High-Performance Acellular Tissue Scaffold Combined with Hydrogel

Article Cite This: ACS Biomater. Sci. Eng. 2019, 5, 3462−3474

pubs.acs.org/journal/abseba

High-Performance Acellular Tissue Scaffold Combined with Hydrogel Polymers for Regenerative Medicine Eunsoo Lee,† Hyun Jung Kim,† Mohammed R. Shaker,† Jae Ryun Ryu,† Min Seok Ham,‡ Soo Hong Seo,‡ Dai Hyun Kim,†,‡ Kiwon Lee,§ Neoncheol Jung,§ Youngshik Choe,∥ Gi Hoon Son,⊥ Im Joo Rhyu,† Hyun Kim,† and Woong Sun*,†

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†

Department of Anatomy and Division of Brain Korea 21 Plus Program for Biomedical Science, Korea University College of Medicine, 73, Inchon-ro, Seongbuk-gu, Seoul 02841, Republic of Korea ‡ Department of Dermatology, Korea University College of Medicine, Seoul, Republic of Korea § Logos Biosystems, Inc., Anyang-si, Gyunggi-do 431-755, Republic of Korea ∥ Department of Neural Development and Disease, Korea Brain Research Institute, Daegu 701-300, Republic of Korea ⊥ Department of Biomedical Sciences and Department of Legal Medicine, Korea University College of Medicine, Seoul 02841, Republic of Korea S Supporting Information *

ABSTRACT: Decellularization of tissues provides extracellular matrix (ECM) scaffolds for regeneration therapy and an experimental model to understand ECM and cellular interactions. However, decellularization often causes microstructure disintegration and reduction of physical strength, which greatly limits the use of this technique in soft organs or in applications that require maintenance of physical strength. Here, we present a new tissue decellularization procedure, namely CASPER (Clinically and Experimentally Applicable Acellular Tissue Scaffold Production for Tissue Engineering and Regenerative Medicine), which includes infusion and hydrogel polymerization steps prior to robust chemical decellularization treatments. Polymerized hydrogels serve to prevent excessive damage to the ECM while maintaining the sophisticated structures and biological activities of ECM components in various organs, including soft tissues such as brains and embryos. CASPERized tissues were successfully recellularized to stimulate a tissue-regeneration-like process after implantation without signs of pathological inflammation or fibrosis in vivo, suggesting that CASPERized tissues can be used for monitoring cell−ECM interactions and for surrogate organ transplantation. KEYWORDS: tissue scaffold, tissue decellularization, hernia repair, tissue regeneration, tissue engineering

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INTRODUCTION

made to decellularize large organs such as the liver, lungs, kidney, and heart within days or weeks.19−24 Although these methods can generate acellular tissues, robust processing of tissues often damages their fine ECM structures, leading to a partial loss of ECM components and a significant reduction in the beneficial biological properties of the materials.11,13,14 However, the gentle procedure to preserve ECM architecture increases the risk posed by the remaining cellular components, which may elicit serious immune reactions after transplantation.25,26 Furthermore, decellularization of soft organs is challenging because of inevitable shrinkage and loss of physical strength of the tissues. Therefore, organ decellulariza-

Three-dimensional bioscaffolds are widely used in tissue engineering and regenerative medicine.1−4 Decellularization is a process to eliminate cellular components from the tissue while preserving the composition and structure of the extracellular matrix (ECM) for further clinical and experimental use.4−7 Since the natural fine architecture of the ECM provides an excellent environmental niche for regeneration and tissue replacement, decellularized tissues are used in clinics for reconstructive surgical applications and transplantable grafts.4−8 Decellularization has been performed through a combination of chemical, physical, and enzymatic treatments.9−13 While upscaling of processes developed in mice to large animal and human organs has been challenging, several successful methods now exist for obtaining whole acellular human organ scaffolds.9,14−18 Recently, technological advances have been © 2019 American Chemical Society

Received: February 12, 2019 Accepted: June 11, 2019 Published: June 11, 2019 3462

