CN103691001A - 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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- CN103691001A CN103691001A CN201310747234.8A CN201310747234A CN103691001A CN 103691001 A CN103691001 A CN 103691001A CN 201310747234 A CN201310747234 A CN 201310747234A CN 103691001 A CN103691001 A CN 103691001A
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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 is because the inorganic constituents of the sclerous tissues with human body is similar, there is good biocompatibility and biological activity, osteoinductive, degradability, after it implants, can form with osseous tissue around good bone bonding, and be widely used as bone renovating material.Clinically they can be generally for the bone defect repair of non-bearing and bearing position, therefore good mechanical property is a basic demand of ceramics bracket.But result of study shows dense calcium phosphate ceramics and can not effectively induce the formation of new bone tissue, only have the calcium phosphate ceramic of loose structure just to there is bone inducibility, therefore, ceramics bracket also should possess loose structure and duct and have good connectivity, to be conducive to growing into of tissue.Based on this, existing more pertinent literature is studied it, but the preparation method of support is all to take calcium phosphate powder body as raw material mostly, adds packed particle as pore creating material.From the output that is 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 guarantee.
The artificial bone tissue engineering bracket material of new type ideal is except possessing good biocompatibility, biological activity and good pore structure and mechanical property, the ability of sticking, breeding and breaking up that also should possess further promotion cell, promote the regeneration of osseous tissue, shorten the time of bone repair process.
Hydrogel is as a kind of important tissue engineering bracket material, and water content is abundant, very similar to tissue, has good biocompatibility and affinity.Somatomedin is a kind of cytokine that can stimulating cellular growth activity, by being combined with specific, high affine cell-membrane receptor, can regulate the growth of cell and the phenotypic expression of albumen.Drug sustained release system refer to a kind of can by control the rate of release of medicine and by medicine targeted release to reach the technology of its optimum efficiency.And drug controlled release system is mainly by a series of chemistry or physical method, specific pharmaceutical pack to be embedded in to macromolecule hydrogel to enter in body by injection or other modes again.In hydrogel, the medicine of load can slowly be discharged into the position that needed by human body is wanted under the combined effect of the slow degradation of self diffusion and macromolecule hydrogel, and can keep drug effect for a long time and stably.In addition, can carry out the degradation rate of suitable regulation and control timbering material by controlling the relative molecular mass of gel rubber material, thereby effectively reach, control the object discharging.In view of the shortcoming of existing technology, be necessary the technology of the three-dimensional porous calcium phosphate ceramic support of a kind of degradable growth factor slow-release of research preparation.
Summary of the invention
The object of this invention is to provide a kind of method of 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 realize by following technical scheme: a kind of method of preparing three-dimensional porous rack composite bed, adopts following step:
Step 1: take the three-dimensional porous rack that interface method is prepared, clean, dry, standby;
Step 2: polylactic acid layers compound, polylactic acid is dissolved in acetone, being made into mass volume ratio is 10% solution, add again 10% hydroxyapatite powder, after ultrasonic dispersion, discard bottom precipitation, three-dimensional porous rack after cleaning is immersed in polylactic acid solution, after one minute, takes out and dry, standby;
Step 3: prepare the oxidized sodium alginate solution of the RGD grafting that mass volume ratio is 10% and 2.5% N-succinyl-chitosan solution with the phosphate buffer solution of pH=7.4, by them, be 9/1～5/5 mixing by volume, 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 that is compounded with polylactic acid is immersed in hydrogel solution, make as early as possible it evenly coated, remove the unnecessary hydrogel in surface after taking out and carry out lyophilization, obtain 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.
In described hydrogel and oxidized sodium alginate microsphere, be all coated somatomedin.
Described somatomedin is at least a kind of in VEGF, cell differentiation somatomedin, transforming growth factor.
The present invention's advantage and effect compared with prior art: adopt polylactic acid to carry out compound to three-dimensional porous ceramics bracket; polylactic acid itself has good biocompatibility; after compound, at rack surface, form one deck protective film and do not affect the connectivity in its duct; to reach the object of fixed support surface particles; thereby prevent that support from having chip to fall into in-vivo tissue in implantation process, reaction causes inflammation.Compare with other support method for coating, the present invention at polylactic acid Solution Dispersion hydroxyapatite powder, hydroxyapatite has bone induction and bone conductibility on the one hand, consistent with the composition of support, on the other hand hydroxyapatite add to a certain extent the support mechanical property improving and there is production technology simple, the advantage that production cost is low.
