CN103212116A - Method for constructing three-dimensional scaffold by polymorphous low-grade adenocarcinoma (PLGA)/calcium carbonate porous composite microsphere - Google Patents

Abstract

The invention discloses a method for nondestructively constructing a three-dimensional scaffold by using PLGA/calcium carbonate porous composite microspheres, comprising the following steps: a. Adding PLGA and calcium carbonate to the organic solvent of dichloromethane, after stirring and ultrasonic treatment, a uniform mixed oil phase of PLGA/calcium carbonate is obtained; b. Disperse the above oil phase into PVA aqueous solution under stirring conditions to obtain a single oil-in-water emulsion; c. Stir the emulsion for a certain period of time to volatilize the dichloromethane in the oil phase droplets to obtain solidified composite microspheres; d. Collect the resulting composite microspheres and wash with deionized water; e. Filling the washed microspheres into the mold, drying at room temperature and low pressure for a certain period of time to obtain the microsphere scaffold; f. Remove the mold and continue drying the brackets to completion. The invention bonds the porous microspheres into a scaffold under mild conditions, and the porous structure on the surface of the microspheres is completely preserved.

Description

A method for constructing three-dimensional scaffolds from PLGA/calcium carbonate porous composite microspheres

technical field

The invention relates to a preparation method of a three-dimensional support, in particular to a method for constructing a three-dimensional support from PLGA/calcium carbonate porous composite microspheres.

Background technique

Bone defect is one of the most common clinical diseases at present, and the repair ability of bone itself is limited. For the treatment of more serious bone defects, bone grafting is needed to assist repair. Autologous bone grafting is the ideal repair method, but its source is limited; allogeneic bone and xenograft bone grafts have risks of disease transmission and immune rejection. Against this background, scaffold-based bone repair has received increasing attention. A scaffold is a three-dimensional structure that provides space for tissue ingrowth and blood vessel regeneration, and can also serve as a carrier for drug and cell delivery. Polymer microspheres are an excellent drug carrier, capable of loading and controlling the release of various drugs. The scaffold constructed of polymer microspheres theoretically has a 100% connected pore structure, and its porosity can also meet the needs of bone repair; the scaffold composed of drug-loaded microspheres can also release targeted therapeutic drugs, which can further Enhance the bone repair ability of the scaffold. Therefore, the microsphere scaffold has a good application prospect in bone repair.

Polylactic glycolic acid (PLGA) is a material with good biocompatibility and biodegradability, and it is one of the few polymer biomaterials approved by the FDA. The research on the bone repair application of PLGA microspheres and microsphere scaffolds has always attracted people's attention. Y Wang et al. (Y Wang, X Shi, L Ren, Y Yao, F Zhang, D-A Wang J Biomed Mater ResPart B201093B84-92) prepared PLGA/titanium composite microsphere scaffolds, on which osteoblasts can grow well and metabolism. A Jaklenec et al. (A Jaklenec, A Hinckfuss, B Bilgen, D Ciombor, RAaron, E Mathiowitz Biomaterials2008291518-1525) prepared a PLGA scaffold capable of sequentially releasing two growth factors.

Currently, there are two main methods for preparing PLGA microsphere scaffolds: temperature treatment and solvent treatment. The temperature treatment is to heat the microspheres to a certain temperature, so that the polymer molecular chains of the microspheres enter the high elastic state from the glass state, the molecular chains have fluidity, and the adjacent microspheres will generate molecular chain entanglement. When the temperature is lowered, the polymer chains re-enter the glassy state, forming a permanent bond between the microspheres. Solvent treatment is to expose the microspheres to a volatile solvent, so that the polymer in the surface area of the microspheres will dissolve to a certain extent, and the surfaces of adjacent microspheres will fuse with each other. When the solvent evaporates, the surface of the microspheres recurs to form a permanent bond between the microspheres. Wei Kun et al. (CN201110243418.1) disclosed a method of using temperature treatment to construct PLGA/nano-calcium carbonate microsphere scaffold; T Jiang (T Jiang, W Abdel-Fatah, C Laurencin Biomaterials2006274894-4903) etc. prepared PLGA/chitosan composite microsphere scaffold. Zhao Liang (CN201110215643.4) disclosed a PLGA drug-loaded microsphere scaffold prepared by volatile absolute ethanol; J Brown et al. (J Brown, L Nair, C Laurencin J Biomed Mater Res Part B200886B396-406) used Mixed solvents were used to prepare PLGA microsphere scaffolds. However, both temperature and solvent treatment will inevitably destroy the microstructure of the microsphere surface, making it impossible to obtain a microsphere scaffold with a finer and more controllable structure. Although people try to use other methods to construct microsphere scaffolds, for example, Gao Changyou et al. (CN201210121194.1) disclosed a method for preparing PLGA microsphere scaffolds by freezing and desolvating, but this method is difficult to construct porous morphology on the surface of the scaffold . It can be seen that how to prepare microsphere scaffolds with fine structures still needs to be further explored.

