BR102014032548A2 - bioactive and bioresorbable fiberglass-based tubular conduit for regeneration of peripheral nervous tissue and process for obtaining same - Google Patents

Abstract

Bioactive and bioresorbable fiberglass-based tubular conduit for regeneration of peripheral nervous tissue and procurement process. A bioactive and bioresorbable glass fiber-based tubular conduit for regeneration of peripheral nervous tissue is described, said conduit comprising from 1 to 1000 bioactive and bioresorbable glass fibers / mm2 with diameters from 1 to 200µm on a non-adherent mold with a diameter between 0.3 and 10 mm, said fibers being joined over said mold with the aid of type i or iv collagen at a concentration of 3-50 mg / ml, and after drying the glass and collagen fibers receive a previously prepared outer layer of collagen type i or iv at a concentration of 5-150 mg / ml or another natural or synthetic polymer, said outer layer being adhered with the aid of thermal processes or chemical crosslinking, and the non-adherent mold is removed. The conduit obtained has an internal diameter between 0.3 and 10 mm, a thickness between 0.1 mm and 2 mm and a length between 2 mm and 60 mm. The process of obtaining the conduit is also described.

Description

BIOACTIVE AND BIORABLEABLE FIBER-BASED TUBULAR CONDUIT FOR REGENERATION OF PERIPHERAL NERVOUS TISSUE AND PROCESS FOR OBTAINING THE SAME FIELD OF THE INVENTION

The present invention pertains to the field of tubular conduits for regenerating peripheral nervous tissue, more specifically, to a tubular conduit comprising bioactive and bioresorbable glass fibers encased in biocompatible and biodegradable polymers.

BACKGROUND OF THE INVENTION

[0002] The use of artificial conduits for nerve tissue regeneration represents a growing alternative to autogenous graft, that is, graft taken from the patient himself. This is a current topic of extreme interest for the repair of peripheral nerve injury.

Injuries to peripheral nervous tissue are quite common, in Europe alone there are over 300,000 cases every year. In the United States, approximately 200,000 patients are referred for surgical treatment each year due to this type of injury, see Ichihara, S. et al., Artificial nerve tubes and their application for repair of peripheral nerve injury: an update of current concepts. Injury, v. 39S4, p. 29-39, 2008.

Peripheral nerve injuries are mostly caused by motor vehicle accidents, invasive surgical procedures such as removal of tumors, sports injuries, syndrome-related injuries, iatrogenics, such as side effects caused by orthopedic interventions, facial cosmetic surgeries, among others. other causes, see Kretschmer, TEA Evaluation of iatrogenic lesions in 722 surgically treated cases of peripheral nerve trauma. J Neurosurg., V. 94, p. 905-912, 2001.

Currently, transplantation of small portions of autogenous nerve represents the “gold standard” procedure for regeneration. This technique has several intrinsic disadvantages such as the need for a second surgical procedure, risk of contamination and other associated problems that require the development of alternatives to this procedure.

[0006] The most commonly used surgical techniques for the repair of nervous tissue, particularly the peripheral nerves (eg, facial nerves), are based on suturing the remaining stumps by gently joining them, ie in the absence of tensions. However, there are some cases in which this binding cannot be done without intending the tissues, which would cause their degeneration. Thus, it is currently known that the use of tubes or conduits in this type of procedure facilitates and speeds up the process of nerve regeneration. Polymeric conduits may be used or experimentally used Schwann cells, which are nerve tissue cells, see Gilchrist, T. E. A. In Vitro nerve repair- in vivo. The reconstruction of peripheral nerves by entubulation with biodegradable glass tubes - a preliminary report. British Journal of Plastic Surgery, v. 51, p. 231-237, 1998.

These conduits can also be used for vascular tissue repair, being a guide for tissue neoformation.

[0008] Many alternatives have been and are being used as conduits for the repair of this type of injury. These conduits can be obtained from artery pieces, veins, muscle fascia and more recently alloplastic materials, including biocompatible polymers, but all have certain restrictions and a still very limited success rate.

[0009] Many types of biomaterials have been proposed over the years as alternatives for grafting nerve tissue damage. At first, silicone tubes were used, however, this material generated reactions of the surrounding tissues that led to the formation of a fibrous capsule around the graft. It is now common sense that the use of biodegradable polymers is a better choice for regeneration of this type of tissue, see Deilon, A.L. J. Hand Surg., V. 19B, p. 271-272, 1994.

The tube or conduit serves as a confinement to the ends (ends) of the nerve bundles and prevents axons and dendrites from being lost in the surrounding tissues.

Another function of these conduits is to prevent other cells from invading the space in which nerve tissue should grow. Eventually fibroblasts may invade this growth site and may hinder or even prevent nerve regeneration.

The microenvironment created at the site of the nerve injury also plays a very important role in advancing the stages of tissue regeneration. The permeability of the conduit used is also presented as a significant factor. Previous studies by Kim, D.H.E. -Comparison of macropore, semipermeable and non-permeable collagen conduits in nerve repair. J. Roconstr. Microsurg., V. 9, p. 415-420, 1993, showed that tubes only made of collagen, but permeable, showed better results than semi or non-permeable tubes of the same material. With this concept, it was later postulated that conduits that have micro and / or macroporosity allow more effective access of neurotrophic factors and macroproteins to the lesion site.

The inner diameter as well as the thickness of the conduit wall are also relevant factors to the nerve regeneration process.

Therefore, the concept is that an ideal tube or conduit for regeneration of nervous tissue would need to be biodegradable, but its rate of degradation would be controlled to coincide with tissue neoformation.

Conduit should also elicit little or no inflammatory response from the host tissue.

During its use and degradation, it should not swell or collapse.

It must be able to be manufactured in different sizes for fine tuning with the respective nerve to be repaired.

And if chemical factors, such as growth factors, are used, they must be released at a rate appropriate to the tissue.

By observing these requirements it is seen that bioactive glass fibers are an excellent option for nerve tissue regeneration, as they have a controlled degradation, release ions that favor tissue neoformation, can be shaped into various sizes and shapes. still, added by growth factors.

Using conduits with bioactive glass fibers would create an optimal microenvironment for nerve bundle regeneration. Anatomy and Physiology To facilitate the follow-up of the text follows a short summary of the anatomy and physiology of this very peculiar tissue.

