软骨脱细胞基质修复兔膝关节软骨缺损的实验研究

目的观察软骨脱细胞基质对兔膝关节软骨缺损的修复作用。方法取兔膝关节软骨片进行脱细胞处理,并通过HE染色、甲苯胺蓝染色、免疫组化染色、扫描电子显微镜评价软骨脱细胞的程度。对兔膝关节软骨面造成直径4 mm、深3 mm骨软骨全层缺损,根据是否在缺损处加入脱细胞软骨分为实验组和对照组,12周后通过HE染色、番红固绿染色来观察膝关节缺损的修复程度。结果 HE染色、甲苯胺蓝染色、免疫组化染色、扫描电子显微镜结果验证了软骨几乎完全脱去细胞和DNA,并基本保留了糖胺聚糖(GAG)和Ⅱ型胶原的含量,为细胞的长入提供了良好的环境。手术12周后实验组缺损部位较对照组修复完整,HE染色、番红固绿染色可见骨与软骨分界明显,连接完整,且新生软骨有较高的GAG含量。结论软骨脱细胞基质对于兔膝关节软骨缺损有一定的修复作用。     

软骨脱细胞基质复合脂肪源性干细胞构建软骨组织的实验研究

[目的] 探讨软骨脱细胞基质支架材料的制备,及其体外复合脂肪源性干细胞构建软骨组织的技术方法.[方法] 成年新西兰大白兔脂肪源性干细胞获得,培养,扩增.成年新西兰大白兔新鲜软骨,低温冻干12 h,后经曲拉通、DNA、RNA酶等处理制备成为软骨脱细胞基质支架材料,终浓度为2×107/L的脂肪干细胞种植于软骨脱细胞基质中于软骨细胞方向诱导培养基中培养2周,构建软骨组织.新鲜制备的软骨脱细胞基质及构建的软骨组织分别行组织学、免疫组织化学及透射电镜检测.[结果] 实验制备的软骨脱细胞基质支架材料内无细胞结构存在,仅残留空白软骨陷窝.具有合适的孔隙率和孔径大小;复合脂肪源性干细胞后细胞向材料内部迁移,粘附,生长良好.部分载体内细胞Ⅱ型胶原免疫组化染色阳性.[结论] 软骨脱细胞基质可作为支架材料应用于软骨组织工程,复合脂肪源性干细胞培养可成功构建软骨组织.     

特殊染色技术在骨关节炎关节软骨形态学研究中的比较

[目的]探讨多种特殊染色技术在骨关节炎关节软骨组织形态学研究中的染色规律及应用中的优缺点。[方法]15个月龄健康C57BL/6J小鼠20只,随机分为正常组和骨关节炎模型组各10只,骨关节炎模型组行单侧膝关节前交叉韧带横断加内侧半月板切除术建立骨关节炎模型,正常组仅切开单侧滑膜囊行假手术,3个月后膝关节取材。对标本固定、脱钙后进行石蜡包埋、切片。分别采用HE染色、番红O-固绿双染色、Masson染色、Van Gieson染色、苦味酸天狼星红染色、SA-β-gal半乳糖苷酶衰老染色和II型胶原免疫组化染色,观察骨关节炎关节软骨组织形态学变化,并对几种染色方法进行比较。[结果]HE染色可以显示骨关节炎关节软骨的一般形态结构,可见模型组出现退行性变化;番红O-固绿双染色和Masson染色在显示结构方面的优势优于HE染色,模型组退变更明显;Masson染色、Van Gieson染色、苦味酸天狼星红染色和II型胶原免疫组化染色可定位II型胶原,模型组显示II型胶原含量减少;SA-β-gal半乳糖苷酶衰老染色可见模型组衰老细胞增多。[结论]在骨关节炎关节软骨的形态学研究中,番红O-固绿双染色和Masson染色有利于获得全面的信息,II型胶原免疫组化染色有利于对II型胶原进行定位定量;SA-β-gal半乳糖苷酶衰老染色有利于分析细胞衰老情况。     

