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Role of chondroitin sulphate tethered silk scaffold in cartilaginous disc tissue regeneration

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2016 Biomed. Mater. 11 025014

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1. Introduction

Engineering a target tissue with complete functional equivalence requires replicating the precise combination of its structure, composition and properties. Replicating the 3D structural hierarchy and surface functionalization of biomimetic scaffolds by covalent conjugation of biomolecules can thus offer fascinating possibilities in tissue engineering. In addition, a systematic investigation to explain the inherently complicated cell–micro-environment interaction will further offer crucial insights for developing tissue engineered constructs.

An intervertebral disc (IVD) is a multilamellar tis-sue, which withstands and dissipates mechanical load and imparts flexibility to the spine. Such challenging biomechanics is mainly dictated by the unique struc-tural organization of collagen fibers in the annulus fibrosus (AF) layer, embedded within a dense fibril-lar matrix of both large (aggrecan, versican) and small (biglycan, decorin, lumican, fibromodulin) aggregating

proteoglycans [1]. In addition, the AF layer is charac-terized by a regional distinction containing outer AF (containing collagen I) and inner AF (containing col-lagen II along with several proteoglycans) layers, which provide an appropriate platform for precisely manag-ing tensile and compressive loads in vivo [2]. Further to this, the disc tissue is known to bear large amounts of load by keeping an appropriate balance between three important factors including the amount of load applied, swelling pressure generated by the negative charges present on the glycosaminoglycan (GAG) chain of proteoglycans and the stress–strain response of the collagen–proteoglycan matrix [3].

Disorganization of the fibrous structural network and depletion of proteoglycans lead to the formation of cracks in the AF layer that ultimately result in a failure to resist complex deformational load during bending and flexion of the spine. So far, several attempts have been made to simulate the composition [4–6] and com-plex structural hierarchy [7–10] of AF tissue in order to develop a functional replacement. In an attempt to

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Role of chondroitin sulphate tethered silk scaffold in cartilaginous disc tissue regeneration

Maumita Bhattacharjee1, Shikha Chawla1, Shibu Chameettachal1, Sumit Murab1, Neel Sarovar Bhavesh2 and Sourabh Ghosh1

1 Department of Textile Technology, Indian Institute of Technology, New Delhi, 110016, India2 Transcription Regulation Group, International Centre for Genetic Engineering & Biotechnology, New Delhi, 110067, India

E-mail: sghosh08@textile.iitd.ac.in

Keywords: intervertebral disc, biofunctionalization, silk fibroin, chondroitin sulfate, extra cellular matrix protein interaction

Supplementary material for this article is available online

AbstractStrategies for tissue engineering focus on scaffolds with tunable structure and morphology as well as optimum surface chemistry to simulate the anatomy and functionality of the target tissue. Silk fibroin has demonstrated its potential in supporting cartilaginous tissue formation both in vitro and in vivo. In this study, we investigate the role of controlled lamellar organization and chemical composition of biofunctionalized silk scaffolds in replicating the structural properties of the annulus region of an intervertebral disc using articular chondrocytes. Covalent attachment of chondroitin sulfate (CS) to silk is characterized. CS-conjugated silk constructs demonstrate enhanced cellular metabolic activity and chondrogenic redifferentiation potential with significantly improved mechanical properties over silk-only constructs. A matrix-assisted laser desorption ionization-time of flight analysis and protein–protein interaction studies help to generate insights into how CS conjugation can facilitate the production of disc associated matrix proteins, compared to a silk-only based construct. An in-depth understanding of the interplay between such extra cellular matrix associated proteins should help in designing more rational scaffolds for cartilaginous disc regeneration needs.

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received 15 November 2015

revised

3 March 2016

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8 March 2016

Published 8 April 2016

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mimic the biochemical composition of native AF tissue, several strategies have been employed including cova-lent coupling of RGD (arginine-glycine-aspartic acid) ligands to silk scaffolds and incorporation of GAGs: collagen II/hyaluronate/chondroitin-6-sulfate scaf-fold, gelatin/chondroitin-6-sulfate copolymer scaffold using 1-ethyl-3,3-dimethyl aminopropyl-carbodiimide (EDC)–N-hydroxysuccinimide (NHS) or glutaralde-hyde crosslinking [4–6]. Simultaneously, attempts have been made to replicate the structural hierarchy of AF tissue. For example, the concentric lamellar architecture of AF tissue was replicated by developing a hybrid algi-nate/chitosan scaffold with unidirectional fiber align-ment. The construct supported AF cell attachment and subsequent extra cellular matrix (ECM) deposition, however the heterogeneity and anisotropic nature of the collagen fibers present within the native AF tissue was lacking in these constructs [7]. Electrospun fibrous scaf-folds with different fiber alignments (partially aligned or random) were developed that supported the attachment of AF cells, their subsequent orientation along the fibers and ECM production. However, the tensile modulus of the scaffolds was found to be lower (7.25 MPa) than the native tissue (28–78 MPa) [11]. In another study, mesen-chymal stem cells were cultured over poly-caprolactone electrospun matrices with anisotropic nanofibrous laminates [9]. These constructs replicated the struc-tural hierarchy of AF tissue, together with an organized ECM, resulting in improved mechanical properties. However, a major challenge in disc tissue engineering is the inability to replicate the exact 3D lamellar organiza-tion and biochemical composition of AF tissue, which often results in the development of constructs with inadequate mechanical properties that face complex and multidirectional loading issues.

