颈椎间盘退变的形态学观察和生物力学研究

目的:观察长时间异常应力环境下兔颈椎间盘的组织形态和生物力学性能变化,为防治颈椎病的研究提供实验依据.方法:选取30只家兔,随机分为对照组、模型组,造模家兔颈椎处于低头屈曲45°位异常应力环境下5小时/次·天.动物处死后,光镜和电镜下观察颈椎间盘组织形态学变化;功能节段生物力学性能测试.比较各组间存在的差异.结果:对照组颈椎间盘组织形态及生物力学性能无明显变化;模型组颈椎间盘发生软骨细胞变性、坏死,髓核皱缩,软骨终板钙化、断裂等组织形态学改变;椎间盘压缩、扭转生物力学性能下降,并随着异常应力作用时间的延长而表现更为显著.结论:长时间处于异常应力环境下能使兔颈椎间盘组织形态和生物力学性能均发生明显退行性改变.     

六味地黄丸含药血清对软骨终板蛋白多糖的影响

背景：软骨终板的结构完整性关系着其生理功能的正常发挥,进而影响整个椎间盘的生理、病理状态;蛋白多糖是软骨终板的主要组分。 目的：观察六味地黄丸含药血清对肿瘤坏死因子α致伤兔软骨终板蛋白多糖含量的影响。 方法：将12只新西兰白兔带终板的24个T12~L2椎间盘随机等分为对照第1,14天组、肿瘤坏死因子α组、肿瘤坏死因子α+中药血清组。后2组培养液中分别含5 μg/L肿瘤坏死因子α,5 μg/L肿瘤坏死因子α和体积分数10%六味地黄丸血清,培养14 d后观察。 结果与结论：随培养时间的延长,软骨终板细胞数量减少、细胞形态变化明显,细胞外基质中胶原水平减少,加入肿瘤坏死因子α,胶原成分下降更快,并出现大量裂隙,含药血清可部分延缓这一过程;随着培养时间延长,软骨终板超微结逐渐损伤,肿瘤坏死因子α可加重损伤,含药血清可部分抑制这种损伤;随着培养时间的延长,各组椎间盘软骨终板的糖胺多糖总量与硫酸软骨素、硫酸角质素和透明质酸水平均降低;肿瘤坏死因子α加速这种结果,而含药血清可延缓生化成分水平的下降。提示六味地黄丸可通过保护软骨终板超微结构,稳定糖胺多糖总量及其各成分水平,对椎间盘软骨终板具有保护作用。     

透明质酸钠溶液复合可注射型骨髓间充质干细胞修复退变椎间盘

背景：透明质酸作为椎间盘组织工程支架的基质材料,可提供蛋白多糖附着点,增加蛋白多糖的沉积,以足够大的孔率允许种子细胞长入。 目的：观察透明质酸钠混合溶液复合骨髓间充质干细胞修复兔退变椎间盘的效果。 方法：以后外侧穿刺抽吸L1/2和L3/4髓核构建日本大耳白兔椎间盘退变模型,造模后2周应用微量注射器向L3/4椎间盘内注射同种异体骨髓间充质干细胞与透明质酸钠混合溶液作为实验组,L1/2椎间盘注射透明质酸钠作为对照组。注射后2,4,8,12 周时行兔椎间盘影像学及病理学检查。 结果与结论：对照组椎间盘高度呈降低趋势,实验组注射后2周椎间盘高度缓慢下降,之后缓慢升高,两组在4个时间点椎间盘高度指数改变差异有显著性意义(P < 0.05)。病理结果显示实验组髓核纤维环形态得到保留,髓核纤维环边界清晰,植入的骨髓间充质干细胞产生增殖分裂,并向周围迁徙规律排列,其病理学评分明显高于对照组。12周内实验组Ⅱ型胶原含量高于对照组(P < 0.05)。说明移植于退变椎间盘内的骨髓间充质干细胞能够存活,且有增殖能力,其与透明质酸钠混合溶液联合移植可以延缓退变椎间盘进一步退变,并促进退变椎间盘修复。     

