CN103638559A - Water-insoluble ultrafine fibroin powder/polylactic acid composite porous scaffold material and preparation method thereof - Google Patents
Water-insoluble ultrafine fibroin powder/polylactic acid composite porous scaffold material and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of biomedicine high-molecular materials, and relates to a water-insoluble ultrafine fibroin powder-enhanced macromolecular composite porous scaffold material for tissue engineering and a preparation method thereof. The preparation method comprises the following steps: dissolving polylactic acid into dioxane or a mixed solvent of dioxane/dimethyl sulfoxide, so as to obtain a polylactic acid solution; adding a certain mass of water-insoluble ultrafine fibroin powder to the polylactic acid solution; ultrasonically defoaming after evenly mixing, and then injecting the polylactic acid solution into a polytetrafluoroethylene mold; freezing at -10 DEG C to -50 DEG C for 1-5 hours; and finally, carrying out freeze drying in a freeze dryer at -45 to -55 DEG C, so as to obtain the water-insoluble ultrafine fibroin powder/polylactic acid composite porous scaffold material. The preparation technology disclosed by the invention is simple, and conditions are easy to control and the prepared porous scaffold material has better biocompatibility, is high in porosity, strong in hydrophilia, and beneficial to conglutination, growth and propagation of cells, has better mechanical strength, and can be widely applied to the field of tissue engineering.
Description
Technical field
The invention belongs to field of biomedical polymer materials, be specifically related to the water-insoluble fibroin powder body/polylactic acid of a kind of used in tissue engineering composite porous support material and preparation method thereof.
Background technology
Tissue engineering bracket material refer to can with organize active somatic cell be combined and can implantable bioartificial the material of body, it is the basic boom of engineered tissue.Biodegradable polymer material is in tissue engineering bracket material, to study at present a more class material, it has good biocompatibility, and after implantable bioartificial body, cell its surface adhesion, propagation formative tissue process in, under the effect of body fluid, enzyme, cell etc., degrade, become small-molecule substance and be absorbed or excrete by metabolism.Polylactic acid is a kind of biodegradable aliphatic polyester, its catabolite is lactic acid, nontoxic and can excrete with human body metabolism, in tissue engineering bracket material, receive much concern, but because of drawbacks limit such as its hydrophilic are poor, degradation rate is slow, cellular affinity is poor its in the application aspect bioengineered tissue.
Natural polymer, as protein, polysaccharide etc. are owing to having good biocompatibility, and biodegradability, has wide practical use at aspects such as clinical repair and tissue engineering brackets.In recent years, people utilize protein and polysaccharide to carry out modification to polylactic acid, prepare various polylactic acid composite porous support materials.Chinese patent notification number CN 101703808A, the day for announcing is on May 12nd, 2010, name is called in ' a kind of porous collagen/polylactic acid bracket ' and discloses a kind of porous collagen/polylactic acid bracket.The method, by the acetone mixed solution lyophilizing at low temperatures of collagen protein, polylactic acid and epithelical cell growth factor EGF, is prepared porous compound support frame.Due to adopt at low temperatures can not be icing acetone, utilize the aperture that lyophilizing can not fine control porous material.Chinese patent notification number CN 1958081A, the day for announcing is on November 12nd, 2008, name is called and in ' Method for preparing 3d porous bracket of chitosan-copolymer of poly lactic acid ', discloses a kind of Method for preparing 3d porous bracket of chitosan-copolymer of poly lactic acid, chitosan is dissolved in lactic acid, add sodium chloride or potassium chloride, then vacuum intensification copolymerization, and solids is carried out to washing by soaking postlyophilization with deionized water, obtain chitosan-polylactic acid copolymer porous support.But the degree of this chemical reaction and the percent grafting of polylactic acid are wayward.Chinese patent notification number CN 101053670; the day for announcing is on October 17th, 2007; name is called ' a kind of fibroin and polymeric lactic acid compound film and preparation method thereof ' and discloses a kind of method of utilizing silk fibroin solution and polylactic acid solution combination drying to prepare fibroin/polymeric lactic acid compound film, and this composite membrane can be used for the aspects such as Wound surface protection film, artificial skin.But need to carry out the processing such as refine and dissolving to fibroin albumen, obtain water-soluble fibroin, fibroin molecular weight is little, easily stripping from prepared composite membrane.
