CN102240415A - Composite porous support material and preparation method thereof - Google Patents
Composite porous support material and preparation method thereof Download PDFInfo
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- CN102240415A CN102240415A CN2011101436423A CN201110143642A CN102240415A CN 102240415 A CN102240415 A CN 102240415A CN 2011101436423 A CN2011101436423 A CN 2011101436423A CN 201110143642 A CN201110143642 A CN 201110143642A CN 102240415 A CN102240415 A CN 102240415A
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Abstract
The invention relates to a composite porous support material prepared from the following raw materials in percentage by mass: 80%-95% of polylactic acid-glycollic acid copolymer, 2.5%-10% of hydroxylapatite and 2.5%-10% of collagen. The porosity of the composite porous support material is adjustable within the range of 45.2-97.4 percent, and the aperture is adjustable within the range of 45-1100 micrometers. According to the invention, the composite porous support material is prepared under the condition of supercritical CO2 through lower reaction pressure and shorter reaction time. Experimental results indicate that cells of a human osteosarcoma cell line MG-63 are adhered to the composite porous support material and have good proliferation status, the composite porous support material cannot cause obvious inflammatory reaction when implanted into the body of a rat, and various inflammatory factors are basically similar to blank groups at postoperative 28th day, and therefore the composite porous support material disclosed by the invention has good biocompatibility.
Description
Technical field
The invention belongs to the material technology field, be specifically related to a kind of porous support materials and supercritical CO
2Technology prepares the method for porous support materials.
Background technology
Tissue engineering bracket material has become the focus of field of tissue engineering technology research.The structure of timbering material and performance directly influence the reparation and the reconstruction of tissue and organ.In field of tissue engineering technology, various timbering materials have been used to the template of cell proliferation and differentiation.Homogenous material is difficult to satisfy the demand of organizational project, and several biomaterial for medical purpose are compound, by the performance that the difference of each constituent content is regulated material monolithic, makes it have better biocompatibility.
PLGA (poly lactate glycollic acid, the polylactic acid-polyglycolic acid copolymer) is a kind of degradable functional polymer organic compound, have excellent biological compatibility, nontoxic, good encystation and filming performance, be widely used in pharmacy, medical engineering material and modernized industrial field.Hydroxyapatite has higher mechanical strength, and its structure is similar to nature bone with character, has obtained using widely in bone tissue engineer.Collagen protein has excellent biological compatibility, can participate in the organization healing process, is stopping blooding, promotes wound healing, is being used widely as aspects such as burn wound's dressing, bone transplantation substitute material, tissue regeneration inducers.Therefore, PLGA, hydroxyapatite, collagen protein three are carried out compound, can remedy the defective of homogenous material.
The aperture of timbering material and structure directly determine the space structure and the form of cell, and the space structure of the cell particularly proximity of cell and cell is then being controlled the propagation and the differentiation of cell.Different field of tissue engineering technology needs the timbering material in different apertures.
In recent years, supercritical CO
2Technology has been applied to prepare various timbering materials.By the control supercritical CO
2Operating condition, as reaction pressure, temperature and time, can regulate the pore structure and the porosity of timbering material.Abroad, have about PLGA at supercritical CO
2In dissolubility, expansion character and use supercritical CO
2Report (J.Supercriti.Fluids 39 (2007) 416-425 of preparation PLGA porous support materials; Biotechnol.Bioeng5 (2008) 998-1009).Domestic, Ren Jie etc. utilize supercritical CO
2Technology has prepared used in tissue engineering porous support materials (patent CN 1749319A).Zhang Run etc. utilize supercritical CO
2Technology has prepared the good polylactic acid porous material (investigation of materials journal 6 (2003) 661-672) of biocompatibility.But in existing report, utilize supercritical CO
2The most aperture ratio of timbering material of preparation is less, and is most below 100 μ m, and preparation pressure height, time are long.And in existing report and the patent, do not use polylactic-co-glycolic acid, hydroxyapatite and collagen protein to prepare porous support materials simultaneously.
Summary of the invention
A technical problem to be solved by this invention is to overcome the shortcoming of above-mentioned timbering material, provides that a kind of aperture is big, porosity is high, the composite porous support material of good biocompatibility.
Another technical problem to be solved by this invention is to provide a kind of preparation method for above-mentioned composite porous support material.
It is that to make the aperture by following quality proportion raw material be that 45～1100 μ m, porosity are 45.2%～97.4% composite porous support material to solve the problems of the technologies described above the technical scheme that adopted:
Polylactic acid-polyglycolic acid copolymer 80%～95%
Hydroxyapatite 2.5%～10%
Collagen protein 2.5%～10%
Above-mentioned polylactic acid-polyglycolic acid copolymer is sold by Mount Tai, Jinan handle of the Big Dipper Bioisystech Co., Ltd, and the mass ratio of lactic acid and glycolic is 80: 20, and molecular weight is 10000 dalton, and glass transition temperature is 45～55 ℃, and viscosity IV is 0.7dl/g.
