CN105521525A - Porous composite scaffold for bone tissue engineering and preparation method therefor - Google Patents
Porous composite scaffold for bone tissue engineering and preparation method therefor Download PDFInfo
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Abstract
The invention relates to a biological field and specifically to a porous composite scaffold for bone tissue engineering and a preparation method therefor. The porous composite scaffold for bone tissue engineering provided by the invention is a hydroxyapatite/nanocrystal cellulose/silk fibroin porous scaffold; the porous composite scaffold is a three-dimensional porous composite scaffold prepared from hydroxyapatite, nanocrystal cellulose and silk fibroin; and the porosity is 90-95% and the aperture is 180-220[mu]m. According to the hydroxyapatite/nanocrystal cellulose/silk fibroin porous scaffold, hydroxyapatite and nanocrystal cellulose are uniformly distributed in the scaffold, so that the scaffold is excellent in mechanical performance; in addition, the scaffold is high in biocompatibility and capable of supporting and promoting osteogenic differentiation of mesenchymal stem cells; and meanwhile, the preparation method is simple in process, wide in raw material sources, high in production efficiency, low in cost, and can be applied to industrial large-scale production.
Description
Technical field
The present invention relates to biological medicine field of material technology, specifically the porous compound support frame and preparation method thereof of bone tissue engineer.
Background technology
Bone is the support of human body, plays protection and supporting function, and play leverage at the volley to human body.Periosteum, sclerotin and bone marrow are the three parts forming bone.But Cranial defect is commonly encountered diseases clinically, according to statistics, the Cranial defect patient that China causes because of disease, wound, tumor etc. every year more than 1,000,000, one of the difficult problem being still orthopaedics therapy at present of the reparation of Cranial defect on a large scale.Although bone has the ability of regeneration and selfreparing, merely by self-regeneration, defect is often difficult to oneself's healing.In this case, then need to treat.At present, the method for the treatment of Cranial defect has bone collection, artificial bone graft's substitution material and tissue engineering technique etc.Wherein, tissue engineering tissue have source not limited, can design shape, low antigenicity and there is the advantages such as vitality in advance.In addition, apply bone tissue engineer technology treatment Cranial defect and can overcome the shortcomings such as autologous bone transplanting donor deficiency and allogenic bone transplantation immunologic rejection.Therefore, utilize bone tissue engineer to carry out Bone Defect Repari to be the focus for the treatment of Cranial defect.
Fibroin albumen (Silkfibroin, SF) is the hydrophobic natural polymer celloglobulin of one extracted from silkworm silk, is made up of 18 seed amino acids, and wherein glycine, alanine and serine account for more than 80% of its total composition.Fibroin albumen abundance, with low cost, having excellent in mechanical performance, Stability Analysis of Structures, good biocompatibility and the advantage of machinability, is a kind of excellent natural macromolecular material.Based on these advantages of fibroin albumen, be used as bone tissue engineering stent material more and more.But because fibroin albumen itself does not have the effect of self-bone grafting and bone conduction, the reparation of Cranial defect is limited to some extent, therefore, prepares a kind of silk fibroin porous scaffold with self-bone grafting and osteoconductive potential, as Bone Defect Repari tissue engineering material, it is the object of this patent.
Hydroxyapatite (Hydroxyapatite, HA) belongs to Ca-P ceramic, because the inorganic constituents inside it and bone is closely similar, is a kind of important bioceramic in bone tissue engineer.Hydroxyapatite has good biocompatibility, biological activity, bone conductibility and osteoinductive, through being usually used in dental restortion, Bone Defect Repari and orthopedic reparation.But hydroxyapatite is not easily absorbed and fragility is large, limit its application in bone tissue engineer.Therefore, a kind of new medicine-carried system must be researched and developed, make hydroxyapatite to be successfully applied in bone tissue engineer.
Nano micro crystal cellulose (NanocrystalCellulose, CNC), pass through chemistry by native cellulose or microcrystalline Cellulose, the method of physics or biochemistry is prepared and obtains, and nano micro crystal cellulose not only has the relevant nature of nanoparticle such as: larger specific surface, high strength and high-crystallinity; Also possess cellulosic basic structure and characteristic, such as: the features such as avirulence, biocompatibility and degradability; In addition, the role that nano-cellulose generally serves as in the middle of complex is reinforced filling, to improve heat stability and the mechanical performance of complex.And, containing abundant hydroxyl in nano micro crystal cellulose, add in complex, the hydrophilicity of complex can be improved.
