羟基丁酸-羟基辛酸共聚物/脱细胞软骨基质复合支架的制备与体外降解

背景:羟基丁酸-羟基辛酸共聚物具有高生物相容性和降解性,但单一材料无法满足组织工程支架的要求。脱细胞软骨基质是制备复合支架的常用材料,具有良好的生物相容性和无抗原性等优点。目的:将羟基丁酸-羟基辛酸共聚物、脱细胞软骨基质以不同比例混合制备复合支架,观察其体外降解速率。方法:取新鲜猪关节软骨,用含有双抗的PBS液浸泡,放入含有苯甲基磺酰氟的tri-HCl缓冲液,加入DNase酶和RNase酶,用D-Hank’s液冲洗等步骤制备脱细胞软骨基质。采用溶剂浇注-颗粒沥滤的方法与羟基丁酸-羟基辛酸共聚物按不同浓度混合,制备出不同比例的复合支架。结果与结论:脱细胞软骨基质的比例不同,复合材料的完全降解时间也不同,8%含量的脱细胞软骨基质最符合软骨组织工程支架的要求。     

等离子体处理胶原锚定PLGA/脱细胞骨基质骨软骨双层支架材料的制备和评估

目的:分别以胶原锚定方法修饰的聚乳酸-聚羟基乙酸共聚物(PLGA)为组织工程软骨支架材料,以脱细胞骨基质为组织工程骨支架材料,将二者结合制备出结合良好的组织工程骨软骨双层支架,并观察结构特征,以评估其作为组织工程化骨软骨复合体支架材料的可行性。方法:实验于2006-02/2007-02在解放军总医院骨科研究所完成。①支架的制备:以犬新鲜松质骨为原料,制备脱细胞骨基质作为骨支架材料;将脱细胞骨基质浸于盛有PLGA溶液的模具中,采用固-液相分离法结合致孔剂溶出法制备出多孔的PLGA/脱细胞骨基质双层支架,然后对PLGA支架部分进行等离子体处理和Ⅰ型胶原锚定修饰。得到的新型组织工程骨软骨双层支架的上层为多孔PLGA,下层为脱细胞骨基质。②支架的特征观察:对支架行扫描电镜检测,并采用乙醇置换法测定PLGA层孔隙率,采用北京亚林公司提供的扫描电镜图像分析系统测定PLGA层支架的平均孔径。结果:扫描电镜显示双层支架的PLGA部分具有良好的孔隙贯通结构,孔径为(211±33)μm,孔隙率为(95.0±1.5)%;脱细胞骨基质骨支架部分具有天然骨的孔径和空隙率;PLGA材料渗入骨支架部分,双层支架结合良好。结论:等离子体处理后胶原锚定修饰的PLGA/脱细胞骨基质双层支架具有良好的结构和孔隙率,结合良好,可作为支架载体应用于组织工程骨软骨复合体的构建。     

脱细胞骨软骨支架的制备及形态学观察

目的：探讨脱细胞骨软骨支架制备的可行性，为关节软骨组织工程提供新的支架材料。方法：3月龄雄性新西兰大白兔，取膝关节髌股关节面的股骨侧，切取4mm×4mm×3mm大小（长×宽X深）骨软骨块，用去垢剂-酶法对骨软骨组织块进行脱细胞脱钙处理，冻干机冻干，制备脱细胞骨软骨支架，并对其进行形态学观察。结果：脱细胞骨软骨支架呈半透明状，弹性良好，组织学评价提示细胞成分已完全祛去，保留了纤维支架。结论：去垢剂-酶法可以祛去骨软骨块中的细胞成分，成为带有空隙的支架材料。[著者文摘]     

