CN101574514A - Product for treating myocardial infarction based on chitosan aquagel and growth factor - Google Patents
Product for treating myocardial infarction based on chitosan aquagel and growth factor Download PDFInfo
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Abstract
The invention discloses an injectable myocardial tissue engineering product based on temperature-sensitive chitosan aquagel. Concretely, the temperature-sensitive chitosan aquagel is used as a support material and carries one or a plurality of different blood vessel growth promotion factors, such as bFGF, PDGF-BB, VEGF, and the like, and the condition that the temperature-sensitive chitosan aquagel restores a myocardial infarction area is observed after the temperature-sensitive chitosan aquagel is injected and transplanted to the specific area of an animal myocardial infarction model. The temperature-sensitive myocardial tissue engineering product established by applying the support material can promote the growth of myocardial capillary vessels and arteriolae, can increase the growth of sarcogenic fibres, can promote the regeneration of myocardial tissues, can increase the locular wall thickness of the infarction area, can remodel the shape of an original ventricle and can improve the function of the heart. The invention has simple operation technology and mild enforcement condition, provides a new product for the myocardial tissue engineering and has an important meaning for the clinical development of treating heart diseases to tissue engineering cardiac muscles.
Description
Technical field
The invention belongs to organizational project and regenerative medicine field, particularly a kind of temperature sensitive property aquagel is the product that carrier carries angiogenic growth factor treatment heart infarction.
Background of invention
The coronary artery disease that causes of atherosclerosis is the main cause of M ﹠ M in the world, wherein takes place the most serious with heart infarction.Although obtaining remarkable progress aspect the heart infarction treatment, myocardial cell can not regeneration behind the heart infarction, the infringement of left ventricular remodeling and cardiac function remains the main cause of heart failure.
Angiogenic growth factor comprises family and their hypotypes such as VEGF, FGF, PDGF, promotes angiogenesis by specific receptor.VEGF mainly acts on endotheliocyte.BFGF is the strong regulator gene of hyperplasia, vigor, differentiation and survival, comprises that in conciliation the growth of the target cell of endotheliocyte has great role.BFGF selects vasostimulant generation in vivo.PDFG mainly acts on the perivascular cell that comprises pericyte and vascular smooth muscle cell, and PDGF-BB plays stable rebirth blood vessel function.
Angiogenic growth factor can promote to be in the tissue regeneration of the different phase of hyperplasia and differentiation, promote the generation of blood vessel, thereby reparation and regeneration to damage location have great role, but this effect does not always reach in vivo, to be somatomedin have the very short half-life and spread faster and can not keep its biological activity at body one of its reason, therefore, must solve somatomedin slow release problem in vivo to strengthen its effect.
Based on material make angiogenic growth factor in vivo the method for slow release be a kind of very promising method.The previous research various carriers of report such as gelatin hydrogel, fibrin hydrogel and Matrigel etc. carry angiogenic growth factor and are applied to the ischemia body and promote the ischemia local vascular to generate and improve the ischemia local function cut much ice.
Chitosan is the copolymer of β-(1-4)-2-acetylaminohydroxyphenylarsonic acid D-glucosan and 2-amino-2-deoxidation-D-glucosan, have excellent biological compatibility and biodegradability, medical domain is many as bio-adhesive agent and wound adjuvant, plays styptic activity and accelerated wound healing.Chitosan can be compound in many ways with a lot of ions, polymer, forms multiple gel, as thermo-responsive hydro gel and heliosensitivity gel etc.There is researcher to be cross-linked into the glue effect by ultraviolet radiation; the aquagel of compound FGF is solidificated in the ischemic myocardium position of rabbit chronic myocardial infarction model; though played certain therapeutic effect; but this method causes the release rate of angiogenic growth factor in heart infarction local necrosis tissue and exist rate lower, can't provide the essence solution for the regeneration of heart infarction slough and vascular tissue's formation.After research report injectable chitosan hydrogen carried bFGF and is injected in S-D rat ischemia hind leg, bFGF slowly released, and local vascular forms and the collateral circulation reinforcement.
Discover chitosan and sodium glycerophosphate complex, the room temperature current downflow is good, gelation takes place during physiology body temperature (37 ℃), form hydrogel, it is a kind of good syringeability carrier, be mainly used in cartilage and bone tissue engineer at present, but as bio-carrier, the product that carries the growth factor for treating heart infarction does not appear in the newspapers so far as yet with it.
