鸡胚营养组合物对衰老大鼠基因损伤修复的影响

目的：观察鸡胚营养组合物对衰老大鼠基因损伤修复的影响，探讨其延缓衰老的可能机制。方法：采用D-半乳糖建立衰老SD大鼠动物模型，每日1次颈背部皮下注射 D-半乳糖500mg/(kg·d)，连续造模90天，对照组颈背部皮下注射等剂量的生理盐水注射液。从第1天起，鸡胚营养组合物组、鸡胚组、营养组合物组、蛋清组，分别以相应剂量对大鼠进行灌胃，每天1次，直至第90天，拉颈处死大鼠。取各组大鼠肝脏、骨髓和脑组织，采用RT-PCR法检测各组织修复基因的表达情况。结果：与衰老模型组相比，鸡胚营养组合物组衰老大鼠肝脏和骨髓组织中错配修复基因MSH2表达量升高（P<0.05）；鸡胚营养组合物组衰老大鼠肝脏组织中同源重组修复基因Xrcc1、Xrcc2、骨髓中同源重组修复基因Rad51以及脑组织中同源重组修复基因Rad51、Xrcc1表达量升高（P<0.05）。结论：鸡胚营养组合物能够修复衰老大鼠肝脏、骨髓和脑组织基因的损伤，此可能为其延缓衰老的作用机制之一。     

藿香蓟叶提取物对大鼠创伤性骨关节炎的治疗作用

摘要 目的：探究藿香蓟叶提取物（ACLE）对创伤性骨关节炎（PTOA）模型大鼠的治疗作用及其可能的分子机制。方法：SPF级雄性SD大鼠采用前交叉韧带切断法构建PTOA大鼠模型,然后将建模成功的大鼠随机分为模型组、藿香蓟叶提取物2.5 g组（ACLE 2.5 g组）、藿香蓟叶提取物5 g组（ACLE 5 g组）和藿香蓟叶提取物10 g组（ACLE 10 g组）,每组21只。另取21只SD大鼠设为假手术组,大鼠只执行前交叉韧带暴露手术,但是不切断。ACLE 2.5 g组、ACLE 5 g组和ACLE 10 g组大鼠在造模结束后分别灌胃给药,剂量分别为2.5 g/kg、5 g/kg和10 g/kg,持续给药处理8周。假手术组和模型组大鼠给予等量生理盐水每日灌胃处理,同样持续干预处理8周。给药结束后,收集各组大鼠的血清、膝关节灌洗液和膝关节软骨组织。采用HE染色（Mankin''s评分）和番红O+固绿染色（OARSI评分）评估大鼠关节损伤情况。采用ELISA试剂盒检测大鼠血清中ALP、BGP和CTX-Ⅰ水平,以及大鼠血清和关节灌洗液中TNF-α、IL-6和IL-1β水平。采用试剂盒检测大鼠血清和关节灌洗液中MDA、SOD、GSH-Px和ROS水平。采用Western blot检测大鼠软骨组织中Aggrecan、SOX-9、Col Ⅱ、MMP-13、Wnt1、β-catenin、Bcl-2和Bax蛋白表达水平。结果：模型大鼠的膝关节软骨组织损伤严重,软骨层状组织结构遭受破坏;ACLE 2.5 g组、ACLE 5 g组和ACLE 10 g组大鼠膝关节软骨组织结构和病理状态均得到不同程度的恢复。与模型组比较,ACLE 2.5 g组、ACLE 5 g组和ACLE 10 g组大鼠Mankin''s和OARSI的评分值均降低（P<0.01）。与模型组比较,ACLE 2.5 g组、ACLE 5 g组和ACLE 10 g组大鼠血清中ALP和BGP水平升高,CTX-Ⅰ水平降低（P<0.05）。与模型组比较,ACLE 2.5 g组、ACLE 5g组和ACLE 10 g组大鼠血清和膝关节灌洗液中IL-6、IL-1β、TNF-?琢、NO和MDA水平均降低（P<0.05）,SOD和GSH-Px水平均升高（P<0.05）。与模型组比较,ACLE 2.5 g组、ACLE 5 g组和ACLE 10 g组大鼠关节软骨组织中Aggrecan、SOX-9、ColⅡ和Bcl-2蛋白表达水平均升高（P<0.05）,MMP-13、Wnt1、β-catenin和Bax蛋白表达水平均降低（P<0.05）。结论：ACLE能够降低PTOA大鼠膝关节软骨组织中炎症反应水平,增强抗氧化能力;抑制Wnt/β-catenin通路的过度激活,通过升高Aggrecan、ColⅡ、SOX-9和Bcl-2的表达,促进软骨细胞外基质的恢复,并抑制软骨细胞的凋亡,从而改善PTOA大鼠软骨组织损伤程度。     

