Autologous platelet-containing plasma gel as well as preparation method and application thereof

Abstract

The invention provides autologous platelet-containing plasma gel and a preparation method and application thereof, and relates to the technical field of biological medicine and tissue engineering.The preparation method comprises the following steps that peripheral venous blood of a patient is extracted and added into a centrifugal tube mixed with a sodium citrate anticoagulant; putting the centrifugal tube into a centrifugal machine for centrifuging, and taking upper-layer plasma; wherein the upper-layer plasma refers to plasma between the upper-layer liquid level and the lower-layer red blood cell layer by 1mm; the centrifugal speed ranges from 1300 r / min to 1700 r / min; the centrifugation time is 3-5 min; under a sterile condition, preparing a calcium gluconate / thrombin mixed solution; and dropping the calcium gluconate / thrombin mixed solution into the upper plasma, mixing, and solidifying to obtain the autologous platelet-containing plasma gel. After one-time centrifugation, upper-layer full-layer plasma is extracted, the volume of the prepared gel is far larger than that of a traditional method, the utilization rate of whole blood platelets and fibrinogen is close to 100%, and regeneration and repair of large-area tissue wounds can be met.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomedicine and tissue engineering, and in particular to an autologous platelet-containing plasma gel and a preparation method and application thereof. Background Art

[0002] Diabetic foot (DF) has a high cumulative prevalence among diabetic patients. DFUs are not only a major cause of disability among diabetic patients, but persistent wounds also impose a significant economic burden on patients, their families, and society. DFU has become a global healthcare problem. Due to the combined effects of multiple risk factors, diabetic skin ulcers often exhibit varying degrees of repair defects, and traditional treatments are ineffective. Since Whitman first proposed the concept of autologous platelet-rich gel, it has been applied to wound treatment in orthopedics, maxillofacial surgery, burns, and plastic surgery, achieving excellent results. Margolis et al. began using autologous platelet-rich gel (APG) for the treatment of DFUs. Subsequent studies have demonstrated that platelet gels have hemostatic, antibacterial, wound-sealing and protective properties, and can release multiple growth factors and cytokines, significantly accelerating wound healing. APG has now become an effective treatment for DFU.

[0003] Studies have found that the mechanism of action of APG in DF disease is primarily related to the release of multiple growth factors that promote ulcer healing, such as platelet-derived growth factor, vascular endothelial growth factor, transforming growth factor, epidermal growth factor, insulin-like growth factor-1, and basic fibroblast growth factor, after platelets in the platelet gel are activated by activators and undergo degranulation. Furthermore, the ratio of growth factors contained in APG is almost identical to that in the normal body, exerting an optimal synergistic effect in promoting ulcer healing, vascular regeneration, and nerve recovery. Secondly, APG is rich in fibrin, which provides a scaffold for cell growth and tissue repair at the ulcer site, ensuring the relatively free chemotaxis of growth factors while also stretching the wound surface to promote wound healing. Furthermore, thrombin acts as a chelating agent for APG, solidifying it into a colloid and applying it to the surface of the ulcer, which not only seals and protects the wound, but also creates a relatively humid and low-oxygen environment, which is conducive to the secretion of cytokines in the wound, the proliferation of capillaries, the regeneration of granulation tissue, and the crawling of epidermal cells at the wound edge, thereby accelerating the healing of the ulcer; in addition, APG is rich in white blood cells and monocytes, and after platelet activation, the α particles in the platelets release a variety of antibacterial active substances, which play an effective anti-infection role.

[0004] Currently, the trauma treatment effect of APG is known in the art. However, traditional autologous platelet-rich plasma technology has the following limitations:

[0005] First, most diabetic foot patients are frail, suffer from heart and kidney dysfunction, and are elderly with hypoproteinemia. Furthermore, local patients have poor medical awareness. By the time they seek medical attention, most diabetic wounds have already developed severe infections and extensive tissue defects. Traditional methods for preparing autologous platelet-rich gels, after secondary centrifugation, yield a small volume of gel: only 6-10 mL of gel can be extracted from 100 mL of blood, making them insufficient for the regeneration and repair of large tissue defects.

[0006] Second, according to the consensus of those skilled in the art, the traditional autologous platelet-rich plasma gel preparation method separates a whole blood sample into multiple samples including a platelet-rich plasma (PRP) sample, a platelet-poor plasma (PPP) sample, and a red blood cell sample. Plasma contains not only platelets and growth factors, but also fibrinogen. The function of fibrinogen is mainly to provide a scaffold for cell growth and tissue repair at the ulcer site, promote platelet aggregation, and thus promote wound healing. In order to achieve better therapeutic effects, the traditional platelet-rich plasma technology only retains the PRP sample, while 90% of the PPP is discarded, resulting in a platelet collection rate of only 80% and a waste of 90% of the fibrinogen.

[0007] Third, traditional APG preparation requires secondary centrifugation and multiple transfers between multiple containers. The process is cumbersome and time-consuming, increasing the risk of external contamination. This is not conducive to the safe and effective preparation of platelet gels and consumes the valuable time of medical workers.

[0008] In view of this, the present invention is proposed. Summary of the Invention

[0009] The purpose of the present invention is to provide an autologous platelet-containing plasma gel and its preparation method and application. The present invention avoids disease transmission and immune rejection reaction, more fully utilizes the intervention of platelets and a large amount of fibrinogen, and greatly reduces the cost of wound treatment.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] The present invention provides a method for preparing an autologous platelet-containing plasma gel, comprising the following steps:

[0012] S1: Draw peripheral venous blood from the patient, add it to a centrifuge tube mixed with sodium citrate anticoagulant, mix it, and obtain a whole blood sample;

[0013] S2: Place the centrifuge tube containing the whole blood sample in a centrifuge and centrifuge. After centrifugation, discard the red blood cell layer at the bottom of the centrifuge tube and take the upper plasma layer;

[0014] The upper plasma refers to the plasma between the upper liquid surface and 1 mm above the lower red blood cell layer;

[0015] The centrifugal speed is 1300-1700 r / min; the centrifugal time is 3-5 min;

[0016] S3: Under sterile conditions, add calcium gluconate injection into the ampoule of lyophilized thrombin powder and mix to obtain a calcium gluconate / thrombin mixture;

[0017] S4: dropping the calcium gluconate / thrombin mixture into the upper layer of plasma, mixing, and obtaining an autologous platelet-containing plasma gel after coagulation.