人关节软骨源性微载体的制备

目的制备一种既能大量扩增人软骨细胞,又能作为细胞支架的人关节软骨源性微载体.方法切取人关节软骨,冷冻干燥后粉碎,筛取150～200μm的软骨颗粒,以质量分数为0.25%的胰蛋白酶37℃消化24 h,加入体积分数为1%的Triton X-100振荡72 h,在倒置相差显微镜下进行观察,并行HE、番红花O染色及软骨蛋白聚糖、Ⅱ型胶原免疫组化染色.将微载体经60Co照射灭菌后与人关节软骨细胞共同培养.结果倒置相差显微镜下可见关节软骨颗粒冻干后呈黄色,形状不一,可呈方形、多角形或不规则形,表面凹凸不平,可见多个陷窝.经胰蛋白酶和Triton X-100处理后,微载体呈淡黄色,关节软骨的基本形态消失,微载体绒毛化,呈绒球状或毛刷状,表面接触面积大幅度增加.微载体与软骨细胞在37℃培养箱中共同培养2 h时即见大量软骨细胞黏附于关节软骨源性微载体上.HE染色显示已无软骨细胞成分;番红花O染色弱阳性,提示仍残留少许糖胺多糖;软骨蛋白聚糖免疫组化染色阴性,提示微载体内软骨蛋白聚糖已被敲除;Ⅱ型胶原免疫组化染色阳性,提示微载体内Ⅱ型胶原仍然存留.结论经胰蛋白酶和Triton X-100处理的关节软骨颗粒,不但脱去了软骨的细胞成分、敲除了软骨蛋白聚糖、使之呈绒球状或毛刷状、增大了表面接触面积,而且保留了对软骨细胞黏附、增殖和再分化起重要作用的Ⅱ型胶原和GAG,经与人关节软骨细胞共培养,证实关节软骨源性微载体与软骨细胞的体外相容性良好.     

鹿茸软骨组织脱细胞基质材料的制备及生物相容性研究

目的探讨鹿茸软骨制备脱细胞基质材料的可行性以及生物相容性,为软骨修复重建探索新材料。方法取梅花鹿鹿茸生长中心间充质层,进行由DNA酶、RNA酶、抑肽酶等介导的脱细胞处理;行组织学和DNA含量检测,评价脱细胞效果。取第2代鹿生茸区骨膜(antlerogenic periosteum,AP)细胞,行荧光干细胞标记明确其干细胞特性后,用PKH26荧光标记并与制备的间充质层脱细胞基质进行复合培养;7 d后取材行HE染色观察以及荧光显微镜下观察PKH26标记的AP细胞在基质表面生长情况。以上观测均以未复合AP细胞的脱细胞基质作为对照。将复合培养7 d的样本移植至裸鼠一侧腹股沟(实验组),取空白培养样本移植于另一侧(对照组)。于移植后7、21 d取材行HE染色,同时对组织进行冰冻切片并在荧光显微镜下观察PKH26标记成功的AP细胞在脱细胞基质表面及内部的生长情况,评价含AP细胞的脱细胞基质在裸鼠体内的组织相容性。结果HE和DAPI染色显示脱细胞处理后材料中无细胞残留,DNA含量为(19.367±5.254)ng/mg,较脱细胞处理前的(3 805.500±519.119)ng/mg显著降低(t=12.630,P=0.000),提示成功制备间充质层脱细胞基质。AP细胞与间充质层脱细胞基质复合培养7 d后,AP细胞主要黏附于材料表面,部分进入脱细胞基质内部。植入裸鼠体内后,随观察时间延长,接种AP细胞可以在脱细胞基质材料中增殖并逐渐进入材料内部,并诱导血管生成。结论实验成功制备鹿茸软骨脱细胞基质,该基质材料在离体和活体情况下适于种子细胞(AP细胞)的黏附和增殖,并具有刺激血管生成的功能,为其用于软骨组织修复提供理论依据。     

