CN105412986B - Small intestinal submucosa carries piece and its preparation method and application of building up one's health by taking tonic - Google Patents
Small intestinal submucosa carries piece and its preparation method and application of building up one's health by taking tonic Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3629—Intestinal tissue, e.g. small intestinal submucosa
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
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- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/602—Type of release, e.g. controlled, sustained, slow
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Abstract
The invention discloses a kind of small intestinal submucosas to carry piece of building up one's health by taking tonic, its microstructure includes SIS collagenous fibres skeleton and loads on the icariin on SIS collagenous fibres skeletons, SIS collagenous fibres skeleton is reticular fiber structure, icariin is graininess, icariin is attached on SIS collagenous fibres skeletons, and the adhesion amount of icariin is 20 ~ 80 μ g/cm2；Small intestinal submucosa of the present invention carries piece of building up one's health by taking tonic, the icariin of attaching particles shape on SIS collagenous fibres skeletons, establish stable drug delivery system, the bone tissue engineer element of SIS collagenous fibres skeleton and icariin the two great potentials is combined, it is reliable that the small intestinal submucosa of formation carries piece composite material of building up one's health by taking tonic, stablize, to icariin safety, it is economical, chemical property is stable and the characteristics such as excellent promoting bone regeneration ability and rush revascularization ability are made full use of, small intestinal submucosa load builds up one's health by taking tonic piece with extremely strong promoting bone regeneration ability, preparation method is simple, it is with a wide range of applications in osteanagenesis field.
Description
Technical field
The invention belongs to drug release systems technology fields in biomedical engineering, and in particular to a kind of small intestinal submucosa
Load is built up one's health by taking tonic piece and its preparation method and application.
Background technology
China is every year there are about 3,500,000 people because bone defect, osseous surgery transplanting about 1,500,000, to substituting occur in different reasons
The demand of bone is very big, the replacement bone of especially big skeletal injury.With the fast development of tissue engineering technique, structure in vitro
It is the important channel for solving this demand to build the artificial replacement bone with osteo-conductive and osteoinductive.Framework material is in group
It knits and is played a crucial role in Engineering Bone structure.Compared to other synthetic materials, natural extracellular matrix (ECM) material by
In its following characteristics：1. excellent biocompatibility；2. closest to the three-dimensional structure of internal cell growth microenvironment；3. it is thin
Born of the same parents' vital movement provides various bioactive molecules, is considered as ideal tissue engineering material always.Intestinal mucosa lower floor
(SIS) be current most study natural extracellular matrix material, ratified by U.S. FDA, be widely used for building artificial
The soft tissues such as bladder, artificial urinary canal, artificial esophagus, artificial blood vessel and artificial skin.SIS material framework ingredient collagens
(collagen) it has been widely used for being used for bone tissue engineer as framework material and pharmaceutical carrier.SIS has compared to collagen
Two big advantages：1.SIS is a natural drug reservoir, it remains many bioactive molecules for being conducive to osteanagenesis；2.SIS exists
More convenient in materials, cost will also be far below collagen.Domestic external application SIS materials are reported as bone tissue engineer framework material
Have, it can not further genralrlization use but be allowed to there are one problem：The deficiency of osteoinductive.By being loaded on SIS
With the drug of osteanagenesis is promoted, and enable to realize slow release, this defect can be made up.If this can be solved
Problem, SIS materials will become ideal engineering material of bone tissue.