[0018] Furthermore, based on the above technical solution, the ratio of the patient's ulcer area to the content of the patient's peripheral venous blood obtained is 1cm 2 :(0.5-2)mL.

[0019] Furthermore, based on the above technical solution, the centrifugal speed is 1500 r / min; the centrifugal time is 4 minutes;

[0020] And / or, in step S2, the centrifugation refers to only one centrifugation, after which the red blood cell layer at the bottom of the centrifuge tube is discarded and the upper plasma layer is taken.

[0021] Furthermore, based on the above technical solution, the concentration of the calcium gluconate injection is 8-12%, preferably 10%; and the added amount is 1-3 mL, preferably 2 mL.

[0022] Furthermore, based on the above technical solution, the thrombin lyophilized powder is 2000U of thrombin lyophilized powder.

[0023] Furthermore, based on the above technical solution, the concentration of the calcium gluconate / thrombin mixture is 1-3 ku / mL, preferably 1 ku / mL.

[0024] Furthermore, based on the above technical solution, the volume ratio of the calcium gluconate / thrombin mixture to the upper plasma is 1:(5-15).

[0025] The present invention also provides an autologous platelet-containing plasma gel prepared by the above-mentioned method for preparing the autologous platelet-containing plasma gel.

[0026] Furthermore, based on the above technical solution, the utilization rate of the autologous platelet-containing plasma gel on platelets in plasma is ≥99.35%, and the utilization rate of fibrinogen in plasma is 100%.

[0027] The present invention also provides an autologous platelet-containing plasma gel prepared by the above-mentioned method for preparing the autologous platelet-containing plasma gel, or the use of the above-mentioned autologous platelet-containing plasma gel in treating or preparing diabetic foot medicine.

[0028] The present invention provides an autologous platelet-containing plasma gel, a preparation method thereof, and an application thereof, which have the following beneficial effects:

[0029] 1. Expanded scope of therapeutic application: Traditional autologous platelet-rich gel is prepared by two centrifugations, which discards 90% of platelet-poor plasma and fibrinogen, resulting in a whole blood platelet and fibrinogen utilization rate of only 70%-92% and 5%-20% respectively. In addition, the prepared gel is small in volume and can only be used for small-scale defects and wounds. The improved autologous platelet-containing gel of the present invention retains the entire layer of plasma, with a platelet utilization rate of nearly 100% and a fibrinogen utilization rate of 100%. The prepared gel volume is much larger than that of the traditional method, thus meeting the needs of regeneration and repair of large-area tissue defects and wounds.

[0030] 2. Greatly improves the utilization rate of fibrinogen in plasma: The platelet-poor plasma discarded after secondary centrifugation during the preparation of traditional autologous platelet-rich gel is rich in fibrinogen, while the fibrinogen utilization rate in the preparation method provided by the present invention is 100%. Fibrinogen can provide a scaffold for cell growth and tissue repair in the ulcer site. Fully utilizing fibrinogen not only ensures the relatively free chemotactic effect of growth factors, but also can stretch the wound surface and shorten the wound healing time.

[0031] 3. Simple and easy operation, reduced contamination risk, and time savings: Traditional preparation of autologous platelet-rich plasma gel requires secondary centrifugation and multiple transfers between multiple containers. This cumbersome and time-consuming process increases the risk of external contamination, hindering safe and effective platelet gel preparation and consuming valuable medical workers' time. The improved technology of the present invention is easy to operate, reduces the risk of contamination, and saves medical workers' time.

[0032] 4. Safe, effective and inexpensive: The autologous platelet-containing gel provided by the present invention is extracted from the patient himself, avoiding disease transmission and immune rejection reaction, and can mobilize the tissue's own repair ability for the organism's own repair. The present invention is based on the original technology for the use of platelets and growth factors. After improvement, it makes more full use of the activation and aggregation of platelets and fibrinogen, and fully utilizes the fibrinogen in plasma to promote wound healing, realizing the autologous platelet-containing gel technology as a safe and effective tissue repair solution; and compared with existing exogenous biomaterials, such as Pinec artificial dermis material (the commercially available 3×3cm Pinec artificial dermis material is about 3,000 yuan), the autologous platelet-containing gel has the advantages of low price and easy acceptance by patients, which can greatly reduce the economic burden on patients, has good social application value, and is a key development project for diabetic wound repair in the future.

[0033] 5. Traditional methods for preparing autologous platelet-rich gels often overemphasize the use of high-concentration platelets as the core treatment method to enhance the treatment effect. The present invention breaks the limitations and constraints of traditional ideas and innovatively proposes a method for preparing autologous platelet-containing gels. It does not rely on high-concentration platelets and can achieve therapeutic effects equivalent to or even better than traditional high-concentration platelet gels. It also significantly improves the utilization rate of fibrinogen and platelets in plasma, and can also meet the treatment needs of large-area wounds or extensive tissue defects. It provides a more feasible and efficient treatment plan for the healing of large-area wounds, greatly broadens the application scope of autologous platelet-containing therapies, and brings revolutionary progress to the field of clinical medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 Schematic diagram of the preparation of autologous platelet-rich gel under the condition of the first centrifugation speed of 1500 r / min for traditional autologous platelet-rich gel;

[0036] Figure 2 A schematic diagram of the method for preparing the autologous platelet-containing gel provided by the present invention;

[0037] Figure 3 This is a comparison chart of the volume and applicable wound area of ​​traditional autologous platelet-rich gel and the autologous platelet-containing gel provided by the present invention;

[0038] Figure 4Comparison pictures of the patient's wound surface taken before treatment and one week after treatment in Example 1;

