电磁辐射对软骨的生物学效应研究进展

作为半刚性的、无血管和神经的结缔组织,软骨具有承重、应力缓冲和辅助运动等多种生理功能。软骨固有的修复能力差,损伤不易逆转且治疗难度大。近年来,大量研究结果显示适宜的电磁辐射对软骨细胞形态的维持及其细胞因子的分泌、软骨细胞外基质的稳态以及骨关节炎的治疗等都具有积极的意义。笔者就电磁辐射对软骨的生物学效应及其可能的作用机制作一综述。     

Degradation in vivo of articular cartilage in rheumatoid arthritis by leucocyte elastase from polymorphonuclear leucocytes

Summary Using a specific substrate, no leucocyte elastase activity could be detected in 55 synovial fluids, including 29 from patients with rheumatoid arthritis (RA). However, a high percentage of samples contained phagocytic inclusions of elastase, ₁-proteinase inhibitor (₁-PI) and ₂-macroglobulin (₂-MG) in both the polymorphonuclear (PMN) and mononuclear phagocytes. Immunofluorescence and indirect peroxidase-antiperoxidase staining of articular cartilage (ACA) from 52% of 21 patients with RA and one with juvenile RA (JRA) showed presence of elastase in the superficial layer of microscopically intact but proteoglycan depleted pannus-free ACA. In histologically altered pannus-free RA-ACA superficial elastase deposits were found in 24% of the cases. Adjacent ACA sections contained IgG, C 3, ₁-PI and rarely ₂-MG. RA-ACA below or surrounded by pannus showed close contact with intact and decaying PMN in 62% and 48% of the cases, respectively. ACA specimens from patients with degenerative disease and systemic lupus were negative. These findings strongly suggest that PMN leucocyte elastase is operative in the degradation of RA-ACA and JRA-ACA, and that this activity is largely dependent upon the presence of entrapped immune complexes in such cartilage.     

Tuning mechanical performance of poly(ethylene glycol) and agarose interpenetrating network hydrogels for cartilage tissue engineering

Hydrogels are attractive for tissue engineering applications due to their incredible versatility, but they can be limited in cartilage tissue engineering applications due to inadequate mechanical performance. In an effort to address this limitation, our team previously reported the drastic improvement in the mechanical performance of interpenetrating networks (IPNs) of poly(ethylene glycol) diacrylate (PEG-DA) and agarose relative to pure PEG-DA and agarose networks. The goal of the current study was specifically to determine the relative importance of PEG-DA concentration, agarose concentration, and PEG-DA molecular weight in controlling mechanical performance, swelling characteristics, and network parameters. IPNs consistently had compressive and shear moduli greater than the additive sum of either single network when compared to pure PEG-DA gels with a similar PEG-DA content. IPNs withstood a maximum stress of up to 4.0 MPa in unconfined compression, with increased PEG-DA molecular weight being the greatest contributing factor to improved failure properties. However, aside from failure properties, PEG-DA concentration was the most influential factor for the large majority of properties. Increasing the agarose and PEG-DA concentrations as well as the PEG-DA molecular weight of agarose/PEG-DA IPNs and pure PEG-DA gels improved moduli and maximum stresses by as much as an order of magnitude or greater compared to pure PEG-DA gels in our previous studies. Although the viability of encapsulated chondrocytes was not significantly affected by IPN formulation, glycosaminoglycan (GAG) content was significantly influenced, with a 12-fold increase over a three-week period in gels with a lower PEG-DA concentration. These results suggest that mechanical performance of IPNs may be tuned with partial but not complete independence from biological performance of encapsulated cells.     

Enzymatic Digestion of Adult Human Articular Cartilage Yields a Small Fraction of the Total Available Cells

We investigated whether different protocols for the digestion of adult human articular cartilage influence the cell yield and capacity to attach and proliferate in culture dishes. Chondrocyte yields were expressed as a percentage of the total number of cells in the tissue, determined both histologically (using the dissector method) and biochemically (measuring the DNA content of tissue digests). Human cartilage specimens ( n = 79) were digested using different protocols based on combinations of collagenase II (CGN), trypsin/EDTA, hyaluronidase, and tosyllysylchloromethane (TLCM). Yields of viable chondrocytes were the highest within a specific range of CGN concentrations and digestion times, but always < 22% of the total available cells. The combination of CGN with trypsin/EDTA or TLCM accelerated the digestion process but did not significantly increase cell yields. The percentage of viable cells that attached to culture dishes ranged 75-85% (< 19% of the total) and was reduced by TLCM. Doubling times of attached cells were comparable in all experimental groups. Our results indicate that chondrocyte yields and capacity to attach and proliferate are not highly sensitive to the specific isolation protocol used. However, typically used cartilage digestion protocols yield only a small fraction of the total available cells, possibly introducing an uncontrolled selection of certain chondrocyte subpopulations.     

