CN106039416A - Chitosan-sericin composite biological scaffold as well as preparation method and application thereof - Google Patents
Chitosan-sericin composite biological scaffold as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a chitosan-sericin composite biological scaffold as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving chitosan by taking an acetic acid solution as a solvent to obtain a chitosan solution; preparing a sericin aqueous solution from the cocoon of bombyx mori of the fibroin-deleted mutant variety; uniformly mixing the chitosan solution and the sericin aqueous solution and pouring into a mould; adding a crosslinking agent for moulding to obtain a chitosan-sericin composite hydrogel; and freeze-drying the chitosan-sericin hydrogel to obtain the chitosan-sericin composite biological scaffold. The invention also provides a composite biological scaffold carrying a nerve growth factor NGF for peripheral nerve entrapment treatment. The composite biological scaffold provided by the invention has good biocompatibility and relatively high porosity and degradability, and the degradation product also has a neurotropic effect; the composite scaffold also has antibacterial and anti-adhesion functions and the like; and compared with the traditional way of relieving sciatic nerve compression simply by operation, the composite biological scaffold provided by the invention can effectively promote the repairing of peripheral nerve entrapment injury.
Description
Technical field
The present invention relates to medical biotechnology field of compound material, prop up in particular to a kind of chitosan sericin compound bio
Frame and the compound bio support carrying nerve growth factor and its preparation method and application.
Background technology
Peripheral nervous card pressure pathological changes is the common disease of Clinical Surgery, and the prevalence crowd is up to 3.8%, and it 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 fiber pipe of juxtra-articular, the plump musculotendinous cuff in particularly neural conduct, variation
Peripheral nervous all can be extruded by ligament and occupying lesion (thecal cyst, lipoma, hematoma or tumor etc.).Outside traditional
Week, Nerve entrapments Therapeutic Method had operative treatment and expectant treatment two kinds, and small part patient symptom is slight, can take conservative controlling
Treat, such as local steroid injection medicine, Boards wall, Yoga etc.；Major part card pressure patient possesses operative treatment indication, generally adopts
Card taking pressure neurolysis carries out operative treatment.There are some researches show in recent years, the mortality of operative treatment reaches 25%, and part is suffered from
Person can not realize long-term efficacy after being treated surgically.This phenomenon is considered to organize the formation of with Post operation local scar and god
After card is pressed, pathology damage is relevant.The effect improving operative treatment the most further is the important topic of hands surgical field in recent years
And hot issue.
Method of Tissue Engineering is widely used in the treatment of peripheral nervous amputation injury in recent years, and achieves preferably effect
Really, peripheral nervous card pressure treatment field but not yet it is applied to.Chitosan (Chitosan) is a kind of natural biologic material, by first
Shell element deacetylation gained, and chitin is widely present in Carapax Eriocheir sinensis, Crusta Penaeus seu Panulirus and arthropodan shell, there is also algae etc. low etc.
In the cell wall of plant, the annual biosynthetic carapace of nature have more than billions of ton, thus chitosan be one very
Abundant natural resources.Because it has sterilization, the antibacterial and function of promotion wound healing, it is developed application for wound
Mouth dressing.Additionally, chitosan has good biocompatibility, immunogenicity is weak, in vivo degradable metabolism.Chitosan at present
Extensively apply in various fields such as food fresh-keeping, biomedicine, environmental protection, cosmetics, agriculturals.By chemical modification effect,
Chitosan molecule structure introduce various functional group or chitosan is blended with other biological material, improving the materialization of chitosan
Matter, so that it each has different functions and effect, is an importance of research after chitosan.
Sericin (Silk Sericin) is wrapped around a kind of natural macromolecular viscous protein on fibroin fiber top layer, about
Account for the 20 ~ 30% of Bombyx bombycis content, be made up of the polypeptide that molecular weight is 24 ~ 400 kDa, its molecule by serine, aspartic acid and
18 kinds of aminoacid compositions such as glycine.The deficiency recognized sericin due to people for a long time and the limitation of research, lead
Causing sericin is taken as refuse to process in filature industry, wastes a large amount of valuable natural resources.It is found that sericin in recent years
Albumen has moisturizing, antibacterial, antioxidation, anticoagulation and promotes the biological nature such as cell adhesion and propagation.Moreover, sericin
There is hydrophilic and degradability, be preferable bio-medical material.Existing report display sericin is usually used in and other materials
(such as elastin laminin, polyvinyl alcohol etc.) copolymerization or simply cross-link, mixing manufacture biological support is with the biological material of excellent
Material.Give full play to sericin application in tissue engineering, there is important social meaning and wide application prospect.
The present invention, with bombyx mori silk fibroin deletion form mutating variety Bombyx bombycis as material, uses lithium bromide extraction method to extract pure silk glue egg
In vain, chitosan is combined by the mode utilizing chemical covalent bonds to cross-link with sericin, is successfully prepared chitosan sericin
Compound rest.This compound rest has chitosan and the respective advantage of sericin concurrently, and compensate for homogenous material application existence
Deficiency.Treatment peripheral nervous card pressure disease application in, the constituent of chitosan of composite can play its sterilization, antibacterial,
The effect of anti-tissue adhesion；And sericin composition can improve the mechanical performance of chitosan, the catabolite of sericin can
Play neurotrophy and protective effect.Additionally, the present invention is directed to peripheral nervous by the main pathological change (Myelin thickness after card pressure
Thinning and disentwining angle velocity, aixs cylinder quantity reduce, axon diameter attenuates), utilize compound bio support to carry and slow release is neural
Somatomedin (NGF), NGF can promote the regeneration of aixs cylinder, the expression of regulation myelin protein, promotes that the regeneration of myelin and myelin are thick
The recovery of degree.
