KR20060018752A - Collagen Conduit Coated with Synthetic Biodegradable Polymer and Manufacturing Method - Google Patents

Abstract

The present invention relates to a collagen conduit coated with a synthetic biodegradable polymer on the outside of the collagen conduit and a method for producing the same.

Neural conduit, biodegradable polymer, nerve regeneration, coating, collagen, nerve cell, PLGA, PLA

Description

Collagen conduit coated with synthetic biodegradable polymer and method for the production example}

1 is a collagen film used to make neural conduits.

2 is an injector used to coat synthetic biodegradable polymers into collagen conduits.

3 is a neural conduit spray coated with a biodegradable polymer that is a biodegradable polymer.

Figure 4 is (1) the surface photograph of the collagen film before coating, (2) the surface photograph of the collagen film coated with 0.1% PLGA, (3) the surface photograph of the collagen film coated with 0.3% PLGA, (4) 0.5% PLGA Surface photo of the collagen film coated with (5) surface photo of the collagen film coated with 0.1% PLA, (6) surface photo of the collagen film coated with 0.3% PLA, (7) collagen film coated with 0.5% PLA Surface photograph of (8) surface photograph of collagen film crosslinked with 0.25% GA, (9) surface photograph of collagen film with 0.5% GA.

5 is a cross-sectional photograph of (1) a pre-coated collagen film, (2) an enlarged cross-sectional photograph of a ○ part of (1), (3) a cross-sectional photograph of a collagen film coated with 0.3% polylacticcoglycolic acid, (4 ) It is cross-sectional photograph which enlarged ○ part of (3).

Figure 6 shows the results of the biodegradability test results of the neural conduit coated with the biodegradable polymer according to the concentration (a) when collagenase was added (b) was not added collagenase. C means no biodegradable polymer, G means PLGA, L means PLA, GA means glutaraldehyde.

Figure 7 shows the elastic modulus of the neural conduit coated with the biodegradable polymer according to the concentration.

8 shows the tensile strength of the neural conduit coated with the biodegradable polymer according to the concentration.

9 shows the cytotoxicity of neural conduits coated with biodegradable polymers at different concentrations.

Figure 10 is a (1) picture of the nerve conduit coated 0.3% PLGA on the left sciatic nerve of the rabbit fetus and (2) a neurophotograph regenerated through the nerve conduit 12 weeks after the implantation.

FIG. 11 is (1) histological picture of low magnification of nerves regenerated through the nerve conduit 12 weeks after embedding the 0.3% PLGA-coated neural conduit into the rabbit femoral left sciatic nerve and (2) histological picture of high magnification.

The present invention relates to a collagen conduit coated with a biodegradable polymer and a method for producing the same, wherein the conduit is composed of collagen.

When the peripheral nerves are cut, the nerve tissue regenerates through a unique process. The axon at the proximal portion of the cut site contracts shortly after the cut but begins to grow a few days later, with growth cones formed at the distal end that grow in all directions. Once one growth cone is reached, the other growth cone is removed and only successful axons continue to the target tissue. However, if the cleavage site is too extensive, the regeneration is not so smooth. In this case, the peripheral nerves, of which function is not important, are partially excised and autologous transplantation is sometimes performed at the nerve cutting sites. However, this method does not fully restore nerve function, and a decrease in function may occur even in the area where the transplanted nerve is collected.

Therefore, there is a neural conduit as a method for restoring its function when the nerve defect is large. Neural conduit refers to a tube that fixes both ends of a cut nerve in an artificial tube and induces nerve connections into the tube. These nerve conduits can prevent the penetration of scar tissue that interferes with nerve regeneration, induce the growth of axons in the right direction, and the substances that interfere with regeneration while maintaining the regeneration materials secreted by the nerve itself It has the advantage of being cut off from the outside.

The most commonly used nerve conduit to date is the collagen conduit. Collagen is a material widely used in the field of biomaterials because of its excellent biocompatibility and weak antigenicity, and has been used in various inventions as a material of neural conduits for nerve regeneration. However, collagen has the disadvantage that the decomposition rate is very fast and the mechanical strength is weak. Although cross-linked collagen was used to compensate for this drawback, cross-linked collagen has a limitation of low biocompatibility due to toxicity and low hydrophilicity due to the cross-linking agent.

