ELBCTROSPliN NERV GUIDES FOR NERVE REGENERATION DESIG ED TO

M DULATE ERVE ARCHITECTURE

DESCRIPTION

BACKGROUND OF THE IN VENTION qft ^' U invention

The in venlion generally relates to nerve guides for use in nerve regeneration. I particular, the invention provides nerve guides which, a e formed . from three dimensional (3D) arrays of highly alj gned electrospun fibers thai are oriented in. parallel with, the l axis of the seamless and cylindrrcally shaped constructs. Gaps and elongated spaces between the stacked fiber arrays provide channel ^' s for directed agonal growth..

Background qf ^' ihe Invention

A peripheral nerve is an enclosed, cable-like bundle of periph eral axons (Song, slender rojections of neurons, see Figure 1). .A nerve provides a common, pathway for the electrochemical nerve impulses that are trarisrrhtted along each of the axons. Each nerve is a cordliFe structure that contains many axons. These axons are often referred, to as "(nerve) ^• fibers". Within, a .nerve, each axon is surrounded by a layer of connective tissue called the endoneuiiuni. The axons are bundled together into groups called fascicles, and each fascicle is wrapped in. a layer of connecti ve tissue called the periiieurium. Finally, the entire nerve is wrapped in a layer of connective tissue called the epineuriurn. Herein, "nerve" and ¾on" my be used interchangeably.

After an injur , peripheral nerves can undergo an astounding degr-ee of regeneration,: When a nerve i severed, all signals distal to the injury site are iniffiediateiy lost. Over tiros, downstream axons undergo Walleri an degeneration [!]. The surviving nerves of the proximal segment subsequently begin, undergo regeneration in response to soluble factors, man of which are produced by Schwann, cells [2,3], If die precipitating ipinry c!eaoly severs the nerve, p-eaim : ma he confined to a surgery that is designed to re-establish the continuit between the tnmcated stomps of the damaged nerve. In thi s surgery the proximal: and the distal aspects of the perineural sheath are sutured together to form an end-to-end anastomosis. In more extreme injuries where a long segment of the erve is cras ed or eonipletely lost., treatment is greatl complicated. Under these conditions a conduit or nerve guide, is used to bridge the gap and direct the regenerating axon to grow towards the distal stump.

Nerv guides in the peripheral nervous system have a relatively long .clinical history; these tubular constructs are designed, to direct the natural processes that lead to re eneration [4,5]. A variety of nainral and. bioengmeered materials have been irsed, in this type of applieationrwiih. mixed success [6], l¾rly synthetic guides consisted, of a simple, hollow tube thai provided little nlore than a protected environment [7.1. With the hollow core desig regenerating: axons literally spill out of the proximal erve: stump: nd grow down the conduit. This not-so subtle "spilling effect ^' ' jumbles normal nerve topograph (this term refers to the relative position of the individual axons to eanh other within the nerve) and. greatly reduce the efficiency, and fidelity, of axon targeting.

Next generation peripheral nerve guides have been lubricated to contain signal molecules [8] and/or structural features [9] that are intended to provide guidance cues to d e regenerating axons. Functional recovery with, these constructs can be quite extensive, as long as the nerve guide is used, to bridge a gap of less than about if) mm in. length. Once ihe injury gap exceeds tins threshold, the regeneration process will be eomproinised to varying degrees. Typically, in these injuries only a limited number of xons will actually traverse the wound bed and th efficiency of targeting to the distal tissues is poor, resulting in. limited functional recovery. Further exacerbating these complications, a series of irreversible degenerati e changes begin to evolve in the distal tissues. As these degenerative changes become entrenched., the prospects of raeaningli functional recovery are greatly diminished, even if a large number of axons are efficiently targeted, to these sites.

Despite extensive and continued, development, the autologous nerve segment represents; the state of the art treatment for long defect nerve injuries. This tissue is highl anisotropic and. its native architecture provides many potential channels to guide

•regenerating axons across, the wound bed. However, use of this autologous tissue comes at the expense of donor site morbidity and results in the transplantati on of a "nerve guide" that is packed with axo.nal ffapients. These fragments must first degenerate before the regenerating axons ears penetrate into the remaining endoneurium of the tissue. This degeneration process slows regeneratio by initially impairing the penetration of the nascent axons into the autograft, a complication that clearly exacerbates the effects -of long term m use le de -innervati o : and. tissue atrophy.

man ways there are similarities between peripheral nerve and the spinal cord. Both ha a highly organized anisotropic structure and axons organised into specific topographical relationships. One cleat; difference that distinguishes spinal cord tissue ..from the peripheral nervous system is the observation that the spinal cord has axons that travel froni the brain downward to the bas of the spinal cord and out into the peripheral ti ssues as well as axons thai travel from, the peripheral tissues upwards towards the brain. For complete regeneration to occur after spinal cord injury ^' both -aspects -of this "two w y trafficking" of axoiis must be restored. Spinal cord injury (SCI), in both human insu!ts and animal models, results in the- Uqu&factive necrosis of the tissue in and around the lesion site-. A fluid-filled cyst commonly emerges as the final consequence of this process (Figure 2), Due to the lack of a solid, substrate, this late-stage pathological e dpoint -represents a physical gap that impedes axonal regeneration and iunciional recovery. Additionally,, astrocytes within. the injury- site proliferate, -hypertrophy and begin to express ehondroitin sulfate proteoglycans (CSPGs), which represent potent inhibitor to axon regeneration. At the neuronal level, asoioiny of motor and sensory neurons results ^' in the loss of trophic support by target tissue, which exasperates neuronal cell death. Additionally, the presence of myelin- debris, as a result of oligodendrocyte death, acts as a potent inhibitor to axon regeneration, i n the spinal cord. Collectively, these cellular responses to spinal cord injury represent major

impediments to axon regeneration and functional recovery

Mono -therapies that address each of these obstacles to regeneration individually have resulted, in only limited, axonal regrowth ami functional recovery. To date, no single intervention has been devised to collectively address all of the known obstacles to axon regeneration in. the spinal cord.

Several, studies -have demonstrated that aligned arrays of eleetrospun fibers can provide the guidance cues necessar to induce axons and glial cells to express a highly polarized phenotype [10-13]. Despite these preliminary and encouraging results, it is difficult to iabdcate a. clinically relevant nerve guide using the conventional electrospinning process which, uses a. rapidly rotating target mandrel to produce aligned fibers. Conventional, eleetrospinfting systems are very effective at producing flat, 2B sheets- with highly anisotropic fibers [14-1.6]. These constructs are easily amenable to experimentation in vitro, however, a 2D sheet is less acceptable, or adaptable, tor use in vivo. A flat 2D sheet can be spun into a very thick structure and then cut into strips thai resemble "square cylinders." These structures are technically seamless,, ^' however, fiber alignment tends to degrade the thicker a sheet becomes when fibers are processed unde conventional 2D clectrospinning conditions, Cutting. : ^' such a sheet also distorts the existing ahgnnient along the cutting plane. Also, in conventional electrospinning systems fiber alignment as induced by a rotating target mandrel is far more dependent upon fiber diameter than in the air gap system. In part this is because air currents and complex electric fields induced by the rotation of the mandrel disturb the trajectory of the charged jet, fiber flight and fiber deposition onto the target, thereby limiting the extent of alignment that can be achieved. Air gap eieetrospinning does not require any mandrel movement to induce fiber alignment, _, making it possible to produce aligned fiber arrays even when the polymer concentration is at the minimal threshold necessary to produce fibers. This allows highly aligned constructs to he fabricated from individual fibers of less than 200 microns in average cross sectional diameter, a size scale tar smaller than can be achieved in conventional systems. Despite these limitations, the efficacy of using the conventional e!ectrospinning process to fabricate hollow, cylindrical nerve guides has been explored to some extent. n these experiments the eleetrospu fibers have been deposited onto a round, rotating mandrel. While the fibers of this type of construct can be induced to exhibit a considerable degree of alignment when produced under these conditions, the fibers, unfortunately, are deposited onto the target mandrel in a

circumferential orientation the axis of alignment is— 90* off with respect to the .resulting _, long axis of the hollow tube). While the nano-to-micron diameter fibers that form the wall of this type of construct do represent a barrier that reduces the risk of intlamrnaior cells penetrating into the hollow lumen of the guide (critical to the regeneration process [17,18]}, this architectural pattern (he, the ar angement of eleetrospun fibers in a circumferenti al pattern) does not lend itself well to pro viding directional guidance cues to regenerating axons.

Other attempts at making nerve guides from eleetrospun (and other) types of fibers generally involve roiling sheets of material to form tubes. Uhforttinately. the resulting rube are hollow and thus fail to mimic the architecture of natural nerve growth. Further, a flat sheet that is rolled mto a tube must, of necessity, have "seams" where the edges of the rolled sheets ar surface exposed, e,g, on the wall of the hollow center of the tube and/or oh. the surface of the tube _v rcsultmg in discontinuities aiid possible weak connective points in the structure, in attempts to produce a more autologous graft-like structure aligned sheets of electrospun materials and films have been prepared and rolled .tightly into more compact structures (32,33 _. ). (The final construct resembles a cinnamon breakfast roll where the electrospu sheets are represented by the dough, and the gaps or seams between die rolled Sheets are represented by the sugar and eimiame-u). While these structures are composed of "aligned fiber arrays" even these structures contain large seams (with respect to the size of the axo s), no matter how tightly they may be rolled during fabrication. These seams represent a potential nexus for mechanical failure and the infiltration by _. unwanted interstitial fibroblasts and or inflammatory cells. Rolling a sheet can not truly integrate the fibers on the nano-scale that is necessary to make a uniform set of "pores" The gaps represent large "eireunrferentially aligned longitudinal pores (geams)" for axons to grow along on the underlying aligned fibers. In a sense this type of design provides a larger ^5< 2Ι " surface area to guide the: gr wth of axons that ar growing along the seams of the rolled sheets.

US patent 6,031 ,148 (Hayes) discloses nerve guides made from roiled sheets of material which have hollow centers.

US patent 6,8 3 ,946 (Goldspinfc et al.) discloses administering growth factors to damaged nerves via a conduit of unidirecti.onal.1y oriented libers containing an alginate matrix. However, the conduits are rolled, sheets which form a tube with a hollow center that contains.: seams.

US patent 7374.774 (Bo iin) teaches electrospun materials with variou uses, e,g. as nerve guides. However, the nerve guides are formed by roiling sheets of fibers, and thus have a hollow center and once again contains seams as a consequence.

US patent application 20.1.0/0047310 (Chen et al,) discloses nerve guides comprised of biodegradable, biocompatible electrospun material However, the guides are made fron< sheets of several layers of conduits which are rolled into cylinders and are thus hollow,

US patent 7,727,441 (Yost et ah) describes a tubular tissue scaffold which comprises a tube having a wall, wherein the wall includes biopoiymer (collagen) fibrils that are aligned in a helical pattern around the longitudinal a is of the t be, _: and where the pitch of the helical pattern changes with the radial position in the tube wall. The scaffold s capable of directing the morpheiogical pattern of attached and growing cells to form a helical pattern around the tube walls, but would not be suitable for use as a nerve guide, where the axons must grow straight down the guide and ot wra or change topology during growth,

SUM ARY OF THE INVENTION

The invention provides nerve guides formed from electrospiin fibers for use in the directed regeneration of damaged or severed peripheral nerves. The technique of static air- gap electrospinning is used to generate cylindrical, seamless three dimensional (3D) arrays of highly aligned elecirospun fibers oriented in parallel with the long axis of the construct. The air-gap technique allows the fabrication of cylinders of aligned fibers without "roiling" a sheet of fibers, and thus «ø potentially weakening or irregular seams or inordinately large "pores" are present in the guide, and the guide does not contain a "hollow ^** centra! channel. Instead, the guide comprises multiple ope channels formed by elongated gaps between the fibers, and the channels are lined with aligned fibers that are oriented along the long axis of the guide and bounded by elongated, oriented fibers. The fibers o f these structures are integrated on a true naao-seale. And, unlike other nerve guides that may contain hundreds to thousands of channels that are designed to support ax n regeneration. A. similar sized cleetrospun 3D nerve guide (depending on its size and fiber characteristics) produced by air gap electrospinning may contain hundreds of thousands to tens of millions of indi vidual channels, each of which is "lined" with nano-tp-micron scale diameter fibers that are designed to provide guidance cues, When the: proximal end of a truncated nerve is introduced into one end o f the nerve guide, the nerve tissue grows into the guide and alon the channels in a directed manner. Thus,, an axon which grows ^" through the guide emerges from the other end of the guide at a desired, predictable location, e.g. in close pro xmn ty o the other, distal end of the severed nerve. This insures that the severed nerve ends will grow with a minimum of excess or random nerve tissue growth, permitting reeonnection of the severed nerve to its distal target tissues, and restoration of functionality. The nerve guides of the invention th s closely mimic and have the structural advantages of natural nerve grafts hut without the undesirable side effects of nerve disintegration _^ Expenmental results presented herein sho that animals treated with the nerve grafts of the invention showed levels of funetio«aS. recovery after 7 weeks that are normally observed in animals treated with autologous grafts after 10-14 weeks. In some embodiments, the nerve guides also somprise gradients of therapeutic substances.

It is an object of this invention to provide an architectural arrangement, that mimics the structure of the ri ls ve autologous graft by deposithig nano o~micron scale eiectrospun fibers into a seamless, cylindrical structure, The ax ns are ^■ guided to gr w in between the aligned fiber ar ays in the channels provided between the individual fibers. This design provides physical guidance cues to direct the axons to grow along a specific direction and along a specific plane order to reconstitute the topography of the nerve that existed before an injury. Maintaining normal topographical relationships is intended to increase the fidelity of axon targeting and increase the likelihood thai an axon will emerge from the distal end of die graft in a position that approximates its position, prior to injury, in turn, this type of regenerative patterns is. designed to increase the .probability that the regenerating axon returns to the disUtf target tissue (sensory and motor) that it innervated prior to injury, in addition, the seamless nature of the guide provides a far more uniform profile of pore spaces { i.e. the channels between the individual fibers) than a rolled sheet of material, reducing; ihe tendency of axons to cl uster and grow as a jumbled disorganized m ss along: the seams, a pattern of gro wth in a regenerating nerve which is associated with reduced axon targeting. Because the air gap eiectrospinning process is very rapid if is possible to place substances of interest, e.g. cells, therapeutic agents, gradient threads (as ^" described herein), etc, within the scaffold as the fibers are depositing into the fiber arrays. The supplementation of the guides with growth factors and/or cells, etc, can be used to further regulat the regenerative:

environment afforded by the scaffolds.

The invention thus provides a nerve guide comprising a plurality of eiectrospun fibers which are seamlessly aligned parallel to a. long axis of said nerve guide; and a ^' plurality of open channels aligned parallel to the long axis of the nerve guide. In some erabodinietns, the nerve guide further comprises a carrier thread comprising one or more therapeutic substances in a gradient, which may include at least one growth factor, in. some

embodiments, an outer sheath is present on the nerve guide.

The invention lso provides a method of facilitating (supporting, inducing, etc,) regeneration of a severed nerve. The method comprises the steps off) attaching a proximal stump of the severed nerv to a first end surface of a nerve guide and a distal end of the severed nerve to a second end. surface of the nerve guide; and ii) allowing (growing) axons of the proximal stom to grow within one or more of said plurality of open channels. The nerve guide comprises: a plurality of electrospun fibers which are seamlessly aligned parallel, to long axis of the. nerve guide, and a. pinrality of pen channels aligned parallel to the long axis of the nerve ^' .guide. The nerve guide further ma include ¾ carrier thread comprising on or more therapeutic substances in. a gradient, such as at least one growth factor, in one embodiment, the severed nerve is a peripheral nerve and the nerve: guide further comprises ari outer sheath, in another embodiment, the severed nerve is a spinal cord nerve.

The invention also p o ides a multi-segmented carrier thread, comprising a plurality of segments, ther in each segment comprises at least one c itiponeftt with a concentration that differs from eonce rations of the cornponent in adjacent segments. The total length of the niultl-segmented carrier thread is from about 5 mm to about 125 nun. A length of any one of the plurality of indi vidual segments is from, about 1 to about 1 ^' 0 mn% in one embodiment, the at least one component is alginate, in another embodiment, the at least one component is a therapeutic agent in another embodimeat, the niulti-seguMmied carrier thread further comprises roierabeads present i at least one of the plurality of segments, the microbeads containing an agent of interest.

The invention also provides a method of forming a polymerized step gradient, comprising the ^; steps of i) adding, to a mold, a first olymerizeable solution comprising at least one substance of interest to a moid; ti) freezing the mold with the first polymerizeable solution therein; iil) adding a second polyinerizeabje soiuiion comprisingtbe at least one substance of interest info the mold, wherein a concentration of the at least one substance of interest in die second polyinefkeable solution, differs from a concentration of the at least one substance of interest in the first polymerizeahle solufioa, and wherein the second, poiymemeable solution comes into direct contact with the fksfpolynieri eable solutio upon introduc tion into the mold; iv) ffeexing said moid with said first and second poiymerizeabie solutions therein; v) repeating steps i) 4v to form, a series of polytnerkeabfe solutions, wherein each successive poiymerizeab solution that is added to the mold in d e adding step comprises «·. different concentration of the at. least one substance of interest than a polymeri eabie solufion thai was added in mi immediately previous adding step; vt) removing the series of poly er&eable solutions from the mold; and viij exposing the series of polyiuerizeable solutions to conditions which -cause polymerisation of the series of polyroerizeabie solutions, thereby forming a polymerized step gradient. .¾ some

embodiments, the polymerizeahle solutions comprise alginate, and the conditions which cause poiyiuerizaiion include exposure to calcium. In other em.bodiments, the method further includes a step of curing said poiymeri¾ed step gradient, e.g, exposing the polymerized step gradient to iiexailuorisopropanol.

The invention also provides a method of fomiing fimctionai blood vessels along a regenerating severed spinal cord, nerve, comprising the steps of i) attaching a proximal stump of the severed spinal, cord nerve to a first end .surface of a nerve guide and a distal, end of the severed spinal cord nerve to a seeoird end surface of said nerve guide; and ii) growing endoiheiial ceils in the vicinity of the nerve guide the endoiheiial cells attaching to the nerve guide and forming functional blood vessels while axons of the proximal stump grow within one off .nio.re of the plurality of open, channels. The nerve guide comprises a plurality of eiectrospnn fibers which are seamlessly al igned para llel to a long axis of the nerve guide, an a plurality of open channels aligned parallel to the long axis of said nerve guide.

