胸椎黄韧带骨化患者黄韧带细胞的体外培养及初步鉴定

目的探讨胸椎黄韧带骨化的发病机理。方法2004年3月-2004年12月对8例行后路减压的下胸椎黄韧带骨化患者（男性5例，女性3例，平均年龄55岁）进行术中取材，采用组织块培养法，体外培养下胸椎黄韧带骨化患者非骨化区域的黄韧带细胞，并进行细胞化学，免疫细胞化学等方面研究；同时对7例急性外伤性下胸椎压缩骨折行胸椎后路减压术的患者（男性5例，女性2例，平均年龄28岁）进行术中取材，体外培养青壮年患者的下胸椎黄韧带细胞作为正常对照。结果体外成功地培养出黄韧带细胞15株，其中骨化患者黄韧带细胞8株（OLF1～OLF8），正常黄韧带细胞7株（NLF1～NLF7），黄韧带细胞可以在体外增殖和传代，通常正常黄韧带生长较慢，而骨化患者的黄韧带细胞则生长较快，并且可形成典型的钙结节样结构；80％以上的细胞呈碱性磷酸酶（ALP）强阳性反应；细胞内的ALP活性及其合成的骨钙素（BGP）含量均较正常对照组明显升高；细胞浆有BMP-2与TGF—betal的阳性表达，表明体外培养的下胸椎黄韧带骨化患者非骨化区域的黄韧带细胞呈现典型的成骨细胞表型特征：而正常黄韧带细胞主要为成纤维细胞表型。结论胸椎黄韧带骨化患者的非骨化区域中存在大量具备典型的成骨细胞表型特征的细胞，可能被骨形成蛋白等骨生长因子所调控。     

骨质疏松症成骨细胞生物学特征的体外研究

目的 建立成人和骨质疏松症成骨细胞培养方法；体外探讨骨质疏松成骨细胞特征性表型的变化。方法 取年轻人和骨质疏松患者，采用骨碎片培养和酶消化技术，分离、培养成骨细胞，体外观察细胞生长和转化状况，并用生物化学、放射免疫和RT—PCR技术检测成骨细胞表型。结果 骨碎片培养和多阶段酶消化相结合，可获得数量多、纯度高的成骨细胞；体外培养条件下，生长中、晚期的骨质疏松症成骨细胞可向成纤维细胞样细胞形态转化，骨钙素、碱性磷酸酶和I型胶原的分泌功能部分丧失，并表达非特异性Ⅲ型胶原，而呈现成纤维细胞样变。结论 骨质疏松成骨细胞可发生退变，并有可能是骨质疏松症发生骨基质成分不足、骨基质钙化不完全、骨组织结构改建不良的重要因素之一。     

胎兔颅骨成骨样细胞体外培养模型的建立

目的：建立成骨细胞体外培养模型,为骨代谢和成骨机理研究提供细胞学基础。方法：用酶消化和组织块移行生长相结合方法,从胎兔颅骨中分离出成骨样细胞群进行原代及传代培养,应用组织学、酶细胞化学、免疫细胞化学和染色体染色对其生物学和遗传学特性进行鉴定。结果：胎兔颅骨成骨样细胞具有体内成骨细胞的形态特征、ALP活性及合成分泌BMP等的功能,并能在体外发生钙生,其遗传学性状稳定。结论：该模型具有体内成骨细胞的生物学和遗传学特性,可用于成骨细胞代谢和成骨作用的体外研究。     

氟化钠对人黄韧带细胞碱性磷酸酶活性及骨钙素合成的影响

目的 探讨氟化钠(NaF)对体外培养条件下人黄韧带细胞碱性磷酸酶(alkaline phosphatase,ALP)活性及骨钙素(bone gla protein,BGP)合成的影响.方法 依据手术标本来源不同,分为正常黄韧带细胞(NLF)组(取自急性外伤性胸腰椎骨折截瘫患者,7例)、退变黄韧带细胞(DLF)组(取自退变性腰椎管狭窄症患者,9例)及骨化黄韧带细胞(OLF)组(取自胸椎黄韧带骨化患者,8例).采用组织块贴壁法进行体外细胞培养,共获得24组传代细胞.取第五代细胞加入不同浓度的NaF,观察细胞形态变化,并测定NaF对各组人黄韧带细胞的ALP活性和BGP合成的影响.结果 不同来源的人黄韧带细胞均可在体外增殖并传代,DLF与OLF组人黄韧带细胞呈多形性表现,并可形成钙结节.体外培养条件下,高浓度(1.0mmol/L)NaF可导致人黄韧带细胞发生中毒反应;低浓度(0.01～0.125 mmol/L)NaF可促进DLF组黄韧带细胞增殖、钙结节形成,ALP活性上调,BGP合成明显增加,而对OLF与NLF组人黄韧带细胞则无明显效应.结论 体外培养条件下,低浓度(0.01～0.125 mmol/L)NaF可促进人退变黄韧带细胞的ALP活性增高及BGP合成增加,提示低浓度NaF可能促进人退变黄韧带细胞向成骨方向分化.     

