3D Printable Collagen-Calcium Phosphate Composites, Their Preparation Methods and Applications

Abstract

This invention belongs to the field of materials and instruments for clinical application in oral medicine, and relates to a method for preparing a 3D-printable collagen-calcium phosphate composite material, comprising: 1) pulverizing type I collagen and type III collagen, and mixing them together in a certain proportion to form a collagen mixture; 2) mixing the collagen mixture with an organic compound crosslinking agent hexamethylene diisocyanate to form a raw material mixture, adding acetone and a catalyst, and heating to form a crosslinked composite collagen; 3) drying the crosslinked composite collagen, and mixing it with calcium phosphate material in a certain proportion after drying, and then adding acetic acid solution to dissolve and prepare a 3D-printable collagen-calcium phosphate composite material. This method can improve the convenience of preparing personalized bone defect repairs with overall mechanical properties, ensure the stability of the morphology and structure of the repair after implantation, and the reliability of guiding / inducing bone regeneration, and is suitable for repairing bone defects of different locations and sizes.

Description

Technical Field

[0001] This invention belongs to the field of materials and devices for clinical application in oral medicine, and relates to a method for preparing a material / device for guiding / inducing bone regeneration, especially a 3D printable collagen-calcium phosphate composite material, its preparation method and application. Background Technology

[0002] The jawbone / alveolar bone is the foundation of dental function and facial appearance. Various diseases commonly occur in the field of oral medicine, leading to damage and defects in the jawbone / alveolar bone, which affect oral function and even facial appearance, requiring timely and effective repair.

[0003] The main clinical treatments for jaw / alveolar bone defects include autologous bone grafting reconstruction and bone substitute implantation. Autologous bone grafting is the traditional technique for bone defect repair and reconstruction and remains the gold standard for its effectiveness. However, autologous bone grafting surgery is relatively complex, involves additional damage to the donor site, carries a higher risk of postoperative infection, and can result in significant losses due to graft failure. Using artificial bone grafts to replace autologous bone implantation into bone defects, guiding / inducing new bone regeneration, and allowing the graft material to degrade and be absorbed simultaneously, can achieve the goals of minimally invasive, rapid, and functional repair of bone defects and reconstruction of bone structure, while avoiding donor site damage. This is an important research direction in contemporary clinical dentistry and basic medicine.

[0004] Currently, commonly used artificial bone graft materials are various granular bone powder materials derived from allogeneic bone or xenogeneic bone such as bovine / porcine bone. After being implanted into the bone defect cavity, these materials can guide blood vessels to grow into the spaces between the bone graft particles and guide / induce osteocytes to attach to the surface of the material, grow, proliferate, and differentiate, thereby forming regenerated new bone to replace the gradually degrading bone graft material, ultimately achieving bone regeneration and repairing bone defects. Bone regeneration requires a stable osteogenic space and environment. In clinical applications, a biomembrane is often used to cover the surface of the bone defect cavity, acting as a barrier separating bone tissue from surrounding soft tissue. This prevents rapidly growing fibroblasts or epithelial cells from growing into the bone defect and competitively inhibits slower-growing osteoblasts, ensuring the stability of the osteogenic environment. Simultaneously, it coats the implanted bone powder material in the bone defect area to prevent its loss, ensuring the stability of the osteogenic space.

[0005] However, granular artificial bone graft materials lack load-bearing capacity, and their bone morphology relies on biomembrane coating and shaping, making it difficult to meet the clinical needs for repairing and reconstructing various large-scale bone defects. With the development of contemporary 3D printing bioengineering technology, the research and development of artificial bone graft materials that can be constructed using 3D printing to have biomimetic natural bone structures and personalized reconstruction morphologies has become a current hot topic in medical research.

[0006] Due to the complexity of bone composition and structure, the application of 3D printing technology in bone tissue engineering still faces numerous material and methodological bottlenecks. Firstly, from the perspective of bone composition, bones are composed of organic and inorganic components. Organic components mainly include proteins such as collagen, which act like fine threads, weaving the basic structure of the bone and giving it elasticity and toughness. Inorganic components are mainly minerals such as calcium and phosphorus, which are deposited in crystalline form on the framework of organic components, enhancing the bone's hardness and strength. This delicate balance between organic combination and inorganic deposition allows the bone to withstand pressure while also possessing a certain degree of deformation capacity to adapt to the complex movement requirements of the body. At the structural level, the surface of the bone is dense and smooth cortical bone, ensuring sufficient bone strength. The interior is composed of cancellous bone made up of crisscrossing trabeculae, which not only reduces bone weight but also houses bone marrow tissue containing numerous cells and blood vessels. Blood vessels and nerves passing through the cortical bone foramina, together with surrounding tissues, maintain the normal physiological function of the bone.

