Fabrication and Characterization of PolymericNerve Conduits

Iris Horng Katelyn Kingiris2k2@gmail.com katelynmking22@gmail.com

Jalen Patel Kartik Pejavara Kaushik Tallamjalenpatel714@gmail.com kartik.pejavara@gmail.com tallam.kaushik@yahoo.com

Dr. Thomas Emge* Dr. N. Sanjeeva Murthy*emge@chem.rutgers.edu murthy@dls.rutgers.edu

New Jersey’s Governor’s School of Engineering and TechnologyJuly 26, 2019

*Corresponding Author

Abstract—Polymeric substances are ideal for the fabrication ofartificial nerve conduits due to their functionality and anatomicsafety. The research presented in this study investigates theproperties of poly(lactic acid) (PLA) and applies these propertiesto the design of various polymeric nerve conduits designed totreat patients that suffer from peripheral nervous system (PNS)injuries through the facilitation of nerve regeneration. Extrusionof PLA fibers, X-ray diffraction, and tensile strength testing wereused to establish the impact of processing methods includingextrusion temperature, spooling speed, annealing, and stretchingon the structural and mechanical properties of PLA. Basedon these trials, it was determined that thin fibers extrudedat high temperatures and spooling speeds would be ideal forproducing a flexible nerve conduit. Fused filament fabrication(FFF), an additive 3D printing process, was utilized to fabricate anerve conduit made of both PLA and thermoplastic polyurethane(TPU).

I. INTRODUCTION

Polymers, macromolecules consisting of repeating subunitsand possessing expansive properties, are extensively used inregenerative medicine. Their properties make manufacturingstraightforward relative to other common materials such asmetals, consequently reducing the cost and time required tofabricate various biomedical devices.

Patients exhibiting peripheral nerve injury (PNI) stand tobenefit greatly from these advancements, which may expeditetheir recovery via accelerated treatment production. A varietyof circumstances may give rise to nerve damage, including mo-tor vehicle trauma and sports injury, so efficient treatment is inhigh demand. Current treatments for PNI involve the suturingof severed nerves that are less than 5 mm apart. Additionally,nerve grafts, segments of neural tissue transferred from otherparts of the body or a foreign donor, are often employed forrepairing larger separations [1]. However, nerve conduits areincreasingly being used as a safer, simpler alternative that doesnot require biological tissue [2]. Instead, the nerve conduit, a

surgically implantable synthetic tube, bridges the severed endsof a nerve in order to facilitate axonal regeneration [1].

This study aims to optimize the production of nerve conduitsthrough the fabrication and characterization of a 3D-printablenerve conduit that would be more economical and allowfor greater personalization. 3D-printing technology delivers adesign capable of meeting the diversity of patients’ needs.Additionally, polymers ensure chemical functionality, forminga device that is both nonkinkable and bioresorbable.

Computer Aided Design (CAD) programs were utilized todesign a nerve conduit suitable for 3D printing. Techniquesincluding X-ray diffraction and tensile strength testing wereemployed to determine the PLA fibers best suited to meetthe demands of the device. Ultimately, a combination of bothpoly(lactic acid) (PLA) and thermoplastic elastomer (TPU)was used to fabricate the final nerve conduit.

II. BACKGROUND

A. Peripheral Nervous System

The peripheral nervous system (PNS) is the network ofnerves that connects the central nervous system (CNS), whichconsists of the brain and spinal cord, to the rest of thebody. Compared to the CNS, the finer nerves of the PNSexhibit greater susceptibility to injury, but also a greatercapacity to regenerate upon injury. Specific characteristics ofthe PNS, including an abundance of growth-promoting factors,relatively faster debris clearance, and greater up-regulation ofregeneration-associated genes (RAGs), contribute to this dis-parity in growth between the two systems [3]. Common to bothsystems are the limitations that the size of the gap betweenthe ends of a severed nerve place on the efficacy repair. As thegap size increases, both the ability of separated ends to locateone another and the availability of growth factors decrease

1

[4]. These factors prevent severed nerve endings separated bymore than 5 mm from regenerating independently.

Fig. 1: Implanted Nerve Conduit [8]

B. Peripheral Nervous System Injuries

Damage to the peripheral nervous system affects a sub-stantial population of civilians and military personnel. Often,the cause is trauma in the form of a motor vehicle accident,occupational injury, athletic injury, crushing force, or, moreinfrequently, an explosion or gunshot wound [5]. Classificationof such injuries is dependent upon the anatomical damagesustained by the affected nerves. Seddon’s classification iden-tifies three categories of damage: neuropraxia, axonotmesis,and neurotmesis. Neuropraxia involves a loss of conductivityof the nerve body, but no permanent damage to the tissue.Axonotmesis encompasses damage to the myelin sheath, orouter covering of the nerve. Damage that affects inner lay-ers of structural tissue, including complete bisection of thenerve, falls under neurotmesis. These broad categories arefurther described by the five degrees of injury outlined bySunderland’s classification. First degree injuries are akin toneuropraxia, while fifth degree damage is the equivalent ofneurotmesis. Second, third, and fourth degree injuries eachrepresent a specific form of axonotmesis [6]. Associated witheach category are specific symptoms ranging from weakness,tingling, and burning to total numbness or chronic pain in theaffected area [7]. Only injuries of the fourth or fifth degreetypically require surgical repair, in which case nerve conduitsmay be useful.

C. Current Treatments of Peripheral Nerve Injuries

There are numerous methods of repairing PNS injuries,though two major interventions currently predominate clinicalpractice. The first is the suturing of nerves that are within 5mm of each other. The scope of this technique is limited, asbridging a gap wider than 5 mm in this fashion overloads thenerve with tension. The second intervention is a nerve graft,used when the separation between the two ends of a severednerve exceeds the 5 mm limit. This procedure necessitatesdonor tissue, of which there are three main types: allogeneicgrafts taken from a human donor, xenogeneic grafts from ananimal, and autologous grafts of the patient’s own tissue.