胎兔颅骨成骨样细胞体外培养模型的建立△

目的建立成骨细胞体外培养模型,为骨代谢和成骨机理研究提供细胞学基础.方法用酶消化和组织块移行生长相结合方法,从胎兔颅骨中分离出成骨样细胞群进行原代及传代培养,应用组织学、酶细胞化学、免疫细胞化学和染色体染色对其生物学和遗传学特性进行鉴定.结果胎兔颅骨成骨样细胞具有体内成骨细胞的形态特征、ALP活性及合成分泌BMP等的功能,并能在体外发生钙化,其遗传学性状稳定.结论该模型具有体内成骨细胞的生物学和遗传学特性,可用于成骨细胞代谢和成骨作用的体外研究.     

微血管周细胞的功能及潜在成骨能力

周细胞由星状间充质细胞所形成，并包在微血管内皮细胞外面。周细胞除有收缩作用，起干细胞作用参与伤口愈合、吞噬及防止渗漏等功能外，尚有成骨能力。应用Monastral蓝作标记，可以观察到周细胞演变为成骨细胞的过程。将周细胞作体外培养，可以具有成骨细胞表型的表达。     

新型双相陶瓷材料复合成骨细胞构建人工骨及其体内成骨的观察

目的 观察骨髓来源的成骨细胞与新型双相陶瓷材料复合培养后细胞生长状况及其体内异位成骨能力。方法 将来源于兔骨髓的成骨细胞与新法制备的HA／TCP共培养2周，通过相差显微镜、扫描电镜观察体外细胞生长情况；然后将复合物自体异位植入体内，6周后观察体内成骨情况。结果 扫描电镜显示材料表面和孔隙内均有成骨细胞生长，有良好增殖活动性和稳定细胞表型，材料中心区未见细胞生长；植入体内6周后取材见内植物有新骨形成、多位于内植物表面，可见成骨细胞、骨细胞、髓腔样结构、板层样骨基质等正常骨组织结构。结论 新型双相陶瓷支架可用作组织工程骨的细胞外基质材料，骨髓来源的成骨细胞可用作骨组织工程的种子细胞。     

骨髓间充质干细胞与成骨

种子细胞研究是骨组织工程学的重要内容。理想的骨种子细胞应该具有以下特点：结构比较简单，是不具有特定机能的原始细胞；取材容易，对机体损伤小；体外培养时增殖力强；自我更新，一定的条件下能向特定的方向分化；稳定表达成骨细胞表型；植入体内后能继续产生成骨活动；无致瘤性。     

珍珠层人工骨促进成骨细胞的体外成骨能力

目的：研究珍珠层人工骨对人成骨细胞体外成骨能力的影响。方法：将珍珠层人工骨、聚乳酸（对照组）与人成骨细胞共同培养，观察细胞形态、细胞活性、碱性磷酸酶（ALP）活性和基质钙化情况。结果：实验组在细胞形态、细胞活性等指标与对照组相比差异无显著性；实验组在培养7d及14d后ALP阳性细胞数多于对照组；培养21d后可观察到骨结节，对照组没有骨结节产生。结论：珍珠层人工骨可促进成骨细胞的体外成骨能力。     

