兔半月板纤维软骨细胞的体外去分化研究

目的:比较研究不同代次间兔半月板纤维软骨细胞生物学特性的改变,为构建组织工程半月板和细胞治疗中的种子细胞优选提供进一步的理论和实践基础。方法:采用机械分离与酶连续消化相结合的方法体外分离兔半月板纤维软骨细胞,单层培养传代至第5代。采用相差倒置显微镜和SEM观察各代细胞的形态变化和超微结构,MTT法检测细胞增殖情况并描绘生长曲线,采用细胞化学染色和免疫组化鉴定细胞分泌的蛋白聚糖和Ⅰ、Ⅱ型胶原蛋白。结果:半月板纤维软骨细胞原代接种后8~12 h开始贴壁,48~72h大部分细胞贴壁,胞质逐渐展开,细胞变大伸长,形成突起,呈多角形,细胞形态规则,轮廓清晰,镜下观察有立体感,7~10 d后,细胞长满传代。传代后细胞贴壁生长能力和增殖速度明显加快,原代至第2代细胞形态较规则,多呈多角形,大小均一。第3代后随着传代次数的增加,细胞中梭形细胞增多,分泌和增殖能力下降,传代周期延长。第5代分裂相少见,密度稀疏,细胞形态不佳,约80%呈长梭形,分泌和增殖能力下降。SEM示第1代半月板细胞形态规则,呈多极性,表面有突起;第5代细胞形态不规则,多呈梭形。生长曲线可见第4代前半月板细胞生长速度及生长周期均相似,第5代后细胞生长速度减慢,增殖减缓。细胞化学和免疫组化染色分析胶原和蛋白聚糖的表达:随传代的进行,Ⅱ型胶原蛋白和蛋白聚糖的表达逐渐减弱而Ⅰ型胶原表达逐渐增强。结论:体外单层培养条件下,随着传代的进行,细胞活力逐渐下降,生长速度减慢,传代周期延长,逐渐变为梭形,形态不规则。体外单层培养系统中半月板纤维软骨细胞表型随传代的进行,Ⅱ型胶原蛋白和蛋白聚糖的表达逐渐减少而Ⅰ型胶原表达逐渐增多,体外单层培养条件下培养的半月板纤维软骨细胞从第3代开始逐渐失去其特异性表型,发生去分化,逐渐变为成纤维细胞的表型。体外单层分离培养的第5代前半月板细胞基本维持纤维软骨细胞的表型和生物学特性,可作为组织工程半月板的种子细胞。     

人关节软骨细胞体外培养的传代特点及鉴定

 目的 研究软骨细胞的传代特点,为软骨细胞移植技术的临床应用提供依据.方法 用胰酶和Ⅱ型胶原酶对软骨进行双重消化得到软骨细胞,通过免疫组化对软骨细胞进行鉴定.结果 软骨细胞贴壁时间为24～36 h,传代培养4～5 d,细胞形态以圆形为主,根据免疫组化鉴定,这是一种切实可行的培养细胞的有效方法.结论 本研究建立了一种简单易行的软骨细胞分离方法,原代及第2代细胞生长情况良好,5代后细胞表型发生变化.     

原代骺板软骨细胞的高效分离和体外培养观察

目的　设计原代骺板软骨细胞的高效分离法 ,并对骺板软骨细胞的体外培养进行初步观察。　方法　改良设计以 1g L的胰蛋白酶 / 1g L的乙二胺四乙酸 (EDTA)、1g L的透明质酸酶和 2g L的Ⅰ型胶原酶系列消化分离原代软骨细胞的三步酶消化分离法 ,观察其对幼兔骺板原代软骨细胞的分离效果 ;倒置显微镜和光镜下观察体外培养条件下细胞生物学特性。　结果　 ( 1)三步酶的消化可使软骨基质完全解离降解 ,细胞均得以分离 ,收获量与组织湿重呈非常显著的正相关 ,每克软骨的细胞收获量平均为 32 .3× 10 6 个 ,细胞存活率平均为 97.6 %。 ( 2 )原代和第 1代细胞附壁生长呈三角形或多角形 ,生长融合时呈卵圆形 ,Ⅱ型胶原免疫组化和甲苯胺蓝 (TB)异染反应均呈阳性 ;第 3代以后细胞逐渐变为梭形 ,Ⅱ型胶原免疫组化为阴性 ,TB异染反应明显减弱。结论　 ( 1)原代骺板软骨细胞三步酶消化分离法具有细胞收获率高、细胞存活率高、操作简便等特点。 ( 2 )随着传代培养次数的增加 ,骺板细胞出现去分化 ,逐渐失去软骨细胞的特异性表型。     

