Traditional Chinese medicine ingredient modified bone graft material and preparation method thereof

Abstract

The invention discloses a traditional Chinese medicine ingredient modified bone graft material and a preparation method thereof, and relates to the technical field of bone graft materials. The feed additive is prepared from the following raw materials: calcined bone meal and a traditional Chinese medicine extract, the traditional Chinese medicine extract comprises a radix salviae miltiorrhizae extract and / or a radix scutellariae extract. The preparation method comprises the following steps: preparing calcined bone meal, preparing simulated body fluid and preparing the modified bone graft material. The product material can be applied to a dental bone grafting material. The traditional Chinese medicine extract is loaded on the bone material, and the bone material is modified, so that the bone graft material with good biocompatibility, osteogenesis promoting capability and better osteogenesis induction potential can be obtained.

Description

Technical Field

[0001] This invention relates to the field of bone graft materials technology, specifically to a bone graft material modified with traditional Chinese medicine ingredients and its preparation method. Background Technology

[0002] Currently, bone graft materials used in clinical practice can be broadly classified into four basic types: autologous bone, allogeneic bone, xenogeneic bone, and synthetic materials. Autologous bone has limited availability, is difficult to shape, requires a separate incision for harvesting, increases trauma, and carries the risk of varying degrees of postoperative complications. Allogeneic bone comes from individuals of the same species as the patient, offering a wider range of sources compared to autologous bone, but it has a longer bone formation time, slower healing, and is more prone to immune rejection. Xenogeneic bone comes from individuals of a different species than the patient, offering a wider range of sources, is more economical, and has more stable composition. Synthetic materials mainly fall into two categories: ceramics and metals. Ceramic materials primarily include calcium phosphate ceramics and bioactive glass, which have strong bioactivity and controllable physicochemical properties, but are brittle and have limited strength. Titanium and titanium alloys are commonly used metallic synthetic bones clinically, but as they are metallic materials, friction and wear are inevitable after implantation, potentially leading to metal particles entering surrounding tissues and causing inflammatory reactions. In a comprehensive comparison, xenogeneic bone, which has a wide range of sources, excellent biocompatibility, and good osteogenic effect, stands out. Previously, the clinical application of xenogeneic bone was greatly limited due to animal diseases and high immunogenicity. However, xenogeneic bone that has been calcined or freeze-dried no longer has this defect, and it is foreseeable that xenogeneic bone will have a wide range of applications in bone graft materials in the future.

[0003] The market for bone graft materials is growing rapidly, with dental materials accounting for a large proportion. In the dental field, materials are typically required to have good osteoconductivity, osteoinductive properties, and biocompatibility, among other core bone biological characteristics. However, conventional bone graft materials currently on the market generally have insufficient performance in these areas. Therefore, it is necessary to develop new bone graft materials to meet the needs of dental clinical practice. Summary of the Invention

[0004] The purpose of this invention is to provide a bone graft material modified with traditional Chinese medicine ingredients and its preparation method. The method involves loading traditional Chinese medicine extracts onto bone materials to modify the bone materials, thereby obtaining bone graft materials with good biocompatibility and osteogenic capacity, and with superior osteogenic induction potential.

[0005] Another objective of this invention is to provide an application of bone graft material modified with traditional Chinese medicine ingredients in dental bone graft materials, particularly for bone repair materials used in alveolar bone defects.

[0006] The technical solution of the present invention is as follows: On one hand, embodiments of the present invention provide a bone graft material modified with traditional Chinese medicine components, which is prepared from raw materials including the following components: calcined bone powder and traditional Chinese medicine extracts; The aforementioned Chinese herbal extracts include Salvia miltiorrhiza extract and / or Scutellaria baicalensis extract.

[0007] Furthermore, in some embodiments of the present invention, the calcined bone powder is prepared by calcining and grinding bovine femur, the tanshinone extract is salvianolic acid B, and the scutellaria extract is baicalin.

[0008] This invention provides a method for preparing the above-mentioned bone graft material, characterized by comprising the following steps: Preparation of calcined bone powder: Fresh bone is taken and impurities such as muscle tissue, periosteum and fat on its surface are removed. After boiling in water, it is soaked in sodium hydroxide solution and hydrogen peroxide solution respectively. After washing, it is calcined, ground and sieved to obtain calcined bone powder for later use. Preparation of simulated body fluid: Take the Chinese herbal extract, prepare the extract solution, and then prepare the extract solution into simulated body fluid to obtain simulated body fluid containing the Chinese herbal extract. Also prepare a blank simulated body fluid without the Chinese herbal extract. The blank simulated body fluid is denoted as SBF solution. Preparation of modified bone graft material: Take the prepared calcined bone powder and immerse it in blank SBF solution. After immersion, take out the bone powder material, wash and dry it, and then immerse it in SBF solution containing Chinese herbal extract. After immersion, take out the solid material, wash and dry it to obtain Chinese herbal modified bone graft material.

[0009] Furthermore, in some embodiments of the present invention, in the above-mentioned calcined bone powder preparation steps, the concentration of sodium hydroxide solution used is 1 mol / L, and the mass concentration of hydrogen peroxide solution used is 30%.

