A multifunctional porous scaffold with capacities of minimally invasive implantation, self-fitting and drug delivery

Materials Today Chemistry 1-2 (2016) 52e62

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Materials Today Chemistry journal homepage: www.journals.elsevier.com/materials-today-chemistry/

A multifunctional porous scaffold with capacities of minimally invasive implantation, self-fitting and drug delivery Shubin Di a, Xian Liu b, Dian Liu a, Tao Gong a, Liuxuan Lu a, Shaobing Zhou a, * a

Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China b State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan 610041, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 November 2016 Received in revised form 13 November 2016 Accepted 13 November 2016 Available online 22 November 2016

Maxillary sinus floor elevation is of frequent occurrence and extremely important for dental implantation. However, how to conveniently implant a synthetic scaffold in the irregular bony cavity of maxillary sinus and simultaneously prompt new bone regeneration is a great challenge in lifting the maxillary sinus floor. Here, we develop a bioactive self-fitting polymer porous scaffold via one-step creation of interconnected pores with controllable size and loading with a bone morphogenetic protein-2 (BMP-2) therapeutic agent inside these pores for the first time. This scaffold can be compressed like a sponge for minimally invasive surgery and return to its original porous structure in response to a temperature of 41
C for self-fitting into the irregular cavity of a maxillary sinus. Moreover, the loaded BMP-2 possesses a great capacity to accelerate new bone regeneration. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Shape memory polymer Self-fitting Drug delivery Bioactive Tissue engineering

1. Introduction Bone tissue engineering scaffolds have been developed for repairing and regenerating damaged bone tissues. However, how to repair the irregular bone defect is still a great challenge. The reason is mainly that the scaffolds based on traditional materials cannot be deformed according to the requirements of surgery; also cannot fill completely in the irregular defect cavity. Among these, the bony cavity of the maxillary sinus is a typically irregular case. Maxillary sinus floor elevation is regarded as an effective method to restore the upper jaw in pre-implantology surgery for successful dental implantation [1]. Implantations of autogenous and/or allogenic bone grafts and synthetic materials have been developed to lift maxillary sinus floor [1,2], but these grafts have some disadvantages, such as potential donor site morbidity, limited donor availability, insufficient osteogenic properties, poor contact with surrounding bone tissue, and large surgical trauma [3e6]. Consequently, the development of the bioactive porous scaffold based on shape memory polymer (SMP), simultaneously with the capacities of minimally invasive surgery and fast regeneration of new bone,

* Corresponding author. E-mail address: [email protected] (S. Zhou). http://dx.doi.org/10.1016/j.mtchem.2016.11.004 2468-5194/© 2016 Elsevier Ltd. All rights reserved.

may be an applicable alternative choice for maxillary sinus floor elevation. SMPs as a smart material have been studied widely due to their unique properties in biomedical applications, including tissue engineering [7e9], cardiovascular stents [10,11], smart sutures [12,13] and drug delivery [14,15]. The shape memory function of SMPs permits them to fill minimally invasive surgeries just like a compressed sponge by starting with a compact scaffold and then recovering to a porous structure upon exposure to body heat. Moreover, the bony cavities of the maxillary sinus generally have an irregular shape, and thus the geometry of the traditionally implanted scaffold needs to be shaped prior to use. The architecture of the scaffolds based on s SMPs can be predesigned, deformed according to the need, and recovered to its initial shape under an environmental stimulus [16e19]. Therefore, SMPs have a great capacity to match the contour of irregular bone structures, which enables them to self-fit into irregular bony defects. An ideal scaffold, in addition to essential characteristics such as biocompatibility, mechanical properties, proper degradation and suitable surface chemistry, should possess a good porosity for bone tissue engineering [20]. Numerous methods have currently been employed to fabricate porous scaffolds, including salt particulate [21], foaming [22], syntactic foams [23], electrospinning [24], and thermally induced phase separation [25]. Although these