脱细胞软骨基质来源的多孔支架复合山羊髓核细胞体内初步构建组织工程髓核的实验研究

[目的]探讨脱细胞软骨基质多孔支架复合PKH26标记的山羊髓核细胞体内异位构建组织工程髓核的可行性.[方法]制备脱细胞软骨基质来源的多孔支架,扫描电镜(scanning electron microscope,SEM)观察、天狼星红染色、HE染色观察、MTT毒性检测;分离山羊髓核细胞,通过倒置显微镜观察、番红O染色、Ⅱ型胶原免疫组化染色进行鉴定;将PKH26标记的山羊髓核细胞接种支架上,体外培养3d后进行LIVE/DEAD活性染色,将细胞支架复合物置入裸鼠皮下,培养6周,病理切片,荧光显微镜下观察,进行番红O、Ⅰ、Ⅱ型胶原免疫组化染色.[结果]扫描电镜观察支架孔隙相连通且分布均匀,天狼星红染色支架呈黄绿相间色,HE支架淡染,MTT检测细胞增殖曲线无统计学差异(P＞0.05);P1代髓核细胞呈软骨样细胞形态,番红O染色、Ⅱ型胶原免疫组化染色均阳性,PKH26标记后的细胞呈红色荧光;体外LIVE/DEAD染色细胞呈绿色荧光,体内培养6周后,带红色荧光的细胞填满支架孔隙,番红O、Ⅱ型胶原免疫组化染色阳性,Ⅰ型胶原免疫组化染色弱阳性.[结论]以脱细胞软骨基质多孔支架复合山羊髓核细胞在体内能够形成组织工程髓核样组织.     

激光打孔脱细胞基质复合骨髓间充质干细胞构建组织工程气管软骨

目的探索激光打孔脱细胞基质诱导骨髓间充质干细胞(BMSCs)于动物体内构建组织工程气管软骨的可行性。方法应用激光打孔联合脱细胞技术制备激光打孔脱细胞基质(LDTM)作为支架材料。抽取兔骨髓,分离培养BMSCs,接种于LDTM支架后植入兔皮下。体内培养12周后,行大体观察、组织学检测、生物力学及生物化学定量检测。结果细胞均匀分布在激光微孔和支架表面,并且该复合物能够较好地维持原始的管状结构,形成了富有弹性的瓷白色软骨样组织。HE染色可见典型的软骨陷窝出现,说明成熟软骨组织形成。Safranin-O染色证实有蛋白聚糖基质产生。生物力学和生物化学检测显示新生气管组织均接近正常气管组织。结论 LDTM可以促进软骨再生并在动物体内诱导BMSCs构建组织工程气管软骨。     

软骨脱细胞基质支架材料的软骨组织工程实验研究

目的应用软骨脱细胞基质作为支架材料，按照组织工程的原理再生软骨，为修复软骨缺损探索新的途径。方法取新西兰大耳白兔1只，体重2．4kg，按改良Courtman法对兔耳软骨行脱细胞处理后用于实验。选用纯种6月龄新西兰大耳白兔18只，雌雄不限，体重2．4～2．6kg。每只耳形成2处1cm×1cm软骨缺损，按修复方式不同随机分为3组，每组24处缺损。A组，软骨脱细胞基质加软骨膜；B组，软骨脱细胞基质；C组软骨膜，作为对照。术后每日观察兔耳修复区的大体变化，并分别于4、12周每组处死3只动物，于修复区切取标本，行HE染色、藏红花红一奥尔新蓝染色、II型胶原基因探针原位杂交实验。结果术后4周内A、B组大体形态无明显改变；术后12周可见修复区轻度增厚，触摸时质地较正常软骨稍硬。C组术后2周，2只2处修复区形成痂皮，术后5周痂皮脱落形成穿孔。术后4周，A、B组HE染色可见软骨脱细胞基质周边有轻度的炎性细胞浸润，以淋巴细胞为主，未形成包囊，藏红花红．奥尔新蓝染色均阴性；C组软骨缺损处的软骨膜塌陷，无细胞增殖现象；各组修复区均未见II型胶原基因探针原位杂交显色。术后12周，A组HE染色示部分软骨脱细胞基质内有新生细胞，细胞排列不规律，ECM嗜碱性增强，藏红花红-奥尔新蓝染色呈阳性，II型胶原基因原位杂交显色示新生细胞内均有大片棕黄色阳性染色区域；B组HE染色示软骨脱细胞基质无吸收现象，周边无包囊，炎性细胞消失，藏红花红一奥尔新蓝染色呈阴性，未见II型胶原基因原位杂交显色；C组HE染色示近软骨断端处可见部分形成新生组织，藏红花红一奥尔新蓝染色呈阳性，II型胶原基因原位杂交显色可见棕黄色阳性染色细胞。结论脱细胞软骨可诱导软骨膜细胞向其中生长而重建软骨，软骨膜和脱细胞软骨复合移植是软骨组织工程的另一种选择。     