Contents of the invention

In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the object of the present invention is to provide a non-destructive preparation method for constructing a three-dimensional scaffold by PLGA/calcium carbonate porous composite microspheres, avoiding temperature and solvent treatment of the microspheres, thereby enabling complete retention Microsphere surface morphology.

The object of the present invention is achieved through the following technical solutions:

A method for constructing a three-dimensional support by PLGA/calcium carbonate porous composite microspheres, comprising the following steps:

a. PLGA and calcium carbonate are added to the methylene chloride organic solvent, and after stirring and ultrasonic treatment, a uniform mixed oil phase of PLGA/calcium carbonate is obtained;

b. Dispersing the oil phase obtained in step a into the PVA water phase under stirring conditions to obtain an oil-in-water single emulsion;

c. Stir the single emulsion at a rate of 200-400rpm for 12-24 hours to volatilize the dichloromethane in the oil phase droplets to obtain solidified composite microspheres;

d. The resulting composite microspheres were collected and fully washed with deionized water;

e. Filling the washed microspheres into a mold, drying at a temperature of 20-25°C and a pressure of 0.8-1MPa for 2-4 hours to obtain an initial scaffold;

f. Remove the mold and continue to dry until complete to obtain a three-dimensional scaffold composed of PLGA/calcium carbonate porous composite microspheres.

In step a, the mass volume ratio of PLGA to methylene chloride is (1/10-1/5) g/ml; the mass ratio of calcium carbonate to PLGA is 1/20-1/2.

The stirring treatment in step a is specifically: the stirring speed is 250-400 rpm, and the stirring time is 5-15 min.

The ultrasonic treatment described in step a specifically includes: the ultrasonic power is 250-350w, and the ultrasonic time is 5-15 minutes.

The molecular weight _Mw of the PLGA is 50-100kDa, wherein LA/GA=75/25.

The concentration of PVA in the PVA aqueous phase in step b is (1/500-1/100) g/ml.

In step b, the volume ratio of the oil phase to the PVA water phase is 1/100˜1/50.

In step c, the stirring rate is 200-400 rpm, and the stirring time is 12-24 hours.

In step f, the specific time for continuing to dry to complete is 36-72 hours.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

In the present invention, the washed wet microspheres are used as three-dimensional scaffold construction units. Since the microspheres still have a certain bonding performance at room temperature, there is no need to perform traditional temperature or solvent treatment on the microspheres, thereby avoiding damage to the surface of the microspheres in the preparation step of the scaffold. The shape is destroyed. Because the present invention completely retains the surface morphology of the microspheres, the microsphere bracket can maintain a finer structure. By preparing microspheres with different shapes, scaffolds with different shapes can also be obtained.

Description of drawings

Fig. 1 is the surface morphology diagram of the composite microsphere prepared in Example 1.

FIG. 2 is a surface topography diagram of the three-dimensional scaffold prepared in Example 1.

3 is a surface topography diagram of the three-dimensional scaffold prepared in Example 2.

FIG. 4 is a surface topography diagram of the three-dimensional scaffold prepared in Example 3.

FIG. 5 is a surface topography diagram of the three-dimensional scaffold prepared in Example 4.

Fig. 6 is a surface topography diagram of the three-dimensional scaffold prepared in Example 5.

Detailed ways

The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto.

