WO2021255701A1 - Biphasic collagen type 1 folding nerve conduits with unidirectional and multidirectional pores and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a method for manufacturing a lamellar type I collagen support consisting of two zones with different pore orientation (unidirectional and multidirectional) which, when folded from the unidirectional zone towards the multidirectional zone, forms a nerve conduit containing a cylindrical collagen I matrix with pores forming unidirectional channels on the inside and a multidirectional wrapping on the outside. The unidirectional phase promotes and directs axonal growth, and the multidirectional phase controls nutrient flow and prevents cell migration that blocks axonal growth. It also relates to the conduit resulting from folding the support, which is indicated in the treatment of peripheral nerve injuries. The novelty of this conduit lies in the fact that, during surgery, it can be adjusted to the exact diameter of the stumps caused by the lesion and to the length of the discontinuity being treated.

Description

NERVOUS DUCTS

TYPE I COLLAGEN BIPHASIC FOLDERS WITH UNIDIRECTIONAL AND MULTIDIRECTIONAL PORES AND THEIR PREPARATION METHOD

Technical sector

[0001] This invention belongs to the field of medical devices in the health sector. It describes the method for producing a type I collagen porous support made up of two continuous zones: one with unidirectionally oriented pores and the other with multidirectionally oriented pores. When the support is moistened and folds from the unidirectional to the multidirectional zone, it forms a conduit that serves to connect the peripheral nerve stumps sectioned adjusting to their diameter; This conduit can be grafted onto injured peripheral nerves in order to guide their regeneration.

Previous technique

[0002] Peripheral nerve injuries are generally associated with direct mechanical trauma. Due to the functional limitations that they produce, they impact the quality of life of the patient and the cost of the health system. This type of injury causes about 8.5 million work disabilities and at least 5 million bedridden people each year [1] Current therapeutic strategies seek neuronal survival, axonal regeneration, and nerve innervation; however, a fully effective therapeutic option is not yet available due to the complexity of the cellular and molecular events that occur immediately after peripheral nerve damage. Surgical repair of the peripheral nerve has partially neuroprotective effects and its results depend on the intervention being carried out in the first 24 hours after the injury [2] However, since it is not a vital emergency, in most cases the cases is not this may happen; therefore, alternatives that ensure the neuroprotection of injured tissues should be explored.

[0003] Annually, peripheral nerve injuries affect more than one million people worldwide [3] They produce functional limitations and neuropathic pain that impact the quality of life of patients and the costs of the health system [4- 6] Complete transection of the axon and connective tissue (neurotmesis) and the injury that maintains continuity of the nerve but degenerates the axon and its myelin sheath (severe axonotmesis) are treated surgically [4,7] Usually, the distal stump ( recipient) is sutured with the proximal (donor) stump of the severed nerve (end-end neurorrhaphy) or coapted to the trunk of an adjacent donor nerve (end-to-side neurorrhaphy). When the distance between the stumps or between the distal stump and the adjacent nerve trunk is great, tension-free sutures cannot be made. In these cases the stumps are connected with peripheral nerve autografts. The major limitations of the autograft are the availability of the donor nerve, the difference that can occur between the diameter of the graft and that of the stumps to be connected, and the size of the lesion. This last parameter is very important since the probabilities of success of autografts decrease when the size of the lesion increases; in humans, they are the treatment of choice for lesions smaller than 30 mm [1]

[0004] An alternative to overcome donor nerve limitation are decellularized allografts obtained by treatments performed on the donor nerve to remove alloantigens (eg Avance® Nerve Graft, FDA approved). These have shown good results promoting nerve regeneration, however, the use of detergents and enzymes to eliminate cellular compounds and the sterilization process, increase their cost and limit the access of the majority of the population to them. In addition, the decellularization process must guarantee the elimination of components that affect axonal activity and growth [1] For this reason, conduits of different natural and synthetic materials have been developed that guide the regeneration of the peripheral nerve when they are grafted into a lesion that Due to its size, it does not allow the nerve stumps to be sutured. [0005] Different types of conduits, made with biodegradable natural or synthetic materials, are used as axonal growth guides and facilitators of nerve reconnection. Natural materials include proteins or peptides (collagen, gelatin, fibrinogen, elastin, keratin, and silk), polysaccharides (chitin, alginate, hyaluronic acid, and other glycosaminoglycans), and some polyesters [poly (3-hydroxybutyrate) or P3HB and poly (3 hydroxybutyric acid-co-3 hydroxyvaleric acid)]. Among the materials of synthetic origin are polylactic acid (PLA), polyglycolic acid (PGA), polyglycolic polylactic acid (PLGA), polycaprolactone (PCL) and polyurethanes (PUs) [8] When the conduits - called Nerve Guidance Conduit or NGC - They request a license from food and drug regulatory agencies in different countries to apply in the treatment of human peripheral nerve injuries, they are evaluated as medical devices [1]

