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Controlled formation of cross-linked collagen fibers for neural tissue engineering applications

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2014 Biofabrication 6 015012

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Biofabrication

Biofabrication 6 (2014) 015012 (15pp) doi:10.1088/1758-5082/6/1/015012

Controlled formation of cross-linkedcollagen fibers for neural tissueengineering applications

Mevan L Siriwardane1,2, Kathleen DeRosa1, George Collins1

and Bryan J Pfister1

1 Department of Biomedical Engineering, New Jersey Institute of Technology, Newark, NJ, USA2 Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey,Newark, NJ, USA

E-mail: pfister@njit.edu

Received 9 September 2013, revised 18 December 2013Accepted for publication 9 January 2014Published 3 March 2014

AbstractFibrous scaffolds engineered to direct the growth of tissues can be important in formingarchitecturally functional tissue such as aligning regenerating nerves with their target.Collagen is a commonly used substrate used for neuronal growth applications in the form ofsurface coatings and hydrogels. The wet spinning technique can create collagen fibers withoutthe use of organic solvents and is typically accomplished by extruding a collagen dispersioninto a coagulation bath. To create well-controlled and uniform collagen fibers, we developedan automatic wet spinning device with precise control over the spinning and fiber collectionparameters. A fiber collection belt allowed the continuous formation of very soft and delicatefibers up to half a meter in length. Wet-spun collagen fibers were characterized by tensile andthermal behavior, diameter uniformity, the swelling response in phosphate buffered saline andtheir biocompatibility with dorsal root ganglion (DRG) neurons and Schwann cells. Fibersformed from 0.75% weight by volume (w/v) collagen dispersions formed the best fibers interms of tensile behavior and fiber uniformity. Fibers post-treated with the cross-linkersglutaraldehyde and genipin exhibited increased mechanical stability and reduced swelling.Importantly, genipin-treated fibers were conducive to DRG neurons and Schwann cell survivaland growth, which validated the use of this cross-linker for neural tissue engineeringapplications.

Keywords: collagen fibers, directed tissue growth, neural tissue engineering, biomaterials

(Some figures may appear in colour only in the online journal)

Introduction

Engineered methods to construct biological tissue substitutesare important for finding new approaches to repairdiseased or damaged tissues [1–6]. Most repair strategiesinclude an engineered biomaterial scaffold that provides aphysical and biochemical substrate for tissue regeneration.Purposeful design of the bulk physical properties of thebiomaterial such as porosity, surface roughness and elasticitycan greatly influence cell proliferation and differentiation[4, 7–9]. In many tissue engineering applications such asnerve regeneration, recapitulation of the native architecture can

be essential to the development of functional tissue [10–12].Accordingly, innovative methods must also consider directedtissue formation where the biomaterial scaffold is designedto provide spatial control in addition to biochemical cues forregenerating cells [13–15]. Directing the growth of tissues todevelop functional tissue architecture, however, remains a keychallenge in tissue engineering including strategies for nerveregeneration.

Spatially oriented scaffolds exhibiting alignedmicrostructures are often used for directed cell alignmentor orientation [13, 14, 16–18]. Scaffold fabrication techniquessuch as gas foaming [19, 20], rapid prototyping [21–23]

1758-5082/14/015012+15$33.00 1 © 2014 IOP Publishing Ltd Printed in the UK

Biofabrication 6 (2014) 015012 M L Siriwardane et al

and thermal induced phase separation [24] mostly producethree-dimensional (3D) scaffolds with random porousstructures. Salt/particulate leaching in conjunction withsolvent casting, melt molding, phase separation anddirectional freeze drying are other common methods forproducing 3D scaffolds and can be used to create alignedchannels [17, 25–31]. Additionally, self-assembled hydrogels[32–38] and methods involving phase separation [39] havealso produced desirable 3D matrices for tissue engineeringapplications. Hydrogels with aligned microstructure or spatialgradients of growth factors, in particular, have also shownpromising results in cell alignment and directional growth[40–46].

Scaffolds exhibiting micro-fiber orientation are ofunique interest for many tissue engineering strategies thatrequire unidirectional alignment of growing cells [11, 16].Biofabrication methods for generating aligned, fibrousscaffolds are limited to electro-spinning [47–51] and a varietyof fiber spinning techniques [11, 52–54]. Electro-spinning iswidely used to create oriented nano-scale scaffolds but requiresthe use of organic solvents, which has been reported to alterthe native structure of natural polymers such as collagen at themolecular scale [49, 55–57]. Several common fiber spinningtechniques in the textile industry such as dry spinning, meltspinning and wet spinning have also been utilized for orientingpolymeric materials into fibrous scaffolds for biomedicalapplications [52–54, 58–64].

Synthetic polymers are readily used for fiber spinningand scaffold fabrication due to the ease of manipulation andreproducibility under various processing conditions. Naturalpolymers, however, are often preferred since they moreclosely mimic the physiochemical properties of the extra-cellular matrix and avoid complications such as inflammationor material toxicity [65]. Unfortunately, conventional fiberspinning techniques are typically not suitable for processingbiological polymers into fibers; e.g. the use of harsh solventsor high temperatures. Among fiber forming techniques, wetspinning conditions are environmentally friendly and lessprone to denature or alter the native molecular orientationof the biopolymer [59, 66].

Wet spinning is often applied manually by injecting apolymer solution through a needle (spinneret) into a coagulantbath [52, 58, 59, 61, 67–70]. pH-dependent precipitation isone coagulation technique used to create single fibers frombiological polymers such as chitosan, hyaluronic acid, silk,alginate, and collagen [58, 59, 62, 71–79]. As the dispersion isextruded into the coagulation bath, a precipitate forms into athin filament. The fiber is then collected from the coagulationbath and dried [62–64, 78]. If this process is not automated,it yields irregular fibers in terms of length, cross-sectionaluniformity, and linearity.

Devices with the capability of controlling parameterssuch as flow rate, pH of the coagulation bath and collectionrate have facilitated the synthesis of continuous and uniformsynthetic fibers [70, 73, 77]. This work describes an automatedwet spinning device, which was custom built to improvereproducibility of collagen fiber fabrication and yield ofcontinuous collagen fibers up to half a meter in length.

