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 invention relates to the technical field of biomedical materials, and in particular to a non-metallic bio-artificial aggregate that is not prone to collapse and its application. Background Art

[0002] Natural bone is divided into cancellous bone and compact bone. Compact bone wraps cancellous bone. Compact bone accounts for about 75% of the weight of human bones and has a porosity of 5-30%; cancellous bone accounts for about 25% of the weight of human bones and has a porosity of 30-90%. In order to meet the requirements of bionics, the internal structure of artificial bone should have a non-uniformly distributed gradient pore structure similar to natural bone. Most traditional artificial bone products are designed with a uniform structure, ignoring the fact that the real bone tissue structure actually contains both compact bone and cancellous bone. This results in the vascularization level, osteogenesis and biomechanical properties of artificial bone scaffold materials that cannot meet clinical bone repair. However, the design of gradient artificial bones is very difficult.

[0003] Patent No. CN108261568B discloses a composite gradient bone repair material and its preparation method, which obtains a gradient scaffold bone repair material with good uniformity and stability through 3D printing. However, this method uses high-temperature calcination to remove the PVA component introduced in the scaffold 3D printing molding process, which reduces the biological activity and osteogenic activity of the material, and the scaffold material only achieves structural biomimetic, without component biomimetic.

[0004] Cristian Parisi et al. added different amounts of calcium hydroxide and phosphoric acid to type I collagen, obtained four gradient solutions with different weight ratios, added the four solutions layer by layer to the mold, and after freeze-drying in one step, carried out thermal cross-linking and NHS / EDC cross-linking in sequence, and freeze-dried after cross-linking. This method achieves the bionics of the structure and components of gradient bone. However, for this method of first obtaining different gradient solutions and then freeze-drying once, the liquid solutions with different gradients will diffuse under the action of their own gravity before and during freeze-drying, causing the four gradient solutions to be mixed to a certain extent, thereby reducing the change of this gradient. In order to reduce the mixing of the aforementioned different gradient solutions under the action of gravity, it is required that the material with the highest density must be in the bottom layer, and the overall material maintains a trend of decreasing density from bottom to top. In addition, it has been found in practice that although the gradient solutions in this study have a certain gradient structure, their porosity is very large, but in terms of porosity, it has a large difference in porosity distribution from natural bone, and cannot achieve true bionics. Summary of the invention

[0005] In order to overcome the above problems, the first aspect of the present invention provides a non-metallic bioartificial aggregate that is not easy to collapse. The non-metallic bioartificial 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 support layer, the second support layer and the third support layer is 1:(1.4-3):(0.9-1.4).

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

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

[0009] Specifically, the ratio of the average porosity of the first support layer, the second support layer and the third support layer is 1:(1.4-2.3):(0.9-1.2).

[0010] Specifically, the ratio of the average porosity of the first support layer, the second support layer and the third support layer is 1:(1.4-2.3):(0.9-1.1).

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

[0012] In some embodiments, the first scaffold layer has an average layer porosity of 30-50%, the second scaffold layer has an average layer porosity of 30-50%, and the third scaffold layer has an average layer porosity of 70-90%.

[0013] In some embodiments, 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.

[0014] Specifically, 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 (3.4-4.5):1.

[0015] In some embodiments, the volume ratio of the first scaffold layer to the third scaffold layer is 1:1.

[0016] In order to obtain a similar structure to natural bone, which is sparse inside and dense outside, the inventors want to design an artificial aggregate with compact bone with a porosity of 5-30% and cancellous bone with a porosity of 30-90%. After a lot of thinking and demonstration, the inventors found that by controlling the porosity, mass and volume of each scaffold layer of the non-metallic bioartificial aggregate, specifically 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); the average porosity of the first scaffold layer is 30-50%, the average porosity of the second scaffold layer is 30-50%, and the average porosity of the third scaffold layer is The average porosity of the frame layer is 70-90%; 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, and the volume ratio of the first scaffold layer and the third scaffold layer is 1:1. Not only can the desired bionic structure be obtained, but the obtained material is not easy to collapse in the direction of bone defect, thus having a good bone repair growth effect. This may be because when the artificial aggregate is in the above structure, it has a certain regulatory effect on 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 guiding the orderly arrangement of trabeculae. It is known that compact bone accounts for about 75% of the weight of human bones, and cancellous bone accounts for about 25% of the weight of human bones. It is speculated that when designing artificial bones, the weight ratio of compact bone to cancellous bone is 75:25, that is, 3:1, which is more in line with the biological bionic structure. However, in the present technical solution, the inventors found that the ratio of the average porosity of the first support layer, the second support layer and the third support layer is (0.9-1.7): (1.4-3): (0.9-1.7); when the average porosity of the first support layer is 30-50%, the average porosity of the second support layer is 30-50%, and the average porosity of the third support layer is 70-90%, the weight of the compact bone is appropriately increased, that is, the total mass of the first support layer and the third support layer and the mass of the second support layer are (3.4-4.8): 1, and the volume ratio of the first support layer and the third support layer is controlled to be 1:1, which not only has good biological properties, but also has good biocompatibility. Stability, and also enable artificial bone materials to quickly repair original defects after implantation, which helps to enhance the new callus and increase its strength. It is speculated that this may be because the increase in compact bone is conducive to the stability of the osteogenic space. The first scaffold layer and the third scaffold layer of the same material with the same volume and similar porosity are of similar thickness, acting on the trauma of the bone defect, which 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 especially meet the clinical demands of early bone repair, accelerate the repair speed of the early bone repair, and the strength of the new bone can continue to remain stable, and the bone is not easily lost.

