[Establishment of a reporter system for estimating activation of human hepatic stellate cells based on COL1A1 promoter and enhanced green fluorescent protein].

OBJECTIVE: To establish a visual reporting system for evaluating the activity of collagen Ⅰ α 1 chain (COL1A1) gene promoter in immortalized human hepatic stellate cells, so as to estimate the activation status of the cells and provide a new cell model for the screening and study of anti-hepatic fibrosis drugs. METHODS: The promoter sequence of human COL1A1 was amplified from the genomic DNA of human hepatocarcinoma cell line HepG2. Based on the pLVX-AcGFP1-N1 plasmid, the recombinant plasmid pLVX-COL1A1-enhanced green fluorescent protein (EGFP) was constructed, in which the enhanced green fluorescent protein gene expression was regulated by the COL1A1 promoter. The monoclonal cell line was acquired by stably transfecting pLVX-COL1A1-EGFP into the immortalized human hepatic stellate cell line LX-2 by the lentivirus packaging system and screening. The cell line was treated with transforming growth factor-ß1 (TGF-ß1) or co-treated with TGF-ß1 and drugs with potential anti-hepatic fibrosis effects. The EGFP fluorescence intensity in cells was analyzed by the fluorescence microscope and ImageJ 1.49 software using a semi-quantitative method. The COL1A1 and EGFP mRNA were detected by reverse transcription real-time quantitative PCR (RT-qPCR), and corresponding proteins were detected by Western blot. RESULTS: The recombinant plasmid pLVX-COL1A1-EGFP with the expression of EGFP regulated by COL1A1 promoter was successfully constructed. Kozak sequence was added to enhance the expression of EGFP, which was identified by double digestion and sequencing. The LX-2 monoclonal cell line LX-2-CE stably transfected with pLVX-COL1A1-EGFP was obtained. After co-treatment with TGF-ß1 and 5 µmol/L dihydrotanshinone Ⅰ with potential anti-hepatic fibrosis effect for 24 h, the total fluorescence intensity and the average fluorescence intensity of LX-2-CE were lower than those in TGF-ß1 single treatment group (P < 0.05), the intracellular mRNA and protein levels of COL1A1 and EGFP were also lower than those in the TGF-ß1 single treatment group (P < 0.05). CONCLUSION: A reporter system for estimating activation of hepatic stellate cells based on COL1A1 promoter regulated EGFP expression is successfully constructed, which could visually report the changes in COL1A1 expression, one of the activation-related markers of hepatic stellate cells, in vitro. It provides a new cell model for the screening and study of anti-hepatic fibrosis drugs.

[Implant prosthesis design scheme for sophisticated congenital partial edentia].

Sophisticated congenital partial edentia are often accompanied by severe shortage of bone height and width due to the absence of permanent teeth; such condition will affect implant placement. This study aimed to display the different typical implant rehabilitation schemes we designed for sophisticated congenital partial edentia cases with severely atrophic alveolar bone.

Crude triterpenoid saponins from Anemone flaccida (Di Wu) exert anti-arthritic effects on type II collagen-induced arthritis in rats.

BACKGROUND: Anemone flaccida Fr . Schmidt (Ranunculaceae) (Di Wu in Chinese) is used to treat punch injury and rheumatoid arthritis (RA). However, the active compounds and underlying mechanism of action mediating the anti-arthritic effects of A. flaccida remain unclear. This study aims to evaluate the underlying action mechanism of A. flaccida crude triterpenoid saponins (AFS) on RA using a type II collagen (CII)-induced arthritis (CIA) rat model, and to assess the anti-inflammatory effects of the main active compounds of AFS, namely flaccidoside II, anhuienoside E, glycoside St-I4a, hemsgiganoside B, hederasaponin B, and 3-O-α-l-rhamnopyranosyl (1 â†’ 2)-ß-d-glucopyranosyl oleanolic acid 28-O-ß-d-glucopyranosyl (1 â†’ 6)-ß-d-glucopyranosyl ester. METHODS: Male Wistar rats (n = 50) were randomly separated into five groups (n = 10) and immunized by CII injection. AFS (200 or 400 mg/kg) and dexamethasone were orally administered for 30 days after establishing the model. The arthritis severity was assessed by paw volume using a plethysmometer. After 30 days of treatment, the right hind paws of the rats were obtained. Paw histology was analyzed by hematoxylin and eosin staining, and radiologic imaging was performed by micro-computed tomography. MTT assays were used to evaluate the cytotoxicity of AFS and its main compounds in RAW264.7 cells. Enzyme-linked immunosorbent assay kits were used to measure interleukin (IL)-6 and tumor necrosis factor (TNF)-α in serum and supernatants from AFS- and main AFS compound-treated RAW264.7 cells stimulated by lipopolysaccharide (LPS). RESULTS: Anemone flaccida crude triterpenoid saponins inhibited redness and swelling of the right hind paw in the CIA model. Radiological and histological examinations indicated that inflammatory responses were reduced by AFS treatment. Moreover, comparing with untreated rats, serum TNF-α (P = 0.0035 and P < 0.001) and IL-6 (P = 0.0058 and P = 0.0087) were lower in AFS-treated CIA rats at the dose of 200 and 400 mg/kg/day. AFS and its main compounds, including hederasaponin B, flaccidoside II, and hemsgiganoside B, significantly inhibited TNF-α (P = 0.0022, P = 0.013, P = 0.0015, and P = 0.016) and IL-6 (P = 0.0175, P < 0.001, P < 0.001, and P < 0.001) production in LPS-treated RAW264.7 cells, respectively. CONCLUSIONS: Anemone flaccida crude triterpenoid saponins and its main bioactive components, including hederasaponin B, flaccidoside II, and hemsgiganoside B, decreased pro-inflammatory cytokine levels in a CIA rat model and LPS-induced RAW264.7 cells.

