KR20090130912A - Porous beads having uniform pore structure for tissue engineering and its manufacturing method - Google Patents

Abstract

PURPOSE: A tissue engineering porous particle and a manufacturing method thereof are provided to manufacture the biodegradable polymer without the use of the organic solvent as a tissue engineering supporter for regenerating various tissues. CONSTITUTION: The method of manufacturing tissue engineering porous particle includes: a step of mixing non-porous particle manufactured with the biodegradable polymer and 1 ~ 50 g ranges water-soluble fine crystal particle with 1 : 5 ~ 30 weight ratio; a step of putting the water-soluble fine crystal particle to inside of the non-porous particle inside of the shielded space pressured with similar pressure; and a step of washing the particle infiltrated with the fine crystal particle and manufacturing porous particle without the use of the organic solvent dissolved the water-soluble fine particle.

Description

Porous particles having uniform pore structure for tissue engineering and its manufacturing method

1 is a scanning electron microscope (SEM) photograph showing the result of adjusting the pore size according to the uniform porosity of the porous particles prepared by Example 1 and various diameters of the water-soluble fine crystal grains.

Figure 2 is a scanning electron microscope (SEM) picture showing the porous particles prepared by Example 1 of the present invention, the results showing that the size of the porous particles is adjusted according to the non-porous particle diameter (surface photograph).

The present invention relates to a porous particle for tissue engineering having a uniform porous structure and a method for manufacturing the same, and more particularly, non-porous particles having various diameters and water-soluble fine crystal particles having a controlled diameter are mixed at a predetermined ratio, The porous particles are prepared by dissolving the water-soluble fine crystal grains uniformly in the non-porous particles by applying temperature and pressure conditions of the same, and then washing and drying them in an aqueous solution to dissolve the water-soluble fine crystal grains. In comparison, the particle size and the pore size can be easily controlled, and uniform porosity is applied to the outside and inside of the porous particles so that the cell culture fluid and the body fluid can be easily penetrated into the porous particles to effectively adhere and proliferate the tissue cells. Not only provide a very effective environment for Furnace relates to porous particles and a method for producing most biodegradable polymers, including polymers with low solubility in solvents that could not be prepared without the use of organic solvents.

Tissue engineering, as established at the first tissue engineering symposium in California in 1988, integrates the basic concepts and techniques of bioscience and engineering to understand and correlate the structure and function of biological tissues. It is an applied science that aims to maintain, improve or restore the function of our bodies by making and transplanting substitutes for tissues.

To summarize the basic tissue engineering techniques, first extract some tissue from the patient's body, separate the cells from the tissue pieces, and then proliferate the separated cells in the required amount through culture and inject them into the porous biodegradable polymer support. After incubation for a period of time, the hybrid cell / polymer construct is transplanted back into the human body. After transplantation, cells are supplied with oxygen and nutrients by the diffusion of body fluid until new blood vessels are formed, and when blood vessels grow and supply blood in the body, cells proliferate and differentiate to form new tissues and organs, and the polymer support is decomposed. It is to apply a technique that is lost.

The history of tissue engineering has been around 15 years since 1988. The history of tissue engineering is one of the new technologies in the field of life science that will lead the future. can do. Important factors in tissue engineering are, above all, the selection of appropriate cells for cultivating necessary tissues, biodegradable materials that provide a framework for tissue formation, and the environment in which the organs manufactured by tissue engineering techniques will be implanted.

The cells to be used in tissue engineering must be healthy, not only perform well the cell's own functions, but also have the following elements: Three-dimensional tissue formation should be possible by interacting with the secretory function of extracellular matrix and interacting with other cells or biomaterials. In addition, it is important to secure a sufficient amount to cultivate the cells in vitro, and careful selection of autologous or heterologous cells in consideration of immunological rejection is important.

Next, an important element in tissue engineering is biodegradable materials that provide a framework for tissue formation. In 1960, it was difficult to process biodegradable polymers such as polylactic acid (PLA) and polyglycolic acid (PGA). It has been neglected to change, but it has been actively studied since the biodegradability of materials could play an important role in tissue engineering. In particular, these biodegradable polymers have been approved by the US Food and Drug Administration (FDA) as non-toxic polymers for human use.

