Effects of Mechanical Stimuli on Adaptive Remodeling of Condylar Cartilage

Abstract

Trabecular bone has been shown to be responsive to low-magnitude, high-frequency mechanical stimuli. This study aimed to assess the effects of these stimuli on condylar cartilage and its endochondral bone. Forty female 12-week-old C3H mice were divided into 3 groups: baseline control (killed at day 0), sham (killed at day 28 without exposure to mechanical stimuli), and experimental (killed following 28 days of exposure to mechanical stimuli). The experimental group was subjected to mechanical vibration of 30 Hz, for 20 minutes per day, 5 days per week, for 28 days. The specimens were analyzed by micro-computed tomography. The experimental group demonstrated a significant decrease in the volume of condylar cartilage and also a significant increase in bone histomorphometric parameters. The results suggest that the low-magnitude, high-frequency mechanical stimuli enhance adaptive remodeling of condylar cartilage, evidenced by the advent of endochondral bone replacing the hypertrophic cartilage.

INTRODUCTION

The sensitivity of bone tissue to alterations in its functional environment is well-recognized in the cranial and post-cranial skeleton. As a result of loading, strain arises in bone tissue, as has been recorded in the mandible of a macaque during chewing (Hylander et al., 1998; Fritton et al., 2000), the ulna of a dog during running (Rubin and Lanyon, 1982), or the humerus of a goose during flying (Rubin, 1984). These studies provide an insight into the mechanical components that control bone modeling. The smallest measurable strains have been quantified during quiet activities such as standing or sitting, as well as the largest strains during vigorous activities including walking, jumping, and flapping, in different species (Fritton et al., 2000). These investigators demonstrated the occurrence of thousands of low-magnitude (<10 μє) daily strain events at high frequencies (40 Hz), compared with few high-magnitude (>1000 μє) strain events. This suggests that activities not typically associated with vigorous locomotion make a predominant contribution to the strain experienced by bone. Other investigators (Rubin and Lanyon, 1987; Qin et al., 1998) have since considered the predominant component of the strain environment “the extremely low level, high frequency mechanical signal”, and they have shown that bone is responsive to these mechanical stimuli, including those non-invasively applied via foot-based whole-body vibration (Fritton et al., 1997) in both short- (Rubin et al., 2001a) and long-term animal studies (Rubin et al., 2001b, 2002a).

Despite the fact that numerous studies have revealed the association between low-magnitude, high-frequency (LMHF) stimuli and responsive remodeling of weight-bearing long bones, little effort has been dedicated to exploration of the impact this specific stimulus may have upon the skeletal components in the craniofacial region, such as the mandibular condyle.

Condylar cartilage has been the target of many studies aiming to examine its adaptation to loading alteration caused by mandibular forward positioning (Rabie et al., 2004; Shen et al., 2006). It is well-accepted that, compared with epiphyseal cartilage in long bone, condylar cartilage demonstrates a distinctive capability to respond to the changes in mechanical loading (Shen and Darendeliler, 2005). Therefore, the objective of this study was to assess the effects of these LMHF stimuli on condylar cartilage and its endochondral bone in adult mice.

MATERIALS & METHOdS

Animal Grouping

We used 40 female 12-week-old C3H mice weighing approximately 20 g each (Westmead Hospital Animal Ethics Committee protocol no. 4023). The mouse C3H strain has high bone mineral density (BMD) compared with that of other mouse strains, and there is a linear association between bone-regenerative capacity and bone density, according to the “drill-hole” model in the tail vertebra (Li et al., 2001). For these reasons, the C3H mouse strain was chosen for this experiment.

The animals were randomly divided into 3 groups. In the Baseline group (n = 10), the animals were designated as baseline controls and were killed at Day 0. In the Sham group (n = 15), the animals were not exposed to mechanical vibration and were killed following 28 days of exposure to mechanical stimuli. In the Experimental group (n = 15), the animals were exposed to mechanical vibration and were killed following 28 days of mechanical exposure.