The design enables user-defined adjustment of the importantprocessing parameters in wet spinning: gauge size of thespinneret, extrusion rate of the collagen dispersion, pH of thecoagulation bath, speed of the fiber collection and the dryingtime. Importantly, the method of fiber collection was essentialin the formation of uniform and straight fibers with consistentproperties. In this study, the effects of collagen dispersionconcentration on fiber size, uniformity, mechanical strengthand swelling response were investigated. In addition, cross-linking reagents were evaluated as a post-processing step toincrease the mechanical stability and to control the swellingresponse of collagen fibers.

Materials and methods

Preparation of collagen dispersions

Type I collagen was extracted and purified from tendonsdissected from Sprague-Dawley rat tails (8–9 weeks old)following a previously established protocol [80]. The extractedtendons were digested in sterile 0.7% acetic acid for 5–7 days at4 ◦C with mechanical agitation (stir bar). The tendon solutionwas transferred to 50 ml centrifuge tubes and spun at 3500 rpmfor 30 min. The collagen supernatant was retained whilediscarding the pellet of tendon debris. Collagen supernatantwas spun again at 2500 rpm for 10–15 min to further separateremaining tendon debris. The supernatant was dialyzed in acellulose membrane tube (MWCO: 12 000–14 000) overnightat 4 ◦C in dialysis buffer (0.5M Na2HPO4, 0.5M NaH2PO4,pH 7.4). After dialysis, the semi-solid gel of collagen wastransferred to 50 ml centrifuge tubes and spun at 3500 rpmfor 30 min to further concentrate the collagen. The collagenpellet was spread onto petri dishes and lyophilized for 48 hto gradually remove water content resulting in a dry poroussponge.

Freeze-dried collagen was weighed and used to prepare0.75, 1.0, 2.0 and 3.4 weight by volume (w/v)% collagendispersions in 0.2% glacial acetic acid. Under these conditions,collagen does not completely solubilize. Collagen dispersionscontain small fiber portions of insoluble collagen, some ofwhich disassemble into smaller fibril subunits in the presenceof acidic solutions below pH 3. This physical transformationof disassembly is regarded as swelling, which results indispersions appearing uniform and opalescent.

The concentration (in mg ml−1) of the collagen within thedispersions was determined by modifying the bicinchoninicacid (BCA) Protein Assay (Pierce, Thermo Scientific,Rockford, IL). Since collagen in dispersion form is notcompletely solubilized, the BCA reactivity is inhibited.This inhibition results in an underestimation of the actualconcentration of collagen, and therefore, cannot be comparedto the supplied albumin protein standards [81]. To create usefulstandards, rat tail collagen with known concentration waspurchased from BD Biosciences (Bedford, MA) and diluted in0.02M acetic acid to 2.0, 1.0 and 0.75 mg ml−1. The samplesof collagen dispersions were diluted ten-fold to within thedetection range of the BCA kit, 0.75–2.0 mg ml−1. 25 μL ofstandards and samples were loaded into the wells of a 96-wellplate.

2

Biofabrication 6 (2014) 015012 M L Siriwardane et al

Collagen Flow

Belt Motion

Bath Level

Motor

Collagen Dispersion

Collagen Fibers

Syringe Pump

(A)

(B)

(C) (D)

Figure 1. Illustrations of the wet spinning system. (A) A syringe pump controls the extrusion rate of collagen dispersion into the device.(B) Wet spinning device with inside view depicting the flow and deposition of collagen; (1) spinneret clamp, (2) spinneret tip, (3) middleconnector, (4) rod connected to gears/motor, (5) collection belt and (6) tension adjustment for belt. (C) Computer drawing of the deviceshowing how the belt is driven with a gear–motor system. (D) Picture of the assembled device.

To assist in solubilizing collagen, 0.2% w/v sodiumdodecyl sulfate (SDS) was added to the BCA reagent. Thisamount of SDS induced negligible non-specific reactivity ofthe BCA. 200 μL of the BCA reagent/SDS solution was addedto each well and the plate was placed on an orbital shakerfor 270 min at 25 ◦C to facilitate BCA reactivity. An Emaxprecision microplate reader spectrophotometer (MolecularDevices, Sunnyvale, CA) was used to measure absorbanceat 570 nm of the protein standards and the unknowns. Astandard curve of the absorbance versus concentration of theprotein standards was used to extrapolate and confirm theconcentrations of the dispersions for wet spinning.

Automatic collagen wet spinning device

The wet spinning device consisted of a syringe pump, acoagulation bath chamber, a fiber collection belt and a gearedvariable speed dc motor for controlling the rate of fibercollection, figure 1. A 10 ml syringe containing the collagendispersions of 0.75, 1.0, 2.0 or 3.4% w/v was loaded into asyringe pump system (Fisher Scientific model no. 78-01001,Dupont, Wilmington, DE) operated at a rate of 12.4 ml h−1.The syringe pump was connected via 9.5 mm ID Viton R©tubingto the spinneret—a blunt end, type 304 stainless steel 22-gaugeneedle (406 μm ID, McMaster-Carr, Elmhurst, IL). A clamp

was built to hold the spinneret submerged into the coagulationbath with slight contact on the surface of the collection belt.

The coagulation chamber was formed from tworectangular sides composed of 2.54 cm thick polypropylene.A polypropylene middle section was used to define theinterior chamber and coagulation bath. Eighteen stainless steelmachine screws and washers (McMaster-Carr, Elmhurst, IL)were used to fasten the three-part chamber and provide aleak-proof tight seal to contain the coagulation bath. The 20◦

incline of the middle section was designed to minimize theamount of coagulation bath needed (150 mL per wet spinningsession) while also allowing ample immersion time for fibercoagulation.

A polytetrafluoroethylene (PTFE) mesh was used as thefiber collection belt (457 μm mesh size, McMaster-Carr,Elmhurst, IL). The PTFE mesh was cut to a length of 28 cmand width of 2.4 cm and sewn together using polypropylenesutures (Ethicon, Somerville, NJ). Three rotating Delrin R©rodssupported the collection belt within the inner chamber. Onerod was directly connected to a gear located on the shaft ofthe motor to drive the belt, a second rod was used to adjust thetension of the belt and a third rod was used to guide the pathof the belt, figure 1(B).

