CN105521525B - A kind of bone tissue engineer porous compound support frame and preparation method thereof - Google Patents
A kind of bone tissue engineer porous compound support frame and preparation method thereof Download PDFInfo
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- CN105521525B CN105521525B CN201510953345.3A CN201510953345A CN105521525B CN 105521525 B CN105521525 B CN 105521525B CN 201510953345 A CN201510953345 A CN 201510953345A CN 105521525 B CN105521525 B CN 105521525B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
- A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
- A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
Abstract
The present invention relates to a kind of biological field, specifically a kind of bone tissue engineer porous compound support frame and preparation method thereof.Bone tissue engineer provided by the invention is hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold with porous compound support frame, the three-dimensional stephanoporate compound stent being made of hydroxyapatite, nano micro crystal cellulose, fibroin albumen, porosity is 90~95%, and aperture is 180~220 μm.Hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold of the present invention, hydroxyapatite and nano micro crystal cellulose are uniformly distributed in bracket, good mechanical performance, and the bracket has good biocompatibility, can support and promote the Osteoblast Differentiation of mesenchymal stem cell.Preparation process of the present invention is simple, material source is extensive, and high production efficiency is at low cost, can be applied to industrialized production.
Description
Technical field
The present invention relates to biological medicine field of material technology, specifically the porous compound support frame of bone tissue engineer and its
Preparation method.
Background technique
Bone is the bracket of human body, plays protection and supporting function to human body, and plays leverage during exercise.Periosteum, bone
Matter and marrow are the three parts for constituting bone.But bone defect is common disease clinically, according to statistics, China every year because disease,
Bone defect patient caused by wound, tumour etc. is more than 1,000,000, and the reparation of a wide range of bone defect is still the problem of orthopaedics therapy at present
One of.Although bone has the ability of regeneration and selfreparing, self-regeneration is leaned on merely, and defect is often difficult self to be cured
It closes.In this case, then it is treated.Currently, the method for the treatment of bone defect has bone collection, artificial bone graft to substitute material
Material and tissue engineering technique etc..Wherein, organizational engineering tissue is with source is unrestricted, can be pre-designed shape, low antigenicity
And have many advantages, such as vitality.In addition, autologous bone transplanting donor can be overcome not using bone tissue engineer technology treatment bone defect
The disadvantages of foot and allogenic bone transplantation immunological rejection.Therefore, it is treatment bone defect that Bone Defect Repari is carried out using bone tissue engineer
Hot spot.
Fibroin albumen (Silk fibroin, SF) is a kind of hydrophobic natural polymer subbundle egg extracted from silk
White matter is made of 18 kinds of amino acid, and wherein glycine, alanine and serine account for about 80% of its total composition or more.Fibroin egg
White abundance, it is low in cost, there is excellent in mechanical performance, stable structure, good biocompatibility and machinability it is excellent
Point is a kind of excellent natural high molecular material.Based on these advantages of fibroin albumen, it is increasingly being used as bone tissue engineer
Timbering material.But since fibroin albumen itself is without the effect of self-bone grafting and osteoacusis, the reparation of bone defect is limited,
Therefore, a kind of silk fibroin porous scaffold with self-bone grafting and osteoconductive potential is prepared, as Bone Defect Repari tissue engineering material,
It is the purpose of this patent.
Hydroxyapatite (Hydroxyapatite, HA) belongs to Ca-P ceramic, since it is non-with the inorganic constituents inside bone
It is often similar, it is a kind of important bioceramic in bone tissue engineer.Hydroxyapatite has good biocompatibility, biology
Activity, osteoconductive and osteoinductive, be frequently used for dental restortion, Bone Defect Repari and orthopedic reparation.But hydroxyapatite is not easy
It is absorbed and brittleness is big, limit its application in bone tissue engineer.Therefore, it is necessary to research and develop a kind of new medicine-carried system, make
Obtaining hydroxyapatite can successfully apply in bone tissue engineer.
Nano micro crystal cellulose (Nanocrystal Cellulose, CNC), is by native cellulose or microcrystalline cellulose
By chemistry, the method for physics or biochemistry is prepared, and nano micro crystal cellulose not only has the relevant nature of nanoparticle
Such as: biggish specific surface, high intensity and high-crystallinity；It has been also equipped with the basic structure and characteristic of cellulose, such as: it is nontoxic
The features such as property, biocompatibility and degradability；In addition, the role that nano-cellulose generally serves as in compound is reinforcement
Filler, to improve the thermal stability and mechanical performance of compound.Moreover, hydroxyl rich in nano micro crystal cellulose, adds
Enter in compound, the hydrophilicity of compound can be improved.
