Cartilage diseases

软骨疾病
  • 文章类型: Journal Article
    背景:探讨和比较3.0T磁共振成像(MRI)T2标测在评估髋关节置换手术中髋臼软骨退变程度的价值。
    方法:纳入26例老年股骨颈骨折患者,采用3.0TMRIT2mapping量化技术扫描。基于MRI图像,根据国际软骨修复协会(ICRS)评分,髋臼软骨退变程度分为0,1,2,3和4级.此外,8例健康志愿者作为对照组。
    结果:与健康人群相比,前部的T2弛豫值,上级,股骨颈骨折患者髋臼软骨后区明显增高(P<0.001)。在股骨颈骨折患者中,1-2级(轻度变性亚组)髋关节16例,3-4级(重度变性亚组)髋关节10例,占61.54%和38.46%,分别。此外,关节软骨前带和上带的T2松弛值与MRI分级呈正相关(P<0.05);而严重退变亚组和轻度退变亚组的关节软骨后部T2松弛值差异无统计学意义(P>0.05)。重要的是,可以通过T2映射伪彩色图像的信号变化来检测髋臼软骨退变。
    结论:3.0TMRIT2标测技术可用于确定髋臼软骨退变的程度,能有效监测病程。
    BACKGROUND: To explore and compare the values of 3.0T magnetic resonance imaging (MRI) T2 mapping in evaluating the degree of acetabular cartilage degeneration in hip replacement surgery.
    METHODS: A total of 26 elderly patients with femoral neck fractures who were scanned in 3.0T MRI T2 mapping quantification technique were included. Basing on MRI images, the degree of acetabular cartilage degeneration was classified into Grade 0, 1, 2, 3 and 4, according to the International Cartilage Repair Society (ICRS) scores. In addition, 8 healthy volunteers were included for control group.
    RESULTS: By comparison with health population, T2 relaxation values in the anterior, superior, and posterior regions of acetabular cartilage in patients with femoral neck fracture were obviously increased (P < 0.001). Among the patients with femoral neck fractures, there were 16 hip joint with Grade 1-2 (mild degeneration subgroup) and 10 hip joints with Grade 3-4 (severe degeneration subgroup), accounting for 61.54% and 38.46%, respectively. Additionally, T2 relaxation values in the anterior and superior bands of articular cartilage were positively related to the MRI-based grading (P < 0.05); while there was no significant difference of T2 relaxation values in the posterior areas of articular cartilage between severe degeneration subgroup and mild degeneration subgroup (P > 0.05). Importantly, acetabular cartilage degeneration can be detected through signal changes of T2 mapping pseudo-color images.
    CONCLUSIONS: 3.0T MRI T2 mapping technology can be used to determine the degree of acetabular cartilage degeneration, which can effectively monitor the disease course.
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  • 文章类型: Case Reports
    我们描述了一名男性重型机械操作员的情况,他在工作中表现出迅速发展的脊髓综合征。脊柱MRI显示胸椎椎体和脊髓梗塞以及由于纤维软骨椎间盘栓塞(FCDE)引起的轻度椎间盘脱垂。在打桩机/重型机械操作人员中,应将纤维软骨椎间盘栓塞视为急性脊髓梗塞的病因机制之一。特别是与相邻椎体梗塞和椎间盘脱出有关。磁共振成像(MRI)变化可能会演变,保证早期随访MRI在适当的情况下。
    We describe the case of a male heavy machinery operator who presented from work with a rapidly evolving spinal cord syndrome. Spinal MRI revealed thoracic vertebral body and cord infarction and evolving mild disc prolapse attributed to fibrocartilaginous disc embolism (FCDE). FCDE should be considered as one of the aetiological mechanisms of acute spinal cord infarction in pile-driver/heavy machinery operators, especially in association with adjacent vertebral body infarction and intervertebral disc prolapse. Magnetic resonance imaging (MRI) changes may evolve, warranting early follow-up MRI in appropriate cases.
