Bioprintable, cell-laden silk fibroinâ€“gelatin hydrogel ...

Acta Biomaterialia xxx (2014) xxx–xxx

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Acta Biomaterialia

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Bioprintable, cell-laden silk fibroin–gelatin hydrogel supportingmultilineage differentiation of stem cells for fabricationof three-dimensional tissue constructs

http://dx.doi.org/10.1016/j.actbio.2014.09.0231742-7061/� 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

⇑ Corresponding authors.E-mail addresses: [email protected] (D.-W. Cho), [email protected]

(S. Ghosh).1 These authors contributed equally to this work.

Please cite this article in press as: Das S et al. Bioprintable, cell-laden silk fibroin–gelatin hydrogel supporting multilineage differentiation of stem cfabrication of three-dimensional tissue constructs. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.09.023

Sanskrita Das a,b,c,1, Falguni Pati b,1, Yeong-Jin Choi d,1, Girdhari Rijal b, Jin-Hyung Shim e, Sung Won Kim f,Alok R. Ray c, Dong-Woo Cho b,⇑, Sourabh Ghosh a,⇑a Department of Textile Technology, Indian Institute of Technology, New Delhi 110016, Indiab Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), 77 Cheong-Am Ro, Nam-gu, Pohang, Kyungbuk 790-784, South Koreac Centre for Biomedical Engineering, Indian Institute of Technology, New Delhi 110016, Indiad Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), 77 Cheong-Am Ro, Nam-gu, Pohang, Kyungbuk 790-784, South Koreae Department of Mechanical Engineering, Korea Polytechnic University, 2121 Jeongwang-dong, Siheung-si, Gyeonggi-do 429-793, South Koreaf Department of Otolaryngology – Head and Neck Surgery, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, South Korea

a r t i c l e i n f o

Article history:Received 22 May 2014Received in revised form 12 September2014Accepted 12 September 2014Available online xxxx

Keywords:BioprintingSilk–gelatin bioinkCytocompatible gelationSelf-standing 3-D constructMultilineage differentiation

a b s t r a c t

Bioprinting has exciting prospects for printing three-dimensional (3-D) tissue constructs by deliveringliving cells with appropriate matrix materials. However, progress in this field is currently extremely slowdue to limited choices of bioink for cell encapsulation and cytocompatible gelation mechanisms. Here wereport the development of clinically relevant sized tissue analogs by 3-D bioprinting, delivering humannasal inferior turbinate tissue-derived mesenchymal progenitor cells encapsulated in silk fibroin–gelatin(SF–G) bioink. Gelation in this bioink was induced via in situ cytocompatible gelation mechanisms,namely enzymatic crosslinking by mushroom tyrosinase and physical crosslinking via sonication. Mech-anistically, tyrosinases oxidize the accessible tyrosine residues of silk and/or gelatin into reactive o-qui-none moieties that can either condense with each other or undergo nonenzymatic reactions withavailable amines of both silk and gelatin. Sonication alters the hydrophobic interaction and acceleratesself-assembly of silk fibroin macromolecules to form b-sheet crystals, which physically crosslink thehydrogel. However, sonication has no effect on the conformation of gelatin. The effect of optimized rhe-ology, secondary conformations of silk–gelatin bioink, temporally controllable gelation strategies andprinting parameters were assessed to achieve maximum cell viability and multilineage differentiationof the encapsulated human nasal inferior turbinate tissue-derived mesenchymal progenitor cells. Thisstrategy offers a unique path forward in the direction of direct printing of spatially customized anatom-ical architecture in a patient-specific manner.

� 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction biomaterial in a defined and organized manner, at the targeted

Despite fascinating advances in the field of tissue engineering,the fabrication of anatomically relevant three-dimensional (3-D)engineered tissues still presents a major hurdle, due to our inabilityto replicate complex tissue architecture, composition and biome-chanical functionality by using pre-formed polymeric scaffolds.Bioprinting has exciting prospects for fabricating complex 3-Dmulticellular tissue analog architecture by delivering a preciselycharacterized progenitor cell population, along with an appropriate

location, in adequate numbers and within the right environment,by dispensing cell suspension or cell-laden hydrogels in a layer-by-layer process [1–6]. But after the initial enthusiasm, progressin effective bioprinting was severely slowed down due to limitedchoices of bioink for cell encapsulation and cytocompatible gelationmechanisms.

During extrusion-based bioprinting, sol-gel transition isinduced for gelatin [7], gelatin/chitosan [8,9], gelatin/alginate [9],gelatin/fibrinogen [10,11], Lutrol F127/alginate [12] and alginate[13,14] by either thermal processes or post-print crosslinking.However, there is tremendous scope for improvement in thedesign of bioink. Alginate-based bioink has major limitations, suchas quick loss of mechanical properties during in vitro culture

ells for

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2 S. Das et al. / Acta Biomaterialia xxx (2014) xxx–xxx

(�40% within 9 days) [15], different responses of human and ani-mal cells [14], lack of bioactive binding sites [13] and resistanceto protein adsorption [14]. Collagen ink suffers from issues of qual-itative batch-to-batch variations, loss of shape and consistency dueto shrinkage and poor mechanical property [16]. Pluronic F-127(Poloxamer 407) dissolves in culture medium, resulting in the col-lapse of the construct architecture. Furthermore, encapsulatedhuman BMP-2 gene-transduced bone marrow stromal cells couldnot grow well within the pluronic gel in vitro and failed to inducebone formation in vivo [17]. Fibrin hydrogels possess poormechanical property and undergo fast disintegration (and henceproteinase inhibitor is needed for stabilizing up to 4 weeksin vitro [10,11]), and fail to induce bone formation in vivo [11].Thus, there is an urgent need to develop a novel bioink for printing3-D tissue analog with tailored mechanical properties, instanta-neous cytocompatible gelation, tailorable degradation rate, tissuespecificity and adaptability to clinical set-up.

In our previous study, an optimized blend ratio of silk fibroinand gelatin was used for the fabrication of microperiodic scaffoldsvia the 3-D printing (direct-write) technique, to induce redifferen-tiation of expanded chondrocytes [18]. The primary structure ofBombyx mori silk fibroin predominantly consists of [GAGAGS]n

(where G is glycine, A is alanine and S is serine) repeat sequences,which form physical crosslinks though b-sheet crystallization. Thecell adhesion mechanism on B. mori silk fibroin is still not properlyunderstood, as it is devoid of known cell adhesion peptides, but itoffers robust mechanical properties and tailorable degradability.On the other hand, the primary sequence of gelatin commonly con-tains GXY (where G is glycine, and X and Y are usually proline andhydroxyproline) and RGD (arginine–glycine–aspertate) amino acidsequences, but it suffers from a faster degradation rate. Thoughtheir blend overcomes the limitation of the individual material,certain points need to be taken into account to develop silk–gelatinbioink. During bioprinting, the bioink should come out of themicronozzle smoothly with minimal pressure. After extrusion, bio-ink should undergo rapid gelation and minimum deformation withdeliverance of excellent cell viability. The gelation process of thehydrogel used as bioink should be mild and cell friendly. The com-position and rheological features of the hydrogel bioink must sup-port cell survival and targeted differentiation [19]. Hence, byconsidering these criteria, we have developed a directly printablebioink of silk fibroin–gelatin (SF–G) blend, which can be cross-linked in situ without any cytotoxic effect. The two different typesof in situ crosslinking strategies evaluated in this study were phys-ical crosslinking using probe-based sonication and enzymaticcrosslinking using mushroom tyrosinase.

