CN1330386C - Method for preparing injectable chitosan hydrogen for tissue engineering - Google Patents
Method for preparing injectable chitosan hydrogen for tissue engineering Download PDFInfo
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- CN1330386C CN1330386C CNB2005100618698A CN200510061869A CN1330386C CN 1330386 C CN1330386 C CN 1330386C CN B2005100618698 A CNB2005100618698 A CN B2005100618698A CN 200510061869 A CN200510061869 A CN 200510061869A CN 1330386 C CN1330386 C CN 1330386C
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Abstract
The present invention discloses a method for preparing injectable chitosan hydrogel for tissue engineering, a carbodiimide condensation method is adopted, methacrylic acid and lactic acid are grafted in a chitosan molecular chain orderly, and the obtained chitosan derivative has polymerisable unsaturation carbon-to-carbon double bond and water solubility simultaneously. A redox initiating system of ammonium peroxodisulfate and tetramethylethylenediamine is adopted to make the chitose derivative to form the chitose hydrogel through double bond cross-linking. The chitose htdrogel with no poison has biodegradability and biocompatibility. The present invention which has simple manufacturing process can be used for an injectable hydrogel bracket in the tissue engineering.
Description
Technical field
The present invention relates to a kind of preparation method that is used for the injectable chitosan hydrogen of organizational project.
Background technology
Syringeability hydrogel support is to have mobile hydrogel prepolymer and allosome or autogenous cell and be injected directly into the body defect after compound a kind of, or not compound and directly inject in the body with cell, material arrive after the defect can be under physics or chemical stimulation original position form have certain mechanical strength, shape and the support that can exchange with body fluid.Wherein not compound and directly inject the cell expansion growing multiplication formative tissue that intravital support can utilize the injected material surrounding tissue with cell.Its advantage mainly is the culturing in vivo environment and the invasive of cell.Simultaneously, hydrogel material has good biocompatibility usually, and its aqueous environment helps protecting the transportation of cell and growing nutrient and secretory product, and easy-to-use cell adhesion part carries out modification.But the operation of hydrogel is difficult with control, and mechanical strength is generally lower.At present, the hydrogel material of employing comprises that natural material such as alginate, collagen, hyaluronic acid, agar, chitosan and gelatin etc. and polymeric material are as poly-fumaric acid propylene glycol ester, Polyethylene Glycol and polyethylene glycol oxide propylene oxide copolymer etc.Gel gel method in vivo has temperature sensitive type, chemical crosslinking type, ionomer type and specificity crosslinked etc.
Chitosan has biological degradability and excellent biological compatibility, is widely used in pharmaceutical carrier and organizational project.Because intermolecular hydrogen bonding is pretended firmly, chitosan only dissolves under acid condition.It can form hydrogel by pH value change, covalent bond or ionic crosslinking.But solubility in acid and gel method have limited chitosan as the injection aquagel application in the tissue repair in vivo.Therefore, to the modification of chitosan molecule structure with promote its under neutrallty condition water solublity and to set up gentle gel method extremely important.At present, bibliographical information the employing glycerophosphate (Glycero1-2-phosphate, β-GP) the chitosan acid solution is adjusted to neutrality form down hydrogels at 37 ℃.Grafting PNIPAM or amino and isobutyric anhydride reaction formation isopropyl amide group on the amino of chitosan molecule chain, gained derivant are not only had a water solublity, and are temperature-sensitive hydrogel.Above-mentioned material can be compound with chondrocyte or stem cell, obtains positive result in animal body in the test.But above aquagel is owing to be physical gel, structural instability, and also the existence of salt and nondegradable PNIPAM can influence cell activity and histocompatibility.
Summary of the invention
The purpose of this invention is to provide a kind of preparation method that is used for the injectable chitosan hydrogen of organizational project.
The preparation method that is used for the injectable chitosan hydrogen of organizational project of the present invention 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 (CML) of grafting methacrylic acid and lactic acid;
3) chitosan with grafting methacrylic acid and lactic acid is dissolved in water or the phosphate buffer (PBS), compound concentration is 1%～2.5% the grafting methacrylic acid and the chitosan solution of lactic acid, add redox initiation system Ammonium persulfate. (APS) and tetramethylethylenediamine (TMEDA) then, the mol ratio of Ammonium persulfate. and tetramethylethylenediamine is 1: 1, the ultimate density of initiator system is 2.5mM～15mM, and 25 ℃～45 ℃ are reacted the formation aquagel down.
