Osteogenic effect in rat osteoporosis model and femur defect model by intraosseous administration of simvastatin microcrystals

Background : Currently, drugs for local bone formation are very limited in clinic. Simvastatin is one of most potential translational drugs by repositioning for its significant osteogenic effect and its history as a safe lipid-lowering medicine. In this study, we prepared the pre-clinical simvastatin microcrystals which could be potentially translated and industrialized. Methods: Simvastatin microcrystals were made by wet media milling method. Characterization of microcrystal particles were evaluated by laser particle size analyzer and high-performance liquid chromatography (HPLC). Furthermore, we verified the osteogenic effect in rat ovariectomy(OVX)-induced osteoporosis model and femur defect model. For osteoporosis model, we delivered simvastatin microcrystals to tibia with poloxamer hydrogel by intraosseous injection. Bone mineral density (BMD) and ultimate force were assessed after treatment. For femur defect model, simvastatin microcrystal was incorporated in clinically used calcium phosphate cements (CPCs) as an implant. Histology and µCT were used for evaluation of the healing. Results: The D 10 , D 50, and D 90 showed size of the most particles ranged from 0.226-3.425 µm. The mean particle size of the microcrystals is determined as 1.365 µm which demonstrated a successful preparation. For osteoporosis model, BMD and the ultimate force of the treated tibia were significantly improved in osteoporosis rat after injection of 0.5 mg and 1 mg simvastatin microcrystals compared with OVX or 0 mg groups. There were no differences observed in BMD and ultimate force between 0.5 mg, 1 mg simvastatin microcrystals group and bone morphogenetic protein (BMP) 5 ug group. For femur defect model, quantitative analysis of bone regeneration by µCT showed bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) were significantly increased in simvastatin microcrystals 50ug (SIM-C 50 ug), SIM-C 100 ug and BMP 5 ug group compared with CPC vehicle group. Trabecular separation (Tb.Sp), and cement volume/tissue volume (CV/TV) were decreased in SIM-C 50 ug, SIM-C 100 ug and BMP 5 ug groups compared with CPC vehicle group. Semiquantitative scale for histology assessment further demonstrated a higher bone regeneration score in drug loaded groups compared with CPC group. Conclusions: Our study shows that simvastatin microcrystals were successfully prepared by wet media milling method and the microcrystals can promote bone formation by local delivery by poloxamer hydrogel or CPC, which is of great translational potential. propeptide tartrate-resistant acid phosphatase 5b;


Background
The healing of bone defects remains a significant clinical orthopedic challenge. Autologous bone grafts are considered as the gold standard. However, these procedures may cause donor-site morbidity, such as hemorrhage, infection, and chronic pain. [1] With the general aging of the population, osteoporosis is a burgeoning public health problem of global concern. However, the ultimate goal of osteoporosis treatment is the prevention of the initial and subsequent occurrences of fragile fractures. Local treatment strategy targeting specific bone regions that are prone to fractures may help to decrease the incidence of osteoporotic fractures. Both prevention of osteoporotic fracture and improvement of bone defect healing could be achieved by locally application of osteogenic drugs.
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. Since Mundy et al. first reported on the anabolic effects of statins on bone formation, [2] emerging pre-clinical studies have demonstrated the ability in promoting bone formation in different in vitro and in vivo studies. [3] However, controversies still remain due to low bioavailability of orally administered statins to the skeleton. [4] After the first-pass hepatic metabolism, only approximately 5% of the active compound remains in the circulation, even less at the defect site. [5] Increasing the dosage is not an option, as high-dose administration of statins may cause adverse side effects. Local delivery of simvastatin is another option that can be used. Our previous studies have shown locally applied statins with sustained release can improve the quality of osteoporotic bone, promote defect healing or enhance pedicle-screw fixation in animals from rats [6], rabbits [7] to minipigs [8,9] and rhesus macaques (unpublished data).
Although these results are promising, the current method for simvastatin delivery in pre-clinical studies is impractical for human application and may impede its clinical translation. Simvastatin is a lipophilic drug that is difficult to dissolve in water. Low aqueous solubility of active pharmaceutical ingredients is a major problem in the design of pharmaceutical. [10] When mixing simvastatin with hydrophilic hydrogel, it's difficult to ensure the homogeneous distribution of drug in vehicle, the large drug particles may cause a precipitation during storage and even application, and also the drug release may not be controlled, due to the heterogeneous particle sizes of drug raw materials. Our study also found that simvastatin in poloxamer hydrogel is not stable and the 60 Co irradiation for sterilization can accelerate its degradation rate.
Pharmaceutical formulation is the process in which different chemical substances, including the active drug, are combined to produce a final medicinal product. Thus, better pharmaceutical preparation process with industrialization and translation benefits is also needed.
As one of the sustained-release drug delivery systems, microcrystals have been increasingly exploited for poorly water-soluble drugs. Microcrystals has a small particle size (1 to 100 μm), a high drug loadings (the drug content can be close to 100%), and are suitable for multiple administration routes and easy to industrial production. The homogeneous particle sizes of microcrystals result in well-controlled drug release, improved physical stability in vehicle, as well as dose homogeneity, besides their long-time therapeutic effect following a single injection. [11] Based on these reasons, here we produced simvastatin microcrystals for intraosseous application by new formulation and preparation method, hoping to achieve an uniform osteogenesis effect which is favorable for its clinical use.
For easy translation, two well-studied delivery methods with according animal models were used to test the osteogenic effect of simvastatin microcrystals. The first is Poloxamer hydrogel as it 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 delivery. [12,13] Among them, Poloxamer 407 is an excipient of various formulations that is approved by the U.S. Food and Drug Administration for pharmaceutical application. [14] The other is calcium phosphate cements (CPCs). CPCs have long been used as osteoconductive bone substitutes in the treatment of bone defects. Calcium phosphate cement consists of one or more calcium orthophosphate powders that are mixed with an aqueous solution. The paste that forms will harden within a few minutes to a solid body. Clinically, antibiotics or bone morphogenic proteins can be added to the CPCs to treat infectious disease or improve bone healing. This study is aimed to test whether simvastatin microcrystals could remain ontogenetic effect delivered by poloxamer or CPC to promote local osteogenesis.

