PLGA

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Article

Electroactive Nanocomposite Porous Scaffolds of PAPn/op-HA/PLGA Enhances Osteogenesis in Vivo Xin Cui Shi, Haitao Wu, Huanhuan Yan, Yu Wang, Zongliang Wang, and Peibiao Zhang ACS Appl. Bio Mater., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019 Downloaded from http://pubs.acs.org on March 25, 2019

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ACS Applied Bio Materials

Electroactive Nanocomposite Porous Scaffolds of PAPn/op-HA/PLGA Enhances Osteogenesis in Vivo Xincui Shi,a#* Haitao Wu,c# Huanhuan Yan,a,b Yu Wang,a Zongliang Wang,a Peibiao Zhanga,* aKey

Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, China.

bSchool

of Applied Chemistry and Engineering, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, P. R. China.

cDepartment

of Orthopedics, Jilin Provincial People's Hospital, 1183 Gongnong Street, Changchun 130021, China.

#

Xincui Shi and Haitao Wu contributed equally to this work and should be considered as the cofirst authors. E-mail: [email protected], [email protected].

KEYWORDS oligoaniline; electroactive; nanocomposite; bone repair; hydroxyapatite; in vivo

ABSTRACT

The three-dimensional (3D) porous nanocomposite biomimic scaffolds with electroactivity and bioactivity were prepared as bone implants by freeze-drying method using 1,4-dioxane as solvent. The multiblock copolymer (PAPn) was synthesized by the condensation polymerization

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of hydroxyl-capped poly(lactide)(PLA) and carboxyl-capped aniline pentamer (AP), which was introduced as electroactive functional polymers. Bioactive component, hydroxyapatite (HA) grafting L-lactic acid oligomer (op-HA), showed better interface compatibility with PAPn and poly(lactide-co-glycolide) (PLGA). Honeycomb-like and interconnected porous structures could be obtained as displayed by scanning electron microscope (SEM). The intramuscular implants showed good biocompatibility and higher osteogenetic activity by promoting cell ingrowth and collagen fibers forming as indicated by SEM, hematoxylin-eosin (H&E) staining and Masson’s trichrome staining. Implantation for repair of rabbit radius defects under 5 v at 100 Hz with 50% duty cycle for 30 minutes every other day were evaluated. And sheep tibia defects were also carried out. The composite scaffold with 1 wt% PAPn exhibited better behaviors, such as distinct bone callus, bridging growth, vague borderlines between newly formed bone at the two defect ends and increased bone density as indicated by radiographic images and Micro-Computed tomography (Micro-CT) images. Electricity stimulation could significantly accelerate the healing of fracture. All in all, the stimuli-responsive electroactive nanocomposites showed a potential application in bone tissue engineering.

1.

INTRODUCTION

Evoking by naturally conductive systems of the body, electrical stimulation have been widely used in the field of tissue engineering.1-3 However, it requires high voltage or currents in the absence of scaffolds resulting in damaging the surround tissue. Accordingly, as a new generation of smart biomaterials, conductive polymers (CPs) have gained considerable attention in the engineering of nerves, cardiac, skeletal muscle and bone etc.

2, 4-10These

applications benefit

from the regulation of cellular behavior and function, such as growth, adhesion, proliferation, migration, elongation and gene expression.11 Actually, electroactive biomaterials have already


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been used to increase the length of neurite outgrowth,12-15 to regulation of osteogenic activities1619

and as a potent trigger to enhance skeletal muscle regeneration.20-23 Numerous CPs such as

polypyrrole (PPy), polyaniline (PANI), polythiophene and their derivatives have been widely investigated.5, 24-25 Nevertheless, the greatest disadvantages of CPs are not degradable and may trigger an inflammatory response in the long term in vivo, needing for a second surgical procedure. It shed light on the aniline oligomers possessing similar electrical conductivities, biodegradable, low toxicity, reducing any long-term adverse effect as well as feasibly functional modification further improving its biocompatibility. However, the limitations of poor mechanical property and poor processability have impeded their further application for clinically biomedical implants and devices. To overcome the drawbacks, attentions have been directed towards form blends or composites combining CPs with elastic, biocompatible polymers.26-29 It will pave the way for improving mechanical performance compatible with native tissues and hardly compromise of their conductivity. Molly M. Stevens and colleagues showed that hemin-doped serum albuminbased fibrous scaffolds could promote neurons exhibiting more branched neurites with electrical stimulation. They also summarized the mechanism proposed for the mediation of electric signals.30 A biohybrid hydrogel composed of collagen, alginate, and electroconductive poly(3,4ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) is developed. The electrical coupling beating frequencies reaching more than 200 beats min−1 endogenous frequencies as well as cardiomyocyte maturation on this substrates.31 As has been reported, flexible conductive materials were obtained with PANIs as the electroactive composites, upon which C2C12 myoblasts were shown to adhere, proliferate and differentiate into myotubes in vitro.21,26,

32-33

Park and coworkers successfully fabricated a polymer-based stretchable electrode from a blend


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of PEDOT: PSS and an aqueous PU dispersion.34 AP-g-GA/PLLA composite nanofiber mats were reported by Liu et al.35 The obtained biomaterials effectively promote the differentiation of MC3T3-E1 cells stimulated by a pulsed electrical signal. Apart from chemical composition, the roughness, porosity and topography are also essential for realizing function of biomaterials. To application in tissue engineering, 3D composite scaffold with proper pore size and porosity, recapitulate the native extracellular matrix (ECM), is vital to form a local niche which could supply temporary support for tissue defects and promote tissue formation.