The preferred mass proportioning of preparation composite porous support material of the present invention is:
Polylactic acid-polyglycolic acid copolymer 90%～95%
Hydroxyapatite 2.5%～5%
Collagen protein 2.5%～5%
The best in quality proportioning of preparation composite porous support material of the present invention is:
Polylactic acid-polyglycolic acid copolymer 95%
Hydroxyapatite 2.5%
Collagen protein 2.5%
The mutual UNICOM of hole in the complex stephanoporate bracket of the present invention.
The preparation method of above-mentioned composite porous support material is: according to the proportion by weight of above-mentioned raw materials, with polylactic acid-polyglycolic acid copolymer, hydroxyapatite, collagen protein mix homogeneously, with tablet machine cold moudling in mould, mould places supercritical CO
2In the reactor, close CO
2Vent valve, setting reaction pressure are that 10～30MPa, reaction temperature are 35～60 ℃, CO
2Flow velocity is 10mL/ minute, opens CO
2Steel cylinder valve, high-pressure pump and temperature control valve reacted 15～90 minutes, opened CO
2Vent valve cuts out CO
2The switch of steel cylinder valve, high-pressure pump and reactor, pressure take out mould after reducing to normal pressure from reactor, obtain composite porous support material.
Preferred reaction pressure is that 14～26Mpa, reaction temperature are that 40～50 ℃, response time are 30～60 minutes in the preparation method of the present invention.
The present invention adopts lower pressure and short time to prepare the compound support frame material of the polylactic acid-polyglycolic acid copolymer of larger aperture and hydroxyapatite, collagen protein, and timbering material is characterized and the inside and outside biocompatibility test.Experimental result shows, this timbering material aperture is even, porosity is high, human osteosarcoma cell line MG-63 cell on this compound support frame material, adhere to and vegetative state good, to not cause tangible inflammatory reaction in this timbering material implantation rat body, 28 days after surgery, each inflammatory factor is similar substantially to blank group, illustrates that this timbering material has excellent biological compatibility.
Description of drawings
Fig. 1 is the composite porous support material SEM figure of embodiment 1 preparation.
Fig. 2 is the composite porous support material SEM figure of embodiment 2 preparations.
Fig. 3 is the composite porous support material SEM figure of embodiment 3 preparations.
Fig. 4 is the composite porous support material SEM figure of embodiment 4 preparations.
Fig. 5 is the composite porous support material SEM figure of embodiment 5 preparations.
Fig. 6 is the composite porous support material SEM figure of embodiment 6 preparations.
Fig. 7 is the composite porous support material SEM figure of embodiment 7 preparations.
Fig. 8 is the composite porous support material SEM figure of embodiment 8 preparations.
Fig. 9 is the composite porous support material SEM figure of embodiment 9 preparations.
Figure 10 is the composite porous support material SEM figure of embodiment 10 preparations.
Figure 11 is the composite porous support material SEM figure of embodiment 11 preparations.
Figure 12 is the composite porous support material SEM figure of embodiment 12 preparations.
Figure 13 is the SEM figure that cultivated behind the inoculating cell for scaffold material 3 days.
Figure 14 is the SEM figure that cultivated behind the inoculating cell for scaffold material 7 days.
Figure 15 is the SEM figure that cultivated behind the inoculating cell for scaffold material 14 days.
Figure 16 is the CLSM figure that cultivated behind the inoculating cell for scaffold material 3 days.
Figure 17 is the CLSM figure that cultivated behind the inoculating cell for scaffold material 7 days.
Figure 18 is the CLSM figure that cultivated behind the inoculating cell for scaffold material 14 days.
Figure 19 is that 7 days timbering material implant sites of blank group postoperative close on muscular tissue HE colored graph.
Figure 20 is that 14 days timbering material implant sites of blank group postoperative close on muscular tissue HE colored graph.
Figure 21 is that 28 days timbering material implant sites of blank group postoperative close on muscular tissue HE colored graph.
Figure 22 is that 7 days timbering material implant sites of test group postoperative close on muscular tissue HE colored graph.
Figure 23 is that 14 days timbering material implant sites of test group postoperative close on muscular tissue HE colored graph.
Figure 24 is that 28 days timbering material implant sites of test group postoperative close on muscular tissue HE colored graph.
The specific embodiment
Below in conjunction with drawings and Examples the present invention is advanced-go on foot detailed description, but the invention is not restricted to these embodiment.
