Article pubs.acs.org/Biomac
Delivery of Growth Factors Using a Smart Porous Nanocomposite Scaffold to Repair a Mandibular Bone Defect Xian Liu,†,‡,§ Kun Zhao,†,‡ Tao Gong,‡ Jian Song,§ Chongyun Bao,§ En Luo,§ Jie Weng,‡ and Shaobing Zhou*,‡ ‡
Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, Sichuan 610031, People’s Republic of China § State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan 610041, People’s Republic of China S Supporting Information *
ABSTRACT: Implantation of a porous scaffold with a large volume into the body in a convenient and safe manner is still a challenging task in the repair of bone defects. In this study, we present a porous smart nanocomposite scaffold with a combination of shape memory function and controlled delivery of growth factors. The shape memory function enables the scaffold with a large volume to be deformed into its temporal architecture with a small volume using hot-compression and can subsequently recover its original shape upon exposure to body temperature after it is implanted in the body. The scaffold consists of chemically cross-linked poly(ε-caprolactone) (c-PCL) and hydroxyapatite nanoparticles. The highly interconnected pores of the scaffold were obtained using the sugar leaching method. The shape memory porous scaffold loaded with bone morphogenetic protein-2 (BMP-2) was also fabricated by coating the calcium alginate layer and BMP-2 on the surface of the pore wall. Under both in vitro and in vivo environmental conditions, the porous scaffold displays good shape memory recovery from the compressed shape with deformed pores of 33 μm in diameter to recover its porous shape with original pores of 160 μm in diameter. In vitro cytotoxicity based on the MTT test revealed that the scaffold exhibited good cytocompatibility. The in vivo micro-CT and histomorphometry results demonstrated that the porous scaffold could promote new bone generation in the rabbit mandibular bone defect. Thus, our results indicated that this shape memory porous scaffold demonstrated great potential for application in bone regenerative medicine.
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INTRODUCTION The worldwide incidence of bone disorders and conditions has been increasing upward and is expected to double by 2020.1 In consideration of the serious limitation in traditional therapies,2−4 tissue engineering provides a novel platform in bone reconstruction, which can incorporate therapies that mimic the critical aspects of natural biological processes.5−7 Tissue engineering utilizes a three-dimensional scaffold containing specific cells that is cultured in vitro or in vivo for a certain period and delivered to the desired site for the purpose of tissue repair.8 The three-dimensional scaffold plays a critical role in the process of tissue engineering, and many approaches have been made to design a suitable scaffold.5,9−11 At the present, tissue engineering generally requires the use of a porous scaffold, which can provide a niche for cell attachment and tissue formation in vitro and in vivo. Moreover, in vivo tissue formation may be more efficient and suitable for tissue reconstruction compared with in vitro tissue formation.12 However, how to implant a porous scaffold with a large volume into the body conveniently and safely still remains a challenging task. A self© 2014 American Chemical Society
setting hydrogel has been recently developed and some advances have been made in bone tissue engineering.13,14 In recent years, shape memory polymers (SMPs) have been demonstrated as a smart material, which has gained great attention for their medical applications, such as tissue engineering,15,16 smart sutures,17 vascular stents,18,19 bladder sensors,20 and mucosa reconstruction.21 They have the ability to recover their original shape from a deformed secondary shape when exposed to an appropriate stimulus such as heat.22 Their shape memory behavior permits their delivery in a compact form via minimally invasive surgeries, and they are able recover into complex final shapes in vivo.17,23−26 Thus, a shape memory porous scaffold is a critical aspect when applying treatment for irregular bone defect disease. Compared with commonly used scaffolds, one of the advantages of the shape memory scaffold is that the architecture of the scaffold can be predesigned, deformed Received: December 26, 2013 Revised: January 23, 2014 Published: January 28, 2014 1019
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Scheme 1. Schematic Fabrication of Shape Memory Porous (SMP) Nanocomposite Scaffold Loaded with and without BMP-2