2,36-37

Usually, the

porous scaffolds were fabricated by techniques such as particle leaching,38 phasef separation,39-40 molecular assembly41 and so on. The interconnecting pores and large surface areas are essential for exchange of nutrients, wastes diffusion and cell in-growth. It remains a long-term and arduous work to develop effective biodegradable scaffolds for tissue replacement and regeneration, which are capable of simultaneously providing topographical and electrical cues. The bone conditions and disorders are frequently clinical tasks, triggering a high demand on the advancement of bone scaffolds. The native bone matrix is inorganic/organic composite. The functional scaffolds should also compose of HA, a bioactive and bioresorbable calcium phosphate, forms the majority of the inorganic component of bone tissue.42-44 HA has been widely used to prepare artificial bonelike ceramic/polymer composites. However, the phase separation could occur between pure HA and the polymer matrix leading to weak interfacial adhesion strength, which suffers from the aggregation of HA nanoparticles. Then, surface modified HA nanoparticles/polymer composites were developed for osteoconduction and osteoblast proliferation. For example, Chen’s group has previously reported the nanocomposites of g-HAP (HA surface-grafted with poly(L-lactide)/PLLA (or g-HAP/PLGA) showed a wide potential application for bone fixation materials.45 Zhang et al investigated 3-D porous scaffold


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composing of g-HAP/PLGA (poly(lactide-co-glycolide) composite in vivo, which exhibited rapid and strong mineralization and osteoconductivity.46 Predictably, biomineralized materials play an important role in the scaffolds for bone regeneration as promoting osteogenesis.47 Although many electroactive biomaterials have been used for cell culture assays in vitro indicating potential application for repair of bone defects, the ultimate goal to fabricate suitable engineering scaffolds for bone grafts in vivo has been reported rarely. With above perspective, we intend to simultaneously utilize features of electrical cues, 3-D porous topographical and modified HA as the bone repair scaffold in vivo. Accordingly, we prepare the elctroactive biodegradable composite based on HA surface modified with oligomeric lactic acid (op-HA),48-49 PLGA and multiblock copolymer PLA-b-AP-b-PLA (PAPn).50 The porous scaffolds of PAPn/opHA/PLGA were fabricated by phase separation method, which was evaluated by intramuscular implantation and replacement for repairing radius defects of rabbits and sheep tibia defects. The effects of PAPn concentration on the porosity, pore structure and morphology as well as mechanical properties were investigated. Materials in vivo mineralization, degradation was also evaluated. Moreover, the osteogenesis was discussed exposed to external electric pulse stimuli after implanted in vivo. 2. EXPERIMENTAL SECTION 2.1. Materials Dicyclohexylcarbodiimide (DCC) with 98% purity was purchased from GL Biochem (Shanghai) Ltd.; N-phenyl-1,4-p-phenylenediamine (98% purity) was bought from Aldrich; pphenylenediamine (98% purity) was obtained from Beijing Beihua Fine Chemical Co., Ltd. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and trypsin were purchased from Gibco USA; penicillin and streptomycin were bought from North China


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Pharmaceutical Co Ltd Shanghai, China; Fluorescein isothiocyanate (FITC), propidium iodide (PI) and 4-[2-hydroxyethyl]-1-Piperazineethanesulfonic acid (HEPES) were purchased from Sigma, USA; Methyl thiazolyl tetrazolium (MTT) was purchased from Solarbio USA; All chemicals were of analytical grade or higher. 2.2. Synthesis of PAPn and op-HA The electroactive block copolymer of PLA-b-AP-b-PLA (PAP), in which the PLA with Mn around 4.8 kDa, was synthesized as reported previously.49-50 In specific, the multiblock copolymer PAPn (Mn=70.0 kDa, PDI =1.30) was synthesized by the condensation polymerization of hydroxyl-capped PLA and carboxyl-capped aniline pentamer (AP).51 The opHA was synthesized by modified the nanohydroxyapatite with oligomeric lactic acid (Mn around 1.5 kDa) according to work reported previously. Scheme 1 outlines the synthesis of PAP and opHA. The op-HA with 9 wt% grafted LAc oligomer was obtained according to our previous work and used in this work.52-53 FT-IR spectrum was carried out to confirm the grafting reaction. The amount of grafted LAc oligomer was analyzed by thermogravimetry analysis (TGA). The PLGA with viscosity-average molecular weight (Mv) about 85 kDa (LA: GA = 80:20) was prepared in our laboratory. The PLAs and PLGA were all synthesized by ring-opening polymerization of the corresponding monomers with stannous octanoate (Sn(Oct)2) as catalyst.


