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Trabecular Bone Structures in the Edentulous Diastema of Osteoporotic Sheep
1 Department of Oral Surgery, Medical University Vienna, Währingerstrasse 25a, A-1090 Vienna, Austria; Correspondence: * corresponding author, reinhard.gruber{at}meduniwien.ac.at
The edentulous ovine diastema represents a suitable region for implantological research. Due to distinctive embryonic origin and mechanical loading, the edentulous diastema may respond differently to osteoporosis than tooth-bearing areas. To test this assumption, we subjected geriatric sheep to ovariectomy, calcium-/vitamin-D-restricted diet, and methylprednisolone administration. Adult control sheep remained untreated. Structural parameters and bone mineral density were determined by microcomputed tomography and conventional computed tomography, respectively. We report that the trabecular microstructure in the diastema was preserved from catabolic changes. In contrast, the premolar maxillary region of osteoporotic sheep had diminished trabecular bone mineral density, with the corresponding structural deteriorations. These results suggest that maxillary trabecular bone of the edentulous diastema does not respond to catabolic changes which occur in the tooth-bearing area in osteoporosis. Our findings imply that regional anatomic domains must be considered in the planning of pre-clinical studies, taking osteoporotic changes into account.
Key Words: maxilla • osteoporosis • sheep • µCT • trabecular bone
With increasing life expectancy in coming decades, the number of elderly people is predicted to increase. Population demographics further implicate an increase in the number of persons with osteoporosis (Riggs and Melton, 1995; Sambrook and Cooper, 2006). Accordingly, the demand of elderly persons with osteoporosis for implant-supported prosthodontics will likely increase over the decades. Aging and osteoporosis, however, are associated with structural changes of the alveolar bone, including the posterior maxilla (Ulm et al., 1997, 1999). Deterioration of the maxillary microstructure requires sophisticated surgical techniques for a stable implant fixation (Martinez et al., 2001; Molly, 2006). Thus, there is a clear demand for pre-clinical models taking into account the physiologically determinant age and clinical conditions that lead to bone loss, such as hypogonadism, malnutrition, and glucocorticoids (Canalis et al., 2007; Holick, 2007). Pre-clinical models must meet at least two requirements: first, a systemic bone mineral density 2.5 standard deviations below peak bone mass of adult animals, according to the clinical WHO definition of osteoporosis (Genant et al., 1999); and second, a compromised maxillary bone microstructure in the region of future augmentation or implant placement. Sheep are considered suitable for pre-clinical testing in oral implant research (Haas et al., 1998; 2002). Sheep show systemic bone loss and reduced mandibular bone mass following ovariectomy (Johnson et al., 2002). However, the magnitude of bone loss does not reach the WHO threshold level (Egermann et al., 2008), and the detrimental effect of ovariectomy is reversible (Sigrist et al., 2007). To achieve a permanent and substantial bone loss, we performed ovariectomies on geriatric sheep, treated them with glucocorticoids, and fed them a calcium-/vitamin-D-restricted diet (Goldhahn et al., 2005). The amount of systemic bone loss satisfies the clinical definition of osteoporosis (Raisz, 2005; Egermann et al., 2008). Whether and how maxillary bone microstructure is influenced in this osteoporosis model remain unknown. The sheep maxilla is different from that in humans, since an edentulous diastema separates incisors from molars (Thomason et al., 2001). In the diastema region, rudimentary tooth anlagen regress and do not develop into teeth (Tucker and Sharpe, 2004). Thus, the diastema region does not require tooth extraction and may represent a suitable area for pre-clinical studies of oral implant research. However, bone from both the diastema and the tooth-bearing area is distinct with regard to embryonic origin and mechanical loading (Thomason et al., 2001; Hovorakova et al., 2005). Bone from the diastema and the tooth-bearing area may respond differently to systemic changes caused by osteoporosis. It can be questioned whether osteoporotic microstructural changes in the diastema region exhibit the same magnitude as in the premolar region of the posterior maxilla. If the diastema region is less subjected to functional loading than the premolars, we would expect that the changes in trabecular bone structure are more pronounced in the diastema. In this study, we report on bone microstructure and bone mass in the diastema and the tooth-bearing region of the maxilla in geriatric ovariectomized sheep treated with glucocorticoids and fed a calcium-/vitamin-D-restricted diet. Using this animal model, we have investigated whether catabolic changes follow regional anatomic domains of the diastema and the premolar region.
