KR102172373B1 - A method for preparation of biodegradable implant with increasing its porosity over time - Google Patents

Abstract

The present invention is a method for producing a biodegradable implant in which a biodegradable polymer melt and biodegradable ceramic particles are mixed by stirring at a temperature higher than the melting point of the biodegradable polymer using a high shear mixer, and a biodegradable polymer prepared accordingly The present invention relates to an implant for bone regeneration including a biodegradable implant in which biodegradable ceramic particles having a diameter of several hundred micrometers are uniformly inserted and molded in a melt of a resin.

Description

A method for preparation of biodegradable implant with increasing its porosity over time}

The present invention is a method for producing a biodegradable implant in which a biodegradable polymer melt and biodegradable ceramic particles are mixed by stirring at a temperature higher than the melting point of the biodegradable polymer using a high shear mixer, and a biodegradable polymer prepared accordingly The present invention relates to an implant for bone regeneration including a biodegradable implant in which biodegradable ceramic particles having a diameter of several hundred micrometers are uniformly inserted and molded in a melt of a resin.

Bone plates or bone screws that are inserted into the body are manufactured using various biocompatible materials, but recently, biodegradable materials are used in that secondary surgery is not required for removal after insertion. Plates and screws made by doing this are in the spotlight. Polymers are widely used as such biodegradable materials.

However, these polymer materials have a disadvantage of having significantly lower elastic modulus and mechanical properties than metal. Therefore, in order to realize the desired mechanical properties, that is, in order to withstand the same load as a plate or screw made of a metal material, a plate and screw of a larger size is used, which makes the procedure difficult and can be a burden on the patient receiving the implant. . On the other hand, in the case of biodegradable polymers such as polylactic acid, which is a material that attracts most attention based on excellent mechanical properties among the polymer materials, the polymer itself does not have active bioactivity, thus loosening the junction between the implant and the bone after implantation. There is a disadvantage that the implant can be separated from the bone.

In order to compensate for these shortcomings due to the use of biodegradable polymer materials, a technique for manufacturing a composite by adding a metal or ceramic material having excellent bioactivity as a reinforcing factor to a polymer substrate is used. Although the complexes have improved physical properties in terms of strength, the structure of the implant before tissue is regenerated due to initial explosive decomposition according to the type of material to be combined for the purpose of these complexes to be transplanted in vivo and used for tissue regeneration. Problems may arise, such as the collapse of the cells, the decomposition being too slow to provide a space to accommodate the newly formed tissue cells. In addition, the shape or size of the particles incorporated into the polymer also affects the structure remaining after its decomposition, so it is one of the factors to be considered when manufacturing the composite.

Accordingly, the inventors of the present invention have a biodegradable polymer as a parent, and in preparing a complex by adding particles of a biocompatible material that degrade faster than this, the present inventors have sufficient to fill the cells of the tissue regenerated by the decomposition of the biocompatible material. As a result of intensive research efforts to discover a composite that provides a space of size but does not collapse the structure of the entire implant and a method for manufacturing the same, the polymer resin melt stirred at a predetermined speed at a temperature higher than the melting point in a high shear mixer, While maintaining the temperature and stirring speed, biodegradable ceramic particles having a size of several hundred micrometers are added, mixed, and molded, thereby providing an implant made of a composite in which ceramic particles having a size of several hundreds of micrometers are uniformly distributed in a polymer resin. This is because the ceramic particles inserted into the polymer resin after implantation in the body are selectively decomposed over time without destroying the structure of the entire implant, so that cells for forming a new tissue adhere and grow, proliferate, and/or differentiate. It was confirmed that sufficient space can be provided, and the present invention was completed.

One object of the present invention is a method for producing a biodegradable implant, which is formed by uniformly inserting biodegradable ceramic particles having an average diameter of 100 to 300 μm into a melt of a biodegradable polymer resin, and having an increased porosity with time, A first step of adding a biodegradable polymer to a high shear mixer and stirring at 3 Hz to 10 Hz while maintaining a temperature of the mixer at 10°C to 50°C higher than the melting point of the biodegradable polymer; A second step of adding a powder containing biodegradable ceramic particles having a higher decomposition rate than the biodegradable polymer to the high shear mixer and mixing while maintaining the temperature and speed; And a third step of molding the mixture to provide a method of manufacturing a biodegradable implant.

Another object of the present invention is that biodegradable ceramic particles having an average diameter of 100 to 300 μm in the melt of the biodegradable polymer resin and having a faster decomposition rate than the biodegradable polymer are uniformly inserted to increase the porosity over time. It is to provide an implant for bone regeneration comprising a biodegradable implant.