2-脱氧葡萄糖诱导大鼠内质网应激预处理模型的最佳剂量

目的探讨2-脱氧葡萄糖(2-deoxy-glucose,2-DG)诱导大鼠内质网应激模型的最佳剂量。方法选择Wistar雄性大鼠108只,体质量240～260 g,采用不同剂量2-DG建立大鼠内质网应激的模型,随机分为6组:2-DG 50、100、150、200 mg/kg组,假手术组,缺血再灌注组。2-DG组按工作浓度为50 mg/mL溶于双蒸水腹腔注射7d,假手术组和缺血再灌注组腹腔注射双蒸水7 d,于脑缺血再灌注后12 h处死,对各组大鼠进行神经行为学评分,采用HE染色观察脑组织的病理形态,免疫组化法和Westernblot法测定GRP78蛋白的表达,用PCR法检测GRP78mRNA的表达。结果与脑缺血再灌注组相比,2-DG各剂量组大鼠的神经行为学评分明显减低(P<0.01),而以100 mg/kg组评分减低最为明显(P<0.05);2-DG各剂量组能不同程度的改善大鼠脑海马CA1区神经细胞的核深染、核固缩程度,减少细胞及间质的水肿,使胞膜趋于清楚、形态接近正常、核仁清晰可见的神经细胞数目增多,而以2-DG100 mg/kg组的效果最为明显。2-DG各剂量组GRP78蛋白表达明显增加,与脑缺血再灌注组比较,差异有显著性(P<0.01),而以100 mg/kg剂量组的GRP78蛋白表达最高,与其他2-DG剂量组比较差异有显著性(P<0.05)。结论 2-DG对脑缺血再灌注所致的神经细胞损伤具有保护作用,其最佳剂量为100 mg/kg。2-DG具有诱导大鼠内质网应激的作用,其最佳剂量是100 mg/kg。     

杜仲提取液对脂多糖诱导的乳腺炎小鼠疗效观察和作用途径

目的研究杜仲提取液对小鼠乳腺炎的治疗效果和作用途径。方法通过乳导管灌注LPS构建小鼠乳腺炎模型,注射LPS 12 h后通过腹腔进行不同浓度梯度杜仲给药,分为6组。给药12 h后对小鼠进行剖杀并取其乳腺组织,利用HE染色观察各组病理变化,利用ELISA方法检测乳腺组织中TNF-2α、IL-6、IL-1β细胞因子的变化情况。结果 1)浓度为50μL 0.2 mg/mL的LPS可成功构建小鼠乳腺炎模型;(2)HE染色结果表明杜仲提取液可减轻LPS诱导的小鼠乳腺炎的损伤程度,随着注射杜仲提取液剂量的增加,乳腺组织的损伤程度减轻,且注射剂量为20 mg/kg和27 mg/kg时效果最佳;(3)ELISA结果表明杜仲提取液可降低乳腺组织中炎性细胞因子IL-1β、IL-6、TNF-α的含量,且注射剂量为27 mg/kg时效果最佳(P0.01)。结论杜仲提取液可显著减轻小鼠乳腺组织病理损伤、减少乳腺组织内炎性细胞因子IL-1β、IL-6、TNF-α的释放,阻断了中性粒细胞向乳腺组织和乳腺腺泡腔募集和迁移过程,从而减轻乳腺组织损伤,其中浓度为27 mg/kg的杜仲提取液抗炎效果最佳。     

