Biomimetic Porous PLGA Scaffolds Incorporating Decellularized

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Biomimetic Porous PLGA Scaffolds Incorporating Decellularized Extracellular Matrix for Kidney Tissue Regeneration Eugene Lih, Ki Wan Park, So Young Chun, Hyuncheol Kim, Tae Gyun Kwon, Yoon Ki Joung, and Dong Keun Han ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03771 • Publication Date (Web): 26 Jul 2016 Downloaded from http://pubs.acs.org on July 28, 2016

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ACS Applied Materials & Interfaces

Biomimetic Porous PLGA Scaffolds Incorporating Decellularized Extracellular Matrix for Kidney Tissue Regeneration

Eugene Lih,†,# Ki Wan Park,†,‡,# So Young Chun,§ Hyuncheol Kim,‡ Tae Gyun Kwon,§ Yoon Ki Joung,*,†,ǁ and Dong Keun Han*,†,ǁ

†

Center for Biomaterials, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea

‡

Department of Chemical and Biomolecular Engineering, Sogang University, Seoul 04107, Republic

of Korea §

Department of Urology, School of Medicine, Kyungpook National University, Daegu 41566,

Republic of Korea ǁ

Department of Biomedical Engineering, Korea University of Science and Technology, Daejeon

34113, Republic of Korea

#

These authors contributed equally to this work. *

Co-correspondences:

Dong Keun Han, Ph.D., E-mail: [email protected] Yoon Ki Joung, Ph.D., E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT Chronic kidney disease is now recognized as a major health problem, but current therapies including dialysis and renal replacement have many limitations. Consequently, biodegradable scaffolds to help repairing injured tissue are emerging as a promising approach in the field of kidney tissue engineering. Poly(lactic-co-glycolic acid) (PLGA) is a useful biomedical material, but its insufficient biocompatibility caused a reduction in cell behavior and function. In this work, we developed the kidney-derived extracellular matrix (ECM) incorporated PLGA scaffolds as a cell supporting material for kidney tissue regeneration. Biomimetic PLGA scaffolds (PLGA/ECM) with different ECM concentrations were prepared by an ice particle leaching method and their physico-chemical and mechanical properties were characterized through various analyses. The proliferation of renal cortical epithelial cells on the PLGA/ECM scaffolds increased with an increase in ECM concentrations (0.2, 1, 5, and 10%) in scaffolds. The PLGA scaffold containing 10% of ECM has been shown to be an effective matrix for the repair and reconstitution of glomerulus and blood vessels in partially nephrectomized mice in vivo, compared with only PLGA control. These results suggest that not only the tissue-engineering techniques can be an effective alternative method for treatment of kidney diseases, but also the ECM incorporated PLGA scaffolds could be one of the promising materials for biomedical applications including tissue engineered scaffolds and biodegradable implants.

Keywords Kidney tissue regeneration, Biomimetic scaffold, Poly(lactide-co-glycolide), Decellularized extracellular matrix, Ice particle leaching method

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INTRODUCTION Chronic kidney disease is increasingly recognized as a global public health problem due to a high mortality worldwide, affecting between 8 and 16% of the global adult population.1 However, therapeutic approaches for appropriate renal care remain limited. The dialysis is expensive, time consuming and exhausting, offering a short and poor quality of life. Kidney transplantation has the potential to dramatically improve the renal function, but it has also problems such as extremely high cost, donor shortages, and immunosuppression-related complications. These limitations have led many researchers to develop alternative treatment of chronic kidney diseases using tissue engineering with scaffolds and cells. A biomaterial scaffold is one of the most important components which interact with cells and growth factors to regenerate a specific tissue. Poly(lactic-co-glycolic acid) (PLGA) has been a typical one among the most attractive polymeric candidates used to fabricate biodegradable scaffolds for tissue engineering applications.2-4 PLGA, which has been approved by the Food and Drug Administration for certain medical applications, exhibits a wide range of erosion times, has tunable mechanical properties and is easily processed into various three-dimensional (3D) structures with the desired shape.5,6 Although PLGA takes a number of advantages as a scaffold material in tissue engineering, the hydrophobicity and insufficient biocompatibility of PLGA caused a reduction in cell behavior and function. In addition, the acidic by-products during in vivo degradation can generate vigorous inflammatory reactions, often leading to clinical failure.7 In recent year, the decellularized extracellular matrix (ECM) derived from various tissues has been used as a scaffold for applications in regenerative medicine.8-10 In particular, many researchers tried to use acellular renal ECM scaffold of mammalian tissues for functional organ recovery, allowing the injured area to recellularize with autologous stem cells or

