CN109091708A

CN109091708A - Collagen-fibroin-chitosan/astragalus polyose engineering skin three-dimensional rack

Info

Publication number: CN109091708A
Application number: CN201810926611.7A
Authority: CN
Inventors: 郑宣; 全仁夫; 高旦华; 冯晓燕; 瞿钢; 许世超; 方伟利; 胡云根
Original assignee: HANGZHOU CITY XIAOSHAN DISTRICT TRADITIONAL CHINESE MEDICAL HOSPITAL
Current assignee: HANGZHOU CITY XIAOSHAN DISTRICT TRADITIONAL CHINESE MEDICAL HOSPITAL
Priority date: 2018-08-15
Filing date: 2018-08-15
Publication date: 2018-12-28

Abstract

The present invention relates to medical instruments fields, more particularly to artificial engineering skin field.Collagen-fibroin-chitosan/astragalus polyose engineering skin three-dimensional rack, the engineering skin three-dimensional rack includes collagen/silk fibroin bracket and astragalus polyose/chitosan microball, for astragalus polyose/chitosan microball uniform load in collagen/silk fibroin bracket, astragalus polyose/chitosan microball and collagen/silk fibroin bracket mass ratio are 1:1 ~ 1:20；Collagen/the silk fibroin bracket is made of collagen and fibroin albumen, and the mass percent of collagen and fibroin albumen is 1:1 ~ 5:1；Astragalus polyose/the chitosan microball is made of astragalus polyose and chitosan, and astragalus polyose is encapsulated in chitosan, and the drugloading rate of astragalus polyose is 10 ~ 30%, and the concentration of astragalus polyose is 0.1ug/ml-200ug/ml.The engineering skin three-dimensional rack is ingenious to construct slow-released system using chitosan microball package astragalus polyose, and is compounded on collagen-fibroin albumen three-dimensional rack, to solve the purpose of the Biocomposite material bracket vascularization promoting.

Description

Collagen-fibroin-chitosan/astragalus polyose engineering skin three-dimensional rack

Technical field

The present invention relates to medical instruments fields, more particularly to artificial engineering skin field.

Background technique

Skin is the maximum human organ of area, has and feels, adjust body temperature, secretion and excretion, prevent moisture evaporation etc. A variety of effects, wherein most important function is the stabilization as human body and the barrier of external environment to maintain interior environment, while its It is also the important component of immune system.With China's economy, society fast development, various acute and chronic Risk Factors, such as Defect of skin caused by mechanical damage, burn, body surface tumor resection, chronic ulcer, traffic accident, building accident etc. is clinically It is very common, physical disabilities very serious are frequently resulted in, or even dead.The treatment method of large skin defect, mainly has Wound covering object, auto-skin grafting, heterogenous skin transplanting etc., but wound covering object does not have physiological function, auto-skin grafting Materials limited area, heterogenous skin trnasplantion immunity rejection is big, skin is easy to fall off necrosis etc., easily causes to the secondary of patient Damage, increases the pain of patient.Therefore, hope is brought to solve this problem using organization engineering skin reparation.

In the latest 20 years, the fast development of organizational engineering provides newly for the reconstruction and reparation of large skin defect Thinking and therapy approach.1987, the bioengineering expert that National Science Foundation (NSF) holds in California In discussion, it has been put forward for the first time the concept of organizational project, and has specified the definition of organizational project: " Tissue Engineering " is the application of principles and methods of engineering and life sciences toward the fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and development of biological Substitutes to restore, maintain, or improve tissue function.Its core is to utilize life section The principle and method of the subjects such as, materialogy, computer science and engineering science, research and development products substitution, improve people at reparation The various tissues of body and organ make its regenerated cross discipline.With the foundation and development of organizational engineering, skin histology Engineering science is rapidly growing, and becomes the hot spot studied nearly ten years.Organization engineering skin in classical meaning includes epidermis substitution Object, dermal substitute and epidermal-dermal bilayer substitute, are related to biologic bracket material, seed cell, internal microenvironment, biology The many aspects such as active factors and stimulus signal.Ideal Graftskin should meet following 4 conditions: (1) having and mutually interconnect Logical three-dimensional porous structure, the transhipment for the growing into of cell, nutriment and metabolite provide convenience；(2) have good Biocompatibility and biodegradability, and degradation rate matches with cambium generating rate；(3) it suitably comes to the surface Structure is learned, sticking, be proliferated and breaking up for cell is conducive to；(4) there is the machinery to match with the destination organization at position to be implanted Intensity.

After decades of development, have multiple organization engineering skin products at present to come out, such as Apligraf^TM、 OrCel^TM、Integra^TMEqual products are successively listed by United States drug and food control office (FDA) approval and are applied to clinical skin In the treatment of skin defect.However, the problem of in conjunction with being encountered during clinical application for many years, by literature search, it is believed that at present Organization engineering skin also faces many shortcoming and defect: 1, since angiogenesis ability is poor, easily leading to the skin early stage of transplanting There is no vascular system to supply nutrition, Graftskin is made to be easy to happen necrosis, causes graft failure；2, in active mass's engineering skin Contained various variant cells easily cause immunological rejection, the cutaneous necrosis transplanted clinically often occur, fall off, or even go out The severe complications such as existing systemic immune response, and there are pathophorous danger；3, organization engineering skin product all at present The part anatomical structure and physiological function that normal skin can only all be restored, can not regenerate the cutaneous appendages knot with critical function Structure, such as: blood vessel, hair follicle, sebaceous glands, sweat gland；4, organization engineering skin reparation at this stage is often accompanied by trauma surface infestation, disunion etc. Complication, survival rate is low, and therapeutic effect is unsatisfactory.To find out its cause, close with the early stage vascularization degree of surface of a wound graft reparation Cut phase is closed.Organization engineering skin, acellular dermal bracket for being studied at present etc., do not have blood vessel web frame, itself does not have Source of nutrition after implanting, need to be grown by inducing peripheral tissue blood vessel, so that it is gradually established blood with surrounding tissue and follow Ring obtains nutrition.Therefore, how to realize that quick vascularization is one of the great difficult problem of current organization engineering skin field face.

