CN104491926B - Preparation method of tissue engineering scaffold with bioactivity - Google Patents
Preparation method of tissue engineering scaffold with bioactivity Download PDFInfo
- Publication number
- CN104491926B CN104491926B CN201410833234.4A CN201410833234A CN104491926B CN 104491926 B CN104491926 B CN 104491926B CN 201410833234 A CN201410833234 A CN 201410833234A CN 104491926 B CN104491926 B CN 104491926B
- Authority
- CN
- China
- Prior art keywords
- tissue engineering
- preparation
- soybean protein
- lyophilization
- sodium alginate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Medicinal Preparation (AREA)
- Manufacture Of Porous Articles, And Recovery And Treatment Of Waste Products (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention discloses a preparation method of a tissue engineering scaffold with bioactivity. The preparation method comprises the following steps: crosslinking thermally treated soybean proteins by using oxidized sodium alginate under the condition of high speed stirring; and freezing and drying to obtain the soybean protein-based tissue engineering scaffold with bioactivity. The porous bracket prepared by the method has a relatively great bore diameter and porosity and good biocompatibility, has certain bioactivity, can be used for promoting secretion of cellular matrixes and directional differentiation of stem cells and can be used as an ideal carrier of the cell in the tissue engineering field.
Description
Technical field
The present invention relates to tissue engineering technical field, particularly relate to a kind of preparation method with bioactive tissue engineering bracket with soybean protein as primary raw material.
Background technology
Tissue engineering bracket is to be the temporary transient substitute of extracellular matrix to a certain extent with tissue biopsy Cell binding the material that can implant organism, is the most basic framework of engineered tissue.Animality derived Protein with collagen protein, fibroin albumen, keratin etc. as representative, with advantages such as the biocompatibility of its excellence and biodegradabilities, is used widely in organizational project.But, animal protein there is also significantly defect as timbering material, there is larger difference, poor wet strength and certain immunogenicity in the property of protein including separate sources, especially it may carry the risk of animal virus, the collagen protein such as extracted by cattle tissue may carry crazy heifer disease virus and foot and mouth disease virus etc., whether brings the science final conclusion endangered still without clear and definite after implanting.
Soybean protein is a kind of vegetable protein being widely used at Material Field, has the cellular affinity of excellence as the animal protein such as collagen, and it does not contains animal virus, uses safer as timbering material.Additionally, still contain the Component of isoflavone of trace in the soybean protein extracted, this phytoestrogen has been shown to promote cytostromatic secretion and the directed differentiation of induction stem cell, therefore has certain biological activity.But the soybean protein of non-modified is a near-spherical albumen, and its water solublity and mechanical property etc. are the most undesirable, this significantly limit its application in field of tissue engineering technology.The method of modifying of soybean protein has a lot, wherein heat treatment is considered as the simplest and effective manner, heat treatment under research display certain condition can destroy the non-covalent bond between soybean protein, form soluble aggregate, thus improve the dissolubility of soybean protein, the mechanical strength of soy protein basis can be improved simultaneously.
English literature " Novel soy protein scaffolds for tissue regeneration:Material characterization and interaction with human mesenchymal stem cells " discloses with the soybean protein through Overheating Treatment as primary raw material, by a kind of porous tissue engineering scaffold obtained after the crosslinked action of T-5398 lyophilization, the degradation rate of support can be regulated and controled by enzyme dosage.But the aperture utilizing support prepared by the method is less than normal, within being generally distributed in 10 ~ 125 μm, is unfavorable for entrance and the propagation of cell.
The Chinese patent " cartilage tissue engineered rack material and preparation method thereof " of Application No. 201310641686.8 then discloses a kind of with sodium alginate and chitosan as base material, by activating the carboxyl of sodium alginate, crosslink reaction with amino of chitosan and form gel, the three-dimensional porous rack that will be formed after gel lyophilizing.It is disadvantageous in that: carbodiimide hydrochloride (EDC) and N-butanimide (NHS) that activated carboxyl is used have certain cytotoxicity, will impact the biocompatibility of support if can not remove completely；The support of preparation lacks biological activity, it is impossible to regulate and control cell behavior, and the specific performance of support is not described in detail by this method in addition.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of preparation method with bioactive tissue engineering bracket, this tissue engineering bracket has preferable aperture porosity and good biocompatibility.
