CN101849850B - Bionic in-situ regeneration repair nano sticking patch and preparation method and application thereof - Google Patents
Bionic in-situ regeneration repair nano sticking patch and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a bionic in-situ regeneration repair nano sticking patch, which is formed by overlapping an layer A with a cationic group material and a layer B with an anionic group material sequentially, wherein the sticking patch comprises 20 to 80 percent by mass of the A layer with the cationic group material and the balance of the layer B with the anionic group material; layer A with the cationic group material comprises at least one of polysaccharide with a cationic group, protein with the cationic group or opypeptide (polypeptide) with the cationic group; layer B with the anionic group material comprises at least one of polysaccharide with an anionic group, protein with the anionic group or opypeptide (polypeptide) with the anionic group; and the sticking patch comprises not more than 10 percent by mass of added functional factors and/or functional polypeptide. The raw materials used by the invention are materials which can be degraded in vitro and have biocompatibility. The sticking patch is flexible, has good tissue adhesivity, is suitable for concave-convex surfaces of visceral organs, has strong stretching resistance and air pressure resistance, is suitable for application of sticking, sealing, leak stoppage, hemostasia, isolation, repair, adhesion prevention and artificial meninges of defect tissues and can also be used as a slowly-releasing carrier of a medicament and a nano-grade tissue engineering support material.
Description
Technical field
The invention belongs to the biological medicine technology field, be specifically related to a kind of nano sticking patch that forms with anionic group material, functional factor and/or functional polypeptide, with the cation group material composite.And the preparation method of this nano sticking patch is provided and the pasting of defective tissue, sealing, leak stopping, hemostasis, isolation in surgical operation, repairs, prevent the purposes aspect adhesion, infection and the artificial meninges, also can be used as slow-released carrier and the nanoscale tissue engineering bracket material of medicine.
Background technology
Often need to be to the sealing of damaged and damaged tissue in surgical operation, repair and prevent adhesion, existing product has film and the gel products of carboxymethyl cellulose, chitosan, polylactic acid, collagen and Fibrin Glue, these products to a certain extent to the sealing of damaged tissue, repair and prevent that adhesion from having played positive effect.But because its product performance of function that had many defective effects, such as collagen protein sponge or film product thickness from 0.5mm to 5mm, cause product not soft, attach poor performance with tissue, product is met tissue fluid and is easily formed sticky shape, product loses hot strength, so that the doctor of Clinical Surgery operates inconvenience, for obtaining certain mechanical property, must add cross-linking agent such as glutaraldehyde etc., cross-linking agent also easily causes calcification to form except certain tissue toxicity is arranged; The untoward reaction such as human allergy that the animal-based protein series products exists larger security risk (such as virus, pathogenic microorganism etc.) and protein macromolecule to cause; Carboxymethyl cellulose, chitosan series products mechanical strength are inadequate, the vivo degradation time or too short or oversize; And the poly-lactic acid products degraded produces acid product etc.; Therefore, a kind of innovative product that overcomes existing film product defects of development has very great clinical value.
In the present invention dexterously biomaterial bionical, promote the nano combined technology of preparing of tissue repair function and biomaterial to combine, the physicochemical property of product is improved, soft, organize adhesion good, adapt to the concavo-convex of organ surface, stretch-proof and anti-air pressure performance are strong, can be used as the pasting of defective tissue, sealing, leak stopping, hemostasis, isolation, repair, prevent the purposes of adhesion, infection and artificial meninges aspect.Also can be used as slow-released carrier and the nanoscale tissue engineering bracket material of medicine.
Summary of the invention
The objective of the invention is the weak point according to above-mentioned existing product, a kind of bionic in-situ regeneration repair nano sticking patch is provided
,Preparation method and use, the present invention at first provides a kind of bionical composite Nano sticking patch that tissue defect is had the in-situ regeneration repairing effect; Next provides a kind of preparation method of this bionical composite Nano sticking patch; Moreover provide this bionical composite Nano sticking patch as the pasting of defective tissue, sealing, leak stopping, hemostasis, isolation, repair, prevent the purposes of adhesion and artificial meninges aspect.Also can be used as slow-released carrier and the nanoscale tissue engineering bracket material of medicine.
