCs in passage 3–5 cultured in five M24 plates (N = 108; n = 6/group) for 7, 14, and 21 days with three different mediums as follows: Control (−) group: α-Modified Eagle Medium; Control (+) group: High-dose glucose Dulbecco's Modified Eagle's Medium (DMEM-HG) + osteogenic medium; and treatment group: DMEM-HG + osteogenic medium + PRF. GMSCs were osteogenic differentiation cultured in vitro in three different mediums by bone alkaline phosphatase (BALP) and osteocalcin (OSC) marker using ICC monoclonal antibody.Statistical Analysis Used:The one-way analysis of variance was performed (P < 0.05) based on Shapiro–Wilk and Levene's tests (P > 0.05).Results:GMSCs were shown to present + CD44, +CD73, +CD90, +CD105 and − CD34, − and CD45 expression as MSCs markers. The treatment group showed the highest BALP expression (16.00 ± 1.732) on day 7, while OSC expression (13.67 ± 2.309) on day 21 showed the statistically significant difference between groups (P < 0.05).Conclusion:GMSCs cultured in PRF demonstrated potential osteogenic differentiation ability capable of accelerating in vitro bone remodeling by enhancing BALP and OSC expression.

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

“…BSP-I in bone tissue regulates cell-to-cell, cell-to-cell matrix interactions and cartilage-to-cell transition to produce compact bone in the repair process of fractures in osteoclast adhesion in bone matrix. miRNA regulates the secretion and expression of BSP-I such as miR181a / b / c / d. BSP-I is strongly expressed in the later stages of osteogenesis from Day 14 to Day 28 and widely secreted by SPCs that regulate osteogenic differentiation ability [16,22,23].…”

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] .…”

“…Mesenchymal Stem Cells have an important role to improve innovative technologies for tissue engineering such as to regenerate or replace damaged, defect or missing tissues by in vitro cell manipulation and extracellular niche design [4] . Stem cell and tissue engineering therapies are expected to be the regenerative medicine strategies in dentistry that provide a novel capability to restore various tissues in orofacial region such as alveolar bone or condylar cartilage of temporomandibular joint [5] . The oral tissues, which are easily accessed by dentists, are a rich source of MSCs.…”

“…The ability of GMSCs' osteogenic differentiation can be accelerated by Platelets Rich Fibrin (PRF) administration in the osteogenic culture medium [5] . PRF contains with abundant and various beneficial growth factor for GMSCs to differentiate and proliferate optimally.…”

“…PRF as a natural biomaterial also serves and acts as a bio-scaffold to support GMSCs. 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] .…”