PL228106B1 - Method for producing biodegradable polymer tubes with filling, preferably intended for regeneration of peripheral nerves - Google Patents

Description

Description of the invention

The subject of the invention is a method of producing biodegradable polymer tubes with a filling, intended in particular for the regeneration of peripheral nerves.

Polymer tubes for biomedical applications, like all medical devices intended to come into contact with the body, apart from properties appropriately selected for a given application, are characterized by biocompatibility. Biocompatible biodegradable polymers can be used as implants when the expected function in the body is required for a limited time, after which the implant is degraded and its products are metabolized and removed from the body or are resorbed. In the latter case, the degradation products of the biomaterial can be used by the body in the natural physiological processes of tissue reconstruction. Biomaterials from biodegradable synthetic polymers are used in the production of medical devices, such as drug release systems, surgical threads, orthopedic fixtures and bone replacement implants, and these polymers are selected for a given application, including on the basis of their physicochemical properties, mechanism and kinetics of biodegradation.

Biodegradable polymers approved by the FDA used in medicine and studied for biomedical applications include poly (lactic acid) (PLA), poly (e-capro-lactone) (PCL) and polycarbonate (PTMC). Their mixtures and copolymers are also used. Tubes of these polymers can be produced by classical methods, for example by extrusion, wet spinning or electro spinning, and the desired strength and flexibility are ensured by the appropriate selection of the components of the mixture and the control of the processing parameters. Examples of applications for such devices are stents used in cardiovascular diseases or tubes to support nerve regeneration. Contrary to products made of non-absorbed biomaterials, for which another surgical intervention is necessary to remove an implant that has already fulfilled its function, products made of biodegradable polymers do not require subsequent surgery.

It is particularly advantageous to use biodegradable materials to produce tubes intended to assist in the regeneration of broken peripheral nerves, the so-called tubulation method, where the torn nerve stumps that cannot be sutured are placed in the tube on both sides and then attached to it. internal walls. The tube provides adequate space for regeneration - the axons of the proximal part of the nerve lengthen as they get closer and consequently connect to the distal end of the nerve. From US 5145943 and US 5145945 there is known a method of producing peripheral nerve regeneration tubes from biodegradable synthetic polymers such as polycarbonate (trimethylene) and polycarbonate copolymers containing lactides and caprolactone by extrusion. From the Journal of Applied Polymer Science, 127, 2259 (2013) there is a known method of producing pipes from synthetic biodegradable polymers, such as PLA, PCL, PTMC, by precipitation of the polymer from its solution deposited on a rod of a certain diameter in a non-solvent. In patents and patent applications relating to devices for supporting the regeneration of peripheral nerves, it is also proposed to use collagen tubes for the production of collagen tubes (US5292802A, WO 2009/084571 A2, EP 2221070 A1, EP 2228036 A1, JP 2009034374 A), chitose (US 7135040 B2, WO 92/13579, JP 4982887 B2, CN 101318035 A, US 2010234863 A1), silk (EP 2465472 A2, EP 2712955 A1, US 8106014 B2) and other proteins (US8153591 B2). In the course of research on tubulation techniques and the nerve bundle regeneration mechanism, it was found that it may be advantageous to use polymer tubing containing an internal scaffold, which will provide an additional basis for the growing neural bundle. This internal scaffold should fill all or part of the tube inside the tube, be structured to be overgrown by the regenerating nerve, or to adapt to the changing shape of the nerve as the nerve cone growing inside the tube pushes up the filling matrix. Hydrogels seem to be an excellent material for the formation of such a matrix.

There are a number of methods of obtaining pipes made of biodegradable polymers, inside which there is a filling based on polymers of natural origin, in particular a hydrogel filling, produced both in a chemical and physical manner.

There is known from patent application P. 391898 A1 an implant supporting the regeneration of damaged nerves in the form of a polymer tube filled with chitosan. Chitosan in the form of an aqueous suspension is placed in the tube and then the whole is freeze-dried. U.S. Patent No. 8,242,076 B2 discloses a method of producing a hyaluronic acid hydrogel by physically cross-linking it and using this hydrogel to regenerate soft tissues, including nervous tissue.

For the regeneration of damaged nerves, keratin polymer tubes containing an inner matrix of hyaluric acid hydrogel, in either hydrated or non-hydrated form, are also used (EP 1991253 B1).

