An injectable composite bone graft material and its preparation method

Abstract

The present application discloses an injectable composite bone graft material and a preparation method thereof, relating to the technical field of bone graft materials. An injectable composite bone graft material is prepared from raw materials comprising the following components: a mixed powder and a composite solution; wherein, the raw materials of the mixed powder include bovine bone powder and pearl powder; the raw materials of the composite solution include a gelatin solution and a citric acid solution. The material has the advantages of good osteogenic performance, appropriate degradation and absorption rate, high mechanical strength, convenient surgical operation, etc. It has low cost and can be applied to maxillary sinus floor elevation surgery, effectively improving the surgical effect.

Description

Technical Field

[0001] This application relates to the technical field of bone graft materials, and particularly relates to an injectable composite bone graft material and a preparation method thereof. Background Art

[0002] With the popularization of oral implant surgery, the internal sinus floor elevation is widely used in patients with insufficient remaining bone mass in the posterior maxilla. This surgery implants bone graft materials between the bottom of the maxillary sinus and the lifted mucosa through the alveolar ridge crest to increase the bone mass between the alveolar ridge crest and the bottom of the maxillary sinus, so as to carry out dental implant surgery.

[0003] For the internal sinus floor elevation, an ideal bone graft material needs to have the following properties: no risk of immune rejection or pathogen infection, be able to adapt to the host bone defect environment and perform bone integration, be gradually degraded and replaced by new bone, and in addition, have some or all of the basic biological properties of bone graft materials: osteoinductivity, osteoconductivity, and bone regeneration.

[0004] Clinically, autologous bone, allogeneic bone, xenogeneic bone, synthetic bone and other bone graft materials are often used in the internal sinus floor elevation to increase the bone mass at the bottom of the maxillary sinus. However, the above-mentioned various bone graft materials are granular, which have the defects that they are not convenient for doctors to operate when implanted into the bottom of the maxillary sinus through the alveolar ridge, and the bone graft materials at the bottom of the maxillary sinus are easily affected by maxillary sinus pneumatization and cause bone resorption and cannot maintain good spatial stability. Therefore, it is urgent to develop a new type of composite bone graft material to improve the surgical effect and reduce the cost. Summary of the Invention

[0005] The purpose of this application is to provide an injectable composite bone graft material and a preparation method thereof. The material has the advantages of good osteogenic performance, appropriate degradation and absorption rate, high mechanical strength, convenient surgical operation, etc. Its cost is low, and it can be applied to the internal sinus floor elevation, and can effectively improve the surgical effect.

[0006] The technical solution of this application is as follows:

[0007] On the one hand, the embodiment of this application provides an injectable composite bone graft material, which is prepared from raw materials including the following components: mixed powder and composite solution;

[0008] Among them, the raw materials of the above-mentioned mixed powder include bovine bone powder and pearl powder;

[0009] The raw materials of the above-mentioned composite solution include gelatin solution and citric acid solution.

[0010] Further, in some embodiments of this application, in the above-mentioned mixed powder, the mass ratio of pearl powder is 25-40%;

[0011] In the above-mentioned composite solution, the volume ratio of gelatin solution is 15-35%.

[0012] Further, in some embodiments of the present application, in the above-mentioned mixed powder, the above-mentioned bovine bone powder is prepared by calcining and grinding bovine femurs, and the above-mentioned pearl powder is pharmaceutical-grade pearl powder.

[0013] Further, in some embodiments of the present application, in the above-mentioned composite solution, the gelatin solution used is prepared from dry gelatin, and its concentration is 11.11%;

[0014] The concentration of the citric acid solution used is 25%.

[0015] Further, in some embodiments of the present application, in the raw material components, the solid-liquid ratio of the above-mentioned mixed powder to the above-mentioned composite solution is 0.35 mL / g.

[0016] On the other hand, the embodiment of the present application also provides a preparation method of the above-mentioned injectable composite bone graft material, which includes the following steps:

[0017] Preparation of bovine bone powder: Remove the surface muscles, periosteum, fat and other impurities from bovine bones, soak them in sodium hydroxide solution and hydrogen peroxide solution respectively after boiling, wash them, then calcine them, and then grind and sieve them to obtain bovine bone powder for standby;

[0018] Preparation of mixed powder: Mix the prepared bovine bone powder and pearl powder in proportion to obtain mixed powder for standby;

[0019] Preparation of gelatin solution: Weigh dry gelatin and add UP water to make the dry gelatin swell to a gel state, and then perform constant temperature treatment to prepare a gelatin solution for standby;

[0020] Preparation of composite solution: Mix the prepared gelatin solution and citric acid solution in proportion to obtain a composite solution for standby;

[0021] Preparation of product material: Mix the prepared mixed powder and composite solution in proportion to obtain a composite slurry, then inject the composite slurry into a mold, and then perform constant temperature curing to obtain the product injectable composite bone graft material.

[0022] Further, in some embodiments of the present application, in the above-mentioned bovine bone powder preparation step, the concentration of the sodium hydroxide solution used is 1 mol / L, and the concentration of the hydrogen peroxide used is 30%.

[0023] Further, in some embodiments of the present application, in the above-mentioned bovine bone powder preparation step, calcination is carried out in a muffle furnace at 850 °C, and then grinding is carried out in a planetary ball mill at a rate of 50 r / min, and bovine bone powder is obtained after sieving.

[0024] Further, in some embodiments of the present application, in the above-mentioned gelatin solution preparation step, the gelatin solution is prepared by subjecting it to a constant temperature treatment in an electrothermal constant temperature oven at 37°C.

[0025] Further, in some embodiments of the present application, in the above-mentioned product material preparation step, the product injectable composite bone graft material is obtained by subjecting it to a constant temperature curing in an electrothermal constant temperature forced air drying oven at a temperature of 37°C and a humidity of 100%.

