Biohybrid for the Use Thereof in the Regeneration of Neural Tracts

Abstract

The invention relates to a biohybrid for the use thereof in the regeneration of neural tracts, comprising an implantable tubular hybrid structure which is degradable and biocompatible and characterized in that it comprises three layers of different porosity: an inner layer a), an intermediate layer b) and an outer layer c), with uninterrupted connection among them, the three layers consisting of the same porous hydrogel based on cross-linked hyaluronic acid, a biohybrid comprising the hybrid tubular structure described, which can contain a fibrous material, preferably poly-L-lactic acid, to a method for producing said tubular hybrid structure and said biohybrid, and to the use of same for regenerating neural tracts in diseases that affect the central nervous system, preferably Parkinson's disease.

Claims

1. A degradable implantable and biocompatible tubular scaffold comprising three layers of different porosity: an inner layer a), an intermediate layer b) and an outer layer c), with uninterrupted connection among them, and the three composed by a same porous hydrogel based on crosslinked hyaluronic acid.

2. The tubular scaffold according to claim 1, wherein the porous hydrogel layers have a porosity: the inner layer a) has micropores of less than 1 μm; the intermediate layer b) has interconnected, honeycomb-like pores, larger than those of the inner and outer layers, of size between 10 to 70 μm and; the outer layer c) has irregular pores smaller than 12 μm in size.

3. The tubular scaffold according to claim 1, wherein said scaffold has an internal diameter with dimensions of about 400 μm and a length of up to 50 mm.

4. A biohybrid comprising a tubular scaffold defined in claim 1, comprising three layers of different porosity: an inner layer a), an intermediate layer b) and an outer layer c), with uninterrupted connection among them, and the three composed by a same porous hydrogel based on crosslinked hyaluronic acid.

5. The biohybrid according to claim 4, that comprises Schwann cells or olfactory envelope glia in its interior.

6. The biohybrid according to claim 4, that further comprises growth factors, or drugs, or a combination of both in its lumen.

7. The biohybrid according to claim 6, wherein the growth factors are selected from neurotrophins NGF, BDNF or GDNF.

8. The biohybrid according to claim 6, wherein the drugs are dopaminergics.

9. The biohybrid according to claim 6, wherein the growth factors and/or drugs are present in the lumen embedded in gels or microparticles.

10. The biohybrid according to claim 9, wherein the gels are injectable and in situ gelifiable peptides or solutions of hydrogels.

11. The biohybrid according to claim 9, wherein the gels are selected from the group consisting of fibrin, collagen and agarose.

12. The biohybrid according to claim 9, wherein the microparticles have a hydrophilic character.

13. The biohybrid according to claim 9, wherein the microparticles have hydrophobic character.

14. The biohybrid according to claim 9, wherein the microparticles are of PLLA or cross-linked gelatin.

15. The biohybrid according to claim 4, that comprises microfilaments of degradable synthetic polyesters of nylon or silk of diameters from microns to tens of microns, arranged in parallel in the lumen, which serve as support for the adhesion and guidance to the migration of cells and the extension of axons.

16. A method for obtaining the tubular scaffold defined in claim 1 comprising three layers of different porosity: an inner layer a), an intermediate layer b) and an outer layer c), with uninterrupted connection among them, and the three composed by a same porous hydrogel based on crosslinked hyaluronic acid, said method comprising: providing a grooved mold for containing said tubular scaffold; introducing into said mold a polymer material in the form of fiber(s); preparing HA solutions and stirring them in the presence of a cross-linking agent; injecting said solutions into the grooves of the mold, obtaining a mold-solutions assembly which cross-links in situ; freezing the mold-solution assembly obtained; and lyophilizing the mold-solution assembly obtaining microporous HA matrices.

17. The method according to claim 16, wherein the mold is of a hydrophobic polymeric material; the polymer material is in the form of fibers is of a hydrophobic polymeric material; and the cross-linking agent is divinyl sulfone, glutaraldehyde or carbodiimide.

18. The method according to claim 16, wherein: the mold is of polytetrafluoroethylene; the polymeric material is in the form of fibers is of poly-ε-caprolactone 5; and the cross-linking agent is divinyl sulfone, glutaraldehyde or carbodiimide.

19. The method according to claim 16 that comprises after the lyophilization step: withdrawing the tubular scaffold from the mold and the rings of material forming the mold itself; removing the fiber of polymeric material, obtaining a duct with a centered inner channel; and hydrating the HA ducts.