牛脱细胞骨基质的生物力学特性及其黏附性

背景:脱细胞骨基质作为一种天然骨生物衍生材料,应用于骨组织工程支架有着其独特的优越性。目的:观察牛松质骨脱细胞后骨基质的生物力学特性、孔隙率及其黏附特性,探讨其作为组织工程骨天然支架材料的可行性。设计、时间及地点:力学实验采用随机对照观察,于2008-02/06在天津医科大学总医院骨科实验室完成。材料:新鲜牛股骨来自16月龄雄性蒙古牛,体质量350kg;新生24h内SD大鼠3只。方法:利用100g/LNaCl联合1%TritonX-100的方法制备脱细胞骨基质。脱细胞骨基质脱钙切片。利用ElectroForce3200力学试验仪对标本进行压力加载测试。利用新生SD大鼠颅骨传代培养第3代成骨细胞与脱细胞骨基质复合培养12h。空白对照组置于9g/LNaCl溶液。主要观察指标:①苏木精-伊红染色观察骨基质平均空隙直径及空隙率。②观察骨基质弹性强度、破坏载荷、弹性模量。③采用细胞计数法计算两者的黏附率。结果:①利用100g/LNaCl与1%TritonX-100联合可达到良好脱细胞效果,与对照组比较,脱细胞后实验组骨小梁无明显破坏。②所测得牛脱细胞骨细胞外基质空隙直径为(376.33±80.91)μm,空隙率为(70.15±2.98)%。③力学测试此脱细胞方法对骨基质力学特性无显著影响。④脱细胞骨基质与体外培养成骨细胞具有良好的黏附性能,黏附率为62.38%。结论:牛松质骨脱细胞骨基质较完整的去除了细胞的免疫原性,具有良好的骨组织工程支架材料力学性质,接近生理结构的空隙直径及孔隙率,黏附性能满足支架材料的要求。     

聚乳酸/聚乙醇酸与脱细胞软骨粒支架及聚乳酸/聚乙醇酸-脱细胞软骨粒支架体外构建组织工程软骨的比较

背景:聚乳酸,聚乙醇酸具有良好的生物力学性能,但是细胞黏附性较差,而脱细胞软骨基质具有较好的细胞黏附性和亲水性,能介导细胞间信号传导及相互作用,但是生物力学性能不好.课题组制备的聚乳酸,聚乙醇酸脱细胞软骨基质支架材料,弥补了2种材料各自的缺点,有望成为一种新型支架材料.目的:比较聚乳酸,聚乙醇酸、脱细胞软骨基质、聚乳酸/聚乙醇酸脱细胞软骨基质3种支架体外构建组织工程软骨的生长情况.设计、时间及地点:对比观察实验,于2008-03/09在山西医科大学实验室完成.材料:聚乳酸,聚乙醇酸平均孔径为100-200μm,孔隙率为94%左右,脱细胞软骨基质支架平均孔径为70-100 μm,孔隙率为85%左右,聚乳酸,聚乙醇酸脱细胞软骨基质平均孔径为100-300 μm,孔隙率为90%左右.方法:根据支架材料的不同,实验分为3组,分别为聚乳酸/聚乙醇酸支架组,脱细胞软骨粒支架组,聚乳酸,聚乙醇酸-脱细胞软骨粒支架组,将猪软骨细胞分离、培养、扩增、接种于3种支架,体外联合培养8周.主要观察指标:免疫组化染色检测Ⅱ型胶原表达;反转录-聚合酶链反应检测组织工程软骨细胞内Ⅱ型胶原mRNA含量;Hoechst 33258荧光法测定DNA含量.结果:软骨细胞在聚乳酸/聚乙醇酸-脱细胞软骨粒支架组生长良好,8周后仍能维持软骨细胞表型,其分泌Ⅱ型胶原的能力优于其他两组.聚乳酸,聚乙醇酸-脱细胞软骨粒支架组DNA含量、Ⅱ型胶原mRNA含量最高,脱细胞软骨基质组次之,聚乳酸,聚乙醇酸组最低,单因素方差分析显示差异有显著性意义(P<0.01).结论:聚乳酸,聚乙醇酸-脱细胞软骨粒支架更适于细胞生长、黏附,更有利于维持细胞表型和促进软骨细胞分泌Ⅱ型胶原.     

