CN113717320B - Preparation method of platelet-rich plasma separation gel, and obtained product and application thereof - Google Patents
Preparation method of platelet-rich plasma separation gel, and obtained product and application thereof Download PDFInfo
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- CN113717320B CN113717320B CN202010447607.XA CN202010447607A CN113717320B CN 113717320 B CN113717320 B CN 113717320B CN 202010447607 A CN202010447607 A CN 202010447607A CN 113717320 B CN113717320 B CN 113717320B
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- rich plasma
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- separation gel
- plasma separation
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F261/00—Macromolecular compounds obtained by polymerising monomers on to polymers of oxygen-containing monomers as defined in group C08F16/00
- C08F261/02—Macromolecular compounds obtained by polymerising monomers on to polymers of oxygen-containing monomers as defined in group C08F16/00 on to polymers of unsaturated alcohols
- C08F261/04—Macromolecular compounds obtained by polymerising monomers on to polymers of oxygen-containing monomers as defined in group C08F16/00 on to polymers of unsaturated alcohols on to polymers of vinyl alcohol
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B04—CENTRIFUGAL APPARATUS OR MACHINES FOR CARRYING-OUT PHYSICAL OR CHEMICAL PROCESSES
- B04B—CENTRIFUGES
- B04B5/00—Other centrifuges
- B04B5/04—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers
- B04B5/0407—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers for liquids contained in receptacles
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B04—CENTRIFUGAL APPARATUS OR MACHINES FOR CARRYING-OUT PHYSICAL OR CHEMICAL PROCESSES
- B04B—CENTRIFUGES
- B04B5/00—Other centrifuges
- B04B5/04—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers
- B04B5/0407—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers for liquids contained in receptacles
- B04B5/0414—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers for liquids contained in receptacles comprising test tubes
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F2/00—Processes of polymerisation
- C08F2/44—Polymerisation in the presence of compounding ingredients, e.g. plasticisers, dyestuffs, fillers
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F251/00—Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F251/00—Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
- C08F251/02—Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof on to cellulose or derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K3/00—Use of inorganic substances as compounding ingredients
- C08K3/34—Silicon-containing compounds
- C08K3/36—Silica
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K7/00—Use of ingredients characterised by shape
- C08K7/22—Expanded, porous or hollow particles
- C08K7/24—Expanded, porous or hollow particles inorganic
- C08K7/26—Silicon- containing compounds
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K2201/00—Specific properties of additives
- C08K2201/011—Nanostructured additives
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a preparation method of platelet-rich plasma separation gel, and an obtained product and application thereof, wherein the platelet-rich plasma separation gel is prepared from polyvinyl alcohol, acrylic acid and derivatives, plasticizers, polysaccharide polymers, emulsifying agents, initiators and silicon dioxide monomers.
Description
Technical Field
The invention relates to a preparation method of gel for separating platelet-rich plasma, and also relates to the prepared gel for separating platelet-rich plasma and application of the gel, belonging to the technical field of blood collection and separation.
Background
Blood is composed of plasma and blood cells, which include three cells, red blood cells, white blood cells and platelets. In 1993 Hood first proposed the concept of platelet rich plasma and found that the number of platelets contained in platelet rich plasma was 4-5 times that of normal blood. Based on the different densities of the components in the blood, the anticoagulated blood is centrifuged by centrifugation means. Wherein the bottom layer is red blood cells and white blood cells with higher density, the uppermost layer is platelet-poor plasma, and the middle layer is platelet-rich plasma. The major mechanisms by which platelet rich plasma contains significant amounts of growth factors to promote repair and regeneration of injured tissue, such as bone regeneration, are based on Platelet Derived Growth Factor (PDGF) and transforming growth factor (TGF- β) present in alpha particles contained in platelet rich plasma. When activated, the platelet-rich plasma can release two growth factors for the bone defect part, thereby promoting the repair and regeneration of injured tissues. In addition, by the development and progress of medical beauty technology, it has been found that the excellent effects of beauty, recovery of self body and treatment are achieved by synthesizing the extracted platelet-rich plasma into gel and then returning the platelet-rich plasma to the patient himself by medical means such as surface coating. The main point is that the defect that rejection reaction occurs by using an external body blood product is effectively avoided by using autologous platelet-rich plasma to repair and regenerate injured tissues.
