Hydrogel-laden paper scaffold system fororigami-based tissue engineeringSu-Hwan Kima,1, Hak Rae Leeb,1, Seung Jung Yub, Min-Eui Hana, Doh Young Leec, Soo Yeon Kimc, Hee-Jin Ahnc,Mi-Jung Hanc, Tae-Ik Leed, Taek-Soo Kimd, Seong Keun Kwonc,2, Sung Gap Imb,2, and Nathaniel S. Hwanga,e,2

aInterdisciplinary Program in Bioengineering, Seoul National University, Seoul 151-742, Republic of Korea; bDepartment of Chemical and BiomolecularEngineering & KI for the NanoCentury, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea; cDepartment ofOtorhinolaryngology–Head and Neck Surgery, Seoul National University Hospital, Seoul 110-744, Republic of Korea; dDepartment of MechanicalEngineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea; and eSchool of Chemical and BiologicalEngineering, Institute of Chemical Processes, Seoul National University, Seoul 151-742, Republic of Korea

Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved October 28, 2015 (received for review March 10, 2015)

In this study, we present a method for assembling biofunctional-ized paper into a multiform structured scaffold system for reliabletissue regeneration using an origami-based approach. The surfaceof a paper was conformally modified with a poly(styrene-co-maleic anhydride) layer via initiated chemical vapor depositionfollowed by the immobilization of poly-L-lysine (PLL) and depositionof Ca2+. This procedure ensures the formation of alginate hydrogelon the paper due to Ca2+ diffusion. Furthermore, strong adhesion ofthe alginate hydrogel on the paper onto the paper substrate wasachieved due to an electrostatic interaction between the alginateand PLL. The developed scaffold system was versatile and allowedarea-selective cell seeding. Also, the hydrogel-laden paper could befolded freely into 3D tissue-like structures using a simple origami-based method. The cylindrically constructed paper scaffold sys-tem with chondrocytes was applied into a three-ring defect tra-chea in rabbits. The transplanted engineered tissues replacedthe native trachea without stenosis after 4 wks. As for the cus-tom-built scaffold system, the hydrogel-laden paper system willprovide a robust and facile method for the formation of tissuesmimicking native tissue constructs.

paper scaffolds | origami | tissue engineering | initiated chemical vapordeposition | hydrogel

The living organ changes its shape from a sheet-like arrange-ment with primitive cells to mature three-dimensional (3D)

structures through morphogenetic processes (1–3). To date, awide range of biomaterials have been used for the total or partialreplacement of damaged organs and/or tissue structures (4–7).As the functions of the living organ are realized by periodicchanges in the spatial arrangement of tissue elements, multiformscaffold systems mimicking the native tissue are desired. Mold-casting and electrospinning, among various other methods,have been introduced to fabricate diverse scaffolds (8, 9). Thesefabrication processes, however, possess limitations for organ-like structure productions. Although recent progress in tissueengineering has focused on using 3D printer schemes, there arestill limitations such as the shortage of appropriate printingmaterials and technical challenges related to the sensitivity ofliving cells (10–12).Paper-based scaffolds have been used previously for cell culture

platforms (13), high-throughput biochemical assay platforms (14),and a point-of-care diagnostic system (15). As a nature-originatedsubstrate, paper has attracted enormous research interest for ap-plications in tissue engineering (16). Cellulose-based paper mayserve as a promising material for tissue engineering as it containsmacroporous structures that allow nutrient transport and oxy-genation (13). In this regard, paper origami is a simple alternativeapproach for fabricating a multiform scaffold. Based on computer-aided design (CAD) planar figures, a variety of shaped scaffoldscould be designed using biofunctionalized paper.In this report, a vapor-phase method, initiated chemical vapor

deposition (iCVD), was used to deposit a functional polymer film

onto the surface of paper substrates. Because the iCVD process isperformed in the vapor phase at a mild substrate temperature, thisprocess preserves the topological microstructures of various sub-strates without damaging them, only rendering the paper surfacebiofunctionalizable (17). Several iCVD-based polymer coatingshave been successfully adopted for surface modification in bio-medical fields (16, 18, 19). For example, the technique was used tofabricate a functional scaffold platform for bone tissue regenera-tion and to immobilize peptides for the enhancement of osteo-genic differentiation (16, 18).In this study, we report and evaluate the development of