[0006] A patent granted in the United States of America [US 4955893, Sep. 11, 1990] described a method of producing an implant to repair and / or regenerate injured nervous tissue, consisting of a cylinder with unidirectional pores and biodegradable made of type I collagen and glycosaminoglycan (GAG), cross-linked or not by vacuum heating or exposure to glutaraldehyde. In the patented method, an aqueous suspension of collagen and GAG is dispensed into cylindrical silicone molds that are placed vertically on a tray of a pre-cooled lyophilizer. The pre-cooled tray and the cooled atmosphere of the lyophilizer establish a freezing gradient in the cylinder that allows the formation of ice crystals that preferentially grow vertically from the part of the suspension in contact with the surface of the tray towards the opposite end of the tray. the suspension. Its lyophilization results in the formation of a collagen-GAG plug with pores or channels oriented preferentially parallel to the axis of the silicone mold that contains it. The resulting porous template is used as a nerve implant and was designed to eliminate the use of autograft in the repair of severed nerves that cannot be sutured.

[0007] Another patent (US 2011/0129515 A1, Jun. 2, 2011), based on the previous one, reported the development of a biodegradable support of collagen l-GAG with unidirectionally oriented pores to promote the migration of axons and cells of Schwann along the axis of a sectioned nerve, during peripheral nerve regeneration in lesions larger than lesions treated with supports made in cylindrical silicone molds. The conduit is made up of a hollow tube made of biodegradable material (eg: NeuraGen®) that contains a porous biodegradable collagen-GAG matrix with pores oriented parallel to the axis of the tube and that go from one end of the tube to the other. conduit, unlike the product of the previous patent. For its preparation, a freezing device is used that thermally insulates the biodegradable tube that contains the l-GAG collagen suspension used to form the internal matrix. This device keeps the tube in a vertical position when it comes into contact with the cooling liquid, prevents the formation of radial thermal gradients and promotes the axial growth of ice crystals that, when lyophilized, produce the pores aligned parallel to the axis of the conduit. The canal can be made of different diameters or sizes, but in surgery these diameters are not adjustable to the diameter of the stumps if they do not match. Using the described procedure, Neuragen 3D is produced, an FDA approved conduit consisting of a type I collagen tube containing an axially oriented internal matrix of type I collagen and chondroitin-6-sulfate. Its internal microstructure provides a guide for axonal growth that controls disordered nerve buds and facilitates guided axonal reconnection. Preclinically, it has been seen that this product increases regeneration to limits similar to those of the autograft in the murine model [9] Also, that when implanted in animal models of peripheral nerve lesions, it induces greater axonal regeneration than conduits that are hollow or that continen filled with multidirected microstructure inside [10]

[0008] Patent CN106267368B describes the application of cetuximab and a cetuximab-loaded collagen carrier for the preparation of a drug that seeks to repair spinal cord injury, and also provides a cetuximab-loaded nerve regenerating collagen carrier material . However, this patent only refers to a unidirectional support for the regeneration of the nerve. [0009] The article "Human Schwann Cells Seeded on a Novel Collagen-Based Microstructured Nerve Guide Survive, Proliferate, and Modify Neurite Outgrowth" by van Neerven et. al., 2014 presents a variety of new bioartificial nerve guides that have been preclinically tested for their safety and properties to support nerve regeneration. Report an in vitro model with human Schwann cells (hSCs) as an intermediate step towards the clinical application of nerve guidance [20]

