Three months after the intraosseous injection, the maximum pull-out strength for the groups treated with 0.5 and 1.0 mg of simvastatin significantly increased by 45.75% and 51.53%, respectively (p ≤ 0.0012), and the stiffness more than doubled compared with that for vehicle alone (p ≤ 0.0012) (Table III).
Histologically, Goldner trichrome staining revealed small amounts of bone surrounding the screws in the vehicle-only group, whereas a large amount of peri-implant trabecular bone and newly formed bone around the implant were found in the simvastatin groups (Fig. 3). Calcein green and alizarin red double-fluorescent labeling confirmed that there was more bone formation and mineralization in the groups injected with simvastatin than those injected with vehicle alone, soon after simvastatin injection (Fig. 4). Significant increases in the dynamic bone formation parameters (MS/BS, MAR, and BFR/BS) were also observed in the simvastatin-treated groups (p ≤ 0.023) (Table IV).
Western blot analysis showed higher expression levels of BMP-2 and VEGF in the simvastatin-treated groups than that in the vehicle-only group (Fig. 5).
Many techniques have been described to augment fixation, including local cement augmentation with PMMA7,35. PMMA can only physically enhance the strength of osteoporotic bone6,7, and exothermic polymerization reaction, potential leakage, and the presence of a permanent foreign body in the vertebra with PMMA may cause osseous or neural tissue injury6,36-38. Local applications of bone anabolic agents, such as BMPs9-11, promote bone formation at a specific site and have numerous advantages compared with systemic treatments in terms of therapeutic efficiency and tolerance5,39. This topical treatment approach could strengthen implant fixation in osteoporotic bone.
Simvastatin has been used safely for many years. A number of studies have demonstrated that simvastatin can significantly increase bone formation18,23,24,40,41. Both a 10-mg/kg intraperitoneal injection of simvastatin daily for 30 days40 and an oral dosage of 5 mg/kg/d for 28 days41 improved osseointegration in osteoporotic rats. The systemic administration of statins necessitates a high daily dose to account for hepatic clearance and likely elicits unwanted side effects, such as increasing the risk of liver failure, kidney disease, rhabdomyolysis, and myalgia42. Local application of simvastatin could promote bone formation23,24,43, and a single intraosseous injection of simvastatin (5 or 10 mg) significantly improved BMD, bone microstructure, and biomechanical strength of osteoporotic bone43 and enhanced implant fixation in osteoporotic rats32. However, the dose required for an intraosseous injection is still high.
Agents delivered through intraosseous injection may reach the circulation and be cleared essentially as quickly as those injected intravenously25-27. Poloxamer 407 is generally considered to be biocompatible and demonstrates low toxicity and weak immunogenicity, and it is cleared by the U.S. Food and Drug Administration (FDA) for ophthalmic, oral, periodontal, and topical use44. At a concentration of ≥20%, it exhibits reversible thermosensitive gelation28-30.
In the current study, we found that an intraosseous injection of simvastatin in poloxamer 407 increased the BMD in ovariectomized minipigs. However, not completely consistent with the finding that bone mass continuously decreases in untreated osteoporosis30,45, the BMD of the vehicle-only group increased slightly, although it had decreased by 3 months after injection in the untreated L3 vertebra compared with before injection (p = 0.066). The injury of a drill-hole itself is known to stimulate bone formation in the short term46,47, so extending the period of observation might have revealed a decrease in the BMD in the vehicle-only group. Considering the sample size and short duration of this study, we postulate that vertebral BMD of calcium-restricted ovariectomized minipigs might decrease substantially over 3 months without treatment.
BMPs and VEGF have been widely investigated for bone regeneration. However, these cytokines are expensive and have a relatively short half-life. Small-molecule drugs that can be processed into sustained-delivery vehicles and produce endogenous BMPs and/or VEGF are valuable in bone-tissue engineering21. A variety of studies have shown that statins increase the expression of BMP-2 by bone cells16,17. In addition, a single intraosseous injection of simvastatin (5 or 10 mg) in ovariectomized rats increased the expression of VEGF and promoted angiogenesis, thereby augmenting bone formation and enhancing implant fixation32. The increased bone formation and implant fixation observed after an intraosseous injection of the simvastatin in poloxamer 407 hydrogel may be related to increases in autogenous VEGF and BMP-2 expression, which play an essential role in bone formation. Additional research is needed to assess whether this simvastatin approach is comparable with or better than exogenous BMPs and/or VEGF. In addition, bone turnover and osteoporosis are associated with inflammation48,49, and statins have anti-inflammatory and antimicrobial properties that are helpful for implant fixation21.
There were several limitations to the present study. First, we only used 0.5 and 1 mg of simvastatin for the local intraosseous injections, and the animals were only observed for 3 months post-injection. The differences in responses to the 2 doses were not significant, suggesting that a lower dose of simvastatin might be effective. Finding the optimal dose of simvastatin was not the goal of the current study, but is an interesting future research topic. Second, we chose bilaterally ovariectomized Guangxi Bama minipigs and investigated the effects of a local simvastatin injection on bone augmentation and pedicle fixation under consistent ovariectomy conditions. However, we did not explicitly test the exact effects of ovariectomy on bone loss in Guangxi Bama minipigs by using a sham ovariectomized group. Although the Guangxi Bama minipig has a body weight that is close to that of an adult human and is widely used in preclinical studies50-52, as an osteoporotic model, it might not respond exactly the same as other minipig strains, such as the Sinclair S-153-55. Our future preclinical studies of osteoporosis will therefore need to include a normal, age-matched cohort as a control.
Given that thermosensitive simvastatin in poloxamer 407 gel is convenient in terms of handling and ease of application, the encouraging results of its response in the present study provide a rationale for evaluating the effect of an intraosseous injection of simvastatin in poloxamer 407 on implant fixation in patients with osteoporosis.
In summary, a single intraosseous injection of a small dose of simvastatin resulted in significant bone augmentation and enhanced the stability of pedicle screws in the vertebrae of calcium-restricted ovariectomized minipigs, a large bone-remodeling species. It therefore potentially provides an adjunctive method for enhancing implant fixation in patients with osteoporosis.
A table showing the distribution of lumbar vertebrae with respect to the randomized treatment assignment as well as photographs showing the “sol-gel” characteristics of the simvastatin in poloxamer 407 hydrogel and the setup used for the biomechanical pull-out testing; a diagram of the titanium alloy pedicle screw used in the study; and an axial radiograph demonstrating intrapedicular screw placement are available with the online version of this article as a data supplement at jbjs.org.
NOTE: The authors thank Guohong Du, BM, for assistance with DXA detection, Hong Wang, MM, for assistance with histology, Hong Cai, MD, for the excellent design of the pedicle screws, and Kuo Zhang, VMD, for assistance with anesthesia for the animals.
Investigation performed at the Departments of Orthopedics and Neurology, Peking University Third Hospital, and Beijing Key Laboratory of Spinal Diseases, Beijing, People’s Republic of China
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