兔腰椎间盘成分抗原性差异的研究

目的 初步了解兔椎间盘不同成分的免疫源性差别,进一步提示人体内不同椎间盘细胞的抗原性差别.方法 根据生物基因相似性原理及在椎间盘不同部位髓核成分含量存在差异的基础上,采用手术切取实验动物不同区域椎间盘组织并埋植于术野中邻近的椎旁肌内,从而建立髓核糖蛋白埋植组(n=20)和纤维环胶原蛋白埋植组(n=20),并且建立埋植自体肌肉的空白对照组(n=20).分别于第1、2、4、6、8周分批处死动物取标本进行HE染色,观察新生血管化和淋巴细胞浸润;进行免疫组织化学染色,观察CD4、CD8 T淋巴细胞阳性率.结果 HE染色显示糖蛋白埋植组标本在术后1周可见淋巴细胞浸润,4周可见典型新生血管化,并且持续至第8周;胶原蛋白埋植组标本在术后4周才可见少量淋巴细胞浸润,不典型新生血管化;肌肉埋植组在各个时间点均未见典型淋巴细胞浸润和新生血管化形成.各组标本总新生血管化率差异有统计学意义.各时间点所取标本进行免疫组织化学染色,计数CD4+和CD8+T淋巴细胞,发现糖蛋白埋植组CD4+和CD8+淋巴细胞计数均高于胶原蛋白埋植组和肌肉埋植组,胶原蛋白埋植组CD4+和CD8+淋巴细胞计数高于肌肉埋植组,差异有统计学意义.结论 兔腰椎间盘成分中髓核糖蛋白相比纤维环胶原蛋白更易诱导T淋巴细胞分化为CD4+和CD8+细胞,进而推论糖蛋白的自身免疫源性强于胶原蛋白,而胶原蛋白强于肌肉组织,具有弱抗原性.     

突出腰椎间盘组织再吸收现象的机制研究

目的探讨椎间盘组织接触有血运组织后能否被吸收缩小及其发生机制。方法(1)将15只大白兔随机分为A、B、C3组。经前外侧入路取出L4-5、L5-6椎间盘,共30个,将这些标本放入通风的无菌操作台内使水分蒸发,当剩余质量为起始质量的一半以下时将容器封口,记录数值备用。在A组兔两侧臀部各做长1cm切口,将椎间盘埋植在皮下。在B组兔两侧臀部各做长1cm切口显露臀肌,将椎间盘埋植在肌肉内。在C组兔下腰部正中做长3cm切口、咬除椎板显露硬脊膜,将2块椎间盘埋植在硬膜外。3个月后取出埋植椎间盘,称重后用体积分数为10%的甲醛溶液固定留作组织学和免疫组化检查。(2)收集LDH患者的椎间盘标本10个,与兔标本一起行组织学及免疫组化检查。组织学检查采用HE染色;免疫组化检查采用抗人巨噬细胞抗体CD68染色。结果埋植在皮下的椎间盘质量无明显变化(P>0.05);埋植在椎管内和肌肉内的椎间盘前后质量有显著性差异,P值分别小于0.01和0.05。组织学检查显示埋植在肌肉内和椎管内的椎间盘均有肉芽组织侵入,免疫组化CD68染色呈阳性,埋植在皮下的椎间盘1例有肉芽组织侵入,CD68染色呈弱阳性;10例患者的椎间盘标本,其中5例破裂型突出HE染色见有明显的血管肉芽组织侵入,另5例膨隆型突出未见肉芽组织侵入,但CD68染色均呈阳性。结论(1)突出     