软骨细胞在松质骨骨基质明胶上生长分化的初步研究

目的 观察软骨细胞在松质骨骨基质明胶（bone matrix gelatin,BMG)上生长分化的状况。方法 分离幼兔关节软骨细胞，以松质骨BMG为支架材料体外培养，于不同时间取材行组织学观察。结果 软骨细胞在松质骨BMG表面及内部孔穴能较好分裂增殖，软骨细胞周围基质Safranin-o及胶原染色阳性。培养到24－42d形成直径为4mm的“圆盘状软骨”。结论 软骨细胞在松质骨BMG上体外培养，能形成软骨组织。     

同种异体软骨细胞直接体内构建组织工程软骨的实验研究

目的：研究兔同种异体软骨细胞直接在体内构建组织工程化软骨的可行性。方法：获取1个月龄兔耳郭软骨，分离消化后将软骨细胞种于PDLLA上形成复合物，6h后植于兔皮下，分别于6，12，18周取材行大体和组织学观察以及苏木精一伊红染色、甲苯胺蓝染色和Masson’s三色染色。结果：体内培养6周后，软骨细胞／PDLLA复合物中有基质分泌，软骨细胞不成熟，12周时，软骨细胞接近成熟并形成凹陷，18周时，新生软骨基本接近正常软骨。结论：利用同种异体软骨细胞直接构建组织工程化软骨的方法可以在动物体内形成新的软骨。     

羟基丁酸-羟基辛酸共聚体/脱细胞软骨基质支架的细胞黏附性

背景:羟基丁酸-羟基辛酸共聚体[poly(hydroxybutyrate-co-hydroxyoctanoate),PHBHOx]是一种新型的多聚羟基烷酸类材料,具有优良的可塑性、生物相容性、生物可降解性等性能,但同其他酯类材料相似,PHBHOx亲水性能较差,单纯PHBHOx材料支架细胞黏附性能明显不足.目的:观察羟基丁酸-羟基辛酸共聚体/脱细胞软骨基质支架的细胞黏附性.方法:取新鲜猪膝关节表面透明软骨,采用非离子型去污剂Triton X-100、低渗Tris-HCl以及Dna酶和Rna酶等进行脱细胞处理.低温粉碎后与PHBHOx以不同比例复合,采用溶剂浇铸-颗粒沥滤技术制备支架.分离培养猪关节软骨细胞,进行细胞-支架黏附实验并通过MTT比色法和扫描电镜观察软骨细胞在支架上的黏附存活情况.结果与结论:PHBHOx/脱细胞软骨基质支架的细胞黏附率较对照组显著提高,软骨细胞在支架上黏附紧密,生长良好.提示复合脱细胞软骨基质可以明显提高单纯PHBHOx支架的细胞黏附性能.     

不同材料构建组织工程软骨及支架的应用

背景：组织工程技术的发展为软骨的再生和修复提供了新的途径,根据软骨自身的结构和特点,作为人工软骨的替代材料和支架材料应具有良好的生物力学性能。目的：总结运动性关节软骨损伤修复材料及其支架材料的应用进展及其生物替代材料的生物力学特征,评价目前组织工程软骨材料应用的性能及发展前景。方法：以＂组织工程;软骨组织;支架材料;生物相容性＂为关键词,应用计算机检索维普数据库和PubMed数据库中1990-01/2011-04关于组织工程软骨应用研究的文章,纳入与有关生物材料与组织工程软骨相关的文章;排除重复研究或Meta分析类文章。以24篇文献为主重点进行了讨论组织工程软骨材料的种类、性能及其应用效果和前景。结果与结论：目前关节软骨修复领域以自体软骨移植效果为最佳,骨髓基质干细胞在离体试验及动物实验中研究较多,在临床应用中较少,尚在探索阶段。支架材料的应用比较繁复,天然材料、人工合成材料以及复合材料都存在一定的不足,虽然复合材料成为研究的热点,但是某些性能并不能很好地符合支架要求,并且在机体内这些材料所带来的长期影响还不能预见,这就迫切需要新材料的出现,来更好地满足组织软骨织支架的要求,达到修复和重建的目的。     

