A bone regeneration formula and preparation method based on bone glue synergy

Abstract

This invention provides a bone regeneration formula based on collagen synergy and its preparation method. The formula uses low-molecular-weight collagen peptides as organic templates and forms hydroxyapatite-like nanostructures through ion-induced mineralization, achieving a tight organic-inorganic interface. The formula can regulate osteoblast differentiation and inhibit osteoclast activity, thereby improving bone density and bone tissue regeneration capacity. The preparation process adopts a low-temperature enzymatic hydrolysis and biomimetic mineralization coupling process to maintain the stability of the active structure. Experimental results show that the formula is superior to conventional formulas in terms of molecular expression, mineralization capacity, and bone metabolism regulation.

Description

Technical Field

[0001] This invention pertains to bone tissue repair formulations and biomimetic mineralization technology, and specifically relates to a bone formation formulation and preparation method based on bone glue synergy. Background Technology

[0002] Bone tissue repair materials often use collagen or gelatin as organic templates, followed by mineralization with calcium salts to mimic the organic-inorganic composite structure of natural bone. However, in existing formulations, the collagen molecular weight is relatively large, making it difficult to form a uniform porous network structure. The inorganic mineralized phase is unevenly distributed in the organic matrix, resulting in weak interfacial bonding and affecting the stability and osteogenic effect of the material.

[0003] Conventional calcium sources such as calcium carbonate and calcium phosphate require complex transformations in the body before they can be absorbed and utilized. The ion release and mineralization processes are difficult to control, the osteoblast differentiation capacity is limited, and the effect of improving bone density is not ideal.

[0004] Existing preparation processes often employ high-temperature or strong acid / alkali treatments, which can easily damage the spatial structure and biological activity of collagen peptides, affecting cell adhesion and proliferation, and also limiting the uniformity and crystallization quality of the mineralized layer.

[0005] In addition, existing formulations lack a systematic consideration of bone metabolism regulation in their ingredient design, and fail to effectively combine functional components that promote osteogenic differentiation and inhibit osteoclast activity, making it difficult to achieve coordination between bone formation and bone resorption in vivo. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a bone formation formula and preparation method based on bone glue synergy, thereby resolving the problems mentioned in the background art.

[0007] This invention is achieved through the following technical solution: a bone formation formula based on collagen synergy, comprising: a collagen polypeptide matrix and a nanoscale calcium salt mineralization phase loaded thereon; The collagen polypeptide has a molecular weight of 300-5000 Da and forms a template backbone with a porous network structure. The mineralized phase is deposited in the form of hydroxyapatite on the surface and internal pores of the template skeleton, forming a tight bond with the template skeleton.

[0008] As a preferred embodiment, it also includes vitamin D3, vitamin K2, magnesium, zinc and hyaluronic acid.

[0009] After adopting the above technical solution, the beneficial effects of the present invention are: 1. Advantages of biomimetic structure: A porous network template is constructed using low molecular weight collagen peptides (300-5000Da), and nano-sized calcium salts are uniformly deposited in the form of hydroxyapatite to form a "collagen synergy" structure that is highly similar to natural bone.

[0010] 2. Synergistic enhancement of ingredients: The introduction of magnesium, zinc ions and hyaluronic acid promotes osteoblast adhesion, proliferation and mineralization; vitamin D3 and K2 further regulate calcium metabolism and bone matrix deposition.

[0011] 3. Excellent biocompatibility and osteogenic activity: Cell experiments showed that ALP activity, mineralized nodule formation, and expression of osteogenic-related proteins such as Runx2 were all superior to the control group.

[0012] A method for preparing the above formulation includes: S1: Low-temperature enzymatic hydrolysis of collagen, at a temperature of 45-55℃; S2: Low molecular weight collagen peptides were isolated; S3: Biomimetic mineralization was carried out at 37℃ and pH 7.4-8.5; S4: The mineralized products are graded, compounded, and homogenized with vitamins, magnesium, zinc, and hyaluronic acid to form a compound formula.

[0013] In a preferred embodiment, the mineralization time in step S3 is 12-48 hours.

[0014] After adopting the above technical solution, the beneficial effects of the present invention are: 1. Low-temperature enzymatic hydrolysis protects activity: Enzymatic hydrolysis at 45-55℃ preserves the biological activity and spatial structure of collagen peptides, which is beneficial for subsequent mineralization.

[0015] 2. Mild biomimetic mineralization conditions: Mineralization is carried out at 37℃ and pH 7.4-8.5, simulating the physiological environment of the human body, to form a stable and uniform nano-mineralized layer.