[0010] Other collagen ducts with hollow tubular morphology are known, unfortunately their efficacy is lower than that of autografts, which are still considered the standard treatment, and that of ducts with unidirectional fillers [11] Preclinical data of tubular ducts Gaps obtained in murine models with excisions of sciatic nerves of different lengths (5, 10 and 14 mm), indicate differences in the repair time and the degree of total sensory or motor recovery [8] The actual efficacy of these NGCs in humans varies. depending on the injury; however, clinical trial results indicate that FDA and CE approved NGCs may be effective in lesions smaller than 30 mm, which is less than the critical size (40 mm) for human peripheral nerve lesions [1,12]

[0011] Another type of biodegradable collagen implant approved for human use uses a sheet to wrap around injured peripheral nerves (e.g., NeuraWrap®). Although this type of product is based on collagen laminar supports, contrary to the product that we claim in this invention, it lacks a directed internal microstructure that guides axonal growth. For this reason, NeuraWrap® and similar products are used as a non-constrictive interface that separates the injured nerve from the surrounding tissue environment [13] and not to direct the regeneration of nerve axons.

Although the tubular conduits with internal collagen-glycosaminoglycan filling developed by the mentioned inventions are manufactured in different diameters, these are fixed diameters that can be greater or less than the diameter of the nerve to be reconnected. This can be an inconvenience because during surgery when the diameters of the canal and the stumps do not coincide, the chances that the graft will fail increases [14], which requires having different diameter canals to find the right that coincides with that of the stumps of the sectioned nerve. The conduit that is made with type I porous, laminar collagen supports -not cylindrical as those described in the two mentioned inventions- and that have two continuous phases with different pore orientation of this invention, is adjusted to the diameter of the stumps that you want to reconnect depending on the number of folds that are made of the unidirectional area of the support. Once the cylinder with unidirectional pores or channels is formed, the adjacent multidirectional zone covers it and forms a barrier that controls the migration of cells, such as fibroblasts, which hinder the process of guided reconnection of the axon. The procedure for obtaining it is based on differential freezing -in two steps- of two type I collagen dispersions of different concentrations served in the same device and not in different devices. In the first step, a dispersion of type I collagen of lower concentration is served in a section of a freezing device that allows the formation of unidirectional ice crystals at -78.5 ° C (or less) and that is separated from the rest with a non-stick material barrier. Unlike what happens in vertical devices, in this procedure the water from the collagen dispersion crystallizes horizontally and in parallel to the surface of the mold where the concentration gradient has been created. Therefore, the nucleation of ice crystals does not occur vertically at an angle of 90 ° to the freezing surface as in the patents initially mentioned. In the second step, after the first freezing, the barrier that separates the first section is removed and the dispersion of type I collagen with the highest concentration is served in the remaining part of the device. After this dispersion is allowed to interact with the collagen dispersion with unidirectional crystals obtained in the first freezing at -78.5 ° C, it is frozen at -20 ° C to form water crystals oriented randomly in different directions (multidirectional). Then, the differentially frozen type I collagen dispersions are lyophilized to obtain porous laminar supports made up of two adjacent and continuous zones with different pore orientation (unidirectional and multidirectional). These supports, when moistened and folded from the unidirectional pore area to the multidirectional pore area, form adjustable ducts. The adjustability of the diameter of the ducts produced With this procedure, it is another big difference with ducts that contain one-way fillers of collagen or other components.

Brief description of the invention

[0013] The present invention relates a method to produce a laminar type I collagen support consisting of two zones with different pore orientation (unidirectional and multidirectional) which, when folding from the unidirectional zone towards the multidirectional zone, forms a nerve conduit that in its The interior contains a cylindrical collagen I matrix with pores that form unidirectional channels, and the exterior contains a multidirectional envelope. The unidirectional phase favors and directs axonal growth, the multidirectional phase controls the flow of nutrients and prevents cell migration that blocks axonal growth. It is also related to the canal that results from folding the support, which is indicated for the treatment of peripheral nerve injuries. The novelty of this canal is given by the fact that during surgery it can be adjusted to the exact diameter of the stumps caused by the injury and to the length of the treated discontinuity.

