CN108553690A - A kind of porous fibroin microballoon and preparation method thereof for mixing strontium - Google Patents

A kind of porous fibroin microballoon and preparation method thereof for mixing strontium Download PDF

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CN108553690A
CN108553690A CN201810333874.7A CN201810333874A CN108553690A CN 108553690 A CN108553690 A CN 108553690A CN 201810333874 A CN201810333874 A CN 201810333874A CN 108553690 A CN108553690 A CN 108553690A
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strontium
fibroin
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microsphere
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CN108553690B (en
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杨明英
雷芳
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Zhejiang University ZJU
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Abstract

The invention discloses a kind of preparation method of porous fibroin microballoon that mixing strontium, the constituent of porous microsphere is fibroin and strontium chloride, and for the porosity of the porous microsphere 80% or more, aperture is 5 40 μm, and strontium chloride weight ratio accounts for 5% the 38% of total weight.Fibroin albumen of the present invention has been commonly used to health food industry as a kind of native protein molecule, will not generate stimulation to human body；Strontium is a kind of indispensable trace element of human body, has the abilities such as osteogenic induction, the effect of Bone Defect Repari can be improved；Main material of the fibroin albumen as cell carrier simultaneously, can simulate the protein ingredient of extracellular matrix；What is be prepared through the invention mixes strontium fibroin porous microsphere, can carry out the sustained release of strontium, effectively overcomes the acquisition growth factor from animal and uses the various problems such as presence.

Description

A kind of porous fibroin microballoon and preparation method thereof for mixing strontium
Technical field
The present invention relates to a kind of preparation methods of porous fibroin microballoon that mixing strontium, belong to biomedical material technology.
Background technology
The effects that bone, cartilaginous tissue play very important support, protection to human body, traditional Bone Defect Repari method is to utilize Autologous bone and allograph bone carry out bone collection treatment, and such method is not merely limited to for bone resource, and expensive, is used simultaneously Allograph bone is also easy to generate the adverse reactions such as immunological rejection.With the development of organizational project and regenerative medicine, in osteanagenesis side Face has achieved immense success, and regenerated bone has normal function and bioactivity.During Bone Defect Repari, need structure negative Cell and the three-dimensional rack of growth factor, such as porous sponge stent, fiber mesh holder and hydrogel are carried, compared to these branch Frame form, microcarrier have the advantages that can direct injection exempt surgical procedures in tissue defects position, while as thin Born of the same parents' carrier, it can expand the dimensional culture of cell, improve cell culture area and maintain cell phenotype.Wherein porous microcarrier There is trepanning, inside to have perforation pore structure, cell can be facilitated to enter inside microcarrier, avoid cell in incubation on surface In cause to damage.In addition, porous microcarrier can also reinforce cytotrophy substance and metabolite transport, provided for cell growth More specific surface areas can greatly improve cell inoculation quantity.
A kind of natural macromolecular albumen that fibroin albumen is made of 18 kinds of human amino acids, can be decomposed again in vivo It is degraded into single amino acid, catabolite is nontoxic, and can be absorbed.Compared to common synthetic material such as polylactic acid etc., fibroin Albumen can with protein component in simulated tissue and function, and with good biocompatibility, degradability and easy processing modification, The advantages that low immunogenicity, it is easier to meet the requirement of biomaterial.Strontium is the important trace element of bone composition, has and promotes The skeletonization effect osteoclastic with inhibition.Strontium can also promote versatile stem cell to osteoblast differentiation, and it is close to improve bone mineral Degree, enhancing bone hardness, self-bone grafting ability and osteoconductive are final to improve bone fine structure and bone mass, promote new osteanagenesis with It repairs.
Invention content
The present invention prepares present in organizational project application that material property is unstable and structure shape for existing microcarrier The main problems such as looks research is incomplete, in conjunction with fibroin albumen advantageous feature, provide one kind is with fibroin albumen and strontium chloride Main material mixes strontium fibroin albumen base porous microsphere using prepared by emulsion method combination Freeze Drying Technique.Prepared by the present invention The problems such as mixing the reparation that strontium fibroin porous microsphere is mainly used for bone, can effectively avoid cell loss, Bone mineral change, enhances to joint The support of bone.The specific technical solution of the present invention is as follows：
The invention discloses a kind of fibroin porous microsphere for mixing strontium, the constituent of porous microsphere is fibroin and strontium chloride, The porosity of the porous microsphere is 80% or more, and aperture is 5-40 μm, and strontium chloride weight ratio accounts for the 5%-38% of total weight.
