Sustained Delivery of BMP-2-Related Peptide from ... - ACS Publications

Article Cite This: ACS Biomater. Sci. Eng. 2018, 4, 211−221

Sustained Delivery of BMP-2-Related Peptide from the True Bone Ceramics/Hollow Mesoporous Silica Nanoparticles Scaffold for Bone Tissue Regeneration Wei Cui,†,‡,∥ Qianqian Liu,§,∥ Liang Yang,† Ke Wang,§ Tingfang Sun,† Yanhui Ji,† Liping Liu,§ Wei Yu,† Yanzhen Qu,† Junwen Wang,‡ Zhigang Zhao,‡ Jintao Zhu,*,§ and Xiaodong Guo*,† †

Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), Wuhan 430022, China ‡ Department of Orthopaedics, Wuhan Puai Hospital, Tongji Medical College, HUST, Wuhan 430032, China § Key Laboratory of Materials Chemistry for Energy Conversion and Storage of Ministry of Education, School of Chemistry and Chemical Engineering, HUST, Wuhan 430074, China S Supporting Information *

ABSTRACT: Bone morphogenetic protein 2 (BMP-2) is one of the most important factors for bone tissue formation. A number of BMP-2 related small molecule bioactive peptides have been designed and shown to be equally effective in osteogenic activity. In this report, we synthesized a novel BMP-2related peptide (designated P28) and designed a delivery system to regulate the controlled release of P28 from true bone ceramics (TBC) combined with an enlarged pore hollow mesoporous silica nanoparticles (HMSNs) composite scaffold. An in vitro release showed that the release of P28 from the TBC/ HMSN scaffold was slower than that from the TBC scaffold. An in vitro cell experiment of the TBC/HMSN/P28 scaffold was tested with MC3T3-E1 cells in comparison to TBC, TBC/HMSN, and TBC/P28 scaffolds. Our results demonstrated that the TBC/HMSN/P28 scaffold had better effects on promoting proliferation and osteogenic differentiation of MC3T3-E1 cells than TBC, TBC/HMSN, and TBC/P28 scaffolds. After four kinds of scaffolds were implanted into a rabbit radius critical bone defect for 6 and 12 weeks, the radiographic and histological examination indicated that this osteogenic delivery system TBC/HMSN/P28 scaffold effectively induced bone regeneration in vivo. Therefore, the TBC/ HMSN/P28 scaffold can promote proliferation and osteogenic differentiation of MC3T3-E1 cells in vitro and new bone tissue generation in vivo. This study provides a promising scaffold for bone tissue engineering and regenerative medicine. KEYWORDS: BMP-2, synthetic peptide, hollow mesoporous silica nanoparticles, true bone ceramics, osteogenic differentiation, bone tissue regeneration

1. INTRODUCTION

DNA technology has simplified BMP production, and the United States Federal Drug Administration (FDA) has allowed the clinical use of rhBMP-2;9 however, large and nonphysiological doses of rhBMP-2 were customarily used because of its short half-life in vivo.10 This led to the occurrence of numerous undesirable side effects such as inflammatory reactions, radiculitis, urinary retention, bone resorption, and cancer risk.11,12 Recently, a number of BMP-2 related small molecule bioactive peptides have been designed and shown to be equally effective in osteogenic activity.13−16 To surmount the shortcomings of rhBMP-2, we have designed a short peptide (designated P28, S[PO4]DDDDDDDKIPKASSVPTELSAISTLYL, MW:3091.20) according to the residues of the

Millions of patients suffer from bone defects annually owing to trauma, cancer, and bone disease, most of which are difficult to repair, bringing challenges for clinical orthopedics worldwide.1 So far, the success rate for autologous bone grafts reaches as high as 80−90%. Therefore, autologous bone grafts are considered the “gold standard” for bone defect repair.2,3 However, autologous bone graft is restricted due to the limited sources of donor materials, new defects at the donor site, and extended surgery duration.4,5 Therefore, the major goal of bone tissue engineering is to develop an ideal bionic bone repair material. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β superfamily (TGF-β) and have exhibited the strongest bone induction activity among bone growth factors.6 Moreover, among BMPs, BMP-2 exhibits the strongest ability in inducing bone-regeneration.7,8 Recombinant © 2017 American Chemical Society

Received: July 20, 2017 Accepted: December 15, 2017 Published: December 15, 2017 211

DOI: 10.1021/acsbiomaterials.7b00506 ACS Biomater. Sci. Eng. 2018, 4, 211−221

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ACS Biomaterials Science & Engineering Scheme 1. Illustration Showing the Synthetic Procedure of the TBC/HMSN/P28 Composite Material

TBC/HMSN/P28). The objectives of this study were to examine the kinetics of P28 release from the TBC/HMSN scaffold; to evaluate the osteogenic differentiation of MC3T3E1 cells on the TBC/HMSN/P28 scaffold in vitro; and to determine the effectiveness of the TBC/HMSN/P28 scaffold in the repair of a radius critical size defect in vivo.

