CN106039416B - Chitosan-sericin compound bio bracket and its preparation method and application - Google Patents
Chitosan-sericin compound bio bracket and its preparation method and application Download PDFInfo
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Abstract
The present invention provides chitosan-sericin compound bio bracket and preparation method and application.The present invention obtains chitosan solution as solvent dissolution chitosan using acetic acid solution；Sericin aqueous solution is prepared using the silk cocoon of fibroin deletion form mutating variety silkworm；It is injected in mold after chitosan solution and sericin aqueous solution are mixed, crosslinking agent molding is added, obtains chitosan-sericin composite hydrogel；By chitosan-sericin hydrogel freeze-drying, chitosan-sericin compound bio bracket is obtained.The present invention also provides a kind of compound bio brackets of carrying nerve growth factor for the pressure treatment of peripheral nerve card.The present invention has good biocompatibility, higher porosity and degradability, catabolite also has neurotrophic effect, compound rest has the effects that antibacterial, prevents adhesion simultaneously, compared with traditional surgery alone releases sciatic nerve crush, the present invention can effectively facilitate the reparation of peripheral nerve card crushing wound.
Description
Technical field
The present invention relates to medical biotechnology field of compound material, in particular to a kind of chitosan-sericin compound bio branch
Frame and the compound bio bracket and its preparation method and application for carrying nerve growth factor.
Background technique
Peripheral nerve card pressure lesion is the common disease of Clinical Surgery, is up to 3.8% in the illness rate of crowd, mainly wraps
Include carpal tunnel syndrome, cubital tunnel syndrome, oar pipe syndrome, common peroneal nerve compression syndrome etc..Nerve entrapments can betide body
Any part of body is most commonly in the bone fibre pipe of juxtra-articular, plump musculotendinous cuff, variation in especially neural conduct
Ligament and occupying lesion (ganglion, lipoma, hemotoncus or tumour etc.) can squeeze peripheral nerve.Traditional is outer
All Nerve entrapments treatment methods have operative treatment and two kinds of conservative therapy, and small part patient symptom is slight, can take conservative control
It treats, such as local steroid injection drug, Boards wall, Yoga；Most of card pressure patient has operative treatment indication, usually adopts
Card taking presses neurolysis to carry out operative treatment.Some researches show that the failure rate of operative treatment reaches 25%, and part is suffered from recent years
Person can not achieve long-term efficacy after being treated surgically.This phenomenon be considered with operation after local scar organize the formation of and mind
Pathology damage is related after card pressure.How further to improve the effect of operative treatment is the important topic of hand surgical field in recent years
And hot issue.
Method of Tissue Engineering is widely used in the treatment of peripheral nerve amputation injury in recent years, and achieves ideal effect
Fruit, but not yet it is applied to peripheral nerve card pressure therapy field.Chitosan (Chitosan) is a kind of natural biologic material, by first
Obtained by shell element deacetylation, and chitin is widely present in crab shell, shrimp shell and the shell of arthropod, and there is also algae etc. is low
In the cell wall of plant, the crust of the annual biosynthesis of nature is known as many as billions of tons, thus chitosan be it is a kind of very
Natural resources abundant.Because its have sterilization, it is antibacterial and promote wound healing function, clinically by development and application be wound
Mouth dressing.In addition, chitosan has good biocompatibility, immunogenicity is weak, in vivo degradable metabolism.Chitosan at present
It is widely applied in various fields such as food fresh-keeping, biomedicine, environmental protection, cosmetics, agriculturals.It is acted on by chemical modification,
Various functional groups are introduced in chitosan molecule structure or chitosan is blended with other biological material, improve the materialization of chitosan
Matter, so that making it respectively has the function of different and effect, it is an importance of research after chitosan.
Sericin (Silk Sericin) is wrapped around a kind of natural macromolecular viscous protein on fibroin fiber surface layer, about
Account for the 20 ~ 30% of silk cocoon content, by molecular weight be 24 ~ 400 kDa polypeptide form, molecule by serine, asparatate and
18 kinds of amino acid compositions such as glycine.The limitation of the deficiency and research that are recognized for a long time due to people sericin, is led
It causes silk gum to be taken as waste to handle in filature industry, wastes the natural resources of a large amount of preciousnesses.In recent years it is found that silk gum
Albumen has moisturizing, antibacterial, anti-oxidant, anticoagulation and promotes the biological natures such as cell adherence and proliferation.Moreover, silk gum
It is ideal bio-medical material with hydrophily and degradability.Existing report display silk gum is usually used in and other materials
(such as elastin laminin, polyvinyl alcohol etc.) copolymerization or simple crosslinking, are made by mixing biological support to obtain the biological material of function admirable
Material.Application of the sericin in organizational engineering is given full play to, there is important social effect and wide application prospect.
The present invention extracts pure silk glue egg using bombyx mori silk fibroin deletion form mutating variety silk cocoon as material, using lithium bromide extraction method
It is white, chitosan is successfully prepared chitosan-sericin in conjunction with sericin in the way of chemical covalent bonds crosslinking
Compound rest.The compound rest has both the advantage of chitosan and sericin respectively, and compensates for homogenous material using presence
Deficiency.Treatment peripheral nerve card pressure disease application in, the constituent of chitosan of composite material can play its sterilization, it is antibacterial,
The effect of anti-tissue adhesion；And sericin ingredient can improve the mechanical performance of chitosan, the catabolite of sericin can
Play neurotrophy and protective effect.In addition, the present invention is directed to the main pathological change (Myelin thickness after peripheral nerve is pressed by card
Thinning and disentwining angle velocity, aixs cylinder quantity are reduced, axon diameter attenuates), nerve is carried and is sustained using compound bio bracket
Growth factor (NGF), NGF can promote the regeneration of aixs cylinder, adjust the expression of myelin protein, promote the regeneration of myelin and myelin thick
The recovery of degree.
