Non-metal bioartificial aggregate not easy to collapse and application thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to non-metal bioartificial aggregate not prone to collapse and application. The non-metal bioartificial aggregate has a gradient three-layer scaffold structure, the obtained non-metal bioartificial aggregate uses a self-assembly technology, collagen molecules in ECM are used as a template, a calcium ion-containing solution and a phosphate ion-containing solution are added, hydroxyapatite grows on the molecular template in an in-situ mineralization manner, and the non-metal bioartificial aggregate is obtained. The structure and composition of a natural compact bone and a cancellous bone are simulated, the bionic structure, composition and function of the stent material are realized, the stent material can well absorb stress after being implanted into a body due to the structural design, and the biomechanical property of the material is improved. Meanwhile, cell permeation and nutrient substance conveying are also facilitated, the growth and proliferation speed of osteoblasts is remarkably increased, and the vascularization level and the speed of conversion from a stent material to a living body bone are increased.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly relates to a non-metallic bio-artificial aggregate that is not easily collapsible and its applications. Background Art

[0002] Natural bone matrix is divided into cancellous bone and cortical bone, and the cortical bone wraps the cancellous bone. Among them, the cortical bone accounts for about 75% of the human bone weight, and the porosity is 5 - 30%; the cancellous bone accounts for about 25% of the human bone weight, and the porosity is 30 - 90%. In order to meet the requirements of bionics, the internal structure of artificial bone should have a gradient pore structure with non-uniform distribution similar to that of natural bone. Most traditional artificial bone products are designed with a uniform structure, ignoring the fact that the actual true bone tissue structure actually includes both cortical bone and cancellous bone in two forms, which results in the inability to better meet the clinical bone repair requirements in terms of the vascularization level, osteogenic performance, and biomechanical performance of artificial bone scaffold materials. However, the design of gradient artificial bone is very difficult.

[0003] The patent with the patent number CN108261568B discloses a composite gradient bone repair material and its preparation method, and a gradient scaffold bone repair material with good uniformity and stability is obtained through 3D printing. However, this method adopts high-temperature calcination to remove the PVA component introduced in the 3D printing forming process of the scaffold, reducing the biological activity and osteogenic activity of the material, and this scaffold material only realizes structural bionics without compositional bionics.

[0004] Cristian Parisi et al. added different contents of calcium hydroxide and phosphoric acid to type I collagen to obtain four gradient solutions with different weight ratios. The four solutions were layered into a mold, and after one-step freeze-drying and forming, thermal cross-linking and NHS / EDC cross-linking were carried out in sequence, and then freeze-dried after cross-linking. This method realizes the bionics of the structure and components of gradient bone. However, for this method of first obtaining different gradient solutions and then freeze-drying at one time, during the process before and during freeze-drying of the different gradient solutions in the liquid state, under the action of its own gravity, the liquid will diffuse, causing a certain degree of mixing of the four gradient solutions, thereby reducing this gradient change. In order to reduce the mixing of the different gradient solutions under the action of gravity, it is required that the densest substance must be at the bottom layer, and the overall material shows a trend of decreasing density from bottom to top. In addition, through practice, it is found that although the gradient solutions in this study have a certain gradient structure, their porosities are all very large, and in terms of porosity, there is a large difference between their porosity distribution and that of natural bone, and true bionics cannot be achieved. Summary of the Invention

[0005] To overcome the above problems, a first aspect of the present invention provides a non-metallic bio-artificial aggregate that is not easily collapsible. The non-metallic bio-artificial aggregate has a gradient three-layer scaffold structure, and the gradient three-layer scaffold structure includes a first scaffold layer, a second scaffold layer, and a third scaffold layer; the ratio of the average porosity of the first scaffold layer, the second scaffold layer, and the third scaffold layer is (0.9 - 1.7):(1.4 - 3):(0.9 - 1.7).

[0006] Specifically, the ratio of the average porosity of the first scaffold layer, the second scaffold layer, and the third scaffold layer is 1:(1.4 - 3):(0.9 - 1.4).

[0007] Specifically, the ratio of the average porosity of the first scaffold layer, the second scaffold layer, and the third scaffold layer is 1:(1.4 - 3):(0.9 - 1.3).

[0008] Specifically, the ratio of the average porosity of the first scaffold layer, the second scaffold layer, and the third scaffold layer is 1:(1.4 - 2.3):(0.9 - 1.3).

[0009] Specifically, the ratio of the average porosity of the first scaffold layer, the second scaffold layer, and the third scaffold layer is 1:(1.8 - 2.3):(0.9 - 1.3).

[0010] Specifically, the ratio of the average porosity of the first scaffold layer, the second scaffold layer, and the third scaffold layer is 1:(1.4 - 2.3):(0.9 - 1.2).

[0011] Specifically, the ratio of the average porosity of the first scaffold layer, the second scaffold layer, and the third scaffold layer is 1:(1.4 - 2.3):(0.9 - 1.1).

[0012] Specifically, the ratio of the average porosity of the first scaffold layer, the second scaffold layer, and the third scaffold layer is 1:(1.8 - 2.3):(0.9 - 1.1).

[0013] Specifically, the ratio of the average porosity of the first scaffold layer, the second scaffold layer, and the third scaffold layer is 1:(1.4 - 2.3):1.

[0014] In some embodiments, the average porosity of the first scaffold layer is 30 - 50%, the average porosity of the second scaffold layer is 70 - 90%, and the average porosity of the third scaffold layer is 30 - 50%.

[0015] Specifically, the average porosity of the first scaffold layer is 31 - 48%, the average porosity of the second scaffold layer is 71 - 88%, and the average porosity of the third scaffold layer is 34 - 50%.