The drug of required promotion osteanagenesis is always a research hotspot in bone tissue engineer, although BMP-2 albumen
It is the most effective Osteoinductive Factor for being recognized and using extensively at present, but since its is very expensive, and use BMP genes
Introduction method is again there are certain safety problem, therefore its Clinical practice also suffers from the restriction of cost and safety.In addition,
BMP-2 and other protein drugs all have that stability is poor, half-life short in vivo, accordingly for drug delivery system
It is it is required that very high.Therefore find low cost, stablize again can the micromolecular compound of efficient bone induction and regeneration substitute BMP right and wrong
It is often significant.The drug for the treatment of osteoporosis mainly realizes the balance of bone metabolism by inhibiting bone information at present, but logical
The compound for crossing promotion osteoblast differentiation and osteanagenesis is considerably less, and very limited report includes：Statins, isoflavones
Derivative, TAK-778 and match jerusalem artichoke flavin derivatives etc..With the development of modern separation technology, extracted from natural products each
Kind compound, difficulty and cost will be well below traditional chemical syntheses.Rarity of the Chinese medicine as Chinese name race, describes big
Treatment of the Chinese herbal medicine for various diseases is measured, validity also increasingly obtains worldwide accreditation.Autonomic drug Herba Epimedii
It is considered to have the effect of strengthening the muscles and bones, function and mechanism of the main active icariin in bone metabolism all the time
Just start within several years gradually to be studied up to date.Chen Ke-Ming et al. have found that rat bone marrow mesenchymal stem cells pass through for the first time
After icariin processing, cell Proliferation accelerates, and breaking up marker ALP, OCN and mineralizing has increase, shows icariin
May have the function of to promote bone metabolism.Our research finds that icariin can be by raising Runx2 expression and activation BMP
Signal path promotes the differentiation of osteoblast；Hsieh et al. it has also been found that icariin by activate BMP and NO signal paths come
Promote osteoblast differentiation, while the gene expressions such as BMP-2, Smad4, Runx2 and OPG are raised.The research of Chung etc. finds excessive
Sheep leaves of pulse plants glycosides can activate MEK/ERK the and PI3K/Akt/eNOS signal paths in endothelial cell to accelerate angiogenesis；In mouse
In critical bone injury scale-model investigation, it has been found that icariin-calcium-phosphate cement composite material can after 6 weeks inducing mouse
Internal cranium injury region forms the area of new bone with blood vessel.Icariin be it is a kind of have very much potential can be used for bone group
The drug of weaver's journey.But there has been no correlation of the icariin as the slow releasing pharmaceutical for promoting osteanagenesis is loaded on SIS at present
Report.
Invention content
First technical problem to be solved by this invention is to provide a kind of small intestine with extremely strong promoting bone regeneration ability
Submucosa carries piece of building up one's health by taking tonic.
Second technical problem to be solved by this invention is to provide a kind of strong small intestinal submucosa of operability and carries medicine
The preparation method of sticking patch.
Third technical problem to be solved by this invention is to provide the application that a kind of small intestinal submucosa carries piece of building up one's health by taking tonic.
Technical solution is used by the present invention solves above-mentioned first technical problem：Small intestinal submucosa carries piece of building up one's health by taking tonic,
The build up one's health by taking tonic microstructure of piece of the load includes SIS collagenous fibres skeleton and loading on the icariin on SIS collagenous fibres skeletons, institute
The SIS collagenous fibres skeleton stated is reticular fiber structure, and the icariin is graininess, and the icariin is attached to
On the SIS collagenous fibres skeletons, the adhesion amount of the icariin is 20~80 μ g/cm2。
Small intestinal submucosa of the present invention carries piece of building up one's health by taking tonic, and the icariin of attaching particles shape, builds on SIS collagenous fibres skeletons
Stable drug delivery system is found, by the bone tissue engineer member of the two great potentials of SIS collagenous fibres skeleton and icariin
Element combines, and the small intestinal submucosa of formation load is built up one's health by taking tonic, and piece composite material is reliable, stablizes, to icariin safety, it is economical, change
It learns stable property and excellent promoting bone regeneration ability and promotees the characteristics such as revascularization ability and made full use of, under the small intestinal mucosa
Layer carries piece of building up one's health by taking tonic with extremely strong promoting bone regeneration ability, is with a wide range of applications in osteanagenesis field.