Though standard, these methods present challenges: allo-geneic and xenogeneic grafts require a tissue donor, whileautologous grafts may sacrifice function at the donor site.

Nerve conduits present an ideal solution to these obstaclesby eliminating the need for donor tissue entirely. Instead, asynthetic tube suffices to connect the ends of a severed axonin such a way that they will rejoin. Once the nerve has healed,the conduit dissolves and a fully functional nerve remains [11].

D. Application of Current Technology

Although nerve conduits already exist, fabricating themthrough 3D printing is yet to be implemented on a largemarket scale. 3D printing provides the benefit of an acceleratedmanufacturing process, consequently making the product moreaffordable for patients. With the deployment of this relativelynew technology, personalized, patient-specific nerve conduitscan be designed and fabricated in very little time either onor off site. This differs greatly from the current method inwhich individual fibers are mechanically braided together intoa tube. This process is extremely time consuming and requiresmedical facilities to stock hundreds of generic conduits thatoften have a limited shelf life. With the use of 3D printing,this problem can be eliminated, cutting business costs.

E. Fabrication of Polymers

Polymers are an ideal choice for the creation of biomedicaldevices as a result of their wide array of mechanical, chemical,and optical properties. Their diverse functionality may beleveraged via 3D printing in order to increase the efficiencyof developing biomedical devices. With 3D printing, a widerange of objects may be created, making the technology aninvaluable asset in the creation of cheaper and more person-alized biomedical devices.

Extrusion is the process of fabricating objects of a specificcross-sectional size. Polymeric resin is placed in an extruder,which melts the material, forces it through a barrel by meansof a screw, and releases it through a die of the desired diameter.This process yields long polymeric fibers that can be used forfurther experimentation. In order to optimize the final product,it is crucial to extrude polymers at an appropriate temperature.

Differential Scanning Calorimetry (DSC) is a method fordetermining a material’s unique thermodynamic properties,including its glass transition, crystallization, and melting tem-perature, and is used to determine the optimal temperaturefor extrusion. The desired temperature is above the material’sglass transition temperature and close to the melting temper-ature so that the polymer can flow easily. DSC data for PLAwere used in this study to determine the optimal temperaturesat which to extrude fibers.

F. Characterization of Polymers

1) X-Ray Diffraction: X-Ray Diffraction (XRD) is a tech-nique used to determine the size and atomic structure of thecrystalline domains of a material. For polymers specifically,X-ray diffraction may be used to determine the percent crys-tallinity of extruded fibers. The process involves shining X-rays at a sample. According to the sample’s particular crystalstructure, these X-rays reflect from periodic planes that diffractthe rays at various angles. These planes consist of associated

2

chains of a polymer. A detector situated posterior to the samplerecords the pattern formed by the diffracted X-rays as theystrike the detector’s surface. The detector remains in a fixedposition as it reads the diffracted X-rays at a solid angle of50°. A computer analyzes these data and displays a digitalimage that resembles concentric circles. The intensities andsections of these circles differ based on the structure of thematerial. These visual data may then be integrated to obtaingraphs of intensity vs. diffraction angle (2θ) or intensity vs.azimuthal angle (χ).

Diffraction patterns can also identify the orientation ofcrystallites within a fiber. Highly oriented fibers produce acharacteristic diffraction pattern that resembles a ring markedby symmetrical areas of high intensity alternating with lowintensity along the ring. As a result of many parallel crystalplanes diffracting light at virtually identical angles, thesediscrete areas of high intensity are produced. Significantorientation indicates a highly organized material, but doesnot indicate the exact quantity of crystalline domains present.Percent crystallinity and crystallite size are quantified based onplots of intensity vs. 2θ. These data are related to a material’smechanical properties, including texture and flexibility [10].

2) Mechanical Testing: The mechanical properties of amaterial indicate how the material will respond when forceis applied to it. They can be used to describe the elasticityand strength of a material. Knowing the mechanical propertiesof a polymer is especially important when integrating thatpolymer into a biomedical device. A biomedical device mustexhibit certain mechanical characteristics depending on itslocation and function within the human body. Often, theoptimal material to be used in a particular device is determinedbased on the material’s mechanical properties [11].

Tensile strength testing is a method of determining apolymer’s mechanical properties. Specifically, tensile strengthtesting determines a polymer’s ability to withstand tension.In this process, strands of polymer are cut and clamped inan electromechanical testing machine. The machine applies aconstant stretching force on the strand until it breaks. Thespeed at which the machine’s arm pulls on the strand isadjustable. The machine records load, or force, and extensionof the fiber. These data can be used to calculate stress, or thetension applied to the fiber, and strain, the percent extensionof the fiber. Young’s modulus is the ratio of stress to strain,with a lower Young’s modulus corresponding to a more pliablematerial [11].

Typically, nerve conduits are flexible in order to allow themto mimic the pliability of biological nerves and tolerate theirdynamic movements. Therefore, flexible polymers are idealin the construction of a nerve conduit. In this study, tensilestrength testing was conducted on PLA fibers extruded atdifferent spooling speeds in order to determine how spoolingspeed affects fiber elasticity and pliability. This information isuseful in determining how to fabricate a nerve conduit.

G. Properties of Polymers Used: TPU and PLA

During the fabrication process, thermoplastic polyurethane(TPU) and poly(lactic acid) (PLA) were considered. They wereselected due to their ability to produce a flexible, nonkinkable,and bioresorbable nerve conduit suitable for 3D printing.