knuckle epitope of BMP-2, and P28 has been proven to play osteoinductive activity similar to BMP-2.17 In general, the osteogenic activity of osteoinductive drugs is significantly affected by rapid release and degradation of the in vivo environment,18 and one promising strategy to overcome these limitations is sustained delivery of drugs encapsulated within nanocarriers. Mesoporous silica nanoparticles (MSNs) have demonstrated great promise in the field of drug delivery due to their versatile surface functionalization,19 excellent mesoporous structure, and an adjustable pore size.20,21 Meanwhile, MSNs also provide large surface area and pore volume,22 in vivo biosafety,23 biodegradation,24 and biodistribution and excretion.25 Recently, MSNs have been proven to be bioactive for bone regeneration.26 The silanol groups on the surface of MSNs react with body fluids to generate bioactive nanosized carbonated apatite, which can bind to natural bone.27,28 This bioactive formation ensures the integration of implanted bone. In addition, MSNs can encapsulate osteoinductive drugs, which promote the bone regeneration in vivo.29,30 Therefore, the ability of MSNs to act as bone regenerators and to carry osteoinductive drugs suggests the potential for designing MSNs with specific medical applications.26 Furthermore, compared with conventional mesoporous silica, hollow mesoporous silica nanoparticles (HMSNs) derived from MSNs with a core−shell structure can be augmented with more favorable features, such as higher storage capacity due to the special hollow core structure, and sustained drug release profiles since drugs can diffuse into the cavities through accessible pore channels on the shell.31 Unfortunately, MSNs do not have the appropriate mechanical properties and osteoconductivity. In our previous study, we prepared a type of true bone ceramic (TBC) scaffold material by high temperature sintering of animal cancellous bone. Our previous reports demonstrated that the TBC scaffold not only can completely retain the fine microporous structure of natural bone but also has excellent biocompatibility, as well as appropriate osteoconductivity and mechanical properties.32,33 In this study, we loaded P28 into HMSN with enlarged mesopores through decane and then combined it with TBC to prepare a new bionic bone repair composite scaffold (e.g.,

2. EXPERIMENTAL SECTION 2.1. Materials. Cetyltrimethylammonium bromide (CTAB), chitosan (CHI), Calcein-AM, propidium iodide (PI), and BCA protein assay were obtained from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Tetraethyl orthosilicate (TEOS), ammonia, decane, and sodium carbonate (Na2CO3) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). BMP-2releated peptide 28 was synthesized from Chinese Peptide Co., Ltd. (Hangzhou, China). Fetal bovine serum (FBS), penicillin-streptomycin, trypsin, and α-MEM were obtained from Gibco Life Technologies Co. (Grand Island, NY). MTT assay kit and ALP protein assay kit were obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Ultrapure milli-Q water was used in all experiments. 2.2. Synthesis of P28. The BMP-2 derived peptide (e.g., P28) was synthesized by Fmoc/tBu solid-phase peptide synthesis (SPSS)33 and preliminarily purified by gel filtration. The higher purity of P28 was determined by high performance liquid chromatography (HPLC, Agilent Technologies Inc., 1100S, USA),34 and the purity was measured to be 97.4% (see the Supporting Information Figures S1 and S2). 2.3. Preparation of TBC. Fresh bovine cancellous bone was cut into pieces, and grease and organic ingredients were removed by sintering. The sintering process mainly consisted of three steps. First, the cancellous bone was heated slowly at a rate of ∼5 °C/min to 800 °C, maintained for 6 h, and then cooled naturally in a muffle furnace. Second, the calcined cancellous bone was soaked in a 0.09 mol/L sodium phosphate solution in a 70 °C water bath for 72 h. Third, the cancellous bone was heated again slowly to 1200 °C, maintained for 1 h, and then cooled naturally to room temperature to obtain the required TBC material. The TBC was sterilized by 70% ethanol for 24 h and washed with deionized water. 2.4. Preparation of Enlarged Pore HMSNs. Enlarged pore hollow mesoporous silica NPs were synthesized through the following pore-expand process (Scheme 1). Briefly, 20 mL of ethanol and 1.6 mL of water were mixed with 0.8 mL of ammonia (∼37−38%), and then 0.6 mL of TEOS was added. The sSiO2 NPs dispersion was obtained after stirring for 6 h at 30 °C. Next, mesoporous silica shell was formed around sSiO2 (sSiO2@mSiO2 core/shell NPs). CTAB and 212