hydrogel-laden paper scaffolds for origami-based tissue engi-neering. A hydrogel layer encapsulated with chondrocytes wasformed on the poly(styrene-co-maleic anhydride) (PSMa)-coatedpaper via iCVD. PSMa was immobilized with poly-L-lysine (PLL)to induce a strong electrostatic adhesion with the thin alginatehydrogel layer, allowing the hydrogel to retain its structureupon folding. Furthermore, through a computer-aided design,the hydrogel-laden paper was folded into multiform organ-likestructures. For further applications in tissue engineering, rabbittrachea was removed and successfully replaced with a cylin-drically shaped scaffold containing rabbit chondrocytes. Thesefindings showed that a simple and bottom-up method to fab-ricate implantable hydrogel-laden paper scaffolds for origami-based tissue engineering is possible.

Significance

This work describes an intriguing strategy for the formation ofhydrogel-laden multiform structures utilizing paper sheets andsuggests a route for trachea tissue engineering. It combinesconcepts extracted from paper origami, functional thin poly-mer coating, and thin hydrogel layering on top of the paperscaffolds. A computer-aided design-based lock-and-key ar-rangement was used for folding the sheets into multiformstructures with spatial arrangements. With encapsulating cellsin hydrogel-laden paper, the scaffold system was able to de-liver biological cues in vivo. In this work, we have successfullyapplied an origami-based tissue engineering approach to thetrachea regeneration model.

Author contributions: S.-H.K., H.R.L., S.G.I., and N.S.H. designed research; S.-H.K., H.R.L.,S.J.Y., M.E.H., and S.K.K. performed research; S.J.Y., D.Y.L., S.Y.K., H.-J.A., M.-J.H., T.-I.L.,and T.-S.K. contributed new reagents/analytic tools; S.-H.K., H.R.L., S.J.Y., T.-S.K., S.K.K.,S.G.I., and N.S.H. analyzed data; and S.-H.K., H.R.L., S.K.K., S.G.I., and N.S.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1S.-H.K. and H.R.L. contributed equally to this work.2To whom correspondence may be addressed. Email: nshwang@snu.ac.kr, otolarynx@snuh.org, or sgim@kaist.ac.kr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1504745112/-/DCSupplemental.

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Results and DiscussionTo develop a hydrogel-laden paper for origami-based tissueengineering applications, a paper substrate was modified by ap-plying a functional polymer thin film via iCVD followed by thin

hydrogel layer formation (Fig. 1). Through iCVD, a polymer filmof PSMa was deposited uniformly on the paper surface (Fig. S1)(20). The scanning electron microscope (SEM) images showedthat the surface morphology of the PSMa-coated paper waspractically identical to that of bare paper (Fig. S2A). The500-nm-thick iCVD PSMa film encapsulated the microfibrousstructure of the paper with proper hydrophobicity (water contactangle of 103°) (Fig. S3) and kept the paper scaffold from rupture/deformation in the wet culture medium, ensuring the mechanicalstability of the paper scaffold under wet conditions. The chem-ical analysis indicated that the deposition of PSMa copolymerfilm altered the surface chemical compositions of the paper. Theoxygen to carbon ratio (O/C) ratio of the PSMa-coated papersurface in the X-ray photoelectron spectrometry (XPS) surveyscan (Fig. S2B) was 1:4, which is fully consistent with thechemical structure of the alternating repeat unit of the PSMapolymer, which is composed of 80% carbon and 20% oxygen(Fig. S4A). Furthermore, functionalities in the PSMa film readilyprovided reactive anhydride groups to form a covalent bond withthe amine functionalities in the PLL, leading to a grafting ofcationic PLL onto the paper surface (Fig. 2A and Fig. S2B) (21).The PLL-immobilized PSMa substrate displayed a newlyemerged, strong nitrogen (N1s) peak at 400 eV (Fig. 2A). Thewell-known reaction between maleic anhydride and nucleophilehas been widely used for biomedical applications including cellchips and microarrays (20, 22). The coupling reaction that formsa strong binding between the amine functionalities in PLL andthe anhydrides in PSMa has also been further confirmed by high-resolution scans of C1s (Fig. S4B). We also checked the PSMacoating uniformity after folding the coated paper by estimatingSEM at three different folding spots (Fig. S5). The SEM analysisshowed that there is no evident change like a flaking-off propertyin the PSMa-coated paper without highly textured surface mor-phology even after folding the coated paper. These results clearlydemonstrated that the PLL was covalently conjugated on thePSMa film. The observation strongly infers that the iCVD PSMaallowed the grafting of the cationic PLL on the substrates.

