Proteomic analysis of mitochondria from infantile hemangioma endothelial cells treated with sodium morrhuate and its liposomal formulation.

Hemangioma is the most common benign tumor of infancy. The aim of this study is to evaluate the biological effects of sodium morrhuate (SM) and its liposomal formulation on infantile hemangioma endothelial cells (IHECs). Morphological analysis revealed that exposure to liposomal sodium morrhuate (LSM) preferentially caused apoptotic death in IHECs, manifested as shrunken configuration and formation of apoptotic bodies. In contrast, necrotic death was prominent in IHECs treated with an equal concentration of SM. By means of proteomic analysis and confirmation experiments, we revealed that the apoptosis-inducing effects of LSM were associated with an upregulation of a set of genes involved in mitochondrial death pathway, including apoptosis-inducing factor, cytochrome c1, caspase-8, and lamin B1. In conclusion, our data highlight the proapoptotic activity of LSM in IHECs through the mitochondrial apoptotic pathway and may provide a promising avenue to treat hemangiomas of infancy.

Cartilage tissue engineering with demineralized bone matrix gelatin and fibrin glue hybrid scaffold: an in vitro study.

To develop a cartilage-like tissue with hybrid scaffolds of demineralized bone matrix gelatin (BMG) and fibrin, rabbit chondrocytes were cultured on hybrid fibrin/BMG scaffolds in vitro. BMG scaffolds were carefully soaked in a chondrocyte-fibrin suspension, which was polymerized by submerging the constructs into thrombin-calcium chloride solution. Engineered cartilage-like tissue grown on the scaffolds was characterized by histology, immunolocalization, scanning electron microscopy, biochemical assays, and analysis of gene expression at different time points of the in vitro culture. The presence of proteoglycan in the fibrin/BMG hybrid constructs was confirmed by positive toluidine blue and alcian blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrices. Chondrogenic properties were further demonstrated by the expression of gene-encoded cartilage-specific markers, collagen type II, and aggrecan core protein. The glycosaminoglycan production and hydroxyproline content of tissue grown on the fibrin/BMG hybrid scaffolds were higher than that of the BMG group. In conclusion, the fibrin/BMG hybrid scaffolds may serve as a potential cell delivery vehicle and a structural basis for cartilage tissue engineering.

Copolymer cell/scaffold constructs for bone tissue engineering: co-culture of low ratios of human endothelial and osteoblast-like cells in a dynamic culture system.

The aim of this study was to compare the effect of different ratios of human umbilical vein endothelial cells (HUVECs) on osteogenic activity of human osteoblast-like cells (HOB) and capillary-like structure (CLS), seeded into copolymer scaffolds in a dynamic culture system. HOB and HUVEC were co-cultured into poly(L-lactide)-co-(1,5-dioxepan-2-one) [poly(LLA-co-DXO)] scaffolds at ratios of 5:1 (5:1 group) and 2:1 (2:1 group). Samples were collected after 5, 15, and 25 days. Cross-sections were processed and the CLS from HUVEC was disclosed in both groups. Cell viability was determined by dsDNA assay. Cells seeded at the ratio of 5:1 had good viability. Total RNA was isolated and the reverse transcription reaction was performed. The influences on the expression of several osteogenic genes were various with regarding to different ratios of HUVEC demonstrated by the PCR array. The RT-PCR results was in consistent with the PCR array results that several osteogenesis related genes had higher expression in the 5:1 group than in the 2:1 group, especially at day 25, such as alkaline phosphatase, insulin-like growth factor 1 (IGF1), and so forth. ELISA showed that the production of IGF1 after 25 days of incubation were higher in cells co-cultured at the 5:1 ratio than at the 2:1 ratio. The results show that under dynamic culture conditions, co-culture of HOB with a low ratio of HUVEC in copolymer scaffolds results in CLS formation and significantly influenced the expression of osteogenic markers.

Comparison of short-run cell seeding methods for poly(L-lactide-co-1,5-dioxepan-2-one) scaffold intended for bone tissue engineering.

Constructs intended for bone tissue engineering are influenced by the initial cell seeding procedure. The seeding method should be rapid, convenient, improve cell spatial distribution, and have no negative effects on cellular viability and differentiation. This study aimed to compare the effect of short-run seeding methods (centrifuge and vortex) with a static method on the scaffolds prepared from poly(L-lactide-co-1,5-dioxepan-2-one) by solvent-casting particulate-leaching (SCPL) technique. Human osteoblast-like cells (HOB) were seeded by the three methods described above. The seeding efficiency was determined by attached cell numbers. Cellular proliferation was analyzed by WST-1 and dsDNA assay. Cell distribution was examined by scanning electron (SEM) and fluorescence microscopy. Expression of alkaline phosphatase (ALP), collagen type I (Col I), osteocalcin (OC) and proliferating cell nuclear antigen (PCNA) were determined by real time RT-PCR. Results indicated that centrifuge and vortex increased seeding efficiency and had no negative effects on cellular viability. The data obtained by the fluorescence microscope confirmed the SEM results that the vortex method improved cell distribution through the scaffolds more than the other two methods (p<0.05). The RT-PCR results showed no significant differences on the expression of mRNA between the three methods of the above markers. The vortex method was found to be a simple and feasible seeding method for the poly(L-lactide-co-1,5-dioxepan-2-one) scaffolds.

