CN110237094B - Preparation method of bottom layer platelet factor paste and sPL platelet factor gel film - Google Patents
Preparation method of bottom layer platelet factor paste and sPL platelet factor gel film Download PDFInfo
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Abstract
The invention discloses a preparation method of a bottom platelet factor paste and an sPL platelet factor gel film, and relates to a preparation method of a bottom platelet factor paste and an sPL platelet factor gel film. The invention aims to solve the problems that a platelet plasma gel product is required to be prepared on site, and a platelet factor sticking film has an irregular shape, uneven thickness and uneven components, wherein the bottom layer platelet factor sticking film comprises a bottom layer lining film and an upper layer covering film, the middle area of the bottom layer lining film is downwards sunken to form a platelet factor bearing area, collagen and an activator are coated on the platelet factor bearing area, and the platelet factor is rapidly contacted with the platelet factor bearing area and is rapidly and uniformly activated. The invention solves the defects that the conventional platelet plasma gel product cannot be standardized and prepared in advance, and the gel adhesive film is not formed, has different thickness, is not shaped and is shrunk in a conventional spraying or mixing mode. The invention is applied to the field of platelet factor gel adhesive films.
Description
Technical Field
The invention relates to a preparation method of a bottom layer platelet factor paste and an sPL platelet factor gel paste film.
Background
The blood of mammals is composed of 55% plasma and 45% blood cells, which can be further classified into three types of red blood cells, white blood cells and platelets. The proportion of the platelets in blood cells is less than 1%, and the platelets play roles in maintaining the integrity of vascular endothelium and participating in hemostasis and coagulation under physiological state, and after body injury, the platelets start a coagulation mechanism and release components such as coagulation factors, pro-vascular active factors, growth factors and the like, thereby playing an important role in regulating early wound healing, thrombosis and tissue repair.
The platelet count in the blood of normal people is 100-300 multiplied by 10 9 And is prone to bleeding when the platelets in the blood are too low per liter. Some diseases are accompanied by thrombocytopenia, so that the transfusion of thrombocytes in clinical disease treatment can be used for treating patients with thrombocytopenia or dysfunction caused by various reasons, such as leukemia, aplastic anemia, lymphoma, pre-bone marrow transplantation and other hematopathy patients and cancer patients with bone marrow suppression caused by radiotherapy and chemotherapy, wherein many patients need to maintain life by continuously transfusing thrombocytes. In addition, studies have shown that many cell growth factors and regulatory factors are present in platelets, which can play a role in repairing organisms under various physiological and pathological conditions, for example, platelet-derived growth factor (PDGF), which is a glycoprotein synthesized from alpha particles of platelets, activates mitosis of aged damaged partial cells, increases cytosolic products, increases regenerative cells, plays an important role in stimulating neovascularization and promoting existing vascular growth, and PDGF is a factor that has a promoting effect on efficient mitogenesis, particularly mesoderm-derived cells, including muscle cells and interstitial cells; transforming growth factor-beta (TGF-beta) is glycoprotein capable of controlling cell proliferation and differentiation, promoting fibroblast expansion, promoting collagen synthesis and fibrinogen to induce bone matrix precipitation and inhibiting bone resorption, and TGF-beta is involved in a plurality of inflammatory reactions in vivo, and is a multifunctional basic anti-inflammatory cytokine in vivo; fibroblast Growth Factor (FGF) can promote angiogenesis, promote fibroblast production, and accelerate damaged tissue repair andembryo development; epidermal Growth Factor (EGF) can repair epithelial cells and accelerate cell proliferation and differentiation; vascular Endothelial Growth Factor (VEGF) can produce collagen and excite hyaluronic acid, so that it has strong tissue repairing action and angiopoiesis promoting action; islet growth factor-1 (IGF-1) is a fibroblast chemotactic agent and can promote protein synthesis and bone formation.
Clinical treatment with trophic factors in platelets can take two forms, one is to enrich platelets directly after centrifugation, which does not activate platelets and therefore has limited effect on the tissue to be repaired. The other method is to crack the prepared platelet-rich plasma containing the least red blood cells by utilizing a high-efficiency induction technology and a freeze thawing principle, and plays an important role in the processes of repairing bones and cartilages, repairing skin injuries, repairing ligament injuries which are difficult to repair by a conventional method, repairing bone defects, maxillofacial reconstruction, gum defects and the like caused by operations. The research and comparison of platelet lysate and platelet-rich plasma show that the platelet-rich plasma has stronger immunogenicity and is mostly used for autograft, and the preparation process of the platelet lysate not only removes residual cell structures and reduces immunogenicity, but also retains various growth factors therein, thereby being capable of creating conditions for allogeneic or xenogeneic transplantation. Thus, as a potential option for constructing tissue engineered tissue, platelet lysates may have a superior application prospect than platelet-rich plasma.