In one embodiment, the elecirospitn fibers of the nerve uide are formed from

polydioxanone,

BRIEF DESCRIPTION Of TH E DRAWINGS Figure 1. Peripheral nerve schematic. The epineurium invests individual axons, forming a collar of Type [V collagen and Iam.in.in. around each axon. The perineurium surrounds a small number of axons, ferming bundles. The tough outer epine ri rn surrounds the enih¾ nerve, (Stroneek and R.eieherf in Frontiers in Neuroscienpe; Indwelling Neural Implants, website located a ucbLnlm..nih,gov},

.Figure 2 A-C. Magnetic resonance imaging (MPJ) showing ihe presence of cyst (denoted by arrow) in. the cord, of a. human SCI patient; B, Schematic illustration of the cyst and axon damage that occurs in SCI (spinal cord. jtyury); C, Cyst present within a rat spina! cord 2 weeks after injury. The cyst is surrounded by astrocytes, cell bodies, and neurite growth inhibitors.

Figure 3A-D. Sc ma ic representations of features of the nerve g de of the inven ion. A, cross-sectional view of the nerv guide is depicted, showing, on the cross-sectional face, openings into the plurality of channels. The alignment of three of the channels parallel to the long axis of ihe guide is show (dashed lines), with arrows indicating the directio of axon growth along (Within) the channels; B, cross-sectional view of a single channel within a nerve guide showing a wall, of the channel; C, nerve guide with sheath; D, nerve guide showing end surfaces.

Figure 4. Fabrication of a gradient thread. A defined quantity of growth factor is prepared in a series of alginate Stock solutions. In this example, during step ( 1 ) a layer composed of 0.005 mg ml alginate thai contains 1 ng of growth factor has been added to the casting vessel. The aliquot is then frozen. In step (2) an aliquot prepared with 0.010 nighni alginate thai has been supplemented with 5 ng GDNF has been added to the easting vessel on top of the frozen aliquot. The two are ffo¾en and the cycle is continued until the construct is completed In the final step _s the frozen column of alginate is extruded info a calcium bath; this induces polymerization of this alginate into a cotitin oiis thread which is then used directly o dried.

Figure 5. Schematic representation of a nerve guide of the invention with a carrier thread containing a therapeutic agent disposed therein.

Figure 6A-B. A. _s Schematic Of the ground targets used in a dynamic two pole air gap eiectros jnni-iig system. Vertical,, hollow piers A ir isra it ground wires to horizontal piers B that project inward towards one another (in this depiction the ground wires are depicted on the outside of the verti cal piers to emphasize that both poles are pounded to a common ground). Fibers€ acc mulate across the gap that separates the two horizontal piers. By rotating the targets depicted as B i his diagram. In unison using a single motor of: a plurality of motors that, are synchronized the ^■ target poles (B) can be slowly rotated to collect fibers in a tar rnore uni rm fashion than can he achieved with a static system (see Figure 6B), Tile positio of the syringe ami eleetrospinning source are relati vely arbitrary and largely dependent upon the cabinet used tor electroprocessing. For example, the eiectrospiunmg source may be positioned parallel to the base of the ground device and. at a height that corresponded to the height of the grounded horizontal piers B; B, close- up of the section of the apparatus where the fiber array is formed (as depicted in Figure 6Af ft is also clear that multiple source solutions positioned in different orientations can be used in this type of system. Typically at least two power supplies are used In the air gap process. The polarity of the air gap systems depicted, in these figures is arbitrary and may need to be reversed for certain polymers. Tins configuration describes die conditions specific to PCL. It may also possible to use alternating currents and or to use a C current that is designed to flash between the two poles back and forth hi (e,g, right to led right to left) in a timing patte n that mateftes fiber deposition as a way fo even further increase alignment and provide even finer control over the process. B ₅ Schematic of a static air ga target array, The static system displayed in thi figure was used to define the etee&ospinn!ng eondtiions necessary to produce the seamless nerve guides and is suitable for the fabrication of guides ^' .less. than about 10 mm in diameter. For larger structures the dynamic system illustrated in Figure 6A allows for the eol!eelio of fibers in a more unifor pattern, on the "hack side" of the graft However, even this type of static system can he modifi ed to increase the diameter o f constructs beyond about 1Q mm by using a second (or more) eiectrospinning source to deposit fibers cm the different surfaces. Also of note, if the electrospmning ground and or target hails (at the end of the inwardly projecting piers in Figure t¾B) ate manipulated it may be possible to ^' roduce (1 ) structures that gradually taper from one end to the other. In addition, the diameter of the balls used to collect the fibe (again depicted in Figure 6B) represents a critical process variable. To produce a 10 mm (or less) diameter co st uct, a 5 mm diameter ball, is used. Without this terminal hall (or equivalent terminal end, e.g. a hall cut in half, a pyramidal shape a cone shape etc. the eiectrospinning stream will pass as a continuous straight jet between the target balls and fail lo collect across the gap to produce a nerve guide. With the halls in lace* the charged eiectrospinning stream passes as a series of ever increasing concentric spir ling rings that result in the deposition of fibers across the target array, in a similar fashion if the steel washer is not used on the eiectrospinning needle, the charged polymer j et leaves the syringe as an unexpectedly long and straight, elongated jet that; passes ^' between the target array gap, and once the jet passes beyond the target it becomes unstable and disintegrates and fibers fail to accumulate on the target. The diameter of the bails is ^' also critical to the process as described, if they are increased to 10 ram in diameter or greater, a hollow cylinder that is composed of longitudinally arrayed fibers . ^' (arrayed ^' along the long axis) forms rather than the solid cylinders disclosed herein (he. which are suitable to use as nerve guides). This type of structure may be useful in the production of some tissue engineering products like blood vessels. One skilled in the art would recognize that, alternating ai gap spinning with conventiona spinning conditions could be used to p duc : structures thai have alternating layers of longitudinally arrayed, radial random and cirenmierent aSiy arrayed fibers. This type of structure would impart unique biological and or mechanical properties to the construct.

Figure 7A-J. Representative Scanning Electron Micrographs iSBM). Panel A. SE of scaffolds, produced from 50 mg ml starting concentrations using air ga eiectrospinning.

Note thai, even heavily beaded scaf h Ids exhibit aligned fibers. Scaffolds produced from D -75 mgniib E-I00 mg/mh F ^■ 125 rog ml., O 1 SO tfeg/mi ^' .:&== 175 nig/ral, I 200 mg/mh J=225 nig/mi, ™2S0 ^' mg/ml, L- ^: 275 .mg/ml. All images captured at 1000 X magnification. Scale in J for Α-, 2 ηι.

figure SA~ Average Fiber Diameter, A, Average PCL cross-sectional fiber diameter varied as a faction, of starting conditions. Of note in the graphical representation the range of fiber diameters also increased with increasing polymer concentration, B, Average fiber diameter as a function of starting PCI., conce tration. From 50 to 200 mgfml FCL average fiber diameter increased in a nearly linear f shion, (linear regression analysis over this specific range of concentrations: R2 ^:::: 0.9I 8), however, at concentrations above 200 mg/ml this relationship markedly deteriorated. Overall the entire range of st rtin concentrations thai we investigated a quadratic equation best described the relationship between starting concentration and average fiber diameter, although even in. that analysis the data represented a poor fit, at best (R2 ^::: 0.ii66). C Summary of pair ise comparisons across ait treatment groups. The broad range of fiber diameters prese t in the scaffolds produced f om 275 mg/ml solutions exhibited substantia! o verlap with a variety of other scaffolds. Scaffolds produced from this starting concentration bad fibers thai were not statistic-ally different than scaffolds produced from, the 125, ISO, 175, 200, 225 and 250 mg/ml.

Figure 9A-E Analysis of fiber alignment b 2D FF ^' L A, average fiber alignment over the entrre length of constructs produced from 50-125 ^' mg/ml solutions:, B< 1:50-200 mg/ ^' rrsi solutions and C, 225-275 tng rn l sola (ions.. A verage fiber alignment is similar in each construct, although there is a trend, towards an increase in the peak FFT value as a function of increasing average fiber diameter, Scaffolds, produced from the 50 and 75 mg/ml starting concentrations exlvibii a broader distribution of alignment values and shoulders on. the FF T plots fifiiistrated at l 8 ^s on the alignment plot) typical of structures that degrade aiignment values (beads and fibers off axis). IX Fiber alignment as a function ^' of initial starting PCL concentration and. E, Fiber alignment as a function: of average fiber diameter. These data indicate that, increasing fiber diameter lias a relatively nominal effect on increasing aiignment in the electrospun constructs.

Figure 10A-B- Materials Testing. A, Peak stress of el ectrospun constructs produced ...from varying concentration of PCI, ( 100, .1-50, 290, and 250 mg/ml). The peak stress of th scaffolds increases with the increasing concentration of PCL from 100 mg/ml to 200 mg/m l, at 250 mg/ml, the strength of the constmct is not si gnificantly deteriorated, but is a little less than that of the scaffold electrospun from PCL at a concentration of 200 mg/ml B, Void space in. elsc r spun constructs produced from varyiag PC!, concentrations (100, 150, 200, and 250 nrg rnl) measured using the liquid intrusion, method. The percentage of void space increased with increasing concentration pf-PCL (P<0,0S).

Figure l-.l A.-G. Cell Culture Experimentation. A, SEM tangential sections of poly

capto lactone based. (PCL: MW of 65 Kda; it was ^' found other: PCL molecular weights do not work as well in this 2 pole eieehOspmning system) scaffolds produced from 125 mg/ml, B, 200 rug/ml and C, 250 rag/ml While there is evidence of fiber damage and some

•compression in the out zones (to some extent mis effect can be mitigated by infusing the grafts with agar or other materials thai fills the voids prior to cutting, the minor damage induced does not represent a serious limitation, in part because the cutting plane is not very long in guid use and it is usually desirable to avoid infusing materials into the grafts that may leave a resid ue after trimming), note the high, porosity of the constructs and the channels that ate formed and presen between each individual fiber, these elongated, pore spaces are arrayed in parallel with the-axis of fiber -alignment. Scaffolds produced f om the 125 mg ml solutions have the beaded structures typical of these constructs (Panel A, asterisks, See also Figure 1.0). Scale bar in C = 10 prn for A-C. D, Fluorescence images of dorsal root ganglia- {DR.G),in scaffold produced from the 125 mg/ml solution that has been cultured for 7 days in the presence of nerve growth factor. DRG: cultured under similar conditions in scaffold produced from 200 mg/ml solution and F, ORG cultured under similar conditions in scaffold produced f om: 250 mg/ml solutions. Note the increasing trend towards fascioulation with increasing PCI. fiber diameter, This effect is particularly evident in the samples cultured in the scaffolds produced from the 250 mg/ml solutions. The axons that are projecting perpendicular to the primary axis of the construct are wi thin the channel used to■ implant the DRGs into the seatlbld (arrows in F and. G}.- G, Corresponds to F but stained to reveal nuclei. Note the extensive overall ceiiu.larity of the cultures and the association of nuclei with, the axons bundles. The asterisks in aad G represent the initial implant site for the DRG, inset in G represents an illustration regarding the method and site of DRG injection into the scaffolds. Bar in. G represents 1.00 μηι-ifc D-G. These data suggest that a critical pore size associated wiilt scaffolds produced Irom the 250 mg/ml solutions that axons will grow along fewer tracks, the guide under these circumstances has begun io function more like a hollow cylinder and is allowing axon aggregation or bundling to occur (again a process that is associated with reduced axon, targeting, an undesirabl result). Figure 12A-P. Nerve Reconstruction: Frozen Sections. A, representative implant recovered after 7 weeks in vivo. Arrow indicates the proximal attachment site of the implant. The guides wer well integrated into the tissue of the damaged nerve. Bar in A~ .10 mm; B, ornarsfci image of frozen sections taken mid-point in guide. Note the anisotropic nature of the tissue, C _? DAFi staining; D, eural filament 68 (NF-68); B; Myelin basic protein (MSP): and F, S100 Schwann cell marker, B-F captured with 40x objective.,. Bar in F ^;::: I00 μιη for 8->F.

Figure 13A-G; Nerve Reconstruction; I ^' Mek Sections. A- : control sciatic nerve samples. Sections are densely populated by myelinated nerves. Scattered blood vessels are present (A, arrow). The epineuriurn is well defined (8, arrow); C~D: samples taken within 1 mm of the proximal nerve stump. Myelinated and non~myelinaied axons of varying caliber are present (C, arrows), (*) in C denotes a ood vessel arrow in D denotes external capsule of impiant. E, sample taker!, within 5.turn of the proximal nerve stump. This domain was characterized by bundles of axons surrounde by a delimiting; band of tissue that resembles; the

perineurium of native nerve (E, arrow). F, samples taken 8-10 mm distal to the proximal nerve stump. Axon caliber and density is reduced with respect to the ^■ . _. proximal domains depicted in C-B. Arrowhead denotes the perineuria!- like structure (precursor to the structure present hi E). Note the nearly uniform alignment of the axons in ali of the cross sections. Ail images captured with a 100X oil immersion lens. Bar i F lor A-F=IG μτη, G, Frequenc of the regener iiug axon 2D eross-seciiona! area at proximal and distal sites of the implanted scaffold. The frequency at which the smaller caliber axons are encountered in the distal domains is greater than the proximal aspects of the _: tissue, typical of the early- to mid-stages of n erve regeneration.

Figure 14A~D. Transmission Electron Microscopy. A and .B, Proximal domains, C and D distal domains of regenerating tissue> Irs these cross sections, ffie roximal and. distal domains exhibit well differentiate myelinated axon (mAX) interspersed with unmyelinated xons (*), Functional blood vessels in the regenerating tissue range from capillarie to small arterioles (B _> BV) with smooth muscle cells (B, Sm), Distal domains (C and D) contain the profiles of jmnierous Sehwami ceils (Se) in association with small caliber axons. The box: in € denotes one of these profiles, in D high magnification, detail of a Schw.arj.ri cell surrounding a sniail caliber axon and. adjacent to a. fibroblast (F) is illustrated. Note the relatively high: degree of maturation in the myelin sheaths. Cyioskeletal elements are clearly

- 1.4 - evident in the axons, interstitial, spaces are filled, with -parallel arrays of collagen fibrils (B, inset shows a tangential section of collagen fibers, also see Col in B and.0). fi sei D illustrates a PCL fiber, note the lack of nryelinafion on these structures.

Figure 1.5A-B. Alginate fibers extruded from a tuberculin syringe: A, wet state, B, afier drying- Bar: in A™ 5 mm.. Blue dye selective added alginate stocks prior to freezing to illustrate the stability of the gradients during processing.

Figure 16. Cross section of an eteetrospun -nerve guide scaffolding. The blue dyed, alginate carrier thread is present in the middl e of the cylindrical construct and. throughout the lifter length of the construct. The depicted fiber is lor illustration and. does not necessarily represent the size of the fiber with respect to -an actual nerve guide., wh ch, i f drawn to actual scale, would likely not be visible in this type of depiction).

Figure 1.7. Schematie depiction of device for fabricating un ^' oro-carrier beads. Alginate Is placed in the syringe, the syringe is charged and directed at the calcium: bath. At charging, the alginate forms fine droplets containing reagents of interest Upon contact with the bath, the -alginate polymerizes and traps the therapeutic agents in. a: small (micro) bead.

Figure 18 A.-E. A, Microscopic ..image of eleetroprocessed alginate beads, which are used to deli ver bioaotive: materials to scaffolds, .showing their uniform, diameter. B, Image of beads hi panel a sho wing the incorporation of NGF; note the nearly ntiifontt diameter of the heads. Arrows paint to same head for reference. Scanning E of .matrix wit

N F ginate beads (black arrow) incorporated within an e!eetrospun scaffold. D, Assay assessing the bioac ivity of NGF-trapped in alginate heads on DRG (dorsal roo ganglion) outgrowth. Beads were: prepared, incubated in irifJuoroethanol or hexafluoisopropariol (electro -spinning solvents) for 10 minutes (to simulate the effects of the eiectrospmmng process), air dried and then placed in a transweli insert witbin a well Overlying an IIS rat D . In the absence of NGF little outgrowth is evident, -however, in- the presence of NGF in the media neoritic outgrowth occurs. This effect is dramatically enhanced with

NGF-dehveted via electrospnn alginate heads. Event incubating DRGs with higher concentration of NGF failed to produce the robust growth effects produced when NGF wa delivered -via the alginate beads E _y Schematic of strip assay. The strips re resent aggreean painted onto a culture dish. This carbohydxaie mimics the effects of the glial scarring that, inhibits axon growth in spinal cord injury. DRGs are placed between the strips and exposed to NGF to induce axon growth. Electrospuu matrix with/ without Chandroitlnase is then placed into each ciiitee plate for 7 days. In the absence of ehondroitinase, DRG aeurites were inhibited from, growing on mggreean lanes: (Sane boundaries -marked by arrows).

Bow/ever, in the presence of eoorfreitittase the growth irfiibilOry properiiea of aggrecan: were neutralized. Controlled release studies with material like NGF ίτέ ρ%4 in the beads have shown that the beads: provide continuous release (as determined by ELiSA.) for at least 13-21 days, far longer than expected. More prolonged: release can be expected if the beads are processed into, a seafibid where tliey become trapped between and within the fibers, or incorporated into a. carrier thread. Beads within the fibers or carrier threads are only avaiiabie to release substances they contain once the fibers or threads begin to egra ^

Figure 19A-B. his panel illustrates surgical procedures used to place: 3D eleetrospun scaffolds produce from, poiyoioxanone (PDS) Into a spinal cord defect. Λ. The rat spinal cord is exposed after laminectomy. B, A 3 mm section is removed by complete spinal cord transaction:, leaving a 3 mm gap in foe tissue. C, This gap i then fi lled with a segment of the eleetrospun seaffo Id either with or without various growth Factors arid or enzymes designed to promote regeneration. The growth., factors are designed t support axon growth ant! survival, the ehroidinase ABC enzyme is present to degrade the sear tissue that inhibits regeneration. D, a ! 0 mm section of spinal cord that has been repaired with an elecirospu matrix is illustrated. The DAP! label revels .a massive infiltration of cells into the implant (border marked by arrows), E, Graph depicting the im rove ent in .himifira mobi lity of SCI rats with varying: implants. A BBS Score of 21 represents complete mobility, a- score of 0 represents complete: paralys s. Animals treated with enhanced matrices exhibit significant improvement in iiinelionai recovery as compared to untreated controls rats. Electron

icrographie surveys revealed dense aecunnilatiens of axons within the implants.