凝血酶法致大鼠血液高凝态模型的建立

目的探索静脉注射凝血酶造成大鼠高凝态模型,为高凝态的研究提供合适动物模型。方法 SD大鼠分为六组,分别于股静脉恒速注射生理盐水和2.5、5、10、20、40 U/kg凝血酶溶液,5 min内采血测活化部分凝血活酶时间(activated partial thromboplastin time,APTT)、凝血酶原时间(prothrombin time,PT)、纤维蛋白原(fibrinogen,FIB),并观察大鼠死亡情况以确定最佳凝血酶剂量。在此基础上,以最佳剂量凝血酶溶液股静脉注射给予大鼠,分别于0、10、30、60、120、180、300 s采血测APTT、PT、FIB以确定最佳采血时间。最后将大鼠分为生理盐水组与凝血酶组(最佳凝血酶剂量和采血时间),采集血样测定APTT、PT、FIB以及全血粘度。结果 10 U/kg凝血酶组大鼠血浆APTT、PT明显缩短,FIB明显升高,且大鼠死亡率低。注射凝血酶后60 s大鼠血浆APTT、PT缩短最多,FIB含量升至最高。同生理盐水组相比,凝血酶组大鼠血浆PT、APTT显著缩短,FIB、全血粘度显著增大,且差异有显著性(P0.05)。结论注射凝血酶溶液可复制大鼠血液高凝态模型,最佳凝血酶剂量为10 U/kg,凝血酶浓度为2U/m L,最佳采血测试时间为60 s。     

雌/雄激素比例对去势大鼠前列腺体积及其脏器系数的影响

目的探讨雌/雄激素比例对去势大鼠前列腺体积及其脏器系数的影响。方法选用雄性SD大鼠随机分为37组,分别为:35个不同雌/雄激素比例组、正常和去势对照组。给药组分别注射不同配伍剂量的雌/雄激素、对照组均注射生理盐水各一个月。最后一次注药后24h取血、处死、取材,称取前列腺重量,测量体积,并计算脏器系数。结果当丙酸睾丸酮给药剂量分别为每只鼠0.02、0.5、2.5和12.5mg时,均未见器官增生程度与苯甲酸雌二醇注射剂量呈负相关。丙酸睾丸酮给药剂量每只鼠为0.1mg时,随着苯甲酸雌二醇注射量增加,前列腺体积及其系数均增大,而当每只鼠苯甲酸雌二醇注射量达到50μg/kg时,器官增生程度不再随苯甲酸雌二醇注射剂量增加而增加。结论雌激素发挥作用需以雄激素存在为前提,雌激素并非总是呈现对抗雄激素促进前列腺增生的作用,而是在一定范围内协同雄激素促进前列腺增生。     

水杨柳根D_(101)树脂提取物保肝降酶作用的实验研究

目的研究水杨柳根D_(101)树脂提取物(HRE)对肝损伤模型动物的保护作用。方法取小鼠行连续灌胃给予50%白酒30天制造酒精性肝损伤模型,于小鼠腹腔注射CCl_4制造急性肝损伤模型,每周2次于大鼠腹腔连续注射CCl_430天制造慢性肝损伤模型,然后分别测定各动物血清ALT和AST水平。结果在酒精性和CCl_4慢性肝损伤动物模型中,HRE高、中剂量组(小鼠为2.7g/kg,1.8g/kg;大鼠为3.9g/kg,2.6g/kg)均能显著降低血清ALT和AST水平(P0.01或P0.05);在CCl_4急性肝损模型小鼠中,HRE高、中剂量组(2.7g/kg,1.8g/kg)也能显著降低血清ALT和AST水平(P0.01或P0.05)。结论水杨柳根的D_(101)树脂提取物的主要成分为黄酮、多糖和皂苷,具有保肝降酶作用,且随剂量增加而增强。     

卡麦角林处理哺乳期小鼠对子代存活和生长的影响

哺乳期是兽类繁殖成功的关键阶段之一,本实验以不同浓度卡麦角林油溶液对哺乳早期雌性小鼠连续灌胃3 d,检测雌性小鼠体重和幼鼠存活及体重变化,以此确定卡麦角林对哺乳雌性小鼠繁殖成功的影响。结果发现,卡麦角林对雌性小鼠在哺乳期的体重相对增长率无显著影响,但处理组幼鼠出现部分死亡,0 μg / kg、50 μg / kg、100 μg/ kg、200 μg/ kg、400 μg/ kg 组幼鼠死亡率分别为1.30% 、3.85% 、4. 08% 、7.38% 、21.19% ,幼鼠死亡数和剂量呈正相关,400 μg / kg 组幼鼠成活率显著下降到78 8% ,其他剂量组幼鼠成活率与对照组差异不显著;给药后7 d 存活幼鼠的生长发育显著被抑制,之后处理组幼鼠体重变化与对照组无显著差异。上述结果表明卡麦角林处理哺乳早期雌性小鼠可降低幼鼠成活率并抑制存活幼鼠的生长发育,进而有可能降低雌性小鼠及其子代的繁殖成功率。     

