Bone regeneration by nanohydroxyapatite/chitosan/poly(lactide-co-glycolide) scaffolds seeded with human umbilical cord mesenchymal stem cells in the calvarial defects of the nude mice.

In the preliminary study, we have found an excellent osteogenic property of nanohydroxyapatite/chitosan/poly(lactide-co-glycolide) (nHA/CS/PLGA) scaffolds seeded with human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro and subcutaneously in the nude mice. The aim of this study was to further evaluate the osteogenic capacity of nHA/CS/PLGA scaffolds seeded with hUCMSCs in the calvarial defects of the nude mice. Totally 108 nude mice were included and divided into 6 groups: PLGA scaffolds + hUCMSCs; nHA/PLGA scaffolds + hUCMSCs; CS/PLGA scaffolds + hUCMSCs; nHA/CS/PLGA scaffolds + hUCMSCs; nHA/CS/PLGA scaffolds without seeding; the control group (no scaffolds) (n = 18). The scaffolds were implanted into the calvarial defects of nude mice. The amount of new bones was evaluated by fluorescence labeling, H&E staining, and Van Gieson staining at 4 and 8 weeks, respectively. The results demonstrated that the amount of new bones was significantly increased in the group of nHA/CS/PLGA scaffolds seeded with hUCMSCs (p < 0.01). On the basis of previous studies in vitro and in subcutaneous implantation of the nude mice, the results revealed that the nHA and CS also enhanced the bone regeneration by nHA/CS/PLGA scaffolds seeded with hUCMSCs in the calvarial defects of the nude mice at early stage.

Xiaotan Sanjie decoction attenuates tumor angiogenesis by manipulating Notch-1-regulated proliferation of gastric cancer stem-like cells.

AIM: To determine the underlying mechanisms of action and influence of Xiaotan Sanjie (XTSJ) decoction on gastric cancer stem-like cells (GCSCs). METHODS: The gastric cancer cell line MKN-45 line was selected and sorted by FACS using the cancer stem cell marker CD44; the stemness of these cells was checked in our previous study. In an in vitro study, the expression of Notch-1, Hes1, Vascular endothelial growth factor (VEGF), and Ki-67 in both CD44-positive gastric cancer stem-like cells (GCSCs) and CD44-negative cells was measured by Western blot. The effect of XTSJ serum on cell viability and on the above markers was measured by MTT assay and Western blot, respectively. In an in vivo study, the ability to induce angiogenesis and maintenance of GCSCs in CD44-positive-MKN-45- and CD44-negative-engrafted mice were detected by immunohistochemical staining using markers for CD34 and CD44, respectively. The role of XTSJ decoction in regulating the expression of Notch-1, Hes1, VEGF and Ki-67 was measured by Western blot and real-time polymerase chain reaction. RESULTS: CD44(+) GCSCs showed more cell proliferation and VEGF secretion than CD44-negative cells in vitro, which were accompanied by the high expression of Notch-1 and Hes1 and positively associated with tumor growth (GCSCs vs CD44-negative cells, 2.72 ± 0.25 vs 1.46 ± 0.16, P < 0.05) and microvessel density (MVD) (GCSCs vs CD44-negative cells, 8.15 ± 0.42 vs 3.83 ± 0.49, P < 0.001) in vivo. XTSJ decoction inhibited the viability of both cell types in a dose-dependent manner in vitro. Specifically, a significant difference in the medium- (82.87% ± 6.53%) and high-dose XTSJ groups (77.43% ± 7.34%) was detected at 24 h in the CD44(+) GCSCs group compared with the saline group (95.42% ± 5.76%) and the low-dose XTSJ group (90.74% ± 6.57%) (P < 0.05). However, the efficacy of XTSJ decoction was reduced in the CD44(-) groups; significant differences were only detected in the high-dose XTSJ group at 48 h (78.57% ± 6.94%) and 72 h (72.12% ± 7.68%) when compared with the other CD44- groups (P < 0.05). Notably, these differences were highly consistent with the Notch-1, Hes1, VEGF and Ki-67 expression in these cells. Similarly, in vivo, XTSJ decoction inhibited tumor growth in a dose-dependent manner. A significant difference was observed in the medium- (1.76 ± 0.15) and high-dose XTSJ (1.33 ± 0.081) groups compared with the GCSCs control group (2.72 ± 0.25) and the low-dose XTSJ group (2.51 ± 0.25) (P < 0.05). We also detected a remarkable decrease of MVD in the medium- (7.10 ± 0.60) and high-dose XTSJ (5.99 ± 0.47) groups compared with the GCSC control group (8.15 ± 0.42) and the low-dose XTSJ group (8.14 ± 0.46) (P < 0.05). Additionally, CD44 expression was decreased in these groups [medium- (4.43 ± 0.45) and high-dose XTSJ groups (3.56 ± 0.31) vs the GCSC control (5.96 ± 0.46) and low dose XTSJ groups (5.91 ± 0.38)] (P < 0.05). The significant differences in Notch-1, Hes1, VEGF and Ki-67 expression highly mirrored the results of XTSJ decoction in inhibiting tumor growth, MVD and CD44 expression. CONCLUSION: Notch-1 may play an important role in regulating the proliferation of GCSCs; XTSJ decoction could attenuate tumor angiogenesis, at least partially, by inhibiting Notch-1.

