CN104258464A - Attapulgite (ATT)-doped PLGA nano-fiber felt, as well as preparation method and application thereof - Google Patents
Attapulgite (ATT)-doped PLGA nano-fiber felt, as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an attapulgite (ATT)-doped PLGA nano-fiber felt, as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving PLGA in a mixed solvent to obtain a PLGA electrospinning solution; uniformly dispersing ATT in the PLGA electrospinning solution while stirring to obtain a PLGA/ATT electrospinning solution; and electrospinning by adopting the PLGA/ATT electrospinning solution for preparation. Nano-fibers promote human mesenchymal stem cells (hMSC) to be differentiated into bone cells. According to the preparation method provided by the invention, the process is simple, the product is easy to obtain, and the cost of used PLGA and ATT is relatively low; the prepared ATT-doped PLGA nano-fibers have good mechanical properties, blood compatibility and biocompatibility, and have broad application prospects in the fields of pharmaceutical carriers, tissue engineering scaffold materials and the like.
Description
Technical field
The invention belongs to nanofiber felt material and Synthesis and applications field thereof, particularly the PLGA nanofiber mats that adulterates of a kind of attapulgite and Synthesis and applications thereof.
Background technology
Electrostatic spinning technique is as a kind of processing method of novel nano-material, have working process parameter can be in harmonious proportion controlled, raw material availability is high, fiber collecting mode is diversified, experimental repeatability is high and be easy to the advantages such as industrialization, the Static Spinning polymer nanofiber of preparation has again controlled, the great specific surface area of fiber size, high porosity and tridimensional network and can well simulate the features such as natural extracellular matrix (ECM), and electrostatic spinning technique and products thereof is widely applied in a lot of field.Especially in biomedical engineering field, natural polymer and synthesis macromolecule all can be used as spinning material, by high molecular copolymerization, method and the different collection modes such as blended, prepare the superfine function fiber of performance and structure diversification, be applied to the aspect such as organizational project and medicament slow release, be not only a developing direction of tissue-engineering fiber material functional, also there is important scientific value and huge application prospect simultaneously.
PLGA is that one is used by FDA (FDA) approval, has good biocompatibility and the high molecular polymer of biological degradability.PLGA can degrade in human body, and product excretes, therefore to human non-toxic by human normal metabolism.And PLGA polymer, as a kind of excellent bio-medical material, has been widely used in implantable material and tissue engineering rack material field.
Human mesenchymal stem cell (human mesenchymal stem cells, hMSC) is a kind of cell with immunoregulation, self replication and multi-lineage potential.In vivo or under external specific inductive condition, hMSC can be divided into the Various Tissues cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium.After continuous passage cultivation and freezen protective, hMSC still has above-mentioned multi-lineage potential, can be used as a kind of desirable seed cell repairing damage, old and feeble or pathological tissues.These good characteristics that hMSC has make it be widely used in the research of osseous tissue injury repairing.Electrostatic spinning nano fiber can analog cell epimatrix structure ECM well, for the adhesion of cell and growth provide good vitro.Build the nano-device based on electrostatic spinning nano fiber and hMSC, and it to be applied to organizational project be a current study hotspot.
Attapulgite ((Attapulgite, ATT) be a kind of tool fiber pattern layer chain transition structure containing Shuifu County's magnesium silicate clay pit.Attapulgite possesses many excellent features, as have good absorbability, unique dispersibility, high temperature resistant, anti-saline and alkaline, have higher plasticity and cohesive force.The light weight of mineral own, property is crisp, water absorption is strong, and dry after-contraction is little, and suspension is met electrolyte and do not flocculated, do not precipitate.Under physiological environment, ATT can be degraded to non-poisonous material.ATT is widely applied in fields such as chemistry painting industry, catalyst, sewage disposal, cosmetics, food additive, biological medicines.
At present, the inorganic nano-particles such as halloysite nanotubes (halloysite nanotubes), LAP, hydroxyapatite (hydroxyapatite), mesoporous silicon oxide (mesoporous silica) all prepare polymer/inorganic particle Hybrid nanofibers by Static Spinning, and they have the mechanical performance of enhancing, good blood compatibility and excellent biocompatibility.But up to now, still do not have bibliographical information to prepare PLGA/ATT composite nano fiber by method of electrostatic spinning, and evaluate further the research report of the mechanical performance of this composite nano fiber, biological activity and blood compatibility and promotion Derived from Mesenchymal Stem Cells thereof.
