微生物发酵牛骨粗胶原蛋白制备胶原多肽

将筛选的安全无毒高产胶原蛋白酶的蜡状芽孢杆菌用于牛骨粗胶原蛋白发酵制备牛骨胶原多肽,结果表明该菌株发酵能显著提高牛骨粗胶原蛋白溶液中游离氨基酸的含量,以水解度和胶原多肽的含量为指标,针对影响发酵的五因素(接种量、pH、牛骨粗胶原蛋白的添加量、发酵温度和发酵时间),在单因素实验确定了较优化的参数范围的基础上,经响应面分析法确定了最佳发酵条件为:接种量1%,pH值6.0,牛骨粗胶原蛋白添加量30.0 g/L,发酵温度27℃,发酵时间42 h,同时获得最大胶原多肽含量的实验值为5.54±0.09 mg/mL。

基因重组人骨形成蛋白-2胶原膜复合物防止大鼠矢状缝扩张后复发的作用

目的 研究基因重组人骨形成蛋白 2 (rhBMP 2 )胶原膜复合物对大鼠顶骨矢状缝扩张后复发的影响。方法 10周龄SD雄性大鼠 32只 ,随机分为空白对照组、单纯扩张组、扩张加胶原膜植入组、扩张加rhBMP 2胶原膜复合物植入组 ,每组 8只。大鼠左右顶骨钻孔 ,建立矢状缝扩张模型 ,第 2 1天时取出扩张装置 ,第 2 8天时处死动物 ;用游标卡尺测量相应时间点骨孔间的距离并计算扩张复发率。动物处死后完整取下顶骨 ,去除表面的软组织 ,采用组织学方法观察各组大鼠矢状缝处组织改建情况 ,用原子吸收分光光度计检测该处的钙含量。结果 施加扩张力后 ,矢状缝中新骨形成活跃 ,rhBMP 2胶原膜复合物植入组形成特有的骨质桥连接 ,钙含量显著高于其他组 (P <0 .0 1) ,扩张复发率则明显降低 (P <0 .0 1)。结论 rhBMP 2胶原膜复合物可以降低扩张力作用下大鼠顶骨矢状缝的扩张复发率 ,为临床上降低扩弓复发率提供新的思路。

新的骨吸收生化指标——血清Ⅰ型胶原羧基端吡啶并啉交联肽

简述Ⅰ型胶原羧基端吡啶并啉交联肽（ＩＣＴＰ）的生化特点、测定方法及临床意义。ＩＣＴＰ是Ⅰ型胶原特异成分，以完整的免疫原性蛋白形式进入血循环，主要反映骨转换，并与尿吡啶肽（ＰＹＲ）相关。目前多采用放射免疫法测定，肝素或ＥＤＴＡ抗凝的血浆ＩＣＴＰ浓度与血清中ＩＣＴＰ浓度无显著差别，相关系数ｒ均大于０．９７。ＩＣＴＰ可能与多种疾病有关，包括：骨质疏松症、特发性生长激素缺乏病、甲亢、甲旁腺功能亢进症、多发性骨髓瘤、类风湿性关节炎等。

骨钙素与代谢性骨病

骨钙素(OC)是一种含量最丰富的骨非胶原蛋白,大部分与骨基质内的羟磷灰石紧密结合,其生理功能尚未完全明确。OC的基因表达受多种激素、维生素和局部细胞因子的调节。OC是成骨细胞的特异性产物,可反映成骨细胞的活性和骨代谢转换水平。了解OC的生物学特征,其表达调控机制以及监测血清OC浓度,对代谢性骨病的诊断、治疗具有重要的临床意义。

骨形成蛋白复合胶原与HA涂层种植体应用的临床研究

目的 观察在人体颌骨植入复合骨形成蛋白 (BMP) \胶原的HA涂层钛种植体后种植体周围新骨形成情况 ,评价BMP \胶原对人体的安全性和诱导骨再生的有效性。方法 将 8个复合有BMP、胶原与HA涂层种植体和 8个HA涂层种植体植入病人颌骨 ,于术后 2、4、8、12周拍摄X线根尖片 ,观察植入区组织反应和利用计算机软件进行骨密度量化分析 ,再将量化的灰度值进行统计检验 ,比较实验组与对照组骨质形成的差别。结果 两组患者均没有出现过敏反应 ;实验组与对照组牙种植体周围骨密度比较有显著性统计学意义 (P <0 .0 5 )。结论 BMP、胶原抗原性低 ,临床上使用安全 ;复合BMP。