1) Properties of Thermoplastic Polyurethane: Thermoplas-tic polyurethane (TPU) is an extremely versatile and flexibleelastomer. A thermoplastic is defined as any plastic polymericmaterial that becomes soft and pliable at elevated tempera-tures. This makes it a great choice for 3D printing. TPU isan ideal polymer for medical applications due to the spectrumof properties it possesses. It can be made soft and pliable orrigid and strong. This is due to the fact that TPU is a blockcopolymer composed of an alternating sequence of rigid andsoft molecular segments. The ratio of hard segments to softones can be modified to increase or reduce the rigidity of thepolymer. Regardless of its high degree of flexibility, TPU isresistant to abrasion, making it capable of withstanding thepressures of the human body.

2) Properties of Poly(lactic Acid): Poly(lactic acid) (PLA)is a biodegradable polymer that differs greatly from thermo-plastics such as TPU in its properties. Additionally, PLA ismade entirely from plant-based renewable resources such assugar cane and corn starch. This property is invaluable asit makes the material bioresorbable and suitable for massproduction. Furthermore, the relative ease with which PLAmelts and cools makes it the most popular filament for 3Dprinting [12]. In view of these benefits, and those of TPU,both PLA and TPU can be utilized in the fabrication of thedevice presented in this study[13].

H. Current Applications of Nerve Conduits

Two of the primary methods used in the biomedical industryfor fabricating tissue scaffolds are self-assembly and fiber-based processes. In self-assembly, the fibers of the scaffoldsare designed using peptides that are synthetically engineeredto possess amphiphilic properties, enabling water to drive thefibers to form a scaffold in solution. In weaving, fibers pro-duced through the extrusion of polymers are woven togetherto create a flexible and nonkinkable nerve conduit [16].

Fibers can be extruded through the use of heat and pressureand then used to make nerve conduits. The draw speed andheat at which a polymer is extruded impacts the organizationof the polymer chains. Taking advantage of this fact, fibers canbe extruded at certain speeds and temperatures to give themspecific properties. Fibers can also be extruded through theuse of electrospinning. Electrospinning produces fibers on thenanoscale by utilizing electric forces to pull charged threadsof melted polymer or polymer in solution. The resulting fibersrange in diameter from 3 nm to 1µm.

While these processes certainly produce suitable conduits,it takes time and makes mass production rather difficult. Onthe other hand, the proposed model for 3D printing a nerveconduit is far less labor-intensive and presents a possibilityfor more efficient and personalized production. While thistechnique is rather limited by the large size of current extrusion

3

nozzles, the advent of smaller extrusion nozzles and moreadvanced extrusion techniques can allow for more refined andfeasible models to be developed at much faster rates thancurrently possible. Additionally, the use of CAD will allowmedical practitioners to 3D print customized conduits designedto target their patients’ needs. The usage of 3D printers will notonly make production faster but also increase the effectivenessof nerve conduits.

I. Cost

The density equation was utilized to estimate the approxi-mate cost of producing a nerve conduit. First, the total volume(0.264 cm3) of the conduit was split in half to find the volumeof each material individually (0.132 cm3). Then, the volumeof each material was multiplied by each material’s respectivedensity. The density of PLA is 1.25 g/cm3 [17]. Additionally,the density of TPU is 1.20 g/cm3 [18]. Therefore, the massof PLA in the model is 0.165 g (0.000165 kg) and the massof TPU in the model is 0.158 g (0.000158 kg). Since the costof PLA is roughly $10 to $20 per kilogram and TPU costsroughly $40 to $50 per kilogram, the cost of the materialsfor a 3D printed conduit would conservatively be $0.01 [19].The actual cost, which includes labor, testing, development,validation, and quality assurance, is significantly higher thanthe cost of the materials.

III. EXPERIMENTAL PROCEDURE

In order to find the most viable material for constructinga nerve conduit, the following tests were performed: tensilestrength testing and X-ray diffraction. These tests were essen-tial in predicting the behavior of the device. Following thisprocess, functional devices of various designs were fabricatedusing a 3D printer and various CAD models.

A. Extrusion of Polymers

The first experiment conducted was the extrusion of PLAfibers. In this study, a capillary rheometer was utilized insteadof an extruder. The polymer resin, PLA 2003D, was placed in aRH2000 Capillary Rheometer. The rheometer heated the resinto its molten state. The plunger then pushed the molten resinout of the die, forming long polymeric fibers. The extrudedfibers were wrapped around spools at variable speeds using afiber winding device (CS-1941 Fiber Take Up). Fibers wereextruded at 160 °C and 170 °C. These two temperatures weredetermined using a DSC scan of PLA (Figure 2).

The temperature at which the PLA begins to transition froma solid to a liquid is around 150 °C, reaching a molten stateat around 180 °C. Thus, the two extrusion temperatures wereselected so that the PLA was malleable enough to be extrudedinto a fiber.

In the experiment, the main variable was the spoolingspeed at which the fiber-winding device wrapped fibers aroundspools. The dial had 24 different speeds, which were convertedto meters per minute using a tachometer, a device used tomeasure the rotational speed of an object.

Fig. 2: DSC scan of PLA [9]

The varying spooling speeds and temperatures affected thediameter of the collected fibers. The diameter of each fiberwas measured using a micrometer.

Throughout the extrusion process, the rate at which themelted polymer was extruded should have ideally remainedconstant. Equation 1 shows how the volume per unit time of asingle cylindrical fiber can be represented by an equation forthe volume of a right cylinder:

Vh = π(d2 )

2 (1)

Where h represents the length of the fiber, V represents thevolume of the fiber, and d represents the diameter of the fiber.Changing the spooling speed directly affects the height of thefiber, allowing more polymer to be extruded per unit of time.Therefore, changing spooling speed is represented by a changein height in the equation above. Based on that equation, it washypothesized that spooling speed and fiber diameter have aninverse-square relationship, meaning that the diameter squaredis inversely proportional to the spooling speed.