Summary of the invention
Technical problem to be solved by this invention is to provide PLGA nanofiber mats and the Synthesis and applications thereof of the doping of a kind of attapulgite, and present invention process is simple, PLGA and ATTA cost used is low, and raw material is easy to get, and can be used for suitability for industrialized production; PLGA/ATT composite nanometer fiber felt can in the Osteoblast Differentiation without any promotion hMSC effective when other inducible factors.
The PLGA nanofiber mats of a kind of attapulgite doping of the present invention, described nanofiber mats is dispersed in Poly(D,L-lactide-co-glycolide PLGA solution by attapulgite ATT, carry out electrostatic spinning to prepare, wherein the mass ratio of ATT and PLGA is 1-3:100.
The preparation method of the PLGA nanofiber mats of a kind of attapulgite doping of the present invention, comprising:
(1) PLGA is dissolved in a solvent, stir and PLGA is dissolved completely, obtain PLGA electrostatic spinning solution; Wherein the mass volume ratio of PLGA and solvent is 1g:4-5mL;
(2) under stirring condition, attapulgite ATT is dispersed in PLGA electrostatic spinning solution, obtains PLGA/ATT electrostatic spinning solution, then carry out electrostatic spinning, dry, to obtain final product.
In described step (1), solvent is THF/DMF mixed solvent, and wherein the volume ratio of THF and DMF is 3:1.
In described step (1), mixing time is 8-12h.
Stir in described step (2) as magnetic agitation, speed is 200-300r/min, and the time is 20-30min.
In described step (2), electrostatic spinning process parameter is voltage 18-22kV, flow velocity 0.8-1.0mL/h, receiving range 15-20cm.
In described step (2), drying is vacuum drying, and wherein the vacuum drying time is 24 – 48h.
The application of the PLGA nanofiber mats of a kind of attapulgite doping of the present invention, the PLGA nanofiber mats of described attapulgite doping promotes that mescenchymal stem cell hMSC is divided into osteoblast.
Described mescenchymal stem cell hMSC is the mescenchymal stem cell of people's Cord Blood-Derived.The PLGA nanofiber mats adulterated by attapulgite utilized growth or inducing culture cultivate mescenchymal stem cell hMSC 14 or 21 days, culture fluid when then getting 14 or 21 days by hMSC lysis, assessment mescenchymal stem cell hMSC is divided into osteoblastic ability.
PLGA and PLGA/ATT is put into 24 well culture plates, cultivate hMSC14 days or 21 days with growth or inducing culture, culture fluid when getting 14 days or 21 days by hMSC lysis;
Get the total protein content that above-mentioned lysate measures every hole hMSC;
The alkaline phosphatase Activity Assessment hMSC getting above-mentioned lysate mensuration hMSC is divided into osteoblastic ability in varied situations;
The content assessment hMSC that culture fluid when getting above-mentioned 14 days or 21 days measures the Bone Gla protein of hMSC secretion is divided into osteoblastic ability in varied situations;
The content assessment hMSC getting the calcium ion of above-mentioned lysate mensuration hMSC is divided into osteoblastic ability in varied situations;
Utilize von-Kossa staining to assess hMSC qualitatively and be divided into osteoblastic ability in varied situations.
Described get 14 days or 21 days culture fluid be cultivate at hMSC within the 13rd day or 20 days, discard original culture fluid, rejoin not containing the DMEM culture medium of FBS, and cultivate 24h.
Described growth medium is pure DMEM culture medium, adds the FBS of 10%, the penicillin/streptomycin of 1%, the ascorbic acid solution of the 5mg/mL of 1%, the β-phosphoglycerol ester solution of the 1mol/mL of 1%.
Described growth medium is pure DMEM culture medium, add the FBS of 10%, the penicillin/streptomycin of 1%, the ascorbic acid (being dissolved in PBS solution) of the 5mg/mL of 1%, the β-phosphoglycerol ester solution (being dissolved in PBS solution) of the 1mol/mL of 1%.Described inducing culture is pure DMEM culture medium, add the FBS of 10%, the penicillin/streptomycin of 1%, the ascorbic acid (being dissolved in PBS solution) of the 5mg/mL of 1%, β-phosphoglycerol the ester solution (being dissolved in PBS solution) of the 1mol/mL of 1%, 1% 10
-5the dexamethasone (derivant) of M.