[0010] Furthermore, in some embodiments of the present invention, in the above-mentioned calcined bone powder preparation steps, the calcination temperature is 600-900°C and the calcination time is 2-6 h.

[0011] Furthermore, in some embodiments of the present invention, in the above-described simulated body fluid preparation step, the blank simulated body fluid SBF solution is prepared from raw materials comprising the following components: NaCl, MgCl2·6H2O, CaCl2, Na2HPO4, Na2CO3, and UP water.

[0012] Furthermore, in some embodiments of the present invention, in the above-mentioned simulated body fluid preparation step, the content of the traditional Chinese medicine extract in the SBF solution containing the traditional Chinese medicine extract is 1-20 μg / ml.

[0013] Furthermore, in some embodiments of the present invention, in the above-mentioned modified bone graft material preparation steps, the calcined bone powder is first immersed in a blank SBF solution, washed and dried, and then immersed in an SBF solution containing traditional Chinese medicine extracts. After the immersion is completed, the solid material is taken out and the liquid material is discarded. Then a new SBF solution with the same composition containing traditional Chinese medicine extracts is taken, and the taken-out solid material is immersed in it again. After the immersion is completed, the solid material is taken out, washed and dried to obtain the traditional Chinese medicine modified bone graft material.

[0014] Furthermore, in some embodiments of the present invention, the single soaking time is 24 hours.

[0015] On the other hand, embodiments of the present invention also provide an application of the above-mentioned modified bone graft material containing traditional Chinese medicine components in dental bone graft materials.

[0016] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects: Regarding the first aspect, embodiments of the present invention provide a bone graft material modified with traditional Chinese medicine components. This material utilizes extracts of Salvia miltiorrhiza and / or Scutellaria baicalensis to modify calcined bone powder, loading the effective components from these extracts onto the calcined bone powder material. This allows the active ingredients of Salvia miltiorrhiza to exert various effects, such as promoting osteoblast proliferation and differentiation and bone formation, inhibiting osteoclast proliferation and differentiation and bone resorption, and anti-inflammation, as well as the active ingredients of Scutellaria baicalensis to exert antibacterial, anti-inflammatory, and periodontal cell repair-promoting effects. Furthermore, loading the effective components of traditional Chinese medicine onto the calcined bone powder material for modification enables the bone material to form a more significantly porous structure, which is conducive to cell ingrowth and nutrient transport. Simultaneously, it significantly enhances the hydrophilicity of the bone powder, creating favorable conditions for promoting protein adsorption and early cell adhesion. Ultimately, the modified bone graft material possesses good biocompatibility, osteogenic capacity, superior osteogenic induction potential, and certain antibacterial and anti-inflammatory capabilities.

[0017] Regarding the second aspect, embodiments of the present invention also provide an application of the above-mentioned modified bone graft material containing traditional Chinese medicine components in dental bone graft materials, particularly for the field of alveolar bone defect repair, which can effectively solve the problem of poor osteogenic capacity of existing materials. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1The image shows the SEM characterization results in the experimental example of this invention; Figure 2 This is a graph showing the XRD test results in an experimental example of this invention; Figure 3 This is a graph showing the FT-IR test results in the experimental example of this invention; Figure 4 This is a graph showing the EDS elemental analysis results in the experimental examples of this invention; Figure 5 This is a graph showing the water absorption time results of the hydrophilicity test in the experimental examples of this invention; Figure 6 This is a diagram showing the cell adhesion test results in an experimental example of the present invention; Figure 7 This is a graph showing the cell proliferation test results in the experimental examples of this invention; Figure 8 This is a graph showing the concentration of standard proteins in the experimental examples of this invention. Figure 9 The graph shows the results of protein concentration and relative ALP activity levels in each group of materials in the experimental examples of this invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0021] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0022] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0023] Example 1 This embodiment provides a calcined bone meal, which is prepared by the following method: Impurity removal: Take fresh bovine femurs, remove visible impurities such as muscle tissue, periosteum and fat from the bone surface, rinse with clean water and boil for 7-8 hours, changing the water during this period until there are no obvious oily floating substances on the surface. Then wash to remove surface grease and dry in a drying oven at 75℃ for 1 hour to remove residual moisture.

[0024] Sodium hydroxide soaking: After the pretreatment step is completed, the treated bovine femur is soaked in 1 mol / L NaOH solution for 36 h. During the soaking, the soaking solution is changed at 30 min and 5 h. After soaking, it is rinsed 2 to 3 times with ultrapure water (UP water) until there is no obvious odor. Then it is dried in a drying oven at 75℃.

[0025] Hydrogen peroxide soaking: After the sodium hydroxide soaking step, the treated bovine femur is soaked in a 30% hydrogen peroxide solution for 36 hours. During this period, the soaking solution is changed at 30 minutes and 5 hours. After soaking, the femur is rinsed 2-3 times with ultrapure water (UP water) to remove the leached oil and residue. Then it is dried in a drying oven at 75°C.