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ACS Biomaterials Science & Engineering decane with a desirable molar ratio were first dissolved in 44 mL of water and 2 mL of ethanol with stirring at 40 °C to obtain a clear micellar solution. The above formed sSiO2 NPs dispersion was poured into the micellar solution, followed by stirring for 30 min. Then, 0.215 mL of TEOS was subsequently added and stirred overnight at 30 °C. The precipitate was collected by centrifugation and redispersed in deionized water (30 mL). Subsequently, the prepared NPs were collected by centrifugation and redispersed in a 0.4 M Na2CO3 aqueous solution and etched for 2 h at 50 °C to remove the solid silica core. Finally, HMSNs were obtained after removal of CTAB micelles and decane in the mesoporous shell by repeated washes with concentrated HCl/ethanol (v/v = 1:10) and water 3 times with the help of sonication, respectively. The structure of the HMSNs was characterized by a transmission electron microscope (TEM, Tecnai G220, FEI Company, Holland) and a N 2 adsorption investigation (TriStar II 3020, Alpha Technologies Company, USA). 2.5. Preparation of TBC/HMSN/P28, TBC/MSN, and TBC/P28 Scaffolds. The TBC/HMSN/P28 scaffold was synthesized according to the procedure shown in Scheme 1. P28-loaded HMSNs (HMSN/ P28) were prepared by a solution-solvent evaporation method. Typically, the mass ratio of P28:HMSNs was 1:3. First, 3 mg (for cell experiment) or 7.5 mg (for animal experiment) of P28 was dissolved in water, and then P28 solution was added into 9 mg or 22.5 mg of HMSNs suspension. After sonication for 5 min, the suspension was evaporated by vacuum. HMSN/P28 was obtained after washing with PBS twice. Subsequently, HMSN/P28 was added into a 2 mg/mL CHI solution (containing 1% acetic acid) and incubated for 24 h. To obtain the TBC/HMSN/P28 scaffold, TBC was put into a 24-well tissue plate, and then 700 μL of HMSN/P28 solutions was added. The compounds were put in a vacuum oven for a certain negative pressure for 30 min twice and then kept at 4 °C for 24 h before freeze-drying. The TBC/HMSN scaffold was made in a similar way except for the addition of the P28 peptide, while the TBC/P28 scaffold was made in a similar route except for the addition of HMSNs. All materials were sterilized by ethanol evaporation and frozen at −20 °C until use. The morphology of the TBC and TBC/HMSN was characterized by a scanning electron microscope (SEM, S-4800, HITACHI Company, Japan). The energy spectrum of the TBC and HMSN/ CHI/TBC porous composite scaffold was characterized by Energy Disperse Spectroscopy (EDS, Inca X-Max, Oxford Instruments plc, UK). 2.6. P28 Release in Vitro. The in vitro release profile of P28 from TBC/P28 and TBC/HMSN/P28 scaffolds was performed. The TBC/ P28 and TBC/MSN/P28 composite scaffolds, which were small cubes with length, width, and height of 5 mm, were incubated in 5 mL of a PBS solution (pH 7.4) at 37 °C for up to 30 days in a water bath shaker at 120 rpm. The eluate of the P28 peptide was detected at 1, 2, 3, 5, 7, 10, 12, 14, 18, 21, and 30 days after incubation. The medium was then removed and replaced with fresh PBS at a specified time. The drug concentration of the P28 was analyzed with HPLC with a UV− vis detector at a wavelength of 220 nm. 2.7. Cell Culture. Mouse MC3T3-E1 cells were purchased from the China Center for Type Culture Collection (Wuhan University). The medium for culturing MC3T3 cells was composed of α-MEM supplemented with 10% fetal bovine serum and a 1% antibioticantimycotic solution. The cells were incubated at 37 °C with 5% CO2, and medium was changed every 3 days. When cells reached 80% confluence, they were detached using 0.25% trypsin/0.03% ethylene diamine tetraacetic acid (EDTA) and seeded in three new cell culture bottles as passage 1 to continue incubation. Cells were seeded on four groups of scaffold and performed related assays on second passage cells after enlarged cultivation. 2.7.1. Cell Culture on Scaffold. The four groups of scaffolds (Group A: TBC; Group B: TBC/HMSN; Group C: TBC/P28; and Group D: TBC/HMSN/P28), which were wafers with a diameter of 10 mm and a thickness of 3 mm, were placed in 24-well culture plates and incubated with medium for 1 h, and then 2 × 105 MC3T3-E1 cells per well were seeded on each scaffold. The cells were incubated at 37 °C with 5% CO2, and the medium was changed every 3 days.