The present invention is easy to operation, and product is easy to get, and the cost of raw material is low, and all has good biocompatibility, and the PLGA/ATT composite nanometer fiber felt of preparation has broad application prospects in the field such as pharmaceutical carrier, tissue engineering bracket material.
The present invention uses the characterization methods such as scanning electron microscope (SEM), thermogravimetry (TGA), measuring mechanical property to verify the feasibility of the inventive method.In addition, the present invention also evaluates the hydrophilic of material, biocompatibility, the characteristic such as blood compatibility and promotion human mesenchymal stem cell Osteoblast Differentiation ability.Concrete test result is as follows:
(1) test result of scanning electron microscope (SEM)
SEM observes display (shown in accompanying drawing 1), PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber pattern prepared by the present invention is regular, surface is regular, and fibre diameter is respectively 1308 ± 296nm, 909 ± 185nm, 560 ± 117nm and 483 ± 133nm.Clearly, when a certain amount of ATT nanoparticle doped is in PLGA spinning liquid, under same spinning condition, the diameter of gained nanofiber decreases, and mainly causes because doping ATT nanoparticle causes PLGA spinning liquid character (as electrical conductivity, surface tension and viscosity etc.) to change.
(2) test result of TGA
The attached TGA curve that Figure 2 shows that PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats.As can be seen from accompanying drawing 2, clearly, when treatment temperature reaches 600 DEG C, in nanofiber, organic principle (PLGA) is by complete calcination (residual mass is 0); And ATT there is no change (residual mass is 98.7wt.%).The residual mass of PLGA/1wt.%LAP, PLGA/2wt.%LAP and PLGA/3wt.%LAP composite nano fiber is respectively 0.98wt.%, 1.87wt.% and 3.17wt.%, and the ATT added in these residual quantities and spinning liquid is similar to.
(3) measuring mechanical property result
Accompanying drawing 3 is the load-deformation curve of PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats.As can be seen from accompanying drawing 3, the fracture strength of composite nanometer fiber felt improves along with the increase of ATT content, and this illustrates that the ATT of doping can improve the fracture strength of fiber.
(4) contact angle test result
Accompanying drawing 4 is the contact angle test pattern of PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats.Because ATT is a kind of hydrophilic material, therefore, the comparatively equal decrease to some degree of PLGA nanofiber mats of the contact angle of distilled water on PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats surface, this illustrates that ATT is doped in the hydrophilic that can improve PLGA nanofiber mats in PLGA fiber to a certain extent, thus, PLGA/ATT nanofiber mats has application prospect widely in fields such as tissue engineering bracket materials.
(5) variety classes nanofiber mats hemolyzing effect compares
The present invention is by the blood compatibility of haemolysis and anticoagulation experimental evaluation PLGA, PLGA/ATT nanofiber mats.Accompanying drawing 5 is the content of hemoglobin in PBS after hemolytic test.Analyze accompanying drawing 5 known, material Dual culture 2h under human red blood cell and each experimental concentration after centrifugal, in supernatant, the content of hemoglobin all also exists significant difference (* * p<0.01) compared with matched group (distilled water), shows that PLGA, PLGA/1%ATT, PLGA/2%ATT, PLGA/3%ATT all haemolysis do not occur.
(6) variety classes nanofiber mats anticoagulant effect compares
PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats prepared by the present invention, except possessing not except hemolysis, can also stop solidifying of blood there being coagulant to deposit in case.Accompanying drawing 6 is the evaluation results to PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats anticoagulation function, and matched group is coverslip.After blood after accompanying drawing 6 gives treated different time (5,10,20,40 and 60min) is dissolved in distilled water, the content of hemoglobin (represents by the OD value at 541nm place, this OD value and content of hemoglobin positive correlation), the content higher explanation blood coagulation phenomenon of hemoglobin is more not obvious, then material stops the blood coagulation ability of solidifying stronger.Analyze accompanying drawing 6 known, in the time period of setting, the content of experimental group hemoglobin is all higher than matched group, and this illustrates that PLGA, PLGA/1%ATT, PLGA/3%ATT and PLGA/5%ATT nanofiber mats all can suppress solidifying of blood to a certain extent.