[0026] Calcination: After the hydrogen peroxide soaking step, the bone meal is calcined at 850°C for 3 hours using an intelligent box-type resistance furnace, and then naturally cooled with the furnace. After that, it is ground for 1 hour using a planetary ball mill at 50 rpm and passed through a 60-mesh sieve to obtain the finished calcined bone meal.

[0027] Example 2 This embodiment provides a bone graft material modified with traditional Chinese medicine ingredients, which is prepared through the following steps: Preparation of calcined bone meal: Same as in Example 1.

[0028] Preparation of simulated body fluids: Prepare 1 mg / mL salvianolic acid solution and 0.5 mg / mL baicalin solution, and prepare them according to the raw material ratios in Table 1: Blank simulated body fluid, denoted as SBF; An SBF solution containing 10 μg / ml salvianolic acid B is denoted as SBF-A(100), where “A(100)” represents that the loading of salvianolic acid B in the corresponding bone graft material is 100 μg / g. An SBF solution containing 20 μg / ml salvianolic acid B is denoted as SBF-A(200), where “A(200)” represents that the loading of salvianolic acid B in the corresponding bone graft material is 200 μg / g. An SBF solution containing 1 μg / ml baicalin is denoted as SBF-B(10), where “B(10)” represents that the baicalin loading in the corresponding bone graft material is 10 μg / g. An SBF solution containing 2 μg / ml baicalin is denoted as SBF-B(20), where “B(20)” represents that the baicalin loading in the corresponding bone graft material is 20 μg / g. An SBF solution containing 10 μg / ml salvianolic acid B and 1 μg / g baicalin is denoted as SBF-A(100)+B(10), where A(100)+B(10) represents that the bone graft material of the corresponding product is simultaneously loaded with 100 μg / g salvianolic acid B and 10 μg / g baicalin.

[0029] Table 1

[0030] Preparation of modified bone graft material: 1 g of the prepared calcined bone powder was immersed in SBF for 24 h, then removed, ultrasonically cleaned for 30 s, and dried at 37 ℃ for 2 h. Then it was immersed in SBF-A(100) for 24 h, and then replaced with new SBF-A(100) for continued immersion. After 24 h, it was removed, ultrasonically cleaned for 30 s, and dried at 37 ℃ for 2 h to obtain 100 μg / g of salvianolic acid B modified bone graft material, denoted as salvianolic acid B-100.

[0031] The soaking process was carried out at a temperature of 37°C.

[0032] Following the same steps, the soaking solutions were replaced with SBF-A(200), SBF-B(10), SBF-B(20), and SBF-A(100)+B(10) respectively, and the following preparations were obtained: 200 μg / g of salvianolic acid B modified bone graft material, denoted as salvianolic acid B-200 (corresponding to soaking solution SBF-A(200)); 10 μg / g baicalin modified bone graft material, denoted as baicalin-10 (corresponding to soaking solution SBF-B(10)); 20 μg / g baicalin modified bone graft material, denoted as baicalin-20 (corresponding to soaking solution SBF-B(20)); The bone graft material modified by 100 μg / g salvianolic acid B and 10 μg / g baicalin was denoted as salvianolic acid B-100+baicalin-10 (corresponding to the soaking solution SBF-A(100)+B(10)).

[0033] Experimental Example 1. SEM characterization Bone powder materials obtained by grinding and sieving directly without calcination in Example 1, calcined bone powder materials obtained in Example 1, bone powder materials obtained by soaking in blank SBF only in Example 2, and modified bone graft materials (tanshinone B-100, tanshinone B-200, baicalin-10, baicalin-20, and tanshinone B-100 + baicalin-10) obtained in Example 2 were subjected to SEM characterization. The test voltage was 5 kV, and the observation magnifications were 500×, 1000×, 5000×, and 10000× to observe the changes in micromorphology before and after calcination and modification.

[0034] The results are as follows Figure 1 As shown.

[0035] SEM image of uncalcined bovine femur bone powder as shown below Figure 1As shown in -A. At magnifications of 500× and 1000×, it can be seen that most bone meal particles have a particle size of 300~900 μm, with an irregular layered structure on the surface and a relatively obvious roughness. At magnifications of 5000× and 10000×, the layered surface is smooth and relatively intact, with no obvious pore structure.

[0036] SEM image of unmodified calcined bovine femur bone powder calcined at 850℃ for 3 h is shown below. Figure 1 As shown in Figure -B, at magnifications of 500× and 1000×, most bone meal particles remain within the 300–900 μm range. Besides the layered structure, the surface of the bone meal exhibits noticeable granules and increased roughness. At magnifications of 5000× and 10000×, numerous irregularly shaped HAP crystals with diameters ranging from 15 μm to 30 μm are observed on the surface of the bone meal, with most exhibiting directional alignment. Larger particles are formed by agglomeration of smaller particles, surrounded by numerous submicron-sized particles and exhibiting significant porosity. Combined with the earlier calcination phenomena, it can be inferred that the porosity arises from the vaporization of organic matter during calcination, leading to the appearance of pores on the bone meal surface.

