Accepted Manuscript Development of Fe3O4 –HA/PU superparamagnetic composite porous scaffolds for bone repair application Yan Yan, Yi Zhang, Yi Zuo, Qin Zou, Jidong Li, Yubao Li PII: DOI: Reference:
S0167-577X(17)31529-X https://doi.org/10.1016/j.matlet.2017.10.067 MLBLUE 23302
To appear in:
Materials Letters
Received Date: Revised Date: Accepted Date:
20 July 2017 21 September 2017 15 October 2017
Please cite this article as: Y. Yan, Y. Zhang, Y. Zuo, Q. Zou, J. Li, Y. Li, Development of Fe3O4 –HA/PU superparamagnetic composite porous scaffolds for bone repair application, Materials Letters (2017), doi: https:// doi.org/10.1016/j.matlet.2017.10.067
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development of Fe3O4–HA/PU superparamagnetic composite porous scaffolds for bone repair application Yan Yan, Yi Zhang,Yi Zuo, Qin Zou, Jidong Li* and Yubao Li* Research Center for Nano Biomaterials, Analytical & Testing Center, Sichuan University, Chengdu 610064, PR China Abstract Magnetic stimulation and magnetically responsive scaffolds can play unique roles in promoting bone repair and regeneration. Here, we designed and fabricated a novel type of magnetic porous scaffold that integrates superparamagnetic, cytocompatibility, and osteostimulation properties by incorporating a spherical core–shell nano-iron oxide–hydroxyapatite (Fe3O4–HA) composite into polyurethane (PU). Its structural, physicochemical and magnetic properties, as well as its preliminary cytocompatibility were evaluated. The results show that the scaffold had a highly interconnected porous structure with a porosity of about 65%，a pore size distribution of 100–600 µm and a compressive strength of 4.16 MPa. The results of the magnetic hysteresis loops suggest that the composite scaffolds possess superparamagnetism and that the saturation magnetization of the scaffold was about 2.19 emu/g. The cell culture indicates that the porous magnetic scaffold had good cell affinity and cytocompatibility. In general, this study demonstrates that the Fe3O4–HA/PU composite scaffold integrating both magnetic properties and biocompatibility may be an ideal candidate material for bone repair. Keywords: Biomaterials; Fe3O4 -HA/PU; magnetic materials; cytocompatibility; porous scaffolds
*Corresponding author. E-mail address: Jidong Li (
[email protected]);Yubao Li (
[email protected]) 1
1. Introduction Mechanical loading is known to be a potent stimulation factor for bone formation and bone regeneration [1]. Nevertheless, in some clinical cases (e.g., in patients who are able to perform less activity or those with paralysis), the insufficient mechanical stimulation that arises from bone defects in the musculoskeletal system may restrain bone regeneration and even result in disuse-induced bone loss. Consequently, increasing research efforts have focused on how to prevent bone loss and improve bone mass by employing external energy to provide sufficient mechanical stimulation. Several evidences suggested mechanical stress signals could be transmitted to nuclear area via intracellular membrane system or cytoskeleton and then activate the downstream genes pathway that closely related to osteogenesis to regulate bone repair. Some mechanical stimulators, such as mechanical vibration [2], low-intensity pulsed ultrasound [3], pulsed electric fields, and pulsed electromagnetic fields [4], were demonstrated to enhance bone restoration and fracture healing. In addition to the aforementioned mechanical force stimuli, magnetic stimulation – as an effective remedy – has constantly piqued researchers’ interests. Inspired by the effects of magnetic stimulation, researchers have introduced magnetic nanoparticles (NPs) into various matrices to fabricate magnetic composites and explore the potential application for bone repair recently [5–6]. Meng et al. reported that the
incorporation
of
γ-Fe2O3
in
the
nanofibers
endows
HA/polylactic
acid
scaffolds
superparamagnetically response and induction of faster osteoblast differentiation [7]. The incorporation of magnetic particles and synergic poly-ε-caprolactone nanofibers enhance cellular adhesion, proliferation, and osteogenic differentiation among mesenchymal stem cells [8]. Zeng et al. fabricated magnetic nanoparticle-loaded HA scaffolds and demonstrated the positive influence on cell behaviors [9]. These studies suggested that the application of magnetic particles holds promise in the field of bone repair.
2
In our previous study, a core–shell Fe3O4-HA spherical composite particle that featured superparamagnetism was successfully synthesized [10]. As we know, HA can enhance the osteoconductivity and bone-bonding bioactivity of bone repair materials. Polyurethane (PU), a versatile elastomeric block copolymer, has exhibited good biocompatibility, and tunable properties [11]. Consequently, we incorporated a superparamagnetic Fe3O4–HA microsphere into the PU matrix to develop a novel magnetic composite scaffold that integrates superparamagnetism, cytocompatibility, and osteostimulation. In this system, the Fe3O4 core is used to give a superparamagnetic response, while the HA shell is applied for bone conductivity and PU is used for foaming porous scaffold. Our hypothesis is that the porous magnetic scaffold can serve as a material and structural template for osteogenesis, while the implanted superparamagnetic material under an external magnetic field will provide effective mechanical stimulation to promote bone regeneration and remodeling. In this preliminary study, the physicochemical properties, magnetic property, and preliminary cytocompatibility of the fabricated magnetic composite scaffold are investigated. 2. Experimental Scaffold fabrication: the magnetic composite microsphere of Fe3O4–HA was prepared via a homogeneous precipitation method, as described previously [10]. Briefly, Fe3O4 NPs were dispersed and used as a substrate in a Ca, P solution to deposit HA on the Fe3O4 spheres by slowly increasing the pH this decomposed urea, while urease was employed as the catalytic. Following that, the Fe3O4–HA/PU composite scaffolds with 15wt% Fe3O4–HA were fabricated via in situ polymerization and a simultaneous foaming method, according to the method previously used to fabricate HA/PU scaffold [12]. The schematic drawing of the fabrication process is presented in Figure 1. Pure PU and the HA/PU scaffolds were also prepared as a control, according to a similar process.
