非接触共培养条件下人BMSCs对氧化应激环境中退变髓核细胞凋亡的保护研究

目的BMSCs移植可修复椎间盘退变,但确切修复机制尚不明确。探讨非接触共培养条件下人BMSCs对氧化应激诱导退变髓核细胞(nucleus pulposus cells,NPCs)凋亡的保护作用,了解BMSCs移植修复椎间盘退变的可能机制。方法密度梯度离心联合贴壁法分离、培养正常人BMSCs,并鉴定CD34、CD45、CD13细胞表面分子。胶原酶消化法分离、培养人退变椎间盘NPCs,HE染色、倒置相差显微镜观察NPCs细胞形态,甲苯胺蓝染色、免疫细胞化学染色鉴定NPCs的类软骨细胞表型。取第3代BMSCs和第1代NPCs按共培养体系不同分为4组:A组单纯NPC(s1×106个)培养,不行凋亡诱导;B组BMSCs(1×106个)与NPCs(1×106个)共培养;C组BMSCs(3×105个)与NPCs(1×106个)共培养;D组单纯NPCs(1×106个)培养,行凋亡诱导。B、C、D组分别于共培养3、7d加入0.1mmolH2O2作用20min诱导NPCs凋亡。胰蛋白酶消化收集各组NPCs,行DAPI染色观察细胞核形态,Annexin-V/碘化丙啶染色、流式细胞仪计算凋亡率,半定量RT-PCR检测Bcl-2、Bax基因转录水平,Western-blot检测Caspase-3蛋白含量。结果成功分离并培养人BMSCs和人退变椎间盘NPCs;BMSCs鉴定呈CD34-、CD45-、CD13+;第1代NPCs呈梭形或多角形,于胞质内表达Ⅱ型胶原和聚集蛋白聚糖。DAPI染色示凋亡的NPCs可见细胞核固缩;共培养3、7d后,B组(29.26%±8.90%,18.03%±2.25%)及C组(37.10%±3.28%,13.93%±1.25%)细胞凋亡率均低于D组(54.90%±5.97%,26.97%±3.10%),高于A组(15.67%±1.74%,8.87%±0.15%),比较差异均有统计学意义(P0.05)。半定量RT-PCR检测示B、D组髓核细胞Bcl-2的转录水平提高(P0.05),Bax的转录水平无显著变化(P0.05);Western blot检测示B、C组NPCs的Cas-pase-3蛋白表达量低于D组,高于A组,差异有统计学意义(P0.05)。结论非接触共培养条件下人BMSCs可一定程度保护氧化应激诱导的退变NPCs凋亡,BMSCs共培养预处理的NPCs可能通过降低Bax/Bcl-2的转录比例来增强抗凋亡能力。     

Age‐related spontaneous lumbar intervertebral disc degeneration in a mouse model

The pathogenesis of intervertebral disc degeneration is unclear, but it is a major cause of several spinal diseases. Animal models have historically provided an appropriate benchmark for understanding the human spine. However, there is little information about when intervertebral disc degeneration begins in the mouse or regarding the relationship between magnetic resonance imaging and histological findings. The aim for this study was to obtain information about age‐related spontaneous intervertebral disc degeneration in the mouse lumbar spine using magnetic resonance imaging and a histological score regarding when the intervertebral disc degeneration started and how rapidly it progressed, as well as how our histological score detected the degeneration. The magnetic resonance imaging index yielded a moderate correlation with our Age‐related model score. The Pfirrmann grade and magnetic resonance imaging index had moderate correlations with age. However, our Age‐related model score had a high correlation with age. Intervertebral disc level was not a significant variable for the severity of disc degeneration. Both Pfirrmann grade and the Age‐related model score were higher in the ≥14‐month‐old group than in the 6‐month‐old group. The present results indicated that mild but significant intervertebral disc degeneration occurred in 14‐month‐old mice, and the degree of degeneration progressed slowly, reaching a moderate to severe condition for 22‐month‐old mice. At least a 14‐month follow‐up is mandatory for evaluating spontaneous age‐related mouse intervertebral disc degeneration. The histological classification score can precisely detect the gradual progression of age‐related spontaneous intervertebral disc degeneration in the mouse lumbar spine, and is appropriate for evaluating it. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:224–232, 2018.     