(7) evaluation of its biocompatibility of variety classes nanofiber mats
Accompanying drawing 7 and 8 is the evaluation result of the biocompatibility to PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats.As shown in Figure 7, hMSC has good adhesion and proliferation activity on PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats surface, illustrates that mesenchymal stem cells derived from human umbilical blood can normally adhere on above-mentioned material surface and divide.Can be found by the propagation of the 3rd day cell, hMSC cell has significant difference (p<0.05) at the proliferative conditions of PLGA/ATT nanofiber surface compared with TCP, demonstrates PLGA/1% ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats and has good biocompatibility.
Accompanying drawing 8 is that (a) PLGA, (b) PLGA/1%ATT, (c) PLGA/2%ATT scheme with the SEM of the hMSC cell adhesion on (d) PLGA/3%ATT nanofiber mats surface after 2.5% glutaraldehyde solution fixes 1-3 hour, the SEM figure of the hMSC cell proliferation on (e) PLGA, (f) PLGA/1%ATT, (g) PLGA/2%ATT and (h) PLGA/3%ATT nanofiber mats surface.As can be seen from the SEM picture of cell, hMSC cell can adhere to and propagation at each group of nanofiber surface well, and the nanofiber of further proved invention report has good cell compatibility.
(8) PLGA/ATT nanofiber mats promotes the evaluation of human mesenchymal stem cell differentiation capability
The present invention is by being determined at ALP activity, the Bone Gla protein of hMSC secretion and the content of calcium ion of hMSC secretion in growth or inducing culture and utilizing von-Kossa staining and ALP staining experiment comprehensive assessment hMSC to be divided into osteoblastic ability in varied situations.Accompanying drawing 9,10,11,12 is the evaluation result of PLGA/ATT nanofiber mats promotion hMSC differentiation capability.
ALP is the early sign of hMSC Osteoblast Differentiation, as shown in Figure 9, in growth medium, the ALP that PLGA/ATT composite nanometer fiber felt is cultivated the hMSC of 21 days is active in PLGA group and TCP group (P<0.05), close to the TCP group of the inducing cultures of 21 days, illustrate that PLGA/ATT composite nanometer fiber felt is when without the Osteoblast Differentiation that can promote hMSC when any other derivant.
Bone Gla protein is the later stage secretions of hMSC Osteoblast Differentiation, osteocalcin secretion thing content measuring result as shown in Figure 10, that growth medium or inducing culture all showed very low osteocalcin secretion amount at the 14th day, and in the middle of 21 days inducing cultures, TCP group, PLGA group or PLGA/ATT group all presents very high osteocalcin secretion amount, and in the middle of growth medium, only have PLGA/ATT group to show very high osteocalcin secretion amount, all there is significant difference (p<0.05) with TCP group and PLGA group.The result of osteocalcin secretion amount further determined that PLGA/ATT can promote the Osteoblast Differentiation of hMSC.
The height of calcium ion content is the important symbol of hMSC Osteoblast Differentiation degree.Calcium ion content testing result as shown in Figure 11, at the 21st day, TCP group in the middle of inducing culture, PLGA group and PLGA/ATT group calcium ion content the 14th day (p<0.05) all under equal conditions, and only have PLGA/ATT group to rise appreciably (p<0.01) from the calcium ion content of 14 to 21 days in the middle of growth medium, all there is significant difference (p<0.05) in the calcium ion content of 21 days growth medium PLGA/ATT groups and TCP group and PLGA group, calcium ion content testing result also proves that PLGA/ATT can promote the Osteoblast Differentiation of hMSC.
The photo that accompanying drawing 12 dyes for Von Kossa, result shows that growth medium PLGA/ATT group produces the phosphate of a large amount of calcium, proves that PLGA/ATT can promote the Osteoblast Differentiation of hMSC further qualitatively.
beneficial effect
(1) present invention process is simple, PLGA and ATT cost used is low, and raw material is easy to get, and can be used for commercial production;
(2) the PLGA/ATT composite nanometer fiber felt that prepared by the inventive method can in the Osteoblast Differentiation without any promotion hMSC effective when other inducible factors;
(3) the PLGA/ATT composite nanometer fiber felt that prepared by the inventive method has good mechanical performance, blood compatibility and biocompatibility, thus has broad application prospects in the field such as pharmaceutical carrier, tissue engineering bracket material.