Chitosan of the present invention-sericin compound bio bracket combines the advantage of two kinds of ingredients respectively, has good
Biocompatibility, excellent mechanical performance, are easy to degrade in vivo, can be used as growth factor, the carrier of drug and cell.This
Chitosan-sericin compound bio bracket is used as the slow-released carrier of nerve growth factor by invention example, is utilized respectively
The effect of the advantages of compound bio bracket and NGF, treatment peripheral nerve card press disease, promote damaged nerve tissue and function assessment
Reparation.
Summary of the invention
The present invention provides a kind of chitosan-sericin compound bio bracket, has good biological activity and bio-compatible
Property, can natural degradation and the reparation for promoting peripheral nerve card crushing to hurt in vivo, while the compound rest has both chitosan
With the advantage of sericin respectively, make up the deficiency of homogenous material, as compound rest have chitosan sterilization, antibacterial, anti-group
The effect of adhesion, the neurotrophy of sericin and protective effect are knitted, the sericin ingredient in bracket can improve chitosan
Mechanical performance.
The present invention also provides a kind of above-mentioned chitosan-sericin compound bio bracket preparation method, simple process,
It is low in cost, while being conducive to the quality control and large-scale production of product.
The present invention also provides chitosan-sericin of the above-mentioned carrying NGF for the pressure treatment of peripheral nerve card is compound
The preparation method of biological support.
Present invention firstly provides a kind of chitosan-sericin compound bio bracket, ingredient is chitosan and silk gum egg
It is white.Chitosan is also known as chitosan, is that the chitin being widely present by nature is obtained by deacetylation.Shell is poly-
Sugar has the effects that sterilizing, promotes wound healing, absorb Wound exudate, anti-tissue adhesion, be not easy syneresis, has been used for making
Standby surgical dressing.Chitosan can be degraded to oligosaccharide by the intracorporal lysozyme of biology, have it is nontoxic, can be fully absorbed by organism
The characteristics of metabolism.Sericin is wrapped around a kind of natural macromolecular albumen on fibroin fiber surface layer, has good biofacies
Capacitive and biological degradability, can degradation in vivo, and catabolite be amino acid (predominantly serine, aspartic acid and sweet ammonia
Acid), catabolite is not only nontoxic to body, can be by organism metabolism, and there is neurotrophic effect.It is multiple that the present inventor studies discovery
Bracket is closed with chitosan and the advantage of sericin respectively, is the ideal biomaterial for the pressure treatment of peripheral nerve card.
Further, chitosan provided by the invention-sericin compound bio bracket is mutated using fibroin deletion form
The silk cocoon of kind silkworm extracts sericin as raw material, and obtains Compound Water after covalent crosslinking agent cross moulding with chitosan
Gel, further freeze-drying is prepared as compound rest.Wherein, chitosan is purchased from SIGMA company, and purity is greater than 75%, domestic silkworm silk
Plain deletion form mutating variety silk cocoon is purchased from Inst. of Silkworm, Chinese Academy of Agricultural Sciences, such variety source is stored in Chinese agriculture section
State, sericulture research institute, institute silkworm resource conservation center.
Due to the excellent biocompatibility of compound rest and bioactivity, constituent of chitosan can play it and sterilize, be antibacterial, anti-
Organize the effect of adhesion；And sericin ingredient can improve the mechanical performance of chitosan, the catabolite of sericin can be sent out
Wave neurotrophy and protective effect.Chitosan-sericin compound bio bracket is prepared as being sustained by an example of the present invention
Carrier carries nerve growth factor (NGF), and the size of bracket is diameter 1cm, thickness 2mm, thin discs shape, for rat ischium
The treatment of Nerve entrapments.
The present invention also provides a kind of above-mentioned chitosan-sericin compound bio bracket preparation method, including it is following
Step:
1) it is crushed and is gone out through ultraviolet light or ethyl alcohol as raw material using the silk cocoon of fibroin deletion form mutating variety silkworm
Then bacterium carries out the extraction of sericin using LiBr or LiCl aqueous solution, it is mass volume ratio that concentration, which is made, in extract
The sericin aqueous solution of 0.005 ~ 0.2g/mL；
2) chitosan is dissolved as solvent using the acetic acid solution of 1%(v/v), adjusts pH to 5.90 ~ 5.95,0.22 μm of filter
Device filtration sterilization obtains the chitosan acetic acid solution of 0.01 g/mL；
3) above-mentioned sericin aqueous solution and chitosan acetic acid solution volume ratio are mixed according to 80:20~20:80 ratio,
Covalent crosslinking agent is added, then injects die for molding after mixing, obtains chitosan-sericin composite hydrogel, it is described covalent
The usage amount of crosslinking agent is that every 1 ml chitosan-sericin mixed solution 333 μ L concentration of addition are mass volume ratio 0.01
The covalent crosslinking agent of g/ml；
4) chitosan-sericin hydrogel is freeze-dried, obtains the chitosan-sericin composite support
Frame.
Wherein, it crushes as scissors can be used silk cocoon shreds or other can make its broken method.Sterilizing methods can be adopted
With ultraviolet irradiation method, silk cocoon is ground into 1 mm2Left and right, front and back sides at least 5 min are irradiated with ultraviolet light respectively；Or use second
Silk cocoon is ground into 1 mm by alcohol infusion method2Left and right, at least impregnates 5 min with the ethyl alcohol of 70% ~ 80% volumetric concentration, in 3500 r/
It is centrifuged 5 minutes under the conditions of min, removes ethyl alcohol；It can also be by above-mentioned ultraviolet irradiation method and ethyl alcohol infusion method use in conjunction, first with purple
Front and back sides are irradiated in outside line, then are impregnated with ethyl alcohol, are then centrifuged for removing ethyl alcohol.Above-mentioned three kinds of methods can reach satisfied sterilizing effect
Fruit.