In recent years, utilize fibroin albumen as three-dimensional rack, become focus at bone tissue engineer application and research.There are some researches show, with fibroin albumen as bone renovating material, obtain reasonable repairing effect for nude mice defect of skull; And the role that nano-cellulose generally serves as in the middle of complex is reinforced filling, to improve the heat stability of complex, hydrophilic and mechanical performance.On the other hand, how improving hydroxyapatite and not easily absorbed and the large shortcoming of fragility, is also the problem that research worker is paid close attention to.
Summary of the invention
The present invention is directed to above-mentioned Problems existing, provide a kind of good mechanical performance, have the bone tissue engineer porous compound support frame of good biocompatibility.
The present invention is achieved in that
A kind of porous compound support frame of bone tissue engineer, it is characterized in that: described bone tissue engineer porous compound support frame is hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold, the three-dimensional stephanoporate compound stent be made up of hydroxyapatite, nano micro crystal cellulose, fibroin albumen, porosity is 90 ~ 95%, and aperture is 180 ~ 220 μm.
Preferably, described hydroxyapatite is needle-like, and particle diameter is 10 ~ 100nm.
Preferably, nano micro crystal cellulose is that its length is 150-350nm, and width is 20-40nm in regular corynebacterium structure.
Another object of the present invention is to provide the method for the porous compound support frame of above-mentioned bone tissue engineer:
Said method comprising the steps of:
(1) Bombyx bombycis is shredded, then add 80-100 DEG C, concentration is 0.2wt%NaCO
3boil 2-4h in aqueous solution and carry out the formation fibroin that comes unstuck, Bombyx bombycis and NaCO
3the ratio of aqueous solution is: 2-5g Bombyx bombycis adds 100ml, 0.2wt%NaCO
3aqueous solution, extremely neutral with washed with de-ionized water fibroin after coming unstuck;
(2) step (1) is repeated;
(3) step (2) is washed till neutral fibroin in 40-60 DEG C of oven dry, obtains fibroin albumen; Get fibroin albumen, adding concentration is in 9.8mol/L lithium bromide water solution, with deionized water dialysis 3 ~ 4 days after 40 ~ 60 DEG C of heating in water bath 4 ~ 6h, and centrifugal twice, obtain 2 ~ 10wt% silk fibroin protein solution;
(4) hydroxyapatite, nano micro crystal cellulose are added in 2 ~ 10wt% silk fibroin protein solution, the ratio of mixing is: every milliliter of silk fibroin protein solution adds 0<m≤60mg hydroxyapatite, 0<m≤60mg nano micro crystal cellulose stirs, wherein m represents quality, then lyophilization, obtains hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold.
Preferably, the amount that every milliliter of silk fibroin protein solution described in step (2) adds hydroxyapatite and nano micro crystal cellulose is 30mg.
Further, described Bombyx bombycis is the Bombyx bombycis of pupa;
Preferably, described dialysis procedure adopt molecular cut off be 10000 ~ 12000 bag filter dialyse.
Preferably, described centrifugal condition is in 25 DEG C, centrifugal 5-15min under 10000r/min condition.
Preferably, the cryodesiccated condition described in step (2) is-50 ~-70 DEG C of lyophilization 18-24h.
The present invention has following advantage and effect relative to prior art:
(1) hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold of the present invention, hydroxyapatite and nano micro crystal cellulose are uniformly distributed in support, good mechanical performance, and this support has good biocompatibility, can support and promote the Osteoblast Differentiation of mesenchymal stem cells MSCs;
(2) this complex stephanoporate bracket has the advantage of porosity high (90-95%), uniform pore diameter (180-220um);
(3) hydroxyapatite, nano micro crystal cellulose and silk fibroin protein solution mix by the present invention, by cryodesiccated method, form hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold, preparation technology is simple, material source is extensive, production efficiency is high, cost is low, can be applicable to industrialized great production.
