J Adv Prosthodont.  2014 Oct;6(5):406-414. 10.4047/jap.2014.6.5.406.

Cellular viability and genetic expression of human gingival fibroblasts to zirconia with enamel matrix derivative (Emdogain(R))

• Kwon YD
• Choi HJ
• Lee H
• Lee JW
• Weber HP
• Pae A

• KMID: 2284741
• DOI: http://doi.org/10.4047/jap.2014.6.5.406

Abstract

PURPOSE
The objective of this study was to investigate the biologic effects of enamel matrix derivative (EMD) with different concentrations on cell viability and the genetic expression of human gingival fibroblasts (HGF) to zirconia surfaces.
MATERIALS AND METHODS
Immortalized human gingival fibroblasts (HGF) were cultured (1) without EMD, (2) with EMD 25 microg/mL, and (3) with EMD 100 microg/mL on zirconia discs. MTT assay was performed to evaluate the cell proliferation activity and SEM was carried out to examine the cellular morphology and attachment. The mRNA expression of collagen type I, osteopontin, fibronectin, and TGF-beta1 was evaluated with the real-time polymerase chain reaction (RT-PCR).
RESULTS
From MTT assay, HGF showed more proliferation in EMD 25 microg/mL group than control and EMD 100 microg/mL group (P<.05). HGFs showed more flattened cellular morphology on the experimental groups than on the control group after 4h culture and more cellular attachments were observed on EMD 25 microg/mL group and EMD 100 microg/mL group after 24h culture. After 48h of culture, cellular attachment was similar in all groups. The mRNA expression of type I collagen increased in a concentration dependent manner. The genetic expression of osteopontin, fibronectin, and TGF-beta1 was increased at EMD 100 microg/mL. However, the mRNA expression of proteins associated with cellular attachment was decreased at EMD 25 microg/mL.
CONCLUSION
Through this short term culture of HGF on zirconium discs, we conclude that EMD affects the proliferation, attachment, and cell morphology of HGF cells. Also, EMD stimulates production of extracellular matrix collagen, osteopontin, and TGF-beta1 in high concentration levels. CLINICAL RELEVANCE: With the use of EMD, protective barrier between attached gingiva and transmucosal zirconia abutment may be enhanced leading to final esthetic results with implants.

Keyword

Enamel matrix derivative (Emdogain(R)); Human gingival fibroblast; Zirconia; Cell proliferation; Cell attachment

MeSH Terms

Cell Proliferation
Cell Survival
Collagen
Collagen Type I
Dental Enamel*
Extracellular Matrix
Fibroblasts*
Fibronectins
Gingiva
Humans
Osteopontin
Real-Time Polymerase Chain Reaction
RNA, Messenger
Transforming Growth Factor beta1
Zirconium
Collagen
Collagen Type I
Fibronectins
Osteopontin
RNA, Messenger
Transforming Growth Factor beta1
Zirconium

Figure

• 

  Fig. 1 MTT assay results of HGF over 4h, 24h and 48h. The data shows higher level of cellular viability on EMD 25 µg/mL group than control and EMD 100 µg/mL group. Significant differences between the control group and EMD 25 µg/mL group were demonstrated (*: P<.05).

• 

  Fig. 2 SEM photomicrographs of HGF on zirconia discs at 4h (×100). (A) control group, (B) EMD 25 µg/mL group, (C) EMD 100 µg/mL group. Human gingival fibroblasts in EMD 25 µg/mL group show more cellular attachment. Spreading of cytoplasm between lamellipodia was observed (white arrow). In control and EMD 100 µg/mL group, stretched cytoplasm was observed.

• 

  Fig. 3 SEM photomicrographs of HGF on zirconia discs at 4h (×500). (A) control group, (B) EMD 25 µg/mL group, (C) EMD 100 µg/mL group. Human gingival fibroblasts were more flattened on EMD 25 µg/mL and EMD 100 µg/mL group compared to control group after 4h culture and the width of cytoplasm increased with EMD concentration (bidirectional arrows).

• 

  Fig. 4 SEM photomicrographs of HGF on zirconia discs at 24h (×100). (A) control group, (B) EMD 25 µg/mL group, (C) EMD 100 µg/mL group. SEM picture shows more cellular attachment observed on EMD 25 µg/mL group than control and EMD 100 µg/mL group.

• 

  Fig. 5 SEM photomicrographs of HGF on zirconia discs at 24h (×500). (A) control group, (B) EMD 25 µg/mL group, (C) EMD 100 µg/mL group. SEM picture shows more flattened cellular morphology in EMD 100 µg/mL group than control and EMD 25 µg/mL group and aggregation of extracellular matrix and EMD vehicles in EMD 100 µg/mL group.

• 

  Fig. 6 SEM photomicrographs of HGF on zirconia discs at 48h (×100). (A) control group, (B) EMD 25 µg/mL group, (C) EMD 100 µg/mL group. More cellular attachments were similar in EMD groups. Cellular processes in EMD 25 µg/mL group were thin and long.

• 

  Fig. 7 SEM photomicrographs of HGF on zirconia discs at 48h (×500). (A) control group, (B) EMD 25 µg/mL group, (C) EMD 100 µg/mL group. More flattened shape of human gingival fibroblast morphology was shown in EMD groups. In EMD 100 µg/mL group, cell to cell attachment was observed.

• 

  Fig. 8 mRNA expression of type I collagen, osteopontin, fibronectin, and TGF-β1. EMD increased the mRNA expression of type I collagen by human gingival fibroblasts dose dependently. The expression of osteopontin and TGF-β1 increased with significant differences in the EMD 100 µg/mL group (*: P<.05). The mRNA expression of osteopontin, fibronectin, and TGF-β1 decreased in the EMD 25 µg/mL group. The decreased amount of osteopontin and TGF-β1 was statistically significant in EMD 25 µg/mL group (*: P<.05).

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