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Scheme 1. Schematic synthesis route and structure of PAPn copolymer (a); and op-HA nanoparticles (b). 2.3. Preparation of Electroactive Nanocomposites 3-D Scaffold Firstly, the PAPn, op-HA, and PLGA were dissolved in chloroform to form a 0.06 g/ml solution with magnetic stirring for 24 h, respectively. Then, they were respectively dispersed under ultrasonic treatment for 30 min. The composite of 10 wt% op-HA/PLGA was prepared by mixing two solutions at volume ratios of op-HA to PLGA at 1:9. The electroactive nanocomposites with 1 wt% and 10 wt % PAPn were obtained by mixing solutions of PAPn, opHA/PLGA (10 wt%, C1) at volume ratios of 1:99 (C2) and 10/90 (C3), respectively. As the 3-D scaffolds were concerned, they all were fabricated using the solid-liquid phase separation method referring to Ma’s work. In detail, the nanocomposites emulsions mentioned above were then transferred into a Teflon vials with 14 mm diameter, 80 mm height and 1mm thickness, respectively. Then, put the vials into a freezer set to 4 oC for overnight. Follow-on to this step, vials were transferred into freeze-drying vessel at 4 oC under vacuum for one week. The dried porous 3-D scaffolds were then stored in a desiccator. With increase of the content of PAP, the scaffolds get darker from intuitive point of view. 2.4. Characterization of Scaffold Differential scanning calorimetry (DSC) measurements were performed on Perkin-Elmer DSC7 at a heating/cooling rate of 10/-10 oC min-1 under N2 atmosphere. Thermo gravimetric analysis (TGA) was run on TA Instrument Q500 to determine the thermal stability under a nitrogen flow. The compressive mechanical properties of samples were performed with an Instron 1121 mechanical tester. The cylinder samples with 5 mm diameter, 5 mm height were prepared for mechanical testing. Three parallel testing specimens for each sample was measured


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and averaged as the results. According to GB/T1041-1993, the scaffolds were subjected to 50% compression at a crosshead speed of 2.0 mm/min. Atomic force microscope (AFM) images were acquired in tapping mode with a SPA400HV instrument with a SPI 3800 controller (Seiko Instruments) with a nominal spring constant of 50 N/m and resonance frequency of ∼300 kHz. The spin-coated films were prepared from CHCl3 followed and dried at room temperature for 24 h. A piece of implant materials was fixed with 4% paraformaldehyde (PFA) in PBS for 1 h at 4oC followed by rinsing three times with PBS. Then, it was dehydrated with ethanol of 60%, 70%, 80%, 90%, 100% for 30 min each concentration. The sample was Freeze-dried. The samples for SEM (XL30 ESEM-FEG, FEI) were immersed in liquid nitrogen for frozen, and then they were rapidly broken off to obtain brittle fracture. Samples were mounted on metal stubs with double sided tape, and coated with Au/Pd in a sputter coater. The characterization of the porosity was carried out by liquid displacement method as published previously.39, 54 The ethanol was used as the displacement liquid. The porosity values of the scaffolds were the average value of three similar samples with 11 mm diameter, 30 mm height. Herein, the samples for testing are PLGA, C1, C2, C3, respectively. In specific, a sample with weight of W1 was put into graduated cylinder containing ethanol (8 mL) and kept in for 5 min, the evacuation–repressurization were carried out duration for about 10 min until no air bubbles emerging from the scaffold. The reading volume of graduated cylinder was recorded as V1. The weight of scaffolds with ethanol was recorded as W2. The rest volume of ethanol in graduated cylinder was recorded as V2. As all known, the density of ethanol was 0.789 g/cm3. The porosity of the scaffold (ξ) was obtained by: ξ = W2-W1/0.789/V1-V2.


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2.5. Proliferation Activity of MC3T3-E1 Cells on Scaffolds with and without Electrical Stimulation (ES) MC3T3-E1 cells were seeded in 24 well plates with 6 mm diameter and 5 mm height scaffolds sterilized by UV light for 30 min. The density was 2×104 cells per well. They were cultured at 37 oC in a humidified 5% CO2 atmosphere with and without ES for 3 or 7 days. MTT assays were introduced to quantitatively assess the cell adhesion and proliferation behaviors in vitro. The solution of MTT (200 µL, 10 mg/mL) was added to each well and incubated for 4 h at 37 oC.