At present, in the skin wound repair process, if autologous peripheral blood platelet trophic factors are adopted for repair, most of the process is that in an operating room, freshly collected blood is concentrated to obtain platelet plasma, then the platelet plasma is rapidly mixed with an activator, and gel formed in a short time after mixing is covered on the surface of a wound. The gel can cover the wound, isolate the wound from the external environment, reduce the infection risk, provide wound repair nutrition and accelerate the healing of the wound. However, because 1) gel needs to be formed quickly in a short time during operation, the gel forming technical requirement on medical staff is high, 2) the shape and the size of the gel cannot be controlled in a spraying or mixing mode, 3) the individual difference exists in the repair factor content in platelet-rich plasma after peripheral blood collection, the repair effect cannot be unified, and 4) under the condition that a great amount of autologous blood cannot be obtained from certain wounds, the quick treatment and the like cannot be performed.
Disclosure of Invention
The invention aims to solve the problems of irregular shape, uneven thickness and uneven components of a platelet factor adhesive film and provides a preparation method of a bottom platelet factor adhesive film and an sPL platelet factor gel adhesive film.
The invention relates to a bottom platelet factor patch which comprises a bottom lining film and an upper covering film, wherein the upper covering film is covered on the bottom lining film; the middle area of the bottom lining film is downwards sunken to form a platelet factor bearing area, and a flow guide convex edge is arranged on the platelet factor bearing area; the upper cover film is provided with a liquid inlet, and the liquid inlet is provided with a sealing cover; wherein the liquid inlet is positioned right above the center of the platelet factor bearing region.
The method for preparing the sPL platelet factor gel patch by using the bottom layer platelet factor patch comprises the following steps:
1. preparing an activating agent: dissolving thrombin freeze-dried powder by using a calcium gluconate solution with the concentration of 0.05-5 mg/mL to ensure that the concentration of thrombin is 500-800IU/mL, thereby obtaining an activator;
2. uniformly spraying an aqueous solution of type I collagen with the mass concentration of 0.005g/mL on the bottom of a platelet factor bearing region, and volatilizing to gel with the water content of 10% in a sterile environment; wherein every 30cm 2 Spraying 1mL of type I collagen aqueous solution on the platelet factor bearing region;
3. uniformly spraying an activator on a platelet factor bearing area coated with collagen at the temperature of 2-4 ℃ under the aseptic condition, pre-cooling a bottom lining film for 2 hours at the temperature of-80 ℃, and freeze-drying for 4 hours in an aseptic freeze dryer;
4. covering the upper covering film on the bottom lining film treated in the third step, and storing for later use;
5. preparation of sPL platelet factor preparation: a. collecting 10-20 parts of anticoagulated umbilical cord blood orMixing the peripheral blood of the anticoagulated healthy person uniformly, wherein the volume of each anticoagulated blood sample is 50-100ml, sub-packaging the mixed anticoagulated blood sample into a centrifuge tube after uniform mixing, and centrifuging for 10min at 20 ℃ and the rotating speed of 200-300 g to remove the lower layer red blood cells; uniformly mixing the residual blood in the centrifuge tube, and centrifuging at 20 ℃ and 1000-2000 g for 15min; transferring the upper plasma to a new centrifuge tube for storage, mixing and counting the platelet plasma at the bottom layer, and regulating the platelet concentration to 1100×10 by using the stored upper plasma 9 ～1400*10 9 a/L; b. c, sequentially carrying out ice bath ultrasonic treatment on the platelet plasma in the step a for 10min and water bath at 37 ℃ for 2h; ice bath ultrasonic treatment is carried out for 10min, and water bath is carried out for 2h at 37 ℃; liquid nitrogen bath for 2h; performing water bath at 37 ℃ for 1min, filtering by a 0.22 mu m filter to obtain a sPL platelet factor preparation, adjusting the concentration of PDGF-AB in the sPL platelet factor preparation to 130-140ng/mL, and then sucking into a sterile screw thread mouth pressure injector for standby at any time;
6. opening a sealing cover of a liquid inlet, connecting a pressure injector with the liquid inlet, injecting sPL platelet factor preparation in the sterile threaded-port pressure injector into a platelet factor bearing area at a pressure of 1.5 kg/square centimeter, and then covering the sealing cover, wherein the sPL platelet factor gel film is obtained after the sPL platelet factor forms gel; wherein the volume ratio of the activator, the aqueous solution of type I collagen and the injected sPL platelet factor preparation is 3:1:6.
