Platelet-rich plasma suspension containing blood cells and preparation method and application of gel of platelet-rich plasma suspension

Abstract

The invention relates to a preparation method and application of a PRP-plus (Platelet Rich Plasma) containing blood cells and a gel of the PRP-plus. According to the PRP-plus prepared by the invention, platelets and leukocytes in blood are 100% efficiently extracted, the concentration of the platelets and the leukocytes is constantly 4 times that of whole blood, and the PRP-plus is particularly characterized in that the concentration of erythrocytes and hemoglobin is the same as that of normal whole blood, and the PRP-plus is rich in cell growth factors and inflammation immune induction factors, so that the PRP-plus has a good application prospect. It is found that PRP-plus can promote proliferation and growth of bone marrow stromal stem cells, fibroblasts, chondrocytes, osteoblasts and vascular endothelial cells, vascular network formation and tissue regeneration and repair by up-regulating protein abundance of a hypoxia inducible factor HIF1alpha and a vascular endothelial cell growth factor VEGF in cells.

Description

Technical Field

[0001] The present invention relates to the technical field of blood products, and in particular to a blood cell-containing platelet-rich plasma suspension and its gel component, preparation method and clinical application. Background Art

[0002] Fractures are the most common diseases in orthopedics. Defective fractures and comminuted fractures are serious types of fractures. Comminuted fractures are often combined with defective fractures. They are one of the common factors causing delayed union and nonunion of fractures. Regardless of delayed union or nonunion, functional recovery is slow and poor, which puts a heavy mental and economic burden on patients and their families, and should be avoided as much as possible. How to prevent nonunion and improve the healing rate of severe fractures is a problem that still needs to be solved in clinical practice.

[0003] Clinically, nonunion of fractures refers to fractures that have not healed for more than 9 months after standardized treatment. The current gold standard for the treatment of nonunion of fractures is to open the fracture again and re-fix it with autologous bone grafting. However, there is still a high risk of nonunion after surgery. Moreover, if the non-healing lesion itself is infected, multiple surgeries are required to clean the lesion and thoroughly control the infection before the above surgical plan can be carried out. At present, the treatment cycle of nonunion of fractures in patients is long, the effect is uncertain, and the functional recovery is not ideal. It is one of the difficult clinical diseases.

[0004] Articular surface osteochondral defect injuries include acute and chronic osteochondral defect injuries, and the causes include traumatic, pathological, and degenerative. Regardless of the cause of the articular surface osteochondral defect injury, the final outcome is irreversible loss of joint function and persistent pain. Clinically, current treatments can use tissue engineering to construct osteocartilage for surgical repair, but this treatment is not only expensive, requires multiple surgeries, and has a long treatment cycle, but the final effect on the joint is also unsatisfactory, and its clinical application is severely limited. Therefore, there is an urgent need to overcome this treatment problem.

[0005] Difficult-to-heal wounds refer to pathological changes caused by infection, cell regeneration and angiogenesis disorders that lead to tissue damage and repair disorders. Difficult-to-heal wounds are common clinical diseases and are very difficult to treat. Many reasons can affect wound healing and repair, the most common of which include diabetes, vasculitis, obesity and smoking. At present, the incidence of difficult-to-heal wounds in clinical practice is on the rise, while the clinical treatment effect is not ideal. The burden on patients and society is heavy, which is a problem that needs to be solved urgently in clinical practice.

[0006] Natural fracture healing is a process of osteogenic differentiation of bone marrow stromal stem cells, reconstruction of vascular networks and bone remodeling. This process can be divided into three stages: the first stage is the hematoma organization stage, the second stage is the callus growth stage, and the third stage is the callus remodeling stage. Among them, the hematoma organization stage and the callus growth stage are the most critical. The main manifestation of fracture at the beginning is local bleeding to form a hematoma, and then the cells in the hematoma gradually absorb and release cytokines that regulate the fibrocartilage differentiation and osteogenic differentiation of bone marrow stromal stem cells and the vascularization and reconstruction of vascular circulation by endothelial cells. Later, the bone remodeling process is completed under stress, that is, bone healing of fractures. Therefore, the production and maintenance of cytokines that regulate the differentiation of bone marrow stromal stem cells and the formation of vascular networks are the key to fracture healing. Moreover, the initial hematoma components of fractures, including blood cells, plasma fibrin and cytokines, are the foundation for initiating the differentiation of bone marrow stromal stem cells and the formation of vascular circulation networks.