Technical solution is used by the present invention solves above-mentioned second technical problem：A kind of small intestinal submucosa load is built up one's health by taking tonic
The preparation method of piece, includes the following steps：
1) preparation of small intestinal submucosa sticking patch：In an aseptic environment, dry sterile SIS materials are taken out, are cut into institute
Need the small intestinal submucosa sticking patch of size, then the small intestinal submucosa sticking patch deionized water cut is infiltrated 12~make for 24 hours
Material fully soaks, and discards and infiltrates 1~2h with 50% ethanol solution after deionized water, is used instead successively after discarding ethanol solution
70%th, 80%, 90% ethanol solution and absolute ethyl alcohol infiltration 1~2h of sticking patch, are finally placed in nothing by small intestinal submucosa sticking patch
It is for use in water-ethanol；
2) preparation of icariin working solution：In an aseptic environment, powdery icariin is added in into dimethyl sulfoxide,
The icariin storing solution of a concentration of 50mg/mL is made in mixing concussion dissolving；The icariin storing solution of 1mL is taken, with anhydrous second
Alcohol mixes, and the icariin working solution of final concentration of 0.5~2.5mg/mL is made；
3) small intestinal submucosa carries the preparation for piece of building up one's health by taking tonic：In an aseptic environment, it is taken out under small intestinal mucosa from absolute ethyl alcohol
Layer sticking patch, according to 400 μ L/cm2Dosage by icariin working solution point several times uniform load to small intestinal submucosa sticking patch
On, each loading capacity is 100 μ L, and the time interval loaded twice is 30~50 minutes, until all icariin working solutions
It is all loaded onto on small intestinal submucosa sticking patch, obtains small intestinal submucosa and carry piece of building up one's health by taking tonic；By obtained load build up one's health by taking tonic piece be placed in it is logical
Load piece of building up one's health by taking tonic then is put into deionized water and rocks 24~48h at room temperature by wind 24~48h of room temperature condition, discard from
Load piece of building up one's health by taking tonic is put into PBS buffer solution after sub- water, concussion at room temperature is rocked, per discarding old PBS buffer solution for 24 hours and add in etc.
New PBS buffer solution is measured, piece of building up one's health by taking tonic will be carried after continuous 5 days and is removed and placed in new PBS buffer solution, it is standby in 2~6 DEG C of preservations
With.
In step 3), the mode that icariin working solution is loaded onto small intestinal submucosa sticking patch includes being added dropwise, spray, note
It penetrates and at least one of impregnates.
Technical solution is used by the present invention solves above-mentioned third technical problem：Above-mentioned small intestinal submucosa load is built up one's health by taking tonic
Application of the piece in osteanagenesis.
Compared with prior art, the advantage of the invention is that：Small intestinal submucosa of the present invention carries piece of building up one's health by taking tonic, in SIS collagens
The icariin of attaching particles shape on fiber reinforcement, establishes stable drug delivery system, by SIS collagenous fibres skeleton and excessive sheep
The bone tissue engineer element of the two great potentials of leaves of pulse plants glycosides combines, and the small intestinal submucosa of formation carries piece composite material of building up one's health by taking tonic
Reliably, stablize, to icariin safety, economy, chemical property stabilization and excellent promoting bone regeneration ability and promote revascularization energy
The characteristics such as power are made full use of, and small intestinal submucosa load builds up one's health by taking tonic piece with extremely strong promoting bone regeneration ability, preparation method letter
It is single, it is with a wide range of applications in osteanagenesis field.
Description of the drawings
Fig. 1 is the SEM pictures for the SIS collagenous fibres skeletons for not loading drug；
Fig. 2 is that the load of embodiment one is built up one's health by taking tonic the SEM pictures of piece；
Fig. 3 is the structure diagram that small intestinal submucosa of the present invention carries piece of building up one's health by taking tonic；
Fig. 4 is that the load of embodiment one is built up one's health by taking tonic the icariin release profiles of piece；
Fig. 5 is that the load of embodiment one is built up one's health by taking tonic piece In vitro biological activity verification result；
HE coloration results of the Fig. 6 for the blank control bone of no implantation material；
Fig. 7 is that the load of embodiment one is built up one's health by taking tonic the HE coloration results of piece group.
