BACKGROUND:The in vitro construction, maturation and differentiation of cellscaffold complexes into tissue-engineered bone is the necessary process of bone tissue engineering construction, but there are no uniform
methods and standards.
OBJECTIVE:To summarize the basic method and technology to build bone tissue engineering at present and to discuss the related development.
METHODS:A compute-based online search was conducted on the PubMed database and CNKI database for the articles related to the in vitro construction and culture of bone tissue engineering bone from January 1997 to
January 2013 with the key words of“bone tissue engineering, cellbiological scaffold, cellinoculation, seeding density, culture in vitro, bioreactor”in English and Chinese. Final y, 44 articles were included according to the inclusion and exclusion criteria.
RESULTS AND CONCLUSION:As the carrier of bone tissue engineering seed cells, the primary prerequisite of biological scaffold is sterile, because the sterile biological scaffold can be able to survive. Sterilization of biological scaffolds includes ultraviolet sterilization, 60Coγ-ray sterilization, soaking in ethanol with the volume fraction of 75%, autoclave method, and ethylene oxide sterilization. 60Coγ-ray sterilization is the common method in the biological scaffold sterilization. The inoculation density of seed cells is the key factors that influence the adhesion growth and proliferation of seed cells on the scaffolds. The adhesion between cells and scaffold materials wil be affected by the affinity of scaffolds, celladhesion and gravity, and other factors. The method for the inoculation of bone tissue engineering seed cells includes static inoculation and dynamic inoculation. Each construction method has its advantages and disadvantages. Overcomeing these disadvantages, forming a uniform construction method and ful y clinical application are the direction of future development.

Objective To investigate the expression of NDRG1 and its relationship to differentiation level and metastasis in prostate cancer. Methods The expression NDRG1 was examined by immunohistochemistry LSAB method in 86 samples of prostate cancer and benigh prostate hyperplasia. Results The NDRG1 was shown to be highly expressed in 30(73.3%)of prostate cancer and 70.0%of benign prostate hyperplasia.There were no evident difference between prostate cancer and prestate hyperplasia(P＞0.05).The expression of NDRG1 was gradually reduction with the Gleason score from 2-4 to 5-7 to 8-10 in prostate cancer, which was statistical significance(P＜0.05).The expression of NDRG1 in lymph node or bone metastasis was significantly lower than that in no metastasis of prostate cancer. There was a negative correlation between the expression of NDRG1 and lymph node or bone metastasis. The age had no correlation with the expression of NDRG1(P＞0.05).Conclusion NDRG1 is involved in the genesis and development of prostate cancer, which may be related to the differentiation level of prostate cancer and serve as a useful marker for early diagnosis of cancer and metastasis.

Objective To explore biological characteristics of chondrogenic differentiation of human periosteum-derived cells and the role of BMP4 in chondrogenic differentiation of these cells.Methods From October 2009 to September 2012,periosteum was obtained from tibia of patients undergoing leg amputation surgery,and isolated periosteum-derived cells by tissue culture method.Cells were cultured in DMEM/F12 containing 10％ fetal bovine serum,and morphology of cells were observed under inverted microscope.Periosteum-derived cells growth and the effect of BMP4 on cells growth examined by cell count using trypan blue,and cells growth curve was made.Experiment was divided into control group,chondrogenic differentiation group and BMP4 group,cells were expanded and differentiated in the presence or absence of BMP4 and complete medium.Then toluidine and immunohistochemical staining analyzed proteoglycan and collagenⅡ expression of these cells after 14 and 21 days.The expression of aggrecan,collagen Ⅱ and SOX9 mRNA of these cells using real-time PCR.Results (1) Periosteumderived cells adhered to growth in vitro,the shape of cell presented fibroblast-like morphology changing into polygonal after 1 week and round cell formation after 2 weeks chondrrogenic differemtiation.Growth curve showed that the passage 3 and 9 cells had similar reproductive activity.The passage 3 cells were positive for CD90 (21.07％) and CD105 (25.84％).(2)Toluidine bule staining and type Ⅱ collagen immunohistochemical staining showed BMP4 group (40.29 ± 4.29,56.74 ± 5.12) and chondrogenic differentiated group (19.27 ± 3.71,38.31 ± 4.25) ccould secrete proteoglycan and collagen Ⅱ,control group were negative (10.24 ± 1.21,15.28 ± 2.23),BMP4 group were significantly than chondrogenic differentiated group.(3) The expression of aggrecan,collagen Ⅱ and SOX9 mRNA of BMP4 group(25.76 ±0.57,6.48 ±0.48,2.91 ±0.18)were significantly higher than that of control group(2.37 ±0.24,1.12 ± 0.31,1.07 ± 0.22)and chondrogenic differentiated group(11.12 ± 0.38,2.24 ± 0.41,1.54 ± 0.35)using real-time PCR.Conclusion Periosteum-derived cells have strong proliferative,and have good potentials of differentiating into chondroblasts like mesenchymal stem cells.BMP4 can promote chondrogenic differentiation of periosteum-derived cells in vitro cultures.