Further, the method for producing the sericin aqueous solution includes:
1) it is crushed and is sterilized as raw material using the silk cocoon of fibroin deletion form mutating variety silkworm, at 25 ~ 50 DEG C,
It uses concentration for LiBr the or LiCl aqueous solution of 6 ~ 8 mol/L, extracts processing 5 ~ 24 hours at 25 ~ 50 DEG C, every gram of silk cocoon
Fragment is extracted using LiBr the or LiCl aqueous solution of 20 ~ 100 mL, 6 ~ 8 mol/L；
2) extract for obtaining step 1) is centrifuged, and is removed insoluble substance, is obtained clear solution；
3) the Tris-HCl buffer of 0.5 ~ 3mol/L, pH 8.0 ~ 11.0 are added into the clear solution that step 2 obtains,
And it dialyses in ultrapure water；
4) the dialyzate centrifugation removal precipitating obtained step 3), is concentrated to get the silk gum that concentration is 0.005 ~ 0.2g/mL
Protein solution.In one embodiment of the invention, the concentration 0.04g/mL of the sericin aqueous solution can be made, to take
Obtain better cross-linking effect.
The preparation process of above-mentioned sericin aqueous solution operates in gnotobasis, the raw materials used and sterilized place of equipment
Reason, for example, ultrapure water used, bag filter are with being before all made of high pressure sterilization, LiBr aqueous solution used, trihydroxy methyl amino first
Heptane hydrochloride salt (Tris-HCl) passes through 0.22 μm of filter filtration sterilization, and ethyl alcohol is by 0.22 μm of filter filtering of hydrophily
Afterwards, it is configured with the ultrapure water of sterilizing.
Further, the method for preparing the chitosan acetic acid solution includes:
1) distilled water dose volume is than 1% glacial acetic acid solution；
2) it weighs chitosan and is dissolved in 1% acetic acid solution obtained by step 1), be placed in 37oIt is sufficiently dissolved in C shaking table 30 minutes；
With 1 mol/L NaOH solution tune pH value to 5.90 ~ 5.95；It is poly- that 0.01 g/mL shell is obtained by 0.22 μm of filter filtration sterilization
Sugared acetic acid solution.
One of glutaraldehyde, malonaldehyde and Geniposide etc. or a variety of may be selected in above-mentioned covalent crosslinking agent.For example, of the invention
One embodiment in can be used concentration be 1wt% Geniposide.
Further, above-mentioned sericin aqueous solution and chitosan acetic acid solution are mixed, in the chitosan-silk gum
The covalent crosslinking agent is added in protein mixed solution, mold is injected after mixing, 37oMaintain be allowed under C at least 8 hours or more
Molding, obtains chitosan-sericin hydrogel.In a specific embodiment, can place it at room temperature overnight at
Type.In the process, chitosan is crosslinked under the action of crosslinking agent with sericin and is reacted, and obtains chitosan-silk gum egg
Plain boiled water gel.
In order to be conducive to increase the porosity of compound rest, before chitosan-sericin hydrogel can be freeze-dried,
In subzero freeze forming, for example, being placed in -20oC to -190o(can generally be placed in liquid nitrogen) freezes at least 4 hours under C,
Growth factor-loaded and drug etc. can be more advantageous to.
Above-mentioned chitosan-sericin compound rest (size can be controlled by the selection of mold), such as can be made
Diameter 1cm, 2 ~ 3mm of thickness, the slow-released carrier of thin discs shape, to adapt to different degrees of peripheral nerve injury.What is obtained is compound
Bracket should be saved aseptically.
In a specific embodiment, chitosan-sericin compound bio bracket preparation method and application is specific
It can comprise the following steps that
1) bombyx mori silk fibroin deletion form mutating variety silk cocoon is weighed, and is cut into 1 mm2Fragment cleans 3 times, goes to remove water
Point, silk cocoon is first layered on an aseptic flat board, after being irradiated five minutes with ultraviolet light, is covered on it with another aseptic flat board
Face, then in turn, the aseptic flat board for taking away starting expose the another side of silk cocoon fragment and are irradiated again with ultraviolet light five minutes, or
Person can by silk cocoon fragment, turn-over, realization irradiate front and back sides with ultraviolet light each 5 minutes respectively one by one with tweezers；
2) the silk cocoon fragment of step 1) is soaked in LiBr the or LiCl aqueous solution that sterile concentration is about 6 mol/L
(LiBr the or LiCl aqueous solution that 55 mL are added in every gram of silk cocoon fragment) (is stirred) when necessary after sufficiently impregnating, and is placed in 35 DEG C
It water-bath 24 hours or so, is extracted the sericin in raw material and is dissolved in LiBr or LiCl aqueous solution；
3) then the extract in step 2 is centrifuged in 3500 r/min, separation removal insoluble substance is clarified
Solution；
4) a quarter volume sterile 1 mol/L, the Tris- of pH 9.0 are added into the clear solution that step 3) obtains
HCl buffer, and dialyse, obtain sericin aqueous solution；
5) step 4) is obtained into sericin aqueous solution centrifugation removal precipitating, being concentrated to get concentration is 0.005 ~ 0.2g/mL
Sericin aqueous solution, preferred concentration 4% is placed in 4 DEG C and saves backup；
6) it weighs chitosan and is dissolved in 1% acetic acid solution of volume ratio, be placed in 37 DEG C of shaking tables and sufficiently dissolve 30 minutes；With 1
Mol/L NaOH solution tune pH value is to 5.90 ~ 5.95；0.01 g/ml chitosan second is obtained by 0.22 μm of filter filtration sterilization
Acid solution is placed in 4 DEG C and saves backup；
7) the sterile sericin aqueous solution that step 5) obtains and the sterile chitosan solution that step 6) obtains is sufficiently mixed
It is even, obtain chitosan-sericin mixed solution；
8) sterile Geniposide is added into the chitosan that step 7) obtains-sericin mixed solution, additional amount is every 1
The Geniposide that 0.33 mL concentration is 0.01 mg/mL is added in ml mixed solution, injects in sterile mold, sets after mixing well
In room temperature 12 hours, chitosan-sericin hydrogel is obtained；
9) manufactured chitosan-sericin hydrogel is put into sterilized petri dishes, then is placed in -80 DEG C and freezes 4 hours.
10) 9) semi-finished product after middle freezing are put into frozen vacuum dryer, are lyophilized overnight in subzero, after freeze-drying
To bracket be removed from the molds.
