CN101053679B - Method for preparing polymer multiporous holder filled with fiber protein gel - Google Patents
Method for preparing polymer multiporous holder filled with fiber protein gel Download PDFInfo
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- CN101053679B CN101053679B CN200710068119A CN200710068119A CN101053679B CN 101053679 B CN101053679 B CN 101053679B CN 200710068119 A CN200710068119 A CN 200710068119A CN 200710068119 A CN200710068119 A CN 200710068119A CN 101053679 B CN101053679 B CN 101053679B
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Abstract
The invention discloses a preparation method of polymer multihole bracket filled with fibrin gel. The process is dissolving fibrinogen powder in physiological saline to prepare fibrinogen solution; dissolving thrombin in calcium chloride solution to prepare thrombin calcium chloride solution; injecting isovolumetric fibrinogen solution and thrombin solution into polymer multihole bracket to get polymer multihole bracket filled with fibrin gel. The polymer multihole bracket filled with fibrin gel of this invention have composite structural feature, wherein polymer multihole bracket provide a certain mechanical strength and space for chondrocyte growth, fibrin gel boosts migration, breeding and differentiation of chondrocyte, accelerates repair of chondrocyte, has very strong application value of repairing organise defect. The preparation method of this invention is simple and has wide material source, high production efficiency.
Description
Technical field
The present invention relates to a kind of preparation method of tissue regeneration support, especially the preparation method of polymer multihole bracket filled with fibrin gel.
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 normal cartilage, surround by a large amount of collagens and proteoglycan extracellular matrix that form, high moisture high-crosslinking-degree around the mature chondrocytes, chondrocyte is spherical in shape.Yet when chondrocyte was cultured on the porous polymer scaffold, chondrocyte was pancake and sticks on the support hole wall.The unusual growing environment that studies show that in the past is disadvantageous for the normal differentiation and the phenotype of chondrocyte.On the other hand, chondrocyte can be kept normal sphere in hydrogel material, keeps normal chondrocyte phenotype simultaneously.But the mechanical strength of hydrogel is generally not high, in cultivation and the implant surgery, is very easy to because of stressed and damaged or cracked in vivo and in vitro.Therefore, in the design of repair of cartilage material, should simulate the spontaneous growth environment of chondrocyte, structure has the recovery support that can promote chondrocyte normal growth and functional expression.
Summary of the invention
The purpose of this invention is to provide a kind of preparation method that can simulate the polymer multihole bracket filled with fibrin gel of human body natural cartilage.
The preparation method of polymer multihole bracket filled with fibrin gel of the present invention may further comprise the steps:
1) 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 1～20U/ml;
2) porous polymer scaffold is immersed in the ethanol, adopts first evacuation, the method for pressure is recovered in the back, and ethanol is imported in the porous polymer scaffold; To import alcoholic acid support and be immersed in the tri-distilled water, and carry out first evacuation repeatedly, the method for pressure is recovered in the back, and ethanol is replaced as water, and is residual until no ethanol;
3) solution with isopyknic fibrinogen solution and thrombin is injected in the porous polymer scaffold simultaneously, to prop up to be placed in 37 ℃ of constant temperature ovens and hatch 5～10 minutes, make the complete gel of Fibrinogen, promptly obtain polymer multihole bracket filled with fibrin gel.
Among the present invention, said porous polymer scaffold can be polylactic acid, polylactic acid one ethanol copolymer, polyurethane or collagen porous support.
Beneficial effect of the present invention is:
The polymer multihole bracket filled with fibrin gel of the present invention's preparation has the composite construction feature, wherein porous polymer scaffold can provide the certain mechanical strength and the space of chondrocyte growth, and fibrin gel is the goods that derive from blood, have excellent biological compatibility and degradation property, wherein contain a large amount of active factorses, have migration, propagation and the differentiation performance of better promotion chondrocyte, quicken the reparation of cartilaginous tissue.Therefore, this compound system has the damaged using value of very strong repair tissue.Preparation method of the present invention is simple, material source is extensive, production efficiency is high.
Description of drawings
Fig. 1 is the laser confocal microscope photo of fibrin gel filling polylactic acid porous support, wherein fibrin gel fluorescent staining;
Fig. 2 is the fibrinogenic gelation curve of variable concentrations;
Fig. 3 is the filling rate of the Fibrinogen filling polylactic acid support of variable concentrations;
Fig. 4 is the mechanical property of pure polylactic acid porous scaffold and fibrin gel filling bracket;
Fig. 5 is the activity of chondrocyte in the fibrin gel filling bracket;
Fig. 6 is the secretion of the GAG of chondrocyte in fibrin gel filling polylactic acid support;
Fig. 7 is distribution and the form (week) of laser confocal microscope observation chondrocyte in fibrin gel filling polylactic acid support, and wherein cell adopts the method for fluorescein(e) diacetate to dye;
Fig. 8 is distribution and the form (week) of scanning electron microscope observation chondrocyte in fibrin gel filling polylactic acid support.
