Magnetic Mesoporous Calcium Sillicate/Chitosan Porous Scaffolds for Enhanced Bone Regeneration and Photothermal-Chemotherapy of Osteosarcoma

The development of multifunctional biomaterials to repair bone defects after neoplasm removal and inhibit tumor recurrence remained huge clinical challenges. Here, we demonstrate a kind of innovative and multifunctional magnetic mesoporous calcium sillicate/chitosan (MCSC) porous scaffolds, made of M-type ferrite particles (SrFe12O19), mesoporous calcium silicate (CaSiO3) and chitosan (CS), which exert robust anti-tumor and bone regeneration properties. The mesopores in the CaSiO3 microspheres contributed to the drug delivery property, and the SrFe12O19 particles improved photothermal therapy (PTT) conversion efficacy. With the irradiation of NIR laser, doxorubicin (DOX) was rapidly released from the MCSC/DOX scaffolds. In vitro and in vivo tests demonstrated that the MCSC scaffolds possessed the excellent anti-tumor efficacy via the synergetic effect of DOX drug release and hyperthermia ablation. Moreover, BMP-2/Smad/Runx2 pathway was involved in the MCSC scaffolds promoted proliferation and osteogenic differentiation of human bone marrow stromal cells (hBMSCs). Taken together, the MCSC scaffolds have the ability to promote osteogenesis and enhance synergetic photothermal-chemotherapy against osteosarcoma, indicating MCSC scaffolds may have great application potential for bone tumor-related defects.

Preparation and characterization of the scaffolds. Morphology, mesoporous structure of calcium sillicate microspheres. Mesoporous calcium sillicate microspheres were prepared by using cetyltrimethyl ammonium bromide (CTAB). The emission scanning electron microscopy (SEM) analysis revealed that the sizes of microspheres were around 200 nm ( Fig. 2A). Additionally, the light-shaded spots within the microspheres could be detected by transmission electron microscopy (TEM) (Fig. 2B), which suggest the mesoporous structure. As depicted in Fig. 2C, the nitrogen adsorption-desorption isotherms of calcium silicate microspheres had the type IV isotherms with type H3 hysteresis loops. Moreover, no limiting adsorption at high P/Po in the type H3 loop demonstrated that the mesopores within the microspheres exhibited the slit-shaped pores with pore size of approximately 2.17 nm (Fig. 2D), which was consistent with the result of TEM analysis. The mesoporous structure significantly increased the BET surface area and pore volume of calcium silicate microspheres up to 291.57 m 2 /g and 0.41 cm 3 /g, respectively (Fig. 2D). Phase structures and property of magnetic mesoporous calcium sillicate/chitosan scaffolds. The crystalline structure of CSC, MCSC 1:7 and MCSC 1:3 scaffolds were characterized by X-ray power diffraction (XRD) assay. As the CaSiO 3 was non-crystalline and CS was semi-crystalline kind of material, therefore, only a broad peak at 2θ = 22° was detected for the CSC porous scaffolds (Fig. 4A). After the incorporation of magnetic particles in the CSC scaffolds, the characteristic peaks due to M-type ferrite were observed for both the MCSC 1:7 and MCSC 1:3 scaffolds. With the increment ratio of magnetic particles, the peak strengths were enhanced at corresponding points (Fig. 4A). Then, fourier transform infrared (FTIR) spectra was used to characterize the functional groups of the CSC, MCSC 1:7 and MCSC 1:3 scaffolds. As shown in Fig. 4B, all these three scaffolds had comparable adsorption peaks. Then, the magnetic property measurement of MCSC scaffolds was evaluated. The saturated magnetization (Ms) and coercivity (Hc) of M-type strontium hexagonal ferrites (SrFe 12 O 19 ) was 61.66 emu/g and 919 Oe, respectively (Fig. 4C). After the ferrite particles were incorporated in scaffolds, the MCSC 1:7 and MCSC 1:3 scaffolds both exhibited good magnetic property (Fig. 4D). The saturated magnetization value of the MCSC 1:3 scaffolds (10.36 emu·g −1 ) were greater than the MCSC 1:7 scaffolds (6.10 emu/g), indicating the ratio of ferrite particles in the scaffolds was positively related to magnetization (Fig. 4D). Moreover, both the MCSC 1:7 and MCSC 1:3 scaffolds exhibited similar coercivities (Hc) (1279 and 1510 Oe, respectively) (Fig. 4D). The higher saturation magnetization and coercivity of MCSC scaffolds resulted in the high magnetic field strength.