[0026] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0027] In view of the above aspects, the embodiments of the present application provide an injectable composite bone graft material and a preparation method thereof. The bone graft material of the product is subjected to injectable modification using bovine bone powder as the base material, and the bovine bone powder / pearl powder mixed powder is combined with the gelatin / citric acid composite solution to form a composite slurry, which can effectively retain organic components such as water-soluble proteins in the pearl powder and improve the biocompatibility and osteoinductivity of the novel composite bone cement; and by compounding the gelatin solution and the citric acid solution, the anti-collapse property and mechanical strength of the product bone graft material can be effectively improved.

[0028] Compared with the commonly used commercial particulate bone graft materials on the market, the product material provided by the present application has more excellent cell compatibility, cell adhesion, and cell osteoinductivity. It has low cost and excellent performance, and has great application potential in the internal sinus floor elevation surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can also be obtained based on these drawings without creative efforts.

[0030] Figure 1 It is a schematic diagram of the degradation rate in the determination of the in vitro degradation rate of Experimental Example 1;

[0031] Figure 2 It is a schematic diagram of the degradation situation in the determination of the in vitro degradation rate of Experimental Example 1;

[0032] Figure 3 It is a schematic diagram of the degradation rate in the determination of the in vitro degradation rate of Experimental Example 2;

[0033] Figure 4 It is a schematic diagram of the degradation situation in the determination of the in vitro degradation rate of Experimental Example 2;

[0034] Figure 5 It is the SEM scanning image in Experimental Example 3;

[0035] Figure 6 It is the EDS detection result graph in Experimental Example 3;

[0036] Figure 7 It is the schematic diagram of the elemental mass percentage in Experimental Example 3;

[0037] Figure 8 It is the infrared spectrum graph in Experimental Example 3;

[0038] Figure 9 It is the XRD detection result graph in Experimental Example 3;

[0039] Figure 10 It is the particle size distribution graph in Experimental Example 3;

[0040] Figure 11 It is the schematic diagram of cell adhesion in Experimental Example 4;

[0041] Figure 12 It is the schematic diagram of cell proliferation in Experimental Example 4;

[0042] Figure 13 It is the standard protein concentration curve graph in Experimental Example 4;

[0043] Figure 14 It is the schematic diagram of protein concentration and relative ALP activity level in Experimental Example 4;

[0044] Figure 15 It is the schematic diagram of the slurry morphology in Experimental Example 5. Detailed implementation manners

[0045] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0046] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other.

[0047] The features and performance of this application will be further described in detail below in combination with the embodiments.

[0048] Embodiment 1

[0049] This embodiment provides a kind of bovine bone powder, which is prepared by the following method:

[0050] Remove organic impurities such as muscle, periosteum, and fat from commercially available bovine femurs. Boil them for 8 hours to completely remove the grease, then immerse them in 1 mol / L NaOH solution for 36 hours. After soaking, use ultra-pure water (UP) to wash off the NaOH on the surface of the bovine femurs. Then immerse the washed bovine femurs in 30% hydrogen peroxide solution for 36 hours to remove the residual grease in the pores of the bovine femurs. After soaking, wash the bovine femurs 3 times with UP water and set aside.

[0051] Then calcine the treated bovine femurs in a muffle furnace at 850 °C for 3 hours. After that, put the calcined bovine femurs into a planetary ball mill and grind them at a rate of 50 r / min for 1 hour. Then sieve them to obtain bovine bone powder.

[0052] Example 2

[0053] This example provides an injectable pearl powder / bovine bone powder composite bone graft material, which is prepared by the following method:

[0054] Preparation of mixed powder: Take the bovine bone powder prepared in Example 1, add pearl powder and mix to obtain mixed powder, and set aside; among them, the mass ratio of the added pearl powder in the mixed powder is 35%.

[0055] Preparation of product material: At a solid-liquid ratio of 0.35 mL / g, add a 25% citric acid solution to the prepared mixed powder to obtain a composite slurry. Then inject the obtained composite slurry into a cylindrical silicone model with a diameter of 6 mm and a height of 5 mm through a 5 mL medical syringe, and place it in an electrothermal constant temperature forced air drying oven at a temperature of 37 °C and a humidity of 100% for curing. After curing, demold and dry to obtain the product bone graft material.

[0056] Example 3

[0057] This example provides an injectable pearl powder / gelatin / bovine bone powder composite bone graft material, which is prepared by the following method:

[0058] Preparation of mixed powder: The same mixed powder as prepared in Example 2.

[0059] Preparation of gelatin solution: Weigh 11.11 g of dry gelatin and add 88.89 mL of UP water. Wait for the dry gelatin to fully swell to a gel state, seal it in a wide-mouth bottle, and place it in an electrothermal constant temperature oven at 37 °C for 12 hours to form an 11.11% gelatin solution, and set aside.

[0060] Preparation of composite solution: Mix the prepared gelatin solution with a 35% citric acid solution to obtain a composite solution, and set aside; among them, the volume ratio of the added gelatin solution in the composite solution is 35%.

[0061] Product material preparation: The mixed powder is mixed with the composite solution at a solid-liquid ratio of 0.35 mL / g to obtain a composite slurry. Then, the obtained composite slurry is injected into a silicone mold through a 5 mL medical syringe and placed in an electrothermal constant temperature blast drying oven at a temperature of 37 °C and a humidity of 100% for curing. After curing, demolding and drying are carried out to obtain the product bone graft material.

[0062] Experimental Example 1

[0063] The purpose of this experimental example is to optimize the ratio of bovine bone powder to pearl powder;

[0064] On the basis of the solution of Example 2, the proportion of pearl powder added in the preparation step of the mixed powder is changed to prepare multiple groups of pearl / bovine bone powder composite bone graft materials, and the performance of each group of product materials is tested.