The main requirement for biodegradable polymer materials in tissue engineering is to play a role that allows cells to adhere to the surface of the material to form a three-dimensional structure, and the transplanted cells adhere to the support evenly. Biodegradable polymeric materials should be able to maintain their shape even after they have been decomposed and destroyed after a certain period of time. In particular, the cells should adhere evenly within the support and grow well without necrosis, which is a very important factor related to the success or failure of tissue engineering.

In addition, an important factor in tissue engineering is the in vivo environment in which artificial organs will be implanted. When cultured cells and polymer materials are transplanted into the body, they are placed in a biochemical and physical environment that is completely different from in vitro. The factors that determine the biochemical environment are the location of the implant, the permeability of the biomaterial, and the material transport capacity. Can be. The environment in which the implant is located plays an important role in the survival and function of the cell and should be carefully considered. Cells can grow well only when there is sufficient supply of surrounding oxygen or nutrients, and the innermost cells in artificial biological tissues are located farthest from these sources, which puts them at risk for growth. Therefore, the permeability of the biomaterial should be properly adjusted or the surrounding environment should be changed according to the permeability of the material.

Porous supports made of biodegradable polymers have played a significant role in the rapid development of tissue engineering to regenerate tissue from desired cells and implant it into the human body to maintain and restore the function and shape of damaged organs. The uniform distribution of cells in the scaffold and the smooth supply of culture medium are recognized as important factors for success and failure in tissue engineering.

In general, biodegradable polymers that are licensed for human use by the FDA, which are used for porous supports, are not only hydrophobic on the porous surface, but also have a disk shape. Therefore, when the cells are cultured there, the culture solution is not smoothly supplied into the polymer support. The necrosis of cells has been observed, and also has the disadvantage that complicated and cumbersome surgery is required for the actual patient. In order to solve this problem, a porous particle having a size of micro units that is capable of injection injection is removed from the existing disc-shaped support (since the particle size is small, the culture medium is easily diffused to the center of the porous spherical particle, Has the advantage of being able to undergo transplantation without manual surgery), lyophilization method (DJ Mooney, et al., Biomaterials , 17 , 1417 (1996)), modified emulsified solvent evaporation method [W / O / W multiple emulsion technique (T. Ehtezazi, et al., J. Control. Rel., 68, 361 (2000)), incorporating gas pocket method [Petra Eiselt, et al., Biomaterials, 21 , 1921 (2000) )], spinning disk atomization method [Y. Senuma, et al., Biomaterials , 21, 1135 (2000)], and their application to various tissue engineering fields (artificial skin, artificial cartilage, bone filler, molded implants, etc.) Is actively underway.

Despite such intensive research and necessity, the porous particles which have been prepared are almost impossible to manufacture without using an organic solvent in the manufacturing process (for low solubility polyglycolic acid, polydioxone, etc., porous particles have been reported). Bar)), which may cause human toxicity due to residual solvents, and do not show uniform porosity in particles, and are suitable for uniform porosity and cell culture inside and outside of particles that have a significant effect on cell adhesion and proliferation. It does not have a degree of pore size, and a method of producing porous particles that can adjust the pore size as needed has not been developed yet.

The present inventors have tried to solve the problems such as the use of the limited polymer of the conventional porous particles (low-solubility polymer can not be produced), residual organic solvent, non-uniform porosity, difficulty in controlling the pore size. As a result,

Non-porous particles made of various biodegradable polymers (including polyglycolic acid and polydioxionone, which were impossible to make by conventional methods) and water-soluble microcrystalline particles are mixed at specific ratios and the non-porous particles at specific temperature and pressure conditions. After infiltrating the fine crystal grains into the porous particles prepared by dissolving the water-soluble fine crystal grains penetrated into the particles in an aqueous solution and drying, the use of the organic solvent in the manufacturing process can be fundamentally suppressed, The space occupied was changed to the empty space, and it was found that the pore size can be controlled by the uniform porosity and the size of the water-soluble fine crystal grains, thereby completing the present invention.