体外镁合金与小鼠成骨细胞联合培养观察

[目的]观察镁合金与小鼠成骨细胞体外联合培养后，小鼠成骨细胞的活力、组织形态变化及生长扩增情况。由此验证镁合金与成骨细胞的生物相容性和骨传导特性，探讨镁合金成为一种新型骨科内置物材料的可行性。[方法]将小鼠成骨细胞体外扩增培养，并应用钙钴法及VonKossa法检测碱性磷酸酶加以验证。传代后将成骨细胞与镁合金金属片体外复合培养，细胞密度为3×10^4／ml。培养24、48、72h后，分别进行扫描电镜（SEM）、共聚焦显微镜观察细胞形态学变化及生长情况。[结果]小鼠成骨细胞体外培养后大量扩增，细胞特性稳定。与镁合金复合培养后，细胞保持良好的活性和分裂增殖能力。SEM观察：细胞粘附明显，增殖分化良好；共聚焦显微镜观察：随时间段的延长，细胞形态完整，轮廓清晰，增殖明显。[结论]镁合金与小鼠成骨细胞具有良好的生物相容性，镁合金对成骨细胞具有良好的骨传导特性。因此，镁合金成为一种新型的骨科内置物材料具有较强的可行性。     

Characterization of cultured human ligamentum flavum cells in lumbar spine stenosis.

To investigate the pathogenesis of the degenerative changes of the ligamentum flavum occurring in lumbar spine stenosis, yellow ligament cells from patients with lumbar spine stenosis were cultured for the first time and subjected to biochemical, histochemical and immunohistochemical study. Stenotic ligamentum flavum (SLF) cells were seen to express high levels of alkaline phosphatase (ALP) activity and to produce a matrix rich in type I and III collagen, fibronectin and osteonectin. The matrix mineralized only following beta-glycerophosphate (betaGP) and ascorbic acid supplementation. Stimulation with human parathyroid hormone (PTH) increased intracellular cAMP concentration. These findings indicate that there was significant evidence of osteoblast-like activity in these cells. SLF cells also stained for S100 protein, type II and type X collagen, and co-localized type II collagen and ALP labelling, reflecting the presence of hypertrophic chondrocyte-like cells. Cultures from control patients showed neither osteoblastic nor chondrocytic features: they expressed type I and type III collagen and fibronectin, but did not stain for osteonectin, nor were bone-like calcifications observed in presence or absence of betaGP. Normal ligamentum flavum (NLF) cells did not synthesized S100 protein or type II or type X collagen, and showed a weaker response to PTH stimulation. Our data demonstrated the presence of hypertrophic chondrocytes with an osteoblast-like activity in the ligamentum flavum of patients with spinal stenosis suggesting that they could have a role in the pathophysiology of the heterotopic ossification of ligamentum flavum (OLF) in lumbar spine stenosis.     

Calcification inhibitors in human ligamentum flavum

To examine the presence of substances which inhibit calcification in human ligamentum flavum, the inhibitory effect of an Na₂HPO₄ extract of the flavum was determined in terms of the in vitro calcium uptake of the ligamentum flavum matrix. Additionally, grafts of extracted and non-extracted dry ligamentum flavum matrices were transplanted into the dorsal muscles of rats, and calcification in the grafts was examined radiologically and histochemically. In order to determine if component cells of human ligamentum flavum produce calcification inhibitors, ligamentum flavum cells were cultured, and the crystal inhibitor activity of the culture medium was measured by a seed test which used hydroxyapatite as the nucleus of precipitation. The calcification reaction system demonstrated that the ligamentum flavum extract contains an inhibitory factor for calcium uptake by the ligamentum flavum matrix. The seed test revealed that human ligamentum flavum cells produce calcification inhibitor activity.     

腰椎退变黄韧带中炎性细胞因子的表达

目的:观察腰椎退变黄韧带中炎性细胞因子的表达情况,探讨其在腰椎黄韧带退变过程中的作用。方法:收集腰椎退变性疾病患者在后路椎板切除减压治疗时取下的黄韧带标本40例(退变组),术前通过CT、MRI证实均有黄韧带肥厚;以10例脊柱外伤行后路手术治疗的青年患者的腰椎黄韧带作为对照(对照组)。将术中切除的黄韧带剔除骨、脂肪及筋膜等组织,修成约2cm×1.5cm×0.3cm,在10%福尔马林溶液中固定24～48h后取出,制备成石蜡切片,采用苏木精伊红(HE)染色,应用光学显微镜观察病理学改变,并用免疫组化技术观察黄韧带标本中白介素-1(IL-1)、IL-6和肿瘤坏死因子-ɑ(TNF-ɑ)的表达情况。结果:对照组黄韧带弹力纤维呈波浪状,排列规则,弹力纤维间可见少量的胶原纤维和散在的成纤维细胞(22.56±6.05个/HP);退变组黄韧带弹力纤维明显减少,胶原纤维增多,并且伴软骨细胞、成纤维细胞、毛细血管的增生,成纤维细胞计数为62.66±18.40个/HP,两组成纤维细胞计数差异有统计学意义(P<0.05)。退变黄韧带组织中IL-1、IL-6、TNF-α表达阳性率分别为75%、77.5%、82.5%,对照组黄韧带组织中的表达阳性率分别为40%、20%、40%,退变组明显高于对照组(P<0.05)。结论:IL-1、IL-6、TNF-ɑ在腰椎退变黄韧带中有较高的表达,这些炎性因子可能参与了腰椎黄韧带退变的过程。     