LPS/D-GalN诱导小鼠急性肝损伤模型的建立

目的建立脂多糖(lipopolysaccharide,LPS)/D-氨基半乳糖(D-galactosamine,D-GalN)诱导小鼠急性肝损伤模型。方法 40只雌性C57BL/6小鼠用于观察8种不同LPS与D-GalN剂量配比联合刺激后小鼠存活时间,以确定模型建立的最佳剂量。使用腹腔注射最佳剂量染毒32只雌性C57BL/6小鼠,分别在0、1、4、8 h处死,每组8只,0 h注射相同剂量生理盐水作为对照。观察染毒后小鼠肝组织病理损伤,检测血清中ALT及炎症因子IL-6、MCP-1和TNF-α表达水平变化。结果通过观察小鼠存活时间,确定腹腔注射最佳染毒剂量为LPS(2.5 mg/kg)/D-GalN(0.3 g/kg);小鼠染毒后肝组织呈进程性病变,最终发展为肝脏弥漫性坏死,肝细胞核崩解。与对照组相比,血清ALT显著升高(P0.001),IL-6、MCP-1、TNF-α均在1 h后达到最高水平(P0.001),然后持续下降。结论成功建立LPS/D-GaIN诱导小鼠急性肝损伤模型,为探索急性肝损伤的致病机制以及药物干预治疗提供有效的动物模型。     

人参皂苷Rg1促进大鼠急性心肌梗死后血管新生

目的:明确人参皂苷Rg1在大鼠发生急性心肌梗死后是否能够促进心脏血管新生。方法:通过结扎SD大鼠左冠状动脉前降支建立大鼠急性心肌梗死模型,并将60只雄性SD大鼠随机分单纯手术组与人参皂苷Rg1治疗组。治疗组的大鼠造模1 h后将预先配成药液的人参皂甙Rgl按5 rag/(kg·d)剂量腹腔注射,1次/日至处死当日。对照组则腹腔注射等量生理盐水1次/日至处死当日。分别于手术后3、7天时对比两组大鼠的基本生命指标,后通过免疫荧光染色CD31对比观察两组大鼠心脏细胞中CD31的表达来评判血管新生水平。结果:①手术后3天、7天,两组大鼠的体重、心脏重量、心重/体重、鼠尾收缩压、心率比较差异均没有统计学意义(P0.05);②手术后3天、7天,人参皂苷Rg1治疗组心脏CD31表达水平明显高于对照组。结论:人参皂苷Rg1能够促进大鼠急性心肌梗后心脏的血管新生。     

Recombinant human midkine stimulates proliferation of articular chondrocytes

Objectives: Midkine, a heparin‐binding growth factor, promotes population growth, survival and migration of several cell types, but its effect on articular chondrocytes remains unknown. The aim of this study was to investigate its role on proliferation of articular chondrocytes in vitro and in vivo. Materials and methods: Bromodeoxyuridine incorporation and MTT assays were performed to examine the proliferative effect of recombinant human midkine (rhMK) on primary articular chondrocytes. Activation of extracellular signal‐regulated kinase (ERK) and phosphatidylinositol 3‐kinase (PI3K) was analysed using western blot analysis. Systemic and local delivery of rhMK into mice and rats was preformed to investigate the proliferative effect of rhMK in vivo, respectively. Histological evaluation, including measurement of articular cartilage thickness, cell density, matrix staining and immunostaining of proliferating cell nuclear antigen was carried out. Results: rhMK promoted proliferation of articular chondrocytes cultured in a monolayer, which was mediated by activation of ERK and PI3K. The proliferative role of rhMK was not coupled to dedifferentiation of culture‐expanded cells. Consistent with its action in vitro, rhMK stimulated proliferation of articular chondrocytes in vivo when it was administered subcutaneously and intra‐articularly in mice and rats, respectively. Conclusion: Our results demonstrate that rhMK stimulates proliferation of primary articular chondrocytes in vitro and in vivo. The results of this study warrant further examination of rhMK for treatment of animal models of articular cartilage defects.     

