Enhanced Osteogenesis of ADSCs by the Synergistic Effect of Aligned

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The enhanced osteogenesis of ADSCs by the synergistic effect of aligned fibers containing collagen I HanBang Chen, YunZhu Qian, Yang Xia, Gang Chen, Yun Dai, Na Li, FeiMin Zhang, and Ning Gu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b08791 • Publication Date (Web): 13 Oct 2016 Downloaded from http://pubs.acs.org on October 14, 2016

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ACS Applied Materials & Interfaces

The Enhanced Osteogenesis of ADSCs by the Synergistic Effect of Aligned Fibers Containing Collagen I Hanbang Chen1, Yunzhu Qian1, 2, Yang Xia1,3, Gang Chen1, Yun Dai4, Na Li1, Feimin Zhang1, 5*, Ning Gu5 1 Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, 210029, China

2 Center of Stomatology, the Second Affiliated Hospital of Soochow University, Suzhou, 215004, China

3 State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory of Biomaterials and Devices, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210009, China.

4 Department of Prosthodontics, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, 210008, China.

5 Suzhou Key Laboratory of Biomaterials and Technologies & Collaborative Innovation Center of Suzhou Nano Science and Technology, Suzhou, 215123, China

*Corresponding author:

Feimin Zhang

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Email: [email protected]

Tel: 86-13801589779

Fax: 86-25-86516414

ABSTRACT

The topographical features and material composition of scaffolds have a powerful influence on cell behaviors such as proliferation and differentiation. Here, scaffolds consisting of aligned fibers with incorporated bioactive collagen I were tested for their ability to enhance osteogenesis in vitro. Rat adipose-derived mesenchymal stem cells (ADSCs) were seeded on the scaffolds and their morphology, proliferation and osteogenic differentiation were examined. Aligned scaffolds with collagen I showed the best osteogenic properties. Also, adhesion-related genes showed the higher


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expression on aligned scaffolds with collagen I. Our findings indicate that fiber alignment combined with incorporation of collagen I increases the capacity of electrospun scaffolds to induce enhanced and directed osteogenesis. Such scaffolds may, therefore, have potential for improving guided oral bone regeneration.

Keywords: scaffold, aligned fibers, collagen, guided bone regeneration, osteogenesis, stem cell



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INTRODUCTION

Effective reconstruction of bone lost by tumors, trauma and periodontal disease is becoming a major challenge in dentistry and maxillofacial surgery.1-5 Bone reconstruction generally requires fabrication of bio-artificial bone grafts that mimic extracellular matrix (ECM) to improve growth and mineralization of nascent bone.

Electrospinning is a popular technique to reproduce fibrous scaffolds with nano- and micro-scale fiber diameter. Electrospun fibers are widely used in skin repair, wound healing and bone regeneration. Key reasons for its popularity and promise in tissue engineering include its technical simplicity and ability to mimic the features of natural ECM and nutrient transport.6 Fibrous scaffolds with different topographical features can be easily obtained by regulating fiber diameter, alignment, distribution and interfiber distance. 7 Biomaterial topography has a great influence on cell biology, 8 with numerous studies showing that cells are able to sense microscale and nanoscale structural features through the mechanism of contact guidance.9,

10

The topography of electrospun

materials has been reported to regulate the spreading, migration, proliferation and differentiation of cells. Different fiber diameters and alignment have direct effects on cellular function and behavior, 11 such as migration and differentiation.12-14 Fibroblasts, Schwann cells, osteoblasts and mesenchymal stem cells (MSCs) have all been reported to exhibit different morphologies on random versus aligned fibers.15-18


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Another feature affecting cell-biomaterial interaction is the composition of the material. Synthetic and natural polymers are most widely used for bone tissue engineering. Synthetic polymers have superior mechanical properties and controllable degradability, whereas natural polymers are reported to be bioactive and may perform specific biofunctions that facilitate tissue engineering.19,20

The excellent cell compatibility and biodegradability of polylactide-co-glycolide (PLGA) and poly-caprolactone (PCL) make them appropriate candidate scaffold materials for tissue engineering.21 Type I collagen (COL1), which is the most important structural protein of natural bone, and also its main organic component, has been extensively investigated and found to be an excellent facilitator of cell attachment, proliferation and differentiation.22 The low immunogenicity and nanofibrillar structure of scaffolds produced from a combination of these polymers and COL1 could provide an ideal microenvironment for cell proliferation and formation of new bone. This makes COL1 one of the most commonly used matrices in bone-tissue engineering.23 COL1-hybrid scaffolds, which mimic the extracellular matrix (ECM) and substitute for natural bone, have demonstrated promise as a platform for bone regeneration.

