Injectable artificial bone matrix as well as preparation method and application thereof

Abstract

The invention provides an injectable artificial bone matrix and a preparation method and application thereof.The method comprises the following steps that S1, a whole blood sample is collected and centrifuged, and a liquid plasma matrix on the upper layer, a plasma matrix albuginea layer on the middle layer and a red blood cell layer on the lower layer are obtained; s2, respectively collecting a liquid plasma matrix and a plasma matrix albuginea layer, heating the liquid plasma matrix, and cooling to obtain plasma matrix gel; s3, uniformly mixing the plasma matrix albuginea layer, the plasma matrix gel and the bone repair material to obtain the injectable artificial bone matrix. The liquid plasma matrix is heated and cooled to form the plasma matrix gel, and the plasma matrix gel is adopted to replace a conventional solid plasma matrix to be mixed with the bone repair material, so that the relative usage amount of the bone repair material and the plasma matrix can be reduced, and the cost is reduced; meanwhile, the plasma matrix albuginea layer is added, a large number of enriched immune cells contained in the plasma matrix albuginea layer are utilized, the local immune regulation function is achieved, and early-stage wound healing can be accelerated.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and specifically relates to an injectable artificial bone matrix and a preparation method and application thereof. Background Art

[0002] As teeth fall out, the alveolar bone in the edentulous area will undergo vertical bone resorption, and the maxillary sinus will gradually become pneumatized, posing a huge challenge to the placement of implants. To solve this problem, it is usually recommended to first perform maxillary sinus floor elevation surgery. Maxillary sinus floor elevation is a predictable and widely used procedure that significantly broadens the indications for implant placement in the maxillary posterior region. A large number of retrospective studies have shown that during maxillary sinus floor elevation surgery, whether implants are implanted simultaneously or in stages, the implants have a high long-term survival rate after surgery.

[0003] In the maxillary sinus floor lift surgery, commonly used transplant materials include: autologous grafts, allogeneic grafts, xenografts, and artificial synthetic materials. At present, although autologous bone is still the gold standard for bone regeneration biomaterials, it also has limitations that cannot be ignored: the opening of a second surgical area, a higher risk of postoperative complications, and faster absorption after transplantation. Therefore, non-autologous finished bone replacement materials are still the first choice of clinicians; at the same time, after the maxillary sinus floor lift surgery, the dynamic air pressure generated by breathing may stimulate the Schneider membrane and promote osteogenesis, but it may also cause excessive compression of the transplant material and new bone, resulting in bone absorption. Therefore, good volume maintenance ability and appropriate absorption rate are important criteria for measuring ideal maxillary sinus floor lift materials.

[0004] Plasma matrix is ​​an autologous product separated and extracted from the patient's peripheral blood. It does not contain additives and has good biosafety. It can not only provide a three-dimensional fibrin scaffold for osteogenesis, but also contains a large number of cells and growth factors, which can provide additional bioactive factors for bone regeneration. It is reported that plasma matrix alone can be used for maxillary sinus floor lifting surgery; and more studies tend to combine plasma matrix with granular bone repair materials to prepare plasma matrix bone blocks for filling. However, for maxillary sinus floor lifting surgery, the field of view is often severely limited, the filling fit of the bone block is limited, and gaps may occur, affecting the surgical effect; at the same time, for some patients with multiple tooth loss, the maxillary sinus area is severely pneumatized, and a large amount of autologous blood is often required to be drawn and combined with a large amount of bone repair materials for transplantation, which brings a huge economic burden to patients. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing an injectable artificial bone matrix, which can at least solve some of the defects existing in the prior art.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A method for preparing an injectable artificial bone matrix comprises the following steps:

[0008] S1. Collect the whole blood sample and centrifuge it to obtain the upper liquid plasma matrix, the middle plasma matrix buffy coat layer and the lower red blood cell layer;

[0009] S2, collecting the liquid plasma matrix and the plasma matrix buffy coat respectively, and heating the liquid plasma matrix and then cooling it to obtain a plasma matrix gel;

[0010] S3. Evenly mix the plasma matrix buffy coat, plasma matrix gel and bone repair material to obtain an injectable artificial bone matrix.

[0011] Furthermore, in step S2, the liquid plasma matrix is ​​heated at a temperature of 60-80° C. and a heating time of 5-20 min.

[0012] Furthermore, in step S2, the liquid plasma matrix is ​​cooled at a temperature of 0-4°C and for a cooling time of 5-40 minutes.

[0013] Furthermore, in step S3, the usage ratio of the bone repair material, the plasma matrix gel and the plasma matrix buffy coat is (1-4 g):(1-20 g):(0.4-8 ml).

