CN1613514A - Three-dimensional porous tissue engineering stand material and preparation thereof - Google Patents
Three-dimensional porous tissue engineering stand material and preparation thereof Download PDFInfo
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- CN1613514A CN1613514A CN 200410094895 CN200410094895A CN1613514A CN 1613514 A CN1613514 A CN 1613514A CN 200410094895 CN200410094895 CN 200410094895 CN 200410094895 A CN200410094895 A CN 200410094895A CN 1613514 A CN1613514 A CN 1613514A
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Abstract
A 3D porous scaffold material for tissue engineering to induce the generation of bone tissue is prepared from chitosan, gelatin, GPSM and calcium nitrate through sol-gel process, chemical treating and freeze drying. Its advantages are high bioactivity and degradability, proper porosity and pore diameter, and controllable properties.
Description
Technical field
The present invention relates to a kind of the have good biological activity and the biomedical material-tissue engineering bracket material of degradability.
Background technology
Since 19th century, bone grafting is devoted to repair always because wound, tumor, bone on a large scale that infection caused are damaged, to recover limb function.But there are many problems in this method, as the shortage of immunologic rejection and donor tissue and organ.An ideal strategy is exactly artificial culture tissue and organ, has proposed the notion of organizational project immediately, and its target is the compound cultivation osteogenic tissue again that utilizes cell and biologic bracket material, reaches the purpose of reparation.Wherein, timbering material as the carrier of signaling molecule or cell, is transported to defect location with it on the one hand, and the support of new bone growth is provided on the other hand.At present existing multiple material is used as biologic bracket material, and as bioabsorbable polymer material, bioceramic/glass etc., but all there is different shortcomings in these material great majority, are difficult to satisfy fully requirements for clinical application.Can produce chronic reaction with body as synthetic high polymer (as PLA etc.), occur aseptic inflammation after the degraded; Natural polymer (as chitosan, collagen etc.) lacks essential space structure and mechanical strength; Bioceramic/glass material (as beta-calcium phosphate class biomaterial, calcium silicates class material etc.) has special biological activity (forming good binding with natural bone), but biological degradability is unsatisfactory.
Summary of the invention
The object of the present invention is to provide a kind of three-dimensional porous tissue engineering bracket material with good biological activity and biological degradability that can adapt to osteoblast adhesion and propagation and bone tissue restoration and reconstruction needs and preparation method thereof.
The said three-dimensional porous tissue engineering bracket material of the present invention is gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material, by chitosan, gelatin, 3-glycerol propyl trimethoxy silicane (GPSM) and lime nitrate (CaNO
3) be composited, each set of dispense is a gelatin 1 than (pressing mass fraction, down together), chitosan 0～0.2, and GPSM 0.15～1.2, lime nitrate 0.006～0.12.
The preparation method of the said three-dimensional porous tissue engineering bracket material of the present invention is as follows:
1) adopt acid solution to prepare the gelatin-chitosan sugar juice as solvent, its proportioning is that chitosan/gelatin is (0～0.2)/1, obtains solution A, and the solid content of solution A is 2.5%～15%; Said acid is a kind of or its mixture in acetic acid, formic acid or the hydrochloric acid, and the pH value of gelatin-chitosan solution is 2～6, and the molecular weight ranges of chitosan is 1～300,000;
2) add 3-glycerol propyl trimethoxy silicane in solution A, its proportioning is that 3-glycerol propyl trimethoxy silicane/(chitosan+gelatin) is (0.15～1)/1, obtains solution B, and the proportioning of said gelatin and chitosan is 1: (0～0.2);
3) press lime nitrate/3-glycerol propyl trimethoxy silicane and be (0.04～0.1)/1, in solution B, add lime nitrate, and mix down, obtain solution C at 4～60 ℃;
4) mixed solution C is injected mould, under 4～60 ℃ temperature,, get gelatine-chitosan-gel of calcium silicate through sol-gel process;
5) in the mould that gelatine-chitosan-gel of calcium silicate is housed, inject the ammonia spirit of 0.5～3M, placed 8～24 hours down, then washing at 20～40 ℃;
6) adopt freezing-lyophilization, cryogenic temperature is-20～-200 ℃, must have the gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material of different pore-size distributions after the lyophilizing.