Adipose-derived mesenchymal stem cells (ADSCs) are becoming an alternative to bone-marrow derived mesenchymal stem cells (BMSCs), especially in clinical use where large cell numbers are required for new bone regeneration. Because they possess a similar immune phenotype, multi-lineage potential and transcriptome.24 Moreover, ADSCs can serve as an abundant source of autologous cells that produce



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lower donor morbidity upon extraction.25 All this makes them highly suitable for tissue engineering, and thus, also for the present study.

Numerous randomly oriented scaffolds have been used for bone reconstruction. Comparatively, aligned fibrous scaffolds are less frequently examined, which is surprising, considering the fact that bone is largely composed of parallel collagen fibers.26 Osteon lamellae are the basic building unit of cortical lamellar bone. The parallel and layered structure is composed of a collagen fibril framework and embedded with mineralized extra-fibrillar matrix and mineral platelets. 27Osteonsare tubular motifs formed by concentrically arranged lamellae around a Haversian canal. 28

Moreover, alignment of the osteon longitudinal-axes parallel to the bone long axis

increases the resistance of the bone to axial forces. Thus, we hypothesized that if bone regeneration was guided along the force direction, improved reconstruction of oral bone could be achieved.

Chen et al observed that osteogenesis was increased in pre-osteoblasts on aligned PCL/COL fibers, 29 compared with those on random ones; however, they ignored the effects of scaffold composition. In this study, aligned and randomly oriented scaffolds were prepared by electrospinning from a PLGA/PCL solution (1:1) with or without COL1. The effect of fiber orientation and collagen addition on osteogenic differentiation of ADSCs was examined to evaluate the potential use of aligned collagen-containing scaffolds in oral bone regeneration.


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EXPERIMENTAL SECTION

Preparation of Electrospun Fibers

PLGA (75:25) with an average molecular weight (Mw) of 12 kDa (Daigang Biomaterial, Jinan, China), and PCL with an average molecular weight of 11 kDa (Daigang Biomaterial, Jinan, China) were dissolved at a 1:1 ratio (0.75g of each) by overnight stirring in 8.5g of 2,2,2-Trifluoroethanol (TFEA, Aladdin, Shanghai, China) to obtain a 15% (w/w) solution. COL1 was extracted from rat tails and dissolved in acetic acid, as described by Rajan et al.30 The resulting COL1 solution (4.2 mg/ml) was mixed into the PLGA/PCL solution at 1:10.

Four types of electrospun fiber were made: random and aligned PLGA/PCL fibers (RPP, APP), and random and aligned COL1/PLGA/PCL fibers (RCPP, ACPP). For electrospinning, the polymer solution was delivered to a rotating cylindrical collector (10 cm in diameter) by a syringe pump with a metal needle (internal diameter, ID = 0.40 mm; Jianpai, Jintan, China) connected to a high-voltage power supply (12–12.5 kV), which created an electric field (60–63 kV/m) between the needle and the collector; the distance between these was 20 cm for all samples. Other electrospinning parameters are described in Table 1.

All the scaffolds were prepared at a thickness of 0.20 mm. For in vitro cell culture tests, the scaffolds were thoroughly dried in a vacuum freeze-drier (Huaxing Technology Develop Co Ltd, Beijing, China) for 48 h, sterilized with 100 IU/mL penicillin-PBS for 2 h, and washed 3 times with PBS at room temperature.


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Table 1

Scaffold

Polymers

Solvent

Rotating

Voltage

speed(rpm)

Polymer flow

Humidity

(ml/h) (kV)

(%)

RPP

PLGA+PCL

TEFA

300

12.5

0.4

50

APP

PLGA+PCL

TEFA

3000

12

0.4

50

RCPP

COL+PLGA+PCL

TEFA

300

12

0.5

55

ACPP

COL+PLGA+PCL

TEFA

3000

12

0.5

50

Characterization of Scaffold Morphology The fiber morphology of the four samples (RPP, APP, RCPP and ACPP) was observed by Field Emission Scanning Electron Microscopy (FESEM; LEO 1530VP, Oberkochen, Germany). All samples were cut with a sharp knife, thoroughly freeze-dried, installed onto imaging stabs and sputter-coated with gold for 30 s (Desk II, Denton Vacuum, Moorestown, NJ, USA). Images were taken at a working voltage of 2.00–5.00 kV. Image-Pro Plus 6.0 (IPP, Media Cybernetics, Rockville, MD, USA) software was used to calculate the fiber diameter and analyze the orientation of the fibers.



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Attenuated Total Reflectance Fourier Transform Infrared Spectroscopic (ATR-FTIR) Analysis Five samples were prepared for ATR-FTIR (NICOLET6700FT-IR, Thermo Scientific, Texas, USA) examination: pure PLGA, RCPP, RPP, APP and ACPP.