In recent years, using fibroin albumen as three-dimensional rack, become hot spot in bone tissue engineer application and research.It grinds
Study carefully and show to use fibroin albumen as bone renovating material, relatively good repairing effect is obtained for nude mice skull defeci；And nanometer
The role that cellulose generally serves as in compound is reinforced filling, to improve the thermal stability of compound, hydrophily and machine
Tool performance.On the other hand, how to improve hydroxyapatite is not easy to be absorbed and brittleness is big disadvantage and researcher is of interest
The problem of.
Summary of the invention
The present invention in view of the above problems, provides a kind of good mechanical performance, with good biocompatibility
Bone tissue engineer porous compound support frame.
The present invention is implemented as follows:
A kind of porous compound support frame of bone tissue engineer, it is characterised in that: the porous composite support of the bone tissue engineer
Frame be hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold, be by hydroxyapatite, nano micro crystal cellulose,
The three-dimensional stephanoporate compound stent of fibroin albumen composition, porosity are 90~95%, and aperture is 180~220 μm.
Preferably, the hydroxyapatite is needle-shaped, and partial size is 10~100nm.
Preferably, nano micro crystal cellulose is in regular corynebacterium structure, and the length is 150-350nm, width 20-
40nm。
It is a further object of the present invention to provide the methods of the porous compound support frame of above-mentioned bone tissue engineer:
It the described method comprises the following steps:
(1) silk cocoon is shredded, 80-100 DEG C, concentration 0.2wt%NaCO is then added32-4h is boiled in aqueous solution to be taken off
Glue forms fibroin, silk cocoon and NaCO3The ratio of aqueous solution are as follows: 100ml, 0.2wt%NaCO is added in 2-5g silk cocoon3Aqueous solution, degumming
Fibroin is cleaned to neutrality with deionized water afterwards；
(2) step (1) is repeated；
(3) step (2) is washed till neutral fibroin to dry in 40-60 DEG C, obtains fibroin albumen；Fibroin albumen is taken, is added
Concentration is to use deionized water dialysis 3~4 days after 40~60 DEG C of 4~6h of heating water bath in 9.8mol/L lithium bromide water solution, centrifugation
Twice, 2~10wt% silk fibroin protein solution is obtained；
(4) hydroxyapatite, nano micro crystal cellulose are added in 2~10wt% silk fibroin protein solution, mixed ratio
Are as follows: 0 < m≤60mg hydroxyapatite is added in every milliliter of silk fibroin protein solution, 0 < m≤60mg nano micro crystal cellulose stirs evenly,
Wherein m indicates quality, is then freeze-dried, obtains hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold.
Preferably, hydroxyapatite and nano micro crystal cellulose is added in every milliliter of silk fibroin protein solution described in step (2)
Amount be 30mg.
Further, the silk cocoon is the silk cocoon of pupa；
Preferably, the dialysis procedure uses molecular cut off to dialyse for 10000~12000 bag filter.
Preferably, the condition of the centrifugation is to be centrifuged 5-15min under the conditions of 25 DEG C, 10000r/min.
Preferably, the condition of freeze-drying described in step (2) is -50~-70 DEG C of freeze-drying 18-24h.
The present invention has the following advantages and effects with respect to the prior art:
(1) hydroxyapatite of the present invention/nano micro crystal cellulose/silk fibroin porous scaffold, hydroxyapatite and nanometer
Microcrystalline cellulose is uniformly distributed in bracket, good mechanical performance, and the bracket has good biocompatibility, Neng Gouzhi
Hold and promote the Osteoblast Differentiation of mesenchymal stem cell；
(2) complex stephanoporate bracket has the advantages that porosity height (90-95%), uniform pore diameter (180-220um)；
(3) present invention mixes hydroxyapatite, nano micro crystal cellulose and silk fibroin protein solution, passes through freeze-drying
Method forms hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold, and preparation process is simple, material source is wide
General, high production efficiency is at low cost, can be applied to industrialized production.