The neuron is the fundamental component cell of the central and peripheral nervous system. Its unique structure allows it to "send" and "receive" signals from the organs to which the neuron is attached.

These signals, which begin in the cell or pericary body, are transported by long cellular projections known as axons to other cells. Dendrites are structures that allow to considerably increase the cell surface, allowing the integration of several impulses brought by numerous axon terminals of other neurons.

The Peripheral Nervous System (PNS) is composed of myelinated and unmyelinated fibers, which can make motor, sensory or autonomic innervation.

Other components of the SNP are Schwann cells. These cells produce the myelin sheath around a segment of an axon, which allows for faster and more effective propagation of electrical signals by axons.

In the attached Figure 1 it is possible to observe the structure of the neurons.

Peripheral Nerve Regeneration Mechanism The regeneration of nervous tissue implies the formation of buds or stumps of axons, and its growth allows a reinnervation of the desired tissue or organ. Functional recovery following the occurrence of a peripheral nerve injury depends on many intrinsic and extrinsic factors to neurons.

First, the neuron must survive the injury and then change its metabolic process from “signaling cell” to a mode of regeneration and growth, also changing protein synthesis, which should be geared towards the production of substances. neurotransmitters necessary for neuronal reconstruction. Secondly, the growth of the distal axon stump should promote the necessary conditioning and tissue regeneration. And thirdly the new tissue must reinnervate the target organ.

When an injury occurs, the distal axon stump gradually degenerates in a process called Wallerian degeneration; This phenomenon occurs between 48 and 160 hours after the injury. For a more effective regenerative response and the return of target tissue functionality, this phenomenon should be strongly minimized.

To this end four factors are relevant: the first is the existence of Schwann cells responsible for the production of myelin sheaths and the formation of a column of these cells is responsible for promoting the environment necessary for axonal regeneration.

The second factor is the presence of neurotrophic photores, such as neural growth factor (NGF), neurotropins, etc.

The third factor is the existence of a basal lamina that acts to support the growth of neuronal cells.

And the fourth factor is the presence of the distal stump, as it provides different neurotrophic factors for axon regeneration.

However, in some cases the lesions caused exceed the critical size at which the tissue would be able to regenerate on its own, thus necessitating artificial grafting and surgical procedures. Classification of Injuries The injuries caused to this tissue can be classified according to their duration or extent. The lesions may therefore be temporary - neuropraxia or axonotmesis - or permanent - neurotmesis.

As for its extent there are 5 degrees of injury: first degree lesions are neuropraxias, thus being a partial conduction rupture at the lesion site, but the axon structure is preserved. This type of injury takes 3 to 4 months for recovery.

Second to fourth degree lesions already refer to axonotmesis, in which the axon is damaged to a degree in which Wallerian degeneration already occurs.

In second-degree injury, conduction is impaired or absent, but Schwann cells and endoneurium (tissue surrounding the myelinated axon) are kept intact. In the third degree lesion this endoneurium is discontinued, but the perineuron (tissue surrounding the nerve fibers) is kept intact. In fourth-degree lesions, epineurium is the only tissue kept intact (tissue that surrounds the fibers forming nerve bundles), in which case surgical intervention is already required.

Already in the fifth degree lesion, known as neurotmesis, there is the complete rupture of the tissues with the formation of scars and great Wallerian degeneration. These cases also require surgical intervention.

In Figure 2 there is a scheme of how these tissues (endoneurium, perineurium and epineurium) form the nerve bundle in the peripheral nervous system.

Autogenous Grafts In current clinical practice the “gold standard” procedure for peripheral nerve regeneration is the use of the patient's own tissue as a graft. These grafts meet the requirements for optimal conduit, but also have many associated disadvantages.

Firstly, the amount of tissue collected is quite limited, there is a need for a second surgical procedure, forming a second site of nerve injury that can lead to sensory loss, increased risk of contamination and costs. And even for this near-ideal conduit, the clinical motor recovery obtained is about 40%, since when using these grafts there is no extra stimulus for tissue regeneration.

These, among other disadvantages, lead researchers to develop synthetic alternatives and attempt more effective regeneration of peripheral nervous tissue.

Nervous Conduit Basics [0044] For a conduit to be clinically usable, it must have three basic characteristics: The first is that it should serve as a guide tube for axon regeneration, as a support site for the conduit The growth of new tissue is fundamental for the efficiency of repair. The conduit can be neither too soft nor too hard, so as not to give in to the pressure of the surrounding tissues or damage those tissues, especially the nerve stump and adjunct blood vessels.

The second is conduit permeability. It must be permeable or semi-permeable to allow the exchange of substances between the medium and the interior of the tube. These exchanges allow the passage of nutrients such as glucose, oxygen and metabolic excreta more efficiently, and also allow diffusion into the conduit of neurotrophic factors and growth factors generated in the outer tissues and retain neurotrophic factors generated by the remaining axons. .

And the third is the ability to control its degradation, and ideally the conduit should dissolve at the same rate as the new nervous tissue is formed. The conduit must maintain its biocompatibility and stability during this process, so it should not swell or collapse, leading to the formation of fibrous tissue around and inside the tube, which would prevent regeneration and maturation of the axon and nerve. Furthermore, it should be considered that very rapid rates of conduit degradation can lead to mechanical and biological axon damage.

Alloplastic Conduits As mentioned above, the first synthetic tube used was made of silicone.

Grafts made from inert and non-biodegradable materials can only regenerate small defects (a few millimeters) and lead to later problems such as fibrous tissue formation, nerve compression and often need future conduit removal.

A graft or conduit made from biodegradable materials is currently shown to be a viable alternative to success in promoting nerve tissue regeneration.

There are several studies using collagen tubes and biodegradable polymers such as PGA (polyglycolic acid), PLGA (poly lactic acid-glycolic acid), PLLA (poly L-lactic acid), PCL (polycaprolactone), PLCL (poly ( L-lactide-co-caprolactone), PHB (beta-hydroxybutyric acid) etc. These conduits are offered in diameters from 1.5 to 10 mm.

Collagen tubes have the advantage of being semipermeable and as a biological substance do not cause adverse effects. Collagen tubes are proven to be effective in accelerating nerve tissue regeneration, and when added with neurotrophic factors are able to regenerate defects of up to 3 cm.

