poly(lactic‑co‑glycolic acid) enhances anti-inflammatory effect

Accepted Manuscript Composite scaffold of micronized porcine cartilage/poly(lacticco-glycolic acid) enhances anti-inflammatory effect

Soomin Kim, Ji Eun Jang, Ju Hee Lee, Gilson Khang PII: DOI: Reference:

S0928-4931(17)32464-5 doi:10.1016/j.msec.2018.02.020 MSC 8413

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

28 June 2017 16 October 2017 22 February 2018

Please cite this article as: Soomin Kim, Ji Eun Jang, Ju Hee Lee, Gilson Khang , Composite scaffold of micronized porcine cartilage/poly(lactic-co-glycolic acid) enhances anti-inflammatory effect. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi:10.1016/ j.msec.2018.02.020

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Composite scaffold of micronized porcine cartilage/ poly(lactic-co-glycolic acid) enhances anti-inflammatory effect

Department of Dermatology, Severance Hospital, Cutaneous Biology Research Institute,

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Soomin Kima,†, Ji Eun Janga,†, Ju Hee Leea,*, and Gilson Khangb,*

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Yonsei University College of Medicine, 03722, Seoul, Republic of Korea Department of BIN Fusion Technology, Department of Polymer Nano Science &

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Technology and Polymer BIN Research Center, Chonbuk National University, Deokjin-gu,

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Jeonju 54896, Republic of Korea

*Corresponding authors:

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Ju Hee Lee

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Department of Dermatology, Severance Hospital, Cutaneous Biology Research Institute, Yonsei University College of Medicine, 03722, Seoul, Republic of Korea

Gilson Khang

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E-mail: [email protected]; Tel: +82-2-2228-2080

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Department of BIN Fusion Technology, Department of Polymer Nano Science & Technology and Polymer BIN Research Center, Chonbuk National University, Deokjin-gu, Jeonju 54896, Republic of Korea E-mail: [email protected]; Tel: +82-63-270-2848

†

These authors contributed equally to this work.

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Abstract The main disadvantage of using poly(lactic-co-glycolic acid) (PLGA), a typical synthetic polymer, as a biomaterial is that it induces inflammation. To overcome this disadvantage, we determined the ability of micronized porcine cartilage (MPC) for alleviating the

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inflammatory effects of a PLGA scaffold. MPC was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and Fourier transform-infrared spectroscopy, and typical

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collagen components were confirmed. The MPC/PLGA scaffolds were fabricated using

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various concentrations of MPC and the compressive strength was evaluated to characterize its physical properties. Although the compressive strength decreased with increasing amounts of

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MPC, the roughness of the surface, assessed by scanning election microscopy, was

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considered to be suitable for facilitating cell attachment. Notably, in vitro experiments showed that the cell adhesion and proliferation rates increased as the MPC content increased.

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MPC further reduced gene expression levels of inflammatory cytokines and cellular reactive

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oxygen species, as determined by real time-polymerase chain reaction and fluorescenceactivated cell sorting, respectively. In addition, in vivo experiments confirmed the interaction between tissues and the scaffolds. Overall, these results confirmed that the MPC/PLGA

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scaffold is superior to the PLGA scaffold in many respects and might be a suitable candidate

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for resolving the disadvantages of PLGA in tissue engineering applications.

Keywords: collagen; micronized porcine cartilage; poly(lactic-co-glycolic acid); proliferation; scaffold; tissue engineering.

Abbreviations: COX-2, cyclooxygenase-2; DCFH-DA, dichlorodihydrofluorescein diacetate; 2

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FACS, fluorescence-activated cell sorting; FT-IR, Fourier transform-infrared; H&E, hematoxylin & eosin; LPS, lipopolysaccharide; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide; MPC, micronized porcine cartilage; PBS, phosphate-buffered saline; PLGA, poly(lactic-co-glycolic acid); ROS, reactive oxygen species; SDS-PAGE,

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sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RT-PCR, real time-polymerase

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chain reaction; TNF-, tumor necrosis factor-alpha.