Tyrosinases belong to the group of oxidative enzymes andoxidize the accessible tyrosine residues of proteins into reactiveo-quinone moieties without breaking the peptide bond. It wasreported that tyrosinase can oxidize �10–11% and 20% of the tyro-sine residues of silk and gelatin, respectively [20,21]. Oxidizedtyrosyl residues (i.e., quinone residues) can either condense witheach other or undergo nonenzymatic reactions with availablenucleophiles, such as amines of both gelatin and silk [20,21]. Thus,tyrosinase can be applied for in situ crosslinking of cell encapsu-lated silk–gelatin hydrogel by sol-to-gel conversion of both silkand gelatin. On the other hand, sonication is a mechanical vibrationthat increases localized dynamics of polymer chains, causing alocalized heating effect, the formation and collapse of bubbles,modulation in pressure and strain rates. Further, sonicationinduces crystalline b-sheets by the alteration of hydrophobichydration and self-assembly of silk macromolecules [22].

To evaluate the cellular responses, we used human nasal infe-rior turbinate tissue-derived mesenchymal stromal cells (hTMSCs),a promising cell source due to their intrinsic capacity to proliferate,thereby providing large cell numbers, and high multilineage

Please cite this article in press as: Das S et al. Bioprintable, cell-laden silk fibroinfabrication of three-dimensional tissue constructs. Acta Biomater (2014), http:

differentiation potential [23,24]. These cells can be isolated fromthe inferior turbinate tissue that was discarded after a frequentlyperformed surgery to relieve nasal obstruction resulting from tur-binate hypertrophy [24]. Most interestingly, passage numbers anddonor age could not affect differentiation characteristics of hTMSCssignificantly, unlike bone-marrow-derived or adipose-derivedmesenchymal stromal cells, [25]. The hTMSCs exhibitedapproximately five times higher proliferation compared to that ofbone-marrow-derived MSCs [26]. Although adipose-derived mes-enchymal stromal cells (ASCs) could easily be isolated by a mini-mally invasive procedure from aspirated fat tissue, hTMSCsdisplayed �30 times higher yield than adipose-derived MSCs atearly passage [27].

In the present study, we explored the strategy of 3-D printinghTMSCs encapsulated within optimized SF–G solution. Further-more, we focused on the effect of secondary conformations andsupramolecular structures developed due to either tyrosinase- orsonication-induced gelation on the printing process and cell sur-vival within the cell-laden 3-D constructs. Targeted multi-lineagedifferentiation of the encapsulated hTMSCs within silk–gelatinconstructs was evaluated by gene expression, immunofluorescenceand histological studies. To evaluate the potential of silk–gelatinbioink compared to the present standard procedure, we made acomparison with alginate because it is a widely used bioink for cellencapsulation and printing due to its easy processability [4,28–30].The outcomes provided a valuable insight into bioprinting ofhTMSCs to engineer custom-made 3-D tissue constructs.

2. Materials and method

2.1. Materials

B. mori cocoons were kindly provided by Yeongdeok SericultureAgricultural Association, South Korea. Gelatin powder (Procineskin, Type A) and sodium alginate (from brown algae, medium vis-cosity) were procured from Sigma-Aldrich, USA. Details of all otherreagents that were used are given with the experimental method.

2.2. Preparation and crosslinking of 8SF–15G bioink

The master SF solution and SF–G blend were prepared asdescribed elsewhere (see Supplementary Materials) [18]. TheSF–G blend was prepared by adding the desired amount (15 wt.%)of ethanol sterilized gelatin powder in 8% w/v autoclaved SF solu-tion. The suspension was kept under agitation at 40 �C for completedissolution of gelatin powder in SF solution. Sterilized alginatesolution (6% w/v) was prepared by adding the required amount ofsodium alginate in deionized water, followed by autoclaving.

For post-printing stabilization of 8SF–15G structures, two dif-ferent crosslinking methods were adapted: enzymatic crosslinkingby mushroom tyrosinase (Sigma-Aldrich) and physical crosslinkingby sonication (Sonic dismembrator model 100, Fisher Scientific).Tyrosinase crosslinked 8SF–15G gel and sonication-induced 8SF–15G gel were designated hereafter as 8SF–15G–T and 8SF–15G–S,respectively. Alginate solution was crosslinked with 100 mM CaCl2

(Kanto Chemical) and 145 mM NaCl (Samchun) solution for 30 min[4].

2.3. Characterization of 8SF–15G bioink

The developed bioink was characterized chemically and physi-cally by Fourier transform infrared spectroscopy (FTIR) andrheology, respectively. FTIR spectra of vacuum-dried uncrosslinked8SF–15G blend as the control, and 8SF–15G–T and 8SF–15G–S,were obtained using a FTIR spectrophotometer (Bruker, Vertex

–gelatin hydrogel supporting multilineage differentiation of stem cells for//dx.doi.org/10.1016/j.actbio.2014.09.023


S. Das et al. / Acta Biomaterialia xxx (2014) xxx–xxx 3

70). Fourier self-deconvolution (FSD) of spectra was performedusing OriginPro 8.0 (OriginLab Corporation, Northampton, MA,USA). Fitting of the FSD FTIR spectra with Gaussian profiles wasdone in the amide I region between 1595 and 1705 cm�1 and itsb-sheet fraction was determined by the integral of Gaussian pro-files (termed ’’bands’’) (see Supplementary Materials for details).

The rheological properties of 8SF–15G–T, 8SF–15G–S and algi-nate were measured at 28 �C and 37 �C using an ARES rheometer(TA instruments, USA). The viscosity of the biopolymer solutionwas evaluated for the shear rate ranging from 0.1 to 1000 s�1. Toevaluate the thermoresponsive behavior, the viscosity of8SF–15G–T, 8SF–15G–S and alginate solution was also measuredat varying temperatures from 10 to 40 �C. The dynamic storage(G0) and loss modulus (G’’) of both 8SF–15G–T and 8SF–15G–Sblends were also measured in oscillatory mode at 18 �C, 28 �Cand 37 �C. In the amplitude sweep measurement, the materialswere tested for strain value of 0.1 to 104% by keeping the frequencyconstant at 1 Hz, and in the frequency sweep measurement, mate-rials were tested from 0.1 to 100 Hz frequency range by keepingthe strain constant at 5% [18].

For the gelation kinetics study, the target strain value of 5% andfrequency value of 1 Hz were selected from one-third of the linearregion, before the crossover points of G0 and G00, from amplitudesweep and frequency sweep results, respectively, to ensure thatthe measurements were conducted within the linear viscoelasticregion and that the G0 and G00 were independent of the strainamplitude and frequency [18]. To evaluate the gelling behaviorinduced via enzymatic and physical crosslinking, gelation kineticsof 8SF–15G–T and 8SF–15G–S were studied with time in oscillationmode at 37 �C.