利用脂肪干细胞构建软骨修复兔膝关节软骨缺损的初步研究

目的 利用兔同种异体软骨脱细胞基质支架和脂肪干细胞体外构建组织工程软骨,探讨其修复关节软骨损伤的可行性.方法 将新西兰大白兔的脂肪干细胞与软骨脱细胞基质支架复合,于软骨细胞方向诱导培养基中培养两周,构建组织工程软骨.兔24只随机分为A、B、C 3组, A组关节软骨缺损处置入经诱导的脂肪源干细胞复合软骨基质支架, B组缺损处只置入软骨基质支架, C组软骨缺损处不做任何处理.分别于术后第12周处死动物,修复处行大体、组织学、Ⅱ型胶原免疫组化染色和透射电镜检测.结果 A组软骨缺损处被类软骨组织填充,修复区表面光滑;Ⅱ型胶原免疫组化染色和甲苯胺蓝染色阳性;电镜下可见软骨陷窝内有细胞结构存在,且有大量均匀颗粒状细胞分泌基质成分存在,细胞周围大量胶原纤维.B组软骨缺损处为纤维组织状物填充,C组软骨缺损处无修复组织填充.结论 脂肪干细胞与软骨脱细胞基质复合并向软骨诱导后可良好地修复关节软骨缺损,具有替代正常软骨的潜力.     

降钙素对兔骨关节炎关节软骨的保护作用

[目的]研究降钙素（ealcitonin，CT）对骨关节炎关节软骨的保护作用。[方法]6个月龄新西兰大白兔20只，随机分为两组均行右膝关节前交叉韧带切断术。1周后实验组开始皮下注射降钙素5 IU／kg，每日1次：对照组注入等剂量生理盐水；术后6周处死动物。取股骨内髁制成切片行HE、番红“O”和Ⅱ型胶原免疫组化染色。HE切片按照Mankin评分表评分。番红“O”染色和Ⅱ型胶原免疫组化染色切片用图像分析仪，测量平均灰度值。[结果]（1）对照组术侧膝关节组织学Mankin评分结果、番红“O”染色和Ⅱ型胶原免疫组化染色平均灰度值测量结果高于健侧，（P〈0．01）。（2）对照组手术膝关节组织学评分、番红“O”染色和Ⅱ型胶原免疫组化染色平均灰度值测量结果高于实验组手术膝关节，（P〈0．05）。[结论]降钙素5IU／kg每日1次皮下注射够明显减轻兔膝关节不稳定诱发的骨关节炎关节软骨的退变。降钙素可能通过提高软骨基质糖胺多糖（glycosaminoglycaris,GAG）和Ⅱ型胶原含量保护关节软骨。     

Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes

OBJECTIVE: The functional integrity of articular cartilage is determined by a balance between chondrocyte biosynthesis of extracellular matrix and its degradation. In osteoarthritis (OA), the balance is disturbed by an increase in matrix degradative enzymes and a decrease in biosynthesis of constitutive extracellular matrix molecules, such as collagen type II and aggrecan. In this study, we examined the effects of the sulfate salt of glucosamine (GS) on the mRNA and protein levels of the proteoglycan aggrecan and on the activity of matrix metalloproteinase (MMP)-3 in cultured human OA articular chondrocytes. DESIGN: Freshly isolated chondrocytes were obtained from knee cartilage of patients with OA. Levels of aggrecan and MMP-3 were determined in culture media by employing Western blots after incubation with GS at concentrations ranging from 0.2 to 200 microM. Zymography (casein) was performed to confirm that effects observed at the protein level were reflected at the level of enzymatic activity. Northern hybridizations were used to examine effects of GS on levels of aggrecan and MMP-3 mRNA. Glycosaminoglycan (GAG) assays were performed on the cell layers to determine levels of cell-associated GAG component of proteoglycans. RESULTS: Treatment of OA chondrocytes with GS (1.0-150 microM) resulted in a dose-dependent increase in aggrecan core protein levels, which reached 120% at 150 microM GS. These effects appeared to be due to increased expression of the corresponding gene as indicated by an increase in aggrecan mRNA levels in response to GS. MMP-3 levels decreased (18-65%) as determined by Western blots. Reduction of MMP-3 protein was accompanied by a parallel reduction in enzymatic activity. GS caused a dose-dependent increase (25-140%) in cell-associated GAG content. Chondrocytes obtained from 40% of OA patients failed to respond to GS. CONCLUSIONS: The results indicate that GS can stimulate mRNA and protein levels of aggrecan core protein and, at the same time, inhibit production and enzymatic activity of matrix-degrading MMP-3 in chondrocytes from OA articular cartilage. These results provide a cogent molecular mechanism to support clinical observations suggesting that GS may have a beneficial effect in the prevention of articular cartilage loss in some patients with OA.     

Influence of aging on the synthesis and morphology of the aggrecans synthesized by differentiated human articular chondrocytes

OBJECTIVE: Synthesis rates of aggrecans by phenotypically stable human articular chondrocytes and the immobilization of these aggrecans in large aggregates were used as variables reflecting the capability of these cells of restoring the extracellular matrix of articular cartilage in vivo in an aging population. DESIGN: Human articular chondrocytes were isolated from articular cartilage obtained from 33 different donors at autopsy. The chondrocytes were cultured in gelled agarose. Synthesis of aggrecans was investigated using Na(2)(35)SO(4)as a radioactive precursor after a 2-week culture period. Electron microscopic study of aggrecan aggregates was done on the macromolecules accumulated over 3 weeks in culture by the chondrocytes obtained from eight other donors with increasing ages. RESULTS: Sulfate incorporation rates into aggrecans correlated inversely with the age of the donor. The value of sulfate incorporation in aggrecans for chondrocytes obtained from mature cartilage of a 20-year-old individual in this system drops to 50% and 25% for chondrocytes obtained from 45- and 69-year-old individuals respectively. Electron microscopic study of aggrecan aggregates showed that the 'de novo' synthesized hyaluronan molecules were fully loaded with aggrecans. Mature human articular cartilage cells were found to synthesize an aggrecan aggregate which carried an average number of 11.7 to 13.1 aggrecans. Cells obtained from immature donors synthesized aggrecan aggregates of which the hyaluronan chain carried twice the amount of aggrecans. These immature human articular cartilage cells were also found to synthesize significant proportions of large aggrecan aggregates with 20 to over 100 aggrecans immobilized on a single hyaluronan chain. The proportions of these large aggrecan aggregates decreased with increasing age of the donors of the chondrocytes. CONCLUSION: The declining aggrecan synthesis rates and the decreased capability of assembling large molecular size aggregates with increasing age in humans illustrates a progressive failure of the repair function of articular cartilage cells in humans.     

Streaming potentials: a sensitive index of enzymatic degradation in articular cartilage

Under physiological conditions, the extracellular matrix of articular cartilage contains a high fixed-charge density, associated with its ionized proteoglycan (PG) molecules. Compression of the highly charged cartilage matrix within the physiologic range leads to the production of electrical streaming potentials. We observed significant changes in the potential response due to chemical modifications of the matrix, such as extraction of PG and glycosaminoglycan (GAG) moieties using chondroitinase-ABC adn trypsin. The streaming potential was a sensitive index of the degradative loss of these matrix constituents and of the kinetics of the enzymatic degradative process.     