Like other alternatives for the regeneration of this type of tissue, studies have shown that the application of other polymers is also effective in defects of 8 to 20 mm. However, they can cause inflammation, irritation and limitations in the effectiveness and speed of axon regeneration.

Therefore, the use of not only biodegradable but also bioactive materials can bring a significant gain in the process of regeneration of nervous tissue. Some bioactive glasses have not only the bioactivity characteristic but also a controlled degradation rate: they are the third generation materials.

F18 bioactive glass, developed through research conducted by the Applicant's Vitreous Materials Laboratory (LaMaV) (filed Brazilian application BR102013020961-9) has the characteristics necessary for the desired application.

Use of Bioactive Glasses in Peripheral Nerve Tissue Injuries The concept of using bioactive glasses in the regeneration of nerve tissue injuries is quite new and little explored. Only a few works have proposed their use in the form of PLGA incorporated powders, see Zhang, X.F. et al. Composition-structure-property (Zn + 2 and Ca + 2 ion release) Evaluation of Si-Na-Ca-Zn-Ce glasses: Potential components for nerve guidance conduits. Materials Science and Engineering C, v. 31, p. 669-676, 2011, dense rigid phosphate glass tubes, see Leniham, D.V. et al. Biodegradable Controlled Release Glass in the Repair of Peripheral Nerve Injuries. J Hand Surg, v. 23B, p. 588-593, 1998 and hand-drawn fibers 150-200μητι diameter of 45S5 bioglass in silicone tubes, see Bunting, S. et al. Bioresorbable Glass Fibers Facilitates Peripheral Nerve Regeneration. J Hand Surg, v. 30B, p. 242-247, 2005.

Therefore, little is known about the effects of using bioactive glasses on this type of injury. However, it is well known that the presence of ions such as Ca + 2, which are largely leached by bioactive glasses, play an extremely important role in the regeneration of nervous tissue. Its presence promotes regulation and stability of the cytoskeleton of nerve cells and stimulates axonal growth.

Other factors relevant to the hypothesis of successful application of these glasses in nerve tissue injuries are their proven effectiveness in soft tissue regeneration, such as Applicant's F18 glass, as mentioned above in this report, and also its degradability. , bioactivity, easy processing and well established safety and efficacy, qualities also presented by F18 glass in various in vitro and in vivo works.

The advantages presented by conduits manufactured from bioactive glass fibers are several, one of which is that they can be shaped to the appropriate size for each type of nerve beam.

And due to the possibility of generating a composite, the conduit has sufficient mechanical strength not to collapse and not give in to the stresses of the surrounding tissues, but at the same time it is flexible enough to cause no damage to the conduits. fabrics around.

Another clear advantage is its biocompatibility, that is, it is not cytotoxic, has high bioactivity and is also completely bioresorbable.

The composite obtained from the bioactive fibers corresponding to the F18 formulation and another polymer, such as collagen, is microporous and semi-permeable, allowing more efficient nutrient and excrete exchange, making tissue regeneration more effective and faster. .

Another extremely positive point regarding this material is the alignment of the fibers within the conduit. Studies have already shown that the alignment of cells within the tube plays a fundamental role in the rate of their regeneration. This alignment allows for faster growth, better regulation of myelin formation and also greater expression of several genes related to earlier production and cell recovery.

Bioactive Glass Fiber Related Properties in Other Applications With respect to cellular interaction and response, a wide variety of cells can be grown on fibers or blankets, as their open structures facilitate the growth of desired organic tissue and improve the process. diffusion of nutrients and excreta from these cells, which enables rapid cure of injuries and tissue defects.

In their study, Clupper, D.C. et al. Bioactive Evaluation of 45S5 bioactive glass fibers and Preliminary Study of Human Osteoblast Attachment. J. Mat Science: Materials in Medicine, v. 15, p. 803-808, 2004 et al. observed a rapid interaction between bone cells (osteoblasts) and the surface of the hand-drawn fibers of the 45S5 glass, after fifteen minutes the cells had already adhered to the material, and their numbers grew over time.

Brown, R.F. et al. Growth and differentiation of osteoblastic cells on 13-93 bioactive glass fibers and scaffolds. Acta Biomaterialia, v. 4, p. 387-396, 2008 found that scaffolds obtained from glass fibers 13-93 combined properties that improve the deposition, binding, differentiation and growth of osteoblasts in the material.

Moimas, L. et al., Rabbit pilot study on the resorbability of three-dimensional bioactive glass fiber scaffolds. Acta Biomaterialia, v. 2, p. 191-199, 2006, found in a preliminary study that scaffolds made from melt spinning fibers when implanted in defects made from rabbit bones showed their full resorption within 6 months and had good results in repair and remodeling of the defect. bony.

Given these great advantages of bioactive fibers when compared to polymeric monolithic devices and other less effective alternatives in regenerating peripheral nervous tissue, it would be interesting if the technique had conduits or tubes for regenerating nerve tissue damage, such tubes or conduits. They are obtained from a set of continuous diameter controlled bioactive fibers, aligned and juxtaposed, adhered to permeable or semi-permeable polymeric tubes. These conduits would have the final characteristics necessary for an ideal conduit for regeneration of this type of tissue, such conduit being suitably flexible, stable, biocompatible, bioactive and bioresorbable.

In the patent literature some documents are found regarding devices for regeneration of peripheral nervous tissue.

[0070] Publication W02005046457-A2 deals with a tube with tapered holes along its body. The tube may contain multiple layers, the first being agarose hydrogel and the outer layer is biocompatible polymers that may or may not be biodegradable. This tube may also contain tubules inside. The product subject of publication W02005046457-A2 is for regeneration of the human sciatic nerve and central nervous system, while the nervous conduit of the invention is for regeneration of peripheral nervous tissue.

The tube of publication W02005046457-A2 may be made of cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, carboxymethyl chitosan, poly-2-hydroxyethyl methacrylate, poly (R-3-hydroxy-co-acid ( R) -3-hydroxyvaleric) -diol (PHB), collagen, keratin, gelatin, glycine, synthetic polymers such as polyesters, including polyhydroxyacids such as polylactic acid (PLA), polyglycolic acid (PGA) and their copolymers such as poly (lactic acid-co-caprolactone), some polyamides and polymethacrylates, as well as non-degradable polymers such as polyurethane, polytetrafluoroethylene, ethylene vinylacetate (EVA), polycarbonates, polyamides and some methyl, or silicone rubber.

