不同强度跑台运动对大鼠软骨下骨结构及成分影响的研究

发布时间：2018-05-15 03:22

本文选题：跑台运动 + 大鼠　；参考：《南方医科大学》2015年硕士论文

【摘要】：研究背景骨性关节炎是一种退行性关节疾病,以渐进性的关节损伤为主要特征,最终往往导致患者关节疼痛和残疾,极大地降低患者生活质量。骨性关节炎的病理过程很复杂,过去的研究主要集中在软骨的退变方面,最近软骨下骨的作用得到了越来越多的重视。事实上,关节软骨与其相邻的软骨下骨是紧密耦合的,作为一个单一的功能性单元共同作用。因此目前很多的研究关注软骨下骨在骨性关节炎进程中的变化,以及在骨性关节炎治疗中以此结构为靶向的必要性和有效性。骨性关节炎是一种多因素疾病。在临床中,不同原因所致的骨性关节炎,其病理过程不尽相同。为了明确软骨下骨在骨性关节炎发展过程中的作用,以往大量的研究采用不同的动物模型观察骨性关节炎发生和发展过程中软骨下骨的变化,结果显示不同类型动物模型中软骨下骨变化不尽相同,甚至会产生完全相反的变化。尽管软骨下骨的变化不尽相同,但最终都会导致骨性关节炎的发生,这些结果提示：软骨下骨可能存在一种稳态的结构,任何破坏这种稳态的行为都会导致软骨的损伤,最终导致骨性关节炎的发生。机械负荷被认为是肌肉骨骼系统中骨和软骨内稳态的一种调节器。以往的证据表明骨和软骨都能对机械负荷具有强度依赖性。跑步运动是一种非常常见的负重运动形式,大量的人体及动物试验探讨了跑步运动对骨的影响,但结论并不一致,运动强度被认为是导致这种不一致结果的重要因素。我们前期的动物实验发现：低强度到中等强度的跑步运动能维持软骨的内稳态,而高强度的运动则会导致软骨退化。然而跑步对软骨下骨的影响,以及与跑步强度的相关性目前尚不明确,因此本实验的第一个目的是探讨不同强度的跑台运动对大鼠软骨下骨显微结构的影响。本部分将在前期探讨不同强度跑步对大鼠关节软骨影响研究的基础上,采用Micro-CT进一步探讨不同强度跑步运动后大鼠胫骨平台软骨下骨(包括软骨下骨板和软骨下骨松质骨)的超微结构,并了解不同跑步强度情况下软骨下骨和软骨变化的相关性。以往很多的研究探讨了不同情况下软骨下骨结构和力学性能的改变,以及其对关节软骨的影响。目前主要是通过Micro-CT扫描骨小梁三维图像来直接观察其显微结构,并定量提供与分析三维结构的参数来对软骨下骨进行研究。Micro-CT对软骨下骨的研究包括对骨的量的分析,即骨密度,同时还包括对骨的质的分析,即骨小梁的空间与形态结构,如骨小梁间隙(trabecularseparation, Tb.Sp);骨体积分数(BV/TV),即骨小梁的体积(bone volume, BV)与样本的总体积(total volume, TV)之比；骨小梁厚度(trabecular thickness, Tb.Th);软骨下骨板孔隙率(porosity);以及骨小梁各向异性的程度(Degree of Anisotropy, DA)等。尽管采用Micro-CT能反映软骨下骨超微结构的变化,但软骨下骨成分如何变化目前尚无报道。拉曼光谱分析法(Raman spectroscopy)是以印度科学家拉曼的名字命名的一项检测材料分子成分的技术。它根据拉曼散射效应原理,对与入射光频率不同的散射光谱进行分析以得到分子振动、转动等方面信息,以获取有机物质和无机样本分子成分的信息,它的特点是能通过微创和无损伤的方法对样品进行定性和定量的分析。它被认为能有效的评估样本在分子水平的信息,并能以最小的损伤和非侵入的方法应用于一些生物样品的检测中,比如检测尿液中葡萄糖含量；体外对人血液中丙肝病毒的检测；颈动脉和冠状动脉粥样硬化；生物材料的骨诱导；一些骨疾病的检测；骨康复过程中骨合成和整合的检测以及对骨组织微观结构成分的检测。以往很多研究都提到应用拉曼光谱定量骨矿化和骨基质来评估骨质量,拉曼光谱也被不少作者证实能作为一种重要的检测手段对骨组织成分进行研究。本研究的第二部分将采用拉曼光谱技术探讨不同强度跑步运动后大鼠软骨下骨成分的变化,从组织成分的角度进-步了解不同跑步强度和软骨下骨、软骨变化之间的相关性。研究目的1.探讨不同强度的跑台运动对大鼠软骨下骨显微结构的影响。2.探讨不同强度跑步运动对大鼠软骨下骨成分和力学特性的影响。研究方法1、实验动物和运动方案24只180-220gSD大鼠随机分成四组,不运动组(sedentary control、低强度运动组(low-intensity running group, LIR)、中强度运动组(moderate-intensity running group, MIR)和高强度运动组(high-intensity running group, HIR) 。以不运动组作为对照,运动组(包括低强度运动组、中强度运动组和高强度运动组)动物分别进行8周强度不同的跑台运动。2.标本处理8周跑台运动结束后,使用0.3%戊巴比妥钠腹腔麻醉后处死,截取每个实验大鼠双侧胫骨并去除软组织,右侧胫骨近端行Micro-CT检测；使用硬组织切片机(EXAKT 3000 CP Band System, Norderstedt,德国)将左侧胫骨近端标本沿矢状面垂直切分成外侧和内侧二部分。每部分分别在冠状面3等分点处继续沿矢状面切开,将之分为3小部分,将中间部分标本放入-80℃冰箱中冻存,具体如图2.1所示。对大鼠左侧胫骨关节软骨的外侧部分和内侧部分分别行拉曼光谱分析,检测前先将标本放入4℃生理盐水中浸泡12h。拉曼光谱检测结束后,将进行过拉曼光谱检测的标本行硬组织切除术后,再进行微硬度检测。3、检测指标3.1 Micro-CT检测：对于软骨下骨板进行分析,承载区域面积1.04×1.04平方毫米作为感兴趣的区域(region of interest, ROI)(图1.2),然后使用CT.vol软件分析和计算,具体分析参数包括：骨密度、孔隙度和软骨下骨板厚度。对软骨下骨松质骨的的分析,通过ROI软件选中一个大小为1.04×1.04×0.52mm3的骨小梁长方体(图1.4)。具体测量参数包括：骨小梁厚度、骨体积分数、骨小梁数量、骨小梁间隔、连接密度、各向异性的程度和结构模型指数。3.2拉曼光谱检测：通过峰值581cm-1与峰值1260cm-1的比率来代表矿物质与基质的比率；通过峰值1070cm-1与960cm-1的比率来代表碳酸盐与磷酸盐的比率；使用960cm-1波峰的半峰全宽的倒数(full-width half-maximal, FWHM)来代表骨的矿物结晶度。3.3微硬度检测：显微硬度测量采用日本岛津公司的HMV-2型号显微硬度计测定。HMV-2显微硬度计分别测量胫骨软骨下骨板和软骨下骨松质骨部位,每个样本每个部位测量3次,取各个部位3次测量的平均值作为该部位的硬度值。结果1.不同强度跑步运动对大鼠软骨下骨板的影响1.1软骨下骨板Micro-CT结果：高强度组软骨下骨板BMD在外侧部位为1.181±0.084g/cm3,内侧部位为1.217±0.076g/cm3,均显著高于不运动组(1.089 ±0.052 g/cm3,1.111±0.084 g/cm3)(p=0.030, p=0.050)。高强度组内侧软骨下骨板厚度(0.271±0.016mm)显著高于不运动组(0.232±0.043mm)((p=0.037),在外侧部分同样发现高强度组软骨下骨板厚度高于不运动组,但是并没有出现显著性差异。高强度组软骨下骨板孔隙率在外侧部位为28.47±2.43%,内侧部位为30.48±1.61%,均显著低于不运动组(34.69±4.39%,47.22±3.63%)(p=0.047,p=0.001)。