Fig. 1. Schematic of fabrication of hydrogel-cell–laden paper. Bare paper wascoated with PSMa via iCVD, followed by dip coating in an aqueous solution ofPLL and calcium chloride. After surface modification, the paper was soaked ina cell-alginate solution for 30 min for gelation. Four different folded paperscaffolds structures were fabricated using paper origami—star, cylindrical,triangular, and square pillar structure—and precisely coated with hydrogel.

Fig. 2. Functionalization of the paper substrate. (A) XPS survey scan spectra for substrates: bare paper, PLL-treated bare paper, PSMa-coated filter paper, andPLL-treated PSMa-coated filter paper. All substrates used were filter papers. (B) Single lap test for adhesion strength of alginate gels (Left). Two substrates coatedwith PSMa-Ca2+ and PSMa-PLL/Ca2+ were glued using alginate gel. Adhesion strength between alginate gel and the two different types of modified substrate:Ca2+ only and Ca2+ and PLL (Right). Forces were loaded at failure points; F, normalized by the width of the joint;w, for lap joints attached using alginate gel (n =10). (C) Tensile strength (Left) and Young’s Modulus (Right) obtained from Universal Testing Machine (UTM) analysis. Tensile strength and Young’s Modulus ofsubstrates were measured under dry and wet conditions (n = 10).

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The grafted PLL layer on the paper surface functions as alinker layer allowing the cell-laden alginate hydrogel to attachstably onto the surface of the paper scaffold. The cell-laden al-ginate solution was dispensed on the cationic PLL-grafted sub-strate, and the negatively charged alginate was bound tightly tothe PLL by the electrostatic attraction. The gelation of the al-ginate was also achieved spontaneously by the Ca2+ initially in-cluded in the PLL solution. When the alginate solution wasdispensed on the PLL surface, the Ca2+ on the surface of thepaper scaffold was released into the alginate solution to form ahydrogel that bound firmly to the surface of the paper scaffold(Movie S1). A single lap shear test showed that the averageadhesion force between the alginate gel and the paper bound tothe PLL layer was about 297 ± 0.18 N/m, which was 49% betterthan that of the specimen without a PLL layer, 200 ± 0.30 N/m,confirming the PLL-mediated attachment of the cell-laden al-ginate gel to the paper scaffold (Fig. 2B). Additionally, thePSMa-coated paper material displayed considerably higher val-ues for tensile strength and Young’s Modulus even under wetconditions, whereas bare paper showed extremely poor me-chanical strength under the same wet conditions (Fig. 2C).Hydrogel thickness and cross-linking density has a significant

influence on cell viability, oxygen supply, and nutrient diffusion(23–25). The hydrogel thickness on the paper-based scaffold wasdirectly governed by the alginate concentration and reactiontime. Increase of the gelation time resulted in the gradual in-crease of the hydrogel thickness, ranging from 517 μm (10 min)to 960 μm (30 min) for a 1% (wt/vol) aqueous alginate solutionand from 231 μm (10 min) to 497 μm (30 min) for a 3% (wt/vol)alginate solution (Fig. 3 A and B). Assuming a constant Ca2+ fluxtoward the alginate solution, the increased alginate concentra-tion would lead to rapid consumption of the Ca2+ ions by cross-linking reactions near the surface of the paper scaffold, resultingin a thinner hydrogel layer. As the gelation time increased, thetotal amount of Ca2+ that diffused in would also increase,leading to a thicker hydrogel on the paper scaffold. Additionally,we hypothesized that increasing the hydrogel concentration re-duced the Ca2+ diffusion length as the diffusion coefficient forCa2+ exponentially declined. To evaluate these hypotheses, wesimulated the scenarios using COMSOL Multiphysics (COM-SOL). The data indicated that the decrease in the diffusion co-efficient occurred due to the high concentration of alginate withgreater cross-linking density. The Ca2+ diffusion length accord-ing to hydrogel concentration showed a precise correlation withthe observed hydrogel thickness pattern (Fig. 3C). Moreover, it