[0016] 3. Graded compounding and homogenization: Through high-pressure homogenization and freeze drying, a porous composite formula with uniform dispersion and controllable structure is obtained, which is suitable for different dosage form requirements. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Preparation flow chart; Figure 2 : SEM microstructure image of the finished product; Figure 3 TEM image of the finished product's nanostructure; Figure 4XRD crystal structure diagram. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] This invention provides a technical solution: a bone formation formula based on collagen synergy, comprising: a collagen polypeptide matrix and a nanoscale calcium salt mineralization phase loaded thereon; The collagen polypeptide has a molecular weight of 300-5000 Da and forms a template backbone with a porous network structure. The mineralized phase is deposited in the form of hydroxyapatite on the surface and internal pores of the template framework, forming a tight bond with the template framework. It also contains vitamin D3, vitamin K2, magnesium, zinc, and hyaluronic acid.

[0021] The calcium source of the nanoscale calcium salt mineralization phase is selected from calcium citrate, milk calcium, amino acid chelated calcium, or a combination thereof.

[0022] Please see Figure 1 A method for preparing a bone regeneration formulation, comprising: S1: Low-temperature enzymatic hydrolysis of collagen, at a temperature of 45-55℃; S2: Low molecular weight collagen peptides were isolated; S3: Biomimetic mineralization was carried out at 37℃ and pH 7.4-8.5 for 12-48 hours. S4: The mineralized products are graded, compounded, and homogenized with vitamins, magnesium, zinc, and hyaluronic acid to form a compound formula.

[0023] Example 1: A bone formation formula based on collagen synergy, comprising 30 parts of collagen peptides, 20 parts of calcium citrate, 0.03 parts of vitamin D3, 0.02 parts of vitamin K2, 3 parts of hyaluronic acid, 2 parts of magnesium, and 1 part of zinc.

[0024] Preparation method Step S1: Low-temperature enzymatic hydrolysis of collagen, specifically: Animal bone (bovine bone or fish bone) was selected and degreased (soaked in 60℃ warm water for 2 hours, then degreased with ethanol) to remove impurities. The bone powder was then pulverized to a particle size ≤1 mm. The bone powder was added to a 0.5 mol / L hydrochloric acid solution at a solid-liquid ratio of 1:10 and stirred at 4℃ for 24 hours to remove inorganic salts and obtain a demineralized bone matrix. The matrix was then washed with deionized water until the pH was approximately 7.

[0025] The demineralized bone matrix was added to a pH 7.5 buffer solution, and a complex protease (papain + trypsin, mass ratio 1:1) was added at a concentration of 2% of the substrate mass. Enzymatic hydrolysis was carried out at a constant temperature of 50℃ for 4 hours with a stirring speed of 200 rpm. After the reaction was completed, the temperature was raised to 90℃ and held for 10 minutes to inactivate the enzyme.

[0026] Step S2: Low molecular weight collagen peptides are separated, specifically: Undissolved matter was removed by centrifugation (8000 rpm, 15 min); the supernatant was fractionated by ultrafiltration membrane (molecular weight cutoff 3000 Da), and the fraction of 500-3000 Da was collected; then vacuum concentration (temperature ≤45℃) was carried out to a solid content of 20%.

[0027] The concentrate was spray-dried at an inlet air temperature of 150°C and an outlet air temperature of 80°C to obtain collagen polypeptide powder.

[0028] Step S3: Perform biomimetic mineralization at 37℃ and pH 7.4-8.5, specifically: Collagen peptides were dissolved in deionized water (5% w / v) and the pH was adjusted to 7.4. A calcium source solution (calcium citrate solution) was slowly added dropwise, along with a phosphorus source (Na2HPO4), with the Ca / P molar ratio controlled at 1.67. The reaction temperature was maintained at 37℃ and the stirring speed at 150 rpm. The mineralization time was 24 hours, during which the pH was maintained at 7.4-8.0 (adjusted using Tris buffer). This resulted in the formation of a hydroxyapatite-like nanodeposit structure.

[0029] After mineralization in step S3, the resulting composite formulation was freeze-dried, and the dried sample was used for microstructure characterization.

[0030] I. Scanning Electron Microscopy (SEM) Analysis After drying, the sample was surface-sprayed with gold, and its microstructure was observed using a scanning electron microscope. The results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the collagen peptide template has a three-dimensional porous network structure with uniform pore size distribution and interconnected pores; nano-sized hydroxyapatite particles are uniformly deposited on the surface of the peptide backbone and the inner wall of the pores, forming a dense and continuous mineralized layer without obvious aggregation, indicating that the mineralized phase and the organic template have excellent interfacial bonding performance.

