In this study, the effects of eight weeks controlled impact loading during the adolescence on bone growth, quality and mechanics have been investigated using a rat tibial compression loading model. Our findings could be used as a basis for future investigation on the impact loading effects during the adolescence for finding a suitable loading protocol which would be beneficial for the overall bone microstructure during the growing period.

High impact loading triggers decreased body weight coupled with a reduced caloric consumption

Body weight was maximum for the control group followed by the shams and other impact groups at the end of the loading period (Fig. 1A). The body weight in HI group was decreased significantly compared to shams after 8 weeks of loading regime (Fig. 1A). Interestingly, food consumption was simultaneously reduced for the same group (HI) at the end of the study (Fig. 1B). The food consumption is generally dependent on the energy expenditure and so is the change in body weight31. Our findings showed that the HI group has less body weight and reduced food intake despite receiving the maximum intensity of the exercise regime. Part of this weight loss could be ascribed to the decreased appetite of the impact groups32, which was evidenced by a significantly lower caloric intake for the HI group (Fig. 1B). Another intriguing fact was the transitory fatigue of the exercised rats. We observed reduced activity limited to less than seven minutes after the forced loading regime. The rats continued their regular cage activity shortly after this phenomenon. It is suggested that the forced exercise can influence the levels of stress hormones and behavior of the animals which may lead to a reduction in caloric intake in the rats33,34. Moreover, bone osteocytes were shown to be sensitive to short term high-impact dynamic loading conditions35. It has also been reported that body weight reduction can activate a sensor dependent on osteocytes, which eventually diminishes caloric intake in the rodents35,36,37. The observed reduced body weight and caloric intake for the high impact group could have resulted from this phenomenon.

Our findings are supportive of other studies on adult animal models. Reduced body weight has been reported in trained animals by Jones et al.38 and Huang et al.31, after 15 and 8 weeks of exercise period in adult rats, respectively. Moreover, simultaneously reduced body weight and reduced caloric consumption have been reported for adult running rats by Crew et al.39, and treadmill exercised in post pubertal rats by Tisuji et al.40 and by Pitts and Bull32.

Medium and high impact loadings decrease longitudinal bone growth despite developing thicker HZ and PZ heights

Both MI and HI groups showed reduced bone growth rates at the proximal metaphysis compared to shams after 8 weeks of loading (Fig. 2B). This phenomenon eventually resulted in significant longitudinal growth retardation for the same two groups (Fig. 2C); it contradicts our hypothesis that longitudinal bone growth rate would remain unaffected under the impact loadings. Some noticeable histomorphometric changes were also concomitant along with this growth retardation. These changes include increased hypertrophic and proliferative zone thicknesses and hypertrophic cell heights (Fig. 3C).

The relationship between applied compression and longitudinal bone growth rate proposed by Hueter-Volkmann law states that increased compression reduces bone growth rate whereas reduced compression increases it41,42. Moreover, large compressive loads can lead to retardation of bone growth or even cease completely the bone growth42,43,44,45. Our findings are also consistent with other studies46,47,48, where rat ulna longitudinal growth was decreased by compressive loading in adolescence.

Bone growth rate is generally correlated to the overall growth plate thickness7,49. Moreover, it is considered to be linearly correlated with the PZ7,31 and HZ27,50 thickness. Hence, the thicker HZ and PZ heights of the MI and HI group were expected to result in elongated bone length. Conversely, bone growth in MI and HI group was depressed even after thickening of growth plates. Previous studies have also reported thicker growth plates under excessive loadings. However, these studies related this phenomenon with dyschondroplasia (osteochondrosis)46,51. Osteochondroses are considered to be disorders of primary and secondary growth centers, or lesions at the apophyseal or epiphyseal growth areas of bones52. Active young athletes are prone to osteochondroses52, although it is not considered to be a permanent disability for diagnosed patients53. In most cases, conservative treatment for such symptoms includes sufficiently long rest52. In one form of such condition known as Scheuermann’s disease54, regular physical exercises are even recommended. In our study, the rats have been sacrificed immediately after the repeated loading regime. So, it remains unclear whether a sufficient amount of unloading period would affect the growth plate thickness and change it accordingly or not.