右归丸对大鼠膝骨关节炎的干预作用及其机制*

目的: 探讨右归丸对膝骨关节炎(KOA)模型鼠关节软骨组织骨诱导因子(OGN)、骨黏连蛋白(ON)和纤维蛋白原2(FBN2)的影响。方法: 将大鼠随机分为假手术组,模型组,硫酸氨基葡萄糖组(硫酸氨基葡萄糖),右归丸(高、中、低剂量)组,每组10只。采用改良Hulth法制备大鼠KOA模型,假手术组和模型组给予等体积生理盐水灌胃,右归丸高、中、低剂量组分别灌胃给予右归丸4.8,2.4,1.2 g/kg,硫酸氨基葡萄糖组灌胃给予硫酸氨基葡萄糖 0.17 g/kg,连续给药8周。干预结束24 h后取鼠膝关节软骨,采用HE染色法观察各组软骨的病理改变,并进行Mankin评分;免疫组化法检测各组关节软骨组织中OGN、ON和FBN2的表达;Western blot法检测各组关节软骨组中糖原合成酶激酶-3β(GSK-3β)的表达。结果: 与假手术组比较,模型组大鼠软骨组织Makin 评分显著升高,软骨组织FBN2蛋白表达水平上显著增加,OGN、ON和GSK-3β蛋白表达水平上的显著降低(P＜0.01);模型组关节软骨边缘严重破坏,软骨细胞排列紊乱。与模型组比较,右归丸高剂量干预组大鼠软骨组织Makin 评分和FBN2蛋白表达水平显著降低,GSK-3β蛋白表达水平上显著增加,且右归丸中、高剂量组OGN、ON蛋白表达水平均显著增加(P＜0.05或P＜0.01),软骨结构趋于正常,软骨细胞分布仅偶见不均,关节软骨表面欠光滑。结论: 右归丸能够延缓关节软骨退变,其可能机制是通过提高骨诱导因子和骨粘连蛋白的表达水平来促进关节软骨的骨化和重构。     

不同频率的振动训练对大鼠早期膝骨关节炎关节软骨的修复作用及其JNK/NF-κB、SOX9机制*

目的: 探讨不同频率的振动训练对大鼠早期膝骨关节炎关节软骨的修复作用及其JNK/NF-κB、SOX9的机制。方法: 成年雄性SD大鼠48只,随机分为6组(n=8):模型对照组(MC组),高频振动1组(GP₁组,频率60 Hz),高频振动2组(GP₂组,频率40 Hz)、中频振动组(ZP组,频率20 Hz)和低频振动组(DP组,频率10 Hz),正常对照组(NC组)。除正常组外,各组大鼠经第1、4、7日双后腿膝关节腔注射2%木瓜蛋白酶溶液和L-半胱氨酸混合液6周后建立早期膝骨关节炎模型后,对振动组大鼠双膝进行4周,每天 40 min,振幅2~5 mm,每周振动5 d的训练。4周后,检测双膝关节股骨外侧髁关节软骨HE染色、番红O染色和Mankin评分形态学观察,股骨内侧髁关节软骨RT-qPCR检测JNK、NF-κB p65、SOX9 mRNA,Western blot 检测JNK、NF-κB p65、SOX9蛋白表达。结果: 与NC比较,其余各组Mankin评分均显著增高(P＜0.01),与MC相比,各振动组Mankin评分均显著降低(P＜0.05),其JNK、NF-κB p65mRNA和蛋白质表达显著降低(P＜0.01),SOX9mRNA和蛋白质表达显著升高(P＜0.01);与高频组相比,低频组的Mankin评分,JNK、NF-κB p65mRNA和蛋白质表达均显著降低(P＜0.05,P＜0.01),而SOX9mRNA和蛋白质表达则显著升高(P＜0.05,P＜0.01)。结论: 不同频率的振动训练对早期膝骨关节炎关节软骨可表现出不同程度的修复效应,低频振动训练软骨修复优于高频振动。可能通过下调关节软骨JNK/NF-κB表达,提高SOX9活性来调控胶原合成。     