took less than 30 min to fabricate the alginate layer as the totalamount of Ca2+ that diffused in became saturated within 30 min(Fig. 3D). Furthermore, alginate concentration-dependent swell-ing ratio changes and corresponding mechanical property changeswere also observed (Figs. S6 and S7). From the change in me-chanical and swelling properties, we determined that 1% (wt/vol)alginate would be suitable for further tissue-engineering applica-tions, as it showed high degrees of swelling ratio as well as stablemechanical properties. These data demonstrate that hydrogelthickness can be controlled in the range of 200 μm to 1 mm.We further explored the use of paper substrates as the tissue-

engineering platform for in vitro and in vivo culture. To this end,a hydrogel layer with rabbit articular chondrocytes (1 × 107 cells/mL)coated on the PSMa-modified paper (1 × 1 cm) was preparedand incubated in two different environments: dynamic culturein a modified bioreactor (9–11 rpm) and static culture. Thechondrocytes survived the encapsulation process and maintainedtheir viability in the 3D alginate hydrogels (Fig. S8 A and B). Inthe dynamic culture, the metabolic activity of cells in hydrogel wasmore up-regulated than in the static culture, and the amount ofDNA, collagen, and glycosaminoglycan (GAGs) in the sampleincreased continuously until day 21 (Fig. S8). The hydrogel-ladenpaper was also able to support cartilaginous tissue formationin vivo (Fig. S9).We applied a prepatterned mold onto an PSMa-modified paper

substrate to fabricate a multi-cell-culture platform that could befurther used in origami-based tissue engineering. Placement of aprepatterned mold with a defined shape on top of the papersubstrate, followed by injection of alginate solution mixed withlabeled cells, resulted in spatially patterned gelation of cell-ladenhydrogel (Fig. 4A). In this regard, using large area paper and aprepatterned mold, this paper-scaffold platform could be de-veloped into a scalable level to build a full-sized organ with multi-arrays of different cell types. Furthermore, by applying alginatesolutions with two different cell types on each side of the papersubstrate, two distinct cell types can be located in a multilevelformat (Movie S2). It is well appreciated that multiform-struc-tured shapes can be readily reconfigured by using CAD (Fig. 4B).To construct arbitrary shapes mimicking various types of tissues,we used the lock-and-key method to fabricate complexly shapedscaffolds without the need for additional adhesives (Fig. 4 B and Cand Movie S3).For further applications of the hydrogel-laden paper scaffold,

we fabricated cylindrically shaped scaffolds via an origami-basedlock-and-key mechanism and applied for tracheal reconstruction.

Fig. 3. Fabrication of hydrogel-laden paper scaffolds. (A) Photographs showing the side view of the hydrogel on paper scaffold. (B) The hydrogel thicknesswas measured at desired time points (10, 20, 30, and 60 min) with various hydrogel percentages [1, 1.5, and 3% (wt/vol) (n = 4)]. Profile of calcium releasefrom paper-based iCVD substrate by (C) COMSOL simulation and (D) experiment (n = 4).