The inventive method operating procedure is simple, implementation condition is gentle.Utilization is in the presence of carbodiimide, but method amino and carboxyl and hydroxyl condensation formation amido link and ester bond prepares polymerisable water-soluble chitosan.Can recently regulate and control the grafting amount of methacrylic acid and lactic acid by the mole that changes carbodiimide and methacrylic acid and lactic acid.Small-molecule substance in the course of reaction can be removed by the method for dialysis.Under the initiation of the Ammonium persulfate. (APS) of biocompatibility and tetramethylethylenediamine (TMEDA), chitosan derivatives can be cross-linked to form aquagel by two keys, and its gel time can be regulated by initiator concentration and reaction temperature.Particularly gel time is longer at normal temperatures, and the short characteristics of gel time are very beneficial for its application as syringeability hydrogel support under body temperature.The inventive method provides a kind of new method of simple possible for preparing polymerisable water-soluble chitosan and aquagel.The chitosan derivatives of gained and aquagel can be used for the syringeability hydrogel support in medicine controlled releasing system and the organizational project.
Description of drawings
Fig. 1 is the sketch map and the molecular structure of the polymerisable water-soluble chitosan of preparation;
Fig. 2 is the infrared spectrum of chitosan, CM and CML;
Fig. 3 is CML
1HNMR spectrogram, solvent are D
2O;
Fig. 4 is the change curve of MA grafting amount under the mol ratio of different EDAC and MA;
Fig. 5 is the change curve of LA grafting amount under the mol ratio of different EDAC and LA;
Fig. 6 is under 37 ℃, the gel time of 1%CML under different initiator concentrations;
Fig. 7 is under the different temperatures, the gel time of 1%CML under the 5mM initiator concentration;
Fig. 8 is under 37 ℃, the gel time of CML solution under the 5mM initiator concentration of variable concentrations;
Fig. 9 is under 37 ℃, the equilibrium swelling ratio of the aquagel that 1%CML prepares under different initiator concentrations in PBS;
Figure 10 is under 37 ℃, and 1%CML is in the degraded of the aquagel for preparing under the 5mM initiator in PBS and 1mg/ml lysozyme/PBS solution;
Figure 11 is the 3T3 cytotoxicity of chitosan derivatives, cell seeding density 2 * 10
4/ hole;
Figure 12 is the 3T3 cytotoxicity of aquagel, cell seeding density 10 * 10
4/ hole;
Figure 13 be chondrocyte in aquagel (a) cell growth curve and (b) dna content with the variation of incubation time, cell seeding density 400 * 10
4/ ml;
Figure 14 adopts CLSM to observe under the different incubation times growing state of chondrocyte in aquagel, cell seeding density 400 * 10
4/ ml adopts the fluorescein diethylester to dye living cells (solid arrow), and ethidium bromide dyes dead cell (dotted arrow); Wherein (a) is 1 day, (b) is 3 days, (c) is 6 days, (d) 9 days, (e) is 12 days.
Figure 15 adopts SEM to observe the form of chondrocyte in aquagel; Wherein (a) (b) is 3 days, (c), (d) is 12 days.
The specific embodiment
Example l: take by weighing chitosan 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 mixed liquor 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 Fig. 2 is seen in its structural change; MA grafting amount is about 23%, sees Fig. 4; Can be in water swelling.Above-mentioned 400mg CM is dissolved in the 50ml tri-distilled water that contains 210 μ l LA (0.2mmol), and the dissolving back adds 460mg EDAC (0.24mmol) fully.After this mixed liquor at room temperature stirred 24h, inserting mixed liquor 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 its structure is seen Fig. 2 and Fig. 3; LA grafting amount is about 52%, sees Fig. 5; In water, can dissolve fully.CML is dissolved in the tri-distilled water, is mixed with 1% (w/v) CML solution.Oxidant Ammonium persulfate. (APS) and Reducing agent tetramethylethylenediamine (TMEDA) are mixed with the aqueous solution of 1mol/l respectively.5 μ l APS solution and 5 μ l TMEDA solution are sequentially added in the 1%CML solution, mix homogeneously, the initiator ultimate density is 5mM.The mol ratio of APS and TMEDA is 1: 1.Mixed liquor moves in the mould by syringe, reacts down at 37 ℃ to form hydrogel.Its gel time is 5.5min, and the equilibrium swelling ratio of this gel in PBS is about 30.See Fig. 6, Fig. 7, Fig. 8 and Fig. 9.
