CN108478882A - A kind of preparation method of compound bone holder material - Google Patents
A kind of preparation method of compound bone holder material Download PDFInfo
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- 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
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- A—HUMAN NECESSITIES
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- 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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- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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Abstract
The present invention relates to a kind of preparation methods of compound bone holder material, first prepare hydroxyethyl chitosan, the precipitation method are used to prepare two-phase phosphorus calcium nanometer powder again, then poly-vinyl alcohol solution is prepared, poly-vinyl alcohol solution and two-phase phosphorus calcium nanometer powder are finally added into hydroxyethyl chitosan solution by extra fine quality ratio, carried out after stirring evenly circulating frozen defrosting processing, freeze-drying to get.Compared with prior art, the present invention selects hydroxyethyl chitosan to improve the cell compatibility of bone holder, phosphorus calcium nanometer powder is added in bone holder, so that bone holder is possessed suitable degradation rate, and use circulating frozen defreezing method, natural polymer is physical crosslinking, chemical cross-linking agent need not be added, bone holder of the invention has better bioactivity, cell can be made preferably to adhere to, it is allowed preferably to be proliferated, which is suitable for bone tissue engineer field.
Description
Technical field
The present invention relates to a kind of preparation methods of compound bone holder material, belong to technical field of biomedical materials.
Background technology
Contain a large amount of phosphorus calcium material, such as tricalcium phosphate, hydroxyapatite, calcium carbonate etc., therefore phosphorus calcium in bone tissue
Material is commonly used for preparing artificial bone scaffold.Due to the compositional similarity with bone, calcium orthophosphate base biomaterial such as hydroxyapatite
(HA, Ca10(PO4)6(OH)2), tricalcium phosphate (β-TCP, Ca3(PO4)2) and biphase calcium phosphor sour (BCP) etc. can be used for bone holder
It prepares.BCP is the mixture of HA and β-TCP, is made of a certain proportion of inorganic substances tricalcium phosphate and hydroxyapatite, two-phase
Phosphorus calcium can overcome tricalcium phosphate to be easy degradation and the not degradable disadvantage of hydroxyapatite, and have good osteoconductive
And osteoinductive.In the various Calcium phosphate biomaterials for bone surgery, BCP is combined with high mechanical properties, and HA's is excellent
The important candidate of the bone substitute of different bioactivity and faster β-TCP degradation rates.The bioceramic made of calcium phosphate
All there is poor mechanical performance, there is certain limitation in weight-bearing areas.
Chitosan has the excellent performances such as biocompatibility, blood compatibility, safety, microbic resolvability, it can
Bioactivity is provided for holder, cell is made to adhere on bone holder, is a kind of preferable bone holder material in biomedical sector.
Chitosan has the shortcomings that mechanical property deficiency as bone holder material, needs through crosslinking, crosslinking agent usually selects glutaraldehyde
Or water-soluble carbodiimide, but the introducing of crosslinking agent can reduce the cell compatibility of holder.
Invention content
Present invention aims to solve the deficiencies of the prior art, and provides a kind of a kind of preparation method of compound bone holder material, systems
The nano bone holder obtained possesses suitable degradation rate, and activity is high, and mechanical property is good, and cell compatibility is good, and cell can be in bone
Preferably adhere to and be proliferated on holder, in terms of being preferably applied to Bone Defect Repari.