No document has been found in the patent literature relating to the manufacture of bioactive glass fiber tubes for regeneration of peripheral nervous tissue.

The Brazilian published application BR1101765-1 relates to a procedure for the preparation of a tissue engineered product oriented to bone tissue regeneration. In it, bone mesenchymal cells are used in fibrin gels.

The Brazilian published application BR0713772-9 provides a new structure made of collagen to improve the promotion of tissue nerve regeneration. The structure is made of collagen in a multilocular thin film formation and thus a new structure other than a colloid shape, a gel shape and a fiber shape. Therefore, it refers to a laminate with different collagen presentations. But it is not necessarily a tube, and the document does not cite the use of bioactive glasses as the biomaterial that encourages tissue regeneration or the presence of glass fibers from such glasses.

The Brazilian published application BR0710174-0 relates to a composition for periodontal soft tissue regeneration that is prepared to contain cells selected from a stem / undifferentiated cell or a biastocyte having the ability to form gums, a blood platelet matrix and plasma material.

The Brazilian published application BR0615905-2 refers to a collagen and gelatin substrate for tissue regeneration that has the ability to release a basic fibroblast growth factor (bFGF) and similar cell growth factors in a Continuous release way into which cells can easily enter, and which is used appropriately for tissue regeneration. In this document, only collagen is used as a substrate and there is no use of bioglass as an incentive for tissue regeneration.

[0077] Brazilian published application BR0601751-7 describes a process for manufacturing a biodegradable biocomposite material using collagen and carbon nanotubes for medical and veterinary applications. There is no mention in this document that the developed material is exclusively indicated for peripheral nerve regeneration and does not mention the use of bioactive glasses.

The Brazilian document BR9707903-0 refers to a membrane with at least three layers, the two outer layers being of a natural material and at least one intermediate layer being of an artificial material. Therefore, this document deals with a membrane and not a tube. There is also no mention of the use of bioactive glasses.

The Brazilian published application BR1005577-0 relates to a biocompatible and bioactive partially synthetic bioceramic material comprising a sintered body of a mixture of bovine hydroxyapatite and bioaviolous niobosphate for bone regeneration. Therefore, it has nothing to do with a glass fiber polymer tube inside as in the present application.

[0080] The Brazilian published application BR0711988-7 relates to a strontium bioactive glass composition for the construction of porous parts for medical application. It does not concern the manufacture of a tube or the use of bioactive glass fibers. The glass composition is also distinct from that proposed in the present application for use in making the tubes.

Published Brazilian document BR0312879-2 refers to a craniomaxillofacial implant material which is made of a macroporous rigid polyethylene resin structure with bioactive glass particles dispersed in the polymeric matrix. Thus, it is quite distinct from the present proposal that it provides a flexible tube with aligned fiberglass bioglass.

The Brazilian published application BR0312907-1 reports that with the aid of bioactive or bone inducing substances a lateral bone support is constructed for an implant that is disposed in an assigned jawbone hole. Bone-inducing or bioactive substances consist of matrix molecules, growth factors and differentiating factors and / or peptides, with growth stimulating properties, all called GSS (Growth Stimulation Substances), disposed in or on the implant. the implant. Therefore, it has nothing to do with a biocomposite tube for regeneration of peripheral nervous tissue.

[0083] Published Brazilian document BR1100608-0 refers to a bioactive conjugate adapted to coat an external metal implant surface. Therefore, it has nothing to do with a biocomposite tube for regeneration of peripheral nervous tissue.

The Brazilian published application BR0712088-5 relates to a method for directed cell growth and controlled tissue regeneration to prevent post-surgical or post-traumatic adhesion, also unrelated to a biocomposite tube for regeneration of peripheral nervous tissue.

The Brazilian published application BR0706921-9 relates to a pharmaceutical composition comprising a bone graft material, a scaffold for tissue engineering applications that has a surface immobilized bone calcification promoting peptide, and more particularly to a bone graft material and a framework for tissue engineering applications. Thus, this invention relates to the creation of a material for bone regeneration and not for regeneration of peripheral nervous tissue. This document also does not report the use of bioactive glasses.

The Brazilian document BR9504554-6 deals with a curative device (flat) which is composed of a flexible material substrate and a solid silicone plate. This device has application in hemostatic treatment and reduction of post-traumatic edema. Therefore, it has nothing to do with a biocomposite tube for regeneration of peripheral nervous tissue.

The Brazilian published application BR0906399-4 refers to the development of a bioactive composite for bone repair, whose polymeric matrix is composed of polysaccharide. As reinforcement, calcium derivatives such as calcium hydroxide, hydroxy apatite and calcium phosphate are used to obtain bioactive composites with specificity for bone tissue. Therefore, it is not related to a biocomposite tube for regeneration of peripheral nervous tissue and does not report the use of bioactive glasses.

International publication WO2013154780A1 is a biphasic device for regeneration of peripheral nervous tissue in which repeated electric fields can be created to stimulate axons. However, this document does not mention the use of bioactive glasses as a nerve regeneration promoter or the presence of glass fibers (bioactive or not). Therefore, it is a multi-layer polymeric tube to support the generation of an electric field, so this document does not anticipate the concept of the present invention.

International publication W02012004627A1 refers to a conduit made of insulating material which has carbon nanotubes on its inner surface. Nor does this document cite the use of bioactive glasses as the nerve regeneration promoter or the presence of glass fibers (bioactive or not).

US Published Application US20020004556A1 deals with a composite with a conductive polymer, matrix of another semicrystalline polymer phase in sufficient quantity to generate a continuous conductive network. Carbon nanotubes or carbon fibers may be added to achieve this effect. In this U.S. patent document no mention is made of making a tube of this polymeric blend, nor is any biological application mentioned. Therefore, this document does not anticipate the concept of the present invention.

The US published application US20080147156A1 relates to devices, systems and methods for stimulating nerves in the spine. Bioactive glass polymer tubules are not mentioned as promoters of nerve regeneration nor the presence of glass fibers (bioactive or not).

