WO2010017496A1

WO2010017496A1 - Biodegradable nerve scaffold conduit for the treatment of nerve injuries

Info

Publication number: WO2010017496A1
Application number: PCT/US2009/053184
Authority: WO
Inventors: Riyi Shi; Jianming Li
Original assignee: Purdue Research Foundation
Priority date: 2008-08-07
Filing date: 2009-08-07
Publication date: 2010-02-11

Abstract

The present application relates to cellular scaffolding operable to direct the growth of cells. According to at least one embodiment, a biodegradable scaffold formed by coating a fiber structure, and thereafter dissolving the fiber structure is disclosed. According to at least one additional embodiment, a biodegradable scaffold formed by the stamping of a scaffold material and forming a multi-walled conduit is disclosed.

Description

Biodegradable Nerve Scaffold Conduit for the Treatment of Nerve Injuries

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority under 35 U.S. C. § 119(e) to United States Provisional Patent Application Serial No. 61/086,929 filed August 7, 2008, entitled Biodegradable nerve Scaffold Conduit for the Treatment of Nerve Injuries, the disclosures of which are hereby incorporated herein by reference.

BACKGROUND

[0002] The biological basis for functional loss after spinal cord injury is the elimination of nerve impulse transmission "up and down" the spinal cord. The basis for a partial functional recovery, independent of how old the injury is, is the restoration of such nerve impulses. Mechanical damage to the nervous system of mammals results in sometimes irreversible functional deficits. Most functional deficits associated with trauma to both the Peripheral Nervous System (PNS) or Central Nervous System (CNS) result from damage to the nerve fiber or axon, blocking the flow of nerve impulse traffic along the nerve fiber. This may be due to a physical discontinuity in the cable produced by axotomy.

[0003] In situations of peripheral nerve transection, end-to-end anastomosis is a preferred method for surgical intervention. However, in cases of large gap defects, anastomosis creates damaging tensile forces and autologous nerve grafts are required. Autologous grafts pose the problems of tissue availability, donor site morbidity, inconsistent functional outcome and neuroma formation (Mackinnon and Dellon, 1990). Multiple attempts have been made to manufacture artificial hipconriuits that bridge the distal and proximal nerve stumps (Doolabh et aL, 1996; Huang and Huang, 2006). However, many such structures have been met with varying success. Peripheral nerve regeneration over long-distances is an incomplete process whereby cells do not receive the appropriate cues for successful outcome. Strategies that attempt to correct the inherent biological deficiencies include the use of stimulatory electric fields/conducting substrates, contact guidance and relevant biochemical signals. However, it is widely accepted that synergisms between stimulatory and inhibitory factors dictate cell fate and the return of proper neural function. Therefore, a multi-faceted approach that more mimics the in vivo environment is crucial for clinical success.

[0004] A first step in the combinatorial strategy is the development of compatible scaffolds for delivery of desired stimuli. Previous attempts to exploit physical cues in the form of nerve guidance conduits or entubulating scaffolds have proved to be challenging. One difficulty in creating topography based nerve guides is the simultaneous control of topography with high void fraction (transparency factor). Incorporation of micro channels or longitudinal fibers along the length of the guides may prove feasible but the significant amount of bulk material generally required to fabricate these structures obstructs regenerating axons. Further, another difficulty in creating compatible scaffolds is finding a compatible medium for producing the scaffolding that results in a desired topography, is found to be utilized for human or animal implantation without significant side effects, and which shows biodegradation and is supportive of additional processing, such as the incorporation of cytokines or phaπnacological agents within the polymer matrix. Thus, a multi-stimuli scaffold would be ideal, yet production of such a scaffold with desired in vivo and in vitro results in a controllable and cost-effective manner has proven difficult.