The change in longitudinal bone growth could also be associated with the caloric intake and body weight of the rodents. It has been reported that the reduction of body weight due to reduced caloric intake can affect cell production in the proliferative zone in a negative manner55, which can eventually slow down longitudinal bone growth. Our observations can be compared with the studies using rat56,57 and swine55 models, where a reduction in body weight coupled with reduction in longitudinal bone growth has been observed. Our overall findings indicate that the generalized claim of the linear relationship between bone growth and growth plate height7,58 might not always be implemented. However, our findings are supportive of other studies7,46,47, where a contrary relationship between the growth rate and height of the growth plate was also observed.

Changes in trabecular bone microstructure are time as well as impact level dependent

Compared to the MI group, the HI group showed load adaptive changes in trabecular microstructure at an earlier (after 4 weeks) period and affecting more structural parameters (Table 1). BMD was significantly increased in both MI and HI groups (Table 1) at the end of loading period. For healthy bone structure, bone mineral content shows an increasing trend during the adolescent period59. Also, higher loading intensity is generally associated with an increased BMD60. In fact, BMD in athletes is elevated under high impact training conditions61. The increase of BMD in our study could be related to hormones triggering mechanisms. Indeed, an increased BMD is controlled by a decreased parathyroid hormone response coupled with an increased calcitonin response62,63, both of which take time to react under favorable loading conditions. Hence, this could explain the significant increment of BMD assessed after 8 weeks instead of 4 weeks of loading. In similar studies, where adult rats have been tested for repetitive jumping exercise64 or treadmill running exercise65, an increased BMD was observed in the loaded limbs.

The BV/TV was also found to significantly increase in the exercised tibiae both after 4 and 8 weeks of loading in the HI group. For a healthy growing bone, an elevated BV/TV is generally correlated with an increased BMD66, as found in this study. The significant BV/TV in HI group after 8 weeks of loading can be explained with the increased BMD for the same group (Table 1). However, the significant increase after 4 weeks of exercise (without simultaneous BMD increase) could be related to triggering of bone metabolism under high impact loading61. Indeed, it has been reported that under controlled loading scenarios, the trabecular structure responds positively through diffusion and active transport of metabolites within the entire microstructure59,67. So, it could be possible that HI loading has accelerated the diffusion and transportation process of the metabolites at an earlier stage and thus elevated the BV/TV in the exercised limb accordingly. Other studies support our findings in growing rats, where swimming exercise was found to significantly increase BV/TV68,69. Also, another study reported a greater BV/TV in the growing rat tibiae after 8-weeks of free fall exercise routine70. The observed increment in Tb.Th during the loading period is a part of normal bone development71. However, a significant increment in the loaded limbs (compared to shams) indicated an additional improvement in trabecular structure under impact loading conditions. The significant reduction in Tb.Sp is often considered as the concomitant increase with BV/TV and Tb.Th72. The significant reduction in Tb.Sp indicates the occurrence of a loading induced bone gain through increased connectivity and gradual thickening of the trabecular structure73. The significant change in Tb.Sp in HI group at an earlier stage can be associated with the significant increase in BV/TV from the same group (Table 1). Our data are supportive of previous findings where an increased Tb.Th was reported in the loaded tibiae of 10-week old adult mice74, as well as with a decreased Tb.Sp reported in loaded tibiae of both growing and adult mice15.

Medium and high impact loadings benefit the cortical bone morphometry in the diaphysis, leading to significantly improved structural- and tissue-level bending mechanical properties