[0016] Specifically, the average porosity of the first scaffold layer is 31-47.8%, the average porosity of the second scaffold layer is 71.4-87.5%, and the average porosity of the third scaffold layer is 34.1-49.2%.

[0017] In some embodiments, the ratio of the total mass of the first and third scaffold layers to the mass of the second scaffold layer is (3.4-4.8):1.

[0018] Specifically, the ratio of the total mass of the first and third scaffold layers to the mass of the second scaffold layer is (3.4-4.5):1.

[0019] Specifically, the ratio of the total mass of the first and third scaffold layers to the mass of the second scaffold layer is (3.4-3.6):1.

[0020] Specifically, in some embodiments, the mass ratio of the first and third scaffold layers is 1:1, and the volumes of the first and third scaffold layers are substantially equal.

[0021] In order to obtain a similar structure with dense outer and porous inner in natural bone, the inventor intends to design an artificial aggregate with a cancellous bone having a porosity of 30 - 90% wrapped by a cortical bone with a porosity of 5 - 30%. After a great deal of thinking and demonstration, the inventor found that by controlling the porosity, mass and volume of each scaffold layer of the non-metallic biological artificial aggregate, specifically, the ratio of the average layer porosity of the first scaffold layer, the second scaffold layer and the third scaffold layer is (0.9 - 1.7):(1.4 - 3):(0.9 - 1.7); the average layer porosity of the first scaffold layer is 30 - 50%, the average layer porosity of the second scaffold layer is 70 - 90%, and the average layer porosity of the third scaffold layer is 30 - 50%; the ratio of the total mass of the first scaffold layer and the third scaffold layer to the mass of the second scaffold layer is (3.4 - 4.8):1, not only can the desired bionic structure be obtained, but also the resulting material is not prone to collapse in the direction of bone defect, thus having a good bone repair and growth effect. This may be because when the artificial aggregate is in the above structure, it plays a certain regulatory role in the secretion and metabolism of calcium ions. At the same time, this structure is conducive to guiding the proliferation of bone cells, thereby balancing the external mechanical stress and facilitating the orderly arrangement of trabecular bone. It is known that cortical bone accounts for about 75% of the weight of the human skeleton, and cancellous bone accounts for about 25% of the weight of the human skeleton. It is speculated that when designing artificial bone, a weight ratio of cortical bone to cancellous bone of 75:25, that is, 3:1, is more in line with the biological bionic structure. However, in this technical solution, the inventor found that when the ratio of the average layer porosity of the first scaffold layer, the second scaffold layer and the third scaffold layer is (0.9 - 1.7):(1.4 - 3):(0.9 - 1.7); the average layer porosity of the first scaffold layer is 30 - 50%, the average layer porosity of the second scaffold layer is 70 - 90%, and the average layer porosity of the third scaffold layer is 30 - 50%, by appropriately increasing the weight of the cortical bone, that is, the ratio of the total mass of the first scaffold layer and the third scaffold layer to the mass of the second scaffold layer is (3.4 - 4.8):1, not only has good biological stability, but also enables the artificial aggregate to quickly repair the original defect after implantation, helps to promote the formation of new bone callus and increase its strength. It is speculated that this may be because the increase in cortical bone is beneficial to the stability of the osteogenic space. The thicknesses of the first scaffold layer and the third scaffold layer of the same material with similar porosities are close. Acting on the wound site of the bone defect, it has a spatial impact on the initial formation and adhesion of the blood clot. At the same time, the stable surface stress of the blood clot also acts on the bone in turn, stimulating the growth of bone cells. The above effects particularly meet the clinical demands in the early stage of bone repair, accelerate the repair speed in the initial stage of bone repair, and the strength of the newly formed bone can be continuously maintained stably, and the bone mass is not easily lost.

[0022] In some embodiments, the preparation method of the non-metallic biological artificial aggregate is as follows: S1. After the acellular matrix that has been defatted, washed out, and antigen-blocked is crushed into acellular matrix particles, it is formulated into an acellular matrix stock solution for standby; a calcium salt aqueous solution and a phosphate aqueous solution are respectively prepared for standby; S2. Adjust the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the acellular matrix particles in the acellular matrix stock solution. After adding the calcium salt aqueous solution to the acellular matrix stock solution, adjust the pH to 9 - 11, and then add the phosphate aqueous solution to obtain the first emulsion; S3. Repeat step S1, adjust the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the acellular matrix particles in the acellular matrix stock solution, and obtain the second emulsion; S4. Repeat step S1, adjust the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the acellular matrix particles in the acellular matrix stock solution, and obtain the third emulsion; S5. Pour the first emulsion into a mold and perform the first freeze-drying; continue to add the second emulsion and perform the second freeze-drying; continue to add the third emulsion and perform the third freeze-drying to obtain a precursor; S6. Immerse the precursor in an aqueous cross-linking agent solution for cross-linking for 12 - 24 h, then rinse with deionized water and perform the fourth freeze-drying; at this time, from the lower part to the upper part of the mold, there are a first scaffold layer, a second scaffold layer, and a third scaffold layer respectively.

[0023] Specifically, in step S1, before defatting, washing out, and antigen-blocking, the acellular matrix is derived from soft tissues of pigs, cows, and humans, including skin, dermis, blood vessels, diaphragms, tendons, ligaments, large intestine, small intestine, and nerve tissues; the aforementioned soft tissues are defatted, washed out, and antigen-blocked by the perfusion-pressure difference method to obtain the acellular matrix, and the specific process of the perfusion-pressure difference method is recorded in the patent text with the patent number CN106075583B.

[0024] In step S1, after the acellular matrix that has been defatted, washed out, and antigen-blocked is physically crushed into acellular matrix particles, 0.4% pepsin aqueous solution is added for standby.