The collection belt was driven by a 12V dc 60 rpm, 3200 gcm torque motor (Jameco Electronics, Belmont, CA). To gear

3

Biofabrication 6 (2014) 015012 M L Siriwardane et al

down the motor speed, two gears with 13 and 1.3 cm diameters(gear ratio of 10:1) were designed using Pro Engineer Wildfire4 (PTC, Needham, MA) software and printed on our rapidprototype, 3D printer (SST 1200es, Dimension, Inc., EdenPrairie), figures 1(C) and (D). Collection of collagen filamentswas optimized at a collection rate of 6 rpm or linear speed of0.008 m s−1.

Collagen dispersions were extruded into a roomtemperature coagulation bath of ammonium hydroxide (AcrosOrganics, Fair Lawn, NJ) and acetone (HPLC grade, FisherScientific, Pittsburgh, PA) at a 1:50 volume ratio. The pH of thebath was monitored and adjusted manually with ammoniumhydroxide to maintain pH 9. The coagulation bath results inthe precipitation of collagen into solid monofilaments. Fiberswere then manually lifted to be cut from the edge of the beltand transferred to a drying rack in which they were air-driedunder the tension of their own weight at room temperature for aminimum of 48 h. The final product is a single continuous fiberof up to half a meter, which is then cut into smaller segmentsfor subsequent characterization and cell studies.

Fiber uniformity analysis

Wet-spun fibers were produced manually and with theautomated device for analysis. For the manual wet spinningprocess, collagen dispersions were gently extruded by handthrough a syringe with a 22-gauge tip into a polypropylenecontainer filled with coagulation bath. Fibers were air-driedon a drying rack at room temperature for a minimum of 48 hprior to diameter measurements.

To determine fiber diameter and uniformity, dried fiberswere cut into 1 cm lengths. A low magnification 4 × objectiveon a Nikon Eclipse TE2000-S inverted microscope was usedto take phase contrast images of the fibers, which were mergedtogether to create a photo montage of the entire 1 cm fiber usingAdobe Photoshop. Each 1 cm segment of fiber was equallydivided into 1 mm intervals in a grid layout using Image Jsoftware (NIH). A measurement was taken at random withineach of these intervals for a total of ten measurements perfiber. Fiber groups used in the study included the following:0.75, 1.0, 2.0 and 3.4% w/v. Diameter measurements for eachfiber group were used to calculate the mean fiber diameter andstandard deviation to determine fiber uniformity.

Cross-linking

In a post-processing step, 0.75 and 2.0% w/v collagen fiberswere cut into 3 cm segments and cross-linked with genipin(Gp) or glutaraldehyde (GA) to increase mechanical strengthand reduce fiber swelling. Fibers were immersed in solutionsof 0.1% and 1.0% Gp (Wako Pure Chemical Industries, Ltd,Japan) in 40% (v/v) ethanol or 0.01%, 0.1% and 1.0% (v/v)of GA in water at 25 ◦C for 24 h. The range of concentrationswas evaluated to establish an optimal cross-linking protocolfor these collagen fibers. The cross-linking reagents wereaspirated from the dishes and then rinsed for 10 min inddH2O, 2 min in phosphate buffered saline (PBS) and 2 minin 70% ethanol. This rinsing procedure with ddH2O and PBSwas repeated three times to ensure that residual cross-linking

reagents are thoroughly removed. Fibers were then air driedinside a sterile tissue culture hood for 24 h.

Mechanical testing

An Instron 3342 universal testing instrument (Instron, UK) wasused to generate uniaxial force–extension data for 0.75, 1.0,2.0 and 3.4% w/v collagen fibers and crosslinked 0.75% w/vfibers. Crosslinking with Gp at 1.0% (w/v) or 10 mg ml−1 andGA at 1.0% (v/v) was used for this study. Air-dried fibers fromeach of the fiber groups (n = 10) were fixed with tape to theInstron grips. Testing was performed on single fibers. Resultsobtained from fibers that broke at contact points or from fibersthat slipped from the clamps were rejected. Based on ASTMstandard D3822-01, fibers were tested under an extension rateof 5 mm min−1 and an initial gauge length of 2 cm. The stressσ was calculated by dividing the measured force by the meancross-sectional area. The strain ε was calculated by dividingextension by the fiber gauge length. A linear regression linewas fitted to the data over the initial 5% strain of the stress–strain curve and the tensile modulus E was calculated from theslope.

Fiber swelling response

The extent of water absorption was evaluated between0.75% w/v non-cross-linked and cross-linked fibers with 1.0%Gp and 1.0% GA. Fibers (n = 10) were incubated in PBS(pH 7.4) at 37 ◦C and imaged on a Nikon TE2000-S invertedmicroscope. A low magnification 4 × objective was used toenable a large field of view over the length of the fibers to assessfiber diameter. Pictures were taken at 30 min, 60 min, and 24 hand diameter measurements were made using ImageJ software.Average fiber diameters were determined from measurementsat ten random locations along the fiber as described above.Here, diameters were measured at the same location along thefiber at each time point.

Differential scanning calorimetry

Differential scanning calorimetry (DSC, Q100, TAInstruments New Castle, DE) was used to characterizethe thermal transitions of collagen fibers spun from eachdispersion including both cross-linked and non-cross-linkedsamples. Fibers were cut into small pieces and weighed into3–6 mg samples. A heat–cool–heat program was implementedover the range of 10 to 250 ◦C with a heating rate of10 ◦C min−1 and a cooling rate of −10 ◦C min−1. Analysisof the thermal transitions can indicate the density of collagenfibril assembly and cross-linking of wet-spun fibers. In thefirst heating cycle, the water is drawn off the sample andthe denaturation temperature can be identified. On the secondheating cycle, a glass transition temperature (Tg) is observeddue to the large segmental motions of denatured collagen.Accordingly, the Tg after thermal collagen denaturation canbe used to evaluate the extent of cross-linking [82–84]. Thismethod is based on a well-known relationship between therestricted chain mobility imposed by cross-linking and theincrease in Tg as a consequence of that restricted mobility [84].