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A cylindrically shaped scaffold (2-cm length and 8-mm diameter)was coated with alginate hydrogel with or without rabbit articularchondrocytes (4 × 107 cells/mL). A three-ring, 120° tracheal ringwas removed, and the tracheal defect was sealed with hydrogel-laden paper scaffold and firmly secured in place via a lock-and-key method (Fig. 5A). In trachea engineering, a tight fixation ofthe tracheal tube is a key point in preventing air leakage, whichcan cause emphysema and inflammation in the surrounding softtissue due to the bacteria inhaled with air (26). Although forminga cylindrical shape appears to be a trivial origami-based method,we have used this simple origami structure and applied to com-plete the tracheal tube structures in an animal model. Moreover,wrapping and folding steps were used to seal the defect areatightly without the use of sutures. This paper scaffold provided airtightness and adequate strength to prevent airway collapse. Paperorigami with a simple lock-and-key method was a facile processand did not require significant surgical time. In comparison, weadditionally performed the trachea model using paper-basedscaffold wrapping with the use of sutures (Fig. S10A). Similar tothe lock-and-key method, the suture around the paper-basedscaffolds was applied to prevent the air leakage and keep it inplace. Accordingly, the sutured sample resulted in trachea tissueregeneration and showed no tissue granulation at week 4 (i.e.,similar to lock-and-key samples).All animals survived without respiratory distress until the

scheduled time for euthanasia. On endoscopic examination, noneof the animals showed an ingrowth of granulation tissue on theinner surface of the grafts, and there was no evidence of infectionor distal accumulation of secretions in the cell-loading samples(Fig. 5B). However, acellular samples showed a narrowing of theinternal lumen (Fig. 5B). In cellular samples, the respiratory mu-cosal layer did not cover the prosthesis completely, but the airwaylumen was well maintained (Fig. 5B). Airway stenosis was minimalin the cellular samples, and the endoscopic airway stenosis grading

is summarized in Fig. S10B. At autopsy, the reconstructed trachealsurface in the animals was clear and smooth without profuse in-flammatory reactions between the tracheal and pretracheal tissuein the cell-loading samples. These samples were also completelyattached to the normal trachea without any granulation or dislo-cation of the membranes (Fig. 5B). Computed tomography (CT)images revealed a luminal contour at the regenerated site, eventhough the tracheal cartilage ring was not fully regenerated in thecellular samples (Fig. 5C). Signs of regenerated cartilage from theimplanted chondrocytes were detected at the edge of the defectedcartilage ring. In histologic evaluation, all samples were integratedinto the surrounding soft tissue (Fig. 5D). Multiple spots of im-mature chondrogenesis were found at the edge of the membrane atweek 2 in the cell-laden samples, and immature chondrogenesiswas up-regulated at week 4 as more chondrogenic spots appeared.All acellular samples showed no tissue granulation at week 4 inhistologic evaluation (Fig. 5D). The cell-laden scaffolds regen-erated the tracheal defect perfectly without granulation tissue in-growth or graft failure.

ConclusionThis work describes a strategy for the formation of hydrogel-ladenmultiform structures starting with paper scaffolds and suggestsa new route for trachea tissue engineering. It combines con-cepts adapted from origami coupled with a vapor-basedfunctional thin-film–coating process, the iCVD process, for pre-serving the morphology of paper microstructure and keeping thepaper robust under wet conditions. This work also identifies asolution in the field of multiform scaffold fabrication: the CAD-based lock-and-key design of planar sheets that can be folded intomultiform structures with spatial arrangements of tissue elements.Additionally, a structured paper scaffold was able to deliver theencapsulated chondrocytes to enhance tissue regeneration in vivo.

Fig. 4. Versatility of multiform constructs based on paper origami. (A) Schematic for the fabrication of patterned coculture platform in 3D. Hydrogel-containing HeLa cells labeled with CellTracker Blue were applied to the substrate, and a prepatterned mold consisting of four equally distributed squarepillars (2-mm radius and 8-mm distance for each pillar) was placed on the sample. After gelation, the mold was removed, and the hole was filled withhydrogel-containing HeLa cells labeled with CellTracker Green. (Scale bar, 1,000 μm.) (B) CAD-based planar figure for precise paper origami and the foldedstructures in 3D coated with hydrogel dyed using water-soluble dyes. Adopting the lock-and-key method, paper origami was completed without additionaladhesive. (C) Assembling paper-building block to fabricate multiform structures. (Scale bar, 2 cm.)

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In summary, an origami-based tissue-engineering approach wassuccessfully applied in the trachea regeneration model.