半月板纤维软骨细胞与壳聚糖/聚磷酸钙体外复合培养的实验研究

目的:观察半月板纤维软骨细胞在不同配比壳聚糖/聚磷酸钙(chitosan/calciumpolyphosphate,CS/CPP)支架材料上的生长状况,优选最佳配比CS/CPP生物材料作为组织工程半月板支架材料。方法:采用机械分离与酶连续消化相结合的方法体外分离兔半月板纤维软骨细胞,单层培养传代至第3代,并对其进行表型鉴定。采用共混-化学交联固化-冷冻干燥法将CS、醛基化海藻酸钠(aldehyde alginate,ADA)、CPP有机地结合起来,制备4种不同配比的新型组织工程半月板复合支架材料。将体外分离培养的第3代半月板细胞,通过二次沉淀接种法将其种植于不同配比的CS/CPP支架上,体外培养7天,采用相差倒置显微镜、SEM、HE染色观察细胞在支架上的形态、黏附、生长情况。结果:体外分离培养的第3代半月板细胞基本维持纤维软骨细胞的表型。相差倒置显微镜下可见,细胞在支架上黏附良好;扫描电镜下可见,细胞在支架上均匀分布,细胞多数呈多角形,并有基质分泌;HE染色结果显示,有细胞长入到三维支架材料内部。其中,半月板细胞在3:7的CS/CPP支架材料上单位面积内数目最多,生长最旺盛,细胞外基质分泌最多。结论:三维多孔的CS/CPP复合材料能促进半月板纤维软骨细胞的黏附、生长和增殖,其中3:7的CS/CPP支架材料细胞相容性和生物活性最好,最适于半月板纤维软骨细胞粘附和生长,并且能促进半月板细胞增殖和维持其表型,有望成为组织工程半月板良好的支架载体。     

纤维软骨细胞-胶原复合物修复半月板损伤的实验研究

为了探讨纤维软骨细胞－胶原复合物对半月板损伤的修复作用，分离提取狗半月板纤维软骨细胞，体外扩增培养后种植于自制胶原模板上形成细胞－胶原复合物，将18只狗随机分为3组，在狗的外侧半月板作一楔形缺损，缺损内分别植入细胞－胶原复合物、胶原或不作处理，在不同时间取材进行大体和组织学观察。结果显示，植入的复合物形成与正常半月板相似的组织，胶原支架逐渐降解，而胶原对照组及空白对照组缺损区仅少部分修复或无修复。提示，这一新的组织工程学方法具有一定的指导意义及实用性。     

大鼠肺微血管内皮细胞原代培养方法的改进

目的：改进大鼠肺微血管内皮细胞（pulmonary microvascular endothelial cells,PMVECs）原代培养方法,以获取纯化的大鼠肺微血管内皮细胞。方法：应用Wistar大鼠,采用改进的组织块贴壁法分离、培养肺微血管内皮细胞;光镜观察细胞形态;Ⅷ因子相关抗原免疫细胞化学法及植物凝集素BSI结合实验鉴定培养的PMVECs。结果：体外培养的原代PMVECs在光镜下呈短梭形或多角形,形成单层后呈铺路石样排列,但随着传代或培养条件的改变,细胞形态发生变化;Ⅷ因子相关抗原染色阴性,异植物凝集素BSI结合实验阳性。结论：通过改进的PMVECs分离、培养方法获得的细胞生长状态良好,纯度高,且能够稳定地传代培养。细胞形态观察结合免疫细胞化学法是目前较为理想的PMVECs鉴定方法。     

体外器官培养系统中纤维软骨组织修复的实验研究

半月板是一种纤维软骨组织，由纤维软骨细胞与以Ｉ型胶原为主的基质构成。以往认为纤维软骨细胞缺乏再生能力，使半月板损伤不能愈合。近来有研究表明脱离了基质的纤维软骨细胞在体外单层培养条件下有很强的增生分化能力。因此我们在体外器官培养并提供纤维蛋白支架的条件...     