B. X-Ray Diffraction

A representative sample of six of the fibers produced viaextrusion were selected for X-ray diffraction. Three of thesefibers were extruded at 160 °C and different spooling speeds(0, 4, 24.4 m/min). The other three fibers were extruded at 170°C and at one of the aforementioned speeds each. Individualmounts for each sample were prepared by punching holes in asmall section of cardboard. Segments of each fiber were thenpositioned across the hole and fastened to the cardboard, asshown in Figure 3.

A sample was then placed vertically in the holder in thediffractometer chamber in order for the X-ray beam to passthrough the sample. The PLA fibers extruded at 160 °C anddifferent spooling speeds (0, 4, 24.4 m/min) were tested intheir initial state and after annealing at 120 °C for 3 hours. ThePLA fibers extruded at 170 °C and extruded at the respectivedifferent speeds were also tested before and after annealing.

4

Fig. 3: Positioning of PLA fibers during X-ray diffraction

The fibers extruded at 160 °C and spooling speeds of 4 m/minand 24.4 m/min were again tested after tensile strength testing.

A Bruker Vantec-500 area detector and a FR571 rotating-anode X-ray generator operating at 40 kV and 45 mA wereutilized to obtain X-ray scattering from each PLA sample. Thesystem used a 1.0 mm pinhole collimation and a Rigaku Osmicparallel-mode (e.g., primary beam dispersion less than 0.01° in2θ) mirror monochromator (Cu Kα; λ = 1.5418A). A heliumenvironment was used to reduce air scatter and absorption.Data collection occurred at room temperature (25 °C) withthe sample remaining stationary and the detector positioned23.5 cm behind it. Spatial calibration and flood-field correctionfor the area detector were performed at this distance prior todata collection. The 2048x2048 pixel images were collectedat the fixed detector (2θ) angle of 19° for 10 min with ω stepof 0.00°. For the intensity versus 2θ plot, a 0.02° step, bin-normalized χ integration was performed on all images withsettings 5° < 2θ < 32° and -130° < χ < -50°.

C. Tensile Strength Testing

Tensile strength testing was performed on two fiber samplesto determine their ability to withstand stressors. The firstsample tested was a PLA fiber that was extruded at 160 °Cand a spooling speed of 4 m/min. Four specimens, measuredto be approximately 50 mm long, were clamped individuallyinto an Instron 5869. This device measured both the extensionof the fiber and the load, or force, being applied upon it.This machine was used to stretch each fiber until failure.The elongation rate of the fiber was 5 mm/min for the firsttwo specimens and 10 mm/min for the fourth specimen. The

third specimen was not pulled properly due a failure of themachine’s clamps.

Load and extension data were collected and plotted in astress vs. strain graph. Although four specimens of the firstsample were tested, the fourth specimen was the only one thatwas tested until failure. The calculations for specimens onethrough three were therefore incomplete.

Another PLA fiber extruded at 160 °C with a spoolingspeed of 24.4 m/min was also tested for tensile strength. Threespecimens of this fiber were measured by means of the sameprocedure employed for the aforementioned specimens. Thefirst specimen of the second sample was tested using a loadcell speed of 10 mm/min while the remaining two were testedat 20 mm/min. It is essential to note that these speeds wereonly altered to accelerate the measurement process, and thischange has no effect on the actual data because the strain ratesused were quite small.

D. Three Dimensional Modeling

In order to 3D print a nerve conduit, multiple different mod-els were fabricated on Fusion360. Multiple concept modelswere created. The first model constructed utilized coils withcircular cross-sectional radii of 0.25 mm. Multiple coils of thesame specification were stacked vertically in a circle with aninner diameter of 5 mm to form a conduit with a height of50 mm. While this structure reduces the risk of kinking, itrequires only a small amount of shear force to break due tothe lack of reinforcement between the coils.

Another model was created with the same specifications;however, instead of utilizing coils, circles were stacked verti-cally on the YZ plane and then revolved around the Z-axisto form rings stacked on top of each other. This formeda ribbed conduit, as shown in Figure 13, which exhibitedincreased flexibility and minimized the amount of materialrequired; however, similar to the first model, the lack ofreinforcement between the rings made the structure too weakto be commercially practical.

These models could only utilize one material due to the factthat each model was created in a singular STL file. 3D printerscan only print multiple materials if multiple STL files withdifferent aspects of the model are created. Each file representsa different material of the model. When printing, the files arestitched together to form a conduit that superimposes bothdesigns into one model with multiple materials. Since it wasdetermined that a combination of polymers would be requiredto fabricate a nerve conduit with all the desired characteristics,new models were designed with this process.

The updated design utilized two different materials, PLAand TPU, to create a helical structure. First, two squareswith side lengths of 0.5 mm were sketched onto the YZplane, both 2.5 mm to the left and right of the Z-axis. Thesesquares were then revolved 90° around the Z-axis. The twobodies were copied, translated 0.5 mm upwards in the Z-axis,and rotated 5° around the Z-axis. This process was repeateduntil a model with a helical structure of height 64 mm wasfabricated. As shown above in Figure 4, the model leaves

5

Fig. 4: Section of CAD model for a dual material, porous, helicalnerve conduit

empty space for another material. An additional variant wasdeveloped by simply rotating the pre-existing model 90 °.The step-like coiling of the model allows the two materialsto lock together so the stitched together STL file does not fallapart when twisted. This torsional rigidity came at the cost ofthe flexibility achieved with the ribbed design; however, thestructural integrity of the conduit was deemed more importantthan the small amount of extra flexibility provided by aribbed structure. If the conduit fails, then the entire surgicalprocedure will be rendered useless. However, a slight decreasein flexibility would not have any large effects. Another modelwhich oriented the two materials as layered rings was created,as shown in Figure 14. This model was developed by sketching50 squares with sides of 0.5 mm in the YZ plane 2.5 mm to theright of the Z axis. Each square was placed above one anotherwith a gap of 0.5 mm between them. The squares were thenrevolved 360° around the Z axis to produce rings with a 0.5mm gap between them. The corresponding model for the othermaterial was created by using the first model and translatingit up 0.5 mm in the Z-axis.

