The Effect And Mechanism Of Treadmill Running With Different Intensities On Bone Mass In A Rat Model

Research backgroundMotion system is composed of bone, bone connection and muscle. Bone plays an important role in sustaining hunman movement. Bone modeling and remodeling are mainly regulated by osteoclasts and osteoblasts. A series of serious complication will be caused when bone resorption exceeds bone formation, including bone loss, osteoporosis and bone fracture.Therefore, the maintenance of normal bone mass is very important. Maximum peak bone is one of the best ways to prevent bone loss and bone fracture. It has been recognized that80%of peak bone mass is detemined by genetic factors. On the other hand, environmental and nutritional factors are other important factors influencing the peak bone mass. As for environmental factor, the mechanical stimulation induced by exercise can significantly promote bone formation and increase bone mass.Mechanical stimulation plays an important role in improving the growth and development of bone. Substantial evidence suggests the effect of mechanical stimulation on bone is closely related to the movement pattern and the types of bone. Treadmill running is a common form of weight-bearing exercise. A large number of human and animal studies have been done to explore the effect of treadmill exercise on bone mass, but the conclusion is inconsistent. Exercise intensity may contribute to this inconsistentcy. However, the relationship between running intensity and bone mass is not yet fully understood. It is therefore of significance and necessity to explore the suitable intensity of running which may benefit bone health.It is thought that there are mainly two sites that regulate bone structure and bone mass in response to mechanical stress. One is the osteocyte itself, surrounded by bone tissue and the osteoblast next to the osteocyte.The other is bone marrow cell. The differentiation of bone marrow cell is suspected to be regulated by hormones, cytokines, and growth factors. Many in vitro experiments showed that many molecules have important roles in promoting BMSCs osteogenic differentiation responding to mechanical stress on bone. However, the effect of mechanical loading in vivo is still unclear. In vitro, low intensity mechanical stimulation and mechanical stretch can promote BMSCs osteogenic differentiation while high intensity reduce the proliferation and osteogenic differentiation abilities. As such, the effect of mechanical stimulation on bone marrow stromal cells (BMSCs) osteogenic differentiation abilities in vitro is intensity dependent. Mechanical loading can increase the pressure acting on the bone marrow and flow capacity, therefore providing mechanical signal to stimulate marrow progenitor cells in vivo. Running with different intensities can produce different intramedullary pressure, flow shear piezoelectric effect and provide different mechanical stimuli to BMSCs. Therefore, we speculated that various mechanical stimuli would lead to different responses in proliferation and differentiation abilities of BMSCs, and bone mass consequently.BMSC is a kind of self-renew multipotent differentiation potential stem cell, which can differentiate into a number of lineages, including osteoblasts, chondroblasts, and adipocytes. Osteogenic differentiation into osteoblasts and osteoblast maturation are two important processes for bone formation. Many transcription factors, including alkaline phosphatase (ALP), osteocalcin (OCN), collagen I (COL I) genes, could reflect the characteristics of osteoblast differentiation phenotypes. Essential to cellular commitment to a differentiation lineage is the activation of defined transcription factors. Osteoblastic differentiation is driven by runx2, followed by osterix, and then characterized by the expression of alkaline phosphatase, osteocalcin, and eventually by the mineralization of the extracellular matrix. Adipogenic differentiation of BMSCs is regulated by PPARy2. Wnt/β-catenin pathway is an important pathway regulating both osteogenic differentiation and adipogenic differentiation. The number of osteoblasts and adipocytes was negatively correlated, and exercise can increase bone mass through promoting osteogenic differention at the expense of adipogenic differentiation.Objectives1. To examine the effect of treadmill running on bone with different intensities using a rat model.2. To investigate the differentiation potential of BMSCs down osteogenic and adipogenic lineages under various mechanical conditions in order to gain insight into the mechanisms responsible for different bone adaptations.Methods1. Experimental animals and exercise protocolsA total of24female SD rats were randomly assigned into groups of control (CON), low-intensity running (LIR), moderate-intensity running (MIR), and high-intensity running (HIR). Rats in LIR, MIR, and HIR groups underwent8weeks’ treadmill running programs, which elicit the respective running intensity of about60%,75%, and90%maximal oxygen consumption (V02max) values based on previous research data.2. Specimen collection and experimental methods Basically, this project can be divided into the following two parts:2.1Eight weeks later, all the animals were sacrificed. Serum biochemical markers were detected by ELISA assays.The osteoclast on the surface of trabecular was evaluated by TRAP staining. Bone mass was measured by MicroCT.2.2In parallel, we cultured BMSCs extracted from bone marrow. Gene expressions of RUNX2, SP7, ALP, OCN, COL1, PPARγ2and β-catenin were analysed by quantitative real-time reverse transcription-polymerase chain reaction; the adipocyte cells in bone marrow was calculated in histological sections with HE staining; Immunohistochemical staining for β-catenin was quantified for β-catenin content in distal ending of femur.Results:1.PINP concentration in MIR group (1.217±0.103ng/ml) was significantly higher than that in CON group (1.122±0.067ng/ml)(p-0.012). Similarly, TGF-β concentration in MIR group (34.55±9.78ng/ml) was significantly higher than that in CON group (24.25±6.42ng/ml)(p=0.035). Conversly, CTX I concentration in MIR group (2.203±0.495ng/ml) was significantly lower than CON group (2.548±0.086ng/ml)(p=0.030). For each biomarker, no significant difference was found in LIR and HIR group, when compared with CON group, respectively.2. Less intensitive TRAP staining was observed in group MIR than group CON. In comparison with CON group, significantly lower TRAP staining IOD was found in MIR group (P=0.000), however, no significant difference was found in LIR and HIR group, respectively.3. The BV/TV (%) in MIR group (42.58±4.81) was significantly higher than that in CON group (27.43±5.03)(p<0.05);Conversly, Tb.Sp(mm) and SMI in MIR group were significantly lower than CON group.For each parameter, no significant difference was found in LIR and HIR group, when compared with CON group, respectively.4. Changes in cell proliferation were evaluated by using an in vitro colorimetric assay. It was found that cell proliferation of BMSCs increased significantly in MIR group, but not in groups LIR and HIR, when compared with CON group.5. The numbers of bone marrow cells that are capable of forming colonies were similar among4groups, whereas, in comparison with CON group, significantly higher number of ALP-positive colony was found in MIR group, but not in groups LIR and HIR.6. The most intensive ALP staining or the highest ALP activity was observed in BMSCs of rats in group MIR. Statistical difference in ALP activity was between groups MIR and CON. Similarly, the most intensive ARS staining or the highest mineral content was found in BMSCs of rats in group MIR. There was statistical difference in mineral content between groups MIR and CON.7. As for the relative mRNA expression of RUNX2, MIR group was significantly higher, whereas, LIR group was significantly lower, than CON group. MIR group exhibited significantly higher expression of SP7than CON group, however, no statistical difference was found in group LIR and HIR, respectively, when compared with group CON. Similarly, in comparison with CON group, significantly higher mRNA expression was found in group MIR in COL la, ALP, and BGLAP, respectively, whereas, no significant difference was found in group LIR and HIR.8. Compared to CON group, a dramatic increase in adipocyte cellularity was observed in MIR group (±)(p<0.001), but not in groups LIR and HIR (p>0.05). In addition, MIR group exhibited significantly lower expression of PPARy2gene than CON group, however, no statistical difference was found in group LIR and HIR, respectively, when compared with group CON.9. β-catenin protein expression in femoral diaphyseal was quantified from immunohistochemical stained sections. In comparison with CON group (2.068±0.597), β-catenin content in group MIR (4.461±0.842) increased significantly (p=0.001), however, these in group LIR and group HIR were not significantly changed. In addition, when compared with CON group, significantly higher mRNA expression of β-catenin was found in group MIR, whereas, no significant difference was found in group LIR and HIR.Conclusion:Our results demonstrated an intensity-dependent effect of running on bone mass. There is likely a biomechanical "window" that is required to maintain optimal bone homeostasis. In addition, such effect on bone mass was closely associated with an intensity-dependent regulation on osteogenic and adipogenic differentiation abilities of BMSCs in response to various running-induced mechanical stimuli through modulating the Wnt/β-catenin pathway.