CN104587525A - Scaffold containing platelets and hyaluronic acid and preparation method of scaffold - Google Patents
Scaffold containing platelets and hyaluronic acid and preparation method of scaffold Download PDFInfo
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- CN104587525A CN104587525A CN201410805755.9A CN201410805755A CN104587525A CN 104587525 A CN104587525 A CN 104587525A CN 201410805755 A CN201410805755 A CN 201410805755A CN 104587525 A CN104587525 A CN 104587525A
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 38
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Landscapes
- Materials For Medical Uses (AREA)
Abstract
The invention is suitable for the technical field of biomedicine materials and provides a biological scaffold containing platelets and hyaluronic acid which are mixed uniformly, wherein the platelets are activated by calcium ions before being mixed with hyaluronic acid. The invention also provides a preparation method of the biological scaffold. The preparation method comprises the following steps: obtaining platelet-rich plasma (PRP); adding calcium ions into the PRP; and uniformly mixing the obtained PRP with hyaluronic acid. The biological scaffold provided by the invention has good biological compatibility and more pore structures and can ensure that the platelets slowly release growth factors.
Description
Technical field
The invention belongs to technical field of biomedical materials, be specifically related to one and comprise platelet and hyaluronic biological support and preparation method thereof and application thereof.
Background technology
Biomaterial for medical purpose is with a wide range of applications in the injury repairing and plastic surgery of tissue.Theoretical basis of its application is physiological environment in analogue body in vitro, on the normal tissue cell of cultured and amplified in vitro is attached on biologic bracket material that the good and degradable of biocompatibility absorbs, and make it increase to cells with nutrient, when cell and biological support form complex, can by the lesion of this complex implanting to human body tissue or organ.Along with timbering material is progressively degraded in vivo, cell is constantly bred and extracellular matrix secretion, obtain cambium thus and this be organized in form, function aspects and corresponding tissue, organ are consistent, thus reach the object of reparation wound and tissue remodeling.
Bio-medical material conventional at present is mainly divided into the biomaterial of natural degradable, the degradable biomaterial of synthetic.But most of biologic bracket material of application at present also exists various defect, and such as immunogenicity is high, poor biocompatibility, and mechanical strength is low.Therefore, the bioactive bracket material of development of new, to promoting that the development of tissue engineering has great importance.
Hyaluronic acid (Haluronic acid, HA) is a kind of acidic mucopolysaccharide, is extensively present in animal and human's body, in the skin of people, synovium of joint liquid, umbilical cord and vitreum, all have distribution.Hyaluronic acid is used widely at field of medicaments, and it can be used for the filler of the operation on joint such as adhered elastomer, osteoarthritis and rheumatic arthritis in the operation of ophthalmology Intraocular implantation.Hyaluronic acid also has the effect impelling medicament slow release and prevention postoperative intestinal adhesion.In addition because HA has good water absorption and humidity-preserving type, be widely used in cosmetics and shaping padding field.Therefore, hyaluronic acid has good histocompatibility.
Platelet rich plasma (Platelet-rich plasma, PRP) be extract from whole blood containing the hematoblastic blood plasma of high concentration.After PRP mixes with calcium chloride and thrombin of beef, platelet wherein can be activated formation gel, and the multiple somatomedin of intra platelet free calcium, comprises transforming growth factor-β (TGF-β), insulin like growth factor (IGF), epidermal growth factor (EGF) etc.But for the simple gel be made up of PRP, wherein platelet is easily shunk, and somatomedin runs off in a large number, this gel can only play a role within a short period of time; Be confined to the hematoblastic time-to-live in addition, PRP gel can only now do existing use, is unfavorable for the large-scale application of PRP.Therefore need badly and provide a kind of carrier bracket, this carrier bracket both can keep the multi-pore structure of PRP inside, can maintain again somatomedin slow releasing wherein, can also keep hematoblastic activity when lyophilizing simultaneously.
Summary of the invention
The object of the present invention is to provide a kind of biological support, this biological support has multi-pore structure, somatomedin slow releasing can be maintained, can biologically active pdgf be kept through long-time cryopreservation simultaneously, be intended to solve the problem that in prior art, biologic bracket material effect is undesirable.
For overcoming the above problems, the invention provides a kind of biological support, comprising platelet and the hyaluronic acid of mixing, this platelet before mixing with hyaluronic acid through calcium ion activated.