was used as the growth factors. A total of 134 μm sucrose microparticles with a size distribution of 10−150 μm and 324 μm sucrose microparticles with a size distribution of 100−850 μm were obtained by sieving, determined by laser diffraction particle size analyzer, as shown in Figure S1 in Supporting Information. All other chemicals and solvents were of reagent grade or better. Fabrication of SMP Scaffolds Loaded with or without BMP-2. Chemically cross-linked poly(ε-caprolactone) (c-PCL) SMP scaffolds were fabricated as shown in Scheme 1. First, 10 g PCL, preweighed allyl alcohol, and 1 g BPO were dissolved in 100 mL of dichloromethane. After that, 1.5 g HA nanoparticles (diameter: 120 nm), 135 g sieved sucrose microparticles (diameter: 324 μm), and 50 g sucrose microparticles (diameter: 134 μm) were dispersed in this solution to yield a homogeneous paste under high-speed stirring. Next, the paste was cast into a mold, placed in an aerator overnight to remove the solvent, and dried at 30 °C. Subsequently, the completely dried composite was press-molded at 130 °C for 15 min for cross-linking to prepare the required block. After cooling, the material was soaked in ethanol to remove residual BPO. Finally, the block-shape composite was soaked in deionized water for 5 days to completely leach out the sucrose microparticles and then dried for 2 days. Thus, the shape memory porous scaffold with two pore sizes (100/300 μm) was obtained, and scaffolds with various weight fractions of allyl alcohol (AL) 2.5, 5, and 10% were fabricated. All scaffolds were sterilized with 70% ethanol prior to use. The fabrication of BMP-loaded SMP scaffold was as follows. First, the c-PCL scaffolds (10 × 10 × 10 mm) were compressed using two flat glass sheets under a pressure of 0.5 MPa at 45 °C and subsequently cooled to 0 °C to fix the temporary shape. Second, the scaffolds were soaked in pure ethanol for 3 days and washed three times with phosphate-buffered saline (PBS) at 4 °C, and sterilized with overnight ultraviolet irradiation. Subsequently, these sterilized SMP scaffolds were soaked in 0.75 wt % solutions of sodium alginate (SA) for 6 h at room temperature (lower than the transition temperature (Ttrans) of the polymer). Thus, the sodium alginate (SA) solution penetrated into the deformed pores of the SMP scaffold and coated the surface of the pores. Finally, 1 mg CaCl2 and 5 μg BMP were dissolved in 1 mL of sterile water to cross-link with the sodium alginate (SA) coat to form the geldrug system. After gelation, the BMP-loaded SMP scaffold was obtained and stored at 4 °C prior to the cell culture and animal experiments. Characterization of the Scaffold. Differential scanning calorimetry (DSC) was employed to measure the thermal properties of the
according to the requirement, and recovered to its original shape by stimulus application. To date, there have been several reports related to the porous scaffold with shape memory function;27−29 however, in most cases of these studies, SMP structure and function in vitro were predominantly investigated. Although the injectable preformed scaffolds displayed excellent shape-memory effect in vivo, the aim of this study was to encapsulate and deliver biomolecules.27 To the best of our knowledge, few investigations have reported the use of the shape memory porous scaffold in vivo for the repair of bone defects. In this study, we fabricated a BMP-2-loaded shape memory porous nanocomposite scaffold (BMP-loaded SMP scaffold) consisting of chemically cross-linked poly(ε-caprolactone) (cPCL) and hydroxyapatite (HA) nanoparticles. The introduction of HA nanoparticles does not only increase the scaffold mechanical stability and provide good ceramic osteoconductivity,30,31 but it can also achieve high quality micro-CT images of the scaffold in vivo. PCL-based SMPs have attracted widespread attention due to its excellent biocompatibility and biodegradability.32,33 We anticipate that a scaffold featuring controlled drug release and shape memory behavior can provide a platform for the design and manufacturing of a tissue-engineering scaffold in bone repair. The scaffold exhibited an excellent shape memory effect from a deformed shape (compressed shape) to a recovered shape at 37 °C. The in vitro cytocompatibility of the scaffold was evaluated by its interaction with rabbit bone marrow stem cells (BMSCs). Most importantly, the scaffold was implanted for the first time in the site of the rabbit mandibular defect for bone tissue generation. The in vivo shape memory recovery and bone generation effect were examined using micro-CT and histological analysis.