International publication WOI999018892A1 refers to bioelectric biopolymer implants for tissue regeneration in general. However, this document does not mention the use of bioactive glass fibers, only the use of metallic fibers. Thus, this international publication is distinct from the present invention.

US6095148 relates to electrically conductive biocompatible polymers which may be used alone or in combination with other polymers for in vitro cultivation of nerve cells, or in vivo to assist in the healing of nerve tissue defects. The voltage or current is applied to the polymer in a way that induces the desired effect on the cells while not damaging them. This patent does not mention the use of bioactive glasses nor does it mention the use of fibers.

[0094] Another document found is the article by Pierucci, A., from Duekm, E.A., and Oliveira A.L. “Peripheral nerve regeneration through biodegradable conduits prepared by solvent evaporation”, Tissue Eng Part A. v. 14 (5), p. 595-606, 2008; where the author compares the effectiveness of PLLA and PCL tubes for peripheral nervous tissue regeneration. However, there is no mention of the use of bioactive glasses as there is no mention of glass fibers.

[0095] It is found that in no recovered document is reported the development of nerve tubes or conduits for regeneration of peripheral nerves using bioactive glass in addition to bioresorbable polymers. Consequently, no document reports the use of highly bioactive glass fibers aligned and capable of inducing and stimulating nerve cell proliferation.

SUMMARY OF THE INVENTION

Broadly, the bioactive and bioresorbable glass fiber-based tubular conduit for regeneration of peripheral nerves according to the invention comprises: a) bioactive and bioresorbable glass fibers with diameters from 1 to 200pm, including between 5 and 75 pm; b) collagen at a concentration of from 3 to 50mg / ml to hold the glass fibers of (a) together over a cylindrical mold of non-adherent material of 0.3 to 10 mm in diameter, including between 0.5 to 4 mm; c) a tubular outer shell previously formed by extrusion, casting, molding or casting to cover the bonded fibers of (b), said coating being made from natural biodegradable polymers selected from collagen type I or IV at a concentration between 5 and 150 mg / ml or synthetic polymers selected from polyethylene glycol (PEG), glycolic polyacid (PGA), lactic-co-glycolic polyacid (PLGA), lactic polyacid PLLA (in the three available forms: L-PLA, PDLA, PDLLA), polycaprolactone (PCL) ) and polyhydroxyalkanoates selected from PHB, with molar weights between 30,000 and 150,000 (g / mol), said coating being bonded to the fibers and collagen (internal conduit) by thermal process at 37-150 ° C or chemical cross-linking to produce a tubular conduit with 1 to 1000 bioactive and bioresorbable glass fibers / mm2, including 10 to 100 fibers / mm2, with an inside diameter between 0.3 and 10 mm, including between 0.5 and 4 mm, es thickness between 0,1 and 2 mm, including 0,3 and 0,7 mm, and length between 2 and 60 mm, including between 5 and 30 mm.

And the process of obtaining the bioactive and bioresorbable glass fiber-based tubular conduit comprises the steps of: a) providing a cylindrical mold of non-adherent material from 0.3 to 10 mm in diameter, including between 0.5 at 4 mm, selected from Teflon or metal; b) winding by any means, manually or mechanically, on the mold of (a) bioactive and bioresorbable glass fibers with diameters from 1 to 200pm, including between 5 and 75 pm; c) pouring on the fibers of b) type I or IV collagen solution at a concentration of 3 to 50mg / ml at a temperature of 30 to 50 ° C to hold said fibers together over said mold and form the inner layer of the conduit; d) drying the inner layer of c) of the tubular conduit at room temperature for at least 1 hour under controlled humidity between 35-55%; e) placing on said dry inner layer d) a previously prepared outer coating of natural biodegradable polymers selected from collagen type I or IV at a concentration between 5 and 150 mg / ml or synthetic polymers selected from polyethylene glycol (PEG), glycolic polyacid (PGA), lactic-co-glycolic polyacid (PLGA), lactic polyacid PLLA (in three available forms: L-PLA, PDLA, PDLLA), polycaprolactone (PCL) and polyhydroxyalkanoates selected from PHB, with molar weights of 30,000 to 150,000 (g / mol); f) joining said outer coating of e) to the fibers and collagen of the inner layer of d) by thermally processing at 37-150 ° C or chemical crosslinking with EDC / NHS, forming the desired tubular conduit over the non-adherent mold of a) ; g) removing the non-adherent mold from a); (h) recover the desired tubular conduit with 1 to 1000 bioactive and bioresorbable glass fibers / mm2 including 10 to 100 glass fibers / mm2, internal diameter 0.3 to 10 mm, including 0.5 to 4 mm, thickness from 0.1 to 2 mm, including 0.3 to 0.7 mm and 2 to 60 mm in length, including 5 to 30 mm; and i) sterilize, pack and store the conduit of h).

Therefore, the invention provides a bioactive and bioresorbable tubular conduit based on peripheral nerve regeneration comprising said glass fibers, an inner layer in biodegradable natural polymers such as collagen and an outer layer also of collagen or polymer Biodegradable synthetic material bonded to said inner layer by thermal process or chemical crosslinking.

The invention also provides an added tubular conduit of growth factors, biofunctionalization molecules or drug delivery systems.

[0100] The invention further provides a process for obtaining said tubular conduit for regeneration of peripheral nerves.

BRIEF DESCRIPTION OF DRAWINGS

The attached FIGURE 1 is a schematic illustration of the structure of a neuron. A - Terminal Button. B - Myelin. C - Ranvier's node. D - Cell body (pericary). E: Dendrite. Adapted from RYU, J., BEIMESCH, C.F., LALLI, T.J. Peripheral Nerve Repair. Orthopedics and Trauma, v. 25, p. 174-180, 2011.

The accompanying FIGURE 2 is a schematic illustration of the structure of a nerve bundle of the peripheral system. A - Axon. B - Ranvier's node. C - Myelin. D - Endoneurium. E - Nervous fiber. F - Perineuro. G - Intrafascicular Epineurium. H - Extrafascicular Epineurium. I - Mesoneurium. Adapted from RYU, J., BEIMESCH, C.F., LALLI, T.J. Peripheral Nerve Repair. Orthopedics and Trauma, v. 25, p. 174-180, 2011..

