CN109260523A - A kind of calcium phosphate bone cement complex microsphere and its preparation method and application - Google Patents
A kind of calcium phosphate bone cement complex microsphere and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of calcium phosphate bone cement complex microspheres and its preparation method and application.It is compounded in the present invention by using fibroin albumen and carboxymethyl chitosan, using orthophosphate skeleton cement solid powder as raw material, and combine Drop Condensation method, calcium phosphate bone cement complex microsphere is prepared, preparation method is simple, is not necessarily to high temperature sintering, the calcium phosphate bone cement complex microsphere mechanical strength with higher, good anti-disintegration, degradability and cell compatibility, have a good application prospect in Bone Defect Repari and pharmaceutical carrier field.
Description
Technical field
The present invention relates to bone renovating material technical fields, and in particular to a kind of calcium phosphate bone cement complex microsphere and its preparation
Methods and applications.
Background technique
Calcium phosphate bone cement (Calcium Phosphate Cement, CPC) is the nineties in last century by Brown and Chow
It succeeds in developing, is a kind of non-ceramic type hydroxy apatites artificial bone with self-curing.It is by solid phase powder and admittedly
Change after liquid mix according to a certain percentage and form the slurry of easy moulding, in a short time self-curing, final hydrated product for
The similar apatite of inorganic constituents and crystal structure of body bone tissue.CPC has good osteoconductive and biocompatibility,
During surgery can random-shaping, bone defect of various shapes is filled, in bone defect healing, decorative sursery reparation and pharmaceutical carrier etc.
Field is widely applied.
Compared with block-like calcium phosphate bone repair materials, calcium phosphate microsphere has better mobility and excellent fillibility
Can, good filling and reparation can be realized in regular shape or irregular bone defect position, meanwhile, calcium phosphate microsphere is being filled out
During filling, microballoon is accumulated to form interconnected pore structure, is conducive to the generation of bone tissue grown into blood vessel.Calcium phosphate
Microballoon can be prepared by the methods of Drop Condensation method, spray drying process, template and emulsion method, traditional calcium phosphate pottery
Porcelain class microballoon need to be handled through high temperature sintering, be unfavorable for carrying medicament and bioactive ingredients, and that treated is micro- through high temperature sintering
Ball degradation rate is slower, and macromolecule is the calcium phosphate microsphere mechanical property deficiency of matrix, and lacks osteoconductive and self-bone grafting
Property.
Summary of the invention
It is an object of the invention to provide a kind of calcium phosphate bone cement complex microsphere in place of overcome the deficiencies in the prior art
And its preparation method and application, improve intensity, degradability and the cell compatibility of calcium phosphate bone cement microballoon.
To achieve the above object, the technical solution adopted by the present invention is as follows:
A kind of preparation method of calcium phosphate bone cement complex microsphere, comprising the following steps:
(1) carboxymethyl chitosan is dissolved in deionized water, and the silk fibroin protein solution that concentration is 10~20wt% is added,
After mixing, mixed solution is obtained；
(2) orthophosphate skeleton cement solid powder, mixed solution and calcium phosphate solid phase are added in the mixed solution of step (1)
The liquid-solid ratio of powder is 1mL:0.4~1g, obtains calcium phosphate bone cement slurry after mixing evenly；
(3) calcium phosphate bone cement slurry is injected in syringe, and be added dropwise in liquid nitrogen, form freezing complex microsphere；
(4) freezing complex microsphere is put into temperature is 30 DEG C~40 DEG C, water in the environment that relative humidity is 95%~100%
After changing 1~4 day, in 30 DEG C~40 DEG C dryings, the calcium phosphate bone cement complex microsphere is obtained.
Compounded in the present invention by using fibroin albumen and carboxymethyl chitosan, using orthophosphate skeleton cement solid powder as
Raw material, and Drop Condensation method is combined, calcium phosphate bone cement complex microsphere is prepared, preparation method is simple, it is not necessarily to high temperature sintering,
The calcium phosphate bone cement complex microsphere intensity with higher, good anti-disintegration, degradability and cell compatibility, in bone
It repairs and pharmaceutical carrier field has a good application prospect.