[0007] In-depth research into natural bone tissue has revealed its complex composition and structure, which play a crucial role in its mechanical properties and bioactivity. Therefore, the development of biomimetic bone tissue components for artificial bone grafts has become a highly anticipated topic in the field of biomedical engineering in recent years. Organic collagen raw materials and inorganic bone powder materials extracted from natural bone tissue have become mature products with extensive clinical applications. However, there is still a lack of tissue engineering products that can recombine organic collagen and inorganic calcium phosphorus to construct structures and functions that mimic natural bone tissue. In particular, the lack of collagen-calcium phosphorus composite materials suitable for 3D bioprinting technology has become a key issue restricting the development of this field. Most materials currently on the market fail to meet the high requirements of 3D bioprinting technology, which demands good biocompatibility, printability, and sufficient mechanical properties.

[0008] Therefore, developing a novel collagen-calcium phosphate composite material is of great significance for promoting the development of 3D bioprinting technology. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a 3D printable collagen-calcium phosphate composite material, its preparation method and application. It can improve the convenience of preparing personalized bone defect repairs with overall mechanical properties, ensure the stability of the morphology and structure of the repair after implantation and the reliability of guiding / inducing bone regeneration, and is suitable for repairing bone defects of different locations and sizes.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] A method for preparing a 3D-printable collagen-calcium phosphate composite material, characterized by comprising the following steps:

[0012] 1) Type I collagen and Type III collagen are pulverized and then mixed together in a certain proportion to form a collagen mixture, wherein the mass percentage of Type III collagen in the collagen mixture is 10-20%;

[0013] 2) The collagen mixture is mixed with the organic compound crosslinking agent hexamethylene diisocyanate to form a raw material mixture, and acetone and catalyst are added to carry out a heating reaction to form crosslinked composite collagen. The weight ratio of the organic compound crosslinking agent hexamethylene diisocyanate to the collagen mixture is 1:14. The amount of acetone added is 100 mL of acetone per 1.8-2.0 g of the raw material mixture, and the amount of catalyst added is 0.002-0.003 g of catalyst per 1.8-2.0 g of the raw material mixture.

[0014] 3) The cross-linked composite collagen is dried and then mixed with calcium phosphate material in a certain proportion. Acetic acid solution is then added to dissolve the mixture to prepare a 3D printable collagen-calcium phosphate composite material. The mass percentage of the calcium phosphate material after mixing with the dried cross-linked composite collagen is 30-50%, the concentration of the acetic acid solution is 0.5-1M, and the concentration of the 3D printable collagen-calcium phosphate composite material is 0.05-0.5% (w / v).

[0015] Preferably, in step 1), the type I collagen is bovine bone collagen or porcine bone collagen, and the type III collagen is bovine collagen or porcine collagen.

[0016] Preferably, in step 2), the catalyst is dibutyltin dilaurate.

[0017] Preferably, in step 2), the heating reaction temperature is 45°C and the heating reaction time is 8 hours.

[0018] Preferably, in step 3), the calcium phosphate material is β-tricalcium phosphate or hydroxyapatite powder.

[0019] Preferably, in step 3), the product is dried in an oven at 35°C for 2 hours.

[0020] In addition, the present invention provides a 3D printable collagen-calcium phosphate composite material, characterized in that it is prepared by the above-described preparation method.

[0021] Furthermore, the present invention also provides the use of the above-mentioned 3D-printable collagen-calcium phosphate composite material to prepare a biomimetic osteogenic scaffold, wherein the biomimetic osteogenic scaffold has an external biomimetic cortical bone morphology and an internal biomimetic cancellous bone porous structure.

[0022] Preferably, the collagen-calcium phosphate composite material is printed and self-cured into the biomimetic osteogenic scaffold using a direct 3D printing method via an extrusion 3D bioprinter.

