research article modification of decellularized goat-lung...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013, Article ID 651945, 11 pageshttp://dx.doi.org/10.1155/2013/651945

Research ArticleModification of Decellularized Goat-Lung Scaffold withChitosan/Nanohydroxyapatite Composite for Bone TissueEngineering Applications

Sweta K. Gupta,1 Amit K. Dinda,2 Pravin D. Potdar,3 and Narayan C. Mishra1

1 Department of Polymer and Process Engineering, Indian Institute of Technology Roorkee, Saharanpur Campus,Saharanpur, UP 247001, India

2Department of Pathology, All India Institute of Medical Sciences, New Delhi 110029, India3 Department of Molecular Medicine & Biology, Jaslok Hospital and Research Center, Mumbai, Maharashtra 400026, India

Correspondence should be addressed to Narayan C. Mishra; [email protected]

Received 2 April 2013; Accepted 26 May 2013

Academic Editor: Ulrich Kneser

Copyright © 2013 Sweta K. Gupta et al.This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Decellularized goat-lung scaffold was fabricated by removing cells from cadaver goat-lung tissue, and the scaffold was modifiedwith chitosan/nanohydroxyapatite composite for the purpose of bone tissue engineering applications. MTT assay with osteoblasts,seeded over the chitosan/nanohydroxyapatite-modified decellularized scaffold, demonstrated significantly higher cell growth ascompared to the decellularized scaffold without modification. SEM analysis of cell-seeded scaffold, after incubation for 7 days,represented a good cell adhesion, and the cells spread over the chitosan/nanohydroxyapatite-modified decellularized scaffold.Expression of bone-tissue-specific osteocalcin gene in the osteoblast cells grown over the chitosan/nanohydroxyapatite-modifieddecellularized scaffold clearly signifies that the cellsmaintained their osteoblastic phenotypewith the chitosan/nanohydroxyapatite-modified decellularized scaffold. Therefore, it can be concluded that the decellularized goat-lung scaffold-modified withchitosan/nanohydroxyapatite composite, may provide enhanced osteogenic potential when used as a scaffold for bone tissueengineering.

1. Introduction

Worldwide, over 2million grafting procedures are performedannually for skeletal reconstruction (e.g., trauma, tumorexcision, failed arthroplasty, and spinal fusion) [1], but due toinadequate availability of ideal bone substitute, the death ratehas been increasing day by day. Autograft and allograft boneswhich are generally used for repairing bone defects resultin secondary trauma and immune repulsion respectively [2].Therefore, to solve the concerns related to current surgicalskeletal reconstruction, there is a necessity to develop an idealbone substitute or biofunctional bone which should mimicthe natural bone and replace the lost/damaged bone in thehuman body. The biofunctional bone can be regenerated bythe approach of tissue engineering, which involves 3 impor-tant elements: (i) cells, (ii) growth factors, and (iii) scaffold(a 3D porous architecture made of biomaterials, whereupon

the cells are seeded) [3]. The interactions among these3 elements (cells, scaffold, and growth factors) control theprocess of tissue regeneration.The design and fabrication of aperfect tissue engineering scaffold is a complex issue becausethe ideal scaffold should be biocompatible, biodegradable,highly porous, and so forth, and the scaffold should alsointermingle with the proximate tissue to permit the scaffoldto be colonized by cells. There are various methods availablefor scaffold fabrication, for example, gas foaming [4, 5],thermally induced phase separation [6], rapid prototyping[7], electrospinning [8, 9], solvent casting/porogen leaching,and decellularization [10]. Amongt these, decellularizationis one of the most promising methods for scaffold fabri-cation, because it generates scaffold from bioorigin (e.g.,cadaver tissue in this study), which consists of various typesof biopolymers required for tissue regeneration. Besides,this method is simple, economical and provides a scaffold

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that retains the original architecture of tissue/extracellularmatrix (ECM). Decellularization is a method where cellsare completely removed from the cadaver organ/tissue with-out adversely affecting its 3D ultrastructure of the tissueskeleton [10]. Decellularization technique has already beenexploited for fabricating scaffold from various xenogenictissues/organs (e.g., urinary bladder [11], small intestinalsubmucosa [12], heart valves [13], pericardium [14], bloodvessels [15], nerves [16], skeletal muscle [17], tendon [18],ligament [19], liver [20], and skin [21]) of rat, pig, buffalo,cattle, and so forth, but, to the best of our knowledge, goat-lungtissue has not yet been applied for decellularization to fabricatescaffold for tissue engineering applications. Here, our aim isto fabricate scaffold from cadaver goat-lung tissue by decellu-larization and to modify it for bone tissue regeneration.Theremight be some important signalling/growth factors embed-ded within the decellularized goat-lung scaffold which canprovide physicochemical cues needed to guide the cellulardevelopment.