Evaluation of in vitro and in vivo osteogenic differentiation of nano-hydroxyapatite/chitosan/poly(lactide-co-glycolide) scaffolds with human umbilical cord mesenchymal stem cells.

We aimed to evaluate the feasibility of the application of the nano-hydroxyapatite/chitosan/poly(lactide-co-glycolide) (nHA/CS/PLGA) scaffold seeded with human umbilical cord mesenchymal stem cells (hUCMSCs) in bone tissue engineering. We prepared the nHA/CS/PLGA, nHA/PLGA, CS/PLGA, and PLGA scaffolds, and tested their mechanical strength. We analyzed the surface antigen markers of hUCMSCs to determine their capability to differentiate into osteoblasts, chondrocytes, and adipocytes. The growth of hUCMSCs on the four types of scaffold was assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay) and observed using scanning electron microscopy (SEM). Quantitative analysis of alkaline phosphatase (ALP) activity and osteocalcin (OCN) content, as well as the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed. After 21 days, the subcutaneous implantations of the scaffolds samples seeded with hUCMSCs into nude mice were analyzed using immunohistochemical staining. The results showed that the mechanical strength of the nHA/CS/PLGA scaffold was enhanced. Furthermore, the nHA/CS/PLGA scaffolds were the most suitable for the adhesion, proliferation, and osteogenic differentiation of hUCMSCs in vitro and nude mouse subcutaneous implantation. The enhanced osteogenic inductivity of the nHA/CS/PLGA scaffolds for hUCMSCs might result from the addition of nHA and CS.

Xiaotan Tongfu granules contribute to the prevention of stress ulcers.

AIM: To investigate the efficacy and potential mechanism of Xiaotan Tongfu granules (XTTF) in stress ulcers. METHODS: One hundred sixty rats were randomly divided into 4 groups (n = 10) as follows: the model group (MP group), the control group (CP group), the ranitidine group (RP group) and the XTTF granule group (XP group). Rats in the MP group received no drugs, rats in the CP group received 0.2 mL of a 0.9% sodium chloride solution via oral gavage, and rats in the RP and XP groups received the same volume of ranitidine (50 mg/kg) or XTTF granule (4.9 g/kg). The cold-restraint stress model was applied to induce stress ulcers after 7 consecutive days of drug administration. Afterwards, rats were sacrificed at 0, 3, 6 and 24 h. Gastric pH was measured by a precise pH meter; gastric emptying rate (GER) was measured by using a methylcellulose test meal; myeloperoxidase activity (MPO), macrophage migration inhibitory factor (MIF), proliferating cell nuclear antigen (PCNA), and heat shock protein 70 (HSP70) were measured by immunohistochemical staining; and mucosal cell apoptosis was measured by transferase dUTP nick end labeling. RESULTS: In the cold-restraint stress model, the development of stress ulcers peaked at 3 h and basically regressed after 24 h. Gastric lesions were significantly different in the RP and XP groups at each time point. Interestingly, although this index was much lower in the RP group than in the XP group immediately following stress induction (7.00 ± 1.10 vs 10.00 ± 1.79, P < 0.05. Concerning gastric pH, between the RP and XP groups, we detected a statistically significant difference immediately after stress induction (0 h: 4.56 ± 0.47 vs 3.34 ± 0.28, P < 0.05) but not at any of the subsequent time points. For GER, compared to the RP group, GER was remarkably elevated in the XP group because a statistically significant difference was detected (3 h: 46.84 ± 2.70 vs 61.16 ± 5.12, P < 0.05; 6 h: 60.96 ± 6.71 vs 73.41 ± 6.16, P < 0.05; 24 h: 77.47 ± 3.17 vs 91.31 ± 4.34, P < 0.05). With respect to MPO and MIF, comparisons between the RP and XP groups revealed statistically significant differences at 3 h (MPO: 18.94 ± 1.20 vs 13.51 ± 0.89, P < 0.05; MIF: 150.67 ± 9.85 vs 122.17 ± 5.67, P < 0.05) and 6 h (MPO: 13.22 ± 1.54 vs 8.83 ± 0.65, P < 0.05; MIF: 135.50 ± 9.46 vs 109.83 ± 6.40, P < 0.05). With regard to HSP70, HSP70 expression was significantly increased in the RP and XP groups at 3 and 6 h compared to the MP and CP groups. In addition, comparing the RP and XP groups also showed statistically significant differences at 3 and 6 h. The expression of PCNA was higher in the RP and XP groups 3 h after stress induction. Between these two groups, small but statistically significant differences were observed at all of the time points (3 h: 69.50 ± 21.52 vs 79.33 ± 15.68, P < 0.05; 6 h: 107.83 ± 4.40 vs 121.33 ± 5.71, P < 0.05; 24 h: 125.33 ± 5.65 vs 128.50 ± 14.49, P < 0.05) except 0 h. With regard to apoptosis, the apoptotic activity in the RP and XP groups was significantly different from that in the MP and CP groups. The XP group exhibited a higher inhibition of cell apoptosis than the RP group at 3 h (232.58 ± 24.51 vs 174.46 ± 10.35, P < 0.05) and 6 h (164.74 ± 18.31 vs 117.71 ± 12.08, P < 0.05). CONCLUSION: The Xiaotan Tongfu granule was demonstrated to be similar to ranitidine in preventing stress ulcers. It exhibited multiple underlying mechanisms and deserves further study.