Accompanying drawing explanation
Fig. 1 is the shape appearance figure of the hydroxyapatite of embodiment 1, and wherein Fig. 1 a is the scanning electron microscope (SEM) photograph of hydroxyapatite, the transmission electron microscope picture of Fig. 1 b hydroxyapatite;
Fig. 2 is the transmission electron microscope picture of the nano micro crystal cellulose of embodiment 1;
Fig. 3 is the scanning electron microscope (SEM) photograph of the silk fibroin porous scaffold of comparative example 1;
Fig. 4 is the scanning electron microscope (SEM) photograph of the nano micro crystal cellulose/silk fibroin porous scaffold of comparative example 2;
Fig. 5 is the scanning electron microscope (SEM) photograph of the hydroxyapatite/silk fibroin porous scaffold of comparative example 3;
Fig. 6 is the scanning electron microscope (SEM) photograph of the hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold of embodiment 1;
Fig. 7 is the cytotoxicity figure of the hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold of the silk fibroin porous scaffold of comparative example 1, the nano micro crystal cellulose/silk fibroin porous scaffold of comparative example 2, the hydroxyapatite/silk fibroin porous scaffold of comparative example 3 and embodiment 1, wherein: SF is the silk fibroin porous scaffold cytotoxicity figure of comparative example 1; CNC/SF is the nano micro crystal cellulose/silk fibroin porous scaffold cytotoxicity figure than embodiment 2; HA/SF is the hydroxyapatite/silk fibroin porous scaffold cytotoxicity figure of comparative example 3; HA/CNC/SF is the cytotoxicity figure of the hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold of embodiment 1;
Fig. 8 is the silk fibroin porous scaffold of comparative example 1, the Proliferation of Bone Mesenchymal Stem Cells differentiation figure than the hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold of the nano micro crystal cellulose/silk fibroin porous scaffold of embodiment 2, the hydroxyapatite/silk fibroin porous scaffold of comparative example 3 and embodiment 1; Wherein: SF is the Proliferation of Bone Mesenchymal Stem Cells differentiation figure of the silk fibroin porous scaffold of comparative example 1; CNC/SF is the nano micro crystal cellulose/silk fibroin porous scaffold Proliferation of Bone Mesenchymal Stem Cells differentiation figure than embodiment 2; HA/SF is the hydroxyapatite/silk fibroin porous scaffold Proliferation of Bone Mesenchymal Stem Cells differentiation figure of comparative example 3; HA/CNC/SF is the Proliferation of Bone Mesenchymal Stem Cells differentiation figure of the hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold of embodiment 1.
Concrete case study on implementation
Elaborate to embodiments of the invention below, the present embodiment is implemented under premised on technical solution of the present invention, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
(1) will the Bombyx bombycis of pupa be gone to shred, the Bombyx bombycis of 2.5g is added 100ml, concentration is the NaCO of 0.2wt%
3carry out the formation fibroin that comes unstuck in aqueous solution, under 100 DEG C of conditions, boil 2h.Extremely neutral with washed with de-ionized water fibroin after coming unstuck;
(2) above-mentioned steps is repeated again once;
(3) by being washed till neutral fibroin in 50 DEG C of oven dry, fibroin albumen is obtained; Get fibroin albumen, adding concentration is in 9.8mol/L lithium bromide water solution, dialysing with deionized water after 50 DEG C of heating in water bath 6h, (molecular cut off of bag filter was 8000 in 4 days, change water every day once), then in 25 DEG C, the centrifugal 10min of 10000r/min, centrifugal twice, obtain the silk fibroin protein solution that concentration is 4wt%;
(4) 30mg hydroxyapatite will be added, 30mg nano micro crystal cellulose adds in the 4wt% silk fibroin protein solution of 1ml step (3), stir ,-60 DEG C of lyophilization 24h, obtain hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold.From Fig. 1 and Fig. 2, hydroxyapatite is needle-like, and particle diameter is 10 ~ 100nm; Nano micro crystal cellulose is that its length is about 150-350nm, and width is about 20-40nm in regular corynebacterium structure.The scanning electron microscope (SEM) photograph of hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold as shown in Figure 6.Can know from Fig. 6, this compound rest has porosity high (90-95%), and uniform pore diameter (180-220um), hydroxyapatite and nano micro crystal cellulose are uniformly distributed in support.