然而,与不运动组相比,低强度运动组和中等强度运动组的BMD、软骨下骨板厚度和孔隙率均无显著性差异。1.2软骨下骨板Microhardness结果显示：在胫骨软骨下骨板外侧部位,HIR组的硬度值为49.7±3.24MPa显著高于SED组46.1±2.61MPa(p=0.001)和MIR组47.3±4.53MPa(p=0.045)；LIR和MIR组分别为47.5±2.53MPa和47.3±4.53MPa,尽管较SED组有所增加,但并无显著性变化。同样的变化出现也出现在软骨下骨板内侧部位,HIR组的硬度值为52.27±2.64MPa显著高于SED组47.92±2.41MPa(p:0.002)；LIR组和MIR组分别为48.51±2.61MPa和46.77±3.18MPa,与SED组相比并无显著性变化。1.3软骨下骨板拉曼结果显示：高强度组外侧和内侧部分矿物质/基质分别显著性低于不运动组外侧和内侧(p=0.021,p=0.028),说明软骨下骨板矿化程度降低。高强度组内侧部分碳酸盐/磷酸盐显著性高于不运动组(p=0.004),磷酸盐/蛋白质则显著性低于不运动组(p=0.032),这说明高强度组软骨下骨板重塑增加。高强度组外侧和内侧部分矿物质结晶度显著性高于不运动组外侧和内侧(p=0.002,p=0.006)。拉曼光谱数据表明与不运动组相比,高强度组重塑和矿物结晶度增加,矿化程度降低。2.不同强度跑步运动对大鼠软骨下骨松质骨的影响2.1软骨下骨松质骨Micr0.CT结果：与不运动组相比,高强度组外侧(p=0.035)和内侧(p=0.002)部分BMD显著性增加。高强度组内侧部位BV/TV显著性高于不运动组(p=0.026),表明运动对松质骨的成骨起到了刺激作用。此外,高强度组外侧和内侧部分松质骨厚度分别显著高于不运动组外侧(p=0.012)和内侧(p=0.027),松质骨分离度在内侧部分低于不运动组(p=0.047)。通过高强度组SMI和CD的降低,我们发现高强度组松质骨发生了板状结构改变。另一方面,除了中等强度组内侧部分BMD显著高于不运动组(p=0.004),外侧部分Tb.N显著高于不运动组(p=0.021)以及低强度组内侧部分Tb.N显著高于不运动组(p=0.032),低强度组和中等强度组均与不运动组无显著性差异。2.2软骨下骨松质骨Microhardness结果显示：在胫骨软骨下骨松质骨外侧部位,H1R组的硬度值为48.26±4.24MPa,显著高于SED组45.42± 2.61MPa(p=O.031),LIR组和MIR组分别46.35±2.53MPa和45.14±3.21MPa,与SED组相比无显著性变化。在软骨下骨松质骨内侧部位,各组间均未出现显著性变化。2.3软骨下骨松质骨拉曼结果显示：高强度组内侧部分矿物质/基质显著低于不运动组(p=0.033),说明高强度组松质骨矿化程度较不运动组低。与不运动组相比,高强度组内侧部分碳酸盐/磷酸盐显著增高(p=0.002),说明高强度组松质骨重塑增加。高强度组外侧和内侧部分矿物质结晶度都显著高于不运动组外侧(p=0.006)和内侧(p=0.002)部分。总的来说,与软骨下骨板类似,高强度组的结果表明相比于不运动组,其重塑和结晶度增加,而矿化程度降低。结论第一部分结论不同强度跑台运动对大鼠软骨下骨显微结构的影响具有强度依赖性：低强度和中强度的跑步运动对大鼠软骨下骨显微结构的影响不显著,高强度的跑步运动则显著改变软骨下骨的显微结构,这种改变被认为与我们前期研究证实的软骨下骨上覆盖的关节软骨退行性改变相关联。第二部分结论不同强度跑台运动对大鼠软骨下骨成分和力学特性的影响也具有强度依赖性。低强度和中强度跑步运动对软骨下骨成分和力学特性无明显影响,但高强度跑步运动导致软骨下骨成分改变,使其变得“硬化和易碎”,进而影响相邻软骨的健康。
[Abstract]:Background osteoarthritis is a degenerative joint disease characterized by progressive joint injury, which eventually leads to patients' joint pain and disability, which greatly reduces the patient's quality of life. The pathological process of osteoarthritis is complex. In the previous study, the cartilage degeneration, and the recent subchondral bone in the main collection. In fact, articular cartilage is closely coupled to its adjacent subchondral bone and acts as a single functional unit. So many studies have focused on the changes in the process of osteoarthritis of subchondral bone and the necessity of targeting this structure in the treatment of osteoarthritis. Osteoarthritis is a multi factor disease. In clinical, the pathological process of osteoarthritis caused by different causes is not the same. In order to clarify the role of subchondral bone in the development of osteoarthritis, a large number of previous studies have adopted different animal models to observe the occurrence and development of osteoarthritis of osteoarthritis. The changes in the lower bone show that the subchondral bone changes in different types of animal models are not the same and even produce completely opposite changes. Although the changes in the subchondral bone are not the same, it will eventually lead to the occurrence of osteoarthritis. These results suggest that the subchondral bone can have a steady state structure and any damage to the stability of the subchondral bone. Behavior can cause damage to cartilage and eventually lead to osteoarthritis. Mechanical