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1. Introduction Biomaterials are materials that can be used in close contact with living organisms, without harmful effects [1]. Therefore, biocompatibility is an indispensable property for the development of biomaterials [2], including lack of side effects such as exothermic reactions,

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inflammatory responses, immune responses (antigenic), toxicity, and carcinogenicity, once transplanted into a living body [3]. The inflammatory response is a particularly important

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factor that determines the success of biomaterial transplantation in the body [4]. Therefore,

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biomaterials should have both excellent biocompatibility and bioactivity to minimize inflammation after implantation [5,6].

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Biomaterials can generally be divided into two major categories: synthetic polymers and

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biological polymers. Poly(lactic-co-glycolic acid) (PLGA), a polylactide and polyglycolide copolymer, is a typical synthetic polymer that shows excellent biodegradability,

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bioabsorbability, and physical properties, making this material particularly useful for

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development of devices in tissue engineering [7,8]. However, PLGA has also been reported to cause inflammatory reactions in surrounding tissues due to the degradation products released during the decomposition process [9]. In addition, owing to its hydrophobic properties,

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nutrient exchange is difficult for PLGA scaffolds, thereby limiting the nutritional supply to

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cells and tissues [10]. Accordingly, the use of natural polymers containing various cytokines might be useful to improve the application of PLGA in tissue engineering [11,12]. Since natural polymers have good biocompatibility, they induce a low inflammatory response after implantation in the body. In addition, they have excellent biodegradability and biofunctional properties and are thus ideal biomaterials for applications in the living body. Collagen is one of the most commonly used natural biomaterials, which accounts for 4

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approximately 30% of all proteins that make up the human body [13]. There are 27 known types of collagen in the body [14], and cartilage is composed of more than 50% collagen type II. In this study, we used collagen as a natural polymer to determine its potential for alleviating

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the inflammatory reaction generated by implantation of a PLGA scaffold. Collagen was obtained from porcine cartilage, which is easily collected as a porcine by-product. The

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porcine cartilage was prepared in powder form and analyzed by sodium dodecyl sulfate-

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polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform-infrared spectroscopy (FT-IR). After confirming that it contained collagen, the micronized porcine cartilage (MPC)

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was used for subsequent analyses to determine its ability for resolving the disadvantages of

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PLGA described above with a particular focus on its anti-inflammatory effect. Inflammation is well known to be induced by necroptosis, and reactive oxygen species (ROS)

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play key roles as activators of necroptosis [15]. Therefore, to elucidate the mechanism

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underlying the anti-inflammatory effects of MPC, ROS production was examined and the expression levels of representative genes related to inflammation such as cyclooxygenase-2 (COX-2) and tumor necrosis factor-alpha (TNF-α) were determined [16,17]. Moreover, we

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sought to determine the optimal amount of MPC to combine with PLGA for minimizing

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inflammatory reactions while improving cell adhesion and proliferation.

2. Materials and methods 2.1. Fabrication of scaffolds PLGA and MPC/PLGA micro-porous three-dimensional scaffolds were fabricated by the solvent-casting/salt-leaching technique using methylene chloride as a solvent (Baker 5

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Analyzed® A.C.S Reagent; JT Baker, Selangor, Malaysia). In brief, the mixture consisted of 1 g of PLGA (molecular weight 90,000 g/mol, 75:25 molar ratio of lactide:glycolide; Resomer® RG 756, Boehringer Ingelheim, Ingelheim am Rhein, Germany); 0.1, 0.2, 0.4, or 0.8 g of MPC (kindly provided by Professor B.H. Min, University of Ajou College of

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Medicine [18]); and 9 g of sieved sodium chloride (NaCl; Orient Chemical Co., Ansan, Korea) particles (180–250 µm) dissolved in 4 ml of methylene chloride (Tedia Co. Inc., Phillipsburg,

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NJ, USA). The blended solution was transferred to cylindrical silicone molds (7 mm in

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diameter and 3 mm in thickness) and pressure was applied (60 kgf/cm2) using a press machine (MH-50Y, CAP 50 tons, Masada, Tokyo, Japan) for 1 d at room temperature. To

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make pores, the scaffolds were immersed in distilled water for 48 h, replaced with new water

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every 6 h, washed with ethanol, and freeze-dried for 24 h using a freeze-dryer (IlShin Lab Co.