The beneficial effects are that: 1. the operation is convenient, and the bottom platelet factor paste and sPL platelet factor preparation can be prepared in advance and stored for a long time. In the treatment site, medical staff only needs to inject sPL platelet factor preparation into the bottom platelet factor patch, and apply the formed sPL platelet factor preparation to the wound surface, so that the invention is particularly convenient for the medical staff to make gel type adhesive films in the operation site;
2. the coagulation promoting factors are fixed at the bottom of the bearing area in advance according to the proportion, so that the platelet factors can be conveniently and rapidly contacted with the bearing area and can be rapidly and uniformly activated.
3. The platelet factor bearing area is provided with the flow guide convex edges, so that the platelet factors can be uniformly distributed on the whole bearing disc after being injected from the middle screw thread mouth, the size and the shape of the adhesive film can be customized according to the shape of the bearing disc, and the defects of non-forming, uneven thickness and unstable shape of the adhesive film in the conventional spraying or mixing mode are overcome.
4. Because the bottom of the bearing area is sprayed with hydrophilic collagen in advance, and the gel film formed by the sPL platelet factor preparation is adsorbed and fixed on the bearing area, the film is not unfixed in shape like a spraying method, a mixing method and a double-gun method, and the gel is shrunken along with the time, so that the phenomenon of different gel thickness is more remarkable.
5. The platelet factors from cord blood or peripheral blood of healthy people are adopted, so that the treatment convenience is provided for patients with larger wounds and incapacity of using autologous blood, long-time sPL platelet factor preparation and gel preparation are not needed, and the emergency treatment is convenient.
6. The method has the advantages that the preparation of the platelet factors is carried out after the mixing of a plurality of blood parts, the problem of uneven content of key factors among single blood parts is reduced, and the more the blood parts are, the closer the content of the key factors is to average and the smaller the fluctuation is, so that the method can well control the quality among batches, and plays a key role in the stability of future application effects.
7. The standardized patch preparation process solves the problem of wound repair result difference caused by different platelet contents, different platelet factor activation efficiency and the like in the process of adopting the platelet factors of patients. Meanwhile, in the preparation process, the following measures are adopted, so that the standardized preparation of the adhesive film is realized, and a foundation is laid for obtaining a uniform treatment effect for future application:
the adoption of the mixed preparation form of a plurality of blood platelets reduces the individuation difference of blood samples, and the concentration of platelet factors among different batches is very similar under the condition that the number of blood parts exceeds 10 parts. Plasma addition is adopted to control the platelet quantity to 1200 x 10 before the platelet factor is released 9 a/L; after platelet factor release, if there is a factor concentration that is too high, PDGF-AB concentration can be controlled to 130-140ng/ml by adding plasma. The patch film has uniform thickness, but various forms, and is convenient for manufacturing square and round shapes according to the shapes of different wounds in futureAnd (5) sticking a film.
Drawings
FIG. 1 is a side view of an underlying platelet factor patch;
FIG. 2 is a top view of an underlying liner film;
FIG. 3 is a graph showing the results of various methods of releasing platelet factors;
FIG. 4 is a graph showing the thickness profile of sPL platelet factor gel patches prepared by various methods;
FIG. 5 is a graph of the shape of a sPL platelet factor patch;
FIG. 6 is a graph showing the change in gel attachment area over time of sPL platelet factor gel patches prepared by different methods; wherein a is the method of the invention, b is the spraying method, c is the mixing method, and d is the double gun method;
FIG. 7 shows cytokine concentrations contained at different locations of sPL platelet factor patches;
FIG. 8 is a graph showing the effect of different blood sample amounts on the factor content of a platelet factor preparation from batch to batch.