[0039] Figure 5 Comparison pictures of the patient's wound surface taken before treatment and one week after treatment in Example 2;

[0040] Figure 6 Comparison pictures of the patient's wound surface taken before treatment and one week after treatment in Example 3;

[0041] Figure 7 Comparison pictures of the patient's wound surface taken before treatment and one week after treatment in Example 4;

[0042] Figure 8 Comparison pictures of the patient's wound surface taken before treatment and one week after treatment in Example 5;

[0043] Figure 9 Comparative Example 1: a comparison of the patient's wound surface taken before treatment and one week after treatment;

[0044] Figure 10 Comparative Example 2: The wound surface of the patient was taken before treatment and the wound surface of the patient was taken one week after treatment;

[0045] Figure 11 Comparative Example 3: The wound surface of the patient was taken before treatment and the wound surface of the patient was taken one week after treatment;

[0046] Figure 12 Comparative Example 4 shows a comparison of the patient's wound surface taken before treatment and one week after treatment;

[0047] Figure 13 Comparative Example 5 shows a comparison of the patient's wound surface taken before treatment and one week after treatment;

[0048] icon:

[0049] 1. Whole blood sample; 2. First plasma; 3. White blood cell layer; 4. Red blood cell layer; 5. Platelets; 6. Platelet-poor plasma (PPP); 7. Platelet-rich plasma (PRP); 8. Upper plasma. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The process parameters for which specific conditions are not specified in the following examples are generally in accordance with conventional conditions.

[0051] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to form one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed in the present invention.

[0052] According to the first aspect of the present invention, Figure 2 As shown, a method for preparing an autologous platelet-containing plasma gel is provided, comprising the following steps:

[0053] S1: Draw peripheral venous blood from the patient, add it to a centrifuge tube mixed with sodium citrate anticoagulant, and mix it to obtain whole blood sample 1;

[0054] Sodium citrate can form a stable complex with calcium ions in the blood, thereby reducing the concentration of free calcium ions in the blood. Since calcium ions are essential for the coagulation process, the reduction in their concentration will prevent the formation of thrombin, thereby inhibiting blood coagulation, keeping the blood liquid during centrifugation and facilitating stratification.

[0055] S2: placing the centrifuge tube containing the whole blood sample 1 in a centrifuge for centrifugation, discarding the red blood cell layer 4 at the bottom of the centrifuge tube, and taking the upper layer of plasma 8;

[0056] The upper plasma refers to the plasma between the upper liquid surface and 1 mm above the lower red blood cell layer;

[0057] The centrifugal speed is 1300-1700 r / min (e.g., 1350 r / min, 1400 r / min, 1450 r / min, 1500 r / min, 1550 r / min, 1600 r / min, 1650 r / min, etc.);

[0058] The centrifugation time is 3-5 min (e.g., 3.5 min, 4 min, 4.2 min, 4.5 min, 4.7 min, 4.9 min, etc.);

[0059] Among them, at a centrifugal speed of 1300-1700 r / min, after centrifugation, the centrifuge tube is separated into layers. Red blood cells are the densest component in the blood and will settle at the bottom of the tube after centrifugation; a small number of white blood cells are present in the middle layer of the centrifuge tube to form a white blood cell layer 3, and plasma containing platelets and fibrinogen is located in the upper layer of the centrifuge tube. Discarding the bottom layer can remove most impurities such as red blood cells, thereby facilitating the purification of platelets. It should be further explained that the centrifuge provided in the present invention has a centrifugal radius of 20 cm, that is, the relative centrifugal force of the centrifuge in the present invention is 378×g-646×g.

[0060] Furthermore, the present invention limits the centrifugal speed to 1300-1700 r / min, which not only improves the purity of the upper plasma but also maintains the activity of platelets, providing a more stable and reliable foundation for the upper plasma in the subsequent gel preparation process;

[0061] If the centrifugal speed is lower than 1300 r / min, heavier impurities such as red blood cells cannot settle to the bottom of the centrifuge tube within 3-5 minutes, and these impurities may potentially contaminate the upper plasma layer.

[0062] Furthermore, if the centrifugation operation is performed at a centrifugal speed lower than 1300 r / min (for example, 1000 r / min) and the centrifugation time is extended (for example, 8 minutes), although the problem of insufficient centrifugal force can be compensated by extending the centrifugation time, if the centrifugation time is too long, the mitochondrial respiratory chain of the platelets will be continuously in a state of stress, resulting in a decrease in superoxide dismutase (SOD) activity and an increase in intracellular ROS levels. When the ROS concentration exceeds the threshold of 50 μmol / L, it will activate the p38MAPK signaling pathway to cause irreversible activation of platelets, prematurely release growth factors, thereby reducing the subsequent concentration of growth factors in the plasma and affecting the therapeutic effect. Moreover, during the relatively low-speed centrifugation process, although the mechanical shear force generated by a single centrifugation is weak, the long-term centrifugation operation will cause the platelets to be continuously exposed to a shear stress environment. This continuous mechanical stimulation will also lead to premature activation of platelets in the plasma.

[0063] If the centrifugal speed is higher than 1700r / min, it will also lead to premature activation of platelets in the plasma. Under the action of high centrifugal force, platelets may be activated prematurely due to mechanical stress or shear force. This activation will cause platelets to release growth factors prematurely, thereby reducing the concentration of growth factors in the subsequent plasma and affecting the treatment effect.

[0064] S3: Under sterile conditions, add calcium gluconate injection into the ampoule of lyophilized thrombin powder and mix to obtain a calcium gluconate / thrombin mixture;

[0065] S4: dropping the calcium gluconate / thrombin mixture into the upper layer of plasma 8, mixing, and obtaining an autologous platelet-containing plasma gel after coagulation.