Technical solution
A kind of preparation method of compound bone holder material, includes the following steps：
(1) hydroxyethyl chitosan is prepared：Take 10g chitosans be added in the 50wt% sodium hydroxide solutions of 15~18g into
Row alkalization, obtains chitosan solution, after standing 1~3h under the conditions of -20 DEG C, 15~25ml solvent isopropanols is added, stirring rises
Temperature is stirred for being cooled to room temperature to 70~80 DEG C, and the mixed of 130~180ml isopropanols and 15~19ml ethylene chlorhydrins is then added
Solution is closed, after stirring evenly, using hydrochloric acid solution tune pH7,
It uses absolute ethyl alcohol to wash again, then dialyses, be finally freeze-dried to get hydroxyethyl chitosan；
(2) two-phase phosphorus calcium nanometer powder is prepared：By Ca (NO3)2.4H2O solution and (NH4)2HPO4Solution is according to calcium phosphorus mole
Than being 1.65:1 is added in container, is uniformly mixed, and pH is then adjusted to 11, obtains the powder precipitation of white, after filtering,
White powder is cleaned with deionized water, is then dried, roasts, grinds successively, 200 mesh sieve is finally crossed, obtains two-phase phosphorus
Calcium nanometer powder；
(3) poly-vinyl alcohol solution is prepared：Polyvinyl alcohol is dissolved in deionized water, poly-vinyl alcohol solution is made；
(4) compound bone holder material is prepared：It takes hydroxyethyl chitosan to be dissolved in the acetum of 1~3v%, obtains hydroxyl
Ethyl chitosan solution, a concentration of 0.4~1.3wt% of hydroxyethyl chitosan, polyethylene is added into hydroxyethyl chitosan solution
Alcoholic solution and two-phase phosphorus calcium nanometer powder, polyvinyl alcohol, hydroxyethyl chitosan and two-phase phosphorus calcium nanometer powder mass ratio be (1~
6):(1~3):1, mixing 20-30h is stirred at room temperature, then deaerate and make to be uniformly dispersed, obtained solution is then poured into 48 holes
In plate circulating frozen thaw processing 3~9 times, be finally putting into freeze drier be dried to get.
Further, in step (1), the alkalization carries out in -20 DEG C of refrigerators, and alkalization time is more than for 24 hours.
Further, in step (1), concentration of hydrochloric acid solution 0.1M.
Further, in step (1), selection of dialysing is bag filter of the molecular cut off in 10000Da, dialysis time 2d
More than.
Further, in step (2), Ca (NO3)2.4H2A concentration of 0.4M, (NH of O solution4)2HPO4Solution it is a concentration of
0.2M。
Further, in step (2), the calcination temperature is 1125 DEG C, time 1h.
Further, in step (3), the mass concentration of the poly-vinyl alcohol solution is 3-8%.
Further, in step (4), polyvinyl alcohol, hydroxyethyl chitosan and two-phase phosphorus calcium nanometer powder mass ratio are 3:2:
1。
Further, in step (4), the circulating frozen defrosting processing refers to：Freezing 24 hours in -20 DEG C of refrigerators are put into,
Then 12 hours of defrosting under room temperature are taken out, freezing in -20 DEG C of refrigerators is then placed into.
Beneficial effects of the present invention：The present invention is compound by polyvinyl alcohol, hydroxyethyl chitosan and biphasic calcium phosphate, by following
Ring freeze-thaw method, physical crosslinking obtain compound rest.Compared with prior art, advantage of the invention is that：1. selecting hydroxyl
Ethyl chitosan improves the cell compatibility of bone holder, and phosphorus calcium, which is added, in bone holder receives
Rice flour end, makes bone holder possess suitable degradation rate, makes it when new bone is grown, the degradation appropriate of bone holder,
The reparation of bone is not interfered with；2. the present invention uses circulating frozen defreezing method, natural polymer can be physical crosslinking, no
It needs to add chemical cross-linking agent；3. the present invention selects ternary material system, freshly prepd bone holder is set to have preferably biological living
Property, cell can be made preferably to adhere to, it is allowed preferably to be proliferated；4. whole preparation process is simple to operation, can carry out a large amount of
Preparation.Compound rest prepared by this method can be used for bone tissue engineer field.
Description of the drawings
Fig. 1 is the scanning electron microscope (SEM) photograph of compound bone holder material made from embodiment 1-6；
Fig. 2 is the porosity of compound bone holder material made from embodiment 1-6；
Fig. 3 is the cell compatibility test result of compound bone holder material made from embodiment 1-6.
Specific implementation mode
The present invention will be illustrated by attached drawing and example below, but these specific embodiments do not limit in any way
The protection domain of the invention.Raw material used in the experimental program can be bought in market.
