CN105056306A - Use of material prepared through cross-linking genipin to intestinal mucosal lower layer - Google Patents
Use of material prepared through cross-linking genipin to intestinal mucosal lower layer Download PDFInfo
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Materials For Medical Uses (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a use of a material prepared through cross-linking genipin to an intestinal mucosal lower layer in the preparation of tissue restoration materials with acid resistance. The cross-linking index of the material is 30-100%. The material prepared through cross-linking genipin to the intestinal mucosal lower layer can resist decomposition of strong acids and pepsins in gastric juice, can be processed to prepare the tissue restoration materials with acid resistance and enzymatic hydrolysis resistance, such as alimentary canal restoration promotion materials, and has a good application prospect.
Description
The application is the applying date: on April 10th, 2014, application number: 201410143074.0, denomination of invention: the divisional application of the patent application of a kind of digestion promoting road cell migration material and its production and use.
Technical field
The present invention relates to the purposes that genipin is cross-linked the material that small intestinal submucosa is prepared from.
Background technology
Digestive tract ulcer, main finger occurs in the duodenal chronic ulcer of harmonization of the stomach, also Meike that (MECKEL) diverticulum around distal esophagus, stomach intestinal anastomosis mouth and containing ectopic gastric mucosa can be betided, the formation of these ulcer is relevant with pepsic Digestion with gastric acid, therefore claims peptic ulcer.Digestive tract ulcer is one of digestive system common disease clinically, and in recent years with the quickening pace of modern life, the change of dietary structure and the irregular of eating time, sickness rate rises gradually.The generation of digestive tract ulcer is relevant with several factors, and the cause of disease common clinically has gastroxia, bile reflux, HP (helicobacter pylori) to infect, degradation under gastric mucosa defence capability.This course of disease is very long, if can not give effective treatment, long-run development can cause gastric cancer, has a strong impact on patient vitals's safety.
The mode of clinical treatment digestive tract ulcer mainly contains expectant treatment mode and operative treatment mode.The drug effect of expectant treatment mode is gentle, painful little, but it is comparatively slow to take effect, and treats usually not thorough, and relapse rate is higher, recur each time, the state of an illness is generally even more serious than the last time, and therefore patient has to strengthen dosage, or use other drug treatment instead, if things go on like this, body drug resistance increases, and clinical effectiveness but might not be significantly improved.Operative treatment mode instant effect, treatment is comparatively thorough, but wound is comparatively large, and complication is comparatively obvious.In recent years along with the development of materialogy and endoscopic technic, emphasis has been transferred to and has been utilized endoscopic technic that biomaterial is delivered to digestive tract ulcer place by domestic and international researcher, reach the object of repairing damaging mucosal, this will be expected to both cure digestive tract ulcer up hill and dale, reduce again the misery that the huge wound of operation is brought, reach diagnoses and treatment integration simultaneously.
Small intestinal submucosa (smallintestinalsubmucosa, SIS) is a kind of natural extracellular matrix class biomaterial, is usually prepared by pig small intestine.SIS is mainly containing compositions such as collagen, aminopolysaccharide, glycoproteins, suitable epithelially stick and grow, VEGF (the vascularendothelialgrowthfactor that it comprises, VEGF), fibroblast growth factor (fibroblastgrowthfactors, etc. FGF) vascularization of repair tissue can also be promoted, the migration of inducing host cell and tissue.SIS is the current natural extracellular matrix class biomaterial being widely used in Tissue Engineering Study, having good cell compatibility, containing multiple somatomedin, non-immunogenicity, have the features such as antimicrobial acivity, anisotropy, tissue specificity regeneration induction, is ideal Mucous rehabilitation material.But still there are some obvious defects, in case of water is yielding in repair materials SIS being used for alimentary canal mucous membrane, easily by strong acid and Pepsin degradation, coefficient of friction is low, causes SIS to be difficult to, with scope conveying, arrive the easy premature breakdown of affected area and lose function.
Summary of the invention
In order to overcome above-mentioned defect, the invention provides the purposes that genipin is cross-linked the material that small intestinal submucosa is prepared from.
Digestion promoting road of the present invention cell migration material, it is the small intestinal submucosa containing genipin, is specifically cross-linked small intestinal submucosa by genipin and is prepared from, and the cross-linking index of submucous layer of small intestine after genipin is cross-linked is 30% ~ 100%.
Cross-linking index, characterizes the crosslinking degree of macromolecular chain.
Genipin of the present invention is cross-linked the material that small intestinal submucosa is prepared from and is preparing the purposes had in the tissue renovation material of Antacid effectiveness; Wherein, the cross-linking index of described material is 30% ~ 100%.
Preferably, described cross-linking index is 30.63% ~ 86.52%.Further preferably, described cross-linking index is 67.28% ~ 86.52%.Again further preferably, described cross-linking index is 67.28% ~ 85.22%.Most preferably, described cross-linking index is 83.74 ~ 85.22% or 67.28 ~ 69.92%.
Described genipin is cross-linked the material that small intestinal submucosa is prepared from and is prepared from by the following method:
(1) get genipin and be dissolved in buffer, make the genipin solution that concentration is not less than 0.01% (w/v);
(2) be soaked in by submucous layer of small intestine in the obtained genipin solution of step (1), soak time is not less than 3h;
(3) take out small intestinal submucosa, cleaning, lyophilization, to obtain final product.
In step (1), the pH of described buffer is 4 ~ 11.Preferably, the pH of described buffer is 7.2.
In step (1), described buffer is PBS solution.
