Interaction of enamel matrix proteins with human periodontal ligament cells

Dorothy Hodgkin Postgraduate Award for research studies (jointly funded by the Engineering and Physical Sciences Research Council, UK, and by Institut Straumann) and the Research Discretionary Funds of the Periodontology Unit, UCL Eastman Dental Institute. Financial support was also provided by the NIHR Comprehensive Biomedical Research Centre and by the WCU Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. R31-10069)

Effects of enamel matrix derivative and transforming growth factor-β1 on human osteoblastic cells

<p>Abstract</p> <p>Background</p> <p>Extracellular matrix proteins are key factors that influence the regenerative capacity of tissues. The objective of the present study was to evaluate the effects of enamel matrix derivative (EMD), TGF-β1, and the combination of both factors (EMD+TGF-β1) on human osteoblastic cell cultures.</p> <p>Methods</p> <p>Cells were obtained from alveolar bone of three adult patients using enzymatic digestion. Effects of EMD, TGF-β1, or a combination of both were analyzed on cell proliferation, bone sialoprotein (BSP), osteopontin (OPN) and alkaline phosphatase (ALP) immunodetection, total protein synthesis, ALP activity and bone-like nodule formation.</p> <p>Results</p> <p>All treatments significantly increased cell proliferation compared to the control group at 24 h and 4 days. At day 7, EMD group showed higher cell proliferation compared to TGF-β1, EMD + TGF-β1 and the control group. OPN was detected in the majority of the cells for all groups, whereas fluorescence intensities for ALP labeling were greater in the control than in treated groups; BSP was not detected in all groups. All treatments decreased ALP levels at 7 and 14 days and bone-like nodule formation at 21 days compared to the control group.</p> <p>Conclusions</p> <p>The exposure of human osteoblastic cells to EMD, TGF-β1 and the combination of factors <it>in vitro </it>supports the development of a less differentiated phenotype, with enhanced proliferative activity and total cell number, and reduced ALP activity levels and matrix mineralization.</p

In vitro wound healing characteristics of amelogenins

Wound healing involves the co-ordinated actions of several cell types, soluble cell mediators and extracellular matrix (ECM). This research project intended to investigate the role of certain ECM proteins in different processes during tissue repair by studying the interaction between dermal cells and ECM. The focus has been on amelogenins, ECM proteins that under physiological conditions aggregate into spherical structures. As a resorbable biomaterial, amelogenins enhance periodontal tissue regeneration and have been introduced in the treatment of hard-to-heal ulcers. However, the mechanisms of action need to be delineated. The aim of this project was to increase the knowledge on the effects of amelogenins on cell behaviour, to further understand the role of this specific ECM protein in tissue repair and regeneration. To study the in vitro effects of amelogenins on wound healing, three human cell types; macrophages, fibroblasts, and endothelial cells, all essential for successful tissue repair, were utilised. The study designs included cell cultures, in monolayer, 3D-culture and an ex vivo model (chick aortic arch assay) for the angiogenesis studies. The evaluation methods included cell quantification, mitogenesis and apoptosis studies by BrdU incorporation and TUNEL measurements respectively, cytokine analysis by ELISA and multiplex bead array, cell surface integrin adhesion assay, gene microarray analysis, phase contrast and fluorescence microscopy for morphology and viability, and ultrastructural studies by electron microscopy. The results demonstrate that amelogenins influence the in vitro cell behaviour of all three cell types investigated. The interaction and uptake of amelogenin aggregates was demonstrated for both macrophages and fibroblasts. In addition, the possible involvement of integrin-dependent adhesion was demonstrated for fibroblasts and endothelial cells, with increased cell binding by multiple integrins subunits and αvβ3, αvβ5 and α5β1. Amelogenin treatment of cultured macrophages displayed anti-inflammatory properties, directing the release of several pro- and anti-inflammatory cytokines. In particular, induced secretion of the specific marker of alternative macrophage activation AMAC-1, along with vascular endothelial growth factor was seen, most probably resulting from a switch of macrophage phenotype to an alternatively activated cell, with tissue repair characteristics. Also, amelogenins increased cell proliferation and induced the expression of genes involved in cellular growth, migration and differentiation in normal dermal fibroblasts. Moreover, amelogenins had the capacity to restore an acute-like phenotype in senescent fibroblasts. Finally, amelogenins displayed pro-angiogenic properties in vitro and ex vivo. In conclusion, the effects of amelogenins on wound healing are plausibly, at least partly, conducted by providing macrophages, fibroblasts, and endothelial cells with tissue repair characteristics. These effects are most probably conducted through cell adhesion via integrin interaction