In order to solve above-mentioned technical problem, the present invention uses following scheme to realize:
A kind of preparation method with bioactive tissue engineering bracket, after the soybean protein after heat treatment utilizes under the conditions of high-speed stirred oxidized sodium alginate crosslinking, lyophilization prepares biological activity tissue engineering bracket based on soybean protein.
Concrete, described heat treatment step is as follows: is dispersed in water by soybean protein and is made into the suspension that mass concentration is 1 ~ 5%, is filtered to remove indissolvable component, obtains the soybean protein after heat treatment after filtrate is freeze-dried under 80 ~ 120 DEG C of constant temperature after stirring 0.5 ~ 2h.
The purpose of heat treatment is to make soybean protein form soluble poly structure set in degeneration after the process of certain time, thus strengthen the dissolubility of soybean protein to a certain extent, needing the soybean protein used in invention to be uniform concentrated solution, the existence of insoluble matter can reduce the intensity of the support of preparation, and the soybean protein dissolubility without this heat treatment step is undesirable.
Concrete, the described step utilizing oxidized sodium alginate to cross-link under high-speed stirred state is as follows:
S21: the soybean protein after heat treatment is dissolved in the calcium chloride solution that concentration is 0.2 ~ 1% and is configured to the solution that concentration is 8 ~ 15%；
S22: the solution prepared in S21 carries out high-speed stirred process and makes solution fully bubble, under keeping equal stir speed (S.S.), dropping concentration is the oxidized sodium alginate solution of 0.5 ~ 2% and continues to react 1 ~ 2h, forms porous gel by crosslinked action.
Certain density calcium chloride solution can promote the mechanical strength dissolving and strengthening the support prepared of soybean protein；By the amino of soybean protein and the aldehyde radical generation schiff base reaction on oxidized sodium alginate, carrying out along with reaction, the degree of cross linking is gradually increased, reactant liquor gradually forms a kind of immobilising gel state, at this moment the bubble formed due to high-speed stirred in reactant liquor will be fixed, reactant liquor can form a kind of gel rich in bubble, and these bubbles can form the loose structure of support after being lyophilized in preparation condition subsequently.
Lyophilization condition after described crosslinking be prior to-4 ~-20 DEG C of freezing 12 ~ 24h, after in freeze dryer lyophilization 12 ~ 24h.
Build-up ice cold for the porous gel after crosslinking, it is subsequently placed in lyophilization in freeze dryer, its purpose is to prepare porous support, though aperture and porosity are had a certain impact by cryogenic temperature, but in actual experiment, find that its impact is minimum, the pore structure of medium-height trestle of the present invention is mainly derived from the bubble in porous gel, so cryogenic temperature, time, the temperature of freeze dryer, freeze-drying times etc. are not the biggest to the aperture in support and Porosity Rate Influence, the present invention does not use orientation Refrigeration Technique, owing to it simply takes some special measures when freezing, in order to meet some special needs of some organizational projects, and the present invention not necessarily uses this technology just can achieve the goal.
Described is filtered into sucking filtration, and because solution viscosity is higher, sucking filtration mode can effectively remove indissolvable component.
The described lyophilization in heat treatment step for filtrate being placed in-4 ~-20 DEG C of freezing 12 ~ 24h, after in freeze dryer lyophilization 12 ~ 24h.
First the soy bean proteinous soln after heat treatment filtration is carried out freezing processing, preferably, soy bean proteinous soln can be put into glaciation in refrigerator, it is placed in lyophilization in cooling driers the most again, the step for can by solution moisture remove, having the soybean protein raw material of soluble structure to obtain, the parameter such as the cryogenic temperature of refrigerator now, cooling time, the temperature of freeze dryer and freeze-drying time does not has much impacts to the soybean protein raw material quality formed.
The speed of described high-speed stirred is 1000 ~ 3000rpm.
Without high-speed stirred, loose structure can also be formed after the simple gel lyophilizing that will be formed after solution crosslinking, but the aperture formed merely with freeze drying process is less and irregular, and combine high-speed stirred, the bubble diameter that soy bean proteinous soln is formed is big and regular, the aperture of the support of final preparation is big, and porosity is high.
Described soybean protein is 10 ~ 100:1 with the mass ratio of oxidized sodium alginate.
Oxidized sodium alginate is to be aoxidized sodium alginate by strong oxidizer, makes the hydroxyl of sodium alginate part aldose unit be transformed into aldehyde radical and a kind of Biological cross-linker of obtaining, and compared with other cross-linking agent, its bio-toxicity is relatively low.