[0007] Nonunion of fractures is caused by events that affect the organization of hematoma and callus growth during fracture healing, such as bacterial infection, bone defect, and insufficient vascular nutrition supply. The cellular and molecular mechanisms of these events are mainly the inability to form a vascular network, insufficient number or function of white blood cells that cannot resist bacterial growth and proliferation, and large bone defects that limit the proliferation of bone marrow stromal stem cells. These three factors interact with each other and cause each other, ultimately resulting in nonunion of fractures, bone atrophy, prolonged infection, inability to restore limb function, and even spread of infection, endangering life. Therefore, the key to reversing, remedying and treating nonunion of fractures is to promote vascular circulation reconstruction, increase the number of white blood cells and immune function, and promote the proliferation and differentiation of stem cells and local tissue cells.

[0008] Articular surface osteochondral defects cannot heal naturally under physiological conditions without human intervention. The main reason is that stable hematoma cannot form naturally in the joint and chondrocyte regeneration is very limited. It can only be gradually replaced by osteoblast proliferation to form ossification, osteophytes, and fibrosis, thereby losing the function of articular surface cartilage, which in turn causes loss of joint function, repeated excessive inflammation and immune response, and constant local pain.

[0009] Studies have shown that a variety of cell growth factors, including vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), transforming growth factor β (TGF-β1), platelet-derived cell growth factor (PDGF), insulin-like cell growth factor (IGF), and inflammatory immune induction factors such as interleukin 1β (IL-1β), tumor necrosis factor α (TNFα), and hypoxia-inducible factor 1α (HIF1α), play a decisive role in tissue cell regeneration and repair. In the early stage of tissue injury, hematoma is formed locally, and inflammatory immune induction factors IL1β and TNFα are secreted, driving white blood cells to reach the lesion area to phagocytose bacteria and remove necrotic tissue. VEGF promotes endothelial cell proliferation and growth and vascular network formation, while FGF, TGF-β1, PDGF, IGF, and HIF1α promote the proliferation and differentiation of bone marrow stromal stem cells and the coordinated growth and proliferation of different tissue cells (chondrocytes, osteoblasts, fibroblasts, and endothelial cells), ultimately completing lesion removal and tissue cell regeneration, also known as tissue regeneration and repair. Therefore, the key factors for tissue regeneration and repair are to increase the number of white blood cells and inflammatory immune-inducing factors in the lesion site, thereby clearing bacteria and necrotic tissue, inducing the formation of vascular networks, rebuilding vascular nutrient supply, and promoting the proliferation, growth, and differentiation of different cells. Summary of the invention

[0010] Based on the above problems, the present invention provides a method for preparing a platelet-rich plasma suspension containing blood cells. The platelet and leukocyte extraction rate of the present invention is 100%, the platelet and leukocyte content is constant at 4 times that of whole blood, and the red blood cell and hemoglobin concentrations are the same as those of whole blood, and the blood suspension is rich in cell growth factors and inflammatory immune induction factors.

[0011] In order to solve the above technical problems, a method for preparing a platelet-rich plasma suspension containing blood cells is provided, characterized in that, a 15ml sterile centrifuge tube is used, 1ml sterile anticoagulant is added in advance, 8ml fresh whole blood is added to the centrifuge tube, and then the centrifuge tube containing a total of 9ml blood is placed in a medical centrifuge, and centrifuged at 3600r / min for 15min at room temperature. After centrifugation, the blood is clearly divided into three levels of components in the centrifuge tube, and the level components from top to bottom are 5.4ml light yellow liquid plasma layer, 0.01ml white flocculent solid buffy coat layer, and 3.59ml dark red solid red blood cell layer, and then the top 3.97ml plasma in the upper light yellow plasma layer is first sucked and discarded with a sterile pipette, and then the bottom 2.43ml red blood cells in the lower red blood cell layer are sucked and discarded, and the remaining 2.6ml blood cell-containing plasma in the middle is gently blown and evenly mixed and suspended. The anticoagulant is any one of ethylenediaminetetraacetic acid, sodium citrate, clopidogrel, aspirin, and low molecular weight heparin.

