The combination of bone tissue scaffolds with osteogenic induction factors is an effective strategy to facilitate bone healing processes. Here, chitosan (CS)/nano-hydroxyapatite (HA) scaffolds containing simvastatin (SIM)-loaded PLGA microspheres were fabricated by combining a freeze-drying technique with a modified water-oil-water emulsion method. The CS/HA weight ratio of 1:2 was selected by analyzing the effect of HA content on the micro-architecture, mechanical strength, and biocompatibility of the scaffold. Drug release kinetics showed that the SIM encapsulated in the scaffold was released in a sustained manner for up to 30 days. In vitro bioactivity study in rat bone marrow-derived mesenchymal stem cells showed that the SIM-loaded scaffolds had a strong ability in accelerating cell proliferation and inducing osteogenic differentiation. Moreover, an in vivo experiment using a rat calvarial defect model also documented that the SIM-loaded scaffolds had a remarkable effect on bone-promoting regeneration. The results of this study suggest that the SIM-loaded CS/HA scaffold is feasible and effective in bone repair and thus may provide a promising route for the treatment of critical-sized bone defects.

Reconstruction of critical-sized bone defects caused by trauma, injury, or congenital malformations remains a difficult problem in orthopedic surgery. The current “gold standard” and clinical approach to repair bone defects is reconstruction using autograft tissue, but it is limited by insufficient tissue supply and additional surgical wounds [Ning et al., 2015]. While using allografts and xenografts may overcome some limitations of the autografts, these bone graft substitutes have the potential risk of immunological rejection and pathogen exposure [David et al., 2015]. Due to the limitations of the current treatments for bone defects, a wide range of bone tissue-engineered scaffolds have been developed to provide better bone graft substitutes for patients. The majority of scaffolds that are currently used for bone repair are naturally derived polymers (such as collagen, chitosan [CS], and fibrin), metals (such as titanium and silicon), and decellularized bone extracellular matrix [Marrelli and Tatullo, 2013; Marrelli et al., 2015; Paduano et al., 2017].

CS, an N-deacetylation product of chitin, is a fiber-like thermoplastic polymer, which can be degraded to glucose by chitosanases and glucosaminidase. In the past few decades, CS has attracted a great deal of attention in bone tissue engineering because of its biocompatibility, biodegradability, low toxicity, and similarity to extracellular matrix [Boukari et al., 2017; Shamekhi et al., 2017]. However, CS has the drawback of weak mechanical properties and quick degradation. Therefore, CS scaffolds are often compounded with hydroxyapatite (HA), a sort of biologically active ceramic that promotes both osteoconductivity and mechanical properties of CS scaffolds [Uswatta et al., 2016]. In previous studies, the CS/HA scaffolds have been shown to support the proliferation and osteogenic differentiation of pre-osteoblasts and enhance bone healing in animal models of bone defects [Chesnutt et al., 2009; Ge et al., 2012; Biazar et al., 2015]. Further advantages of the CS/HA scaffolds are their low costs. They are easy to fabricate and can be processed into various shapes.

The osteogenic potential of bone tissue-engineered scaffolds can be enhanced by functionalizing them with osteogenic induction factors and optimizing their mechanical properties [Gentile et al., 2016; Sun et al., 2016; Tatullo et al., 2016]. In particular, bone morphogenic proteins are regarded as potent osteogenic growth factors and have been applied in the clinical treatment of bone defects [Kim et al., 2017]. However, these growth factors are costly and readily biodegradable, and may aggravate inflammatory reactions [Shields et al., 2006]. Recently, simvastatin (SIM), a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, has attracted increased attention in the field of bone tissue engineering for its strong bone-inducing potential and good safety profile. SIM has been reported to increase osteogenesis by increasing BMP-2 and Runt-related transcription factor 2 (RUNX2) expression as well as protecting osteoblasts from apoptosis, and its beneficial effects on bone metabolism have already been confirmed in animal models of osteoporosis and bone fractures [Jadhav and Jain, 2006; Jia et al., 2015; Oryan et al., 2015].

In this work, the CS/HA scaffold was functionalized with SIM-loaded PLGA microspheres in order to deliver a controlled and prolonged release of SIM. The amount of HA within the scaffolds was first optimized by investigating the effect of increasing amounts of HA on the micro-architecture, mechanical strength, and biocompatibility of the scaffolds. Then, SIM release from the composite scaffold was determined, and its in vitro osteogenic effect was evaluated using bone marrow-derived mesenchymal stem cells (BMSCs). In addition, the in vivo osteogenic potential of the scaffolds was evaluated using a rat calvarial defect model.