丹参注射液对颈椎病家兔血液流变性的影响

目的观察家兔颈椎间盘退变过程中血液流变性变化和丹参注射液对这些变化的影响.方法通过破坏家兔颈椎动、静力平衡,建立家兔颈椎间盘退变的模型.60只兔子,随机分成3组,颈椎间盘退变组(A)40只,分4个亚组(A1、A2、A3、A4),即为造模后1、3、5、7月组,每组各10只;退变加丹参治疗组(B)10只,造模后饲养6个月后,每日腹腔内注射丹参注射液2 ml,1个月后处死;正常对照组(C)10只.通过测定血液流变学(全血低切黏度、高切黏度、血浆黏度、红细胞比容、血沉、血浆纤维蛋白原),比较各组之间的差异.结果 7月组(A4)家兔全血高切黏度、低切黏度、血浆黏度、血沉与正常对照组(C)比较有极显著差异(P＜0.01),红细胞比容同正常组比较P＜0.05;丹参治疗组(B)家兔全血高切黏度、低切黏度、血沉与A4组比较P＜0.01,红细胞比容同A4组比较(P＜0.05),以上均有显著性差异.结论丹参能改善家兔血液流变性和微循环的灌注,促进组织的修复,阻止椎间盘的进一步退变.     

针刺损伤兔椎间盘退变模型组织病理变化及软骨样细胞的迁移规律**☆

背景：兔椎间盘退变模型间盘退变表现为随时间进展脊索细胞将被软骨样细胞逐渐替代,但兔针刺纤维环间盘退变模型中软骨样细胞的来源和移行规律尚不明确。 目的：观察针刺兔纤维环间盘退变模型椎间盘病理变化过程,并初步探讨软骨样细胞来源及移行规律。 方法：将24只新西兰大白兔随机分为手术组与假手术组。手术组使用16 G穿刺针针刺L2/L3、L3/L4、L4/L5及L5/L6椎间盘纤维环,假手术组暴露至相同椎间盘前方后冲洗闭合伤口。 结果与结论：针刺损伤椎间盘退变过程中的软骨样细胞来源于终板。在髓核与上下终板交界处,软骨细胞脱离终板成串向髓核中心迁移;在髓核与内层纤维环交界处,软骨细胞沿纤维走行迁移并随之向皱缩的髓核边缘迁移。椎间盘退变过程中非钙化层逐渐变薄,非钙化层/钙化层比值逐渐降低。 关键词：椎间盘退变;软骨细胞;软骨终板;纤维环穿刺;新西兰大白兔  doi:10.3969/j.issn.1673-8225.2012.09.018     

腰椎终板Modic的改变:生化组织学特点

背景:1988年Modic等系统描述了在退变的腰椎间盘终板及终板下骨质MRI信号改变的类型、分型标准及组织学变化,并将其命名为Modic改变。各型Modic改变的退变程度尚不清楚。目的:通过对各型Modic改变的组织学观察和生化成分的检测,了解各型终板组织学特点及生化成分,研究各型Modic改变退变程度。方法:手术取得所需终板36例,手术时根据影像定位,取出所需终板,按Modic改变分型分组:无Modic改变的12例,ModicⅠ型12例,ModicⅡ型12例。将标本行苏木精-伊红染色,在光镜下观察软骨终板组织学特点,用免疫组织化学方法观察Ⅱ型胶原表达,用间苯三酚法测定蛋白多糖的含量。结果与结论:免疫组织化学染色结果显示,各组在腰椎终板基质中可见棕黄色细颗粒状阳性表达,在有Modic改变的椎体终板中,Ⅱ型胶原积分灰度值比无Modic改变者明显升高(P0.05),ModicⅠ型积分灰度值明显高于ModicⅡ型(P0.05)。蛋白多糖检测:在有Modic改变的椎体终板中,蛋白多糖的含量比无Modic改变者明显升高(P0.05),ModicⅠ型蛋白多糖的含量高于ModicⅡ型(P0.05)。组织学特点:ModicⅠ型(水肿型)软骨下血管化的纤维组织以及与此相关的软骨终板出现裂隙或破裂;ModicⅡ型(脂肪型)脂肪组织替代正常的软骨或骨组织;Modic(-)病理学无上述变化。结果可发现,Modic改变是腰椎终板退变逐渐加重的连续性过程。     