Another object of the present invention is to the preparation method that a kind of biological support is provided, comprise the following steps:
(1) hematoblastic blood plasma PRP is rich in acquisition;
(2) calcium ion is added, to activate platelet to being rich in hematoblastic blood plasma PRP;
(3) the hematoblastic blood plasma that is rich in that the platelet that step (2) obtains is activated is mixed homogeneously with hyaluronic acid solution.
The present invention also provides the store method of biological support, comprising: biological support is placed in liquid nitrogen and places 1.5-2.5 hour, is then placed in freezer dryer lyophilizing 40-50 hour.
Biological support provided by the invention contains platelet and hyaluronic acid, and this kind of biomaterial is not mentioned in the prior art.Hematoblastic blood plasma is rich in the present invention's utilization and hyaluronic acid prepares biological support, and this biological support has good biocompatibility, can fully degrade in vivo.Through electron-microscope scanning checking, the support that biological support of the present invention is made relative to the simple support be made up of platelet and hyaluronic acid has more pore structure, and this support can also make platelet energy slow releasing somatomedin simultaneously.Simultaneously in biological support store method of the present invention, through freezing and frozen dried, the platelet in this biological support can keep active fully, and therefore this store method extends the pot-life.Preparation method for biological support of the present invention, easy and simple to handle, production efficiency is high, with low cost, has good prospects for commercial application.
Accompanying drawing explanation
Fig. 1 is the scanning electron microscopic picture of display PRP supporting structure;
Fig. 2 is the scanning electron microscopic picture of display HA supporting structure;
The scanning electron microscopic picture of the PRP/HA mixed biologic supporting structure that Fig. 3 provides for the display embodiment of the present invention;
Fig. 4 shows the adhesion situation of HeLa cell in different stent materials and compares;
Fig. 5 shows the proliferative conditions of HeLa cell in different stent materials and compares.
Detailed description of the invention
In order to make the technical problem to be solved in the present invention, technical scheme and beneficial effect clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The embodiment of the present invention provides a kind of biological support, comprises platelet and the hyaluronic acid of mixing, wherein platelet before mixing with hyaluronic acid through calcium ion activated.
In embodiments of the present invention, platelet for mixing with hyaluronic acid comes from platelet rich plasma (Platelet-rich plasma, PRP), the implication of platelet rich plasma is known in the art, wherein platelet rich plasma is the platelet concentrate that whole blood obtains after centrifugal, wherein includes the platelet of in whole blood more than 70%.
Platelet rich plasma is that fresh blood is prepared from through low-speed centrifugal.Common preparation method is: by the whole blood of collection at room temperature with 27.5 ~ 37.5 turns/min low-speed centrifugal, 15 ~ 20min (or the centrifugal 5min of 1220 turns/min), make erythrocyte, leukocyte sinks substantially, get upper plasma, due to platelet light specific gravity, major part is retained in upper plasma, obtains platelet rich plasma thus.
In the embodiment of the present invention, adopt secondary centrifuging legal system for platelet rich plasma, that is, carry out two times centrifugal, the centrifugal rear upper serum of second time is carried out resuspended to being enriched with hematoblastic sediment fraction.
The embodiment of the present invention can come from calcium chloride or calcium gluconate for activating hematoblastic calcium ion.In activation platelet process, by adding calcium chloride or calcium gluconate solution makes calcium ion final concentration in final mixed solution be 0.1% ~ 0.5% in platelet rich plasma.
Preferably, after add calcium chloride or calcium gluconate in PRP, this mixed solution is placed 5-20min in 30 DEG C-37 DEG C and fully react to make it, obtain best activation effect thus.
The platelet be activated can keep good activity, sustainable release somatomedin in application process in follow-up body, is conducive to playing best therapeutic effect.
In the application, as do not explicitly not pointed out, then the solution of indication is aqueous solution, and such as calcium chloride solution refers to the aqueous solution of calcium chloride.Mass percent concentration is interpreted as the concentration represented with percent.
In the embodiment of the present invention, the hyaluronic acid mixed with the PRP that platelet is activated is the hyaluronic acid solution of 0.5-2%.Preferably, be 1:1-1:4 for the preparation of the PRP of biological support of the present invention and the volume ratio of hyaluronic acid solution.