The present invention innovation is also, the three-dimensional porous rack after compound to polylactic acid is compounded with the hydrogel that is written into somatomedin again, and in hydrogel, has added the sodium alginate micro ball of coated somatomedin to carry out modification.The somatomedin that sodium alginate micro ball is written into mainly plays a role in the bone reparation later stage, is conducive to prevent the release in advance of somatomedin after sodium alginate is coated, and ineffective.Along with hydrogel and sodium alginate degraded in vivo, the substep of realizing somatomedin discharges, and promotes tissue blood vessel and endothelialization growth and osteoblastic differentiation and proliferation, shortens the time of bone repair 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 growth factor slow-release that performance is better undoubtedly, and this also will become timbering material and the bone renovating material of better 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
(A) support photo before compound wherein; (B) the compound support photo of polylactic acid; (C) the support photo of polylactic acid and hydrogel two-layer compound (amplification is 50 times)
(D), (E), (F) are respectively the photo (amplification is 1000 times) of above-mentioned support corresponding position
Fig. 4 is that the present invention implanted in experimental animals after 4 weeks, bone reparation contrast situation different multiples photo
The specific embodiment
Preparation technology's flow process of the present invention as shown in Figure 1, except special statement, the raw materials used chemical pure that is, with embodiment, explain in detail the present invention below:
Embodiment 1: take 2.0g polylactic acid and be dissolved in 20mL to be made into mass volume ratio be 10% solution, then add the hydroxyapatite dry powder of mass fraction 10%, discard bottom precipitation after ultrasonic dispersion.Standby three-dimensional porous rack is placed in to this solution, after one minute, takes out and dry; With the phosphate buffer solution of pH=7.4, prepare the oxidized sodium alginate solution of arginine-glycine-aspartic acid sequence (RGD) grafting that mass volume ratio is 10% and 2.5% N-succinyl-chitosan solution.After sterilizing, 9mL N-succinyl-chitosan solution and 1mL RGD grafting oxidized sodium alginate solution are mixed, and rapid stirring is even.The three-dimensional porous rack that is compounded with before polylactic acid is put into this mixed solution, after being coated fast evenly, remove surperficial superfluous water gel; After lyophilization, obtain having the three-dimensional porous rack composite bed of growth factor slow-release.
Embodiment 2: take 2.0g polylactic acid and be dissolved in 20mL to be made into mass volume ratio be 10% solution, the hydroxyapatite dry powder that adds mass fraction 10%, after ultrasonic dispersion, discard bottom precipitation, standby three-dimensional porous rack is placed in to this solution, after one minute, take out and dry; With the phosphate buffer solution of pH=7.4, prepare the oxidized sodium alginate solution of the RGD grafting that mass volume ratio is 10% and 2.5% N-succinyl-chitosan solution.After sterilizing, 9mL N-succinyl-chitosan solution and 1mL RGD grafting oxidized sodium alginate solution are mixed, add the VEGF of 10ng/mL concentration
165the BMP-2 of (VEGF), 10ng/mL concentration (cell differentiation somatomedin) and 10mg be written into TGF-beat1 (transforming growth factor) sodium alginate micro ball and rapid stirring even, the three-dimensional porous rack that is compounded with before polylactic acid is put into this mixed solution, after being coated fast evenly, remove surperficial superfluous water gel; After lyophilization, obtain having the three-dimensional porous rack composite bed of growth factor slow-release.
Embodiment 3: take 2.0g polylactic acid and be dissolved in 20mL to be made into mass volume ratio be 10% solution, add the hydroxyapatite dry powder of mass fraction 10%, discard bottom precipitation after ultrasonic dispersion, porous support is placed in to dispersion liquid, after one minute, take out and dry; With the phosphate buffer solution of pH=7.4, prepare the oxidized sodium alginate solution of the RGD grafting that mass volume ratio is 10% and 2.5% N-succinyl-chitosan solution, after sterilizing, 8mLN-succinyl-chitosan solution and 2mL RGD grafting oxidized sodium alginate solution are mixed, add the VEGF of 10ng/mL concentration
165, 10ng/mL concentration BMP-2 and 10mg is written into TGF-beat1 sodium alginate micro ball and rapid stirring is even, the three-dimensional porous rack that is compounded with before polylactic acid is put into mixed solution, fast coated evenly after, remove surperficial superfluous water gel; After lyophilization, obtain having the three-dimensional porous rack composite bed of growth factor slow-release.