Specific embodiment
The present invention is described in further detail below in conjunction with attached drawing embodiment.
The small intestinal submucosa of embodiment one carries piece of building up one's health by taking tonic, and microstructure includes SIS collagenous fibres skeleton and loads on
Icariin on SIS collagenous fibres skeletons, SIS collagenous fibres skeleton be reticular fiber structure, icariin for graininess (
Granularity is 0.3~0.6 μm), icariin is attached on SIS collagenous fibres skeletons, and the adhesion amount of icariin is 33.2 ± 5.6
μg/cm2, preparation method includes the following steps：
1) preparation of small intestinal submucosa sticking patch：In an aseptic environment, dry sterile SIS materials are taken out, are cut into 1cm
The small intestinal submucosa sticking patch of × 1cm, then the small intestinal submucosa sticking patch deionized water cut is infiltrated 12~makes material for 24 hours
Material fully wetting discards ethanol solution 1~2h of infiltration with 50% after deionized water, is used instead successively after discarding ethanol solution
70%th, 80%, 90% ethanol solution and absolute ethyl alcohol infiltration 1~2h of sticking patch, are finally placed in nothing by small intestinal submucosa sticking patch
It is for use in water-ethanol；
2) preparation of icariin working solution：In an aseptic environment, powdery icariin is added in into dimethyl sulfoxide,
The icariin storing solution of a concentration of 50mg/mL is made in mixing concussion dissolving；The icariin storing solution of 1mL is taken, with anhydrous second
Alcohol mixes, and the icariin working solution of final concentration of 0.5mg/mL is made；
3) small intestinal submucosa carries the preparation for piece of building up one's health by taking tonic：In an aseptic environment, it is taken out under small intestinal mucosa from absolute ethyl alcohol
Layer sticking patch, by the 0.5mg/mL icariin working solution of 400 μ L in four times uniform load to small intestinal submucosa sticking patch, often
Secondary loading capacity is 100 μ L, and the time interval loaded twice is 30~50 minutes, until 400 μ L icariin working solutions are whole
It is loaded onto on small intestinal submucosa sticking patch, the small intestinal submucosa for obtaining embodiment one carries piece of building up one's health by taking tonic；Obtained load is built up one's health by taking tonic piece
Ventilation 24~48h of room temperature condition is placed in, then load piece of building up one's health by taking tonic is put into deionized water and rocks 24~48h at room temperature, is abandoned
It goes after deionized water load piece of building up one's health by taking tonic being put into PBS buffer solution, concussion at room temperature is rocked, per discarding old PBS buffer solution for 24 hours simultaneously
The new PBS buffer solution of equivalent is added in, piece of building up one's health by taking tonic will be carried after continuous 5 days and is removed and placed in new PBS buffer solution, in 2~6 DEG C of guarantors
It deposits spare.