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  • 文章类型: Journal Article
    目的:为“髌股疼痛”(PFP)/“髌骨软化症”的诊断价值确定最适当的功能评估方法,(有效性,可靠性,灵敏度,特异性,预测价值和临床适用性);概述问卷的初步解释及其适当性,通过根据体格检查和磁共振成像(MRI)在其分数中确定的截止点;确定应将哪些方法相互结合使用以获得可靠有效和高效的临床诊断。
    方法:(1)验证了观察者内部和观察者之间的可靠性以及PFP问卷/物理测试之间的关系。(2)问卷的预测能力。主题:113膝盖患有PFP,使用“膝关节损伤和骨关节炎结果评分-髌股疼痛和骨关节炎”(KOOS-PF)进行评估,“Kujala-髌股评分”(KPS),“维多利亚-体育研究所-髌骨肌腱评估问卷”(VISA-P),和身体检查:“髌骨触诊”,“髌骨倾斜”,\“髌骨忧虑\”,“克拉克”和“蹲下”。
    结果:问卷本身的相关性为0.78结论:KOOS-PF,KPS和VISA-P证明了它们在PFP/软骨软化症中的诊断价值(有效性,可靠性,灵敏度,特异性,预测价值和临床适用性)。KOOS-PF是最通用的,最适合轻度病例和早期发现和预防。深蹲是最好的,因为它的可靠性和与问卷的临床关系,正确地预测了它。所讨论的功能评估工具应通过相互结合来应用。
    OBJECTIVE: To determine the most appropriate method of functional assessment for \"patellofemoral pain\" (PFP)/\"chondromalacia patella\" for its diagnostic value, (validity, reliability, sensitivity, specificity, predictive value and clinical applicability); to outline initial interpretations of the questionnaires and their appropriateness, through the cut-off points determined in their scores based on physical test and Magnetic Resonance Imaging (MRI); to establish which methods should be used in conjunction with each other to obtain clinical diagnoses that are robust effective and efficient.
    METHODS: (1)Intra- and inter-observer reliability and of the relationship among PFP questionnaires/physical tests validated. (2)Predictive capacity of the questionnaires. Subject: 113 knees with PFP, assessed using \"Knee-injury-and-Osteoarthritis Outcome-Score-for-Patellofemoral-pain-and-osteoarthritis\" (KOOS-PF), \"Kujala-Patellofemoral-Score\" (KPS), \"Victorian-Institute-of-Sports-Assessment-for-Patellar-tendons-questionnaire\" (VISA-P), and the physical tests: \"patellar-palpation\", \"patellar-tilt\", \"patellar-apprehension\", \"Clarke\" and \"squat\".
    RESULTS: Questionnaires correlations themselves was 0.78CONCLUSIONS: KOOS-PF, KPS and VISA-P demonstrated their diagnostic value in PFP/chondromalacia (validity, reliability, sensitivity, specificity, predictive value and clinical applicability). KOOS-PF was the most versatile, and the most appropriate in mild cases and for early detection and prevention. Squat was the best due to its reliability and clinical relationship with the questionnaires, which predicted it correctly. The functional assessment tools discussed should be applied by combining them with each other.
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  • 文章类型: Case Reports
    鼻塞是耳鼻咽喉科门诊部常见的问题。在这种情况下,一名青少年男孩从小就长期患有右侧鼻塞问题,寻求咨询。诊断性鼻内窥镜检查显示鼻中隔向左偏曲,再加上右下鼻甲肥大,全部覆盖健康粘膜。鼻和鼻旁窦的CT扫描进一步确定了骨性高密度病变,毛玻璃衰减,局限在右下鼻甲。随后的活检证实了青少年小梁骨化性纤维瘤(JTOF)。患者接受了内窥镜右内侧上颌骨切除术,最后的组织学证实了JTOF的诊断。
    Nasal obstruction is a commonly reported issue in the Otorhinolaryngology Outpatient Department. In this case, an early adolescent boy with a long-standing problem of right-sided nasal obstruction since childhood sought consultation. Diagnostic nasal endoscopy revealed a deviation of the nasal septum to the left, coupled with right inferior turbinate hypertrophy, all overlying healthy mucosa. A CT scan of the nose and paranasal sinuses further identified a bony hyperdense lesion with ground glass attenuation, confined to the right inferior turbinate. Subsequent biopsy confirmed juvenile trabecular ossifying fibroma (JTOF). The patient underwent endoscopic right medial maxillectomy, and the final histology affirmed the diagnosis of JTOF.