Th17/Treg细胞相关因子IL-17及TGF-β在兔腰椎间盘退变髓核中的变化

目的探讨兔腰椎间盘退变髓核中Th17/Treg细胞相关因子IL-17及TGF-β的变化情况。方法新西兰大白兔50只,随机取20只作为对照组;剩余30只采用腹前外侧入路穿刺L3～4、L4～5椎间盘建立腰椎退变动物模型,MRI检查确定椎间盘退变,选取造模成功的20只作为实验组。第4、8周两组分别处死10只动物,观察椎间盘的大体观。第4、8周HE染色对两组动物的椎间盘髓核进行病理观察,从炎性细胞浸润、纤维组织增生及新生血管形成3个方面加以评估。第4、8周采用ELISA检测两组椎间盘中IL-17及TGF-β含量。结果实验组与对照组第4、8周炎性细胞浸润、新生血管形成、纤维组织增生比较差异有统计学意义(P 0. 01),实验组第4、8周炎性细胞浸润、纤维组织增生及新生血管形成比较差异有统计学意义(P 0. 01)。第4、8周实验组中IL-17、TGF-β含量比对照组明显增多(P 0. 01)。结论腰椎间盘突出可能与自身免疫反应有关。     

腰椎间盘突出症的免疫病理学研究

目的从免疫组织化学角度研究破碎型和完整型腰椎间盘突出症的病理机制和病理过程，比较其差异，探讨腰椎间盘突出症的不同病理学分型。方法选取40例腰椎间盘突出症患者的椎间盘手术标本，依术中所见分为两组：（1）破碎型腰椎间盘突出组（切开突出病变部浅层后纵韧带及纤维环可见破碎椎间盘组织与椎间盘母体分离，突出病变质软，自行溢出或较易钳出）。（2）完整型腰椎间盘突出组（切开突出病变部浅层后纵韧带及纤维环无破碎椎间盘组织溢出，突出病变质硬，必须以器械切除）。所获得的椎间盘标本均行常规HE染色；以鼠抗人CD43RO、CD20单克隆抗体进行免疫组织化学标记，双盲法半定量计数阳性细胞，Ridit等级分析；以FITC标记的兔抗人IgM、IgG抗体进行免疫荧光标记，双盲法半定量计数荧光量，Ridit等级分析。结果两组形态学有显著差异：（1）HE染色可见破碎组标本边缘灶性炎性细胞浸润，血管化；完整组髓核面积减少，纤维环增厚，软骨基质增生；（2）破碎组标本CD。sRO免疫组织化学阳性反应与完整组比较差异有统计学意义（P〈0．05）；（3）破碎组标本IgG和kM免疫荧光阳性反应与完整组比较差异有统计学意义（P〈0．01）。结论（1）破碎型腰椎间盘突出症标本中有T淋巴细胞浸润和免疫球蛋白IgG、IgM沉积，因而其病理机制可能是在损伤基础上的自免疫炎症反席过程。（2）完整犁腰椎间鼎突出以髓核很蛮、软骨某质及纤维环增牛为丰兽表现．     

破裂型椎间盘突出动物模型中新生血管因子与炎性反应的研究

目的 探讨破裂型椎间盘突出后重吸过程中新生血管的长入与炎性反应的存在.方法 将20只SD雄性大鼠,经手术切除尾椎椎间盘.埋植在背部硬膜外.造成破裂型椎间盘突出模型.动物30d后处死,取埋植的髓核组织,进行HE染色,免疫组化检测.结果 30d后椎间盘组织的形态结构发生明显改变,椎间盘组织中新生血管与巨噬细胞的免疫组化染色均呈阳性.结论 破裂型椎间盘突出的髓核组织经过一段时间确实有新生血管的长入与炎性反应的存在,其可能在椎间盘突出重吸收过程中发挥重要作用.     

破裂性椎间盘突出重吸收机制的研究

目的 探讨破裂性椎间盘突出发生重吸收的作用机制.方法 将20只远交群雄性大鼠随机分成对照组、实验组.实验组经手术切除尾椎椎间盘,埋植在硬膜外.30 d后处死,取埋植髓核组织,进行HE染色及免疫组化检测.对照组将线团埋植在背部肌肉内,30 d后处死作试验对照.结果 与对照组比较实验组埋植组织的质量明显缩小,实验组的TNF-α、VEGF免疫组化染色呈阳性.结论 椎间盘突出后发生的重吸收与其游离接触血运有关,新生血管与炎性反应在重吸收过程中发挥很大作用.     