Animals Approval for this animal study was obtained from the Animal Experimentation Commission of the Veterinary Office of the Canton of Grison, Switzerland, and strictly followed the guidelines of the Swiss Federal Veterinary Office for the use and care of laboratory animals. Osteoporosis induction took place at the AO Research Institute in Davos, Switzerland. Diagnostic processing of the sheep heads was performed at the Medical University of Vienna, Austria. Twelve female Swiss White Mountain sheep were divided into two groups. The animals of the control group were between 3.2 and 5.2 yrs old (mean, 3.8 ± 0.9), with an average weight of 62.8 ± 12.2 kg. The sheep of the osteoporosis group were between 6.9 and 9.0 yrs old (mean, 7.5 ± 1.0) and weighed 65.6 ± 9.6 kg on average (Table 1  ).
Osteoporosis Induction Bilateral ovariectomy (OVX) was performed through a low median laparotomy under general anesthesia with Isofluran (Halocarbon Laboratories, River Edge, NJ, USA) and intravenous Temgesic (buprenorphine HCl, 0.3 mg/mL, Reckitt & Colman Pharmaceutics, Hull, UK). The control group was fed a standard diet with normal calcium and vitamin D levels (5 g calcium and 1000 IU vitamin D3/day) and received no surgical intervention. The diet for the osteoporosis group contained just 1.5 g calcium and 100 IU vitamin D3 a day (Eberle Nafag AG, Gossau, Switzerland). The total amount of 2000 mg methylprednisolone solution (Streuli & Co AG Pharmazeutika, 8730 Uznach, Switzerland) was given to the animals by intramuscular injection. Steroid application and the calcium-/vitamin-D-restricted diet began 2 wks following ovariectomy over a period of 4 to 8 mos (Egermann et al., 2008) (Table 1  ). The clinical condition of all animals was documented daily. Animals were killed by intravenous administration of 20 mL Pentobarbital 162 mg/mL (Vetanarcol®, Intervet, Vienna, Austria).
Quantitative CT Imaging
MicroCT Imaging MicroCT scanning was performed with an industrial device (RayScan 250E, Wälischmiller, Meersburg, Germany). The scans were performed on the right maxilla (150 kV, 1 sec integration time, 900 projections, 1024 x 1024 a-Si flat panel detector, cone-beam reconstruction). A region of 330 mm³ volume above the highest root of the 2nd premolar and the edentulous diastema was chosen for evaluation (Fig. 2 ). Nominal isotropic resolution was 70 µm, the dataset was around 1000 x 1000 x 1000 voxels, and the analysis region was represented by 100 microtomographic slices. Trabecular and cortical regions were separated by semi-automatically drawn contours, and the complete trabecular structure of the chosen region was evaluated. The grey-scale images were Gaussian-filtered ( = 1.2, support = 2), with a threshold (7.0% of maximum grey value) to form binary images on which morphological analyses were performed. We used three-dimensional analysis techniques (Image Processing Language, Scanco Medical, Bassersdorf, Switzerland) to assess bone volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), and structural model index (SMI). The structure model index quantifies the characteristic form of the cancellous bone in terms of plate-like to rod-like. For an ideal plate and rod structure, this index is 0 and 3, respectively (Hildebrand and Ruegsegger, 1997).
Statistical Analysis Descriptive statistics of all variables were determined, including means and standard deviations. Results were considered to be significant at p < 0.05. The results of individual animals and group averages were plotted. We used a one-way ANOVA to analyze the differences between groups.
Clinical Condition of the Sheep Healing after ovariectomy was uneventful in all animals. During osteoporosis induction, some adverse side-effects appeared. Three animals from the osteoporosis group developed abscesses at the injection sites, which were caused by an infection with Mycobacterium pseudotuberculosis. After incision and regular wound care, these healed uneventfully after a few days.