以3种材料为载体体外构建组织工程软骨

背景:纤维胶原蛋白被公认是菲常理想的载体,但其胶性易流动,降解较快,且通透性有待置疑.而海绵状胶原蛋白多微孔,三维立体结构,吸附性好,临时基质与细胞基质相近(Ⅱ型胶原),作者所查文献中作为骨髓间充质干细胞载体用于软骨形成的报道较少.目的:对比观察骨髓间充质干细胞在纤维蛋白海绵、生物蛋白海绵、明胶海绵载体中软骨形成的能力和可行性.设计、时间及地点:体外对比观察实验,于2004 12/2008-08在海南省人民医院中心实验室完成.材料:纤维胶原蛋自海绵由美国Sigma公司提供,生物蛋白海绵(主要成分为Ⅱ型胶原,并吸附定量的碱性成纤维细胞生长因子)由辽宁绿谷制药有限公司提供,明胶海绵由南京金陵制约有限公司提供.方法:抽取猪髂骨骨髓血,用密度梯度离心法分离出有核细胞,在含转化生长因子β 1的DMEM培养液中扩增骨髓间充质干细胞,将第3代骨髓间充质干细胞以载体内多点注射和表面点滴法植于Ⅱ型纤维胶原蛋白海绵、生物蛋白海绵及明胶海绵3种载体上,继续以含有转化生长凶子β 1的DMEM培养液体外培养.主要观察指标:观察细胞的生长情况,于4周取材进行大体组织观察,组织学分析及超微结构观察.结果:骨髓间充质干细胞存体外分离后可在转化生长因子β 1培养液下扩增.纤维胶原蛋白海绵组可见少量的软骨基质与细胞:生物蛋白海绵组可见较多的基质与软骨细胞,细胞生长活跃,明胶海绵组可见少量散在的胶原基质,但无细胞生长.生物蛋白海绵组软骨细胞阳性数量多于纤维胶原蛋白海绵组(P<0.01),两组都明显多于明胶海绵组(P<0.01).结论:骨髓间充质干细胞在生物蛋白海绵载体中软骨形成能力最强,纤维蛋白海绵次之,而明胶海绵与细胞生长不同步,软骨形成能力最差.     

脱细胞真皮基质预防Frey综合征的系统评价

目的系统评价脱细胞真皮基质(AMD)预防Frey综合征的有效性和安全性。方法计算机检索Cochrane图书馆(2010年第1期)、MEDLINE、EMbase、SIGLE、GreyNet、NTIS、CBMdisc、VIP、CNKI和万方数据库,查找有关应用脱细胞真皮基质预防Frey综合征的研究。检索时限均为1995～2010年。由4名评价者独立选择试验、提取资料和评估方法学质量,然后采用RevMan 5.0.0软件对资料进行Meta分析。结果共纳入15个研究,共计472例受试患者。Meta分析结果显示,植入ADM能有效降低Frey综合征发生,其主观和客观差异均有统计学意义[RR=0.11,95%CI(0.06,0.18),P<0.01;RR=0.14,95%CI(0.10,0.19),P<0.01];暂时性面神经麻痹的发生率低于对照组,其差异无统计学意义[OR=0.78,95%C(I0.37,1.66),P=0.53];血清肿及粘液囊肿的发生率高于对照组,其差异也无统计学意义[OR=2.63,95%CI(0.09,79.25),P=0.58],并可以通过放置引流和局部加压包扎解决;涎漏的发生率低于对照组,其差异有统计学意义[OR=0.24,95%CI(0.08,0.69),P=0.009]。结论本系统评价结果显示,应用ADM能有效降低Frey综合征的发生率,安全性较好。建议术前行碘酒/碘伏过敏检查,术中行快速冰冻切片明确肿瘤性质,尽量固定牢固,术后放置负压引流及行局部加压包扎。     