Detailed Description
The first embodiment is as follows: the bottom platelet factor patch of the embodiment comprises a bottom lining film 1 and an upper covering film 2, wherein the upper covering film 2 is covered on the bottom lining film 1; the middle area of the bottom lining film 1 is sunken downwards to form a platelet factor bearing area 4, and a flow guide convex edge is arranged on the platelet factor bearing area; the upper cover film 2 is provided with a liquid inlet 3, and the liquid inlet 3 is provided with a sealing cover 5; wherein the liquid inlet 3 is positioned right above the center of the platelet factor bearing region.
The second embodiment is as follows: the first difference between this embodiment and the specific embodiment is that: the liquid inlet 3 is connected with a sterile threaded port pressure injector in a threaded way. The other is the same as in the first embodiment.
And a third specific embodiment: this embodiment differs from the first or second embodiment in that: the depth of the platelet factor bearing region 4 is 3mm. The other is the same as the first or second embodiment.
The specific embodiment IV is as follows: this embodiment differs from one of the first to third embodiments in that: the platelet factor bearing region 4 is square or circular. The other is the same as in one of the first to third embodiments.
Fifth embodiment: the method for attaching the sPL platelet factor gel membrane in the embodiment is carried out according to the following steps:
1. preparing an activating agent: dissolving thrombin freeze-dried powder by using a calcium gluconate solution with the concentration of 0.05-5 mg/mL to ensure that the concentration of thrombin is 500-800IU/mL, thereby obtaining an activator;
2. uniformly spraying type I collagen aqueous solution with mass concentration of 0.005g/mL on the bottom of the platelet factor bearing region 4, and volatilizing to gel with water content of 10% in sterile environment, wherein every 30cm 2 Spraying 1mL of type I collagen aqueous solution on the platelet factor bearing region 4;
3. uniformly spraying an activator on the platelet factor bearing region 4 coated with collagen at the temperature of 2-4 ℃ under the condition of sterility, pre-cooling the bottom lining film 1 at-80 ℃ for 2 hours, and putting the bottom lining film into a sterile freeze dryer for freeze drying for 4 hours;
4. covering the upper cover film on the bottom lining film 1 treated in the third step, and storing for later use;
5. preparation of sPL platelet factor preparation: a. collecting 10-20 parts of anticoagulated umbilical cord blood or anticoagulated peripheral blood of healthy people, uniformly mixing, wherein the volume of each anticoagulated blood sample is 50-100ml, packaging into a centrifuge tube after uniformly mixing, centrifuging for 10min at 20 ℃ at the rotating speed of 200-300 g, and removing lower-layer red blood cells; uniformly mixing the residual blood in the centrifuge tube, and centrifuging at 20 ℃ and 1000-2000 g for 15min; transferring the upper plasma to a new centrifuge tube for storage, mixing and counting the platelet plasma at the bottom layer, and regulating the platelet concentration to 1100×10 by using the stored upper plasma 9 ～1400*10 9 a/L; b. c, sequentially carrying out ice bath ultrasonic treatment on the platelet plasma in the step a for 10min and water bath at 37 ℃ for 2h; ice bath ultrasonic treatment is carried out for 10min, and water bath is carried out for 2h at 37 ℃; liquid nitrogen bath for 2h; performing water bath at 37 ℃ for 1min, filtering by a 0.22 mu m filter to obtain a sPL platelet factor preparation, adjusting the concentration of PDGF-AB in the sPL platelet factor preparation to 130-140ng/mL, and then sucking into a sterile screw thread mouth pressure injector for standby at any time;
6. opening a sealing cover 5 of the liquid inlet, connecting a pressure injector with the liquid inlet 3, then injecting sPL platelet factor preparation in the sterile threaded-port pressure injector into a platelet factor bearing area 4 at a pressure of 1.5 kg/square centimeter, then covering the sealing cover 5, and obtaining the sPL platelet factor gel film after the sPL platelet factor forms gel; wherein the volume ratio of the activator, the aqueous solution of type I collagen and the injected sPL platelet factor preparation is 3:1:6.
Specific embodiment six: the fifth difference between this embodiment and the third embodiment is that: in the third step, the sterile freeze dryer sets the temperature of a cold well to be minus 60 ℃ and the vacuum degree to be 0.1mbar. The other is the same as in the fifth embodiment.
