Intraosseous Injection of Simvastatin in Poloxamer 407 Hydrogel Improves Pedicle-Screw Fixation in Ovariectomized Minipigs

Fu, X. MD; Tan, J. MD; Sun, C.G. MD; Leng, H.J. PhD; Xu, Y.S. MD, PhD; Song, C.L. MD, PhD

Journal of Bone & Joint Surgery - American Volume:
doi: 10.2106/JBJS.15.00937
Scientific Articles
Abstract

Background: Osteoporosis leads to poor osseointegration and reduces implant stability. Statins have been found to stimulate bone formation, but the bioavailability from oral administration is low. Local application may be more effective at augmenting bone formation and enhancing implant stability. This study was performed to evaluate the efficacy of an intraosseous injection of simvastatin in thermosensitive poloxamer 407 hydrogel to enhance pedicle-screw fixation in calcium-restricted ovariectomized minipigs.

Methods: Nine mature female Guangxi Bama minipigs underwent bilateral ovariectomy and were fed a calcium-restricted diet for 18 months. Simvastatin (0, 0.5, or 1 mg) in thermosensitive poloxamer 407 hydrogel was injected into the lumbar vertebrae (L4-L6), and titanium alloy pedicle screws were implanted. Bone mineral density (BMD) measurements of the lumbar vertebrae were determined by dual x-ray absorptiometry (DXA) before and 3 months after treatment. The lumbar vertebrae were harvested and analyzed with use of microcomputed tomography, biomechanical pull-out testing, histological analysis, and Western blot analysis for bone morphogenetic protein (BMP)-2 and vascular endothelial growth factor (VEGF) expression.

Results: Evaluation over a 3-month study period demonstrated that the BMD of the vertebrae injected with 0.5 and 1.0 mg of simvastatin had increased by 31.25% and 31.09%, respectively, compared with vehicle-only injection (p ≤ 0.00014 for both) and increased by 32.12% and 28.16%, respectively, compared with the pre-treatment levels (p < 0.0001 for both). A single injection of simvastatin in poloxamer 407 increased trabecular volume fraction, thickness, and number and decreased trabecular separation (p ≤ 0.002). The bone formation and mineral apposition rates significantly increased (p ≤ 0.023). The percentage of osseointegration in the simvastatin 0.5 and 1-mg groups was 46.54% and 42.63% greater, respectively, than that in the vehicle-only group (p ≤ 0.006), and the maximum pull-out strength was 45.75% and 51.53% greater, respectively, than in the vehicle-only group (p ≤ 0.0005). BMP-2 and VEGF expressions were higher than for the vehicle-only injection.

Conclusions: A single intraosseous injection of simvastatin in thermosensitive poloxamer 407 hydrogel stimulated bone formation, increased BMD, improved bone microstructure, promoted osseointegration, and significantly enhanced the stability of pedicle screws in calcium-restricted ovariectomized minipigs.

Clinical Relevance: These results provide rationale for evaluating intraosseous injection of simvastatin in poloxamer 407 to enhance implant fixation in patients with osteoporosis.

Author Information

1Departments of Orthopedics (X.F., J.T., C.G.S., H.J.L., and C.L.S.) and Neurology (Y.S.X.), Peking University Third Hospital, Beijing, People’s Republic of China