NIR photothermal conversion efficiency of magnetic mesoporous calcium sillicate/chitosan scaffolds. Compared with CSC scaffolds, the MCSC scaffolds possessed better photothermal conversion efficiency (Fig. 5A). With the irradiation of NIR laser, the temperature in both the MCSC 1:7 and MCSC 1:3 scaffolds in mediums was gradually increased (Fig. 5A). In addition to PTT, controlled drug delivery therapy is an effective approach to kill malignant cells. Doxorubicin (DOX), a widely used chemotherapy medication, was employed to evaluate the efficacy of MCSC as a drug carrier. The drug release profiles showed that DOX was gradually released from its carriers and the MCSC 1:7/DOX scaffolds had a similar drug release profile to that of the MCSC 1:3/DOX scaffolds (Fig. 5B,C). Notably, the NIR laser irradiation accelerated the DOX release ratio and the drug cumulative release ratio at 24 h for the MCSC 1:7/DOX, MCSC 1:3/DOX, MCSC 1:7/DOX/NIR and MCSC 1:3/DOX/NIR was 55.0%, 58.6%, 76.0% and 79.3%, respectively (Fig. 5B,C). The rapid release of DOX from MCSC scaffolds may have the potential to reduce systemic cytotoxicity for the higher local drug concentrations in tumors.

Application of the MCSC scaffolds in photothermal and anti-cancer therapy. In vitro analyses
for anti-tumor effect. In order to evaluate the synergism of MCSC scaffolds combined with PTT in combating tumor proliferation, their anti-tumor effects were tested both in vitro and in vivo. After incubation with MCSC 1:7 and MCSC 1:3 scaffolds for 24 h, MG-63 cells exhibited high cell viability indicating that both MCSC 1:7 and MCSC 1:3 scaffolds had good biocompatibility (Fig. 6A). MCSC 1:7/DOX and MCSC 1:3/DOX scaffolds treatment showed moderate anti-proliferative impacts on MG-63 cells (Fig. 6A). Next, we examined the synergy between MCSC scaffolds and PTT. MG-63 cells were incubated with MCSC scaffolds for 24 h, followed by exposure to laser illumination. As expected, laser irradiation combined with MCSC scaffolds exhibit potent anti-proliferative effects on MG-63 cells in a dose dependent manner, and laser irradiation enhanced the anti-tumor response of MCSC/DOX scaffolds as indicated by lower cell viability compared with cells treated with MCSC/DOX alone (Fig. 6A). Among them, the proliferative arrest effect in MCSC 1:7/DOX and MCSC 1:3/DOX scaffolds was stronger than those of MCSC 1:7 and MCSC 1:3 scaffolds when exposed to laser irradiation (Fig. 6A). Furthermore, cells irradiated twice exhibited a more significant proliferative arrest response and the MCSC 1:3/DOX scaffolds had a more profound cytotoxicity effect than that of MCSC 1:7/DOX scaffolds (Fig. 6B). The live/dead assay was used to validate the phenomenon. In consistent with previous findings, the MCSC 1:7/DOX and MCSC 1:3/DOX scaffolds had moderate cytotoxic effect on MG-63 cells. When cells was exposed to NIR, the MCSC scaffolds showed strong cytotoxic effect on MG-63 cells as almost no green signals was detected in MCSC 1:3 and MCSC 1:3/DOX treated groups (Fig. 6C). In vivo assay for anti-tumor effects. To further understand the synergistic effect of PTT in combination with MCSC 1:3 or MCSC 1:3/DOX scaffolds on anti-tumor effects, in vivo analyses were conducted