Previously we have developed chondroitin sulfate (CS) conjugated silk fibrous scaffolds having lamellar architecture, using Antheraea mylitta silk fibers (due to its extraordinary strength and presence of RGD peptide sequences) using a custom-made winding machine [2, 12]. The parallel alignment of the CS-conjugated silk fibers guided the attachment, proliferation and ECM deposition of the goat articular chondrocytes along the direction of the fibers. Covalent crosslinking of CS molecules to a silk fibrous scaffold could provide certain cues to using human nasal chondrocytes to synthesize disc specific matrix components, so as to achieve the bio-mechanical functionality of the construct akin to native goat AF tissue. Moreover, the dynamic culture condi-tions provided for 28 d formed a cartilaginous tissue gradient with collagen I in the exterior (fibrocartilage) and collagen II in the interior (hyaline cartilage), simi-lar to the anatomical features of native IVD. However, the underlying mechanism as to why silk-CS constructs could impart higher mechanical property over silk-only based constructs remaine largely unexplained [12].

Chondroitin sulfate, a sulphated GAG, consisting of repeating units of glucuronic acid and N-acetylgalactosa-mine, has been chosen as it plays a regulatory role in the

biological and mechanical functionality of disc tissue. In addition, CS has been reported to promote the synthesis of proteoglycans during cyclic loading and also reduces the level of cell death and matrix damage, thus preventing the process of degeneration during acute overload [13]. Moreover, CS also elicits cellular reparatory processes and anti-inflammatory activity by reducing the levels of IL-1β induced NF-kβ nuclear translocation, subse-quently reducing the production of pro-inflammatory cytokines/enzymes [14, 15]. However, sulphation pat-terns of chondroitin-4-sulfate change in human IVD with ageing, together with an increase in ratio of 6- to 4-sulphated N-acetyl glucosamine residues. Hence, an increase in chondroitin-4- sulfate content may prevent tissue degeneration to a certain extent due to its reported therapeutic functionality [16].

The main goal of this study is not to develop a tis-sue engineered AF disc construct but to improve the function of CS conjugation to the silk scaffolds, to investigate cellular responses in terms of differential expression of ECM proteins at varied culture times, and to study protein–protein interaction and how this modulates the mechanical properties of the engineered constructs. We have also tried to elucidate the combined effect of controlled fiber alignment and biochemical composition in improving the functionality of these constructs towards AF tissue engineering.

2. Materials and method

2.1. Preparation of lamellar fibrous scaffolds and characterizationMultilamellar scaffolds were fabricated using Antheraea mylitta silk fibers obtained from the textile industry. Degummed silk fibers were used to fabricate a silk fibrous scaffold, with a multilamellar criss-cross pattern in successive layers, using a custom-designed fiber winding machine reported previously [17]. The resultant multilamellar silk scaffolds, measuring 0.3 cm in height and 0.5 cm in diameter with an inter-fiber spacing of 45.16 ± 19 μm, had a criss-cross orientation with an angle of +30° in one layer and −30° in the following layer. Some of the multilamellar silk scaffolds were biofunctionalized with CS as described earlier [12].

The crosslinking reaction was then characterized by attenuated total reflectance-Fourier transformed infrared spectroscopy (ATR-FTIR) spectroscopy (Alpha P Spectroscope, Bruker), a ninhydrin assay (to estimate the amount of free amine groups before and after crosslinking) and solution state nuclear magn-etic resonance (NMR) spectroscopy (2D [1H,1H] nuclear Overhauser effect spectroscopy; NOESY) [12]. The IR spectra were deconvoluted using Peak-fit v4.12 software (Systat Software Inc. San Jose, CA, USA). After normalizing, the relative area under each peak of the spectra was used to estimate the efficiency of the reaction by calculating the percentage of alde-hyde formation after the first reaction and Schiff’s

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base formation after the second reaction. For the nin-hydrin assay, the optical absorbance value of the test solution was measured using an ultraviolet–visible spectrophotometer (BioRad iMARK Microplate reader, US) at 560 nm, using various known concentrations of glycine as standard. NMR samples of oxidized CS, silk fibroin and silk-CS were prepared in deionized water containing 10% D2O (v/v) and trace of 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS). NMR experi-ments were conducted at 25 °C on a Bruker Avance III spectrometer equipped with a 5 mm cryogenic triple- resonance TCI probe, operating at a field strength of 700 MHz. DSS was used for 1H chemical shift referencing. Temperature calibration was carried out using a 100% d4-methanol sample [18]. 2D [1H,1H] NOESY spectra were measured using a mixing time (τm) of 100 ms.

2.2. Cell cultureArticular chondrocytes were isolated from the goat cartilage by enzymatic treatment with 0.1% w/v type II collagenase (Gibco) for 16 h at 37 °C [2]. Once isolated the cells were resuspended in a culture medium containing Dulbecco’s modified Eagle medium (Cellclone) with 10% fetal bovine serum (Biological Industries), 100 U ml−1 penicillin-streptomycin, 2.5 μg ml−1 gentamycin and 5 μg ml−1 amphotericin, 5 ng ml−1 fibroblast growth factor-2 (FGF-2) (Millipore) and 1 ng ml−1 transforming growth factor β1 (TGFβ1) (Millipore).

For culturing cells, the scaffolds were pre-sterilized in 70% ethanol overnight followed by pre-wetting in complete culture medium. Primary chondrocytes (P2) were subsequently seeded on these pre-conditioned scaffolds at a density of 2 × 105 cells/scaffold. Post-seeding, constructs were statically cultured for up to 6 weeks with the medium changed twice a week. Admin-istration of FGF was subsequently stopped after 1 week of culture. Cell-seeded constructs were harvested after 1 week, 3 weeks and 6 weeks for analysis.