(5), after the porous support that step (4) obtains being carried out ultraviolet sterilization, 496 well culture plates are put into.By 0.25% pancreatin/PBS solution by preosteoblast MC3T3-E1 from culture plate digestion, centrifugal condition be: 5min, 1500rpm, after removing supernatant, add the fresh α-MEM culture medium that concentration is the hyclone of 10%, concentration of cell suspension is adjusted to 2.5 × 10
6/ mL.Then add in porous support by 10 μ L cell suspension, after hatching 2h, every hole adds 100 μ L culture medium, in 37 DEG C of incubators, be cultured to required time.Change liquid every other day, to keep the nutrition supply of cell.
In 96 orifice plates, every hole adds 100 μ LMTT solution, is placed in 37 DEG C of incubators and continues to hatch 4h.Above taking out with tweezers, the compound rest that obtains is put in 2mL centrifuge tube, adds 1mLDMSO, blows and beats to crystal and dissolve completely.Drawing 200 μ L adds in ELISA Plate, at the upper absorbance measuring 570nm of microplate reader (Bio-Rad550).As shown in Figure 7, cell quantity in hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold increases along with the prolongation of time, and the amount increased is maximum in four kinds of supports, shows that hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold prepared by this patent has good biocompatibility.
(6), after the porous support that step (4) obtains being carried out ultraviolet sterilization, 496 well culture plates are put into.By 0.25% pancreatin/PBS solution by preosteoblast MC3T3-E1 from culture plate digestion, centrifugal condition be: 5min, 1500rpm, after removing supernatant, add the fresh α-MEM culture medium that concentration is the hyclone of 10%, concentration of cell suspension is adjusted to 2.5 × 10
6/ mL.Then 10 μ L cell suspension are added in porous support, after hatching 2h, every hole adds 100 μ L culture medium, be changed to after 24h containing dexamethasone culture medium (containing the ascorbic acid of 10%FCS, 50mM, the DMEM in high glucose of 10mM β-phosphoglycerol disodium and 100nM dexamethasone), in 37 DEG C of incubators, be cultured to required time.Change liquid every other day, to keep the nutrition supply of cell.
Taken out from culture plate by sample, after PBS cleans 3 times, add 100 μ L cell pyrolysis liquids, 4 DEG C of cracking are spent the night, ultrasonication.Add 500 μ L alkaline phosphatase substrate reactant liquors, after 37 DEG C of water-bath 30min, add the NaOH solution 500 μ L cessation reaction of 0.1M, measure the absorption value at 405nm place immediately at ultraviolet-visible spectrophotometer.As shown in Figure 8, on hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold, the alkaline phosphatase activities of cell extends continuous increase in time, and the amount increased is maximum in four kinds of supports, show that hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold prepared by this patent can support propagation and the differentiation of mesenchymal stem cells MSCs.
Embodiment 2
(1) will the Bombyx bombycis of pupa be gone to shred, the Bombyx bombycis of 5g is added 100ml, concentration is the NaCO of 0.2wt%
3carry out the formation fibroin that comes unstuck in aqueous solution, under 80 DEG C of conditions, boil 4h, extremely neutral with washed with de-ionized water fibroin after coming unstuck;
(2) above-mentioned steps is repeated again once;
(3) by being washed till neutral fibroin in 40 DEG C of oven dry, fibroin albumen is obtained; Get fibroin albumen, adding concentration is in 9.8mol/L lithium bromide water solution, dialysing with deionized water after 40 DEG C of heating in water bath 6h, (molecular cut off of bag filter was 8000 in 3 days, change water every day 2 times), then in 25 DEG C, the centrifugal 5min of 10000r/min, centrifugal twice, obtain the silk fibroin protein solution that concentration is 10wt%;
(4) 60mg hydroxyapatite will be added, 60mg nano micro crystal cellulose adds in the 10wt% silk fibroin protein solution of 1ml step (3), stir ,-70 DEG C of lyophilization 18h, obtain hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold.