Embodiment 1
A kind of preparation method of compound bone holder material, includes the following steps：
(1) hydroxyethyl chitosan is prepared：It takes 10g chitosans to be added in the 50wt% sodium hydroxide solutions of 16.7g to carry out
Alkalization, obtains chitosan solution, and the alkalization carries out in -20 DEG C of refrigerators, and alkalization time is for 24 hours, to be stood under the conditions of -20 DEG C
After 1h, be added solvent isopropanol, stirring be warming up to 78 DEG C, be stirred for being cooled to room temperature, then be added 133ml isopropanols and
The mixed solution of 18.3ml ethylene chlorhydrins after stirring evenly, is washed using the hydrochloric acid solution tune pH7 of 0.1M, then using absolute ethyl alcohol
It washs 3 times, then selects molecular cut off in the bag filter dialysis 2d of 10000Da, be finally freeze-dried poly- to get ethoxy shell
Sugar；
(2) two-phase phosphorus calcium nanometer powder is prepared：By the Ca (NO of 0.4M3)2.4H2(the NH of O solution and 0.2M4)2HPO4Solution
It is 1.65 according to calcium phosphorus molar ratio:1 is added in three-neck flask, is uniformly mixed, and is then adjusted to pH using 25% ammonium hydroxide
11, the powder precipitation of white is obtained, after filtering, white powder is cleaned with deionized water, then 100 DEG C of dryings, place into horse
Not kiln roasting, calcination temperature are 1125 DEG C, and time 1h then drops to room temperature with stove, then takes out grinding, finally cross 200
Mesh sieves, and obtains two-phase phosphorus calcium nanometer powder；
(3) poly-vinyl alcohol solution is prepared：Polyvinyl alcohol is dissolved in deionized water, obtained mass concentration is
4% poly-vinyl alcohol solution；
(4) compound bone holder material is prepared：It takes hydroxyethyl chitosan to be dissolved in the acetum of 1v%, obtains ethoxy
Poly-vinyl alcohol solution and double is added into hydroxyethyl chitosan solution by chitosan solution, a concentration of 0.7wt% of hydroxyethyl chitosan
Phase phosphorus calcium nanometer powder, polyvinyl alcohol, hydroxyethyl chitosan and two-phase phosphorus calcium nanometer powder mass ratio are 1:1:1, at room temperature
It is stirred for 24 hours, then deaerates and make to be uniformly dispersed, obtained solution, which is then poured into circulating frozen defrosting processing in 48 orifice plates, (puts
Enter freezing 24 hours in -20 DEG C of refrigerators, then takes out 12 hours of defrosting under room temperature, then place into cold in -20 DEG C of refrigerators
Freeze) 9 times, be finally putting into freeze drier dry 48h to get.
Embodiment 2
A kind of preparation method of compound bone holder material, includes the following steps：
(1) hydroxyethyl chitosan is prepared：It takes 10g chitosans to be added in the 50wt% sodium hydroxide solutions of 16.7g to carry out
Alkalization, obtains chitosan solution, and the alkalization carries out in -20 DEG C of refrigerators, and alkalization time is for 24 hours, to be stood under the conditions of -20 DEG C
After 1h, be added solvent isopropanol, stirring be warming up to 78 DEG C, be stirred for being cooled to room temperature, then be added 133ml isopropanols and
The mixed solution of 18.3ml ethylene chlorhydrins after stirring evenly, is washed using the hydrochloric acid solution tune pH7 of 0.1M, then using absolute ethyl alcohol
It washs 3 times, then selects molecular cut off in the bag filter dialysis 2d of 10000Da, be finally freeze-dried poly- to get ethoxy shell
Sugar；
(2) two-phase phosphorus calcium nanometer powder is prepared：By the Ca (NO of 0.4M3)2.4H2(the NH of O solution and 0.2M4)2HPO4Solution
It is 1.65 according to calcium phosphorus molar ratio:1 is added in three-neck flask, is uniformly mixed, and is then adjusted to pH using 25% ammonium hydroxide
11, the powder precipitation of white is obtained, after filtering, white powder is cleaned with deionized water, then 100 DEG C of dryings, place into horse
Not kiln roasting, calcination temperature are 1125 DEG C, and time 1h then drops to room temperature with stove, then takes out grinding, finally cross 200
Mesh sieves, and obtains two-phase phosphorus calcium nanometer powder；
(3) poly-vinyl alcohol solution is prepared：Polyvinyl alcohol is dissolved in deionized water, be made mass concentration be 4% it is poly-
Glycohol solution；
(4) compound bone holder material is prepared：It takes hydroxyethyl chitosan to be dissolved in the acetum of 2v%, obtains ethoxy
Poly-vinyl alcohol solution and double is added into hydroxyethyl chitosan solution by chitosan solution, a concentration of 0.9wt% of hydroxyethyl chitosan
Phase phosphorus calcium nanometer powder, polyvinyl alcohol, hydroxyethyl chitosan and two-phase phosphorus calcium nanometer powder mass ratio are 2:1:1, at room temperature
It is stirred for 24 hours, then deaerates and make to be uniformly dispersed, then will obtain
Solution pours into circulating frozen defrosting processing in 48 orifice plates and (is put into freezing 24 hours in -20 DEG C of refrigerators, then takes out
Thaw under room temperature 12 hours, then place into freezing in -20 DEG C of refrigerators) 9 times, dry 48h is finally putting into freeze drier,
To obtain the final product.
