Digital light processing 3D printed silk fibroin hydrogel for cartilage tissue engineering

Journal Pre-proof Digital Light Processing 3D printed Silk Fibroin Hydrogel for Cartilage Tissue Engineering

Heesun Hong, Ye Been Seo, Do Yeon Kim, Ji Seung Lee, Young Jin Lee, Hanna Lee, Olatunji Ajiteru, Md. Tipu Sultan, Ok Joo Lee, Soon Hee Kim, Chan Hum Park PII:

S0142-9612(19)30778-1

DOI:

https://doi.org/10.1016/j.biomaterials.2019.119679

Reference:

JBMT 119679

To appear in:

Biomaterials

Received Date:

17 June 2019

Accepted Date:

10 December 2019

Please cite this article as: Heesun Hong, Ye Been Seo, Do Yeon Kim, Ji Seung Lee, Young Jin Lee, Hanna Lee, Olatunji Ajiteru, Md. Tipu Sultan, Ok Joo Lee, Soon Hee Kim, Chan Hum Park, Digital Light Processing 3D printed Silk Fibroin Hydrogel for Cartilage Tissue Engineering, Biomaterials (2019), https://doi.org/10.1016/j.biomaterials.2019.119679

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

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Digital Light Processing 3D printed Silk Fibroin Hydrogel for Cartilage Tissue Engineering

Heesun Hong, 1* Ye Been Seo,1*Do Yeon Kim,1 Ji Seung Lee,1 Young Jin Lee,1 Hanna Lee,1 Olatunji Ajiteru,1 Md. Tipu Sultan,1 Ok Joo Lee,1 Soon Hee Kim1, and Chan Hum Park1,2

DOI: 10.1038/s41467-018-03759-y 1

Nano-Bio Regenerative Medical Institute, College of Medicine, Hallym University, Chuncheon 24252, Republic of Korea.

2

Departments of Otorhinolaryngology-Head and Neck Surgery, Chuncheon Sacred Heart Hospital, School of Medicine, Hallym University, Chuncheon 24252, Republic of Korea.

*These authors contributed equally to this work

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Address for correspondence: Chan Hum Park

Nano-Bio Regenerative Medical Institute Hallym University 1 Hallymdaehak-gil, Chuncheon Gangwon-do, 24252 Republic of Korea

Department of Otorhinolaryngology-Head and Neck Surgery Chuncheon Sacred Heart Hospital School of Medicine Hallym University 77 Sakju-ro, Chuncheon Gangwon-do, 24253 Republic of Korea

E-mail: [email protected] Tel: 82-33-240-5181 Fax: 82-33-241-2909

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Abstract

Three-dimensional printing with Digital Lighting Processing (DLP) printer has come into the new wave in the tissue engineering for regenerative medicine. Especially for the clinical application, it needs to develop of bio-ink with biocompatibility, biodegradability and printability. Therefore, we demonstrated that Silk fibroin as a natural polymer fabricated with glycidyl-methacrylate (Silk-GMA) for DLP 3D printing. The ability of chondrogenesis with chondrocyte-laden Silk-GMA evaluated in vitro culture system and applied in vivo. DLP 3D printing system provided 3D product with even cell distribution due to rapid printing speed and photopolymerization of DLP 3D printer. Up to 4 weeks in vitro cultivation of Silk-GMA hydrogel allows to ensure of viability, proliferation and differentiation to chondrogenesis of encapsulated cells. Moreover, in vivo experiments against partially defected trachea rabbit model demonstrated that new cartilage like tissue and epithelium found surrounding transplanted Silk-GMA hydrogel. This study promises the fabricated Silk GMA hydrogel using DLP 3D printer could be applied to the fields of tissue engineering needing mechanical properties like cartilage regeneration.

Keywords: Digital Light Processing (DLP) 3D printing, Silk fibroin – glycidyl methacrylate (Silk-GMA), Chondrogenesis, Cartilage tissue engineering

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1. Introduction 3D bio printing technology is one of the effective ways for creating scaffolds that can contain cells with appropriate microenvironmental condition for tissue engineering and regenerative medicine [1, 2]. In the past few years, the interests of Digital Light Processing (DLP) using as a 3D bio printing technology for the creation of more complex tissue and organ structure has been increased [3-6]. DLP printer is one of the light-assisted direct 3D bioprinting technologies can provide the solution for some disadvantages of inkjet printing and extrusion printing, such as large mechanical stresses on the encapsulated cells in high-viscosity hydrogels or application with high cell dose for various shape of 3D tissue structures or reduction of cell viability due to long printing time such as indirect 3D printing system [7-9]. DLP 3D bioprinter is based on a local photo polymerization process with cross-section of the product to cure the liquid photocurable resin layer by layer induced by UV projection [8, 10]. DLP 3D bioprinter provide speedy printing time at 1 mm3 per every second with high resolution about 1 um. due to short printing time without nozzle system and printing in the aqueous process, higher cell viability could be served compare to other 3D printers like inkjet, extrusion and laser assisted type printer [6, 11-13]. Hydrogels as a scaffold formed 3D crosslinked hydrated fibers in one of the importance materials in 3D bioprinting. For application of tissue engineering and regenerative medicine, it can be used as a cell matrix with biocompatible and biodegradable support, and the properties as native tissue that has extracellular matrix reinforced by 3D network [14-16]. The hydrogel must have mechanical stability against the physical impact and mimicking conditions, which lead to tissue regeneration for implantation purposes [17-19]. However, despite of these advantages of 3D printed hydrogel as a scaffold, in vivo applications such as transplantation still are a few. 1

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Silk from Bombyx mori is a natural fibrous polymer composed of two proteins, fibroin and sericin. Silk fibroin (SF) has been used for a various biomedical and biotechnological applications due to biocompatibility, biodegradability, high tensile strength, and excellent biological characteristics such as proliferation and adherence of various cells, and low inflammation [20-24]. In particular, SF can be processed into various forms and structures of amino-acid modifications with the addition of methacrylate groups to amine residue groups of hydrogels such as the gel, membrane, powder and porous sponges under all aqueous processing conditions and light polymerization [29-32]. Success of deposition and repair process for cartilage depend on the activity of chondrocyte. Chondrocytes from cartilage include knee joint, trachea, and other tissues can grow on the artificial matrices and implanted subcutaneously or direct injection with modified autologous chondrocytes. In vivo maturation, exhibiting morphological and biological functions like cartilage formation with chondrocyte in lacunae which regulate extracellular matrix (ECM) turnover include glycosaminoglycan (GAG) production and maintain tissue homeostasis similar to native cartilage [25-27]. Although many surgical procedures include micro-fracture, or transplantation of manipulated autologous chondrocytes with growth factors, cytokines or gene therapies attempts have been tried for low self-regeneration capacity of chondrocyte, successful treatments for large cartilage defects still have some limitation to over-come for potential cartilage regeneration with tissue engineering technologies [28-30]. In a previous study [8], we chemically modified bio-ink for DLP 3D printing of SF with glycidyl methacrylate (GMA) (Silk-GMA) and set the final concentration of Silk-GMA in hydrogel. From these results, we suggested that 3D printed Silk-GMA hydrogel with chondrocyte by DLP printing under in vitro culture system can provide a new strategy for tissue 2

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engineering. In this study, we demonstrated that fabricated human and rabbit derived chondrocyte laden SF hydrogel modified with GMA preparing by DLP 3D printing and evaluated its applicability as an artificial trachea with in vivo transplantation for the first trial. Moreover, we confirmed cytocompatibility, mechanical properties and biodegradability of the hydrogel, glycosaminoglycan (GAG) production, histological chondrogenic expression and new cartilage formation in vitro and in vivo transplantation.