Therefore, the present invention, when used for tissue engineering, the culture medium essential for the growth of cells can be easily diffused to the center of the porous particles having a diameter of micro units, as well as suitable for cell growth, uniform porosity and pore size control, The present invention relates to a porous particle and a method for manufacturing the same, which can be produced with most biodegradable polymers including low solubility polymers that cannot be manufactured by the conventional method because no organic solvent is used in the manufacturing process.

The present invention is mixed with non-porous particles made of biodegradable polymer and water-soluble microcrystalline particles having a solubility in water (20 ° C., 100 g solvent) in the range of 1 to 50 g in a ratio of 1: 5 to 30,

The non-porous nature of the biodegradable polymer in a confined space in which the temperature of ± 50 ° C and the pressure of 1 to 50 MPa of the softening point (Ts) or melting point (Tm) are equally applied through the three-dimensional omnidirectional direction. Infiltrate the water-soluble fine crystal grains into the particles,

The method of preparing biodegradable porous particles for tissue injection capable of injection injection comprising the process of preparing the porous particles by dissolving the microcrystalline particles penetrated into an aqueous solution to dissolve the water-soluble microcrystalline particles.

In addition, the present invention is a particle produced by the method is a porous particle having a porosity of 80 to 96%, a diameter of 100 ~ 5,000 ㎛ range, the pores having a diameter of 25 ㎛ ~ 500 ㎛ range inside and outside of the particle The uniformly distributed, there is another feature in the biodegradable porous particles for tissue engineering that can be injected injection, the pore size is adjusted to the range of 0.01 ~ 0.6 per particle diameter.

Hereinafter, the present invention will be described in more detail.

In the present invention, the culture medium essential for cell growth is easily diffused to the center of the porous particles through the pores having a diameter of micro-units, It is possible to control the pore and pore size uniformly distributed on the outside, and it is possible to manufacture most biodegradable polymers including low solubility polymer which was impossible to manufacture by conventional method because organic solvent is not used in the manufacturing process. It relates to a porous particle that can be implanted into the body by injection.

In general, the porous particles prepared for tissue engineering are non-uniform porosity, lack of multi-space connectivity, and the diffusion of the culture medium and the movement of cells, which are essential for cell growth, are limited, and the use of polymers having low solubility in solvents is limited. The organic solvent used in the process has a human toxicity, and it is difficult to control the pore size, and thus, it is difficult to form a pore size suitable for each cell growth according to the cell type. In order to overcome the limitations of the conventional porous particles, the present invention applies specific water-soluble fine crystal grains having various particle sizes to specific centers, that is, inside the center of the softened or melted non-porous particles by applying heat and pressure in a closed space. It is possible to control the uniform porosity and pore size by infiltrating the inside of the non-porous particles by applying conditions that can uniformly penetrate the water-soluble microcrystalline particles, and then dissolving it in an aqueous solution. It was intended to produce a porous particle that can be suppressed at the source.

Looking at the manufacturing method of the porous particles for tissue engineering of the present invention in more detail.

The present invention mixes non-porous particles made of biodegradable polymers with water-soluble microcrystalline particles, and then applies specific temperature and pressure conditions in an enclosed space that is equally applied through three-dimensional omnidirectional interiors of the non-porous particles. After uniformly penetrating the water-soluble fine crystal particles into, and then dissolved in an aqueous solution it is characterized in that to produce particles capable of controlling the uniform porosity and pore size.

Since the non-porous particles made of the biodegradable polymer are constituent materials of the porous particles for human use, they should exhibit biocompatibility, and the biodegradable polymer has a molecular weight of 1,000 to 1,000,000 g / mol, and a lactic acid and a glycol. Homopolymers or copolymers selected from acid, caprolactone, dioxanone, hydroxybutyric acid, hydroxybalic acid, phosphoester, ethylene oxide can be used. Specifically, poly (lactic acid), polyglycolic acid (poly (glycolic acid)), polycaprolactone (poly-ε-caprolactone), polydioxanone, polylactic acid-glycolic acid copolymer (poly (lactic acid-co-glycolic acid)), polydioxanone-co-ε-caprolactone), polylactic acid-caprolactone copolymer (poly (lactic acid-co-ε-caprolactone) caprolactone)), polyhydroxybutyric acid-co-hydroxyvaleric acid, poly (phosphoester), polyethylene oxide-polylactic acid copolymer, polyethylene oxide-poly One or two or more selected from the lactic glycolic acid copolymer and the polyethylene oxide-polycaprolactone copolymer can be selected and used.

