Impact of isolation method on doubling time and the quality of chondrocyte and osteoblast differentiated from murine dental pulp stem cells

Rohaya Megat Abdul Wahab, Nur Akmal Mohamed Rozali, Sahidan Senafi, Intan Zarina Zainol Abidin, Zaidah Zainal Ariffin, Shahrul Hisham Zainal Ariffin

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Background. Stem cells are normally isolated from dental pulps using the enzymatic digestion or the outgrowth method. However, the effects of the isolation method on the quality of the isolated stem cells are not studied in detail in murine models. The aim of this study was to compare the matrices secreted by osteoblast and chondrocytes differentiated from dental pulp stem cells isolated through different means. Method. DPSC from murine incisors were isolated through either the outgrowth (DPSC-OG) or the enzymatic digestion (DPSC-ED) method. Cells at passage 4 were used in this study. The cells were characterized through morphology and expression of cell surface markers. The cells' doubling time when cultured using different seeding densities was calculated and analyzed using one-way ANOVA and Tukey's multiple comparison post-test. The ability of cells to differentiate to chondrocyte and osteoblast was evaluated through staining and analysis on the matrices secreted. Results. Gene expression analysis showed that DPSC-OG and DPSC-ED expressed dental pulp mesenchymal stem cell markers, but not hematopoietic stem cell markers. The least number of cells that could have been used to culture DPSC-OG and DPSC- ED with the shortest doubling time was 5 × 102 cells/cm2 (11.49 ± 2.16 h) and 1 × 102 cells/cm2 (10.55 h ± 0.50), respectively. Chondrocytes differentiated from DPSC- ED produced 2 times more proteoglycan and at a faster rate than DPSC-OG. FTIR revealed that DPSC-ED differentiated into osteoblast also secreted matrix, which more resembled a calvaria. Discussion. Isolation approaches might have influenced the cell populations obtained. This, in turn, resulted in cells with different proliferation and differentiation capability. While both DPSC-OG and DPSC-ED expressed mesenchymal stem cell markers, the percentage of cells carrying each marker might have differed between the two methods. Regardless, enzymatic digestion clearly yielded cells with better characteristics than outgrowth.

Original languageEnglish
Article number3180
JournalPeerJ
Volume2017
Issue number6
DOIs
Publication statusPublished - 2017

Fingerprint

tooth pulp
Dental Pulp
osteoblasts
chondrocytes
Osteoblasts
isolation techniques
Chondrocytes
Stem cells
Pulp
stem cells
Stem Cells
mice
Digestion
digestion
cells
Cells
Mesenchymal Stromal Cells
Proteoglycans
Analysis of variance (ANOVA)
Gene expression

Keywords

  • Chondrocyte
  • Dental pulp
  • Enzymatic digestion
  • Osteoblast
  • Outgrowth
  • Stem cell

ASJC Scopus subject areas

  • Neuroscience(all)
  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Impact of isolation method on doubling time and the quality of chondrocyte and osteoblast differentiated from murine dental pulp stem cells. / Megat Abdul Wahab, Rohaya; Rozali, Nur Akmal Mohamed; Senafi, Sahidan; Abidin, Intan Zarina Zainol; Ariffin, Zaidah Zainal; Zainal Ariffin, Shahrul Hisham.

In: PeerJ, Vol. 2017, No. 6, 3180, 2017.

Research output: Contribution to journalArticle

Megat Abdul Wahab, Rohaya ; Rozali, Nur Akmal Mohamed ; Senafi, Sahidan ; Abidin, Intan Zarina Zainol ; Ariffin, Zaidah Zainal ; Zainal Ariffin, Shahrul Hisham. / Impact of isolation method on doubling time and the quality of chondrocyte and osteoblast differentiated from murine dental pulp stem cells. In: PeerJ. 2017 ; Vol. 2017, No. 6.
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T1 - Impact of isolation method on doubling time and the quality of chondrocyte and osteoblast differentiated from murine dental pulp stem cells