MI and HI loadings significantly affected tibial diaphysis, modifying both its cortical microstructure and its mechanical properties (Tables 2 and 3); it supports the hypothesis that bone morphometry and biomechanics are improved by impact loadings and that higher impact intensity has greater positive influence on bone morphometry and biomechanics. Indeed, stiffness was significantly increased in these groups compared to the sham group (Table 3). This improved stiffness eventually triggered the bones to reach a significantly higher ultimate load (Table 3). Interestingly, MI and HI groups exhibited significantly increased cortical area, simultaneously coupled with periosteal perimeter expansion and endocortical perimeter reduction (Table 2) after 8 weeks of loading regime. The medullary area (Ma.Ar) in the HI group also decreased significantly (Table 2). Cortical bone area at the mid-diaphysis and the corresponding ultimate load (F ult ) are highly correlated to each other75,76. Hence, the increased ultimate loads for MI and HI groups can be justified from their significantly increased cortical area. Our findings agree with other studies on adult rodents, where an increased ultimate strength have been associated with exercised tibiae in 15 and 10 week old rats31 and mice74, respectively. Bone stiffness can be related to its morphology and cross-sectional geometry in growing rats77. More specifically, stiffness can be associated with the increase or decrease in cortical thickness (Ct.Th), total area (Tt.Ar) and cortical area (Ct.Ar)78,79. Ct.Ar has been increased significantly for both MI and HI groups (Table 2). Also, Ct.Th and Tt.Ar significantly increased for the HI group (Table 2). Hence, a strengthened diaphysis associated with an increased stiffness can be justifiable for the MI and HI groups. Increased ultimate load and stiffness was also observed in a study31 of 15-week old swimming and running rat groups along with an increased cortical area in the swimming groups. Another study with 10 week old mice reported an increased cortical thickness and stiffness, along with the cortical and total area increment in the loaded limbs74.

No effects were found on Young’s modulus when comparing LI, MI and HI loadings to shams (Table 3). Young’s modulus or bone rigidity is an intrinsic mechanical property of the bone80,81. With the greater structural strength found in the MI and HI groups (Table 3), an increase was also expected in the Young’s modulus. In a bending test, the evaluation of rigidity depends linearly on stiffness and cubicly on the span length of the tibiae (Equation 2)82. In the MI and HI groups, tibial lengths decreased by 4% and 5%, respectively. Even though their stiffnesses increased, the decreased span lengths in the cubic form might have counteracted this change, yielding unchanged Young’s moduli. Another study also reported non-significant changes in Young’s modulus in growing mice limbs74 undergoing 2 weeks of axial loading regime generating a maximum 2400 με at the mid-diaphysis of the tibiae.

Energy to failure load increased significantly for the MI and HI groups (Table 3). Greater energy to failure loads implies that the MI and HI tibiae sustained more deformation or strain before failure. Strengthening of bone tissue in the MI and HI groups is associated with its adaptation in response to the applied loading regime. During the loading period, compositional alterations might have occurred within the newly forming and pre-existing bone cellular matrix83, possibly involving type I collagen, which is known to affect the post-yield behavior of the bone84,85. Biochemical changes might have also interfered with collagen fibers orientation within the bone matrix and thus altered the toughening-mechanism in the bone microstructure86. Ultimate stress (σ ult ) and failure stress (σ fail ) are significantly increased and decreased respectively in both MI and HI groups (Table 3). The ultimate/failure load is the controlling factor in the corresponding ultimate/failure stress for the same group of tibiae (Equation 3)31,87. The respective significant increase and decrease in F ult and F fail for the MI and HI groups can explain the significant change in σ ult and σ fail for the same groups. Our results agree with other studies, where 15-week old rats exhibited increased stress and failure loads in the trained limbs31,88. Moreover, studies using 8-week old mice also observed increased ultimate stress in the loaded limbs after 2 weeks of axial loading regime74.

Limitations

The current study has limitations involving some methodological aspects. For the trabecular VOI, only the proximal metaphysis was analyzed. It was chosen over the distal section because of the large amount of trabeculae in this region. Also, it has been reported that the proximal tibia has greater bone volume compared to the distal tibia89 and has also been used more often in bone remodeling studies16,19,74. As for bone growth rate, it was only measured at the proximal site. This choice was justified as proximal metaphysis is responsible for blood supply and vascular stasis in growing bone and the contribution of the proximal tibial growth plate in the total longitudinal growth was found to reach approximately 80% in adolescents90. So, any loading effects on proximal bony region could presumably be considered as to have the main effects between the proximal and distal parts as well. Also, the biological effects of bone remodeling were not investigated as part of this study. A future study could investigate bio-markers to infer on bone formation and resorption for better understanding of bone growth mechanism involved in impact loading regimes. Moreover, rats were sacrificed immediately after the last in vivo loading regime at 81 days old. A detraining period before the sacrifice, which might have modified the growth plate histomorphometry47,91, was not evaluated as our primary objective was to investigate the bone growth rate and histomorphometry while the growth plate was still active92,93.