Technical problem

[0014] Most of the type I collagen-based guided nerve regeneration conduits that have been approved by the United States Food and Drug Administration (FDA) and are European Conformity (CE) certified for compliance with European health, safety and environmental protection standards are hollow tubular structures that, once sutured to the proximal and distal stumps of a severed nerve, reconnect it. Therefore, they lack an internal microstructure that facilitates adhesion and guided migration of cells associated with the peripheral nerve stumps that they connect. This hinders the axonal growth required to promote proper reconnection of the sectioned axons. To a lesser extent, ducts with internal microstructure with unidirectional pores have been recorded that favor Schwann cell adhesion, proliferation and differentiation and guided axonal growth. All these canals, with or without internal microstructure, are limited by the size of the lesion, since at the time of surgery they cannot be adapted to the diameter of the lesions. Injured stumps [1] Therefore, there is a need to develop biphasic laminar supports that allow the diameter of the canal formed during surgery to be adjusted to the diameter of the stumps of the injured peripheral nerve.

Solution to problem

[0015] To solve this technical problem, the invention provides biphasic laminar type I collagen supports with two zones with different pore orientation: one with unidirectional pores designed to form the matrix or internal filling of the conduit; another, with multidirectionally oriented pores designed to surround the unidirectional interior of the duct and constitute the external surface thereof. When the supports are folded from the unidirectional to the multidirectional zone, they form a cylindrical matrix of type I collagen with unidirectional pores or channels oriented along the length of the duct and not interconnected. The unidirectional matrix, after being formed, is covered by the support with multidirectional pores that acts as a migratory barrier for unwanted cells and provides mechanical resistance. The invention also provides the method for making these supports

Advantageous effects of the invention

[0016] This design allows a conduit to be formed that can be tailored to the diameter of the peripheral nerve stumps cut during peripheral nerve repair.

Brief description of the figures Fig. 1

[0017] [Fig.1] Representative image of the analysis with scanning electron microscopy of the biphasic laminar supports of type I collagen. Clearly, the area with unidirectional pores / channels (A), the inferred area (B) and the area with randomly oriented pores (C).

Fig. 2

[0018] [Fig.2] Scanning electron microscopy of the unidirectional area of biphasic laminar supports of type I collagen. (A) Cross section showing shows non-interconnected pores; (B) Longitudinal section showing the formation of non-interconnected unidirectional channels.

Fig. 3

[0019] [Fig.3] Sequence of images that illustrate the folding of the supports from the unidirectional zone to the multidirectional zone. (A) 1cm2 laminar support moistened; (B) Rolling of the support from the edge of the unidirectional zone to the multidirectional zone; (C) Covering the free edge of the multidirectional zone with natural glue; (D) Conduit filled with a type I collagen matrix with unidirectional pores covered by a multidirectional laminar envelope.

Fig. 4

[0020] [Fig.4] Appearance of the canal hydrated with culture medium and folded so that the phase with unidirectional channels remains inside and the multidirectional phase remains outside. (A) Cross-sectional view; (B) Side view.

Fig. 5

[0021] [Fig.5] Determination of glutaraldehyde remaining in biphasic type I collagen laminar supports

Fig. 6

[0022] [Fig.6] Percentage of enzymatic degradation of the biphasic laminar support of type I collagen cross-linked with 0.06% v / v glutaraldehyde.

Fig. 7

[0023] [Fig.7] Evaluation of Young's modulus of biphasic laminar supports of type I collagen, not cross-linked (NC) and cross-linked with different concentrations (0.02 to 0.1 v / v) of glutaraldehyde (P1-P5).

Fig. 8

[0024] [Fig.8] Determination of the surface electrical charge of biphasic laminar supports of type I collagen not cross-linked (0%) and cross-linked with different concentrations (0.02 to 0.1 v / v) of glutaraldehyde. Multidirectional zone (M) and unidirectional zone (U). Fig. 9

[0025] [Fig.9] Implantation of the canal made with the biphasic laminar support of type I collagen cross-linked with 0.06% v / v of glutaraldehyde in a murine model of sciatic nerve injury greater than the critical size (1 cm). (A) Lateral view of the canal made with the biphasic type I collagen laminar support during surgery to reconnect the rat sciatic nerve stumps; (B) Front view of the duct; (C) Appearance of the sciatic nerve reconnected with the canal 12 weeks after grafting; (D) Appearance of the sciatic nerve reconnected with the inverted sciatic nerve 12 weeks after grafting (positive control-gold standard); (E) Appearance of the ungrafted sciatic nerve 12 weeks after the procedure (negative control, critical size of the lesion).