Natural bone tissue consists of organic-inorganic nano-composite in which hydroxyapatite (HA, Ca

10(PO4)6(OH)2)

crystals are deposited on fibrous collagen [22]. The hybridof chitosan (organic) and hydroxyapatite (inorganic) com-posite scaffolds has received great attention for bone tissueengineering applications, because they canmimic the naturalbone composition [23].Therefore, from the biomimetic pointof view, before applying goat-lung scaffold for bone tissueregeneration, we aim to modify the decellularized goat-lungscaffold with chitosan/nanohydroxyapatite composite andevaluate the modified (with chitosan/nanohydroxyapatitecomposite) decellularized scaffold for bone tissue engineer-ing applications.

2. Materials and Methods

2.1. Materials. All the chemicals employed in this workwere of analytical grade and purchased from different firms:Sigma Aldrich, Himedia, and Rankem. Chitosan (CS) fromcrab shells (C

12H24N2O9, deacetylation degree ≥75%) was

purchased from Sigma Aldrich Inc., USA. Hydroxyapatite(calcium phosphate-tribasic-nanopowder, <200 nm particlesize, ≥97% synthetic) was supplied by Sigma. Hydroxyapatitenanopowder will be called nHAp in this paper. Ammoniasolution (NH

4OH, molecular weight 35.05 g/mol) was pur-

chased from Rankem. Autoclaved double-distilled water wasused throughout the experiment.

2.2. Fabrication and Characterization of Decellularized Scaf-fold from Goat-Lung Tissue. The decellularization of goat-lung tissue was done by enzymatic method using trypsin.In brief, the goat-lung tissue was sectioned and treatedwith 0.25% Trypsin-EDTA in 1X PBS (Dulbecco’s phosphate-buffered saline, Himedia, India) supplemented with 1% Pen-Strep solution (Himedia, India) for 24 h at 37∘C. They werethen further treated with 0.1% SDS in 1X PBS at 37∘C for6–8 h, followed by agitating in solution containing RNase(20𝜇g/mL, Himedia, India) and DNase (0.2mg/mL, Hime-dia, India) for 24 h at 37∘C, followed by intensive wash cycles

with 1X PBS. The goat-lung decellularized scaffolds weredisinfected by agitating in an aqueous solution of 0.1% (v/v)hydrogen peroxide and 4% (v/v) ethanol. The decellularity ofthe fabricated scaffold was assessed by DNA quantificationand hematoxylin and eosin staining. The decellularized scaf-fold was then morphologically characterized by SEM (scan-ning electron microscopy), and chemical analysis of scaffoldsamples was performed with LEO 1525 scanning electronmicroscope equipped with an EDS (energy dispersive spec-troscopy) detector (Carl Zeiss SMT Ltd., Hertfordshire, UK).To quantify the chemical content of small areas (∼60𝜇m2)on the sample surface, distinctly different topographical areaswere chosen from the sample and examined at 10 kV usingINCA Energy 3000 software.

2.3. Preparation and Characterization of CS/nHAp CompositeParticles. The coprecipitation method has been widely usedto prepare composite material involving nHAp [24]. Here,in this study, nHAp has been mixed with chitosan, by usingcoprecipitation technique, to develop CS/nHAp composite.For preparing CS/nHAp composite particles, a clear andhomogenous solution of chitosan was made in dilute aceticacid. Chitosan (1 wt%) solution was prepared by dissolving1 g of chitosan in 100mL of 1% (v/v) acetic acid and stirringovernight. Simultaneously, 1 g of nanohydroxyapatite powder(nHAp) was accurately weighed and mixed with 100mL ofautoclaved double-distilledwater to form the suspension.ThenHAp suspension was then added to the chitosan solution inthe ratio of 4 : 6 wt% (CS : nHA) under vigorous stirring toobtain a uniform dispersion of hydroxyapatite in the solutionmixture. The solution mixture was then stirred at 45∘C for10min. This was followed by addition of NH

4OH dropwise

into the dispersed mixture with vigorous stirring. Eventuallythe solution turned opalescent, suggesting the formation ofCS/nHAp composite particles. Finally, the precipitate was fil-tered, and the excess acetic acid and ammonia were removedfrom the resultant mixture through dialysis. The CS/nHApcomposite particle, so formed, was characterized by FTIR(Fourier transform infrared spectroscopy) and AFM (atomicforce microscopy). FTIR for CS, nHAp, and CS/nHAp com-posites was carried out using Nexus Thermo spectropho-tometer (ThermoNicolet, USA) over a range between 4000and 400 cm−1 at 2 cm−1 resolution, averaging 100 scans. Sam-ples were processed to form compressed potassium bro-mide disks. Samples were taken in the ratio of 1mg per900mg potassium bromide. AFMwas also performed to findCS/nHAp composite’s particle size and its morphology.