[0025] In step S1, a calcium salt aqueous solution and a phosphate aqueous solution are respectively prepared for standby, where the calcium salt is selected from one or more of calcium nitrate, calcium acetate, calcium chloride, calcium bicarbonate, or calcium citrate; the phosphate is selected from one or more of diammonium hydrogen phosphate, dipotassium hydrogen phosphate, or disodium hydrogen phosphate.

[0026] Specifically, the calcium salt is calcium acetate or calcium chloride. At this time, the hydroxyapatite in-situ generated with the phosphate has a rod-like or columnar structure, which is more conducive to the stability of the osteogenic space and further ensures the stable strength of the artificial aggregate.

[0027] The phosphate is diammonium hydrogen phosphate.

[0028] In some embodiments, in the first emulsion, the mass ratio of the calcium salt in the aqueous calcium salt solution, the phosphate in the aqueous phosphate solution, and the acellular matrix particles of the acellular matrix feed liquid is (7.89 - 16.81):(3.44 - 7.51):0.5; in the third emulsion, the mass ratio of the calcium salt in the aqueous calcium salt solution, the phosphate in the aqueous phosphate solution, and the acellular matrix particles of the acellular matrix feed liquid is (7.89 - 16.81):(3.44 - 7.51):0.5.

[0029] In some embodiments, in the second emulsion, the mass ratio of the calcium salt in the aqueous calcium salt solution, the phosphate in the aqueous phosphate solution, and the acellular matrix particles of the acellular matrix feed liquid is (1.62 - 2.63):(0.73 - 1.18):0.5.

[0030] In some embodiments, the first freeze-drying, the second freeze-drying, the third freeze-drying, and the fourth freeze-drying are all stepwise freeze-drying, and the stepwise freeze-drying includes six stages.

[0031] In some embodiments, the specific process conditions of the stepwise freeze-drying are as follows: The first stage: -80°C to -40°C, keep warm for 3 - 10 h; The second stage: -40°C to -20°C, keep warm for 10 - 18 h; The third stage: -20°C to -10°C, keep warm for 12 - 18 h; The fourth stage: -10 to 0°C, keep warm for 10 - 15 h; The fifth stage: 0 to 20°C; keep warm for 2 - 5 h; The sixth stage: 20 to 30°C, keep warm for 2 - 5 h.

[0032] Based on the research of Cristian Parisi et al., the inventors tried to pour the first emulsion, the second emulsion and the third emulsion into a mold in sequence and then freeze-dry them at one time. As described in the background art, the different-gradient solutions in the liquid state diffuse under the action of their own gravity before and during freeze-drying, causing a certain degree of mixing and reducing the gradient change. Therefore, the inventors initially tried stepwise freeze-drying, but the result was not ideal. Each scaffold layer could not be well combined together, that is, each scaffold layer was easily peeled off or separated. Finally, the inventors adjusted the mass ratio of calcium salts in the calcium salt aqueous solution, phosphates in the phosphate aqueous solution, and decellularized matrix particles in the decellularized matrix stock solution, that is, in the first emulsion, the mass ratio of calcium salts in the calcium salt aqueous solution, phosphates in the phosphate aqueous solution, and decellularized matrix particles in the decellularized matrix stock solution was (7.89-16.81):(3.44-7.51):0.5; in the second emulsion, the mass ratio of calcium salts in the calcium salt aqueous solution, phosphates in the phosphate aqueous solution, and decellularized matrix particles in the decellularized matrix stock solution was (1.62-2.63):(0.73-1.18):0.5; in the third emulsion, the mass ratio of calcium salts in the calcium salt aqueous solution, phosphates in the phosphate aqueous solution, and decellularized matrix particles in the decellularized matrix stock solution was (7.89-16.81):(3.44-7.51):0.5; combined with specific staged freeze-drying, which affected the formation speed of local crystal nuclei being extruded to form specific ice crystal morphologies, balanced the integrity of the product fiber structure, and at the same time combined with the hygroscopicity of hydroxyapatite formed after heating, overcoming the problem of easy separation of the scaffold layers. This not only improved the interfacial bonding performance of the three-layer scaffold and prevented material delamination during the repair process, but more importantly, by increasing the peel strength between layers, the handling feel was improved, making the material easier to be used for implantation.

[0033] Specifically, in step S6, the cross-linking agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) or glutaraldehyde, and the concentration of the cross-linking agent aqueous solution is 0.1-1 mol / L.

[0034] The second aspect of the present invention provides an application of a non-metallic bio-artificial aggregate that is not easily collapsible in the preparation of dental repair materials, jaw repair materials, spinal repair materials, joint repair materials, and pelvic repair materials.