[0014] Furthermore, the bone repair material is in granular form and is any one of autologous bone, allogeneic bone, xenogeneic bone, and artificial bone repair material.

[0015] Furthermore, the particle size of the bone repair material is 0.25-1 mm.

[0016] Furthermore, in step S3, the bone repair material and the plasma matrix gel are first mixed evenly, and then the plasma matrix buffy coat is added thereto and mixed evenly.

[0017] Furthermore, in step S1, the centrifugal acceleration of the centrifugation is 50-3000g, and the centrifugal time is 2-15min.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The preparation method of the injectable artificial bone matrix provided by the present invention heats and cools the liquid plasma matrix to form a plasma matrix gel, and uses the plasma matrix gel instead of the conventional solid plasma matrix to mix with the bone repair material, which can reduce the relative usage of the bone repair material and the plasma matrix and reduce the cost; at the same time, the plasma matrix buffy coat is added, and the large amount of immune cells enriched in it are utilized to exert the local immune regulation function, which can accelerate early wound healing.

[0020] (2) The preparation method of the injectable artificial bone matrix provided by the present invention is to first evenly mix the plasma matrix gel and the bone repair material, and then add the plasma matrix white film layer. The design of this adding sequence can make the mixture solidify and form a jelly-like shape that fits the morphology of the defect area, which is conducive to retention.

[0021] (3) The injectable artificial bone matrix provided by the present invention is composed of bone repair material, plasma matrix gel and plasma matrix buffy coat, which can provide the bone conduction, bone induction and bone regeneration elements necessary for the osteogenesis process, and can directly inject the bone matrix material into the defect area when the clinical surgical field of view is limited, thus facilitating clinical operation.

[0022] The present invention will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention is a flow chart of the method for preparing the injectable artificial bone matrix.

[0024] Figure 2 is a picture of the plasma matrix gel in Example 1 of the present invention;

[0025] Figure 3 is a picture of the plasma matrix buffy coat layer in Example 1 of the present invention;

[0026] Figure 4 is a comparison chart of the quality of the artificial bone matrix prepared in Example 1 of the present invention before and after 30 days;

[0027] Figure 5 : is a comparison diagram of the effects before and after the maxillary sinus floor lift using the artificial bone matrix prepared in Example 2 of the present invention, wherein (a) is before the maxillary sinus floor lift treatment, and (b) is after the maxillary sinus floor lift treatment;

[0028] Figure 6 1 is a comparison diagram of the effects of using the artificial bone matrix prepared in Example 2 of the present invention before and after alveolar ridge preservation, wherein (a) is before site preservation treatment, and (b) is after site preservation treatment;

[0029] Figure 7 It is a comparison chart of the degradation time of the artificial bone matrix prepared in Example 2 of the present invention and Comparative Example 1. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The present invention provides an injectable artificial bone matrix, such as Figure 1 As shown, the preparation method of the injectable artificial bone matrix specifically comprises the following steps:

[0032] S1. Collect whole blood samples and centrifuge them to obtain the upper liquid plasma matrix, the middle plasma matrix buffy coat layer and the lower red blood cell layer.

[0033] Specifically, a whole blood sample is collected using a liquid plasma matrix blood collection tube, and in combination with a balancing tube, the collected whole blood sample is quickly placed in a plasma matrix preparation platform and centrifuged at a centrifugal acceleration of 50-3000g for 2-15min; for example, the centrifugal acceleration can be 60g, 100g, 200g, 500g, 700g, 1000g, 1500g, 2000g, etc., and the centrifugal time can be 3min, 6min, 8min, 10min, 12min, etc. The specific centrifugal process conditions are determined according to the composition and content of the whole blood sample.

[0034] Among them, the liquid plasma matrix after centrifugation is rich in albumin; the plasma matrix buffy coat layer after centrifugation is the liquid layer between the upper liquid plasma matrix and the lower red blood cells, and the plasma matrix buffy coat layer contains a large number of enriched immune cells.

[0035] S2. Collect the liquid plasma matrix and the plasma matrix buffy coat layer separately, heat the liquid plasma matrix and then cool it to obtain a plasma matrix gel.

[0036] Among them, collecting and processing the liquid plasma matrix and the plasma matrix buffy coat separately can avoid the destruction of immune cells in the plasma matrix buffy coat during the heating process of the liquid plasma matrix.

