UNIVERSIDADE ESTADUAL PAULISTA

“JÚLIO DE MESQUITA FILHO”

Instituto de Ciência e Tecnologia

Campus de São José dos Campos

ORIGINAL ARTICLE DOI: https://doi.org/10.4322/bds.2023.e3847

Braz Dent Sci 2023 Oct/Dec; 26 (4): e3847

Potential eﬀect of platelet rich ﬁbrin prepared under diﬀerent

centrifugation protocols on stem cells from the apical papilla

O potencial efeito da ﬁbrina rica em plaquetas preparada sob diferentes protocolos de centrifugação nas células

tronco da papila apical

Lina Samir SHALABY

1,2

, Sahar SHAWKAT

, Iman FATHY



1 - Newgiza University, School of Dentistry, Oral Biology Department, Giza, Egypt.

2 - Cairo University, Faculty of Dentistry, Oral Biology Department, Cairo, Egypt.

3 - Ain Shams University, Faculty of Dentistry, Oral Biology Department, Cairo, Egypt.

How to cite: Shalaby LS, Shawkat S, Fathy I. Potential effect of platelet rich brin prepared under different centrifugation protocols on

stem cells from the apical papilla. Braz Dent Sci. 2023;26(4):e3847. https://doi.org/10.4322/bds.2023.e3847

ABSTRACT

Objectives: The present work was designed to evaluate the proliferation and differentiation potential of stem

cells from the apical papilla (SCAP) seeded along with platelet rich brin (PRF) scaffolds prepared under two

different centrifugation protocols. Materials and Methods: Standard and advanced PRF protocols were used.

Cells were divided into 4 groups: negative control, positive control, standard (L-PRF) and advanced (A-PRF)

groups. Cell count and cell viability assays were carried out to assess the proliferation capacity. Alizarin red S

(ARS) stain, Alkaline phosphatase (ALP) activity and Receptor activator of nuclear factor-kappa B ligand (RANKL)

immunouorescence staining were used to evaluate the osteogenic potential in the differentiated cells. Results:

Both types of platelet rich brin increased the cell count, cell viability with no cytotoxicity that was reected on

increased proliferation and differentiation in terms of the performed tests. Conclusion: A-PRF group showed

signicant increase in proliferation and differentiation potentials compared to L-PRF group.

KEYWORDS

Alkaline phosphatase; Centrifugation; Platelet rich brin; RANKL ligand; Stem cells.

RESUMO

Objetivo: Este estudo objetivou avaliar o potencial proliferativo e de diferenciação das células tronco da papila

cultivadas conjuntamente com brina rica em plaquetas (PRF) preparados sob dois protocolos de centrifugação

distintos. Material e Métodos: Protocolos padrão e avançado de PRF foram utilizados. As células foram

divididas em 4 grupos: controle negativo, controle positivo, padrão (L-PRF) e avançado (A-PRF). A contagem

de células e ensaio de viabilidade foram realizados para vericar a capacidade proliferativa. Coloração

vermelho de alizarina S, atividade de fosfatase alcalina e imunouorescência para o receptor ativador do fator

nuclear kappa-B (RANKL) foram utilizados para avaliar o potencial osteogênico e de diferenciação celular.

Resultados: Ambos os tipos de PRF aumentaram o número de células, viabilidade celular sem toxicidade o que

reetiu no aumento da proliferação e diferenciação de acordo com os testes realizados. Conclusão: O grupo

A-PRF aumentou signicativamente a proliferação e diferenciação comparado com o grupo L-PRF.

PALAVRAS-CHAVE

Células-tronco; Centrifugação; Fibrina rica em plaquetas; Fosfatase alcalina; RANKL.

Braz Dent Sci 2023 Oct/Dec; 26 (4): e3847

Shalaby et al.

Diﬀerent centrifugation protocols of PRF on SCAP

Shalaby et al.