100g is that used raw material of example and proportioning thereof are with the preparation composite porous support material:
Polylactic acid-polyglycolic acid copolymer 95g
Hydroxyapatite 2.5g
Collagen protein 2.5g
Its preparation method is as follows:
Get polylactic acid-polyglycolic acid copolymer 95g, hydroxyapatite 2.5g, collagen protein 2.5g, mix homogeneously, being cold-pressed into diameter with tablet machine is that 3cm, thickness are the disk of 1.5cm, and the concrete size of disk can be that 2～5cm, thickness are that the size according to reactor is determined in the scope of 1～2cm at diameter.Tabletting is put into mould, and mould places supercritical CO
2In the reactor, close CO
2Vent valve, setting reaction pressure are that 18MPa, reaction temperature are 45 ℃, CO
2Flow velocity is 10mL/ minute, opens CO
2Steel cylinder valve, high-pressure pump and temperature control valve make pressure and temperature reach setting value, react 45 minutes, open CO
2Vent valve cuts out CO
2The switch of steel cylinder valve, high-pressure pump and reactor, pressure take out mould after reducing to normal pressure from reactor, be prepared into composite porous support material.
Prepared composite porous support material carries out pattern with the Quanta200 environmental scanning electron microscope and characterizes, and the results are shown in Figure 1, and according to the porosity of following method test bracket material: with known volume (V
1) dehydrated alcohol place graduated cylinder, support (dry weight is W) was immersed wherein 5 minutes, the cumulative volume of the timbering material that dehydrated alcohol and dehydrated alcohol are saturated is V
2Take out the saturated timbering material of dehydrated alcohol, the volume of remaining dehydrated alcohol is V
3The cumulative volume of timbering material is: V=(V
2-V
1)+(V
1-V
3)=V
2-V
3, V
2-V
1Be the volume of polymeric stent material, V
1-V
3Be the volume of dehydrated alcohol in the timbering material, then the porosity of timbering material is: ε (%)=(V
1-V
3)/(V
2-V
3) * 100.The result shows that the hole of this complex stephanoporate bracket is open, and better connective between the Kong Yukong, average pore size is 205 μ m, and porosity is 88.9%.
In embodiment 1, mould is placed supercritical CO
2In the reactor, close CO
2Vent valve, setting reaction pressure are that 12MPa, reaction temperature are 60 ℃, CO
2Flow velocity is 10mL/ minute, opens CO
2Steel cylinder valve, high-pressure pump and temperature control valve make pressure and temperature reach setting value, react 30 minutes, and other steps are identical with corresponding embodiment, are prepared into composite porous support material.
Prepared composite porous support material carries out pattern with the Quanta200 environmental scanning electron microscope and characterizes, and the results are shown in Figure 2, and tests its porosity according to the method among the embodiment 1.The result shows, is interconnected between the part hole of this complex stephanoporate bracket, and average pore size is 730 μ m, and porosity is 83.6%.
Embodiment 3
In embodiment 1, mould is placed supercritical CO
2In the reactor, close CO
2Vent valve, setting reaction pressure are that 12MPa, reaction temperature are 35 ℃, CO
2Flow velocity is 10mL/ minute, opens CO
2Steel cylinder valve, high-pressure pump and temperature control valve make pressure and temperature reach setting value, react 30 minutes, and other steps are identical with corresponding embodiment, are prepared into composite porous support material.
Prepared composite porous support material carries out pattern with the Quanta200 environmental scanning electron microscope and characterizes, and the results are shown in Figure 3, and tests its porosity according to the method among the embodiment 1.The result shows, is interconnected between the part hole of this complex stephanoporate bracket, and average pore size is 300 μ m, and porosity is 48.6%.
In embodiment 1, mould is placed supercritical CO
2In the reactor, close CO
2Vent valve, setting reaction pressure are that 12MPa, reaction temperature are 40 ℃, CO
2Flow velocity is 10mL/ minute, opens CO
2Steel cylinder valve, high-pressure pump and temperature control valve make pressure and temperature reach setting value, react 30 minutes, and other steps are identical with corresponding embodiment, are prepared into composite porous support material.
Prepared composite porous support material carries out pattern with the Quanta200 environmental scanning electron microscope and characterizes, and the results are shown in Figure 4, and tests its porosity according to pycnometric method.The result shows, is interconnected between the part hole of this complex stephanoporate bracket, and average pore size is 327 μ m, and porosity is 60.7%.
Embodiment 5
In embodiment 1, mould is placed supercritical CO
2In the reactor, close CO
2Vent valve, setting reaction pressure are that 12MPa, reaction temperature are 50 ℃, CO
2Flow velocity is 10mL/ minute, opens CO
2Steel cylinder valve, high-pressure pump and temperature control valve make pressure and temperature reach setting value, react 30 minutes, and other steps are identical with corresponding embodiment, are prepared into composite porous support material.