Suddenly, in (1), the concentration of genipin solution is 0.01 ~ 0.6% (w/v).Preferably, the concentration of described genipin solution is 0.05 ~ 0.6% (w/v).Further preferably, the concentration of described genipin solution is 0.05 ~ 0.3% (w/v).Again further preferably, the concentration of described genipin solution is 0.3% (w/v).
In step (2), the ratio of submucous layer of small intestine surface area and genipin solution volume is (200 ~ 3): 1.
In step (2), soak time is 3 ~ 24h.
In step (2), the temperature of immersion is 4 DEG C ~ 65 DEG C.Preferably, the temperature of described immersion is 37 DEG C.
Genipin of the present invention is cross-linked the material that small intestinal submucosa is prepared from, there is the characteristic of strong acid stomach function regulating proteases for decomposing in anti-gastric juice, the tissue renovation material with antiacid resistance to enzymolysis performance can be made, as digestion promoting road repair materials, and its good mechanical performance, no cytotoxicity, hemolysis rate is low, histocompatibility is good, pore size is suitable, be suitable for Growth of Cells, simultaneously containing the raised growth factor, repair without obvious calcification phenomenon in body, use safety, for gastrointestinal mucosal reparation provides a kind of selection newly, preparation method is simple, have broad application prospects.
Obviously, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
The detailed description of the invention of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
Fig. 1 SIS film.Wherein, A: the SIS film of fresh preparation is translucent shape; B: after lyophilizing, SIS film is white paper-like;
Fig. 2 genipin is cross-linked cross-linking index after SIS.(* P < 0.05 represents that each group has significant difference compared with SIS; #P < 0.05, represents that each crosslinked group has significant difference compared with 0.01% crosslinked group).
SIS different time sections degraded situation in simulated gastric fluid after Fig. 3 genipin is crosslinked.1:SIS+ simulated gastric fluid; 2:SIS+PBS; 3:0.01%GP is cross-linked SIS+ simulated gastric fluid; 4:0.05%GP is cross-linked SIS+ simulated gastric fluid; 5:0.1%GP is cross-linked SIS+ simulated gastric fluid; 6:0.3%GP is cross-linked SIS+ simulated gastric fluid; 7:0.5%GP is cross-linked SIS+ simulated gastric fluid; 8:0.6%GP is cross-linked SIS+ simulated gastric fluid
SIS in simulated gastric fluid 2 weeks after Fig. 4 genipin is crosslinked, 4 weeks, the macroscopic observation of degrading for 8 weeks.
The relation of Fig. 5 SIS cross-linking index and genipin crosslinking time.(* P<0.05 represents that the cross-linking index being cross-linked crosslinked 12h group in 24h group and genipin solution in genipin solution has significant difference)
The relation of Fig. 6 SIS cross-linking index and crosslinking temperature.(* P<0.05 represents that 37 DEG C of genipin solution are cross-linked the cross-linking index that group and 25 DEG C of genipin are cross-linked group and have significant difference)
Fig. 7 SIS and variable concentrations genipin are cross-linked the swelling ratio of SIS.(* P<0.05 represents there is significant difference compared with uncrosslinked SIS group; #P<0.05 represents to there is significant difference compared with 0.05% group)
Fig. 8 SIS and variable concentrations genipin are cross-linked the infrared scan collection of illustrative plates of SIS.
SIS ultimate tensile strength test after Fig. 9 genipin is crosslinked.(* P < 0.05 represents that each group has significant difference compared with SIS, #P < 0.05, represents that each crosslinked group has significant difference compared with 0.5% crosslinked group).
Figure 10 SIS and variable concentrations genipin are cross-linked SIS and in simulated gastric fluid, soak 0 week, 2 weeks, 4 weeks and ultimate tensile strength test after 8 weeks.(* P < 0.05 represents that each group has significant difference compared with SIS, and #P<0.05 represents there is significant difference with corresponding 0 week group).
Figure 11 SIS and variable concentrations genipin are cross-linked SIS and in simulated gastric fluid, soak 0 week, 2 weeks, 4 weeks and 8 weeks rear rigidity tests.
Figure 12 ELISA method detects SIS and concentration is VEGF and TGF-β 1 release conditions in 14 days in 0.3% genipin crosslinked group SIS.
SIS scanning electron microscope (SEM) photograph after Figure 13 genipin is crosslinked.A: uncrosslinked SIS; B:0.05%GP cross-linked material; C:0.1%GP cross-linked material; D:0.3%GP cross-linked material; The material that E:0.5%GP is crosslinked.
Figure 14 people's gastric epithelial cell GES-1 is inoculated into living cells dyeing after SIS and variable concentrations genipin crosslinked SIS upper 3 day.A-e is the cell observed under 40 times.A: uncrosslinked SIS; B:0.05%GP cross-linked material; C:0.1%GP cross-linked material; D:0.3%GP cross-linked material; The material that e:0.5%GP is crosslinked.F-j is the cell observed under 100 times.F: uncrosslinked SIS; G:0.05%GP cross-linked material; H:0.1%GP cross-linked material; I:0.3%GP cross-linked material; The material that j:0.5%GP is crosslinked.
Figure 15 digestion promoting road cell migration material lixiviating solution haemolysis situation.1: negative control; 2: positive control; 3: uncrosslinked SIS lixiviating solution; 4:0.05%GP cross-linked material lixiviating solution; 5:0.1%GP cross-linked material lixiviating solution; 6:0.3%GP cross-linked material lixiviating solution; 7:0.5%GP cross-linked material lixiviating solution.