2Beijing Key Laboratory of Spinal Diseases, Beijing, People’s Republic of China

E-mail address for C.L. Song: schl@bjmu.edu.cn

Article Outline

Osteoporosis is highly prevalent and often undiagnosed or untreated in the elderly and postmenopausal women1. In orthopaedic surgery, osteoporosis leads to poor osseointegration and reduces implant stability, which is a well-known complication occurring most frequently in patients with osteoporosis2-4. Although various surgical techniques and implants have been designed to increase load-sharing, fixation in osteoporotic bone remains a major challenge5. Bone cements such as polymethylmethacrylate (PMMA) improve fixation physically but not physiologically6,7. Bioresorbable calcium phosphate does not improve fixation and provides only temporary improvement during early rehabilitation8. The authors of several recent studies have reported that locally used recombinant human bone morphogenetic proteins (rhBMPs)9-11 and bisphosphonates12,13 can augment targeted bone and increase the pull-out strength of screws implanted in osteoporotic bone. However, as biological agents, rhBMPs are expensive. Bisphosphonates do not have a direct effect on bone formation. Bone remodeling suppression by bisphosphonates is closely linked to microdamage accumulation and impairs the targeted repair of damage14,15.

Statins, which are inhibitors of HMG-CoA (3-hydroxy-3-methylglutaryl-coenyzyme A) reductase, have been used for many years to treat hyperlipidemia and to prevent cardiovascular events. Mundy et al. first reported on the anabolic effects of statins on bone formation16. Simvastatin was demonstrated to stimulate osteoblastic differentiation in vitro17, and a high dosage with a long duration of oral administration promoted bone formation in ovariectomized rats18. However, the anabolic effect of statins on bone formation remains controversial19-21. Possibly, this is either because the dosage used for cardiovascular effects is suboptimal for bone formation or because the concentration available to bone via oral delivery is limited21. The primary organ that is targeted by statins is the liver. Less than 5% of orally administered statins reach systemic circulation, and an even lower concentration reaches the skeleton22. Locally applied simvastatin was shown to promote the healing of calvarial bone defects and fractures in rats23,24. We hypothesized that local intraosseous injection of simvastatin may improve its bioavailability for bone formation, thereby enhancing pedicle-screw fixation.

Intraosseous injection is an established, rapid, safe, and effective alternative to peripheral intravenous drug delivery. Agents injected via the intraosseous route can reach the circulation as quickly as those administered intravenously25-27. Poloxamer 407 demonstrates biocompatibility, low toxicity, and weak immunogenicity. It also demonstrates reversible thermosensitive gelation (with the “sol-gel” characteristics of being a liquid at low temperature but a semisolid gel at body temperature), which makes it suitable as a vehicle for drug delivery28-30.

To our knowledge, no data exist regarding the effects of local intraosseous injection of simvastatin on implant fixation in a large animal model. Therefore, the aim of the present study was to verify the efficacy of a single intraosseous injection of simvastatin in thermosensitive poloxamer 407 hydrogel to augment bone formation and enhance the stability of pedicle screws in ovariectomized minipigs.

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Materials and Methods

Experimental Design

Nine mature female Guangxi Bama minipigs (45 to 50 kg) were used in this study. After a 2-week acclimation, all animals were anesthetized and underwent bilateral ovariectomy. The animals were fed a calcium-restricted (0.75% calcium) diet throughout the study4. Eighteen months after ovariectomy, 27 lumbar vertebrae (L4-L6) were randomly assigned to receive an intraosseous injection of 0 mg (vehicle), 0.5 mg, or 1.0 mg of simvastatin (see Appendix). Calcein green (Sigma; 20 mg/kg) and alizarin red (Sigma; 30 mg/kg) were administered intravenously 2 and 3 weeks after treatment, respectively, as double-fluorescent labeling.

Animals were killed 3 months after the injection, and the lumbar vertebrae were resected en bloc and stored at −80°C until further use. The harvested spine was evaluated using portable animal radiography to ensure intrapedicular screw placement. All specimens were evaluated with use of micro-computed tomography (micro-CT), and 6 specimens from each group were randomly selected for biomechanical pull-out testing and Western blot analysis of BMP-2 and vascular endothelial growth factor (VEGF) expression. The remaining 3 specimens from each group were used for histological analysis. To minimize experimental variability, 1 technician, who was blinded to the specimen treatment group, conducted all tests. The study protocols were approved by the Animal Ethics Committee of Peking University Third Hospital and were carried out under the guideline of the Association for Assessment and Accreditation of Laboratory Animal Care.