The cleavage of biglycan by aggrecanases

OBJECTIVE: Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4] and aggrecanase-2 (ADAMTS-5) have been named for their ability to degrade the proteoglycan aggrecan. While this may be the preferred substrate for these enzymes, they are also able to degrade other proteins. The aim of this work was to determine whether the aggrecanases could degrade biglycan and decorin. METHODS: Biglycan, decorin and aggrecan were purified from human and bovine cartilage and subjected to degradation by recombinant aggrecanase-1 or aggrecanase-2. In vitro degradation was assessed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and immunoblotting, and the cleavage site in biglycan was determined by N-terminal amino acid sequencing. SDS/PAGE and immunoblotting were also used to assess in situ degradation in both normal and arthritic human articular cartilage. RESULTS: Both aggrecanase-1 and aggrecanase-2 are able to cleave bovine and human biglycan at a site within their central leucine-rich repeat regions. Cleavage occurs at an asparagine-cysteine bond within the fifth leucine-rich repeat. In contrast, the closely related proteoglycan decorin is not a substrate for the aggrecanases. Analysis of human articular cartilage from osteoarthritic (OA) and rheumatoid arthritic (RA) joints showed that a biglycan degradation product of equivalent size is present in the extracellular matrix. No equivalent degradation product was, however, detectable in normal adult human articular cartilage. CONCLUSION: Biglycan, which is structurally unrelated to aggrecan, can act as a substrate for aggrecanase-1 and aggrecanase-2, and these proteinases may account for at least part of the biglycan degradation that is present in arthritic cartilage.     

Proteoglycan depletion of intact articular cartilage by retinoic acid is irreversible and involves loss of hyaluronate

Intact sesamoid bones from bovine metacarpophalangeal joints were cultured with retinoic acid for 9 days and allowed to recover in control medium for up to 17 days. Retinoic acid (300 ng/ml) induced 91.8% inhibition of glycosaminoglycan (GAG) synthesis and 50.6% loss of sulfated GAGs from the cartilage. Retinoic acid also induced 38.2% loss of hyaluronate from the matrix. The synthesis and content of the large aggregating proteoglycan (aggrecan) were preferentially decreased compared with that of the small nonaggregating species. The aggrecan synthesized was similar to control aggrecan in size, aggregation capacity, and composition of its GAGs. GAG synthesis was almost completely restored in control medium within the next 6 days. The GAGs synthesized during recovery were slightly shorter than control GAGs and showed a higher ratio of chondroitin-6-sulfate over chondroitin-4-sulfate. Neither the proteoglycan content nor the hyaluronate content recovered within 17 days. The aggregation capacity of newly synthesized aggrecan was normal. However, the retention of proteoglycans synthesized in the recovery period was much lower in treated cartilage than in control cartilage (T1/2 of 17 and 38 days, respectively). In conclusion, the retinoic-acid-induced proteoglycan depletion was irreversible in spite of the restored synthesis of aggrecan with a normal aggregation capacity. The reduced retention of newly synthesized aggrecan during recovery might be caused by a lack of hyaluronate. This model seems suitable to study aspects of cartilage destruction and repair.     

Development and characterization of a highly specific and sensitive sandwich ELISA for detection of aggrecanase-generated aggrecan fragments

OBJECTIVE: To develop an enzyme linked immunosorbent assay (ELISA) to quantify the levels of specific aggrecan fragments generated by aggrecanase-mediated cleavage at the 373Glu-374 Ala bond within the aggrecan interglobular domain. METHODS: The ELISA employs a commercially available monoclonal antibody to capture aggrecan fragments containing keratan sulfate (KS). Aggrecan fragments generated by cleavage at the Glu-Ala bond were then detected using a monoclonal neoepitope antibody (mAb OA-1) that specifically recognizes the N-terminal sequence 'ARGSVIL'. RESULTS: The mAb OA-1 antibody was highly specific for the immunizing neoepitope peptide since neither peptides spanning the cleavage site nor mutated peptides were detected. Aggrecan fragments generated by ADAMTS-4 digested human aggrecan monomers and from IL-1-stimulated human cartilage explants were quantified by the ELISA, and we observed increased sensitivity of the ELISA compared to mAb OA-1 Western analysis. We also observed that the basal, as well as IL-1-stimulated production of ARGS aggrecan fragments from human articular cartilage explants was blocked by a selective aggrecanase inhibitor, consistent with generation of the ARGS neoepitope in human articular cartilage being mediated by aggrecanase. Using purified human aggrecan digested by ADAMTS-4 as standard to quantify ARGS aggrecan fragments in human synovial fluids, we determined that the calculated amount of ARGSVIL-aggrecan fragments by ELISA measurement is in agreement with the published levels of these fragments, supporting its potential utility as a biomarker assay for osteoarthritis. CONCLUSION: We have developed an assay that detects and quantifies specific aggrecan fragments generated by aggrecanase-mediated cleavage. Because aggrecanase mediates degradation of human articular aggrecan in joint disease, the KS/mAb OA-1 ELISA may serve as a biomarker assay for evaluation of preclinical and clinical samples.     

