猪髋动脉内皮细胞与猪小肠黏膜下基质的联合培养

目的:将猪小肠黏膜下基质与猪髋动脉内皮细胞的复合培养,证实小肠黏膜下基质是否可以成为组织工程化血管的良好载体材料。方法:实验于2005-09/2006-01在同济大学生命科学与技术学院生物医学工程研究所完成了猪小肠黏膜下基质的制备与处理,在复旦大学医学院附属中山医院肿瘤中心完成了猪小肠黏膜下基质的射线照射及消毒,在同济大学生命科学与技术学院实验室完成了猪髋动脉内皮细胞与猪小肠黏膜下基质的复合培养。实验材料:猪小肠黏膜下基质取自屠宰场获取的新鲜家猪空肠,猪髋动脉内皮细胞(PIEC,中国科学院上海细胞生物学研究所细胞库)。实验分组:分为猪髋动脉内皮细胞单独培养组与猪小肠黏膜下基质-猪髋动脉内皮细胞复合培养组。实验方法:①小肠黏膜下基质采用物理和化学方法处理。②猪髋动脉内皮细胞单独培养。③猪小肠黏膜下基质-猪髋动脉内皮细胞复合培养。实验评估:采用倒置相差显微镜观察细胞的黏附和生长,猪小肠黏膜下基质的组织结构;测定单纯培养猪髋动脉内皮细胞和猪小肠黏膜下基质-猪髋动脉内皮细胞复合培养细胞的生长曲线,每日取3孔培养细胞经消化后细胞计数,取其均值,总共8日;以评价猪小肠黏膜下基质与猪髋动脉内皮细胞的细胞相容性。结果:①小肠黏膜下层表面形态:猪小肠黏膜下基质为浅白色薄膜状,厚度约80μm,光镜下可见处理后膜的上下两侧结构不同,一面较粗糙,孔径较大,用于细胞种植,另一面较平坦。单经物理方法处理后的猪小肠黏膜下基质表面有大量残留的细胞碎屑,再行化学处理后基质表面无细胞碎屑残留,胶原纤维未受损。②单独培养与复合培养的猪髋动脉内皮细胞形态对比:倒置显微镜观察,猪小肠黏膜下基质与猪髋动脉内皮细胞复合培养接种细胞24h贴壁,第1～4天时细胞增殖不明显,第5天有较多细胞直接贴附于材料边缘,随时间延长,猪髋动脉内皮细胞在猪小肠黏膜下基质表面黏附生长良好,细胞均匀分布于基质,细胞形态多为梭形及多角形。与猪髋动脉内皮细胞单独培养细胞相比,伪足不明显,细胞形态也相对规则。③细胞生长曲线:复合培养组的细胞计数前4d较单纯培养组少,第5天后时细胞计数高于单纯培养组,复合培养组在接种的后1d数量最少,两组细胞计数均在最后1d达到最大值。结论:猪髋动脉内皮细胞可在猪小肠黏膜下基质上黏附生长,猪小肠黏膜下基质可促进猪髋动脉内皮细胞的增殖,猪小肠黏膜下基质有良好的细胞相容性,其孔径、结构有助于猪髋动脉内皮细胞的黏附和生长,是良好的组织工程生物衍生材料。     