After removing the medium, hydrochloric acid/isopropanol solution (1 mL, 1/50, v/v) was

added to each well. The optical density (OD492) was measured using a Microplate Spectrophotometer. Results were shown with mean standard deviation of triplicates for each type of scaffold. 2.6. Intramuscular Implantation (a)PLGA PLGA

(b)C1 C1

(c) C2 C2

(d)C3C3

Figure 1. Samples of porous scaffolds of (a) PLGA, (b) op-HA/PLGA(C1), (c)1 wt% PAP/opHA/PLGA(C2), and (d) 10% wt PAP/op- HA/PLGA(C3). The cylinder samples with 6 mm diameter and 5 mm height were prepared as shown in Figure 1. They were exposured to UV radiation for 1 h, which were implanted intramuscularly for in vivo assessment. They were embedded into dorsal muscle of 12 rabbits by the surgery. Three different samples (PLGA, C1, C2, C3) were embedded for each rabbit. Three rabbits were sacrificed with an air injection at 4, 12 and 20 weeks, respectively. That is to say, each group experiment have three parallel samples. After taken out, the scaffolds were observed with a digital camera (Fujifilm FinePix S602, China). The specimens for SEM were fixed with


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glutaraldehyd, dehydrated with ethanol and then freeze-dried. With regard to specimens for histological analysis, they were fixed with 4% paraformaldehyde for 2 h at room temperature. Samples were then dehydrated through ethanol and embedded in paraffin. Sections were taken using a microtome. The surrounding tissues of specimens were stained with hematoxylin and eosin (H & E) to exam the inflammatory responses of materials. Moreover, masson’s trichrome staining was performed to evaluate ostegenesis. All stained slides were mounted and scanned using a histology slide scanner. 2.7 Implantation for Rrepair the Radius Defects of Rabbit The porous scaffolds (n = 24) with the dimension of 20 (L) ×4 (W) ×2 (H) mm were fabricated and sterilized with ethylene oxide. After anesthesia with 10 mg/kg sodium pentobarbital and 0.2 ml Lumianning, the New Zealand white rabbits were suffered to create bilateral critical-sized radius defects (20 mm) by surgery. Subsequently, twenty-four different scaffolds, six parallel scaffolds for each sample (PLGA, C1, C2, C3, respectively), were placed in critical-sized radius defects of rabbits without any fixation. The wounds were closed in routine ways of surgery. One group (12 scaffolds with three parallel scaffolds for each sample) was suffered to stimulate by a pulsed electrical signal under 5v at 100 Hz for 30 minutes every other day. These rabbits were taken care of by the Experimental Animal Centre of Jilin University. 2.8 Implantation for Repair of Sheep Tibia Defects The porous cylinder scaffolds (n = 9) with 15 mm diameter and 25 mm height were fabricated for repair of sheep tibia defects and sterilized with ethylene oxide. After anesthesia with 10 mg/kg sodium pentobarbital and 0.6 ml Lumianning, the sheep were suffered to create bilateral critical-sized tibia defects (25 mm) by surgery. Subsequently, the steel plates were placed in the outside of the tibia forming bone defect models. And then, these animal models


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were divided into four groups to implant different scaffolds (Blank, C1, C2, C3), for each group with three parallel samples. They were suffered to stimulate by a pulsed electrical signal under 10 v at 100 Hz for 30 minutes every other day from 4 weeks to 8 weeks. The wounds were closed in routine ways of surgery. 2.9 X-ray Examination To statically evaluate the healing degree, the Lane−Sandhu radiographic scoring system was introduced.55 Grading is assigned to five professionals who scored for parallel samples of a group of samples at all postoperative time points. The specific scoring criteria of Lane−Sandhu radiographic scoring system are shown in Table 1. Table 1. Lane−Sandhu Radiographic Scoring System Degree of Bone Formation no new born formed the area of new bone accounts for 25% of the defect area the area of new bone accounts for 50% of the defect area the area of new bone accounts for 75% of the defect area the area of new bone accounts for 100% of the defect area Degree of Union fracture line is fully visible fracture line is partially visible fracture line is not visible Degree of Medullary Cavity Remodeling no sign of remodeling recanalization of medullary cavity cortical bone structure forms after recanalization of medullary cavity

Scorces 0 1 2 3 4 0 2 4 0 2 4

3．RESULTS and DISCUSSION The bone defect was beyond a critical size (15 mm for rabbit), the self-healing capabilities of bone would be impeded.56,57 It was due to the interruption or descent of mechanical properties and signal transduction. Correspondingly, the tissue engineering scaffolds should be introduced to supply temporary mechano-induction and interconnected pores for cell ingrowth. We intend to