Therefore, the present inventors can control the mechanical properties and biodegradability of the conduit according to the concentration of the coated biodegradable polymer solution when coating the outside of the collagen conduit with the biodegradable polymer, so that the desired mechanical strength and decomposition rate are not toxic. The present invention has been completed and found that it can be usefully used for the repair of peripheral nerve defects.

One object of the present invention is to provide a collagen conduit in which the outside of the collagen conduit is coated with a synthetic biodegradable polymer.

Still another object of the present invention is to provide a biodegradable polymer-coated collagen conduit manufacturing process comprising rolling and drying a porous collagen film and spraying and coating a synthetic biodegradable polymer solution on the outside of the collagen film.

In one embodiment, the present invention relates to a collagen conduit wherein the outside of the collagen conduit is coated with a synthetic biodegradable polymer.

"Conduit" in the present invention is a tubular object that is used as a neural conduit for biotransplantation, more specifically for nerve regeneration. Since a conduit for living transplantation, a material having high biocompatibility should be used. In the present invention, collagen was used.

In the present invention, "collagen" includes all forms of collagen extracted from nature or produced recombinantly. Collagen is preferred as a medical material because it has excellent biocompatibility and tissue adhesion, low antigenicity, functions to promote differentiation and proliferation of host cells, and has a characteristic of being completely degraded and absorbed in vivo. Natural collagen can be extracted and used from connective tissues of various organs such as skin, bone, cartilage, tendons and organs of animals by acid solubilization, alkali solubilization, neutral solubilization and enzyme solubilization. To date, Type I to XIX have been discovered, and Type I to V type collagen is the most used. In the present invention, all types of collagen may be used alone or in combination of one or more components. Preferably, they are natural type I, II, III, IV and V collagen, and most preferably bovine-derived type I collagen.

However, collagen has a disadvantage in that it is difficult to maintain the strength for a period of time when the collagen is in contact with water, body fluid or blood and the decomposition rate is fast. In order to solve this problem, when crosslinking with a crosslinking agent such as glutaraldehyde (GA) or epoxy, the toxicity of the crosslinking agent itself is not only a problem for the living body, but also a promoting effect on the biochemical properties of collagen, in particular, cell proliferation. Will be lost. In addition, the crosslinking agent-treated collagen has the disadvantage that the biodegradation hardly occurs and the mechanical strength is too strong. In the case of performing physical crosslinking treatment with radiation, electron beam, ultraviolet rays or heat instead of the crosslinking agent treatment, the crosslinking rate is unstable, and it is difficult to perform crosslinking treatment so that the absorption rate in vivo can be controlled without providing sufficient physical properties.

Collagen of the present invention is characterized in that the non-crosslinked collagen. Instead, to improve the mechanical strength of the collagen conduit while reducing the decomposition rate of the conventional crosslinked collagen in the present invention, the outside of the collagen conduit was coated with a synthetic biodegradable polymer.

As used herein, the term "biodegradable polymer" refers to a polymer that is gradually converted into a non-toxic decomposition product after a certain time in vivo. In the present invention, the synthetic biodegradable polymer was used as a conduit jacket coating to increase the mechanical strength of the collagen conduit, and the collagen conduit coated with the synthetic biodegradable polymer is preferably present until the nerve is regenerated.

Synthetic biodegradable polymers that can be used as skin coatings in the present invention include all biodegradable polymers that have properties that enhance the mechanical strength of collagen conduits and do not interfere with neuronal regeneration. For example, polylactic acid (PLA), polyglycolic acid (PGA), and copolymers thereof poly (lactic-co-glycolic acid) (PLGA) ), Polyesters such as poly (lactic-co-glycolic acid) -glucose (PLGA-glucose), star polymers thereof, polyorthoesters, polyanhydrides, and polydioxa Biodegradable polymers such as, but not limited to, polydioxanone, polyamino acid, polyhydroxybutyric acid, polycaprolactone, polyalkylcarbonate, and the like. The biodegradable polymers may be used as derivatives thereof, or may be used alone or in combination of two or more components. Preferably it is PLA, PGA, PLGA, or a mixture thereof, More preferably, it is PLA, PLGA.

