人工生物膜修复椎间盘纤维环的研究

目的 用不同方法处理椎间盘纤维环,观察人工生物膜对椎间盘纤维环缺损的修复作用.方法 成年山羊8只,雌雄各4只,对每只羊的腰椎(腰2/3、腰3/4、腰4/5)椎间盘纤维环进行随机处理,处理方法包括:(1)暴露出椎间盘纤维环,不作任何处理;(2)暴露出椎间盘纤维环后,将其切开,用人下生物膜填充缺口;(3)暴露出椎间盘纤维环,尖刀将其切开,作为对照组.12周后,通过生物力学测试、核磁共振(MRI)及脱钙病理切片染色观察椎间盘纤维环的修复效果.结果 12周后,测试纤维环承受最大压力,单纯暴露组为(4.92±0.17)MPa,纤维环单纯切开组为(2.48±0.39)MPa,人工生物膜修复组为(3.76±1.56)MPa.人工生物膜修复组与单纯切开组,椎间盘纤维环完整性及生物强度均不如单纯暴露组,人工生物膜修复组优于单纯切开组,两组间的差异有统计学意义(P＜0.05),人工生物膜可与椎间盘纤维环的胶原纤维良好融合.结论 人工生物膜可对椎间盘纤维环缺口起到修复作用,能使其生物力学强度平均恢复76.4%,人工生物膜可以促进椎间盘结构完整性的恢复.     

组织工程学方法培养的同种异体纤维环细胞移植与椎间盘再生的实验研究

椎间盘是人体中最缺乏血供的组织,它的再生能力与关节软骨一样低下。在行髓核摘除术后,纤维环的再生很少,其结果导致椎间盘的变性将不可避免。该实验采用组织工程学方法。利用薄膜封闭的蜂窝状发育不全的胶原基质支架(ACHMS支架)作为纤维环细胞的载体,将纤维环细胞移植于经激光气化切除的椎间盘内。研究移植的纤维环细胞对兔椎间盘的再生能力。从20只日本白兔上分离出纤维环细胞。用PKH-26萤光染色标记后将其种植于蜂窝状发育不全的胶原基质支架内,支架周围用薄膜包绕封闭。纤维环细胞在薄膜封闭的蜂窝状发育不全的胶原基质支架内培养1周后移植于受体白兔的椎间盘空隙内。这些受体白兔的椎间盘髓核组织已被吲哚花青绿染料加强的激光所气化。分别于术后2、4、8、12周对白兔麻醉后摄腰椎软组织X线片,测量椎间盘高度,并取出腰椎标本。对移植同种异体纤维环细胞的兔椎间盘组织进行连续冰冻切片、番红精-O染色后组织学检查,观察PKH-26荧光标记的同种异体纤维环细胞的增殖情况。结果显示,同种异体纤维环细胞能存活,并具有增殖能力,在接受细胞移植的椎问盘内形成     

磁共振椎间盘造影术在尸体椎间盘纤维环撕裂中的应用研究

纤维环撕裂通常被认为是椎间盘退化的一种主要特征,该疾病甚至在无症状人群中也常出现。目前常规造影术的应用价值在临床上虽有争议,但该方法对于探查纤维环撕裂,以及通过造影所诱发的疼痛来确定下腰疼痛原因,或许有所帮助。对于椎间盘内注射造影剂后进行磁共振显像(磁共振椎间盘造影术,即MR椎间盘造影术)在这方面的应用,很多临床医生了解不多。该研究通过对照传统的MRI,调查MR椎间盘造影术对椎间盘纤维环撕裂的诊断价值。研究所用的标本(下胸椎和腰椎)系5具未经防腐处理的尸体,标本未曾经过外科治疗,去除过多的脊周软组织后于—60℃低温冷冻,实验时在室温条件下解冻24小时后观察。标本中有22/24个(另两个椎间盘间隙缺失)椎间盘可用于观察。观察分三     