Fig. 10

[0026] [Fig.10] Representative image of an in vitro culture of Schwann cells isolated from the sciatic nerve of neonatal mice seeded in the unidirectional zone of the biphasic support of type I collagen crosslinked with 0.06% glutaraldehyde. After 7 days of culture the cells were immunostained with the peripheral nerve cell marker S100b.

Fig. 11

[0027] [Fig.11] Fourier transform infrared spectroscopy. (A) Representative spectra of the multidirectional areas of the supports cross-linked with different concentrations of glutaraldehyde (P1-P5), non-cross-linked supports (NC) and gelatin (G). (B) Representative spectra of the unidirectional regions of the supports cross-linked with different concentrations of glutaraldehyde (P1-P5), non-cross-linked supports (NC) and gelatin (G).

Description of some way to carry out the invention

[0028] The method object of the invention serves to elaborate laminar supports constituted by two continuous zones or phases (unidirectional and multidirectional) and an interface between them (Figure 1).

[0029] The freezing device used to crystallize the water from the dispersion of type I collagen (5 mg / g) unidirectionally, is sealed with a lid and is kept in a closed adiabatic environment until unidirectional freezing of the collagen dispersion occurs.

[0030] The area of the biphasic supports can be equal to or greater than 10 cm2. The unidirectional part forms non-interconnected channels throughout its microstructure (Figure 2).

[0031] When folded from the unidirectional zone to the multidirectional zone, the supports form ducts constituted by an inner filling with unidirectional pores and an outer covering with multidirectional pores (Figure 3).

[0032] The diameter of the ducts depends on the number of bends of the unidirectional support and the thickness of the sheet, therefore, it can be adjusted to match the diameter of the stumps to be connected (between 1 and 8 mm ). For example, Figure 4 shows a cylindrical, non-hollow conduit with a diameter of 3mm.

The manufactured conduits were stabilized by crosslinking with glutaraldehyde preferably but compounds or treatments such as genipin, carbodiimide, hydrothermal treatment, ultraviolet radiation, epoxy derivatives, citric acid or glyoxal can also be used. The residual levels of the crosslinking agent were less than 0.6 ng / g of support (Figure 5).

[0034] The ducts are reabsorbed because the biphasic supports with which they are made are biodegradable. These data suggest that in vivo the duct is reabsorbed in periods of time greater than a month, guaranteeing that as this occurs the proximal axons migrate guided until reconnect (Figure 6).

[0035] The evaluation of the Young's Modulus of the channels made with the supports crossed with different concentrations of glutaraldehyde (0.02 to 0.1% v / v) showed that the channel crossed with 0.06%, chosen to evaluate the growth and differentiation of cells Schwann primaries, has a modulus (0.437 MPa) that is similar to Young's Modulus of the rat sciatic nerve (0.576 ± 0.16 MPa) [15] Which suggests that the canal has the flexibility and resistance required to avoid the compression of the axons in vivo (Figure 7). [0036] The surface electrical charge of type I collagen biphasic laminar supports, not cross-linked or cross-linked with different concentrations of glutaraldehyde, indicates that the Z potential of the support cross-linked with 0.06% glutaraldehyde is negative in the multidirectional zone and positive in the unidirectional zone (Figure 8). This suggests that in vivo the multidirectional covering with random pores of the ducts does not favor the adhesion of fibroblasts, while the unidirectional zone promotes the adhesion and proliferation of Schwann cells [16,17]

[0037] When in vivo the canal is formed and adjusted to the diameter of the stumps of the severed nerves, the multidirectional zone of the support envelops the unidirectional internal filling only once; then it is fixed with suture or tissue adhesives (Figure 9).

[0038] The biphasic supports are cytocompatible because the Schwann cells that are seeded in the unidirectional zone adhere and grow oriented in the same direction of the pores / channels that characterize the microstructure of this zone (Figure 10).

[0039] The process of making biphasic laminar supports, not cross-linked (NC) or cross-linked with different concentrations of glutaraldehyde (P1-P5), does not affect the characteristics of native type I collagen (Figure 10).