The small intestinal submucosa of embodiment two carries piece of building up one's health by taking tonic, and microstructure includes SIS collagenous fibres skeleton and loads on
Icariin on SIS collagenous fibres skeletons, SIS collagenous fibres skeleton be reticular fiber structure, icariin for graininess (
Granularity is 0.3~5 μm), icariin is attached on SIS collagenous fibres skeletons, and the adhesion amount of icariin is 56.2 ± 8.8 μ
g/cm2, preparation method includes the following steps：
1) preparation of small intestinal submucosa sticking patch：In an aseptic environment, dry sterile SIS materials are taken out, are cut into 1cm
The small intestinal submucosa sticking patch of × 1cm, then the small intestinal submucosa sticking patch deionized water cut is infiltrated 12~makes material for 24 hours
Material fully wetting discards ethanol solution 1~2h of infiltration with 50% after deionized water, is used instead successively after discarding ethanol solution
70%th, 80%, 90% ethanol solution and absolute ethyl alcohol infiltration 1~2h of sticking patch, are finally placed in nothing by small intestinal submucosa sticking patch
It is for use in water-ethanol；
2) preparation of icariin working solution：In an aseptic environment, powdery icariin is added in into dimethyl sulfoxide,
The icariin storing solution of a concentration of 50mg/mL is made in mixing concussion dissolving；The icariin storing solution of 1mL is taken, with anhydrous second
Alcohol mixes, and the icariin working solution of final concentration of 1.5mg/mL is made；
3) small intestinal submucosa carries the preparation for piece of building up one's health by taking tonic：In an aseptic environment, it is taken out under small intestinal mucosa from absolute ethyl alcohol
Layer sticking patch, by the 1.5mg/mL icariin working solution of 400 μ L in four times uniform load to small intestinal submucosa sticking patch, often
Secondary loading capacity is 100 μ L, and the time interval loaded twice is 30~50 minutes, until 400 μ L icariin working solutions are whole
It is loaded onto on small intestinal submucosa sticking patch, the small intestinal submucosa for obtaining embodiment two carries piece of building up one's health by taking tonic；Obtained load is built up one's health by taking tonic piece
Ventilation 24~48h of room temperature condition is placed in, then load piece of building up one's health by taking tonic is put into deionized water and rocks 24~48h at room temperature, is abandoned
It goes after deionized water load piece of building up one's health by taking tonic being put into PBS buffer solution, concussion at room temperature is rocked, per discarding old PBS buffer solution for 24 hours simultaneously
The new PBS buffer solution of equivalent is added in, piece of building up one's health by taking tonic will be carried after continuous 5 days and is removed and placed in new PBS buffer solution, in 2~6 DEG C of guarantors
It deposits spare.
Piece of building up one's health by taking tonic is carried for the small intestinal submucosa of embodiment one, carries out following experiment detection and performance verification：
1st, microstructure
The small intestinal submucosa for the embodiment one being stored in PBS buffer solution is carried to piece of building up one's health by taking tonic to take out, is placed in ventilation room temperature
Condition, with scanning electron microscopic observation patch faces feature, as a comparison, does not load the SIS collagenous fibres of drug up to being completely dried
For skeleton as blank control group, SEM pictures are shown in Fig. 1 and Fig. 2, and Fig. 1 is the SEM pictures of blank control group sticking patch, and Fig. 2 is implements
The SEM pictures of the sticking patch of example one.Fig. 3 is that small intestinal submucosa of the present invention carries the structure diagram of piece of building up one's health by taking tonic, and in Fig. 3,1 is SIS
Collagenous fibres skeleton, 2 be granular icariin.From Fig. 2 and Fig. 3 as it can be seen that being attached on SIS collagenous fibres skeletons a large amount of
Drug molecule, i.e., granular icariin.
2nd, the In-vitro release curves of icariin SIS sticking patch
The small intestinal submucosa for the embodiment one being stored in PBS buffer solution is carried into piece of building up one's health by taking tonic and is removed and placed in 24 orifice plates,
The 400 fresh PBS buffer solution of μ L are added in, concussion at room temperature is rocked for 24 hours, is then placed in sticking patch taking-up in new hole, while again
Add in the 400 fresh PBS buffer solution of μ L.It collects the PBS buffer solution in old hole and measures its volume (V1), existed with spectrophotometer
Absorption value is measured under 268nm, and its icariin concentration (C contained is calculated according to working curve1), the excessive sheep of release in first day
Leaves of pulse plants glycosides amount is C1V1.This process was repeated within the 2nd, 4,6,9,12,15,18,21,24,27,30,33,38,43 and 48 day, it will
The volume and icariin concentration of the PBS buffer solution of collection in N days are denoted as V respectivelyNAnd CN, this period interior excessive sheep discharged
Leaves of pulse plants glycosides amount is CNVN, Cumulative release amount ∑ CNVN(N=1,2,4,6,9,12,15,18,21,24,27,30,33,38,43,48),
The build up one's health by taking tonic icariin release profiles of piece of the load of formation are shown in Fig. 4.