Embodiment 3
(1) will the Bombyx bombycis of pupa be gone to shred, the Bombyx bombycis of 2g is added 100ml, concentration is the NaCO of 0.2wt%
3carry out the formation fibroin that comes unstuck in aqueous solution, under 90 DEG C of conditions, boil 3h.Extremely neutral with washed with de-ionized water fibroin after coming unstuck;
(2) above-mentioned steps is repeated again once;
(3) by being washed till neutral fibroin in 60 DEG C of oven dry, fibroin albumen is obtained; Get fibroin albumen, adding concentration is in 9.8mol/L lithium bromide water solution, dialysing with deionized water after 60 DEG C of heating in water bath 4h, (molecular cut off of bag filter was 8000 in 4 days, change water every day once), then in 25 DEG C, the centrifugal 15min of 10000r/min, centrifugal twice, obtaining concentration is 2wt% silk fibroin protein solution;
(4) 5mg hydroxyapatite will be added, 5mg nano micro crystal cellulose adds in the 2wt% silk fibroin protein solution of 1ml step (3), stir ,-70 DEG C of lyophilization 24h, obtain hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold.
Comparative example 1
(1) will the Bombyx bombycis of pupa be gone to shred, the Bombyx bombycis of 5g is added 200ml, concentration is the NaCO of 0.2wt%
3aqueous solution comes unstuck formation fibroin, boils 3h in 100 DEG C, after coming unstuck with washed with de-ionized water fibroin to neutral, and then repeat above-mentioned steps once, be finally just washed till neutral fibroin in 50 DEG C of oven dry, obtain fibroin albumen; Get fibroin albumen, adding concentration is in 9.8mol/L lithium bromide water solution, 4 days (molecular cut off of bag filter is 8000) is dialysed with deionized water after 50 DEG C of heating in water bath 6h, wherein change water every day twice, then in 25 DEG C, the centrifugal 10min of 10000r/min, centrifugal twice, obtain the silk fibroin protein solution that concentration is 4wt%;
(2) by the 4wt% silk fibroin protein solution of step (1) gained, in-60 DEG C of lyophilization 24h, silk fibroin porous scaffold is obtained.The scanning electron microscope (SEM) photograph of silk fibroin porous scaffold as shown in Figure 3.Can know from Fig. 3, it is 80-90% that this compound rest has porosity, and aperture is 200-300um.
(3), after the porous support that step (2) obtains being carried out ultraviolet sterilization, 496 well culture plates are put into.By 0.25% pancreatin/PBS solution by preosteoblast MC3T3-E1 from culture plate digestion, centrifugal condition be: 5min, after 1500rpm, removal supernatant, add the fresh α-MEM culture medium that concentration is the hyclone of 10%, concentration of cell suspension is adjusted to 2.5 × 10
6/ mL.Then add in porous support by 10 μ L cell suspension, after hatching 2h, every hole adds 100 μ L culture medium, is cultured to required time, changes liquid every other day in 37 DEG C of incubators, to keep the nutrition supply of cell.
In 96 orifice plates, every hole adds 100 μ LMTT solution, is placed in 37 DEG C of incubators and continues to hatch 4h.Above taking out with tweezers, the compound rest that obtains is put in 2mL centrifuge tube, adds 1mLDMSO, blows and beats to crystal and dissolve completely.Drawing 200 μ L adds in ELISA Plate, at the upper absorbance measuring 570nm of microplate reader (Bio-Rad550).As shown in Figure 7, cell quantity in silk fibroin porous scaffold increases along with the prolongation of time, but silk fibroin porous scaffold prepared by comparative example 1 is minimum, and hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold prepared by this patent has best biocompatibility.
(4), after the porous support that step (2) obtains being carried out ultraviolet sterilization, 496 well culture plates are put into.By 0.25% pancreatin/PBS solution, preosteoblast MC3T3-E1 is digested from culture plate, centrifugal condition is: 5min, 1500rpm, after removing supernatant, add the fresh α-MEM culture medium that concentration is the hyclone of 10%, concentration of cell suspension is adjusted to 2.5 × 10
6/ mL.Then 10 μ L cell suspension are added in porous support, after hatching 2h, every hole adds 100 μ L culture medium, be changed to after 24h containing dexamethasone culture medium (containing the ascorbic acid of 10%FCS, 50mM, the DMEM in high glucose of 10mM β-phosphoglycerol disodium and 100nM dexamethasone), in 37 DEG C of incubators, be cultured to required time.Change liquid every other day, to keep the nutrition supply of cell.