The inside of the conduit of the invention is a collagen and the outside is a non-uniform layer having a collagen layer coated with a synthetic biodegradable polymer. The inside of the conduit consisting of collagen alone has the advantages of collagen, ie, cell adhesion, which is advantageous for the growth of nerve cells. Conduits using conventional synthetic biodegradable polymers have low hydrophilicity, making cell adhesion difficult (DJ Mooney., Biomaterials , 17: 115-124, 1996). The present invention improves the mechanical strength by coating the synthetic biodegradable polymer on the outside of the conduit while using the properties of collagen, a natural biodegradable polymer.

Synthetic biodegradable polymers coated outside the conduit may be coated one or more times, for example two or three times.

The conduit of the present invention is freely adjustable the inner diameter and length. When used as a nerve conduit, the inner diameter and length are adjusted according to the size of the missing nerve. The inner diameter is preferably 0.5 mm to 5 mm, more preferably 0.8 mm to 1.2 mm, and the length is preferably 5 mm to 200 mm, more preferably 10 mm to 20 mm.

Collagen conduits coated with synthetic biodegradable polymers of the present invention are medical alternatives, such as artificial neural conduits, artificial spinal cord. Artificial esophagus, artificial organs. It can be used for artificial blood vessels, artificial valves, etc., but is not limited thereto. Application to artificial nerve channels also used for nerve regeneration.

In a specific experimental embodiment, the collagen conduit of the present invention can maintain its shape for a period of time, compared with the conventional GA crosslinked collagen neural conduit shows little biodegradation and high cytotoxicity. Biodegradable biodegradable polymer coatings have adequate mechanical strength, but are safe without in vivo toxicity, have no loss of promoting effect on cell proliferation, and degradation rates are adequately reduced compared to uncoated collagen. In addition, the strength and decomposition rate of the collagen conduit coated with a synthetic biodegradable polymer was freely adjustable according to the type, concentration and number of coating layers of the polymer used as a coating agent. As a result of in vivo experiments, when the rabbit left sciatic nerve was surgically cut about 15 mm in length and the collagen conduit coated with 0.3% PLGA of the present invention was implanted and the suture was opened after 12 weeks, the nerve was completely regenerated. The implanted neural conduit was fully biodegradable.

In still another aspect, the present invention provides a method for producing a collagen conduit coated with a synthetic biodegradable polymer comprising rolling and drying a porous collagen film and spraying and coating the synthetic biodegradable polymer solution outside the collagen film. It is about.

The size of the pores is very important for the nutrients from outside the catheter to enter the catheter and the nerves regenerated inside the catheter to prevent the penetration of external scar tissue. The higher the concentration in the sprayed coating solution, the smaller the size of the pores in the conduit.

As the concentration of the coating solution increases, the rate of biodegradation of the conduit is slowed. It is important to control the rate of decomposition so that it remains in vivo until the nerve is regenerated.

Preferably it is to be degraded within 4 to 20 weeks, more preferably 8 to 16 weeks.

In addition, as the concentration of the coating solution increases, the mechanical strength of the conduit increases, and in order to maintain the shape for a certain period in the living body, it preferably has a tensile strength of 0.1 to 0.6 MPa and an elastic modulus of 0.2 to 1.2 MPa.

Depending on the size of the cut nerves, all of the above considerations should be used to use a coating solution of the appropriate concentration. The biodegradable polymer concentration is preferably 0.05% to 1.0%, more preferably 0.1% to 0.5%.

If the coating layer is too thick, peeling phenomenon will occur, and if too thin, a homogeneous coating layer will be formed. The thickness of the coating is determined according to the concentration of the coating liquid, the pressure at the time of injection, the amount of injection, and the distance between the injection nozzle and the conduit. In a specific embodiment, the distance between the spray nozzle and the conduit is 3 cm and spray coated. The thickness of the coating layer is preferably 1 μm to 50 μm, more preferably 5 μm to 20 μm, and most preferably 8 μm to 12 μm.