2.3. Metabolic activityAn MTT assay was carried out to determine cellular metabolic activity. Constructs (n = 4/group) were harvested after 1 week, 3 weeks and 6 weeks, rinsed in phosphate buffered saline (PBS) and stained with tetrazolium salt MTT at 37 °C. After 6 h, an equal volume of dimethyl sulfoxide was added to the constructs to solubilize the crystals. The absorbance was measured at 560 nm using an iMark microplate Absorbance reader (Biorad). The experiment was repeated three times.

2.4. Immunohistochemical analysisAfter 1, 3 and 6 weeks respectively, constructs (n = 4/group) were rinsed thoroughly in PBS and fixed in 4% formalin. The fixed constructs were processed for immunohistochemistry (IHC). The constructs were permeabilized with triton X-100 (0.1%), followed by three consecutive washes with PBS and blocked with 1% bovine serum albumin (BSA diluted in PBS). For staining, the constructs were

incubated with primary antibodies: rhodamine-phalloidin (200 μg ml−1, 1 : 100, Millipore), anti-collagen II (200 μg ml−1, 1 : 100, Millipore), alexa fluor 546 goat anti-mouse IgG (1 : 200, Invitrogen) by incubating for 1 h at room temperature (RT). Nuclear staining was performed using 4′,6-diamidino-2-phenylindole (DAPI 300 nM, 2 : 100, Invitrogen) at RT after 5 min incubation. Images were captured using a confocal microscope (Leica TCS SP5) by scanning the constructs from the outer to inner layers. The composite image was generated by stacking together all the scans pertaining to each respective layer.

2.5. Gene expression studiesRNA was isolated from the engineered constructs using trizol (Invitrogen) and an Rneasy mini kit (Qiagen) as per the manufacturer’s instructions. The RNA concentration and purity were estimated using a nanodrop (Thermo Scientific, Wilmington, USA). Extracted RNA samples were reverse-transcribed into cDNA using a first strand cDNA synthesis kit (Fermentas). A quantitative real time polymerase chain reaction (qRT-PCR) was carried out using a SYBR green master mix and Rotor-Gene Q thermocycler (Qiagen). All reactions were carried out in triplicate, with each one comprising of a total reaction volume of 25 μl containing 1 μl of cDNA and 2.5 μl of primers. The QuantiTect primers (Qiagen) used were glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cat no. QT00079247), RhoA (Cat no. QT00044723), Sox-9 (Cat no. QT00001498), Collagen II (Cat no. QT00049518), Collagen I (Cat no. QT00037793), Aggrecan (Cat no. QT00001365), Biglycan (Cat no. QT01870029). Rotor-Gene Q software was used for analysis and the relative expression levels were calculated using the 2−(ΔΔc(t)) method with GAPDH as a housekeeping gene.

2.6. Biochemical analysisConstructs (n = 6/group) were harvested after 1, 3, and 6 weeks, and digested with proteinase K. The GAG content was spectrophotometrically measured after reacting with dimethylmethylene blue, using CS (from bovine trachea, 100 kDa MW, Sigma) as a standard.

A hydroxyproline assay was used to determine the total collagen produced by the cells. Constructs (n = 6/group) were digested with 500 μl of 1 mg ml−1 proteinase K containing a proteinase inhibitor and incubated over-night at 56 °C. The temperature was elevated to 100 °C for 15 min, then samples were hydrolysed by adding 1.5:1 volume ratio of 6N HCl and incubated overnight at 110 °C. The samples were dried and analyzed using a hydroxyproline assay [19], using hydroxyproline as a standard. The experiment was repeated three times.

2.7. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis of ECM proteins2.7.1. Isolation and SDS-PAGE of ECM proteins Engineered constructs were harvested after 3 and 6 weeks and minced followed by treatment with 1.5 ml

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of 7 M urea buffer (7 M urea, 2 mM sodium EDTA, 50 mM tris (hydroxymethyl) amino-methane, 0.5% triton X-100, 1% protease inhibitor mixture, 2 mM dithiothreitol (DTT), 1 mM phenyl methyl sulfonyl fluoride) with constant rotation at 4 °C overnight. The solution was centrifuged at 12 000 rpm for 10 min to extract the protein components. The resultant protein solution was mixed with 4 volumes of ice cold acetone, kept at −20 °C for 60 min and centrifuged at 10 000 rpm for 10 min to obtain a protein pellet that was air dried and resuspended in a tris buffer, pH 8.0. The proteins thus obtained were separated by electrophoresis under reducing conditions on an 8% SDS polyacrylamide gel. The gel was stained with coomassie brilliant blue R 250 for the 3 week constructs. For the 6 week samples, an additional silver staining was performed to obtain more clear protein bands. Molecular weights were determined using a molecular weight marker range 3.5–97 kDa.

2.7.2. In gel tryptic digestion The gels were given two consecutive washes of water for 10 min each following which the band of interest was harvested using a clean scalpel. Each gel piece was cut into 1 mm cubes and washed thoroughly with water. Destaining was performed by incubating the bands in 50/50 (acetonitrile ACN/50 mM ammonium bicarbonate, NH4HCO3) for 30 min. After this, the bands were dehydrated in 100% ACN for 5 min and dried in air for 10 min. Freshly prepared 10 mM DTT/100 mM NH4HCO3 was added to each sample and incubated for 30 min at 56 °C. The bands were washed with 50/50 (ACN/50 mM NH4HCO3). The bands were incubated in 50 mM iodoacetamide in 50 mM NH4HCO3 for 30 min at RT in the dark. The gel pieces were again washed and dehydrated. Proteins were digested by adding 10 μl of a 20 ng μl−1 trypsin solution for 16 h at 37 °C. Peptides were extracted with 1% trifluoroacetic acid by sonication for 5 min.

