Background technology
China approximately has 3,500,000 people to occur that bone is damaged because of different reasons every year, and osseous surgery is transplanted and is about 1,500,000 examples, very large to the demand of replacement bone, the replacement bone of especially large skeletal injury. Along with the fast development of tissue engineering technique, structure has into the important channel that the artificial replacement bone of bone conductibility and osteoinductive is this demand of solution in vitro. Framework material plays vital effect in tissue engineered bone builds. Than other synthetic materials, n cell epimatrix (ECM) material is due to its following characteristics: 1. the biocompatibility of excellence; 2. close to the three-dimensional structure of cells in vivo growth microenvironment; 3. for cell activities provides various bioactive molecules, be considered to desirable tissue engineering material always. Intestinal mucosa lower floor (SIS) is the n cell extracellular matrix materials of current most study, ratifies by U.S. FDA, is widely used for building the soft tissues such as artificial bladder, artificial urethra, artificial esophagus, artificial blood vessel and artificial skin. SIS material framework ingredient collagen (collagen) has been widely used for as framework material and pharmaceutical carrier for bone tissue engineer. It is natural drug reservoir that SIS has two large advantage: 1.SIS than collagen, and it has retained many bioactive molecules that are conducive to osteanagenesis; 2.SIS is more convenient on drawing materials, and cost also will be far below collagen. Existing as bone tissue engineer framework material report with SIS material both at home and abroad, but have a problem to make it further to promote the use of: the deficiency of osteoinductive. There is by being written into the medicine that promotes osteanagenesis on SIS, and enable to realize slow release, can make up this defect. If can solve this difficult problem, SIS material will become desirable engineering material of bone tissue.
In bone tissue engineer, the medicine of needed promotion osteanagenesis is a study hotspot always, although BMP-2 albumen is the most effective osteogenic induction factor of extensively being approved and adopting at present, but because it is very expensive, and adopt BMP method of gene introduction to have certain safety problem, therefore its clinical use is still subject to the restriction of cost and security. In addition, BMP-2 and other protein drugs are poor, the short problem of half-life in vivo of existence and stability all, and the corresponding requirement for drug delivery system is very high. Therefore find low cost, stable again efficiently the micromolecular compound of bone induction and regeneration to substitute BMP be very significant. The osteoporotic drug main for the treatment of will absorb to realize by suppressing bone the balance of bone metabolism at present, but by promoting the compound of osteoblast differentiation and osteanagenesis considerably less, very limited report comprises: statins, isoflavone derivative, TAK-778 and match jerusalem artichoke flavin derivatives etc. Along with the development of modern separation technology, from natural products, extract various compounds, difficulty and cost all will be well below traditional chemical syntheses. Chinese medicine, as the rarity of name family of China, has been recorded the treatment of a large amount of Chinese herbal medicines for various diseases, and its validity also more and more obtains worldwide accreditation. Autonomic drug barrenwort is considered to have the effect of strong muscle key bone all the time, and function and the mechanism of its main active icariin in bone metabolism just starts to be studied gradually for several years up to date. After the people such as ChenKe-Ming find that rat bone marrow mesenchymal stem cells is processed through icariin first, cell proliferation is accelerated, and differentiation mark ALP, OCN and the increase that mineralized show that icariin may have the effect that promotes bone metabolism. Our research finds that icariin can Runx2 expresses and activation BMP signal path promotes osteoblastic differentiation by raising; The people such as Hsieh also find that icariin promotes osteoblast differentiation by activating BMP and NO signal path, and the gene expression such as BMP-2, Smad4, Runx2 and OPG is simultaneously raised. The research of Chung etc. finds that MEK/ERK and PI3K/Akt/eNOS signal path that icariin can activate in endothelial cell accelerate Angiogenesis; In the critical bone injury scale-model investigation of mouse, we find icariin-calcium-phosphate cement composite can be after 6 weeks in inducing mouse body cranium injury region form the area of new bone with blood vessel. Icariin is due to safety, economy, and stable chemical nature, promoting bone regeneration ability and short revascularization ability are that a kind of very tool is potential can be for the medicine of bone tissue engineer. But not yet have at present and on SIS, be written into the relevant report of icariin as the slow releasing pharmaceutical of promotion osteanagenesis.
Summary of the invention
Technical problem to be solved in the utility model is, for the deficiencies in the prior art, to provide a kind of icariin-small intestinal submucosa sticking patch with extremely strong promoting bone regeneration ability.
The utility model solves the problems of the technologies described above adopted technical scheme: icariin-small intestinal submucosa sticking patch, the microstructure of this sticking patch comprises SIS collagenous fibres skeleton and loads on the icariin on SIS collagenous fibres skeleton, described SIS collagenous fibres skeleton is reticular fiber structure, described icariin is graininess, and described icariin is attached on described SIS collagenous fibres skeleton.