The present invention is a kind of method for preparing and mixing strontium porous fibroin microballoon, and used specific preparation process is as follows：
1) silk cocoon shredded, degumming, after dissolving, using filtering, dialyse, be condensed into silk fibroin protein solution；
2) it takes petroleum ether in beaker, 3.5h or more is pre-chilled at -30 DEG C--50 DEG C；In this temperature range and in the time It can just work it out, the too low too high pattern of temperature can all change, and the time, too short refrigerating effect was bad, can not also be molded.
3) it separately takes petroleum ether in new beaker again, emulsifier is added, the two is made to be uniformly mixed；
4) 2%-6.5% (w/v) silk fibroin protein solution, 0.1%-4% (w/v) strontium chloride are uniformly mixed, in being added 3) Solution, stir into uniform-spherical；Mixing strontium is advantageous in that：Research mainly adds BMP-2 in fibroin material at present Deng with stem cell body extroversion osteoblast differentiation and skeletonization and VEGF etc. therapeutic effect is improved at the related cell factor of blood vessel. However, cell factor still faces in clinical application：Half-life short, release concentration is not easy to control, is mainly obtained from animal, Cost is high, perishable, is often accompanied by the major issues such as immunological rejection.Therefore, it finds new vascularization promoting and promotes into soma Cell is the research hotspot using tissue engineering technique repairing bone defect to the organizational project source factor of Osteoblast Differentiation；
5) it in the petroleum ether being pre-chilled in pouring into 4) solution 2) rapidly, then is placed in freezing environment and staticly settles；
6) petroleum ether in removing 5) solution under freezing atmosphere obtains and mixes strontium fibroin albumen microballoon containing ice crystal, by this Fibroin albumen microballoon is freezed, then can get the dry porous fibroin protein microsphere for mixing strontium by being freeze-dried.
As a further improvement, the strontium porous fibroin protein microsphere of mixing in step 6) of the present invention passes through follow-up alcohol Class processing enhances its mechanical property.Without alcohols handle fibroin can soluble in water, while this walk be conducive to wash away it is remaining Emulsifier.
As a further improvement, strontium porous fibroin protein microsphere of the present invention of mixing is obtained according to biomineralization principle The silk fibroin porous microballoon of strontium carbonate mineralising with strontium ion stable sustained-release ability.By mineralising, strontium ion can reach Slow release effect, into the water without mineralising, strontium ion can release quickly, be unable to reach desired effect.
Compared with prior art, the present invention has following prominent characteristics：
1) excellent biocompatibility：Fibroin albumen has been commonly used to health food row as a kind of native protein molecule Industry will not generate stimulation to human body；
2) syringeability：Fibroin albumen microballoon, can be with direct injection in tissue compared to hydrogel, porous sponge stent etc. Rejected region, the problems such as bone defect caused by privileged sites, labyrinth etc. can be overcome not easy to operate；
3) promote bone repair：Strontium is a kind of indispensable trace element of human body, has the abilities such as osteogenic induction, can carry The effect of high Bone Defect Repari；Main material of the fibroin albumen as cell carrier simultaneously, can simulate the protein ingredient of extracellular matrix；
4) what is be prepared through the invention mixes strontium fibroin porous microsphere, can carry out the sustained release of strontium, effectively overcome Growth factor is obtained from animal uses the various problems such as presence；
5) of low cost, it is environment friendly and pollution-free.
Specific implementation mode
Technical scheme of the present invention is described in further detail below by specific embodiment, following embodiment is pair The present invention explanation and the invention is not limited in following embodiments.
Embodiment 1
1) silk cocoon is shredded, degumming, after dissolving, it is molten using filtering, the fibroin albumen dialysed, be condensed into 2% (w/v) Liquid；
2) it takes 450ml petroleum ethers in 500ml beakers, 4h is pre-chilled at -50 DEG C；
3) it takes 40ml petroleum ethers in the new beakers of 100ml again, 0.7ml span-80 is added, the two is made to be uniformly mixed；
4) silk fibroin protein solution of 10ml, 0.01g strontium chlorides are uniformly mixed, the solution in being added 3) stirs into uniformly It is spherical；
5) in the petroleum ether being pre-chilled in pouring into 4) solution 2) rapidly, then -50 DEG C is placed in and is staticly settled；
6) petroleum ether in removing 5) solution under freezing atmosphere obtains and mixes strontium fibroin albumen microballoon containing ice crystal, by this Fibroin albumen microballoon is freezed, then can get the dry porous fibroin protein microsphere for mixing strontium by being freeze-dried.
The constituent of the porous microsphere of gained is fibroin and strontium chloride, the porosity of the porous microsphere 80% with On, aperture is 5 μm -40 μm, and strontium chloride weight ratio accounts for the 5%-38% of total weight.