[0012] To solve the above technical problems, the present invention also provides PRP-plus gel. Preparation method 1: Add medical sterile 10% CaCl2 and sterile 500u thrombin to the prepared PRP-plus, wait for more than 5 minutes, and the PRP-plus gradually coagulates into a blood-red gel. Preparation method 2: PRP-plus gel can also be prepared in this way. 1ml sterile physiological saline is added to a 15ml sterile centrifuge tube in advance, and 8ml fresh whole blood is added to the centrifuge tube. Then, the centrifuge tube containing 9ml of blood is centrifuged at 3600r / min for 15min at room temperature within 3 minutes. After centrifugation, the blood is clearly divided into three layers in the centrifuge tube. From top to bottom, the layers are pale yellow liquid plasma layer (5.4ml), white flocculent solid buffy coat layer (0.01ml) and dark red solid red blood cell layer (3.59ml). Then, within 2 minutes, the top 3.97ml of plasma in the upper plasma layer is first sucked and discarded with a sterile pipette, and the bottom 2.43ml of red blood cells in the lower red blood cell layer are sucked and discarded. The 2.6ml blood cell-containing plasma in the middle is left. After gently blowing and mixing, let it stand for 5 minutes to obtain 2.6ml blood-red PRP-Plus gel.

[0013] Compared with the prior art, the beneficial effects of the present invention are: the PRP-plus prepared by the present invention can efficiently extract 100% of the platelets and leukocytes in the blood without waste, and its platelet and leukocyte concentration is constant at 4 times that of whole blood. The particularly prominent feature is that the red blood cell and hemoglobin concentrations are the same as normal whole blood, and it is rich in cell growth factors and inflammatory immune induction factors. It is found that PRP-plus can promote the proliferation and growth of bone marrow stromal stem cells, fibroblasts, chondrocytes, osteoblasts and vascular endothelial cells, the formation of vascular networks and tissue regeneration and repair by upregulating the abundance of intracellular HIF1α and VEGF protein factors. The preparation of the present invention clarifies the blood cell components, extraction methods and application scenarios of PRP-plus, and provides a new solution for the clinical solution of difficult and thorny problems such as defective fractures, fracture nonunion, osteochondral defects, and difficult-to-heal wounds, which has important clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A , Figure 1B , Figure 1C This is a graph showing the experimental results of the preparation of PRP-plus and the determination of the content of its components according to an embodiment of the present invention;

[0015] Figure 2A , Figure 2B , Figure 2C , Figure 2D This is a result diagram of detecting the concentration of cell growth factors and the abundance of inflammatory immune inducing factor proteins according to an embodiment of the present invention;

[0016] Figure 3 This is a graph showing the experimental results of PRP-plus on proliferation and growth of different tissue cells according to an embodiment of the present invention;

[0017] Figure 4A , Figure 4B , Figure 4C This is a diagram showing the experimental results of regulating vascular endothelial cell vascular network formation according to an embodiment of the present invention;

[0018] Figure 5A , Figure 5B , Figure 5C , Figure 5D , Figure 5E This is a diagram showing the treatment process and results of PRP-plus for treating difficult-to-heal wounds according to an embodiment of the present invention;

[0019] Fig. 6A , Figure 6B , Figure 6C It is a diagram showing the experimental treatment process and results of PRP-plus in treating defective fracture healing according to an embodiment of the present invention;

[0020] Fig. 7A , Figure 7B , Figure 7C , Fig.7D The treatment process and result diagram of PRP-plus for treating nonunion of fractures according to an embodiment of the present invention;

[0021] Fig. 8A , Figure 8B , Figure 8C , Fig.8D This is a diagram showing the experimental treatment process and results of using PRP-plus to treat articular surface osteochondral defects according to an embodiment of the present invention. DETAILED DESCRIPTION

[0022] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0023] Example:

[0024] In order to solve the difficult problems of clinical defective fractures, fracture nonunion, osteochondral defects, and difficult-to-heal wounds, this embodiment has invented a formula and preparation method of platelet-rich plasma plus blood cells (PRP-plus) and its gel. The white blood cell and platelet content in the PRP-plus prepared in this embodiment is constant at 4 times that of whole blood, and the red blood cell and hemoglobin concentrations are the same as normal whole blood. It is rich in cell growth factors and inflammatory immune induction factors. It has been found through experiments that PRP-plus can promote the proliferation, growth, and differentiation of bone marrow stromal stem cells by upregulating the abundance of intracellular VEGF and HIF1α proteins, and also promote the proliferation and growth of different tissue cells fibroblasts, chondrocytes, osteoblasts, and vascular endothelial cells and the formation of vascular networks, thereby inducing tissue regeneration and repair to treat defective fractures, fracture nonunion, osteochondral defects, difficult-to-heal wounds, and other diseases. The specific method is shown below.

[0025] See Figure 1, where Figure 1A and Figure 1B The following is a schematic diagram of the preparation process of PRP in this embodiment. The specific preparation process is as follows:

[0026] 1.1. Blood collection: ① Blood collection from New Zealand rabbit veins: Blood is collected from the ear marginal veins of adult New Zealand rabbits (weighing about 3kg). Surgery can be performed if there is no redness, swelling or bleeding in the surgical area; 3ml / kg of medical 5% chloral hydrate is injected intraperitoneally for anesthesia; disinfection is performed after anesthesia takes effect, hair is plucked in the surgical area (note that hair cannot be shaved), alcohol disinfection is performed, the rabbit ear marginal vein is found, and the scalp needle is inserted into the ear marginal vein. After blood returns, the tail end of the scalp needle is connected to a 10ml sterile syringe containing 1ml ethylenediaminetetraacetic acid to collect about 8ml of blood, and then transferred to a 15ml sterile tube for use. ② Human venous blood collection: 8ml of human elbow venous blood is directly drawn with a 10ml sterile syringe containing 1ml ethylenediaminetetraacetic acid, and then transferred to a 15ml sterile tube for use.

[0027] 1.2. Preparation of PRP-plus: The collected blood was centrifuged only once, at room temperature and 3600r / min for 15 minutes. After centrifugation, the blood was clearly separated into three layers in the centrifuge tube. From top to bottom, the layers were pale yellow liquid plasma layer (5.4ml), white flocculent solid buffy coat layer (0.01ml) and dark red solid red blood cell layer (3.59ml). Then, the top 3.97ml of plasma in the upper layer was sucked and discarded with a sterile pipette, and the bottom 2.43ml of red blood cells in the lower layer were sucked and discarded. The 2.6ml of blood cell-containing plasma in the middle was left, and gently blown and evenly mixed. After suspension, 2.6ml of sterile blood-red PRP-Plus was obtained.

[0028] 1.3. Preparation of PRP (platelet-rich plasma): The collected blood is centrifuged for the first time. The first centrifugation parameters are as follows: centrifugation at 2000r / min for 10 minutes at room temperature. After centrifugation, the blood is clearly separated into three layers of components in the centrifuge tube, from top to bottom, namely, a light yellow plasma layer, a white flocculent buffy coat layer, and a dark red red blood cell layer. Then, a 5ml sterile syringe is connected to a disposable sterile lumbar puncture needle with a scale and carefully aspirated to the surface of the buffy coat layer, and transferred to a new 15ml sterile centrifuge tube. The remaining cellular components are discarded. This part only contains all the upper plasma. The second centrifugation parameters are as follows: centrifugation at 2000r / min for 10 minutes at room temperature and the blood is clearly separated into two parts, namely, a light yellow plasma layer and a white flocculent mass layer. Use a sterile pipette to carefully aspirate and discard the upper plasma layer, and only retain 1.43ml of plasma in the bottom layer and transfer it to a medical sterile 5ml syringe. After repeated aspiration and suspension, about 1.43ml of light yellow PRP can be obtained.