Embodiment 3
A kind of preparation method of compound bone holder material, includes the following steps：
(1) hydroxyethyl chitosan is prepared：It takes 10g chitosans to be added in the 50wt% sodium hydroxide solutions of 16.7g to carry out
Alkalization, obtains chitosan solution, and the alkalization carries out in -20 DEG C of refrigerators, and alkalization time is for 24 hours, to be stood under the conditions of -20 DEG C
After 1h, be added solvent isopropanol, stirring be warming up to 78 DEG C, be stirred for being cooled to room temperature, then be added 133ml isopropanols and
The mixed solution of 18.3ml ethylene chlorhydrins after stirring evenly, is washed using the hydrochloric acid solution tune pH7 of 0.1M, then using absolute ethyl alcohol
It washs 3 times, then selects molecular cut off in the bag filter dialysis 2d of 10000Da, be finally freeze-dried poly- to get ethoxy shell
Sugar；
(2) two-phase phosphorus calcium nanometer powder is prepared：By the Ca (NO of 0.4M3)2.4H2(the NH of O solution and 0.2M4)2HPO4Solution
It is 1.65 according to calcium phosphorus molar ratio:1 is added in three-neck flask, is uniformly mixed, and is then adjusted to pH using 25% ammonium hydroxide
11, the powder precipitation of white is obtained, after filtering, white powder is cleaned with deionized water, then 100 DEG C of dryings, place into horse
Not kiln roasting, calcination temperature are 1125 DEG C, and time 1h then drops to room temperature with stove, then takes out grinding, finally cross 200
Mesh sieves, and obtains two-phase phosphorus calcium nanometer powder；
(3) poly-vinyl alcohol solution is prepared：Polyvinyl alcohol is dissolved in deionized water, be made mass concentration be 4% it is poly-
Glycohol solution；
(4) compound bone holder material is prepared：It takes hydroxyethyl chitosan to be dissolved in the acetum of 2v%, obtains ethoxy
Poly-vinyl alcohol solution and double is added into hydroxyethyl chitosan solution by chitosan solution, a concentration of 0.9wt% of hydroxyethyl chitosan
Phase phosphorus calcium nanometer powder, polyvinyl alcohol, hydroxyethyl chitosan and two-phase phosphorus calcium nanometer powder mass ratio are 3:2:1, at room temperature
It is stirred for 24 hours, then deaerates and make to be uniformly dispersed, obtained solution, which is then poured into circulating frozen defrosting processing in 48 orifice plates, (puts
Enter freezing 24 hours in -20 DEG C of refrigerators, then takes out 12 hours of defrosting under room temperature, then place into cold in -20 DEG C of refrigerators
Freeze) 9 times, be finally putting into freeze drier dry 48h to get.