2. Materials and Methods Preparation of glycidyl methacrylated Silk Fibroin (Silk-GMA). Glycidyl methacrylated silk fibroin (Silk-GMA) bio-ink was prepared from our previous established method [8]. In brief, Bombyx mori cocoons were collected from the Rural Development Administration (Jeonju, Korea). To remove the sericins, 40 g of sliced cocoons by four pieces were boiled in 1 L of 0.05 M Na2CO3 solution at 100 °C for 30 min, and washed with distilled water several times. Then, degummed silk fibroin (SF) was dried at dry oven for 36 hrs. For the synthesis of SF and GMA, 40 g of degummed SF was dissolved in 9.3M of lithium bromide (LiBr) solution at 60°C for 1 hour, and 6ml of glycidyl methacrylate (211.5 mM, GMA, Sigma-Aldrich, USA) and were mixed with stirring at 1000 rpm for 6 hours at 60 °C to make reaction between SF and GMA. Then, the solution was filtered through a miracloth (Calbiochem, San Diego, USA) and to remove salts, dialyzed with distilled water using dialysis membranes (MWCO 12–14 kDa, Spectrumlabs, USA) for 7 days. Resulted Silk-GMA solutions were frozen at −80 °C for 12 h and freeze-dried for 36 hours. Silk-GMA formed like the sponge-textured was stored 4 °C for further use. Figure 1-A illustrated a schematic presentation for methacrylation of SF with GMA. 3

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Digital Light Processing (DLP) 3D Printing. From our previous published study [8], we designed and fabricated a high-quality DLP printer for laboratory scale printing to reduce of over-cured layering for the best-quality after printing (Fig. 1-B). Our DLP printing system consists of three major components : 1) UV Digital Micro-mirror Device™ with a resolution of 30 µm (Texas Instruments, Dallas, USA), 2) UV-LED (365 nm, LG Innotec, Seoul Korea), and 3) a lens module with two UV-grade biconvex lenses. The build plate is made for an adjustable thickness of various layer. The system was professionally customized (NBRTech. Ltd, Chuncheon, Korea). Before the application to DLP printer, the patterns for printing was designed by CAD software CADian3D (IntelliKorea, Seoul, Korea) and sliced to a layer file. Prints were carried out by repeating the image projecting into the Silk-GMA hydrogel. Printing conditions were used as follows: 1) for in vitro and in vivo experiment, printing thickness, 40 μm; number of base layer, 3; curing time of base layer, 5 s; number of buffer layer, 7; curing time of buffer layer, 4.5 s; and size, 10 x 10 x 2 mm and 5 x 5 x 2 mm (W x L x H), respectively; 2) for in vitro experiments printing thickness, 50 μm; number of base layer, 3; curing time of base layer, 4 s; number of buffer layer, 1; curing time of buffer layer, 3 s; size 7 x 5 x 2 mm (external diameter x internal diameter x height), respectively (Table 1).

In vitro experiment Preparation of Cells. NIH/3T3 mouse fibroblast cells were purchased from ATCC (USA). Cells were cultured for 3 days in 100 mm culture dish (Corning Inc., Corning, NY, USA) containing DMEM-high glucose (Dulbecco’s modified Eagle medium-high glucose, Welgene, Korea) supplemented with 10% Fetal Bovine Serum (FBS, Atlas Biologicals, USA) and 1% antibiotic-antimycotic (A/A, Thermo Fisher Scientific, USA). Human chondrocytes 4

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from septoplasty patients were isolated from nasal septal cartilage with informed consent. The procedures described in this study were approved by the Institutional Review Board of Chuncheon Sacred Heart Hospital of Hallym University (IRB No. 2017–87). The cartilage was washed several times with sterile phosphate buffered saline (PBS, GIBCO, USA) and chopped with a scalpel blade under aseptic conditions. Then the minced cartilage digested with 0.6% collagenase A (Roche Applied Science, Germany) at 37 °C for 6 h. Isolated chondrocytes were cultured into 100 mm culture dishes until 85% confluency and subcultured up to two passages. The cell medium consists of DMEM with 10% FBS and 1% penicillin-streptomycin (P/S, GIBCO, USA). All cultures were maintained in 5% CO2 incubator at 37 °C with the medium changing every 3–4 days. Cells are detached using trypsin-EDTA 0.25%, counted with 0.4% trypan blue (GIBCO, USA) exclusion by LUNA II automatic cell counter (Logos, Korea), and prepared for the DLP 3D printing.

Printing of cell-laden Silk-GMA hydrogel. Firstly, to prepare of bio ink with Silk-GMA (Fig 1-B), freeze-dried Silk-GMA was dissolved in serum free DMEM-F12 with the photoinitiator, 0.6% of Lithium phenyl(2,4,6-trimethylbenzoyl) phosphinate (LAP, Tokyo chemical industry, Tokyo, Japan) in hydrogel prepolymer. And then, the Silk-GMA hydrogels were filtered with 40 μm of cell strainer (93040, SPL Life Sciences, Korea), and sterilize at 60° C for 30 minutes. For the cell printing, 1 x 106 cell of human chondrocytes at passage 4 and NIH/3T3 cells were mixed with Silk-GMA bio-ink respectively, and these cell-laden Silk-GMA bio-ink hydrogels (5 x 5 x 2 mm) were printed using DPL 3D printer. After printing, the chondrocyte-laden SilkGMA hydrogels were washed with sterile 1x PBS and cultured in a humidified atmosphere of 5% CO2 at 37 °C in a 6-well and 24-well plate (Corning Inc., Corning, NY, USA) containing DMEM-F12 media with 10% FBS and 1% A/A, respectively. 5

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In vitro experiments

Assessment of cell distribution in Silk-GMA hydrogel. On culture day 0, 4, and 7, each piece of Silk-GMA hydrogel with NIH 3T3 was counted by program in IN Cell Analyzer 2200 system to confirm of distribution after printing. NIH/3T3 cell line was stained with PKH26 kit (Red fluorescence cell linker, Sigma-Aldrich, USA) and Masson’s Trichrome staining (MT, Sigma-Aldrich, USA) according to the manufacturer’s instruction. In brief, in PKH26, cells were culture and harvested, and then mixed with Silk-GMA solution at a density of 2 × 107 cells per ml for printing out through DLP printer. Silk-GMA hydrogels with NIH 3T3 were cultured in an incubator at 37 °C in a humidified atmosphere of 5 % CO2 for 0 to 7 days. Then, the collected hydrogels were fixed in 4% paraformaldehyde. The hydrogels were treated with 1 % poly vinyl alcohol (PVA, Sigma-Aldrich St. Louis, USA) and incubated overnight at 4 °C. The hydrogels were embedded with Optimum Cutting Temperature compound (OCT compound, Leica Biosystems Melbourne Pty Ltd) and frozen at -80 °C. The samples were sectioned at 14 μm thickness using cryosections (Leica CM3050 S, Leica Biosystems, Germany). Fluorescence images were obtained at InCell Analyzer 2200 (InCell Analyzer 2200 Cell Imaging System, GE Healthcare Life Sciences) and cell counting was done by InCell Developer Toolbox (v1.6) using InCell Analyzer workstation.