The object of the invention realizes being finished by following technical scheme:
A kind of bionic in-situ regeneration repair nano sticking patch, the constituted mode that it is characterized in that described sticking patch is to form by superposeing successively with the A layer of cation group material and with the B layer of anionic group material, wherein said successively stack refers to, by the constituted mode of [(A+B) n+A] or [(B+A) n+B], wherein stacking fold n can be positive integer.
The thickness of described A layer or B layer is nanoscale.
The mass percent that described A layer with the cation group material accounts for described sticking patch is 20%-80%, and all the other are the B layer with the anionic group material.
Described B layer is band anionic group material, but this group material has a biocompatible materials for vivo degradation, and described band anionic group material comprises at least a in band anionic group polysaccharide, band anionic group albumen or the band anionic group polypeptide (poly-peptide).
Describedly include but are not limited in carboxymethyl cellulose, carboxymethyl chitosan, hyaluronic acid, alginic acid, chondroitin sulfate and heparin polysaccharide and the derivant thereof one or more with the anionic group polysaccharide, and some macromolecular materials and the derivant such as polylactic acid, polyglycolic acid.
Described A layer is band cation group material, but this group material has a biocompatible materials for vivo degradation, and described band cation group material comprises at least a in band cation group polysaccharide, band cation group albumen or the band cation group polypeptide (poly-peptide).
Described band cation group polysaccharide includes but are not limited to chitosan and derivant thereof.
Described band cation group polypeptide (poly-peptide) includes but are not limited to polylysine and derivant thereof.
Described group albumen and polypeptide (poly-peptide) include but are not limited to polyampholyte collagen, elastin laminin and hydrolyzate thereof.
Contain the functional factor and/or the functional polypeptide that are no more than 10% mass percent in the described sticking patch.
Described functional polypeptide include but are not limited to contain-arginine-glycine-aspartic acid-polypeptide, contain-valine-glycine-valine-Ala-Pro-glycine-polypeptide or contain-isoleucine-lysine-valine-alanine-valine-polypeptide in one or more mixture.
Described functional factor includes but are not limited to fibronectin (FN), laminin，LN (LN), VEGF (VEGF), Fibrinogen (fg), epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGFs), bone morphogenetic protein (BMPs), insulin like growth factor (IGF), platelet derived growth factor (PDGF), thrombin and molecular weight are less than one or more the mixture in 10000 the oligochitosan.
A kind of preparation method of bionic in-situ regeneration repair nano sticking patch is characterized in that described method adopts following steps:
1) respectively preparation with the A solution of cation group material with the B solution of anionic group material;
2) at a flat board (plate material must be nontoxic, and water insoluble or sour, alkaline solution are processed except thermal source through degerming) upper spraying A or B solution, drying and forming-film; Spraying thereon or immersion B or A solution wash with water for injection again, replace so respectively spraying or soak A liquid or B liquid, wash with water for injection, and repetitive operation n-1 time sprays or immersion A or B solution, at last more thereon with water for injection washing, drying and forming-film; The thickness of controlling every layer of A or B is nanoscale, makes the described nano sticking patch such as claim 1-2;
3) interpolation of functional factor and polypeptide can be added on 1) in described A or the B solution, quality is 0% ~ 10% than concentration; Also can to prepare separately quality be the spraying of 0% ~ 10% solution C than concentration or be immersed in 2) the nano sticking patch surface that makes;
4) as developer, 1) preparation A and/or B solution in can add an amount of riboflavin;
Described nano sticking patch is used for the pasting of defective tissue, sealing, leak stopping, hemostasis, isolation, repairs, prevents the purposes of adhesion and artificial meninges aspect, also can be used for the slow-released carrier as medicine.