Taken out from culture plate by sample, after PBS cleans 3 times, add 100 μ L cell pyrolysis liquids, 4 DEG C of cracking are spent the night, ultrasonication.Add 500 μ L alkaline phosphatase substrate reactant liquors, after 37 DEG C of water-bath 30min, add the NaOH solution 500 μ L cessation reaction of 0.1M, measure the absorption value at 405nm place immediately at ultraviolet-visible spectrophotometer.As shown in Figure 8, on silk fibroin porous scaffold, the alkaline phosphatase activities of cell extends continuous increase in time, and the amount increased is minimum in four kinds of supports, but the alkaline phosphatase activities of cell is the highest on hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold prepared by this patent, show that hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold prepared by this patent can support propagation and the differentiation of mesenchymal stem cells MSCs, and effect is best.
Comparative example 2
(1) will the Bombyx bombycis of pupa be gone to shred, the Bombyx bombycis of 5g is added 200ml, concentration is the NaCO of 0.2wt%
3aqueous solution, boils 3h and carries out the formation fibroin that comes unstuck at 100 DEG C.Extremely neutral with washed with de-ionized water fibroin after coming unstuck, and then repeat above-mentioned steps once, be finally just washed till neutral fibroin in 50 DEG C of oven dry, obtain fibroin albumen; Get fibroin albumen, adding concentration is in 9.8mol/L lithium bromide water solution, 4 days (molecular cut off of bag filter is 8000) is dialysed with deionized water after 50 DEG C of heating in water bath 6h, wherein change water every day twice, then in 25 DEG C, the centrifugal 10min of 10000r/min, centrifugal twice, obtain the silk fibroin protein solution that concentration is 4wt%;
(2) will add 60mg nano micro crystal cellulose adds in the 4wt% silk fibroin protein solution of 1ml step (1), stir,-60 DEG C of lyophilization 24h, obtain the scanning electron microscope (SEM) photograph of nano micro crystal cellulose/silk fibroin porous scaffold nano micro crystal cellulose/silk fibroin porous scaffold as shown in Figure 4.Can know from Fig. 4, the porosity of this compound rest is 80-90%, and aperture is 200-250um, and nano micro crystal cellulose is uniformly distributed in support.
(3), after the porous support that step (2) obtains being carried out ultraviolet sterilization, 496 well culture plates are put into.By 0.25% pancreatin/PBS solution by preosteoblast MC3T3-E1 from culture plate digestion, centrifugal condition be: 5min, after 1500rpm, removal supernatant, add the fresh α-MEM culture medium that concentration is the hyclone of 10%, concentration of cell suspension is adjusted to 2.5 × 10
6/ mL.Then add in porous support by 10 μ L cell suspension, after hatching 2h, every hole adds 100 μ L culture medium, in 37 DEG C of incubators, be cultured to required time.Change liquid every other day, to keep the nutrition supply of cell.
In 96 orifice plates, every hole adds 100 μ LMTT solution, is placed in 37 DEG C of incubators and continues to hatch 4h.Above taking out with tweezers, the compound rest that obtains is put in 2mL centrifuge tube, adds 1mL dimethyl sulfoxide (DMSO), blows and beats to crystal and dissolve completely.Drawing 200 μ L adds in ELISA Plate, at the upper absorbance measuring 570nm of microplate reader (Bio-Rad550).As shown in Figure 7, cell quantity in nano micro crystal cellulose/silk fibroin porous scaffold increases along with the prolongation of time, but the cell concentration of hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold prepared by this patent embodiment 1 is maximum, shows that hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold prepared by this patent has best biocompatibility.
(4), after the porous support that step (2) obtains being carried out ultraviolet sterilization, 496 well culture plates are put into.By 0.25% pancreatin/PBS solution, preosteoblast MC3T3-E1 is digested from culture plate, centrifugal condition is: 5min, 1500rpm, after removing supernatant, add the fresh α-MEM culture medium that concentration is the hyclone of 10%, concentration of cell suspension is adjusted to 2.5 × 10
6/ mL.Then 10 μ L cell suspension are added in porous support, after hatching 2h, every hole adds 100 μ L culture medium, be changed to after 24h containing dexamethasone culture medium (containing the ascorbic acid of 10%FCS, 50mM, the DMEM in high glucose of 10mM β-phosphoglycerol disodium and 100nM dexamethasone), in 37 DEG C of incubators, be cultured to required time.Change liquid every other day, to keep the nutrition supply of cell.