Accompanying drawing explanation
Fig. 1 be the present invention relates to (a) PLGA, (b) PLGA/1%ATT, the SEM figure of (c) PLGA/2%ATT and (d) PLGA/3%ATT nanofiber mats and the nanofiber of correspondence thereof diameter distribution profile;
Fig. 2 is the TGA curve of PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats that the present invention relates to;
Fig. 3 is the stress-strain diagram of PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats that the present invention relates to;
Fig. 4 is the contact angle test picture of PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats that the present invention relates to; Wherein (a), (b), (c), (d) are respectively: the contact angle test result of PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats;
Fig. 5 is the haemolysis test result of PLGA, PLGA/1%ATT, PLGA/32%ATT and PLGA/3%ATT nanofiber mats that the present invention relates to; Wherein illustration is that human red cell is respectively at H
2hatch in O, PBS, PLGA (1), PLGA/1%ATT (2), PLGA/2%ATT (3) and PLGA/3%ATT (4) solution 2h centrifugal after photo;
Fig. 6 is the anticoagulation test result of PLGA, PLGA/1%ATT, PLGA/32%ATT and PLGA/3%ATT nanofiber mats that the present invention relates to;
Fig. 7 is the test result that PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats that the present invention relates to affects hMSC cell adhesion vigor and proliferation activity;
Fig. 8 is that hMSC cell cultivates the form of 8 hour cells adhesions on (a) PLGA, (b) PLGA/1%ATT, (c) PLGA/2%ATT and (d) PLGA/3%ATT nanofiber mats surface; E the form of 3 days cell proliferation is cultivated on () PLGA, (f) PLGA/1%ATT, (g) PLGA/2%ATT and (h) PLGA/3%ATT nanofiber mats surface;
Fig. 9 is the ALP active testing result that PLGA and the PLGA/3%ATT nanofiber mats that the present invention relates to affects hMSC differentiation;
Figure 10 is the osteocalcin secretion measurement test result that PLGA and the PLGA/3%ATT nanofiber mats that the present invention relates to affects hMSC differentiation;
Figure 11 is the calcium ion content test result that PLGA and the PLGA/3%ATT nanofiber mats that the present invention relates to affects hMSC differentiation;
Figure 12 is the Von Kossa coloration result that PLGA and the PLGA/3%ATT nanofiber mats that the present invention relates to affects hMSC differentiation.
Detailed description of the invention
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1
Get 4 parts of each 0.8g PLGA, mix with 2.4mL THF and 0.8mL DMF respectively, stirred at ambient temperature 8h, treat that PLGA dissolves completely.8mg, 16mg and 24mg ATT powder is added respectively in the 2nd, 3 and 4 part of PLGA solution.Use magnetic agitation 20min, stir speed (S.S.) is 200r/min, obtains PLGA and PLGA/ATT spinning liquid.PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats is prepared by method of electrostatic spinning.Wherein, receiving range is 15cm voltage be 20kV flow velocity is 0.8mL/h, and the composite nanometer fiber felt of preparation puts drying baker inner drying 48h to remove residual moisture and solvent in vacuum.
SEM observed result shows, PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber pattern obtained is regular, surface is regular, and fibre diameter is respectively 1308 ± 296nm, 909 ± 185nm, 560 ± 117nm and 483 ± 133nm (see accompanying drawing 1).Relatively the diameter of four kinds of different fibers can find, when a certain amount of ATT nanoparticle doped is in PLGA spinning liquid, under same spinning condition, the diameter of gained nanofiber decreases, and mainly causes because doping ATT nanoparticle causes PLGA spinning liquid character (as electrical conductivity, surface tension and viscosity etc.) to change.
Embodiment 2
PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats taking preparation in the embodiment 1 of certain mass (about 5mg) respectively carries out TGA test, the attached TGA curve that Figure 2 shows that PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats.As can be seen from Figure 2, clearly, when treatment temperature reaches 600 DEG C, in nanofiber, organic principle (PLGA) is by complete calcination (residual mass is 0); And ATT there is no change (residual mass is 98.7wt.%).The residual mass of PLGA/1wt.%LAP, PLGA/2wt.%LAP and PLGA/3wt.%LAP composite nano fiber is respectively 0.98wt.%, 1.87wt.% and 3.17wt.%, and the ATT added in these residual quantities and spinning liquid is similar to.