The preferred embodiment of preparation method as calcium phosphate bone cement complex microsphere of the present invention, the injection
The needle diameter of device is 0.5~1mm.
The partial size that the present invention passes through the control adjustable calcium phosphate bone cement microballoon of needle diameter.
The preferred embodiment of preparation method as calcium phosphate bone cement complex microsphere of the present invention, the mixing
The mass fraction of fibroin albumen is 2%~5% in solution；The mass fraction of carboxymethyl chitosan is in the mixed solution
0.5%~2%.
The preferred embodiment of preparation method as calcium phosphate bone cement complex microsphere of the present invention, the mixing
The mass ratio of fibroin albumen and carboxymethyl chitosan is 2:1 in solution.
The preferred embodiment of preparation method as calcium phosphate bone cement complex microsphere of the present invention, the mixing
The mass ratio of fibroin albumen and carboxymethyl chitosan is 3:1 in solution.
On the one hand the addition of fibroin albumen and carboxymethyl chitosan is capable of forming three-dimensional space network structure, effectively improves
The mechanical strength of calcium phosphate bone cement complex microsphere enhances the anti-disintegration ability of microballoon, on the other hand, fibroin albumen of the invention
And carboxymethyl chitosan, attachment site can be provided for the growth of mesenchymal stem cell, it is dry thin to be conducive to medulla mesenchyma
The growth of born of the same parents, plays the role of induction of bone growth.
The preferred embodiment of preparation method as calcium phosphate bone cement complex microsphere of the present invention, the step
(2) in, the liquid-solid ratio of mixed solution and calcium phosphate solid phase powder is 1mL:0.6g.
The preferred embodiment of preparation method as calcium phosphate bone cement complex microsphere of the present invention, the solid phase
Powder includes tetracalcium phosphate, bata-tricalcium phosphate, type alpha tricalcium phosphate, calcium carbonate, calcium monohydrogen phosphate, calcium dihydrogen phosphate, calcium octahate phosphate, nothing
At least one of sizing calcium phosphate
The preferred embodiment of preparation method as calcium phosphate bone cement complex microsphere of the present invention, the solid phase
Powder includes type alpha tricalcium phosphate, mixes strontium calcium octahate phosphate and/or mix strontium amorphous calcium phosphate；In the solid phase powder, strontium and calcium
Molar ratio are as follows: strontium: calcium=0.1~1.6:1；The molar ratio of the sum of the calcium and strontium and phosphorus are as follows: calcium+strontium: phosphorus=1.1~1.4:1.
The preferred embodiment of preparation method as calcium phosphate bone cement complex microsphere of the present invention, the solid phase
In powder, the molar ratio of strontium and calcium are as follows: strontium: calcium=0.7~1.0:1；The molar ratio of the sum of the calcium and strontium and phosphorus are as follows: calcium+strontium:
Phosphorus=1.3:1.
Strontium calcium octahate phosphate (Sr-OCP) is mixed as Sr2+Source can improve the intensity of calcium phosphate bone cement complex microsphere.Different Sr2 +Source doping modified phosphate calcium bone cement complex microsphere, can slowly release Sr in Tris buffer solution2+。Sr2+Source to from
Sub- release performance has an impact, with mixing strontium calcium octahate phosphate (Sr-OCP) or mix strontium amorphous calcium phosphate (Sr-ACP) modified phosphate calcium bone
Cementitious composite microballoon shows more stable, suitable ion release rate, promotes cell Proliferation and Osteoblast Differentiation.
Using the calcium phosphate bone cement complex microsphere of mentioned component, the molar ratio of the sum of strontium amount, calcium and strontium with phosphorus is not only mixed
((calcium+strontium)/phosphorus) is adjustable, no side effect and and there are the advantages such as strontium ion of sustained release tool special pharmacological function, also there is nothing
The features such as impurity and miscellaneous phase ion, preparation process are relatively easy, cost is relatively low is suitble to the reparation of human body non-bearing position bone, replaces
The fields such as generation, correction and Surgical reconstruction.