[0065] The addition amount of pearl powder in each group is shown in Table 1:

[0066] Table 1

[0067] Group Pearl powder / wt% Remarks 1 0 Pearl powder not added 2 25 / 3 30 / 4 35 Same as Example 2 5 40 / 6 45 / 7 50 /

[0068] The injectability of each group of product materials was measured, and the specific method is as follows:

[0069] A 5 mL syringe filled with the pearl / bovine bone powder composite slurry was vertically fixed on the base of a universal mechanical testing machine, and a force with a speed of 1 mm / min was applied to the push rod of the syringe. The test ended when the force reached 100 N. The injectability formula of the material is as follows:

[0070]

[0071] M 0 is the mass of the syringe itself, M 1 is the mass of the syringe after loading the composite slurry, M 2 is the mass of the syringe after the test is completed.

[0072] Each group of samples was repeatedly detected 3 times, and the average value was taken. The composite slurry with a high injectability was selected for subsequent research.

[0073] The results are shown in Table 2:

[0074] Table 2

[0075]

[0076]

[0077] Note: a: P < 0.05 when the Nth group is compared with the 1st group; b: P < 0.05 when the Nth group is compared with the (N + 1)th group

[0078] As can be seen from the results, when the addition ratio of pearl powder is 25%-35%, there is no significant difference in the injection rate between the pearl / bovine bone powder composite slurry and the injectability of the bovine bone powder slurry (P>0.05); when the addition ratio of pearl powder is 40%-50%, as the addition amount of pearl powder increases, the injectability of the pearl / bovine bone powder composite slurry decreases accordingly, and the difference in injectability among groups is statistically significant (P<0.05).

[0079] The injectability of the slurry is related to factors such as the solid-phase particle spacing and the solid-liquid ratio: when the addition ratio of pearl powder is 40%-50%, pearl powder increases the distance between the composite powders, thus increasing the cross-linking entanglement distance between citrate ions and Ca 2+ . Therefore, at a solid-liquid ratio of 0.35 mL / mg, the composite powders cannot be completely cross-linked and entangled, resulting in solid-liquid separation of the pearl / bovine bone powder composite slurry within this range. In this experiment, when the addition ratio of pearl powder is 0-35%, the composite slurry exhibits good injectability. The 2nd, 3rd, and 4th groups of injectable pearl / bovine bone powder composite bone graft materials were selected for key research.

[0080] The setting time of each group of product materials was measured, and the specific method is as follows:

[0081] The Gillmore double-needle method was used: a self-made thick needle with a mass of 113 g and a diameter of 2.1 mm was used to measure the initial setting time, and a self-made thin needle with a mass of 445 g and a diameter of 1.1 mm was used to measure the final setting time. The injectable bone graft material was cured in an electrothermal constant-temperature hot air dryer. It was taken out of the hot air dryer every 2 minutes for needle penetration measurement. When there were no obvious needle penetration marks on the material surface, the initial setting time (initial setting time, T I ) and the final setting time (final setting time, T F ) of each group of materials were recorded. Each group of samples was detected 3 times, and the average value was taken. According to clinical requirements: 3 min ≤ T I ≤ 8 min, T F ≤ 15 min was used to evaluate each group of injectable pearl / bovine bone powder composite bone graft materials.

[0082] The results are shown in Table 3:

[0083] Table 3

[0084]

[0085]

[0086] Note: a: P<0.05 when the Nth group vs the 1st group, b: P<0.05 when the Nth group vs the N+1th group

[0087] It can be seen from the results that when the mass ratio of pearl powder added is 0%-35%, the T of the injectable pearl / bovine bone powder composite bone graft material I and T F both decrease with the increase of the addition ratio of pearl powder, and the difference between adjacent groups is statistically significant (P<0.05); when the mass ratio of pearl powder added is 35%-50%, the T of the pearl / bovine bone powder composite slurry I and T F both increase with the increase of the addition ratio of pearl powder, and the difference between adjacent groups is statistically significant (P<0.05).

[0088] The setting time of the injectable bone graft material is also related to factors such as the distance between solid-phase particles, the solid-liquid ratio, viscosity, and the type of additive. In this experiment, the viscosity of the bovine bone powder bone slurry is small and the setting time is long; the viscosity of the injectable pearl / bovine bone powder composite slurry is large, and the setting times of the injectable pearl / bovine bone powder composite bone graft materials in groups 2-7 are all less than that of the injectable bovine bone powder bone graft material (P<0.05). With the increase of the mass ratio of pearl powder, the distance between pearl / bovine bone powder particles increases, so the setting time of the 40%-50% injectable pearl / bovine bone powder composite bone graft material increases. The setting times of the injectable bone graft materials in groups 2-7 all meet the clinical requirements, but the curing times of the injectable composite materials in groups 2, 3 and 4 are shorter, and they can quickly solidify to maintain a stable spatial structure at the transplantation site, showing good application potential.

[0089] The in vitro degradation rate of the product materials of each group was measured, and the specific method is as follows:

[0090] Weigh the initial mass N of the solidified injectable bone graft material of each group 0 , add the injectable pearl / bovine bone powder composite bone graft material to the PBS solution (pH = 7.4) according to the specification of 0.2 g / ml, and transfer it to an electrothermal constant temperature hot air drying oven at a temperature of 37°C and a humidity of 100%. Observe the degradation of the injectable pearl / bovine bone powder composite bone graft material at 1, 3, 5 and 7 days respectively, and take out the material after drying and weigh it as N x at the above 5 time points. Calculate the degradation rate of each group at different times through the following formula. Each group of samples was detected 3 times and the average value was taken.

[0091]

[0092] The results are as Figure 1 and Figure 2 shown:

[0093] Figure 1Schematic diagram of the degradation rate of 1 - 7 groups of injectable bone graft materials within 7 days in PBS solution; in the figure, a indicates P < 0.05 when the Nth group is compared with the 1st group, and b indicates P < 0.05 when the Nth group is compared with the (N + 1)th group.

[0094] Figure 2 Schematic diagram of the degradation of the solidified 1 - 7 groups of injectable bone graft materials within 7 days; in the figure, A - D on the left respectively represent the degradation of each group of injectable bone graft materials on the 1st, 3rd, 5th, and 7th days.

