Osteoporosis as a skeletal disorder is characterized by compromised bone strength, which leads to an increased risk of fracture. Patients with femoral fracture account for a substantial volume of orthopaedic hospitalizations. Thus, strengthening femurs is a keen healthcare issue particularly among the aging population [1]. Load-imposing exercises are recommended as a protective measure [2], but they are often limited to healthy individuals who can afford physical activities [3]. Varying loading modalities have therefore been developed in an attempt to enhance femoral bone formation, and biomechanical factors such as loading frequencies, strain and strain-induced fluid flow, and alteration in intramedullary pressure are investigated to understand load-driven anabolic responses [4–7]. Whole body vibrations, for instance, are effective in elevating bone mass in femoral trabecular bone [8]. Few mild, non-invasive loading modalities, however, have been shown to stimulate formation of cortical bone in the femur. In this report, one form of joint loading – knee loading – is described as a potential means to provoke anabolic responses in femoral cortical bone.

Joint loading is a relatively new form of mechanical stimulus in which loads are laterally applied to the epiphyseal bone. Long bones such as an ulna and a femur are apparently less stiff in the lateral direction than in the axial direction, and the epiphysis rich in trabecular bone is softer than the diaphysis composed of cortical bone. Therefore, our rationale behind joint loading is that exposing the least rigid part of bone – epiphyseal bone – to lateral loads might effectively trigger load-driven anabolic responses in the diaphysis. In fact, elbow loading in our previous mouse study [9, 10] revealed enhanced cortical bone formation in the ulnar diaphysis. Compared to an axial ulna-bending modality [11], elbow loading required at most one fourth of the loads (0.5-N forces) to induce bone formation with the same loading timetable. The current study extends elbow loading to knee loading and examines the anabolic effects on femoral cortical bone.

We have previously shown that knee loading stimulates bone formation in the tibia [12, 13], but no study has been performed to evaluate its effects on the femur. A knee is anatomically a complex structure and application of loads to the knee does not imply that the same effects are expected to the tibia and the femur. With knee loading, loads are applied to the lateral and medial epicondyles of the femur as well as the lateral and medial condyles of the tibia. Since the tibial condyle is positioned closer to the loader than the femoral epocondyle, the femoral epiphysis is expected to receive fewer loads than the tibial epiphysis. In addition, anabolic responses to knee loading at different frequencies may vary between the tibia and the femur. If knee loading is effective in the femur, it provides a possibility in future to prevent bone loss throughout the femur including the femoral neck. A substantial bone loss or damage to the femoral neck could result in a serious healthcare problem particularly in the aging population.

Two specific questions addressed herein are (1) whether lateral loads applied to the epiphysis enhance cortical bone formation in the femoral diaphysis; and (2) if so, whether bone formation is dependent on loading frequencies. Using femurs ex vivo, we previously showed that molecular transport in diaphyseal lacunae was activated with knee loading [14]. However, any in vivo consequences or potential mechanisms of enhanced molecular transport were not examined. In this study, we address the question of whether loads applied to the joint site would enhance bone formation in the remote diaphyseal bone.

To answer that question we used a custom-made piezoelectric mechanical loader for knee loading. Dependence of loading frequencies on anabolic responses has been pointed out in varying loading studies [11, 15], and we employed loading frequencies at 5, 10, 15 and 20 Hz. The results in this study clearly support that knee loading can effectively enhance bone formation in a loading frequency-dependent manner.

The animals used for bone histomorphometry tolerated the procedures, and any abnormal behavior including weight loss or diminished food intake was not observed. No bruising or other damages were detected at the loading site.

Dependence of anabolic responses on loading frequencies

Dependence on the loading frequencies was also prominent in the relative parameters defined on the periosteal surface (Fig. 4). In order to further evaluate the effects of knee loading on the periosteal and endosteal surfaces, rMS/BS, rMAR, and rBFR/BS were determined. Compared to the loading at 5 Hz, the loading at 15 Hz resulted in a significant increase in three morphometric parameters (p < 0.01 for rMS/BS and rBFR/BS; and p < 0.05 for rMAR). Furthermore, compared to the loading at 20 Hz, the loading at 15 Hz resulted in an increase in these parameters (p < 0.01 for rMS/BS and rBFR/BS; p < 0.05 for rMAR). In contrast, no significant alterations were observed on the endosteal surface at any loading frequency (p = 0.75 – 0.99; data not shown).