Detailed description of the invention
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, figure
The transmission electron microscope picture of 1b 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 nano micro crystal cellulose/silk fibroin porous scaffold scanning electron microscope (SEM) photograph of comparative example 2；
Fig. 5 is hydroxyapatite/silk fibroin porous scaffold scanning electron microscope (SEM) photograph of comparative example 3；
Fig. 6 is hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold scanning electron microscope (SEM) photograph of embodiment 1；
Fig. 7 is nano micro crystal cellulose/fibroin egg of the silk fibroin porous scaffold of comparative example 1, comparative example 2
Hydroxyapatite/nanometer of white porous support, hydroxyapatite/silk fibroin porous scaffold of comparative example 3 and embodiment 1
Microcrystalline cellulose/silk fibroin porous scaffold cytotoxicity figure, in which: SF is the silk fibroin porous branch of comparative example 1
Frame cytotoxicity figure；CNC/SF is nano micro crystal cellulose/silk fibroin porous scaffold cytotoxicity figure than embodiment 2；HA/
SF is hydroxyapatite/silk fibroin porous scaffold cytotoxicity figure of comparative example 3；HA/CNC/SF is the hydroxyl of embodiment 1
Base apatite/nano micro crystal cellulose/silk fibroin porous scaffold cytotoxicity figure；
Fig. 8 is the silk fibroin porous scaffold of comparative example 1, nano micro crystal cellulose/fibroin albumen than embodiment 2
Hydroxyapatite/nanometer of porous support, hydroxyapatite/silk fibroin porous scaffold of comparative example 3 and embodiment 1 is micro-
Crystalline cellulose/silk fibroin porous scaffold Proliferation of Bone Mesenchymal Stem Cells differentiation figure；Wherein: SF is comparative example 1
The Proliferation of Bone Mesenchymal Stem Cells of silk fibroin porous scaffold breaks up figure；CNC/SF is the nano microcrystalline fiber than embodiment 2
Element/silk fibroin porous scaffold Proliferation of Bone Mesenchymal Stem Cells differentiation figure；HA/SF be comparative example 3 hydroxyapatite/
Silk fibroin porous scaffold Proliferation of Bone Mesenchymal Stem Cells differentiation figure；HA/CNC/SF be embodiment 1 hydroxyapatite/receive
Rice microcrystalline cellulose/silk fibroin porous scaffold Proliferation of Bone Mesenchymal Stem Cells differentiation figure.
Case is embodied
It elaborates below to the embodiment of the present invention, the present embodiment carries out under the premise of the technical scheme of the present invention
Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation
Example.
Embodiment 1
(1) silk cocoon of pupa will be gone to shred, by the NaCO that 100ml is added in the silk cocoon of 2.5g, concentration is 0.2wt%3Aqueous solution
Middle progress degumming forms fibroin, boils 2h under the conditions of 100 DEG C.Fibroin is cleaned to neutrality with deionized water after degumming；
(2) it is primary that above-mentioned steps are repeated；
(3) neutral fibroin will be washed till to dry in 50 DEG C, obtains fibroin albumen；Fibroin albumen is taken, concentration, which is added, is
In 9.8mol/L lithium bromide water solution, with deionized water dialysis 4 days, (molecular cut off of bag filter was after 50 DEG C of heating water bath 6h
8000, it is primary that water is changed daily), it then is centrifuged 10min in 25 DEG C, 10000r/min, twice, obtaining concentration is 4wt%'s for centrifugation
Silk fibroin protein solution；
(4) 30mg hydroxyapatite will be added, the 4wt% fibroin egg of 1ml step (3) is added in 30mg nano micro crystal cellulose
It in white solution, stirs evenly, -60 DEG C of freeze-dryings for 24 hours, obtain hydroxyapatite/nano micro crystal cellulose/silk fibroin porous
Bracket.From Fig. 1 and Fig. 2 it is found that hydroxyapatite be it is needle-shaped, partial size be 10~100nm；Nano micro crystal cellulose is in regular
Corynebacterium structure, length are about 150-350nm, and width is about 20-40nm.Hydroxyapatite/nano micro crystal cellulose/fibroin
The scanning electron microscope (SEM) photograph of albumen porous support is as shown in Figure 6.From Fig. 6 it is recognised that the compound rest has porosity height (90-
95%), uniform pore diameter (180-220um), hydroxyapatite and nano micro crystal cellulose are uniformly distributed in bracket.
