FIBROUS POLYMER MATERIAL COMPRISING FIBROIN AND POLYMER SCAFFOLDS COMPRISING THEREOF

Abstract

The application pertains to the field of biomaterials and their use, in particular for the production of implants to support the growth and recovery of biological tissues. More particularly, a fibrous polymer material is described comprising a first layer of aligned fibroin fiber, a second layer of fibroin fibers, wherein the fibroin fibers are randomly oriented within said second layer, a third layer of aligned fibroin fibers; and polymer scaffolds comprising thereof.

Claims

1. A fibrous polymer material, comprising: a first layer of aligned fibroin fiber; a second layer of fibroin fibers, wherein the fibroin fibers are randomly oriented within said second layer; and a third layer of aligned fibroin fibers.

2. The fibrous polymer material of claim 1, wherein the fibroin is from silkworms.

3. The fibrous polymer material of claim 1, wherein the fibroin fibers have a diameter of between 100 nm and 900 nm.

4. The fibrous polymer material of claim 1, wherein the fibrous polymer material is functionalized by the addition of at least one biological molecule.

5. A method for producing a fibrous polymer material according to claim 1, comprising preparing a solution of fibroin and poly(ethylene oxide), and forming fibroin fibers by electrospinning said solution.

6. The method according to claim 5, wherein the solution comprises between 4% and 15% fibroin and; wherein forming fibroin fibers by electrospinning comprises: a) first dispersing at a collector rotation speed of more than 2,000 RPM, in order to produce a layer of aligned fibers, b) then dispersing the solution at a collector rotation speed of less than 1,000 RPM, in order to produce a layer of randomly organized fibers, and; c) finally dispersing the solution in the same condition as in step a).

7. A polymer scaffold, comprising the fibrous polymer material of claim 1.

8. The polymer scaffold of claim 7, wherein the polymer scaffold has a tubular shape.

9. The polymer scaffold of claim 7, wherein the fibroin fibers of the first layer of the fibrous polymer material of the invention are aligned with an axis of the tube.

10. The polymer scaffold of claim 7, wherein the polymer scaffold has a tubular shape which inner part has a multi-channeled structure.

11. The polymer scaffold of claim 7, wherein the polymer scaffold comprises 2 or more sheets of the fibrous polymer material, wherein said 2 or more sheets are combined.

12. The polymer scaffold of claim 7, wherein the polymer scaffold has a tubular shape, wherein an inner part has a multi-channeled structure, and an external part formed of two or more sheets of the fibrous polymer material, wherein said sheets are combined.

13. The polymer scaffold of claim 7, wherein the polymer scaffold has a tubular shape, wherein an inner part has a multi-channeled structure, and an external part formed of two or more sheets of the fibrous polymer material, wherein said 2 or more sheets are combined, and wherein said external part formed of two or more sheets of the fibrous polymer material extends on each end of the tube beyond the multi-channeled structure.

14. The polymer scaffold of claim 13, wherein the fibrous polymer material is functionalized by the addition of at least one biological molecule.

15. A method to produce a polymer scaffold comprising folding the fibrous polymer material of claim 1 into a shape of interest, and treating said fibrous polymer material so as to induce -sheet formation.

16. The fibrous polymer material of claim 2, wherein the silkworms are chosen form a species selected from the group consisting of Bombyx mori, Anthroea Yama-Ma and Anthroea Perny.

17. The fibrous polymer material of claim 3, wherein the fibroin fibers have a diameter of between 250 nm and 650 nm.

18. The fibrous polymer material of claim 4, wherein the at least one biological molecule is selected from the group consisting of neuronal growth factor (NGF), ciliary neurotrophic factor (CNTF) and epidermal growth factor (EGF), neurotrophin 3 (NT-3), brain derived neurotrophic factor (BDNF) and Neurotrophin 4/5 (NT-4/5).

19. The polymer scaffold of claim 14, wherein the at least one biological molecule is selected from the group consisting of NGF, CNTF and EGF, NT-3, and BDNF and NT-4/5.

20. The polymer scaffold of claim 15, wherein treating said fibrous polymer material so as to induce -sheet formation includes treating the fibrous polymer material with a methanol aqueous solution or water vapor annealing.

Description

FIGURE LEGEND

[0066] FIG. 1: Typical electro spinning set up; a) syringe with polymer solution; b) syringe pump; c) spinneret; d) Taylor cone; e) rotating drum collector; f) high voltage power supply.