Prepared composite porous support material carries out pattern with the Quanta200 environmental scanning electron microscope and characterizes, and the results are shown in Figure 5, and tests its porosity according to the method among the embodiment 1.The result shows, is interconnected between the part hole of this complex stephanoporate bracket, and average pore size is 540 μ m, and porosity is 75.8%.
In embodiment 1, mould is placed supercritical CO
2In the reactor, close CO
2Vent valve, setting reaction pressure are that 12MPa, reaction temperature are 45 ℃, CO
2Flow velocity is 10mL/ minute, opens CO
2Steel cylinder valve, high-pressure pump and temperature control valve make pressure and temperature reach setting value, react 15 minutes, and other steps are identical with corresponding embodiment, are prepared into composite porous support material.
Prepared composite porous support material carries out pattern with the Quanta200 environmental scanning electron microscope and characterizes, and the results are shown in Figure 6, and tests its porosity according to the method among the embodiment 1.The result shows, is interconnected between the part hole of this complex stephanoporate bracket, and average pore size is 537 μ m, and porosity is 58.4%.
In embodiment 1, mould is placed supercritical CO
2In the reactor, close CO
2Vent valve, setting reaction pressure are that 12MPa, reaction temperature are 45 ℃, CO
2Flow velocity is 10mL/ minute, opens CO
2Steel cylinder valve, high-pressure pump and temperature control valve make pressure and temperature reach setting value, react 60 minutes, and other steps are identical with corresponding embodiment, are prepared into composite porous support material.
Prepared composite porous support material carries out pattern with the Quanta200 environmental scanning electron microscope and characterizes, and the results are shown in Figure 7, and tests its porosity according to the method among the embodiment 1.The result shows, is interconnected between the part hole of this complex stephanoporate bracket, and average pore size is 375 μ m, and porosity is 83.9%.
In embodiment 1, mould is placed supercritical CO
2In the reactor, close CO
2Vent valve, setting reaction pressure are that 12MPa, reaction temperature are 45 ℃, CO
2Flow velocity is 10mL/ minute, opens CO
2Steel cylinder valve, high-pressure pump and temperature control valve make pressure and temperature reach setting value, react 90 minutes, and other steps are identical with corresponding embodiment, are prepared into composite porous support material.
Prepared composite porous support material carries out pattern with the Quanta200 environmental scanning electron microscope and characterizes, and the results are shown in Figure 8, and tests its porosity according to the method among the embodiment 1.The result shows, is interconnected between the part hole of this complex stephanoporate bracket, and average pore size is 430 μ m, and porosity is 87.2%.
In embodiment 1, mould is placed supercritical CO
2In the reactor, close CO
2Vent valve, setting reaction pressure are that 10MPa, reaction temperature are 45 ℃, CO
2Flow velocity is 10mL/ minute, opens CO
2Steel cylinder valve, high-pressure pump and temperature control valve make pressure and temperature reach setting value, react 45 minutes, and other steps are identical with corresponding embodiment, are prepared into composite porous support material.
Prepared composite porous support material carries out pattern with the Quanta200 environmental scanning electron microscope and characterizes, and the results are shown in Figure 9, and tests its porosity according to the method among the embodiment 1.The result shows, is interconnected between the part hole of this complex stephanoporate bracket, and average pore size is 1100 μ m, and porosity is 45.2%.
In embodiment 1, mould is placed supercritical CO
2In the reactor, close CO
2Vent valve, setting reaction pressure are that 14MPa, reaction temperature are 45 ℃, CO
2Flow velocity is 10mL/ minute, opens CO
2Steel cylinder valve, high-pressure pump and temperature control valve make pressure and temperature reach setting value, react 45 minutes, and other steps are identical with corresponding embodiment, are prepared into composite porous support material.
Prepared composite porous support material carries out pattern with the Quanta200 environmental scanning electron microscope and characterizes, and the results are shown in Figure 10, and tests its porosity according to the method among the embodiment 1.The result shows, is interconnected between the part hole of this complex stephanoporate bracket, and average pore size is 358 μ m, and porosity is 83.5%.
In embodiment 1, mould is placed supercritical CO
2In the reactor, close CO
2Vent valve, setting reaction pressure are that 26MPa, reaction temperature are 45 ℃, CO
2Flow velocity is 10mL/ minute, opens CO
2Steel cylinder valve, high-pressure pump and temperature control valve make pressure and temperature reach setting value, react 45 minutes, and other steps are identical with corresponding embodiment, are prepared into composite porous support material.
