KR100856135B1 - Tissue Engineering Implants for Neuronal Regeneration Using Biocompatible Injectable Hydrogels - Google Patents

Abstract

The present invention relates to a tissue engineering implant for nerve regeneration using a biocompatible injectable hydrogel, and more particularly, biocompatibility capable of biocompatible and temperature sensitive polyethylene glycol / polyester block copolymer or sol-gel phase transition. Using hydrogel of small intestinal mucosa powder as a scaffold for tissue engineering, adult stem cells or nervous system cells and bioactive substances were complexed to the scaffold and implanted into the damaged spinal cord nerves. It relates to a tissue engineering implant capable of regenerating or restoring a role.

Polyethylene glycol / polyester block copolymer, small intestinal submucosa powder, support for tissue engineering, implant for tissue engineering

Description

Development of tissue engineered scaffolds for nerve regeneration using biocompatible and injectable hydrogel

Figure 1 shows an inverted micrograph of the stem cells cultured in Examples 1 and 2 [(A) muscle-derived stem cells, (B) fat-derived stem cells, (C) bone marrow-derived stem cells, (D) nerves Derived stem cells, (E) olfactory envelope cells, (F) Schwann cells (magnification: 40)].

Figure 2 is a phase change behavior picture of the biocompatible hydrogel for nerve regeneration, (A) and (B) is a copolymer synthesized in Example 3 (A) is a photo of the sol state taken at room temperature 25 ℃, ( B) is a picture showing the phase transition to gel at 37 ℃, (C) and (D) is a small intestinal submucosa powder prepared in Example 4, (C) is a photo of the sol state taken at room temperature 25 ℃, (D) shows a picture of the phase transition to the gel at 37 ℃.

Figure 3 is a photograph showing the surgical procedure performed by Example 5 (A) is a surgical appearance, (B) is a complete cut by the length of the spinal cord to be cut during surgery (B1) 1 mm, (B2) 3 mm, (B3) 5 mm, (C) and (D) is a small intestinal submucosa gel of Example 4, (E) and (F) is a synthetic hydrogel of Example 3 It is a photograph showing.

4 is a photograph showing a scene of the BBB score for evaluating the exercise force of the surgical model of Example 6, Figure 4a is a circular plate for observing the movement, Figure 4b is a graph showing the score of the damage model by length 4C shows the score of each implant group.

Figure 5 is a photograph showing the rehabilitation training for improving the exercise power, Figure 5A is a four-week operation of the 1 mm surgical model implanted with the hydrogel implant of the stem cells and growth factors complexed on the rotating cylinder after 4 weeks 5B is a photograph showing a swimming state.

FIG. 6 is a photograph showing rehabilitation training for improving athletic performance. FIG. 6A is a rotary cylinder after 4 weeks of a 1 mm surgical model in which a small intestinal submucosa gel implant of Example 4 in which stem cells and growth factors are combined. It is a photograph of exercise, Figure 6B is a photograph showing a state of swimming.

7 is a photograph showing the exercise induced potential test to quantitatively evaluate the regenerated nerves mentioned in Example 7.

FIG. 8 is an immunochemical staining photograph for histological evaluation of the extent of regenerated nerves performed in Example 8. Spinal nerve tissue photograph (A) taken 4 weeks after transplantation of the hydrogel implant of Example 3. FIG. , 4 weeks after transplantation of the small intestinal submucosa gel implant of Example 4 (B) is extracted from the spinal nerve tissue (B), (C1) is a normal model H & E, (C2) is a normal model NF, (C3) is normal NSE of the model, (D1) is H & E of the hydrogel transplantation model of Example 3, (D2) is NF of the hydrogel transplantation model of Example 3, (D3) NSE of the hydrogel transplantation model of Example 3, ( D4) is the GFAP of the hydrogel transplantation model of Example 3, (D5) is BrdU of the hydrogel transplantation model of Example 3, (E1) is H & E of the submucosal tissue gel transplantation model of Example 4, (E2) is NF, (E3) of the small intestinal submucosa gel transplantation model of Example 4, NSE, (E4) of the small intestinal submucosa gel transplantation model of Example 4, GFAP, (E5) of the small intestinal submucosa gel transplantation model of 4 is a BrdU staining image of the small intestinal submucosa gel transplantation model of Example 4.

The present invention relates to a tissue engineering implant for nerve regeneration using a biocompatible injectable hydrogel, and more particularly, biocompatibility capable of biocompatible and temperature sensitive polyethylene glycol / polyester block copolymer or sol-gel phase transition. Using hydrogel of small intestinal mucosa powder as a scaffold for tissue engineering, adult stem cells or nervous system cells and bioactive substances were complexed to the scaffold and implanted into the damaged spinal cord nerves. It relates to a tissue engineering implant capable of regenerating or restoring a role.