Diﬀerent centrifugation protocols of PRF on SCAP

INTRODUCTION

Dental trauma and caries both of which are

prevalent dental problems that leads to pulp

exposure, infection and necrosis. Under these

conditions, endodontic treatment is the most

common clinical treatment through which the

dental pulp is removed. In pediatric dentistry

and endodontics, the management of immature

permanent teeth remains a challenge. As once

the tooth lost its vitality, the root development

halts, leaving behind a weak tooth that unable to

withstand the normal physiological masticatory

forces. The consequences will be a high rate

of root fracture with poor prognosis in the

medium to the long term. Most of the studies

revealed that, teeth were lost in the rst 10 years

following trauma, despite being endodontically

treated in more than 50% of cases [1]. Tissue

engineering has been a topic of extensive

research over the past decade, involves a triad

(stem cells, scaffold and growth factors), aiming

to form a new tissue to restore the anatomy

and function as the original one. Regenerative

endodontic techniques (RETs) have been

recently introduced with the ultimate goal

of stimulating further root development and

thickening of root dentinal walls [2]. Stem-cell

biology has become an important eld for the

understanding of tissue regeneration. A variety

of dental MSCs have been isolated including stem

cells from bone marrow (BMSCs), dental pulp

(DPSCs), exfoliated deciduous teeth (SHED),

periodontal ligament stem cells (PDLSCs),

dental follicle precursor cells (DFPCs), stem cells

from apical papilla (SCAP) and gingiva- derived

mesenchymal stem cells (GMSCs). SCAP

are particularly relevant and significant in

regenerative endodontic procedures since they

are the cells suggested to populate the root

canal area following regenerative endodontics.

Hence, they should be targeted for maximum

benet of stem cell research and translational

medicine [3].

Platelets, isolated from a peripheral blood,

showed the ability of concentrated platelets to

provide 6–8 times supraphysiological doses of

growth factors. Earlier studies, demonstrated

the ability of several key growth factors, found

in platelets, to stimulate the recruitment and

differentiation of mesenchymal stem cells and

other target cells which markedly support tissue

regeneration [4]. The Platelet rich plasma

(PRP) was introduced to the world of dentistry

in 1997 by Whitman and co-workers. It was

suggested that PRP can attract stem cells from

surrounding periapical tissues. PRP was referred

as a first-generation platelet concentrate,

followed by the platelet rich brin (PRF) as a

second-generation platelet concentrate that was

developed rst by Choukroun et al. in 2001 and

the third- generation, called concentrated growth

factors (CGF) that was developed and described

in 2006 by Sacco [5].

Since PRF introduction in 2001, various

protocols utilizing the low-speed centrifugation

concept for PRF preparation, such as advanced

platelet rich fibrin (A-PRF) and injectable

platelet rich brin (i-PRF), have been proposed

with different amounts of growth factors

and other biomolecules necessary for tissue

regeneration and wound healing. The alteration

in centrifugation parameters, such as speed

and time, was showed to have a direct impact

on growth factors release within the PRF

matrix [4,5]. However, reference data about

potential effect of centrifugation parameters

modication on PRF matrix and its impact on

tissues regeneration still not properly covered

and needs further research. Hence, the present

work was designed to evaluate the proliferation

and differentiation potential of the stem cells

from apical papilla seeded along with platelet

rich brin scaffolds prepared under two different

centrifugation protocols which are standard and

advanced/ Low speed centrifugation concept

(LSCC) protocols.

MATERIALS AND METHODS

Stem cells isolation, characterization and culture

This study was approved by the research

ethics committees Faculty of Dentistry Cairo

University, number 19515. All experiments were

performed in accordance with the committee

guidelines of the stem cells experiment.

The current study was performed by using

human sound impacted third molars (n=12)

collected from healthy young patients

(18 to 21 years old) with incompletely formed

roots. The extracted teeth were immediately

rinsed with sterile PBS (PH 7.4) and transferred

in transfer solution (PBS + 10000 U penicillin/

streptomycin + preservative media) until being

transferred to the laboratory for further work.