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Simvastatin in Poloxamer 407 Gel Formulation

Poloxamer 407 (25% w/w; BASF) was added to isotonic phosphate-buffered saline (PBS) solution (pH 7.4, 4°C) with gentle mixing until it dissolved, and simvastatin in poloxamer 407 gels were prepared by adding simvastatin (National Institutes for Food and Drug Control, Beijing, China) to the poloxamer 407 solution as previously described30. The final simvastatin concentrations were 0, 0.5, and 1 mg/mL. The simvastatin in poloxamer 407 hydrogels were thermosensitive, which meant that they were liquid at 4°C and formed a semisolid gel at 37°C (see Appendix).

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Pedicle Screws

The titanium alloy pedicle screw used in this study was designed for suitability with the lumbar vertebrae of cadaveric specimens from pilot adult minipigs, and their dimensions were calculated using a scanned CT image (see Appendix). The pedicle screws were made and provided by Beijing Fito Lanvie Medical.

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Surgical Procedure

Eighteen months after ovariectomy, the minipigs were anesthetized and the L4-L6 vertebrae were exposed with a standard posterior approach. The pedicle-screw starting points were determined before surgery using the pilot adult minipig cadaveric specimens and CT images. At the base of the superior articular facet near the midpoint of the transverse process, the cortical bone at the insertion site was removed with a rongeur, and a guide hole was formed with a probe7,31. Each pedicle at the 3 vertebral levels was prepared and tapped, simvastatin (0, 0.5, or 1 mg) in 1 mL of poloxamer 407 hydrogel was injected into each vertebra via the guide hole on the left pedicle, and the screw was inserted into the pedicle. All operations were performed by a senior spinal surgeon.

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Bone Mineral Density (BMD) Quantification

The BMD of L3 (untreated) and L4-L6 was measured before and 3 months after treatment. Each vertebra was scanned using dual x-ray absorptiometry (DXA) (Discovery system; Hologic), and using the “metal-removal” software, the BMD of each vertebra, excluding the implanted screws, was analyzed by 1 technician blinded to the specimen treatment group (Fig. 1).

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Micro-CT Scanning and Bone Microstructure Analysis

The specimens were placed in a sample holder with PBS solution and scanned (Inveon; Siemens Medical Solutions U.S.A.) at a spatial resolution of 36 μm, 80 kV/500 μA, a 900-ms integration time, and 360 projections per 360°, as previously described30. A volume of interest (VOI) was defined on the reconstructed images as the trabecular bone region 7 mm in diameter and 10 mm in length starting from the pedicle screw tip along the long axis of the screw. Inveon software was used to automatically calculate 3-dimensional (3D) parameters for the bone in the VOI of each specimen32, which included the bone volume per total volume (BV/TV), trabecular number (Tb.N), average trabecular thickness (Tb.Th), average trabecular separation (Tb.Sp), and percentage of osseointegration (%OI).

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Biomechanical Pull-Out Testing

Six specimens randomly selected from each group were tested to pull-out failure using a Bionix Servohydraulic Test System (MTS Systems). The specimens were thawed for 12 hours to reach room temperature. The screw was fixed in a 100-mm cylindrical rod with an inner thread that matched the outer thread of the screw tail, and was attached such that the axis of the testing machine was oriented along the screw axis (see Appendix). Each screw was pulled out at a constant rate of 5 mm/min until purchase failure33. The force and displacement data were recorded. The maximum pull-out strength and stiffness were measured for comparison.