Seventh embodiment: the present embodiment differs from the fifth or sixth embodiment in that: the bottom lining film 1 is made of polyurethane material or polypropylene material. Other features and advantages of the present invention are the same as those of the fifth or sixth embodiment
Eighth embodiment: this embodiment differs from one of the fifth to seventh embodiments in that: the upper cover film 2 is made of high-density polyethylene or medical release paper. The others are the same as in one of the fifth to seventh embodiments. The high-density polyethylene and medical release paper in the implementation method are resistant to irradiation and have the function of preventing microorganism permeation.
Detailed description nine: this embodiment differs from one of the fifth to eighth embodiments in that: if the concentration of PDGF-AB in sPL platelet factor preparation is less than 130ng/mL, it is at 1100 x 10 9 ～1400*10 9 Increasing the platelet concentration in step a within a range of individual/L; if the concentration of PDGF-AB in sPL platelet factor preparation is higher than 140ng/mL, the supernatant plasma remaining from step a is used for dilution. Other features are the same as those of the fifth to eighth embodiments
The beneficial effects of the invention are verified by the following examples:
embodiment one: the bottom platelet factor patch of the embodiment comprises a bottom lining film 1 and an upper covering film 2, wherein the upper covering film 2 is covered on the bottom lining film 1; the middle area of the bottom lining film 1 is sunken downwards to form a platelet factor bearing area 4, and a flow guide convex edge 6 is arranged on the platelet factor bearing area 4; the upper cover film 2 is provided with a liquid inlet, and the liquid inlet is provided with a sealing cover. Wherein a side view of the underlying platelet factor patch is shown in FIG. 1; a top view of the underlying liner film is shown in fig. 2;
the method for preparing the sPL platelet factor gel patch by using the bottom layer platelet factor patch comprises the following steps:
1. preparing an activating agent: dissolving thrombin freeze-dried powder by using a calcium gluconate solution with the concentration of 0.05-5 mg/mL to ensure that the concentration of thrombin is 500-800IU/mL, thereby obtaining an activator;
2. uniformly spraying type I collagen aqueous solution with mass concentration of 0.005g/mL on the bottom of the platelet factor bearing region 4, and volatilizing to gel with water content of 10% in sterile environment, wherein every 30cm 2 Spraying 1mL of type I collagen aqueous solution on the platelet factor bearing region 4;
3. uniformly spraying an activator on the platelet factor bearing region 4 coated with collagen at the temperature of 2-4 ℃ under the condition of sterility, pre-cooling the bottom lining film 1 at-80 ℃ for 2 hours, and putting the bottom lining film into a sterile freeze dryer for freeze drying for 4 hours;
4. covering the upper cover film on the bottom lining film 1 treated in the third step, and storing for later use;
5. preparation of sPL platelet factor preparation: a. collecting 10-20 parts of anticoagulated umbilical cord blood, uniformly mixing, wherein the volume of each anticoagulated blood sample is 50-100ml, packaging in a centrifuge tube after uniformly mixing, centrifuging for 10min at 20 ℃ and the rotating speed of 200-300 g, and removing lower red blood cells; uniformly mixing the residual blood in the centrifuge tube, and centrifuging at 20 ℃ and 1000-2000 g for 15min; transferring the upper plasma to a new centrifuge tube for storage, mixing and counting the platelet plasma at the bottom layer, and regulating the platelet concentration to 1100×10 by using the stored upper plasma 9 ～1400*10 9 a/L; b. c, sequentially carrying out ice bath ultrasonic treatment on the platelet plasma in the step a for 10min and water bath at 37 ℃ for 2h; ice bath ultrasonic treatment is carried out for 10min, and water bath is carried out for 2h at 37 ℃; liquid nitrogen bath for 2h; performing water bath at 37deg.C for 1min, filtering with 0.22 μm filter to obtain sPL platelet factor preparation, adjusting PDGF-AB concentration in sPL platelet factor preparation to 130-140ng/mL, and sucking into sterile screw thread pressure syringeStandby;
6. opening a sealing cover 5 of the liquid inlet, connecting a pressure injector with the liquid inlet 3, then injecting sPL platelet factor preparation in the sterile threaded-port pressure injector into a platelet factor bearing area 4 at a pressure of 1.5 kg/square centimeter, then covering the sealing cover 5, and obtaining the sPL platelet factor gel film after the sPL platelet factor forms gel; wherein the volume ratio of the activator, the aqueous solution of type I collagen and the injected sPL platelet factor preparation is 3:1:6.