The embodiment of the present invention also provides a kind of preparation method of biological support, and the method comprises the following steps:
S01 prepares PRP: obtain venous blood, by secondary centrifuging legal system for platelet rich plasma PRP, wherein secondary centrifuging method is described above;
S02 platelet activation: add calcium chloride solution or calcium gluconate solution in the PRP that step S01 obtains, make the final concentration of calcium ion in gained mixture be 0.1% ~ 0.5%, then place 5-20min in 30 DEG C-37 DEG C;
The hyaluronic acid solution of PRP and the 0.5-2% that above-mentioned platelet is activated by S03 is with the volume ratio mix homogeneously of 1:1-1:4.
The biological support prepared by the embodiment of the present invention can keep hematoblastic activity, can slow releasing somatomedin, plays hyaluronic biocompatibility simultaneously, impels effect of medicament slow release and prevention postoperative intestinal adhesion.
Particularly, in above-mentioned steps S03, the PRP/ hyaluronic acid mixed liquor mix homogeneously of gained is carried out preferably by shaking table.Particularly, this mixed liquor is positioned over 30-37 DEG C of shaking table, with 150-200rpm/min rotating speed, vibration 20-40min.
In a particular embodiment, above-mentioned mixed liquor can be directly applied to medical field, such as that above-mentioned mixed liquor is subcutaneous as being injected between implant, also can be further processed, apply widely to obtain.Particularly, can by the mixed liquor of above-mentioned mix homogeneously in left at room temperature 2-3 hour, to obtain PRP/ hyaluronic acid mixed gel.
Further, the gel of above-mentioned acquisition can be placed in liquid nitrogen and place 1.5-2.5 hour, then be placed in freezer dryer lyophilizing 40-50 hour, namely obtain PRP/ hyaluronic acid scaffold.This kind of frozen dried can improve the pot-life of biological support of the present invention, and with lyophilizing before gel phase than the activity not reducing biological support.
Particularly, the temperature of the biological support Ying Yu after lyophilizing-20 DEG C or lower is preserved, to obtain the best pot-life.
Gel prepared by the embodiment of the present invention, can pre-determine the shape of gel as required, such as, poured into by PRP/ hyaluronic acid mixed liquor and have in the container of reservation shape; Alternatively, also the support after lyophilizing can be cut into predetermined shape, to be applied to different fields.
At the biological support of the present invention after frozen dried, normal saline should be utilized before the use to reduce and to make it play maximum bioactivity to activate platelet.
Preparation method of the present invention is easy and simple to handle, and cost is low, and the biological support of preparation maintains activity and the function of two kinds of main components simultaneously, and through frozen dried, substantially prolongs the pot-life not reduce activity simultaneously.
Below by way of specific embodiment, the preparation method of biological support of the present invention and the performance parameter of this biological support of gained are after testing described.
Embodiment one PRP/HA compound bio support preparation method
S01 obtains venous blood, and by secondary centrifuging legal system for platelet rich plasma PRP, wherein secondary centrifuging method concrete operation step can be see: Jin Zhe etc., " secondary centrifuging legal system is for the comparison of platelet rich plasma centrifugal condition ", Chinese Medical Sciences University's journal, the 41st volume, 3rd phase, in March, 2012.
Add calcium chloride solution in the platelet rich plasma that S02 obtains to step S01, make the final concentration of calcium chloride in gained plasma mixtures be 0.5%, mixing, then place 5min in 37 DEG C, activating reaction is fully carried out.
What step S02 obtained by S03 mixes with the ratio of 1:4 containing being chlorinated the hyaluronic acid solution that calcium-activated hematoblastic PRP and concentration are 0.5%.
The mixture that step S03 obtains by S04, in left at room temperature 3 hours, makes the full cross-linked molding of hyaluronic acid in mixture, obtains PRP/ hyaluronic acid mixed gel.
The gel that step S04 obtains by S05 is placed in liquid nitrogen and places taking-up after 1.5 hours, puts into freezer dryer lyophilizing 40 hours, namely obtains PRP/ hyaluronic acid scaffold of the present invention.
Embodiment two PRP/HA compound bio support preparation method
S01 obtains venous blood, and by secondary centrifuging legal system for platelet rich plasma PRP, wherein secondary centrifuging method concrete operation step is with embodiment one.
Add calcium gluconate solution in the platelet rich plasma that S02 obtains to step S01, make the final concentration of calcium gluconate in gained plasma mixtures be 0.1%, mixing, then place 20min in 30 DEG C, calcium ion is fully activated for platelet.