[0095] Figure 1 The degradation rate of the 7 groups of solidified injectable bone graft materials increased with time after adding PBS solution (P < 0.01). Figure 2 Shown is the degradation of the 7 groups of injectable bone graft materials under macroscopic observation: the degradation rate of the 1st group of injectable bovine bone powder bone graft material was small (P < 0.05), and the degradation rate on the 7th day was 15.41 ± 0.27%; in the 2nd - 7th groups of injectable pearl / bovine bone powder composite bone graft materials, the degradation rate decreased with the increase in the pearl mass ratio (P < 0.05). The degradation rate of the 2nd group of injectable pearl / bovine bone powder composite bone graft material reached 63.98 ± 0.70% on the 7th day, and the degradation rate of the 7th group of injectable pearl / bovine bone powder composite bone graft material was 24.14 ± 0.38% on the 7th day.

[0096] The degradation rate of injectable bone graft materials is related to factors such as the internal structure of the material, solidification time, and the spacing of solid-phase particles, etc.: in this experiment, pore structures were generated during the solidification process of injectable bone graft materials, so the 1 - 7 groups of solidified injectable bone graft materials all had a certain degradation rate. In the 1st, 5th, 6th, and 7th groups of injectable bone graft materials, there was enough time during the solidification process to crosslink and precipitate a tightly structured surface, which could prevent the infiltration of PBS and result in less degradation of the bone graft material. In the 2nd, 3rd, and 4th groups of injectable bone graft materials, the solidification time was short, and there was not enough time during the solidification process to crosslink and precipitate a tightly structured surface to effectively prevent the infiltration of PBS solution, resulting in obvious degradation of the bone graft material. The degradation rate of an ideal injectable bone graft material should be consistent with the new bone formation rate at the transplantation site, providing a stable space for the formation of new bone and then being biodegradable and completely replaced by new bone. The degradation rate of the injectable bone graft materials in this experiment is relatively large and still cannot meet the clinical requirements for the degradation rate of bone graft materials. The 4th group of injectable composite bone graft material with a relatively low degradation rate can be selected for optimization.

[0097] The compressive strength of the product materials of each group was measured, and the specific method is as follows:

[0098] Before the test, the upper and lower ends of the cured cylindrical injectable pearl / bovine bone powder composite bone graft material were ground flat using sandpaper. Then, the samples were placed on a universal mechanical testing machine to test their compressive strength, with the instrument moving at a speed of 0.1 mm / min. When the pressure curve reached a peak and cracks appeared in the sample material, the operation of the testing machine was stopped, and the maximum pressure F (N) borne by the sample material and the compressive strength were recorded. Each group of materials was tested 3 times, and the average value was taken. Clinical data showed that the pressure range borne by the alveolar bone in the posterior maxillary molar region was 4 - 13 MPa, and based on this, injectable composite bone graft materials meeting clinical requirements were selected for subsequent research.

[0099] The results are shown in Table 4:

[0100] Table 4

[0101]

[0102]

[0103] Note: a: P < 0.05 when comparing the Nth group with the 1st group; b: P < 0.05 when comparing the Nth group with the (N + 1)th group

[0104] As can be seen from the results, the compressive strength of the injectable bovine bone powder bone graft material in the 1st group was greater than that of the injectable pearl / bovine bone powder composite bone graft materials in the 2nd - 7th groups (P < 0.05). For the compressive strength of the injectable pearl / bovine bone powder composite bone graft materials in the 2nd - 7th groups: as the mass ratio of pearl powder increased, the compressive strength of the injectable pearl / bovine bone powder composite bone graft material increased accordingly. The compressive strength of the injectable bone graft material is affected by factors such as its internal structure and setting time: an injectable bone graft material with a longer setting time can provide sufficient time for the precipitation of solid-phase particles and the growth of crystals, which helps to generate large crystal substances with a complete structure inside the injectable bone graft material. Therefore, in this experiment, it was found that the compressive strength of the injectable bone graft materials with a long setting time (the 1st, 5th, 6th, and 7th groups) was greater than that of the injectable bone graft materials with a short setting time (the 2nd, 3rd, and 4th groups): 25% - 35% injectable pearl / bovine bone powder composite bone graft material < 45 - 50% injectable pearl / bovine bone powder composite bone graft material < injectable bovine bone powder bone graft material, but the current mechanical strength of the injectable bone graft materials in the 1st - 7th groups did not reach the compressive strength range (4 - 13 MPa) that should be borne by the posterior maxillary teeth region clinically. The 4th group of injectable pearl / bovine bone powder composite bone graft material with relatively excellent comprehensive performance can be optimized in process to improve its compressive strength.

[0105] In summary, through experimental methods such as injectability, setting time, degradation rate, and compressive strength, 7 groups of injectable pearl / bovine bone powder composite bone graft materials with different pearl powder mass ratios (0%, 25%, 30%, 35%, 40%, 45%, 50%) were tested. The results showed that the composite bone graft material with 35wt% pearl powder had better comprehensive performance: the composite slurry had high viscosity, the injectability reached 98.5±0.10%, the initial setting time was 4.40±0.11min, and the final setting time was 10.44±0.33min. However, its degradation rate was 45.40±0.29% and the compressive strength was 0.75±0.01MPa, which did not yet meet the clinical requirements. The physical and chemical properties of the composite bone graft material could be improved through liquid phase optimization.

[0106] Experimental Example 2

[0107] Based on the conclusion of Experimental Example 1, the optimal pearl / bovine bone powder ratio (the volume ratio of pearl powder in the mixed powder was 35%, the same as in Example 2) was used for liquid phase optimization.

[0108] That is, on the basis of the scheme of Example 3, the proportion of the gelatin solution added in the preparation step of the composite solution was changed to prepare multiple groups of pearl / gelatin / bovine bone powder composite bone graft materials, and the performance of each group of product materials was tested.