猪小肠黏膜下层移植修复兔膝关节内侧副韧带缺损

目的:观察猪小肠黏膜下层修复兔膝关节内侧副韧带缺损的作用效果。方法:实验于2006-01/08在上海交通大学医学院附属第三人民医院动物房及中心实验室完成。选用新西兰大白兔24只,动物左右两膝分别作为猪小肠黏膜下层移植组及自体韧带移植组,分别制作兔膝关节内侧副韧带缺损模型。猪小肠黏膜下层移植组植入猪小肠黏膜下层,猪小肠黏膜下层作为细胞外基质,无免疫原性;自体韧带移植组植入对侧副韧带。分别于术后2,4,8和12周取白兔膝关节内侧副韧带,行大体形态观察及组织学和力学检查,两组间进行对比观察。结果:24只白兔均进入结果分析,无脱失。①内侧副韧带移植段大体形态观察结果:术后4周,猪小肠黏膜下层移植组有大量的肉芽增生,与周围粘连;自体韧带移植组肉芽组织与周围粘连相对少。术后8周,猪小肠黏膜下层移植组组织梭形增生;自体韧带移植组组织平行增生,直径略小于猪小肠黏膜下层移植组。术后12周,猪小肠黏膜下层移植组增生组织平行于自体韧带,大小一致;自体韧带移植组增生组织直径与猪小肠黏膜下层移植组相一致。②内侧副韧带移植段光镜检查结果:术后2周,猪小肠黏膜下层移植组少量炎性细胞浸润,成纤维细胞数目不多,血管增生明显;自体韧带移植组炎性细胞稀少。术后4周,猪小肠黏膜下层移植组有大量的成纤维细胞,胶原增多;自体韧带移植组成纤维细胞和胶原比猪小肠黏膜下层移植组多。术后8周,猪小肠黏膜下层移植组纤维细胞为主,纤维纵向排列;自体韧带移植组也以成纤维细胞为主。术后12周,两组均有大量的胶原,排列整齐,细胞和纤维密度相一致。③内侧副韧带力学检查结果:术后4周猪小肠黏膜下层移植组白兔内侧副韧带的最大拉伸负荷大于自体韧带移植组[(36.8±8.9,25.3±7.3)N(P<0.05)],术后2,8,12周,两组差异无显著性意义(P>0.05)。结论:猪小肠黏膜下层是一种良好的生物材料,可以用来移植修复膝关节内侧副韧带缺损和损伤,移植后期具有与自体韧带相似的替代修复作用。     

兔许旺细胞与猪小肠黏膜下层的体外复合培养☆

背景:国内外研究以大鼠许旺细胞与小肠黏膜下层复合修复神经缺损,取得了较好结果. 目的:观察兔许旺细胞与猪小肠黏膜下层体外共培养的生物相容性. 方法:采用分步酶消化法分离培养新西兰大白兔许旺细胞,取第3代许旺细胞接种在猪小肠黏膜下层上. 结果与结论:①苏木精-伊红染色:复合培养24 h后细胞在材料上良好黏附.1周时部分细胞在猪小肠黏膜下层基质层表面呈单层生长,细胞扁平,细胞核呈长梭形,细胞间连接紧密.2周后,细胞呈多层生长.②扫描电镜:复合培养2 d,细胞黏附于材料表面并伸展,细胞体呈纺锤形,从胞体伸出两根细长突起,相邻细胞的突起首尾相接连成细胞链或融合或交联或在纤维的侧方平行生长;1周后细胞在材料上大量增殖,呈串珠链黏附于支架上,类似于神经中的Bunger带.表明小肠黏膜下层与许旺细胞有良好的相容性.     

猪小肠黏膜下层基质支架材料复合脂肪基质干细胞的生物相容性

背景:小肠黏膜下层具有良好的细胞、组织相容性和降解性,是一种理想的组织工程支架材料,将脂肪基质干细胞与小肠黏膜下层复合后进行定向诱导,可构建靶组织,具有一定的临床应用潜能.目的:制备脱细胞猪小肠黏膜下层基质,并检验其与家兔脂肪基质干细胞的生物相容性.方法:使用酶消化-高盐水脱细胞法处理猪小肠黏膜下层,石蜡切片检测脱细胞效果,扫描电镜观察小肠黏膜下层表面结构.体外分离培养家兔脂肪基质干细胞,分别将第3代脂肪基质干细胞单面复合和双面复合至小肠黏膜下层培养1周观察材料上下表面细胞复合情况.结果与结论:小肠黏膜下层材料为白色半透明膜状物,石蜡切片显示细胞去除彻底,扫描电镜显示小肠黏膜下层黏膜结构紧密,浆膜面纤维结构松散.脂肪基质干细胞与材料复合后,通过相差显微镜观察到细胞在小肠黏膜下层上附着生长,扫描电镜检测提示单面复合脂肪基质干细胞的小肠黏膜下层,仅在上表面有大量细胞生长,下表面无细胞或仅有少量细胞生长,双面复合脂肪基质干细胞的小肠黏膜下层上下表面均可见大量细胞融合生长,石蜡切片可见细胞贴附于材料表面生长.提示酶消化-高渗盐水法可彻底去除小肠黏膜下层表面细胞成分,小肠黏膜下层对脂肪基质干细胞的生长具有良好的支持作用.     