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EXPERIMENTAL SECTION
Materials. Raw PCL and HA nanoparticles were synthesized as previously described.34,35 The weight-average molecular weight (Mw) of PCL determined using gel permeation chromatography (GPC; Waters 2695 and 2414) was 112 kDa. Benzoyl peroxide (BPO), allyl alcohol (AL), and sodium alginate (SA) were purchased from Chengdu Kelong Chemical Reagent Company (China). BMP-2 from the InductOs kit 1020
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polymers on a TA Instruments (Q100, American). The specimens were heated from 10 to 80 °C at a rate of 10 °C min−1 in nitrogen gas with a flow rate of 20 mL min−1. Dynamic mechanical analysis (DMA) was performed on a DMA983 analyzer (TA Instruments, America), using a tensile resonant mode at a heating rate of 5 °C min−1 from 0 to 60 °C and at a frequency of 1 Hz. The storage modulus E′, loss factor tan, and one-way shape memory cycles for specimen size 50 × 12 × 1 mm (length × width × thickness) were tested as previously reported.36 The static compression test for specimens with a dimensional size of 10 × 10 × 10 mm (length × width × thickness) was achieved at a crosshead speed of 0.5 mm/min at room temperature using a universal testing machine Instron 5567 (Instron Co., Massachusetts). The gel fraction estimate was performed according to a prior method.37 To test whether HA nanoparticles in the SMP scaffolds leaked out during its use, thermogravimetric analysis (TGA) was performed for the SMP scaffold samples before and after being soaked in PBS for 1 week on a TA Instruments (Netzsch STA 449C, Bavaria, Germany) at a scan rate of 10 °C min−1, to 700 °C. In Vitro Shape Memory Recovery. The resulting porous scaffold with a dimension of 5 × 5 × 7.5 mm (length × width × thickness) was first deformed by compression at 0.5 MPa to obtain a temporary shape (5 × 5 × 2 mm), and the shape was fixed at 0 °C for 1 h. Next, the shape recovery was observed using a stereo-optical microscope (OLYMPUS SZX16) in water at 37 °C. To observe the changes in the interior porosities, the scaffold was freeze-fractured in liquid nitrogen and a cross-section was observed using scanning electron microscopy (SEM, KYKY-2800) at different recovery stages. The shape recovery ratio and shape fixed ratio were defined as
30 min and rinsed twice with PBS. The samples were incubated with rhodamine-phalloidin (diluted as 1:100, Invitrogen, U.S.A.) to stain the actin cytoskeleton for 15 min, and the nuclei were subsequently stained with DAPI. Prior to visualization under a fluorescence microscope (IX70; Olympus, Tokyo), the samples were washed four times with PBS. The samples were also dried and sputter-coated with gold for scanning electron microscopy (KYKY-2800) after 7 days of culture. The cell proliferation on the scaffolds was measured using the 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. After 1−7 d of culture, the samples were harvested. The absorbance was measured using a Beckman DU7400 spectrophotometer at a wavelength of 570 nm. In Vivo Shape Memory Recovery. The original scaffold (15 × 10 × 5 mm) was prepared and deformed in a compressed shape (12 × 7 × 3 mm) prior to use in vivo. All of the animals in this study were cared for according to the protocol approved by the Animal Care Committee of Sichuan University. Rabbits were anesthetized via intravenous administration of ketamine hydrochloride (20 mg/kg) and xylazine (5 mg/kg) prior to all experimental procedures. A total of 12 New Zealand white rabbits were randomly divided into three groups: SMP scaffold group, BMP-loaded SMP scaffold group, and untreated control group. Two of these groups were randomly applied on two sides of one rabbit mandible. One scaffold group was inserted into one side of the mandibular defect, while another scaffold group was implanted on the other side of the defect, or the side was kept empty as the control group. The bone defects were performed according to previous reports.38,39 Briefly, a 2 cm incision was made approximately 1 cm lower from the edge of the mandible body to expose the bone. Next, 15 × 10 mm2 critical-sized bicortical bone defects were formed on both sides of the mandible body, and the periosteum was removed during this process. The incision was closed in layers using 4−0 resorbable sutures. These animals received prophylactic i.m. antibiotic at the time of surgery and for 3 days postoperatively. All rabbits were sacrificed after a recovery period of 8 weeks following micro-CT and histological examination. To measure the recovery ability of the SMP scaffold in vivo, we scanned the scaffold with a cone beam computed tomography (Morita Company, Japan). We examined the temporal shape of the scaffold at 1 and 10 min after implantation. The surgery was performed on a temperature/pump at 42 °C (TP700, Gaymar, American), and the animals were kept warm during anesthesia. Micro-CT Measurement. The animals were sacrificed after a recovery period of 8 weeks and half of the sample from each group (4 samples each group) were harvested for micro-CT testing. The morphology of the mandibular defect was assessed using a micro-CT 80 scanner (Scanco Medical, Bassersdorf, Switzerland). The parameters of the micro-CT were set to 70 kV, 114 mA, 700 ms of integration time, a resolution of 2048 × 2048 pixels and an isotropic voxel size of 18 μm for the scanning region. The volume of interest was selected as the mandibular defect and extended for a total of 500 slices. The following microarchitectural parameters were assessed in volume of interest images: bone volume to total volume ratio (BV/TV), bone mineral density (BMD), trabecular thickness (Tb.Th.), and trabecular number (Tb.N.). Histological Examination. After the rabbits were sacrificed at 8 weeks, the other half of the samples from each group were fixed in 4% paraformaldehyde solution for histological examination. The samples were dehydrated with alcohol and embedded in methylmethacrylate without decalcification. A model SP1600 microtome (Leica Microsystems, Wetzlar, Germany) was used to cut the sections at a thickness of 100 μm and polished to approximately 15−20 μm for hematoxylin and eosin (HE) and trichrome-masson staining. A microscope (DF 490, Leica, Switzerland) was used to examine the histological images, and images were captured using imaging software (Leica). The ratios between the mineralized new bone area and the total defect area of the images were calculated. Statistical Analysis. All data were expressed as mean ± SD, and the statistical analyses were performed using a paired t-test with SPSS 10.0 (SPSS, Chicago, IL, U.S.A.). A probability value (p) of less than 0.05 was considered to be statistically significant.