Summary of the invention
This product adopts temperature sensitive property aquagel to carry angiogenic growth factor as bio-carrier first and makes up syringeability organizational project cardiac muscle product, it is expelled in the dancing heart ischemic injuries cardiac muscle, slowly discharge in vivo to reach angiogenic growth factor, and keep better biological activity, promote heart infarction position capillary network and arteriolar generation, and promote into myofibrillar generation, improve the blood flow of ischemic tissue, repair large tracts of land heart infarction cicatrix and improve the purpose of cardiac function.
Therefore, first goal of the invention of the present invention is for the heart infarction treatment provides a kind of new product, specifically, provides a kind of syringeability tissue engineering product of angiogenic growth factor.
In a specific embodiments, the syringeability tissue engineering product of described angiogenic growth factor is based on the angiogenic growth factor mixture of aquagel.Wherein, injectable aquagel is temperature sensitive property aquagel, it is characterized in that under the room temperature condition for liquid, be expelled in the body after, be condensed into solid-state in the short time.
In another embodiment, angiogenic growth factor can be existing any angiogenic growth factor, for example, comprises all angiogenic growth factors of FGF, VGEF, PDGF.Wherein, FGF comprises aFGF, bFGF etc. in interior FGF family; VEGF is the VEGF family that comprises VEGF-A, VEGF-B etc.; PDGF comprises the PDGF family of PDFG-AA, PDGF-AB, PDGF-BB etc.Also in another embodiment, both can use single angiogenic growth factor and aquagel to prepare the syringeability tissue engineering product, and also can unite and use above-mentioned two or more angiogenic growth factors arbitrarily and aquagel to prepare the syringeability tissue engineering product.
Second goal of the invention of the present invention provides the method for the syringeability tissue engineering product of the above-mentioned angiogenic growth factor of preparation, comprising:
(1) preparation of temperature sensitive property aquagel
Raw material is prepared as follows: 1%-3% chitosan (C) solution; 10%-50% sodium (GP) aqueous solution; 1%-4% hydroxyethyl-cellulose (HEC) solution.
Temperature sensitive property aquagel preparation method: with above-mentioned three kinds of solution according to C: GP: HEC=4: 1: 1 ratio at room temperature is mixed into liquid state, can be condensed into solid-state under 37 ℃ of conditions.
(2) preparation of angiogenic growth factor solution:
A.bFGF solution: with the PBS dissolving of pH value 7.2-7.8;
B.PDGF-BB: be dissolved in aseptic 100mM acetic acid and 0.1%BSA, can be further diluted by other solution;
C. if VEGF solution is dissolved in deionized water, can further be diluted by other solution;
D. the preparation of other angiogenic growth factor solution sees detailed description for details.
(3) based on the preparation of temperature sensitive property aquagel and angiogenic growth factor treatment heart infarction product:
Get a certain amount of (for example, the angiogenic growth factor solution for preparing 20ng-500ug), with the abundant mix homogeneously of temperature sensitive property aquagel for preparing, hybrid mode is as follows:
A. single angiogenic growth factor solution and temperature sensitive property aquagel
As: the VEGF solution of the PDGF-BB solution of the bFGF solution of 20ng-500ug and the mixing of temperature sensitive property aquagel, 20ng-500ug and the mixing of temperature sensitive property aquagel, 20ng-500ug and the mixing of temperature sensitive property aquagel etc.
B. angiogenic growth factor solution is united and temperature sensitive property aquagel
As: the bFGF combined PD GF-BB solution of the bFGF associating VEGF solution of 20ng-500ug and the mixing of temperature sensitive property aquagel, 20ng-500ug and the mixing of temperature sensitive property aquagel etc.
Should be understood that, the concrete injection rate of angiogenic growth factor should be decided according to different animals kind and its myocardial infarction damaged area size, in view of different manufacturers with the source angiogenic growth factor purity different with effectiveness, the Different Individual case is different with sensitivity to the dependency of medicine, and the injection rate of angiogenic growth factor should according to circumstances be decided.Among the present invention, the scope of selecting for use usually is 250ng-50ug.
