CN103691001B - Method for preparing three-dimensional porous stent composite layer - Google Patents
Method for preparing three-dimensional porous stent composite layer Download PDFInfo
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- CN103691001B CN103691001B CN201310747234.8A CN201310747234A CN103691001B CN 103691001 B CN103691001 B CN 103691001B CN 201310747234 A CN201310747234 A CN 201310747234A CN 103691001 B CN103691001 B CN 103691001B
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Abstract
The invention discloses a method for preparing a three-dimensional porous stent composite layer, and belongs to the technical field of biomaterials. In order to prevent inflammatory reaction caused by drop of scraps of a stent due to a dynamic action, a coating film is required on the surface of a porous ceramic stent, and addition of hydroxyapatite powder fills up a voidness defect of the surface layer of the stent so as to improve the strength of the stent; a porous calcium phosphate ceramic stent is compounded by a polylactic acid solution added with hydroxyapatite and a growth factor-loaded hydrogel solution; growth factors loaded in the hydrogel can be slowly released to a human part needing the growth factors under the joint action of own diffusion and slow degradation of polymeric hydrogel so as to promote vascular endothelial growth of a tissue and proliferation and differentiation of an osteoblast; and the stent is gradually degraded and eventually transformed into a bone; and the method is mainly used for preparing the three-dimensional porous stent composite layer.
Description
Technical field
The invention belongs to technical field of biological materials, especially bone renovating material and engineering material of bone tissue composite bed and preparation method thereof.
Background technology
Calcium phosphate ceramic due to the inorganic constituents of the sclerous tissues with human body similar, there is good biocompatibility and biological activity, osteoinductive, degradability, good bone bonding can be formed with the osseous tissue of surrounding after it implants, and be widely used as bone renovating material.They can be commonly used to the bone defect healing of non-bearing and bearing position clinically, therefore good mechanical property is a basic demand of ceramics bracket.But result of study shows that dense calcium phosphate ceramics effectively can not induce the formation of new bone tissue, the calcium phosphate ceramic of loose structure is only had just to have self-bone grafting ability, therefore, ceramics bracket also should possess loose structure and there is good connectivity in duct, to be conducive to growing into of tissue.Based on this, existing more pertinent literature is studied it, but mostly the preparation method of support be all with calcium phosphate powder body for raw material, add packed particle as pore creating material.From the output being blended into support of powder, operation is comparatively loaded down with trivial details, often in order to obtain high strength, just must sacrifice the porosity of support, and the connectivity in duct also cannot effectively ensure.
The artificial bone tissue engineering bracket material of new type ideal is except should possessing excellent biocompatibility, biological activity and good pore structure and mechanical property, also should possess the ability of sticking, breed and breaking up promoting cell further, promote the regeneration of osseous tissue, shorten the time of Bone Defect Repari process.
Hydrogel is as a kind of important tissue engineering bracket material, and water content is enriched, very similar to tissue, has good biocompatibility and affinity.Somatomedin be a kind of can the cytokine of stimulating cellular growth activity, by being combined with specific, high affine cell-membrane receptor, the growth of cell and the phenotypic expression of albumen can be regulated.Drug sustained release system refer to a kind of can by control medicine rate of release and by medicine targeted release to reach the technology of its optimum efficiency.And drug controlled release system is mainly embedded in macromolecule hydrogel by a series of chemistry or physical method specific pharmaceutical pack and is entered in body by injection or other modes.The medicine of load in hydrogel can be released slowly into the position that needed by human body is wanted under the combined effect of the slow degraded of self diffusion and macromolecule hydrogel, and can keep permanent and stable drug effect.In addition, the degradation rate of suitable regulation and control timbering material can be carried out by the relative molecular mass controlling gel rubber material, thus effectively reach the object of Co ntrolled release.In view of the shortcoming of existing technology, be necessary the technology of the three-dimensional porous calcium phosphate ceramic support studying a kind of degradable growth factor slow-release of preparation.
Summary of the invention
The object of this invention is to provide a kind of method preparing three-dimensional porous rack composite bed, it can realize growth factor slow-release effectively, promotes the proliferation and differentiation of blood vessel and endothelialization growth and osteocyte.