软骨终板形态与椎间盘退变的相关性

背景：以往研究证明多种内环境因素共同作用引发椎间盘退变,最重要的机制为椎间盘软骨终板的退变。 目的：分析椎间盘退变与终板形态的关系。 方法：回顾性分析62例因椎间盘源性慢性下腰痛和79例因髓核脱出致神经根性症状患者的腰椎MRI正中矢状位图像资料。根据腰椎MRI正中矢状位T1W1图像确定终板形态,T2W1图像确定椎间盘退变程度分级。 结果与结论：平坦型和不规则型终板最常见于椎间盘退变人群下腰椎,L5/S1平坦型最多见。髓核脱出组与椎间盘源性慢性下腰痛组中凹陷型终板椎间盘退变程度均较平坦型、不规则型低,平坦型终板椎间盘退变程度较不规则型低(P < 0.01)。两组间凹陷型与不规则型终板椎间盘退变程度差异无显著性意义,髓核脱出组平坦型椎间盘退变程度较椎间盘源性慢性下腰痛组高(P < 0.05)。提示随着椎间盘退变程度的加重,软骨终板形态有由凹陷型向平坦型、不规则型依次转变的趋势。     

制作兔腰椎终板下缺血模型 MRI与病理学变化验证的可行性

背景:目前的椎间盘退变动物模型主要通过改变椎间盘生物力学环境、损伤椎间盘自身结构以及应用基因技术改变动物的遗传性状等诱发退变,但这些方法均为外界人为因素直接作用于椎间盘,与椎间盘退变的自然病程差别较大。目的:评价经皮穿刺兔腰椎终板下椎体注射平阳霉素制作椎体终板下缺血模型的可行性。方法:选取新西兰大白兔46只,每兔设L5为实验组、L4为对照组。穿刺腰椎终板下椎体,L5注射平阳霉素(2g/L)1mL,,L4注射生理盐水1mL。其中4只兔子术前行腰动脉造影。术后第1,2,3,4,5周,2个月及3个月随机选取6只行MRI检查,并取病理送检。测量对比第4周实验组MRI与病理切片的缺血面积。结果与结论:对照组MRI和病理学检查均无特异性变化,实验组MRI第1,2周变化不明显,第3周出现FST1WI低信号,T2WI及FST2WI呈稍高信号,第4周信号变化更明显;实验组病理第1,2周无特异性变化;第3,4周骨小梁排列杂乱,骨细胞逐渐减少,椎体骨髓内血细胞减少,脂肪细胞增多融合,终板软骨细胞减少、结构紊乱,纤维环及髓核无明显变化;第5周出现椎间盘退变表现;第2,3个月终板下椎体缺血持续存在、椎间盘退变更加明显。实验组第4周MRI与病理切片缺血面积之间有显著正相关性(r=0.965,P0.001)。结果证实,采用经皮穿刺终板下椎体注射平阳霉素的方法可成功制作兔腰椎终板下缺血动物模型,具有创伤小、操作简便、重复性好和成功率高的特点。该模型是研究腰椎及椎间盘退变较理想的一种动物模型。     