2.7.2. Cell Adhesion Assay. After MC3T3-E1 cells were treated with TBC, TBC/HMSN, TBC/P28, TBC/HMSN/P28 groups for 24 h, the medium was removed, and the scaffolds were gently rinsed three times with a PBS solution to ensure that the cells did not adhere to the surface of the scaffold. To calculate the cell adhesion rate of each scaffold, the cells were collected and counted.35 2.7.3. Confocal Laser Scanning Microscopy (CLSM) Investigation. After MC3T3-E1 cells were treated with TBC, TBC/HMSN, TBC/ P28, TBC/HMSN/P28 groups for 3 days, the medium was removed, and the scaffolds were gently rinsed three times with a PBS solution. To label living and dead cells, MC3T3-E1 cells treated with different groups were incubated with Calcein-AM/PI solutions36 for 15 min at 37 °C with 5% CO2. Finally, the living cells and dead cells were observed respectively using a LSCM (Leica, Germany) at the excitation wavelengths of 490 and 525 nm. 2.7.4. Cell Proliferation Assay. MTT assay was used to evaluate the cell proliferation.37 After MC3T3-E1 cells were treated with TBC, TBC/HMSN, TBC/P28, TBC/HMSN/P28 groups for 2, 4, 6, and 8 days, the medium was removed, and the scaffolds were gently rinsed with a PBS solution. Then, 200 μL of a MTT solution (5 mg/mL, pH 7.4) and 1.2 mL of α-MEM were added to each well, followed by incubation for 4 h at 37 °C with 5% CO2. To dissolve MTT formazan sufficiently, 1 mL of dimethyl sulfoxide (DMSO, Sigma, USA) was added to each well. Finally, quantification of the formazan formed was measured using a microplate reader (Thermo Fisher Scientific, USA) at the wavelength of 570 nm. 2.7.5. Determination of Alkaline Phosphatase (ALP) Activity. ALP activity of MC3TE-E1 cells on the four groups of scaffolds was measured using the ALP protein assay kit and the BCA protein assay kit.38 After MC3T3-E1 cells were cultured with TBC, TBC/HMSN, TBC/P28, TBC/HMSN/P28 groups for 5, 10, and 15 days, the ALP activity of the supernatant in the cell lysate was measured using the microplate reader at the wavelength of 405 nm. The results of the four types of scaffolds were normalized by protein content. 2.8. Radial Bone Defect in Vivo. Animal experiments were performed as preapproved by the Institutional Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). Twenty male New Zealand white rabbits (body weight 2 kg) were purchased from the Animal Center of Tongji Medical College. All rabbits had a bilateral radial bone defect. All 20 rabbits were randomly divided into two groups (10 rabbits per group). The left and right radii of 10 rabbits were groups A (TBC) and B (TBC/HMSN), respectively, and the left and right radii of the other 10 rabbits were groups C (TBC/P28) and D (TBC/HMSN/ P28), respectively. After each rabbit was anaesthetized with an intramuscular injection of ketamine (10 mg/kg body weight), surgery was performed under sterile conditions. After lateral longitudinal skin was incised in the midshaft of the radius, the radial bone was exposed, and a 15 mm diaphysis was cut off using a burr drill with sterile saline irrigation. Then, the prepared cylindrical scaffold was embedded within the defect region, and the soft tissue and skin were closed by suturing. All rabbits were individually caged and fed after the operation. 2.8.1. Radiographic Examination. At 6 and 12 weeks postoperation, all rabbits were euthanized according to the intravenous injection of a phenobarbital solution, and radius specimens were obtained. Then, these specimens were observed by X-ray (Carestream Health, USA) and 3-dimensional-computerized tomography (3D-CT) (General Electric, USA). The Lane-Sandhu X-ray criteria39 were used to evaluate the treatment of the radial bone defect in the four groups. 2.8.2. Histological and Histomorphometric Analysis. For histological examination, the specimens were fixed in 10% neutral formalin, subsequently decalcified in 10% EDTA (pH 7.0), dehydrated in alcohol, and embedded in paraffin. Serial slices of 5 μm were then sectioned using a microtome and subsequently stained with hematoxylin/eosin (HE) as well as Masson’s trichrome for microscopic observation. The slices were then placed on an Olympus BX51 light microscope connected with a change coupled device camera (Olympus, Japan), and the histological analyses of the new bone 213


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ACS Biomaterials Science & Engineering

Figure 1. TEM images of the HMSNs synthesized through varying molar ratios of decane/CTAB: (a) 0.0, (b) 0.5, (c) 1.0, (d) 2.0, and (e) 4.0. regeneration were performed using Image-Pro Plus software (Media Cybernetics, USA). 2.9. Statistical Analysis. All numerical data were reported as mean ± standard deviation (SD) and tested for significance (p < 0.05) using analysis of variance (ANOVA) with GraphPad Prism 6 (GraphPad Software, USA). Tukey’s test using a posthoc multiple comparison was performed to compare individual pairs of groups.