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differentiated.1,11 The ECM components including structural proteins and bioactive growth factors display inherent bioactivity with the cell-ECM binding domains in promoting cell adhesion, migration, proliferation, and differentiation.12 Moreover, the ECM scaffold provided importantly three dimensional structures made of collagen and other biopolymers for reconstruction, which create specific cellular niches within body tissues.13-15 However, not only ECM presents weak mechanical characteristics and a rapid degradation rate as a scaffold, but also its processing like as chemical or morphological modification is also difficult due to poor solubility in organic solvents.16 In the present study, we developed the kidney-derived ECM incorporated PLGA scaffolds as a cell supporting material for kidney regeneration. Such biomimetic PLGA scaffolds (PLGA/ECM) with different ECM concentrations were prepared by an ice particle leaching method. The morphology of microstructures, porosity, thermal and mechanical properties, water contact angle, and degradation rate of the scaffolds were evaluated through various analyses. The cell viability and proliferation on the PLGA/ECM scaffolds were assessed with human renal cortical epithelial cell in comparison with only PLGA control. To evaluate the regenerative activity in vivo of the biomimetic scaffolds, the PLGA scaffold containing ECM was implanted into a partially nephrectomized area in mouse, and evaluated by histological stains and real-time polymerase chain reaction (PCR) analysis.

MATERIALS AND METHODS Materials Poly(D,L-lactide-co-glycolide) (PLGA, lactide:glycolide = 50:50, MW 40,000) was purchased

from

Evonik

Ind.

(Essen,

Germany).

Dichloromethane

3


(DCM)

and

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deoxyribonuclease (DNase) were obtained from Sigma-Aldrich Co. (St. Louis, MO). Human renal cortical epithelial (HRCE) cells, renal epithelial cell basal medium, and renal epithelial cell growth kit were purchased from American Type Culture Collection (ATCC, Manassas, VA). Penicillin, streptomycin, hematoxylin and eosin (H&E), 4’,6-diamidino-2-phenylindole (DAPI), phosphate-buffered saline (PBS) solution, and Triton X-100 were purchased from Invitrogen (Rockville, MD). Cell Counting Kit-8 (CCK-8) assay was obtained from Dojindo Molecular Technologies, Inc. (Rockville, MD). The extracellular matrix (ECM) powder was prepared by decellularization of porcine kidneys (Yorkshire pigs, female, 2-3 months, 22-30 kg).17 In brief, kidney sections were washed with PBS and incubated in a decellularizing solution (Triton X-100 1% (v/v) containing peniciliin 100 U/mL and streptomycin 100 µg/mL) at 4 °C, 200 rpm for 14 days. After washing, tissues were treated with DNase solution (30 µg/mL) in PBS for 1 h. Decellularized kidney ECM was sufficiently rinsed with PBS to remove residual DNase and other chemicals, and then lyophilized. The ECM powder was obtained by freeze milling using a Freezer/Mill 6750 from SPEX CertiPrep (Metuchen, NJ). The ECM powder was used after confirmation of decellularization with H&E and DAPI stain analysis and sterilization with ethylene oxide using a PERSON-EO35 sterilizer from Person Medical Co. (Gunpo, Korea) (Figure S1).

Fabrication of PLGA/ECM scaffolds and films The PLGA/ECM scaffolds were fabricated using ice microparticles as porogen.18,19 Ice microparticles (100-200 µm) were prepared by spraying cold deionized water into liquid nitrogen. A 13 wt% solution of PLGA dissolved in DCM was gently mixed with different concentrations of ECM powder (0.2, 1, 5, and 10 wt% compared to PLGA). The PLGA only 4



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group without ECM was used as a control. Ice microparticles were added in these polymer solutions with a ratio of 1:1 in cold chamber (-20 °C). The slurries were thoroughly agitated until a homogeneous paste-like mixture using a frosty spatula (at -20 °C). The mixtures were then transferred to the round silicone mold (8 × 5 mm2), prior to freezing in liquid nitrogen. The samples were freeze-dried for 4 days to remove the ice-microparticles and organic solvent, and the biomimetic porous scaffolds were finally obtained; they are denominated PLGA, PLGA/ECM0.2, PLGA/ECM1, PLGA/ECM5, and PLGA/ECM10 with the following concentrations of ECM from 0.2 to 10 wt%. To evaluate the surface properties of PLGA and PLGA/ECM groups, the film type of samples were fabricated by solvent-casting process. The PLGA was dissolved in DCM and then ECM with different concentrations was added into each polymer solutions. The homogeneously dispersed suspensions were poured into Teflon mold (3 × 9 cm2) and allowed to evaporate slowly at room temperature for 48 h in a fume hood. The samples were dried under vacuum to completely remove residual organic solvent. All samples were kept in a freezer at -20 °C to prevent the hydrolytic degradation of PLGA and denaturation of ECM.