The object of the invention is to be realized by following technical scheme: a kind of method preparing three-dimensional porous rack composite bed, adopts following step:
Step one: take the three-dimensional porous rack that interface method is prepared, cleaning, oven dry, for subsequent use;
Step 2: the compound of polylactic acid layers, polylactic acid is dissolved in acetone, being made into mass volume ratio is 10% solution, add the hydroxyapatite powder of 10% again, bottom precipitation is discarded after ultrasonic disperse, three-dimensional porous rack after cleaning is immersed in PLA solution, takes out after one minute and dry, for subsequent use;
Step 3: preparing mass volume ratio with the phosphate buffer solution of pH=7.4 is the oxidized sodium alginate solution of the RGD grafting of 10% and the N-succinyl-chitosan solution of 2.5%, be 9/1 ~ 5/5 mixing by volume by them, obtain hydrogel solution, in this solution, add the somatomedin of 5 ~ 10ng/mL concentration and the sodium alginate micro ball of coated somatomedin, be uniformly mixed rapidly;
Step 4: the three-dimensional porous rack being compounded with polylactic acid is immersed in hydrogel solution, makes it evenly coated as early as possible, remove the hydrogel of excess surface after taking out and carry out lyophilization, obtaining the three-dimensional porous rack composite bed of growth factor slow-release.
Described composite bed is surperficial two-layer compound, and can be degradable.
Described hydrogel is the mixture of oxidized sodium alginate and N-succinyl-chitosan.
Described oxidized sodium alginate is the oxidized sodium alginate after arginine-glycine-aspartic acid sequence grafting.
All coated somatomedin in described hydrogel and oxidized sodium alginate microsphere.
Described somatomedin is at least the one in VEGF, cell differentiation somatomedin, transforming growth factor.
The present invention's advantage compared with prior art and effect: adopt polylactic acid to carry out compound to three-dimensional porous ceramics bracket; polylactic acid itself has good biocompatibility; form one deck protective film at rack surface after compound and do not affect the connectivity in its duct; to reach the object of fixed support surface particles; thus prevent support from implantation process, having chip to fall into in-vivo tissue, cause inflammation reaction.Compared with other support method for coating, the present invention has disperseed hydroxyapatite powder at PLA solution, hydroxyapatite has self-bone grafting and bone conductibility on the one hand, consistent with the composition of support, on the other hand hydroxyapatite add the support mechanical property that improves to a certain extent and to have production technology simple, the advantage that production cost is low.
The present invention's innovation is also, the three-dimensional porous rack after polylactic acid compound is compounded with again to the hydrogel being loaded into somatomedin, and the sodium alginate micro ball adding coated somatomedin in hydrogel carries out modification.The somatomedin that sodium alginate micro ball is loaded into mainly plays a role in the Bone Defect Repari later stage, is conducive to the release in advance preventing somatomedin after sodium alginate is coated, and ineffective.Along with the degraded in vivo of hydrogel and sodium alginate, realize the substep release of somatomedin, promote that tissue blood vessel and endothelialization grow and osteoblastic differentiation and proliferation, shorten the time of Bone Defect Repari process, and can be degradable, finally transform skeletonization completely.Therefore, the present invention provides the preparation method of the two-layer compound three-dimensional porous rack of the more excellent growth factor slow-release of performance undoubtedly, and this also will become timbering material and the bone renovating material of more excellent bone tissue engineer.