and MNNG xenograft mouse model was established. Upon the NIR irradiation, the temperature in the tumor loci injected with the MCSC 1:3 scaffolds increased to approximately 44 °C (Fig. 7A). However, the temperature was comparable before and after the treatment of MCSC 1:3 scaffolds alone around the tumor loci (Fig. 7A,B). Next, the anti-tumor effects of MCSC 1:3 and MCSC 1:3/DOX scaffolds were evaluated. Compared with MCSC 1:3 scaffolds, MCSC 1:3/DOX scaffolds significantly inhibited tumor proliferation, indicating that MCSC 1:3/DOX scaffolds had anti-tumor responses in vivo (Fig. 7C,D). After NIR laser irradiation, the tumor volumes of MCSC 1:3-NIR mice and MCSC 1:3/DOX-NIR mice were significantly decreased (Fig. 7D). The tumor volumes in MCSC 1:3/ DOX-NIR mice were the smallest among others (Fig. 7C,D). Furthermore, MNNG cells were transfected with lentivirus containing enhanced green fluorescent protein genes (EGFP) (Fig. 7E) and again xenograft mouse model was established. Compared to day 0, the tumor volume was increased in mice treated with MCSC 1:3 and remained comparable in mice treated with MCSC1:3/DOX (Fig. 7F,G). In contrast, both the fluorescence intensity and area were significantly reduced in mice treated with the MCSC 1:3-NIR and MCSC1:3/DOX-NIR at day 12 and mice treated with MCSC1:3/DOX-NIR showed a more remarkable decrease (Fig. 7F,G). Hematoxylin and eosin (H&E) staining revealed that the MCSC 1:3-NIR and MCSC 1:3/DOX-NIR induced significantly higher cell necrosis ratio compared with MCSC 1:3 and MCSC 1:3/DOX scaffolds (Fig. 7H,I). These findings indicate that PTT could synergize with MCSC to achieve potent anti-tumor effects both in vitro and in vivo.

Application of the scaffolds in bone tissue regeneration. The evaluation of MCSC scaffolds in bone
regeneration in vitro. Next, we detected whether MCSC scaffolds were appropriate for bone regeneration. The attachment and morphology of hBMSCs cultured on CSC, MCSC 1:7 and MCSC 1:3 scaffolds were observed by SEM. Three days after incubation, the hBMSCs were seen to be attached on the surface of the pore struts and well-distributed ( Fig. 8A-C). Then the cell proliferation of cultured hBMSCs on scaffolds was determined by CCK-8. As shown in Fig. 8D, all tested scaffolds promoted hBMSCs proliferation, and the MCSC 1:7 and MCSC 1:3 scaffolds had significantly higher efficacy than CSC scaffolds at day 1 and day 7 in promoting cell proliferation. Notably, hBMSCs cultured on MCSC 1:3 scaffolds had the highest proliferation rate at all time points (Fig. 8D). Moreover, the expression of osteogenic genes was significantly higher in cells cultured on MCSC 1:3 scaffolds than that on MCSC 1:7 and CSC scaffolds ( Fig. 8E-H). Compared with CSC scaffolds, the expression of bone morphogenetic protein (BMP)-2, phosphorylated Smad1/5 and Runx2 at the protein level was remarkably upregulated in hBMSCs cultured on the MCSC 1:7 and MCSC 1:3 scaffolds (Fig. 8I), indicating BMP/Smad signaling was, at least in part, involved in promoting osteogenesis. Bone regeneration in vivo. To validate the osteogenesis effects of magnetic scaffolds, a bone defect rat model was established. Compared with rats in the control group, rats treated with