肿瘤坏死因子受体和配体超家族的新成员

骨原蛋白(OPG)/核因子_κB受体激活剂的配体(RANKL)/核因子_κB的受体激活剂(RANK)是肿瘤坏死因子受体和配体起家族成员。RANKL由成骨细胞前体/骨髓基质细胞和激活的T淋巴细胞合成,通过结合破骨细胞或树突状细胞表面的RANK受体,促进破骨细胞的形成、融合、激活和存活,并有助于树突状细胞对抗原的提呈作用,OPG作为RANKL的假受体,对此过程具有抑制作用。此外,OPG/RANKL/RANK系统在调节淋巴系统发育、哺乳期动物乳腺腺泡的形成以及大动脉钙化中也起着重要的作用,是一组多功能的细胞因子系统。

Morphological and serum hyaluronic acid, laminin and type Ⅳ collagen changes in dimethylnitrosamine-induced hepatic fibrosis of rats

AIM： To study the morphological and serum hyaluronic acid （HA）, laminin （LN）, and type IV collagen changes in hepatic fibrosis of rats induced by dimethylnitrosamine （DMN）.METHODS： The rat model of liver fibrosis was induced by DMN. Serum HA, type IV collagen, and LN were measured by ELISA. The liver/weight index and morphological changes were examined under electron microscope on d 7, 14, 21, and 28 by immunohistochemical alpha smooth muscle actin α-SMA staining as well as Sirius-red and HE staining.RESULTS： The levels of serum HA, type IV collagen and LN significantly increased from d 7 to d 28 （P = 0.043）. The liver/weight index increased on d 7 and decreased on d 28. In the model group, the rat liver stained with lie and Sirius-red showed evident hemorrhage and necrosis in the central vein of hepatic 10 Iobules on d 7. Thin fibrotic septa were formed joining central areas of the liver on d 14. The number of α-SMA positive cells was markedly increased in the model group. Transitional hepatic stellate cells were observed under electron microscope. All rats in the model group showed micronodular fibrosis in the hepatic parenchyma and a network of α-SMA positive cells. Typical myofibroblasts were embedded in the core of a fibrous septum. Compared to the control group, the area-density percentage of collagen fibrosis and pathologic grading were significantly different in the model group （P〈0.05） on different d （7, 14, and 28）. The area-density percentage of collagen fibrosis in hepatic tissue had a positive correlation with the levels of serum HA, LN, and type IV collagen.CONCLUSION： The morphological and serum HA, type IV collagen, and LN are changed in DMN-induced liver fibrosis in rats.

Enrichment of putative human epidermal stem cells based on cell size and collagen type IV adhesiveness

The enrichment and identification of human epidermal stem cells （EpSCs） are of paramount importance for both basic research and clinical application. Although several approaches for the enrichment of EpSCs have been established, enriching a pure population of viable EpSCs is still a challenging task. An improved approach is worth developing to enhance the purity and viability of EpSCs. Here we report that cell size combined with collagen type IV adhesiveness can be used in an improved approach to enrich pure and viable human EpSCs. We separated the rap- idly adherent keratinocytes into three populations that range in size from 5-7 μm （population A）, to 7-9 μm （population B）, to ≥9μm （population C） in diameter, and found that human putative EpSCs could be further enriched in population A with the smallest size. Among the three populations, population A displayed the highest density of plintegrin receptor, contained the highest percentage of cells in G0/G1 phase, showed the highest nucleus to cytoplasm ratio, and possessed the highest colony formation efficiency （CFE）. When injected into murine blastocysts, these cells participated in multi-tissue formation. More significantly, compared with a previous approach that sorted putative EpSCs according to pl-integrin antibody staining, the viability of the EpSCs enriched by the improved approach was significantly enhanced. Our results provide a putative strategy for the enrichment of human EpSCs, and encourage further study into the role of cell size in stem cell biology.