[0109] The added amounts of the gelatin solution in each group are shown in Table 5:

[0110] Table 5

[0111]

[0112]

[0113] The injectability of each group of product materials was measured, and the method was the same as the injectability measurement part in Experimental Example 1;

[0114] The results are shown in Table 6:

[0115] Table 6

[0116] Grouping Gelatin / vol% Injectability / % A 0 97.41±0.07 B 15 97.37±0.04 C 25 97.35±0.02 D 35 <![CDATA[97.14±0.04 b > E 45 <![CDATA[96.83±0.02 a,b > F 55 <![CDATA[95.41±0.04 a,b >

[0117] Note: a: P<0.05 when group x vs group A; b: P<0.05 when group x vs group x+1

[0118] The results showed that there was no significant difference in the injectability of the B-D pearl / gelatin / bovine bone meal composite slurry (P>0.05). When the volume ratio of the gelatin solution was 15%-25%, there was no significant difference in the injectability between the pearl / bovine bone meal composite slurry and the pearl / gelatin / bovine bone meal composite slurry (P>0.05). When the volume ratio of the gelatin solution was 35%-55%, there was a significant difference in the injectability between the pearl / bovine bone meal composite slurry and the pearl / gelatin / bovine bone meal composite slurry (P<0.05). Gelatin could improve the viscosity of the pearl / gelatin / bovine bone meal composite slurry. With the increase of the volume ratio of gelatin, the viscosity of the pearl / gelatin / bovine bone meal composite slurry increased, thus reducing the injectability of the pearl / gelatin / bovine bone meal composite slurry. The injectability of the pearl / gelatin / bovine bone meal composite slurry in this experiment was all greater than 95%, and they were all easily injectable slurries, and subsequent studies could be carried out.

[0119] The setting time of the product materials of each group was measured, and the method was the same as the part of the setting time measurement in Experimental Example 1;

[0120] The results are shown in Table 7:

[0121] Table 7

[0122]

[0123]

[0124] Note: a: P<0.05 when the x group is compared with the A group; b: P<0.05 when the x group is compared with the x+1 group

[0125] The results showed that with the increase of the volume ratio of gelatin mixture, both the initial setting time and the final setting time of the injectable bone graft material optimized by gelatin were prolonged. This may be because gelatin increased the distance between the pearl / bovine bone meal particles, thus increasing the setting time of the injectable pearl / gelatin / bovine bone meal composite bone graft material. Therefore, four groups of injectable pearl / gelatin / bovine bone meal composite bone graft materials of A, B, C, and D with an initial setting time ≤ 8 min and a final setting time ≤ 15 min were selected for subsequent experimental studies.

[0126] The in vitro degradation rate of the product materials of each group was measured, and the method was the same as the part of the in vitro degradation rate measurement in Experimental Example 1;

[0127] The results are as Figure 3 、 Figure 4 shown:

[0128] Figure 3 It is a schematic diagram of the degradation rate of the injectable pearl / gelatin / bovine bone meal composite bone graft materials of groups A-F within 7 days; in the figure, a represents P<0.05 when the Nth group is compared with the 1st group, and b represents P<0.05 when the Nth group is compared with the N+1th group.

[0129] Figure 4 Schematic diagram of the degradation of injectable pearl / gelatin / bovine bone powder composite bone graft materials in groups A - F within 7 days; in the figure, a - d on the left represent the degradation of each group of composite materials on the 1st, 3rd, 5th, and 7th days respectively.

[0130] As can be seen from the results, the injectable pearl / bovine bone powder composite bone graft material in group A degraded significantly in PBS solution, and the degradation rate was 46.49 ± 0.74% after 7 days. When the volume ratio of the gelatin solution was 15% - 35%, with the increase of the volume ratio of the gelatin solution, the degradation rate of the injectable pearl / gelatin / bovine bone powder composite bone graft material in PBS solution decreased accordingly. Among them, the degradation rate of the injectable pearl / gelatin / bovine bone powder composite bone graft material in group D was 4.52 ± 0.35% on the 7th day. When the volume ratio of the gelatin solution was 45% - 55%, the degradation rate of the injectable pearl / gelatin / bovine bone powder composite bone graft material increased. The sample material in group E began to show obvious degradation on the 3rd day, and the sample material in group F showed obvious degradation on the 5th day. Both groups of materials were completely degraded on the 7th day. Analyzing the reason for the change in the degradation rate of the injectable pearl / gelatin / bovine bone powder composite bone graft material: when the volume ratio of the gelatin solution was 15% - 35%, gelatin in the gelatin - citric acid composite liquid had strong viscosity, increasing the bond strength between injectable pearl / bovine bone powder particles and improving the anti - degradation property of the injectable pearl / gelatin / bovine bone powder composite bone graft material; but when the volume ratio of the gelatin solution was 45% - 55%, the injectable pearl / gelatin / bovine bone powder composite bone graft material showed obvious degradation, probably because the gelatin solution adhered to a large amount of pearl / bovine bone powder, increasing the cross - linked entanglement distance of the ester bonds between Ca 2+ and the citric acid solution, resulting in the complete degradation of the cured injectable pearl / gelatin / bovine bone powder composite bone graft material in PBS solution. Therefore, select the injectable pearl / gelatin / bovine bone powder composite bone graft materials in groups B, C, and D with low degradation rates for subsequent experimental research.