[0023] Preferably, the negative mold of the biomimetic bone scaffold is first printed using printing wax via an extrusion 3D printer, and then the collagen-calcium phosphate composite material is injected into the negative mold. After it solidifies, the negative mold is removed by hot water dissolving and rinsing to obtain the biomimetic bone scaffold.

[0024] Compared with the prior art, the 3D printable collagen-calcium phosphate composite material of the present invention, its preparation method and application have one or more of the following beneficial technical effects:

[0025] 1. This invention is the first to use hexamethylene diisocyanate, a high molecular crosslinking agent, to enhance the viscoelasticity of collagen. By precisely controlling the degree and distribution of crosslinking of hexamethylene diisocyanate, the adhesive raw material of this invention exhibits excellent tensile strength and toughness, overcoming the shortcomings of traditional adhesive raw materials that are brittle and have poor elasticity. Thus, while maintaining the biocompatibility and biodegradability of the adhesive raw material, its mechanical properties are significantly improved.

[0026] 2. This invention is the first to use β-calcium triphosphate (βTCP) or hydroxyapatite (HA) powder mixed with hexamethylene diisocyanate crosslinked collagen acetic acid solution. The resulting viscoelastic composite material has self-curing ability, thus realizing the printability of collagen-calcium phosphate composite material. This is a breakthrough in the material bottleneck of 3D printing technology in the field of bone tissue engineering.

[0027] 3. This invention is the first to successfully apply collagen-calcium phosphate composite material to 3D bioprinting, creating personalized bone defect repair bodies with overall mechanical properties. At the same time, by adjusting the material composition and printing parameters, the porosity, mechanical properties and degradation rate of the scaffold can be precisely controlled to meet the needs of bone tissue regeneration and repair in different parts of the body.

[0028] 4. This invention is the first to design an indirect 3D printing method for preparing bone tissue engineering products. A negative mold of a specific bone graft material scaffold can be printed using conventional printing wax and a general-purpose 3D printer. Then, a collagen-calcium phosphate composite material is poured in and allowed to solidify. After solidification, the negative mold material is removed by hot water dissolving and rinsing to obtain the collagen-calcium phosphate composite bone scaffold. This method significantly improves the preparation efficiency of biological products, ensures that the quality of biological products is not affected, avoids the high standard requirements of 3D bioprinting production equipment, effectively reduces production costs, and improves the convenience and popularity of 3D printing methods for preparing bone tissue engineering products.

[0029] 5. This invention is the first to design and prepare a collagen-calcium phosphate composite bone scaffold with a specific external biomimetic cortical bone morphology and an internal biomimetic porous cancellous bone structure. The external biomimetic cortical bone is closely connected to the natural cortical bone around the bone defect, which is conducive to the fixation of the bone graft material and the formation of a closed barrier. The internal biomimetic cancellous bone is closely attached to the medullary cavity around the bone defect, which is conducive to the ingrowth of blood vessels and bone cells into the bone graft material scaffold. It is suitable for the repair and treatment of various complex bone defects. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments. The content of the embodiments is not intended to limit the scope of protection of the present invention.

[0031] Most materials currently on the market fail to meet the high requirements of 3D bioprinting technology, which demands not only good biocompatibility but also printability and sufficient mechanical properties. To address this challenge, the inventors have actively invested in research and development, aiming to create an ideal 3D bioprinting bone graft material. Through in-depth research into the molecular structure and interactions of collagen and calcium phosphate, and by employing biopolymer chemical cross-linking technology and precise control of material composition, they have successfully prepared a novel collagen-calcium phosphate composite material. This material not only possesses excellent biocompatibility but also printability. After self-curing, the printed product exhibits excellent strength and elasticity. By combining it with medical imaging technology, personalized bone repair scaffolds can be custom-made according to the patient's specific condition and needs. These scaffolds are highly similar to native tissue in morphology, structure, and function, providing a good scaffold and microenvironment for bone regeneration and effectively promoting osteoblast adhesion and proliferation.

[0032] The method for preparing the 3D-printable collagen-calcium phosphate composite material of the present invention includes the following steps:

[0033] 1. Type I and Type III collagen are pulverized and then mixed together in a certain proportion to form a collagen mixture.

[0034] In the collagen mixture, the mass percentage of type III collagen is 10-20%. Preferably, the mass percentage of type III collagen is 15%.