[0067] FIG. 2: SEM images of A) beginning of third aligned layer on top of middle random layer; B) final top aligned layer; C&D) cross section of tri-layered material after snap frozen in liquid nitrogen and cut longitudinally.

[0068] FIG. 3: Average maximum stress measurements (left) and average Young's Modulus measurements (right) of silk materials and a native sciatic nerve.

[0069] FIG. 4: SEM images of first layer of aligned fibers.

[0070] FIG. 5: Cross section of a tubular polymer scaffold according to the invention, which inner part has a multi-channeled structure.

[0071] FIG. 6. Implantation process. (A) the rat sciatic nerve was exposed and secured; (B) the implant was sutured twice at 180 at the distal nerve segment; (C) the proximal nerve segment was placed inside implant cavity; (D) the implant was sutured twice at 180 at the proximal nerve segment

[0072] FIG. 7: Tear strength analysis of material formed of either a sheet comprising a single layer of aligned fibers, a sheet of the fibrous polymer material of the invention (comprising three layers of fibers), and a combination of three sheets of the fibrous polymer material of the invention.

[0073] FIG. 8: Average force-displacement at maximum tensile force of material formed of either a sheet comprising a single layer of aligned fibers, a sheet of the fibrous polymer material of the invention (comprising three layers of fibers), and a combination of three sheets of the fibrous polymer material of the invention.

[0074] FIG. 9: cross section of the rat sciatic nerve distal to the implantation site 4 months after surgery using the presented silk fibroin device (not functionalized with any biomolecules). Immunostaining includes DAPI (stains the cell nuclei), -III Tubulin (stains axons of neurons), and Phalloidin (stains actin, extracellular matrices).

[0075] FIG. 10: cross section of a healthy rat sciatic nerve. Immunostaining includes DAPI (stains the cell nuclei), -III Tubulin (stains axons of neurons), and Phalloidin (stains actin, extracellular matrices).

EXAMPLES

Methods

[0076] 1. Preparation of Silk Fibroin Solution

[0077] Silk cocoons from the Bombyx mori silkworm were cut into small pieces and boiled for 30 minutes in a 0.02 M Na2CO3 (Sigma-Aldrich) aqueous solution. The silk fibroin (SF) fibers were rinsed three times in cold DI water then allowed to dry for 24 hours at room temperature. The dry SF fibers were dissolved in a 9.3 M solution of LiBr (Sigma-Aldrich) for up to 4 hours at 60 C.

[0078] The SF solution was dialyzed against DI water using Slide-a-Lyzer dialysis cassettes (Thermo Fisher Scientific) for 3 days to remove salts. The SF solution was centrifuged twice to remove solid particles. The final concentration was about is between 6 and 8 wt %. A 10 wt % SF solution was achieved by allowing water to evaporate from the solution overnight. The resulting 10 wt % SF solution was stored at 4 C. for up to 6 weeks.

[0079] 2. Electro spinning

[0080] A 10 wt % SF solution was mixed at a ratio of 4:1 with a 5 wt % solution of poly(ethylene oxide) (PEO, average My 900,000, Sigma-Aldrich) resulting in a 8 wt % SF (1 wt % PEO) spinning solution. The spinning solution was dispensed through a 19 G stainless steel spinneret (Ram-Hart Instrument Co.), at a flow rate of 1 mL/hr while a voltage between 10-15 kV is administered to the needle. The electrospinning rotating drum collector made in-house had a diameter of 12.8 cm, a thickness of 3 cm, and was positioned on the same axis as the spinneret at a distance of 12.5 cm from the tip of the spinneret. The entire system was enclosed in a plexiglass containment area with a controlled humidity range of 25-35%. To obtain an aligned fiber material (SF-A), the spinning solution was dispensed continuously for 90 minutes while the collector rotated at 4,000 RPM. To obtain a tri-layered (aligned-random-aligned) fiber material (SF-ARA), the spinning solution was dispensed for a total of 90 minutes: 30 minutes with a collector rotation speed at 4,000 RPM, followed immediately by 30 minutes at 400 RPM, then 30 minutes at 4,000 RPM. To obtain a random fiber material (SF-R), the spinning solution was dispensed continuously for 90 minutes with a collector rotation speed at 400 RPM. Standard aluminum foil was used to cover the collector surface before each process to allow for easier sample recovery.