Figure 20 A-G, Structure of rat spinal cord approx imately 6 weeks after comp lete spinal cord transection. These images were captured from within the body of a 3D Polydioxartonc (FDS) nerve guide/graft A, illustrates the typical structure of the regenerating tissue. Samples were embedded in plastic and cut i cross section (perpends eulat to axis of fiber alignment) and stained, with tr5¾>aii blue. Images were captured with a bright field microscope. The grafts are packed with dense arrays of axons< As evidenced by the circular profiles of foe axon the vast majority of the regenerating axons present in this nascent tissue ar cut in eire iar profil es, demonstrating the potent directional cues provided by the fibers, Arrows denote PDS fiber in both panels. B illustrates a simi lar region of the regenerating spinal cord,

- 1,6. - images captured by tr smission .electron microscopy. The circular structures surrounded by dense material caw. be definitively identified as axons, jnteoningling these myelinated axons are un- yleinated axons. The box in B outimes a PDS fiber, shows at higher magnification in parse! C. Note the PDS fibers do not become myelinated. Panel C illustrates the nature of PDS fibers, and the defect within the fiber is believed to represent a degenerative process that is associated with PDS resorption. This pattern is most likely ^■■ similar to that structure ohservedin the light rHicrographic irnages in the first pane! and marked by the arrowhead in the upper middle aspects of the image. T he arrowhead, in C denotes an endothelial cell. Figure 21 A-.E, This series of panels (A-E) documents the use of PDS fibers by endotheli al cells as a .scaffolding in. the formation of functional blood vessels. In A., the arrow indicates a PDS fiber in cross section within a spinal: cord: implant, in panels B and C arrows point to PDS fibers surrounded by endothelial cells, early in the process the endothelial cells appear to be closely applied to the surface of the fibers. Wi th: time they appear less closely applied, this may be associated with the degeneration of the PDS fibers and or some process intrinsic to the endothelial cell biology. Panels ( ^' € _> D and E) illustrate unprecedented results. As can. be seen in these panels, the PDS fibers are surrounded by endothelial cells that have fermed a functional blood vessel. Under all known circumstances, that we are aware of the presence, the contact of bloo cells with a PDS fiber should induce a coagulation cascade and the formation of a clot,. However, this is clearly not the ease with the spinal cord grafts of the invention. In the spinal cord the PDS fibers . co-exist within the i a mens of functional blood vessels as indicated by the .BC observed in sections adjacent and. encompassed by endothelial cells that surround the PDS fiber. This result oray be unique to the spinal cord environment and points to a unique interaction between the endothelial cells and eiectrospun PDS fibers.

DETAILED DESCRIPTION

lire invention provides nerve guides formed from air-gap el ectrospun fibers for use in the directed regeneration of damaged or severed peripheral nerves and/or spinal cord injuries, hi particular, the technique of air-gap eieetrosptrmin.g is. used to generate three dimensional (3D) arrays or bundles of highly aligned electrospun fibers that are oriented in parallel with the long axis of the construct. When the end of a truncated nerve or spinal cord is introduced info, approximated or otherwise encounters one end of the nerve guide, the nerve tissue grows into the guide and along channels formed y elongated gaps between the individual fibers, the channels ^' being oriented parallel to and along the length of the libera, i.e. along the long axis of the guide. The construct thus guides unidirectional growth of the proximal end of a nerve toward a desired location, e.g. toward -and directly up to the distal end of the severed . nerve or damaged spina! cord, Herein, "proxnna!" refers to the end of a severed nerve that, is closest to the center of the body or the brain and "'distal" refers to the end that is timbe from the center of the body or further from the brain. These terms may also be applied to the nerve guides described herein;, with respect to a view of the guide in. a. figure (the end closest to the viewer is the proximal end) and with respect to the connection to a. severed nerve ^' (the end of the guide thai is connected to the proximal nerve stump is considered to be the proximal end of the guide}, even though; when out of this contex t, the two ends of the guide are substantially identical. Also,, nerve- refers to axons present in peripheral nerve and or spina! eord.

The inventio thus provides a 3D, ''aerni-solid" nerve guide produced by air gap eleetrosp ming. Air gap spinning makes it possible to produce microscopic (e.g. < 5-10 mm ^' in diameter) to macroscopic (e.g. >S~10 mm. in diameter), cylindrical constructs comprised of dense anisotropic arrays (bundles) of nano-to-rnieron scale diameter fibers. These fibers are aligned in parallel with the long axis of the constructs, an architectural, feature that provides thousands of individual channels (in turn, each is channel i lined with "aligned" aftays of fibers) that can. be used to support, and direct, axon growth. With the use of the nerve guides of the invention, fractional recovery in nerve inj ury is greatly improved because regenerating axons are confined to a specific tissue plane that mimics their original position within an intact nerve. The nerve guides of the invention thus cause regenerating axons t emerge from the distal aspect of the nerve guide in the near vicinity of where they existed prior to injury, increasing the proba ility of proper rejoining to the residual distal nerve stump _*

Air-gap electrospinning is described in detail In the Examples section below. Briefly, this technique involves the use of two . grounded targets which are static (not. rotated) during the eleetrospinning procedure (although it should be noted that a. device can be produced in which the targets are designed to rotate in synchrony to allow fibers to be deposited on all. sides of the forming fiber array). An "air" gap (e.g. adjustable from.2-6 inches) separates the terminal ends of the two grounded, targets. The electrospinning stream of charged polymers is directed into trie air gap. As the charged polymer jet reaches the gap, h is laid out in a series of loops that pass back and forth between the grounded targets and collects as a parallel array of fibers, forming a seamless cylindrical construct This process can induce fiber alignmen over a wide range of spinning conditions (individual fibers of <200 nm to at least 3-4 μιη in average cross sectional diameter) and depositing them in a static air gap system into macroscopic (up to at least it) mm in diameter) 3D cylindrical arrays. Larger diameter structures can be produced in dynamic air gap systems. The packed fiber arrays of these constructs, in many ways, resemble the structure of an autologous nerve graft (e.g. compare Figures 1 and 2A). A complete discussion of this process is provided in Jha ei al., 2010 In. press Acta Biomalerials (reference 34),

White eleetrospun PCL ma he used for the reconstruction of peripheral nervous system, and e!ectrospun FDS for use in the reconstruction of the central nervous system,, u is recognized that other polymer (tor example, PG , FLA eo-poiymers of PGA PL A, polyesters, and native proteins such as collagens fihronectin, fibrinogens and other natural and synthetic proteins) ma also be used in these applications, it is further recognized that, through the use of multipl eieetrospmnm sources, different polymers and/or different polymer concentrations can be used to produce the guides. This may be particularly effective for use in spinal cord injuries. For example, synthetic fibers Coated with specific materials (ECM including but not limited to collagen, lamirhn, fihroneeiiu, Type IV collagen, other proteins such as fibrinogen, and/or ceil surface proteins and intracellular proteins, pharmaceutical., growth f ctors etc) may be spun into the system for a defined interval of time, then a second source of material (different polymer, different concentration of same polymer, or polymer supplemented with different material) can be used to deposit additional arrays of fibers in a specific pattern, onto the target, A similar pattern of fabrication, can. he used to produce arrays of different nati ve proteins and or combinations of native eleetrospun proteins and synthetic fibers. For example, fibers in the core of the device may be composed of PDS (with or without additional materials added to the elcctrospinning solvent (as disclosed above), then the next outer fibers may be of a different polymer (with or without additional materials) and so on. An number of different layers can be produced in this way. By simultaneously eiectrospinning from multiple-sources fibers of specific characteristics can he intermingle with one another in a seamless fashion on an individual fiber scale, in most eiectrospinning system average pore dimension correlates roughly with average fiber U 2010/048744 size, Small fibers results in small pores, large fibers tend to produce larger pores ( 14). By intenningling different fiber sizes it is possible to regulate the number and si of pores present in a construct. It is desirable to match the d ameter of the nerve guide and the number of pores with the number of axons present in a nerve that is being reconstructed as a strategy to suppress axon sprouting. Excessive axon sprouting can: lead decreased axon targeting. By limiting the number ^' of pores available for growth it may be possible to physically constrain this process and thereb increase the fidelity .of re eneration. It is also recognized thai materials can be dripped and or sprayed onto the forming device before during or after the fabrication process.

In addition, the data provided herein shows that an eleetrospun matrix produced by air gap eiectrospinning can be supplemented with proteins and e zy es that can promote neuronal survival and neutraliz the growth inhibitory proteins associated with gliotic scar (or mesenchymal scar).. Specifically, in vitro assays have demonstrated that ne ve growth factor (NGF) delivered via alginate beads can be incorporated in an eiecirospnn matrix and remai bioactive, as demonstrated by enhancing dorsai root ganglion (DRG) nenritic outgrowth. These data also suggest that delivery of this growth factor (and others) potentiates the biological, activity of the growth factor, possible because it releases and/or delivers th growth factor in a more suitable tertiary structure for receptor binding. n addition, the data demonstrates how Choudroibnase ABC. (ChABC) can be incorporaied into the matrix and released in a bioactive form that neutralizes die axon growth inhibitory properties of CSP ^' G, Collectively, these experiments point toward, a novel approach by which biocompatible matrix i used t bridge the fl id- IOed cavity and provide

regenerating axons with a directional matrix, containing trophic and inhibitor neutralizing support, This muiti-faetorial approach is designed to address each of the specific defects known to inhibit axon regeneration in the cen tra! nervous system. Of note, the scaffolds can be prepared with specific growth factors placed in. specific positions within the graft in order to selectively promote the growth of specific axon populations. For example some axons grow in response to nerve gro wth factor,, other axon tracks grow in response to neiux>tTop ln 3 (NT-3), By positioning the growth f ctors in specific locatkms and in. gradients . fabricated as described herein, the outgrowth of specific axon populations can be fa more subtlety controlled than if the factors were present throughout the implanted scaffolds.

A cross-sectional view of an exemplary nerve snide Is depicted schematically in igure 3 A. This figures shows nerve guide 10 with cross sectional surface 11. Nerve guide 10.may he substantially cyl.irs.drie a! and comprises a plurality of electrospun fibers, proximal cross-sectional ends 31 of which are shown. For the sake of clarity,, only one fiber 30 is depicted in phantom (dotted lines, near the bottom of the figure) as extending within and parallel to the long axi of the guide. Flowe-ven one of skill i the art will understand that the guide co.niai.ns hundreds of thousands to millions of suc aligned fibers which extend from one end of tire guide to the other, parallel to the long axis of nerve guide 10, interspersed amongst the elongated fibers are hundreds of thousands to millions of channels 20 (i.e. elongated gaps or spaces, only three of which are shown in phantom by pairs of parallel dashed lines in Figure 3 A). Channels 20 extend continuously along ami through the interior of the nerve guide substantially parallel to the long axis oidhe guide., and emerge at the other end of the guide. Channel openings 21 are shown on cross sectional surface 11 , The channels do not contain seams (i.e. the edges of a rolled sheet of electraspun material is not present) and are pot formed by rolling a 2D sheet of electrospu material. The channels in effect form a void space within the guide and the outer hounds of each channel (i.e. the wall of the channel) is formed by the outside surfaces of elongated fibers which traverse the length of the guide.

A single channel with channel wall 22 is depicted schematical ly in cross section in Figure 3B. As can be seen, channel wail 22 is formed by the sides of fibers 30 which are arranged, in a parallel array or bundle. As an. axon grows along channel 22, it comes into direct contact with and in effect grows directly on and/or adjacent to the aligned fibers which define the channel. The pores or channels are depicted in Figure 3 A are: approximatel circular in cross section.. In reality, they may have a more complex profile e.g. as in Fi ure 3B, or may be even rnore complex. The "walls' ^* that form the channel, (which are represented as continuous or solid in Figure 38) are defined b a series of individual fibers that may or may not be fully !ongitudinaHy connected id one another in the fashion depicted in this schematic, and thus the channels may also be interconnected.

The aligned fibers provide a directional cue to the regenerating axons, which will tend to grow down the ehanfiel in the direction of fiber alignment* ie. along the long axis of the guide. Conversely, one can consider the channels a _: series of aligned open spaces established and bordered by the fibers. As can. be seen, this structure closely resembles the architecture of a nerve (see Figure 1 ), with the open, channels providing a path for axon growth (e;.g. in, the direction of the arro s m ^' Figure 3A} the ibets pro idin a solid or semi-solid support _: by filling ibe space which, in a natural nerve, is occupied by the epinerium and or perineummt. Signiffcan iy, he nerve guides of the invention are not:

formed by rolling flat, 2--dimensiona1 sheets of fibers into a tube and thus do not have single hollow center, nor do they have seams at any surface. In other words,; the guide has a. true 3 -dimensional architecture as originally synthesized.

In some embodiments, the nerve guide also includes a sheath 40 (see Figure 3C), Sheath 40 (which may also he referred to a. coating or covering) may be formed b

eiectrospinnmg: (fibers) or eiectrospraying (droplets) a polymer or eo-polyrner directly onto the surface of the guide after it is constructed. The electrospuu .fibers of the sheath ma be aligned, with the long axis of the guide, or at an angle (e.g. 90") with respect to the long axis of the guide, or may he non-aligned., randomly configured (disordered) in nature. Figure 3€ shows a: schematic view of nerve guide 10 of the invention having an internal arra of fibers longitudinally aligned with tbe long axis of the guide, and with outer sheath 40 ^' formed f om, a mass of non-aligned eleetrospun fibers. Generally, ibe coating or sheath covers the entire outer surface of the guide except for the proximal and distal ends of the guide where the regenerating axons enter and. leave the guide, respectively although this need not always be the ease, lb. some -embodiments, the sheath covers only a portion of the guide or may be absent ail together. Jn.: other-embodiments, it. may be designed to overhang the ends of the guide and form a "sock- like" shueture designed to slip ove the ends of a: cut or otherwise damaged nerve to facilitate placement of the guide. Experiments described herei showed very unexpected results with the outer sheath. When, used in the peripheral nervous ^' system, ibis sheath was very effective at reducing interstitial and inflammatory cell infiltration into the guides. However, the use of a similar sheath (or collar) in spinal cord reconstruction experiments produced, tbe opposite result, the use of the sheath inhibited regeneration. When, used, the sheath or covering is generally present at a depth of from about 50 mrcrons to about 200 microns. The -coating provides mechanical support to suppress compression or deformation of the guide (e.g. during manipulation and when in place in a subject), and is able to support sutures which connect the guide to a severed nerve end. In addition, the sheath m y induce the formation of a fibrofie capsule which suppresses infiltration of inflammatory cells into the core of the guide (Telerneco et ah 2005). Exemplary materials for ^• forming ^' n eleetrospun coating include but are not limited to po!ygalaetic acid (PGAjtPCL eo-poiyrners _s PGA, PDS, PLA, PDS and denatured eoHagens, e.g. gelatin which can: be used to ind ce the formation of the tferoiic capsule necessary to protect the internal environment of the ^■ ■nerve guides, in some embodiments, the coating is not electros un but instead is formed by coating the guide in some other maimer e.g, adhering a suitable substance onto the external surface of the guide, fa this embodiment.,, the substance that is chosen is generally of low or no toxicity, ("biQcompatible"), and is usually designed to induce a mild inflammatory response to generate the subsequent formation of the fibrotic capsule For example, a PC based ne ¾ guide can he prepared and dipped into a solution of gelatin prepared in water (this solution can be prepared with, or without additional therapeutic reagents and may be applied uniforml or in a gradient fashion over the outside surface of the guide, e.g. by dipping the devke repeatedly and preferefttiaily on one end into the solution). The PCI, will largely be stable in the water for the short inter al needed to coat the outer surfece. Or alternativel the exterior may be coated with various materials simply by spraying or aeroselrag the material onto the exterior of the device. The fibrotic capsule can. also be induced to form: through the ^■ application of specific reagents such as TGF-I or other pro- inflammatory substances; this g owth factor is a potent infianitnatory agent. The use of this type of strategy will produce a capsule in the absence of any additional outside sheath through just the use of this growth factor. One skilled: in the arts will also be cogni ant that it is also possible to use a ti-iiannnatory reagents where the formation of a fibrotic capsule is ttndesirah!e, to ther embodiments, an electrospun sheath m y be first attached to the guide, and then the electrospun or elecirosprayed coatfa may he further "coated" with, one or more layers of a suitable material or materials, such, as PCI..,. PDS _S PGA, PLA. and selected proteins such as gelatin eoliagens nil or fibrinogen). The sheaths or coatings on fife guide may further comprise therapeutic substances as well, e,g. antibiotics, growth factors., antidnfiainrnatory agents, pro- nilarnmatory agents, pharmaceuticals designed to promote axon regenerahon/s rviva] (cAMi* and analogs of this signal reagent which promote axon growth and or regents designed to m nip late the. ISTQOO ^• §ystem--wb.k-b.-inhibits axon growth, and can he blocked with pharmaceutical reagents and or antibodies-are of particular interest for use in spinal cord injuries); These factors may foe added as a more or les tu ifprrn distribution on ⁽ lie guides or m specific patterns as required by specific circumstances.

