ORIGINAL ARTICLE

Collagen 3 D fleece as scaffold for cardiac tissue engineering

Aishwarya Srinivasan & Alan Mathew Punnoose &

Nithya Nagarajan & Sarah Kuruvilla &

Praveen Kumar Sehgal & Komarakshi Balakrishnan

Received: 30 December 2011 /Accepted: 21 January 2012 /Published online: 16 February 2012# Indian Association of Cardiovascular-Thoracic Surgeons 2012

AbstractBackground The limited ability of cardiac muscle to regenerateafter myocardial infarction motivates studies aimed at curativetreatment options through cell engraftment. The purpose of thisstudy was to test woven collagen fibres for possible use as 3 Dscaffold for cardiac tissue engineering.Methods Neonatal ventricular rat Cardiomyocytes were iso-lated and characterized using specific antibodies by immu-nocytochemistry and confocal microscopy following whichthey were seeded on collagen scaffolds.Results Collagen fleece fabricated out of woven collagenfibres harbored metabolically active cardiomyocytes. Thevideo of the beating cells on the scaffold is substantialevidence that the collagen fleece could be used for thepreparation of tailor made cardiac tissue construct.Conclusion Collagen fleece possesses the necessary tensilestrength as well as the elasticity for maintaining pulsatingcardiomyocytes. Their flexibility and compatibility project

them as promising candidates for use in tailor made cardiactissue constructs.

Keywords Myocardial infarction . Collagen . Cardiac tissueengineering

Introduction

Myocardial infarction if not fatal, undergo healing withfibrous scar formation in the infarcted zone which resultsin impaired pump function as cardiac tissue lacks intrinsicregenerative capability [1]. The limited ability of cardiacmuscle to regenerate after myocardial infarction motivatesstudies aimed at curative treatment options. Most studies inanimal models of myocardial infarction support the notionthat cell engraftment can improve contractile function [2].Efforts to regenerate functional myocardial tissue have beenpursued through cell grafting by syringe injection [3] directlyin the ventricular wall or in the coronary vessels. Amore promising approach however, is the developmentof tissue-engineering strategies using biomatrices to success-fully engraft new cells into the myocardium [4]. The 3 Denvironment of a scaffold would provide orientation to cellsand improve cell-cell interaction. Hence, engineering a 3 Dcardiac tissue construct in vitro would offer novel perspectivesfor basic cardiovascular research and for tissue replacementtherapy [5]. Awide variety of biodegradable and biocompatiblepolymers [6] have been investigated to fabricate micro andnano fibrous membranes [3, 7–12]. The connective tissueframe work of the human heart is essentially a three-dimensional mesh of collagen fibrils. Hence, an attempt wasmade to mimic native cardiac Extracellular Matrix (ECM)using collagen for an in vitro cardiac patch. In the earlier studyreported by us [13], we described a process of collagen fibre

Aishwarya Srinivasan and Alan Mathew Punnoose have contributedequally to the manuscript.

Praveen Kumar Sehgal passed away during the course of this study.

A. Srinivasan : P. K. SehgalBioproducts Laboratory, Central leather Research institute,Chennai, India

A. M. Punnoose :N. Nagarajan : S. KuruvillaCell and Tissue Engineering Laboratory,Sri Ramachandra University,Chennai, India

K. Balakrishnan (*)Cardiac Sciences, Malar Fortis Hospital,Adyar,Chennai 600020, Indiae-mail: cvskrb@gmail.com

Indian J Thorac Cardiovasc Surg (January–March 2012) 28(1):1–5DOI 10.1007/s12055-012-0134-8

extraction by microbial treatment and found these fibres topossess enough mechanical strength to suit the requirement ofa scaffold that would be subjected to mechanical handling. Inthe present study, we have woven these collagen fibres into a 3D fleece (CF) for use as a possible tissue construct.

Materials and methods

Extraction of collagen

The extraction of collagen was carried out according to theprocedure of our US patent application No.20070117176.The same has been reported by us in our earlier publication[13]. Briefly, Achilles tendon of bovine origin was placed ina nutrient broth containing Staphylococcus aureus ATCC29213 in its log phase for 96 h. The collagen fibres thatremained in the broth after exposure were disinfected withrepeated washing with n-propanol. CF was prepared andNeonatal Ventricular Rat Cardiomyocytes (NVRCMs) wereseeded on them and characterized using specific markers.

Collagen scaffold preparation

Collagen fibres obtained by microbial treatment of tendonwere opened mechanically and punch woven into a fleeceusing a Mag Sitra Eletrash, fibre opening machine, India.CF was cut into 1 cm×1 cm×1 mm (l×b×t), packed inpolythene sachets, hermetically sealed and sterilized usingethylene oxide for 4 h.