[0017] In some embodiments, the method for preparing the non-metallic bio-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.

[0018] Specifically, in step S1, the decellularized matrix is ​​derived from soft tissues of pigs, cows and humans, including skin, dermis, blood vessels, diaphragms, tendons, ligaments, large intestine, small intestine and nervous tissues, before defatting, washing out and antigen blocking; the above-mentioned soft tissues are defatted, washed out and antigen blocked by the perfusion-pressure difference method to obtain the decellularized matrix, and the specific process of the perfusion-pressure difference method is recorded in the patent text of patent number CN106075583B.

[0019] In step S1, the decellularized matrix that has been defatted, washed out and antigen-blocked is physically crushed into decellularized matrix particles, and then a 0.4% pepsin aqueous solution is added for later use.

[0020] In step S1, a calcium salt aqueous solution and a phosphate aqueous solution are respectively prepared for standby use, wherein the calcium salt is selected from one or more of calcium nitrate, calcium acetate, calcium chloride, calcium bicarbonate or calcium citrate; and the phosphate is selected from one or more of diammonium hydrogen phosphate, dipotassium hydrogen phosphate or disodium hydrogen phosphate.

[0021] Specifically, the calcium salt is calcium acetate or calcium chloride. At this time, the hydroxyapatite subsequently generated in situ with the phosphate has a rod-like or columnar structure, which is more conducive to the stability of the bone formation space and further ensures the stable strength of the artificial aggregate.

[0022] The phosphate is diammonium hydrogen phosphate.

[0023] In some embodiments, 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.

[0024] In some embodiments, 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 solution is (1.62-2.63):(0.73-1.18):0.5.

[0025] In some embodiments, 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.

[0026] In some embodiments, the specific process conditions of 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.

[0027] Based on the research of Cristian Parisi et al., the inventor tried to pour the first emulsion, the second emulsion and the third emulsion into the mold in sequence and freeze-dry them once. The result is as described in the background technology. The solutions with different gradients in the liquid state diffuse under the action of their own gravity before and during freeze-drying, causing a certain mixing and reducing the gradient change. Therefore, the inventor initially tried step-by-step freeze-drying, but the result was not ideal. Each support layer could not be well combined, that is, each support layer was easily peeled off or separated. Finally, the inventor combined 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 with a specific staged freeze-drying, affecting the speed at which the local nucleus was squeezed and formed, and combining the hygroscopicity of the hydroxyapatite formed after heating, overcame the problem of easy separation of the support layer.

[0028] 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.

[0029] The second aspect of the present invention provides a non-metallic bio-artificial aggregate that is not easy to collapse and is used in tooth repair materials, jaw repair materials, spine repair materials, joint repair materials, and pelvic repair materials.