In recent years, with the continuous development of science and technology, the research of organization engineering skin vascularization is also gradually goed deep into, and takes Obtained very big success.Studies have shown that effectively living by building composite material slow-released system, screening seed cell type, addition The modes such as sex factor, cytogene modification, can promote the early stage vascularization of organization engineering skin.And composite biological material is with it Superior performance and by favor, wherein collagen (collagen, COL)-fibroin albumen (silkfibroin, SF) material Bracket is found to have good hydrophily, histocompatbility and biological degradability.Collagen is the master of animal connective tissue Constituent is wanted, the collagen in various animals source has closely similar chemistry and biology feature.Collagen is nontoxic With lower immunogenicity, good biocompatibility and biodegradability, and it is from a wealth of sources, it is using earliest day Right biomaterial can provide microenvironment for the adherent of cell, growth, proliferation and directed differentiation；Fibroin albumen is a kind of containing someone The native protein of body essential amino acid has good biocompatibility and mechanical property, and it is slow to degrade.Fibroin albumen by Silk is made by degumming, is a kind of natural polymer fibrin of no physiological activity, accounts for the 70%~80% of silk, contains 18 Kind of amino acid, wherein glycine (Gly), which accounts for about 46%, alanine (Ala) and accounts for about 29%, serine (Ser), accounts for about 12%.Fibroin Albumen is by heavy chain (H chain, relative molecular mass 350kD), light chain (L chain, relative molecular mass 25.8kD) and glycoprotein p25 (phase To molecular mass 23.55kD), 3 oligonucleotide chain compositions.In addition, there are the two different structures of SilkI and SilkII for fibroin albumen As SilkI conformation includes a random ball of string and a- spiral, and SilkII conformation is then anti-parallel ss-sheet.Wherein SilkI structure is not It is enough to stablize, stable SilkII structure can be transformed into through polar solvent, heat treatment etc..Vepari etc. and Altman etc. thinks fibroin Albumen major advantage is as follows: 1, the mechanical property and flexibility incomparable with other natural fibers；2, to water and oxygen permeability It is good；3, a variety of different forms can be obtained by relatively simple processing；4, be conducive to histocyte to grow into, the inside and outside degradation of body Slowly.Based on above-mentioned advantage, application study of the porous fibroin albumen in skin tissue engineering scaffold is developed increasingly.Cause This, is added a certain proportion of fibroin albumen in collagen, can effectively improve the mechanical and physical performance under its dry state, such as improves mechanics With water repelling property, reduce hot water dissolve-loss ratio, enhance its adhesion to fibroblast, skin epidermal cells, provided for cell Permanent support preferably to match the histiocytic speed of growth, and can reach different by different processing methods Form and hole requirement, enhance the elastic and more sky shape structure of bracket.

As the rarity of motherland's medicine, traditional Chinese medicine is in terms of set a broken bone section, cutaneous diseases, and activating blood and removing stasis Method is always It is considered as one of most important therapy.And blood circulation promoting medicine promotes the pharmacological action of angiogenesis to be confirmed by many scholars, It is widely used in the clinical treatment of the diseases such as cerebral arterial thrombosis, coronary heart disease, fracture.Radix Astragali is common Chinese herbal medicine, sweet in flavor, property Tepor has the effect of QI invigorating qi-restoratives, can give birth to use, can also process use.Raw use can table, and energy toxin expelling, myogenic admittedly；Toast is mended with energy QI invigorating In；Its micromicro diuresis is relieved oedema or abdominal distension through diuresis or purgation wet, and the duration is longer.It is Chinese medicine tonifying Qi key medicine.Modern pharmacological research finds that it has enhancing Immune system resists disease, builds up health, resisting oxidation and delaying senility, improves cardiac status, antiviral, the functions such as anticancer.In Doctor thinks tonifying Qi with green blood, and Li Gao " differentiation on endogenous " the Shou Zai party reuses Radix Astragali, it is believed that Radix Astragali can " big tonifying spleen lung it Gas makes Yin grows while yang is generating with the source ... of abundant green blood, and the prosperous blood of gas is raw ".Having scholar to study discovery proves that Radix Astragali has very strong rush blood vessel Palingenesis, and the effect be mainly attributed to Radix Astragali effective component-astragalus polyose (Astragalus Polysacharin, APS).Astragalus polyose has facilitation to the Rat Intestinal Mucous Microvascular Endothelial Cells proliferation of in vitro culture, and research finds APS The proliferation of cell can be obviously promoted in low concentration.

The astragalus polyose that Chinese invention patent (publication number: CN104586775A) discloses a kind for the treatment of radiation pneumonia is slow Microballoon is released, by weight, comprising: 1 part of astragalus polyose, 3~7 parts of chitosan, wherein the degree of polymerization of chitosan is 30CPS, 50CPS, 130CPS also disclose the preparation method of the astragalus polyose sustained-release micro-spheres, the astragalus polyose prepared using this method Sustained-release micro-spheres, have ideal Targeting Effect, slow release after administration, and long action time enhances astragalus polyose in target organ Concentration, and do not cause local blood concentration high；Product safety, environmental protection, side effect high to the therapeutic effect of radiation pneumonitis It is small；It is easy to operate, process stabilizing, easy to industrialized production；The encapsulation rate and drugloading rate of product are high；Skin smooth is fine and smooth.