Microsphere and Scaffold Fabrication

PLGA microspheres were fabricated utilizing a water-oil-water (W/O/W) emulsion method. Briefly, 250 mg of granular PLGA (actide:glycolide 50:50, molecular weight 38-54 kDa) and 12 mg of SIM were added to 3 mL of dichloromethane (Aladdin, Shanghai, China) and sonicated for 30 s. The resulting mixture was slowly dropped into 15 mL of 3% (w/v) aqueous PVA solution in a 50-mL beaker and agitated magnetically at 200 rpm. Crushed ice was placed around the beaker to keep the temperature stable. After 6 h, the SIM-loaded PLGA microspheres were washed 3 times with excess amount of deionized water, recovered by centrifugation (500 g, 5 min), prefrozen at -80°C for 2 h, and lyophilized in a freeze-dryer (YiBei, Shanghai, China) for 48 h. The blank PLGA microspheres were fabricated using the same approach, except that no SIM was added to the dichloromethane. The CS/nano-HA scaffold was prepared using a freeze-drying process. Briefly, 100 mg of granular CS (degree of acetylation ≤40%; Sigma-Aldrich, St. Louis, MO, USA) were mixed with different dosages of HA (nanopowder, <200-nm particle size; Sigma-Aldrich) in 10 mL of 0.1 M acetic acid solution (Aladdin). Then, the CS/HA slurry was transferred to plastic molds, degassed under vacuum for 30 min, prefrozen at -80°C for 2 h, and lyophilized for 48 h. To obtain microsphere-loaded CS/HA scaffolds, 7 mg of PLGA microspheres were added to 10 mL of CS/HA slurry, and the mixture was fully blended and freeze-dried as above. The CS/HA scaffolds with different HA contents were produced and designated as follows: HA-0.5, HA-1, HA-2, and HA-3, corresponding to CS/HA weight ratios of 2/1, 1/1, 1/2, and 1/3, respectively.

Morphological Observations

A scanning electron microscope (Hitachi, Ibaraki, Japan) was used to examine the morphology of the microspheres and scaffolds. The samples were fixed on double-sided tape, coated with gold, and imaged at 10-kV acceleration potential.

Encapsulation Efficiency

The amount of SIM in the microspheres was determined using a UV spectrophotometer (Beckman Coulter, Brea, CA, USA). Briefly, 1 mg of SIM-loaded PLGA microspheres was dissolved in 5 mL of acetonitrile and sonicated for 40 s. The content of SIM in the solution was quantified by a standard curve generated from known concentrations using a UV spectrophotometer at a wavelength of 240 nm. Encapsulation efficiency of the SIM-loaded PLGA microspheres was calculated according to the following formula:

Pore Size and Porosity

The pore size of the scaffolds was assessed using the method described by O'Brien et al. [2004] previously. Briefly, each sample was embedded in glycol methacrylate and sectioned serially at 10 μm using a Leica RM2165 histotome (Mannheim, Germany), stained with aniline blue, and visualized on a microscope (Nikon Optiphot, Japan). The pore sizes of the slices were imaged and analyzed using Scion Image (Scion Corporation, Frederick, MD, USA).

The porosity of the scaffolds was calculated according to the following equation:

where V1 is the initial volume of the alcohol solution, V2 is the total volume of the alcohol solution and the scaffold immersed in alcohol solution for 1 h, and V3 is the residual volume of the alcohol solution after removal of the immersed scaffold.

Mechanical Property Test

Compressive testing was conducted on an Instron 5542 universal tester (Instron Corp., Norwood, MA, USA) with a 5-N load cell. In brief, samples with a diameter of 10 mm and a height of 3 mm were hydrated in PBS for 30 min prior to mechanical testing. After that, the hydrated samples were tested in unconfined compression at a loading rate of 0.5 mm/min to 40% strain. The slope of the initial linear elastic region of stress-strain curves was used to calculate the compressive modulus.