2.4. Preparation of bioink

The hTMSCs were isolated from discarded inferior turbinatetissue (�0.03 g) from a patient (male; age 24) with informed con-sent, who underwent septoplasty and partial turbinectomy, in theDepartment of Otolaryngology – Head and Neck Surgery at theCatholic University of Korea with approval of the institutionalreview board (KC08TISS0341). The hTMSCs isolatation processwas reported elsewhere [24]. Briefly, the inferior turbinate tissueobtained after surgery was washed immediately with saline solu-tion containing gentamicin. Further, the tissue was washed withantibiotic–antimycotic solution followed by washing with phos-phate buffered saline (PBS). The turbinate tissue was then cut into�1 mm3 pieces and was placed into a culture dish. Dulbecco’smodified Eagle’s medium (DMEM) containing 10% fetal bovineserum (FBS) was then added and the cut tissues were incubatedat 37 �C and 5% CO2. After 3 weeks, the culture dish was washedwith PBS and the hTMSCs were detached using 0.25% trypsin con-taining 1 mM ethylenediaminetetraacetic acid (EDTA). The hTMSCswere subcultured for further use in experiments.

The cell encapsulated hydrogel (bioink) was prepared by encap-sulating hTMSCs (cell density, 2–5 � 106 cells ml�1) at passageP4-P5 in 8SF–15G–T, 8SF–15G–S and alginate solution. For enzy-matic crosslinking, a concentration of 500 units of tyrosinase wasincorporated in one batch of 8SF–15G hydrogel prior to cell encap-sulation. Similarly, in physical crosslinking using a probe-basedsonicator, 8SF–15G hydrogel was sonicated at 50% amplitude for10 s. Thereafter, a 10� concentrated cell culture medium wasadded to the bioink (1:10 ratio) to maintain the same osmoticpressure within the bioink prior to cell encapsulation.

2.5. Printing of cell-laden constructs with SF–G bioink

Self-standing cell-laden 8SF–15G–T and 8SF–15G–S constructswere prepared by using an in-house-developed multi-head


deposition system (MHDS) [29]. The bioink was fed into a sterilesyringe and was fixed in the barrel of the MHDS. The temperatureof the syringe barrel was maintained at 28 �C using a temperaturecontroller and the printing chamber temperature was maintainedat 18 �C. The bioink was dispensed through a sterile metal needle(nozzle diameter 250 lm), using a pneumatic pressure of 200–250 kPa and a deposition speed of 60 mm min�1. The depositionspeed and pressure were controlled by customized softwaredeveloped in house. Subsequent layers were deposited at a 90�angle to the underlying layer. For surface characterization ofthe printed construct, the constructs were observed using scan-ning electron microscopy (SEM; JSM-5300, JEOL, Tokyo, Japan).

Printed hTMSC encapsulated 8SF–15G–T, 8SF–15G–S, alginateconstructs were allowed for crosslinking under sterile conditions.The crosslinked constructs were washed with sterile PBS thricefor 5 min each and were dipped in DMEM (Hyclone, USA) supple-mented with 10% FBS (Gibco, USA), 5% penicillin/streptomycin(Gibco, USA) and incubated at 37 �C, 5% CO2. For the cell prolifera-tion study, constructs were cultured in cell proliferating media(DMEM) for 3 weeks. For the differentiation study, the constructswere initially cultured in cell proliferating media for 24 h, followedby replacement with the differentiation media. Osteogenic mediacontained DMEM with 10 nM dexamethasone (Sigma-Aldrich,USA), 10 mM b-glycerol phosphate (Sigma-Aldrich, USA), and50 lg ml�1 ascorbic acid-2-phosphate (Sigma-Aldrich, USA). Chon-drogenic media contained 10 ng ml�1 transforming growth factorb1 (BD Biosciences, USA), 50 lg ml�1 ascorbate-2-phosphate,10 nM dexamethasone and 6.25 lg ml�1 insulin transforming sele-nite (ITS) (Sigma-Aldrich, USA). Adipogenic media contained1 ll ml�1 dexamethasone, 3.3 mM biotin (Sigma-Aldrich, USA),17 mM pantothenic acid (D-pantothenic acid hemicalcium salt,Sigma-Aldrich, USA), 250 mM isobutyl-1-methylxanthine (IBMX,Sigma-Aldrich, USA) and 10 mg ml�1 insulin (Sigma-Aldrich,USA). The media were changed every 3 days.

2.6. DNA quantification

To verify the cellular content and the cell proliferation within aconstruct, the dsDNA content was measured. For DNA quantifica-tion, all the constructs were digested in 1 ml of papain solution(125 lg ml�1 papain in 0.1 M sodium phosphate with 5 mMNa2-EDTA and 5 mM cysteine-HCl at pH 6.5) for 16 h at 60 �C.Papain solution without sample for using as a diluent in biochem-ical assays was also incubated. The DNA content was determinedvia Hoechst 33258 assay using a fluorescence spectrophotometer(TECAN, Switzerland; excitation wavelength: 360 nm, emissionwavelength: 450 nm) [31]. The standard curve for DNA was gener-ated using calf thymus DNA and used for quantifying the DNA inconstructs.

2.7. Live/dead assay of the constructs

Cell viability of the constructs at days 1, 7, 14 and 30 were con-ducted using a live/dead viability kit (LIVE/DEAD� Viability/Cyto-toxicity Kit, Invitrogen, USA). The assay dye was prepared bymixing ethidium homodimer (EthD-4 lM) and calcein AM (2 lM)in PBS. The printed cell encapsulated 8SF–15-T constructs werewashed with PBS, incubated for 30 min in the live/dead dye solu-tion at 37 �C and visualized by a confocal laser scanning micro-scope (Olympus FV 1000, USA). Four representative images weretaken from different areas of a single constructs. For each timepoint, samples were analyzed in triplicate. The live and dead cellsat the respective days were counted from the images by Image Jsoftware (NIH) and compared [32].




2.8. Long-term stability of the constructs

Long-term stability of the printed 8SF–15G–T and 8SF–15G–Sconstructs was evaluated by incubating them in cell culture media.Images were captured of the printed constructs soon after fabrica-tion and considered as day 0. Subsequently, images of the con-structs were captured after incubation in culture media at days 7and 21. Additionally, alginate constructs were also printed using6% alginate solution following the same procedure and a stabilitytest was carried out in a similar fashion.

2.9. Evaluation of cell proliferation rate

The proliferation rate of hTMSCs encapsulated 8SF–15G–T, 8SF–15G–S and 6% w/v alginate constructs were obtained by using theCell Counting Kit-8 (CCK-8 kit, Dojindo Molecular Technologies,Kumamoto, Japan) following the manufacturer’s protocol. At thescheduled time period (days 1, 4, 7, 14 and 21), the constructs werewashed with PBS and incubated in the CCK-8 kit along with cellculture media in a ratio of 1:10. After 2 h, the absorbance was readusing a plate reader (Wallac 1420 VICTOR3, PerkinElmer, USA).

