甲状旁腺激素1-34对DH豚鼠原发性膝骨性关节炎的潜在保护作用及其作用机制
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摘要
第一部分DH豚鼠原发性膝骨关节炎模型的实验研究
目的：探讨Dunkin Hartley(DH)豚鼠原发性骨性关节炎的发生及其特点，对不同月龄的DH豚鼠不同原发osteoarthritis(OA)阶段关节软骨大体改变、关节软骨结构、组织化学成分的变化进行动态观察，了解软骨组织病理变化，为后续试验提供科学可用的OA原发模型。
方法：32只雌性1月龄DH豚鼠随机分为4组，各组动物分别在1月龄、3月龄、6月龄、9月龄时处死，打开膝关节腔后，观察各组豚鼠膝关节软骨面并拍照记录。后腿去皮及肌肉组织，避免损伤膝关节，切取胫骨到股骨之间的关节软骨组织后，70％酒精固定豚鼠股骨，固定72小时后用EDTA-2Na制成脱钙液行脱钙处理，约8周后骨组织变软，钙盐彻底脱失后行常规梯度酒精脱水、二甲苯透明并进行石蜡包埋切片，将厚度约为6-8gm的骨组织石蜡切片行Masson染色，用于关节软骨组织形态学观察，采用Mankin评分方法定量分析关节软骨损伤情况；另取部分关节软骨标本立即按程序制成电镜标本行扫描电镜观察软骨面改变情况及透射电镜观察软骨细胞改变情况。采用免疫组化方法检测各组大鼠膝关节软骨中蛋白多糖（glycosaminoglycans, GAG）、基质金属蛋白酶-3(matrixmetalloproteinase-3, MMP-3）表达情况，应用Image pro-Plus6.0软件对免疫组化阳性蛋白表达情况进行积分光密度(integrated optical density, IOD）计算，定量分析各组大鼠膝关节软骨阳性细胞表达差异。采用ELISA法测量不同月份豚鼠血清中雌二醇的含量，进行统计分析。
结果：
1各组DH豚鼠体重变化随着月龄的增高，豚鼠体重逐渐升高，与前一月龄比较，差异具有统计学意义(P<0.05)。
2不同月龄DH豚鼠膝关节软骨表面大体观察发现：1月龄组豚鼠膝关节软骨面光滑完整；而随着月龄的增长，3月龄时观察可见豚鼠膝关节软骨面晦涩，偶见关节面轻度破溃；6月龄时关节面晦涩加重，破溃面积增大；9月龄时破溃面积明显增大，周围可见较小骨赘形成。
3关节软骨Masson染色形态学观察发现：1月龄组豚鼠关节软骨面完整，软骨层厚，软骨与软骨下骨界限清晰，基质着色均匀，细胞有序排列；3月龄组关节软骨面较完整，软骨略薄，软骨与软骨下骨界限较清晰，基质着色局部不一致，细胞排列出现簇集现象；6月龄组关节软骨面完整性欠佳，软骨更薄浅，软骨与软骨下骨界限模糊，中层部分失染，细胞数量减少，簇集现象较重；9月龄组关节软骨面完整性更差，软骨变薄明显，软骨与软骨下骨界限模糊不清，基质着色不均，软骨细胞变形坏死，且排列紊乱，深层有增殖细胞团生成。
4关节软骨Mankin评分显示：随着DH豚鼠月龄增高，Mankin评分也不断升高，各组之间评分统计均有显著差异，具有统计学意义(P<0.05)。
5扫描电镜结果：1月龄组软骨面光滑，似覆盖一层无定形物质，典型的垄形结构平行，无胶原暴露。3月龄组部分软骨表面无定形物质有所缺失，偶见圆形突起，部分暴露表层胶原纤维，排列较不规则，可见流水冲蚀样改变。6月龄组部分胶原纤维网增粗、断裂、剥脱样改变,圆形突起较3月龄组增多。部分软骨面垄形结构完全消失，表面粗糙，见散在凹塌，凹塌为缺失的表面软骨细胞所在。9月龄组软骨面胶原集合成粗枝状并翻起，裂缝中可见胶原纤维，凹塌样改变增多，软骨表面正常结构消失。
6透射电镜结果：1月龄组分布在表层及深层的软骨细胞大多呈梭形或椭圆形，细胞结构、轮廓清晰，胞质内有丰富的线粒体、高尔基复合体和粗面内质网，且结构清晰。细胞外见纵横交错的胶原纤维，表层胶原纤维多与表面平行，深层多与软骨下骨垂直，不具明显横纹。3月龄组细胞基本清晰，少见不规则形，核膜基本清晰，染色质分布在核周边，部分细胞质结构不清，偶见粗面内质网肿胀。6月龄组细胞较不清晰，不规则形增多，核膜较不清晰，部分细胞质结构不清，可见粗面内质网肿胀，胞质中可见钙结晶体，胶原纤维部分增粗。9月龄组细胞不清晰且不规则，核膜不清晰，胞质结构不清，有大量钙结晶体，粗面内质网肿胀，胶原纤维增粗，出现明显横纹。
7关节软骨免疫组化检测显示：随着DH豚鼠月龄增高，豚鼠软骨中GAG表达1月与3月间表达无明显差异，6月开始起显著降低，比较具有统计学差异（P<0.05)；MMP-3表达3月开始显著升高，各组之间评分统计均有显著差异，具有统计学意义（P<0.05)。
8随着DH豚鼠月龄的升高，血清中雌二醇含量呈增高趋势，1月至3月，3月至6月升高明显，有统计学意义（P<0.05)，6月至9月雌二醇含量变化较平稳，无明显改变（P>0.05)。
结论：雌性DH豚鼠随着月龄的增长，膝关节软骨发生了退行性变，且病变程度岁月龄增长而加重。 DH豚鼠作为一种原发性的OA动物模型，其发生过程与人OA的形成具有一定的相似之处，因此可以利用此模型来探讨OA发生的病因和机制。
第二部分甲状旁腺激素1-34对豚鼠原发性膝骨关节炎潜在的保护作用
目的：探讨早期应用甲状旁腺激素1-34（Parathyroid Hormone (1-34)，PTH(1-34)）干预对DH豚鼠原发性膝骨关节炎模型的保护作用。
方法：48只雌性1月龄DH豚鼠随机分为6组，其中2组为给药组，在3月龄时开始给予PTH(1-34)皮下注射干预（15μg/kg/d，每周5天），分别干预3个月和6个月。在1月龄、3月龄、6月龄、9月龄时处死各组动物，打开膝关节腔后，观察各组豚鼠膝关节软骨面并拍照记录。后腿去皮及肌肉组织，避免损伤膝关节，切取胫骨到股骨之间的关节软骨组织后，70％酒精固定豚鼠股骨，固定72小时后用EDTA-2Na制成脱钙液行脱钙处理，约8周后待钙盐彻底脱失骨组织变软后行常规梯度酒精脱水、二甲苯透明并进行石蜡包埋切片，将厚度约为6-8gm的骨组织石蜡切片行Masson染色，用于关节软骨组织形态学观察，采用Mankin评分方法定量分析关节软骨损伤情况；采用免疫组化方法检测各组大鼠膝关节软骨中II型胶原（Type-II Collagen）、金属基质蛋白酶-13（matrix metalloproteinases-13,MMP-13）、骨硬化蛋白（sclerostin,SOST）表达情况，应用Image pro-Plus6.0软件对免疫组化阳性蛋白表达情况进行积分光密度(integrated optical density, IOD）计算，定量分析各组大鼠膝关节软骨阳性细胞表达差异。
结果：
1不同月龄DH豚鼠膝关节软骨表面大体观察发现：1月龄组豚鼠膝关节软骨面光滑完整；而随着月龄的增长，3月龄时观察可见豚鼠膝关节软骨面晦涩，偶见关节面轻度破溃；6月龄时关节面晦涩加重，破溃面积增大；9月龄时破溃面积明显增大，周围可见较小骨赘形成。6月PTH(1-34)给药组关节面破溃程度较轻，与3月龄组相仿；9月PTH(1-34)给药组关节面破溃程度较相同月龄对照组轻，且小骨赘形成较少。
2关节软骨Masson染色形态学观察发现：1月龄组豚鼠关节软骨面完整，软骨厚，软骨与软骨下骨界限清晰，基质着色均匀一致，细胞排列有序；3月龄组关节软骨面较完整，软骨略薄，软骨与软骨下骨界限较清晰，基质着色局部不一致，细胞排列出现簇集现象；6月龄组关节软骨面完整性欠佳，软骨更薄浅，软骨与软骨下骨界限模糊，中层部分失染，细胞数量减少，簇集现象较重；6月PTH(1-34)给药组关节软骨面较完整，软骨厚度较相同月份对照组厚，基质着色较均匀，入骨细胞形态、排列均有所改善；9月龄组关节软骨面完整性更差，软骨变薄明显，软骨与软骨下骨界限模糊不清，基质着色不均，软骨细胞变形坏死，且排列紊乱，深层有增殖细胞团生成。9月PTH(1-34)给药组无论在软骨面完整性、软骨厚度、细胞形态、排列方面均较相同月龄对照组有改善。
3关节软骨Mankin评分显示：随着DH豚鼠月龄增高，Mankin评分也不断升高，各组之间评分统计均有显著差异，具有统计学意义（P<0.05)。PTH(1-34)能显著降低Mankin评分，各给药组与同期对照组相比均有显著性差异（P<0.05)。
4关节软骨免疫组化检测显示：随着DH豚鼠月龄增高，豚鼠软骨II型胶原表达显著降低，MMP-13与SOST显著升高，各组之间统计均有显著差异，具有统计学意义（P<0.05)。与同月龄对照组比较，PTH(1-34)能显著升高II型胶原表达，降低MMP-13与SOST的表达，各给药组与同期对照组相比均有显著性差异（P<0.05)。
结论：在DH豚鼠原发性骨关节炎动物模型中，早期应用PTH(1-34)皮下注射能够刺激软骨细胞II型胶原合成，提高II型胶原表达，并降低MMP-13与SOST的表达，延缓DH豚鼠原发性膝关节软骨退变。
第三部分甲状旁腺激素1-34对豚鼠原发性膝骨关节炎的作用机制