2.7.3. MALDI-TOF analysis 1 μl peptide extract after tryptic digestion was mixed with 1 μl of a suitable matrix α-cyano hydroxycinnamic acid in 1 : 2 v/v of ACN: 0.1% TFA. 1 μl of this mix was spotted on a MALDI target plate and air dried at RT. MALDI-TOF spectra were acquired using AB SCIEX 5800 MALDI TOF–TOF (AB SCIEX, Shanghai, China). Calibration was carried out using a peptide mass standards kit (AB SCIEX). 1000 laser shots per MS spectra were acquired at 400 Hz pulse frequency, while in the case of MS/MS, 2000 laser shots were acquired at 1 kHz pulse frequency. Further, an MS analysis was carried out using protein pilot software V 3.0 (AB SCIEX). A subsequent MS/MS data analysis was carried out using easy access wizard™ (AB SCIEX) and Quan Tis™ (AB SCIEX). Protein identification was carried out using a MASCOT search engine (Matrix Science) against the UniProtKB-SwissProt database in mammalian taxonomy. The protein sequences were

then analyzed for similarity searches by searching the sequences against the NCBI non-redundant protein database (nr) using the BLAST tool.

The protein–protein interactions were studied using a database of known and predicted protein inter-actions STRING 10.0 (Search Tool for the Retrieval of Interacting Genes/Proteins) software.

2.8. Biomechanical characterization of constructs2.8.1. Unconfined compression test of AF tissue and constructs Compression tests of native goat AF tissue (n = 6) and engineered constructs (n = 6) after 1, 3 and 6 weeks were conducted on a computer-controlled universal testing machine (UTM) model H5KS (Tinius Olsen, single stand machine, UK) with QMAT 5.37 software. The compression device consisted of two non-rotating stainless steel jaws with cross head adjustments. The area of tissue was measured using a vernier caliper. The samples were mounted onto the jaws of the UTM under a compressive load range of up to 900 N, test speed of 1 mm min−1, and compressed to 20% strain without applying any preload [12]. The experiment was repeated six times.

2.8.2. Tensile testing Tensile testing for native AF layers (n = 6, repeated three times) was performed in the computer-controlled UTM model H5KS (Tinius Olsen, single stand machine, England) with QMAT 5.37 professional software with a strain rate of 1 mm min−1. The engineered constructs were mounted on the jaws and stretched vertically at a rate of 1 mm min−1 with an applied load range of up to 900 N (n = 6) [12].

2.9. Statistical analysisStatistical significance was calculated by independent t-tests using SPSS software (SPSS Inc., Chicago, USA) and data was considered significant if p < 0.05. Data was presented as mean ± standard deviation (SD). The dependence of tensile strength and compressive strength on the amount of collagen and GAG production in silk and silk-CS constructs was determined by calculating the correlation coefficient.

3. Results

3.1. Biofunctionalization of multilamellar silk scaffolds with CSIn an attempt to optimize the architecture and biochemical composition of the constructs, firstly concentric lamellar, criss-crossed silk fibrous scaffolds were developed. The crosslinking reaction was carried out between silk and CS in a two-step mechanism, in which firstly sodium periodate oxidizes CS by glucuronic acid ring opening and converts its vicinal hydroxyls into aldehyde groups, followed by Schiff’s base formation between the amine groups of silk protein and aldehyde groups of oxidized CS [12]. The crosslinking reaction was

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characterized as previously described [12] and validated by ATR-FTIR and Fourier self-deconvolution (FSD) spectra, which showed 49.4% of the aldehyde formation in oxidized CS and 75.6% Schiff’s base formation with respect to the available free amine content in silk fibroin. A decrease in the intensity of the saccharide hydroxyl groups clearly indicated the formation of an aldehyde group. Moreover, clear evidence of the formation of Schiff’s base provided the necessary evidence of the silk-CS crosslinking (supplementary figure S1, stacks.iop.org/BMM/11/025014/mmedia). Further, the biofunctionalization of CS was validated by a ninhydrin assay which quantitatively estimated the efficiency of the reaction to be 80% (figures 1(A) and (B). Direct evidence of complex formation between silk and CS was observed from the 2D [1H,1H] NOESY spectra where silk backbone amide proton resonance at 7.85 ppm and 8.16 ppm showed an NOE cross peak to the proton of CS at 2.05, 4.06, 3.83 and 4.60 ppm thereby validating the crosslinking reaction between silk and CS (figure 1(C)).

3.2. Multilamellar scaffolds supporting the metabolic activity of cells, and cellular and newly synthesized ECM alignmentEstimation of metabolic activity indicative of cell viability demonstrated a 1.37 fold increase in silk-CS constructs over silk-only based constructs after the first week. Moreover, after 3 and 6 weeks the viability was found to be 1.54 fold and 2.4 fold higher in silk-CS and silk-only constructs (p < 0.05), respectively. Within silk-only constructs, the rate of metabolic activity remained almost the same over time (figure 2(A)). The topographical guidance of silk fibers on cellular orientation was confirmed by rhodamine-phalloidin staining which revealed that an actin cytoskeleton was organized in co-ordination with the nucleus, and the cells were aligned along the silk fibers (figure 2(B)). After 6 weeks, the immunostaining for collagen II revealed the presence of collagen fibrils distributed around the cells, parallel to the aligned fibers on the scaffold. Relatively higher intensity of staining and

Figure 1. (A) The blue colour indicates the presence of free amine groups in silk solution and the colourless solution indicates the absence of free amine groups in silk-CS solution. (B) The graph shows that 80% of the free amine groups present in silk are involved in reactions between silk and CS. (C) A selected region of a 2D NOESY spectra of silk fibroin (green contours), chondroitin sulphate (red contours) and silk-CS complex (blue contours) showing cross peaks of amide protons. NOE cross peaks of amide protons of silk to CS in the complex are shown with black arrows.