Example 1

Weigh 0.5g PLGA (LA/GA=75/25, Mw=100kDa) and 0.2g calcium carbonate powder into 5ml dichloromethane, stir at 400rpm for 5min, then stir at 300rpm for 10min under 300w ultrasonic power , to obtain a uniform mixed oil phase of PLGA/calcium carbonate. Weigh 2g of PVA and add it to 250ml of deionized water, stir for 15min, heat to 90°C to dissolve the PVA, and obtain the PVA water phase after cooling. The oil phase was added dropwise into the PVA aqueous solution under the stirring condition of 400 rpm to obtain an oil-in-water single emulsion, wherein the volume ratio of the oil phase to the water phase was 1/50. The single emulsion was continuously stirred for 24 hours to obtain PLGA/calcium carbonate porous composite microspheres. The solidified composite microspheres were collected and washed 3 times with deionized water. Then the microspheres were filled into a polytetrafluoroethylene mold, and dried at a temperature of 20° C. and a pressure of 0.8 MPa for 4 hours to obtain an initial scaffold. Remove the mold and continue to dry for 72 hours to obtain a three-dimensional scaffold composed of PLGA/calcium carbonate porous composite microspheres.

Fig. 1 is the surface topography diagram of the PLGA/calcium carbonate porous composite microsphere prepared in this example.

Fig. 2 is the surface topography diagram of the three-dimensional scaffold composed of PLGA/calcium carbonate porous composite microspheres prepared in this example. It can be seen from the figure that the porous structure on the surface of the scaffold remains intact and is not affected by the preparation process of the scaffold.

Example 2

Weigh 0.5g PLGA (LA/GA=75/25, Mw=100kDa) and 0.025g calcium carbonate powder into 5ml dichloromethane, stir at 250rpm for 15min, then stir at 250rpm for 15min under 250w ultrasonic power , to obtain a uniform mixed oil phase of PLGA/calcium carbonate. Weigh 1g of PVA and add it to 500ml of deionized water, stir for 15min, heat to 90°C to dissolve the PVA, and obtain the PVA water phase after cooling. The oil phase was added dropwise into the PVA aqueous solution under the stirring condition of 200 rpm to obtain an oil-in-water single emulsion, wherein the volume ratio of the oil phase to the water phase was 1/100. The single emulsion was continuously stirred for 24 hours to obtain PLGA/calcium carbonate porous composite microspheres. The solidified composite microspheres were collected and washed 3 times with deionized water. Then the microspheres were filled into a polytetrafluoroethylene mold, and dried at a temperature of 25° C. and a pressure of 1 MPa for 2 hours to obtain an initial scaffold. Remove the mold and continue to dry for 36 hours to obtain a three-dimensional scaffold composed of PLGA/calcium carbonate porous composite microspheres.

Fig. 3 is the surface topography diagram of the three-dimensional scaffold composed of PLGA/calcium carbonate porous composite microspheres prepared in this example. Similarly, the surface morphology of the stent was not damaged.

Example 3

Weigh 1g of PLGA (LA/GA=75/25, Mw=50kDa) and 0.05g of calcium carbonate powder into 5ml of dichloromethane, stir at 300rpm for 10min, then stir at 250rpm for 5min under 350w ultrasonic power, A homogeneous mixed oil phase of PLGA/calcium carbonate was obtained. Weigh 5g of PVA and add it to 500ml of deionized water, stir for 5min, heat to 90°C to dissolve the PVA, and obtain the PVA water phase after cooling. The oil phase was added dropwise into the PVA aqueous solution under the stirring condition of 250 rpm to obtain an oil-in-water single emulsion, wherein the volume ratio of the oil phase to the water phase was 1/100. The single emulsion was continuously stirred for 20 hours to obtain PLGA/calcium carbonate porous composite microspheres. The solidified composite microspheres were collected and washed 3 times with deionized water. Then the microspheres were filled into a polytetrafluoroethylene mold, and dried at a temperature of 23° C. and a pressure of 1 MPa for 4 hours to obtain an initial scaffold. Remove the mold and continue to dry for 36 hours to obtain a three-dimensional scaffold composed of PLGA/calcium carbonate porous composite microspheres.

Fig. 4 is a surface topography diagram of a three-dimensional scaffold composed of PLGA/calcium carbonate porous composite microspheres prepared in this example. Similarly, the surface morphology of the stent was not damaged.

Example 4

This embodiment prepares the process of the single emulsion of oil-in-water with embodiment 1, continues to carry out following steps:

The single emulsion was continuously stirred (rotating at 250 rpm) for 24 hours to obtain PLGA/calcium carbonate porous composite microspheres. The solidified composite microspheres were collected, washed three times with deionized water, and freeze-dried at -20°C for 48 hours. The dried microspheres were taken, filled into a polytetrafluoroethylene mold, heated at 70° C. for 1 hour, and then cooled and demolded to obtain a microsphere scaffold.