Beneficial effect
This product, carries one or more angiogenic growth factors and carries out the site of myocardial infarction injection for curing as bio-carrier with temperature sensitive property aquagel.In vivo, angiogenic growth factor can slowly discharge along with the aquagel degraded, thereby efficiently solve it in vivo because of spreading too fast and the too fast problems such as activity reduction that cause of degraded, promoted the vascularization of heart infarction district, increasing blood supplies, increase ventricle wall volume and infarcted region chamber wall thickness, reinvent original ventricle shape, improve cardiac function.This product has the feature of syringeability, and invasive is little, has directionality, is convenient to the treatment operation, can avoid the risks that operation brought such as cardiac arrest, extracorporeal circulation.Operating procedure of the present invention is simple, implementation condition is gentle, for cardiac muscle tissue engineering provides a kind of new product, engineered cardiac muscle treatment cardiac disorder clinical is carried out significant.
Description of drawings
Fig. 1. solid gel shape aquagel
Fig. 2. the histocompatibility of aquagel detects (HE dyeing)
A.1 all, * 40; B.2 all, * 40; C.3 all, * 40; D.4 all, * 40;
Fig. 3. rat heart infarction Preparation of model
A. rat coronary artery anterior descending branch ligation;
B. electrocardio diagram ST section is raised the formation of prompting heart infarction;
Fig. 4. back infarction middle part ventricle wall thickness situation (HE dyeing) around aquagel injection and the simple heart infarction
A. aquagel injection, * 4; B. simple heart infarction, * 4;
Fig. 5. aquagel carries angiogenic growth factor injection, aquagel injection and infarction middle part angiogenesis situation (VIII factor immunohistochemical staining) after 4 weeks
A. aquagel carries angiogenic growth factor injection, * 10; B. the aquagel injection * 10; C.PBS injection * 10;
The specific embodiment
Only further describe the present invention now with mode with reference to following non-restrictive example.But should be appreciated that the following examples only as illustration, should be by any way when doing the restriction overall to the invention described above.Unless other explanation is arranged, the traditional molecular biology in embodiments of the invention use this area, cytobiology, tissue engineering or the like.These technology are that the technical staff knows, and detailed explanation is arranged in the literature.Referring to, for example, Lanza, Langer and Vacanti " Principlesof Tissue Engineering " (2006); Atala and Lanza " Methods of Tissue Engineering " (2006).
Embodiment 1: the preparation of aquagel
The 200mg chitosan places the 10ml small beaker, add 0.1% acetic acid 9ml, after treating that chitosan dissolves fully, high pressure steam sterilization (121 ℃, 20min), the 5g sodium places the 10ml small beaker, add 10ml culture medium and 0.22 μ m membrane filtration degerming, after the 250mg hydroxyethyl-cellulose irradiation degerming, be dissolved into 2.5% solution with the 10ml culture medium, the three can make pH value for neutral by 4: 1: 1 mixed, the liquid gel of room temperature good fluidity can be solid gel shape (Fig. 1) in about 10 minutes in the placement incubator.
Embodiment 2: the histocompatibility of aquagel detects
Aquagel 0.1ml injection is implanted in the leg muscle of S-D rat, respectively at putting to death in 1,2,3,4 weeks, gets 2 at every turn, carries out paraffin section after the taking-up specimen, makes the situation that inflammatory reaction is observed in HE dyeing.The visible aquagel of postoperative 1 week section extensively is distributed in the cardiac muscular tissue gap, a little inflammatory cell that mixes (Fig. 2 A) therebetween; There is comparatively significantly inflammatory cell infiltration (Fig. 2 B) around the visible aquagel of postoperative 2 week group; Postoperative 3 week, group failed to see tangible aquagel, but inflammatory cell infiltration still obviously (Fig. 2 C); Do not see that tangible aquagel sample material is residual in 4 weeks of postoperative, but the still visible a small amount of inflammatory cell infiltration (Fig. 2 D) in injection site.
Embodiment 3: the selection of animal and the preparation of heart infarction
S-D rat (about 225-300g) is used in experiment, and pentobarbital sodium (40mg/kg body weight) carries out intraperitoneal anesthesia, is connected with animal trace respirator.The row thoracotomy is cut off pericardium, fully exposes heart.At left auricle lower edge 2mm place, prick with 0-7 silk thread seam.Left locular wall loses color after the ligation, and ventricular wall motion weakens (Fig. 3 A).Cardiac monitoring sees that the ST section that I, II lead obviously raises, and shows that coronary artery infarction animal model prepares successfully (Fig. 3 B).