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) 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, puts into 37 ℃ of waters bath with thermostatic control and hatched 10 minutes, be mixed with the solution of 1U/ml;
2) polylactic acid porous scaffold is immersed in the ethanol, adopts first evacuation, the method for pressure is recovered in the back, and ethanol is imported in the porous polymer scaffold, fully is immersed in the ethanol up to support; To import alcoholic acid support and be immersed in the tri-distilled water, and adopt first evacuation, the method for pressure is recovered in the back, and ethanol is replaced as water, replaces repeatedly 10 times, and is each 10 minutes, residual to there not being ethanol;
3) isopyknic fibrinogen solution and the thrombin solution with the step 1) preparation is injected in the polylactic acid porous scaffold simultaneously, to prop up to be placed in 37 ℃ of constant temperature ovens and hatch 10 minutes, make the complete gel of Fibrinogen, promptly obtain the polylactic acid porous scaffold that fibrin gel is filled.Fig. 1 is seen in the distribution of gel in polylactic acid bracket.
Example 2:
1) prepare Fibrinogen and thrombin solution with the method for example 1 step 1), but the concentration of fibrinogen solution is 40mg/ml.
Step 2) and 3) with the step 2 of example 1) and 3), the fibrin gel polylactic acid porous scaffold of filling.
Example 3:
1) prepares Fibrinogen and thrombin solution with the method for example 1 step 1).But the concentration of thrombin solution is 20U/ml.
Step 2) and 3) with the step 2 of example 1) and 3), the fibrin gel polylactic acid porous scaffold of filling.
Example 4:
Step 1) is with example 1 step 1).
Step 2) with the step 2 of example 1), but what adopt is polylactic acid one ethanol copolymer porous support.
Step 3) gets the polylactic acid porous scaffold that fibrin gel is filled with example 1 step 3).
Example 5:
Step 1) is with example 1 step 1).
Step 2) with the step 2 of example 1), but what adopt is the polyurethane porous support.
Step 3) gets the polylactic acid porous scaffold that fibrin gel is filled with example 1 step 3).
Example 6:
Step 1) is with example 1 step 1).
Step 2) with the step 2 of example 1), but what adopt is the collagen porous support.
Step 3) gets the polylactic acid porous scaffold that fibrin gel is filled with example 1 step 3).
Example 7:
Step 1) and 2) with example 1 step 1) and 2).
Step 3) is with example 1 step 3), but incubation time is 5 minutes, gets the polylactic acid porous scaffold that fibrin gel is filled.
Example 8:
1) with the method preparation Fibrinogen and the thrombin solution of example 1 step 1), fibrinogenic concentration is respectively 10mg/ml, 20mg/l and 40mg/ml, and the concentration of thrombin solution is 10U/ml.
2) isopyknic fibrinogen solution and thrombin solution are mixed, the gel time during utilized ultraviolet spectroscopy different fibrinogen concentration is seen Fig. 2.
Example 9:
1) prepares the Fibrinogen and the thrombin solution of various concentration with the method for example 8 step 1).
Step 2) and 3) with the step 2 of example 1) and 3).
4) utilize the method for weighing to calculate the filling rate of fibrin gel in polylactic acid bracket.Fig. 3 is the filling rate of the Fibrinogen filling polylactic acid support of variable concentrations (ultimate density behind the Fibrinogen gel).
Example 10:
1) step 1) is with example 1 step 1), but the concentration of fibrinogen solution is 40mg/ml, and the concentration of thrombin solution is 10U/ml.
Step 2) and 3) with example 1 step 2) and 3).Utilize the mechanical test instrument to measure the mechanical property of gel-free filling polylactic acid support and fibrin gel filling polylactic acid support, the results are shown in Figure 4, the mechanical strength of as seen filling after-poppet is improved.
Example 11:
Step 1) is with example 1 step 1), but the concentration of fibrinogen solution is 40mg/ml, and the concentration of thrombin solution is 10U/ml.Fibrinogen solution and thrombin solution are at first used 220nm filter membrane filtration sterilization.Then with chondrocyte and fibrinogen solution uniform mixing.The ultimate density of chondrocyte is 1,000,000/ml.
Step 2) and 3) with example 1 step 2) and 3).
The polylactic acid porous scaffold that the fibrin gel of preparation is filled is put in 24 well culture plates, adds culture fluid, carries out In vitro culture in incubator.Cytoactive adopts 3-(4, the 5-dimethylthiazole)-2, and 5-diphenyl tetrazole bromine salt (MTT) method detects.Fig. 5 is the variation of the activity of chondrocyte in the fibrin gel filling bracket with incubation time, and visible chondrocyte has higher activity in the support that fibrin gel is filled.Adopt 1, the method for the inferior blue chromogenic reagent-ultraviolet detection of 9-dimethyl is measured the mucoitin sulfuric acid (GAG) of emiocytosis, the results are shown in Figure 6, and visible chondrocyte can be secreted more GAG in the support that fibrin gel is filled.The form of cell culture after one week adopts laser confocal microscope and scanning electron microscope to observe, and the result sees Fig. 7 and Fig. 8 respectively.
