Preparation method and application of hard bone source biomimetic bone hydrogel based on enzymatic acid salt extraction

Abstract

The application discloses a preparation method and application method of hard bone source biomimetic bone hydrogel based on enzymatic acid salt extraction, and belongs to the technical field of biomaterial preparation.The method comprises the following steps: pretreating hard bone raw materials to obtain hard bone powder; performing a deashing treatment on the hard bone powder under an acid condition, so that bone minerals are converted into a soluble state; then, through in-situ dilution and pH adjustment, synchronous extraction of collagen fibers is realized under a low-temperature condition by using pepsin as an auxiliary; the extraction liquid is subjected to ultrafiltration purification and concentrated to a gel concentration; finally, a crosslinking agent is introduced, and the system is adjusted to neutral, so that the formation of nano-hydroxyapatite and the crosslinking of collagen fibers are simultaneously induced, and a three-dimensional collagen network structure inlaid with nano-hydroxyapatite is formed.The application realizes efficient co-extraction and collaborative utilization of collagen and mineral components in hard bone in the same process through a synergistic process of "acid-enzyme-salt", and the obtained hydrogel has a calcium-phosphorus ratio close to that of natural bone, and is suitable for biomedical fields such as bone defect repair.

Description

Technical Field

[0001] This invention relates to a method for preparing biomimetic bone hydrogels derived from hard bone and its application based on enzymatic acidic salt extraction, belonging to the field of biomaterial preparation technology. Background Technology

[0002] Natural bone is a composite system composed of organic type I collagen fibers and inorganic nano-hydroxyapatite, highly ordered and interwoven at the micro- and nano-scale. The synergistic effect of these two components endows bone tissue with exceptional mechanical load-bearing capacity and structural stability. In the fields of biomaterials and tissue engineering, the construction of biomimetic bone hydrogels utilizing this natural matrix characteristic has become a research hotspot in areas such as bone defect repair and medical dressings due to its excellent biocompatibility, osteoconductivity, and tunable degradation.

[0003] Currently, the mainstream strategy for preparing such collagen / hydroxyapatite composite hydrogels is usually the "in vitro recombination method": that is, collagen is first extracted and purified from animal tissues, and then artificially synthesized hydroxyapatite (usually prepared by dry precipitation, wet precipitation or high-temperature solid-phase method) is added for physical blending, and chemical cross-linking methods such as glutaraldehyde and EDC / NHS are used to construct a network. However, artificially synthesized hydroxyapatite is not only energy-intensive and prone to particle agglomeration, but also lacks the trace elements unique to natural bone minerals; more importantly, this simple recombination breaks the original spatial topological relationship and strong interfacial bonding force between collagen and mineral phases in natural bone, resulting in the mechanical strength and osteogenic induction activity of the composite hydrogel often being difficult to match that of natural bone tissue.

[0004] To overcome the shortcomings of artificial reconstitution, the comprehensive development of directly utilizing biologically derived bone raw materials has extremely high added value potential. However, bone tissue has a dense structure with highly interlocked organic and inorganic components. Existing bone processing technologies generally adopt a "divide and conquer" approach with a single extraction method: on the one hand, when extracting collagen fibers, high-concentration strong acids are often used for prolonged deliming, directly converting valuable calcium and phosphorus minerals into waste liquid for discharge, resulting in significant resource waste and environmental pollution; on the other hand, when preparing natural bone powder or extracting hydroxyapatite, high-temperature calcination or strong alkali removal is often used, causing irreversible denaturation and thermal degradation of the precious type I collagen fiber triple helix structure.

[0005] In summary, existing technologies for processing bone, a natural composite material, generally face technical bottlenecks such as cumbersome process steps, high reagent consumption, and difficulty in simultaneously retaining and synergistically utilizing collagen components and hydroxyapatite. Therefore, there is an urgent need in this field to develop a mild, efficient, and integrated preparation process that can achieve efficient in-situ co-extraction of organic and inorganic components from bone without collagen denaturation, thereby expanding the in-depth application of aquatic and even livestock bone resources in the field of high-value-added biomaterials. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a biomimetic bone hydrogel derived from bone that is universally applicable and retains its components intact. Furthermore, this invention also provides the application of the obtained biomimetic bone hydrogel in the preparation of bone defect repair materials.