Braz Dent Sci 2023 Oct/Dec; 26 (4): e3847

Shalaby et al.

Diﬀerent centrifugation protocols of PRF on SCAP

Shalaby et al.

Diﬀerent centrifugation protocols of PRF on SCAP

Patient recruitment

The current study was performed by

using human sound impacted third molars

(n=12) collected from healthy young patients

(18 to 21 years old) in the Oral and Maxillofacial

Surgery Department, Ain Shams University.

▪ Patient’s inclusion criteria were:

- Age ranges from 18 to 21 years old

- Sex, males and females

- Medically t and well

- Has the capacity to give an informed

consent

- Partially sound or fully impacted wisdom

teeth with incompletely formed roots

▪ Patient’s exclusion criteria were:

- Radiographic examination showed

completely formed roots

- Carious wisdom teeth

- Strategic tooth or not causing harm

during or upon its eruption

▪ Patients were informed about the nature of

the study, and they were asked to sign an

informed consent.

▪ The blood sample was collected later from

another adult healthy volunteer with normal

blood picture.

Isolation of SCAP

Apical papilla (soft tissue loosely attached

to the apices of incompletely formed roots) was

detached with a pair of forceps. Tissue obtained

from apical papillae of all teeth were mixed all

together. SCAP were isolated from this tissue

using enzymatic digestion method.

Characterization of SCAP

Characterization was done using flow

cytometric analysis. The immunoassaying

stains [CD45-PC5 (Phycoerythrin Cynin),

CD44-FITC (uorescein Isothiocyanate) and

CD73-PE (phycoerythrin)] were used to label

the isolated cells.

Culture Protocol of SCAP

The cells were cultured in T-75 culture

ask, in complete culture media (Dulbecco’s

modified Eagle’s medium (DMEM) (Gibco,

Invitrogen) containing 10% fetal bovine serum

(FBS) (Gibco) and 1% penicillin/streptomycin

(Gibco). Flask was incubated at 37 °C in an

atmosphere of 5% CO2. The media was changed

every 24 hours. When the cells reached 80%

confluency, the cells were harvested and

passaged. Cells from the 3rd passage were used

in the following assays.

PREPARATION TECHNIQUES OF PRF

A sample of blood was collected from an adult

healthy volunteer with normal blood picture in a

plain glass tube “without anticoagulants”, then

immediately centrifuged at room temperature

(20-25ºC) to prepare the PRF gel according to the

selected protocols in the current study design [6]

as follows:

▪ Standard Leucocytes PRF (L-PRF), sterile

plain glass-based vacuum tubes (10 ml;

2700 rpm for 12 minutes).

▪ Advanced PRF (A-PRF), sterile plain

glass-based vacuum tubes (10 ml; 1500

rpm for 14 minutes).

SEEDING OF THE SCAP AND GROUPING:

Seeding of the SCAP in the different groups

was done according to the study design for

7 days for osteogenic differentiation [7,8], into

4 different groups as follows;

1. Negative control (NC): SCAP + conventional

culture media*

2. Positive control (PC): SCAP + osteogenic

culture media (OM)**

3. Leukocyte PRF (L-PRF): SCAP + OM + L-PRF

4. Advanced PRF (A-PRF): SCAP + OM + A-PRF

* Conventional culture media: (Dulbecco’s

modied Eagle’s medium (DMEM) (Gibco,

Invitrogen) containing 10% fetal bovine

serum (FBS) (Gibco) and 1% penicillin/

streptomycin (Gibco).

** Osteogenic culture media (OM): low

glucose DMEM containing 10% fetal

bovine serum (FBS) and 1% penicillin/

streptomycin, 50ng/ml Ascorbic acid

and 10mM β-Glycerophosphate (Gibco,

Thermosientic, Germany).

Braz Dent Sci 2023 Oct/Dec; 26 (4): e3847

Shalaby et al.

Diﬀerent centrifugation protocols of PRF on SCAP

Shalaby et al.

