The purpose of this study was to evaluate the effects of concentrated growth factor exudate (CGFe) on human periodontal ligament cells (hPDLCs) stimulated by tumor necrosis factor (TNF)-α. From the peripheral blood of healthy donors, CGFe was prepared according to the Sacco protocol after 7 days of incubation. The hPDLCs were cultured by a tissue explant method and identified with anti-vimentin and anti-cytokeratin antibodies. Cells were subjected to four different treatments: i) Control; ii) TNF-α (10 ng/ml); iii) CGFe (concentration 50%); and iv) CGFe+TNF-α. The proliferation of hPDLCs was measured with Cell Counting Kit-8 assays. Osteogenic differentiation and mineralization were determined by Alizarin Red S staining, alkaline phosphatase activity, western blotting and reverse transcription-quantitative polymerase chain reaction. CGFe enhanced cell proliferation and upregulated ALP activity, the mineralization level, and osteogenic-associated osteocalcin, runt-related transcription factor 2 and Osterix gene expression in hPDLCs under inflammatory conditions induced by TNF-α. The present study demonstrated that CGFe enhanced hPDLC proliferation and osteogenic differentiation in the presence of TNF-α-induced inflammation. As CGFe can be obtained from the venous blood of patients, it generates no immune reaction. Thus, it is more economical and beneficial to use CGFe in clinical periodontal regeneration practice than synthetic growth factors.

The purpose of the present study was to evaluate the effects of platelet-rich fibrin (PRF) exudate on the proliferation, osteogenic differentiation and mineralization of human periodontal ligament cells (hPDLCs) in vitro. In the present study PRF was obtained with permission, from the peripheral blood of healthy donors and PRF exudates were collected on the 7th day of incubation. hPDLCs were obtained from healthy premolars, cultured by a tissue explant method and identified with anti-vimentin and anti-cytokeratin antibodies. PRF exudates were added to hPDLCs in different concentrations to evaluate cell proliferation and osteogenic differentiation. The proliferation of hPDLCs was measured using a colorimetric assay. Osteogenic differentiation and mineralization were determined by Alizarin red staining, alkaline phosphatase activity (ALP), western blotting and reverse transcription-quantitative polymerase chain reaction. Cell proliferation was enhanced by addition of the PRF exudate, which also promoted the formation of mineralized matrix nodules and upregulated ALP activity and osteoblast-associated levels of osteocalcin, runt-related transcription factor and osterix gene expression. As these stimulatory effects occurred in a dose-dependent manner, it was concluded that high concentrations of the PRF exudate served an essential role in the proliferation, osteogenic differentiation and mineralization of hPDLCs in vitro. The present study demonstrated that PRF exudate enhanced hPDLC proliferation, induced the osteoblastic differentiation of hPDLCs into mineralized tissue-formation cells in vitro, and may therefore provide potential benefits for periodontal tissue engineering; contributing to the primary processes of periodontal tissue regeneration. From the perspective of both economics and biology, PRF has greater clinical benefits than analogous growth factors.

ABSTRACT. We established a necrotizing enterocolitis (NEC) rat model and explored the role of bifidobacteria in the intestines of the rats and its regulation on intestinal Toll-like receptors (TLRs). Seventyfive newborn Sprague-Dawley rats were randomly divided into 5 groups (15 rats/group): group A, artificial feeding group (formula-fed); group B, NEC model (LPS + formula-fed); group C, bifidobacterium (LPS + formula-fed + bifidobacterium microcapsules, intragastric administration); group D, artificial feeding + bifidobacterium (formulafed + bifidobacterium microcapsules gavage); group E, rat breastfeeding group (rat breast-feeding). After 3 days of feeding, rats were placed in incubators, fasted for 12 h, and killed by decapitation. The ileocecal proximal segment ileum was fixed and sliced; pathological examination was conducted, and TLR2, TLR4, and nuclear factorkB p65 protein expression in the intestinal tissue was detected by immunohistochemistry. There was a statistically significant difference 11506 W. Zhou et al. ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (3): 11505-11514 (2015) in pathological scores between groups C and B (H = 21.789, P = 0.000), and the former was lower than the latter. TLR2, TLR4, and nuclear factor-kB p65 expression in intestinal tissue was determined in groups A-E. There were statistically significant differences between groups C and B (P = 0.001; P = 0.000; P = 0.000). Bifidobacteria had a protective effect on the intestines of newborn rats with NEC, which showed reduced NEC and intestinal damage severity. This observation may be related to the reduced levels of TLR2, TLR4, and nuclear factor-kB P65 observed during the inflammatory response.