[0066] Specifically, platelets and fibrinogen both play an important role in the ulcer repair process. As described in the background art, platelets, upon activation, can release a variety of growth factors to promote ulcer healing, while fibrinogen can provide a scaffold for cell growth and tissue repair at the ulcer site, ensuring the relatively free chemotaxis of growth factors while also pulling the wound surface to promote wound healing. Traditional autologous platelet-rich gel preparations, in order to obtain relatively pure and highly concentrated platelet-rich plasma, are typically centrifuged twice. However, this also discards 90% of the platelet-poor plasma and fibrinogen, resulting in a platelet utilization rate of only 70%-92% in the whole blood sample, and an even lower fibrinogen utilization rate of only 5%-20%. Furthermore, the gel prepared using traditional methods is small in volume; only 6-10 mL of gel can be extracted from 100 mL of blood. This makes it suitable only for small-scale wounds and cannot meet the regeneration and repair needs of large-scale tissue wounds.

[0067] On the premise of taking completely consistent peripheral venous blood from the patient, the present invention extracts the upper layer of plasma after one centrifugation and adds a calcium gluconate / thrombin mixture. The volume of the prepared gel is much larger than the gel volume prepared by the traditional method. The utilization rate of whole blood platelets is close to 100%, and the utilization rate of fibrinogen is 100%. Not only is the repair effect good and the time is short, but it can also meet the regeneration and repair of large-area tissue defects.

[0068] like Figure 3 As shown, the volume of gel obtained by the traditional method is small, so it is only suitable for the treatment of small wounds; while the volume of gel obtained by the preparation method provided by the present invention is significantly larger than that of the traditional method, and can be used for the regeneration and repair of large-area tissue defects and wounds.

[0069] As an optional embodiment of the present invention, the ratio of the patient's ulcer area to the content of the patient's peripheral venous blood obtained is 1 cm 2 :(0.5-2)mL, such as 1cm 2 :0.75mL, 1cm 2 :0.8mL, 1cm 2 :1mL, 1cm 2 :1.5mL, etc.

[0070] Specifically, if Figure 3 As shown, compared with the traditional preparation method, the autologous platelet-containing plasma gel provided by the present invention can prepare a gel with a much larger volume using the same amount of blood as the traditional method, which can meet the regeneration and repair of large-area tissue defects and wounds.

[0071] As an optional embodiment of the present invention, the centrifugal speed is 1500 r / min; the centrifugal time is 4 minutes;

[0072] And / or, in step S2, the centrifugation refers to only one centrifugation, after which the red blood cell layer at the bottom of the centrifuge tube is discarded and the upper plasma layer is taken.

[0073] Specifically, traditional preparation of autologous platelet-rich plasma gel requires secondary centrifugation and multiple transfers between multiple containers. This cumbersome and time-consuming process increases the risk of external contamination, hindering the safe and effective preparation of platelet gel and consuming valuable medical workers' time. The preparation method provided by the present invention is easy to operate, reduces the risk of contamination, and saves medical workers' time.

[0074] Furthermore, in the traditional method, the first centrifugation is a light centrifugation, and its centrifugal speed is usually 500-1000 r / min; the whole blood sample 1 can be separated into two layers by the first centrifugation, the upper layer is the first plasma 2, and the lower layer is the white blood cell and red blood cell sedimentation layer. By using a lower speed for the first centrifugation, the platelets 5 can be evenly dispersed in the first plasma 2 without precipitation, forming a platelet-rich first plasma 2 layer.

[0075] If the first centrifugation in the traditional method adopts a higher centrifugal speed (greater than 1000r / min, such as 1500r / min) for centrifugation for 4min, Figure 1 As shown, the whole blood sample 1 is divided into three layers, namely the first plasma 2, the white blood cell layer 3 and the red blood cell layer 4. At a higher centrifugal speed, the platelets 5 will be precipitated and deposited at the boundary between the white blood cell layer 3 and the first plasma 2. In order to obtain a higher purity first plasma 2, the platelets 5 deposited at the boundary are usually discarded, resulting in the loss of platelets 5 in the first plasma 2, thereby reducing the volume of the final gel.

[0076] Furthermore, in the traditional method, the second centrifugation is a heavy centrifugation, and its centrifugal speed is usually 2000-2500r / min. The second centrifugation is performed at an ultra-high speed (2500r / min) for 6 minutes to precipitate the platelets 5 in the first plasma 2, forming platelet-rich plasma (PRP) 7 and platelet-poor plasma (PPP) 6 layers. The platelet-rich plasma (PRP) 7 of the lower layer is taken, and the platelet-poor plasma (PPP) 6 of the upper layer is discarded to obtain a high concentration of platelet-rich plasma (PRP) 7, which is further used to prepare the gel.

[0077] The present invention directly adopts a centrifugal speed of 1300-1700 r / min for only one centrifugation, although it also causes platelets 5 to be deposited at the boundary between the white blood cell layer 3 and the first plasma 2, but the upper plasma 8 collected by the present invention is as follows. Figure 2 As shown, the upper plasma 8 refers to the plasma between the upper liquid surface and 1 mm above the lower red blood cell layer 4. That is, the upper plasma 8 collected in the present invention includes the first plasma 2 and the white blood cell layer 3. Therefore, the high-speed centrifugation operation will not affect the gel to be prepared in the present invention. In addition, the high-speed centrifugation can more quickly precipitate heavier components such as red blood cells and white blood cells to the bottom of the centrifuge tube, thereby improving the separation efficiency and further reducing impurities in the upper plasma 8.

[0078] Furthermore, the present invention limits the centrifugation time to 3-5 minutes, aiming to ensure that red blood cells can effectively and fully settle to the bottom of the centrifuge tube, thereby avoiding unnecessary interference with plasma components; however, if the centrifugation time exceeds 5 minutes, the excessively long centrifugation time will lead to further deposition of platelets and fibrinogen, making the concentration of the collected upper plasma layer uneven. More importantly, this uneven concentration of the upper plasma will directly affect the final concentration of the gel prepared by the method of the present invention, affecting its effectiveness in medical applications. Gels with uneven concentrations may not provide a consistent healing environment, affecting cell growth and differentiation, thereby reducing their ability to repair wounds.