Study design
The pre-clinical simvastatin microcrystals were prepared through wet media milling method. The lyophilized sample was re-dissolved by 1 mL of sodium chloride injection then undergoes sonication (in a water bath). Further particle size determination was then implemented. The moisture and impurities were also determined.

Preparation of simvastatin microcrystals
A solution of Tween 80 in sterile water for injection was prepared under stirring until its complete dissolution. Simvastatin was added to Tween 80 solution and agitated for 1 h to produce the simvastatin suspension. Then, the obtained simvastatin suspension was transferred to the grinding miller and grinded for 1 h at 3000 rpm. Mannitol was added to the grinding solution and stirred to dissolve. The resultant solution was diluted to the corresponding concentration with sterile water for injection. The finished product was obtained by freeze-drying from the solution. Table 1 shows the preparation of simvastatin microcrystals. Freeze-drying process and parameters: the freezing temperature: -40℃; the freezing time: 300min; primary drying: -20℃-150min; -8℃-240min-210min; -6℃-120min-300min; 0℃-180min-0min, ; secondary drying: 10℃-90min-0min; 20℃-120min-0min.

Particle size determination
The particle size and distribution was measured by a laser particle size analyzer (BT-9300S, Bettersize Co., Ltd., Dandong, China). 1 mL of sodium chloride injection was added to the simvastatin microcrystals and sonicated (in a water bath) for 2 min. The distilled water was added into 2/3 of the cuvette for background calibration. 3-5 drops of the prepared sample solution was added into the sizing cuvette and stirred for 30 s.
The sample was analyzed for 3 times. The D10, D50, and D90 as well as the obtained span were correlated with 10%, 50%, and 90% of the cumulative particle size distribution.

Determination of moisture and impurities
The water content was determined by moisture determination method according to the Pharmacopoeia of the

Sterilization of simvastatin microcrystal
The drug substance and excipients are sterilized in the manufacturing process. The manufacturing plant, the vial, rubber stopper and aluminum cap are sterilized. Therefore, there is no special sterilization method for the final microcrystals. Shanghai Rebone Biomaterials Co., Ltd.) 5 ug. Rats received bilateral OVX to induce osteoporosis under isoflurane, as described previously. [15] Three months later after OVX the animals will demonstrated osteoporosis and receive the intraosseous injection. A month later after the intraosseous injection, the animals were sacrificed for further assessment.

Local intraosseous injection of simvastatin microcrystals / Poloxamer 407 gel
We located the entry point at the front of the tibial intercondylar eminence and inserted the needle parallel to the long axis of the tibia as previously describe. [16] A tibial radiograph ensured that the needle tip was in the medullary cavity (this validation step can be omitted after sufficient procedural skill is gained) and 100 ul solution was given to the right tibia marrow cavity for each animal.

BMD
Bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry (DXA) for smallanimal high-resolution (UltraFocus DXA, Faxitron). The region of interest (ROI) of BMD was set to the whole bone.