Chitosan sericin compound bio support of the present invention combines two kinds of respective advantages of composition, has good
Biocompatibility, excellent mechanical performance, be prone to degraded in vivo, can be as somatomedin, medicine and the carrier of cell.This
Chitosan sericin compound bio support is used as the slow-released carrier of nerve growth factor by invention example, is utilized respectively
The advantage of compound bio support and the effect of NGF, treatment peripheral nervous card pressure disease, promote that damaged nerve tissue is learned and function assessment
Reparation.
Summary of the invention
The present invention provides a kind of chitosan sericin compound bio support, has good biological activity and bio-compatible
Property, can natural degradation promote the reparation that peripheral nervous card crushing hinders in vivo, the most described compound rest has chitosan concurrently
Advantage respective with sericin, makes up the deficiency of homogenous material, as compound rest have the sterilization of chitosan, antibacterial, anti-group
Knitting the effect of adhesion, the neurotrophy of sericin and protective effect, the sericin composition in support can improve chitosan
Mechanical performance.
The present invention also provides for the preparation method of a kind of above-mentioned chitosan sericin compound bio support, and technique is simple,
With low cost, the most beneficially quality control of product and large-scale production.
Present invention also offers the above-mentioned chitosan sericin for the lift-launch NGF of peripheral nervous card pressure treatment to be combined
The preparation method of biological support.
Present invention firstly provides a kind of chitosan sericin compound bio support, its composition is chitosan and sericin egg
In vain.Chitosan, also known as chitosan, is that the chitin being widely present by nature obtains through deacetylation.Shell gathers
Sugar has sterilizing, promotion wound healing, absorption Wound exudate, anti-tissue adhesion, is difficult to the effects such as syneresis, has been used for system
Standby surgical dressing.Chitosan can be degraded to oligosaccharide by the lysozyme in organism, have nontoxic, can be fully absorbed by organism
The feature of metabolism.Sericin is wrapped around a kind of natural macromolecular albumen on fibroin fiber top layer, has good biofacies
Capacitive and biological degradability, can degradation in vivo, and catabolite is aminoacid (predominantly serine, aspartic acid and sweet ammonia
Acid), catabolite is not only nontoxic to body, by organism metabolism, and can have neurotrophic effect.It is multiple that the present inventor studies discovery
Conjunction support, with chitosan and the respective advantage of sericin, is the ideal biomaterial for the pressure treatment of peripheral nervous card.
Further, the chitosan sericin compound bio support that the present invention provides is to use the sudden change of fibroin deletion form
The Bombyx bombycis of kind silkworm extracts sericin as raw material, and obtains Compound Water after covalent crosslinking agent cross moulding with chitosan
Gel, further lyophilization is prepared as compound rest.Wherein, chitosan is purchased from SIGMA company, and purity is more than 75%, domestic silkworm silk
Element deletion form mutating variety Bombyx bombycis is purchased from Inst. of Silkworm, Chinese Academy of Agricultural Sciences, and such variety source is stored in Chinese agriculture section
Can Ye institute state of institute silkworm resource conservation center.
The biocompatibility excellent due to compound rest and biological activity, constituent of chitosan can play its sterilization, antibacterial, anti-
The effect of tissue adhesion；And sericin composition can improve the mechanical performance of chitosan, the catabolite of sericin can be sent out
Wave neurotrophy and protective effect.Chitosan sericin compound bio support is prepared as slow release by an example of the present invention
Carrier, carries nerve growth factor (NGF), and the size of support is diameter 1cm, thickness 2mm, and thin discs shape, for rat ischium
The treatment of Nerve entrapments.
Present invention also offers the preparation method of a kind of above-mentioned chitosan sericin compound bio support, including following
Step:
1) use the Bombyx bombycis of fibroin deletion form mutating variety silkworm as raw material, pulverized and through ultraviolet or ethanol sterilizing,
Then use LiBr or LiCl aqueous solution to carry out the extraction of sericin, extract is made concentration be mass volume ratio 0.005 ~
The sericin aqueous solution of 0.2g/mL；
2) use 1%(v/v) acetic acid solution dissolve chitosan as solvent, adjust pH to 5.90 ~ 5.95,0.22 μm filter mistake
Filter bacterium, obtain the chitosan acetic acid solution of 0.01 g/mL；
3) above-mentioned sericin aqueous solution is mixed according to 80:20～20:80 ratio with chitosan acetic acid solution volume ratio, add
Inject die for molding after covalent crosslinking agent, then mixing, obtain chitosan sericin composite aquogel, described covalent cross-linking
The usage amount of agent is that to add 333 μ L concentration be mass volume ratio 0.01 g/ml to every 1 ml chitosan sericin mixed solution
Covalent crosslinking agent；
4) by described chitosan sericin hydrogel lyophilization, described chitosan sericin compound rest is obtained.
Wherein, pulverize as shears being used to be shredded by Bombyx bombycis or other can make its broken method.Sterilizing methods can be adopted
Use ultra-vioket radiation method, Bombyx bombycis is ground into 1 mm2Left and right, irradiates positive and negative at least 5 min respectively with ultraviolet；Or employing second
Alcohol infusion method, is ground into 1 mm by Bombyx bombycis2Left and right, at least soaks 5 min with the ethanol of 70% ~ 80% volumetric concentration, at 3500 r/
It is centrifuged 5 minutes under the conditions of min, removes ethanol；Can also be by above-mentioned ultra-vioket radiation method and soak with ethanol method use in conjunction, first with purple
Positive and negative is irradiated in outside line, then by soak with ethanol, is then centrifuged for removing ethanol.Above-mentioned three kinds of methods all can reach satisfied sterilizing effect
Really.
