Cross-Reference to Related Application

This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62/958,527, filed on January 8, 2020, the entire disclosure of which is incorporated herein by reference.

Field of the Invention

The invention relates to the treatment and repair of damaged nerves. In particular, it relates to promoting recovery of damaged nerves through artificial connection means.

Background of the Invention

Neurological diseases have driven market growth and demand for nerve regeneration technologies to combat such disease. Peripheral nerve regeneration that is achieved by implanting a scaffold is a common strategy to treat peripheral nerve injury.

Summary of the Invention

In accordance with embodiments of the present invention, a scaffold can be used as an artificial nerve graft to bridge nerve injury gaps and promote peripheral nerve regeneration. More particularly, the scaffold is constructed from a sheet made from chitosan, or another fibrous material, and including nanofibers as its nano feature and microchannels as its micro feature. To this end, a chitosan solution is frozen in a mold and then lyophilized to provide the sheet, in which the microchannels are formed. Thereafter, the nanofibers are electrospun and deposited directly into the microchannels of the chitosan sheet. Finally, the chitosan sheet can be rolled into a spiral shaped scaffold including microchannels, at least some of the microchannels having one or more nanofibers therein. Using the process of the present invention, microchannels having diameters as small as 20 pm can be created. Depending upon the temperature gradient established during the freezing process, the microchannels can have diameters varying from, for example, about 20 pm to about 100 pm. Depending on the chosen material of the nanofibers, the nanofibers can range from, for example, about 70 nm to about 900 nm in diameter. Brief Description of the Figures

For a more complete understanding of the present disclosure, reference is made to the following detailed description of exemplary embodiments considered in conjunction with the accompanying drawings, in which: FIG. 1 is a diagram showing the bridging of a gap in an injured nerve via a scaffold constructed in accordance with one embodiment;

FIG. 2 is a micrograph of a scaffold constructed in accordance with one embodiment, illustrating its rolled construction and microchannels;

FIG. 3 is a micrograph showing various microchannels of a scaffold constructed in accordance with one embodiment;

FIG. 4 is a micrograph of a scaffold constructed in accordance with one embodiment, illustrating nanofibers (as indicated by white arrows) and microchannels (indicated by black arrows);

FIG. 5 is a schematic diagram illustrating a process for making a scaffold in accordance with one embodiment;

FIGS. 6A-6D are views of a mold unit used in fabricating a chitosan sheet in accordance with one embodiment;

FIG. 7 is a top plan view of a mold constructed by combining a pair of mold units in accordance with one embodiment; FIG. 8 is an elevational view of the mold shown in FIG. 7;

FIG. 9 is a micrograph illustrating microchannels passing through a chitosan sheet constructed in accordance with one embodiment;

FIG. 10 is a schematic diagram illustrating an electrospinning process using a spinning disk setup in accordance with one embodiment; FIG. 11 is a diagram of a side view of a spinning disk during the performance of an electrospinning process in accordance with one embodiment, illustrating the orientation of a chitosan sheet’s microchannels relative to the direction of the disk’s spinning; FIG. 12 is a graph illustrating experimental results demonstrating cell adherence on a scaffold constructed in accordance with one embodiment, relative to other growth media;

FIG. 13 is an immunostaining image, illustrating cell adhesion on a scaffold constructed in accordance with one embodiment and demonstrating that micro-channels regulate the orientation of the cell bodies; and

FIG. 14 is an immunostaining image, illustrating cell migration on a scaffold constructed in accordance with one embodiment.

Detailed Description of Exemplary Embodiments

Reference will now be made to several embodiments of the present invention(s), examples of which are illustrated in the accompanying figures. Wherever practicable similar or like reference numbers may be used in the figures and may indicate similar or like functionality. The figures depict embodiments of the present invention for purposes of illustration only. One skilled in the art will readily recognize from the following description that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the invention described herein.

All terms defined herein should be afforded their broadest possible interpretation, including any implied meanings as dictated by a reading of the specification as well as any words that a person having skill in the art and/or a dictionary, treatise, or similar authority would assign thereto.