Summary of the invention
The object of the present invention is to provide a kind of water-insoluble fibroin powder body to strengthen polylactic acid composite porous support material and preparation method thereof, the method is that the water-insoluble superfine silk powder body obtaining by physical grinding is mixed with polylactic acid solution, through lyophilization, prepare water-insoluble superfine silk powder body/polylactic acid composite porous support material.
The present invention is realized by the following technical solutions, water-insoluble superfine silk powder body/polylactic acid composite porous support material and preparation method thereof, and its preparation method comprises the following steps:
1) at room temperature, polylactic acid is dissolved in the mixed solvent of dioxane or dioxane/dimethyl sulfoxide, obtains weight percentage and be 3 ~ 10% polylactic acid solution;
2) water-insoluble superfine silk powder body is added in polylactic acid solution, mix homogeneously, after ultrasonic deaeration, inject politef mould, freezing 1 ~ 5h at-10 ~-50 ℃, finally lyophilizing at-45 ~-55 ℃ in freeze dryer, obtains water-insoluble superfine silk powder body/polylactic acid composite porous support material of the present invention.
Above-mentioned dioxane/dimethyl sulfoxide mixed solvent refers to that dioxane and dimethyl sulfoxide are mixed to get for 99:1 ~ 50:50 by volume.
The addition of above-mentioned water-insoluble fibroin powder body is to be 5:95 ~ 50:50 metering by the mass ratio of water-insoluble fibroin powder body and polylactic acid.
Above-mentioned polylactic acid refers to poly (l-lactic acid), poly-D-ALPHA-Hydroxypropionic acid or poly-D, Pfansteihl, and the molecular weight of polylactic acid is 1 * 10
5~ 5 * 10
5.
The mean diameter of above-mentioned water-insoluble superfine silk powder body is 2.4 μ m.
Owing to adopting technique scheme, preparation method of the present invention compared with prior art, tool has the following advantages and beneficial effect: fibroin is native protein, there is good biocompatibility and biodegradability, the superfine silk powder body that method by fibroin fiber by physical grinding obtains has retained the original biochemical structure of fibroin albumen and active integrity, and good mechanical property, overcome water-soluble fibroin molecular weight little, undercapacity and the easily shortcoming of stripping.This preparation method technique is simple, mild condition, the composite porous support material obtaining has good mechanical property, good hydrophilic property, porosity is high, has improved the hydrophilic of polylactic acid bracket, has increased cell adhesion forces, for cell provides good microenvironment in its superficial growth, propagation and differentiation, can be widely used in tissue engineering bracket material.
Accompanying drawing explanation
Fig. 1 is the particle size distribution figure of composite porous support material of the present invention water-insoluble fibroin powder body used.
Fig. 2 is that the water absorption rate of composite porous support material of the present invention is schemed over time.
Fig. 3 is the compressive strength figure of composite porous support material of the present invention.
Fig. 4 is the scanning electron microscope (SEM) photograph of composite porous support material of the present invention.
The specific embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail.
embodiment 1
take respectively the poly-D of 0.3g, (molecular weight is 1 * 10 to Pfansteihl
5) and 9.7g dioxane, be added in 100ml beaker magnetic agitation 1h.After polylactic acid dissolves completely, taking 0.045g fibroin powder body adds in polylactic acid solution, continue to stir 3h, after mix homogeneously, ultrasonic 30min deaeration, pours in politef mould, and the refrigerator and cooled of putting into-20 ℃ is frozen 1 hour, then lyophilizing at-45 ~-55 ℃ of temperature, the demoulding obtains superfine silk powder body/polylactic acid composite porous support material.The porosity of prepared composite porous support material is 90.5%.Fig. 2 a is the water absorption rate curve over time of prepared composite porous material, and Fig. 3 a is prepared composite porous support material compressive strength, and Fig. 4 a is the scanning electron microscope (SEM) photograph of prepared composite porous support material.
