Platelet vesicle-decellularized cancellous bone composite scaffold as well as preparation and application methods thereof

Abstract

The invention discloses a platelet vesicle-decellularized cancellous bone composite scaffold as well as a preparation method and an application method thereof. The platelet vesicle-acellular cancellous bone composite scaffold is formed by crosslinking platelet vesicles with fibrinogen gel as a scaffold background and cancellous bone extracellular matrix particles. The preparation method of the composite scaffold comprises the following steps: firstly, separating cancellous bone blocks from bone tissues of animals, and then carrying out decellularization and demineralization treatment to obtain the cancellous bone extracellular matrix particles. Then extracting platelets, preparing vesicles through membrane extrusion, and resuspending the vesicles in normal saline containing fibrinogen; finally, under the action of thrombin or calcium ions, cross-linking the platelet vesicle and the acellular cancellous bone to form the platelet vesicle-acellular cancellous bone composite scaffold. According to the composite scaffold, platelet vesicle fibrinogen hydrogel serves as a substrate, rapid cell recruitment and vascularization of a tissue repair part are achieved, osteogenic differentiation of a specific part is completed through a decellularized cancellous bone matrix, and a new thought and new work are provided for the field of bone regeneration.

Description

Technical Field

[0001] The present invention belongs to the technical field of surgical materials, relates to bone tissue regeneration, and specifically relates to a platelet vesicle - acellular cancellous bone composite scaffold and its preparation and application methods. Background Art

[0002] Fractures and severe bone defects caused by high - energy trauma are difficult to recover relying on the self - repair mechanism, greatly affecting the quality of life of patients and becoming a clinical problem. At present, although autologous bone transplantation is the gold standard for bone regeneration, it has many risks, such as potential infection, nerve injury, and chronic pain at the donor site.

[0003] In the field of bone tissue engineering, scaffold materials such as hydroxyapatite (HAP), bioactive glass (BS), and gelatin methacrylate (GelMa) are commonly used for bone repair. Although they have certain osteoconductivity, they cannot simulate the extracellular matrix (ECM) to provide an ideal three - dimensional microenvironment for the biological behavior of cells. Decellularized ECM (dECM) is a natural candidate for simulating ECM. dECM from specific sources can guide the differentiation of stem cells, especially dECM from bone tissue (DBM), which has strong osteogenic ability.

[0004] Prior art 1 (202411425082.4) discloses a preparation method of a decellularized bone matrix combined with local anticoagulation, which mixes an anticoagulant with the decellularized bone matrix to form an anticoagulant complex, so that the decellularized bone matrix composite material can reduce discomfort after being implanted into the human body and solves the problem of the coagulation risk of the decellularized bone matrix. Prior art 2 (202310554324.9) discloses a preparation method of a drug - loaded microsphere / decellularized bone matrix composite material. This composite material uses mesoporous materials and amino - modified biodegradable polyesters as the matrix materials of the drug - loaded microspheres, achieving a good drug - slow - release effect.

[0005] However, bone regeneration is a complex and coordinated process. Initially, fibrinogen is activated by thrombin to form a gel - like clot hematoma. The hematoma contains abundant bioactive factors such as growth factors and inflammatory factors, which can quickly construct a temporary microenvironment conducive to cell migration and angiogenesis. Removing the hematoma may lead to a significant delay in bone healing. Therefore, the reconstruction during the hematoma stage is also the key to promoting bone regeneration. Therefore, developing materials for promoting bone repair not only requires creating a microenvironment conducive to osteogenesis but also needs to simulate the three - dimensional environmental changes of the entire bone tissue repair from the time dimension. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides a platelet vesicle - decellularized cancellous bone composite scaffold and its preparation and application methods. By using platelet vesicle hydrogel as a substrate, rapid cell recruitment and vascularization at the tissue repair site are achieved. Through the decellularized cancellous bone matrix, osteogenic differentiation at a specific site is completed, simulating the microenvironment changes in different stages of bone regeneration and promoting bone tissue remodeling.

[0007] The platelet vesicle - decellularized cancellous bone composite scaffold is formed by cross - linking platelet vesicles and cancellous bone extracellular matrix microparticles with fibrinogen gel as the scaffold background.

[0008] The preparation method of the platelet vesicle - decellularized cancellous bone composite scaffold: obtaining cancellous bone particles from animal tissues, performing decellularization with an optimized decellularization protocol to prepare specific demineralized cancellous bone ECM microparticles, and combining them with platelet vesicle fibrinogen hydrogel to obtain the platelet vesicle - decellularized cancellous bone composite scaffold. The specific steps are as follows:

[0009] Step 1: Isolate cancellous bone blocks from the bone tissues of animals.

