Tracking Endogenous Amelogenin and Ameloblastin In Vivo

Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While therapeutic application of EMPs has been widely documented, their expression and biological function in non-enamel tissues is unclear. Our first aim was to screen for amelogenin (AMELX) and ameloblastin (AMBN) gene expression in mandibular bones and soft tissues isolated from adult mice (15 weeks old). Using RT-PCR, we showed mRNA expression of AMELX and AMBN in mandibular alveolar and basal bones and, at low levels, in several soft tissues; eyes and ovaries were RNA-positive for AMELX and eyes, tongues and testicles for AMBN. Moreover, in mandibular tissues AMELX and AMBN mRNA levels varied according to two parameters: 1) ontogenic stage (decreasing with age), and 2) tissue-type (e.g. higher level in dental epithelial cells and alveolar bone when compared to basal bone and dental mesenchymal cells in 1 week old mice). In situ hybridization and immunohistodetection were performed in mandibular tissues using AMELX KO mice as controls. We identified AMELX-producing (RNA-positive) cells lining the adjacent alveolar bone and AMBN and AMELX proteins in the microenvironment surrounding EMPs-producing cells. Western blotting of proteins extracted by non-dissociative means revealed that AMELX and AMBN are not exclusive to mineralized matrix; they are present to some degree in a solubilized state in mandibular bone and presumably have some capacity to diffuse. Our data support the notion that AMELX and AMBN may function as growth factor-like molecules solubilized in the aqueous microenvironment. In jaws, they might play some role in bone physiology through autocrine/paracrine pathways, particularly during development and stress-induced remodeling

Recombinant Amelogenin Regulates the Bioactivity of Mouse Cementoblasts in Vitro

Amelogenin (AMG) is a cell adhesion molecule that has an important role in the mineralization of enamel and regulates events during dental development and root formation. The purpose of the present study was to investigate the effects of recombinant human AMG (rhAMG) on mineralized tissue-associated genes in cementoblasts. Immortalized mouse cementoblasts (OCCM-30) were treated with different concentrations (0.1, 1, 10, 100, 1000, 10,000, 100,000 ng · mL-1) of recombinant human AMG (rhAMG) and analyzed for proliferation, mineralization and mRNA expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen type I (COL I), osteopontin (OPN), runt-related transcription factor 2 (Runx2), cementum attachment protein (CAP), and alkaline phosphatase (ALP) genes using quantitative RT-PCR. The dose response of rhAMG was evaluated using a real-time cell analyzer. Total RNA was isolated on day 3, and cell mineralization was assessed using von Kossa staining on day 8. COL I, OPN and lysosomal-associated membrane protein-1 (LAMP-1), which is a cell surface binding site for amelogenin, were evaluated using immunocytochemistry. F-actin bundles were imaged using confocal microscopy. rhAMG at a concentration of 100,000 ng · mL-1 increased cell proliferation after 72 h compared to the other concentrations and the untreated control group. rhAMG (100,000 ng · mL-1) upregulated BSP and OCN mRNA expression levels eightfold and fivefold, respectively. rhAMG at a concentration of 100,000 ng · mL-1 remarkably enhanced LAMP-1 staining in cementoblasts. Increased numbers of mineralized nodules were observed at concentrations of 10,000 and 100,000 ng · mL-1 rhAMG. The present data suggest that rhAMG is a potent regulator of gene expression in cementoblasts and support the potential application of rhAMG in therapies aimed at fast regeneration of damaged periodontal tissue., A protein with its roots in dental development stimulates the proliferation and gene expression of cells linked to regeneration. Amelogenin is a mediator of enamel and tooth root formation, and the main component of a recently-developed medicine for periodontal regeneration. An international research group led by Sema Hakki, of Selcuk University, Turkey, has now elucidated the effects of amelogenin on cementoblasts, a type of cell responsible for producing the vital, mineralized layer on surface of the tooth root. Hakki’s team found that the bacteria-derived amelogenin increased the rate of mouse cementoblast proliferation and mineralization in vitro, and increased the expression of genes related to bone and tissue generation. The team also demonstrated the presence of a likely amelogenin receptor on the cells used in their study. These findings support further investigation into amelogenin’s therapeutic potential.PubMedWoSScopu