Isolation of neonatal ventricular rat cardiomyocytes(NVRCMs)

NVRCMs were isolated from 2 to 5 day old Wistar rat pupsusing the enzymatic dissociation protocol established bySadoshima [14] with minor modifications. This study con-forms to the guiding principles of Institutional AnimalEthics Committee, Committee for the Purpose of Controland Supervision of Experiments on Animals (CPCSEA),and the Guide for the Care and Use of Laboratory Animalspublished by the National Institutes of Health (NIH Publi-cation No. 85–23, revised 1996). Registration Number: 466/01/a/CPCSEA dated 24th August 2001. Briefly, pups weredecapitated, hearts were removed and placed in ice coldPhosphate Buffered Saline (PBS) (without Ca2+, Mg2+).Hearts were washed, twice with ice cold PBS and atria wereremoved using scissors. Ventricles were quickly choppedwith a sterile scalpel (within 5 min). The minced ventricleswere subjected to repeated rounds of enzymatic digestions(0.2% Trypsin, 0.03% Pancreatin, and 0.015% DNase I) in around bottom glass tube maintained at 37°C and with amagnetic stirrer at 300 rpm for 6 to 8 min. The supernatant

from the first digestion of 3 min was discarded. Followingeach round of digestion the tube was briefly removed fromthe magnetic stirrer. The supernatant was allowed to settlefor a minute and was filtered through a sterile 100 μm nylonmesh (BD BioSciences) into a fresh 50 ml falcon tubecontaining 1 ml of Fetal Calf Serum (FCS) to arrest enzy-matic action. The tubes were centrifuged at 1,200 rpm for3 min at 4°C and the pellet was resuspended in 10 ml ofpreplating medium (Dulbecco’s modified eagle medium/F12- DMEM/F12+10% FCS). 30 ml of cell suspensionwas plated out onto 15 cm cell culture dishes and incubatedfor 80 min at 37°C. A second preplating step for 60 minensured an enriched culture of 95% NVRCMs.

Cell culture dishes were prepared by treatment with sterile2% gelatin in PBS at 37°C for 6 h. The coating solution wasdiscarded and dishes dried under UV exposure in a clean airbench. Cells were counted using trypan blue exclusion andresuspended in cardiomyocyte medium containing DMEM/F12+10% Horse Serum (HS) supplemented with end concen-tration of 1.2 μM Bromodeoxyuridine (BrdU) for eliminationof non cardiomyocytes. Cells were washed vigorously withPBS 24 h later and media changed to DMEM+5% HS+BrDU. Roughly 5×105 cells were seeded onto 18 mm coverslips for immuno- cytochemistry and 2×106 cells on CF.Pretreatment of scaffolds using 2% gelatin in PBS wasfollowed by soaking in FBS \ HS one hour prior to cellseeding. The cells were added drop wise on the scaffolds,allowed to settle for half an hour and excess medium wasadded drop wise later.

Immuno-cytochemistry

Sterile autoclaved glass coverslips (18 mm in diameter)were placed into wells of 12 well cell culture plates. Thecoverslips were washed with 2 N HCl followed by 70%ethanol for 15 min respectively and allowed to dry in asterile air bench for roughly an hour under UV exposure.They were then coated with 2% gelatin avoiding air bubblesand seeded with NVRCMs; roughly 5 X 105 cells per well.Cells were then stained with various antibodies. All solu-tions were freshly prepared with PBS and steps were carriedout at room temperature. Briefly wells were rinsed with0.5 ml DMEM/F12 followed by two washes with PBS.Cells were then fixed in 3.7% formaldehyde for 10 minand rinsed twice with PBS. Permeabilization was achievedusing a 0.1% triton X-100 solution for 10 min followed by asingle wash with PBS. The cells were later blocked for anhour with blocking solution containing 3% Bovine SerumAlbumin (BSA) and 0.2% tween 20 in PBS. They were thenincubated with primary antibody in blocking solution over-night at 4°C with mild agitation. The wells were rinsedtwice with PBS and incubated at room temperature withfluorescence conjugated secondary antibody for 1 h with

2 Indian J Thorac Cardiovasc Surg (January–March 2012) 28(1):1–5

mild agitation and rinsed twice with PBS. The followingconcentrations were used. Mouse anti actinin (1:250),Mouse anti myosin (1: 500), Mouse anti Alpha CardiacMuscle Actin (1: 250), Mouse anti connexin-43 (1:500),SIGMA, Mouse anti desmin (1: 500), BIOGENEX, Goatanti mouse FITC 1:500, Santacruz Biotech was used assecondary antibody. Cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI) 1 μg/ml for nuclearstaining. Coverslips were washed well in distilled water,mounted onto slides using anti fade mounting mediumand viewed under UV and ~540 nm filter. The scaffoldwith cells were formalin fixed, paraffin mounted and20 μm sections were stained according to standard protocols.The slides were stored at 4°C. The images were capturedusing a fluorescence microscope (NIKON TE 2000i Eclipse)

and digital images were analyzed using Image ProPlussoftware.

Results

NVRCMs were grown on gelatin coated culture ware. Cultureswere monitored by microscopy to ensure viability. Cellsshowed characteristic beating up to 18 days in culture whichwas video captured using Image ProPlus software (Fig. 1).