R f = T1/Ts1,F R r = Trex /Ts1,F where Rf is the shape fixity ratio, Rr is the shape recovery ratio, Ts1,F is the compressed strain experience during deformation, T1 is the retention strain at 0 °C, and Trex is the strain after recovery. In Vitro Degradation and BMP Release from the Scaffolds. The scaffold was placed in a test tube containing 20 mL of phosphatebuffered saline (PBS, 0.1 M, pH 7.4) for the in vitro degradation study. The tubes were placed in a heated shaking air bath that was maintained at 37 °C and 120 cycles min−1 and observed for 14 weeks. The in vitro degradation was evaluated from the loss of the scaffold mass and the reduction of the polymer number-average molecular weight. The mass loss was determined gravimetrically by comparing the dry weight remaining at a specific time with the initial weight. The molecular weight was determined using GPC as previously described. To examine whether shape memory actuation affected the release of growth factors, temporary and full-recovery scaffolds of the same size (7.5 × 5 × 5 mm) were prepared and loaded with BMP-2. The in vitro drug release from these BMP-2-loaded SMP scaffolds was performed under similar conditions with in vitro degradation. At the appropriate intervals, 1.0 mL of release medium was collected and 1.0 mL of fresh buffer solution was added back. The amount of BMP release was determined using a UV−visible spectrophotometer (Shimadzu UV2551, Japan). Cell/Scaffold Interactions In Vitro. We used New Zealand white rabbit BMSCs for the cell and scaffold interaction examination. Rabbit BMSCs were obtained from tibiae bone marrow using density gradient centrifugation and then suspended in α-minimum essential medium with 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD) and incubated at 37 °C with 5% CO2 in humidity. The BMSCs were split, purified, and resuspended for passage until they reached 90% confluence. Cells of passage 3 were used for the following in vitro study. To examine BMSC cellular morphology on the SMP scaffolds, F-actin was stained for fluorescent visualization. Briefly, the round fullrecovered scaffold (diameter = 10 mm, high = 5 mm) was bathed in α-MEM culture medium for 24 h and subsequently in fetal serum for 4 h prior to use. The BMSCs were seeded at a density of 5 × 104/mL on the surface of the scaffold. After 3 d of incubation, the samples were fixed in 4% formaldehyde for 30 min at room temperature and washed twice with PBS. Next, the samples were treated with trinitrotoluene in PBS for 1021
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Figure 1. (A) DSC thermograms of pure PCL film, AL/PCL film containing 2.5, 5, and 10 wt % of AL, and SMP scaffolds containing 10% AL with pore size of 100 and 100/300 μm; (B) DMA curves of flex storage modulus of AL/PCL films with AL/PCL weight ratios of 2.5, 5.0, and 10%; (C) Gel fraction of SMP scaffolds with different pore size; (D) Curve of compression deformation and compressive strength of SMP scaffold with pore size of 300/100 μm loaded with or without BMP.