Mechanical Vibration Loading

In the current study, we used an oscillating device (Juvent®™ Inc., Somerset, NJ, USA) similar to that specified by Fritton et al. (1997) and recently used by Rubin et al. (2004). We achieved mechanical stimulation to the animals by placing them, while still in their cages with bedding removed, directly on a device which generated vertical ground-based vibration. This machine generated 30-Hz pulses, creating peak-to-peak accelerations of 2.9 m/ sec², referred to as a fraction of earth’s gravitational field, 0.3 g (1 g = 9.8 m/sec²). Based on Rubin’s previous research, it is believed that this produces peak strains of approximately 5 μє (Fig. 1). Animals in the Experimental group were subjected to 20 minutes’ vibration per day for 5 days per week for a total of 28 days, a protocol similar to that used in other studies (Rubin et al., 2002a,b).

Specimen Preparation

Mice were killed by CO₂ asphyxiation, and their decapitated heads were fixed in 10% buffered formalin. The left hemimandibles were dissected and sectioned to isolate the condylar process. The samples were then rinsed in 0.2 M phosphate buffer 5 times at five-minute intervals. They were immersed in 0.1 M osmium tetroxide (24°C) for 4 days. To make the condylar cartilage visible by x-ray absorption, we developed a staining protocol that involved the en bloc application of the osmium tetroxide solution (2% w/v OsO₄). Osmium tetroxide is a strong oxidant that cross-links lipids in cell membranes. The osmium is a heavy metal which is embedded onto the cell membrane to provide contrast to the cartilage.

Microtomographic Imaging

The x-ray microtomographic scans were acquired by means of the SkyScan 1172 high-resolution desktop x-ray microtomograph (SkyScan, Aartselaar, Belgium). The exposure was set at 110 kV/93 μA. The data were then processed with standard cone-beam reconstruction software (Feldkamp et al., 1984) to generate a series of 1024 8-bit axial slices that had Z-dimensional spacing equal to the within-slice pixel spacing. The three-dimensional data were subsequently digitally rendered as 3D visualizations with the use of commercially available software (VGStudio Max, Volume Graphics GmbH, Heidelberg, Germany).

Analysis of Condylar Cartilage

The osmium tetroxide enhancement further allowed the condylar cartilage to be segmented and reconstructed in a 3D context to show the spatial configuration and the volume of the cartilage. During this segmentation process, axial slices were interactively partitioned by means of a 2D dynamic region growing tool. This algorithm allowed regions of interest to be selected consecutively from individual sections based on a selected grey value and tolerance. The region of condylar cartilage was subsequently visualized as a separate 3D rendering (Fig. 2).

Bone Histomorphometric Analysis

We used CT Analyzer software (Version 1.02, SkyScan®), which utilizes ‘marching cubes’ algorithms, to perform three-dimensional histomorphometric analyses on condylar trabecular bone. The bone histomorphometric parameters were: trabecular bone pattern factor (TbPf), which is an index of connectivity of trabecular bone (Hahn et al., 1992); mean trabecular separation (MnTbSp); and trabecular number (TbN), indicating the density and quantity of bone tissue (Appendix Table). Previous studies have shown that structural metrics measured in 3D morphometry by micro-CT correlate closely with those measured by standard histomorphometry (Muller et al., 1998; Hildebrand et al., 1999). Volumes of interest (VOI) of 0.0452 mm³ were localized in the condylar bone that was beneath the midpoint of the condylar cartilage and lying within the superior third of the condylar process (Fig. 3a). Using sagittal sections, we extracted a portion of the dataset of only trabecular bone as a separate object, and exported it as a voxel dataset for histomorphometric analysis (Fig. 3b ).

RESULTS

Changes in Volume of Condylar Cartilage

We used one-way analysis of variance to compare the volumes of condylar cartilage among the baseline, the sham, and the treated groups. The volume measurements were significantly different among the 3 groups (p = 0.000) (Table ).

Post hoc comparisons of the total cartilage volume showed a significant reduction in cartilage volume at 4 wks with vibration, compared with volume in the baseline sham and the controls. Post hoc tests, however, showed no significant differences in cartilage volume at day 0 in the baseline sham and four-week control groups (Table ).