Advantage of the present invention is, the present invention is compound by bioabsorbable polymer material, the analog cell epimatrix, for the berthing of cell, grow, breed and vivosphere is provided and obtains nutrition, carry out metabolism, and the vivo degradation speed of the optimum organization controlled material by material; Being added with of functional factor and functional structure polypeptide helps sticking, stretching of cell, can regulate and control between the cell-matrix and cell-intercellular interaction, thereby realizes that material is at the multifunctional usage of the aspects such as the reparation of damaged tissue, regeneration; Make the physicochemical property of product be beneficial to adhesion and the gene expression of cell by alternately the superpose preparation method gained nanometer materials of polyelectrolyte of material, particularly strengthened the mechanical property of material, avoided the toxic and side effects of cross-linking agent.
The specific embodiment
By the following examples feature of the present invention and other correlated characteristic are described in further detail, so that technical staff's of the same trade understanding:
Embodiment 1. prepares one 12 cm diameter culture dishs, a built-in same diameter polymer material film (processing through washing, sterilization and depyrogenation), drying; Preparation is with the A solution (1mg/ml chitosan, 0.1mol/L acetic acid, 0.2mol/L NaCl, pH=4) of cation group material with the B liquid (1mg/ml carboxymethyl chitosan, 0.15mol/L NaCl, pH=6) of anionic group material respectively; Get part B liquid and add functional polypeptide (1 * 10
-4Mg/ml) be mixed with C liquid, the functional polypeptide amino acid sequence is: lysine-lysine-cysteine-Vitro By Serine/arginine-Gly-Asp-serine-cysteine-lysine-lysine; Mentioned solution is filled to respectively in the instantaneous spraying equipment of high pressure, at first toward the instantaneous spraying C of culture dish inner high voltage liquid, drying and forming-film; Then the instantaneous spraying A of high pressure liquid washes with water for injection; Then the instantaneous spraying B of high pressure liquid with the water for injection flushing, so alternately sprays A liquid, B liquid, flushing, repetitive operation 800 times; Follow the again instantaneous spraying A of high pressure liquid, wash with water for injection; The last instantaneous spraying C of high pressure liquid, with the water for injection flushing, drying is taken off film, and veneer washes with water for injection, and dried gets finished product.
Embodiment 2. prepares one 12 cm diameter culture dishs, a built-in same diameter polymer material film (processing through washing, sterilization and depyrogenation), drying; Preparation is with the A solution (2mg/ml chitosan, 0.1mol/L acetic acid, 0.2mol/L NaCl, pH=4) of cation group material with B liquid (alginic acid of 2mg/ml, 0.15mol/L NaCl, pH=6) the abbreviation B liquid of anionic group material respectively; Getting part B liquid adds epidermal growth factor (EGF) and (0.01mg/ml) is mixed with C liquid; Mentioned solution is filled to respectively in the instantaneous spraying equipment of high pressure, at first toward the instantaneous spraying C of culture dish inner high voltage liquid, drying and forming-film; Then the instantaneous spraying A of high pressure liquid washes with water for injection; Then the instantaneous spraying B of high pressure liquid with the water for injection flushing, so alternately sprays A liquid, B liquid, flushing, repetitive operation 200 times; Follow the again instantaneous spraying A of high pressure liquid, wash with water for injection; The last instantaneous spraying C of high pressure liquid, with the water for injection flushing, drying is taken off film, and veneer washes with water for injection, and dried gets finished product.
Embodiment 3. prepares one 12 cm diameter culture dishs, a built-in same diameter polymer material film (processing through washing and sterilization depyrogenation), and the water for injection flushing is dry; Preparation band cation group material (1mg/ml collagen, 0.1mol/L acetic acid, 0.2mol/L NaCl, pH=3.5) and with the B liquid (1mg/ml hyaluronic acid, 0.15mol/L NaCl, pH=6) of anionic group material respectively; Mentioned solution is filled to respectively in the instantaneous spraying equipment of high pressure, at first toward the instantaneous spraying A of culture dish inner high voltage liquid, drying, then the instantaneous spraying B of high pressure liquid with the water for injection flushing, so alternately sprays A liquid, B liquid, flushing, repetitive operation 500 times; The last instantaneous spraying A of high pressure liquid again, with the water for injection flushing, drying is taken off film, and veneer washes with water for injection, and dried gets finished product.