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Ltd., Gyeonggi-do, South Korea).

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2.2. SDS-PAGE

SDS-PAGE was performed to determine the molecular weight of MPC according to the Laemmli method [19]. MPC (25 µg and 50 µg) was loaded on a 7.5% polyacrylamide gel at

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30 mA/gel. The molecular weight of MPC was analyzed using molecular weight standards

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(Bio-Rad, Hercules, CA, USA). The transferred protein gel was stained with Coomassie Brilliant Blue R250 solution for detection.

2.3. Cell culture NIH/3T3 cells (mouse embryo fibroblasts, KCLB216480) and a mouse leukemic monocyte macrophage cell line (RAW 264.7, KCLB40071) were purchased from the Korean Cell Line 6

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Bank (KCLB, Seoul, Korea). Both cell types were used between passages 3 and 6 for the experiments. NIH/3T3 cells were cultured with RPMI 1640 medium (Lonza, Walkersville, MD, USA) containing 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA) and 1% penicillin/streptomycin (Gibco) at 5% CO2 and 37 °C in a humidified incubator. RAW 264.7

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cells were cultured with Dulbecco’s modified Eagle medium (Lonza) under the same conditions used for the culture of NIH/3T3 cells. At 80% confluence, the cells were sub-

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cultured using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA; Gibco). The collected

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cells were centrifuged at 1200 rpm for 3 min and re-suspended in new medium. Before seeding, the scaffolds were sterilized by immersion in 70% ethanol for 30 min and

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washed with phosphate-buffered saline (PBS) three times. The scaffolds were immersed in

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cell culture medium for 30 min and transferred to a 24-well plate. For cell culture on the scaffold, cells were counted using a hemocytometer. The cells (NIH/3T3: 1 × 105, RAW

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264.7: 5 × 105) were seeded on each scaffold at 100 µl. After 1 h, 900 µl of cell culture

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medium was added. During the experiment, the medium was replaced every 3 days. All processes were performed on an aseptic bench.

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2.4. Morphological observation of scaffolds and cell attachment

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Scanning electron microscopy (SEM, S-2250N; Hitachi, Tokyo, Japan) was used to observe the morphology of the constructed scaffolds and the attachment of cells on the scaffolds. The dried pure scaffolds were used immediately. The scaffolds with cells were washed with PBS and fixed with 4% glutaraldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 24 h at room temperature. The samples were dehydrated in a graded ethanol series (50%, 60%, 70%, 80%, 90%, and 100%) for 10 min each on a clean bench. For SEM observations, dried 7

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scaffolds were mounted on a metal stub and coated with a thin layer of platinum using a plasma-sputtering apparatus (Emitech K575 Sputter Coater, Emitech Ltd., Ashford Kent, UK) under an argon atmosphere. The morphologies of constructed scaffolds and cells on scaffolds

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were examined by SEM.

2.5. FT-IR

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An FT-IR spectrometer (JASCO FT/IR-4200; Jasco Inc., Tokyo, Japan) was used to analyze

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the chemical differences between the pure PLGA scaffold and PLGA scaffolds containing MPC. In this experiment, dried pure scaffolds were sliced and analyzed immediately in the

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spectral range of 2500–500 cm-1.

2.6. Compressive strength

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To examine the mechanical strength of the fabricated scaffolds, conventional open-

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sided/unconfined analyses were conducted using a texture analyzer (FTC, Sterling, VA, USA). For this test, the scaffolds (n = 3) were immersed in cell culture medium for 3 days and measured at a 1.0 mm/min loading rate. The compressive strength was recorded and defined

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as the slope of the linear section of a stress-strain curve.

2.7. Proliferation assay To analyze the proliferation of NIH/3T3 on the scaffolds, a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT; Sigma) assay was used. At 4 h, and at 1, 2, and 3 days of culture, the scaffolds (n = 3) were transferred to new 24-well plates with 1 ml of new medium and 100 µl of MTT solution (5 mg/ml stock in PBS), and samples were then incubated at 8

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37°C for 4 h. When violet crystals were created, they were melted using 1 ml of dimethyl sulfoxide solution. Then, 100 µl of the solution was transferred to a 96-well plate. The optical density was measured at 570 nm using a microplate reader (E-Max; Molecular Devices,

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Sunnyvale, CA, USA).