bFGF对人退变椎间盘髓核细胞影响的临床研究

目的:探讨bFGF对人退变髓核细胞合成细胞外基质的影响,观察退变髓核细胞基因表达量高于正常髓核细胞的生长刺激因子对退变髓核细胞的影响。方法:分离、培养人退变椎间盘髓核细胞,取第2代髓核细胞,随机将退变椎间盘髓核细胞分为5组。A组:加入100μg/L bFGF;B组:加入200μg/L bFGF;C组:500μg/L bFGF;D组:1 000μg/LbFGF,E组:对照组,不加干扰因素。通过对试验组和对照组髓核细胞测定细胞II型胶原和糖胺多糖的mRNA表达;检测细胞外基质II型胶原和糖胺多糖表达水平。结果:bFGF刺激退变髓核细胞,II型胶原、糖胺多糖mRNA,细胞外液Ⅱ型胶原和糖胺多糖含量在第7天较对照组增加明显(P<0.05)。14 d、21 d II型胶原、糖胺多糖mRNA明显低于对照组(P<0.05)。但细胞外液Ⅱ型胶原和糖胺多糖含量在14 d、21 d两个时间点仍高于正常组(P<0.05)。结论:根据本研究结果结合文献调研,bFGF在椎间盘髓核细胞中存在着双重效应(促进或改善椎间盘退变)。     

丁香苷抑制大鼠椎间盘退变

背景:椎间盘退变是由于椎间盘内部髓核和纤维环组织发生损伤和退化导致的椎间盘结构和功能发生变化,目前尚无有效的治疗药物。目的:探讨丁香苷抑制大鼠椎间盘退变的作用。方法:取10只雄性SD大鼠,将每只大鼠的尾椎Co_(4)/Co_(5)椎间盘设为模型组、Co_(5)/Co_(6)椎间盘设为丁香苷组、Co_(6)/Co_(7)椎间盘设为对照组,对照组不进行任何处理,模型组、丁香苷组采用微型穿刺针进行纤维环全层穿刺建立椎间盘退变模型,造模后即刻,模型组、丁香苷组椎间盘分别注射2.5μL的生理盐水、丁香苷溶液(5μmol/L)。注射4周后取材,采用苏木精-伊红和番红O-固绿染色观察大鼠椎间盘退变程度,免疫组化染色分析大鼠椎间盘组织内Ⅱ型胶原、聚集蛋白聚糖及基质金属蛋白酶3,13的表达。结果与结论:①苏木精-伊红染色显示,模型组椎间盘高度降低,软骨终板变薄且有裂隙出现,纤维环结构紊乱且出现裂隙,髓核消失;丁香苷组椎间盘高度正常或略低于对照组,软骨终板退变程度较模型组轻,纤维环排列较模型组相对规整且无裂隙,髓核部分皱缩。②番红O-固绿染色显示,模型组椎间盘软骨终板出现缺损且软骨钙化层变薄,出现明显退变;丁香苷组椎间盘软骨终板结构形态有一定程度恢复。③免疫组化染色显示,与对照组比较,模型组椎间盘软骨组织内Ⅱ型胶原、聚集蛋白聚糖的表达降低(P<0.0001),基质金属蛋白酶3,13的表达升高(P<0.0001);与模型组比较,丁香苷组椎间盘软骨组织内Ⅱ型胶原、聚集蛋白聚糖的表达升高(P<0.001,P<0.0001),基质金属蛋白酶3,13的表达降低(P<0.001,P<0.0001)。④结果表明,丁香苷可通过抑制基质金属蛋白酶3,13的表达、提高Ⅱ型胶原和聚集蛋白聚糖的表达来改善椎间盘的结构和功能,预防和减缓椎间盘退变过程。     

In vivo study of novel biodegradable and osteoconductive CaO-SiO2-B2O3 glass-ceramics

To evaluate the possibility of novel CaO-SiO2-B2O3 glass-ceramics (CS10B) as a new bone replacement material, we compared the biodegradation and osteoconduction properties of CS10B, hydroxyapatite (HA), and tricalcium phosphate (TCP). Porous CS10B implants were prepared by the polymer sponge method. L5-6 single-level posterolateral spinal fusions were performed on 30 New Zealand white male rabbits. The animals were divided into three groups by implant material: CS10B, HA, and TCP. Radiographs were performed every 2 weeks. All animals were euthanized 12 weeks after surgery. The ratio of the area occupied by the ceramics by final and initial radiographs was calculated using radiomorphometric analysis. Uniaxial tensile strength was determined from seven cases in each group. The ratio of the area occupied by HA (88.7%+/-16.1%) was significantly higher than the others (p<0.005), and the ratio of the area occupied by CS10B (28.2%+/-9.3%) was significantly lower than those of HA and TCP (37%+/-9.6%, p<0.05). The mean values of the tensile strengths of the CS10B (182.7+/-19.9 N) and HA (191.4+/-33.5 N) were significantly higher (p<0.05) than that of TCP (141.1+/-28.2 N). CS10B had a fusion mass tensile strength similar to that of HA. Histological analysis confirmed that CS10B was well incorporated into the fusion mass. These findings suggest that CS10B is a possible bone replacement material.     