脱矿脱细胞骨基质环支架体外构建组织工程化椎间盘纤维环

目的:探计以脱矿脱细胞骨基质环为支架、纤维环细胞为种子细胞体外培养构建组织工程化椎间盘纤维环的可行性.方法:取兔椎间盘纤维环细胞培养,应用甲苯胺蓝染色和Ⅰ型、Ⅱ型胶原免疫组织化学染色进行鉴定.用纤维蛋白凝胶接种技术将兔椎间盘纤维环细胞接种到经脱矿脱细胞制备的骨基质环支架材料上,体外培养3个月.每月取培养的细胞支架复合体进行大体形态、HE染色光镜检查和扫描电镜观察,并用生化方法榆测羟脯氨酸、氨基葡聚糖(GAG)、脱氧核糖核酸(DNA)含量;逆转录聚合酶链反应(RT-PCR)方法检测Ⅰ、Ⅱ型胶原信使核糖核酸(mRNA)表达;免疫组化和蛋白质印迹方法检测Ⅰ、Ⅱ犁胶原蛋白表达.结果:培养的第1代细胞甲苯胺蓝染色呈异染性,Ⅰ型、Ⅱ型胶原免疫组化染色均可见阳性表达,表明培养的第1代细胞具有椎间盘纤维环细胞的表型特点.构建的复合体体外培养1、2、3个月时大体呈白色半透明样环状,有光泽,质韧,具有一定弹性,可扭曲;HE染色光镜下见支架孔洞被红染的组织填充,且空洞内的细胞密度逐渐增加;扫描电镜观察材料表面逐渐被组织填充;Ⅰ型、Ⅱ型胶原免疫组化染色均为阳性;培养2个月时复合体羟脯氨酸、GAG、DNA含量明显高于1个月时(P<0.01),3个月时与2个月时比较无显著性差异(P>0.05).各时间点复合体羟脯氨酸、GAG、DNA含量均低于正常纤维环(P<0.05或<0.01);培养1个月时复合体可检测到Ⅰ、Ⅱ型胶原mRNA和蛋白的表达.2个月时与1个月时比较有显著性差异(P<0.01),3个月时与2个月时比较无显著性差异(P>0.05).结论:以脱矿脱细胞骨基质环为支架、纤维环细胞为种子细胞构建的复合体在体外培养时,细胞能够保持表型特点、逐渐增殖和行使功能,此复合体可被鉴定为类纤维环组织,用其构建组织工程化椎间盘纤维环可行.     

兔关月板纤维软骨细胞的培养及生物学特性研究

目的：通过半月板纤维软骨细胞的分离、培养、鉴定,观察其生长特点,研究半月板生物学特性及其损伤修复的细胞学基础。方法：机械分离兔半月板软骨,胰蛋白酶、胶原酶联合消化,１０％ＦＢＳＥ的ＤＭＥＭ中原代和传代培养,例置显微镜动态观察细胞形态及生长情况,ＧＡＧ、Ⅱ型胶原免疫组化染色,电镜观察细胞超微结构。结果：培养细胞呈多角形,有突起,富含分泌颗粒,ＧＡＧ、Ⅱ型胶原染色阳性,细胞线粒体和内质网发达。结论：培养的细胞保持了体内纤维软骨细胞的基本特性。     

纤维环膨隆在CT椎间盘造影图象上的表现

通过73例95个椎间盘的CAT椎间盘造影图像分析，作者认为纤维环膨隆绝大多数是向后方的膨隆，只有当椎间盘退变到相当严重程度时才有普遍超过椎体边缘均匀一致有膨隆。     

不同接种方法构建组织工程化椎间盘纤维环细胞支架复合体的对比研究

目的 在以脱矿脱细胞骨基质环为支架以纤维环细胞为种子细胞构建组织工程化椎间盘纤维环细胞支架复合体过程中,探索最佳的构建复合体方法.方法 分别使用纤维蛋白凝胶接种技术和传统的直接接种技术一静置法构建技术构建组织工程化椎间盘纤维环细胞支架复合体.对构建产物进行倒置显微镜观察、扫描电镜观察和细胞计数,比较两方法的效果.结果 纤维蛋白凝胶接种技术构建的细胞支架复合体.细胞粘附更多、增殖更迅速.结论 纤维蛋白凝胶接种技术在以脱矿脱细胞骨基质环为支架以纤维环细胞为种子细胞构建组织工程化椎间盘纤维环细胞支架复合体过程中,比静置法效果更好.     

纤维环穿刺法建立兔椎间盘退变模型

[目的]通过纤维环穿刺法建立兔椎间盘退变模型,分析其特点.[方法]将16只新西兰大白兔随机分为2组.经腹膜外入路暴露X2、3、L3、4椎间隙.A组为纤维环穿刺组,采用16号针头穿刺,B组为假手术对照组.术后2,4,6,8周通过MRI和组织病理学检查观察髓核变性及组织病理情况.[结果)纤维环穿刺组髓核信号强度在术后呈现逐渐降低趋势,髓核面积逐渐缩小,椎间隙高度也逐步下降,从术后4周开始两组差异有统计学意义(p＜0.O5 ).随着时间的进展,纤维环穿刺组髓核内细胞含量逐渐减少.[结论]纤维环穿刺法可成功建立椎间盘退变模型,比较真实地模拟了人类椎间盘损伤后的退变过程.     