2.8. Quantification of inflammatory cytokines

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Real-time polymerase chain reaction (RT-PCR) was used to investigate the expression of

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inflammatory cytokines. Cells were collected from the PLGA scaffolds and MPC/PLGA scaffolds. Total RNA was isolated using an RNeasy Plus Mini Kit (Qiagen, Hilden, Germany)

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and quantified using a NanoDrop 2000 spectrophotometer (Nano-Drop Technologies,

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Wilmington, DE, USA). PCR was conducted using primers for GAPDH, TNF-α, and COX-2 and extended using TOPscript One-step RT-PCR DryMIX (Enzynomics, Daejeon, Korea).

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All PCR products were loaded on an agarose gel (1%) for electrophoresis at 100 V and

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visualized under ultraviolet light (ETX-20.M; Vilber Lourmat, CollmatLo, France) at 360 nm. The primers were purchased from Bioneer (Daejeon, Korea) and the primer sequences are listed in Table 1. All reactions were performed in triplicate and the intensity of the band was

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confirmed using Image J, and normalized and plotted with respect to the GAPDH levels.

Table 1. Primer sequences used in RT-PCR.

Gene

Primer sequence

Annealing

Size (bp)

temperature TNF-

F: CCCTCACACTCACAAACCAC

60C

133 9

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R: ACAAGGTACAACCCATCGGC COX-2

F: CCTCTGCGATGCTCTTCC

60C

136

60C

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R: TCACACTTATACTGGTCAAATCC GAPDH

F: GGAGAGTGTTTCCTCGTCCC

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R: ATGAAGGGGTCGTTGATGGC

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2.9. Flow cytometry

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To measure ROS, RAW 264.7 cells (1 × 106 cells/scaffold) were cultured. As a negative control, cells were cultured in a cell culture dish. As a positive control, lipopolysaccharide

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(LPS; 1 mg/ml), which is an inflammation-inducing substance, was added to the cultured

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cells 1 day after seeding. At 2 days after seeding, the cells were treated with 20 μM dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich) for 40 min in a 37 °C

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incubator. After washing two times with PBS, the cells were detached using trypsin-EDTA.

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The cells were centrifuged and suspended in PBS, and then flow cytometry was performed using a fluorescence-activated cell sorter (FACS; Becton-Dickinson, Franklin Lakes, NJ,

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USA).

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2.10. Animal surgeries

Wister rats (female, 100–150 g; Han-Il Laboratory Animal Center, Jeonju, Korea) were anesthetized by intramuscular injection. Sterilized scaffolds were implanted subcutaneously under the armpit. For histological analyses, the animals were euthanized at 1 and 4 weeks after transplantation, and the scaffolds with surrounding tissues were cut out and fixed in a 10% formalin solution for 24 h. After fixation, the samples were embedded in paraffin blocks. 10

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2.11. Histological analysis The paraffin blocks were cut into 4.5-μm-thick sections using a microtome (Thermo Scientific, Waltham, MA, USA). The sections were stained and collected on coated slides and

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dried. Sectioned samples were deparaffinized and dehydrated using a xylene and ethanol series at room temperature. The sections were stained using hematoxylin & eosin (H&E) and

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CD-68 staining protocols. Stained sections were observed using an optical microscope

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(Nikon Eclipse TE2000-U-Inverted; Tokyo, Japan). The stained tissue sections were

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quantitatively analyzed using MATLAB (VER.7.0; MathWorks, Natick, MA, USA).

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2.12. Statistical analysis

All data are expressed as means ± standard deviation. Student’s t-tests (Excel 2010;

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Microsoft, Redmond, WA, USA) were used for statistical analysis, and significant

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differences were judged at *p < 0.05 and **p < 0.005.