应用自体血清培养大鼠关节软骨细胞生物学行为研究

目的:比较自体血清与胎牛血清体外培养大鼠关节软骨细胞生物学行为的差异。方法:分离培养雄性8周Sprague-Dawley(SD)大鼠软骨细胞,分别在5%、10%自体血清和10%胎牛血清中进行单层传代培养,采用光镜观察细胞形态变化,绘制生长曲线评估细胞增殖速度,通过甲苯胺蓝染色,Ⅰ、Ⅱ型胶原免疫组织化学染色以及流式细胞仪分析细胞CD26、CD44及Ⅰ、Ⅱ型胶原表达以检测软骨细胞生物学行为的变化。结果:(1)在培养3代内,原代培养的软骨细胞形态呈多角形,而3代培养的细胞都为梭形,三组无明显差别;(2)5%自体血清培养的细胞的生长速度与10%胎牛血清培养的细胞接近,10%自体血清培养的软骨细胞的生长速度快于前两组;(3)甲苯胺蓝染色结果证明三组细胞均随传代数增加酸性粘多糖蛋白分泌量下降,且10%胎牛血清组比10%和5%自体血清培养组下降更明显,而10%与5%自体血清培养组之间无明显差异;(4)免疫组化染色和流式细胞仪检测结果表明10%与5%自体血清培养的软骨细胞Ⅱ型胶原表达高于10%胎牛血清培养的细胞(P<0.05),10%与5%自体血清组无显著性差异(P>0.05);随体外培养时间延长,三组细胞合成的Ⅱ型胶原均逐渐减少,Ⅰ型胶原表达逐渐增多。(5)流式细胞仪观察显示三组CD26表达虽有增加但无显著性差异,CD44表达均随传代数增加而逐渐增加,且在1、3代细胞之间具有显著性差异(P<0.05)。在3种培养条件下,相同代数的软骨细胞CD26和CD44表达无显著性差异。结论:本研究发现10%自体血清培养大鼠软骨细胞生长速度快,且仍可以较好保持细胞表型,推测在可能条件下使用浓度较高的自体血清可以缩短体外培养时间而达到较多细胞保持表型的目的。     

成纤维细胞生长因子对关节软骨细胞转化生长因子β1作用的免疫组织化学观察

目的观察成纤维细胞生长因子对培养兔关节软骨细胞转化生长因子β１的表达。方法培养１月龄新西兰兔关节软骨细胞，在每次换液时加入成纤维细胞生长因子１００ｎｇ／ｍｌ，铺满后收集细胞，作关节软骨细胞转化生长因子β１常规及电镜免疫组织化学观察。结果光镜下实验组细胞明显呈棕色，电镜下细胞膜表面有明显的胶金颗粒黏附。结论成纤维细胞生长因子能促进关节软骨细胞转化生长因子β１的表达。     

逼尿肌细胞的原代培养和鉴定

目的建立逼尿肌细胞的原代培养及鉴定方法。方法应用Ⅱ型胶原酶消化分离逼尿肌细胞,在含20%胎牛血清DMEM培养液中培养,观察细胞形态和扩增情况,用a-SM-Actin进行免疫组化鉴定细胞类型。结果 逼尿肌细胞在培养18 h后开始贴壁在瓶底,4~5 d后可见细胞融合,8~10 d后可见细胞覆盖瓶底80%以上。a-SM-Actin免疫组化染色鉴定,光镜下观察梭形细胞胞浆内见纵行排列的棕黄色丝状物。结论该方法简单、易于掌握,短期内可获得大量高纯度的逼尿肌细胞。     

Immunolocalization of types I, II, and X collagen in the tibial insertion sites of the medial meniscus