NVRCMs seeded on glass coverslips were characterizedusing antibodies targeting cardiac specific markers such asActinin, Connexin 43, Desmin, Alpha Cardiac Muscle Actinand also unspecific marker such as Skeletal Myosin. Sincethe primary antibodies were specific for cardiomyocytes, the

Fig. 1 Snap shot of NVRCMsin culture. a Screen shot of invitro cultured beatingNVRCMs on culture ware (2 D)X 200. b Screen shot of in vitrocultured NVRCMs beatingtogether on culture ware 1 weekpost seeding X 400. c, dContinuous screen shot of invitro cultured NVRCMsbeating in between collagenfibres X 600. The beatingcardiomyocytes were foundstretched between the fibres.The arrows indicate the positionof cells in the grid to revealpulsation. These myocytes wereviable 10 days post seeding

Fig. 2 Immunofluorescentimage of NVRCMs stainedwith cardiac specific antibodies.a anti-Actinin—showing thecharacteristic striations X 400.b anti-Alpha Cardiac MuscleActin—X 400. c anti-Connexin43 showing the gap junctionbetween two cells—X 400.d anti-Desmin, X 400. eanti-Myosin X 400. f CLSMimage—anti-Myosin showingthe characteristic striationsof NVRCMs X 630

Indian J Thorac Cardiovasc Surg (January–March 2012) 28(1):1–5 3

cytoskeletal proteins of the fibroblasts were not stained andonly their nuclei are visible.

As observed in (Fig. 2a), anti-actinin staining revealedthe typical striated pattern characteristic of NVRCMs.(Fig. 2b) shows long filamentous structures when stainedwith anti-Alpha Cardiac Muscle Actin antibody. Gap junc-tions were observed among bundles of cardiomyocytes whichgrouped together and they were connexin 43 positive asshown in (Fig. 2c). NVRCMs also exhibited the characteristicpattern of fibre orientation of Desmin seen in (Fig. 2d) asgreen striations, which is a trademark of cardiac muscle. Westained NVRCMs with anti-skeletal-myosin antibody whichalso recognizes cardiac myosin as specified by SIGMA(Fig. 2e). We were able to observe strong signals depictingthe well studied banding pattern of cardiac muscle throughCLSM as shown in (Fig. 2f). The excitation and emissionwavelengths of FITC are 480 nm/540 nm.

Since collagen fluoresces under UV light, backgroundstaining complicated microscopic analysis and CLSMimage of NVRCMs on CF could not be captured. But byphase contrast microscopy, beating NVRCMs were observedstretched between CF, which was recorded, indicating that thecells are viable and metabolically active.

Discussion

An ideal scaffold for a cardiac patch should be biocompat-ible, biodegradable, enable growth of seeded cells, flexibleto support beating cells, resistant to deformation while beingmechanically handled and should not produce excessiveinflammatory reaction on implantation. Most scaffolds tryto mimic the ECM and provide a near normal environmentfor optimal growth. The ECM of the heart is chieflycomposed of collagen, a weak immunogen, and since itis available in many physical forms and can be moldedinto a desired shape, it was the obvious choice for anideal cardiac construct. The collagen fibres used in thisstudy, have already been characterized by spectroscopicand electron microscopic analysis and their mechanicalstrength established. These findings have been reportedby us in our earlier publication [13]. Hence, we hypothesizedthat a fleece fabricated out of CF would suit the requirementsof a scaffold that could be subjected to mechanical handlingand would be resistant to deformation. We found that therandom organization of the CF creates pockets of varied sizesthereby providing adequate porosity for cell seeding. Thescaffold also provides more surface area for adherence. Cul-tured NVRCMs adhered well to 2D surface of the culturedishes and were characterized by specific antibodies. Theywere found to be healthy under normal culture conditions.NVRCMs seeded on CF were observed to beat 10 days afterseeding and pulsation was present for a maximum of 18 days

in culture. Our results indicate that CF has the potentialto be molded as a 3D cell delivery vehicle particularlyas a cardiac construct. We are currently quantifying theNVRCMs on CF using SQUID magnetometer and theearly results are promising, comparable to those observed byTanaka [15]. The recording of beating cells betweencollagen fibres is sufficient evidence that the NVRCMsgrown on CF are viable and maintain their contractilefunction which is a potentially favorable achievementtowards the future goal of designing a pliable regenerativepatch.

Considerable research though remains to be carried out onseeding a heterogeneous population of cells on the scaffoldand validate the suitability of the fibres as a thicker patch.

Conclusion

Our efforts to mimic the 3- dimensional architecture of theECM, which provides the essential milieu for cell organiza-tion, survival and function, have proven fruitful. We havedesigned CF which provides plasticity and supports thegrowth of NVRCMs. The fibres are comparable to the ECMenvironment of the heart as they harbor metabolically activecardiomyocytes. CF also possesses the necessary tensilestrength for mechanical handling as well as the elasticity formaintaining pulsating cardiomyocytes. Their flexibility andcompatibility project them as promising candidates for use intailor made cardiac tissue constructs.

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