Embodiment 4
A kind of preparation method of compound bone holder material, includes the following steps：
(1) hydroxyethyl chitosan is prepared：It takes 10g chitosans to be added in the 50wt% sodium hydroxide solutions of 16.7g to carry out
Alkalization, obtains chitosan solution, and the alkalization carries out in -20 DEG C of refrigerators, and alkalization time is for 24 hours, to be stood under the conditions of -20 DEG C
After 1h, be added solvent isopropanol, stirring be warming up to 78 DEG C, be stirred for being cooled to room temperature, then be added 133ml isopropanols and
The mixed solution of 18.3ml ethylene chlorhydrins after stirring evenly, is washed using the hydrochloric acid solution tune pH7 of 0.1M, then using absolute ethyl alcohol
It washs 3 times, then selects molecular cut off in the bag filter dialysis 2d of 10000Da, be finally freeze-dried poly- to get ethoxy shell
Sugar；
(2) two-phase phosphorus calcium nanometer powder is prepared：By the Ca (NO of 0.4M3)2.4H2(the NH of O solution and 0.2M4)2HPO4Solution
It is 1.65 according to calcium phosphorus molar ratio:1 is added in three-neck flask, is uniformly mixed, and is then adjusted to pH using 25% ammonium hydroxide
11, the powder precipitation of white is obtained, after filtering, white powder is cleaned with deionized water, then 100 DEG C of dryings, place into horse
Not kiln roasting, calcination temperature are 1125 DEG C, and time 1h then drops to room temperature with stove, then takes out grinding, finally cross 200
Mesh sieves, and obtains two-phase phosphorus calcium nanometer powder；
(3) poly-vinyl alcohol solution is prepared：Polyvinyl alcohol is dissolved in deionized water, be made mass concentration be 4% it is poly-
Glycohol solution；
(4) compound bone holder material is prepared：It takes hydroxyethyl chitosan to be dissolved in the acetum of 1v%, obtains ethoxy
Poly-vinyl alcohol solution and two-phase are added into hydroxyethyl chitosan solution by chitosan solution, a concentration of 1wt% of hydroxyethyl chitosan
Phosphorus calcium nanometer powder, polyvinyl alcohol, hydroxyethyl chitosan and two-phase phosphorus calcium nanometer powder mass ratio are 4:2:1, it stirs at room temperature
Mix mixing for 24 hours, then deaerate and make to be uniformly dispersed, then by obtained solution pour into circulating frozen defrosting processing in 48 orifice plates (be put into-
Then 12 hours of defrosting under room temperature are taken out in freezing 24 hours in 20 DEG C of refrigerators, then place into freezing in -20 DEG C of refrigerators) 3
It is secondary, be finally putting into freeze drier dry 48h to get.
Embodiment 5
A kind of preparation method of compound bone holder material, includes the following steps：
(1) hydroxyethyl chitosan is prepared：It takes 10g chitosans to be added in the 50wt% sodium hydroxide solutions of 16.7g to carry out
Alkalization, obtains chitosan solution, and the alkalization carries out in -20 DEG C of refrigerators, and alkalization time is for 24 hours, to be stood under the conditions of -20 DEG C
After 1h, be added solvent isopropanol, stirring be warming up to 78 DEG C, be stirred for being cooled to room temperature, then be added 133ml isopropanols and
The mixed solution of 18.3ml ethylene chlorhydrins after stirring evenly, is washed using the hydrochloric acid solution tune pH7 of 0.1M, then using absolute ethyl alcohol
It washs 3 times, then molecular cut off is selected to exist
The bag filter dialysis 2d of 10000Da, is finally freeze-dried to get hydroxyethyl chitosan；
(2) two-phase phosphorus calcium nanometer powder is prepared：By the Ca (NO of 0.4M3)2.4H2(the NH of O solution and 0.2M4)2HPO4Solution
It is 1.65 according to calcium phosphorus molar ratio:1 is added in three-neck flask, is uniformly mixed, and is then adjusted to pH using 25% ammonium hydroxide
11, the powder precipitation of white is obtained, after filtering, white powder is cleaned with deionized water, then 100 DEG C of dryings, place into horse
Not kiln roasting, calcination temperature are 1125 DEG C, and time 1h then drops to room temperature with stove, then takes out grinding, finally cross 200
Mesh sieves, and obtains two-phase phosphorus calcium nanometer powder；
(3) poly-vinyl alcohol solution is prepared：Polyvinyl alcohol is dissolved in deionized water, be made mass concentration be 4% it is poly-
Glycohol solution；
(4) compound bone holder material is prepared：It takes hydroxyethyl chitosan to be dissolved in the acetum of 2v%, obtains ethoxy
Poly-vinyl alcohol solution and two-phase are added into hydroxyethyl chitosan solution by chitosan solution, a concentration of 1wt% of hydroxyethyl chitosan
Phosphorus calcium nanometer powder, polyvinyl alcohol, hydroxyethyl chitosan and two-phase phosphorus calcium nanometer powder mass ratio are 5:1:1, it stirs at room temperature
Mix mixing for 24 hours, then deaerate and make to be uniformly dispersed, then by obtained solution pour into circulating frozen defrosting processing in 48 orifice plates (be put into-
Then 12 hours of defrosting under room temperature are taken out in freezing 24 hours in 20 DEG C of refrigerators, then place into freezing in -20 DEG C of refrigerators) 3
It is secondary, be finally putting into freeze drier dry 48h to get.