CSC, MCSC 1:7 and MCSC 1:3 scaffolds showed obvious signs of bone formation and decreased defect area as indicated by micro-CT scanning (Fig. 9A). Moreover, bone formation in rats treated with MCSC 1:3 scaffolds was more impressive compared to that in rats treated with MCSC 1:7, and CSC scaffolds (Fig. 9A). The trabecular bone parameters, such as bone mineral density (BMD) and bone volume per tissue volume (BV/TV) were also measured. The BV/TV in rats treated with MCSC 1:3 scaffolds was 57.32 ± 3.53% which is significantly higher than that of the MCSC 1:7 scaffolds group (36.54 ± 2.08%), CSC scaffolds group (27.63 ± 4.09%) and control group (6.33 ± 1.2%) (Fig. 9B). Additionally, the BMD in rats treated with MCSC 1:3 scaffolds was significantly higher than that in rats treated with other scaffolds (Fig. 9C). Furthermore, Van Gieson's picrofuchsin staining showed that MCSC 1:3 and MCSC 1:7 scaffolds both exhibited osteogenic induction ability, and the effects MCSC 1:3 was more prominent than the latter (Fig. 9D). Additionally, the histomorphometric assay showed that the percentage of new bone area in MCSC 1:3 scaffolds and MCSC 1:7 scaffolds groups was significantly higher than that in CSC scaffolds and control groups (Fig. 9E). Bone formation and mineralization were also determined by calcein fluorescence assay. The fluorescence signaling located near the scaffolds indicated the new bone formation of the loci around the scaffolds (Fig. 9F). Notably, the fluorescence signaling in the MCSC 1:3 scaffolds group was higher over other scaffolds (Fig. 9F,G). These data demonstrate that the MCSC 1:3 scaffolds could effectively promote bone formation in vivo.

Discussion
Surgical resection of osteosarcoma may cause bone defects, and residual tumor tissues induce tumor recurrence 30 . The fabrication of multifunctional scaffolds with potent anti-tumor activity and osteogenesis property is a critical and promising strategy for treatment of the tumor-related bone defects. Herein, we, for the first time, fabricated the MCSC scaffolds combining the ability of enhanced new bone regeneration with the excellent capacity of chemo-photothermal synergetic therapy against osteosarcoma (Fig. 1).
The multifunctional MCSC porous scaffolds were fabricated by a freeze-drying method using SrFe 12 O 19 , CaSiO 3 and CS as original materials. The pore sizes of interconnected macropores were mainly distributed around 100~300 μm, which were formed due to the distillation of ice crystals during the freeze-drying process (Fig. 3) 31 . These macropores not only supported the adhesion and spreading of hBMSCs (Fig. 8), but also promoted the ingrowth of newly formed bone tissues (Fig. 9). The CS and mesporous CaSiO 3 microspheres played a vital role in cell performance and bone regeneration, too. The CSC scaffolds without magnetic particles possessed excellent cytocompatibility, and exhibited better bioactivity than blank control group (Fig. 9). On one hand, CS was an important osteoconductive matrix due to its excellent biocompatibility and biodegradability 32 ; on the other hand the Ca and Si from CaSiO 3 particles might have the potential to improve the stem cell viability and osteogenic differentiation 33,34 .