[0131] Measure the compressive strength of each group of products, and the method is the same as the part of measuring the compressive strength in Experimental Example 1;

[0132] The results are shown in Table 8:

[0133] Table 8

[0134] Grouping Gelatin / vol% Maximum pressure / N Compressive strength / MPa A 0 20.83±0.26 0.74±0.01 B 15 <![CDATA[153.73±0.40 a,b > <![CDATA[5.49±0.01 a,b > C 25 <![CDATA[210.92±0.20 a,b > <![CDATA[7.53±0.01 a,b > D 35 <![CDATA[234.62±0.92 a,b > <![CDATA[8.38±0.03 a,b > E 45 <![CDATA[224.73±3.03 a,b > <![CDATA[8.03±0.11 a,b > F 55 <![CDATA[181.15±0.14 a,b > <![CDATA[6.47±0.01 a,b >

[0135] Note: a: P < 0.05 when group x VS group A; b: P < 0.05 when group x VS group x + 1

[0136] The results showed that the compressive strength of the injectable pearl / gelatin / bovine bone powder composite bone graft material was greater than that of the injectable pearl / bovine bone powder composite bone graft material (P<0.05). When the volume ratio of the gelatin solution was between 15% and 35%, the compressive strength that the injectable pearl / gelatin / bovine bone powder composite bone graft material could withstand increased with the increase of the volume ratio of the gelatin solution; when the volume ratio of the gelatin solution was between 45% and 55%, the compressive strength that the injectable pearl / gelatin / bovine bone powder composite bone graft material could withstand decreased with the increase of the volume ratio of the gelatin solution. The possible reason is that when the added volume ratio of the gelatin solution was between 15% and 35%, the adhesiveness of the pearl / bovine bone powder was increased, the movement of the pearl / bovine bone powder particles was restricted, and the compressive strength of the injectable pearl / gelatin / bovine bone powder composite bone graft material was improved. However, the gelatin solution with a larger volume ratio occupied too many gaps in the pearl / bovine bone powder particles, and the gelatin-citric acid composite solution could not build a strong cross-linked structure with the composite powder, thus causing the decrease of the compressive strength of the injectable pearl / gelatin / bovine bone powder composite bone graft material. Therefore, the D-group injectable pearl / gelatin / bovine bone powder composite bone graft material with the maximum compressive strength was selected for subsequent experimental research.

[0137] In summary, the physicochemical properties of 6 groups of injectable pearl / gelatin / bovine bone powder composite bone graft materials with different gelatin volume ratios (0%, 15%, 25%, 35%, 45%, 55%) were measured by methods such as injectability, setting time, degradation rate and compressive strength. The results showed that the comprehensive performance of the 35 vol% gelatin group was relatively excellent: the injectability reached 97.14±0.04%, the initial setting time was 7.21±0.04 min, the final setting time was 12.89±0.26 min, the degradation rate decreased from 45.40±0.29% to 4.52±0.35% (P<0.05), and the compressive strength increased from 0.75±0.01 MPa to 8.38±0.03 MPa.

[0138] Experimental Example 3

[0139] Three groups of injectable bone graft materials, namely the bovine bone powder group (same as Example 1), the 35 wt% pearl powder composite group (same as Example 2) and the 35 vol% gelatin optimized group (same as Example 3), were selected, and their physicochemical properties were characterized by detection methods such as SEM, ESD, FT-IR, XRD and particle size. The specific test items, test methods and test results are as follows:

[0140] SEM test:

[0141] The above three groups of injectable bone graft materials were placed on the conductive glue of the sample stage, and the surface morphology of the materials was observed and the pore size range was measured at an acceleration voltage of 10 kV with magnification multiples of 500 and 3500.

[0142] The results are as Figure 5 shown:

[0143] Figure 5 SEM scanning images of three groups of injectable bone graft materials. In the figures, A / B are 500x and 3500x SEM images of the bovine bone meal group (same as Example 1), C / D are 500x and 3500x SEM images of the pearl / bovine bone meal group (same as Example 2), and E / F are 500x and 3500x SEM images of the pearl / gelatin / bovine bone meal group (same as Example 3). The blue arrows indicate disc-shaped crystals, and the red arrows indicate needle-shaped crystals.

[0144] As can be seen from the results, when observing the three groups of injectable bone graft materials at a magnification of 500, there are pore structures inside all of them. The pore size range of the injectable bovine bone meal bone graft material ( Figure 5 A) is 42.73um - 99.01um, the pore size range of the injectable pearl / bovine bone meal composite bone graft material ( Figure 5 C) is 96.88um - 288.31um, and the pore size range of the injectable pearl / gelatin / bovine bone meal composite bone graft material ( Figure 5 E) is 75.94um - 208.62um. The pore size of the injectable bone graft material is related to factors such as reaction temperature and setting time. The reaction degree of bovine bone meal with citric acid solution is low and the heat release is small, so the internal pore size of the injectable bovine bone meal bone graft material is the smallest. The reaction degree of pearl / bovine bone meal with citric acid solution is high and the heat release is large, so the pore sizes of the two groups of injectable composite bone graft materials are larger than that of the injectable bovine bone meal bone graft material. In addition, the gelatin in the injectable pearl / gelatin / bovine bone meal composite bone graft material increases the distance between pearl / bovine bone particles and increases its setting time, allowing CO 2 bubbles to have enough time to escape, so the formed pore size is smaller than that of the injectable pearl / bovine bone meal composite bone graft material. It is considered that pores larger than 50um are beneficial for blood to flow into the internal part of the bone graft material and improve the osteoinductive ability of the bone graft material, and pores in the range of 200um - 300um can also maintain a certain mechanical strength. The pore sizes of the injectable composite bone graft materials prepared in this scheme meet the above specified pore size range.

[0145] When observing at a magnification of 3500, disc-shaped crystals precipitate inside the pores of the injectable bovine bone meal bone graft material ( Figure 5 B), disc-shaped crystal clusters precipitate inside the pores of the injectable pearl / bovine bone meal composite bone graft material ( Figure 5 D), and there are gelatin fibers adhering to the surface of the optimized injectable pearl / gelatin / bovine bone meal composite bone graft material, and needle-shaped crystals precipitate around the gelatin fibers ( Figure 5F). The discoid crystals that appear may be carbonate apatite crystals, and the needle-shaped crystals may be calcium-deficient apatite crystals. The reasons for the formation of crystals with different morphologies in the pore structures of the three groups of injectable bone graft materials may be as follows: The discoid crystals in the injectable bovine bone powder bone graft material and the injectable pearl / bovine bone powder composite bone graft material may be discoid carbonate apatite crystals. During the process of dissolving the composite powder in the citric acid liquid phase, CO 3 2- , CO 3 2- can react with PO in hydroxyapatite 4 3- and precipitate discoid carbonate apatite crystals in the pore structure through substitution. The number of carbonate apatite crystals may be related to the amount of CO 3 2- and the substitution amount of HA: The HA in the calcined bovine bone powder dissolves in the citric acid liquid phase to produce CO 3 2- in a small amount, resulting in fewer carbonate apatite crystals obtained by substitution with HA. The composite solid phase of pearl / bovine bone powder dissolved in the citric acid solution can produce more CO 3 2- , and more carbonate apatite crystals can be displaced in the injectable pearl / bovine bone powder composite bone graft material. In the injectable pearl / gelatin / bovine bone powder composite bone graft material, there is COO- in the gelatin, which can preferentially react with Ca 2+ in the pearl / bovine bone powder during the curing process, promoting the precipitation of needle-shaped calcium-deficient apatite crystals around the gelatin fibers.