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RESULTS AND DISCUSSION Characterization of the SMP Scaffold Loaded with or without BMP. In this study, we fabricated a shape memory porous nanocomposite scaffold consisting of chemically crosslinked PCL (c-PCL) and HA nanoparticles using hot-pressing technology. The cross-linking occurred via a free radical reaction between the PCL macromolecular chains and AL molecular chains in the presence of benzoperoxide as an initiator under 130 °C. Highly interconnected pores were obtained using a sugarleaching method. Here, the pore size of these scaffolds was set to the range of 100−900 μm based on previous reports that the porous architecture plays a significant role in tissue regeneration between 10 and 1000 μm, which can preserve tissue volume, provide temporary mechanical function, deliver biofactors,40 and promote bone formation in vivo.41 Next, the BMP-loaded SMP scaffold was also fabricated by coating the calcium alginate layer mixed with BMP-2 on the surface of the pore wall of the SMP scaffold. To regulate the transition temperature of the SMP scaffolds closely to body temperature, we adopted allyl alcohol (AL) as a plasticizer. On the basis of these DSC curves in Figure 1A, we determined that by varying the concentration of allyl alcohol (AL), SMP scaffolds were obtained with different transition temperatures. The melting temperatures (Tms) of pure cross-linked PCL, PCL cross-linked with 10% AL (AL/ PCL) and its scaffolds with a pore diameter of 100 μm, 100−300 μm were 58 ± 0.3, 39 ± 0.4, 38.9 ± 0.2, and 40 ± 0.4 °C, respectively. Thus, when the allyl alcohol (AL) concentration reached 10%, the transition temperature of the shape memory polymer was close to the physical temperature. Dynamic mechanical analysis (DMA) was also performed to evaluate the shape memory properties of the composites. As
shown in Figure 1B, the tensile storage modulus as a function of temperature for AL/PCL with an allyl alcohol (AL) weight ratio of 2.5, 5.0, and 10% are demonstrated. We determined that that there were more gaps in the storage modulus (E′), which could be essentially attributed to the presence of the chemically crosslinking structure according to a previous report.42 Moreover, the E′ below 30 °C initially experienced a plateau (>200 MPa) and then a decrease of up to 3 orders of magnitude in the temperature range of 30−50 °C, and the E′ reached a low modulus plane of 0.01 MPa. The large change in E′ indicated that the material exhibited an excellent shape memory effect. The transition temperature (Ttrans) of the AL/PCL decreased from 48 to 37 °C when the concentration of allyl alcohol (AL) increased from 2.5 to 10%. Thus, the concentration of allyl alcohol in AL/PCL was optimized at 10%, and subsequent investigations of both the in vitro and in vivo shape memory effect were focused on this material. As shown in Figure 1C, changes in the gel fraction of the SMP scaffolds with different micropore sizes are demonstrated. Importantly, the scaffold with the pore size of 100/900 μm was not investigated due to its poor mechanical properties. The chemical cross-linking was successfully performed, and the gel fraction was more than 90%. There was no significant difference among these samples with various micropore sizes. The curve of compression deformation and compressive strength of SMP scaffold with pore size of 100/300 μm loaded with or without BMP is shown in Figure 1D. The compressive strength increased from 70 to 90 N, while the compressive modulus increased from 700 to 1079 MPa with the addition of Ca-alginate coating. With the addition of the Ca-alginate layer, the size of the pores decreased. For the porous structure, the compressive modulus of 1022
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Figure 2. In vitro BMP-2 release from the temporary and fully recovered BMP-2-loaded SMP scaffolds in PBS at pH 7.4 at 37 °C (A); In vitro degradation evaluated from the loss of scaffold mass (B) and the reduction of the number-average molecular weight of polymer (C).
Figure 3. The shape memory recovery process of the scaffold at 37 °C. Images “a” to “d” are the recovery process observed using an optical microscope, and images “A−D” correspond to the “a−d” images, which show the recovery process of the interior pores of the scaffold observed using a scanning electron microscope.
alginate layer was uniform. Also, it demonstrated that these pores were interconnected. In Vitro Degradation and Release. Tissue engineering not only includes the use of a biodegradable scaffold but also refers to the promotion of bone formation by supplying bone related growth factors. BMP-2 is one of the most important growth factors, which can stimulate bone growth.46,47 The in vitro BMP2 release from the Ca-alginate coated temporary and fullrecovery shape memory scaffolds is shown in Figure 2A. The release profiles show a parallel and nearly complete overlapping result for the two types of scaffolds, suggesting that the shape memory actuation virtually had no effect on BMP-2 release. We also found that, in the initial 15 h, BMP-2 release was rapid and the total release amount was approximately 65%, and in the
scaffolds with small pores was significantly higher than large-pore scaffolds.43 The mechanical property was sufficient to maintain the porous structure of the scaffold in vivo,44,45 and thus, the pore size of 100/300 μm was selected as an optimized size for this scaffold. The porosity of the scaffolds with a pore size of 100 and 100/300 μm was 82 ± 3.4 and 90.6 ± 5.2%, respectively. To measure whether sodium alginate was infiltrated into the pores of the scaffold and these pores of the scaffold were interconnected, the solution (0.75%) mixed with tea polyphenol which is pale yellow was employed. From the image of the cross-section of the scaffold observed with optical microscope (Figure S2 in Supporting Information), we can find that the surface inside the scaffold is also pale yellow and the color is uniform, indicating that the solution was infiltrated into of SMP scaffolds and the 1023
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Figure 4. (A) Comparison of shape recovery ratio of SMP scaffolds hibernated for different periods of time; (B) The shape recovery ratio of c-PCL sheet and SMP scaffolds heated in air and in water; (C) Quantitative thermal mechanical cycle of c-PCL/AL sheet.