Materials and MethodsFabrication of Chemically Modified Substrates. Chemically modified paper wasprepared from filter paper (Whatman; Grade 3, GE Healthcare Life Science)commonly available in the laboratory. The filter paper was used as received,with no pretreatment or cleaning process before the application of the iCVDprocess. The PSMa polymer film was deposited in an iCVD vacuum reactorchamber (Daeki Hi-Tech). Maleic anhydride (Ma) (99%) and styrene (99%)were used as monomers. Tert-butyl peroxide (TBPO, 99%) was used as theinitiator. All monomers and initiator were purchased from Sigma-Aldrichand used without any further purification. Ma was heated to 55 °C whereasstyrene and TBPO were maintained at room temperature for addition intothe chamber as vapor. The flow rate of Ma, styrene, and TBPO was 1.6, 6.0,and 1.2 standard cubic centimeter per minute (sccm), respectively. The filterpaper was placed on the substrate, which was maintained at 25 °C, for ad-sorption of monomers. The iCVD chamber pressure was fixed at 600 mTorr.

The filament temperature was maintained at 180 °C to initiate the freeradical polymerization of PSMa. The deposition rate was monitored in situwith the He-Ne laser (JDS Uniphase). The calcium chloride (Sigma-Aldrich,96%) was solubilized in distilled water at a concentration of 0.25 M. The PLLsolution [Sigma-Aldrich, 0.1% (wt/vol) in H2O] and calcium chloride solutionwere mixed in a 1:10 volumetric ratio. The PSMa-deposited filter paper wasdip-coated in PLL and calcium chloride solution for 2 h.

Organ-Shaped 3D Hydrogel Structure Fabrication and Cell Encapsulation. Forprecise design of the structure, a planar figure was created using a CADprogram. Tubular, rectangular, parallel-piped, or star-shaped structures werereadily constructed through the classic paper origami method. The structurewas then soaked in alginate. After 30 min of gelation, the paper-basedstructure was coated with alginate hydrogel and washed three times withPBS (Gibco-BRL). For cell encapsulation, rabbit articular chondrocytes weretrypsinized and resuspended in alginate hydrogel at a density of 1 ∼4 × 106

cells/mL depending on the experiment. Other steps for fabricating the 3Dhydrogel structure encapsulated with cells were the same as for the methoddescribed above. The medium was changed every 2 d.

Sustained Release of Calcium Ion from Paper-Based iCVD Substrate. The releaseprofile for calcium ion from the paper-based iCVD substrate was measuredusing Pointe Scientific Calcium Liquid Reagents (Pointe Scientific). The paper-based iCVD substrate (1 × 1 cm) was soaked in distilled water at roomtemperature. ddH2O purified for the desired time points (0, 5, 10, 15, 20, 25,and 30 min) were collected and quantified using Infinite 200 PRO TECAN Ltd.Relative absorbance intensity values were converted into the actual con-centration of calcium ion by correlation with a standard curve.

Hydrogel Thickness Depending on Time and Hydrogel Concentration. The paper-based iCVD substrate (1 × 1 cm) was soaked in 1, 1.5, and 3% (wt/vol) sodiumalginate (FMC biopolymer), respectively. Samples at the desired time points(10, 20, 30, and 60 min) were collected and imaged using AM413MT Dino-Lite X(Dino-Lite). The hydrogel thickness was analyzed as three random points werechosen, and the average was calculated by DinoCapture 2.0 (Dino-Lite).