From fig. 4, it can be seen that different layers of loads are built up one's health by taking tonic, similar release behavior is presented in piece, and in preceding 2 days drugs, there are one higher
Burst size, the release of icariin later tends towards stability, and this stable release behavior can continue 50 days or so.Single-layer repairing
Stabilization release amount of medicine be 0.3~0.4 μ g/cm2/ day, the stabilization release amount of medicine of bilayer patch mesh is 0.6~0.8 μ g/cm2/
Day, it is seen that the drugloading rate for carrying piece of building up one's health by taking tonic is stablized, and drug release is only related with the area of sticking patch.
3rd, piece In vitro biological activity verification result of building up one's health by taking tonic is carried
3.1st, cell culture and passage：α-MEM the culture mediums for preparing to contain 5~10% fetal calf serums are as culture solution；It will
It is dense that 14 cells of MC3T3-E1Subclone detached from mouse calvarium prebone MC3T3-E1 cell lines are placed in 5% carbon dioxide
It is cultivated in degree, 37 DEG C of cell incubator, the dosage of culture solution is 200 μ L/cm2, old culture solution and addition are discarded every 48h
The new culture solution of equivalent, it is ensured that the dosage of culture solution is 200 μ L/cm2, when culture to 14 cells of MC3T3-E1Subclone converges
Right to carry out cell passage when being more than 90%, the process of cell passage is：Old culture solution is discarded, addition dosage is 200 μ L/
cm2PBS buffer solution, clean 2~3 times；Then it is 40~50 μ L/cm to add in dosage20.25% pancreatin, incubated at 37 DEG C
Educate 1min；It collects cell and adds in centrifuge tube, 2min is centrifuged under 1000rpm rotating speeds；It discards supernatant, then adds in 1mL trainings
Cell is resuspended in nutrient solution, obtains cell suspension, carries out cell count to this cell suspension and calculates the cell density of the cell suspension；
According to the cell density being calculated, by cell suspension with (1~2) × 104Cell/cm2Dosage add in new cell culture
Then bottle adds in the culture solution mixing of 5mL in the Tissue Culture Flask, is placed in cell incubator and cultivates；Repeat above-mentioned cell
Succeeding generations are until obtain 14 cells of MC3T3-E1 Subclone of sufficient amount；
3.2nd, 14 cells of MC3T3-E1 Subclone that degree of converging is taken to be 70~80%, add in centrifuge tube,
2min is centrifuged under 1000rpm rotating speeds, is discarded supernatant, 1mL culture solutions is added and cell is resuspended, cell suspension is obtained, to this cell
Suspension carries out cell count and calculates the cell density of the cell suspension, then according to the cell density being calculated by the cell
Suspension is diluted to 1 × 105The final concentration of cell/mL is prepared and obtains cell suspension；By 5 × 104The cell density of cells/well will
It prepares obtained diluted cell suspension to drop evenly in 24 orifice plates, completes cell seeding, be put into cell culture immediately after
It is incubated in case；After being incubated 4~6h, 24 orifice plates for being implanted with cell are taken out, with 1~2mL/cm2The culture solution of dosage cleans 2~3 times,
The cell that surface is not sticked is removed, adds in new culture solution with the dosage in 1mL/ holes later, this is finally implanted with to 24 holes of cell
Plate is placed in cell incubator incubation；
3.3rd, icariin-SIS sticking patch determination of activity：24 orifice plates of cell are implanted with after cell incubator is incubated 16h, from
It is taken out in cell incubator, discards old culture solution, all holes are divided into 4 groups, be separately added into culture medium, culture medium+SIS sticking patch
(transwell), culture medium+icariin-SIS sticking patch (transwell) and containing 1 × 10-5The culture medium of M icariin, often
Culture medium is replaced every two days.After culture 3 days, the RNA of each group cell is extracted, OCN is measured using real-time RT-PCR technologies
Expression quantity, the expression of OCN is higher to illustrate that Osteoblast Differentiation is stronger.Fig. 5 is carries piece In vitro biological activity verification result of building up one's health by taking tonic, figure
In 5, control：Control medium group；SIS：Control medium+SIS sticking patch (transwell) group；SIS+IC：Control culture
Base+icariin-SIS sticking patch (transwell) group；IC：Containing 1 × 10-5The culture medium group of M icariin.It is from figure 5 it can be seen that single
The OCN expression of pure SIS groups is similar with control group, does not have skeletonization facilitation, and the OCN for carrying piece group of building up one's health by taking tonic is expressed with directly cultivating
The effect that base adds in icariin is similar, has and significantly facilitates bone differentiation.