Embodiment 6
A kind of preparation method of compound bone holder material, includes the following steps：
(1) hydroxyethyl chitosan is prepared：It takes 10g chitosans to be added in the 50wt% sodium hydroxide solutions of 16.7g to carry out
Alkalization, obtains chitosan solution, and the alkalization carries out in -20 DEG C of refrigerators, and alkalization time is for 24 hours, to be stood under the conditions of -20 DEG C
After 1h, be added solvent isopropanol, stirring be warming up to 78 DEG C, be stirred for being cooled to room temperature, then be added 133ml isopropanols and
The mixed solution of 18.3ml ethylene chlorhydrins after stirring evenly, is washed using the hydrochloric acid solution tune pH7 of 0.1M, then using absolute ethyl alcohol
It washs 3 times, then selects molecular cut off in the bag filter dialysis 2d of 10000Da, be finally freeze-dried poly- to get ethoxy shell
Sugar；
(2) two-phase phosphorus calcium nanometer powder is prepared：By the Ca (NO of 0.4M3)2.4H2(the NH of O solution and 0.2M4)2HPO4Solution
It is 1.65 according to calcium phosphorus molar ratio:1 is added in three-neck flask, is uniformly mixed, and is then adjusted to pH using 25% ammonium hydroxide
11, the powder precipitation of white is obtained, after filtering, white powder is cleaned with deionized water, then 100 DEG C of dryings, place into horse
Not kiln roasting, calcination temperature are 1125 DEG C, when
Between be 1h, then drop to room temperature with stove, then take out grinding, finally cross 200 mesh sieve, obtain two-phase phosphorus calcium nano powder
End；
(3) poly-vinyl alcohol solution is prepared：Polyvinyl alcohol is dissolved in deionized water, be made mass concentration be 4% it is poly-
Glycohol solution；
(4) compound bone holder material is prepared：It takes hydroxyethyl chitosan to be dissolved in the acetum of 1v%, obtains ethoxy
Poly-vinyl alcohol solution and two-phase are added into hydroxyethyl chitosan solution by chitosan solution, a concentration of 1wt% of hydroxyethyl chitosan
Phosphorus calcium nanometer powder, polyvinyl alcohol, hydroxyethyl chitosan and two-phase phosphorus calcium nanometer powder mass ratio are 6:3:1, it stirs at room temperature
Mix mixing for 24 hours, then deaerate and make to be uniformly dispersed, then by obtained solution pour into circulating frozen defrosting processing in 48 orifice plates (be put into-
Then 12 hours of defrosting under room temperature are taken out in freezing 24 hours in 20 DEG C of refrigerators, then place into freezing in -20 DEG C of refrigerators) 9
It is secondary, be finally putting into freeze drier dry 48h to get.
Performance test
1. the scanning electron microscope (SEM) photograph of compound bone holder material made from embodiment 1-6 is shown in Fig. 1, wherein Figure 1A is embodiment 6,
Figure 1B is embodiment 5, and Fig. 1 C are embodiment 4, and Fig. 1 D are embodiment 3, and Fig. 1 E are embodiment 2, and Fig. 1 F are embodiment 1.
2. the porosity of the compound bone holder material of testing example 1-6, porosity test uses alcohol displacement method, test
As a result see Fig. 2.The porosity of these types of holder is 60~85% as seen from Figure 2, is hole connection structure, is being propped up conducive to cell
Growth is attached on frame.
3. cell compatibility is tested
Cell compatibility testing process is as follows：Sample is carried out disinfection with 75% alcohol, it is then dry thin with medulla mesenchyma
Born of the same parents cultivate, and are evaluated using CCK-8.Fig. 3 is the cell compatibility test of compound bone holder material prepared by the present invention
As a result, as seen from Figure 3 cell on holder as time increases, cell survival rate be in increase trend, for preparing answers
Closing bone holder material has good cell compatibility.