The: nerve guides of the invention are typically substantiall (roughly, largely) cylindrical in shape and have a diameter in the range of .from a out 1 till to about 20 mm (e.g. about 1, 2, 3, 4, 5. 6, 7, % 9, i ll 11, ! 2, i.3 _; 1 I S, 16, 17, I S, 19, 20 mm,, or In some cases even longer, e.g. up about 25 or 30 mm). Average fiber diameters for PCL based constructs may range from about 100 nra (e.g. about 50, 100, or 150 mm or even about 20 - 250 ran) up to about 5 microns (e.g. about i , 2, 3, , or 5, or more, microns). These values encompass the fiber diameters most, suitable for eleetrospinmng _. in the air gap system and tor use ip providm giiidanee cues to axons. H wever, typicall these values will range from about 500 nm to- about, 2000 nm (e.g. about 500, 600, 7O0, SCO, 900, i000, _: 1200, 1300, 1400, 1500, 1000, 1700, 1800, or 1900 mn), -depending upon specific applications. The nerve guides thai we have used in .reconstruction experiments n the rodent sciatic nerve and rodent spinal cord have mean: fiber diameter of about 1 micron but those skilled in the arts will realize thai eleetrospun constructs are usually composed of range of fiber diameters. At 200 mg:½l. PCL. produced fibers ranging from about 50 nm all the way to 5 microns but the mean and median, values are approximately 1 micron. The- channels within the nerve guide generally occupy a total void volume of from, about 50% -to about 95% (e.g. about 50, 55, 60, 65, 70, 75, 80, 85, 9», or 95%), and most fre uently from about 80% to about 95% (e.g. about 80, 85, 90 or 95%). A 10 mm diameter guide composed of i di dual fibers that are nominally 1 micron. In diameter and void volume of approximately 90%. will contain 10 million fibers- (e-afculateo ^* {romrwhete (3.I: )(5rom)(5rnm) ^::: 7S _; 5 mnr. For a circular structure with 90% open space the total volume occupied, by the fibers is

(0 J)(7S,Smra2:) ^:::; 7.B5 mm*. A 1 micron diameter fiber (assuming a circular profile) occupies (3.1 )(0.5)(0>5H).785 \im ^% o area. Total area occupied by the fibers is =7,850,000 οι ^? . Total area occupied b 1 fiber=0.785 μηΛ Dividing total area occupied by fibers by the total area occupied by 1 fiber yields approximatel 10,000,000 fibers). Assumin that pore spaces between the individual fiber approximate the diameter of the fibers, a similar ^■ calculation will pro vide ars estimate of the total number of individual chartnels present in this type, of construct, (total area occupied by open spaces: 90% or: 77,? 15,000 μηΓ for a 10 mm diameter nerve guide.) if the pores spaces are approximately the di ameter of the fibers the total number of pores in this construct will be: 77,715,000 μηι¼.7§5 ηι ³ - , 000,000 potential cfimmels; assuming 2 micron diameter channels yieids=77,?i5,0OO μιη¾.14 μ m ² -=2 ,750, 000 potential channels, assuming 5 micron diameter channels

yields^??,? 1 ,000 μηι ² /10.625 ίΒ: ² -3,960,(Κ)ί} channels). One skilled, in. the art will recognise that it may be desirable to match the total, number of fibers or the total number of pore spaces to the estiroated iiumber of nerves present in an injury s te, in addition,, th :

length of the guides are generally i the range of from about 10 mm to. about 125 mm. for peripheral nerve: injuries and from. 2 m to 50 mm for spinal eord. Those of skill m the art will reeogiike thai the guides may be of my suitable length and cimu«ference, and i fact may be fnhrieaied or tailored for particular purposes, eg, for use in different nerves, in particular individuals, for pamcuiar locations in the body, according to the type of lesio being treated, etc.

Those of skill in the art will recognize: that a wide range of sizes of guides and, numbers of channels might be suitable, depending for example on the type of nerve that is to grow through: the guide, the distance the regenerating nerve should grow, the limit of the air-gap eleetrospinning method. In air gap eleetrospinning the total length of a construct that can be produced is depended upon the specific polymer to be spun and the starting concentratio of that polymer. For example, for PCL prepared at SOmg ml in TFE the ideal distance between, the target poles may be on the order of about.30-60 mm. At 200 rag/rai the distance between the target poles may be on order of 50-100 mm. We note that for PCL, anil other polymers that ver defined elect-rosphmlng conditions may be necessary to produce ≠. collect fibers in the air gap system. For examples of specific conditions used to process PCL see table below, ambient conditions s eb temperature and humidity can alter these documented spinning conditions. The values provided in Table f are suitable for PGL in TFE at ffimperatures of about 680F and a relative humidity of about 30- 50 %.

Ί able 1 , Eleetrospinning conditions

150mg PCL/mI ^' IPE 1 mm -7kV 12mS/hr 21 cm 6 cm

I?5n¾ PCL/:ml ^' n¾ 23 mm -7kV t Omi hr 18 cm f 6 cm

2CJ0mg PCIiml TFE 2 mm •7kV !0 ^: roS/¾.r I S cm 1 6 cm

225mg PCL/ml TFE 32 mm. -7k.V 12ml¾i 1 cm j 5 cm

2S0mg PCL/mi TEE 2 -5kV 10ml hr 15 cm 5 cm

275 nig PCL/ml T E 19 mil; ~3kV 8ml hr 13 cm. { 5 cm

Table 1 emphasizes that the conditions thai must be used to eleetrospm in the air gap system can be very specific: and require(d) empirical analysis to define. For e m le,; the use of a conductive ^' washer fadii es spinning in this system, whereas in conventional systems a needle will often suffice as a source for charging the system, This discussion documents specific conditions used to produce PCL based scaffolds. Washer diameter refers to the diameter of washer placed over the needle used to deliver the polymer solution to the electric field. This washer is necessary to overcome the point charge effects observed in the air gap system, without these round, washers the PCI, polymer fa ls to target properly (Other polymers, such as PDS under certain starting conditions spin better without the washer). Ground voltage refers to the common voltage applied to the paired target poles. These value were optimized based, on fi er collection. For PCL the source solution was held constant and charged to 2ilkV+ for all. concentrations (ideal voltages may vary b polymer and solvent or melt condi lions). Polymer deli ery rate refers to the rate at which, the polymer solutio was delivered to the electric field. Too low or too fast of a. delivery rate can result in. the failure to: collect fibers across the target array. Spin gap is the distance- from the source solution to the target array. This interval is determined largely by the circular path of the charged

electrospmning jet and is set. so- that the diameter o f the spiral path coincides with the diameter of the gap between, the target array (different distances will work, but often requires simultaneous adjustments in flow rate and eieeirospinning voltages). The gap between poles: is a. measure of the distance between the two target poles. These vafues were optimized based on fiber formation, but values outside these will work, however the accumul ion of fibers i less efficient.

Those of skili in the art will recognize that the nerve guides of the invention will generally contain at least two suitable end surface (a first end surface and a second end. ends) which, are substantially fiat and substantially circular (beihg roughly the cross-section of the . ey&driea] guide) and which contain or include openings into the channels located withiij the guide, thereby providing means of ingress and egress For the regenerating nerve, in some embodiments it may be necessary to hav a guide shaped in the .confi uration, which

5 contains: a branch pointfs) to accommodate injuries that occur near where nerves ^' may ^' branch.

(¾¾r example near the separation of the tibial and. commo pe on al nerves from the ^. sciatic nerve). Figure 3D shows a schematic depiction of first end surface 70 and second end surface. 71 (in phantom), with a single open, channel 20 (also in phantom) traversing the guide there between, Channel openings 21 a and 21 b open on. firs end surface 70 and second

Q end surface 71 , respectively,. The guides may contain these suitable end surfaces directly after eiectrospmning, or the guide may be further processed, if necessary, e.g. by "trimming ¹ ' the ends to form such end surfaces- Suitable surfaces may or may not have a diameter that is substantially the same as that of the guide itself, he. the diameter of an end surface may he somewhat larger, but usually ma be somewhat smaller than that of the guide itself e.g. if

S: the di tal and proximal end sections of the guide are somewhat tapered or sloped. In general, the diameter of each end surface of the nerve guides of the invention will he substantially the same and can be prepared to match the diameter of the speci fic nerve to be repaired.

Typically, in the peripheral nervous system nerves tha might be repaired range from about 1 -15 fflffl in cross sectional diameter, although in some cases the diameter may be greater;

Q this value will, vary by individual and the exact position of the injury to be repaired. Values for the spina! cord may be similar. Most injuries in the spinal cord cause damage to a portion of the cord and not complete transection, rather by their nature these inj ries usually d mag a fraction of the total cross sectional diameter of the cord. To repair these injuries the damaged are ^'■ (i,e _# the cyst) may be excised and. reconstructed wi h a nerve guide that fits the

B surgically induced deficit. However, it may be necessary to repair a large full diamete

defect Under these circumstances the nerve guide will be on the order of about 10-20 mm in diameter.

Typical relationships between average fiber diameter, estimated void space and average number of fibers per ram ² of a cross section is as follows.: Average Fiber Diameter Estimated Void space Average ^: Number of Pi ers mni ⁱ

100 ran. 0.55 45000000

200 ran 0,55 ! 1250000

300 m 0.6 4444444

500 rar: 0,83 600000

; 000 am 0.85 150000

2000 nrri 0.9 2S000

3000 m 0.92 S889

5000 mil 0.95 2000

These numbers are calculated with the fbliowing assuirrpiions; A. All fibers are uniform in diameter; B, (Mentations based on given void space values liieb are extrapolated from studies described herein; and C, assumption of circular cross sections for all fibers.

Similarly, the averag channel (pore) size and numbers per rnnr in a cross section of the construct are as follows:

Average Pore Size Average Number of Fores pet 1 mm ²

100 nm 55000000

200 rmr 13750000

300 rim $666661

500 nm 3400000

100.0 nm 850000

2000 nm 22500

3000 nm 5 2222.2

5000 nm 38000

10900 n 9500

These.nurnbers are calculated w th the following assumptions; A, All pores are circular in profile and uniform; B. Calculations are based on given void space values which are extrapolated from studies presented, herein; C, Total pore area varies directly as a function of void volume- assumptions, While the nerve guides of the invention ate generally ^' configured as a single guide with: multiple channels tor fostering the directional growth of a single nerve stump, usually in a substantially straight line (as the shortest distance to be traversed by the regenerating nerve), this need not always be the ease. The invention also encompasses other

configurations of nerve guides, e.g. guides which are curved or bent at an angle, multiple guides connected end to end (e.g. to extend across longer distances), two or more guides which are attached side by side and/or on to of one another, etc. All such confi urations are encompassed by the present invention.

Those of skill in the art will recognize that the eiectrospim fibers from which the guide if fbraied. may be made of a suitable non-toxic material which is amendable to air-gap electrospinniug, and which is capable of being slowly absorbed or dissolved unde physiological conditions. The material should possess sufficient tensile strength and stability to permit manipulation f e.g. surgical suturing to a nerve end or other tissue), and to remain intact for a period of time snfficient to support nerv grow th. However, the material should not be overly rigid, but should be somewhat flexible and conformable to the contours of the physiological environment in which it is used, and should ultimately be absorbed (resorbed, dissolved, etc,} within the: body. Examples of such materials include but are not limited to olyeaprolactone (PCL), Examples of such materials include but are not limited to polycapro lactone (PCI,), Polydioxone (PDS} ₅ polylachdes (PLA), polygiycolic aeid (PGA), co-polymers of PGA PLA, polyvinyl alcohol (PVA), Polyethylene glycol (PEG). Poly ethylene oxide (ΡΒΘ).

Other potential eaudidates include; poly(urethanes), poly(siioxanes), silicones, poly(ethylene), poiy(vinyi pyrrolidone), poly(2-hydrosyeti-ryl n ethacrylate), poiy(N-vinyi pyrrolidoue), polyfmethyi methaerylate), polyCacryiic acid), poiyaeryi mide,

poSy|ethylene~eo~vin;yl acetate), polv(eihylene glycol), polyfmethacrylie acid),

po!yanhydndes and poiyorihoesicrs. Natural proleinsit luding collagens, fibrinogens, fihroneciin may also be used in the air gap eleetrospimiing system to produce 3D nerve guides ft is should be noted that some polymers can be eiectropun as melts and do not require solvents. Also, while we have disclosed the use ^' of electric fields in the fabrication of our aligned guides if is possible to process some polymers-ihvery- $tron ^' g _. m¾gneiic ^: -fields to produc fibers, n most biological, systems materials that cars, be degraded or dissolved are believed to be highly desirable for use in tissue engineering applications. With the: advent of materials■ that cad reside m m. inert stale and not induce adverse effects: it may be desirable -to use: those materials in the discussed applications. Materials that are pemianently indwelling may be used to support tissues that are too delicate to otherwise be .maintained ^' dtie to mechanical damage. For example rrylon _. and other more or less penrwneni: materials may be needed to support tissues that can not generate sufficient material strength to resist damage from mechanical nauraas originating with, movement or other msalts.

Typically, a nerv-fe guide is likely t he formed .from FCL with. a MW ^■ ø! 65000 daltons lor the peripheral nervous system, Electrospun BG ^' L produced from a staffing;

concentration of about 200 :mgi ^: mi in ΊΈΕ produces fibers that are approximately 1 microti i average cross sectional diameter. This particular fbrnra icm has a eonfluence of

characteristics that provide a unique architectural and. structural environment ibat fosters nerve regeneration. This formulation spirts very well and targets into the 2 pole system efficiently (the spinning conditions thai produce good, fibers at lower PCL concentrations are far more restrictive and difficult to achieve, at higher concentrations the inherent limitations of the 2 pole eleetrospinning process restricts the overall length, that can be achieved in the constructs), ^' The 200 _: mg/mi fo midation produces scaftbids that exhibit nearly no fiber defects/These constructs have approximately 90% void space . between the fibers yet it suppresses axon aggregation and has excellent materialpro erties thai are sufficient to withstand mechanical, insults. The exterior sheath used in the fabrication of these devices is typkaOy composed of a PGA/PLA co-polymer coating that induces a. mild, inflammatory response and the formation, of a fibrotic capsule, The guide is pia.eed in contact with the stumps of. the damaged nerve and sutured and glued in place. Specific growth faciots that have been identified for use i . these guides include nerve growth factor, and or glial derived neurotrophic facfof (GD ^' P),

In spinal cord injuries the nerve guide is composed of PDS (the parent polymer of this specific formulation is polydioxanone or PDO, PI30 is not available commercially but is available lor purchase as suture material or PDS ..sutu e _* Ideally, the PDS suture is soaked, in methylene chloride to remove the blue dye that is incorporated into it during the suture iabricanon process. This dye reduces the ''spinabilit ^? of the PDS. PDS is subject to the fonrtation of peaks and ripples in the air ga process as it deposits across the target array he» the blue and apparently charged dye Is retained in the material PDS is eiectrospun at concentration ..ranging from about 100 rag/ml HHP (fibers a pro imately 1. micron, i average cross sectional diameter} to about 175 mg mi HFIP (fibers thai are approxim t !y 1.54.? microns in average cross sectional diameter). Specific factors that have been incorporated into these devices include cAMP analogs, to drive axon growth and overcome the inhibitory effects of ^' ..myelin on ¾xon outgrowth, ehondroitinase ABC enz me (e.g.. abotU G.2S - 1.0 units per 3 mm implant) to degrade glial sear associated with spinal cord injury that inhibits axon, growth, nerve growth factor as a tropic agent lor axons,. T-3

(neuroirophin 3} as a tropic factor for axons, and. BDNF (brai derived neurotrophic factor) as a tropic factor tor axons. All growth factors were used at an. estimated concentration of approximately 0.5-5 -micrograms per 3 ram -segment implanted .into the rat, for human use these values can be scaled accordingly.

Of particular note fo eon.stn.icts designed to reconstruct spinal eord injures, we ha ve found thai endothelial cells migrate along and -attempt to surround PDS fibers that are about 0,5 to about 1.0 microns in. diameter. With time as the fiber degrades, the endothelial cellsremain and a capillary develops,: with the- lumen existing where the PD fiber had at first been sirrrounded by the endothelial cells, This unexpected effect has not been, observed in: the peripheral, nervous system to date in animals reconstructed with PC based gratis and appears to be unique to the CNS (Figure 20). This effect in the CNS is of interest fo at least two reasons. First, the endothelial cells clearly preferentially appear to use these fibers as preferred path upon win eh. to penetrate into: the -scaffolds to provide noirtent and waste exchange support to the regenerating tissues. Second, it has been observed thai capillaries formed by endothelial ceils in the CNS form a template that directs the growth/of CNS axons (35, 36). This growth occurs in parallel with those capillaries, thus the growth of endothelial cells along, a defined track established by the PDS fibers will represent yet another cue to direct _; :axons -to grow down the uides. We also note that fiber alignment is crucial for axon, penetration and gr wth. An excessive disturbance in fiber alignment at the .-terminal ends of the grafts can inhibit axon, penetration, data that clearly demonstrates the uti ty and desirability of the fiber alignment possible using the air gap electrospinning system.

Specific additional reagents that may be desirable include extracellular matrix proteins mixed uniforml through out the scaffolds. These proteins, including laminin.

eollagerss of various; types including Type 1A" and Type I. fibronecttn, RDG peptides, other

AH - binding moieties mid fibrinogen (which are a potent promoter of axon growth), These .materials may be mixed with the eleetrospinning reagents or alternatively the nerve guides can be immersed into ttief¾. or they ma be aerosoled into the constructs during the fabrication process in the presence or absence of an electric field. Other reagents may include an i roiics, a w raraatof agents, pro-infiarnrnatory agents, biologically active peptides, inert peptides, various cell types including Sch ann cells, Glial cells, astrocytes, oiigodentrocytes, endothelial cells and other cells thai can naturally provide trophic support or those cells that have been engineered to provide trophic support to regenerating axons. For spinal cord treatments the incorporation of glial cells is of particular interest as thes cells provide trophic support to regenerating axons while providing a functional barrier tha inhibits the infiltration of unwanted interstitial fibroblasts into a nerve guide. Ceils may be added simply by dribbling a highly concentrated solution of cells gradually into the forming .fiber arrays or soaking the constructs in cells after the .fabrication process. Or alternatively, cells may be introduced into the constructs by aerosol deposition in the prescenee or absence of an electric field.

The nerve guides of the invention are gradually absorbed (resorbed, dissolved, in, etc.) the bi!ogieal fluid that siirrotiKds tJiem in the body. The rate of dissolution is calibrated {e.g. by the selection of polyrneris},, by the thickness or si¾e of the guide, by the presence of a covering or coating, etc.) so that the guide is present for at least about 5 weeks and usually for at least, about 52 weeks, (and can be fabricated s as t be present for variou times in between, e.g. about 1.0, 15, 20. 25, 30, 35, 40, 45, or 50 weeks) in order to facilitate -nerve regeneration for a period, of time thai is long enough for the growing anions to reach and join to the distal nerve end. The precise tinre that is required may vary somewhat f om

circuni stance to circumstance, depending e.g. on the type of lesion that needs to be repaired and the di tance between the two severed nerve ends.

In some embodiments, the nerve guides of the invention also comprise gradients of soluble substances or agents that are therapeutically beneficial to regenerating nerves. The gr dients are precise and spatially regulated signaling gradients which permit highly regulated delivery of a substance of interest to a particular location of interest over very short distances (e.g; distances ranging from about 5 mm to about 12,5 nun, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 _; 59, 55 _: , 60, 65, ^" 0. 75, 80. 85, 90, 95, 100, 105, 1 10, 1 15, 120. or 125 mm, or even greater). The incorporation of signaling gradients into the nerve guides of the -invention addresses two critical issues concerning the processes that drive nerve regeneration. For example, growth ..factor gradients c r; be expected to accelerate axon elongation and growth along the axis of the bioactive gradient:, an intervention that should greatly improve functional outcomes, in nerve injury.

