PLGA scaffolds for enhanced vascularized bone formation

Journal Pre-proof Bilayer pifithrin-␣ loaded extracellular matrix/PLGA scaffolds for enhanced vascularized bone formation Xiaobo Xie, Wanshun Wang, Jing Cheng, Haifeng Liang, Zefeng Lin, Tao Zhang, Yao Lu, Qi Li

PII:

S0927-7765(20)30133-8

DOI:

https://doi.org/10.1016/j.colsurfb.2020.110903

Reference:

COLSUB 110903

To appear in:

Colloids and Surfaces B: Biointerfaces

Received Date:

22 October 2019

Revised Date:

3 February 2020

Accepted Date:

24 February 2020

Please cite this article as: Xie X, Wang W, Cheng J, Liang H, Lin Z, Zhang T, Lu Y, Li Q, Bilayer pifithrin-␣ loaded extracellular matrix/PLGA scaffolds for enhanced vascularized bone formation, Colloids and Surfaces B: Biointerfaces (2020), doi: https://doi.org/10.1016/j.colsurfb.2020.110903

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier.

Bilayer pifithrin-α loaded extracellular matrix/PLGA scaffolds for enhanced vascularized bone formation

Xiaobo Xie,a Wanshun Wang,b Jing Cheng,a Haifeng Liang,a Zefeng Lin,b Tao Zhang,b

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Yao Lu,a,b,c,* and Qi Li,a,*

Department of Orthopedics, Zhujiang Hospital, Southern Medical University, 253

Gongye Road, Guangzhou, Guangdong, P. R. China, 510282

Guangdong Key Lab of Orthopedic Technology and Implant Materials, Key

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b

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Laboratory of Trauma & Tissue Repair of Tropical Area of PLA, Hospital of Orthopedics, General Hospital of Southern Theater Command of PLA, 111 Liuhua

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Road, Guangzhou, Guangdong, P. R. China, 510010

Clinical Research Centre, Zhujiang Hospital, Southern Medical University, 253

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Gongye Road, Guangzhou, Guangdong, P. R. China, 510282

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* Corresponding author: [email protected] (Y. L), [email protected] (Q. L).

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Graphic Abstract

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Highlights

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1. Bilayer pifithrin-α loaded extracellular matrix/PLGA scaffold was prepared. 2. The SIS-ECM/PLGA/PFTα scaffold could promote osteogenesis in vitro.

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3. The SIS-ECM/PLGA/PFTα scaffold could enhance new bone formation in vivo.

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Abstract Small intestinal submucosa extracellular matrix (SIS-ECM) composite materials are catching eyes in tissue engineering but have been rarely studied in bone repair. In this study, we developed the unique bilayer bone scaffolds by assembling decellularized SIS-ECM and poly(lactic-co-glycolic acid) (PLGA) nanofibers through the

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electrospinning technique. To strengthen the bioactivity of the scaffolds, pifithrin-α (PFTα), a p53 inhibitor that can reduce the repressive function of p53 in osteogenesis,

was preloaded in the PLGA electrospinning solution. We found that the resultant SIS-

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ECM/PLGA/PFTα scaffolds exhibited porous morphology, good biocompatibility, and enhanced osteoinductivity. Specifically, the SIS-ECM/PLGA/PFTα scaffolds

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could promote the osteogenic differentiation and mineralization of the preosteoblasts MC3T3-E1 in a PFTα does dependent manner in vitro. Furthermore, the SIS-

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ECM/PLGA/PFTα scaffolds were better than the pure SIS-ECM and SIS-ECM/PLGA scaffolds in terms of vessel and new bone tissue formation after 4 weeks post-

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implantation in vivo. These overall findings indicated that the bilayer PFTα loaded SIS-ECM/PLGA scaffolds facilitated vascularized bone regeneration, showing

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promising potential for bone tissue engineering.