Evaluation of cell proliferation and viability in Silk-GMA hydrogel. After DLP 3D printing of Silk-GMA hydrogels containing 1 × 106 cells of human chondrocyte, each chondrocyteladen Silk-GMA hydrogels were cultured for 0, 3, 5, 7, and 14 days at 37 °C in a humidified atmosphere of 5 % CO2. As a control was used Cell-free-Silk-GMA hydrogel. Cell Counting Kit-8 assay (CCK-8, EnzoBiochem, NY, USA) evaluated for cell proliferation according to 6

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the manufacturer’s protocol. Live & Dead assay kit (Invitrogen, Carlsbad, USA) was used for cell viability according to manufacturer’s protocol. In brief, each Silk-GMA hydrogel of culture time was washed with D-PBS (Dulbecco’s phosphate buffered saline, GIBCO, USA). And then incubated D-PBS containing 2uM Calcein-AM and 4 uM ethidium homodimer-1 for 30 min at 37 °C and 5% CO2 incubator. After incubating, stained hydrogels were washed with D-PBS, and results were visualized and imaged using a fluorescent microscope (Eclipse 80i, Nikon, Tokyo, Japan).

Cartilage tissue engineering. 30% of Silk-GMA solution containing 1 × 107 cells per ml of human chondrocytes was printed in a trachea-ring shape (7 mm of external diameter, 5 mm of internal diameter, and 2 mm of height) by DLP printer for in vitro cartilage tissue engineering (Table 1). For in vitro test, the chondrocyte-laden Silk-GMA hydrogels were kept in a CO2 incubator up to 4 weeks with changing media every 2 days. All constructs were evaluated at each time point of 1, 2, 3, and 4 weeks by histological analysis.

Sulfated glycosaminoglycan (sGAG) assay. The sulfated glycosaminoglycan (sGAG) analysis for evaluation of the chondrogenic regeneration in the hydrogel was performed according to the protocol from the Sulfated Glycosaminoglycan Assay Kit (Biocolor, Carrickfergus, U.K.). Briefly, the chondrocyte-laden Silk-GMA hydrogels were digested by papain extraction reagent at 65°C overnight. The papain extraction reagent is prepared by dissolving 400 mg of sodium acetate (Sigma-Aldrich), 200 mg of EDTA-disodium dihydrate (Duchefa Biochemie, Netherland), and 40 mg of L-cysteine hydrochloride (Sigma-Aldrich, USA) in 50 ml of a 0.2 M

sodium phosphate buffer (Na2HPO4 – NaH2PO4, Sigma-Aldrich, USA) and 250 μl of a papain (Papain from papaya latex, P3125, Sigma-Aldrich, USA). Samples were treated with papain 7

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extract reagent and centrifuged at 1000 xg for 10 minutes, some of the supernatants were used to measure the amount of dsDNA, and then the remaining supernatants were mixed with blyscan dye reagent. Absorbance was measured at 656 nm with microplate readers (Absorbance Reader, BioTek, Winooski, USA).

Chondrogenic gene expression analysis with RT-PCR. The chondrocyte-laden Silk-GMA hydrogels were cultured for 2 to 4 weeks, then washed once with PBS and frozen in liquid nitrogen for 1 minute. Subsequently, the frozen hydrogel in the sterilized tube containing beads was crushed twice for 20 seconds using a Hand Held Homogenizer (SuperFastPrep-2, MP Biomedicals, Santa Ana, California, USA). Total RNA was extracted using Trizol Reagent (Invitrogen, Carlsbad, Calif.). In brief, after minced of frozen hydrogel with Trizol, the aqueous phase containing RNA was isolated with chloroform/isopropyl alcohol. The RNA pellets were washed with 75 % ethanol, and then air-dried for 10 min. And after re-dissolved in 20 µl RNase-free DEPC water, isolated RNA was stored in deep freezer (−80 °C). 25 ng of RNA was used to synthesize cDNA using Go Script ™ Reverse Transcription Mix (Promega, Madison, USA). Primers were used for the amplification of cartilage specific genes; (1) Collagen type II, (2) Collagen type X, (3) Sox-9, (4) Aggrecan, and (5) GAPDH as a commonly endogenous gene (Table 2). Amplification of specific genes was performed in a SimpliAmp Thermal Cycler (Thermo Scientific, California, USA) with Maxime PCR premix kit (Intron Biotechnology, Korea). PCR amplification conditions were 35 cycles of 95° C for 30s, 55 ° C for 30s and 72° C for 30s. Gel electrophoresis was performed 1% agarose (LE agarose, Genomic one, Korea).

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Histological analysis of in vitro cartilage tissue formation. The printed chondrocytes-laden Silk-GMA hydrogels were cultured for 1, 2, 3, and 4 weeks incubated at 37 °C in a humidified atmosphere of 5% CO2. Cell labeling was performed using the PKH-26 kit (Sigma-Aldrich, USA). Fluorescence images were obtained with a fluorescence microscope (Eclipse 80i, Nikon, Tokyo, Japan) at a wavelength of 565 nm. Cultured chondrocyte-laden Silk-GMA hydrogels from in vitro experiments were fixed with 10% formalin, immersed in sucrose solution for 1 day and molded with Tissue-Teck Optimum Cutting Temperature compound (Tissue Tek, Elkhart, IN, USA), and frozen with liquid nitrogen, and cut to 10 μm thickness. For the detection of the PKH26-labeled chondrocyte, structure of tissue and chondrogenesis in SilkGMA hydrogels were stained with Dapi (Vector Lab., Burlingame, USA) and observed under a fluorescence microscope (Eclipse 80i, Nikon Co., Japan). Hematoxylin and eosin (H & E) to confirm tissue and cell morphologies, Safranin-O staining for the identification of the proteoglycan-rich matrix, and Masson’s Trichrome (MT) staining to assess the collagen production were performed. Stained sections were analyzed under a light microscope (Eclipse 80i, Nikon Co., Japan).

In vivo experiments All animal experiments in this study was approved by the institutional animal care and use committee of Hallym University (IACUC No. Hallym 2018-10), Chuncheon, Korea.

Implantation of chondrocyte-laden Silk-GMA hydrogel. For observation of hydrogel degradation, chondrocyte-laden or chondrocyte-free Silk-GMA hydrogel was implanted. In brief, 8-week old, female athymic mice (BALB/c-nude mice; Kangwon bio, Korea) were 9

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anaesthetized with xylazine and ketamine. Dividing based on the backbone of mouse, 1 of chondrocyte-laden or chondrocyte-free Silk-GMA hydrogel was implanted into the dorsal subcutaneous spaces of the mice, each site of right and left, respectively, (n = 4). 8 weeks after implantation, the implanted Silk-GMA hydrogels were removed, and then fixed in 4% PFA, embedded in paraffin, and sectioned transversely into 14 um-thickness sections for histological analysis.