[0040] In one embodiment, the type I collagen with which the biphasic laminar support is made is obtained from bovines, pigs, sheep, goats, murines, humans, fish or other animal sources by acid hydration using organic acids (acetic, citric, ascorbic , propanoic, formic, lactic) or inorganic acids (hydrochloric, phosphoric, sulfuric) any other weak acid; pepsin or trypsin or another enzyme may or may not be used for their extraction. Also, from other natural and synthetic biomaterials.

[0041] In one embodiment, the freezing method uses liquid nitrogen, a mixture of solid C02 with isopropanol, any other subcooled liquid system that allows reaching -78.5 ° C, as well as any other cooling method. [0042] In one modality of obtaining the biphasic laminar support, the collagen is crosslinked with glutaraldehyde, genipin, carbodiimide, hydrothermal treatment and other crosslinking procedures for a period between 12 and 36 hours at a temperature between 15 ° C and 39 ° C with horizontal agitation at a frequency between 100-200 rpm.

[0043] In one embodiment, the process for obtaining the biphasic laminar support includes sterilization with ethylene oxide, ionizing radiation (alpha, beta, gamma) or non-ionizing radiation (UV, microwave, X-ray), with supercritical fluids or other methods of sterilization.

[0044] In another aspect the biphasic type I collagen laminar support is cut according to the required size, moistened with sterile water for injection or sterile saline or PBS, autologous fluids such as blood plasma and other biosecure wetting solutions or suspensions . Then, it is folded leaving the phase with unidirectional channels on the inside and the multidirectional phase on the outside according to the diameter and length of the desired duct.

Examples

[0045] Experimental Example 1. Preparation of folding type I collagen biphasic laminar supports.

[0046] -For the preparation of the biphasic laminar supports of type I collagen, molds of non-stick material of 10 x 10 cm were used. Type I collagen dispersions of 5 mg / g were prepared in a 0.05M acetic acid solution, pH 2 to 3, for the unidirectional phase and 8 mg / g for the multidirectional phase, which were cross-linked with glutaraldehyde (0.02 to 0.1% v / v) for 24 h at 37 ° C on an orbital shaker at 150 rpm. The 5 mg / g collagen dispersion was first served in a section of the mold delimited by a separator of non-stick material, with one of its walls in contact with a surface that provides a temperature lower than -78.5 ° C. After this freezing, the non-stick material separator was removed, the 8 mg / g collagen dispersion was added, it was allowed to interact with the previously frozen dispersion and it was frozen at -20 ° C. Finally, the two frozen dispersions they were differentially lyophilized at 100 mTorr for 48 hours. The appearance of the support with the two zones, unidirectional and multidirectional, is presented in Figure 1.

[0047] Experimental Example 2. Analysis with scanning electron microscopy of the microstructure of the unidirectional zone of the biphasic laminar supports of type I collagen.

[0048] Scanning electron microscopy analysis of the unidirectional zone evidenced the formation of unidirectional non-interconnected pores or channels (Figure 2).

[0049] Experimental Example 3. Formation of cylindrical ducts containing an internal matrix with unidirectional pores or channels surrounded by an envelope with multidirectional pores.

[0050] The biphasic laminar supports (10 cm2) were trimmed to obtain supports with an area of 1 cm2. The one-way zone of the samples was moistened with sterile saline and rolled. Once the winding was finished, the multidirectional area was moistened and folded over the cylindrical support formed by the winding of the unidirectional area. The edge of the multidirectional zone was fixed to the surface with a natural glue (Figure 3, Figure 4).

[0051] Experimental Example 4. Evaluation of residual glutaraldehyde.

[0052] For the evaluation of residual glutaldehyde, samples (1 cm2) were taken from the unidirectional and multidirectional zones of biphasic laminar supports of type I collagen cross-linked with different concentrations of the cross-linking agent (0.02 to 0.1% v / v) and incubated (12 h, 60 ° C) with 1 mL of a 1 M NaOH, 0.1 M glycine and 0.1 M Na2S03 solution. After centrifugation (400 X g, 5 min), aliquots of the supernatants (100 mL) were taken and their absorbance at 238 nm was measured. The data obtained were interpolated in a calibration curve made with known concentrations of glutaraldehyde (Figure 5).

[0053] Experimental Example 5. Evaluation of the enzymatic degradation of a biphasine laminar support cross-linked with 0.06% v / v of glutaraldehyde.