In order to achieve the above object, the first aspect of the present invention is formed by uniformly inserting biodegradable ceramic particles having an average diameter of 100 to 300 μm into a melt of a biodegradable polymer resin, and increasing the porosity with time, biodegradable As a method of manufacturing an implant, a biodegradable polymer is added to a high shear mixer, and the temperature of the mixer is stirred at 3 Hz to 10 Hz while maintaining a temperature of 10 to 50° C. higher than the melting point of the biodegradable polymer. The first step; A second step of adding a powder containing biodegradable particles having a higher decomposition rate than the biodegradable polymer to the high shear mixer and mixing while maintaining the temperature and speed; And it provides a method for producing a biodegradable implant comprising a; and a third step of molding the mixture.

The present invention is designed to discover a composite material for providing an implant capable of compensating for the poor strength of a biodegradable polymer implant and promoting tissue regeneration, and a bioceramic that can be biodegradable in the body while having excellent strength. It is based on the confirmation that a remarkably increased strength and elasticity can be realized by uniformly inserting a biodegradable material, such as, in the form of particles having a predetermined size, into a polymer matrix to form a composite. Accordingly, the present invention is to prepare a biodegradable implant having improved strength and elasticity by rapidly mixing and molding biodegradable particles of a predetermined size while maintaining a molten state of a biodegradable polymer using a temperature-controllable high shear mixer. It is characterized by the first development of the method.

At this time, the biodegradable ceramic particles to be added preferably have a size of 100 to 300 μm in average diameter. For example, when the biodegradable implant of the present invention prepared as described above is implanted into the body, the biodegradable ceramic particles are decomposed first compared to the polymer matrix due to the difference in the decomposition rate over time, and the position where they occupied Empty space, that is, pores are formed. On the other hand, considering that the purpose of the implant is to be implanted in damaged and/or deleted tissue to replace it and preferably to aid in the creation of new tissue, a sufficient space to be filled with newly generated tissue cells such as bone cells It is desirable to be able to provide.

The manufacturing method of the present invention is characterized in that it is carried out using a high shear mixer. The high shear mixer is a machine capable of effectively mixing, emulsifying, and dispersing a reactant with a higher shear force by using a nozzle shape that can increase the maximum effect of a higher speed and shear force than a general mixer. Specifically, the manufacturing method of the present invention is to uniformly distribute ceramic particles having a relatively low density in a high-density polymer melt. By using the high shear mixer, a large size of several hundred micrometers in a high-viscosity polymer melt Ceramic particles can be evenly dispersed.

As the biodegradable polymer, polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, or poly-ε-caprolactone may be used alone, or two or more of them may be used in combination. For example, among these polymers, polylactic acid having excellent mechanical properties may be used alone, but is not limited thereto.

As the biodegradable ceramic particles, tricalcium phosphate (TCP), calcium monohydrogen phosphate, falcalcium phosphate, particles of amorphous calcium phosphate or calcium sulfate, particles of a combination thereof, or a mixture of these particles may be used. However, it is not limited thereto. For example, considering that the manufacturing method of the present invention is performed at a temperature higher than the melting point of the biodegradable polymer, in order to maintain the biodegradable ceramic particles in the form of intact microspheres in the composite to be finally provided, the glass transition temperature is It may be advantageous to choose a material higher than the melting point of the polymer.

Meanwhile, in order to impart strength to the implant to be manufactured, in addition to the biodegradable ceramic, bioactive ceramics such as hydroxyapatite (HA), bioglass, and crystallized glass apatite may be additionally included, but are not limited thereto. .

The biodegradable ceramic particles may be added in an amount of 30 to 50% by volume based on the total amount of the mixture. When the content of the biodegradable ceramic particles is less than 30% by volume, the connection between the particles is insufficient, and decomposition of the isolated ceramic particles is inhibited, and it is difficult to achieve a continuous increase of the porosity to the desired level, and when it is more than 50% by volume However, since the physical properties of the mixture are poor, it may be difficult to ensure the stability of the mixture itself.

The biodegradable implant of the present invention is characterized in that pores are formed as the biodegradable ceramic particles inserted into the polymer matrix dissolve at a faster rate compared to the biodegradable polymer over time during implantation in the body. In addition, since the implant is made of a biocompatible material, cells penetrate into the formed pores, attach to the implant, and fill the pores while growing, proliferating and differentiating into tissue cells, and thus can be usefully used for tissue regeneration.

