CN106039416B

CN106039416B - Chitosan-sericin compound bio bracket and its preparation method and application

Info

Publication number: CN106039416B
Application number: CN201610477686.2A
Authority: CN
Inventors: 王琳; 王征; 杨文�; 张磊; 谢洪建; 王健; 李晓麟
Original assignee: Union Hospital Tongji Medical College Huazhong University of Science and Technology
Current assignee: Union Hospital Tongji Medical College Huazhong University of Science and Technology
Priority date: 2016-06-27
Filing date: 2016-06-27
Publication date: 2019-05-17
Anticipated expiration: 2036-06-27
Also published as: CN106039416A

Abstract

The invention provides a chitosan-sericin composite biological scaffold and a preparation method and application. In the invention, acetic acid solution is used as solvent to dissolve chitosan to obtain chitosan solution; silkworm cocoons of silk fibroin-deficient mutant varieties are used to prepare sericin aqueous solution; chitosan solution and sericin aqueous solution are mixed uniformly and then injected into a mold , adding a cross-linking agent to form to obtain a chitosan-sericin composite hydrogel; freeze-drying the chitosan-sericin hydrogel to obtain a chitosan-sericin composite biological scaffold. The invention also provides a composite biological scaffold carrying nerve growth factor NGF for peripheral nerve compression therapy. The present invention has good biocompatibility, high porosity and degradability, the degradation product also has neurotrophic effect, and at the same time, the composite scaffold has the functions of antibacterial and anti-adhesion. It can effectively promote the repair of peripheral nerve entrapment injury.

Description

Chitosan-sericin compound bio bracket and its preparation method and application

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 37^oIt 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, 37^oMaintain 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 -20^oC to -190^o(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, 37^oIt 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 follows_sFor the weight under swelling state, W_dFor 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 V_m, it is small to be dipped into dehydrated alcohol 24 Shi Hou, weighing are denoted as M_t, 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, 37^oIt 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, 37^oIt 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 37^oC。

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.

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

1. A preparation method of a chitosan-sericin composite biological scaffold, comprising the following steps:

1) Using silk fibroin-deficient mutant silkworm cocoons as raw materials, pulverized and sterilized by ultraviolet light or ethanol, and then used LiBr or LiCl aqueous solution to extract sericin, and the extract was made into a concentration of 0.005~0.2 g /mL of sericin aqueous solution;

2) Dissolve chitosan with 1% acetic acid solution by volume as a solvent, adjust the pH to 5.90~5.95, filter and sterilize with a 0.22 μm filter to obtain 0.01 g/mL chitosan acetic acid solution;

3) Mix the above-mentioned sericin aqueous solution and chitosan acetic acid solution according to the volume ratio of 80:20～20:80 to obtain a chitosan-sericin mixed solution, add a covalent cross-linking agent, mix well and then inject Molding in a mold to obtain a chitosan-sericin composite hydrogel, the use amount of the covalent cross-linking agent is to add 333 μL of 0.01 g/mL chitosan-sericin mixed solution per 1 mL of chitosan-sericin mixed solution. covalent crosslinking agent;

4) Freeze drying the chitosan-sericin hydrogel to obtain the chitosan-sericin composite scaffold.

2. preparation method according to claim 1, is characterized in that: wherein, pulverizing is to adopt scissors or other methods that can make it broken and the cocoon is cut into pieces to 1 mm ² ; Irradiate the front and back of the shredded silkworm cocoons for at least 5 minutes; or use the ethanol soaking method, soak the shredded silkworm cocoons with 70%~80% volume concentration ethanol for at least 5 minutes, centrifuge at 3500 r/min for 5 minutes, and remove the ethanol; Or combined application of the above-mentioned ultraviolet irradiation method and ethanol soaking method, first irradiate the front and back sides of the chopped silk cocoons with ultraviolet rays, then soak in ethanol, and then centrifuge to remove the ethanol.