The oxidizability of described oxidized sodium alginate is 20 ~ 80%.Different oxidizabilities has certain impact to the intensity of cross-linking reaction and the support of preparation.
Compared with prior art, there is advantages that
1, this method is with the soybean protein of plant origin for preparing the primary raw material of support, with animal protein such as traditional collagen, fibroin albumen, albumin as compared with raw material, its property of protein is the most homogeneous, and will not carry animality virus, uses safer.
2, this method is simple to operate, and reaction is carried out in aqueous, and used reagent is the most nontoxic, does not pollutes the environment, it is possible to the biohazard brought due to toxic chemical substance residual after avoiding stenter to implant body interior.
3, the soybean protein brace aperture rate that prepared by this method is more than 80%, and pore size is 200 ~ 400 μm and is evenly distributed, and support has good cellular affinity and has certain biological activity promoting stem cell directional differentiation.
Detailed description of the invention
In order to allow those skilled in the art be more fully understood that technical scheme, below the present invention is further elaborated.
Embodiment 1:
A kind of preparation method of biological activity tissue engineering bracket based on soybean protein, including following preparation process:
S1. heat treatment: soybean protein is dispersed in water and is made into the suspension that mass concentration is 1%, after stirring 0.5h under 80 DEG C of constant temperature, sucking filtration removes indissolvable component, filtrate is placed in-20 DEG C of freezing 12h, after soybean protein after lyophilization 12h obtains heat treatment in freeze dryer；
S2. oxidized sodium alginate crosslinking: the soybean protein after heat treatment is dissolved in the calcium chloride solution that concentration is 0.2% and is configured to the solution that concentration is 8%, after the solution of preparation is stirred with the stir speed (S.S.) of 1000rpm so that it fully bubbles, under keeping equal stir speed (S.S.), dropping concentration is the oxidized sodium alginate solution of 0.5% and continues to react 1h, porous gel is formed by crosslinked action, described oxidized sodium alginate oxidizability is 20%, and soybean protein is 10:1 with the mass ratio of oxidized sodium alginate；
S3. lyophilization: by the porous gel in S2 prior to-20 DEG C of freezing 12h, after in freeze dryer lyophilization 12h i.e. obtain porous support.
Embodiment 2:
A kind of preparation method of biological activity tissue engineering bracket based on soybean protein, including following preparation process:
S1. heat treatment: soybean protein is dispersed in water and is made into the suspension that mass concentration is 5%, after stirring 2h under 120 DEG C of constant temperature, sucking filtration removes indissolvable component, filtrate is placed in-4 DEG C of freezing 24h, after soybean protein after lyophilization 24h obtains heat treatment in freeze dryer；
S2. oxidized sodium alginate crosslinking: the soybean protein after heat treatment is dissolved in the calcium chloride solution that concentration is 1% and is configured to the solution that concentration is 15%, after the solution of preparation is stirred with the stir speed (S.S.) of 3000rpm so that it fully bubbles, under keeping equal stir speed (S.S.), dropping concentration is the oxidized sodium alginate solution of 2% and continues to react 2h, porous gel is formed by crosslinked action, described oxidized sodium alginate oxidizability is 80%, and soybean protein is 100:1 with the mass ratio of oxidized sodium alginate；
S3. lyophilization: by the porous gel in S2 prior to-4 DEG C of freezing 24h, after in freeze dryer lyophilization 24h i.e. obtain porous support.
Comparative example 1
In addition to calcium chloride concentration is 0.05%, other condition is with embodiment 1.
Comparative example 2
In addition to calcium chloride concentration is 5%, other condition is with embodiment 2.
Comparative example 3
Except in S2, the mass ratio of soybean protein and oxidized sodium alginate is in addition to 5:1, and other condition is with embodiment 1.
Comparative example 4
Except in S2, the mass ratio of soybean protein and oxidized sodium alginate is in addition to 300:1, and other condition is with embodiment 2.
Comparative example 5
In addition in S2, stir speed (S.S.) is 500rpm, other condition is with embodiment 1.
Comparative example 6
In addition in S2, stir speed (S.S.) is 5000rpm, other condition is with embodiment 2.