Accordingly, the present invention, provides, a simple and effective method to fabricate gradien ts of soluble materials and even cells in to various tissue engineering constructs. This method incorporates various therapeutic reagents into dissolvable libers and makes it. possible t build very precise and spatially regulated gradients over very short linear distances. As added advantage, the method makes it. possible t fabricate gradients with virtually no loss of reagent during the fabrication of the device. While this particular description concentrates on a discussion of nerve guides designed for peripheral nerve regeneration, die use of highly precise growth factor gradients has applications in the treatment of injuries and diseases i the CNS and in other tissues and organs and in. vascular construction.

There are tw main types o f gradients that can be generate using the methods of the invention, hi one embodiment, the exemplary substance alginate is used as a carrier of therapeutic agents since this carbohydrate Is largely inert and has. been safely used as a carrier/delivery platform tor a. variety of .materials, hi the following discussion, methods rising the exemplary carrier alginate are described, Ifewever, one of skill in the art will recognize that the description would apply to other types of carriers as well excep when reference is made to particular properties specific to alginate.

In the first technique, a constant concentration of carrier (e.g, alginate) is used to fabrica te the entire length of a fiber and the concentration- of the therapeutic reagent th at is placed at specific intervals long the construct is varied. This design is referred to as a ^Concentratio - Dependent Gradient" (CDG ^' K since the concentration of the. reagent varies along the length of die fiber. In the second technique, the concentration of carrier varies along the length of the thread and a constant concentration of therapeutic reagent is present along the entire thread. This type of construct is referred to as -a "Dissolution Dependent Gradient" (DDG) because, release is regulated by die differential dissolution of the carrier, not by the concentration, of agent, which is constant, in either design, the effective concentration of agent thai is released from the thread is varied with precision.

In the fabrication of a COG gradient a stock solution of carrier (e.g, alginate} is

^• - 3.3 " prepared in nominally calcium f ee dd¾0. The specific coueemTation of alginate varies as a ftsnctlon of the specific application under consideration in some embodiments, a 10 mg/rhl solution is used , A reagent of interest is mixed with the alginate stock solution at different concentrations. Specific concentrations will vary as a function of the type of gradient to be prepared and the nature of the reagent to be incorporated into that gradient (e.g.

FSiamiaeeuticai vs growth factor etc.).

Fabrication of a thread or fiber having varying concentrations of reagent (in this ease, GDNF) is illustrated schematically in Figure 4.» To fabricate a CDG gradient an aliquot of alginate containing the reagent of interest at a. specific concentration is added to a casting vessel or mold; for example, a small bore diameter tubing or a tuberculin syringe may be used, the diameter of which varies depending upon the fiber size that is needed, initially, one discrete volume (aliquot) of liquid alginate containing a particular concentration of agent is placed into the vessel and frozen (1 of Figure 4). After freezing, the next aliquot in the series (he. the second volume of liquid alginate containing a second concentration- 0-f agent, which differs ^' from the concentration of agent in the .first aliquot) is added to the vessel (2 of Figure 4) and the vessel and its contents are again frozen. These sequential cycles (he,, the repetitive addition of liquid with: a particular concentration of agent, followed by freezing) is continued until all desired different concentrations of agent hav been added and frozen along the length of the mold. Figure 4 illustrates 4 of such cycles. The addition of the I squid alginate solutions to die frozen aliquots in the casting vessel likely induces a very slight amount of melting in the frozen samples. This slight -.melting likely helps each section of the gradient t freeze into a solid continuous structure that undergoes polymerization into a thread, a fo lows: once the gradient is completed, -the frozen alginate column is extruded, from the easting vessel (e.g. using air or a plunger) and polymerisation is induced, for example _* by contact with: calcium, e.g. via extrusion directly into a 2% calcium bath (typically at room temperature), ("are is required so that the frozen alginate gradient does not rneli prior to entering the calcium bath. Upon contact with the calcium bath, the frozen alginate begins to thaw and immediately or: simultaneously polymerizes into a. fiber or thread. This fiber is rinsed (e.g. in a 2% calcium bath supplemented with 5-10% propauoi); dried; and then "cured ^* ' in a final rinse of, for example,, hexaSluorisopropanol (HF!P) to fftrrrt a solid yet flexib le fiber of alginate. Fabrication of a DDG gradient is carried out in a .similar manner, except that each. ^subsequent solution that is added contain, a different concentration of carrier, & some segments, the concen ration, of the incorporated one or more agents may be ¾ero, i.e one or more of the agents may be absent from on or more of the segments of the thread.

The carrier threads are substantially cylindrical, and the final dimensions of the camer thread are in the range of from about ^" 5mm to 1,25 mm in length ( .g, about 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, SO, 85 0, 95, 100, 105, M 0, 115, 120, or 125 mm in length) and once completed, and ready for implantation may be on the order of about 0.5 to 2.5-3.0 mm in diameter, and usually about 1 -2 mm in diameter. Bad) carrier thread generally comprises at least two segments (e.g. segments A, B, .. , n., where n ranges ^• from about 2 to about I OC) (e.g. about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 6 , 70, 75« SO, 85, 0, 5, or 100. or even more) and where each segment contains a different coneentration of at least one substance of interest (e.g. a therapeutic agent, a poiymerizealiie polymer, etc:). However, single segment threads are also encompassed by the invention. The segments may be of equal or different lengths, For example, a 15 torn, thread may include 3 to 5 segments containing distinct concentrations of a therapeutic substance, and each segment; may be the same length along the longitudinal axis of the thread, or the lengths of individual segments (which may be determined by specific applications) may var front one t another. Individual segments of a thread will; generally range from about 0.5 mm to about 5 mm. in length, i.e. about 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3,5, 4, 4.5 or 5 mm, but may he longer e.g. up to abont 10, 15, 20, 25 or 30 rum. or more in length. In one embodiment, the carrier or gradient threads of the invention may be used in the nerve guides described herein. However, this need not always be the case. The earner threads y be used for many other applications- where it is ^' desired to place a solid yet flexible, miero-aized biologically compatible gradient carrier, for exam le, to gradually release therapeutic agents within a relatively small targeted area, e.g. within the brain, the eye, the heart, at or near the gums, etc, Otfier applications include the use of carrier materi als cast in the form of sheets.

Fabrication of gradient sheets f the inve tion is essentially the same as that of threads, except tha the moid is wider in one dimension.

Threads may be prepared to direct the formation of biood vessels along a defined path, as tor example in -the heart where it ma be desirable to "ti ue engineer an artery in situ" to repair a blocked coronary artery. This may be accomplished by fabricating a gradient thread containing the appropriate signaling molecules and positioning it adjacent to an existing vessel, hi essence the signaling gradient, is designed to coax existing endothial cells to migrate along the _. gradient. The threads may also be com ine within or adjacent; to a small diameter (2-4 m) PDS based cylindrical construct produced by eleetrospinning or other suitable method (e.g. extrusion and or easting). By laying track of PDS; containing (o next to) the alginate signaling thread the mnnatlon of a blood vessel can be directed to form, along a specific axis of orientation (cg, see Example 6). Other polymers, including specifically, ^■ collagen, flbroneetia and ^' fibrinogen ^' may be used in this application as well as other natuxai and synthetic polymers, In applications using PDS, the unique characteristics of PDS fibers are exploited, e.g. that they ar■ generally 0.5-1.0 (but not restricted to) .microns in average diameter:, and thai they promote the formation of capillaries and in some

embodiments larger diameter blood vessels. The PDS cylinder can include various therapeutic agents or cells, e,g; it may be p.re~seeded with stem cells, endothelial cells and/or other desirable cell types for use in thi type of application.

Sheets of gradients can be used in. treating larger scale. in uries suc as bfctrns and other dermal injuries to promote skin regeneration. Gradients can be man factured into these sheets such that the signals run fforn a. low concentration along the edges of the sheet to a higher concentration in the middle. This type of gradient wonld be intended to promote and accelerate the mi gration of cells across the surface. This type of sheet might also he applied to transplanted (including by not restricted to the heart and. kidney) or tissne engineered organs (liver, and bladder represent two candidate sites for this type of application) to promote nascent blood vessel formation and or differentiation of stem cells or engineered, cells. The; purpose of these constructs is deliver gradients over a suf face that is too large to be effectively treated with gradient threads. Fabrication of gradient sheets of the Invention is essentially th same as that of threads, except that the mold is wider hi; one dirnension.

Gradients may also be used Or for the targeted release of toxic substances withi tumors; or other areas where an excessive cell, proliferation or migration is undesirable. The gradient threads may be used for targeted slow release of substances of interest in. any liquid environment in which the polymerized carrier will degrade or dissolve, thereby releasing (delivering) the entrapped subsianeefs) of interest (e.g. within the body of a bird or animal, wtthin a plant, etc.), when one or more: micro-sized carrier threads are placed or implanted at a targeted area of i erest.

In the coarse of developing this technique, it was discovered that rinsing alginate constructs wit HFfP greatly increases th ir stability in aqueous buffers, As a direct result, this increased stability also serves to drastically slow the overall release of agents such as functional growth factors front alginate based materials. For example, release ean be detected up to and even beyond about 2 weeks. This is in contrast to conventional alginate systems, in which agent release is substantially complete within 3-4 days. Additional control over release kinetics can be achieved: by adding such, agents as heparin -sulfate to the alginate, this specific factor is highly charged and binds peptides and other charged agents, thu slo wing release.

After curing, the ill anient is ready for ineorporation into a tissue engineering scaffol or other construct. As can be seen, this ^' maaiufaciiwing method allows ihbrieafion of specific gradients across very precisely defined spatial domains along the length of th fiber. In addition, upon hydration, the cored fiber is very flexible and does not swell appreciably, and thus is. ell suited to inclusion in the nerve guides of the invention,. Typically, the cured alginate fiber is introduced, into a. nerve guide during the spinning process. A polymer ^' is span and induced to fonn a guide with a diameter of approximately Imnr, the gradient threads are applied to the ekctrospun construct and the spinning process is resumed. In some

embodiments, more than one thread per construct may be incorporated into the nerve guides, and, especially in the central nervous system, it may he desirable to have the gradients precisely positioned to more efficiently provide signals to speci fic cell populations. Also, the orientation of the gradient may be positioned in any direction (e.g. parallel with the long axi of the ui e^ at right angles to the long axis, or at an angle to the long axis), multiple threads of the same (or different} gradients may be placed in opposite directions or the same direction, etc, A gradient may be prepared such -that he middle of the thread contains a high concentration of material or a low concentration of material. Individual segment lengths thai make up the gradient threads that contain the different amounts of therapenfie reageuts can he of equal length or of different unequal lengths (some short some long etc.) as determined from the specific conditions encountered in an injur -bed.■ Alternat ely, a uniform coneenp¾tipn of material m.ay be desirable in. some clinical applications. Any number of possible configurations can be produced with this method.

In some embodiments, the alginate threads may be processed at room temperature and not frozen. For exam le, this may he done when cells are to he incorporated into the fibers, tinder these circumstances, the gradient may be produced by manipulating the

- 3.7 - ^• viscosity of -the carrier soluti ns, This can often be easily achie ed by increasing the concentration of alginate or through the addition of other substances, e.g. sugars. By carefully preparing fee gradient through the gentle addition of each subsequen aliquot, a gradient of material, that has not been froze can be prepared. The gradient is theft polymerized by extrusion as. described. We note that gradients cars also he prepared by placing aliquots of material into a trough and adding polymerization, agents to the trough. I this way, liquid (or frozen gradients) may be less disturbed during the polymerization step because extrusion is avoided, ft : is also clear that gradient fibers do not have to be exposed to drying prior to- use and in some applications, for example those containing cells, it wilt he desirable to keep them in a hydrateei state. Alginate threads are relatively stiff when dried; it may be desirable in some applications to incorporate plastizing agents, (for example, PE j- polyethylene glycol) into these structures to adjust flexibility, e.g. to make them more flexible, hi all of these embodiments, therapeutic agents incorporated into the alginate threads are released as the alginate dissolves:.

A: schematic representation of a cross sectional view of a electrospnn nerve guide with a carrier thread is presented in Figure 5. As can be seen, this schematic contains a nerve guide TO with one exemplary open channel 20 (not to scale as die channel would ordinarily be on the same size scale as the individual fibers that comprise the grafi) with channel, opening 21 at the surface of the cross section, and a centrally located carrier thread 50 with end 54 at the surface of the cross section (tMs fiber is also not drawn t scale). Carrier thread: 50 is comprised .of a plurality of contiguous yet distinct segments SI, 5,2 and 53, each, of which contains a diiferent concentration of active agent 60 therein (e.g. carrier thread 50 provides a step-gradient of active agent 00)· With tune, carrier thread 50 dissolves,, releasing active agent 60 into the surroundin fibers of the guide (not shown, for the sake of clarity), and into open channel 20,. As axons grow along open channel 20, they are exposed to active agent 60, Further, the concentration of active agent 00 will be greater in the vicinity of segment 53, which has a relatively high concentration of active agent 60,. than in. the vicinity of segment 51, which, has a relati vel lo concentration. Such differences in the

concentration of an acti ve agent may he used to modulate ax nal growth. For example, in this illustration, if the active agent is a growth factor, the growth rate of the axon would he more rapid (would accelerate) as they progress through the guide and encounter the higher concentrations of active agent near the distal end of the guide. The advantage of this type of structure is that the threads.. of alginate- can be laid directly Into the forming fiber arrays of a nerve guide . For spinal eord reconstruction i t is possible to place the fibers hi specific sites to direct the reeons itution of nennaX spinal, c rd topography. In the spinal eord axons originating above an injury as well as below an injury must grow across the injury site (i.e. two way traffic is effect). The alginate threads make it possible to produce gradients over very short distances (e.g. less than 100mm) and in a. manner thai can be optimized for axons growing up or down across the injury site. For example growth factor A (or pharmaceutical reagent) ma be required in gradient that runs from, below and injury to above the injury, growth factor B (or pharmaceutical reagent) may need to be in a gradient that runs from above the injury to below the injury. To achieve this th gradient threads are prepared and placed in appropriate orientation. It should be emphasize that these gradients can be produced and customized to specific injuries over the •relative short distances (<l -2 rnrn, 2-5 mm, 5-10 mm, 10-30 rani, and > 30-1.00 aim) that may be encountered in an injury in the spinal cord or in the peripheral, nervous system.

Also, while in addition to signaling and pharmaceutical gradients, it is also possible to incorporate various other proteins such as extracellular matrix (ECM) proteins or ^■ specific peptide factors like RGD (i.e. the Arg-Gly~Asp peptide, the activity binding moiety for many cell surface receptors) in gradients into these threads. By doing so as the thread begins to break -down that incorporated ECM proteins are released in a gradient fashion and become attached io the fibers in the immediate vicinity. In thi s was a gradient of binding elements on the fibers can. be produced t© enhance ceil migration, and or axon elongation down, the protein gradient. Cells will grow along a binding gradient in preferential fashion from a low concentration of ECM protein to high concentratiou.(or densi ty) of ECM protein. This provides yet another wa to provide guidance cues to cells to direct them to grow down the elongated pore spaces present in the nerve guides.

Variations in the constructio of carrier threads may be made so as to tailor the threads to particular purposes and/or to improve the results that are obtained. For exa rsple, more than one substance may be prepared in a given gradient thread, and the gradients of these multiple substances may be nniqnely tailored and ma be uniquely different f orn one anther or even running in completely opposite directions. In another example, the release kinetics of alginate threads can: also be modulated, b coating the exterior of the threads with various materials including such biodegradable polymers as PGA, PLA, P.D.S or other Suitable polymers to slow the br ak o n of the alginate and r to increase the material strength, of the construct The surface of the carrie fiber can be modified by electro spraying or electrospinnin various natural and or synthetic polymers onto the surface of the structure. This provides far mor subtle control over the structure of the construct than simply dipping the material into: a polymer bath. Multiple reagents can be added to sin le alginate thread in any number of different configurations id concentrations. Some may e placed into the fiber as a, gr dient, while others may be present at a continuous concentration, or even just at one specific site in. the fiber (e.g. one of the ends or in a middle position}:. Further, mul tiple fibers con.ta.Mag identical or different doses or gradients of materials might be used to ^• fabricate a construct. For example, by electrospinning a cylindrical construct and positioning alginate fibers in specific positions within the .scaffold, it is possible to deliver specific ^• factors to different sections of the guide in a highly precise ^. .manner.

In one embodiment, alginate is used in. the fabrication of carri er threads. Reasons for this include that alginate is water soluble and thus easy to work with, is relatively ^■ non-toxic, and dissolves or is absorbed, slowly by the body in a manner that can be controlled by adjusting the concentration.. However, those of ski.il in the art will recognize; that other substances may be used to fabricate the carrier threads, examples of which include but are not limited to various carbohydrates, proteins such as the ECM proteins of the collagen family, fibroneetin, fibrinogen, intracellular proteins such as actin, and or synthetic materials such as hydrogeis and other bibeompatiahle polymers (e.g. PDS, PEG, PGA, FLA,

J.GA/PLA copolymers and others listed herein). As .an. example, PDS can be. dissolved in HFIP and mixed with, varying amounts of therapeutic agents and extruded and allowed to dry to produce a fiber, using methods much like those used in, the production of sutures and fishing, line. ^' Therapeutic agents/substances/reagents that may be incorporated int the carrier threads of the invention include but are not limited to anesthetics, hypnotics, sedatives, s!eep inducers, antipsychotics, antidepressants, an iailergics, antianginals, antiarthrities, antiasthmatics, antidiabetics, anttdiarrheai drugs, anticonvulsants, antigout drugs, ^• antihistamines, antipruritics, emetics, antiemetics, aniispasmondies, appetite suppressants, neuroactive substances, neurotransmitter agonists, antagonists,- eceptor blockers, reuptake .modulators, beta-adrenergic blockers, calcium channel blockers^ disulfarirn, nniscle relaxants, analgesics, antipyretics, stimulants, ^' anticholinesterase agents,

parasympathomimetic agents hormones, anticoagulants, arm ^' fhrombotics, thrombolytics, imi meglobulins, i mu osup ressants, hormone agonists, hormone antagonists, vitamins, antimicrobial agents, antineoplastics, antacids, digestants, ta&aiives, cathartics, antiseptics, diuretics, disinfectants, fungicides, ectoparasiticides, antiparasitics, heavy metals, heavy metal antagonists, chelating agents, alkaloids, salts, ions, autaeoids, digitalis, ^■ cardiac ^' glycosides, antiarthyihmics, antihypertensives, vasodilators, vasoconstrictors,

antimuscarinics, gangiionic stimulating agents, ganglionic blocking agents, neuromuscular blocking agents, adrenergic nerve inhibitors, antioxidants, anti-inilanitnatories, wound care products, amithrombogemc agents, antititfnoral agents, aniithrombogenic agents,

antiangiogeuic agents, antigenic agents, wound healing agents, plant extracts, growth factors, growth hormones, cytokines, immimoglohuHos, osteoblasts, myoblasts, neuroblasts, fibroblasts, glioblasis; germ cells, hepaioeyt.es, chondrocytes, keratinoeytes, smooth muscle cells, cardiac muscle cells, connective tissu cells, epithelial cells, endothelial cells, hormone-secreting ceils, neurons, emollients, humectants, anti-rejection drugs, spermicides, conditioners, antibacterial agents, antifungal agents, antiviral agents, antibiotics,

tranquilizers, cholesterol-reducing dregs, antitussives, hisfamine-bloeking drugs and monoamine oxidase inhibitor, nucleic acids such as DNA. RNA,(e.g. siRNA), etc.