The medial meniscus of the rabbit knee joint attaches to the tibial plateau via anterior and posterior insertions. Intact meniscal tibial insertions are essential for meniscal function. In the present study the distributions of types I, II, and X collagen in meniscal tibial insertions were investigated by indirect immunohistochemistry in a rabbit model. Four tissue zones were histologically identified in the anterior insertion site, including the ligamentous zone, uncalcified and calcified fibrocartilaginous zones and bone; the ligamentous zone was not observed in the posterior insertion site. Labeling for type I collagen was found to be strong in the ligament tissue and bone, and weak in the fibrocartilages which were also labeled for type II collagen. Tissues positive for different types of collagen overlapped and formed an irregular interface with various angles and depths, especially at the interface between the calcified fibrocartilage and bone. Positive labeling for type X collagen was identified only in the calcified fibrocartilage zone. The coexistence of types I and II collagen in the meniscal tibial insertions may indicate that this structural unit is subjected to both compressive and tensile loads. Type X collagen may play a role in maintaining the calcifying status of this tissue zone, so that its mechanical stiffness is kept between that of uncalcified fibrocartilage and hard bone. Restoration of the insertional structure including the distinct collagen distribution should be considered for a functional meniscal substitution. Received: 22 June 1999/Accepted: 27 September 1999     

Pulsed holmium:yttrium-aluminum-garnet (Ho:YAG) laser ablation of fibrocartilage and articular cartilage

A new, near-infrared, pulsed holmium laser (wavelength, 2.1 microns; pulse duration, 400 microseconds) was used to ablate bovine articular cartilage and meniscal fibrocartilage. Microscopic examination revealed zones of thermal damage extending 550 microns from ablation sites. Ablation rates were measured with a mass loss technique. Above threshold, mass removal rates were proportional to laser radiant exposure. Threshold radiant exposure for ablation was 50 J/cm2 for articular cartilage and 11 J/cm2 for meniscal fibrocartilage. Because the holmium laser can precisely and rapidly resect cartilaginous tissues with only moderate necrosis, function in a saline environment in direct contact with tissue, and be transmitted through conventional optical fibers, it has the potential to become a useful tool for the precise arthroscopic removal of intraarticular tissue.     

The effect of magnetization transfer in meniscal fibrocartilage

Magnetic resonance imaging of the knee was performed in 28 patients (ages 15–72 years), using a 1.5-T unit. Volume gradient echo (3D GRASS) acquisition with and without presaturation off-resonance RF pulse was used to evaluate magnetization transfer (MT) effects, determined by placing regions of Interest on muscle, fat, hyaline, and fibrocartilage; the percent change in signal intensity was calculated and compared using a paired two-sample t test. An in vitro study of the normal meniscus from a cadaver containing a scalpel cut extending to an articular surface was performed to observe the relative improvement in contrast in the presence of a small meniscal defect. MR imaging of the specimen was performed using an Omega CSI 2.0–T system (General Electric Medical Systems, Fremont, CA). Analysis of clinical images resulted in signal loss, compared to that of the identically timed and tuned non-MT images of 47 ± 5, 8 ± 5, 49 ± 5, and 57 ± 7% for muscle, fat, articular cartilage and fibrocartilage, respectively. Application of MT improved the depiction of the artificially introduced meniscal defect. Meniscal fibrocartilage demonstrates significant MT effect after application of off-resonance RF presaturation, which may improve visualization of meniscal defects.     

Comparison of biochemical characteristics of cultured fibrochondrocytes isolated from the inner and outer regions of human meniscus

In order to evaluate the ability of fibrochondrocytes to synthesize collagen and proteoglycan, human medial meniscal cells were cultured in a monolayer. Meniscal cells were prepared from the two regions (outer 1/3 and inner 2/3) in consideration of the difference in vascular supply, and articular chondrocytes were also obtained from the same knee joint. Regarding total collagen synthesis, regional differences were not found, but age-related differences were found in human medial meniscus. In contrast, proteoglycan synthesis revealed significant regional differences; meniscal cells from the inner 2/3 synthesized a greater amount of proteoglycan. After long-term monolayer culturing, proteoglycan synthesis by meniscal cells decreased in a time-dependent manner, and morphological changes to fibroblast-like cells were found. In the presence of transforming growth factor (TGF)-β, proteoglycan synthesis increased in a dose-dependent manner. These findings suggest that the inner regions of the human meniscus contain cells with a chondrocytic phenotype. Received: 11 February 1998 Accepted: 14 September 1998     

