CN101041088A - Method for producing compound frame of injection type polyester micro-carrier and fibrin gel - Google Patents
Method for producing compound frame of injection type polyester micro-carrier and fibrin gel Download PDFInfo
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- CN101041088A CN101041088A CN 200710068118 CN200710068118A CN101041088A CN 101041088 A CN101041088 A CN 101041088A CN 200710068118 CN200710068118 CN 200710068118 CN 200710068118 A CN200710068118 A CN 200710068118A CN 101041088 A CN101041088 A CN 101041088A
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- 229920000728 polyester Polymers 0.000 title claims abstract description 35
- 102000009123 Fibrin Human genes 0.000 title claims abstract description 23
- 108010073385 Fibrin Proteins 0.000 title claims abstract description 23
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 title claims abstract description 23
- 229950003499 fibrin Drugs 0.000 title claims abstract description 23
- 238000002347 injection Methods 0.000 title claims abstract description 14
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- 238000002360 preparation method Methods 0.000 claims abstract description 10
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Abstract
The invention discloses a method for preparing a composite support of injection polyester micro carrier and fibrin gel, wherein the composite support is formed by composed fibrin gel and polyester micro carrier. The micro carrier supply physical strength and space for cell growth and expansion, the fibrin gel simulates the cell epimatrix of cartilage cell, to support the transmission and original injection shaping of micro carrier, and accelerate the transfer, increment and differentiation of cartilage cell, and accelerate the healing of cartilage cell. The inventive composite support can effectively load micro carrier from free movement in body and loss in plant process, with better biological compatibility, simple preparation, wide resource, high producing efficiency and wide application.
Description
Technical field
The present invention relates to the preparation method of a kind of injection type polyester class microcarrier and fibrin gel compound rest.
Background technology
The cartilage injury is present common disease, because arthritis or articular cartilage damage that athletic injury caused bring misery for many patients.The metabolism of cartilage is active and repair ability is limited, does not have blood vessel, can not form the fiber grumeleuse after damage, does not have the inflammatory cell migration to enter, and does not also have the blood vessel undifferentiated cell to enter the position of damage, so difficultly repair voluntarily.In addition, this lacks undifferentiated cell in damage location cartilage, does not have the chondrocyte migration to grow among the cartilage of damage after the damage.And with advancing age, the splitting ability of chondrocyte reduces gradually, and the ability that produces extracellular matrix also decreases.Up to now, still lack effective method clinically and repair impaired cartilaginous tissue.Using the method for regenerative medicine and the reparation that principle is carried out cartilaginous tissue is a present important means, and has obtained good effect.Wherein, the repair of cartilage support plays crucial effect in regenerating bone or cartilage.
The cartilage of human body belongs to connective tissue, wherein contains a spot of chondrocyte and a large amount of intercellular substance.Chondrocyte is dispersed in the cartilage lacuna in cartilage matrix, and the substrate dyeing around the lacuna is called cartilage capsule more deeply.Inmature chondrocyte is less, and the normal single marginal zone that is distributed in articular cartilage is oblate.Increased gradually by the volume of edge to central chondrocyte, ovalize or circle have significantly cartilage capsule.Immature chondrocyte can divide, and normal 2～8 of deep mature chondrocytes distributes in groups.The chemical constituent of matter mainly comprises collagen protein, proteoglycan and a spot of noncollagen protein between chondrocyte.Collagen in the cartilaginous tissue is mainly the II Collagen Type VI, accounts for more than 90% of collagen total amount.The II type collagen fiber is three-dimensional netted distribution in cartilage, for articular cartilage provides tensile strength.Proteoglycan combines the huge proteoglycan aggregate of formation by connecting albumen with the non-covalent bond form with hyaluronic acid.Contain abundant negative acid ion on the proteoglycan aggregate strand, can form hydrogel in conjunction with a large amount of water, for cartilage provides comprcssive strength and elasticity.75% of water accounts cartilaginous tissue full weight wherein contains a large amount of cations with the negative charge in the balance proteoglycan molecule, and contains required nutrient substance of many chondrocytes and products of cellular metabolism.No blood vessel in the cartilaginous tissue, but because cartilage matrix is rich in moisture, nutrient substance is easy to infiltration, so chondrocyte still can obtain necessary nutrition.