The preferred embodiment of preparation method as calcium phosphate bone cement complex microsphere of the present invention, the fibroin
Protein solution is prepared by the following method gained:
By silk in the Na of 0.02~0.05mol/L2CO320~60min is boiled in solution, is repeatedly washed by rubbing with the hands with deionized water
To eliminate silk gum；9~10mol/L LiBr solution is dissolved in after drying, after 50 DEG C~60 DEG C 3~5h of dissolution, deionized water dialysis
24~48h, centrifugation removal fibroin condensation product, it is molten to be made the fibroin albumen that mass fraction is 10~20wt% after concentrated or dilution
Liquid.
The present invention also provides a kind of calcium phosphate bone cement complex microspheres being prepared according to the above method.
The present invention also provides the applications of above-mentioned calcium phosphate bone cement complex microsphere, and the microballoon is as medical bone defect
The application of packing material or drugs with function carrier material.
Microballoon of the invention mechanical strength with higher and excellent anti-disintegration, suitable degradability, to mouse
The cell Proliferation and Osteoblast Differentiation of mesenchymal stem cell have facilitation, can be used as medical bone defect packing material or work
With drug carrier material, have a good application prospect in Bone Defect Repari and pharmaceutical carrier field.
Compared with prior art, the invention has the benefit that
The present invention, as raw material, is compounded by using calcium phosphate bone cement using fibroin albumen and carboxymethyl chitosan, knot
It closes Drop Condensation method and prepares calcium phosphate bone cement complex microsphere, the calcium phosphate bone cement complex microsphere intensity with higher, with
And excellent anti-disintegration, degradability and biocompatibility, it has a good application prospect in Bone Defect Repari and pharmaceutical carrier field.
Detailed description of the invention
Fig. 1 is the Cell proliferation results of the calcium phosphate bone cement complex microsphere of Examples 1 to 4 and comparative example 1~2.
Fig. 2 is the ALP Activity Results of the calcium phosphate bone cement complex microsphere of Examples 1 to 4 and comparative example 1~2.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with specific embodiment to the present invention
It further illustrates.It will be appreciated by those skilled in the art that described herein, specific examples are only used to explain the present invention, not
For limiting the present invention.
In embodiment, used experimental method is conventional method unless otherwise specified, material used, reagent etc.,
It is commercially available unless otherwise specified.
Embodiment 1
A kind of embodiment of preparation method as calcium phosphate bone cement complex microsphere of the present invention, the present embodiment institute
The preparation method of the calcium phosphate bone cement complex microsphere stated the following steps are included:
(1) carboxymethyl chitosan is dissolved in deionized water, and the fibroin albumen that mass fraction is 10~20wt% is added
Solution obtains mixed solution after mixing, and the mass fraction of fibroin albumen is 2% in the mixed solution；The mixing is molten
The mass fraction of carboxymethyl chitosan is 1% in liquid；
(2) orthophosphate skeleton cement solid powder, mixed solution and calcium phosphate solid phase are added in the mixed solution of step (1)
The liquid-solid ratio of powder is 1mL:0.6g, obtains calcium phosphate bone cement slurry after mixing evenly；
(3) calcium phosphate bone cement slurry is injected in syringe, and be added dropwise in liquid nitrogen, form freezing complex microsphere；
(4) freezing complex microsphere is put into temperature is 37 DEG C, in the environment that relative humidity is 98% after aquation 3 days, 37
DEG C drying, obtains the calcium phosphate bone cement complex microsphere.
Silk fibroin protein solution described in the present embodiment is prepared by the following method gained:
By silk in the Na of 0.02~0.05mol/L2CO320~60min is boiled in solution, is repeatedly washed by rubbing with the hands with deionized water
To eliminate silk gum；9~10mol/L LiBr solution is dissolved in after drying, after 50 DEG C~60 DEG C 3~5h of dissolution, deionized water dialysis
24~48h, centrifugation removal fibroin condensation product, it is molten to be made the fibroin albumen that mass fraction is 10~20wt% after concentrated or dilution
Liquid.
Solid phase powder described in the present embodiment is made of tetracalcium phosphate and calcium monohydrogen phosphate 1:1 mixing in molar ratio.