目的：PTH(1-34)在软骨和骨调控中起重要作用，目前它作为一种治疗OA的潜在药物受到关注。本实验在DH豚鼠原发性骨关节炎动物模型基础上，早期给予PTH(1-34)进行干预，观察PTH(1-34)对豚鼠膝关节软骨及软骨下骨相关因子及骨组织形态结构的影响，初步探讨其作用机制。
方法：48只雌性1月龄DH豚鼠随机分为6组，其中2组为给药组,在3月龄时开始给予PTH(1-34)皮下注射干预（15μg/kg/d，每周5天），分别干预3个月和6个月。在1月龄、3月龄、6月龄、9月龄时处死各组动物。后腿去皮及肌肉组织，避免损伤膝关节，采集的左右胫骨髁软骨下骨行Micro-CT检测，检测之后的远端标本放入4%多聚甲醛中固定72小时以上，用EDTA-2Na制成脱钙液行脱钙处理，约8-10周后骨组织变软待钙盐彻底脱失后行常规梯度酒精脱水、二甲苯透明并行石蜡包埋切片,将厚度约为6-8gm的骨组织石蜡切片分别行免疫组化检测各组豚鼠膝关节软骨及软骨下骨中骨保护素（osteoprotegerin, OPG）、核因子κB受体活化因子配基（receptor activator of nuclear factor-κB ligand, RANKL）、甲状旁腺激素受体（Parathyroid hormone-related peptide,PTH1R）表达情况,并计算软骨及软骨下骨中OPG/RANKL的比值变化。应用Image pro-Plus6.0软件对免疫组化阳性蛋白表达情况行积分光密度(integrated opticaldensity, IOD）计算，定量分析各组大鼠膝关节软骨阳性细胞表达差异。对各组胫骨软骨下骨行Micro-CT检测，计算软骨下骨骨小梁结构数据：骨密度（BMD）、相对体积（bone volume/trabecular volume, BV/TV)、骨小梁厚度(trabecular thickness, Tb.Th)、骨小梁数量（trabecular number,Tb.N)、软骨下骨骨板厚度（Plate thickness, Pl.Th)及结构模型指数（Struc-ture Model Index, SMI），定量分析软骨下骨结构参数。
结果：
1关节软骨免疫组化检测显示：随着DH豚鼠月龄的增高，豚鼠软骨RANKL表达显著升高，OPG、PTH1R表达降低，各组之间统计均有显著差异，具有统计学意义（P<0.05)。与同期对照组比较，PTH(1-34)能显著降低MMP-13、SOST、RANKL的表达，升高OPG、PTH1R的表达，各给药组与同期对照组相比均有显著性差异（P<0.05)。
2软骨下骨免疫组化检测显示：随着DH豚鼠月龄增高，豚鼠软骨下骨OPG、PTH1R表达降低，RANKL表达升高，各组之间统计均有显著差异，具有统计学意义（P<0.05)。与同期对照组比较，PTH(1-34)能显著升高软骨下骨OPG、PTH1R表达，降低RANKL表达，各给药组与同期对照组相比均有显著性差异（P<0.05)。
3DH豚鼠软骨OPG/RANKL比值及软骨下骨OPG/RANKL比值均随月龄增高而降低，各组之间统计均有显著差异，具有统计学意义（P<0.05)。与同期对照组比较，PTH(1-34)能显著升高软骨及软骨下骨OPG/RANKL比值，各给药组与同月龄对照组相比均有显著性差异（P<0.05)。
4Micro-CT检测软骨下骨示：随着月龄的增高，对照组软骨下骨BMD升高，较前一月龄组相比，差异均具有统计学意义（P<0.05），6月给药组较相同月龄对照组BMD升高不明显，差异无统计学意义（P＞0.05），9月给药组较相同月龄对照组BMD升高明显，差异具有统计学意义（P<0.05）。对照组软骨下骨BV/TV在3月龄时升高明显，6月龄和9月龄时无明显变化，给药组较同月龄对照组能显著升高BV/TV值，差异有统计学意义（P<0.05）。Tb.Th及Pl.Th随月龄升高而升高，较前一月龄组比较均有统计学意义（P<0.05），给药组较同月龄对照组能显著降低Tb.Th及Pl.Th值，差异有统计学意义（P<0.05）。对照组SMI在1月龄和3月龄无显著差异，在6月龄和9月龄时降低明显，差异有统计学意义（P<0.05），9月给药组较同月龄对照组能显著升高SMI值，差异有统计学意义（P<0.05）。
结论：早期给予PTH(1-34)干预能通过降低RANKL在软骨及软骨下骨的表达，升高OPG、PTH1R在软骨及软骨下骨的表达，升高OPG/RANKL在软骨及软骨下骨的比值,改善软骨下骨骨小梁微观结构,维持软骨下骨生物力学性能发挥治疗OA的作用。
Part one Experimental study on guinea pigs with spontaneous kneeosteoarthritis
Objective: To investigate the characteristics of Dunkin Hartley guineapigs with spontaneous knee osteoarthritis, and observe age-related changes inarticular cartilage, subchondral bone mineral density and estradiol levels inblood serum in this model over the entire course of osteoarthritis progression,and provide a scientific model of primary osteoarthritis.
Methods: We studied spontaneous knee osteoarthritis in32femaleDunkin Hartley guinea pigs. Animals were randomly sacrificed at1,3,6,9, or12months of age (eight animals at each time point). Knee joints spaces wereopened. Then, cartilage surfaces of femur and tibia were observed andrecorded by digital camera. After disarticulation, femurs were fixed for72hours in70%ethanol and decalcified for6weeks with15%EDTA-2Na(pH7.4, at4°C). Tissues were regularly dehydrated, embedded in paraffin, andcut into6-8um-thick sections. The sections were stained with Masson. Usingwith light microscope (Olympus BX61, Japan), three color digital imageswere recorded to analysis articular cartilage lesions from each section indifferent regions. Mankin’s score system was properly adjusted and applied tomeasure morphological changes of cartilage. The expressions ofglycosaminoglycans and matrix metalloproteinase-3antibody were detectedusing immunohistochemistry method. Positive expression was quantified byoptical density method, using with Image pro-Plus (IPP) software. Bloodsamples were harvested before sacrifice for enzyme-linked immunosorbentassay (ELISA) analysis of estradiol concentration.
Results:
1As the animals’ age increased, they gained significantly more weight. The weight changes in each group were compared, and there weresignificantly different from each group animals(P<0.05).