The second aspect of the present invention is formed by uniformly inserting and forming biodegradable ceramic particles having an average diameter of 100 to 300 μm in the melt of the biodegradable polymer resin and having a faster decomposition rate than the biodegradable polymer, and increasing the porosity with time. It provides an implant for bone regeneration comprising a biodegradable implant.

The biodegradable implant of the present invention has excellent strength and elasticity and can be used as an implant for bone regeneration, but is not limited thereto.

For example, the implant for bone regeneration of the present invention may be manufactured by the method of the first aspect, but is not limited thereto.

As described above, the bone regeneration implant of the present invention maintains the polymeric skeleton over time after implantation, but pores are formed and expanded according to the decomposition of the biodegradable ceramic particles, so that tissue regeneration can be promoted by accommodating new tissue cells. It may be a material.

The method of manufacturing a biodegradable implant of the present invention uses a temperature-controllable high shear mixer to maintain the biodegradable polymer in a molten state, while adding biodegradable ceramic particles of a predetermined size, mixing evenly, and molding, thereby improving strength and elasticity. An implant having an improved biodegradable polymer as a parent can be provided, and the implant prepared in this way not only significantly improves strength and elasticity, but also proliferates by penetrating tissue cells into pores formed as the inserted biodegradable ceramic particles are decomposed. It can be differentiated, so it can be usefully used as an implant for bone regeneration.

1 is a diagram schematically showing a method of manufacturing a tricalcium phosphate (TCP) microsphere and a polylactic acid composite according to an embodiment of the present invention.
2 is a diagram schematically showing a process of forming surface pores by dissolving TCP microspheres acting as a porogen after implanting a TCP microsphere-polylactic acid complex into the body.
3 is a scanning electron microscope (SEM) image showing the surface morphology of (a) TCP microspheres (TCPms) and (b) TCP powder (TCPpw), and (c) X- of TCP microspheres and TCP powder. It is a diagram showing X-ray diffraction (XRD).
Figure 4 is (a) a hand press machine used in the manufacture of a cylindrical specimen and (b) a real photo of the prepared pure PLA and PLA-40% by volume TCP composite, and (c) pure PLA, PLA- It is a diagram showing SEM images of the 40% by volume TCPms composite and the PLA-40% by volume TCPpw composite. Energy-dispersive X-ray spectroscopy (EDS) mapping images of Ca and P are shown in the inset of PLA-TCPms and PLA-TCPpw.
5 is a diagram showing SEM images of a pre-osteoblast cell line (MC3T3-E1) after 1 day of culture on (a) pure PLA, (b) PLA-TCPms and (c) PLATCPpw, respectively. Red arrows indicate cells.
6 is a diagram showing (a) MTS (methoxyphenyl tetrazolium salt) assay results after 5 days of culture and (b) ALP (alkaline phosphatase) activity after 10 days of culture (*p <0.05).
7 is a view showing the water contact angle of pure PLA and PLA composite, TCPms and TCPpw.
8 is a diagram showing the decomposition characteristics of pure PLA, TCPms and TCPpw over time. (a) shows a three-dimensional μ-CT image of each specimen at different decomposition time points, and (b) shows a cross-sectional SEM image of each specimen 30 days after accelerated decomposition in HCl.
9 is a cross-sectional μ-CT image of (a) pure PLA, (b) TCPms and (c) TCPpw after 16 weeks of in vivo transplantation, and (d) the bone volume around the implant calculated using the μ-CT program. Indicates (*p <0.05).
FIG. 10 is a histological image of (a) pure PLA, (b) TCPms and (c) TCPpw stained with Goldner's trichrome stain 16 weeks after transplantation in vivo, and (d) contact with the transplanted bone. It is a diagram showing the bone-to-implant contact ratio (I: implant, NB: new bone, CT: connective tissue). SEM images of each specimen are shown in the insets of each of (a) to (c) (scale bar in the inset = 100 μm, *p<0.05).
FIG. 11 is a diagram showing changes in size and shape due to decomposition of a comparative example magnesium particle (Mg)-PLA complex over time after immersion in a simulated body fluid (SBF).
12 is a diagram showing a change in pH due to decomposition of the Mg-PLA complex of Comparative Example over time after immersion in SBF.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited by these examples.

