Locally anticoagulant acellular bone matrix composite material and preparation method thereof

Abstract

The invention relates to the technical field of biological materials, in particular to a local anticoagulation acellular bone matrix composite material and a preparation method thereof.The preparation method comprises the following steps that S1, bone tissue is obtained and treated, and an acellular bone matrix is prepared; s2, mixing an anticoagulant with the acellular bone matrix to form an anticoagulant compound; s3, mixing the anticoagulant compound with a biocompatible carrier to form a final composite material; and S4, preparing the composite material into an implant or a stent with a required shape through a molding and curing process. By arranging the acellular bone matrix, the natural structure and biological activity of the bone matrix are reserved to the maximum extent, and meanwhile, all cell components are removed, so that immunogenicity and pathogen transmission are reduced; by adding the anticoagulant, discomfort can be reduced after the local anticoagulant acellular bone matrix composite material is implanted into a human body, and the problems that in the prior art, an acellular bone matrix has the blood coagulation risk, and incompletely-treated bone tissue has immunological rejection are solved.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to a local anticoagulation acellular bone matrix composite material and a preparation method thereof. Background Art

[0002] Clinically, bone defects caused by various reasons are very common, and their repair has always been a difficult problem in orthopedics. The ideal method of bone defect repair is constantly being explored. Tissue engineering bone, a regenerative repair method, brings new hope to bone defect repair. After 20 years of development, it has achieved rich results in three major research areas. However, after the seed cells and scaffold complex are implanted into the body, angiogenesis takes several days, and cells tolerate hypoxia for several hours; the oxygen and nutrients required for cell survival can only be supplied by the body's tissue fluid infiltration, and the diffusion distance of oxygen is limited to 0.2mm. This determines that the survival of seed cells and the in vitro construction of tissue engineering bone cannot be pre-ordered according to the actual size of the bone defect, especially for large bone defects, that is, the thickness of the constructed tissue engineering bone cannot be greater than 0.5mm, otherwise the seed cells therein will die due to lack of oxygen and nutrients. After the existing tissue engineering bone is implanted into the body, its blood supply is carried out in an infiltration manner; and when the material comes into contact with blood, the body's coagulation mechanism will be activated, causing blood clots to form around the implant, further exacerbating the oxygen and nutrient supply barriers of the implanted tissue engineering bone.

[0003] Although traditional decellularized bone matrix has good biocompatibility, it has the risk of blood coagulation in clinical applications, and unprocessed bone tissue has the problem of immune rejection. Therefore, it is of great significance to develop a new type of anticoagulant decellularized bone matrix composite material. Summary of the invention

[0004] The present invention aims to solve one of the technical problems in the above-mentioned related art at least to a certain extent.

[0005] In order to achieve the above object, one aspect of the present invention discloses a local anticoagulation decellularized bone matrix composite material, comprising a decellularized bone matrix and an anticoagulant, wherein the decellularized bone matrix and the anticoagulant are loaded on a biocompatible carrier.

[0006] In addition, the local anticoagulation acellular bone matrix composite material according to the above embodiment of the present invention may also have the following additional technical features:

[0007] Furthermore, biologically active factors are also included.

[0008] Furthermore, the bioactive factor is a growth factor or a cytokine.

[0009] According to the locally anticoagulated decellularized bone matrix composite material of the present invention, the natural structure and biological activity of the bone matrix can be retained to the maximum extent by setting up the decellularized bone matrix, while removing all cellular components to reduce the risk of immunogenicity and pathogen transmission; by adding an anticoagulant, the locally anticoagulated decellularized bone matrix composite material can reduce and lower discomfort after being implanted into the human body; by providing multiple cavities on the set carrier, full consideration is given to the integration with the host tissue, and the design of the porous structure allows cell migration and vascularization, thereby accelerating the tissue repair process.

[0010] On the other hand, the present invention discloses a method for preparing a local anticoagulant decellularized bone matrix composite material, comprising the following steps: S1: obtaining and processing bone tissue to prepare a decellularized bone matrix; S2: mixing an anticoagulant with the decellularized bone matrix to form an anticoagulant complex; S3: mixing the anticoagulant complex with a biocompatible carrier to form a final composite material; S4: preparing the composite material into an implant or scaffold of a desired shape through a molding and curing process.