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more uniform distribution of collagen II was noticed in silk-CS constructs as compared to silk-only constructs (figures 2(C) and (D)).

3.3. Biofunctionalization of silk with CS upregulated expression of disc specific genes and biochemical componentsTo further understand the role of CS on developing a chondro-inductive environment, we studied differential gene expression patterns in both the constructs (figure 3).

The level of RhoA was upregulated 3.48 fold at 3 weeks and 2.87 fold at 6 weeks in a CS-tethered con-struct compared to a silk-only matrix. The expression of collagen type I could not be detected in either construct (data not shown). A significant expression of an early chondrogenic marker Sox-9 (2.96 fold higher) and other important cartilage markers such as collagen type II (1.8 fold higher), aggrecan (2.56 fold higher) and biglycan (2.4-fold higher) were found to be higher (p < 0.05) in silk-CS constructs after 6 weeks.

The effect of the biofunctionalization of the silk scaffold on synthesis and accumulation of impor-tant disc matrix components was then investigated through biochemical estimation of collagen and GAG (figures 4(A) and (B)). Negligible levels of collagen and GAG were detected after 1 week in both silk-CS and silk-only constructs (data not shown). After 6 weeks the collagen content was 2.6 fold higher (p < 0.05) in the case of silk-CS (79.1 ± 5.7 μg mg−1 wet weight)

compared to silk-only constructs (30.5 ± 2.8 μg mg−1 wet weight) (figure 4(A)). The GAG content was 1.7 fold higher (p < 0.05) in silk-CS (300 ± 8.07 μg mg−1 wet weight) as compared to silk-only constructs (180 ± 3.2 μg mg−1 wet weight) (figure 4(B)). The ratio of GAG/collagen was determined to be 3 and 6 (due to lower collagen content) in silk-CS and silk-only con-structs after 6 weeks, respectively.

3.4. Expression of disc associated proteins in the presence of CSWe further analyzed the ECM proteins in the constructs through proteomic analysis using the MALDI-TOF technique. The selection of bands was based on the differential protein expression profiles of the 1D SDS PAGE, and a number of bands were selected based on the exclusive presence in the silk-CS constructs, while we also selected some bands that were expressing in both the silk-only and silk-CS lanes. The selected bands taken further for the MALDI analysis are shown in figure 4(C) (3 weeks) and figure 4(D) (6 weeks). The proteins identified through MALDI analysis are listed

in table 1.A total of 18 proteins were specifically expressed

in the case of silk-CS while 11 proteins were expressed in both silk-only and silk-CS constructs. The protein–protein interaction network as analyzed by STRING revealed a high degree of connection between the iden-tified proteins in both cases (figure 5), but the number of hubs (a hub was identified as a gene having more

Figure 2. (A) MTT assay of silk-only and silk-CS constructs after 1 week, 3 weeks and 6 weeks; data represented as mean ± SD (Abv: w-weeks). (B) DAPI and rhodamine-phalloidin staining of a silk-CS construct showing the nucleus and actin cytoskeleton of the cell; the arrow represents silk fiber and the star represents the cell. (C) Collagen II staining in a silk-only construct after 6 weeks. (D) Collagen II staining in a silk-CS construct after 6 weeks.

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than four connections) was found to be higher in silk-only based constructs. In silk-only constructs the maxi-mum number of connections was found to be present around genes related to a MAP kinase (MAPK) path-way, while in silk-CS constructs two such major net-works were found. The first one included Annexin 6 and genes involved in a MAPK pathway, while the second one included ribosomal proteins genes, which indi-cates relatively higher synthetic activity. The interac-tion between the different identified proteins expressed in the two groups revealed clear involvement of p38-MAPK, ERK-MAPK, Mitogen activated protein kinase- extracellular signal regulated kinases (MEK) MEK-ERK and TGFβ signaling in chondrogenesis (figure 6).

3.5. Mechanical parity with native disc tissueWe then examined the effect of replication of anatomical hierarchy and biochemical composition on the compressive and tensile properties of the constructs. The compressive strength of the silk-only constructs was found to be 3.76 MPa, 6.8 MPa and 11.3 MPa after 1, 3, 6 weeks, respectively. By contrast, the compressive strength of silk-CS constructs were 6.85 MPa, 11.5 MPa and 20.6 MPa after 1, 3, 6 weeks, respectively (p < 0.05); values corresponding to compressive strength akin to goat AF tissue (figure 7(A)). Native human AF tissue

displays tensile strength of 3.9 ± 1.8 MPa (outer anterior AF) and 8.6 ± 4.3 MPa (outer posterior AF) [20]. The tensile strength increased from 4.75 MPa to 7.5 MPa for the silk-only construct and 7 MPa to 11.3 MPa for the silk-CS construct from 1 to 6 weeks of culture (figure 7(B)). The relation between the mechanical behavior and biochemical composition of the constructs was determined by calculating the correlation between tensile strength and collagen content, and compressive strength and GAG content of silk-only and silk-CS constructs. The correlation coefficient of tensile strength and collagen production in silk-only and silk-CS constructs (6 weeks) was found to be r 2 = 0.75 and r 2 = 0.77, respectively (figures 7(C) and (D)). The correlation coefficient of compressive strength and GAG production in silk-only and silk-CS constructs (6 weeks) was found to be r 2 = 0.63 and r 2 = 0.79, respectively (figures 7(E) and (F)). This suggests that a strong positive correlation exists between the tensile strength and collagen production, and the compressive strength and GAG production.

4. Discussion

AF tissue is known to be a highly organized collagen fiber reinforced tissue with heterogeneous distribution

Figure 3. Gene expression in silk-only and silk-CS constructs after 3 weeks and 6 weeks. (A) RhoA, (B) Collagen type II (Col-II), (C) Sox-9, (D) Aggrecan, (E) Biglycan.