人胎关节软骨细胞体外培养的生物学特性

目的:研究软骨组织工程中传代软骨细胞与原代的差异。方法:用5个月人胎关节软骨分离培养细胞,观察细胞存活率、贴壁率、生长曲线和组织形态学的改变。结果:(1)软骨块在4℃下,3d 内细胞存活率可达 93.4％～97.6％。(2)原代细胞为圆形或三角形;第4代有一半转变成梭形,到第6代全部变为长梭形。(3)传代细胞贴壁时间(2～3h)短于原代(4～7h)。玻璃瓶内贴壁率传代细胞为78.7％～85.5％,原代8.8％。(4)生长曲线表明,从原代到第4代都有高增殖力;到第8代时增殖降低;到第12代几乎丧失细胞增殖。结论:软骨块4℃冷藏,3d内对细胞存活率无明显影响。第4代细胞贴壁快、增殖快,适于作为组织工程用细胞。第8代以后不适合。     

Establishment of an in vitro three‐dimensional model for cartilage damage in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads to progressive joint destruction. To further understand the process of rheumatoid cartilage damage, an in vitro model consisting of an interactive tri‐culture of synovial fibroblasts (SFs), LPS‐stimulated macrophages and a primary chondrocyte‐based tissue‐engineered construct was established. The tissue‐engineered construct has a composition similar to that of human cartilage, which is rich in collagen type II and proteoglycans. Data generated from this model revealed that healthy chondrocytes were activated in the presence of SFs and macrophages. The activated chondrocytes subsequently displayed aberrant behaviours as seen in a disease state such as increased apoptosis, decreased gene expression for matrix components such as type II collagen and aggrecan, increased gene expression for tissue‐degrading enzymes (MMP‐1, ‐3, ‐13 and ADAMTS‐4, ‐5), and upregulation of inflammatory mediator gene expression (TNF‐α, IL‐1β, IL‐6 and IKBKB). Additionally, the inclusion of SFs and macrophages in the model enabled both cell types to more closely replicate an in vivo role in mediating cartilage destruction. This is evidenced by extensive matrix loss, detected in the model through immunostaining and biochemical analysis. Subsequent drug treatment with celecoxib has shown that the model was able to respond to the therapeutic effects of this drug by reversing cartilage damage. This study showed that the model was able to recapitulate certain pathological features of an RA cartilage. If properly validated, this model potentially can be used for screening new therapeutic drugs and strategies, thereby contributing to the improvement of anti‐rheumatic treatment. Copyright © 2017 John Wiley & Sons, Ltd.     

A novel engineered dermis for in vitro photodamage research

The realization of biologically relevant human tissue equivalents as an in vitro model to investigate human diseases, as well as to test the efficacy or toxicity of novel compounds, is emerging as a new challenge in tissue engineering. Currently, the in vitro three‐dimensional (3D) dermis model mainly involves the use of cells embedded in exogenous non‐human matrices. However, such models feature biological and functional disparities with native dermis, therefore limiting their relevance to the in vivo situation. The purpose of this study was to provide a reliable endogenous human dermal equivalent (HDE) able to recapitulate the extracellular matrix (ECM) remodelling of the native dermis occurring after external damage. To this end, UVA irradiation was used to induce photodamage to both the HDE and to a fibroblast‐populated collagen matrix. The photodamage was investigated at the cellular and ECM level and the results showed that, although a cellular response was detected in both systems, no ECM reorganization characteristic of the in vivo photo‐aged dermis could be detected in the fibroblast‐populated collagen matrix. In contrast in the HDE, the neosynthesized ECM recapitulated the characteristic ageing behaviour of the dermis found in vivo, in terms of collagen and hyaluronic acid synthesis as well as collagen organization remodelling. This study therefore demonstrates the role of the endogenous ECM in recapitulating in vitro the functionality of the human dermis and the proposed HDE as a novel tool for photoprotection trials. Copyright © 2016 John Wiley & Sons, Ltd.     

