[Effects of low level laser irradiation on the osteogenic capacity of sodium alginate/gelatin/human adipose-derived stem cells 3D bio-printing construct].

OBJECTIVE: To explore the effects of low level laser irradiation (LLLI) on the osteogenic capacity of three-dimensional (3D) structure by 3D bio-printing construct used human adipose-derived stem cells (hASCs) as seed cells. METHODS: Using hASCs as seed cells, we prepared sodium alginate/gelatin/hASCs 3D bio-printing construct, and divided them into four groups: PM (proliferative medium), PM+LLLI, OM (osteogenic medium) and OM+LLLI, and the total doses of LLLI was 4 J/cm². Immunofluorescence microscopy was used to observe the viability of the cells, and analyze the expression of the osteogenesis-related protein Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN). RESULTS: The 3D constructs obtained by printing were examined by microscope. The sizes of these 3D constructs were 10 mm×10 mm×1.5 mm. The wall thickness of the printed gelatin mold was approximately 1 mm, and the pores were round and had a diameter of about 700 µm. The cell viability of sodium alginate/gelatin/hASCs 3D bio-printing construct was high, and the difference among the four groups was not significant. On day 7, the expression of OCN from high to low was group OM+LLLI, PM+LLLI, OM and PM. There were significant differences among these groups (P<0.01), but there was no significant difference between group PM+LLLI and OM. On day 14, the expression of OCN in each group was higher than that on day 7, and there was no significant difference between group OM+LLLI and OM. The expression of Runx2 in group OM+LLLI was more than 90%, significantly higher than that in group OM (P<0.01). But the expression of Runx2 in group PM+LLLI and OM+LLLI were significantly lower than that in the non-irradiated groups. The expression of osteogenesis-related protein Runx2 and OCN were higher in OM groups than in PM groups. Furthermore, the irradiated groups were significantly higher than the non-irradiated groups. CONCLUSION: LLLI does not affect the cell viability of sodium alginate/gelatin/hASCs 3D bio-printing construct, and may promote the osteogenic differentiation of hASCs.

[Role of vitamin K-dependent protein Gas6 in the expression of endothelial cell adhesion molecule-1 and chemokines induced by Porphyromonas gingivalis lipopolysaccharide].

OBJECTIVE: Growth-arrest-specific protein 6 (Gas6) is a vitamin K-dependent protein and involved in cell proliferation, survival, adhesion and migration . Also it has been shown to play an important role in the inflammatory response .The aim of present study was to investigate the role of Gas6 in the process of the expression of adhesion molecules and chemokines of human umbilical vein endothelial cells (HUVECs) induced by Porphyromonas gingivalis lipopolysaccharide(P.g-LPS). METHODS: After up-regulation and down-regulation of the expression of Gas6, the vascular endothelial cells were stimulated with 1 mg/L P.g-LPS for 3 h and 24 h. Real-time quantitative polymerase chain reaction(real-time PCR) was taken to detect the expression of the cell adhesion molecules:intercellular adhesion molecule-1 (ICAM-1) and E-selectin, as well as chemokines:interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1). Wound healing assay was taken to observe the migration ability of endothelium cells in different groups. RESULTS: After 3 h of P.g-LPS stimulation, the expression of adhesion molecules and chemokine in the down-regulation group was not significantly different from that in the control group,while in the up-regulation group the decrease of E-selectin, ICAM-1, IL-8 and MCP-1 was 81%±0%, 47%±3%, 76% ± 3%, 26% ± 6% respectively. After 24 h of P.g-LPS stimulation, the expression of adhesion molecules and chemokine in down-regulation group was significantly higher than that in control group (2.06±0.07, 1.99±0.11, 3.14±0.15, 1.84±0.03 flod), while these molecules in the down-regulation group was significantly lower than in the control group (29%±1%, 62%±3%, 69%±1%, 41%±2%). Differences were statistically significant (P<0.01). Wounding healing assay showed that down-regulation of Gas6 enhanced migration ability of endothelial cells while up-regulation of Gas6 weakened this ability,which was consistent with the trend of real-time PCR result. CONCLUSION: Down-regulation of the Gas6 gene enhanced the expression of ICAM-1, E-selectin, IL-8 and MCP-1 in HUVECs after P.g- LPS stimulating, while up-regulaiton of the Gas6 gene weakened the expression of ICAM-1, E-selectin, IL-8 and MCP-1 in HUVECs after P.g-LPS stimulating,suggesting that Gas6 may play a role in the process of endothelial cell adhesion.

[Effect of low-level laser irradiation on proliferation and osteogenic differentiation of human adipose-derived stromal cells].

OBJECTIVE: To examine the in vitro effects of low-level laser irradiation (LLLI) on proliferation and differentiation of human adipose-derived stromal cells (hASCs). METHODS: Cultured cells were exposed to different doses of LLLI with a semiconductor diode laser (980 nm; 100 mW-12 W power output). The effects of laser on proliferation were assessed daily up to seven days of culture in cells irradiated for four consecutive days with laser doses of 2, 4, 6 or 8 J/cm2, the cells without irradiation were used as controls. Half of the cells were changed to osteogenic medium (OM) when they had grown to 70% confluence. The hASCs both with and without osteogenic supplements were divided into three groups, and each group was irradiated at doses of 0, 2 and 4 J/cm2. In order to examine the in vitro effects of LLLI on osteogenic differentiation of hASCs, the alkaline phosphatase activity was assessed on day 7, and alizarin red staining (AR-S) and quantitative detection were assessed on days 14 and 21. The expression of osteoblast master genes (ALP and Runx2) were tested on days 7 and 14. RESULTS: The proliferation medium(PM)+LLLI4 J/cm2 group had the highest multiplication rate. In the groups with osteogenic supplements, LLLI increased alkaline phosphatase activity and mineralized nodule formation, and stimulated the expression of ALP and Runx2. Furthermore, the effect became more obvious at high dose. CONCLUSION: Our data demonstrated that hASCs proliferation and osteogenic differentiation were enhanced by LLLI. With the increase of laser dose, the effect of LLLI would be enhanced at first, and then be decreased after reaching a peak.