The current methods for separating platelet rich plasma in the market are as follows: 1) A secondary separation method; 2) Gel separation method. The secondary separation method is to separate blood cells and blood plasma of the whole blood by first low-speed centrifugation according to different densities of blood components. Wherein the plasma contains platelet rich plasma and platelet poor plasma, and is mixed with a little red blood cells and other impurities, and then the lower blood component including red blood cells is extracted, and then suspended. And then carrying out secondary centrifugation and suspension to finally obtain platelet-rich plasma. In this method, the first centrifuged blood component needs to be removed, and this process requires the injection of a long needle into a centrifuge tube to aspirate the component that needs to be removed. This process has two potential effects on platelets: 1) The pollution probability of platelets is increased; 2) Platelets are easy to activate and aggregation of platelets is easy to occur; in addition, the method has low recovery rate of platelet rich plasma and low rejection rate of red blood cells. The method is used for extracting platelet-rich plasma from blood in the market at present. Gel separation is also a separation method based on differences in the density of blood components, the density of the gel being between that of red blood cells and platelet rich plasma. The red blood cells and the platelet-rich plasma are isolated by the centrifugal gel, secondary centrifugation is not needed, and the whole operation process is in closed-loop operation. The method has the advantages of reduced activation probability of platelet-rich plasma, simple operation, and high platelet-rich plasma recovery rate. However, this method has specific requirements on the gel, such as thixotropic properties, viscosity, biocompatibility, etc., so that less platelet rich plasma extraction is commercially available. There are few patents reporting on blood separation gels, but there are still respective drawbacks.
Patent CN 106366426a discloses a separation gel system for purifying and purifying platelet-rich plasma and a preparation method thereof, the separation gel system mainly comprises polyisobutylene, chlorinated paraffin, nano silicon dioxide and modified white carbon black, wherein the chlorinated paraffin is a chlorinated derivative of paraffin, and chloride generated during treatment can cause a certain influence on the environment.
Patent CN 101570637B discloses a preparation method of an organosilicon separation gel. The separation gel system mainly comprises organopolysiloxane, hydroxy silicone oil and silane coupling agent. The system stability of the organosilicon is poor, and the problem of failure frequently occurs in the actual use process. In addition, small molecules in the organosilicon can easily form oil globules on the surface of the separated plasma, and the performance of the product is affected.
The patent CN 100341599C discloses an inert gel for separating serum from blood cells, which mainly comprises fumed silica, silica and a stabilizer, and has a specific gravity of 1.04-1.05g/cm at 20deg.C 3 Can be used only for separating serum, and the density of platelet-rich plasma is 1.05g/cm 3 On the left and right, the purity and recovery rate of the platelet-rich plasma are affected to a low extent, and therefore, the method is not suitable for separating and extracting the platelet-rich plasma with high concentration.
Disclosure of Invention
Aiming at a plurality of defects existing in the separation of platelet-rich plasma in the prior art, the invention provides a preparation method of a platelet-rich plasma separation gel. The preparation method is simple to operate, easy to implement and convenient for industrialized production. The obtained separation gel has the advantages of good biocompatibility, good thixotropic property, good platelet separation effect, high platelet recovery rate, low operation difficulty and the like.
Furthermore, the invention also provides a platelet-rich plasma separating tube and a method for extracting platelet-rich plasma by using the separating tube based on the separating gel. The separation tube contains the platelet-rich plasma separation gel, collected blood directly enters the separation tube and is separated in the separation tube, the whole separation process is in a closed sterile environment, the operation is convenient, stable and safe, the separated platelet-rich plasma is not easy to be infected, the recovery rate is high, the direct extraction operation can be directly carried out, secondary transfer is not needed, and the separation efficiency is improved.
The following describes the technical scheme of the invention in detail:
a method of preparing a platelet rich plasma separation gel, the method comprising the steps of:
(1) Uniformly mixing 30-50 parts of polyvinyl alcohol, 8-15 parts of acrylic acid, 1-5 parts of plasticizer and 10-15 parts of polysaccharide polymer, and heating under the protection of gas to fully dissolve the added substances;
(2) Cooling after the reaction, adding 10-20 parts of acrylic acid derivative, 0.1-0.4 part of initiator and 0.1-1 part of emulsifier into the reaction system in the step (1), stirring and mixing uniformly, and heating to perform free radical initiation polymerization reaction;
(3) Cooling after the reaction, adding 0.1-0.4 part of initiator and 0.1-1 part of emulsifier into the system in the step (2), stirring and mixing uniformly, and heating to perform free radical initiated polymerization reaction;
(4) Cooling after the reaction, adding 5-10 parts of silicon dioxide monomer into the reaction system in the step (3), and reacting under stirring and temperature control to obtain platelet-rich plasma separation gel; wherein, each raw material is added in parts by weight.
Further, in the above preparation method, the polyvinyl alcohol is EG-40 grade for medical use, and the viscosity is 40-60mPa.s.
Further, in the above preparation method, the polysaccharide polymer is two or more of chitosan, sodium alginate, starch, calcium alginate, glucomannan and cellulose. During the polymerization reaction, the polysaccharide polymer plays a role in certain emulsification and adhesion.
In the preparation method, the plasticizer is at least one of polylactic acid, tributyl citrate and propylene glycol. The plasticizer can adjust the plasticity of the separation gel. The plasticizer weakens acting force among polymer molecules by being inserted among the polymer molecular chains, and increases the mobility of the polymer molecular chains, so that the influence of the separation gel caused by excessive viscosity of the polymer is avoided.
Further, in the above preparation method, the acrylic acid derivative is at least one of glycidyl acrylate, butyl acrylate, hydroxyethyl methacrylate, methyl acrylate and polyacrylic acid. Wherein the acrylic acid derivative further enhances the polymerization effect of acrylic acid and polyvinyl alcohol in the reaction system.