If the nervous system is damaged in the central nervous system, not only will it lose its function, but it is known that it is almost impossible to restore or regenerate the damaged nervous system. However, as neuroscience by molecular biology has been developed in recent years, the structure and function of neurons have been elucidated at the molecular level, and the importance of neurotransmitters and neurotrophic growth factors has emerged. It has been proven that it can reverse orthodoxy and regenerate the central nervous system.

The first way to regenerate nerves is to improve the damaged area into a biological environment suitable for regeneration, and the second way is to strengthen the regeneration of nerve axons themselves. In order to improve the biological environment, cells that are known to be essential for inducing regeneration and blocking neuronal regeneration inhibitory proteins secreted by glial cells from the damaged area, such as Schwann's cells, olfactory nerve cells, and neural stem cells, etc. The method of administration is the best known method to date. In addition, as a method of strengthening the regeneration ability of nerve axons, the method of administering nerve growth factor or neurotrophic factor is most effective. Nerve growth factors (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4 / 5 (neurotrophin-4 / 5) are the growth and differentiation of central nervous system cells, It is a nerve growth factor that regulates various functions such as survival and death. In other words, as it is known that nerve growth factors for the treatment of neurological diseases induce tissue and cell restoration and promote growth, treatment with spinal cord injuries is effective.

Stem cells have unique characteristics that can be precisely replaced by specific tissues or cells at any location in the body and can be differentiated into specific cell types of progenitor cells, which can be a very good source of regenerative medicine. It offers many possibilities in the treatment or restoration of damaged organs. Stem cells are largely divided into embryonic stem cells from early embryonic or fetal germ cells and adult stem cells, immature cells that are separated from adult bone marrow, fat, and muscle. It has great potential in therapy and gene therapy. However, since embryonic stem cells cannot be ruled out ethical problems, they are further accelerating the study of adult stem cells, and many studies have been carried out on the neural cell therapy for the treatment of neurological diseases using adult stem cells derived from bone marrow. It is becoming. The total number of mesenchymal stem cells in the bone marrow is known to be 1 X 10 ⁶ , and the technology of culturing mesenchymal stem cells by collecting a small amount of bone marrow has already been accumulated at home and abroad and is actually used in clinical trials. come. Treatment with mesenchymal stem cells has been reported in patients with amyotrophic lateral sclerosis and there are examples of hematopoietic stem cell injection and mononuclear cell injection. Clinical studies of self-derived mesenchymal stem cell therapy in traumatic spinal cord injury patients are ongoing in Korea, and mesenchymal stem cells are injected into patients with severe limb paralysis. The movement of the wrist, fingers and elbow motor nerves are showing some positive effects.

As mentioned above, cell therapy methods have been widely researched and developed as a technique for regenerating damaged spinal cord nerves, but it is generally known that the regeneration of stem cells does not allow long-term survival. In order to overcome these problems, it is necessary to study long-term survival of neuronal regenerative cell sources and to improve neuronal regenerative capacity. This problem can be overcome by histological engineering.

Tissue engineering consists of three important elements: scaffolds, cells, and bioactive substances. Generally, biocompatible biodegradable scaffolds capable of long-term survival of cells by improving the engraftment ability of various cells such as bone cells, tissue cells, chondrocytes, and stem cells, and affecting the proliferation and differentiation of cells. Tissue-engineered implants are prepared by complexing various bioactive substances such as cytokines and growth factors.

Although the transplantation of various body organs has been actively carried out with the help of recently developed medicine, the increase in the waiting time for transplantation due to the shortage of organ donors has been increasing day by day. Various biocompatible biodegradable supports have been developed to regenerate or replace tissue. For the successful development of such biotissue engineering, a support for reconstructing tissue cells separated in the body into a tissue having a three-dimensional structure in vitro is essential. The present invention invented a biocompatible support for transplantation for central nerve regeneration.

The present inventors are hydrophilic polymers, have high solubility in water and organic solvents, are non-toxic, non-responsive to immunity, and chemically bond with hydrophobic, biodegradable ester-based polymers when applied to a living body. It is biocompatible because it uses polyethylene glycol which can control the decomposition period by increasing the amount of phosphate, and it is decomposed into biological metabolites through the dissolution in the human body, chemical hydrolysis, and decomposition by enzymes and is discharged to the outside of the body. Caprolactone (CL), a hydrophobic, biodegradable ester family that can control the decomposition period by controlling the molecular weight and chemical composition, is an essential component, and paradioxanone (PDO), trimethylene carbonate (TMC), or these are simultaneously identified. Sol-gel phase according to temperature and concentration in aqueous solution This behavior was studied through changes in the type and chemical structure of hydrophobic moieties, and injection of sol copolymers into mice confirmed gel formation near body temperature, hydrophilic moieties composed of polyethylene glycol, and caprolactone (CL). A block copolymer comprising segments as essential components, paradioxanone (PDO) segments, trimethylenecarbonate (TMC) segments, or biodegradable polyester-based hydrophobic moieties containing these segments simultaneously, wherein the copolymer has a molecular weight of 2,000. A biocompatible and temperature sensitive polyethylene glycol / biodegradable polyester block copolymer having a ˜700 g / mole has been developed and patented (Patent Application 2006-0023991).