[0035] Meanwhile, in this invention, preferably, the type I collagen is bovine bone collagen or porcine bone collagen, and the type III collagen is bovine collagen or porcine collagen.

[0036] 2. The collagen mixture is mixed with the organic compound crosslinking agent hexamethylene diisocyanate to form a raw material mixture, and acetone and catalyst are added to carry out a heating reaction to form crosslinked composite collagen.

[0037] The hexamethylene diisocyanate is an organic compound cross-linking agent that can increase collagen viscoelasticity after cross-linking with collagen molecules.

[0038] The weight ratio of the organic crosslinking agent hexamethylene diisocyanate to the collagen mixture is 1:14. The amount of acetone added is 100 mL for every 1.8-2.0 g of the raw material mixture. The amount of catalyst added is 0.002-0.003 g for every 1.8-2.0 g of the raw material mixture.

[0039] Preferably, the catalyst is dibutyltin dilaurate.

[0040] More preferably, the temperature of the heating reaction is 45°C, and the heating reaction time is 8 hours.

[0041] 3. The cross-linked composite collagen is dried, and after drying, it is mixed with calcium phosphate material in a certain proportion. Then, acetic acid solution is added to dissolve it to prepare a collagen-calcium phosphate composite material that can be 3D printed.

[0042] The calcium phosphate material is natural or synthetic hydroxyapatite powder or β-tricalcium phosphate powder, which is combined with collagen to construct a biomimetic material, accounting for 30-5% of the composite material, and can increase the material's supporting strength.

[0043] In this process, after the dried cross-linked composite collagen is mixed with the calcium phosphate material, the calcium phosphate material accounts for 30-50% by mass. The concentration of the acetic acid solution is 0.5-1M, and the concentration of the 3D-printable collagen-calcium phosphate composite material is 0.05-0.5% (w / v). That is, in the 3D-printable collagen-calcium phosphate composite material, the concentrations of the cross-linked composite collagen and calcium phosphate material are 0.05-0.5% (w / v).

[0044] Preferably, the product is dried in an oven at 35°C for 2 hours.

[0045] Through the above steps, a 3D-printable collagen-calcium phosphate composite material can be prepared. It is a liquid with adjustable viscosity, which can be used for 3D printing or for negative mold casting. Moreover, it is a self-curing material that can be cured at room temperature and has a certain supporting strength and bending elasticity after curing.

[0046] The aforementioned 3D-printable collagen-calcium phosphate composite material can be used to prepare a biomimetic osteogenic scaffold, which has an external biomimetic cortical bone morphology and an internal biomimetic cancellous bone porous structure.

[0047] Preferably, the outer biomimetic cortical bone is planar with a thickness of 1-3 mm, and the inner biomimetic cancellous bone is a cubic structure with a porous, trabecular-like structure and a thickness of 1-2 cm. The outer biomimetic cortical bone is tightly connected to the natural cortical bone surrounding the bone defect, which is beneficial for the fixation of the bone graft material and the formation of a closed barrier; the inner biomimetic cancellous bone is tightly attached to the medullary cavity surrounding the bone defect, which is beneficial for the ingrowth of blood vessels and bone cells from the medullary cavity into the bone graft material scaffold.

[0048] One method involves using direct 3D printing to print the collagen-calcium phosphate composite material using an extrusion 3D bioprinter and then allowing it to self-cure into the biomimetic bone scaffold. Alternatively, a negative mold of the biomimetic bone scaffold can be printed using an extrusion 3D printer first, then the collagen-calcium phosphate composite material can be poured into the negative mold. After solidification, the negative mold can be removed by hot water dissolving and rinsing to obtain the biomimetic bone scaffold.

[0049] In practical applications, standardized biomimetic osteogenic scaffolds of different sizes are prefabricated according to the expected application site, such as alveolar bone defects and mandibular bone defects, and sterilized for later use. During the operation, the prepared osteogenic scaffolds are shaped and modified according to the actual size and shape of the bone defect using a scalpel or dental drill, and then implanted into the bone defect. This allows the external biomimetic cortical bone to be tightly connected with the natural cortical bone around the bone defect, which is conducive to the fixation of the bone graft material and the formation of a closed barrier. The internal biomimetic cancellous bone adheres to the medullary cavity around the bone defect.