Biomechanical test
A three-point bending test was used to evaluate the mechanical properties of tibia using a mechanical

SEM
The fresh fractured surfaces of the cements were examined under scanning electron microscopy (FE-SEM, S-4800, Hitachi, Japan). The samples were sputter coated with a 20-nm-thick layer of gold prior to examination.

Serum assay
Whole blood was collected by cardiac puncture at euthanasia and subsequently centrifuged at 2500 × g for 20 min at 4°C to separate serum for measurement of serum bone formation marker, intact N-terminal propeptide of type-1 procollagen (P1NP) and bone resorption marker, tartrate-resistant acid phosphatase 5b (TRACP 5b) by enzyme-linked immunosorbent assay (Shanghai Jianglai Biotech, Shanghai, China).

Histology
Histologic examinations were performed to verify the situation of the implant and bone tissue ingrowth.
For the decalcification protocol, the specimens were decalcified by 10% citric acid after fixation and then

Statistical analysis
Data are expressed as mean ± SD (standard deviation) with annotations in legends. Comparisons between groups were assessed with one-way ANOVA. Statistical analyses were performed using prism software (version 8.0, San Diego, California, USA). Differences were considered significant at P<0.05.

Characterization of microcrystals
The particle size of simvastatin microcrystals is shown in Table 3. The mean particle size is 1.365 µm. The D10, D50, and D90 showed size of the most particle ranged from 0.226-3.425 µm. The moisture and related substances were controlled well. These data showed we successfully made simvastatin microcrystals. As demonstrated in Fig 1, precipitations can be seen in solutions of 5 mg or 10 mg/L simvastatin after intense shaking. However, solutions of simvastatin microcrystals look clear even without shaking.

Establishment of the rat model of osteoporosis
Surgery was successful in all animals and recovery was uneventful. The body mass is significantly increased after OVX surgery compared with Sham group (Fig. 2A). Uterus weight at autopsy demonstrated significant decrease in all groups receiving OVX compared with Sham group (Fig. 2B). The increased body mass and decreased uterus weight indicated that we successfully removed ovaries. Furthermore, left tibia (untreated side) BMD and ultimate force was significantly decreased in all OVX groups compared with Sham group (Fig. 2C, D).  Fig. 3A showed that BMD of right tibia in all drug loaded groups is significantly increased compared with OVX group and SIM-C 0 mg group. Furthermore, ultimate force of tibia in drug loaded groups also improved significantly compared with OVX group and SIM-C 0 mg group (Fig. 3B). And there is no difference in BMD or ultimate force between sham group and drug loaded groups, indicating the BMD of drug loaded groups recovered to a normal level. No differences were observed between BMP and SIM-C groups.  The VOI quantitative analysis confirmed that bone regeneration values were significantly improved by the supplement of simvastatin microcrystals or BMP in CPC. BV/TV, Tb.Th, Tb.N were significantly higher in SIM-C 50ug, 100ug and BMP 5ug group compared with CPC group (Fig. 6A-C). Tb.Sp, CV were also decreased in drug group compared with CPC group (Fig. 6D-E).

Histology
Bone regeneration of these histological sections was scored on a semiquantitative scale by an individual blinded observer, using a modified scoring method similar to that described previously (Table 4). [18] Representative histological findings of bone formation further proved the results from µCT analysis.
Decalcification removed all minerals and CPC implant while kept the tissue. Revealed by HE and Masson staining, abundant newly bone formation and bone matrix with collagen deposition in simvastatin microcrystals and BMP loaded CPC, just a little bone formation in CPC group (Fig. 7). Semiquantitative scale demonstrated a higher bone regeneration score in drug loaded CPC group (Fig. 8). Table 4 Semiquantitative scale for evaluation of bone regeneration.

Score Extent of new bone in defect 0
No bone formation 1 Minimal bone formation (only very small portion in the defect) 2 Low bone formation (less than one-fourth of the defect) 3 Moderate bone formation (less than one-half and more than one-fourth of the defect) 4 Abundant bone formation (less than three-quarter and more than one-half of the defect) 5 Extensive bone formation (more than three-quarter of the defect)

Serum assay
There is no significant difference in serum P1NP and TRACP5b between groups at the same time point (Fig. 9A, B) Fig. 9. Serum bone formation marker, intact N-terminal propeptide of type-1 procollagen (P1NP) and bone resorption marker, tartrate-resistant acid phosphatase 5b (TRACP 5b) in different groups. All data are expressed as mean ± SD.