2.4. Modification of Decellularized Scaffold with CS/nHApComposite and Its Characterization. Decellularized goat-lung tissue was modified with CS/nHAp composite to formthe CS/nHAp-modified decellularized scaffold. Modifica-tion of decellularized scaffold was achieved by chemicallyattaching the decellularized scaffold andCS/nHAp compositewith the help of EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide) andNHS (N-hydroxysuccinimide).The cross-linking solution contains 1X PBS (pH 7.4) with a final con-centration of 2mM EDC and 5mMNHS. The decellularized

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tissues were reacted with this modified/crosslinking solutionat room temperature for 15min to activate the carboxylgroups present on the collagen molecules of the decellu-larized goat-lung tissue (collagen-based biomaterial) [25].After incubation, the CS/nHAp composite was added to thecrosslinking solution containing decellularized scaffold. Theincubation continues for 24 h at room temperature on anorbital shaker table at 80–100 rpm. Following incubation, theresultant crosslinked tissues were rinsed with 1X PBS for 2 hon an orbital shaker table with several changes of 1X PBS solu-tion to remove residual crosslinkers and unbound CS/nHApcomposite particles. The CS/nHAp-modified decellularizedscaffold was then characterized by EDS and FTIR.

The decellularized goat-lung scaffolds were modified/dipcoated with chitosan and nanohydroxyapatite by immers-ing in a solution of chitosan (1 wt%) in acetic acid (1% v/v)and nHA (1 wt%) in distilled water, respectively, for 5 hat 37∘C. The chitosan-modified decellularized scaffold andnanohydroxyapatite-modified decellularized scaffold werethen rinsed with 1X PBS to remove residual chitosan andnanohydroxyapatite before use.

2.5. Human Osteoblast Cell Culture. Human osteoblast cellswere obtained from Jaslok Hospital and Research Center,Mumbai, India. The osteoblast cells were cultured in Dul-becco’s modified Eagle’s medium (DMEM, Himedia, India)supplementedwith 10% fetal bovine serum (FBS,GIBCO), 1%penicillin-streptomycin solution (Himedia, India), 1 𝜇L/mLInsulin (Sigma, USA), and 2 𝜇L/mL glutamine (Himedia,India). Osteoblast cells were grown as monolayer cultures inT-75 flasks (Nunc, India) and were subcultured twice a weekat 37∘C in an atmosphere containing 5%CO

2in air and >95%

relative humidity.

2.6. Evaluation of In Vitro Biocompatibility Using Human Os-teoblast Cells. Human osteoblast cell behaviors with decel-lularized goat-lung scaffold, chitosan-modified decellular-ized scaffold, nHAp-modified decellularized scaffold, andCS/nHAp-modified decellularized scaffold were individu-ally determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Briefly, all the scaffolds(1mm × 1mm pieces) were plated, in triplicate, in a 96-wellculture plate to recondition them by incubating in DMEMovernight at 37∘C in CO

2incubator. The next day, osteoblast

cells were released from the flask by trypsinization andresuspended at a density of 5× 103 cells per well, in triplicate,and incubated for 7 days at 37∘C in CO

2incubator. After

a respective time period of 7 days, 90𝜇L of fresh completeDMEM media and 10 𝜇L of MTT solution (5mg/mL stockin PBS) were added to each well (final volume of 100 𝜇L).The plate was incubated at 37∘C for 4 h until purple formazancrystals were formed. The medium was then discarded and200𝜇L of DMSO (dimethyl sulphoxide) was added to thewells and mixed properly to dissolve the formazan crystals.The plate was then swirled gently. Absorbance values wereblanked against DMSO, and the absorbance of cells exposedto medium only (i.e., no scaffold added) was taken as control.The absorbance was read at 490 nm using a Microplate

Reader Model Sunrise (TECAN, India) with the subtractionof plate absorbance at 650 nm. The absorbance was plottedagainst the number of days.

2.7. Histological Examination of the CS/nHAp-Modified Recel-lularized Scaffold. Osteoblast cells were seeded onto theCS/nHAp-modified decellularized scaffold and were main-tained at 37∘C under 5%CO

2in a humidified atmosphere.

CS/nHAp-modified recellularized scaffolds were collectedafter 7 days, in duplicate, washed in 1X PBS, and fixed in 2.5%glutaraldehyde at room temperature. The samples were thenembedded in paraffin and longitudinally cut into 5 𝜇m thicksections. The sections were mounted on slides and stainedwith hematoxylin and eosin staining to identify the cellularcomponents present in the CS/nHAp-modified recellularizedscaffold.