4th, skeletonization verification of the piece in Mice Body of building up one's health by taking tonic is carried
The load of embodiment one piece of building up one's health by taking tonic is applied particularly to calvaria deficiency model Bone Defect Repari, specific experiment process
For：Choice experiment object：58 week old male C57BL/6 mouse are chosen as experimental subjects, establish cranium deficiency model：
The Biopsy Punch equipment that calvaria both sides position is produced with Miltex companies removes the bone tissue of a diameter of 4mm, so
Implantation is onesize at the missing of side afterwards carries piece of building up one's health by taking tonic, and another place's implantation blank is as control；Mouse is normal after operation stitching
It is put to death after raising 8 weeks, obtains skull bone tissue and carry out following skeletonization status analysis in vivo：
4.1st, Histological results：HE is dyed and MTS coloration results such as Fig. 6 and Fig. 7.Blank controls of the Fig. 6 for no implantation material
The HE coloration results of bone, Fig. 7 are that the load of embodiment one is built up one's health by taking tonic the HE coloration results of piece group.Comparison diagram 6 and Fig. 7 are as it can be seen that in blank
It compares at bone lacks, almost without area of new bone after 8 weeks, and in the position for being implanted into integration engineering bone of the present invention, occurs more
Ripe freshman bone tissue comprising blood vessel, pulp cavity, and bone bridge almost covers half absent region；
4.2nd, statistical calculations result：Using NIH software Is magej to the new bone area ratio at mouse bone lacks into
Row calculates, and each sample takes the slice of 5 different cross sections, finds that new bone area ratio is in blank group by statistical analysis
4.7% ± 5.9% (Mean ± S.D.), and in experimental group new bone area ratio for 29.9% ± 16.4% (Mean ±
S.D.), the two has significant difference (p<0.001), it was demonstrated that the present invention carries piece significant effect during Bone Defect Repari in vivo of building up one's health by taking tonic.