loads are considered as a regulator of bone and cartilage homeostasis in the musculoskeletal system. Previous evidence suggests that bone and cartilage can be strongly dependent on mechanical loads. Running is a very common form of weight bearing movement. The human and animal experiments have explored the effect of running on bone, but the conclusion is not consistent. Exercise intensity is considered an important factor in this disagreement. Our previous animal experiments found that low to medium intensity running could maintain the internal stability of the cartilage, while high intensity exercise could lead to cartilage degradation. However, the effect of running on subchondral bone and the correlation with running intensity is not clear. Therefore, the first aim of this experiment is to explore the effect of different intensity running on the microstructure of subchondral bone in rats. This part will discuss the effects of different intensity running on the articular cartilage of rats in the earlier period, and use M Icro-CT further explored the ultrastructure of the subchondral bone (including subchondral bone plate and subchondral cancellous bone) in the tibial plateau of rats with different intensity of running, and the correlation of the changes of subchondral bone and cartilage under different running intensity. A lot of previous studies have explored the structure and mechanical properties of the subchondral bone under different conditions. Change, and its effect on articular cartilage. The main purpose is to directly observe the microstructure of the bone trabecular 3D images by Micro-CT scanning, and provide and analyze the parameters of the three-dimensional structure to study the subchondral bone. The study of the subchondral bone by.Micro-CT includes the analysis of the bone mass, that is, bone density, and also includes bone density. The qualitative analysis is the spatial and morphological structure of the trabecular bone, such as bone small Liang Jianxi (trabecularseparation, Tb.Sp); bone volume fraction (BV/TV), the ratio of the volume of bone trabecular (bone volume, BV) to the total volume of the sample (total volume, TV); the thickness of the bone trabecula (trabecular thickness, Tb.Th); the porosity of the subchondral bone plate; and bone The degree of trabecular anisotropy (Degree of Anisotropy, DA). Although Micro-CT can reflect the changes in the ultrastructure of the subchondral bone, there is no report on how the subchondral bone composition is changed. The Raman spectroscopy (Raman spectroscopy) is a technique for detecting the molecular components of the material, named by the Raman of India scientist. It is based on the principle of Raman scattering effect to analyze the scattering spectra different from the incident light frequency to obtain information on molecular vibration and rotation to obtain the information of organic and inorganic sample molecules. It is characterized by a qualitative and quantitative analysis of the samples by a minimally invasive and noninvasive method. Effective assessment of samples at molecular level, and the use of minimal damage and noninvasive methods in the