Estudo morfológico no músculo gastrocnêmio de camundongos C57 BL10 submetidos à ingestão prolongada de etanol Study of ultrastructural alterations in gastrocnemius muscle of C57 BL10 mice after prolonged ethanol ingestion

Os efeitos do alcoolismo crônico no músculo gastrocnêmio de camundongos bem nutridos foram estudados morfologicamente com a finalidade de se avaliar a hipótese de que o etanol exerce um papel tóxico direto sobre o músculo esquelético. Foram usados 30 camundongos C57BL10 machos, adultos jovens, divididos em dois grupos: Grupo A (controle) consistindo de 10 camundongos que beberam água e Grupo B (alcoólico) de 20 camundongos que beberam etanol a 25%. Todos os animais receberam" uma ração balanceada de laboratório e foram mantidos nesse regime ad libitum nas mesmas condições ambientais durante 48 semanas e pesados uma vez por semana. O consumo diário de dieta e a ingestão calórica foram calculados tendo os animais apresentado substancial ganho de peso, não mostrando qualquer sinal de desnutrição. Ao final do experimento os animais foram sacrificados para estudos morfológicos. Pela microscopia óptica não foram encontradas alterações. Importantes anormalidades foram observadas pela microscopia eletrônica em todos os espécimens.O retículo sarcoplasmático mostrou-se freqüentemente dilatado, resultando na formação de grandes vesículas e envolvendo as cisternas terminais com deslocamento das tríades. Áreas de estreitamento, ruptura e perda de miofibrilas ocorreram assim como zonas de completa desorganização de miofibrilas com perda do padrão estriado normal. As mitocondrias estavam em linhas gerais dentro dos limites da normalidade. Agregados tubulares peculiares vistos comumente na paralisia periódica e outras condições patológicas humanas, foram encontrados tanto no músculo dos camundongos controles como no dos alcoólicos. Os nervos intramusculares assim como as junções neuromusculares mostravam-se normais. Importantes anormalidades nos capilares musculares foram observadas, consistindo de tumefação das células endoteliais e de espessamento da lâmina basal. Difusa infiltração lipídica microvesicular foi vista no citoplasma dos hepatócitos parecendo constituir uma evidência a mais da ação tóxica do etanol sobre o organismo. Concluímos que a ingestão prolongada de etanol, representando 14,4% de calorias totais, produz no músculo gastrocnêmio de camundongos C57BL10 bem nutridos um elenco de alterações ultraestruturais que refletem um efeito tóxico direto sobre o músculo esquelético. As alterações constatadas são semelhantes àquelas descritas na miopatia alcoólica crônica humana<br>The effects of chronic alcoholism on gastrocnemius muscle of well-nourished mice were morphologically studied to test the direct toxic role of ethanol on skeletal muscle. Thirty male young adult C57BL10 mice were divided in two groups: Group A (control) consisting of ten mice that drank water and Group B (alcoholic) consisting of twenty mice that drank 25% ethanol. All mice were allowed a balanced laboratory chow. The animals were kept on this ad libitum regimen under the same conditions of environment for 48 weeks and were weighed once a week. The daily dietary consumption and caloric intake were estimated, the animals having had a substantial weight gain, showing no signs of malnutrition. At the end of the experiment the animals were killed for morphological studies. No abnormalities were observed by conventional microscopy.Striking deviations from normal were verified by electron microscopy in all specimens. Dilatation of sarcoplasmic reticulum was a common feature, sometimes resulting in the formation of large vesicles and involving the terminal cisternae with the displacement of the triads. Areas of narrowing, splitting and loss of myofibrils were seen. Zones of complete disorganization of miofibrils could be occasionally observed. Mitochondria were generally normal. Peculiar tubular aggregates seen commonly in periodic paralysis and other human pathological conditions, were encountered in both control and alcoholic mice. Intramuscular nerves and neuromuscular junctions were normal. Important abnormalities in muscle capillaries were observed, consisting of swelling of endothelial cells and thickening of the basal lamina. A diffuse microvesicular lipid infiltration was seen in the cytoplasm of the hepatocytes which seems to be a further evidence of the toxic role played by ethanol. We concluded that prolonged ingestion of ethanol, representing 14.4% of total calories, produces in the gastrocnemius muscle of well-nourished C57BL10 mice a distinct spectrum of ultrastructural changes which reflects a direct toxic effect on the skeletal muscle. These alterations are similar to those described in human chronic alcoholic myopathy