3. The preparation method according to claim 1, wherein the method for preparing the sericin aqueous solution comprises:

1) Using silk fibroin-deficient mutant silkworm cocoons as raw materials, pulverize and sterilize them, and use 20-100 mL of LiBr or LiCl aqueous solution with a concentration of 6-8 mol/L per gram of silkworm cocoon fragments at 25-50 °C , perform extraction treatment at 25~50℃ for 5~24 hours;

2) Centrifuge the extract obtained in step 1) to remove insoluble substances to obtain a clear solution;

3) Add 0.5~3 mol/L, pH 8.0~11.0 Tris-HCl buffer to the clear solution obtained in step 2), and conduct dialysis in ultrapure water;

4) Centrifuge the dialysate obtained in step 3) to remove the precipitate, and concentrate to obtain a sericin aqueous solution with a concentration of 0.005-0.2 g/mL;

The preparation process of the above-mentioned sericin aqueous solution is performed in a sterile environment, and the raw materials and equipment used are all sterilized. Among them, the ultrapure water and dialysis bags used are all autoclaved before use. Methylaminomethane hydrochloride was filtered and sterilized through a 0.22 μm filter, and ethanol was prepared with sterilized ultrapure water after being filtered through a hydrophilic 0.22 μm filter.

4. preparation method according to claim 1 is characterized in that: the method for preparing described chitosan acetic acid solution comprises:

1) Dilute glacial acetic acid into a 1% volume ratio of acetic acid solution;

2) Dissolve chitosan in the acetic acid solution obtained in step 1) and place it in a shaker at 37°C for 30 minutes; adjust the pH to 5.90~5.95 with 1 mol/L NaOH solution; then filter and sterilize it through a 0.22 μm filter, A 0.01 g/mL chitosan acetic acid solution was obtained.

5. The preparation method according to claim 1, wherein the covalent crosslinking agent can be selected from one or more of glutaraldehyde, malondialdehyde and genipin.

6 . The preparation method according to claim 1 , characterized in that: in step 3), after the mixing, injection into the mold and molding conditions are maintained at 37 ^° C. for at least 8 hours. 7 .

7. The preparation method according to claim 1, characterized in that: step 4) freezing at -20 ^o C to -190 ^o C for at least 4 days before the chitosan-sericin hydrogel freeze-drying Hour.

8. The chitosan-sericin composite bioscaffold prepared by the method of claim 1.

9. The application of the chitosan-sericin composite bioscaffold prepared by the method of claim 1 for the preparation of degradable grafts in vivo, or the application of the nerve growth factor composite bioscaffold for peripheral nerve compression therapy preparation.

10. A composite biological scaffold carrying nerve growth factor, prepared by the following method:

1) Using silk fibroin-deficient mutant silkworm cocoons as raw materials, pulverize it and sterilize it with ultraviolet light or ethanol, and then use LiBr or LiCl aqueous solution to extract sericin, and make the extract into a concentration of 0.005~0.2g /mL of sericin aqueous solution;

2) Use 1% acetic acid solution by volume as solvent to dissolve chitosan, adjust pH to 5.90~5.95, filter and sterilize 0.22 μm filter to obtain 0.01 g/ml chitosan acetic acid solution;

3) According to the volume ratio of chitosan acetic acid solution and sericin aqueous solution is 1:1, mix the above sericin aqueous solution and chitosan acetic acid solution to obtain 200 μL of chitosan-sericin mixed solution, add 20 μL of NGF solution with a concentration of 10 ng/μL, and mixed thoroughly; add a covalent cross-linking agent, and then inject it into a mold to form after mixing to obtain a chitosan-sericin composite hydrogel. The covalent cross-linking agent The dosage of the agent is to add 333 μL of covalent cross-linking agent with a concentration of 0.01 g/ml per 1 mL of chitosan-sericin mixed solution;

4) Freeze-drying the chitosan-sericin hydrogel to obtain the composite bioscaffold carrying the nerve growth factor.