Characterizations of PLGA/ECM matrix The microstructure morphology of the scaffolds was analyzed by field emission-scanning electron microscopy (FE-SEM; Hitachi S-4800, Japan) at an acceleration voltage of 10 kV, after sputter-coating with gold. The average diameters of the pore size were calculated by measuring no less than 50 randomly selected fibers from SEM micrographs using an image analysis software (Image J, National Institutes of Health, USA). The porosity of the scaffolds was estimated using the following equation: Porosity (%) = (1

5


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- ρ/ρ0) × 100, where ρ is the density of the porous scaffold and ρ0 is the density of the bulk PLGA (1.347 g/cm3).20 Five samples were measured per sample type. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR; 4100, JASCO, MD, USA) was performed with a spectral range of 400-4000 cm-1 in transmittance mode to characterize the composition and molecular conformation of the scaffolds. The amount of the ECM in the scaffolds was measured via the thermogravimetric analysis (TGA; TA Instruments Hi-Res TGA 2950, USA). The scaffolds were kept under vacuum for 24 h prior to analysis. The precisely weighed specimen was heated to 800 °C at heating rate of 10 °C/min under a constant nitrogen flow rate of 50 mL/min and the weight loss was obtained. The mechanical compressive properties of the scaffolds were determined using a universal testing machine (Instron 4464, MA, USA) equipped with a 10 N load cell at a cross-head speed of 10 mm/min. A rod with a ball-shaped (diameter 4.5 mm) tip was hammered vertically on cylindrical scaffolds (8 × 5 mm2), in triplicate.17,21 The water contact angle (WCA) was measured using an optical bench-type contact angle goniometry (VCA Optima XE Video Contact Angle System, Crest Technology, Singapore) at room temperature. The films were cut into rectangular shape (10 × 10 mm2). Droplets of distilled water (2 µL) were added onto the surface of the substrates. Since the PLGA matrix containing ECM, which is more hydrophilic with increasing concentration of ECM, usually absorbed water quickly, the instantaneous WCA was recorded within 1 sec. Ten points of each samples were tested to calculate average values.

In vitro cell culture 6



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HRCE cells were used to determine the biocompatibility of the biomimetic scaffolds. For cell preparation, HRCE cells were cultured in renal epithelial cell growth medium containing 0.5% FBS, 10 nM triiodothyronin, 10 ng/mL rhEGF, 100 ng/mL hydrocortisone, 5 ug/mL rh insulin, 1 uM epinephrine, 5 ug/mL trasferrin, 2.4 mM L-alanyl-L-glutamine, and 1% penicillin/streptomycin in an atmosphere of humidified 5% CO2 at 37 °C. The films (1 × 1 cm2) were transferred to 24 well plates after treated by ultraviolet irradiation. One hundred microliter of cell suspension with a concentration of 2 × 105 cells/mL was seeded onto the PLGA and PLGA/ECM films in a drop-wise manner and then incubated for 2 h for cellular attachment to the substrates. After each time point (1, 3, 5 and 7 days), the effect of ECM on the proliferative activity of HRCE cells on the samples was assessed by CCK-8 assay. The PLGA and PLGA/ECM samples were transferred to a new 24 well plates and rinsed twice with sterilized PBS, and serum-free media containing CCK-8 solution in a ration of 1:10 was added. After incubation under 5% CO2 at 37 °C for 2 h, the absorbance of the solution was measured using a microplate reader (Multiskan Spectrum, Thermo Electron Co., Vantaa, Finland) at 450 nm.22,23

In vivo animal study The animal study was approved by the Kyungpook National University Hospital Institutional Animal Care and Use Committee (IACUC). The injured kidney cortex model was produced in mouse (ICR, 5 weeks old, 20 g, male) by partial nephrectomy (excision volume 2 × 4 × 5 cm3) to assess the therapeutic potential of ECM-contained scaffolds for kidney tissue regeneration. Sixty mice were divided into 3 groups: native control group, PLGA, and biomimetic PLGA/ECM10 scaffolds. The renal artery and vein of the left kidney were occluded with a vascular clamp for 20 min, and then the scaffold was implanted on the 7


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injured left kidney. The right kidney was removed to properly evaluate the regenerative ability of implanted scaffolds for functional recovery as well as histological regeneration. Mice were sacrificed at 3, 7, and 28 days post-operation (each term, n=3). The extracted scaffold and surrounding tissue were divided into two equal parts for histological analysis and real-time PCR analysis. For histological and immunohistochemical (IHC) analysis, the half of scaffold implanted region was fixed with 4% paraformaldehyde after operation. The paraffin embedded samples were cut into 5-µm sections for hematoxylin and eosin (H&E) and IHC staining. The details of each antibody used for IHC are given in Table S1. The regenerated glomerular number per unit area (×100 magnification, 4860 µm2) was measured at 28 days. Real-time PCR analysis was carried out on the remaining half of scaffold. The primer sequences of the candidate genes and GAPDH are shown in Table S2. SYBR Green PCR conditions were 95 °C for 10 min, followed by 45 cycles of 95 °C for 10 sec, 58 °C for 50 sec, and 72 °C for 20 sec. To analyze the relative changes in gene expression, 2-∆∆Ct method was used.17

Statistical analysis All values were presented as mean ± standard deviation (SD). The statistically significant of the differences was analyzed by Student’s t test. The difference was considered to be significant at p value of less than 0.05.