Figure 16 SD rat back subcutaneous implantation SIS and variable concentrations genipin are cross-linked SIS material structure and observe.Inflammatory cell situation during figure (a)-(e) material the 2nd week.(a): uncrosslinked SIS; (b): 0.05%GP cross-linked material; (c): 0.1%GP cross-linked material; (d): 0.3%GP cross-linked material; E material that (): 0.5%GP is crosslinked.Inflammatory cell situation during figure (f)-(j) material the 2nd week.(f): uncrosslinked SIS; (g): 0.05%GP cross-linked material; (h): 0.1%GP cross-linked material; (i): 0.3%GP cross-linked material; J material that (): 0.5%GP is crosslinked.* represent SIS position, # represents that genipin is cross-linked SIS position.
The subcutaneous composite implant material of Figure 17 SD rat back, application Image – Proplus6.0 software inflammatory cell counting.(*: compare with SIS group in same time point, P < 0.05 has significant difference; #: compare with SIS group in same time point, P < 0.05 has significant difference; ☆: compare with SIS group in same time point, P < 0.05 has significant difference).
Detailed description of the invention
Abbreviation:
SIS: submucous layer of small intestine; GP: genipin; The Eagle culture medium of DMEM:Dulbecco improvement; FBS: hyclone; MTT:3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt
The preparation of embodiment 1 digestion promoting road of the present invention cell migration material
One, the preparation of digestion promoting road of the present invention cell migration material
1, the preparation of SIS
SIS of the present invention can buy commercially available product, also can be prepared as follows.
(1) cleaning arranges: get the fresh pig small intestinal of butchering rear half an hour, wash away small intestine contents with water, and upset small intestinal, adds salt and rub rear water and repeatedly rinse 3 times, cut small intestinal open, be then cut into the intestinal segment of 15 centimeter length with scalpel.
(2) isolate SIS: strike off muscle layer with spatula, placenta percreta, be placed in normal saline 4 DEG C of preservations and spend the night.
(3) defat: be filtered dry water with gauze with after deionized water rinsed clean.Immerse in the mixed liquor of chloroform-methanol, chloroform: the ratio of methanol is 1:1, to be placed in fume hood 4 hours, within average 2 hours, to change a not good liquor, and per half an hour, stirs once.
(4) de-cell: by the rinsed with deionized water 20 times of the SIS after defat, repeatedly clean, drift is to tasteless.Then putting into concentration is in the trypsin solution of 0.25%, and 4 DEG C of de-cell process are spent the night.
(5) with rinsed with deionized water 10 times afterwards with 0.5% SDS process at least 4 hours, clean, obtain fresh SIS film, as shown in Figure 1A.
(6) lyophilizing: be placed in-20 DEG C successively after cleaning ,-40 DEG C, in-80 DEG C successively after pre-freeze in vacuum freeze drier lyophilizing preserve, the SIS film after obtained lyophilizing, as shown in Figure 1B.
2, the preparation of gastrointestinal mucosal repair materials of the present invention
(1) take Powdered GP to be dissolved in PBS (pH=7.2) solution, fully stir, GP is dissolved completely, making mass volume ratio is respectively 0.01%, 0.05%, the GP solution of 0.1%, 0.3%, 0.5%, 0.6%.
(2) SIS of preparation is dipped in GP solution, crosslinked 24h on the constant-temperature table of 37 DEG C.
(3) SIS after crosslinked is taken out from GP solution, repeatedly rinse by PBS solution, at least 5 times, and be immersed in 24h in PBS solution.
(4) SIS after crosslinked is taken out from PBS solution, repeatedly rinse with distilled water, at least 5 times, and be immersed in 24h in distilled water.
(5) taken out from distilled water by SIS, namely lyophilization obtains gastrointestinal mucosal repair materials of the present invention.
Two, the detection of digestion promoting road of the present invention cell migration material
1, detection method
(1) mensuration of cross-linking index (CI):
1. the preparation of ninhydrin reaction liquid: by the stannous chloride (SnCl of NaOH and 0.04g of the citric acid of 1.05g and 10ml (1.0M)
22H
2o) mix, add deionized water and be adjusted to 25ml, separately the 1,2,3-indantrione monohydrate of 1g is added in 25ml ethylene glycol monomethyl ether, more above-mentioned two liquid mixing are stirred 45min, make ninhydrin solution, be stored in black bottle for subsequent use.
2. each crosslinked group of SIS and uncrosslinked SIS sample 1.5mg is taken respectively, add ninhydrin solution 1ml, at 100 DEG C, heating in water bath 20min, is cooled to room temperature, add 15ml isopropyl alcohol (isopropylglyeoho1), under 570nm, measure absorbance with spectrophotometer.Free amino group in sample heats afterwards and 1,2,3-indantrione monohydrate reacts, and the bluish violet pigment of generation is directly proportional to absorbance.The content of free amino group in different sample is checked in the standard curve that the glycine of variable concentrations obtains.Institute responds and all carries out in darkroom.With M
sIScontaining free a-amino acid molal quantity in the SIS that group expression is uncrosslinked, M
gPgroup represents a-amino acid molal quantity in each group of sample, calculates the degree of cross linking of each group of material as follows:
CI＝(M
SIS－M
GP)/M
SIS×100％
(2) antiacid resistance to enzymolysis Performance Detection
For determining that the GP be suitable for is cross-linked concentration further, the SIS that obtained variable concentrations GP is crosslinked is dipped in simulated gastric fluid, observes the situation of its antiacid resistance to enzymolysis.