S. Di et al. / Materials Today Chemistry 1-2 (2016) 52e62

techniques have achieved certain success with respect to fabricating porous scaffolds, their shortcomings are still obvious, including limited control of three-dimensional substrate structures for salt particulate [26] and containing toxic heavy metal cations for foaming. Additionally, for achieving osteoinductivity and an enhancement of the repair of bone defects, utilizing BMP-2 therapies has been documented as being effective [27e29]. However, BMP-2 will lose bioactivity due to the short half-life in vivo, leading to a poor efficacy in bone defect repair. Accordingly, in this study, we provide a representative example of developing a bioactive multifunctional porous scaffold simultaneously with capacities of minimally invasive implantation, selffitting and drug delivery for prompting the repairing of irregular bone tissue via the one-step creation of interconnected pores and loading with BMP-2 protein inside the pores. Different from traditional pore-forming strategies, BMP-2 loaded calcium alginate (CA) hydrogel microspheres, fabricated using electro-spraying technology, are selected as the pore-forming agent (Scheme 1A). Calcium alginate hydrogel is a non-toxic material and widely used as a biomaterial in medicine. Poly(ethylene glycol)-poly(ε-caprolactone)-based polyurethane (PEG-PU), with excellent shape memory function, is used as the scaffold matrix (Scheme 1B). The BMP-loaded shape memory porous scaffold is achieved first by accumulating the BMP-2-loaded CA hydrogel microspheres, then by pouring the PEG-PU solution into the spaces among these microspheres and evaporating organic solvent, and finally by freezedrying these hydrogel microspheres (Scheme 1C). The pores of the scaffold are formed through the contraction of these hydrogel microspheres due to the removal of water; moreover, the pore size can be easily controlled by adjusting the original diameter of the hydrogel microspheres. Simultaneously, drug loading into these contractive microspheres is accomplished during the pore forming and endows the scaffold with bioactivity for accelerate the new bone regeneration. The approach to fabricating a porous scaffold is reported here for the first time. The scaffold can be readily deformed into a small volume scaffold for minimally invasive implantation and recovered to its original porous structure in response to a close body temperature, consequently fitting inside the cavity of a maxillary sinus (Scheme 1D). 2. Experimental section 2.1. Materials Poly(ethylene glycol) (PEG, molecular weight (Mw): 4000) from Sigma, and 2,2-Bis (hydroxymethyl) butyric Acid(DMPA), 4,4diphenylmethane diisocyanate (MDI) from Tokyo Chemical Industry Co.Ltd., were used without further purification. ε-caprolactone (ε-CL, 99.9%, Aldrich) and N,N-Dimethylformamide (DMF, 99%, Kelong in Chengdu (China)) were distilled over freshly powdered CaH2 under a reduced pressure. Stannous octoate (Sn(Oct)2, 95%) and Stannous chloride (SnCl2) were separately purchased from Aldrich and Kelong chemical reagent factory in Chengdu (China). BMP-2 and sodium alginate (SA) were purchased from Sigma-Aldrich, USA. 2.2. Preparation of calcium alginate hydrogel microspheres Different concentrations calcium solution and sodium alginate solution were prepared by dissolving pre-weighed amounts of calcium chloride (CaCl2) and SA, respectively, in ultrapure water. Calcium alginate (CA) hydrogel microspheres were prepared by using electro-spraying technology. In brief, SA solution was dropwise added into a petri dish containing CaCl2 solution using a micro syringe pump with High Voltage Direct Current (HVDC) (Scheme