Age-related changes in the glycosaminoglycans of human meniscal aggrecan

Introduction Meniscal damage and degradation, which are strongly correlated with subsequent OA, have been identified in approximately 60% of people over 60 years of age. Age‐related changes in articular cartilage glycosaminoglycans (GAGs) have been described, and used to facilitate the study of pathology‐related changes ( Plass et al. 1998 ). However, such data do not yet exist for the meniscus. Materials and methods Undamaged human menisci were obtained following leg amputations, and the vascular and avascular zones of each lateral and medial meniscus were extracted into 4 m GuHCl. Aggrecan was recovered in the A1 fraction following CsCl density gradient centrifugation, and the relative abundance of chondroitin, dermatan and keratan sulphates (CS, DS and KS) was examined by NMR spectroscopy at 400 MHz and 43 °C. Results Human meniscal aggrecan was shown to contain CS, DS and KS, and our data show age‐related changes in the relative abundance of these GAGs. The change was similar for medial and lateral menisci and for the vascular and avascular zones within these. The KS abundance in aggrecan from young menisci (<15 years) was found to be 15–20% of the total GAGs. However, in older samples, it comprised only 7–12% of the GAGs. We have confirmed the presence of DS in human meniscal aggrecan and show that the abundance of DS gradually falls from approximately 16% at 10 years to 2–4% at 75 years. There is some variability between humans, although the trend is clear and for each human there is good agreement between medial and lateral menisci and vascular and avascular locations. The levels of CS comprise the remainder of the GAG attached to aggrecan and contribute the remainder of the GAG abundance. This can be seen to increase from 67 to 72% at 10 years to approximately 90% at 75 years. Discussion Our data show a clear age‐related change in the relative abundance CS, DS and KS from human meniscal aggrecan. The data show a decrease in the abundance of KS and DS and a concomitant increase in CS levels. These observations differ from those widely seen for articular cartilage, in which the levels of CS are seen to fall with age. We have confirmed that DS is a component of human meniscal aggrecan in agreement with previous work ( McNicol & Roughley 1980 ). However, previously reported levels of DS, approximately 20%, are those found only in younger menisci. Absolute levels of these GAGs have not yet been determined, and hence the mechanisms which bring about this relative increase in CS with age may include either changes in biosynthetic output and/or widespread GAG loss in which KS and DS loss increases with age.     

Glycosaminoglycan hydrogel films as bio-interactive dressings for wound healing

Chemically-crosslinked glycosaminoglycan (GAG) hydrogel films were prepared and evaluated as bio-interactive wound dressings. Hyaluronan (HA) and chondroitin sulfate (CS) were first converted to the adipic dihydrazide derivatives and then crosslinked with poly(ethylene glycol) propiondialdehyde to give a polymer network. The crosslinking occurred at neutral pH in minutes at room temperature to give clear, soft hydrogels. After gelation, a solvent-casting method was used to obtain a GAG hydrogel film. A mouse model was used to evaluate the efficacy of these GAG films in facilitating wound healing. Full-thickness wounds were created on the dorsal side of Balb/c mice and were dressed with a GAG film plus Tegaderm' or TegadermT' alone. A significant increase in re-epithelialization was observed on day 5 (p < 0.001) and day 7 (p < 0.05) for wounds treated with a GAG film plus Tegaderm versus those treated with Tegaderm alone. While no significant differences in wound contraction or inflammatory response were found, wounds treated with either HA or CS films showed more fibro-vascular tissue by day 10. The GAG hydrogel films provide a highly hydrated, peri-cellular environment in which assembly of other matrix components. presentation of growth and differentiation factors, and cell migration can readily occur.     

