CN101045913A - Extracorporeal blood vessel regenerating model and its application in evaluating the vascularizing function of biomaterial - Google Patents
Extracorporeal blood vessel regenerating model and its application in evaluating the vascularizing function of biomaterial Download PDFInfo
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- CN101045913A CN101045913A CN 200710048530 CN200710048530A CN101045913A CN 101045913 A CN101045913 A CN 101045913A CN 200710048530 CN200710048530 CN 200710048530 CN 200710048530 A CN200710048530 A CN 200710048530A CN 101045913 A CN101045913 A CN 101045913A
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Abstract
The present invention discloses one kind of extracorporeal blood vessel regenerating model and its application in evaluating vascularizing function of biomaterial. The extracorporeal blood vessel regenerating model is made through inoculating ECV304 cell to the surface of collagen gel, which is compounded with collagen solution, culture liquid and neutralizing solution and through mixing, and culturing to make ECV304 cell form blood vessel-like structure spontaneously on the surface of collagen gel. The extracorporeal blood vessel regenerating model may be used for simultaneous detection of great amount of samples, and has high result repeatability, easy standardizing, no interference of added growth factor and low experiment cost.
Description
Technical field:
The present invention relates to biomaterial and estimate the field, more particularly, relate to the effect of the surface properties of extracorporeal blood vessel regenerating model research and evaluation of tissue engineering materials and degraded product revascularization, the material of screening promotion vascularization, control degradation speed and revascularization are complementary.
Background technology:
The evaluation of its biocompatibility of biomaterial is the important content and the key link of biomaterial research and application.Evaluation of its biocompatibility in the past mainly lays particular emphasis on the biological safety aspect.But along with the development of organizational project and the proposition of third generation biomaterial notion, require material at biodegradable also necessary simultaneously biologically active, be material when guaranteeing Biosafety, also answer the performance of sustenticular cell specific function, reparation that helps organizing and regeneration.Therefore, used in tissue engineering degradable biomaterial biological activity (or biological function) is studied and evaluation becomes more and more important.
Organizational project is a complicated system engineering, needs the problem of solution a lot.Wherein, the vascularization problem that how to solve timbering material is one of organizational project key link of achieving success.This is because when the volume of regenerating tissues reaches several cubic millimeters, and its inner cell will be difficult to obtain by osmosis the support of nutrition and oxygen, and must rely on growing into of capillary vessel just can keep its normal metabolism.Therefore, the application organizes engineering successfully to make up biological tissue, used biomaterial should be able to promote the revascularization in the repair tissue.Because material is that surface topography, physico-chemical property or degraded product all can exert an influence to revascularization, therefore, should carry out from material surface and two aspects of degraded product the evaluation of material vascularization function.Promote in the research of tissue engineering material of revascularization in screening, filter out suitable blood vessel experimental model with research material to angiopoietic influence, and then filter out and promote that the material of revascularization is very important.The blood vessel experimental model is the important tool of research screening bioengineered tissue material vascularization function.The research screening of having set up at present promotes the correlation model of the tissue engineering material of revascularization that following two big classes are arranged.
(1) body inner model
Though this class model and human body are more approaching, but still there are a lot of problems, as a large amount of laboratory animal of needs, complex operation, the cycle is long, and the plant and instrument costliness is difficult to obtain fast experimental result.And because complex physical environment in the animal body makes the research specific factor become difficult to angiopoietic influence, the result also is difficult to repeat.
(2) external model
The research of the outer evaluation method of biomaterial vascularization functive still is in the starting stage at present.A large amount of useful exploration work have been done in the James Kirkpatrick laboratory of Germany in this respect.They adopt endotheliocyte and collagen gel, and research biomaterial (as polysulfone membrane) surface and metal ion are (as Cr
3+, Co
2+) to angiopoietic influence.
Biomaterial polysulfone membrane surface to the concrete research process of vascularization influence is: human dermis's capillary endothelium (HDMEC) of turning out voluntarily in film surface seeding laboratory, after for some time, adding concentration on endotheliocyte is the type i collagen solution (containing 2% NaHCO3, the NaOH of 0.05N and the HEPES of 200mM) of 1.5mg/ml.Hatch the formation gel in the incubator, add the formation that the nutrient solution that contains 2ng/ml alkaline fiber cell growth factor (bFGF) stimulates capillary structure (TLS).After TLS formation situation was used the Calcein-AM fluorescent dye, laser confocal microscope was observed down.Observed TLS after 5 days as a result and formed, TLS is just formed after promptly 5 days.