Further, the method producing described sericin aqueous solution includes:
1) use the Bombyx bombycis of fibroin deletion form mutating variety silkworm as raw material, pulverized and sterilizing, at 25 ~ 50 DEG C, use
Concentration is LiBr or the LiCl aqueous solution of 6 ~ 8 mol/L, carries out extraction process 5 ~ 24 hours at 25 ~ 50 DEG C, every gram of Bombyx bombycis fragment
LiBr or the LiCl aqueous solution using 20 ~ 100 mL 6 ~ 8 mol/L extracts；
2) extract that step 1) obtains is centrifuged, removes insoluble substance, obtain settled solution；
3) to step 2) settled solution that obtains adds 0.5 ~ 3mol/L, the Tris-HCl buffer of pH 8.0 ~ 11.0, and
Ultra-pure water is dialysed；
4) dialysis solution centrifugal segregation precipitation step 3) obtained, is concentrated to give the sericin that concentration is 0.005 ~ 0.2g/mL
Aqueous solution.In one embodiment of the invention, the concentration that can make described sericin aqueous solution is 0.04g/mL, to obtain more
Good cross-linking effect.
The preparation process of above-mentioned sericin aqueous solution operates in gnotobasis, the raw materials used and the most sterilized place of equipment
Reason, such as, ultra-pure water used, bag filter with the front autoclaving that all uses, LiBr aqueous solution used, trihydroxy methyl amino first
Heptane hydrochloride salt (Tris-HCl) is all through 0.22 μm filter filtration sterilization, and ethanol is to filter through hydrophilic 0.22 μm filter
After, configure with the ultra-pure water of sterilizing and form.
Further, the method preparing described chitosan acetic acid solution includes:
1) distilled water dose volume is than 1% glacial acetic acid solution；
2) weigh chitosan and be dissolved in step 1) gained 1% acetic acid solution, be positioned over 37oC shaking table fully dissolves 30 minutes；With 1
Mol/L NaOH solution adjusts pH value to 5.90 ~ 5.95；0.01 g/mL chitosan second is obtained through 0.22 μm filter filtration sterilization
Acid solution.
Above-mentioned covalent crosslinking agent may select one or more in glutaraldehyde, malonaldehyde and genipin etc..Such as, the present invention
An embodiment in can concentration be the genipin of 1wt%.
Further, above-mentioned sericin aqueous solution is mixed with chitosan acetic acid solution, at described chitosan sericin
Protein mixed solution adds described covalent crosslinking agent, injects mould after mixing, 37oMaintain under C more than at least 8 hours and be allowed to
Molding, obtains chitosan sericin hydrogel.In a detailed description of the invention, overnight at room temperature can be placed on
Type.In the process, there is cross-linking reaction in chitosan and sericin under the effect of cross-linking agent, obtains chitosan sericin egg
Plain boiled water gel.
In order to be beneficial to increase the porosity of compound rest, can by before chitosan sericin hydrogel lyophilization,
At subzero freeze forming, such as, it is placed in-20oC to-190oFreezing at least 4 hours (typically can be placed in liquid nitrogen) under C,
Growth factor-loaded and medicine etc. can be more beneficial for.
Above-mentioned chitosan sericin compound rest (size can be controlled by the selection of mould), such as, can make
Diameter 1cm, thickness 2 ~ 3mm, the slow-released carrier of thin discs shape, to adapt to peripheral nerve injury in various degree.Obtain is compound
Support should aseptically preserve.
In a detailed description of the invention, the preparation method and application of chitosan sericin compound bio support is concrete
Can comprise the following steps that
1) weigh bombyx mori silk fibroin deletion form mutating variety Bombyx bombycis, and be cut into 1 mm2Fragment, cleans 3 times, removes moisture removal, first
Bombyx bombycis is layered on an aseptic flat board, irradiates after five minutes with ultraviolet, with another aseptic flat board cover it face, then
In turn, take away initial aseptic plate exposure and go out the another side ultraviolet of Bombyx bombycis fragment and irradiate again five minutes, or permissible
With tweezers by Bombyx bombycis fragment turn-over one by one, it is achieved irradiate positive and negative respectively with ultraviolet each 5 minutes；
2) the Bombyx bombycis fragment of step 1) is soaked in aseptic concentration and is about in LiBr or the LiCl aqueous solution of 6 mol/L (every gram
Bombyx bombycis fragment adds LiBr or the LiCl aqueous solution of 55 mL) fully soak after (being stirred if desired), be placed in 35 DEG C of water-baths 24
About hour, the sericin in raw material is made to be extracted and be dissolved in LiBr or LiCl aqueous solution；
3) then by step 2) in extract 3500 r/min be centrifuged, separate remove insoluble substance, obtain clarification molten
Liquid；
4) in the settled solution that step 3) obtains, 1 mol/L, the Tris-HCl of pH 9.0 that four/mono-volumes are aseptic is added
Buffer, and dialyse, obtain sericin aqueous solution；
5) step 4) is obtained sericin aqueous solution centrifugal segregation precipitation, is concentrated to give the silk that concentration is 0.005 ~ 0.2g/mL
Glue protein aqueous solution, preferred concentration is 4%, is placed in 4 DEG C and saves backup；
6) weigh chitosan and be dissolved in volume ratio 1% acetic acid solution, be positioned in 37 DEG C of shaking tables and fully dissolve 30 minutes；Use 1 mol/L
NaOH solution adjusts pH value to 5.90 ~ 5.95；0.01 g/ml chitosan acetic acid solution is obtained through 0.22 μm filter filtration sterilization,
It is placed in 4 DEG C to save backup；
7) the aseptic chitosan solution that aseptic sericin aqueous solution and step 6) step 5) obtained obtains fully mixes,
To chitosan sericin mixed solution；
8) adding aseptic genipin in the chitosan sericin mixed solution that step 7) obtains, addition is that every 1 ml mixes
Close in solution and add the genipin that 0.33 mL concentration is 0.01 mg/mL, fully inject in aseptic mould after mixing, be placed in room
Temperature 12 hours, obtains chitosan sericin hydrogel；
9) the chitosan sericin hydrogel made is put in sterilized petri dishes, then be placed in-80 DEG C of freezings 4 hours.