Example 1: biodegradable ceramic Microsphere -Preparation of polymer complex

Tricalcium phosphate powder (TCPw; Ca ₃ (PO ₄ ) ₂ , Sigma-Aldrich, USA), reagent-grade polyvinyl butyral (PVB, Butvar) as a binder B-98, Sigma) and KD6 (Hypermer KD-6, UniQema, Everberg, Belgium) as a dispersant was used to prepare a slurry. The slurry was spray-dried to prepare TCP microspheres (TCPms), and the collected microspheres were sintered at 500° C. for 10 hours, and then at 1350° C. for 2 hours, followed by sieving 50 from the sintered microspheres. Microspheres of 100 to 250 μm size were separated with% efficiency. In order to obtain a complex containing TCP at 40% by volume, the separated TCPms were melted at 180°C using a high-shear mixer, and a mass ratio of the molten PLA and TCPms:PLA=1.63:1 (mass ratio) ratio) was mixed homogeneously. As a comparative example, in order to prepare a PLA-TCPpw composite containing the same amount of TCPpw, TCPpw was mixed in the same ratio as the molten PLA under the same conditions. Cylindrical molds and a hand press machine (hand press machine, hydraulic unit model 3925, Carver, USA) were used to form a non-viscous or completely fluid complex at 170°C to prepare a cylindrical sample.

Experimental example 1: Characterization

TCPms and TCPpw were analyzed by scanning electron microscopy (SEM, JSM-6360, JEOL, Japan) and X-ray diffraction (XRD; D8-Advance, BRUKER, Germany) using monochromatic Cu Kα radiation I did. Samples were scanned over a diffraction angle ranging from 20 to 50° at a scan rate of 2°/min.

A cylindrical sample was cut with a low-speed saw to obtain a cross section of pure PLA and PLA-TCP composites, and the shape of the cross section was analyzed with energy-dispersive X-ray spectroscopy (EDS). Confirmed by combined SEM.

The contact angle of distilled water droplets on the surface was measured using a contact angle analyzer (Phoenix 300, Surface Electro Optics Co. Ltd, Korea) to confirm the surface hydrophilicity of pure PLA and PLA-TCP composites.

The SEM images and XRD patterns of TCPms and TCPpw are shown in FIG. 3. TCPms showed a distinct spherical shape with a size of 100 μm or more (FIG. 3(a)). TCPpw showed an irregular shape of about 2 μm in size, and the shape was completely different from TCPms (FIG. 3(b)). The appearance of the XRD characteristic peak indicated that the properties of TCP did not change by the production of microspheres using a high shear mixer (FIG. 3(c)).

A photograph of the hand press machine used for the production of the cylindrical specimen and the resulting pure PLA and PLA composite specimens are shown in FIGS. 4(a) and (b), respectively. Unlike pure PLA, which is yellowish and translucent, both complexes were opaque due to the incorporation of TCP. Furthermore, spherical microspheres were visually identified in the PLA-TCPms specimen, while the surface of the PLA-TCPpw specimen was uniform white.

The surface morphology of pure PLA, PLA-TCPms composite, and PLA-TCPpw composite is shown in FIG. 4(c). The surface of pure PLA was uniform and was a flawless surface where only grinding lines appeared. Conversely, in the case of the PLA-TCPms specimen, TCPms having various sizes uniformly distributed were observed in the PLA matrix. At this time, the distinct spheres observed indicate that the shear mixing process did not distort its structure. As shown in Figure 4(c), EDS mapping of Ca and P additionally confirmed the presence of TCPms. Although the surface morphology of PLA-TCPpw is uniform and flawless, and quite different from that of pure PLA, where several polishing lines appear, EDS mapping of Ca and P confirmed the presence of TCPpw.

Experimental example 2: In vitro biocompatibility (in vitro biocomparibility )

Cell adhesion, proliferation and differentiation tests were performed using the pre-osteoblast cell line MC3T3-E1 (ATCC, CRL-2593). For the cell adhesion test, pre-incubated cells were dispensed on the surface of pure PLA, PLA-40 vol% TCPms and PLA-40 vol% TCPpw specimens at a cell density of 3×10 ⁴ cells/mL, Incubated for one day in a wet incubator at 37° C. and air containing 5% CO ₂ . The attached cells were observed by SEM. For the cell proliferation and differentiation test, the precursor osteoblasts were dispensed on the surface of the specimen at a density of 0.25×10 ⁴ cells/mL. Cell proliferation on pure PLA and PLA complex was evaluated using a methoxyphenyl tetrazolium salt (MTS) proliferation assay 5 days after culture. After incubation, the specimen was washed twice with Dulbecco's phosphate-buffered saline (PBS). The washed specimen was immersed in a culturing medium (fetal bovine serum (FBS)-absent) mixed with a 10% cell proliferation assay kit solution (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA). . After incubation for 2 hours, the absorbance of the solution was measured at 490 nm using a microplate reader (Model 550; Biorad, USA). Cell differentiation was evaluated using an alkaline phosphatase (ALP) activity assay. Differentiation of cells on each specimen was confirmed by ALP assay after culturing for 10 days. For the ALP test, one day after dispensing, the culture medium was exchanged with a fresh medium containing β-glycerophosphate (10 mM) and ascorbic acid (50 mg/mL). ALP activity was measured by quantifying the level of p-nitrophenol (pNP) converted from p-nitrophenyl phosphate in the presence of ALP during the reaction. The pNP level was measured at a wavelength of 405 nm using a microplate reader.

