CN100431624C - Method for preparing injectable polyletic acid micro-carrier/chitosan hydrogel composite scaffold - Google Patents
Method for preparing injectable polyletic acid micro-carrier/chitosan hydrogel composite scaffold Download PDFInfo
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- CN100431624C CN100431624C CNB2005100618683A CN200510061868A CN100431624C CN 100431624 C CN100431624 C CN 100431624C CN B2005100618683 A CNB2005100618683 A CN B2005100618683A CN 200510061868 A CN200510061868 A CN 200510061868A CN 100431624 C CN100431624 C CN 100431624C
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- 238000000034 method Methods 0.000 title abstract description 7
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- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims abstract description 27
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- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 claims abstract description 21
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 8
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- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 12
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Abstract
The present invention discloses a method for preparing an injectable polyletic acid micro-carrier/a chitosan hydrogel composite scaffold. The methacrylic acid and the lactic acid are orderly grafted in a molecule chain of the chitosan by adopting a carbodiimide condensation method to obtain a water solubility chitosan derivative which can be polymerized. A polylactic acid microcarrier is mixed with the chitosan hydrogel prepolymer, the thickener konjak glucomannan is added simultaneously to increase the viscosity of prepolymer solution to ensure the suspension of the polylactic acid microcarrier, which is convenient for the operation of injection process. A redox initiating system is oxidized by adopting ammonium peroxodisulfate and tetramethylethylenediamine, causing the compound to form the composite scaffold of a cell micro-carrier/ the chitosan hydrogel in situ gelation under the body temperature. The present invention has simple manufacturing process, and the made composite scaffold not only solves problems that the injectable micro-carrier which is not easy to shape and to move, but also can increase the intensity of the hydrogel obviously, and the present invention has the synergy. The composite scaffold has no poision, biodegradable performance and biological compatibility, and the present invention can be used as an injectable scaffold in organization engineering.
Description
Technical field
The present invention relates to the preparation method of a kind of injectable polylactic acid microcarrier/aquagel compound rest, in particular for the syringeability support in the organizational project.
Technical background
The syringeability support is with cell and a kind of to have a material mobile, good biocompatibility compound, directly by injector to inject to the body defect.Material can form in position have certain mechanical strength, definite shape and the support that can exchange with body fluid, cell is grown in support and final formative tissue.Also material directly can be injected in the body, utilize the cell expansion growing multiplication formative tissue of injected material surrounding tissue.Owing to have characteristics such as invasive, culturing in vivo environment and low expense, the syringeability support has important clinical application value and bright development prospect.The syringeability support mainly is divided into syringeability hydrogel support and syringeability cell microcarrier.Syringeability hydrogel support mainly by sol-gel transition molding in vivo, have characteristics such as the good cell compatibility and easy-formation, but intensity is low, and degraded is fast.And the syringeability cell microcarrier need adopt liquid to pile up molding as transporting vector injection in vivo to damaged part, and is easy to operate, but is not easy molding, and deposits the problem of migration in vivo.At present, all pay attention to the raising of one pack system performance both at home and abroad basically, do not see both combinations to obtain optimized performance.
Summary of the invention
The objective of the invention is to provide the preparation method of a kind of injectable polylactic acid microcarrier/aquagel compound rest for organizational project.