[0029] 1.4. Preparation of L-PRP (leukocyte-rich platelet-rich plasma): The collected blood is centrifuged for the first time at 2000 r / min at room temperature for 10 min. After centrifugation, the blood is clearly divided into three parts in the centrifuge tube, which are a light yellow plasma layer, a white flocculent buffy coat layer and a dark red red red blood cell layer from top to bottom. Then, a 5 ml sterile syringe is connected to a disposable sterile lumbar puncture needle with a scale and the blood is carefully aspirated to 1 mm below the buffy coat layer. The blood is transferred to a new 15 ml sterile centrifuge tube and the remaining cell components are discarded. The blood in the new centrifuge tube must contain all the upper plasma layers and all the buffy coat layers. The blood is then centrifuged for the second time at 2000 r / min at room temperature for 10 min. The upper plasma layer is carefully aspirated and discarded with a sterile pipette, leaving only the bottom 1.43 ml plasma and white flocculent mass layer, which are transferred to a medical sterile 5 ml syringe. After repeated aspiration and suspension, about 1.43 ml of yellow-white L-PRP can be obtained.

[0030] See attached Figure 1C In this example, the platelet, leukocyte, erythrocyte and hemoglobin concentrations in PRP, L-PRP and PRP-plus were measured. The average platelet concentration in human whole blood was 279.33±7.41 (10 9 / L), the average platelet concentration in PRP was 1055.33±119.20 (10 9 / L); the average platelet concentration in L-PRP was 1086.67±62.87 (10 9 / L); the average platelet concentration in PRP-plus was 1116.33±22.03 (10 9 / L). The platelet content of PRP, L-PRP and PRP-plus increases in sequence, and their platelet content is significantly higher than the platelet level in whole blood (about 4 times), which meets the conditions of platelet-rich plasma. Figure 1B The average concentration of white blood cells in human whole blood is 6.84±0.22 (10 9 / L), the average concentration of white blood cells in PRP was 0.0117±0.0005 (10 9 / L). The average concentration of leukocytes in L-PRP was 14.42±3.84 (10 9 / L), the average white blood cell concentration in PRP-plus was 28.08±1.75(10 9 / L). The average white blood cell content in PRP was significantly lower than the detection threshold of the complete blood cell counter, while the average white blood cell content in L-PRP was significantly higher than that in whole blood and PRP. In addition, the average white blood cell content in PRP-plus was significantly higher than that in whole blood and L-PRP. The average white blood cell content in L-PRP was twice that in whole blood, while that in PRP-plus was twice that in L-PRP. The difference was statistically significant (P < 0.01). See Appendix Figure 1B The average concentration of red blood cells in whole blood was 5.35±0.24 (10 12 / L), the red blood cells in PRP are very few and can be ignored. The average concentration of red blood cells in L-PRP is 0.0013±0.0005 (10 12 / L), the average red blood cell concentration in PRP-plus was 5.40±0.4 (10 12 / L). Similarly, the average hemoglobin concentration in whole blood is 160.66±3.09 (g / L), and the hemoglobin in PRP is very small and can be ignored. The average hemoglobin concentration in L-PRP is 0.0183±0.0063 (g / L), and the average hemoglobin concentration in PRP-plus is 161.66±3.30 (g / L). PRP extraction contains almost no red blood cells and hemoglobin. L-PRP is contaminated with red blood cells and hemoglobin, but the content is relatively small, while the average content of red blood cells and hemoglobin in PRP-plus is basically consistent with that in whole blood. From the above data, it can be seen that the platelet extraction rate in PRP is 67.59%, the platelet extraction rate in L-PRP is 69.58%, and the platelet extraction rate in PRP-plus is 100% (due to the swelling of red blood cells, the actual calculated value is 130%). In addition, the leukocyte extraction rate in L-PRP is 37.68%, while the leukocyte extraction rate in PRP-plus is 100% (due to the swelling of red blood cells, the actual calculated value is 133%). Will attach Figure 2A and 2B, Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of cell growth factors and inflammatory immune induction factors IL-1β and TNFα in adult whole blood, PRP, L-PRP and PRP-plus. The average concentrations of vascular endothelial growth factor, fibroblast growth factor, transforming growth factor β1, platelet-derived growth factor and insulin-like growth factor in whole blood were 101.28±18.82pg / ml, 98.99±14.24pg / ml, 15.02±2.09ng / ml, 23.12±5.19ng / ml and 171.13±15.07ng / ml, respectively. The average concentrations of vascular endothelial growth factor, fibroblast growth factor, transforming growth factor β1, platelet-derived growth factor, and insulin-like growth factor in PRP were 340.32±43.57pg / ml, 339.92±52.35pg / ml, 54.23±14.91ng / ml, 68.12±13.25ng / ml, and 719.15±103.17ng / ml, respectively. The average concentrations of vascular endothelial growth factor, fibroblast growth factor, transforming growth factor β1, platelet-derived growth factor, and insulin-like growth factor in L-PRP were 342.21±36.28pg / ml, 443.84±32.61pg / ml, 65.68±16.82ng / ml, 59.01±15.37ng / ml, and 727.23±88.47ng / ml, respectively. Finally, the average concentrations of vascular endothelial growth factor, fibroblast growth factor, transforming growth factor β1, platelet-derived growth factor, and insulin-like growth factor in PRP-plus were 819.22±77.83pg / ml, 739.17±83.17pg / ml, 84.21±7.91ng / ml, 88.31±7.72ng / ml, and 837.81±93.24ng / ml, respectively. It can be seen that compared with whole blood, the concentrations of vascular endothelial growth factor, fibroblast growth factor, transforming growth factor β1, platelet-derived growth factor, and insulin-like growth factor in various PRP extracts were significantly increased, and the differences were statistically significant (P<0.01). There was no significant difference between PRP and L-PRP, but PRP-plus was higher than PRP and L-PRP, and the difference was statistically significant (P<0.01). The average concentrations of interleukin-1β and tumor necrosis factor α in whole blood were 60.17±11.71 pg / ml and 6.33±2.34 ng / ml, respectively. The average concentrations of interleukin-1β and tumor necrosis factor α in PRP were 30.38±4.51 pg / ml and 3.73±0.77 ng / ml, respectively.The average concentrations of interleukin-1β and tumor necrosis factor α in L-PRP were 179.41±39.41pg / ml and 11.23±2.33ng / ml, respectively. The average concentrations of interleukin-1β and tumor necrosis factor α in PRP-plus were 277.29±89.11pg / ml and 24.31±7.93ng / ml, respectively. Compared with whole blood, the average concentrations of interleukin-1β and tumor necrosis factor α in PRP were the lowest, and the concentrations in PRP-plus were the highest. The differences were statistically significant (P<0.01).