Three-point bending test. Three-point bending test was performed to evaluate the mechanical strength as a support to be implanted in the rabbit trachea. The flexural modulus was measured using a general-purpose tester (QM 100SS, Qmesys Corp., Kyounggi-do, Korea). The length of the specimen was 12 mm, and the distance between two points of contact was 10 mm. The normal trachea tissues of the rabbit as a control, modified gelatin with glycidyl methacrylate as a comparative, the cell-free and the cell-containing Silk-GMA hydrogels were cultured up to 2 weeks and their strength was compared (Fig 7-A).

Preparation of Rabbit chondrocytes. 15 weeks old, New Zealand white rabbit’s ear (Kangwon Science, Korea) were used for isolation of chondrocyte. In brief, the extracted ear cartilage tissue was washed with 1x PBS (pH 7.4 Solution) and chopped in small pieces. Then choppedcartilage was placed in serum free DMEM-F12 (1:1) containing 0.001% of Collagenase type II (250 U/mg, Worthington Biochemical Corporation, USA) for 24 hours in a humidified incubator with 5% CO2 at 37°C. On the next day, cultured ear cartilage tissues were centrifuged at 2000 rpm for 5 minutes. After the supernatant was removed, and the remnants incubated for 24 hours with collagen type II treatment with supplied 5% CO2 at 37°C. On day 3, the 10

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impurities were removed, then, the cells were collected and seeded in cell culture dish (Corning Inc., Corning, NY, USA) containing DMEM-F12 (1:1) supplemented with 10% FBS and 1% A/A solution. Cells were cultured for 3 days in 100 mm culture dish (Corning Inc., Corning, NY, USA). After culturing up to 4 passages, the rabbit chondrocytes were used for in vivo artificial trachea transplantation.

Artificial trachea transplantation. Printed Silk-GMA hydrogel with rabbit chondrocytes were cultured for 6 weeks. New Zealand white rabbits, 15 weeks age (n = 2) were used for traches partial defect model. The rabbits were anesthetized with 2:1 mixture of ketamine (250 mg/5 ml, Huons, Korea) and Rompun injection (23.32 mg/ml, Bayer Korea, Korea). A partial defected trachea model was prepared on the normal trachea by the cutting estimated area of trachea with size of 10 x10 mm, and printed artificial trachea was transplanted into the cutting area with Silk-GMA hydrogel with rabbit chondrocytes cultured for 6 weeks. After the operation, steroids (5 mg/ml, Ilsung Pharmaceuticals CO., LTD, Korea) has been injected to the rabbits and the status was observed through an endoscope every 2 weeks (Fig 8).

Histological analysis of in vivo cartilage tissue formation. Every implanted Silk-GMA hydrogel harvested from dorsal subcutaneous spaces of nude mouse on implanted time 1, 2, 4, and 8 weeks. 14 μm-thickness sections of Silk-GMA hydrogels were prepared for PKH26 fluorescence, H & E staining, Safranin-O, and MT staining for observation of Silk-GMA hydrogel degradation. And, to detect cartilage generation of transplanted chondrocyte-laden Silk-GMA hydrogel to partial trachea defected rabbit model, rabbit has sacrificed 6 weeks after transplantation. Whole rabbit trachea include transplanted Silk-GMA hydrogel was harvested, and then 10 μm-thickness cross-sections were prepared for H & E and MT 11

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staining. All stained trachea sections were analyzed under a light microscope (Eclipse 80i, Nikon Co., Japan), and PKH26 stained samples observed under a fluorescence microscope.

Statistical analysis. All data were expressed as mean and standard deviation and compared using t- test. The difference was considered statistically significant at p < 0.05. Data were analyzed using the Graph Pad Prism 6 (Graph Pad Software, Sandie go, CA).

3. Results 3.1. Analysis of Cell distribution in Silk-GMA hydrogel after DLP 3D printing For the confirmation of cell distribution in the Silk-GMA hydrogel during printing and culture periods, printed NIH 3T3-laden Silk-GMA hydrogel was sectioned, and detected with InCell Analyzer and fluorescence microscope after stained with PKH26 in red fluorescence and Masson's trichrome (MT). Distribution of cells in Silk-GMA hydrogel after printing (0 d) was confirmed that cells were evenly distributed over the Silk-GMA hydrogel. In addition, during cultivation for 4~7 days, the cells in Silk-GMA were evenly proliferated in Silk-GMA hydrogels with detection in MT and PHK26 staining (Fig 2-A). In the results of cell counting depending cultivation time from 0, 4 and 7 days, cells distribution in Silk-GMA hydrogels became even of top and the bottom portion of Silk-GMA hydrogel (Fig 2-B).

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3.2. Evaluation of cell viability and proliferation in Silk-GMA hydrogel To determine the cell viability and proliferation in the 3D printed hydrogel, human derived chondrocytes-laden Silk-GMA hydrogels were cultured for 0 ~ 14 days. Live / Dead assay and Cell Counting Kit-8 (CCK-8) assay were used. The graph of the CCK-8 results presents the cell viability at each time point (3, 5, 7, 14 days). The averages of the proliferation activity were 1.33 ± 0.027, 1.436 ± 0.023, 1.608 ± 0.059, 1.73 ± 0.089, and 2.874+0.11 in culture time 0, 3, 5, 7, and 14 days, respectively. The proliferative activity of the cells in Silk-GMA hydrogel gradually increased up to 14 days, and moreover significant cell growth was observed from the 7 days to 14 days (Fig. 3-A). For the confirmation of cell viability in Silk-GMA hydrogel, we performed the cell viability using Live and Dead assay at each culture time. Images of Live & Dead assay resulted by confocal microscope, live cells in green fluorescence with Calcein-AM showed no significant difference among 0 day to 5 days. However, the chondrocytic cell body was detected after 5 days of culture, and on 14 days, culture showed significantly more active cell growth was found compared to other time point (Fig. 3-B).

3.3. In vitro histological analysis for cartilage regeneration Hematoxylin and eosin (H & E), Masson's trichrome (MT), Safranin O (SFO) and PKH67 staining were performed of 1, 2, 3, and 4 weeks cultivation of human derived chondrocyteladen Silk-GMA hydrogel. Every histological result showed that the cartilage cell structure and neo-cartilage formation appeared in the Silk-GMA hydrogel with human derived chondrocytes of 1-week cultures. In H & E staining, the number of chondrocyte increased culture periods from 1 to 4 weeks, and chondrocyte population gradually increased according to culture period. In MT staining, the portion that was differentiated into cartilage was specifically stained with 13

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blue color when differentiated into cartilage, and cytoplasm and nuclei in light red and cell in dark blue respectively. Especially, on 4th-week of cultivation was stained darker than the 1 week. Chondrocytes were observed in the pores on Silk-GMA with red color, and the surrounding matrix gradually stained according to culture period in SFO staining. In addition, PKH staining showed that cells are labeled with yellow-orange within pores of the hydrogel and observed for up to 4 weeks. From these results, Silk-GMA hydrogel can provide an excellent environment for growth and maintenance of chondrocytes (Fig. 4).

