KR20190001630A - Method of Producing Scaffolds for Tissue Regeneration - Google Patents

Abstract

The present invention relates to a method for manufacturing a tissue regeneration support comprising a step of sintering a PCL microsphere in a constant temperature water bath. Specifically, the method comprises a step of sintering the PCL microsphere having a particle size of 500 μm or less in a constant temperature water bath at 52-54°C. The tissue regeneration support manufactured according to the method of the present invention has high strength and can stimulate angiogenesis to an excellent degree.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a method for producing a support for tissue regeneration,

The present invention relates to a support for tissue regeneration comprising a step of sintering the PCL microspheres in a constant temperature water bath.

In tissue engineering, tissue regeneration supports frequently fail to regenerate tissue because integration with host tissues by cellular infiltration is difficult. Because of insufficient environmental factors that prevent penetration into cells and tissues, biomaterials need to provide a biological signal that maintains and regulates cellular activity through the combination of the signal of the parental cell and the isolation and transmission of growth factors.

The ideal support for restoring bone tissue has the following characteristics: (1) (3-D) porous structure, (2) interconnected pore network, (3) biocompatibility and absorbency at controlled degradation and resorption, and Mechanical properties (Laurencin CT et al. Annu Rev Biomed Eng. 1999; 1: 19-46).

Of these, the long-term survival and function of the tissue regeneration support depends on the development of new blood vessels that supply nutrients and oxygen to the cells in the middle of the tissue graft. Inadequate blood supply is a major cause of fracture nonunion, as reabsorption of necrotic bone in the early stages of the fracture recovery process requires angiogenesis at the site of injury (Brownlow HC et al. (2): 145-50). Inactive bone grafts have relatively low angiogenic potential and more persistent difficulties. Long bone fractures are prone to bone necrosis by causing a decline in normal blood supply due to peripheral soft tissue damage. This means that increasing vascular supply directly affects bone repair and growth.

In ossification of the endothelium and cartilage, angiogenesis precedes osteogenesis. Microvessels are known to stimulate bone formation even before blood flow appears. In addition, blood vessels are involved in the transport of osteoclasts and osteoclasts. It is also known that the existing vascular network serves as a skeleton for subsequent osteoblast differentiation (Kanczler JM et al., Eur Cell Mater. 2008; 15: 100-14). It is also known that the existing vascular network serves as a skeleton for subsequent osteoblast differentiation.

Thus, the selection of a suitable support to stimulate and assist bone graft through the formation of microvessels is very important. Rentsch and colleagues developed PCL scaffolds coated with collagen I and chondroitin sulfate and showed a relative increase in bone volume in rats with a long bone-size-deficient support (Rentsch C et al. J Biomed Mater Res A.95 (3): 964-72).

On the other hand, so far most sintering techniques for microspheres have been used in drug delivery systems (Catauro M et al. J Appl Biomater Biomech. 2010; 8 (1): 42-51; Hernan Perez de la Ossa D, et al J Control Release 2012; Fan M et al J Biomater Appl. 2012).

However, it is not known whether or not the tissue regeneration support prepared by sintering the microspheres having a small particle size can exhibit excellent characteristics. Specifically, it is known that sintering a small microsphere having a particle diameter of several hundreds of micrometers (for example, 500 탆 or less) disturbs the shape or weakens the strength, and thus researches for sintering PCL microspheres having a particle size of at least 500 탆 or more It is only in progress.

Under these circumstances, the present inventors have found that when a support for tissue regeneration is manufactured using a constant temperature water bath in the process of sintering PCL microspheres having small particle diameters, the spherical shape of the microspheres is maintained, It was confirmed that a support for tissue regeneration could be produced. In this case, it was confirmed that the support for tissue regeneration using the microspheres having a small particle size is superior to the microsphere having a large particle size, and has an excellent angiogenesis effect and is suitable for tissue regeneration.

One object of the present invention is to provide a method for producing a support for tissue regeneration comprising sintering a PCL microsphere having a particle diameter of 500 탆 or less in a constant temperature water bath at 52 to 54 캜.

