CN104211982B - It is adapted to the manufacture method of the multiple aperture organizational project scaffold of cell growth - Google Patents
It is adapted to the manufacture method of the multiple aperture organizational project scaffold of cell growth Download PDFInfo
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- CN104211982B CN104211982B CN201310664598.XA CN201310664598A CN104211982B CN 104211982 B CN104211982 B CN 104211982B CN 201310664598 A CN201310664598 A CN 201310664598A CN 104211982 B CN104211982 B CN 104211982B
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Abstract
The present invention discloses the manufacture method of the multiple aperture organizational project scaffold for being adapted to cell growth.The method comprises the following steps：(1) 1.0g of gelatin 0.3, the 8mg of Sodium Hyaluronate 1 and the 0.5g of 6 sodium ascorbyl phosphate of chondroitin 0.05 are dissolved in 5 10ml distilled waters and constitute solution；(2) mixed in adding the solution with 1 5mL0.5 5%EDC；(3) 1 10min is placed in room temperature, then freezes 0.5 3h at 20 DEG C, 60~80 DEG C freeze 0.5 3 hours again；(4) most afterwards through freezing 48 96 hours under the conditions of 50 DEG C of freeze dryers to obtain preliminary scaffold；(5) the preliminary scaffold and 5 20mL0.1 0.5%EDC are acted on into 12 48h in room temperature；(6) again after 20~30 DEG C of condition freezes 12 48h, finally, freezed 48 96 hours under the conditions of 50 DEG C of freeze dryers, scaffold is obtained, such scaffold has that porous spongy space multistory framework, histocompatbility are good, of good quality, pore size is suitable and absorption of water is good, no toxicity also can be degradable.
Description
Technical field
The present invention relates to the medical tissue engineering material of organizational engineering, more particularly to suitable cell growth multiple aperture group
The manufacture method of weaver's journey scaffold.
Background technology
Organizational project refers to design, set up, safeguard human body cell with the principle and method of engineering science using life science
With the growth of tissue, the tissue or the function of organ being damaged with recovery.The reparation for developing into tissue or organ of organizational project
Possibility is provided with building again.Tissue engineering material is also increasingly subject to the attention of people, and research and development have good organization's compatibility
Material turned into organizational project development foundation stone.
Scaffold in the past is generally graininess, line-like structures, without porous space comprehensive architecture.In addition, in the past
Scaffold usually histocompatbility is bad or with certain toxicity or can not be degradable, or absorption of water is bad.So
Scaffold be unfavorable for that cell grows in certain Spatial infrastructure, be unfavorable for their interaction and cell communication, be not inconsistent
Close actual nature in body.
The content of the invention
The problem that the present invention is solved is problem of the existing scaffold without porous stereoeffect.
To solve the above problems, the present invention provides a kind of manufacture of the multiple aperture organizational project scaffold of suitable cell growth
Method, the method comprises the following steps：(1), gelatin 0.3-1.0g, Sodium Hyaluronate 1-8mg and chondroitin 6- sodium ascorbyl phosphates
0.05-0.5g is dissolved in 5-10ml distilled waters and constitutes solution；(2) mixed in, adding the solution with 1-5mL0.5-5%EDC
It is even；(3) 1-10min, is placed in room temperature, then 0.5-3h is freezed at -20 DEG C, -60~80 DEG C freezes 0.5-3 hours again；(4)、
48-96 hours is most freezed under the conditions of -50 DEG C of freeze dryers of warp afterwards to obtain preliminary scaffold；(5), by the preliminary scaffold and 5-
20mL 0.1-0.5%EDC act on 12-48h in room temperature；(6), again after -20~-30 DEG C of condition freezing 12-48h, finally,
Freezed 48-96 hours under the conditions of -50 DEG C of freeze dryers, scaffold is obtained.
In further scheme, the number range of the gelatin is that 0.4~0.7g, the number range of Sodium Hyaluronate are 3
~8mg, the best scope of chondroitin 6- sodium ascorbyl phosphates are that the number range of 0.2~0.4g or distilled water is 7.0~8.5ml.
In further scheme, the number range of the EDC is 1.5~2.5ml in step 2.
It is in step 3, described to be 2~5min, placed in -20 DEG C of freezings in room temperature standing time in further scheme
The number range of time is 1.5~2.5h or is 1.5~2.5h in -80 DEG C of number ranges for freezing standing time.
In further scheme, the number range of freeze-drying time is 64~80h under conditions of -50 DEG C in step 4.
In further scheme, in steps of 5, the number range of EDC is 8~13ml, the numerical value in room temperature standing time
Scope is 18~30h.