indicating the presence of open-ended mesopores in HMSNs (Figure 2a). With the increase of the molar decane/surfactant ratio (0.0, 0.5, and 1.0), the average pore diameter increases (2.5, 3.4, and 4.2 nm). During the micellar solution preparation, it is possible that the decane molecules occupy the core of the surfactant micelles.42 The volume of the micelle is increased, whereas the effective cross-sectional area of one surfactant molecule remains constant.42 Thereafter, it remains constant around 4.1 nm until a decane/surfactant molar ratio of 2.0. The incorporation of more decane has no further effect on the value of the pore diameter. When the decane/surfactant molar ratio increased to 4.0, the average pore diameter decreased to 3.6 nm. This thus indicated that more of the expanded agent presumably led to a destruction of the formed micelles.42 We thus can conclude that the optimal value for the molar decane/ surfactant ratio to enlarge the pore size of HMSNs is between 1.0 and 2.0. According to the morphology and pore size of HMSNs with different molar decane/surfactant ratios, the poreexpand HMSNs with the decane/surfactant molar ratio of 1.0 were chosen in the following experiments. Figure 3 depicts TEM images (Figure 3a, b, c) with different magnifications and the X-ray diffraction (XRD) patterns (Figure 3d) of samples prepared using a decane/surfactant molar ratio of 1.0. The resulting HMSNs displayed homogeneous spherical morphology and a uniform hollow structure with a diameter of ∼150− 200 nm and a shell thickness of ∼30−40 nm. The powder XRD pattern of the as-prepared HMSNs, a single broad diffraction peak 2θ = 22.5°, indicates that the particles are amorphous silica. 3.2. Characteristics of the Scaffolds. To meet the growth of new bone tissue, the scaffold material for repairing bone defect must have good biocompatibility, bone conductivity, and porosity.43 The main component of TBC is hydroxyapatite which is the major inorganic component of bone. The structures of the TBC and TBC/HMSN scaffolds were analyzed by SEM (Figure 4), and the results confirmed that the TBC scaffold contained abundant pores with natural round or oval structures, which were connected with each other. Moreover, the trabecular bone was arranged in an ordered fashion, and the surface was rough. Pore diameters were

3. RESULTS AND DISCUSSION 3.1. Generation of Enlarged Pore HMSNs. The preparation route for enlarged pore HMSNs is shown in Scheme 1. First, silica (sSiO2) NPs were synthesized through a modified Stöber method.40 Next, a mesoporous silica shell was coated on the silica core by a surfactant templating sol−gel approach to form the core/shell-structured sSiO2@mSiO2 NPs, using the surfactant CTAB as a template and decane as a poreexpand additive. The degree of condensation of the mesoporous shell was higher than the solid core due to the mediation of an ammonia solution and the influence of surfactants. 42 Therefore, the solid silica core can be subsequently removed by a mild treatment with a Na2CO3 aqueous solution at relatively low temperature (50 °C) to form the hollow mesoporous silica nanoparticles (HMSNs, Figure 1). In addition, the more open structure of the shell facilitated the penetration of the −OH ions to selectively etch the core part.41 To eliminate the potential poisonous effects of cationic surfactant CTAB on cells, CTAB in HMSNs was removed by the HCl/ethanol mixture with the aid of ultrasonication. Decane and CTAB were mixed at molar ratios of 0.0, 0.5, 1.0, 2.0, and 4.0 to control the pore size. HMSNs, with the decane/ CTAB molar ratios of 0.0, 0.5, and 1.0, had a smooth surface (Figure 1a and 1b). However, when the molar ratios of decane/ CTAB increased to 2.0 and 4.0, the surface of HMSNs became rough (Figure 1c and 1d). The specific surface area and pore structure of HMSNs were investigated by N2 adsorption investigation. Figure 2 depicts the nitrogen adsorption− desorption isotherm (Figure 2a) and the pore size distribution (Figure 2b) of the material obtained with decane/surfactant molar ratios of 0.0, 0.5, 1.0, 2.0, and 4.0. A classical type IV adsorption curve was observed for HMSNs, and the desorption curve declined rapidly near the middle of the relative pressure, 214


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Figure 3. (a-c) TEM images of HMSNs with a molar ratio of decane/ CTAB of 1.0 and (d) an X-ray diffraction pattern of the HMSNs.