The preparation method of injectable polylactic acid cell microcarrier of the present invention/aquagel compound rest may further comprise the steps:
1) at normal temperatures, chitosan (CS) is dissolved in the solution that contains methacrylic acid (MA), add water-soluble carbodiimide (EDAC) then, under room temperature, react, the mol ratio of water-soluble carbodiimide and methacrylic acid is 0.25～1.5, after reaction finishes, place the tri-distilled water dialysis to remove unreacting substance and by-product, lyophilizing obtains the chitosan (CM) of grafting methacrylic acid;
2) at normal temperatures, the chitosan of grafting methacrylic acid is dissolved in the solution that contains lactic acid (LA), add water-soluble carbodiimide then, under room temperature, react, the mol ratio of water-soluble carbodiimide and lactic acid is 0.3～1.8, after reaction finishes, place the tri-distilled water dialysis to remove unreacting substance and by-product, lyophilizing obtains the chitosan derivatives (CML) of grafting methacrylic acid and lactic acid;
3) at room temperature, the chitosan derivatives of grafting methacrylic acid and lactic acid is dissolved in water or the phosphate buffer (PBS), be mixed with concentration and be 1～2.5% chitosan derivative solution, add Rhizoma amorphophalli glucomannan (KGM) powder then, the concentration of Rhizoma amorphophalli glucomannan in chitosan derivative solution is 0.6～1%.After stirring, add 2.5～15mM initiator system Ammonium persulfate. (APS) and tetramethylethylenediamine (TMEDA), the mol ratio of Ammonium persulfate. and tetramethylethylenediamine is 1: 1, mix homogeneously;
4) the polylactic acid microcarrier is joined in the mixed liquor of step 3), wherein the w/v of polylactic acid microcarrier in mixed liquor is 2.5%～10%, behind the mix homogeneously, place 25 ℃～45 ℃ reaction formation polylactic acid microcarrier/aquagel compound rests down.
Above-mentioned polylactic acid microcarrier is the polylactic acid microsphere that the surface has collagen, can directly carry out compoundly, also can carry out compound behind the required cell of its surface load in advance (as chondrocyte, osteoblast or hepatocyte etc.).
The inventive method operating procedure is simple, implementation condition is gentle.Utilize the viscosity of full-bodied Rhizoma amorphophalli glucomannan as the chitosan solution of thickening agent raising grafting methacrylic acid and lactic acid, guarantee the suspension of polylactic acid microcarrier in solution, what be beneficial to inject carries out smoothly.With the gel prepolymer is that transport agent is expelled to desired area with microcarrier, and gelation forms the microcarrier/hydrogel compound rest with definite shape in position, hydrogel helps the molding of microcarrier, and with its parcel, can prevent its migration in vivo; Simultaneously microcarrier can effectively improve the intensity of hydrogel, and the two is compound to have certain cooperative effect.This compound rest is nontoxic, tool biological degradability and biocompatibility.The inventive method provides cytoskeleton of a kind of new construction and preparation method thereof, can be used in organizational project and the shaping reparation.
Description of drawings
Fig. 1 is the change curve of MA grafting amount under the mol ratio of different EDAC and MA;
Fig. 2 is the change curve of LA grafting amount under the mol ratio of different EDAC and LA;
Fig. 3 is under 37 ℃, the photo after the polylactic acid microcarrier suspended 15 minutes in KGM/CML solution; Wherein CML concentration is 1%, and labelling is KGM concentration among the figure;
Fig. 4 is under 37 ℃ and the 5mM APS/TMEDA, adds the gel time of the 1%CML of different K GM content;
Fig. 5 is the equilibrium swelling ratio of 1%CML gel 24h in water of the different K GM content for preparing under 37 ℃ and 5mM APS/TMEDA;
Fig. 6 is the dynamic modulus of elasticity of the 1%CML gel of the different K GM content for preparing under 37 ℃ and 5mM APS/TMEDA, and what mark among the figure is KGM content;
Fig. 7 be 37 ℃ with 5mM APS/TMEDA under the dynamic modulus of elasticity of 0.6%KGM/1%CML compound rest of the different polylactic acid microcarrier content that prepare, be labeled as the bulking value content of microcarrier among the figure;
Fig. 8 be 37 ℃ with 5mM APS/TMEDA under the dried internal structure of 0.6%KGM/1%CML compound rest of the different polylactic acid microcarrier content that prepare, figure is labeled as the bulking value content of microcarrier down, (a) being 2.5%, (b) is 5%, (c) is 10%;
Fig. 9 is the cytoactive of chondrocyte in 5% microcarrier/0.6%KGM/1%CML compound rest, and cell seeding density is 600 * 10
4/ ml;
Figure 10 adopts laser confocal microscope (CLSM) to observe the growth behavior of chondrocyte in 5% microcarrier/0.6%KGM/1%CML compound rest, and (a) is 1 day among the figure, (b) is 6 days, (c) is 12 days;
Figure 11 adopts scanning electron microscope (SEM) to observe the form of chondrocyte in 5% microcarrier/0.6%KGM/1%CML compound rest, among the figure (a), (b) is 1 day, (c), (d) is 6 days, (e), (f) is 12 days.