[0081] 3. Scanning Electron Microscope Imaging

[0082] Electrospun fiber materials were gold sputter coated and analyzed using a scanning electron microscope (SEM) at the surface and the edge of the material. To observe the three layers of the SF-ARA samples, the material was snap frozen in liquid nitrogen before being cut for a clean, blunt edge for analyses.

[0083] 4. Tensile Strength Testing

[0084] Electrospun SF-R, SF-A, and SF-ARA samples were rolled (perpendicular to fiber alignment for samples SF-A and SF-ARA) 4 rotations to produce a hollow tube. The tubes were water vapor annealed for 4 hours then immersed in Milli-Q water overnight at 37 C. to extract the PEO from the fibers. The tubes were subsequently rinsed then immersed in PBS for storage.

[0085] To perform tensile strength tests, each sample with a gauge length of 3 mm was consecutively secured lengthwise between the upper and lower holding grips. Each trial was carried out at a cross--head speed of 0.06 mm/s while recording load measurements in N every 100 ms. Assays for each material were done in triplicate.

[0086] 5. Implant Fabrication-Layered, Multi-Channel Design

[0087] The tri-layered electrospun material was slowly peeled from the aluminum foil and a 5 mm (parallel to aligned fibers) by 3 cm (perpendicular to aligned fibers) rectangle of the material was cut using a clean scalpel blade. The long side of the material was then folded in half in order to enclose the material surface previously in contact with the aluminum foil obtaining a 5 mm by 1.5 cm rectangle. The material was then rolled while adding a Teflon-coated stick (0.2 mm diameter) every half-rotation. Once completely rolled, the free edge of the material was tightly pressed to the outside of the rolled tube and the tube was immediately immersed in methanol for 5 min to induce -sheet formation. The tube was allowed to air-dry for 1 hr and the Teflon-coated sticks were then removed resulting in a 5 mm long multi-channel tube.

[0088] From the tri-layered electrospun material, a 7 mm (parallel to aligned fibers) by 3 cm (perpendicular to aligned fibers) rectangle was cut and placed so that the surface previously in contact with the aluminum foil is face down. The multi-channel tube was placed at the bottom center on this rectangle (aligned fibers in the same direction). The larger material was then rolled around the tube for 3 rotations to create a jacket. The edge was tightly pressed to the tube and the entire system was water vapor annealed at room temperature for 4 hours to induce -sheet formation. The implants were immersed in Milli-Q water overnight at 37 C. then rinsed three times in order to eliminate traces of PEO. The implants were then sterilized in 70% ethanol overnight. They were finally rinsed three times with sterile water and immersed in sterile PBS for storage.

[0089] 6. Surgery

[0090] Male Sprague Dawley rats of 12 six week old were each anesthetized with vetflurane throughout the entire operation. The rats' left hind limbs were each shaved and sterilized. For each rat, an incision parallel to the femur was made and the sciatic nerve was exposed, isolated and fixed with two micro clips 10 mm apart. The nerve was severed at the distal end of the fixed section and the implant's outer layer was sutured twice to the distal segment's epineurium at 180 enveloping the epineurium and nerves fascicles. From the proximal nerve segment, a 3-4 mm portion of nerve was extracted and discarded. The exposed perineurium and fascicles of the proximal nerve segment was inserted into the opening of the implant, and the implant's outer layer was sutured twice to the epineurium at 180. The operated area was then cleaned and the wound was closed and sutured.

Results

[0091] Electrospinning

[0092] After a total of 90 minutes of electrospinning, a fibrous silk fibroin material 3 cm wide, 40 cm long, and 0.08 mm (0.01 mm) thick was obtained. Fiber diameters were analyzed using ImageJ software and varied between 250 nm and 650 nm with an average of 390 nm. The SF-ARA material clearly presented three distinct layers visible through SEM analyses (FIG. 2). The aligned-fiber surface of the SF-ARA was also shown to completely cover the random fiber layer in the center (FIG. 2B) verifying that the guidance factor of this material was not compromised.

[0093] The electrospinning setup is shown in FIG. 1

[0094] Tensile Strength Testing

[0095] Average tensile strength results of material samples SF-A, SF-ARA, and SF-R are represented in the stressstrain graph displayed in FIG. 3. Stress and strain were calculated using equations (1) and (2) respectively:

[00001]$\begin{array}{cc}=\frac{F}{A}& \left(1\right)\\ \mathrm{.Math.}=\frac{.Math..Math.l}{l_0}& \left(2\right)\end{array}$

Where is tensile stress, F is force, A is cross sectional area (calculated by material thickness multiplied by the width of material: A=0.08 mm30 mm), is strain, l is the change in length, and l.sub.0 is initial length.