Embodiment 2
1) silk cocoon is shredded, degumming, after dissolving, it is molten using filtering, the fibroin albumen dialysed, be condensed into 4% (w/v) Liquid；
2) it takes 450ml petroleum ethers in 500ml beakers, 4h is pre-chilled at -50 DEG C；
3) it takes 40ml petroleum ethers in the new beakers of 100ml again, 0.7ml span-80 is added, the two is made to be uniformly mixed；
4) silk fibroin protein solution of 10ml, 0.01g strontium chlorides are uniformly mixed, the solution in being added 3) stirs into uniformly It is spherical；
5) in the petroleum ether being pre-chilled in pouring into 4) solution 2) rapidly, then -50 DEG C is placed in and is staticly settled；
6) petroleum ether in removing 5) solution under freezing atmosphere obtains and mixes strontium fibroin albumen microballoon containing ice crystal, by this Fibroin albumen microballoon is freezed, then can get the dry porous fibroin protein microsphere for mixing strontium by being freeze-dried.
Embodiment 3
1) silk cocoon is shredded, degumming, after dissolving, it is molten using filtering, the fibroin albumen dialysed, be condensed into 6.5% (w/v) Liquid；
2) it takes 450ml petroleum ethers in 500ml beakers, 4h is pre-chilled at -50 DEG C；
3) it takes 40ml petroleum ethers in the new beakers of 100ml again, 0.7ml span-80 is added, the two is made to be uniformly mixed；
4) silk fibroin protein solution of 10ml, 0.01g strontium chlorides are uniformly mixed, the solution in being added 3) stirs into uniformly It is spherical；
5) in the petroleum ether being pre-chilled in pouring into 4) solution 2) rapidly, then -50 DEG C is placed in and is staticly settled；
6) petroleum ether in removing 5) solution under freezing atmosphere obtains and mixes strontium fibroin albumen microballoon containing ice crystal, by this Fibroin albumen microballoon is freezed, then can get the dry porous fibroin protein microsphere for mixing strontium by being freeze-dried.
Embodiment 4
1) silk cocoon is shredded, degumming, after dissolving, it is molten using filtering, the fibroin albumen dialysed, be condensed into 2.5% (w/v) Liquid；
2) it takes 450ml petroleum ethers in 500ml beakers, 4h is pre-chilled at -50 DEG C；
3) it takes 40ml petroleum ethers in the new beakers of 100ml again, 0.7ml span-80 is added, the two is made to be uniformly mixed；
4) silk fibroin protein solution of 10ml, 0.01g strontium chlorides are uniformly mixed, the solution in being added 3) stirs into uniformly It is spherical；
5) in the petroleum ether being pre-chilled in pouring into 4) solution 2) rapidly, then -50 DEG C is placed in and is staticly settled；
6) petroleum ether in removing 5) solution under freezing atmosphere obtains and mixes strontium fibroin albumen microballoon containing ice crystal, by this Fibroin albumen microballoon is freezed, then can get the dry porous fibroin protein microsphere for mixing strontium by being freeze-dried.
Embodiment 5
1) silk cocoon is shredded, degumming, after dissolving, it is molten using filtering, the fibroin albumen dialysed, be condensed into 2.5% (w/v) Liquid；
2) it takes 450ml petroleum ethers in 500ml beakers, 4h is pre-chilled at -50 DEG C；
3) it takes 40ml petroleum ethers in the new beakers of 100ml again, 0.7ml span-80 is added, the two is made to be uniformly mixed；
4) silk fibroin protein solution of 10ml, 0.05g strontium chlorides are uniformly mixed, the solution in being added 3) stirs into uniformly It is spherical；
5) in the petroleum ether being pre-chilled in pouring into 4) solution 2) rapidly, then -50 DEG C is placed in and is staticly settled；
6) petroleum ether in removing 5) solution under freezing atmosphere obtains and mixes strontium fibroin albumen microballoon containing ice crystal, by this Fibroin albumen microballoon is freezed, then can get the dry porous fibroin protein microsphere for mixing strontium by being freeze-dried.