仿生脉冲磁场联合同种异体骨髓间充质干细胞移植治疗股骨头坏死的实验研究

目的研究仿生脉冲磁场（BEMF）联合同种异体骨髓间充质干细胞（BMSCs）对兔早期股骨头坏死（ONFH）模型的促血管再生、骨再生的修复作用。方法取成功造模的液氮型ONFH模型24只,并随机分为4组,A组为制作模型而不填充任何材料,B组为单纯明胶海绵和PBS液填充,C组为填充复合骨髓间充质干细胞的明胶海绵,D组为填充复合骨髓间充质干细胞的明胶海绵,并予仿生脉冲磁场照射4周（2h/d）。于治疗后2、4、8周对各组股骨头进行解剖学、X线片、CT、组织切片观察及免疫组化血管染色,并进行血管计数。结果 （1）股骨头大体及CT观察：A组2周时可见典型的骨坏死;B组2周时可见明胶海绵吸收,4、8周骨坏死缺损清晰可见;C、D组：2周可见明胶海绵吸收,4周有新骨形成伴髓腔再通,8周新生骨与宿主骨界限模糊;（2）血管计数：术后4、8周C、D组新生血管计数大于A、B组（P〈0.05）,C、D两组比较,D组新生血管计数大于C组（P〈0.05）。结论 BEMF联合同种异体骨髓间充质干细胞移植可促进坏死股骨头内血管再生和骨修复作用。     

直肠癌新辅助治疗对组织驻留细胞浸润的影响及意义

目的探究新辅助治疗对局部进展期直肠癌(LARC)患者肿瘤微环境中CD103+CD8+T细胞的影响。方法选取接受新辅助治疗的LARC患者58例和未接受新辅助治疗的LARC患者82例。利用免疫荧光染色对两组患者手术切除标本的石蜡切片进行抗CD103+和CD8+染色, 使用卡方检验比较两组标本中CD103+CD8+T细胞的浸润情况、Spearman相关性分析探索新辅助治疗敏感性与CD103+CD8+T细胞浸润的关系。计数资料组间差异采用χ2检验, 相关性分析采用Spearman相关系数。结果实验组CD103+CD8+T细胞高浸润率高于对照组(58.62%比40.24%, χ2=4.597, P<0.05)。两组患者CD103+CD8+T细胞浸润程度与T分期、临床分期、分化程度有关(P<0.05);与患者年龄、性别、淋巴结转移及肿瘤大小无关(P>0.05)。根据CD103+CD8+T细胞浸润程度, 将实验组分为高低浸润组:高浸润组新辅助治疗的敏感率高于低浸润组(44.11%比12.50%, χ2=6.571, P<0.05)。CD103+CD8+T细胞浸润程度与新辅助治...     

抑制Tregs增强DC/HCC融合疫苗诱导抗肝癌免疫反应的研究

目的 通过抑制荷瘤小鼠体内调节性T细胞(TregFoxp3+),探讨其增强DC/HCC融合细胞疫苗诱导抗肝癌免疫作用的相关机制.方法 皮下建立小鼠肝癌模型,24只小鼠随机分为4组:(1)环磷酰胺联合疫苗组[环磷酰胺(CTX)+ VAC];(2)疫苗组(VAC);(3)单纯环磷酰胺组(CTX);(4)对照组.各组治疗后,取外周血测定CD8+及CD4+淋巴细胞比例,同时取肿瘤组织及脾脏组织分别行苏木素-伊红(HE)染色和免疫组织化学观察肿瘤坏死和CD8+细胞及Treg+细胞的浸润,并用细胞计数试剂盒(CCK-8)检测脾脏淋巴细胞特异性细胞毒T淋巴细胞(CTL)活性.结果 与其他各组比较,CTX+ VAC组外周血CD4+及CD8+细胞比例增高[ CTX+ VAC:17.50％、14.69％;VAC:16.17％、13.07％;CTX:12.17％、11.59％;对照组:12.24％、11.16％];肿瘤组织可见大量肿瘤细胞坏死,脾脏组织中Foxp3+细胞浸润减少,CD8+细胞浸润增高,CTL对肿瘤细胞杀伤作用明显增强(P＜0.05).结论 通过CTX抑制荷瘤小鼠微环境及外周血中的Treg细胞,可以明显增强DC/HCC融合疫苗对肿瘤的杀伤作用.     