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fabricate the PAPn/op-HA/PLGA composite porous scaffolds to provide osteoinductivity and electro-activity for ultimate bone remodeling. Previously, we have reported the composites of PAP/op-HA/PLGA act as matrix for cell evaluation in vitro.49 The results indicated that these composites have good biocompatibility and osteogenic activity, which may be a new generation of smart biomaterials used in tissue engineering scaffolds and tissue repair. Therefore, we prepared 3-D porous scaffolds based on PAPn/op-HA/PLGA to act as artificial ECM used to implant the bone defects in vivo in this report. 3.1 Characterization of Scaffolds

Figure 2. TGA curves of the composites: (a) PLGA, (b) C1, (c) C2, (d) C3. The FT-IR spectra were given in the revised manuscript Figure S2. The absorption peaks at 604 and 565cm-1 were designated to the bending vibration of PO43-. The benzenoid unit and the quinoid unit of the PAPn appeared at 1510 and 1600 cm-1, repectively. The absorption at1614 cm-1 in the spectra was attributed to the –COO-vibration of the formed calcium carboxylate on the surface of n-HA. In the field of repairing and replacing of scleroses tissue, the mechanical and processing properties of materials should be taken into consideration. The thermal stability of composites has vital effect on the processing conditions. Herein, the stability of PLGA, C1,


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C2 and C3 was investigated after being dried and heated under identical conditions. As shown in Figure 2, the samples were heated from 50 oC to 600 oC. They all showed obvious weight loss for these composites. As for PLGA, the obvious weight loss occurred at 329 oC. In contrast, it occurred at 350 oC for C1. With increase of the content PAP in the composites from C2 to C3, the thermal decomposition temperature decreased but were still high than that of PLGA. The decomposition of the biomaterials was mainly due to the decomposition of PLGA chains. The results indicated that all the composites showed good thermal stability. As for the mechanical properties, compressive strength of the composite C2 with 1.0 wt% of PAPn (4.08±0.78 MPa) was higher than that of C1 (3.69±0.34 MPa) and C3 (2.52±0.17 MPa). It may be due to the different morphology of the scaffold, such as the porosity and the pore wall surfaces as investigated below. The micro-structures of the PLGA, C1, C2 and C3 were further investigated by AFM observations (Figure 3). Figure 3c and 3d exhibited triangular pyramidal protuberance morphologies, which grow in number and bulk with increasing the content of PAPn. It may be ascribed to the aggregate and self-assembly of PAPn due to the π-π interaction of aniline pentamer segments.


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Figure 3. AFM height images of the film surface topography of PLGA (a), op-HA/PLGA(b), and PAPn/op-HA/PLGA with the PAPn content of 1.0 wt % (c) and 10 wt % (d).


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Figure 4. SEM micrographs of porous scaffolds of PLGA (a), C1 (b), C2 (c) and C3 (d) fabricated with freeze- drying method. The scale bars are 200 µm (-1) and 50 µm (-2). The SEM micrographs of different scaffolds were displayed in Figure 4. All scaffolds showed clearly pore structures. Honeycomb-like and interconnected porous structures could be observed, which was fabricated by freeze-drying method. Arrangement direction of the tubular pores is in


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line with crystal’s conformation direction. Pores became gradually larger approaching to the central position of scaffolds. It may be due to the heat conduction resulting in lower freezing rate in central position, which tends to form larger crystal. The diameter of pores was range from 100 to 300 nm. The arrangement of the pores was relatively regular, the pore walls were rough. Compared with the other scaffolds, C3 with 10 wt% PAP was looser accompanied by irregularity of the pore wall. According to liquid displacement method, the porosity of scaffolds was increase with the increase of the content of conductive materials. It was highest for C3 (up to 95.0±0.9) compared with PLGA (81.0±0.5), C1(84.4±0.7) and C2(88.4±0.4). It indicates that the content of PAPn in the composites has influence on the interface of scaffold. The relatively looser morphology and higher porosity may responsible for the poor mechanical property of C3. SEM micrographs of pore wall surfaces were displayed in Figure 5. The op-HA and PAP nanoparticles with diameter around 100 nm were uniformly dispersed on the surface of pore walls and formed few aggregates, which may supply favorable interface for attachment and proliferation of osteoblast.


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Figure 5. SEM micrographs of pore wall surfaces of PLGA, C1 ，C2 and C3 fabricated with freeze-drying method. The scale bars are 5 µm. 3.2. Biocompatibility Assessment of 3D Porous Scaffolds with and without ES in vitro To evaluate the biocompatibility of different scaffolds, the MC3T3-E1 cell were seeded and cultured for 3 and 7 days with and without electrical stimulation. MTT assays were introduced to quantitatively assess the cell adhesion and proliferation behaviors in vitro, as shown in Figure 6. The scaffolds (C1, C2 and C3) exhibited good biocompatibility which were comparable to that of PLGA. Relative to without electrical stimulation (ES), all of the scaffolds showed obviously better osteoblast adhesion and proliferation behaviors with ES. Additionally, the osteoblast metabolic activity of electroactive scaffolds (C2) with 1.0 wt% PAPn was significantly improved especially applied pulse ES. Polymers could accelerate the proliferation of osteoblasts. The DAPI staining was used to determinate the cell adhesion and penetration into the inner pore of scaffolds. As can be seen from the Figure S2, osteoblasts can infiltrate into the scaffold and distribute evenly throughout the scaffold. The number of cells on the conductive scaffold material was significantly higher than that in the control group. It is demonstrated that porous scaffolds can provide enough interstitial space to facilitate cell creeping and growth.