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of water and proteoglycans, which together dictate the stress–strain responses generated within the AF tissue during motion and loading of the spine. Thus with the aim of replicating the anatomic features and chemical constitution of native AF tissue, we developed a multi-lamellar assembly of silk fibrous scaffold conjugated with CS. In a previous attempt [12], we showed that conjugating CS with silk could lead to higher compressive strength compared to silk-only based constructs. However, we could not successfully demonstrate a significant difference in the chondrogenic redifferentiation of human nasal chondrocytes cultured on silk-only and silk-CS scaffolds, which made the success of the CS attachment strategy questionable. A probable reason could be that the nasal chondrocytes have approximately a four-fold higher proliferation rate and significantly higher ECM synthesis over articular chondrocytes [21], which may have masked the subtle effect of CS tethered with silk fibroin.

In our earlier report [12], the peaks for C = N were masked to some extent by the prominent peaks of silk, and thus failed to provide convincing proof for the

formation of Schiff’s base. However, in the present study, we validated the crosslinking reaction by curve fitting of the FSD spectrum (supplementary figure S1), a ninhydrin assay and NMR NOESY. The broadening of the Hβ protons of tyrosine residues of silk fibroin (2.76 and 2.89 ppm) in NMR could possibly depict the formation of the complex, which is an outcome of the slow conformational exchange in the complex [12]. Such strong cross peaks could be seen only when two protons were close in space within 5 Ǻ, which strongly indicated covalent linkage of CS to the silk polymer.

Further, we successfully developed multilamel-lar silk and silk-CS constructs with uniform tissue formation. The presence of CS increased the cellular metabolic activity of silk-CS constructs, as it is known to enhance the proliferation of chondrocytes [22, 23]. The importance of the scaffold’s architectural guidance to the cells was conspicuous as the cells were aligned along the fiber direction. Collagen-specific staining illustrated the synthesis of collagen type II by redif-ferentiated chondrocytes, which was found to be rela-tively higher in silk-CS constructs; confirming better

Figure 4. (A) Collagen estimation after 3 and 6 weeks (Abv: w-weeks); data represented as mean ± SD. (B) GAG estimation after 3 and 6 weeks (Abv: w-weeks); data represented as mean ± SD. SDS-PAGE of ECM proteins produced in silk-only and silk-CS constructs (C) after 3 weeks, (D) after 6 weeks.

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chondrogenic response after functionalization (figures 2(C) and (D)). Hence, a lamellar silk scaffold function-alized with CS provided an efficient template to simu-late not only the structural hierarchy but also to guide the cell attachment, proliferation, orientation, and production of ECM proteins in the direction of the silk fibers, thus simulating the structural features of the AF region of native disc tissue.

Differential gene expression patterns in chondro-cytes were studied to access the role of CS in improving the chondrogenic environment. RhoA regulates Sox-9 activity by controlling the expression of Sox-5 and Sox-6 and thus controls chondrogenesis [24]. RhoA directly controls the post translational modification of the SOX-9 gene via its downstream effector Rho kinase (ROCK), which further regulates chondrogenesis by promoting gene expression of the cartilage-specific matrix [25]. Thus, the relatively higher expression of RhoA in CS conjugated matrices was conspicuous, indicating positive control over cartilage specific gene expression as depicted by the higher gene expression of SOX-9, collagen II and biglycan. Sox-9 is involved in the production of collagen II, however with disc aging and degeneration, the expression diminishes. Thus CS-tethering to scaffold may aid in Sox-9 synthesis, which in turn will help in the production of collagen II [26]. Collagen I, which is a crucial component of AF tissue, was not expressed in the present study. The develop-ment of a differential expression of collagen between the inner (collagen II) and outer (collagen I) layers in our previously reported study [2] was most likely due

to the CS gradient present in silk constructs, as well as dynamic culture conditions. Flow-induced shear force promoted expression of collagen I in peripheral layers of engineered constructs. By contrast, in our present study CS was uniformly attached throughout the construct, which consequently led to the deposi-tion of mainly collagen II uniformly throughout the construct.

Aggrecan is a large proteoglycan and is the main component of cartilage ECM after collagens [27]. Being highly hydrophilic, it interacts with the meshwork of collagen and gets embedded in the tissue network, and provides the necessary compressive strength. The small proteoglycans, such as biglycan, enhance aggregation of collagen fibers to form a network of collagen, thus increasing the overall tensile strength of the cartilage [28]. Thus, enhanced gene expression of these pro-teoglycans highlighted that the tethering of CS at the cell-biomaterial interface could successfully enhance the expression of important cartilage specific markers, facilitating chondrogenic redifferentiation. The bio-chemical estimated ratio of GAG/collagen in our study was found to be 3 which is close to the GAG/collagen ratio of 2.8 in native AF tissue [29], thus proving that the silk-CS constructs closely resemble the ECM com-position of the native tissue.

After 3 weeks, cells on both the constructs expressed several proteins possessing specialized roles in cell–matrix interactions and therefore indicate a transition from proliferating to redifferentiating chondrocytes. These proteins are namely laminin subunit-alpha-2

Table 1. Proteins expressed in both the constructs as well as exclusively in silk-CS constructs (combined list for 3 weeks and 6 weeks).