Prepared composite porous support material carries out pattern with the Quanta200 environmental scanning electron microscope and characterizes, and the results are shown in Figure 11, and tests its porosity according to the method among the embodiment 1.The result shows that the hole of this complex stephanoporate bracket is open, and better connective between the Kong Yukong, average pore size is 100 μ m, and porosity is 95.7%.
In embodiment 1, mould is placed supercritical CO
2In the reactor, close CO
2Vent valve, setting reaction pressure are that 30MPa, reaction temperature are 45 ℃, CO
2Flow velocity is 10mL/ minute, opens CO
2Steel cylinder valve, high-pressure pump and temperature control valve make pressure and temperature reach setting value, react 45 minutes, and other steps are identical with corresponding embodiment, are prepared into composite porous support material.
Prepared composite porous support material carries out pattern with the Quanta200 environmental scanning electron microscope and characterizes, and the results are shown in Figure 12, and tests its porosity according to the method among the embodiment 1.The result shows that the hole of this complex stephanoporate bracket is open, and better connective between the Kong Yukong, average pore size is 45 μ m, and porosity is 97.4%.
Embodiment 13
100g is that used raw material of example and proportioning thereof are with the preparation composite porous support material:
Polylactic acid-polyglycolic acid copolymer 80g
Hydroxyapatite 10g
Collagen protein 10g
Its preparation method is identical with enforcement 1.
100g is that used raw material of example and proportioning thereof are with the preparation composite porous support material:
Polylactic acid-polyglycolic acid copolymer 90g
Hydroxyapatite 5g
Collagen protein 5g
Its preparation method is identical with enforcement 1.
In order to prove beneficial effect of the present invention, the inventor has carried out various performance tests to the composite porous support material of the embodiment of the invention 1 preparation, and various test situation are as follows:
Experiment material: human osteosarcoma cell line MG-63 cell is purchased in China typical culture collection center (Wuhan); Healthy male SD rat, body weight 200～220g purchases in Xi'an Jiaotong University Medical College's Experimental Animal Center (Xi'an); Hyclone, Dulbecco ' s Modified Eagle Medium (DMEM) cell culture fluid, tetramethyl azo azoles salt (MTT), Hirst fluorescent dye (Hoechst 33342), type i collagen, pancreatin, dimethyl sulfoxine are purchased the Sigma-Aldrich company in the U.S.; Glutaraldehyde, paraformaldehyde are produced by U.S. Amersco company; Interleukin 1 β test kit, interleukin-6 test kit, interleukin 10 test kit, tumor necrosis factor TNF-alpha test kit are by U.S. R﹠amp; D company produces.
Experimental apparatus: Nikon TE2000-s inverted phase contrast microscope, produce by Japanese Nikon company; The Zenyth3100 microplate reader is produced by Austrian Anthos company; Leica TCS SP5CLSM is produced by German Leica company.
1, measures the water absorption rate and the weight-loss ratio of timbering material
The timbering material back of weighing is immersed in 37 ℃ the phosphate buffer solution (0.01mol/L, pH are 7.4), inhaled with filter paper and go the rack surface redundant moisture, replication in selected interval until constant weight, is the weight in wet base of timbering material.
In the formula, w
wBe the weight in wet base of support, w
dInitial dry weight for support.
6 timbering materials back of weighing is immersed in 37 ℃ the phosphate buffer solution (0.01mol/L, pH are 7.4), taken out wherein 1 timbering material respectively, inhale with filter paper and go the rack surface redundant moisture, weigh after the lyophilization at the time point of each setting.
In the formula, w
tBe the dry weight of lyophilization after-poppet material, w
dInitial dry weight for timbering material.
The water absorption rate of timbering material and weight-loss ratio test result see Table 1.
The water absorption rate of table 1 timbering material and weight-loss ratio test result
| 3 |
7 |
14 |
21 days | 28 days | |
| Water absorption rate (%) | 33.93 | 40.51 | 43.75 | 42.48 | 40.54 |
| Weight-loss ratio (%) | 0.08 | 0.36 | 0.58 | 0.64 | 0.75 |
By table 1 as seen, timbering material can reach maximum water absorption rate at 14 days, and after 21 days, the weight-loss ratio of timbering material increases to more than 0.6%.Experimental result shows that timbering material has good hydrophilicity, biological stability and moderate biological degradability.
2, estimate the external biological compatibility of timbering material
(1) cultivate on the plane of cell
The MG-63 cell is placed 25cm
2Tissue Culture Flask in, with containing the DMEM cell culture fluid of 10% (v/v) hyclone at 37 ℃, 5%CO
2, 95% relative humidity CO
2Cultivate in the incubator, changed liquid once in per 2 days.