Example 2: take by weighing chitosan 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 232.5mg EDAC (0.12mmol), then at room temperature, stirring reaction 24h.For removing unreacted MA and other micromolecule products, inserting mixed liquor 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. 4.
Example 3: MA grafting amount is about 23% 400mg CM and is dissolved in the 50ml tri-distilled water that contains 210 μ l LA (0.2mmol), the dissolving back adds 115mg EDAC (0.06mmol) fully.After this mixed liquor at room temperature stirred 24h, inserting mixed liquor 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. 5.
Example 4: CML (MA grafting amount 23% and LA grafting amount 52%) is dissolved in the water, is mixed with 2.5% CML solution, adding concentration then is the APS of 5mM and the TMEDA of 5mM, and the mol ratio of APS and TMEDA is 1: 1.37 ℃ of following placements are about 8min and can form not flowable aquagel, see Fig. 8.
Example 5: adding concentration in 1%CML (MA grafting amount 23% and LA grafting amount 52%) solution is the APS of 5mM and the TMEDA of 5mM, and the mol ratio of APS and TMEDA is 1: 1., form aquagel down at 37 ℃.Aquagel is immersed among the PBS behind the balance 24h, and (W weighs after the lyophilizing
1).Then gel is immersed in 1mg/ml lysozyme/PBS solution, every 1d or 3d sampling, lyophilizing, (W weighs
2).The surplus anharmonic ratio of lyophilizing hydrogel is calculated as follows:
Surplus anharmonic ratio=the W of hydrogel
2/ W
1* 100%
Chitosan gel rubber is degraded in PBS slowly, and in the presence of lysozyme, degraded in 8 days is seen Figure 10 fully.
Example 6: after taking by weighing 200mg CML (MA grafting amount 23% and LA grafting amount 52%) usefulness irradiation under ultraviolet ray 3h sterilization, be dissolved in 10ml and contain among the DMEM of 10% calf serum, and under 37 ℃, hatch 24h, standby.After with blood counting chamber the 3T3 cell being counted, plantation 3T3 cell in 96 well culture plates, every hole 2 * 10
4Individual cell, culture medium 200 μ l.After cultivating 24h,, add the culture medium continuation cultivation that 200 μ l contain CML with the culture medium sucking-off in the hole.The MTT activity of results of regular determination 3T3 cell is calculated the relative rate of increase.Estimate the cytotoxicity of CML.Employing contains the DMEM of 10% calf serum as negative control.Rate of increase computing formula is as follows relatively:
The relative rate of increase=OD
Sample/ OD
ContrastOD represents the absorbance of MTT test fluid in * 100% formula.
The 3T3 cytotoxicity of CML is 1 grade of toxicity, meets in the organizational project requirement to material.See Figure 11.
Example 7: the cytotoxicity that adopts the lye mensuration hydrogel of hydrogel.Take by weighing a certain amount of CML (MA grafting amount 23% and LA grafting amount 52%) with irradiation under ultraviolet ray 3h sterilization after, the tri-distilled water that adds sterilization earlier is mixed with 2% CML solution, adds double PBS then and is mixed with 1% CML/PBS solution.Take by weighing a certain amount of APS and TMEDA respectively with the solution that PBS is mixed with 1mol/l, sterilize with 0.22 μ m membrane filtration.Adding concentration is the APS of 5mM and the TMEDA of 5mM, and the mol ratio of APS and TMEDA is 1: 1, causes the preparation hydrogel down at 37 ℃, adds the DMEM that contains 10% calf serum then, hatches 24h under 37 ℃, and gel/DMEM is 100mg/ml, and is standby.Behind the 3T3 cell counting of collecting, every hole plantation 10 * 10
4Cell, every hole add the fresh DMEM of 200 μ l, behind the cultivation 24h, inhale and go culture medium, add lye, measure the cytoactive of different incubation times, calculate the relative rate of increase.With the DMEM that contains 10% calf serum as negative control.The cytotoxicity of aquagel is 1 grade of toxicity, meets in the organizational project requirement to material.See Figure 12.