Taken out from culture plate by sample, after PBS cleans 3 times, add 100 μ L cell pyrolysis liquids, 4 DEG C of cracking are spent the night, ultrasonication.Add 500 μ L alkaline phosphatase substrate reactant liquors, after 37 DEG C of water-bath 30min, add the NaOH solution 500 μ L cessation reaction of 0.1M, measure the absorption value at 405nm place immediately at ultraviolet-visible spectrophotometer.As shown in Figure 8, on nano micro crystal cellulose/silk fibroin porous scaffold, the alkaline phosphatase activities of cell extends continuous increase in time, but the alkaline phosphatase activities of cell is the highest on hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold prepared by this patent, show that hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold prepared by this patent can support propagation and the differentiation of mesenchymal stem cells MSCs, and effect is best.
Comparative example 3
(1) will the Bombyx bombycis of pupa be gone to shred, the Bombyx bombycis of 5g is added 200ml, concentration is the NaCO of 0.2wt%
3aqueous solution, boils 3h and carries out the formation fibroin that comes unstuck at 100 DEG C.Extremely neutral with washed with de-ionized water fibroin after coming unstuck, and then repeat above-mentioned steps once, be finally just washed till neutral fibroin in 50 DEG C of oven dry, obtain fibroin albumen; Get fibroin albumen, adding concentration is in 9.8mol/L lithium bromide water solution, 4 days (molecular cut off of bag filter is 8000) is dialysed with deionized water after 50 DEG C of heating in water bath 6h, wherein change water every day twice, then in 25 DEG C, the centrifugal 10min of 10000r/min, centrifugal twice, obtain the silk fibroin protein solution that concentration is 4wt%;
(2) 60mg hydroxyapatite will be added add in the 4wt% silk fibroin protein solution of 1ml step (1), stir ,-60 DEG C of lyophilization 24h, obtain hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold.The scanning electron microscope (SEM) photograph of hydroxyapatite/silk fibroin porous scaffold as shown in Figure 5.Can know from Fig. 5, the porosity of this compound rest is 70-85%, and aperture is 150-180um, and hydroxyapatite is uniformly distributed in support.
(3), after the porous support that step (2) obtains being carried out ultraviolet sterilization, 496 well culture plates are put into.By 0.25% pancreatin/PBS solution, preosteoblast MC3T3-E1 is digested from culture plate, centrifugal condition is: 5min, 1500rpm, after removing supernatant, add the fresh α-MEM culture medium that concentration is the hyclone of 10%, concentration of cell suspension is adjusted to 2.5 × 10
6/ mL.Then add in porous support by 10 μ L cell suspension, after hatching 2h, every hole adds 100 μ L culture medium, in 37 DEG C of incubators, be cultured to required time.Change liquid every other day, to keep the nutrition supply of cell.
In 96 orifice plates, every hole adds 100 μ LMTT solution, is placed in 37 DEG C of incubators and continues to hatch 4h.Above taking out with tweezers, the compound rest that obtains is put in 2mL centrifuge tube, adds 1mLDMSO, blows and beats to crystal and dissolve completely.Drawing 200 α L adds in ELISA Plate, at the upper absorbance measuring 570nm of microplate reader (Bio-Rad550).As shown in Figure 7, cell quantity in hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold increases along with the prolongation of time, but the cell concentration of hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold prepared by this patent embodiment 1 is maximum, shows that hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold prepared by this method has best biocompatibility.
(4), after the porous support that step (2) obtains being carried out ultraviolet sterilization, 496 well culture plates are put into.By 0.25% pancreatin/PBS solution by preosteoblast MC3T3-E1 from culture plate digestion, centrifugal condition be: 5min, after 1500rpm, removal supernatant, add the fresh α-MEM culture medium that concentration is the hyclone of 10%, concentration of cell suspension is adjusted to 2.5 × 10
6/ mL.Then 10 μ L cell suspension are added in porous support, after hatching 2h, every hole adds 100 μ L culture medium, be changed to after 24h containing dexamethasone culture medium (containing the ascorbic acid of 10%FCS, 50mM, the DMEM in high glucose of 10mM β-phosphoglycerol disodium and 100nM dexamethasone), in 37 DEG C of incubators, be cultured to required time.Change liquid every other day, to keep the nutrition supply of cell.