Effect of alendronate on post-traumatic osteoarthritis induced by anterior cruciate ligament rupture in mice

IntroductionPrevious studies in animal models of osteoarthritis suggest that alendronate (ALN) has antiresorptive and chondroprotective effects, and can reduce osteophyte formation. However, these studies used non-physiologic injury methods, and did not investigate early time points during which bone is rapidly remodeled prior to cartilage degeneration. The current study utilized a non-invasive model of knee injury in mice to investigate the effect of ALN treatment on subchondral bone changes, articular cartilage degeneration, and osteophyte formation following injury.MethodsNon-invasive knee injury via tibial compression overload or sham injury was performed on a total of 90 mice. Mice were treated with twice weekly subcutaneous injections of low-dose ALN (40 μg/kg/dose), high-dose ALN (1,000 μg/kg/dose), or vehicle, starting immediately after injury until sacrifice at 7, 14 or 56 days. Trabecular bone of the femoral epiphysis, subchondral cortical bone, and osteophyte volume were quantified using micro-computed tomography (μCT). Whole-joint histology was performed at all time points to analyze articular cartilage and joint degeneration. Blood was collected at sacrifice, and serum was analyzed for biomarkers of bone formation and resorption.ResultsμCT analysis revealed significant loss of trabecular bone from the femoral epiphysis 7 and 14 days post-injury, which was effectively prevented by high-dose ALN treatment. High-dose ALN treatment was also able to reduce subchondral bone thickening 56 days post-injury, and was able to partially preserve articular cartilage 14 days post-injury. However, ALN treatment was not able to reduce osteophyte formation at 56 days post-injury, nor was it able to prevent articular cartilage and joint degeneration at this time point. Analysis of serum biomarkers revealed an increase in bone resorption at 7 and 14 days post-injury, with no change in bone formation at any time points.ConclusionsHigh-dose ALN treatment was able to prevent early trabecular bone loss and cartilage degeneration following non-invasive knee injury, but was not able to mitigate long-term joint degeneration. These data contribute to understanding the effect of bisphosphonates on the development of osteoarthritis, and may support the use of anti-resorptive drugs to prevent joint degeneration following injury, although further investigation is warranted.     

Transforming growth factor beta superfamily members: role in cartilage modeling

Normal and abnormal extracellular matrix turnover is thought to result, in part, from the balance in the expression of metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). The clinical manifestations of an imbalance in these relationships are evident in a variety of pathologic states, including osteoarthritis, deficient long-bone growth, rheumatoid arthritis, tumor invasion, and inadequate cartilage repair. Articular cartilage defects commonly heal as fibrocartilage, which is structurally inferior to the normal hyaline architecture of articular cartilage. Transforming growth factor-beta 1 (TGF-beta1), a cytokine central to growth, repair, and inflammation, has been shown to upregulate TIMP-1 expression in human and bovine articular cartilage. Additionally, members of the TGF-beta superfamily are thought to play key roles in chondrocyte growth and differentiation. Bone morphogenetic protein-2 (BMP-2), a member of this superfamily, has been shown to regulate chondrocyte differentiation states and extracellular matrix composition. It was proposed that, by optimizing extracellular matrix composition, BMP-2 would enhance articular cartilage healing. After determining the release kinetics of BMP-2 from a collagen type I implant (Long-Evans male rats; two implants/rat, n = 14), it was found that, in a tissue engineering application, BMP-2 induced a hyaline-like repair of New Zealand White rabbit knee articular cartilage defects (3-mm full-thickness defects in the femoral trochlea; 2 defects/rabbit, n = 36). The quality of cartilage repair with BMP-2 (with or without chondrocytes) was significantly better than defects treated with BMP-2, as assessed by a quantitative scoring scale. Immunohistochemical staining revealed TIMP-1 production in the cartilage defects treated with BMP-2. When studied in vitro, it was found that BMP-2 markedly increased TIMP-1 mRNA by both bovine articular and human rib chondrocytes. Additionally, increased TIMP-1 mRNA was translated into increased TIMP-1 protein production by bovine chondrocytes. Taken together, these data suggest that BMP-2 may be a useful cytokine to improve healing of cartilaginous defects. Furthermore, these data suggest that the beneficial effects of BMP-2 may be, in part, related to alterations in extracellular matrix turnover.     