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Histological Analysis

The remaining 3 vertebrae from each group were fixed in 10% formalin for 2 weeks, dehydrated in a graded series of ethanol solutions for 48 hours under vacuum, and embedded in PMMA. Sections were cut parallel to the screw axis and were ground to a thickness of 50 to 70 μm using a sawing and polishing system (EXAKT). Sections were stained with Goldner trichrome and observed with light microscopy. The bone mineral apposition, as labeled by the fluorescence, was observed with fluorescence microscopy (DM3000; Leica). Dynamic indices of bone formation, including the rates of mineralizing surface per bone surface (MS/BS), mineral apposition (MAR), and bone formation per bone surface (BFR/BS), were measured by 1 technician blinded to the specimen treatment group using image analysis software (BIOQUANT OSTEO; BIOQUANT Image Analysis) according to previously published standard guidelines34.

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Western Blot Analysis

After biomechanical pull-out testing, Western blot analysis was performed. Approximately 1 g of the lyophilized sample and 2 stainless steel balls (10 mm in diameter) were introduced into a 35-mL milling jar. After being capped, the jar was placed on the adapters and shaken for 5 minutes at 30 Hz in a mixer mill (MM400; Retsch Technology). The powdered samples were added into RIPA (radioimmunoprecipitation assay) lysis buffer (Cell Signaling Technology), homogenized for 1 minute, kept on ice for 30 minutes, and centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected, and the protein concentration was determined. Equal amounts of protein were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) (10% gels) and incubated with mouse monoclonal anti-VEGF, rabbit polyclonal anti-BMP-2, and anti-β-actin (Santa Cruz Biotechnology). After incubation with the fluorescent secondary antibodies (Santa Cruz Biotechnology) for 2 hours at room temperature, the signals were measured using an Odyssey infrared imaging system (LI-COR Biosciences) as previously described30.

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Data Analysis

The data are presented as the mean and standard deviation. One-way analysis of variance was used to assess the differences among the groups. When significant effects were detected, the differences among the groups were evaluated with Fisher protected least significant difference (LSD) post-hoc comparisons. The BMD measurements before and after treatment for the same dose of simvastatin were evaluated with a paired t test. Statistical analyses were performed using SPSS software (version 20.0; IBM). P values of <0.05 were considered significant.

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Results

All 9 animals were instrumented successfully without neurovascular complications. There were no signs of side effects or morbidity associated with the treatment. A post-harvest radiograph confirmed intrapedicular screw placement (see Appendix).

Three months after treatment, the BMD measurements for the lumbar vertebrae injected with 0.5 and 1.0 mg of simvastatin had increased by 31.25% and 31.09%, respectively, compared with the vehicle-only group (p ≤ 0.00014 for both), and by 32.12% and 28.16%, respectively, compared with the pre-treatment levels (p < 0.0001 for both) (Table I). The BMD measurement in the untreated L3 decreased from a mean of 0.60 ± 0.08 g/cm2 to 0.53 ± 0.07 g/cm2 (p = 0.066).

The 3D micro-CT images clearly indicated improvements in the peri-implant trabecular bone after the intraosseous injection of simvastatin in poloxamer 407 hydrogel (Fig. 2). Compared with vehicle-only injection, 0.5 and 1.0 mg of simvastatin significantly increased the BV/TV by 47.49% and 43.42%, the Tb.Th by 66.09% and 54.28%, and the Tb.N by 49.23% and 39.01%, respectively, and decreased the Tb.Sp by 55.85% and 52.80%, respectively (p ≤ 0.006 for all). The %OI was also 46.54% and 42.63% greater for 0.5 and 1.0 mg of simvastatin, respectively, compared with that of the vehicle-only group (p ≤ 0.006) (Table II).

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).

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Discussion

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.

Bone microstructure also influences strength4. In the present study, we found that a single intraosseous injection of simvastatin in poloxamer 407 hydrogel significantly improved the trabecular microstructure and osseointegration in minipigs. More importantly, the pull-out strength increased significantly.

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.

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Appendix Cited Here...

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

Disclosure: Funding for this study was provided by the National Natural Science Foundation of China (Nos. 81171693, 81100895) and the National High Technology Research and Development Program of China (SS2015AA020304). The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article.