Other noncollagen protein in the cartilaginous tissue such as anchor albumen, fiber adhesion albumen etc. by and cell surface receptor and other macromole between interaction chondrocyte is sticked on the cartilage matrix and the structure of stable cartilage matrix.
Different according to cartilage matrix composition and organizational structure, cartilage is divided into hyaline cartilage, fibrous cartilage and elastic cartilage.Articular cartilage belongs to hyaline cartilage, and its feature is that fiber content lacks than fibrous cartilage and elastic cartilage, and proteoglycan hydrogel content is more relatively, and cartilage matrix is a transparence.Sophisticated articular cartilage can be divided into shallow top layer, middle level, deep layer and calcification layer according to the variation of its cell and substrate.Wherein the proteoglycan content in middle level and the deep layer is higher.
The intravital cartilage of people has extremely special mechanical property.For example articular cartilage has good elasticity and toughness, can bear bigger load, has slick surface simultaneously, and the frictional force when making joint motion is minimum.Cartilaginous tissue is in a single day damaged, will bring huge misery and inconvenient to the patient.
In recent years, injectable type cell microcarrier and injection-type hydrogel material are because advantage such as its culturing in vivo and Wicresoft's reparation and extensively being paid close attention to.The injection-type cell microcarrier needs liquid carrier to transport.Traditional liquid carrier comprises glycerol, gelatin etc.Because it is base material that the injectable microcarrier generally adopts the Biodegradable polyester base polymer, density is bigger, so precipitation easily takes place and the influence use in injection process.Therefore, the performance of liquid carrier still need improve, and as should having enough viscosity or density guaranteeing the suspension of cell microcarrier in solution, and guarantees can not precipitate in the certain hour.In addition, even if simple cell microcarrier can inject and randomly in vivo pile up molding, but support loosely organized, mechanical strength is lower, microcarrier in vivo may migration or migration and lose shape.Hydrogel is the cytoskeleton that an other class can be injected, and the predecessor of hydrogel is solution state, and very easily injection in vivo can rapid shaping under physics or chemical stimulation.But the mechanical strength of hydrogel is lower, and degradation speed is very fast, causes fragmentation or caves in, thereby lose due shape and the growing space that can not continue to keep cell.
Injectable cell microcarrier and injection aquagel are carried out compound, utilize liquid hydrogel predecessor for transporting vector injection behind the interior damaged part of body, predecessor is fast setting formation gel in vivo.Gel and microcarrier form the compound rest with definite shape and intensity.Microcarrier is wrapped in the hydrogel, thereby has limited its migration or migration; Simultaneously, microcarrier and gel are compound, are equivalent to particle filled composite material, can improve the intensity of hydrogel greatly.In addition, the degradation speed of microcarrier is slower, and after hydrogel degraded fully, piling up of microcarrier still can be kept the space that cell is grown, and the exchange of cell desired nutritional and metabolite is convenient in the interconnected pore between ball and the ball.Therefore, the compound of the two can remedy its shortcoming mutually, has certain synergism.
Summary of the invention
The purpose of this invention is to provide a kind of microcarrier of load effectively, prevent microcarrier migration in vivo, prevent that simultaneously cell runs off in planting process, the injection-type microcarrier of good biocompatibility and the preparation method of fibrin gel compound rest.