Metal ion Co
2+Concrete research process to the vascularization influence is: human dermis's capillary endothelium (HDMEC) that experimental cell is still cultivated voluntarily for the laboratory.With containing CoCl
2Individual layer HDMEC cell on the solution-treated culture plate is as the research cell.On cell, add 3mg/ml by I type carpenter's glue original solution and DMEM nutrient solution 1/1 composite mixing solutions by volume, in incubator, hatch and make into gel.Under laser confocal microscope, observe endotheliocyte and under the stimulation of somatomedin, form the TLS situation.After TLS formation situation was still used the Calcein-AM fluorescent dye, laser confocal microscope was observed down.Observed TLS after 20 hours as a result and formed, TLS is just formed after promptly 20 hours.
Above-mentioned capillary structure (TLS) external model all adds collagen gel and sets up on endotheliocyte, they are used for biological engineering material influence evaluation angiopoietic, they also have following deficiency: a. jointly, and not adopt building of standard be cell, and the endotheliocyte of employing laboratory separation and Culture, there is the endotheliocyte purification difficult, limited amount, culture cycle is long, big for differences, problems such as stdn difficulty; B. cultivate the stimulation that needs the appositional growth factor, mixed into the influence of somatomedin the result; C. need fluorescence dye and laser confocal microscope, complex operation, to the requirement for experiment condition height, the also corresponding rising of cost; D. experimental period is long.
Summary of the invention:
The deficiency that the extracorporeal blood vessel regenerating experimental model of setting up at prior art exists, purpose of the present invention aims to provide a kind ofly uses standard to build to be cell strain to set up, need not the appositional growth factor, need not the operation of fluorescence dye and laser confocal microscope, experimental implementation is simple, cost is low, experimental period is short, be easy to large sample repetition and the standardized extracorporeal blood vessel regenerating experimental model that is used for research evaluation bioengineered tissue material to the vascularization influence.
Another object of the present invention provides a kind of extracorporeal blood vessel regenerating model with the present invention's preparation and estimates the method that bioengineered tissue influences revascularization with material surface character and material degradation thing.
In order to solve the defective that above-mentioned prior art exists, the ECV304 cell (the Human umbilical vein endothelial cells strain of spontaneous transformation) that the present invention uses standard to build and is has been set up a kind of novel extracorporeal blood vessel regenerating model.This model is that the contriver is based on setting up on the following phenomenon basis of finding in bioengineered tissue material vascularization functional study, promptly on lower concentration collagen surface, the ECV304 cell can spontaneously form TLS at short notice under the no appositional growth factor stimulates.
The extracorporeal blood vessel regenerating model that is used for bioengineered tissue material vascularization functional evaluation research disclosed by the invention is prepared by following method:
(1) be that the collagen solution of 0.1～1.5mg/ml and nutrient solution that cycles of concentration is 5～10 times and pH value are 6.5～7.5 neutralizer by (5～7) with concentration: (2～1): the volume ratio of (3～2) is mixed with collagen gel;
(2) with the ECV304 cell inoculation to the collagen gel surface, under culture environment, cultivate, make the ECV304 cell at the spontaneous formation capillary structure in collagen gel surface, promptly be prepared into extracorporeal blood vessel regenerating model.And the capillary structure of preparation has tube chamber sample gap (seeing accompanying drawing 2).
In technique scheme, described collagen solution can be dissolved in mass concentration 0.1～2% acetate by type i collagen and be mixed with; Described neutralizer can be by HEPES, NaHCO
3Be mixed with NaOH; Collagen solution and nutrient solution and neutralizer preferably are mixed with collagen gel in the mode of quick ice bath; The ECV304 cell preferably is inoculated on the collagen gel in the mode of individual cells suspension; Cell suspension can be added the RPMI1640 nutrient solution and be made into by the ECV304 cell of logarithmic phase behind 0.25% tryptic digestion; The culture environment of cell inoculation on collagen gel be, every hole 50～100 μ l of culture plate, and temperature is 37 ℃, atmosphere is 5%CO
2, humidity is saturated humidity.
The extracorporeal blood vessel regenerating model that the present invention discloses can be applicable to bioengineered tissue material vascularization functional evaluation, as bioengineered tissue material surface character revascularization is influenced, and bioengineered tissue material degradation product is to the revascularization influence etc.The extracorporeal blood vessel regenerating model postgraduate fabric texture engineering materials surface properties that utilization the present invention discloses is to the influence of revascularization, and its process mainly may further comprise the steps:
(1) with tryptic digestion ECV304 cell, adds nutrient solution and make cell suspension, be inoculated in the bioengineered tissue material surface of being studied, be cultured to cytogamy and form individual layer;
(2) with the ECV304 cell of tryptic digestion material surface growth, add nutrient solution and make cell suspension, be inoculated into the collagen gel surface, be cultured to capillary structure and form, promptly make blood vessel regenerating model;
(3) examine under a microscope the ECV304 intradermal cell at the capillary structure that the collagen surface forms, calculate the tubular structure number.