2The articular cartilage in1-month old animals was smooth and withoutany evidence of degeneration. At3months of age, mild discontinuous fibrosisoccasionally appeared. By6months of age, cartilage ulcerations and matrixloss were obvious and osteophytes had begun to develop when animalsreached9months of age.
3As seen by Masson’s trichrome staining, articular cartilage in1-monthold animals had normal cellularity and extracellular matrix. Early histologicalchanges were observed in3-month old animals, including focal proteoglycanloss and fibrillation. At6months old, animals displayed chondrocytehypertrophy,“cloning” and surface cartilage lesions, and more obviouschondrocyte death/loss. Fibrillation and proteoglycan loss were observed in9-month old animals, and these degenerative changes extended into deeperzones.
4All of the aforementioned changes were semi-quantitatively confirmedby age-dependent increased histological score according to the Mankinscoring system. There was a significant difference of Mankin scores betweenin each groups (P <0.05).
5The cartilage surface of normal group was smooth as if it was coveredwith some amorphous substance, and the stria structure was intact, and nocollagen was exposed. The collagenous fibers on the cartilage surface of3-month group were exposed, and they arranged irregularly as if they wereeroded by running water, and in6month group some collagenous fiber retiabecame coarse, broken, exfoliative and even were turned over. In9monthgroup, there were some scattering on the surface of chondrocyte, collagenousfiber retia became coarser than6month group.
6TEM revealed more details regarding cellular degenerations during OAprogression in this model: more chondrocytes were observed in1-month oldanimals and no apoptotic or necrotic chondrocytes were found; cell nucleiand membranes were integrated and rich in cytoplasmic organoids, chromatin was distributed uniformity, and few abnormal chondrocytes were detected in3-month old animals, with irregular shapes and loss of cytoplasmic organoids.Age-related increase in apoptotic or necrotic chondrocytes were detected, asshown in Figure5, the cavity around the cell became smaller with cellular andnuclear contraction, cytoplasmic organoid loss or disappearance, and loss ofnormal chondrocytes occurred. For each age group, normal and degeneratingcell numbers were calculated, as shown in Figure6, and the percentage ofdegenerating cells increased in an age-related manner.
7We used immunohistochemistry to examine the distribution of MMP-3and GAG expression in knee joint cartilage from guinea pigs from each agegroup. The IOD values in each group showed that expression of GAG wasintense in younger animals, with a significant age-related decrease in GAGexpression beginning from6months of age(P<0.05). In contrast, an increasein MMP-3expression was detected with increasing age of the animals(P<0.05).
8From months1to6, serum estradiol levels increased markedly(P<0.05), before remaining stable with no significant differences between6-month old and9-month old animals.
Conclusion: Age-related articular cartilage degeneration occurred inDunkin Hartley guinea pigs beginning at3months of age in parallel withactivate cellular degeneration and matrix catabolism, while no directlypositive or negative correlation between osteoarthritis progression andestradiol serum level. Dunkin Hartley guinea pigs is a scientific model ofprimary osteoarthritis.
Part two The protential effects of PTH(1-34) on knee joints of DH guineapigs with spontaneous osteoarthritis.
Objective: To investigate the effect of PTH(1-34) on articular cartilagedegeneration in knee joints of DH guinea pigs with spontaneous osteoarthritis.