2.10. Biochemical assay

The hTMSCs encapsulated 8SF–15G–T, 8SF–15G–S and 6% w/valginate constructs were cultured in chondrogenic and osteogenicmedia and were analyzed for an estimation of generated sulfatedGAGs (sGAGs) and total collagen content after 14 and 21 days ofculture. Samples were digested in papain digestion buffer for16 h at 60 �C and assayed for total sGAGs content via spectropho-tometry (ASYS UVM340) with 1,9-dimethylmethylene blue chlo-ride (Sigma-Aldrich, USA). Chondroitin sulfate A sodium salt frombovine trachea (Sigma-Aldrich, USA) (0–60 lg ml�1) was used asa standard. The total collagen content was measured using ahydroxyproline assay. The hydroxyproline content of the speci-mens subsequent to acid hydrolysis was quantified by treatmentwith p-dimethylaminobenzaldehyde (PDAB, Sigma-Aldrich, USA)and chloramine-T (Sigma-Aldrich, USA) according to the manufac-turer’s protocol. The hydroxyproline content in samples was calcu-lated by referring to the standard curve generated in advance.

2.11. Evaluation of osteogenic, chondrogenic and adipogenicdifferentiation

After printing, constructs were cultured for 24 h in cell prolifer-ation media, followed by replacement by osteogenic, chondrogenicand adipogenic media. Total messenger RNA (mRNA) was isolatedfrom the engineered constructs using RNAiso plus (Takara, Seoul,Korea) following the manufacturer’s protocol. RNA concentrationand the purity were determined using a Nanodrop ND 1000 Spec-trophotometer (NanoDrop Technologies, Wilmington, DE).Extracted RNA was reverse-transcribed into cDNA using a firststrand cDNA synthesis kit (Thermo Scientific, USA). Quantitativereal-time polymerase chain reaction (qRT-PCR) was conductedusing a SYBR green master mix (Takara, Seoul, Korea) and Rochelightcycler 2.0 (Roche Applied Science, USA). The primers andprobes for the reference gene glyceraldehyde 3-phosphate dehy-drogenase (GAPDH) and target genes aggrecan (ACAN), collagenII (COL-II), runt-related transcription factor 2 (RUNX2), alkalinephosphatase (ALP), osteopontin (OPN), peroxisome proliferator-activated receptor c (PPARc), CCAAT/enhancer binding protein a(C/EBPa) and lipoprotein lipase (LPL) were designed on the basisof published gene sequences (NCBI and PubMed). The analysiswas carried out with lightcycler software 4.0 (LCS4) and the rela-tive expression levels were calculated using the 2�(DDc(t)) method


with GAPDH as a control. For each time point, samples were ana-lyzed in triplicate.

2.12. Immunohistochemical analysis

At the requisite time period (days 14 and 30), cell-laden 8SF–15G–T constructs were rinsed with PBS and fixed in 10% formalinfor 30 min. They were treated with 0.2% bovine serum albuminfor blocking the non-specific background staining. They were thenprocessed for immunohistochemistry using antibodies againstRUNX2 (Santa Cruz Biotechnology, USA), ALP (Abcam, USA) forosteogenesis and ACAN (Abcam, USA), COL-II (Abcam, USA) forchondrogenesis and counterstained with 40,6-diamidino-2-phenyl-indole (Gibbs, USA). Samples were visualized by a confocal micro-scope (Olympus FluoView™ FV1000, Tokyo, Japan).

2.13. Histological analysis

Constructs were rinsed in PBS, fixed in 4% formaldehyde,embedded in OCT, sectioned (10 lm thick) and stained withMayer’s haematoxylin and eosin (H&E) for studying cell morphol-ogy. Sections were also stained with alcian blue (Sigma-Aldrich,USA) and alizarin red dye (Sigma-Aldrich, USA) for assessing thepresence of sGAG for chondrogenic differentiation and mineraliza-tion for osteogenic differentiation, respectively.

2.14. Statistical analysis

Each test was carried out in triplicate. Unless otherwise men-tioned, data are presented as mean ± standard deviation. Statisticalanalyses were performed using a two-tailed Student’s t test withp < 0.05 as the criterion for statistical significance.

3. Results and discussion

3.1. Preparation and crosslinking of silk–gelatin bioink

The overall bioprinting process of 3-D cell-laden constructsusing SF–G bioink is shown in Fig. 1. Principal to our approach isthe selection of suitable ratios of silk and gelatin blend based onthe rheological parameters and ease of extrusion. For extrusion-based dispensing, printing fidelity generally increases withincreasing viscosity, as bioinks with higher viscosities and instantgelation (crosslinking) would facilitate the maintenance of the fil-amentous shape after deposition [28]. But highly concentratedhydrogel bioinks would provide a restrictive environment for masstransport, cell proliferation, migration, and extracellular matrix(ECM) accumulation [33]. Thus, based on our previous optimiza-tion of rheological characteristics [18] of various SF–G bioinks itseemed logical to select a blend of 8% SF and 15% G for preparationof the bioink.

The SF–G bioink displays thermoresponsive behavior withoutcrosslinking, exhibiting liquid phase at physiological temperature(37 �C) and transformation into gel at temperatures below 30 �C.We efficiently utilized this phenomenon to print cell-laden con-structs with engineered porosity. Crosslinking of the cell printedSF–G constructs is necessary to stabilize them in physiological con-ditions. Commonly used crosslinking agents such as methanol,glutaraldehyde, EDC-NHS [34], photoinitiator and exposure to UVlight [35] would not be suitable for cell encapsulation. Hence,two innocuous in situ crosslinking methods were chosen forSF–G hydrogel: enzymatic crosslinking by mushroom tyrosinase[21,36] and physical crosslinking via sonication [22,37]. Differentconcentrations of tyrosinase and different sonication regimes weretried to optimize precise conditions for in situ crosslinking of the



Fig. 1. (a) Schematic elucidating the bioprinting of 3-D cell-laden constructs using in situ crosslinkable silk–gelatin (8SF–15G) bioink. (b) Crosslinking mechanism of silk andgelatin by tyrosinase, where three major steps are involved.


SF–G blend. After evaluating those conditions, sonication with 50%amplitude for 10 s (Supplementary Tables S.1 and S.2) and tyrosi-nase concentration of 500 units (Supplementary Table S.3) wereultimately chosen for physical and chemical crosslinking, respec-tively, as they formed a stable gel within 1 h (SupplementaryFig. S.1). The performance of 8SF–15G–T and 8SF–15G–S bioinkswas compared with alginate solution, as it is commonly used asa bioink for cell encapsulation [4,28–30].