DOI: 10.1021/acsbiomaterials.9b00219 ACS Biomater. Sci. Eng. 2019, 5, 3462−3474

Article

ACS Biomaterials Science & Engineering

For traditionally decellularized (TD) skin sheet preparation (Figure 7, Figure S7), pig skin was frozen at −80 °C for 30 min and thawed at room temperature for 1 h. After three freeze−thaw cycles, the epidermal side of the skin was gently scraped with a scalpel to remove the pig stratum corneum, and the processed skin was incubated in 0.25% Trypsin-EDTA (Gibco, MA, U.S.A.) for 6 h, followed by washes in deionized water four times for 20 min. Processed skin sheet was then incubated in 70% ethanol for 10−12 h, 3% H2O2 (Sigma-Aldrich, MO, U.S.A.) for 15 min, and washed with deionized water four times for 20 min each. Next, the processed skin sheet was incubated in 1% Triton X100 (Sigma-Aldrich, MO, U.S.A.) in 0.26% EDTA/0.69% Tris overnight, 0.1% peracetic acid/4% ethanol three times for 2 h.32 Finally, the thickness of the processed skin sheet was 2−2.5 mm, which was washed in sterile PBS two times for 15 min, deionized water six times for 20 min each. All steps of skin decellularization were performed at room temperature with gentle shaking. ̈ DNA Quantification. To assess total DNA contents in the naive and CASPERized samples, the DNeasy Blood and Tissues kit was used according to the manufacturer’s manual (Qiagen, Hilden, Germany). Briefly, tissues were digested with proteinase K overnight. DNA was purified from the samples using buffers provided by the company and measured spectrophotometrically (Thermo Fisher Scientific, MA, U.S.A.). Optical densities at 260 and 280 nm were used to estimate the purity and yield of nucleic acids, respectively. ̈ and Collagen Quantification. The collagen contents of naive CASPERized samples were quantified using a Sircol collagen assay kit (Biocolor, Carrickfergus, U.K.) according to the manufacturer’s instructions. This step was done following collagen extraction by incubation in a pepsin-containing solution at 65 °C overnight. The absorbance of each sample was determined at 550 nm using a microplate reader (Molecular Devices, CA, U.S.A.), and the collagen quantity was calculated using a standard curve generated with watersoluble denatured collagen from bovine skin. GAG Quantification. The GAG contents of native and decellularized tissues were quantified using the Blyscan glycosaminoglycan assay (Biocolor, Carrickfergus, U.K.) according to the manufacturer’s instructions after digestion in a papain-containing solution overnight at 65 °C. The absorbance of each sample was determined at 656 nm using a microplate reader (Molecular Devices, CA, U.S.A.), and chondroitin 4-sulfate was used as a standard. Physical Strength Measurements. For measurement of compressive property, the samples were prepared at sizes close to 5 × 5 × 5 mm (mouse tissues) or 10 × 10 × 5 mm (polymer-only) (n = 5, in each group) for use in the mechanical testing machine (5900 series, Instron Corporation, MA, U.S.A.). Tissues were loaded between two parallel steel plates of a compression testing machine. Next, the force was applied to the tissue by moving the crossheads together. During the test, a plot of stress (σ) versus strain (ε) was constructed using the manufacturer-provided software. For tensile testing, pig skin samples of 15 mm × 60 mm × 2 mm in size were prepared (n = 5, in each group). Skin samples were aligned in the direction of loading and secured at each end using grips, and three preconditioning cycles were applied to each sample using the following parameters (5900 series, Instron Corporation, MA, U.S.A.): crosshead velocity v = 20 mm/min, minimum and maximum strain 0% to 5% of the initial length. Subsequently, the tensile tests were performed with v = 20 mm/min and a strain corresponding to material failure. Manufacturer software (Bluehill, Instron, MA, U.S.A.) was used to control strain rate and record force/elongation data. Mean Linear Intercept Analyses. Images of equal magnification from collagen type IV immunolabeled liver tissues were captured using a confocal microscope. All image processing was performed using Fiji. The RGB images were converted to 8-bit gray scale and binary images. The binary images were further processed as skeletonized structures. A grid of horizontal lines was superimposed on the skeletonized image, and the points where the liver structure intercepted the probes were recorded and exported using Fiji and the Grid Overlay plug-in. Mean linear intercept equaled the number of intercepts divided by the total probe length.

tion is mostly applicable to rigid organs that can endure harsh decellularization processes.27,28 This unmet need requires a better decellularization procedure that provides several important features, including better preservation of the structure and function of ECM components, maintenance of physical strength after removal of cellular components from the tissues, and biocompatibility for clinical use.29,30 In this study, we describe a highly flexible technique, CASPER (Clinically and Experimentally Applicable Acellular Tissue Scaffold Production for Tissue Engineering and Regenerative Medicine), for producing decellularized organs or tissues in a perfusion-free manner, while maintaining fine structures and biological activities of the ECM with complete removal of cellular components. CASPERized organs support highly porous architectures, allowing recellularization in vitro and in vivo. Therefore, CASPER offers unique opportunities for examination of the cell and ECM interactions in a wide range of organs as experimental tools in vitro and for transplantation of processed organs or tissues for regeneration in vivo.