(molecular weight is 1.8 * 10 to take respectively 0.5g poly (l-lactic acid)
5) and 9.5g dioxane and dimethyl sulfoxide mixed solvent (80/20, volume ratio), be added in 100ml beaker magnetic agitation 1h.After polylactic acid dissolves completely, taking 0.1g fibroin powder body adds in polylactic acid solution, continue to stir 3h, after mix homogeneously, ultrasonic 30min deaeration, pours in politef mould, and the refrigerator and cooled of putting into-30 ℃ is frozen 2 hours, then lyophilizing at-45 ~-55 ℃ of temperature, the demoulding obtains superfine silk powder body/polylactic acid composite porous support material.The porosity of prepared composite porous support material is 88.6%.
embodiment 3
(molecular weight is 1 * 10 to take respectively the poly-D-ALPHA-Hydroxypropionic acid of 0.6g
5) and 5.4g dioxane and dimethyl sulfoxide mixed solvent (90/10, volume ratio), be added in 100ml beaker magnetic agitation 2h.After polylactic acid dissolves completely, taking 0.18g fibroin powder body adds in polylactic acid solution, continue to stir 3h, after mix homogeneously, ultrasonic 40min deaeration, pours in politef mould, and the refrigerator and cooled of putting into-40 ℃ is frozen 4 hours, then lyophilizing at-45 ~-55 ℃ of temperature, the demoulding obtains superfine silk powder body/polylactic acid composite porous support material.The porosity of prepared composite porous support material is 89.7%.Fig. 2 b is the water absorption rate curve over time of prepared composite porous material, and Fig. 3 b is prepared composite porous support material compressive strength, and Fig. 4 b is the scanning electron microscope (SEM) photograph of prepared composite porous support material.
(molecular weight is 3.2 * 10 to take respectively the poly-D-ALPHA-Hydroxypropionic acid of 0.6g
5) and 6.9g dioxane and dimethyl sulfoxide mixed solvent (60/40, volume ratio), be added in 100ml beaker magnetic agitation 2h.After polylactic acid dissolves completely, taking 0.3g fibroin powder body adds in polylactic acid solution, continue to stir 3h, after mix homogeneously, ultrasonic 30min deaeration, then pours in politef mould, and the refrigerator and cooled of putting into-50 ℃ is frozen 2 hours, finally lyophilizing at-45 ~-55 ℃ of temperature, the demoulding obtains superfine silk powder body/polylactic acid composite porous support material.The porosity of prepared composite porous support material is 87.3%.Fig. 4 d is the scanning electron microscope (SEM) photograph of prepared composite porous support material.
embodiment 5
Take respectively the poly-D of 0.4g, (molecular weight is 5 * 10 to Pfansteihl
5) and 9.6g dioxane, be added in 100ml beaker magnetic agitation 3h.After polylactic acid dissolves completely, taking 0.16g fibroin powder body adds in polylactic acid solution, continue to stir 3h, after mix homogeneously, ultrasonic 20min deaeration, then pours in politef mould, and the refrigerator and cooled of putting into-30 ℃ is frozen 4 hours, finally lyophilizing at-45 ~-55 ℃ of temperature, the demoulding obtains superfine silk powder body/polylactic acid composite porous support material.The porosity of prepared composite porous support material is 91.6%.
embodiment 6
Take respectively the poly-D of 0.6g, (molecular weight is 1.2 * 10 to Pfansteihl
5) and 5.4g dioxane and dimethyl sulfoxide mixed solvent (50/50, volume ratio), be added in 100ml beaker magnetic agitation 2h.After polylactic acid dissolves completely, taking 0.3g fibroin powder body adds in polylactic acid solution, continue to stir 3h, after mix homogeneously, ultrasonic 40min deaeration, pours in politef mould, and the refrigerator and cooled of putting into-40 ℃ is frozen 4 hours, then lyophilizing at-45 ~-55 ℃ of temperature, the demoulding obtains superfine silk powder body/polylactic acid composite porous support material.The porosity of prepared composite porous support material is 88.5%.Fig. 2 c is the water absorption rate curve over time of prepared composite porous material, and Fig. 3 c is prepared composite porous support material compressive strength, and Fig. 4 c is the scanning electron microscope (SEM) photograph of prepared composite porous support material.