[0030] Beneficial effects: (1) The non-metallic bio-artificial aggregate obtained by the present invention utilizes self-assembly technology, takes the collagen molecule in ECM as a template, adds a solution containing calcium ions and a solution containing phosphate ions, and in situ mineralizes and grows hydroxyapatite on the molecular template. After the mineralized nucleated hydroxyapatite is combined with 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 biological activity of the material after implantation in the body. (2) The structure and composition of natural compact bone and cancellous bone are simulated to achieve the biomimetic structure, composition and function of the scaffold material. This structural design enables the scaffold material to absorb stress better after implantation in the body and improve the biomechanical properties of the material. At the same time, it is also conducive to cell penetration and nutrient delivery, significantly improving the growth and proliferation rate of osteoblasts, and accelerating the vascularization level and the speed of conversion of the scaffold material to living bone. (3) Compared with other artificial calcium phosphate artificial bones, allogeneic bones or xenogeneic bones (especially decalcified bone matrix DBM), the non-metallic bio-artificial aggregate obtained by the present invention can better absorb endogenous growth factors and improve osteogenic activity. (4) By controlling the average porosity ratio 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); when the average porosity of the first scaffold layer is 30-50%, the average porosity of the second scaffold layer is 30-50%, and the average porosity of the third scaffold layer is 70-90%, the weight of the compact bone is appropriately increased, that is, the total mass of the first scaffold layer and the third scaffold layer and the mass of the second scaffold layer The ratio is (3.4-4.8): 1, and the volume ratio of the first scaffold layer and the third scaffold layer is controlled to be 1:1. Not only does it have good biological stability, but it also enables the artificial aggregate to quickly repair the original defects after implantation, which helps to enhance the new bone callus and increase its strength, accelerate the repair speed in the early stage of bone repair, and the strength of the new bone can continue to remain stable, and bone loss is not easy, especially meeting the clinical demands of early bone repair. DETAILED DESCRIPTION

[0031] Example 1

[0032] This example provides a non-metallic bioartificial aggregate that is not easy to collapse. The non-metallic bioartificial aggregate has a gradient three-layer support structure. 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 porosity of the first support layer, the second support layer and the third support layer is 1:1.97:0.92.

[0033] The average porosity of the first support layer is 41.7%, the average porosity of the second support layer is 82.3%, and the average porosity of the third support layer is 38.5%. The average porosity is measured by mercury intrusion.

[0034] 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 3.6:1.

[0035] The volume ratio of the first support layer to the third support layer is 1:1.

[0036] The preparation method of non-metallic biological artificial aggregate is: S1. After the delipidated, washed and antigen-blocked decellularized matrix is ​​crushed into decellularized matrix particles, 20 mL of 0.4% pepsin aqueous solution (purchased from Qingdao Jieshikang) is added to prepare the decellularized matrix solution for use; calcium salt (calcium chloride) aqueous solution and phosphate (ammonium hydrogen phosphate) aqueous solution are separately prepared for use.

[0037] In step S1, the decellularized matrix is ​​derived from the soft tissue of the porcine small intestine before defatting, washing out and antigen blocking. The soft tissue of the porcine 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 patent No. CN106075583B.

[0038] 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 solution, add the calcium salt aqueous solution to the decellularized matrix solution, adjust the pH to 9.8, add the phosphate aqueous solution, and prepare a 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 solution is 7.89:3.55:0.5.

[0039] 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 solution 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 decellularized matrix particles in the decellularized matrix solution is 2.05:0.92:0.5.

[0040] 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 solution 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 decellularized matrix particles in the decellularized matrix solution is 7.95:3.55:0.5.

[0041] 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 a 0.1 mol / L cross-linking agent (specifically glutaraldehyde) aqueous solution for 12 hours of cross-linking, 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.

[0042] The first freeze-drying, the second freeze-drying, the third freeze-drying and the fourth freeze-drying are all staged freeze-drying, which includes six stages. The specific process conditions of staged freeze-drying are: Stage 1: -50℃, keep warm for 5h; The second stage: -20℃, keep warm for 10h; Stage 3: -10℃, keep warm for 12h; Stage 4: -5℃, keep warm for 10h; Stage 5: 10℃; keep warm for 5h; Stage 6: 25℃, keep warm for 3h.

[0043] Comparative Example 1 This example provides a non-metallic bioartificial aggregate that is not easy to collapse. The non-metallic bioartificial aggregate has a gradient three-layer support structure. The difference from Example 1 is that 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 porosity of the first support layer, the second support layer and the third support layer is 1:2.04:1.54.

[0044] The average porosity of the first scaffold layer is 40.5%, the average porosity of the second scaffold layer is 82.6%, and the average porosity of the third scaffold layer is 62.3%.

[0045] The method for preparing 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 solution is 3.11:1.4:0.5.

[0046] Comparative Example 2 This example provides a non-metallic bioartificial aggregate that is not easy to collapse. The non-metallic bioartificial aggregate has a gradient three-layer support structure. The difference from Example 1 is that 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 porosity of the first support layer, the second support layer and the third support layer is 1:1.42:1.09.

[0047] The average porosity of the first scaffold layer is 61.5%, the average porosity of the second scaffold layer is 87.6%, and the average porosity of the third scaffold layer is 67.2%.