It is also known, from the patent application EP 2255833 A2, a polymer tube made of a biodegradable polymer containing an inner matrix of biodegradable polymer with longitudinally arranged pores. The matrix-forming polymers, such as classical proteins, aminoglucans, cellulose or synthetic biodegradable polymers, are cross-linked by the classical method by heating under vacuum or with the aid of a cross-linking agent. From the patent description WO 89/10728 there is known a method of producing an internal matrix of polymer tubes from a composition of proteins and polysaccharides by introducing a suspension of this material into a cylindrical mold and then its solidification and lyophilization. The patent application WO 95/20359 A1 describes the use of polymer pipes made of a biodegradable polymer, such as poly (glycolic acid), poly (lactic acid) or their copolymers, filled with a matrix of polysaccharide (methylcellulose), which is a carrier of biologically active substances. The matrix is manufactured using the classical method. In the embodiments described above, the polysaccharide fillers introduced into the polymer tubing are usually stabilized by physical action or contain additives in the form of other chemical cross-linking agents. The process of producing these fillings is multi-stage - the creation of the filling and sterilization of the product must be carried out in separate production stages.

There are also known methods for producing hydrogels from water-soluble polysaccharides, using ionizing radiation, in particular gamma radiation and accelerated electron beams. Ionizing radiation affects polysaccharides, such as water-soluble or aqueous derivatives of cellulose, starch, chitin, carrageenan, having hydrophilic substituents, especially non-ionic ones, i.e. hydroxypropyl cellulose, hydroxyethyl cellulose, methyl cellulose and copolymers containing the same, copolymers containing the same, having substituents dissociating into ions, i.e. carboxymethyl starch, carboxymethyl chitin, carboxymethyl chitosan, carboxymethyl carrageen (US patents 6,875,862, US 6,617,448, US 7,208,593, US 7,307,157, US patent applications JP-289006-A1 2005-8796- A1 A-2001-2703). According to these methods, water is added to the polysaccharide and mixed to form a (thixotropic) paste - a pseudogel, and the resulting paste is sealed in a package and exposed to ionizing radiation. The polysaccharide concentration in the pseudogel is above 5%, in particular it is 5-65% w / w, the used ionizing radiation can be an accelerated electron beam or gamma radiation, the absorbed radiation dose is higher than 5 kGy. The description of the patent application US 20080070997 A1 discloses the formation of polymer hydrogels from CMC, consisting in the preparation of a physical gel with an acid or an acid solution and irradiation with ionizing radiation, or additionally subsequent immersion of the obtained gel in the development of an acid, or additionally adding a water-insoluble metal compound in especially alumina.

Carboxymethyl cellulose (CMC) is a water-soluble derivative of a naturally derived polymer - cellulose. It may be in the form of a monovalent metal salt, in particular the sodium salt. Carboxymethylcellulose is a biocompatible and biodegradable polymer that does not accumulate in the environment as it is hydrolyzed and decomposed into simple sugars by enzymes. It swells in water and then dissolves to form highly viscous solutions depending mainly on the molecular weight and degree of substitution. CMC is used in the food industry, mainly as a thickening agent, as well as a filler, emulsifier, dietary fiber, dispersant, in the pharmaceutical industry as a drug controlled release agent, as a tablet disintegrating agent, and in the form of aqueous solutions of its sodium salt as a binder in wet granulation, for the production of ointments and as a hydrogel base.

Carboxymethyl chitosan (CMCS) is a water-soluble derivative of a polymer of natural origin - chitin, or chitosan, which is rare in nature. CMCS may be in the form of a monovalent metal salt, in particular a sodium salt. CMCS is soluble in water and in aqueous solutions with strong acidic, neutral and alkaline reaction. The range of solubility depends on the values of physicochemical parameters such as: DDA, DS and molecular weight. CMCS is a biodegradable polymer that does not harm the environment as it is hydrolyzed and decomposed by enzymes. Due to its biocompatibility and its bacteriostatic properties, CMCS is being researched towards pharmaceutical and biomedical applications, incl. as a thickening agent for solutions, for the preparation of ointments and gels.

A method for the production of filled biodegradable polymer tubes, especially for the regeneration of peripheral nerves, consisting of biodegradable polymer tubes filled with a polysaccharide hydrogel prepared by cross-linking the polysaccharide in these tubes using the method of cross-linking polysaccharides by ionizing radiation, according to the invention, a polymer tube made of poly (lactic acid), poly (e-caprolactone), polycarbonate trimethylene), their mixtures or copolymers, with an internal diameter of 0.5-10 mm, a solution of polysaccharide or polysaccharides, such as carboxymethyl chitosan, carboxymethylcellulose or their mixtures, is introduced, water or phosphate buffer, or a physical gel of this polysaccharide or polysaccharides formed by mixing this polysaccharide or polysaccharides with water or phosphate buffer, with a polysaccharide content of 4-20% w / w, closes the filled tube in a beam-proof packaging ionizing and sterilizing the tubes and the polysaccharide inside the tube is cross-linked by irradiating with a dose of 15-50 kGy ionizing radiation. Polymer tubes made by extrusion, wet spinning, electrospinning, polymer precipitation or forming from a film or foil are used. As ionizing radiation, an accelerated electron beam or gamma radiation is used.