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Figure 1. Schematic representation of fabricating the porous PLGA/ECM scaffolds by an ice microparticle leaching method.

RESULTS AND DISCUSSION Characteristics of biomimetic PLGA/ECM scaffolds The biomimetic porous scaffolds, composed of PLGA and decellularized ECM from porcine kidney, were fabricated by using an ice particle leaching method (Figure 1), so that the scaffold and film types of PLGA/ECM were obtained. Figure 2a showed the microstructure of the porous PLGA and PLGA/ECM scaffolds prepared with ice particles, respectively. As observed, all scaffolds exhibited a highly open-porous structure, and the large pores were connected to each other with interconnected porous walls. The size of the pores in the scaffolds prepared with ice particulates having diameters 100−200 µm was from 108.21 to 189.64 µm. The porosity was higher than 95%. All scaffolds showed the similar high porostiy (Table 1). This is because the same ratio of ice particles was used to prepare the PLGA scaffolds with and without ECM. The pore structure, including pore size, porosity, and interconnectivity, is very important factor to developing an ideal porous scaffold for tissue engineering. The interconnected pores and high porosity could facilitate cell migration,

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nutrient diffusion, and metabolic waste removal. The pore size of 100−200 µm was usually used for tissue engineering, because the scaffolds with pore size of 100−200 µm had proper mechanical property and good cell distribution.24-26 However, many reports are not in good consensus, because there are various pore structures and these scaffolds are used in different fields of tissue engineering. The ice particle leaching method offers many advantages for the easy manipulation and precise control of pore size without remaining porogen in the scaffold after porogen leaching.

Table 1. Physical characteristics of PLGA/ECM scaffolds Porosity

Compressive stress at break

Ti

Tm

(%)

(MPa)

(°C)

(°C)

PLGA

95.87 ± 0.21

1.836 ± 0.044

268

361

PLGA/ECM0.2

95.95 ± 0.19

1.398 ± 0.040

282

369

PLGA/ECM1

95.83 ± 0.18

1.267 ± 0.035

285

371

PLGA/ECM5

95.67 ± 0.24

0.703 ± 0.015

304

383

PLGA/ECM10

96.44 ± 0.37

0.194 ± 0.003

313

388

ECM

-

-

275

420

The surface wettability of PLGA and PLGA/ECM scaffolds was evaluated by water contact angle. The water spreading time of all scaffolds was 1 sec to inhibit water absorption into the substrate. As shown in Figure 2b, water contact angles on PLGA, PLGA/ECM0.2, PLGA/ECM1, PLGA/ECM5, and PLGA/ECM10 scaffolds were 83.63, 69.07, 63.43, 51.50, and 44.03°, respectively. It can be seen that contact angle values of PLGA/ECM were lower than that of PLGA indicating more hydrophilic, and the values slightly decreased with the increase in ECM loading amount. Hydrophilic scaffold has been reported to allow for fast spreading property, high-efficient cell seeding and better attachment of cells.27 Most 10



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modifications in scaffold, including plasma treatment, physical adsorption, and chemical immobilization, were complex and inconvenient. The hydrophilic modification of scaffold with ECM molecules is very simple, and it also enhances cell behavior such as cell adhesion, growth, proliferation, and differentiation as a potential application in tissue engineering.23,28

Figure 2. (a) SEM images (×100; scale bar = 300 µm and ×1000 magnification; scale bar = 30 µm) and (b) water contact angles of PLGA/ECM scaffold with different concentrations of ECM (mean ± SD, n = 10, *p < 0.01 and **p < 0.001).

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Figure 3. (a) ATR-FTIR spectra and (b) TGA thermograms of the composite scaffolds.

The chemical composition and incorporated amount in dependence of ECM were determined by ATR-FTIR. The FTIR spectra of PLGA and PLGA/ECM were depicted in Figure 3a. The O−H stretching vibrations of hydroxyl group and carboxyl groups appeared as a wide and weak band at 3550 cm−1. The sharp peak at 1750 cm−1 was assigned to the stretching vibration of C=O in carboxyl groups. The C=O peak of PLGA/ECM and ECM at 1750 cm−1 was wider than that of PLGA. The peaks of stretching vibration at 2849 and 2920 cm−1 were assigned to O−H, NH2, and N−H in the amide group, which were not found in the