1. the preparation of simulated gastric fluid
Take the pepsin (come from the gastric mucosa of pig, pepsic enzyme is lived as 800-2500U/mg) of NaCl and 3.2g of 2.0g, be dissolved in the concentrated hydrochloric acid of 7ml, be diluted with water to 1000ml, pH is about 1.2.
2. overall degraded situation
The SIS of uncrosslinked SIS and each crosslinked group is made the sample of 2cm × 2cm, often group chooses 3, is immersed in simulated gastric fluid, changes liquid every three days, observes the degraded situation of 3 weeks under 37 DEG C of conditions.
Result as shown in Figure 3,1 group: SIS+ simulated gastric fluid; 2 groups: SIS+PBS; 3 groups: 0.01%GP is cross-linked SIS+ simulated gastric fluid; 4 groups: 0.05%GP is cross-linked SIS+ simulated gastric fluid; 5 groups; 0.1%GP is cross-linked SIS+ simulated gastric fluid; 6 groups: 0.3%GP is cross-linked SIS+ simulated gastric fluid; 7 groups: 0.5%GP is cross-linked SIS+ simulated gastric fluid; 8 groups: 0.6%GP is cross-linked SIS+ simulated gastric fluid.
3. degradation rate measures
Be that the SIS that 0.05%, 0.1%, 0.3% and 0.5% genipin is cross-linked makes the disk that diameter is 16mm by uncrosslinked SIS and concentration, every 3 are immersed in simulated gastric fluid, change liquid every three days, process 8 weeks under 37 DEG C of conditions.Measure 2 weeks respectively, 4 weeks, the residual mass percentage ratio of material when 8 weeks.
Statistical method: adopt SPSS16.0 software, with independent sample T inspection statistics.
2, testing result
Result is as shown in Fig. 2 ~ 4 and table 1:
The cross-linking index of table 1 digestion promoting of the present invention road cell migration material and antiacid resistance to enzymolysis performance
By statistical analysis cross-linking index, each crosslinked group, compared with uncrosslinked SIS, all has significant difference (P < 0.05); In crosslinked group, 0.05% to 0.6% crosslinked group all has significant difference (P < 0.05) compared with 0.01% group."-" represents and does not detect." # " represents and have significant difference (P<0.05) compared with 0.5% group.
As can be seen from Fig. 2 and table 1, adopt repair materials prepared by the inventive method, the cross-linking index of submucous layer of small intestine is 30.63% ~ 86%, along with the increase of genipin concentration, interdigital number first increases rear reduction, and when genipin concentration is 0.5%, cross-linking index is the highest.
As can be seen from Fig. 3 ~ 4 and table 1, simple SIS is just all degraded in 1 day, and the present invention through the crosslinked repair materials of genipin process 2 weeks time, material residual rate is 49.93% ~ 96.5%, can effective antiacid resistance to enzymolysis.Wherein, when genipin concentration is 0.05% ~ 0.6%, cross-linking index is 67.28% ~ 86.52%, the antiacid resistance to enzymolysis function admirable of material, and when processing 8 weeks, material residual rate is up to 89.91% ~ 93.51%; When genipin concentration is 0.05% ~ 0.3%, cross-linking index is 67.28% ~ 85.22%, the antiacid resistance to enzymolysis function admirable of material, when processing 8 weeks, material residual rate also up to 89.91% ~ 93.51%, and the amount of genipin less; When genipin concentration is 0.05%, cross-linking index is 68.6 ± 1.32%, the antiacid resistance to enzymolysis function admirable of repair materials, and genipin consumption is little; When genipin concentration is 0.3%, cross-linking index is 84.48 ± 0.74%, and the antiacid resistance to enzymolysis performance of repair materials is best.
Experimental result illustrates, digestion promoting road of the present invention repair materials can effective antiacid resistance to enzymolysis, can be used for preparing digestive tract repair materials.
The preparation of embodiment 2 digestion promoting road of the present invention cell migration material
One, the preparation of digestion promoting road of the present invention cell migration material
1, the preparation of SIS
SIS of the present invention can buy commercially available product, also can be prepared as follows.
(1) cleaning arranges: get the fresh pig small intestinal of butchering rear half an hour, wash away small intestine contents with water, and upset small intestinal, adds salt and rub rear water and repeatedly rinse 3 times, cut small intestinal open, be then cut into the intestinal segment of 15 centimeter length with scalpel.
(2) isolate SIS: strike off muscle layer with spatula, placenta percreta, be placed in normal saline 4 DEG C of preservations and spend the night.
(3) defat: be filtered dry water with gauze with after deionized water rinsed clean.Immerse in the mixed liquor of chloroform-methanol, chloroform: the ratio of methanol is 1:1, to be placed in fume hood 4 hours, within average 2 hours, to change a not good liquor, and per half an hour, stirs once.
(4) de-cell: by the rinsed with deionized water 20 times of the SIS after defat, repeatedly clean, drift is to tasteless.Then putting into concentration is in the trypsin solution of 0.25%, and 4 DEG C of de-cell process are spent the night.
(5) with rinsed with deionized water 10 times afterwards with 0.5% SDS process at least 4 hours, clean, obtain fresh SIS film, as shown in Figure 1A.
(6) lyophilizing: be placed in-20 DEG C successively after cleaning ,-40 DEG C, in-80 DEG C successively after pre-freeze in vacuum freeze drier lyophilizing preserve, the SIS film after obtained lyophilizing, as shown in Figure 1B.