3.2. Characterization of 8SF–15G bioink

The secondary conformations of the 8SF–15G–T and 5SF–15G–Sbioinks, as well as uncrosslinked 8SF–15G, were characterizedthrough FTIR. There are three structural morphologies in which silkfibroin can exist: silk I, II and III, where silk I is a structural formconsisting of more water and less b-sheet content, silk II is a struc-tural form consisting of b-sheets and silk III is a three-fold helicalstructure that is observed at the air–water interface [38]. The


absorbance data of 8SF–15G portray the bands for amide I, amideII and amide III at 1655, 1561 and 1265 cm�1, respectively, repre-senting characteristic peaks for silk and gelatin (Fig. 2). In the caseof 8SF–15G–T, the amide I band at 1656 cm�1 (with a shoulder at1640 cm�1), amide II band at 1543 and 1563 cm�1 and amide IIIat 1263 cm�1 were noticed. However, in the case of 8SF–15G–S,amide I band at 1698 cm�1 and 1650 cm�1, amide II at1558 cm�1 and amide III at 1229 cm�1 were observed. The 1460–1300 cm�1 spectral range appearing in 8SF–15G–T was more sim-ilar to Silk I than to Silk II [36]. However, along with the spectralpattern of Silk I (bands observed at 1050, 1033, 1018 and1265 cm�1), the component at 1078 cm�1 revealed the presenceof a certain amount of Silk II in 8SF–15G–T. The peak at1460 cm�1 of 8SF–15G–T was not observed in 8SF–15G–S, buttwo new peaks at 1451 and 1437 cm�1 appeared, probably dueto sonication-induced b-sheet crystallization [22]. Altogether,these findings highlighted the presence of predominant Silks Iand II in 8SF–15G–T and 8SF–15G–S, respectively [39].



Fig. 2. Characterization of silk–gelatin (SF–G) bioink by FTIR. (a) FTIR spectra of the 8SF–15G, 8SF–15G–T and 8SF–15G–S, (b) representative deconvoluted spectra of 8SF–15G–S and (c) b-sheet content of 8SF–15G, 8SF–15G–T and 8SF–15G–S.


Further, curve-fitting of the FSD spectrum (Fig. 2b and Supple-mentary Fig. S.3) was used to calculate the relative ratios of b-sheetfraction from the amide I region (Fig. 2c). The 8SF–15G–S samplepossessed higher b-sheet content compared to uncrosslinked8SF–15G and 8SF–15G–T (Table 1). The b-sheet content of 8SF–15G–S was in the range of the b-sheet obtained from methanoltreated SF–G blends [18]. We could not exclude the possibility thatnominal b-sheet content of uncrosslinked 8SF–15G and 8SF–15G–Tmight result from vacuum drying at room temperature conductedprior to carrying out a FTIR study.

The flow behavior of 8SF–15G–T, 8SF–15G–S and alginate attwo different temperatures, 28 �C and 37 �C (Fig. 3a), was investi-gated and correlated with the generated shear rate during printingof the structure. The calculated shear rate on the nozzle was 193.7–386.7 s-1 and accordingly the viscosity of 8SF–15G–T and 8SF–15G–S was evaluated (Table 2). The viscosity of both 8SF–15G–Tand 8SF–15G–S is nearly six-fold lower at 37 �C than that at28 �C at the generated shear rate in the micronozzle, as both8SF–15G–T and 8SF–15G–S were in the sol state at 37 �C; henceit was difficult to print any structure at this temperature. Unlikealginate, a significant decrease in viscosity over 30 �C was noticedwith both 8SF–15G–T and 8SF–15G–S (Fig. 3d), illustrating theinability to print any structure over 30 �C. Hence, we have decidedto print the structure at 28 �C as both 8SF–15G–T and 8SF–15G–Swere in the semi-gel state at this temperature and the viscosityof the bioink was adequate for printing. The 6% alginate solutionwas frequently used as a bioink for printing structure [4]. The vis-cosity of both 8SF–15G–T and 8SF–15G–S at 28 �C was found to besimilar to that of 6% alginate solution at the generated shear rateon the micronozzle wall (Fig. 3a), which also exemplifies the print-ing fidelity of our bioinks at 28 �C.

The storage and loss modulus of 8SF–15G–T and 8SF–15G–Swere investigated at three different temperatures, i.e. syringe bar-rel temperature (28 �C), printing chamber temperature (18 �C) andat physiological temperature (37 �C) to ensure fabrication of theself-standing construct. The bioinks were dispensed at 28 �C whilemaintaining the printing chamber temperature at 18 �C as the

Table 1Secondary structure of silk-fibroin in 8SF-15G, 8SF-15G-T, and 5SF-15G-S.

Conformation 8SF-15G (%) 8SF-15G-S (%) 8SF-15G-T (%)

b-sheet 17.3 25.4 14.2Random coils 15.3 14.5 19.5Silk I, type II b-turns 25.1 12.9 21.7a-helices 2.8 1.9 2.3Aggregated b-strands 39.5 25.3 42.3


storage modulus of both 8SF–15G–T and 8SF–15G–S was signifi-cantly higher at 18 �C than at 28 �C (Fig. 3b), which ensured theshape retention following fabrication. The storage modulus of both8SF–15G–T and 8SF–15G–S was nearly half at 37 �C than that at28 �C. Hence, we assumed that the filamentous shape of the struc-ture following fabrication could be well maintained after deposit-ing at 28 �C.

The gelation kinetics of 8SF–15G was evaluated following tyros-inase incorporation (8SF–15G–T) or sonication (8SF–15G–S). At28 �C, both 8SF–15G–T and 8SF–15G–S exhibited a solid like phaseprior to complete gelation due to its thermoresponsive property,and hence the gelation kinetics was investigated only at 37 �C.Gelation occurred in both cases at 37 �C with increment in complexmodulus significantly during the 40 min study period (Fig. 3e).Rapid gelation was noticed in the case of sonication-induced gela-tion with the significant increment of complex modulus comparedwith that of tyrosinase-induced gelation. Relatively stiffer gel wasformed in 8SF–15G–S (complex modulus was almost two timeshigher than the 8SF–15G–T), which can be explained by the pres-ence of a significantly higher percentage of b-sheet in 8SF–15G–Sthan that in 8SF–15G–T, as evidenced in FTIR analysis.

3.3. Preparation of hTMSCs laden bioink, fabrication of constructs,long-term stability and viability

The movement, temperature and pressure of each printing headof in-house-developed MHDS (Fig. 4a) [40] can be controlled indi-vidually, and four different bioinks or cell suspensions can be dis-pensed simultaneously through a programmed fashion using CAD/CAM-based software developed by our laboratory. We have suc-cessfully produced self-standing cell-laden 8SF–15G–T and 8SF–15G–S constructs by depositing hTMSCs encapsulated hydrogelthrough a 250 lm diameter nozzle and printing in a layer-by-layermanner (Fig. 4). Various printing parameters such as dispensingspeed, line width, spacing between lines, height of each layersand printing temperature were optimized. During fabrication, thetemperature of the syringe barrel was maintained at 28 �C andthe printing chamber was maintained at 18 �C, so that cell-laden8SF–15G bioink remained in a semi-gel state in the syringe andthe bioink smoothly extruded while applying pressure. Immedi-ately after extrusion, low temperature enabled us to preventspreading or deformation of the deposited cell-laden filamentousstructures and ensured shape retention (Fig. 4d and e). A highdegree of control over the deposition process is required forfabrication of self-standing cell-laden construct. With the 250 lmnozzle, the obtained line width of the construct was 280-320 lm.Furthermore, the change in the filament diameter during



Fig. 3. Characterization of silk–gelatin (SF–G) bioink by rheology. (a) Viscosity at varied shear rates and storage (G’) and loss (G’’) modulus of (b) 8SF–15G–T and (c) 8SF–15G–S. (d) Viscosity at varied temperatures and (e) gelation kinetics of 8SF–15G–T and 8SF–15G–S at 37 �C (⁄p < 0.05).