(2) inoculation of cell on timbering material
Before the MG-63 cell inoculation, be that 15mm, thickness are the timbering material Co of 5mm with diameter
60Irradiation sterilization.Inoculate 5 * 10 on each timbering material
4Individual MG-63 cell.In order to study the suitable time of cell adhesion, there is the timbering material of MG-63 cell to leave standstill respectively 2,4,6 hours inoculation, then timbering material is moved in the centrifuge tube, jolting makes and does not adhere to or adhere to untight MG-63 cell and come off from timbering material, utilizes blood counting chamber to measure after the jolting MG-63 cell quantity in the culture fluid.The cell inoculation rate is calculated as follows:
The cell adhesion result of the test shows, behind the MG-63 cell inoculation, leaves standstill when adding culture fluid in 2 hours, rate of vaccination has only 16.4%, leaves standstill when adding culture fluid in 4 hours, and rate of vaccination can reach 93.7%, yet when leaving standstill 6 hours adding culture fluid, rate of vaccination reduces to 79.6%.Therefore, for the adhesion of MG-63 cell on timbering material, 2 hours too short, and not adding culture fluid in 6 hours then can be owing to the more MG-63 of the making cell of the active component metabolism in the culture fluid can not get enough nutrition, thereby breaks away from support and dead.Therefore, inoculation time the best of MG-63 cell is 4 hours.
(3) propagation of cell on timbering material
With the MG-63 cell to inoculate 5 * 10 on each support
4Individual being inoculated on the timbering material, cell inoculation added 3mL DMEM cell culture fluid after 4 hours, changed liquid once in per 2 days, cultivated respectively 3,7,14 days, utilized MTT test determination cell survival rate, and the result represents with absorbance.Assay method is: the support after cultivating with the DMEM cell culture fluid flushing of serum-free, remove not adherent cell, then it is moved in the new aseptic culture plate, every hole adds the DMEM cell culture fluid of 1mL serum-free and the MTT aqueous solution of 0.1mL 5mg/mL, 37 ℃ are continued to cultivate 4 hours in incubator, have the purple material to form on support in this process.Draw culture fluid, add the 0.5mL dimethyl sulfoxine, and constantly vibration is rocked, make all dissolvings of purple precipitation, get the 0.2mL lysate and change in 96 orifice plates, measure its optical density value (OD) with microplate reader, every group of parallel assay three holes at the 570nm place.Establish in the test that not add the hole that cell only adds culture fluid be the blank hole, other operations are identical with experimental group.During last colorimetric, return to zero with the blank hole.
The MTT test result shows, cultivates the support after 3 days, 7 days and 14 days, and its absorbance is respectively 0.29,0.43 and 0.97, and this shows that timbering material is fit to the cell growth, can promote cell proliferation.
(4) form of cell on timbering material characterizes
Sem analysis: cultivated respectively 3,7,14 days behind the support inoculation MG-63 cell, take out support, with pH value is that 7.4 phosphate buffer cleans support 3 times, be that 2.5% glutaraldehyde water solution is fixed 30 minutes at 4 ℃ with mass fraction, the reuse pH value is 7.4 phosphate buffer cleaning, cut into slices with surgical blade, with volume fraction is 50%, 60%, 70%, 80%, 90%, 95%, 100% ethanol water gradient dehydration (each gradient was washed 30 minutes), observe with SEM behind the metal spraying, the results are shown in Figure 13～15.As seen from the figure, the MG-63 cell is evenly distributed in the timbering material, in the hole of timbering material and the bore edges cell that all distributing uniformly.The pseudopodium of cell surface and microvillus have shown that cell growth state is good.
CLSM analyzes: cultivated respectively 3,7,14 days behind the support inoculation MG-63 cell, take out support, with pH value is that 7.4 phosphate buffer cleans support 3 times, with mass fraction is that 3.7% paraformaldehyde aqueous solution is fixed 30 minutes at 4 ℃, the reuse pH value is 7.4 phosphate buffer cleaning, cut into slices with surgical blade, Hoechst 33342 dyeing liquors with 10 μ g/mL under the lucifuge condition dyeed 10 minutes, be that 7.4 phosphate buffer thorough washing is removed unnecessary dyeing liquor with pH value at last, with the fluorescence imaging of CLSM observation of cell, the results are shown in Figure 16～18.As seen from the figure, the MG-63 Premeabilisation of cells has also been moved in the internal network of timbering material, in the surface and hole of timbering material, can be observed the nucleus of circle, and expression cell growth conditions on support is good.
3, estimate the interior biocompatibility of body of timbering material
(1) laboratory animal and grouping
Healthy male SD rat, body weight 200～220g presses the experimental group sub-cage rearing, and adaptability is raised after 7 days and is experimentized.Rat is divided into two groups of blank group and experimental grouies, 9 every group at random.