7) three-dimensional porous tissue engineering bracket material is cut into required form, size, sterilization packages spare, and that shape can be is bar-shaped, tabular, membranaceous, tubulose or specific shape.
In step 6), said freezing-lyophilization can adopt earlier freezingly, cryogenic temperature be-20～-80 ℃, after the lyophilizing again the second time freezing, cryogenic temperature is-120～-200 ℃, obtains after the lyophilizing having gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material that diplopore directly distributes.
In step 6), gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material is put into growth factor solution, wherein the volume ratio of gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material and growth factor solution is 1/4.Evacuation after the swelling, negative pressure are 0.1～0.5MPa, get gelatine-chitosan-calcium silicates-somatomedin composite with distill water dialysis at last.Said somatomedin is fibroblast growth factor (FGF), transforming growth factor (TGF-ρ), insulin like growth factor (IGF), platelet derivation somatomedin (PDGF) or bone morphogenetic protein (BMP) etc.Gelatine-chitosan-calcium silicates-somatomedin the composite that obtains is carried out lyophilizing once more handle, cryogenic temperature is-196 ℃, and lyophilization at least 48 hours can get gelatine-chitosan-calcium silicates-somatomedin three-dimensional porous tissue engineering bracket material.
The three-dimensional porous tissue engineering bracket material that is made by the present invention can adopt following method to carry out performance evaluation:
One, the porosity of three-dimensional porous tissue engineering bracket material and pore structure
The three-dimensional porous tissue engineering bracket material that the present invention obtains is tested porosity, applying electronic scanning microscope (SEM) observation port form and pore size distribution with Archimedes's method.
Two, degradability evaluation
The three-dimensional porous tissue engineering bracket material that the present invention is obtained is earlier after carry out the external degradation experimental evaluation after deionized water and washing with acetone, the oven dry.Is to soak the Ca that discharges behind the different time in 7.4 three (methylol) amido methane (Tris) buffer solution by such three-dimensional porous tissue engineering bracket material at pH value
2+Percentage composition or the weightlessness of the material degradability of coming evaluating material.
Three, evaluated biological activity
The three-dimensional porous tissue engineering bracket material that the present invention is obtained is earlier after deionized water and washing with acetone, carry out external solution biological activity test after drying.Solutions employed be human body simulation body fluid SBF (Simulated Body Fluid, SBF).SBF contains ion identical with human plasma and ion cluster concentration.SBF consists of:
NaCl：7.996g/L
NaHCO
3：0.350g/L
KCl：0.224g/L
K
2HPO
4·3H
2O。0.228g/L
MgCl
2·6H
2O：0.305g/L
HCl：1.0mol/L
CaCl
2：0.278g/L
Na
2SO
4：0.071g/L
NH
2C(CH
2OH)：6.057g/L
Three-dimensional porous tissue engineering bracket material is in SBF, and reaction condition is in 1.5cm * 1.5cm porous material, 20.0ml/daySBF, the 37 ℃ of calorstats.After respectively three-dimensional porous tissue engineering bracket material being soaked 1,3,5 and 7 day, take out sample and process deionized water wash.Use electronics scanning electron microscope (SEM), fourier infrared conversion spectrum analyser (FTIR) and X-ray diffractometer (XRD) etc. that analytical test is carried out on the three-dimensional porous tissue engineering bracket material surface respectively then.
Four, the evaluation of biocompatibility
The three-dimensional porous tissue engineering bracket material that the present invention is obtained is earlier after deionized water and washing with acetone, dry back gamma-radiation illumination-based disinfection.By detecting such three-dimensional porous tissue engineering bracket material comes evaluating material to the synthetic influence of collagen of MC3T3-E1 mice analogy osteoblast cell proliferation rate, alkali phosphatase (ALP) activity and cell biocompatibility and osteoinductive.