[0031] II. Transmission Electron Microscopy (TEM) Analysis The sample was ultrasonically dispersed and then placed on a copper mesh. The nanoscale structure was observed using a transmission electron microscope. The results are as follows: Figure 3 As shown. By Figure 3As can be seen, the hydroxyapatite-like crystals are needle-like or plate-like in shape, with a length of 50–150 nm and a width of 10–30 nm. They are oriented along the long axis of collagen polypeptide fibers, forming an oriented mineralization structure similar to that of hydroxyapatite in natural bone. Selected area electron diffraction (SAED) shows a diffuse ring pattern, indicating that the crystal size is in the nanometer range and also has a certain degree of crystallinity.

[0032] III. X-ray Diffraction (XRD) Analysis The dried sample was subjected to X-ray diffraction analysis. The test conditions were: Cu Kα rays, tube voltage 40 kV, tube current 40 mA, scanning range 2θ = 10°~60°, and scanning speed 2° / min. The results are as follows: Figure 4 As shown.

[0033] Depend on Figure 4 It can be seen that the sample has characteristic diffraction peaks at 2θ=31.8°, 34.6°, 42.8°, and 46.7°, which are basically consistent with the standard spectrum of hydroxyapatite (JCPDS No.09-0432). Among them, the characteristic peaks of the (002) and (211) crystal planes are obvious, indicating that the mineralized phase is a well-crystallized hydroxyapatite-like phase without other calcium salt impurities.

[0034] comprehensive Figures 2 to 4 It is known that the formulation of the present invention constructs an organic-inorganic composite system with a three-dimensional porous network structure through low-temperature enzymatic hydrolysis and biomimetic mineralization process of bone collagen polypeptide template. Nanoscale hydroxyapatite is uniformly deposited on the surface and inside of the template, forming a multi-level structure that is highly similar to natural bone tissue, providing an ideal microenvironment for cell adhesion, proliferation and osteogenic differentiation.

[0035] Step S4: The mineralized products are graded, compounded, and homogenized with vitamins, magnesium, zinc, and hyaluronic acid to form a compound formula. Specifically: After mineralization, cool to room temperature; add vitamin K2 and vitamin D3 sequentially (under light-protected conditions), and stir for 30 minutes; then add hyaluronic acid, magnesium salt, and zinc salt, and stir until homogeneous. Use a high-pressure homogenizer (80 MPa) to cycle the mixture three times to ensure uniform dispersion and form a stable composite system. Freeze-dry (-50℃, 48 hours) to obtain a porous structure formulation; or, if necessary, compress into tablets or package into powder.

[0036] As an embodiment of the present invention, Example 1, Comparative Example 1, and Comparative Example 2 were compared experimentally. Specifically: Comparative Example 1: The formula uses traditional calcium carbonate + collagen, that is, traditional inorganic calcium sources (such as calcium carbonate) are used to replace nano-sized calcium salts, and the collagen is not subjected to low molecular weight treatment.

[0037] Comparative Example 2: High-temperature process, i.e., high-temperature enzymatic hydrolysis is used in the bone collagen processing.

[0038] 1. Experimental Design 1.1 Cell Experiments 1.1.1 Cell source and culture Mouse osteogenic progenitor cells MC3T3-E1 were selected and cultured in α-MEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. The culture was placed in a 37°C, 5% CO2 incubator.

[0039] 1.1.2 Group Design The experiment was divided into four groups: the extract group of the formulation of the example (concentration 100 μg / mL), the extract group of comparative example 1, the extract group of comparative example 2, and the blank control group.

[0040] 1.1.3 Osteogenesis Induction Osteogenesis induction solution containing 10 mmol / L sodium β-glycerophosphate, 50 μg / mL ascorbic acid, and 10 μg / mL dexamethasone was added to the culture system. -8 mol / L.

[0041] 2. ALP activity detection On day 7 of culture, the culture medium was removed, and the cells were washed with PBS and lysis buffer was added to collect cell proteins. Using an ALP detection kit, the reaction was carried out at 37°C for 15 minutes, and the absorbance was measured at 405 nm. ALP activity (U / L) was calculated, and the results are shown in Table 1. It can be seen that the alkaline phosphatase activity in the example group was higher, indicating an increased level of osteoblast differentiation.

[0042] Table 1. Osteogenesis-related markers (ALP activity) 3. Alizarin Red staining (mineralized nodules) After 21 days of culture, the samples were fixed with 4% paraformaldehyde for 30 minutes, washed with distilled water, and then stained with 2% Alizarin Red S staining solution (pH 4.2) for 20 minutes. The samples were then observed and photographed under a microscope. The dye was then dissolved with 10% cetylpyridine chloride, and quantitative analysis was performed at a wavelength of 562 nm. The results are shown in Table 2. Compared with the control group, the number of mineralized nodules increased and the calcium salt deposition capacity was enhanced in the example group.