The cross-linking of polysaccharides by ionizing radiation used in the method according to the invention has numerous advantages over the classic methods used hitherto for cross-linking polysaccharides inside polymer tubes for peripheral nerve regeneration. The most important advantages are: no need to use additional chemicals as cross-linking agents, often toxic and difficult to remove from the final product, durability of the hydrogel obtained by this method, and the possibility of combining in one technological step the radiation cross-linking of the polysaccharide filling with radiation sterilization of the entire product, which leads to to shorten and reduce the cost of the production process of such biomaterials. The polysaccharide gel according to the invention, of the polysaccharide chains used interconnected by covalent bonds, is insoluble in whole or in part in water and aqueous solutions and forms the inner filling of the polymer tube (hydrogel matrix inside the tube), which is integrated with the tube, not sliding out of it.

Polymer tubes with hydrogel filling, obtained by the method according to the invention, can be used as medical devices, in particular as implants supporting the regeneration of peripheral nerves. They can be used for implantation immediately after being produced with the method according to the invention, without an additional separate sterilization process, if a dose of ionizing radiation was used to produce it, ensuring sterilization, and the product was placed in a bacterial and virus-impermeable packaging.

The method according to the invention is illustrated by the following examples with reference to the drawing showing the tube with a filling in a perspective view and a photo of a fragment of the tube with a filling made with a scanning electron microscope.

Example 1

Carboxymethyl cellulose (CMC) in the form of sodium salt (Daicel Co. Ltd., Japan) with the degree of substitution DS 2.34 and weight-average molecular weight 7.4-105 mol-g was used to prepare the hydrogel filling of the polymer tubes. 1. A mixture of CMC with water, with a concentration of CMC equal to 6.0% w / w, was prepared by mixing appropriate amounts of CMC and water and leaving the mixture thus prepared for 24 hours, ie until the CMC was completely dissolved in water and the mixture was homogenous. The resulting mixture was a very viscous solution. This mixture was introduced by means of a syringe with a needle into tubes made of a mixture of PLA / PTMC 50/50 w / w polymers, with an internal diameter of 1.2 mm and a length of 2 cm, made by precipitation of polymers from their solution deposited on a rod of a specific diameter, in sometimes with a solvent (described in the Journal of Applied Polymer Science, 127, 2259 (2013)). The tubes were filled so that the mixture of polysaccharide and water completely filled the tube lumen (inner volume of the tubes). The amounts of filling were determined by measuring the mass of the empty tube and after filling with the CMC-water mixture. The tubes filled with the mixture were sealed individually in a foil resistant to ionizing radiation. Additionally, samples of the mixture were sealed in a foil resistant to ionizing radiation and used for further research. Then the tubes and samples of the mixture were irradiated with a beam of accelerated electrons from the accelerator. Absorbed doses ranged from 1 to 50 kGy. After irradiation, the tubes containing the hydrogel packing were assessed for the integrity of the resulting hydrogel with the tube by immersing the tubes in a phosphate buffered saline (approx. 0.15 mol / L total salt) solution at 37 ° C for 5 days. The lack of integrity was considered to wash out the gel from inside the tube and dissolve the gel due to non-cross-linking or insufficient cross-linking of its chains. The presence of the gel inside the tube was considered good integrity after the test time had elapsed. A tube filled with the mixture but not irradiated (dose 0 kGy) was used as a control sample. The integrity results of the resulting tubular hydrogel are shown in Table 1. Minus (-) means no or no gel in the tube after incubation, plus (+) means integrity of the hydrogel with the tube.

The irradiated mixtures after being removed from the foil (contained in the foils) were transferred to beakers with deionized water in order to extract the soluble fraction, i.e. the sol. The water was changed initially every 12 hours, and after two days every 24 hours. During extraction, the hydrogel network absorbed water, dissolving the non-cross-linked mixture or, in the case of the presence of an insoluble fraction (gel), washing out the non-cross-linked and degraded parts (sol) of the polysaccharide. The non-irradiated mixture (dose 0 kGy) was tested in the same way as a control. The swollen gel (ms) was dried to constant weight (mg). GF gel fraction (%) - the amount of fraction insoluble in the sample, i.e. after extraction of the sol - was calculated using the equation (1), in which (mg) is the mass of the cross-linked gel fraction and (mP) is the initial mass of CMC in the sample.