11) volume ratio of chitosan and sericin in the chitosan for obtaining step 7)-sericin mixed solution
1:1 is added the NGF solution that 20 uL concentration are 10 ng/uL, mixes well, and subsequent step is constant, and the shell for obtaining carrying NGF is poly-
Sugar-sericin compound bio bracket.
The present invention also provides above-mentioned chitosan-sericin compound bio bracket answering in the treatment of periphery Nerve entrapments
With.Chitosan provided by the invention-sericin compound bio bracket can be used for preparing degradable in vivo graft, can be used for
Nerve growth factor is carried, the neural restoration after operation decompression is pressed for peripheral nerve card.
The beneficial effects of the present invention are:
1, the present invention extracts sericin as raw material using the silk cocoon of fibroin deletion form mutating variety silkworm, and poly- with shell
The crosslinked molding of sugar, prepares chitosan-sericin composite hydrogel as crosslinking agent using Geniposide for the first time, goes forward side by side one
Step obtains chitosan-sericin compound bio bracket, further, such compound rest is applied to periphery mind for the first time
Field is repaired through card pressure disease.
2, chitosan provided by the invention-sericin compound bio bracket has good biocompatibility, is damaging
It can be degradable after the completion of neural restoration.Degradation of chitosan product is oligosaccharide, and with antibacterial, prevent adhesion effect, can be prevented
Adhesion and cicatrization are organized after operation decompression operations；Sericin catabolite is amino acid, nerve cell can be supported to deposit
Living and proliferation, has neurotrophy and protective effect.Chitosan-sericin compound bio bracket is a kind of excellent biology
Material is applied to the treatment of peripheral nerve card pressure.
3, above-mentioned chitosan provided by the invention-sericin compound bio bracket also has good mechanical performance, such as
Intensity and flexibility will not cause excessive compression to tissue in vivo.
4, according to above-mentioned chitosan made of the method for the present invention-sericin compound bio bracket also have hole it is more, ratio
The big feature of surface area has good drug or cell factor slow-release capability, and bracket can carry cell factor or drug,
Make its slow release, to promote to block the reparation of pressure neurotrosis.Example provided by the invention is that compound bio bracket carries nerve
Growth factor NGF is used for the treatment of peripheral nerve card pressure.
5, compared with traditional surgery alone releases sciatic nerve crush, the method for the present invention is for the first time by nerve growth factor
For the treatment of peripheral nerve card pressure, NGF can promote the regeneration of aixs cylinder, adjust the expression of myelin protein, promote myelin again
Raw and Myelin thickness recovery, the pathological change for Nerve entrapments damage are treated.
6, above-mentioned chitosan provided by the invention-sericin compound bio bracket preparation process is simple, low in cost,
Be conducive to the quality control and large-scale production of product simultaneously.
Detailed description of the invention
Wherein, CS represents chitosan, and SS represents sericin.
Fig. 1 is different proportion (volume ratio) sterile chitosan, sericin mixed solution formation hydrogel effect picture；Scale
It is 1 centimetre.
Fig. 2 is different proportion chitosan-sericin composite hydrogel freezing of preparation of the embodiment of the present invention and low temperature is true
The freeze-drying bracket outside drawing that sky is dried to obtain；Scale is 1 centimetre.
Fig. 3 is that different proportion chitosan-sericin compound bio bracket cross section of preparation of the embodiment of the present invention is swept
Retouch electron microscopic picture；Scale is 500 microns.
Fig. 4 is different proportion chitosan-sericin compound bio bracket expansion rate of water absorption of preparation of the embodiment of the present invention
Versus time curve.
Fig. 5 is different proportion chitosan-sericin compound bio bracket fourier conversion of preparation of the embodiment of the present invention
Infrared spectrum analysis.
Fig. 6 is schwann cells in different proportion chitosan-sericin compound bio bracket of preparation of the embodiment of the present invention
Proliferative conditions comparison.
Fig. 7 A is to carry neurotrophic factor (NGF) different proportion chitosan-sericin compound bio bracket NGF afterwards
Daily burst size situation comparison.Fig. 7 B is that different proportion chitosan-sericin is compound afterwards for carrying neurotrophic factor (NGF)
The comparison of biological support NGF cumulative release amount situation.
Fig. 8 A is that different proportion chitosan-sericin compound bio bracket is sustained the undifferentiated PC12 cell differentiation of NGF rush
Microscope difference figure；Wherein a is negative control group (NGF is not added), and b is CS100/SS0 group, and c is CS80/SS20 group, and d is
CS50/SS50 group, e are CS20/SS80 group, and f is CS0/SS100 group, and scale is 50 microns.Fig. 8 B is poly- for different proportion shell
Sugar-sericin compound bio bracket sustained release NGF promotees undifferentiated PC12 cell differentiation situation column statistical chart.
Fig. 9 is that rat sciatic nerve card pressing mold type decompresses postoperative merging carrying NGF compound bio bracket, compound bio bracket
Internal degradation situation in different time points.
Figure 10 A, Figure 10 B are respectively different disposal group the 2nd week, the 4th week after rat sciatic nerve card pressure model surgery decompression
Card splenium position sciatic nerve MBP, β -3 tubulin, DAPI colored graph, scale are 50 microns.
Figure 11 is that rat sciatic nerve card presses different disposal group the 2nd week, the 4th week gastrocnemius muscle fibre after model surgery decompression
Masson colored graph, scale are 20 microns.
Figure 12 is that rat sciatic nerve card presses different disposal group the 2nd week after model surgery decompression, the 4th week sura myocyte straight
Diameter statistics.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with attached drawing, to the embodiment of the present invention
In technical solution be clearly and completely described, it is clear that described embodiments are some of the embodiments of the present invention, without
It is whole embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not before making creative work
Every other embodiment obtained is put, shall fall within the protection scope of the present invention.
The method comprises the steps of firstly, preparing sterile chitosan solutions, while being original with bombyx mori silk fibroin deletion form mutating variety silk cocoon
Material, using the method for sterile extracting, successfully extracts pure silk glue protein using lithium bromide (LiBr) or lithium chloride (LiCl) extraction method,
And by chemical crosslinking and freezing drain in the way of, be successfully prepared for chitosan-sericin compound bio bracket, this is multiple
Closing bracket and having prevents tissue adhesion, antibacterial action, while its catabolite is nontoxic to body and there is neurotrophy to make
With.