Claims (9)
1. a kind of preparation method of compound bone holder material, which is characterized in that include the following steps：
(1) hydroxyethyl chitosan is prepared：It takes 10g chitosans to be added in the 50wt% sodium hydroxide solutions of 15~18g and carries out alkali
Change, obtain chitosan solution, after standing 1~3h under the conditions of -20 DEG C, 15~25ml solvent isopropanols is added, stirring is warming up to
It 70~80 DEG C, is stirred for being cooled to room temperature, 130~180ml isopropanols is then added and the mixing of 15~19ml ethylene chlorhydrins is molten
Liquid after stirring evenly, is washed using hydrochloric acid solution tune pH7, then using absolute ethyl alcohol, then dialyses, be finally freeze-dried,
Up to hydroxyethyl chitosan；
(2) two-phase phosphorus calcium nanometer powder is prepared：By Ca (NO3)2·4H2O solution and (NH4)2HPO4Solution is according to calcium phosphorus molar ratio
It is 1.65:1 is added in container, is uniformly mixed, and pH is then adjusted to 11, obtains the powder precipitation of white, will after filtering
White powder is cleaned with deionized water, is then dried, roasts, is ground successively, is finally crossed 200 mesh sieve, is obtained two-phase phosphorus calcium
Nanometer powder；
(3) poly-vinyl alcohol solution is prepared：Polyvinyl alcohol is dissolved in deionized water, poly-vinyl alcohol solution is made；
(4) compound bone holder material is prepared：It takes hydroxyethyl chitosan to be dissolved in the acetum of 1~3v%, obtains ethoxy
It is molten that polyvinyl alcohol is added into hydroxyethyl chitosan solution by chitosan solution, a concentration of 0.4~1.3wt% of hydroxyethyl chitosan
Liquid and two-phase phosphorus calcium nanometer powder, polyvinyl alcohol, hydroxyethyl chitosan and two-phase phosphorus calcium nanometer powder mass ratio are (1~6):(1
~3):1, mixing 20-30h is stirred at room temperature, then deaerate and make to be uniformly dispersed, then obtained solution is poured into 48 orifice plates and is followed
Ring freeze-thaw handle 3~9 times, be finally putting into freeze drier be dried to get.
2. the preparation method of compound bone holder material as described in claim 1, which is characterized in that in step (1), the alkalization
It is carried out in -20 DEG C of refrigerators, alkalization time is more than for 24 hours.
3. the preparation method of compound bone holder material as described in claim 1, which is characterized in that in step (1), hydrochloric acid solution
A concentration of 0.1M.
4. the preparation method of compound bone holder material as described in claim 1, which is characterized in that in step (1), dialysis is selected
Be bag filter of the molecular cut off in 10000Da, dialysis time is 2d or more.
5. the preparation method of compound bone holder material as described in claim 1, which is characterized in that in step (2), Ca
(NO3)2·4H2A concentration of 0.4M, (NH of O solution4)2HPO4A concentration of 0.2M of solution.
6. the preparation method of compound bone holder material as described in claim 1, which is characterized in that in step (2), the roasting
Temperature is 1125 DEG C, time 1h.
7. the preparation method of compound bone holder material as described in claim 1, which is characterized in that in step (3), the poly- second
The mass concentration of enolate solution is 3-8%.
8. the preparation method of compound bone holder material as described in claim 1, which is characterized in that in step (4), polyethylene
Alcohol, hydroxyethyl chitosan and two-phase phosphorus calcium nanometer powder mass ratio are 3:2:1.
9. such as the preparation method of the compound bone holder material of claim 1 to 8 any one of them, which is characterized in that step (4)
In, the circulating frozen defrosting processing refers to：Freezing 24 hours in -20 DEG C of refrigerators are put into, then takes out and thaws 12 under room temperature
Then a hour places into freezing in -20 DEG C of refrigerators.
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Citations (2)
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|---|---|---|---|---|
| CN106188442A (en) * | 2016-08-02 | 2016-12-07 | 西安交通大学 | A kind of chitosan derivatives hydrogel and preparation method thereof |
| CN106620872A (en) * | 2017-01-18 | 2017-05-10 | 中国人民解放军第四军医大学 | Formula and preparation method of engineered bone scaffold |
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2018
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106188442A (en) * | 2016-08-02 | 2016-12-07 | 西安交通大学 | A kind of chitosan derivatives hydrogel and preparation method thereof |
| CN106620872A (en) * | 2017-01-18 | 2017-05-10 | 中国人民解放军第四军医大学 | Formula and preparation method of engineered bone scaffold |
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| 王晶彦等: ""天然HA 与双相磷酸钙复合支架修复骨缺损对比研究"", 《稀有金属材料与工程》 * |
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Application publication date: 20180904 |