Claims (2)
1. small intestinal submucosa load is built up one's health by taking tonic piece, it is characterised in that the build up one's health by taking tonic microstructure of piece of the load includes SIS collagenous fibres skeletons
With load on the icariin on SIS collagenous fibres skeletons, the SIS collagenous fibres skeleton is reticular fiber structure, described
Icariin for graininess, the icariin is attached on the SIS collagenous fibres skeletons, the icariin
Adhesion amount be 20 ~ 80 μ g/cm2；The small intestinal submucosa carries the preparation method for piece of building up one's health by taking tonic, and includes the following steps：
1）The preparation of small intestinal submucosa sticking patch：In an aseptic environment, dry sterile SIS materials are taken out, are cut into required ruler
Very little small intestinal submucosa sticking patch, then 12 ~ 24 h of the small intestinal submucosa sticking patch deionized water cut infiltration are made into material
Fully wetting is discarded after deionized water with the ethanol solution of 50 %, 1 ~ 2 h of infiltration, discard use instead successively after ethanol solution 70 %,
80 %, the ethanol solution of 90 % and absolute ethyl alcohol infiltration 1 ~ 2 h of sticking patch, are finally placed in absolute ethyl alcohol by small intestinal submucosa sticking patch
In it is for use；
2）The preparation of icariin working solution：In an aseptic environment, powdery icariin is added in into dimethyl sulfoxide, mixing
The icariin storing solution of a concentration of 50 mg/mL is made in concussion dissolving；The icariin storing solution of 1mL is taken, with absolute ethyl alcohol
The icariin working solution of final concentration of 0.5 ~ 2.5 mg/mL is made in mixing；
3）Small intestinal submucosa carries the preparation for piece of building up one's health by taking tonic：In an aseptic environment, small intestinal submucosa is taken out from absolute ethyl alcohol to mend
Piece, according to 400 μ L/cm2Dosage by icariin working solution point several times uniform load to small intestinal submucosa sticking patch,
Each loading capacity is 100 μ L, and the time interval loaded twice is 30 ~ 50 minutes, until all icariin working solutions are whole
It is loaded onto on small intestinal submucosa sticking patch, obtains small intestinal submucosa and carry piece of building up one's health by taking tonic；Obtained load piece of building up one's health by taking tonic is placed in draft chamber
Load piece of building up one's health by taking tonic then is put into deionized water and rocks 24 ~ 48 h at room temperature, discards deionized water by warm 24 ~ 48 h of condition
Load piece of building up one's health by taking tonic is put into PBS buffer solution afterwards, concussion at room temperature is rocked, and every 24 h discards old PBS buffer solution and adds in equivalent
New PBS buffer solution will carry piece of building up one's health by taking tonic and be removed and placed in new PBS buffer solution, saved backup in 2 ~ 6 DEG C after continuous 5 days.
The piece 2. small intestinal submucosa load according to claim 1 is built up one's health by taking tonic, it is characterised in that small intestinal submucosa load is built up one's health by taking tonic
The step 3 of the preparation method of piece）In, icariin working solution be loaded onto small intestinal submucosa sticking patch mode include be added dropwise,
At least one of spray, injection and immersion.
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| CN106620878B (en) * | 2016-11-11 | 2019-12-31 | 广州医科大学附属口腔医院 | Collagen-chitosan composite material capable of slowly releasing icariin and preparation method thereof |
| CN107308495A (en) * | 2017-07-06 | 2017-11-03 | 苏州期佰生物技术有限公司 | A kind of artificial blood vessel based on trees-Osima jacoti, Osima excavata and preparation method thereof |
| CN111840650B (en) * | 2020-07-28 | 2022-03-18 | 中国医学科学院生物医学工程研究所 | Collagen-based modified citrus pectin composite material and preparation method and application thereof |
| CN114425104B (en) * | 2021-12-21 | 2023-03-03 | 中国人民解放军空军军医大学 | Medicine-carrying bone guiding/inducing composite structure and preparation method and application thereof |
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| US20070122388A1 (en) * | 2000-12-08 | 2007-05-31 | Kropp Bradley P | Tissue graft compositions and methods for producing same |
| CN102462561A (en) * | 2010-11-19 | 2012-05-23 | 北京迈迪顶峰医疗科技有限公司 | Small intestinal submucosa (SIS) soft tissue repair patch and preparation method thereof |
| CN102698319A (en) * | 2012-03-02 | 2012-10-03 | 首都医科大学附属北京朝阳医院 | Anti-tumor biological patch |
| CN104874024B (en) * | 2015-05-20 | 2017-05-03 | 宁波大学 | Cell assembling small-intestinal submucosa bionic composite engineering bone and preparation method thereof |
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