Fig. 5 is a surface topography diagram of the microsphere scaffold of this embodiment. It can be seen from the figure that the pore structure on the surface of the microsphere basically disappears, leaving only sporadic pores.

Example 5

This embodiment prepares the process of the single emulsion of oil-in-water with embodiment 1, continues to carry out following steps:

The single emulsion was continuously stirred (rotating at 250 rpm) for 24 hours to obtain PLGA/calcium carbonate porous composite microspheres. The solidified composite microspheres were collected, washed three times with deionized water, and freeze-dried at -20°C for 48 hours. Take the dried microspheres, fill them into polytetrafluoroethylene molds, and place them in an airtight container containing dichloromethane for 2 minutes. Take out the mold and place it under low pressure for 24h to remove residual solvent. Release the mold to obtain the microsphere scaffold.

Fig. 6 is a surface topography diagram of the microsphere scaffold of this embodiment. It can be seen from the figure that the pore structure on the surface of the microspheres was severely damaged.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the embodiment, and any other changes, modifications, substitutions and combinations made without departing from the spirit and principle of the present invention , simplification, all should be equivalent replacement methods, and are all included in the protection scope of the present invention.

Claims (9)

1. A method for building a three-dimensional support by PLGA/calcium carbonate porous composite microspheres, is characterized in that, comprises the following steps: a. PLGA and calcium carbonate are added to the methylene chloride organic solvent, and after stirring and ultrasonic treatment, a uniform mixed oil phase of PLGA/calcium carbonate is obtained; b. Dispersing the oil phase obtained in step a into the PVA water phase under stirring conditions to obtain an oil-in-water single emulsion; c. Stir the single emulsion at a rate of 200-400rpm for 12-24 hours to volatilize the dichloromethane in the oil phase droplets to obtain solidified composite microspheres; d. The resulting composite microspheres were collected and fully washed with deionized water; e. Filling the washed microspheres into a mold, drying at a temperature of 20-25°C and a pressure of 0.8-1MPa for 2-4 hours to obtain an initial scaffold; f. Remove the mold and continue to dry until complete to obtain a three-dimensional scaffold composed of PLGA/calcium carbonate porous composite microspheres. 2. The method for constructing a three-dimensional scaffold by PLGA/calcium carbonate porous composite microspheres according to claim 1, wherein the mass volume ratio of PLGA to methylene chloride in step a is (1/10～1/5) g/ml; the mass ratio of calcium carbonate to PLGA is 1/20～1/2. 3. the method for building three-dimensional support by PLGA/calcium carbonate porous composite microsphere according to claim 1, it is characterized in that, described in step a stirring process, be specifically: stirring speed 250～400rpm, stirring time is 5～ 15min. 4. The method for constructing a three-dimensional scaffold by PLGA/calcium carbonate porous composite microspheres according to claim 1, characterized in that the ultrasonic treatment described in step a is specifically: ultrasonic power is 250～350w, ultrasonic time is 5 ~15min. 5. The method for constructing a three-dimensional scaffold from PLGA/calcium carbonate porous composite microspheres according to claim 1, wherein the molecular weight of the PLGA is _Mw =50-100kDa, wherein LA/GA=75/25. 6. The method for constructing a three-dimensional scaffold by PLGA/calcium carbonate porous composite microspheres according to claim 1, wherein the concentration of PVA in the PVA water phase in step b is (1/500-1/100) g/ ml. 7. The method for constructing a three-dimensional scaffold by PLGA/calcium carbonate porous composite microspheres according to claim 1, characterized in that, in step b, the volume ratio of the oil phase to the PVA water phase is 1/100 to 1/50. 8. The method for constructing a three-dimensional support by PLGA/calcium carbonate porous composite microspheres according to claim 1, characterized in that, the single emulsion is stirred as described in step c, and the specific conditions are: the speed of stirring is 200～400rpm, stirring The time is 12-24 hours. 9. The method for constructing a three-dimensional scaffold by PLGA/calcium carbonate porous composite microspheres according to claim 1, characterized in that the specific time for continuing to dry to complete in step f is 36 to 72 hours.

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