Embodiment 2
A kind of embodiment of preparation method as calcium phosphate bone cement complex microsphere of the present invention, the present embodiment institute
The preparation method of the calcium phosphate bone cement complex microsphere stated the following steps are included:
(1) carboxymethyl chitosan is dissolved in deionized water, and the fibroin albumen that mass fraction is 10~20wt% is added
Solution obtains mixed solution after mixing, and the mass fraction of fibroin albumen is 3% in the mixed solution；The mixing is molten
The mass fraction of carboxymethyl chitosan is 1% in liquid；
(2) orthophosphate skeleton cement solid powder, mixed solution and calcium phosphate solid phase are added in the mixed solution of step (1)
The liquid-solid ratio of powder is 1mL:0.6g, obtains calcium phosphate bone cement slurry after mixing evenly；
(3) calcium phosphate bone cement slurry is injected in syringe, and be added dropwise in liquid nitrogen, form freezing complex microsphere；
(4) freezing complex microsphere is put into temperature is 37 DEG C, in the environment that relative humidity is 98% after aquation 3 days, 37
DEG C drying, obtains the calcium phosphate bone cement complex microsphere.
Silk fibroin protein solution described in the present embodiment is prepared by the following method gained:
By silk in the Na of 0.02~0.05mol/L2CO320~60min is boiled in solution, is repeatedly washed by rubbing with the hands with deionized water
To eliminate silk gum；9~10mol/L LiBr solution is dissolved in after drying, after 50 DEG C~60 DEG C 3~5h of dissolution, deionized water dialysis
24~48h, centrifugation removal fibroin condensation product, it is molten to be made the fibroin albumen that mass fraction is 10~20wt% after concentrated or dilution
Liquid.
Solid phase powder described in the present embodiment includes type alpha tricalcium phosphate, mixes strontium calcium octahate phosphate and/or mix strontium amorphous calcium phosphate；
In the solid phase powder, the molar ratio of strontium and calcium are as follows: strontium: calcium=0.7:1；The molar ratio of the sum of the calcium and strontium and phosphorus are as follows: calcium+
Strontium: phosphorus=1.3:1.
Embodiment 3
A kind of embodiment of preparation method as calcium phosphate bone cement complex microsphere of the present invention, the present embodiment institute
The preparation method of the calcium phosphate bone cement complex microsphere stated the following steps are included:
(1) carboxymethyl chitosan is dissolved in deionized water, and the fibroin albumen that mass fraction is 10~20wt% is added
Solution obtains mixed solution after mixing, and the mass fraction of fibroin albumen is 4% in the mixed solution；The mixing is molten
The mass fraction of carboxymethyl chitosan is 1% in liquid；
(2) orthophosphate skeleton cement solid powder, mixed solution and calcium phosphate solid phase are added in the mixed solution of step (1)
The liquid-solid ratio of powder is 1mL:0.6g, obtains calcium phosphate bone cement slurry after mixing evenly；
(3) calcium phosphate bone cement slurry is injected in syringe, and be added dropwise in liquid nitrogen, form freezing complex microsphere；
(4) freezing complex microsphere is put into temperature is 37 DEG C, in the environment that relative humidity is 98% after aquation 3 days, 37
DEG C drying, obtains the calcium phosphate bone cement complex microsphere.
Silk fibroin protein solution described in the present embodiment is prepared by the following method gained:
By silk in the Na of 0.02~0.05mol/L2CO320~60min is boiled in solution, is repeatedly washed by rubbing with the hands with deionized water
To eliminate silk gum；9~10mol/L LiBr solution is dissolved in after drying, after 50 DEG C~60 DEG C 3~5h of dissolution, deionized water dialysis
24~48h, centrifugation removal fibroin condensation product, it is molten to be made the fibroin albumen that mass fraction is 10~20wt% after concentrated or dilution
Liquid.
Solid phase powder described in the present embodiment includes type alpha tricalcium phosphate, mixes strontium calcium octahate phosphate and/or mix strontium amorphous calcium phosphate；
In the solid phase powder, the molar ratio of strontium and calcium are as follows: strontium: calcium=0.7:1；The molar ratio of the sum of the calcium and strontium and phosphorus are as follows: calcium+
Strontium: phosphorus=1.3:1.