Summary of the invention

In order to solve the above technical problems, it is poly- that the first purpose of the invention is to provide a kind of collagen-fibroin albumen-shells Sugar/astragalus polyose engineering skin three-dimensional rack, the engineering skin three-dimensional rack is ingenious to wrap up astragalus polyose using chitosan microball Slow-released system is constructed, and is compounded on collagen-fibroin albumen three-dimensional rack, to solve the Biocomposite material bracket vascularization promoting Purpose.A second object of the present invention is to provide the preparation methods of above-mentioned engineering skin three-dimensional rack.

In order to realize first above-mentioned purpose, present invention employs technical solutions below:

Collagen-fibroin-chitosan/astragalus polyose engineering skin three-dimensional rack, the engineering skin three-dimensional rack include Collagen/silk fibroin bracket and astragalus polyose/chitosan microball, astragalus polyose/chitosan microball uniform load is in collagen/fibroin On albumen bracket, astragalus polyose/chitosan microball and collagen/silk fibroin bracket mass ratio are 1:1~1:20；The glue Original/silk fibroin bracket is made of collagen and fibroin albumen, the mass percent of collagen and fibroin albumen be 1:1~ 5:1；Astragalus polyose/the chitosan microball is made of astragalus polyose and chitosan, and astragalus polyose is encapsulated in chitosan, yellow The drugloading rate of astragalus polysaccharides is 10~30%, and the concentration of astragalus polyose is 0.1ug/ml-200ug/ml.

Preferably, astragalus polyose/the chitosan microball and collagen/silk fibroin bracket mass ratio be 1:5~ 1:10。

Preferably, astragalus polyose/chitosan microball average grain diameter is 20~50 μm, collagen/fibroin albumen The porosity of bracket is 85~95%.

Preferably, the mass percent of the collagen and fibroin albumen is 2:1~3:1, the Radix Astragali is more The drugloading rate of sugar is 15~25%.

Due to the adoption of the above technical solution, the three-dimensional structure constructed using collagen-fibroin albumen is matrix system to the present invention Standby organization bracket, recombination chitosan wraps up traditional Chinese medicine monomer astragalus polyose slow-released system, come the new bio composite skin material constructed Material.The engineering skin three-dimensional rack have the advantages that it is following uniqueness: 1, COL-SF timbering material have good biocompatibility, Biodegradability and suitable surface chemical structure, no overt toxicity will not cause intradermal immunization rejection, have good Good biological safety, and explanation is conducive to tissue inner cell and grows into bracket within the regular hour, meets its as a group weaver The primary condition of journey skin will greatly improve clinical treatment efficiency；2, the COL-SF bracket for being sustained APS is big in preparation process and SD Its activity is still kept in mouse body, can significantly promote vascularization and regeneration in COL-SF bracket；3, the COL-SF tri- prepared Dimensional scaffold has interconnected three-dimensional porous structure, can be the migration of tissue inner cell, growth, adherent, proliferation, nutriment Transhipment with metabolite provides convenience；4, zoografting is postoperative can form early stage neovasculature, must solve to move by fine Skin of making skin graft is easy problem downright bad, that survival rate is low；5, relative to current organization engineering skin product, this novel skin composite wood Material will greatly restore and the anatomical structure and physiological function of regeneration normal skin tissue.

Engineering skin three-dimensional rack of the present invention has good biocompatibility, can promote neoplastic skin blood vessel network again It is raw, the speed and efficiency of neoplastic skin reparation are improved, enhances the bioactivity of reconstruction, is conducive to the weight of neoplastic skin tissue Modeling is the treatment of large skin defect caused by mechanical damage, body surface tumor resection, traffic accident and building accident etc., mentions It is orthopaedic trauma, burns unit and Reconstructive surgery to big for continually and efficiently novel tissue engineering skin source The treatment of areas of skin defect opens a kind of brand-new approach for combining organization engineering skin and traditional Chinese medical theory.

Detailed description of the invention

Fig. 1 is influence of the APS to HUVEC cell Proliferation.Cell is analyzed with mtt assay after cultivating 12,24,36,48 and 72h Proliferation.

Fig. 2 is influence of the APS to HUVEC cell migration.A: control group；B:0.1 μ g/mL APS；C:1 μ g/mL；D:10 μ g/mL；E:50 μ g/mL；F:100 μ g/mL；G:200 μ g/mL；H:20ng/mL VEGF.

Fig. 3 is that flow cytomery HUVEC is processed correspondingly the cell cycle after 24 hours.A: control group；B:0.1 μ g/mL APS；C:1 μ g/mL；D:10 μ g/mL；E:50 μ g/mL；F:100 μ g/mL；G:200 μ g/mL；H:20ng/mL VEGF.

Fig. 4 is that flow cytomery HUVEC is processed correspondingly the Apoptosis after 24 hours.A: control group；B:0.1 μ g/mL APS；C:1 μ g/mL APS；D:10 μ g/mL APS；E:50 μ g/mL APS；F:100 μ g/mL APS；G:200 μ g/mL APS；H:20ng/mL VEGF.

Fig. 5 is flow cytomery natural death of cerebral cells rate figure in Fig. 4.