[0010] The animal is a large mammal, specifically cattle or pigs. The bone tissues of the animal include the spine, ribs, scapula or pelvis.

[0011] Step 2: Prepare decellularized cancellous bone:

[0012] s2.1: Cut the cancellous bone blocks obtained in Step 1 into smaller cancellous bone particles.

[0013] s2.2: After rinsing the cancellous bone particles obtained in s2.1 with water, soak them in 0.6% (v / v) peracetic acid ultrapure water.

[0014] s2.3: Transfer the soaked cancellous bone particles to a sterile 1% (v / v) Triton - X100 solution and vibrate at 100 rpm and 4°C for 24 - 48 h.

[0015] s2.4: Wash the cancellous bone particles repeatedly with sterile water.

[0016] s2.5: Add the cancellous bone particles to a 1% (w / v) sodium dodecyl sulfate (SDS) aqueous solution and vibrate at 100 rpm and 4°C for 24 - 48 h to obtain decellularized cancellous bone particles.

[0017] s2.6: Immerse the decellularized cancellous bone particles in a 10% EDTA decalcifying solution, put them into a rapid ultrasonic decalcifying machine, and perform decalcification at 4°C for 3 - 6 h.

[0018] s2.7. Rinse the decellularized cancellous bone particles after demineralization with tap water, dry them in ventilation, then grind them and filter through a filter screen with a diameter of 500 μm.

[0019] s2.8. Perform radiation sterilization on the filtered powder to obtain cancellous bone extracellular matrix microparticles, and store them in a freeze-dried manner.

[0020] Step 3. Construct a platelet vesicle - decellularized cancellous bone composite scaffold:

[0021] s3.1. Mix venous blood and citrate - dextrose solution A (ACD - A) with a volume ratio of 9:1, and then centrifuge at 200 g for 10 - 20 min.

[0022] s3.2. Take the upper platelet layer, centrifuge at 800 g for 10 - 20 min, collect the precipitate to obtain platelets.

[0023] s3.3. Resuspend the platelets in physiological saline containing 5 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM prostaglandin E1 (PGE1), and sonicate for 10 min.

[0024] s3.4. Set the heating temperature of the constant - temperature module to 20 °C, and extrude it through a PES membrane with a pore size of 200 nm at a speed of 0.1 - 1.0 ml / s to obtain a platelet vesicle suspension.

[0025] s3.5. Centrifuge the platelet vesicle suspension obtained in s3.4 at 15000 g for 30 - 60 min, collect the precipitate and resuspend it in physiological saline containing 10 mg / ml fibrinogen.

[0026] s3.6. Add an equal volume of cancellous bone extracellular matrix microparticles to the fibrinogen hydrogel containing platelet vesicles obtained in s3.5, then add thrombin, mix well and let it stand for 5 - 10 min to obtain a platelet vesicle - decellularized cancellous bone composite scaffold.

[0027] The method of the platelet vesicle - decellularized cancellous bone composite scaffold in bone defects simulates the three - dimensional environmental changes in the whole process of bone tissue repair through the slow release of early platelet vesicles and the osteogenic differentiation of cancellous bone ECM microparticles.

[0028] The present invention has the following beneficial effects:

[0029] 1. Using bone matrix and platelet materials from natural sources, it has good biocompatibility and bioactivity.

[0030] 2. Perform specific demineralization on the bone matrix to enhance bioactivity while retaining mechanical strength.

[0031] 3. Platelet vesicles using fibrinogen gel as the scaffold background can achieve rapid release of early platelet vesicles at the regeneration site.

[0032] 4. In vitro experiments verified that the modification of platelet vesicle gel significantly promoted the migration and vascularization of stem cells at the regeneration site;

[0033] 5. In vivo experiments verified that the platelet vesicle - acellular cancellous bone composite scaffold had a good osteogenic promotion effect and had a definite improvement effect on the repair of bone defects, providing new ideas and methods for promoting the repair of bone implants; Description of the Drawings

[0034] Figure 1 Scanning electron micrograph of the platelet vesicle - acellular cancellous bone composite scaffold in Test Example 1;

[0035] Figure 2 Rheological properties of the composite scaffold in Test Example 1;

[0036] Figure 3 Compressive stress - strain curve of the composite scaffold in Test Example 1;