detection of some biological samples, such as detection of glucose in urine; detection of HCV in human blood in vitro; carotid and coronary atherosclerosis; bone induction of biomaterials; detection of some bone diseases. Measurement of bone synthesis and integration in the process of bone rehabilitation and the detection of microstructure in bone tissue. A lot of previous studies have mentioned quantitative bone mineralization and bone matrix using Raman spectroscopy to evaluate bone mass. Raman spectroscopy has also been proved by many authors to study the composition of bone tissue as an important test method. The second part of the study will use Raman spectroscopy to explore the changes in the subchondral bone composition of rats after running at different intensities. From the point of view of tissue composition, the correlation between different running intensity and subchondral bone and cartilage changes is studied. Objective 1. to study the microstructure of subchondral bone of rats with different intensity of running table. Influence of.2. on the subchondral bone composition and mechanical properties of rats with different intensity of running. Methods 1, 24 180-220gSD rats were randomly divided into four groups, no exercise group (sedentary control, low intensity exercise group (low-intensity running group, LIR), middle strength exercise group (moderate-intensity runni). Ng group, MIR) and high intensity exercise group (high-intensity running group, HIR). The exercise group (including low intensity exercise group, middle strength exercise group and high intensity exercise group) was treated with 0.3% pentobarbital sodium (0.3% pentobarbital) after 8 weeks of running stage, respectively. After drunken death, the bilateral tibia of each experimental rat was intercepted and the soft tissue was removed. Micro-CT was detected at the proximal end of the right tibia. The proximal tibia specimens were vertically cut into two parts of the lateral and internal sides along the sagittal plane using the hard tissue slicer (EXAKT 3000 CP Band System, Norderstedt, Germany). Each part was followed at the 3 equal points of the coronal plane, respectively. The lateral sagittal section was divided into 3 small parts, and the middle part of the specimen was stored in the refrigerator at -80 C. As shown in Figure 2.1, the lateral and medial part of the articular cartilage of the left tibia of the rat was analyzed by Raman spectrum respectively. Before the test, the specimens were put into the physiological salt water of 4 degrees centigrade before the end of 12h. Raman spectrum. After the specimens performed by Raman spectroscopy, the microhardness was detected by.3, and the detection index was 3.1 Micro-CT: the subchondral bone plate was analyzed, the area of the bearing area was 1.04 x 1.04 square millimeter as the region of interest (region of interest, ROI) (Figure 1.2), and then the CT.vol software was used to analyze and calculate, specific points. The parameters included bone density, porosity, and subchondral bone plate thickness. Analysis of subchondral bone cancellous bone. A small Liang Changfang body size of 1.04 x 1.04 x 0.52mm3 was selected