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spectrum of PLGA (Figure S2a). The absorbance peaks at 1450, 1380, and 1190 cm−1 were attributed to C−H wagging of CH2 and CH3 and deformation of PLGA. The 1090 cm−1 was also found to be bands relative to the C−O stretching of PLGA. The peaks at 1630, 1535, and 1230 cm−1, respectively, were ascribed to the stretching vibration of protein amide C=O (amide I), bending vibration of N−H in amide group (amide II), and the C−N stretching and N−H plane bending from amide linkages (amide III), which were also not found in the spectrum of PLGA (Figure S2b and c). The bands corresponding to the amide absorption displayed an increase in intensity with the increasing the ECM content in PLGA/ECM samples.29 Thermal properties of the scaffolds were studied using TGA. Figure 3b shows the comparative thermogravimetric curves of PLGA, ECM and PLGA/ECM blended scaffolds. It was found that after the large weight loss of PLGA decomposition, the residual weight increased with the addition of ECM in the PLGA scaffolds. When the temperature arrived at 600 °C, there was still some char content existing in ECM powder. The remained char contents of the PLGA/ECM5 and PLGA/ECM10 scaffolds at around 600 °C were about 5 and 10% respectively, which were very close to the weight of ECM content. Also, the TGA results exhibited a slight increase in the initial decomposition temperature (Ti) and melting temperature (Tm) from 268 to 313 °C and from 361 to 388 °C, respectively, when ECM increased from 0 to 10% in the PLGA scaffolds (Table 1). The differences in Ti and Tm of the samples might be arisen from the increase of secondary interactions between ECM and PLGA with the increase of ECM into PLGA. The TGA results demonstrated that the addition of ECM to PLGA increased their thermal stability. Thermal stability of material is an important property to produce bioabsorbable scaffolds. In the degradation and cell culture studies, the thermal energy was continuously supplied to the scaffolds to maintain a constant

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body temperature of approximately 37 °C. However, during heat-treating process of the material, thermal degradation can cause the generation of smaller molecular fragments such as byproducts of degradation that can interfere with the chemical composition of the material and alter its cytotoxicity and biocompatibility.30

Figure 4. (a) Compressive stress-strain curves and (b) compressive modulus of the PLGA/ECM scaffolds (mean ± SD, n = 3, *p < 0.01 and **p < 0.001).

The mechanical properties of PLGA and PLGA/ECM scaffolds were characterized by 14



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compression test. Figure 4a displays the compressive stress-strain curves for an elastomeric scaffold with porous structure showing three distinct regimes, which are composed of a linear elastic regime (strain 0−10%; bending of pore edges/struts), a collapse plateau regime (strain 10−60%; distortion of pore struts and pore collapse) and a densification regime (strain 60−100%; complete pore collapse and clogging).31,32 The PLGA/ECM scaffolds were soft and elastic and the overall strength is generally low, compared with PLGA. By incorporation of ECM, the stress of the PLGA/ECM scaffolds was significantly lower than that of PLGA only at all the tested strains. The decreasing trend of stress is also proportional to the loading of ECM from 0.2 to 10%. The stress at break for PLGA/ECM10 scaffold was 0.194 MPa, which is about 9.5 times lower than that of the PLGA only group (Table 1). The slope in the linear elastic region gave the compressive modulus. As shown in Figure 4b, the compressive moduli of PLGA, PLGA/ECM0.2, PLGA/ECM1, PLGA/ECM5, and PLGA/ECM10 scaffolds significantly decreased from 8.11 to 0.50 MPa. It is known that the mechanical properties of polymer composites including ECM decreased due to poor mechanical property of ECM consisting of natural polymers such as collagen, laminin, and fibrinogen. Such mechanical properties of the delicate PGLA/ECM scaffolds can be tailored to match the properties of soft tissues and be used in a wide range of applications. The mechanical properties of the microenvironment have been known to be important in the determination of cell proliferation and differentiation via the mechanotransduction pathways, where cells convert mechanical stimuli into chemical signals that affect cellular responses. Engler et al. demonstrated for the first time the sensitivity of mesenchymal stem cells (MSCs) to the stiffness of substrates; MSCs showed neuronal phenotype on a soft matrix to mimic brain tissue, whereas the cells on the rigid substrate comparable to collagenous bone were differentiated into osteoblast.33 Many researchers have studied the effect of matrix stiffness similar to in vivo environment on cell behavior and significant implications for the design of 15


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scaffolds with appropriate mechanical property for tissue regeneration. For examples, endothelial cells on compliant substrates adopted more spindle-shaped morphology than those on stiffer substrates,34 the gelatin scaffold with 800 kPa (like cartilage ECM) had significantly high cell proliferation of articular chondrocytes and synthesis of sulfated glycosaminoglycan,35 and cardiomyocytes from heart muscle cells were shown to beat best on a matrix with heart-like elasticity whereas cells on rigid matrix to mechanically mimic a post-infarct fibrotic scar stopped beating.36 In particular, Chen et al. and Szeto et al. proved that the soft matrix has inherent advantage toward tissue engineering of new kidney by retaining tubular-like morphology of primary proximal tubular epithelial cells and preventing transforming growth factor β1 (TGF-β1) induced epithelial-mesenchymal transition (EMT), which is a major cause of renal fibrosis.37,38 The PLGA/ECM10 scaffold was less stiff or softer compared to other scaffolds. These results proposed that the soft-mechanical property of PLGA/ECM10 scaffold has a benefit for kidney tissue regeneration because the compressive stress of kidney is around 0.25 MPa.39

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Figure 5. Cell viability of HRCE cells cultured on PLGA/ECM scaffolds with culture times of 1, 3, and 5 days. Values are expressed as mean ± SD (n = 6). *p < 0.01 and **p < 0.001.