COL1 Immunofluorescence. The samples were washed three times with PBS and fixed with 3.7% paraformaldehyde for 15 min at room temperature. Paraformaldehyde was removed by washing twice with PBS. The samples were then permeated in 0.5% Triton X-100 in PBS for 3 min and blocked with 3% rabbit serum albumin (BSA) for 30 min, prior to incubation with the primary antibody (Collagen Type I, Abcam plc) (1:50 dilution)for overnight. The samples were further maintained with the secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (Beyotime, Shanghai, China) (1:200 dilution) for 1 h at room temperature. Images were taken with a Zeiss CLSM 710 microscope.

Cell Culture and Cell Seeding OriCellTM Sprague–Dawley (SD) rat adipose-derived mesenchymal stem cells (ADSCs; Passage [P] 2), and OriCellTM SD ADSCs growth medium were purchased from Cyagen Biosciences (Guangzhou, China). The culture medium was supplemented with 10-7-M dexamethasone, 50-µM ascorbate-2-phosphate and 10-mM


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β-glycerol phosphate (Sigma-Aldrich, St. Louis, MO, USA) to make osteogenic medium. The ADSCs were cultured in a 5% CO2 atmosphere at 37°C. Adherent cells were passaged at confluency of approximately 80%. P 3–5 cells were used in our experiments. For proliferation examination, the cells were seeded at 5×103 cells/sample (6 mm in diameter) and cultured in OriCellTM SD ADSCs growth medium in a 96-well plate. At day 1, 3, 5 and 7, cell number was measured using cell counting kit-8 (CCK-8, Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, after 1 h in 10% kit medium, the absorbance at 450 nm was measured on a plate reader. The average result expressed as relative fluorescence units (RFUs) was taken for each sample of five replications. For differentiation assessment, the cells were seeded at 1.5×104 cells/sample (14 mm in diameter) in osteogenic medium in 6-well plates. The scaffolds with seeded cell were cultured in an incubator for 2 h at 37°C and 5% CO2 to allow cell attachment, and then 2.0 ml of culture medium was added to each well. The medium was replaced and supplemented every second day. The cells were cultured for up to 2 weeks on scaffolds, according to the requirements of the experiment.

Cell Morphology and Distribution

The morphology and distribution of ADSCs on the scaffolds were observed by FESEM and confocal laser scanning microscopy (CLSM; Zeiss-LSM510, Carl Zeiss,



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Oberkochen, Germany). The methods were reported in our previous work.31

Cell morphology of adhesion and spreading was observed and analyzed after culturing for 3 days.

Real-time (quantitative) Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Analysis of Osteogenic Markers and Adhesion-related Genes. The method of qRT-PCR was similar to our previous report.32 To be brief, total cellular RNA was obtained with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The concentrations and purity of the RNA samples were calculated with NanoVue Plus spectrophotometer (GE Healthcare, Piscataway, NJ, USA). Reverse transcription reactions were performed with 1000 ng total RNA of each sample, using PrimeScript RT reagent kit (TaKaRa Bio Co., Ltd., Otsu, Japan). The primer sequences are given in Supporting Information. The data represents the average of three independent experiments.

Statistical Analysis Quantitative data were given as the mean ± the standard deviation (SD). SPSS ver17.0 (SPSS, Chicago, IL, USA)was used for data analyses. Repeated measures analysis of variance (rANOVA) was used for the proliferation (CCK-8) and qRT-PCR results. Conventional analysis of variance (ANOVA) was chosen for contact angle, tensile strength and fiber-diameter measurements. The threshold for significance was set at


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p 0.05).



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In our experience, fiber diameter is mainly related to the electrospinning parameters, including voltage, collection distance, solution concentration and feeding speed. The above results demonstrate this, with similar processing parameters leading to similar fiber diameters (Table 1). Although the addition of COL1 solution did modify the polymer concentrations, the change was evidently small enough not to have an effect on fiber diameter. The hydrophilic and tensile properties were shown in Supporting Information.

ATR-FTIR Analysis of Electrospun PLGA, PLGA/PCL and COL1/PLGA/PCL Scaffolds and COL1 Immunofluorescening

Figure

2.

ATR-FTIR

spectra

of

electrospun

PLGA,

PLGA/PCL

and

COL1/PLGA/PCL scaffolds (A); COL1 immunofluorescening of RCPP and ACPP: green (B and C).

Typical spectra of electrospun PLGA, PLGA/PCL and COL1/PLGA/PCL are shown in Figure 2A. Compared with PLGA, PLGA/PCL composites showed a peak at 1721


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cm-1, representing the typical stretching of the free-bonded urethane C=O group found in PCL. The peaks ascribing to amide I (N-H2 stretching at 1650 cm-1) and amide II (N-H stretching at 1550 cm-1) are clearly present in the spectrum of electrospun COL1/PLGA/PCL (RCPP and ACPP). And the presence of a broad peak around 3320 cm-1 is corresponded to the vibration of the hydroxyl (O-H) and amide (-NH, -NH2) groups in COL1. Combined, these results indicate the presence of COL1 in the RCPP and ACPP scaffolds.