兔许旺细胞与猪小肠黏膜下层的体外复合培养

背景：国内外研究以大鼠许旺细胞与小肠黏膜下层复合修复神经缺损,取得了较好结果。目的：观察兔许旺细胞与猪小肠黏膜下层体外共培养的生物相容性。方法：采用分步酶消化法分离培养新西兰大白兔许旺细胞,取第3代许旺细胞接种在猪小肠黏膜下层上。结果与结论：①苏木精-伊红染色：复合培养24h后细胞在材料上良好黏附。1周时部分细胞在猪小肠黏膜下层基质层表面呈单层生长,细胞扁平,细胞核呈长梭形,细胞间连接紧密。2周后,细胞呈多层生长。②扫描电镜：复合培养2d,细胞黏附于材料表面并伸展,细胞体呈纺锤形,从胞体伸出两根细长突起,相邻细胞的突起首尾相接连成细胞链或融合或交联或在纤维的侧方平行生长;1周后细胞在材料上大量增殖,呈串珠链黏附于支架上,类似于神经中的Bunger带。表明小肠黏膜下层与许旺细胞有良好的相容性。     

脱细胞猪小肠黏膜下层支架复合软骨细胞构建组织工程软骨

背景:关节软骨损伤后无论是否施加干预,都难以达到满意的修复效果.目的:观察以猪自体软骨细胞为种子细胞复合脱细胞猪小肠黏膜下层构建组织工程软骨的可行性.方法:将培养至第3代的猪膝关节软骨细胞接种于小肠黏膜下层膜上,复合培养48 h,构建细胞-载体复合物,光学显微镜、扫描电镜观察软骨细胞在小肠黏膜下层膜上的生长情况.结果与结论:苏木精-伊红染色见细胞在小肠黏膜下层基质层表面呈单层或复层生长;免疫组织化学染色结果显示软骨细胞与小肠黏膜下层表面之间形成一条连续阳性表达条带;扫描电镜见软骨细胞在支架孔隙内贴壁良好生长.     

脱细胞猪小肠黏膜下层支架复合软骨细胞构建组织工程软骨

背景：关节软骨损伤后无论是否施加干预,都难以达到满意的修复效果。目的：观察以猪自体软骨细胞为种子细胞复合脱细胞猪小肠黏膜下层构建组织工程软骨的可行性。方法：将培养至第3代的猪膝关节软骨细胞接种于小肠黏膜下层膜上,复合培养48h,构建细胞-载体复合物,光学显微镜、扫描电镜观察软骨细胞在小肠黏膜下层膜上的生长情况。结果与结论：苏木精-伊红染色见细胞在小肠黏膜下层基质层表面呈单层或复层生长;免疫组织化学染色结果显示软骨细胞与小肠黏膜下层表面之间形成一条连续阳性表达条带;扫描电镜见软骨细胞在支架孔隙内贴壁良好生长。     

半月板纤维软骨细胞与小肠黏膜下层的组织相容性

目的:观察小肠黏膜下层与半月板纤维软骨细胞的组织相容性,评价其作为组织工程半月板支架材料的可行性及应用价值。方法:实验于2005-06/12在华中科技大学同济医学院附属协和医院骨科实验室完成。①物理和化学方法处理猪小肠黏膜下层支架材料。②体外复合培养兔半月板纤维软骨细胞与小肠黏膜下层。③通过苏木精-伊红染色观察小肠黏膜下层的组织学结构表现;倒置相差显微镜观察细胞生长及与生物材料附着的情况;扫描电镜观察半月板纤维软骨细胞与小肠黏膜下层复合情况。结果:①小肠黏膜下层组织学检查:经物理方法处理后的小肠黏膜下层表面有大量残留的细胞碎屑;化学方法处理后的小肠黏膜下层表面没有残留的细胞碎屑,胶原纤维未受损。②细胞生长及附着情况:相差显微镜观察,复合培养第1～4天时细胞增殖不明显,第5天有较多细胞直接贴附于材料边缘,第7天有大量细胞向材料边缘聚集,细胞形态多为梭形或多角形,连成一片。③细胞与支架复合情况:扫描电镜观察,单纯小肠黏膜下层支架材料可见黏膜表面较粗糙,孔隙较多,大小不一。半月板纤维软骨细胞与小肠黏膜下层复合培养7d可见细胞间通过突起相互连接附着在材料表面,细胞在小肠黏膜下层支架材料上生长、黏附、增殖良好,并分泌大量的细胞外基质成分。结论:小肠黏膜下层具有良好的组织相容性,可以用作组织工程半月板的支架材料。     