[0037] Figure 4 Early release efficiency of platelet vesicles in the composite scaffold in Test Example 1;

[0038] Figure 5 Biocompatibility results of the composite scaffold in Test Example 1;

[0039] Figure 6 Effect of different materials on the recruitment ability of bone marrow mesenchymal stem cells (BMSCs) in Test Example 2;

[0040] Figure 7 Effect of different materials on the ring - forming ability of human umbilical vein endothelial cells (HUVECs) in Test Example 2;

[0041] Figure 8 Micro - CT examination results of the critical - sized bone defect model in Test Example 3. Detailed Embodiments

[0042] The present invention will be further explained below with reference to the accompanying drawings; obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0043] Embodiment 1

[0044] This embodiment provides a preparation method for a platelet vesicle - acellular cancellous bone composite scaffold, and the specific steps are as follows:

[0045] Step 1: Obtain cancellous bone from fresh porcine spinal bone using a hollow bone drill with a diameter of 4 mm.

[0046] Step 2: Prepare acellular cancellous bone:

[0047] s2.1: Cut the cancellous bone blocks obtained in Step 1 into cancellous bone particles with a thickness of 2 mm, then rinse with tap water for 1 h, subsequently soak in 0.6% (v / v) peracetic acid ultrapure water for 1 h, and then transfer to a sterile 1% (v / v) Triton-X100 solution and shake at 100 rpm and 4 °C for 24 h.

[0048] s2.2: Wash the cancellous bone particles with 500 ml of sterilized water and keep stirring for 1 h, repeating twice. Then place them in a 1% (w / v) sodium dodecyl sulfate (SDS) aqueous solution and shake at 4 °C and 100 rpm for 24 h to obtain acellular cancellous bone particles.

[0049] s2.3: Soak the acellular cancellous bone particles in 10% EDTA decalcifying solution and decalcify in a rapid ultrasonic decalcifier at 4 °C for 4 h. After decalcification is completed, rinse with tap water, air dry and grind, and pass through a filter with a diameter of 500 μm to screen out acellular cancellous bone extracellular matrix microparticles with a diameter less than 500 μm; irradiate for disinfection and freeze-dry for storage.

[0050] Step 3: Construct a platelet vesicle - acellular cancellous bone composite scaffold:

[0051] s3.1: Mix 9 ml of venous blood with 1 ml of citrate dextrose solution, transfer to a 50 mL centrifuge tube, and centrifuge at 200 g for 10 min. Take the upper platelet layer, transfer to a 15 mL centrifuge tube, and centrifuge at 800 g for 20 min to collect platelets. Resuspend the platelets in physiological saline containing 5 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM prostaglandin E1 (PGE1), and sonicate for 10 min, set the horn No. 2, on for 2 s, off for 3 s, and the temperature is 20 °C.

[0052] s3.2: Set the heating temperature of the constant temperature module to 20 °C, and manually extrude at a constant speed through a PES membrane with a pore size of 200 nm, and the extrusion speed is 0.1 - 1.0 ml / s to obtain a platelet vesicle suspension. Then, centrifuge the platelet vesicle suspension at 15000 g for 60 min, collect the precipitate and resuspend it in physiological saline containing 10 mg / mL fibrinogen, and the platelet vesicle concentration is 100 μg / mL.

[0053] S3.3. Add an equal volume of cancellous bone extracellular matrix microparticles to 1 mL of fibrinogen solution containing platelet vesicles, then add 20 U of thrombin, mix well and let stand for 5 min. After coagulation, a platelet vesicle - acellular cancellous bone composite scaffold is obtained.

[0054] Example 2

[0055] This example provides a method for preparing a platelet vesicle - acellular cancellous bone composite scaffold. On the basis of Example 1, cancellous bone is obtained from the femur of porcine origin.

[0056] Example 3

[0057] This example provides a method for preparing a platelet vesicle - acellular cancellous bone composite scaffold. On the basis of Example 1, cancellous bone is obtained from the humerus of porcine origin.

[0058] Example 4

[0059] This example provides a method for preparing a platelet vesicle - acellular cancellous bone composite scaffold. On the basis of Example 1, cancellous bone is obtained from the scapula of porcine origin.

[0060] Example 5

[0061] This example provides a method for preparing a platelet vesicle - acellular cancellous bone composite scaffold. On the basis of Example 1, cancellous bone is obtained from the hip bone of porcine origin.

[0062] Example 6

[0063] This example provides a method for preparing a platelet vesicle - acellular cancellous bone composite scaffold. On the basis of Example 1, cancellous bone is obtained from the tibia of bovine origin.