Release of agen from the carrier thread generally occurs over a period of time ranging from at least about 1. hr to about 35 days, and usually from at least about 1 day to about 21. days, However, the use of coaling and oar alternative polymers in the ' ^' fabrication of the threads can be itsed to modulate these intervals to shorter and longer times,

in one embodiment, the carrier of therapeutic substances is a thread or fiber, it is also possible to use a modified form of this fabrication method to produce heads, disks, sheets, or other shapes of the carrier containing reagents. ^' These disks might be placed i various places to provide local reagent delivery. For example, disks might be prepared and applied on or into the brain. This would, make it possible to deliver various: reagents over a prolonged period of time f om a position, behind, the blood brain barrier (many therapeutic reagents can not pass this barrier to reach the brain or other components of the CNS). ^' To fabricate a disk a reagent of interest is mixed with a specific carrier (e.g. alginate;

concentration. The supplemented alginate Is dropped into a calcium bath to induce polymerization, as described; the resulting bead ½ gently compressed, and then frozen. This produces a thin disk of alginate supplemented with the reagent of choice. The size and final shape of the bead can be regulated by the: volume- of alginate: used i:o prepare: the bead and/or by using different surfaces with, varying hydrophobic ro erti s:. In: addliiotg such structures could be layered and or coated as described for thg alginate fibers to differentially delivery different or th same reagents.

In yet: other embodiments, very long term release kinetics (e..g, for periods o ^' time rar ging from about 35 days to about 1 year can be achieved by encapsulating various agents of interest (e.g.- therapeutic materials) into microb ds ( "microearri rs", e.g. of alginate or some oilier suitable substance) using electro spray technology; and, optionally, incorporating the mierobeads into a earner thread. This treatment sequesters the re-agent of interest within the nticrobead and. away f om, the -harmful effects of organic solvents. The beads are then be mixed with another slowly degrading polymer, for example typically biocompatible polymers could include PGL, PDS. FLA., PG A and or copolymers of PG A, PLA and. or other polymers. The mixture is then, extruded to produce a carrier thread or fiber containing the alginate mierocarriers and the: encapsulated materials, which are effectively immobilized within the mierobeads. As- the polymer fiber breaks down the incorporated alginate mierocarriers are exposed and. begin to breakdown, releasing, the eageni:. A similar approach •could be used to produce disks and or other shapes that are tailored to specific applications, e.g. for use in the nerve guides of the invention, or for other purposes. See Figure IB and the figure legend for one method of producing micro carrier beads by eiectrospray.

The invention further provides methods ¹ for guiding or facilitating the regenerative growth of severed or truncated nerves in a patient in need thereof The method involves attaching (e.g. suturing) a first end surface of a nerve guide of the invention to th -proximal end of a severed nerve and attaching a second end of the nerve guide to the distal end of the severed nerve. In. other words, _, in the peripheral nervous system, the guide is interposed between the two nerve stumps and is used to bridge the gaps between the two. Irs. the process of joining the nerve ends to the guide, the guide and nerve ending may be moved or manipulated in order■¾ achieve a suitable orientation of all three, entities, within the limits imposed by the length of th e gap bet ween the ends of the nerve. Since the natural tend ency is for the proximal nerve end to begin to regenerate, when this end is attached to the guide, the regenerating tissue will encounter the guide and be able to preferentially grow ^' only into tile void space of .the guide, i.e. into the open channels, since the rest of the space is filled with the fibers which make up the guide, in order to insure proper entry of the regenerating axons into the guide, the nerve stumps are sutured or otherwise attached to the guide a closely as possible, and may even be partially inserted into ie guide. In spinal cord injuries axons will grow into the . guides from both ends as there are ascending and descending axons in the centra! nervous system. In these injuries it is ^■ unlikely the guides will be sutured in place, rather they may be glued or he d in place by the surrounding tissue so as not to disrupt the regeneration process.

The nerves that are regenerated in the peripheral, nervous system using the methods of the invention are generally peripheral nerves, examples of which include but are not limited to sciatic nerve at its terminal branches including the sural branches and die tibial, common fibular, superficial and deep fibular, and plantar nerves. he femoral nerve including motor and sensory subdivisions. The roots, divisions and terminal nerves of the brachial plexus, this includes the major divisions such as the niuseulotaneous, median, ulnar and radial nerves, aid the axial nerve. Also included axe more distal branches including the anterior interdsseus and posterior interosseas, the ulnar recurrent deep and superficial radial, and other named and im-named terminal branches. In the central nervous system potential repair sites include the spinal cord proper and nerves of the cauda equine and nerve rootlets exiting the spinal canal Generally, for the methods of the invention to be suitable for use, the nerve will have been, injured in some manner, e.g. severed, partially severed, crushed, o otherwise damages so that the integrity of the nerve is compromised. The injury is usually accidental,, but may also occur as the result of another necessary procedure, e.g. a. surgical procedure.

The subjects in whom the nerve guides of the invention are used are generally mammals, although this need not always be the ease. The mammals are generally humans, but this also need not always be the ease as veterinary used are also contemplated,.

EXAMPLES EXAMPLE 1.

An ideal nerve guide configuration would cfoseiy mimic the structure of an.

autologous nerve graft. This idealised guide would display a cylindrical: shape and be coniposed of dense three dimensional (3D) arrays of highly aligned e!ectrospun fibers that were Oriented in parallel with the long axis of the construct Gaps and. elongated spaces between the stacked, fiber arrays would provide thousands of channels for directed axonal growth. This type Of configuration can be approximated, by rolling a 2D sheet of aligned fibers into a cylinder. Unfortunately, rolling a 2D sheet will result in a seam Irs. the construct that could provide an avenue for the infiltration and penetration oiirrOammatory and intersti rial celts into the construct. The seam could also represent a nexus, for mechanical failure. Air gap eiectrospinning makes it possible to circumvent" these limitations and produce cylindrical, seamless, and. truly, 3D constructs composed of aligned arrays of electtospun fibers that are oriented in parallel with the long axis of die cylindrieai construct. in this study, we characteris the air gap eiecirospinuing process and examine how this fabrication technique can be exploited to produce nerve guides that .facilitate the regeneration of peripheral: nerve fibers, Poly¾-caprolaetone (PCL) was chosen as the polymer for the manufacture of die nerve guide doe to its slow rate of degradation [19], thus it ca ac as a guide for the new axons throughout ¾e nerve regeneration, process. PCL was dectrospnn at -varying concentrations for in vitro testing to determine the most appropriate air gap eiectrospinning variables for the manirtacture of constructs suitable for directing tlie axons in peripheral nerve injuries,

METHODS

eiectmspimingi PCL (Sigma; PCL 65,000 M,W.) was dissolved in. inoroethanol f ^' fFE) at various concentrations, including 50, 75, 100, 125, 150, .175, 200, 225, 250 and 275 mg/rat .Solutions were loaded into a 1.0 rui. syringe that was capped with an 18 gauge blunt-tipped needle. Conductive, circular washers of varying diameters were placed over the blunt tipped needle; several different eiectrospinning configurations were tested at each concentration of PCL,

The air gap electrospinoiug system, used, in thi study consists of two vertical piers grounded to a common voltage, typically set to -4.0 to-16.0 kV (Figure 6, point A). From each vertical pier an additional set: of horizontal piers project inwards at 90° with respect t the upright piers (Figure 6, point BL A gap (which can be adjusted from about 1-0 inches) separates the terminal ends of these projecting piers.

Figure 6B shows a close-tip view of the part of die apparatus wherein the ne e: gihde is formed. This figure illustrates a simple set-up to produce cylindrical semi-solid nerve guides composed of 3D fibers arrays. More sophisticated systems can be iabrieated ihat use rotating targets,, but in this simple example the target 'arrays i static. This system uses 2 grounded targets. This static (no rotation of targets) system is constructed of 2: parallel vertical piers (A of Figure 6B) grounded to a common voltage (-3. to -10 kV). Each pier is capped ith a (grounded and. insulated) metal sphere (B of Figure 6B), from each grounded sphere additional (grounded and also insulated) metal piers project inward at 90° with respect to the upright piers inwards towards one another (C of Figure 6B}. The inwardly projective piers ar capped with small metal (non-insulated but grounded) targets, these intensity th electric field. An '' ir ' gap (adjustable from about 2-6. inches) separates the terminal ends of these projecting target piers, live eleetrospinning stream is directed into She air gap separating the grounded piers. As.. the charged polymer stream reaches the target it is kid out ip a series of loops that pass back and forth between the tenslnal portions of the grounded piers and collects as a parallel array of fibers (D of Figure 68). The elliptical nature of the fibers, illustrated in the schematic are greatly exaggerated, i reality of the fiber are deposited in a nearly linear cylindrical bundle.: Air gap eleetrospinning is very effective at in.dueing the alignment in small diameter fibers, which can range from. <200 nn\ to several microns in average cross sectional- diameter, and depositing them into macroscopic- _. Structures (u to at least 10 mm in diameter) 3D cylindrical arrays. The densely packed and highly aligned fibers of the 3D nerve guide of the invention that are produced in this manner resemble the anisotropic structure present within an autologous nerv graft. The fibers ar arrayed in parallel with the long axis of the constructs and in cross section, these cylindrical scaffolds exhibit dense bundles of fibers Alignment in conventional, eleetrospinning systems can be highly dependent upon fiber diameter ( 14, 15)

in. this Example, eleetrospinning solutions were charged to +22 kV-and directed into the gap separating the grounded horizontal piers. Polymer solutions were metered into the air gap system using a syringe driver with rates of delivery varying from 2-20 ni l/hour (see Table 1 for specific conditions). The distance between, the solution reservoir and ground target array was varied f om: 1.0 em to 30 cm. Once the charged eleetrospinning jet forms in this type of spinning, the polymer stream reaches the: target and is laid out in a. serie of loops that pass back and forth, between the terminal portions of th " grounded piers, resulting in the formation of a bundle of parallel fiber arrays (Figure: 6A, point C), Conditions -.for eleetrospinning at each PCL concentration were optimized to maximize fiber formation and collection onto the mandrel. The eleetrospinning conditions disclosed in. Table 2 are optimized to the specific laboratory environment (e.g., 68 ^a F with approximately 40% humidity) and eleetrospinning cabinet. Adjustments; m.ay be necessary to account for varying: ambient conditions and differeut po!ymers, Tabic 2. Summary of specific efec mspir ing conditions fo POL in the two pole elect ospinning system. With increasing PC concentration the target voltages, the electrospimiiug distance between the syringe reservoir and target as well as the distance between the poles o f t ie air gap system -must be reduced in order to induce fiber deposition across the poles.

Routine Scanning. Eteei n Microscopy ΕΜ}, A Zeiss EVO XVP scanning: electron microscope equipped with digital acquisition was used lor image capture. Electrospun constructs, were remo ved from the target and cut into 3 sections of equal length. Bach of the 3 segments was mounted onto a. scanning electron microscope stud and sputter coated.

A erage fiber diameter was . ^' determined from ^::: 3-5 SEM images captured at magnifications ran in from 4S - 5O0OX fkftn each of the 3 sections using the NIH IniageTool software

Fiber gnni u analysis. Using the digital SEM images captured -for fiber diameter analysis, the relative degree of fiber alignment s measured in each guide segment .using the NIH ImageJ 2D fast Fourier transform (2D EFT) function f 14,20). By using the 2D FI approach an. alignment plo can be generated. The height of the resulting peaks, read from the Y axis, reports the degree- of orientation, in the dat image. The position of the peak ^' s on the X axis reports the principal angle of fiber orientation■ [ 14,20 . The FFT ^■ alignment data was _:

normalized to ihe lateral edges of the cylindrical extracts which were arbitrarily assigned a value of 900 on the unit circle. The relative degree of fiber alignment and the principal, angl of fiber orientation were . determined for each of the construct formulations using the 2D FFT. ^' method.

Materials ^' Testing. ά Physical Properties, -Materials -strength, was -measured b nniaxiaiiy testing the construct to failure at an extension rate of 100 mm/rnin using a Bionix 200 Mechanical Testing Systems instrument (MTS Systems Corp., Eden S½airie, MN).

Cylindrical elecirospun samples were prepared from -variable concentrations of PCL (100, 150, 200 and 250 rag ml; N ~ 4-6). Testing the material properties of an eieetrospun sample iSi: typically done wit a flat sheet of material, whereas our constructs are cylindrical in nature. ^' To address this limitation, we elected, to flatten the scaffolds and the cut out

"dog-bone" shapes using a die punch (2 J? mm wide, gauge length of 0.295- mm), This approach, at least, allows Us to use a configuration. that controls for grip and geometry effects -to evaluate how fiber diameter in each of our scaffolds contributes to their material properties, -Specimen thickness was estimated using Mitutoyo ΪΡ54 digital micrometer ( dnioyo American Corp., Aurora, It). In this study, stress at failure lor these scaffold is reported [13.]. Data sets were screened by one-way analysis of variance (ANOV A ; p < 0. 1 }· The physical properties of the scaffolds were quantified in terms -of "their overall void space. Cylindrical, eleetros un sanvplcs prepared from- variable concentrations of PCI ^, ( 100, 150, 200 and 250 nig/ml; -4-6) were tested . using ihe liquid intrusion method o Phara el al 121 j. Scaffolds were elecirospun, put under vacuum for 10 .minutes and weighed £ Wl }. Then, they- were soaked in 90% ethano! for 10 minutes for initial, hydration, and.

subsequently in distilled water for 3-0 minutes, Bydrated scaffolds were weighed (W2). The void space as calculated by dividing the volume of intruded water (as determined by the change in mass due to intrusion of water h aving density of 1 g/nil) by th total volume after intrusion. Percent Void Space 100

Data sets were screened by Student- Newman-Ken is multiple pairwise comparison; p<0,05.

- 4 Cell Culture. orsa root ^" ganglion (DRG) explantx were prepared from embryonic da 15 rats as described previously [22,23]. P£L scaffolds for eel! culture experiments were prepared from representative starting concentrations ( 125, 200, and 250 mg/ml). Scaffolds were soaked In 100% e-thanol overnight, then rinsed in 70% ethanoi, and then rinsed 3 in sterile Phosphate buttered saline (PBS), A 25 gauge needle was used to prepare an openin into the dorsal surface of each scaffold, and a single DRG was inserted into the resulting cavity, DRG explams were maintained for 7-10 days in media supplemented/wit 0.1 μ§/ΐηί H ^' GF: Media was exchanged every other day.

Immumifhiorescmce Miercmcopy: DRG expiarm. DRG exptant were rinsed, in PBS and fixed in 4% paraformaldehyde prepared in. PBS. Samples were extracted in 0.1% triian and inrmunostained with the neuron specific marker Tuj I (Tubulin. J I : M S-43SP, Goyanee, l r-500). Antibodies were dilated in PBS supplemented with 1 % BSA and incubated with all samples overnight at 40C. Scaffolds were rinsed and eountefsfained with Goat anti-mouse antibodies conjugated- with Texas Red (1 :200). Ail samples were stained with DAT! to reveal nuclei, A Mikon TE300 microscope equipped with a iO objective and. a DXM 1200 digital camera was used to capture images at a. ixel resolution: of 3840 X 3072. Individual images of the DRG exp fan is were assembled into montages using. Adobe Photoshop software.

Et ctr&spimung for in viva -experimentation. For implant studies, nerve guides were eleeirospun from a starting concentration of 200 mg/ i PCLusmg optimized conditions (Table 2). To reduce inflammatory cell infiltration into the fiber arrays of the guide, an exterior coating of PGA; FLA (50:50) copolymer was electro-sprayed (TOO mg/rni in TFE) onto the outside of the completed- constructs. This was achieved by placing a circular, 120 mm diameter steel plate directly behind the completed nerve guide. To process the

PGA/PLA copolymer an alligator clip was attached to the electrospinning source syringe (no washer was used) and charged to +221 VVthe steel plate was charged, to -2kV tor collection. By placing the completed nerve- guide between the electrospinning source solution and the grounded steel plate, the fibers of PGA/PLA copolymer deposit on the -surface of the nerve guide construct terming a bam er thai is designed to limit the penetration of interstitial cells into the reconstructed nerve in vivo [17],

Surgery, All surgical and postoperative care procedures were performed in accordance with the Virginia Comntonwealih University Institutional Animal Care and Use Committee.

- 4:8.~ Nerve guides were prepared from starting concentrations* of 200 mg/inl for preliminary implant studies. Adult (70-90 day) Lang Evans Hooded rats (Ν^3 ^' ) were intubated and brought to a surgical plane with 2.5% isofinrane. Body temperature was kept BonBothermie using a homeother ie blanket. Hair was removed from, trie hindquarters, skin was swabbed wiihbetadine. Using sterile techniques, skin aad muse ie overlaying the sciatic nerve were mobilized., and a 10 ram segment of the nerve was excised. This interval represents a critical threshold short term regeneration experiments that can be used to characterize the efficacy o -a candidate guide in the rodent model Elecirospim nerve guides were sutured to the distal, and proximal stomps of the injured tissue using 10-0 nylon sutures (Bthicon, Inc. USA}. At the conclusion of surgery, skin incisions were stapled arid the animals were allowed to. recover on warming ad. After surgery all animals were given free access to food and wate and housed 2 per cage. Analgesic .medication (Tylenol oral suspension, 2ing mi) was mixed nto the drinking water and administered for the first 3 days post surgery. After ? weeks, animals were sacrificed with a lethal dose of pentobarbital (Sigma- Aldnch, St Louis, MO), and the nerve Implants were harvested for microscopic analysis.