[0007] The core of this process lies in utilizing acidic conditions to form soluble ions from bone salts. Subsequently, a low-salt environment is created through appropriate dilution, stimulating the targeted hydrolysis of collagen telopeptides by pepsin. During this process, the presence of salt ions helps regulate the system's osmotic pressure and ionic strength, facilitating the dissolution of collagen fibers and preventing excessive acid denaturation of collagen. Simultaneously, it ensures that nano-hydroxyapatite can be mineralized in situ and adhere to the collagen fiber network during subsequent pH adjustment.

[0008] The method for preparing biomimetic bone hydrogel using bone material provided by this invention specifically includes the following steps: (1) Hard bone pretreatment: The hard bone raw material is washed, degreased at low temperature, washed again and dried in sequence, and then crushed to obtain hard bone powder.

[0009] (2) Acid-induced deashing treatment: The bone powder is deashed under acidic conditions. The deashing treatment uses an inorganic acid or organic acid (preferably hydrochloric acid) solution with a concentration of 0.5 to 1.5 mol / L (preferably 1.0 mol / L) and a solid-liquid ratio of 1:5 to 1:80 (preferably 1:10). The mixture is stirred at 20 to 30°C (preferably 25°C) for 1 to 6 h (preferably 3 h) to convert the mineral components in the bone into soluble ionic states.

[0010] (3) In-situ low-salt enzymatic extraction: The deashed system is diluted with water (preferably by a dilution factor of 0.5 to 2 times) to create a low ionic strength reaction environment, and the pH of the system is adjusted to 1.5 to 3.0 (preferably 2.0). Pepsin is added at 0 to 10°C (preferably 4°C) for in-situ synergistic extraction. The amount of pepsin added is 1% to 5% (preferably 2%, based on the mass of bone meal), and the extraction time is 6 to 72 h (preferably 48 h) to obtain a mixed extract containing type I collagen and soluble calcium phosphate components.

[0011] (4) Filtration and tangential ultrafiltration purification: The extract obtained in step (3) is filtered to remove unreacted residues, and then desalted and concentrated by tangential ultrafiltration. The ultrafiltration membrane has a molecular weight cutoff of 30-100 kDa (preferably 50 kDa). The concentration of collagen in the extract is increased to the critical concentration for gelation (preferably not less than 9.0 mg / mL) by ultrafiltration, and the conductivity of the system is monitored in real time to ensure the desalting effect.

[0012] (5) In-situ mineralization crosslinking and non-thermal sterilization: A crosslinking agent is added to the concentrated solution obtained in step (4), and the pH of the system is adjusted to neutral (preferably 7.2-7.4). Under the drive of the crosslinking agent and the induction of the neutral environment, the in-situ mineralization of nano-hydroxyapatite and the chemical crosslinking of collagen fibers are realized simultaneously, thereby constructing a three-dimensional collagen network structure embedded with nano-hydroxyapatite. The formed wet gel is freeze-dried and non-thermal sterilized to obtain a biomimetic bone hydrogel dried product. The crosslinking agent is preferably an EDC / NHS system (preferably with a mass ratio of 3:1), and the final addition concentration of EDC is 1.0-3.0 g / L.

[0013] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The present invention realizes the comprehensive utilization of collagen and mineral components in bone in the same process system, which avoids the waste of resources caused by the traditional bone processing method of "divide and conquer" and overcomes the defects of the traditional "in vitro recombination method" for preparing collagen / hydroxyapatite composite hydrogel, which requires the addition of artificially synthesized hydroxyapatite and the weak interfacial bonding between collagen and minerals. (2) Structural biomimicry: By controlling the ultrafiltration concentration factor and cross-linking parameters, the micropores and mechanical properties of the hydrogel can be precisely adjusted to make it highly compatible with natural cancellous bone; (3) Green and efficient: The entire process is carried out at low temperature, which preserves the natural activity of biological macromolecules to the maximum extent, and the reaction reagents are controllable and easy to scale up industrially. Attached Figure Description

[0014] The technical solution and beneficial effects of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, wherein: Figure 1 This invention relates to the effects of different types of acids on the dissolution rate of calcium and phosphorus in bone during the deashing process. Figure 1 A) and the calcium-phosphorus molar ratio ( Figure 1 B) Comparison curves of the effects.