Optical Density(OD492）

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0.8 0.6

PLGA C1 C2 C3 *

3d 7d * **

0.4 0.2 0.0

Stimuli- Stimuli+ Stimuli- Stimuli+


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Figure 6. MC3T3-E1 cell Optical Density (OD492) analysis on different substrates analysis by MTT assay with and without electrical stimulation (ES) for 3 and 7 days. (*) represents statistical significance with p < 0.05. (**) represents statistical significance with p < 0.01. 3.3. Intramuscular Implantation All animal experiments were performed in strict accordance to the Chinese regulations of Animal Welfare and permission granted by the Ethical committee granted by the Institutional Animal Care Committee of China-Japan Union Hospital, Jilin University. The animals were anesthetized through intramuscular injection of ketamine (10 mg/kg).The porous scaffolds of samples (PLGA, C1, C2 and C3) were embedded into the dorsal muscle of rabbit for assessment in vivo. The implants were examined at 4, 12 and 20 weeks post-surgery. All the implants kept their original shapes, which indicate that the composite implants could supply scaffold for defects more than 20 weeks. The distinct mineralization could be observed as time goes on after the surgery as shown in Figure 7.


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a-2

b-1

b-2

c-1

c-2

d-1

d-2

C2

C1

PLGA

a-1

C3

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Figure 7. SEM analysis of intramuscular implants of porous scaffolds at 4 weeks post-surgery PLGA (a-1-2), C1 (b-1-2), C2 (c-1-2) and C3 (d-1-2).The scale bars are 200 μm and 50 μm. The SEM photographs of the intramuscular implants of PLGA, C1, C2, and C3 at 4 weeks post-surgery were shown in Figure 7. The interfaces between scaffolds and the tissue muscles could be observed clearly (Figure 7 a-1, b-1, c-1 and d-1). They were linked by tight junction. The regular porous structure became irregular with the biodegradation the scaffolds and the


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formation of new tissue. The scaffolds were wrapped up by fibers. It also could be observed that the cells and capillaries grew into the scaffolds (Figure 7 a-2, b-2, c-2 and d-2). Cells were widely distributed inside of the scaffolds. Especially for C3 with 1.0 wt% PAP, more cells showed unfurled lamella shape with fully extended filopodia could be observed. These phenomena indicate that the scaffolds with better interconnectivity could be benefit for the material exchange and mineral deposition and favorable for cell ingrowth. PLGA

C1

C2

C3

a-1

b-1

c-1

d-1

a-2

b-2

c-2

d-2

a-3

b-3

c-3

d-3

4w 12 w

20 w

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Figure 8. Histological analysis showing the representative micrographs of porous scaffolds implanted intramuscularly at 4w，12w and 20w post-surgery. PLGA (a-1-3), C1 (b-1-3), C2 (c1-3) and C3 (d-1-3). Arrows indicate the inflammatory cells, all the Bars are 25 μm. H&E staining and Masson’s trichrome staining were carried out for histomorphology analysis at different intervals. Typical photographs are shown in Figure 8 and Figure 9. In Figure 8, few inflammatory cells could be seen, which indicates that the implants showed acceptable


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biocompatibility in vivo. Cell migration was allowed to the inside of scaffolds due to the interconnected pores. At 12 weeks after implantation, all the scaffolds were filled with the newly formed tissue produced by the ingrowth cells. The amount of capillaries and connective fibers was more on the C2 with 1.0 wt% PAPn. At 20 weeks, the scaffold implanted was almost degraded. The connective tissue with vessels refilled the disintegration position. Masson’s trichromen staining (Figure 6) shows that collage was formed obviously. Consistent with the results of H&E staining, scaffold C2 exhibited better behaviors. Hence, we concluded that introducing PAPn and op-HA to PLGA could improve the osteogenic activity. PLGA

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Figure 9. Masson’s trichrome staining showing the micrographs of PLGA (a-1-3), C1 (b-1-3), C2 (c-1-3) and C3 (d-1-3) implants at 4w, 12w, 20w post-surgery embedded in rabbit dorsal muscles. Red color shows muscle tissue, dark green color shows collagen fiber, and red yellow shows blood cells. All the Bars are 50 μm.