(5) it after the obtained porous support of step (4) being carried out ultraviolet sterilization, is put into 496 well culture plates.With 0.25%
Pancreatin/PBS solution by preosteoblast MC3T3-E1 from culture plate digestion, centrifugal condition are as follows: 5min, 1500rpm, remove supernatant
After night, the fresh α-MEM culture medium for the fetal calf serum that concentration is 10% is added, concentration of cell suspension is adjusted to 2.5 × 106/
mL.Then 10 μ L cell suspensions are added in porous support, after hatching 2h, 100 μ L culture mediums are added in every hole, in 37 DEG C of incubators
It is middle to cultivate to required time.Liquid is changed, every other day to keep the nutrition supply of cell.
In 96 orifice plates, 100 μ L MTT solution are added in every hole, are placed in 37 DEG C of incubators and are continued to be incubated for 4h.It is taken with tweezers
Acquired compound rest is put into 2mL centrifuge tube above out, and 1mL DMSO is added, and piping and druming to crystal is completely dissolved.It draws
200 μ L are added in ELISA Plate, and the absorbance of 570nm is measured on microplate reader (Bio-Rad 550).As shown in fig. 7, hydroxy-apatite
Cell quantity in stone/nano micro crystal cellulose/silk fibroin porous scaffold increases with the extension of time, and increased
Amount be in four kinds of brackets at most, show this patent preparation hydroxyapatite/nano micro crystal cellulose/silk fibroin porous branch
Frame has good biocompatibility.
(6) it after the obtained porous support of step (4) being carried out ultraviolet sterilization, is put into 496 well culture plates.With 0.25%
Pancreatin/PBS solution by preosteoblast MC3T3-E1 from culture plate digestion, centrifugal condition are as follows: 5min, 1500rpm, remove supernatant
After night, the fresh α-MEM culture medium for the fetal calf serum that concentration is 10% is added, concentration of cell suspension is adjusted to 2.5 × 106/
mL.Then 10 μ L cell suspensions are added in porous support, after hatching 2h, 100 μ L culture mediums are added in every hole, are changed to contain afterwards for 24 hours
Dexamethasone culture medium (ascorbic acid containing 10%FCS, 50mM, 10mM β-phosphoglycerol disodium and 100nM dexamethasone height
Sugared DMEM), it cultivates in 37 DEG C of incubators to required time.Liquid is changed, every other day to keep the nutrition supply of cell.
Sample is taken out from culture plate, after PBS is cleaned 3 times, 100 μ L cell pyrolysis liquids are added, 4 DEG C of cracking overnight, surpass
Sound is broken.It is added 500 μ L alkaline phosphatase substrate reaction solutions, after 37 DEG C of water-bath 30min, the NaOH solution of 0.1M is added
500 μ L terminate reaction, immediately the absorption value at ultraviolet-visible spectrophotometer measurement 405nm.As shown in figure 8, hydroxyl phosphorus
The alkaline phosphatase activities of cell extend at any time on lime stone/nano micro crystal cellulose/silk fibroin porous scaffold is continuously increased,
And increased amount be in four kinds of brackets at most, show this patent preparation hydroxyapatite/nano micro crystal cellulose/fibroin egg
White porous support can support the proliferation and differentiation of mesenchymal stem cell.
Embodiment 2
(1) silk cocoon of pupa will be gone to shred, by the NaCO that 100ml is added in the silk cocoon of 5g, concentration is 0.2wt%3In aqueous solution
It carries out degumming and forms fibroin, boil 4h under the conditions of 80 DEG C, clean fibroin to neutrality with deionized water after degumming；
(2) it is primary that above-mentioned steps are repeated；
(3) neutral fibroin will be washed till to dry in 40 DEG C, obtains fibroin albumen；Fibroin albumen is taken, concentration, which is added, is
In 9.8mol/L lithium bromide water solution, with deionized water dialysis 3 days, (molecular cut off of bag filter was after 40 DEG C of heating water bath 6h
8000, water is changed daily 2 times), it then is centrifuged 5min in 25 DEG C, 10000r/min, centrifugation twice, obtains the silk that concentration is 10wt%
Fibroin solution；
(4) 60mg hydroxyapatite will be added, the 10wt% fibroin of 1ml step (3) is added in 60mg nano micro crystal cellulose
It in protein solution, stirs evenly, it is more to obtain hydroxyapatite/nano micro crystal cellulose/fibroin albumen by -70 DEG C of freeze-drying 18h
Hole bracket.