[0037] SEM image of bovine femur bone powder after SBF soaking is shown below. Figure 1 As shown in Figure C, at magnifications of 500× and 1000×, no significant change was observed in the particle size range of the bone powder. The surface exhibited a small, irregular layered structure with no obvious large particles. At magnifications of 5000× and 10000×, numerous tiny particles were observed, some densely packed, others more sparsely packed, with relatively few pores. A distinct layered structure was also observed, indicating that SBF solution immersion forms a denser, more regularly arranged particle layer on the bone powder surface, and this layer covers some of the original porous structure. A small number of columnar crystals were also observed on the bone powder surface, presumably residual reagents from the SBF solution.

[0038] SEM image of the modified bone graft material Tanshinone B-100 is shown below. Figure 1 As shown in Figure D, the SEM image of the modified bone graft material, Tanshinone B-200, is as follows. Figure 1 As shown in Figure -E, the surface morphology of the bone powder before and after modification with salvianolic acid B differs significantly. Observation of the salvianolic acid B-modified bone powder at 500× and 1000× magnification reveals numerous irregular structures on the surface, with a significantly increased surface roughness compared to SBF solution immersion, likely due to drug loading. Observation at 5000× and 10000× magnification reveals a large number of particles <30 μm in diameter, with irregular shapes and smooth surfaces; the bone powder surface still exhibits numerous porous structures. Although the coating surface after salvianolic acid B loading does not show similar plate-like crystals, it still exhibits significant morphological changes, indicating successful loading of salvianolic acid B.

[0039] SEM image of baicalin-10, a modified bone graft material, is shown below. Figure 1 As shown in Figure -F, the SEM image of the modified bone graft material baicalin-20 is as follows. Figure 1 -G. Observation of baicalin-modified bone powder at 500× and 1000× magnification reveals a large number of layered structures. Observation at 5000× and 10000× magnification shows a small number of particles with a diameter <30 μm. The bone powder modified with high concentration of baicalin has a greater number of large-diameter particles on the surface, indicating that different concentrations of baicalin modification affect the particle size of HAP crystals in biomimetic co-deposition. At the same time, under 10000× magnification, unidentified plaque-like accumulation structures were observed on the surface of bone powder modified with both salvianolic acid B and baicalin. The morphology of bone powder modified with salvianolic acid B and baicalin also showed significant differences. The latter showed fewer and smaller large-diameter particles, indicating that the addition of drugs changes the morphology of the coating, and different drugs have different effects.

[0040] SEM images of the modified bone graft material, Danshensu B-100 + Baicalin-10, are shown below. Figure 1 As shown in -H, its surface is based on a layered arrangement, and its overall roughness is higher than that of the single modified group, with the particle size generally less than 30 μm.

[0041] In addition, both the bone powder modified with salvianolic acid B and baicalin showed a lamellar accumulation structure on the surface, and the two had significantly different morphologies.

[0042] 2. XRD Testing Bone powder material soaked only in blank SBF as described in Example 2, modified bone graft materials (tanshinone B-100, tanshinone B-200, baicalin-10, baicalin-20), and tanshinone B-100 + baicalin-10 obtained in Example 2 were subjected to XRD testing. The bone powder was dry and uniformly dispersed. The instrument light source was Cu-Ka, the scanning range was 10-70°, and the scanning mode was continuous scanning. The results are as follows Figure 2 As shown.

[0043] The X-ray diffraction pattern of unmodified bone powder (soaked in blank SBF solution) was compared with the X-ray diffraction pattern of hydroxyapatite (HAP) from JCPDS (74-0565). It was found that the calcined bone powder could observe diffraction peaks (002), (210), (211), (300), etc., which are the main diffraction peaks of HAP. This indicates that the main component of the calcined bovine femur bone powder is HAP and it is a single phase.

[0044] It should be noted that HAP decomposes into β-TCP under high-temperature conditions. The higher the temperature, the lower the proportion of HAP and the higher the proportion of β-TCP. Furthermore, β-TCP is more degradable than HAP. Therefore, the proportion of β-TCP can be controlled by adjusting the temperature, thereby controlling the degradability of the bone meal. In this experiment, the calcination temperature was 850 ℃, which did not reach the minimum temperature for HAP decomposition. Therefore, a single HAP phase appeared on the surface of the bone meal after calcination. Considering the poor degradability of single HA, if the addition of the β-TCP phase is required later, a muffle furnace with a higher maximum temperature should be used for calcination, and the optimal proportions of each phase for degradability should be investigated.

[0045] The addition of salvianolic acid B and baicalin did not alter the coating structure. Furthermore, due to the biomimetic co-deposition method used to composite the traditional Chinese medicine components, a new HAP coating could be formed on the bone powder surface. Figure 2 As can be seen from the results, the diffraction peaks become sharper after the addition of the drug components, and the relative intensity of some characteristic peaks also increases significantly, indicating that the doping of salvianolic acid B and baicalin can improve the crystallinity of the newly formed HAP coating.