[0037] Specifically, the heating temperature of the liquid plasma matrix is ​​60-80°C, and the heating time is 5-20 minutes. The albumin in the liquid plasma matrix is ​​denatured by heating treatment to form a plasma matrix gel. After the heating is completed, cooling can reduce the damage of heat to immune cells in the subsequent plasma matrix buffy coat layer and tissues or cells in the transplantation area; the cooling method can be natural cooling or cooling using a device with a refrigeration function. In this embodiment, cooling is performed by using a device with a refrigeration function to reduce clinical operation time. Specifically, the cooling temperature of the liquid plasma matrix is ​​0-4°C, and the cooling time is 5-40 minutes.

[0038] Since the amount of plasma matrix gel prepared using a whole blood sample is higher than the amount of solid plasma matrix prepared using the same volume of whole blood sample, in this embodiment, the liquid plasma matrix is ​​heated to form a plasma matrix gel form, which can relatively reduce the amount of plasma matrix and bone repair material used, improve the raw material utilization rate of the plasma matrix, and reduce costs.

[0039] S3. Evenly mix the plasma matrix buffy coat, plasma matrix gel and bone repair material to obtain an injectable artificial bone matrix.

[0040] Specifically, the bone repair material is a conventional granular medical bone repair material, including but not limited to autologous bone, allogeneic bone, xenogeneic bone, artificial bone repair material, etc. The particle size of the bone repair material is selected to be 0.25-1 mm.

[0041] In some embodiments, the usage ratio of bone repair material, plasma matrix gel and plasma matrix buffy coat is (1-4 g):(1-20 g):(0.4-8 ml), preferably 1 g:(1-4 g):(0.5-2) ml.

[0042] As for the mixing order of the plasma matrix buffy coat layer, the plasma matrix gel and the bone repair material, the bone repair material, the plasma matrix gel and the plasma matrix buffy coat layer can be mixed evenly at the same time, or the bone repair material and the plasma matrix gel can be mixed evenly first, and then the plasma matrix buffy coat layer is added thereto and mixed evenly. The latter is preferred in this embodiment, where the plasma matrix buffy coat layer is added after the bone repair material and the plasma matrix gel are mixed evenly, so that the plasma matrix buffy coat layer can solidify the mixture 2-5 minutes after being added, forming a jelly-like structure that fits the morphology of the defect area, which is conducive to fixation, while the plasma matrix buffy coat layer, the plasma matrix gel and the bone repair material are mixed at the same time and then injected, and a jelly-like structure cannot be formed.

[0043] Therefore, the preparation method of the injectable artificial bone matrix provided by the present invention can reduce the relative usage amounts of bone repair materials and plasma matrix while retaining the biologically active components of the plasma matrix itself, thereby preparing an injectable artificial bone matrix with slow degradation rate, convenient use, high enrichment of growth factors and immune cells, which is applied to bone tissue regeneration of favorable type bone defects, can maintain a good space locally, promote blood vessel regeneration and new bone formation, improve treatment effect, and reduce costs.

[0044] The preparation process and application effect of the injectable artificial bone matrix of the present invention are described below through specific examples.

[0045] Embodiment 1:

[0046] This embodiment provides an injectable artificial bone matrix, and the preparation method is as follows:

[0047] (1) Use a liquid plasma matrix blood collection tube to collect 10 mL of human blood, combine with a balancing tube, and quickly place it in the plasma matrix preparation platform and centrifuge at 700g for 8 minutes.

[0048] (2) Use a disposable syringe to extract the liquid plasma matrix on the upper layer of the blood collection tube, and use another new disposable syringe to extract 0.5 mL of the plasma matrix white film layer between the upper liquid plasma matrix and the lower red blood cells. Figure 3 As shown; the liquid plasma matrix in the syringe was heated at 75°C for 15 minutes, and then cooled in an ice bath at 0°C for 10 minutes to obtain a plasma matrix gel, as shown Figure 2 shown.

[0049] (3) Take 0.5 g of Bio-Oss bone graft material, 2 g of plasma matrix gel, and 0.5 mL of plasma matrix buffy coat, mix them evenly, and transfer them into an injection device to form an injectable artificial bone matrix.

[0050] The degradation time of the injectable artificial bone matrix prepared in this example was tested, and the testing method was as follows:

[0051] The injectable artificial bone matrix prepared in this example was added with 10 mL of DMEM cell culture medium and placed in a 37° C. incubator. After 30 days, the integrity of the plasma matrix gel was observed and the quality of the artificial bone matrix was recorded. The quality of the artificial bone matrix before and after 30 days of degradation test was statistically compared. Figure 4 As shown, three parallel samples of artificial bone matrix were taken and the test results were averaged.

[0052] Depend on Figure 4 It can be seen that the injectable artificial bone matrix provided in this embodiment can maintain the stability of quality after degradation for 30 days.

[0053] Embodiment 2:

[0054] This embodiment provides an injectable artificial bone matrix, and the preparation method is as follows:

[0055] (1) Use a liquid plasma matrix blood collection tube to collect 10 mL of human blood, combine with a balancing tube, and quickly place it in the plasma matrix preparation platform and centrifuge at 1000g for 6 minutes.

[0056] (2) Use a disposable syringe to extract the liquid plasma matrix on the upper layer of the blood collection tube, and use another new disposable syringe to extract 1 mL of the plasma matrix buffy coat layer between the upper liquid plasma matrix and the lower red blood cells; heat the liquid plasma matrix in the syringe at 70°C for 20 minutes, and then cool it in an ice bath at 0°C for 10 minutes to obtain a plasma matrix gel.

[0057] (3) Take 1 g of Bio-Oss bone graft material and 1 g of plasma matrix gel, mix them evenly, transfer them to the injection device, and inject them into the defect area in advance; then take 1 mL of the plasma matrix white film layer and inject it into the defect area. After standing for 5 minutes, it solidifies to form a jelly-like artificial bone matrix.

[0058] The artificial bone matrix prepared according to the method of this embodiment was tested for the therapeutic effect of maxillary sinus floor lifting, and the testing method was as follows:

[0059] After communicating 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 the STA painless anesthesia device was used to inject Bilanma for local infiltration anesthesia. After extraoral iodine disinfection, a sterile surgical towel was laid. A transverse incision and a distal vertical incision were made on the top of the alveolar ridge, and the flap was fully opened to expose the lateral 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 lateral 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. Take 1g of the plasma matrix gel after heating and cooling in this embodiment, mix it with 1g of Bio-Oss bone graft material and inject it under the maxillary sinus mucosa, then inject 1mL of plasma matrix white film layer, and let it stand for 5 minutes to solidify. Then, the autologous bone slices were backfilled to cover the bone window, fully reduce tension and tightly suture the wound. The postoperative medical instructions were given as required, and a CBCT was taken to observe the height of the maxillary sinus floor bone after a follow-up examination 6 months later. The results are as follows Figure 5 shown.

[0060] Depend on Figure 5 It can be seen that the injectable artificial bone matrix provided in this embodiment has a significant regeneration-promoting effect in the clinical scenario of maxillary sinus floor augmentation.

[0061] In addition, the artificial bone matrix prepared according to the method of this embodiment was tested for the therapeutic effect of alveolar ridge preservation, and the testing method was as follows:

[0062] After communicating the treatment plan with the patient, measure the patient's blood pressure and heart rate, ask the patient to rinse with compound chlorhexidine for 2-3 minutes, disinfect the surgical area with iodine in the mouth, use STA painless anesthesia instrument to inject Bilanma for local infiltration anesthesia, and spread sterile surgical towels after extraoral iodine disinfection. After extracting the affected tooth, take 0.5g of the plasma matrix gel that has been heated and cooled in this embodiment, mix it with 0.5g of Bio-Oss bone grafting material and inject it into the extraction socket, then inject 0.5mL of plasma matrix white film layer, let it stand for 5 minutes to wait for solidification, cover it with commercial collagen membrane, and suture the wound tightly. Give postoperative medical advice as required, and review it after 6 months. The results are as follows Figure 6 shown.

[0063] Depend on Figure 6 It can be seen that the injectable artificial bone matrix provided in this embodiment has a significant regeneration-promoting effect in the clinical scenario of alveolar ridge preservation.

[0064] Comparative Example 1:

[0065] This comparative example provides an artificial bone matrix, and its preparation process is substantially the same as that of the above-mentioned Example 2, except that in this comparative example, a solid plasma matrix is ​​collected by a solid plasma matrix collection tube after centrifugation, and the solid plasma matrix is ​​compressed with a pressure of 1N for 2 minutes to form a plasma matrix membrane, and the plasma matrix membrane is cut into pieces with a diameter of 1-2 mm, and the cut plasma matrix membrane is used to replace the plasma matrix gel in Example 2 and mixed with the Bio-Oss bone graft material.

[0066] The same mass of the artificial bone matrix prepared in this comparative example and the artificial bone matrix prepared in Example 2 were respectively subjected to degradation time tests. The test method was as follows: the artificial bone matrix was added to the DMEM cell culture medium and placed in a 37°C incubator. The degradation time of the artificial bone matrix was recorded, that is, the time required for the artificial bone matrix to degrade to lose its complete shape. The results are shown in FIG. Figure 7 shown.