混编肌腱重建兔膝前交叉韧带关节腔内愈合的组织学研究

[目的]比较自体-异体混编肌腱和同种异体肌腱重建兔前交叉韧带在关节腔内的重塑过程。[方法]取新西兰大白兔的双侧后腿趾长伸肌腱作为移植材料装入无菌塑料瓶,深低温-80℃保存14 d,放入-20℃冰箱保存备用。将40只新西兰兔随机平均分成自体异体肌腱混编组(混编组)和同种异体肌腱组(异体组),各20只。取兔右膝关节内侧切口,切除前交叉韧带后,按照正常兔前交叉韧带位置,选取胫骨与股骨骨道进行重建。分别于术后3、8、12、24周对重建肌腱行大体观察后应用HE染色、甲苯胺蓝染色、CD31免疫组织化学染色进行组织学评估比较。[结果]术后3周,两组均有炎性细胞浸润,肌腱内部有坏死现象。术后8周,两组可见大量圆形新生细胞由肌腱表面向中心推进,胶原纤维排列无序,细胞浸润程度以混编组相对较高。12周时,混编组内部大量圆形新生细胞,胶原纤维开始出现有序排列,异体组纤维排列仍紊乱,肌腱中心偶可见无细胞区。24周时,混编组新生细胞呈梭形,胶原纤维沿韧带长轴方向排列有序。异体组细胞仍呈圆形,胶原纤维出现有序排列。甲苯胺蓝染色结果显示:3、8周时,两组均未见异染。12周时混编组甲苯胺蓝染色出现异染,异体组未见异染,术后24周时两组均出现异染。CD31免疫组织化学染色血管计数及HE染色细胞计数在术后3、8、12周均高于异体组(P≤0.05),24周时低于异体组(P≤0.05)。[结论]混编肌腱关节腔内愈合经历缺血坏死期、再血管化及细胞增殖期和韧带重塑期,其塑型改建过程明显快于同种异体移植物。     

Phenotypic characteristics of rabbit intervertebral disc cells. Comparison with cartilage cells from the same animals.

STUDY DESIGN: Intervertebral disc cells were extracted from the surrounding matrix, and their metabolic activities and phenotypes were studied. OBJECTIVES: To compare the metabolic activities and phenotypes of cell populations extracted from the intervertebral discs of young rabbits with those of articular and growth plate chondrocytes from the same animals. SUMMARY OF BACKGROUND DATA: The phenotype of intervertebral disc cells has been poorly studied and still is debated. METHODS: The intervertebral discs as well as articular and vertebral growth plate cartilage of rabbits were digested enzymatically. The morphology of freshly isolated cells was examined. Their contents of collagen II and X mRNAs were determined by Northern blot analysis, and their sulfation activity by 35S-sulfate incorporation as chondrocytic markers. Cells were cultured at high density or low density and grown in primary culture. The stability of their phenotype was monitored by evaluating the collagen I and II mRNA ratio. The proteoglycans newly synthesized by the cells also were quantified, and their elution profile analyzed on Sepharose 2B columns. RESULTS: The anulus fibrosus cells were morphologically undistinguishable from articular chondrocytes. The nucleus pulposus contained mainly large vacuolated cells and a few smaller cells. All freshly extracted cells expressed different levels of collagen II mRNA. Anulus fibrosus and nucleus pulposus cells contained, respectively, 22% and 8% of collagen II mRNA compared with that found in articular or growth plate chondrocytes from the same animal. Only growth plate chondrocytes expressed collagen X. When anulus fibrosus cells were incubated for 48 hours at high density, they had collagen II mRNA contents similar to those of articular and growth plate chondrocytes, but synthesized five to six times fewer sulfated proteoglycans. When seeded at low density, anulus fibrosus cells divided more slowly than articular chondrocytes and incorporated four times fewer 35S-sulfate into proteoglycans. Their collagen II mRNA content was 2.75-fold lower than that of chondrocytes, and the procollagen alpha 1II/alpha 1I mRNA ratio was 3.1 for anulus fibrosus cells and 7 for chondrocytes. No collagen X mRNA was detected. When incubated for 48 hours at high density, the nucleus pulposus giant cells had four times less collagen II mRNA content than cartilage cells but synthesized the same amounts of sulfated proteoglycans. They did not divide during 21 days in culture and still contained collagen II mRNA but no collagen X mRNA. CONCLUSIONS: Findings showed that intervertebral disc cells all express cartilage-specific matrix proteins with quantitative differences, depending on their anatomic situation. It is suggested that anulus fibrosus cells are chondrocytic cells at a different stage of differentiation than articular and growth plate chondrocytes. The phenotype of nucleus pulposus cells still is unclear. They could be chondrocytic or notochordal. A definitive answer to this important question requires differentiating markers of notochordal cells.     