Embodiment 3
(1) silk cocoon of pupa will be gone to shred, by the NaCO that 100ml is added in the silk cocoon of 2g, concentration is 0.2wt%3In aqueous solution
It carries out degumming and forms fibroin, boil 3h under the conditions of 90 DEG C.Fibroin is cleaned to neutrality with deionized water after degumming；
(2) it is primary that above-mentioned steps are repeated；
(3) neutral fibroin will be washed till to dry in 60 DEG C, obtains fibroin albumen；Fibroin albumen is taken, concentration, which is added, is
In 9.8mol/L lithium bromide water solution, with deionized water dialysis 4 days, (molecular cut off of bag filter was after 60 DEG C of heating water bath 4h
8000, it is primary that water is changed daily), it then is centrifuged 15min in 25 DEG C, 10000r/min, twice, obtaining concentration is 2wt% for centrifugation
Fibroin solution；
(4) 5mg hydroxyapatite will be added, the 2wt% fibroin albumen of 1ml step (3) is added in 5mg nano micro crystal cellulose
It in solution, stirs evenly, -70 DEG C of freeze-dryings for 24 hours, obtain hydroxyapatite/nano micro crystal cellulose/silk fibroin porous branch
Frame.
Comparative example 1
(1) silk cocoon of pupa will be gone to shred, by the NaCO that 200ml is added in the silk cocoon of 5g, concentration is 0.2wt%3Aqueous solution is de-
Glue forms fibroin, boils 3h in 100 DEG C, cleans fibroin to neutrality with deionized water after degumming, and it is primary then to repeat above-mentioned steps,
It is finally just washed till neutral fibroin to dry in 50 DEG C, obtains fibroin albumen；Fibroin albumen is taken, addition concentration is 9.8mol/L bromination
In lithium aqueous solution, with deionized water dialysis 4 days (molecular cut off of bag filter is 8000) after 50 DEG C of heating water bath 6h, wherein often
It changes water twice, is then centrifuged 10min in 25 DEG C, 10000r/min, and centrifugation twice, obtains the fibroin albumen that concentration is 4wt%
Solution；
(2) it is more for 24 hours in -60 DEG C of freeze-dryings to be obtained into fibroin albumen for the resulting 4wt% silk fibroin protein solution of step (1)
Hole bracket.The scanning electron microscope (SEM) photograph of silk fibroin porous scaffold is as shown in Figure 3.From Fig. 3 it is recognised that the compound rest has hole
Rate is 80-90%, aperture 200-300um.
(3) it after the obtained porous support of step (2) being carried out ultraviolet sterilization, is put into 496 well culture plates.With 0.25%
Pancreatin/PBS solution by preosteoblast MC3T3-E1 from culture plate digestion, centrifugal condition are as follows: 5min, 1500rpm, removal supernatant
After night, the fresh α-MEM culture medium for the fetal calf serum that concentration is 10% is added, concentration of cell suspension is adjusted to 2.5 × 106/
mL.Then 10 μ L cell suspensions are added in porous support, after hatching 2h, 100 μ L culture mediums are added in every hole, in 37 DEG C of incubators
Middle culture changes liquid, every other day to required time to keep the nutrition supply of cell.
In 96 orifice plates, 100 μ L MTT solution are added in every hole, are placed in 37 DEG C of incubators and are continued to be incubated for 4h.It is taken with tweezers
Acquired compound rest is put into 2mL centrifuge tube above out, and 1mL DMSO is added, and piping and druming to crystal is completely dissolved.It draws
200 μ L are added in ELISA Plate, and the absorbance of 570nm is measured on microplate reader (Bio-Rad 550).As shown in fig. 7, fibroin albumen
Cell quantity in porous support increases with the extension of time, but silk fibroin porous branch prepared by comparative example 1
Frame be it is minimum, this patent preparation hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold have best life
Object compatibility.
(4) it after the obtained porous support of step (2) being carried out ultraviolet sterilization, is put into 496 well culture plates.With 0.25%
Pancreatin/PBS solution digests preosteoblast MC3T3-E1 from culture plate, centrifugal condition are as follows: 5min, 1500rpm remove supernatant
After night, the fresh α-MEM culture medium for the fetal calf serum that concentration is 10% is added, concentration of cell suspension is adjusted to 2.5 × 106/
mL.Then 10 μ L cell suspensions are added in porous support, after hatching 2h, 100 μ L culture mediums are added in every hole, are changed to contain afterwards for 24 hours
Dexamethasone culture medium (ascorbic acid containing 10%FCS, 50mM, 10mM β-phosphoglycerol disodium and 100nM dexamethasone height
Sugared DMEM), it cultivates in 37 DEG C of incubators to required time.Liquid is changed, every other day to keep the nutrition supply of cell.
Sample is taken out from culture plate, after PBS is cleaned 3 times, 100 μ L cell pyrolysis liquids are added, 4 DEG C of cracking overnight, surpass
Sound is broken.It is added 500 μ L alkaline phosphatase substrate reaction solutions, after 37 DEG C of water-bath 30min, the NaOH solution of 0.1M is added
500 μ L terminate reaction, immediately the absorption value at ultraviolet-visible spectrophotometer measurement 405nm.As shown in figure 8, fibroin egg
The alkaline phosphatase activities of cell extend be continuously increased at any time on white porous support, and increased amount is minimum in four kinds of brackets
, but this patent preparation hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold on cell alkaline phosphatase
Enzymatic activity be it is highest, show this patent preparation hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold can
Support the proliferation and differentiation of mesenchymal stem cell, and effect is best.
Comparative example 2
(1) silk cocoon of pupa will be gone to shred, by the NaCO that 200ml is added in the silk cocoon of 5g, concentration is 0.2wt%3Aqueous solution,
3h is boiled at 100 DEG C carries out degumming formation fibroin.Fibroin is cleaned to neutrality with deionized water after degumming, then repeats above-mentioned steps
Once, it is finally just washed till neutral fibroin to dry in 50 DEG C, obtains fibroin albumen；Fibroin albumen is taken, addition concentration is 9.8mol/
In L lithium bromide water solution, with deionized water dialysis 4 days (molecular cut off of bag filter is 8000) after 50 DEG C of heating water bath 6h,
Water is wherein changed daily twice, is then centrifuged 10min in 25 DEG C, 10000r/min, and centrifugation twice, obtains the silk that concentration is 4wt%
Fibroin solution；
(2) 60mg nano micro crystal cellulose will be added to be added in the 4wt% silk fibroin protein solution of 1ml step (1), stirring is equal
Even, -60 DEG C of freeze-dryings for 24 hours, obtain nano micro crystal cellulose/silk fibroin porous scaffold nano micro crystal cellulose/fibroin egg
The scanning electron microscope (SEM) photograph of white porous support is as shown in Figure 4.From Fig. 4 it is recognised that the porosity of the compound rest is 80-90%, hole
Diameter is 200-250um, and nano micro crystal cellulose is uniformly distributed in bracket.
(3) it after the obtained porous support of step (2) being carried out ultraviolet sterilization, is put into 496 well culture plates.With 0.25%
Pancreatin/PBS solution by preosteoblast MC3T3-E1 from culture plate digestion, centrifugal condition are as follows: 5min, 1500rpm, removal supernatant
After night, the fresh α-MEM culture medium for the fetal calf serum that concentration is 10% is added, concentration of cell suspension is adjusted to 2.5 × 106/
mL.Then 10 μ L cell suspensions are added in porous support, after hatching 2h, 100 μ L culture mediums are added in every hole, in 37 DEG C of incubators
It is middle to cultivate to required time.Liquid is changed, every other day to keep the nutrition supply of cell.
In 96 orifice plates, 100 μ L MTT solution are added in every hole, are placed in 37 DEG C of incubators and are continued to be incubated for 4h.It is taken with tweezers
Acquired compound rest is put into 2mL centrifuge tube above out, is added 1mL dimethyl sulfoxide (DMSO), is blown and beaten complete to crystal
Fully dissolved.It draws 200 μ L to be added in ELISA Plate, the absorbance of 570nm is measured on microplate reader (Bio-Rad 550).Such as Fig. 7 institute
Show, the cell quantity in nano micro crystal cellulose/silk fibroin porous scaffold increases with the extension of time, but this patent
Embodiment 1 prepare hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold cell concentration be it is most, show
Hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold of this patent preparation has best biocompatibility.
(4) it after the obtained porous support of step (2) being carried out ultraviolet sterilization, is put into 496 well culture plates.With 0.25%
Pancreatin/PBS solution digests preosteoblast MC3T3-E1 from culture plate, centrifugal condition are as follows: 5min, 1500rpm remove supernatant
After night, the fresh α-MEM culture medium for the fetal calf serum that concentration is 10% is added, concentration of cell suspension is adjusted to 2.5 × 106/
mL.Then 10 μ L cell suspensions are added in porous support, after hatching 2h, 100 μ L culture mediums are added in every hole, are changed to contain afterwards for 24 hours
Dexamethasone culture medium (ascorbic acid containing 10%FCS, 50mM, 10mM β-phosphoglycerol disodium and 100nM dexamethasone height
Sugared DMEM), it cultivates in 37 DEG C of incubators to required time.Liquid is changed, every other day to keep the nutrition supply of cell.