ABOUT).

The calculated amounts of the gel fraction in the samples are also presented in Table 1. The average weight of the mixture introduced into the tube was determined on the basis of testing two samples.

Table 1

The results presented in Table 1 show the formation of a gel fraction for doses above 15 kGy. Despite the presence of the insoluble fraction, no integrity of the hydrogel with the tube was found for the 25 kGy dose, while integration of the hydrogel with the tube was observed for the 50 kGy dose. The above demonstrates the possibility of producing a CMC hydrogel inside the casing tube by cross-linking it with ionizing radiation after introducing the CMC as a mixture with water inside the tube.

Example 2

Carboxymethylchitosane (CMCS) in the form of sodium salt (Kraeber & Co GmbH) (DDA = 93.8%, DS = 1.9%), with DDA 94% deacetylation degree, degree of substitution, was used to produce the hydrogel filling of polymer tubes. DS 96%, and a weight-average molecular weight of 3.4-105 mol-g-1. CMCS mixtures with water were prepared with CMCS concentrations equal to 7.0, 10.0 and 15.0% w / w by mixing the appropriate amounts of CMCS and water and leaving the prepared mixtures for 48 hours (7% mixture) and 4 days (10 - and 15% mixture), i.e. until the CMCS is completely dissolved in water and the mixture is homogenized. The 7% CMCS mixture was a very viscous solution, the 10- and 15% mixtures were pseudogel. Then the procedure of Example 1 was followed. Tables 2, 3 and 4 show, for samples containing 7, 10 and 15% CMCS, respectively, the amounts of tube filling determined as in Example 1 and the results of the integrity evaluation of the formed hydrogel with the tubes and the amount of gel fraction. in the samples determined as in example 1.

Table 2

The results presented in Table 2 show the formation of a gel fraction for doses of 15 kGy and above as a result of cross-linking of the CMCS chains by irradiating a mixture of the polysaccharide with water, in which the CMCS concentration was 7.0% w / w. The integrity of the hydrogel with the tubes was found in the case of using doses showing the presence of the insoluble fraction (GF> 0). The above indicates the possibility of producing a hydrogel from the CMCS polysaccharide inside the sheath tube by cross-linking it with ionizing radiation after introducing this polysaccharide as a mixture with water inside the tube.

Table 3

The results presented in Table 3 show the formation of a gel fraction for doses of 15 kGy and above, as a result of cross-linking of the CMCS chains by irradiating a mixture of this polysaccharide with water, in which the CMCS concentration was 10.0% w / w. The integrity of the hydrogel with the tubes was found in the case of using doses showing the presence of the insoluble fraction (GF> 0). The above demonstrates the possibility of producing a hydrogel from the CMCS polysaccharide inside the casing tube by cross-linking it with ionizing radiation after introducing the polysaccharide as a mixture with water into the tube.

The figure shows a tube with a CMCS hydrogel filling in a perspective view and a photo of a fragment of the tube with this CMCS hydrogel filling at a concentration of 10% w / w, taken with a scanning electron microscope.

Table 4

The results presented in Table 4 show the formation of a gel fraction for doses of 15 kGy and above, as a result of cross-linking of the CMCS chains by irradiating the mixture of this polysaccharide with water, in which the CMCS concentration was 15.0% w / w. The integrity of the hydrogel with the tube was found in the case of using doses showing the presence of the insoluble fraction (GF> 0). The above demonstrates the possibility of producing a CMCS hydrogel inside the casing tube by cross-linking it with ionizing radiation after introducing the CMCS as a mixture with water inside the tube.

Example 3

The CMCS used in Example 2 was used to prepare the hydrogel filling of the polymer tubes. A mixture of CMCS with phosphate buffer (PBS) with a CMCS concentration equal to 10.0% w / w was prepared by mixing the appropriate amount of CMCS and PBS and leaving the mixture prepared in this way for 4 days. , i.e. until the CMCS is completely dissolved and the mixture is homogenous. The mixture was in the form of a pseudogel. The mixture was introduced by means of a syringe with a needle into polymer tubes made of the following synthetic biodegradable polymers: a. PLA / PTMC of the composition 25/75 (w / w) with an internal diameter of 1.2 mm and a length of 20 mm; b. PLA with an inside diameter of 2 mm and a length of 30 mm; c. PCL with an inside diameter of 4 mm and a length of 40 mm; d. d) PCL / PTMC composed of 75/25 (w / w) with an internal diameter of 3 mm and a length of 30 mm.