Discussion
Approaches to regenerate bone include specific grafting surgical techniques with autogenous bone, substitutes, barrier membranes, growth factors, stem cell therapy, and lately the osteopromotive pharmacological compounds. The pharmacological approaches have gained popularity owing to their convenience and the advantageous cost-effectiveness when applied with other grafting techniques. [19] A number of studies have demonstrated that simvastatin can significantly increase bone formation. [7,15] In addition to direct effect on osteoblasts and osteoclast, the underlying mechanisms are complexed by statins' pleiotropic effects including anti-inflammation, angiogenesis and antibacterial activity which could also play an important role in bone formation. [3,20] Despite of the unclear mechanism, abundant in vivo studies have shown the positive effect of simvastatin on bone formation. Locally application of simvastatin can improve osteoporosis in ovariectomized rats [6] and minipigs. [8] Also, local delivery of simvastatin through controlled drug delivery systems with much lower doses and dosages are effective in fracture healing. [20] Both a 10 mg/kg intraperitoneal injection of simvastatin daily for 30 days [21] and an oral dosage of 5 mg/kg/d for 28 days [22] improved osseointegration in osteoporotic rats.
As simvastatin has been used as lipid-lowering medicine safely for many years. It seems very promising that statins could be translated into clinic. However, after decades of studying, we are still stay in preclinical phases. Only in the scope of dentistry, clinical studies have investigated the potential of simvastatin it is necessary to maintain the effective concentrations of simvastatin in the circulating blood for a long time to be sure that simvastatin is able to continuously affect the bone. Therefore, in the systemic approach, simvastatin needs to be continuously administered for a long period of time, which increases the total dose. [20] A high daily dose is likely elicits unwanted side effects, such as increasing the risk of liver failure, kidney disease, rhabdomyolysis, and myalgia. [3] Local delivery of simvastatin is another option that can be used. Carries that have been reported to deliver simvastatin includes PLGA (poly Lactic-coglycolic acid), collagen, gelatin, TCP (three calcium phosphate), hydrogen. However, these studies, including our previous studies, usually used raw chemicals or oral formulations while additional formulation process is needed for locally application. In this study, we have successfully create simvastatin microcrystals with a mean particle size of 1.365 µm. And microcrystals significantly increase water solubility of simvastatin witch allow us to dissolve microcrystals easily even in concentration of 10 mg/ml.
What's more, microcrystals can be easily industrialized as a mature pharmaceutical preparation process.
However, due to the unclear mechanism how simvastatin promotes bone formation, it's necessary to verify whether simvastatin microcrystals remain the osteogenic effect.
We showed single intraosseous injection of simvastatin microcrystals with 0.5 mg or 1 mg could improve bone quality of tibia as well as BMP 5 ug. Both BMD and strength were increased in treated tibia.
However, we did not observe any difference in BMD and strength between SIM-C 0.5 mg and SIM-BPC 0.5 mg group although the water solubility is increased in microcrystal form. Despite all this, we proved simvastatin remains the ability to promote bone formation after making it into microcrystals. In bone defect experiment, 50 ug and 100 ug simvastatin microcrystals also promoted defect healing compared with the vehicle group. And BMP 5 ug group did not show better healing than simvastatin microcrystal group. Thus, this study showed formulation of simvastatin microcrystals could be effective with poloxamer or CPC which are both potential to be approved by China Food and Drug Administration.
There are some limitations to the present study. First, we did not study simvastatin microcrystals release in vitro and retention in vivo, although we previously showed no initial burst release was observed during the first 24 h in simvastatin/poloxamer 407 hydrogels. [8] To further translation, a precise pharmacodynamic study for microcrystals is needed. Second, the optimal dose for local usage is still not clear due to the limited groups. It seems the dose of simvastatin could be still decreased to some extent. Our big animal study showed 0.5 and 1 mg injection of simvastatin in minipig vertebrae is effective to enhance implant fixation and improve bone quality. [8,9] Our unpublished data showed 3D-printed porous cages with 0.5 mg simvastatin in hydrogel can promote bone ingrowth and spinal fusion in adult male rhesus macaques.
The possible overdose in this study may also explain the phenomenon that we did not find any difference between SIM-C 0.5 mg and SIM-BPC 0.5 mg group in osteoporosis model.

Conclusion
We successfully prepared simvastatin microcrystals with a mean particle size of 1.365 µm by wet media milling method. And simvastatin microcrystals remain the osteogenic ability both in osteoporosis model and bone defect model. It might provide an adjunctive method to augment local osteoporotic bone, reduce the incidence of osteoporotic fractures, or improve bone defect healing.

Ethics approval and consent to participate
All experimental procedures and protocols were approved by Peking University Third Hospital Committee on Ethics in the Care and Use of Laboratory Animals (LA2016304).