Changes in Histomorphometrics of Endochondral Bone

We used univariate analysis of variance to compare the multiple histomorphometric parameters among the 3 groups, using a model in which group is a fixed factor and animal is a random factor nested within each group. Post hoc comparisons were carried out with no adjustment for multiple comparisons, since the comparison of interest was the control group at 4 wks and the treated animals at 4 wks. Pairwise comparisons between the 2 groups yielded significant differences for variables used in the three-dimensional histomorphometric analysis. The variables with significant group differences were: Volume of Object (Vobj) (p = 0.027), Surface of Object (Sobj) (p = 0.002), TbPf (p = 0.003), Structure Model Index (SMI) (p = 0.005), MnTbSp (p = 0.001), and TbN (p = 0.001). The other variables [VOI, Relative Volume (RVol), Bone surface/bone volume (BS_BV), and mean trabecular thickness (MnTbTh)] showed no significant differences. This showed an increase in TbPf, in TbN, and in MnTbSp between the treated animals and the controls.

DISCUSSION

The combination of en bloc heavy metal staining of condylar cartilage and high-resolution x-ray microtomography that was applied in this study has shown considerable potential for detecting changes in cartilage thickness. The field of heavy metal staining has a long history of application in electron beam microscopy; however, its use in x-ray microtomography is only beginning to be explored. In this study, the prolonged post-fixation of specimens in osmium tetroxide allowed osmium to be deposited within the cartilage, making it distinguishable from the underlying bone tissue.

The bone histomorphometric parameters of particular interest were measured via a micro-CT imaging system. The increase in trabecular bone pattern factor (TbPf) between the treated animals and the controls indicated a more disconnected trabecular structure with more free-ending trabeculae. The concurrent increase in trabecular number (TbN) seen in the treated animals indicated an increase in bone quantity. The increase in mean trabecular separation (MnTbSp) with the less-well-connected trabecular bone in this area of the condyle was representative of newly formed bone. The decrease in the relative volume of condylar cartilage in the treated animals, along with the increase in bone quantity, showed that the LMHF stimuli induced osteogenesis, leading to adaptive growth of the condyle in adult mice.

The biological mechanism behind the phenomenon that increased bone formation associated with a decrease in cartilage thickness has been well-documented. In their series of studies elucidating the changes in condylar cartilage in response to mandibular advancement, Rabie et al.(2004) and Shen et al.(2006) have clearly reported that the adaptive modeling of condylar cartilage is characterized by enhanced transition from chondrogenesis to osteogenesis. The vibrating mechanical stimulation in the present study was shown as a possible mechanism for the acceleration of the modeling. However, the clinical significance and the clinical implications of this application remain to be evaluated. Histologically, this transition is realized by the breakdown of hypertrophic cartilage, which is replaced by the advent of new bone beneath it. In another study (Xiong et al., 2005), microcomputed tomography was used to explore the relationship between hypertrophic cartilage and the microstructural properties of cancellous bone produced by mandibular advancement in the condyles of adult rats. The authors characterized new bone formation in the condyle by thinner trabecular thickness, more trabecular number, and increased trabecular space. They concluded that mechanical strain produced by mandibular advancement induced adaptive growth of the condyle.

A questionable aspect of this study design is the degree of transmission of the vertical ground-based oscillation, since it diminishes as the signal travels proximally through the skeleton. In a study measuring transmissibility in the hips and spines of humans standing on the oscillating plate, the authors showed that approximately 80% of a 30-Hz ground-based signal reached the hip and spine (Pope, personal communication). To date, there has been little work to show the portion of strain that reaches the mandible. However, it is reasonable to anticipate that the strain signal may be greater toward the distal elements of the skeleton, but that a majority of these signals are transmitted to the craniofacial skeleton.

Calculation of the strain in cancellous bone due to 0.3-g oscillation is beyond the scope of the present research. By attaching strain gauges to the midshaft of tibia, some authors have shown that 30-Hz, 0.3-g oscillation stimulated peak strains of approximately 5 μe on the cortex (Sommerfeldt and Rubin, 2001). They also further assumed that the existing interrelationship between cortical and trabecular bone strain during the high-magnitude, low-frequency events is not disrupted in this lower-strain-magnitude domain.