In further scheme, in step 6, the number range that room temperature standing time is frozen at -20 DEG C is 18~
30min is 64~80h in the best number range of freeze-drying time.
Compared with prior art, the present invention has advantages below：
Due to the present invention using above-mentioned preparation condition and by each Composition Control within the above range, so, obtained engineering
Scaffold has that porous spongy space multistory framework, histocompatbility be good, of good quality, pore size suitable and absorption of water
It is good, it is additionally, since using the material, so, there is no the toxicity can be degradable yet.
Brief description of the drawings
Fig. 1 is the flow chart of the manufacture method of the multiple aperture organizational project scaffold of the suitable cell growth of the present invention；
Fig. 2 is a kind of structure chart of the engineering scaffold manufactured by the method for the present invention, and the figure shows engineering scaffold
SEM amplifies 2000 times of photos after platinum spraying plating, and the photo shows that this material is in smooth porous spongy space
Framework, scale 20um, P are sponge loose structure, and uniform pore diameter, surface is smooth, are 60-80um through measuring pore size.
Specific embodiment
To describe technology contents of the invention, structural feature, institute's reached purpose and effect in detail, below in conjunction with embodiment
And coordinate accompanying drawing to be described in detail.
Fig. 1 is referred to, the multiple aperture spongy tissue engineering scaffold of the suitable cell growth of the present invention comprises the following steps：
(1), gelatin 0.3-1.0g, Sodium Hyaluronate 1-8mg and chondroitin 6- sodium ascorbyl phosphates 0.05-0.5g are dissolved in 5-
Solution is constituted in 10ml distilled waters；
In this step, when gelatin is less than 0.3g, scaffold quality can be too soft, is difficult lyophilized shaping, it is impossible to formed many
Cavernous structure, pore size is suitable；When gelatin is more than 1.0g, scaffold quality is too hard and crisp, and absorption of water is deteriorated.In the present invention
In, gelatin be 0.3g, 0.35g, 0.4g, 0.42g, 0.47g, 0.5g, 0.53g, 0.57g, 0.59g, 0.65g, 0.7g, 0.75g,
0.78g, 0.81g, 0.84g, 0.89g, 0.92g, 0.95g, 0.97g or 1.0g, the best number range of gelatin is 0.4~
0.7g, scaffold quality and absorption of water are suitable within the range.
In this step, when Sodium Hyaluronate is less than 1mg, scaffold quality is too hard and crisp, and absorption of water is deteriorated, and is more than
During 8mg, aperture diminishes, and quality softens.Such as, can be 1mg, 1.5mg, 1.8mg, 2mg, 2.5mg, 2.7mg, 2.9mg, 3mg,
3.4mg、3.6mg、4mg、4.1mg、4.2mg、4.9mg、5mg、5.7mg、5.9mg、6mg、6.3mg、6.5mg、6.8mg、
6.9mg, 7mg, 7.2mg, 7.8mg or 8mg.In the present invention, the best number range of Sodium Hyaluronate is 3~8mg.
In this step, the chondroitin 6- sodium ascorbyl phosphates can be 0.05g, 0.1g, 0.13g, 0.15g, 0.2g,
0.23g, 0.27g, 0.3g, 0.35g, 0.4g, 0.41g, 0.43g, 0.47g or 0.5g.In the present invention, chondroitin 6- phosphoric acid
The best scope of sodium salt is 0.2~0.4g.
In this step, when the amount of distilled water is less than 5ml, quality becomes fragile；When the amount of distilled water is more than 10ml, it is difficult
It is lyophilized.Such as, 5ml, 5.5ml, 5.8ml, 6ml, 6.2ml, 6.7ml, 6.9ml, 7ml, 7.1ml, 7.3ml, 7.7ml, 8ml,
8.5ml, 8.9ml, 9ml, 9.1ml, 9.3ml, 9.6ml, 9.9ml or 10ml.The best number range of distilled water is 7.0~
8.5ml。
(2), with 1-5mL 0.2%1-ethyl-3- (3-dimethylaminopropyl)-carbodiimide (EDC)
Add mixing in the solution；
In this step, when EDC is less than 1ml, scaffold quality can be too soft, is difficult lyophilized shaping, it is impossible to form porous
Shape structure；When EDC is more than 5ml, scaffold quality is too hard and crisp, and absorption of water is deteriorated.In the present invention, EDC be 1ml,
1.3ml, 1.6ml, 1.9ml, 2ml, 2.2ml, 2.5ml, 2.8ml, 3.1ml, 3.4ml, 3.7ml, 4.0ml, 4.3ml, 4.6ml or
Person 5ml, EDC best number range is 1.5~2.5ml in the present invention, and scaffold quality and absorption of water are suitable.