Quantitative CT Imaging
MicroCT Imaging
Pre-clinical models reflecting the compromised situation of maxillary bone in adult persons with osteoporosis are critical for progress in oral implant research. However, the impact of catabolic bone turnover on different regions in the maxilla is unknown. To determine the suitability of the edentulous diastema for pre-clinical studies in an osteoporotic background, we examined whether catabolic changes become apparent in the diastema region, and if the tooth-bearing regions respond differently. We therefore performed microCT analysis of the maxillary trabecular compartment and compared the structural parameters from geriatric osteoporotic sheep with those of adult sheep. The main observation of this study is that aging and osteoporosis induction considerably reduced the structural parameters of the premolar region, but not of the diastema region. Ovariectomy, malnutrition, and glucocorticoid application in sheep cause a rapid and sustained decline in bone mass of the appendicular skeleton, according to the WHO definition of osteoporosis (Lill et al., 2002a,b). The osteoporotic sheep have a reported decrease in structural parameters of bone volume (26%, 30%, 57%) and trabecular thickness (24%, 68%, 50%) in the femur head, vertebra, and iliac crest, respectively (Lill et al., 2002b). Using the same animal model, we obtained evidence that these catabolic changes also occurred in the premolar region of the maxilla, with a 64% decrease in bone volume ratio accompanied by a 33% thinning of trabecular thickness. Structural changes of the trabeculae were associated with a shift from a plate-like to a rod-like structure, based on the structure model index algorithm (Hildebrand and Ruegsegger, 1997). Moreover, a considerable decrease in premolar trabecular bone mineral density was observed. The results of our study are consistent with the assumption that osteoporosis is a systemic disease affecting all bones, at least in the trabecular compartment. However, our finding that the diastema region in osteoporotic sheep is protected from the catabolic changes suggests that regional anatomic domains respond in their individual way to changes in systemic bone remodeling. The mechanisms that underlie the absence of catabolic changes in the diastema remain unknown. Supported by the concept of Urist and Reddi proposing that the response of bone is individually affected by the embryologic origin and the specific mechanical input (Reddi, 1975), a potential explanation may be that the different embryologic origins and local biomechanical strain are responsible for the preservation of trabecular structure. This concept is in accord with observations in ovariectomized rats in which bone loss was locally increased in the tibia, compared with bone loss in the mandible (Mavropoulos et al., 2007). Furthermore, bone loss was increased in the mandible, lacking the antagonist, as a response to diminished loading by the extracted antagonist (Elovic et al., 1995). Nevertheless, these studies do not necessarily support our findings that trabecular bone reacts differently, depending on the localization, in the same skeletal structure. Studies will be required to assess whether and by which mechanism trabecular bone in the appendicular skeleton may also be protected from systemic bone loss in osteoporosis. Although the posterior maxilla is a critical anatomical region in implant dentistry, clinical evidence about an association with bone loss in axial and appendicular skeleton is rather weak (von Wowern and Worsaae, 1988; Drage et al., 2007). Most studies were performed on mandibles with the more cortical appearance (von Wowern, 2001; Lindh et al., 2004; Devlin et al., 2007). The reason for this may be related to the difficulties of densitometric evaluation in maxillary bone compared with the different regions of the mandible, and the limited potential of CT to differentiate trabecular from cortical bone (Stoppie et al., 2006). Moreover, like ovariectomy, aging, glucocorticoid administration, and vitamin D deficiency are associated with systemic bone loss and structural changes in the alveolar bone (von Wowern et al., 1992; Hildebolt et al., 2004). Therefore, the impact of each factor on the overall structural changes observed in the premolar region of our animal model requires further investigation. We do not know if the edentulous premolar region shows the same response and resembles the diastema region by preserving the trabecular structure. Eventually, the impact of osteoporosis is different in the human edentulous situation and more pronounced than in the ovine diastema model. In conclusion, our results demonstrate that the diastema region of sheep undergoing ovariectomy, and experiencing malnutrition and glucocorticoid application, resists the catabolic changes that cause bone loss in the premolar region. The preservation of trabecular bone structure in the diastema suggests that regional anatomic domains respond differently to systemic changes in bone turnover. It will be important to reveal the underlying mechanism to determine a target that helps to preserve bone strength in osteoporosis. Also, studies will be required to determine if the data are exclusive to the diastema region, which is restricted to animals, or if edentulous regions may behave similarly. Finally, it has been suggested that the diastema region in the sheep model does not represent a tool to simulate the structural changes required for a relevant pre-clinical model.
The authors acknowledge Prof. Andre Gahleitner for valuable comments on the manuscript. This study was supported by the Department of Oral Surgery, Medical University of Vienna, Austria. Received for publication January 29, 2008. Revision received June 3, 2008. Accepted for publication June 18, 2008.
Journal of Dental Research, Vol. 87, No. 9,
866-870 (2008)
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