Keywords: Small intestinal submucosa extracellular matrix; PLGA; pifithrin-α; Composite scaffolds; Electrospinning; Bone tissue engineering

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1. Introduction

Small intestinal submucosa extracellular matrix (SIS-ECM) derived from porcine intestinal submucosal layer is a FDA-approved biomaterial that attracts growing interests in different researches of tissue engineering, such as skin injuries, vessel repair and hernia repair [1-4]. SIS-ECM mainly consists of glycoprotein, proteoglycan,

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glycosaminoglycan, and collagen, especially type I and type III collagen. Moreover, various cytokines including vascular endothelial growth factor (VEGF), transforming

growth factor-β (TGF-β), basic fibroblast growth factor (bFGF) are found residual in

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SIS-ECM even though the original sample is treated by multiple-step process [1, 5]. These cytokines can facilitate the recruitment of progenitor cells, promotion of

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differentiation, and ingrowth of vascular endothelial cells, and thus induce site-

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specific remodeling of damaged tissues [6]. In light of the bioactive properties, SISECM is considered as a desirable implanted material for tissue engineering [7].

are limited.

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However, as to the repair of bone defects, a global health event, studies of SIS-ECM

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Composite materials can obtain multifunction from the constituent materials with

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different physical or chemical properties [8-10]. Specifically, combination of synthetic and natural polymers could not only exhibit good biomechanical but also different biofunctions, favoring bone tissue engineering [11-13]. Synthetic polymers such as poly(lactic-co-glycolic acid) (PLGA), poly(ε -caprolactone) (PCL), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) can be used in bone repair application due to their biocompatibility, biochemical strength, and drug delivering 4

property [14, 15]. Very recently, Parmaksiz et al. have prepared a bone scaffold by assembling bovine SIS-ECM with synthetic PCL and hydroxyapatite microparticles, and found that this composite scaffold could promote the osteogenic differentiation of bone marrow mesenchymal stem cells [16]. However, the biofunctions of such kind of composite scaffolds are still needed to improve. Moreover, the in vivo osteoinductivity of these composite scaffolds remains unclear. Hence, the aim of this study is to develop a SIS-ECM/synthetic polymer scaffold with enhanced in vivo

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osteoinductivity.

Towards this goal, here we fabricated a unique bilayer bone scaffolds consisted of

SIS-ECM, PLGA and pifithrin-α (PFTα). PLGA was chosen because it can

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encapsulate biomolecules to enhance the bioactive of the scaffolds. Compared with

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PCL and PGA, PLGA shows excellent solubility and can be dissolved in common solvents to reduce toxicity. Moreover, PLGA offers better biological degradation and

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mechanical property of the bone scaffolds [17, 18]. On the other hand, PFTα is a chemical compound that suppresses p53 transcription to antagonize the repressive

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effects on bone remodeling [19-21]. In our approach, the SIS-ECM scaffolds were first obtained by typical decellularization and freeze-drying. Then the PFTα loaded

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PLGA nanofibers were electrospined on the surface of the SIS-ECM scaffolds (Figure 1a). We found the resultant scaffolds, termed SIS-ECM/PLGA/PFTα, had no

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cytotoxity and supported cell adhesion of preosteoblasts MC3T3-E1. Moreover, adding of PFTα into the scaffolds significantly enhanced osteogenic differentiation and mineralization of MC3T3-E1 cells. Most importantly, in vivo study showed that the SIS-ECM/PLGA/PFTα scaffolds remarkably promoted vascular bone formation as compared to the pure SIS-ECM and SIS-ECM/PLGA scaffolds at 4 weeks post5

implantation. In addition, the scaffolds exhibited good histocompatibility and did not caused adverse influences on the main organs after implantation. Therefore, the SISECM/PLGA/PFTα scaffolds with excellent biocompatibility and osteoinductivity can serve as potential filling biomaterials for bone tissue engineering.