In order to eradicate the residual tumor cells to avoid tumor recurrence, we employed the strategies of drug delivery system and photothermal therapy. It was noted that NIR promoted the DOX release from MCSC scaffolds and the cumulative release ratio is 79.3% at 24 h, indicating anti-tumor drugs could be rapidly released locally to combat malignant cells and could potentially minimize the side effects caused by chemo-regimens. This phenomenon could possibly due to accelerated Brownian movement and the accelerated degradation of the chitosan matrix due to the increased temperatures in the particle surrounding, as suggested by a previous study 21 . In addition, DOX could also be gradually released from scaffolds at relatively low ratio, one potential translational usage of this property is that other potent anti-tumor drugs or drug combination could be carried to designated area and released at optimal ratio to combat tumors. Compared with MCSC, MCSC/DOX induced a higher cell death ratio and irradiation further enhanced their anti-tumor effects in a dose dependent manner. When irradiated twice, MG-63 cells treated with MCSC 1:3/DOX exhibited pronounced cell viability decrease compared with MCSC 1:7/DOX. This could be elucidated by that the temperature elevation in MCSC 1:3/DOX scaffolds was higher than that in MCSC 1:7/DOX scaffolds and the amount of DOX released by MCSC 1:3/DOX scaffolds was more than that of MCSC 1:7/DOX scaffolds (Fig. 5A-C). In vivo analyses further proved that NIR significantly promoted the temperature in tumors treated with MCSC1:3 scaffolds and the tumor volume was significantly smaller than that treated with MCSC1:3 scaffolds alone. Xenograft mouse model validated these results, which showed that NIR synergized with MCSC1:3 scaffolds to exert proliferative arrest effects and NIR enhanced the anti-tumor effects of MCSC1:3/DOX scaffolds. These findings strongly suggest that the MCSC1:3/DOX scaffolds had potent anti-tumor effects. However, the establishment of in situ bone metastasis animal model is very difficult and the xenograft mouse model of bone cancer could reflect the anti-tumor effects of PTT synergy with MCSC scaffolds as indicated by studies in solid tumors [35][36][37] . We also admit that the fact that NIR could be more difficult in reaching bone tissues. In future studies, we will use a more suitable mouse model to test the NIR penetration in bone tissues and its combination with MCSC scaffolds in combating malignant cells in bones. Interestingly, in the present study, we could see that the more loading of magnetic field, the higher anti-tumor effects. The phenomenon could possibly be explained by the fact that PTT induced a higher local temperature in tumor cells, thereby increasing the expression of reactive oxygen species (ROS) and heat shock proteins 38 as well as decreasing concentrations of metabolites associated with cell proliferation and tumor growth 39 . Further studies are needed to explore the mechanism of PTT synergy with MCSC scaffolds in inhibiting tumor cell proliferation. The CSC could support the adhesion, spreading and proliferation of hBMSCs (Fig. 8), but its property for enhancing new bone regeneration was limited (Fig. 9). Recently, It was reported that strong static magnetic field (SMF) not only up-regulated the stem cell differentiation, but also stimulated ectopic bone formation 40 . In this study, we incorporated SrFe 12 O 19 ferrite particles into the CSC scaffolds, resulting in the formation of MCSC porous scaffolds. COL1, OCN, Runx2 and ALP are essential for osteogenesis and their upregulation is a positive indicator for osteogenesis 41,42 . Interestingly, the MCSC scaffolds significantly augmented the expression of these indicators for hBMSCs, suggesting MCSC scaffolds have a potential in promoting bone formation. In vivo analysis revealed that the markers for bone formation such as BV/TV and BMD were increased. Micro-CT scan, Van Gieson's picrofuchsin staining and calcein fluorescence assay further proved that hBMSCs loaded MCSC scaffolds could effectively promote osteogenesis. The role of BMP/Smad signaling pathway in bone formation has been widely acknowledged in bone formation 43,44 and Runx2 has been reported to the downstream mediator for Smad1/5 45 . The present study revealed that the expression of BMP, Smad1/5 and Runx2 was upregulated in hBM-SCs loaded MCSC scaffolds. These data indicate that BMP/Smad/Runx2 signaling was, at least in part, involved in the magnetic scaffolds promoted osteogenic differentiation in hBMSCs. Furthermore, we observed that the higher ratio of ferrite particles, the higher expression of osteogenic proteins and bone formation. It was reported that composites containing SrFe 12 O 19 can potentially better facilitate bone formation 46 , which was in line with this study. Therefore, we could reasonably infer that M-type ferrite particles, at least SrFe 12 O 19 , had the potential to contribute to osteogenesis, and more studies are warranted to unveil the underlying mechanism.

Conclusions
This study suggested that the MCSC scaffolds could load anti-tumor drugs and synergy with PTT to combat tumor cells. Additionally, MCSC scaffolds could enhance the proliferation of hBMSCs and promote bone formation. These results strongly suggest that MCSC scaffolds are bi-functional and may have a potential role in patients with surgical resection of malignant bone tumors treatment. In summary, we have derived pre-clinical  data supporting a role for MCSC scaffolds in harbouring potent anti-tumor and bone regeneration capability both in vitro and in vivo. Nevertheless, more studies are needed to evaluate the efficacy and application of MCSC in clinics for patients with bone defects due to bone tumor resection.