Combination of IL-6, IL-10, and MCP-1 with traditional serum tumor markers in lung cancer diagnosis and prognosis.

Early detection and treatment is critically important for lung cancer patients. Inflammatory mediators such as IL-6, IL-10, and MCP-1 participate in lung cancer regulation. CEA, CA125, and ProGRP are commonly used serum tumor markers for lung cancer. In this study, we assessed the sensitivity and specificity of CEA, CA125, and ProGRP when used in combination with IL-6, IL-10, and MCP in lung cancer diagnosis. Serum from three different groups (healthy controls, individuals with high risk for lung cancer, and lung cancer patients) was collected. Electrochemiluminescence was used to detect expressions of CEA, CA125, and ProGRP; ELISA was used to examine serum levels of IL-6, IL-10, and MCP-1. Specificity and sensitivity of single as well as combination markers in lung cancer diagnosis were determined. Results indicated that CEA, CA125, ProGRP, and MCP-1 were significantly up-regulated in lung cancer patients as compared to those in controls and high risk individuals. Higher IL-6 and IL-10 levels were observed in both lung cancer patients and high-risk individuals as compared to those in controls. Highest sensitivity (95.2%) in cancer diagnosis was achieved when all six markers were used. This was followed by a combination of IL-6, IL-10, CEA, CA125, and ProGRP (92.6%). The most sensitive (88.6%). Four-marker combination was composed of IL-6, CEA, CA125, and ProGRP. As the combined usage of CEA, CA125, ProGRP, IL-6, IL-10, and MCP-1 significantly improved sensitivity of lung cancer detection; this biomarker arrangement may be beneficial for early diagnosis, treatment, and prognosis of lung cancer.

[Quantitative evaluation of fabricating complete denture by computer numerical control in manufacturing dentition and baseplate separately plus adhesive molding].

OBJECTIVE: To quantitatively evaluate the assembly precision of fabricating complete denture by computer numerical control (CNC) in manufacturing dentition and baseplate separately plus adhesive molding. METHODS: The 3D surface data of a standard edentulous maxilla plaster cast model and the temporary base-plate were obtained using an Activity 880 3D scanner. The data (data1) of a complete denture were designed using a set of computer aided design (CAD) software developed by the research group of this study. The pins without undercut were designed as 3D shape of the joining area of the dentition and the baseplate by using the software of Imageware 13.2 and Geomagic Studio 2013. Zero in the top and 0.05 mm in the rest surfaces of the retention pins were set for adhesive clearance. Zenotec T1 (5-axis milling machine) was employed to manufacture polymethyl methacrylate (PMMA) dentition and baseplate. Double sides posterior and one anterior "union teeth" were got. The teeth were inserted into the retention pins in the baseplate and cemented with self-curing resin (Huge Dental Material Co., Ltd). The denture was scanned with the 3D scanner to obtain dataset Data4. Data2 and Data3 registration was set in Data4, Data2 and Data3 were united to gain Data 5. The adhesive clearance on the top of the retentional pins was measured, which was originally designed into 0 mm, and the assembly precision of dentition and baseplate obtained. RESULTS: The average clearance measurements between the dentition and the baseplate: left molar teeth (0.44±0.04) mm, max 0.52 mm, min 0.29 mm; right molar teeth (0.52±0.07) mm, max 0.64 mm, min 0.28 mm; anterior teeth (0.60±0.10) mm, max 0.81 mm, min 0.40 mm; total average clearance (0.52±0.10) mm. CONCLUSION: The adhesive clearance can be controlled to the level of 0.5 mm when the joining part of the artificial teeth and the base was designed into the shape of retentional pins and the artificial dentition divided into 3 parts. We succeeded in using the CAD/ computer aided manufacturing (CAM) technology to fabricate the complete denture. Although the assembly precision of the dentition and the baseplate is not perfect, the results have proved that the technical routes are workable.

[A preliminary study for the effect of nano hydroxyapatite on human adipose-derived mesenchymal stem cells mixture 3D bio-printing].

OBJECTIVE: To study the effect of nano hydroxyapatite on human adipose-derived mesenchymal stem cells(hASCs) mixture 3D bio-printing for cells' proliferation and osteogenesis. METHODS: P5 hASCs were used as seed cells, 10 g/L nano hydroxyapatite was added into the cell-sodium alginate-gelatin mixture (concentration: 20 g/L sodium alginate, 80 g/L gelatin; cell density: 1×106/mL), then the mixture was printed by 3D bio-printer as the experimental group. And the cell-sodium alginate-gelatin mixture without nano hydroxyapatite was printed as the control group. Respectively, both the experimental and control groups were detected by microscope, CCK-8, Western blot and PCR at certain time pointsafter being printed, whose cells' proliferation and osteogenic differentiation were analyzed. RESULTS: The microscopic observation and CCK-8 results showed that the cells of the experimental group and the control group both had a good proliferation 24 h and 7 d after being printed. The Western blot results showed that 14 d after printing, the expression of Runt-related transcription factor 2 (RUNX2) had no statistical difference between the experimental group and control group. The PCR results showed that 14 d after printing, the expression of osteogenesis-related genes (RUNX2, osterix, and osteocalcin) was significantly higher in the experimental group than in the control group. CONCLUSION: Nano hydroxyapatite can increase osteogenic differentiation of the hASCs mixture after bio-printing, in which the cells still have a good proliferation.