In addition, the small intestinal mucosa tissue prepared by mixing the subintestinal mucosa tissue with an acidic solution and pepsin is titrated to a pH suitable for living body with a basic solution and freeze-dried to prepare a submucosal tissue powder, and without using a polymer Patents have been applied for the development of an injectable hydrogel that can form a gel in situ when the tissue powder is injected into an aqueous solution in vivo [Patent Application 2006-100430].

Therefore, the present invention is based on the development of a tissue-engineered implant containing stem cells and physiologically active substances for the purpose of central nerve regeneration based on the above technology.

Accordingly, the inventors of the present invention, based on the above-described techniques for applying biocompatible and temperature-sensitive polyethylene glycol / polyester block copolymer or a hydrogel of biocompatible small intestinal submucosal tissue powder capable of sol-gel phase transition for tissue engineering The present invention has been completed by developing a tissue engineering implant in which adult stem cells or nervous system cells and bioactive substances are combined for the purpose of central nerve regeneration.

Accordingly, the present invention provides an implant for tissue engineering using a biocompatible and temperature sensitive polyethylene glycol / polyester block copolymer or a biocompatible small intestinal submucosal tissue powder capable of sol-gel phase transition as an improved invention of the patent. Its purpose is to.

It is also an object of the present invention to provide a method for inducing nerve regeneration by transplanting the implant after partial or complete incision in the site of acute or chronic spinal cord injury.

The present invention

In a tissue engineering implant comprising an adult stem cell or a nervous system cell and a bioactive material in a biocompatible support,

The biocompatible support is characterized by a tissue engineering implant which is an injectable hydrogel of biocompatible and temperature sensitive polyethyleneglycol / biodegradable polyester block copolymer or biocompatible small intestinal submucosa powder capable of sol-gel phase transition.

The biocompatible and temperature sensitive polyethylene glycol / biodegradable polyester block copolymer contains a hydrophilic portion composed of polyethylene glycol and caprolactone (CL) segments as essential components, and a paradioxanone (PDO) segment and trimethylene carbonate. The biocompatible small intestinal submucosal tissue powder composed of (TMC) segments or biodegradable polyester-based hydrophobic moieties containing these segments simultaneously and having a molecular weight of 2,000 to 7,000 g / mole and capable of sol-gel phase transition The tissue was mixed with pepsin and an acidic solution, titrated to a pH of 5.5 to 7.8 with a basic solution, and then lyophilized to be powdered. The copolymer or powder was prepared as an injectable hydrogel with a phosphate buffer solution and used as a transplant. do.

The present invention will be described in more detail as follows.

The present invention uses a hydrogel of biocompatible and temperature sensitive polyethylene glycol / polyester block copolymer or biocompatible small intestinal submucosa tissue powder capable of sol-gel phase transition as a support for tissue engineering and biological activity with adult stem cells or nervous system cells. The present invention relates to a tissue engineering implant capable of regenerating or restoring the role of the central nerve by connecting axons over time by incorporating a substance into the support and implanting the damaged spinal nerve.

First, the adult stem cells and nervous system cells used in the present invention will be described.

Stem cells have unique characteristics that can be precisely replaced by specific tissues or cells at any location in the body and can be differentiated into progenitor cells of specific cell types, which make stem cells a very good source of regenerative medicine. This suggests a number of possibilities for the treatment or restoration of damaged organs.

Bone marrow-derived mesenchymal stem cells exhibit complex characteristics of stem cell populations. In other words, they have excellent expandability in vitro and can be differentiated into complex mesodermal forms, and their differentiation into neurons is a very significant result in the treatment of neurological diseases, which can be isolated and cultured in large quantities relatively easily. The use of cells suggests new treatments for various neurological diseases.

In addition, skeletal muscle-derived stem cells and adipose-derived stem cells exhibit the complex characteristics of a group of stem cells that can differentiate into a variety of cells depending on the surrounding environment, can be easily isolated and can be grown in large quantities in vitro . Neural stem cells are differentiated into blood, bones, chondrocytes, etc., showing multipotent ability beyond the germ layers. Neural stem cells can proliferate and restore neural tissues by differentiating into neural cells or glial cells.

Olfactory neuroenvelope cells, which are neuronal cell sources, are the cells surrounding the olfactory nerve and are widely known to play a decisive role in nerve regeneration as part of the central nerve. .

Isolation and culture of these stem cells and nervous system cells are described in detail in the Examples below.