3
Figure 1. Schematic illustration of the fabrication of Fe3O4–HA/PU composite scaffolds
Scaffold characterization: The microstructure of the samples was observed using SEM (JSM-6510LV, Jeol, Japan) and TEM (JEM-100CXП; Jeol, Japan). FTIR (Nicolet 6700, US) and XRD (INCA 8129, UK) were employed to determine the chemical groups and components. The hysteresis loop was measured by a vibrating sample magnetometer (LS 7410, US). Porosity was calculated using the Archimedes method [13]. The mechanical test was performed according to the ASTM D 5024-95 standard. Cell activity was observed by SEM and live/dead assay. Cell proliferation on scaffolds was evaluated by MTT assay. 3. Results and discussion Figure 2a shows the XRD patterns of the Fe3O4–HA/PU composite scaffolds. The main characteristic peaks of Fe3O4 (JCPDS no.85-1436) and HA (JCPDS no.9-432) are present. In Figure 2b, the absorption peaks at 1,720 cm–1 confirms the formation of the PU. The absorption band of PO4 stretching at 1,040 cm–1 is attributed to the n-HA component in the scaffold, while the band at 555 cm–1 of Fe-O also confirms the existence of Fe3O4 in the composite scaffolds. Fe3O4 NPs are uniform in both shape and size, as they are spherical and approximately 20 nm in diameter (Figure 2c). The Fe3O4–HA magnetic composite also exhibited a spherical shape, and its size is nearly 3–4 times larger than that of
4
Figure 2. XRD patterns of Fe3O4–HA/PU (a); FTIR spectra of Fe3O4–HA/PU (b); TEM images of the Fe3O4 nanoparticle (c); and Fe3O4/HA composite (d). SEM images of pure PU (e); Fe3O4–HA/PU (f) and HA/PU scaffolds (g).
the Fe3O4 NPs, which may be due to the fact that the composite consists of an Fe3O4 core and an HA shell (Figure 2d). From Fgures 2e-2g, it can be observed that the three resulting scaffolds all exhibit a uniform porous structure, with a pore size distribution of 100–600 µm, which is beneficial to both the cells and bone tissue ingrowth. It can be observed that more micropores are located on the walls, while the Fe3O4–HA/PU and HA/PU scaffolds have a rougher surface when compared with the pure PU scaffolds. The three-dimensional micro-computed tomography image of the Fe3O4–HA/PU composite scaffold in Figure 1 also confirms its uniform and interconnective porous structure. The porosity and compressive strength of Fe3O4–HA/PU scaffolds are about 65% and a 4.16 MPa respectively, which are similar with those of HA/PU scaffolds and higher than those of PU scaffolds (Table 1). Table 1 Porosity and compressive strength of three kinds of porous scaffolds Scaffolds
Porosity (%)
Compressive strength (MPa)
PU
45.51±4.34
2.55±0.13
Fe3 O4-HA/PU
65.59±5.14
4.16±0.16
HA/PU
67.14±3.02
3.93±0.24
5
The magnetic hysteresis loops pass through the origin, and the low remanence and coercivity of all
the samples (Figure 3) indicate that superparamagnetic nano-Fe3O4 endows the Fe3O4–HA and Fe3O4–HA/PU composite scaffolds with superparamagnetism. The superparamagnetic properties are crucial for the biomedical applications of magnetic composites, which can provide effective mechanical stimulation under an external magnetic field to promote tissue regeneration [14], while the saturation magnetization value decreases after incorporating Fe3O4 into the HA and PU matrix. This can be attributed to the fact that the magnetic domains of the nano-Fe3O4 particles were covered and formed chemical bonds with HA and PU.
Figure 3. Magnetic hysteresis loop, saturated magnetization (Mss/emu· g–1), remanence (Mremu·g–1) and coercivity (Hc/Oe) of Fe3O4 (a); the Fe3O4–HA composite (b); and the Fe3O4–HA/PU composite (c).
Figure 4. Fluorescent images of MG63 cells cultured with different scaffolds for 7 days: PU (a); Fe3O4–HA/PU (b); and HA/PU (c). SEM images of MG63 cells cultured with different scaffolds at 7 days: PU (d, d’); Fe3O4–HA/PU (e, e’); and HA/PU (f, f’). MTT assay of MG-63 cells cultured with various scaffolds (g). [n=4]. ***P