[0079] As an optional embodiment of the present invention, the concentration of the calcium gluconate injection is 8-12% (such as 9%, 10%, 11%, etc.), preferably 10%; the added amount is 1-3 mL (such as 1.5 mL, 2 mL, 2.5 mL, etc.), preferably 2 mL.

[0080] As an optional embodiment of the present invention, the thrombin lyophilized powder is 2000U of thrombin lyophilized powder.

[0081] As an optional embodiment of the present invention, the concentration of the calcium gluconate / thrombin mixture is 1-3 ku / mL (such as 1.5 ku / mL, 2 ku / mL, 2.5 ku / mL, etc.), preferably 1 ku / mL.

[0082] Specifically, the calcium ions provided by calcium gluconate can activate the conversion of prothrombin into active thrombin, play a promoting role in the conversion of fibrinogen into fibrin, and can optimize platelet activation and growth factor release. The present invention limits the concentration of the calcium gluconate / thrombin mixture to 1-3 ku / mL in order to ensure that the mixture remains relatively stable during preparation and use, thereby playing multiple roles such as coordinating coagulation, enhancing gel stability through fibrin formation, and promoting tissue repair through platelet activation.

[0083] As an optional embodiment of the present invention, the volume ratio of the calcium gluconate / thrombin mixture to the upper plasma is 1:(5-15), such as 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, etc., preferably 1:10.

[0084] The present invention also provides an autologous platelet-containing plasma gel prepared by the above-mentioned preparation method of the autologous platelet-containing plasma gel.

[0085] As an optional embodiment of the present invention, the utilization rate of the autologous platelet-containing plasma gel on platelets in plasma is ≥99.35%, and the utilization rate of the fibrinogen in plasma is 100%.

[0086] The present invention also provides an autologous platelet-containing plasma gel prepared by the above-mentioned method for preparing the autologous platelet-containing plasma gel, or the use of the above-mentioned autologous platelet-containing plasma gel in treating or preparing diabetic foot medicine.

[0087] Specifically, the autologous platelet-containing gel provided by the present invention is extracted from the patient himself, avoiding disease transmission and immune rejection reaction, and can mobilize the tissue's own repair ability to be utilized for the organism's own repair. The present invention is based on the original technology for utilizing platelets and growth factors. After improvement, it makes more full use of platelets and a large amount of fibrinogen, realizing the autologous platelet-containing gel technology as a safe and effective tissue repair solution; and compared with existing exogenous biomaterials, such as Pinec artificial dermis material, autologous platelet-containing gel has the advantages of low price and easy acceptance by patients, which can greatly reduce the economic burden of patients and has good social application value. It is a key development project for future diabetic wound repair.

[0088] The present invention will be further described in detail below with reference to specific examples and comparative examples.

[0089] Example 1

[0090] We receive patients with diabetic wounds. After comprehensive treatment including early debridement and dressing change, negative pressure drainage, blood sugar monitoring, anti-infection, multi-organ function support, and nutritional support, the wounds are transformed from infected wounds to repair wounds. We prepare autologous platelet-containing gels to promote wound healing.

[0091] Step 1: The wound area of ​​the patient in Case 1 was measured with a ruler to be 90 cm 2 , 60 mL of peripheral venous blood was collected from the patient, divided into sodium citrate tubes, and mixed to obtain whole blood samples;

[0092] Step 2: At room temperature, centrifuge the centrifuge tube containing the whole blood sample at a speed of 1500 r / min for 4 minutes. After one centrifugation, draw the upper plasma to 1 mm above the red blood cell layer and transfer it to another sterile plastic container to obtain approximately 30 mL of plasma.

[0093] Step 3: Under sterile conditions, add 2 mL of 10% calcium gluconate injection to a 2000 U thrombin lyophilized powder ampoule and shake thoroughly until dissolved into a calcium gluconate / thrombin mixture. The prepared liquid concentration is 1 ku / mL.

[0094] Step 4: Add 3 mL of the calcium gluconate / thrombin mixture to the autologous platelet-containing plasma, mix gently, and wait for the mixture to coagulate to form an autologous platelet-containing plasma gel.

[0095] Step 5: Fill the wound surface and sinus with the prepared autologous platelet-containing plasma gel, suture the skin edges to minimize the wound area, and cover with vaseline gauze;

[0096] Step 6: Continue with negative pressure drainage treatment. Replace the upper negative pressure material after 1 week. The wound at the suture site heals well with no obvious exudation. Continue with negative pressure drainage treatment. Remove the negative pressure after 1 week and continue with iodine tincture and dressing treatment. Remove the sutures at 4 weeks (25-30 days).

[0097] The comparison of the patient's wound surface before treatment and the patient's wound surface after treatment for one week is shown in the figure below. Figure 4 As shown, it can be seen that the patient's wound healed well.

[0098] Example 2

[0099] We receive patients with diabetic wounds. After comprehensive treatment including early debridement and dressing change, negative pressure drainage, blood sugar monitoring, anti-infection, multi-organ function support, and nutritional support, the wounds are transformed from infected wounds to repair wounds. We prepare autologous platelet-containing gels to promote wound healing.

[0100] Step 1: The wound area of ​​the patient in Case 2 was measured with a ruler to be 80 cm 2 , 60 mL of peripheral venous blood was collected from the patient, divided into sodium citrate tubes, and mixed to obtain whole blood samples;

[0101] Step 2: At room temperature, centrifuge the centrifuge tube containing the whole blood sample at a speed of 1500 r / min for 4 minutes. After one centrifugation, draw the upper plasma layer to 1 mm above the red blood cell layer and transfer it to another sterile plastic container to obtain approximately 25 mL of plasma.

[0102] Step 3: Under sterile conditions, add 2 mL of 10% calcium gluconate injection to a 2000 U thrombin lyophilized powder ampoule and shake thoroughly until dissolved into a calcium gluconate / thrombin mixture. The prepared liquid concentration is 1 ku / mL.

[0103] Step 4: Add 3 mL of the calcium gluconate / thrombin mixture to the autologous platelet-containing plasma, mix gently, and wait for the mixture to coagulate to form an autologous platelet-containing plasma gel.