[0035] Beneficial effects: (1) The non-metallic bio-artificial aggregate obtained in the present invention utilizes self-assembly technology. Taking collagen molecules in ECM as templates, a calcium ion-containing solution and a phosphate ion-containing solution are added, and hydroxyapatite is in-situ mineralized and grown on the molecular templates. After the hydroxyapatite formed by mineralization nucleation binds to ECM, it has a structure similar to that of natural bone tissue, which is conducive to the recognition of human cells and macromolecules, and improves the bioactivity of the material after implantation in the body. (2) By simulating the structure and composition of natural cortical bone and cancellous bone, the bionic design of the structure, composition and function of the scaffold material is realized. This structural design enables the scaffold material to better absorb stress after implantation in the body, improving the biomechanical properties of the material. At the same time, it is also beneficial to cell penetration and nutrient delivery, significantly improving the growth and proliferation rate of osteoblasts, as well as accelerating the level of vascularization and the conversion rate of the scaffold material into living bone. (3) Compared with other synthetic calcium phosphate artificial bones, allogeneic bones or xenogeneic bones (especially decalcified bone matrix DBM), the non-metallic bio-artificial aggregate obtained in the present invention can better adsorb endogenous growth factors and improve osteogenic activity. (4) By controlling the ratio of the average porosity of the first scaffold layer, the second scaffold layer and the third scaffold layer to be (0.9-1.7):(1.4-3):(0.9-1.7); the average porosity of the first scaffold layer is 30-50%, the average porosity of the second scaffold layer is 70-90%, and the average porosity of the third scaffold layer is 30-50%, and appropriately increasing the weight of the cortical bone, that is, the ratio of the total mass of the first scaffold layer and the third scaffold layer to the mass of the second scaffold layer is (3.4-4.8):1, not only has good biological stability, but also enables the artificial aggregate to quickly repair the original defect after implantation, helps to enhance the newly formed callus and increase its strength, speeds up the repair speed in the early stage of bone repair, and the strength of the newly formed bone can be continuously maintained stably, and the bone mass is not easily lost, especially meeting the clinical demands in the early stage of bone repair.(5) Control the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the acellular matrix particles in the acellular matrix feed liquid in the first emulsion to be (7.89 - 16.81):(3.44 - 7.51):0.5; in the second emulsion, the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the acellular matrix particles in the acellular matrix feed liquid is (1.62 - 2.63):(0.73 - 1.18):0.5; in the third emulsion, the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the acellular matrix particles in the acellular matrix feed liquid is (7.89 - 16.81):(3.44 - 7.51):0.5; combined with specific staged freeze-drying (that is, freeze-dry three times separately and then cross-link together and then perform the fourth freeze-drying, and each freeze-drying includes six stages, and each stage is set with a specific temperature range), not only can improve the interfacial bonding performance of the three-layer scaffold and avoid the problem of material delamination during the repair process, more importantly, by significantly increasing the peel strength between layers, thereby improving the operating feel, making the material easier to be used for implantation, and can be implanted at any angle during operation to ensure that the product does not delaminate. Detailed implementation mode

[0036] Example 1

[0037] This example provides a non-metallic bio-artificial aggregate that is not easily collapsed. The non-metallic bio-artificial aggregate has a gradient three-layer scaffold structure, and the gradient three-layer scaffold structure includes a first scaffold layer, a second scaffold layer, and a third scaffold layer; the ratio of the average layer porosity of the first scaffold layer, the second scaffold layer, and the third scaffold layer is 1:2.0:0.9.

[0038] The average layer porosity of the first scaffold layer is 41.7%, the average layer porosity of the second scaffold layer is 82.3%, and the average layer porosity of the third scaffold layer is 38.5%. The average layer porosity is measured by the mercury intrusion method.

[0039] The ratio of the total mass of the first scaffold layer and the third scaffold layer to the mass of the second scaffold layer is 3.6:1.

[0040] The mass ratio of the first scaffold layer and the third scaffold layer is 1:1. (In this embodiment, the volumes of the first scaffold layer and the third scaffold layer are basically equal).

[0041] The preparation method of the non-metallic bio-artificial aggregate is as follows: S1. After the acellular matrix that has been defatted, washed out, and antigen-blocked is crushed into acellular matrix particles, 20 mL of 0.4% pepsin aqueous solution (purchased from Qingdao Jieshikang) is added to prepare an acellular matrix feed liquid for standby; an aqueous solution of calcium salt (calcium chloride) and an aqueous solution of phosphate (ammonium hydrogen phosphate) are respectively prepared for standby.

[0042] In step S1, before defatting, washing out, and antigen blocking, the acellular matrix is derived from the soft tissue of porcine small intestine. The soft tissue of porcine small intestine is defatted, washed out, and antigen blocked by the perfusion-pressure difference method to obtain the acellular matrix. The specific process of the perfusion-pressure difference method refers to the patent with the patent number CN106075583B.

[0043] S2. Adjust the mass ratio of calcium salt in the calcium salt aqueous solution, phosphate in the phosphate aqueous solution, and acellular matrix particles in the acellular matrix feed liquid. After adding the calcium salt aqueous solution to the acellular matrix feed liquid, adjust it to pH = 9.8, then add the phosphate aqueous solution to prepare the first emulsion; in the first emulsion, the mass ratio of calcium salt in the calcium salt aqueous solution, phosphate in the phosphate aqueous solution, and acellular matrix particles in the acellular matrix feed liquid is 7.89:3.55:0.5.

[0044] S3. Repeat step S1, adjust the mass ratio of calcium salt in the calcium salt aqueous solution, phosphate in the phosphate aqueous solution, and acellular matrix particles in the acellular matrix feed liquid to prepare the second emulsion; in the second emulsion, the mass ratio of calcium salt in the calcium salt aqueous solution, phosphate in the phosphate aqueous solution, and acellular matrix particles in the acellular matrix feed liquid is 2.05:0.92:0.5.

[0045] S4. Repeat step S1, adjust the mass ratio of calcium salt in the calcium salt aqueous solution, phosphate in the phosphate aqueous solution, and acellular matrix particles in the acellular matrix feed liquid to prepare the third emulsion; in the third emulsion, the mass ratio of calcium salt in the calcium salt aqueous solution, phosphate in the phosphate aqueous solution, and acellular matrix particles in the acellular matrix feed liquid is 7.95:3.55:0.5.

[0046] S5. Pour the first emulsion into a mold and perform the first freeze-drying; continue to add the second emulsion and perform the second freeze-drying; continue to add the third emulsion and perform the third freeze-drying to obtain a precursor. S6. After the precursor is immersed in an aqueous solution of 0.1 mol / L cross-linking agent (specifically glutaraldehyde) for cross-linking for 12 h, it is rinsed with deionized water and subjected to the fourth freeze-drying; at this time, from the lower part to the upper part of the mold, there are a first support layer, a second support layer, and a third support layer respectively.