灌注法构建小肠黏膜全层去细胞外基质

背景：对于解剖层次复杂的细胞外基质,单纯浸泡法则很难达到理想的去细胞效果.目的：观察灌注法去细胞技术制备小肠黏膜全层去细胞外基质的效果. 方法：采用灌注法去细胞技术,经成年雄性新西兰大白兔小肠黏膜上动脉灌注去离子剂后处理获得小肠黏膜全层去细胞外基质.采用MTT法检测小肠黏膜全层去细胞外基质浸提液（实验组）或含体积分数20%小牛血清的DMEM（对照组）与1月龄新西兰大白兔骨髓间充质干细胞共培养后的细胞相对增殖率. 结果与结论：①大体观察：小肠黏膜上动脉经灌注30 min后变得苍白透明,经2 h灌注后肠段变得苍白剔透,清楚可见脉管纹理.②组织学与扫描电镜观察：灌注法构建的小肠黏膜全层去细胞外基质的去细胞程度均匀,胶原纤维未受损,孔隙多,孔隙率为（86.72±2.98）%.③M TT 检测实验：培养2,4,7 d,实验组细胞A值明显高于对照组细胞A值（P〈0.05）,且实验组细胞相对增殖率均〉1.表明采用灌注法构建小肠黏膜全层去细胞外基质可操作性强,安全性好,且小肠黏膜全层去细胞外基质对骨髓间充质干细胞无毒性,并有一定的促生长作用.     

猪小肠黏膜下层作为组织工程肌腱支架的强度

背景:小肠黏膜下层是一种能够适合于细胞迁移、生长和增殖的良好的支架材料,已被应用于组织工程研究。目的:分析猪小肠黏膜下层作为组织工程肌腱支架的可行性。方法:将24只左腿屈趾肌腱缺损罗曼鸡随机分组,实验组于缺损处植入猪小肠黏膜下层与鸡胚趾深屈肌肌腱细胞复合物,对照组于缺损处植入单纯猪小肠黏膜下层,植入后3,6,9周检测两组植入材料肌腱最大拉伸强度。结果与结论:植入后第3周时,实验组和对照组植入材料肌腱最大拉伸强度差异无显著性意义。植入后第6,9周时,实验组植入材料肌腱最大拉伸强度高于对照组(P<0.05)。表明肌腱细胞-小肠黏膜下层材料复合构建的组织工程肌腱力学强度强于单纯小肠黏膜下层材料修复肌腱,可作为组织工程肌腱支架材料。     

Guns and Knives in New Mexico: Patterns of Penetrating Trauma, 1978–1993

Objective: To identify patterns of nonfatal and fatal penetrating trauma among children and adults in New Mexico using ED and medical examiner data.
Methods: The authors retrospectively sampled in 5-year intervals all victims of penetrating trauma who presented to either the state Level-1 trauma center or the state medical examiner from a 16-year period (1978–1993). Rates of nonfatal and fatal firearm and stabbing injury were compared for children and adults.
Results: Rates of nonfatal injury were similar (firearm, 34.3 per 100,000 person-years; stabbing, 35.1). However, rates of fatal injury were significantly different (firearm, 21.9; stabbing, 2.7; relative risk: 8.2; 95% confidence interval: 5.4, 12.5). From 1978 to 1993, nonfatal injury rates increased for children (p = 0.0043) and adults (p < 0.0001), while fatal penetrating injury remained constant. The increase in nonfatal injury in children resulted from increased firearm injury rates. In adults, both stabbing and firearm nonfatal injury rates increased.
Conclusions: Nonfatal injury data suggest that nonfatal violence has increased; fatal injury data suggest that violent death rates have remained constant. Injury patterns vary by age, mechanism of trauma, and data source. These results suggest that ED and medical examiner data differ and that both are needed to guide injury prevention programs.     