BACKGROUND:Study confirms that bone morphogenetic protein can induce osteogenesis;however the ultrastructure of periosteal cells induced by bone morphogenetic protein-7 remains poorly reported.
OBJECTIVE:To study the bioactivity and ultrastructure of periosteal cells induced by bone morphogenetic protein-7 in vitro.
METHODS:The primary periosteal cells isolated from adult tibial bone were in vitro cultured, and then divided into experimental group and control group. In the experimental group, cells were cultured with bone morphogenetic protein-7 and culture adjuvant;while cells in the control group were only cultured with the adjuvant. Three samples in each group were tested at 5, 10, 15 days, respectively. The general structure of cultured cells was observed using von Kossa staining, and the ultrastructure was observed under transmission electron microscopy.
RESULTS AND CONCLUSION:The periosteal cells in the two groups grew wel in vitro, showing uniform morphology. Early cells were spindle-shaped, with strong three-dimensional sense and ful transparency;mitotic cells were short columnar or cubic shaped, there were a lot of rough endoplasmic reticulum and Golgi complex in osteoblasts under electron microscope. Later stage of cells developed from long fusiform into wide shuttle and irregular shape, there were a large number of matrix vesicles within the cells under the electron microscope. The membrane coating, alkaline phosphatase and calcium-binding protein in the cytoplasm, as wel as calcium crystals were found. The osteogenesis basement and lateral sides appeared projections, which were connected with adjacent bone cells. Induction of bone morphogenetic protein-7 in vitro promotes the osteoblasts proliferation, division and bone formation speed. The results suggest that bone morphogenetic protein-7 can significantly enhance the proliferation ability of osteoblasts in vitro.

Objective To investigate the expressions of carbohydrate antigen 19-9(CA19-9), carbohydrate antigen 15-3(CA15-3) and carbohydrate antigen 125(CA125) and their clinical significance in papillary thyroid carcinoma (PTC). Methods The expressions of CA19-9, CA15-3 and CA125 were detected by immunohistochemical MaxVision method in 80 cases of PTC and 80 cases of benign thyroid lesions (BTL), including 34 cases of nodular goiter, 26 cases of Hashimoto's thyroiditis and 20 cases of follicular adenoma. The relationship between expressions of CA19-9, CA15-3 and CA125 and the clinical pathological characteristics were analyzed. Results The expression rates of CA19-9, CA15-3 and CA125 in 80 cases of PTC were 85%, 100%and 43.8%respectively, compared with BTL, the difference was statistically significant (P < 0.01). There was no significant correlation between expressions of CA19-9, CA15-3 and CA125 and age, gender, number and diameter of tumor nodules, lymph node metastasis, and TNM staging (P > 0.05). The sensitivity of CA19-9, CA15-3 and CA125 in the differential diagnosis of PTC and BTL were 85%, 100% and 43.8% respectively, and the specificity were 91.3%, 36.3%and 91.3%, respectively. Conclusion The expressions of CA19-9, CA153 and CA125 are helpful for the differential diagnosis of PTC and BTL.