Embodiment 4
A kind of embodiment of preparation method as calcium phosphate bone cement complex microsphere of the present invention, the present embodiment institute
The preparation method of the calcium phosphate bone cement complex microsphere stated the following steps are included:
(1) carboxymethyl chitosan is dissolved in deionized water, and the fibroin albumen that mass fraction is 10~20wt% is added
Solution obtains mixed solution after mixing, and the mass fraction of fibroin albumen is 2% in the mixed solution；The mixing is molten
The mass fraction of carboxymethyl chitosan is 2% in liquid；
(2) orthophosphate skeleton cement solid powder, mixed solution and calcium phosphate solid phase are added in the mixed solution of step (1)
The liquid-solid ratio of powder is 1mL:0.6g, obtains calcium phosphate bone cement slurry after mixing evenly；
(3) calcium phosphate bone cement slurry is injected in syringe, and be added dropwise in liquid nitrogen, form freezing complex microsphere；
(4) freezing complex microsphere is put into temperature is 37 DEG C, in the environment that relative humidity is 98% after aquation 3 days, 37
DEG C drying, obtains the calcium phosphate bone cement complex microsphere.
Fibroin albumen described in the present embodiment is prepared by the following method gained:
Silk fibroin protein solution described in the present embodiment is prepared by the following method gained:
By silk in the Na of 0.02~0.05mol/L2CO320~60min is boiled in solution, is repeatedly washed by rubbing with the hands with deionized water
To eliminate silk gum；9~10mol/L LiBr solution is dissolved in after drying, after 50 DEG C~60 DEG C 3~5h of dissolution, deionized water dialysis
24~48h, centrifugation removal fibroin condensation product, it is molten to be made the fibroin albumen that mass fraction is 10~20wt% after concentrated or dilution
Liquid.
Solid phase powder described in the present embodiment includes type alpha tricalcium phosphate, mixes strontium calcium octahate phosphate and/or mix strontium amorphous calcium phosphate；
In the solid phase powder, the molar ratio of strontium and calcium are as follows: strontium: calcium=1:1；The molar ratio of the sum of the calcium and strontium and phosphorus are as follows: calcium+
Strontium: phosphorus=1.3:1.
Embodiment 5
A kind of embodiment of preparation method as calcium phosphate bone cement complex microsphere of the present invention, the present embodiment institute
The preparation method of the calcium phosphate bone cement complex microsphere stated the following steps are included:
(1) carboxymethyl chitosan is dissolved in deionized water, and the fibroin albumen that mass fraction is 10~20wt% is added
Solution obtains mixed solution after mixing, and the mass fraction of fibroin albumen is 4% in the mixed solution；The mixing is molten
The mass fraction of carboxymethyl chitosan is 2% in liquid；
(2) orthophosphate skeleton cement solid powder, mixed solution and calcium phosphate solid phase are added in the mixed solution of step (1)
The liquid-solid ratio of powder is 1mL:0.4g, obtains calcium phosphate bone cement slurry after mixing evenly；
(3) calcium phosphate bone cement slurry is injected in syringe, and be added dropwise in liquid nitrogen, form freezing complex microsphere；
(4) freezing complex microsphere is put into temperature is 30 DEG C, in the environment that relative humidity is 95% after aquation 1 day, 30
DEG C drying, obtains the calcium phosphate bone cement complex microsphere.
Silk fibroin protein solution described in the present embodiment is prepared by the following method gained:
By silk in the Na of 0.02~0.05mol/L2CO320~60min is boiled in solution, is repeatedly washed by rubbing with the hands with deionized water
To eliminate silk gum；9~10mol/L LiBr solution is dissolved in after drying, after 50 DEG C~60 DEG C 3~5h of dissolution, deionized water dialysis
24~48h, centrifugation removal fibroin condensation product, it is molten to be made the fibroin albumen that mass fraction is 10~20wt% after concentrated or dilution
Liquid.