The sPL platelet factor gel adhesive film prepared in the embodiment is compared with the platelet factor gel prepared by a spraying method, a mixing method and a double gun method in terms of film forming speed, shape, thickness and factor content uniformity,
1. preparation process has sufficient platelet factor release
The invention adopts the operations of sequentially carrying out ice bath ultrasound for 10min, 37 ℃ water bath for 2h, liquid nitrogen bath for 2h and 37 ℃ water bath for 1min on concentrated platelet plasma to obtain the sPL platelet factor preparation, and compared with a conventional repeated freeze thawing mode and a simple ultrasound method, the method adds two 37 ℃ water bath incubations after ultrasound pyrolysis, and obviously increases the concentration of cytokines. The same concentrated platelet plasma was divided into three parts, one part was subjected to the method of the present invention, one part was subjected to 3 times of freeze thawing (-80C refrigerator freezing for 2 hours, 37C resuscitation for 1min was one time of freeze thawing), and one part was subjected to simple ultrasonic method (two times of ice bath ultrasonic treatment, 10min each), and the difference in comparison results was significant P <0.001 (Table 1, FIG. 3)
TABLE 1 platelet factor PDGF-AB release comparison (ng/ml)
| Test batch | The invention is that | 3 times freeze thawing method | Simple ultrasonic method |
| 1 | 152.3 | 91.3 | 109.4 |
| 2 | 131.3 | 86.7 | 99.3 |
| 3 | 140.7 | 78.7 | 87.0 |
| 4 | 142.1 | 90.1 | 113.9 |
| Average of | 141.6±8.6 | 86.7±5.7 | 102.4±11.9 |
2. The sPL platelet factor film forming speed is high
TABLE 2 in situ operation time and gel film Forming speed
Because the activator is freeze-dried on the film in advance, and the sPL platelet factor preparation is prepared by adopting allogeneic umbilical blood or healthy human peripheral blood, only the sPL platelet factor is injected into the film in an operating room according to the needs of patients at any time, the operation is simple and convenient, and the whole time and the gel forming time are far lower than those of other methods (Table 2). Because the radial flow guide convex edges are embedded on the bearing disc, the injected sPL platelet factor preparation can be uniformly distributed in different sectors, and the phenomena of more local preparations and less local preparations can not be generated. And the sPL platelet factor preparation rapidly reaches the far end of the bearing area under the action of the pressure injector, so that the gel forming speed is high and the gel forming time of different positions is close.
3. sPL platelet factor patch film thickness is uniform
Table 3 multipoint thickness measurement of platelet factor patches using a thickness gauge:
by carrying out thickness analysis on gels at different positions in a random dot taking mode, as can be seen from table 3 and fig. 4, the gel formed by the method has uniform thickness, the standard deviation of the gel is 0.3, the spraying method is 0.9, the mixing method is 1.6, and the double gun method is 1.8. Therefore, the gel patch film can be formed to be uniform in thickness and easy to control, and the effect of future treatment is stable. 4. sPL platelet factor patch shape fixation
The shape of the sPL platelet factor film is shown in figure 5, because the bottom of the bearing disc is sprayed with hydrophilic collagen in advance, the gel film formed by the sPL platelet factor preparation is absorbed and fixed on the gel film, and the position where the platelet factor reaches is influenced by the shape of the guide convex edge and the bearing disc, so that the fixed film shape is formed. On the surface of the purchased pigskin, wounds with an area of 4.5cm by 4.5cm and a depth of 0.3cm were formed, the wounds were covered with gels made by four methods respectively, and after different times, the shrinkage and hydration of the gels were measured (Table 4), and it was seen that the patch of the present invention did not have a non-fixed shape as in the spray coating method, the mixing method and the double gun method, and that the gel did not generate significant hydration on the surface of the attached pigskin and shrinkage of the attached area over time (FIG. 6).