Chitosan is a kind of biodegradable natural polycation polysaccharide (pKa=6.3), and has excellent biological compatibility, biological degradability.It can present non-special interaction with cell membrane negative electricity composition, and the release of adjustable bioactive molecule, is widely used at biomedical aspect.Gelatin is the partial hydrolysate of collagen protein, and biocompatibility is good, and does not have the antigen reaction.The construction unit of gelatin is glycine one proline and hydroxyproline and lysine (Gly-Pro-Hyl-Hly), is flexible ampholyte, and isoelectric pH=4.7 help cytoadherence.The present invention introduce GPSM behind colloidal sol-gel reaction with the compound calcium silicates class material that obtains of lime nitrate as inorganic constituents, can give timbering material good biological activity.Discover when calcium silicates class material is exposed in the ambient body fluid, its surperficial generable chemical reaction: ion exchange and irregular surface dissolution effect can produce a hydrated silica gel layer (Si-OH), present area and have promoted at coarse formation one deck crystallite apatite layer biology on glass thereby increased.This layer can be in vivo forms being short in the time of a few hours, not only with synosteosis, also combines with collagen fiber.In addition, GPSM can also be as the cross-linking agent of gelatin, chitosan, thereby can strengthen the mechanical property of porous compound support frame material on the one hand, and can regulate and control the degradation rate of compound support frame material by GPSM, makes it to be complementary with growing into of cambium.More be applicable to the three-dimensional active porous material of inducing the bone/cartilage tissue growth in order to make, the present invention also can be in material compound somatomedin with different inducing functions, as fibroblast growth factor (FGF), transforming growth factor (TGF-ρ), insulin like growth factor (IGF), platelet derivation somatomedin (PDGF), bone morphogenetic protein (BMP) etc.In application, somatomedin can be wrapped in swollen porous material inside equably, is released along with the degraded of porous material.
In a word, compared with prior art, material source of the present invention is extensive, and is with low cost.And adopt factors such as blend ratio that sol-gel process can be by controlling collagen/chitosan/GPSM/ lime nitrate, reaction temperature, time to come physics, chemical property and the degradation rate of control material; Adopt freezing-lyophilization can be by regulating the micro structure that factors such as cryogenic temperature are come control material, thereby prepare three-dimensional porous tissue engineering bracket material with suitable aperture and porosity.Gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material by the present invention's preparation has good biological activity and degradability, and the compound somatomedin that tissue construction is played most important effect of energy, with the induced osteogenesis cell differentiation, help the formation of osseous tissue, can satisfy the needs of cartilage of new generation and bone tissue engineering stent material development fully.
Description of drawings
Fig. 1 is the SEM photo of the present invention's three-dimensional porous tissue engineering bracket material of precooling preparation under-20 ℃ of temperature.
The composite porous SEM shape appearance figure of immersion after 3 days in SBF that Fig. 2 obtains for the present invention.
Fig. 3 is the SEM shape appearance figure of MC3T1-E1 analogy osteoblast after the surface of the three-dimensional porous tissue engineering bracket material that the present invention obtains is cultivated 7 days.
Fig. 4 is the SEM photo of the present invention's three-dimensional porous tissue engineering bracket material of precooling preparation under-196 ℃ of temperature.
Fig. 5 is the SEM photo with three-dimensional porous tissue engineering bracket material that diplopore directly distributes of the present invention preparation.
The specific embodiment
The present invention is further illustrated below by embodiment.