Embodiment 4. prepares one 12 cm diameter culture dishs, a built-in same diameter polymer material film (processing through washing, sterilization and depyrogenation), drying; Preparation is with the A solution (5mg/ml chitosan, 0.1mol/L acetic acid, 0.2mol/L NaCl, pH=4) of cation group material with the B liquid (1mg/ml hyaluronic acid, 0.15mol/L NaCl, pH=6) of anionic group material respectively; Get part B liquid and add thrombin (concentration: 2000IU/ml) be mixed with C liquid; Mentioned solution is filled to respectively in the instantaneous spraying equipment of high pressure, at first toward the instantaneous spraying C of culture dish inner high voltage liquid, drying and forming-film; Then the instantaneous spraying A of high pressure liquid washes with water for injection; Then the instantaneous spraying B of high pressure liquid with the water for injection flushing, so alternately sprays A liquid, B liquid, flushing, repetitive operation 300 times; Follow the again instantaneous spraying A of high pressure liquid, wash with water for injection; The last instantaneous spraying C of high pressure liquid, with the water for injection flushing, drying is taken off film, and veneer washes with water for injection, and dried gets finished product.
Embodiment 5. prepares one 12 cm diameter culture dishs, a built-in same diameter polymer material film (processing through washing, sterilization and depyrogenation), drying; Preparation is with A solution (1mg/ml polylysine, 0.1mol/L acetic acid, the 0.2mol/L NaCl of cation group material respectively, pH=4) with the B liquid (collagen of 1mg/ml of anionic group material, the VEGF of 10ug/ml, 0.15mol/L NaCl, pH=8) abbreviation B liquid; At first toward the interior dip-coating B liquid of culture dish, drying and forming-film is followed dip-coating A liquid, washes with water for injection, so alternately dip-coating B liquid, A liquid, flushing, repetitive operation 100 times, last dip-coating B liquid again, flushing, drying is taken off film, and veneer washes with water for injection, and dried gets finished product.
Press the prepared sample of embodiment (hereinafter referred to as test sample), its safety and efficacy carried out biology and Evaluating Mechanical Properties test:
1.Cell toxicity test:
Reference/technical standard: GB/T 16886.5-2003
Cell strain: L-929 cell (l cell)
Culture fluid: contain 10%(V/V) MEM of calf serum
Blank: with batch cell culture fluid
Negative control: high density polyethylene (HDPE)
Positive control: 5g/L phenol solution
Lixiviate medium: with criticizing the MEM that does not contain calf serum
Extraction time: 24 hours
Test sample lixiviate ratio: 1g/5ml
Test method: lixiviating solution test (mtt assay)
At 27 ℃, 5%CO
2Blank, negative control, positive control and test sample lixiviating solution contact the cell of adherent growth, cultivate 72h after, add MTT liquid, hatch 4h.After the absorption, add DMSO, under wavelength 630nm, each group is carried out absorbance measurement by spectrophotometer, and calculate the relative rate of increase of cell.
Result: the cytotoxicity of test sample: 0 grade
Conclusion: with reference to GB/T 16886.5-2003, this test sample no cytotoxicity.
2.The Intradermal irritant test
Reference/technical standard: GB/T 16886.10-2005
Experimental animal: healthy new zealand rabbit
Lixiviate medium: 0.9% sodium chloride injection
Test sample: material lixiviating solution
Negative control: with batch lixiviate medium
Route of exposure: intradermal injection
Judging quota: observe 24h, 48h, 72h erythema, the edema extent of reaction
The result: 24h, 48h, 72h injection site and surrounding skin are organized all and are reacted without erythema, edema after the injection, and test side dermoreaction is not more than the control sides dermoreaction.
Conclusion: with reference to GB/T 16886.10-2005, this test sample is without the Intradermal irritant reaction.
3.Acute systemic toxicity
Reference/technical standard: GB/T 16886.11-1997/ASTM F 750
Experimental animal: healthy mice
Lixiviate medium: 0.9% sodium chloride injection
Test sample: material lixiviating solution
Negative control: with batch lixiviate medium
Route of exposure: tail vein injection
Judging quota: observe 4h, 24h, 48h, 72h animal general state, toxicity performance and dead animal number.