Further, in the above preparation method, the emulsifier is at least one of glycerin, sodium dodecyl sulfate, sodium dodecyl benzene sulfonate and sorbitol.
Further, the molecular weight of each polymer used in the present invention is not particularly limited, and those usually used in the market may be used.
In the preparation method, the initiator is ammonium persulfate, laurylamine peroxide, azobisisobutyronitrile or benzoyl chloride.
In the preparation method, the silicon dioxide monomer is hydrophobic fumed silica or/and nano silica. The silica monomers are all nano-sized and have a particle size in the range of 45-55nm. The thixotropic property and viscosity of the whole separation gel can be adjusted by the silica monomer, and the thixotropic property and the adhesiveness of the separation gel can be improved to a great extent by the combination of the silica monomer and the hydroxyl bond in the system.
Further, in the step (1), the reaction is performed under a gas atmosphere, and the shielding gas may be nitrogen or an inert gas. The raw materials react at 80-120 ℃ for 2-3h. So that the added substances are sufficiently dissolved.
Further, in the step (2), the temperature is reduced to 55-60 ℃, and after the acrylic acid derivative, the initiator and the emulsifier are added, the reaction is carried out for 2-4 hours at 75-90 ℃.
Further, in the step (3), the temperature is reduced to 40-60 ℃, and after the emulsifier and the initiator are added, the reaction is carried out for 1-3 hours at 70-90 ℃.
Furthermore, the step (2) and the step (3) are added with the emulsifier and the initiator in a sectional manner, which are equivalent to the boosting effect in the reaction system, and the sectional addition aims to ensure that the polymerization reaction system can react completely and improve the yield of the separation gel.
Further, in the step (4), the silica monomer is added and then stirred at 30-40 ℃ for 2-3 hours. The reaction is carried out at a relatively high stirring speed, and preferably under vacuum.
The invention synthesizes the platelet-rich plasma separation gel by the combination mode of the raw materials and the reaction process, the separation gel belongs to biological grade gel, has biological safety performance and high safety, is milky white at normal room temperature, and has the density range of 1.05-1.08g/cm 3 The separation gel has good stability, thixotropic property and separation effect, good platelet separation effect, high platelet recovery rate and low operation difficulty, and therefore, the separation gel is also within the protection scope of the invention.
Furthermore, the invention also provides a platelet-rich plasma separation tube, which comprises the platelet-rich plasma separation gel prepared by the method. Preferably, the separating tube is a vacuum separating tube, i.e. the inside of the separating tube is in a vacuum state. Therefore, through the cooperation with the blood taking needle, the blood of a patient can be directly collected into the separating tube for centrifugal separation based on the vacuum condition in the separating tube, and the cross contamination of the blood is avoided. Through centrifugation, the separation gel forms an obvious boundary between platelet-rich plasma and red blood cells, is not mutually dissolved with the platelet-rich plasma, and has good separation effect.
Further, the separation tube is a blood collection tube.
Further, the separating tube also contains an anticoagulant.
Further, the anticoagulant may be a conventional anticoagulant reported in the prior art, such as heparin, sodium citrate, potassium fluoride, ethylenediamine tetraacetic acid, etc., preferably sodium citrate.
Further, the ratio of anticoagulant to separation gel is preferably 1:8.
furthermore, the invention also provides a method for extracting the platelet-rich plasma, which comprises the steps of collecting whole blood by adopting the platelet-rich plasma separating tube containing the separating gel, and centrifuging, separating and extracting the platelet-rich plasma from the platelet-rich plasma separating tube.
Further, when the platelet-rich plasma separation tube is used for extracting platelet-rich plasma, the method comprises the following specific steps:
(1) Firstly, filling a certain amount of separation gel into a separation tube in a filling mode, adding a certain amount of anticoagulant in proportion, and then performing treatment modes such as vacuum treatment, sterilization and the like to obtain the vacuum separation tube containing the separation gel and the anticoagulant. When the blood taking device is used, based on the negative pressure advantage of the separating tube, the whole blood is taken together with the blood taking needle, and then the separating tube is gently shaken for a plurality of times, so that the whole blood and the anticoagulant are uniformly mixed;
(2) Setting centrifugal speed and time, and centrifuging the separating tube after blood collection;
(3) After centrifugation, layering blood, namely a red blood cell layer, a separation gel layer, a platelet rich plasma layer and an anemic platelet plasma layer from bottom to top;
(4) And slowly sucking out the platelet-rich plasma by adopting a sterile device to obtain the platelet-rich plasma. The platelet-rich plasma extraction method is simple and convenient to operate, has low technical requirements on operators, can be carried out by only once separation in the separation tube, is subjected to whole-process airtight operation, does not need secondary transfer, and is high in safety, and platelet-rich plasma is not easy to be infected. The separation gel is arranged between platelet-rich plasma and red blood cells, has clear interface, good platelet-rich plasma separation effect and high recovery rate.