[0104] Step 5: Fill the wound surface and sinus with the prepared autologous platelet-containing plasma gel, suture the skin edges to minimize the wound area, and cover with vaseline gauze;

[0105] Step 6: Continue with negative pressure drainage treatment. Replace the upper negative pressure material after 1 week. The wound at the suture site heals well with no obvious exudation. Continue with negative pressure drainage treatment. Remove the negative pressure after 1 week and continue with iodine tincture and dressing treatment. Remove the sutures at 4 weeks (25-30 days).

[0106] The comparison of the patient's wound surface before treatment and the patient's wound surface after treatment for one week is shown in the figure below. Figure 5 As shown, it can be seen that the patient's wound healed well.

[0107] Example 3

[0108] We receive patients with diabetic wounds. After comprehensive treatment including early debridement and dressing change, negative pressure drainage, blood sugar monitoring, anti-infection, multi-organ function support, and nutritional support, the wounds are transformed from infected wounds to repair wounds. We prepare autologous platelet-containing gels to promote wound healing.

[0109] Step 1: The wound area of ​​the patient in Case 3 was measured with a ruler to be 100 cm 2 , 60 mL of peripheral venous blood was collected from the patient, divided into sodium citrate tubes, and mixed to obtain whole blood samples;

[0110] Step 2: At room temperature, centrifuge the centrifuge tube containing the whole blood sample at a speed of 1500 r / min for 4 minutes. After one centrifugation, draw the upper plasma layer to 1 mm above the red blood cell layer and transfer it to another sterile plastic container to obtain approximately 34 mL of plasma.

[0111] Step 3: Under sterile conditions, add 2 mL of 10% calcium gluconate injection to a 2000 U thrombin lyophilized powder ampoule and shake thoroughly until dissolved into a calcium gluconate / thrombin mixture. The prepared liquid concentration is 1 ku / mL.

[0112] Step 4: Add 3 mL of the calcium gluconate / thrombin mixture to the autologous platelet-containing plasma, mix gently, and wait for the mixture to coagulate to form an autologous platelet-containing plasma gel.

[0113] Step 5: Fill the wound surface and sinus with the prepared autologous platelet-containing plasma gel, suture the skin edges to minimize the wound area, and cover with vaseline gauze;

[0114] Step 6: Continue with negative pressure drainage treatment. Replace the upper negative pressure material after 1 week. The wound at the suture site heals well with no obvious exudation. Continue with negative pressure drainage treatment. Remove the negative pressure after 1 week and continue with iodine tincture and dressing treatment. Remove the sutures at 4 weeks (25-30 days).

[0115] The comparison of the patient's wound surface before treatment and the patient's wound surface after treatment for one week is shown in the figure below. Figure 6 As shown, it can be seen that the patient's wound healed well.

[0116] Example 4

[0117] We receive patients with diabetic wounds. After comprehensive treatment including early debridement and dressing change, negative pressure drainage, blood sugar monitoring, anti-infection, multi-organ function support, and nutritional support, the wounds are transformed from infected wounds to repair wounds. We prepare autologous platelet-containing gels to promote wound healing.

[0118] Step 1: The wound area of ​​the patient in Case 4 was measured with a ruler to be 72 cm 2 50 mL of peripheral venous blood was collected from the patient and placed in a sodium citrate tube and mixed to obtain a whole blood sample;

[0119] Step 2: At room temperature, centrifuge the centrifuge tube containing the whole blood sample at a speed of 1500 r / min for 4 minutes. After one centrifugation, draw the upper plasma to 1 mm above the red blood cell layer and transfer it to another sterile plastic container to obtain approximately 20 mL of plasma.

[0120] Step 3: Under sterile conditions, add 2 mL of 10% calcium gluconate injection to a 2000 U thrombin lyophilized powder ampoule and shake thoroughly until dissolved into a calcium gluconate / thrombin mixture. The prepared liquid concentration is 1 ku / mL.

[0121] Step 4: Add 3 mL of the calcium gluconate / thrombin mixture to the autologous platelet-containing plasma, mix gently, and wait for the mixture to coagulate to form an autologous platelet-containing plasma gel.

[0122] Step 5: Fill the wound surface and sinus with the prepared autologous platelet-containing plasma gel, suture the skin edges to minimize the wound area, and cover with vaseline gauze;

[0123] Step 6: Continue with negative pressure drainage treatment. Replace the upper negative pressure material after 1 week. The wound at the suture site has healed well with no obvious exudation. Continue with negative pressure drainage treatment. Remove the negative pressure after 1 week and continue with iodine tincture and dressing treatment. Remove the sutures after 4 weeks (25-30 days).

[0124] The comparison of the patient's wound surface before treatment and the patient's wound surface after treatment for one week is shown in the figure below. Figure 7 As shown, it can be seen that the patient's wound healed well.

[0125] Example 5

[0126] We receive patients with diabetic wounds. After comprehensive treatment including early debridement and dressing change, negative pressure drainage, blood sugar monitoring, anti-infection, multi-organ function support, and nutritional support, the wounds are transformed from infected wounds to repair wounds. We prepare autologous platelet-containing gels to promote wound healing.

[0127] Step 1: The wound area of ​​the patient in Case 5 was measured with a ruler to be 75 cm 2 50 mL of peripheral venous blood was collected from the patient and placed in a sodium citrate tube and mixed to obtain a whole blood sample;

[0128] Step 2: At room temperature, centrifuge the centrifuge tube containing the whole blood sample at a speed of 1500 r / min for 4 minutes. After one centrifugation, draw the upper plasma layer to 1 mm above the red blood cell layer and transfer it to another sterile plastic container to obtain approximately 25 mL of plasma.

[0129] Step 3: Under sterile conditions, add 2 mL of 10% calcium gluconate injection to a 2000 U thrombin lyophilized powder ampoule and shake thoroughly until dissolved into a calcium gluconate / thrombin mixture. The prepared liquid concentration is 1 ku / mL.

