Characterization of enamel developmental defects in patients with orofacial clefts and their relationship to surgical procedures.

AIMS: This study aimed to characterize the prevalence of development defects of enamel (DDE) in patients with cleft based on the cleft phenotype and explore the relationship between surgical procedures and different types of DDE. MATERIAL AND METHODS: In this cross-sectional study, 290 standardized orthodontic documentation and medical records from a reference hospital were evaluated, which treated patients with: cleft lip (CL), cleft lip with alveolar bone involvement (CLa), cleft lip and palate (CLP), cleft palate (CP), cleft median (CM), and considering laterality as unilateral or bilateral. DDE was assessed using the Ghanim Index (2015). Information on surgical intervention periods was obtained from medical records. Statistical analyses were performed using prevalence ratio (PR) for DDE comparisons between cleft phenotypes and surgical procedures. RESULTS: The prevalence of DDE was 77.2%. Demarcated hypomineralization was associated with CP and CLP, while hypoplasia was associated with CLa, especially when bilateral. Hypoplasia was also associated with the labial adhesion surgery. CONCLUSION: Demarcated hypomineralization was the most common DDE in this population, and the cleft phenotype influenced the type of DDE manifested. The lip adhesion surgery increased the chances of hypoplasia manifestation. CLINICAL RELEVANCE: The type of DDE in patients with cleft depends on the cleft phenotype. Understanding this susceptibility enables the multidisciplinary team to monitor dental development, thus allowing early diagnosis and timely referral to the pediatric dentist and better prognoses.

Isolation and characterization of myofibroblast cell lines from oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) invasion is followed by several stromal events such as inflammatory and immune cell infiltration, neo-vascularization, fibroblast activation and occasionally myofibroblast emergence. Our previous studies demonstrated that myofibroblasts in the stroma of OSCC are associated with a more aggressive behavior, leading to shorter patient overall survival. Therefore, we evaluated whether OSCC-associated myofibroblasts have different characteristics compared to OSCC-associated fibroblasts. OSCC myofibroblast cell lines were isolated, cultured and characterized on the basis of the expression of specific isoform α of smooth muscle actin (α-SMA) and of the excessive production of type I collagen. To assess the proliferative potential of the cell lines, growth curves were constructed, whereas the production and activity of matrix metalloproteinases (MMP) were analyzed by ELISA and enzymography, respectively. Myofibroblast clones were positive for α-SMA and vimentin, and negative for pan-cytokeratin and CD34. In long time cultures, western blotting, flow cytometry and ELISA analysis revealed constant α-SMA expression and elevated production of type I collagen. There were no differences on proliferative potential between fibroblast and myofibroblast clones, but myofibroblast cells secreted significantly higher levels of MMP-1, -2, -9 and -13. Furthermore, MMP-2 gelatinolytic activity was significantly higher in myofibroblast clones. The results of this study suggest that myofibroblasts may contribute to OSCC invasion through elevation of MMP synthesis.

Smad7 blocks transforming growth factor-ß1-induced gingival fibroblast-myofibroblast transition via inhibitory regulation of Smad2 and connective tissue growth factor.

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1), its downstream signaling mediators (Smad proteins), and specific targets, including connective tissue growth factor (CTGF), play important roles in tissue remodeling and fibrosis via myofibroblast activation. We investigated the effect of overexpression of Smad7, a TGF-ß1 signaling inhibitor, on transition of gingival fibroblast to myofibroblast. Moreover, we analyzed the participation of CTGF on TGF-ß1-mediated myofibroblast transformation. METHODS: To study the inhibitory effect of Smad7 on TGF-ß1/CTGF-mediating gingival fibroblast transition into myofibroblasts, we stably overexpressed Smad7 in normal gingival fibroblasts and in myofibroblasts from hereditary gingival fibromatosis (HGF). Myofibroblasts were characterized by the expression of the specific marker isoform α of the smooth muscle actin (α-SMA) by Western blot, flow cytometry, and immunofluorescence. Enzyme-linked immunosorbent assay for type I collagen was performed to measure myofibroblast activity. CTGF's role on myofibroblast transformation was examined by enzyme-linked immunosorbent assay and small interference RNA. RESULTS: TGF-ß1 induced the expression of α-SMA and CTGF, and small interference RNA-mediating CTGF silencing prevented fibroblast-myofibroblast switch induced by TGF-ß1. In Smad7-overexpressing fibroblasts, ablation of TGF-ß1-induced Smad2 phosphorylation marked decreased α-SMA, CTGF, and type I collagen expression. Similarly, HGF transfectants overexpressing Smad7 demonstrated low levels of α-SMA and phospho-Smad2 and significant reduction on CTGF and type I collagen production. CONCLUSIONS: CTGF is critical for TGF-ß1-induced gingival fibroblast-myofibroblast transition, and Smad7 overexpression is effective in the blockage of myofibroblast transformation and activation, suggesting that treatments targeting myofibroblasts by Smad7 overexpression may be clinically effective in gingival fibrotic diseases, such as HGF.

Serum-starved apoptotic fibroblasts reduce blastocyst production but enable development to term after SCNT in cattle.

Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.

Ovariectomy delays alveolar wound healing after molar extractions in rats.

PURPOSE: This study was conducted to investigate the morphological effects of the absence of estrogen on alveolar wound healing of young female rats after tooth extraction. MATERIALS AND METHODS: A total of 60 4- to 6-week-old female rats underwent bilateral ovariectomy (OVX) or sham operations. Three weeks later, the first mandibular molars were extracted. Subsequently, the animals were killed by cervical dislocation 3, 5, 7, 14, 21, or 28 days after tooth extraction. The mandibles were removed, and serial transversal sections of mesial alveolus of the first mandibular molars were obtained for histometric analysis. RESULTS: OVX sockets showed significant increases in fibroblasts and collagen content 3 and 5 days after the extractions, followed by significant decreases in these parameters in the subsequent periods. In accordance with the decreased collagen content in the latest period of healing, new bone formation was significantly reduced in the OVX animals. CONCLUSION: These findings suggest that the initial molecular changes observed in the absence of estrogen lead to delayed alveolar wound healing.

A new method for extraction of mandibular first molars in rats

The aim of the present study is to describe a new technique for the mandibular first molar extraction based on tooth section in rats. One hundred and forty Wistar rats, from three different researches underwent general anaesthesia. Each animal was then positioned on a specific board and hollemback 3ss was used to make the syndesmotomy. The mandibular first molars were extracted after tooth section with carbide » drills in a high-speed hand piece under constant irrigation with sterile saline solution. The mesial portion of the first molar was removed with a modified curved mosquito forceps. Thedistal portion was removed and the socket was closed with 5-0 nylon thread sutures using non-traumatic needles. During the first week after tooth extraction, animals were fed with regular pressed food to avoid post-operatory trauma. This technique is careful and avoid unnecessary trauma, with minimal numbers of fractures (9.3%) and accidents such as haemorrhage (2%). All the reminiscent roots could be removed by the described method. Our technique proved to be anefficient model for future researches on alveolar wound healing with minimal fractures and accidents and provided better post-extraction outcomes for the rats.