丝素蛋白应用于椎间盘纤维环组织工程的研究进展

[目的]综述国内外有关丝素蛋白应用于椎间盘纤维环组织工程的研究进展,为下一步的实验研究提供理论依据。[方法]广泛查阅近年来国内外有关丝素蛋白的研究成果,尤其是应用于椎间盘纤维环组织工程的相关文献,归纳总结其发展现状和研究进展。[结果]目前丝素蛋白提纯工艺已臻成熟,并且在多个领域得到应用,比如纤维环、软骨、肌腱、韧带组织工程和药物缓释材料等方面。以丝素蛋白为材料制成的支架,结构稳定,力学强度高,还具有良好的生物相容性和可控的生物降解速率,并且能够通过简单的化学修饰来改进各方面的性能。[结论]丝素蛋白作为椎间盘纤维环组织工程支架的理想材料,表现出了良好的发展前景,值得进一步研究和推广。     

经皮穿刺纤维环建立兔腰椎间盘退变模型

目的探讨使用自制穿刺针经皮穿刺纤维环制备兔腰椎间盘退变模型的可行性。方法将18只新西兰大白兔分为实验组、假手术组及空白对照组。实验组及假手术组使用自制穿刺针穿刺L_(3~4)、L_(4~5)和L_(5~6)椎间盘位置,实验组穿刺椎间盘深度为5 mm,假手术组钝性穿刺但不损伤椎间盘,空白对照组不作处理。术后3、6、9周每组取2只兔麻醉后行腰椎MRI检查,处死行大体观察并取椎间盘行HE染色及髓核蛋白多糖含量测定。结果术后3周开始实验组MRI信号强度、髓核蛋白多糖含量与其他两组相比明显下降(P0.05),其后呈逐渐下降趋势,大体观察及HE染色显示实验组髓核及纤维环呈逐渐退变趋势。结论自制穿刺针经皮穿刺纤维环法能成功建立兔腰椎间盘退变模型,具有操作简单、损伤小、动物存活率高等优点。     

Platelet-rich plasma induces annulus fibrosus cell proliferation and matrix production

Purpose

Platelet-rich plasma (PRP) contains growth factors and creates a 3D structure upon clotting; PRP or platelet lysate (PL) might be considered for annulus fibrosus (AF) repair.

Methods

Bovine AF cells were cultured with 25 % PRP, 50 % PRP, 25 % PL, 50 % PL, or 10 % FBS. After 2 and 4 days, DNA, glycosaminoglycan (GAG), and mRNA levels were analyzed. Histology was performed after injection of PRP into an AF defect in a whole disc ex vivo.

Results

By day 4, significant increases in DNA content were observed in all treatment groups. All groups also showed elevated GAG synthesis, with highest amounts at 50 % PL. Collagen I and II expression was similar between groups; aggrecan, decorin, and versican expression was highest at 25 % PL. Injection of PRP into the AF defect resulted in an increased matrix synthesis.

Conclusions

Platelet-rich preparations increased the matrix production and cell number and may therefore be considered to promote AF repair.     

Galectins‐1 and ‐3 in Human Intervertebral Disc Degeneration: Non‐Uniform Distribution Profiles and Activation of Disease Markers Involving NF‐κB by Galectin‐1

Degeneration of the human intervertebral disc (IVD) is assumed to underlie severe clinical symptoms, in particular chronic back pain. Since adhesion/growth‐regulatory galectins are linked to arthritis/osteoarthritis pathogenesis by activating a pro‐degradative/‐inflammatory gene expression signature, we hypothesized a similar functional involvement of galectins in IVD degeneration. Immunohistochemical evidence for the presence of galectins‐1 and ‐3 in IVD is provided comparatively for specimens of spondylochondrosis, spondylolisthesis, and spinal deformity. Immunopositivity was detected in sections of fixed IVD specimens in each cellular compartment with age‐, disease‐, and galectin‐type‐related differences. Of note, presence of both galectins correlated with IVD degeneration, whereas correlation with age was seen only for galectin‐3. In addition, staining profiles for these two galectins showed different distribution patterns in serial sections, an indication for non‐redundant functionalities. In vitro, both galectins bound to IVD cells in a glycan‐dependent manner. However, exclusively galectin‐1 binding triggered a significant induction of functional disease markers (i.e., IL6, CXCL8, and MMP1/3/13) with involvement of the nuclear factor‐kB pathway. This study thus gives direction to further network analyses and functional studies on galectins in IVD degeneration. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2204–2216, 2019     