The immunofluorescence analysis shown in Figure 2B-C indicate the uniform distribution of COL on the surface of the fibers. The local existence of COL may indicate the interaction between scaffolds and cells after seeding.

Stem Cell Morphology on Scaffolds



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Figure 3.Representative CLSM images of ADSCs on scaffolds after 1 day: RPP (A, E); RCPP (B, F); APP (C, G); ACPP (D, H). Actin is stained red and nuclei are stained blue; Representative SEM images of ADSCs on scaffolds after 1 day: RPP (I); RCPP (J); APP (K); ACPP (L).

As seen in the CLSM images (Figure 3 A-H), ADSCs on the RPP and RCPP scaffolds exhibited wide and randomly oriented spreading after 1 day, whereas cells on APP and ACPP scaffolds were directionally-oriented and displayed a bipolar morphology with oval nuclei along the dominant direction of the fibers. No obvious morphological differences were observed between the two random scaffolds or between the two aligned scaffolds, indicating that cell morphology was not influenced by the addition


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of COL1. However, fiber alignment did have an influence on cell morphology.

Prabhakaran et al used CLSM to generate 3D images showing the morphology of the cells that infiltrated the scaffold bulk. The cells maintained their directed and elongated morphology after infiltration of aligned electrospun scaffolds. We were unable to perform 3D reconstruction due to the limitations of our microscope; however, we plan to do this in the near future and expect to observe a similar phenomenon.

SEM imaging (Figure 3 I-L) showed phenomena similar to those above. For all scaffolds, ADSCs adhered and grew well on the scaffolds, and interaction and association between cells and surrounding fibers were observed. ADSCs seeded on randomly oriented scaffolds exhibited multipolar phenotype with flatter cell bodies, while cells on aligned scaffolds presented an extended, bipolar morphology.

Both CLSM and SEM demonstrated the relationship between cell morphology and fiber orientation, with cell orientation and migration corresponding to the specific topography of the substrate through adhesion-cytoskeleton interactions. Randomly oriented fibers provide multiple focal adhesion sites while the aligned fibers guide cell growth in a parallel direction. Contact guidance33 is the most likely explanation for the tendency of the ADSCs to grow along the circumferential direction of the parallel fibers, leading to enhanced cellular alignment in the direction of the fibers. Furthermore, on the aligned scaffolds, it could be seen that some filamentous processes extended out from the ADSCs and penetrated into the surface layer of the scaffold (between fibers). This phenomenon was less frequent on the randomly



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oriented scaffolds. Cell infiltration (into the scaffold bulk), which is essential for a range of tissue-engineering applications, especially for bone tissue engineering, could be observed clearly in both random and aligned scaffolds. Pore size and pore interconnectivity are crucial for cell infiltration. Furthermore, they allow rapid diffusion of nutrients, metabolites and waste products, which is particularly important for cell proliferation and bone regeneration.34

Stem-cell Proliferation and Differentiation Tests

Stem-cell Proliferation Tests

Figure 4. A. Proliferation of ADSCs on scaffolds measured using CCK-8 assays after 1, 3, 5 and 7 days; B. Flow cytometric analysis of the cell cycle distribution among cells seeded on different scaffolds at day 7 (n = 3). (S + G2/M) % means proliferation index. *, p< 0.05 vs RPP; &, p< 0.05 vs RCPP; #, p< 0.05 vs APP.


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Proliferation of stem cells (ADSCs) cultured on scaffolds was assessed by optical density (OD) using a CCK-8 kit, and the results are shown in Figure 5A. Cell numbers were increased from days 1 to 7 in all groups. However, no significant differences were observed between the four scaffold types after 1 d (p>0.05). Cells proliferated slowly in the first 3 days, but the rate increased after this. Cell density on the APP scaffold was significantly lower than that on the other three scaffolds at day 5 and 7. ADSCs proliferated well over the 7 days on both random and aligned scaffolds. The biological properties of electrospun scaffolds are highly dependent on fiber diameter and pore size, which affect transport of nutrients and metabolic products during cell growth.35 Random scaffolds, with their interwoven structure, have more pores than aligned scaffolds, since the latter exhibit a dense structure. The highly porous random scaffolds offer a more favorable environment for the growth and proliferation of ADSCs. Thus, cell proliferation in the RPP group was greater than that for APP.

On day 3 and 7, ODs for the collagen-incorporated scaffolds were significantly higher than those for the corresponding collagen-free scaffolds (p

Enhanced Osteogenesis of ADSCs by the Synergistic Effect of Aligned

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