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References

1. van den Bergh JP, van Geel TA, Geusens PP. Osteoporosis, frailty and fracture: implications for case finding and therapy. Nat Rev Rheumatol. 2012 ;8(3):163–72. Epub 2012 Jan 17.
2. Virdi AS, Irish J, Sena K, Liu M, Ke HZ, McNulty MA, Sumner DR. Sclerostin antibody treatment improves implant fixation in a model of severe osteoporosis. J Bone Joint Surg Am. 2015 ;97(2):133–40.
3. Virdi AS, Liu M, Sena K, Maletich J, McNulty M, Ke HZ, Sumner DR. Sclerostin antibody increases bone volume and enhances implant fixation in a rat model. J Bone Joint Surg Am. 2012 ;94(18):1670–80.
4. Borah B, Dufresne TE, Chmielewski PA, Gross GJ, Prenger MC, Phipps RJ. Risedronate preserves trabecular architecture and increases bone strength in vertebra of ovariectomized minipigs as measured by three-dimensional microcomputed tomography. J Bone Miner Res. 2002 ;17(7):1139–47.
5. Torstrick FB, Guldberg RE. Local strategies to prevent and treat osteoporosis. Curr Osteoporos Rep. 2014 ;12(1):33–40.
6. Paré PE, Chappuis JL, Rampersaud R, Agarwala AO, Perra JH, Erkan S, Wu C. Biomechanical evaluation of a novel fenestrated pedicle screw augmented with bone cement in osteoporotic spines. Spine (Phila Pa 1976). 2011 ;36(18):E1210–4.
7. Yi X, Wang Y, Lu H, Li C, Zhu T. Augmentation of pedicle screw fixation strength using an injectable calcium sulfate cement: an in vivo study. Spine (Phila Pa 1976). 2008 ;33(23):2503–9.
8. Mattsson P, Larsson S. Calcium phosphate cement for augmentation did not improve results after internal fixation of displaced femoral neck fractures: a randomized study of 118 patients. Acta Orthop. 2006 ;77(2):251–6.
9. Seeherman HJ, Li XJ, Smith E, Parkington J, Li R, Wozney JM. Intraosseous injection of rhBMP-2/calcium phosphate matrix improves bone structure and strength in the proximal aspect of the femur in chronic ovariectomized nonhuman primates. J Bone Joint Surg Am. 2013 ;95(1):36–47.
10. Seeherman HJ, Li XJ, Smith E, Wozney JM. rhBMP-2/calcium phosphate matrix induces bone formation while limiting transient bone resorption in a nonhuman primate core defect model. J Bone Joint Surg Am. 2012 ;94(19):1765–76.
11. Seeherman HJ, Li XJ, Bouxsein ML, Wozney JM. rhBMP-2 induces transient bone resorption followed by bone formation in a nonhuman primate core-defect model. J Bone Joint Surg Am. 2010 ;92(2):411–26.
12. Qi M, Hu J, Li J, Li J, Dong W, Feng X, Yu J. Effect of zoledronate acid treatment on osseointegration and fixation of implants in autologous iliac bone grafts in ovariectomized rabbits. Bone. 2012 ;50(1):119–27. Epub 2011 Oct 17.
13. Alghamdi HS, Bosco R, Both SK, Iafisco M, Leeuwenburgh SCG, Jansen JA, van den Beucken JJ. Synergistic effects of bisphosphonate and calcium phosphate nanoparticles on peri-implant bone responses in osteoporotic rats. Biomaterials. 2014 ;35(21):5482–90. Epub 2014 Apr 14.
14. Mashiba T, Hirano T, Turner CH, Forwood MR, Johnston CC, Burr DB. Suppressed bone turnover by bisphosphonates increases microdamage accumulation and reduces some biomechanical properties in dog rib. J Bone Miner Res. 2000 ;15(4):613–20.