2, the preparation of gastrointestinal mucosal repair materials of the present invention
(1) take Powdered GP to be dissolved in PBS (pH=4.0) solution, fully stir, GP is dissolved completely, make the GP solution that mass volume ratio is 0.3% respectively.
(2) be dipped in GP solution by the SIS of preparation, the ratio of submucous layer of small intestine surface area and genipin solution volume is 200:1 (cm
2: ml), crosslinked 24h on the constant-temperature table of 37 DEG C.
(3) SIS after crosslinked is taken out from GP solution, repeatedly rinse by PBS solution, at least 5 times, and be immersed in 24h in PBS solution.
(4) SIS after crosslinked is taken out from PBS solution, repeatedly rinse with distilled water, at least 5 times, and be immersed in 24h in distilled water.
(5) taken out from distilled water by SIS, namely lyophilization obtains gastrointestinal mucosal repair materials of the present invention.
The preparation of embodiment 3 digestion promoting road of the present invention cell migration material
One, the preparation of digestion promoting road of the present invention cell migration material
1, the preparation of SIS
SIS of the present invention can buy commercially available product, also can be prepared as follows.
(1) cleaning arranges: get the fresh pig small intestinal of butchering rear half an hour, wash away small intestine contents with water, and upset small intestinal, adds salt and rub rear water and repeatedly rinse 3 times, cut small intestinal open, be then cut into the intestinal segment of 15 centimeter length with scalpel.
(2) isolate SIS: strike off muscle layer with spatula, placenta percreta, be placed in normal saline 4 DEG C of preservations and spend the night.
(3) defat: be filtered dry water with gauze with after deionized water rinsed clean.Immerse in the mixed liquor of chloroform-methanol, chloroform: the ratio of methanol is 1:1, to be placed in fume hood 4 hours, within average 2 hours, to change a not good liquor, and per half an hour, stirs once.
(4) de-cell: by the rinsed with deionized water 20 times of the SIS after defat, repeatedly clean, drift is to tasteless.Then putting into concentration is in the trypsin solution of 0.25%, and 4 DEG C of de-cell process are spent the night.
(5) with rinsed with deionized water 10 times afterwards with 0.5% SDS process at least 4 hours, clean, obtain fresh SIS film, as shown in Figure 1A.
(6) lyophilizing: be placed in-20 DEG C successively after cleaning ,-40 DEG C, in-80 DEG C successively after pre-freeze in vacuum freeze drier lyophilizing preserve, the SIS film after obtained lyophilizing, as shown in Figure 1B.
2, the preparation of gastrointestinal mucosal repair materials of the present invention
(1) take Powdered GP to be dissolved in alkaline buffer (pH=11) solution, fully stir, GP is dissolved completely, make the GP solution that mass volume ratio is 0.3% respectively.
(2) be dipped in GP solution by the SIS of preparation, the ratio of submucous layer of small intestine surface area and genipin solution volume is 3:1 (cm
2: ml), crosslinked 24h on the constant-temperature table of 37 DEG C.
(3) SIS after crosslinked is taken out from GP solution, repeatedly rinse by PBS solution, at least 5 times, and be immersed in 24h in PBS solution.
(4) SIS after crosslinked is taken out from PBS solution, repeatedly rinse with distilled water, at least 5 times, and be immersed in 24h in distilled water.
(5) taken out from distilled water by SIS, namely lyophilization obtains gastrointestinal mucosal repair materials of the present invention.
The choice of parameters of embodiment 4 digestion promoting road of the present invention cell migration material
1, experimental technique
(1) screening of digestion promoting road cell migration crosslink material time
The preparation method of digestion promoting road of the present invention cell migration material is that in the genipin solution of 0.3%, soak time is respectively 0h except SIS is immersed in concentration, and beyond 3h, 6h, 12h, 24h, 48h, 72h, 96h and 120h, all the other conditions are same with embodiment 1.
(2) screening of digestion promoting road cell migration crosslink material temperature
The preparation method of digestion promoting road of the present invention cell migration material, be in the genipin solution of 0.3% except SIS is immersed in concentration, soaking temperature is respectively 4 DEG C, 16 DEG C, 25 DEG C, and beyond 37 DEG C and 56 DEG C, all the other conditions are same with embodiment 1.
2, experimental result
(1) crosslinking time
Result as shown in Fig. 5 and table 2,
The cross-linking index of SIS under the different crosslinking time of table 2
Experimental result shows, the concentration of genipin and temperature certain, when crosslinking time is less than 24h, cross-linking index increases gradually along with the prolongation of crosslinking time; Be more than or equal to 24 hours when crosslinked, the cross-linking index of SIS no longer rises arrival plateau, no difference of science of statistics (P>0.05) between the cross-linking index of each time point.
Experimental result illustrates, in the method for gastrointestinal mucosal repair materials of the present invention, crosslinking time can be more than 3h, is preferably 24 ~ 72h, most preferably is 24h.
(2) crosslinking temperature
Result such as Fig. 6 and Biao prepares shown in 3,
SIS cross-linking index under table 3 different temperatures
Experimental result shows, and in concentration and crosslinking time one timing of genipin, the cross-linking index of SIS increases gradually along with the rising of temperature; When crosslinking temperature is more than 37 DEG C, cross-linking index rises not obvious.At 37 DEG C and 56 DEG C, the cross-linking index of SIS does not have obvious significant difference (P>0.05).