[0130] Step 4: Add 3 mL of the calcium gluconate / thrombin mixture to the autologous platelet-containing plasma, mix gently, and wait for the mixture to coagulate to form an autologous platelet-containing plasma gel.

[0131] Step 5: Fill the wound surface and sinus with the prepared autologous platelet-containing plasma gel, suture the skin edges to minimize the wound area, and cover with vaseline gauze;

[0132] Step 6: Continue with negative pressure drainage treatment. Replace the upper negative pressure material after 1 week. The wound at the suture site has healed well with no obvious exudation. Continue with negative pressure drainage treatment. Remove the negative pressure after 1 week and continue with iodine tincture and dressing treatment. Remove the sutures after 4 weeks (25-30 days).

[0133] The comparison of the patient's wound surface before treatment and the patient's wound surface after treatment for one week is shown in the figure below. Figure 8 As shown, it can be seen that the patient's wound healed well.

[0134] Comparative Examples 1-5

[0135] Comparative Examples 1-5 all adopted the traditional method for preparing autologous platelet-rich plasma gel, and the specific steps are as follows:

[0136] Step 1: Collect peripheral venous blood from the patient, place it in a sodium citrate tube, mix it, and obtain a whole blood sample;

[0137] Step 2: At room temperature, centrifuge the centrifuge tube containing the whole blood sample at a speed of 800 rpm for 4 minutes. After the centrifugation, collect the first plasma layer and transfer it to another sterile centrifuge tube.

[0138] Step 3: The centrifuge tube containing the first plasma was centrifuged for a second time at a centrifugal speed of 2500 r / min for 6 min. After the second centrifugation, the lower platelet-rich plasma (PRP) layer was collected and transferred to another sterile plastic container;

[0139] Step 4: Under sterile conditions, add 2 mL of 10% calcium gluconate injection to a 2000 U thrombin lyophilized powder ampoule and shake thoroughly until the mixture dissolves into a calcium gluconate / thrombin solution with a concentration of 1 ku / mL.

[0140] Step 5: Add 1 mL of the calcium gluconate / thrombin mixture to the autologous platelet-rich plasma, mix gently, and wait for the mixture to coagulate to form a traditional autologous platelet-rich plasma gel.

[0141] Step 6: Fill the wound surface and sinus with the prepared autologous platelet-rich plasma gel, suture the skin edges to minimize the wound area, and cover with vaseline gauze;

[0142] Step 7: Continue with negative pressure drainage treatment. Replace the upper negative pressure material after 1 week. The wound at the suture site heals well with no obvious exudation. Continue with negative pressure drainage treatment. Remove the negative pressure after 1 week and continue with iodine tincture and dressing change treatment. Remove the sutures at 4 weeks (25-30 days).

[0143] The specific operating parameters in Comparative Examples 1-5 are shown in Table 1.

[0144] The comparison pictures of the patient wounds taken before treatment and after treatment for 1 week in Comparative Examples 1-5 are as follows: Figures 9-13 shown.

[0145] Comparative Example 6

[0146] This comparative example refers to patent CN 114209806 B. The main difference from Examples 1-5 is that the platelet-rich fibrin in the middle layer is used to treat patients with diabetic foot ulcers. The specific steps are as follows:

[0147] Step 1: The wound area of ​​the patient in comparative example 6 was 5 cm measured by a ruler. 2 5 mL of peripheral venous blood was collected from the patient and placed in a tube without sodium citrate within 1 minute. The blood was mixed to obtain a whole blood sample.

[0148] Step 2: At room temperature, immediately centrifuge the whole blood sample at a speed of 1500 r / min for 14 min. After centrifugation, the blood is divided into three layers. Discard the supernatant, remove the red blood cell fragments at the bottom layer, and collect the 3cm middle gel layer. 3 , obtaining platelet-rich fibrin;

[0149] The prepared platelet-rich fibrin was placed statically in a dry and sterilized dressing tray, and squeezed between two sterile gauze layers to form a platelet-rich fibrin membrane, which was evenly spread or stuffed on the diabetic foot wound; the surface was covered with sterile gauze soaked in 1% ethacridine, and the outer layer was bandaged with sterile gauze. The platelet-rich fibrin membrane was replaced every 10 days.

[0150] At the fourth week, 15 mL of peripheral venous blood was drawn from the patient in Comparative Example 6, and about 15 cm 3 The middle gel layer had a fibrin utilization rate of 80.37%, a platelet utilization rate of 83.55%, and a blood draw volume / wound area value of 3. The patient's wound was sutured in the first week, but had not healed in the fourth week, and the healing time was 47 days.

[0151] Comparative Example 7

[0152] Compared with Example 1, the main difference between this comparative example is that the centrifugal speed in step 2 is 1000 r / min and the centrifugal time is 4 min. The other steps and technical parameters are the same as those in Example 1.

[0153] Comparative Example 8

[0154] Compared with Example 1, the main difference between this comparative example is that the centrifugal speed in step 2 is 2000 r / min and the centrifugal time is 4 min. The remaining steps and technical parameters are the same as those in Example 1.

[0155] Comparative Example 9

[0156] Compared with Example 1, the main difference between this comparative example is that the centrifugal speed in step 2 is 1500 r / min and the centrifugal time is 8 min. The remaining steps and technical parameters are the same as those in Example 1.

[0157] Comparative Example 10

[0158] Compared with Example 1, the main difference between this comparative example is that the centrifugal speed in step 2 is 1000 r / min and the centrifugal time is 8 min. The remaining steps and technical parameters are the same as those in Example 1.

[0159] Test method:

[0160] Platelet concentration was tested using the Sysmex XN2000 hematology analyzer, and the reagents used were those supplied by the Sysmex manufacturer.

[0161] The fibrinogen concentration test was performed using the Sysmex CS5100 fully automatic coagulation analyzer. The reagents used were those provided by the Sysmex manufacturer, and the coagulation method was used for detection.

[0162] Table 1

[0163]

[0164]

[0165]

[0166] Among them, the larger the blood draw volume / wound area data value is, the lower the utilization rate of the patient's systemic venous blood drawn is.