Lineage plasticity and cell biology of fibrocartilage and hyaline cartilage: its significance in cartilage repair and replacement

Cartilage repair is a major goal of modern tissue engineering. To produce novel engineered implants requires a knowledge of the basic biology of the tissues that are to be replaced or reproduced. Hyaline articular cartilage and meniscal fibrocartilage are two tissues that have excited attention because of the frequency with which they are damaged. A basic strategy is to re-engineer these tissues ex vivo by stimulating stem cells to differentiate into the cells of the mature tissue capable of producing an intact functional matrix. In this brief review, the sources of cells for tissue engineering cartilage and the culture conditions that have promoted differentiation are discussed within the context of natural cartilage repair. In particular, the role of cell density, cytokines, load, matrices and oxygen tension are discussed.     

Menisci are efficiently transduced by recombinant adeno-associated virus vectors in vitro and in vivo

BACKGROUND: Meniscal tears remain an unsolved problem in sports medicine. Gene transfer is a potential approach to enhancing meniscal repair. Recombinant adeno-associated virus is a method of gene transfer that has advantages over previously used approaches to this problem. HYPOTHESIS: Direct gene transfer to meniscal cells can be accomplished using recombinant adeno-associated virus in vitro and in vivo. STUDY DESIGN: Controlled laboratory study. METHODS: Recombinant adeno-associated viruses containing the reporter gene lacZ were tested for their ability to achieve gene transfer into lapine and human meniscal cells in vitro and into lapine meniscal defects in vivo. Results were assessed by detecting beta-galactosidase, the enzyme encoded by the lacZ gene. RESULTS: Maximal efficiency of gene transfer was 81.6% +/- 6.6% for lapine and 87.2% +/- 14.8% for human meniscal cells in vitro. Expression of the transferred gene continued for the 28-day duration of the study. When the recombinant adeno-associated virus vector was injected into meniscal tears in a lapine meniscal tear model, transgene expression continued in meniscal cells adjacent to the tear for at least 20 days in vivo. CONCLUSIONS: The data suggest that recombinant adeno-associated virus vectors can directly and efficiently transfer and stably express foreign genes in isolated lapine and human meniscal cells in vitro and in lapine meniscal defects in vivo. CLINICAL RELEVANCE: This direct gene transfer approach may form a basis for improved treatments of meniscal tears.     

Do asymptomatic marathon runners have an increased prevalence of meniscal abnormalities? An MR study of the knee in 23 volunteers

Excessive repetitive musculoskeletal loads and stresses associated with intense physical activity may lead to deterioration of the menisci of the knee. Therefore, MR imaging was performed on the knees of 23 asymptomatic marathon runners (eight men, 15 women; average age, 40 years; average number of years training, 10; average training distance per week, 41 miles) to determine the prevalence of meniscal signal abnormalities. None of the runners had previous knee injuries or surgery and each of them regularly competes in 26-mile, 50-mile, or 100-mile marathon races. T1-weighted coronal MR images and proton density-weighted and T2-weighted sagittal images were obtained with a 1.5-T MR system and a transmit/receive extremity coil. The medial and lateral menisci were divided into four portions, or horns, and a total of 92 horns were evaluated (i.e., four horns per knee: medial posterior, medial anterior, lateral posterior, and lateral anterior). Two meniscal horns (2%) had grade 3 signal (grade 3 indicates a meniscal tear), 12 (13%) had grade 2 signal, 29 (32%) had grade 1 signal (grades 1 and 2 are indicative of meniscal degeneration), and 49 (53%) had grade 0 signal (grade 0 is normal). Overall, the prevalence of meniscal tears was 9% (two meniscal tears found in 23 runners). This is lower than the prevalence of MR signal abnormalities indicative of meniscal tears reported for asymptomatic, nonrunner athletes (20% of 20 athletes) and for asymptomatic nonathletes (16% of 74 subjects). Fifty-three percent of the meniscal horns of the nonrunner athletes had grade 1 or 2 signal, indicative of meniscal degeneration. Our results indicate that the prevalence of meniscal tears in marathon runners is no higher than the prevalence reported for sedentary persons, and the runners have the same amount of meniscal degeneration as do nonrunner athletes.     