2.8. Morphological Examination of the CS/nHAp-ModifiedRecellularized Scaffold after a 7-Day Cell Attachment Study.The human osteoblast cells were seeded onto the CS/nHAp-modified decellularized scaffold and incubated for 7 days.The recellularized scaffolds, after 7 days, were harvestedand fixed in 2.5% glutaraldehyde at room temperature. Thesamples were then washed in 1X PBS and observed underSEM at the saturation pressure of water vapour (1 torr) andan accelerating voltage of 15 kV. Images showing surfacemorphologies of osteoblast cells-CS/nHAp-modified decel-lularized scaffold constructs were taken at 250x, 500x, and1000x magnifications.

2.9. Gene Expression Study. The in vitro cellular response ofhuman osteoblast cells to the CS/nHAp-modified decellular-ized scaffold has been demonstrated by gene expression stud-ies. Molecular expression of mesenchymal stem cell marker(CD105, CD73, CD44), pluripotency (Nanog), differentiation(Lif), and bone-tissue-specific marker (OCN), which areresponsible for repair and regeneration process of bone, wascarried out for osteoblast cells in presence of CS/nHAp-modified decellularized scaffold, for 7 days, to determinethe differences in transcriptional response of cells. OCN issecreted solely by the osteoblast cells [26], and therefore,was used as a tissue-specific biomarker in bone formation,bone mineralization, and calcium ion homeostasis [27, 28].𝛽-Actin marker (housekeeping gene) was used as internalcontrol.

2.10. Statistical Analysis. Cell viability and cell proliferationexperiments were performed in triplicate (𝑛 = 3), andthe experimental results are represented as mean values ±standard deviations (SD). Using MINITAB statistical soft-ware (MINITAB Release 13.32), statistical analysis (one-wayANOVA test) was performed with 𝑃 < 0.05 considered asbeing statistically significant.

3. Results

3.1. Fabrication and Characterization of Decellularized Scaf-fold from Goat-Lung Tissue. Goat-lung scaffold was fabri-cated by decellularizing cadaver goat lung by employing


Decellularized goat-lung scaffold surface morphology

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Figure 1: (a) Scanning electron micrograph of decellularized goat-lung tissue at 250X showing porous architecture. (b) DNA content innative lung tissue and the decellularized tissue. Native tissue served as a positive control. (c) Hematoxylin and eosin staining of decellularizedlung tissue section showing collagenous protein structure with negative cell nuclei staining. No nuclear counterstaining was detectable,indicating a total cell removal. (d) EDS spectrum of the decellularized scaffold, showing carbon and oxygen as the major constituents ofscaffold, confirming its organic nature. (∗) stands for significant difference between native lung tissue and decellularized tissue (𝑃 < 0.05).

trypsin-SDS-based decellularization method as discussed inSection 2.2. The scaffold was characterized by SEM, DNAquantification, H&E staining, and EDS (Figure 1). Mor-phological examination of the surface of decellularized scaf-fold, by SEM, confirms the porous nature of the goat-lungtissue scaffold, as expected for a porous lung (Figure 1(a)).The DNA quantification assay indicated significant removalof the cellular material from the native goat-lung tissue(Figure 1(b)). H&E staining of decellularized-scaffold sectionshows collagenous ECM structure with negative cell nucleistaining (Figure 1(c)), which indicates the nonexistence ofany cell in the scaffold. The EDS spectrum (Figure 1(d)) ofthe decellularized scaffold indicates that the tissue containsmainly carbon and oxygen with small amount of sodium,chlorine, magnesium, and sulphur. The presence of carbonand oxygen elements indicated the organic nature of the goat-lung tissue, and therefore, it is predicted to be biodegradableinside the body.

3.2. Characterization of CS/nHAp Composite Particles. CS/nHAp composite was characterized by comparing theab-sorption bands arising from the IR spectrum of nHApand CS standard sample. The FTIR spectrum of chitosan(Figure 2(a)) shows a broad –OH (3450–3100 cm−1) andaliphatic C–H (2990–2850 cm−1) stretching bands. Anothermajor absorption band between 1220 and 1020 cm−1 repre-sents the free primary amino group (–NH

2) at C2position of

glucos-amine, a major group present in chitosan. The bandat 1647 cm−1 represents acetylated amino group of chitin,which indicates that the sample is not fully deacetylated. Inthe FTIR spectra of nHAp (Figure 2(a)), the broader band at1000–1040 cm−1 represents the stretching vibration of P–O.The two absorption bands, which appeared at 603 cm−1 and566 cm−1, correspond to the bending vibration of P–O. Theabsorption bands at 3425 cm−1 and 1641 cm−1 correspond tothe stretching and bending vibration of H–O, respectively.


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Figure 2: (a) FTIR spectrum of chitosan, hydroxyapatite, and CS/nHAp composite. (b) AFM image of CS/nHAp composite particles.