[0005] Although natural biomaterials for scaffolding for PNS and/or CNS use more closely mimic the structure/function relationship, acellularized constructs have limited customization potential, possible antigeniciy, or may be the source of human/zoonotic disease. Synthetic polymers offer the advantage of biodegradability, precise property control and timed drug/cytokine release capability. However, incorporating appropriate multiple stimuli and necessary biomechanical properties for successful outcome remains a challenge. Further, due to the exceedingly high aspect ratios and thin walled nature of tubule shapes employed by many scaffolds, manufacture has been a challenge. Likewise, Factors such as conduit geometry, biomaterial topography, internal matrix composition, cross sectional transparency factor, porosity/permeability, electrical conductivity and the presence of cytokines all play roles in dictating the degree of axonal regeneration.

[0006] As such, providing a multi-stimuli biodegradable neural scaffold that promotes the growth of PNS and/or CNS tissue in vivo that is able to be produced in a cost-effective manner, and is reasonably customizable would be appreciated by those in the art.

SUMMARY OF THE INVENTION

[0007] In accordance with the above-stated objects, the instant application relates to scaffolds for cell growth, and the manufacture of such scaffolds comprising of intralumenal walls and defined topography or through the use of soluble fibers.

[0008] The parameters of thickness, diameter, layering complexity and topography are controllable. Thickness can be altered by polymer-solvent concentration whereas diameter, number of intralumenal walls and conduit length are dictated during assembly. The imparted topography is template dependent and can be fine-tuned based on in vitro data. Conduit mechanical properties can be improved via inclusion of additional polymer strips as reported. Spacers add an element of structural stability to withstand in vivo forces and decrease the probability of conduit collapse. The parent biomaterial can be substituted as well. Therefore, the conduits can be optimized to match the size, geometry and type of tissue defect. Attachment of the PLLA would be accomplished with an auxilliary sleeve (i.e. collagen) to assist proximal and distal stump suturing.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Fig. 1 illustrates a process for forming a neural scaffold according to at least one embodiment herein.

DETAILED DESCRIPTION

[0010] According to the present application, neural scaffolding incorporating topographical cues that control cellular behavior to promote nerve regeneration, including bridging distal and proximal nerve stumps are provided. As provided herein, poly 1-lactic acid (PLLA) is utilized as an exemplary material for producing such a scaffolding, although it will be appreciated that other materials may be employed. For instance, poly-lactic glycolic acid (PLGA) copolymer, poly-caprolactone (PCL) and even non-biodegradeable polydimethylsiloxame (PDMS) or other materials known in the art.

[0011] According to at least one embodiment, the exemplary neural scaffold is tubular in nature, and is optionally characterized as having micro fibers of the biodegradable scaffold material extending from the periphery of the tubule into the core of the tubule. In at least one exemplary embodiment, such a tubular scaffold is created through first creating a fiber core or template, then coating the template with a scaffold material, and finally selectively dissolving the core or template. [0012] For instance, in at least one exemplary embodiment, carmehzed table sugar (sucrose) may be employed as the mateπal for the core or template Optionally, the caimehzed suciose coie may be turned into a miciofibei template thiough the heating of gianulated suciose to caimehzation tempeiatuies (above about 200 C), which induces a browning ieaction and cieates a bypioduct suitable foi manipulation Theieaftei, the iesultmg suciose fibei is optionally spun by placing a glass micropipette into the sucrose melt and w ithdi aw ing it at iates of appioximalely 2-1 5 m/s Accoiding to at least one exemplaiy embodiment, by spinning the fibeis at this late, suciose libeis in the iange of 8-10 μm in diameter may be cieated, with an optional length of up to approximately tens of centimeteis may be created For instance in at least one exemplary embodiment, it was found that a length to diameter ratio of up to approximately 10 may be realized Optionally, a small spinneret (e g , one with a tip radius of approximately 200 μm), faster draw rates and reduced melt viscosity results in thinner fibers The resulting fibers have an glassy texture, high modulus, low fiber stiffness due to thin fibπl diameters Further, diameter, wall thickness, surface roughness, and porosity of the resulting surface can be controlled for each sucrose fiber by changing the diaw rate, melt viscosity, and creation parameters