[0031] This example uses Western-blot experiments to study the effect of PRP-plus on the abundance of hypoxia-inducible factor HIF1α and vascular endothelial growth factor VEGF in chondrocytes and vascular endothelial cells. The experimental results are shown in the attached Figure 2C , chondrocytes and endothelial cells were expressed at 2.5×10 5 / ml plate, after the cells adhered, the DMEM cell culture medium containing 10% whole blood, 10% PRP, 10% L-PRP and 10% PRP-plus was replaced, and 10% fetal bovine serum was used as the control. On the third day, the cells in the multiple phase were collected for Western blotting to detect the abundance of hypoxia-inducible factor HIF1α and vascular endothelial growth factor VEGF. The protein abundance was expressed as protein gray value / β-actin gray value. The results showed that in chondrocytes, compared with the control group, the abundance of hypoxia-inducible factor HIF1α protein factor increased slightly after whole blood, PRP, and L-PRP treatment, while the abundance of hypoxia-inducible factor HIF1α protein increased significantly after PRP-plus treatment, and the difference was statistically significant (P < 0.01). At the same time, in vascular endothelial cells, compared with the control group, there was no significant change in the abundance of vascular endothelial growth factor VEGF protein after whole blood treatment, and the abundance of vascular endothelial growth factor VEGF protein increased significantly after PRP and PRP-plus treatment, and the abundance of vascular endothelial growth factor VEGF protein increased most significantly after PRP-plus treatment, with statistically significant differences (P < 0.01). The results showed that PRP-plus can significantly increase the abundance of hypoxia-inducible factor HIF1α and vascular endothelial growth factor VEGF protein factors in tissue cells.

