Characterization of Cre recombinase mouse lines enabling cell type-specific targeting of postnatal intervertebral discs

Cre/loxP technology is an important tool for studying cell type-specific gene functions. Cre recombinase mouse lines, including Agc1-CreER^T2, Col2a1-Cre; Col2a1-CreER^T2, Shh-Cre, Shh-CreER^T2, and Osx-Cre, have been proven to be valuable tools to elucidate the biology of long bones, yet the information for their activity in postnatal intervertebral disc (IVD) tissues was very limited. In this study, we used R26-mTmG fluorescent reporter to systematically analyze cell specificity and targeting efficiency of these six mouse lines in IVD tissues at postnatal growing and adult stages. We found that Agc1-CreER^T2 is effective to direct recombination in all components of IVDs, including annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplate (CEP), upon tamoxifen induction at either 2 weeks or 2 months of ages. Moreover, Col2a1-Cre targets most of the cells in IVDs, except for some cells in the outer AF (OAF) and NP. In contrast, the activity of Col2a1-CreER^T2 is mainly limited to the IAF of IVD tissues at either stage of tamoxifen injection. Similarly, Shh-Cre directs recombination specifically in all NP cells, whereas Shh-CreER^T2 is active only in a few NP cells when tamoxifen is administered at either stage. Finally, Osx-Cre targets cells in the CEP, but not in the NP or AF of IVDs tissues at these two stages. Thus, our data demonstrated that all these Cre lines can direct recombination in IVD tissues at postnatal stages with different cell type specificity and/or targeting efficiency, and can, therefore, serve as valuable tools to dissect cell type-specific gene functions in IVD development and homeostasis.     

椎间盘退变动物模型的研究进展

椎间盘退变是腰痛发生的主要原因,严重影响了人们的生活和工作。尽管具体发病机制尚不明确,但近年来其相关动物模型的研究有了很大的进步。造模方法包括结构损伤、应力改变及基因敲除等,本文综述并讨论了这些方法的优缺点和应用方向,以期为后续的研究奠定理论基础。     

经皮靶点穿刺臭氧注射术治疗伴有纤维环后方高信号的腰椎间盘突出症

目的:观察采用经皮靶点穿刺臭氧注射术治疗伴有纤维环后方高信号(HIZ)的腰椎间盘突出症(LDH)的临床疗效。方法:136例伴有纤维环后方HIZ的LDH患者根据治疗方法分为2组:75例患者为经皮靶点穿刺臭氧注射治疗组(A组),61例患者为保守治疗组(B组)。A组患者在C型臂X光机引导下对靶点成功穿刺后注射浓度为40μg/mL的O_3~O_2混合气体2~5 mL。采用MacNab腰腿痛手术评价标准和Oswestry功能障碍指数(ODI)评分比较两组患者的治疗效果。结果:136例患者除24例外均获随访,时间18~44个月。在术后第1、2、3、6、9、12和18个月,根据MacNab腰腿痛手术评价标准,A组有效率分别为88.00%、90.67%、93.33%、89.39%、84.85%、78.13%和73.44%,B组的有效率分别为68.85%、62.30%、55.74%、61.82%、58.12%、54.17%和47.92%,各个时间点两组间比较差异均有显著性(P0.05)。术后第12和18个月,A组ODI评分较低,两时间点组间比较差异无显著性(P0.05),但与术前及B组比较差异均有显著性(P0.05)。结论:经皮靶点穿刺臭氧注射是一种有效的治疗伴有纤维环后方HIZ的LDH的方法,其临床疗效比较稳定。     

The chemokine,CXCL16, and its receptor,CXCR6, are constitutively expressed in human annulus fibrosus and expression of CXCL16 is up-regulated by exposure to IL-1ß in vitro

Chemokines are an important group of soluble molecules with specialized functions in inflammation. The roles of many specialized chemokines and their receptors remain poorly understood in the human intervertebral disc. We investigated CXCL16 and its receptor, CXCR6, to determine their immunolocalization in disc tissue and their presence following exposure of cultured human annulus fibrosus cells to proinflammatory cytokines. CXCL16 is a marker for inflammation; it also can induce hypoxia-inducible factor 1α (HIF-1α), which is a phenotypic marker of heathy nucleus pulposus tissue. We found CXCL16 and CXCR6 immunostaining in many cells of the annulus portion of the disc. Molecular studies showed that annulus fibrosus cells exposed to IL-1ß, but not TNF-α, exhibited significant up-regulation of CXCL16 expression vs. control cells. There was no significant difference in the percentage of annulus cells that exhibited immunolocalization of CXCL16 in grade I/II, grade III or grade IV/V specimens. The presence of CXCL16 and its receptor, CXCR6, in the annulus in vivo suggests the need for future research concerning the role of this chemokine in proinflammatory functions, HIF-1α expression and disc vascularization.     