人骨髓间充质干细胞体外培养方法的改良

目的改良人骨髓间充质干细胞(HBMSCs)体外分离培养的方法。方法采用含体积比为10%胎牛血清的α-MEM培养体系,通过密度梯度离心和贴壁筛选法从人骨髓中分离培养贴壁细胞,流式检测该细胞表面标志物,体外诱导成脂、成骨、成软骨分化并鉴定该细胞的多项分化能力,集落克隆形成和MTT试验检测该细胞的活力和增殖能力,并分别与经典的Dexter长期培养体系比较。结果采用改良方法从人骨髓中分离、培养出具有塑料底物粘附性的细胞,目的细胞不表达或低表达CD14、CD34、CD45,高表达CD73、CD90、CD105;体外成功诱导脂肪细胞、骨细胞、软骨细胞分化;Dexter-LTC体系培养的集落个数(为8.0±1.0)个、单个集落细胞数为(59.2±8.2)个,改良法培养的集落个数为(11.3±1.53)个、单个集落中细胞数为(66.5±14.4)个(P<0.05);在Dexter-LTC培养体系下,BMSCs于培养d10进入对数生长期、d19进入平台期,改良法培养BMSCs d4进入对数生长期、d15进入平台期。结论成功建立了改良的HBMSCs体外分离培养体系,且该方法培养的HBMSCs体外扩增速度、增殖能力、克隆形成能力明显改善。     

首次体外受精胚胎移植夫妇术前心理应激状态的比较

目的:探讨首次体外受精-胚胎移植(in vitro fertilization and embryo transfer,IVF-ET)夫妇术前心理应激状况。方法:入选首次行IVF-ET的278对夫妇,于进入治疗周期前(30±1)d进行压力感知量表(perceived stress scale,PSS)、抑郁自评量表(self-rating depression scale,SDS)、焦虑自评量表(self-rating anxiety scale,SAS)和事件影响量表修订版(impact of event scale-revised,IES-R)测量,对比男性和女性的压力感知、抑郁、焦虑和创伤应激症状。结果:首次接受IVF-ET治疗的夫妇术前女性、男性抑郁症状检出率分别为28.8%、31.3%,焦虑症状检出率分别为14.4%、19.4%,男女间差异无统计学意义;术前女性、男性IVF-ET治疗相关的创伤应激症状检出率分别为58.3%、47.5%,女性高于男性(P0.05)。男性和女性患者的焦虑、抑郁和压力感知评分均高于常模(P0.05),男女性间差异无统计学意义。女性患者IVF-ET治疗相关的创伤应激症状评分为(23.1±13.1)分,高于男性[(20.5±13.1)分,P0.05]。结论:首次接受IVF-ET治疗的夫妇术前双方心理应激水平均升高;男性和女性压力感知、抑郁和焦虑症状相似,女性IVF-ET治疗相关的创伤应激症状更显著,需采取针对性处理。     

A template model for studying anticancer drug efflux transporter inhibitors in vitro

Efflux transporters play an important role in drug absorption and also in multidrug resistance. ABCG2 (BCRP) is an efflux transporter conferring cross‐resistance to mitoxantrone (Mit), irinotecan (CPT11), and its active metabolite SN38. MBLI87, a new ABCG2 inhibitor has proven its efficacy against ABCG2‐mediated efflux in vitro and in vivo. This work aimed at modeling and quantifying the cellular interaction between MBLI87 and different substrates using a mechanistic template model. An in vitro competition experiment study was carried out with HEK293 cells overexpressing ABCG2 exposed to fixed concentrations of substrates (Mit, CPT11, SN38) and to MBLI87 at several concentration levels. A nonlinear mixed‐effects transport inhibition model was developed to fit intracellular drug concentrations. In this model, drugs cross the cell membrane through passive diffusion, active drug efflux is ABCG2 mediated, interaction between substrates and inhibitor occurs within the transporter. The interaction was found to be noncompetitive. The MBLI87 Ki was estimated to 141 nm for Mit, 289 nm for CPT11, and 1160 nm for SN38. The ratio of intrinsic transport clearance divided by diffusion clearance was estimated to 2.5 for Mit, 1.01 for CPT11, and 5.4 for SN38. The maximal increase in the intracellular substrate concentration that is possible to achieve by inhibition of the transporter was estimated to 1.5 for Mit, 0.1 for CPT11, and 4.4 for SN38. This mechanistic template model describes both drug accumulation and cellular transport, and the mixed‐effects approach allows an estimation of intra‐ and interassay variability. This model is of great interest to study cytotoxic cellular pharmacokinetics.     