The accompanying FIGURE 3 is a bar graph showing fibroblast proliferation (MTT assays) with L929 fibroblasts with extracts obtained from a solution where the material was immersed for 24, 72 and 144 hours.

The attached FIGURE 4 is a photomicrograph showing a human fibroblast in close contact with the biomaterial of the invention.

The attached FIGURE 5 is a photograph of a bioactive fiber silicone conduit within it.

The attached FIGURE 6 shows a picture of an in vivo test. A) Gait record; B) Registered footprints. [Adapted from Monte-Rasol, V. V. et al. sciatic functional index in sciatic nerve crush injuries in rats. Evaluation of method reproducibility among examiners. Acta Ortop. Bras. 14 (3); 2006].

The attached FIGURE 7 is a series of micrographs of peripheral nerve tissues for all groups evaluated. Left column: Toluidine Blue stained tissue (for structural observation). Right column: immunofluorescence, arrows: nerve fiber tissue, * fibrosis, Fatty tissue. 20pm range.

DETAILED DESCRIPTION OF THE INVENTION

The bioactive and bioresorbable glass fiber-based tubular conduit of the invention comprises glass fibers in principle of any bioactive and bioresorbable glass formulation, i.e. a third generation biomaterial, which has the characteristic of permitting obtaining of continuous, aligned fibers and of varied and controlled diameters is useful for the manufacture of this conduit.

[0109] The manufacture of conduits or guide tubes from bioactive glass fibers is a treatment alternative that may lead to increased rates of nerve tissue regeneration and increased success rate, improving the prognosis of the clinical case.

[0110] The use of a biodegradable and additionally bioactivity biomaterial leads to easier repair of the axonal defect.

Furthermore, it overcomes some drawbacks and limitations of other embodiments of this biomaterial, for example, particulates and / or particulates incorporated into polymers, which may result in fragmentation and swelling of the polymeric material by detachment of particles and crystals from the composite, causing sterile inflammatory reactions and disrupting the regeneration process of new nervous tissue.

A preferred glass composition material is that designated as formulation F18 cited above owned by the Applicant and whose composition corresponds to that of Table 1 below. TABLE 1 [0113] This composition gives glass high reactivity and low chemical durability, as expected for highly bioactive glass. Its greater glass stability allows the glass to be manipulated and heat treated and / or sintered without uncontrolled crystallization, thus allowing simple but low crystallization processes to be used.

Therefore, since the conduit in question uses bioactive glass fibers in its interior, the F18 formulation is usable as it allows the manufacture of continuous, controlled diameter, industrial-scale fibers.

Once the glass composition is determined, the fibers must be obtained.

[0116] The simplest and least costly aligned shape-making process known today is downdrawing. This process is widespread and widely used in the industry for the manufacture of fiberglass.

To obtain the fibers from composition F18, the procedures previously described in Applicant's BR10 2013020961-9 were used.

Generally, the glass is heated in a crucible inside a melting furnace at temperatures above 1200 ° C for melting, then its temperature is adjusted until the viscosity of the melt is obtained. glass mass between 102 5 to 104 dPa.s. After this step, the glass seeps through the holes in the crucible, forming the fibers that are collected at a constant speed, which will determine their final diameter.

[0119] Once the bioactive fibers are prepared, the next step is the making of tubular conduit.

To this end, the bioactive fibers are aligned and juxtaposed with the aid of a cylindrically shaped general mold. The mold useful for the purposes of the invention is of various metallic or non-metallic materials. An especially useful material is Teflon.

The bioactive fibers to be positioned over said cylindrical mold comprise diameters from 1 to 200pm, including between 5 and 75 pm.

[0122] The cylindrical mold is of variable diameter. The ideal diameter will depend on the size of the final structure to be obtained, as it delimits the internal diameter of the conduit to be manufactured, ranging from 0.3 to 10 mm in diameter, including 0.5 to 4 mm

[0123] At this stage, collagen or any biodegradable polymer is useful for ensuring more stability between bioactive fibers and the cylindrical mold. Collagen is preferably type I (including animal collagen) in different concentrations depending on the final viscosity desired. A preferred concentration range is between 3 and 50mg / ml.

Collagen poured onto the bioactive fibers coiled in the cylindrical mold forms the inner layer of the conduit.

After drying for at least 1 hour at room temperature and humidity controlled at 35-55%, the inner layer of the tubular conduit is ready to receive the outer layer.

[0126] The outer layer of the conduit is pre-prepared, either in the form of flat film or already cylindrical in shape and fitted over the ready-made inner layer. For this the outer layer is formed on the inner layer (bioactive fibers + collagen) by thermal process or by chemical crosslinking, forming the conduit over the mold.

[0127] The outer layer of the conduit is obtained from various natural or synthetic biodegradable polymers.

In the case of natural biodegradable polymers collagen is selected, preferably of type I or type IV.

For synthetic polymers intended to form the outside of the conduit, various types of polymers or combinations thereof are useful, selected from polyethylene glycol (PEG), glycolic polyacid (PGA), lactic-co-glycolic acid (PLGA), lactic polyacid PLLA (in the three available forms: L-PLA, PDLA, PDLLA), polycaprolactone (PCL) or polyhydroxyalkanoates such as PHB.

Each of these polymers may undergo various types of forming processes to be manufactured in tubes intended to form the outside of the conduit of the invention.

And for making the conduit, one or more types of synthetic and / or natural biodegradable polymers are useful. For polymers such as PGA, PLLA, PCL or PHB the processes to be used are selected from extrusion, casting, molding or casting.

[0132] Polymers such as collagen type I, type IV, PGA and PEG are shaped by solvent bonding, forming films or drawn, forming wires that are later shaped into tubes or extrusion, injection or molding processes.

Each of these techniques has its particularities in relation to the processing parameters; and the choice thereof will also depend on the polymer selected.

In general, regardless of the processing technique used to obtain the tubes intended to form the outer layer of the conduit, the polymers are heated above the glass transition temperature and usually above 50 ° C (this temperature will vary to each polymer formulation) so that they behave like a rubber or viscous liquid. The viscous mass is then introduced into a mold or matrix to produce the desired shape, usually under pressure.

[0135] In the case of collagen, casting processing is widely used because it is practical and less expensive.