Age-related changes in fibromodulin and lumican in human intervertebral discs.

STUDY DESIGN: An analysis of proteoglycans of the intervertebral disc using immunoblotting of tissue extracts. OBJECTIVES: To investigate the changes in structure and abundance of fibromodulin and lumican in human intervertebral discs during aging and degeneration. SUMMARY OF BACKGROUND DATA: Fibromodulin and lumican are keratan sulfate proteoglycan constituents of the disc's extracellular matrix, whose interaction with collagen fibrils may contribute to the mechanical properties of the tissue. Changes in their abundance and/or structure that occur with aging and degeneration therefore may have an impact on disc function. METHODS: Lumbar intervertebral discs were obtained from individuals of different ages, and extracts of anulus fibrosus and nucleus pulposus were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using antibodies specific for fibromodulin and lumican. RESULTS: The major changes in abundance observed with age were a decrease in fibromodulin in the adult nucleus pulposus and an increase in lumican in anulus fibrosus during early juvenile development. In addition, fibromodulin in the anulus fibrosus exhibited a structural change with increasing age, characterized by a shift toward the predominance of its glycoprotein form lacking keratan sulfate. Fibromodulin was more abundant in the anulus fibrosus than in nucleus pulposus at all ages, whereas lumican was much more abundant in nucleus pulposus than in anulus fibrosus in the young juvenile; in the adult, however, lumican was present in comparable levels in both tissues. With increasing degrees of degeneration, fibromodulin exhibited an increase in abundance. CONCLUSIONS: Growth, aging, and degeneration of the intervertebral disc are associated with changes in the abundance and structure of fibromodulin and lumican, which presumably influence the functional properties of the tissue.     

Water, fixed charge density, protein contents, and lysine incorporation into protein in chymopapain-digested intervertebral disc of rabbit

Chymopapain (Discase) was injected at a dose of 0.125 nanokatal unit into the intervertebral discs of rabbits, and sequential changes in the metabolism of water, proteoglycan, collagen, and noncollagenous protein were investigated separately in the nucleus pulposus, anterior, and posterior anulus fibrosus. One week after chymopapain injection, the water and proteoglycan content was lower in all of the fractionated tissues of the anterior and posterior anulus and nucleus pulposus of the discs than in the control discs. In the anterior and posterior anulus, the proteoglycan content recovered after 12 weeks, but there was no recovery in the nucleus pulposus. The collagen content continued to increase up to the 12th week in the nucleus pulposus, while the noncollagenous protein content decreased in all tissue fractions after 1 week. In the anterior and posterior anulus, the content of noncollagenous protein recovered after 3 to 6 weeks, but there was no recovery in the nucleus pulposus. The lysine incorporation in collagen and noncollagenous protein was inhibited in all tissue fractions after 12 weeks, suggesting a decrease in synthetic activity. The intradiscal pressure calculated from proteoglycan hydration at 1 to 6 weeks after chymopapain injection showed a marked decrease to 0.8 to 0.9 atm, but it recovered to 1.6 atm after 12 weeks.     