间充质干细胞移植治疗椎间盘退变疾病的研究进展

目的综述间充质干细胞（mesenchymal stem cells,MSCs）移植治疗椎间盘退变疾病的研究现状。方法查阅近年来有关MSCs移植治疗椎间盘退变疾病的国内外相关文献,进行回顾及综合分析。结果椎间盘移植MSCs在一定的条件下能表达类软骨细胞表型,增加基质合成,缓解椎间盘退变。结论MSCs移植治疗椎间盘退变疾病是一种很有前途的方法。     

骨髓间充质干细胞-壳聚糖凝胶复合体移植治疗椎间盘退变

目的 观察兔骨髓间充质干细胞.壳聚糖凝胶复合体移植修复椎间盘髓核缺损退变的效果.方法 建立兔椎间盘髓核缺损退变模型,将兔骨髓间充质干细胞-壳聚糖凝胶复合体注射移植入缺损退变模型中,兔继续培养4周后处死,取出移植修复的椎间盘进行组织HE染色、Aggre-can番红O染色及Ⅱ-collagen免疫组织化学染色,与正常椎间盘及未行移植的缺损退变椎间盘进行随机对照,检测移植修复的效果.结果 骨髓间充质干细胞-壳聚糖凝胶复合体可以在缺损的椎间盘中正常生长,并呈现向类髓核细胞分化的趋势,合成分泌Ⅱ-collagen和Aggrecan,维持原椎间盘髓核组织的生物学特性,而缺损退变组髓核组织纤维化、完整性丧失,水分丢失,Ⅱ-collagen合成明显减少(P<0.05).结论 兔骨髓间充质干细胞-壳聚糖凝胶复合体能够修复椎间盘缺损退变.     

MRI evaluation of spontaneous intervertebral disc degeneration in the alpaca cervical spine

Animal models have historically provided an appropriate benchmark for understanding human pathology, treatment, and healing, but few animals are known to naturally develop intervertebral disc degeneration. The study of degenerative disc disease and its treatment would greatly benefit from a more comprehensive, and comparable animal model. Alpacas have recently been presented as a potential large animal model of intervertebral disc degeneration due to similarities in spinal posture, disc size, biomechanical flexibility, and natural disc pathology. This research further investigated alpacas by determining the prevalence of intervertebral disc degeneration among an aging alpaca population. Twenty healthy female alpacas comprised two age subgroups (5 young: 2–6 years; and 15 older: 10+ years) and were rated according to the Pfirrmann‐grade for degeneration of the cervical intervertebral discs. Incidence rates of degeneration showed strong correlations with age and spinal level: younger alpacas were nearly immune to developing disc degeneration, and in older animals, disc degeneration had an increased incidence rate and severity at lower cervical levels. Advanced disc degeneration was present in at least one of the cervical intervertebral discs of 47% of the older alpacas, and it was most common at the two lowest cervical intervertebral discs. The prevalence of intervertebral disc degeneration encourages further investigation and application of the lower cervical spine of alpacas and similar camelids as a large animal model of intervertebral disc degeneration. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1776–1783, 2015.     

Reliability of a magnetic resonance imaging-based grading system for cervical intervertebral disc degeneration

STUDY DESIGN: This was a radiographic reliability study of a novel grading system for cervical intervertebral disc degeneration. OBJECTIVES: This study aimed to develop and test the reliability of a reproducible grading system for cervical intervertebral disc degeneration on the basis of the routine magnetic resonance imaging (MRI). SUMMARY OF BACKGROUND DATA: Cervical disc degeneration is common after middle age, and the morphology of cervical disc degenerative disease has often been studied using MRI. There are few specific MRI-based grading systems for cervical intervertebral disc degeneration despite the clinical importance of this problem. This study proposes a novel reproducible grading system for cervical disc degeneration and demonstrates the reliability of this classification scheme. METHODS: A grading system for cervical intervertebral disc degeneration was developed based on relevant previous literature. MRI grading of 300 cervical intervertebral discs using T2-weighted sagittal images was performed by 4 spinal surgeons (observers) in a blinded fashion. Intraobserver and interobserver reliabilities were assessed by calculating kappa statistics. RESULTS: Grade I degeneration was observed in 27 discs (9.0%); grade II, in 56 (18.7%); grade III, in 124 (41.3%); grade IV, in 67 (22.3%); and grade V, in 26 (8.7%) discs. Kappa coefficients for intraobserver and interobserver agreements ranged from substantial to excellent (intraobserver, 0.907 to 0.950 and interobserver, 0.730 to 0.826). Complete agreement was obtained, on an average, in 72.1% of the discs. A difference of 1, 2, and 3 grades was observed in 22.4%, 3.3%, and 0.2% discs, respectively. CONCLUSIONS: This grading system is comprehensive and easily applicable with sufficient reproducibility. It can be used as a common nomenclature for research and discussions.     