10) by 9) in after freezing semi-finished product put in frozen vacuum dryer, in subzero overnight lyophilizing, after lyophilizing
To support be removed from the molds.
11) volume ratio of chitosan and sericin in the chitosan sericin mixed solution that step 7) obtains is made to be
1:1, adds the NGF solution that 20 uL concentration are 10 ng/uL, fully mixes, and subsequent step is constant, and the shell obtaining carrying NGF gathers
Sugar sericin compound bio support.
The present invention also provides for above-mentioned chitosan sericin compound bio support answering in periphery Nerve entrapments is treated
With.The chitosan sericin compound bio support that the present invention provides can be used for preparing degradable in vivo graft, can be used for
Carry nerve growth factor, the CO2 laser weld after peripheral nervous card pressure operation decompression.
The beneficial effects of the present invention is:
1, the present invention uses the Bombyx bombycis of fibroin deletion form mutating variety silkworm to extract sericin as raw material, and with chitosan warp
Cross moulding, utilizes genipin to prepare chitosan sericin composite aquogel as cross-linking agent first, and obtains further
Obtain chitosan sericin compound bio support, further, first such compound rest is applied to peripheral nervous card
Pressure disease repairs field.
2, the chitosan sericin compound bio support that the present invention provides has good biocompatibility, in damage
CO2 laser weld can be degradable after completing.Degradation of chitosan product is oligosaccharide, has antibacterial, anti effect, can prevent
Tissue adhesion and cicatrization after operation decompression operations；Sericin catabolite is aminoacid, it would be preferable to support neurocyte is deposited
Live and propagation, there is neurotrophy and protective effect.Chitosan sericin compound bio support is a kind of excellent biology
Materials application is in the treatment of peripheral nervous card pressure.
3, the above-mentioned chitosan sericin compound bio support that the present invention provides also has good mechanical performance, as
Intensity and pliability, will not cause excessive compression to tissue in vivo.
4, the above-mentioned chitosan sericin compound bio support made according to the inventive method also have hole many, ratio
The feature that surface area is big, has good medicine or cytokine slow-release capability, and support can carry cytokine or medicine,
It is made slowly to discharge, to promote the reparation of card pressure nerve injury.The example that the present invention provides is that compound bio support carries nerve
Somatomedin NGF is for the treatment of peripheral nervous card pressure.
5, compared with releasing sciatic nerve crush with tradition surgery alone, the inventive method is first by nerve growth factor
For the treatment of peripheral nervous card pressure, NGF can promote the regeneration of aixs cylinder, the expression of regulation myelin protein, promotes myelin again
The raw recovery with Myelin thickness, the pathological change for Nerve entrapments damage is treated.
6, the above-mentioned chitosan sericin compound bio support preparation technology that the present invention provides is simple, with low cost,
Be conducive to quality control and the large-scale production of product simultaneously.
Accompanying drawing explanation
Wherein, CS represents chitosan, and SS represents sericin.
Fig. 1 is different proportion (volume ratio) aseptic chitosan, sericin mixed solution formation hydrogel design sketch；Scale
It it is 1 centimetre.
Fig. 2 is that the freezing also low temperature of different proportion chitosan sericin composite aquogel prepared by the embodiment of the present invention is true
The lyophilizing support outside drawing that sky is dried to obtain；Scale is 1 centimetre.
Fig. 3 is that the cross section of different proportion chitosan sericin compound bio support prepared by 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 support expansion rate of water absorption prepared by the embodiment of the present invention
Versus time curve.
Fig. 5 is the fourier conversion of different proportion chitosan sericin compound bio support prepared by the embodiment of the present invention
Infrared spectrum analysis.
Fig. 6 is Scs in different proportion chitosan sericin compound bio support prepared by the embodiment of the present invention
Proliferative conditions contrast.
Fig. 7 A is for carrying neurotrophic factor (NGF) different proportion chitosan sericin compound bio support NGF afterwards
Burst size situation contrast every day.Fig. 7 B is combined for carrying neurotrophic factor (NGF) different proportion chitosan sericin afterwards
Biological support NGF cumulative release amount situation contrasts.
Fig. 8 A is that different proportion chitosan sericin compound bio support slow release NGF promotees the differentiation of undifferentiated PC12 cell
Microscope difference figure；Wherein a is negative control group (not adding NGF), and b is CS100/SS0 group, and c is CS80/SS20 group, and d is
CS50/SS50 group, e is CS20/SS80 group, and f is CS0/SS100 group, and scale is 50 microns.Fig. 8 B is that different proportion shell gathers
Sugar sericin compound bio support slow release NGF promotees undifferentiated PC12 cell differentiation situation column cartogram.
Fig. 9 is that rat sciatic nerve card pressing mold type decompression is postoperative inserts lift-launch NGF compound bio support, compound bio support
In different time points vivo degradation situation.
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 decompresses
Card splenium position sciatic nerve MBP, β-3 tubulin, DAPI colored graph, scale is 50 microns.
Figure 11 is different disposal group the 2nd week, the 4th week gastrocnemius muscle fiber after rat sciatic nerve card pressure model surgery decompression
Masson colored graph, scale is 20 microns.