颈椎不稳对邻近节段椎间盘蛋白多糖及Ⅱ型胶原的影响

背景：颈椎行减压融合内固定术后邻近节段椎间盘加速退变,单个节段不稳是否也会加速邻近节段椎间盘退变还不清楚。 目的：研究颈椎不稳动物模型邻近节段椎间盘形态学、蛋白多糖及Ⅱ型胶原的变化。 方法：16只新西兰大白兔,随机分为实验组及对照组,每组8只。实验组通过颈椎前路穿刺破坏纤维环及抽吸C5/6髓核组织建立兔颈椎不稳动物模型,12周X射线证实退变后处死动物取材,切取C4/5椎间盘组织,从矢状面切开,取其髓核组织10 mg,间苯三酚法测定髓核中蛋白多糖的量,另取椎间盘组织制作石蜡切片后进行苏木精-伊红染色和SABC免疫组化染色观察。 结果与结论：实验组C4/5椎间盘髓核脊索细胞减少,被成纤维细胞样细胞取代,偶见圆形的软骨细胞,且椎间盘纤维环变得粗糙,排列紊乱,玻璃样变性及色素沉着,可见纤维软骨细胞,内外层纤维环之间形成裂隙。髓核中蛋白多糖的含量降低,与对照组相比差异有显著性意义。退变椎间盘髓核及纤维环中Ⅱ型胶原也较对照组明显减少。结果表明颈椎不稳可诱发邻近节段颈椎退变,表现为椎间盘发生形态学变化,蛋白多糖、Ⅱ型胶原含量下降。     

Influence of chondroitin sulfate and hyaluronic acid on structure, mechanical properties, and glioma invasion of collagen I gels

To mimic the extracellular matrix surrounding high grade gliomas, composite matrices composed of either acid-solubilized (AS) or pepsin-treated (PT) collagen and the glycosaminoglycans chondroitin sulfate (CS) and hyaluronic acid (HA) are prepared and characterized. The structure and mechanical properties of collagen/CS and collagen/HA gels are studied via confocal reflectance microscopy (CRM) and rheology. CRM reveals that CS induces fibril bundling and increased mesh size in AS collagen but not PT collagen networks. The presence of CS also induces more substantial changes in the storage and loss moduli of AS gels than of PT gels, in accordance with expectation based on network structural parameters. The presence of HA significantly reduces mesh size in AS collagen but has a smaller effect on PT collagen networks. However, both AS and PT collagen network viscoelasticity is strongly affected by the presence of HA. The effects of CS and HA on glioma invasion is then studied in collagen/GAG matrices with network structure both similar to (PT collagen-based gels) and disparate from (AS collagen-based gels) those of the corresponding pure collagen matrices. It is shown that CS inhibits and HA has no significant effect on glioma invasion in 1.0?mg/ml collagen matrices over 3 days. The inhibitory effect of CS on glioma invasion is more apparent in AS than in PT collagen gels, suggesting invasive behavior in these environments is affected by both biochemical and network morphological changes induced by GAGs. This study is among the few efforts to differentiate structural, mechanical and biochemical effects of changes to matrix composition on cell motility in 3D.     