Embodiment 3
PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats of preparation in embodiment 1 is cut into the rectangular of 1cm × 5cm, and 3 Duplicate Samples got by each sample.With the thickness of 3 diverse locations of the every bar fiber felt of miking, average as the thickness of each sample.By the mechanical performance of universal material test machine test fiber felt, draw load-deformation curve (see accompanying drawing 3).As can be seen from load-deformation curve, after a certain amount of ATT is doped in PLGA fiber, the fracture strength of PLGA nanofiber can be improved significantly.
Embodiment 4
PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats of preparation in embodiment 1 is cut into the square tiles of 1cm × 1cm, 3 Duplicate Samples got by each sample.Use research type contact angle measurement DSA 30 tests the water contact angle of different nanofiber mats.Clearly, the comparatively equal decrease to some degree of PLGA nanofiber mats of the contact angle of distilled water on PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats surface, this illustrates that ATT is doped in the hydrophilic (see accompanying drawing 4) that can improve PLGA nanofiber mats in PLGA fiber to a certain extent.
Embodiment 5
The normal adults whole blood 5mL that taking heparin lithium is stable, centrifugal 3min (rotating speed is 3000r/min), with PBS washing precipitation 3 times, obtain erythrocyte.With the proportional arrangement adult blood erythrocyte suspension of PBS according to 1:100, for subsequent use in 4 DEG C of refrigerators.
Take PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats of preparation in the embodiment 1 of certain mass (about 5mg) respectively, 3 Duplicate Samples got by often kind of material.Then, according to fiber mat quality: the ratio of human red cell suspension volume is that by different fiber felt immersion human red cell suspension, (matched group arranges 0.3mL human red cell suspension and is dissolved in 1.2mLPBS 2mg:1mL, and 0.3mL human red cell suspension is dissolved in 1.2mL distilled water), and 2h is hatched under 37 DEG C of environment.Then, take out fiber felt, and will the centrifugal 1min of human red cell suspension (1000rpm) of fiber felt be soaked, get supernatant and test the light absorption value of supernatant at 540nm place, with the hemolytic of evaluating material with Lambda 25 type ultraviolet spectrophotometer.Experimental result shows that haemolysis (see accompanying drawing 5) does not all occur for PLGA, PLGA/1%ATT, PLGA/2%ATT, PLGA/3%ATT.
Embodiment 6
PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats of preparation in embodiment 1 is cut into the square tiles of 1cm × 1cm, 3 Duplicate Samples (matched group is: the coverslip of diameter 1.8mm) got by each sample.Test group and matched group are placed in 12 porocyte culture plates.Then, in every hole, fiber felt and matched group coverslip drip the normal adults whole blood prepared in 20 μ L embodiments 6, add 10 μ LCaCl simultaneously
2solution (0.2mol/L), hatches the different time under being placed in 37 DEG C of environment.After each incubation time terminates, add 5mL distilled water to every hole and hatch 5 minutes, then with the light absorption value of Lamada 25 type ultraviolet spectrophotometer test solution at 540nm place, with the anticoagulant property of evaluating material.Experimental result shows that PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats all can suppress solidifying (see accompanying drawing 6) of blood to a certain extent.
Embodiment 7
PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats of preparation in embodiment 1 is cut into 2cm × 2cm fritter, the coverslip spreading over diameter 1.8cm is placed in 24 porocyte culture plates.Carry out ultraviolet sterilization to paving fiber on the cover slip and soak 12h by DMEM α-12 culture medium, then every hole supplements 400 μ L culture medium and inoculates 2 × 10
4individual hMSC cell.Every block culture plate arranges different incubation times (8h and 3d).After each incubation time terminates, in culture hole, add 40 μ L MTT, continue to cultivate 4h, living cells and MTT are acted on completely and generates MTT first a ceremonial jade-ladle, used in libation crystal.Discard the culture medium in every hole, add 400 μ L DMSO, MTT first a ceremonial jade-ladle, used in libation crystal is dissolved completely and records the OD value at 570nm place, under analyzing different incubation time, cell, at the adhesion of nanofiber surface or proliferative conditions, evaluates the impact of PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats on cell adhesion and propagation.
The adhesion of hMSC cell on PLGA, PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats surface and proliferation activity, illustrate that mesenchymal stem cells derived from human umbilical blood can normally adhere on above-mentioned material surface and break up.Can be found by the propagation (see accompanying drawing 7) comparing the 3rd day cell, hMSC cell has significant difference (p<0.05) at the proliferative conditions of PLGA/ATT nanofiber surface compared with pure TCP, further demonstrates PLGA/1%ATT, PLGA/2%ATT and PLGA/3%ATT nanofiber mats and has good cell compatibility.