Such non-porous particles can be prepared using a mill or freezer mill generally used in the art.

At this time, the diameter of the prepared non-porous particles is preferably in the range of 50 to 2,500 µm, preferably 100 to 500 µm. If the diameter is less than 50 ㎛ has a problem that it is difficult to form a uniform porosity while having a pore size necessary for cell culture, and if the diameter exceeds 2,500 ㎛ there is a problem that particles with low porosity uniformity is formed.

In addition, the water-soluble fine crystal particles can be easily dissolved and removed in the aqueous solution during the manufacturing process, as well as biocompatibility, solubility in water (20 ℃, 100g solvent) is in the range of 1 to 50 g specifically, sodium chloride (sodium chloride), sodium citrate, sodium acetate, sodium tetraborate, sodium sulfate, citric acid, potassium chloride, potassium phosphate Phosphate), dextran, sugar can be used to select one or two or more selected. The diameter of such water-soluble fine crystal grains is preferably 25 to 500 µm, preferably 25 to 100 µm. If the diameter is less than 25 ㎛ pore size is not enough for cell culture, the water-soluble microcrystalline particles are difficult to penetrate into the non-porous spherical particles, if the diameter exceeds 500 ㎛ porosity uniformity There is a problem that low particles are formed.

The non-porous particles and the water-soluble microcrystalline particles are preferably maintained in a weight ratio of 1: 5 to 30, preferably in a weight ratio of 1: 10 to 20, and more preferably in a weight ratio of 1: 20. If the mixing ratio of the water-soluble fine crystal grains is less than 5 weight ratio, there is a problem in that no pores are formed in the center portion of the particles or the particles stick to each other. If the mixing ratio exceeds 30 weight ratio, the water-soluble crystals are unnecessarily wasted. That is, when maintaining the said range, the porosity aimed at by this invention can maintain 80 to 96%.

Next, in the process of infiltrating the water-soluble microcrystalline particles into the non-porous particles by applying a specific temperature and pressure to the mixture, the temperature is applied to the inside of the non-porous particles of the water-soluble microcrystalline particles by softening and melting the biodegradable polymer Conditions that can be penetrated include ± 50 ° C of softening point (Ts) or melting point (Tm) of biodegradable polymer, preferably ± 20 ° C of softening point (Ts) or melting point (Tm) of biodegradable polymer It is good to keep it. If it is less than 50 ℃ than the softening point (Ts) or melting point (Tm), there is a problem that the water-soluble crystals do not penetrate into the interior of the non-porous particles, if the temperature exceeds 50 ℃ than the softening point (Ts) or melting point (Tm) There is a problem that the polymer is pyrolyzed. In addition, the pressure is preferably in the range of 1 to 50 MPa, preferably in the range of 10 to 15 MPa. If the pressure is less than 1 MPa, water-soluble crystals do not penetrate into the interior of the non-porous particles. In the case of exceeding 50 MPa, water-soluble crystals are broken, so that a porous particle having a desired pore size cannot be produced.

This temperature and pressure should be performed in a confined space that is applied equally through three-dimensional directions, so that the water-soluble microcrystalline particles penetrate deeply into the non-porous particles so that pores are uniformly formed on the surface and inside of the non-porous particles. Is formed.

Porous particles prepared by the above method has a porosity of 80 to 96%, and the pores of the same size as the water-soluble microcrystalline particles used for forming the pore are uniformly distributed inside and outside the particles, and the pore size formed is 25 Since the pore size is less than 25 μm, small pores are insufficient for cell growth, and the pore size is 500 μm or per porous particle diameter. If it exceeds 0.6, it is preferable to maintain the above range in order to form particles having low porosity uniformity and to apply it as a support for tissue cell and stem cell culture, including chondrocytes derived from human tissues, in particular, human-derived chondrocytes.