Figure 12 is that different disposal group the 2nd week, the 4th week gastrocnemius cell are straight after rat sciatic nerve card pressure model surgery decompression
Footpath is added up.
Detailed description of the invention
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing, to the embodiment of the present invention
In technical scheme be clearly and completely described, it is clear that described embodiment is a part of embodiment of the present invention, and not
It it is whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art are not before making creative work
Put the every other embodiment obtained, broadly fall into the scope of protection of the invention.
The method comprises the steps of firstly, preparing aseptic chitosan solution, be former with bombyx mori silk fibroin deletion form mutating variety Bombyx bombycis simultaneously
Material, the method applying aseptic extracting, use lithium bromide (LiBr) or lithium chloride (LiCl) extraction method successfully to extract pure silk glue protein,
And utilize chemical crosslinking and the freezing mode drained, and successfully it being prepared for chitosan sericin compound bio support, this is multiple
Closing support to have and prevent tissue adhesion, antibacterial action, its catabolite is nontoxic to body simultaneously and has neurotrophy and makees
With.
The chitosan sericin compound bio support that the present invention provides can be used for preparing internal degradable transplanting
Thing, the present invention utilizes chitosan sericin compound bio support to carry nerve growth factor, can effectively facilitate periphery god
Reparation through card pressure disease injury.
The preparation of embodiment 1 chitosan sericin compound bio support
1, the selection of Bombyx bombycis:
(being purchased from Inst. of Silkworm, Chinese Academy of Agricultural Sciences, this bombyx mori silk fibroin lacks the Bombyx bombycis of selection bombyx mori silk fibroin deletion mutation kind
Lose mutating variety and be stored in state of Inst. of Silkworm, Chinese Academy of Agricultural Sciences silkworm resource conservation center, deposit number (or commodity compile
Number)185Nd-s, 140Nd-s,139Nd-sFor raw material, main chemical composition is: sericin.
2, the selection of chitosan:
Chitosan is purchased from Sigma company, and purity is more than 75%.
3, the extraction of sericin with separate
1) weigh the Bombyx bombycis 1g of bombyx mori silk fibroin deletion mutation kind respectively and be cut into 1 mm2The fragment of left and right, is placed in the burning of cleaning
In Bei.Being cleaned 3 times by above-mentioned Bombyx bombycis fragment ultra-pure water, 3500 r/min are centrifuged 5 minutes and remove moisture removal.
In gnotobasis, Bombyx bombycis fragment use 75% ethanol soak respectively 5 minutes, then centrifugal under 3500r/min
After within 5 minutes, removing ethanol standby.
2) the Bombyx bombycis fragment after sterilizing adds the LiBr aqueous solution that concentration is 6 mol/L of 55 mL, beaker is put into
35 DEG C of water-baths 24 hours in thermostat water bath, dissolve sericin；
3) by step 2) 3500 r/min are centrifuged 5 minutes in the extract that obtains proceeds to centrifuge tube, and remove insoluble substance, obtain
The solution of clarification；
4) in the settled solution that step 3) obtains, add Tris-HCl buffer (1 mol/L, the pH of 1/4th volumes
9.0)；
5) solution obtained in step 4) is transferred in the good bag filter of pretreatment (MWCO 3500), be then placed into containing
In the beaker of ultra-pure water；This beaker being placed in low rate mixing dialysis on agitator, changed a water every 2 hours, dialysis 24 is little altogether
Time；
6) forwarding in centrifuge tube by the dialysis solution obtained in step 5), 3500 r/min are centrifuged 5 minutes, remove precipitation；
7) solution that step 6) obtains is reinstalled in bag filter and by bag filter two ends clamp, then bag filter is put
Concentrate in PEG6000 solution (through autoclaving before Shi Yonging) that mass percentage concentration is 20%, sericin aqueous solution is concentrated
To 0.005 ~ 0.2 g/mL.
4, the preparation of aseptic chitosan solution
1) distilled water dilutes 100% glacial acetic acid and is configured to the acetic acid solution of volume ratio 1%；
2) weigh chitosan and be dissolved in step 1) gained 1% acetic acid solution, obtain 0.01 g/mL chitosan acetic acid solution, be positioned over
37 DEG C of shaking tables fully dissolve 30 minutes；
3) prepare 1 mol/L NaOH solution, add step 2) gained 0.01 g/mL chitosan acetic acid solution, regulate solution ph
To 5.90 ~ 5.95；
4) chitosan solution that step 3) obtains is through 0.22 μm filter filtration sterilization, i.e. can get aseptic chitosan solution, standby
With.
5, the preparation of chitosan sericin compound bio support
1) 5 clean beakers are taken, respectively according to volume ratio: chitosan: sericin=1:0, chitosan: sericin=4:1, shell
Polysaccharide: sericin=1:1, chitosan: sericin=1:4, chitosan: sericin=0:1 is by aseptic chitosan acetic acid solution
Join in beaker with sericin aqueous solution, fully mix, obtain CS100/SS0, CS80/SS20, CS50/SS50, CS20/
The chitosan sericin mixed solution of five kinds of variable concentrations ratios of SS80, CS0/SS100；
2) every kind of ratio adds genipin aqueous solution, and addition is to add 330 uL 0.01 g/mL in every 1 mL mixed solution
Inject in aseptic mould, 37 after genipin aqueous solution, fully mixingoReact 8 hours under C, obtain chitosan sericin
Composite aquogel, its outward appearance (scale is 1 centimetre) as shown in Figure 1；By 2) in the chitosan sericin compound water congealing that obtains
Glue is put in sterile petri dish, puts into-80 DEG C of freezings 4 hours, places into overnight lyophilizing in frozen vacuum dryer, after lyophilizing from
Mould takes out and obtains aseptic chitosan sericin compound bio support, its outward appearance (scale is 1 centimetre) as shown in Figure 2；
Embodiment 2 chitosan sericin compound bio support performance evaluation
The chitosan sericin compound bio support of preparation in embodiment 1 is carried out following performance evaluation, particularly as follows:
1, macrostructure
Use selected mould so as to get chitosan sericin compound rest be prepared to diameter 1cm, high 2 ~ 3mm's
Flaky texture, as shown in Figure 2.