As a physiologically active substance used in the present invention, neurotrophic neuron growth factor NGF (Nerve growth factor), BDNF (brain-derived neurotrophic factor), NT-3 (neurotrophin-3), NT-4 / 5 (neurotrophin -4/5) is a type of polypeptide that is a water-soluble molecule with a specific structure that affects the biological activity of cells. It promotes cell division to increase cell number, change morphology and induce differentiation. It works. In addition, the preparation of tissue engineering implants including stem cells and growth factors will be described in detail in the following Examples.

The role of the scaffold to isolate the damaged spinal cord from the outside and to maintain the implanted cells or administered neuronal regeneration inducers for regeneration after nerve injury is very important. Biodegradable synthetic polymers and natural materials are widely used for tissue engineering scaffolds. Recently, small intestinal submucosal tissues have been used alone or in combination with synthetic polymers. In the present invention, a tissue support was developed using a temperature sensitive polyethylene glycol / biodegradable polyester block copolymer and a small intestinal submucosa as a biocompatible support.

Referring to the biocompatible and temperature-sensitive polyethylene glycol / biodegradable polyester block copolymer used as a support in the present invention.

Polyethylene glycol (PEG), which is used as a polymerization initiator of polyethylene glycol / biodegradable polyester block copolymers, has many advantages in the field of drug delivery, so it can be easily entrapped and released as a drug carrier and has high solubility in water and organic solvents. It is non-toxic and has no rejection of immune action. It has excellent biocompatibility and is approved by the US Food and Drug Administration for use in humans. In addition, since PEG has the greatest protein adsorption inhibitory effect among hydrophilic polymers and improves the biocompatibility of blood contact substances, many applications have been made as biomaterials. However, there has been a problem that biodegradation does not occur while using biomaterials containing PEG. Because PEG is non-degradable and accumulates in the body, it has been reported to induce increased toxicity of plasma cholesterol and triglycerides after intraperitoneal injection. Therefore, in order to solve this problem, low molecular weight PEG having a molecular weight of 5,000 g / mole or less that can be easily removed from the body by filtration of the kidney and biodegradable esters that can be decomposed into biocompatible products by metabolism of the human body Copolymerization with the monomers resulted in the production of polyethylene glycol / biodegradable polyester block copolymers.

Biodegradable polymers of the ester series have the advantage of controlling the decomposition period by controlling the molecular weight and chemical components. Block copolymers of polyethylene glycol (PEG) and polycaprolactone (PCL), which have become basic models, have already been applied to biomaterials as temperature-sensitive copolymers having sol-gel phase transition properties. However, caprolactone is biodegradable, compatible with many polymers, and has a feature of easily crystallizing. However, high crystallinity has a disadvantage of decreasing compatibility with tissues and showing long-term dissociation behavior when applied in vivo.

Thus, to caprolactone, paradeoxanone (PDO), trimethylene carbonate (TMC) of biodegradable ester series or these were added at a constant content ratio to lower crystallinity and control the biodegradation period. That is, it is represented by the following formula (1), each segment forms a random hydrophobic portion at random.

In Formula 1, x, y and z are each segments constituting the hydrophobic polyester portion, x is 50 to 95 mol%, y + z is 5 to 50 mol% (including when y or z is 0) .

Paradioxanone (PDO) and trimethylene carbonate (TMC), or these are simultaneously ring-opened to ester-based caprolactone (CL) using low molecular weight polyethylene glycol (Mn = 350 to 2000 g / mole) as a hydrophilic part in the present invention. Synthesis via copolymerization. Synthesis method is carried out by azeotropic distillation of polyethylene glycol, followed by addition of an ester monomer, methylene chloride (MC) or toluene as a reaction solvent, using an acid catalyst as the monomer activator, the reaction temperature is -40 ~ 130 The polymerization is carried out in the range of ° C. The acid catalyst is preferably one or more selected from HCl, HBr, CF ₃ COOH, CCl ₃ COOH, BrCH ₂ COOH, CH ₃ COOH, BCl ₃ , BBr ₃ and Camphorsulfonic acid.

The aqueous solution phase of the synthesized polyethylene glycol / biodegradable polyester block copolymer maintains the sol state having flow characteristics at room temperature and forms a gel over a certain temperature range (30 to 46 ° C.), and the critical temperature (44 to 47 ° C.) In the above, a thermosensitive material exhibiting a new sol-gel phase transition behavior showing flow characteristics was prepared, and thermal properties and crystallinity were analyzed by using a differential scanning calorimeter and an X-ray diffractometer. Injected into the rat to confirm the formation and integrity of the gel.

In addition, in order to apply such a temperature-sensitive block copolymer as an injectable drug carrier or a functional support for tissue regeneration, it is necessary to have a low viscosity, fast gel formation, and a low molecular weight to be easily discharged out of the body. Paradioxanone (PDO), trimethylene carbonate (TMC) or by introducing a constant content ratio at the same time was able to reduce the crystallinity by lowering the viscosity, and to obtain a molecular weight close to the expected value, biocompatibility and temperature sensitivity One of the requirements for hydrogels was the low viscosity and low molecular weight.