Accompanying drawing is described as follows:
Fig. 1 is preparation technology's flow process of the present invention
Fig. 2 is the electron scanning micrograph of sodium alginate micro ball of the present invention
Fig. 3 is electron scanning micrograph of the present invention
Support photo wherein before (A) compound; (B) the support photo of polylactic acid compound; (C) the support photo (amplification is 50 times) of polylactic acid and hydrogel two-layer compound
(D), (E), (F) are respectively the photo (amplification is 1000 times) of above-mentioned support corresponding position
Fig. 4 is that the present invention implants in experimental animals after 4 weeks, Bone Defect Repari contrast situation different multiples photo
Detailed description of the invention
Preparation technology's flow process of the present invention as shown in Figure 1, except special statement, is raw materials usedly chemical pure, states the present invention in detail below by embodiment:
Embodiment 1: take 2.0g polylactic acid and be dissolved in 20mL and be made into the solution that mass volume ratio is 10%, then add the hydroxyapatite dry powder of mass fraction 10%, discard bottom sediment after ultrasonic disperse.Three-dimensional porous rack for subsequent use is placed in this solution, takes out after one minute and dry; Preparing mass volume ratio with the phosphate buffer solution of pH=7.4 is the oxidized sodium alginate solution of arginine-glycine-aspartic acid sequence (RGD) grafting of 10% and the N-succinyl-chitosan solution of 2.5%.By 9mL N-succinyl-chitosan solution and the mixing of 1mL RGD grafting oxidized sodium alginate solution after sterilizing, and rapid stirring is even.The three-dimensional porous rack being compounded with polylactic acid is before put into this mixed solution, quick coated evenly after, remove excess surface hydrogel; The three-dimensional porous rack composite bed with growth factor slow-release is obtained after lyophilization.
Embodiment 2: take 2.0g polylactic acid and be dissolved in 20mL and be made into the solution that mass volume ratio is 10%, add the hydroxyapatite dry powder of mass fraction 10%, discard bottom sediment after ultrasonic disperse, three-dimensional porous rack for subsequent use is placed in this solution, take out after one minute and dry; Preparing mass volume ratio with the phosphate buffer solution of pH=7.4 is the oxidized sodium alginate solution of the RGD grafting of 10% and the N-succinyl-chitosan solution of 2.5%.By 9mL N-succinyl-chitosan solution and the mixing of 1mL RGD grafting oxidized sodium alginate solution after sterilizing, add the VEGF of 10ng/mL concentration
165the BMP-2 (cell differentiation somatomedin) of (VEGF), 10ng/mL concentration and 10mg is loaded into TGF-beat1 (transforming growth factor) sodium alginate micro ball and rapid stirring is even, the three-dimensional porous rack being compounded with polylactic acid is before put into this mixed solution, quick coated evenly after, remove excess surface hydrogel; The three-dimensional porous rack composite bed with growth factor slow-release is obtained after lyophilization.
Embodiment 3: take 2.0g polylactic acid and be dissolved in 20mL and be made into the solution that mass volume ratio is 10%, add the hydroxyapatite dry powder of mass fraction 10%, discard bottom sediment, porous support is placed in dispersion liquid after ultrasonic disperse, takes out after one minute and dries; Preparing mass volume ratio with the phosphate buffer solution of pH=7.4 is the oxidized sodium alginate solution of the RGD grafting of 10% and the N-succinyl-chitosan solution of 2.5%, by 8mLN-succinyl-chitosan solution and the mixing of 2mL RGD grafting oxidized sodium alginate solution after sterilizing, add the VEGF of 10ng/mL concentration
165, 10ng/mL concentration BMP-2 and 10mg to be loaded into TGF-beat1 sodium alginate micro ball rapid stirring even, the three-dimensional porous rack being compounded with polylactic acid is before put into mixed solution, quick coated evenly after, remove excess surface hydrogel; The three-dimensional porous rack composite bed with growth factor slow-release is obtained after lyophilization.
Embodiment 4: take 2.0g polylactic acid and be dissolved in 20mL and be made into the solution that mass volume ratio is 10%, add the hydroxyapatite dry powder of mass fraction 10%, discard bottom sediment, porous support is placed in dispersion liquid after ultrasonic disperse, takes out after one minute and dries; Preparing mass volume ratio with the phosphate buffer solution of pH=7.4 is the oxidized sodium alginate solution of the RGD grafting of 10% and the N-succinyl-chitosan solution of 2.5%, by 7mL N-succinyl-chitosan solution and the mixing of 3mL RGD grafting oxidized sodium alginate solution after sterilizing, add the VEGF of 10ng/mL concentration
165, 10ng/mL concentration BMP-2 and 10mg to be loaded into TGF-beat1 sodium alginate micro ball rapid stirring even, the three-dimensional porous rack being compounded with polylactic acid is before put into mixed solution, quick coated evenly after, remove excess surface hydrogel; The three-dimensional porous rack composite bed with growth factor slow-release is obtained after lyophilization.