Elevation in cortical bone area and thickness

A frequency-dependent enhancement of the cortical area and the cortical thickness was observed (Fig. 5). First, the cross-sectional cortical area was increased from 0.66 ± 0.01 mm² (control) to 0.74 ± 0.01 mm² (loading) at 15 Hz (p < 0.001), and from 0.66 ± 0.01 mm² (control) to 0.72 ± 0.02 mm² (loading) at 10 Hz (p < 0.05). No significant alteration was observed at 5 Hz (p = 0.43) or 20 Hz (p = 0.56). Second, the cortical thickness was also elevated from 0.188 ± 0.007 mm (control) to 0.213 ± 0.008 mm (knee loading) at 15 Hz (p < 0.05), and from 0.183 ± 0.005 mm (control) to 0.199 ± 0.005 mm (knee loading) at 10 Hz (p < 0.05). No significant changes were observed at 5 Hz (p = 0.45) or 20 Hz (p = 0.06). Among the four loading frequencies, the loading at 15 Hz generated a cross-sectional cortical area significantly greater than that at 5 Hz (p < 0.05). Regarding the cortical thickness, there was no statistical difference among the four loading frequencies (p = 0.19).

Bone porosity in the tibia and the femur

Bone porosity in the cortical bone was significantly different between the tibia and the femur (Fig. 6). Regarding intracortical porosity, the tibia (1.79 ± 0.10 in %) was more porous than the femur (1.18 ± 0.04) (p < 0.001). Similarly, the porosity density (number/mm²) was higher in the tibia (831 ± 42) than the femur (666 ± 20) (p < 0.01).

The histomorphometric results reveal that knee loading – lateral loads applied to the femoral distal epiphysis – increase formation of diaphyseal cortical bone in the femur. The enhancement was most significant with a 15 Hz loading frequency. Unlike a spring loading device [17], hip loading with insertion of a pair of wires [18], or femoral vein ligation [19], the described knee loading modality is non-invasive. Strain has been considered a potential determinant for load-driven bone formation [20, 21], and a strain threshold required to induce anabolic responses was modeled as a function of the number of daily loading cycles [4]. With 0.5-N loads in the current study, an in situ strain was measured as small as 12 μstrain at the site of bone formation in the femoral mid-diaphysis. Since loads were applied 3 min per day, the number of daily loading cycles was 2,700 with the most effective loading frequency at 15 Hz. These results indicate that cortical bone formation can be enhanced with an in situ strain of ~10 μstrain at 1,000 – 10,000 loading cycles. Given the same loading cycles, the estimated in situ strain was approximately 1/10 of the predicted threshold value of 1000 μstrain [4].

Knee loading was reported as an effective means to enhance bone formation in the tibia without inducing significant in situ strain at the site of bone formation [12, 13, 22]. In the present study we demonstrated that it is effective to stimulate bone formation in the femoral diaphysis, although the most effective loading frequency differs. Many factors can contribute to the observed bone formation including load-driven blood perfusion, muscle contraction, and strain induced fluid flow (Fig. 7). One of the hypotheses is that the applied loads generate an oscillatory alteration in intramedullary pressure and the induced pressure gradient stimulates fluid flow and anabolic responses. Trabecular bone in the epiphysis is less stiff than cortical bone in the diaphysis [23, 24], and Young's modulus in the lateral direction is smaller than that in the axial direction [25]. Therefore, lateral loads with knee loading to the femoral epiphysis may effectively modulate intramedullary pressure in the femoral bone cavity. Furthermore, modulation of intramedullary pressure may produce a local pressure gradient in the diaphysis and induce interstitial fluid flow on the cortical bone surface.