[0067] Depend on Figure 7 It can be seen that the degradation time of the artificial bone matrix prepared by using plasma matrix gel in the present invention is much longer than the degradation time of the artificial bone matrix prepared by using solid plasma matrix in the comparative example. Therefore, the degradation rate of the artificial bone matrix can be greatly reduced by heating the liquid plasma matrix and then cooling it into a gel state.

[0068] Comparative Example 2:

[0069] This comparative example provides an artificial bone matrix, and its preparation process is substantially the same as that of the above-mentioned Example 2, except that the artificial bone matrix of this comparative example does not contain a plasma matrix buffy coat layer.

[0070] The artificial bone matrix prepared in this comparative example and Example 2 was injected into the defect area for bone repair. It can be observed that the artificial bone matrix prepared in Example 2 solidified into a jelly state in the defect area, while the artificial bone matrix in this comparative example was in a gel state in the defect area. In addition, the artificial bone matrix prepared in Example 2 had a relatively faster postoperative tissue healing and a reduced postoperative pain compared with the artificial bone matrix prepared in this comparative example. This is because the plasma matrix buffy coat layer contains rich immune cell components, which play an important role in the immune regulation process of the local surgical area. In addition, the platelet content of the plasma matrix buffy coat layer prepared by high-speed centrifugation can exceed the platelet concentration in normal blood by more than 15 times, and has significant advantages in promoting tissue healing, alleviating postoperative pain, and releasing growth factors.

[0071] Comparative Example 3:

[0072] This comparative example provides an artificial bone matrix, and its preparation process is substantially the same as that of the above-mentioned Example 2, except that in this comparative example, the Bio-Oss bone graft material, the plasma matrix gel and the plasma matrix buffy coat are mixed simultaneously.

[0073] By comparison, it can be seen that the different mixing orders in this comparative example and Example 2 result in different morphologies after injection into the defect area. In Example 2, the plasma matrix white film layer is injected later, and the mixture solidifies into a jelly state after standing in the defect area, which can well fit the morphology of the defect area and is conducive to retention; while the Bio-Oss bone graft material, plasma matrix gel and plasma matrix white film layer in this comparative example are mixed and injected into the defect area at the same time in a gel state, unable to form a block structure, which is not conducive to retention.

[0074] The above examples are merely illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. All designs that are the same or similar to the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing an injectable artificial bone matrix, characterized in that: The steps include: S1. Collect the whole blood sample and centrifuge it to obtain the upper liquid plasma matrix, the middle plasma matrix buffy coat layer and the lower red blood cell layer; S2, collecting the liquid plasma matrix and the plasma matrix buffy coat respectively, and heating the liquid plasma matrix and then cooling it to obtain a plasma matrix gel; S3. Evenly mix the plasma matrix buffy coat, plasma matrix gel and bone repair material to obtain an injectable artificial bone matrix.

2. The method for preparing the injectable artificial bone matrix according to claim 1, characterized in that: In the step S2, the liquid plasma matrix is ​​heated at a temperature of 60-80°C and a heating time of 5-20 minutes.

3. The method for preparing the injectable artificial bone matrix according to claim 1, characterized in that: In step S2, the liquid plasma matrix is ​​cooled at a temperature of 0-4°C and for a cooling time of 5-40 minutes.

4. The method for preparing the injectable artificial bone matrix according to claim 1, characterized in that: In the step S3, the usage ratio of the bone repair material, the plasma matrix gel and the plasma matrix buffy coat is (1-4 g):(1-20 g):(0.4-8 ml).

5. The method for preparing the injectable artificial bone matrix according to claim 1, characterized in that: The bone repair material is in granular form and is any one of autologous bone, allogeneic bone, xenogeneic bone and artificial bone repair material.

6. The method for preparing the injectable artificial bone matrix according to claim 1, characterized in that: The particle size of the bone repair material is 0.25-1 mm.

7. The method for preparing the injectable artificial bone matrix according to claim 1, characterized in that: In the step S3, the bone repair material and the plasma matrix gel are first mixed evenly, and then the plasma matrix buffy coat is added thereto and mixed evenly.

8. The method for preparing the injectable artificial bone matrix according to claim 1, characterized in that: In the step S1, the centrifugal acceleration of the centrifugation is 50-3000g, and the centrifugal time is 2-15min.

9. An injectable artificial bone matrix, characterized in that: The method is prepared according to any one of claims 1 to 8.

10. Use of the injectable artificial bone matrix according to claim 9 in materials for treating bone defects.

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