After obtaining the outer layer of the tube-shaped conduit, it receives inside it the adhesion of bioactive glass fibers by thermal processes (ranging from 37-150 ° C) and / or with the aid of polymeric solutions, preferably collagen type I or type IV.

[0137] The collagen solution useful for the outer conduit layer has concentrations between 5 and 150mg / ml. This solution may or may not be cross-linked by different means. For example: using EDC / NHS (1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide / N-hydroxysuccinimide), transglutaminase, glutaraldehyde, genepicin, sulfonates, polyethylene glycol, formaldehydes or enzymes.

If the EDC system is selected, an example of the cross-linking process can be used as a solution of 1-60mM EDC and 1-30mM NHS in 1-100mM MES. A suitable solution comprises 30mM EDC and 10mM NHS in 50mM MES. This step is followed by a wash with 0.1-0.2 M NaH 2 PO 4 and deionized water 4 times or more for a period of two hours. After these steps, collagen can be dried or other freeze-drying processes for up to 48 hours, so this collagen is useful for making the outside of the conduit.

The final conduit comprises from 1 to 1000 fibers per mm2, with a preferred range between 10 and 100 fibers per mm2, this concentration being determined with respect to the desired reactivity for the final conduit.

[0140] The wall thickness for this type of material is between 0.1 and 2 mm, with a preferred range between 0.3 and 0.7 mm.

[0141] The invention also contemplates adding growth factors, biofunctionalizing molecules or drug delivery systems to the tubular conduit of the invention.

[0142] For completion, the tubular conduit is sized to the desired length, from millimeters to centimeters. An especially useful range is between 2 mm and 60 mm, including 5 mm to 30 mm.

[0143] At the end of the process the cylindrical mold, being non-adherent in nature, is easily separated from the tubular conduit.

Following these procedures, the tubular conduit should be sterilized, preferably using ethylene oxide or gamma radiation, packaged and packaged for storage and marketing.

In view of its intended purpose, tubular conduit for regeneration of peripheral nerves according to the invention should be tested for cytotoxicity.

To verify the non-cytotoxicity of the material, in vitro and in vivo assays were performed.

[0147] And for the investigation of the actuation of glass fibers in nervous tissue, in vivo assays were performed (at the moment in pilot scale).

The tests performed so far have shown in vitro cell viability with very favorable results and similar to the most traditional bio glass on the market, the 45S5, ie the new bioactive glass developed allows the adhesion and viability of bone tissue cells. and soft tissue such as fibroblasts.

In vivo assays showed non-cytotoxicity of local and systemic material as well as non-genotoxicity of material by the Comet assay. Therefore, the material does not generate changes in the cellular genetic code.

[0150] Test results as shown in Figure 3 confirm non-cytotoxicity of the material.

[0151] The graph shown in Figure 3 shows the increased cell proliferation of fibroblasts when exposed to material leached ions at different dilutions in DMEM medium (100%, 50%, 25% and 12.5%) (Dulbecco-Modified Eagle Medium), being (#) p <0.05 versus CG, (^) p <0.05 versus CG, and (*) p <0.05 versus 50%, 25%, and 12.5%. It can be noted that in the extract with 100% of the ions the proliferation is increased with a statistically significant difference when compared to the control.

Another assay performed to verify human cell viability was the cytotoxicity assay on fibroblasts collected from a 65-year-old patient. Cells were cultured together with the material in RPMI-1640 culture medium for 7 days. After this experimental time, the cells were counted by optical microscopy. Cell proliferation was quite satisfactory, starting with 5-104 and ending with 3-105 cells, with good biocompatibility and close contact with the biomaterial, as can be seen in Figure 4.

Another factor that evidences the efficacy of the material in soft tissues is its angiogenic potential verified by in vivo immunohistochemistry assays in rats (skin wound assays). The VEGF and TGF-β factors were evaluated by optical microscope for control animals and animals treated with the biomaterial of the invention.

Vascular endothelial growth factor (VEGF) is a signaling protein that, among other things, promotes the growth of new blood vessels. Therefore its presence demonstrates the amount of formation of new vessels and the greater the presence of this factor the greater the angiogenic potential of the material. It was observed that the characteristic color intensity of this growth is more prominent in the generated slide of the animal treated with the biomaterial of the invention.

The growth transforming factor beta (TGF-β) is a protein that controls proliferation, cell differentiation and other functions in most cells. Therefore, their presence generally shows the rate of cell proliferation. Again, this factor was stained and is quantified by histological slides. It was possible to observe, once again, higher color intensity in the generated slide of the animal treated with the biomaterial.

With regard to in vivo assays, to date, in vivo assays related to peripheral nervous tissue are pilot scale and were performed with an earlier version of the conduit that was silicone with the bioactive fibers inside (Figure 15). 5). The medical grade silicone tube was selected because it is considered an inert material, and thus only the action of bioactive fibers would be evaluated.

In this study Functional Analysis or Sciatic Functional Index assays were performed as well as histological analyzes. The animals were divided into three groups (n = 2) where one group received the biomaterial for 2 months (group 1), another received the biomaterial for 4 months (group 2) and the other underwent autogenous grafting (group 3 - but this one group was only analyzed after 4 months of surgery).

[0158] The surgical procedure was based on the making of 3 mm long lesions on the sciatic nerve of male Wistar rats and then the suture of the tube with the biomaterial or the implantation of the autogenous nerve removed during the procedure itself (the cut portion was inverted and sutured).

[0159] The Sciatic Functional Index Assay is a gait test in which the anatomical footprints of the analyzed animals are measured, generating a functionality index. Figure 6 exemplifies how this test is performed. In A) the gait record is seen and in B) the recorded footprints.

[0160] The results obtained through this trial showed that for animals that underwent autogenous graft (group 3), the expected pattern was observed, that is, gait worsening soon after injury and an improvement at the end of the experiment (period 4 months). This finding attests that nerve regeneration has occurred at least in part because some degree of gait recovery has been observed.

In the animal that received the biomaterial 2 months (group 1), a maintenance of the function was observed very close to the values found for the animals of group 3, after 4 months. However, at 4 months (group 2) function deteriorated due to fibrous tissue formation; This phenomenon was due to the presence of silicone in the conduits.