[0167] Comparative Examples 1-5 use the traditional method for preparing autologous platelet-containing plasma gel. As shown in Table 1, the blood draw volume / wound area data values ​​in Comparative Examples 1-5 are significantly higher than those in Examples 1-5. On this basis, if the gel preparation method provided by Comparative Examples 1-5 is to treat large wounds, such as 90 cm in Example 1, the blood draw volumes in Comparative Examples 1-5 are 225 mL, 149.4 mL, 450 mL, 450 mL, and 299.7 mL, respectively, which are much larger than the 60 mL in Example 1. Therefore, the present invention can achieve a significant therapeutic effect of healing large wounds with a small amount of blood draw. The volume of the APG gel prepared by the present invention is much larger than that of the traditional method, and can meet the regeneration and repair of large-area tissue defects.

[0168] According to Table 1, in Comparative Example 6, since the anticoagulant sodium citrate is not added, the blood will coagulate during the centrifugation process. In theory, the serum separated from the plasma after the blood coagulation floats on the top, and the serum does not contain fibrinogen, so it is discarded. However, Comparative Example 6 is difficult to implement in actual operation. First, it is necessary to ensure that the blood is centrifuged in the centrifuge within 1 min. The experimental operation is difficult and the operability is low, which greatly increases the experimental failure rate. Secondly, the blood will still coagulate during the centrifugation process, resulting in unclear stratification of the supernatant, the middle gel layer and the red blood cell layer at the bottom. In addition to serum and red blood cells, the discarded supernatant and the red blood cell layer at the bottom also include some fibrinogen and platelets, resulting in the loss of platelets and fibrinogen. Therefore, the utilization rate of platelets and fibrinogen is lower than that of Examples 1-5, and red blood cells will be mixed into the middle gel layer, which will affect the healing effect of the wound, thereby delaying healing.

[0169] According to Table 1, compared with Example 1, in Comparative Example 7, since the centrifugal speed in Comparative Example 7 was 1000 r / min, the red blood cells did not completely settle to the bottom of the centrifuge tube within 4 minutes, resulting in a higher content of the red blood cell layer, that is, a decrease in the plasma content in the upper layer, further resulting in a decrease in the gel volume and a prolonged treatment time.

[0170] According to Table 1, compared with Example 1, in Comparative Example 8, the centrifugal speed is 2000 r / min, which is too high, resulting in premature release of growth factors by platelets, affecting the therapeutic effect.

[0171] According to Table 1, compared with Example 1, Comparative Example 9 has a longer centrifugation time of 8 minutes, which leads to further deposition of platelets and fibrinogen, making the concentration of the collected upper plasma uneven, affecting the treatment effect.

[0172] According to Table 1, compared with Example 1, the centrifugal speed of Comparative Example 10 is 1000 r / min and the centrifugation time is 8 min, which is equivalent to extending the centrifugation time at a lower centrifugal speed. The longer centrifugation time causes the platelets in the plasma to be activated prematurely and releases the growth factors in advance, thereby affecting the treatment effect.

[0173] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an autologous platelet-containing plasma gel, characterized in that: The steps include: S1: Draw peripheral venous blood from the patient, add it to a centrifuge tube mixed with sodium citrate anticoagulant, mix it, and obtain a whole blood sample; S2: Place the centrifuge tube containing the whole blood sample in a centrifuge and centrifuge. After centrifugation, discard the red blood cell layer at the bottom of the centrifuge tube and take the upper plasma layer; The upper plasma refers to the plasma between the upper liquid surface and 1 mm above the lower red blood cell layer; The centrifugal speed is 1300-1700 r / min; the centrifugal time is 3-5 min; S3: Under sterile conditions, add calcium gluconate injection into the ampoule of lyophilized thrombin powder and mix to obtain a calcium gluconate / thrombin mixture; S4: dropping the calcium gluconate / thrombin mixture into the upper layer of plasma, mixing, and obtaining an autologous platelet-containing plasma gel after coagulation.

2. The method for preparing the autologous platelet-containing plasma gel according to claim 1, characterized in that: The ratio of the patient's ulcer area to the content of the patient's peripheral venous blood obtained is 1cm 2 :(0.5-2)mL.

3. The method for preparing the autologous platelet-containing plasma gel according to claim 1, characterized in that: The centrifugal speed is 1500 r / min and the centrifugal time is 4 min; And / or, in step S2, the centrifugation refers to only one centrifugation, after which the red blood cell layer at the bottom of the centrifuge tube is discarded and the upper plasma layer is taken.

4. The method for preparing the autologous platelet-containing plasma gel according to claim 1, wherein: The concentration of the calcium gluconate injection is 8-12%, preferably 10%; the added amount is 1-3 mL, preferably 2 mL.

5. The method for preparing the autologous platelet-containing plasma gel according to claim 1, characterized in that: The thrombin lyophilized powder is 2000U of thrombin lyophilized powder.

6. The method for preparing the autologous platelet-containing plasma gel according to claim 1, characterized in that: The concentration of the calcium gluconate / thrombin mixture is 1-3 ku / mL, preferably 1 ku / mL.

7. The method for preparing the autologous platelet-containing plasma gel according to claim 1, characterized in that: The volume ratio of the calcium gluconate / thrombin mixture to the upper plasma is 1:(5-15).

8. An autologous platelet-containing plasma gel prepared by the method for preparing an autologous platelet-containing plasma gel according to any one of claims 1 to 7.

9. The autologous platelet-containing plasma gel according to claim 8, characterized in that The utilization rate of the autologous platelet-containing plasma gel on platelets in plasma is ≥99.35%, and the utilization rate of the fibrinogen in plasma is 100%.

10. Use of the autologous platelet-containing plasma gel prepared by the method for preparing the autologous platelet-containing plasma gel according to any one of claims 1 to 7 or the autologous platelet-containing plasma gel according to any one of claims 8 to 9 in treating or preparing a drug for diabetic foot.

Images