As described above, the porous particles of the present invention exhibit a tendency to increase the diameter by 2-3 times compared to the diameter of the non-porous particles used as the material, that is, the range of 50 to 2,500 μm. This is due to the expansion of the volume of the polymer due to the penetration of the water-soluble microcrystalline particles into the non-porous particles in the manufacturing process is to increase the diameter as described above.

Although this invention is demonstrated in detail based on an Example, this invention is not limited to an Example.

Example  One

In order to prepare porous particles, polycaprolactone (PCL), which is a biodegradable polymer, is first pulverized into micro particles using a freezer mill (SPEX 6750, USA), and then micro sieves are used. To obtain a non-porous polymer particle having a non-uniform shape separated by size (100 <d <200, 200 <d <300, 300 <d <425, 425 <d <500 μm).

Salt crystals having a diameter of 25 to 50 and 50 to 100 µm were added to the nonporous particles in a 1/20 weight ratio, respectively, and uniformly mixed using a vortex mixer. An experiment in which the non-porous particles / salt crystals were mixed evenly in a frame made of brass (inner diameter; 18 mm, height; 12 mm), covered with a brass top plate, and preheated to 80 ° C. It was placed in a utility press. First, after pressing for 3 minutes at a pressure of 10 ~ 15 MPa so that heat is uniformly transferred to the inside of the frame of brass material, it was immediately pressed for 1 minute 30 seconds at a pressure of 15 ~ 20 MPa. This process allows salt crystals to penetrate into soft nonporous particles. In this case, since the mixture is subjected to temperature and pressure in a sealed frame, the mixture forms the same temperature and pressure conditions through three-dimensional omnidirectional directions.

In order to remove salt crystals penetrated inside the non-porous particles, the specimen prepared above was washed in ultrapure water for 12 hours and dried to obtain various particle sizes (250 to 450, 450 to 650, 650 to 850, 850 to 1050 μm). ) And porous particles having various pore sizes (25-50, 50-100 μm) were easily prepared.

       The form of the porous particles prepared above was observed through an electron scanning electron microscope (SEM), and the results are shown in FIGS. 1 and 2, and their pore sizes and porosities are shown in Table 1.

[Separation and Analysis Method]

1. Separation of salt crystals and non-porous particles: use of fine particle separation sieve

2. Form of nonporous and porous particles (pore size, pore uniformity and diameter)

Analysis: Electronic scanning microscope measurement and image analysis program of measured photo

3. Porosity:

(Porous particle volume-nonporous particle volume) / porous particle volume x 100

Diameter of Salt Crystals (㎛) Diameter of Nonporous Particles (μm) Pore size of porous particles (㎛) Diameter of Porous Particles (㎛) Porosity (%) 25 to 50 100 to 200 25 to 50 250 to 450 85 ～ 96 200 ～ 300 450 to 650 300 to 425 650 ～ 850 425 ～ 500 850 ～ 1050 50 to 100 100 to 200 50 to 100 250 to 450 85 ～ 96 200 ～ 300 450 to 650 300 to 425 650 ～ 850 425 ～ 500 850 ～ 1050

As shown in Table 1 and Figures 1 and 2, the porous particles prepared by Example 1 of the present invention showed a uniform pore distribution, the size of the pore size and non-porous particles of the same size as the salt crystal used It can be observed that the size is controlled by.

In addition, the porosity of the prepared porous particles was confirmed to have a 85 ~ 96% suitable for use in tissue engineering.

Comparative example  One

In the same manner as in Example 1, the porous particles were prepared by placing a mixture of non-porous particles / salt crystals uniformly between the open plate and the flat plate instead of the frame forming a closed space. At this time, the pressure applied to the mixture exists in only one direction (the direction in which the pressure is applied).

When produced by the above method, it was observed that the porous particles were formed instead of the flat porous support in the form of discs.

Example  2

In the same manner as in Example 1, porous particles were prepared using polydioxanone which is a biodegradable polymer (low solubility in solvents, thus preventing the production of porous particles by conventional methods).