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3.4 Implantation for Repair the Radius Defects of Rabbit Pulsed electromagnetic field (PEMF) has been clinically used to treat delayed fracture healing. The positive effects of EMFs on new-bone formation have been well established. Inoue et al revealed the enhancing effects of PEMF on callus formation and maturation in the late-phase of bone healing.58 Yonemori and coworkers in vivo demonstrated that the proliferative activity of osteoblasts in the bone marrow increased significantly after electrical stimulation.59 Enlightened by these works, we intend to in vivo evaluate bone regeneration using electroactive composites as bone substitute with electro-stimulation. The New Zealand white rabbits were suffered to create bilateral critical-sized radius defects (20 mm, longer than that healing spontaneously) by surgery. The defects are clearly and the same with each other, which is benefit for comparing under the same condition. Scaffolds with and without electroactive component were stimulated by a pulsed electrical signal under 5 v at 100 Hz with 50% duty cycle for 30 minutes every other day. At 4 weeks post-surgery, few bone callus formation appeared in the blank group (Figure 10a-2). In the presence of implants, we could observe obvious bone callus and the formation of bone bridges (Figure 10b-2, c-2, d-2, e2). Compared to the scaffold of C3 with 10 wt % PAPn, scaffold C2 with 1 wt% PAPn exhibits better behaviors, such as distinct bone callus, bridging growth, vague borderlines between newly formed bone and the two defect ends and increased bone density (Figure 10d-2). At 10 weeks post-surgery, the densities of newly formed bone increased obviously for the groups with implants. In the blank group (Figure 10a-3), the bone callus grew creepingly. The fracture healing only formed in the end of bone leading to non-union repairs. For the other groups with different scaffolds, the density of bone-bridge increased obviously. The gap between the implants and the neighboring bone disappeared. Among them, the group of PLGA performed


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worse with lower bone density and less new bone formation. The radiograph of C1 only with opHA showed higher density, which indicated that op-HA could promote more rapid mineralization (Figure 10b-3). In the group of C2 with 1 wt% PAPn as electroactive materials, the bone bridges fused well with the bone fracture end and were smooth (Figure 10d-3). Moreover, clear outlines of cortex and bone marrow cavities could be observed. This phenomenon confirmed that electroactive material was more benefit for the mineralization and osteogenesis. It attributes to PAPn could reinforce the conductivity of extracellular signals. In contrast, C3 with 10 wt% PAPn was worse (Figure 10e-3). The density of newly formed bone was less. Not only the cortex was faint, but also it was noncontinuous. The osteoinductivity is correlated with the content of PAPn. To statically evaluate the healing degree, the Lane−Sandhu radiographic scoring system was introduced as shown in Figure S3. There was a statistically significant difference (p < 0.05) scores of C2 compared to that of C3 at 10 weeks after operation. However, no statistically significant difference was observed between the average score in the rest groups (p > 0.05).


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Figure 10. Under electricity stimulation, Representive digital radiographs (DR). blank control (a), PLGA (b),C1 (c), C2 (d) and C3 (e) at 0 weeks (a-1,b-1,c-1,d-1and e-1), 4 weeks (a-2,b-2,c2,d-2 and e-2) and 10 weeks (a-3, b-3,c-3, d-3 and e-3). 3.5 Implantation for Repair Tibia Defects of Sheep The bone defects of small animals, mouse or rabbit, are relatively less than that of demic bone. Furthermore, there is a different in light of the osteogenic potential. Therefore, some researches were carried out on man-sized animal models such as goat and sheep. Herein, the critically sized bone defects were created in the sheep models. The photographs of scaffold and the surgical procedure are shown in Figure 10. The steel plate with plastic property was firstly embedded in


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the outboard of tibia. Holes on the steel plates except for the fourth one were fixed with screw. Then, critically sized bone defects (25 mm) were created by sawing the periosteum and tibia between the third and the fifth screw (Figure 11a-1-4). Scaffolds with or without electroactive composition were implanted into the tibia defects of sheep. Bone substitutes with different components were implanted to the defects except for the blank group.


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Figure 11. Representive radiographic images of the sheep tibia defects (25 mm). Blank (a-1-4), C1 (b-1-4), C2 (c-1-4) and C3 (d-1-4) at 0 w, 4 w, 12 w and 24w.


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The typical radiographs at different intervals are displayed in Figure 11. Obvious bone growth only could be observed at 12 weeks. This is different from that of rabbits because of different animal species. Few bone calluses could be seen at the two end of defect segment in the blank group (Figure 11a-3). As for the C1 without electroactive composite, bone bridge could be observed, but it was noncontinuous (Figure 11b-3). The scaffolds of C2 and C3 with 1 wt% and 10 wt% PAPn, respectively exhibited better bone healing behaviors, which successfully bridged the segmental defect (Figure 11c-3 and d-3). At 24 weeks, the osseous callus became bigger, but the defect was still obvious for the blank group (Figure 11a-4). The ability of osteoinduction is related to the content of PAPn. Although bone bridge formed with C1 and C2 as scaffolds, nonunion healing of defect occurred in the defect segment (Figure 11b-4, d-4). In contrast, the group with C3 as the substitute showed the best osteointegration (Figure 11c-4). The x-ray density of neobone tissue area was similar to that of normal tibia. Furthermore, the bordelines are vague and bone bridge was continuous. The sheep with C3 scaffold fully recovered the wound and could move the operated limbs normally. The Lane−Sandhu scores was performed on the samples of blank and experimental groups as shown in Figure S4. At 12 weeks after operation, C2 with 1.0 wt% PAPn showed significant difference than C1 without any electroactive component. The C2 group exhibited better bone repair effect at 24 weeks. There was a statistically significant difference among the scores obtained from C2 group compared to blank and C3. Although without significant difference, the average score was higher than C1 groups. Electroactive composite PAPn plays an important role in the bone healing process.