Embodiment 1
With 1M acetic acid solutions concentration is that 15% gelatin solution and concentration are 1% chitosan solution, then by 1: 1 with its even blend, obtain the gelatin/chitosan mixed solution that gelatin, chitosan content are respectively (by quality) 7.5%, 0.5%.Add GPSM and lime nitrate in above-mentioned solution respectively, wherein the GPSM/ lime nitrate is (by quality) 1/0.05; GPSM/ (gelatin+chitosan) is (by quality) 0.5/1.Above-mentioned solution mixed 4 hours down fully at 40 ℃, to fully evenly (not having phase-splitting), then its 25ml was injected the mould of 5cm * 8cm, sealed, and was placed in 40 ℃ the calorstat 3 days, gelatine-chitosan-gel of calcium silicate.The ammonia spirit 10ml that adds 1M in above-mentioned mould placed 16 hours down at 25 ℃, used 1M NaCl solution and distilled water thorough washing to neutral then.Gel after the ammonia treatment is placed-20 ℃ of precoolings 24 hours, more than 48 hours, can obtain gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material in lyophilization under the 5KPa condition after the lyophilizing at-40 ℃ afterwards.The timbering material that obtains is cut into diameter 10mm, and the circular disc of thick 1.5mm places at the bottom of the hole of 24 well culture plates, and the alcohol solution dipping with 75% 1 hour washes repeatedly with distilled water, removes moisture.Efficient ultra violet lamp sterilization is arranged in closed environment.Timbering material after the sterilization is stand-by with polyethylene film sealing back.
The three-dimensional porous tissue engineering bracket material that obtains is tested porosity, applying electronic scanning microscope (SEM) observation port form and pore size distribution with Archimedes's method.The porosity of the three-dimensional porous tissue engineering bracket material that the present invention obtains is 92.7%, and the size distribution in hole is at 300～500 microns, and being evenly distributed, being communicated with of hole, referring to Fig. 1.Behind deionized water and washing with acetone, ready 1.5cm * 1.5cm timbering material is carried out external biological degraded test and biological activity test respectively: (1) biodegradation test: above-mentioned ready material is soaked in the Tris solution (pH7.4) of 20ml, and places 37 ℃ of constant temperature oscillators (rotating speed is 100rpm) to 60 days.Took out that material is weighed (weight-loss ratio that calculates material) in per two days and with Ca in plasma emission spectroscopy method (ICP) the detection soak
2+Release content, thereby determine the degradability of material.The degraded result of the test shows that the degradation rate of this timbering material after soaking 60 days is 45.3%.(2) biological activity test: above-mentioned ready material is soaked in the SBF solution of 20ml, and places 37 ℃ of calorstats to 7 day.Carry out SEM, fourier infrared conversion spectrum (FTIR) and XRD test after taking out sample and process deionized water wash in per two days.Test result finds that the surface of gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material can sedimentary new thing be a class bone hydroxyapatite layer (referring to Fig. 2) mutually.Therefore, this timbering material biologically active.
With density is 1 * 10
41.5ml MC3T3-E1 analogy osteoblast suspension (10%FCS, α-MEM culture medium) place 24 well culture plates of sterilization in 37 ℃, 5%CO
2Incubator in spend the night, in each hole, add the timbering material of above-mentioned sterilization then, at 37 ℃, 5%CO
2Incubator in cultivated 7 days.Respectively at the growth rate (MTT method) that detected cell in 1,3,5,7 day after cultivating, the ALP activity (Nitrobenzol cellophane method) of cell, experimental result is carried out data statistics with the t-test method.In addition, with SEM observation of cell sticking and growing state (referring to Fig. 3) on the timbering material surface.Test result shows, such porous gelatine-chitosan-calcium silicates tissue engineering bracket material has good biocompatibility, the MC3T3-E1 analogy osteoblast is after cultivating 7 days on the support, the blank relatively group of its ALP activity has increased by 47.6%, but inoperative to MC3T3-E1 analogy osteoblast propagation.Think that at present the active height of ALP is an important symbol of reflection osteoblast maturity state.Therefore, the gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material that is compounded with BMP can the induced osteogenesis cell differentiation, helps the formation of osseous tissue.