Methods: Forty-eight1-month-old guinea pigs were divided into sixgroups: four groups were untreated and sacrificed at1,3,6and9months ofage; the other two groups received PTH (1-34)(15ug/kg/day,5days weekly) from3months of age, and were sacrificed at6and9months. Knee jointsspaces were opened. Then, cartilage surfaces of femur and tibia were observedand recorded by digital camera. After disarticulation, femurs were fixed for72hours in70%ethanol and decalcified for6weeks with15%EDTA-2Na(pH7.4, at4°C). Tissues were regularly dehydrated, embedded in paraffin, andcut into6-8um-thick sections. The sections were stained with Masson. Usingwith light microscope (Olympus BX61, Japan), three color digital imageswere recorded to analysis articular cartilage lesions from each section indifferent regions. Mankin’s score system was properly adjusted and applied tomeasure morphological changes of cartilage. The expressions of Type-IICollagen, matrix metalloproteinases-13and sclerostin antibody were detectedusing immunohistochemistry method. Positive expression was quantified byoptical density method, using with Image pro-Plus (IPP) software.
Results:
1The gross morphology at1month of age showed that the guinea pigs’articular surfaces were smooth, with an absence of osteophyte formation in thefemoral condyles and tibial plateaus. By3months of age, mild discontinuousfibrosis could be seen at the femoral condyles and tibial plateaus. The animalsdisplayed greater severity of OA with increasing age,6-month-old guinea pigsshowed obvious cartilage ulceration, and at9months of age, cartilage loss andtypical osteophyte were developed, the degeneration was particularlypronounced in the medial condyle. Although animals in the PTH(1-34)-treated group had rougher surfaces, ulceration and osteophytes, thesewere not as serious as those in the control groups.
2With the Masson stain, the articular cartilage in1-month-old animalshad a smooth surface and normal cellularity, and no abnormalities were notedin the chondrocytes or extracellular matrix. Early histological changes wereobserved in3-month-old animals, including focal proteoglycan loss andfibrillation. With increasing age,6-month-old animals displayed more obviouschondrocyte death/loss, fibrillation and proteoglycan loss, and thesedegenerative changes extended into deeper zone and cell cloning is prominent in9-month-old animals. The cartilage samples in the PTH (1-34)-treatedgroups showed marked differences when compared with the control groups,with slight articular cartilage degeneration.
3The Mankin score reflected pathological changes to the cartilage. Forthe age-matched groups, the Mankin scores increased with age (P <0.05).PTH (1-34) treatment reduced the Mankin score at6and9months of ageseparately, in contrast to the control group (P <0.05).
4The analysis found a significant decrease in type-II collagen expressionand an increase in the expression of MMP-13and SOST in the control groupsas the age of the animals increased (P <0.05). Guinea pigs treated with PTH(1-34) showed a increase in type-II collagen expression, and a decrease inMMP-13and SOST expression at6and9months of age respectively, ascompared with the control groups(P <0.05).
Conclusion: Early intervention with PTH (1-34) stimulated typeⅡc ollagen synthesis and inhibited expressions of SOST and MMP-13, andprevention of further aggravation of cartilage degradation in a naturallyoccurring OA modelPart three The mechanism of PTH(1-34) on knee joints of DH guinea pigs
with spontaneous osteoarthritis.
Objective: PTH (1-34) was a potent bone-turnover agent and appliedinto treatments of bone metabolic diseases. This study aimed to study themechanism of parathyroid hormone (1-34) on the micro-structure ofsubchondral bone, and protect against cartilage degradation in an animalmodel of naturally occurring OA.
Methods: Forty-eight1-month-old guinea pigs were divided into sixgroups: four groups were untreated and sacrificed at1,3,6and9months ofage; the other two groups received PTH (1-34)(15ug/kg/day,5days weekly)from3months of age, and were sacrificed at6and9months. Knee jointsspaces were opened. Then, cartilage surfaces of femur and tibia were observedand recorded by digital camera. After soft tissues removed, femurs wereplaced under the probe and followed same position. The distal femur was scanned by micro computed tomography(Micro-CT). Static parameters anddynamic parameters, including bone mineral density(BMD), bone volume(BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), Platethickness(Pl.Th) and Structure Model Index(SMI). After Micro-CT test,femurs were fixed for72hours in70%ethanol and decalcified for6weekswith15%EDTA-2Na (pH7.4, at4°C). Tissues were regularly dehydrated,embedded in paraffin, and cut into6-8um-thick sections. The expressions ofosteoprotegerin, receptor activator of nuclear factor-κB ligand and Parathyroidhormone-related peptide antibody in cartilage and subchondral bone weredetected using immunohistochemistry method. Positive expression wasquantified by optical density method, using with Image pro-Plus (IPP)software.
Results:
1The expressions of OPG, RANKL and PTH1R in cartilage: Theanalysis found a significant decrease in OPG and PTH1R expression and anincrease in the expression of RANKL in the control groups as the age of theanimals increased (P <0.05). Guinea pigs treated with PTH (1-34) showed aincrease in OPG and PTH1R expression, and a decrease in RANKLexpression at6and9months of age respectively, as compared with the controlgroups (P <0.05).
2The expressions of OPG, RANKL and PTH1R in subchondral bone:The analysis found a significant decrease in OPG and PTH1R expression andan increase in the expression of RANKL in the control groups as the age of theanimals increased (P <0.05). Guinea pigs treated with PTH (1-34) showed aincrease in OPG and PTH1R expression, and a decrease in RANKLexpression at6and9months of age respectively, as compared with the controlgroups (P <0.05).
3With increasing age, the control group had a gradually decreasing ratioof OPG/RANKL in both the cartilage and subchondral bone. Compared withthe control animals, increases in the OPG/RANKL ratio for cartilage andsubchondral bone in the PTH (1-34)-treated groups (P <0.05).
4Bone mineral density increased with age and reached constant levelsafter6months. PTH (1-34)-treated animals showed higher values comparedwith those of the control groups, with a significant difference between animalsaged9months. Tb.Th increased with age, and BV/TV was highest at3months,and then remained constant. With advanced age, the SMI were markedlydecreased. Compared with the control groups, PTH (1-34) treatment increasedBV/TV at both6months and9months, and SMI at9months of age. From1to6months of age, subchondral plate thickness of control group wereincreased markedly (P<0.05), and remained fairly constant thereafter. ThoughPTH treated group has slightly increased subchondral plate thickness, there isno difference when compared with control group.
Conclusion: PTH (1-34) can increase OPG and PTH1R expression,OPG/RANKL ratio in both cartilage and subchondral bone, decrease RANKLexpression in both cartilage and subchondral bone, and retard the deteriorationof subchondral trabecular bone.