As described above, in vitro biocompatibility assays including cell adhesion, proliferation and differentiation tests were performed using MC3T3-E1 cells. The pure PLA and PLA-TCPms and PLA-TCPpw complexes all exhibited excellent cellular responses after 1 day of culture, as confirmed by the number of attached cells shown in the SEM image of FIG. 5.

As observed in Fig. 5(a), cells on pure PLA were spherical instead of having a widespread filopodia. On the other hand, the presence of TCPms and TCPpw favored the spreading of cells with filopodia-like extensions, indicating that biocompatibility was improved (Figs. 5(b) and (c)). . In addition, MTS and ALP activity assays of pure PLA and PLA-TCPms and PLA-TCPpw complexes were performed. After 5 days of culture, PLA-TCPms and PLA-TCPpw showed significantly higher cell viability levels than pure PLA, as shown in FIG. 6(a).

The presence of bioactive TCPms and TCPpw provided a favorable environment for growth and proliferation of cells. In addition, the complex with these, after 10 days of culture, showed a significantly higher ALP activity level compared to pure PLA (Fig. 6 (b)). In vitro cell tests clearly showed that incorporation of bioactive TCPms improved the biocompatibility of pure PLA to a level equal to or higher than that of PLA-TCPpw.

In the art, in order to improve the hydrophilicity of the matrix and improve cell adhesion, there have been attempts to introduce a hydrophilic material into a relatively hydrophobic matrix. In order to confirm the effect of the TCP incorporated therein on the hydrophilicity of the composite, the water contact angle was measured on the pure PLA and PLA-TCPms and PLA-TCPpw composites, and the results are shown in FIG. 7.

Determination of the wettability of the material is essential for the interaction between the material and the cell in the early stages of contact. The water contact angle of pure PLA was about 80.8°, which was reduced to 65.5 and 67.6° when TCPms and TCPpw were mixed, respectively. Therefore, the incorporation of hydrophilic TCP significantly increased the hydrophilicity of the complex compared to pure PLA, and thus, as shown in FIGS. 5 and 6, it was possible to provide an environment favorable for adhesion and proliferation of cells during the initial stage of contact. Furthermore, the increased hydrophilicity was able to accelerate the decomposition rate of the complex relative to pure PLA by providing a more hydrophilic surface.

Experimental example 3: Test of decomposition properties in vitro

Accelerated degradation tests were performed to confirm the connectivity of the microspheres. Since PLA is stable even at a relatively low pH, a 3×3×3 mm ³ size specimen was immersed in a 1 M HCl solution at 37°C. After 30 days of decomposition, the cross-sectional surface shape of pure PLA, PLA-TCPms and PLA-TCPpw was confirmed using SEM (JSM-6360, JEOL, Japan). Furthermore, the degradation behavior, pore generation and pore connectivity were examined by micro-computed tomography with a 0.5 mm aluminum filter at 8.17 μm resolution, 130 kV voltage and 60 mA current. -computed tomography; μ-CT, Skyscan, 1172 micro-tomography system; Skyscan, Kontich, Belgium).