Imm&Mtfiuorescence Microscopy: I phmted Nerve Guides. Tissue was immersed for 2 hr in 4% paraformaldehyde prepared in PBS at room temperature and. then incubated overnight in 30% sucrose prepared in PBS at 4 ^H C. The samples were frozen and cut into 30 μηι thick sections for staining _* Primar antibodies including, S 100 (Schwann cell marker, Dak , Denmark, 1 :S00 MBP (Myelin basic protein, SMl- 9, Covan.ee, 1:1000), and NF-dS (Neiirotikment 68, Sigma, 1 : 1500) were incubated on the sections overnight at 4 C.

Samples were rinsed in FBS-triion nd eounterstained with secondary antibodies (Invitrogen €>r Alexis) ^' for 60 minutes at room temperature. DAPlAvas used to image the nuclei.

Electron Microscopy. Tissue was recovered and immersed in 2% glutaraklehyde plus 2% paraformaldehyde for 12 hr at 40C arid post fixed in. 1 ,0% osmium plus 2.5% potassium ferricyanide. Samples were subjected to a graded series of dehydration and embedded in Poly/Bed (Polysetences). It is difficult to get good plastic infiltration into the dense matrix of .an. electrospuu scaffold that has been densely populated by cells; air bubbles are often trapped and interfere with subsequent processing. To overcome: thi limitation,, samples are placed under a vacuum during the final infiltration step. The sections were stained with toluidine blue/crystal violet and imaged on a Nikon TE300 microscope. All images were captured and stored in TIFF format. To determine the total number of axons passing through the implants, a montage of the ^■■ omplete cross sectional area of the graft was prepared from images captured with I0x; brighl&e objective km and a Nikon TB3C)u microscope. The images were imported mto the NIH ImageTooi to measure the area encompassed by the fibroiic capsule (induced by the PGA/PLA coating). Total axon number was determined, for a series of images captured at systematic intervals thfougliouf the nerye cross section using a l OOX oil immersion lens. Each raw data image covered an area of 125 μιη x 1.25 μτη and was subdivided into a series of 25 pro. X 25 μη* grid squares. The number of myelinated axons present in each data imag was determined by noti-bias sampling methods. To extrapolate the total number of axons present in the cross sectional area, the density of myelinated axons in the sam led images was multiplied by the total cross sectional area of the grafts. Data, images used to determine myelinated axon number were used to deteraune ^' individual ^' atonal cross sectional areas usin HIH ImageTooi. All measurements were calibrated with a stage micrometer.

mSVLTS

Ek irospinning ch ra ierisiics and fiber properties: The discussion concerning fiber properties is subdivided int three sections based, on starting conditions and eleeirospinnrag characteristics. Overall, the average fiber diameter of the scaffolds produced varied as a Junction of the starting conditions.

Starling Solutions 50-100 mg mL At concentrations ranging from. 50 to, 100 mg/m.i PCL, mere was nominal to poor fiber formation and fiber collection. Fibers appeared to coalesce out of an amorphous-cloud, of material that collected the vicinity of the target grounds. These scaffolds exhibited extensive heading; these beads were interconnected with an array of small diameter fibers thai were oriented in parallel with the: long: axis of the ..cylindrical scaffolds (Figure 7A-C). Maeroseopicajly, these cylindrical construct were predominately composed of aligned eiernepts.

Beaded structures can form in m eieeirospinning field as a consequence of Rayleigli instabilities that develop in the charged jet as a result of: (A) inadequate flow rates of the eleetfospmning solution; (B) electric field effects; iC) inadequate polymer chain

entanglements in the solvated spinning solution and; (B) too low of a starting polymer concentration [24].. Increasing the flow rate of the .polymer solution into the electric field did not suppress bead formation in these scaffolds, instead, the increased flow rat resulted in the accumulation of a dense c loud of solvent in f he vicinity of the target which prevented libers from forming .altogether. Nor was the format on of the header! structures visibly altered by directly altering the potential of the electri _. field,, manipulating the ^' distance between: the syringe reservoir and ground targets, or by changing She diameter o f the washer used to direct the charged electrospirmiog jet at the bi-polar target (key el rnents of specific electrospinaing conditions are summarized in 1 able 2). Gi en. these results it is clear that -a SO rng/ml starting solution represents the near absolute minimal threshold concentration for fiber formation to take place in this particular air gap eleetrosprrmiug system,

Starting Solutions 125-200 rng/mi Over this range of starting concentrations average fiber diameter increased and. was more heterogeneous with, respect to the fibers electrospun from: the 50-275 nig/mi solutions (Figure SB). As the starting PCL concentration was increased beyond 125 mg mi the beaded structures present in scaffolds produced from the lower starting concentrations became increasingly less apparent (Figure 7E-J), Average f ber diameter in scaffolds produced from the 125 rog/nd. solutions was 382 nm, these fibers ranged Irom 90 nm to i .3 μηι in cross sectional diameter. Scaffolds produced, from the 200 nig/m! solutions had an average fiber diameter of 90:6 nrn with a range of 60 n to more than 5.1 u.m in average cross-section a! diameter.

The visible components of the eleetrospiniung field changed, dramatically over this range of starting concentrations. At 125 mg mf the charged eiectrospinning jet was distinct and composed of a continuous jet of material that emanated from the syringe tip; this jet was several centimeters i length. As the charged jet approached the targets ¾ became unstable and fibers appeared to form out of an. amorphous e!oud of material. As the starting concentration was increased to 150-200 rag rn! the charged, jet appeared as a distinct series of spiraling loops that originated irom a prominent Taylor cone. The appearance of th s stable jet coincided with a marked reduction in bead formation, a physical property indicating that sufficient polymer chai entanglements are present i the solvated. spinning solution to suppress the Rayleigh instabilities observed at lower polymer concentrations, The spiraling jet of materia! was directed towards the gap between the two poles of the air gap system. Fibers .formed in the immediate vicinity of the grounded piers and were visibly collected across the gap separating the two grounded targets. As additional fibers collected across the targets, the: re-existing fibers collapsed into a much: more cornpa i cylindrical structure. With the formation of the spiraling jet of charged material it was necessary to reduce the distance between the eiectrospinning source reservoir (syringe) and the grounded targets

-SI - (Table 2 _· >, Fiber deposition was optimized when the ^■ apparent diameter of the spiraling jet coincided witli the distance between the piers of the bi-polar ground, ^" tinder these conditions fibers were observed to rapidly accrue on the ^' .ground targets.

Starting Solutions 225-2$ 5 mg ml. The relation ships betw en the starting solution properties an final fiber properties was less well defined m scaffolds produced from ..starting concentrations greater than 200 rog ml (Figitrs SB), Scaffolds spun from, the 225, 250, and 275 nig/rni solutions exhibited marked heterogeneity in fiber size (Figures 7H-J and Figure 6). For example, scaffolds produced .ftotn the 225 nig rol solutions had an average fiber diameter of approximately Sib m» with a range from 120 ma ^' to approximately 3.5 \im. At 250 mg/ml average fiber diameter increased to 1.7 tm and the scaffolds exhibited fibers ranging from 240am to 6, μω. Average diameter in scaffolds prepared from the 275 mg/ml solutions decreased to 750 nm. substantially less than the average fiber diameter observe in scaffolds prepared from starting solutions ranging from 125-250 mg/ml (Figure 6).

in order to fabricate scaffolds from the 225 and 250 mg/ml solutions it was necessary io increase the diameter of the washer used to direct the electrospinning jet towards the grounded target. We suspect that the mass and momentum of the electrosp inning jet at. these higher starting concentrations restricts its lateral deviations as the jet passes from the syringe to the air gap targets. As we increased the diameter of the washer placed o ver the syringe,, the lateral deviations in the spiral jet increased and fibers collected across the grounded ^■ piers forming a cylindrical construct. In parallel with this change, it was also necessary to (A) move the grounded targets closer to the electraspinning reservoir, and (B) reduce the distance between the piers of the bi-polar ground (Table 2), These modifications appear to bring the targets within a domain of the electrospinning field that corresponded to the diameter of the spira!ing jet of charged polymer,

Fiber alignment , 2D FFT analysis of die different scaffolds produced In this stud demonstrate that fiber alignment is similar, regardless of fiber diameter, when scaffolds are produced using a bi-polar grounded target tin contrast to scaffolds produced in a

conventional eleetrospijining systems where fiber diameter and rates of mandrel rotation interact to determine the extent of .alignment). In contrast to conventional systems, varying the PC concentration and/or increasing the average fiber diamete had little effect on fiber alignment in. scaffolds produced by the air gap system (Figure 9D-E), Not surprisingly, the principal mgU of fiber alignment k _\ all of these scaffolds was in parallel with tir long axis of the c lindrical constructs (Figure 9). However, there were some subtle differences across the samples that we examined. Scaffolds produced from the 50-100 ing/mi solutions contained structural elements, that partially degraded alignment valises. These scaffolds each exhibited. a characteristic shoulder in the 2D FFT alignment plot (occurring at about 1S00). The headed stmctures arid the off axis fiber that are present in this family of scaffolds undoubtedly contribute to these results (Figure 7), Even, so, these scaffolds still exhibit considerable fiber alignment, and for this family of constructs the 2D ^' FFT analysis generated alignment values ranging fom 0, 14 to approximately 0, 1.6 units (Figure 9A), The 2D FFT analysis reported that constructs produced front starting concentrations of 125^200 tng/ml lacked the distinct shoulder detected in scaffolds produced from lower starting

coiicentraiions of PCL. Overall, these scaffolds were incrementally more aligned than scaffolds produced from, the 50-100 m gVml solutions (Figure 9B). A similar trend was observed in scaffolds prepared from the starting solutions of 225-275 rng/ntl and these samples displayed the highest degree of fiber alignment (Figure 9C).

Materials lasting and Physical Properties. To verify the structural properties of scaffolds produced in the air gap process we conducted materials testing experiments (stress at failure increases as a ^' function of fiber alignment in eiectrosptin materials). 2D FFT analysis indicates thai fiber alignment incrementally increased as a function of starting PCL concentration (and increasing fiber diameter), A similar trend was noted in the bulk material properties of the scaffolds (Figure 1 ). For example, peak stress in scaffolds produced from the 100 mg ml solutions was - 2.00 Mpa (Figure lOAf This value increased to 3,25 MPa m scaffolds produced from the _: 150 mg/mi solutions, and to 4.25 MPa in scaffolds produced from 200 mg/ml solutions. Peak stress was 3.5 MPain scaffolds produced ironl the 250 tng/ml solutions. Statistical analysis of these data indicates that scaffolds produced from 150, 200 and, 250 mg/mi PCL exhibited, similar material properties at failure. Peak stress in each of these constructs was greater than what was observed in scaffolds produced .from. 100 mg/ml PCL (P< 0O7) TLe«e values, with the exception of scaffolds produced f om the WQ mg mi sohufons, compare favorably to the material properties of native rodent scia ic nerve

While each scaffold is composed of highly aligned fibers, considering the variable fiber diameters, the spaces between each of the individual fibers can be expected to be very different which. would influence the penetration of regenerating axons. Our analysis, as determined by liquid intrusion- measurements, revealed significant differences in. scaffold void space ("porosity") develop as a function of starting conditions (P<0,05; Figure 10B). Average void space for scaffolds produced from 1.00 nig/rnl solutions was approximately 58% and _. ranged to greater than 95% for scaffolds produced from the 250 mg/ml starting solutions.

Cell mitur experiments. While each of the scaffolds produced in this study di d. exhibit similar degrees of fiber alignment, there are additional considerations that must ^' be accounted for in. the selection of a candidate eonstmet that is to be used in nerve reconstruction. For example, scaffolds produced, from 50-100 mg ml PCL exhibit extensive ^' l ding and tensile properties that are below that of the rat sciatic nerve [25] . The beaded structures could represent obstacles to regenerating axons and or disrupt guidance cues present in the aligned fibers. At the other extreme, scaffolds produced from the 275 mg/ml solutions have highly aligned arrays of fibers and material bulk material properties that exceed the sciatic nerve of the rodent. However, the e!ecirosptnning configuration thai is necessary to process these solutions into a scaffold results in very . hort constructs, limiting their utility in a clinical applications,

An additional consideration regarding the .physical properties of the constructs concerns bow the functional porosity of the caffolds af eets the axonal regeneration, In these assays we tested, how effective different scaffold, configuration were at supporting the penetration of axons into the fiber arrays using explaine dorsal root ganglia (DRG) as a model system. Scaffolds produced from the 1.25 (average fiber diametep ^:: 383.ηηττ/-228 nm S.D.), 200 (906 am +/~923 mil} and 250 (1 ,66 ^" nni ^■ ÷/- 1 J 65 mn) mg/ml PC I, starting concentrations were tested i these assays (Figure 1 1 A),

D Gs were implanted directly into the cylindrical seafi lds (scai!elds were prepared with a length of 10-15 mm and 5-7 mm in diameter ) and cultured for 7 days in the presence of NGF, At the ^' conclusion of this experimental interval all the scaffolds exhibited axons that projected into and parallel with the surrounding fiber arrays (Figure 1 1D-F). These experiments suggest that constructs composed of smaller diameter fibers foster the penetration of numerous individual axon along individual PCL fiber tracks or perhaps more accurately within the pore spaces). This pattern was particularly evident in samples cultured in the scaffolds prepared from the 325 mg/ml starting solutions. At the other extreme, DRGs plated into scaffolds prepared from the 250 rng ml solutions exhibited pronounced iaseiculatiom (Figure 1 1 F), Irs these cultures the axons appeared to interact with one ano ther, form handles and then pass along far fewer fiber tracks. A .nuclear stain of the expiarited cultures revealed thai cells associated. (Schwann cells arid interstitial fibroblasts) with the DRGs heavily infiltrated the scaffolds arid tracked in. association with the DEG neurons (Figure 1 IG%

Preliminary peripheral nerve reconstruction experiments. For in vivo testing, we prepared nerve guides from the 200 mg/ml starting concentrations of PGL, This construct fomHilaiion exhibited highly aligned fibers, excellent material properties apd, extensive void spaces to support the penetration of a ons along individual tracks (which in cell culture experiments suppressed axon iaseieulation). ^' In. these ex eriments, a 10 mm section of the right sciatic nerve was removed and replaced with an eleetrospun nerve guide of the invention,.

During the 1 -2 weeks after surgery, the lesioned animals walked on. 3 limbs and earned the injured limb. Motion at the knee and distal joints was substantially absent. After 3-4 weeks the animals carried weight on the injured leg, but gait in this limb was

characterized by a pronounced abduction at the hip. By 6 weeks, gait improved and the exaggerated abduction at the hip was replaced with motion in more normal planes as mobility increased at the knee and ankle joints. At this time point, the animals also responded to a sensory stimulation in the guise of a withdrawal relies in response to afoot pinch. Given these results, implants were recovered ? weeks after the initial .surgical reconstruction.

At recovery the implanted nerve guides were visibly well integrated with the stumps of the transected ..nerves (See Figure 12A). The guides lifted easily out of the surrounding tissue, nd there as very little evidence of fibrosis on, or in the vicinity of the implant sites. Frozen sections taken Ixom the mid-point of th regenerating tissue had a pronounced anisotropic structure and were densely populated with ceils (Figure 12B--C). Staining for MF-68 revealed a dense population of axons in the regenerating tissue. These axons were aligned in. parallel with the long axis of the cylindrical nerve guides (Figure 12D). Myelin basic p otein was present in. a dense Bbriilar pattern (Figure 12E). Staining for 100 revealed elongated Schwann cells that were aligned in parallel with the long axis of the regenerating tissue (Figure 12F). Together, the staining patterns ^' for S I 00 and MBP suggest thai a subset of the axons present in these central domams (of the implants) is activel undergoing:

myelination.

To verity the architectural organization of the reconstructe nerves, we embedded representative samples for light and transmission electron niicroscepy, and cut than in cross section. Thick sections imaged by light .microscopy revealed that the proximal segments of the nerve guides that were densel packed with myelinated xons. These axon were more variable in size than axons present in a normal sciatic nerve (see Figure 13 A-B).

Non- yelinated axons were interspersed with the rnyelina led axons (Pignre I3C, arrows and Figure 14A). The ΡβΑ/ΡΙ,Α coating mat we nsed on the implants induced the formation of an epmenraum-llke capsule (Figure 1:313). Samples taken from the mid-point of the grafts exhibited numerous bundles of axons thai were surrounded by a delimiting band of tissue that was reminiscent of the perraemiurn (Figure I 3B). Cells with an elongated, spindle-like morphology were present in association wuh the periphery of these feci cole- like structures (Figure I C, arrow:). Functional blood vessels, as indicated by the presence of red blood cells (Figures 1 €, asterisk and I4B), were observed at scattered intervals within the reconstructed tissue.

Overall the extent of inyelinatlon present on individual axons is ^' surprisingly mature within the regenerating tissue. The myelin sheath surrounding the axons was considerably thicker than anticipated given the relatively short period of time allowed for regeneration (Figure 14). Also surprisingly, the regenerating: tissue was packed with highly aligned fibrils of collagen thai were predominately oriented in parallel with the long axi of the developin tissue (Figure 14B, inset):. At this tune the role of these _: fibers in directing axon growth remains an open question. Conversely, these fibers do not appear to have adversely affected the regeneration process; guides composed of collagen have exhibited efficacy in nerve reconstruction [26],

Using quantitative tnorphometrks we estimated that the proximal section of the grafts contained approximately S,40 ^' 0 myelinated axons, a Figure that is comparable to the estimates given in the intact sciatic nerve [27). At the distal end., and again based on our light microscopy survey, we estimated that there were about 1,900 myelinated axons present in the regenerating tissue. Consistent with the early stages of regeneration that we have, examined i this study, we observed that the average 20 myelinated axon eross-sectional area decrease as a function of increasing distance from the proximal nerve stum * and that majority of axons present in the distal domains were, on average, smaller in caliber and mare uniform in size distribution s compared to the proximal, d m in (Figure 136).