15. Allen MR, Burr DB. Bisphosphonate effects on bone turnover, microdamage, and mechanical properties: what we think we know and what we know that we don’t know. Bone. 2011 ;49(1):56–65. Epub 2010 Oct 16.
16. Mundy G, Garrett R, Harris S, Chan J, Chen D, Rossini G, Boyce B, Zhao M, Gutierrez G. Stimulation of bone formation in vitro and in rodents by statins. Science. 1999 ;286(5446):1946–9.
17. Song C, Guo Z, Ma Q, Chen Z, Liu Z, Jia H, Dang G. Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells. Biochem Biophys Res Commun. 2003 ;308(3):458–62.
18. Li X, Song QS, Wang JY, Leng HJ, Chen ZQ, Liu ZJ, Dang GT, Song CL. Simvastatin induces estrogen receptor-alpha expression in bone, restores bone loss, and decreases ERα expression and uterine wet weight in ovariectomized rats. J Bone Miner Metab. 2011 ;29(4):396–403. Epub 2010 Nov 10.
19. Patil S, Holt G, Raby N, McLellan AR, Smith K, O’Kane S, Beastall G, Crossan JF. Prospective, double blind, randomized, controlled trial of simvastatin in human fracture healing. J Orthop Res. 2009 ;27(3):281–5.
20. Tsartsalis AN, Dokos C, Kaiafa GD, Tsartsalis DN, Kattamis A, Hatzitolios AI, Savopoulos CG. Statins, bone formation and osteoporosis: hope or hype? Hormones (Athens). 2012 ;11(2):126–39.
21. Shah SR, Werlang CA, Kasper FK, Mikos AG. Novel applications of statins for bone regeneration. Natl Sci Rev. 2015 ;2(1):85–99. Epub 2014 Aug 16.
22. Schachter M. Chemical, pharmacokinetic and pharmacodynamic properties of statins: an update. Fundam Clin Pharmacol. 2005 ;19(1):117–25.
23. Yueyi C, Xiaoguang H, Jingying W, Quansheng S, Jie T, Xin F, Yingsheng X, Chunli S. Calvarial defect healing by recruitment of autogenous osteogenic stem cells using locally applied simvastatin. Biomaterials. 2013 ;34(37):9373–80. Epub 2013 Sep 7.
24. Fukui T, Ii M, Shoji T, Matsumoto T, Mifune Y, Kawakami Y, Akimaru H, Kawamoto A, Kuroda T, Saito T, Tabata Y, Kuroda R, Kurosaka M, Asahara T. Therapeutic effect of local administration of low-dose simvastatin-conjugated gelatin hydrogel for fracture healing. J Bone Miner Res. 2012 ;27(5):1118–31.
25. Hoskins SL, do Nascimento P Jr, Lima RM, Espana-Tenorio JM, Kramer GC. Pharmacokinetics of intraosseous and central venous drug delivery during cardiopulmonary resuscitation. Resuscitation. 2012 ;83(1):107–12. Epub 2011 Aug 25.
26. Buck ML, Wiggins BS, Sesler JM. Intraosseous drug administration in children and adults during cardiopulmonary resuscitation. Ann Pharmacother. 2007 ;41(10):1679–86. Epub 2007 Aug 14.
27. Paxton JH, Knuth TE, Klausner HA. Proximal humerus intraosseous infusion: a preferred emergency venous access. J Trauma. 2009 ;67(3):606–11.
28. Pillai O, Panchagnula R. Transdermal delivery of insulin from poloxamer gel: ex vivo and in vivo skin permeation studies in rat using iontophoresis and chemical enhancers. J Control Release. 2003 ;89(1):127–40.
29. Djekic L, Krajisnik D, Martinovic M, Djordjevic D, Primorac M. Characterization of gelation process and drug release profile of thermosensitive liquid lecithin/poloxamer 407 based gels as carriers for percutaneous delivery of ibuprofen. Int J Pharm. 2015 ;490(1-2):180–9. Epub 2015 May 19.