Although the degradation of TCP proceeded faster than that of PLA under physiological conditions, the degradation rate of the complexes was very slow. TCP was degraded by about 55% 24 weeks after implantation in vivo, and the molecular weight of PLA decreased to 23% at the same time point. Therefore, in order to evaluate the interconnectivity between microspheres, an accelerated degradation test of PLA-TCPms was performed in a 1 M HCl solution at 37° C. that mimics the temperature of a physiologically active condition, that is, body temperature. Considering that the solubility of the calcium phosphate-based material is greatly improved in an acidic environment, the test was performed in HCl. Large-scale bulk erosion of PLA occurred due to hydrolytic degradation, and the number of carboxylic acid chain ends, known to autocatalyze ester hydrolysis, decreases as the decomposition progresses. Increased. As known in the art, the pKa of lactic acid is 3.84, so the decomposition rate of the PLA solution is the lowest at pH 4. At pH>4, lactic acid appears predominantly in dissociated form, and at pH<4, lactic acid is present in the form of an acid bound to the end of the chain, accelerating the hydrolysis reaction through autocatalysis. Thus, despite the fact that PLA undergoes large-scale erosion, the rapid chain scission that occurs at low pH can be explained by the surface erosion behavior of PLA. On the other hand, the rate of decomposition of solid PLA was mostly independent of the pH of the medium. Therefore, in the present invention, since the used HCl medium accelerates only the decomposition of TCPms, the interconnectivity of the microspheres in the complex could be clearly observed. The decomposition behavior of pure PLA, PLA-TCPms and PLA-TCPpw after different decomposition periods were analyzed by μ-CT, and the results are shown in FIG. 8.

3D μ-CT images of the specimens disassembled for different times were obtained. Since PLA in the image could not be detected due to the different contrast modes between ceramic and polymer, the shape of pure PLA and PLA composite specimens before and after decomposition in FIG. 8(a) is shown by red dotted lines. Although no difference was observed in pure PLA until the 30th day, decomposition of TCPms and TCPpw in PLA-TCPms and PLA-TCPpw was clearly observed. The average size of the microspheres before digestion was about 106 μm. After 30 days of immersion, the average pore size of the PLA-TCPms complex was about 83 μm, and showed 97.52% interconnectivity to the decomposed portion of the complex. The pore size was smaller than the original size of the added microspheres due to the TCP remaining without completely dissolving during the immersion period. However, the size of the pores formed was still large enough to allow for the ingrowth of the cells. Since the resolution limit of μ-CT was only 7 μm, the average pore size of the PLA-TCPpw composite could not be calculated. However, as shown in FIG. 8(b), a distinct morphological difference appeared in the SEM image after 30 days of decomposition. The PLA-TCPms specimen contained round, interconnected pores formed by decomposition of TCPms, whereas the PLA-TCPpw specimen contained small pores of about 2-10 μm in size, consistent with the size of the powder and its aggregates. The small pore size of the PLA-TCPpw complex restricts the flow of fluid, and unlike the larger pores of the PLA-TCPms complex, it is not suitable for intracellular growth. The total amount of degraded TCP after 30 days was higher in the PLA-TCPms complex than in the PLA-TCPpw complex, as the larger interconnected pores formed by the microspheres promote fluid flow into the complex. The relative masses of PLA-TCPms and PLA-TCPpw remaining after 30 days of decomposition were about 50 and 60%, respectively. The successful formation of interconnected micropores in the PLA-TCPms composite is due to the size effect of the space holder, as increasing the size of the space holder in the manufacture of the porous scaffold increases the interlinkability and the decomposition rate of the space holder. Can be explained. In the present invention, TCPms and TCPpw can be regarded as space holders of different sizes. Unlike the two PLA composites, no morphological change was observed for pure PLA. Although the accelerated degradation test is suitable for confirming the interconnectivity of microspheres in the complex, it does not accurately represent the actual degradation behavior of pure PLA and PLA complexes under in vivo conditions. Therefore, by performing an additional in vitro disintegration test in a simulated body fluid (SBF) or PBS solution for a longer period of time, it was possible to observe the disintegration behavior of the complex under physiological conditions.

Experimental example 4: In vivo bone response evaluation

In order to confirm the potential of the PLA-TCPms complex for bone regeneration, an in vivo animal experiment using a rabbit femoral defect model was performed. For comparison, pure PLA and PLA-TCPpw were also tested. Specifically, a healthy New Zealand white rabbit (male, 10 weeks old, weight about 2.0 kg, KOSA Bio Inc., Seongnam, Korea) was used for the animal experiment. All rabbits contained 0.5 mL of 2% xylazine hydrochloride (xylazine HCl, Rompun, Bayer Korea, Korea), 1 mL of tiletamine HCl, Zoletil, Vicbac Laboratories, France, and lidocaine (Yuhan Corporation, Korea). A combination of was anesthetized by intramuscular injection.

For the in vivo experiment, a cylindrical specimen (5 mm diameter, 5 mm height) made of pure PLA, PLA-TCPms composite, and PLA-TCPpw composite was used. A hole was previously drilled in the intercondyle notch using a hand drill, and then two different types of specimens were implanted into the left and right femoral defects. The wound was sutured, and gentamicin (5 mg/kg) was injected subcutaneously into the experimental animals as an antibiotic prophylaxis post operation after the operation. In vivo animal experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee of Genoss (GEN-IACUC, no. 1703-03).