Experimental result illustrates, in the method for gastrointestinal mucosal repair materials of the present invention, crosslinking temperature can be more than 4 DEG C, is preferably 4 DEG C ~ 56 DEG C, more preferably 37 DEG C ~ 56 DEG C, most preferably is 37 DEG C.
The Performance Detection of embodiment 5 digestion promoting road of the present invention cell migration material
1, detection method
Experiment material: SIS prepared by embodiment 1 and gastrointestinal mucosal repair materials of the present invention.
(1) mensuration of swelling ratio
Be immersed in respectively in 10mlPBS by different materials (2cm × 2cm), measure material weight before soaking, after 37 DEG C of immersion 24h, blotting material surface moisture measures the weight of material again.Swelling ratio calculates by following formula:
S
w(％)＝(W
s-W
i)/W
i×100％
S
wrepresent swelling ratio, W
srepresent the weight after material water suction, W
iit is the initial weight of material.
(2) infrared spectrum analysis
Different materials U.S. Nicolet company 170SX type Fourier transform infrared spectrometer tested, scanning wave-number range is 4000 ~ 650cm
-1.
(3) mechanical property
1. the crosslinked rear SIS Mechanics Performance Testing of variable concentrations genipin
Get each material respectively and be cut into 1cm × 4cm size, each sample is got 8 groups and is carried out uniaxial tension, determination limit tensile strength and rigidity.
2. after simulated gastric fluid process different time, the mechanical property of digestion promoting road cell migration material
Each material is immersed in simulated gastric fluid, respectively at 2 weeks, sample was taken out in 4 weeks and 8 weeks, sample is also cut into 1cm × 4cm size by washing lyophilizing, each sample is got 8 groups and is carried out uniaxial tension, measures ultimate tensile strength and the rigidity of digestion promoting road cell migration material after simulated gastric fluid process different time.
(4) VEGF, TGF-β 1 growth factor release situation
Get 0.1gSIS film and 0.1g0.3% genipin is cross-linked each 3 groups of the repair materials of the present invention obtained, lower 37 DEG C of aseptic condition is infiltrated in 3mlPBS, within every 48 hours, takes out 1ml material lixiviating solution, and covers 1ml fresh sterile PBS, continuous sampling 14 days.ELISA method measures VEGF and TGF-β 1 content in material lixiviating solution.
(5) scanning electron microscope detects the crosslinked rear SIS structure of GP
By the uncrosslinked SIS of scanning electron microscopic observation and the collagen fiber of repair materials of the present invention and the change of hole.
(6) cytotoxicity experiment
1. the preparation of lixiviating solution.Uncrosslinked SIS and repair materials of the present invention are cut into 1cm × 3cm, and (surface area is about 6cm
2), ethylene oxide sterilizing.According to subject material surface area/lixiviating solution volume=3:1, getting surface area is 18cm
2material be immersed in the DMEM (10%FBS) of 6mL, aseptically, be placed in 37 DEG C of water isolation type constant incubator lixiviate 24h and take out, obtained lixiviating solution stock solution.
2. people's gastric epithelial cell (GES-1) that the phase of taking the logarithm grows, by 2 × 10
3individual cells/well is inoculated in 3 96 orifice plates (be respectively 3 different time point), and cultivate 24h and make cell fully adherent, discard stock solution, add lixiviating solution, negative control is normal DMEM group, and positive control is the DMEM culture fluid containing 1% phenol.Often organize 3 porocytes and be placed in cultivation in incubator, change liquid every other day.
3. respectively at 1,3, each taking-up one piece of culture plate after 5d, every hole adds 10 μ L5g/LMTT, hatch 4h for 37 DEG C, inhale the dimethyl sulfoxide adding 150 μ L after abandoning culture fluid and be placed in 15 ~ 20min, concussion 10min under room temperature, adopt microplate reader to measure each hole absorbance value at 553nm wavelength, and calculate the average of each group.By formula RGR=experimental group A
553/ negative control group A
553× 100%, calculate the relative appreciation rate (RGR) of cell, and carry out the evaluation of material toxicity grading according to National Medical Devices biological assessment standard.
(7) direct toxicity
Uncrosslinked SIS and repair materials of the present invention are cut into the disk that diameter is 16mm, and use ethylene oxide sterilizing.Soaked in the medium by rounded material, by culture medium sucking-off after 24h, every hole inoculates 1 × 10 equably
4individual gastric epithelial cells GES-1 is at material surface.Contain at 1ml in the DMEM culture medium of the FBS of 10% and cultivate 3 days, sucking-off culture medium, washs with PBS, and interpolation 200ul concentration is that the calcein of 2 μMs dyes to living cells.Under 37 DEG C of conditions, lucifuge hatches 30min, observes the form of cell on material with laser confocal microscope.
(8) cell hemolytic experiment
1. (surface area is about 6cm uncrosslinked SIS and repair materials of the present invention to be cut into 1cm × 3cm
2), ethylene oxide sterilizing.According to subject material surface area/lixiviating solution volume=3:1, getting surface area is 18cm
2material be immersed in the normal saline of 6ml, aseptically, be placed in 37 DEG C of water isolation type constant incubator lixiviate 24h and take out, obtained lixiviating solution stock solution.
2. healthy rabbits auricular vein anticoagulation 10ml is separately got, then in fresh anticoagulation: the ratio of normal saline=4:5 makes dilution Sanguis Leporis seu oryctolagi.