Silk-CS Silk-only and silk-CS

Name Symbol UniProt ID Name Symbol UniProt ID

Ankyrin repeat domain 17 ANKRD17 O75179 Laminin alpha 2 LAMA2 F1MMT2

Annexin 6 ANX6 P79134 Insulin-like growth factor 1 IGF1 P07455

Ras-related protein Rab-14 RAB14 P61106 Bone morphogenetic protein 1 BMP1 O18958

Decorin DCN P21793 Toll like receptor-1 TLR1 A7YK65

Integrins ITG P53712, P61625 Mitogen activated protein kinase MAPK P46196

Perlecan PLC Q6DTL8 Protein kinase C-alpha PKCA P04409

Thrombospondin 1 THBS1 Q28178 Lactoferrin LTF P24627

Zinc finger protein ZAC 1 ZAC1 Q52NW6 Alpha-1-acid glycoprotein AGP Q3SZR3

Ribosomal protein SA RPSA P26452 Transglutaminase 2 TGM2 P51176

SRY (sex determining

region Y)-box 9

SOX9 Q03255 Ghrelin/obestatin prepropeptide GHRL Q9BDJ6

Pituitary-specific positive

transcription factor 1

POU1F1 P10036 Procollagen-lysine, 2-oxoglutarate

5-dioxygenase 1

PLOD1 O77588

DNA (cytosine-5-)-

methyltransferase 1

DNMT1 Q24K09

Hyaluronan and

proteoglycan link protein 1

HAPLN1 A8E4P9

Collagen 12 alpha 1 COL12A1 P25508

Collagen 2 alpha 1 COL2A1 P02459

SH3 and multiple ankyrin

repeat domains 3

SHANK3 Q9BYB0

Superoxide dismutase 3 SOD3 A3KLR9

Laminin alpha 1 LAMA1 O19174

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protein, insulin like growth factor-1, bone morpho-genic protein, map kinase and protein kinase C, which participate in chondrocytes signaling, modulating chondrocyte phenotype and chondrogenesis of mes-enchymes [30–32]. Interestingly, few important pro-teins were identified exclusively in silk-CS constructs, namely decorin, which is a small leucine rich proteo-glycan (SLRP) found in the disc matrix and is known to guide proper collagen assembly during fibrillogenesis. It has been reported that incorporation of decorin dur-ing collagen I fibrillogenesis, results in an increase in the tensile strength and modulus of the collagen gels, showing a possible role of decorin in modulating the mechanical behavior of silk-CS constructs [33]. Per-lecan is a heparin sulfate proteoglycan which interacts with ECM proteins, growth factors and receptors, and plays a role in cartilage development by influencing cell signaling [34]. In addition, perlecan has been reported to be localized within the pericellular matrix of cartilage tissue to which fibroblast growth factor (FGF2) remains

bound, which plays important role during mecha-notransduction by activating extracellularly regulated kinase (ERK) [35].

After 6 weeks of culture, the protein commonly identified in both silk-only and silk-CS constructs was procollagen lysine 2 oxoglutarate 5 dioxygenase 1, which is involved in regulating fibrillar collagen crosslinks. Interestingly, cells on silk-CS constructs exclusively produced major protein hyaluronan and proteoglycan link protein (cartilage link protein), which assembles and stabilizes the aggregates of pro-teoglycan monomers with hyaluronic acid. It is also known to upregulate aggrecan and collagen II and thus helps in shock absorption, resistance against com-pression, and maintains homeostasis [36, 37]. Colla-gen XII helps in the organization and stabilization of collagen fibrils, increasing the load bearing capacity of the cartilage [38]. Collagen II provides structural stability to cartilage and is responsible for providing resistance against tensile forces [39]. Procollagen lysine

Figure 5. Protein–protein interaction network of the proteins identified in (A) silk-only constructs, (B) silk-CS constructs. Gene ontology classification of the genes responsible for the synthesis of the proteins identified in (C) silk-only constructs, (D) silk-CS constructs. The signaling pathways depicted in (C) and (D) (colour coding represents individual pathways) are numbered as follows: (1) angiogenesis (P00005), (2) angiotensin II-stimulated signaling through G proteins and β-arrestin (P05911), (3) B cell activation (P00010), (4) EGF receptor signaling pathway (P00018), (5) endothelin signaling pathway (P00019), (6) FGF signaling pathway (P00021), (7) gonadotropin releasing hormone receptor pathway (P06664), (8) inflammation mediated by chemokine and cytokine signaling pathway (P00031), (9) insulin/IGF pathway-mitogen activated protein kinase kinase/MAP kinase cascade (P00032), (10) insulin/IGF pathway-protein kinase B signaling cascade (P00033), (11) integrin signaling pathway (P00034), (12) ionotropic glutamate receptor pathway (P00037), (13) PDGF signaling pathway (P00047), (14) Ras pathway (PO4393), (15) T cell activation (P00053), (16) TGF-β signaling pathway (P00052), (17) toll receptor signaling pathway (P00054), (18) vascular endothelial growth factor (VEGF) signaling pathway (P00056).

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2 oxoglutarate 5 dioxygenase 1 regulates the conver-sion of lysine to hydroxylysine and induces formation of pyridinoline collagen cross-links [40, 41]. These intermolecular and intramolecular pyridinoline col-lagen cross-links are known to stabilize the collagen network and also help in resisting tensile stresses [3]. Further, the expression of toll-like receptors (TLRs) probably reflects the anti-inflammatory properties of CS. CS uses TLRs for inhibiting the activation of inflammatory cytokines, MyD88, by inhibiting NF-kβ [42]. The results thus suggest that incorporation of CS modulated the chondrocytes to release several impor-tant proteins, which could successfully impart a high level of biological and mechanical functionality to the silk-CS constructs.