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Figure 12. Micro-CT images of sheep tibia regeneration as followed C1, C2 and C3 as bone substitute (a) front view, (b) sectional view. Tibia specimens obtained at 24 weeks post-surgery were subjected to 3D CT reconstruction images. They were used to further assess the reconstruction of defects with C1, C2, and C3 as the substitute, respectively. The micro-CT results are coincident with that of radiographic findings. The bone volume fraction defined as the ratio of bone volume to the total volume of the defect (BV/TV) was measured using Medraw Print V1 software. The BV/TV value of C2 Group was 45.6%, which was higher than that of C1 group (41.4%) and C3 group (32.4%). Representative three dimensional reconstructions of each group are presented in Figure 12. We could observe that the defects were all bridged in all the groups investigated. But there are still small differences between them. As can be seen, the medullary cavity was incompletely filled using C1 as scaffold (Figure 12a). The specimen with C2 (Figure 12a) was the best, which showed more smooth surface then that of C3. The borderlines between newly formed bone and the two defect ends were invisible. Besides, the osteogenesis was eccentric, namely, the osteogenesis in the side far from the plates was more obvious than that was near the plates (Figure 12b).


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4．CONCLUSION Scaffolds based on PLGA, op-HA and PAPn were successfully fabricated by freeze-drying method. The properties and morphology of scaffolds were investigated, which exhibited good thermal stability, honeycomb-like and interconnected porous structures. The scaffolds all showed biodegradation, biocompatibility and osteoinductivity in vivo experiments. Furthermore, experiments for repairing the bone defects of rabbit and sheep demonstrated that electroactive composite PAPn plays an important role in the bone healing process. The osteoinductivity is correlated with the content of PAPn. Bone substitute with 1.0 wt% PAPn has more positive effect on the defect repairing process than that of scaffold with 10.0 wt% PAPn. We propose that positively charged surface provids bound areas for phosphate which is very important for nucleation mineralization. On the other hand, electroactive substrates could pinpointing the effect of electrical stimulation on the scaffold enhancing extracellular signal transduction and bone conductivity. This stimulus-responsive smart nanocomposite scaffold showed potential application in bone tissue engineering. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website …... Further experimental details and results, including the FT-IR spectra of the composites in Figure S1, DAPI staining of MC3T3-E1 cell on different scaffolds with and without electrical stimulation (ES) for in Figure S2. Lane−Sandhu radiographic scores of rabbit radius defects and sheep tibia defects in Figure S3 and S4, respectively.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. Author Contributions Xincui Shi and Haitao Wu contributed equally to this work and should be considered as the cofirst authors.The manuscript was written through contributions of all authors. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS Funding Sources This work was financially supported by the Program of Scientiﬁc Development of Jilin Province 20170520141JH, National Natural Science Foundation of China (Projects. 51473164 and 51673186), the joint funded program of Chinese Academy of Sciences and Japan Society for the Promotion of Science (GJHZ1519). and the Special Fund for Industrialization of Science and Technology Cooperation

between

Jilin

Province

and

Chinese

Academy of

Sciences (2017SYHZ0021), and Scientiﬁc Development of Jilin Province 20170520121JH.

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(57) Mokbel, N.; Naaman, N.; Nohra, J.; Badawi, N. Healing Patterns of Critical Size Bony Defects in Rats after Grafting with Bone Substitutes Soaked in Recombinant Human Bone Morphogenetic Protein-2: Histological and Histometric Evaluation. Brit. J. Oral. Max. Surg. 2013, 51, 545-549. (58) Inoue, N.; Ohnishi, I.; Chen, D. G.; Deitz, L. W.; Schwardt, J. D.; Chao, E. Y. S. Effect of Pulsed Electromagnetic Fields (Pemf) on Late-Phase Osteotomy Gap Healing in a Canine Tibial Model. J. Orthop. Res. 2002, 20, 1106-1114. (59) Yonemori, K.; Matsunaga, S.; Ishidou, Y.; Maeda, S.; Yoshida, H. Early Effects of Electrical Stimulation on Osteogenesis. Bone 1996, 19, 173-180.


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