TBC/HMSN composite scaffold show that the element Si ratio of quality was 1.77 (Figure 5b). This further confirmed that HMSNs are successfully incorporated into TBC. 3.3. Release Kinetics of P28 in Vitro. The cumulative release of P28 over time from TBC and TBC/HMSN scaffolds was shown in Figure 6. After incubation for 30 days, 81.48 ± 0.53% and 68.83 ± 2.17% of P28 were respectively released from TBC and TBC/HMSN scaffolds. However, the amount of P28 that could be ultimately loaded by TBC and TBC/HMSN was about 82% and 98%, respectively. In other words, about all of P28 was released from the TBC scaffold, and about 2/3 of P28 was released from the TBC/HMSN scaffold after incubation for 30 days. On the first day, the release of P28 from TBC and TBC/HMSN scaffolds was 34.45 ± 1.62% and 14.79 ± 1.91%, respectively. The release of P28 began to decrease on the third day and then gradually became constant. After incubation for 12 days, 72.82 ± 1.35% and 55.10 ± 2.07% of P28 were respectively released from TBC and TBC/HMSN scaffolds. The release profiles indicated the more sustained release of P28 from TBC/HMSN scaffolds than from TBC scaffolds, which was attributed to the encapsulation of P28 by HMSNs. The slow release of osteogenic growth factor can promote osteogenesis.18 Previous studies showed that repetitive glutamic acid or aspartic acid has a high affinity for HA.15,44,45 Thus, to enhance the affinity of P28 with the scaffold material, the amino acid sequence of P28 was added into the repetitive amino acid sequence. The release profiles indicated that the addition of HMSNs greatly reduced the release of P28 and achieved an improved sustained release effect rather than the neat TBC scaffold. 3.4. Cell Adhesion and Viability Assay. The composite materials for bone formation must have good biocompatibility and should be beneficial for the adhesion of cells on their surfaces.1 The cell adhesion rates were measured for 24 h after MC3T3-E1 cells had been seeded on the four groups of scaffolds (Figure 7a). The percentage of cells attached to TBC/ HMSN/P28 and TBC/P28 scaffolds was 66.68 ± 4.73% and 72.78 ± 4.90% respectively, which was greater than that of the TBC/HMSN and TBC scaffolds (48.25 ± 6.51% and 51.41 ± 5.23%, respectively). Clearly, cell adhesion rates on TBC/

Figure 2. Plots show the (a) nitrogen adsorption−desorption isotherms and (b) pore size distribution for the HMSNs produced from various molar ratios of decane/CTAB: 0.0, 0.5, 1.0, 2.0, and 4.0.

measured to be 220−800 μm based on the SEM images. The SEM images of the TBC/HMSN scaffold showed that the freeze-dried chitosan was filamentous distribution in the internal structure of TBC, and the surface of the trabecular bone became rougher, indicating that HMSNs were well composited with TBC with the help of chitosan. The TBC material retains the natural pore structure of cancellous bone to ensure the growth of new bone tissue, blood vessels, and other soft tissue, which has also been demonstrated in previous studies.32,33 In this study, HMSNs were successfully incorporated into the TBC scaffold, and the combination of TBC and HMSNs had no significant influence on the pore structure of the TBC scaffold. In addition, the presence of Si in the TBC/HMSN composite scaffold was confirmed by EDS with mapping element analysis. The EDS spectra showed mainly the Ca and P peaks in the TBC scaffold with no Si peak; hence, the main elements were Ca and P (Figure 5a). The EDS results of the 215


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Figure 4. SEM images of (a) TBC and (c) TBC/HMSN scaffolds and (b) and (d) the magnified images for (a) and (c), respectively. (Yellow arrows in (d) indicate HMSNs.)

Figure 5. Energy spectrum analysis of (a) TBC and (b) TBC/HMSN scaffolds.

After incubation for 3 days, the MC3T3-E1 cells adhered to the scaffolds were examined using CLSM (Figure 7b). The living cells, which adhered to the scaffolds staining Calcein-AM, showed a green fluorescence signal, while the dead cells (stained by PI) displayed a red fluorescence signal. Clearly, there was a large number of living cells adhering to the TBC/ P28 and TBC/HMSN/P28 scaffolds, which were significantly more than TBC and TBC/HMSN scaffolds. This can be ascribed due to the following three reasons. First, because of the chemotactic effect of P28 on cells, the number of cells adhered to the TBC/P28 and TBC/HMSN/P28 scaffolds was more than those on the TBC and TBC/HMSN scaffolds after planting for 24 h. Second, P28 could play a role in promoting the proliferation of MC3T3-E1 cells. Third, TBC and HMSN have good biocompatibility for cell adhesion and proliferation. Remarkably, there were only a few dead cells on the four groups of scaffolds. In addition, to assess cellular activity, the proliferation of the MC3T3-E1 of each group of cells was tested by using the MTT assays after incubation for 2, 4, 6, and 8 days. As shown in Figure 7c, the results indicated that the MC3T3-E1 cells had great enhancement of proliferation on four materials, and the cell proliferation on TBC/HMSN/P28 and TBC/P28 scaffolds was significantly higher than those on TBC/HMSN and TBC scaffolds in the whole culture period (* p < 0.05, ** p < 0.01). These results demonstrated that TBC/HMSN/P28 possessed

Figure 6. Cumulative release of P28 from TBC/P28 and TBC/ HMSN/P28 scaffolds.