It is preferred to synthesize polyethyleneglycol / biodegradable polyester block copolymers having a total molecular weight in the range of 2,000 to 7,000 g / mole, but when the total molecular weight is less than 2,000 g / mole, the sol-gel in the human body temperature range for the purpose of the present invention is used. This is because there is a problem that the phase transition does not occur and is kept in a sol state, and when it exceeds 7,000 g / mole, there is a problem that it takes a long time for biodegradation to occur due to its large molecular weight.

As a representative example of the polyethylene glycol / biodegradable polyester block copolymer used in the present invention it can be represented by the following scheme 1.

In Scheme 1, n is an integer representing a repeating unit of polyethylene glycol constituting the hydrophilic portion, x, y and z are each segment constituting the hydrophobic polyester portion, x is 50 to 95 mol%, y + z is 5 ˜50 mol% (including when y or z is zero).

The polyethylene glycol / biodegradable polyester block copolymer prepared in this way has lower crystallinity than the polyethylene glycol-polycaprolactone block copolymer based on the basic model, so that the gel can be formed quickly and have a low viscosity that is easy to handle. It has a low molecular weight for easy discharge to the outside and can be applied as a drug carrier in injection form or a porous support for tissue engineering. In addition, the biodegradation period can be controlled by adding a biodegradable ester-based paradioxanone (PDO), trimethylene carbonate (TMC), or these simultaneously in a constant content ratio to caprolactone showing long-term biodegradability. In addition, since the aqueous solution of polyethylene glycol / biodegradable polyester block copolymer shows sol-gel phase transition at various temperature ranges according to various kinds of biodegradable polymers such as paradioxanone (PDO) and / or trimethylene carbonate (TMC) As a biomaterial that is an object of the present invention, it can be applied to the purpose of gelling at a temperature slightly higher or lower than the biological temperature as well as the gelling property at the biological temperature when applied to the human body.

In addition, the biocompatible small intestinal submucosal tissue powder applicable as another support in the present invention will be described as follows.

When preparing the powder, the small intestine submucosa of mammals except humans is used. Preferably the mammal is selected from cattle, pigs, rabbits and rats. The small intestinal submucosa is a non-cellular tissue, most of which is composed of collagen, and in addition, various growth factors, glucosaminoglycans and fibronectin are included. The growth factors include transforming growth factor (TGF), insulin-like growth factor (IGF), acidic and basic fibroblast growth factor (aFGF, bFGF), and vascular endothelial cells. Vascular endothelial growth factor (VEGF) and nerve growth factor (NGF), and glucosaminoglycans include hyaluronic acid, chondroitin, and chondroitin-4-sulfate. (chondroitin-4-sulfate), heparan sulfate, heparin, alginic acid, dextran, starch, chitin and chitosan. .

The presence of extracellular matrix and cytokines in the small intestinal mucosa is involved in the functional aspects of cells such as cell adhesion, migration, proliferation and growth, and is very useful for tissue regeneration and wound healing. It is possible.

First, the submucosal tissue of the mammal is separated, and the small intestine of the mammal is separated to remove the plant, and then immersed in saline and phosphate buffer solution to remove the mesentery and then cut to a constant length. The small intestine thus cut is inverted to remove the mucosal layer by friction and then reversed to separate the intestinal and muscle layers to separate the small intestinal submucosa.

The separated small intestinal mucosa is pulverized after lyophilization and the ground small intestinal mucosa is mixed with an acidic solution and pepsin. Using a basic solution to the mixed solution is titrated to pH 5.5 ~ 7.8, preferably pH 6.5 ~ pH 7.5 to prepare a biocompatible small intestinal submucosal tissue powder capable of gel formation.

The acidic solution may be a solution that can be adjusted to pH 2.5 to 4.5, preferably, an aqueous solution containing 1 to 5% by weight of an acid selected from acetic acid, paratoluenesulphonic acid and maleic acid is used. Do.

In addition, the mixing ratio of the small intestinal submucosa, the acidic solution and pepsin is preferably 1: 1 to 5: 0.1 to 2 weight ratio because pepsin forms the appropriate concentration and conditions for releasing the collagen chain of the small intestinal submucosa.

The basic solution is used to titrate acidity and to reduce the inflammatory response of surrounding cells when used as a subcutaneous injection or tissue engineering support, for example sodium hydroxide (NaOH), sodium carbonate (Na ₂ CO ₃ ), Sodium hydrogen carbonate (NaHCO ₃ ), disodium hydrogen phosphate (Na ₂ HPO ₄ ), calcium hydrogen carbonate (Ca (HCO ₃ ) ₂ ), calcium hydroxide (Ca (OH) ₂ ), calcium hydroxide (Ca (OH) NO ₃ ) 1 or 2 selected from calcium hydroxide (Ca (OH) Cl), calcium cyanide hydroxide (Ca (OH) CN), potassium hydroxide (KOH), ammonium hydroxide (NH ₄ OH) and sodium acetate (CH ₃ COONa) More than one species can be used. This basic solution is titrated to adjust to an in vivo pH of pH 5.5-7.7.