[0047] The first freeze-drying, the second freeze-drying, the third freeze-drying, and the fourth freeze-drying are all staged freeze-dryings. The staged freeze-drying includes six stages. The specific process conditions of the staged freeze-drying are: The first stage: -50 °C, keep warm for 5 h; The second stage: -20 °C, keep warm for 10 h; The third stage: -10 °C, keep warm for 12 h; The fourth stage: -5 °C, keep warm for 10 h; The fifth stage: 10 °C; heat preservation for 5 h; The sixth stage: 25 °C, heat preservation for 3 h.

[0048] Example 2

[0049] This example provides a non-metallic bio-artificial aggregate that is not easily collapsed. The non-metallic bio-artificial aggregate has a gradient three-layer scaffold structure, and the gradient three-layer scaffold structure includes a first scaffold layer, a second scaffold layer, and a third scaffold layer; the ratio of the average porosity of the first scaffold layer, the second scaffold layer, and the third scaffold layer is 1:2.3:1.1.

[0050] The average porosity of the first scaffold layer is 31.0%, the average porosity of the second scaffold layer is 71.4%, and the average porosity of the third scaffold layer is 34.1%. The average porosity is measured by the mercury intrusion method.

[0051] The ratio of the total mass of the first scaffold layer and the third scaffold layer to the mass of the second scaffold layer is 3.4:1.

[0052] The mass ratio of the first scaffold layer and the third scaffold layer is 1:1. (In this example, the volumes of the first scaffold layer and the third scaffold layer are basically equal).

[0053] The preparation method of the non-metallic bio-artificial aggregate is as follows: S1. After the decellularized matrix that has been defatted, washed out, and antigen-blocked is crushed into decellularized matrix particles, 20 mL of 0.4% pepsin aqueous solution (purchased from Qingdao Jieshikang) is added to prepare a decellularized matrix liquid, and it is reserved; an aqueous solution of calcium salt (calcium chloride) and an aqueous solution of phosphate (ammonium hydrogen phosphate) are respectively prepared and reserved.

[0054] In step S1, before defatting, washing out, and antigen-blocking, the decellularized matrix is derived from the soft tissue of pig small intestine. The soft tissue of pig small intestine is defatted, washed out, and antigen-blocked by the perfusion-pressure difference method to obtain the decellularized matrix. The specific process of the perfusion-pressure difference method refers to the patent with the patent number CN106075583B.

[0055] S2. Adjust the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the decellularized matrix particles of the decellularized matrix liquid. After adding the calcium salt aqueous solution to the decellularized matrix liquid, adjust it to pH = 9.8, and then add the phosphate aqueous solution to prepare the first emulsion; in the first emulsion, the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the decellularized matrix particles of the decellularized matrix liquid is 8.82:6.33:0.5.

[0056] S3. Repeat step S1, adjust the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the acellular matrix particles in the acellular matrix feedstock solution, and configure to obtain a second emulsion; in the second emulsion, the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the acellular matrix particles in the acellular matrix feedstock solution is 1.62:0.73:0.5.

[0057] S4. Repeat step S1, adjust the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the acellular matrix particles in the acellular matrix feedstock solution, and configure to obtain a third emulsion; in the third emulsion, the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the acellular matrix particles in the acellular matrix feedstock solution is 8.82:6.33:0.5.

[0058] S5. Pour the first emulsion into a mold and perform the first freeze-drying; continue to add the second emulsion and perform the second freeze-drying; continue to add the third emulsion and perform the third freeze-drying to obtain a precursor; S6. After the precursor is immersed in an aqueous solution of 0.1 mol / L cross-linking agent (specifically glutaraldehyde) for cross-linking for 15 h, rinse with deionized water and perform the fourth freeze-drying; at this time, from the lower part to the upper part of the mold, there are a first scaffold layer, a second scaffold layer, and a third scaffold layer respectively.

[0059] The first freeze-drying, the second freeze-drying, the third freeze-drying, and the fourth freeze-drying are all stagewise freeze-drying. The stagewise freeze-drying includes six stages. The specific process conditions of the stagewise freeze-drying are: The first stage: -45°C, keep warm for 10 h; The second stage: -25°C, keep warm for 15 h; The third stage: -15°C, keep warm for 12 h; The fourth stage: -5°C, keep warm for 10 h; The fifth stage: 10°C; keep warm for 3 h; The sixth stage: 20°C, keep warm for 5 h.

[0060] Example 3

[0061] This example provides a non-metallic bio-artificial aggregate that is not easily collapsible. The non-metallic bio-artificial aggregate has a gradient three-layer scaffold structure. The gradient three-layer scaffold structure includes a first scaffold layer, a second scaffold layer, and a third scaffold layer; the ratio of the average layer porosity of the first scaffold layer, the second scaffold layer, and the third scaffold layer is 1:1.8:1.

[0062] The average layer porosity of the first scaffold layer is 47.8%, the average layer porosity of the second scaffold layer is 87.5%, and the average layer porosity of the third scaffold layer is 49.2%. The average layer porosity is measured by the mercury intrusion method.

[0063] The ratio of the total mass of the first support layer and the third support layer to the mass of the second support layer is 3.4:1.

[0064] The mass ratio of the first support layer to the third support layer is 1:1. (In this embodiment, the volumes of the first support layer and the third support layer are substantially equal).

[0065] The preparation method of the non-metallic biological artificial aggregate is as follows: S1. After the acellular matrix that has been defatted, washed out, and antigen-blocked is crushed into acellular matrix particles, 20 mL of 0.4% pepsin aqueous solution (purchased from Qingdao Jieshikang) is added to prepare an acellular matrix stock solution for standby; calcium salt (calcium chloride) aqueous solution and phosphate (ammonium hydrogen phosphate) aqueous solution are respectively prepared for standby.