HMSN/P28 and TBC/P28 scaffolds were significantly higher than those on TBC/HMSN and TBC scaffolds (* p < 0.05, ** p < 0.01). In addition, no significant difference existed between the TBC/HMSN/P28 and TBC/P28 scaffolds (p > 0.05), and no significant difference existed between the TBC/HMSN and TBC scaffolds (p > 0.05). This result proved that the TBC/ HMSN/P28 and TBC/P28 scaffolds were suitable for the adhesion of MC3T3-E1 cells, and P28 could play an important role in chemotaxis for MC3T3-E1 cells in order to promote more cell adhesion on the materials. 216


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Figure 7. (a) Adhesion rates of MC3T3-E1 in TBC, TBC/HMSN, TBC/P28, and TBC/HMSN/P28 groups; (b) fluorescence microscopy images of cell viability of MC3T3-E1 in (b1) TBC, (b2) TBC/HMSN, (b3) TBC/P28, and (b4) TBC/HMSN/P28 groups; and (c) proliferation of MC3T3-E1 in TBC, TBC/HMSN, TBC/P28, and TBC/HMSN/P28 groups (* p < 0.05, ** p < 0.01).

Figure 8. ALP activity of MC3T3-E1 in TBC, TBC/HMSN, TBC/ P28, and TBC/HMSN/P28 groups (* p < 0.05, ** p < 0.01).

good biocompatibility, and the proliferation ability of MC3T3E1 was obviously increased with the existence of P28 in the growth environment. Although there was no significant difference between TBC/HMSN/P28 with TBC/P28 after incubation for 2 and 4 days (p > 0.05), the cell proliferation on TBC/HMSN/P28 was significantly higher than that on TBC/ P28 after incubation for 6 and 8 days (* p < 0.05, ** p < 0.01). Since the release of P28 from the TBC/HMSN/P28 scaffold was slower than that from the TBC/P28 scaffold, the effective concentration of P28 could be maintained for a longer period of time. It is very important to promote MC3T3-E1 cell proliferation and osteogenic differentiation. Between 5 to 7 and 7 to 10, the slope of the release curve of TBC/P28 and TBC/ HMSN/P28 was 2.48, 2.61 and 4.44, 4.85. Their release rates were very similar. However, compared with TBC/P28, the TBC/HMSN/P28 composite contained chitosan, which improved the cell compatibility of the TBC/P28 scaffold to some extent. This might be the other reason why cells could proliferate better on the TBC/HMSN/P28 scaffold. 3.6. ALP Activity Analysis. Generally, ALP activity is a key index for the detection of osteogenic differentiation.3 As shown in Figure 8, the ALP activity of four groups was tested after

Figure 9. (a) X-ray images and (b) 3D-CT images of the rabbits with different implants for 6 and 12 weeks and (c) Lane-Sandhu X-ray scores of four groups at 6 and 12 weeks (* p < 0.05, ** p < 0.01).

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Figure 10. Histological evaluation with (a) HE and (b) Masson staining of TBC, TBC/HMSN, TBC/P28, and TBC/HMSN/P28 groups at 6 and 12 weeks. NB represents the new bone in the images. The scale bar in the upper left image can be applied to all the others.

there were not enough silicon ions released after incubation for 15 days. Thus, we conclude that there was a significant difference between TBC/HMSN/P28 with TBC/P28 after incubation for 15 days, possibly due to the sustained release of P28 in HMSN to induce osteogenic differentiation. 3.7. Radial Bone Defect in Rabbit. In general, a small range of bone defect will heal spontaneously without further intervention.48 However, spontaneous recovery cannot occur when the bone defect reaches or even exceeds the critical-size bone defect (CSD),49,50 which was defined as the minimum size of the bone defect of which the spontaneous healing range was less than 10%.51,52 Meanwhile, the 15 mm defect in the radial bone of rabbit was also considered to be a reliable CSD.53,54 Thus, it is necessary to evaluate the osteogenic ability of TBC/HMSN/P28 using a CSD model. In this study, all rabbits recovered well after the operation. The wounds in the implant areas of the rabbits healed well without any infection, and no rabbit was excluded from the analysis. As shown in X-ray images (Figure 9a), after postoperation for 6 weeks, scaffolds were still visible in all four groups, and their density was close to or higher than that of the surrounding bone tissue. Furthermore, the new bone tissue formation around the scaffolds was not obvious in the TBC and TBC/ HMSN groups, although the TBC/P28 and TBC/HMSN/P28 groups showed slightly better results than the other two groups. At 12 weeks postoperation, the density of the four groups of scaffolds increased further compared with that at the 6 weeks time point. The formation of new bone tissue around the scaffolds was obvious, and the scaffolds were tightly integrated with the host bone, especially in the TBC/P28 and TBC/ HMSN/P28 groups. In addition, the scaffolds in all the groups showed certain degradation. In terms of the Lane-Sandhu X-ray scores (Figure 9c), the TBC/P28 and TBC/HMSN/P28 groups were significantly higher than that of the TBC and TBC/HMSN groups at 12 weeks postoperation (* p < 0.05, ** p < 0.01).