The preparation method of injection type polyester class microcarrier of the present invention and fibrin gel compound rest may further comprise the steps:
1) polyester polymer is dissolved in the dichloromethane, is mixed with the homogeneous solution that concentration is 50mg/ml; This solution is joined in the aqueous solution that mass concentration is 0.1～0.5% polyvinyl alcohol, and the aqueous solution of polyvinyl alcohol and the volume ratio of dichloromethane are 5: 1, stir volatilization down and remove dichloromethane, filter to isolate the polyesters microsphere, drying;
2) to be immersed in mass concentration be in 6% the hexamethylene diamine normal propyl alcohol solution to the polyesters microsphere that step 1) is obtained, in 40 ℃ of water-baths, reacted 3～15 minutes, with the deionized water thorough washing to remove unreacted hexamethylene diamine, then at 35 ℃ of vacuum dryings to constant weight, obtain the surface and contain amino polyesters microsphere;
3) fibrinogen powder is dissolved in the normal saline, compound concentration is the fibrinogen solution of 10～40mg/ml; Thrombin is dissolved in the calcium chloride solution, and compound concentration is the thrombin calcium chloride solution of 20～40U/ml;
4) with step 2) surface that obtains contains amino polyesters microsphere to immerse mass concentration is in 1% glutaraldehyde solution, reacts 3h at least under 4 ℃, removes unreacted glutaraldehyde with deionized water rinsing, obtains the polyesters microsphere that aldehyde radical is contained on the surface; The polyesters microsphere that then this surface is contained aldehyde radical immerses in the fibrinogen solution of 10mg/ml, reacts 24h at least under 4 ℃, and is freezing-lyophilizing, obtains the surface and contains fibrinogenic polyesters microcarrier;
5) be 3%～15% to mix polyesters microcarrier and fibrinogen solution by mass ratio, concussion is dispersed in the fibrinogen solution polyesters microcarrier on agitator, to be dispersed with the fibrinogen solution and the thrombin solution equal-volume uniform mixing of polyesters microcarrier then, 37 ℃ form gel down, obtain injection type polyester class microcarrier and fibrin gel compound rest.
Among the present invention, said polyester polymer can be polylactic acid or polylactic-co-glycolic acid.
Beneficial effect of the present invention is:
Injection-type microcarrier of the present invention and fibrin gel compound rest have the composite construction feature, and wherein microcarrier can and increase for the cell growth certain mechanical strength and space is provided; Fibrin gel then helps realizing the conveying and the in-situ injection molding of microcarrier.Fibrin gel is the goods that derive from blood, has excellent biological compatibility and degradation property, wherein contains a large amount of active factorses, has migration, propagation and the differentiation performance of better promotion chondrocyte, accelerates to repair impaired cartilaginous tissue.This compound rest is the load microcarrier effectively, prevents microcarrier migration in vivo, prevents that cell runs off in planting process, good biocompatibility, and preparation method is simple, material source is extensive, production efficiency is high, has a good application prospect.
Description of drawings
Fig. 1 is the stereoscan photograph of polylactic acid-glycollic acid copolymer microspheres.
Fig. 2 is the stereoscan photograph that amino polylactic acid-glycollic acid copolymer microspheres is contained on the surface.
Fig. 3 is the scanning electron microscope sheet that fibrinogenic polylactic acid-glycollic acid copolymer microspheres is contained on the surface.
Fig. 4 is the laser confocal microscope photo that fibrinogenic polylactic acid-glycollic acid copolymer microspheres is contained on the surface.Fibrinogen is at first used the fluorescein isothiocyanate labelling.
Fig. 5 is the stereoscan photograph of the compound polylactic-co-glycolic acid microcarrier of fibrin gel support.
Fig. 6 is the laser confocal microscope photo of the compound polylactic-co-glycolic acid microcarrier of fibrin gel support.Fibrinogen is at first used the fluorescein isothiocyanate labelling.
Fig. 7 is an In vitro culture after one week, chondrocyte in the compound microcarrier support of fibrin gel form and the laser confocal microscope photo of distribution.Wherein cell adopts the method for fluorescein(e) diacetate to dye.
Specific implementation method
Further specify the present invention below in conjunction with example, but these examples are not used for limiting the present invention.