Fig. 6 is that fluorescence inverted microscope detects the vegf expression (200X) after HUVEC is processed correspondingly 24 hours.A: control Group；B:0.1 μ g/mL APS；C:1 μ g/mL APS；D:10 μ g/mL APS；E:50 μ g/mL APS；F:100 μ g/mL APS；G: 200μg/mL APS；H:20ng/mL VEGF.

Fig. 7 is astragalus polyose/chitosan microball light microscopic observation result.

Fig. 8 is to observe result under astragalus polyose/chitosan microball scanning electron microscope.

Fig. 9 is the external elution profiles of astragalus polyose/chitosan microball.

Figure 10 is to observe result under compound rest scanning electron microscope.

Specific embodiment

The influence that 1 astragalus polyose of embodiment is proliferated huvec

1. experiment purpose

(1) astragalus polyose (APS) influence to Human umbilical vein endothelial cells (HUVEC) cell cycle in vitro is studied.

(2) influence of the external Endothelial Cells Growth factor (VEGF) expression of research astragalus polyose (APS).

2. experiment reagent and instrument

2.1 experiment reagent

Reagent needed for testing is shown in Table 1-1.

Table 1-1 experiment reagent

2.2 laboratory apparatus

Reagent needed for testing is shown in Table 1-2.

Table 1-2 laboratory apparatus

3. experimental procedure

3.1 mtt assay detect cell Proliferation

HUVEC is washed through PBS, and cell suspension is made with the M200 culture medium containing 0.5%FBS in digestion.Through cell count, By every hole 5x103/150 μ L inoculation and 96 orifice plates.Full culture medium discards culture solution after culture 24 hours, changes and contains only 0.5% The culture medium of FBS pre-processes 24 hours, to reach cell synchronization.It is separately added into control group, experimental group and positive control tissue culture Support base.After continuing routine culture 12,24,36,48,72 hours, 50 μ L of MTT solution is added, is measured respectively using microplate reader by mtt assay The absorbance (0D value) of hole 490nm and 690nm.Every group sets 3 multiple holes, at least tests in triplicate.

Detect cell migration in 3.2 cells Transwell

It is detected using the cell transwell transfer method.The cell Transwell is 8.0 μm of films.HUVEC is washed through PBS, is disappeared Change, cell suspension is made with the M200 culture medium containing 0.5%FBS.After blood counting chamber counts, the density for adjusting HUVEC is 1x105/ml；Take 200 μ L HUVEC cells that 24 orifice plate transwell upper chambers are added, lower room is separately added into 600 μ L blank groups, real Test group and positive controls.3 multiple holes of every group of setting.Culture plate is put into the incubator of 37 DEG C of 5%C02 and is incubated for for 24 hours.Training After supporting, take out upper layer cell, wipe small indoor cell away with cotton swab, after with the fixed cell 10min of 20% ethyl alcohol.PBS is washed Remove loose cell.With cell 20min, PBS washing 3 times of 0.1% lower surface violet staining Transwell.In 100x Each hole randomly selects 5 visuals field under microscope, counts pigmented cells.It is analyzed with Image ProPus software.3.3 cell Cycle detection

Using flow cytomery.HUVEC washes overflow through PBS, and digestion is made carefully with the M200 culture medium containing 0.5%FBS Born of the same parents' suspension.Through cell count, 6 orifice plates are inoculated in by every hole 1x105/2ml.Full culture medium discards culture solution after culture 24 hours, Culture medium pretreatment 24 hours for containing only 0.5%FBS are changed, to reach cell synchronization.It is separately added into 2ml control group, it is real Test group and positive controls culture medium.After continuing routine culture 24 hours, cell is collected by centrifugation, PBS is washed 2 times, is abandoned supernatant, is added Enter 70% ethyl alcohol of 1ml pre-cooling, cell piping and druming is fixed at single cell suspension, 4 DEG C of storages.Cell 2 times of PBS washing fixation, 2000rpm is centrifuged 5min, n (final concentration of 20 μ g/ml) and RNaseA (final concentration of 50 μ g/ml) is added, 37 DEG C are protected from light dyeing 30min mixes cell before upper machine, crosses 200 mesh nylon wires.Upper machine testing in 30 minutes.Using the stream of U.S. company BD production Formula cell instrument carries out DNA measurement, and excitation wavelength 480mn detects cell cycle distribution situation.It is provided using BD company corresponding soft Part program carries out data processing.

The detection of 3.4 Apoptosis

Using flow cytomery.HUVEC is washed through PBS, and digestion is made carefully with the M200 culture medium containing 0.5%FBS Born of the same parents' suspension.Through cell count, by every hole 1x 105/2ml inoculation and 6 orifice plates.Full culture medium discards culture solution after culture 24 hours, Culture medium pretreatment 24 hours for containing only 0.5%FBS are changed, to reach cell synchronization.It is separately added into 2ml control group, it is real Test group and positive controls culture medium.After continuing routine culture 24 hours, cell is collected by centrifugation, PBS is washed 2 times, collects each group 1x106, cell in centrifuge tube.Cell is resuspended in 500 μ L Buffer liquid, 5 μ L PI and 5 μ L are separately added into AnnexinV-FITC, mixing is gone in streaming pipe for detecting after room temperature is protected from light incubation 30min.