[0146] EDS test:

[0147] Switch to the EDS test mode in the Thermo Fisher scanning electron microscope instrument, analyze the element ratios of the above three groups of injectable bone graft materials in the surface area, and calculate the calcium-phosphorus ratios of the three groups of injectable bone graft materials.

[0148] The results are as Figure 6 , Figure 7 shown:

[0149] Figure 6 are the EDS test result diagrams of the three groups of injectable bone graft materials. In the figure, A is the bovine bone powder group (the same as Example 1), B is the pearl / bovine bone powder group (the same as Example 2), and C is the pearl / gelatin / bovine bone powder group (the same as Example 3).

[0150] Figure 7 are the schematic diagrams of the element mass ratios of the three groups of injectable bone graft materials.

[0151] As Figure 6 can be seen, there are mainly four elements in each group of injectable bone graft materials, namely Ca, P, C, and O elements. Statistical analysis of the element ratios of the three groups of injectable bone graft materials is as Figure 7As shown: The Ca / P of the injectable bovine bone powder bone graft material is 2.01 ± 0.17, which is similar to the calcium-phosphorus ratio of human bones. The injectable pearl / bovine bone powder composite bone graft material contains added pearl powder, and the mass percentage of Ca element is 54.30 ± 0.53%, which is higher than that of the injectable bovine bone powder bone graft material (P < 0.01), and the Ca / P is increased to 3.02 ± 0.42. There is a gelatin solution in the injectable pearl / gelatin / bovine bone powder composite bone graft material. Therefore, the mass ratios of C element and O element are 13.82 ± 0.09% and 51.20 ± 0.38% respectively, which are higher than those of the other two groups of injectable bone graft materials (P < 0.01). The Ca / P of this group of injectable composite bone graft material is 4.05 ± 0.18, showing a significant difference in calcium-phosphorus ratio from the other two groups (P < 0.05). The calcium-phosphorus ratio has a certain influence on the mechanical strength of the injectable bone graft material, and the mechanical strength increases with the increase of the calcium-phosphorus ratio. Therefore, the injectable pearl / gelatin / bovine bone powder composite bone graft material shows better compressive ability than the other two groups of injectable bone graft materials.

[0152] FT-IR test:

[0153] After adding potassium bromide to the above three groups of injectable bone graft materials and grinding them into fine powder, tablets were pressed, and then the components of each group of injectable bone graft materials were tested in the scanning range of 4000 - 450 cm -1 below.

[0154] The results are as Figure 8 shown:

[0155] Figure 8 are the infrared spectra of the three groups of injectable bone graft materials. In the figure, red represents the bovine bone powder group (same as Example 1), green represents the pearl / bovine bone powder group (same as Example 2), and black represents the pearl / gelatin / bovine bone powder group (same as Example 3).

[0156] As can be seen from the figure, stretching vibration peaks of Ca - O can be observed at 470 cm -1 , 473 cm -1 . The peak at 712 cm -1 represents the vibration of CO 3 2- which is a unique marker of aragonite. The peak at 980 cm -1 is the stretching vibration peak of HPO 4 2- . The vibration peak of the carboxyl bond in citric acid can be observed at 1276 cm -1 , which is an important feature of citric acid. The peak at 1303 cm -1 represents the C - O bond where the hydroxyl group is connected to the carbon atom. The peak at 1787 cm -1 represents CO 3 2-The C=O group in it, 1988 cm -1 The triplet at is the stretching vibration of O-H in carbonated hydroxyapatite, 2546 cm -1 The peak at is attributed to v(C-H) of the organic matrix in pearl powder, 2922 cm -1 There is a stretching vibration peak of C-H in HA at, 3080 cm -1 The weak peak at is derived from v(N-H) of amide B, representing the presence of gelatin, 3400 cm -1 , 3570 cm -1 The peak at represents the stretching vibration of O-H. The main components of each group of injectable bone graft materials need to be confirmed in combination with the XRD test results.

[0157] XRD test:

[0158] The above three groups of injectable bone graft materials were subjected to XRD detection: the instrument light source was Cu-Kα, the scanning range was 10-80°, and the scanning mode was continuous scanning.

[0159] The results are as Figure 9 shown:

[0160] Figure 9 is the XRD test result diagram of the three groups of injectable bone graft materials. In the figure, red represents the bovine bone meal group (same as Example 1), green represents the pearl / bovine bone meal group (same as Example 2), and black represents the pearl / gelatin / bovine bone meal group (same as Example 3).

[0161] As can be seen from the figure, compared with the standard card PDF, it is obtained that: the three groups of injectable bone graft materials all show (002), (121), (120) and (300) diffraction peaks, which are the characteristic peaks of HA (JCPDF84-1998). The introduction of the (300) diffraction peak at 2θ = 32.1° indicates that PO in HA 4 3- is replaced by CO 3 2- to generate carbonated apatite. The two groups of injectable pearl / bovine bone meal composite bone graft materials show (111), (102), (112) and (221) diffraction peaks, which are the characteristic peaks of aragonite (JCPDF75-2230). The (100), (111) and (120) diffraction peaks are the characteristic peaks of brushite (JCPF83-1888).