Histological analyzes (Figure 7) showed that for group 3 animals, after 4 months, the nerve structure is not as uniform as a normal (non-injured) nerve, showing its smaller fibers compared to one normal tissue and also with many flaws. Neurofilaments corroborate the general morphological findings, showing axonal discontinuity ranges (within an expected pattern of tissue repair after a serious injury) and the immunofluorescence reaction is weaker when compared to normal peripheral nervous tissue.

The animals that received the biomaterial for 2 and 4 months (group 1 and 2) presented disorganized tissue with fibrosis and less expression of neurofilaments compared to group 3. These findings are indicative of a rejection of the organism to the material. may be from the tube, which in this case was silicone. For with the 2-month animals (group 1) fibers were no longer found, as they had completely dissolved (Figure 7).

[0164] New in vitro and in vivo tests should be conducted with the silicone free conduit version in its composition.

[0165] However, the results found in the functional analysis assays lead us to believe that the presence of bioactive glass fibers was a great stimulus for nervous beam formation, since at two months the animals showed a significant improvement in gait, and this It only happens for 4-6 months for animals with autogenous grafts, but once this material dissolved (about two months) the formation of a fibrous tissue around the silicone was inevitable, impairing the regeneration process of the nervous tissue. .

Advantageously, the bioactive glass fibers included in the tubular conduit of the invention constitute a material that stimulates the faster regeneration of peripheral nervous tissue.

[0167] Another advantage is the alignment of the glass fibers, making the new fabric form much faster and more organized.

[0168] Cost is another important factor in the development of this new biomaterial-based conduit. These are very high value-added products that would be of great interest to Brazilian companies, as the existing conduits in the market are imported and can cost between 1,200 and 1,800 euros each. In Brazil, the 1.5 mm diameter Neurolac tube costs R $ 9,735.00 each.

[0169] The developed tubular conduit has a lower manufacturing cost compared to the market, being possible its sale at more affordable prices.

[0170] Another clear advantage is that it is made from fully biocompatible and bioresorbable materials, making an additional surgical procedure unnecessary for removal.

[0171] The new conduit has flexibility, which is relevant for not damaging the surrounding tissues and is also permeable or semipermeable, which ensures that the exchange of nutrients and cellular excreta is done more effectively, thus accelerating the tissue regeneration process.

Claims (8)

1. Bioactive and bioresorbable glass fiber-based tubular conduit for regeneration of peripheral nervous tissue, said conduit comprising: (a) 1 to 1000 bioactive and bioresorbable glass fibers with diameters from 1 to 200pm; b) collagen at a concentration of from 3 to 50mg / ml to hold the glass fibers of a) together over a cylindrical mold of non-adherent material from 0.3mm to 10mm in diameter; and c) a tubular outer shell previously formed by extrusion, casting, molding or casting to cover the bonded fibers of (b) said shell made of natural biodegradable polymers selected from collagen type I or IV at a concentration between 5 and 150 mg / ml or synthetic polymers selected from polyethylene glycol (PEG), glycolic polyacid (PGA), lactic-co-glycolic polyacid (PLGA), lactic polyacid PLLA (in the three available forms: L-PLA, PDLA, PDLLA), polycaprolactone (PCL) ) and polyhydroxyalkanoates selected from PHB, with molar weights between 30,000 and 150,000 (g / mol), said coating being bonded to the fibers and collagen (internal conduit) by thermal process at 37-150 ° C or chemical cross-linking to produce a tubular conduit with an inner diameter between 0.3 mm and 10 mm, a thickness between 0.1 and 2 mm, and a length between 2 mm and 60 mm, Tubular conduit according to Claim 1, characterized in that the bioactive and bioresorbable fiberglass is F18 fiber. Tubular conduit according to Claim 1, characterized in that the inside diameter of the cylindrical mold of a) is between 0.5mm and 4mm. Tubular conduit according to claim 1, characterized in that it comprises from 10 to 100 bioactive and bioresorbable glass fibers / mm2 with diameters between 5 pm and 75 pm, the internal diameter of the conduit being between 0.5 mm and 4 mm; the thickness between 0.3 mm and 0.7 mm and its length between 5 mm and 30 mm. Method for obtaining conduit according to claim 1, characterized in that it comprises the steps of: a) providing a cylindrical mold of non-adherent material of 0.3 to 10 mm in diameter, selected from Teflon or metal; b) winding by any means, manually or mechanically, on the mold of (a) between 1 and 100 fibers / mm2 of bioactive and bioresorbable glass with diameters from 1 to 200pm; c) pouring on the fibers of b) type I or IV collagen solution at a concentration of 3 to 50mg / ml at a temperature of 30 to 50 ° C to hold said fibers together over said mold and form the inner layer of the conduit; d) drying the inner layer of c) of the tubular conduit at room temperature for at least 1 hour with controlled humidity between 35-55%; e) placing on said dry inner layer d) a previously prepared outer coating of natural biodegradable polymers selected from collagen type I or IV at a concentration between 5 and 150 mg / ml or synthetic polymers selected from polyethylene glycol (PEG), glycolic polyacid (PGA), lactic-co-glycolic polyacid (PLGA), lactic polyacid PLLA (in three available forms: L-PLA, PDLA, PDLLA), polycaprolactone (PCL) and polyhydroxyalkanoates selected from PHB, with molar weights of 30,000 to 150,000 (g / mol); f) joining said outer coating of e) to the fibers and collagen of the inner layer of d) by thermally processing at 37-150 ° C or chemical crosslinking with EDC / NHS, forming the desired tubular conduit over the non-adherent mold of a) : g) removing the non-adherent mold from a); h) recovering the desired tubular conduit having an inside diameter of 0.3 to 10 mm, a thickness of 0.1 to 2 mm, and 2 to 60 mm in length; and i) sterilize, pack and store the conduit of h). Process according to Claim 5, characterized in that the bioactive and bioresorbable fiberglass is F18 fiber. Process according to Claim 5, characterized in that the internal diameter of the cylindrical mold of a) is between 0.5 mm and 4 mm. Process according to Claim 5, characterized in that it comprises between 10 and 100 bio-resorbable bioactive glass fibers / mm 2 with diameters between 5 pm and 75 pm, the internal diameter of the conduit being between 0.5 mm and 4 mm, thickness between 0.3 mm and 0.7 mm and its length between 5 mm and 30 mm.