4

Biofabrication 6 (2014) 015012 M L Siriwardane et al

Thermal analysis was performed on dry fiber samples in thisstudy. Hence, the denaturation temperatures of the collagenfibers were higher compared to fibers in the wet state in theliterature [49, 85, 86].

Neuronal cell culture

Dorsal root ganglia (DRG) were isolated from embryonic day15 rat pups taken from pregnant Sprague-Dawley rats (CharlesRiver, Wilmington, MA) and held in L-15 medium on wet ice.Explants were dissociated using 0.25% trypsin for 1 h at 37 ◦C.Neurobasal medium +1% fetal bovine serum (FBS) was thenadded, and the tissue was triturated followed by centrifugationat 1000 rpm for 5 min. The supernatant was aspirated and thecells were re-suspended at 5 × 106 cells mL−1 in Neurobasalmedium supplemented with 1% B-27, 0.5 mM L-glutamine,1% penicillin/streptomycin, 1% FBS, 20% glucose (Sigma,St Louis, MO), 20 ng mL−1 nerve growth factor, 20 μM FdU(Sigma), and 20 μM Uridine (Sigma). This cell suspension(5 μL) was added directly onto the end of the collagen fibersamples (0.75% w/v non-cross-linked, 1.0% Gp, and 1.0%GA cross-linked fiber groups). The cultures were placed in ahumidified tissue culture incubator (37 ◦C and 5% CO2) for2 h at which point 2 mL of media was added to each culture.The culture media was changed every 2–3 days in vitro byreplacing with fresh media pre-warmed to 37 ◦C.

Schwann cell culture

Embryonic Schwann cells were also used to assess thecytocompatibility of the wet-spun collagen fibers in this study.Schwann cells from E9 rat pups of timed-pregnant Sprague-Dawley rats (Charles River, Wilmington, MA) were obtainedat passage 5–7 from Dr Haesun Kim, Rutgers University,Department of Biological Sciences (Newark, NJ). Schwanncells at 80% confluency on a 100 mm dish coated with100 μg ml−1 poly-L-lysine were collected using 0.25% trypsinfor 5 min at 37 ◦C. DMEM with 10% FBS was then added, andthe tissue was triturated followed by centrifugation at 1000 rpmfor 5 min. Schwann cells were re-suspended in DMEMmedium supplemented with 10% FBS, 0.05% forskolin, 0.01%neuregulin, and 1% penicillin/streptomycin. Schwann cells atdensity of 2.5 × 104 cells per 10 μL suspension were platedon the ends of the wet-spun collagen fibers and placed in ahumidified tissue culture incubator (37 ◦C and 5% CO2) for2 h at which point 2 mL of media was added to each culture.The culture media was changed every 2–3 days in vitro byreplacing with fresh media pre-warmed to 37 ◦C.

Collagen fiber treatment and sterilization

Neuronal and Schwann cell cultures were generated withinpolystyrene cell culture dishes or multi-well plates. Driedcollagen fibers were cut into 1 cm segments, rinsed for 10 minin ddH2O, 2 min in 70% ethanol and air dried inside a sterilelaminar flow hood. Fibers were adhered to the bottom of theculture well base by applying 50 μL of 2 mg ml−1 collagen tothe wells of a 12-well polystyrene tissue culture treated platewhile allowing to air dry for 1–2 h prior to cell plating.

Cell viability and immunocytochemistry

At day 10 in vitro, cells were labeled using fluorescentprobes to distinguish live and dead cells (LIVE/DEADViability/Cytotoxicity Kit; Molecular Probes, Eugene, OR).Cells were rinsed in PBS and incubated with 2 μM calcein AMand membrane impermeable propidium iodide (5 μg mL−1,stains DNA of dead cells only) in PBS at 37 ◦C for 30 min andthen rinsed three times in PBS. Neuronal adhesion and neuriteoutgrowth on fibers was analyzed from fluorescent imaging ofaxons with labeled neurofilaments. For immunocytochemistry,the cultures were fixed in 4.0% paraformaldehyde (Fisher,Fairlawn, NJ) for 1 h, rinsed in PBS and permeabilized using0.1% Triton X100 (Kodak, Rochester, NY) +4% goat serum(Invitrogen) for 1 h. Neurofilament antibody (NF-200, Sigma-Aldrich, St Louis, MO) was added (in 0.1% Triton X100,4% goat serum in PBS) for 18–24 ◦C for 1 h at 1:400dilution. Secondary fluorophore-conjugated antibody (Alexa488-conjugated IgG, Molecular Probes) was added in PBS at18–24 ◦C for 2 h at 1:1000 dilution.

Results

Quantification of collagen dispersion concentration

Collagen dispersions induced minimal colorimetric changeafter 2 h of incubation following the BCA protein assaykit protocol. This is likely due to dispersions consistingprimarily of insoluble components of collagen that inhibitthe colorimetric reaction. Hence, any assay results fromthe standard protocol could not be compared to the BSAprotein standards for quantification. Here, we modified theBCA protein assay to help solubilize collagen to improve thecolorimetric reaction by adding 0.2% w/v SDS to the BCAreagent. We also found that physical agitation of the plate withan orbital shaker greatly enhanced the reaction.

To test this protocol, colorimetric absorbancemeasurements were compared between the kit BSAstandards and standards made from a commercially purchasedcollagen at a known concentration. Regression analysis wasused to compare the absorbance versus concentration curvesfor the BSA standards and collagen standards, figure 2. Theresults also show that using 0.2% w/v SDS and shaking thesamples up to 270 min enhanced the colorimetric reaction.No significant changes in absorbance values were detectedbeyond 270 min. The collagen standards, however, didnot match the BSA standards. Accordingly, using collagenstandards at known concentrations were more suitable forinterpolating the concentration of the unknown collagendispersion samples.