Embodiment 8
The hMSC cell PBS buffer solution cultivating 8 hours and 3 days in embodiment 8 is washed 3 times, and fixes 2h with the glutaraldehyde solution of 2.5%.Then, above-mentioned fixing hMSC cell graded ethanol (30%, 50%, 70%, 80%, 90%, 95% and 100%) is carried out processed successively, and process 10min, ethanol consumption is 1mL at every turn.By after the hMSC cell natural drying that is disposed, observed the cell morphology (see accompanying drawing 8) of nanofiber surface by SEM.Result shows, hMSC cell can adhere to and propagation at each group of nanofiber surface well, and the nanofiber of further proved invention report has good biocompatibility.
Embodiment 9
PLGA and the PLGA/3%ATT nanofiber mats of preparation in embodiment 1 is cut into 2cm × 2cm fritter, the coverslip spreading over diameter 1.8cm is placed in 24 porocyte culture plates.Ultraviolet sterilization is carried out to paving fiber on the cover slip and soaks 12h by DMEM α-12 culture medium, then original culture medium is sucked, every Kong Xin adds 1000 μ L growth medium (pure DMEM culture medium, add the FBS of 10%, the penicillin/streptomycin of 1%, the ascorbic acid (being dissolved in PBS solution) of the 5mg/mL of 1%, β-phosphoglycerol the ester solution (being dissolved in PBS solution) of the 1mol/mL of 1%) or inducing culture (pure DMEM culture medium, add the FBS of 10%, the penicillin/streptomycin of 1%, the ascorbic acid (being dissolved in PBS solution) of the 5mg/mL of 1%, β-phosphoglycerol the ester solution (being dissolved in PBS solution) of the 1mol/mL of 1%, the dexamethasone (derivant) of the 10-5M of 1%), and inoculate 2 × 10
4individual hMSC cell.Within every three days, change once corresponding culture medium, cultivate 14 days or 21 days.
After the cultivation of 14 days or 21 days, by cell Reporter LysisBuffer cell pyrolysis liquid cracking in above-mentioned each hole, cleavage method carries out according to Reporter Lysis Buffer lysate description method.After cracking, get lysate and test every hole ALP activity according to ALKALINE PHOSPHATASE ACTIVITY description.
Get above-mentioned lysate tests total protein in every hole content according to BCA protein reagent box description.Therefore can the ALP of unit of account albumen active.Experimental result shows that PLGA/ATT can promote the Osteoblast Differentiation (see accompanying drawing 9) of hMSC.
Embodiment 10
People's Bone Gla protein test kit (Intact Human Osteocalcin EIA Kit) is utilized to measure the amount of hMSC secretion Bone Gla protein in each hole after the cultivation of 14 days and 21 days.Because FBS can disturb the mensuration of BGP content, therefore hMSC needs in the growth not containing FBS or inducing culture, to cultivate 24h in advance.After cultivation terminates, respectively by the media transfer in every hole in Eppendorf pipe, the operating procedure provided according to people's Bone Gla protein test kit description tests the BGP content of each group of hMSC secretion.Experimental result shows, that growth medium or inducing culture all showed very low osteocalcin secretion amount at the 14th day, and in the middle of 21 days inducing cultures, TCP group, PLGA group or PLGA/ATT group all presents very high osteocalcin secretion amount, and in the middle of growth medium, only have PLGA/ATT group to show very high osteocalcin secretion amount, all there is significant difference (p<0.05) with TCP group and PLGA group.The result of osteocalcin secretion amount further determined that PLGA/ATT can promote the Osteoblast Differentiation (see accompanying drawing 10) of hMSC.
Embodiment 11
In Example 9 cell pyrolysis liquid with QuantiChrom calcium ion concentration kit measurement after 14 days or 21 days are cultivated the calcium ion concentration of hMSC.Method of testing is carried out to specifications.Experimental result shows, at the 21st day, TCP group in the middle of inducing culture, PLGA group and PLGA/ATT group calcium ion content the 14th day (p<0.05) all under equal conditions, and only have PLGA/ATT group to rise appreciably (p<0.01) from the calcium ion content of 14 days to 21 days in the middle of growth medium, all there is significant difference (p<0.05) (see accompanying drawing 11) in the calcium ion content of 21 days growth medium PLGA/ATT groups and TCP group and PLGA group, calcium ion content testing result also proves that PLGA/ATT can promote the Osteoblast Differentiation of hMSC.