Mechanical stretching force promotes collagen synthesis by cultured cells from human ligamentum flavum via transforming growth factor-beta1.

Although mechanical stress as a result of spinal instability is known to cause hypertrophy of the ligamentum flavum resulting in degenerative spinal canal stenosis, the mechanism of the ligament hypertrophy is not well understood. In the present study, we investigated the effect of mechanical stretching force on collagen synthesis and transforming growth factor-beta1 (TGF-beta1) production using ligament cells isolated from human ligamentum flavum in vitro. Ligamentum flavum cells (LFCs) were isolated from human ligamentum flavum obtained from patients who underwent lumbar spine surgery. The LFCs were subjected to a mechanical stretching force using a commercially available stretching device that physically deformed the cells. Collagen synthesis and TGF-beta1 production levels in the LFCs were then examined. Notable increases were observed in the gene expressions of collagen types I, III, and V in LFCs subjected to mechanical stretching force. The increase in collagen gene expression of LFCs was inhibited in the presence of anti-TGF-beta1 antibodies. Production of TGF-beta1 by the LFCs also increased significantly by the mechanical stretching force. Exogenous application of TGF-beta1 was confirmed to increase collagen synthesis of the LFCs. This data indicated that mechanical stretching force can promote TGF-beta1 production by LFCs, resulting in hypertrophy of the ligament.     

氟与黄韧带骨化

目的：探讨氟与黄韧带骨化的关系。方法：采用氟离子电极等方法测定１１ 例胸椎黄韧带骨化症（ 包括５ 例氟骨症） 患者血清及黄韧带标本中氟、钙含量,分别选取胸腰椎急性外伤性截瘫及腰椎管狭窄患者为正常及退变对照。结果：氟骨症骨化患者与非氟骨症骨化患者黄韧带中氟、钙含量均显著增高（ Ｐ＜ ０－０１） 。结论：氟在黄韧带骨化中起重要作用,可能是诱导退变黄韧带进一步骨化的重要诱因。     

中央型腰椎管狭窄症患者黄韧带组织生化成分变化的研究

目的:通过测定中央型腰椎管狭窄症患者腰椎黄韧带胶原蛋白和蛋白多糖含量变化,观察组织显微结构改变,探讨黄韧带组织生化成分改变与中央型腰椎管狭窄症发病的相关性方法:收集中央型腰椎管狭窄症患者的腰椎黄韧带65块作为退变组,正常腰椎黄韧带27块作为对照组,采用微量羟脯氨酸测定法和硫酸-咔唑法分别测定两组黄韧带中羟脯氨酸及糖醛酸吸光度,并计算胶原蛋白和蛋白多糖含量;游标卡尺测量黄韧带厚度;并行HE染色和Masson染色,显微镜下观察各组组织结构改变。结果:退变组黄韧带厚度、胶原蛋白及蛋白多糖含量均高于对照组,差异有显著性(P0.05);病理学观察退变黄韧带弹性纤维排列紊乱,数量减少,胶原纤维增生。结论:退变腰椎黄韧带中胶原蛋白和蛋白多糖含量增加,黄韧带生化成分改变可能引起黄韧带厚度增加,参与中央型腰椎管狭窄症的发生。     

Lumbar canal stenosis caused by amyloidosis of the yellow ligament]