Although the strain within the trabeculae is not necessarily high, the increase in frequency may elevate other components of the physical environment. It has been shown that increasing the frequency from 0.1 to 10 Hz elevates intramedullary pressure and increases fluid flow from bone (Xiong et al., 2005). The shear stresses resulting from the fluid flow may be a key component in regulating the response (Sommerfeldt and Rubin, 2001). Further, there is a consistent differential response to these low-level signals, with cortical bone not influenced and trabecular bone more responsive in both short-term rat (Rubin et al., 2001a) and longer-term mouse (Judex et al., 2001) studies. The exact nature of this difference in response is unclear at this time. One hypothesis proposed for this enhanced metabolic activity in trabecular bone is that there is a greater cell density because of a greater surface area:volume ratio, or, alternatively, the fact that the endosteal compartment may be exposed to a distinct set of cellular signals (Sommerfeldt and Rubin, 2001).

Another proposed mechanotransduction pathway from tissue to the cellular level is related to bone fluid flow. Theoretical models that estimate the fluid shear stress on the osteocytic process during loading indicate that shear stresses of up to 3.0 x 10⁻⁶ MPa are produced when cortical bone is loaded, with the resulting shear stress nearly proportional to the product of the applied stress and frequency when below 25 Hz (Weinbaum et al., 1994; Zeng et al., 1994). In these models, similar shear stresses were found if a stress of 20–40 MPa was applied at a frequency of 1–2 Hz, or if a stress of 2–4 MPa was applied at a frequency of 20–30 Hz. Extrapolating these results to an applied loading of 10 μє at either 1 Hz or 30 Hz gives osteocytic shear stresses of the magnitude of 1.0 x 10⁻⁸ MPa. This is below the 0.6 x 10⁻⁶ to 3.0 x 10⁻⁶ MPa range of stresses that have elicited an intracellular Ca²⁺ response in bone cells in vitro. For the parameters in current use, the theoretical models suggest that such low-magnitude strains would not be stimulatory, but the mechanotransduction mechanism in bone remains poorly understood (Fritton et al., 2000).

Table.

Post hoc Multiple Comparisons of Cartilage Volume among Baseline, Control (Con), and Experimental (Exp) Groups (dependent variable: total cartilage volume mm³)

					95% Confidence Interval
(I) Group	(J) Group	Mean Difference (I-J)	Std. Error	Sig.	Lower Bound	Upper Bound
* The mean difference is significant at the 0.05 level.
1 Baseline	2 Sham	−0.043	0.026	0.114	−0.097	0.010
	3 Exp	0.088(*)	0.026	0.002	0.035	0.141
2 Sham	1 Baseline	0.043	0.026	0.114	−0.010	0.097
	3 Exp	0.131(*)	0.023	0.000	0.084	0.178
3 Exp	1 Baseline	−0.088(*)	0.026	0.002	−0.141	−0.035
	2 Sham	−0.131(*)	0.023	0.000	−0.178	−0.084

Figure 1.

Oscillating vibration machine. Mice were placed on the vibrating platform with the oscillating machine connected to power through its power supply and the transformer. The control module allowed for the onset of vibrations and monitored their duration.

Figure 2.

Condylar process rendered into a three-dimensional view, with the segmented cartilage (top wavy section) distinguished from the underlying bone tissue.

Figure 3.

Extraction of bone volume of interest for histomorphometric analysis. (a) Localization of a Volume of Interest (VOI, 0.0452 mm³ dark gray) of trabecular structure derived from the endochondral bone beneath condylar cartilage. (b) Voxel dataset of the VOI trabecular bone obtained with CT Analyzer software for histomorphometric measurement.

Footnotes

Notes

Acknowledgements

This work was supported by the Discipline of Orthodontics, University of Sydney, and the Australian Society of Orthodontists. We thank Associate Professor Gang Shen for his help in the preparation of this manuscript.

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