[0015] Figure 2 The different solid-liquid ratios of the deashing system in this embodiment of the invention affect the dissolution rate of calcium and phosphorus in bone. Figure 2 A) and the calcium-phosphorus molar ratio ( Figure 2 B) Comparison curves of the effects.

[0016] Figure 3 This is a kinetic effect diagram of different hydrochloric acid concentrations during the deashing process in an embodiment of the present invention; wherein, Figure 3 A represents the calcium dissolution rate curve. Figure 3 B represents the phosphate dissolution rate curve. Figure 3 C represents the curve showing the change in the calcium-to-phosphorus molar ratio.

[0017] Figure 4 This is a diagram showing the optimized process parameters for the in-situ low-salt enzymatic extraction stage in an embodiment of the present invention; wherein, Figure 4 A shows the kinetic effect curves of different pepsin addition amounts on collagen dissolution rate. Figure 4 B is the curve showing the effect of stirring speed on collagen dissolution rate. Figure 4 C is a bar chart showing the effect of different dilution ratios on collagen dissolution rate when water is added to create a low-salt environment.

[0018] Figure 5 This is a graph showing the amino acid composition (number of residues per 1000 residues) of the purified collagen component in the extract prepared in an embodiment of the present invention.

[0019] Figure 6 The SDS-PAGE gel electrophoresis pattern of the collagen fibers in the extract prepared in this embodiment of the invention is used to verify that it retains the typical α-chain characteristics of natural type I collagen.

[0020] Figure 7 The circular dichroism (CD) spectrum of the collagen prepared in this embodiment of the invention verifies the integrity of its triple helix structure.

[0021] Figure 8 For based on Figure 7 The relative content analysis diagram of the secondary structure of collagen in the circular dichroism spectrum.

[0022] Figure 9 This is a physicochemical characterization diagram of the ultrafiltration concentration stage of the present invention; wherein, Figure 9 A is the curve showing the relationship between collagen concentration and soluble calcium and phosphate ion concentration in the system during ultrafiltration concentration. Figure 9 B is a comparison diagram of the macroscopic gelation state of hydrogels under different concentration gradients.

[0023] Figure 10 This is a comprehensive characterization diagram of the nano-sized hydroxyapatite precursor in the mixed extract prepared by the process of this invention; wherein... Figure 10 a is a macroscopic diagram illustrating the Tyndall effect, which demonstrates the stability of colloids. Figure 10 b is a field emission transmission electron microscope (TEM) topography image. Figure 10 c, 10d, and 10e are dynamic light scattering spectra, Zeta potentials, and hydration radius distributions, respectively.

[0024] Figure 11 This is an optimized diagram of the biomimetic bone hydrogel crosslinking process of the present invention; wherein, Figure 11 A represents the effect of crosslinking agent (EDC / NHS) concentration on gelation properties. Figure 11 B represents the effect of crosslinking time on gelation properties.

[0025] Figure 12 The figures show the physicochemical and biomechanical properties of the biomimetic bone hydrogel product obtained by this invention; wherein, Figure 12 A represents the time-swelling rate change curve of the hydrogel in simulated body fluid. Figure 12 B represents the in vitro enzymatic degradation rate curve. Figure 12 C represents the uniaxial compressive strength test curve.

[0026] Figure 13 This is a field emission scanning electron microscope (SEM) cross-sectional image of the lyophilized biomimetic bone hydrogel prepared in this invention; wherein, Figure 13 a is a diagram of the three-dimensional porous network structure formed by collagen fiber crosslinking at a scale of 100 μm. Figure 13 b is a magnified microscopic image of nano-hydroxyapatite uniformly embedded on the surface of collagen fibers, generated in situ at a scale of 10 μm.