Chitosan provided by the invention-sericin compound bio bracket can be used for preparing intracorporal degradable transplanting
Object, the present invention carry nerve growth factor using chitosan-sericin compound bio bracket, can effectively facilitate periphery mind
Reparation through card pressure disease injury.
The preparation of 1 chitosan of embodiment-sericin compound bio bracket
1, the selection of silk cocoon:
The silk cocoon of bombyx mori silk fibroin deletion mutation kind is selected (to be purchased from Inst. of Silkworm, Chinese Academy of Agricultural Sciences, the domestic silkworm silk
Plain deletion mutation kind is stored in state, Inst. of Silkworm, Chinese Academy of Agricultural Sciences silkworm resource conservation center, deposit number (or quotient
Product number)185Nd-s, 140Nd-s,139Nd-sFor raw material, main chemical component are as follows: sericin.
2, the selection of chitosan:
Chitosan is purchased from Sigma company, and purity is greater than 75%.
3, the extraction of silk gum with separate
1) the silk cocoon 1g of bombyx mori silk fibroin deletion mutation kind is weighed respectively and is cut into 1 mm2The fragment of left and right, is placed in cleaning
Beaker in.Above-mentioned silk cocoon fragment is cleaned 3 times with ultrapure water, 3500 r/min are centrifuged 5 minutes and remove moisture removal.
In gnotobasis, silk cocoon fragment is impregnated 5 minutes respectively using 75% alcohol, is then centrifuged at 3500r/min
Remove within 5 minutes spare after alcohol.
2) concentration that 55 mL are added into the silk cocoon fragment after sterilizing is the LiBr aqueous solution of 6 mol/L, and beaker is put into
Sericin is dissolved in 35 DEG C water-bath 24 hours in thermostat water bath；
3) extract for obtaining step 2 is transferred to 3500 r/min in centrifuge tube and is centrifuged 5 minutes, removes insoluble substance,
Obtain clear solution；
4) into the clear solution that step 3) obtains be added a quarter volume Tris-HCl buffer (1 mol/L,
PH 9.0)；
5) solution obtained in step 4) is transferred in the bag filter (MWCO 3500) pre-processed, is then placed into
In beaker containing ultrapure water；The beaker is placed on blender and mixes slowly dialysis, a water was changed every 2 hours, is dialysed altogether
24 hours；
6) dialyzate obtained in step 5) is gone in centrifuge tube, 3500 r/min are centrifuged 5 minutes, removal precipitating；
7) solution that step 6) obtains is reloaded into bag filter and by bag filter both ends clamp, then will dialysis
Bag is placed in concentration in the PEG6000 solution (using preceding through high pressure sterilization) that mass percentage concentration is 20%, by sericin aqueous solution
It is concentrated to 0.005 ~ 0.2 g/mL.
4, the preparation of sterile chitosan solution
1) distilled water dilutes the acetic acid solution that 100% glacial acetic acid is configured to volume ratio 1%；
2) it weighs chitosan and is dissolved in 1% acetic acid solution obtained by step 1), obtain 0.01 g/mL chitosan acetic acid solution, put
It is placed in 37 DEG C of shaking tables and sufficiently dissolves 30 minutes；
3) 1 mol/L NaOH solution is prepared, 0.01 g/mL chitosan acetic acid solution obtained by step 2 is added, adjusts solution
PH value is to 5.90 ~ 5.95；
4) chitosan solution that step 3) obtains passes through 0.22 μm of filter filtration sterilization, and it is molten that sterile chitosan can be obtained
Liquid, it is spare.
5, chitosan-sericin compound bio bracket preparation
1) 5 clean beakers are taken, respectively according to volume ratio: chitosan: sericin=1:0, chitosan: sericin=4:
1, chitosan: sericin=1:1, chitosan: sericin=1:4, chitosan: sericin=0:1 is by sterile chitosan acetic acid
Solution and sericin aqueous solution are added in beaker, are mixed well, obtain CS100/SS0, CS80/SS20, CS50/SS50,
Chitosan-sericin the mixed solution of five kinds of various concentration ratios of CS20/SS80, CS0/SS100；
2) Geniposide aqueous solution is added in every kind of ratio, and additional amount is that 330 uL, 0.01 g/ is added in every 1 mL mixed solution
The Geniposide aqueous solution of mL, injects in sterile mold after mixing well, 37oIt is reacted 8 hours under C, obtains chitosan-silk gum
Albumen composite hydrogel, appearance is as shown in Figure 1 (scale is 1 centimetre)；2) chitosan-sericin obtained in is compound
Hydrogel is put into sterile petri dish, is put into -80 DEG C and is freezed 4 hours, place into frozen vacuum dryer and be lyophilized overnight, is lyophilized
After be removed from the molds to obtain sterile chitosan-sericin compound bio bracket, (scale is 1 li to appearance as shown in Figure 2
Rice)；
2 chitosans of embodiment-sericin compound bio bracket performance evaluation
Following performance evaluation is carried out to the chitosan prepared in embodiment 1-sericin compound bio bracket, specifically:
1, macrostructure
Using selected mold, the chitosan-sericin compound rest made is prepared to diameter 1cm, high by 2 ~
The flaky texture of 3mm, as shown in Figure 2.
2, microstructure.
Chitosan-silk gum egg is observed under scanning electron microscope (ULTRA PLUS-43-13, Zeiss, Germany produce)
White compound bio bracket.As shown in figure 3, five kinds of different proportion chitosans-sericin compound bio bracket cross section SEM
Figure, scale are 500 microns, and internal stent is multiple-void structure, CS100/SS0, CS80/SS20, CS50/SS50, CS20/
Five kinds of compound rest aperture averaging values of SS80, CS0/SS100 are respectively 157.69 μm, 130.55 μm, 146.27 μm,
136.35 μm, 151.99 μm, a large amount of micropores can sertoli cell growth and promotion nutriment and metabolite exchanges well.