Diﬀerent centrifugation protocols of PRF on SCAP

ASSESSMENT METHODS

Proliferation assessment

Cell counting ‘Trypan blue’ by Hemocytometer

The cells were counted by automated

hemocytometer to estimate the total number

of cells according to the following protocol [7].

The samples were loaded as suspension on

hemocytometer and observed. Nonviable cells

were stained; however, the viable cells did not

take up the stain.

Assessment of cell viability by cell cytotoxicity and

proliferation assay (MTT)

MTT assay was used to monitor the response

and health of cells in culture. The optical density

was assayed by spectrophotometer. The cell

cytotoxicity assay was performed according to

manufacturer instructions using the Vybrant®

Methyl-tetra-zolium (MTT; 3-(4, 5 dimethylthiazol-

2-yl)-2, 5-diphenyltetrazolium bromide) Cell

Proliferation Assay Kit, cat no: M6494 (Thermo

Fisher, Germany).

Differentiation assessment

Assessment of inorganic deposition using Alizarin

red S (ARS) stain

ARS stain was used to assess the deposition of

inorganic content in differentiated SCAP, according

to the following protocol [9]. The microscopic

examination was performed by LABOMED

microscope with suitable magnication, cat no:

9126000; USA. Then the area fraction percentage

was calculated. The staining intensity was scored

according to a fourtier system: 0, no staining; 1+,

weak; 2+, moderate; and 3+, strong. In brief,

the H-score of each sample was calculated as

the sum of each intensity (0-3) multiplied by the

percentage of positive cells (0-100%). The score

ranged from 0-300. The median value of H-score

was calculated.

Assessment of Alkaline phosphatase (ALP) activity

The Alkaline phosphatase activity was

measured according to manufacturer instructions

in supernatant of differentiated SCAP using an

ALP assay kit (Sigma)® with para-nitrophenyl

phosphate (p-NPP) as substrate. 100 µL of

each p-nitrophenol standard and 50 µL of

each test sample was added to a 96-well plate.

After incubation at 37 ºC the absorbance was

measured immediately at 405 nm using a on a

spectrophotometer using an ELx800 absorbance

microplate reader (ELx 800; Bio-Tek Instruments

Inc., Winooski, VT, USA). A standard curve of

absorbance versus concentration was generated

and used to determine the ALP activity (U/L).

Assessment of RANKL expression in SCAP using

immunouorescence staining

Cells from different groups were harvested

and cultured for 24 hours on cover slips and

examined for the expression of Receptor activator

of nuclear factor kappa-Β ligand (RANKL) for

SCAP using specic polyclonal antibody [10].

The microscopic examination was performed

by LABOMED Fluorescence microscope with

suitable magnication, cat no: 9126000; USA.

The immunouorescence (IF) staining intensity

was scored according to a fourtier system: 0,

no staining; 1+, weak; 2+, moderate; and 3+,

strong. In brief, the H-score of each sample

was calculated as the sum of each intensity

(0-3) multiplied by the percentage of positive

cells (0-100%). The score ranged from 0-300.

The median value of H-score was calculated.

STATISTICAL ANALYSIS METHOD

All experiments were performed in triplicate.

All assays were repeated three times to ensure

reproducibility. Data were analysed using the

GraphPad prism version 9.3.1. (San Diego, US)

and used also for graph plotting. Each value

represents the mean ± standard deviation (SD).

Statistical significance was determined using

one-way analysis of variance (ANOVA) followed

by multiple comparison Tukey’s post-hoc test

to explore differences between multiple groups

means while controlling the experiment-wise

error rate. P-value: level of significance,

a p-value ≥ 0.05: means statistically insignicant,

p-value < 0.05 mean statistically significant,

p-value < 0.01: high statistically signicant.

RESULTS

Cell characterization (Flow cytometry)

Characterization of the isolated cells via

immunoassaying with stem cell markers CD44,

CD73 versus CD45 using ow cytometry revealed

that most of the cells showed double bright