社区获得性肺炎15种常见菌对22种抗菌药体外活性的研究

目的掌握我省社区获得性肺炎（CAP）细菌谱和药敏谱,为我省提供治疗CAP的准确资料。方法细菌的分离与鉴定采用常规法和仪器法．最小抑菌浓度的检测采用琼脂稀释法。结果121例CAP检出病原体52例（42．90％）．居前5位分别是肺炎链球菌、流感嗜血杆菌、肺炎克雷伯菌、铜绿假单胞菌、葡萄球菌和卡他莫拉菌（并列）。药敏中阳性菌和大多数阴性菌分别测14种抗菌素．嗜血杆菌和莫拉菌测9种抗菌素,基本上确定了常用药的药敏谱。结论CAP病原谱和药敏谱南于地区不同、人群不同、季节不同、诊治水平等不同,其组成和结果都有所不同,这个参数并不能判断每一种抗生素的最终疗效。     

A new automated method for continuous registration of factor VII activation in vitro. Activation is accelerated by the concentration of factor VII and the activity state of the protein

When a plasma sample is exposed to tissue factor, single-chain factor VII (FVII) is gradually converted to the active two-chain form (FVIIa). In the present study, we have constructed a measurement system, which allows continuous registration of the activation of FVII to FVIIa in vitro. In this system, FVII activation follows parabolic kinetic after an initial lag-phase. The slope of the linear phase is a measure of the protein concentration of factor VII (FVII_totai), while the length of the non-linear phase represents the velocity of FVII activation. The time required for complete activation of FVII is inversely related to both FVII,_otai and the relative amount of FVIIa in the sample. In future studies, this new measurement system will make it possible to study the process of FVII activation in different samples, and to examine how varying concentrations of exogenuous added components affect the activation process in vitro.     

Enhancement of Calcium/Vitamin D Supplement Efficacy by Administering Concomitantly Three Key Nutrients Essential to Bone Collagen Matrix for the Treatment of Osteopenia in Middle-Aged Women: A One-Year Follow-Up

Two vitamins and proline (CB₆Pro), three nutrients essential for bone collagen, were used in combination to a 1000 mg calcium/250 IU vitamin D (Ca/D) daily supplement to treat osteopenia as a preventive measure against osteoporosis later in life. Middle-aged women not using estrogen were screened for osteopenia using the WHO criteria and divided into three groups (n = 20 each): 1) placebo healthy controls with normal bone mineral density (BMD); 2) control Ca/D-treated osteopenic patients; and 3) Ca/D + CB₆Pro-treated osteopenic patients. The three groups were comparable at baseline except for BMD. After one-year treatment, cortical diaphyseal BMD remained constant in each group, but trabecular bone loss persisted (at 5 lumbar sites) in osteopenic group 2. No further bone loss was detected in osteopenic group 3. A loss of 2% was evidenced in the placebo group at one lumbar site. Markers of bone formation (which increase in coupling to resorption) decreased significantly in both osteopenic groups. Although biomarkers of resorption did not change, hormone (PTH and 1,25(OH)₂D₃)-induced osteoclastic activity was significantly reduced. No decline in BMD occurred at any bone site in osteopenic group 3, highlighting the importance of improving the quality of bone matrix concomitantly to mineral replacement.