Modification of osteoarthritis by pulsed electromagnetic field--a morphological study

OBJECTIVE: Hartley guinea pigs spontaneously develop arthritis that bears morphological, biochemical, and immunohistochemical similarities to human osteoarthritis. It is characterized by the appearance of superficial fibrillation by 12 months of age and severe cartilage lesions and eburnation by 18 months of age. This study examines the effect of treatment with a pulsed electromagnetic field (PEMF) upon the morphological progression of osteoarthritis in this animal model. DESIGN: Hartley guinea pigs were exposed to a specific PEMF for 1h/day for 6 months, beginning at 12 months of age. Control animals were treated identically, but without PEMF exposure. Tibial articular cartilage was examined with histological/histochemical grading of the severity of arthritis, by immunohistochemistry for cartilage neoepitopes, 3B3(-) and BC-13, reflecting enzymatic cleavage of aggrecan, and by immunoreactivity to collagenase (MMP-13) and stromelysin (MMP-3). Immunoreactivity to TGFbeta, interleukin (IL)-1beta, and IL receptor antagonist protein (IRAP) antibodies was examined to suggest possible mechanisms of PEMF activity. RESULTS: PEMF treatment preserves the morphology of articular cartilage and retards the development of osteoarthritic lesions. This observation is supported by a reduction in the cartilage neoepitopes, 3B3(-) and BC-13, and suppression of the matrix-degrading enzymes, collagenase and stromelysin. Cells immunopositive to IL-1 are decreased in number, while IRAP-positive cells are increased in response to treatment. PEMF treatment markedly increases the number of cells immunopositive to TGFbeta. CONCLUSIONS: Treatment with PEMF appears to be disease-modifying in this model of osteoarthritis. Since TGFbeta is believed to upregulate gene expression for aggrecan, downregulate matrix metalloprotease and IL-1 activity, and upregulate inhibitors of matrix metalloprotease, the stimulation of TGFbeta may be a mechanism through which PEMF favorably affects cartilage homeostasis.     

三步酶消化法高效分离兔原代关节软骨细胞及体外培养观察

目的　观察设计的三步酶消化法分离原代关节软骨细胞的效果,并对体外培养不同代次细胞的生物学活性进行评价。方法　三步酶消化法设计为以培养液配制的1g/L胰蛋白酶及1g/LEDTA混合液、1g/L透明质酸酶和2g/LⅠ型胶原酶顺序消化关节软骨分离细胞,检测细胞收获效率和存活率;体外传代培养观察细胞形态、生长及胞外基质中Ⅰ型和Ⅱ型胶原、蛋白多糖聚集体等的变化。结果　(1)关节软骨经三步酶消化基质逐步解离和降解,细胞得以完全释放和分离,每克软骨的细胞收获量平均为50 3×106 个细胞,细胞存活率平均为98. 8%。(2)原代和第一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异染反应均呈阳性,原代细胞外基质有高的硫酸糖胺多糖含量为(92±10)μg/cm2;第三代后细胞逐渐变为梭形,Ⅱ型胶原免疫组化为阴性,甲苯胺蓝异染反应明显减弱,第四代细胞外基质的硫酸糖胺多糖含量为(48±12)μg/cm2。结论　(1)三步酶消化法可使关节软骨基质完全消化降解,具有高细胞收获率、高细胞存活率和操作简便等特点。(2)原代和第一代软骨细胞具有良好的生物学活性,而第三代以后的细胞生物学活性低下。