When the intestinal submucosal tissue powder prepared without using the polymer was injected into the aqueous phase in vivo, a gel was formed on the spot and a hydrogel for injection capable of slowly releasing the drug was obtained.

Surgery and regeneration of the spinal cord injury animal model in the present invention is as follows.

In the embodiment of the present invention, but selected and used as an animal model, it is possible for all mammals except humans.

Fischer rats were used as an example to reduce the immune response of the cells used for regeneration in the selection of mice in the spinal cord injury model. The rat's central nerve injury first removes the skin in the middle of the spine, then removes the fascia and muscles, exposing the T8-T9 region of the vertebral bone and then removing the exposed vertebral bone. At this time, careful attention is required so as not to damage the spinal cord. When the spinal cord was exposed, a microsurgical instrument was used to carefully remove the dura and the spinal cord was completely cut to a length of 1 mm, 3 mm, and 5 mm to establish a nerve injury model. Herein, undifferentiated adult stem cells alone or olfactory nerve cells and Schwann cells were mixed and a tissue engineered implant containing a growth factor as a bioactive substance was implanted.

After surgery, rats with spinal cord injury are paralyzed from the lower part of the body and require special attention and care. First of all, it lays down on the heating bed to maintain the body temperature after surgery, and it helps oxygen to breathe irregularly due to the stress after the operation. It moves to a clean cage and new bedding and warms the floor. For a week after surgery, urination was helped twice a day for smooth urination and a dose of antibiotics was injected once a day. Daily massages, walking exercises, and swimming were conducted regularly to improve athletic performance.

In the present invention, the exercise force evaluation, exercise developmental potential test and histological evaluation used as an index for evaluating the regeneration of nerve axons are as follows.

The performance rating was assessed by BBB (Basso, Beattie and Bresnahan) scores and the scores scored for each model were visualized graphically. Quantitative evaluation of neuronal axons in normal or spinal nerve injury model and spinal nerve regeneration model was performed by measuring exercise-induced potential test. Histological staining was used to investigate inflammatory reactions, new tissue formation and neural axon generation at the cutting site. By confirming, it confirmed that the role as a central nerve was restored or regenerated.

Accordingly, the present invention has proposed a method of more effective nerve regeneration by the regenerative cell source, the bioactive material and the tissue engineering implant for nerve regeneration secured based on the above experiment.

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples. In particular, the pre-filed 2006-23991 and 2006-100430 are the mains of biocompatible and temperature sensitive polyethylene glycol / biodegradable polyester block copolymers or biocompatible small intestinal submucosa powders capable of sol-gel phase transition. Includes a full description of the death penalty hydrogels.

Example 1 Isolation and Culture of Adult Stem Cells Used as Cell Sources for the Treatment of Spinal Cord Injury

The present invention is an embodiment of the isolation and culture of four kinds of adult stem cells as a cell source for transplantation to the spinal cord injury site.

The first cell source is the isolation of skeletal muscle-derived stem cells and differentiation into neurons. In order to separate the stem cells derived from skeletal muscle, muscle was separated from the thigh of 60-80 g Fischer rats, and the cells were separated using collagenase. The isolated cells were suspended in Dulbecco's modified eagle medium (DMEM) containing 5% fetal calf serum, 5% horse serum and 2% antibiotics, and then dispensed into collagen-coated cell culture flasks. After 1 hour, the supernatant was collected from the cell culture flask, centrifuged, washed with the culture medium, and then dispensed into a new cell culture flask. Within 1 hour, most of the fibroblasts adhered to the bottom of the flask. When the fibroblasts were about 30 to 40% filled in the cell culture flask, the supernatant was collected, centrifuged, washed with the culture solution, and dispensed into a new flask. After 2 hours and 1, 2, and 3 days, the same process was repeated to isolate skeletal muscle-derived stem cells, and the cells were cultured in a cell culture flask at a concentration of 10 ³ to 10 ⁴ cells / cm ² . After sowing to 37 ℃, it was incubated in a 5% CO ₂ incubator. The cultured cells were replaced with culture medium every 3 days and subcultured once every 15 days.

The second cell source is the isolation and neural differentiation of adipose derived stem cells. In order to separate adipose-derived stem cells, abdominal fat of 60-80 g of Fischer rats was isolated and subjected to collagenase treatment. Cells were cultured using DMEM medium containing 10% fetal calf serum and 1% antibiotics and subcultured in the same manner as the stem cells derived from skeletal muscle.

The third cell source is the isolation and neuronal differentiation of bone marrow-derived stem cells. In order to separate the bone marrow-derived stem cells, the femur and the lower femur of 60-80 g Fischer rats were isolated, and a phosphate buffered saline was perfused using a 1 ml syringe to obtain cells in the bone lumen and separated by centrifugation. It was. Cells were cultured using DMEM medium containing 10% fetal calf serum and 1% antibiotics and subcultured in the same manner as the stem cells derived from skeletal muscle.