Embodiment 1 ~ 2 and comparative example 1 ~ 6 are carried out performance test, and acquired results is as follows:
| Project | Aperture/μm | Porosity/% | Modulus of compressibility/KPa |
| Embodiment 1 | 320~400 | 85 | 30 |
| Embodiment 2 | 200~280 | 88 | 48 |
| Comparative example 1 | 300~420 | 87 | 22 |
| Comparative example 2 | 280~550 | 80 | 42 |
| Comparative example 3 | 320~480 | 76 | 24 |
| Comparative example 4 | 210~360 | 88 | 36 |
| Comparative example 5 | 380~570 | 74 | 32 |
| Comparative example 6 | 120~180 | 90 | 54 |
In general, support pore diameter range is between 200 ~ 400 μm, and porosity is beneficially the growing into and breed of cell more than 80%.In comparative example 1 and 2, calcium chloride concentration mainly produces impact to the modulus of compressibility of support, in comparative example 1, modulus of compressibility is less than normal relative to embodiment 1, and the dissolubility that calcium chloride concentration too high in comparative example 2 makes soybean protein is deteriorated, there is pore size distribution in the support therefore prepared uneven, the problem that pore size distribution range is wider；By embodiment 1 and 2 and comparative example 3 and 4, soybean protein can affect the degree of cross linking with the mass ratio of oxidized sodium alginate, and then affecting the modulus of compressibility of support, the modulus of compressibility in comparative example 3 and comparative example 4 is less than normal, illustrates that too high or too low mass ratio all reduces the mechanical property of support；By embodiment 1 and 2 and comparative example 5 and 6, stir speed (S.S.) significantly affects the size of bubble in preparation process, the number of bubbles that relatively low stir speed (S.S.) produces is less and is relatively large in diameter, cause comparative example 5 mesoporous distribution width and porosity too small, although comparative example 6 porosity and modulus of compressibility obtain certain lifting, but aperture is too small.Support prepared by the present invention has considered the impact on support performance of each index, and support can not be brought significant benefit by the raising of single index, can cause dying down of another index performance on the contrary, and the condition and range of the present invention has the effect of optimum.
Above-described embodiment is only the wherein specific implementation of the present invention, and it describes more concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that, for the person of ordinary skill of the art, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, these obvious alternative forms belong to protection scope of the present invention.
Claims (5)
1. a preparation method with bioactive tissue engineering bracket, it is characterised in that its step is as follows:
S1: be dispersed in water by soybean protein and be made into the suspension that mass concentration is 1 ~ 5%, is filtered to remove indissolvable component after stirring 0.5 ~ 2h, obtains the soybean protein after heat treatment after filtrate is freeze-dried under 80 ~ 120 DEG C of constant temperature；
S2: the soybean protein after heat treatment is dissolved in the calcium chloride solution that mass concentration is 0.2 ~ 1% and is configured to the solution that mass concentration is 8 ~ 15%；
S3: the solution prepared in S2 carries out high-speed stirred process and makes solution fully bubble, under keeping equal stir speed (S.S.), dropping mass concentration is the oxidized sodium alginate solution of 0.5 ~ 2% and continues to react 1 ~ 2h, by being cross-linked to form porous gel；Described high-speed stirred speed is 1000 ~ 3000rpm；Described soybean protein is 10 ~ 100:1 with the mass ratio of oxidized sodium alginate；
S4: porous gel described in lyophilization prepares biological activity tissue engineering bracket based on soybean protein.
A kind of preparation method with bioactive tissue engineering bracket the most according to claim 1, it is characterised in that the lyophilization condition after described crosslinking be prior to-20 ~-4 DEG C of freezing 12 ~ 24h, after in freeze dryer lyophilization 12 ~ 24h.
A kind of preparation method with bioactive tissue engineering bracket the most according to claim 1, it is characterised in that described is filtered into sucking filtration.
A kind of preparation method with bioactive tissue engineering bracket the most according to claim 1, it is characterised in that the lyophilization in described heat treatment step for filtrate being placed in-20 ~-4 DEG C of freezing 12 ~ 24h, after in freeze dryer lyophilization 12 ~ 24h.