目的：探讨Dunkin Hartley(DH)豚鼠原发性骨性关节炎的发生及其特点，对不同月龄的DH豚鼠不同原发osteoarthritis(OA)阶段关节软骨大体改变、关节软骨结构、组织化学成分的变化进行动态观察，了解软骨组织病理变化，为后续试验提供科学可用的OA原发模型。
方法：32只雌性1月龄DH豚鼠随机分为4组，各组动物分别在1月龄、3月龄、6月龄、9月龄时处死，打开膝关节腔后，观察各组豚鼠膝关节软骨面并拍照记录。后腿去皮及肌肉组织，避免损伤膝关节，切取胫骨到股骨之间的关节软骨组织后，70％酒精固定豚鼠股骨，固定72小时后用EDTA-2Na制成脱钙液行脱钙处理，约8周后骨组织变软，钙盐彻底脱失后行常规梯度酒精脱水、二甲苯透明并进行石蜡包埋切片，将厚度约为6-8gm的骨组织石蜡切片行Masson染色，用于关节软骨组织形态学观察，采用Mankin评分方法定量分析关节软骨损伤情况；另取部分关节软骨标本立即按程序制成电镜标本行扫描电镜观察软骨面改变情况及透射电镜观察软骨细胞改变情况。采用免疫组化方法检测各组大鼠膝关节软骨中蛋白多糖（glycosaminoglycans, GAG）、基质金属蛋白酶-3(matrixmetalloproteinase-3, MMP-3）表达情况，应用Image pro-Plus6.0软件对免疫组化阳性蛋白表达情况进行积分光密度(integrated optical density, IOD）计算，定量分析各组大鼠膝关节软骨阳性细胞表达差异。采用ELISA法测量不同月份豚鼠血清中雌二醇的含量，进行统计分析。
结果：
1各组DH豚鼠体重变化随着月龄的增高，豚鼠体重逐渐升高，与前一月龄比较，差异具有统计学意义(P<0.05)。
2不同月龄DH豚鼠膝关节软骨表面大体观察发现：1月龄组豚鼠膝关节软骨面光滑完整；而随着月龄的增长，3月龄时观察可见豚鼠膝关节软骨面晦涩，偶见关节面轻度破溃；6月龄时关节面晦涩加重，破溃面积增大；9月龄时破溃面积明显增大，周围可见较小骨赘形成。
3关节软骨Masson染色形态学观察发现：1月龄组豚鼠关节软骨面完整，软骨层厚，软骨与软骨下骨界限清晰，基质着色均匀，细胞有序排列；3月龄组关节软骨面较完整，软骨略薄，软骨与软骨下骨界限较清晰，基质着色局部不一致，细胞排列出现簇集现象；6月龄组关节软骨面完整性欠佳，软骨更薄浅，软骨与软骨下骨界限模糊，中层部分失染，细胞数量减少，簇集现象较重；9月龄组关节软骨面完整性更差，软骨变薄明显，软骨与软骨下骨界限模糊不清，基质着色不均，软骨细胞变形坏死，且排列紊乱，深层有增殖细胞团生成。
4关节软骨Mankin评分显示：随着DH豚鼠月龄增高，Mankin评分也不断升高，各组之间评分统计均有显著差异，具有统计学意义(P<0.05)。
5扫描电镜结果：1月龄组软骨面光滑，似覆盖一层无定形物质，典型的垄形结构平行，无胶原暴露。3月龄组部分软骨表面无定形物质有所缺失，偶见圆形突起，部分暴露表层胶原纤维，排列较不规则，可见流水冲蚀样改变。6月龄组部分胶原纤维网增粗、断裂、剥脱样改变,圆形突起较3月龄组增多。部分软骨面垄形结构完全消失，表面粗糙，见散在凹塌，凹塌为缺失的表面软骨细胞所在。9月龄组软骨面胶原集合成粗枝状并翻起，裂缝中可见胶原纤维，凹塌样改变增多，软骨表面正常结构消失。
6透射电镜结果：1月龄组分布在表层及深层的软骨细胞大多呈梭形或椭圆形，细胞结构、轮廓清晰，胞质内有丰富的线粒体、高尔基复合体和粗面内质网，且结构清晰。细胞外见纵横交错的胶原纤维，表层胶原纤维多与表面平行，深层多与软骨下骨垂直，不具明显横纹。3月龄组细胞基本清晰，少见不规则形，核膜基本清晰，染色质分布在核周边，部分细胞质结构不清，偶见粗面内质网肿胀。6月龄组细胞较不清晰，不规则形增多，核膜较不清晰，部分细胞质结构不清，可见粗面内质网肿胀，胞质中可见钙结晶体，胶原纤维部分增粗。9月龄组细胞不清晰且不规则，核膜不清晰，胞质结构不清，有大量钙结晶体，粗面内质网肿胀，胶原纤维增粗，出现明显横纹。
7关节软骨免疫组化检测显示：随着DH豚鼠月龄增高，豚鼠软骨中GAG表达1月与3月间表达无明显差异，6月开始起显著降低，比较具有统计学差异（P<0.05)；MMP-3表达3月开始显著升高，各组之间评分统计均有显著差异，具有统计学意义（P<0.05)。
8随着DH豚鼠月龄的升高，血清中雌二醇含量呈增高趋势，1月至3月，3月至6月升高明显，有统计学意义（P<0.05)，6月至9月雌二醇含量变化较平稳，无明显改变（P>0.05)。
结论：雌性DH豚鼠随着月龄的增长，膝关节软骨发生了退行性变，且病变程度岁月龄增长而加重。 DH豚鼠作为一种原发性的OA动物模型，其发生过程与人OA的形成具有一定的相似之处，因此可以利用此模型来探讨OA发生的病因和机制。
第二部分甲状旁腺激素1-34对豚鼠原发性膝骨关节炎潜在的保护作用
目的：探讨早期应用甲状旁腺激素1-34（Parathyroid Hormone (1-34)，PTH(1-34)）干预对DH豚鼠原发性膝骨关节炎模型的保护作用。
方法：48只雌性1月龄DH豚鼠随机分为6组，其中2组为给药组，在3月龄时开始给予PTH(1-34)皮下注射干预（15μg/kg/d，每周5天），分别干预3个月和6个月。在1月龄、3月龄、6月龄、9月龄时处死各组动物，打开膝关节腔后，观察各组豚鼠膝关节软骨面并拍照记录。后腿去皮及肌肉组织，避免损伤膝关节，切取胫骨到股骨之间的关节软骨组织后，70％酒精固定豚鼠股骨，固定72小时后用EDTA-2Na制成脱钙液行脱钙处理，约8周后待钙盐彻底脱失骨组织变软后行常规梯度酒精脱水、二甲苯透明并进行石蜡包埋切片，将厚度约为6-8gm的骨组织石蜡切片行Masson染色，用于关节软骨组织形态学观察，采用Mankin评分方法定量分析关节软骨损伤情况；采用免疫组化方法检测各组大鼠膝关节软骨中II型胶原（Type-II Collagen）、金属基质蛋白酶-13（matrix metalloproteinases-13,MMP-13）、骨硬化蛋白（sclerostin,SOST）表达情况，应用Image pro-Plus6.0软件对免疫组化阳性蛋白表达情况进行积分光密度(integrated optical density, IOD）计算，定量分析各组大鼠膝关节软骨阳性细胞表达差异。
结果：
1不同月龄DH豚鼠膝关节软骨表面大体观察发现：1月龄组豚鼠膝关节软骨面光滑完整；而随着月龄的增长，3月龄时观察可见豚鼠膝关节软骨面晦涩，偶见关节面轻度破溃；6月龄时关节面晦涩加重，破溃面积增大；9月龄时破溃面积明显增大，周围可见较小骨赘形成。6月PTH(1-34)给药组关节面破溃程度较轻，与3月龄组相仿；9月PTH(1-34)给药组关节面破溃程度较相同月龄对照组轻，且小骨赘形成较少。
2关节软骨Masson染色形态学观察发现：1月龄组豚鼠关节软骨面完整，软骨厚，软骨与软骨下骨界限清晰，基质着色均匀一致，细胞排列有序；3月龄组关节软骨面较完整，软骨略薄，软骨与软骨下骨界限较清晰，基质着色局部不一致，细胞排列出现簇集现象；6月龄组关节软骨面完整性欠佳，软骨更薄浅，软骨与软骨下骨界限模糊，中层部分失染，细胞数量减少，簇集现象较重；6月PTH(1-34)给药组关节软骨面较完整，软骨厚度较相同月份对照组厚，基质着色较均匀，入骨细胞形态、排列均有所改善；9月龄组关节软骨面完整性更差，软骨变薄明显，软骨与软骨下骨界限模糊不清，基质着色不均，软骨细胞变形坏死，且排列紊乱，深层有增殖细胞团生成。9月PTH(1-34)给药组无论在软骨面完整性、软骨厚度、细胞形态、排列方面均较相同月龄对照组有改善。
3关节软骨Mankin评分显示：随着DH豚鼠月龄增高，Mankin评分也不断升高，各组之间评分统计均有显著差异，具有统计学意义（P<0.05)。PTH(1-34)能显著降低Mankin评分，各给药组与同期对照组相比均有显著性差异（P<0.05)。
4关节软骨免疫组化检测显示：随着DH豚鼠月龄增高，豚鼠软骨II型胶原表达显著降低，MMP-13与SOST显著升高，各组之间统计均有显著差异，具有统计学意义（P<0.05)。与同月龄对照组比较，PTH(1-34)能显著升高II型胶原表达，降低MMP-13与SOST的表达，各给药组与同期对照组相比均有显著性差异（P<0.05)。
结论：在DH豚鼠原发性骨关节炎动物模型中，早期应用PTH(1-34)皮下注射能够刺激软骨细胞II型胶原合成，提高II型胶原表达，并降低MMP-13与SOST的表达，延缓DH豚鼠原发性膝关节软骨退变。
第三部分甲状旁腺激素1-34对豚鼠原发性膝骨关节炎的作用机制