3.4. Cartilage expression of in vitro Silk-GMA hydrogel culture GAG synthesized from chondrocytes in the 3D hydrogels was measured blyscan dye exclusion at 1, 2 (5.125 ± 0.38 and 7.042 ± 0.0168, respectively) and 4 weeks (15.3 ± 0.77) cultivation. The GAG contents were not significantly changed on 1 week and 2 weeks cultivation. However, on culture period 4 weeks, GAG contents in cultured chondrocytes-laden Silk-GMA hydrogel was remarkably increased about 3 times more than that of week 1 (Fig. 5-A). Expression of cartilage specific genes such as collagen type II, collagen type X, Sox-9, and aggrecan in 3D hydrogels with chondrocytes were detected by reverse transcription-polymerase chain reaction (RT-PCR) and gel-electrophoresis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control gene (Table 2). Fig 5-B shows that cartilage genes include collagen type II, collagen type X, Sox-9, and aggrecan did not express on culture period from 1 week to 2 weeks. However, at the end of 4 weeks cultivation, cartilage specific genes were expressed on collagen type II, collagen type X, Sox-9, and aggrecan. These data shows that Silk-GMA as a hydrogel resulted to provide sufficient condition for cartilage matrix production.

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3.5.

Histological analysis of in vivo Silk-GMA hydrogel to cartilage tissue formation

and observation of Silk-GMA hydrogel degradation We performed to exhibit in vivo cartilage like tissue formation of chondrocyte Silk-GMA hydrogels with implantation to nude mice (Fig 6). Depend on cultivation time from 1 ~ 8 weeks in dorsal subcutaneous part of nude mice, disk type (size 7 x 5 x 2 mm) of chondrocyte-laden Silk-GMA hydrogel group showed histological characteristics of cartilage like tissue, such as cell organization and extracellular matrix distribution, including proteoglycan and collagen with H&E, MT, SFO and PKH26 staining compare to chondrocyte-free Silk-GMA hydrogel with H&E and PKH26 staining (Fig 6-A). H & E staining results showed that implanted chondrocyte-laden Silk-GMA hydrogel showed embedded chondrocyte in Silk-GMA. In Safranin O staining demonstrated that chondrocyte in Silk-GMA hydrogel could produce chondrogenic matrix with detection by orange to red color, and collagen matrix in blue staining in MT staining. In fluorescence microscopic examinations with PKH26, chondrocytes detected in red color for up to 4 weeks in vivo (Fig 6-B). Encapsulated chondrocytes in Silk-GMA hydrogel and their matrix surrounded by hydrogel were observed with getting darker stained over the cultivation time in nude mice. Observation of Silk-GMA up to 8 weeks after in vivo cultivation, the Silk-GMA hydrogel provided an excellent environment for the growth of chondrocytes and cartilage formation in vivo. Moreover, encapsulated chondrocyte sits in the pores of silk-GMA, and smaller size of pores compare to earlier periods were evenly spread due to tissue ingrowth of transplanted hydrogel.

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3.6. Three-point bending test for the evaluation of mechanical property To confirm the mechanical strength of the artificial trachea model, 3-point bending test by general-purpose tester was investigated on chondrocyte-laden, -free Silk-GMA hydrogel, -free Gel-GMA (printing size = 12 mm x 10 mm, L x W) with up to 2 weeks cultivation and native trachea tissue from rabbit as a normal control (Fig. 7-B). Firstly, chondrocyte-free GelatinGMA hydrogel at 1 week and 2 weeks cultivation were weaker than the native rabbit trachea tissue. Also, the mechanical strength of Silk-GMA hydrogel without chondrocytes at 1 week showed same results with chondrocyte-free Gel-GMA hydrogel. However, the strength of chondrocyte-free Silk-GMA increased depending on cultivation time. In addition, the SilkGMA hydrogel containing cells had a greater mechanical strength than hydrogel itself and the strength was increased over the time. The mechanical strength of hydrogel could be improved by cell encapsulation. We transplanted chondrocyte-laden Silk-GMA hydrogel cultured 1 week for in vivo experiments because of similar stiffness with normal tissue.

3.7. In vivo transplantation of rabbit chondrocytes-laden Silk-GMA hydrogel for trachea regeneration For regeneration of the artificial trachea, rabbit derived chondrocyte-laden Silk-GMA hydrogels (size 10 x 10 x 2 mm) were printed, culture for 1 week, and transplanted into the partially defected model of rabbit trachea (Fig. 8-A & -B). Endoscopy performed every 2 weeks after transplantation. Endoscopic result of first 2week showed narrowed the luminal by unbalanced integration of transplanted Silk-GMA hydrogel to the surrounding tissues. However, as transplanted periods go on, the internal diameter of defected portion gradually increased, and the surrounding tissues grow into the surgical site and became part of the trachea 16

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(Fig. 8-C). Rabbit was sacrificed at 6 weeks after transplantation of chondrocyte-laden SilkGMA hydrogel for in vivo histological detection of artificial trachea (Fig 9). H & E and MT staining at 6 weeks after transplantation showed characteristics of new cartilage formation in the regenerated tissue over the transplanted Silk-GMA hydrogel. Several matured chondrocytes in lacunae are found in hydrogel, and also partially extension of epithelial layer from native epithelium of trachea was formed below the hydrogel with chondrocytes in lacunae, indicating regeneration of ciliate layer in trachea. Moreover, the blood vessel formation and collagen matrix found the extended epithelium matrix surrounding of transplanted chondrocyte-laden Silk-GMA hydrogel. As a result of transplantation of the artificial trachea composed of hydrogel and chondrocytes it demonstrated that, the Silk-GMA hydrogel replaced as the defective part of trachea serving as a guide for the regeneration of trachea.

4. Discussion DLP printer is one of the light-supportive 3D bioprinting system can contribute for the progression of layering scaffold with fabricated complex with desired forms and expected cell types for tissue engineering and regenerative medicine. For the successful application to tissue engineering, there are some elements have to be overcome include damaging of cells in hydrogel during printing with light. DLP 3D bioprinter provide high resolution under rapid printing speed, and has advantages such as nozzle-free system and printing in the aqueous condition with variously designed forms by layering technologies [8, 14, 31]. From the results of our previous study with distinct advantages; 1. layering technology of DLP 3D printing system with LAP as a photoinitiator, 2. functions of modified SF with glycidyl methacrylate (Silk-GMA) [8], and 3. positive element of SF itself containing the typical cellular appearances 17