A kind of preparation method with bioactive tissue engineering bracket the most according to claim 1, it is characterised in that the oxidizability of described oxidized sodium alginate is 20 ~ 80%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410833234.4A CN104491926B (en) | 2014-12-29 | 2014-12-29 | Preparation method of tissue engineering scaffold with bioactivity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410833234.4A CN104491926B (en) | 2014-12-29 | 2014-12-29 | Preparation method of tissue engineering scaffold with bioactivity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104491926A CN104491926A (en) | 2015-04-08 |
| CN104491926B true CN104491926B (en) | 2017-01-11 |
Family
ID=52933436
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410833234.4A Active CN104491926B (en) | 2014-12-29 | 2014-12-29 | Preparation method of tissue engineering scaffold with bioactivity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104491926B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106237386A (en) * | 2016-08-29 | 2016-12-21 | 东华大学 | A kind of natural fibroin base macropore support for bone defect healing and preparation method thereof |
| US9783777B1 (en) | 2016-10-18 | 2017-10-10 | King Saud University | Method of making three-dimensional, leaf-based scaffold for three-dimensional cell culture |
| CN108144122A (en) * | 2018-01-24 | 2018-06-12 | 中山大学 | A kind of organizational project 3D stents based on bean curd and its preparation method and application |
| CN111979180A (en) * | 2020-09-02 | 2020-11-24 | 杭州优渡生物科技有限公司 | Three-dimensional amplification method for maintaining cell phenotype |
| CN112316210B (en) * | 2020-11-09 | 2021-10-12 | 上海大学 | Mixed hydrogel containing soy protein, preparation method and application of mixed hydrogel, vascularized network stent and preparation method of vascularized network stent |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1300397C (en) * | 2005-04-13 | 2007-02-14 | 武汉大学 | Blended fiber of sodium alginate/soybean protein isolate. preparation method and application thereof |
| CN101766842B (en) * | 2010-03-22 | 2013-11-06 | 四川大学 | Biological tissue material for artificial organs and preparation method thereof |
| CN102940906B (en) * | 2012-12-17 | 2014-04-16 | 江苏科技大学 | Preparation method of biological active bone tissue engineering material |
-
2014
- 2014-12-29 CN CN201410833234.4A patent/CN104491926B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104491926A (en) | 2015-04-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104491926B (en) | Preparation method of tissue engineering scaffold with bioactivity | |
| Cao et al. | Fish collagen-based scaffold containing PLGA microspheres for controlled growth factor delivery in skin tissue engineering | |
| Chien et al. | Novel soy protein scaffolds for tissue regeneration: Material characterization and interaction with human mesenchymal stem cells | |
| Balaji et al. | Characterization of keratin–collagen 3D scaffold for biomedical applications | |
| JP5368476B2 (en) | Collagen collagen burn dressings made from jellyfish | |
| WO2014079198A1 (en) | Degradable wound-repairing material and preparation method thereof | |
| US20210207098A1 (en) | Drying formulation for hydrogel microcarriers | |
| CN106243376A (en) | A kind of silk fibroin nano-fiber porous support and preparation method thereof | |
| JP6223474B2 (en) | Silk-based porous scaffold and method for its preparation | |
| CN103816573A (en) | Preparation method of porous gelatin/hyaluronic acid composite microspheres | |
| CN102228695A (en) | Method for preparing basic fibroblast growth factor sustained-release carrier | |
| CN106367460A (en) | Method for preparing collagen sponge under acid condition | |
| TW201442747A (en) | Albumin tissue scaffold | |
| CN105251052A (en) | Cartilage extracellular matrix and silk fibroin composite orientation cartilage support and preparation method thereof | |
| CN113842502A (en) | Injection filler containing deproteinized bone and preparation method thereof | |
| CN107715181B (en) | Preparation method of biodegradable tissue engineering skin scaffold | |
| CN108273132A (en) | A kind of fibroin albumen/keratin composite porous material and preparation method thereof | |
| CN106421902A (en) | Quick-gelatinization silk fibroin solution and preparation method thereof | |
| CN102836462A (en) | Preparation method for collagen tissue engineering skin | |
| CN103768654B (en) | Water-soluble fibroin powder and preparation method thereof | |
| CN102008740B (en) | Absorbable growth factor composite dressing | |
| CN105879101B (en) | Outer compress material of a kind of feather keratin base boiomacromolecule and preparation method thereof | |
| CN105879102B (en) | A kind of feather keratin grafting alginic acid sponge dressing and preparation method thereof | |
| KR102615727B1 (en) | Hydrogel for vascular embolization with adjustable decomposition time and manufacturing method thereof | |
| CN109867811B (en) | Porous-solid composite silk material and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Inventor after: Xu Honglei Inventor after: Tian Ye Inventor after: He Xiaofan Inventor after: Tian Xiaojun Inventor before: Xu Honglei Inventor before: Tian Ye Inventor before: He Xiaofan |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |