An injectable plasma matrix gel and its preparation method and application

Abstract

The present invention provides an injectable plasma matrix gel and a preparation method and application thereof, belonging to the technical field of biomedical materials. The preparation method of the injectable plasma matrix gel comprises: centrifuging a blood sample in a liquid plasma matrix blood collection tube; collecting the upper liquid plasma matrix and the brown-yellow layer of erythrocyte sedimentation at the junction of the upper and lower layers; heating the liquid plasma matrix to convert it into a gel state to obtain a plasma matrix gel; mixing the plasma matrix gel with the brown-yellow layer of erythrocyte sedimentation to obtain an injectable plasma matrix gel. Compared with a solid plasma matrix, the injectable plasma matrix gel provided by the present invention has a better shaping function, is conducive to minimally invasive implantation, and can reduce the demand for whole blood. Compared with a liquid plasma matrix, the injectable plasma matrix gel provided by the present invention has a fast molding speed and a slow degradation speed, can maintain a certain space in the injection area, delays absorption, and has outstanding advantages in promoting the regeneration of soft tissue and favorable bone defects.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to an injectable plasma matrix gel and a preparation method and application thereof. Background Art

[0002] In order to maintain the long-term stability of implant restoration in the aesthetic area of ​​the anterior teeth, operations such as soft tissue thickening, widening of keratinized gingiva, and reconstruction of gingival papillae are often required. Studies have shown that the width of keratinized gingiva plays a key role in the patient's comfort when brushing teeth, and can significantly reduce the discomfort when brushing teeth and reduce marginal bone loss. Therefore, some scholars believe that retaining keratinized gingiva of appropriate width can play a protective role in the health of implant restoration. At the same time, studies on the thickness of labial soft tissue and the effect of implant restoration have also shown that soft tissue thickness can affect the color matching degree between the soft tissue around the implant and the gums of the adjacent natural teeth, and at the same time, it plays a vital role in the stability of the marginal bone level. Based on the long-term research results of natural tooth root surface coverage, soft tissue thickening can improve the long-term stability of the soft tissue edge around the implant.

[0003] Soft tissue thickening surgery can be performed with autologous grafts or soft tissue substitutes. Among them, autologous grafts including autologous connective tissue and keratinized gingiva are considered the "gold standard" for the use of materials. In addition, soft tissue substitutes such as acellular dermal matrix and xenogeneic collagen matrix are currently becoming the mainstream of clinical use. Compared with autologous grafts, soft tissue substitutes can avoid opening up a second surgical area and reduce the overall morbidity of surgery. However, soft tissue substitutes lack growth factors and cellular components and have potential biosafety issues, which is still a defect that soft tissue substitutes cannot overcome.

[0004] To solve this problem, some scholars have used plasma matrix for soft tissue augmentation related surgeries, which to a certain extent avoids the opening of a second surgical area and reduces the occurrence of postoperative complications. At the same time, autologous growth factors and cell components can play a good role in promoting regeneration and achieve relatively satisfactory results. Existing autologous plasma matrices include two types, namely solid plasma matrix and liquid plasma matrix. However, solid plasma matrix requires a larger surgical incision when used, which does not conform to the concept of minimally invasive surgery; liquid plasma matrix is ​​used by injection, which solves this problem to a certain extent, but the liquid plasma matrix after injection is absorbed quickly and cannot maintain a stable space locally, which brings new difficulties to the application of plasma matrix in soft tissue augmentation surgery. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide an injectable plasma matrix gel and its preparation method and application. Compared with the traditional solid plasma matrix, the injectable plasma matrix gel provided by the present invention has better shaping function, is conducive to minimally invasive implantation, and can greatly reduce the demand for whole blood. Compared with the traditional liquid plasma matrix, the injectable plasma matrix gel provided by the present invention has a fast molding speed, a slow degradation rate of coagulation products, can maintain a certain space in the injection area, delay absorption, and has outstanding advantages in promoting soft tissue regeneration and favorable bone defect regeneration.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing an injectable plasma matrix gel, the preparation method comprising the following steps:

[0008] (1) centrifuging a blood sample in a liquid plasma matrix blood collection tube;

[0009] (2) collecting the upper liquid plasma matrix and the buffy coat at the junction of the upper liquid plasma matrix and the lower red blood cells;

[0010] Heating the liquid plasma matrix to transform it into a gel state, thereby obtaining a plasma matrix gel;

[0011] (3) Mixing the plasma matrix gel with the buffy coat to obtain the injectable plasma matrix gel.

[0012] It should be noted that the buffy coat described in the present invention is also called the white film layer, which is located at the junction of the upper liquid plasma matrix and the lower red blood cells, and contains a large number of enriched immune cells and platelets (containing a large number of growth factors). The liquid plasma matrix is ​​rich in albumin.

[0013] The method provided by the present invention heats the liquid plasma matrix so that the albumin inside it is thermally denatured and the structure is changed, thereby converting the liquid plasma matrix into a gel state. The liquid plasma matrix is ​​first separated from the buffy coat, and then the plasma matrix gel is mixed with the buffy coat, so that the heating process can avoid damaging the immune cells and platelets in the buffy coat. The injectable plasma matrix gel thus obtained retains a large amount of growth factors, platelets and immune cells in the blood.

[0014] Compared with the traditional solid plasma matrix, the injectable plasma matrix gel provided by the present invention has better shaping function and is conducive to minimally invasive implantation. In addition, when the solid plasma matrix is ​​transformed into the traditional solid plasma matrix membrane, its volume loss is as high as about 75%; while when the liquid plasma matrix in the present invention is transformed into the plasma matrix gel, its volume retention rate can reach more than 95%. Therefore, compared with the traditional solid plasma matrix, the injectable plasma matrix gel provided by the present invention can also significantly reduce the demand for whole blood.

[0015] Compared with traditional liquid plasma matrix, the injectable plasma matrix gel provided by the present invention has a fast molding speed (it can solidify within 10 minutes), the degradation rate of the coagulation product is slow, it can maintain a certain space in the injection area and delay absorption, and it is rich in immune cells, which can exert local immune regulation function and accelerate early wound healing. Therefore, it has greater advantages in promoting soft tissue regeneration and favorable bone defect regeneration.

[0016] In the present invention, there is no particular limitation on the ratio between the plasma matrix gel and the buffy coat, and those skilled in the art can adjust it according to actual needs. In order to make full use of the blood sample, the present invention preferably uses all the plasma matrix gel and all the buffy coat prepared from the blood sample to prepare the injectable plasma matrix gel.

[0017] In the present invention, there is no particular limitation on the centrifugal method. As non-limiting examples, angular centrifugation or horizontal centrifugation may be used.

[0018] In some embodiments of the present invention, the centrifugal acceleration of the centrifuge is 50-3000g; for example, it can be 50g, 60g, 80g, 100g, 200g, 300g, 500g, 600g, 800g, 1000g, 1200g, 1500g, 1800g, 2000g, 2200g, 2500g, 2800g or 3000g, etc., where "g" represents the acceleration due to gravity. However, the present invention is not limited to the listed values, and other values ​​not listed in this range are also applicable.

[0019] In some embodiments of the present invention, the centrifugation time is 2-15 min, for example, 2 min, 3 min, 5 min, 6 min, 8 min, 10 min, 12 min, 13 min or 15 min, etc. However, the present invention is not limited to the listed values, and other values ​​not listed within the range are also applicable.

[0020] In the present invention, in order to collect the entire buffy coat, the liquid within a certain distance above and below the junction can be collected together. As a non-limiting example, the method for collecting the buffy coat in step (2) is: extracting the mixed liquid from 1 to 2 mm above the junction to 1 to 2 mm below the junction.

[0021] In the present invention, when collecting the brown-yellow layer of the erythrocyte sedimentation unit, the extracted mixed liquid is limited to the above-mentioned range, which can not only ensure that the entire brown-yellow layer of the erythrocyte sedimentation unit is collected, but also avoid extracting too much upper liquid plasma matrix and lower red blood cells, resulting in too little plasma matrix gel and mixing in a large number of red blood cells.

[0022] In some embodiments of the present invention, the heating temperature is 60-80°C; for example, it can be 60°C, 62°C, 63°C, 65°C, 66°C, 68°C, 70°C, 72°C, 73°C, 75°C, 76°C, 78°C or 80°C, etc. However, the present invention is not limited to the listed values, and other values ​​not listed in the range are also applicable.

[0023] In some embodiments of the present invention, the heating time is 5-20 min, for example, 5 min, 6 min, 8 min, 10 min, 12 min, 13 min, 15 min, 16 min, 18 min or 20 min, etc. However, the present invention is not limited to the listed values, and other values ​​not listed in the range are also applicable.

[0024] In the present invention, the heating temperature and time are controlled within the above ranges, which is helpful to obtain a plasma matrix gel in good condition. If the heating temperature is too high or the heating time is too long, the albumin in the liquid plasma matrix may be excessively denatured or even charred; if the heating temperature is too low or the heating time is too short, the liquid plasma matrix may not be able to form a gel.

[0025] In some embodiments of the present invention, the plasma matrix gel is cooled to below 37° C. before the mixing in step (3).

[0026] In the present invention, cooling is performed before mixing the plasma matrix gel with the buffy coat to prevent the high-temperature plasma matrix gel from destroying the immune cells in the buffy coat. In the present invention, there is no particular limitation on the cooling method. As a non-limiting example, cooling may be performed at 0-4°C for more than 5 minutes.

[0027] In a second aspect, the present invention provides an injectable plasma matrix gel prepared by the preparation method described in the first aspect.

[0028] In a third aspect, the present invention provides a plasma matrix gel block, which is a coagulation product of the injectable plasma matrix gel described in the second aspect.

[0029] The present invention provides

[0030] In a fourth aspect, the present invention provides a use of the injectable plasma matrix gel as described in the second aspect or the plasma matrix solid as described in the third aspect as a soft tissue filling material.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] Compared with traditional solid plasma matrix, the injectable plasma matrix gel provided by the present invention has better shaping function, is conducive to minimally invasive implantation, and can significantly reduce the demand for whole blood.

[0033] Compared with traditional liquid plasma matrix, the injectable plasma matrix gel provided by the present invention has a fast molding speed (it can solidify within 10 minutes), the degradation rate of the coagulation product is slow, it can maintain a certain space in the injection area and delay absorption, and it is rich in immune cells, which can exert local immune regulation function and accelerate early wound healing. Therefore, it has greater advantages in promoting soft tissue regeneration and favorable bone defect regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a photo of the injectable plasma matrix gel provided in Example 1 after solidification;

[0035] Figure 2 This is a comparison chart of the quality of the injectable plasma matrix gel provided in Example 1 before and after 30 days of degradation after solidification;

[0036] Figure 3 This is a comparison diagram of the effects of the injectable plasma matrix gel prepared by the method of Example 1 before and after thickening the labial soft tissue;

[0037] Figure 4 This is a comparison diagram of the effects of the injectable plasma matrix gel prepared by the method of Example 1 before and after filling the gingival papilla;

[0038] Figure 5 This is a comparison chart of the effects of maxillary sinus floor lifting before and after the injectable plasma matrix gel prepared by the method of Example 2. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below with reference to the accompanying drawings and through specific implementations. It should be understood by those skilled in the art that the specific implementations are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0040] Example 1

[0041] This embodiment provides an injectable plasma matrix gel, and the preparation method thereof is as follows:

[0042] (1) Collect 20 mL of human blood using two liquid plasma matrix blood collection tubes, each with 10 mL, and quickly place them in the plasma matrix preparation platform and centrifuge at 1500 g for 5 minutes;

[0043] (2) Use a disposable syringe to draw liquid plasma matrix 5 mm above the junction of red blood cells and liquid plasma matrix (2 blood collection tubes, a total of 8 mL);

[0044] (3) heating the liquid plasma matrix in the syringe at 80°C for 5 minutes, and then cooling in an ice bath at 0°C for 7 minutes to obtain a plasma matrix gel (about 8 mL);

[0045] (4) Use another new disposable syringe to draw out the mixed liquid containing the Buffy coat layer between 1 mm above and below the junction of red blood cells and liquid plasma matrix (a total of 0.5 mL for two blood collection tubes);

[0046] (5) Use a two-way tube to connect two syringes and mix them evenly to form an injectable plasma matrix gel. After standing for 10 minutes, it solidifies to form a plasma matrix gel block, as shown in the photo. Figure 1 shown.

[0047] Example 2

[0048] This embodiment provides an injectable plasma matrix gel, and the preparation method thereof is as follows:

[0049] (1) Collect 40 mL of human blood using four liquid plasma matrix blood collection tubes, each with 10 mL, and quickly place them in the plasma matrix preparation platform and centrifuge at 800 g for 7 minutes;

[0050] (2) Use a disposable syringe to extract the liquid plasma matrix 3 mm above the junction of red blood cells and plasma matrix;

[0051] (3) heating the liquid plasma matrix in the syringe at 70° C. for 10 minutes, and then cooling in an ice bath at 0° C. for 5 minutes to obtain a plasma matrix gel;

[0052] (4) Injecting the plasma matrix gel into the space that needs regeneration;

[0053] (5) Use another new disposable syringe to extract the mixed liquid containing the Buffy coat layer between 1 mm above and below the junction of red blood cells and liquid plasma matrix, and inject it into the regenerated space to cover the plasma matrix gel (in situ formation of injectable plasma matrix gel). After standing for 10 minutes, it solidifies and forms a plasma matrix gel block.

[0054] Example 3

[0055] This embodiment provides an injectable plasma matrix gel, and the preparation method thereof is as follows:

[0056] (1) Collect 20 mL of human blood using two liquid plasma matrix blood collection tubes, each with 10 mL, and quickly place them in the plasma matrix preparation platform and centrifuge at 1000 g for 6 minutes;

[0057] (2) Use a disposable syringe to draw out the liquid plasma matrix 5 mm above the junction of red blood cells and liquid plasma matrix;

[0058] (3) heating the liquid plasma matrix in the syringe at 60° C. for 20 minutes, and then cooling in an ice bath at 0° C. for 7 minutes to obtain a plasma matrix gel;

[0059] (4) Use another new disposable syringe to extract the mixed liquid containing the Buffy coat layer between 2 mm above and below the junction of red blood cells and liquid plasma matrix;

[0060] (5) Use a two-way tube to connect two syringes and mix them evenly to form an injectable plasma matrix gel.

[0061] Comparative Example 1

[0062] This comparative example provides a solid plasma matrix membrane, and the preparation method is as follows:

[0063] (1) Collect 20 mL of human blood using two solid plasma matrix blood collection tubes, each with 10 mL, and quickly place them in the plasma matrix preparation platform and centrifuge at 1500 g for 5 minutes;

[0064] (2) After centrifugation, the upper solid plasma matrix (approximately 4 mL per blood collection tube) was obtained and compressed at 1 N pressure for 2 minutes to obtain a solid plasma matrix membrane (approximately 1 mL per sheet).

[0065] Comparison of Example 1 and Comparative Example 1 shows that after the liquid plasma matrix in Example 1 is converted into the plasma matrix gel, the volume retention rate is 100%, while after the solid plasma matrix in Comparative Example 1 is converted into the solid plasma matrix membrane, the volume retention rate is only 25%. It can be seen that when the product volume is the same, the consumption of whole blood by the injectable plasma matrix gel provided by the present invention is much lower than that of the solid plasma matrix membrane.

[0066] Degradation test

[0067] The injectable plasma matrix gel provided in Example 1 was allowed to stand for 10 minutes to solidify to form a plasma matrix gel block, and then 10 mL of DMEM cell culture medium was added and placed in a 37°C incubator. After 30 days, the integrity of the plasma matrix gel block was observed, and the mass before and after degradation was recorded. The mass of the plasma matrix gel block before and after the degradation test was statistically compared. Figure 2 shown.

[0068] from Figure 2 It can be seen that after the plasma matrix gel provided in Example 1 solidifies, it can basically maintain stable quality in the first 30 days of degradation. It can be seen that its degradation rate is slow, which is beneficial to maintain a certain space in the injection area, delay absorption, and promote tissue regeneration.

[0069] Clinical Application

[0070] 1. Thickening of the labial soft tissue

[0071] After communicating the treatment plan with the patient, the patient's blood pressure and heart rate were measured, the surgical area was disinfected with iodine intraorally, and the STA painless anesthesia device was used to inject Bilanma for local infiltration anesthesia of the area with insufficient soft tissue thickness. After extraoral iodine disinfection, a sterile surgical towel was laid. The patient's blood was collected, and an injectable plasma matrix gel was prepared according to the method of Example 1. A 25G black needle was used to inject the injectable plasma matrix gel into the defect area until the fullness of the lip side met the requirements. The injection was given once every 2 weeks for three consecutive injections. After the treatment was completed, it was observed for 3 months, and the changes in the effects before and after were recorded. Comparison of the effects before and after the thickening of the lip soft tissue Figure 3 shown.

[0072] from Figure 3 It can be seen that compared with before treatment, the fullness of the labial contour of the edentulous area increased after soft tissue thickening treatment, indicating that the injectable plasma matrix gel provided by the present invention has a significant regeneration-promoting effect in the clinical scenario of labial soft tissue thickening.

[0073] 2. Gingival papilla filling

[0074] After communicating the treatment plan with the patient, the patient's blood pressure and heart rate were measured, the surgical area was disinfected with iodine intraorally, and the STA painless anesthesia device was used to inject Bilanma to locally infiltrate and anesthetize the surrounding area of ​​the retracted gingival papilla. After extraoral iodine disinfection, a sterile surgical towel was laid. The patient's blood was collected, and an injectable plasma matrix gel was prepared according to the method of Example 1. A 25G black needle was used to inject the injectable plasma matrix gel into the black triangle root. The injection was performed once every 2 weeks for three consecutive injections. After the treatment was completed, it was observed for 3 months, and the changes in the effects before and after were recorded. Comparison of the effects before and after gingival papilla filling Figure 4 shown.