[0048] The method for preparing 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 decellular matrix particles of the decellular matrix solution 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 decellular matrix particles of the decellular matrix solution 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 decellular matrix particles of the decellular matrix solution is (1.73:0.75:0.5.

[0049] Comparative Example 3 This example provides a non-metallic bioartificial aggregate that is not easy to collapse. The non-metallic bioartificial 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 porosity of the first support layer, the second support layer and the third support layer is 1:2.2:1.07.

[0050] The average porosity of the first scaffold layer is 39.5%, the average porosity of the second scaffold layer is 87.2%, and the average porosity of the third scaffold layer is 42.4%.

[0051] 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 3:1.

[0052] Comparative Example 4 This example provides a non-metallic bioartificial aggregate that is not easy to collapse. Different from Example 1, the preparation method of the non-metallic bioartificial aggregate is prepared by a one-step freeze-drying method, specifically, step S5 is changed to pouring the first emulsion into a mold, continuing to add the second emulsion, continuing to add the third emulsion, and freeze-drying at -30°C for 24 hours to obtain a precursor.

[0053] 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 microscopy image.

[0054] Comparative Example 5 This example provides a non-metallic bio-artificial aggregate that is not easy to collapse. 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 1:1.91:1.07.

[0055] 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%.

[0056] The method for preparing non-metallic bio-artificial aggregate is different from that of Example 1 in that: step S5 is to pour the first emulsion into a mold and freeze-dry it at -30°C for 10 hours; continue to add the second emulsion and freeze-dry it at -30°C for 10 hours; continue to add the third emulsion and freeze-dry it at -30°C for 10 hours to obtain a precursor.

[0057] Results Test 1. A beagle dog alveolar bone with gingival defect model (removal of part of the mandibular alveolar bone) was prepared. The non-metallic bioartificial aggregates obtained in Example 1, Comparative Examples 1-3, and Comparative Example 5 were respectively filled into the bone defects of the same models. The mandibular alveolar bone of the beagle dog was tested by CBCT at 3 months and 6 months after surgery to record the alveolar bone height (measured three times and the average value was taken). At the same time, the DPX-L dual-energy X-ray absorptiometry was used to measure the bone density of the mandibular alveolar bone at the defect to be built and the new alveolar bone 6 months after surgery before building the model and compare them before and after. Nuclear magnetic resonance detection was performed at 1 month, 3 months, and 6 months after surgery, respectively, and whether there was collapse was judged by observing whether the surface of the repaired part was smooth. The results are shown in Table 1: Table 1 As can be seen from Table 1, by observing the nuclear magnetic resonance spectrum, the surface of the repaired area in Example 1 is smooth at 1 month, 3 months, and 6 months after surgery, and it can be inferred that it is not easy to collapse and the bone formation space is stable; the height of the new alveolar bone in Example 1 reaches 3.78 mm three months after surgery, which is significantly greater than that of Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 5, indicating that the non-metallic bioartificial aggregate obtained in Example 1 can quickly repair the original defects after implantation, help to enhance the new bone callus and increase its strength, accelerate the repair speed in the early stage of bone repair, and the strength of the new bone can continue to remain stable, especially meeting the clinical demands of early bone repair; the alveolar bone density in Example 1 is close to that before modeling, indicating that the material obtained in Example 1 has a good repair effect after implantation, and bone loss is not likely to occur in the repair area.

[0058] 2. Peel strength test: According to YY / T 0729.2-2009 Tissue Adhesive Bonding Performance Test Method Part 2: T-Peel Tensile Strength Test, the non-metallic bio-artificial aggregates obtained in Example 1 and Comparative Examples 1-5 were swollen in water for 1 hour, and then the peel strength test was performed. Each test was performed at the interface of the first scaffold layer, the second scaffold layer, and the third scaffold layer. Each location was tested three times in parallel, and the average value was finally taken. The results are shown in Table 2: Table 2 3. The in vivo osteoinductivity of the non-metallic bioartificial aggregates obtained in Example 1 and Comparative Example 2 was tested in accordance with YY∕T 1680-2020. The results are shown in Table 3. It can be seen that the non-metallic bioartificial aggregate obtained in Example 1 has obvious osteoinductivity, and is significantly greater than the non-metallic bioartificial aggregate obtained in Comparative Example 2.

[0059] Table 3 Example New bone formation element content (%) Example 1 ＞30 Comparative Example 2 10-20

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 30-50%, and the average porosity of the third support layer is 70-90%.

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 1 or 2, characterized in that: The volume ratio of the first support layer to the third support layer is 1:

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 tooth repair materials, jaw repair materials, spine repair materials, joint repair materials, and pelvic repair materials.

Images