by ROI software (Figure 1.4). The specific parameters included bone trabecular thickness, bone volume, bone trabecular number, bone trabecular interval, connection density, anisotropy. The degree of sex and the structural model index.3.2 Raman spectroscopy: the ratio of the peak 581cm-1 to the peak 1260cm-1 to represent the ratio of the mineral to the matrix; the ratio of the peak 1070cm-1 to the 960cm-1 to represent the ratio of the carbonate to the phosphate; the reciprocal of the full width of the half peak of the 960cm-1 wave peak (full-width half-maximal, FWHM) The mineral crystallinity.3.3 microhardness test was used to represent the bone mineral. Microhardness measurement was measured by the HMV-2 microhardness tester of Shimadzu Corporation. The.HMV-2 microhardness tester was used to measure the subchondral bone plate and the subchondral bone of the subchondral bone of the tibia respectively. Each part of the sample was measured 3 times, and the average value of 3 measurements at each part was taken as the Department. Results 1. the effect of running exercise on the subchondral bone plate of rats at different intensity 1. 1.1 subchondral bone plate results: the subchondral bone plate BMD in the high strength group was 1.181 + 0.084g/cm3 in the lateral part and 1.217 + 0.076g/cm3 in the medial part, which was significantly higher than that in the non motor group (1.089 + 0.052 g/cm3,1.111 + 0.084 g/cm3) (p=0.030, p=0.050). The thickness of the medial subchondral bone plate in the high strength group (0.271 + 0.016mm) was significantly higher than that in the non motor group (0.232 + 0.043mm) (p=0.037). In the lateral part, the thickness of subchondral bone plate in the high intensity group was higher than that in the non motor group, but there was no significant difference. The porosity of subchondral bone plate in the high strength group was 28.47 + 2.43% in the lateral part. The site was 30.48 + 1.61%, which was significantly lower than that in the non motor group (34.69 + 4.39%, 47.22 + 3.63%) (p=0.047, p=0.001). However, there was no significant difference in the thickness and porosity of the subchondral bone plate between the low intensity exercise group and the moderate intensity exercise group, compared with the non exercise group. The Microhardness result of the subchondral bone plate of the.1.2 soft bone showed that the subchondral bone plate of the tibia was in the tibial plate. In the lateral part, the hardness of group HIR was 49.7 + 3.24MPa significantly higher than that in group SED 46.1 + 2.61MPa (p=0.001) and MIR group 47.3 + 4.53MPa (p=0.045), LIR and MIR groups were 47.5 + 2.53MPa and 47.3 + 4.53MPa, although the group increased, but there was no significant change. The same change appeared in the medial part of the subchondral bone plate. The value of 52.27 + 2.64MPa was significantly higher than that in group SED (47.92 + 2.41MPa (p:0.002), and LIR and MIR groups were 48.51 + 2.61MPa and 46.77 + 3.18MPa, respectively, and there was no significant change in the subchondral bone plate of.1.3 in the SED group. The lateral and medial minerals / matrix were significantly lower than the outside and inside of the non motor group (p=). 