Embodiment 4: the short angiogenesis of cardiac muscle Function detection of aquagel
20 of S-D rats about 240g are divided into aquagel injection group, simple myocardial infarction model group, PBS injection group at random.8 of picked at random S-D rats, male and female are not limit, pentobarbital sodium 35mg/kg body weight intraperitoneal injection of anesthesia.By the rat coronary artery ligation, cause myocardial infarction after 30 minutes, myocardial ischemia place injection aquagel.In 1,2,4 weeks of postoperative, put to death respectively and carry out histology, immunohistochemical detection.The experimental group that as seen result injects aquagel has improved the ventricle wall thickness at heart infarction position than other two matched groups, and (Fig. 4) vessel density at infarction position increases, and the myocardium quantity of survival increases, and fibrosis alleviates.
Embodiment 5: aquagel carries somatomedin and promotes angiogenesis of cardiac muscle and improve the cardiac function detection
18 of S-D rats about 240g are divided at random aquagel carries somatomedin injection group, PBS carries somatomedin injection group, PBS injection group.6 of picked at random S-D rats, male and female are not limit, pentobarbital sodium 35mg/kg body weight intraperitoneal injection of anesthesia.By the rat coronary artery ligation, cause myocardial infarction 30min after, somatomedin (as 1ug) aquagel is carried in myocardial ischemia place injection.Carry out the ultrasoundcardiogram monitoring after the injection two days later.In 4 weeks of postoperative, carry out putting to death animal after ultrasoundcardiogram and hemodynamics detect respectively and carry out histology, immunohistochemical detection.As seen the result injects experimental group that aquagel carries somatomedin and obviously improves than the cardiac function of other two matched groups, heart infarction position ventricle wall thickness obviously improves, the vessel density at infarction position obviously increases (Fig. 5), and the myocardium quantity of survival increases, and fibrosis alleviates.
Claims (10)
1. syringeability tissue engineering product that is used for the treatment of myocardial infarction, it is characterized in that described product is based on the angiogenic growth factor mixture of aquagel, wherein aquagel is injectable temperature sensitive property aquagel, be liquid at ambient temperature, after being expelled in the body, the short time is internal energy be condensed into solid-state.
2. by the syringeability tissue engineering product described in the claim 1, wherein angiogenic growth factor both can be any among FGF, VGEF, the PDGF, also can be among FGF, VGEF, the PDGF any two kinds or more than.
3. by the syringeability tissue engineering product described in the claim 2, wherein said FGF comprises aFGF, bFGF etc. in interior FGF family.
4. by the syringeability tissue engineering product described in the claim 2, wherein said VEGF is the VEGF family that comprises VEGF-A, VEGF-B etc.
5. by the syringeability tissue engineering product described in the claim 2, wherein said PDGF comprises the PDGF family of PDFG-AA, PDGF-AB, PDGF-BB etc.
6. by the arbitrary described syringeability tissue engineering product of claim 1-5, wherein myocardial infarction is an acute myocardial infarction, or old myocardial infarction.
7. be used for preparing the method for the arbitrary described syringeability tissue engineering product of claim 1-6, comprise:
(1) preparation of temperature sensitive property aquagel:
Prepare 1%-3% chitosan (C) solution; 10%-50% sodium (GP) aqueous solution; 1%-4% hydroxyethyl-cellulose (HEC) solution;
Above-mentioned then three kinds of solution are according to C: GP: HEC=4: 1: 1 ratio at room temperature is mixed into liquid state, can be condensed into solid-state under 37 ℃ of conditions;
(2), select suitable buffer preparation angiogenic growth factor solution according to common practise;
(3) based on the preparation of temperature sensitive property aquagel and angiogenic growth factor treatment heart infarction product:
Get the angiogenic growth factor solution for preparing, with the abundant mix homogeneously of temperature sensitive property aquagel for preparing.
8. the described method of claim 7, wherein in step (2),
A. if bFGF solution, the PBS of available pH value 7.2-7.8 dissolving;
B. if PDGF-BB solution dissolves in aseptic 100mM acetic acid and 0.1%BSA, can further be diluted by other solution;
C. if VEGF solution is dissolved in deionized water, can further be diluted by other solution;
D. if other angiogenic growth factor solution can use suitable conventional soln to dissolve.
9. claim 7 or 8 described methods, wherein in step (3):
BFGF solution and the temperature sensitive property aquagel of 20ng-500ug can be mixed;
Or the PDGF-BB solution of 20ng-500ug and temperature sensitive property aquagel mixed;
Or the VEGF solution of 20ng-500ug and the mixing of temperature sensitive property aquagel etc.