What step S02 obtained by S03 is 2% containing the hematoblastic PRP be activated and concentration, and hyaluronic acid solution mixes with the ratio of 1:1.
The mixture that step S03 obtains by S04, in left at room temperature 2 hours, makes the full cross-linked molding of hyaluronic acid in mixture, obtains containing hematoblastic PRP/ hyaluronic acid mixed gel.
The gel that step S04 obtains by S05 is placed in liquid nitrogen and takes out after freezing 2.5 hours, puts into freezer dryer lyophilizing 50 hours, namely obtains PRP/ hyaluronic acid scaffold of the present invention, and this support is in-20 DEG C of preservations.
Embodiment three PRP/HA compound bio support Electronic Speculum detects
PRP/HA biological support obtained to PRP biological support, HA biological support (preparation method slightly) and above-described embodiment one is individually fixed in aluminum stake, gold-plated to three kinds of different biological support sputterings by injection apparatus, all adopt the gold-plated 90sec of 15mA electric current, then detect under being placed in scanning electron microscope (also referred to as scanning electron microscope, SEM).
Fig. 1 shows the scanning result of the biological support be made up of PRP, and Fig. 2 shows the scanning result of the biological support be made up of HA, and Fig. 3 shows the scanning result of the PRP/HA biological support that the embodiment of the present invention provides.Can find out through comparing, the configuration of surface (Fig. 1) of PRP support presents block or graininess, aperture is large and irregular, configuration of surface (Fig. 2) compact structure of HA support, porosity is low, and the configuration of surface (Fig. 3) of PRP/HA support prepared by embodiment two has hole not of uniform size, interlock and arrange in length and breadth and there is the range scale from nanoscale to micron.The result of scanning electron microscope shows, PRP/HA mixed biologic support has higher porosity compared to PRP support and HA support.
There are some researches show that the biological support of micro-meter scale will be more conducive to the propagation of cell and external material exchange, the biological support that therefore prepared by embodiment one is more conducive to playing hematoblastic effect and the reparation carrying out in-vivo tissue.
Embodiment four PRP/HA compound bio support biocompatibility detects
In order to evaluate the biocompatibility of PRP/HA support, HeLa cell is placed on biological support prepared by above-described embodiment one and cultivates, detect cell sticking and proliferative conditions on this support, simultaneously with PRP support and HA support in contrast, same HeLa cell culture processes is taked.Concrete operation step is as follows:
1. by HeLa cell with 2 × 10
4/ cm
2density be inoculated in 48 orifice plates, be placed with in advance in each hole PRP support, HA support or embodiment one preparation timbering material, wherein 3 Duplicate Samples established by each sample, do not have the blank well of inoculating cell for negative control with corresponding matrix of materials type.
2., after cell culture 3h, suck culture fluid, with PBS rinsing once.
3. add the cell culture medium of 300 μ l and the mixed liquor (volume ratio 9:1) of CCK-8 in every hole, in cell culture incubator, hatch 2h.
4. every hole is got 200 μ l cell culture fluids and is detected in 96 well culture plates, moves in liquid process and avoids bubble to produce as far as possible.
5. survey the light absorption value (OD) of cell by microplate reader, carry out under A450nm wavelength.
Fig. 4 shows HeLa cell and stick situation on three kinds of timbering material, as shown in the figure, the PRP/HA support that the HeLa cell after cultivation is prepared in embodiment one sticks degree with close on PRP support, but higher than HA support.Fig. 5 shows HeLa cell at the proliferative conditions on three kinds of timbering materials, and as shown in the figure, the propagation degree on the PRP/HA support that HeLa cell is prepared in embodiment one a little more than the propagation degree on PRP support, and is significantly higher than HA support.HeLa cell sticking on three kinds of timbering materials shows with the result of proliferation experiment, and the mixing of PRP and HA obviously can promote sticking and breeding of cell, also namely improves the biocompatibility of timbering material of the present invention.
Embodiment five PRP/HA compound bio support biologically active pdgf detects
Research display, after platelet is activated agent (such as thrombin) activation, the P-selective factor B be present in Cytoplasm on α membrana granulosa can be expressed in platelet cell film surface, wherein P-selective factor B is a kind of glycoprotein, therefore measure platelet by the change of expression of P-selective factor B before activated by thrombin and after activation, platelet can be measured by the degree activated.