[0066] In step S1, before defatting, washing out, and antigen-blocking, the acellular matrix is derived from the soft tissue of porcine small intestine. The soft tissue of porcine small intestine is defatted, washed out, and antigen-blocked by the perfusion-pressure difference method to obtain the acellular matrix. The specific process of the perfusion-pressure difference method refers to the patent with the patent number CN106075583B.

[0067] S2. Adjust the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the acellular matrix particles of the acellular matrix stock solution. After adding the calcium salt aqueous solution to the acellular matrix stock solution, adjust it to pH = 9.8, then add the phosphate aqueous solution to prepare the first emulsion; in the first emulsion, the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the acellular matrix particles of the acellular matrix stock solution is 16.81:7.44:0.5.

[0068] S3. Repeat step S1, adjust the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the acellular matrix particles of the acellular matrix stock solution to prepare the second emulsion; in the second emulsion, the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the acellular matrix particles of the acellular matrix stock solution is 2.63:1.18:0.5.

[0069] S4. Repeat step S1, adjust the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the acellular matrix particles of the acellular matrix stock solution to prepare the third emulsion; in the third emulsion, the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the acellular matrix particles of the acellular matrix stock solution is 16.34:7.81:0.5.

[0070] S5. Pour the first emulsion into a mold and perform the first freeze-drying; continue to add the second emulsion and perform the second freeze-drying; continue to add the third emulsion and perform the third freeze-drying to obtain a precursor; After the precursor is immersed in an aqueous solution of 0.1 mol / L crosslinking agent (specifically glutaraldehyde) for 24 h of crosslinking, it is rinsed with deionized water and subjected to the fourth freeze-drying; at this time, from the lower part to the upper part of the mold, there are a first support layer, a second support layer, and a third support layer respectively.

[0071] The first freeze-drying, the second freeze-drying, the third freeze-drying, and the fourth freeze-drying are all stage freeze-dryings. The stage freeze-drying includes six stages, and the specific process conditions of the stage freeze-drying are as follows: The first stage: -60 °C, keep warm for 4 h; The second stage: -30 °C, keep warm for 10 h; The third stage: -15 °C, keep warm for 12 h; The fourth stage: -5 °C, keep warm for 10 h; The fifth stage: 10 °C, keep warm for 2 h; The sixth stage: 25 °C, keep warm for 5 h.

[0072] Comparative Example 1 This example provides a non-metallic bio-artificial aggregate that is not easily collapsed. The non-metallic bio-artificial aggregate has a gradient three-layer support structure. Different from Example 1, the gradient three-layer support structure includes a first support layer, a second support layer, and a third support layer; the ratio of the average layer porosity of the first support layer, the second support layer, and the third support layer is 1:2.0:1.5.

[0073] The average layer porosity of the first support layer is 40.5%, the average layer porosity of the second support layer is 82.6%, and the average layer porosity of the third support layer is 62.3%.

[0074] The preparation method of the non-metallic bio-artificial aggregate is different from that of Example 1 in that: in the third emulsion, the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the decellularized matrix particles in the decellularized matrix liquid is 3.11:1.4:0.5.

[0075] Comparative Example 2 This example provides a non-metallic bio-artificial aggregate that is not easily collapsed. The non-metallic bio-artificial aggregate has a gradient three-layer support structure. Different from Example 1, the gradient three-layer support structure includes a first support layer, a second support layer, and a third support layer; the ratio of the average layer porosity of the first support layer, the second support layer, and the third support layer is 1:1.4:1.1.

[0076] The average layer porosity of the first support layer is 61.5%, the average layer porosity of the second support layer is 87.6%, and the average layer porosity of the third support layer is 67.2%.

[0077] The preparation method of the non-metallic bio-artificial aggregate is different from that in Example 1 in that: in the first emulsion, the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the decellularized matrix particles of the decellularized matrix liquid is 3.01:3.58:0.5. In the third emulsion, the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the decellularized matrix particles of the decellularized matrix liquid is 3.01:3.58:0.5. In the second emulsion, the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the decellularized matrix particles of the decellularized matrix liquid is (1.73:0.75:0.5).

[0078] Comparative Example 3 This example provides a non-metallic bio-artificial aggregate that is not easily collapsible. Different from Example 1, the preparation method of the non-metallic bio-artificial aggregate is prepared by one-step freeze-drying method. Specifically, step S5 is changed to pour the first emulsion into a mold, then continue to add the second emulsion, and then continue to add the third emulsion, and freeze-dry at -30°C for 24 h to obtain a precursor.

[0079] The non-metallic bio-artificial aggregate obtained in this example was characterized by scanning electron microscopy, and no obvious porosity gradient change was observed in the electron micrograph.

[0080] Comparative Example 4 This example provides a non-metallic bio-artificial aggregate that is not easily collapsible. Different from Example 1, the gradient three-layer scaffold structure includes a first scaffold layer, a second scaffold layer, and a third scaffold layer; the ratio of the average porosity of the first scaffold layer, the second scaffold layer, and the third scaffold layer is 1:1.9:1.1.

[0081] The average porosity of the first scaffold layer is 48.2%, the average porosity of the second scaffold layer is 92.3%, and the average porosity of the third scaffold layer is 51.4%.

[0082] The preparation method of the non-metallic bio-artificial aggregate is different from that in Example 1 in that: step S5 is to pour the first emulsion into a mold and freeze-dry at -30°C for 10 h; then continue to add the second emulsion and freeze-dry at -30°C for 10 h; then continue to add the third emulsion and freeze-dry at -30°C for 10 h to obtain a precursor.

[0083] Comparative Example 5 This example provides a non-metallic bio-artificial aggregate that is not easily collapsible. Different from Example 1, anhydrous ethanol is used instead of aqueous solution to dissolve calcium salt and phosphate. The specific differences in the preparation process are as follows: S1. In step S1, prepare an aqueous solution of calcium salt (calcium chloride) and an anhydrous ethanol solution of phosphate (ammonium hydrogen phosphate) separately for later use.

[0084] S2. Adjust the mass ratios of the calcium salt in the calcium salt absolute ethanol solution, the phosphate in the phosphate absolute ethanol solution, and the decellularized matrix particles in the decellularized matrix stock solution. After adding the calcium salt absolute ethanol solution to the decellularized matrix stock solution, adjust the pH to 9.8, and then add the phosphate absolute ethanol solution to prepare the first emulsion. In the first emulsion, the mass ratio of the calcium salt in the calcium salt absolute ethanol solution, the phosphate in the phosphate absolute ethanol solution, and the decellularized matrix particles in the decellularized matrix stock solution is 7.89:3.55:0.5.

[0085] S3. Repeat step S1, adjust the mass ratios of the calcium salt in the calcium salt absolute ethanol solution, the phosphate in the phosphate absolute ethanol solution, and the decellularized matrix particles in the decellularized matrix stock solution, and prepare the second emulsion. In the second emulsion, the mass ratio of the calcium salt in the calcium salt absolute ethanol solution, the phosphate in the phosphate absolute ethanol solution, and the decellularized matrix particles in the decellularized matrix stock solution is 2.05:0.92:0.5.

[0086] S4. Repeat step S1, adjust the mass ratios of the calcium salt in the calcium salt absolute ethanol solution, the phosphate in the phosphate absolute ethanol solution, and the decellularized matrix particles in the decellularized matrix stock solution, and prepare the third emulsion. In the third emulsion, the mass ratio of the calcium salt in the calcium salt absolute ethanol solution, the phosphate in the phosphate absolute ethanol solution, and the decellularized matrix particles in the decellularized matrix stock solution is 7.95:3.55:0.5.

[0087] S5. Pour the first emulsion into a mold and perform the first freeze-drying. Then continue to add the second emulsion and perform the second freeze-drying. Next, continue to add the third emulsion and perform the third freeze-drying to obtain the precursor. S6. Immerse the precursor in an aqueous solution of 0.1 mol / L cross-linking agent (specifically glutaraldehyde) for cross-linking for 12 h, then rinse with deionized water and perform the fourth freeze-drying. At this time, from the lower part to the upper part of the mold, there are the first support layer, the second support layer, and the third support layer respectively.

[0088] The first freeze-drying, the second freeze-drying, the third freeze-drying, and the fourth freeze-drying are all staged freeze-dryings. The staged freeze-drying includes six stages, and the specific process conditions of the staged freeze-drying are as follows: The first stage: -50 °C, keep warm for 5 h; The second stage: -20 °C, keep warm for 10 h; The third stage: -10 °C, keep warm for 12 h; The fourth stage: -5 °C, keep warm for 10 h; The fifth stage: 10 °C, keep warm for 5 h; The sixth stage: 25 °C, keep warm for 3 h.

[0089] The obtained non-metallic bio-artificial aggregate has a gradient three-layer scaffold structure, which includes a first scaffold layer, a second scaffold layer and a third scaffold layer; the ratio of the average layer porosity of the first scaffold layer, the second scaffold layer and the third scaffold layer is 1:1.5:0.7.

[0090] The average layer porosity of the first scaffold layer is 38.6%, the average layer porosity of the second scaffold layer is 71.3%, and the average layer porosity of the third scaffold layer is 27.5%.

[0091] It can be seen that compared with the preparation method of Example 1, in Comparative Example 5, only the calcium salt and phosphate aqueous solution was changed to an absolute ethanol solution of calcium salt and phosphate. The average layer porosity of each layer in Comparative Example 5 differed greatly from that in Example 1, which also directly led to a huge difference in the ratio of the average layer porosity of the first scaffold layer, the second scaffold layer and the third scaffold layer. This may be because the polarity of ethanol is much weaker than that of water. Therefore, it is more difficult to form stable hydrogen bonds during freeze-drying, which affects the freeze-drying behavior of the aggregate treated with Ca-P, resulting in the pores of the finally obtained aggregate being irregular and denser (the denseness is manifested as a significant decrease in the average layer porosity of Comparative Example 5 compared with Example 1; the pore irregularity is manifested as a significant increase in the difference in the average layer porosity between the first scaffold layer and the third scaffold layer in Example 5 compared with Example 1), further affecting the performance of the product.

[0092] Result test 1. Make a beagle alveolar bone with gingival defect model (resected part of the mandibular alveolar bone). Fill the non-metallic bio-artificial aggregates obtained in Example 1 and Comparative Examples 1-3 and Comparative Example 5 into the bone defects of each same model. Perform CBCT tests on the mandibular alveolar bone of the beagle at 3 months and 6 months after surgery to record the alveolar bone height (measure three times and take the average value). At the same time, use a DPX-L dual-energy X-ray bone densitometer to measure the bone density of the mandibular part of the alveolar bone at the defect site to be built before building the model and the newly formed alveolar bone at 6 months after surgery and compare them before and after; and perform nuclear magnetic resonance tests at 1 month, 3 months and 6 months after surgery respectively. Judge whether there is collapse by observing whether the surface of the repaired area is smooth. The results are shown in Table 1: Table 1

[0093] As can be seen from Table 1, by observing the nuclear magnetic resonance spectrum, the surface of the repair site in Example 1 was smooth at 1 month, 3 months, and 6 months after surgery. It can be speculated that it is not easy to collapse and the osteogenic space is stable; the height of the newly formed alveolar bone in Example 1 reached 3.78 mm at 3 months after surgery, which was significantly greater than that of Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 5. This shows that the non-metallic biological artificial aggregate obtained in Example 1 can quickly repair the original defect after implantation, help to enhance the newly formed callus and increase its strength, accelerate the repair speed in the initial stage of bone repair, and the strength of the newly formed bone can be continuously maintained stably, especially meeting the clinical demands in the early stage of bone repair; the alveolar bone density in Example 1 was close to that before modeling, indicating that the material obtained in Example 1 had a good repair effect after implantation and the repair area was not prone to bone loss.