Example 1:
1) polylactic-co-glycolic acid is dissolved in the dichloromethane, is mixed with the homogeneous solution that concentration is 50mg/ml; It is that the aqueous solution of polyvinyl alcohol and the volume ratio of dichloromethane are 5: 1 in 0.1% the poly-vinyl alcohol solution that this solution is joined mass concentration.Dichloromethane was removed in volatilization in 8 hours under mechanical agitation; Filter to isolate polylactic acid-glycollic acid copolymer microspheres, drying;
2) to be immersed in mass concentration be in 6% the hexamethylene diamine normal propyl alcohol solution to the polylactic acid-glycollic acid copolymer microspheres that step 1) is obtained, reaction is 3 minutes in 40 ℃ of water-baths, with the deionized water thorough washing to remove unreacted hexamethylene diamine, in 35 ℃ of vacuum drying ovens, be dried to constant weight then, promptly obtain the surface and contain amino polylactic acid-glycollic acid copolymer microspheres.Fig. 2 is the stereoscan photograph that amino polylactic acid-glycollic acid copolymer microspheres is contained on the surface.
3) fibrinogen powder is dissolved in the normal saline, hatched 10 minutes in 37 ℃ of waters bath with thermostatic control, Fibrinogen is fully dissolved, the concentration of fibrinogen solution is 10mg/ml; Thrombin is dissolved in the 40mM calcium chloride solution, in 37 ℃ of waters bath with thermostatic control, hatched 10 minutes, be mixed with the solution of 20U/ml.
4) with step 2) surface that obtains contains amino polylactic acid-glycollic acid copolymer microspheres to immerse mass concentration is in 1% glutaraldehyde solution, 4 ℃ are reacted 3h down, remove unreacted glutaraldehyde with a large amount of deionized water rinsings, obtain the polylactic acid-glycollic acid copolymer microspheres that aldehyde radical is contained on the surface; The polylactic acid-glycollic acid microsphere that then this surface is contained aldehyde radical immerses in the fibrinogen solution of 10mg/ml, and 4 ℃ are reacted 24h down, every vibration in 2 hours once.After freezing-lyophilizing, obtain the surface and contain fibrinogenic polylactic acid-glycollic acid copolymer microspheres, i.e. polylactic-co-glycolic acid microcarrier is shown in the laser confocal microscope photo of the stereoscan photograph of Fig. 3 and Fig. 4.
5) be 3% to mix polylactic-co-glycolic acid microcarrier and fibrinogen solution by mass ratio, concussion is dispersed in the fibrinogen solution polyesters microcarrier on agitator, to be dispersed with the fibrinogen solution and the thrombin solution equal-volume mix homogeneously of polylactic-co-glycolic acid microcarrier then, putting into 37 ℃ of constant temperature ovens hatched 10 minutes, make Fibrinogen crosslinked, form gel, promptly obtain the compound rest of injection-type polylactic-co-glycolic acid microcarrier and fibrin gel.Fig. 5 and Fig. 6 are respectively the stereoscan photograph and the laser confocal microscope photos of the compound polylactic-co-glycolic acid microcarrier of fibrin gel support.
Example 2:
Step 1) with example 1 prepares polymer microballoon, but the polymer that uses is polylactic acid, and all the other steps obtain the compound rest of injection-type polylactic acid microcarrier and fibrin gel with example 1.
Example 3:
Step 1) with example 1 prepares polylactic acid-glycollic acid copolymer microspheres, but the mass concentration of poly-vinyl alcohol solution is 0.5%, and all the other steps are with example 1.
Example 4:
Step 1) is with the step 1) of example 1.
Step 2) with the step 2 of example 1), but the time of aminolysis be 15 minutes, surperficially contain amino polylactic acid-glycollic acid copolymer microspheres.All the other steps are with example 1.
Example 5:
Step 1)～3) with step 1)～3 of example 1), but cofabrication fibrinogen concentration is the solution of 40mg/ml in step 3).
Step 4) is with the step 4) of example 1, but the concentration of fibrinogen solution is 40mg/ml.All the other steps are with example 1.