Compared with the prior art, the adhesive has the following advantages:
1. the silica monomer selected in the platelet rich plasma separation gel of the present invention is nanosilica and hydrophobic fumed silica, preferably both of which are used in combination. The nano silica is based on the characteristics of the nano silica, the density of the separation gel can be regulated by being distributed in the middle of a molecular chain of the polymer, the structure of the hydrophobic fumed silica is different from that of the nano silica, and the hydrophobic fumed silica has the characteristics of large specific surface area, surface hypoxia and the like, is easy to bond and act with oxygen in the polymer, and improves intermolecular force. In addition, the addition amount of the hydrophobic silicon dioxide has little influence on the viscosity coefficient of the system, can exist on the surface of the polymer to play a role of bonding, can exist in the molecular chain of the polymer, shows strong fluidity, and improves the stability, the thickening property and the thixotropic property of the polymer.
2. The invention prepares the bioseparation gel with hydrophobicity and thixotropy through the reaction of polysaccharide polymers and other raw materials which are nontoxic and harmless and have no pollution to the environment. Polysaccharide polymers play two main roles in this system. Firstly, the effect of emulsification and adhesion is achieved, secondly, the polysaccharide has hydroxyl groups, a large number of hydroxyl groups can be provided, the polysaccharide can form an inter-transmission network with silicon hydroxyl groups on the surface of a silicon dioxide monomer, and hydrogen bonds and physical adsorption acting force forming the network are relatively weak acting forces. Under the action of mechanical force, the reticular structure is destroyed to become low-viscosity fluid, the fluidity of colloid can be improved, when the mechanical force disappears, the chain particles of the silica aggregate in the separation colloid form the reticular structure again by hydrogen bonds, the initial viscosity state is restored, and the components such as red blood cells in blood are isolated from the components of platelet-rich plasma, so that the effect of separating platelet-rich plasma is successfully achieved.
3. The separation gel is inert, has biological safety characteristics from the selected materials, can be directly packaged into the separation tube for sale, is convenient to use and carry, and has reliable safety guarantee on the medical level through biocompatibility and no heat source detection, thereby solving the problem of potential safety hazard of the separation gel and the current blood collection tube (namely the separation tube) in the aspect of biocompatibility.
4. The invention can directly extract the platelet-rich plasma in the separating tube, the separating gel can well isolate the platelet-rich plasma from red blood cells after centrifugal separation, and the platelet-rich plasma can be obtained by directly extracting the platelet-rich plasma without fusion interference of the platelet-rich plasma with blood. The method has the advantages of simple operation, high separation efficiency, no need of secondary transfer separation in the whole separation process, difficult infection of the separated platelet-rich plasma, high purity, good separation effect and high recovery rate, and can be used for direct extraction operation.
5. The preparation method of the platelet-rich plasma separation gel is simple and safe, is convenient to develop and apply, and has important significance in the fields of medical science and the like.
Detailed Description
The following detailed description of the invention is merely exemplary in nature and is not intended to limit the scope of the invention, as defined by the appended claims.
Unless otherwise indicated, the amounts of the following raw materials are parts by weight.
The particle size of the hydrophobic fumed silica and the nanosilica used in the examples described below are both 45-55nm. The polyvinyl alcohol is EG-40 grade for medical use, and the viscosity is 40.0-60.0 Pa.s.
Example 1
The raw materials of the platelet-rich plasma separation gel comprise (by weight parts): polyvinyl alcohol: 30 parts; acrylic acid: 8 parts; and (3) a plasticizer: 1 part of polylactic acid; polysaccharide polymer: 6 parts of sodium alginate and 4 parts of chitosan; acrylic acid derivative: 10 parts of butyl acrylate; and (3) an initiator: 0.2 parts of ammonium persulfate; emulsifying agent: 0.1 part of glycerin and 0.1 part of sodium dodecyl sulfate; silica monomer: 2 parts of nano silicon dioxide and 3 parts of hydrophobic gas phase nano silicon dioxide.
The preparation method comprises the following steps:
1. mixing 30 parts of polyvinyl alcohol, 8 parts of acrylic acid, 1 part of polylactic acid, 6 parts of sodium alginate and 4 parts of chitosan, and placing into a four-necked flask, N 2 Keeping the temperature at 80 ℃ under protection, and reacting for 2 hours;
2. cooling to 55 ℃, adding 10 parts of butyl acrylate, 0.1 part of ammonium persulfate, 0.05 part of glycerol and 0.05 part of sodium dodecyl sulfate, stirring, mixing uniformly, heating to 75 ℃, and reacting for 2 hours;
3. cooling to 40 ℃, adding 0.1 part of ammonium persulfate, 0.05 part of glycerol and 0.05 part of sodium dodecyl sulfate, heating to 70 ℃, and reacting for 1 hour;
4. cooling to 30 ℃, adding 2 parts of nano silicon dioxide and 3 parts of hydrophobic gas phase nano silicon dioxide, placing in a vacuum state of-0.086 Pa, strongly stirring, and reacting at constant temperature for 2 hours to obtain the platelet-rich plasma separation gel. The separation gel has a density of 1.058g/cm at 25deg.C 3 。
Example 2
The raw materials of the platelet-rich plasma separation gel comprise (by weight parts): polyvinyl alcohol: 50 parts; acrylic acid: 15 parts; and (3) a plasticizer: 5 parts of tributyl citrate; polysaccharide polymer: 8 parts of sodium alginate and 7 parts of chitosan; acrylic acid derivative: 20 parts of glycidyl acrylate; and (3) an initiator: 0.8 parts of ammonium persulfate; emulsifying agent: 1 part of sodium dodecyl benzene sulfonate and 1 part of sorbitol; silica monomer: 5 parts of nano silicon dioxide and 5 parts of hydrophobic gas phase nano silicon dioxide.