from the length of the scaffold between the original and final shapes. We also examined the effect of storage on the shape recovery ratio. The hibernation time had a small effect on the shape recovery ratio (Figure 4A). To investigate whether water had any effect on the SMP scaffolds, the shape recovery effect of the SMP scaffolds was performed by heated the samples in air and water, respectively, and no obvious differences in the shape recovery ratio were found (Figure 4B). This may be because PCL is a hydrophobic polymer and water molecules cannot penetrate into the PCL matrix.48 Furthermore, the shape-memory fixation and recovery cycles, as well as the shape recovery ratio is approximately 90%, and the shape fixed ratio was approximately 94%, suggesting that the SMP scaffold exhibited good and stable shape memory function at near physiological temperatures (Figure 4C). The corresponding recovery process of the interior pores of the scaffold were observed using SEM (Figure 3A−D). The pore diameter of the original shape was approximately 160 μm with the porosity of about 90.6 ± 5.2% and converts to 33 μm with the porosity of 30.1 ± 4.5% after the scaffold was compressed. However, stimulation with heat at 37 °C resulted in a recovery of the deformed pore size to approximately 160 μm in 60 s. This result was consistent with the recovery process of the bulk scaffold. The scaffold had an anticipated shape memory recovery in vitro from a small, compact structure to a voluminous structure, which is an important feature for minimal-invasive surgery and tissue engineering applications. In Vitro Cytotoxicity Analysis of SMP Scaffold. BMSCs are an important source of osteogenic cells for bone tissue engineering.49 Herein, rabbit BMSCs were employed to assess the in vitro cytotoxicity of the SMP scaffold based on the MTT
following period, the release was slow. This result indicated that the scaffold could be used to deliver BMP-2 in vivo. The in vitro degradation of the scaffold matrix evaluated from the loss of the scaffold mass and the reduction of the numberaverage molecular weight of polymer is shown in Figure 2B,C. The weight loss proceeded slowly during the entire degradation period. After 14 weeks of incubation, scaffolds with a pore size of 100 and 100/300 μm lost approximately 7 and 12% of its initial mass, respectively. With the introduction of Ca-alginate coating, the weight loss of all the composites increased, indicating a preferential dissolution of the Ca-alginate component and an increase in the hydrophilicity of the scaffold. As shown in Figure 2C, the reduction of the molecular weight was faster than the mass loss of the scaffolds, which was also consistent with the typical characteristic of bulk degradation. Overall, the trend was nearly consistent with mass loss. The degradation was attributed to the hydrolysis of the ester bonds of the PCL molecular chains. In addition, to investigate whether HA nanoparticles in the SMP scaffolds leaked during handling, delivery, and use, thermogravimetric analysis (TGA) was performed for the SMP scaffolds before and after being soaked in PBS for 1 week. As shown in Figure S3 (Supporting Information), we found that the content of HA in the SMP scaffolds was nearly unchanged after being soaked in PBS for one week, which indicated that the HA nanoparticles were stably integrated with the PCL polymer matrix in the SMP scaffolds. In Vitro Shape Memory Recovery. The shape memory recovery process of the SMP scaffold in water at 37 °C is shown in Figure 3. In addition, the temporary shape (compressed) recovered to its original shape in 60 s (from Figure 2a−d), and the recovery ratio was approximately 91%, which was calculated 1024
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Figure 5. BMP-2-loaded SMP scaffolds with rabbit bone marrow mesenchymal stem cells (BMSCs) in vitro. (A−C) Fluorescence microscope images of BMSCs combined with the scaffolds. The round mark shows assembled BMSC nuclei (blue), which suggests that the BMSCs grew into the deep porosity of the scaffolds. (D, E) SEM images of BMSCs in the pores and on the surface of the SMP scaffolds, respectively. (F) MTT result of the BMSCs cultured on the SMP scaffolds, which indicate that there was no significant difference in the proliferation between the control and SMP scaffold group (p > 0.05).
Figure 6. In vivo shape memory recovery process of the BMP-2-loaded SMP scaffold observed using cone beam computed topography. (A) Compressed scaffold and (B) the scaffold implanted in the rabbit mandibular bone defect; the scaffold can recovery from its temporary shape (C) to the original shape (D) in vivo after 10 min of implantation.
test. We performed F-actin staining and SEM for morphological observation. Nuclear staining of the cells on the scaffold and the
dense blue sites are in the scaffold pores, indicating that more BMSCs grew into the pores compared to cells on the surface of 1025
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Figure 7. Three-dimensional micro-CT images of the bone defect area. The “a−c” images were viewed from the lateral side of the mandible and “A−C” are viewed from the lower edge of the mandible. The red squares show the defect area and the white arrows show the defect bottom. The BMP-2-loaded scaffold group (C, c) had the most bone generation, and half of the defect area was filled with calcified structure. The SMP scaffold group (B, b) had more neonatal bone than the control group (A, a). There were more trabecula (Tb)-like structures (red arrow shown) in the SMP scaffold group and BMP-2loaded scaffold group than in the control group.