移植骨髓间充质干细胞对兔退变椎间盘髓核细胞凋亡的影响

目的 观察骨髓间充质干细胞(MSCs)移植对兔退变椎间盘髓核细胞凋亡的影响.方法 以各兔L2/3、L3/4、L4/5、L5/6节段分为正常组、退变组、成纤维细胞(SFs)移植对照组、MSCs移植治疗组.MSCs和SFs分别经绿色荧光蛋白(GFP)转染后,注射植入退变椎间盘的髓核.通过透射电镜观察退变椎间盘凋亡髓核细胞形态;用实时定量聚合酶链反应(PCR)检测退变组织中髓核细胞凋亡相关基因bcl-2和box mRNA的表达;免疫荧光法标记髓核细胞凋亡相关蛋白Caspase-3,并通过TUNEL法标记凋亡髓核细胞,激光共聚焦显微镜检测髓核细胞凋亡蛋白表达率和细胞凋亡比率.结果 透射电镜下,退变椎间盘中凋亡髓核细胞呈现出核染色质边集,空泡形成,核膜断裂,凋亡小体形成等变化.MSCs移植治疗组bcl-2 mRNA的表达量高于退变组和SFs移植对照组(P<0.05),bax mRNA的表达量与退变组差异无统计学意义(P>0.05).MSCs移植治疗组细胞凋亡率和Caspase-3表达率均高于正常组[细胞凋亡率分别为(16.75±2.14)%和(6.86±1.08)%;Caspase-3表达率分别为[(20.34±1.03)%和(6.09±0.77)%](P<0.05),低于退变组和SFs移植对照组[细胞凋亡率分别为(31.87±4.16)%和(29.02±2.16)%;Caspase-3表达率分别为(31.50±3.78)%和(30.20±4.93)%](P<0.05).结论 髓核细胞凋亡在椎间盘退变过程中起重要作用.MSCs移植能有效抑制椎间盘髓核细胞凋亡,延缓椎间盘退变过程.     

骨髓间充质干细胞治疗椎间盘退行性病变的研究进展

对椎间盘退变机制、BMSCs生物学特性、支架材料及细胞因子认识的深入,为BMSCs修复退变的椎间盘组织奠定了基础。研究表明,BMSCs治疗椎间盘退行性病变具有可行性,临床应用前景广阔。本文就BMSCs治疗椎间盘退行性病变的研究进展进行综述。     

Using notochordal cells of developmental origin to stimulate nucleus pulposus cells and bone marrow stromal cells for intervertebral disc regeneration

Purpose

Bone marrow stromal cells (BMSCs) have been proposed to complement the declining population of nucleus pulposus cells (NPCs) found in a degenerative intervertebral disc. Although able to stop degeneration, they could not produce enough matrix to restore a healthy state. Looking at development, when a large amount of matrix is produced, the disc also contains notochordal cells (NCs), potential progenitors or regulators of NPCs. The aim of the study was, therefore, to combine NCs to a BMSC/NPC mix and evaluate their effects on cell phenotype and matrix production, in long-term culture.

Methods

In a 3D hydrogel, NCs were co-cultured in different ratios with BMSCs and/or NPCs. Matrix production, cell morphology, and gene expression of disc markers were assessed after 4 weeks of culture.

Results

At day 28, BMSCs/NPCs highly expressed disc matrix markers (type II collagen and aggrecan) and produced disc matrix up to 30 % of values obtained for the positive control (BMSCs under TGFβ stimulation). The addition of NCs only slightly up-regulated marker expression (6–12× increase); an up-regulation not reflected at the matrix level. During the 4 weeks of culture, however, the NC phenotype changed drastically (morphology, disc marker expression).

Conclusion

In contrast to previously reported short-term studies, long-term co-cultures with NCs had no substantial effects on BMSCs and NPCs, most likely due to the loss of the NC native phenotype during culture. It, therefore, appears critical to maintain this specific phenotype for a long-term effect of the NCs.     

Nondestructive,indirect assessment of the biomechanical properties of the rat intervertebral disc using contrast‐enhanced μCT