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MATERIALS AND METHODS

Animals and Tissue Collection. For organ decellularization and implantation, 2-month-old male mice were used (purchased from Koatech, Inc., Pyeongtaek, Korea). Pregnant C57BL/6 dams were obtained from Koatech. Embryos from day 18.5 were extracted after laparotomy. Further, 5xFAD AD model mice (age, 6 months) were kindly gifted by Dr. Youngshik Choe (Korea Brain Research Institute). Pig tissues (heart, kidney, liver, and skin) were obtained from the local market. The human liver, lung, kidney, and skin were dissected from a fresh cadaver donated to the Korea University College of Medicine through a body donation program. All experiments were carried out in accordance with the regulations and approval of the Institutional Animal Care and Use Committee of the Korea University. CASPER Decellularization Method. For tissue CASPERization, tissues were incubated in a hydrogel monomer solution containing 2− 20% acrylamide and 0.1% bis-acrylamide supplemented with 0.25% photoinitiator 2,2′-azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride and 0.01% sodium azide in PBS for 2 h at 4 °C with gentle shaking. Hydrogel-infused specimens were degassed and polymerized for 2 h at 37 °C using the X-Clarity Polymerization system (Logos Biosystems, Korea). Subsequently, the specimens were placed in a bottle filled with 4% SDS solution and gently shaken in a shaker incubator at 37 °C. After decellularization, specimens were washed in deionized water overnight at room temperature while shaking to remove SDS. For electrophoretic tissue decellularization, polymer infusion/polymerization samples were processed in the same manner as above. Next, the polymerized samples were placed on a tissue container in the electrophoretic tissue decellularization chamber (Figure 3a) and processed under the following conditions: 1.2 A for mouse samples, 0.8 A for pig samples, 37 °C, time dependent on sample thickness (Logos Biosystems, Korea). Supplementary Table 1 provides detailed information on the acrylamide monomer concentration, infusion time, SDS concentration, and decellularization processing times (shaking/electrophoresis), according to the samples. Supplementary Table 2 provides Supporting Information regarding materials used for the process. Traditional Decellularization (TD) Method. Mouse liver decellularization was achieved using a method similar to the whole mouse kidney decellularization described previously with modifications (Figure 2b, Figure S2a).31 Briefly, the sample was kept frozen at −80 °C. Before decellularization, the sample was thawed at room temperature for 30 min. The sample was incubated with deionized water for 30 min, 1% Triton X-100 for 16 h, and 0.1% SDS solution for 2 h at room temperature with gentle shaking. In some experiments, decellularization was executed similarly to the CASPER procedure, but without the hydrogel embedding step. 3463