Figure 12 adopts SEM to observe the form of chondrocyte in 5% microcarrier/0.6%KGM/1%CML compound rest, and (a) is sphaerocyst among the figure, (b) is cell mass, (c) cellular layer for sprawling.
The specific embodiment
Example 1
Take by weighing chitosan (CS) 800mg and place the 250ml conical flask, add 100ml tri-distilled water and 420 μ lMA (0.48mmol), treat that CS dissolves fully after, add 930mg EDAC (0.48mmol), then at room temperature, stirring reaction 24h.For removing unreacted MA and other micromolecule products, inserting reaction mixture by molecular weight is 10, and in the 000Da bag filter, the 3d that dialyses under a large amount of tri-distilled waters and room temperature changes tri-distilled water every day 2～3 times.At last this liquid is freezed, lyophilizing obtains the grafted chitosan of MA (CM).The CM yield is all greater than 90%, and MA grafting amount is about 23%, sees Fig. 1; Can be in water swelling.Above-mentioned 400mgCM is dissolved in the 50ml tri-distilled water that contains 210 μ l LA (0.2mmol), and the dissolving back adds 460mgEDAC (0.24mmol) fully.After this mixed liquor at room temperature stirred 24h, inserting reaction mixture by molecular weight was 10, and in the 000Da bag filter, the 3d that dialyses under a large amount of tri-distilled waters and room temperature changes tri-distilled water every day 2～3 times.At last this liquid is freezed, lyophilizing obtains the chitosan (CML) of grafting MA and LA.The yield of CML is all greater than 90%, and LA grafting amount is about 52%, sees Fig. 2.With the 100mg percent grafting is that 23% methacrylic acid and percent grafting are that the chitosan derivatives (CML) of 52% lactic acid is dissolved in the water, is mixed with 10ml 1%CML solution, adds 60mg KGM powder then, mix homogeneously.Add 50 μ l 1M Ammonium persulfate .s (APS) and 50 μ l 1M and tetramethylethylenediamine (TMEDA) solution, mix homogeneously.Add 500mg polylactic acid microcarrier then, the w/v of microcarrier is 5% in mixed liquor, behind the mix homogeneously, is expelled in the mould with 1ml syringe (needleless).Place under 37 ℃, obtain microcarrier/hydrogel compound rest behind the 8min.The dynamic modulus of elasticity of this compound rest is 0.12～1.15MPa when frequency is 0.1～100rad/s, sees Fig. 7, and its internal structure of dry back is seen Fig. 8 b.
Example 2
Take by weighing chitosan 800mg and place the 250ml conical flask, add 100ml tri-distilled water and 420 μ l MA (0.48mmol), treat that CS dissolves fully after, add 232.5mg EDAC (0.12mmol), then at room temperature, stirring reaction 24h.For removing unreacted MA and other micromolecule products, inserting reaction mixture by molecular weight is 10, and in the 000Da bag filter, the 3d that dialyses under a large amount of tri-distilled waters and room temperature changes tri-distilled water every day 2～3 times.At last this liquid is freezed, lyophilizing obtains the grafted chitosan of MA (CM).The CM yield is all greater than 90%, and MA grafting amount is about 11.74%, sees Fig. 1.
