Human osteoblastic cells discriminate between 20‐kDa amelogenin isoforms

Abstract

Enamel matrix derivative (EMD) is used to stimulate healing of alveolar bone after destructive marginal periodontitis; however, the roles of the different EMD constituents are unclear. The aim here was to compare the effect of two EMD fractions (A1 and A2) on primary human osteoblasts cultured in the presence of 50 μg ml(-1) of A1, A2, or EMD. SDS-PAGE showed that A1 and A2 were comprised of amelogenins migrating at around 20 kDa. Fourier transform infrared (FTIR) analysis revealed that A1 and A2 had different secondary structures, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) identified different peptide mass values. Osteoblasts responded differently to A1 and A2. Whereas A1 enhanced the proliferation [measured by the incorporation of 5-bromo-2'-deoxyuridine (BrdU)] of osteoblasts, the expression of runt-related transcription factor-2 (RUNX2) mRNA, and the secretion of interleukin 6 (IL-6) into the cell culture medium, exposure to A2 resulted in increased alkaline phosphatase (ALP) activity, increased expression of CD44 mRNA, and increased secretion of osteoprotegrin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL). The level of osteocalcin in the cell culture medium was increased after all treatments, while A2 stimulated the expression of dentin matrix protein 1 (DMP1) mRNA. The results suggest that both A1 and A2 participate in the observed effect of EMD, but have different effects on the expression of osteoblast mRNA and the secretion of osteoblast protein, and thus might facilitate the differentiation of a different phenotype.

Cathodic Polarization Coats Titanium Based Implant Materials with Enamel Matrix Derivate (EMD)

“…The peaks observed in the MALDI spectrum of pure EMD in the 5 kDa and 16 kDa region concurred with the peaks that have previously been reported in the literature [40,52–54]. Moreover, sequencing of pure EMD MALDI-ISD revealed fragments with the sequence YEVLTPLKWYQNM that were corresponding to the amino acid stretch 33–45.…”

“…While pure EMD was dissolved in 0.1% acetic acid at pH 3.1, the ACN+TFA solution used for detaching EMD from the coated coins had a pH of 1.8. It was believed that the results obtained by Riksen et al [54] were measured at a pH that was different to the pH used for the samples of this study. Hence, an influence of the different pH values of the single samples could not be excluded.…”

“…The peak found at 16.9 kDa for pure EMD was also observed for TiZr EMD and Ti EMD. Riksen et al [54] showed a peak at 16.6 kDa for two different amelogenin fractions separated from EMD by size-exclusion high-performance liquid chromatography. As Riksen et al [54] used 0.05% acetic acid for dissolving EMD, the pH should have been slightly higher than for the 0.1% acetic acid solution used in this study.…”

“…Riksen et al [54] showed a peak at 16.6 kDa for two different amelogenin fractions separated from EMD by size-exclusion high-performance liquid chromatography. As Riksen et al [54] used 0.05% acetic acid for dissolving EMD, the pH should have been slightly higher than for the 0.1% acetic acid solution used in this study. Cohen et al [55] described a pH dependency of the mass peak of a MALDI measurement when using a HCCA matrix like it was used in this study.…”

Cytokine expression in response to root repair agents

“…Similar results were reported by Riksen et al . (), who observed high RANKL mRNA levels in osteoblasts when they were cultured in the presence of derivative fractions of enamel matrix. Conversely, Nishiguchi et al .…”