2, microstructure.
Descend to observe chitosan sericin egg in scanning electron microscope (ULTRA PLUS-43-13, Zeiss, Germany produces)
White compound bio support.As it is shown on figure 3, the SEM of five kinds of different proportion chitosan sericin compound bio support cross sections
Figure, scale is 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 be respectively 157.69 μm, 130.55 μm, 146.27 μm,
136.35 μm, 151.99 μm, a large amount of micropores can be supported cell growth well and promote nutrient substance and metabolite exchange.
3, mechanical property
Utilize Universal micro test machine (Instron5848 MicroTester, Instron, USA) to test shell at normal temperatures to gather
The mechanical property of sugar sericin compound bio support.Record CS100/SS0, CS80/SS20, CS50/SS50, CS20/
The elastic modelling quantity of five kinds of compound rests of 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.Can
Seeing in five kinds of compound bio supports, along with the increase of sericin concentration, the mechanical performance of compound rest is improved.Its
In, CS50/SS50 ratio compound bio support has enough suitably mechanical performance can support repairing of neural injury.
4, expansion rate
The compound bio support of lyophilizing is soaked in the phosphate buffered solution (pH7.4) approximated with internal pH, when difference
Between point take out, measure its expansion rate of water absorption (wherein W as followssFor the weight under swelling state, WdFor dry weight).
Experimental result as shown in Figure 4, as a example by CS50/SS50, as shown in Figure 4, after immersing PBS solution 24 hours,
Sericin nerve trachea expands 9.6 times, tends towards stability, at about 14 times, it is possible to be applicable to internal milieu after 48 hours.
5, porosity
Compound bio support after lyophilizing is weighed and is designated as M0, measure its volume and be designated as Vm, it is dipped in dehydrated alcohol 24 hours
After, weigh and be designated as Mt, porosity (the dehydrated alcohol density of sericin nerve trachea is calculated according to below equation=0.789 g/
cm3)
In embodiment 1, CS100/SS0, CS80/SS20, CS50/SS50, CS20/SS80, CS0/SS100 five kinds of preparation is combined
Biological support porosity is respectively 83.30%, 84.13%, 85.34%, 82.47%, 90.73%, containing the most apertures, favorably
Embedding and the lift-launch of cell in cytokine.
6, infrared spectrum analysis
Fourier transform infrared spectrometer (Nexus, Thermal Nicolet, USA) is utilized to measure five kinds of compound bios respectively
Support is at 4000-650 cm-1Characteristic peak, spectrogram is as shown in Figure 5.
The amide I peak (1600-1690cm that in Fig. 5, in visible CS0/SS100, sericin is unique-1), amide II peak
(1480-1575 cm-1) and amide III peak (1229-1301 cm1)；In CS100/SS0, chitosan is positioned at 1656 cm1、1490
cm1Two distinctive absworption peaks.After two kinds of composition mixing, according to S100/SS0, CS80/SS20, CS50/SS50, CS20/
SS80, CS0/SS100 ratio order, along with the increase of sericin concentration, sericin characteristic absorption peak grows out of nothing also
Gradually strengthening, the characteristic absorption peak of chitosan the most gradually weakens last disappearance, illustrates that compound bio support possesses shell really
Polysaccharide and two kinds of compositions of sericin, and mixed compound bio support had the excellent of chitosan and sericin concurrently
Point.
Embodiment 3 compound bio support supports Neuronal Survival and the assessment of multiplication capacity
Use the aseptic chitosan solution of preparation in embodiment 1 and sericin aqueous solution according to CS100/SS0, CS80/SS20,
Five kinds of ratios of CS50/SS50, CS20/SS80, CS0/SS100 are applied directly in cell orifice plate, and every kind of ratio adds genipin water
Solution, addition is to add the genipin aqueous solution that 330 uL concentration are 0.01 g/mL in every 1 mL mixed solution, 37oUnder C
Reacting 8 hours, then freezing 4 hours at-80 DEG C, are then placed in lyophilizing in vacuum freeze drier, and the shell obtaining lyophilizing gathers
Sugar sericin compound bio support；By the Rat Schwann Cells (RSC96) collected from Tissue Culture Flask through suspending, dispel,
Plant on the Tissue Culture Plate being covered with lyophilizing support.It is DMEM(Dulbecco's Modified that cell cultivates used medium
Eagle's Medium) culture medium, culture medium adds 5% hyclone, cell be put in cell culture incubator (37 DEG C, CO2Concentration
Being 5%, humidity is 100%) middle cultivation.
As shown in Figure 6, after planting, the detection cell increasing of cell after planting 1,3,5,7 and 9 day of CCK-8 method is used
Grow situation.It will be appreciated from fig. 6 that the quantity of Rat Schwann Cells (RSC96) increases in time, the therefore chitosan sericin egg of lyophilizing
White compound bio support can support long-term surviving and the propagation of RSC96 cell, and CS50/SS50 concentration ratio group is better than it
His four kinds of concentration ratio groups.