[0050] For the repair of large-scale bone defects, a personalized digital 3D prosthesis is reconstructed based on preoperative medical imaging data. High-precision direct or indirect 3D printing technology is used to transform the digital model of the prosthesis into a physical personalized collagen-calcium phosphate composite biomimetic bone scaffold.

[0051] Preferably, the personalized biomimetic bone scaffold is designed to fit the specific condition of the patient's bone defect site, featuring a biomimetic cortical bone morphology and thickness adapted to the defect area, as well as the porous structure and thickness of the internal biomimetic trabecular cancellous bone. This design ensures a perfect match between the scaffold and the patient's bone defect site, providing excellent biomechanical support. After implantation, it forms a good bio-interface with surrounding tissues, making it suitable for repairing maxillary bone defects with complex three-dimensional morphology and large-scale mandibular bone defects. Through the reconstruction and implantation of personalized digital 3D prostheses, more precise and effective treatment methods can be provided to patients. The design ensures that the bone repair material scaffold is highly similar to the original tissue in morphology, structure, and function; the relatively smooth cortical bone surface contacts the surrounding mucoperiosteum and other soft tissues, which is conducive to the soft tissues closing the wound through suturing. At the same time, it acts as a barrier, preventing fibroblasts, epithelial cells, and other cells in the soft tissues from growing into the bone regeneration area, while ensuring the growth of osteoblasts, blood vessels, and other cells with slower proliferation and regeneration rates on the surface of the artificial bone particles; the porous cancellous bone inside provides ample space for blood to permeate and fill, forming blood clots, as well as further osteoblast and blood vessel ingrowth, providing a good foundation for subsequent bone regeneration activities.

[0052] The above embodiments of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the implementation of the present invention. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing a 3D-printable collagen-calcium phosphate composite material, characterized in that, Includes the following steps: 1) Type I collagen and Type III collagen are pulverized and mixed together in a certain proportion to form a collagen mixture, wherein the mass percentage of Type III collagen in the collagen mixture is 10-20%; the Type I collagen is bovine bone collagen or porcine bone collagen, and the Type III collagen is bovine collagen or porcine collagen. 2) The collagen mixture is mixed with the organic compound crosslinking agent hexamethylene diisocyanate to form a raw material mixture, and acetone and a catalyst are added. The mixture is then heated to form crosslinked composite collagen. The weight ratio of the organic compound crosslinking agent hexamethylene diisocyanate to the collagen mixture is 1:

14. The amount of acetone added is 100 mL per 1.8-2.0 g of the raw material mixture, and the amount of catalyst added is 0.002-0.003 g per 1.8-2.0 g of the raw material mixture. The catalyst is dibutyltin dilaurate. The heating temperature is 45°C, and the heating time is 8 hours. 3) The cross-linked composite collagen is dried and then mixed with calcium phosphate material in a certain proportion. Acetic acid solution is then added to dissolve the mixture and prepare a 3D printable collagen-calcium phosphate composite material. The mass percentage of the calcium phosphate material after mixing with the dried cross-linked composite collagen is 30-50%, the concentration of the acetic acid solution is 0.5-1M, and the concentration of the 3D printable collagen-calcium phosphate composite material is 0.05-0.5% (w / v). The mixture is dried in an oven at 35°C for 2 hours.

2. The method for preparing the 3D printable collagen-calcium phosphate composite material according to claim 1, characterized in that, In step 3), the calcium phosphate material is β-tricalcium phosphate or hydroxyapatite powder.

3. A 3D-printable collagen-calcium phosphate composite material, characterized in that, It is prepared by the preparation method described in any one of claims 1-2.

4. The use of the 3D-printable collagen-calcium phosphate composite material of claim 3 to prepare a biomimetic osteogenic scaffold, wherein the biomimetic osteogenic scaffold has an external biomimetic cortical bone morphology and an internal biomimetic cancellous bone porous structure.

5. The use according to claim 4, characterized in that, The collagen-calcium phosphate composite material was printed and self-cured into the biomimetic osteogenic scaffold using a direct 3D printing method via an extrusion 3D bioprinter.

6. The use according to claim 4, characterized in that, First, a negative mold of the biomimetic bone scaffold is printed using an extrusion 3D printer with printing wax. Then, the collagen-calcium phosphate composite material is poured into the negative mold. After it solidifies, the negative mold is removed by hot water dissolving and rinsing to obtain the biomimetic bone scaffold.