3. cleaned glass test tube numbering 1-7 is got, wherein No. 1 test tube adds 5ml normal saline as negative control, No. 2 test tubes add 5ml distilled water as positive control, 3-7 test tube add respectively SIS and 0.05%, 0.1%, 0.3%, the crosslinked SIS lixiviating solution 5ml of 0.5%GP, every test tube adds dilution Sanguis Leporis seu oryctolagi 0.2m1, mixing, 37 DEG C of constant water bath box insulation 60min.All test tubes through 1500rpm, centrifugal 5min.
4. observe its haemolysis situation and draw supernatant to 96 orifice plate, spectrophotometer surveys absorbance at 545nm wavelength place, calculating hemolysis rate of averaging.Hemolysis rate=(experimental group A value-negative control group A value)/(positive controls A value-negative control group A value) × 100%.
(9) tissue compatible Journal of Sex Research
Respectively by SIS film, repair materials SIS of the present invention (1cm × 1cm) with implanting male SD rat (150-160g) dorsal sc after ethylene oxide sterilizing, in 1 after implantation, 2,4, within 8 weeks, draw materials respectively (n=3) carry out histological observation, and with Image – Proplus6.0 software, often organize material selection 6 zoness of different and carry out inflammatory cell counting under 400 powered microscope fields scopes.
(10) calcification research
Atomic absorption spectrometry quantitative analysis is adopted to implant the subcutaneous rat SIS calcification situation that after 4 weeks, genipin is crosslinked.Material before implantation and the material lyophilizing of 4 weeks of implantation subcutaneous rat are weighed, and is used nitric acid/Perchloric Acid Digestion degraded, adopt atomic absorption spectrophotometry to measure the content of calcium atom.Calcium content in the material quality of calcium constituent contained in every milligram of dry weight material represents.
2, testing result
(1) swelling ratio
Result as shown in Fig. 7 and table 4,
Table 4 swelling ratio
Pass through statistical analysis, compared with simple SIS, the swelling ratio of repair materials of the present invention significantly declines (P<0.05) and declines gradually along with the rising swelling ratio of genipin concentration, and when the concentration of genipin is more than 0.1%, the swelling ratio of SIS drops to about 100%.
Experimental result illustrates, in repair materials of the present invention, SIS and genipin there occurs cross-linking reaction.
(2) infrared spectrum analysis
As shown in Figure 8, compared with simple SIS, repair materials of the present invention is at 1549cm for result
-1having there is decline in the amino absworption peak at place, and slowly declines along with this absworption peak of rising of genipin concentration.
Experimental result illustrates, in repair materials of the present invention, free amino group is consumed, and SIS and genipin there occurs cross-linking reaction.
(3) mechanical property
1. the crosslinked rear SIS Mechanics Performance Testing of variable concentrations genipin
Result is as shown in Fig. 9 and table 5:
The mensuration of table 5 ultimate tensile strength
By statistical analysis ultimate tensile strength, compared with uncrosslinked SIS group, concentration is that 0.05%, 0.1%, 0.3%, 0.5%GP is cross-linked the repair materials group of the present invention obtained and all has significant difference (P < 0.05).In four groups of cross-linked materials, compared with 0.5% crosslinked group, 0.05 and 0.1% group have significant difference (P < 0.05), and no difference of science of statistics (P>0.05) compared with 0.5% crosslinked group and 0.3% crosslinked group.
2. after simulated gastric fluid process different time, the mechanical property of digestion promoting road cell migration material
Result is as shown in Figure 10, Figure 11:
By statistical analysis ultimate tensile strength, and without compared with before simulated gastric fluid process, repair materials of the present invention through simulated gastric fluid process after 2 weeks ultimate tensile strength significantly decline (P<0.05), drop to untreated before 1/5.But in 6 subsequently week, repair materials ultimate tensile strength change of the present invention is little.And simulated gastric fluid process 8 weeks rear rigidity is without significant change.Uncrosslinked group of SIS dissolves completely in 24h, cannot obtain Mechanical Data.
Experimental result shows, repair materials of the present invention has good mechanical property, after gastric juice process, also still maintain certain ultimate tensile strength and rigidity, is conducive to material and plays a role in chronic tissue repair process.
(4) VEGF, TGF-β 1 growth factor release situation
As shown in figure 12,0.3% genipin is cross-linked the repair materials sustained release VEGF of the present invention and TGF-β 1 somatomedin that obtain to result, and the 14th day time, VEGF cumulative release amount reaches 72pg, and the cumulative release amount of SIS film is 84pg; The cumulative release amount of TGF-β 1 reaches 50pg, the cumulative release amount of SIS film 74pg.
Experimental result illustrates, the release growth factor VEGF that repair materials of the present invention can continue and TGF, can promote that damaged tissue regenerates in the repair process of gastrointestinal mucosal.
(5) scanning electron microscope detection architecture
As shown in figure 13, no matter repair materials of the present invention, compared with uncrosslinked SIS material, is that low concentration is crosslinked or high concentration is cross-linked, does not all occur the change of collagen fiber fusion and obvious pore size and porosity, be suitable for cell adhesion and growth result.
Experimental result illustrates, repair materials of the present invention is suitable for cell adhesion and growth, can be used for in-vivo tissue reparation.
(6) cytotoxicity experiment
Result is as shown in table 6 and table 7:
MTT experiment result shows, and the lixiviating solution of repair materials of the present invention cultivates people's gastrointestinal mucosal epithelial cell 1 day, and 3 days, after 5 days, cell proliferation rate was all more than 90%, and material toxicity grading is 0 grade or 1 grade, shows material no cytotoxicity.