30. Tan J, Fu X, Sun CG, Liu C, Zhang XH, Cui YY, Guo Q, Ma T, Wang H, Du GH, Yin X, Liu ZJ, Leng HJ, Xu YS, Song CL. A single CT-guided percutaneous intraosseous injection of thermosensitive simvastatin/poloxamer 407 hydrogel enhances vertebral bone formation in ovariectomized minipigs. Osteoporos Int. 2016 ;27(2):757–67. Epub 2015 Jul 30.
31. Upasani VV, Farnsworth CL, Tomlinson T, Chambers RC, Tsutsui S, Slivka MA, Mahar AT, Newton PO. Pedicle screw surface coatings improve fixation in nonfusion spinal constructs. Spine (Phila Pa 1976). 2009 ;34(4):335–43.
32. Tan J, Yang N, Fu X, Cui Y, Guo Q, Ma T, Yin X, Leng H, Song C. Single-dose local simvastatin injection improves implant fixation via increased angiogenesis and bone formation in an ovariectomized rat model. Med Sci Monit. 2015;21:1428–39. Epub 2015 May 18.
33. Kim YY, Choi WS, Rhyu KW. Assessment of pedicle screw pullout strength based on various screw designs and bone densities-an ex vivo biomechanical study. Spine J. 2012 ;12(2):164–8. Epub 2012 Feb 14.
34. Parfitt AM. Bone histomorphometry: proposed system for standardization of nomenclature, symbols, and units. Calcif Tissue Int. 1988 ;42(5):284–6.
35. Dodwad SM, Khan SN. Surgical stabilization of the spine in the osteoporotic patient. Orthop Clin North Am. 2013 ;44(2):243–9. Epub 2013 Feb 12.
36. Chen LH, Tai CL, Lai PL, Lee DM, Tsai TT, Fu TS, Niu CC, Chen WJ. Pullout strength for cannulated pedicle screws with bone cement augmentation in severely osteoporotic bone: influences of radial hole and pilot hole tapping. Clin Biomech (Bristol, Avon). 2009 ;24(8):613–8. Epub 2009 May 29.
37. Shea TM, Laun J, Gonzalez-Blohm SA, Doulgeris JJ, Lee WE 3rd, Aghayev K, Vrionis FD. Designs and techniques that improve the pullout strength of pedicle screws in osteoporotic vertebrae: current status. Biomed Res Int. 2014;2014:748393. Epub 2014 Mar 3.
38. Belkoff SM, Molloy S. Temperature measurement during polymerization of polymethylmethacrylate cement used for vertebroplasty. Spine (Phila Pa 1976). 2003 ;28(14):1555–9.
39. Stadelmann VA, Gauthier O, Terrier A, Bouler JM, Pioletti DP. Implants delivering bisphosphonate locally increase periprosthetic bone density in an osteoporotic sheep model. A pilot study. Eur Cell Mater. 2008;16:10–6. Epub 2008 Jul 31.
40. Ayukawa Y, Okamura A, Koyano K. Simvastatin promotes osteogenesis around titanium implants. Clin Oral Implants Res. 2004 ;15(3):346–50.
41. Du Z, Chen J, Yan F, Xiao Y. Effects of simvastatin on bone healing around titanium implants in osteoporotic rats. Clin Oral Implants Res. 2009 ;20(2):145–50. Epub 2008 Dec 1.
42. Park JB. The use of simvastatin in bone regeneration. Med Oral Patol Oral Cir Bucal. 2009 ;14(9):e485–8. Epub 2009 Sep 1.
43. Yang N, Cui Y, Tan J, Fu X, Han X, Leng H, Song C. Local injection of a single dose of simvastatin augments osteoporotic bone mass in ovariectomized rats. J Bone Miner Metab. 2014 ;32(3):252–60. Epub 2013 Aug 10.