Expression of Collagen Ⅳ, Fibronectin, Laminin in Non-small Cell Lung Cancer and Its Correlation with Chemosensitivities and Apoptosis~*

Objective： To study the expression of extracellular matrix （ECM） proteins including Collagen Ⅳ （Co Ⅳ）, Fibronectin, Laminin in human non-small cell lung cancer （NSCLC） specimens and the relationship between ECM and cell adhesion, proliferation, apoptosis and drug sensitivity in NSCLC cell line. And to investigate the role of phosphatidylinositol 3-kinase （PI3-K） in signal transduction of Co Ⅳ in NSCLC. Methods： The expression of ECM proteins was detected by using immunohistochemical staining （Envision＇s）. Adherent cells were stained with 1% methylene blue. Cell proliferation and cytotoxic effects were monitored by MTT assay. Cell apoptosis was analyzed by FITC-Annexin V/PI double staining variables flow cytometry （FCM）. Results： The expression rate of Co Ⅳ （93%） was the highest compared to others in NSCLC stroma. After treated with Co Ⅳ, the adhesion of H1299 cells was increased and the cytotoxicity of cis-platinum （DDP） against H1299 cells was decreased compared to the control （P〈0.05）. After treated with Co Ⅳ both survival and proliferation rates were higher and apoptosis rate was lower than without Co Ⅳ （P〈0.05）. PI3-K inhibitor LY294002 decreased both survival and proliferation rates （82.7%±2.0% and 75.2%±6.8%, respectively）, even on Co Ⅳ-coated surface （92.2%±2.8% and 84.6%±9.2%, respectively）. And it also helped DDP increase apoptosis. Conclusion： ECM remodeling existed in NSCLC. Co Ⅳ protected NSCLC cells from DDP-induced apoptosis and weakened the cytotoxicity of DDP. PI3-K pathway might be the crucial mechanism of apoptosis impairment and drug resistance.

The Use of Bone Morphogenetic Protein-7 and Resveratrol in Collagen Type II of Articular Cartilage

This study aimed to investigate the effects of resveratrol and bone morphogenetic protein 7 on type II collagen from superficial and middle zone of porcine articular chondrocytes. Articular cartilage was isolated from dissected porcine knee joint n = 12. Isolated cells were plated as monolayers at a density of 1 × 105 cells/well in 12-well culture plates and incubated at 37℃ in a humid atmosphere of 5% carbon dioxide and 95% air. Cell cultures were treated for four days with various concentrations of bone morphogenetic protein-7 and resveratroL Cells were then collected and analysed for collagen type II expression by real time polymerase chain reaction and protein level quantification by enzyme-linked immunosorbent assay. Cartilage tissue sections were localised for collagen type II by immunohistochemistry. Moreover, resveratrol and bone morphogenetic protein-7 effects on cartilage matrix contents were analysed by histology. Resveratrol and bone morphogenetic protein-7 stimulates expression of collagen type II mRNA and protein level accumulation in the surface zone and middle zone at 50μM ＋ 300 ng/ml （RSV ＋ BMP-7）. Immunohistochemistry results confirmed the presence of collagen type II on articular cartilage. Histological tissue sections confirmed that chondrocytes were obtained from different zones of articular cartilage. The study suggests that a combination of bone morphogenetic protein-7 and resveratrol up-regulate the expression and synthesis of collagen type II.

Application of collagen-chitosan/fibrin glue asymmetric scaffolds in skin tissue engineering

To create a scaffold that is suitable for the construction of tissue-engineered skin, a novel asymmetric porous scaffold with different pore sizes on either side was prepared by combining a collagen-chitosan porous membrane with fibrin glue. Tissue-engineered skin was fabricated using this asymmetric scaffold, fibroblasts, and a human keratinocyte line (HaCaT). Epidermal cells could be seen growing easily and achieved confluence on the fibrin glue on the upper surface of the scaffold. Scanning electron microscopy showed typical shuttle-like fibroblasts adhering to the wall of the scaffold and fluorescence microscopy showed them growing in the dermal layer of the scaffold. The constructed composite skin substitute had a histological structure similar to that of normal skin tissue after three weeks of culture. The results of our study suggest that the asymmetric scaffold is a promising biologically functional material for skin tissue engineering, with prospects for clinical applications.