[0027] Figure 14 Effects of biomimetic bone hydrogel on cell proliferation and osteogenic differentiation: A) Effect of hydrogel on chondrocyte proliferation; B) Effect of hydrogel on alkaline phosphatase (ALP) activity in MC3T3-E1 cells.

[0028] Figure 15 Table of femoral injury recovery grading evaluation results in experimental animals of the control group and the biomimetic bone hydrogel treatment group.

[0029] Figure 16 Appearance and magnified view of the femoral injury site in experimental animals. A: Control group; B: Biomimetic bone hydrogel treatment group; The injured area is marked by a red circle, and the corresponding area is magnified on the right.

[0030] Figure 17 Three-dimensional reconstruction and sagittal images of the femoral injury site in experimental animals. A: Control group; B: Biomimetic bone hydrogel treatment group; including 3D reconstruction image of the femur ( Figure 1 , Figure 2 (and sagittal images, with the lesion area marked in red.)

[0031] Figure 18 Figure 1 shows the results of measurements of relevant indicators for femoral defect repair in experimental animals. A: Trabecular bone thickness; B: Bone mineral density; Comparison between the control group and the biomimetic bone hydrogel treatment group. Detailed Implementation

[0032] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0033] Example 1: Optimal process for preparing bone-derived biomimetic bone hydrogels via enzymatic acidic salt method This embodiment provides a method for preparing highly biomimetic collagen / hydroxyapatite hydrogels with excellent properties by coupling acid deliming with low-salt enzymatic extraction. The specific steps are as follows: (1) Pretreatment: The bone raw material is cleaned, degreased at low temperature, dried, and then crushed by a pulverizer to obtain bone powder.

[0034] (2) Acid-induced deashing: Bone powder was added to a 1.0 mol / L hydrochloric acid solution at a solid-liquid ratio of 1:10 (g / mL), and the mixture was stirred and deashed for 3 h at room temperature (25℃). During this stage, a large amount of bone minerals were converted into a soluble state, and the pH of the system was approximately 2.0. Figures 1-3 As shown, under these optimal parameters, the dissolution rate of calcium and phosphorus elements reaches its maximum, and the calcium-to-phosphorus molar ratio in the extract is very close to the theoretical ratio of natural hydroxyapatite.

[0035] (3) In-situ low-salt enzymatic extraction: Add one volume of pure water to the deashed system for appropriate dilution to reduce ionic strength, then add 2% pepsin by weight of bone meal, and extract by stirring at 4°C for 48 h. Figure 4 As shown, under this optimal enzyme amount and time, the collagen dissolution rate reaches its best and excessive enzymatic hydrolysis is avoided.

[0036] (4) Ultrafiltration purification: After filtering the obtained extract to remove residue, desalting and concentration were carried out using a 50 kDa tangential flow ultrafiltration device until the collagen concentration in the system reached the critical concentration for gelation of 9.47 mg / mL.

[0037] (5) In-situ mineralization and cross-linking molding: Add 1 g / L of EDC / NHS cross-linking agent to the concentrate and adjust the pH of the system to 7.4. React at room temperature for 12 h to induce the soluble calcium phosphate component to mineralize in-situ into nano-hydroxyapatite, while promoting the cross-linking of collagen fibers. After the system forms an inverted, non-flowing hydrogel, it is freeze-dried and sterilized without heat to obtain the biomimetic bone hydrogel product.

[0038] Example of efficacy verification: Evaluation of the physicochemical and biological properties of the biomimetic bone hydrogel prepared in this invention. To verify the technical advantages of the biomimetic bone hydrogel prepared in Example 1 above, conventional analytical methods were used to characterize the performance of its intermediate products and final product at key stages. The results are as follows: 1. Verification of the fidelity of extract components and molecular structure ① Amino acid and molecular weight distribution: The extracted collagen was analyzed using a fully automated amino acid analyzer (…). Figure 5 ) and SDS-PAGE electrophoresis ( Figure 6The test results showed that it was rich in glycine, proline and hydroxyproline, and the electrophoretic pattern clearly showed the α1 and α2 chains characteristic of natural type I collagen, indicating that the in situ enzymatic acid extraction did not destroy the peptide backbone of collagen.