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1A). The SA solution was put into a 20-mL syringe, which was attached to a polyethylene catheter (1.4 m) and a circular shaped metal needle. The needle has an inner diameter of 0.8 mm and a distance from the collector of 15 cm. A voltage of 20 kV and a flow rate of 60 mL/h were chosen as the experimental conditions. After that, the CA hydrogel microspheres were left in the gelling medium for 48 h, then separated from the solution using a stainless steel grid and left at room temperature before further studies. The diameters of the calcium alginate hydrogel microspheres were determined by using a Zoom-stereo microscope. The mean size and size distribution of the CA hydrogel microspheres were determined by using a laser diffraction particle size analyzer (LA-9200, HORIBA). BMP-2-loaded CA hydrogel microspheres were also prepared, using a process similar to that mentioned above. The only difference was that 160 mL of the stock solution of BMP-2, with a concentration of 50 mg/100 mL in phosphate buffer (80 mg), was added to the SA solution. 2.3. Synthesis of PEG-PU polymer PEG-PCL was synthesized according to our previous report [30]. In brief, ε-CL (10 g, 87.7 mmol), PEG (6.67 g, 1.67 mmol), and SnCl2 (0.167 g) were added into a 50 mL bottom flask with a stopcock. Then, the reaction system was kept under vacuum for 4 h and kept at 140
C for an additional 6 h. The product was sequentially dissolved in dichloromethane and precipitated in cold ethyl alcohol. The precipitate was dried under vacuum. PEG-PU was synthesized using a two-step solution polymerization [31], as displayed in Scheme 1B. To regulate the transition temperature of the SMP scaffolds close to body temperature, we synthesized a series of polymers with three different ratios of the soft segments and hard segments. Take the stoichiometry of PEGPU with a PEG-PCL: MDI: DMPA ratio of 2:2:1 as an example; firstly, PEG-PCL (2.00 g, 0.15 mmol) was added into a 100 mL threenecked round flask equipped with a magnetic stirrer and dewatered at 80
C for 2 h under vacuum to remove the moisture. Then, MDI and DMF were successively added into the flask, with a reaction time of 3 h at 60
C, under magnetic stirring to prepare the prepolymer. After that, pre-weighed chain extender DMPA and MDI were added into the reaction system, with Sn(Oct)2 as the catalyst, for 6 h at 60
C. The whole reaction system was under the Ar gas protection condition. Finally, a pale yellow viscous liquid was obtained. FT-IR was carried out using a Nicolet 5700 IR spectrometer. 1 H NMR spectra were recorded using a Bruker AM-300 spectrometer. 2.4. Fabrication of SMP scaffolds P PEG-PU SMP scaffolds were fabricated as shown in Scheme 1C. The PEG-PU polymer was dissolved in a mixed solvent of DMF and dichloromethane (20:80 v/v%). CA hydrogel microspheres were assembled in an ordered structure in a plastic syringe. Then, polymer solution was poured into the syringe with a specific mass ratio and oscillated in the ultrasonic oscillator for 30 min to maintain solution penetration into the space among these microspheres. As the organic solvent evaporated, the syringe was left with a polymer mixed into the gaps of accumulated CA hydrogel microspheres. After that, the syringe was processed using the freeze-drying technique for 24 h and a porous scaffold was obtained. 2.5. Characterization of the scaffolds Surface topographies of the scaffolds were erved under scanning electron microscopy (SEM, FEI, Quanta 200, Philips, The

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Scheme 1. Schematic illustration of the engineering of the bioactive shape memory polymer (SMP) porous scaffold. (A) The fabrication of BMP-2-loaded CA hydrogel microspheres using an electro-spraying technology; (B) the synthesis of the shape memory polymer, PEG-PU; (C) the fabrication of the BMP-2-loaded SMP porous scaffold; (D) the shape deformation and recovery of the scaffold.

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Netherlands) at each stage of the shape memory cycle. The porosity of scaffolds was determined using ethanol replacement method [32]. Differential scanning calorimetry (DSC) was used to measure the thermal properties of the polymers on a TA Instruments (Q100, American). Dynamic mechanical analysis (DMA) was performed on a DMA983 analyzer (TA Instruments, America). The test specimen dimensions were ~8 mm diameter  ~24 mm height. The static compression test for specimens with a dimensional size of (~8 mm diameter  ~4 mm height) was achieved using a universal testing machine Instron 5567 (Instron Co., Massachusetts). The compressive stress-strain curve was measured until the specimen was compressed to a maximum. 2.6. In vitro shape memory recovery Hape memory fixity ratio (Rf) and recovery ratio (Rr) of the scaffold were measured using clubbed-shaped specimens on a DMA (TA, DMA-Q 800), using a controlled force mode according to previous report [7]. The Rf and Rr were defined as

Rf ¼

T1 Tm

(1)

Rr ¼

T2 Tm

(2)

where Rf is the shape fixity ratio, Rr is the shape recovery ratio, T1 is the retention strain of deformed scaffold after 0
C fixed, T2 is the strain after recovery, and Tm is the compressed strain experience during deformation. The results represent the averages of at least three specimens. The macroscopic characterization of the shape memory effect is performed as follows. Cylindrical porous scaffold (~8 mm diameter x~4 mm height) was first deformed by compression to obtain a temporary shape (~4 mm diameter x~12 mm height) and the shape was fixed at 0
C for 10 min. Second, the fixed samples were immersed in 41
C warm water. Then, the images of the shape recovery were recorded using a video camera. The shape recovery ratio was defined as Images of the shape recovery of the SMPs scaffold was defined as [(d-d1)/(8-d1)]  100, where d refers to the diameter of the compressed scaffold at a given time at 41
C and d1 indicates the diameter at 0 min. The results represent the averages of at least three scaffolds. 2.7. In vitro degradation To quantify scaffold hydrolytic degradation, dry scaffolds were weighed and immersed in 10 mL of phosphate-buffered saline (PBS, 0.1 M, pH 7.4). The degradation was conducted at 37
C in a water bath with the solution exchanged every 7 days. The degree of degradation was evaluated according to the change of the weights of scaffolds. 2.8. Cell/scaffold interactions in vitro Dog BMSCs were obtained from iliac bone marrow according to a previous method [33]. The bone marrow were cultured in aminimum essential medium supplemented with 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD) for four days, then nonadherent cells were dropped and the adherent cells were cultured for passage one. The third passage cells were used for the following experiments. The sterilized scaffolds (prepared as 10 mm diameter and 5 mm height discs) were incubated in a small volume of growth medium for 4 h prior to use. Cell proliferation on scaffold was analyzed by Alamar Blue (Trek Diagnostics Systems Inc.,