Illustrating the conduit manufacturing process as a specific embodiment is as follows. The hydrated collagen film is conduit shaped using Teflon rods and then dried in a 30 ° C. dryer. Remove the Teflon rods and wash at least once with distilled water. The collagen conduit is cut to the desired length. The polymer solution prepared by dissolving the biodegradable polymer, preferably PLGA or PLA in methyl chloride (MC) at a concentration of 0.01% to 1%, preferably 0.1% to 0.5%, is spray coated on the cut conduit. The biodegradable polymer coated conduit is washed one or more times with distilled water and then dried in a vacuum dryer for about 24 hours. Sterilize with ion gas and pack and store.

Hereinafter, the present invention will be described in detail by experimental examples. However, the following examples are merely to illustrate the invention, the present invention is not limited to the following examples.

Example 1 Preparation of Biodegradable Collagen Conduit Coated with Biodegradable Polymer

It was used as a basic material for making a: (Integra LifeSciences Coporation, Plainsboro, NJ, USA CollaTape ®) nerve conduit (1) a commercially available dental porous collagen film. The collagen film was soaked in distilled water and then made a conduit shape using a Teflon rod. This was dried in a 30 ℃ drier, the Teflon rod was removed and washed again with distilled water. The collagen neural conduit thus made was cut to the required length and spray coated with a biodegradable polymer. The spray coating used an injector (Richpen, Tokyo, Japan) having a tip diameter of 0.3 mm (FIG. 2). Methyl chloride (MC; Duksan, Kyoungkido, South Korea) contains 0.1%, 0.3% 0.5% of biodegradable polymer PLGA (Sigma, St. Louis, MO, USA) or PLA (Sigma, St. Louis, MO, USA). It was prepared by melting to concentration. Spray coating was spray-coated for about 5 seconds to be evenly coated on all sides of the conduit using a sprayer while rotating the collagen neural conduit made using a rotator. The neural conduit coated with the biodegradable polymer was washed and dried in a vacuum dryer for about 24 hours. Then sterilized with ion gas again, sterilized neural conduit was packaged and used (Fig. 3).

In addition, crosslinked collagen neural conduits were prepared using glutaraldehyde (GA: Sigma, St. Louis, Mo., USA) in order to compare the properties of the coated collagen neural conduits with those of existing collagen neural conduits. The pre-coated collagen conduit made by the method presented above was stored at 0.25 ° C. and 0.5% GA solution at 4 ° C. for about 24 hours, and then taken out and washed with distilled water. After washing, the crosslinked neural conduit was lyophilized to preserve the crosslinked properties. Then, sterilized with ion gas again, the sterilized neural conduit was packaged and used.

The neural conduit of the present invention can be prepared by adjusting the inner diameter and the length according to the size of the missing nerve. In the rabbit nerve reconstruction used in this experiment, a conduit with a diameter of 1 mm and a length of 20 mm is preferable, and thus the size of the rabbit nerve reconstruction was used.

Example 2 Surface Observation by Electron Microscopy After Coating

Collagen films not coated with biodegradable polymers, collagen films coated with biodegradable polymers, crosslinked collagen films were observed by scanning electron microscopy (Hitachi, Tokyo, Japan).

As can be seen in Figure 4 both the PLGA and PLA, the higher the concentration in the coating solution, the smaller the size of the pores. The size of these pores is very important for the nutrients from outside the catheter to enter the catheter and the nerves regenerated inside the catheter to prevent external scar tissue from infiltrating. Conduit is most appropriate. In addition, the cross section of the neural conduit coated with 0.3% PLGA showed that the biodegradable polymer was coated with a thickness of about 10 μm, unlike before the coating (FIG. 5). The thickness was adjustable by adjusting the concentration of the coating solution and the distance between the pressure and the specimen during spray coating.

