| Research backgroundOsteoarthritis (OA) is a degenerative joint disease characterized by progressive joint damage. It causes joint pain and disability, thus leading to a reduced quality of life in OA patients. OA has traditionally been thought as a primary articular cartilage disorder; however, the role of subchondral bone (SB) is currently believed to be of particular importance in the complex pathogenesis of this disease. In fact, the conditions of articular cartilage and its supporting SB are tightly coupled and should be viewed as an authentic functional unit. As such, more and more studies are trying to understand the changes of SB during the pathogenic processes, and even the necessity to carefully consider this structure in the treatment of OA.OA is thought to be a multifactorial disease. In clinics, different alterations of SB can be observed in various OA conditions. In order to understand the role of SB in the development and progression of OA, a large number of animal models have been developed to examine the changes of SB. However, these results are in consistent, and even opposite. The fact that different changes in SB lead to similar cartilage damage probably indicates that, only when the adaptative changes of the underlying SB are stabilized within a certain range can cartilage integrity be maintained. Mechanical loading is considered to be a critical regulator of skeletal homeostasis, including bone and cartilage. It is suggested that both cartilage and bone may respond to mechanical loading in an intensity-dependent manner. As a common form of weight-bearing exercise, the effect of running on bone has been documented extensively with inconsistent findings, which clearly imply that intensity may be an important factor influencing the effect of running on bone mass. In a rat model, we previously demonstrated that treadmill running with low-to-medium intensity maintains cartilage homeostasis, whereas, high-intensity running may cause cartilage degradation. We therefore hypothesize that the response of SB may differ under different running-induce loadings, and would like to test this hypothesis using the same animal model in the present study. So the first objective of this study was to explore the effect of treadmill running with different intensities on the microstructure of subchondral bone in a rat model. In this part, three-dimensional Microfocal Computed Tomography (Micro-CT) was applied to examine the proximal end of tibia of rats which were subjected to treadmill running with different intensities. Three-dimensional images and microarchitectural parameters of subchondral bone (including the subchondral bone plate and the subchondral trabecular bone) were obtained and compared among different groups. A number of parameters may be obtained to reflect the changes of bone qualitatively and quantitatively, including bone volume fraction, which describes the ratio of bone volume over tissue volume (BV/TV); trabecular separation (Tb.Sp, mm); trabecular thickness (Tb.Th, mm); trabecular number (Tb.N). Nevertheless, this technology could only provide information on structural and material properties of bone tissue, but not on the composition on the molecular level.Raman spectroscopy is a vibrational technique that provides information on the chemical composition of organic and inorganic samples, allowing less invasive and nondestructive qualitative and quantitative analysis. It has been considered effective in assessing sample information at the molecular level, and has been used for several minimally and non-invasive applications of biological samples such as detecting glucose in urine, atherosclerosis in coronary and carotid arteries, hepatitis C in human blood serum in vitro, lactate identification in blood, osteoinduction in biomaterial implants, several bone diseases, and bone synthesis and osteointegration after healing, as well as evaluating the microstructure of human cortical bone (osteon). Several works have proposed the use of Raman spectroscopy to determine the mineralization and organic matrix to assess bone quality. The study of Raman spectroscopic on molecular level enables us to understand the effect of different intensity running exercise on the composition of SB, which is the second objective of this experiment.Objectives1. To investigate the effect of treadmill running with different intensities on the ultrastructure of subchondral bone in a rat model.2. To understand the effect of treadmill running with different intensities on composition and mechanical property of rat subchondral bone.Methods1. Experimental animals and exercise protocolsA total of 24 female SD rats were randomly assigned into groups of sedentary (SED), low-intensity running (LIR), moderate-intensity running (MIR), and high-intensity running (HIR). Rats in LIR, MIR, and HIR groups underwent 8 weeks’treadmill running programs, which elicit the respective running intensity of about 60%,75%, and 90% maximal oxygen consumption (VO2max) values based on previous research data.2. Sample preparationEight weeks later, all animals were sacrificed under anesthesia by cervical dislocation. Bilateral tibias from each animal were dissected free of soft tissues. The proximal end of the right tibia was used for micro-CT scanning, while the proximal end of the left tibia was collected for further preparation. Briefly, the proximal end of tibia was cut sagittally into lateral and medial side, using a high speed, water cooled saw with a fine diamond coating (EXAKT 300 CP Band System, Norderstedt, Germany). For each side, three sections were obtained by two sagital cuts at 1/3 and 2/3 of its width, respectively (Figure 1).Afterwards, the middle sections were stored at -80℃ immediately.Samples were soaked in physiological saline for 12 h under 4 C before Raman measurements (on one sagital surface) and microhardness testing (on the other surface).3. Specimen collection and experimental methods3.1. Micro-CT study:For analysis of subchondral plate, the load bearing region with an area of 1.04×1.04 mm2 was selected as Region of Interest (ROI) (Figure 2A). Bone mineral density (BMD), porosity, and thickness of subchondral plate were measured and calculated using CT-analyser software. For analysis of subchondral trabecular bone, a cuboid of