目的：PTH(1-34)在软骨和骨调控中起重要作用，目前它作为一种治疗OA的潜在药物受到关注。本实验在DH豚鼠原发性骨关节炎动物模型基础上，早期给予PTH(1-34)进行干预，观察PTH(1-34)对豚鼠膝关节软骨及软骨下骨相关因子及骨组织形态结构的影响，初步探讨其作用机制。
方法：48只雌性1月龄DH豚鼠随机分为6组，其中2组为给药组,在3月龄时开始给予PTH(1-34)皮下注射干预（15μg/kg/d，每周5天），分别干预3个月和6个月。在1月龄、3月龄、6月龄、9月龄时处死各组动物。后腿去皮及肌肉组织，避免损伤膝关节，采集的左右胫骨髁软骨下骨行Micro-CT检测，检测之后的远端标本放入4%多聚甲醛中固定72小时以上，用EDTA-2Na制成脱钙液行脱钙处理，约8-10周后骨组织变软待钙盐彻底脱失后行常规梯度酒精脱水、二甲苯透明并行石蜡包埋切片,将厚度约为6-8gm的骨组织石蜡切片分别行免疫组化检测各组豚鼠膝关节软骨及软骨下骨中骨保护素（osteoprotegerin, OPG）、核因子κB受体活化因子配基（receptor activator of nuclear factor-κB ligand, RANKL）、甲状旁腺激素受体（Parathyroid hormone-related peptide,PTH1R）表达情况,并计算软骨及软骨下骨中OPG/RANKL的比值变化。应用Image pro-Plus6.0软件对免疫组化阳性蛋白表达情况行积分光密度(integrated opticaldensity, IOD）计算，定量分析各组大鼠膝关节软骨阳性细胞表达差异。对各组胫骨软骨下骨行Micro-CT检测，计算软骨下骨骨小梁结构数据：骨密度（BMD）、相对体积（bone volume/trabecular volume, BV/TV)、骨小梁厚度(trabecular thickness, Tb.Th)、骨小梁数量（trabecular number,Tb.N)、软骨下骨骨板厚度（Plate thickness, Pl.Th)及结构模型指数（Struc-ture Model Index, SMI），定量分析软骨下骨结构参数。
结果：
1关节软骨免疫组化检测显示：随着DH豚鼠月龄的增高，豚鼠软骨RANKL表达显著升高，OPG、PTH1R表达降低，各组之间统计均有显著差异，具有统计学意义（P<0.05)。与同期对照组比较，PTH(1-34)能显著降低MMP-13、SOST、RANKL的表达，升高OPG、PTH1R的表达，各给药组与同期对照组相比均有显著性差异（P<0.05)。
2软骨下骨免疫组化检测显示：随着DH豚鼠月龄增高，豚鼠软骨下骨OPG、PTH1R表达降低，RANKL表达升高，各组之间统计均有显著差异，具有统计学意义（P<0.05)。与同期对照组比较，PTH(1-34)能显著升高软骨下骨OPG、PTH1R表达，降低RANKL表达，各给药组与同期对照组相比均有显著性差异（P<0.05)。
3DH豚鼠软骨OPG/RANKL比值及软骨下骨OPG/RANKL比值均随月龄增高而降低，各组之间统计均有显著差异，具有统计学意义（P<0.05)。与同期对照组比较，PTH(1-34)能显著升高软骨及软骨下骨OPG/RANKL比值，各给药组与同月龄对照组相比均有显著性差异（P<0.05)。
4Micro-CT检测软骨下骨示：随着月龄的增高，对照组软骨下骨BMD升高，较前一月龄组相比，差异均具有统计学意义（P<0.05），6月给药组较相同月龄对照组BMD升高不明显，差异无统计学意义（P＞0.05），9月给药组较相同月龄对照组BMD升高明显，差异具有统计学意义（P<0.05）。对照组软骨下骨BV/TV在3月龄时升高明显，6月龄和9月龄时无明显变化，给药组较同月龄对照组能显著升高BV/TV值，差异有统计学意义（P<0.05）。Tb.Th及Pl.Th随月龄升高而升高，较前一月龄组比较均有统计学意义（P<0.05），给药组较同月龄对照组能显著降低Tb.Th及Pl.Th值，差异有统计学意义（P<0.05）。对照组SMI在1月龄和3月龄无显著差异，在6月龄和9月龄时降低明显，差异有统计学意义（P<0.05），9月给药组较同月龄对照组能显著升高SMI值，差异有统计学意义（P<0.05）。
结论：早期给予PTH(1-34)干预能通过降低RANKL在软骨及软骨下骨的表达，升高OPG、PTH1R在软骨及软骨下骨的表达，升高OPG/RANKL在软骨及软骨下骨的比值,改善软骨下骨骨小梁微观结构,维持软骨下骨生物力学性能发挥治疗OA的作用。
Part one Experimental study on guinea pigs with spontaneous kneeosteoarthritis
Objective: To investigate the characteristics of Dunkin Hartley guineapigs with spontaneous knee osteoarthritis, and observe age-related changes inarticular cartilage, subchondral bone mineral density and estradiol levels inblood serum in this model over the entire course of osteoarthritis progression,and provide a scientific model of primary osteoarthritis.
Methods: We studied spontaneous knee osteoarthritis in32femaleDunkin Hartley guinea pigs. Animals were randomly sacrificed at1,3,6,9, or12months of age (eight animals at each time point). Knee joints spaces wereopened. Then, cartilage surfaces of femur and tibia were observed andrecorded by digital camera. After disarticulation, femurs were fixed for72hours in70%ethanol and decalcified for6weeks with15%EDTA-2Na(pH7.4, at4°C). Tissues were regularly dehydrated, embedded in paraffin, andcut into6-8um-thick sections. The sections were stained with Masson. Usingwith light microscope (Olympus BX61, Japan), three color digital imageswere recorded to analysis articular cartilage lesions from each section indifferent regions. Mankin’s score system was properly adjusted and applied tomeasure morphological changes of cartilage. The expressions ofglycosaminoglycans and matrix metalloproteinase-3antibody were detectedusing immunohistochemistry method. Positive expression was quantified byoptical density method, using with Image pro-Plus (IPP) software. Bloodsamples were harvested before sacrifice for enzyme-linked immunosorbentassay (ELISA) analysis of estradiol concentration.
Results:
1As the animals’ age increased, they gained significantly more weight. The weight changes in each group were compared, and there weresignificantly different from each group animals(P<0.05).