3.6 immunofluorescent staining

HUVEC is washed through PBS, and cell suspension is made with the M200 culture medium containing 0.5%FBS in digestion.Through cell count, By every hole 1x105/2ml inoculation and 6 orifice plates.Full culture medium discards culture solution after culture 24 hours, changes and contains only 0.5%FBS Culture medium pre-process 24 hours, to reach cell synchronization.It is separately added into 2ml control group, experimental group and positive control tissue culture Support base.After continuing routine culture 24 hours, PBS is cleaned 3 times.4% poly formic acid fixes cell 60min, and PBS is cleaned 3 times.It is added dropwise Rabbit VEGF monoclonal antibody (1:100 dilution) directly is added dropwise after closing 30min in 5% Normal Goat Serum, and room temperature is incubated for 90min.The goat antirabbit fluorescence secondary antibody (1:100 dilution) of FITC label is added dropwise in PBS after cleaning 3 times, room temperature is protected from light incubation 60min.PBS is cleaned 3 times, and DAPI is added dropwise and marks nucleus, room temperature, which is protected from light, is incubated for 10min.PBS is cleaned 3 times, direct fluorescence microscopy The number of sem observation cell green fluorescence.DAPI marks nucleus, and fluorescence microscope is blue.

4. experimental result

Influence of 4.1 APS to HUVEC cell Proliferation

Mtt assay shows that APS can promote huvec cell Proliferation in a certain range (0.1-100ug/ml), in dosage according to Lai Xing.APS effect 24,48, after 72h, which is above control group, difference it is statistically significant (P < 0.05, P<0.01).Act on the most obvious after 72h, 100 μ g/ml APS groups are compared with blank control group cell proliferation rate up to 158.80%.

As shown in Figure 1, influence of the APS to HUVEC cell Proliferation.With mtt assay point after cultivating 12,24,36,48 and 72h Analyse the proliferation of cell.

The effect that 4.2 APS migrate HUVEC

As shown in Fig. 2, compared with the control group, APS is directly proportional to concentration to migration to HUVEC, and in 100 μ g/mL Shi Zuoyong reaches highest.200 μ g/mL APS reduce the migration of HUVEC compared with 100 μ g/mL.Control group is compared, and APS exists HUVEC mobility is respectively increased when 0.1 μ g/ml, 1 μ g/ml, 10 μ g/ml, 50 μ g/ml, 100 μ g/mL, 200 μ g/mL 12.092%, 39.328%, 57.835%, 72.253%, 112.875%, 76.923%.

Influence of 4.3 APS to the HUVEC cell cycle

APS group, VEGF group can make HUVEC phase cell cycle G2/M and S phase ratio as the result is shown for fluidic cell cycle detection Example increases.Blank control group G0/G1 phase, G2/M phase and S phase are respectively 50.67%, 23.49%, 10.21%.With APS concentration Increase, the ratio of G2/M phase and S phase increase, and when 10ug/ml is statistically significant compared with blank control group.APS concentration exists Reach highest when 100ug/ML, G2/M phase and S phase are respectively 40.67% and 13.01%.After when VEGF group effect small 24, G0/G1 Phase, G2/M phase and S phase are respectively 32.87%, 40.67% and 15.59%, and effect has statistical difference compared with blank control group. As shown in figure 3, showing to change with the inducing cell cell cycle to G2/M phase and S phase after handling cell.

4.4 detection natural death of cerebral cells

Flow cytomery natural death of cerebral cells rate is as shown in Figure 5.Q2 after blank control group, APS group and the effect for 24 hours of VEGF group Phase and Q3 phase tune are died rate and are not substantially change.As shown in figure 4, showing APS to HUVEC without apparent toxic effect.

4.5 immunofluorescent staining

EGF is that HUVEC cell-specific promotees growth factor, can promote HUVEC cell and forms rete vasculosum [3].Such as Fig. 6 institute Show, blank control group HUVEC cell only a small amount of VEGF expression.APS, VEGF group can stimulate the expression of HUVEC cell more VEGF.With the increase of APS concentration, vegf expression amount increases, and the vegf expression amount highest in 100 μ g/mL, is blank control Times of group expression quantity, there is significant statistical difference.100 μ g/mL APS increase compared with blank group vegf expression, than VEGF group expression quantity It is slightly lower, it is the 88.9% of VEGF group vegf expression amount.If Fig. 6 shows that APS can promote HUVEC cell VEGF expression, this for HUVEC cell early proposes vascularization and is even more important.

5. conclusion

Experiment in vitro shows that this astragalus polyose/chitosan microball obtained can more steadily discharge drug in 48h, And phenomenon of burst release is not present.This experiment discovery APS has the function of promoting HUVEC proliferation within the scope of a certain concentration, promote The HUVEC cell cycle changes from the G0/G1 phase to G2/M phase and S phase, and and dose proportional, reach most in 100 μ g/mL It is high.APS effect for 24 hours after, 100 μ g/mL groups and VEGF group G2/M phase and S phase be respectively 40.67% and 13.01% and 40.67% and 15.59%, and blank control group G2/M phase and S phase are respectively 23.49% and 10.21%.And after acting on for 24 hours, stream Formula Apoptosis detection display APS does not increase HUVEC apoptosis rate, APS promote endothelial cell proliferation and vascularization without Generate side effect.Immunofluorescent staining the experimental results showed that, APS effect for 24 hours after, the vegf expression of HUVEC also with APS agent Amount is positively correlated, and the vegf expression of 100 μ g/mL APS promotion HUVEC compares blank control group and is significantly improved.200μg/mL When APS effect slightly decline compared with 100 μ g/mL, this phenomenon may be due to the slight cell that occurs when APS concentration is 200 μ g/mL Toxicity.

The above result of study confirms that APS has good promotion HUVEC cell Proliferation, increases G2/M in the HUVEC cell cycle The ratio of phase and S phase.APS can raise the expression of HUVEC cell VEGE simultaneously, can further promote the generation of new vessels.

