This study was designed to investigate the impact of the simvastatin delivery system (gel injection or gel/dome implants) on bone growth and inflammation, and the histologic and mechanical characteristics of new bone induced by these applications after various times. This can help determine how topical simvastatin could be applied clinically to increase bone thickness. For this purpose, a bilateral mandible model using mature female Sprague Dawley rats was used and bone surface was stimulated without making a surgical defect, to exclude natural bone healing and to simulate adding to thin bone. Mature rats do not show active bone growth and only represent remodeling similar to adult humans.
Local application has a therapeutic advantage by preventing systemic side-effects and focusing the drug dose. 0.5 mg SIM in rats which weigh approximately 300 g is equal to 1.7 mg/kg. The clinically effective dosage range to treat hypercholesterol in humans by oral administration is up to 1.0 mg/kg/day. However, a topical dose affects a localized area of bone, whether in a 70 kg human or 0.3 kg rat. Even with weekly 0.5 mg injections, the 1.5 mg/kg/week compares favorably with the 7 mg/kg/week in human oral regimens. In addition, simvastatin has a half-life of 1–3 hours [21
] and tends to concentrate at very low levels in peripheral tissue within the first two hours after administration [22
]. Therefore, local doses in the studied range should be well tolerated in the human. Animals in the current study tolerated the implanted simvastatin dome and injection of simvastatin gel with minimal adverse effects. All animals looked normal and active immediately after recovery from the anesthesia used for the application of drug. Weight loss at 3 days was 14–21 g; however, after 24 days weight loss was reduced significantly to a minimal 7–9 g.
The soft tissue swelling noted in this study was less following injections than dome implantations. The swellings were firm and histologically comprised overwhelmingly of fibroblasts and collagen concentrated around the periosteum with very few inflammatory cells (lymphocytes, neutrophils, macrophages). It is possible that simvastatin particles themselves in gel cause irritation which could stimulate bone formation. This surely has some impact, as the distance between the fibrous response in the soft tissue and the new bone growth (area) at 7 days was negatively correlated (r = −0.52, p < 0.0001).
A single injection of SIM showed limited bone formation, suggesting it was unable to maintain the needed concentration or that multiple pulsing doses give an additive or synergistic effect (). The cumulative and integrated effects of the three injections resulted in equivalent or greater bone growth and periosteal cellular activity compared to surgical dome implantation, but with much less swelling (). In fact, the three-injection SIM-INJ protocol nearly doubled the width of the mandible compared to the GEL-INJ, adding an average of one millimeter of new bone in this small animal. While multiple injections to the rat mandible gave an additive effect on increasing bone thickness, a 58% increase in bone thickness occurred in mouse calvaria after a single injection of 2.2 mg SIM-GEL in a previous study, but the dimension of new bone thickness was much less (< 0.1 mm) and was associated with considerable inflammation [23
]. In addition, the amount of new bone thickness appeared to be associated with original bone size, suggesting that the amount of new bone using the same technique on human-sized alveolar bone should be dramatically thicker than one millimeter and therefore clinically relevant.
Significantly higher osteoblast surface at 7 days and BFR between 15 and 22 days () on the GEL-DOME side compared to the GEL-INJ side (single injection; p = 0.03) indicated that surgical trauma played a role in dome stimulation of bone formation. The possibility that a systemic effect from locally applied simvastatin carried over to the control side was discounted in an earlier study [17
], where data used to calculate mandibular area showed that bone growth on the gel alone side was not different in animals treated on the contralateral side with simvastatin versus untreated mandibles. Swelling and infiltration were still present on the gel side only, presumably due to surgical trauma.
Multiple injections of simvastatin caused more fibroblast-like cells and collagen-like tissue near new bone than SIM–DOME, often contiguous with the periosteum (). Statins have been shown to stimulate bone formation in bone marrow-derived mesenchymal stem cells [24
], and induced osteoblastic differentiation in bone marrow cells [25
]. It was not possible with the histologic approach used in this study to determine how many of the fibroblast-like cells were osteoblast precursors or stem cells, from where the fibroblast-like cells originated, or what is the precise role of these fibroblast-like cells.
Judging from the decalcified bone data, osteoblast activity was on the decline after 7 days. Therefore, the week between calcein incorporation (days 15 and 22) in the undecalcified specimens was catching bone formation on the downswing after a single injection of SIM. It is noted, however, that bone formation was still a predominant activity during that period on the lateral bone surface. There was almost no resorption activity preceding bone formation at day 3 or 7. This result is consistent with in vitro studies showing a decreased measure of osteoclastic potential (RANKL/osteoprotegrin mRNA) caused by simvastatin in mouse bone cell culture [27
]. In a recent report, twice weekly injections of simvastatin appeared to reduce bone resorption in a ligature-induced rat periodontitis model [28
]. However, in our studies, osteoclast surface was 4% of the total bone surface at 24 days, implying a remodeling process at that later time. Bone modeling can be defined by continuous bone formation without the necessity of a previous resorption phase, so we consider simvastatin-induced bone formation as a modeling, not remodeling process. However, later remodeling resulted in either bone resorption and reshaping of the new bone. This remodeling response and effects were evaluated in long–term studies of the fate of the new bone in a non-defect, non-implant model at 90 days after application of drug. About 45–75% of the ratio of new bone to old bone at 24 days was reduced at 90 days after simvastatin application (). This resorption may be caused by the tension of overlying tissue and the non-physiologic shape without functional loading. This implies that stressing bone with an implant or root structure providing proper loading is an essential requisite for protection of functional bone. In spite of the limitations of the current model, however, 55% of the maximum new bone formed following multiple SIM-INJ was retained at 90 days, significantly greater than controls.
We measured mechanical properties of mandibles at 24 and 90 days to characterize the new bone, which was woven bone at 24 days with a highly increased bone formation rate (). Increased bone volume was expected to increase both fracture force and stiffness. This was observed at 24 days for the injection sites, where these mechanical properties were 1.4–1.5 times higher than controls. Bone remodeling by 90 days reduced bone volume, lowered fracture force by 30% and stiffness by 25%, with values not significantly different than controls. However, the observation that injection sites maintained mechanical properties that were 1.3 times higher that controls implies that new mature bone was present and exerting mechanical advantage to existing bone structure. Dome placement, on the other hand, was ineffective at producing improved mechanical performance at either 24 or 90 days.