Embodiment 4: take 2.0g polylactic acid and be dissolved in 20mL to be made into mass volume ratio be 10% solution, add the hydroxyapatite dry powder of mass fraction 10%, discard bottom precipitation after ultrasonic dispersion, porous support is placed in to dispersion liquid, after one minute, take out and dry; With the phosphate buffer solution of pH=7.4, prepare the oxidized sodium alginate solution of the RGD grafting that mass volume ratio is 10% and 2.5% N-succinyl-chitosan solution, after sterilizing, 7mL N-succinyl-chitosan solution and 3mL RGD grafting oxidized sodium alginate solution are mixed, add the VEGF of 10ng/mL concentration
165, 10ng/mL concentration BMP-2 and 10mg is written into TGF-beat1 sodium alginate micro ball and rapid stirring is even, the three-dimensional porous rack that is compounded with before polylactic acid is put into mixed solution, fast coated evenly after, remove surperficial superfluous water gel; After lyophilization, obtain having the three-dimensional porous rack composite bed of growth factor slow-release.
Embodiment 5: take 2.0g polylactic acid and be dissolved in 20mL to be made into mass volume ratio be 10% solution, add the hydroxyapatite dry powder of mass fraction 10%, discard bottom precipitation after ultrasonic dispersion, porous support is placed in to dispersion liquid, after one minute, take out and dry; With the phosphate buffer solution of pH=7.4, prepare the oxidized sodium alginate solution of the RGD grafting that mass volume ratio is 10% and 2.5% N-succinyl-chitosan solution, after sterilizing, 6mL N-succinyl-chitosan solution and 4mL RGD grafting oxidized sodium alginate solution are mixed, add the VEGF of 10ng/mL concentration
165, 10ng/mL concentration BMP-2 and 10mg is written into TGF-beat1 sodium alginate micro ball and rapid stirring is even, the three-dimensional porous rack that is compounded with before polylactic acid is put into mixed solution, fast coated evenly after, remove surperficial superfluous water gel; After lyophilization, obtain having the three-dimensional porous rack composite bed of growth factor slow-release.
Embodiment 6: take 2.0g polylactic acid and be dissolved in 20mL to be made into mass volume ratio be 10% solution, add the hydroxyapatite dry powder of mass fraction 10%, discard bottom precipitation after ultrasonic dispersion, three-dimensional porous rack is placed in to dispersion liquid, after one minute, take out and dry; With the phosphate buffer solution of pH=7.4, prepare the oxidized sodium alginate solution of the RGD grafting that mass volume ratio is 10% and 2.5% N-succinyl-chitosan solution, after sterilizing, 5mL N-succinyl-chitosan solution and 5mL RGD grafting oxidized sodium alginate solution are mixed, add the VEGF of 10ng/mL concentration
165, 10ng/mL concentration BMP-2 and 10mg is written into TGF-beat1 sodium alginate micro ball and rapid stirring is even, the three-dimensional porous rack that is compounded with before polylactic acid is put into mixed solution, fast coated evenly after, remove surperficial superfluous water gel; After lyophilization, obtain having the three-dimensional porous rack composite bed of growth factor slow-release.
Embodiment 7: take 2.0g polylactic acid and be dissolved in 20mL to be made into mass volume ratio be 10% solution, add the hydroxyapatite dry powder of mass fraction 10%, discard bottom precipitation after ultrasonic dispersion, porous support is placed in to dispersion liquid, after one minute, take out and dry; With the phosphate buffer solution of pH=7.4, prepare the oxidized sodium alginate solution of the RGD grafting that mass volume ratio is 10% and 2.5% N-succinyl-chitosan solution, after sterilizing, 8mL N-succinyl-chitosan solution and 2mL RGD grafting oxidized sodium alginate solution are mixed, add the VEGF of 5ng/mL concentration
165, 5ng/mL concentration BMP-2 and 10mg is written into TGF-beat1 sodium alginate micro ball and rapid stirring is even, the three-dimensional porous rack that is compounded with before polylactic acid is put into mixed solution, fast coated evenly after, remove surperficial superfluous water gel; After lyophilization, obtain having the three-dimensional porous rack composite bed of growth factor slow-release.