Cell culture of the intervertebral disc of rats: factors influencing culture, proteoglycan, collagen, and deoxyribonucleic acid synthesis.

To establish cell culture of the nucleus pulposus and anulus fibrosus of rat intervertebral disc, the effects of culture conditions on the growth of cells and the synthesis of DNA, proteoglycan, and collagen were studied. For cell culture of the nucleus pulposus, the use of 3-week-old rats and a medium adjusted to pH 7.0 was optimal. There was almost no difference in growth between cells in Ham's F12 medium and those in Dulbecco's Modified Eagle Medium. In cells isolated from the anulus fibrosus, a medium adjusted to pH 7.0-7.6 was preferable, but irrespective of rat age. Culture cells of the nucleus pulposus were composed of large cells with vacuoles and small polygonal cells. These cells had a slight growth activity and a fair capability of proteoglycan and collagen synthesis. Culture cells of the anulus fibrosus were composed of polygonal and spindle-shape cells, and the growth was more vigorous with the potentials for proteoglycan and collagen synthesis than the nucleus cells.     

实验性脊柱内固定后相应区域椎间盘超微结构观察

目的　观察脊柱内固定后相应区域椎间盘的超微结构变化。　方法　日本大耳白兔2 4只,随机分成实验组和对照组,每组12只。实验组骨膜下游离T1 0 ～L3棘突和关节突,克氏针制成“L”形,将钢丝横行穿过T1 1、1 2 ,L1、2 的关节突关节,并与置于T1 1 ～L3棘突两旁的克氏针系紧,对相应区域的脊柱行内固定术。对照组未行手术,仅喂养至实验完成。术后6个月,对两组动物摄X线片观察1次,随后处死动物。取两组动物的L1 椎间盘组织(髓核、纤维环内侧及纤维环外侧)行透射电镜观察,对两组T1 2 、L2 椎间盘组织分别行水平面和矢状面透射电镜及扫描电镜观察。　结果　X线片显示,实验组与对照组椎体及椎间隙差别不明显;透射电镜与扫描电镜观察,实验组椎间盘的髓核、纤维环内层细胞的结构改变较纤维环外层早;对照组的髓核、纤维环内层细胞的结构改变与纤维环外层差别不明显。在退变的椎间盘基质中,蛋白多糖颗粒和特殊结构明显减少。髓核与纤维环基质内有蛋白多糖颗粒和一种特殊结构,而特殊结构在髓核与纤维环内层的形态不一致。　结论　脊柱内固定术后6个月,实验组在异常应力环境下发生椎间盘退变。髓核、纤维环内层基质内的特殊结构分布有特殊规律,与蛋白多糖颗粒在椎间盘退变中的生物学行为密切相关。     

Cyclic mechanical stretch stress increases the growth rate and collagen synthesis of nucleus pulposus cells in vitro

STUDY DESIGN: A rabbit model designed to investigate the effects of applied cyclic tensile stress on the cell division rate and the collagen synthesis in the rabbit nucleus pulposus cells in vitro. OBJECTIVE: To evaluate the effects of mechanical stress on nucleus pulposus cells, thus adding to the understanding of the adaptation of the intervertebral disc to mechanical stress. SUMMARY OF BACKGROUND DATA: Intervertebral disc cells in vivo are exposed to a multitude of physical forces during physical motion. Although it is known that in intervertebral disc disease, a common pathway of disc degeneration is mechanical stress on the nucleus pulposus or the anulus fibrosus or both, the underlying mechanism has been less well defined. METHODS: Nucleus pulposus cells were isolated from 4-week-old Japanese white rabbits. These cells were subjected to the mechanical cyclic stretch stress using a computerized, pressure-operated instrument that physically deformed the cells. The DNA synthesis rate, collagen synthesis rate, and cell cycle progression were measured. RESULTS: Cyclic tensile stretch increased the DNA synthesis rate in nucleus pulposus cells and in the population of cells in the S phase of the cell cycle during 1 to 2 days of subjugation to stress. Cyclic tensile stretch also increased collagenous protein synthesis in nucleus pulposus cells during 1 to 4 days of stress. CONCLUSIONS: Mechanical stress on nucleus pulposus cells promotes the proliferation of cells and alters the properties of intervertebral disc cells. This study may reflect the adaptation of the intervertebral disc to increased motion and stress.     