[0011] In addition, the local anticoagulation acellular bone matrix composite material according to the above embodiment of the present invention may also have the following additional technical features:

[0012] Furthermore, it also includes pretreatment of bone tissue to remove impurities and residual soft tissue on the bone tissue.

[0013] Furthermore, it also includes adding bioactive factors into the anticoagulant complex.

[0014] Furthermore, the molding process adopts 3D printing.

[0015] Furthermore, the decellularization treatment includes physical methods and chemical methods.

[0016] According to the method for preparing a local anticoagulant decellularized bone matrix composite material of the present invention, the cell components that may cause immune rejection reactions are removed through decellularization treatment, while the natural structure of the bone matrix is ​​retained, which helps to improve the biocompatibility of the material, and the thorough decellularization step reduces the immunogenicity of the material and reduces the immune response that may occur after implantation, thereby improving the safety of the material; by adding anticoagulants, such as heparin or heparin sulfate, the composite material can significantly reduce the risk of coagulation after implantation, which is particularly important for applications that require rapid healing and reduced postoperative complications; by selecting a suitable biocompatible carrier material and adjusting its composition, the mechanical properties of the composite material can be customized to meet different clinical application requirements; by utilizing advanced molding technologies such as 3D printing, the shape and structure of the implant or scaffold can be precisely controlled to achieve personalized customization and the manufacture of complex shapes.

[0017] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0019] Figure 1 It is a schematic diagram of the structure of the local anticoagulation acellular bone matrix composite material of the present invention;

[0020] Figure 2 This is a schematic diagram of the structure of the locally anticoagulated acellular bone matrix composite material after molding;

[0021] Figure 3 is a line graph comparing fracture healing rates of the rat fracture model of the present invention;

[0022] Figure 4 It is a line graph comparing the volume fraction of new bone formation in the rat fracture model of the present invention.

[0023] In the figure: 1. carrier; 2. decellularized bone matrix; 3. anticoagulant; 4. pores; 5. bioactive factors. DETAILED DESCRIPTION

[0024] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0025] The locally anticoagulated acellular bone matrix composite material according to an embodiment of the present invention will be described below with reference to the accompanying drawings.

[0026] A method for preparing a local anticoagulation acellular bone matrix composite material comprises the following steps:

[0027] S1: Obtain and process bone tissue to prepare decellularized bone matrix 2. The selection of bone tissue requires the selection of suitable animal or human bone tissue, such as bovine bone or human bone, to ensure the legality and traceability of the source. The decellularization treatment includes physical methods and chemical methods. Physical method: Use high-speed centrifugation, mechanical stirring or ultrasonic treatment to physically break cells. Specifically, high-speed centrifugation: By controlling the centrifugal force and time, bone cells and matrix can be effectively separated. Mechanical stirring: Use stirring of different intensities and frequencies to physically destroy the cell structure. Ultrasonic treatment: Use the cavitation effect generated by ultrasound to further break up cells, while controlling the treatment time to avoid damage to the matrix.

[0028] Chemical method: Use enzymes such as collagenase or deoxyribonuclease (DNase) to digest cell components, and use surfactants to help remove cell membranes. Specifically, enzyme treatment includes selecting appropriate enzyme concentrations and action time, using enzymes such as collagenase or DNase to digest cell components while maintaining the integrity of the matrix. When using surfactants, use non-ionic or cationic surfactants to help remove cell membranes and reduce cell residues.

[0029] In the actual operation process, a multi-step combined treatment process is carried out by combining physical and chemical methods to achieve a more thorough decellularization effect. And ensure that all chemical substances used are completely removed after decellularization to avoid affecting the subsequent addition of bioactive factors 5 and anticoagulants 3. Through experiments, decellularized bone matrix 2 was co-cultured with peripheral blood mononuclear cells, and the levels of inflammatory factors IL-6 and TNF-α were detected by enzyme-linked immunosorbent assay (ELISA). The levels of IL-6 and TNF-α induced by decellularized bone matrix 2 were 23.5pg / mL and 15.2pg / mL, respectively, which were much lower than the levels induced by untreated bone tissue (185pg / mL and 120pg / mL, respectively).