Local stimulation of articular cartilage repair by transplantation of encapsulated chondrocytes overexpressing human fibroblast growth factor 2 (FGF-2) in vivo

BACKGROUND: Defects of articular cartilage are an unsolved problem in orthopaedics. In the present study, we tested the hypothesis that gene transfer of human fibroblast growth factor 2 (FGF-2) via transplantation of encapsulated genetically modified articular chondrocytes stimulates chondrogenesis in cartilage defects in vivo. METHODS: Lapine articular chondrocytes overexpressing a lacZ or a human FGF-2 gene sequence were encapsulated in alginate and further characterized. The resulting lacZ or FGF-2 spheres were applied to cartilage defects in the knee joints of rabbits. In vivo, cartilage repair was assessed qualitatively and quantitatively at 3 and 14 weeks after implantation. RESULTS: In vitro, bioactive FGF-2 was secreted, leading to a significant increase in the cell numbers in FGF-2 spheres. In vivo, FGF-2 continued to be expressed for at least 3 weeks without leading to differences in FGF-2 concentrations in the synovial fluid between treatment groups. Histological analysis revealed no adverse pathologic effects on the synovial membrane at any time point. FGF-2 gene transfer enhanced type II collagen expression and individual parameters of chondrogenesis, such as the cell morphology and architecture of the new tissue. Overall articular cartilage repair was significantly improved at both time points in vivo. CONCLUSIONS: The data suggest that localized overexpression of FGF-2 enhances the repair of cartilage defects via stimulation of chondrogenesis, without adverse effects on the synovial membrane. These results may lead to the development of safe gene-based therapies for human articular cartilage defects.     

The Effect of Exercise on the Early Stages of Mesenchymal Stromal Cell-Induced Cartilage Repair in a Rat Osteochondral Defect Model

The repair of articular cartilage is challenging owing to the restriction in the ability of articular cartilage to repair itself. Therefore, cell supplementation therapy is possible cartilage repair method. However, few studies have verified the efficacy and safety of cell supplementation therapy. The current study assessed the effect of exercise on early the phase of cartilage repair following cell supplementation utilizing mesenchymal stromal cell (MSC) intra-articular injection. An osteochondral defect was created on the femoral grooves bilaterally of Wistar rats. Mesenchymal stromal cells that were obtained from male Wistar rats were cultured in monolayer. After 4 weeks, MSCs were injected into the right knee joint and the rats were randomized into an exercise or no-exercise intervention group. The femurs were divided as follows: C group (no exercise without MSC injection); E group (exercise without MSC injection); M group (no exercise with MSC injection); and ME group (exercise with MSC injection). At 2, 4, and 8 weeks after the injection, the femurs were sectioned and histologically graded using the Wakitani cartilage repair scoring system. At 2 weeks after the injection, the total histological scores of the M and ME groups improved significantly compared with those of the C group. Four weeks after the injection, the scores of both the M and ME groups improved significantly. Additionally, the scores in the ME group showed a significant improvement compared to those in the M group. The improvement in the scores of the E, M, and ME groups at 8 weeks were not significantly different. The findings indicate that exercise may enhance cartilage repair after an MSC intra-articular injection. This study highlights the importance of exercise following cell transplantation therapy.     

Release kinetics of transforming growth factor-beta1 from fibrin clots

In any therapeutic model involving a tissue-engineering approach to the repair of partial-thickness articular cartilage defects, a chondrogenic differentiation factor is required to ensure tissue-specific healing. Transforming growth factor-beta1 (TGF-beta1) is known to act in this capacity, but at such high concentrations as to render its direct injection into the joint cavity inadvisable. This situation calls for a delivery system that can be applied directly to the defect site and that will release the drug gradually over a period of some weeks. Liposome encapsulation represents one such system, and has been recently implemented with some success in an animal model for cartilage repair. However, the kinetics of TGF-beta1 release have not been determined, it was the purpose of the present study to characterize these. The liberation of [(125)I]-labeled TGF-beta1 from fibrin matrices containing this agent in either a free or liposome-encapsulated form was monitored by liquid scintillation counting for 25 days in vitro. During the initial 5 days, fibrin clots containing liposome-encapsulated TGF-beta1 released this cytokine at a slower rate (2% to 4% per day) than did those containing the free molecules (10% to 20% per day); thereafter, the release rates were similar. At the end of the incubation period, only 40% of the liposome-encapsulated TGF-beta1 had been released from the fibrin clots, as compared with 68% from those containing the free molecules. Liposome encapsulation thus represents a suitable means of establishing a slow-delivery system in tissue-engineering approaches to articular cartilage repair.     