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of chondrocyte [20, 32]. Not only 3D printing, some elements should be considered for regeneration of the tissue or organ. Especially, bio ink must provide biocompatibility, biodegradability, and biological microenvironment with extracellular matrix condition as originated native tissue [32, 33]. Additionally, it also needs mechanical stability such as high tensile strength to resist for the physical stress during 3D printing [33]. Silk fibroin (SF) is the powerful biocompatible, biodegradable, and non-cytotoxic fibrous polymer from the natural source with excellent mechanical properties [20, 32]. And it also provides the diversity of methods for crosslink or solution-to-gel [33], ease to structure modification [34], and embedded hydration properties [35, 36]. Recently, Cheng et al [37] reported that SF has capability of proliferation and differentiation of chondrocytes due to reversion of dedifferentiation process and promote the expression of the cartilaginous extracellular matrix. Moreover, Fabricated SF hydrogel maintain the chondrocyte phenotype and promote the cartilaginous ECM [38]. And, the high density of chondrocyte in scaffolds leads to the regeneration of cartilage with more mechanical stiffness compare to low or cell free scaffolds [39]. Therefore, we hypothesized that Silk-GMA will be suitable as a bio-ink for DLP 3D bioprinting, and furthermore Silk-GMA with chondrocyte can be differentiated to cartilage tissue formation in vitro and in vivo system for the clinical trials. Na et al. [40] reported that while 3D printing process, cells in 10% GelMA hydrogels solution started to sediment of the plate due to gravity. We demonstrated that labeled NIH 3T3 cells in thirty percent of Silk-GMA were dispersed homogeneity right after 3D printing with DLP printer. And depending on in vitro culture period from 4 to 7 days, cells were distributed equally over the Silk-GMA hydrogels (Fig 2). With this result, 3 seconds per layer of rapid printing speed with DLP printing and viscosity of Silk-GMA provide appropriate 3D printing condition and highly biocompatibility for cell based in vitro or in vivo application. Additionally, cell proliferation 18

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and viability with Cell Counting Kit-8 and Live/Dead assay was performed with human nasal septal cartilage derived chondrocyte 30% Silk-GMA hydrogel (Fig 3). Encapsulated human derived chondrocytes in 30% Silk-GMA hydrogel proliferated gradually from culture day 0 to day 7, and at the end of cultivation on day 14, proliferation of human chondrocytes remarkably increased about double times compare to day 0 or day 3 of cultivation. The confocal microscopic observation with Live/Dead assay was used to detect for cell viability of chondrocytes in 30% Silk-GMA hydrogel with labelled Calcein-AM in green (live cells) and ethidium homodimer-1 in red fluorescence (dead cells). Chondrocytes in green fluorescence were increased depend on cultivation period from day 0 to day 14 with excellent viability. And cell-to-cell connections were observed with rapidly increasing of viable chondrocytes in SilkGMA hydrogel on day 14. It was determined the characterization of Silk-GMA for cellular stability and 3D printing condition with DLP printer. These results showed that Silk-GMA provided enhancing proliferation and viability of chondrocytes in Silk-GMA hydrogel throughout the in vitro long term cultivation condition. Moreover, DLP 3D printing technology could provide high cell viability because printing speed of DLP 3D printing is faster than other 3D printing system. The earliest stage of chondrogenesis developed with condensation of pre-cartilage cells from the consequences of mesenchymal cell recruitment, migration, and proliferation.

These

processes are followed and functionally controlled by cellular interactions with the surrounding extracellular matrix (ECM), chondrogenic cell growth/differentiation factors, and transcriptional/microenvironmental factors [41]. Chondrocytes are the functional cells that synthesize ECM components such as collagen, glycoproteins, proteoglycans and hyaluronan, and regulate ECM turnover and tissue homeostasis. The potential of a biomaterial matrix for chondrogenesis could be evaluated with providing temporary constructs for adhesion, 19

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proliferation, and differentiation of chondrocytes under the same condition with cartilage ECM [42, 43]. Based on these backgrounds, we performed that ring-shaped Silk-GMA hydrogel with human derived chondrocytes induced into the cartilage tissue formation and detected ability of chondrogenesis under in vitro condition (Fig 4). The histological characteristics of chondrocytes in the Silk-GMA hydrogel showed that typical chondrocytes with cartilage tissue formation over 4 weeks cultivation. From the results of Masson’s trichrome (MT) staining, cytoplasm and nuclei of chondrocyte with Silk-GMA showed in light red and dark blue. And collagen embed matrix in blue appeared 1 week and became darker at 4 weeks of in vitro cultivation.

The differentiation into cartilage was specifically stained with blue color when

differentiated into cartilage. Therefore, 4 weeks cultivation of chondrocytes with Silk-GMA in vitro condition could lead to differentiation of chondrogenesis and neo-cartilage formation. In Hematoxylin and Eosin (H & E) results showed that gradually increased chondrocyte population in Silk-GMA hydrogel depending on culture periods. Safranin O (SFO) staining results showed that cell nuclei and granules of chondrocyte in Silk-GMA hydrogel were expressed in typical red color. PKH labelled chondrocytes in Silk-GMA hydrogel were indicated with yellow to orange color in porous hydrogel. These results suggested the in vitro culture system for 3D printing with Silk-GMA hydrogel can provide positive circumstances for proliferation and induction of differentiation with encapsulated human derived chondrocyte. And moreover, it leads to in vivo application of artificial tissue or organ with long-term cultivation. For the artificial cartilage development, the various hydrogels have been developed as scaffolds for manipulated chondrocyte transplantation with periosteum in cartilage defects [16-19]. The hydrogel for cartilage generation not only provides mechanical supports for the tissue instead of native ECM, but also interacts with the resident chondrocytes and support the differentiation 20

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of chondrocyte [43, 44]. Cartilage is composed of chondrocytes within a compact structure of ECM composed with collagen Type II (Col II) and proteoglycans. These ECM compositions are very important to maintain for the mechanical and functional properties of the tissue in vivo. Proteoglycans are composed of protein that are associated with one or more varieties of glycosaminoglycan (GAG) chains [45, 46]. Assessment of secreted GAG contents from chondrocytes in hydrogel can evaluate bio-characteristic function of chondrogenesis. For evaluation of ECM production in vitro condition, human derived chondrocyte-laden Silk-GMA cultured up to 4 weeks (Fig 5-A).

The results of cultivation for 4 weeks contained the higher

amount of GAG compared to 1 and 2 weeks of incubation. It presents that Silk-GMA hydrogel provide an ideal environmental condition for maintenance of chondrocytic phenotype and function in vitro condition. And it also showed that repairing of defect cartilage with encapsulated chondrocyte in Silk-GMA can be modulated of growth factors, adhesion, migration, proliferation, and differentiation under in vivo condition. Chondrogenic gene expression with human chondrocyte in Silk-GMA performed with Collagen Type II for cartilage specific ECM, Collagen Type X for chondrogenic hypertrophic marker, SOX9 for chondrogenic transcriptional factor, and Aggrecan for cartilage specific proteoglycan major protein.

Each gene marker did not express on week 1 or weak signal

showed on Collagen Type X and SOX9 at 2 weeks in vitro cultivation, however, on 4 weeks cultivation, every chondrogenic specific genes expressed (Fig. 5-B). These results showed that encapsulated chondrocytes in Silk-GMA hydrogel can be re-differentiated with providing of an attractive environment for the proliferation and differentiation to chondrocyte. Dedifferentiation of chondrocyte in 2D monolayer culture system leads to loss of the chondrogenic phenotyping of collagen Type II and a low level of proteoglycan synthesis, and maintenance of fibroblast-like morphology. These dedifferentiated chondrocytes can be re21