The terms, “for example”, “e.g ”, “optionally”, as used herein, are intended to be used to introduce non-limiting examples. The phrases “in one embodiment” and “in some embodiments” as used herein do not necessarily refer to the same embodiment(s), though they may. Furthermore, the phrases “in another embodiment” and “in some other embodiments” as used herein do not necessarily refer to a different embodiment, although they may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

In addition, as used herein, the term “or” is an inclusive “or” operator, and is equivalent to the term “and/or,” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a,” “an,” and “the” includes plural references. The meaning of “in” includes “in” and “on.” In addition, the terms “comprises” and “comprising” when used herein specify that certain features are present in that embodiment, however, this phrase should not be interpreted to preclude the presence or addition of additional steps, operations, features, components, and/or groups thereof.

With reference to FIG. 1, a scaffold 10 is provided in accordance with one embodiment for repairing damaged nerve 12, including peripheral nerve injury. More particularly, the scaffold 10 is adapted to be connected to ends of 14 of the injured nerve 12 so as to bridge a gap formed therebetween, thereby facilitating functional recovery of the injured nerve 12.

The scaffold 10 of the present invention has microchannels 16 and nanofibers 18 as its micro and nano features, respectively (see FIGS. 2-4). The microchannels 16 are included to promote neurite extension and are conducive to formation of myelinated axons. To this end, the microchannels 16 extend in a direction generally along a longitudinal axis of the scaffold 10 (i.e., in the direction of desired cell growth). To further facilitate healing, at least some of the nanofibers 18 are deposited within the microchannels 16 of the scaffold 10. In doing so, the nanofibers 18 function to aid Schwann cell migration.

FIG. 5 illustrates a fabrication method for making the scaffold 10 in accordance with one embodiment. More particularly, a chitosan solution is placed in a mold 20 and undergoes a freezing step to create a chitosan sheet 22 having microchannels 16. The sheet 22 then undergoes a 3D printing (e.g., electrospinning) step to deposit nanofibers 18 into the microchannels 16. Finally, the resultant chitosan sheet 24 with both the microchannels 16 and nanofibers 18 undergoes a rolling step to achieve the spiral scaffold 10. With the methods of the present invention, the microchannels 16 can be provided with diameters as small as 20 pm.

In accordance with one embodiment, the mold 20 is formed by combining a pair of mold units 26 (see FIGS. 6A-6D and 7). Each of the mold units 26 includes a plate 28 and a raised stair 30 at one end thereof. In one embodiment, each of the mold units 26 is coated with a non-stick material, such as the material commercially available under the name TEFLON®. In one embodiment, each of the mold units 26 is about 15 cm long, about 10 cm wide and about 4 cm thick. In another embodiment, the mold units 26 can be provided with other suitable dimensions.

With reference to FIGS, 7 and 8, the mold units 26 are combined together such that the raised stair 30 of each mold unit 26 abuts against the plate 28 of the other mold unit 26, thereby providing a gap 32 in the mold 20. In one embodiment, the stairs 30 separate the two plates 28 of the mold units 26 by about 200 mih. In one embodiment, the gap 32 is about 13 cm long and about 10 cm wide. In other embodiments, the gap 32 can be provided with other suitable dimensions. Once formed, the mold 20 is provided with open top and bottom ends 34, 36.

To make the chitosan sheet 22, the mold 20 is placed in a freezer. In one embodiment, the open bottom end 36 of the mold 20 is placed directly on a cooling or freezing surface 38 (see FIG. 8) of the freezer (not shown). A chitosan solution is used to fabricate the sheet. In an embodiment the chitosan solution is made by dissolving about 4% weight per volume (W/V) chitosan in about 2% volume per volume (V/V) acetic acid. The chitosan solution is then poured into the gap 32 of the mold 20 through the open top end 34. Due to its viscosity, the chitosan solution is retained within the gap 32 (i.e., it does not flow out from the mold 20 through the open bottom end 36). A freezing temperature is applied to the chitosan solution through the open bottom end 36 of the mold 20 via the freezing surface 38. As a result, the bottom end 36 and its adjacent portions of the mold 20 (and corresponding portions of the chitosan solution in the mold 20) are subjected to the freezing temperature, while the top end 34 and its adjacent portions of the mold 20 (and corresponding portions of the chitosan solution) remain, at least initially, at room temperature (i.e., the freezing temperature applied to the bottom end 36 of the mold is not applied directly to the top end 34). Through this differential application of temperature, a positive temperature gradient is created between the bottom end 36 and top end 34 of the mold 20 and hence the chitosan solution. Consequently, ice crystals form within the chitosan solution and then precipitate out in the direction of the positive temperature gradient (i.e., from the bottom end 36 towards the top end 34 of the mold 20). The precipitation of the ice crystals ultimately produces the chitosan sheet 22 with microchannels 16 (see FIG. 9).