Embodiment 4 compound bio support carries the controlled release detection of NGF
Use the aseptic chitosan solution of preparation in embodiment 1 and sericin aqueous solution according to CS100/SS0, CS80/SS20,
Five kinds of ratio mix homogeneously of CS50/SS50, CS20/SS80, CS0/SS100, adding 20 uL concentration is the aseptic of 10 ng/uL
NGF solution, every kind of ratio adds genipin aqueous solution, and addition is to add 0.33mL mass concentration in every 1 mL mixed solution to be
The genipin aqueous solution of 0.01 g/mL, injects after mix homogeneously in mould, 37oReact 8 hours under C, then at-80 DEG C
Freezing 4 hours, it is then placed in lyophilizing in vacuum freeze drier, obtains carrying the chitosan sericin compound bio of NGF
Support.
Carefully support is removed from the molds, puts in six orifice plates of cleaning, hole adds PBS (pH 7.4) 1 mL
Fully submergence lyophilizing support, 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 days, carefully take out supernatant, and
Rejoin 1 mL PBS (pH 7.4).
Using euzymelinked immunosorbent assay (ELISA) (ELISA) to measure the content of NGF in supernatant, Fig. 7 A show each detection time point NGF
Burst size, Fig. 7 B show NGF and adds up release rate, by calculating in each time point supernatant accumulative NGF content with total
The ratio of slow release amount i.e. obtains NGF and adds up release rate.Propped up by the five kinds of compound bios being equipped with NGF knowable to Fig. 7 A, Fig. 7 B result
Frame all can go out NGF by slow release, and the NGF amount that every day plateau of slow release, slow release went out is about 0.2 ng, can reach to treat peripheral nervous
Therapeutic dose needed for damage.Compare between the sustained release performance of five kinds of compound bio supports and there is no obvious difference.
Embodiment 5 carries the assessment of the chitosan sericin compound bio support reparation card pressure neural capacity of NGF
Choose the SD rat of 32 200-250 g, inject the dosage of 0.3 ml 10% chloral hydrate according to every 100g big by 32
After Mus anesthesia, take right arm reclining, expose right hind sciatic nerve, one internal diameter 1.3 mm external diameter 2.0 of cutting ferrule on sciatic nerve
The silica gel sleeve pipe of mm length 1.0 cm.Left sciatic is played tricks surgical procedure, i.e. careful exposes and free sciatic nerve.Then use
7-0 nylon wire is sewed up.Card pressure processes and 32 rat stochastic averagina was divided into after 1 month four groups: surgery alone decompression group,
Emptied out supports group, card splenium position direct injection NGF solution group, lift-launch NGF compound bio support group.Still according to every 100 g injections
The dosage of 0.3 mL10% chloral hydrate, by after 32 rat anesthesias, takes right arm reclining, exposes right hind sciatic nerve.Simple hands
Art decompression group only remove card be pressed in supraneural silica gel sleeve pipe do not do other process；Inserting emptied out supports group after operation decompression is:
Remove after card is pressed in supraneural silica gel sleeve pipe and place the unloaded compound bio support not carrying NGF；Card splenium position direct injection
NGF solution group is: removes card and is pressed in direct injection NGF solution after supraneural silica gel sleeve pipe；Carry NGF compound bio support group
For: remove the chitosan sericin compound bio support placing lift-launch NGF after card is pressed in supraneural silica gel sleeve pipe, then
Sew up with 7-0 nylon wire.Wherein, chitosan sericin compound bio support specification is diameter 1.0 cm, high 2 mm,
Carrying NGF amount is 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 decompression is postoperative inserts lift-launch NGF compound bio support, compound bio support
In different time points vivo degradation situation.
At operation decompression and carry out above-mentioned 4 kinds of packet transaction 2 weeks, after 4 weeks, therapentic part sciatic nerve be MBP and β
3-tubulin immunohistochemical staining, MBP is Nogo protein, and β 3-tubulin is the mark of the micro-pipe of neural axon, result
Respectively as shown in Figure 10 A, Figure 10 B.Compared with other three process groups, after operation decompression, insert lift-launch NGF compound bio support
Group therapentic part sciatic nerve Nogo protein and neural axon micro-tubular structure density significantly increase, and illustrate that operation decompression is rearmounted
Enter to carry NGF compound bio support group and preferably can promote the reparation of injured nerve after operation decompression.
The gastrocnemius simultaneously taking sciatic nerve domination does horse pine dyeing statistics diameter of muscle fiber, assesses muscle fiber atrophy feelings
Condition.As shown in figure 11, in four process groups, lift-launch NGF compound bio support group diameter of muscle fiber is inserted after operation decompression
Greatly, and sham operated rats no significant difference, insert lift-launch NGF compound bio support group after operation decompression is described and can preferably repair and be subject to
Damage atrophy that is neural and that reduce gastrocnemius cell.Figure 12 is the statistics picture of Figure 11.To sum up, in rat sciatic nerve card pressing mold type
In, operation uses, after releasing card pressure, the chitosan sericin compound bio stent in the treatment carrying NGF, has good nerve
Injury repairing effect.
Last it is noted that various embodiments above is only in order to illustrate technical scheme, it is not intended to limit；To the greatest extent
The present invention has been described in detail by pipe with reference to foregoing embodiments, it will be understood by those within the art that: it depends on
So the technical scheme described in foregoing embodiments can be modified, or the most some or all of technical characteristic is entered
Row equivalent；And these amendments or replacement, do not make the essence of appropriate technical solution depart from various embodiments of the present invention technology
The scope of scheme.