Cell viability on PLGA/ECM matrix To evaluate in vitro cytocompartibility and cell proliferation on the PLGA and PLGA/ECM films, the cell viability of cultured HRCEs on the films was observed by CCK-8 assay. Cell proliferation for each material is illustrated in Figure 5. The PLGA group showed the lowest cell proliferation as compared to the degraded PLGA/ECM films. The degradation byproducts from the PLGA caused a harmful effect on the cell viability in the scaffold. In contrast, cell proliferation was significantly higher on PLGA/ECM when compared to PLGA control for 1, 3, and 5 days. From 3 days on, cells grew faster on the PLGA/ECM with an increase of ECM content in scaffolds. Cells on the PLGA/ECM10 group also displayed highly increased cell proliferation activity compared to other groups at 5 days. Although the degradation by-product of PLGA affected cell viability, the cell proliferation was enhanced

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by bioactive components of the ECM. In our previous study,17 we quantitatively assessed ECM components in the decellularized kidney consisting collagen type I, collagen type IV, fibronectin, and laminin. These ECM proteins are the major components of the glomerular basement membrane and can accelerate ECM protein synthesis and secretion of cells.40,41 In addition, the levels of growth factors were evaluated to confirm the preserved endogenous growth factors in the acellular ECM, including vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), and hepatocyte growth factor (HGF). These growth factors play a critical role in the cell proliferation. The functional ECM proteins incorporated PLGA scaffolds had a more favorable effect on kidney tissue regeneration.

Renal reconstruction after partial nephrectomy To evaluate in vivo host cell recruitment to the scaffold-grafted regions as an indicator of renal tissue regeneration, the appearance of glomerular structures was surveyed by H&E staining and visualization at low (×40) and high (×400) magnifications (Figure 6a). For in vivo animal study, the PLGA/ECM10 scaffold was selected as an experimental group among the scaffolds containing ECM based on in vitro cell proliferation assay, compared with PLGA control. At 3 days, red blood cells were observed abundantly in the PLGA and PLGA/ECM10 scaffolds. At 7 days, several pores were appeared by scaffold degradation, and PLGA/ECM10 scaffold showed tighter cell density than that of PLGA control. At 28 days, the scaffolds were significantly degraded and newly formed glomeruli were located in the regenerated tissue. The PLGA/ECM10 scaffold exhibited enhanced numbers and sized of mature glomeruli and the formation of distal and proximal convoluted tubules compared with PLGA control. The 18



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glomerular number per unit area of the PLGA/ECM10 was 10.0 and that of PLGA control was 1.8 at 4 weeks (native, 11.4 ± 1.1) (Figure 6b). At 28 days, the scaffolds were significantly degraded and newly formed glomeruli were located in the regenerated tissue. In particular, number of kidney-related cells is found higher for PLGA/ECM group as compared to PLGA control, and the scaffold degradation rate was also found faster in PLGA/ECM scaffolds than that of PLGA scaffolds. It seems that ECM-derived bioactive molecules in PLGA/ECM scaffolds stimulated cell growth and metabolic acids and enzymes secreted by active living cells accelerated hydrolytic degradation of PLGA and enzymatic degradation of ECM, respectively. The biocompatible and rapidly biodegradable PLGA/ECM scaffold might provide a favorable microenvironment for cell proliferation, migration and recruiting, compared with PLGA.

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Figure 6. Reconstruction of PLGA/ECM scaffolds compared with native renal tissue and PLGA scaffold: (a) H&E staining of scaffolds at 3, 7, and 28 days, (b) the number of regenerated glomeruli (black arrows) per unit area (×100 magnification, 4860 µm2) at 28 days, and (c) real-time PCR analysis of expression of mesenchymal and renal-related markers in scaffolds at 3, 7, and 28 days; Native (black), PLGA (blue), and PLGA/ECM10 (red). Gene expression analysis of the mesenchymal marker (Wnt4), the renal marker (Pax2), the initiation of nephrogenesis marker (Wt1), the glomerular endothelial marker (vWF), the renal epithelial marker (Emx2 and Krt10), the epithelial and collecting duct marker (Cadherin), and the cortex marker (Laminin) were performed. Values are expressed as mean ± SD (n = 3). *p 20



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< 0.01 and **p < 0.001, compared with PLGA control.