Sample is taken out from culture plate, after PBS is cleaned 3 times, 100 μ L cell pyrolysis liquids are added, 4 DEG C of cracking overnight, surpass
Sound is broken.It is added 500 μ L alkaline phosphatase substrate reaction solutions, after 37 DEG C of water-bath 30min, the NaOH solution of 0.1M is added
500 μ L terminate reaction, immediately the absorption value at ultraviolet-visible spectrophotometer measurement 405nm.As shown in figure 8, nanometer is micro-
The alkaline phosphatase activities of cell extend be continuously increased at any time on crystalline cellulose/silk fibroin porous scaffold, but this patent
The alkaline phosphatase activities of cell are highests on hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold of preparation
, show that hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold of this patent preparation can be supported to fill between marrow
The proliferation and differentiation of matter stem cell, and effect is best.
Comparative example 3
(1) silk cocoon of pupa will be gone to shred, by the NaCO that 200ml is added in the silk cocoon of 5g, concentration is 0.2wt%3Aqueous solution,
3h is boiled at 100 DEG C carries out degumming formation fibroin.Fibroin is cleaned to neutrality with deionized water after degumming, then repeats above-mentioned steps
Once, it is finally just washed till neutral fibroin to dry in 50 DEG C, obtains fibroin albumen；Fibroin albumen is taken, addition concentration is 9.8mol/
In L lithium bromide water solution, with deionized water dialysis 4 days (molecular cut off of bag filter is 8000) after 50 DEG C of heating water bath 6h,
Water is wherein changed daily twice, is then centrifuged 10min in 25 DEG C, 10000r/min, and centrifugation twice, obtains the silk that concentration is 4wt%
Fibroin solution；
(2) 60mg hydroxyapatite will be added to be added in the 4wt% silk fibroin protein solution of 1ml step (1), will stir evenly ,-
60 DEG C of freeze-dryings for 24 hours, obtain hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold.Hydroxyapatite/silk
The scanning electron microscope (SEM) photograph of fibroin porous support is as shown in Figure 5.From Fig. 5 it is recognised that the porosity of the compound rest is 70-
85%, aperture 150-180um, hydroxyapatite is uniformly distributed in bracket.
(3) it after the obtained porous support of step (2) being carried out ultraviolet sterilization, is put into 496 well culture plates.With 0.25%
Pancreatin/PBS solution digests preosteoblast MC3T3-E1 from culture plate, centrifugal condition are as follows: 5min, 1500rpm remove supernatant
After night, the fresh α-MEM culture medium for the fetal calf serum that concentration is 10% is added, concentration of cell suspension is adjusted to 2.5 × 106/
mL.Then 10 μ L cell suspensions are added in porous support, after hatching 2h, 100 μ L culture mediums are added in every hole, in 37 DEG C of incubators
It is middle to cultivate to required time.Liquid is changed, every other day to keep the nutrition supply of cell.
In 96 orifice plates, 100 μ L MTT solution are added in every hole, are placed in 37 DEG C of incubators and are continued to be incubated for 4h.It is taken with tweezers
Acquired compound rest is put into 2mL centrifuge tube above out, and 1mL DMSO is added, and piping and druming to crystal is completely dissolved.It draws
200 α L are added in ELISA Plate, and the absorbance of 570nm is measured on microplate reader (Bio-Rad 550).As shown in fig. 7, hydroxy-apatite
Cell quantity in stone/nano micro crystal cellulose/silk fibroin porous scaffold increases with the extension of time, but this patent
Embodiment 1 prepare hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold cell concentration be it is most, show
Hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold of this method preparation has best biocompatibility.