Example 3 : Biodegradability Experiment

Biodegradability experiments were carried out with neuroconduits for 8 weeks in PBS solution at pH 7.4 with or without 10 μg / ml collagenase type I (Sigma, St. Louis, MO, USA). The neural conduit was taken out and dried at the point of time, followed by specifying the mass change. The mass change is calculated by the following equation (1).

W (%) = (Wf / Wi) × 100

Where Wf is the weight of the conduit after biodegradation and Wi is the initial weight of the conduit before biodegradation.

From the results shown in FIG. 6, it can be seen that the biodegradation rate decreases as the concentration of the coating solution coating the neural conduit increases. However, the crosslinked neural conduit showed little biodegradation compared to the coated neural conduit.

Example 4 Mechanical Properties Experiment

Mechanical properties were measured by pulling the jig until the collagen film before coating, the collagen film after coating, the cross-linked collagen film was cut at a rate of 1mm / min using a universal testing machine. In order to experiment similarly to the coated conduit, two 20 mm × 8 mm collagen films were subjected to a tensile test by overlapping the coated parts to the outside. The pre-coated film and the crosslinked film were subjected to a tensile test by overlapping the two before and after. Mechanical properties are manifested by modulus of elasticity and tensile strength. We also examined the mechanical properties of both neural conduits in both dry and wet conditions.

As can be seen from the results shown in Table 1, 7 and 8, the elastic modulus and tensile strength increased as the concentration of the coating liquid coating the nerve conduit increased regardless of the dry or wet condition. In addition, the crosslinked collagen film showed much greater mechanical properties than the coated collagen film. However, such a large mechanical property is not similar to the mechanical properties of the neural tissue, so it is thought that it will adversely affect the nerve regeneration because it gives a great stress on the connected neural tissue when connecting the damaged and conduit.

The values are expressed as mean ± standard deviation (n = 3).

Example 5 : Cytotoxicity Experiment

Cytotoxicity experiments were carried out through the WST-1 assay. WST-1 assay is a method of measuring cell viability by quantifying cell proliferation ability or cell viability by colorimetric measurement. Tetrazolium salt (WST-1) by mitochondrial dehydrogenase in living cells is transformed into formazan pigment. The absorbance of the medium depends on the degree of conversion. The tetrazolium salt added to the medium is converted into formazan pigment by succinate-tetrazolium-reductase (EC 1.3.99.1), which is present in the respiratory chain of mitochondria and is active only in living cells. . As the number of live cells increases, the activity of the entire mitochondrial dehydrogenase in the sample increases, which increases the production of formazan pigment, which is linear with the number of formazan pigment and metabolic cells in the medium. The correlation is This assay does not require washing or binding of cells, and has the advantage that it can be performed in the same microfilter plate from initiation of microculture to data interpretation by an ELISA reader. Cell proliferation and viability can also be quantified non-RI spectrophotometrically in proliferative or drug sensitivity assays.

The collagen neural conduit, the coated collagen neural conduit, and the crosslinked collagen neural conduit before elution were eluted in distilled water at 37 ° C. for 3 days, and the eluate was used for cytotoxicity experiments. Fibroblasts (L929) and neurons (PC12) were used for cytotoxicity experiments. L929 and PC12 were purchased from KCLC (Korea Cell Line Bank), 10% Fetal Bovine Serum (FBS; Gibco, Grand Island, NY, USA) and 1% Penicilis-streptomycin (PS; Gibco, Grand Island, NY, USA) Cells were cultured in an incubator at 37 ° C. 5% CO 2 concentration using RPMI Medium 1640 (Gibco, Grand Island, NY, USA) medium. Each cell with a concentration of 1 × 10 ⁵ cells / ml was incubated in 96 wells for one day, and the eluate for each neural conduit prepared above was added. After 3 days of addition, the solution was stained by adding a WST-1 solution and the absorbance of the solution was measured. Cytotoxicity experiments for each neural conduit were performed five times and averaged. According to FIG. 9, the coated collagen neural conduit exhibited almost the same cell viability as before the coating regardless of the cells, and thus there was little toxicity by the coated biodegradable polymer. However, GA-crosslinked collagen neural conduits showed very low cell viability compared to the coated collagen neural conduits, which was found to have cytotoxicity by GA used as a crosslinking agent.