[0039] ② Three-dimensional structural integrity: Circular dichroism (CD) scanning ( Figure 7 , Figure 8 The results confirmed that the mild extraction environment of this invention allows collagen molecules to perfectly retain their natural triple helix structure, which is the material basis for maintaining the high mechanical strength of the hydrogel.

[0040] 2. In-situ mineralization mechanism and nanoparticle characterization In ultrafiltration concentration ( Figure 9 During the pre-gelation stage (pH adjustment), dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to monitor the system. Figure 10 As shown, the system exhibits a clear Tyndall effect, confirming the presence of a stable colloid. The hydration radius of the nanoparticles is mainly distributed around 200 nm, and the Zeta potential indicates that they possess good dispersibility, proving that the process of this invention successfully achieves in-situ retention and particle size control of the hydroxyapatite precursor.

[0041] 3. Macroscopic and microscopic property characterization of biomimetic bone hydrogel products ① Crosslinking degree and network formation: Free amino consumption colorimetric method ( Figure 11 This study confirmed that the introduction of EDC / NHS crosslinking agent significantly improved the crosslinking degree of the hydrogel, forming a stable three-dimensional covalent network.

[0042] ② Fluid adaptability and controllable degradation: The hydrogel was placed in simulated body fluid and a buffer solution containing collagenase ( Figure 12 A, 12B). The results show that the hydrogel prepared by this invention absorbs water rapidly in the initial stage and then tends to stabilize, exhibiting excellent tissue fluid absorption potential; at the same time, by adjusting the degree of crosslinking, it can effectively resist enzyme degradation and achieve controllable regulation of the degradation cycle to adapt to the growth rate of new bone tissue.

[0043] ③ Compressive strength matching: uniaxial compression test ( Figure 12 C) indicates that the hydrogel prepared under the optimal crosslinking parameters not only has good elasticity, but its maximum compressive strength is significantly better than that of traditional physical blends, and it can provide the mechanical support required in the early stage of bone defects.

[0044] ④ Biomimetic microstructure: Field emission scanning electron microscopy (SEM) Figure 13Observations revealed that the finished product exhibited a highly interconnected three-dimensional porous network of collagen fibers and particles. The in-situ generated nano-hydroxyapatite particles were uniformly embedded and anchored within the surface and pores of the collagen fiber scaffold. This microstructure highly replicated the extracellular matrix structure of natural cancellous bone, providing an ideal microenvironment for osteoblast adhesion, proliferation, and tissue vascularization.

[0045] 4. Effects of biomimetic bone hydrogels on cell proliferation and osteogenic differentiation ① Chondrocyte proliferation: via OD 450 Cell proliferation was measured at different time points (24 h, 48 h, 72 h). Figure 14 A). The results showed that, compared with the control group, the chondrocyte proliferation level in the biomimetic bone hydrogel treatment group remained similar at each time point, indicating that the hydrogel itself has good biocompatibility with chondrocytes and does not inhibit cell proliferation.

[0046] ②Alkaline phosphatase (ALP) activity in MC3T3-E1 cells: determined by alkaline phosphatase activity assay ( Figure 14 B) Assessment of osteogenic differentiation. The results showed that the ALP activity in the biomimetic bone hydrogel-treated group was significantly higher than that in the control group, suggesting that the hydrogel promotes the differentiation of osteogenic progenitor cells.

[0047] 5. Evaluation of the effect of biomimetic bone hydrogel on femoral injury repair ① Evaluation of femoral injury repair grading: based on the femoral injury recovery grading ( Figure 15 In the control group, all experimental animals had a femoral injury score of 0, indicating no significant repair; while in the biomimetic bone hydrogel treatment group, all animals had a score of 2, suggesting that the hydrogel significantly promoted the healing of femoral defects.

[0048] ② Observation of the appearance of the injured site: through postoperative femoral specimen and magnified local images ( Figure 16 Observation showed that the biomimetic bone hydrogel treatment group formed continuous bone-like tissue at the defect site, and the damaged area was significantly filled; while the control group still showed obvious defects and gaps, and no new bone formation was observed.