DiSCUSSfON

In this study, two pole eleeirospirrnifig was used to fabricate ^!1 sem.i -solid", ID- cylindrical constructs composed of fibers that have been deposited into highly aligned arrays that are oriented parallel to the long axis of constructs. In contrast to conventional electrospinnhig systems, til© extent of fiber alignment in air gap spinning is clearly much less dependent upo the size of th fibers, Using the present air gap system, it is possible to eleen-ospin scaffolds composed of aligned PCL fibers ranging from less than. 200 mn to approximately 1 ,5- L 8 μνα in average cross-sectional diameter. The uncoupling of fiber alignment from its normal dependence on fiber diameter has allowed, us to develop and test a, family of -scaffolds with distinct material properties and intrafiber spacing (void volume) to select the best configuration- for a nerve conduit.

Several studies already have demonstrated that 2D sheets composed of anisotropic eiectrospun fibers can effectively induce the alignment of neuronal, cells [ 10- 12] as well as the directed, and accelerated migration of a variety of cell, types [13,28,29]. This type of 2 surface is particularly well suited for in vitro applications; cells plated onto a Hat sheet are readily accessible for analysis by a variety of microscopy techniques [30]. These 2D constructs are less well suited: for use in the actual reconstruction of a damaged nerve.

Sheets of deetrospun. materials have been described as "3D" in the Htera tore; however, the 3D aspect of such, materials may he-overstated and is largely confined to a description of the surface topography of the scaffold. Air gap electrospinning makes it possible to produce a truly 3D cylindrical construct composed of linear arrays of fibers oriented along a common axis. Cylindrical scaffolds which are! 5-20 mm. in cross-sectional diameter have been produced using this system and virtually no degradation in fiber alignment has been observed in the large scale constructs.

A.s noted; the fabrication of nerve guides using conventional eleetrospinnm

processes has been explored to some degree. Electrospnn PCL-based. devices have been fabricated as hollow, cylindrical tubes that are designed to confine regenerating nerves to the lumen of the device: while restricting the penetration of inflammatory cells into the injury: site [IB], These hollo devices appeared to initiate only a! nominal inflammatory response whe used in sciatic nerve reconstraetion. Additionally, PCL breaks down slowly, and as a result, has displayed good biocom.patibiliiy in a variety of hioenglneerin - applications. Our experiments yielded similar results and we did ot observe evidence of nflammat r e ^' eils or scarring within our guides, Morphoraetfic analysis would suggest that hollow nerve guides produced by conventional eleeirospinuing processes using PCX can direct perhaps 25% of the axons (based on volume of tissue, not the actual number of axons) present hi the proximal stump to regenerate down the length of 10 mm tube over a 1 week interval [ 18]. While the metrics between this study and our study var to some degree, our estimates, suggest that we achieved a similar degree of axon regeneration (25% of the axons present in the proximal section reached the distal end of the guides as determined by nu^horoetrie analysis) in our 3D guides over a 7 week interval (we believe the regeneration process has not reached its penultimate extent b any means in our experiments-given, the very brief time ^' interval of these preliminary experiments.

The extent of functional recovery that can be achieved after nerve injury is limited by the nature of the precipitating Injury and by processe that exist downstream to the actual wound bed. For example, in peripheral nerv injuries, fiaictional recovery m essentially be complete if the continuity of the endoneurmm is spared during precipitating event

(neuropraxia). In. this type of inj ury the myelin sheath thai is distal to the wound bed.

represents a "'thlhiengilv ^* guidance conduit. The regenerating axons are confined to returning to the end organs associated with the surviving endon curiam, These observ tio s sugges that guidance cues incor orated into a synthetic nerve guide ears play a critical role in directing the regeneration process [9]. The extent, and fidelity, of nerve regeneration and subsequent junctional recovery is greatly reduced once the endoneuriurn has been

compromised (axonotniesis and neurotmesisj.

Whil hollow- electrospun devices afford a measure of impro vement over earlier nerve guide designs fabricated by more conventional processes, they represent a relatively early evolutionary stage in nerve guide design. The autologous nerve guide remains the gold standard treatment for nerv injuries that require reconstruction, and the unique 3D design of the present nerve guides is modeled on the architecture of these "natural guides". The use of autologous Implants is not without its limitations. For example, the action of harvesting the autologous tissue obviously results in morbidity at the donor site. The axonai debris that is present within the harvested tissue also must largely degrade before regenerating axons can penetrate the tissue, a time delay that can exacerbate the onset of degenerative changes in. the

^• - S 8 - distal tissues {e.g. loss of motor end plates itv.ftiuscie, muscle disuse atrophy). While regenerative processes in. the peripheral nervous system can restore a considerable degree of function to end, organ tissues, the recovery of fin motor skills, unfortunately, is limited, It is likely that, functional recovery can be improved by (A) accelerating axon growth across the injury bed and simultaneously (B) confining these regenerating o to a spatial, domain {tissue plane} that mimics their original position within the nerve _* The present synthetic guides are designed to provide the guidance cues that are inherently present i autologous grafts without the time delay needed to degrade the axonal fragments present i the harvested tissues, to addition, it is likely that the present 3D guides can he used to confine regenerating axons to the tissue plane where they existed prior to the precipitating event that damaged the nerve. This can be expected to increase the probability thai the axons passing down the guide structure will emerge closer t their original position, a circumstance that should improve targeting and thereby increase functional recovery.

in summary, air gap e!eetrospinning makes it possible to directly incorporate guidance cues into die structure of a. truly 3D construct , The pom spaces present between the aligned, fibers in these nerve guides readily supported axonal growth.. Somewhat surprisingly, even scaffolds composed of the smallest diameter fibers supported the penetration of axons and Schwann cells in in vitro studies. Physical and biochemical cues that accelerate axonal regeneration ca toe expected, to further improve functional recovery in distal tissues by limiting end-organ complications associated with derimiemitiou (Le. atrophy), [29,31 ], CONCLlffiQW

In peripheral nerve mjuri.es, the extent of recovery depends upon the appropriate targeting and the time taken by the regenerating axons to reach and re-innervate the target tissues, hi contrast to - conventional hollow nerve conduits, the present novel 3D nerve guides with dense arrays of anisotropic fibers are very effective at directing axon elongation, along a defined axis, thereby providing the spatial cues for proper targeting. These guides provide a straight path for the regenerating axons which, is the shortest distance to reach their targets. Also, in contrast to the autologous grails, the use of air gap ekctrospun nerve guide in addition to avoiding the donor site morbidity, eliminates the time lag that occurs due to the requirement for degradation, of pre-existing axons from the autologous graft before the regenerating axons can penetrate the graft. These factors can be expected to accelerate regeneration across the injury she, and thereby reduce associated complications, and improve

-- ¾ ^: Sf - fundi oiial rec every

EXAMPLE 2,· Gradients of Therapeutic Substance

In human .-nerve injuries* where a segment of issue has been injured -to the point where a nerve guide must be used to re-establish, continuity of the injured tissue (or otherwise direct the regeneration process) the actual physical -dimensions of the nerve segment that has to be reconstructed may be relatively nominal in length. However, even the loss of -a relatively short segment of tissue can represent a nearly nsiumouwtable barrier ^" to. regeneration. There is considerable evidence, that growth iaetor gradients established by various cells that migrate into hie injury .site play a critical role in directing the nerve regeneration process. Clearly _* in circumstances where a. nerve guide is used to reconstruct damaged tissue the regeneration process could be enhanced, and accelerated, If a . specific growth factor gradient could be directly meorporated into a nerve guide prior to

reeonstmetive surgery, However, recapitulating a growth factor gradien -over the relatively short distances typically observed in nerve - injuries is a daunting problem.

This can, however, be accomplished using the methods of the invention. Figures 15 A and show CDG alginate fibers made as described above hut with different

concentrations of dye rather than a therapeutic agent As can be seen, the procedure of adding aliquots and freezing between each addition,, then polymerizing the thread that is formed, results in discrete bands of differing concentrations of dye molecules along the length of the thread.

In one example, a highly aligned scafioid ofPCL fibers was produced ^' by

eleetrosp nmng a small volume of ^' PCL into an air gap eleetrospitnhng system to produce a t!un, tubular shaped scaffold, A thread of alginate supp.Imtet.ned with a dye gradient was then added to this scaffold, .-and the eieeirospinning process was then re -started and the alginate- fiber was trapped in the middle of the construct. Figure 16 shows a cross section of the eleetrosptsrs nerve guide with the dyed carrier thread disposed therein (indicated b the arrow s.

EXAMPLE 3. Dissolution Dependent: Gradients

As described above, the second type of gradient, the, _. Dissolution Dependent Gradient (DOG) -is fabricated by varying the concentration of carrier (e.g. alginate) along the length of the thread like construct. This type of gradient can 5e fabricated using a constant

concentration of therapeutic reagent or a variable concentration of therapeutic reagent, Th

_ \ basis for this type of gradient lies in the observation that alginate threads composed of low concentrations of this carbohydrate dissolve faster than alginate threads composed of higher concentrations of this -carbohydrate. Thus, the rate of release of an active agent incorporated into the thread can be controlled by placing the agent within a segment of the gradient thread tot will dissolve at or wit iin a desired tmw frame, 0;g, a first segment of the thread may dissolve over a period of days (e.g. 1-7 days), a second segnierit. may dissol ve o ver a period of e.g. 1-2 weeks, a third segment may dissolve over a period of e.g. 2-4 weeks, and. so on for additional fourth, fifth, sixth, seventh, eighth, ninth, and tenth, etc. segments, with segments containing higher concentration: of alginate (or other sui able carrier) dissolving more slowly. As described; for the CDC gradient, aliquots of alginate of varying

concentrations are sequentially added to the moid, whic is frozen after each addition. The frozen (hot not yet polymerized) thread is then extruded into a calcium bath to polymerize and the construct, which is then solid at room or body temperature. The subsequent steps in processing the thread are identical- Representative alginate concentrations that might he used in the fabrication of an exemplary DOG construct containing 7 different segments (i.e. segments 1 -7) each ith a different alginate concentration are presented in Table 3.

Table 3, Exemplary Concentrations of alginate (mg/roi) In different segments of a fiber

EXAMPLE 4. Incorporation of the growth factor glial ceil line derived neurotrophic factor (GDNF) into a earner thread.

A. gro wth factor gradient composed of GDi F ^' that is targeted to promote, and accelerate, peripheral nerve generation is fabricated and used in a nerve gude fo the invention. GDNF is used since this peptide growth factor accelerates axon elongation hi the peripheral nervous system . Two methods are used to build precision gradients; both are based on trapping bio active agents in alginate "threads". In the first method, GDNF is directly mixed ("direct capture") into a. graded series of alginate solutions (0,0025/0.005, 0.0100, 0.0150, 0.0200,. 0.0250, and 0.050 mg0ni alginate) and. individual aliquots are fabricated into a continuous "thread" as described herein. The carrier thread is incorporated into a nerve guide. Release of the GDNF from the carrier thread is regulated by the differential dissoiuiion of the alginate (which occurs as a function of alginate concefttration}, in th e second method, the concentration of alginate is held constant and the concentration of GDMF is varied, Table 4 i llustrates representati ve concentrations of GDNF that cars be nsed, o prepare an exemplary 15 mm long gradient thread. This exemplar gradient is designed to contain 7 differen t segments in the fiber. The nihnber of segments can be varied, a necessary, as can the _: fractionai volume of each specific component or region of the gradient.

Table 4. Exemplary Concentrations of GDN (eg) in different segments of an alginate fiber

EXAMPLE 5. Mierobeads i threads

In. yet another embodiment bioactive reagents such as GDNF is mixed with a. solutio.n-^falgin&t¾ ^' ( ^a: isi ^nai ^ ^: '@ 0.05 mgs/ml) and the solution is electro-aerosoled.

(eleetrosprayed) into a 2% ealciurn bath. The electro-aerosol process produces a ver fine mist (analogous, to the mist produced by a btnnidifier) of alginate droplets that are associated with the bio-reactive reagent. On: contact with the ealciurn bath the alginate polymerizes and: forms a. bead that captures the associated bloactive ^■ reagent The heads are recovered, rinsed in. hexaflaonsopropanol (HTTP) and, lyophil zed. Dry micro-camer heads are mixed with various concentrations of alginate and a thread is produced, as described herein. The final construct contains alginate niiero^arrier beads supplemented with GDNF thai is (rapped wtthio. ntierbbeads in segments of alginate thread. Release is dependent upon the breakdown of the alginate thread and the exposure of the micro-carrier beads to the surrounding environment

hi an exemplar preparation, 10.0 nig dry GDNF was suspended in 200 microliters of sodium alginate (0.025 mg/ml) and protease free BSA (0.025 rog/ l) in Dl water. Figaro 1 ? shows art exemplary setup: of an apparatus for use in producing micro-carrier beads. The solution is loaded into 1 mi syringe and placed in a vertical position.25 cm -away from a met l plate and charged to +2t¾fv', the metal plate is charged to -20k V. A glass culture dish with a .2.0% calcium chloride solution, was placed on the grounded metal plate. With charging, a fine aerosol was produced, nd. on contact with th calcium hath, alginate beads formed, trapping the growth factor i . the alginate. Beads were collected, washed in ilFiP and !yophiiized. Beads can be stored at -70 *C and/or added directly to electrospinning solutions with PCL and processed into 3D nerve guides or into aiignate threads to produce unique gradient compositions of therapeutic substances, it is also possible to trap cells into these miroeartier beads by carrying out die processing is solntio.na that are physiologically relevant

To raanuitict re threads which contai the heads, aiio.uot containin different concentrations of alginate are mixed with micro-carrier beads containing an agen of interest, usually a therapeutic or pharmaceutical agent, a growth, factor. The solutions of alginate are then, added in a sequential: manner to a cylindrical "casting" vessel as described above; for small: diameter threads (approximately 1.00 ηι in diameter) small bore sections of Teflon: tubing may be used, and for larger sixes a tuberculin syringe is used. The initial volume of alginate is frozen. After freezing, the next aliquot in. the gradient series (higher concentration of alginate also eontaining .microbeads) is dded to the vessel and frozen.; these sequential cycles are continued until the final gradient is achieved. A continuous thread is produced by extruding the frozen alginate■column into a 2% calcium hath. The resulting thread, is dried aid "cured ^' ' in a rinse of HFIP to form a filament of alginate with very precisely defined spatial domains of mi crobeads. A s is the ease with other embodiments of the carrier thread described herein, the domains may be varied by alginate concentration, by the number of heads added to a domain, and/or by the concentration of acti e agent present, in the microbeads that are added, to a domain (which may be different lor each domain}, or by using combinations of these techniques. This: r eess and the resulting beads, m w&lte-e pentii&atal. results ^' obtained' usin _. the. beads alone, is presented In Figure lEA-E. The results demonstrate that alginate encapsulated proteins ate protected from damage induced by organic eiectrospiftning solvents (and potentially Oilier protein damaging solvents) and thai the encapsulated material is released into the surroitndiftg environment in a bioaetlve form.

EXAMPLE 6. Treatment of Spina? Cord Injuries

Treatment of spinal cor injuries using the nerve gukfes of the invention was carried out using a rat mammalian model. The spinal cord was exposed after laminectomy, a 3 mm section was removed by complete spinal cord transaction (Figure 19A), leaving a 3 mm gap i the tissue (Figur W&). The gap was then filled with a segment of the eieetrospun PDS scaffold either with or without various growth factors and or enzymes designed to promote regeneration (Figure 19C), The growth factors are designed to support axon growth and survi val, the ehroidmase ABC enzyme ^' is present to degrade the scar tissue that: inhibits regeneration. Figure ! 9D shows a 10 mm. section, f spina! cord that was repaired with an eiectrospun matrix after about 3 weeks. The tissue was:fixed and cut by frozen section.. The reconstructed spina! cord is shown, at the top of the panel, the arrows denote the portio of the cord that was sectioned in longitudinal section and then tained with DA PI (labels; all cell nudes). DAP1 label revealed a massive infiltration of cells into the implant, the border of which, is marked by arrows. At later time points the implants are completely infiltrated b cells and xon . The ceil population revealed by DAI ¹ ! in this: sample consisted mainly of Oligodendrocytes and glial, cells.

Treated and control, rata were tested for hindlitnb mobility and a graph depleting the improvement in hmdlimh mobility is shown in Figure 19E. _: A: B ^' BB Score of 21. represent complete ^' mobility; a score of Ό represents complete paralysis:. At earl time points amn als subjected to spinal cord .transaction move by dragging their paralysed: hind limbs. With time animals treated with, the nerve guide begin to develop movement in the lower extremities, beginning at the proximal joints (hip) and. the moving more distally to the feet with time. We also observe that animals earl i the recovery process have a very flaccid midsection, presumabl due t the spinal cord lesions:: impact on muscles in the abdominal area. Again, animal treated with the guides begin to recover (control untreated animals do: not) and tone begins to return to these muscles. The animals loolc less "flattened" and more cylindrical shaped as these muscles gain tone. As can be seen, animals treated with enhanced m trice exhibit sigdi fie ant impro ement irs. functional recover m compared ^' to untreated control rats and rats treated ^■■ with control scaffolds that' contain no supplementation with growth, factors or enzymes designed to degrade scar tissue. After as little ^' s 30 days animals treated with the enhanced (supplemented with growth factors and scar degrading enzyroes) exhibited 8BB scores approaching § ₅ . a value that reflects motion in ^' mote than one distal joint. The extent and rate of recovery ;seen in this lrial is unprecedented. In additio to the functional . recovery observed in the lower■extremities, all animals treated witb implants recovered bladder hmetion a forther indication of functional regeneration,

Electron roierograplne surveys revealed dense accumulations of axons within the implants (Figure.20A-C and Figure 21 A-E., These figures document the axonal

organization that occtus after regeneration (see figur legends), The data demonstrates that in spina! card injuries, a 3D nerve guide, aud/or one or more fibers thereof supports

( acilitates, promotes, induces, etc.) the formation and penetration of functional blood vessels and regenerating axons,

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While the invention ^" has been described in terims of its preferred embodiments, those skilled in the art will reeogrhxe that the invention can be practiced with modification within the spirit and scope of ih¾ appended claims. Accordingly, the present invention shoaMwoi he limited, to the embodiments as described above, but should further include all modifications and eqravalerrts thereof within the spirit and ^' scope of the description provided herein.