(4) calculate the length of capillary structure or the area that is covered with the ScionImage image analysis software;
(5) compare with control group, the evaluating material surface properties filters out the material that promotes revascularization to the effect of revascularization.
The extracorporeal blood vessel regenerating model postgraduate fabric texture engineering materials degraded product that utilization the present invention discloses is to the influence of revascularization, its process steps and research material degraded product to the process steps of revascularization influence different places be (1), (2) step, other steps are basic identical.Postgraduate's fabric texture engineering materials degraded product to (1), (2) step of revascularization influence is:
(1) the ECV304 cell inoculation is produced extracorporeal blood vessel regenerating model in the collagen gel surface;
(2) mixing solutions of RPMI1640 nutrient solution and institute's research material degraded product is put on extracorporeal blood vessel regenerating model and cultivate, make the ECV304 cell at the spontaneous formation capillary structure in collagen gel surface.
The present invention selects the ECV304 endothelial cell strain for use, has overcome the culturing cell purification difficult, limited amount, culture cycle is long, and is big for differences, and the result is difficult to relatively wait problems between the different experiments chamber, detect when can be used for great amount of samples, favorable reproducibility is easy to stdn as a result.And extracorporeal blood vessel regenerating model of the present invention, there is not the appositional growth factor, got rid of the interference of somatomedin to interpretation of result, also reduced experimental cost.The ECV304 cell can form TLS at short notice, and experimental period is short, can obtain experimental result fast.The ECV304 cell can be observed under common opticmicroscope at the TLS that gel surface forms, and plant and instrument is not had particular requirement, is easy to popularize in most of laboratories.Of the present invention open, be the influence that research evaluation bioengineered tissue material forms revascularization, screening can promote the bioengineered tissue material of revascularization that an effective tool is provided.
Description of drawings
Accompanying drawing 1 is that the ECV304 cell is cultivated the form that forms on polystyrene culture plate surface.
Accompanying drawing 2 is that the ECV304 cell is cultivated the capillary structure that forms on the collagen gel surface.The arrow indication is tube chamber sample gap
Embodiment:
The preparation embodiment of collagen gel:
I type ox-hide collagen (or other type i collagens) is dissolved in mass concentration 0.1% acetate (acetic acid concentration 0.1-2% all can), is made into concentration and is the collagen solution of 0.8mg/ml (concentration 0.1～1.5mg/ml all can).Get 5 times of spissated RPMI1640 nutrient solutions (cycles of concentration 5-10 doubly all can) with collagen solution by 5/2 volume ratio (volume ratio be 5/2 or 7/1 all can) rapid mixing in ice bath, the neutralizer that adds 3 parts of volume parts again is mixed to solution and is orange red uniformly.Neutralizer by 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) (200mmol/L), NaHCO
3(0.2619mol/L), NaOH (50mmol/L) is mixed with, pH=7.4 (pH=6.5-7.5 all can).Add in 96 orifice plates mixture is careful, 50 μ l/ holes, under 37 ℃ condition, cultivate be no less than 30 minutes and make collagen gel standby.
ECV304/ collagen gel blood vessel regenerating model is made embodiment
The ECV304 cell of taking the logarithm vegetative period, mass concentration 0.25% tryptic digestion, it is 0.5 * 10 that RPMI1640 nutrient solution (containing 10% foetal calf serum, 100U/ml penicillin, 100U/ml Streptomycin sulphate) is made density
5～1.5 * 10
5The individual cells suspension of individual/ml is inoculated on the collagen gel in 96 well culture plates, at every hole 100～50 μ l, 37 ℃, 5%CO
2, cultivated 8 hours under the saturated humidity, under the inverted phase contrast microscope, can be observed the capillary structure (TLS) that the ECV304 endotheliocyte forms on the collagen surface.
Used in tissue engineering material surface character is to the detection embodiment of revascularization influence
(D L-PLA) is example with poly-dl-lactide
The preparation of film: D, L-PLA is made into the solution (concentration 0.5-2% can) of 1% mass concentration with trichloromethane dissolving, at cover glass (2.6 * 2.2cm
2) surface filming, every cover glass surface-coated D, L-PLA solution 100～500ul.After placement was spent the night in the air, vacuum-drying was to weight.Uv sterilisation 2h is above standby.