[0046] 3. FT-IR test The calcined bone powder obtained in Example 1, the bone powder material in Example 2 after only blank SBF soaking, the modified bone graft material obtained in Example 2 (tanshinone B-100, baicalin-10, and tanshinone B-100 + baicalin-10) were subjected to FT-IR testing. Samples were prepared using potassium bromide pellets, and the scanning range was 450-4000 cm⁻¹. -1 The following tests were conducted to determine the composition of the bone meal.

[0047] The results are as follows Figure 3 As shown.

[0048] The calcined bone powder from Example 1 was used as a control group, and its infrared spectrum showed obvious HAP characteristic absorption peaks.

[0049] Among them, 3571 cm -1 The peak is the contraction vibration peak of -OH; 961 cm⁻¹ -1 CO3 2- Characteristic absorption peak; 1048 cm⁻¹ -1 PO4 3- The stretching vibration peak; 571 cm -1 This is generated by the deformation vibration of PO, which indicates that the control group contains HAP. The infrared spectrum of bone powder soaked in SBF solution contains the same characteristic peak as HAP, indicating that the coating component formed on the surface of bone powder by biomimetic co-deposition method is also HAP. At the same time, tanshinone B and baicalin did not change the composition of the bone powder itself and the formed coating.

[0050] CO3 2-The presence of CO3 in the simulated body fluid may be due to the presence of Na2CO3 in the raw materials used to prepare the simulated body fluid, with some ions remaining on the surface of the bone powder after washing; alternatively, it could be due to the reaction of organic matter in the bovine femur with O2 during calcination, producing CO3 under high-temperature conditions. 2- Compared with the control group, the infrared spectra of bone powder modified with 100 μg / g salvianolic acid B (salvianolic acid B-100) and 10 μg / g baicalin (baicalin-10) also showed characteristic peaks belonging to the two drugs. (1660 cm⁻¹) -1 and 1610 cm -1 For the vibrational peak of the aromatic skeleton [70-71] The calcined bone powder did not contain aromatic hydrocarbons, indicating that baicalin successfully entered the coating; and at 1264 cm -1 1610 cm -1 The characteristic peak of salvianolic acid B appeared at the location, indicating the successful loading of salvianolic acid B.

[0051] Meanwhile, both the SBF-modified group and the two groups of modified traditional Chinese medicines reached a depth of 1415 cm. -1 and 1456 cm -1 A peak appears, along with free CO3. 2- The infrared peaks are different; these two peaks represent CO3. 2- The entry of CO2 into the HAP lattice may occur during the immersion modification process, where CO2 from the air dissolves into the SBF solution and replaces the anions in the HAP, thus causing the peak value. Biomimetic co-deposition forms different calcium-phosphorus phases on the material surface, mostly a mixture of octacalcium phosphate and hydroxyapatite. However, the addition of different drugs may alter the chemical reactions occurring during deposition, thereby affecting the formation of the calcium-phosphorus phase. In the experiment, the coating composition was HAP before and after drug addition, possibly due to the influence of the bone powder's own composition and internal residual ions (such as calcium and sodium). Meanwhile, the coating composition remained unchanged after the addition of salvianolic acid B and baicalin, indicating that the two drugs did not affect the deposition reaction.

[0052] 4. EDS Elemental Analysis The bone powder material obtained without calcination in Example 1, the calcined bone powder material obtained in Example 1, the bone powder material after being soaked in blank SBF only in Example 2, and the modified bone graft material obtained in Example 2, namely, salvianolic acid B-100, salvianolic acid B-200, baicalin-10, baicalin-20, and salvianolic acid B-100 + baicalin-10, were used to determine the atomic percentage of the main elements in the micro-region.

[0053] The results are shown in Table 2 and Figure 4 As shown in Table 2, which compares the atomic percentages of various bovine bone meal materials.

[0054] Table 2

[0055] Figure 4 middle: Figure 4 -A is the EDS elemental analysis diagram of the bone powder material obtained without the calcination step in Example 1; Figure 4 -B is the EDS elemental analysis diagram of the calcined bone meal material in Example 1; Figure 4 -C is the EDS elemental analysis diagram of the bone meal material after only being soaked in blank SBF in Example 2; Figure 4 -D is the elemental analysis diagram of the modified bone graft material, salvianolic acid B-100, in Example 2; Figure 4 -E is the EDS elemental analysis diagram of the modified bone graft material Danshensu B-200 in Example 2; Figure 4 -F is the EDS elemental analysis diagram of baicalin-10, the modified bone graft material in Example 2; Figure 4 -G is the EDS elemental analysis diagram of baicalin-20, the modified bone graft material in Example 2; Figure 4 -H is the EDS elemental analysis diagram of the modified bone graft material tanshinone B-100 + baicalin-10 in Example 2.

[0056] The results show that the bovine bones contained elements such as Ca, P, O, Na, and C before calcination. After calcination, the C element in the bovine femur disappeared, indicating that the C-containing substances combined with O2 and escaped during the calcination process, and the bovine femur was fully burned. The Na element may have been partially absorbed during the initial NaOH soaking process. + The significantly higher O content before and after calcination is likely due to residual oxygen. This could be caused by higher air humidity during testing, leading to greater water absorption by the bone meal.