(2) plantation in the body of timbering material
Before in timbering material (diameter is that 15mm, thickness are 5mm) is implanted the rat body, use C
o60 irradiation sterilizations.Be that 5% pentobarbital sodium water solution is by 30mg/kg body weight intraperitoneal anesthesia rat with mass fraction, fixing rat, choosing the back both sides is implant site, cut off surperficial fine hair, sterilization, respectively cut off 1 little otch that is about 1.5cm in its spinal column both sides, will be tried material and implant subcutaneous rat, skin suture.Blank group is not implanted any material, just carries out conventional operation technique by the experimental group method.Rat is placed under the ventilation condition, and all animals all can freely drink water and diet.
(3) test method
Clinical follow activities in rats situation, wound healing and material repel situation.Respectively 7 days, 14 days and 28 days pentobarbital sodium execution animals by the injection fatal dose.Carefully cut with 1cm that timbering material links to each other in muscular tissue, cut in half, after half soaks a week with neutral formalin, the tissue that fixes is made wax stone after the dehydration of ethanol gradient, waxdip, embedding, and (interruption is cut into slices 5 times to cut out thick about 4 microns section, 3 of each serial section), with scribbling mass fraction is that the microscope slide of 10% poly-D-lysine aqueous solution is fished for tissue slice, paraffin section was baked 3 hours for 65 ℃, after the dewaxing entry, carry out conventional H E dyeing, last capping, naturally dry, under optical microscope, observe.Second half uses liquid nitrogen freezing rapidly, the inflammatory factor of closing on muscular tissue (content of interleukin 1 β, interleukin-6, interleukin 10 and tumor necrosis factor TNF-alpha) with ELSA kit measurement timbering material implant site, dynamic quantitative research support is implanted biocompatibility in the body of back, the results are shown in Table 2.
(4) result of the test
Bad phenomenon such as all none death of laboratory animal experimental session, incision do not have redness, fester, diet and activity are all normal.Wound healing is good.Estimate classification according to the tissue reaction's stage division among GB/T 16175-2008 and the GB/T 16886.11-1997.
Blank group postoperative observation by light microscope the results are shown in Figure 19～21, and test group postoperative observation by light microscope the results are shown in Figure 22～24.As seen from the figure, blank group postoperative 7 days is seen a small amount of inflammatory cell infiltration in the muscular tissue, mainly be lymphocyte, inflammatory cell extent of reaction II level; Postoperative 14 days, myocyte's sarolemma is more smooth smooth, and myofilament, muscle segment are arranged more neat, and the mitochondrion form is no abnormal, and muscle corpuscle size, form are normal, and kernel does not have pyknosis, inflammatory cell extent of reaction I level; Postoperative 28 days, myocyte's sarolemma is smooth smooth, myofilament, muscle segment marshalling, the mitochondrion form is no abnormal, and muscle corpuscle size, form are normal, and kernel does not have pyknosis.Test group postoperative 7 days, no significant change before material appearance form and volume and the implantation, its surrounding tissue has many point-like hyperemia, the fibrous material hypertrophy is arranged around the material, beginning to form loose cyst wall, see inflammatory cell infiltration in the muscular tissue, mainly is neutrophilic granulocyte and lymphocyte, cyst wall forms IV level, inflammatory cell extent of reaction III level; Postoperative 14 days, material and surrounding tissue begin suddenly to connect, and easily peel off, and the fibrous capsule parcel is not too obvious, the point-like hyperemia of its surrounding tissue still exists, tangible fibrous tissue parcel is arranged around the timbering material, visible fibroblast, fibrocyte and collagen fiber, and formed the fibrous capsule cavity configuration, inflammatory cell is than the preceding obviously minimizing of two weeks, a small amount of lymphocyte is arranged in the muscular tissue, and cyst wall forms III level, inflammatory cell extent of reaction II level; Postoperative 28 days, material is chimeric good with muscular tissue on every side, combines closely and does not have sharp interface.The densification of fibrosis blister cavities, the thickness of wall are thin than the formation initial stage, and the minute quantity lymphocyte is only arranged in the muscular tissue, the muscle fiber marshalling, and the no abnormality seen structure, cyst wall forms II level, inflammatory cell extent of reaction I level.
The inflammatory factor of different time after the table 2 timbering material implantation subcutaneous rat
By table 2 as seen, the tumor necrosis factor TNF-alpha of blank group and test group, interleukin 1 β, interleukin-6 are along with the increase of time, and content reduces gradually.In addition, its content in the time of 7 days after surgery test group organize apparently higher than blank, but 28 days after surgery the time, test group is then near blank group.For interleukin 10, test group is starkly lower than blank group in the time of 7 days after surgery, but 28 days after surgery the time, test group is then near blank group.The minimizing of tumor necrosis factor TNF-alpha, interleukin 1 β and interleukin-6, the expression inflammation alleviates; And the increase of interleukin 10 represents that inflammation alleviates.Therefore, ELSA result shows, being implanted in postoperative and can causing slight inflammation in the time of 7 days of timbering material, and inflammation weakens gradually afterwards, disappears substantially in the time of 28 days after surgery, illustrate that timbering material has the interior biocompatibility of good body.