AU - Megat Abdul Wahab, Rohaya

AU - Rozali, Nur Akmal Mohamed

AU - Senafi, Sahidan

AU - Abidin, Intan Zarina Zainol

AU - Ariffin, Zaidah Zainal

AU - Zainal Ariffin, Shahrul Hisham

PY - 2017

Y1 - 2017

N2 - Background. Stem cells are normally isolated from dental pulps using the enzymatic digestion or the outgrowth method. However, the effects of the isolation method on the quality of the isolated stem cells are not studied in detail in murine models. The aim of this study was to compare the matrices secreted by osteoblast and chondrocytes differentiated from dental pulp stem cells isolated through different means. Method. DPSC from murine incisors were isolated through either the outgrowth (DPSC-OG) or the enzymatic digestion (DPSC-ED) method. Cells at passage 4 were used in this study. The cells were characterized through morphology and expression of cell surface markers. The cells' doubling time when cultured using different seeding densities was calculated and analyzed using one-way ANOVA and Tukey's multiple comparison post-test. The ability of cells to differentiate to chondrocyte and osteoblast was evaluated through staining and analysis on the matrices secreted. Results. Gene expression analysis showed that DPSC-OG and DPSC-ED expressed dental pulp mesenchymal stem cell markers, but not hematopoietic stem cell markers. The least number of cells that could have been used to culture DPSC-OG and DPSC- ED with the shortest doubling time was 5 × 102 cells/cm2 (11.49 ± 2.16 h) and 1 × 102 cells/cm2 (10.55 h ± 0.50), respectively. Chondrocytes differentiated from DPSC- ED produced 2 times more proteoglycan and at a faster rate than DPSC-OG. FTIR revealed that DPSC-ED differentiated into osteoblast also secreted matrix, which more resembled a calvaria. Discussion. Isolation approaches might have influenced the cell populations obtained. This, in turn, resulted in cells with different proliferation and differentiation capability. While both DPSC-OG and DPSC-ED expressed mesenchymal stem cell markers, the percentage of cells carrying each marker might have differed between the two methods. Regardless, enzymatic digestion clearly yielded cells with better characteristics than outgrowth.

AB - Background. Stem cells are normally isolated from dental pulps using the enzymatic digestion or the outgrowth method. However, the effects of the isolation method on the quality of the isolated stem cells are not studied in detail in murine models. The aim of this study was to compare the matrices secreted by osteoblast and chondrocytes differentiated from dental pulp stem cells isolated through different means. Method. DPSC from murine incisors were isolated through either the outgrowth (DPSC-OG) or the enzymatic digestion (DPSC-ED) method. Cells at passage 4 were used in this study. The cells were characterized through morphology and expression of cell surface markers. The cells' doubling time when cultured using different seeding densities was calculated and analyzed using one-way ANOVA and Tukey's multiple comparison post-test. The ability of cells to differentiate to chondrocyte and osteoblast was evaluated through staining and analysis on the matrices secreted. Results. Gene expression analysis showed that DPSC-OG and DPSC-ED expressed dental pulp mesenchymal stem cell markers, but not hematopoietic stem cell markers. The least number of cells that could have been used to culture DPSC-OG and DPSC- ED with the shortest doubling time was 5 × 102 cells/cm2 (11.49 ± 2.16 h) and 1 × 102 cells/cm2 (10.55 h ± 0.50), respectively. Chondrocytes differentiated from DPSC- ED produced 2 times more proteoglycan and at a faster rate than DPSC-OG. FTIR revealed that DPSC-ED differentiated into osteoblast also secreted matrix, which more resembled a calvaria. Discussion. Isolation approaches might have influenced the cell populations obtained. This, in turn, resulted in cells with different proliferation and differentiation capability. While both DPSC-OG and DPSC-ED expressed mesenchymal stem cell markers, the percentage of cells carrying each marker might have differed between the two methods. Regardless, enzymatic digestion clearly yielded cells with better characteristics than outgrowth.

KW - Chondrocyte

KW - Dental pulp

KW - Enzymatic digestion

KW - Osteoblast

KW - Outgrowth

KW - Stem cell

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