The preparation method comprises the following steps:
1. 50 parts of polyvinyl alcohol, 15 parts of acrylic acid, 5 parts of tributyl citrate, 8 parts of sodium alginate and 7 parts of chitosan are mixed and placed in a four-neck flask, N 2 Keeping the temperature at 120 ℃ under protection, and reacting for 3 hours;
2. cooling to 60 ℃, adding 20 parts of glycidyl acrylate, 0.4 part of ammonium persulfate, 0.5 part of sorbitol and 0.5 part of sodium dodecyl benzene sulfonate, stirring, mixing, heating to 90 ℃, and reacting for 4 hours;
3. cooling to 60 ℃, adding 0.4 part of ammonium persulfate, 0.5 part of sorbitol and 0.5 part of sodium dodecyl benzene sulfonate, heating to 90 ℃, and reacting for 3 hours;
4. cooling to 40 ℃, adding 5 parts of nano silicon dioxide and 5 parts of hydrophobic gas phase nano silicon dioxide, placing in a vacuum state of-0.078 Pa, strongly stirring, and reacting at constant temperature for 3 hours to obtain the platelet-rich plasma separation gel. The separation gel has a density of 1.080g/cm at 25deg.C 3 。
Example 3
The raw materials of the platelet-rich plasma separation gel comprise (by weight parts): polyvinyl alcohol: 40 parts; acrylic acid: 8 parts; and (3) a plasticizer: 2 parts of tributyl citrate and 3 parts of polylactic acid; polysaccharide polymer: 5 parts of calcium alginate and 8 parts of starch; acrylic acid derivative: 19 parts of hydroxyethyl methacrylate; and (3) an initiator: 0.3 parts of azodiisobutyronitrile; emulsifying agent: 0.5 part of glycerin and 0.4 part of sodium dodecyl sulfate; silica monomer: 3 parts of nano silicon dioxide and 4 parts of hydrophobic gas phase nano silicon dioxide.
The preparation method comprises the following steps:
1. 40 parts of polyvinyl alcohol, 8 parts of acrylic acid, 2 parts of tributyl citrate, 3 parts of polylactic acid, 5 parts of calcium alginate and 8 parts of starch are mixed and placed in a four-necked flask, N 2 Keeping the temperature at 110 ℃ under protection, and reacting for 2 hours;
2. cooling to 60 ℃, adding 19 parts of hydroxyethyl methacrylate, 0.15 part of azobisisobutyronitrile, 0.25 part of glycerol and 0.2 part of sodium dodecyl sulfate, stirring, mixing, heating to 85 ℃, and reacting for 3 hours;
3. cooling to 50 ℃, adding 0.2 part of sodium dodecyl sulfate, 0.25 part of glycerol and 0.15 part of azodiisobutyronitrile, heating to 90 ℃, and reacting for 3 hours;
4. cooling to 40 ℃, adding 3 parts of nano silicon dioxide and 4 parts of hydrophobic gas phase nano silicon dioxide, placing in a vacuum state of-0.08 Pa, strongly stirring, and reacting at constant temperature for 2.5 hours to obtain the platelet-rich plasma separation gel. The separation gel has a density of 1.065g/cm at 25deg.C 3 。
Example 4
The raw materials of the platelet-rich plasma separation gel comprise (by weight parts): polyvinyl alcohol: 50 parts; acrylic acid: 10 parts; and (3) a plasticizer: 2 parts of propylene glycol; polysaccharide polymer: 6 parts of cellulose and 4 parts of glucomannan; acrylic acid derivative: 15 parts of methyl acrylate; and (3) an initiator: 0.8 parts of ammonium persulfate; emulsifying agent: 2 parts of sodium dodecyl benzene sulfonate; silica monomer: 2 parts of nano silicon dioxide and 6 parts of hydrophobic gas phase nano silicon dioxide.