Biologically active pdgf detecting step is as follows:
1. get the PRP/HA support 0.5g prepared respectively at-20 DEG C of trimestral PRP supports of cryopreservation and embodiment one, add 200 μ l normal saline reduction;
2. add thrombin, wherein the ratio of thrombin and normal saline is 1:1 (v/v);
3. utilize ELISA kit to detect the expression value of P-selective factor B, testing result is as shown in table 1 below.
Table 1 P-selective factor B expression testing result
The testing result of table 1 shows, the platelet in the PRP/HA support after rehydration reducing activity has more biological activity than the platelet in PRP support.As can be seen here, hematoblastic activity in PRP can be improved by adding HA.Can find out, in the PRP/HA support that after freezing three months prepared by the embodiment of the present invention, biologically active pdgf is still very high simultaneously.
Embodiment six PRP/HA biological support PDGF (platelet derived growth factor) and EGF release detects
The slow releasing of somatomedin has important effect in organizational project application, if growth factor release is too fast, they may be removed rapidly by from transplantation site, and therefore they will reduce greatly to the effect that tissue injury repairs.Desirable load has the biological support of somatomedin generally to need within a couple of days or time several weeks, and somatomedin is discharged lentamente.
Detecting step for PDGF and EGF release in PRP/HA biological support is as follows:
In order to detect rehydration reduction PRP/HA biological support in hematoblastic biological activity, being placed in of the preparing PRP support that low temperature (-20 DEG C) preserves and PRP/HA support are mixed with normal saline respectively, add thrombin afterwards, wherein the ratio of normal saline and thrombin is 1:1 (v/v).After steeping the 1st, 3,5, within 7 days, get the normal saline solution of the immersion support of appropriate amount respectively, detected the release conditions of somatomedin PDGF and EGF by elisa assay method.
After rehydration, in PRP support, the somatomedin testing result of intra platelet free calcium is as shown in table 2 below, and the testing result of PRP/HA support of the present invention is as shown in table 3.
The somatomedin testing result of PRP support release after table 2 rehydration
The somatomedin testing result of PRP/HA support release after table 3 rehydration
As can be seen from the result of above-mentioned table 2 and table 3, after rehydration, the persistent period of PRP/HA support release somatomedin will obviously be longer than PRP support, and therefore this result shows that PRP/HA support has excellent growth factor slow-release effect.
PRP/HA biological support prepared by above-described embodiment two has also carried out the test as described in embodiment three to six, and result shows that PRP/HA biological support of the present invention has better performance relative to PRP support and HA support equally.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a biological support, comprises platelet and the hyaluronic acid of mixing, described platelet before mixing with hyaluronic acid through calcium ion activated.
2. biological support as claimed in claim 1, it is characterized in that, described platelet comes from platelet rich plasma.
3. biological support as claimed in claim 1, it is characterized in that, described calcium ion comes from calcium chloride or calcium gluconate.
4. biological support as claimed in claim 1, it is characterized in that, in described activation process, the final concentration of calcium ion is 0.1% ~ 0.5%.
5. a preparation method for biological support, comprises the following steps:
(1) hematoblastic blood plasma is rich in acquisition;
(2) calcium ion is added, to activate platelet to described being rich in hematoblastic blood plasma;
(3) the hematoblastic blood plasma that is rich in that the platelet that step (2) obtains is activated is mixed homogeneously with hyaluronic acid solution.
6. the preparation method of biological support as claimed in claim 5, it is characterized in that, the final concentration of the calcium ion added in described step (2) is 0.1% ~ 0.5%.
7. the preparation method of biological support as claimed in claim 5, is characterized in that, described step (2) makes gained mixture place the operation of 5-20min in 30 DEG C-37 DEG C after being also included in and adding calcium ion.
8. the preparation method of biological support as claimed in claim 5, is characterized in that, in described step (3), in hyaluronic acid solution, hyaluronic concentration is 0.5-2%.
9. the preparation method of biological support as claimed in claim 5, is characterized in that, be rich in hematoblastic blood plasma and mix with the volume ratio of 1:1-1:4 with hyaluronic acid solution in described step (3).
10. the store method of the biological support according to any one of claim 1-4, comprises the following steps: described biological support is placed in liquid nitrogen and places 1.5-2.5 hour, is then placed in freezer dryer lyophilizing 40-50 hour.
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