Embodiment 5: take 2.0g polylactic acid and be dissolved in 20mL and be made into the solution that mass volume ratio is 10%, add the hydroxyapatite dry powder of mass fraction 10%, discard bottom sediment, porous support is placed in dispersion liquid after ultrasonic disperse, takes out after one minute and dries; Preparing mass volume ratio with the phosphate buffer solution of pH=7.4 is the oxidized sodium alginate solution of the RGD grafting of 10% and the N-succinyl-chitosan solution of 2.5%, by 6mL N-succinyl-chitosan solution and the mixing of 4mL RGD grafting oxidized sodium alginate solution after sterilizing, add the VEGF of 10ng/mL concentration
165, 10ng/mL concentration BMP-2 and 10mg to be loaded into TGF-beat1 sodium alginate micro ball rapid stirring even, the three-dimensional porous rack being compounded with polylactic acid is before put into mixed solution, quick coated evenly after, remove excess surface hydrogel; The three-dimensional porous rack composite bed with growth factor slow-release is obtained after lyophilization.
Embodiment 6: take 2.0g polylactic acid and be dissolved in 20mL and be made into the solution that mass volume ratio is 10%, add the hydroxyapatite dry powder of mass fraction 10%, discard bottom sediment, three-dimensional porous rack is placed in dispersion liquid after ultrasonic disperse, takes out after one minute and dries; Preparing mass volume ratio with the phosphate buffer solution of pH=7.4 is the oxidized sodium alginate solution of the RGD grafting of 10% and the N-succinyl-chitosan solution of 2.5%, by 5mL N-succinyl-chitosan solution and the mixing of 5mL RGD grafting oxidized sodium alginate solution after sterilizing, add the VEGF of 10ng/mL concentration
165, 10ng/mL concentration BMP-2 and 10mg to be loaded into TGF-beat1 sodium alginate micro ball rapid stirring even, the three-dimensional porous rack being compounded with polylactic acid is before put into mixed solution, quick coated evenly after, remove excess surface hydrogel; The three-dimensional porous rack composite bed with growth factor slow-release is obtained after lyophilization.
Embodiment 7: take 2.0g polylactic acid and be dissolved in 20mL and be made into the solution that mass volume ratio is 10%, add the hydroxyapatite dry powder of mass fraction 10%, discard bottom sediment, porous support is placed in dispersion liquid after ultrasonic disperse, takes out after one minute and dries; Preparing mass volume ratio with the phosphate buffer solution of pH=7.4 is the oxidized sodium alginate solution of the RGD grafting of 10% and the N-succinyl-chitosan solution of 2.5%, by 8mL N-succinyl-chitosan solution and the mixing of 2mL RGD grafting oxidized sodium alginate solution after sterilizing, add the VEGF of 5ng/mL concentration
165, 5ng/mL concentration BMP-2 and 10mg to be loaded into TGF-beat1 sodium alginate micro ball rapid stirring even, the three-dimensional porous rack being compounded with polylactic acid is before put into mixed solution, quick coated evenly after, remove excess surface hydrogel; The three-dimensional porous rack composite bed with growth factor slow-release is obtained after lyophilization.
Embodiment 8: take 2.0g polylactic acid and be dissolved in 20mL and be made into the solution that mass volume ratio is 10%, add the hydroxyapatite dry powder of mass fraction 10%, discard bottom sediment, three-dimensional porous rack is placed in dispersion liquid after ultrasonic disperse, takes out after one minute and dries; Preparing mass volume ratio with the phosphate buffer solution of pH=7.4 is the oxidized sodium alginate solution of the RGD grafting of 10% and the N-succinyl-chitosan solution of 2.5%, by 8mL N-succinyl-chitosan solution and the mixing of 2mL RGD grafting oxidized sodium alginate solution after sterilizing, add the VEGF of 7.5ng/mL concentration
165, 7.5ng/mL concentration BMP-2 and 10mg to be loaded into TGF-beat1 sodium alginate micro ball rapid stirring even, the three-dimensional porous rack being compounded with polylactic acid is before put into mixed solution, quick coated evenly after, remove excess surface hydrogel; The three-dimensional porous rack composite bed with growth factor slow-release is obtained after lyophilization.