Effect of Decorin and Dermatan Sulfate on the Mechanical Properties of a Neocartilage

Decorin is known to influence the size of collagen fibrils in ligaments and tendons and it has been hypothesized to provide a structural link between collagen fibrils in connective tissues, including cartilage. Coincidently, mechanical properties of skin, ligament, and tendons are altered in decorin knockout mice, suggesting it may influence the structural properties of tissue or tissue matrix organization. To further examine the role of decorin in the extracellular matrix development and subsequent material properties of cartilage, tissue (neocartilage) was grown in a 3D culture model using a pure population of genetically modified chondrocytes stably overexpressing decorin (DCN) or decorin lacking dermatan sulfate (MDCN). An empty vector (CON) served as a virus control. Following generation of the cartilage-like tissues, mechanical properties in tension and compression, collagen fibril diameter, matrix organization, and biochemistry of the tissue were determined. There were no differences between CON and DCN tissues in any parameter measured. In contrast, tissue generated in MDCN cultures was thinner, had higher collagen density, and higher elastic moduli as compared to both CON and DCN tissues. Considering there was no difference in stiffness between CON and DCN tissues, the notion that decorin contributes to the mechanical properties via load transfer was refuted in this model. However, contrasts in the mechanical properties of the MDCN tissue suggest that the dermatan sulfate chains on decorin influences the organization/maturation and resultant mechanical properties of the matrix by as an yet-unidentified regulatory mechanism.     

Tissue Engineered Cartilage Integration to Live and Devitalized Cartilage: A Study by Reflectance Mode Confocal Microscopy and Standard Histology

This study investigated the in vivo formation of engineering cartilage within living or devitalized cartilage discs using reflectance mode confocal microscopy and conventional light microscopy. Pig articular chondrocytes were suspended in fibrin glue and placed between two cartilage discs. Four experimental groups were prepared: in groups 1 and 2, the cell-hydrogel composite was placed between two live or between two devitalized cartilage discs, respectively; in groups 3 and 4, acellular fibrin glue was placed between two live or between two devitalized cartilage discs, respectively. Samples were implanted in the back of nude mice and analyzed after 2, 5, and 8 weeks. Results showed that engineered cartilage seems to grow more homogenously when the cell-seeded gel was placed between devitalized cartilages than when it was placed between live cartilage matrices. Confocal microscopy provides valuable information on the integration of tissue-engineered cartilage with native tissue and could be useful for nondestructive imaging in vivo.     

Inhibition of Proteoglycan Biosynthesis Decreases the Calcification of Chondrocyte Cultures

In order to determine the role of proteoglycan in the calcification of cartilage, the effects on calcifying chondrocyte cultures of treatments that disrupt proteoglycan biosynthesis have been studied. Treatment of secondary cultures of embryonic chick chondrocytes with non-toxic concentrations of the β-xyloside p-nitrophenyl β-D-xylopyranoside (PNPX) resulted in dose-dependent inhibition of both proteoglycan and mineral deposition. Based on the expression of Type X collagen, however, PNPX is also a potent inhibitor of chondrocyte differentiation. Under-sulfation of proteoglycans was effected by growth of chondrocyte cultures in sulfate-depleted medium. Growth in low-sulfate medium did not significantly affect the growth or differentiation of these cultures, but caused an approximate two-fold decrease in mineral content compared to cultures grown in normal medium. These findings indicate that disruption of proteoglycan biosynthesis in chondrocyte cultures results in decreased levels of calcification. Therefore, proteoglycans appear to function as promoters of chondrocyte calcification.     