[Construction and identification of BIRC5 shRNA lentiviral expression vector].

AIM: Design and synthesis complementary DNA sequences of 3 pairs of short hairpin structure and a pair of negative control sequence by RNA interference technique and construct and identify a lentiviral interference vector with human BIRC5 gene as target gene. METHODS: The designed and synthesised Single-Stranded primer were annealed to Double-Stranded oligo sequences and subcloned into linear pMAGic lentiviral plasmid vector digested by enzyme Age I and EcoR I. Screening positive clone after transformed into DH5α competent cells and identified by PCR amplification and DNA sequencing. RESULTS: 335 bp straps of positive clone and 298 bp straps of negative clone form PCR amplification production have been obtained after gel electrophoresis, the designed and synthesised sequences have been contained in these clone straps confirmed by the result of DNA sequencing. CONCLUSION: Four pairs of BIRC5 shRNA recombinant lentiviral expression vector were constructed successfully, which laid the foundation for researching the inhibition of BIRC5 siRNA target against tumor cells proliferation, induction apoptosis and gene therapy.

[Killing effect of cytotoxicty T lymphocyte primed by two different methods preparation of dendritic cells pulsed with antigen on Tca8113 cells in vitro].

OBJECTIVE: To study the feasibility of dendritic cells (DCs) vaccine on the therapy of tongue carcinoma and find the better way of antigen load. METHODS: The antigen peptides of Tca8113 cells were obtained by acid eluted technique and repetitive freeze thaw method. Separating T cell and inducing dendritic cells were obtained from human peripheral blood monocyte. Divided into three groups: Weak acid elution method antigen group, anti-freeze-thaw method antigen group, and the control group (without tumor antigen). T cells and UCs were mixed to culture by different effector-target ratio. Using MTT assay measured the quantities of absorbance and calculated stimulation index. Dendrtic cells pulsed with antigen were mixed with T cells by different effector-target ratio. MIT assay was used to measure the quantities of absorbance and calculate killing rate. RESULTS: DCs vaccine was constructed successfully. DCs vaccine can induce T lymphocytes to kill Tca8113 cells and display the dose-effect relationship. There was significant difference among the three groups. The acid eluted and repetitive freeze thaw groups were better than the control group. The acid eluted group was better than repetitive freeze thaw group. CONCLUSION: DCs vaccine can induce T lymphocytes to kill Tca8113 cells. The antigen peptides obtained by acid eluted technique is better than repetitive freeze thaw method in immunotherapy of tongue cancer.

[Diagnosis and surgical approach of parapharyngeal space neoplasms].

PURPOSE: The aim of this study is to investigate the diagnosis and reasonable surgical approach for parapharyngeal space neoplasms. METHODS: A retrospective study was carried out from the data of 24 patients, who suffered from paralaryngeal space neoplasms and underwent surgical treatment from 1998 to 2006. The follow- up was carried out from 1 to 10 years. RESULTS: The patients had no specific symptoms initially. Most patients visited the doctor with cervical or submandibular masses and some of them with disorders of swallowing or speech. 87.5% of the neoplasms were benign and 12.5% were malignant. Neurogenic tumors and tumors originating from the parotid gland accounted the most. CT or MRI provided useful information such as their sizes, shapes, extents and the relationship between the neoplasm and surrounding tissues. The trans-cervical approach was used in 10 cases, the trans-parotid approach in 10 cases, the trans-mandible approach in 4 cases. One patient was dead, two patients were lost to follow up, the others had been cured without recurrence. CONCLUSIONS: CT and/or MRI are essentially useful tools for diagnosis and presurgical planning. Based on the trans-cervical approach, according to the size, origination and nature of the neoplasms, choose of the trans-parotid or the trans- mandibular approach for surgical treatment is reasonable. Supported by Natural Scientific Research Plan of Shanxi Province of China [Grant No.2006k09-G3(5) and 2005K14-G8(2)].

[Arthroscopic investigation of the status of temporomandibular joint in patients with temporomandibular joint pain].

OBJECTIVE: To investigate the status of the temporomandibular joint (TMJ) in patients with complaints of joint pain for improving clinical therapy. METHODS: Twenty three joints in twenty patients who complained of TMJ pain were examined radiographically and arthroscopically. RESULTS: There were at least one and more to six pathological changes could be found arthroscopically in the 23 temporomandibular joints, which were different in some respects with radiographic findings. CONCLUSION: TMJ related pain may be associated with pathological alterations in the TMJ, and synovitis may be one of the causes of TMJ pain.