聚醚醚酮/羟基磷灰石/碳纤维复合材料的生物力学分析

目的利用有限元分析法比较聚醚醚酮/羟基磷灰石/碳纤维复合材料（75PEEK/10HA/15CF）与钛合金的生物力学。方法建立C4～C6有限元模型,于C5～C6间植入75PEEK/10HA/15CF或钛合金人工椎间盘和椎间融合器,计算在前屈、后伸、侧弯和旋转时临近椎体、椎间盘的wonMises应力变化和C5～C6节段的活动度及植入物上的应力分布。结果在正常情况下,钛合金人工椎间盘置换模型在前屈时C5椎体、C4~5椎间盘平均won Mises应力改变率分别为75PEEK/10HA/15CF人工椎间盘模型的1.50倍和1.67倍;在侧弯时C6椎体的平均won Mises应力改变率为75PEEK/10HA/15CF人工椎间盘置换模型的1.33倍。融合器模型,在前屈时C5椎体、C4~5椎间盘应力改变率前者为后者的1.48倍和1.87倍;在侧弯时C6椎体应力改变率,前者为后者的1.67倍。钛合金植入物的最大应力为75PEEK/10HA/15CF的4.5倍,并出现应力集中。结论与钛合金相比,75PEEK/10HA/15CF能更好地将负荷传递,增加融合率,能有效地减少临近椎体的应力,减少植入物沉降的发生。     

Identification of glycosaminoglycans in human airway secretions

Glycosaminoglycans (GAGs), known to be present in airway mucus, are macromolecules with a variety of structural and biological functions. In the present work, we used fluorophore-assisted carbohydrate electrophoresis (FACE) to identify and relatively quantify GAGs in human tracheal aspirates (HTA) obtained from healthy volunteers. Primary cultures of normal human bronchial epithelial (NHBE) and submucosal gland (SMG) cells were used to assess their differential contribution to GAGs in mucus. Distribution was further assessed by immunofluorescence in human trachea tissue sections and in cell cultures. HTA samples contained keratan sulfate (KS), chondroitin/dermatan sulfate (CS/DS), and hyaluronan (HA), whereas heparan sulfate (HS) was not detected. SMG cultures secreted CS/DS and HA, CS/DS being the most abundant GAGs in these cultures. NHBE cells synthesized KS, HA, and CS/DS. Confocal microscopy showed that KS was exclusively found at the apical border of NHBE cells and on the apical surface of ciliated epithelial cells in tracheal tissues. CS/DS and HA were present in both NHBE and SMG cells. HS was only found in the extracellular matrix in trachea tissue sections. In summary, HTA samples contain KS, CS/DS, and HA, mirroring a mixture of secretions originated in surface epithelial cells and SMGs. We conclude that surface epithelium is responsible for most HA and all KS present in secretions, whereas glands secrete most of CS/DS. These data suggest that, in diseases where the contribution to secretions of glands versus epithelial cells is altered, the relative concentration of individual GAGs, and therefore their biological activities, will also be affected.     