Immunohistochemical Characterization of Rapid Dentin Formation Induced by Enamel Matrix Derivative

The purpose of this study was to examine the pulpal expression of dentin-related proteins during enamel matrix derivative (EMD)–induced reparative dentin formation in a pulpotomy model in pig incisors. Pulpotomies were performed on 72 lower incisors in 24 adult miniature swine. The exposed pulp tissue was treated with EMD or covered with a calcium hydroxide paste (Dycal ® ). At predefined time-points, ranging from 4 days to 12 weeks, experimental teeth were extracted and examined by use of light microscopy, and expression of dentin-related proteins in the pulps was investigated by immunohistochemistry, using antibodies against type I collagen, dentin sialoprotein (DSP), sheathlin, and EMD. In all EMD-treated teeth a substantial amount of reparative dentin formation was observed. The amount of reparative dentin in calcium hydroxide–treated teeth was significantly smaller than in EMD-treated teeth ( P < 0.005) and was less effective in bridging the pulpal wounds. Immunohistochemistry demonstrated that enamel matrix proteins were present in detectable amounts at the application site for about 4 weeks. Moreover, the expression of proteins related to dentin formation in the wounded pulp tissue was about 2 weeks advanced in EMD-treated teeth. These findings demonstrate that enamel matrix molecules have the capacity to induce rapid pulpal wound healing in pulpotomized teeth, and suggest that the longevity and continued presence of enamel matrix macromolecules at the application site can be utilized to stimulate growth and repair of dentin over a period consistent with a favorable clinical outcome.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/48011/1/223_2003_Article_153.pd

Floral divergence in closely related Leucospermum tottum (Proteaceae) varieties pollinated by birds and longproboscid flies

The Proteaceae are renowned for their floral diversity but surprisingly the role of pollinators in driving evolutionary divergence in this family has been underexplored. Here we focus on recently diverged taxa to gain insight into the processes that generate diversity by testing whether two varieties of Leucospermum tottum might have originated by pollinator mediated adaptive divergence. L. tottum var. tottum has pale salmon-coloured horizontally-oriented flowers, long nectar tubes, and small volumes of concentrated nectar. L. tottum var. glabrum has red and yellow vertically oriented flowers, short nectar tubes, and large volumes of dilute nectar. Despite the morphological divergence, the varieties are indistinguishable using eight molecular markers, indicating a very early stage of differentiation. Consistent with their morphologies, L. tottum var. tottum is pollinated by long-proboscid flies (Philoliche rostrata and Philoliche gulosa), Cape sugarbirds (Promerops cafer), and, to a lesser extent, by Orange-breasted sunbirds (Anthobaphes violacea), whereas, L. tottum var. glabrum is pollinated only by Orange-breasted sunbirds. A. violacea visits both varieties, but makes more frequent contact with pollen presenters when foraging on L. tottum var. glabrum. The exclusion of birds caused a steeper reduction in seed production in L. tottum var. glabrum than in L. tottum var. tottum, consistent with specialization for bird-pollination in this variety. Additionally, L. tottum var. glabrum exhibits autogamy, whereas L. tottum var. tottum does not. Floral divergence between the two L. tottum varieties corresponds with divergence in pollinator use