(3) 1-10min is placed in room temperature, then freezes 0.5-3h at -20 DEG C, -80 DEG C freeze 0.5-3 hours again；
In this step, when 1min is less than in room temperature standing time, scaffold quality can be too soft, is difficult lyophilized shaping,
Vesicular texture cannot be formed；When EDC is more than 10min, scaffold quality is too hard and crisp, and absorption of water is deteriorated.In the present invention,
It is 1min, 2min, 3min, 4min, 5min, 6min, 7minl, 8min, 9min or 10min in room temperature standing time, is put in room temperature
It is 2~5min to put time best number range, and scaffold quality and absorption of water are suitable within the range.
In this step, when when freezing standing time and being less than 0.5h for -20 DEG C, ice crystal will is formed, it is impossible to using lyophilized
Machine is freezed；When -20 DEG C freeze standing time is more than 3h, scaffold quality is too hard and crisp, and absorption of water is deteriorated.In the present invention,
It is 0.5h, 1.0h, 1.5h, 2h, 2.5h, 3h in room temperature standing time, is in -20 DEG C of freezing standing time best number ranges
1.5~2.5h, scaffold quality and absorption of water are suitable within the range.
In this step, when when freezing standing time and being less than 0.5h for -80 DEG C, scaffold freezing is not enough, it is impossible to uses and freezes
Dry machine is freezed；When -80 DEG C freeze standing time is more than 3h, scaffold quality is too hard and crisp, and absorption of water is deteriorated.In the present invention
In, it is 0.5h, 1.0h, 1.5h, 2h, 2.5h, 3h in room temperature standing time, freeze standing time best numerical value model at -80 DEG C
It is 1.5~2.5h to enclose, and scaffold quality and absorption of water are suitable within the range.
(4) 48-96h, is most freezed under the conditions of -50 DEG C of freeze dryers of warp afterwards to obtain preliminary scaffold；
In this step, when freeze-drying time is less than 48h, the lyophilized deficiency of scaffold, also in thick, it is impossible to obtain preliminary
Scaffold；When freeze-drying time is more than 96h, scaffold quality is too hard and crisp, and absorption of water is deteriorated.In the present invention, when lyophilized
Between be 48h, 56h, 64h, 72h, 80h, 88h, 96h, be 64~80h in the best number range of freeze-drying time, within the range
Scaffold quality and absorption of water are suitable.
(5) the preliminary scaffold and 5-20mL0.2%EDC, are acted on into 12-48h in room temperature；
In this step, when EDC is less than 5ml, scaffold quality can be too soft, and aperture will be too small；When EDC is more than 20ml
When, scaffold quality is too hard and crisp, and absorption of water is deteriorated.In the present invention, EDC be 5ml, 5.2ml, 5.6ml, 6.3ml, 7ml,
7.5ml, 8ml, 10ml, 11ml, 13ml, 16ml, 20ml, EDC best number range are 8~13ml, within the range pin hand
Frame quality and absorption of water are suitable.
In this step, when 12h is less than in room temperature standing time, scaffold quality can be too soft；When EDC is more than 48h,
Scaffold quality is too hard and crisp, and absorption of water is deteriorated.In the present invention, room temperature standing time be 12h, 18h, 24h, 30h, 36h,
42h, 48h, are 18~30h in room temperature standing time best number range, and scaffold quality and absorption of water are closed within the range
It is suitable.
(6), finally, 48-96 is freezed under the conditions of -50 DEG C of freeze dryers small after -20 DEG C of condition freezing 12-48h again
When, scaffold is obtained.
In this step, when when freezing standing time and being less than 12h for -20 DEG C, ice crystal will is formed, it is impossible to use freeze dryer
It is lyophilized；When -20 DEG C freeze standing time is more than 48h, scaffold quality is too hard and crisp, and absorption of water is deteriorated.In the present invention,
12h, 18h, 24h, 30h, 36h, 42h, 48h, are 18~30min in room temperature standing time best number range, in the scope
Interior scaffold quality and absorption of water are suitable.
In this step, when 48h is less than in -50 DEG C of freeze-drying times, the lyophilized deficiency of scaffold, quality softens；When lyophilized
When time is more than 96h, scaffold quality is too hard and crisp, and absorption of water is deteriorated.In the present invention, freeze-drying time be 48h, 56h,
64h, 72h, 80h, 88h, 96h, are 64~80h in the best number range of freeze-drying time, within the range scaffold quality and
Absorption of water is suitable.
