Mechanically Robust Shape Memory Polyurethane Nanocomposites

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Mechanically Robust Shape Memory Polyurethane Nanocomposites for Minimally Invasive Bone Repair Yuanchi Zhang, Jinlian Hu, Xin Zhao, Ruiqi Xie, Ting-Wu Qin, and Fenglong Ji ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00655 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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Mechanically Robust Shape Memory Polyurethane Nanocomposites for Minimally Invasive Bone Repair Yuanchi Zhanga, Jinlian Hu*a, b, Xin Zhaoc, Ruiqi Xiea, Tingwu Qind, Fenglong Jie

a Institute

of Textiles and Clothing, The Hong Kong Polytechnic University, Hung Hom,

Hong Kong, China b Smart Biomaterial Research Center, The Hong Kong Polytechnic University, Shen Zhen

Base, China c Department

of Biomedical Engineering, The Hong Kong Polytechnic University, Hung

Hom, Kowloon, Hong Kong, China d Institute of Stem Cell and Tissue Engineering, West China Hospital, Sichuan University,

Chengdu, China e School

of Textiles Materials and Engineering, Wuyi University, Jiangmen, China

ABSTRACT Shape memory polymers (SMPs) have great potential utility in the area of minimally invasive surgery, however, insufficient mechanical properties hinder their applications for bone defect repair, particularly in high load-bearing locations. In this study, hydroxyapatite (HA)/reduced graphene oxide (rGO) nanofillers were incorporated into a shape memory polyurethane (SMPU) to enhance its mechanical properties. Then the nanocomposite was further modified using Arginyl-glycyl-aspartic acid (RGD peptide) to improve its cellular adhesion towards promoting neotissue formation and integration with surrounding bone tissue. The physical and biological properties in terms of their

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chemical structure, surface wettability, mechanical behaviors, shape memory performance and cell adhesion were systematically investigated. The results demonstrated that the multi-modified SMPU/HA/rGO/RGD nanocomposite significantly enhanced mechanical properties (e.g., ~200% increase in Young’s modulus and >300% enhancement in tensile strength compared with the unmodified SMPU), which might be attributed to the intercalated structure and metal-affinity inside the nanocomposite. Adhesion of rabbit bone mesenchymal stem cells was clearly demonstrated on RGDimmobilized SMPU nanocomposite surface. Having excellent shape memory behavior (e.g. 97.3% of shape fixity ratio and 98.2% of shape recovery ratio), we envision that our SMPU/HA/rGO/RGD nanocomposite can be implanted into a bone defect with a minimally invasive surgery. KEYWORDS: Shape memory polyurethane nanocomposite; hydroxyapatite, reduced graphene oxide; Arginyl-glycyl-aspartic; enhanced mechanical properties; boosted cell adhesion; minimally invasive bone repair.

INTRODUCTION Bone defect has become one of the most concerning issues in public health area due to its high cause of disability and morbidity1-2. In the United States alone, more than 1.5 million cases of osteoporotic vertebral fracture costs US$ 12-18 billion every year3. Particularly, the need for load-bearing bone repair is continuously growing due to the increase of elderly population and large bone damages. Several approaches like autografts and

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allograft have been explored for decades to repair defected bones4, however, the insufficient availability, high donor site morbidity and graft resorption demand the development of new materials

4-5.

For load-bearing bones, the requirement of suitable

mechanical strength should involve improved biomimetic design and fabrication of repair biomaterials6. Traditional rigid metallic materials such as stainless steel, titanium alloys employed as load-bearing implants, present much higher elastic modulus than human cancellous bones even cortical bones, leading to unexpected implantation behaviors like stress shielding effect, loosening, wear and improper loading that can result in instability, inflammation even the bone resorption7. At the same time, the terrible processability also limits their employments in minimally invasive surgery to a great extent. Extensive surgical exposure should be controlled in a small degree since it could negatively affect the biological environment around the damaged site8. Therefore, a smart polymeric biomaterial with adaptable functions (e.g. tunable mechanical properties, excellent bioactivity and cytocompatibility) is highly desired for minimally invasive bone repair. Shape memory polymers (SMPs) such as shape memory polyurethane (SMPU) can be programmed and then maintain a temporary structure under certain conditions9-10, then the permanent shape can be recovered upon an appropriate external stimulus such as high temperature11-12, water environment13-14, or light15-16. Thermal sensitive SMPs have a thermal transition temperature (Ttrans): the materials could be deformed at T>Ttrans and fixed at TTtrans. Hence, SMPs could expand itself upon a certain trigger after being implanted as a compacted, small size in

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human body17. Such unique property of SMPs make it extraordinarily popular as minimum invasive therapy e.g. the vascular graft18, heart scaffold19. Notwithstanding the popularity of SMPs in treating bond defect, demands for rigidity and strength of materials are still growing especially for defects located in load bearing regions. For instance, polyurethane generally has the modulus of ~10-50 MPa while that of cancellous bone is usually higher than 70 MPa7,

20.

Besides, pure polymer materials lack bioactivity,

osteoconductive and osteogenetic properties for the application in bone repair. A SMP possessing simultaneously the sufficient mechanical properties and biocompatibility is accordingly sought after. Previously our group has developed a series of SMPUs with polycaprolactone (PCL) as the soft segment (contribute to shape fixity and recover process), accompanied by isocyanate e.g. 4, 4’-methylenebis(phenyl isocyanate) (MDI) and 1,6-hexamethylene diisocyanate (HDI), as well as chain extender e.g. 1,4-butanediol (BDO) as the hard segment (contribute to permanent shape)21-23. Based on these PCL-SMPUs, composites incorporated with graphene oxide (GO) or hydroxyapatite (HA) have also been studied for biomedical applications24-25. For instance, Tan et al. published a SMPU composite with graphene oxide (GO) using PCL (Mn=2000 g/mol) and MDI26. The Young’s modulus of pristine SMPU was only 2.8 MPa. After modification, the mechanical performance (~12.9MPa in Young’s modulus) was still not satisfactory for bone repair. However, terrible processability extremely limited the diversified employment of this SMPU form as bone repairing materials. We believed that the insufficient mechanical

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performances (strength & modulus) of these materials were due to the unstable internal structures as well as the limited interactions between nanofillers and SMPU matrix. Herein, we developed a multi-modified SMPU nanocomposite using incorporated HA/rGO nanoparticles as well as bonded RGD on surface to achieve enhanced performance for minimally invasive bone repair. As the main inorganic component of natural bone, HA provides excellent biocompatibility, osteoinduction capability27-29 and has been used as a filler to improve polymer osteoconductivity and mechanical properties30-31. rGO is also usually considered as the fillers to modify the SMPs to improve mechanical and thermal properties, even promote the osteogenic differentiation and bone regeneration32-33. On one hand, less functional groups on the surface of GO could alleviate the toxicity34. On the other hand, rGO showed a relatively good interfacial bonding with SMP matrix35-36. Considering mesenchymal stem cells (MSCs) are a promising cell source for bone tissue engineering and their high sensitivity to the environment, it is crucial to provide a robust platform for their adhesion, proliferation and even differentiation. In order to further promote cell adhesion properties, RGD, a kind of widely accepted binding motif for integrin receptors, composed of L-arginine, glycine, and aspartate, was selected as the cell attracting tool on the surface, which has been proved in many studies 37-39. We expect that the incorporated HA/rGO will enhance the nanocomposite’s mechanical properties through forming a stable structure and improved interactions between the nanofillers and SMPU via the metal affinity. For comparison, pristine SMPU and single-modified SMPU nanocomposites (SMPU/HA, SMPU/rGO)

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were prepared as control groups. As a proof of concept, ultraviolet (UV)–vis experiments, Fourier-transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and contact angle tests were performed to demonstrate the successful modifications. In addition, tensile tests and bending tests have been carried out to confirm the mechanical performances and shape memory properties, respectively. Improved ratios were listed for comparison. For further understanding the origin of these enhanced performances, energy dispersive X-ray spectroscopy (EDS) attached SEM, wide-angle X-ray diffraction (WAXD) and computer simulation were used to revel the underlying mechanism. Finally, cell adhesion morphologies were conducted to demonstrate the cytocompatibility of the nanocomposite. EXPERIMENTAL SECTION Materials. PCL-diol (Wn~550, CAPA2054) was purchased from Perstorp. MDI, 1,4-Butanediol (BDO), phosphate buffered saline (PBS), 1,1’-Carbonyldiimidazole (CDI), (3-Aminopropyl) triethoxysilane (APTES), GO sheet, RGD sequence were all obtained from Sigma-Aldrich. HA nanoparticles was commercial products from Emperor Nano Material Co. Ltd. Rabbit bone mesenchymal stem cells (MSCs) and growth medium were purchased from the Cyagen Biosciences Company. The BDO were in advance dried under vacuum at 100 oC for 24 hours to remove the moisture. Preparation of SMPU and HA/rGO. The PCL-diol was firstly vigorously stirred at 100oC for 1 hour to remove the moisture. After the temperature was cooled down to 85oC, MDI was added slowly into the PCL-diol. In order to form SMPU oligomer, the 6



reaction was maintained at 85oC for 2 hours with mechanical rabbling. Then, certain dried BDO was added with rapid mixing about 40 seconds. The ratio of soft segment (PCL)/the hard segment (MDI+BDO) was 56/44 and the SMPU polymer chain were ended with hydroxy groups. Following sufficient polymerization, the solution was poured into the mold and then placed in oven to dry. The whole reactions were processed under the vacuum environment. During the process of synthesizing SMPU, pre-treatment of HA and GO were conducted (Figure 1A and 1B). Briefly, HA was modified by APTES through ultrasonic dispersion in acetone. Then GO was reduced to rGO firstly using sodium citrate as the reductant according to the reference40. After that, the dried HA-APTES was mixed with rGO in the organic solvent to obtain the HA/rGO nanoparticles where the APTES acted as the linker to combine the hydroxy groups in HA and rGO respectively. Figure 1D shows the potential structure of the HA/rGO nanoparticle.

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Figure 1. Schematic diagram of SMPU/HA/rGO/RGO nanocomposite and its cell adhesion: (A) modification of HA with APTES; (B): reduction of GO; (C) immobilization of RGD on the surface; (D) potential structure of HA/rGO; (E) molecular structure of SMPU/HA/rGO/RGD nanocomposite; (F) cell adhesion through coupling between RGD on surface and the integrin in cell. Preparation of SMPU nanocomposites. Synthesized SMPU was solved in the dimethylacetamide (DMAc) followed by dispersing different nanofillers (HA, rGO, HA/rGO) respectively in the solvent to acquire various SMPU nanocomposites. Remaining DMAc was removed at 80oC for 24 hours in the oven. Molecular structure of SMPU/HA/rGO was shown in Figure 1E. Due to preceding incorporation of HA and rGO, the intercalated structure between polymer chain and HA/rGO might exist inside the

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nanocomposite. The content of HA and rGO as well as HA/rGO was 2wt%, relative to corresponding SMPU nanocomposites. Furthermore, 0.1g/ml RGD sequence was treated on acquired SMPU/HA/rGO nanocomposite surface through being dipped in the PBS buffer with adding CDI as the linker. Figure 1C presented the immobilization of RGD. Therefore, cell adhesion could be achieved through the coupling between RGD and integrin in cell (Figure 1F). Characterizations of SMPU nanocomposites. Ultraviolet (UV)–vis experiments were performed with a UV-2100S spectrophotometer (Shimadzu, Japan) to compare the difference of nanoparticles before and after treatments in UV absorption. SEM (FEI Inspect F50) at an accelerating voltage of 5 kV was used to recognize the surface morphologies of nanofillers e.g. rGO, SMPU matrix and nanocomposites e.g. SMPU/HA, respectively. Elemental analysis of the selected area in nanocomposites was obtained using an EDS attached to SEM. Perkin-Elmer (2000 FT-IR) spectrometer with attenuated-total-reflectance accessories was employed in the wavenumber range of 6504000 cm-1 to confirm the modifications of SMPU at room temperature. Contact angles of these SMPU nanocomposites were checked by the standard water contact angle goniometer. Data was recorded after the 4μL deionized (DI) water being dropped on the surface of samples. Tensile tests of SMPU and nanocomposites were enforced at room temperature by using an INSTRON 5566 tester with an extension rate of 5 mm min-1. Young’s Modulus and tensile strength at 50% elongation were further derived from the stress-strain curves to compare the mechanical properties. WAXD patterns of the samples

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were recorded using a Rigaku Smartlab XRD instrument using Cu Ka radiation source (1.54 Å). Sample was scanned from 2θ = 5 oC to 90 oC. Shape memory performance was investigated through recording changes of the angle under different conditions. In original state, the angle of composite was noted as θ0. Firstly, the sample was placed in oven at 80oC for 5 min and then bended it by angle θm. After being kept for 10 min, the fixed angle of sample was recorded as θf. Afterwards, the sample was recovered at 80oC and the final angle was indicated as θr. Shape fixity ratio (Rf) and shape recovery ratio (Rr) were defined from the equations: 𝑅f =

θm ― θf θm ― θ0

× 100%

(1);

θr

𝑅r (𝑁) = θm ―

θ0

× 100% (2)

Where θ0 =0 and θm =180 in this case. Cell adhesion. To investigate improved MSCs adhesion on SMPU/HA/rGO/RGD’s surface, unmodified SMPU and single-modified SMPU nanocomposites were utilized as control groups. Prior to cell seeding, the samples were sterilized through UV light for 60 min followed by being placed at the bottom of every well in a 24-well culture plate. Subsequently, the MSCs were seeded onto the surfaces of prewetted samples with a density of 5000 cells/well. These cell-seeded specimens were incubated in the 5% CO2 atmosphere at 37oC for 24 hours for cell attachment. Then, the specimens were washed twice by the PBS solution to remove unattached cells prior to being fixed with glutaraldehyde for 3 hours. The dehydration process was conducted with serial ethanol solutions (25%, 50%, 75%, 90% and 100%) for 100 min totally followed by being dried at room temperature. At last, SEM was performed for capturing the images of cell 10



adhesion and spreading behavior. Statistical analysis. SPSS 22.0 software was used to analyze statistically all data. Results were compared using one-way ANOVA and significant difference was set as p

Mechanically Robust Shape Memory Polyurethane Nanocomposites

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