Theband at 1422 cm−1 and 874 cm−1 is stretching andbendingvibration of C=O, respectively. The FTIR spectrum of CS/nHAp composite (Figure 2(a)) contains the entire charac-teristic absorption bands of CS and nHAp. In comparisonto chitosan, CS/nHAp composite is characterized by twoabsorption bands at 635 cm−1 and 955 cm−1, correspondingto stretching vibration bands of P–O from PO

4

3− andbending deformation mode of O–H from nHAp, confirmingthe successful formation of CS/nHAp composite scaffold[29]. AFM study (Figure 2(b)) demonstrated spherical shapeof CS/nHAp composite particles with size in the range of61–97 nm diameters.

3.3. Modification of Decellularized Goat-Lung Scaffold withCS/nHApComposite and Its Characterization. Decellularizedgoat-lung scaffold was modified/crosslinked with CS/nHApcomposite using EDC and NHS. In this study, EDC and NHSwere added to increase the rate and amount of crosslinkingbetween –COOH groups present in the decellularized goat-lung tissue (collagen-based biomaterial) and –NH

2groups

present in the CS/nHAp composite, thereby increasing thecompressive stiffness of the scaffold [30–32].

3.3.1. Characterization of CS/nHAp-Modified DecellularizedScaffold by EDS andFTIR. To confirm themodification of thedecellularized scaffold, CS/nHAp-modified decellularizedscaffold was characterized by energy dispersive spectroscopy(EDS), which demonstrated the presence of phosphate (P)and calcium (Ca) peaks within the CS/nHAp-modified decel-lularized scaffold (Figure 3), whereas, the respective peak ofP and Ca was absent in the EDS spectrum (shown in theFigure 1(d)) of decellularized goat-lung scaffold. This resultsuggested that the decellularized scaffold was modified by

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Figure 3: EDS spectrum of the CS/nHAp modified decellularizedscaffold showing the presence of Phosphorous and Calcium peaksdue to the presence of nHAp composite in the scaffold. Arrowsrepresent the Phosphorous and Calcium peaks in the CS/nHApmodified decellularized scaffold.

CS/nHAp composite. Peaks corresponding to Sodium (Na)and chlorine (Cl) might be present in the spectrum becausethe scaffolds were stored in saline solution. As the CS/nHAp-modified decellularized scaffold was made of natural mate-rials (calcium, phosphorous, and collagen) found in bonetissue, it makes an excellent biocompatible/biodegradablematerial for bone tissue regeneration.

FTIR analysis has also been performed as a complemen-tary study to characterize the CS/nHAp-modified decellular-ized scaffold formation. If we compare the FTIR of CS/nHApcomposite and CS/nHAp-modified decellularized scaffold,


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Figure 4: FTIR spectra of (a) CS/nHAp composite and (b) CS/nHAp-modified decellularized scaffold and decellularized scaffold. Peakcorresponding to the free amino group (between 1220 and 1020 cm−1) is present in the CS/nHAp composite, but got reduced in theCS/nHAp-modified decellularized scaffold, indicating the crosslinking of –NH

2group of CS/nHAp composite with the –COOH group of

the decellularized scaffold.

it is evident that the peak intensity of free amino group(–NH

2) at C

2position of glucosamine (1220–1020 cm−1)

for CS/nHAp composite has got significantly reduced inthe IR spectra of CS/nHAp-modified decellularized scaffold(Figure 4). The IR spectrum of CS/nHAp-modified decellu-larized scaffold and decellularized scaffold (Figure 4(b)) doesnot exhibit much change, which leads to the conclusion thatcrosslinking of CS/nHAp composite does not disturb theoriginal matrix structure of the decellularized scaffold.

3.4. Osteoblast Cell Behavior on the Scaffold

3.4.1. Cell Viability, Proliferation, and Attachment over theScaffold. MTT assay was performed to assess the osteoblastcell viability and proliferation over goat-lung decellularizedscaffold, chitosan-modified decellularized scaffold, nHAp-modified decellularized scaffold, and CS/nHAp-modifieddecellularized scaffold. Osteoblast cells seeded on a tissueculture plate without scaffold were taken as control. Theabsorbance value, that is, O.D., a measure of cell viability,was determined for the cell-seeded scaffolds. The resultshowed that osteoblast cells were viable in all the scaffolds,but a significant increase was observed in the cell viabilityof osteoblasts with CS/nHAp-modified decellularized scaf-fold as compared to the other scaffolds tested (Figure 5).Though there was no significant difference in the prolif-eration of osteoblast cells in decellularized scaffold andnHAp-modified decellularized scaffold, chitosan-modifieddecellularized scaffold shows less osteoblast cell proliferationbecause chitosan lacks bone-bonding bioactivity [24]. Thisresult is also supported by a study that demonstrates thatosteoblasts exhibit significantly higher proliferation capacityon the CS/nHAp scaffold as compared to that on purechitosan scaffolds [29].