[0057] After soaking in blank SBF, the P content decreased and the C content increased. Combined with the infrared test results, some PO4 in hydroxyapatite was found to be present. 3- CO3 2- replace.

[0058] The types of elements contained in bone powder modified with salvianolic acid B and baicalin remained unchanged. However, the element content of bone powder varied under different types and concentrations of traditional Chinese medicine modification. This may be because the traditional Chinese medicine affected the chemical reaction during the deposition of the calcium-phosphorus coating, thus affecting the ionic composition of the coating.

[0059] Although both salvianolic acid B and baicalin contain carbon in their chemical structures, Table 2 shows that the carbon content on the surface of the modified bone powder is not significantly related to the type and concentration of the modified drug. This is likely because the solution formulation contains CO3 when using the biomimetic co-deposition method for drug loading. 2- Ions, some of which may remain on the surface and in the pores of bone powder, may result in the bone powder containing some carbon elements before the drug is loaded. Since the concentration of loaded drug is low and there is also the possibility of external contamination, it is impossible to infer the amount of drug loaded based on the content of carbon elements.

[0060] 5. Hydrophilicity test The calcined bone powder material obtained in Example 1, the bone powder material after being soaked in blank SBF only in Example 2, and the modified bone graft materials of salvianolic acid B-100, salvianolic acid B-200, baicalin-10, baicalin-20, and salvianolic acid B-100 + baicalin-10 obtained in Example 2 were compressed into tablets using a tablet press. Three tablets of each type of bone powder were prepared as parallel samples. A quantitative amount of UP water was added to the bone powder tablets using a water contact angle meter. Then, the time required for an equal volume of UP water to be completely absorbed by the tablets was tested. The shorter the water absorption time, the better the hydrophilicity of the bone powder and the more porous structures it has.

[0061] The results are shown in Table 3 and Figure 5 As shown in the figure. Table 3 is a comparison table of water absorption time for each bone meal material.

[0062] Table 3

[0063] Figure 5 middle: Group A represents the water absorption time of the calcined bone meal material in Example 1; Group B represents the water absorption time of the blank SBF-soaked bone meal material in Example 2; Group C represents the water absorption time of the bone graft material, Danshensu B-100, in Example 2; Group D represents the water absorption time of the bone graft material Tanshinone B-200 in Example 2; Group E represents the water absorption time of baicalin-10, the bone graft material in Example 2; Group F represents the water absorption time of baicalin-20, the bone graft material in Example 2; Group G represents the water absorption time of the bone graft material Danshensu B-100 + Baicalin-10 in Example 2.

[0064] Figure 5 There was no statistically significant difference between groups labeled with the same lowercase letter (a / b) (P>0.05), while there was a statistically significant difference between groups labeled with different lowercase letters (a / b) (P<0.001).

[0065] The results show that the hydrophilicity of bone meal is significantly improved after soaking in SBF. Based on the SEM observation results, it is speculated that the irregular layered structure on the surface of bone meal increases and the interlayer pores increase after soaking, thus accelerating the water absorption rate.

[0066] Compared with bone powder after simple calcination, the water absorption time of each modified group was significantly shortened (P<0.001), and the hydrophilicity was significantly improved. Combined with SEM morphology analysis, it is speculated that this is related to the increased layered structure and porosity of the surface. The water absorption time of bone powder modified with traditional Chinese medicine was significantly shorter than that of the SBF soaking group (P<0.001), which can be attributed to the abundance of hydrophilic groups such as -COOH and -OH in the active ingredients of the two traditional Chinese medicines, and may also be related to the increased surface roughness of the material after drug modification. The hydrophilicity of the salvianolic acid B group was significantly better than that of the baicalin group (P<0.001), which may be related to its molecular structure and effective loading characteristics on the bone powder surface. There was no difference in hydrophilicity between salvianolic acid B (100, 200) and baicalin (10, 20) under different loading concentrations (P>0.05). The three groups of salvianolic acid B (100, 200) and salvianolic acid B-100 + baicalin-10 showed the best hydrophilicity (P < 0.001) and there was no significant difference among the groups (P > 0.05).

[0067] The hydrophilicity of a material affects the types and structures of specific proteins that are adsorbed, thereby influencing the type of adsorbed cells and their differentiation direction, ultimately affecting the osteogenic properties of the material. Further biological evaluations should be conducted to further elucidate the impact of bone meal hydrophilicity on osteogenic properties.