44. Li C, Li C, Liu Z, Li Q, Yan X, Liu Y, Lu W. Enhancement in bioavailability of ketorolac tromethamine via intranasal in situ hydrogel based on poloxamer 407 and carrageenan. Int J Pharm. 2014 ;474(1-2):123–33. Epub 2014 Aug 17.
45. Jones G, Nguyen T, Sambrook PN, Kelly PJ, Gilbert C, Eisman JA. Symptomatic fracture incidence in elderly men and women: the Dubbo Osteoporosis Epidemiology Study (DOES). Osteoporos Int. 1994 ;4(5):277–82.
46. Uchida S, Sakai A, Kudo H, Otomo H, Watanuki M, Tanaka M, Nagashima M, Nakamura T. Vascular endothelial growth factor is expressed along with its receptors during the healing process of bone and bone marrow after drill-hole injury in rats. Bone. 2003 ;32(5):491–501.
47. He YX, Zhang G, Pan XH, Liu Z, Zheng LZ, Chan CW, Lee KM, Cao YP, Li G, Wei L, Hung LK, Leung KS, Qin L. Impaired bone healing pattern in mice with ovariectomy-induced osteoporosis: a drill-hole defect model. Bone. 2011 ;48(6):1388–400. Epub 2011 Mar 21.
48. Koh JM, Khang YH, Jung CH, Bae S, Kim DJ, Chung YE, Kim GS. Higher circulating hsCRP levels are associated with lower bone mineral density in healthy pre- and postmenopausal women: evidence for a link between systemic inflammation and osteoporosis. Osteoporos Int. 2005 ;16(10):1263–71. Epub 2005 Feb 9.
49. Duggan SN, Purcell C, Kilbane M, O’Keane M, McKenna M, Gaffney P, Ridgway PF, Boran G, Conlon KC. An association between abnormal bone turnover, systemic inflammation, and osteoporosis in patients with chronic pancreatitis: a case-matched study. Am J Gastroenterol. 2015 ;110(2):336–45. Epub 2015 Jan 27.
50. Brown AL, Farhat W, Merguerian PA, Wilson GJ, Khoury AE, Woodhouse KA. 22 week assessment of bladder acellular matrix as a bladder augmentation material in a porcine model. Biomaterials. 2002 ;23(10):2179–90.
51. Jiang G, Li L, Fan J, Zhang B, Oso AO, Xiao C, Yin Y. Dietary soy isoflavones differentially regulate expression of the lipid-metabolic genes in different white adipose tissues of the female Bama mini-pigs. Biochem Biophys Res Commun. 2015 ;461(1):159–64. Epub 2015 Apr 10.
52. Wang X, Cao C, Huang J, Yao J, Hai T, Zheng Q, Wang X, Zhang H, Qin G, Cheng J, Wang Y, Yuan Z, Zhou Q, Wang H, Zhao J. One-step generation of triple gene-targeted pigs using CRISPR/Cas9 system. Sci Rep. 2016;6:20620. Epub 2016 Feb 9.
53. Mosekilde L, Weisbrode SE, Safron JA, Stills HF, Jankowsky ML, Ebert DC, Danielsen CC, Sogaard CH, Franks AF, Stevens ML, et al.. Calcium-restricted ovariectomized Sinclair S-1 minipigs: an animal model of osteopenia and trabecular plate perforation. Bone. 1993 ;14(3):379–82.
54. Lafage MH, Balena R, Battle MA, Shea M, Seedor JG, Klein H, Hayes WC, Rodan GA. Comparison of alendronate and sodium fluoride effects on cancellous and cortical bone in minipigs. A one-year study. J Clin Invest. 1995 ;95(5):2127–33.
55. Bonjour JP, Ammann P, Rizzoli R. Importance of preclinical studies in the development of drugs for treatment of osteoporosis: a review related to the 1998 WHO guidelines. Osteoporos Int. 1999;9(5):379–93.
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