embodiment 7
(molecular weight is 4 * 10 to take respectively 0.4g poly (l-lactic acid)
5) and 7.6g dioxane and dimethyl sulfoxide mixed solvent (99/1, volume ratio), be added in 100ml beaker magnetic agitation 2h.After polylactic acid dissolves completely, taking 0.12g fibroin powder body adds in polylactic acid solution, continue to stir 3h, after mix homogeneously, ultrasonic 30min deaeration, then pours in politef mould, and the refrigerator and cooled of putting into-30 ℃ is frozen 2 hours, finally lyophilizing at-45 ~-55 ℃ of temperature, the demoulding obtains superfine silk powder body/polylactic acid composite porous support material.The porosity of prepared composite porous support material is 89.4%.
As can be seen from Figure 1, the mean diameter of the superfine silk powder body that the method by physical grinding obtains is in 2.4 μ m left and right, from Fig. 2 and Fig. 3, can find out, water-insoluble fibroin powder body add and along with the increase of addition, hydrophilic and the mechanical strength of prepared composite porous support material obviously strengthen.Therefore, superfine silk powder body add the cell adhesion forces of having improved composite porous material, for cell provides good microenvironment in its superficial growth, propagation and differentiation.
Claims (5)
1. water-insoluble superfine silk powder body/polylactic acid composite porous support material and preparation method thereof, its preparation method comprises the following steps:
1) at room temperature, polylactic acid is dissolved in the mixed solvent of dioxane or dioxane/dimethyl sulfoxide, obtains weight percentage and be 3 ~ 10% polylactic acid solution;
2) water-insoluble superfine silk powder body is added in polylactic acid solution, mix homogeneously, after ultrasonic deaeration, inject politef mould, freezing 1 ~ 5h at-10 ~-50 ℃, finally lyophilizing at-45 ~-55 ℃ in freeze dryer, obtains water-insoluble superfine silk powder body/polylactic acid composite porous support material of the present invention.
2. water-insoluble superfine silk powder body/polylactic acid composite porous support material according to claim 1 and preparation method thereof, is characterized in that described dioxane/dimethyl sulfoxide mixed solvent refers to that dioxane and dimethyl sulfoxide are mixed to get for 99:1 ~ 50:50 by volume.
3. water-insoluble superfine silk powder body/polylactic acid composite porous support material according to claim 1 and preparation method thereof, the addition that it is characterized in that water-insoluble fibroin powder body is to be 5:95 ~ 50:50 metering by the mass ratio of water-insoluble fibroin powder body and polylactic acid.
4. water-insoluble superfine silk powder body/polylactic acid composite porous support material according to claim 1 and preparation method thereof, is characterized in that described polylactic acid refers to poly (l-lactic acid), poly-D-ALPHA-Hydroxypropionic acid or poly-D, Pfansteihl, and the molecular weight of polylactic acid is 1 * 10
5~ 5 * 10
5.