[0054] Samples (1 cm2) were taken from the unidirectional and multidirectional areas of the biphasic laminar support of type I collagen cross-linked with 0.06% of glutaraldehyde. The samples were incubated with type I collagenase for one month and measurements of collagen degradation products were made at different times (Figure 6).

[0055] Experimental Example 6. Evaluation of the Young's modulus of biphasic laminar supports of type I collagen cross-linked with different concentrations of glutaradehyde.

[0056] Non-cross-linked and cross-linked supports (4 cm2) with different concentrations of glutaraldehyde, were moistened and rolled from the unidirectional zone to the multidirectional zone to form the non-cross-linked (NC) and cross-linked (P1-P5) ducts, each zone it had the same area. The Young's modulus of the ducts was calculated from the slope of the stress-strain curve (Figure 7).

[0057] Experimental Example 7. Determination of the surface electrical charge of biphasic laminar supports of type I collagen.

[0058] The electrical potential (Zeta potential) of the surface of the unidirectional and multidirectional zones of the biphasic laminar supports of type collagen was measured with a Zetasizer Nano ZS (Figure 8).

[0059] Experimental Example 8. Implantation of a biphasic folding nerve conduit in a rat sciatic nerve injury model.

[0060] A nerve conduit made with the biphasic collagen type I collagen crosslinked with 0.06% v / v gluraldehyde was grafted in a murine model of sciatic nerve injury greater than the critical size 1cm (Figure 9). The plastic surgeon who performed the surgical procedure was able to fold the PBS-moistened support from the unidirectional area to the multidirectional area and adjust the diameter of the canal to the diameter of the severed sciatic nerve stumps. The support was fixed on the injured ends of the operated nerve with a natural glue and surgical suture. Four months after the graft was performed, the canal made with the support restored the continuity of the sectioned sciatic nerve and was remodeled. In the injured nerve grafted with the inverted fragment obtained when creating the lesion (positive control), restoration of continuity was also observed in a similar way as occurred with the conduit made with the support object of this invention. In the group of animals whose lesions were not grafted (negative control) to demonstrate that the lesion caused by their size could not be repaired spontaneously, the presence of disorganized axonal shoots was observed that were not observed in the groups of autografted animals with inverted sciatic nerve or grafted with the conduit produced with the support claimed in this invention.

[0061] Experimental Example 9. In vitro growth of Schwann cells in a collagen type I biphasic collagen nerve duct with unidirectional and multidirectional pores

[0062] Schwann cells - isolated from the sciatic nerve of neonatal mice - were seeded in the unidirectional zone of the biphasic support of type I collagen cross-linked with 0.06% glutaraldehyde and cultured for 7 days. The cultures were fixed, immunostained with the S100b antibody to detect the expression of this characteristic marker of peripheral nerve cells. The adhesion and alignment of the cells to the unidirectional fibers of the unidirectional pores of the analyzed area is clearly appreciated (Figure 10).

[0063] Experimental Example 10. FTIR analysis of biphasic type I collagen laminar supports.

[0064] Fourier transform infrared spectroscopy (FTIR) analysis of the unidirectional and multidirectional zones of the biphasic laminar supports was made to evaluate the chemical characteristics of type I collagen after its processing. The spectra of the dry samples (2 mg) were obtained with a FT / IR-4200 spectrophotometer (Jasco, Germany), in the range of 4000-500 cm-1 with a resolution of 8 cm-1 and 150 runs / sample ( Figure 11). The results indicate that the chemical properties of collagen were not affected by the process of making the supports.

Industrial applicability

[0065] The conduit that is part of this invention is different from other conduits authorized by regulatory agencies for human use. For example, the Neuragen 3D ™ conduit consists of an external matrix of type I collagen and an internal matrix of type I collagen and chondroitin-6-sulfate oriented axially, is not made from a single support and cannot be adjusted during surgery to the diameters of the nerve stumps that it reconnects [9] NeuraGen®, Neuromatñx ™ and NeuroFlex ™ collagen type I conduits are hollow collagen I tubes that present a multidirectional microstructure in the walls because they are made with multidirectional supports of this material [18] These conduits, like Neurogen 3D ™, have fixed diameters. NeurawrapTM is a non-restrictive resorbable laminar support because it has a lateral opening whose edges can be sutured together after adjusting to the diameter of the injured nerve; Being an external envelope, it acts as an interface between the injured nerve and the surrounding tissue [13] In this same type of product is NeuroMend TM, a type I collagen envelope that is used in the treatment of minimally damaged peripheral nerves [19]

