Novel biological activity of ameloblastin in enamel matrix derivative

Objective Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration. However, the effects of EMD on gingival epithelial cells during regeneration of periodontal tissues are unclear. In this in vitro study, we purified ameloblastin from EMD and investigated its biological effects on epithelial cells. Material and Methods Bioactive fractions were purified from EMD by reversed-phase high-performance liquid chromatography using hydrophobic support with a C18 column. The mouse gingival epithelial cell line GE-1 and human oral squamous cell carcinoma line SCC-25 were treated with purified EMD fraction, and cell survival was assessed with a WST-1 assay. To identify the proteins in bioactive fractions of EMD, we used proteome analysis with two-dimensional gel electrophoresis followed by identification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Results Purified fractions from EMD suppressed proliferation of GE-1 and SCC-25. LC-MS/MS revealed that ameloblastin in EMD is the component responsible for inhibiting epithelial cell proliferation. The inhibitory effect of ameloblastin on the proliferation of GE-1 and SCC-25 was confirmed using recombinant protein. Conclusion The inhibitory effects of EMD on epithelial cell proliferation are caused by the biological activities of ameloblastin, which suggests that ameloblastin is involved in regulating epithelial downgrowth in periodontal tissues. .

Osteoblast response (initial adhesion and alkaline phosphatase activity) following exposure to a barrier membrane/enamel matrix derivative combination.

BACKGROUND AND OBJECTIVE: The enamel matrix derivative (EMD) has been used in combination with barrier membranes to optimize regeneration in vertical osseous defects. However, the osteoblast response when exposed to the EMD/barrier membrane combination has not yet been evaluated. The osteoblast behavior when exposed to a combination of regenerative materials must be evaluated to fully understand their effect on bone regeneration. Therefore, the present study was undertaken to estimate the initial adhesion and alkaline phosphatase (ALP) activity of an osteoblast cell line (SaOS-2) when exposed to four commercially available resorbable membranes and determine if the addition of EMD had any modulatory effect on osteoblast behavior. MATERIALS AND METHODS: 5 x 104 SaOS-2 cells between passages 7-10 were cultured in two 24-well culture plates. Plate A was used for the adhesion assay and Plate B was used for the ALP assay. A MTT (3-[4, 5-dimethylthiazolyl-2]-2, 5-diphenyltetrazolium bromide) assay was done after 24 hours to determine the adhesion of the osteoblastic cells to four barrier membranes: 1) a non cross-linked porcine Type I and III collagen membrane (BG), 2) a weakly cross-linked Type I collagen membrane (HG), 3) a glutaraldehyde cross-linked bovine Type I collagen (BM), and 4) a resorbable polymer membrane (CP). Osteoblast differentiation was studied using an ALP assay with p-nitro phenyl phosphate as the substrate at 24 hours, 72 hours, and 1 week. A total of 50 microg/ml of EMD dissolved in 10 mM acetic acid was added into each well and the entire experimental protocol outlined above was repeated. RESULTS: The osteoblast adhesion to collagen barriers showed a statistically insignificant reduction following the addition of EMD. Adhesion to the polymer barrier, although significantly lower when compared with collagen barriers, was unaffected by the addition of EMD. ALP activity after 1 week among the various groups was as follows: EMD alone (75.59+/-2.5)>EMD/BG(64.78+/-3.04)>EMD/HG(55.40+/-3.89) approximately EMD/BM(54.75+/-4.17)>BG (51.32+/-2.76)>HG(49.92+/-2.4)>BM(48.14+/-1.4)>Control(46.29+/-1.39)>EMD/CP (37.46+/-3.54)>CP(32.12+/-1.49) CONCLUSION: There was no additive effect on osteoblast adhesion/ALP activity following exposure to an EMD/polymer combination. EMD/collagen positively influences osteoblast differentiation in a time dependent manner.

Effect of enamel matrix proteins on the treatment of intrabony defects: a split-mouth randomized controlled trial study

The objective of this split-mouth, double-blind, randomized controlled trial was to compare the clinical effect of treatment of 2- or 3-wall intrabony defects with open flap debridement (OFD) combined or not with enamel matrix proteins (EMP). Thirteen volunteers were selected with one pair of or more intrabony defects and probing pocket depth (PPD) > 5 mm. All individuals received instructions regarding oral hygiene and were submitted to scaling and root planing. Each participant received the two treatment modalities: test sites were treated with OFD and EMP, and control sites received only OFD. After 6 months, a significant reduction was observed in PPD for the EMP group (from 6.42 ± 1.08 mm to 2.67 ± 1.15 mm) and for the OFD group (from 6.08 ± 1.00 mm to 2.00 ± 0.95 mm) (p < 0.0001), but with no significant difference between groups (p = 0.13). A significant gain in relative attachment level (RAL) was observed in both groups (EMP: from 13.42 ± 1.88 mm to 10.75 ± 2.26 mm, p < 0.001; OFD: from 12.42 ± 1.98 mm to 10.58 ± 2.23 mm, p = 0.013), but with no significant difference between groups (p = 0.85). Gingival recession (GR) was higher in the EMP group (from 1.08 ± 1.50 mm to 2.33 ± 1.43 mm; p = 0.0009) than in the OFD group (from 0.66 ± 1.15 mm to 1.16 ± 1.33 mm; p = 0.16), but this difference was not significant (p = 0.06). In conclusion, the results showed that OFD combined with EMP was not able to improve treatment of intrabony defects compared to OFD alone.

O objetivo deste estudo clínico controlado, randomizado, duplo-cego, tipo boca-dividida foi comparar o efeito clínico do tratamento de defeitos infra-ósseos de 2 ou 3 paredes com retalho de espessura total (RET) associado ou não com a proteína da matriz do esmalte (PME). Treze voluntários com 1 par ou mais de defeitos infra-ósseos foram selecionados com profundidade clínica de sondagem (PCS) > 5 mm. Todos receberam instruções de higiene bucal, raspagem e alisamento radicular. Cada participante recebeu os dois tipos de tratamento: o lado teste foi tratado com RET e PME, e o lado controle recebeu somente RET. Após 6 meses, foi observada uma redução significante na PCS para o grupo PME (de 6,42 ± 1,08 mm para 2,67 ± 1,15 mm) e para o grupo RET (de 6,08 ± 1,00 mm para 2,00 ± 0,95 mm) (p < 0,0001), mas não houve diferença significante entre os grupos (p = 0,13). Um ganho significante de nível clínico de inserção relativo (NCIR) foi observado em ambos os grupos (PME: de 13,42 ± 1,88 mm para 10,75 ± 2,26 mm, p < 0,001; RET: de 12,42 ± 1,98 mm para 10,58 ± 2,23 mm, p = 0,013), mas não houve diferença significante entre os grupos (p = 0,85). A retração gengival (RG) foi maior para o grupo PME (de 1,08 ± 1,50 mm para 2,33 ± 1,43 mm; p = 0,0009) do que para o grupo RET (de 0,66 ± 1,15 mm para 1,16 ± 1,33 mm; p = 0,16), mas essa diferença não foi significante (p = 0,06). Concluiu-se que o tratamento de defeitos infra-ósseos com RET associado à PME não mostrou resultados melhores que o uso de RET sozinho.