The preparation method comprises the following steps:
1. 50 parts of polyvinyl alcohol, 10 parts of acrylic acid, 2 parts of propylene glycol, 6 parts of cellulose and 4 parts of glucomannan are mixed and placed in a four-neck flask, N 2 Keeping the temperature at 100 ℃ under protection, and reacting for 2.5 hours;
2. cooling to 60 ℃, adding 15 parts of methyl acrylate, 0.4 part of ammonium persulfate and 1 part of sodium dodecyl benzene sulfonate, stirring, mixing, heating to 85 ℃, and reacting for 3 hours;
3. then cooling to 40 ℃, adding 1 part of sodium dodecyl benzene sulfonate and 0.4 part of ammonium persulfate, heating to 80 ℃, and reacting for 2 hours;
4. cooling to 40 ℃, adding 2 parts of nano silicon dioxide and 6 parts of hydrophobic gas phase nano silicon dioxide, placing in a vacuum state of-0.085 Pa, strongly stirring, and reacting at constant temperature for 3 hours to obtain the platelet-rich plasma separation gel. The separation gel has a density of 1.058g/cm at 25deg.C 3 。
Example 5
The raw materials of the platelet-rich plasma separation gel comprise (by weight parts): polyvinyl alcohol: 40 parts; acrylic acid: 15 parts; and (3) a plasticizer: 2 parts of polylactic acid and 2 parts of propylene glycol; polysaccharide polymer: 2 parts of cellulose and 8 parts of calcium alginate; acrylic acid derivative: 15 parts of polyacrylic acid; and (3) an initiator: 0.6 parts of laurylamine peroxide; emulsifying agent: 2 parts of sodium dodecyl benzene sulfonate; silica monomer: 4 parts of nano silicon dioxide and 6 parts of hydrophobic gas phase nano silicon dioxide.
The preparation method comprises the following steps:
1. 40 parts of polyvinyl alcohol, 15 parts of acrylic acid, 2 parts of propylene glycol, 2 parts of polylactic acid, 2 parts of cellulose and 8 parts of calcium alginate are mixed and placed in a four-necked flask, N 2 Keeping the temperature at 120 ℃ under protection, and reacting for 3 hours;
2. cooling to 55 ℃, adding 15 parts of polyacrylic acid, 0.3 part of laurylamine peroxide and 1 part of sodium dodecyl benzene sulfonate, stirring, mixing, heating to 85 ℃, and reacting for 2.5 hours;
3. cooling to 50 ℃, adding 1 part of sodium dodecyl benzene sulfonate and 0.3 part of laurylamine oxide, heating to 85 ℃, and reacting for 2 hours;
4. cooling to 30 ℃, adding 4 parts of nano silicon dioxide and 6 parts of hydrophobic gas phase nano silicon dioxide, placing in a vacuum state of-0.095 Pa, strongly stirring, and reacting at constant temperature for 3 hours to obtain the platelet-rich plasma separation gel. The separation gel has a density of 1.068g/cm at 25deg.C 3 。
Comparative example 1
Platelet rich plasma separation gel was prepared as in example 1, except that: sodium alginate and chitosan are not added. The separation gel has a density of 1.052g/cm at 25deg.C 3 。
Verification example
1. Physicochemical testing
The platelet-rich plasma separation gels prepared in examples 1 to 5 and comparative example 1 were degassed and sterilized in a vacuum state, and the sterilized separation gel and anticoagulant were filled into separation tubes in a certain ratio and order, respectively, and then the separation tubes were subjected to vacuum and sterilization treatment to obtain a vacuum blood collection tube containing the platelet-rich plasma separation gel. The separation test tube can be a centrifuge tube or a blood collection tube, and finally is a vacuum tube.
The index performance of the platelet-rich plasma separation gel has very important significance for separating plasma, and the thixotropic property and the separation effect of the separation gel are verified in order to evaluate the performance of the platelet-rich plasma separation gel prepared by the invention.
1. Viscosity measurement
Thixotropic refers to the property of a fluid that, under a certain shear rate, reduces with time the shear stress of the fluid. The gel is a stable relatively high viscous state under the condition of standing still, and becomes a mobile phase in the process of being stirred by external force. Viscosity is an important index for measuring thixotropy, the viscosity of the separation gel is too large, the time for separating plasma components is too long, the viscosity is too small, the adhesion of the separation gel to a blood collection tube is insufficient, and a flowing phenomenon exists. The thixotropic properties of the separator gel can be indirectly reflected by the values of the viscosity of the separator gel before and after different rotational speeds.
(1) Influence of temperature
The separation gels of examples 1 to 5 and comparative example 1 were tested with an NXS-11 type rotational viscometer in a stationary state, and the test temperatures were set at 20℃at 23℃at 26℃at 30℃at 35 ℃. The test results are shown in Table 1.
As can be seen from the table, the viscosity of the separation gels of examples 1 to 5 changed correspondingly with the change in temperature, and the viscosity tended to decrease with the increase in temperature, but the range of the viscosity change of the separation gel was not very large in the temperature range of 20 to 35℃and the viscosity in this temperature range did not affect the use of the separation function. From this, it can be seen that the viscosity of the separation gels of examples 1 to 5 was stable over a certain temperature range. Whereas the viscosity of the separation gel in comparative example 1 was in the temperature range of 20 to 30 c, the range of variation in the viscosity of the separation gel was large, probably due to the decrease in the hydrogen bond component contained in the polymer, resulting in the decrease in the force on the gel surface and thus the influence of temperature was large. The separation gel of comparative example 1 has a certain selectivity to the separation temperature of platelet-rich plasma in the practical application process, thereby having a certain influence on the separation effect and separation application.