[0068] Summary of physicochemical properties: As can be seen from the experimental results in sections 1-5 above, SEM (scanning electron microscopy) revealed that compared with unmodified bone powder, the bone powder modified with traditional Chinese medicine formed a more significant macroporous structure on its surface. This optimized three-dimensional structure not only facilitates cell adhesion, spreading, and migration but also provides channels for the transport of nutrients and metabolites, creating a better osteogenic microenvironment. XRD and FT-IR analyses confirmed that the main component of calcined bone powder is HAP, and the crystallinity of HAP increased after modification with traditional Chinese medicine, reaching a higher concentration at 1660 cm⁻¹. -1 With 1610 cm -1Characteristic peaks of the two traditional Chinese medicine extracts were observed at various locations, indicating that salvianolic acid B and baicalin were successfully loaded onto the bone powder surface. Hydrophilicity testing showed that the modification with traditional Chinese medicine significantly improved the hydrophilicity of the bone powder (P<0.001). Compared with the SBF soaking group (water absorption time approximately 4.28 seconds), the water absorption time of the salvianolic acid B and baicalin synergistic modification group was significantly shortened to approximately 1.45 seconds (P<0.001). The improved hydrophilicity helps promote the adsorption of early serum proteins on the material surface, thereby mediating and accelerating the subsequent adhesion and activation of osteoblasts, laying a good foundation for the osteogenic process. Based on the above physicochemical performance test results and combined with the advantages of the two traditional Chinese medicine components, the salvianolic acid B-100 + baicalin-10 group was selected for subsequent in vitro biological evaluation.

[0069] 6. Co-culture of mouse preosteoblasts with composite materials Groups: blank group (MC3T3-E1 cells only, Cell Culture and Preservation Center of Chinese Academy of Sciences), control group (commercial bone graft material, Beijing Paisheng Biotechnology Co., Ltd.), and experimental group (tanshinone B-100 + baicalin-10). After sterilization with 3kGy γ-rays, the cell compatibility and cell-induced osteogenicity of the above materials were compared and studied.

[0070] MC3T3-E1 cells were revived and cultured in a cell incubator at 37°C and 100% humidity (FORMA 371, Thermo Fisher Scientific, USA). They were then seeded into culture plates containing materials for each group according to experimental requirements, in preparation for subsequent in vitro cell experiments.

[0071] 7. Cell adhesion test Based on the grouping described in Section 6 above (Co-culture of mouse preosteoblasts and composite materials), MC3T3-E1 cells were seeded at a density of 2.5 × 10⁻⁶ cells in 48-well cell culture plates containing materials from each group. 4 pcs / cm 2 Add 150 μL of complete cell culture medium to each well. Observe the cell adhesion on the surface of the control group (commercial bone powder) and the experimental group (Example 2, Danshensu B-100 + Baicalin-10) using SEM (500×, 3500× magnification).

[0072] The adhesion of MC3T3-E1 cells to the surfaces of the control group (commercial bone meal) and the experimental group (Example 2, Danshensu B-100 + Baicalin-10) is shown in the figure. Figure 6 As shown. Among them, Figure 6 -A and Figure 6 -B indicates the control group. Figure 6 -C and Figure 6 -D indicates the experimental group. Figure 6 The left column shows cell cluster images at 500x magnification, and the right column shows cell cluster images at 3500x magnification. Figure 6The red arrow in -D indicates the MC3T3-E1 cell cluster under 3500x magnification.

[0073] As shown in the figure, cells adhered well in both groups. The white granules on the surface of the control group were cells or cell clusters, while the black shading on the surface of the experimental group was adhered cell clusters.

[0074] 8. Cell proliferation test Based on the grouping described in Section 6 above (co-culture of mouse preosteoblasts and composite materials), the cells were cultured at a density of 5 × 10⁶ cells / year. 3 cells / cm 2 The cells were seeded at the appropriate density into 48-well cell culture plates containing materials from each group, with 150 μL of complete cell culture medium added to each well. The cell counts of the control group (commercial bone meal) and the experimental group (Example 2, Danshensu B-100 + Baicalin-10) were measured at days 1, 3, 5, and 7 using CCK-8 reagent (APExBIO Technology Co., Ltd., USA). The OD value at 450 nm was measured using a microplate reader (Synergy H1, Berten Instruments Co., Ltd., USA). The cell count at each time point was represented by the OD value of the blank group after standardization. Each group was repeated three times.

[0075] The results are as follows Figure 7 As shown, Figure 7 The bar chart on the left shows the standardized OD values, and the line chart on the right shows the cell proliferation curves of each group of materials over 7 days.

[0076] The results showed that the number of cells in all three groups increased with increasing culture time. By day 7, compared with the blank group and the control group, the cell proliferation rate in the experimental group reached 32.53±1.76 times, which was significantly higher than that in the other two groups (P<0.05).

[0077] 9. Results of relative activity level determination of alkaline phosphatase (ALP) Based on the grouping described in Section 6 (Co-culture of mouse pre-osteoblasts and composite materials), the protein concentrations in the control and experimental groups were tested on days 5 and 10 using the BCA protein kit; ALP activity was detected using an ALP activity kit (Shanghai Beyotime Biotechnology Co., Ltd.). A protein standard curve was plotted to calculate cell protein concentrations. Using 0.5 mmol / L phenol standard solution as a control, 50 μL of phenol standard solution, cell lysis buffer, and buffer (blank control) were added to 96-well plates, followed by 150 μL of chromogenic substrate. The plates were incubated at 37°C for 2 hours, and the absorbance at 405 nm was measured to calculate relative ALP activity. Each group was repeated three times.