Other embodiments and above-mentioned steps similar, the dosage just adding somatomedin is different.Find by comparing N-succinyl-chitosan and the RGD grafting oxidized sodium alginate volume proportion composite aquogel from 9/1 to 5/5, the compressive strength of the N-succinyl-chitosan/oxidized sodium alginate hydrogel obtained when proportioning is 8/2 is for the highest, and the pattern of hydrogel is also even.
Can be in accompanying drawing 3 and find out, the three-dimensional porous rack surface before compound is more coarse, and calcium phosphate ceramic granule is exposed on surface, and after being compounded with polylactic acid, rack surface is coated with thin film, and surface also becomes opposed flattened.After being compounded with the hydrogel being loaded into somatomedin, obviously can find out the coated hydrogel that three-dimensional porous rack intimate surface is complete, and the granule of support itself and microsphere are well fixed on three-dimensional porous rack.
Can find out in accompanying drawing 4, be loaded in hydrogel and promote self-bone grafting proliferation and differentiation and promote two-layer compound three-dimensional porous rack prepared by the somatomedin of vascularization, in implantation animal body after 8 weeks, Cranial defect position has been repaired complete, and promote that vascularization growth result is remarkable, and still there is defect at the Cranial defect position not being loaded into somatomedin support, and more material is had still to be present in defect.
Claims (4)
1. prepare a method for three-dimensional porous rack composite bed, it is characterized in that:
Step one: take the three-dimensional porous rack that interface method is prepared, cleaning, oven dry, for subsequent use;
Step 2: the compound of polylactic acid layers, polylactic acid is dissolved in acetone, being made into mass volume ratio is 10% solution, add the hydroxyapatite powder of 10% again, bottom precipitation is discarded after ultrasonic disperse, three-dimensional porous rack after cleaning is immersed in PLA solution, takes out after one minute and dry, for subsequent use;
Step 3: preparing mass volume ratio with the phosphate buffer solution of pH=7.4 is the oxidized sodium alginate solution of the RGD grafting of 10% and the N-succinyl-chitosan solution of 2.5%, be 9/1 ~ 5/5 mixing by volume by them, obtain hydrogel solution, in this solution, add the somatomedin of 5 ~ 10ng/mL concentration and the oxidized sodium alginate microsphere of coated somatomedin, be uniformly mixed rapidly;
Step 4: the three-dimensional porous rack being compounded with polylactic acid is immersed in hydrogel solution, makes it evenly coated as early as possible, remove the hydrogel of excess surface after taking out and carry out lyophilization, obtaining the three-dimensional porous rack composite bed of growth factor slow-release.
2. a kind of method preparing three-dimensional porous rack composite bed according to claim 1, is characterized in that, described composite bed is surperficial two-layer compound, and can be degradable.
3. a kind of method preparing three-dimensional porous rack composite bed according to claim 1, is characterized in that, all coated somatomedin in described hydrogel and oxidized sodium alginate microsphere.
4. a kind of method preparing three-dimensional porous rack composite bed according to claim 1, is characterized in that, described somatomedin is at least the one in VEGF, cell differentiation somatomedin, transforming growth factor.
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| CN110507852A (en) * | 2019-08-28 | 2019-11-29 | 中南大学湘雅二医院 | A kind of osseous tissue renovating material |
| CN112759788B (en) * | 2019-11-06 | 2023-06-30 | 天津理工大学 | Heat-conducting composite hydrogel with solid-liquid interpenetrating network structure and preparation method thereof |
| CN115105630B (en) * | 2022-08-08 | 2023-08-18 | 池州学院 | 3D printing material embedded with chitosan/gelatin composite hydrogel and preparation method thereof |
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