The tubes, individually packed in foil resistant to ionizing radiation, were irradiated with a beam of accelerated electrons from the accelerator with a dose of 25 kGy. Table 5 shows the amounts of tube filling determined as in Example 1 and the results of the integrity assessment of the resulting hydrogel with tubes made as in Example I.

Table 5

The results presented in Table 5 indicate the possibility of producing a CMCS hydrogel inside polymer pipes of different internal diameter and length, made of synthetic biodegradable polymers, as a result of cross-linking with ionizing radiation, after introducing CMCS in the form of a mixture with an aqueous salt solution (PBS) into inside the tube. It was found that in the case of using a 10% w / w solution of CMCS in phosphate buffer, a gel is formed after absorbing a dose of 25 kGy (from Table 1: GF = 27%), and the hydrogel obtained inside the tube is integral with the tube made of various synthetic biodegradable polymers. The above shows the possibility of producing a CMCS hydrogel inside casing pipes made of synthetic biodegradable polymers by cross-linking it with ionizing radiation after introducing CMCS in the form of a mixture with water inside the pipes.

Example 4

The CMC and CMCS used in Example 1 and 2 were used to prepare the hydrogel filling of the polymer tubes. A mixture of CMCS and CMC with water was prepared, with a polysaccharide concentration of 8%, in which the content of CMC and CMCS was 4% w / w, by mixing appropriate amounts CMC, CMCS and water and leaving the prepared mixture for 48 hours, ie until the polysaccharides are completely dissolved in water and the mixture is homogenous. The mixture was a very sticky pseudogel. The mixture was introduced, using a syringe with a needle, into polymer tubing made of a mixture of PLA / PTMC polymers 50/50% w / w. The tubes with the filling and the batches of the mixture were placed in a tightly closed foil and irradiated with a beam of accelerated electrons from the accelerator. The absorbed doses ranged from 1-50 kGy. Table 6 shows the amounts of tube filling, the amounts of gel fraction determined as in Example 1 and the results of the integrity evaluation of the resulting hydrogel with tubes also performed as in Example 1.

Table 6

The results presented in Table 6 indicate the formation of a gel fraction for doses of 15 kGy and above, as a result of cross-linking, by irradiation, of the mixture of CMC and CMCS with water, in which the total concentration of CMC and CMCS was 8.0%. The integrity of the hydrogel with the tube was found when using doses showing the presence of an insoluble fraction (GF> 0). The above indicates the possibility of producing a hydrogel from a mixture of CMC and CMCS inside the casing tube by cross-linking them with ionizing radiation, after introducing CMC and CMCS into the tube as a mixture with water.

On the basis of the conducted research, it is concluded that water and water-soluble polysaccharides, such as CMC and CMCS, introduced into pipes made of synthetic biodegradable polymer or a mixture of such polymers, in the form of a very viscous solution or pseudogel, undergo cross-linking inside the tube due to the irradiation of the tube from polysaccharide with ionizing radiation, in particular with an accelerated electron beam, forming an internal hydrogel filling integrated with the tube.

Claims (3)

Patent claims A method of producing biodegradable polymer tubes with a filling, especially for the regeneration of peripheral nerves, consisting of biodegradable polymer tubes filled with a polysaccharide hydrogel produced by cross-linking the polysaccharide in these tubes, using the method of cross-linking polysaccharides by ionizing radiation, characterized in that a solution of a polysaccharide or polysaccharides, such as carboxymethyl chitosan, carboxymethylcellulose or their mixtures, is introduced into a polymer tube made of poly (lactic acid), poly (s-caprolactone), polycarbonate, and their mixtures or copolymers, with an internal diameter of 0.5-10 mm, in water or phosphate buffer, or the physical gel of this polysaccharide or polysaccharides formed by mixing this polysaccharide or polysaccharides with water or phosphate buffer, with a polysaccharide content of 4-20% w / w, closes the filled tube in a package resistant to ionizing radiation and ensuring sterility of the tubes, and the polysaccharide inside the tube is cross-linked by irradiation with a dose of ionizing radiation of 15-50 kGy. 2. The method according to p. The process of claim 1, wherein the polymer tubes are made by extrusion, wet spinning, electrospinning, polymer solution precipitation or film or foil forming. 3. The method according to p. The method of claim 1, characterized in that an accelerated electron beam or gamma radiation is used as the ionizing radiation.