Meniscal tear characteristics in young athletes with a stable knee: arthroscopic evaluation

BACKGROUND: There has been great interest in the literature regarding meniscal tears in unstable knees, but there is not as much information available on stable knees. PURPOSE: To report the characteristics of isolated meniscal tears (type and location) in athletes with intact cruciate ligaments. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Arthroscopic surgery was performed on 314 (83.1%) knees in the acute phase ( < 6 weeks) of injury and on 64 (16.9%) knees more than 6 weeks after injury for a total of 364 athletes (378 knees). Cooper's classification was used to classify the meniscal tears according to the type and location. RESULTS: Overall, 262 of 378 tears (69.3%) were located in the medial meniscus and 116 (30.7%) in the lateral meniscus. Vertical tears (77.5%) were significantly more frequent than were horizontal tears (22.5%; chi(2) test, P < .001). A total of 23.2% of tears involved the peripheral zones (zone 0 or 1), and tears that extended into the posterior horn accounted for 75.7%. Regarding the tear shape between male and female athletes, on both sides there were no statistically significant differences in the percentage of horizontal, bucket-handle, longitudinal, or radial tears. CONCLUSION: The characteristics of isolated meniscal tears differ with regard to the sport, sex, and tear location and type from those seen in unstable knees. This knowledge is useful in knee injury management.     

Accuracy of MRI patterns in evaluating anterior cruciate ligament tears

 The purpose of this study was to determine the different patterns of anterior cruciate ligament (ACL) tears on MRI and the prevalence and accuracy of these patterns. Images were obtained on high-tesla and low-tesla units and the results compared to determine whether field strength affects the interpretation using the grading system. In 172 patients who underwent knee MRI (109 knees with high-tesla units and 63 knees with low-tesla units) and arthroscopy, there was a total of 91 arthroscopically proven ACL tears. Five patterns of ACL tears were observed and designated as type 1 (diffuse increase in signal on T2-weighted images and enlargement of the ligament, 48%); type 2 (horizontally oriented ACL, 21%); type 3 (nonvisualization of the ACL, 18%); type 4 (discontinuity of the ACL, 11%); and type 5 (vertically oriented ACL, 2%). The positive predictive value (PPV) for type 2, 4, and 5 patterns was 100% for both field strengths; for type 3 PPV was just above 80% for both field strengths. The PPV value for type 1 was 90% for the high-tesla unit and 79% for the low-tesla, unit, which was not statistically significant. Combining the results of both field strengths, the overall sensitivity and specificity were 93% and 89%, respectively. Arthroscopic results were also used to determine the association between meniscal and ACL tears. Only 13% of ACL tears were isolated, the rest being associated with meniscal tears. Forty-five percent of medial meniscal and 50% of lateral meniscal tears were associated with an ACL tear, and 94% of ACLs were torn when both menisci were torn.     

膝关节半月板损伤的MRI与关节镜对照研究

目的评价MRI在膝关节半月板撕裂诊断和评级中的价值。方法参照Mesgarzadeh标准对76位患者78个撕裂半月板的MR影像作回顾性分析,3位MRI主治医师在不知道关节镜检查结果的情况下独自阅片,按Mesgarzadeh的分级标准确定半月板撕裂的类型并记录评定结果,包括联合的前交叉韧带撕裂。结果MRI诊断半月板撕裂的敏感性和特异性分别为92%和87%,Ⅵ型是半月板撕裂中最常见的类型,尤其在发生移位的半月板撕裂中最常见。结论MRI是半月板撕裂伤和交叉韧带损伤的可靠诊断工具。