Fluorine-induced apoptosis and lipid peroxidation in human hair follicles in vitro.

Fluoride is an essential trace element for human body; however, exposure to high amounts of fluoride has been documented to be correlated with an increasing risk of hair loss. To date, little is known about the mechanism(s) of how fluoride affects hair follicles. Here, we demonstrated that middle (1.0 mmol/L) and high (10.0 mmol/L) concentrations of sodium fluoride (NaF) significantly inhibited hair follicle elongation in vitro, but low NaF (0.1 mmol/L) showed little influence. Moreover, treatment with high levels of NaF resulted in a marked increase in terminal dUTP nick end labeling-positive cells in the outer layer of the outer root sheath, the dermal sheath, and the lower bulb matrix surrounding dermal papilla. Furthermore, the enhanced apoptosis was coupled with an increased oxidative stress manifested as higher malondialdehyde content. Additionally, the presence of selenium considerably antagonized the effects of middle NaF on hair follicles, with regard to either the suppression of hair growth or the induction of oxidative stress and apoptosis. In conclusion, exposure to high levels of fluoride compromises hair follicle growth and accelerate cell apoptosis in vitro. The toxicity of fluoride can be reduced by selenium, at least partially via the suppression of intracellular oxidative stress.

[Study of immunolipo-sodium morrhuate applied on human hemangioma endothelial cells in vitro].

PURPOSE: To explore the method of preparing immunolipo-sodium morrhuate and evaluate its effect on human hemangioma endothelial cells in vitro. METHODS: Using SPDP((N-Succinimidyl-3-(2-pyridyldithio)) propionate) as cross-linker, anti-VEGFR2/KDR monoclone antibody was combined to the liposome surface to prepare immunolipo-sodium morrhuate by extruding method, and then its effect on human hemangioma endothelial cells in vitro was observed by laser scanning confocal microscope, inverted microscope, Gimsa staining, transmission electron microscope, MTT and flow cytometry. RESULTS: The average diameter of the immunoliposome was 122.9 nm, which had a very good stability when compared with normal liposome, it had stronger and faster combining ability, its potential to induce apoptosis was much more prominent, and its toxic effect on human hemangioma endothelial cells was gentle, which was similar to normal liposome. CONCLUSION: We have prepared immunolipo-sodium morrhuate successfully, which has very good specific initiative targetting ability in vitro and can induce pervasive apoptosis of human hemangioma endothelial cells.

[Three-dimensional finite element analysis of three conjunctive methods of free iliac bone graft for established mandibular body defects].

OBJECTIVE: To analyze the stress and displacement distribution of 3D-FE models in three conjunctive methods of vascularized iliac bone graft for established mandibular body defects. METHODS: Using computer image process technique, a series of spiral CT images were put into Ansys preprocess programe to establish three 3D-FE models of different conjunctions. RESULTS: The three 3D-FE models of established mandibular body defects by vascularized iliac bone graft were built up. The distribution of Von Mises stress and displacement around mandibular segment, grafted ilium, plates and screws was obtained. CONCLUSION: It may be determined successfully that the optimal conjunctive shape be the on-lay conjunction.

[Apoptotic study on the effect of fluorine and selenium on the human hair follicle in vitro].

OBJECTIVE: The aim of this study was to observe the human hair follicle apoptosis status affected by fluorine and the antagonism effect by selenium in vitro. METHODS: The single hair follicles were separated and cultured, then they were added in different concentrations of sodium fluoride and sodium selenite. Chosen the appropriate concentrations, they were divided into 7 groups. The TUNEL was used to investigate the apoptotic cells of different parts. The morphous of hair follicles was observed consecutively and electron microscope was used. RESULTS: We found that in 1 mmol/L and 10 mmol/L sodium fluoride groups, when the human hair follicles in vitro were cultured on the 5th day, the apoptotic cells of outer root sheath (ORS), dermal sheath and hair papilla, hair bulb were obviously increased. But 0.01 mmol/L sodium selenite weakened the toxicity of 1 mmol/L sodium fluoride at the outer root sheath and hair bulb (P < 0.05). CONCLUSIONS: Different concentrations of sodium fluoride had different effect on the growth of human hair follicle in vitro which were cultured on 5th day. Sodium fluoride of certain concentration could accelerate the apoptosis of human hair follicle in vitro. Sodium selenite of certain concentration could act antagonism to the toxicity of sodium fluoride.