BACKGROUND: Because of complicated physiological environment and difficulty to control experimental conditions, it is difficult to get satisfactory results from in vivo studies of cell mechanics.OBJECTIVE: To study the action and mechanism of p38MAPK signaling pathways on myoblast apoptosis based on successful construction of in vitro mechanical stimulation models.METHODS: The C2C12 cells cultured in vitro were divided into control group and SB203580 treatment group. Cyclic tensile stress was applied on the C2C12 myoblast cells for 0, 6, 12 and 24 hours in each group. The Flexcell Strain Unit-5000T was used to expose C2C12 myoblast cell to an equiaxial cyclic of 15% magnitude and a frequency of 10 cycles/min, each cycle including the 3 s stretch and 3 s relaxation. Hoechst 33258 fluorescent staining and optical microscope were used to detect cell apoptosis. RT-PCR, flow cytometric analysis were used to observe the apoptosis of C2C12 myoblast cells and Western blotting were used to detect the activity of p38MAPK and p-p38MAPK. RESULTS AND CONCLUSION: The optical microscope tested the change in the morphology. Hoechst 33258 staining showed that after treatment with cyclic stress, the cell took the typical appearance of apoptosis with chromatin condensation and apoptotic bodies. RT-PCR and flow cytometry showed that with the extension of time the rate of the apoptosis of C2C12 myoblast cell increased. And cells imposed SB203580 before imposing cyclical tensile stress, the results showed that the apoptosis was markedly affected, and the p-p38MAPK expression declined apparently. These findings demonstrate that p38MAPK signaling pathways in stress mediated into C2C12 myoblast cell apoptosis plays an important role.

Objective:To analyze the correlation between serum claudin-1(Cla-1) level and the risk of papillary thyroid carcinoma(PTC) in patients with thyroid nodules.Methods:The clinical data of 345 patients with thyroid nodules were retrospectively analyzed. According to the pathological results, they were divided into PTC group and benign thyroid nodule(BTN) group. The difference of serum Cla-1 level between 2 groups and its correlation with the risk of PTC were analyzed.Results:In groups of PTC( n=225) and BTN( n=120), the median value of serum Cla-1 level was 14.03(10.30, 20.40) ng/mL. The differences in the median value[17.90(14.00, 22.93)ng/mL vs 9.40(8.15, 11.20) ng/mL] of serum Cla-1 level in the PTC and BTN were statistically significant by Wilcoxon signed-rank test. The prevalence of PTC in thyroid nodules increased gradually with increasing of serum Cla-1 level. Receiver operated characteristic curve analysis showed that the best diagnostic cut-off value of the PTC was 13.02 ng/ml of which the sensitivity was 81.8%, the specificity was 89.2%, and the area under curve(AUC) was the largest(AUC=0.944, P<0.01, 95% CI 0.922-0.965). Logistic regression analysis showed that elevated serum Cla-1 level increased the risk of PTC, and it was statistically significant( OR=4.334, 95% CI 1.662-11.303, P=0.003). There was a significant correlation among the serum Cla-1 level and gender, age, location of involvement, number and diameter of cancer nodules, extracapsular invasion of thyroid, lymph node metastasis, tumor stage and combined with Hashimoto′s thyroiditis( P<0.01). Conclusion:The serum level of Cla-1 may be one of risk factors to predict PTC, and it is related to the total amount of PTC tumor cells in vivo, but it was not related to the aggressive behavior of tumor.