Embodiment 2
With 0.1M hydrochloric acid solution compound concentration is 10% gelatin solution, adds GPSM and lime nitrate in above-mentioned solution respectively, and wherein the GPSM/ lime nitrate is (by quality) 1/0.05; The GPSM/ gelatin is (by quality) 0.5/1.Above-mentioned solution mixed 4 hours down fully at 40 ℃, to fully evenly (not having phase-splitting), then its 25ml was injected the mould of 5cm * 8cm, sealed, and was placed in 40 ℃ the calorstat 3 days, gelatin-gel of calcium silicate.The ammonia spirit 10ml that adds 1M in above-mentioned mould placed 16 hours down at 25 ℃, used 1M NaCl solution and distilled water thorough washing to neutral then.Gel after the ammonia treatment is placed-20 ℃ of precoolings 24 hours, more than 48 hours, can obtain gelatin-calcium silicates three-dimensional porous rack in lyophilization under the 5KPa condition after the lyophilizing at-40 ℃ afterwards.The porous material that obtains is tested porosity, applying electronic scanning microscope (SEM) observation port form and pore size distribution with Archimedes's method.The porosity of the three-dimensional porous tissue engineering bracket material that the present invention obtains is 89.2%; The size distribution in hole is at 300～500 microns, and being evenly distributed, being communicated with of hole.Employing is carried out external biological degraded test and biological activity test with the method for embodiment 1, and experimental result shows this timbering material biologically active, and the degradation rate after soaking 60 days is 70.2%.
Embodiment 3
With 1M acetic acid solutions concentration is that 15% gelatin solution and concentration are 1% chitosan solution, then by 1: 1 with its even blend, obtain the gelatin/chitosan mixed solution that gelatin, chitosan content are respectively (by quality) 7.5%, 0.5%.Add GPSM and lime nitrate in above-mentioned solution respectively, wherein the GPSM/ lime nitrate is (by quality) 1/0.05; GPSM/ (gelatin+chitosan) is (by quality) 0.5/1.Above-mentioned solution mixed 4 hours down fully at 40 ℃, to fully evenly (not having phase-splitting), then its 25ml was injected the mould of 5cm * 8cm, sealed, and was placed in 40 ℃ the calorstat 3 days, gelatine-chitosan-gel of calcium silicate.The ammonia spirit 10ml that adds 1M in above-mentioned mould placed 16 hours down at 25 ℃, used 1M NaCl solution and distilled water thorough washing to neutral then.Gel after the ammonia treatment is placed liquid nitrogen (196 ℃) precooling 2 hours, more than 48 hours, can obtain gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material in lyophilization under the 5KPa condition after the lyophilizing at-40 ℃ afterwards.The porous material that obtains is tested porosity, applying electronic scanning microscope (SEM) observation port form and pore size distribution with Archimedes's method.The porosity of the porous material that the present invention obtains is 64.5%; The size distribution in hole is at 3～10 microns, referring to Fig. 4.Employing is carried out external biological degraded test and biological activity test with the method for embodiment 1, and experimental result shows this timbering material biologically active, and the degradation rate after soaking 60 days is 34.6%.
Embodiment 4
With 1M acetic acid solutions concentration is that 15% gelatin solution and concentration are 1% chitosan solution, then by 1: 1 with its even blend, obtain the gelatin/chitosan mixed solution that gelatin, chitosan content are respectively (by quality) 7.5%, 0.5%.Add GPSM and lime nitrate in above-mentioned solution respectively, wherein the GPSM/ lime nitrate is (by quality) 1/0.05; GPSM/ (gelatin+chitosan) is (by quality) 0.5/1.Above-mentioned solution mixed 4 hours down fully at 40 ℃, to fully evenly (not having phase-splitting), then its 25ml was injected the mould of 5cm * 8cm, sealed, and was placed in 40 ℃ the calorstat 3 days, gelatine-chitosan-gel of calcium silicate.The ammonia spirit 10ml that adds 1M in above-mentioned mould placed 16 hours down at 25 ℃, used 1M NaCl solution and distilled water thorough washing to neutral then.Gel after the ammonia treatment was placed-20 ℃ of precoolings 24 hours, more than 48 hours, can obtain gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material in lyophilization under the 5KPa condition at-40 ℃ afterwards with embodiment 1.This timbering material is placed the ammonia spirit of 10ml, 1M again, and under 25 ℃, placed 16 hours, use 1M NaCl solution and distilled water thorough washing then to neutral.Porous material after the ammonia treatment was placed liquid nitrogen (196 ℃) precooling 2 hours, afterwards at-40 ℃ in lyophilization under the 5KPa condition more than 48 hours, can obtain having gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material that diplopore directly distributes: size is evenly distributed on the hole wall of 300-500 micron macropore for the micropore of 3-10 micron, referring to Fig. 5.The porosity of testing this support with Archimedes's method is 95.5%.Employing is carried out external biological degraded test and biological activity test with the method for embodiment 1, and experimental result shows this timbering material biologically active, and the degradation rate after soaking 60 days is 53.4%.