Taken out from culture plate by sample, after PBS cleans 3 times, add 100 μ L cell pyrolysis liquids, 4 DEG C of cracking are spent the night, ultrasonication.Add 500 μ L alkaline phosphatase substrate reactant liquors, after 37 DEG C of water-bath 30min, add the NaOH solution 500 μ L cessation reaction of 0.1M, measure the absorption value at 405nm place immediately at ultraviolet-visible spectrophotometer.As shown in Figure 8, on hydroxyapatite/silk fibroin porous scaffold, the alkaline phosphatase activities of cell extends continuous increase in time, but the alkaline phosphatase activities of cell is the highest on hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold prepared by this patent, show that hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold prepared by this patent can support propagation and the differentiation of mesenchymal stem cells MSCs, and effect is best.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (9)
1. a bone tissue engineer porous compound support frame, it is characterized in that: described bone tissue engineer porous compound support frame is hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold, the three-dimensional stephanoporate compound stent be made up of hydroxyapatite, nano micro crystal cellulose, fibroin albumen, porosity is 90 ~ 95%, and aperture is 180 ~ 220 μm.
2. bone tissue engineer porous compound support frame according to claim 1, is characterized in that: described hydroxyapatite is needle-like, and particle diameter is 10 ~ 100nm.
3. bone tissue engineer porous compound support frame according to claim 1, is characterized in that: nano micro crystal cellulose is that its length is 150-350nm, and width is 20-40nm in regular corynebacterium structure.
4., for the preparation of a method for the bone tissue engineer porous compound support frame one of claims 1 to 3 Suo Shu, it is characterized in that: said method comprising the steps of:
(1) Bombyx bombycis is shredded, then add 80-100 DEG C, concentration is 0.2wt%NaCO
3boil 2-4h in aqueous solution and carry out the formation fibroin that comes unstuck, Bombyx bombycis and NaCO
3the ratio of aqueous solution is: 2-5g Bombyx bombycis adds 100ml, 0.2wt%NaCO
3aqueous solution, extremely neutral with washed with de-ionized water fibroin after coming unstuck;
(2) step (1) is repeated;
(3) step (2) is washed till neutral fibroin in 40-60 DEG C of oven dry, obtains fibroin albumen; Get fibroin albumen, adding concentration is in 9.8mol/L lithium bromide water solution, with deionized water dialysis 3 ~ 4 days after 40 ~ 60 DEG C of heating in water bath 4 ~ 6h, and centrifugal twice, obtain 2 ~ 10wt% silk fibroin protein solution;
(4) hydroxyapatite, nano micro crystal cellulose are added in 2 ~ 10wt% silk fibroin protein solution, the ratio of mixing is: every milliliter of silk fibroin protein solution adds 0<m≤60mg hydroxyapatite, 0<m≤60mg nano micro crystal cellulose stirs, wherein m represents quality, then lyophilization, obtains hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold.
5. the preparation method of a kind of bone tissue engineer porous compound support frame according to claim 4, is characterized in that: the amount that every milliliter of silk fibroin protein solution described in step (2) adds hydroxyapatite and nano micro crystal cellulose is 30mg.
6. the preparation method of a kind of bone tissue engineer porous compound support frame according to claim 4, is characterized in that: described Bombyx bombycis is the Bombyx bombycis of pupa.
7. the preparation method of a kind of bone tissue engineer porous compound support frame according to claim 4, is characterized in that: described dialysis procedure adopt molecular cut off be 10000 ~ 12000 bag filter dialyse.
8. the preparation method of a kind of bone tissue engineer porous compound support frame according to claim 4, is characterized in that: described centrifugal condition is in 25 DEG C, centrifugal 5-15min under 10000r/min condition.
9. the preparation method of a kind of bone tissue engineer porous compound support frame according to claim/4, is characterized in that: the cryodesiccated condition described in step (2) is-50--70 DEG C of lyophilization 18-24h.
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| CN107789667B (en) * | 2017-10-12 | 2020-12-04 | 广州贝奥吉因生物科技有限公司 | Porous composite scaffold for promoting cartilage regeneration and preparation method thereof |
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