[0064] Test Example 1

[0065] This test example conducts physical property detection, platelet vesicle release efficiency evaluation, and cytotoxicity evaluation on the platelet vesicle - acellular cancellous bone composite scaffold obtained in Example 1.

[0066] Step 1. Use a scanning electron microscope (SEM) to observe the microscopic appearance of the platelet vesicle - acellular cancellous bone composite scaffold prepared in Example 1. As Figure 1 shown, the scale bar = 200 μm. Abundant pore structures can be seen between the acellular cancellous bone microparticles, which means that the internal structure of the scaffold is conducive to cell migration activities.

[0067] Step 2. Study the rheological properties of the platelet vesicle - acellular cancellous bone composite scaffold prepared in Example 1. As Figure 2As shown, throughout the entire time and frequency range, the G′ of the composite scaffold is always greater than G″, which is consistent with the solid-like behavior of the three-dimensional network in the cementitious system, indicating that the three-dimensional network inside the scaffold has good stability.

[0068] Step 3: Perform a compression test on the platelet vesicle - acellular cancellous bone composite scaffold prepared in Example 1. As Figure 3 shown, when the compression deformation of the composite scaffold reaches 50%, its compression modulus can reach 100 Kpa. Compared with traditional hydrogel materials, the compression modulus of the composite scaffold prepared in Example 1 is significantly improved because the mechanical properties of the composite scaffold are determined by the new network composed of interconnected hard cancellous bone particles. In addition, although the modulus of the composite scaffold is not yet sufficient to reach the strength of intact cortical bone, the relatively small modulus is more conducive to the mechanical signal transduction and osteogenic differentiation of cells.

[0069] Step 4: In vitro, place the platelet vesicle - acellular cancellous bone composite scaffold prepared in Example 1 and the acellular cancellous bone particles (DBM) prepared in Example 1 in a 6-well plate, add 1 ml of PBS solution, and collect the supernatants on days 0, 1, 4, 7, 10, and 13, and measure their concentrations by the BCA method. As Figure 4 shown, the platelet fibrinogen gel component in the composite scaffold group can be rapidly released within one week. This means that platelet vesicles play an early role at the regeneration site, creating favorable conditions for the recruitment and rapid vascularization of stem cells.

[0070] Step 5: In vitro, co-culture the composite scaffold prepared in Example 1 with BMSCs and HUVECs for 1 day and 3 days respectively, and evaluate the toxicity of the composite scaffold by live / dead staining. As Figure 5 shown, the composite scaffold has good biocompatibility and promotes the proliferation of BMSCs and HUVECs.

[0071] Test Example 2

[0072] This test example evaluates the stem cell recruitment performance and angiogenic performance of the platelet vesicle - acellular cancellous bone composite scaffold obtained in Example 1 respectively.

[0073] Step 1: In vitro, use the Transwell system to co-culture the acellular cancellous bone particles (DBM) and the composite scaffold prepared in Example 1 with BMSCs respectively, and evaluate the recruitment effect of the composite scaffold on BMSCs. As Figure 6 shown, scale bar = 100 μm. Compared with the acellular cancellous bone particles, the composite scaffold significantly enhances the stem cell recruitment performance by introducing platelet vesicles.

[0074] Step 2: Immerse the acellular cancellous bone microparticles and composite scaffolds prepared in Example 1 in high-glucose medium for 3 days. Subsequently, collect the leachate for the vascular ring formation assay and take pictures. As Figure 7 shown, scale bar = 50 μm. Compared with the acellular cancellous bone microparticles, the composite scaffold significantly enhanced the angiogenesis ability by introducing platelet vesicles.

[0075] Test Example 3

[0076] Evaluation of the bone repair ability of the platelet vesicle-acellular cancellous bone composite scaffold obtained in Example 1 in this test example

[0077] Step 1: Establish a skull defect model in SD rats. Create circular bone defects with a diameter of 5 mm on both sides of the skull of each rat and divide them into 3 groups:

[0078] (1) Control group: Administer normal saline (200 μL);

[0079] (2) DBM group: Administer the acellular cancellous bone microparticles prepared in Example 1 (200 μL);

[0080] (3) Composite scaffold group: Administer the composite scaffold prepared in Example 1 (200 μL).

[0081] Step 2: After 4 weeks, collect the rat skull tissues, perform three-dimensional reconstruction by micro-CT, and observe the bone repair ability of the composite scaffold. As Figure 8 shown, compared with the control group and the DBM group, the composite scaffold group significantly enhanced the bone repair ability of the composite scaffold by introducing platelet vesicles.