Embodiment 5
With 1M acetic acid solutions concentration is that 15% gelatin solution and concentration are 1% chitosan solution, then by 1: 1 with its even blend, obtain the gelatin/chitosan mixed solution that gelatin, chitosan content are respectively (by quality) 7.5%, 0.5%.Add GPSM in above-mentioned solution, wherein GPSM/ (gelatin+chitosan) is 0.5/1, and fully mixes 4 hours down at 40 ℃, to complete evenly (not having phase-splitting).The solution 25ml of mix homogeneously is injected the mould of 5cm * 8cm, seals, be placed in 40 ℃ the calorstat 3 days, gelatine-chitosan-gel of calcium silicate.In mould, add the ammonia spirit 10ml of 1M, placed 16 hours down at 25 ℃, extremely neutral with 1M NaCl solution and distilled water thorough washing.Gel after the ammonia treatment is placed-20 ℃ of precoolings 24 hours, more than 48 hours, can obtain gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material in lyophilization under the 5KPa condition at-40 ℃ afterwards.The BMP lyophilized powder is dissolved in 4M guanidine hydrochloride (GuHCl)-0.05M CaCl
2In, solid content is 100mg/ml, places 12 hours in 4 ℃ of environment.With 1cm
3Above-mentioned porous support materials is put into the ready BMP solution of 4ml, and swelling is after 1 hour, evacuation 2 times, and negative pressure is 0.2MPa, each 5 minutes, last distill water dialysis 24 hours.The porous material that is compounded with BMP that obtains is carried out lyophilizing once more handle, the pre-freeze temperature is-196 ℃, and the time is 1 hour, and lyophilization can get gelatine-chitosan-calcium silicates-BMP three-dimensional porous tissue engineering bracket material more than 48 hours more afterwards.The timbering material that obtains is cut into diameter 10mm, and the circular disc of thick 1.5mm places at the bottom of the hole of 24 well culture plates, and the alcohol solution dipping with 75% 1 hour washes repeatedly with distilled water, removes moisture.Efficient ultra violet lamp sterilization is arranged in closed environment.Timbering material after the sterilization is stand-by with polyethylene film sealing back.
With density is 1 * 10
41.5ml MC3T3-E1 analogy osteoblast suspension (10%FCS, α-MEM culture medium) place 24 well culture plates of sterilization in 37 ℃, 5%CO
2Incubator in spend the night, in each hole, add the timbering material of above-mentioned sterilization then, at 37 ℃, 5%CO
2Incubator in be cultured to 7 days.Respectively at the growth rate (MTT method) that detected cell in 1,3,5,7 day after cultivating and the ALP activity (Nitrobenzol cellophane method) of cell, experimental result is carried out data statistics with the t-test method.In addition, with SEM observation of cell sticking and growing state on the timbering material surface.Test result shows, such porous gelatine-chitosan-calcium silicates tissue engineering bracket material has good biocompatibility, the MC3T3-E1 analogy osteoblast is after cultivating 7 days on the support of the porous gelatine-chitosan-calcium silicates that combines BMP, the blank relatively group of its ALP activity has increased by 213.7%, but inoperative to osteoblastic proliferation.Therefore, the gelatine-chitosan-calcium silicates three-dimensional porous rack that is compounded with BMP can promote the induced osteogenesis cell differentiation greatly, helps the formation of osseous tissue.