20. The method according to claim 16, that comprises after the hydration step, the insertion of PLLA fibers in its interior.

21. The tubular scaffold defined in claim 2, that is obtained by a method as defined in claim 16.

22. A method for using the tubular scaffold defined in claim 1 comprising inducing the regeneration of neural tracts and the reconnection of damaged or degenerate neuronal populations.

23. The method for using the tubular scaffold according to claim 22 comprising regenerating tracts in diseases affecting the central nervous system.

24. The method for using the tubular scaffold according to claim 22 comprising regenerating tracts in Parkinson's disease or spinal cord injuries.

25. A method for using the biohybrid defined in claim 4 comprising inducing the regeneration of neural tracts and the reconnection of damaged or degenerate neuronal populations.

26. The method for using the biohybrid, according to claim 25 comprising regenerating tracts in diseases affecting the central nervous system.

27. The method for using for using the biohybrid, according to claim 25 comprising regenerating tracts in Parkinson's disease or spinal cord injuries.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0059] FIG. 1 shows the pore size distribution (μm) and porosity (fraction of the total pore volume, %) of the different layers of the tubular scaffold.

[0060] FIG. 2 shows the diffusion of glucose through the hybrid duct (small molecule)

[0061] FIG. 3 shows the diffusion of bovine serum albumin (BSA, larger molecule) through the tubular duct.

[0062] FIG. 4 shows, through a confocal microscopy image, the results of diffusion through Schwann cells cultured 10 days inside the duct, whose cytoskeleton is marked in gray falcidin. The dashed line shows the limits of the channel: Cells cannot pass through it.

[0063] FIG. 5 is a scanning electron microscopy image of the same Schwann cell culture as in FIG. 4, showing the channel with adhered cells, and the longitudinal section structure of the tube. No cells are detected either in the exterior or in the middle layer of the duct.

EXAMPLES

1. Preparation of Materials

[0064] A thin block of polytetrafluoroethylene (PTFE) with perforated grooves 1.5 mm wide was used as the mold for the ducts. A poly-ε-caprolactone (PCL, PolySciences) fiber of 400-450 μm in diameter was provided in each groove using PTFE washers with a 1.5 mm outer diameter every 3 cm of fiber to keep it centered. These fibers acted as a negative for the lumen of the ducts. HA solutions (Sigma-Aldrich) at 1,3 and 5 wt % HA, were prepared in a sodium hydroxide solution (NaOH, Scharlab) and were gently stirred. Divinyl sulfone was used as a crosslinking agent (DVS, Sigma-Aldrich) (by a 1,4 Michael addition) in a molar ratio of DVS: HA, monomer units, of 9:10. After addition, the solutions were stirred for additional 10 seconds and were injected into the grooves of the mold. Once the solution was gelled, the mold was placed in a Petri dish to avoid evaporation and was cooled to −20° C. The mold-solution assembly was then lyophilized (Lyoquest-85, Telstar) for 24 h at 20 Pa and −80° C. to generate microporous HA matrices due to water sublimation. The fiber duct was then carefully withdrawn from the mold and the PTFE rings were removed. In order to extract the PCL fiber from each of the HA ducts, said fiber was stretched from its ends to reduce its diameter. Finally, the ducts were cut into 6 mm portions and stored at 4-8° C. in 30 sterile distilled water until use (up to 4 weeks).

[0065] HA ducts were obtained after lyophilization of HA solutions at 1,3 and 5 wt % of HA, injected into the molds together with the cross-linking agent. The result was a soft, stable duct with dimensions of 5.384±0.246 mm in length and 1.251±0.117 mm in width. This duct had a centered inner channel of 0.406±0.056 mm in diameter. The central channel was sufficiently wide under wet conditions to allow insertion of PLLA fibers in its interior.

[0066] The central channel extends from one end to the other of the scaffold. In the case of HA-PLLA the soft fibers are arranged parallel to the surface of the channel to favor the extension of the cells on them.

[0067] A structural study using scanning electron microscopy (SEM) images of the porosity in different zones of the wall of HA ducts at 5% by weight, revealed a unique permeable substrate, in which three pore topologies were observed. The surface of the channel had a continuous and homogeneous layer with micropores; the internal structure showed larger interconnected honeycomb-like pores, and the outer surface was rough with a random cavities distribution.