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2. Materials and Methods

2.1. Preparation of SIS-ECM scaffolds

The SIS-ECM scaffolds were prepared by a classic method [22]. Briefly, intestines

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of pathogen-free pig were rinsed and cut into 10 cm sections, and immersed in an aqueous solution (0.5% peracetic acid and 10% ethanol) for 1 h. Then the sample was

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rinsed with phosphate buffered saline (PBS), scraped off the mucosa, muscle layer

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and serosal membrane, and rinsed with PBS to obtain intestinal submucosa. For decellularization, the submucosa was placed in a 0.5 M NaOH solution for 30 min at 4 °C to dissolve the cells, and then neutralized with PBS. The decellularized

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submucosa was placed in a mixed solution containing 40 U/mL trypsin and 20 U/mL a-galactosidase for 3 h at 37 °C to remove residual DNA and a-gal natural antigen

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components in order to reduce immunogenicity. Finally, the sample was immersed in

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cryoprotectant for 30 min and freeze-dried to form the SIS-ECM scaffolds. 2.2. Preparation of SIS-ECM/PLGA/PFTα Scaffolds

PLGA (LA/LG 85/15, Mw=200,000, 10% w/v, PURASORB PDLG8531, Purac, Netherlands) was dissolved in trifluoroethanol and stirred for 3 h at room temperature (RT) to obtain a homogeneous solution. Then PFTα (Macklin, China) with various 6

concentrations (20 and 40 μM, respectively) was added into the PLGA solution and stirred for 30 min. Then the SIS-ECM scaffolds were placed on a metal operation plate and the PLGA/PFTα solution was spinned on the surface of SIS-ECM scaffolds by an electrospinning machine (Tongli, TL-BM-300, China), with a high-voltage power of 15 kV, spinning rate of 3 mL/h, and the distance between the needle and the receiving SIS-EMC scaffolds was 15 cm. After spinning, the scaffolds were placed in a 25 °C vacuum oven for 3 d to obtain the final scaffolds, termed SIS-

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ECM/PLGA/PFTα 20 and SIS-ECM/PLGA/PFTα 40, respectively. The SISECM/PLGA scaffolds were fabricated in the same manner but in the absence of PTFα in the initial spinning solution.

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2.3. Characterization of the scaffolds

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The morphology of the as-prepared scaffolds was observed using scanning electron

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microscopy (SEM). The samples were dried under vacuum and coated with gold. Both sides of the samples were examined with SEM (S-3000N, Hitachi, Japan).

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2.4. Cell Culture

Preosteoblasts MC3T3-E1 (ATCC, CRL-12424) were used to test the

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biocompatibility and osteoinductivity of the scaffolds. Cells were cultured in α-

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Minimum Essential Medium (α-MEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) at 37 °C in a humidified atmosphere of 5% CO2. For osteoinductivity study, cells were cultured with osteogenic medium (α-MEM with 10% FBS, supplemented with 0.01 μM dexamethasone, 50 μg/mL ascorbic acid and 10 mM Na-β-glycerophosphate, Sigma, USA) at 37 °C, 5% CO2.

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2.5. Cell Proliferation

Cell Counting Kit-8 (CCK-8) assay was used to test the proliferation of MC3T3-E1 cells co-cultured with the scaffolds. Cell suspensions (5 × 103 cells per well) were seeded in the different scaffolds. At day 1, 3 and 5, CCK-8 solution (10%) was added into each well according to the manufacturer’s instructions and incubated for 2 h. Optical densities (ODs) at 450 nm was measured using a microplate reader (Multiskan

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GO, Thermo Scientific, USA).

2.6. Live/dead staining

Live/dead staining was also performed to determine the cytotoxicity of scaffolds.

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MC3T3-E1 cells were seeded in the scaffolds as described above. At day 1, 3 and 5,

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cells were washed with PBS and followed by staining with 1 μM calcein-AM (Beyotime Biotechnology, China) and 2 μM propidium iodine (PI) for 30 min. Then

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2.7. Hemolysis Test

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cells were observed by a fluorescence microscope (BX51, Olympus, Japan).

Healthy human blood containing EDTA was collected and diluted with PBS in a

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ratio of 4:5 by volume. The scaffolds were dipped in 5 mL PBS and incubated at 37 °C for 30 min. Then 0.2 mL diluted blood was added into the samples and co-

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incubated for additional 1 h. Then all samples were centrifuged at 3000 rpm for 5 min. The supernatant was collected and ODs at 545 nm were determined. PBS and deionized (DI) water were set as the negative control and positive control, respectively. The hemolysis ratio (HR) was calculated as followed:

HR = (ODsample - ODnegative control) / (ODpositive control - ODnegative control) × 100 8

2.8. Cell adhesion and morphology

MC3T3-E1 cells were co-cultured with the scaffolds for 24 h and then fixed with 2.5% glutaraldehyde overnight at 4 °C. The samples were dehydrated in a graded ethanol series (30%, 50%, 70%, 90% and 100%, respectively) and dried in hexamethyldisilazane. The samples were dried under vacuum, coated with gold and the cell adhesion and morphology were observed by SEM.

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2.9. Alkaline phosphatase (ALP) activity

ALP activity was tested to analyze the osteogenic differentiation of MC3T3-E1

cells in the scaffolds. Cell suspensions (5 × 103 cells per well) were co-cultured with

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the scaffolds using osteogenic medium. The osteogenic medium was half-renewed

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every 48 h. At day 7 and 14, cells in the scaffolds were washed twice with PBS and lysed using 0.2% Triton X-100. ALP concentration was determined by ALP detection

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kit (Beyotime Biotechnology, China) according the manufacturer’s instructions. The total intracellular protein was determined by the BCA protein assay kit (Pierce, USA).

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Finally, ALP activity was normalized to the corresponding total protein content.

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2.10. Alizarin red S staining

The mineralization of the MC3TC-E1 cells was observed by alizarin red S staining.

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After co-cultured for 14 days, the media were removed and cells were washed twice by PBS. The scaffolds and cells were fixed in 4% polyformaldehyde for 30 min at RT. The scaffolds and cells were then washed by PBS and stained with alizarin red S (Cyagen Biosciences, USA, pH=8.3) for 5 min. After frequently washing by PBS, the cells were observed using a microscope. 9

2.11. Ectopic Osteogenesis Rat Model The MC3T3-E1 cells (5 × 103) were seeded in the scaffolds and cultured with osteogenic medium for 7 days before implantation. A classic ectopic osteogenesis model was established [23, 24] and all animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of General Hospital of Southern Theater Command of PLA. Adult male Sprague-Dawley rats (250−300 g)

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were randomly divided into 4 groups (n=6). The rats were anesthetized with intraperitoneal injection of chloral hydrate and the cell-loaded scaffolds were implanted into the muscle pouch of the lateral thigh. The rats were sacrificed after 4

weeks of implantation, and then the scaffolds were extracted and fixed in 10%

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2.12 Histological Study

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paraffin for further examination.

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The samples were cut into 5-μm-thick sections and dyed with hematoxylin and eosin (H&E). Masson's trichrome staining was also performed to evaluate the

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formation of bone tissue in the scaffolds. The pathological sections were observed with microscope.

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2.13 Histocompatibility

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To study the histocompatibility of the scaffolds, main organs including heart, liver,

spleen, lung and kidney were collected after 4 weeks of implantation. The samples were dyed with H&E staining and observed using microscope.

2.14 Statistics

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Statistical analysis was performed by SPSS 22.0 software (IBM, USA). Statistical comparisons among groups were evaluated by one-way ANOVA followed by post hoc multiple comparison (LSD). A p-value less than 0.05 was considered statistically significant. All data were expressed as mean ± standard deviation.

3.1. Characterization of SIS-ECM/PLGA/PFTα Scaffolds

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3. Results and Discussion

We prepared primary SIS from porcine jejunum by mechanical abrasion,

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degreasing, decellularization, and freeze-drying. SEM images (Fig. 1b) showed that the SIS-ECM had a film-like structure with crisscross fibers arranged on the surfaces.

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Moreover, small pores were found in the scaffolds, which were in consistent with

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previous reports [22, 25]. Then we prepared the SIS-ECM/PLGA and SISECM/PLGA PFTα scaffolds using the electrospinning technique (Fig. 1a). As shown in Fig. 2b, the SIS-ECM layer of the hybrid scaffolds remained its typical morphology,

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suggesting that electrospinning of the PLGA had mild effects on the SIS-ECM layer. In addition, the electrospinning layer showed continuous nanofibrous morphology and

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the nanofibers did not contain beads. Highly porous and interconnected structure was

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also constructed by the nanofibers, enabling oxygen and nutrition diffusion for maintaining cell proliferation and viability [26-29].

3.2. In Vitro Biocompatibility and Cell Adhesion

Biocompatibility is the highest priority of bone scaffolds. SIS-ECM and PLGA have been well proved to be biocompatible [22, 30], but the adding of PFTα may 11

affects cell viability. We first investigate cytotoxicity and cells proliferation by introducing CCK-8 assay. As presented in Fig. 2a, the OD values at 450 nm among all scaffolds were similar without statistical differences at all-time intervals (day 1, 3 and 5), showing that the as-prepared scaffolds were not toxic towards preosteoblasts. Moreover, the OD values increased along with the culture time, suggesting that cells proliferated well in the scaffolds.

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To further confirm of biocompatibility of the scaffolds, live/dead staining was carried out. As shown in Fig. S1 (Supporting Information), cells in all scaffolds were alive (green fluorescence) at day 1 and 3, and only very few dead cells (red fluorescence) were observed at day 5. Meanwhile, it was obvious that the number of

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the cells in the scaffolds increased overtime, indicating that the scaffolds supported

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cell proliferation. These results were consistent with the CCK-8 assay.

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Hemolytic test was performed to study the blood compatibility of different scaffolds. The OD values at 545 nm of the positive control group (pure water) were significantly higher due to the destruction of erythrocytes and the release of heme (Fig.

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2b). In contrast, OD values of the scaffolds and the negative control group (PBS) were

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very low, demonstrating the scaffolds caused negligible influence on erythrocytes.

To investigate cell adhesion and morphology in the scaffolds, we seeded MC3T3-

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E1 cells in both sides of the scaffolds and cultured for 24 h, and then observed by SEM. scaffolds. Interestingly, we found that the cells adhered in the SIS-ECM layer rather than the PLGA layer (Fig. 3), which was probably due to the hydrophobicity of PLGA [31]. Additionally, SEM images showed that the cells grew well in all scaffolds. The cells had typical spindle morphology and stably attached to the 12

scaffolds by the extended pseudopods, indicating that the scaffolds were suitable for cell adhesion.

3.3. In Vitro Osteoinductivity

To evaluate the in vitro osteoinductivity of the scaffolds, ALP assay was performed and results were displayed in Fig. 4a. After 7 days of osteoinduction, the ALP activity level of cells in the pure SIS-ECM and SIS-ECM/PLGA scaffolds were similar,

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whereas it significantly enhanced in the SIS-ECM/PLAG/PFTα 20 and SISECM/PLAG/PFTα 40 scaffolds. ALP is a typical marker at the early stage of

osteogenesis and the beginning of osteogenic differentiation [32]. Hence, doping of

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PFTα in the scaffolds had promoted osteogenic differentiation of the preosteoblasts. Moreover, ALP levels significantly decreased and had no differences among all

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scaffolds began to mineralization [33].

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groups after 14 days of osteoinduction, suggesting that the preosteoblasts in the

Subsequently, we performed Alizarin red S staining to determine mineralization of

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the preosteoblasts. Alizarin red S is an indicator of mature osteocytes by dying the calcified nodules into dark red matter [34]. As presented in Fig. 4b, only few calcified

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nodules (blue arrows) were found in the cells of the pure SIS-ECM and SISECM/PLGA groups. In the contrary, more calcified nodules were observed in the SIS-

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ECM/PLAG/PFTα 20 scaffolds. Moreover, we found that the number of the calcified nodules increased in a PFTα does dependent manner. The calcified nodules were scattered throughout the SIS-ECM/PLAG/PFTα 40 scaffolds, indicating that PFTα loaded SIS-ECM/PLGA scaffolds could enhance mineralization of the preosteoblasts.

3.4. In Vivo Bone Formation Induced by SIS-ECM/PLGA/PFTα Scaffolds 13

To evaluate the in vivo osteoinductivity, we established an ectopic osteogenesis rat model and implanted the MC3T3-E1 cells loaded scaffolds into the model. After 4 weeks post-implantation, the samples were collected to evaluate the formation of new bone tissue. The H&E staining (Fig. 5a) illustrated that there were no inflammatory cells in all scaffolds, indicating that the implantation of scaffolds was safety and had not caused persistent inflammatory reaction or rejection. However, there were only some loose fibrous tissues in the SIS-ECM scaffolds, while new bone-like tissues

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were observed in the SIS-ECM/PLGA and SIS-ECM/PLGA/PFTα scaffolds. Furthermore, we found that there were many capillaries (red arrows) in SISECM/PLGA/PFTα 40 group, showing that the adding of PFTα in the scaffolds could

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promote vascularization.

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It is generally believed that p53 can inhibit angiogenesis by regulating the growth arrest, senescence and apoptosis of vascular smooth muscle cells (VSMCs) [35, 36].

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As a p53 inhibitor, PFTα could down-regulate p53 expression and contribute to the blood vessels formation. Vascularization plays an important role in bone formation

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[37], which generates vascular signals to participate in bone tissue growth and homeostasis regulation [38, 39]. Moreover, endothelial cells can regulate bone

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development and promote osteoblasts proliferation [40]. The formation of vascular networks is also conducive to bone regeneration because it provides oxygen and

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nutrients and remove metabolic waste [41, 42].

As expected, Masson's trichrome staining (Fig. 5b) showed that SIS-

ECM/PLGA/PFTα 40 group had the largest and darkest blue staining area (collagen, orange arrows), followed by SIS-ECM/PLGA/PFTα 20 and SIS-ECM/PLGA groups. Hence, these overall results indicated that the SIS-ECM/PLGA/PFTα 40 significantly 14

promoted vascularized new bone formation and exhibited the best osteoinductivity in vivo.

3.5. In Vivo Histocompatibility

Theoretically, SIS-ECM will not cause severe host immune response by the process of decellularization. However, decellularization cannot eliminate whole innate cells within SIS-ECM, and thus mild immune reaction does exist and mainly depends on

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the number of resident cells [43, 44]. Hence, we collected the main organs of the rats after 4 weeks implanted with SIS-ECM/PLGA/PFTα 40 scaffolds and performed

H&E staining to study the histocompatibility of the scaffolds. Overall, the main

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organs including heart, liver, spleen, lung and kidney showed no inflammatory and

histological abnormalities in the scaffold group as compared to the healthy control

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(Fig. S2, Supporting Information). Specifically, the myocardial fibers were normal

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columnar shape and deep stained moisturizing disk structure could be seen. The liver was lobular and the hepatocytes radially arranged around the central vein. The spleen section showed dense lymphoid tissue composed of lymphocytes. The alveolus

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pulmonis and bronchioles were complete in lung section. The kidney showed typical glomeruli and tubules. These above-mentioned results demonstrated that the SIS-

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ECM/PLGA/PFTα scaffolds had good histocompatibility in vivo.

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In addition to the promising advantages of the SIS-ECM/PLGA/PFTα scaffolds, the

detailed signaling and molecular mechanisms of the scaffolds involved in bone formation remain to be studied. Moreover, the in-situ bone repair efficiency of the scaffolds needs to be further studied in bone defect model, especially in critical-size long-bone defects. 15

4. Conclusion In conclusion, a novel bilayer SIS-ECM/PLGA/PTFα bone scaffolds were successfully fabricated. The in vitro results indicated that the as-prepared scaffolds could support the proliferation and adhesion of preosteoblasts, and significantly enhanced cell osteogenic differentiation and mineralization. Furthermore, the SIS-

promoted

vascularized

bone

formation

in

vivo.

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ECM/PLGA/PTFα scaffolds not only exhibited good biocompatibility, but also Consequently,

the

SIS-

Conflicts of interest

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There are no conflicts to declare.

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ECM/PLGA/PTFα scaffolds have a potential application in bone tissue engineering.

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Author contributions

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Xiaobo Xie, Yao Lu and Qi Li conceived and designed the experiments. Xiaobo Xie and Wanshun Wang prepared the materials and performed the characterization. Wanshun Wang, Jing Cheng and Haifeng Liang performed the in vitro experiments. Xiaobo Xie, Zefeng Lin and Tao Zhang performed the in vivo studies. The manuscript was drafted by Xiaobo Xie and Yao Lu. All authors read and approved the manuscript.

Acknowledgements 16

This study was supported by National Natural Science Foundation of China (81902198),

China

Postdoctoral

Innovation

Talent

Supporting

Program

(BX20190150), China Postdoctoral Science Foundation (2019M662980), President Foundation of Zhujiang Hospital, Southern Medical University (yzjj2018rc09), Scientific Research Foundation of Southern Medical University (C1051353, PY2018N060), Science and Technology Planning Project of Guangdong Province (2014A020215025, 2017B030314139) and Medical Research

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Foundation of Guangdong Province (A2019228).

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Appendix A. Supplementary data

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Supplementary material related to this article can be found, in the online version.

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Figure 1. Preparation and characterization of the scaffolds. (a) Schematic illustration of the preparation and application of the SIS-ECM/PLGA/PTFα scaffolds. (b) SEM images of the scaffolds.

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Figure 2. Biocompatibility of the scaffolds. (a) Cell proliferation of MC3T3-E1 in the

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scaffolds at day 1, 3 and 5 measured by CCK-8 assay. (b) Hemolytic test of the

scaffolds. PBS and DI water were set as the negative control and positive control,

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respectively. Data were represented as mean ± standard deviation (n = 3); *p < 0.05,

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**p < 0.01.

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Figure 3. Cell adhesion. Morphology of MC3T3-E1 cells (blue arrows) in the

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scaffolds after 1 day of co-culture observed by SEM. The results showed that cells

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only adhered in the SIS-ECM layer and had no difference among all groups.

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Figure 4. In vitro osteogenic differentiation and mineralization. (a) ALP activity of

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MC3T3-E1 cells in different scaffolds at day 7 and 14. The ALP levels were

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significantly higher in the SIS-ECM/PLGA/PFTα 20 and SIS-ECM/PLGA/PFTα 40 scaffolds at day 7. (b) Alizarin red S staining of the MC3TC-E1 cells after co-cultured with different scaffolds for 14 days. More calcified nodules (blue arrows) were observed in the SIS-ECM/PLGA/PFTα 40 scaffolds. Data were represented as mean ± standard deviation (n = 3); *p < 0.05, **p < 0.01.

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Figure 5. In vivo osteoinductivity. (a) H&E staining of the scaffolds. Vessel formation (red arrows) was only observed in the SIS-ECM/PLGA/PFTα 40 scaffolds. (b) Masson's trichrome staining of the scaffolds. More collagen (orange arrows) was found in the SIS-ECM/PLGA/PFTα 40 scaffolds.

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