Bone remodeling plays a key role to maintain the skeletal functional integrity. It is a site and time-specific event combining bone resorption and formation. Higher mammals with the harversian system undergo both cancellous and intracortical bone remodeling, while lower mammals without harversian system, such as mice and rats, do not normally undergo intracortical remodeling [
Ovariectomized (OVX) rodents are a well-established animal model in osteoporosis studies [
The intracortical remodeling rate of human jawbones is around 20 times higher than that of iliac crest [
This study investigated the stimulating effect of ovariectomy onto intracortical bone remodeling in rat mandibles.
The animal experiment was approved by the Committee on Use Live Animal for Teaching and Research, The University of Hong Kong. All rats were held in a 12:12 h light-dark circle with each rat separated in a metal cage at 25 degree Celsius room temperature in the Laboratory Animal Unit of the University of Hong Kong. All animals were allowed free access to water and standard rodent diet. Their general well-being was monitored by a 24 h closed circuit television in the holding room during the entire experimental period.
Sixteen 12-week-old, female Spraque-Dawly (SD) rats were randomly assigned into an OVX or a control group, with 8 in each. After a 7-day acclimatization period, the rats of the OVX group underwent a bilateral ovariectomy according to our standardized protocol already published elsewhere [
Fluorochrome labeling using calcein green (15 mg/kg; C0875, Sigma-Aldrich, Saint Louis Mo, USA) and alizarin complexone (30 mg/kg; A3882, Sigma-Aldrich, Saint Louis, MO, USA) was performed 10 days and 1 day before the sacrifice, respectively. The reagents were dissolved in sodium bicarbonate solution and administrated subcutaneously [
All rats were scarified 18 weeks postoperatively. Mandibles were harvested entirely and the attached soft tissue was carefully removed. All samples were fixed in 10% neutral buffered formalin solution for 2 days and then transferred to 70% ethanol for further use.
The changes of trabecular microarchitecture were assessed by micro-CT (SkyScan-1076 X-ray microtomography, SkyScan, Kontich, Belgium) according to the manufacturer’s instructions. The X-ray source was supplied with 88 kV voltage and 100
Trabecular bone in the interradicular septum of the first molar (M1) was selected as the region of interest (ROI) (Figure
ROI selection in micro-CT and histomorphometry assessment. (a) Trabecular bone inside the circle is selected as the ROI in micro-CT assessment. (b) The bone area above the yellow line is defined as alveolar cortical bone; nonalveolar cortical bone is defined as the area below the yellow line except for trabecular bone (square).
Histomorphometric assessment was performed to evaluate the intracortical remodeling. Nondecalcified mandibles were dehydrated in graded ethanol (70%, 95%, and 100%) and embedded in methyl methacrylate (MMA, Technovit 7500, Kulzer, Hamburg, Germany). Mandibles were sectioned (~100
To ensure the measurement was performed at a similar region in all the animals, sections containing the deepest root of first molar in each animal were selected. The sections were examined under fluorescence microscope (Nikon, Tokyo, Japan).
The alveolar cortical bone was defined as the area above the root at M1 region; nonalveolar cortical bone was defined as the area below the root except for trabecular bone (Figure
To assess the intracortical remodeling, the total bone area (B.Ar), the bone surface (BS), the total length of the labeled bone surface (LS), the single labeled bone surface (sLS), the double labeled bone surface (dLS), and the mean interlabel width (Ir.L.Wi) were measured. The mineralizing bone surface (MS) was calculated as 0.5sLS + dLs. Mineral apposition rate (MAR,
Periodontal ligament attached bone surfaces were selected to determine whether bone modeling was affected by OVX. The parameters including MAR and BFR (
All data were presented as mean values ± SD and analyzed by independent
General well-being was defined as normal daily food and water intake, unremarkable behavioral pattern, and normal daily activity of rats during entire experimental period.
In the control group, the trabecular bone presented as a well-connected network, while in the OVX group the trabecular bone withered and became separated. Quantitative analysis revealed that the BMD (
OVX changes the microarchitecture in rat mandibles. Micro-CT images show remarkable trabecular bone loss in OVX rats (a) compared with that in the control (b) group. BMD and BV/TV in OVX group are significantly decreased compared to those in control group. Tb·Th decreases and Tb·Sp increases significantly in OVX group. Tb·N shows no significant difference in both groups. BMD: bone mineral density; BV/TV: bone volume/tissue volume; Tb.Th: trabecular thickness; Tb.N: trabecular number; and Tb.Sp: trabecular separation.
In the alveolar bone region, there was no significant difference related to the bone area between the OVX rats (
Photomicrograph of mandible in OVX rats viewed with fluorescent light. Labeled osteons (indicated by arrow) are found.
Ovariectomy stimulates remodeling activity in mandible of rat. In the alveolar cortical region (a), MAR, surface-based BFR (
In nonalveolar regions, no significant difference in the bone area between OVX rats (
The MAR (
Primates and dogs represent ideal animal models to investigate diseases characterized by abnormal intracortical remodeling. Both models show intracortical remodeling throughout the skeleton, just like humans [
Some studies reported that intracortical remodeling in rodents could be stimulated under pathological conditions [
Jawbones have a unique structure and undergo the highest intracortical remodeling rate throughout the skeleton [
This study investigated radiographic and histomorphometric changes in mandibular cancellous and cortical bones of OVX rats. Consistent with previous studies, both trabecular bone volume and bone mineral density decreased significantly after surgery [
Histomorphometry is considered to be the gold standard in dynamic bone research. Better than other methods which estimate the remodeling status detecting humeral biomarkers, it may provide site-specific information directly by measuring MAR and BFR at different time points using the fluorochrome sequential labeling approach [
As mature mammals routinely undergo remodeling in cortical bones, the place where bone resorption and bone formation occur simultaneously, BFR evaluation might provide insight into the intracortical remodeling rate. In this study, active intracortical bone remodeling characterized by labeled osteons in the cortical bone region was found in both OVX rats and the control group. Rats after sham surgery manifested a very low-rate mandibular remodeling, whereas OVX rats disclosed significantly increased remodeling rates. In the alveolar region, ovariectomy stimulated a six-time higher intracortical BFR compared with that in control animals. These findings were similar to a previous report by Kubek et al. which so far is the only study addressing this issue in mandibles in a mouse model [
In the bone surface attached to the periodontal ligament, where bone modeling primarily takes place [
The study of Kubek et al. [
This study demonstrated that ovariectomy can stimulate intracortical remodeling in rat jawbones, to the authors’ knowledge, for the first time. It might be taken into consideration that OVX rats are useful to study various bone diseases associated with abnormal intracortical bone remodeling processes, such as BRONJ.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by the General Research Fund (HKU785412), Research Grant Council of Hong Kong. The authors appreciate the valuable technical assistance provided by Orthopedic Research Centre, Department of Orthopedics and Traumatology of the Li Ka Shing Faculty of Medicine, The University of Hong Kong.