[0013] Further according to at least one embodiment, the resulting sucrose fibers aie encapsulated by a selected scaffold mateπal According to at least one exemplary embodiment, the scaffold material may comprise a biodegradable scaffold material, which may comprise a polymeric mateπal According to at least one exemplary embodiment, the sucrose fiber is encapsulated by dipping the fibei into a 2% PLLA/chloroform solution or by spraying or otherwise applying such a solution to the fibei Coated fibers are then optionally air-dπed to remove residual solvent It will be appreciated that when the scaffold material is selected, that a solvent for the scaffold material should optionally be a good solvent foi the scaffold material, but a poor solvent for the sucrose fiber, thereby allowing encapsulation of the sucrose fiber without changing its structure. According to at least one exemplary embodiment, the resulting neural scaffold may be completed by removing the sucrose fiber core from within the encapsulation material by providing a solvent that is a good solvent for the sucrose fiber, but is a poor solvent for the scaffold material. In the present exemplary embodiment, immersion of the PLLA-coated sucrose fiber core in distilled water resulted in the rapid dissolution and outflow of the sucrose fiber core from the scaffold material thai remained as the resulting neural scaffold.

[0014] Scanning electron micrographs of an exemplary embodiment indicates smooth surfaces punctuated by random pores, with a tendency toward submicrometer diameters. It will be appreciated that smaller submicrometer particles can readily diffuse across the boundaries while transmembrane transport of larger objects is inhibited, thereby allowing nutrient and waste exchange of cellular materials across the surface of the resulting neural scaffold. It will be appreciated that the resultant surface allows for permeability of the neural scaffold despite the use of scaffold materials that may have a low gas permeability (e.g., PLLA), thereby increasing the biocompatibility of the selected scaffold materials.

[0015] According to at least one exemplary embodiment, cross-sectional scans of resultant PLLA tubule neural scaffolding revealed individual tubes with external diameters on the order of about 8-100 μm, with tube walls generally between about 0.75-3 μm thick in single tube entities. However, these diameters and thicknesses may be customized as necessary by changing the size of the sucrose fibers as noted above, and by increasing or decreasing the concentration of the scaffold material to solvent or reducing the amount of overall scaffold material added to the sucrose fibers.

[0016] It will be appreciated that more complex neural scaffolds may be created through combining multiple sucrose fiber cores of similar or varied length, diameter, and surface characteristics prior to coating with a scaffold material. For example, multiple sugar fibers (for example, 100-200 fibers) may be placed in parallel and simultaneously coated with the PLLA solution as noted above. However, such a technique has proven to result in a greater thickness of the resultant scaffold material between tubules, than at the periphery of the combined tubules, thereby reducing the ability to tune than the thickness of individual tubules consistently. As such, maximum control of the scaffold tubule thickness, porosity, and surface is generally achieved when each tubule is fabricated individually and then later assembled.

[0017] Turning now to Fig. 1, According to at least one additional embodiment, a neural scaffolding may be prepared by creating a patterned film or substrate manufactured via a polymer casting and/or stamping technique over a selected template, and thereafter assembling the resulting substrate into a conduit for neural formation. As shown in Fig. 1 , a film of scaffold material may be created by coating or spreading an amount of the dissolved scaffold material over a template having a desired surface pattern. Thereafter, as an optional step, a second template having a second desired surface pattern may be stamped into the scaffold material such that both sides of the resultant scaffold material retains a desired surface pattern. After any necessaiy curing of the scaffold material, the resulting film may be rolled or otherwise assembled into a conduit resulting in a neural scaffold. [0018] By way of non-limiting example, a film of PLLA may be formed by PLLA of inherent viscosity of .99-1.17 (available from Birmingham Polymers) initially dissolved in chloroform in a 5% w/v concentration. An exemplary template may include brushed stainless steel stock (available from McMaster-Carr), holographic diffraction gratings (available from Edmunds Scientific, Optometries), or any other acceptable template for the casting process that has the desired surface topography. Optionally, templates may be cleaned with a stream of compressed air and ethanol. Thereafter, according Io at least one exemplary embodiment, 2mL of the PLLA solution is applied over a selected portion of the template (for example, a 10cm x 10cm portion). After approximately 10 minutes of solvent evaporation (in a fume hood), a secondary template is optionally placed over the PLLA film that forms, and aligned in the same direction as the casting template. Pressure thereafter may be applied to the secondary template to stamp the backside of the resulting film. Following stamping, the residual polymer and underlying master are optionally cured in a fume hood for a predetermined time (for example, approximately 1 hour). After proper curing, PLLA film may optionally cut to size and carefully dissociated from the casting template, and may thereafter be utilized as a sheet to assemble a conduit assembly to be utilized as a biodegradable scaffold.