Automated wet spinning of collagen

Wet-spun fibers are controlled through the adjustment of fourparameters: collagen dispersion concentration, spinneret size,speed of the collection belt and dispersion flow rate. The abilityof the device to extrude dispersions through the syringe pumpsystem and form fibers was compared for 0.5, 0.75, 1.0, 2.0and 3.4% w/v dispersions. Spinnerets with gauge sizes of

5

Biofabrication 6 (2014) 015012 M L Siriwardane et al

y = 0.8799x + 0.2961 R = 0.99806

y = 0.1591x + 0.1155 R = 0.95241

y = 0.3913x + 0.3093 R = 0.99919

0

0.5

1

1.5

2

2.5

0 1 2 3 4

Abs

orba

nce

@ 5

70nm

(O

.D.)

Concentration (mg/mL)

Standard Curves

BSA

BD Collagen

BD Collagen 270 min Shake

Figure 2. Quantification of collagen by modifying BCA protein assay. Comparison of standard curves from bovine serum albumin (BSA),Becton Dickinson (BD) rat tail type I collagen and BD collagen with 270 min of shaking to enhance the reaction.

21 and 22 were required to produce small diameter fibers.Spinnerets with gauges above 22, however, were too narrowand dispersions above 1.0% w/v resulted in obstruction due toits high viscosity and the likely presence of insoluble collagenparticulates. 22-gauge spinnerets produced fibers in the targetrange of 20–50 μm for this study.

Using a 22-gauge spinneret, dispersions at 0.5% w/vproduced brittle two-dimensional films while 0.75% w/vconsistently generated fibers. 0.75 and 1.0% w/v dispersionswere readily extruded into the coagulation bath, whereas theviscosity of 2.0 and 3.4% w/v dispersions made it difficultto load into the syringe, flow through the Viton R© tubing andextrude out of the spinneret. At high concentrations (3.4–5.0%w/v), collagen dispersions contained large insoluble collagenparticulates that would clog and disrupt the extrusion. Thiswas resolved by warming the 2.0 and 3.4% w/v in a 40 ◦Cwater bath for 10 min prior to wet spinning. Warming enabledthe 2.0 and 3.4% w/v dispersions to be extruded throughthe tubing and into the coagulation bath. 3.4% w/v was themaximum concentration that resulted in dispersions visuallyclear of particles.

Collagen fibers formed in a coagulation bath were initiallyfragile and required a minimum of 1 min exposure to thecoagulation reagents in order to exhibit sufficient strength tosupport their own weight. The 20◦ incline, length and speed ofthe belt was designed to accommodate coagulation time andproduce proper fiber formation prior to removal from the bath.Furthermore, we found that fiber uniformity was dependent onmatching the speed of the collection belt to the extrusion rate.The speed of the motor (60 rpm) was too fast to collect fibers.Therefore, we used a 10:1 gear ratio to slow down the speedto 6 rpm (linear speed of 0.008 m s−1) where 0.75–3.4% w/vdispersions consistently formed fibers on the collection belt atan extrusion rate of 12.4 ml h−1. Best results were achievedwhen the spinneret was in slight contact with the surface ofthe moving collection belt.

A small range of syringe pump flow rates for thegiven speed of the collection belt was established to adjustfiber diameter. Using a collagen dispersion concentration

of 0.75% w/v, flow rates above 12.4 ml h−1 resulted inprecipitated collagen with irregular, poorly defined fibermorphology. Flow rates below 2.0 ml h−1 produced thin, flat2D films of precipitated collagen. As flow rates were steadilydecreased from 12.4 to 2.0 ml h−1, fibers were produced withdecreasingly smaller diameters ranging from 46.5 ± 10.9 μmto 23.2 ± 10.9 μm, respectively. Hence, the flow rate can bevaried for applications requiring different diameter fibers.

An analysis of diameter and uniformity revealed that0.75 and 1.0% w/v dispersions produced fibers that were46.5 ± 10.9 μm and 55.9 ± 14.9 μm, respectively, butwere not significantly different, p > 0.05, figure 3. Furtherincreases in collagen dispersion concentration led to increasedfiber diameters. 2.0 and 3.4% w/v collagen dispersionsproduced fibers with mean diameters of 105.02 ± 24 μm and193.4 ± 38.9 μm, respectively. Standard deviation in fiberdiameter remained between 20–25% of the mean fiberdiameter for each concentration.

To demonstrate the improvement in fiber formation, handspun fibers (n = 10) were compared to fibers producedautomatically (n = 10) using our device. Fibers werewet-spun from 0.75% w/v dispersions manually using thesame syringe, tubing and 22-gauge needle used in the wetspinning device. Mean fiber diameters of wet-spun collagenfibers produced from the automated wet spinning device(46.5 ± 10.9 μm) were smaller, more uniform andsignificantly different (p < 0.05) compared to fibers producedmanually (57 ± 31.1 μm).

Instron tensile analysis

For non-cross-linked fiber samples, fiber stiffness decreased asthe concentration of dispersion increased, figure 4. The 0.75%and 1.0% w/v dispersions had tensile moduli of 1265 ±171 MPa and 800 ± 143 MPa, respectively, table 1. The 2.0and 3.4% w/v dispersions had much lower moduli of 286 ±58 MPa and 236 ± 98 MPa, respectively (n = 10 for eachgroup).

To investigate the effect of cross-linking on tensilemodulus, fibers wet-spun from 0.75% w/v dispersions were

6

Biofabrication 6 (2014) 015012 M L Siriwardane et al

0

50

100

150

200

250

Mea

n F

iber

Dia

met

er (

µm)

Collagen Dispersions (w/v)%

Fiber Diameter Uniformity Dependence on Collagen Concentration

0.75wt 1.0%

2.0%

3.4%

*

**

** 0.75%

1.0%

2.0%

3.4%

Figure 3. Fiber diameter increases with collagen dispersion concentration. ∗ denotes p > 0.05 or statistically insignificant, ∗∗ denotesp < 0.05 or statistically significant difference.