A study of force distribution of loading stresses on implant-bone interface on short implant length using 3-dimensional finite element analysis.

OBJECTIVE: The purpose of this study is to evaluate the biomechanics of short dental implants. STUDY DESIGN: Three-dimensional finite element analysis was used to simulate stress distribution of 8-mm implants with 6 different diameters in I to IV types of bone densities; meanwhile, axial and oblique loads were applied in this study. RESULTS: It was found that the maximum Von-Mises stress varied significantly when the diameter was within 3.3 mm and 5 mm, whereas the change of peak stress was not obvious when the diameter was within 5.5 mm to 7.1 mm. The peak stress on the implant-bone interface increased with the reduction of bone density. The stress in types I and II had similar distribution and the same was true for types III and IV. CONCLUSIONS: These results revealed that implants with larger diameter (<5.5 mm) and bone quality enhancement may be preferable to get better clinical effects. Prospective clinical studies are required to confirm this.

Expression of cellular fibronectin mRNA in adult periodontitis and peri-implantitis: a real-time polymerase chain reaction study.

Cellular fibronectin (cFn) is a type of bioactive non-collagen glycoprotein regarded as the main substance used to maintain periodontal attachment. The content of cFn in some specific sites can reflect the progress of periodontitis or peri-implantitis. This study aims to evaluate the expression of cFn messenger RNA (mRNA) in tissues of adult periodontitis and peri-implantitis by real-time fluorescent quantitative polymerase chain reaction (PCR) and to determine its clinical significance. A total of 30 patients were divided into three groups of 10: healthy, adult periodontitis and peri-implantitis. Periodontal tissue biopsies (1 mm×1 mm×1 mm) from each patient were frozen in liquid nitrogen. Total RNA was extracted from these tissues, and the content, purity and integrity were detected. Specific primers were designed according to the sequence, and the mRNA expression levels of cellular fibronectin were detected by real-time PCR. The purity and integrity of the extracted total RNA were both high, and the specificity of amplified genes was very high with no other pollution. The mRNA expression of cFn in the adult periodontitis group (1.526±0.441) was lower than that in the healthy group (3.253±0.736). However, the mRNA expression of cFn in the peri-implantitis group (3.965±0.537) was significantly higher than that in the healthy group. The difference revealed that although both processes were destructive inflammatory reactions in the periodontium, the pathomechanisms were different and the variation started from the transcription level of the cFn gene.

Chitosan-phosphorylated chitosan polyelectrolyte complex hydrogel as an osteoblast carrier.

To simulate extra-cellular matrix, a novel three-dimensional scaffold of polyelectrolyte complex (PEC) hydrogel as an osteoblast carrier was synthesized. First, chitosan, a natural glycosaminoglycan, was modified by phosphorylation to obtain a water-soluble phosphorylated chitosan (P-content: 10.7 mass%). The PEC hydrogel was then formed from equal volumes of 0.173 mass% phosphorylated chitosan in water and 1 mass% chitosan in 1% (V/V) acetic acid solution. Rat osteoblasts were seeded in the hydrogel. The PEC hydrogel had a three-dimensional hierarchically-porous structure and good cytobiocompatibility for osteoblasts in vitro. It is concluded that the PEC hydrogel is a promising material as an osteoblast carrier.

[A clinical study of immediate dental implant and immediate restoration].

OBJECTIVE: To evaluate the clinical results of immediate implant and immediate restoration and to discuss the applying principles of these techniques. METHODS: Fourteen cases underwent immediate implant surgery for 37 dental implants immediately after the teeth or roots were extracted. Among them, 6 cases (14 implants) received immediate restoration after implant placement. The second stage operation and final restoration were performed 4 months on average postoperatively, and the mean follow-up time was 22 months. RESULTS: Two implants from 1 case were lost at 3 weeks after immediate implant and immediate restoration. The rest cases achieved good clinical results. Accumulative 4-year survival rate was 94.6%. CONCLUSIONS: The result of immediate implant and immediate restoration is predictable if the cases are properly selected.