Example 3
The MA grafting amount that example 1 is obtained is about 23% 400mg CM and is dissolved in the 50ml tri-distilled water that contains 210 μ l LA (0.2mmol), and the dissolving back adds 115mg EDAC (0.06mmol) fully.After this reaction mixture at room temperature stirred 24h, inserting by molecular weight was 10, and in the 000Da bag filter, the 3d that dialyses under a large amount of tri-distilled waters and room temperature changes tri-distilled water every day 2～3 times.At last this liquid is freezed, lyophilizing, obtaining product is MA and LA grafted chitosan (CML).The yield of CML is all greater than 90%, and LA grafting amount is about 43.2%, sees Fig. 2.
Example 4
The 100mg CML that example 1 is obtained is dissolved in the water, is mixed with 10ml 1%CML solution, adds 100mg KGM powder then, and mix homogeneously, KGM content are 1%.Add 250mg polylactic acid microcarrier then, place 37 ℃ of water-baths, visible polylactic acid microcarrier suspension helps the carrying out of injecting after 15 minutes in 1%KGM/1%CML solution.See Fig. 3.
Example 5
The 100mg CML that example 1 is obtained is dissolved in the water, is mixed with 10ml 1%CML solution, adds 60mg KGM powder then, mix homogeneously.Add 50 μ l 1M Ammonium persulfate .s (APS) and 50 μ l 1M tetramethylethylenediamine (TMEDA) solution, behind the mix homogeneously, directly place under 37 ℃.Its gel time is 5.4min, sees Fig. 4.The equilibrium swelling ratio is 65, sees Fig. 5.The dynamic modulus of elasticity is 1.2KPa～24.4KPa when frequency is 0.1～100rad/s, sees Fig. 6.
Example 6
The 100mg CML that example 1 is obtained is dissolved in the water, is mixed with 10ml 1%CML solution, adds 60mg KGM powder then, mix homogeneously.Add 50 μ l 1M Ammonium persulfate .s (APS) and 50 μ l 1M tetramethylethylenediamine (TMEDA) solution.Add 250mg polylactic acid microcarrier, its microcarrier content is 2.5%, behind the mix homogeneously, is expelled in the mould with 1ml syringe (needleless), places under 37 ℃, can get microcarrier/hydrogel compound rest behind the 8min.The dynamic modulus of elasticity of this compound rest is 13KPa～258KPa when frequency is 0.1～100rad/s, sees Fig. 7, and its internal structure of dry back is seen Fig. 8 a.
Example 7
The 100mg CML that example 1 is obtained is dissolved in the water, is mixed with 10ml 1%CML solution, adds 60mg KGM powder then, mix homogeneously.Add 50 μ l 1M Ammonium persulfate .s (APS) and 50 μ l 1M tetramethylethylenediamine (TMEDA) solution.Add 1g polylactic acid microcarrier, its microcarrier bulking value content is 10%, behind the mix homogeneously, is expelled in the mould with 1ml syringe (needleless), places under 37 ℃, can get microcarrier/hydrogel compound rest behind the 8min.The dynamic modulus of elasticity of this compound rest is 0.87～2.15MPa when frequency is 0.1～100rad/s, sees Fig. 7, and its internal structure of dry back is seen Fig. 8 c.
Example 8
CML, KGM that example 1 is obtained adopt the irradiation under ultraviolet ray sterilization; The polylactic acid microcarrier adopts 75% alcohol-pickled sterilization; After APS and TMEDA and phosphate buffer (PBS) were mixed with the solution of 1M, adopting the aperture was 0.22 μ m cellulose acetate membrane filter-sterilized.Chondrocyte is planted in 250mg polylactic acid microcarrier surface in advance, and it is standby that cell covers with the back after 7 days.50mg CML is dissolved in earlier among the 5ml PBS, be mixed with 1%CML/PBS solution after, add 30mg KGM powder, mix homogeneously.Add the PBS solution of initiator A PS/TMEDA, ultimate density is 5mM.Cell suspension with the 0.5ml high concentration joins in the gel prepolymer earlier, mix homogeneously, and cell seeding density is 600 * 10
4/ ml.The microcarrier that adds the load cell then, content are 5%.With the 1ml syringe this mixture is expelled in the mould, places under 37 ℃, behind the 8min, form compound rest.This compound rest is transferred in 24 well culture plates, added the culture medium contain 20% calf serum and cultivates, changed liquid in per 2～3 days.The active increase earlier of chondrocyte back is constant, sees Fig. 9.Cell has part death in gelation and mixed process, cell can be to the microcarrier surface migration, adhere to, sprawl and breed, and has cell mass to exist, and sees Figure 10 and Figure 11.Cell in the gel is circular, and has cell mass, and the cell on microcarrier surface is the cellular layer of sprawling, and sees Figure 12.