Claims (2)
1. the preparation method of a polymer multihole bracket filled with fibrin gel, its step is as follows:
1) 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 1～20U/ml;
2) porous polymer scaffold is immersed in the ethanol, adopts first evacuation, the method for pressure is recovered in the back, and ethanol is imported in the porous polymer scaffold; To import alcoholic acid support and be immersed in the tri-distilled water, and carry out first evacuation repeatedly, the method for pressure is recovered in the back, and ethanol is replaced as water, and is residual until no ethanol;
3) be injected into isopyknic fibrinogen solution and thrombin calcium chloride solution in the porous polymer scaffold simultaneously, to prop up to be placed in 37 ℃ of constant temperature ovens and hatch 5～10 minutes, make the complete gel of Fibrinogen, promptly obtain polymer multihole bracket filled with fibrin gel.
2. by the preparation method of the described polymer multihole bracket filled with fibrin gel of claim 1, it is characterized in that said porous polymer scaffold is polylactic acid, polylactic-co-glycolic acid, polyurethane or collagen porous support.
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| CN200710068119A CN101053679B (en) | 2007-04-17 | 2007-04-17 | Method for preparing polymer multiporous holder filled with fiber protein gel |
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Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101352581B (en) * | 2008-09-19 | 2012-09-26 | 中国人民解放军第三军医大学 | Adhesion agent for fixing articular cartilage repair implant and use of human fibrinogen in preparing the adhesion agent |
| CN101716382B (en) * | 2009-12-02 | 2015-07-15 | 浙江大学 | Preparation method of trinary composite stent of plasmid DNA / fibrin gel / polymer |
| CN102634978A (en) * | 2011-02-11 | 2012-08-15 | 傅亚 | Preparation method of fibrin glue reinforced polylactic acid fiber |
| PL218235B1 (en) * | 2011-09-12 | 2014-10-31 | Henryk Bursig | Allogeneic and autologous fibrin carrier, method for its preparation and the use of a fibrin carrier for transplantation of cells, especially human cells |
| CN103372233A (en) * | 2012-04-13 | 2013-10-30 | 杭州龙禧生物医药科技有限公司 | Preparation method and product of tissue-engineered cartilage graft |
| RU2533457C1 (en) * | 2013-04-19 | 2014-11-20 | Федеральное государственное бюджетное учреждение "Федеральный научный центр трансплантации и искусственных органов имени академика В.И. Шумакова" Министерства здравоохранения Российской Федерации | Bioactive resorbed porous 3d-matrix for regenerative medicine and method for preparing it |
| CN105107027A (en) * | 2015-07-17 | 2015-12-02 | 深圳爱生再生医学科技有限公司 | Fibrin gel-polylactic acid composite scaffold and preparation method thereof |
| CN105194731B (en) * | 2015-10-27 | 2016-07-13 | 上海科医联创生物科技有限公司 | The gel support formula of a kind of external structure tissue engineering bone/cartilage and preparation method |
| CN106039420B (en) * | 2016-05-30 | 2019-09-10 | 浙江大学 | Fibrin material and preparation method thereof for cartilage reparation integrated with subchondral bone |
| CN106620873A (en) * | 2016-11-17 | 2017-05-10 | 太原理工大学 | Composite hydrogel cartilage repair material and preparation method thereof |
| CN107715175B (en) * | 2017-09-30 | 2018-10-23 | 南京师范大学 | A kind of compound porous bone tissue engineering scaffold of PLGA/ amphoteric ions and preparation method thereof |
| CN108144127B (en) * | 2018-01-25 | 2020-12-22 | 南京医科大学附属口腔医院 | Fibrin gel/polylactic acid-glycolic acid microsphere scaffold and preparation method and application thereof |
| CN112656816A (en) * | 2021-01-15 | 2021-04-16 | 浙江大学 | Exosome-loaded fibrin glue and application thereof |
| CN114191615B (en) * | 2021-12-21 | 2022-07-08 | 浙江大学 | Multilayer composite material for promoting repair of articular cartilage-calcified layer-subchondral bone and preparation method thereof |
| WO2023115295A1 (en) * | 2021-12-21 | 2023-06-29 | 浙江大学 | Multi-layer composite material for promoting articular cartilage-calcified layer-subchondral bone repair and preparation method therefor |
| CN114805917B (en) * | 2022-03-30 | 2023-06-16 | 浙江大学 | Preparation method of three-level porous material based on fibrin fiber bridging |
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| WO2000078370A1 (en) * | 1999-06-22 | 2000-12-28 | The Brigham And Women's Hospital, Inc. | Biologic replacement for fibrin clot for intra-articular use |
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