Solid phase powder described in the present embodiment includes type alpha tricalcium phosphate, mixes strontium calcium octahate phosphate and/or mix strontium amorphous calcium phosphate；
In the solid phase powder, the molar ratio of strontium and calcium are as follows: strontium: calcium=0.1:1；The molar ratio of the sum of the calcium and strontium and phosphorus are as follows: calcium+
Strontium: phosphorus=1.1:1.
Embodiment 6
A kind of embodiment of preparation method as calcium phosphate bone cement complex microsphere of the present invention, the present embodiment institute
The preparation method of the calcium phosphate bone cement complex microsphere stated the following steps are included:
(1) carboxymethyl chitosan is dissolved in deionized water, and the fibroin albumen that mass fraction is 10~20wt% is added
Solution obtains mixed solution after mixing, and the mass fraction of fibroin albumen is 3.5% in the mixed solution；The mixing
The mass fraction of carboxymethyl chitosan is 0.5% in solution；
(2) orthophosphate skeleton cement solid powder, mixed solution and calcium phosphate solid phase are added in the mixed solution of step (1)
The liquid-solid ratio of powder is 1mL:1g, obtains calcium phosphate bone cement slurry after mixing evenly；
(3) calcium phosphate bone cement slurry is injected in syringe, and be added dropwise in liquid nitrogen, form freezing complex microsphere；
(4) freezing complex microsphere is put into temperature is 40 DEG C, in the environment that relative humidity is 100% after aquation 4 days, 40
DEG C drying, obtains the calcium phosphate bone cement complex microsphere.
Silk fibroin protein solution described in the present embodiment is prepared by the following method gained:
By silk in the Na of 0.02~0.05mol/L2CO320~60min is boiled in solution, is repeatedly washed by rubbing with the hands with deionized water
To eliminate silk gum；9~10mol/L LiBr solution is dissolved in after drying, after 50 DEG C~60 DEG C 3~5h of dissolution, deionized water dialysis
24~48h, centrifugation removal fibroin condensation product, it is molten to be made the fibroin albumen that mass fraction is 10~20wt% after concentrated or dilution
Liquid.
Solid phase powder described in the present embodiment includes type alpha tricalcium phosphate, mixes strontium calcium octahate phosphate and/or mix strontium amorphous calcium phosphate；
In the solid phase powder, the molar ratio of strontium and calcium are as follows: strontium: calcium=1.6:1；The molar ratio of the sum of the calcium and strontium and phosphorus are as follows: calcium+
Strontium: phosphorus=1.4:1.
Comparative example 1
The preparation method of calcium phosphate bone cement complex microsphere described in this comparative example the following steps are included:
(1) orthophosphate skeleton cement solid powder is added in the silk fibroin protein solution that mass fraction is 3%, fibroin albumen is molten
The liquid-solid ratio of liquid and calcium phosphate solid phase powder is 1mL:0.6g, obtains calcium phosphate bone cement slurry after mixing evenly；
(2) calcium phosphate bone cement slurry is injected in syringe, and be added dropwise in liquid nitrogen, form freezing complex microsphere；
(3) freezing complex microsphere is put into temperature is 37 DEG C, in the environment that relative humidity is 98% after aquation 3 days, 37
DEG C drying, obtains the calcium phosphate bone cement complex microsphere.
Silk fibroin protein solution described in this comparative example is prepared by the following method gained:
By silk in the Na of 0.02~0.05mol/L2CO320~60min is boiled in solution, is repeatedly washed by rubbing with the hands with deionized water
To eliminate silk gum；9~10mol/L LiBr solution is dissolved in after drying, after 50 DEG C~60 DEG C 3~5h of dissolution, deionized water dialysis
24~48h, centrifugation removal fibroin condensation product, it is molten to be made the fibroin albumen that mass fraction is 10~20wt% after concentrated or dilution
Liquid.
Solid phase powder described in this comparative example includes type alpha tricalcium phosphate, mixes strontium calcium octahate phosphate and/or mix strontium amorphous calcium phosphate；
In the solid phase powder, the molar ratio of strontium and calcium are as follows: strontium: calcium=0.7:1；The molar ratio of the sum of the calcium and strontium and phosphorus are as follows: calcium+
Strontium: phosphorus=1.3:1.