[0019] According to at least another exemplary embodiment, a neural scaffold may be created wherein a multi-wall scaffold is formed. For example, a partially cured PLLA polymer film may be placed on a glass surface. Thereafter, a small stainless steel wire (.005 in diameter) may be placed along an edge of the PLLA film, with small droplets of dissolved PLLA solution placed on this initiator wire near the edges of the film and allowed to solidify. This drop of cured polymer fuses the bottom PLLA film to the metal wire, allowing the rolling of the conduit from the PLLA film. For the rolling process, two methods were utilized to space the internal walls. Stainless steel wires (.005 diameter) and cut strips of patterned PLLA polymer were chosen as the wall spacing material (Figure IA, IB). Either stainless steel spacing wires or PLLA filmstrips were then intermittently placed along the polymer sheet (for better reproducibility, the wires or strips could be fused with more PLLA solution to the parent polymer sheet at defined distances). The initiator wire was then carefully rolled down the remainder of the PLLA sheet. Once the PLLA sheet had been fully wound, a small swab of PLLA solution was brushed along the final edge, fusing the PLLΛ end to the wound roll. The PLLA cylinder was then cut at points between the fused joints. The central portion of the tube was kept, with the stainless steel initiator and spacing wires (if applicable) carefully removed. After drying, the PLLA film may optionally be additionally cured by placement in a vacuum chamber at a selected pressure for a selected period of time (e.g., at 25 in Hg pressure for 48 hours).

[0020] Optionally, templates are chosen based on the presence of unidirectional surface grooves. Atomic force microscopy (AFM) scans of replica PLLA from brushed stainless steel surfaces show a highly varied pattern. The surface features created thereby are predominately unidirectional but the undulations in height are relatively large. Ridge heights ranged from 0.2-0.8 microns with irregular width spacing. In contrast, AFM data with diffraction gratings as templates demonstrate more controllable and repeatable results. PLLA replica films containing 500 lines/mm, 1000 lines/mm, 1200 lines/mm were lifted from the originals. 500 lines/mm samples possessed grooves of 250nm in average height, while 1000 lines/mm specimens showed peak to valley groove heights averaging 125nm. 1200 lines/mm spaced grooves showed ridge heights of only 60nm. [0021] It will be appreciated that the presence of intra lumenal walls increases the available surface area for cell adhesion. Since topography also increases surface area, the true change in surface area in utilizing stamped PLLA films can range from approximately 4 to 8 times greater than hollow conduits of the same peπmeter When fully cured in the formed state, the PLLA conduits also possessed material elasticity (memory) and conduits would stiuctuially iebound even aftei slight mechanical distuibance This w as demonstiated w hat an application of 0 2N (oi 30% compiession) did not iesull in peimancnt defoimation [0022] In application, the diiectional cues piox ided by the biodegi adable scaffold substi ates discussed e effect iegenei atmg cells in an actπ e mannei Testing suggests that neui ite emei gencc angles w eit biased toi both sympathetic and PC 12 cell types based upon the dπ eelional cues piovided by the scaiiold substi ates 1 ui theimoie neui al piocesses turned tow aids the dii ection of any undei lying giooves oi tubes piovided by the neuial scaffold This turning iesponse could be seen diiectly and via the population diffeiences between eventual neuπte alignment and emanation angles Fmthei , sympathetic nemons cultmed the neuial scaffold also displayed neuntes that weie 1030% longer than contiols