0.021, p=0.028), indicating that the mineralization of subchondral bone plates decreased. The medial part carbonate / phosphate in the high strength group was significantly higher than that in the non exercise group (p=0.004), and the phosphate / protein was significantly lower than the non exercise group (p=0.032), which indicated that the subchondral bone plate remodeling increased in the high intensity group. The mineral crystallinity of the lateral and medial part of the high strength group was significant. The work was higher than the lateral and medial (p=0.002, p=0.006) of the non exercise group. The Raman spectrum data showed that the high strength group remodeling and the mineral crystallinity, the mineralization degree decreased, the effect of.2. on the subchondral cancellous bone of the rat 2.1, compared with the non exercise group, and the Micr0.CT result of the subchondral cancellous bone of the subchondral bone: high strength compared with the non exercise group. The lateral (p=0.035) and the medial (p=0.002) part of the BMD increased significantly. The BV/TV in the medial part of the high intensity group was significantly higher than that in the non exercise group (p=0.026), indicating that the exercise played a stimulating role in the osteogenesis of the cancellous bone. In addition, the thickness of the lateral and medial part of the cancellous bone in the high strength group was significantly higher than that in the outside (p=0.012) and the medial (p=0.), respectively (p=0.). 027) the separation degree of cancellous bone was lower than that in the non motor group (p=0.047). Through the decrease of SMI and CD in the high intensity group, we found that the cancellous bone in the high intensity group had a plate structure change. On the other hand, the medial part of the BMD was significantly higher than the non exercise group (p= 0.004), and the lateral part of the lateral part was significantly higher than that of the non exercise group (p=0.021). The Tb.N in the medial part of the low intensity group was significantly higher than that in the non exercise group (p=0.032). The low intensity group and the medium strength group had no significant difference with the non exercise group. The Microhardness results of.2.2 subchondral cancellous bone showed that in the lateral part of the subchondral bone of the tibia, the hardness of the H1R group was 48.26 + 4.24MPa, which was significantly higher than that in the group of SED (p= 45.42 + 2.61MPa (p=). O.031), the LIR group and the MIR group were 46.35 + 2.53MPa and 45.14 + 3.21MPa respectively. There was no significant change in the medial part of the subchondral bone of the cancellous bone in the subchondral bone. The Raman results of the subchondral cancellous bone of the subchondral bone were not found in each group. The mineral / matrix of the medial part of the high strength group was significantly lower than that of the non motor group (p=0.033), indicating high strength. The degree of mineralization of the cancellous bone in the degree group was lower than that in the non exercise group.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R684.3

【参考文献】