A study on differentiation potency of adult stem cells from pulp, periodontal ligament, and dental follicle to osteoblast

Joong-Kyou Lee; Jae-Hoon Lee

doi:10.5125/jkaoms.2010.36.1.7

Journal List > J Korean Assoc Oral Maxillofac Surg > v.36(1) > 1032448

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Lee and Lee: A study on differentiation potency of adult stem cells from pulp, periodontal ligament, and dental follicle to osteoblast

Original Article

J Korean Assoc Oral Maxillofac Surg 2010;36(1):7-15.

Published online: 15 January 2010

DOI: https://doi.org/10.5125/jkaoms.2010.36.1.7

A study on differentiation potency of adult stem cells from pulp, periodontal ligament, and dental follicle to osteoblast

Joong-Kyou Lee, Jae-Hoon Lee

Department of Oral and Maxillofacial Surgery, College of Dentistry, Dankook University

Jae-Hoon Lee Department of Oral and Maxillofacial Surgery, College of Dentistry, Dankook University 29 Anseodong, Choenan, Chungnam, 330-714, Korea Tel: +82-41-550-1991~3 Fax: +82-41-551-8988 E-mail: lee201@dankook.ac.kr

Received 18 December 2009 Revised 20 January 2010 Accepted 29 January 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Complex human tissues harbor stem cells and precursor cells, which are responsible for tissue development or repair. Recently, dental tissues such as dental pulp, periodontal ligament (PDL), dental follicle have been identified as easily accessible sources of undifferentiated cells.

These tissues contain mesenchymal stem cells that can be differentiate into bone, cartilage, fat or muscle by exposing them to specific growth conditions. In this study, the authors procured the stem cell from pulp, PDL, and dental follicle and differentiate them into osteoblast and examine the bone induction capacity.

Dental pulp stem cell (DPSC), periodontal ligament stem cell (PDLSC), and dental follicle precursor cell (DFPC) were obtained from human 3rd molar and cultured. Each cell was analyzed for presence of stem cell by fluorescence activated cell sorter (FACs) against CD44, CD105 and CD34, CD45. Each stem cell was cultured, expanded and grown in an osteogenic culture medium to allow formation of a layer of extracellular bone matrix. Osteogenic pathway was checked by alizarin red staining, alkaline phosphatase (ALP) activity test and RT-PCR for ALP and osteocalcin (OCN) gene expression.

According to results from FACs, mesenchymal stem cell existed in pulp, PDL, and dental follicle. As culturing with bone differentiation medium, stem cells were differentiated to osteoblast like cell. Compare with stem cell from pulp, PDL and dental follicle-originated stem cell has more osteogenic effect and it was assumed that the character of donor cell was able to affect on differential potency of stem cell. From this article, we are able to verify the pulp, PDL, and dental follicle from extracted tooth, and these can be a source of osteoblast and stem cell for tissue engineering.

Keywords: Stem cells, Osteoblasts, Dental pulp, Periodontal ligament (PDL), Dental follicle

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Fig. 1.

Morphology of stem cells from dental tissues.(2-3 passages)

A, B: DPSC (x 200, x 100), C, D: PDLSC (x 200, x 100), E, F: DFPC (x 200, x 100).

(DPSC: dental pulp stem cell, PDLSC: periodontal ligament stem cell, DFPS: dental follicle precursor cell)

Fig. 2.

Characterization of DPSC, PDLSC, and DFPC immunophenotype in vitro. (DPSC: dental pulp stem cell, PDLSC: periodontal ligament stem cell, DFPS: dental follicle precursor cell)

Fig. 3.

Morphology of cells in osteogenic culture media after 21 days (x 100).

A, C, E: DPSC, PDLSC, DFPC. In universal culture media after 21 days (X 100). B, D, F: DPSC, PDLSC, DFPC.

(DPSC: dental pulp stem cell, PDLSC: periodontal ligament stem cell, DFPS: dental follicle precursor cell)

Fig. 4.

Alizarin red staining of cultured cells in osteogenic media after 21 days.

A, C, E: DPSC, PDLSC, DFPC. In universal culture media. B, D, F: DPSC, PDLSC, DFPC.

(DPSC: dental pulp stem cell, PDLSC: periodontal ligament stem cell, DFPS: dental follicle precursor cell)

Fig. 5.

ALP activity test after 7, 14, 21 days of culture. A: Experimental group (osteogenic media), B: Control group (universal media).

(DPSC: dental pulp stem cell, PDLSC: periodontal ligament stem cell, DFPS: dental follicle precursor cell)

Fig. 6.

RT-PCR analysis for expression of osteoblast markers after 7, 14, 21 days of culture (exp: osteogenic media, control: universal media). A: ALP, B: OCN.

(ALP: alkaline phosphatase, OCN: osteocalcin, DPSC: dental pulp stem cell, PDLSC: periodontal ligament stem cell, DFPS: dental follicle precursor cell, GAPDH: glyceraldehyde 3-phosphate dehydrogenase)

Table 1.

Characteristics of samples. Sample number, sex and age, location of extracted teeth, and remark.

Sample	Sex/Age	Extracted tooth	Remark
1	F/21	#18, #48	Pulp, PDL, Dental follicle
2	F/22	#28, #38	Infection
3	M/26	#18, #48	Pulp, PDL
4	M/28	#18, #48	Pulp, PDL
5	F/25	#18, #48	Pulp, PDL
6	M/20	#48	Pulp, Dental follicle
7	F/18	#28, #38	Pulp, PDL, Dental follicle
8	M/30	#18, #48	Pulp, PDL

(PDL: periodontal ligament)

Table 2.

RT-PCR primer list and sequence.

ALP	forward 5’ -CCTCCTCGGAAGACACTCTG-3’
	reverse 5’ -AGACTGCGCCTGGTAGTTGT-3’
OCN	forward 5’ -GGCAGCGAGGTAGTGAAGAG-3’
	reverse 5’ -CTGGAGAGGAGCAGAACTGG-3’
GAPDH	forward 5’ -CTGTCTTCACCACCATGGAGA-3’
	reverse 5’ -CGGCCATCACGCCACAGTTT-3’

(ALP: alkaline phosphatase, OCN: osteocalcin, GAPDH: glyceraldehyde 3-phosphate dehydrogenase)

Table 3.

ALP activity test (unit: concentration mM).

Cell	Group	7 days	14 days	21 days
DPSC	Exp	0.064612	0.183676	0.23865
DPSC	Control	0.002982	0.005191	0.008953
PDLSC	Exp	0.618275	0.671693	0.683715
PDLSC	Control	0.193169	0.16455	0.138428
DFPS	Exp	0.640952	0.581677	0.64924
DFPS	Control	0.142449	0.144931	0.138428

(ALP: alkaline phosphatase, DPSC: dental pulp stem cell, PDLSC: periodontal ligament stem cell, DFPS: dental follicle precursor cell)

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