Table 4 change in shape of film over time (length x width = cm x cm)
| Time (min) | The invention is that | Spray coating process | Mixing method | Double gun method |
| 0 | 4.5cm*4.5cm | 4.5cm*4.5cm | 4.7cm*4.1cm | 4.4cm*4.5cm |
| 30 | 4.5cm*4.5cm | 4.1cm*4.2cm | 4.7cm*3.8cm | 4.0cm*4.2cm |
| 60 | 4.5cm*4.5cm | 3.5cm*4.0cm | 3.9cm*2.2cm | 3.6cm*3.1cm |
| 120 | 4.4cm*4.3cm | 3.4cm*4.0cm | 3.3cm*2.1cm | 3.5cm*3.0m |
5. The sPL platelet factor adhesive film contains uniform cell factors at different positions
TABLE 5 cytokine content at different positions
In order to detect the cytokine content at different positions of the film, gels at different positions of the film are randomly selected, and the ELISA method is used for analyzing the cytokine content. As can be seen from Table 5 and FIG. 7 (PDGF-AB, TGF-beta are left ordinate axis and FGFb is right ordinate axis), the sPL platelet factor patches prepared by the present invention have uniform cytokines at different positions.
6. The platelet factor preparations of different batches obtained by the invention have uniform factor content
As can be seen from Table 6 and FIG. 8, the method for preparing the platelet factor preparation after mixing a plurality of umbilical cord blood reduces the problem of obvious individuation difference caused by preparing one sample, and the obtained factor preparation has uniform content before preparing a film, so that the factor content between batches is very similar, and the consistency of future treatment effect is objectively maintained.
TABLE 6 different batches of PDGF-AB concentrations (ng/ml)
In conclusion, the invention is convenient to operate and convenient for medical staff to manufacture gel type adhesive films on the operation site; the coagulation promoting factors are fixed at the bottom of the bearing area in advance according to the proportion, so that the platelet factors can be conveniently and rapidly contacted with the bearing area and can be rapidly and uniformly activated. The platelet factor bearing area is provided with the flow guide convex edges, so that the platelet factors can be uniformly distributed on the whole bearing disc after being injected from the middle screw thread mouth, the size and the shape of the adhesive film can be customized according to the shape of the bearing disc, and the defects of non-forming, uneven thickness and unstable shape of the adhesive film in the conventional spraying or mixing mode are overcome. Because the bottom of the bearing area is sprayed with hydrophilic collagen in advance, and the gel film formed by the sPL platelet factor preparation is adsorbed and fixed on the bearing area, the film is not unfixed in shape like a spraying method, a mixing method and a double-gun method, and the gel is shrunken along with the time, so that the phenomenon of different gel thickness is more remarkable. In the platelet factor release stage, a staged 37 ℃ 2h incubation method is adopted, and the nutritional ingredients in the platelets are maximally released. The platelet factors from cord blood or peripheral blood of healthy people are adopted, so that the treatment convenience is provided for patients with larger wounds and incapacity of using autologous platelets. The method has the advantages that the preparation of the platelet factors is carried out after the mixing of a plurality of blood samples, the problem of uneven content of key factors among single blood samples is reduced, and the more the number of the blood samples is adopted, the closer the content of the key factors is to be average and the smaller the fluctuation is, so that the quality among batches can be well controlled, and the method plays a key role in the stability of future application effects. The standardized patch preparation process solves the problem of wound repair result difference caused by different platelet contents, different platelet factor activation efficiency and the like in the process of adopting the platelet factors of patients. Meanwhile, in the preparation process, the following measures are adopted, so that the standardized preparation of the adhesive film is realized, and a foundation is laid for obtaining a uniform treatment effect for future application:
the preparation method has the advantages that the preparation method adopts the mixed preparation form of multiple cord blood or peripheral blood platelets of healthy people, the individuation difference of blood samples is reduced, and under the condition that the number of blood samples exceeds 10, the concentration of platelet factors in different batches is relatively close. Plasma addition is adopted to control the platelet quantity to 1200 x 10 before the platelet factor is released 9 a/L; after release of platelet factorsIf there is too high factor concentration, PDGF-AB concentration can be controlled between 130ng/ml and 140ng/ml by adding plasma. The patch film has uniform thickness, but various forms, and is convenient for manufacturing square and round patches according to the shapes of different wounds in the future.