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Figure 5:Osteoblast cell viability and proliferation assessed byMTTassay for 7 days. Absorbance was measured in replicates of threeand the calculated standard error of the mean (SEM) plotted aserror bars. Here, absorbance (490 nm) is directly proportional tocell viability. Osteoblast cell proliferation with CS/nHAp modifieddecellularized scaffold is shown to be highest after the 7 daysof incubation. (∗) stands for insignificant difference (𝑃 > 0.05)between the scaffolds. (∗) unmarked bars show significant differencebetween the scaffolds (𝑃 < 0.05).

Figure 6 shows the scanning electron microscopicimage of osteoblast cell adhesion and cell interaction withCS/nHAp-modified decellularized scaffold. After 7 daysof incubation, it was observed that cells get adhered to


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Figure 6: SEM images of the recellularized CS/nHAp-modified decellularized scaffold after 7 days of osteoblast cell seeding at 250x (a), 500x(b), and 1000x (c) magnifications. Arrows represent the osteoblast cells over the CS/nHAp-modified decellularized scaffold.

the scaffold and have polygonal morphology with the cellmembrane being somewhat flattened onto the rough surfaceof the CS/nHAp-modified decellularized scaffold, created byCS/nHAp particles. SEM image of osteoblast cell-scaffold(CS/nHAp-modified decellularized scaffold) construct alsodepicts that osteoblast cells have spread all around the porousstructure of the scaffold, and it becomes more clear whencompared to control image (decellularized scaffold withoutcells, Figure 1(a)). This result was also supported with MTTassay, which confirmed the potential of CS/nHAp-modifieddecellularized scaffold towards bone tissue regeneration.

3.4.2. Histological Examination of the Recellularized CS/nHAp-Modified Scaffold. Figure 7 illustrates H&E stainingimage of CS/nHAp-modified recellularized scaffold incu-bated with the osteoblast cells for a period of 7 days.H&E staining shows the presence of osteoblast cells inthe CS/nHAp-modified recellularized scaffold. The nuclearcounterstaining of multiple cellular groups of osteoblast cells(stained blue) surrounded by collagenous protein structure(stained pink) was detectable in the CS/nHAp-modifiedrecellularized scaffold; this indicates that the osteoblast cellsmight have got infiltrated through the surface of the scaffold

to the interior side, as expected from a highly porous scaffold.The result of H&E staining further supports the growthand proliferation of osteoblast cells over CS/nHAp-modifieddecellularized scaffold, thereby proving great prospective ofthis scaffold for bone tissue engineering applications.

3.4.3. Gene Expression Study:MolecularMarker Expressions inCell-Scaffold Construct. Gene expression profiling of CD105,CD73, CD44, Nanog, Lif, and OCN has been demonstratedfor in vitro cellular responses of human osteoblast cellsto the CS/nHAp-modified decellularized scaffold (Figure 8).CD105, CD73, and CD44 genes are found to be expressedin the osteoblast cells seeded over CS/nHAp-modified decel-lularized scaffold. The expression of mesenchymal stem cellmarkers (CD105, CD73, and CD44) in the osteoblast cellsdoes not exhibit any significant change in expression, whichindicates that cells are viable and proliferating inside theCS/nHAp-modified scaffold. It was found that Nanog and Lifgenes were not expressed in the osteoblast cells seeded overCS/nHAp-modified scaffold. Osteocalcin (OCN), a noncol-lagenous protein, is responsible for calcium ion binding and isa marker of bone mineralization [33]. OCN was expressed inthe osteoblast cells seeded over CS/nHAp-modified scaffold.


Figure 7: Hematoxylin and eosin staining of osteoblast recellu-larized CS/nHAp-modified scaffold. Images at magnification 10xvisualize the scaffoldmatrix after a successful 7-day recellularizationof osteoblast cells. Nuclear counterstaining was detectable, iden-tifying the cells growing inside the CS/nHAp-modified scaffold.Hematoxylin stains the nuclei of osteoblast cells blue, and theeosin stains the scaffold ECM structures pink. Arrows represent theosteoblast cells within the CS/nHAp-modified scaffold.

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Figure 8: Gene expression profile of osteoblast cells after incubatingwith CS/nHAp-modified decellularized scaffold for 7 days. Here,actin has been used as a housekeeping gene (internal control).

4. Discussion

In this paper, we evaluated the suitability of the decellularizedgoat-lung scaffold modified with CS/nHAp composite forbone tissue engineering applications. We employed trypsin-SDS-based decellularization method for removing the cel-lular components from the goat-lung tissues. The resultsof SEM (Figure 1(a)) of decellularized goat-lung scaffoldindicated cell removal, good porosity, and pore-to-poreinterconnectivity within the scaffold. The porous nature ofthe goat-lung tissue scaffold was expected for the scaffoldas it was derived from a highly porous cadaver lung. DNA

quantification assay andH&E staining of decellularized goat-lung scaffold (Figures 1(b) and 1(c)) confirmed the significantremoval of cellular components from the native goat-lungtissues, demonstrating the use of decellularized goat-lungscaffold as a template for the growth and proliferation ofosteoblast cells. The EDS spectrum of decellularized goat-lung tissue (Figure 1(d)) indicated carbon and oxygen as themajor components, which verifies that the scaffold will begradually degraded in the body after implantation.