While these models produced the correct combination offlexibility and biodegradability with the simultaneous use ofPLA and TPU, they neglected to address the need for thenerve conduit to be porous. The existing models would notallow the exchange of nutrients to the regenerating axons, thusrendering the conduit ineffective. To address this issue, thenext model included small holes that would allow the exchangeof nutrients, but still provide a clear path of growth for thedamaged nerve. The model started the same way as the modelpresented in Figure 4, with two squares with sides of 0.5 mmrevolved 90° around the Z-axis. However, instead of simplycopying this structure, translating it up 0.5 mm and offsettingit 5°, another two squares with sides of 0.5 mm were sketchedonto the YZ plane directly above the previous squares drawn.These squares were revolved 85° around the Z-axis instead of90, and then offset 5°. This pattern of long and slightly shorterbodies were layered on top of each other until a height of 64mm was reached. The complementary STL file used for theother material was, however, the same as the model displayedin Figure 4. The interlocking of the alternating step patternallows for the formation of small pores in alternating layerswhich permits the diffusion of oxygen and nutrients into theregenerating region.

IV. RESULTS

In the fabrication of biomedical devices, detailed analysisof the used materials is crucial. The various tests performed inthis study, including X-ray diffraction, polymer extrusion, andtensile strength testing, are extremely important in determininghow the device responds to its surrounding environment andany possible strains that might be placed upon it by the humanbody.

A. Extrusion - Processing

Five polymer fibers were extruded using PLA 2003D with-out being wound onto a spool. Seven fibers were extrudedat 160 °C and wrapped around spools at varying speeds,which were measured using a tachometer. Four fibers wereextruded at 170 °C and spun around spools at varying speedsto determine how these factors affect fiber diameter.

Fig. 5: Linearized graph of diameter vs. spooling speed at 160◦C and170◦C

Extrusion temperatures, spooling speeds, and fiber diam-eters were recorded. The data were graphed using Excel toshow the mathematical relationship between spooling speedand diameter. It was determined from the data that the inversespooling speed is proportional to the square of fiber diameter.This relationship is confirmed by the R2 value of the linearline of best fit being close to 1. The equivalence of theseexperimental data and the predicted relationship derived priorto the trials provided further confirmation of the relationship.

Additionally, the data indicated that an increase in theextrusion temperature resulted in a decreased fiber diameterwhen spooling speed was held constant. This is due to thereduction in the melted polymer’s viscosity when a highertemperature is applied. Consequently, when the fibers wereextruded at a higher temperature, the fluidity of the PLAincreased. This resulted, as shown in Figure 5, in a smalleraverage fiber diameter. These data are associated with a smallmargin of uncertainty due to slight variations in the spoolingspeed, plunger speed, and air pockets between the PLA pellets.

B. X-Ray Diffraction - Structure

The degree of crystallinity and degree of orientation of eachextruded PLA fiber was determined through X-ray diffractiontesting. The extrusion temperature, spooling speed, stretching,and annealing processes each fiber was subjected to produceddifferences in its crystallite size, percent crystallinity, andorientation. All as-spun fibers, fibers neither stretched nor an-nealed, produced nearly identical scattering images consistingof a single, low-intensity ring. This pattern, as depicted in

6

Figure 6, revealed that all such fibers were amorphous. Bycontrast, the annealed PLA fibers produced multiple, highintensity rings, as seen in Figure 7, indicating a greater degreeof crystallinity. The drawn fibers, which were stretched duringtensile strength testing, exhibited a medial degree of crys-tallinity, producing a different low intensity diffraction patternindicative of highly oriented crystallites within the fiber, asshown in Figure 8. The pattern consisted of two distinctregions of high intensity which corresponded to two clearlydefined peaks on the χ azimuthal angle vs. intensity plot forthis fiber, as shown in Figure 12. Also displayed in Figure12 are χ angle vs. intensity plots of as-spun and annealedfibers. The substantially smaller peaks produced by these fiberscorresponded to the uniform rings depicted in their diffractionpatterns. This comparison identified the drawn fibers as havingthe most organized arrangement of crystalline domains, whilethose of the as-spun and annealed fibers appeared randomlydispersed.

Both percent crystallinity and crystallite size were cal-culated to compare the quantity of crystalline domains ineach fiber. Percent crystallinity was determined by integrat-ing diffraction patterns to produce graphs of intensity vs.diffraction angle (2θ) for each sample. For each plot, the areabeneath the peaks was calculated as a percentage of the totalarea beneath the curve to quantify percent crystallinity. Thegraphs corresponding to the as-spun and drawn fibers exhibitedweaker, broader peaks, as shown in Figure 9. These fibers areamorphous. The sharp peaks of the annealed fibers, as shownin Figure 11, amounted to roughly 40% crystallinity for thesesamples, making them highly crystalline.