3. Results and discussion

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3.1. MPC analysis

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The protein composition of the MPC powder was analyzed by SDS-PAGE and is shown in Fig. 1A. As an animal-derived collagen, MPC was found to possess all three typical animal collagens: type I (α2 chain, 97.4 kDa), type II (β chain, 205 kDa), and type III (α1 chain, 116 kDa). In particular, type II collagen showed strong expression, as expected, because MPC is derived from cartilage. The compressive strength of the pure PLGA scaffold (without MPC) was 56 ± 4.1 N, and 11

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decreased with an increase in the MPC amount added in a dose-dependent manner (Fig. 1B). Natural polymers such as collagen, chitosan, and silk fiber are hydrophilic substrates, whereas the PLGA polymer acts as a hydrophobic substrate; accordingly, the physical properties of PLGA improve with increasing amounts [20]. Thus, mixing a hydrophilic and

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hydrophobic substrate reduces the compressive strength due to weak interfacial bonding [21]. FT-IR analysis was also used to determine the chemical compositions of MPC, PLGA, and

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MPC/PLGA scaffolds, and the results are summarized in Fig. 1C. A specific peak for PLGA

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was detected at 1700–1800 cm-1 (corresponding to carbonyl and ester groups). Peaks for MPC were detected at 1650–1655 cm-1 (amide I) and 1234–1450 cm-1 (amide III), which are

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specific for collagens [22]. Both the MPC peaks and PLGA peaks were observed in the

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spectra of MPC/PLGA scaffolds. In particular, the intensity of the peak at 1450–1750 cm-1 became stronger with increasing contents of MPC. MPC was distributed evenly in the PLGA

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scaffold and the chemical properties of each material were not affected by the mixture.

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Figure 1. (A) SDS-PAGE results indicating that MPC contained three types of collagen. The

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expression intensity increased as the amount of loaded MPC increased. (B) Compressive

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strength of PLGA scaffolds and MPC/PLGA scaffolds immersed in media after 3 days. (C) FT-IR spectra for MPC, PLGA scaffolds, and MPC/PLGA scaffolds.

3.2. Morphology of scaffold surfaces and NIH/3T3 cells on scaffolds Scaffolds should have a high porosity and surface roughness for facilitating cell attachment and proliferation [23]. The surface properties of the scaffolds and morphology of NIH/3T3 13

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cells on scaffolds were observed by SEM (Fig. 2A–B). Each scaffold exhibited uniform porosity according to the size of NaCl crystals (180–250 µm) and the interconnectivity of the pores. In addition, we clearly observed a dose-dependent effect of increasing roughness of the

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surface of PLGA scaffolds mixed with MPC at increasing concentrations.

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Figure 2. (A) Surface characterization of PLGA and MPC/PLGA scaffolds by SEM (magnification ×300, ×100 scale bar = 100 µm, 500 µm). (B) SEM images of NIH/3T3 cells

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cultured on PLGA and MPC/PLGA scaffolds for 1 and 3 days. Magnification ×1000, scale bar = 50 µm; white arrows indicate attached cells.

NIH/3T3 cells are commonly used to examine the cellular compatibility of biomaterials owing to their high rates of proliferation and extracellular matrix secretion. To visually observe the cell shape and adhesion pattern, NIH/3T3 cells were seeded on the scaffolds. Cell 14

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attachment was observed at 1 and 3 days of cell culture. The cells adhered not only on the surface of the scaffold but also inside the pores of the scaffold. The initial cell attachment pattern was spherical, and then the morphology of cells changed into a long spindle shape during growth, covering both the surface and pores of the MPC/PLGA scaffolds at 3 days

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after seeding. In particular, more cell attachment was observed in the group with the highest MPC concentration (80 wt%) than in other groups. However, cell morphology did not differ

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at any MPC concentration compared to that observed on the pure PLGA scaffold, even after 3

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days. This enhancement of cell adhesion is attributed to the effect of MPC on increasing the

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hydrophilicity of the scaffold in a dose-dependent manner [24].