Chitosan and nHAp have been studied extensively andused as a scaffold/biomaterial for bone tissue engineeringapplications [23]. A composite biomaterial CS/nHAp is ex-pected to show an increased osteoconductivity and biodegra-dation together with sufficientmechanical strength for ortho-pedic use due to the synergistic effect of chitosan and hydro-xyapatite [34]. Therefore, the decellularized goat-lung scaf-fold was modified/crosslinked with CS/nHAp composite formaking the scaffold suitable for bone tissue regeneration.

A composite of CS and nHAp was synthesized andcharacterized for studying the interaction between CS andnHApmolecules.The FTIR spectrumof CS/nHAp composite(Figure 2(a)) demonstrated no shifting of peaks of any groupin the composite spectrum and no new peak formation,which shows that CS/nHAp composite is only a mixture andthat no chemical reaction had taken place between CS andnHApduring the composite formation.This indicates that theoriginal characteristics of CS and nHApwere not lost, and theCS/nHAp composite, as a whole, will be an effective osteoin-ductive material. The AFM study (Figure 2(b)) indicated thatthe CS/nHAp composite particles were in the nanometerrange (67–91 nm diameter), which will pro-vide more surfacearea for osteoblast cells for adhesion and proliferation.

Decellularized goat-lung scaffold was modified/cross-linked with CS/nHAp composite using EDC and NHS.Figure 9 explains the possible interaction between –COOH groups present in the decellularized goat-lung tissue(collagen-based biomaterial) and –NH

2groups present in the

CS/nHAp composite. The carboxyl groups of decellularizedtissues are activated to give O-acylurea ester groups whichultimately crosslink with free amine groups of CS/nHApcomposite (Figure 9). EDC and NHS helped in acceleratingthe cross-linking reaction.

The EDS spectrum of CS/nHAp-treated decellularizedscaffold (Figure 3) result indicated that the decellularizedscaffold was modified by CS/nHAp composite. Peaks corre-sponding to sodium (Na) and chlorine (Cl) might be presentbecause the scaffolds were stored in saline solution.

The FTIR analysis of CS/nHAp-modified decellularizedscaffold (Figure 4) showed a significant reduction in thefree amino group (–NH

2) when compared with CS/nHAp

composite, which might be due to crosslinking of (–NH2)

groups of CS/nHAp composites with the (–COOH) groupsof the decellularized scaffold (Figure 9) during the formationof CS/nHAp-modified decellularized scaffold. The EDSspectrum (Figure 3) of modified decellularized scaffold(with CS/nHAp composite) shows high oxygen contentalong with adequate mineralization of CS/nHAp-modifieddecellularized scaffold, which can help in maintaining


+N

CNN

O

O

HO

SO

O NH

NO

NO

O

S

OO

O

COOH

DS

DSDS

DS

+

NH

O CS/nHApcomposite

CS/nHApcomposite

Intermediate compound,unstable,

acylurea ester

Semistableamine reactive NHS ester

Decellularized scaffold crosslinked with CS/nHAp composite

EDC

NHS

+NHCl−

Cl− N+

H

NH2

reactive O ,−O−

O−

O−

O−

Figure 9: Schematic representing possible crosslinking reaction between decellularized scaffold and CS/nHAp composite in the presence ofEDC and NHS. Nucleophilic attack by NH

2group of chitosan leads to formation of amide bond between COOH group of decellularized

scaffold and NH2group of CS/nHAp composite. EDC and NHS catalyze the covalent bindings between carboxylic acid and amino

groups, thus, crosslinking CS/nHAp composite with the decellularized scaffold and forming CS/nHAp-modified decellularized scaffold. DS:decellularized scaffold, EDC: 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide, NHS: N-hydroxy succinimide.

the necessary bone mineral density and balance for the lossof oxygen transported to the osteoblast cells. Moreover, asthe CS/nHAp-modified decellularized scaffold was madeof natural materials (calcium, phosphorous, and collagen)found in bone tissue, it makes an excellent biocompati-ble/biodegradable material for bone tissue regeneration.