[0030] S2: Mix the anticoagulant 3 with the decellularized bone matrix 2 to form an anticoagulant complex. Specifically, when in use, the user selects a suitable anticoagulant 3, such as heparin or heparin sulfate, according to the expected clinical application and anticoagulant effect, taking into account factors including anticoagulant effect, biocompatibility, cost-effectiveness and availability. And through in vitro experiments and animal models, determine the optimal dose of different anticoagulants 3 to achieve the best anticoagulant effect and minimal side effects. Evaluate the compatibility of the proposed anticoagulant 3 with the decellularized bone matrix 2 to ensure that the structural integrity and biological activity of the matrix will not be affected after mixing. Finally, the selected anticoagulant 3 is mixed with the decellularized bone matrix 2, and the anticoagulant 3 is fixed by physical mixing or chemical cross-linking to ensure its uniform distribution and stability in the composite material. In this embodiment, the anticoagulant 3 is evenly dispersed in the decellularized bone matrix 2 in the form of a solution, and stirred or ultrasonicated to ensure uniform mixing.

[0031] S3: Mix the anticoagulant complex with the biocompatible carrier 1 to form a final composite material. When screening the carrier 1 material, the present invention selects a carrier 1 material with good biocompatibility, mechanical properties and degradation characteristics, such as porous polylactic acid-glycolic acid copolymer PLGA, polycaprolactone (PCL) or polylactic acid (PLA). In this embodiment, a polycaprolactone carrier 1 with a porous structure is used. The porous structure is used to increase the surface area, promote cell attachment, migration and tissue growth. Then, the anticoagulant complex and the carrier 1 material are dissolved in a suitable solvent to form a uniform mixed solution, and then the mixed solution is poured into a mold, and the desired shape is formed through a suitable curing process.

[0032] S4: The composite material is formed into an implant or stent of a desired shape through a molding and curing process. Specifically, in the actual application process, the user selects a suitable molding process, such as 3D printing, compression molding or electrospinning, according to the shape and structural requirements of the final product. In this embodiment, 3D printing is used, and the molded composite material is subjected to a chemical crosslinking agent or a photoinitiator to promote the crosslinking reaction between molecules inside the composite material, thereby enhancing its structural stability and mechanical strength.

[0033] In one embodiment, the surface of the composite material may be further processed, such as by plasma treatment, chemical etching or mechanical polishing, to increase the roughness of the surface of the composite material and promote cell attachment and proliferation.

[0034] In one embodiment, a bioactive coating, such as growth factors, antimicrobial agents, or anti-inflammatory drugs, is applied to the surface of the composite material to impart additional biological functions to the composite material.

[0035] In one embodiment, 3D printing or photolithography is used to create specific micropatterns on the surface of the composite material to guide the growth and differentiation of cells.

[0036] In one embodiment, before step S1, the bone tissue is also pretreated, and the bone tissue is thoroughly cleaned to remove impurities and residual soft tissue on the bone tissue. The pretreatment cleaning step specifically includes: using sterile water or saline to preliminarily rinse the bone tissue to remove blood, grease and other visible impurities on the surface; then using an ultrasonic cleaner to further remove tiny impurities on the surface of the bone tissue and in the micropores, and the cavitation effect of ultrasound helps to loosen and remove difficult-to-clean areas; using proteases or other biological enzymes to enzymatically treat the bone tissue to help remove residual soft tissue and cell fragments; using mild chemical detergents, such as neutral detergents, to further clean the bone tissue and remove residual biomacromolecules; after cleaning, using a large amount of sterile water or saline to rinse the bone tissue to neutralize the residual chemical detergents or enzymes; finally, under sterile conditions, using sterile air or a drying oven to dry the surface of the bone tissue to prepare for subsequent decellularization. Use a flow cytometer to count cells in bone tissue samples before and after decellularization. The experimental results showed that the cell count of bone tissue before decellularization was 2.5\times 10^62.5×106 cells / mL, and after decellularization it dropped to 1.5\times 10^41.5×104 cells / mL, with a removal rate of 99.4%.

[0037] The following steps are also included: adding bioactive factor 5 to the anticoagulant complex. Select bioactive factor 5 that can promote cell growth and tissue repair, such as bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), and use adsorption, microencapsulation or covalent binding techniques to fix the bioactive factor 5 to the anticoagulant complex during loading. In the rat fracture model experiment, the composite material was implanted into the fracture site, and the healing condition was evaluated by X-ray and micro-CT. The results are as follows: Figure 3 As shown in Figure 2, the fracture healing rate of the composite implant group was 85% (after 12 weeks) and that of the control group was 65%. Figure 4 As shown, the volume fraction of new bone formation was 40% in the control group and 25% in the control group.