[0096] The SF-A and SF-ARA samples were shown to possess similar average maximum stress measurements of 2.98 N/mm.sup.2 and 2.63 N/mm.sup.2 respectively which were slightly higher than that of a native sciatic nerve (2.55 N/mm.sup.2) and significantly higher than that of SF-R demonstrating an average maximum stress of 0.49 N/mm.sup.2. However, the SF-A samples were found to be significantly less resistant to elongation than both the SF-ARA and SF-R samples exhibiting an average strain of 0.85 mm/mm at the average maximum stress occurring immediately before rupture. This behavior is comparable to the native nerve which had a strain value of 0.80 mm/mm. SF-ARA and SF-R samples had similar strain measurements at their respective average maximum stress points (2.63 mm/mm and 2.50 mm/mm respectively). SF-ARA and SF-R samples also showed some elasticity before permanent deformation whereas the average SF-A curve does not present any apparent elastic characteristics. Finally, in contrast to the SF-A samples, SF-ARA samples presented a gradual decline in stress measurements following the rupture point (immediately after the maximum average stress point); SF-A samples experienced an abrupt/sharp decline in stress corresponding to a more violent rupture. This result suggests that, contrary to SF-A samples, SF-ARA samples preserve some continuity after initial rupture.

[0097] Average tensile strength results of sutured SF-ARA and SF-A material samples are represented in FIGS. 7 and 8. A single layer or 3 layers of the SF-A or SF-ARA material were sutured (without a knot) at 1 mm from the longitudinal edge of each sample. The non-sutured edge and the two suture ends were secured within the clamps of the apparatus. While the average sutured single layer of the SF-ARA material resisted more than 3.5 times the tensile force of the average sutured single layer of the SF-A material before tearing, the average sutured 3-layered SF-ARA material resisted more than 2.5 times the tensile force of the sutured single layer of the SF-ARA material before tearing. The sutured single and 3-layer SF-ARA material samples, despite reaching their maximum force, conserved substantial continuity until the maximum displacement reached during the experiment (4 mm). The sutured single layer of the SF-A material lost continuity or virtually all continuity before the maximum displacement reached during the experiment (4 mm). The 3-layered SF-ARA material therefore presents a superior resistance to tearing than the sutured single layer samples.

[0098] Implant Design/Preparation

[0099] After SEM analyses of the tri-layered material, it was discovered that the act of peeling the material from the aluminum foil collector caused many of the aligned fibers to stretch, break, and coil due to the slight adhesion of the fibers to the foil (FIG. 4). This is an undesirable consequence since the coiling of the fibers negates/nullifies the alignment of the fibers and therefore eliminates the surface guidance factor. Therefore the material was folded in half in order to conceal the flawed surface before rolling the material into a multi-channel tube (FIG. 5). The implants had a final diameter of approximately 1.1 mm.

[0100] Surgery

[0101] Surgery was successfully carried out on the right sciatic nerve of all 12 rats by following a 4-step process depicted in FIG. 6. The implant was easily sutured to the distal nerve segment while avoiding any disturbance to the nerve fascicles due to the small hollow pocket provided by the jacket enveloping the multichannel conduit. The implant was then readily positioned for the sutures to the proximal nerve segment as the hollow pocket secured the nerve in the correct position for the final sutures (demonstrated in FIG. 6). The tri-layered material was found to have sufficient tear strength to withstand suture punctures which was not the case with the SF-A material. The results of the implantation was assessed 4 and 8 months after surgery. The rat sciatic nerve was fixed and cross-section samples were stained using DAPI, anti-III tubulin antibodies and Phalloidin (FIG. 9). A healthy rat sciatic nerve was treated similarly, and used as control (FIG. 10).

[0102] The observation confirms that there was a high level of nerve regeneration at 8 months after the implantation of the silk guidance conduit. As there exists several types of neurons within the sciatic nerve, and they all vary in axon diameter, 50 of the largest axons in a 37500 m.sup.2 section area were measured in the 8 month sample as well as in the control sample. The average diameter of axons found in the 8 month cross section was found to be 1.34 m 0.24 m while the average diameter of axons found in the control cross section was found to be 1.48 m0.24 m. This therefore demonstrates a clear improvement of nerve regeneration between after 8 months supported by a silk guidance conduit that approaches the results obtained from healthy nerve control samples.