Claims (7)
1, a kind of three-dimensional porous tissue engineering bracket material, it is characterized in that said three-dimensional porous tissue engineering bracket material is gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material, be composited by chitosan, gelatin, 3-glycerol propyl trimethoxy silicane and lime nitrate, it is gelatin 1 that each components in mass portion is counted proportioning, chitosan 0～0.2,3-glycerol propyl trimethoxy silicane 0.15～1.2, lime nitrate 0.006～0.12.
2. the preparation method of a kind of three-dimensional porous tissue engineering bracket material as claimed in claim 1 is characterized in that its step is as follows:
1) adopt acid solution to prepare the gelatin-chitosan sugar juice as solvent, its proportioning is that chitosan/gelatin is 0～0.2/1, obtains solution A, and the solid content of solution A is 2.5%～15%; Said acid is a kind of or its mixture in acetic acid, formic acid or the hydrochloric acid, and the pH value of gelatin-chitosan sugar juice is 2～6;
2) add 3-glycerol propyl trimethoxy silicane in solution A, its proportioning is that 3-glycerol propyl trimethoxy silicane/(chitosan+gelatin) is 0.15～1/1, obtains solution B;
3) be 0.04～1/1 by lime nitrate/3-glycerol propyl trimethoxy silicane, in solution B, add lime nitrate, and mix down, obtain solution C at 4～60 ℃;
4) solution C is injected mould, under 4～60 ℃ temperature,, get gelatine-chitosan-gel of calcium silicate through sol-gel process;
5) in the mould that gelatine-chitosan-gel of calcium silicate is housed, inject the ammonia spirit of 0.5～3M, placed 8～24 hours down, then washing at 20～40 ℃;
6) adopt freezing-lyophilization, the pre-freeze temperature is-20～-200 ℃, and the time is 1～24 hour, and lyophilization afterwards at least 48 hours gets gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material;
7) three-dimensional porous tissue engineering bracket material is cut into required form, size, sterilization packages spare, and that shape is is bar-shaped, tabular, membranaceous, tubulose or specific shape.
3, the preparation method of a kind of three-dimensional porous tissue engineering bracket material as claimed in claim 2, it is characterized in that in step 6), said freezing-lyophilization adopts earlier freezing, cryogenic temperature is-20～-80 ℃, for the second time freezing again after the lyophilizing, cryogenic temperature is-120～-200 ℃, obtains gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material after the lyophilizing.
4, the preparation method of a kind of three-dimensional porous tissue engineering bracket material as claimed in claim 2, it is characterized in that in step 6), gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material is put into growth factor solution, wherein the volume ratio of gelatine-chitosan-calcium silicates three-dimensional porous tissue engineering bracket material and growth factor solution is 1/4, evacuation after the swelling, negative pressure is 0.1～0.5MPa, gets gelatine-chitosan-calcium silicates-somatomedin composite with distill water dialysis at last.
5, the preparation method of a kind of three-dimensional porous tissue engineering bracket material as claimed in claim 4 is characterized in that said somatomedin is fibroblast growth factor, transforming growth factor, insulin like growth factor, platelet derivation somatomedin or bone morphogenetic protein.
6, the preparation method of a kind of three-dimensional porous tissue engineering bracket material as claimed in claim 4, it is characterized in that said gelatine-chitosan-calcium silicates-somatomedin composite is carried out lyophilizing once more to be handled, cryogenic temperature is-196 ℃, lyophilization at least 48 hours gets gelatine-chitosan-calcium silicates-somatomedin three-dimensional porous tissue engineering bracket material.
7, the preparation method of a kind of three-dimensional porous tissue engineering bracket material as claimed in claim 2, the molecular weight ranges that it is characterized in that chitosan is 1～300,000.
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| CN110869065A (en) * | 2017-04-13 | 2020-03-06 | 塞普托东专业股份有限公司 | Connective tissue, such as bone, dentin or pulp regeneration material, comprising calcium silicate |
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| CN113144294B (en) * | 2021-02-20 | 2023-01-24 | 北京邦塞科技有限公司 | Preparation method of three-dimensional porous collagen scaffold, double-layer collagen scaffold, preparation method and application |
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