The fourth cell source is the isolation and neuronal differentiation of neuronal stem cells. In order to separate neuron-derived stem cells, the skull of 60-80 g Fischer rats was incised and only the brain part was purely separated. The tissue was cut with surgical scissors, washed twice, and cultured using a neuronal cell culture medium, and passaged in the same manner as the stem cells derived from skeletal muscle.

Example 2 Isolation and Culture of Nervous System Cells Used as Cell Sources for the Treatment of Spinal Cord Injury

The present invention is an embodiment of the isolation and culture of olfactory nerve envelope cells Schwann cells as neuronal cells to regenerate the damaged spinal nerve.

As the first cell source, olfactory epithelial cells were removed from the skull of 60-80 g Fischer rats to remove olfactory bulbs, washed twice with HBSS (Hanks buffer), and cells were separated from tissues using 0.125% trypsin. The isolated cells were incubated in F12 / DMEM medium containing 10% fetal calf serum and passaged for 3 days with medium exchange and 15 days.

As a second cell source, Schwann cells were isolated from 60-80 g Fischer rat sciatic nerve, put in Leibovitiz-15 culture, peeled epithelium, and cut the nerve into 1 mm size and cultured in DMEM culture containing 10% fetal bovine serum. . The cultures were replaced twice a week and incubated for 5 weeks, transferring nerve fragments into new containers at weekly intervals.

Example 3 Synthesis of Polyethyleneglycol / Biodegradable Polyester Block Copolymer

To synthesize an MPEG-PCL / PPDO block copolymer with a molecular weight of 3150 g / mole, 1.67 g (2.24 mmol) of methoxypolyethylene glycol (MPEG) and 80 mL of toluene were placed in a well-dried 100 mL round flask and Dean Stark trap. Azeotropic distillation was carried out at 130 ° C. for 3 hours. After distillation, all of toluene was removed, and methoxy polyethylene glycol (MPEG) was cooled to room temperature, and 5.11 g (2.24 mmol) of purified caprolactone (CL) and 0.38 ml (2.24 mmol) of paradioxanone (PDO) were injected. Then, 25 mL of methylene chloride (MC) purified beforehand as a reaction solvent was added thereto, followed by 4 mL of HCl as a polymerization catalyst, and the mixture was stirred at room temperature for 24 hours. All procedures were carried out under high purity nitrogen. In order to remove unreacted monomer or initiator after the reaction, the reactant was slowly dropped to 600 mL of hexane and 150 mL of heptane. The precipitate was dissolved in methylene chloride (MC), filtered through a filter paper, and the solvent was removed through a rotary evaporator and dried under reduced pressure. The synthesized block copolymer was sterilized with EO gas, dissolved in phosphate buffer solution at 15 to 20 wt%, uniformly dispersed, and then prepared an injectable gel formulation.

Example 4 Preparation of Biocompatible Small Intestinal Submucosa Tissue Gel

After washing the pig factory within 4 hours after death, cut it to a certain size and apply physical force using tweezers to remove the outermost layers, the vertical yarn, the dense layer and the mucosal mucosa, and wash them again with saline to clean the tubular submucosa. Tissues were prepared and stored in -80 ° C. cooler for use.

After freeze-drying the stored small intestinal mucosa through the above process, it is pulverized with a blender to obtain a powder of 10 ~ 30 ㎛ size using a freeze micro grinder, 1% by weight of the pulverized submucosal tissue with 3% by weight of acetic acid 1 The mixture was stirred for 48 hours using pepsin and tertiary distilled water. Subsequent formation of the submucosal tissue solution was adjusted to pH 7.4 using 1 N sodium carbonate (Na ₂ CO ₃ ), sodium bicarbonate (NaHCO ₃ ), and sodium hydroxide (NaOH). The pH-adjusted solution was lyophilized and then pulverized using a lyomicrograph to obtain a final biocompatible submucosal tissue powder. The final powder thus obtained was mixed with a phosphate buffer solution of pH 7.4 to confirm that an injectable gel was formed.

Example 5 Transplantation of Implants Complicated with Stem Cells and Bioactive Substances

This embodiment is a method for implanting the prepared nerve regeneration support in an animal model.

Gels for neuronal regeneration support prepared in Examples 3 and 4 were mixed with a predetermined amount of olfactory nerve envelope cells and Schwann cells, stem cells alone or neuronal cells isolated in Examples 1 and 2 so that the total number of cells is 2 × 10 ⁶ Implants containing a nerve growth factor BDNF (200 ng / scaffold) was prepared. The thoracic vertebrae (T8-T9) of 70-80 g 5 week-old female Fischer rats were completely injured by each size as shown in FIG. Oxygen was supplied for rapid recovery after surgery and rested in a heating bed at 40 ° C. until the anesthesia awoke, helped with urination twice a day for a week, and injected a certain amount of antibiotics once a day [FIG. 3].