Figure 8. Quantification analysis of the micro-CT results in the bone defect area. The indices of bone volume to total volume ratio (BV/TV), bone mineral density (BMD), trabecular thickness (Tb.Th.), and trabecular number (Tb.N.) were used to assess the new bone formation in the mandibular defect (*p < 0.05).
the scaffold (Figure 5A). Moreover, the F-actin cytoskeleton spread along the cell membrane and demonstrated typical BMSC morphology (Figure 5B). An overlaid image of Figure 5A,B is shown in Figure 5C. The BMSCs grew into the deep porosity of
the scaffolds and attached well onto the pore wall. Furthermore, the SEM images in Figure 5D,E demonstrated that the BMSCs grew into the scaffold pores and the cells attached and spread very well on the wall surface of these pores, suggesting that the 1026
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Figure 9. Histomorphometric results of the bone defect area: (A−C) HE staining and (D−F) Masson staining. The defect in the control group was full of granulation tissue (A), and there was no mature trabecula near the defect sides (D). The scaffold group showed much neonatal bone in the defect area (B, E). The collagen fibers filled in the defect and small cavity-like (SC) structures were found in the center of the defect. The BMP-2-loaded scaffold group (C, F) showed the highest level of new bone formation, and more matured bone formed in the defect area. High magnification of Masson staining in image “F” near the small cavity (G), which showed that many multinuclear cells were found around the cavity; and quantification of the histological results (H). The scaffold group had more bone formation and showed a significant difference compared to the control group. The BMP-2-loaded scaffold group had the highest level of new bone area percentage; *p < 0.05.
scaffold exhibited good cell compatibility. In addition, the MTT results indicated that there was no significant difference in the proliferation between the control and SMP scaffold groups (Figure 5F). Taken together, these results demonstrated that the SMP scaffold showed excellent cytocompatibility with rabbit BMSCs. It is well-known that the static PCL scaffold used in tissue engineering has good biocompatibility,50 and in this study, the shape memory scaffold maintains this feature. In Vivo Shape Memory Recovery. To keep the animal warm after anesthesia, the implantation operation was performed on a temperature/pump at 42 °C. The in vivo shape memory recovery process of the BMP loaded SMP nanocomposite scaffold is shown in Figure 6. The introduction of HA nanoparticles can improve the quality of the micro-CT images of the scaffold similarly as a contrast agent. We immediately scanned the scaffold using cone beam computed topography and found that the scaffold could recover from the compressed shape to the original shape at 10 min after operation. In contrast to the in vitro recovery process, more time was needed for the scaffold to return to its final shape in vivo. This may be due to the heat transfer in the air, which is lower than in water. Moreover, it can
be beneficial that this recovery takes more time; thus, we have sufficient time to implant the scaffold into the body. Micro-CT Results. The three-dimensional micro-CT images of the bone defect area and the corresponding quantification analysis of bone formation were used to determine the feasibility of SMP as a bone tissue engineering scaffold. The threedimensional rendering of the entire bone defect area is shown in Figure 7. Furthermore, Figure 7a−c is viewed from the lateral side of the mandible. The SMP scaffold group demonstrated more bone formation compared to the control group. In the control group, only less neonatal bone appeared along the defect sides (Figure 7a), while the SMP scaffold group filled more than 1/3 of the defect area (Figure 7b). The BMP-2-loaded scaffold group demonstrated the most neonatal bone in the defect area and filled more than half of the bone defect area after 8 weeks of implantation (Figure 7c). Moreover, Figure 7A−C are viewed from the lower side of the mandible, and the results were consistent with those shown in Figure 7a−c. The control group demonstrated less neonatal bone and few mature bone trabeculae in the defect area, while the SMP scaffold group had many neonatal bone trabeculas in the defect area. Moreover, the 1027
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demonstrated almost 20% osteogenesis area ratio in histological examination.52 Moreover, the BMP-2-loaded scaffold could significantly promote new bone generation.
BMP-loaded SMP scaffold group has more bone trabeculas and was denser than the SMP scaffold group. The three-dimensional images were also supported by the micro-CT quantification analysis results. As shown in Figure 8A−D, the scaffold with or without BMP-2 group had more bone formation in the defect area. The two groups showed higher BMD, BV/TV, Tb.N., and Tb.Th. compared to the control group. In addition, the micro-CT data showed significant differences between these groups. The BMP-2-loaded SMP scaffold group exhibited the highest enhancement in bone formation, with an increase in BMD (4.69-fold), BV/TV (2.91fold), Tb.N. (2.95-fold), and Tb.Th. (1.48-fold) compared to the control group. The SMP scaffold group exhibited a relatively lower enhancement in bone formation, with an increase in BMD (3.17-fold), BV/TV (2.28-fold), Tb.N. (1.71-fold), and Tb.Th. (1.09-fold) compared to the control group. These results indicated that the SMP scaffold favored bone generation in the defect area, and the scaffold with the incorporation of BMP-2 delivery and shape memory function revealed a more obvious effect in promoting new bone formation. Histomorphometric Analysis. To further confirm the increase in new bone formation in the SMP scaffold group observed using micro-CT, histomorphometric analysis of the bone defect area was also performed (Figure 9). The HE staining and masson staining for the control group are shown in Figure 9A and D, respectively. At 8 weeks, less new bone formation was detected near the defect sides and no mature bone trabecula was formed in the defect area. The defect area was full of granulation tissue (Figure 9A). Moreover, the defect area was loose and only filled with blue staining collagen fibers (Figure 9D). However, the SMP scaffold group had newly formed bone within the defect (Figure 9B). Enhanced red staining representing mature or premature bone was formed between the collagen fibers, and a trabecula-like structure was also formed in the defect (Figure 9E). These results suggested that the SMP scaffold could improve bone formation compared to the control group. On the basis of the HE staining image of the BMP-2-loaded scaffold group (Figure 9C), we further found that new bone formation was greater than the SMP scaffold group. The bone defect area was nearly full of new bone and trabecula-like structures. The partial red staining in Figure 9F suggests that more mature or premature bone mineralization was formed. The high magnification of the masson staining near the small cavity in BMPloaded SMP scaffold group is shown in Figure 9G. We found that many multinuclear cells (osteoclast) were located around the small cavity, which is known as scaffold resorption in bone tissue engineering.51 The result was also consistent with the micro-CT results. Furthermore, BMP-2 delivery using the nanocomposite scaffold could further enhance new bone regeneration in contrast to a blank scaffold. To demonstrate the promotion of new bone formation using the BMP-2-loaded scaffold, quantification of neonatal bone in the defect area at 8 weeks was plotted in Figure 9H. A significant difference was found between the SMP scaffold group and control group, which also indicated that the SMP scaffold could improve the bone formation in the defect area. The BMP-2loaded scaffold group also demonstrated the highest level of new bone area percentage, which was significantly different from the other groups. The new bone area percentage in the SMP scaffold group and BMP-2-loaded scaffold group is 21.83 ± 5.13 and 42.5 ± 5.13%, respectively. The ability to generate new bone using the SMP scaffold was consistent with previous reports of PCL/HA composites implanted in the rat femur for bone generation and
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CONCLUSIONS In this study, we successfully fabricated one type of shape memory porous nanocomposite scaffold, and used it to deliver BMP-2 to enhance bone ingrowth and regeneration for the treatment of bone defects. The scaffold was implanted for the first time in the rabbit mandibular defect for bone tissue regeneration. Our findings revealed an excellent shape memory effect from a temporal shape (compressed) to a recovered porous shape in vivo in response to body temperature, which enables it to be conveniently implanted via minimally invasive surgery. The micro-CT and histomorphometry results showed that the BMP2-loaded SMP scaffold could significantly promote bone defect repair. The smart scaffold also has great potential in resolving the problem of implantation of a scaffold with a large volume in an in vivo environment, which is very complex and dynamically changing. Thus, the study provides a facile engineered strategy toward the design of multifunctional tissue engineering scaffold for the treatment or repair of diseased or destroyed human organs and tissues.
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ASSOCIATED CONTENT
S Supporting Information *
The size distribution determined by laser diffraction particle size analyzer (Figure S1). The cross-section of the SMP scaffold observed by optical microscope (Figure S2). TGA curves (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected];
[email protected]. Author Contributions †
These authors contributed equally to this work (X.L. and K.Z.).
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was partially supported by National Basic Research Program of China (973 Program, 2012CB933600), National Natural Science Foundation of China (51173150, 51373138), National Key Project of Scientific and Technical Supporting Programs Funded by MSTC (2012BAI17B06), and Research Fund for the Doctoral Program of Higher Education of China (20120184110029).
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