Example 6 Animal Feeding Experiment

Five rabbits of 3 kg were injected with ketamine hydrochloride into the intraperitoneal cavity of the experimental animals to induce deep anesthesia, and then the area around the surgery was shaved and sterilized using an iodine sponge. After placing the animal lying down, a 3 cm incision was made parallel to the posterior femur. The left sciatic nerve was exposed from the sciatic notch to the popliteal site, and the nerve of about 15 mm in length was cut. Nerve and distal nerve cuts were connected to a 17 mm long 0.3% PLGA coated neural conduit. Sutures were performed at 3 to 4 sites using 10-0 nylon so that about 1 mm of nerves in each nerve cutting portion entered the conduit. The collagen gel prepared after closure was inserted into the conduit using a syringe. A photograph of the neural conduit inserted after all procedures is shown in FIG. 10 (1). After disinfecting the site, the rabbit was left intact for 12 weeks after the absolute site was completely sealed.

6-1 : Visual findings

As shown in FIG. 10 (2), after 12 weeks after transplantation of the nerve conduits, the sutures were re-dissected and opened. The nerves were completely regenerated, and the nerve conduits that had been implanted were completely biodegraded and not visually observed.

6-2 : Histological Examination

In order to perform histological examination, the regenerated sciatic nerve was removed and collected, fixed at 2.5% G, embedded in Epon, and cut into 0.5 μm thickness using a microcutter. Each tissue piece was stained using toluidine blue. The prepared slides were observed under an optical microscope and an electron microscope, respectively. As shown in FIG. 11, axons surrounded by a well-distributed thick horseshoe were observed in the cross-sectional view of the regenerated nerve. These results indicate that nerve regeneration was effective.

Since the collagen conduit coated with the biodegradable polymer of the present invention has no toxicity due to the crosslinking agent, it is a safe and biocompatible conduit that can control the rate of decomposition and mechanical strength, and thus is effective for regeneration of nerves damaged by surgery, tumors and trauma. Can be used

Claims (14)

Collagen nerve conduit coated on the outside of the collagen conduit with synthetic biodegradable polymers. The method of claim 1, wherein the synthetic biodegradable polymer is polylactic acid (PLA), polyglycolic acid (PGA), poly (lactic-co-glycolic acid) ): PLGA), poly (lactic-co-glycolic acid) -glucose (PLGA-glucose), polyorthoester, polyanhydride, polydioxanone, polyamino acid acid), polyhydroxybutyric acid, Polycaprolactone, Polyalkylcarbonate, and derivatives and mixtures thereof. The collagen conduit of claim 2, wherein the synthetic biodegradable polymer is PGA, PLA, PLGA or a mixture thereof. The collagen conduit of claim 3, wherein the synthetic biodegradable polymer is PLA or PLGA. The collagen conduit according to claim 1, wherein the collagen is type I, II, III, IV, V or mixtures thereof. 6. The collagen conduit of claim 5, wherein the collagen is type I. Rolling a porous collagen film (rolling) drying method of producing a collagen neural conduit coated with a synthetic biodegradable polymer comprising the step of spraying and coating the synthetic biodegradable polymer solution to the outside of the collagen film. The method of claim 7, wherein the concentration of the synthetic biodegradable polymer is 0.05% to 1.0%. The method of claim 8, wherein the concentration of the synthetic biodegradable polymer is 0.1% to 0.5%. The method of claim 7, wherein the synthetic biodegradable polymer is PLA, PGA, PLGA, PLGA-glucose, polyorthoester, polyanhydride, polydioxanone, polyamino acid, polyhydroxybutyric acid, polycaprolactone, polyalkylcarbonate And derivatives and mixtures thereof. The method of claim 10, wherein the synthetic biodegradable polymer is PGA, PLA, PLGA or a mixture thereof. The method of claim 11, wherein the synthetic biodegradable polymer is PLA or PLGA. 8. A process according to claim 7, wherein the collagen is type I, II, III, IV, V or mixtures thereof. The method of claim 13, wherein the collagen is type I.

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