(4) it after the obtained porous support of step (2) being carried out ultraviolet sterilization, is put into 496 well culture plates.With 0.25%
Pancreatin/PBS solution by preosteoblast MC3T3-E1 from culture plate digestion, centrifugal condition are as follows: 5min, 1500rpm, removal supernatant
After night, the fresh α-MEM culture medium for the fetal calf serum that concentration is 10% is added, concentration of cell suspension is adjusted to 2.5 × 106/
mL.Then 10 μ L cell suspensions are added in porous support, after hatching 2h, 100 μ L culture mediums are added in every hole, are changed to contain afterwards for 24 hours
Dexamethasone culture medium (ascorbic acid containing 10%FCS, 50mM, 10mM β-phosphoglycerol disodium and 100nM dexamethasone height
Sugared DMEM), it cultivates in 37 DEG C of incubators to required time.Liquid is changed, every other day to keep the nutrition supply of cell.
Sample is taken out from culture plate, after PBS is cleaned 3 times, 100 μ L cell pyrolysis liquids are added, 4 DEG C of cracking overnight, surpass
Sound is broken.It is added 500 μ L alkaline phosphatase substrate reaction solutions, after 37 DEG C of water-bath 30min, the NaOH solution of 0.1M is added
500 μ L terminate reaction, immediately the absorption value at ultraviolet-visible spectrophotometer measurement 405nm.As shown in figure 8, hydroxyl phosphorus
The alkaline phosphatase activities of cell extend be continuously increased at any time on lime stone/silk fibroin porous scaffold, but prepared by this patent
Hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold on the alkaline phosphatase activities of cell be highest, table
Hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold of bright this patent preparation can support that medulla mesenchyma is dry
The proliferation and differentiation of cell, and effect is best.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (5)
1. a kind of method for bone tissue engineer porous compound support frame, it is characterised in that: the bone tissue engineer is with porous
Compound rest is hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold, is by hydroxyapatite, nano microcrystalline
The three-dimensional stephanoporate compound stent of cellulose, fibroin albumen composition, porosity are 90~95%, and aperture is 180~220 μm；
The hydroxyapatite be it is needle-shaped, partial size be 10~100nm；
The nano micro crystal cellulose is in regular corynebacterium structure, and the length is 150-350nm, width 20-40nm；
It the described method comprises the following steps:
(1) silk cocoon is shredded, 80-100 DEG C, concentration 0.2wt%NaCO is then added32-4h is boiled in aqueous solution carries out degumming shape
At fibroin, silk cocoon and NaCO3The ratio of aqueous solution are as follows: 100ml, 0.2wt%NaCO is added in 2-5g silk cocoon3Aqueous solution is used after degumming
Deionized water cleans fibroin to neutrality；
(2) step (1) is repeated；
(3) step (2) is washed till neutral fibroin to dry in 40-60 DEG C, obtains fibroin albumen；Fibroin albumen is taken, concentration is added
To be used deionized water dialysis 3~4 days after 40~60 DEG C of 4~6h of heating water bath in 9.8mol/L lithium bromide water solution, centrifugation two
It is secondary, obtain 2~10wt% silk fibroin protein solution；
(4) hydroxyapatite, nano micro crystal cellulose are added in 2~10wt% silk fibroin protein solution, mixed ratio are as follows: every
30mg hydroxyapatite is added in milliliter silk fibroin protein solution, 30mg nano micro crystal cellulose stirs evenly, and is then freeze-dried, obtains
To hydroxyapatite/nano micro crystal cellulose/silk fibroin porous scaffold.
2. a kind of preparation method of bone tissue engineer porous compound support frame according to claim 1, it is characterised in that: institute
The silk cocoon stated is the silk cocoon of pupa；
3. a kind of preparation method of bone tissue engineer porous compound support frame according to claim 1, it is characterised in that: institute
The dialysis procedure stated uses molecular cut off to dialyse for 10000~12000 bag filter.
4. a kind of preparation method of bone tissue engineer porous compound support frame according to claim 1, it is characterised in that: institute
The condition for the centrifugation stated is to be centrifuged 5-15min under the conditions of 25 DEG C, 10000r/min.
5. a kind of preparation method of bone tissue engineer porous compound support frame according to claim 1, it is characterised in that: step
Suddenly the condition of freeze-drying described in (4) is -50--70 DEG C of freeze-drying 18-24h.
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Application publication date: 20160427 Assignee: HANGZHOU HUIBO TECHNOLOGY Co.,Ltd. Assignor: GUANGZHOU CHUANGSEED BIOMATERIALS CO.,LTD. Contract record no.: X2022440000233 Denomination of invention: A porous composite scaffold for bone tissue engineering and its preparation method Granted publication date: 20190125 License type: Common License Record date: 20221013 |