[0043] Table 2, Mineralization capacity (alizarin red quantitative analysis) 4. Western blot detection Total protein was extracted from cells, and protein concentration was determined using the BCA method. SDS-PAGE electrophoresis (10% separating gel) was performed, and the samples were transferred to a PVDF membrane and blocked with 5% skim milk for 1 hour. Runx2, ALP, OCN, RANKL, and OPG primary antibodies were added, and the mixture was incubated overnight at 4°C. Then, HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour. ECL staining and imaging analysis were performed to analyze protein expression levels. The results are shown in Table 3. Osteogenesis-related proteins showed increased expression, while osteoclast-related proteins showed decreased expression.

[0044] Table 3, Protein Expression (Western blot) 5. qPCR detection Total RNA was extracted using the Trizol method, and cDNA was synthesized by reverse transcription. Real-time quantitative PCR was performed using the SYBR Green system with the following reaction conditions: 95℃ pre-denaturation for 30 seconds, 40 cycles (95℃ for 5 seconds, 60℃ for 30 seconds). The relative expression levels were calculated, and the results are shown in Table 4. It can be seen that the expression of osteogenic genes is upregulated, while the expression of osteoclast-related genes is downregulated.

[0045] Table 4, Gene Expression (qPCR) As an embodiment of the present invention, Example 1, Comparative Example 1, and Comparative Example 2 were compared experimentally. Specifically: 1. Laboratory animals and grouping Eight-week-old SD rats, weighing 200±20 g, were randomly divided into three groups (n=30): Example Group, Comparative Example 1, and Comparative Example 2.

[0046] 2. Establishment of an osteoporosis model An osteoporosis model was established by bilateral oophorectomy (OVX), and medication was started one week after the operation.

[0047] 3. Administration method The example group was given the formulation suspension (200 mg / kg) by gavage daily according to body weight, while the control group was given the corresponding formulation. The dosing period was 12 weeks.

[0048] 4. Bone mineral density test (DEXA) Femoral bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry at weeks 0, 4, 8, and 12 of the experiment. The average values ​​were then statistically analyzed, and the results are shown in Table 5.

[0049] 5. Histological analysis Femur sections were harvested, fixed in 4% formaldehyde, decalcified in EDTA solution for 2 weeks, and then embedded in paraffin. Sections were prepared with a thickness of 5 μm. HE and TRAP staining were performed to observe the trabecular structure and osteoclast count. The results are shown in Table 6.

[0050] Table 6, Histological Analysis 6. Serum biochemical marker detection Serum was collected to test ALP (osteogenic marker), OCN (osteogenic marker), and TRAP (osteoclast marker).

[0051] The results are shown in Table 5. Table 5. Comparison of Animal Experiments This formula demonstrates a high level of performance in osteogenic differentiation, mineralization capacity, and bone density enhancement. It increases the expression of osteogenic-related proteins and genes, while decreasing osteoclast-related indicators. Bone tissue structure improves, and trabecular bone arrangement becomes more regular. Overall, it exhibits a regulatory effect on bone formation and bone resorption processes.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A bone regeneration formula based on collagen synergy, characterized in that, include: Bone collagen polypeptide matrix and nanoscale calcium salt mineralization phase loaded thereon; The collagen polypeptide has a molecular weight of 300-5000 Da and forms a template backbone with a porous network structure. The mineralized phase is deposited in the form of hydroxyapatite on the surface and internal pores of the template skeleton, forming a tight bond with the template skeleton.

2. The bone regeneration formula based on bone glue synergy as described in claim 1, characterized in that: The calcium source of the nanoscale calcium salt mineralization phase is selected from calcium citrate, milk calcium, amino acid chelated calcium, or a combination thereof.

3. The bone regeneration formula based on bone glue synergy as described in claim 2, characterized in that: It also contains vitamin D3, vitamin K2, magnesium, zinc, and hyaluronic acid.

4. A method for preparing the formulation as described in claim 3, characterized in that: include: S1: Low-temperature enzymatic hydrolysis of collagen, at a temperature of 45-55℃; S2: Low molecular weight collagen peptides were isolated; S3: Biomimetic mineralization was carried out at 37℃ and pH 7.4-8.5; S4: The mineralized products are graded, compounded, and homogenized with vitamins, magnesium, zinc, and hyaluronic acid to form a compound formula.

5. The preparation method of the formulation according to claim 4, characterized in that: In step S3, the mineralization time is 12-48 hours.

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