[The experimental study of lipo-sodium morrhuate promoting apoptosis of ECV-304 cells].

OBJECTIVE: To evaluate the effect of lipo-sodium morrhuate on ECV-304 cell line. METHODS: The effect lipo-sodium morrhuate was evaluated by toxicology trial (MTT), electron microscope, DNA electrophoresis and flow cytometer. RESULTS: The toxicology results showed, that the number of vital cells in lipo-sodium morrhuate group decreased slowly. The electron microscope exhibited apoptosis in the lipo-sodium morrhuate group. And there were typical DNA ladder in DNA electrophoresis and typical apoptosis peak in flow cytometer. The apoptosis rate was 22.23%. CONCLUSIONS: Unlike the normal preparation of sodium morrhuate, lipo-sodium morrhuate could induce apoptosis of ECV-304 cell line.

[Mechanical force distribution on cleft maxillary finite-element models after alveolar and hard palate bone graft].

OBJECTIVE: To investigate the biomechanical force distribution of three different cleft maxillary finite-element models pre- and post-bone graft with specified load to certain area of the models. METHODS: Developing a cleft palate bony model from a 15-year cleft palate male CT scan DICOM data and generating alveolar bone-grafted, alveolopalatal bone-grafted cleft maxillary finite-element model was set up through gluing the graft model. Also the pre-grafted model as compared, vector lip force on the anterior and anteriolateral face of the alveolar ridge was applied, and studied the press distribution properties and localized area. RESULTS: The press principal spread along the alveolar ridge and focused on anterior wall of maxillary prior to graft. But the press tended to be evenly distributed after bone grafted, whether alveolar or/and hard palate bone grafted. The grafted bone could resisted the medially deformation of alveolar crest and decreased the shear press to the nasal base bony structure. The map showed no significant differences along with alveolar or/and hard palate bone graft. CONCLUSION: The postoperative lip pressure plays an important role for the deformation and deviation of alveolar ridge. Alveolar bone graft could even the distribution of the stresses and should be emphasized. But the grafted bony palatal appears superior to but no significant mapping and anti-deformation difference with alveolar bone graft.

[Stress distribution analysis of the surface of condyle during simulated fracture healing of the neck of condyle by miniplate osteosynthesis].

OBJECTIVE: To find the reasons why patients always have temporomandibular disorders (TMD) after condylar fracture by analyzing the stress distribution change of the condylar surface whose subcondylar fracture ware fixed by miniplate during the whole healing process. METHODS: Analyzing the stress distribution change of the condylar surface whose subcondylar fracture were fixed by miniplate during the whole healing process by three-dimension finite element method (3D FEM). RESULTS: During the whole healing process of the fracture, the miniplate osteosynthesis was helpful to the biomechanics environment rehabilitation of the condylar local, but it still had difference with normal after fixing 12 weeks long. CONCLUSION: The difference of stress distribution of condylar surface that fractured and fixed by miniplate with normal may be part of the reason of TMD after the subcondylar fracture miniplate osteosynthesis.

Dental fluorosis and dental caries in permanent teeth: rural schoolchildren in high-fluoride areas in the Shaanxi province, China.

OBJECTIVES: To study the dental fluorosis and caries in the permanent teeth of 12 to 13-year-old children in fluorosis-endemic areas; to assess the relationship between fluorosis and the fluoride content of the drinking water and the relationship between caries and the fluoride content of the water; finally, to analyze the effect of fluoride intake and water stored in clay pots on dental fluorosis. MATERIAL AND METHODS: 477 children were divided into 5 groups (A to E) according to the fluoride concentration of the waters, i.e. by 0.4, 1.0, 1.8, 3.5, and 5.6 mg F/l, respectively. Dental fluorosis was assessed by TF score and caries by the DMF-T index. A questionnaire was used to obtain information about water storage and other information relevant to children's fluoride intake. RESULTS: A positive relationship was found between the mean TF scores and the water fluoride concentration. In groups B and D, the TF score was higher in 13-year-olds than in 12-year-olds. Caries prevalence and mean DMF-T ranged from 2.6% and 0.03 (group E) to 22.1% and 0.38 (group A). Storage of water in clay pots seemed to increase the severity of fluorosis slightly, and to decrease the caries prevalence. CONCLUSIONS: Defluoridation of drinking water, or--alternatively--the provision of low-fluoride water sources, should be given high priority in the examined Shaanxi rural areas. Fluoride concentration of drinking water should be maximum 0.6 mg/l. Storage of water in the local clay pots may increase the severity of dental fluorosis.