Comparative example 2
The preparation method of calcium phosphate bone cement complex microsphere described in this comparative example the following steps are included:
(1) carboxymethyl chitosan is dissolved in deionized water, obtains carboxymethyl chitosan solution, the carboxymethyl chitosan
The mass fraction of carboxymethyl chitosan is 3% in solution；
(2) orthophosphate skeleton cement solid powder, carboxymethyl chitosan are added in the carboxymethyl chitosan solution of step (1)
The liquid-solid ratio of solution and calcium phosphate solid phase powder is 1mL:0.6g, obtains calcium phosphate bone cement slurry after mixing evenly；
(3) calcium phosphate bone cement slurry is injected in syringe, and be added dropwise in liquid nitrogen, form freezing complex microsphere；
(4) freezing complex microsphere is put into temperature is 37 DEG C, in the environment that relative humidity is 98% after aquation 3 days, 37
DEG C drying, obtains the calcium phosphate bone cement complex microsphere.
Solid phase powder described in this comparative example includes type alpha tricalcium phosphate, mixes strontium calcium octahate phosphate and/or mix strontium amorphous calcium phosphate；
In the solid phase powder, the molar ratio of strontium and calcium are as follows: strontium: calcium=0.7:1；The molar ratio of the sum of the calcium and strontium and phosphorus are as follows: calcium+
Strontium: phosphorus=1.3:1.
The calcium phosphate bone cement complex microsphere prepared to Examples 1 to 6 and comparative example 1~2 carries out following performance detection.
1, anti-disintegrating property measurement
1g calcium phosphate bone cement complex microsphere is taken to be added in the beaker equipped with 30mL SBF solution, in 120rpm, 37 DEG C
It is shaken in constant-temperature table, timing sampling, record microballoon obviously starts the time of disintegration, and the results are shown in Table 1.
2, external degradation
The calcium phosphate bone cement complex microsphere for weighing certain mass is immersed in the PBS solution of 30mL, and microballoon is placed at
Microballoon is taken out after shaking 4 weeks in 37 DEG C of constant-temperature tables and is dried, calculates the mass loss rate of microballoon, as a result 1 institute of table by 120rpm
Show.
3, compression strength
The microballoon for taking 8 particle diameters almost the same respectively, is put into mold, passes through universal testing machine test sample
Compression strength, loading speed 1mm/min, the results are shown in Table 1.
4, porosity
Using dehydrated alcohol as liquid phase medium, by specific gravity balance, the hole of sample is measured according to Archimedes's drainage
Rate, the results are shown in Table 1.
5, cell Proliferation
Using the calcium phosphate bone cement complex microsphere sample of Examples 1 to 4 as experimental group, made with the sample of comparative example 1~2
For control group, the proliferation feelings of cell (Marrow Mesenchymal Stem Cells, ATCC CRL-12424) are detected using CCK-8 kit
Condition places a sample into 48 orifice plates, and inoculating cell quantity is 1 × 104The hole cell/, changes liquid every other day, at 37 DEG C, 5%CO2Culture
Cultivate 1 in case respectively, 3, after 7d, by absorbance value of the microplate reader at 450nm, as a result as shown in Figure 1.
6, Osteoblast Differentiation
Using the calcium phosphate bone cement complex microsphere sample of Examples 1 to 4 as experimental group, made with the sample of comparative example 1~2
For control group, microsphere sample is placed in 48 orifice plates, Marrow Mesenchymal Stem Cells, inoculating cell are cultivated in microsphere sample
Quantity is 1 × 104The hole cell/, changes liquid every other day, at 37 DEG C, 5%CO2Incubator in cultivate 7,10d respectively after, according to kit
ALP determination of activity is carried out, as a result as shown in Figure 2.
Table 1
By embodiment 1 and 1~2 result of comparative example it is found that the addition of carboxymethyl chitosan and fibroin albumen can be mentioned significantly
Compression strength, the anti-disintegration of high bone cement microballoon promote cell Proliferation and Osteoblast Differentiation.By Examples 1 to 5 result it is found that
By adjusting the composition, solid phase powder composition and liquid-solid ratio of mixed solution, it is capable of compression strength, the hole of Effective Regulation bone cement
Rate and degradation property.