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ACS Biomaterials Science & Engineering Scanning Electron Microscopy (SEM). CASPERized tissues were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer and stored for 24 h at 4 °C. After the samples were washed with 0.1 M phosphate buffer, they were cut into segments of approximately 1 cm length and cryoprotected in 25% sucrose, 10% glycerol in 0.05 M PBS (pH 7.4) for 2 h. Further, they were fast-frozen in nitrogen slush and fractured at approximately −160 °C. Next, samples were placed back into the cryoprotectant at room temperature and allowed to thaw. After they were washed in 0.1 M phosphate buffer (pH 7.4), the samples were fixed in 1% OsO4/0.1 M phosphate buffer (pH 7.3) at 3 °C for 1.5 h and washed again in 0.1 M phosphate buffer (pH 7.4). After they were rinsed with dH2O, specimens were dehydrated in a graded ethanol− water series to 100% ethanol, critical point dried using CO2, and finally mounted on aluminum stubs using sticky carbon tapes. The fractured material was mounted to present fractured surfaces across the parenchyma to the beam and coated with a thin layer of Au/Pd (approximately 2 nm thick) using a Gatan ion beam coater. Images were recorded with a 7401 FEG scanning electron microscope (Jeol, MA, U.S.A.). Histology and Immunostaining Analysis. CASPERized tissues were fixed with 4% paraformaldehyde. CASPERized tissues were then cryoprotected in 30% sucrose in PBS, embedded in OCT compound, and sectioned (100 μm) using a cryotome (Figure 1g, Figure 3e, Figure S4c). Sectioned tissues were stained with Eosin or Picrosirius red for 3 min. The stained samples were washed with dH2O several times at room temperature. Nuclei counterstaining was carried out using Hoechst33342. Slides were imaged and captured on a slide scanner Axio Scan.Z1 (Zeiss, Oberkochen, Germany). For immunolabeling, CASPERized samples were fixed with 4% paraformaldehyde. Samples were sectioned (100 μm) using a cryotome and then incubated with 6% bovine serum albumin (BSA), 0.1% (v/v) Triton X-100, and 0.01% (w/v) sodium azide in PBS for 30 min. Subsequently, tissues were incubated with primary antibodies for 2 h at room temperature with gentle shaking. The stained tissues were washed with PBS several times at room temperature and then stained with the secondary antibodies for 1 h at room temperature with gentle shaking. Details of antibodies and imaging are provided in the Supplementary Table 3. Then, stained samples were washed with PBS several times and immersed in the CUBIC-mount solution for 1 h at room temperature. All stained tissues were imaged using a Leica SP8 confocal laserscanning microscope and processed using ImageJ software. Cell Culture and Repopulation. HeLa cells were cultured in DMEM (Gibco, MA, U.S.A.) medium, supplemented with 10% fetal bovine serum (Gibco, MA, U.S.A.) and 1% penicillin and streptomycin (Gibco, MA, U.S.A.). Cells were infected with the GFP-expressing retrovirus 3 days prior to the experiment. Fixed CASPERized tissues were incubated in 70% ethanol for sterilization. Next, the tissues were kept for 1 h in the complete medium after they were washed with HBSS. Cells were resuspended at a concentration of 5 × 104 cells per CASPERized tissue. Cells were seeded on the CASPERized tissue slice and a 6-well plate. CASPERized tissue slices were kept for 2 h in a humidified environment at 37 °C with 5% CO2, allowing cell attachment after seeding followed by addition of complete culture medium. Implantation. CASPERized tissue scaffolds (4 mm cubic size) were sterilized three times in 70% ethanol and 100% ethanol for 20 min in each step, rinsed twice in distilled water for 30 min, 0.2% peracetic acid/ 4% ethanol solution under shaking for 6 h, and rinsed in sterile PBS for 3 h. CASPERized tissue scaffolds were surgically implanted to the luminal side of the abdominal wall of 2-month-old male mice. After the operating area of the mouse was shaved, the skin was cleaned with 70% ethanol (Merck Millipore, MA, U.S.A.) and 10% povidone-iodine (Firson, Cheonan, Korea). Alfaxalone and xylazine were used to induce anesthesia. A small midline abdominal incision was made, and the CASPERized tissue block was adhered to the abdominal wall, secured in place using 6−0 silk sutures for completion of the skin closure. Mice were euthanized after 3 and 7 weeks, and the implants along with the surrounding tissues were harvested and fixed in 4% paraformaldehyde for immunohistochemistry.

Measuring Depth of Cell Infiltration. The depth of cell infiltration was measured using images of tissue sections with Hoechst33342 counterstaining, by measuring the total distance of the innermost cells from the periphery of the CASPERized tissues along an axis perpendicular to the midline of the CASPERized tissue using ImageJ software. Measurements were made on implanted CASPERized tissue from three different animals (n = 3). The data were expressed as mean ± SD. RNA Isolation. Total RNA from implanted tissues was extracted using TRIzol (Invitrogen, MA, U.S.A.) and the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The purity and integrity of the total RNA were checked using the NanoDrop (Thermo Fisher Scientific, MA, U.S.A.) and 2100 Bioanalyzer (Agilent, CA, U.S.A.), respectively. Microarray and Raw Data Preparation and Statistical Analysis. The microarray service was provided by Macrogen Inc. (Macrogen, Seoul, Korea). Five micrograms of total RNA was used for labeling. Probe synthesis from the total RNA samples, hybridization, detection, and scanning was performed according to standard protocols from Affymetrix Inc. (Santa Clara, CA, U.S.A.). Briefly, cDNA was synthesized using the GeneChip WT (Whole Transcript) Amplification kit as described by the manufacturer. Single-stranded cDNA was synthesized using Superscript II reverse transcriptase and T7-oligo primers at 42 °C for 1 h. Double-stranded (ds)-cDNA was obtained using DNA ligase, DNA polymerase I, and RNase H at 16 °C for 2 h, followed by T4 DNA polymerase at 16 °C for 5 min for gap filling. After cleanup with a Sample Cleanup Module (Affymetrix, CA, U.S.A.), dscDNA was used for in vitro transcription (IVT). cDNA was transcribed using the GeneChip WT Terminal Labeling Kit (Affymetrix). Approximately 5.5 μg of labeled DNA target was hybridized to the Affymetrix GeneChip Array at 45 °C for 16 h according to the Affymetrix standard protocol. After hybridization, hybridized arrays were washed, stained on a GeneChip Fluidics Station 450, and scanned on a GCS3000 Scanner (Affymetrix, CA, U.S.A.). Raw data were extracted automatically from the Affymetrix data extraction protocol using the Affymetrix GeneChip Command Console Software (AGCC). After importing CEL files, the data were summarized and normalized using the robust multiaverage (RMA) method implemented in the Affymetrix Expression Console Software (EC). We exported the results of gene level RMA analysis and performed differentially expressed gene (DEG) analysis. The comparative analysis between the test sample and control sample was carried out using fold change. For a DEG set, hierarchical cluster analysis was performed using complete linkage and Euclidean distance as a measure of similarity. Gene-enrichment and functional annotation analyses for significant probe list were performed using gene ontology (http://geneontology.org/) and KEGG (http:// kegg.jp), respectively. All statistical tests and visualization of differentially expressed genes were conducted using R statistical language v. 3.1.2. (www.r-project. org). We identified differentially expressed genes with at least a 1.5-fold increase or decrease in the implanted tissue between 3 and 7 weeks. Quantitative Real-Time PCR. Total RNA was prepared using the Trizol Reagent (Invitrogen, MA, U.S.A.) through the RNA isolation protocol. cDNA synthesis was carried out using a Superscript kit (Invitrogen, MA, U.S.A.). Real-time PCR was performed on an Applied Biosystems 7500 Real-Time PCR System using iQTM SYBR green supermix (Bio-Rad, CA, U.S.A.), and gene expression levels were determined relative to GAPDH levels (for detailed primer information, see Supplementary Table 4). Surgery for Hernia Repair. Eleven male Sprague−Dawley rats (average weight = 360 g) (Orientbio, SungNam, Korea) were divided into two groups, receiving either traditionally decellularized or CASPERized pig skin sheet scaffolds. Under the effects of anesthesia (2.5−3.0% Isoflurane/O2 gas mixture through a nonrebreather mask), animals were shaved, and the operative site was prepped with a 2% chlorhexidine acetate solution. Ventral hernias were created by incising the skin, subcutaneous tissues, and full thickness of the abdominal wall at the linea alba for a length of 3 cm. After carefully excising the hernia sac using sharp dissection to define hernia borders, a scaffold (3 cm × 3 cm) was fitted in the resultant 3464


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Figure 1. CASPER: polymer-based and high precision decellularized tissue scaffold. (a) Schematic illustration showing the CASPERization process. First, the organ was immersed in a 5−10% acrylamide/0.1% bis-acrylamide and thermal initiator-containing solution for 2 h. Second, the infused acrylamide was polymerized at 37 °C for 2 h. Third, cellular components were removed in a 2−4% SDS solution with shaking for 1−3 days. (b) Macroscopic appearance of a CASPERized mouse embryo (E18) and various organs. Scale bar, 1 cm. (c) Physical properties of different acrylamide concentration and CASPERized mouse kidney with infusions of different acrylamide concentrations. (n = 5, in each group). (d) Changes in the kidney size and loss of DNA contents with different percentages of acrylamide after 12 h of decellularization (n = 4, in each group). (e) Comparison of ̈ organs (n = 3). (f) Analysis of DNA contents in the naive ̈ and CASPERized organs (n = 5, ***p < mechanical properties of CASPERized and naive ̈ and CASPERized tissues were stained with eosin (red) and Hoechst (blue). Scale bar, 0.001). (g) The absence of nuclei in CASPERized organs. Naive 100 μm. defect with approximately 5 mm of circumferential overlap. The skin sheet was placed intraperitoneally and secured to the abdominal wall across the defect in a bridging fashion using eight interrupted 5−0 Prolene (Ethicon, Somerville, NJ) sutures. The overlying skin was closed with skin staples. To minimize contamination of the surgery site, aseptic technique and draping were used during surgery. After 40 and 90 days, all rats were euthanized by inhalation of 70% carbon dioxide. Following euthanasia, the implantation site was harvested and embedded in 10% formalin buffer, for 48 h, and then embedded in paraffin, according to established standard protocols. Subsequently, 5μm-thick sections were deparaffinized twice in fresh xylene for 8−10 min and rehydrated sequentially with decreasing ethanol concentrations (100%, 95%, 90%, 80%, 70%, 50%) and distilled water (8−10 min) for each step (Figure 7e, Figure S7c). Tissue slices were stained using a kit for Masson’s trichrome staining (Abcam, Cambridge, U.K.), according to the manufacturer’s protocol. For H&E staining, slides were thawed, hydrated, washed, and stained with hematoxylin and eosin according to the manufacturer’s protocol (Sigma-Aldrich, MO, U.S.A.). CD31 immunolabeling was performed using the avidin−biotin complex (ABC) technique for antibody detection. Slides were incubated in 3% H2O2 in methanol for 1 h to quench endogenous peroxidase activity. The sections were washed twice with PBS for 5 min each and then incubated with normal blocking serum for 1 h. This was followed by incubation with CD31 primary antibodies overnight at 4 °C in a humidity chamber. Subsequently, sections were washed with PBS,

again twice for 5 min each, and incubated with biotinylated goat antirabbit secondary antibody (Vectastain ABC Elite kit; Vector Laboratories, CA, U.S.A.) for 1 h. Sections were then treated with ABC solution (Vectastain ABC Elite kit) for 1 h, washed with PBS, and incubated with DAB substrate for 10 min. Counterstaining was carried out with Harris hematoxylin (Sigma-Aldrich, MO, U.S.A.). Controls were routinely included. Stained slices were mounted with VectaMount (Vector Laboratories, CA, U.S.A.) mounting medium and then imaged with a Slide Scanner Axio Scan.Z1 (Zeiss, Oberkochen, Germany). Images were processed and analyzed using the ZEN software (Zeiss, Oberkochen, Germany). Adhesion Severity Scoring. The severity of peritoneal adhesions was quantified using Hopkins adhesion Score.33 A score from 0 to 4 was assigned with equal weighting to five different parameters of adhesion formation, frequency, size, and width. Statistical Analysis. All quantitative data are provided as mean ± standard deviation. A two-tailed Student’s t-test was used for all statistical analyses, with a P value 1 mm deep by week 7 (Figure 5b). All implanted specimens were highly vascularized with strong recruitment of host blood vessels (Figure 5c). Immunostaining of markers specific for endothelial cells, CD31, and VEGF revealed that new blood vessels were integrated into the implanted CASPERized tissues and closely associated with the basement membrane, as visualized by collagen type IV (Figure 5d). Fibroblasts were identified by staining alpha smooth muscle actin (α-SMA), and these fibroblasts remained in extra glomeruli structures in the CASPERized kidney tissue, suggesting that fibroblasts may 3469


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Figure 6. Transcriptome analysis of CASPERized tissues after implantation. (a) Clustered heat map of differentially expressed genes in 3- and 7-week CASPERized kidney tissue implants. The abscissa represents the sample, and the ordinate represents the different genes. Yellow indicates high gene expression, whereas blue indicates low gene expression. (b) Biological processes of the differentially expressed genes identified in 3- and 7-week implants using ClueGo plugin in Cytoscape. The 3-week DEGs were grouped into 136 groups, according to their biological processes (GO term with p-value < 0.05). The 7-week DEGs were grouped into six categories, according to their biological processes (GO term with p-value < 0.05). (c) Charts showing the proportions of ECM and immune response gene clusters with up-regulated (pink) or down-regulated (light green) expression in CASPERized tissues 3 vs 7 weeks postimplantation. (d,e) Real-time PCR analysis of genes selected for ECM, fibrosis-related factors (d), and immune response targets (e) in the implanted CASPERized tissues postimplantation 3 vs 7 weeks (n = 3). (f) Immunohistochemical staining for CD11b+ macrophage cells (M1 phage, red) and CD163+ macrophage cells (M2 phage, red) in CASPERized tissue implants. Nuclei were counterstained with SYTO16 (green). Scale bar, 25 μm. Quantitative analysis of CD11b+ and CD163+ cell densities (n = 3, the number of cells in 1 mm2, **p < 0.01 and ***p < 0.001).

phages (CD11b) to anti-inflammatory M2 macrophages (CD163) over time, which aligned with normal tissue regeneration processes (Figure 6f). These results suggest that tissue-regeneration-like processes progressed in CASPERized tissue implants. Application of CASPERized Tissue for Surgical Mesh. Finally, we applied the CASPERized tissue to the rat hernia model to test its effectiveness and applicability for surgical tissue repair. The introduction of supporting materials to enhance hernia repairs improved surgical outcomes; however, pain from organ/tissue adhesions, mechanical mismatch, and potential scar tissue formation, infection, and recurrence are still common complications. Considering that CASPERized tissues exhibited high preservation of structural and biological features of native tissues with enhanced physical strength, we tested their potential use as a hernia mesh. We prepared CASPERized pig skin and compared its physical strength with traditionally decellularized (TD) tissue materials. The CASPERized tissue had a tensile strength four times greater than the decellularized materials (Figure 7a). We implanted CASPERized or TD materials to the animal model of an abdominal hernia. Samples were harvested at

40 and 90 days postoperatively, and we compared the outcomes of peritoneal adhesions, hernia recurrence, and residual implant thickness. We utilized a modified Hopkins Adhesion Score (Figure S7a) to assess the number and severity of adhesions encountered at the time of the sample harvest. The animals receiving CASPERized tissue exhibited only marginal enhancement of peritoneal adhesion, while animals receiving TD exhibited a gradual increase in Hopkins adhesion scores (Figure 7b,c and Figure S7b). Furthermore, hernia recurrences frequently occurred in 2 out of 6 rats treated with TD at 40 days after surgery and 2 out of 3 rats at 90 days. However, in the CASPERized tissue-treated group, reherniation did not occur at both 40 days (n = 5) and 90 days (n = 5) after surgery. High incidences of adhesion and hernia recurrence appeared to be associated with rapid degradation of the ECM component. Accordingly, CASPERized tissue was well preserved until 90 days after implantation, while TD was progressively degraded (Figure 7d and Figure S7b). Histological analysis showed complete regeneration of the omentum on the peritoneal surface of CASPERized tissues, although regeneration of the muscular layer was absent (Figure 7e and Figure S7c). Furthermore, 3470


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Figure 7. Repairing effect of CASPERized tissue in rat hernia model (a) Comparison of tensile strength of traditionally decellularized (TD) and CASPERized pig skins (n = 5 in each group, **p < 0.01, *p < 0.05). Scale bar, 1 cm. (b) Representative images on postoperative day 40 and 90 of hernia repair using TD (upper) and CASPERized tissue (down). (c) Adhesion scores of TD and CASPER groups at 40 and 90 days after surgery (n = 5 for 40 days, n = 3 for 90 days in both groups, ***p < 0.001). (d) Measurement of residual implant thickness. The average thickness of each implanted sheet was obtained after five measurements at the tissue midpoint (n = 5 for 40 days, n = 3 for 90 days in both groups, ***p < 0.001). (e) Histological analysis of Masson’s trichrome- and H&E-stained implanted tissue. Scale bar, 1 mm. Inset, 25 μm. Note the significant differences in residual sheet thickness and omentum regeneration (yellow arrow) following CASPERized tissue implantation. CD31 and FSP1 immunohistochemistry confirmed fibroblasts and neovasculature formation (red arrows) at the anastomotic sites in both groups on postoperative day 40. Scale bar, 25 μm.

perfusion step can be eliminated and substituted by electrophoresis in the CASPER protocol, damaged large organs or parts of large organs can be used for decellularization, which will greatly increase the potential use of damaged human organs for regeneration. Our protocol is inspired by hydrogel-based tissue-clearing techniques, such as CLARITY and ACT, which include acrylamide embedding and electrophoretic tissue processing steps.46,47 CLARITY includes tissue fixation with paraformaldehyde, which immobilizes protein components with a hydrogel polymer, allowing selective removal of lipid components. Further, because CASPER is executed using unfixed organs, the infused hydrogel monomer does not interact with cellular components and selectively provides physical support of the ECM. The infiltrated hydrogel efficiently prevents the ECM from excess damage during the decellularization step, and thus, unfixed cellular components can be robustly eliminated by passive diffusion or active electrophoretic supply of chemical detergents. Therefore, complete removal of the cellular component is easily achieved, which reduces potential immunological responses upon implantation. In addition, the

neovascularization (Figure 7e, CD31) and the presence of fibroblasts (Figure 7e, FSP1) in CASPERized tissues were evident (Figure 7e); yet, signs of fibrosis and calcification were not found. Furthermore, we did not find any signs of long-term inflammatory foreign body responses, as examined by macrophage immunocytochemical staining (Figure S7d). These results suggest that CASPERized tissue can be used as a biomaterial for regenerative medicine.

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DISCUSSION Tissue engineering with the use of decellularized scaffolds has rapidly progressed during the last decades.9−11,14−20,22,24 However, the properties and composition of the ECM and cells are highly diverse, depending on the species, ages, and type of organs, and there is no standard protocol for universal decellularization. Therefore, protocol optimization for each organ is very sophisticated and often challenging. In particular, it is difficult to preserve the detailed structure and ECM quality after decellularization of soft organs. Our CASPER protocol is applicable to decellularization of various types and sizes of organs/tissues, from small to large animals. Because the 3471


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of ECM architectures. Therefore, CASPER offers unique opportunities to examine cell and ECM interactions in a wide range of organs as experimental tools in vitro and for transplantation of processed organs or tissues for regeneration in vivo.

hydrogel polymer in CASPERized materials prevents rapid degradation of the ECM after in vivo implantation, providing desirable properties as biomaterials for tissue restoration surgery. Bioscaffolds can be produced via synthetic or natural polymers as bioinks.48,49 Synthetic polymers can be synthesized with appropriate physical, chemical, and biomemetic properties and designed with a structure suitable for applications such as joint prostheses, vascular catheters, heart valves, ligaments, wound dressings, and surgical meshes.49−53 However, they often cause complications such as adverse inflammatory foreign body responses, postimplantation infections, poor tissue ingrowth and vascularization of implanted materials, and low integration with the host tissue.54,55 Natural polymers are usually obtained from natural tissues, which have assured biocompatibility and bioactivity. However, natural polymers have disadvantages such as poor consistency, loss of some biological activity features, weak mechanical strength, and higher cost.56−58 The combination of synthetic and natural polymers may increase advantages of the high-performance desirable mechanical characteristics, consistency of synthetic properties with lower cost, multifunctionality, biodegradability, and biocompatibility of natural polymers.59−61 In this respect, our CASPER strategy, which is a fusion of tissue ECM with a synthetic hydrogel polymer, may offer a wide range of biomedical applications by obtaining new materials with a broader range of physical and biological properties. We found that CASPERized tissues were well-adapted to the host environment and last for extended periods (>10 weeks), recruiting blood vessels and host cells (fibroblasts, immune cells, etc.). There was considerable remodeling of ECM structures after implantation, primarily because of random angiogenesis and preferential infiltration of fibroblasts. In addition to the continuous ECM remodeling, pro-inflammatory to antiinflammatory conversions of macrophages were detected, which is a common regenerative response of the host immune system.62−64 Furthermore, CASPERized tissue sheets showed the superior mechanical compatibility and ability to promote omentum tissue reconstruction, reducing postoperative complications and hernia recurrences.56,57 Considering the physical and functional preservation of native tissues after the process, CASPERized tissues can be used in various medical applications, in addition to hernia repair, especially when strong mechanical strength and high durability are desirable.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.9b00219. Mouse kidney decellularization with different concentrations of acrylamide infusion, digital and confocal images of CASPER organs, physical and biological characteristics of CASPER, microarray data analysis, hernia repair using traditionally decellularized (TD) skin sheet and CASPERized skin sheet, sources and information on materials, reagents, condition of CASPERization, sequence information for real-time PCR (PDF) High-resolution 3D imaging of skin and hair structures through collagen type IV and keratin double-labeling (MP4)

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Neuronal cells, HT22 cells, in CASPERized mouse liver tissues along with the ECM structure (MP4)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +82-2-2286-1404. Fax: +82-2-929-5696. ORCID

Woong Sun: 0000-0003-1792-4894 Author Contributions

W.S. and E.L designed the study. E.L. performed most of the tissue decellularization and tissue immunolabeling. H.J.K. and J.R.R. contributed to the in vitro 3D cell culture experiments. M.R.S. and G.H.S. performed the gene-based computational analysis. M.S.H., S.H.S., and D.H.K. performed the surgery for hernia repair in the rat model. K.L. and N.J. manufactured the electrophoretic tissue decellularization system. I.J.R., H.K., and Y.C. provided experimental materials, including human tissue. W.S. and E.L. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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CONCLUSIONS The CASPER (Clinically and Experimentally Applicable Acellular Tissue Scaffold Production for Tissue Engineering and Regenerative Medicine) protocol is a highly flexible tissue decellularization technology that combines a synthetic polymer and an electrophoretic process. CASPERized tissues preserve the sophisticated microstructures of the extracellular matrix (ECM), comparable to that in the original tissue, even though the combined robust cell removal step completely removes cellular compartments from the samples. Because the polymer protects the ECM from excess damage during the decellularization step, a robust procedure such as electrophoretic tissue processing can be applied. Even without perfusion, decellularization of large organs can be achieved. CASPERized organs have a high porosity to allow repopulation of cells in vitro and in vivo. In particular, CASPERized tissue implants stimulate normal tissue-regeneration-like tissue remodeling, including angiogenesis, pro- to anti-inflammatory reactions, and remodeling

Notes

The authors declare the following competing financial interest(s): N.J. is founder and shareholder of Logos Biosystems, Inc.

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ACKNOWLEDGMENTS We greatly appreciate the technical support of Ms. Jieun Na for SEM analysis. We also thank the Korea Institute of Science and Technology (KIST) for helping to measure physical strength. Instruments (IMARIS) was supported by Brain Research Core Facilities in Korea Brain Research Institute (KBRI). This research was supported by the Brain Research Program through the National Research Foundation (NRF) funded by the Korean Ministry of Science, ICT & Future Planning (NRF2015M3C7A1028790, 2018R1D1A1A02086190), KBRI basic research program through the Korea Brain Research Institute funded by the Ministry of Science and ICT (19-BR-02-01). 3472


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High-Performance Acellular Tissue Scaffold Combined with Hydrogel

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