Symptomatic lumbar canal stenosis without bony stenosis has previously been described. We describe the pathological modifications of ligamentum flavum among such operated patients. Ten patients were prospectively included in this study. Their mean age was 74, ranges: from 52-90. Clinical manifestation was a radicular claudication (sciatic or crural). Neuroradiology confirmed in all cases the ligamentum flavum thickness as the main cause of the symptomatology. This feature was also confirmed operatively and complete resection of the ligamentum flavum was performed. Resolution of the radicular pain was obtained in all cases at last follow-up. Pathological examination of the ligamentum flavum displayed characteristic features of degenerative modifications and elastic fibers fragmentation caused by numerous amorphous deposits. The deposits were studied using red Congo staining, polarized light and immunostaining methods. Such technique showed evidence of amyloid origin of the deposits. Immunodetection was positive for the P component in the amyloid deposits and for beta-2-microglobulin in one case (chronic renal failure and hemodialysis). The deposits did not express antitransthyretin antibodies. In parallel, control ligamentum flavum were obtained from 10 operated patients affected by bony lumbar stenosis. Moderate degenerative features were observed but small amounts of amyloid deposits were found in only 3 of those cases, without thickening of the ligamentous structure. This study correlates the presence of thickened ligamentum flavum caused by amyloid deposition, with symptomatic non-osseous lumbar canal stenosis. Association with degenerative modifications of the spine in the studied cases is suggestive of a microtraumatic origin.     

Pseudocystic degeneration of the lumbar ligamentum flavum: a little known entity

OBJECTIVE: The objective of this work was to investigate the clinical and histologic features of patients with pseudocystic lesions of the ligamentum flavum in the lumbar region of the spinal canal and ascertain the existence of genuine ligamentum flavum pseudocysts. METHODS: Retrospective chart and histologic study of a patient cohort with lumbar radiculopathy due to a cystic intraspinal lesion and who had undergone decompressive surgery was conducted. Intraoperatively, the stenosing process had been found to be different from common etiologic entities and had been submitted for histologic examination. RESULTS: The 33 patients with symptoms and signs of lumbar radiculopathy were between 48 and 85 years of age (mean 63.5 years). Twenty (61%) of them were women. All patients showed degenerative changes of the bony structures of the spine by conventional radiography. Segmental instability due to degeneration of the lumbar spine was present in 45%. Computed tomography and/or magnetic resonance imaging showed a cystic lesion. Clinical and histologic examination confirmed their origin from within the severely degenerated ligamentum flavum. CONCLUSIONS: Radiologic, surgical, and histologic findings suggest that the pseudocystic degeneration of the ligamentum flavum represents a genuine entity that is associated with degenerative changes of the structures of the respective lumbar spine segment. These pseudocystic lesions may compress the adjacent nerve roots, provoking symptoms and signs of radiculopathy. The findings suggest that the surgical treatment not only must consist of removal of the pseudocyst but must also include a radical extirpation of the ligamentum flavum surrounding the pseudocyst to avoid recurrence of such a lesion.     

合并腰椎疾患的下胸椎黄韧带骨化临床诊治

目的探讨合并腰椎疾患的下胸椎黄韧带骨化临床特点及诊治方法。方法下胸椎黄韧带骨化同时存在腰椎疾患的患者23例,诊断结合X线、椎管造影、CT、MRI检查,体征以肌张力增高和深反射异常为特点;患者均采用病变节段全椎板减压手术治疗。结果23例均获随访,时间10-36个月,手术减压1-3节胸椎椎板,患者在末次随访时都有不同程度的神经功能改善。术后功能恢复优4例,良13例,可6例。术后到末次随访时无一例患者因腰椎疾病而再次接受手术。结论合并腰椎疾患的下胸椎黄韧带骨化需要注意将客观体征与多种影像学检查相结合,尽早诊断、早期手术。     

胸椎黄韧带骨化与腰椎黄韧带退变的病理及基本代谢元素的比较研究

目的：探讨胸椎黄韧带骨化的病因。方法：对１４例胸椎黄韧带骨化（包括５例氟骨症）及１４例腰椎管狭窄症患者手术切除的黄韧带标本作病理研究；对患者血清及黄韧带采用雾化原子吸收法等方法测定钙、磷、镁、锌、铜、锰、钼、氟含量，取急性外伤性截瘫患者为对照。结果：（１）骨化黄韧带初期的病理改变与黄韧带退变性质类似；（２）除氟元素外，７种基本代谢元素在骨化与退变患者血清及黄韧带中含量均呈基本一致的变化规律；（３）非氟骨症骨化患者黄韧带中氟含量显著增高（Ｐ＜０．０１）。结论：本文证实胸椎黄韧带骨化发生于黄韧带退变的基础之上，但退变不直接导致骨化，元素氟是诱导退变黄韧带进一步骨化的重要诱因。