Rabbits were killed 16 weeks after transplantation. The femur including the graft was extracted and fixed in 10% buffered formaldehyde for later analysis. In order to study pure PLA, PLA complex and bone tissue, bone tissue was measured at 24.86 μm resolution, 130 kV voltage and 60 mA current, μ-CT with 0.5 mm filter (Skyscan, 1172 micro-tomography System; Skyscan, Kontich, Belgium). After scanning, the 2D model was reconstructed using NRecon software (Skyscan, Kontich, Belgium), and using the CTAn program (Skyscan, Kontich, Belgium), a specific threshold for image analysis ( specific threshold) to determine the bone volume around the implant.

For histological evaluation, the extracted bone tissue including the implanted specimen was fixed with a neutral 10% formaldehyde solution and embedded in a resin (resin, Technovit, 7200 VLC, Kulzer, Germany). A nondecalcified thin bottom surface was prepared and reduced in thickness to about 50 μm using a cutting and polishing system (Exakt, Germany). The tissue was stained with Goldner's trichrome stain, and a microscope image of the stained part using a panoramic digital slide scanner (Pannoramic 250 Flash III, 3DHISTECH Ltd, Hungary) and SEM (JSM-6360, JEOL, Japan) Got it. The mature bone matrix, immature new bone and calcified cartilage were stained green, red and light green, respectively. The bone-to-implant contact (BIC) ratio was calculated from histological images using the ImageJ program (National Institutes of Health, Maryland, USA).

In vivo experiments using a rabbit femoral injury model evaluated the initial stage bone response of pure PLA, PLA-TCPms and PLA-TCPpw. 16 weeks after transplantation, the cross-sectional shape and the amount of newly formed bone were confirmed by μ-CT analysis. 2D images of trans-axial sections along the central axis of pure PLA, PLA-TCPms and PLA-TCPpw are shown in Figs. 9(a) to (c), respectively, and the calculated amount of new bone around the implant is shown. It is shown in Fig. 9(d).

From the 2D μ-CT image, it was clearly confirmed that the amount of new bone around the graft was more in the two composite specimens than in the pure PLA sample. This was also supported by quantitative analysis of the bone volume of each specimen. New bone volumes of pure PLA, PLA-TCPms and PLA-TCPpw were 0.73, 5.21 and 5.98 mm ³ , respectively. The incorporation of TCP significantly improved the bone response of PLA by providing a substrate similar to that of actual bone, and as a result, stimulated the growth of osteoblasts, as confirmed in in vitro experiments.

Histological cross-sectional images of pure PLA, PLA-TCPms and PLA-TCPpw stained with Goldner's tricolor staining are shown in FIGS. 10(a) to (c), respectively. Successful new bone (stained dark green) formation was clearly observed around the implant. However, since the connective tissue is in direct contact with the pure PLA, the contact between the new bone and the implant is blocked, and thus implant loosening can be caused (FIG. 10(a)). On the other hand, in both the PLA-TCPms complex (FIG. 10(b)) and the PLA-TCPpw complex (FIG. 10(c)), the new bone tissue was in contact with the implant more directly. Incorporation on bioactive TCP has been shown to induce new bone formation on the graft. One major morphological difference was observed in the newly formed bones for the PLA-TCPms complex and the PLA-TCPpw complex. Consistent with the in vitro disintegration analysis, certain crevices ('pores') in which new bones can grow were formed in the edges of the PLA-TCPms specimen, and thus between the implant and the bone. Osteointegration was improved. Unlike the new bones being absorbed into the pores of the PLA-TCPms complex (integrated), the PLA-TCPpw complex, as shown in the red dotted line and the inserted SEM images in Figs. 10(b) and (c), of any decomposition It showed clear boundaries without signs. This was demonstrated by the quantitative BIC ratio results shown in Fig. 10(d). BIC ratios calculated from histological images were about 16, 60 and 32% for pure PLA, PLA-TCPms and PLA-TCPpw, respectively. The BIC ratio of PLA-TCPms was significantly higher compared to the values for pure PLA and PLA-TCPpw specimens, which is consistent with histological observations, that the contact length between the bone and the graft of the new bone grown in the pore. This indicates that the (contact length) has been significantly increased. Histological images and BIC ratio data successfully indicate that the incorporation of TCPms significantly improved osteosynthesis. Furthermore, it is shown that the formation of new bone tissue inside the complex leads to a result of improving the mechanical integrity between the bone and the implant.