Claims (3)
1. the preparation method of injectable polylactic acid microcarrier/aquagel compound rest may further comprise the steps:
1) at normal temperatures, chitosan is dissolved in the solution that contains methacrylic acid, add water-soluble carbodiimide then, under room temperature, react, the mol ratio of water-soluble carbodiimide and methacrylic acid is 0.25～1.5, after reaction finishes, place the tri-distilled water dialysis to remove unreacting substance and by-product, lyophilizing obtains the chitosan of grafting methacrylic acid;
2) at normal temperatures, the chitosan of grafting methacrylic acid is dissolved in the solution that contains lactic acid, add water-soluble carbodiimide then, under room temperature, react, the mol ratio of water-soluble carbodiimide and lactic acid is 0.3～1.8, after reaction finishes, place the tri-distilled water dialysis to remove unreacting substance and by-product, lyophilizing obtains the chitosan derivatives of grafting methacrylic acid and lactic acid;
3) at room temperature, the chitosan derivatives of grafting methacrylic acid and lactic acid is dissolved in water or the phosphate buffer, be mixed with concentration and be 1～2.5% chitosan derivative solution, add the Rhizoma amorphophalli glucomannan powder then, the concentration of Rhizoma amorphophalli glucomannan in chitosan derivative solution is 0.6～1%, after stirring, adds 2.5～15mM initiator system Ammonium persulfate. and tetramethylethylenediamine, the mol ratio of Ammonium persulfate. and tetramethylethylenediamine is 1: 1, mix homogeneously;
4) the polylactic acid microcarrier is joined in the mixed liquor of step 3), wherein the w/v of polylactic acid microcarrier in mixed liquor is 2.5%～10%, behind the mix homogeneously, reacts formation polylactic acid microcarrier/aquagel compound rest down in 25 ℃～45 ℃.
2. the preparation method of injectable polylactic acid microcarrier according to claim 1/aquagel compound rest is characterized in that the polylactic acid microcarrier is the polylactic acid microsphere that the surface has collagen.
3. the preparation method of injectable polylactic acid microcarrier according to claim 2/aquagel compound rest is characterized in that at polylactic acid microcarrier area load cell.
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| CN100431623C (en) * | 2006-10-23 | 2008-11-12 | 天津大学 | Method for preparing 3D porous bracket of chitosan - copolymer of poly lactic acid |
| CN101225123B (en) * | 2007-12-07 | 2010-10-13 | 北京化工大学 | Water-soluble chitosan derivatives as well as preparation method and uses thereof |
| CN101322858B (en) * | 2008-07-14 | 2013-06-12 | 昆明理工大学 | Degradable stephanoporate stent material for inducting osseous tissue regeneration and repair and preparation thereof |
| CN101791432B (en) * | 2010-03-19 | 2013-04-03 | 浙江大学 | Method for preparing galactose chitosan/polyester polymer composite stent |
| CN103958666B (en) * | 2011-11-29 | 2020-01-10 | 加利福尼亚大学董事会 | Glucomannan scaffold for three-dimensional tissue culture and engineering |
| CN110478529A (en) * | 2019-09-24 | 2019-11-22 | 广西医科大学 | A kind of 3D printing konjac glucomannan hydrogel scaffold and preparation method and application |
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