The utility model icariin-small intestinal submucosa sticking patch, the icariin of attaching particles shape on SIS collagenous fibres skeleton, set up stable drug delivery system, SIS collagenous fibres skeleton and these two bone tissue engineer elements that have potentiality of icariin are combined, icariin-small intestinal submucosa sticking patch the composite forming is reliable, stable, to icariin safety, economical, stable chemical nature and the excellent characteristic such as promoting bone regeneration ability and short revascularization ability are made full use of, this icariin-small intestinal submucosa sticking patch is a kind of medicine carrying sticking patch, there is extremely strong promoting bone regeneration ability, be with a wide range of applications in osteanagenesis field.
As preferably, the granularity of described icariin is 0.3～5 μ m; As further preferred, the granularity of described icariin is 0.3～0.6 μ m, to guarantee the even releasing effect of icariin.
As preferably, the adhesion amount of described icariin is 20～80 μ g/cm2, to guarantee giving full play to of icariin drug effect.
Compared with prior art, the utility model has the advantage of: the utility model icariin-small intestinal submucosa sticking patch, the icariin of attaching particles shape on SIS collagenous fibres skeleton, set up stable drug delivery system, SIS collagenous fibres skeleton and these two bone tissue engineer elements that have potentiality of icariin are combined, icariin-small intestinal submucosa sticking patch the composite forming is reliable, stable, to icariin safety, economical, stable chemical nature and the excellent characteristic such as promoting bone regeneration ability and short revascularization ability are made full use of, this icariin-small intestinal submucosa sticking patch is a kind of medicine carrying sticking patch, there is extremely strong promoting bone regeneration ability, be with a wide range of applications in osteanagenesis field.
Embodiment bis-: icariin-small intestinal submucosa sticking patch, as shown in Figure 1, its microstructure comprises SIS collagenous fibres skeleton 1 and loads on the icariin 2 on SIS collagenous fibres skeleton 1, SIS collagenous fibres skeleton 1 is reticular fiber structure, icariin 2 is graininess, granularity is 0.3～5 μ m, and icariin 2 is attached on SIS collagenous fibres skeleton 1, and the adhesion amount of icariin 2 is 56.2 ± 8.8 μ g/cm2。
For icariin-small intestinal submucosa sticking patch of embodiment mono-, carry out following experiment and detect and performance verification:
1, microstructure
Icariin-small intestinal submucosa the sticking patch that is kept at the embodiment mono-in PBS buffer solution is taken out, be placed in ventilation room temperature condition until bone dry, by scanning electron microscopic observation sticking patch surface characteristics, as a comparison, do not load the SIS collagenous fibres skeleton of medicine as blank group. From the SEM picture of the sticking patch of embodiment mono-, on SIS collagenous fibres skeleton, adhere to a large amount of drug molecules, i.e. granular icariin.
2, the release in vitro curve of icariin SIS sticking patch
Icariin-small intestinal submucosa the sticking patch that is kept at the embodiment mono-in PBS buffer solution is taken out and puts into 24 orifice plates, add the fresh PBS buffer solution of 400 μ L, under room temperature, 24h is rocked in concussion, then sticking patch is taken out and is placed in new hole, rejoins the fresh PBS buffer solution of 400 μ L simultaneously. Collect in old hole PBS buffer solution and measure its volume (V1), under 268nm, measure absorption value with spectrophotometer, and calculate according to working curve the icariin concentration (C that it contains1), the icariin amount that first day discharges is C1V1. Within the 2nd, 4,6,9,12,15,18,21,24,27,30,33,38,43 and 48 days, repeat this process, volume and the icariin concentration of the PBS buffer solution of collection in N days are designated as respectively to VNAnd CN, the icariin amount discharging in this time period is CNVN, Cumulative release amount ∑ CNVN(N=1,2,4,6,9,12,15,18,21,24,27,30,33,38,43,48), the icariin release profiles of icariin-small intestinal submucosa sticking patch of formation is shown in Fig. 2.
As seen from Figure 2, icariin-small intestinal submucosa sticking patch of the different numbers of plies presents similar release behavior, has a higher burst size at first 2 days medicines, and the release of icariin afterwards tends towards stability, and this Stable Release behavior can continue about 50 days. The stable release amount of medicine of single-layer repairing is 0.3～0.4 μ g/cm2/ day, the stable release amount of medicine of bilayer patch mesh is 0.6～0.8 μ g/cm2/ day, the medicine carrying amount of visible icariin-small intestinal submucosa sticking patch is stable, and medicine discharges only relevant with the area of sticking patch.