The results of bone formation in the current study are consistent with the reported enhancement of molecular transport ex vivo with knee loading [14]. Load-driven interstitial fluid flow was monitored using dye migration in histological bone sections [5] and visualized through fluid displacements in cortical and trabecular bones [26]. Recently a fluorescence-based technique, "fluorescence after photo-bleaching," was employed to evaluate molecular transport in a lacuna [14, 27, 28]. In order to examine the possibility of induction of molecular transport in the diaphysis with knee loading, the photo-bleaching experiments were conducted using a mouse femur ex vivo[14]. A fluorescently labeled lacuna in cortical bone was photo-bleached and a rate of fluorescence recovery was measured with and without loads on the epiphysis. It was reported that knee loading shortened the fluorescent recovery approximately by one quarter [14]. A further linkage analysis between alteration in intramedullary pressure and shortening of the recovery time would contribute to our understanding of the mechanism behind the anabolic responses with knee loading.

Differential sensitivity of the periosteal and endosteal surfaces was observed in response to knee loading. Although bone formation increased on both the periosteal and the endosteal surfaces, in contrast to the marked enhancement of bone formation on the periosteal surface, the endosteal surface exhibited no significant increase in any of the morphometric parameters. Our observations are consistent with some of the previous studies using other loading modalities such as tibia bending [29–31], ulna axial loading [32], and loading with a spring device [17], although strain distributions are different among loading modalities. The results may suggest a differential role of alterations in intramedullary pressure on the periosteal and endosteal surfaces, and differences in anatomical and physiological microenvironments. For instance, the non-uniform osteogenic potentials could also result from local variations in cellular and molecular compositions, and/or heterogeneity in load-driven pressure gradient and interstitial fluid flow on these two surfaces [5–7].

We examined porosity of cortical bone in the tibia and the femur as a potential cause of differential frequency dependence in response to knee loading. As reported by Qin et al. with their pressure experiments, we postulated that the observed frequency dependence with knee loading could be caused by interactions of microstructures in the lacunocanalicular network and intramedullary pressure [6, 7]. The histological analysis revealed that cortical bone in the tibia is more porous than that in the femur. Our porosity observation indicates that femoral microstructure might be sensitive to higher frequencies that tibial microstructure. A further bone microstructures analysis would help to elucidate the role of load frequency in the mechanism of joint loading.

Limitations inherent in the present study include the restricted loading frequencies and varying numbers of daily loading cycles among the loading groups. In whole-body vibrations, it has been suggested that higher frequencies (> 30 Hz) can stimulate bone formation with a lower level of strain [33, 34]. Although the anabolic responses at 20 Hz were insignificant, we do not have any information on the effects of loading frequencies above 20 Hz. Dependence of the observed anabolic responses on loading frequencies requires further investigations since a daily loading cycle and a strain rate are considered important factors, and mechanical testing should be conducted to examine strengthening of bones with knee loading. Note that although loading at 20 Hz has the highest cycle number as well as the strain rate, it did not exhibit superior capability of bone formation. We did not detect any morphological damage on loaded knees, but more careful inspection using molecular markers such as expression and activities of matrix metalloproteinases will be useful for detecting the maximum loads that do not cause any inflammation or evaluating potential regional acceleratory phenomena [35–37]. Our preliminary studies indicate that knee loading is able to enhance bone formation throughout the femur including the femoral neck, and it can stimulate healing of wounded bone. Our future analyses include the role of osteoclastic activities as well as osteoblastic activities with knee loading. A prototype human knee joint loading supporter, designed and fabricated for mechanical characterization, could be used in future for a clinical trial [38].

In summary, the current mouse femur study demonstrates that knee loading is an effective means to enhance cortical bone formation on the periosteal surface in the femoral diaphysis with an in situ strain as small as ~10 μstrain at the site of bone formation. Furthermore, the efficacy of the observed anabolic responses was dependent on the loading frequency. The results here extend our knowledge of load-driven bone formation and the interplay between the epiphysis and the diaphysis in the femur. Strengthening femurs contributes to preventing femoral fractures, and the described knee loading might provide a novel strategy to develop mechanical therapies.