In Vitro Effects of Enamel Matrix Derivative on Microvascular Cells

Abstract

Periodontal regeneration requires a coordinated series of events that includes not only the recruitment of periodontal ligament (PDL)-specific cells, but vascular cells as well. The mechanisms of action of enamel matrix derivative (EMD) are poorly understood, and its effects on vascular cells are unknown. The objective of this study was to examine the extent to which EMD affects angiogenesis and PDL cell recruitment. The effects of EMD on human microvascular endothelial cells (HMVECs) were determined by examining proliferation, chemotaxis, angiogenesis, and migration. Proliferation was determined using water-soluble tetrazolium salt (WST)-1 reagent. Chemotaxis was determined using microporous-culture well inserts. Angiogenesis was assessed on plates containing matrigel. The effects of HMVECs on the migration of PDL cells were assessed by evaluating PDL cell outgrowth from collagen gels cultured in the presence of HMVECs on fibrin matrix and surrounded by fibronectin-containing fibrin clots at 24 hours. Effects of EMD on PDL expression of vascular endothelial cell (VEGF) types (A, B, C, and D) and isoforms were determined using reverse transcription-polymerase chain reaction (RT-PCR). Production of VEGF, platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-AB, and transforming growth factor (TGF)-beta1 by EMD-stimulated PDL cells was assessed quantitatively in conditioned media using specific enzyme-linked immunosorbent assays (ELISAs). EMD at concentrations <50 microg/ml resulted in significant (P <0.05) stimulation of HMVEC proliferation. Compared to baseline, EMD also stimulated a 100% increase in HMVEC chemotaxis when PDL cells were present (P <0.05). All doses of EMD tested (25, 50, and 100 microg/ml) increased angiogenesis in vitro. HMVECs, in combination with EMD at a concentration of 100 microg/ml, stimulated a 750% increase in migration of PDL cells from collagen gels into fibrin clots compared to controls when neither was present. RT-PCR results indicated that PDL cells expressed VEGF-A, -B, and -C and multiple isoforms of VEGF-A, including VEGF(121), VEGF(165), and VEGF(189), whether or not EMD was present in the culture media. ELISAs determined a 400% increase in VEGF concentration by PDL C cells in EMD-stimulated conditioned media and a similar increase in TGF-beta(1)-stimulated media. It is likely that EMD stimulates angiogenesis directly by stimulating endothelial cells and indirectly by stimulating the production of angiogenic factors (VEGF) by PDL cells. Importantly, the data are consistent with the concept that EMD enhances bidirectional communication between HMVEC and PDL cells during angiogenesis associated with healing.