Crystallite size was determined based on the relative widthof the peaks shown on each diffraction graph. All as-spunfibers were amorphous, exhibiting roughly equivalent peaksslightly larger than 10° in width. These wide peaks, as shownin Figure 9, indicated that the unprocessed fibers had crys-talline domains smaller than 1 nm. Slightly larger crystallitesformed within the drawn fibers, which produced peaks roughly5.2° wide, as depicted in Figure 10. The annealed fibersshowed much slimmer, more defined peaks, as seen in Figure11, giving an average crystallite size of 26 nm and evidencingfairly good growth of crystalline regions. The presence of thispattern across all samples showed that the effect of annealingwas approximately the same for all fibers.

The processing methods that each fiber was subject toproduced differences in crystallite size and orientation betweenthem. Annealing produced fibers with the greatest number ofcrystalline domains, since heating provided ample time forthe folding processes by which crystalline regions form. As-spun and stretched fibers were not reheated, and thus remainedamorphous. Despite the relatively few crystallites present inthe drawn fibers, the stretching process aligned these crystal-lites in the direction of the applied force, producing orientedfibers. The extrusion process produced a similar effect, asrapid spooling speeds produced a slight pulling force whichcaused quickly spun fibers to display more orientation, if notcrystallinity, than fibers extruded at slower speeds. Orientation

TABLE I: Tensile Strength Testing Data.

Test Young’s modulus Yield FailureE(MPa) σ(MPa) ε(%) σ(MPa) ε(%)

4-4 0.048 1.19 0.078 1.41 10.4Sample 6 0.042 0.30 0.084 0.65 8.3

was also more pronounced in thinner fibers, as the crystallineregions that aligned represented a greater proportion of thetotal fiber volume, thus producing a greater effect relative tothicker fibers.

Taken together, these data revealed that annealing producedthe most crystalline fibers, with stretching producing morealignment than as-spun fibers, which were the most amor-phous. For the purposes of designing a nerve conduit, moreamorphous fibers were needed to achieve a design both pliantand nonkinkable. Thus, it was determined that fibers wouldbe used as-spun. Fibers extruded at 160 °C and spun atmoderate speeds (∼ 4 m/min) fit these parameters, makingthese processing conditions ideal for nerve conduit material.Fibers extruded in this way could mimic the range of motionof a nerve in order to best facilitate its regeneration.

C. Tensile Strength Testing - Properties

During tensile strength testing, two fibers extruded at 160 °Cand spun at different spooling speeds were used. These fiberswere chosen for their uniform thickness. Fiber 4, extruded at aspooling speed of 4 m/min, and fiber 6, extruded at a spoolingspeed of 24.4 m/min, were tested for tensile strength. Thosedata were recorded; however, tests 4-1, 4-2, and 4-3 were notincluded because of incomplete data. Tests 4-4, 6-1, 6-2, and6-3 were completed, and the data for fiber 6 were averagedfor further analysis. Figures 15-18 display the data pertainingto the following discussion.

Hooke’s Law (2) was applied in order to determine Young’smodulus,

σ = Eε (2)

where σ is stress, ε is strain, and E is Young’s modulus, theratio of stress to strain. The data were plotted as σ vs. ε , andYoung’s modulus was determined from the slope of the initiallinear portion of the curve. The slopes for sample 6 averaged0.042 MPa, while Young’s modulus for 4-4 was 0.048 MPa, asshown in Table 1. Young’s modulus is a measure of stiffness,indicating that sample 4 was stiffer compared to sample 6,which had a lower Young’s modulus.

The yield stress and yield elongation were determined fromthe yield point. At the yield point, the fiber ceases to be elasticand plastic deformation begins to occur. The relation betweenyield stress and spooling speed was analyzed. Sample 4-4 hada yield stress of 1.19 MPa, while sample 6 had an average yieldstress of 0.30 MPa. The average yield stress of sample 4-4,which was spun at 4 m/min, was higher than that of sample6, which was spun at 24.4 m/min. This indicates that lowerspooling speed results in higher yield stress, which means thatfor a fiber extruded at lower spooling speed, more force is

7

needed for elongation, supporting the conclusion that sample4-4 is stronger than sample 6.

Furthermore, the behavior of the stress vs. strain curvefollowing the yield point suggests information about theelastic behavior of the PLA fiber used. All four specimensshowed characteristics of a ductile material because the yieldpoint was followed by significant elongation. This indicatesthat the fibers did not immediately break upon yielding, butstretched to an extent and thinned out, a phenomenon knownas “necking,” before breaking.

The major event following the yield point is the point offailure, also known as the breaking point. This failure point iscomprised of the failure stress and failure elongation. Thesedata indicate that lower spooling speed produces fibers withhigher failure stress. Sample 4-4 had a failure stress of 1.41MPa, while sample 6 had an average failure stress of 0.65MPa. In other words, fibers spun at lower spooling speed arestronger. In addition, the failure strain of specimen 4-4 wassignificantly higher relative to that of sample 6, signifying thatsample 4-4 is stronger because more force and elongation wasneeded to trigger failure [14].

It was observed that for all tests, the break point of thePLA fiber was higher than the yield point. This phenomenonby which a material breaks at a higher stress and higher strainis known as strain hardening, in which there is a completeorientation of the polymer chains [15]. This demonstrated theability of PLA to remain strong under load far beyond its yieldpoint, making it capable of withstanding pressures within thehuman body.

D. Structure-Property Correlation

The structure of PLA fibers determined the specific mechan-ical properties they exhibited during tensile strength testing.As such, the features of the stress vs. strain plots generatedduring tensile strength testing could be explained using the X-ray diffraction patterns produced by as-spun fibers and drawnfibers. The values for Young’s modulus calculated from theinitial portion of stress vs. strain graphs indicated that as-spunfibers were ductile. Accordingly, the uniform, low-intensityrings displayed in the diffraction patterns of these materialsshowed that they were amorphous, explaining their behaviorat this point in the trial.

