the effects of dental pulp stem cells on

The effects of dental pulp stem cells on bone regeneration in rat calvarialdefect model: Micro-computed tomography and histomorphometricanalysis

Asutay, Fatih, et al. Archives of oral biology 60.12 (2015): 1729-1735.

Pawornwan RittipakornOral and Maillofacial Surgury, Prince of Songkla University

Introduction• The use of human dental pulp stem cells (hDPSC) is a great

interest in bone reconstruction because they can be easily isolated and expanded in culture; moreover, they have shown in vitro and in vivo multipotential plasticity

• In previous study showed that human DPSC transplanted into large rat calvarial defects were capable of differentiating into osteogenic cells without any graft rejection.

(Gronthos, Mankani,Brahim, Robey, & Shi, 2000).(de Mendonça Costa, André, et al. 2008.)

IntroductionWhy do they not use other substitutional graft materials?It’s because some limitations

Autologous bone grafts• Limit availability of tissue• Donor site morbidity

Allografts/Xenografts• Risk of immunoreaction• Transmission of infections

Synthetic grafts• Do not have osteo-inductive properties

Introduction

(Mao et al., 2004 de lange et al.,2014)

HA and calcium phosphate are essential components within hard tissues and calcium phosphorus elements can be easily absorbed.

This study aimed to evaluate the osteogenic effects of DPSCs in

combination with HA/TCP in rat calvarialmodel using histomorphometry and µ-CT.

Material and methodsIsolation and characterization of human dental pulp stem cells

Animal and surgical procedure

Micro-computer tomography examination of samples

Histological examine of samples

Statistical analysis

Material and methods

Digested in collagenase type I (1% (w/v),(Sigma–Aldrich) to generate single cell suspensionssingle cell

suspensions

5 Human third molar obtained from adults 17-25 years old


15% (v/v) fetal bovine serum (FBS; Invitrogen/GIBCO)

Low glucose Dulbecco’s modified Eagle’s medium (LDMEM;Invitrogen/GIBCO, Paisley, UK)

100 IU/mL penicillin–100 mg/mL streptomycin (Invitrogen/GIBCO).

Material and methodsUndifferentiated SCs were subjected to flow cytometryanalyses.

Confirm DPSCs characterization.

Material and methodsImmunophenotyping the human DPSC was performed with antibodies against the following human antigens

CD45

CD73

CD90VCAM-1

HLA-GCD44

CD166

CD34

HA/TCPDPSCs



Micro-computer tomography examination of samplesHistological examine of samples


Material and methods15 adult female Wistar albino rats

(de Mendonca Costa et al., 2008).

Group 1 Unfilled(control).

Group 2 both defects filled with HA/TCP paste.

Group 3both defects filled

with HA/TCP + human DPSCs.

Bilateral calvarial bone defects, 4 mm in diameter

Material and methods• Evaluate bone mineral density (BMD) in the defect

area

(de Mendonca Costa et al., 2008).


10% formaldehyde for 5 days.

bone samples

10% formic acid for 10 days at room temperature.

6 randomly selected cross sections from each sample of 6 µm thickness were cut, mounted on slides

The total newly calcified area and total defect area were calculated for each group.

hematoxylin-eosin (HE).

Material and methodsStatistical analysis

• One-way analysis of variance (ANOVA) using SPSS 13.0 for Windows software was applied to all m-CT and histomorphometry data.

• Groups were compared using Tukey HSD test after testing by ANOVA.

• All data are presented as mean ` standard deviation (SD). A value of p < 0.05 was accepted as statistically significant

ResultsMicro-CT

ResultsHistomorphometry

ResultsHistomorphometry: 8 weeks post implantation.

ResultsFlow cytometry

CD45

CD73

CD90VCAM-1

HLA-GCD44

CD166

CD34

mesenchymal stem cell markers

DiscussionWhy this study use rat calvarias ?

(Schmitz & Hollinger, 1986). Therefore, rats were chosen as the experimental animal and bilateral defects allow fewer animals to be used. Moreover, the immune system of rats does not reject human DPSCs.

(de Mendonca Costa et al., 2008)

The bilateral rat calvarial full-thickness defect model was chosen in the current study. This technique has the advantages of reproducibility, high throughput and economic considerations

DiscussionWhy this study use DPSCs in third molar?

Wisdom tooth extraction is one of the most routine procedures in oral surgery. These cells can be promoted as a useful source, since they are acquired from extracted teeth. Results from various studies indicate that DPSCs have good potential to achieve the ultimate targets of tissue engineering with a suitable carrier platform .

(Kanafi, Ramesh, Gupta, & Bhonde, 2014; Kuo et al., 2015).

DiscussionHA/TCP

HA/TCP is currently used in clinical practice and its excellent mechanical, biodegradation and biocompatibility characteristics are previously highlighted (Bagher, Rajaei, & Shokrgozar, 2012)

A previous study has shown that HA/TCP is an absorbable and biocompatible material, which in combination with MSCs induces mineralization to a greater extent than scaffold alone or the control group (Vahabi et al., 2012)

ConclusionThe results of this study demonstrated the potential of DPSC-loaded-HA/TCP scaffolds to benefit of healing process.

In conclusion, the present study suggests that the treatment of craniofacial defects may be carried out with DPSCs combined with a suitable scaffold

Critique paper1. Archives of oral biology 60.12 (2015 ) impact

factor = 1.472. “The effects of dental pulp stem cells on bone

regeneration in rat calvarial defect model: Micro-computed tomography and histomorphometricanalysis “ specific and clearly

3. This study does not provide evidence of short (<8 weeks) or long-term (>8 weeks) effects of treatment.

4. No immunohistochemical evaluation was made of osteopontin, osteonectin, and osteocalcin, all of which play an important role in the mineralization and regeneration of bone tissue.

Thank you

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