[0078] The results are as follows Figure 8 , Figure 9 As shown, the standard protein concentration curve was obtained by measurement ( Figure 8The protein concentrations of the materials in the blank group, control group, and experimental group were calculated at 5 and 10 days. Figure 9 -A), the OD values ​​of the three groups of materials were detected by the ALP reagent detection kit and the relative activity levels of ALP in the three groups of materials were calculated. Figure 9 The results showed that the relative activity levels of ALP in all three groups of materials increased over time. The relative activity level of ALP in the experimental group was higher than that in the control group and the blank group (P<0.01), indicating that its osteogenic induction ability was superior.

[0079] Summary of in vitro biological evaluation: The in vitro cell experiments described in sections 6-9 above demonstrate that the herbal compound bone graft material provided by this invention exhibits good biocompatibility and osteogenic capacity. Regarding biocompatibility, MC3T3-E1 cells adhered well to the material surface and exhibited a relaxed morphology, indicating good cell compatibility. In terms of cell proliferation, by day 7 of culture, the experimental group (tanshinone B-100 + baicalin-10) showed a cell proliferation fold of 32.53 ± 1.76 times, significantly superior to the blank control group and the commercial bone powder material (P < 0.05), indicating that the material can continuously support highly active cell growth and expansion. In the detection of alkaline phosphatase (ALP) activity, a key indicator of early osteogenic differentiation, the experimental group showed significantly higher relative activity than the control group on days 5 and 10 (P < 0.01). This significant increase in ALP activity clearly indicates that the herbal compound bone powder can effectively promote the differentiation process of pre-osteoblasts into osteoblasts, possessing significant early osteogenic induction potential.

[0080] In summary, this invention provides a bone graft material modified with traditional Chinese medicine ingredients and its preparation method. By loading traditional Chinese medicine extracts onto bone material and modifying the bone material, a bone graft material with good biocompatibility, osteogenic capacity, and superior osteogenic induction potential can be obtained. This product material can be used in dental bone grafting, particularly for bone repair of alveolar bone defects.

[0081] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A bone graft material modified with traditional Chinese medicine ingredients, characterized in that, It is prepared from raw materials including the following components: calcined bone powder and traditional Chinese medicine extracts; The herbal extracts include Salvia miltiorrhiza extract and / or Scutellaria baicalensis extract.

2. The bone graft material according to claim 1, characterized in that, The calcined bone powder is prepared by calcining and grinding bovine femur, the tanshinone extract is salvianolic acid B, and the scutellaria baicalensis extract is baicalin.

3. A method for preparing a bone graft material as described in any one of claims 1 to 2, characterized in that, It includes the following steps: Preparation of calcined bone meal: Fresh bone was taken and its surface muscle tissue, periosteum, fat and other impurities were removed. After boiling in water, it was soaked in sodium hydroxide solution and hydrogen peroxide solution respectively. After washing, it was calcined and then ground and sieved to obtain calcined bone meal for later use. Preparation of simulated body fluid: Take the Chinese herbal extract, prepare the extract solution, and then prepare the extract solution into simulated body fluid to obtain simulated body fluid containing the Chinese herbal extract. Also prepare a blank simulated body fluid without the Chinese herbal extract. The blank simulated body fluid is denoted as SBF solution. Preparation of modified bone graft material: Take the prepared calcined bone powder and immerse it in blank SBF solution. After immersion, take out the bone powder material, wash and dry it, and then immerse it in SBF solution containing Chinese herbal extract. After immersion, take out the solid material, wash and dry it to obtain Chinese herbal modified bone graft material.

4. The preparation method according to claim 3, characterized in that, In the calcined bone powder preparation step, the sodium hydroxide solution used has a concentration of 1 mol / L, and the hydrogen peroxide solution used has a mass concentration of 30%.

5. The preparation method according to claim 3, characterized in that, In the calcined bone powder preparation step, the calcination temperature is 600-900℃ and the calcination time is 2-6 h.

6. The preparation method according to claim 3, characterized in that, In the simulated body fluid preparation step, the blank simulated body fluid SBF solution is prepared from raw materials comprising the following components: NaCl, MgCl2·6H2O, CaCl2, Na2HPO4, Na2CO3, and UP water.

7. The preparation method according to claim 3, characterized in that, In the simulated body fluid preparation step, the content of the traditional Chinese medicine extract in the SBF solution containing the traditional Chinese medicine extract is 1-20 μg / ml.

8. The preparation method according to claim 3, characterized in that, In the preparation steps of the modified bone graft material, the calcined bone powder is first immersed in a blank SBF solution, washed and dried, and then immersed in an SBF solution containing traditional Chinese medicine extracts. After the immersion is completed, the solid material is taken out and the liquid material is discarded. Then, a new SBF solution with the same composition containing traditional Chinese medicine extracts is taken, and the taken-out solid material is immersed in it again. After the immersion is completed, the solid material is taken out, washed and dried to obtain the traditional Chinese medicine modified bone graft material.

9. The preparation method according to claim 8, characterized in that, The soaking time for a single soaking is 24 hours.

10. The application of a bone graft material modified with traditional Chinese medicine components as described in any one of claims 1 to 2 in dental bone graft materials.

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