TLS detects: with the ECV304 cell of 0.25% tryptic digestion culture plate surface logarithmic phase, it is 1 * 10 that adding RPMI1640 nutrient solution (adding 10% foetal calf serum in addition, 100U/ml penicillin, 100U/ml Streptomycin sulphate) is made density
4～5 * 10
4The individual cells suspension of individual/ml is inoculated in D, L-PLA film surface, 37 ℃, 5%CO
2, be cultured to cytogamy under the saturated humidity and form individual layer.With the ECV304 cell on 0.25% tryptic digestion film surface, it is 0.5 * 10 that the RPMI1640 nutrient solution (adding 5% foetal calf serum in addition) that adds low serum is made density
5～1.5 * 10
5The individual cells suspension of individual/ml is inoculated on the collagen gel in 96 well culture plates every hole 100 μ l, 37 ℃, 5%CO
2, cultivated 8 hours under the saturated humidity.Inverted phase contrast microscope is observed the ECV304 intradermal cell down at the TLS that the collagen surface forms, and calculates the tubular structure number.Calculate the length of TLS or the area that is covered with the ScionImage image analysis software.With culture plate (polystyrene) is contrast.Detected result and control group are compared, estimate.The result shows, at D, the L-PLA superficial cell forms the number of TLS and length all greater than the cell on control group surface, with remarkable (p＜the 0.05) (D of control group comparing difference, the L-PLA surface is respectively 22 ± 2,7963 ± 438.23um, and the control group surface is respectively 18 ± 4.1,6897.92 ± 344.90um).Promptly show D, the L-PLA surface can promote the formation of TLS than the vascularization of polystyrene surface.
Used in tissue engineering material degradation product is to the detection embodiment of revascularization influence
(D L-PLA) is example with poly-dl-lactide
Degraded product preparation: with the solvent evaporation method casting film.Solvent is a trichloromethane, and concentration is 5%, and mould is the tetrafluoroethylene mould.After the drying at room temperature, vacuum-drying is to weight.Co
60Standby behind the irradiation.With reference to ISO 1099313 degraded D, L-PLA film.The degraded medium is a stroke-physiological saline solution.After degraded for some time (1,7,14,30,60,90,120 day), take out D, the L-PLA film obtains degradation solution.
TLS detects: press preceding method and make ECV304/ collagen gel blood vessel regenerating model.With the mixing solutions of RPMI1640 nutrient solution and degraded product as nutrient solution (volume ratio is 1/1).Inverted phase contrast microscope is observed the ECV304 endotheliocyte down at the TLS that the collagen surface forms after 8 hours, calculates the tubular structure number.Calculate the length of TLS or the area that is covered with image analysis software.With physiological saline is control group.Measuring result and control group are compared, estimate.Measuring result sees attached list 1.Measurement shows in the subordinate list 1, D, the degraded product in preceding 1 week of L-PLA slightly promote the formation of TLS, slightly suppress in the time of 14 days, but there was no significant difference (p＜0.05).30 days restraining effect remarkable (p＜0.05), restraining effect strengthens gradually afterwards, slightly take a favorable turn to 120 days, but still has slight inhibition (p＜0.05).
Table 1
| Degradation time (my god) | TLS (individual) | TLS length (μ m) |
| 17 14 30 60 90 120 control groups | 18±2.6 18±3.7 17±3.1 15±2.8 15±2.2 16±3.9 16±3.6 18±4.1 | 7677.93±691.01 ** 7876.49±78.76 ** 6781.04±88.15 * 6114.99±409.70 ** 5491.42±307.51 ** 5890.73±483.03 ** 6337.88±500.69 ** 6897.92±344.90 ** |
Annotate:
*Compare there was no significant difference (p＜0.05) with control group;
*With control group comparing difference remarkable (p＜0.05)
Claims (10)
1, a kind of extracorporeal blood vessel regenerating model that is used for bioengineered tissue material vascularization functional evaluation is characterized in that this model is prepared by following method:
(1) be that the collagen solution of 0.1～1.5mg/ml and nutrient solution that cycles of concentration is 5～10 times and pH value are 6.5～7.5 neutralizer by (5～7) with concentration: (2～1): the volume ratio of (3～2) is mixed with collagen gel;
(2) with the ECV304 cell inoculation to the collagen gel surface, under culture environment, cultivate, make the ECV304 cell at the spontaneous formation capillary structure in collagen gel surface, promptly be prepared into extracorporeal blood vessel regenerating model.