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  • 文章类型: Evaluation Study
    目的:本研究旨在探讨两种手术方法的有效性,自体髌腱移植重建和胫骨平台拔出修复,使用猪模型。主要重点是评估内侧半月板后部(MMPP)缺陷的修复能力,弯月面的整体结构完整性,以及两个手术组之间股骨和胫骨软骨的保护。总体目标是使用这些发现为临床研究提供实验指南。
    方法:选择12头猪,建立距胫骨平台插入点10mm的MMPP损伤模型。他们被随机分为三组,每组四只动物:重建(MMPP的自体肌腱移植重建),拔出修复(通过胫骨平台骨隧道缝合修复MMPP),和对照(使用正常内侧半月板作为阴性对照)。术后12周对动物实施安乐死,以评估半月板,肌腱骨愈合评估,膝关节软骨的大体观察。使用国际软骨修复学会(ICRS)分级和Mankin评分评估胫骨和股骨软骨损伤。对半月板-肌腱连接区进行组织学和免疫组织化学染色,初生半月板,和肌腱。Ishida评分用于评估重建组的再生半月板。磁共振成像(MRI)用于评估半月板愈合。
    结果:所有12头猪手术后恢复良好;所有切口均愈合,无感染,无明显并发症发生。总体观察显示,与对照组相比,重建和拔出修复组的效果更好。在胫骨软骨中,重建组有ICRSI级损伤,而拔出修复组和对照组有ICRSII级和III级损伤,分别。Mankin评分在重建组和对照组之间有明显差异;组织学染色显示,重建组再生半月板的结构与原始半月板相似。免疫组化染色显示,重建组再生半月板与原始半月板的Ⅰ型和Ⅱ型胶原染色程度类似。在重建组中,再生半月板与正常初生半月板之间的Ishida评分没有显着差异。MRI显示重建和拔除修复组的MMPP已完全愈合,而对照组尚未愈合。
    结论:自体髌腱移植重建MMPP可产生纤维软骨样再生半月板。重建和拔出修复都可以保持半月板的结构完整性,促进MMPP的愈合,延迟半月板变性,保护膝盖软骨.
    OBJECTIVE: This study was performed to investigate the effectiveness of two surgical procedures, autologous patellar tendon graft reconstruction and trans-tibial plateau pull-out repair, using a pig model. The primary focus was to assess the repair capability of medial meniscus posterior portion (MMPP) deficiency, the overall structural integrity of the meniscus, and protection of the femoral and tibial cartilage between the two surgical groups. The overall aim was to provide experimental guidelines for clinical research using these findings.
    METHODS: Twelve pigs were selected to establish a model of injury to the MMPP 10 mm from the insertion point of the tibial plateau. They were randomly divided into three groups of four animals each: reconstruction (autologous tendon graft reconstruction of the MMPP), pull-out repair (suture repair of the MMPP via a trans-tibial plateau bone tunnel), and control (use of a normal medial meniscus as the negative control). The animals were euthanized 12 weeks postoperatively for evaluation of the meniscus, assessment of tendon bone healing, and gross observation of knee joint cartilage. The tibial and femoral cartilage injuries were evaluated using the International Society for Cartilage Repair (ICRS) grade and Mankin score. Histological and immunohistochemical staining was conducted on the meniscus-tendon junction area, primary meniscus, and tendons. The Ishida score was used to evaluate the regenerated meniscus in the reconstruction group. Magnetic resonance imaging (MRI) was used to evaluate meniscal healing.
    RESULTS: All 12 pigs recovered well after surgery; all incisions healed without infection, and no obvious complications occurred. Gross observation revealed superior results in the reconstruction and pull-out repair groups compared with the control group. In the tibial cartilage, the reconstruction group had ICRS grade I injury whereas the pull-out repair and control groups had ICRS grade II and III injury, respectively. The Mankin score was significantly different between the reconstruction and control groups; histological staining showed that the structure of the regenerated meniscus in the reconstruction group was similar to that of the original meniscus. Immunohistochemical staining showed that the degree of type I and II collagen staining was similar between the regenerated meniscus and the original meniscus in the reconstruction group. The Ishida score was not significantly different between the regenerated meniscus and the normal primary meniscus in the reconstruction group. MRI showed that the MMPP in the reconstruction and pull-out repair groups had fully healed, whereas that in the control group had not healed.