Claims (7)
1. the manufacture method of the multiple aperture organizational project scaffold of cell growth is adapted to, it is characterized in that：The method includes following step
Suddenly：
(1) it is double that gelatin 0.3-1.0g, Sodium Hyaluronate 1-8mg and chondroitin 6- sodium ascorbyl phosphates 0.05-0.5g are dissolved in 5-10ml
Solution is constituted in steaming water；
(2) mixed in adding the solution with 1-5mL0.5-5%EDC；
(3) 1-10min is placed in room temperature, then freezes 0.5-3h at -20 DEG C, -60~-80 DEG C freeze 0.5-3 hours again；
(4) 48-96 hours is most freezed under the conditions of -50 DEG C of freeze dryers of warp afterwards to obtain preliminary scaffold；
(5) the preliminary scaffold and 5-20mL0.1-0.5%EDC are acted on into 12-48h in room temperature；
(6) finally, 48-96 is freezed under the conditions of -50 DEG C of freeze dryers small after -20~-30 DEG C of condition freezing 12-48h again
When, scaffold is obtained.
2. the manufacture method of the multiple aperture organizational project scaffold of cell growth is adapted to as claimed in claim 1, it is characterized in that：
In step 1, the number range of the gelatin is that 0.4~0.7g, the number range of Sodium Hyaluronate are 3~8mg, chondroitin 6-
The best scope of sodium ascorbyl phosphate is that the number range of 0.2~0.4g or distilled water is 7.0~8.5ml.
3. the manufacture method of the multiple aperture organizational project scaffold of cell growth is adapted to as claimed in claim 1, it is characterized in that：
The number range of the EDC is 1.5~2.5ml in step 2.
4. the manufacture method of the multiple aperture organizational project scaffold of cell growth is adapted to as claimed in claim 1, it is characterized in that：
In step 3, it is described room temperature standing time be 2~5min, the number range that freezes standing times at -20 DEG C be 1.5~
2.5h is 1.5~2.5h in -80 DEG C of number ranges for freezing standing time.
5. the manufacture method of the multiple aperture organizational project scaffold of cell growth is adapted to as claimed in claim 1, it is characterized in that：
The number range of freeze-drying time is 64~80h under conditions of -50 DEG C in step 4.
6. the manufacture method of the multiple aperture organizational project scaffold of cell growth is adapted to as claimed in claim 1, it is characterized in that：
In steps of 5, the number range of EDC is that 8~13ml, the number range in room temperature standing time are 18~30h.
7. the manufacture method of the multiple aperture organizational project scaffold of cell growth is adapted to as claimed in claim 1, it is characterized in that：
In step 6, the best number range of freeze-drying time is 64~80h under the conditions of -50 DEG C.
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| CN201310664598.XA CN104211982B (en) | 2013-05-31 | 2013-11-30 | It is adapted to the manufacture method of the multiple aperture organizational project scaffold of cell growth |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1342722A (en) * | 2001-09-26 | 2002-04-03 | 天津大学 | Process for preparing dual-layer combined chitosan-gelatin-mucinase scaffold material |
| CN102772823A (en) * | 2012-07-25 | 2012-11-14 | 华南理工大学 | Preparation method of hyaluronic acid/gelatin/chondroitin sulfate bone repair bionic scaffold |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6696074B2 (en) * | 2000-12-04 | 2004-02-24 | Tei Biosciences, Inc. | Processing fetal or neo-natal tissue to produce a scaffold for tissue engineering |
| CN1141150C (en) * | 2001-02-28 | 2004-03-10 | 中国医学科学院生物医学工程研究所 | Compound collagen stroma tissue engineering support and preparation method thereof |
| JP2005046538A (en) * | 2003-07-31 | 2005-02-24 | Jms Co Ltd | Porous body for medical treatment and method for manufacturing it |
| CN1255479C (en) * | 2003-09-23 | 2006-05-10 | 中国医学科学院生物医学工程研究所 | Composite bone tissue engineering rack material and its prepn |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1342722A (en) * | 2001-09-26 | 2002-04-03 | 天津大学 | Process for preparing dual-layer combined chitosan-gelatin-mucinase scaffold material |
| CN102772823A (en) * | 2012-07-25 | 2012-11-14 | 华南理工大学 | Preparation method of hyaluronic acid/gelatin/chondroitin sulfate bone repair bionic scaffold |
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Effective date of registration: 20180309 Address after: 102200 room No. 29, life Garden Road, Changping District science and Technology Park, Beijing City, Beijing, 1 room B316-81 Patentee after: Beijing origin love Biotechnology Co., Ltd. Address before: 323000 Xueyuan Road, Liandu, Zhejiang, No. 1, Patentee before: Lishui University |