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Cleveland, OH) on day 2, 4, and 6. The methods was performed according to the Alamar Blue manufactures protocol. F-actin was used to stain the SMP scaffolds to determine cellular fluorescence morphology. Cells were seeded at a density of 5  104/ mL onto each scaffold. After 4 d of incubation, the cells on scaffolds were fixed using 4% formaldehyde and treated with trinitrotoluene before actin cytoskeleton staining. The samples were incubated in PBS containing rhodamine-phalloidin (diluted as 1:100, Invitrogen, U.S.A.) for 15 min, and then cell nuclei were counterstained using 4,6-diamidino-phenylindole (DAPI, Sigma; 2 mg/mL in PBS). The samples were washed four times before being viewed under a fluorescence microscope (IX70; Olympus, Tokyo). The red fluorescence was the actin cytoskeleton and blue staining showed the nuclei. The samples were also dried via treatment using a series of graded ethanol solutions (75%, 85%, 95% and 100%) at room temperature and sputter-coated with gold for scanning electron microscopy (KYKY-2800) after 6 days of cultivation. 2.9. In vivo study of SMP scaffold in beagle maxillary sinus Six healthy adult beagles (age: 24 months; weight: 10e15 Kg), with intact dentition and healthy periodontium, were bred before performing bilateral sinus augmentation. All experimental protocols were approved by the Animal Care and Experiment Committee of Sichuan University. Twelve maxillary sinus floor augmentations in six animals were made bilaterally and randomly applied according to the following three study groups: i) untreated control group (n ¼ 4, group A), ii) SMP scaffold group (n ¼ 4, group B), iii) BMP-2 loaded SMP scaffold group (n ¼ 4, group C). The original cylindrical scaffold (diameter ¼ 8 mm, length ¼ 4 mm) was first deformed via compression to obtain a compressed shape (d ¼ 4 mm, l ¼ 12 mm). The surgical process was modified according to a previous report [34]. All dogs were sacrificed after 12 weeks, and the sinus explants were fixed overnight in 4% paraformaldehyde solution (PBS, pH 7.4) and then analyzed by using the CT technique and histological examination. 2.10. CT measurement The relevant part of the skull was removed and fixed in neutral buffered 4% formalin solution. The harvested bone specimens were imaged with Micro-CT -CT (Scanco Medical, Bassersdorf, Switzerland) with 70 kV voltage tension, 114 mA tube current, and 700 ms of integration time. The dog's sinus between nasal sides of internal bone wall and infraorbital nerve canal internal sides wall was selected as interesting place. The bone mineral density (BMD) in sinus cavity and bone volume to total sinus volume (BV/TV) was calculated. 2.11. Histological examination After Mic-CT scanning, the samples were used for histological examination. Blinded analyses were performed. One dog maxillary sinus was divided into two samples (left and right side) carefully. The samples were prepared without decalcification of embedding in methylmethacrylate for hard tissue sections. The sections were designed vertical to the sagittal plane and the samples were polished from premolar to first molar as shown in Fig. S1 in the Support Information (SI); the schematic drawing of red rectangles denotes the section place. The first section was collected until polished to the first molar plane. 100-mm-thick sections were cut by using the model SP1600 microtome (Leica Micro-systems, Wetzlar, Germany), and then the sections were polished to approximately 15e20 mm for trichrome Masson staining. The histological images were captured under a microscope (DF 490, Leica, Switzerland),

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and new bone volume and total histological images in the sinus were calculated for quantification. 2.12. Statistical analysis Quantitative data were expressed as mean ± standard deviation. To use the LSD for statistical analysis when the homogeneity of variance, and using the Dunnet test for statistical analysis when the heterogeneity of variance. Statistical analysis was performed using SPSS 19.0 (SPSS, Chicago, IL, U.S.A.) and a probability value (p) of less than 0.05 was considered statistically significant. 3. Results and discussions 3.1. Characterization of the SMP scaffold The composition and chemical structure of the PEG-PU copolymer were confirmed using FT-IR and 1H NMR, as shown in Fig. S2 in SI. The BMP-2-loaded CA hydrogel microspheres were successfully fabricated using electro-spraying technology, and their encapsulation efficiency was approximately 98%. Their morphology was observed using a Zoom-stereo microscope and appeared spherical, uniform, and smooth (Fig. 1A). Moreover, the size of the CA hydrogel microspheres could be easily manipulated through changing the concentration of alginate sodium solution. From Fig. S3, we can find that the average size of CA microspheres increased from approximately 216 to 334, 425, and 588 mm as the alginate sodium concentration (w/v) increased from 0.25% to 0.5%, 1%, and 1.5%. The size of the microspheres was also consistent with the result determined via laser diffraction particle size analysis (Fig. 1B). To verify whether the hydrogel microspheres can be

employed as a pore-forming agent, CA microspheres at the original size of 334 mm were freeze-dried and contracted from a wellspherical shape to an irregular shape with an average size of 48 mm (Fig. 1B and C) due to the removal of water inside these microspheres. The porous scaffold was fabricated by pouring PEG-PU polymer solution into the space among the accumulated CA hydrogel microspheres and subsequently freeze-drying these microspheres. The pores were automatically formed after these microspheres shrank, with space remaining due to the removal of water. Simultaneously, BMP-2 remained in the shrunken microspheres. The pore size almost matched the original size of the pore-forming agent, i.e., the CA hydrogel microspheres, and could be easily controlled via changing the size of the microspheres. By measuring the pore size of the cross-section of the scaffold using SEM, we found that the average pore sizes were ~228, ~345, ~439 and ~601 mm (Fig. 2A), corresponding, respectively, to 216, 334, 425 and 588 mm of the CA microspheres. Moreover, the porosity increased as the pore size increased; however, the compressive modulus (E) decreased gradually (Fig. 2B). The pore size of the scaffold can be easily tuned by the addition of the different sizes of CA microspheres. It has been reported that a scaffold with pore size ranging between 300 and 350 mm is very suitable for bone tissue engineering [35]. Pores with sufficient space allow for attachment and proliferation of the diverse cells responsible for the formation of a tissue or organ. Therefore, the CA hydrogel microspheres with a size of 334 mm were selected as the pore-forming agent for the following investigations. The porosity was also an important parameter for the bone tissue engineering scaffold. The effect of the mass ratio of PEG-PU polymer to CA hydrogel microspheres on the porosity of

Fig. 1. (A) Calcium alginate hydrogel microspheres prepared using a micro syringe pump with High Voltage Direct; (B) the size distribution determined by using a laser diffraction particle size analyzer; (C) image “c” is the calcium alginate hydrogel microspheres after freeze-drying. a and c were observed by using a Zoom-stereo microscope.

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Fig. 2. (A) Influence of the sodium alginate concentration (wt.%) on the pore size of a scaffold; (B) compressive modulus (E) and % porosity values of different pore sizes. Standard deviations are calculated based on three measurements (n ¼ 3) for each composition.

the resulting scaffolds was investigated. From Fig. S4A, we can find that the porosities of the scaffolds with PEG-PU/microspheres mass ratios of 1:3, 1:4, and 1:5 are 72%, 82% and 87%, respectively. A porosity higher than 80% is conducive to the growth of new bone tissue. Additionally, the bone tissue engineering scaffold should maintain sufficiently high mechanical properties. When the porosity of the scaffold increased, its mechanical properties generally decreased. From Fig. S4A, we can also see that the E remained approximately 2.0 MPa, sufficiently maintaining the porous structure when the porosity was 82%, corresponding to the PEG-PU/microspheres mass ratio of 1:4.

3.2. In vitro shape memory recovery To confirm whether the porous scaffold has a good shape memory function in response to near body temperature, the main parameters, including Rf, Rr and recovery temperature, were investigated. From the DSC curves of PEG-PU polymer films with different soft/hard segment ratios and a porous scaffold (Fig. S5A), we found that the transition temperature of the polymer increased from 38
C to 43
C as the soft segment content increased from 70% to 80%. Moreover, the scaffold from the PEG-PU polymer with a soft/hard segment ratio of 75:25 had the same transition temperature close to body temperature as its polymer film. To further understand the viscoelastic properties, DMA of each sample was employed. Tensile storage modulus (E') and tan (d) as a function of temperature are shown in Fig. 3A. The PEG-PU film and scaffolds possessed an obvious transition near the melting temperature (Tm), which is in agreement with the DSC results. A drop in E' was observed across the Tm of the scaffold, going from a plateau of 200e480 MPa in the glassy state to 5e15 KPa in the rubbery state. The tremendous change in E0 indicated that the PEGPU film, porous scaffold, and deformation scaffold have an excellent shape memory effect [36,37]. The E' of the porous scaffold was lower than that of the PEG-PU film because of the porous structure, which influences its intrinsic performance. The same DMA was used to quantitatively demonstrate the shape memory properties of the porous scaffold (Fig. 3B). A typical shape memory response is seen in a cycle of four key steps. Starting from the asterisk, samples were loaded to a tensile strain of 25% at 41
C, cooled to 10
C, unloaded to reveal fixing, and continuously heated to 41
C, revealing the shape recovery. Both the Rf and Rr values of the porous scaffold exceeded 95% and 83%, respectively, demonstrating a good shape memory function. Moreover, the porosity of the scaffold influenced the shape memory function. The Rf and Rr values of the scaffold with the porosity of 82% and PEG-PU/

microspheres mass ratio of 1:4 were 92% and 87%, respectively (Fig. S4B). The curves of compressive strength of the porous scaffolds with temporary shape, original shape and recovery shape are shown in Fig. S5B. The compressive strength of the temporary shape was the highest, owing to the decrease of porous structure. The compressive strength of the recovery shape was similar to that of the original shape (approximately 30 N), indicating that shape recovery cannot have an effect on mechanical properties. The mechanical property is sufficient for maintaining the porous structure of a scaffold in vivo [38,39]. The recovery processes of the porous scaffold in the form of a cylindrical shape (~8-mm diameter  ~4 mm height), viewed from side and top, are shown in Fig. 3C. At first, it was compressed into a temporary shape (~4-mm diameter  ~12 mm height) at 50
C and cooled to 0
C to fix the temporary shape. The arrows indicate the compressing directions. Later, the compressed scaffold was submerged into a 41
C water bath and recovered to its original shape in ~30 s. The result showed that the Rf and Rr were 96% and 81%, respectively. The corresponding recovery process of the interior pores of the scaffold was observed using SEM (Fig. 3D1D4). We found that the original interior pores were uniform and interconnected, which is suitable for bone tissue growth; they could be deformed under compression at 50
C and fixed at 0
C, and they returned to their original shapes with a size of 310 mm, again, at 41
C. The results suggest that the porous scaffold possesses good shape memory function at a near physiological temperature for potential minimally invasive surgery. To confirm whether these pores of the scaffold were interconnected, the permeation of red Rhodamine B water solution from the top to bottom of the scaffold was a good proof (Fig. S6). Additionally, from the magnified SEM image in Fig. 3E, we can see that every shrunken BMP-2-loaded CA microsphere was attached to the surface of each corresponding pore, which was proven by the EDX spectra (Fig. 3F).

3.3. In vitro degradation The in vitro degradations of the PEG-PU film and its scaffold matrix evaluated from the mass loss of materials, the change of pH value of the degradation medium and the decrease of the molecular weight of the polymer are shown in Fig. S7AeC. We can see that the PEG-PU polymer had good degradability via hydrolysis. The porous scaffold degraded more rapidly than the PEG-PU film because the water molecules penetrate into the interconnected pores more rapidly.

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Fig. 3. (A) DMA curves of PEG-PU film, porous scaffold, and deformed porous scaffold; (B) the cyclic tensile curve of the porous scaffold. The beginning of each cycle is marked by an asterisk. Sample was loaded, cooled to 10
C and unloaded (fixing), followed by continuous heating to 41
C (recovery). The arrows denote the various stages, specifically (1) deformation, (2) fixing, (3) unloading, and (4) recovery; (C) the shape memory recovery process of the scaffold at 41
C. Images C1eC4 and C1’C4' represent the recovery process, observed using an optical microscope (the blue arrows indicate the compression direction); (D) images D1-D400 show the recovery process of the interior pores of the scaffold, observed using a scanning electron microscope; (E) the magnified SEM images of the interior pores, with the red arrows indicating the shrunken CA microspheres; (F) EDX spectrum of the shrunken CA microspheres. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.4. In vitro cytotoxicity The in vitro cytocompatibility of the porous scaffold was further assessed via culturing with dog bone marrow mesenchymal stem cells (BMSCs). PEG-PU film, porous scaffold and its corresponding deformed scaffold were selected as experimental samples. From

the confocal laser scanning microscope images of BMSCs in Fig. 4AeC, we can see that these cells grew healthily and expanded approximately on the scaffold. In the SEM image (Fig. 4D), we can find that the BMSCs grew into the pore of the scaffold and spread well on the surface of the pore. The Alamar Blue analysis is consistent with these results, and the cell viability of the three

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samples became more than 85% after 6 days of cultivation (Fig. 4E). In Fig. 4F, we can see that the cell proliferation, which is determined by MTT method, is obvious after cultured with the scaffolds for 6 days. Therefore, the porous scaffold has good cytocompatibility.

3.5. CT measurement results To evaluate whether the scaffold can integrate into the host tissue of a maxillary sinus, maxillofacial Micro-CT images were acquired and the quantification analysis of bone regeneration of the maxillary sinus area was further performed. The group without implanting any material was used as control. The implantation of the compressed porous scaffold into the dog bony cavities of the maxillary sinus is schematically illustrated in Fig. 5A. The scaffold was readily implanted via a minimally invasive surgery; moreover, in the bony cavity of the maxillary sinus, it could return to its original porous structure and subsequently self-fit into the irregular cavity. To further evaluate the new bone formation prompted by the BMP-2 loaded shape memory polymer (SMP) porous scaffold sinus at 12 weeks post-operation, coronal maxillofacial CT images were employed (Fig. 5B). Compared with the control group

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(Fig. 5Be1) and SMP porous scaffold group (Fig. 5Be2), the BMP-2 loaded SMP porous scaffold group had the most neonatal bone in the bony cavity of the dog maxillary sinus (Fig. 5Be3). From the corresponding quantification analysis of bone formation, we can also find that the BMP-2-loaded SMP scaffold possessed the highest bone mineral density (BMD) among the three groups; the BMD value was 2.611-fold as high as that of the blank scaffold, with a significant difference between the two groups (P < 0.05) (Fig. 5C). The bone volume to total volume ratio (BV/TV) analysis was similar to the BMD. The BMP-2 loaded scaffold group had more BV/TV than the scaffold alone group, having an increase of nearly 8.89-fold (Fig. 5D). These results demonstrated that the BMP-2-loaded SMP scaffold could promote new bone regeneration in the maxillary sinus and, in turn, lift the maxillary sinus floor.

3.6. Histomorphometric analysis To gain a better understanding of the increase of bone regeneration in the maxillary sinus area by the BMP-2-loaded SMP scaffold, histomorphometric analysis was performed at 12 weeks. The sections vertical to the sagittal plane at the first molar place

Fig. 4. SMP scaffolds cultured with dog bone marrow mesenchymal stem cells (BMSCs) in vitro. (A-C) show the fluorescence microscope images of BMSCs. The blue staining, shown by the arrow, represents nuclei (A); F-actin stained with red fluorescence (B); (C) the combination of (A) and (B). (D) SEM image showing the BMSCs on the surface of the SMP scaffolds; (E) Alamar Blue result of the BMSCs cultured with the samples, which indicates that there was no significant difference in the proliferation among the PEG-PU films, deformed scaffold, and original scaffold (p > 0.05); (F) Cell proliferation of the SMP scaffolds. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. (A) Schematic illustration of the implantation of the compressed porous scaffold into the dog bony cavity of the maxillary sinus and the shape recovery and self-fitting into the bony cavity; (B) micro-CT analysis of the bone formation in the maxillary sinus of a dog at 14 weeks. The control group (i.e., with no material implanted) was observed with a dark region in the maxillary sinus (1). The SMP scaffold group (2) had some highlighted neonatal bone regions (as indicated by yellow arrows) and some relatively lower density connective tissue-like images in the maxillary sinus. The BMP-2 loaded scaffold group (3) had more new bone highlighted in the maxillary sinus, and nearly one-third of the sinus area was filled with a calcified structure. (C), (D) The quantitative morphometric analysis of bone mineral density (BMD) and bone volume to total sinus volume (BV/TV) (*p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

were stained with Masson's trichrome and shown in Fig. 6AeE. As Fig. 6A and D shows, connective tissue and some new bone formations can be found near the bottom area of the sinus in both the blank SMP scaffold and BMP-2-loaded SMP scaffold groups, and in the upper area, part of the scaffold remains. This can be ascribed to the fact that in the bottom area near the surgery place, the aggregation of blood and trauma of bone may mobilize MSCs, which exert the reparative effects of MSCs for bone formation [40]. The BMP-2 loaded scaffold group had more bone formation than the scaffold alone group, indicating that the BMP-2 is effective in enhancing new bone regeneration. Fig. 6B and E shows the high magnifications of black rectangle areas corresponding to Fig. 6A and D, respectively. Fig. 6B shows a large vessel-like structure and granulation tissue. Fig. 6E shows more active new bone formation in the form of an osteoid with osteocytes and blood vessels, as well as a newly formed bone line by the osteoblast. Angiogenesis plays an important role in bone formation, offering the delivery of oxygen and nutrients and clearing away cellular debris [41]. Therefore, the new bone formation firstly occurred around the good revascularization area in Fig. 6E. Interestingly, the high magnification of the granulation tissue structure of the green triangle star is shown in Fig. 6C. It seems that inflammatory granules grew inside the scaffold pores and that many cells recruited and many osteoblast-like cells collected near

the boundary of the granules, which may potentially subsequently form the new bone. Both osteoblast and fiber collagen were necessary for the new bone formation, and the aggregation of osteoblast and fiber collagen near the boundary of granules in Fig. 6C may mean that the mineralization of new bone began at those areas. The new bone formation of histomorphometric images was quantified using the new bone area fraction and plotted in Fig. 6F. A significant difference was found between the blank scaffold and BMP-2 loaded scaffold group. The BMP-2 loaded group had more bone formation than the scaffold alone group and approximately a 3.12-fold increase at the 12th week. The histomorphometric quantification results supported the Micro-CT results. Delivering BMP-2 by using hydrogel microspheres in the scaffold enables prompting new bone formation due to the BMP-2 function [42e44]. 4. Conclusion In summary, we developed a new bioactive self-fitting polymer scaffold for maxillary sinus floor elevation via the one-step creation of interconnected pores with controllable size and loading with BMP-2 therapeutic agent inside the pores. Different from traditional pore-forming strategies, CA hydrogel microspheres were not only a drug carrier but also a pore-forming agent, which has many

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Fig. 6. Masson staining results of the maxillary sinus area treated with a blank scaffold and BMP-2 loaded scaffold. (A)e(C) correspond to the blank scaffold group and (D), (E) correspond to the BMP-2 loaded scaffold group. Connective tissue and some new bone formation are seen near the bottom area of the sinus in the two groups, and some residual none-degraded scaffold is seen in the upper area. The magnification is a large vessel-like structure and granulation tissue in the scaffold alone group (B). A high magnification image of the BMP-2 loaded group is shown in (E); new bone, stained red around the blood vessels, and osteocyte are found inside the new bone. In the high magnification image (C), it seems that inflammatory granules grew inside the scaffold pores and that many cells recruited and many osteoblast-like cells collected near the boundary of the granules, which may potentially subsequently form new bone. (F) The quantitative analysis of the new bone regeneration in the blank scaffold and BMP-2 loaded scaffold groups (*p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

advantages, such as avoiding a sacrifice of the pore-forming material and non-toxicity to body tissues. Moreover, the porosity and pore size of the scaffold can be readily adjusted by varying the original size of the CA hydrogel microspheres, and the poreforming approach is very simple and repeatable. More importantly, the scaffold has an excellent shape memory function in response to a temperature of 41
C, being able to undergo compression like a sponge for convenient implanting via a minimally invasive surgery and later return to its original porous structure upon a temperature stimulus for self-fitting into the irregular cavity of a maxillary sinus. The loaded BMP-2 induces a fast osteogenetic capability in the maxillary sinus area. Therefore, the bioactive smart scaffold is an effective resolution for lifting the maxillary sinus floor for successful dental implantation. Acknowledgements S.D and X.L contributed equally to this work. This work was partially supported by National Natural Science Foundation of China (Nos.51373138, 21574105, 31400829) and the Sichuan Province Youth Science and Technology Innovation Team (Grant No.2016TD0026). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.mtchem.2016.11.004.

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