It is another object of the present invention to provide a support for tissue regeneration, which is produced according to the above production method.

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

On the other hand, each description and embodiment disclosed herein can be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein are within the scope of the present invention. Further, the scope of the present invention can not be said to be limited by the following detailed description.

In addition, those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described in this application. Further, such equivalents are intended to be included in the present invention.

One embodiment for implementing the present invention is a method for producing a support for tissue regeneration comprising sintering a PCL microsphere having a particle diameter of 500 탆 or less in a constant temperature water bath at 52 to 54 캜.

When the microspheres having a small particle size (for example, 500 탆 or less) are sintered, the spherical shape is not maintained and the contact area between the microspheres is narrowed, which makes it difficult to obtain excellent strength. According to the present invention, it was confirmed that a microsphere having a small particle diameter was sintered using a constant temperature water bath for fine temperature control, and as a result, it was possible to produce a tissue regeneration support having excellent characteristics under specific conditions. It was confirmed that the support for tissue regeneration prepared by the above method is very suitable for tissue regeneration because it is a new blood vessel in vivo.

The term "PCL microspheres" as used herein is small particles comprising PCL. PCL is an acronym for Polycaprolactone and is a widely known substance as a biodegradable polyester. The microspheres may include a particle size of 1 占 퐉 -1 mm, but the microspheres disclosed herein may have a particle size of, for example, 10-500 占 퐉, a particle diameter of 100-300 占 퐉, and a particle diameter of 100-200 占 퐉. When a PCL having a particle size of a uniform size is used, a support for tissue regeneration having physically excellent characteristics can be manufactured by reducing the size of pores. The microspheres may be substantially uniform in size in the form of spheres in general. Also, some may be flat, bent, elliptical, or irregular in shape, while others may be spherical.

As used herein, the term "water bath" means a bath that is kept constant at a certain temperature. Generally, a container in a constant-temperature water tank is filled with water maintained in a heated state. Other materials such as oil, silicone, or sand may be used instead of water as the temperature-keeping material.

The constant temperature water bath is for sintering the PCL microspheres, and it is important that the temperature is kept constant. In one embodiment of the present invention, the PCL microspheres were sintered at 53 deg.

The term "sinter" as used in the present invention means a process of compressing and molding a solid material by heat, pressure or the like without melting it to a liquefaction point.

In the production method of the present invention, the support for tissue regeneration can be produced through the step of sintering the PCL microspheres at a temperature of 52 to 54 ° C for 30 minutes to 2 hours. Specifically, it can be sintered while maintaining the temperature at 52.5 to 53.5 캜. More specifically, sintering can be performed while maintaining the temperature at 53 캜. When the PCL microspheres having a size of 100-200 탆 are sintered at a temperature of 52 캜 or below to produce a tissue regeneration support, the compressive strength may be low. When the support for tissue regeneration is manufactured by sintering at a temperature of 54 캜 or higher, The gap between the spheres decreases, and the shape may be disturbed.

The sintering may be performed for 30 minutes to 2 hours. If the sintering is performed within 30 minutes, the compressive strength may be low. If the sintering is performed for 2 hours or more, the shape may be disturbed or the contact area may be excessively widened.

The PCL microspheres may be formed by mixing PCL with an organic solvent, a water-soluble polymer, or the like. Specifically, the PCL can be dissolved in an organic solvent and added to an aqueous solution of a water-soluble polymer to be mixed. The mixing can be effected by gentle agitation. Solid microspheres can be formed through the mixing.

The organic solvent is a solvent which is a commonly used organic material. Specifically, at least one selected from the group consisting of dichloromethane, chloroform, carbon tetrachloride, acetone, dioxane, and tetrahydrofuran may be used, but is not limited thereto. In one embodiment of the present invention, dichloromethane was used.