    CONCLUSIONS: Autologous patellar tendon graft reconstruction of the MMPP can generate a fibrocartilage-like regenerative meniscus. Both reconstruction and pull-out repair can preserve the structural integrity of the meniscus, promote healing of the MMPP, delay meniscal degeneration, and protect the knee cartilage.
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  • 文章类型: Journal Article
    IVA型粘多糖贮积症(MPSIVA;MorquioA综合征)是一种罕见的常染色体隐性遗传溶酶体贮积症(LSD),由水解酶缺乏引起,N-乙酰半乳糖胺-6-硫酸盐硫酸酯酶,临床特征主要是肌肉骨骼表现。人类骨骼受累的机制通常是使用侵入性技术如骨活检来探索的。这使得人类的分析变得复杂。我们在野生型和MPSIVA敲除小鼠(UNT)中使用DDA和SWATH-MS比较了骨蛋白质组,以获得有关该疾病的机制信息。我们的发现揭示了基因敲除小鼠中超过1000种失调的蛋白质,包括那些与氧化磷酸化有关的,氧化应激(活性氧),DNA损伤,和铁运输,并提示乳酸脱氢酶可能是一个有用的预后和随访生物标志物。确定反映MPSIVA临床过程的生物标志物,严重程度,和进展对疾病管理有重要意义。
    Mucopolysaccharidosis type IVA (MPS IVA; Morquio A syndrome) is a rare autosomal recessive lysosomal storage disease (LSD) caused by deficiency of a hydrolase enzyme, N-acetylgalactosamine-6-sulfate sulfatase, and characterized clinically by mainly musculoskeletal manifestations. The mechanisms underlying bone involvement in humans are typically explored using invasive techniques such as bone biopsy, which complicates analysis in humans. We compared bone proteomes using DDA and SWATH-MS in wild-type and MPS IVA knockout mice (UNT) to obtain mechanistic information about the disease. Our findings reveal over 1000 dysregulated proteins in knockout mice, including those implicated in oxidative phosphorylation, oxidative stress (reactive oxygen species), DNA damage, and iron transport, and suggest that lactate dehydrogenase may constitute a useful prognostic and follow-up biomarker. Identifying biomarkers that reflect MPS IVA clinical course, severity, and progression have important implications for disease management.
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  • 文章类型: Journal Article
    关节软骨缺损受到软骨再生能力不足的挑战。Catalpol(CA),地黄的主要活性成分,可以对各种疾病发挥保护作用。然而,CA对关节软骨损伤治疗的影响尚不清楚。在这项研究中,通过手术在小鼠模型中诱导全层关节软骨缺损。用CA腹膜内注射动物4或8周。根据宏观观察的结果,显微计算机断层扫描CT(μCT),组织学和免疫组织化学染色,CA治疗可促进小鼠软骨修复,导致软骨再生,骨结构改善和基质合成代谢。具体来说,间充质干细胞(MSCs)的标志物CD90的表达增加,在软骨中观察到。此外,我们评估了CA对MSCs的迁移和软骨形成作用。向C3H10T1/2细胞中加入不同浓度的CA。结果表明,CA增强了细胞的迁移和软骨形成,而不影响增殖。总的来说,我们的发现表明CA可能通过刺激内源性MSCs治疗软骨缺损。
    Articular cartilage defect is challenged by insufficient regenerative ability of cartilage. Catalpol (CA), the primary active component of Rehmanniae Radix, could exert protective effects against various diseases. However, the impact of CA on the treatment of articular cartilage injuries is still unclear. In this study, full-thickness articular cartilage defect was induced in a mouse model via surgery. The animals were intraperitoneally injected with CA for 4 or 8 weeks. According to the results of macroscopic observation, micro-computed tomography CT (μCT), histological and immunohistochemistry staining, CA treatment could promote mouse cartilage repair, resulting in cartilage regeneration, bone structure improvement and matrix anabolism. Specifically, an increase in the expression of CD90, the marker of mesenchymal stem cells (MSCs), in the cartilage was observed. In addition, we evaluated the migratory and chondrogenic effects of CA on MSCs. Different concentration of CA was added to C3H10 T1/2 cells. The results showed that CA enhanced cell migration and chondrogenesis without affecting proliferation. Collectively, our findings indicate that CA may be effective for the treatment of cartilage defects via stimulation of endogenous MSCs.