3, icariin-small intestinal submucosa sticking patch In vitro biological activity the result
3.1, cell is cultivated and is gone down to posterity: α-MEM culture medium of preparing to contain 5～10% hyclones is as nutrient solution; By the MC3T3-E1Subclone14 cell separating from mouse calvarium prebone MC3T3-E1 clone be placed in 5% gas concentration lwevel, the cell culture incubator of 37 DEG C is cultivated, the consumption of nutrient solution is 200 μ L/cm2, discard old nutrient solution and add the nutrient solution that equivalent is new every 48h, the consumption of guaranteeing nutrient solution is 200 μ L/cm2, carry out passage when being cultured to when MC3T3-E1Subclone14 cell degree of converging is greater than 90%, the process of passage is: discard old nutrient solution, adding consumption is 200 μ L/cm2PBS buffer solution, clean 2～3 times; Then adding consumption is 40～50 μ L/cm20.25% pancreatin, at 37 DEG C, hatch 1min; Collecting cell is also added to centrifuge tube, centrifugal 2min under 1000rpm rotating speed; Supernatant discarded, then adds 1mL nutrient solution re-suspended cell, obtains cell suspension, and this cell suspension is carried out cell count and calculates the cell density of this cell suspension; According to the cell density calculating, by cell suspension with (1～2) × 104Cell/cm2Consumption be added to new Tissue Culture Flask, then in this Tissue Culture Flask, add the nutrient solution of 5mL to mix, be placed in cell culture incubator and cultivate; Repeat above-mentioned passage process until obtain the MC3T3-E1Subclone14 cell of q.s;
3.2, getting degree of converging is 70～80% MC3T3-E1Subclone14 cell, be added to centrifuge tube, centrifugal 2min under 1000rpm rotating speed, supernatant discarded, add again 1mL nutrient solution re-suspended cell, obtain cell suspension, this cell suspension is carried out cell count and calculates the cell density of this cell suspension, then according to the cell density calculating, this cell suspension is diluted to 1 × 105The final concentration of cell/mL, i.e. preparation obtains cell suspension; By 5 × 104The cell suspension of the dilution that the cell density of cells/well obtains preparation evenly drips in 24 orifice plates, completes cell seeding, then puts into immediately cell culture incubator and hatches; Hatch after 4～6h, take out 24 orifice plates that are implanted with cell, with 1～2mL/cm2The nutrient solution of consumption cleans 2～3 times, removes the cell that does not stick on surface, adds new nutrient solution afterwards with the consumption in 1mL/ hole, and 24 orifice plates that finally this are implanted with to cell are placed in cell culture incubator and hatch;
3.3, icariin-SIS sticking patch determination of activity: 24 orifice plates that are implanted with cell are hatched after 16h in cell culture incubator, from cell culture incubator, take out, discard old nutrient solution, be divided into 4 groups by porose institute, add respectively culture medium, culture medium+SIS sticking patch (transwell), culture medium+icariin-SIS sticking patch (transwell) and contain 1 × 10-5The culture medium of M icariin, every other day changes culture medium. Cultivate after 3 days, extract the RNA of each group of cell, utilize real-timeRT-PCR technology to measure the expression of OCN, the higher explanation Osteoblast Differentiation of expression of OCN is stronger. Fig. 3 is icariin-small intestinal submucosa sticking patch In vitro biological activity the result, in Fig. 3, and control: control medium group; SIS: control medium+SIS sticking patch (transwell) group; SIS+IC: control medium+icariin-SIS sticking patch (transwell) group; IC: containing 1 × 10-5The culture medium group of M icariin. As seen from Figure 3, the OCN of simple SIS group expresses with control group similar, there is no skeletonization facilitation, and the OCN of medicine carrying sticking patch group expresses and directly adds the effect of icariin similar at culture medium, has and obviously facilitates bone differentiation.
4, the skeletonization checking of icariin-small intestinal submucosa sticking patch in Mice Body
Icariin-small intestinal submucosa sticking patch of embodiment mono-is specifically applied to the reparation of calvaria disappearance model bone, specific experiment process is: choice experiment object: choose 58 week age male C57BL/6 mouse as experimental subjects, set up cranium disappearance model: the BiopsyPunch equipment of producing in position, calvaria both sides Miltex company is removed the bone tissue that diameter is 4mm, then implant icariin-small intestinal submucosa sticking patch at a side disappearance place, another place implants blank in contrast; After operation stitching, normal the raising after 8 weeks of mouse put to death, and obtains cranium tissue and carries out as skeletonization status analysis in lower body:
4.1, histology result: contrast visible without the HE coloration result of blank bone of implant and the HE coloration result of the medicine carrying sticking patch group of embodiment mono-, at blank bone lacks place, after 8 weeks, almost there is no area of new bone, and in the position of implanting the utility model integration engineering bone, there is the more ripe freshman bone tissue that comprises blood vessel, pulp cavity, and bone bridge nearly cover half disappearance region;
4.2, statistical calculations result: adopt NIH software I magej to calculate the area of new bone area ratio at Mouse Bone disappearance place, each sample is got the section of 5 different cross sections, find that by statistical analysis in blank group, area of new bone area ratio is 4.7% ± 5.9% (Mean ± S.D.), and area of new bone area ratio is 29.9% ± 16.4% (Mean ± S.D.) in experimental group, both have significant difference (p < 0.001), prove the utility model icariin-small intestinal submucosa sticking patch in vivo in bone repair process effect remarkable.