Cell-Laden–Based Paper Scaffold for Tracheal Implantation and Surgical Procedures.All protocols (no. 14–0199-S1A0) were performed in accord with the guidelinesof the Animal Care Ethics Committee of Seoul National University Hospital. NewZealand White rabbits (Koatech Laboratory Animal Company) weighing 2.6 to∼3.0 kg were placed under anesthesia with an intramuscular injection of zoletile(50 mg/kg) and xylazine (4.5 mg/kg). Surgery was performed as follows. Theanimal was placed in a supine position. The relevant area was shaved and dis-infected. A vertical incision was made at the midline of the neck. The strapmuscles were separated at the midline until the tracheal cartilages were en-countered. A three-ring, 120° anterior tracheal wall defect was thenmade in thetracheal rings, and the paper-based tubular structure was placed in the trachea.The paper-based tubular structure (4-mm radius and 2-cm height) was coatedwith 1% (wt/vol) alginate hydrogel containing P1 rabbit chondrocytes (celldensity, 2.8 × 107 cells/mL). This tracheal defect size has been shown to result insevere tracheal stenosis and subsequent death in rabbits. The tracheal defectwas covered by wrapping the paper around the trachea and inserting the keyinto the lock to finish the implantation. The skin incision was closed using 4–0 nylon (Johnson & Johnson). Postoperatively, the animals were observed for 2 hbefore being returned to their cages where water and standard feed wereavailable. For the following 4 d, the rabbits were given 20 mg/kg kanamycin asprophylaxis. Clinical signs were monitored daily with special attention to weight,cough, sputum production, wheezing, and dyspnea.

Immunohistochemical Staining with Aggrecan and Collagen Type II. Tissuesamples were fixed in 4% paraformaldehyde for 24 h and dehydrated with agraded ethanol series. The fixed samples were embedded in paraffin, and theparaffin sections were stained with aggrecan and collagen type II. Immuno-histochemical staining was performed on 4-μm-thick serial sections mountedon poly-L-lysine–coated slides. The sections were deparaffinized and subjectedto antigen retrieval using pressure cooking in 1× citrate buffer (pH 6.0) (Invi-trogen) for 10min followed by slow cooling for 30min. The sections were thenincubated with monoclonal anti-aggrecan antibody (1:500 dilution; NovusBiological) and monoclonal anti-collagen type II antibody (1:500 dilution;Novus Biological) for 1 h and developed using the Peroxidase Substrate Kit(Vector) and Mayer’s hematoxylin (Sigma) as a counterstain.

ACKNOWLEDGMENTS. This research was supported in part by the Ministry ofHealth and Welfare of Korea (Grants HI13C1789010014, HI13C0451020013,and HI14C15410100); by the National Research Foundation of Korea

Fig. 5. Functionally defined paper scaffold transplantation for trachealreconstruction. (A) Procedure for tracheal reconstruction using the cylindri-cally shaped scaffold using lock and key. After a three-ring, 120° trachea walldefect was made on the tracheal ring (i), the sample was used to cover thedefect (ii), and the key was inserted into the lock (iii). (B) Endoscopic imagesat 1, 2, 3, and 4 wks. Double asterisk (**) indicates the paper scaffold. (C )Computed tomographic (CT) images at 4 wks. Asterisks show signs ofregenerated chondrocytes. No obvious stenosis was observed on the CTimages. White, red, and green colors indicate the cartilage, airway, andmuscles, respectively. (D) Histologic evaluation of cross-sections of speci-mens taken after implantation. All samples were collected at 4 wks andanalyzed by hematoxylin-eosin, Safranin-O, Aggrecan, and collagen type II.Double asterisks (**) and arrowheads indicate the paper scaffold and immaturechondrogenesis, respectively. (Scale bar, X 12.5: 1,600 μm; X 40: 500 μm.)

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funded by the Ministry of Education, Science and Technology (Grant NRF-2012M3A9C6050102); and by the Advanced Biomass R&D Center of the Global

Frontier Project funded by the Ministry of Science, Information & Communica-tion Technology (ICT), and Future Planning (ABC-2011-0031350).

1. Bryant DM, Mostov KE (2008) From cells to organs: Building polarized tissue. Nat RevMol Cell Biol 9(11):887–901.

2. Lutolf MP, Hubbell JA (2005) Synthetic biomaterials as instructive extracellular mi-croenvironments for morphogenesis in tissue engineering. Nat Biotechnol 23(1):47–55.

3. Stevens MM, George JH (2005) Exploring and engineering the cell surface interface.Science 310(5751):1135–1138.

4. Langer R, Tirrell DA (2004) Designing materials for biology and medicine. Nature428(6982):487–492.

5. Khademhosseini A, Langer R, Borenstein J, Vacanti JP (2006) Microscale technologiesfor tissue engineering and biology. Proc Natl Acad Sci USA 103(8):2480–2487.