The shape and porosity of the porous particles prepared using the polydioxanone showed the same tendency as in Example 1.

Example  3

In the same manner as in Example 1, porous particles were prepared using polylactic acid as a biodegradable polymer.

The form and porosity of the porous particles prepared using the polylactic acid showed the same tendency as in Example 1.

Example  4

In the same manner as in Example 1, a porous particle was prepared using polyglycolic acid (a low solubility in solvents, which prevents the production of porous particles) by using a biodegradable polymer.

The form and porosity of the porous particles prepared using the polyglycolic acid showed the same tendency as in Example 1.

As described above, the present invention mixes non-porous particles having various diameters made of biodegradable polymers with water-soluble fine crystal grains of controlled diameter, and applies heat and pressure thereto to uniformly distribute the water-soluble fine crystal grains in the non-porous particles. After permeation, the present invention relates to porous particles obtained by dissolving, washing, and drying the water-soluble fine crystal particles in an aqueous solution, and a method for preparing the same. Having a uniform porosity on the outside, when used for tissue engineering, cell culture fluids and body fluids can penetrate smoothly into particles, providing a very effective environment for efficient adhesion and proliferation of tissue cells, and could not be manufactured by conventional methods. Most biodegradable polymers, including polymers with low solubility in solvents, can be prepared without the use of organic solvents, and can be easily introduced into the body through injection, which can be very useful as a tissue engineering support for various tissue regeneration. have.

Claims (6)

Non-porous particles made of biodegradable polymers and water-soluble microcrystalline particles having a solubility in water (20 ° C., 100 g solvent) in the range of 1 to 50 g are mixed at a weight ratio of 1: 5 to 30, The non-porous nature of the biodegradable polymer in a confined space in which the temperature of ± 50 ° C and the pressure of 1 to 50 MPa of the softening point (Ts) or melting point (Tm) are equally applied through the three-dimensional omnidirectional direction. Infiltrate the water-soluble fine crystal grains into the particles, The method for producing biodegradable porous particles for tissue engineering, comprising the step of preparing the porous particles without the use of an organic solvent, washing the particles infiltrated with the fine crystal particles in an aqueous solution and dissolving the water-soluble fine crystal particles. According to claim 1, wherein the biodegradable polymer is a polylactic acid (poly (lactic acid)), polyglycolic acid (poly (glycolic acid)), polycaprolactone (poly-ε-) having a molecular weight of 1,000 ~ 1,000,000 g / mol caprolactone), polydioxanone, polylactic acid-co-glycolic acid (poly (lactic acid-co-glycolic acid)), polydioxanone-ca-prolactone copolymer (polydioxanone-co-ε-caprolactone), Polylactic acid-caprolactone copolymer (poly (lactic acid-co-ε-caprolactone)), polyhydroxybutyric acid-hydroxybalic acid copolymer (polyhydroxybutyric acid-co-hydroxyvaleric acid), polyphosphoester (poly (phosphoester)), a polyethylene oxide-polylactic acid copolymer, a polyethylene oxide-polylactic glycolic acid copolymer, a polyethylene oxide-polycaprolactone copolymer, or a biocompatible biodegradable polymer which is a mixture of two or more kinds thereof. By Article methods The method of claim 1, wherein the water-soluble fine crystal grains are selected from sodium chloride, sodium citrate, sodium acetate, sodium tetraborate, sodium sulfate, citric acid, potassium chloride, potassium phosphate, dextran and sugar having a diameter of 25 to 500 ㎛ or A method for producing a mixture, characterized in that two or more. The method of claim 1, wherein the non-porous particles have a diameter ranging from 50 to 2,500 µm. According to claim 1, wherein the prepared porous particles are characterized in that the diameter ranges from 100 to 5,000 ㎛. Prepared by the method of claim 1, the porosity is 80 to 96%, the porous particles having a diameter of 100 ~ 5,000 ㎛ range, the pores having a diameter of 25 ~ 500 ㎛ range inside and outside the particles uniformly Distribution, but the pore size is biodegradable porous particles for tissue engineering, characterized in that adjusted to the range of 0.01 ~ 0.6 per spherical particle diameter.

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