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expressed the differentiated phenotype in an appropriate 3D circumstance with increasing of cartilage-specific proteoglycan and collagen synthesis [47-50]. For the clinical application of engineered human cartilage, in vivo degradation of the hydrogel has been proposed a very important element as a matrix, and the consequences of uneven cell distribution after 3D printing might cause some problems of resorption [51, 52]. High-speed DLP 3D printing was used to get better homogeneity of cell distribution all over the Silk-GMA as a hydrogel. Many study groups suggested that higher cell density provides better cell to cell interaction, faster tissue formation, and acceleration of extracellular matrix remodeling depend on culture periods, the other side, it can allow changing of viscosity, printability, and mechanical stiffness of the hydrogel [53-55]. Therefore, we performed that cell-laden or -free Silk-GMA hydrogel implanted dorsal subcutaneous part of nude mice (Fig. 6) before rabbit trachea transplantation for evaluation of mechanical properties, and culture up to 8 weeks under in vivo condition. In vivo cultivation performed up to 8 weeks after transplantation into nude mice by the subcutaneous method and also implanted cell free silk-GMA hydrogel as a control at the other side of the nude mouse to provide the same condition. Implanted human chondrocyte laden silk-GMA hydrogel showed in vivo cultivation-time dependent patterns, like chondrocytes enveloped by fibrous pores in Silk-GMA. The degradation of Silk-GMA hydrogels happened with chondrocyte spread out in small pores on Silk-GMA hydrogels. And moreover, cellular analysis exhibits that chondrocyte-laden Silk-GMA hydrogel group showed histological characteristics of cartilage tissue. It also showed tissue ingrowth with cell organization and extracellular matrix distribution, including proteoglycan and collagen with cultivation period. From these results, in vivo implantation to the nude mouse model as a preclinical trial could be served the positive aspect for confirmation of safety, stability and toxicity of the 3D printed Silk-GMA hydrogel. 22

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The mechanical properties of hydrogel such as stiffness and durability are one of the important elements for improvement of tissue engineering and clinical applications [55]. Especially, mechanical properties on cartilage tissue engineering require the long-term structural stability after transplantation. Moreover, high cell density of chondrocyte induces the increasing of cellcell connection, preventing a dedifferentiation of cells, enhancement of chondrogenic proliferation and ECM deposition[56]. Recently, Conventional synthetic biodegradable polymers such as polylactic acid (PLA), polyglycolic acid (PGA), and polylactic-co-glycolic acid (PLGA) combined natural source of polymer are popular for cartilage generation with various cell type, include mesenchymal stem cell (MSC), MSC aggregate and cartilage-resident chondroprogenitor cells [57-59]. These combinations of various scaffolds can provide appropriate mimic like native cartilage ECM and cell spreading homogeneously in 3D biomimic condition due to high fluidic content [60]. We compared the time-dependent differences of mechanical strength with Silk-GMA hydrogel itself, cultured chondrocyte-laden, -free Silk-GMA, modified gelatin with glycidyl methacrylate (Gel-GMA) and native trachea tissue from rabbit (Fig. 7). 1 and 2 weeks cultured chondrocyte-laden Silk-GMA hydrogels showed the strong mechanical strength among groups. This result shows that the mechanical strength of Silk-GMA hydrogel could be improved with cell encapsulation in hydrogel with fulfilled excellent stiffness and durability [61, 62]. These results lead to apply for in vivo transplantation of cultured chondrocyte-laden Silk-GMA hydrogel due to better stiffness compared to native rabbit trachea tissue or another source of hydrogel, such as Gel-GMA. Furthermore, comparison with mechanical strength of trachea from human or larger animal models is needed to develop the hydrogel for cartilage regeneration with controlling of mechanical strength of Silk-GMA hydrogel.

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With endoscopically monitoring for 6 weeks after transplantation to partially trachea defected rabbit model (Fig 8), firstly, the result of endoscopic observation showed that the internal diameter of transplanted chondrocyte-laden Silk-GMA hydrogel gradually increased and surrounding tissues grew into the surgical part of the transplanted artificial trachea on 6 weeks. Additionally, histological results with H&E and MT staining showed that new cartilage was regenerated on the transplanted part (Fig. 9). Chondrocyte in lacunae and the mucosal epithelium were observed upper and lower section of transplanted chondrocytes-laden SilkGMA hydrogel. And new blood vessel formation and collagen in matrix were found in epithelial layer under the transplanted chondrocyte-laden Silk-GMA hydrogel. These histological results suggest that Silk-GMA hydrogel has strong advantages for replacement as the part of defected trachea. In vivo cultivation of Silk-GMA hydrogel guides the possibility to clinical application for cartilage regeneration using human derived chondrocyte with 3D printed hydrogel. Altogether, we focused on the capabilities and abilities of reconstruction for cartilage defect with chondrocytes-laden Silk-GMA hydrogel by in vitro chondrogeneic differentiation abilities and in vivo application by transplantation for the first trial.

Briefly, 1)

capability of chondrocytes-laden Silk-GMA hydrogel for chondrogenesis under in vitro condition, 2) mechanical properties of Silk-GMA with or without chondrocytes compare to native tissues and another source, such as Gelatin-GMA, 3) transplanted cultured Silk-GMA hydrogel with chondrocytes into the trachea defect rabbit model, 4) evaluation of possibilities under in vivo condition for differentiation to cartilage tissue of chondrocytes-laden Silk-GMA with histological analysis.

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5. Conclusion Despite of our previous paper presented that modified Silk-GMA by 3D DLP printing has good printability with detailed structure by DLP 3D printing method, capability as a bio-ink, good mechanical properties and biocompatibility with cell viability, still more qualified potential of Silk-GMA for the chondrogeneic differentiation is needed to apply for clinical defected model. In this study, we focused on the capabilities and abilities of reconstruction for cartilage defect with chondrocytes-laden Silk-GMA hydrogel by in vitro chondrogeneic differentiation abilities and in vivo application by transplantation for the first trial. And moreover, we used modified Silk-GMA with biomechanical properties comparable to native rabbit trachea for the application of cartilage tissue engineering. Especially, using 3D DLP printing can obtain precise structural construction like native tissue and good cellular compatibilities due to rapid printing speed. Therefore, our study focused on the clinical application for trachea injury patients for the first time as per our best knowledge. And we present that the in vivo transplantation with chondrocytes-laden Silk-GMA by 3D DLP printing enables to produce functional and efficient properties of engineered cartilage with Silk-GMA. These results show the possibility that Silk-GMA with DLP 3D printer technology provided not only strong effectiveness for chondrogenesis in vitro and in vivo transplantation, but also suggested excellent biocompatibilities with strong mechanical advantages for defected tissue regeneration. Further studies are needed for the regeneration of defected cartilage with various cell type include mesenchymal stem cell and the combination of hydrogel based on Silk-GMA.

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Acknowledgement This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP; grant No.: 2016R1E1A1A01942120) and Hallym University research fund.

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Figure captions

Fig 1. Schematic presentation for methacrylation of silk fibroin (SF) with glycidyl methacrylate (GMA) (Silk-GMA) and bioprinting of chondrocyte with Silk-GMA by digital light processing (DLP) 3D printer. A. As a pre-hydrogel, fabricated SF was chemically modified with GMA. Degummed silk was dissolved in 9.3M LiBr and the solution were reacted with GMA for 6 h at 60 °C. To removed salts, it was dialyzed with distilled water at room temperature for 7 days and then freeze-dried. B. Diagram of DLP 3D bioprinting procedure using Silk-GMA with chondrocytes. Human or rabbit-derived chondrocytes were mixed with Silk-GMA including LAP as a photopolymer reagent. Chondrocyte-laden SilkGMA was printed in a layering type with DLP printer. The liquid Silk-GMA bio-ink with LAP harden due to UV (365nm) exposed when the build plate moved down to bio-ink in reservoir repeatedly till model is complete. Printed chondrocyte-laden Silk GMA hydrogels were cultured up to 1 week under in vitro condition. LiBr; Lithium bromide. DMD; digital micromirror device, LAP; Lithium phenyl(2,4,6-trimethylbenzoyl) phosphinate.

Table 1. Condition of DLP 3D printing with Silk-GMA hydrogel. Table 2. Primer sequences used for RT-PCR analysis. 31

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Fig 2. Analysis of cell distribution and changing cell dose depend on cultivation period in the Silk-GMA hydrogel after DLP 3D printing was performed with NIH 3T3, mouse fibroblast cell line. A. (1) PKH26 labelled NIH 3T3 cells were detected on upper and lower portion of 3D printed Silk-GMA hydrogel with IN Cell Analyzer, (2) Masson's trichrome (MT) staining and (3) PKH26 labelled NIH 3T3 cells with fluorescence microscopic detection. Scale bar = 1mm. B. Cell counting results of NIH 3T3 cells in Silk-GMA hydrogel with IN Cell Developer for culture time at 0, 4, and 7 days (ns; no significant).

Fig 3. In vitro Cell proliferation and viability assay for human chondrocyte in Silk-GMA hydrogel. A. CCK 8 assay for cell proliferation rate increased according to culture period, gradually. Data are shown as the mean ± SD (*p<0.05, **p<0.005 and *** p>0.0005, respectively). B. Confocal microscopic images for Live & Dead assay with Calcein-AM (live cells, green fluorescence) and ethidium homodimer-1 (dead cells, red fluorescence) staining showed that human chondrocytes were proliferated well in 30% of Silk-GMA hydrogel up to 2 weeks cultivation (Scale bar = 500um).

Fig 4. In vitro histological detection of human chondrocytes-laden Silk-GMA hydrogel for cartilage tissue formation. Silk-GMA hydrogel with human chondrocytes shows histological characteristics of cartilage tissue with time in vitro cultivation. The morphologies of chondrocytes in the Silk-GMA hydrogel were found as typical chondrocytes by histological analysis over 4 weeks cultivation such as Masson’s trichrome (MT, collagen : blue, cytoplasm: light red and cell nuclei in black), Hematoxylin and eosin (H & E), Safranin O (SFO, nuclei : black, cytoplasm : blue, and cartilage : orange to red) and PKH labelled cells before printing in yellow-orange: chondrocytes, green: Silk-GMA hydrogel. Scale bars present 500 μm. 32

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Fig 5. Analysis of GAG concentration and expression of chondrocytic specific gene for human chondrocyte-laden Silk GMA hydrogel in vitro culture. A. The data shows the GAG content in vitro culture time. Error bars indicate the standard deviation of the mean. *p<0.05 and **p<0.005, respectively. B. Gel electrophoresis after RT-PCR with chondrocytic specific genes, Collagen Type II, Sox9, Aggrecan and Collagen Type X. GAG, Glycosaminoglycan.

Fig 6. In vivo histological evaluation of chondrocytes-laden Silk-GMA hydrogel for cartilage tissue engineering in dorsal subcutaneous part of nude mice. Chondrocyte-laden Silk-GMA hydrogel group showed histological characteristics of cartilage tissue. Cell organization and extracellular matrix distribution, including proteoglycan and collagen with cultivation period compare to chondrocyte-free Silk-GMA hydrogel. A. each of chondrocytefree or chondrocyte-laden Silk-GMA hydrogel (disk type, 7 x 5 x 2 mm) was implanted to right and left into the dorsal subcutaneous spaces of nude mice. B. Silk-GMA hydrogel without chondrocyte stained with H&E (b), PKH26 (c) and unstained (a). C. Chondrocyte-laden SilkGMA hydrogel was unstained (a) or stained with H&E (b), MT (c), SFO (d) and PKH26 (e). H&E; Hematoxylin & Eosin, MT; Masson’s trichrome, SFO; Safranin-O. Scale bars represent 5mm (white color) and 500 μm (grey color).

Fig 7. Three-point bending test performed for confirmation of mechanical strength of Silk-GMA hydrogel during 3D culture up to 2 weeks with General-purpose tester. The strength of human chondrocytes-laden Silk-GMA hydrogel, chondrocyte-free Silk-GMA hydrogel, native rabbit trachea tissue as a control, and 20% Gelatin modified with glycidyl methacrylate (Gel-GMA) as a comparative sample were used for comparison of flexural 33

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modulus. A. To confirm the mechanical strength of the 3D printed hydrogel to be transplanted in the rabbit trachea was performed using general-purpose tester (specimen size = 10 mm x 10 mm, L x W). B. The human chondrocytes-laden Silk-GMA hydrogel at 1 & 2 weeks shows better mechanical strength compare to chondrocyte-free Silk-GMA hydrogel, Gel-GMA hydrogel and native rabbit trachea tissue. gf ; loading force.

Fig 8. Schematic summaries of chondrocytes-laden Silk-GMA hydrogel transplantation and endoscopic observation of rabbit trachea for 6 weeks after transplantation. A. Artificial trachea was printed by DLP printer (10 x 10 x 2 mm, W x D x H) with chondrocyte from rabbit ear, and cultured for 1 week. B. (a & b) After cutting and removing the part of trachea by 10 x10 mm, (c) artificial trachea was implanted. Scale bars represent 5mm. C. Endoscopy at 2, 4, and 6 weeks after trachea transplantation. Transplanted chondrocyte-laden Silk-GMA hydrogel showed the internal diameter gradually increased after transplantation and surrounding tissues grow into the surgical part of the trachea at 6 weeks after transplantation.

Fig. 9. In vivo histological analysis of chondrocyte-laden Silk-GMA hydrogel for development of cartilage tissue formation on 6 weeks after transplantation. H & E and MT staining at 6 weeks post-transplantation showed new cartilage tissue formation in the regenerated tissue over the implanted Silk-GMA hydrogel. A. Cross-sectioned whole rabbit trachea stained with H&E staining after 6 weeks of transplantation of chondrocyte-laden SilkGMA (Scale bar = 1mm, x200). B & B-1. Extension of native epithelium of trachea covered the chondrocyte-laden Silk-GMA hydrogel, and inside of extended epithelium has chondrocytes in lacunae (H&E staining and MT staining) (Scale bar = x40). C & C-1. Arrows 34

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indicate chondrocytes sit in lacuna and are surrounded by the matrix they have secreted.

D

& D-1. The new blood vessel formation (asterisk) with light-red and brief red detected in H&E staining & MT staining, respectively. Collagen matrix in light pink color (H&E staining) and blue color (MT staining) (bold arrow). And the epithelium of rabbit trachea also can be found on 6 weeks after chondrocyte-laden Silk-GMA hydrogel transplantation.

Native epithelium

of trachea partially extended to the transplanted chondrocyte-laden Silk-GMA hydrogel (arrowhead). L Lumen, E Esophagus, H&E Hematoxylin and Eosin, and MT Masson’s trichrome (Scale bars = 500 μm, x100).

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Fig 1.

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