Claims (5)
1. A preparation method of sPL platelet factor gel adhesive film is characterized by comprising the following steps:
the sPL platelet factor gel patch is prepared by using a bottom platelet factor patch, wherein the bottom platelet factor patch comprises a bottom lining film (1) and an upper covering film (2), and the upper covering film (2) is covered on the bottom lining film (1); the middle area of the bottom lining film (1) is sunken downwards to form a platelet factor bearing area (4), and a flow guide convex edge is arranged on the platelet factor bearing area; the upper cover film (2) is provided with a liquid inlet (3), and the liquid inlet (3) is provided with a sealing cover (5); wherein the liquid inlet (3) is positioned right above the center of the platelet factor bearing area;
1. preparing an activating agent: dissolving thrombin freeze-dried powder by using a calcium gluconate solution with the concentration of 0.05-5 mg/mL to ensure that the concentration of thrombin is 500-800IU/mL, thereby obtaining an activator;
2. uniformly spraying type I collagen aqueous solution with mass concentration of 0.005g/mL on the bottom of the platelet factor bearing region (4), and volatilizing to gel with water content of 10% in sterile environment, wherein every 30cm 2 Spraying 1mL of type I collagen aqueous solution on the platelet factor bearing region (4);
3. uniformly spraying an activator on a platelet factor bearing region (4) coated with collagen at the temperature of 2-4 ℃ under the condition of sterility, pre-cooling the bottom lining film (1) at the temperature of-80 ℃ for 2 hours, and then putting the bottom lining film into a sterile freeze dryer for freeze drying for 4 hours;
4. covering the upper cover film (2) on the bottom lining film (1) treated in the third step, and storing for later use;
5. preparation of sPL platelet factor preparation: a. collecting 10-20 parts of anticoagulated umbilical cord blood or anticoagulated peripheral blood of healthy person, mixing uniformly, wherein the volume of each anticoagulated blood sample is 50-100ml, packaging into centrifuge tube after mixing uniformly, and then placing at 20deg.C and rotating speed of 20Centrifuging for 10min under the condition of 0g-300g, and removing lower layer red blood cells; uniformly mixing the residual blood in the centrifuge tube, and centrifuging at 20 ℃ and 1000-2000 g for 15min; transferring the upper plasma to a new centrifuge tube for storage, mixing and counting the platelet plasma at the bottom layer, and regulating the platelet concentration to 1100×10 by using the stored upper plasma 9 ～1400*10 9 a/L; b. c, sequentially carrying out ice bath ultrasonic treatment on the platelet plasma in the step a for 10min and water bath at 37 ℃ for 2h; ice bath ultrasonic treatment is carried out for 10min, and water bath is carried out for 2h at 37 ℃; liquid nitrogen bath for 2h; performing water bath at 37 ℃ for 1min, filtering by a 0.22 mu m filter to obtain a sPL platelet factor preparation, adjusting the concentration of PDGF-AB in the sPL platelet factor preparation to 130-140ng/mL, and then sucking into a sterile screw thread mouth pressure injector for standby at any time;
6. opening a sealing cover (5) of the liquid inlet, connecting a pressure injector with the liquid inlet (3), then injecting sPL platelet factor preparation in the sterile threaded-port pressure injector into a platelet factor bearing area (4) at a pressure of 1.5 kg/square centimeter, and then covering the sealing cover (5), and obtaining the sPL platelet factor gel patch after the sPL platelet factor forms gel; wherein the volume ratio of the activator, the aqueous solution of type I collagen and the injected sPL platelet factor preparation is 3:1:6.
2. The method for preparing a gel patch of sPL platelet factor according to claim 1, wherein in the third step, the sterile freeze dryer is set at-60 ℃ and the vacuum degree is 0.1mbar.
3. The method for preparing the sPL platelet factor gel patch according to claim 1, wherein the bottom lining film (1) is made of polyurethane material or polypropylene material.
4. The method for preparing the sPL platelet factor gel patch according to claim 1, wherein the upper cover film (2) is high-density polyethylene or medical release paper.
5. An sPL platelet according to claim 1A method for preparing a factor gel patch, characterized in that the concentration of PDGF-AB in sPL platelet factor preparation is 1100 x 10 if it is lower than 130ng/mL 9 ～1400*10 9 Increasing the platelet concentration in step a within a range of individual/L; if the concentration of PDGF-AB in sPL platelet factor preparation is higher than 140ng/mL, the supernatant plasma remaining from step a is used for dilution.
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| CN112121229B (en) * | 2020-09-27 | 2022-09-09 | 天晴干细胞股份有限公司 | Tissue engineering material for inhibiting inflammation and promoting alveolar bone repair and preparation method and application thereof |
| CN112206345B (en) * | 2020-10-13 | 2022-12-16 | 天晴干细胞股份有限公司 | Sustained-release multi-crosslinking hydrogel dressing and preparation method and application thereof |
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