The result: the reaction of test sample treated animal is not more than the negative control treated animal in the 72h observation period.
Conclusion: with reference to GB/T 16886.11-1997/ASTM F 750, this test sample acute systemic toxicity is the result meet the requirements.
4.Hemolytic test
Reference/technical standard: GB/T 16886.4-2003/GB/T 16175-1996
Experimental animal: healthy new zealand rabbit
Dilution anticoagulant Sanguis Leporis seu oryctolagi preparation: fresh anticoagulant Sanguis Leporis seu oryctolagi+0.9% sodium chloride injection
Negative control: 0.9% sodium chloride injection
Positive control: distilled water
Route of exposure: tail vein injection
Test method: test sample is immersed in 0.9% sodium chloride injection by a certain percentage, behind 37 ℃ of water bath heat preservation 30min, add 0.2ml and dilute fresh anticoagulant Sanguis Leporis seu oryctolagi, 37 ℃ of water bath heat preservation 60min.Get supernatant behind the centrifugal 5min of 2500rpm, measure its absorbance at the 545nm place with ultraviolet spectrophotometer, calculate hemolysis rate.
The result: the hemolysis rate of this test sample is 0.2%.
Conclusion: with reference to GB/T 16886.4-2003, the hemolytic test result of this test sample meets the requirement of medical material.
5.Mechanical property test
Reference/technical standard: M.J. Smith, M.J. McClure, S.A. Sell, et al. Suture-reinforced electrospun polydioxanone – elastin small-diameter tubes for use in vascular tissue engineering:A feasibility study. Acta Biomaterialia, 2008,4 (1): 58 ~ 66
Test method: at the tensile property of the omnipotent mechanical test aircraft measurements of M350-20KN sample: sensor 250 kgf, draw speed 10 mm/min, sample size is 3.
The result: the hot strength of this test sample is 589 ~ 612KPa, and elongation at break maintains in the 140-160% scope.
Conclusion: reference literature, this test sample mechanical property satisfy the demand of human body interior mechanics environment fully.
Can be used for the damaged aspect of tissue by above evaluation test and data declaration product of the present invention, and safe and effective.
Below can further prove result of use of the present invention by animal experiment:
30 of new zealand rabbits, body weight (2500 ± 40) gram, male and female are regardless of, and are divided at random two groups: 15 of A groups (being used for anatomic observation wound angiogenesis), 15 of B groups (being used for anatomic observation wound repair situation); Be ready to specification and be 1.5X1.5 centimetre product of the present invention; All animals adopts the anesthesia of injection urethane, anaesthetizes safe rear dorsal position, is fixed on the operating-table; Make the wound of 3 millimeters of diameters in the roughly the same position of each new zealand rabbit pulmonary; Product of the present invention is attached on the wound of 3 millimeters of experiment new zealand rabbit pulmonary diameters, sews up and dissect otch, postoperative sub-cage rearing, another day anatomic observation.
Result of the test: the intensity of product of the present invention can withstand the air pressure when breathing.Average 8 days, pulmonary's wound of A group experiment new zealand rabbit began to generate blood vessel, wound healing after B organizes average 27.5 days.Behind the wound healing, product of the present invention is natural degradation in vivo.Product of the present invention can adapt to the concavo-convex of organ surface, can touch wound, and not can with other tissue adhesions.Product of the present invention can be used for the softness such as stomach, liver and the internal organs that are difficult to sew up or easy hemorrhage internal organs, helps to shorten the surgical operation time, and shortens rehabilitation duration.
Claims (6)
1. bionic in-situ regeneration repair nano sticking patch, the constituted mode that it is characterized in that described sticking patch is to form by superposeing successively with the A layer of cation group material and with the B layer of anionic group material, wherein said successively stack refers to, constituted mode by [(A+B) n+A] or [(B+A) n+B], wherein stacking fold n is positive integer, described B layer is band anionic group material, described band anionic group material comprises band anionic group polysaccharide, with anionic group albumen or at least a with in the anionic group polypeptide, described A layer is band cation group material, described band cation group material comprises band cation group polysaccharide, with cation group albumen or at least a with in the cation group polypeptide, but described B layer with anionic group material and described A layer with the have biocompatible materials of cation group material for vivo degradation.