Embodiment 6
1) silk cocoon is shredded, degumming, after dissolving, it is molten using filtering, the fibroin albumen dialysed, be condensed into 2.5% (w/v) Liquid；
2) it takes 450ml petroleum ethers in 500ml beakers, 4h is pre-chilled at -50 DEG C；
3) it takes 40ml petroleum ethers in the new beakers of 100ml again, 0.7ml span-80 is added, the two is made to be uniformly mixed；
4) silk fibroin protein solution of 10ml, 0.2g strontium chlorides are uniformly mixed, the solution in being added 3) stirs into uniform ball Shape；
5) in the petroleum ether being pre-chilled in pouring into 4) solution 2) rapidly, then -50 DEG C is placed in and is staticly settled；
6) petroleum ether in removing 5) solution under freezing atmosphere obtains and mixes strontium fibroin albumen microballoon containing ice crystal, by this Fibroin albumen microballoon is freezed, then can get the dry porous fibroin protein microsphere for mixing strontium by being freeze-dried.
Embodiment 7
1) silk cocoon is shredded, degumming, after dissolving, it is molten using filtering, the fibroin albumen dialysed, be condensed into 2.5% (w/v) Liquid；
2) it takes 450ml petroleum ethers in 500ml beakers, 4h is pre-chilled at -50 DEG C；
3) it takes 40ml petroleum ethers in the new beakers of 100ml again, 0.7ml span-80 is added, the two is made to be uniformly mixed；
4) silk fibroin protein solution of 10ml, 0.4g strontium chlorides are uniformly mixed, the solution in being added 3) stirs into uniform ball Shape；
5) in the petroleum ether being pre-chilled in pouring into 4) solution 2) rapidly, then -50 DEG C is placed in and is staticly settled；
6) petroleum ether in removing 5) solution under freezing atmosphere obtains and mixes strontium fibroin albumen microballoon containing ice crystal, by this Fibroin albumen microballoon is freezed, then can get the dry porous fibroin protein microsphere for mixing strontium by being freeze-dried.
Embodiment 8
1) silk cocoon is shredded, degumming, after dissolving, it is molten using filtering, the fibroin albumen dialysed, be condensed into 2.5% (w/v) Liquid；
2) it takes 450ml petroleum ethers in 500ml beakers, 4h is pre-chilled at -30 DEG C；
3) it takes 40ml petroleum ethers in the new beakers of 100ml again, 0.7ml span-80 is added, the two is made to be uniformly mixed；
4) silk fibroin protein solution of 10ml, 0.2g strontium chlorides are uniformly mixed, the solution in being added 3) stirs into uniform ball Shape；
5) in the petroleum ether being pre-chilled in pouring into 4) solution 2) rapidly, then -50 DEG C is placed in and is staticly settled；
6) petroleum ether in removing 5) solution under freezing atmosphere obtains and mixes strontium fibroin albumen microballoon containing ice crystal, by this Fibroin albumen microballoon is freezed, then can get the dry porous fibroin protein microsphere for mixing strontium by being freeze-dried.
Below table provides comparative example 1-2 and is compared with embodiment 2, comparative example 1-2 only change fibroin concentration this One technical characteristic, remaining technical characteristic is same as Example 2, to obtain the selection pair of fibroin concentration by morphology observation In the importance of technical solution of the present invention.
Fibroin concentration Chlorination content of strontium Morphology observation
Comparative example 1 1% 0.01g Microballoon is easy avalanche, and adhesion is serious
Embodiment 2 4% 0.01g Surface is in cellular, internal hole
Comparative example 2 7% 0.01g Aperture and porosity are too small
Below table provides comparative example 3 and is compared with embodiment 4,7, and comparative example 3 only changes chlorination content of strontium, Remaining technical characteristic is same as Example 4, to obtain the selection of chlorination content of strontium for the technology of the present invention by morphology observation The importance of scheme.
Fibroin concentration Chlorination content of strontium Morphology observation
Embodiment 4 2.5% 0.01g Surface is in cellular, internal hole
Embodiment 7 2.5% 0.4g Surface is in cellular, internal hole
Comparative example 3 2.5% 0.8g Porous microsphere cannot be obtained
Below table provides comparative example 4-5 and is compared with embodiment 5, comparative example 3 only change precooling temperature this Technical characteristic, remaining technical characteristic is same as Example 5, to obtain precooling temperature selection for this by morphology observation The importance of inventive technique scheme.