Example 6:
Step 1)～3) with step 1)～3 of example 1), but cofabrication concentration of thrombin is the calcium chloride solution of 40U/ml in step 3).
Step 4) is with the step 4) of example 1, but the concentration of thrombin solution is 40U/ml.All the other steps are with example 1.
Example 7:
Step 1)～4) with step 1)～4 of example 1).
Step 5) is with the step 5) of example 1, but the mass ratio of polylactic-co-glycolic acid microcarrier and fibrinogen solution is 15%.
Example 8:
Step 1)～2) with step 1)～2 of example 1).
Step 3) is with example 1 step 3), but cofabrication fibrinogen concentration is the solution of 40mg/ml, and concentration of thrombin is the solution of 30U/ml.
Step 4) is with the step 4) of example 1.
Fibrinogen solution (40mg/ml) and thrombin (30U/ml) solution at first are the filter membrane filtration sterilization of 220nm with the aperture.To be the polylactic-co-glycolic acid microcarrier and the fibrinogen solution uniform mixing of example 1 preparation then, wherein the mass percentage concentration of polylactic-co-glycolic acid microcarrier is 15%, and then this mixture mixed with chondrocyte, put into 37 ℃ of incubators and hatched 10 minutes, obtain containing the compound microcarrier support of fibrin gel of chondrocyte.The ultimate density of chondrocyte is 3,000,000/ml.This compound rest that contains chondrocyte is put in 24 well culture plates, adds culture fluid, in incubator, carry out In vitro culture.Fig. 7 is an In vitro culture after 7 days, chondrocyte in the compound microcarrier support of fibrin gel form and the laser confocal microscope photo of distribution.
Claims (2)
1. the preparation method of injection type polyester class microcarrier and fibrin gel compound rest, its preparation process may further comprise the steps:
1) polyester polymer is dissolved in the dichloromethane, is mixed with the homogeneous solution that concentration is 50mg/ml; This solution is joined in the aqueous solution that mass concentration is 0.1～0.5% polyvinyl alcohol, and the aqueous solution of polyvinyl alcohol and the volume ratio of dichloromethane are 5: 1, stir volatilization down and remove dichloromethane, filter to isolate the polyesters microsphere, drying;
2) to be immersed in mass concentration be in 6% the hexamethylene diamine normal propyl alcohol solution to the polyesters microsphere that step 1) is obtained, in 40 ℃ of water-baths, reacted 3～15 minutes, with the deionized water thorough washing to remove unreacted hexamethylene diamine, then at 35 ℃ of vacuum dryings to constant weight, obtain the surface and contain amino polyesters microsphere;
3) fibrinogen powder is dissolved in the normal saline, compound concentration is the fibrinogen solution of 10～40mg/ml; Thrombin is dissolved in the calcium chloride solution, and compound concentration is the thrombin calcium chloride solution of 20～40U/ml;
4) with step 2) surface that obtains contains amino polyesters microsphere to immerse mass concentration is in 1% glutaraldehyde solution, reacts 3h at least under 4 ℃, removes unreacted glutaraldehyde with deionized water rinsing, obtains the polyesters microsphere that aldehyde radical is contained on the surface; The polyesters microsphere that then this surface is contained aldehyde radical immerses in the fibrinogen solution of 10mg/ml, reacts 24h at least under 4 ℃, and is freezing-lyophilizing, obtains the surface and contains fibrinogenic polyesters microcarrier;
5) be 3%～15% to mix polyesters microcarrier and fibrinogen solution by mass ratio, concussion is dispersed in the fibrinogen solution polyesters microcarrier on agitator, to be dispersed with the fibrinogen solution and the thrombin solution equal-volume uniform mixing of polyesters microcarrier then, 37 ℃ form gel down, obtain injection type polyester class microcarrier and fibrin gel compound rest.
2. by the preparation method of described a kind of injection type polyester class microcarrier of claim 1 and fibrin gel compound rest, it is characterized in that said polyester polymer is polylactic acid or polylactic-co-glycolic acid.
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