Parametric convergence sensitivity and validation of a finite element model of the human lumbar spine

The primary objective of this study was to generate a finite element model of the human lumbar spine (L1–L5), verify mesh convergence for each tissue constituent and perform an extensive validation using both kinematic/kinetic and stress/strain data. Mesh refinement was accomplished via convergence of strain energy density (SED) predictions for each spinal tissue. The converged model was validated based on range of motion, intradiscal pressure, facet force transmission, anterolateral cortical bone strain and anterior longitudinal ligament deformation predictions. Changes in mesh resolution had the biggest impact on SED predictions under axial rotation loading. Nonlinearity of the moment-rotation curves was accurately simulated and the model predictions on the aforementioned parameters were in good agreement with experimental data. The validated and converged model will be utilised to study the effects of degeneration on the lumbar spine biomechanics, as well as to investigate the mechanical underpinning of the contemporary treatment strategies.     

Embedding of human vertebral bodies leads to higher ultimate load and altered damage localisation under axial compression

Computer tomography (CT)-based finite element (FE) models of vertebral bodies assess fracture load in vitro better than dual energy X-ray absorptiometry, but boundary conditions affect stress distribution under the endplates that may influence ultimate load and damage localisation under post-yield strains. Therefore, HRpQCT-based homogenised FE models of 12 vertebral bodies were subjected to axial compression with two distinct boundary conditions: embedding in polymethylmethalcrylate (PMMA) and bonding to a healthy intervertebral disc (IVD) with distinct hyperelastic properties for nucleus and annulus. Bone volume fraction and fabric assessed from HRpQCT data were used to determine the elastic, plastic and damage behaviour of bone. Ultimate forces obtained with PMMA were 22% higher than with IVD but correlated highly (R² = 0.99). At ultimate force, distinct fractions of damage were computed in the endplates (PMMA: 6%, IVD: 70%), cortex and trabecular sub-regions, which confirms previous observations that in contrast to PMMA embedding, failure initiated underneath the nuclei in healthy IVDs. In conclusion, axial loading of vertebral bodies via PMMA embedding versus healthy IVD overestimates ultimate load and leads to distinct damage localisation and failure pattern.     

Diurnal variations in intervertebral disc height affect spine flexibility,intradiscal pressure and contact compressive forces in the facet joints

Diurnal changes of intervertebral disc height are caused by high compressive loading during the day, which expulses fluid from the disc, and by osmotic pressure, which imbibes fluid into the disc at low loading. The aim of the present study was to determine the magnitude of diurnal changes in spine flexibility, intradiscal pressures and contact forces in the facet joints. A validated osseoligamentous finite element model of the lumbar spine was used to determine these quantities for morning and evening situations. Disc height varied by 10% for these two situations. Spine flexibility and facet joint forces were markedly higher in the evening than in the morning. Intradiscal pressures were higher in the morning than in the evening. The different spine flexibilities in the morning and evening should be taken into account during kinematical measurements. Predicted facet joint forces may be used for the designing and pre-clinical testing of artificial facet joint replacements.     

Finite elements/Taguchi method based procedure for the identification of the geometrical parameters significantly affecting the biomechanical behavior of a lumbar disc

The aim of this work is to show a quick and simple procedure able to identify the geometrical parameters of the intervertebral disc that strongly affect the behavior of the FEM model. First, we allocated a selection criterion for the minimum number of geometrical parameters that describe, with a good degree of approximation, a healthy human vertebra. Next, we carried out a sensitivity analysis using the ‘Taguchi orthogonal array’ to arrive at a quick identification of the parameters that strongly affect the behavior of the Fem model.