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  • 文章类型: English Abstract
    UNASSIGNED: To discuss the application of anterior region suture of the popliteal hiatus (PH) under arthroscopy in the treatment of discoid lateral meniscus (DLM) injury with instability in the popliteal tendon region.
    UNASSIGNED: The clinical data of 53 patients (56 knees) with DLM injury who met the selection criteria between March 2014 and November 2022 were retrospectively analyzed. There were 15 males and 38 females, aged 8-55 years with an average age of 36.5 years. Fourteen cases had a history of trauma, while the remaining 39 cases had no clear history of trauma. The disease duration ranged from 1 day to 6 years, with an average duration of 15.6 months. According to the Watanabe classification, there were 40 knees of complete type and 16 knees of incomplete type. The preoperative International Knee Documentation Committee (IKDC) knee joint score was 51.2±8.3, the Lysholm score was 59.6±11.2, and the visual analogue scale (VAS) score was 4.7±1.3. After the arthroscopic meniscal plasty, the instability of the popliteal tendon region meniscus was checked by probing traction. Subsequently, the Out-inside technique or a combination of Out-inside and All-inside techniques was used to suture the anterior region of the PH. The stability of the meniscus after suturing was assessed, and if necessary, further suturing using the All-inside technique at the posterior region of the PH, the posterior horn of the meniscus, and using the Out-inside technique at the anterior horn of the meniscus was performed. Postoperative complications were recorded. The effectiveness was evaluated using pre- and post-operative IKDC scores, Lysholm scores, and VAS scores.
    UNASSIGNED: After operation, knee joint pain, crepitus, and locking disappeared, with McMurray and grinding tests turning negative. All patients were followed up 12-93 months with an average of 57.5 months. There was no complication such as common peroneal nerve injury, deep vein thrombosis of the lower limbs, joint infection, or joint stiffness. At last follow-up, the IKDC knee joint score was 76.7±5.5, the Lysholm score was 94.0±4.1, and the VAS score was 1.1±0.8. The differences compared with preoperative scores were significant ( t=-22.090, P<0.001; t=-23.704, P<0.001; t=19.767, P<0.001).
    UNASSIGNED: Suturing of the anterior region of the PH is crucial in the treatment of DLM injury with instability in the popliteal tendon region.
    UNASSIGNED: 探讨关节镜下腘肌腱裂孔(popliteal hiatus,PH)前区缝合在外侧盘状半月板(discoid lateral meniscus,DLM)损伤并腘肌腱区不稳治疗中的应用。.
    UNASSIGNED: 回顾分析2014年3月—2022年11月收治且符合选择标准的53例(56膝)DLM损伤患者临床资料。男15例,女38例;年龄8~55岁,平均36.5岁。14例有外伤史,余39例无明确外伤史。病程1 d~6年,平均15.6个月。根据Watanabe分型:完全型40膝,不完全型16膝。术前国际膝关节文献委员会(IKDC)膝关节评分为(51.2±8.3)分,Lysholm评分为(59.6±11.2)分,疼痛视觉模拟评分(VAS)为(4.7±1.3)分。行关节镜下半月板成形术后,用探钩牵拉检查腘肌腱区半月板均存在不稳,进而采用Out-inside技术或Out-inside技术联合All-inside技术缝合PH前区,检查缝合后半月板稳定性,必要时再采用All-inside技术缝合PH后区、半月板后角和Out-inside技术缝合半月板前角。记录并发症发生情况;采用手术前后IKDC评分、Lysholm评分和VAS评分评价疗效。.