Patent bibliography

[0066] patcitl:

[0067] US 4955893

[0068] US 2011/0129515 A1

[0069] CN106267368B

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Claims

Claims [Claim 1] i A porous laminar support of type I collagen with two adjacent zones CHARACTERIZED BECAUSE IT INCLUDES i) a zone with a unidirectional pore orientation ¡i) a zone with a multidirectional pore orientation [Claim 2] The porous laminar support of type I collagen with two adjacent zones of claim one CHARACTERIZED BECAUSE IT INCLUDES i) a zone with unidirectional pore orientation: with an orientation index between 0.9 and 1.0, porosity greater than 80% and size of pore between 100 and 400 um. I) a zone with multidirectional pore orientation: with an orientation index between -1.0 and 1.0, porosity greater than 90% and pore size between 100 and 500 um. [Claim 3] A method for making the support of claim 1 CHARACTERIZED IN WHICH IT INCLUDES A first phase of unidirectional freezing with the following stages: a) In a mold of non-stick material that comprises two separate sections with a barrier of the same material, a dispersion of collagen I with a concentration between 2-7 mg / g is served in a of the sections, which on one of its sides is in contact with a thermoconductive material; b) The free end of the conductive material is put in contact with a surface that provides a temperature lower than -78.5 ° C; c) The system is sealed with a lid and kept in a closed adiabatic environment until one-way freezing of the collagen dispersion; d) Then the system is removed from the closed adiabatic environment and kept at -20 ° C; e) The lid and the barrier are removed. A second multidirectional freezing stage with the following stages: f) A collagen I dispersion with a concentration between 7-10 mg / g is served in the free section of the mold, ensuring that there is direct contact with the frozen collagen I dispersion unidirectionally; g) The dispersion is frozen at -20 ° C, to obtain multidirectional freezing; h) The system with the two differentially frozen dispersions is lyophilized to obtain the porous sheet of type I collagen. [Claim 4] The method of claim 3 CHARACTERIZED BECAUSE the collagen I used is of bovine, murine, human or other animal source obtained by acid hydration, using or not pepsin or trypsin or another enzyme for its extraction. [Claim 5] The method of claim 4 CHARACTERIZED BECAUSE the acid dispersion of collagen I is obtained using organic acids (acetic, citric, ascorbic, propanoic, formic, lactic) or inorganic acids (hydrochloric, phosphoric, sulfuric) any other weak acid; said dispersion must have a pH between 2-4; said dispersion contains between 0.01 and 0.1% v / v of glutaraldehyde or other crosslinking agent. [Claim 6] The method of claim 5 CHARACTERIZED BECAUSE it has been cross-linked with glutaraldehyde, genipin, carbodiimide, hydrothermal treatment, ultraviolet radiation, epoxy derivatives, citric acid, glyoxal for a period between 12 and 36 hours at a temperature between 15 ° C and 39 ° C with horizontal agitation at a frequency between 100-200 rpm. [Claim 7] The process of any of the preceding claims CHARACTERIZED BECAUSE the cross-linked collagen dispersion is centrifuged between 2000 to 3000 rpm / 30 minutes, or is subjected to ultrasound for 30 minutes, or is subjected to a vacuum between 500-100 mTorr during 5 minutes to degas. [Claim 8] The process of any of the preceding claims CHARACTERIZED BECAUSE the system used for freezing is selected from the group consisting of liquid nitrogen, solid C02 mixture with isopropanol that allows reaching -78.5 ° C. [Claim 9] The method of any of the preceding claims CHARACTERIZED BECAUSE the biphasic laminar support of type I collagen is sterilized with ethylene oxide, ionizing radiation (alpha, beta, gamma) or non-ionizing (UV, microwave, X-ray) or with supercritical fluids. [Claim 10] The method of any of the preceding claims CHARACTERIZED BECAUSE the biphasic laminar support of type I collagen is cut according to the required size, moistened with sterile water for injection or saline or sterile PBS or autologous fluids such as plasma blood and folds leaving the phase with unidirectional channels on the inside and the multidirectional phase on the outside according to the diameter and length of the desired duct j