[0075] from Figure 4It can be seen that compared with before treatment, the labial gingival papilla regenerated and filled the black triangle area after gingival papilla filling treatment, reducing the size of the black triangle area, indicating that the injectable plasma matrix gel provided by the present invention has a significant regeneration-promoting effect in the clinical scenario of gingival papilla filling.

[0076] 3. Maxillary sinus lift

[0077] After discussing the treatment plan with the patient, the patient's blood pressure and heart rate were measured, and the patient was asked to rinse with compound chlorhexidine for 2-3 minutes. The surgical area was disinfected with iodine in the mouth, and local infiltration anesthesia was performed by injecting Bilanma using the STA painless anesthesia device. After extraoral iodine disinfection, a sterile surgical towel was laid. A transverse incision and a distal vertical incision were made on the alveolar ridge top, and the flap was fully opened to expose the side wall of the maxillary sinus. An ultrasonic bone knife was used to carefully cut and separate a piece of bone about 2×1cm on the side wall of the maxillary sinus and temporarily stored in normal saline. The maxillary sinus mucosa and bone wall were carefully peeled off with a stripper.

[0078] Collect blood from patients and prepare a mixed liquid containing plasma matrix gel and Buffy coat layer according to the method of Example 2. Inject plasma matrix gel into the lower part of maxillary sinus mucosa, then inject mixed liquid containing Buffy coat layer, let stand for 10 minutes to wait for solidification, then fill the bone window with autologous bone chips, fully reduce tension and suture the wound tightly. Give postoperative medical advice as required, and take CBCT to observe the height of maxillary sinus floor bone after 6 months. Comparison of the effects before and after maxillary sinus floor lift Figure 5 shown.

[0079] from Figure 5 It can be seen that compared with before treatment, the bone height of the maxillary posterior edentulous area increased after the maxillary sinus floor lift treatment, allowing the implantation of the implant, indicating that the injectable plasma matrix gel provided by the present invention has a significant regeneration-promoting effect in the clinical scenario of maxillary sinus floor lift.

[0080] Based on the above experimental results, it can be seen that compared with the traditional solid plasma matrix, the injectable plasma matrix gel provided by the present invention has better shaping function, is conducive to minimally invasive implantation, and can greatly reduce the demand for whole blood. Compared with the traditional liquid plasma matrix, the injectable plasma matrix gel provided by the present invention has a fast molding speed, slow degradation of coagulation products, can maintain a certain space in the injection area, delay absorption, and has outstanding advantages in promoting soft tissue regeneration and favorable bone defect regeneration.

[0081] The above description is only a specific embodiment of the present disclosure, so that those skilled in the art can understand or implement the present disclosure. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure will not be limited to the embodiments described herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing an injectable plasma matrix gel, characterized in that: The preparation method comprises the following steps: (1) centrifuging a blood sample in a liquid plasma matrix blood collection tube; (2) collecting the upper liquid plasma matrix and the buffy coat at the junction of the upper liquid plasma matrix and the lower red blood cells; Heating the liquid plasma matrix to transform it into a gel state, thereby obtaining a plasma matrix gel; (3) Mixing the plasma matrix gel with the buffy coat to obtain the injectable plasma matrix gel.

2. The preparation method according to claim 1, characterized in that: The centrifugal acceleration of the centrifugation is 50-3000g.

3. The preparation method according to claim 1 or 2, characterized in that: The centrifugation time is 2-15 min.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The method for collecting the buffy coat in step (2) is: extracting the mixed liquid from 1 to 2 mm above the junction to 1 to 2 mm below the junction.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The heating temperature is 60-80°C.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The heating time is 5-20 minutes.

7. The preparation method according to any one of claims 1 to 6, characterized in that: Prior to the mixing in step (3), the plasma matrix gel was cooled to below 37°C.

8. An injectable plasma matrix gel, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 7.

9. A plasma matrix gel block, characterized in that: The plasma matrix gel block is a coagulation product of the injectable plasma matrix gel according to claim 8.

10. Use of the injectable plasma matrix gel as claimed in claim 8 or the plasma matrix solid as claimed in claim 9 as a soft tissue regeneration material or a favorable bone defect regeneration material.

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