    UNASSIGNED: 术后膝关节疼痛、关节弹响及交锁症状消失,McMurray试验及研磨试验转为阴性。53例患者均获随访,随访时间12~93个月,平均57.5个月。无腓总神经损伤、下肢深静脉血栓形成、关节感染和关节僵硬等并发症发生。末次随访时IKDC膝关节评分为(76.7±5.5)分,Lysholm评分为(94.0±4.1)分,VAS评分为(1.1±0.8)分,与术前比较差异均有统计学意义( t=−22.090, P<0.001; t=−23.704, P<0.001; t=19.767, P<0.001)。.
    UNASSIGNED: PH前区缝合是DLM损伤并腘肌腱区不稳治疗的关键。.
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  • 文章类型: Journal Article
    目的:是否以及如何治疗局部软骨缺损的决定仍然在术中根据软骨的视觉表现和关节镜探头凹陷的发现做出。然后,治疗决策通常基于根据已建立的分类系统进行分级,which,因此,需要具有较高的可靠性和准确性。本研究的目的是评估Outerbridge分类在分期软骨缺损中的可靠性和准确性。
    方法:我们使用Outerbridge分类对七个新鲜冷冻的人类尸体膝盖进行了观察关节镜研究,共表现出九种软骨缺损。为了评估准确性,缺陷的严重程度通过组织学检查得到证实.使用Cohen'skappa和类内相关系数(ICC3.1)计算了评分者和内部可靠性。
    结果:Outerbridge分类的评估者间和内部可靠性范围从差到实质性,分别为0.24≤κ≤0.70和κ=0.55至κ=0.66。通过与组织学检查比较评估的准确性总体为63%。错误的评估是,然而,仍然经常处于2级和3级的歧视。我们没有发现更高的经验与准确性或观察者内部可靠性之间的任何关系。一起来看,这些结果鼓励外科医生进一步使用诊断性关节镜检查来评估软骨损伤。然而,特别是在2级和3级,观察到与组织学的偏差。这是,然而,决定是否通过手术解决缺陷的点。
    结论:诊断性关节镜检查是软骨损伤评估的标准,然而,观察者间的可靠性是公平的。在解释不同的观察者结果时需要谨慎。与组织学检查相比,“更简单”的Outerbridge分类的准确性不足,强调在基于指南的术中决策中需要改进技术。
    OBJECTIVE: The decision on whether or not and how to treat a local cartilage defect is still made intraoperatively based on the visual presentation of the cartilage and findings from indentations with an arthroscopic probe. The treatment decision is then usually based on grading according to established classifications systems, which, therefore, need to have high reliability and accuracy. The aim of the present study was to evaluate the reliability and accuracy of the Outerbridge classification in staging cartilage defects.
    METHODS: We performed an observer arthroscopic study using the Outerbridge classification on seven fresh-frozen human cadaveric knees, which collectively exhibited nine cartilage defects. To evaluate accuracy, defect severity was verified through histological examination. Interrater and intrarater reliabilites were calculated using Cohen\'s kappa and the intra-class correlation coefficient (ICC 3.1).
    RESULTS: The interrater and intrarater reliability for the Outerbridge classification ranged from poor to substantial, with 0.24 ≤ κ ≤ 0.70 and κ = 0.55 to κ = 0.66, respectively. The accuracy evaluated by comparison with the histological examination was 63% overall. The erroneous evaluations were, however, still often at the discrimination of grade 2 and 3. We did not find any relationship between higher experience and accuracy or intraobserver reliability. Taken together, these results encourage surgeons to further use diagnostic arthroscopy for evaluating cartilage lesions. Nevertheless, especially in grade 2 and 3, deviations from the histology were observed. This is, however, the point where a decision is made on whether to surgically address the defect or not.
    CONCLUSIONS: Diagnostic arthroscopy is the standard for cartilage lesion assessment, yet interobserver reliability is fair to substantial. Caution is warranted in interpreting varied observer results. The accuracy of the \"simpler\" Outerbridge classification is insufficient compared to histological examinations, highlighting the need for improved techniques in guideline-based intraoperative decision-making.