[0038] like Figure 1 , Figure 2As shown, in this embodiment, a Y-shaped bracket is taken as an example. According to an embodiment of the present invention, the locally anticoagulated decellularized bone matrix composite material comprises a decellularized bone matrix 2 and an anticoagulant 3, and the decellularized bone matrix 2 and the anticoagulant 3 are loaded on a biocompatible carrier 1. By setting the decellularized bone matrix 2, the natural structure and biological activity of the bone matrix can be retained to the maximum extent, while removing all cellular components to reduce the risk of immunogenicity and pathogen transmission. By adding the anticoagulant 3, the locally anticoagulated decellularized bone matrix composite material can reduce and reduce discomfort after being implanted into the human body. The locally anticoagulated decellularized bone matrix composite material also includes a bioactive factor 5. Specifically, the bioactive factor 5 adopts a growth factor or a cytokine. By introducing the bioactive factor 5, cell proliferation, differentiation and tissue regeneration can be promoted. When in use, the composite material has a clear hierarchical structure: the decellularized bone matrix 2 serves as the base layer, providing the necessary mechanical support and biocompatibility; the anticoagulant 3 is evenly dispersed in the bone matrix to form the second layer, ensuring blood compatibility after implantation; the bioactive factor 5 serves as the third layer, and its release rate in the body is controlled through a specific release system.

[0039] The carrier 1 is provided with multiple holes 4, which fully considers the integration with the host tissue. The design of the porous structure allows cell migration and vascularization, thus accelerating the tissue repair process.

[0040] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements, unless otherwise clearly defined. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0041] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0042] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A local anticoagulation acellular bone matrix composite material, characterized in that: The invention comprises a decellularized bone matrix (2) and an anticoagulant (3), wherein the decellularized bone matrix (2) and the anticoagulant (3) are loaded on a biocompatible carrier (1).

2. The locally anticoagulated acellular bone matrix composite material according to claim 1, characterized in that: Also included are bioactive factors (5).

3. The locally anticoagulated acellular bone matrix composite material according to claim 2, characterized in that: The bioactive factor (5) is a growth factor or a cytokine.

4. A method for preparing the locally anticoagulated acellular bone matrix composite material according to any one of claims 1 to 3, characterized in that: The steps include: S1: Obtain and process bone tissue and prepare acellular bone matrix (2); S2: mixing the anticoagulant (3) with the decellularized bone matrix (2) to form an anticoagulant complex; S3: mixing the anticoagulant complex with the biocompatible carrier (1) to form a final composite material; S4: The composite material is prepared into an implant or scaffold of a desired shape through a molding and curing process.

5. The method for preparing a local anticoagulated acellular bone matrix composite material according to claim 4, characterized in that: It also includes the pretreatment of bone tissue to remove impurities and residual soft tissue on the bone tissue.

6. The method for preparing a local anticoagulated acellular bone matrix composite material according to claim 4, characterized in that: It also includes adding a biologically active factor (5) to the anticoagulant complex.

7. The method for preparing a local anticoagulated acellular bone matrix composite material according to claim 4, characterized in that: The molding process adopts 3D printing.

8. The method for preparing a local anticoagulated acellular bone matrix composite material according to claim 4, characterized in that: The decellularization treatment includes physical methods and chemical methods.

9. The method for preparing a local anticoagulated acellular bone matrix composite material according to claim 5, characterized in that: The pretreatment cleaning step specifically includes: using sterile water or physiological saline to initially rinse the bone tissue to remove blood, grease and other visible impurities on the surface; using an ultrasonic cleaner to further remove tiny impurities on the surface of the bone tissue and in the micropores; using protease or other biological enzymes to enzymatically treat the bone tissue; using chemical detergents to further clean the bone tissue to remove residual biomacromolecules; after cleaning, rinsing the bone tissue with a large amount of sterile water or physiological saline to neutralize the residual chemical detergent or enzyme; and finally drying the surface of the bone tissue under sterile conditions using sterile air or a drying oven.

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