Objective To discuss the application of gelatin sponge-hemocoagulase plugging agent in patients with pulmonary puncture bleeding.Methods The clinical data of 43 patients with hemorrhage caused by DSA-guided lung puncture biopsy,who received gelatin sponge-hemocoagulase plugging agent treatment at the Jining Municipal First People's Hospital of China between September 2021 and May 2023,were collected,and the hemostatic effect of gelatin sponge-hemocoagulase plugging agent was analyzed.Results Successful lung puncture needle biopsy was achieved in all the 43 patients.The puncture needle channel occlusion was accomplished by using gelatin sponge-hemocoagulase plugging agent.Five minutes after occlusion treatment,in one patient,whose moderate hemoptysis with moderate bleeding shadow before puncture needle biopsy changed to bloody sputum,the intrapulmonary bleeding shadow displayed on image became slightly enlarged when compared the size five minutes ago,while in all the remaining patients successful hemostasis was achieved,the hemoptysis disappeared and the pulmonary hemorrhage shadow was similar to that five minutes ago.No occlusion-related complications occurred in all patients.Conclusion For the treatment of pulmonary hemorrhage caused by DSA-guided lung puncture biopsy,gelatin sponge-hemocoagulase plugging agent is clinically safe and effective.

Objective To investigate the difference of iNOS and NQO1 in breast cancer with different mo-lecular subtypes and the correlation between clinical parameters,and to explore the clinical treatment of breast can-cer. Methods The data on 100 patients with breast cancer who had undergone modified radical mastectomy was retrospectively reviewed.Expressions of iNOS and NQO1 were detected by immunohistochemistry.Numeration data was processed using χ2test or Fisher′s exact test.Spearman correlation was applied to analyze different clinical mo-lecular pathological features.Results NQO1 expression was correlated with TNM staging and lymph node metasta-sis(χ2=6.603,4.938,P<0.05),and the expression rate of NQO1 in overexpressing of HER-2 was higher than that in type luminal A(χ2=8.341,P < 0.008). Expression of iNOS was related to expression of Ki-67,stage of TNM and lymph node metastasis(χ2=5.243,8.157,and 5.929,P<0.05),the expression rate of iNOS in lumi-nal A was significantly lower than that in type luminal B(χ2=7.990,P<0.008).Conclusions The expression of NQO1 and iNOS may be involved the process of oxidative stress associated with breast cancer,and it plays an im-portant role in the development of breast cancer. Different molecular level of oxidative stress between the types of breast cancer may be different,whose molecular mechanism needs to be further studied.

Objective To observe potential effect of radon hot springs on the changes of cell cycle and its regulatory proteins of CDK1,CDK2,CDK4,CDK6,CyclinD1,CyclinE1,WEE1,CDC25A in the peripheral blood lymphocytes of residents.Methods A random sampling method was used to persons 46 persons from the residents around radon hot spring in Wentang town,and 39 persons were selected from the control area without radon exposure.Flow cytometry was used to detect the changes of cell cycle and the expressions of cell cycle-related regulatory proteins.Multiple linear regression method was used to analyze the relationship between the expressions of cell cycle regulatory proteins and radon exposure.Results The percentages of cells at G0/G1 phase and S phase in lymphocytes were different in the two groups (t =2.250,-2.382,P ＜ 0.05).The expression levels of CDK1,CDK6 and CyclinE1 in the peripheral blood lymphocytes of radon hot spring group were significantly decreased (t =4.770,11.419,5.238,P ＜ 0.05) and negatively correlated with radon exposure (t =-5.097,-11.128,-5.117,P ＜0.05).The expression levels of CDK2,CDK4,CyclinD1,WEE1 and CDC25A in the peripheral blood lymphocytes of radon hot spring group were increased but not significantly(P ＞ 0.05).Conclusions The incidences of a higher ratio of S-phase cells and lower expression levels of CDK1,CDK6 and CyclinE1 in the peripheral blood lymphocytes of residents in Wentang town may be related to long-term radon exposure.