Isolation and Culture of BMSCs

BMSCs were collected from the bone marrow of femurs of male Wistar rats. All procedures were approved by the Animal Care and Use Committee of the Shanghai Ninth People's Hospital (Shanghai, China). Briefly, both ends of the femurs were cut off by sterile operation scissors, and the bone marrow was flushed out with Dulbecco's modified Eagle's medium (DMEM; Hyclone, UT, USA) containing 10% fetal bovine serum (FBS; Gibco, CA, USA) and 1% penicillin-streptomycin (D10 media). The resultant suspension was centrifuged at 500 g for 5 min, and the cell pellet was diluted with D10 media. The cells were cultured in T-75 flasks (BD Biosciences, Franklin Lakes, NJ, USA) at 37°C in a humidified incubator containing 5% CO2, and culture media were changed twice a week.

Cell Proliferation

For cell proliferation assays, the scaffolds were sterilized by epoxyethane and placed in 24-well plates. BMSCs were seeded on scaffolds at a concentration of 5 × 104 cells/scaffold. At preset time points, cell activity was evaluated using a cell counting kit (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer's instructions, and absorbance was measured at 450 nm using a microplate reader (Thermo Electron Corporation, Waltham, MA, USA).

Release Properties

To evaluate the in vitro SIM release from the PLGA microspheres, 1 mg of PLGA microspheres was dispersed in 5 mL of PBS and incubated on a reciprocal shaker at 100 rpm and 37°C. At preestablished times, supernatant was collected, and the amount of SIM in the supernatant was determined by the UV spectrophotometer method. To determine the in vitro release kinetics of SIM from the scaffold, each sample was immersed in 2 mL of PBS and agitated on a shaker. At certain time points, the supernatant was collected, and 2 mL of fresh PBS was added. The content of SIM in the supernatant was analyzed using a UV spectrophotometer.

In vitro Osteogenesis Study

In order to analyze osteogenesis of the scaffolds, BMSCs were seeded in 6-well culture plates (BD Biosciences) at a density of 6 × 105 cells/well and incubated in osteogenic induction media. Then, inserts covered with 0.8-μm-pore membrane along with the different scaffolds were added to the wells of the culture plates. The culture media were changed twice a week, but the scaffolds were not exchanged.

Alkaline phosphatase (ALP) activity of the BMSCs in the different groups were measured using a commercial ALP activity assay kit (Beyotime, Shanghai, China). Seven and 14 days after cell seeding, ALP expression in BMSCs was determined according to the manufacturer's instructions. The mineralized matrix nodules formed by BMSCs were evaluated using alizarin red staining. On day 21, cells were washed 3 times with PBS, fixed in 4% paraformaldehyde for 30 min, and incubated with alizarin red (Beyotime) for 10 min at room temperature. After that, the nonspecific alizarin red staining was removed by washing 3 times with distilled water. For quantification of the mineralization, the mineralized matrix nodules were dissolved in 10% (w/v) cetylpyridinium chloride (Aladdin), and absorbance was measured at 540 nm using a microplate reader.

Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction

Cellular mRNA was isolated from BMSCs in different groups using TRIzol reagent (Beyotime). Complementary DNA was synthesized using a reverse transcription kit (Invitrogen) according to the manufacturer's instructions. mRNA expression of bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), and RUNX2 were examined using rat-specific primers (Table 1). The amplification procedure included 40 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 45 s. Relative mRNA expression was calculated using the 2−ΔΔCt method, and the GAPDH gene was used as an internal control.

Table 1

Primer sequences for RT-qPCR (see text for gene abbreviations)

Primer sequences for RT-qPCR (see text for gene abbreviations)
Primer sequences for RT-qPCR (see text for gene abbreviations)

Surgical Procedures

All in vivo experiments were conducted according to the protocols approved by the Animal Care and Use Committee of the Shanghai Ninth People's Hospital (Shanghai, China). Twenty 12-week-old male Wistar rats (mean weight 330 ± 19 g) were used in this study, and rats were randomly assigned to a control group (no scaffold, n = 5), a blank scaffold group (scaffolds without SIM, n = 5), and a SIM-loaded scaffold group (SIM-loaded scaffolds, n = 5). General anesthesia was induced by intraperitoneal injection of a mixture of ketamine (80 mg/kg; Bayer Korea, Seoul, Korea) and xylazine (8 mg/kg; Bayer Korea). After the anesthetic procedure, the cranial area of the rat was shaved and disinfected with 75% alcohol. Then, the flap was raised, and a trephine drill was used under copious saline solution to create a circular bone defect of 5 mm. Thereafter, the scaffold was gently implanted (5 mm in diameter and 2 mm thick) into the bone defect, and the skin incision was sutured with 4-0 threads. After 8 weeks, all rats were sacrificed, and the defect sites were dissected and soaked in 4% paraformaldehyde.

Micro-CT Measurements

The specimens were examined using a micro-CT scanning system (GEe Xplore Locus SP Micro-CT: GE Healthcare, Milwaukee, WI, USA), which was operated at 45 kV and 80 mA with a resolution of 20 μm. 3D images of the specimens were reconstructed from 2D slices, and morphometric parameters such as the bone volume per total volume (BV/TV) were analyzed using CTAn software.

Histological Analysis

After micro-CT imaging, all specimens were decalcified for 4 weeks by immersing them in 10% EDTA (Beyotime). Following decalcification, the specimens were dehydrated with graded ethanol solutions and embedded in paraffin (Aladdin). Sections of 5-μm thickness were cut using a histotome, stained with hematoxylin and eosin (H&E) (Beyotime) and examined by light microscopy.

Statistical Analysis

All data in our study are expressed as means ± SD, and differences between groups were examined for statistical significance by analysis of variance using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA). For all comparisons, the level of significance was set at p < 0.05.

Characterization of PLGA Microspheres

Figure 1 shows scanning electron microscopy images of the PLGA microspheres. The blank PLGA microspheres exhibited fine and round morphology, and their particle size distribution was in the range of 1-21 μm (Fig. 1a). There was no difference in particle size or morphology between the blank PLGA microspheres and SIM-loaded PLGA microspheres (Fig. 1b). Moreover, the encapsulation efficiency of SIM in PLGA microspheres achieved was about 85.6%.

Fig. 1

Scanning electron microscope images of blank PLGA microspheres (a) and simvastatin-loaded PLGA microspheres (b).

Fig. 1

Scanning electron microscope images of blank PLGA microspheres (a) and simvastatin-loaded PLGA microspheres (b).

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Characterization of Scaffolds with Different HA Contents

The morphology of the CS/HA scaffolds with different HA contents is shown in Figure 2. All scaffolds exhibited an interconnected porous structure and the microspheres were evenly distributed in the scaffolds. With increasing HA content, scaffold pore size decreased, but porosity did not remarkably change (Fig. 3a, b). The mean size of HA-0.5, HA-1, and HA-2 scaffolds was >100 μm, which was within the recommended pore size range for bone regeneration [Rogina et al., 2016]. The addition of HA induced a concentration-dependent increase in the compressive modulus of the scaffolds (Fig. 3c). The compressive modulus of the HA-3 scaffold was about 3 times higher than that of the HA-0.5 scaffold. To examine biocompatibility of the 4 scaffolds, BMSCs were seeded on the scaffolds, and their proliferation was determined using the CCK-8 assay. On day 3, no significant difference in the optical density (OD) value was found among the 4 groups; however, the OD value of the HA-3 group was significantly lower than that of the HA-1 group on day 7 (Fig. 3d).

Fig. 2

Scanning electron microscope images of the hydroxyapatite (HA)-0.5 (a), HA-1 (b), HA-2 (c), and HA-3 (d) scaffolds.

Fig. 2

Scanning electron microscope images of the hydroxyapatite (HA)-0.5 (a), HA-1 (b), HA-2 (c), and HA-3 (d) scaffolds.

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Fig. 3

Pore diameter (a), porosity (b), compressive modulus (c), and biocompatibility (d) of the hydroxyapatite (HA)-0.5, HA-1, HA-2, and HA-3 scaffolds. * p < 0.05 vs. the HA-0.5 group, #p < 0.05 vs. the HA-1 group.

Fig. 3

Pore diameter (a), porosity (b), compressive modulus (c), and biocompatibility (d) of the hydroxyapatite (HA)-0.5, HA-1, HA-2, and HA-3 scaffolds. * p < 0.05 vs. the HA-0.5 group, #p < 0.05 vs. the HA-1 group.

Close modal

Taken together, in the HA-2 scaffold, the compressive modulus was higher, and micro-architecture and biocompatibility were comparable to the HA-0.5 and the HA-1 scaffold. Thus, the HA-2 scaffold was used in the following study.

SIM Release from Free Microspheres and Scaffolds

The cumulative SIM release profile was determined using UV spectrophotometry. The SIM-loaded PLGA microspheres exhibited a sharp initial burst, and approximately 37.16% of the loaded SIM was released during the first 3 days. Thereafter, SIM was released in a sustained manner between days 3 and 30. The average release rate of SIM was 90.12% after 30 days (Fig. 4a). The SIM release from the scaffold showed a similar pattern to that from the free microspheres. Initially, a high rate of SIM release was observed during the first 3 days, with a slower rate of SIM release over the next 27 days. The rate of SIM release from the microspheres incorporated in the scaffolds was steadier than that from the free PLGA microspheres. At the end of the assay, the SIM-loaded microspheres embedded within the scaffolds had released 83% of the encapsulated content (Fig. 4b).

Fig. 4

Cumulative in vitro release profile of simvastatin (SIM) from free PLGA microspheres (a) and scaffolds (b).

Fig. 4

Cumulative in vitro release profile of simvastatin (SIM) from free PLGA microspheres (a) and scaffolds (b).

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Proliferation of BMSCs on SIM-Loaded Scaffolds

The effect of SIM-loaded scaffolds on cell proliferation was assessed by CCK-8 assay. Rat BMSCs were seeded onto the scaffolds at the same initial density and incubated for different periods of time. As shown in Figure 5, the OD value increased with increasing culture time in all groups, and the SIM-loaded scaffold group displayed a significantly higher OD value than the control group and the blank scaffold group. However, there was no significant difference in cell proliferation between the control group and the blank scaffold group.

Fig. 5

Cell proliferation of bone marrow-derived mesenchymal stem cells cultured on different scaffolds was assessed with CCK-8 after 1, 3, 7, and 14 days of incubation. SIM, simvastatin. * p < 0.05 vs. the control group. #p < 0.05 vs. the bank scaffold group.

Fig. 5

Cell proliferation of bone marrow-derived mesenchymal stem cells cultured on different scaffolds was assessed with CCK-8 after 1, 3, 7, and 14 days of incubation. SIM, simvastatin. * p < 0.05 vs. the control group. #p < 0.05 vs. the bank scaffold group.

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ALP Activity and Calcium Deposition

Cellular ALP activity, a marker of early osteoblastic differentiation and phenotype, was examined after cells were seeded for 7 or 14 days. As shown in Figure 6a, ALP activity of BMSCs increased in all groups with increasing incubation time. On day 7, ALP activity of BMSCs cultured in the SIM-loaded group was significantly higher than that in the control and blank scaffold groups, and osteogenesis induction was more evident on day 14. On day 21, the mineralized matrix nodules formed by BMSCs were evaluated using alizarin red staining. As shown in Figure 6b, the SIM released from the scaffold significantly enhanced the mineralization of BMSCs. Furthermore, quantitative results also showed that the amount of alizarin red in the SIM-loaded scaffold group was significantly higher than that in the other 2 groups (Fig. 5c).

Fig. 6

a Alkaline phosphatase (ALP) activity of bone marrow-derived mesenchymal stem cells in different treatment groups 7 and 14 days after seeding. b The formation of mineralized matrix nodules in different groups was evaluated using alizarin red staining (ARS). c The amount of ARS was quantified by measurement of absorbance at 562 nm. SIM, simvastatin. * p < 0.05 vs. the control group, #p < 0.05 vs. the blank scaffold group.

Fig. 6

a Alkaline phosphatase (ALP) activity of bone marrow-derived mesenchymal stem cells in different treatment groups 7 and 14 days after seeding. b The formation of mineralized matrix nodules in different groups was evaluated using alizarin red staining (ARS). c The amount of ARS was quantified by measurement of absorbance at 562 nm. SIM, simvastatin. * p < 0.05 vs. the control group, #p < 0.05 vs. the blank scaffold group.

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Osteogenic Gene Expression

Figure 7 shows mRNA expression of BSP, OPN, RUNX2, and OCN in rat BMSCs cultured in the different groups. Although there was no significant difference in BSP and OCN levels among the 3 groups on day 7, OPN and RUNX2 expression levels were significantly higher in the SIM-loaded scaffold group than in the control and the blank scaffold groups. After 2 weeks of incubation, the SIM-loaded scaffold group showed a significantly higher expression of the 4 osteogenic-related genes than the other 2 groups.

Fig. 7

Gene expression analyses of bone sialoprotein (BSP) (a), osteopontin (OPN) (b), runt-related transcription factor 2 (RUNX2) (c), and osteocalcin (OCN) (d) of bone marrow-derived mesenchymal stem cells in different groups after 7 and 14 days of culture. The results are presented as relative ratios to the chitosan/hydroxyapatite group on day 7. SIM, simvastatin. * p < 0.05 vs. the control group, #p < 0.05 vs. the blank scaffold group.

Fig. 7

Gene expression analyses of bone sialoprotein (BSP) (a), osteopontin (OPN) (b), runt-related transcription factor 2 (RUNX2) (c), and osteocalcin (OCN) (d) of bone marrow-derived mesenchymal stem cells in different groups after 7 and 14 days of culture. The results are presented as relative ratios to the chitosan/hydroxyapatite group on day 7. SIM, simvastatin. * p < 0.05 vs. the control group, #p < 0.05 vs. the blank scaffold group.

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Micro-CT Measurements

The amount of new bone formed within the calvarial defect was evaluated using micro-CT. Eight weeks postoperatively, there was almost no newly formed bone in the control group, whereas the other 2 groups demonstrated remarkable new bone formation within the defect (Fig. 8a). Besides, bone formation was increased in the SIM-loaded group compared to the blank scaffold group. Furthermore, the degree of bone repair was quantified by calculating the BV/TV ratio. After 8 weeks, the BV/TV ratio of the blank scaffold group was markedly higher than that of the control group, but significantly lower than that of the SIM-loaded group (Fig. 8b).

Fig. 8

a 3D reconstruction micro-CT images of different groups 8 weeks after operation. Black circles indicate the defect sites. b Morphometric analyses of bone volume/total tissue volume (BV/TV) for each group at 8 weeks. * p < 0.05 vs. the control group, #p < 0.05 vs. the blank scaffold group.

Fig. 8

a 3D reconstruction micro-CT images of different groups 8 weeks after operation. Black circles indicate the defect sites. b Morphometric analyses of bone volume/total tissue volume (BV/TV) for each group at 8 weeks. * p < 0.05 vs. the control group, #p < 0.05 vs. the blank scaffold group.

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Histological Analysis

Representative sections of H&E staining are shown in Figure 9. Consistent with the micro-CT results, the SIM-loaded scaffold demonstrated the most robust osteogenic activity, and most of the defect area in the SIM-loaded scaffold group was filled with eosin-stained newly formed bone tissue (Fig. 9). Although the amount of new bone was higher than in the control group, it was much less in the blank scaffold group than in the SIM-loaded scaffold group.

Fig. 9

Histological analysis of bone regeneration in the defect area of the different groups at 8 weeks. Black arrows indicate the edges of the host bone. SIM, simvastatin.

Fig. 9

Histological analysis of bone regeneration in the defect area of the different groups at 8 weeks. Black arrows indicate the edges of the host bone. SIM, simvastatin.

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Optimal bone tissue engineering scaffolds should not only support cell adhesion and proliferation but also have the ability to carry and release osteogenic induction factors [Garg et al., 2012]. In the present study, the CS/HA scaffolds were used to incorporate SIM-loaded microspheres for the controlled delivery of SIM to enhance their osteogenic potential. The extensive characterization carried out in our study demonstrated that the SIM-loaded CS/HA scaffold is a safe and effective bone graft substitute. In addition, the superior regenerative capability of the SIM-loaded scaffold was also confirmed in vivo, as shown by the enhanced bone regeneration within a 5-mm rat calvarial defect.

SIM is a cholesterol-reducing drug and has been extensively used to treat hyperlipidemia [Owens et al., 2014]. In recent decades, scientists found that SIM also has other pharmacological actions which are beneficial to bone regeneration [Park, 2009]. Previous studies indicated that SIM can enhance bone formation by increasing the expression of osteogenesis-related genes in MSCs and reducing osteoblast apoptosis, as well as inhibit bone resorption via the inhibition of osteoclast proliferation and differentiation [Noronha Oliveira et al., 2017; Sequetto et al., 2017]. However, systemic administration of SIM is limited by low drug availability, and local injection at effective doses may cause excessive inflammation [Zhang et al., 2016]. To improve drug bioavailability and avoid side effects, SIM was incorporated into PLGA microspheres, nontoxic and biodegradable drug carriers which can release SIM in a sustained and controlled manner.

The single emulsion-solvent evaporation method was used to prepare PLGA microspheres due to its advantages of good controllability and simple processing. The PLGA microspheres obtained were spherical in shape and had a smooth outer surface without any cracks, which was consistent with previous studies [Naito et al., 2014]. In addition, drug encapsulation efficiency was high at 85.6%, which can greatly reduce the costs of SIM. More importantly, a sustained drug release of SIM from PLGA microspheres for >30 days was achieved, which can provide a sustained osteogenesis induction effect during the process of bone healing.

Since the micro-architecture and mechanical strength of biomaterials have a great influence on bone regeneration, the HA content in the scaffold was first optimized. In our study, all the HA-0.5, HA-1, and HA-2 scaffolds had a high porosity (>80% with mean pore size >100 μm, which provided a suitable pore space for cell infiltration and subsequent vascular ingrowth [Murphy and O'Brien, 2010; Di Liddo et al., 2014]. The increased HA content reduced the pore size, suggesting that HA may accelerate the ice crystal formation in the process of freeze-drying. Although the HA-3 scaffold had the highest compressive modulus, BMSCs cultured on the HA-3 scaffold showed decreased proliferation. Moreover, when incorporated into the scaffold, the initial burst release of the PLGA microspheres was markedly improved, probably because the scaffold provided an additional diffusive barrier.

The in vitro biological properties of the SIM-loaded scaffolds were investigated using BMSCs. The CCK-8 assay revealed that the number of BMSCs on the SIM-loaded scaffold is greater than that on the control and the blank scaffold groups, suggesting that the SIM-loaded composite scaffold provided a more suitable microenvironment for cell proliferation. Furthermore, the SIM released from the scaffold also significantly increased different osteogenesis-related markers, including ALP activity, BSP, OCN, OPN, and RUNX2. ALP contributes to the mineralization of bone matrix at an early stage through hydrolyzing pyrophosphate [Sardiwal et al., 2013]. BSP, a small integrin-binding ligand N-linked glycoprotein, can mediate mineral deposition, bind type I collagen, and promote cell attachment [Kruger et al., 2013]. OPN, a highly phosphorylated sialoprotein, plays an important role in cell adhesion and matrix mineralization [Zhou et al., 2015]. RUNX2 can elevate the expression levels of bone matrix protein genes and increase the number of immature osteoblasts, and is usually highly expressed in the early stage of osteogenic differentiation [Komori, 2010]. OCN, an osteoblast-specific secreted protein, is one of the most abundant extracellular matrix proteins in the bone, and it usually contributes to osteogenic differentiation in the late stage [Zoch et al., 2016]. The results documented that the SIM incorporated in PLGA microspheres was able to promote osteogenic differentiation of BMSCs, and the drug dosage used in our study was appropriate.

The in vivo osteogenic potential of the SIM-loaded scaffold was evaluated using a critical-sized calvarial defect in a rat model, since it is a simple, reproducible, and reliable method to investigate the effect of bone graft materials on repairing critical-sized bone defects [Quinlan et al., 2015]. Eight weeks after operation, obvious bone regeneration was found in the blank scaffold and the SIM-loaded scaffold. Quantitative results based on micro-CT clearly indicated the positive effect of SIM on bone repair. The BV/TV ratio within the defect was the highest for the SIM-loaded scaffold group, implying a synergistic effect of the CS/HA scaffold and the SIM released from the microspheres on bone repair. These results confirmed the feasibility and effectiveness of developing a functional bioactive CS/nano-HA scaffold containing PLGA microspheres as a drug delivery platform for bone repair.

Our study also has several limitations. The osteogenic potential of bone graft substitutes may be affected by some systemic conditions, especially old age [Matsumoto et al., 2017]. To provide a comprehensive reference for clinical application, the osteogenic potential of the SIM-loaded scaffold should be extended to aged rats, such as Wistar rats >20 months [Ardura et al., 2016]. In addition, only 1 SIM dosage was evaluated in this study, and the in vivo release profile of SIM from the scaffold was also unclear; thus, further experiments are needed to identify the optimal SIM content in the scaffold.

The present study was supported by National Natural Science Foundation of China (grant Nos. 81371964 and 81572137).

The authors declare that they have no competing interests.

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