Embodiment 2 loads astragalus polyose/chitosan microball collagen/silk fibroin bracket preparation and quality evaluation

1. experiment purpose

(3) astragalus polyose/chitosan microball is prepared using emulsification-cross-linking method, and optimizes preparation method；

(4) building load astragalus polyose/chitosan microball collagen/silk fibroin bracket.

2. experiment reagent and instrument

2.3 experiment reagent

Reagent needed for testing is shown in Table 2-1.

Table 2-1 experiment reagent

2.4 laboratory apparatus

Reagent needed for testing is shown in Table 2-2.

Table 2-2 laboratory apparatus

3. experimental procedure

3.5 astragalus polyoses/chitosan microball preparation

Astragalus polyose/chitosan microball is prepared using emulsification-cross-linking method, the specific steps are as follows:

(1) it accurately weighs 2g chitosan solid, under 50 DEG C of water bath conditions, adds in the glacial acetic acid solution of 100mL 2%, 40rpm stirring, is completely dissolved chitosan, obtains chitosan-glacial acetic acid solution.

(2) 1g astragalus polyose accurately being weighed, added in the chitosan-acetic acid solution prepared, stirring makes it completely dissolved, Then 20mL mixed solution is measured.

(3) atoleine of the 90mL containing 2mLSpan-80 is added in beaker as oily phase, by chitosan mixed solution by It is added dropwise to oily phase, under 50 DEG C of water bath conditions, 40rpm stirring 30min emulsifies it and maintains 20min.

(4) after to be emulsified, the glutaraldehyde of 2mL 25% is added into solution.Under 50 DEG C of water bath conditions, 40rpm stirring After 3h, reactant is transferred to centrifuge tube, stands centrifugation, abandon upper liquid, petroleum ether precipitates 3 times.

(5) mixture washed is filtered by vacuum, while is eluted with isopropanol, it is contemplated that obtain crocus solid powder End, as astragalus polyose/chitosan microball.It will take out, be placed on clean surface plate in drug bearing microsphere, 50 DEG C of constant temperature oven Middle drying is for 24 hours.

3.6 load astragalus polyose/chitosan microball collagen/silk fibroin bracket preparations

Using solution casting-Freeze Drying Technique, specific preparation process is as follows:

(1) collagen and fibroin albumen are dissolved in respectively in the distilled water of certain volume, are made into 3.5% collagen Aqueous solution and 1.5% silk fibroin water solution in.

(2) collagen aqueous solution is mixed with silk fibroin water solution according to solute albumen quality ratio 7:3, will be being prepared Good astragalus polyose/chitosan microball is added in mixed liquor by a certain amount, and mixing is sufficiently stirred.

(3) the above-mentioned mixed solution containing microballoon is added in set mould container, -20 DEG C of pre-freeze moldings, so - 60 DEG C of vacuum drying 48h to 72h afterwards, the bracket of acquisition are cylinder, and it is highly 0.50cm, each that bottom surface radius, which is 0.70cm, Bracket contains astragalus polyose/chitosan microball 0.1g.

4. performance test

The observation of 4.1 pattern partial sizes

Using ion sputtering instrument to astragalus polyose/chitosan microball and load astragalus polyose/chitosan microball collagen/silk Fibroin bracket is conductive processing, plating current 10mA, time 40s.Then, application scanning Electronic Speculum is to the sample handled well Surface topography observation is carried out, and by image measurement and analysis, calculates microsphere average grain diameter.

4.2 carrying drug ratios and encapsulation rate

Using Anthrone Sulphuric acid Colorimetry astragalus polyose/chitosan microball carrying drug ratio and encapsulation rate.Firstly, weighing anthracene Ketone 0.2g is added in the 100mL concentrated sulfuric acid, and dissolution is sufficiently stirred, obtains anthrone reagent.Then, 6 test tube with ground stoppers are taken, precision is matched Make a series of glucose solutions, concentration is followed successively by 0,10,20,30,40,50 μ g/mL, every test tube 1mL, and each concentration is done 3 times It repeats.Then, anthrone reagent 4mL is added into every test tube rapidly, after oscillation mixes, respectively manages while being placed in boiling water and heat 10min.It takes out, immerses in ice water rapidly and cool down and place 10min.Using 0 μ g/mL pipe as blank, uv-spectrophotometric is used respectively The measurement absorbance A at 620nm is counted, and is abscissa, absorbance A as ordinate using concentration (μ g/mL), draws standard curve.

It after taking a certain amount of astragalus polyose/chitosan microball to be fully ground in mortar, moves in beaker, distilled water is added Sufficiently dissolution astragalus polyose (can ultrasonic dissolution assisting), centrifuging and taking supernatant, after distilled water constant volume, the measurement absorbance A in 620nm at, and Astragalus polyose concentration is calculated according to standard curve.The calculation formula of microballoon carrying drug ratio and encapsulation rate is as follows:

Carrying drug ratio=(dose/microballoon gross mass in microballoon) × 100%

Entrapment efficiency=(the total dose of dose/addition in microballoon) × 100%

The outer release behavior of 4.3 microspheres

A certain amount of Radix Astragali chitosan microball is accurately weighed in the phosphate buffer of pH 7.4, release in aids drug body Condition, setting revolving speed are 200rpm, and temperature is 37 DEG C ± 5 DEG C.Respectively at 0,0.5,1,1.5,2,2.5,3,3.5,4,4.5,5, 5.5,6,8,10,12,20,25,30,48h respectively samples 5.0mL, and supplements 5.0mL fresh medium solution.Then, using ultraviolet point Light photometer measures sample absorbance A at 620nm, and releases according to standard curve calculating astragalus polyose concentration and drug are accumulative Rate is put, formula is as follows:

Accumulative release rate=drug release total amount/(drug bearing microsphere weight × carrying drug ratio) × 100%

4.4 porosity measurement

Brace aperture rate is measured using medium infusion method.The bottom surface radius (r) and height (h) of sample are measured first, and Calculate bracket volume.Then, claim the aerial quality of drying sample is denoted as m1, by bracket immerse atoleine (density After it is saturated (can pass through negative pressure degasification), to further take out sample in ρ), gently wiping the medium of sample surfaces.Sample is weighed again Quality is denoted as m2, and is calculated from the formula porosity:

Porosity (%)=(m2-m1)/ρ/(h π r^2) × 100%

5. experimental result

5.1 astragalus polyoses/chitosan microball drugloading rate and encapsulation rate

(1) standard curve

Respectively with drug concentration (C) and absorbance (A) for abscissa and ordinate, astragalus polyose dissipating in the solution is drawn Point diagram and linear fit curve, and calculate that linear fit equation is Y=0.0056X-0.0007, R²At=0.9996,620nm, Astragalus polyose linear relationship in the 10 μ g/ml concentration ranges of μ g/ml~50 is good.

(2) sample encapsulation rate and carrying drug ratio

According to the absorbance of the drug bearing microsphere measured in the solution, linear fit equation is substituted into, calculates drug concentration, often A test point is measured in parallel 3 times, show that astragalus polyose/chitosan microball carrying drug ratio is 13.7%, encapsulation rate 70.6%.

5.2 astragalus polyoses/chitosan microball light microscopic and scanning electron microscope result

(1) observed result under light microscopic

Astragalus polyose/chitosan microball appearance is uniformly dispersed in crocus powder, granule, no adhesion.Microballoon under microscope In crocus spherical shape, microballoon size is shown in Fig. 7 than more uniform.

(2) observed result under Scanning Electron microscope

The spherical morphology of chitosan drug-loading microballoon is preferable, microsphere average grain diameter be 35 μm, dispersibility preferably, a small amount of microballoon it Between have adhesion phenomenon, see Fig. 8.

The external releasing result of 5.3 astragalus polyoses/chitosan microball

Radix Astragali/chitosan microball of preparation is placed in the PBS buffer solution of pH 7.4 and tests its external sustained release property, institute It is as shown in Figure 9 to obtain elution profiles.It can be seen that the astragalus polyose burst size in 2h from the cumulative release time graph of drug bearing microsphere Reaching 50%, astragalus polyose burst size reaches 70% in 8h, illustrate that drug bearing microsphere rate of release early period is slightly fast, but without prominent Release phenomenon appearance.Drug bearing microsphere rate of release slows down after 10h, and slow release effect is obvious, and astragalus polyose cumulative release connects after 48h Nearly 100%, it is basically completed release.Entire sustained release process, accumulation elution profiles are gradually increasing, and drug bearing microsphere has apparent slow Release effect.

5.4 load astragalus polyose/chitosan microball collagen/silk fibroin bracket analysis of porosity

It is homogeneous to load astragalus polyose/chitosan microball collagen/silk fibroin bracket.According to being measured in parallel three times Result known to (table 2-3), load astragalus polyose/chitosan microball collagen/silk fibroin bracket porosity be 90.8%, It is relatively reasonable, be conducive to the absorption of cytotrophy substance and the discharge of waste.

Table 2-3 compound rest analysis of porosity

5.5

5.6 load astragalus polyose/chitosan microball collagen/silk fibroin bracket Electronic Speculum results

Tissue engineering bracket surface micro-structure (size in such as hole, distribution, hole form and porosity) influence cell Stick, be proliferated and break up.It is shown according to surface sweeping Electronic Speculum result (Figure 10), loads astragalus polyose/chitosan microball collagen/silk Fibroin brace aperture is larger, and form is good, and microballoon is evenly distributed wherein.

6. conclusion

(1) it is poly- to prepare carrying drug ratio 13.7%, astragalus polyose/shell of encapsulation rate 70.6% for repeated optimum experimental processing Sugared microballoon, average grain diameter are 35 μm.It is observed under light microscopic and scanning electron microscope, astragalus polyose/chitosan microball form is preferable, dispersion Property is preferable, has adhesion phenomenon between only a small amount of microballoon.

(2) experiment in vitro is shown, this astragalus polyose/chitosan microball obtained can more steadily discharge medicine in 48h Object, and phenomenon of burst release is not present.

(3) load astragalus polyose/chitosan microball collagen/silk fibroin bracket is homogeneous, porosity 90.8%. Scanning Electron microscopically observation, compound rest hole is larger, and form is good, and microballoon is evenly distributed wherein, is conducive to Radix Astragali Polysaccharide is also beneficial to the material exchange and waste discharge of cell in the intracorporal slowly long-acting stable release of people.