3, mechanical property
It is tested at normal temperature using Universal micro test machine (Instron5848 MicroTester, Instron, USA)
Chitosan-sericin compound bio bracket mechanical property.Measure CS100/SS0, CS80/SS20, CS50/SS50,
The elasticity modulus of five kinds of compound rests of CS20/SS80, CS0/SS100 be respectively 0.0254 ± 0.0019 MPa, 0.0310 ±
0.0011 MPa,0.0347 ± 0.0022 MPa,0.0360 ± 0.0008 MPa,0.0478 ± 0.0023 MPa.It can
See in five kinds of compound bio brackets, with the increase of sericin concentration, the mechanical performance of compound rest is improved.Its
In, there is CS50/SS50 ratio compound bio bracket mechanical performance suitable enough can support repairing of neural injury.
4, expansion rate
By the compound bio bracket of freeze-drying be soaked in in the approximate phosphate buffer solution of internal pH (pH7.4), not
It is taken out with time point, measures its expansion rate of water absorption (wherein W as followssFor the weight under swelling state, WdFor dry weight).
Experimental result is as shown in figure 4, by taking CS50/SS50 as an example, as shown in Figure 4, after immersing PBS solution 24 hours,
Sericin nerve trachea tends towards stability after expanding 9.6 times, 48 hours, at 14 times or so, can be suitable for vivo environment.
5, porosity
Compound bio bracket weighing after freeze-drying is denoted as M0, measure its volume and be denoted as Vm, it is small to be dipped into dehydrated alcohol 24
Shi Hou, weighing are denoted as Mt, porosity (the dehydrated alcohol density of sericin nerve trachea is calculated according to the following formula=0.789
g/cm3)
Five kinds of CS100/SS0, CS80/SS20, CS50/SS50, CS20/SS80, CS0/SS100 prepared in embodiment 1
Compound bio brace aperture rate is respectively 83.30%, 84.13%, 85.34%, 82.47%, 90.73%, containing more aperture,
Be conducive to the embedding of cell factor and the carrying of cell.
6, infrared spectrum analysis
Measured respectively using Fourier Transform Infrared Spectrometer (Nexus, Thermal Nicolet, USA) five kinds it is compound
Biological support is in 4000-650 cm-1Characteristic peak, spectrogram is as shown in Figure 5.
The unique I peak (1600-1690cm of amide of sericin in visible CS0/SS100 in Fig. 5-1), II peak of amide
(1480-1575 cm-1) and III peak of amide (1229-1301 cm−1)；Chitosan is located at 1656 cm in CS100/SS0−1、1490
cm−1Two characteristic absorption peaks.After two kinds of ingredient mixing, according to S100/SS0, CS80/SS20, CS50/SS50, CS20/
SS80, CS0/SS100 ratio sequence, with the increase of sericin concentration, sericin characteristic absorption peak is from scratch simultaneously
It gradually increases, and the characteristic absorption peak of chitosan then gradually weakens last disappearance, illustrates that compound bio bracket has shell really
Two kinds of ingredients of glycan and sericin, and mixed compound bio bracket has had both the excellent of both chitosan and sericin
Point.
The assessment of 3 compound bio bracket of embodiment support Neuronal Survival and proliferative capacity
Using the sterile chitosan solution and sericin aqueous solution prepared in embodiment 1 according to CS100/SS0, CS80/
Five kinds of ratios of SS20, CS50/SS50, CS20/SS80, CS0/SS100 are applied directly in cell orifice plate, and capital Buddhist nun is added in every kind of ratio
Par solution, additional amount are the Geniposide aqueous solution that 330 uL concentration are added in every 1 mL mixed solution and are 0.01 g/mL,
37oIt is reacted 8 hours under C, is then freezed 4 hours at -80 DEG C, be then placed in vacuum freeze drier and be lyophilized, be lyophilized
Chitosan-sericin compound bio bracket；By the Rat Schwann Cells (RSC96) collected from Tissue Culture Flask through suspending,
It dispels, plants on the tissue culture plate for being covered with freeze-drying bracket.Cell culture used medium is DMEM(Dulbecco's
Modified Eagle's Medium) culture medium, 5% fetal calf serum is added in culture medium, cell is put in cell incubator (37
DEG C, CO2Concentration is 5%, humidity 100%) in culture.
As shown in fig. 6, after planting, using the increasing of CCK-8 method detection cell cell after plantation 1,3,5,7 and 9 day
Grow situation.It will be appreciated from fig. 6 that the quantity of Rat Schwann Cells (RSC96) increases at any time, therefore the chitosan-silk gum egg being lyophilized
White compound bio bracket can support the long-term surviving and proliferation of RSC96 cell, and CS50/SS50 concentration ratio group is better than it
His four kinds of concentration ratio groups.
4 compound bio bracket of embodiment carries the controlled release detection of NGF
Using the sterile chitosan solution and sericin aqueous solution prepared in embodiment 1 according to CS100/SS0, CS80/
Five kinds of ratios of SS20, CS50/SS50, CS20/SS80, CS0/SS100 are uniformly mixed, and it is 10 ng/uL's that 20 uL concentration, which are added,
Geniposide aqueous solution is added in sterile NGF solution, every kind of ratio, and additional amount is that addition 0.33mL mass is dense in every 1 mL mixed solution
Degree is the Geniposide aqueous solution of 0.01 g/mL, is injected in mold after mixing, 37oIt is reacted 8 hours under C, then -80
It is freezed 4 hours at DEG C, is then placed in vacuum freeze drier and is lyophilized, the chitosan-sericin for obtaining carrying NGF is compound
Biological support.
Carefully bracket is removed from the molds, is put into six clean orifice plates, PBS (pH 7.4) 1 mL is added in hole
Sufficiently submergence freeze-drying bracket, is placed in 37oC。
At the 1st, 2,3,4,5,6,7,8,9,10,12,14,16,20,25,30,35 and 40 day, supernatant is carefully taken out, and
Rejoin 1 mL PBS (pH 7.4).