Dissociable correlates of two classes of retrieval processing in prefrontal cortex

Although substantial evidence suggests that the prefrontal cortex (PFC) implements processes that are critical for accurate episodic memory judgments, the specific roles of different PFC subregions remain unclear. Here, we used event-related functional magnetic resonance imaging to distinguish between prefrontal activity related to operations that (1) influence processing of retrieval cues based on current task demands, or (2) are involved in monitoring the outputs of retrieval. Fourteen participants studied auditory words spoken by a male or female speaker and completed memory tests in which the stimuli were unstudied foil words and studied words spoken by either the same speaker at study, or the alternate speaker. On "general" test trials, participants were to determine whether each word was studied, regardless of the voice of the speaker, whereas on "specific" test trials, participants were to additionally distinguish between studied words that were spoken in the same voice or a different voice at study. Thus, on specific test trials, participants were explicitly required to attend to voice information in order to evaluate each test item. Anterior (right BA 10), dorsolateral prefrontal (right BA 46), and inferior frontal (bilateral BA 47/12) regions were more active during specific than during general trials. Activation in anterior and dorsolateral PFC was enhanced during specific test trials even in response to unstudied items, suggesting that activation in these regions was related to the differential processing of retrieval cues in the two tasks. In contrast, differences between specific and general test trials in inferior frontal regions (bilateral BA 47/12) were seen only for studied items, suggesting a role for these regions in post-retrieval monitoring processes. Results from this study are consistent with the idea that different PFC subregions implement distinct, but complementary processes that collectively support accurate episodic memory judgments.     

Interprofessional approaches to creating safe,high quality health care

Three supplementary perspectives are presented arguing that interprofessional collaboration is both necessary and desirable. Nonetheless, there are often too many serious intra-professional barriers and obstacles to interprofessional collaboration to make it successful. Some of these barriers, it is argued and illustrated, are found in the multiple ways in which professional identity is tacitly acquired and embodied in the practitioners' habitual, everyday practice. The paper then explores ways in which reflection, especially Second order reflection, can help to elucidate and overcome these obstacles, as well as increasing professional adaptability and competence.     

Systematic Reviews Published in the January 2011 Issue of The Cochrane Library

ABSTRACT

The Cochrane Library of Systematic Reviews is published quarterly as a DVD and monthly online. The January 2011 issue (first quarterly DVD for 2011) contains 4515 complete reviews, 1985 protocols for reviews in production, and 13,521 one-page summaries of systematic reviews published in the general medical literature. In addition, there are citations of 641,000 randomized controlled trials, and 14,018 cited papers in the Cochrane methodology register. The health technology assessment database contains over 9300 citations. One hundred and seven new reviews have been published in the last 3 months, of which five have potential relevance for practitioners in pain and palliative medicine.     

Modified micro-superior percutaneously-assisted total hip: early experiences & case reports

Because of the extensile nature and familiarity of the standard posterior-lateral approach to the hip, a family of "micro-posterior" approaches has been developed. This family includes the Percutaneously-Assisted Total Hip (PATH) approach, the Supercapsular (SuperCap) approach and a newer hybrid approach, the Supercapsular Percutaneously-Assisted Total Hip (SuperPATH) approach. Such approaches should ideally provide a continuum for the surgeon: from a "micro" (external rotator sparing) posterior approach, to a "mini" (external rotator sacrificing) posterior approach, to a standard posterior approach. This could keep a surgeon within his comfort zone during the learning curve of the procedure, while leaving options for complicated reconstructions for the more practiced micro-posterior surgeons. This paper details one author's experiences utilizing this combined approach, as well as permutations of this entire micro-posterior family of approaches as applied to more complex hip reconstructions.     

Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity. Activity kinetics and diagnostic significance in myocardial infarction.

This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.     

Structure and function of "metalloantibiotics"

Although most antibiotics do not need metal ions for their biological activities, there are a number of antibiotics that require metal ions to function properly, such as bleomycin (BLM), streptonigrin (SN), and bacitracin. The coordinated metal ions in these antibiotics play an important role in maintaining proper structure and/or function of these antibiotics. Removal of the metal ions from these antibiotics can cause changes in structure and/or function of these antibiotics. Similar to the case of "metalloproteins," these antibiotics are dubbed "metalloantibiotics" which are the title subjects of this review. Metalloantibiotics can interact with several different kinds of biomolecules, including DNA, RNA, proteins, receptors, and lipids, rendering their unique and specific bioactivities. In addition to the microbial-originated metalloantibiotics, many metalloantibiotic derivatives and metal complexes of synthetic ligands also show antibacterial, antiviral, and anti-neoplastic activities which are also briefly discussed to provide a broad sense of the term "metalloantibiotics."