trabecular bone with size of 1.04×1.04×0.52 mm3 at beneath the ROI of subchondral plate was selected. BMD (g/cm3), percent bone volume (BV/TV,%), trabecular thickness (TbTh, mm), trabecular separation (Tb.Sp, mm), trabecular bone pattern factor (TbPf, 1/mm), structure model index (SMI), and degree of anisotropy (DA) were calculated for subchondral trabecular bone.3.2. Raman microscopy study:the relative peak intensities of selected pairs of bands were calculated in this study. The mineral to matrix ratio, which indicates the amount of mineralization, was calculated from the intensity of the phosphate V4 (mineral,580cm-1) peak divided by Amide Ⅲ (matrix,1280cm-1) peak in the present study. For remodeling evaluation, the ratios between carbonate-phosphate (1070/960 cm-1) and phosphate-protein (960/1660 cm-1) were calculated using the Raman band intensities. In addition, the width of the primary phosphate band (near 959 cm-1) was measured, and the inverse full-width half-maximal (FWHM) was obtained to give an indication of the degree of crystallinity of the mineral part of the bone. The ratios and FWHM were calculated on a pixel-by-pixel basis, and then averaged for each specimen, resulting in a single value for each specimen.3.3 After Raman microscopy study, Microhardness analysis was carried out on the specimen.Vickers microhardness test was performed using a HMV-2T microhardness test machine fitted with a pyramidal diamond indenter (Shimadzu, Japan).For each bone section,3 microhardess measurements were taken randomly for subchondral plate and subchondral trabecular bone, respectively.Results:1. Effect of running on subcdhondral plate1.1 Results from Micro-CT:In comparison with group SED, a significantly higher BMD was observed in group HIR in both lateral (1.181±0.084g/cm3 for HIR group and 1.089±0.052 g/cm3 for SED group, p=0.030), and medial sides (1.217± 0.076 g/cm3 for HIR group and 1.111 ±0.084 g/cm3 for SED group, p=0.050), respectively. In medial side, subchondral plate was significantly thicker in HIR group (0.271±0.016 mm) than that in SED group (0.232±0.043 mm) (p=0.037). In lateral side, thicker subchondral plate was also found in group HIR compared with group SED, but without statistical difference. A significantly lower porosity of subchondral plate was defined in HIR group than SED group in either lateral side (28.47±2.43% vs.34.69±4.39%, p=0.047) or medial side (30.48±1.61% vs.47.22± 3.63%, p=0.001). Nevertheless, compared with group SED, there was no statistical difference in groups LIR and MIR, in either BMD, thickness or porosity, respectively.1.2Results from microhardness test:Group HIR exhibits significantly higher microhardness than group SED in medial (52.27 ± 2.64 GPa vs.47.92±2.42 GPa, p=0.001) and lateral side (49.7±3.25 GPa vs.46.1±2.61 GPa, p=0.002), respectively. When comparing with group SED, there is no statistical difference in either group LIR or MIR.1.3Results from Raman spectra:HIR led to a decrease in mineral-matrix ratio in either medial (p=0.021) or lateral (p=0.028) side, indicating a less mineralized subchondral plate following HIR. There was a significantly higher carbonate-phosphate ratio (p=0.004) and a significantly lower phosphate-protein ratio (p=0.032) in the medial side in group HIR when compared with group SED, which suggested that HIR resulted in an increased remodeling of subchondral plate. Crystallinity in group HIR was significantly higher than that group SED in medial (p=0.002) and lateral side (p=0.006), respectively. Results from Raman spectra indicate that HIR led to an increased remodeling and less mineralized subchondral plate with increased mineral crystallinity.2. Effects of running on Subchondral trabecular bone2.1 Results from micro-CT:Compared with group SED,signficant increase in BMD is detected in group HIR in both lateral (p=0.035) and medial (p=0.002) sides. HIR leads to significantly higher BV/TV than controls in medial side (p=0.026), indicating a stimulatory effect on trabecular bone formation. In addition, HIR also leads to an increase in trabecular bone thickness in both medial (p=0.012) and lateral (p=0.027) sides, as well as a decrease in trabecular separation in medial side (p=0.047). Together with the decreased values of SMI and CD induced by HIR, our results indicate that HIR led to more and denser trabecular bone with a more plate-like microarchitecture. On the other hand, there are no obvious changes in either LIR or HIR groups, except for an increase in BMD in group MIR in medial side (p=0.004), as well as in Tb.N in group MIR in lateral side (p=0.021), and group LIR in medial side (p=0.032).2.2 Significant higher microhardness in HIR group (48.26±4.24 GPa) was observed in lateral side when compared with SED group (45.43±2.61 GPa)(p=0.031).2.3 HIR led to a decrease in mineral-matrix ratio in medial side (p=0.033), indicating a less mineralized subchondral trabecular bone following HIR. When compared with group SED, a significantly higher carbonate-phosphate ratio (p=0.002) was observed in the medial side in group HIR, suggesting that HIR resulted in an increased remodeling of subchondral trabecular bone. Crystallinity in HIR group was significantly increased than that in SED group in either medial (p=0.002) or lateral side (p=0.006). Taken together, similar to subchondral plate, HIR resulted in an increased remodeling and less mineralized subchondral trabecular bone with increased mineral crystallinity.Conclusion:Our findings suggested an intensity-dependent effect of running on the microarchitecture of SB. No obvious changes were observed in rat subjected to treadmill running with low or medium intensity. Nevertheless, running with high-intenstiy led to considerable alteration in SB microarchitecture, which was thought to contribute to the degenerative changes in overlying cartilage demonstrated in our previous study. An intensity-dependent effect of running was also suggested on composition and mechanical property of SB. Both low- and medium-intensity running led to little changes in composition and mechanical property of SB, whereas, high-intensity running was found to make the SB "brittler and stiffer" and adversely affecte the integrity of the overlying articular cartilage. |
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