Embodiment 8: take 2.0g polylactic acid and be dissolved in 20mL to be made into mass volume ratio be 10% solution, add the hydroxyapatite dry powder of mass fraction 10%, discard bottom precipitation after ultrasonic dispersion, three-dimensional porous rack is placed in to dispersion liquid, after one minute, take out and dry; With the phosphate buffer solution of pH=7.4, prepare the oxidized sodium alginate solution of the RGD grafting that mass volume ratio is 10% and 2.5% N-succinyl-chitosan solution, after sterilizing, 8mL N-succinyl-chitosan solution and 2mL RGD grafting oxidized sodium alginate solution are mixed, add the VEGF of 7.5ng/mL concentration
165, 7.5ng/mL concentration BMP-2 and 10mg is written into TGF-beat1 sodium alginate micro ball and rapid stirring is even, the three-dimensional porous rack that is compounded with before polylactic acid is put into mixed solution, fast coated evenly after, remove surperficial superfluous water gel; After lyophilization, obtain having the three-dimensional porous rack composite bed of growth factor slow-release.
Other embodiment and above-mentioned steps are similar, just add the dosage of somatomedin different.By comparing the composite aquogel of N-succinyl-chitosan and RGD grafting oxidized sodium alginate volume proportion from 9/1 to 5/5, find, the compressive strength of the N-succinyl-chitosan/oxidized sodium alginate hydrogel obtaining when proportioning is 8/2 is for the highest, and the pattern of hydrogel is also even.
In accompanying drawing 3, can be 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, the surface opposed flattened that also becomes.After being compounded with the hydrogel that is written into somatomedin, obviously can find out the closely complete hydrogel that has been coated of three-dimensional porous rack surface, and the granule of support itself and microsphere are well fixed on three-dimensional porous rack.
In accompanying drawing 4, can find out, in hydrogel, be written into the two-layer compound three-dimensional porous rack that promotes prepared by bone induction proliferation and differentiation and the somatomedin that promotes vascularization, implant animal body interior after 8 weeks, bone defect has been repaired complete, and promote that vascularization growth result is remarkable, and that the bone defect that is not written into somatomedin support still has is damaged, and there is more material to be still present in defect.
Claims (6)
1. a method of preparing three-dimensional porous rack composite bed, is characterized in that:
Step 1: take the three-dimensional porous rack that interface method is prepared, clean, dry, standby;
Step 2: polylactic acid layers compound, polylactic acid is dissolved in acetone, being made into mass volume ratio is 10% solution, add again 10% hydroxyapatite powder, after ultrasonic dispersion, discard bottom precipitation, three-dimensional porous rack after cleaning is immersed in polylactic acid solution, after one minute, takes out and dry, standby;
Step 3: prepare the oxidized sodium alginate solution of the RGD grafting that mass volume ratio is 10% and 2.5% N-succinyl-chitosan solution with the phosphate buffer solution of pH=7.4, by them, be 9/1～5/5 mixing by volume, 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 that is compounded with polylactic acid is immersed in hydrogel solution, make as early as possible it evenly coated, remove the unnecessary hydrogel in surface after taking out and carry out lyophilization, obtain the three-dimensional porous rack composite bed of growth factor slow-release.
2. a kind of method of 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 of preparing three-dimensional porous rack composite bed according to claim 1, is characterized in that, described hydrogel is the mixture of oxidized sodium alginate and N-succinyl-chitosan.
4. a kind of method of preparing three-dimensional porous rack composite bed according to claim 3, is characterized in that, described oxidized sodium alginate is the oxidized sodium alginate after arginine-glycine-aspartic acid sequence grafting.
5. a kind of method of preparing three-dimensional porous rack composite bed according to claim 1, is characterized in that, in described hydrogel and oxidized sodium alginate microsphere, has all been coated somatomedin.
6. a kind of method of preparing three-dimensional porous rack composite bed according to claim 5, is characterized in that, described somatomedin is at least a kind of in VEGF, cell differentiation somatomedin, transforming growth factor.
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| 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 |
| CN115105630A (en) * | 2022-08-08 | 2022-09-27 | 池州学院 | 3D printing material embedded with chitosan/gelatin composite hydrogel 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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