Species differences in cell culture of mammalian articular chondrocytes

Summary Articular chondrocytes from eight mammalian species (rabbit, opossum, woodchuck, cat, dog, sheep, rhesus and cebus monkeys) were grown in monolayer culture using a single regimen. The animals were immature or young adult. ham's F12 medium supplemented with 10% fetal bovine serum was employed for the primary cultures and Dulbecco-Vogt medium, for the secondary. Marked species differences were found with respect to cell morphology, growth in primary and secondary cultures, incorporation of radiosulfate into macromolecules, adhesion to the flask surface, response to vitamin C, and chondroid expression in spinner bottles. Under these particular conditions, rabbit chondrocytes grew most rapidly and incorporated several times more sulfate than did the others. Additional experiments carried out with other media on four of the species indicate that optimal conditions for culturing mammalian chondrocytes must be determined for each species individually.     

脱细胞纤维环基质对兔纤维环源干细胞分化的影响

目的观察脱细胞纤维环基质(DAFM)对纤维环源干细胞(AFSC)分化行为的影响,为新型纤维环组织工程支架材料的开发提供依据。方法将从新西兰大白兔获得的AFSC接种于由猪纤维环组织制备的DAFM膜和无DAFM膜培养皿,分别进行成脂、成骨、成软骨分化诱导,考察DAFM对AFSC分化行为的影响。结果 AFSC在DAFM膜上生长良好。DAFM膜上AFSC成脂、成软骨诱导分化水平低于无DAFM膜者,而成骨诱导分化水平显著高于无DAFM膜者;DAFM膜上的成脂、成骨及成软骨诱导比值(诱导组基因表达量/对照组基因表达量)均高于无DAFM膜者。结论猪DAFM有利于促进兔AFSC成骨分化,抑制其成脂及成软骨分化,较好地维持AFSC的差异性分化潜能。     

Determination of annulus fibrosus cell response to tensile strain as a function of duration,magnitude, and frequency

As clinical evidence suggests that mechanical forces can have both reparative and traumatic effects on the spine, we investigated responses to different magnitudes, frequencies, and durations of applied tensile strain in an in vitro system. We examined the interactions of inflammatory and mechanical stimuli on cells isolated from the annulus fibrosus. Rabbit annulus fibrosus fibrochondrocytes were cultured in the presence or absence of an inflammatory stimulus. Cells were exposed to various magnitudes and frequencies of tensile strain for 4 or 24 h, and mRNA expression of catabolic mediators of inflammation and matrix degradation was measured by quantitative real time PCR and compared to control cells. Conditioned media were analyzed for matrix metalloprotease activity and production of prostaglandin E₂. Application of low magnitudes and frequencies of tensile strain resulted in down‐regulation of catabolic mediators, particularly under inflammatory stress. However, loss of this protective effect was observed at higher frequency and magnitude, and after prolonged duration. These in vitro data confirm the existence of magnitude, frequency, and duration based effects, which determine biochemical response of disc tissue resulting in either anti‐ or pro‐catabolic outcomes. This may help to explain the beneficial effects of motion‐based therapies as well as the destructive effect of traumatic levels of applied strain. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1275–1283, 2011     

腰椎间盘纤维环切口不同修复方法的生物力学研究

【摘要】 目的：通过生物力学测试研究腰椎间盘纤维环切口不同修复方法的生物力学强度。方法：选取小牛腰椎标本制成50个脊柱功能节段,每个节段均在纤维环上作一10mm横切口。按照不同修复方法随机平均分为5组：粘合剂组（A组,选用DermaBond粘合切口）、简单缝合组（B组,采用常规U型缝合法处理切口）、MPSS组（C组,采用改良荷包缝合法处理切口）、粘合剂+简单缝合组（D组）及粘合剂+MPSS组（E组）,然后进行纤维环抗静水压强度测试及疲劳测试,根据各组的泄露压力和极限转数来评价其修复效果。结果：A～E组的泄露压力分别为（0.76±0.11）MPa、（1.66±0.11）MPa、（1.84±0.15）MPa、（1.88±0.13）MPa、（2.16±0.24）MPa。E组泄露压力明显高于其他组,差异有显著性（P＜0.05）。在疲劳测试中,从A～E组各组最后的极限转数分别为（4.42±0.59）万次、（8.06±0.75）万次、（8.44±1.01）万次、（9.76±0.23）万次、（9.88±0.21）万次。E组与D组比较差异无显著性（P＞0.05）,与其他各组比较差异均有统计学意义（P＜0.05）。结论：应用粘合剂+改良荷包缝合法处理纤维环切口,具有较高的生物力学强度。