A novel keratanase-generated keratan sulphate antibody and its applications

Introduction The sequencing of the genome has provided us with important information regarding the primary structure of many matrix proteins. This in turn has lead to advances in studies of the functions of post‐translational modifications on connective tissue proteoglycans (PGs). Changes in GAG structure with ageing and disease have been well documented ( Thonar et al. 1986 ; Brown et al. 1998 ). However, little is known about the exact sites of and differential substitution of GAGs on the aggrecan core protein and how these substitutions facilitate normal function or the changes seen with disease. The CS : KS ratio of substitution change significantly, with KS levels increasing with age and decreasing with the onset of disease. Objective was to produce monoclonal antibody (MAb) reagents to keratanase (k'ase) generated stub epitopes, that could be used to help identify, characterize and quantify sites of KS substitution on PGs, providing the potential to determine how the arrangement of such substitutions change with development, ageing and pathology. Methods Bovine Nasal Cartilage aggrecan (BNC A1D1) was trypsin digested, generating a range of glycosaminoglycan (GAG) fragments. The sample was then subjected to anion‐exchange and size exclusion chromatography to separate KS from CS fragments. Fractions collected were analysed by SDS‐PAGE and Western blotting. Fractions positive for KS were pooled and kinase digested to expose the KS stub antigens. Immunization and fusions were carried out as previously described ( Nieduszynski et al. 1990 ). Initial screenings were carried out using ELISA. Briefly, 96‐well microtitre plates were coated with the immunizing antigen overnight at 37 °C. The plates were then blocked prior to the addition of hybridoma media for 1–2 h at 37 °C. Binding was detected using an alkaline phosphatase‐conjugated secondary antibody for 1 h at 37 °C prior to the addition of the substrate. Positive wells were further screened by ELISA and SDS‐PAGE using the immunizing antigen, chondroitinase‐digested BNC and an A1D1 BNC preparation to establish the kinase stub specificity of the hybridomas. Further screenings by Western blotting was carried out on positive hybridomas selected. Antigens used included keratanase‐digested bovine corneal KS‐PGs, keratanase‐II‐digested KS‐PGs and a nonkeratanase‐digested corneal KS‐PG sample. Results Screening: Screening identified two positive hybridomas, B‐KS‐I and B‐KS‐II, which were specific for kinase‐generated KS stub. On screening, these antigens showed reactivity specifically for kinase‐digested BNC abc core, with no reactivity to the nonkinased linear KS GAG epitopes. Reactivity to kinase‐digested corneal KS‐PGs indicated that the MAbs generated were indeed to a stub structure in the KS chain and not to some linkage region epitope, amino acid sequence or oligosaccharide present on the core protein. Application: Immunohistochemistry utilizing B‐KS‐I was used to localize KS in a range of tissues along side anti‐KS 5D4. In human articular cartilage engineered grafts, labelling showed B‐KS‐I and 5D4 to have broadly overlapping labelling patterns for KS; however, label for B‐KS‐I had a much more restricted and subtle tissue distribution than that of antibody 5D4. Discussion These new KS stub MAbs have potential to be used in many different areas of research. They may be used in analysis of trypsin‐digested purified aggrecan from cattle joints of different ages to determine sites of KS substitution, which remain common or change with development and ageing. They may also be used in analysis of cartilage explant culture metabolites to assess KS substitution on the aggrecan fragments generated after stimulation of these cultures with cytokines such as IL‐1 or TNF‐α. Collectively it will provide important new information on the changing pattern of KS substitution in connective tissue PGs with development, ageing and the onset of pathology.     

Comparative study on Lewis lung tumour lines with ‘low’ and ‘high’ metastatic capacity III. Glycosaminoglycan synthesis,transport and degradation in cell lines

The glycosaminoglycans (GAGs) of low (LM) and highly metastatic (HM) cell lines of the Lewis lung tumour (3LL) were compared using [³H]glucosamine labelling techniques. The GAGs isolated from nuclei, cytoplasm, pericellular fractions and medium were analysed by cellulose acetate electrophoresis and by digestion with specific enzymes, and the following conclusions were drawn. 1. Increased cellular uptake and incorporation of [³H]glucosamine into glycoconjugates of the cytoplasm was a typical feature of the highly metastatic cell line after a 48-h labelling. However, there was no elevated radioactivity in glycolipids. 2. Radioactivity of the purified GAGs was two and three times higher in nuclear and cytoplasmic fractions of HM cells than in those of LM cells. There was much less difference between the two cell lines in the pericellular fractions. 3. A definite change from chondroitin sulphate to dermatan sulphate dominancy was recorded in each GAG fraction. Higher heparan sulphate labelling was observed in the cytoplasmic and pericellular GAGs of HM cultures. 4. In the post-labelling period about three times more GAG was present in the extracellular compartment of the HM cultures compared with the LM cultures. 5. In the LM cultures the total GAG-associated radioactivity decreased by 73 per cent in the 48-h chase period whereas in the HM cultures it decreased by only 30 per cent. This indicates a higher rate of GAG degradation in the LM cultures.Abbreviations GAG glycosaminoglycan - 3LL Lewis lung tumour - HM highly metastatic - LM low metastatic - FCS foetal calf serum - PBS phosphatebuffered saline - HP heparin - HS heparan sulphate - HA hyaluronic acid - CS chondroitin sulphate - DS dermatan sulphate - PCA perchloric acid - TCA trichloroacetic acid - CPC cetylpyridinium chloride - E extracellular - P pericellular - C cellular - CY cytoplasmic - N nuclear compartment - ECM extracellular matrix