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  • 文章类型: Journal Article
    背景:基于CRISPR-Cas9的基因组工程代表了软骨组织工程和理解驱动软骨疾病的分子途径的强大治疗工具。然而,原代软骨细胞难以转染并在单层(2D)细胞培养过程中迅速去分化,使单细胞来源的编辑克隆群体的长时间扩增不可行。出于这个原因,专注于软骨和风湿性疾病的功能遗传学研究长期以来一直在细胞模型中进行,这些模型很难概括人类软骨组织的天然分子特性(例如,细胞系,诱导多能干细胞)。这里,我们着手开发一种适用于来自不同软骨来源的软骨细胞群体的非病毒CRISPR-Cas9批量基因编辑方法.
    方法:我们筛选了用于原代多指软骨细胞中核糖核蛋白(RNP)递送的电穿孔和脂质纳米粒,和优化的RNP试剂组装。我们淘汰了RELA(也称为p65),核因子κB(NF-κB)的亚基,在多指软骨细胞中,并通过RT-qPCR和Western印迹进一步表征敲除(KO)细胞。我们测试了来自不同软骨来源的软骨细胞中的RELAKO,并通过RT-qPCR表征了它们的表型。我们检查了在存在和不存在白介素1β(IL-1β)的情况下野生型(WT)和KO细胞颗粒的软骨形成潜力。
    结果:我们确立了电穿孔作为软骨细胞增强转染和编辑效率的最佳转染技术,同时保持高细胞活力。我们以前所未有的90%的效率淘汰了RELA,证实与未编辑的细胞相比,IL-1β刺激后的炎症途径激活较低。我们的方案可以很容易地转移到从骨关节炎(OA)患者收获的原代人软骨细胞,人FE002软骨祖细胞,牛软骨细胞,和人类软骨细胞系,使用相同的协议实现相当的平均RELAKO编辑水平。来自原代人软骨细胞的所有KO颗粒保留了与WT细胞相当的软骨形成能力,并在发炎条件下显示出增强的基质保留。
    结论:我们展示了我们的批量基因编辑方法的适用性,以开发有效的自体和同种异体现成的基因治疗策略,并使人类软骨细胞的功能遗传学研究能够揭示软骨疾病的分子机制。
    BACKGROUND: CRISPR-Cas9-based genome engineering represents a powerful therapeutic tool for cartilage tissue engineering and for understanding molecular pathways driving cartilage diseases. However, primary chondrocytes are difficult to transfect and rapidly dedifferentiate during monolayer (2D) cell culture, making the lengthy expansion of a single-cell-derived edited clonal population not feasible. For this reason, functional genetics studies focused on cartilage and rheumatic diseases have long been carried out in cellular models that poorly recapitulate the native molecular properties of human cartilaginous tissue (e.g., cell lines, induced pluripotent stem cells). Here, we set out to develop a non-viral CRISPR-Cas9, bulk-gene editing method suitable for chondrocyte populations from different cartilaginous sources.
    METHODS: We screened electroporation and lipid nanoparticles for ribonucleoprotein (RNP) delivery in primary polydactyly chondrocytes, and optimized RNP reagents assembly. We knocked out RELA (also known as p65), a subunit of the nuclear factor kappa B (NF-κB), in polydactyly chondrocytes and further characterized knockout (KO) cells with RT-qPCR and Western Blot. We tested RELA KO in chondrocytes from diverse cartilaginous sources and characterized their phenotype with RT-qPCR. We examined the chondrogenic potential of wild-type (WT) and KO cell pellets in presence and absence of interleukin-1β (IL-1β).
    RESULTS: We established electroporation as the optimal transfection technique for chondrocytes enhancing transfection and editing efficiency, while preserving high cell viability. We knocked out RELA with an unprecedented efficiency of ~90%, confirming lower inflammatory pathways activation upon IL-1β stimulation compared to unedited cells. Our protocol could be easily transferred to primary human chondrocytes harvested from osteoarthritis (OA) patients, human FE002 chondroprogenitor cells, bovine chondrocytes, and a human chondrocyte cell line, achieving comparable mean RELA KO editing levels using the same protocol. All KO pellets from primary human chondrocytes retained chondrogenic ability equivalent to WT cells, and additionally displayed enhanced matrix retention under inflamed conditions.
    CONCLUSIONS: We showcased the applicability of our bulk gene editing method to develop effective autologous and allogeneic off-the-shelf gene therapies strategies and to enable functional genetics studies in human chondrocytes to unravel molecular mechanisms of cartilage diseases.
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