2The articular cartilage in1-month old animals was smooth and withoutany evidence of degeneration. At3months of age, mild discontinuous fibrosisoccasionally appeared. By6months of age, cartilage ulcerations and matrixloss were obvious and osteophytes had begun to develop when animalsreached9months of age.
3As seen by Masson’s trichrome staining, articular cartilage in1-monthold animals had normal cellularity and extracellular matrix. Early histologicalchanges were observed in3-month old animals, including focal proteoglycanloss and fibrillation. At6months old, animals displayed chondrocytehypertrophy,“cloning” and surface cartilage lesions, and more obviouschondrocyte death/loss. Fibrillation and proteoglycan loss were observed in9-month old animals, and these degenerative changes extended into deeperzones.
4All of the aforementioned changes were semi-quantitatively confirmedby age-dependent increased histological score according to the Mankinscoring system. There was a significant difference of Mankin scores betweenin each groups (P <0.05).
5The cartilage surface of normal group was smooth as if it was coveredwith some amorphous substance, and the stria structure was intact, and nocollagen was exposed. The collagenous fibers on the cartilage surface of3-month group were exposed, and they arranged irregularly as if they wereeroded by running water, and in6month group some collagenous fiber retiabecame coarse, broken, exfoliative and even were turned over. In9monthgroup, there were some scattering on the surface of chondrocyte, collagenousfiber retia became coarser than6month group.
6TEM revealed more details regarding cellular degenerations during OAprogression in this model: more chondrocytes were observed in1-month oldanimals and no apoptotic or necrotic chondrocytes were found; cell nucleiand membranes were integrated and rich in cytoplasmic organoids, chromatin was distributed uniformity, and few abnormal chondrocytes were detected in3-month old animals, with irregular shapes and loss of cytoplasmic organoids.Age-related increase in apoptotic or necrotic chondrocytes were detected, asshown in Figure5, the cavity around the cell became smaller with cellular andnuclear contraction, cytoplasmic organoid loss or disappearance, and loss ofnormal chondrocytes occurred. For each age group, normal and degeneratingcell numbers were calculated, as shown in Figure6, and the percentage ofdegenerating cells increased in an age-related manner.
7We used immunohistochemistry to examine the distribution of MMP-3and GAG expression in knee joint cartilage from guinea pigs from each agegroup. The IOD values in each group showed that expression of GAG wasintense in younger animals, with a significant age-related decrease in GAGexpression beginning from6months of age(P<0.05). In contrast, an increasein MMP-3expression was detected with increasing age of the animals(P<0.05).
8From months1to6, serum estradiol levels increased markedly(P<0.05), before remaining stable with no significant differences between6-month old and9-month old animals.
Conclusion: Age-related articular cartilage degeneration occurred inDunkin Hartley guinea pigs beginning at3months of age in parallel withactivate cellular degeneration and matrix catabolism, while no directlypositive or negative correlation between osteoarthritis progression andestradiol serum level. Dunkin Hartley guinea pigs is a scientific model ofprimary osteoarthritis.
Part two The protential effects of PTH(1-34) on knee joints of DH guineapigs with spontaneous osteoarthritis.
Objective: To investigate the effect of PTH(1-34) on articular cartilagedegeneration in knee joints of DH guinea pigs with spontaneous osteoarthritis.