Mechanical characterization of the intervertebral disc involves labor‐intensive and destructive experimental methodology. Contrast‐enhanced micro‐computed tomography is a nondestructive imaging modality for high‐resolution visualization and glycosaminoglycan quantification of cartilaginous tissues. The purpose of this study was to determine whether anionic and cationic contrast‐enhanced micro‐computed tomography of the intervertebral disc can be used to indirectly assess disc mechanical properties in an ex vivo model of disc degeneration. L3/L4 motion segments were dissected from female Lewis rats. To deplete glycosaminoglycan, samples were treated with 0 U/ml (Control) or 5 U/ml papain. Contrast‐enhanced micro‐computed tomography was performed following incubation in 40% Hexabrix (anionic) or 30 mg I/ml CA⁴⁺ (cationic) for 24 h (n = 10/contrast agent/digestion group). Motion segments underwent cyclic mechanical testing to determine compressive and tensile modulus, stiffness, and hysteresis. Glycosaminoglycan content was determined using the dimethylmethylene blue assay. Correlations between glycosaminoglycan content, contrast‐enhanced micro‐computed tomography attenuation, and mechanical properties were assessed via the Pearson correlation. The predictive accuracy of attenuation on compressive properties was assessed via repeated random sub‐sampling cross validation. Papain digestion produced significant decreases in glycosaminoglycan content and corresponding differences in attenuation and mechanical properties. Attenuation correlated significantly to glycosaminoglycan content and to all compressive mechanical properties using both Hexabrix and CA⁴⁺. Predictive linear regression models demonstrated a predictive accuracy of attenuation on compressive modulus and stiffness of 79.8–86.0%. Contrast‐enhanced micro‐computed tomography was highly predictive of compressive mechanical properties in an ex vivo simulation of disc degeneration and may represent an effective modality for indirectly assessing disc compressive properties. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2030–2038, 2018.

     

Therapeutic effect of co-culture of rat bone marrow mesenchymal stem cells and degenerated nucleus pulposus cells on intervertebral disc degeneration

BackgroundAfter non-contact co-culture of bone marrow mesenchymal stem cells (BMSCs) with nucleus pulposus cells (NPCs), exosomes secreted by BMSCs were able to ameliorate the degree of disc degeneration. The reason for this is, at least in part, that exosomes from BMSCs achieve by affecting the level of autophagy in NPCs, while the components in exosomes are diverse and their specific mechanism of action is still unclear.PurposeHere, we aimed to explore the therapeutic effect of co-culture of BMSCs and NPCs on NPCs and explore its specific mechanism of action.Study design/SettingIn vitro study.MethodsRat NPCs and BMSCs were isolated and cultured in vitro. The serum deprivation experiment (using oxygen, glucose, and serum deprivation [OGD]) simulates the pathological state of low blood supply of the intervertebral disc in vivo. We used apoptotic cell staining and flow cytometry to study the effect of BMSCs on the apoptosis rate of rat NPCs, and the apoptotic proteins active-caspase-3, active-caspase-9, autophagy marker proteins LC3 and Beclin 1 were further detected using Western blot analysis. The expression levels of the pro-apoptotic protein Bax and the apoptosis-inhibiting protein Bcl2 were measured. The differentially expressed miRNAs were screened in a gene expression profiling chip. Then qRT-PCR was used to detect the effect of different treatment methods on miR-155 expression. The effect of anti-miR-155 antibodies on autophagy was studied by flow cytometry and transmission electron microscopy. A luciferase reporter assay was used to study the direct interaction between miR-155 and BACH1 mRNA, which was analyzed by TargetScan software, and the results were verified by Western blotting.ResultsCompared with the OGD group, the expression level of miR-155 and the NPC autophagy level significantly increased; the HO-1 protein expression increased; and the Bach1 protein expression, degeneration index, and apoptosis index all significantly decreased in the co-culture group. After BMSCs transfected with anti-miR-155 were co-cultured with NPCs, the miR-155 expression in the cells was significantly reduced, the HO-1 protein expression and the level of cell autophagy was reduced. However, Bach1 protein expression, NPC degeneration index, and apoptosis index increased. After being inhibited by the autophagy inhibitor wortmannin, the cell degeneration index and apoptosis rate significantly improved.ConclusionIn the OGD model, BMSCs can significantly increase the viability, the level of autophagy, and reduce the level of apoptosis in rat NPCs. BMSC exosomes increase miR-155 expression in NPCs, which targets Bach1 and in turn upregulates HO-1 expression, activates autophagy in NPCs, inhibits the apoptosis level, and improves intervertebral disc degeneration.Clinical SignificanceOur experiment shows that it is maybe feasible to treat disc degeneration with drugs. At the same time, compared with BMSC injection method of treatment, side effects of drug therapy are smaller, and can be controlled, it also provides a new way for intervertebral disc degeneration drug treatment.     