0

20

40

60

80

100

120

140

160

180

200

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

Stre

ss (

MP

a)

Strain

3.4%

0.75%

1.0%

2.0%

0

10

20

30

40

50

60

70

0 0.01 0.02 0.03 0.04 0.05

Stre

ss (

MP

a)

Strain

0.75%

1.0%

2.0% 3.4%

(A)

(B)

Figure 4. Stress–strain curves for wet-spun fibers from increasingcollagen dispersion concentrations (w/v). (A) Stress–strain responseto failure. (B) Stress–strain response within the initial 5% strain,where the best-fit line was drawn to estimate the slope or modulus ofelasticity, E.

treated in 1.0% GA or 1.0% Gp. Cross-linking was a post-treatment process, where dried fibers were rehydrated witha cross-linking solution and then dried for a minimum of24 h prior to mechanical testing. Since this procedure couldaffect fiber mechanical properties, a sham group was made bysoaking 0.75% w/v wet-spun fibers in dH2O only and dried

0

50

100

150

200

250

300

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2

Stre

ss (

MP

a)

Strain

1.0% Gp

1.0% GA

Control (in Water)

Dry

Figure 5. Stress–strain curves for different cross-linking treatmentsfor 0.75% w/v wet-spun collagen fibers.

Table 1. Effects of concentration and cross-linking on the tensileproperties of wet-spun collagen fibers. Errors indicate one standarddeviation.

%w/v Collagendispersion Tensile modulus UTS % Elongation

Dry 0.75 1265 ± 171 MPa 262 ± 62 MPa 18.4 ± 4.9Dry 1.0 800 ± 143 MPa 240 ± 25 MPa 36.7 ± 8.0Dry 2.0 286 ± 58 MPa 57 ± 15 MPa 43.6 ± 9.1Dry 3.4 236 ± 98 MPa 25 ± 2 MPa 11.2 ± 2.0

0.75 Sham-treated, 707 ± 68 MPa 59 ± 18 MPa 10.9 ± 1.6non-cross-linked0.75 Cross-linked 2394 ± 148 MPa 222 ± 74 MPa 16.4 ± 1.31.0% genipin0.75 Cross-linked 2821 ± 168 MPa 136 ± 2.6 MPa 10.8 ± 1.91.0% glutaraldehyde

in parallel with fiber cross-linking treatments (n = 10 for eachtreatment group).

For cross-linked fiber samples, the stress–strain curvesrevealed that the tensile modulus increased with 1.0% GAand 1.0% Gp treatments compared to the sham and dry fibergroups, figure 5. The procedure of rehydrating and dryingcollagen fibers lowered tensile strength from a mean tensile

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Biofabrication 6 (2014) 015012 M L Siriwardane et al

0

5

10

15

20

25

30

35

40

45

Dry 30min 60min 24hr 6wk

Fib

er D

iam

eter

(µm

)

Incubation Time

Swelling Response of Collagen Fibers

Non-crosslin

1.0% Glutaraldeh1.0% Genipi

**

* Non-crosslinked

1.0% Glutaraldehyde

1.0% Genipin

Figure 6. Swelling behavior for non-cross-linked and cross-linked 0.75% w/v collagen fibers in PBS. ∗ denotes p > 0.05 or statisticallyinsignificant, ∗∗ denotes p < 0.05 or statistically significant difference.

modulus of 1265 ± 171 to 707 ± 68 MPa. Collagen fiberstreated with 1.0% GA and 1.0% Gp, however, had greatlyincreased tensile moduli of 2821 ± 168 MPa and 2394 ±148 MPa, respectively, table 1. Interestingly, the GA cross-linked fibers would consistently break at a strain of 10%whereas the Gp fibers demonstrated more toughness allowingstrains up to 20% before breaking.

Swelling response

Collagen fibers like other natural polymers are known toundergo water absorption under physiological conditions. Thedegree of swelling was quantified based on fiber diameterbefore (dry) and after incubation in PBS for 30 min, 60 min,24 h and 6 weeks at 37 ◦C. Non-cross-linked 0.75% w/vfibers swelled to slightly more than twice its diameter within30 min of incubation and remained constant for the next 24 h,figure 6. 1.0% GA and 1.0% Gp cross-linked fibers showedgreatly reduced swelling that also occurred within the first30 min with minimal change over 24 h. At 6 weeks, non-cross-linked fibers continued to swell whereas the cross-linked fibersshowed limited swelling after the first 30 min. At 6 weeks,the swelling response between 1.0% GA and 1.0% Gp wasstatistically insignificant.

Differential scanning calorimetry

DSC was performed on collagen fibers spun from eachdispersion including both cross-linked and non-cross-linkedsamples and the thermal transitions were identified. Duringthe first heat step, a broad endotherm in the graph was dueto water being driven off from the collagen fibers, figure 7.A second small endothermic peak marked denaturation at180–230 ◦ C. On the second heating cycle, the observation of Tg

Table 2. Comparison of thermal properties for wet-spun collagenfibers.

Denaturation Glass transitionw/v% temperature (Td) temperature (Tg)

0.75 223.02 ± 1.5 ◦C 208.87 ± 1.5 ◦C1 213.41 ± 2.0 ◦C 201.93 ± 0.5 ◦C2 187.24 ± 0.5 ◦C 185.11 ± 0.1 ◦C3.4 186.35 ± 0.5 ◦C 182.96 ± 0.5 ◦C0.75, Cross-linked 0.1% GA 232.93 ± 0.5 ◦C 206.63 ± 2.0 ◦C2.0, Cross-linked 0.1% GA 231.83 ± 2.0 ◦C 203.26 ± 0.5 ◦C

occurred at 185–210 ◦ C. As the concentration of the collagendispersions increased, both denaturation temperature (Td) andTg of wet-spun fibers decreased, table 2. This is consistentwith our mechanical and swelling data that suggests betterorganization of collagen fibrils in fibers wet-spun from lowercollagen dispersion concentrations. Fiber samples from 2%w/v dispersions treated with cross-linkers had higher Td andTg than non-cross-linked 2% w/v fibers indicating more energyrequired to break cross-link bonds. The change in Tg was notseen for the fibers from 0.75% w/v dispersions.