Matrix replenishment by intervertebral disc cells after chemonucleolysis in vitro with chondroitinase ABC and chymopapain

BACKGROUND CONTEXT: One of the advantages of chemonucleolysis for the treatment of a herniated intervertebral disc is the potential for the disc to self-repair. It has been suggested that the enzymes used for chemonucleolysis differentially affect the potential of the disc cells to promote repair. PURPOSE: To test the ability of nucleus pulposus and anulus fibrosus cells to repair the extracellular matrix degraded in vitro by either chondroitinase ABC or chymopapain. STUDY DESIGN: An alginate cell culture system was used to monitor the progress of matrix repair after chemonucleolysis in vitro. METHODS: Rabbit nucleus pulposus or anulus fibrosus cells precultured for 10 days in alginate gel were briefly exposed to low concentrations of chondroitinase ABC or chymopapain and then returned to normal culture conditions for up to 4 weeks. At each time point, the contents of DNA and matrix macromolecules and proteoglycan synthesis were measured. RESULTS: The DNA content of enzyme-treated alginate beads during the following 4 weeks of culture was higher in the chondroitinase ABC group than in the chymopapain group (NP, p<.01, and AF, p<.05). The content of proteoglycan in beads containing nucleus pulposus and anulus fibrosus cells in the chondroitinase ABC group was higher than that in the chymopapain group (NP and AF, p<.001). The rate of proteoglycan synthesis and the content of collagen did not, however, differ between those two groups. CONCLUSIONS: Intervertebral disc cells exposed to chondroitinase ABC reestablish a matrix richer in proteoglycan than cells exposed to chymopapain. This may be because of differences in the substrate spectrum of each enzyme. Although these results cannot be translated directly to the in vivo situation, they suggest the possibility that cells in discs subjected to chondroitinase ABC-induced chemonucleolysis retain a greater ability to replenish their extracellular matrix with proteoglycans than cells in discs exposed to chymopapain.     

Viscoelastic properties of intervertebral disc cells. Identification of two biomechanically distinct cell populations

STUDY DESIGN: A combined experimental and theoretical biomechanical study to quantify the mechanical properties of living cells of the porcine intervertebral disc. OBJECTIVES: To quantify zonal variations in the mechanical properties and morphology of cells isolated from the intervertebral disc. SUMMARY OF BACKGROUND DATA: Cellular response to mechanical stimuli is influenced by the mechanical properties of cells and of the extracellular matrix. Significant zonal variations in intervertebral disc matrix properties have been reported. No information is currently available on the corresponding regional variations in the mechanical properties of intervertebral disc cells, despite evidence of significant differences in cellular phenotype and biologic response to loading. METHODS: The micropipette aspiration test was used in combination with a three-parameter viscoelastic solid model to measure the mechanical properties of cells isolated from the anulus fibrosus, transition zone, and nucleus pulposus. RESULTS: Intervertebral disc cells exhibited viscoelastic solid behaviors. Highly significant differences were observed in the morphology, cytoskeletal arrangement, and biomechanical properties of the nucleus pulposus cells as compared with anulus fibrosus or transition zone cells. Cells of the nucleus pulposus were approximately three times stiffer and significantly more viscous than cells of the anulus fibrosus or transition zone. CONCLUSIONS: The findings of this study provide new evidence for the existence of two biomechanically distinct cell populations in the intervertebral disc. These differences in mechanical behavior may be related to observed differences in the cytoskeletal architecture between these cells, and may further play an important role in the development, maintenance, and degeneration of the intervertebral disc.