2. Cells and Hybrid Duct

[0068] Primary cultures of Schwann rat cells (SCs, Innoprot) were used. SCs were grown in flasks and were grown to converge at 37° C., 5% CO2, in a complete medium containing essential and non-essential amino acids, vitamins, organic and inorganic compounds, hormones, growth factors, trace minerals and 10% of fetal bovine serum (P60123, Innoprot). All experiments were performed with cells in passage 4 to 6. 5% HA ducts were disinfected with their hollow lumen or occupied by PLLA fibers, and their films were disinfected and preconditioned for cell culture experiments in an enclosure of laminar flow by means of two successive rinses with 70° ethanol for 1 hour. The samples were rinsed with ethanol at 50° and 30° for 10 minutes at a time, and then rinsed thoroughly with deionized water. The viability and proliferation of SCs were evaluated by the MTS assay (CellTiter 96 Aqueous One Solution, Promega). In HA ducts and 5 HA ducts with PLLA fibers in their lumen there was a significant increase in absorbance with the culture time, with respect to two-dimensional materials. The results obtained in both three-dimensional structures were of the same order for each culture and similar to those found for PLLA films on day 10. Viable and dead cells were stained and photographed by fluorescence microscopy; The images after 5 and 10 days of culture show a considerable amount of calcein-stained live cells for the three structures: HA ducts, HA-PLLA ducts and PLLA bundles. Cell mortality was greater on PLLA fibers than inside HA ducts, with or without such fibers. Quantitatively, flow cytometry analysis revealed a decrease in the percentage of dead cells inside the ducts with the time of culture (LIVE/DEAD Cell Viability Assay, Life Technologies), whereas this decrease of dead cells did not occur when the cells were cultured with the control (well plate culture well). The study of cell distribution inside the ducts by immunohistochemistry and the image processing revealed a uniform cell population after 10 days of culture along one end of the lumen, irrespective of whether or not it was filled with fibers. In those ducts containing PLLA fibers, the cells appeared to be better distributed along the lumen section, while the cells were rather wound up as a leaf in the empty lumen; this fact is reflected as the deviation of the mean intensity along the ducts, which is greater in the first case. Finally, the identity of the cells was confirmed as SCs by staining of anti-GFAP, anti-p75 and anti-S100 antibodies, and their morphology was revealed by falcidin in ducts with or without fibers. In HA ducts SCs achieved a high degree of confluence after 10 days of culture and had cellular processes, often branched. The cells spread and proliferated as a layer and migrated along the lumen. However, on the PLLA fibers the SCs cells were aligned with respect to the long axis of the fibers and showed a bipolar morphology, with a mainly spindle shape and established cell-cell contacts. In multiphoton imaging it was possible to observe, without the need of any cut, that the cells were accommodated coating the lumen of the HA and HA-PLLA ducts. Similar results could be confirmed by scan electron microscopy (SEM) images, in which the details allow to assess the different degree of adhesion of the cells depending on the substrate: the cells showed a round conformation forming aggregates and establishing adhesions located on the surface of the HA lumen, but elongated and completely adhered to the fibers, revealing intimate PLLA-cells contact. Expression of myelin glycoprotein (p0) after 10 days of culture increased compared to 1 day in HA and HA-PLLA ducts. The expression of myelin zero protein (p0), which encodes the major myelin protein (constituting more than 50% of the total protein in mature Schwann cells) and is involved in the adhesion of membranes in spiral wrappings of myelin sheaths, in processes of compaction, interestingly increased in a 3D environment without addition of any axonal signal. This expression p0 is barely detectable after 1 day, but its presence is massive in the ducts after 10 days, both with fibers and without fibers.

[0069] FIGS. 2 and 3 show that both small molecules of physiological interest, such as glucose, as proteins (such as BSA), can diffuse easily through the walls of the tube. FIGS. 4 and 5 show the effectiveness of channel confinement in cells that were seeded in the interior. This shows, at the same time, that cells from the outside cannot penetrate the channel. This property protects the cells inside the tube from possible aggressions from the environment.

[0070] As evidence that the three-layer membrane is not an obstacle to the passage of bioactive nutrients and molecules, but prevents migration of cells from the inside out, or penetration from outside, diffusion results are also presented through the duct of Schwann cells culture, FIGS. 4 and 5.