The water-soluble polymer may serve as a binder for uniformly diluting PCL in the present invention. Specifically, any one or more selected from the group consisting of water-soluble water-soluble polymers such as polyvinyl alcohol, polyethylene glycol, polyvinyl alcohol, polyethylene glycol, polyvinyl butyral, gelatin and chitosan, and polyethylene oxide may be used. Do not. In one embodiment of the present invention, polyvinyl alcohol has been used.

The microspheres may or may not further comprise "bioactive factors" in addition to PCLs, organic solvents, water soluble polymers. A "physiologically active factor" may be a small organic molecule, nucleic acid, or polypeptide capable of stimulating or promoting cell penetration, cell growth, angiogenesis, angiogenesis, nerve regeneration, or cell differentiation.

Even when a supporter for tissue regeneration is produced without using a physiologically active factor, the supporter is suitable for tissue regeneration because it induces angiogenesis, and further, it can be used homogeneously and stably.

The formed PCL organic solvent can be separated according to the particle size through the sieve. In one embodiment of the present invention, microspheres of 100-200 탆, 200-300 탆, and 300 탆 or more were separated.

The term "support" as used herein may refer to a substance that replaces or partially replaces damaged organs or tissue in vivo. In particular, the polymer scaffold may include a biodegradable polymer material, and the scaffold may be completely decomposed in vivo after the scaffold is maintained until its function and role are sufficiently performed.

It is preferable that the "support" of the present invention is physically stable in order to perform a function of replacing the living body for a certain period of time. Although PCL microspheres having a small particle size are used in the present invention, they can have a sufficient contact area through proper sintering and can have a high compressive strength. In particular, a biologically active substance other than PCL is not added, so that a stable tissue regeneration support having a constant arrangement can be produced.

In the present invention, the term "tissue regeneration" may mean any phenomenon that treats, alleviates, or improves tissue damage through processes such as proliferation of damaged cells or differentiation of tissues by angiogenesis. In particular, but not exclusively, the tissue regeneration can be promoted by angiogenesis. The blood vessels can be infiltrated into the pores of the tissue regeneration support and regenerated.

Accordingly, it is recognized that it is desirable to have a suitable volume of pores for the support to be used for tissue regeneration. In general, a support using a microsphere having a large particle diameter can have a larger volume of air than when using a microsphere having a small particle diameter. However, if the pore is excessively large, it is problematic because it can not be a physically strong support. In order to produce physically strong supporters having pores suitable for vascular tissue regeneration, detailed manipulation is required in the selection of microspheres and the degree of sintering.

In one embodiment of the present invention, a PCL microspheres having particle diameters in the range of 100-200 μm, 200-300 μm, and> 300 μm were sintered in a constant temperature water bath at 53 ° C. to prepare a tissue regeneration support, It was confirmed that blood vessels were formed as the pores of the three regeneration supports. In particular, when a support for tissue regeneration was prepared using PCL microspheres having a particle size in the range of 100-200 占 퐉, compared with the case of using PCL microspheres having a particle size in the range of 200-300 占 퐉 and > 300 占 퐉, It was confirmed that there is excellent angiogenesis ability.

In the present invention, the term "support for tissue regeneration" does not necessarily correspond to the shape of the tissue damage site to be implanted, regardless of its shape, However, if necessary, the shape of the tissue damage site to be transplanted may be grasped in advance, and a shape suitable for the shape may be produced. In addition, the structure may be shaped in advance by a membrane structure or a band structure so as to be generally applicable to various situations.

Various materials for increasing the moldability of the above-described tissue regeneration support may be further added. That is, a linear material, a tubular material, a particle-like material, and an indefinite material excellent in biocompatibility can be further added to improve physical properties of the tissue regeneration support. Materials of this type may be selected from materials such as collagen, carboxymethylcellulose (CMC) or animal-derived gelatin, and there is no particular limitation as long as they do not cause an immune response to the recipient.

Another aspect for implementing the present invention is a support for tissue regeneration produced according to the above-described production method. Specifically, it may be a tissue regeneration support made of spherical PCL microspheres having a particle diameter of 500 탆 or less. The tissue regeneration support has high strength and can stimulate angiogenesis to an excellent degree.