Example 6 Evaluation of Regeneration by Improving Exercise

In order to improve the performance of the spinal cord injury model after the surgery, muscle strength was improved through the massage and rotary chamber every day from 1 week. In addition, regular swimming exercises helped improve hind limb movement. The exercise power evaluation of the model undergoing rehabilitation was evaluated by BBB (Basso, Beattie, Bresnahan) score every week after surgery [FIG. 4]. Before evaluation, the movement was observed on a circular plate after training on a rotating cylinder to improve athletic performance. For exercise measurement, a method of scoring along the BBB scale was chosen. The score was scored by the movement of the rat's hind legs, the shape of the foot, the angle of the foot, and the stability of the torso. Compared to the non-transplanted blank group, the grafted surgical group showed higher exercise power. The smaller the damage, the greater the improvement in the exercise power. In addition, in the 1 mm model, the group using the natural small intestinal submucosa gel showed a slightly higher exercise power than the group using the synthetic hydrogel, but both groups confirmed the regeneration of the central nerve by improving the exercise power.

Example 7 Assessment of Regeneration Through Exercise-Induced Potential Testing

In this study, exercise-induced dislocation test was performed to determine the degree of regeneration of spinal cord nerves [FIG. 7]. Motor Evoked Potential (MEP) is a record that amplifies the minute electrical action potential that occurs when muscles contract to move. This exercise-induced potential test is a method that is widely used for the examination of the motor pathway by the potential recorded in the muscle, peripheral nerve or spinal cord by stimulation of the motor cortex or the motor pathway of the cerebrum. After 4 weeks and 8 weeks, exercise-induced potential test was performed to measure the delay time (Latency, m / s) and amplitude (Amplitude, μ V), and to observe the regeneration of nerve axons. Transplantation animal models for nerve regeneration showed about 30-40% nerve regeneration compared to the control group without the implant.

Example 8 Histological Evaluation

Tissues were harvested 4 and 8 weeks after surgery for histologic evaluation of regenerated nerves. The tissues were cut longitudinally to determine the number of regenerated neuroaxons, and H & E staining was used to determine whether inflammatory reactions in the scaffolds and cell grafts, the formation of new tissues, and whether axons grew. As a result of H & E staining, the inflammatory response due to the transplantation of the scaffold and the cells could not be confirmed, and new tissue was formed around the cut spinal nerve. To more clearly demonstrate neuronal regeneration, neuronal specific markers (neurofilament; SCYTEK, USA), NSE (neuron-specific enolase; Serotec, UK) and glial specific markers, glial fibrillary acidic protein; Dako, Regeneration was assessed by histological immunostaining with antibodies to Denmark. In addition, in order to investigate the effects on the survival and regeneration of the transplanted cells, the cells were labeled with Brdu (bromodeoxyuridine; Dako, Denmark) and transplanted before cell transplantation. As a result of the staining, the survival of the transplanted cells was confirmed and the nerve regeneration was induced through the growth of neuronal axons and glial cells around the damaged spinal cord nerve [Fig. 8].

As described above, a tissue engineering implant comprising a neuronal regenerative cell source and a physiologically active substance according to the present invention is implanted into an injured spinal cord nerve and the nerve axons are connected over time to regenerate or restore the role of the central nerve. As a result, it is possible to improve the engraftment ability of neuronal regenerative cell sources and various cells such as bone cells, tissue cells, and chondrocytes to develop biocompatible biodegradable scaffolds that can survive the cells for a long time, and to influence the proliferation and differentiation of cells. The development of a tissue-engineered implant in which bioactive substances such as eggplant cytokines and growth factors are combined contributes to the improvement of nerve regeneration ability. To replace or regenerate biotissue-deficient organs or tissues to replace Various three-dimensional structure made from, is also expected to replace will very useful to develop biocompatible biodegradable support having the ease of use in an injection mold.

Claims (7)

In a tissue engineering implant comprising an adult stem cell and a bioactive material in a biocompatible support, The biocompatible support is a tissue engineering implant, characterized in that the injection-type hydrogel of biocompatible small intestinal submucosa powder capable of sol-gel phase transition. delete delete delete The biocompatible small intestinal submucosa powder capable of sol-gel phase transition is mixed with pepsin and an acidic solution, titrated to a pH of 5.5 to 7.8 with a basic solution, and then lyophilized. Characterized by an implant. 2. The implant of claim 1, wherein the injectable hydrogel is gelled by mixing a biocompatible small intestinal submucosa powder capable of sol-gel phase transition with a phosphate buffer solution. A method of inducing nerve regeneration by implanting the implant of any one of claims 1, 5 and 6 after partial or complete incision in a site of acute or chronic spinal cord injury in a mammal other than human.

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