Nucleus pulposus cells expressing hBMP7 can prevent the degeneration of allogenic IVD in a canine transplantation model

We have previously explored the possibilities of allogenic intervertebral disc (IVD) curing disc degeneration disease in clinical practice. The results showed that the motion and stability of the spinal unit was preserved after transplantation of allogenic IVD in human beings at 5‐year follow‐up. However, mild degeneration was observed in the allogenic transplanted IVD cases. In this study, we construct the biological tissue engineering IVD by injecting the nucleus pulposus cells (NPCs) expressing human bone morphogenetic protein 7 (hBMP7) into cryopreserved IVD, and transplant the biological tissue engineering IVD into a beagle dog to investigate whether NPCs expressing hBMP7 could prevent the degeneration of the transplanted allogenic IVDs. At 24 weeks after transplantation, MRI scan showed that IVD allografts injected NPCs expressing hBMP7 have a slighter signs of degeneration than IVD allografts with NPCs or without NPCs. The range of motion of left–right rotation in the group without NPCs was bigger than that of two cells injection group. PKH‐26‐labeled cells were identified at IVD allograft. The study demonstrated that NPCs expressing hBMP7 could survive at least 24 weeks and prevent the degeneration of the transplanted IVD. This solution might have a potential role in preventing the IVD allograft degeneration in long time follow‐up. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1366–1373, 2013     

Regeneration potential and mechanism of bone marrow mesenchymal stem cell transplantation for treating intervertebral disc degeneration

Intervertebral disc degeneration is a primary cause of low back pain and has a high societal cost. The pathological mechanism by which the intervertebral disc degenerates is largely unknown. Cell-based therapy especially using bone marrow mesenchymal stem cells as seeds for transplantation, although still in its infancy, is proving to be a promising, realistic approach to intervertebral disc regeneration. This article reviews current advances regarding regeneration potential in both the in vivo and vitro studies of bone marrow mesenchymal stem cell-based therapy and discusses the up-to-date regeneration mechanisms of stem cell transplantation for treating intervertebral disc degeneration.     

细胞衰老及衰老相关分泌表型在椎间盘退变中的研究进展

椎间盘细胞在不断分裂增殖和外界不利因素的持续刺激下极易发生老化。衰老细胞在影响椎间盘结构和功能同时异常表达衰老相关分泌表型(SASP),通过分泌促炎因子、趋化因子、生长因子和蛋白酶类等物质加速椎间盘退变的进程。目前已证实抗衰老治疗在椎间盘退变疾病中有很好的应用前景,包括减少衰老细胞发生、细胞移植抗衰老治疗等,特别是近些年靶向干预SASP抗衰老治疗受到学者广泛关注。本文就近年来细胞衰老和SASP在椎间盘微环境中的作用机制及相关干预措施进行综述。     

Cell therapy for disc degeneration--potentials and pitfalls

Disc degeneration is considered a major source of pain in patients with chronic low back pain. Novel strategies to cure or decrease the symptoms and increase the patient's quality of life and function are under development. Until recently conservative treatment and fusion surgery were the main therapeutic options. Disc prostheses are undergoing clinical evaluation. The potential for cell transplantation to the intervertebral disc with mature autologous disc cells, chondrocytes, or stem cells is in early stages of investigation. Cell transplantation potentially can increase proteoglycan production and induce disc regeneration or slow down the degeneration process. In animal models, transplantation of autologous disc cells and chondrocytes (derived from costal cartilage) has been demonstrated to be feasible and may slow disc degeneration.     

Spinal hernia tissue autofluorescence spectrum

The laser intervertebral disc decompression may provide appropriate relief in properly selected patients with contained disc herniations. The present investigation aims to characterise intervertebral disc material by autofluorescence induced by laser light. Degeneration of the intervertebral disc is associated with progressive biochemical changes in disc material. Percutaneous laser disc decompression has become rather popular for the treatment of lumbar disc herniation, but there are problems in the selection of patients. For this purpose, recognition of the disc composition is necessary. We propose a new type of spectroscopic investigation. It is advantageous to the characterization of intervertebral disc material. Intervertebral disc specimens were removed during open surgery from different disc locations. Preoperative patients’ MRI was evaluated using the Pfirrmann disc degeneration and Komori scale for migrating of herniated nucleus pulposus. Adjacent slices of stained disc sections were evaluated by histology/histochemistry and autofluorescence spectra. Comparison of the MRI, spectral, histological and histochemical data was performed. The MRI Komori scale correlated with the histology Boos degeneration index. In the histochemistry, collagens other than collagens I and II of the disc were distinguished with best positive correlation coefficient (0.829) and best negative one (?0.904) of proteoglycans of sequester to Boos index. A correlation of the IV Gaussian component of the hernia spectra with the Boos index was established. The Gaussian component correlation with different collagen types and proteoglycan was determined for the disc and sequester. “Autofluorescence-based diagnosis” refers to the evaluation of disc degeneration by histological and histochemical evaluation; it can provide additional data on the degeneration of an intervertebral disc.