In the cell behavior study, MTT assay (Figure 5) showsthat all the scaffolds (decellularized scaffold, chitosan-mod-ified decellularized scaffold, nHAp-modified decellularizedscaffold, and CS/nHAp-modified decellularized scaffold)supported the growth and proliferation of osteoblast cells.However, osteoblast cells exhibited higher proliferative ten-dency over CS/nHAp-modified decellularized scaffold. Oste-oblasts are highly sensitive to chemical-physical propertiesof the scaffolds. The better proliferation of osteoblasts overCS/nHAp-modified decellularized scaffold is believed to bedue to higher attachment of osteoblasts, which might be dueto the presence of chitosan, nHAp, and ECM of the decellu-larized scaffold. The synergistic effect of chitosan and nHAp[34] in the CS/nHAp-modified decellularized scaffold mighthave provided bone-like microenvironment to the osteoblastcells, which may also lead to an increase in osteoblastactivity, including cell growth and proliferation. Surface com-position (chitosan and nHAp) and roughness provided byCS/nHAp particle in the CS/nHAp-modified decellularizedscaffold might have provided necessary chemical cues to theosteoblast cells, which are influenced in higher cell prolif-eration. Though there was no significant difference in theproliferation of osteoblast cells in decellularized scaffold andnHAp-modified decellularized scaffold, chitosan-modifieddecellularized scaffold shows less osteoblast cell proliferation

because chitosan lacks bone-bonding bioactivity [24]. Thisresult is also supported by a study that demonstrates thatosteoblasts exhibit significantly higher proliferation capacityon the chitosan/hydroxyapatite scaffold as compared tothat on pure chitosan scaffolds [29]. When the surface ofthe CS/nHAp-modified osteoblast recellularized scaffold wasanalyzed by SEM, osteoblast polygonal morphology with thecell membrane being somewhat flattened onto the roughsurface (created by CS/nHAp particles) of the CS/nHAp-modified decellularized scaffold, was observed (Figure 6).This type of morphology of osteoblast cells was reportedpreviously on the collagen-hydroxyapatite composite scaffoldfor bone tissue engineering [35].TheCS/nHApparticles, withtheir rough surface and nanofeatures, are believed to be themain enhancing factor for good cell attachment and well-spread morphology of the osteoblast cells over CS/nHAp-modified decellularized scaffold. H&E staining of CS/nHAp-modified osteoblast recellularized scaffold (Figure 7) provedthat osteoblast cells not only get adhered to the surface of thedecellularized goat-lung tissue but are also infiltrated throughthe surface of the decellularized scaffold to the interiorside, as expected from a highly porous scaffold. The geneexpression study (Figure 8) demonstrated that the osteoblastcells maintained their phenotype after getting incubated for7 days in vitro with the CS/nHAp-modified decellularizedscaffold. The absence of the expression of Nanog and Lifgenes in the osteoblast cells with CS/nHAp-modified scaffoldindicates the nonpluripotency of the osteoblast cells inpresence of the scaffold. This may be due to the combinedeffect of CS, nHAp, and ECMof scaffold. Osteocalcin (OCN),a non-collagenous protein, is responsible for calcium ion


binding and is a marker of bone mineralization [33]. OCNwas expressed in the osteoblast cells seeded over CS/nHAp-modified scaffold, which indicates the potentiality of thescaffold to act as osteoblast cell carrier for bone tissue engi-neering application. It also signifies that the cells maintainedtheir osteoblastic phenotype within the CS/nHAp-modifieddecellularized goat-lung scaffold, which can be applied forbone tissue regeneration.

The present study underscores that the modification ofdecellularized goat-lung scaffold with CS/nHAp compositehas the potential to act as osteoblast cell carrier and favors thegrowth and proliferation of osteoblast cells, thereby, provinggreat prospective of this scaffold for bone tissue engineeringapplications.

5. Conclusions

Goat-lung tissue was successfully decellularized and modi-fied with CS/nHAp composite for bone tissue engineeringapplication. The CS/nHAp-modified decellularized scaffoldis biocompatible and forms a favorable three-dimensionalmatrix that leads to improved osteoblast activity includingcell adhesion, growth, and proliferation. The good prolifer-ation of the osteoblasts over the CS/nHAp-modified decellu-larized scaffold is believed to be due to the higher attachmentof cells over the scaffold. The higher attachment might bedue to the presence of nHAp particles (inorganic cues)and the roughness provided by nHAp particles along withchitosan, which positively affected the growth and prolif-eration of osteoblasts. The osteoblast cells maintained theirosteoblastic phenotype within the CS/nHAp-modified decel-lularized goat-lung scaffold.Thus, CS/nHAp-modified decel-lularized goat-lung scaffold can be applied for bone tissueregeneration.

Acknowledgments

The authors are thankful to the Council of Scientific andIndustrial Research (Grant no. 27(0222)/1 O/EM R-II dated31.05.10), India, for the financial support of this work.The authors are also thankful to the Institute Instrumen-tation Centre, IIT Roorkee, and Department of MolecularMedicine, Jaslok Hospital and Research Centre, Mumbai,India, for providing stem cell and molecular research facilityfor this work.

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