[0094] 2. Peel strength test: According to YY / T 0729.2-2009 Test methods for the bonding properties of tissue adhesives - Part 2: T-peel tensile load-bearing strength test, after the non-metallic biological artificial aggregates obtained in Example 1 and Comparative Examples 1-5 were swollen in water for 1 h, the peel strength test was carried out. Each test was carried out at the interface of the first support layer, the second support layer, and the third support layer respectively, and parallel experiments were carried out 3 times at each place, and the average value was finally taken. The results are shown in Table 2: Table 2

[0095] 3. According to YY∕T 1680-2020, the in vivo osteogenic induction of the non-metallic biological artificial aggregates obtained in Example 1 and Comparative Example 2 was tested. The results are shown in Table 3. It can be seen that the non-metallic biological artificial aggregate obtained in Example 1 has obvious osteogenic induction potential, and is significantly greater than that of the non-metallic biological artificial aggregate obtained in Comparative Example 2.

[0096] Table 3

Claims

1. A non-metallic bio-artificial aggregate that is not prone to collapse, characterized in that: The non-metallic bio-artificial aggregate has a gradient three-layer scaffold structure, which includes a first scaffold layer, a second scaffold layer and a third scaffold layer; the ratio of the average porosity of the first scaffold layer, the second scaffold layer and the third scaffold layer is (0.9-1.7): (1.4-3): (0.9-1.7).

2. The non-collapse-resistant non-metallic bio-artificial aggregate according to claim 1, characterized in that: The average porosity of the first support layer is 30-50%, the average porosity of the second support layer is 70-90%, and the average porosity of the third support layer is 30-50%.

3. The non-collapse-resistant non-metallic bio-artificial aggregate according to claim 1 or 2, characterized in that: The ratio of the total mass of the first support layer and the third support layer to the mass of the second support layer is (3.4-4.8):

1.

4. The non-collapse-resistant non-metallic bio-artificial aggregate according to claim 2, characterized in that: The ratio of the total mass of the first support layer and the third support layer to the mass of the second support layer is (3.4-3.6):

1.

5. The non-collapse-resistant non-metallic bio-artificial aggregate according to claim 1, characterized in that: The preparation method of the non-metallic biological artificial aggregate is: S1. After the delipidated, washed and antigen-blocked decellularized matrix is ​​crushed into decellularized matrix particles, the decellularized matrix liquid is prepared for standby use; a calcium salt solution and a phosphate solution are prepared for standby use; S2. adjusting the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the decellularized matrix particles in the decellularized matrix feed solution, adding the calcium salt aqueous solution to the decellularized matrix feed solution, adjusting the pH to 9-11, adding the phosphate aqueous solution, and configuring to obtain a first emulsion; S3. Repeat step S1 to adjust the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the decellularized matrix particles in the decellularized matrix feed solution to obtain a second emulsion; S4. Repeat step S1 to adjust the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the decellularized matrix particles in the decellularized matrix feed solution to obtain a third emulsion; S5. Pour the first emulsion into a mold and perform the first freeze-drying; continue to add the second emulsion and perform the second freeze-drying; continue to add the third emulsion and perform the third freeze-drying to obtain a precursor; S6. After the precursor is immersed in the cross-linking agent aqueous solution for cross-linking for 12-24 hours, it is rinsed with deionized water and freeze-dried for the fourth time; at this time, from the bottom of the mold to the top of the mold, there are the first scaffold layer, the second scaffold layer and the third scaffold layer respectively.

6. The non-collapse-resistant non-metallic bio-artificial aggregate according to claim 5, characterized in that: In the first emulsion, the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the decellularized matrix particles in the decellularized matrix solution is (7.89-16.81):(3.44-7.51):0.5; in the third emulsion, the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the decellularized matrix particles in the decellularized matrix solution is (7.89-16.81):(3.44-7.51):0.

5.

7. The non-collapse-resistant non-metallic bio-artificial aggregate according to claim 5, characterized in that: In the second emulsion, the mass ratio of the calcium salt in the calcium salt aqueous solution, the phosphate in the phosphate aqueous solution, and the decellularized matrix particles in the decellularized matrix liquid is (1.62-2.63):(0.73-1.18):0.

5.

8. The non-collapse-resistant non-metallic bio-artificial aggregate according to any one of claims 5 to 7, characterized in that: The first freeze-drying, the second freeze-drying, the third freeze-drying and the fourth freeze-drying are all staged freeze-drying, and the staged freeze-drying includes six stages.

9. The non-collapse-resistant non-metallic bio-artificial aggregate according to claim 8, characterized in that: The specific process conditions of the staged freeze-drying are: The first stage: -80℃--40℃, keep warm for 3-10h; The second stage: -40℃--20℃, keep warm for 10-18h; The third stage: -20℃--10℃, keep warm for 12-18h; The fourth stage: -10-0℃, keep warm for 10-15h; Stage 5: 0-20℃; keep warm for 2-5h; Stage 6: 20-30℃, keep warm for 2-5h.

10. Use of the non-collapse-resistant non-metallic bio-artificial aggregate according to claim 1 in preparing tooth repair materials, jaw repair materials, spine repair materials, joint repair materials, and pelvic repair materials.

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