Table 2Rheological properties of 8SF-15G-T, 5SF-15G-S, and 6% alginate solution.

Samples Viscosity (Pa.s) Storage modulus (Pa) Loss modulus (Pa)

28 �C 37 �C 18 �C 28 �C 37 �C 18 �C 28 �C 37 �C

200 s-1

(Shear rate)400 s-1

(Shear rate)200 s-1

(Shear rate)400 s-1

(Shear rate)1% strain 1% strain 1% strain 1% strain 1% strain 1% strain

8SF-15G-T 1.71 0.95 0.37 0.34 530000 415000 166000 58700 65900 313008SF-15G-S 3.25 1.58 0.57 0.54 751000 504000 221000 84500 32800 422006% Alginate 4.45 3.31 3.04 2.32 – – – – – –


incubation in cell culture media was evaluated by swelling study.The swelling behavior of printed constructs was evaluated(Supplementary Fig. S.4). The 8SF–15G–T constructs revealedhigher swelling percentage (55–60%) than that of 8SF–15G–Sconstructs (30-35%). This might be due to higher percentage ofb-sheets in 8SF–15G–S than 8SF–15G–T, as evident from the FTIRresults. The rigid and hydrophobic b-sheet crystals result in lowerswelling of 8SF–15G–S.

To apply printed cellular constructs for tissue regeneration, themost important requirements would be the long-term survival andpreservation of functionality of the encapsulated cells within thehydrogel bioink [41]. The effect of shear force due to the pneumaticpressure applied during printing might be detrimental to theencapsulated cells. The pressure and nozzle diameter were opti-mized to obtain maximum cell viability. Fig. 5 illustrates represen-tative images for the live/dead assay of the cell-laden 8SF–15G–Tconstructs at days 1, 7, 14 and 30. Remarkably, hTMSCs were viablein 8SF–15G–T constructs over 1 month in in vitro culture condi-tions, maintaining a stable 3-D structure. Predominant green fluo-rescence evidenced the dominated population of live cells and


clearly indicated that cells were viable even on day 30. Takentogether, the optimized dispensing parameters did not have anyharmful effect on the encapsulated cells and also 8SF–15G–Tmatrix chemistry supported cell viability at least up to 1 monthwithout disruption of constructs, emphasizing the potential ofSF–G bioink as a cell encapsulating material.

Long term stability of the constructs during their culture inmedia is an indispensable requirement for potential applicationsin 3-D tissue regeneration. The stability of the printed 8SF–15G–T and 8SF–15G–S was evaluated by incubating them in cellculture media and comparing them to that with alginate con-structs (Fig. 6). The 8SF–15G–T and 8SF–15G–S constructs werefound to be stable for 7 days without any disruption of the struts.However, alginate constructs were found to degrade a little. Atday 21, 8SF–15G–S constructs were found to be unstable withdeformation of the structure. Sonication caused transformationof the a-helix to the b-sheet of silk fibroin, which in turn cross-links the SF–G hydrogel with the tightly packed b-sheet. How-ever, sonication does not crosslink gelatin (SupplementaryTable S.1) and the gelatin can be released slowly in a sustained



Fig. 4. 3-D bioprinted silk–gelatin constructs through a MHDS. (a) Actual view and (b) schematic diagram of MHDS for printing of cell-laden construct. (c, d) Dimensions ofthe printed structures showing line width, line gap and spacing between layers. Representative images of self-standing SF–G constructs: (e) sonication-induced b-sheetcrystallized 8SF–15G–S and (f) tyrosinase crosslinked 8SF–15G–T constructs (scale bar, 5 mm). SEM images of the 8SF–15G–T constructs: (g) top view (scale bar, 5 mm) and(h) inclined view (scale bar, 1 mm).

Fig. 5. Live/dead images of hTMSCs encapsulated in tyrosinase crosslinked 8SF–15G–T construct at day 1 (a, e), day 7 (b, f), day 14 (c, g) and day 30 (d, h) (scale bar, upperpanel, 400 lm and lower panel, 100 lm). (i) Quantitative analysis of cell viability within 8SF–15G–T constructs over 30 days.


manner from the 8SF–15G–S, leaving behind the voids. This hasalso been validated by carrying out the gelatin release profilestudy for a period of 28 days, which shows �18% release of gela-tin (Supplementary Fig. S.5). Hence, the 8SF–15G–S constructswere unstable in culture media for longer duration. On the otherhand, 8SF–15G–T constructs maintain their shape and structureat day 21. Covalent crosslinking by tyrosinase enhances the sta-bility of 8SF–15G–T constructs. Alginate structure completely dis-solves away at day 21. This might be because of the ionicexchange of the Ca2+ with Na+ and others and solubilizing ofuncrosslinked alginate in cell culture media. Hence, we used only8SF–15G–T constructs for further studies as these constructswere stable for over 1 month in cell culture media.


3.4. Evaluation of cell proliferation rate and biochemical assay

Comparative evaluation of 8SF–15G–T, 8SF–15G–S and alginatebioinks was done in terms of cell proliferation, ECM productionand gene expression analysis. The proliferation rate of cells encapsu-lated in alginate, 8SF–15G–T and 8SF–15G–S was evaluated up to3 weeks (Fig. 7a). The rate of cell proliferation was lowest in the caseof alginate, probably due to the lack of bioactive molecules in algi-nate [12]. Though both 8SF–15G–T and 8SF–15G–S have gelatin con-taining cell adhesive RGD motifs, the cell proliferation rate washighest in 8SF–15G–T. The presence of a tightly packed b-sheet crys-talline environment in 8SF–15G–S might have slowed down theproliferation rate of encapsulated hTMSCs. On the contrary, the



Fig. 6. Long-term stability of the 8SF–15G–T, 8SF–15G–S, and alginate (control) constructs in cell culture media.