Embodiment 3 prepares collagen-fibroin-chitosan (COL-SF-CTS) three-dimensional rack

1, the extraction and purification of type i collagen

Fresh ox abdomen is taken, muscle, fat and fascia tri-distilled water repeated flushing are rejected.The Niu Jian handled in right amount is taken to cut At fragment, 2h is impregnated with 0.1% Na2C03, then rinsed 5 times with tri-distilled water.Ox key fragment is moved into mortar, pours into appropriate liquid Nitrogen sufficiently freezes, and is then broken into beef tendon powder with the even poly- machine of tissue, saved backup at -20 DEG C.Appropriate ox key powder is taken, is added Enter 0.5% acetic acid solution and add sufficiently be swollen, digested more than for 24 hours at 4 DEG C of 1% trypsin solution, a few drop H2O2 are then added, fill Divide after mixing and stands 10-15min.3% acetic acid 1000ml is added, after mixing well, filtered on buchner funnel, the beef tendon that will be swollen Particle filtering falls.Swelling object is centrifuged (2000rpm/min, 15min) in centrifuge to remove insoluble matter.It is molten with 5%NaCI afterwards Salt solution analysis, centrifugation (2000rpm/min, 5min), collect precipitating.Sediment is sufficiently swollen in 3% acetic acid, is not swollen beef tendon Above-mentioned steps after grain is swollen again.Dialyse in tri-distilled water 72h at 4 DEG C, and it is primary that every 12h changes water.Collagen after dialysis, chilled freeze-drying It is saved backup at 4 DEG C afterwards.

2, prepared by chitosan microball (CTS)

(1) 10g chitosan, which is dissolved in 100ml distilled water, is configured to 100ml gelatin solution (concentration 10%), fast at 95 DEG C Soybean oil is placed in water-bath preheats therebetween by fast uniform stirring until dissolution.

(2) 100ml beaker is put on magnetic stirring apparatus, inside puts magneton, 100ml soybean oil is first poured into beaker, then pour into 5ml Chitosan solution mixes the two, 45 DEG C of stirring 10min, and mixing speed is as maximum as possible.

(3) cooling of reactant ice-water bath (is prepared 500ml beaker, is placed on blender, inside puts ice water and magnetic by stirring simultaneously Gelatin and soybean oil blend are poured slowly into cooling by son).Chitosan is cooling to be condensed.

(4) separatory funnel liquid separation removes lower layer's water phase, is added and precools acetone, sufficiently washs.

(5) reactant is poured into beaker, three times with acetone washing, removes remaining soybean oil.

(6) 0.5% glutaric acid solution, crosslinking curing 2h is added.

(7) add suitable glycine, to remove unreacted crosslinking agent, products obtained therefrom centrifuge is at 3000r/min Centrifugation, sample filter, and three times, naturally dry, obtain lurid powdered product is chitosan microball to acetone washing.

(8) astragalus polyose solution or VEGF solution of dosage needed for testing directly are added dropwise to the dry chitosan of 2mg It is micro- to be coated in chitosan to APS after natural drying and VEGF for microballoon (liquor capacity is in the expansion ratio range of gelatine microsphere) In ball, to be used for subsequent experimental.

3, collagen-fibroin albumen (COL-SF) three-dimensional rack and collagen-fibroin-chitosan microballoon (COL-SF-CTS) Compound rest preparation

Collagen, fibroin albumen are configured to the swelling solution of 0.5% (w/v), collagen and chitosan respectively with the acetic acid of 0.5M The mass ratio of swelling solution 9:1 by volume is blended, and searches to mix and uniformly obtains collagen/fibroin albumen mixed liquor, in 4 after vacuum defoamation It DEG C saves backup.Prepare need before collagen/fibroin-chitosan microballoon compound rest by collagen/fibroin albumen mixed liquor with it is upper 2mg obtained by step includes the chitosan microball (chitosan microball amount in a compound rest) of required APS or VEGF dosage Mixing.By the collagen of preparation/fibroin albumen mixed liquor or prepare in collagen/fibroin-chitosan microballoon mixed liquor dropwise The glutaric acid aqueous solution of 0.5% (w/v) of addition, normal temperature crosslinked 2 hours.After be uniformly injected into mold, -70 DEG C of freeze overnight postpositions are frozen It is lyophilized in dry machine and obtains collagen/fibroin albumen three-dimensional rack or collagen/fibroin-chitosan microballoon compound rest for 24 hours.

Claims

1. collagen-fibroin-chitosan/astragalus polyose engineering skin three-dimensional rack, which is characterized in that the engineering skin is three-dimensional Bracket includes collagen/silk fibroin bracket and astragalus polyose/chitosan microball, and astragalus polyose/chitosan microball uniform load exists In collagen/silk fibroin bracket, astragalus polyose/chitosan microball and collagen/silk fibroin bracket mass ratio are 1:1 ~ 1:20； Collagen/the silk fibroin bracket is made of collagen and fibroin albumen, the quality percentage of collagen and fibroin albumen Than for 1:1 ~ 5:1；Astragalus polyose/the chitosan microball is made of astragalus polyose and chitosan, and astragalus polyose is encapsulated in shell In glycan, the drugloading rate of astragalus polyose is 10 ~ 30%, and the concentration of astragalus polyose is 0.1ug/ml-200ug/ml.

2. collagen-fibroin-chitosan according to claim 1/astragalus polyose engineering skin three-dimensional rack, feature It is, astragalus polyose/chitosan microball and collagen/silk fibroin bracket mass ratio are 1:5 ~ 1:10.

3. collagen-fibroin-chitosan according to claim 1/astragalus polyose engineering skin three-dimensional rack, feature Be, astragalus polyose/chitosan microball average grain diameter be 20 ~ 50 μm, collagen/silk fibroin bracket porosity be 85 ~ 95%。

4. collagen-fibroin-chitosan according to claim 1/astragalus polyose engineering skin three-dimensional rack, feature It is, the mass percent of collagen and fibroin albumen is 2:1 ~ 3:1, and the drugloading rate of the astragalus polyose is 15 ~ 25%.