6. Steinke M, et al. (2014) An engineered 3D human airway mucosa model based on anSIS scaffold. Biomaterials 35(26):7355–7362.

7. Wang J, et al. (2014) Phage nanofibers induce vascularized osteogenesis in 3D printedbone scaffolds. Adv Mater 26(29):4961–4966.

8. Jakab K, Neagu A, Mironov V, Markwald RR, Forgacs G (2004) Engineering biologicalstructures of prescribed shape using self-assembling multicellular systems. Proc NatlAcad Sci USA 101(9):2864–2869.

9. Jang JH, Castano O, Kim HW (2009) Electrospun materials as potential platforms forbone tissue engineering. Adv Drug Deliv Rev 61(12):1065–1083.

10. Gross BC, Erkal JL, Lockwood SY, Chen C, Spence DM (2014) Evaluation of 3D printingand its potential impact on biotechnology and the chemical sciences. Anal Chem86(7):3240–3253.

11. Murphy SV, Atala A (2014) 3D bioprinting of tissues and organs. Nat Biotechnol 32(8):773–785.

12. Schubert C, van Langeveld MC, Donoso LA (2014) Innovations in 3D printing: A 3Doverview from optics to organs. Br J Ophthalmol 98(2):159–161.

13. Derda R, et al. (2009) Paper-supported 3D cell culture for tissue-based bioassays. ProcNatl Acad Sci USA 106(44):18457–18462.

14. Deiss F, et al. (2013) Platform for high-throughput testing of the effect of solublecompounds on 3D cell cultures. Anal Chem 85(17):8085–8094.

15. Martinez AW, Phillips ST, Whitesides GM, Carrilho E (2010) Diagnostics for the de-veloping world: Microfluidic paper-based analytical devices. Anal Chem 82(1):3–10.

16. Park HJ, et al. (2014) Paper-based bioactive scaffolds for stem cell-mediated bonetissue engineering. Biomaterials 35(37):9811–9823.

17. Ozaydin-Ince G, Dubach JM, Gleason KK, Clark HA (2011) Microworm optode sensorslimit particle diffusion to enable in vivo measurements. Proc Natl Acad Sci USA 108(7):2656–2661.

18. Kim MJ, et al. (2013) BMP-2 peptide-functionalized nanopatterned substrates forenhanced osteogenic differentiation of human mesenchymal stem cells. Biomaterials34(30):7236–7246.

19. Kang BJ, et al. (2014) Umbilical-cord-blood-derived mesenchymal stem cells seededonto fibronectin-immobilized polycaprolactone nanofiber improve cardiac function.Acta Biomater 10(7):3007–3017.

20. Tenhaeff WE, Gleason KK (2007) Initiated chemical vapor deposition of alternatingcopolymers of styrene and maleic anhydride. Langmuir 23(12):6624–6630.

21. Fernandes TG, et al. (2010) Three-dimensional cell culture microarray for high-throughput studies of stem cell fate. Biotechnol Bioeng 106(1):106–118.

22. Lee MY, et al. (2008) Three-dimensional cellular microarray for high-throughputtoxicology assays. Proc Natl Acad Sci USA 105(1):59–63.

23. Matsunaga YT, Morimoto Y, Takeuchi S (2011) Molding cell beads for rapid con-struction of macroscopic 3D tissue architecture. Adv Mater 23(12):H90–H94.

24. Napolitano AP, et al. (2007) Scaffold-free three-dimensional cell culture utilizingmicromolded nonadhesive hydrogels. Biotechniques 43(4):494, 496–500.

25. Mosadegh B, et al. (2014) Three-dimensional paper-based model for cardiac ischemia.Adv Healthc Mater 3(7):1036–1043.

26. Kwon SK, et al. (2014) Tracheal reconstruction with asymmetrically porous poly-caprolactone/pluronic F127 membranes. Head Neck 36(5):643–651.

27. Farndale RW, Buttle DJ, Barrett AJ (1986) Improved quantitation and discriminationof sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim BiophysActa 883(2):173–177.

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