(2) Influence of the rotation speed on the separation of the gel viscosity (25 ℃ C.)
And under the temperature condition of 25 ℃, setting different rotating speed gradients to carry out viscosity test on the separation gel, gradually reading a stable first numerical value after the viscometer starts working until the viscosity of the separation gel does not change along with the change of the rotating speed, and then gradually regulating the rotating speed down according to the gradient set at the beginning, wherein under the condition of reducing the test rotating speed, the viscosity of the separation gel changes. The viscosity results of the separation gel at different rotational speeds are shown in tables 2 and 3 below.
It can be seen from the table that under certain temperature conditions, the viscosity of the separation gel gradually decreases with increasing rotational speed. The more complete the shearing force is applied as the rotational speed increases. After a certain rotational speed, the viscosity is sheared to reach equilibrium, and is gradually stabilized. As can be seen from the table, examples 1 to 5 and comparative example 1 reached a stable state of viscosity under the corresponding maximum shear force, indicating that the shear force applied to the separation gel reached a complete state. As can be seen from comparison of tables 2 and 3, when the rotation speed of the test apparatus is gradually reduced, the viscosity of the separation gel is also gradually recovered, which means that the molecular chain damaged by the external force in the gel is repaired and recovered to the previous state during stopping or reducing the rotation speed, so the viscosity is gradually increased. It is noted that the viscosity of the separator gel of comparative example 1 was gradually lowered as the rotation speed was increased, and reached a stable value at 1000r/min, but in the experiment of gradually lowering the rotation speed, the viscosity did not recover to the previous value as the rotation speed was decreased, that is, the external force damaged the linkage of the molecular chains of the separator gel, and the viscosity of the separator gel could not recover to the original state.
In addition, the formulations of examples 1-5 differ in the viscosity of the separation gel and thus the centrifugal force used to finally reach a stable viscosity value, but this result does not affect the final separation effect but is related to the maximum centrifugal force selected during centrifugation.
2. Separation observation
The separation gels of examples 1 to 5 and comparative example 1 were subjected to a blood separation effect test. And respectively taking a plurality of vacuum blood collection tubes containing separation gel and anticoagulant, and then taking 9mL of blood of volunteers by utilizing the negative pressure advantage of the blood collection tubes, and centrifuging the blood. Centrifugation conditions: 1600r/min,7 minutes, after centrifugation was completed, the following points were observed:
1) The separation gel was observed to effectively isolate the components in the blood (mainly three layers, in order from bottom to top: red blood cells (red) -separation gel (milky white) -platelet rich plasma (yellow));
2) Observing whether the interface between the separation gel and the platelet-rich plasma has hemolysis;
3) The separated system was left in a cool area for 24 hours, and whether the whole system was in a stable state was observed. The results obtained are shown in Table 4 below.
2. Verification of separation Effect
For comparison, a blood collection tube containing the separation gel of example 1, a blood collection tube containing the separation gel of comparative example 1, and a polyacrylate system separation gel blood collection tube collected from the market were separately selected for the simultaneous test.
1. Test sample: blood collection tubes containing anticoagulant and the separation gel of example 1, other products were used as controls. Control 1: blood collection tubes containing anticoagulant and the separation gel of comparative example 1. Control 2: a blood collection tube containing anticoagulant and polyacrylate system separation gel is available in the market.
The blood collection apparatuses mentioned above have all been subjected to sterilization treatment.
2. The experimenter: 30 volunteers were randomly drawn, with a male-female ratio of 1:1, the age is between 28 and 45, and the average age is 35 years. Volunteers were randomly divided into 3 groups, group a, group B, and group C, with a ratio of 1 for men and women: 1. group a uses the test sample to perform platelet-rich plasma separation on whole blood, group B uses control 1 to perform platelet-rich plasma separation on whole blood, and group C uses control 2 to perform platelet-rich plasma separation on whole blood.
3. 9mL of blood at the positive elbow of the volunteer is automatically extracted based on the vacuum degree of the blood collection tube respectively, and the volunteer turns back and forth, so that the anticoagulant is fully mixed with the blood.
4. Placing centrifuge tubes containing blood into a centrifuge for centrifugation, wherein the conditions set by the centrifuge are as follows: rotational speed: 1600r/min, time: 7min.
5. After centrifugation, the blood collection tube is taken out, slowly suspended, the blood collection tube is respectively connected with a 10mL syringe, and Platelet Rich Plasma (PRP) after centrifugation is extracted for detection.
6. Test results
6.1 PRP volume for each group as shown in table 5 below.
As can be seen from Table 5, the volumes of platelet rich plasma obtained using the separation gel of the present invention and the control separation gel are different. By using the separation gel of the invention, 5.06mL of platelet-rich plasma can be averagely separated from 9mL of whole blood, and 4.11mL and 4.48mL of platelet-rich plasma can be averagely separated from the separation gel of the control 1 and the control 2. Meanwhile, the platelet rich plasma separated by the separation gel of comparative example 1 lacking the polysaccharide polymer component was hemolyzed.