To sum up, the present invention prepares calcium phosphate bone water in conjunction with Drop Condensation method by using calcium phosphate bone cement as raw material
Mud complex microsphere, the calcium phosphate bone cement complex microsphere intensity with higher and excellent anti-disintegration are suitable to degrade
Property, cell Proliferation and Osteoblast Differentiation to Marrow Mesenchymal Stem Cells have facilitation, can be used as medical bone defect and fill out
Material or drugs with function carrier material are filled, is had a good application prospect in Bone Defect Repari and pharmaceutical carrier field.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Claims (10)
1. a kind of preparation method of calcium phosphate bone cement complex microsphere, which comprises the following steps:
(1) carboxymethyl chitosan is dissolved in deionized water, and the silk fibroin protein solution that mass fraction is 10~20wt% is added,
After mixing, mixed solution is obtained；
(2) orthophosphate skeleton cement solid powder, mixed solution and calcium phosphate solid phase powder are added in the mixed solution of step (1)
Liquid-solid ratio be 1mL:0.4~1g, obtain calcium phosphate bone cement slurry after mixing evenly；
(3) calcium phosphate bone cement slurry is injected in syringe, and be added dropwise in liquid nitrogen, form freezing complex microsphere；
(4) freezing complex microsphere is put into temperature is 30 DEG C~40 DEG C, aquation 1 in the environment that relative humidity is 95%~100%
After~4 days, in 30 DEG C~40 DEG C dryings, the calcium phosphate bone cement complex microsphere is obtained.
2. the preparation method of calcium phosphate bone cement complex microsphere according to claim 1, which is characterized in that the mixing is molten
The mass fraction of fibroin albumen is 2%~5% in liquid；The mass fraction of carboxymethyl chitosan is 0.5% in the mixed solution
~2%.
3. the preparation method of calcium phosphate bone cement complex microsphere according to claim 2, which is characterized in that the mixing is molten
The mass ratio of fibroin albumen and carboxymethyl chitosan is 2:1 in liquid.
4. the preparation method of calcium phosphate bone cement complex microsphere according to claim 2, which is characterized in that the mixing is molten
The mass ratio of fibroin albumen and carboxymethyl chitosan is 3:1 in liquid.
5. the preparation method of calcium phosphate bone cement complex microsphere according to claim 1, which is characterized in that the step
(2) in, the liquid-solid ratio of mixed solution and calcium phosphate solid phase powder is 1mL:0.6g.
6. the preparation method of calcium phosphate bone cement complex microsphere according to claim 1, which is characterized in that the solid phase powder
End is fixed including tetracalcium phosphate, bata-tricalcium phosphate, type alpha tricalcium phosphate, calcium carbonate, calcium monohydrogen phosphate, calcium dihydrogen phosphate, calcium octahate phosphate, nothing
At least one of type calcium phosphate.
7. the preparation method of calcium phosphate bone cement complex microsphere according to claim 6, which is characterized in that the solid phase powder
End includes type alpha tricalcium phosphate, mixes strontium calcium octahate phosphate and/or mix strontium amorphous calcium phosphate；In the solid phase powder, strontium and calcium rub
That ratio are as follows: strontium: calcium=0.1~1.6:1；The molar ratio of the sum of the calcium and strontium and phosphorus are as follows: calcium+strontium: phosphorus=1.1~1.4:1.
8. the preparation method of calcium phosphate bone cement complex microsphere according to claim 7, which is characterized in that the solid phase powder
In end, the molar ratio of strontium and calcium are as follows: strontium: calcium=0.7~1.0:1；The molar ratio of the sum of the calcium and strontium and phosphorus are as follows: calcium+strontium: phosphorus
=1.3:1.
9. a kind of calcium phosphate bone cement complex microsphere that the method for any one according to claim 1~8 is prepared.
10. the application of calcium phosphate bone cement complex microsphere according to claim 9, which is characterized in that the microballoon conduct
The application of medical bone defect packing material or drugs with function carrier material.
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