Figure 11. Quantification of the percent area of newly formed bone for each group at 6 and 12 weeks after surgery (* p < 0.05, ** p < 0.01).

incubation for 5, 10, and 15 days. Our results indicated that the ALP activities of MC3T3-E1 cells on TBC/HMSN/P28 and TBC/P28 scaffolds increased with culture periods from 5 to 15 days, and there was no significant increase for the ALP activities between TBC/HMSN and TBC scaffold groups. There were significant differences between TBC/HMSN/P28 and TBC/ P28 with TBC/HMSN and TBC in the whole culture period (* p < 0.05, ** p < 0.01). This finding proved that P28 had the ability to induce osteogenic differentiation for MC3T3-E1 cells. It is worth noting that there was no significant difference between TBC/HMSN/P28 with TBC/P28 after incubation for 5 and 10 days, but there was a significant difference after incubation for 15 days (* p < 0.05, ** p < 0.01). Previous studies proved that the release of silicon ions from MSN could contribute to osteoblast differentiation.46,47 However, there was no significant difference of ALP activities between TBC/ HMSN and TBC scaffold groups from 5 to 15 days. The probable reason is that there is an electrostatic interaction between chitosan and silicon ions in the aqueous solution. With the aid of CHI, the degradation rate of HMSNs decreased, and 218



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As shown in 3D-CT images (Figure 9b) for the 6 weeks postoperation, the gap between the scaffold and host bone could be observed in the TBC and TBC/HMSN groups, while the gap was not obvious in the TBC/P28 and TBC/HMSN/ P28 groups. At 12 weeks postoperation, the scaffolds were wrapped by the new bone tissue and were completely integrated with the host bone in the TBC/P28 and TBC/ HMSN/P28 groups, while the scaffolds in the TBC and TBC/ HMSN groups were still not integrated with host bone. 3.9. Histological Analyses. To confirm the formation of new bone tissue, we employed histological analysis. As shown in HE staining (Figure 10a) and Masson staining (Figure 10b), in TBC and TBC/HMSN groups, the pores of the scaffolds were filled with fibrous connective tissues with no evident new bone formation at 6 weeks postoperation, and a very small amount of new bone tissue formation was seen at 12 weeks postoperation. In contrast, there were large quantities of new bone tissue formation in TBC/P28 and TBC/HMSN/P28 groups at 6 weeks postoperation, which was even more than that of TBC and TBC/HMSN groups at 12 weeks postoperation. Of note, the pores of the scaffolds were completely filled with new bone tissue in TBC/P28 and TBC/HMSN/P28 groups at 12 weeks postoperation, which gradually grew into lamellar bone. Quantitative bone regeneration was determined by histomorphometric analysis (Figure 11). At 6 weeks postoperation, the measured areas of the new bone tissue formation in TBC/ P28 and TBC/HMSN/P28 groups were significantly higher than the TBC and TBC/HMSN groups (* p < 0.05, ** p < 0.01), and there was no significant difference between TBC/ P28 with TBC/HMSN/P28 groups (p > 0.05). At 12 weeks postoperation, the measured areas of the new bone tissue formation in the TBC/P28 and TBC/HMSN/P28 groups were still significantly higher than TBC and TBC/HMSN groups, and the TBC/HMSN/P28 group was significantly higher than the TBC/P28 group (* p < 0.05, ** p < 0.01). By comparing the results of radiographic and histological examinations, it was worth noting that the effect of TBC/HMSN/P28 to repair radial bone defect was better than P24/TBC/Collagen I which was reported in a previous experiment by Li et al.33 Consequently, TBC combined with HMSN for sustained release of P28 could promote the formation of new bone tissue and ultimately achieve the purpose of repairing critical size bone defects.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00506. MS and HPLC spectrum of the BMP-2 derived peptide (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*J.Z. Phone: +86-27-87793240. E-mail: [email protected]. cn. *X.G. Phone: +8615327216660. E-mail: xiaodongguo@hust. edu.cn. ORCID

Jintao Zhu: 0000-0002-8230-3923 Xiaodong Guo: 0000-0001-9429-457X Author Contributions ∥

W.C. and Q.L. contributed equally to this paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (81371939, 81672158, and 51473059) and the National Key Research and Development Program of China (2016YFC1100100).

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REFERENCES

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4. CONCLUSIONS In summary, we have successfully developed the TBC/HMSN/ P28 composite scaffold for bone tissue engineering. P28 was encapsulated into HMSNs with enlarged mesopores and then combined with TBC to form the drug delivery system TBC/ HMSN/P28 composite scaffold. Our results demonstrated that the TBC/HMSN/P28 composite scaffold could promote proliferation and osteogenic differentiation of MC3T3-E1 cells in vitro. Moreover, based on the results of radiographic examination images and histological examination, the TBC/ HMSN/P28 scaffold delivery system could significantly promote the formation of new bone tissue and repair of critical size defect in vivo. In summary, the TBC/HMSN/P28 composite scaffold possesses sustained release property, excellent biocompatibility, and strong bone conduction and regeneration ability. We thus believe that the TBC/HMSN/ P28 composite scaffold is a promising material for bone tissue engineering and regenerative medicine. 219


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