Methods: Forty-eight1-month-old guinea pigs were divided into sixgroups: four groups were untreated and sacrificed at1,3,6and9months ofage; the other two groups received PTH (1-34)(15ug/kg/day,5days weekly) from3months of age, and were sacrificed at6and9months. Knee jointsspaces were opened. Then, cartilage surfaces of femur and tibia were observedand recorded by digital camera. After disarticulation, femurs were fixed for72hours in70%ethanol and decalcified for6weeks with15%EDTA-2Na(pH7.4, at4°C). Tissues were regularly dehydrated, embedded in paraffin, andcut into6-8um-thick sections. The sections were stained with Masson. Usingwith light microscope (Olympus BX61, Japan), three color digital imageswere recorded to analysis articular cartilage lesions from each section indifferent regions. Mankin’s score system was properly adjusted and applied tomeasure morphological changes of cartilage. The expressions of Type-IICollagen, matrix metalloproteinases-13and sclerostin antibody were detectedusing immunohistochemistry method. Positive expression was quantified byoptical density method, using with Image pro-Plus (IPP) software.
Results:
1The gross morphology at1month of age showed that the guinea pigs’articular surfaces were smooth, with an absence of osteophyte formation in thefemoral condyles and tibial plateaus. By3months of age, mild discontinuousfibrosis could be seen at the femoral condyles and tibial plateaus. The animalsdisplayed greater severity of OA with increasing age,6-month-old guinea pigsshowed obvious cartilage ulceration, and at9months of age, cartilage loss andtypical osteophyte were developed, the degeneration was particularlypronounced in the medial condyle. Although animals in the PTH(1-34)-treated group had rougher surfaces, ulceration and osteophytes, thesewere not as serious as those in the control groups.
2With the Masson stain, the articular cartilage in1-month-old animalshad a smooth surface and normal cellularity, and no abnormalities were notedin the chondrocytes or extracellular matrix. Early histological changes wereobserved in3-month-old animals, including focal proteoglycan loss andfibrillation. With increasing age,6-month-old animals displayed more obviouschondrocyte death/loss, fibrillation and proteoglycan loss, and thesedegenerative changes extended into deeper zone and cell cloning is prominent in9-month-old animals. The cartilage samples in the PTH (1-34)-treatedgroups showed marked differences when compared with the control groups,with slight articular cartilage degeneration.
3The Mankin score reflected pathological changes to the cartilage. Forthe age-matched groups, the Mankin scores increased with age (P <0.05).PTH (1-34) treatment reduced the Mankin score at6and9months of ageseparately, in contrast to the control group (P <0.05).
4The analysis found a significant decrease in type-II collagen expressionand an increase in the expression of MMP-13and SOST in the control groupsas the age of the animals increased (P <0.05). Guinea pigs treated with PTH(1-34) showed a increase in type-II collagen expression, and a decrease inMMP-13and SOST expression at6and9months of age respectively, ascompared with the control groups(P <0.05).
Conclusion: Early intervention with PTH (1-34) stimulated typeⅡc ollagen synthesis and inhibited expressions of SOST and MMP-13, andprevention of further aggravation of cartilage degradation in a naturallyoccurring OA modelPart three The mechanism of PTH(1-34) on knee joints of DH guinea pigs
with spontaneous osteoarthritis.
Objective: PTH (1-34) was a potent bone-turnover agent and appliedinto treatments of bone metabolic diseases. This study aimed to study themechanism of parathyroid hormone (1-34) on the micro-structure ofsubchondral bone, and protect against cartilage degradation in an animalmodel of naturally occurring OA.
Methods: Forty-eight1-month-old guinea pigs were divided into sixgroups: four groups were untreated and sacrificed at1,3,6and9months ofage; the other two groups received PTH (1-34)(15ug/kg/day,5days weekly)from3months of age, and were sacrificed at6and9months. Knee jointsspaces were opened. Then, cartilage surfaces of femur and tibia were observedand recorded by digital camera. After soft tissues removed, femurs wereplaced under the probe and followed same position. The distal femur was scanned by micro computed tomography(Micro-CT). Static parameters anddynamic parameters, including bone mineral density(BMD), bone volume(BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), Platethickness(Pl.Th) and Structure Model Index(SMI). After Micro-CT test,femurs were fixed for72hours in70%ethanol and decalcified for6weekswith15%EDTA-2Na (pH7.4, at4°C). Tissues were regularly dehydrated,embedded in paraffin, and cut into6-8um-thick sections. The expressions ofosteoprotegerin, receptor activator of nuclear factor-κB ligand and Parathyroidhormone-related peptide antibody in cartilage and subchondral bone weredetected using immunohistochemistry method. Positive expression wasquantified by optical density method, using with Image pro-Plus (IPP)software.
Results:
1The expressions of OPG, RANKL and PTH1R in cartilage: Theanalysis found a significant decrease in OPG and PTH1R expression and anincrease in the expression of RANKL in the control groups as the age of theanimals increased (P <0.05). Guinea pigs treated with PTH (1-34) showed aincrease in OPG and PTH1R expression, and a decrease in RANKLexpression at6and9months of age respectively, as compared with the controlgroups (P <0.05).
2The expressions of OPG, RANKL and PTH1R in subchondral bone:The analysis found a significant decrease in OPG and PTH1R expression andan increase in the expression of RANKL in the control groups as the age of theanimals increased (P <0.05). Guinea pigs treated with PTH (1-34) showed aincrease in OPG and PTH1R expression, and a decrease in RANKLexpression at6and9months of age respectively, as compared with the controlgroups (P <0.05).
3With increasing age, the control group had a gradually decreasing ratioof OPG/RANKL in both the cartilage and subchondral bone. Compared withthe control animals, increases in the OPG/RANKL ratio for cartilage andsubchondral bone in the PTH (1-34)-treated groups (P <0.05).
4Bone mineral density increased with age and reached constant levelsafter6months. PTH (1-34)-treated animals showed higher values comparedwith those of the control groups, with a significant difference between animalsaged9months. Tb.Th increased with age, and BV/TV was highest at3months,and then remained constant. With advanced age, the SMI were markedlydecreased. Compared with the control groups, PTH (1-34) treatment increasedBV/TV at both6months and9months, and SMI at9months of age. From1to6months of age, subchondral plate thickness of control group wereincreased markedly (P<0.05), and remained fairly constant thereafter. ThoughPTH treated group has slightly increased subchondral plate thickness, there isno difference when compared with control group.
Conclusion: PTH (1-34) can increase OPG and PTH1R expression,OPG/RANKL ratio in both cartilage and subchondral bone, decrease RANKLexpression in both cartilage and subchondral bone, and retard the deteriorationof subchondral trabecular bone.
引文
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