Using the content of NGF in enzyme-linked immunization (ELISA) measurement supernatant, Fig. 7 A show each detection time point NGF
Burst size, Fig. 7 B show NGF and adds up release rate, by calculating the NGF content added up in various time points supernatant and total
The ratio of sustained release amount obtains NGF and adds up release rate.Five kinds of compound bio branch of NGF are equipped with known to Fig. 7 A, Fig. 7 B result
Frame can be sustained out NGF, and the NGF amount that the plateau of sustained release is sustained out daily is about 0.2 ng, can achieve treatment peripheral nerve
Therapeutic dose needed for damage.Compared to relatively without apparent difference between the sustained release performance of five kinds of compound bio brackets.
Embodiment 5 carries chitosan-sericin compound bio bracket reparation card pressure neural capacity assessment of NGF
The SD rat for choosing 32 200-250 g, according to the dosage of every 100g injection 0.3 ml, 10% chloraldurate by 32
After rat anesthesia, right lateral position, exposure right hind sciatic nerve, one internal diameter of cutting ferrule, the 1.3 mm outer diameter on sciatic nerve are taken
The silica gel sleeve pipe of 2.0 mm long, 1.0 cm.Left sciatic is played tricks surgical procedure, i.e. carefully exposure and free sciatic nerve.So
It is sutured afterwards with 7-0 nylon wire.32 rat stochastic averaginas are divided into four groups: surgery alone solution after card pressure processing 1 month
Pressure group, card splenium position direct injection NGF solution group, carries NGF compound bio bracket group at emptied out supports group.Still according to every 100 g
The dosage for injecting 0.3 mL10% chloraldurate after 32 rat anesthesias, will take right lateral position, exposure right hind sciatic nerve.It is single
Pure operation decompression group only removes card and is pressed in supraneural silica gel sleeve pipe and does not do other processing；Emptied out supports group is placed in after operation decompression
Are as follows: removal card places the unloaded compound bio bracket for not carrying NGF after being pressed in supraneural silica gel sleeve pipe；It directly infuses card splenium position
Penetrate NGF solution group are as follows: removal card is pressed in direct injection NGF solution after supraneural silica gel sleeve pipe；Carry NGF compound bio bracket
Group are as follows: removal card places the chitosan-sericin compound bio bracket for carrying NGF after being pressed in supraneural silica gel sleeve pipe, so
It is sutured afterwards with 7-0 nylon wire.Wherein, chitosan-sericin compound bio bracket specification is 1.0 cm of diameter, high by 2
Mm, carrying NGF amount are 200 ng, and chitosan used and sericin volume ratio are 1:1, i.e. CS50/SS50.
Fig. 9 is that rat sciatic nerve card pressing mold type decompresses postoperative merging carrying NGF compound bio bracket, compound bio bracket
Internal degradation situation in different time points.
After operation decompresses and carries out above-mentioned 4 kinds of packet transactions 2 weeks, 4 weeks, MBP and β are done to therapentic part sciatic nerve
3- tubulin immunohistochemical staining, MBP are Nogo protein, and β 3- tubulin is the mark of neural axon micro-pipe, as a result
Respectively as shown in Figure 10 A, Figure 10 B.Compared with other three processing groups, NGF compound bio bracket is carried in merging after operation decompression
Group therapentic part sciatic nerve Nogo protein and neural axon micro-tubular structure density significantly increase, and illustrate operation decompression postposition
The reparation of injured nerve can preferably be promoted after operation decompression by entering to carry NGF compound bio bracket group.
The gastrocnemius for taking sciatic nerve to dominate simultaneously does horse pine dyeing statistics diameter of muscle fiber, assesses muscle fiber atrophy feelings
Condition.As shown in figure 11, merging carries NGF compound bio bracket group diameter of muscle fiber most after operation decompression in four processing groups
Greatly, with sham-operation group no significant difference, illustrate operation decompression after merging carry NGF compound bio bracket group can preferably repair by
Damage nerve and the atrophy for reducing sura myocyte.Figure 12 is the statistics picture of Figure 11.To sum up, in rat sciatic nerve card pressing mold type
In, operation uses the chitosan-sericin compound bio stent in the treatment for carrying NGF after releasing card pressure, has good nerve
Injury repair effect.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (10)
1. a kind of chitosan-sericin compound bio bracket preparation method, comprising the following steps:
1) it using the silk cocoon of fibroin deletion form mutating variety silkworm as raw material, is crushed and is sterilized through ultraviolet light or ethyl alcohol,
Then the extraction that sericin is carried out using LiBr or LiCl aqueous solution, it is 0.005 ~ 0.2 g/mL's that concentration, which is made, in extract
Sericin aqueous solution；
2) chitosan is dissolved as solvent using the acetic acid solution of volume ratio 1%, adjusts pH to 5.90 ~ 5.95,0.22 μm of filter
Filtration sterilization obtains the chitosan acetic acid solution of 0.01 g/mL；
3) by above-mentioned sericin aqueous solution and chitosan acetic acid solution according to the volume ratio of 80:20~20:80 mix shell gathers
Sugar-sericin mixed solution is added covalent crosslinking agent, then injects die for molding after mixing, and obtains chitosan-silk gum egg
White composite hydrogel, the usage amount of the covalent crosslinking agent are that 333 μ L are added in every 1 mL chitosan-sericin mixed solution
Concentration is the covalent crosslinking agent of 0.01 g/mL；
4) chitosan-sericin hydrogel is freeze-dried, obtains the chitosan-sericin compound rest.
2. preparation method according to claim 1, it is characterised in that: wherein, crush for using scissors or other can make it
Broken method shreds silk cocoon to 1 mm2；Ultraviolet irradiation method can be used in sterilizing methods, irradiates the silkworm shredded respectively with ultraviolet light
Cocoon front and back sides at least 5 min；Or ethyl alcohol infusion method is used, the silkworm shredded is at least impregnated with the ethyl alcohol of 70% ~ 80% volumetric concentration
5 min of cocoon, is centrifuged 5min under the conditions of 3500 r/min, removes ethyl alcohol；Or above-mentioned ultraviolet irradiation method and ethyl alcohol infusion method are joined
Application is closed, the silk cocoon front and back sides shredded is first irradiated with ultraviolet light, then impregnated with ethyl alcohol, is then centrifuged for removing ethyl alcohol.