5. according to water-insoluble superfine silk powder body/polylactic acid composite porous support material described in claims 1 and preparation method thereof, it is characterized in that the mean diameter of described water-insoluble superfine silk powder body is 2.4 μ m.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104258466A (en) * | 2014-10-28 | 2015-01-07 | 罗卫华 | Nano-cellulose/polylactic acid porous scaffold and preparation method thereof |
| CN104548200A (en) * | 2015-02-06 | 2015-04-29 | 武汉纺织大学 | Method of preparing highly branched polysaccharide-fibroin hydrogel bracket |
| CN113144289A (en) * | 2021-04-27 | 2021-07-23 | 武汉理工大学 | Silk fibroin/polylactic acid composite scaffold with function of directionally inducing peripheral nerve regeneration and preparation method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002146084A (en) * | 2000-11-16 | 2002-05-22 | National Institute Of Advanced Industrial & Technology | Method for producing polymer porous material |
| CN101053670A (en) * | 2007-04-03 | 2007-10-17 | 浙江理工大学 | Silk fibroin and polymeric lactic acid compound film and its preparation method |
| WO2011075516A2 (en) * | 2009-12-18 | 2011-06-23 | President And Fellows Of Harvard College | Active scaffolds for on-demand drug and cell delivery |
| CN102219844A (en) * | 2011-04-02 | 2011-10-19 | 武汉纺织大学 | Method for preparing water-insoluble fibroin nanometer microcrystal powder |
| CN102441173A (en) * | 2010-09-30 | 2012-05-09 | 武汉纺织大学 | Preparation method of a drug release system with water-insoluble protein powder as drug carrier |
| CN102698321A (en) * | 2012-06-29 | 2012-10-03 | 上海大学 | Preparation method of bone repairing body with drug loading function |
-
2013
- 2013-12-16 CN CN201310686094.8A patent/CN103638559A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002146084A (en) * | 2000-11-16 | 2002-05-22 | National Institute Of Advanced Industrial & Technology | Method for producing polymer porous material |
| CN101053670A (en) * | 2007-04-03 | 2007-10-17 | 浙江理工大学 | Silk fibroin and polymeric lactic acid compound film and its preparation method |
| WO2011075516A2 (en) * | 2009-12-18 | 2011-06-23 | President And Fellows Of Harvard College | Active scaffolds for on-demand drug and cell delivery |
| CN102441173A (en) * | 2010-09-30 | 2012-05-09 | 武汉纺织大学 | Preparation method of a drug release system with water-insoluble protein powder as drug carrier |
| CN102219844A (en) * | 2011-04-02 | 2011-10-19 | 武汉纺织大学 | Method for preparing water-insoluble fibroin nanometer microcrystal powder |
| CN102698321A (en) * | 2012-06-29 | 2012-10-03 | 上海大学 | Preparation method of bone repairing body with drug loading function |
Non-Patent Citations (3)
| Title |
|---|
| BANANI KUNDU ETAL.: ""Silk fibroin biomaterials for tissue regenerations"", 《ADVANCED DRUG DELIVERY REVIEWS》, vol. 65, no. 4, 5 November 2012 (2012-11-05), pages 457 - 470, XP028593336, DOI: 10.1016/j.addr.2012.09.043 * |
| JOSHUA R. MAUNEY ETAL.: ""Engineering adipose-like tissue in vitro and in vivo utilizing human bone marrow and adipose-derived mesenchymal stem cells with silk fibroin 3D scaffolds"", 《BIOMATERIALS》, vol. 28, no. 35, 31 August 2007 (2007-08-31), pages 5280 - 5290, XP022290568, DOI: 10.1016/j.biomaterials.2007.08.017 * |
| 郉帅等: ""聚乳酸/丝素蛋白复合组织工程支架的制备及生物学性能的评价"", 《吉林大学学报（医学版）》, vol. 34, no. 3, 31 May 2008 (2008-05-31), pages 405 - 410 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104258466A (en) * | 2014-10-28 | 2015-01-07 | 罗卫华 | Nano-cellulose/polylactic acid porous scaffold and preparation method thereof |
| CN104258466B (en) * | 2014-10-28 | 2015-11-25 | 中南林业科技大学 | The preparation method of nano-cellulose/polylactic acid porous scaffold |
| CN104548200A (en) * | 2015-02-06 | 2015-04-29 | 武汉纺织大学 | Method of preparing highly branched polysaccharide-fibroin hydrogel bracket |
| CN113144289A (en) * | 2021-04-27 | 2021-07-23 | 武汉理工大学 | Silk fibroin/polylactic acid composite scaffold with function of directionally inducing peripheral nerve regeneration and preparation method thereof |
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