[0032] This example studies the effect of PRP-plus on the proliferation and growth of different tissue cells through cell proliferation experiments. The experimental results are shown in the attached Figure 3Different tissue cells (bone marrow stromal stem cells, chondrocytes, osteoblasts, fibroblasts, and vascular endothelial cells) were plated in 96-well plates, with 3,000 cells per well. After 24 hours of cell attachment, the DMEM cell culture medium containing 10% PRP, 10% L-PRP, and 10% PRP-plus was replaced once for 5 days. The DMEM culture medium containing 10% fetal bovine serum was used as the control. The OD value of cell proliferation and growth was measured by a cell counting kit (CCK-8) at different time points, i.e., on the 1st day, 3rd day, and 5th day. The results showed that on the 1st and 3rd day, various tissue cells proliferated and grew significantly, but on the 5th day, only the PRP and PRP-plus treatment groups were still proliferating and growing significantly, and the proliferation and growth of PRP-plus was the most obvious, indicating that PRP-plus has a stronger and more lasting effect on the proliferation and growth of tissue cells. Therefore, the results show that PRP-plus promotes cell proliferation for a longer time and has a better effect than PRP and L-PRP, and the difference is statistically significant (P < 0.01).

[0033] This example studies the effect of PRP-plus on the ability of endothelial cells to form vascular venation through a vascular endothelial cell vascular venation formation experiment. The experimental results are shown in Figure 4. Endothelial cells were plated in a 24-well plate, and after the cells adhered to the wall, the DMEM cell culture medium containing 10% PRP-plus was replaced (see Figure 4). Figure 4A ), and the formation of vascular endothelial cells into vascular networks was observed on the 2nd and 4th days. The results showed that vascular endothelial cells proliferated significantly on the 2nd day and initially aggregated to form clusters (see attached). Figure 4B ), and on the 4th day, obvious vascular network formation was observed (see Appendix Figure 4C ). The results showed that PRP-plus significantly promoted the formation of vascular networks by endothelial cells.

[0034] This example studies the effect of PRP-plus prepared in this example on the treatment of patients with difficult-to-heal wounds. In this example, a patient suffered a severe trauma to his left thigh due to a car accident and had an infection for 2 years, leaving a difficult-to-heal wound with tissue defects. Figure 5A The patient had a difficult-to-heal wound with tissue loss on his left thigh and was treated with autologous PRP-plus gel. Figure 5B , fill the wound with autologous PRP-plus gel. Figure 5C On the third day of treatment, granulation tissue grew and filled the wound surface. Figure 5D On the fifth day of treatment, the wound surface was dry and scabbed. Figure 5E On the 7th day of treatment, the wound surface was completely epithelialized and healed.

[0035] This example studies the effect of PRP-plus prepared in this example on the treatment of defect fractures. In this example, defect fractures were modeled on the left lateral femoral condyle of New Zealand white rabbits. The size of the defect fracture accounted for one third of the diameter of the femoral condyle (see attached Fig. 6A ). Four weeks after PRP-plus gel packing treatment, the defect fracture lesion was filled with regenerated tissue (see Appendix Figure 6B ), 12 weeks after PRP-plus gel packing treatment, the defect fracture lesion was found to have completely regenerated and achieved bone healing (see Appendix Figure 6C ).

[0036] This example studies the treatment of nonunion of fractures in patients with PRP-plus prepared in this example. In this example, a patient suffered multiple fractures in the whole body (right femoral neck, right femoral shaft, right fibula, right tibia distal end, right ankle joint, right ulna shaft) due to a car accident. Two years after the surgery, the fractures of the right femoral neck, right fibula, right tibia distal end, and right ankle joint healed, leaving nonunion of the right femoral shaft fracture and right ulna fracture (see attached Fig. 7A and 7C After three local injections of autologous PRP-plus, the patient achieved complete bone healing of the right femoral shaft and right ulna 12 months later (see Appendix Figure 7B and 7D ).

[0037] This example studies the effect of PRP-plus prepared in this example on the treatment of osteochondral defect in the articular surface. In this example, an osteochondral defect was modeled on the right femoral articular surface of the knee joint of New Zealand white rabbits. The defect had a diameter of 4 mm and a depth of 4 mm (see attached). Fig. 8A ). Three weeks after the PRP-plus gel packing treatment, the lesion area was filled with regenerated tissue (see Appendix Figure 8B ); 6 weeks after PRP-plus gel filling treatment, the defect lesion was significantly reduced (see Appendix Figure 8C ); 12 weeks after PRP-plus gel packing treatment, the joint surface had completely regenerated and healed (see Appendix Fig.8D ).