Neuronal growth response

During peripheral nerve regeneration, facilitating the sustainedmigration of Schwann cells and extension of regeneratingaxons are critical. We first examined the neurocompatibilityof cross-linked wet-spun collagen fibers produced from 0.75%w/v dispersions. Dissociated dorsal root ganglia neurons wereplated on the ends of cross-linked fibers treated with 1.0%Gp and 1.0% GA. DRG neurons on Gp-treated fibers had aviability of >95% at 10 days in vitro. In contrast, the GA-treated fibers displayed <5% viability during the same time

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Biofabrication 6 (2014) 015012 M L Siriwardane et al

(C )

(A)

(D)

(B)

Figure 7. Differential scanning calorimetry thermograms for wet-spun collagen fibers. (A) 0.75% w/v non-cross-linked fiber, (B) 0.75% w/vfiber cross-linked with 0.1% GA, (C) 2.0% w/v non-cross-linked fiber and (D) 3.4% w/v non-cross-linked fiber. (1) Glass transitiontemperature, (2) denaturation temperature, (3) broad peak representing water loss from sample.

point in culture. Furthermore, the cultures were assessed forneuronal adhesion to the fibers and stained for neurofilamentto investigate the morphology of neurite outgrowth on thefibers. GA cross-linked fibers resulted in widespread cell deathcompared to robust neurite outgrowth on the Gp cross-linkedfibers, figure 8. We assessed the migration of embryonicSchwann cells towards and along wet-spun collagen fibers.Schwann cells were plated at one end of a non-cross-linkedcollagen fiber mounted on the bottom of a culture well. Withinday 1 in culture, Schwann cells attached and migrated alongthe surface of fibers and exhibited dense morphology alongthe length of the fiber edge (figures 9(A) and 10(A)). Calceinstain confirmed viable cells in the presence of collagen fibers(figure 9(A)). Schwann cells plated on the ends of cross-linkedfibers treated with 1.0% Gp exhibited long extending processeswith oriented growth parallel to the fiber as early as day 1(figure 10(B)). Schwann cells were often present along thesurface or edge of the fiber.

Discussion

Device and technique development

A wet spinning device was engineered for the controlledfabrication of collagen fibers. Unlike electro-spinning, the

wet spinning approach enables the production of orientedmonofilaments from biological polymers without the use ofharsh organic solvents, which can compromise their nativestructure [49, 55–57]. Dispersions made from collagen inacidic solvents can produce electro-spun mats, however,the slow evaporation rate and high affinity of acids tocollagen result in partially welded scaffolds with low porosity[55]. Hence, solvents such as the fluoroalcohols with highevaporation rates and moderate affinity to collagen are widelyused for electro-spinning collagen. Interestingly, many ofthe fluoroalcohols used in electro-spinning not only denaturecollagen but also lower its denaturation temperature [87].Indeed, electro-spinning collagen from fluoroalcohols resultsin denatured collagen that closely resembles gelatin atthe molecular scale [49, 57]. In this study, pure collagendispersions can be prepared in acidic solutions up to 1.0%w/v for wet spinning without any processing limitations.

We found that wet spinning of 0.75% w/v collagen fibersrequires a minimum of 1 min to coagulate sufficiently tosupport their own weight. The key feature that allowed theformation of long uniform collagen fibers with consistentdiameters and mechanical properties from batch to batchwas the moving collection belt supporting fibers during theperiod of coagulation. Indeed, best results were achieved

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Biofabrication 6 (2014) 015012 M L Siriwardane et al

(A)

(B)

(D)

(E)

(C)

Figure 8. Viability and axon growth of embryonic DRGs on cross-linked collagen fibers at Day 10. (A) Negative neurofilament staining on1.0% GA-treated fibers. (B) Neurofilament staining shows axon growth along 1.0% Gp-treated fibers. (C) Live dead staining shows celldeath (red) and few neurons (green) associating with the 1.0% GA-treated fibers. (D) Little cell death and a high level of neuronalassociation are seen on the 1.0% Gp-treated fibers. Dashed lines denote edges of fiber. (E) Long neuronal processes extending along 1.0%Gp-treated fibers, Calcein stain (green). Scale bars: (A), (B), (C), (D) 100 μm and (E) 50 μm.

Figure 9. Growth morphology and response of embryonic Schwann cells on wet-spun collagen fibers. (A) Calcein stain of Schwann cellsmigrating towards a collagen fiber, day 1. (B) Phase contrast image of Schwann cells adhering and covering a collagen fiber, day 7,20 × magnification. Lines denote edges of fiber and dashed lines represent longitudinal axis of fiber. Scale bars: (A) 100 μm and (B) 50 μm.

when the spinneret was in close contact with the belt,extruding the collagen dispersion onto the moving belt. Thecollagen dispersion extrusion rate could then be adjusted from2–12 ml h−1. This mismatch in velocities induced a controlledstretching of the wet-spun fiber along its axis resulting in arange of collagen fiber diameters from 23.2–45.5 μm. Outsidethis range fibers could not be formed.

Another important feature of this device is the geometryof the device’s interior chamber. The design incorporates thecorrect length of collection belt arranged at a 20◦ angle tobalance adequate coagulation and drying time. In addition, theinterior volume was minimized for the efficient use of wet

spinning reagents. This compact design works well in sanitaryenvironments such as a cell culture hood.

Properties of wet-spun fibers

Wet spinning studies have shown that fiber diameter varies withcollagen concentration [88, 89]. Accordingly, our hypothesiswas that increasing the collagen dispersion concentrationwould create fibers of larger sizes and higher strengths.Indeed, higher dispersion concentrations resulted in largerdiameter fibers, see figure 3. In contrast, the tensile modulusand ultimate tensile strength of the fibers decreased as the

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Biofabrication 6 (2014) 015012 M L Siriwardane et al

(A)

(B)

Figure 10. Migration and orientation of embryonic Schwann cellson wet-spun collagen fibers. (A) Phase contrast image of Schwanncells on untreated collagen fibers, day 1, 20 × magnification.(B) Phase contrast image of Schwann cells on Gp cross-linkedcollagen fibers, day 1, 20 × magnification. Scale bar = 100 μm.

collagen concentration in the dispersions increased, table 1.It is believed that at higher concentrations, viscosity andturbulent flow during extrusion leads to the formation of largeand irregular diameters [86]. It is likely that the high viscosityof the dispersion inhibits the movement of collagen fibrils toorient against the shear forces exerted during extrusion [90].Consequently, as the fiber diameter increases, collagen fibrillarpacking is likely reduced due to poor orientation of the fibrils.In contrast, extrusion of low concentration dispersions (0.75–1.0% w/v) produces small diameter fibers of higher strengthand stiffness. During extrusion, the reduction in viscosity mayresult in increased longitudinal alignment and fibrillar packingdensity, leading to stronger interactions within or betweenindividual collagen fibrils [90–94]. The low tensile modulusin larger fibers seen in our results could be explained by thestraightening of misaligned fibrils during deformation [86].As the fibrils extend and orient longitudinally, tensile stresswould become unevenly distributed with regions of weakpoints where failure may occur. In this study, we found thatthe 0.75% w/v dispersions consistently formed the most well-defined fibers with the highest modulus and tensile properties.