As can be seen by comparison, the separation gel of the invention has good platelet-rich plasma separation effect and high recovery rate, and the whole blood with the same quantity can be separated to obtain more platelet-rich plasma.
6.2 Example 1 pH of PRP isolated from separation gel
The PRP separated from the separation gel of example 1 was tested for pH at 0h and 6h using MP220 acidometers calibrated with calibration solutions having pH values of 4, 7, and 9, respectively. The results of the pH measurements are shown in Table 6.
The platelet-rich plasma showed weak acidity in pH, and it was found from the results of measuring the pH values of the separated platelet-rich plasma for 0h and 6h using the separation gel of example 1 that the platelet-rich plasma remained stable and hardly changed in pH. The results show that the platelet-rich plasma isolated by the method has good pH stability.
6.3 Conditions of PRP concentration for each group
The platelet concentrations of 0h and 6h in each group of platelet rich plasma were measured by performing routine blood analysis using a fully automatic blood analyzer, and the results are shown in Table 7 below.
As can be seen from Table 7, the platelet-rich plasma separated by the separation gel of the present invention has significantly higher platelet concentration than that of each control sample, and the platelet concentration in the sample is stable within 6 hours.
Claims (11)
1.A preparation method of platelet-rich plasma separation gel is characterized by comprising the following steps:
(1) Uniformly mixing 30-50 parts of polyvinyl alcohol, 8-15 parts of acrylic acid, 1-5 parts of plasticizer and 10-15 parts of polysaccharide polymer, and heating for reaction under the protection of gas so that the added substances are fully dissolved;
(2) Cooling after the reaction, adding 10-20 parts of acrylic acid derivative, 0.1-0.4 part of initiator and 0.1-1 part of emulsifier into the reaction system in the step (1), stirring and mixing uniformly, and heating to perform free radical initiation polymerization reaction;
(3) Cooling after the reaction, adding 0.1-0.4 part of initiator and 0.1-1 part of emulsifier into the system in the step (2), stirring and mixing uniformly, and heating to perform free radical initiated polymerization reaction;
(4) Cooling after the reaction, adding 5-10 parts of silicon dioxide monomer into the reaction system in the step (3), and reacting under stirring and temperature control to obtain platelet-rich plasma separation gel; wherein, each raw material is added in parts by weight.
2. The preparation method according to claim 1, characterized in that: the polysaccharide polymer is more than two of chitosan, sodium alginate, starch, calcium alginate, glucomannan and cellulose; the plasticizer is at least one of polylactic acid, tributyl citrate and propylene glycol; the acrylic acid derivative is at least one of glycidyl acrylate, butyl acrylate, hydroxyethyl methacrylate, methyl acrylate and polyacrylic acid.
3. The preparation method according to claim 1, characterized in that: the emulsifier is at least one of glycerol, sodium dodecyl sulfate, sodium dodecyl benzene sulfonate and sorbitol; the initiator is ammonium persulfate, azodiisobutyronitrile or benzoyl chloride; the silicon dioxide monomer is hydrophobic gas phase silicon dioxide or/and nano silicon dioxide.
4. The preparation method according to claim 1, characterized in that: the viscosity of the polyvinyl alcohol is 40.0-60.0 Pa.s.
5. The preparation method according to claim 1, characterized in that: the particle size of the silica monomer is 45-55nm.
6. The preparation method according to any one of claims 1 to 5, characterized in that: in the step (1), the reaction temperature is 80-120 ℃ and the reaction time is 2-3h; in the step (2), the temperature is reduced to 55-60 ℃, and after acrylic acid derivatives, an initiator and an emulsifier are added, the mixture is reacted for 2-4 hours at 75-90 ℃; in the step (3), the temperature is reduced to 40-60 ℃, and after an emulsifier and an initiator are added, the mixture is reacted for 1-3 hours at 70-90 ℃; in the step (4), the silica monomer is added and then stirred and reacted for 2 to 3 hours at the temperature of between 30 and 40 ℃.
7. A platelet-rich plasma separation gel prepared according to the method of preparing a platelet-rich plasma separation gel according to any one of claims 1 to 6.
8. The platelet rich plasma separation gel according to claim 7, wherein: the density of the platelet-rich plasma separation gel is 1.05-1.08g/cm 3 。
9. A platelet rich plasma separation tube, characterized by: a platelet rich plasma separation gel comprising the platelet rich plasma separation gel of claim 7 or 8.
10. The platelet rich plasma separation tube according to claim 9 wherein: the interior of the platelet-rich plasma separation tube is in a vacuum state.
11. A method for extracting platelet-rich plasma is characterized by comprising the following steps: a step of collecting whole blood using the platelet-rich plasma separation tube according to claim 9 or 10, and then subjecting the platelet-rich plasma separation tube to centrifugation to extract platelet-rich plasma.
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| CN116715801B (en) * | 2023-06-30 | 2024-02-23 | 珠海朗泰生物科技有限公司 | PRP (platelet-derived polymer) separating gel with low cytotoxicity and preparation method and application thereof |
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