The incorporation of TCPms and TCPpw successfully improved the cellular response of the PLA complex. In particular, PLA-TCPms provided the additional advantage of generating pathways for improved osteosynthesis compared to PLA-TCPpw. Although the accelerated degradation behavior does not accurately mimic the degradation behavior under physiological conditions, it clearly demonstrates the potential for successful conversion from TCPms to pores for improved osteosynthesis. This possibility was also confirmed by the results of in vivo experiments at an early stage. The newly formed bone inside the pores of the implant can help maintain the implant structure even after PLA erosion has begun. PLA-TCPms may be more useful than PLA-TCPpw, where the risk of large-scale erosion still exists due to its ability to form interconnected pores.

Experimental example 5: statistical analysis

All experiments were performed three or more times, and the experimental results were expressed as mean±standard deviation. The difference between each group (pure PLA, PLATCPms and PLA-TCPpw) was determined using a one-way analysis of variance with a p value of less than 0.05, considered to be a statistically significant difference (*p <0.05).

Comparative example 1: biodegradable metal Microsphere -Preparation of polymer complex

As biodegradable particles, a 4 mm×4 mm×6 mm scale Mg containing magnesium particles at 25% by volume in a similar manner to Example 1, except that magnesium particles were used instead of TCP, which is a kind of ceramic. -PLA complex was prepared. In order to confirm the decomposition characteristics of the prepared complex upon implantation in vivo, the prepared sample was immersed in a simulated body fluid (SBF), and after 3 and 5 days elapsed, the size and shape of the solution were observed with the naked eye. The pH was also observed and the measured results are shown in FIGS. 11 and 12, respectively. As shown in Figs. 11 and 12, magnesium in the complex decomposes rapidly within a few days and cannot maintain its shape, as well as excessively increases the surrounding pH due to the decomposition of magnesium, which adversely affects maintaining the pH balance in vivo. It was confirmed to be possible. This indicates that the polymer composite containing magnesium particles is not suitable for use as an implant for tissue regeneration.

The incorporation of TCPms into the PLA substrate improved biocompatibility to a level equivalent to that of the PLA complex containing TCPpw, a well-known complex used in orthopedic applications. The potential of PLA-TCPms to form pores with excellent interconnectivity for improved osteosynthesis was successfully confirmed through in vitro accelerated degradation tests and in vivo analysis using rabbit femoral defect models. Due to the excellent biocompatibility and improved osteo-adhesion ability of the PLA-TCPms complex, it may be a more promising candidate for bone tissue regeneration compared to the conventional PLA-TCP complex.

Claims (8)

As a method for producing a biodegradable implant in which the porosity increases with time, the biodegradable ceramic particles having an average diameter of 100 to 300 μm are uniformly inserted into the melt of the biodegradable polymer resin,
A first step of adding a biodegradable polymer to a high shear mixer and stirring at 3 Hz to 10 Hz while maintaining a temperature of the mixer at 10°C to 50°C higher than the melting point of the biodegradable polymer;
A second step of obtaining a mixture by adding a powder containing biodegradable ceramic particles having a higher decomposition rate than the biodegradable polymer to the high shear mixer and mixing while maintaining a temperature and speed; And
Including; a third step of molding the mixture,
The biodegradable polymer is selected from the group consisting of polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, poly-ε-caprolactone, and combinations thereof,
The biodegradable ceramic particles are tricalcium phosphate (TCP) microspheres, the method of manufacturing a biodegradable implant.
The method of claim 1,
The biodegradable polymer is polylactic acid, a method for producing a biodegradable implant.
The method of claim 1,
The biodegradable ceramic particles are added in an amount of 30 to 50% by volume based on the total amount of the mixture.
The method of claim 1,
A method of manufacturing a biodegradable implant, wherein the biodegradable ceramic particles dissolve at a faster rate than the biodegradable polymer over time during implantation into the body, and pores are formed.
In the melt of the biodegradable polymer resin, the average diameter is 100 to 300 μm, and the decomposition rate is faster than the biodegradable polymer. As an implant for bone regeneration comprising a biodegradable implant that increases porosity over time, formed by uniformly inserting biodegradable ceramic particles,
The biodegradable implant is a bone regeneration implant prepared by the method of any one of claims 1 to 4.
The method of claim 5,
Bone regeneration implants that maintain the skeleton over time, but form and expand pores.
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