[0038] In summary, the platelet and leukocyte extraction rate of PRP-plus prepared in this embodiment is 100%, and its average content is always 4 times that of whole blood, and the concentration of red blood cells and hemoglobin is the same as that of normal whole blood. It is rich in cell growth factors and inflammatory immune induction factors, and it is found that PRP can promote the proliferation and growth of various tissue cells by upregulating the abundance of intracellular hypoxia-inducible factor HIF1α and vascular endothelial growth factor VEGF protein. The PRP-plus and its gel prepared in this embodiment, and the research on them have refined the classification and practical application of PRP, and discovered a new mechanism for PRP to promote bone, cartilage, and soft tissue regeneration and repair, providing a new solution for clinical practice, which has important clinical significance.

[0039] The above is an embodiment of the present invention. The above embodiments and the specific parameters in the embodiments are only for the purpose of clearly describing the invention verification process, and are not intended to limit the patent protection scope of the present invention. The patent protection scope of the present invention is still subject to its claims. Any equivalent structural changes made by using the contents of the description and drawings of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a platelet-rich plasma suspension containing blood cells, characterized in that: Add 1ml of sterile anticoagulant in a 15ml sterile centrifuge tube in advance, and add 8ml of fresh whole blood to the centrifuge tube to make a total of 9ml of blood; then place the centrifuge tube in a medical centrifuge and centrifuge at 3600r / min for 15min at room temperature. After centrifugation, the blood is clearly separated into three layers of components in the centrifuge tube, and the upper, middle and lower layers are 5.4ml of light yellow liquid plasma layer, 0.01ml of white flocculent solid buffy coat layer, and 3.59ml of dark red solid red blood cell layer, respectively; then use a sterile pipette to carefully absorb and discard the top 3.97ml of plasma in the upper light yellow plasma layer, and then absorb and discard the bottom 2.43ml of red blood cells in the lower red blood cell layer, and the remaining 2.6ml of blood cell-containing plasma in the middle is gently blown and evenly mixed, and then suspended.

2. The method for preparing a platelet-rich plasma suspension containing blood cells according to claim 1, wherein the anticoagulant is any one of ethylenediaminetetraacetic acid, sodium citrate, clopidogrel, aspirin, and low molecular weight heparin.

3. A method for preparing a blood cell platelet-rich plasma suspension gel, characterized in that: Add medical sterile 10% CaCl2 and sterile 500u thrombin to the platelet-rich plasma suspension containing blood cells prepared in claim 1, wait for more than 5 minutes, and the platelet-rich plasma suspension containing blood cells gradually coagulates into a blood-red gel.

4. A method for preparing a blood cell platelet-rich plasma suspension gel, characterized in that: Pre-add 1ml of sterile saline into a sterile 15ml centrifuge tube, add 8mL of fresh whole blood into the centrifuge tube, and then place the centrifuge tube in a medical centrifuge within 3 minutes, and centrifuge at 3600r / min for 15 minutes at room temperature. After centrifugation, the blood is clearly separated into three layers in the centrifuge tube. The upper, middle and lower layers are 5.4ml of light yellow liquid plasma layer, 0.01ml of white flocculent solid buffy coat layer, and 3.59ml of dark red solid red blood cell layer. Then, within 2 minutes, use a sterile pipette to absorb and discard the top 3.97ml of plasma in the upper light yellow plasma layer, and then absorb and discard the bottom 2.43ml of red blood cells in the lower red blood cell layer. The remaining 2.6ml of blood cell-containing plasma in the middle is gently blown and evenly mixed, and then allowed to stand for 5 minutes to gradually coagulate into a blood-red gel.

5. Use of a blood cell platelet-rich plasma suspension gel prepared by the preparation method according to claim 3 or 4, characterized in that Application in the treatment of osteochondral defects, fractures, nonunion of fractures, and difficult-to-heal wounds.

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