Experimental result illustrates, repair materials no cytotoxicity of the present invention, is suitable for repairing in body.
(7) direct toxicity
Result as shown in figure 14, excites the gastric epithelial cell for people of lower display green fluorescence at laser confocal microscope 488nm wavelength, 638nm wavelength excites the collagen structure for repair materials of the present invention of lower display red fluorescence.After cultivating 3 days, there is a large amount of cells on uncrosslinked SIS and repair materials of the present invention surface, and cell is in the state that significantly extends.
Experimental result illustrates, repair materials avirulence of the present invention, is suitable for repairing in body.
(8) cell haemolysis
Shown in haemolysis result Figure 15, repair materials supernatant liquid of the present invention is water white transparency, and erythrocyte all precipitates.After calculating, hemolysis rate is in table 8.
Experimental result shows, and the GP of variable concentrations is cross-linked the repair materials hemolysis rate of the present invention that obtains all lower than 5%, meets country's " BiologicalEvaluationofMedicalDevice-with blood interaction test and Selection " standard.
Experimental result illustrates, repair materials of the present invention, without obvious hemolytic, is suitable for repairing in body.
(9) tissue compatible Journal of Sex Research
Result is as shown in Figure 16, Figure 17, and Figure 16 is the 2nd week and the 8th week different materials inflammatory cell infiltration situation.Figure 17 is different time points, the number of inflammatory cell under 400 times of visuals field around SIS and repair materials of the present invention.
Experimental result shows, postoperative 1st week and the 2nd week, and SIS group inflammatory reaction is comparatively serious relative to repair materials of the present invention, and inflammatory cell number is significantly higher than repair materials of the present invention.Postoperative 8th week, uncrosslinked SIS substantially all degraded, and repair materials structure of the present invention is relatively complete.Obviously declining appears in SIS group inflammatory cell for the 4th week after surgery, within postoperative 8th week, substantially disappears.Repair materials group number of inflammatory cells of the present invention maintains reduced levels, substantially constant.
Experimental result illustrates, repair materials of the present invention has good histocompatibility, can be used for repairing in body.
(10) calcification research
Result is as shown in table 9,
Table 9SIS and repair materials SIS of the present invention implants subcutaneous rat 4 weeks rear calcium contents
Experimental result shows, and uncrosslinked SIS and repair materials of the present invention implanted subcutaneous rat after 4 weeks, and the content of calcium atom has a small amount of increase, but without obvious calcification phenomenon.
Experimental result illustrates, is used for repairing in body by repair materials of the present invention, without obvious calcification, can be used for gastrointestinal mucosal reparation.
To sum up, genipin of the present invention is cross-linked the material that small intestinal submucosa is prepared from effectively can resist acid and pepsic degraded, the tissue renovation material with antiacid resistance to enzymolysis performance can be made, digestion promoting road repair materials, good mechanical performance, no cytotoxicity, hemolysis rate is low, and histocompatibility is good, pore size is suitable, be suitable for Growth of Cells, simultaneously containing the raised growth factor, repair without obvious calcification phenomenon in body, can be used for facilitating digestion road cell migration, use safety, preparation method is simple, has good clinical value.
Claims (9)
1. genipin is cross-linked the material that small intestinal submucosa is prepared from and is preparing the purposes had in the tissue renovation material of Antacid effectiveness; Wherein, the cross-linking index of described material is 30% ~ 100%.
2. purposes according to claim 1, is characterized in that: described repair materials is digestion promoting road cell migration material.
3. purposes according to claim 1 and 2, is characterized in that: described cross-linking index is 30.63% ~ 86.52%.
4. purposes according to claim 3, is characterized in that: described cross-linking index is 67.28% ~ 85.22%.
5. purposes according to claim 4, is characterized in that: described cross-linking index is 83.74 ~ 85.22% or 67.28 ~ 69.92%.
6. purposes according to claim 1 and 2, is characterized in that: described material is prepared from by the following method:
(1) get genipin and be dissolved in buffer, make the genipin solution that concentration is not less than 0.01% (w/v);
(2) be soaked in by submucous layer of small intestine in the obtained genipin solution of step (1), soak time is not less than 3h;
(3) take out small intestinal submucosa, cleaning, lyophilization, to obtain final product.
7. purposes according to claim 1 and 2, is characterized in that:
In step (1), the pH of described buffer is 4 ~ 11; Preferably, the pH of described buffer is 7.2;
In step (1), described buffer is PBS solution.
8. repair materials according to claim 6, is characterized in that: in step (1), and the concentration of genipin solution is 0.01 ~ 0.6% (w/v);
Preferably, the concentration of described genipin solution is 0.05 ~ 0.6% (w/v);
Further preferably, the concentration of described genipin solution is 0.05 ~ 0.3% (w/v);
Again further preferably, the concentration of described genipin solution is 0.3% (w/v) or 0.05% (w/v).
9. repair materials according to claim 6, is characterized in that:
In step (2), the ratio of submucous layer of small intestine surface area and genipin solution volume is (200 ~ 3): 1;
In step (2), soak time is 3 ~ 24h;
In step (2), the temperature of immersion is 4 DEG C ~ 65 DEG C; Preferably, the temperature of described immersion is 37 DEG C.
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| CN109529120A (en) * | 2018-11-30 | 2019-03-29 | 广州新诚生物科技有限公司 | A kind of small intestinal submucosa matrix repairs the preparation method of gel |
| CN116531567A (en) * | 2023-05-09 | 2023-08-04 | 成都睿科美医疗科技有限公司 | Endometrial repair material |
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