[0082] The above is only the preferred embodiment of the present invention. It should be noted that although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention, without deviating from the scope defined by the claims of the present invention.

Claims

1. Platelet vesicle - acellular cancellous bone composite scaffold, characterized in that: The composite scaffold is formed by crosslinking platelet vesicles and cancellous bone extracellular matrix microparticles with fibrinogen gel as the scaffold background.

2. Preparation method of platelet vesicle - acellular cancellous bone composite scaffold, characterized in that: Cancellous bone particles are obtained from the bone tissue of animals, decellularized, and demineralized cancellous bone extracellular matrix microparticles are prepared. These are then added to the platelet vesicle fibrinogen hydrogel and crosslinked under the action of enzymes or calcium ions to obtain a platelet vesicle - decellularized cancellous bone composite scaffold.

3. The preparation method of the platelet vesicle - acellular cancellous bone composite scaffold according to claim 2, characterized in that: The method for preparing demineralized cancellous bone extracellular matrix microparticles is as follows: s2.

1. Isolate cancellous bone blocks from the bone tissue of animals, cut them into particles, rinse with water, and soak in peracetic acid ultrapure water. s2.

2. Transfer the soaked cancellous bone particles to a sterile Triton - X100 solution, shake, and then repeatedly wash with sterile water. s2.

3. Add the cancellous bone particles to an aqueous sodium dodecyl sulfate solution and shake to obtain decellularized cancellous bone particles. s2.

4. Perform ultrasonic demineralization on the decellularized cancellous bone particles, wash, dry, and grind them into powder to obtain cancellous bone extracellular matrix microparticles.

4. The preparation method of the platelet vesicle - acellular cancellous bone composite scaffold according to claim 3, characterized in that: Soak the decellularized cancellous bone particles in 10% EDTA decalcifying solution and perform decalcification at 4°C for 3 - 6 h in a rapid ultrasonic decalcifier.

5. The preparation method of the platelet vesicle - acellular cancellous bone composite scaffold according to claim 3, characterized in that: Screen the cancellous bone extracellular matrix microparticles using a filter with a diameter of 500 μm.

6. The preparation method of the platelet vesicle - acellular cancellous bone composite scaffold according to claim 2, characterized in that: Extract platelets from venous blood, prepare platelet vesicles by membrane extrusion, and resuspend them in physiological saline containing fibrinogen to obtain a platelet vesicle fibrinogen hydrogel.

7. The preparation method of the platelet vesicle - acellular cancellous bone composite scaffold according to claim 6, characterized in that: The method for preparing platelet vesicles is as follows: s3.

1. Mix venous blood and citrate - glucose solution A in a volume ratio of 9:1, and then centrifuge at 200 g for 10 - 20 min. s3.

2. Take the upper platelet layer, centrifuge at 800 g for 10 - 20 min, collect the precipitate to obtain platelets. s3.

3. Resuspend the platelets in physiological saline containing 5 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM prostaglandin E1 (PGE1) and sonicate for 10 min. s3.

4. Set the heating temperature of the constant - temperature module to 20°C and extrude through a PES membrane with a pore size of 200 nm at a speed of 0.1 - 1.0 ml / s to obtain a platelet vesicle suspension. s3.

5. Centrifuge the platelet vesicle suspension obtained in s3.4 at 15000 g for 30 - 60 min, collect the precipitate, and resuspend it in physiological saline containing 10 mg / ml fibrinogen.

8. The preparation method of the platelet vesicle - acellular cancellous bone composite scaffold according to claim 6, wherein: In the platelet vesicle fibrinogen hydrogel, the concentration of platelet vesicles is 100 μg / mL.

9. The preparation method of the platelet vesicle - acellular cancellous bone composite scaffold according to claim 2, wherein: Add an equal volume of cancellous bone extracellular matrix microparticles to the platelet vesicle fibrinogen hydrogel, then add thrombin, mix well, and let it stand to obtain a platelet vesicle - decellularized cancellous bone composite scaffold.

10. Method for applying platelet vesicle - acellular cancellous bone composite scaffold, characterized in that: Apply the platelet vesicle - decellularized cancellous bone composite scaffold as described in claim 1, or the platelet vesicle - decellularized cancellous bone composite scaffold prepared by any of the methods described in claims 2 - 9, to the preparation of bone defect drugs to simulate the three - dimensional environmental changes during the entire process of bone tissue repair.

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