The tissue regeneration support prepared according to the method for producing a tissue regeneration support comprising a step of sintering the PCL microspheres in a constant temperature water bath at 52 to 54 DEG C has a high strength and can stimulate angiogenesis to an excellent degree.

1A to 1F are SEM images of a support sintered at 51 ° C, 52 ° C, 53 ° C, 54 ° C, 55 ° C and 57 ° C, respectively, with PCL microspheres having particle sizes in the range of 100-200 μm. FIG. 1G is a view showing the compressive strength of the support.
2A, 2B, 2C and 2D, and 2E and 2F show the SEM (A, C, and E) of the support using PCL microspheres having particle diameters in the range of 100-200, 200-300, B, D, and F are enlarged 500 times).
3A-3C show PCL microspheres having particle diameters ranging from 100-200 [mu] m, 200-300 [mu] m, and > 300 [mu] m respectively and transplanted into subcutaneous tissues of rats for 4 weeks. The grafts were then separated and fixed with VEGF immunohistochemistry (IHC) and observed under a microscope. Figure 1G is a graphical representation of the number of new microvessels.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Manufacturing example  One.

(1) Material

Poly (ε-caprolactone) (PCL), polyvinyl alcohol (PVA) (MW: 13-23 kDa, 87-89% hydrolysis) and dichloromethane were used as received from Sigma-Aldrich . VEGF R2 was purchased from Cell Signaling (Beverly, MA). ABC universal kits were purchased from Vector (Burlingame, Calif.).

(2) Preparation of a single microsphere

PCL (24% w / v) was dissolved in 20 mL of dichloromethane, added to 1 liter of an aqueous solution of 0.25% w / v PVA and stirred at 400 rpm for 3 hours at room temperature. The resulting emulsion was gently agitated until the organic solvent was extracted from the PCL to form solid microspheres. Next, the microspheres were washed with deionized water to remove residual organic solvent. Commercial spheres (Sieves IG / 3-EXP, Retsch, Germany) were used to selectively isolate the microspheres having particle diameters in the following ranges: 100-200 μm; 200-300 탆; > 300 μm.

(3) Sintered microspheres Scaffold  Ready

PCL microspheres having a specific range of particle sizes were poured into aluminum molds and heated in a constant temperature water bath for 1 hour, respectively. The solid matrix was recovered and cooled to room temperature and finally separated from the mold to obtain a microsphere scaffold.

Experimental Example  1. Effect of Sintering Temperature on Shape and Strength of Support

In (3) of Production Example 1, the PCL microspheres having a particle size in the range of 100-200 mu m were sintered at a temperature of 51 DEG C, 52 DEG C, 53 DEG C, 54 DEG C, 55 DEG C, 57 < [deg.] ≫ C, thereby obtaining six supports, respectively. The shape and strength of the support for the six supports were measured.

(1) Scanning Electron Microscopy Image measurement

For scanning electron microscopy image measurements of the support, the scaffold was fixed with 2.5% glutaraldehyde. The scaffolds of Examples 1 to 3 were dried at critical points using CO ₂ and then gold sputtered in vacuum and scanned under a scanning electron microscope (SEM) (S-4300, HITACHI, Japan) after dehydration with a series of staged alcohols. Were used.

SEM images of the supports sintered at 51 DEG C, 52 DEG C, 53 DEG C, 54 DEG C, 55 DEG C, and 57 DEG C are shown in Figs. 1A to 1F, respectively. The support sintered at 52 ℃ maintained a circular shape, but the bonding area was narrow. As the sintering temperature increased, the adhesion area increased. However, the spheres of the support sintered at 54 캜 became inactive and the pores between the microspheres decreased. Spherical shape was maintained in the support sintered at 53 캜 and had an appropriate bonding area.

(2) Compression test of support

Each support was formed into a column having a diameter of 5 mm and a length of 8 mm. The compressive strength was measured using an Instron 5567 machine. Five samples were measured for each support and the highest and lowest values discarded (n = 3). The compressive strength was calculated by dividing the fracture load by the specimen below the bed area of the circular cylinder.