Histologic results were confirmed by real-time PCR analysis (Figure 6c). The number of mesenchymal stem cells and the expression of differentiation-related marker increased till 7 days, and then subsequently decreased. The PLGA/ECM10 scaffold indicated a relatively early and high expression of mesenchymal stem cells and renal differentiation markers, compared with PLGA scaffold. After the scaffold was implanted into kidney, surrounded and circulated stem cells are recruited into the scaffold, at 3 days, these cells are found inside of scaffold (by H&E and IHC), at 7 days, these stem cells are showing differentiation into early renal lineage cells, and at 28 days, the differentiated cells formed glomerular-like structures with mature renal cells. Therefore, Wnt4 (a kidney MSC–like cell marker) and Pax2, Wt1, vWF, Emx2, and Krt10 (early renal lineage specific markers)42 are relatively high expressed at 7 days and those early markers gradually decreased at late period. In IHC analysis, the expression of T cell markers was mainly found at the implanted PLGA scaffold at 3 days, whereas PLGA/ECM10 scaffold exhibited relatively weak staining for CD4, CD8, dendritic cell, ED-A, and MCP-1 (Figure 7a and Figure S3). The PLGA/ECM10 scaffold usually displayed undetectable staining level for these antibodies from 7 days, but the PLGA scaffold showed a later diminishment. In the analysis of pro-inflammatory (Figure 7b) and fibrosis-related genes expression (Figure 7c), the PLGA demonstrated increased expression of these genes as compared with that of PLGA/ECM10 scaffold till 7 days, and then subsequently decreased.

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Figure 7. (a) Immunohistochemical analysis of the scaffolds implanted region with T cell markers (×200 magnification) and Gene expression analysis by real-time PCR of (b) proinflammatory markers and (c) fibrosis-related markers. Values are expressed as mean ± SD (n = 3). *p < 0.01 and **p < 0.001, compared with PLGA control.

The scaffold is a major component in tissue engineering and regenerative medicine which provides ideal microenvironment to support cells growth and specific tissue regeneration in the reconstruction.43 The porous scaffold was fabricated with high surface area to volume ratio to enhance cell adhesion and migration and to facilitate more efficient supply of oxygen and nutrients to the cells.44,45 Even though PLGA as a scaffold material has several benefits 22



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such as tunable biodegradability and mechanical properties, its hydrophobicity and insufficient biocompatibility causes a reduction in its ability of cell adhesion, migration, proliferation, and differentiation. The biochemical signals in decellularized renal ECM can critically influence cellular survival, cell behaviors, and phenotypic modulation. The kidney ECM, including glomerular basement membrane (GBM) and mesangial matrix, is composed of four main macromolecules such as laminin, collagen type IV, nidogen and heparan sulphate proteoglycans. Of these molecules, laminin is to play an important role in regulating cell proliferation during reconstruction of glomerular epithelium. Laminins has been critically involved in the polarization of the developing kidney tubular epithelium.46 Furthermore, Salas et al. reported that laminins affected cell attachment, spreading and polarization of Madin-Darby canine kidney cells through the use of function-blocking antibodies.47,48 Collagen type IV, a major collagenous component of GBM, can promote the repair of physiological functions in injured renal proximal tubular cells by restoring integrin polarity and stimulating repair of mitochondrial function and active Na+ transport in kidney regeneration.49 Collagen type VI as a major structural protein in glomerulus is involved in podocyte maintenance,50 and collagen type I and fibronectin, respectively, can support renal cortical cells and podocyte differentiation which may recapitulate the developing kidney in vivo.40 Moreover, the growth factors such as VEGF, IGF, EGF, and HGF remained in the decellularized ECM can enhance renal tissue regeneration. It is well known that VEGF not only controls the differentiated properties of the glomerular but also shows autocrine and paracrine functions to stimulate proliferation of renal tubular epithelial cells and to recruit endothelial cells in tubulogenesis.51 IGF and EGF, which administrate against acute ischemia renal injury activated renal regeneration, are known to be mitogenic for renal proximal

tubules.52 In renal failure, HGF induces mitogenic and morphogenetic responses of renal tubular cells as well as reconstruction of the normal renal tissue structure.53 23


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Figure 8. Gene expression analysis of anti-inflammatory markers by real-time PCR. Values are expressed as mean ± SD (n = 3). *p < 0.01 and **p < 0.001, compared with PLGA control.