Larger differences in temperature between the top and bottom ends 34, 36 of the mold 20 result in faster freezing, larger ice crystals, and microchannels with larger diameters, thereby providing a mechanism for controlling the size of the microchannels 16. For instance, a bottom temperature at -20 °C and a top temperature at room temperature yields microchannels 16 of about 20 pm in diameter. In contrast, when the bottom and top ends are -80 °C and room temperature, respectively, the freezing step yields microchannels 16 of about 100 pm in diameter.

In some embodiments, there can be as many as 400 microchannels 16 in a single nerve scaffold 10 that has a diameter of 2.0 mm. In an embodiment, the nanofibers 18 are as small as 70 nm, and consist of pure chitosan. Due to the precipitation freezing-based manufacturing process of the present invention, in some embodiments, the microchannels 16 can be present throughout the chitosan sheet 22, including in the middle of the chitosan sheet 22 as well as on the sheet’s outer surfaces.

After the chitosan solution is completely frozen, the frozen sheet is removed from the mold 20 and lyophilized for a predetermined time period (e.g., twenty-four hours) in a conventional manner. After the completion of the lyophilization step, the lyophilized sheet is washed with a suitable solution (e.g., 200 proof alcohol) to remove acetic acid from the sheet 22 without disrupting or removing the chitosan materials in the sheet 22. In the case of 200 proof alcohol, the chitosan material in the lyophilized sheet is insoluble in ethanol, enabling the absorption of any acetic acid in the sheet. The washed sheet is then air-dried to form the completed chitosan sheet, which may be cut into a desired size dictated by the final application of the scaffold 10.

In accordance with one embodiment, an electrospinning step is performed on the chitosan sheet 22 to deposit nanofibers 18 in the microchannels 16 by employing an electrospinning apparatus 40 (see FIG. 10). In one embodiment, a polymer solution inside a spinneret 42 is ejected by a syringe pump 44 through a positively-charged needle 46, depositing the material that will constitute the nanofibers 18. To this end, the chitosan sheet 22 is adhered to a spinning collector disk 48, which is negatively-charged, via a suitable mechanism (e.g., double-sided tape). The chitosan sheet 22 is fixed such that the microchannels 16 are oriented parallel to the direction of the disk’s spinning (see FIG. 11). In one embodiment, the spinning disk 48 rotates at about 950 rpm. In another embodiment, the voltage between the positively-charged needle 46 and the negatively-charged disk 48 is about 16 kV. In one embodiment, the needle 46 and the disk 48 may be about 10 cm apart. In one embodiment, the spinning solution comprises 5% Chitosan, weight by volume, in a solution of Trifluoroacetic acid and Hexafluoro-2-propanol in a 60:40 ratio, volume by volume, such that there is about 5 g of polymer for every 100 ml of solution. In one embodiment, the mixture of polymer and solution can be deposited at a rate of about 0.25 ml/h. In one embodiment a second wash with a cleaning solution (e.g., a saturated sodium carbonate water solution) is preferred in order to neutralize the Trifluoroacetic acid.

In one embodiment, the electrospinning material is Polycaprolactone (PCL). Nanofibers 18 deposited using PCL are typically from about 500 nm in diameter to about 900 nm in diameter. In another embodiment, the nanofibers 18 are made of chitosan. In one embodiment, the chitosan nanofibers 18 have diameters from about 70 nm to about 200 nm, with an average of approximately 110 nm in diameter. The smaller diameter chitosan nanofibers 18 allow for the deposition of multiple nanofibers in a single microchannel, increasing the available surface area on which cells can grow and proliferate. In another embodiment, the nanofibers 18 are made of collagen.