The protein–protein interaction network analysis revealed the expression of Annexin 6 which interacts with protein kinase C α (PKCα) and controls the pro-liferation, differentiation and ECM production by the chondrocytes [43]. PKCα is reported to activate the MEK-ERK pathway which directly promotes chondro-genesis and modulates GAG content and the degree of sulfation [44]. A second pathway for GAG produc-tion that was found to be active in only silk-CS con-structs included p38-MAPK signaling via the TGF-β pathway. In this case too, Annexin 6 plays a pivotal role in triggering the pathway by regulating the expression of β1-integrin on the cell surface [44]. These integrin receptors present on the cell surface can interact with the exogenously provided TGF-β for chondrogenic differentiation [45, 46]. TGF-β regulates WNT-5A, which in turn regulates PKC-α. PKC-α then triggers

the p38-MAPK pathway for chondrogenic differen-tiation and GAG production [44, 47]. This signaling pathway also triggers the expression of SLRP molecules such as decorin and perlecan. Decorin helps in the for-mation of a fully functional extracellular matrix (by helping in the assembly of collagen II and aggrecan) for load bearing [48]. Perlecan on the other hand is very important for the expression of chondrogenic markers during the later stages of chondrogenic development and any abnormality may cause defective tissue forma-tion [49].

Hyaluronan and proteoglycan link protein 1 (HAPLN1) that was only expressed in silk-CS con-structs, is a very stable macromolecule present in car-tilage that imparts compression resistance and shock absorption. The abnormal production or absence of HAPLN1 can result in degenerated nonfunctional car-tilage tissue [50]. Thus the expression of this proteogly-can molecule in the silk-CS constructs confirmed the formation of a functional cartilage construct. THBS1 (Thrombospondin 1) was also specifically expressed in the silk-CS system. THBS1 has been reported to activate both the small and large forms of latent TGF-β [51]. This must have further helped in the upregulation of the TGF-β pathway for GAG production due to ample availability of activated TGF-β in the system. Thus, the CS-conjugated silk fibroin created a suitable chondro-genic environment through the activation of Annexin 6 directing the proliferation and differentiation of chondrocytes. The integrins were found to be upregu-lated only in the case of silk-CS crosslinked samples, thus emphasizing the role of p38-MAPK signaling as

Figure 6. A schematic diagram explaining the cellular signaling pathways and biochemical steps occurring in the cells as a result of CS conjugation to the silk scaffolds. CS interacts with Annexin 6 receptors present on the cellular surface, triggering the signaling cascade through PKC-α, which in turn activates a MEK-ERK pathway and regulates integrin expression. Integrin can further trigger a WNT-5A pathway, which activates the p38-MAPK pathway. The combined effect of these signaling cascades is an enhancement in the production of ECM proteins and GAGs.

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an additional upregulating pathway for GAG produc-tion in these constructs, in addition to the ERK-MAPK pathway that was also active in silk-only constructs.

Further, we tried to understand the effect of incor-porating chemical cues to the lamellar silk scaffold in the expression of various proteins, which might have played role in modulating the functional properties of the construct. In terms of mechanical properties, the tensile strength of silk-CS constructs was 11.3 MPa after 6 weeks of culture, which was found to be even higher than in native human AF tissue (3.9–8.6 MPa) [20], depicting achievement of in vivo like mechani-cal properties in our study (figure 7). The achievement of mechanical properties of engineered tissues akin to

their complex native counterparts resulted from a syn-ergistic contribution of anisotropic lamellar organiza-tion of the silk fibers as well as CS-tethering. This is the uniqueness of this study that elucidates the role of CS in the production of ECM proteins and their interac-tions controlling the functional properties of the con-struct.

The current study was thus successful in eluci-dating how biochemical and orientational cues elicit optimal cellular and biomechanical responses in order to develop cartilage constructs, which helped in gen-erating insights into the neotissue formation process. Hence we believe that this strategy has a broader util-ity in studying the developmental aspects of cartilage

Figure 7. (A) Compressive strength of silk-only and silk-CS constructs at regular intervals. (B) Tensile strength of silk-only and silk-CS constructs at regular intervals (Abv: w-weeks). (C) Correlation between tensile strength and collagen content in the silk-only construct after 6 weeks. (r 2 = 0.756). (D) Correlation between the tensile strength and collagen content in the silk-CS construct after 6 weeks. (r 2 = 0.774). (E) Correlation between compressive strength and GAG content in the silk-only construct after 6 weeks. (r 2 = 0.632). (F) Correlation between the compressive strength and GAG content in the silk-CS construct after 6 weeks (r 2 = 0.79).

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tissue engineering. This same strategy could be fur-ther extrapolated for attachment of other functional domains, such as cellular transmembrane receptor binding domains to silk biomaterials to simulate key niche components to establish in vitro model of degen-erated disc tissue.

5. Conclusions

Conjugation of bioactive molecules to scaffolds has led to the fabrication of advanced biomaterials with optimal biophysical and biochemical cues to develop a more efficient tissue engineering strategy, which can activate the innate reparative mechanism for tissue regeneration. We have developed a multilamellar silk scaffold functionalized by CS in an attempt to study the dynamics of the attachment of this bioactive molecule to a silk scaffold. Covalent attachment of CS ligands with silk fibers led to the enhancement of ECM associated protein expression and subsequently improved the biomechanics of the constructs. This unique correlation between the biochemical composition and mechanical properties was established by cellular guidance of primary chondrocytes along the fiber axis and their subsequent ECM deposition. This combination of lamellar silk fibrous architecture and functionalization with CS could act synergistically to replicate biochemical composition, leading to a high analogy to the structure–function relationship of disc tissue, offering potential for IVD tissue engineering.

Acknowledgment

The authors would like to acknowledge financial support from the Indian Council of Medical Research project (09/NCD-1) and the Department of Biotechnology’s financial support for the high-field NMR spectrometers at ICGEB, New Delhi and NII, New Delhi.

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