2, the extracorporeal blood vessel regenerating model that is used for bioengineered tissue material vascularization functional evaluation as claimed in claim 1 is characterized in that described collagen solution is dissolved in mass concentration 0.1～2% acetate by type i collagen to be mixed with.
3, the extracorporeal blood vessel regenerating model that is used for bioengineered tissue material vascularization functional evaluation as claimed in claim 1 is characterized in that described neutralizer is by HEPES, NaHCO
3Be mixed with NaOH.
4, the extracorporeal blood vessel regenerating model that is used for bioengineered tissue material vascularization functional evaluation as claimed in claim 1 is characterized in that collagen solution and nutrient solution and neutralizer are mixed with collagen gel in the mode of quick ice bath.
5, the extracorporeal blood vessel regenerating model that is used for bioengineered tissue material vascularization functional evaluation as claimed in claim 1 is characterized in that the ECV304 cell that described ECV304 cell is a logarithmic phase.
6, the extracorporeal blood vessel regenerating model that is used for bioengineered tissue material vascularization functional evaluation as claimed in claim 5 is characterized in that the ECV304 cell is digested by trypsinase, adds to be inoculated on the collagen gel after nutrient solution is made cell suspension.
7, the extracorporeal blood vessel regenerating model that is used for bioengineered tissue material vascularization functional evaluation as claimed in claim 6, it is characterized in that the ECV304 cell of described cell suspension, behind 0.25% tryptic digestion, add the RPMI1640 nutrient solution by logarithmic phase.
8, the extracorporeal blood vessel regenerating model that is used for bioengineered tissue material vascularization functional evaluation as claimed in claim 6 is characterized in that the culture environment of cell inoculation on collagen gel is, every hole 50～100 μ l of culture plate, and temperature is 37 ℃, atmosphere is 5%CO
2, humidity is saturated humidity.
9, about the application of the described extracorporeal blood vessel regenerating model of each claim in the claim 1 to 8 in bioengineered tissue material vascularization functional evaluation, it is characterized in that application process mainly may further comprise the steps:
(1) with tryptic digestion ECV304 cell, adds nutrient solution and make cell suspension, be inoculated in the bioengineered tissue material surface of being studied, be cultured to cytogamy and form individual layer;
(2) with the ECV304 cell of tryptic digestion material surface growth, add nutrient solution and make cell suspension, be inoculated into the collagen gel surface, be cultured to capillary structure and form, promptly make blood vessel regenerating model;
(3) examine under a microscope the ECV304 intradermal cell at the capillary structure that the collagen surface forms, calculate the tubular structure number.
(4) calculate the length of capillary structure or the area that is covered with the ScionImage image analysis software;
(5) compare with control group, the evaluating material surface properties filters out the material that promotes revascularization to the effect of revascularization.
10, with the method for the described extracorporeal blood vessel regenerating model of each claim in the claim 1 to 8, it is characterized in that mainly may further comprise the steps to tissue engineering material vascularization functional evaluation:
(1) the ECV304 cell inoculation is produced extracorporeal blood vessel regenerating model in the collagen gel surface;
(2) mixing solutions of RPMI1640 nutrient solution and institute's research material degraded product is put on extracorporeal blood vessel regenerating model and cultivate, make the ECV304 cell at the spontaneous formation capillary structure in collagen gel surface;
(3) examine under a microscope the ECV304 intradermal cell at the capillary structure that the collagen surface forms, calculate the tubular structure number.
(4) calculate the length of capillary structure or the area that is covered with the ScionImage image analysis software;
(5) compare with control group, the evaluating material surface properties filters out the material that promotes revascularization to the effect of revascularization.
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|---|---|---|---|
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115516077A (en) * | 2020-06-22 | 2022-12-23 | 凸版印刷株式会社 | Gel composition and method for producing same, and three-dimensional structure and method for producing same |
| US11859210B2 (en) | 2012-09-28 | 2024-01-02 | Scripps Health | Methods of differentiating stem cells into chondrocytes |
-
2007
- 2007-02-27 CN CNB2007100485303A patent/CN100554408C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11859210B2 (en) | 2012-09-28 | 2024-01-02 | Scripps Health | Methods of differentiating stem cells into chondrocytes |
| CN115516077A (en) * | 2020-06-22 | 2022-12-23 | 凸版印刷株式会社 | Gel composition and method for producing same, and three-dimensional structure and method for producing same |
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