Embodiment 12
The formaldehyde of 3.7% (solvent is PBS solution) is utilized by the hMSC cultivated 21 days in embodiment 9 under 4 DEG C of conditions, to fix 2 hours then with the formaldehyde that PBS solution cleaning removing is residual.Calcification situation is observed by von Kossa staining, first 2.5% silver nitrate process (every hole 300 μ L) is used, be placed in 60min under ultraviolet light simultaneously, with deionized water rinse 3 times, then use the hypo solution of 5% (every hole 300 μ L) to continue process 3min.Finally, after sodium thiosulfate washes clean, digital camera is used to take pictures to the fiber sample after dyeing.Experimental result (see accompanying drawing 12) shows, growth medium PLGA/ATT group produces the phosphate of a large amount of calcium, proves that PLGA/ATT can promote the Osteoblast Differentiation of hMSC further qualitatively.
Claims (10)
1. the PLGA nanofiber mats of an attapulgite doping, it is characterized in that: described nanofiber mats is dispersed in Poly(D,L-lactide-co-glycolide PLGA solution by attapulgite ATT, carry out electrostatic spinning to prepare, wherein the mass ratio of ATT and PLGA is 1-3:100.
2. a preparation method for the PLGA nanofiber mats of attapulgite doping, comprising:
(1) PLGA is dissolved in a solvent, stir and PLGA is dissolved completely, obtain PLGA electrostatic spinning solution; Wherein the mass volume ratio of PLGA and solvent is 1g:4-5mL;
(2) under stirring condition, attapulgite ATT is dispersed in PLGA electrostatic spinning solution, obtains PLGA/ATT electrostatic spinning solution, then carry out electrostatic spinning, dry, to obtain final product.
3. the preparation method of the PLGA nanofiber mats of a kind of attapulgite doping according to claim 2, is characterized in that: in described step (1), solvent is THF/DMF mixed solvent, and wherein the volume ratio of THF and DMF is 3:1.
4. the preparation method of the PLGA nanofiber mats of a kind of attapulgite doping according to claim 2, is characterized in that: in described step (1), mixing time is 8-12h.
5. the preparation method of the PLGA nanofiber mats of a kind of attapulgite doping according to claim 2, is characterized in that: stir in described step (2) as magnetic agitation, speed is 200-300r/min, and the time is 20-30min.
6. the preparation method of the PLGA nanofiber mats of a kind of attapulgite doping according to claim 2, is characterized in that: in described step (2), electrostatic spinning process parameter is voltage 18-22kV, flow velocity 0.8-1.0mL/h, receiving range 15-20cm.
7. an application for the PLGA nanofiber mats of attapulgite doping as claimed in claim 1, is characterized in that: the PLGA nanofiber mats of described attapulgite doping promotes that mescenchymal stem cell hMSC is divided into osteoblast.
8. the application of the PLGA nanofiber mats of a kind of attapulgite doping according to claim 7, it is characterized in that: the PLGA nanofiber mats adulterated by attapulgite utilized growth or inducing culture cultivate mescenchymal stem cell hMSC 14 or 21 days, culture fluid when then getting 14 or 21 days by hMSC lysis, assessment mescenchymal stem cell hMSC is divided into osteoblastic ability.
9. the application of the PLGA nanofiber mats of a kind of attapulgite doping according to claim 7, it is characterized in that: described growth medium is pure DMEM culture medium, add the FBS of 10%, the penicillin/streptomycin of 1%, the ascorbic acid solution of the 5mg/mL of 1%, the β-phosphoglycerol ester solution of the 1mol/mL of 1%.
10. the application of the PLGA nanofiber mats of a kind of attapulgite doping according to claim 7, it is characterized in that: described inducing culture is pure DMEM culture medium, add the FBS of 10%, the penicillin/streptomycin of 1%, the ascorbic acid of the 5mg/mL of 1%, β-phosphoglycerol the ester solution of the 1mol/mL of 1%, 1% 10
-5the dexamethasone of M.
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| CN114990724A (en) * | 2022-06-20 | 2022-09-02 | 苏州卡彭新材料科技有限公司 | Diamond-doped PLGA nanofiber composite material |
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