关节镜辅助下可吸收软骨钉固定急性髌骨脱位合并膝关节骨软骨骨折的临床疗效观察

[摘要] 目的 探讨关节镜辅助下可吸收软骨钉固定急性髌骨脱位合并膝关节骨软骨骨折的临床疗效。方法 回顾性分析西宁市第一人民医院2016年3月至2020年1月通过关节镜辅助下可吸收软骨钉固定治疗的33例急性髌骨脱位合并膝关节骨软骨骨折患者,随访7 ～ 28个月,平均（15.15±5.54）个月,术前及术后6个月时完善X线、CT评价骨折复位愈合情况,并用Lsyholm评分、HSS评分评价膝关节功能。结果 术后33例骨折均获得满意的复位,术后6个月复查时均达到骨性愈合,膝关节屈伸活动良好,未发生感染、骨块移位、关节僵硬等并发症,Lsyholm评分从术前（19.33±6.86）分提高到术后6个月（89.72±5.11）分,差异有统计学意义（P<0.01）;HSS评分从术前（38.63±7.40）分提高到术后6个月（92.15±4.42）分,差异有统计学意义（P<0.01）。结论 关节镜辅助下可吸收软骨钉固定急性髌骨脱位合并膝关节骨软骨骨折,可微创、准确、安全地恢复关节面完整性,无需二次手术取出内植物,治疗效果确切,是髌骨脱位合并骨软骨骨折的良好的治疗方式。     

组织工程软骨植入缺损后修复区力学特性分析

目的利用组织工程技术建立体外软骨缺损实验模型,研究修复区人工软骨和宿主软骨的力学特性。方法采用一种琼脂糖凝胶作为人工软骨,制作猪软骨深层缺损,在缺损处仿临床植入人工软骨,用生物胶黏接,建立组织工程修复膝关节软骨缺损的体外模型;在压缩载荷作用下,通过数字图像相关技术研究组织工程软骨植入缺损后修复区即刻力学行为。结果压缩过程中界面处没有出现开裂现象,压缩分别为软骨层厚度的3.5%、5.6%、7.04%和9.0%时获得了修复区中间层应变分布图和应变变化曲线。压缩量从3.5%增加到9%时,在垂直软骨面方向上宿主软骨最大压应变增加75.9%,人工软骨最大拉应变增加226.99%;在平行软骨表面方向,交界面处最大拉应变增加116.9%,增加量远高于宿主软骨区和人工软骨区;对于修复区剪应变,随着压缩量增加交界处剪应变方向发生相反的改变。结论软骨组织工程修复缺损效果有很大的不确定性,这与修复区的力学环境有关。组织工程软骨植入缺损后,修复区受到复杂应变状态,随着压缩量增加,界面处、宿主软骨、人工软骨都发生较大的应变变化,界面处垂直软骨面方向的应变由压应变可转化为拉应变,平行软骨表面方向的拉应变有显著增加,交界处剪应变方向甚至发生了相反的改变,而且剪应力数值迅速增加。这种复杂应变状态造成修复区细胞力学环境的较大变化,还可能引起界面的开裂,影响缺损修复过程,这些力学环境变化应受到临床治疗的重视。     

以转染CDMP1基因的BMSCs修复软骨缺损

 目的 探讨CDMP1基因转染的骨髓间充质干细胞（BMSCs）负载于聚乳酸-羟基乙酸（PLGA）支架上修复喉软骨缺损的能力,并对其修复效果做出初步评估。方法 用反转录聚合酶链式反应（RT-PCR）和免疫印迹法(Western blot)检测hCDMP1mRNA和蛋白的表达;用免疫组织化学方法检测Ⅱ型胶原蛋白(ColⅡ)以及糖胺聚糖(GAG)的表达;将转染前后的细胞支架培养体系移植入兔甲状软骨全层缺损处,从大体、组织学方面观察其对软骨缺损的修复作用。结果 腺病毒感染方法可以将外源hCDMP1基因成功转入BMSCs,并使其获得稳定表达;和对照组比较,转染hCDMP1基因的BMSCs分泌ColⅡ、GAG等软骨特异性基质的能力增强,有促进软骨分化趋势;转染细胞支架复合物可更加有效地修复喉软骨缺损。结论 转染hCDMP1基因的BMSCs/PLGA三维生物支架复合物移植动物体内可修复喉软骨缺损。