Claims (10)
1. a preparation method for chitosan sericin compound bio support, comprises the following steps:
1) use the Bombyx bombycis of fibroin deletion form mutating variety silkworm as raw material, pulverized and through ultraviolet or ethanol sterilizing,
Then using LiBr or LiCl aqueous solution to carry out the extraction of sericin, it is 0.005 ~ 0.2 g/mL's that extract is made concentration
Sericin aqueous solution；
2) acetic acid solution using volume ratio 1% dissolves chitosan as solvent, adjusts pH to 5.90 ~ 5.95,0.22 μm 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, injects die for molding after adding covalent crosslinking agent, then mixing, obtains chitosan sericin egg
White composite aquogel, the usage amount of described covalent crosslinking agent is that every 1 mL chitosan sericin mixed solution adds 333 μ L
Concentration is the covalent crosslinking agent of 0.01 g/mL；
4) by described chitosan sericin hydrogel lyophilization, described chitosan sericin compound rest is obtained.
Preparation method the most according to claim 1, it is characterised in that: wherein, pulverize as using shears or other can make it
Bombyx bombycis is shredded to 1 mm by broken method2；Sterilizing methods can use ultra-vioket radiation method, irradiates the silkworm shredded respectively with ultraviolet
Cocoon positive and negative at least 5 min；Or use soak with ethanol method, at least soak, with the ethanol of 70% ~ 80% volumetric concentration, the silkworm shredded
Cocoon 5 min, under the conditions of 3500 r/min, centrifugal 5min, removes ethanol；Or above-mentioned ultra-vioket radiation method and soak with ethanol method are joined
Close application, first irradiate the Bombyx bombycis positive and negative shredded with ultraviolet, then by soak with ethanol, be then centrifuged for removing ethanol.
Preparation method the most according to claim 1, it is characterised in that: produce the method bag of described sericin aqueous solution
Include:
1) use the Bombyx bombycis of fibroin deletion form mutating variety silkworm as raw material, pulverized and sterilizing, at 25 ~ 50 DEG C, every gram
LiBr or the LiCl aqueous solution that Bombyx bombycis fragment uses 20 ~ 100 mL concentration to be 6 ~ 8 mol/L, carries out extraction process 5 at 25 ~ 50 DEG C
~ 24 hours；
2) extract that step 1) obtains is centrifuged, removes insoluble substance, obtain settled solution；
3) to step 2) settled solution that obtains adds the Tris-HCl buffer of 0.5 ~ 3 mol/L, pH 8.0 ~ 11.0, and
Ultra-pure water is dialysed；
4) dialysis solution centrifugal segregation precipitation step 3) obtained, is concentrated to give 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 the most sterilized process of equipment,
Wherein, ultra-pure water used, bag filter with the front autoclaving that all uses, LiBr aqueous solution used, trishydroxymethylaminomethane
Hydrochlorate is all through 0.22 μm filter filtration sterilization, and ethanol is after hydrophilic 0.22 μm filter filters, with surpassing of sterilizing
Pure water configuration forms.
Preparation method the most according to claim 1, it is characterised in that: prepare the method bag of described chitosan acetic acid solution
Include:
1) glacial acetic acid is diluted to the acetic acid solution of 1% volume ratio；
2) chitosan is dissolved in step 1) gained acetic acid solution, is positioned in 37 DEG C of shaking tables and fully dissolves 30 minutes；Use 1 mol/L
NaOH solution adjusts pH value to 5.90 ~ 5.95；It is then passed through 0.22 μm filter filtration sterilization, obtains 0.01 g/mL chitosan acetic acid
Solution.
Preparation method the most according to claim 1, it is characterised in that: described covalent crosslinking agent may select glutaraldehyde, the third two
One or more in aldehyde and genipin.
Preparation method the most according to claim 1, it is characterised in that: inject die for molding bar after mixing described in step 3)
Part is 37oAt least 8 hours are maintained under C.
Preparation method the most according to claim 1, it is characterised in that: step 4) is at described chitosan sericin water-setting
First-20 before glue lyophilizationoC to-190oFreezing at least 4 hours under C.
8. the chitosan sericin compound bio support prepared by method described in claim 1.
9. the chitosan sericin compound bio support that method described in claim 1 prepares is for preparing degradable in vivo
The application of graft, or the preparation carrying nerve growth factor compound bio support for the pressure treatment of peripheral nervous card.
10. carry the compound bio support of nerve growth factor, purgation prepare:
1) use the Bombyx bombycis of fibroin deletion form mutating variety silkworm as raw material, pulverized and through ultraviolet or ethanol sterilizing,
Then using LiBr or LiCl aqueous solution to carry out the extraction of sericin, it is 0.005 ~ 0.2g/mL's that extract is made concentration
Sericin aqueous solution；
2) acetic acid solution using volume ratio 1% dissolves chitosan as solvent, adjusts pH to 5.90 ~ 5.95,0.22 μm filter mistake
Filter bacterium, obtain the chitosan acetic acid solution of 0.01 g/ml；
3) in the ratio that volume ratio is 1:1 of chitosan acetic acid solution Yu sericin aqueous solution, by water-soluble for above-mentioned sericin
Liquid and chitosan acetic acid solution mix to obtain 200 μ L chitosan sericin mixed solutions, and adding 20 μ L concentration is 10 ng/ μ L
NGF solution, fully mix；Inject die for molding after adding covalent crosslinking agent, then mixing, obtain chitosan sericin
Composite aquogel, it is dense that the usage amount of described covalent crosslinking agent is that every 1 mL chitosan sericin mixed solution adds 333 μ L
Degree is the covalent crosslinking agent of 0.01 g/ml；
4) by described chitosan sericin hydrogel lyophilization, the compound bio of described lift-launch nerve growth factor is obtained
Support.
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| CN116640452A (en) * | 2023-04-28 | 2023-08-25 | 华中科技大学同济医学院附属协和医院 | Preparation method and application of sericin micro-stent |
| CN116640452B (en) * | 2023-04-28 | 2024-06-11 | 华中科技大学同济医学院附属协和医院 | Preparation method and application of sericin micro-stent |
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