Fig. 7. Cellular responses within silk–gelatin (SF–G) constructs. (a) Cell proliferation analysis in cell-laden alginate, tyrosinase incorporated 8SF–15G–T and sonicated 8SF–15G–S at various time points. (b) DNA contents of the printed constructs in chondrogenic and osteogenic condition, (c) glycosaminoglycans content (lg ng–1 of DNA), (d) totalcollagen content (lg ng�1 of DNA) of cell-laden alginate, 8SF–15G–T and 8SF–15G–S constructs for assessing chondrogenic and osteogenic capacity of respective bioink(⁄p < 0.05).


presence of covalent crosslinking and a lower percentage of b-sheetin 8SF–15G–T might have provided a stable yet favorable environ-ment for the encapsulated hTMSCs. The reduced proliferation rateafter 2 weeks probably indicates a pivotal balance between prolifer-ation and differentiation of stem cells [42]. We cannot exclude thepossibility that acreduced proliferation rate in the case of 8SF–15G–S after 21 days might also be due to slow release of the uncross-linked gelatin component, as we noticed gradual disintegration ofconstructs. This outcome further highlights the important role ofthe microenvironment in cell proliferation. In comparison, earlierstudies reported that the cells encapsulated in 8% silk hydrogels pro-liferated over the first 6 days, followed by stabilization of cell num-bers, indicating the limit of cell proliferation in the encapsulatedstate [22]. Also, cells were found to be largely deformed and aggre-


gated with time-poor cell viability after 6 days, probably due tothe predominant presence of tightly packed b-sheets in sonicatedsilk hydrogel. However, the 8SF–15G blend used in this studyprovided a supportive microenvironment for the encapsulatedhTMSC for the 21 day study period.

The amounts of accumulated GAG normalized to the DNA con-tent increased in all experimental groups from 2 to 3 weeks of cul-ture, indicating chondrogenic differentiation and temporalaccumulation of cartilaginous matrix. Interestingly, the level ofGAG content was lower at both 2 (p = 0.008) and 3 (p = 0.005)weeks in 8SF–15G–S than that of 8SF–15G–T (Fig. 7c). But GAGcontent level did not differ significantly between 2 and 3 weeksin alginate constructs (p = 0.212). There was no significant changein DNA contents at different time points (between 2 and 3 weeks;



Fig. 8. Evaluation of differentiation of hTMSCs into chondrogenic, osteogenic and adipogenic lineages. 8SF–15G–T supported better chondrogenic and adipogenicdifferentiation, whereas 8SF–15G–S supported better osteogenic differentiation of encapsulated hTMSCs.


Fig. 7b), which indicates that the chondrogenic differentiation wasindirectly regulated through differential cell proliferation.

The level of total collagen content normalized to the DNA con-tent was highest in 8SF–15G–T at 3 weeks in the chondrogeniccondition, compared to alginate (p = 0.028) and 8SF–15G–S(p = 0.005) constructs (Fig. 7d). In the case of the osteogenic condi-tion, the total collagen production was significantly higher at2 weeks in 8SF–15G–S constructs than others. The presence oftightly packed b-sheets in 8SF–15G–S might have provided a stiffmatrix environment to the cells, which in turn has promoted


higher collagen production in the osteogenic condition at 2 weeks.However, at 3 weeks the level of collagen content was reduced in8SF–15G–S compared with that of 2 weeks of the same constructs.This could be because of the slow release of the uncrosslinked gel-atin portion from the tightly packed b-sheet in the case of 8SF–15G–S as discussed earlier. There were no significant differencesin the levels of collagen content at 2 and 3 weeks in alginate con-structs in osteogenic conditions (p = 0.098). This emphasizes thesuperiority of 8SF–15G constructs over alginate as a cell encapsu-lating material for preserving the cellular functionality.



Fig. 9. Confocal images for assessing the chondrogenesis and osteogenesis of hTMSC laden tyrosinase-induced crosslinked silk–gelatin constructs. For chondrogenesis, ACANand COL-II expressions and for osteogenesis, RUNX2 and ALP expressions are shown at days 14 and 30.


To date, alginate has been extensively used as a bioink for bio-printing due to ease of processing and translating into 3-Dstructures by crosslinking with Ca2+ ions [4,12–14,30]. Interest-ingly, in the present study both hTMSCs-laden 8SF–15G–T and8SF–15G–S constructs have outperformed alginate bioink in termsof cell proliferation and ECM production. Due to the absence of cellrecognizable motifs, alginate does not favor cell attachment;rather, it only passively encapsulates cells [12,19]. Furthermore,the reason behind better cellular proliferation in the 8SF–15G–Tconstruct could be the difference in the matrix microenvironmentcompared to 8SF–15G–S. Comparatively less b-sheet content in8SF–15G–T would result in a less compacted matrix structure,which will allow easier matrix remodeling by cells, due to easierdiffusion of proteolytic enzymes and faster hydrolysis. The rheo-logical analysis also further corroborates this claim, which clearlyrevealed that the 8SF–15G–S gel was two times stiffer than thatof the 8SF–15G–T gel.

3.5. Evaluation of chondrogenic, osteogenic and adipogenicdifferentiation in SF–G constructs

The multilineage differentiation capability of encapsulatedhTMSCs was assessed by respective differentiation marker geneexpression analysis during culture in chondrogenic, osteogenicand adipogenic differentiation media for 3 weeks (Fig. 8). At day21, hTMSCs encapsulated in 8SF–15G–T showed greater expressionof early chondrogenic transcription factor SOX9 (five-fold higherthan alginate) than that of 8SF–15G–S and alginate in the presenceof chondrogenic differentiation factors. The level of SOX9 expres-


sion was 5.5-fold upregulated in 8SF–15G–S at day 14 comparedto alginate. 8SF–15G–T constructs revealed a higher expressionlevel of ACAN at all the time points, especially at day 21 with a13-fold increase compared with that of alginate. COL-II expressionlevel was also higher in 8SF–15G–T constructs than that of 8SF–15G–S and alginate at all the time points, with 2.5- and 5-foldchanges at days 7 and 14, respectively. However, there were no sig-nificant differences in COL-II expression levels between 8SF–15G–Tand 8SF–15G–S constructs at day 21. Altogether these results indi-cate the highest chondrogenic potential of 8SF–15G–T constructs.

Interestingly, at all time points except day 21, hTMSCs encapsu-lated in 8SF–15G–S revealed higher osteogenic gene expressionlevel for transcription factor RUNX2, early differentiation markerALP and OPN, compared to that of alginate and 8SF–15G–T(Fig. 8). There was a 6- and 9-fold increase of RUNX2 level in8SF–15G–S compared with that of alginate at days 7 and 14,respectively. However, at day 21, the level was similar to that of8SF–15G–T, with a 3-fold increase in RUNX2 expression level. Inthe case of ALP expression level, a 3- and 6-fold increase wasobserved in 8SF–15G–S constructs at days 7 and 14, respectively.A significant increase in the expression level of OPN was observedat day 14 in 8SF–15G–S constructs with a 6-fold increase. However,at day 21, the OPN expression level was higher in 8SF–15G–T con-structs than that of others. Overall the results suggest that 8SF–15G–S constructs supported the highest osteogenic differentiationof hTMSCs until the second week. At day 21, the expression levelsof osteoblast specific genes were low for 8SF–15G–S constructs.The instability of 8SF–15G–S constructs in cell culture media forprolonged duration, as discussed earlier, might be responsible for



Fig. 10. Histological images showing the osteogenesis and chondrogenesis of hTMSCs laden tyrosinase induced cross-linked silk–gelatin constructs. H&E staining of (a, b)osteogenic and (c, d) chondrogenic constructs. Overall, hTMSCs retained a round shape in the gel and proliferated throughout the gel (b, d). Accumulations of mineral-likephase (red dots) were observed in the osteogenic construct by alizarin red staining (e, f). Collection of GAGs in the chondrogenic constructs were observed by alcian bluestaining (g, h). Osteogenic and chondrogenic acellular controls are represented by (i, j) and (k, l), respectively.


down-regulation in the expression level of transcription factors at3 weeks.