Fibroin concentration Chlorination content of strontium Precooling temperature Morphology observation
Comparative example 4 2.5% 0.05g -20℃ Porous microsphere cannot be obtained
Embodiment 5 2.5% 0.05g -50℃ Surface is in cellular, internal hole
Comparative example 5 2.5% 0.05g -80℃ Aperture and porosity are too small
Embodiment 9
1) silk cocoon is shredded, degumming, after dissolving, it is molten using filtering, the fibroin albumen dialysed, be condensed into 2.5% (w/v) Liquid；
2) it takes 450ml petroleum ethers in 500ml beakers, 4h is pre-chilled at -50 DEG C；
3) it takes 40ml petroleum ethers in the new beakers of 100ml again, 0.7ml span-80 is added, the two is made to be uniformly mixed；
4) silk fibroin protein solution of 10ml, 0.05g strontium chlorides are uniformly mixed, the solution in being added 3) stirs into uniformly It is spherical；
5) in the petroleum ether being pre-chilled in pouring into 4) solution 2) rapidly, then -50 DEG C is placed in and is staticly settled；
6) petroleum ether in removing 5) solution under freezing atmosphere obtains and mixes strontium fibroin albumen microballoon containing ice crystal, by this Fibroin albumen microballoon is freezed, then can get the dry porous fibroin protein microsphere for mixing strontium by being freeze-dried.
7) the porous fibroin microballoon and NH that will 6) obtain4HCO3Mixing place 2d after, after fixing 30min with ethyl alcohol, spend from Sub- water rinses ethyl alcohol and NH well4HCO3, the strontium carbonate mineralising with strontium ion stable sustained-release ability is obtained using freeze-drying Silk fibroin porous microballoon.
Embodiment 10
1) silk cocoon is shredded, degumming, after dissolving, it is molten using filtering, the fibroin albumen dialysed, be condensed into 2.5% (w/v) Liquid；
2) it takes 450ml petroleum ethers in 500ml beakers, 4h is pre-chilled at -50 DEG C；
3) it takes 40ml petroleum ethers in the new beakers of 100ml again, 0.7ml span-80 is added, the two is made to be uniformly mixed；
4) silk fibroin protein solution of 10ml, 0.05g strontium chlorides are uniformly mixed, the solution in being added 3) stirs into uniformly It is spherical；
5) in the petroleum ether being pre-chilled in pouring into 4) solution 2) rapidly, then -50 DEG C is placed in and is staticly settled；
6) petroleum ether in removing 5) solution under freezing atmosphere obtains and mixes strontium fibroin albumen microballoon containing ice crystal, by this Fibroin albumen microballoon is freezed, then can get the dry porous fibroin protein microsphere for mixing strontium by being freeze-dried.
7) 6) after the porous fibroin microballoon obtained being mixed fixed 30min with ethyl alcohol, ethyl alcohol is rinsed well with deionized water, The silk fibroin porous microballoon for mixing strontium not soluble in water is obtained using freeze-drying.
Finally, it should also be noted that it is listed above be only the present invention several specific embodiments and comparative example Son.It is clear that the invention is not restricted to above embodiment, acceptable there are many deformations.Those skilled in the art can be from this All deformations that disclosure of invention is directly exported or associated, are considered as protection scope of the present invention.

Claims (4)

1. a kind of porous fibroin microballoon for mixing strontium, which is characterized in that the constituent of the porous microsphere is fibroin and chlorination Strontium, the porosity of the porous microsphere is 80% or more, and aperture is 5-40 μm, and strontium chloride weight ratio accounts for the 5%- of total weight 38%.
2. a kind of preparation method of porous fibroin microballoon as described in claim 1 that mixing strontium, which is characterized in that used tool Preparation step is as follows：
1) silk cocoon is shredded, degumming, after dissolving, using filtering, dialyse, to be condensed into 2%-6.5% (w/v) fibroin albumen molten Liquid；
2) take petroleum ether in beaker, at -30 DEG C --- -50 DEG C of precooling 3.5h or more；
3) it takes petroleum ether in new beaker again, emulsifier is added, the two is made to be uniformly mixed；
4) 2%-6.5% (w/v) silk fibroin protein solution, 0.1%-4% (w/v) strontium chloride are uniformly mixed, it is molten in being added 3) Liquid stirs into uniform-spherical；
5) it in the petroleum ether being pre-chilled in pouring into 4) solution 2) rapidly, then is placed in freezing environment and staticly settles；
6) petroleum ether in removing 5) solution under freezing atmosphere obtains and mixes strontium fibroin albumen microballoon containing ice crystal, by the fibroin Protein microsphere is freezed, then can get the dry porous fibroin protein microsphere for mixing strontium by being freeze-dried.
3. the preparation method according to claim 2 for mixing strontium porous fibroin microballoon, which is characterized in that in the step 6) Mix strontium porous fibroin protein microsphere pass through follow-up alcohols processing enhance its mechanical property.
4. the preparation method according to claim 2 or 3 for mixing strontium porous fibroin microballoon, which is characterized in that described mixes strontium Porous fibroin protein microsphere obtains the fibroin of the strontium carbonate mineralising with strontium ion stable sustained-release ability according to biomineralization principle Albumen porous microsphere.
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