Cellular effects of enamel matrix derivative are associated with different molecular weight fractions following separation by size-exclusion chomatography

Dwight L. Johnson, David Carnes, Bjorn Steffensen, David L Cochran

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

Background: Enamel matrix derivative (EMD) was shown to enhance soft tissue healing and regeneration of the periodontium; however, the mechanisms of this action are unknown. It is assumed that ameiogenin, the most abundant protein in EMD, is the protein primarily responsible for the effects of EMD. The purpose of this study was to fractionate EMD and associate its specific cellular effects with different molecular weight fractions following size-exclusion chromatography. Methods: Freshly dissolved EMD was fractionated by gel filtration, and forty-five 7-ml fractions were collected, desalted, lyophilized, and resuspended. These fractions were analyzed for their effects on the differentiation of osteoprogenitor cells (C2C12) and the proliferation and differentiation of human microvascular endothelial cells (HMVECs). Alkaline phosphatase activity (C2C12) was measured as a marker for osteogenic differentiation before and after preincubation of the fractions with the bone morphogenetic protein (BMP) decoy receptor, noggin. Angiogenesis (HMVEC) was evaluated as a marker for endothelial cell differentiation. Enzymographic assays used polyacrylamide gels copolymerized with denatured type I collagen to determine gelatinolytic activities in each fraction. Results: EMD fractionated into three major protein peaks following size exclusion chromatography with cross-linked dextran particle matrix. Peak 1 was associated with the column void volume, whereas peak III eluted near the salt volume. Peak II eluted between these two peaks. Proliferation and angiogenic activities were associated with peaks II and III for the microvascular cells. The differentiation of osteoprogenitor cells, indicated by alkaline phosphatase activity, was induced by EMD components present in peak I and the leading edge of peak IL The additional observation that this differentiation was inhibited by prior treatment of the fractions with noggin suggested the activity was induced by BMP rather than ameiogenin or other unknown proteins, Gelatinolytic activities were detected in the early fractions of peaks I and II of gel-fractionated EMD. Conclusions: The cellular activities stimulated by EMD are not associated with a single molecular weight species. The fact that noggin abolishes C2C12 alkaline phosphatase activity suggests that effects on osteoprogenitor cell differentiation are the result of a BMP-like protein(s), whereas effects on proliferation and angiogenesis are associated with lower molecular weight species present in peaks II and III. Finally, unhealed EMD displays gelatinolytic activities that are also detectable following size-exclusion separation of its constituents. The masses of these activities were consistent with those reported for latent and active matrix metal-ioproteinase-20.

Original languageEnglish (US)
Pages (from-to)648-656
Number of pages9
JournalJournal of Periodontology
Volume80
Issue number4
DOIs
StatePublished - Apr 2009

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Dental Enamel
Molecular Weight
Cell Differentiation
Gel Chromatography
Alkaline Phosphatase
Bone Morphogenetic Proteins
Endothelial Cells
Proteins
Bone Morphogenetic Protein Receptors
Periodontium
Differentiation Antigens
Collagen Type I
Dextrans
Regeneration
Salts
Gels
Metals
Cell Proliferation

Keywords

  • Angiogenesis
  • Bone morphogenetic protein
  • Cell differentiation
  • Cell proliferation

ASJC Scopus subject areas

  • Periodontics

Cite this

Cellular effects of enamel matrix derivative are associated with different molecular weight fractions following separation by size-exclusion chomatography. / Johnson, Dwight L.; Carnes, David; Steffensen, Bjorn; Cochran, David L.

In: Journal of Periodontology, Vol. 80, No. 4, 04.2009, p. 648-656.

Research output: Contribution to journalArticle

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abstract = "Background: Enamel matrix derivative (EMD) was shown to enhance soft tissue healing and regeneration of the periodontium; however, the mechanisms of this action are unknown. It is assumed that ameiogenin, the most abundant protein in EMD, is the protein primarily responsible for the effects of EMD. The purpose of this study was to fractionate EMD and associate its specific cellular effects with different molecular weight fractions following size-exclusion chromatography. Methods: Freshly dissolved EMD was fractionated by gel filtration, and forty-five 7-ml fractions were collected, desalted, lyophilized, and resuspended. These fractions were analyzed for their effects on the differentiation of osteoprogenitor cells (C2C12) and the proliferation and differentiation of human microvascular endothelial cells (HMVECs). Alkaline phosphatase activity (C2C12) was measured as a marker for osteogenic differentiation before and after preincubation of the fractions with the bone morphogenetic protein (BMP) decoy receptor, noggin. Angiogenesis (HMVEC) was evaluated as a marker for endothelial cell differentiation. Enzymographic assays used polyacrylamide gels copolymerized with denatured type I collagen to determine gelatinolytic activities in each fraction. Results: EMD fractionated into three major protein peaks following size exclusion chromatography with cross-linked dextran particle matrix. Peak 1 was associated with the column void volume, whereas peak III eluted near the salt volume. Peak II eluted between these two peaks. Proliferation and angiogenic activities were associated with peaks II and III for the microvascular cells. The differentiation of osteoprogenitor cells, indicated by alkaline phosphatase activity, was induced by EMD components present in peak I and the leading edge of peak IL The additional observation that this differentiation was inhibited by prior treatment of the fractions with noggin suggested the activity was induced by BMP rather than ameiogenin or other unknown proteins, Gelatinolytic activities were detected in the early fractions of peaks I and II of gel-fractionated EMD. Conclusions: The cellular activities stimulated by EMD are not associated with a single molecular weight species. The fact that noggin abolishes C2C12 alkaline phosphatase activity suggests that effects on osteoprogenitor cell differentiation are the result of a BMP-like protein(s), whereas effects on proliferation and angiogenesis are associated with lower molecular weight species present in peaks II and III. Finally, unhealed EMD displays gelatinolytic activities that are also detectable following size-exclusion separation of its constituents. The masses of these activities were consistent with those reported for latent and active matrix metal-ioproteinase-20.",
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