3. preparation method according to claim 1, it is characterised in that: produce the method packet of the sericin aqueous solution
It includes:
1) it is crushed and is sterilized, at 25 ~ 50 DEG C, every gram as raw material using the silk cocoon of fibroin deletion form mutating variety silkworm
Silk cocoon fragment uses 20 ~ 100 mL concentration for LiBr the or LiCl aqueous solution of 6 ~ 8 mol/L, extracts processing 5 at 25 ~ 50 DEG C
~ 24 hours；
2) extract for obtaining step 1) is centrifuged, and is removed insoluble substance, is obtained clear solution；
3) the Tris-HCl buffer of 0.5 ~ 3 mol/L, pH 8.0 ~ 11.0 is added into the clear solution that step 2 obtains, and
It dialyses in ultrapure water；
4) the dialyzate centrifugation removal precipitating obtained step 3), is concentrated to get the sericin that concentration is 0.005 ~ 0.2 g/mL
Aqueous solution；
The preparation process of above-mentioned sericin aqueous solution operates in gnotobasis, the raw materials used and sterilized processing of equipment,
Wherein, ultrapure water used, bag filter are with being before all made of high pressure sterilization, LiBr aqueous solution used, trishydroxymethylaminomethane
Hydrochloride passes through 0.22 μm of filter filtration sterilization, and ethyl alcohol is after 0.22 μm of filter filtering of hydrophily, with surpassing for sterilizing
Pure water configures.
4. preparation method according to claim 1, it is characterised in that: prepare the method packet of the chitosan acetic acid solution
It includes:
1) glacial acetic acid is diluted to the acetic acid solution of 1% volume ratio；
2) chitosan is dissolved in acetic acid solution obtained by step 1), is placed in 37 DEG C of shaking tables and sufficiently dissolves 30 minutes；With 1 mol/L
NaOH solution tune pH value is to 5.90 ~ 5.95；Then pass through 0.22 μm of filter filtration sterilization, obtain 0.01 g/mL chitosan acetic acid
Solution.
5. preparation method according to claim 1, it is characterised in that: glutaraldehyde, the third two may be selected in the covalent crosslinking agent
One of aldehyde and Geniposide are a variety of.
6. preparation method according to claim 1, it is characterised in that: inject die for molding item after mixing described in step 3)
Part is 37oIt is maintained under C at least 8 hours.
7. preparation method according to claim 1, it is characterised in that: step 4) is in the chitosan-sericin water-setting
First -20 before glue freeze-dryingoC to -190oIt is freezed under C at least 4 hours.
8. the chitosan as made from claim 1 the method-sericin compound bio bracket.
9. chitosan made from claim 1 the method-sericin compound bio bracket is used to prepare degradable in vivo
The application of graft, or the preparation of the carrying nerve growth factor compound bio bracket for the pressure treatment of peripheral nerve card.
10. carrying the compound bio bracket of nerve growth factor, it is made by laxative remedy:
1) it using the silk cocoon of fibroin deletion form mutating variety silkworm as raw material, is crushed and is sterilized through ultraviolet light or ethyl alcohol,
Then the extraction that sericin is carried out using LiBr or LiCl aqueous solution, it is 0.005 ~ 0.2g/mL's that concentration, which is made, in extract
Sericin aqueous solution；
2) chitosan is dissolved as solvent using the acetic acid solution of volume ratio 1%, adjusts pH to 5.90 ~ 5.95,0.22 μm of filter mistake
Bacterium is filtered out, the chitosan acetic acid solution of 0.01 g/ml is obtained；
3) ratio for being 1:1 in the volume ratio of chitosan acetic acid solution and sericin aqueous solution, above-mentioned sericin is water-soluble
Liquid and chitosan acetic acid solution mix to obtain 200 μ L chitosans-sericin mixed solution, and 20 μ L concentration of addition are 10 ng/ μ L
NGF solution, mix well；Covalent crosslinking agent is added, then injects die for molding after mixing, obtains chitosan-sericin
Composite hydrogel, the usage amount of the covalent crosslinking agent are that every 1 mL chitosan -333 μ L of sericin mixed solution addition are dense
Degree is the covalent crosslinking agent of 0.01 g/ml；
4) chitosan-sericin hydrogel is freeze-dried, obtains the compound bio for carrying nerve growth factor
Bracket.
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| WO2019047043A1 (en) * | 2017-09-06 | 2019-03-14 | 南通纺织丝绸产业技术研究院 | Silk fibroin/chitosan composite smart hydrogel and preparation method therefor |
| CN108543112B (en) * | 2018-03-30 | 2020-06-30 | 西南大学 | Preparation method of sericin-agarose composite gel with cell proliferation promoting activity and product thereof |
| CN109701065B (en) * | 2019-01-03 | 2020-07-17 | 中国人民解放军总医院 | Preparation process of chitosan biological dressing coated with micromolecular lithium chloride |
| CN109984857B (en) * | 2019-04-10 | 2021-09-24 | 华中科技大学同济医学院附属协和医院 | Method for establishing accurate peritoneal adhesion animal model and application |
| CN111150887B (en) * | 2019-12-31 | 2021-11-30 | 广州医科大学附属第一医院（广州呼吸中心） | Antibacterial composite bracket CS/SF/CMs-CIP for promoting healing of seawater-soaked wound and preparation method thereof |
| CN111166931A (en) * | 2020-01-21 | 2020-05-19 | 海南卓瑞生物医药有限公司 | Methacrylic acid sericin/chitosan quaternary ammonium salt hydrogel and preparation method and application thereof |
| CN112028980B (en) * | 2020-09-14 | 2022-04-22 | 暨南大学 | Sericin, extraction method, application of sericin as cellular antiviral immunopotentiator and application of sericin |
| CN114773692B (en) * | 2022-06-20 | 2022-09-27 | 北京智枢生物科技有限公司 | Active biogel capable of controlling NGF release for long term and application thereof |
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