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3.3. Cell viability on scaffolds

MTT assays were conducted to evaluate the proliferative ability of NIH/3T3 cells on

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scaffolds at 4 h, and 1, 2, and 3 days after cell seeding. As shown in Fig. 3, the proliferation

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rates of both the pure PLGA scaffold and MPC/PLGA scaffolds increased over time. However, the cell proliferation rate for the MPC/PLGA scaffolds increased rapidly, while that for the pure PLGA scaffold increased slowly. In particular, the cells showed the highest

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growth rate on the 80 wt% MPC/PLGA scaffold. These results suggest that MPC increases

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the hydrophobicity of the hydrophobic surface of PLGA to improve water absorption and increase roughness, resulting in a high proliferation rate [25]. Moreover, the various cytokines produced by MPC could further promote proliferation in a dose-dependent manner.

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Figure 3. Cell viability of NIH/3T3 cells on PLGA and MPC/PLGA scaffolds analyzed by an

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MTT assay after 4 h, and 1, 2, and 3 days (**p < 0.005).

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3.4. Inflammatory response

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RT-PCR was performed to analyze the expression of genes involved in inflammation in RAW 264.7 cells seeded on the scaffolds. Inflammatory cytokines such as TNF-α and COX-2 were

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investigated and the results were normalized against levels of GAPDH, a housekeeping gene. TNF-α and COX-2 are commonly used markers to examine inflammatory immune responses

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[26,27]. From day 1 to day 3, the expression levels decreased gradually as the MPC content increased (Fig. 4). The expression levels of both TNF-α and COX-2 were lowest for scaffolds containing 80 wt% MPC. These results suggest that MPC can decrease the expression of proinflammatory cytokines, and that scaffolds containing MPC may be suitable for implantation. Furthermore, the secretion of inflammatory cytokines decreased over time, confirming that MPC has a positive anti-inflammatory effect. 16

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Figure 4. Gene expression profiles of TNF-α and COX-2 in cells seeded on PLGA and

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MPC/PLGA scaffolds (**p < 0.005).

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3.5. In vitro release of ROS from cells on scaffolds

ROS causes oxidative damage by acting on DNA or cell membranes in the body [28]. The

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ROS generated during the oxidation process is eliminated by antioxidant enzymes in the body;

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however, excessive production of free radicals is a major contributor to many diseases such as rheumatoid arthritis, degenerative diseases, cancer, and arteriosclerosis [29]. A FACS assay was performed using DCFH-DA to measure the degree of oxidative stress in cells cultured on

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the PLGA and MPC/PLGA scaffolds. DCFH-DA is hydrolyzed in cells, separated into DCFH

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as nonpolar and polar fractions, and then oxidized to DCF, a fluorescent substance. Therefore, ROS in the cell can be quantified by flow cytometry based on the fluorescence intensity. The optimal MPC content was determined by measuring the ROS content after incubation of RAW264.7 macrophages on the PLGA and MPC/PLGA scaffolds for 2 days. The intensity of fluorescence was weakest in the negative control group (cell culture alone) and was highest in the positive control (stimulated by LPS) (Fig. 5). The peak value for the RAW264.7 cells 17

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cultured on the pure PLGA scaffold was similar to that of the positive control group, indicating the production of ROS. However, the peak fluorescence of the scaffold with 80 wt% MPC was similar to that of the negative control group, indicating a protective effect of MPC against ROS accumulation. These results are in line with the other findings for the in vitro

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experiments described above, indicating that the MPC 80 wt% scaffold is an optimal

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biomaterial to minimize inflammation.

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Figure 5. Reduction of oxidative stress for PLGA and MPC/PLGA scaffolds. Fluorescence spectra from flow cytometry analysis showing the effects of MPC/PLGA on the generation of ROS (A: negative control, B: cells cultured on PLGA scaffolds, C: cells cultured on MPC 80 wt%, D: positive control).

3.6. Histological changes of implanted scaffolds To evaluate the in vivo effects of MPC on PLGA scaffolds, H&E staining was performed to 18

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determine the infiltration of inflammatory cells via interactions between scaffolds and tissues. Sterile scaffolds were implanted in the axillary region of Wister rats for 1 and 4 weeks. As shown in Fig. 6A, in the pure PLGA scaffold, extensive macrophages and giant cells were observed around the scaffold, indicating an acute inflammatory reaction. Although these cells

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were also detected on the MPC/PLGA scaffolds, they were found in far fewer numbers than those observed for pure PLGA scaffolds, and the number of inflammatory cells decreased

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over time.