An evaluation of the maximum compressive strength is shown in Fig. 1G. The strength increased with increasing sintering temperature. The results of this compressive strength measurement were more supported by the SEM image.

Experimental Example  2. Effect of particle size of microspheres on shape and angiogenesis of supporter

In (3) of Production Example 1, PCL microspheres having particle diameters in the range of 100 to 200 mu m, 200-300 mu m, and > 300 mu m were each sintered at a temperature of 53 DEG C in a constant temperature water bath, . SEM images of the three supports and their effects on bioactivity were measured.

(1) Scanning Electron Microscopy Image measurement

SEM images were measured on the three kinds of supports in the same manner as in (2) of Experimental Example 1, and they are shown in Fig. SEMs of the supports using PCL microspheres having particle diameters in the range of 100-200 urn, 200-300 urn and> 300 urn are shown in FIGS. 2A and 2B, 2C and 2D, and 2E and 2F, respectively (A, Fold increase, and B, D and F increase 500 times).

The three kinds of supports were spherical, and the empty space was wide according to the particle diameter of the microspheres.

(2) Evaluation of vascular innervation in vivo

Microvascular infiltration of the microsphere support was investigated in the subcutaneous tissues of the mice over 4 weeks. BALB / c mice were anesthetized by intramuscular injection of a ketamine-rompun mixture (1.5 mg / 1.5 mg / kg body weight, respectively). A dermal incision of sufficient size to accommodate the test sample was made on the dorsal side of the mice. After making the pockets with blunt dissection, the microsphere support was gently inserted into each pocket and the skin was sutured with overlapping sutures. Four weeks after transplantation, the mice were sacrificed and the entire graft was removed from the dorsal subcutaneous tissue. Each graft sample was fixed with 4% paraformaldehyde and subjected to VEGF immunohistochemical staining (IHC) staining followed by microscopic observation.

As a result, as shown in Fig. 3, many blood vessels penetrated into the support. Through this, it was found that the support sintered using a small microsphere was well integrated with the host tissue. In particular, it has been confirmed that the supporter using 100-200 mu m microspheres induces vascular tissue growth and angiogenesis better than supporters using microspheres larger than 100 mu m.

 Statistical analysis

All values are expressed as standard error ± standard error. Data were analyzed by one-way analysis of variance (ANOVA) and student t-test (Sigma plot, San Rafael, CA). In all study experiments, n = 5 samples were used. Quantitative data were reported as mean ± standard deviation (SD). All results were initially assessed using one-way ANOVA and then a Tukey (HSU) analysis of differences between groups was performed to obtain a 95.0% confidence interval.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (9)

And sintering the PCL microspheres having a particle size of 500 탆 or less in a constant temperature water bath at 52 to 54 캜.
The method of manufacturing a support for tissue regeneration according to claim 1, wherein the PCL microspheres are formed by mixing PCL with an organic solvent and a water-soluble polymer.
The method of producing a support for tissue regeneration according to claim 1, wherein the particle size of the PCL microspheres is 100 to 300 탆.
The method of manufacturing a support for tissue regeneration according to claim 1, wherein the particle size of the PCL microspheres is 100 to 200 탆.
The method of claim 2, wherein the organic solvent is at least one selected from the group consisting of dichloromethane, chloroform, carbon tetrachloride, acetone, dioxane, and tetrahydrofuran.
The method according to claim 2, wherein the water-soluble polymer is at least one selected from the group consisting of polyvinyl alcohol, polyethylene glycol, polyvinyl alcohol, polyethylene glycol, polyvinyl butyral, gelatin, and chitosan .
The method according to claim 1, wherein the temperature of the constant temperature water bath is 52.5 to 53.5 ° C.
The method of claim 1, wherein the sintering step is performed for 30 minutes to 2 hours.
9. A support for tissue regeneration, produced according to the method of any one of claims 1 to 8.

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