[0049] ③ Imaging analysis: Through three-dimensional reconstruction and cross-sectional observation of the femur ( Figure 17 The biomimetic bone hydrogel treatment group showed that the bone tissue in the defect area was continuous and dense, and the defect was completely filled; while in the control group, gaps and unhealed bone tissue were still clearly visible in the defect area, further confirming the effect of hydrogel in promoting bone repair.

[0050] ④ Quantitative index analysis: Through the measurement of trabecular thickness and bone mineral density ( Figure 18The results of the biomimetic bone hydrogel treatment group were significantly higher than those of the control group, indicating that the hydrogel not only promotes bone tissue formation, but also improves the microstructure and mineralization of new bone, providing good mechanical support and biological environment for bone defect sites.

[0051] Conclusion: The above results fully demonstrate that the enzymatic acidic salt method proposed in this invention breaks through the barriers of existing bone separation and extraction technologies, and successfully achieves efficient co-extraction and in-situ biomimetic composite of organic collagen and inorganic minerals in the same system. The resulting biomimetic bone hydrogel has excellent bone repair properties and shows extremely high industrial application and clinical translation value.

[0052] The above embodiments only illustrate and describe the basic principles, main features, and beneficial effects of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. Various changes and modifications can be made to the present invention without departing from its spirit and scope, and all such changes and modifications should fall within the protection scope of the present invention.

Claims

1. A method for preparing a bone-derived biomimetic bone hydrogel based on enzymatic acidic salt extraction, characterized in that, Includes the following steps: (1) After cleaning, degreasing, drying and pulverizing the bone raw material, bone powder is obtained; (2) The bone powder is deashed under acidic conditions; (3) Dilute the deashed system with water and adjust it to acidity. Add pepsin under low temperature conditions for in-situ extraction to obtain a mixed extract containing type I collagen and mineral components. (4) The extract is filtered and subjected to tangential flow ultrafiltration, and then concentrated to a gel-forming concentration; (5) Add a cross-linking agent to the concentrated extract, adjust the pH of the system to neutral, and simultaneously induce the formation of nano-hydroxyapatite and the cross-linking of collagen fibers, thereby forming a three-dimensional collagen network structure embedded with nano-hydroxyapatite. After freeze-drying and non-thermal sterilization, the biomimetic bone hydrogel dried product is obtained.

2. The method according to claim 1, characterized in that, The bone material is selected from the skeletal tissue of vertebrates.

3. The method according to claim 2, characterized in that, The bone material is at least one of fish bone, mammal bone, or poultry bone.

4. The method according to claim 1, characterized in that, The deashing treatment in step (2) uses an inorganic acid or organic acid solution with a concentration of 0.5 to 1.5 mol / L, a deashing temperature of 20 to 30°C, and a deashing time of 2 to 5 h.

5. The method according to claim 1, characterized in that, The collagen extraction process described in step (3) is as follows: dilute the deashing solution with water by 1 to 6 times, adjust the pH of the system to 1.5 to 3.0, add pepsin, and stir and extract for 24 to 72 h at 0 to 10°C.

6. The method according to claim 1, characterized in that, The ultrafiltration purification in step (4) uses a tangential flow ultrafiltration device with a molecular weight cutoff of 30-100 kDa to concentrate the collagen in the system to a concentration of not less than 9.0 mg / mL.

7. The method according to claim 1, characterized in that, The crosslinking agent mentioned in step (5) is an EDC (1-[3-dimethylaminopropyl]-3-ethylcarboimide) / NHS (N-hydroxysuccinimide) crosslinking system, wherein the mass fraction of EDC is 1% to 3% and the mass fraction of NHS is 0.5% to 1%.

8. The biomimetic bone hydrogel prepared by the method according to any one of claims 1 to 6, characterized in that, The hydrogel has a calcium-to-phosphorus molar ratio of 1.61 to 1.71, a pore size distribution of 50 to 200 μm, and a compressive strength of not less than 1.5 MPa.

9. The application of the biomimetic bone hydrogel according to claim 8 in the preparation of bone defect repair materials.

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