The DSC results support this notion that higher collagendispersion concentrations may lead to less organized collagenfibril assembly. Collagen fibers made from 2.0 and 3.4%w/v dispersions had reduced Td and Tg, figure 7. Thermaldenaturation of collagen can be effected by reduced fibrilpacking as well as interstitial water content [95–98]. Withheat, the water content within collagen fibers facilitates theprogressive dissociation into three randomly coiled peptideα-chains characteristic of gelatin. Accordingly, more waterpresent between collagen fibrils may reduce the temperature

required to break bonds between α-chains. The increasedpresence of water may also be correlated to the reduction inmechanical properties of the fibers. For thicker fibers, the watercontent within large spaces between fibril packing inhibitsproper longitudinal alignment and orientation, which resultsin reduced tensile properties. According to the thermochemicalproperties among the dispersions, the 0.75% w/v fibersexhibited the most thermal stability as evident by the higherhelix–coil transition temperature.

Fiber cross-linking

Based on the mechanisms described above, cross-linkingcollagen fibrils within the fibers should increase the tensileproperties and limit the swelling or water uptake into the fibers.The stability of these properties is important if long-termperformance in an aqueous environment is required for thetissue engineering application. In this study, Gp was evaluatedas a biocompatible cross-linker using GA as a positive control.The higher denaturation temperatures confirm cross-linkingsince more energy is required to break bonds formed betweenα chains in the presence of cross-linkers. The formation ofcross-links between collagen chains can also reduce pore sizeand spaces for water penetration between fibrils. Both GA andGp were very effective in reducing swelling and enhancing thetensile properties of the fibers. Another potentially importantfiber property may be the ability to deform without breaking.Here we found that the GA cross-linked fibers consistentlybroke at strains of 10% whereas the Gp fibers demonstratedmore toughness allowing strains up to 20% before breaking.

The thermal analysis approach used in this study toassess cross-linking of collagen was based on the relationshipbetween restricted chain mobility within collagen imposedby cross-linking and the increase in Tg due to the restrictedmobility [84]. Tg is determined by a combination of chainstiffness and cohesive forces within the polymer. The reductionof large-scale chain motions by increasing the amount ofcross-linking can be assessed by the inception of Tg [84].Collagen in its native form does not have a Tg since large-scalesegmental motion is restricted by the non-bonded interactionsthat hold the protein in its secondary and tertiary structures.When collagen is denatured, those interactions are eliminated,and large-scale chain mobility is possible, which makes itlikely to observe Tg. Chemical cross-links are preservedduring denaturing because of their covalent nature, and asa consequence, changes in cross-linking can be probed byobserving changes in Tg. An increase in Tg due to cross-linkingwas seen for all fibers except from those spun from 0.75%w/v dispersions. The results were similar for both GA and Gpverifying that cross-linking was achieved.

The cytotoxicity effect of GA has been a substantialdrawback for this commonly used cross-linker [99–101].Furthermore, physical cross-linking achieved by otherprocesses such as dehydrothermal treatment (DHT) and UV-irradiation compromise the stability of collagen due to thermaldegradation [56, 102, 103]. A few studies have previouslydemonstrated that Gp has the potential to be used as asubstitute cross-linking agent [83, 104–108], however, its

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Biofabrication 6 (2014) 015012 M L Siriwardane et al

cyto-compatibility with neuronal tissue has only recentlybegun to be explored [99]. Here, we found that Gp-treatedfibers supported DRG neuron survival and axon outgrowthwhere most neurons died on GA-treated fibers. Importantly,neurocompatibility is confirmed by the robust growth of axonsand migration of Schwann cells along the Gp-treated fibers andvalidates the future use of Gp for neural tissue engineeringapplications.

Although this work elucidates the efficacy andbiocompatibility of the end-product scaffolds in 2D tissueculture systems, the end goal is to translate this technologytowards the fabrication of a 3D engineered nerve guidanceconduit for in vivo neural applications. Hence, the end-product collagen monofilaments produced from the deviceare to be cut into desired lengths and stacked into a 3Dmulti-filament bundle, which can then be incorporated into acomposite nerve guidance channel. The multi-filament bundleand hydrogel are to be utilized as filler substrate materialsin a nerve conduit design as a strategy for peripheral nerverepair. The presence of an aligned multi-filament bundle withinthe conduit will provide critical physical cues to allow robustaxonal growth throughout the proposed conduit. The successof Gp-treated cross-linking in this work renders its potentialuse in binding individual collagen fibers comprising the multi-filament bundle via inter-, intra-helical and inter-microfibrillarcross-links [104].

The wet spinning device created here has the ability toform long continuous, straight and uniform collagen fibers[62, 70, 73, 76, 77]. In particular, the design facilitates theformation of very soft and delicate fibers, which makes itsuitable for producing wet-spun fibers from a variety ofnaturally-derived polymers. Other important advantages of ourdesign are the small quantity of chemical reagents required foreach production cycle and its small size enables portability andtransport into sterile working environments (i.e. tissue culturehoods). The self-contained chamber could also support variouscoagulation baths and could even be used for cross-linkingtreatments. These features make the device ideal for most anywet spinning process required for biological application.

Acknowledgments

Financial support provided by an NSF CAREER CBET-0747615. The authors thank John Hoinowski for his expertise,design contributions and fabrication of device.

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