Claims (6)
1. composite porous support material is characterized in that it is that to make the aperture by following quality proportion raw material be that 45～1100 μ m, porosity are 45.2%～97.4% composite porous support material:
Polylactic acid-polyglycolic acid copolymer 80%～95%
Hydroxyapatite 2.5%～10%
Collagen protein 2.5%～10%
The molecular weight of above-mentioned polylactic acid-polyglycolic acid copolymer is 10000 dalton.
2. composite porous support material according to claim 1 is characterized in that it is that to make the aperture by following quality proportion raw material be 45～1100 μ m, and porosity is 45.2%～97.4% composite porous support material:
Polylactic acid-polyglycolic acid copolymer 90%～95%
Hydroxyapatite 2.5%～5%
Collagen protein 2.5%～5%
3. composite porous support material according to claim 1 is characterized in that it is that to make the aperture by following quality proportion raw material be 45～1100 μ m, and porosity is 45.2%～97.4% composite porous support material:
Polylactic acid-polyglycolic acid copolymer 95%
Hydroxyapatite 2.5%
Collagen protein 2.5%
4. composite porous support material according to claim 1 is characterized in that: the mutual UNICOM of the hole in the complex stephanoporate bracket.
5. the preparation method of the described composite porous support material of claim 1, it is characterized in that: according to the proportion by weight of above-mentioned raw materials, with polylactic acid-polyglycolic acid copolymer, hydroxyapatite, collagen protein mix homogeneously, with tablet machine cold moudling in mould, mould places supercritical CO
2In the reactor, close CO
2Vent valve, setting reaction pressure are that 10～30MPa, reaction temperature are 35～60 ℃, CO
2Flow velocity is 10mL/ minute, opens CO
2Steel cylinder valve, high-pressure pump and temperature control valve reacted 15～90 minutes, opened CO
2Vent valve cuts out CO
2The switch of steel cylinder valve, high-pressure pump and reactor, pressure take out mould after reducing to normal pressure from reactor, obtain composite porous support material.
6. the preparation method of composite porous support material according to claim 5, it is characterized in that: described reaction pressure is 14～26MPa, and reaction temperature is 40～50 ℃, and the response time is 30～60 minutes.
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| CN108686270A (en) * | 2018-06-04 | 2018-10-23 | 杭州电子科技大学 | A kind of high porosity tissue engineering bracket and preparation method thereof |
| CN110198746A (en) * | 2016-09-19 | 2019-09-03 | 坦佩林大学注册基金会 | It is composite porous |
| CN110251730A (en) * | 2019-07-05 | 2019-09-20 | 遵义医科大学 | A kind of porous compound skeleton and preparation method thereof |
| CN111763506A (en) * | 2020-07-09 | 2020-10-13 | 上海浦景化工技术股份有限公司 | Temporary plugging diverting agent and preparation method and application thereof |
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| CN102552984A (en) * | 2012-02-29 | 2012-07-11 | 中国科学院长春应用化学研究所 | Bone tissue engineering bracket material and preparation method thereof |
| CN102552984B (en) * | 2012-02-29 | 2014-08-06 | 中国科学院长春应用化学研究所 | Bone tissue engineering bracket material and preparation method thereof |
| CN103785059A (en) * | 2014-01-03 | 2014-05-14 | 朱小龙 | Bone repair regeneration material and preparation method thereof |
| CN103785059B (en) * | 2014-01-03 | 2015-12-02 | 朱小龙 | A kind of Bone Defect Repari regrown material and preparation method thereof |
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| CN107722331A (en) * | 2017-09-15 | 2018-02-23 | 浙江大学 | The step pressure release foaming technique of supercritical carbon dioxide two prepares the method with double-pore structure bone tissue engineering scaffold |
| CN108686270A (en) * | 2018-06-04 | 2018-10-23 | 杭州电子科技大学 | A kind of high porosity tissue engineering bracket and preparation method thereof |
| CN110251730A (en) * | 2019-07-05 | 2019-09-20 | 遵义医科大学 | A kind of porous compound skeleton and preparation method thereof |
| CN111763506A (en) * | 2020-07-09 | 2020-10-13 | 上海浦景化工技术股份有限公司 | Temporary plugging diverting agent and preparation method and application thereof |
| CN111763506B (en) * | 2020-07-09 | 2023-01-13 | 上海浦景化工技术股份有限公司 | Temporary plugging diverting agent and preparation method and application thereof |
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