Calcium citrate： a new biomaterial that can enhance bone formation in situ

Objective： To investigate the effect of a new biomaterial combining calcium citrate and recombinant human bone morphogenetic protein-2 （rhBMP-2） on bone regeneration in a bone defect rabbit model. Methods： Totally 30 male New Zealand white rabbits were randomly and equally divided into calcium citraterhBMP-2 （CC-rhBMP-2） group and rhBMP-2 only group. Two 10 ram-long and 5 ram-deep bone defects were respectively created in the left and right femoral condyles of the rabbits. Subsequently 5 pellets of calcium citrate （10 mg） combined with rhBMP-2 （2 rag） or rhBMP-2 alone were implanted into the bone defects and compressed with cotton swab. Bone granules were obtained at 2, 4 and 6 weeks after procedure and received histological analysis. LSD t-test and a subsequent t-test were adopted for statistical analysis. Results： Histomorphometric analysis revealed newlyformed bones, and calcium citrate has been absorbed in the treatment group. The percent of newly formed bone area in femoral condyle in control group and CC-rhBMP-2 group was respectively 31.73%±1.26% vs 48.21%±2.37% at 2 weeks; 43.40%±1.65% vs 57.32%±1.47% at 4 weeks, and 51.32%±7.80% vs 66.74%±4.05% at 6 weeks （P〈0.05 for all）. At 2 weeks, mature cancellous bone was observed to be already formed in the treatment group. Conclusion： From this study, it can be concluded that calcium citrate combined with rhBMP-2 signifcantly enhances bone regeneration in bone defects. This synthetic gelatin matrix stimulates formation of new bone and bone marrow in the defect areas by releasing calcium ions.

Treatment of osteoarthritis with mesenchymal stem cells

Osteoarthritis(OA)is one of the most prevalent joint diseases with prominent symptoms affecting the daily life of millions of middle aged and elderly people.Despite this,there are no successful medical interventions that can prevent the progressive destruction of OA joints.The onset of pathological changes in OA is associated with deviant activity of mesenchymal stem cells(MSCs),the multipotent precursors of connective tissue cells that reside in joints.Current therapies for OA have resulted in poor clinical outcomes without repairing the damaged cartilage.Intra-articular delivery of culture-expanded MSCs has opened new avenues of OA treatment.Pre-clinical and clinical trials demonstrated the feasibility,safety,and efficacy of MSC therapy.The Wnt/β-catenin,bone morphogenetic protein 2,Indian hedgehog,and Mitogen-activated protein kinase signaling pathways have been demonstrated to be involved in OA and the mechanism of action of MSC therapies.

The relationship between porosity, collagen fiber orientation and strength of plated bone after rigid plate fixation and removal

Objective: To understand the relationship between porosity, collagen fiber orientation and strength of the plated bone after rigid plate fixation and removal. Methods: Seventy two New Zealand white rabbits were used in this experiment. Eight animals served as control and the other sixty four were plated on their intact left tibiae with stainless steel (316L) 4 hole plates to induce early osteoporosis. The plates were removed 2 months after internal fixation in 40 plated animals, 8 of which were sacrificed immediately following plate removal and the other 32 were killed in successive groups with 8 in each group 1,2,3 and 4 months after plate removal. The remaining 24 plated animals were killed at 3,4 and 6 months after plate fixation. After sacrifice, the samples of plated bone were prepared for light microscope, quantitative histological analysis, polarized light microscope and biomechanical test. Results: The internal fixation with a rigid plate could induce the regional osteoporosis which manifested both bone loss and disorganized bone structure (loss of the orientation of the collagen fibers) leading to decreased strength of the plated bone. Although the regional osteoporosis could recover gradually after plate removal, the bone structure remained disorderly even when the bone mass returned to normal. Delayed restoration of bone structure was related to delayed restoration of bone strength. Conclusions: Besides the bone loss, the disorganized bone structure is the main cause of decrease of bone strength after rigid plate fixation and removal.