Treatment of cartilage defects by Low-intensity pulsed ultrasound in a sheep model

Aim of this study was to evaluate effects of Low-intensity pulsed ultrasound on repair of articular cartilage defects. Low-intensity pulsed ultrasound (Lipus) can induce the differentiation and activation of chondrocytes. This study was designed to evaluate the effect of Lipus on articular cartilage defects in a sheep. Eight sheep were divided in to two groups. The animals received bilateraly, articular cartilage defects 4 mm in diameter and 2 mm in deep on the patellar groove and experimental groups were treated with intensity 200 mW/cm², 20 min/day with low-intensity pulsed ultrasound for 2 month. Then both knee joints underwent surgery for remove of formed tissue sample from defects.The samples were evaluated by Quantitative real-time polymerase chain reaction (qRT-PCR), Safranin-o staining, Immunofluorescence Staining and Morphological characterization. The best and worst sample per group according to Macroscopic and micriscopic scoring were icentified. The results showed that the operated groups with-Lipus-treatment and without-Lipus treatment had considered statistically significant. Gross photography revealed that the defects in experimental groups were filled with proliferative tissue, while in control groups, a thin layer of proliferative tissue was formed in defects. qRT-PCR results showed the expression of coll2, sox9, aggrecan and Osteocalcin in experimental groups. Intense safranin-O staining show the formation cartilage tissue in ultrasound treated group, while loose safranin-o-staining were observed at the control groups. Immunofluorescence staining showed the type 2 Collagen protein expression. We suggest that low-intensity pulsed ultrasound provide the mechanistic basis force for articular cartilage repair and effective treatment modality for improving of articular cartilage defects.

     

Quantitative ultrasound can assess the regeneration process of tissue-engineered cartilage using a complex between adherent bone marrow cells and a three-dimensional scaffold

Articular cartilage (hyaline cartilage) defects resulting from traumatic injury or degenerative joint disease do not repair themselves spontaneously. Therefore, such defects may require novel regenerative strategies to restore biologically and biomechanically functional tissue. Recently, tissue engineering using a complex of cells and scaffold has emerged as a new approach for repairing cartilage defects and restoring cartilage function. With the advent of this new technology, accurate methods for evaluating articular cartilage have become important. In particular, in vivo evaluation is essential for determining the best treatment. However, without a biopsy, which causes damage, articular cartilage cannot be accurately evaluated in a clinical context. We have developed a novel system for evaluating articular cartilage, in which the acoustic properties of the cartilage are measured by introducing an ultrasonic probe during arthroscopy of the knee joint. The purpose of the current study was to determine the efficacy of this ultrasound system for evaluating tissue-engineered cartilage in an experimental model involving implantation of a cell/scaffold complex into rabbit knee joint defects. Ultrasonic echoes from the articular cartilage were converted into a wavelet map by wavelet transformation. On the wavelet map, the percentage maximum magnitude (the maximum magnitude of the measurement area of the operated knee divided by that of the intact cartilage of the opposite, nonoperated knee; %MM) was used as a quantitative index of cartilage regeneration. Using this index, the tissue-engineered cartilage was examined to elucidate the relations between ultrasonic analysis and biochemical and histological analyses. The %MM increased over the time course of the implant and all the hyaline-like cartilage samples from the histological findings had a high %MM. Correlations were observed between the %MM and the semiquantitative histologic grading scale scores from the histological findings. In the biochemical findings, the chondroitin sulfate content increased over the time course of the implant, whereas the hydroxyproline content remained constant. The chondroitin sulfate content showed a similarity to the results of the %MM values. Ultrasonic measurements were found to predict the regeneration process of the tissue-engineered cartilage as a minimally invasive method. Therefore, ultrasonic evaluation using a wavelet map can support the evaluation of tissue-engineered cartilage using cell/scaffold complexes.