Example 8: after 100mg CML (MA grafting amount 23% and LA grafting amount 52%) sterilization, be mixed with 1%CML/PBS solution, standby.Behind the cell counting of collecting, centrifugal, abandoning supernatant.Add 0.5ml PBS, blow and beat to cell and disperse fully, form the cell suspension of high concentration, standby.Add the 50 μ l 1M APS/PBS solution and the 50 μ l 1M TMEDA/PBS solution of the sterilization of 0.22 μ m membrane filtration in 10ml 1%CML/PBS solution in order respectively, the mol ratio of APS and TMEDA is 1: 1, mix homogeneously.Cell suspension is joined in this mixed solution mix homogeneously.Celliferous mixed liquor is expelled in the mould each mould 0.25ml with the 1ml syringe.After finishing, the incubator of at once mould being put into 37 ℃ is hatched.Behind the 10min, take out, visible solution loses flowability, and forms hydrogel.The hydrogel that will coat cell is at once transferred in 24 well culture plates, after adding 2ml contains the DMEM of 10% calf serum, puts into incubator.Behind the 30min, change liquid once.Behind 90min, abandoning supernatant, every hole adds the DMEM that 2ml contains 20% calf serum, puts into incubator and cultivates.Changed liquid once in per two days.Chondrocyte can keep active at hydrogel, but does not breed, and there is the part cell death in the later stage, sees Figure 13.Cellular morphology is circular, sees Figure 14 and Figure 15.
Claims (1)
1. be used for the preparation method of the injectable chitosan hydrogen of organizational project, 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 of grafting methacrylic acid and lactic acid;
3) chitosan with grafting methacrylic acid and lactic acid is dissolved in water or the phosphate buffer, compound concentration is 1%～2.5% the grafting methacrylic acid and the chitosan solution of lactic acid, add redox initiation system Ammonium persulfate. and tetramethylethylenediamine then, the mol ratio of Ammonium persulfate. and tetramethylethylenediamine is 1: 1, the ultimate density of initiator system is 2.5mM～15mM, and 25 ℃～45 ℃ are reacted the formation aquagel down.
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| JP5725862B2 (en) * | 2007-10-30 | 2015-05-27 | ヴィスコゲル アーベーＶｉｓｃｏｇｅｌ Ａｂ | Chitosan composition |
| CN102321248B (en) * | 2011-06-10 | 2013-04-24 | 冯淑芹 | Injectable temperature sensitive gel used for filling and repairing damaged tissues |
| CN102344523B (en) * | 2011-07-05 | 2013-12-25 | 金陵科技学院 | Preparation method of hydrogel for drug-loaded contact lens |
| CN102921046A (en) * | 2012-10-31 | 2013-02-13 | 川北医学院第二临床医学院 | Preparation method of chitosan hydrogel stent for tissue engineering |
| CN103601894B (en) * | 2013-10-25 | 2016-03-23 | 上海应用技术学院 | CP pearl of a kind of surface grafting chitosan and preparation method thereof |
| CN108310460B (en) * | 2018-02-02 | 2021-08-03 | 武汉大学 | Injectable high-strength temperature-sensitive modified chitin-based hydrogel and preparation method and application thereof |
| CN116671516A (en) * | 2023-08-04 | 2023-09-01 | 云南熙乐科技有限公司 | Preparation method of aqueous self-degradation antibacterial spray |
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|---|---|---|---|---|
| WO2005054440A2 (en) * | 2003-12-01 | 2005-06-16 | Tissue Engineering Consultants, Inc. | A biomimetic composition reinforced by a polyelectrolytic complex of hyaluronic acid and chitosan |
| WO2005097871A1 (en) * | 2004-03-22 | 2005-10-20 | Universite De Geneve | Pseudo-thermosetting neutralized chitosan composition forming a hydrogel and a process for producing the same |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005054440A2 (en) * | 2003-12-01 | 2005-06-16 | Tissue Engineering Consultants, Inc. | A biomimetic composition reinforced by a polyelectrolytic complex of hyaluronic acid and chitosan |
| WO2005097871A1 (en) * | 2004-03-22 | 2005-10-20 | Universite De Geneve | Pseudo-thermosetting neutralized chitosan composition forming a hydrogel and a process for producing the same |
Non-Patent Citations (3)
| Title |
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| 水溶性壳聚糖的制备与应用 赵文伟等，化学通报，第4期 1994 * |
| 水溶性壳聚糖的制备与应用 赵文伟等，化学通报，第4期 1994;组织工程用海藻酸盐水凝胶的研究进展 何淑兰等，化工进展，第23卷第11期 2004 * |
| 组织工程用海藻酸盐水凝胶的研究进展 何淑兰等，化工进展，第23卷第11期 2004 * |
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