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In addition, inflammatory responses were estimated based on the thickness of the fibrous wall, and the quantitative results are summarized in Fig. 6B [30]. Thickly formed fibrous walls

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could be identified in the pure PLGA scaffold. The thickness of the fibrous wall was slightly

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reduced over time, but was still greater than that of the PLGA scaffolds containing MPC.

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Figure 6. (A) Hematoxylin & eosin-stained tissue section for the PLGA scaffold (0 wt% MPC) and MPC/PLGA scaffolds (10, 20, 40, and 80 wt% MPC) after 1 and 4 weeks of

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implantation in vivo and (B) fibrotic wall thickness quantification. Magnification ×200, scale

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bar = 100 µm. **p < 0.005.

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In the experimental groups, the thickness of the fibrous wall decreased as the MPC content increased. In particular, the fibrous wall was the thinnest in the scaffold with 80 wt% MPC.

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Immunohistochemistry was performed using CD68 as a marker to identify macrophages in surrounding tissues. As shown in Figure 7, 1 week after the transplantation of pure PLGA

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scaffolds, many CD68-positive cells were found in the tissues surrounding the scaffold. However, a low density of positive cells was observed in the peripheral tissue for the PLGA scaffolds containing MPC. In particular, the 80 wt% MPC scaffold showed the lowest CD68positive cell density. After 4 weeks, the number of CD68-positive cells decreased in all groups. However, in the tissues transplanted with the pure PLGA scaffold, the number of positive cells was still higher than that for tissues transplanted with the PLGA scaffold 20

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containing MPC. In the group containing 80 wt% MPC, the number of CD68-positive cells decreased remarkably. This is consistent with the in vitro results, indicating that the

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MPC/PLGA scaffold has the same anti-inflammatory effect in vitro and in vivo.

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Figure 7. (A) CD68 immunohistological tissue section for the PLGA scaffold (0 wt% MPC) and MPC/PLGA scaffolds (10, 20, 40, and 80 wt% MPC) after 1 and 4 weeks of implantation in vivo and (B) CD68-positive cells density. Magnification ×200, scale bar = 100 µm. *p < 0.05 and **p < 0.005.

4. Conclusions 21

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MPC extracted from porcine cartilage contains collagen and can be used to compensate for the disadvantages of PLGA as a functional biomaterial, such as its hydrophobic properties, difficulties in nutrient exchange, and induction of inflammation. In this study, scaffolds were prepared by adding MPC to PLGA. We confirmed the physical properties of MPC/PLGA

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scaffolds and the superior cell attachment ability, proliferation rate, and secretion of cytokines related to inflammation for cells cultured on MPC/PLGA scaffolds compared to those

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cultured on the pure PLGA scaffold. The scaffold containing 80 wt% MPC had the most

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positive effects in all tests. Moreover, we investigated the optimal amount of MPC for minimizing the inflammatory responses in vitro and in vivo; the secretion of inflammatory

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cytokines was reduced with increasing amounts of MPC. In addition, the infiltration of

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inflammatory cells and the thickness of the fibrous membrane decreased during transplantation.

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Our results suggest that bioactive compounds in MPC have positive effects for improving the

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biocompatibility of a PLGA scaffold. The use of such naturally derived biomaterials may be useful for tissue engineering applications. However, their mechanical strength will need to be

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improved prior to application in a wide range of fields such as for bone generation.

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Acknowledgments

Funding: This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea [grant number: HI15C2996].

Conflict of interest: None 22

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Highlights

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Porcine cartilage was added to poly(lactic-co-glycolic acid) (PLGA) scaffolds

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Micronized porcine cartilage (MPC) improved cell attachment and proliferation in vitro MPC reduced inflammatory cytokines and oxidative stress in vitro and in vivo

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MPC/PGLA scaffolds can serve as useful biomaterials for tissue engineering

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