Other diffraction patterns of as-spun and drawn fibersshowed differences that explained the structural changes oc-curring within the fibers during the subsequent period ofelongation. As shown in Figure 19, the drawn fibers exhibiteddiffraction patterns consisting of discontinuous rings, indicat-ing orientation of crystallites within the fiber. This is consistentwith the drawing process, which introduces tension in theamorphous strands of the fiber such that its crystalline domainsalign. This additional ordering within the fiber explains theincrease in strength the fiber showed just before its breakingpoint. At the yield point, the amorphous, randomly orientedfibers yielded relatively quickly since only weak dispersionforces acting between the chains were holding the fiber to-gether. Once elongation oriented the fibers in the direction

of the load, the force acted against the strong covalent bondsbetween units in the polymer chain which required more forceto overcome. Hence, the increase in the fiber’s strength duringtensile strength testing correlates with the X-ray diffractiondata and the structural differences between the as-spun anddrawn fibers.

In the same way, the X-ray diffraction patterns producedby the annealed fibers explained their behavior under stressby simple handling. Uniform, high intensity rings marked thediffraction patterns of annealed fibers, indicating that thesefibers formed crystalline domains that were not oriented. Thishigh degree of crystallinity explained the hardness of thefiber. When held, annealed fibers felt more brittle than as-spunfibers of the same sample. Annealed fibers also broke underpressure, whereas as-spun fibers bent. From these data, tensilestrength testing results can be predicted for annealed samples.Compared to the as-spun fibers, annealed fibers would havea higher Young’s modulus, indicating a greater degree ofstiffness. The yield point of an annealed fiber would also bemuch higher than that of an as-spun fiber spun at the samespeed, indicating increased strength. However, it would breakalmost immediately after its yield point due to its increasedcrystallinity and, consequently, decreased ductility, behavingsimilar to glass rather than plastic.

E. Final Conduit

The final design, as shown in Figure 20, employs a helicalstructure which utilizes the flexibility of TPU and biodegrad-ability of PLA to fabricate a nerve conduit that will breakaway from the nerve and be absorbed by the body upon thecompletion of the regeneration process. This staggered helicaldesign also allows for the printing of pores around the entiretyof the nerve conduit. Relative to previous models, this designis the most efficient due to these benefits. The present problemwith printing this nerve conduit is the relatively large size ofcurrent extrusion nozzles used by 3D printers compared tothe conduit. The small size of the model makes it difficult toproduce a conduit that adheres to the parameters establishedby the CAD model to a high degree of accuracy.

In spite of these challenges, data from PLA testing wereused to achieve maximum efficiency during the 3D printingprocess. Although the 3D-printed conduit was not made outof extruded PLA fibers, the extrusion process is similar to 3Dprinting. The two main variables that affected fiber propertiesin this study, extrusion temperature and spooling speed, canalso be altered in a 3D printer. The resin can be heated upto various temperatures, as it is in extrusion. Furthermore,changing spooling speed during extrusion can be equated tochanging the speed of the 3D-printing hand. Changing pressureof the 3D printer’s nozzle is also an option, which is similar tochanging the plunger speed in the extrusion, but plunger speedwas not a variable considered in this study Although mostlow-end 3D printers have fixed settings, many 3D printerscan change settings, allowing for the fabrication of morecustomizable conduits.

8

V. CONCLUSIONS

The data obtained from the trials suggested that thin, as-spun PLA fibers extruded at high temperatures are suitablefor nerve conduits with the desired pliability. X-ray diffrac-tion showed that as-spun fibers are amorphous. Tensile testsshowed that the fibers with small diameters were the mostpliable. Extrusion experiments demonstrated that extrusion athigher temperatures and speeds produces thinner fibers.

The final design of the nerve conduit integrated PLA inways that best leveraged its properties. Using TPU in con-junction with PLA was one method of doing so, as the twomaterials complemented each other in ways that provided bothstrength and flexibility. The structure of the conduit incorpo-rated elements that achieved the remaining specifications of anideal conduit: nonkinkability and porosity. The coiled structureof the conduit ensured that the device would be nonkinkable,while also having small gaps that would allow for the passageof nutrients.

A. Future Work

In view of the limited timeline and resources available forthis project, further procedures that could enhance the outcomestill remain. With more time, it would be possible to conductmore extensive testing on the final nerve conduit. Observingthe device’s response to different pHs and compression test-ing would provide more concrete methods of predicting itscompatibility with the human body and ample opportunity torefine any shortcomings.

Additionally, medical grade polymers, such as Strataprene3534 which is inherently semi-porous, could be utilized toeliminate the need to produce complex structures such as thehelical design. This would effectively address the issue createdby the limited resolution of current 3D printers.

Although the final conduit may have some limitations, itrepresents the next frontier of regenerative medicine. The useof developing technologies will be crucial in decreasing thecost of treatment while also maintaining the same level ofefficacy. Innovation is key in improving the prognosis of allpatients across all groups.