After the performance of the electrospinning step, excess nanofibers are cut, removing nanofibers on the surface of the chitosan sheet 24 that are outside the microchannels 16. This step ensures the remaining nanofibers 18 are unidirectional, extending through the microchannels 16 so as to facilitate functional nerve recovery. In one embodiment, the chitosan sheet 24 is rolled around a thin rod, which is subsequently removed. As a consequence of this rolling step, the resulting scaffold 10 includes multiple layers with spacing therebetween. After the roll up, the chitosan sheet can be inserted into an electrospun PCL nanofibrous tube to hold the spiral structure. In an embodiment, the PCL nanofibrous tube is electrospun with the same parameters as the chitosan sheet. In an embodiment, it is electrospun on a metal tube, which is subsequently removed.

In one embodiment, the scaffold 10 is stored in an autoclaved deionized water environment before implantation so as to inhibit deformation of the microchannels 16. To repair damaged nerve 12, ends of the scaffold 10 are connected (e.g., sutured) to the nerve’s ends 14 to bridge them together. The scaffold 10 facilitates connecting of the nerve ends 12 to one another and hence functional recovery of the nerve 12. The scaffold 10 is adapted to degrade after the healing of the nerve.

Example 1: Schwann Cell Viability

A chitosan scaffold made in accordance with an embodiment of the present invention was compared to a tissue culture plate and a Polycaprolactone (PCL) scaffold in order to confirm that the chitosan scaffold of the present invention can serve as a platform for cell growth and proliferation. The control scaffolds are PCL scaffolds made from porous PCL sheets and aligned PCL nanofibers, and were made by a process described in Chang et al., Tissue-Engineered Spiral Nerve Guidance Conduit for Peripheral Nerve Regeneration, Acta Biomaterialia, vol. 73, pages 302-311 (June, 2018), the entire contents of which are incorporated herein by reference. On the other hand, the chitosan scaffold was made by the method described hereinabove. The control scaffolds and chitosan scaffolds were of the same dimensions.

The tissue culture plate was purchased from VWR International, LLC (VWR Catalog Number 10062-892). To conduct the test, Schwann cells were seeded on the scaffolds and the tissue culture plate and cultured 7 days. After day 7, a MTS assay (3-(4,5-dimethylthiazol -2-yl)- 5-(3- carboxymethoxyphenyl)- 2 - (4 - sulfophenyl) - 2Htetrazolium) was used to measure the number of live cells on the scaffolds. Briefly, the MTS solution was added into the cell culture and incubated for 2 hours. Then the optical density of the cell culture medium was measured and put into the standard curve to calculate the cell number. The results, which are illustrated FIG. 12, show a greater cell population on the chitosan scaffold of the present invention than on the control scaffold made from Polycaprolactone (PCL). The same cell culture sample was fixed with 4% paraformaldehyde and immunostained for S-100 (represented in green in FIG. 13 of U.S. Provisional Patent Application No. 62/958,527, which has been incorporated by reference into the present application), and nucleus (represented in blue in FIG. 13 of U.S. Provisional Patent Application No. 62/958,527) for a visual representation of cell adhesion (see accompanying FIG. 13 and FIG. 13 of U.S. Provisional Patent Application No. 62/958,527). In FIG. 13 of the present application, black arrows point to microchannel walls, while white arrows point to cells adhered thereto. The circles in FIG. 13 of the present application demarcate some instances of cells that are attached on the top edge of the microchannel walls.

Cell migration facilitated by chitosan nanofibers was illustrated by a cell migration study, as shown in FIG 14. Cells were seeded in the middle point of the nerve conduit with or without chitosan nanofibers. After 7-days of cell culture, the horizontal length of the area covered by cells was measured and compared. The length of cell-covered area increased from 738± 28 um to 833±37 um in 7 days without the help of chitosan fibers and increased from 877±39 um to 1194±60 um in 7 days with the help of chitosan fibers, which is 12.87% increase without nanofibers and 36.15% with nanofibers.

It should be understood that the embodiments described herein are merely exemplary in nature and that a person skilled in the art may make many variations and modifications thereto without departing from the scope of the present invention. All such variations and modifications, including those discussed above, are intended to be included within the scope of the invention.