With H&E staining for histological analysis, the PLGA scaffold incorporating renal ECM induced the effective recruitment of tissue-specific host progenitor cells, and the recruited cells were proliferated and differentiated into the targeted cell types and regenerated functional glomerulus tissues. IHC analysis demonstrated that the PLGA/ECM scaffold showed minimally inflammatory reaction, whereas the PLGA scaffold displayed a high immunogenicity due to acidic degradation byproducts from PLGA. Detection of immune cells using CD4 and CD8 antibodies revealed a reduced T cell infiltration into the PLGA/ECM10 scaffold, compared with the PLGA scaffold. The real-time PCR result indicated the PLGA/ECM10 scaffold provided a more cell-friendly microenvironment as compared with the PLGA scaffold. The mesenchymal stem cell marker, Wnt4 expression, was higher in the PLGA/ECM10 scaffold at early period of regeneration (7 days), and the expression level was usually high in PLGA/ECM10 scaffold in renal differentiation related markers for epithelium and endothelium. The PLGA/ECM10 scaffold also revealed the suppression of pro-inflammatory and fibrosis-related cytokines by reduced TNF-α, IL-6, IL24



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1β, vimentin, Col1, and α-SMA expression levels. The ECM contained PLGA scaffolds were found to exert anti-inflammatory effect as well as immunosuppressive effect. In the analysis of anti-inflammatory gene expression, the PLGA/ECM scaffold also exhibited an enhanced expression of TGF-β and IL-4 genes (Figure 8). After grafting the scaffold into a partially nephrectomized mouse kidney, the newly formed tubular and glomerular structures were observed within the regenerated tissue and host-graft interfaces, and especially neovascularity was found in the PLGA scaffold incorporating renal ECM (Figure S4). Based on these histological results, such biomimetic PLGA/ECM10 scaffolds are considered to provide a suitable cellular environment for cell proliferation and differentiation towards renal lineages in comparison with PLGA scaffolds. The obtained results showed that the restored kidney in structural and functional regeneration might depend on the cellular recognition and interaction between renal cells and the bioactive molecules of decellularied ECM components.

Conclusions Biomimetic and biodegradable PLGA scaffolds incorporating acellular renal ECM were successfully fabricated by an ice particle leaching method. The physic-chemical properties of the PLGA/ECM scaffolds were characterized as well as their thermo-mechanical properties were also evaluated by using various analysis techniques with different concentrations of ECM. The porous scaffolds showed interconnected pore structure, and they had uniform pore size distribution and porosity to be 100-200 µm and 95.5%, respectively. The scaffolds tended to be hydrophilic and mechanically soft with increasing concentrations of ECM. The cellular result demonstrated that the renal ECM contained materials were cytocompatible and stimulated the in vitro proliferation of renal cortical epithelial cells, compared with PLGA only. Following murine in vivo implantation, the newly formed tubule- and glomeruli-like

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structures and neovascularization were observed in the regenerated tissue in the PLGA/ECM scaffold. The ECM components in PLGA scaffold improved the biocompatibility and reconstructivity of PLGA materials by stimulating cell proliferation and recruiting host stem cells in the partially injured kidney. In addition, the PLGA/ECM scaffolds depicted the suppression of the inflammatory response and elicited anti-inflammatory activity, compared with only PLGA scaffold. The present investigation suggested that the biomimetic PLGA scaffold incorporating decellularized renal ECM could be used as a physiological support system to provide 3D cellular environment for new tissue formation and to promote regeneration in partial defects of the kidney tissue. Furthermore, the acellular ECM components obtained from specific tissue were expected to utilize as a customized matrix in targeted tissue regeneration. More extensive investigations on therapeutic potential of these scaffolds that can restore functional kidney will be necessary to suggest a new therapeutic approach in the treatment of chronic kidney disease.

Supporting Information. Table S1. Antibody information for immunohistochemical analysis Table S2. Primer Sequences Figure S1. DAPI and H&E staining of porcine kidney tissue after decellularization, showing no remnant nuclear structures with maintained ECM (*spontaneous fluorescence by collagen). Figure S2. ATR-FTIR spectra of PLGA, ECM, and PLGA/ECM materials. The ranges of (a) 3300-2500, (b) 1700-1400, and (c) 1500-1200 cm-1; PLGA (black), PLGA/ECM0.2 (purple), PLGA/ECM1 (green), PLGA/ECM5 (blue), PLGA/ECM10 (red), and ECM (brown). Figure S3. Immunohistochemical analysis of the scaffolds for dendritic cell, ED-A, and MCP-1 as pro-inflammatory markers. Figure S4. Histological analysis (H&E staining) identified primitive glomeruli (white asterisk) 26



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and newly formed neovascular (red arrow) in the PLGA/ECM10 scaffold at 28 days after implantation. This material is available at free of charge via the Internet at http://pubs.acs.org

Acknowledgments: This work was supported by Bio & Medical Technology Development Program (2014M3A9D3033887), Cell Regeneration Program (2013M3A9C6049717), Pioneer Research Center Program (2014M3C1A3056052), and New Generation Medical Device Platform Program (2015M3A9E2028580) through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (MSIP), Republic of Korea.

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