VI. ACKNOWLEDGMENT

The authors gratefully acknowledge Dr. Sanjeeva Murthyand Dr. Thomas Emge of Rutgers University for the entiretyof their guidance throughout the experimentation process. Dr.Sanjeeva Murthy, Associate Research Professor at the NewJersey Center for Biomaterials at Rutgers University, providedguidance and knowledge on past and current research ofbiomedical devices. His wide array of expertise in the fieldwas crucial during both the conception and development ofour device. Furthermore, his lectures on the various propertiesand applications of polymers were essential in the selection ofthe polymers used for this project. Additionally, Dr. Emge pro-vided a myriad of information on X-ray diffraction techniques.The analysis performed upon our selected polymer using thisprocess helped to determine its properties and ascertain that itwould meet the physical demands that would be placed upon

the product. The authors would also like to thank their projectliaison, Megan Brown, an aerospace engineering student atPurdue University, for her assistance, planning skills, andadvice. Her constant support of our endeavours has beeninvaluable throughout the process of completing this researchpaper. Lastly, the authors of this paper gratefully acknowledgethe following: Dean Jean Patrick Antoine, the Director ofGSET for his management and guidance; Research Coordi-nator and residential teaching assistant Helen Sagges for herassistance in conducting proper research; Michael Higgins,head residential teaching assistant, Rutgers University, RutgersSchool of Engineering, and the State of New Jersey forthe chance to advance knowledge, explore engineering, andopen up new opportunities; Lockheed Martin and the NewJersey Space Grant Consortium for funding of our scientificendeavours; and lastly NJ GSET Alumni, for their continuedparticipation and support.

9

VII. APPENDIX

Fig. 6: As-spun PLA fiber extruded at 160 °C with speed 4 m/min

Fig. 7: Annealed PLA fiber extruded at 160 °C with speed 4 m/min

Fig. 8: Drawn PLA fiber extruded at 160 °C with speed 4 m/minshowing preferred orientation

Fig. 9: X-ray diffraction of as-spun PLA fibers

Fig. 10: X-ray diffraction of drawn PLA fiber 4-4

Fig. 11: X-ray diffraction of annealed PLA fibers

Fig. 12: Chi plot of as-spun (black), annealed (blue), and drawn (red)PLA fibers

10

Fig. 13: Zoomed view of coil model

Fig. 14: Midsection of dual material ring model design

Fig. 15: Stress vs. strain graph for sample 6-1 with Young’s modulus

Fig. 16: Stress vs. strain graph for sample 6-2 with Young’s modulus

Fig. 17: Stress vs. strain graph for sample 6-3 with Young’s modulus

Fig. 18: Stress vs. strain graph for sample 4-4 with Young’s modulus

Fig. 19: Stress vs. strain graph for sample 4-4 with X-ray diffractionfor samples that were as-spun (shown after yield point) and drawn(shown where strain hardening occurs)

11

Fig. 20: CAD models of porous, helical nerve conduit made of PLA(pictured left) and TPU (pictured right) that were overlapped to formthe final conduit

REFERENCES

[1] Zhu, W., Tringale, K. R., Woller, S. A., You, S., Johnson, S., Shen,H., ... Chen, S. (2018). Rapid continuous 3D printing of customizableperipheral nerve guidance conduits. Materials Today, 21(9), 951-959.https://doi.org/10.1016/j.mattod.2018.04.001.

[2] Griffin, M. F., Malahias, M., Hindocha, S., and Khan, W. S. (2014).Peripheral Nerve Injury: Principles for Repair and Regeneration. TheOpen Orthopaedics Journal, 8, 199-203.

[3] Huebner, E. A., and Strittmatter, S. M. (2009). Axon Regeneration inthe Peripheral and Central Nervous Systems. Results Probl Cell Differ,48, 339-351.

[4] Gaudet, A. D., Popovich, P. G., and Ramer, M. S. (2011). Wallerian de-generation: gaining perspective on inflammatory events after peripheralnerve injury. Journal of Neuroinflammation, 8.

[5] Miranda, G. E., and Torres, R. Y. (2016). Epidemiology of TraumaticPeripheral Nerve Injuries Evaluated with Electrodiagnostic Studies in aTertiary Care Hospital Clinic. PRHSJ, 35(2), 76-80.

[6] Tezcan, A. H. (n.d.). Peripheral Nerve Injury and Current TreatmentStrategies. InTech, 1-31. http://dx.doi.org/10.5772/intechopen.68345

[7] Mayo Clinic Staff. (2017, November 18). Peripheral nerve injuries.Retrieved July 15, 2019, from Mayo Clinic website:0355631.

[8] Powers, D. (n.d.). Implantation of a nerve conduit [Photograph].[9] Differential scanning calorimetry (DSC) comparison of PLA spool

material and printed PLA. [Illustration]. (2017, Jun).[10] ”Basics of X-Ray Powder Diffraction,” Scott A. Speakman, 2011. PPT

file available from MIT and the author.[11] Murthy, S. (Presenter). (2019, July 2). Polymers in regenerative

medicine: Fabrication of scaffolds and optimization of their structure.Lecture presented in Rutgers University, New Brunswick, NJ.

[12] What is thermoplastic polyurethane (TPU) [Fact sheet]. (n.d.). RetrievedJuly 17, 2019, from Lubrizol website.

[13] Rogers, T. (2015, October 7). Everything You Need To Know AboutPolylactic Acid (PLA) [Fact sheet]. Retrieved July 17, 2019, fromCreative Mechanisms website.

[14] Christensen, R. M. (2011). Defining Yield Stress and Fail-ure Stress (Strength). The Theory of Materials Failure, 118-132.doi:10.1093/acprof:oso/9780199662111.003.0009

[15] Crow. (2015). Polymer Properties Database.[16] Norman, J. J.; Desai, T. A. (January 2006). ”Methods for fabrication

of nanoscale topography for tissue engineering scaffolds”. Annals ofBiomedical Engineering. 34 (1): 89–101.

[17] Flynt, J. (2019, March 19). What is the Density of PLA. Retrieved July24, 2019, from 3DINSIDER website.

[18] TPU 1.75 mm 3D Printer Filament. (n.d.). Retrieved July 24, 2019, fromRigid Ink website.

[19] How Much is 3D Printing Filament Cost. (n.d.). Retrieved July 24, 2019.

12