2. a kind of bionic in-situ regeneration repair nano sticking patch according to claim 1, the thickness that it is characterized in that described A layer or B layer is nanoscale.
3. a kind of bionic in-situ regeneration repair nano sticking patch according to claim 1 is characterized in that the mass percent that described A layer with the cation group material accounts for described sticking patch is 20%-80%, and all the other are the B layer with the anionic group material.
4. a kind of bionic in-situ regeneration repair nano sticking patch according to claim 1 is characterized in that described band anionic group polypeptide is band anion base reunion peptide, and described band cation group polypeptide is band cation radical reunion peptide.
5. a kind of bionic in-situ regeneration repair nano sticking patch according to claim 1 and 2, it is characterized in that described nano sticking patch is used for the pasting of defective tissue, sealing, leak stopping, hemostasis, isolation, repairs, prevents the purposes of adhesion and artificial meninges aspect, or be used for slow-released carrier and nanoscale tissue engineering bracket material as medicine.
6. the preparation method of a bionic in-situ regeneration repair nano sticking patch is characterized in that described method adopts following steps:
(1) respectively preparation with the A solution of cation group material with the B solution of anionic group material;
(2) at flat board spraying A or B solution, drying and forming-film, wherein plate material must be nontoxic, and water insoluble or sour, alkaline solution are processed except thermal source through degerming; Spraying thereon or immersion B or A solution wash with water for injection again, replace so respectively spraying or soak A liquid or B liquid, wash with water for injection, and repetitive operation n-1 time sprays or immersion A or B solution, at last more thereon with water for injection washing, drying and forming-film; The thickness of controlling every layer of A or B is nanoscale, makes the described nano sticking patch such as claim 1-2;
(3) add functional factor and polypeptide in (1) described A or B solution, mass percent concentration is 0%-10%; Or separately the function equipment factor and polypeptide mass percent concentration are the solution C spraying of 0%-10% or are immersed in the nano sticking patch surface that (2) make.
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| US10245034B2 (en) | 2015-08-31 | 2019-04-02 | Ethicon Llc | Inducing tissue adhesions using surgical adjuncts and medicants |
| US10569071B2 (en) | 2015-08-31 | 2020-02-25 | Ethicon Llc | Medicant eluting adjuncts and methods of using medicant eluting adjuncts |
| CN105902523A (en) * | 2016-03-14 | 2016-08-31 | 苏州博创康源生物技术有限公司 | Drug-loaded tissue adhesive film and preparation method thereof |
| CN105687165A (en) * | 2016-03-14 | 2016-06-22 | 苏州博创康源生物技术有限公司 | Tissue adhesive film loaded with protein or polypeptide drug and preparation method of tissue adhesive film |
| CN106038517A (en) * | 2016-03-14 | 2016-10-26 | 苏州博创康源生物技术有限公司 | Polysaccharide medicine loading tissue adhesion film and preparing method thereof |
| CN106038516A (en) * | 2016-03-14 | 2016-10-26 | 苏州博创康源生物技术有限公司 | Compound drug loading tissue pasting membrane and preparation method thereof |
| CN107913436A (en) * | 2016-10-09 | 2018-04-17 | 刘英芹 | Act on the bone and soft tissue synchronizing regeneration derivant of disability tissue in situ |
| CN110064074A (en) * | 2018-01-23 | 2019-07-30 | 苏州博创同康生物工程有限公司 | A kind of compound support frame material and its preparation method and application |
| CN109276759B (en) * | 2018-11-27 | 2020-11-24 | 北京航空航天大学 | Bionic antibacterial abdominal wall repair material |
| CN109731141B (en) * | 2019-01-17 | 2021-12-21 | 广州润虹医药科技股份有限公司 | Composite membrane for guiding tissue repair and preparation method and application thereof |
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