A significant increase in the expression of the master adipogenicregulators PPARc and C/EBP together with an increase in early adi-pogenic marker LPL was observed when hTMSCs were cultured in8SF–15G–T compared with that of 8SF–15G–S and alginate in adi-pogenic condition (Fig. 8). The gene expression levels show that8SF–15G–T clearly outperformed alginate and 8SF–15G–S at alltime points for PPARc with a 3-, 4- and 6-fold upregulation in geneexpression level at 7, 14 and 21 days, respectively. In the case of8SF–15G–T constructs, 3-, 5- and 6-fold increments in C/EBP geneexpression were observed at 7, 14 and 21 days, respectively, com-pared to alginate constructs. Although the level of LPL expressionwas not significantly different at day 7 among three constructs,at 14 (2-fold) and 21 days (3-fold), a significant increase of LPLexpression level was witnessed. Altogether these results indicatethe better adipogenic potential of 8SF–15G–T constructs. Adipo-genic differentiation was reported to improve under high densityconditions where the cells have a more rounded morphology,and is also related to their growth arrest [43]. Thus, it could beassumed that 8SF–15G–T constructs provide a favorable environ-ment for the encapsulated hTMSCs for adipogenic differentiation.The stiffness of the matrix has been shown to direct the differenti-ation of stem cells when the cells were either adhered to substrates[44] or encapsulated in a matrix [45]. The reason behind higherlevels of chondrogenic/adipogenic and osteogenic differentiationin 8SF–15G–T and 8SF–15G–S constructs, respectively, might bea factor of the physical properties of the construct, which in turncreate a difference in microenvironment for the encapsulated cells.

3.6. Immunohistochemical and histological evaluation of 8SF–15G–Tconstructs

Immunohistochemical analysis was performed on the cell-ladenconstructs for RUNX2 and ALP for assessing the osteogenic


differentiating capacity, and ACAN and COL-II for assessing thechondrogenic differentiating capacity only in 8SF–15G–Tconstructs, as 8SF–15G–S constructs were unstable after long-termculture. There was nominal expression of ACAN and COL-II at day 14in the case of chondrogenic differentiation of encapsulated hTMCSs(Fig. 9). The expression of both ACAN and COL-II increased signifi-cantly at day 30. Similarly, a higher intensity of ALP and RUNX2staining was noticed at day 30, compared to day 14 during osteo-genic differentiation of hTMSCs, (Fig. 9), which was in agreementwith the gene expression analysis. Thus, the 8SF–15G–T constructssupport both osteogenic and chondrogenic potential of the encapsu-lated hTMSCs when respective differentiation factors wereprovided.

Histological analysis (H&E) revealed that encapsulated hTMSCsin 8SF–15G–T constructs retained a round shape and were non-aggregated throughout the study, while some cell-to-cell connec-tions were visualized (Fig. 10). Accumulations of mineral-likephase were observed in the osteogenic constructs by alizarin redstaining (Fig. 10e and f). Intense staining by alcian blue confirmedGAGs production by the encapsulated hTMSCs in the chondrogenicconstructs (Fig. 10g and h). Cellular aggregation is thought to be animportant step for inducing chondrogenic differentiation in MSCs,but in this case most of the cells are separated from each other andsurrounded by SF–G matrix. However, qRT-PCR studies showedupregulation in chondrogenic transcription factors with time, anddependence on matrix composition. Future attempts will be madeto encapsulate clusters of defined numbers of cells, and evaluatewhether that strategy further enhances the extent of differentia-tion, or induces faster differentiation compared to the same num-ber of scattered single cells encapsulated in SF–G bioink. Overall,observations from histological study further validate the findingof biochemical and immunohistochemical analysis.

Taken together, in the present study, for the first time, bioprint-ing with SF–G bioink that encompasses living hTMSCs is demon-strated. We have formulated a directly printable and in situ




crosslinkable SF–G bioink and provided a protocol to significantlyaccelerate progress in direct printing of cell-laden constructs. Here,we obtain a synergistic effect through combining the tailorablemechanical properties of silk fibroin with higher cellular respon-siveness of gelatin for the fabrication of tissue construct. Further,implementation of innocuous in situ crosslinkable SF–G bioinkallows the addition of cells directly to the deposition process forthe fabrication of self-standing cell-laden constructs with engi-neered porosity. This way, complex tissue-like architectures canbe produced by the deposition of various types of cells at specifiedlocations and by controlling the cell densities. Moreover, being arobust dispensing method, this cell printing process can result inconstructs that easily reach anatomically relevant (centimeterscale) sizes for tissue replacement. There is a prospect to fabricateheterogeneous tissue constructs using the two kinds of in situcrosslinking methods of SF–G hydrogels having different stiffness,as especially required for osteochondral tissue regeneration. Thestability of the 8SF–15G–S constructs can be extended by addi-tional crosslinking with either tyrosinase or riboflavin [46] subse-quent to the gelation of printed constructs. Further studiesshould be undertaken to investigate the heterogeneous tissue gra-dient formation. Hence, it is not an overstatement that this studyaddresses an unmet requirement of novel bioink for the biofabrica-tion of tissue analog consisting of live cells for tissue engineeringpurposes [12,47]. Optimized dispensability of silk–gelatin bioinkwould have the potential to establish simpler, practical and eco-nomical options for bioprinting tissue analogous structure withhigh resolution, reproducibility and, more importantly, long termcell viability. Nevertheless, there is potential utility in broaderareas like cell-based sensors [48], drug/toxicity screening [48]and in vitro diseased models, where a tissue analog structuredeveloped using our bioprinting method is worthwhile.

4. Conclusions

These results provide a proof-of-principle for the biofabricationof 3-D tissue constructs using novel silk–gelatin based bioinkencompassing living progenitor cells and in situ crosslinkingthrough cytocompatible gelation mechanisms. The developed con-structs supported multilineage differentiation of encapsulated stemcells and specific tissue formation. Tissue printing for targeted tissueregeneration in a patient-specific and site-specific manner would bethe answer for personalized therapy and our study is a promisingstep in this direction.

Acknowledgements

This work was supported by the National Research Foundation ofKorea (NRF) grant funded by the Korea government (MSIP) (No.2010-0018294 and 2011-0030075) and the Indo-Korean ResearchInternship Program (INT/ROK/IKRI-5/2013) from the Indo-Koreajoint program of cooperation in Science & Technology, organizedby the Department of Science and Technology, Government of Indiaand Ministry of Education, Science and Technology, Republic ofKorea.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.actbio.2014.09.023.


Appendix B. Figures with essential colour discrimination

Certain figures in this article, particularly Figs. 1–10, are diffi-cult to interpret in black and white. The full colour images canbe found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2014.09.023.

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Bioprintable, cell-laden silk fibroinâ€“gelatin hydrogel ...

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