10. claim 7 or 8 described methods, wherein in step (3):
BFGF associating VEGF solution and the temperature sensitive property aquagel of 20ng-500ug are mixed;
Or the bFGF combined PD GF-BB solution of 20ng-500ug and the mixing of temperature sensitive property aquagel etc.
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| CN102038977A (en) * | 2010-11-29 | 2011-05-04 | 中国人民解放军军事医学科学院基础医学研究所 | Tissue engineered cardiac muscle using OPF (Oligo(Poly(Ethylene Glycol) Fumarate) hydrogel as a carrier and preparation method thereof |
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| CN111214706A (en) * | 2018-11-25 | 2020-06-02 | 中国科学院大连化学物理研究所 | Temperature-sensitive composite gel emulsion and application thereof |
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- 2008-05-06 CN CNA2008100941531A patent/CN101574514A/en active Pending
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| CN102038977A (en) * | 2010-11-29 | 2011-05-04 | 中国人民解放军军事医学科学院基础医学研究所 | Tissue engineered cardiac muscle using OPF (Oligo(Poly(Ethylene Glycol) Fumarate) hydrogel as a carrier and preparation method thereof |
| CN102139096A (en) * | 2011-03-22 | 2011-08-03 | 华东理工大学 | Injectable biological active factor composition and preparation method and application thereof |
| CN103087533A (en) * | 2013-01-17 | 2013-05-08 | 黑龙江省重生生物科技有限公司 | Biological gel membrane for cell culture as well as preparation method and application thereof |
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| US10589002B2 (en) | 2014-04-14 | 2020-03-17 | University of Pittsburgh—of the Commonwealth System of Higher Education | Biodegradable, thermally responsive injectable hydrogel for treatment of ischemic cardiomyopathy |
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| US11123454B2 (en) | 2014-04-14 | 2021-09-21 | University of Pittsburgh—of the Commonwealth System of Higher Education | Biodegradable, thermally responsive injectable hydrogel for treatment of ischemic cardiomyopathy |
| CN105148322A (en) * | 2015-06-16 | 2015-12-16 | 深圳大学 | Injectable hydrogel and method for preparing same |
| CN106667899B (en) * | 2015-11-11 | 2020-11-10 | 南开大学 | Injectable hydrogels incorporating IGF-1C polypeptides |
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| US11590162B2 (en) | 2016-10-07 | 2023-02-28 | University of Pittsburgh—of the Commonwealth System of Higher Education | Biodegradable, antioxidant, thermally responsive injectable hydrogel and uses therefor |
| US11389569B2 (en) | 2017-04-03 | 2022-07-19 | University of Pittsburgh—of the Commonwealth System of Higher Education | Biodegradable, porous, thermally responsive injectable hydrogel as soft tissue defect filler |
| CN108404207A (en) * | 2018-05-28 | 2018-08-17 | 浙江大学 | The preparation method of temperature sensitive aquagel cell factor compound rest |
| CN111214706A (en) * | 2018-11-25 | 2020-06-02 | 中国科学院大连化学物理研究所 | Temperature-sensitive composite gel emulsion and application thereof |
| CN110721344B (en) * | 2019-11-21 | 2020-12-11 | 北京帝康医药投资管理有限公司 | Injectable biological gel for promoting myocardial repair and preparation method thereof |
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| CN114585396B (en) * | 2020-05-08 | 2023-02-28 | 四川大学 | Injectable hydrogel with anti-inflammatory and repair promoting functions, preparation method thereof and application thereof in heart repair |
| CN115887622A (en) * | 2023-01-30 | 2023-04-04 | 东莞市人民医院 | Puerarin composite hydrogel material, and preparation method and application thereof |
| CN115957181A (en) * | 2023-02-08 | 2023-04-14 | 江苏省人民医院（南京医科大学第一附属医院） | Injectable temperature-sensitive hydrogel suitable for myocardial infarction repair and preparation method thereof |
| CN115957181B (en) * | 2023-02-08 | 2023-09-15 | 江苏省人民医院（南京医科大学第一附属医院） | Injectable thermosensitive hydrogel suitable for myocardial infarction repair and preparation method thereof |
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