Preparation of magnetic calcium silicate/chitosan porous (MCSC) scaffolds. Mesoporous calcium
silicate microspheres were prepared by using organic template method. In brief, cetyltrimethyl ammonium bromide (CTAB, 1.76 g) and ammonium hydroxide (NH 3 ·H 2 O, 3.20 ml) were dissolved in deionized water followed by stirring for 30 min. Under vigorous agitation, tetraethyl orthosilicate (TEOS, 9.33 ml) and 9.73 g calcium nitrate tetrahydrate were successively dissolved in the above solution, and were further aged for 20 h. The precipitates (mesoporous calcium silicate microspheres) were washed by deionized water, dried at 80 °C and calcined at 550 °C for 4 h.

NIR laser-induced temperature increase and in vitro drug release test.
A PBS solution with pH value of 7.4 was prepared by adding 5.8032 g Na 2 HPO 4 ·12H 2 O and 0.8894 g NaH 2 PO 4 ·2H 2 O into 1000 mL distilled water. A NIR laser was fixed 15 cm higher than the center of the scaffolds. Under the irradiation of NIR (λ = 808 nm, 4.6 W/cm 2 ), the temperatures of the media with the scaffolds were detected by thermocouple thermometer (Brannan, Cleator Moor, England). The NIR-light-triggered drug release tests of MCSC 1:3/DOX and MCSC 1:7/DOX scaffolds were performed by the immersion of DOX loaded scaffold in 5 ml PBS solution under orbital shaking of 80 rpm. In brief, the probe of NIR laser (λ = 808 nm, 4.6 W·cm −2 ) was fixed 15 cm higher from the center of the scaffolds. At given time intervals, the scaffolds were irradiated by NIR laser for 6 min. Then 1.0 ml DOX-release medium was extracted and supplemented with 1.0 ml fresh PBS. The DOX concentrations of the DOX-release medium were characterized by fluorescence spectrophotometer (F-4600, Hitachi, Tokyo, Japan) using a xenon lamp as excitation source (λ = 495 nm). The in vitro drug release of the MCSC 1:7/DOX or MCSC 1:3/DOX scaffolds without NIR irradiation was performed with similar procedures.
Cell Culture. The human MG-63 and MNNG osteosarcoma cell line was obtained from ATCC, cultured with high glucose DMEM (Gibco) containing 10% FBS (Gibco) and 1/100 penicillin-streptomycin (Gibco). All cells were kept in humidified atmosphere containing 5% CO 2 at 37 °C and subject for anti-tumor analyses.

PTT in tumors in vivo.
The xenograft mouse model bearing osteosarcoma cells was constructed as previously described 33 . In brief, the osteosarcoma MNNG (5 × 10 6 ) cells with or without EGFR transfection were subcutaneously inoculated into the left flank of nude mice (4-6 weeks old, 20 ± 2 g). When tumors were palpable, the mice were randomly divided into MCSC 1:3 scaffolds, MCSC 1:3/DOX scaffolds with laser irradiation, MCSC 1:3 scaffolds and MCSC 1:3/DOX scaffolds without irradiation groups (n = 5/group). A skin incision was made at the edge of the tumor and then the scaffold (diameter: 4 mm, height: 2 mm) was implanted.
For laser irradiation, the mice were exposed to the NIR laser (0.3 W/cm 2 ) for 6 min post scaffolds implantation. Meanwhile, the temperature in the tumor was monitored by real-time IR thermal imaging system. The first NIR treatment time was regarded as day 0. From day 0, tumor sizes were measured with calipers and their volumes were calculated by the following the formula: (length × width 2 )/2. V 0 is the initial tumor volume-scaffold volume at day 0. The relative tumor size = V/V 0 . Whole-body fluorescent imaging was performed at day 0 and day 12. The mice were sacrificed at the 12 th day, and their tumors were immersed in 4% neutral buffered formalin for 24 h, then the tissues were embedded in paraffin and cut to 5 μM sections. Afterwards, the sections were dehydrated with gradient alcohol and xylene, then they were subjected for hematoxylin-eosin (H&E) staining according to manufacturer's instructions (Beyotime, China).
The tumor cell necrosis rate was also measured. Five slices were selected, and five random visions were measured. Tumor cell necrosis rate (TCNR) was measured according to the formula, TCNR = the number of necrotic cells/the total cell number × 100%. The size of MCSC scaffolds for all in vitro analyses was 6 mm X 2 mm and the amount of the DOX was 300 ng.
Morphology assay. A number of 1.0 × 10 4 hBMSCs at its third passage were seeded in a 24-well culture plate and incubated scaffolds. To evaluate the cell attachment and morphology in CSC, MCSC 1:7 and MCSC 1:3 scaffolds at 1, 3 and 7 days, the scaffolds were fixed in 2.5% glutaraldehyde for 20 min, washed with PBS for three times, dehydrated in a graded ethanol and hexamethyldisilizane. Then dehydrated cellular scaffolds were coated with gold and observed by SEM (Hitachi, S-4700, Tokyo, Japan).

Real-time polymerase chain reaction (RT-PCR).
After hBMSCs were treated with the scaffolds for 14 days, then total RNA was extracted using the TRIzol reagent (Invitrogen, #15596-026). cDNA was reverse-transcribed using SuperScript First-Strand Synthesis System (Invitrogen, #1209992, USA). The expression of ALP, Runx2, OCN and COL1 was measured by RT-PCR using FastStart Universal SYBR Green Master (Roche, #04913914001, USA) in a ViiA 7 Real-Time PCR System (Applied Biosystems, USA). The procedure for this reaction is 95 °C, 5 min; 34 cycles for 95 °C, 5 min and 60 °C, 40 s; and 72 °C, 5 min. The data was analyzed by 2 −ΔΔCt method and actin was used as the reference gene. The primers for RT-PCR are listed in Table 1.
Bilateral critical-sized calvarial-defect rat model. Twenty Sprague-Dawley male rats (300-350 g) obtained from Silaike (Shanghai, China) were used to establish the bilateral critical-sized calvarial-defect rat model (n = 5/group). Two parietal defects were created (diameter: 5 mm, height: 2 mm), then either CSC, MCSC 1:7 and MCSC 1:3 scaffolds were implanted into the defect and the incisions were closed. A polychrome sequential fluorescent labeling method was performed to observe bone formation and mineralization on rats at week 12 post-treatment. At 2, 4 and 6 weeks after implantation, fluorochromes [25 mg/kg tetracycline (TE; Sigma, USA), 30 mg/kg alizarin reds (AL; Sigma, USA) and 20 mg kg 1 calcein (CA; Sigma, USA)] were injected (i.p.) to rats. At 12 weeks post-implantation, the rats were sacrificed and micro-CT (Skyscan 1176, Kontich, Belgium). Three dimention (3-D) images were reconstructed using the 3-D Creator software. Besides bone volume to total bone volume (BV/TV) and local bone mineral densities (BMD). After dehydration in ascending concentrations of alcohol the undecalcified specimens were embedded in poly-methylmethacrylate and 150 mm thick sections in the orientation of the sagittal surface were obtained using a microtome (Leica, Hamburg, Germany). The sections were observed for fluorescent labeling using a confocal microscope (Leica, Heidel-berg, Germany). The excitation/emission wavelengths of the chelating fluorochromes used were 405/560-590 nm (tetracycline, yellow), 543/580-670 nm (alizarin red, red) and 488/500-550 nm (calcein, green). Histological analyses were carried out by Van Gieson's picrofuchsin staining. With a computer-based image analysis system (Image-Pro Plus 6.0, Media Cybernetics, Silver Springs, MD), the area of new bone was quantified as a percentage of the total bone defect area.
All the data were expressed as means ± standard deviation (SD) and were analyzed using one-way ANOVA with a post hoc test. A two-tailed p-value < 0.05 was considered statistically significant.