[0023] Optionally any of the piecedmg neuial scaffolding may be cieated by utilizing scaffold matei ial compi ismg neurologically active compounds including 4-aminopyi idmes and 4- aminopyπdme denvatives, mosme, oi othei biologically active matenals known to aid oi impiove neuial iegeneiation Fuithei , it will be appieciated that the scaffold disclosed heiein may be utilized to act as a scaffold foi a variety of diveise tissues and cell giowth, and is not limited to use only as neive scaffold

Claims

What is claimed is:

1. A biodegradable scaffold formed by a process comprising the steps of : a. providing one or more fibers soluble in a first solvent; b. substantially encapsulating the one or more fibers with a scaffold material that is substantially insoluble in the first solvent: and c. dissolving the one or more fibers, thereby leaving the scaffold material.

2. The biodegradable scaffold of claim 1 , further comprising the steps of: a. providing a prcdescribed amount of sugar; b. carmelizing the sugar; and c. spinning the carmelized sugar to form one or more fibers.

3. The biodegradable scaffold of claim 2, wherein the first solvent is water.

4. The biodegradable scaffold of claim 3, wherein the scaffold material is selected from the group consisting of poly 1-lactic acid, poly-lactic-glycolic acid, and polycapro lactone.

5. The biodegradable scaffold of claim 3, wherein the scaffold material remaining after the dissolution of the one or more fibers results in one or more tubules.

6. The biodegradable scaffold of claim 5, wherein the one or more tubules have an external diameter on the order of about 8-100 μm, with walls of the tubules generally between about 0.75-3 μm thick.

7. The biodegradable scaffold of claim 3, wherein the sugar is sucrose.

8. The biodegradable scaffold of claim 3, wherein the spinning of the carmelized sugar is performed by placing a glass rod into the carmelized sugar and withdrawing the glass rod at a rate of approximately .2 to approximately 1.5 m/s.

9. The biodegradable scaffold of claim 3, wherein the step of substantially encapsulating the one or more fibers with a scaffold material is performed by immersing the one or more fibers in a solution of poly 1-lactic acid dissolved in chloroform.

10. A biodegradable scaffold formed by the process comprising the steps of: a. providing a first template having a preselected topographical surface; b. substantially covering the template with a scaffold material operable to confonn to the preselected topographical surface of the template with a first surface of the scaffold material; c. providing a second template having a preselected topographical surface; d. imprinting a second surface of the scaffold material with the second template; e. removing the imprinted scaffold material from the first and second templates; and f. forming a conduit from the resulting imprinted scaffold material.

1 1. The biodegradable scaffold of claim 10, wherein the scaffold material is selected from the group consisting of poly 1-lactic acid, poly-lactic-glycolic acid, and polycaprolactonc.

12. The biodegradable scaffold of claim 10, wherein the conduit is formed by rolling the imprinted scaffold material into a substantially tubular structure operable to direct the growth of neural cells when the biodegradable scaffold is utilized in vivo.

13. The biodegradable scaffold of claim 12, wherein the first template is selected from the group consisting of brushed stainless steel stock and holographic diffraction gratings.

14. A biodegradable scaffold formed by a process comprising the steps of : a. providing a predescribed amount of sugar; b. carmelizing the sugar; c. spinning the carmelized sugar to form one or more fibers providing one or more fibers soluble in a first solvent; d. substantially encapsulating the one or more fibers with a scaffold material that is substantially insoluble in the first solvent; and e. dissolving the one or more fibers, thereby leaving the scaffold material.

15. The biodegradable scaffold of claim 14, wherein the scaffold material is selected from the group consisting of poly 1-lactic acid, poly-lactic-glycolic acid, and polycaprolactone.

16. The biodegradable scaffold of claim 15, wherein the first solvent is water.

17. The biodegradable scaffold of claim 16, wherein the scaffold material is poly 1-lactic acid dissolved in chloroform.