[0162] The characterization results reflect the hydrolysis and solidification mechanism of the optimized injectable composite bone graft material prepared in this application: the citric acid solution, as a commonly used mixing solution for the solid phase of injectable bone graft materials, is acidic and can dissolve pearl powder and bovine bone meal to generate Ca 2+ , HPO 4 2- , PO4 3- and CO 3 2- plasma, and promote the recombination of these ions to form new hydroxyapatite, calcium hydrogen phosphate and carbonated apatite crystals, which finally solidify into pearl / bovine bone powder composite bone graft materials as the crystals grow.

[0163] Particle size test:

[0164] Dry the bovine bone powder (same as Example 1) and pearl / bovine bone powder (same as Example 2), put them into a four-way optical quartz cuvette with a 10 mm optical path, add 1 mL of PBS solution to disperse evenly, and put it into a Zeta particle size analyzer to detect the particle size of the solid powder material.

[0165] The results are as Figure 10 shown:

[0166] Figure 10 are the particle size distribution diagrams of bovine bone powder (same as Example 1) and pearl / bovine bone powder (same as Example 2). In the figure, the yellow diagonal area represents bovine bone powder, and the green area represents pearl / bovine bone powder.

[0167] It can be seen from the results that the particle size of the calcined bovine bone powder is 1.16 ± 0.05 μm, and the particle size of the pearl / bovine bone powder is 1.8 ± 0.03 μm. Adding pearl powder can significantly increase the particle size of the composite powder (P < 0.05). The particle size of the powder will also affect the injection rate of the injectable bone graft material: when the solid phase particle size is in the micron range, the injection rate of the injectable bone graft material is high. The micron-sized pearl / bovine bone powder prepared in this application can be used for the research and development of injectable bone graft materials.

[0168] In summary, through the characterization of the physicochemical properties of the materials, it can be seen that the specifications of the composite materials are in the micron range. The optimized injectable pearl / gelatin / bovine bone powder composite bone graft material has a pore structure inside, the pore size range is 75.94 μm to 208.64 μm, calcium-deficient apatite crystals precipitate around the gelatin fibers adhered to the surface, and there are mainly four elements: C, O, P and Ca. The Ca / P is 4.05 ± 0.18, and the main components are HA, pearl powder, carbonated apatite and brushite. The injectability rate reaches 97.14 ± 0.04%, the initial setting time is 7.21 ± 0.04 min, the final setting time is 12.89 ± 0.26 min, the degradation rate is 4.52 ± 0.35%, and the compressive strength is 8.38 ± 0.03 MPa. All performance indicators meet the clinical physicochemical performance requirements and can be used for subsequent in vitro biological evaluation.

[0169] Experimental Example 4

[0170] In this experimental example, a commercial bone graft material (produced by Beijing Parsen Biotechnology Co., Ltd.) was selected as the control group, and an injectable pearl / gelatin / bovine bone powder composite bone graft material (the same as in Example 3) was used as the experimental group to comparatively study their in vitro biological properties. Each group of materials was sterilized by γ-ray (3 kGy) before the experiment.

[0171] Mouse pre-osteoblasts (MC3T3-E1) were used to study the cytocompatibility and cell-induced osteogenicity of the above materials. The revived cells were transferred to a 100 mm culture dish, and then 7 ml of complete cell culture medium (formula: 85% high-glucose cell culture medium + 10% fetal bovine serum + 5% dual antibiotics) was added to the culture dish. Then, it was placed in a cell incubator at a temperature of 37 °C and a humidity of 100% for cultivation, and the cell culture medium was changed every 3 days. When the cell growth adherent coverage rate in the culture dish reached about 80%, a certain number of cells were inoculated into the cell culture plate containing the material according to the experimental requirements for incubation to prepare for subsequent in vitro cell experiments.

[0172] It should be noted here that the following experimental measurement data were analyzed by one-way analysis and LSD test using SPSS 26 statistical software (IBM, USA). P < 0.05 indicates that the difference is statistically significant. The analyzed data were expressed in the form of mean ± standard deviation (Mean ± SD). The data graphs were plotted using GraphPad Prism 9.5 software (GraphPad Software, Inc., USA).

[0173] Cell adhesion assay:

[0174] The adhesion of cells on the surface of each group of sample materials was observed by SEM electron microscopy scanning. The experimental steps are as follows:

[0175] (1) The density of MC3T3-E1 cells inoculated in the 48-well cell culture plate containing each group of materials was 2.5×10 4 cells / cm 2 , and then 150 μl of culture medium was added to each well and placed in a cell incubator at a constant temperature for 24 h.

[0176] (2) After culturing for 24 h, the materials were gently rinsed 3 times with PBS solution to remove the cells and residual culture medium on the surface of the non-adherent cells.

[0177] (3) 100 μl of 4% paraformaldehyde solution was added to each well. React at room temperature for 15 minutes to fix the cells on the material surface.

[0178] (4) The surface of the material was washed with PBS solution to remove the excess paraformaldehyde solution, and the process was repeated 3 times.

[0179] (5) Dehydrate with ethanol solutions of different concentrations in a gradient manner (20%, 50%, 70%, 100%), dehydrate for 10 min at each gradient concentration, and dehydrate twice in 100% ethanol solution.

[0180] (6) After dehydration, place it in an electrothermal constant temperature forced air drying oven at 37 °C and dry for 24 h.

[0181] (7) Place the dried material with adhered cells on the surface of the conductive adhesive with forceps, sputter gold for 300 s, and observe under SEM at an accelerating voltage of 15 kV. Observe the overall adhesion situation on the surface of the material at a magnification of 500, and observe the situation on the surface of the cell-adhered material at a magnification of 3500.

[0182] The results are as Figure 11 shown:

[0183] Figure 11 It is a schematic diagram of the adhesion of cells in each group of bone graft materials. In the figure, A / B are the SEM images of cell adhesion at 500 and 3500 times of the control group (commercially available products), and C / D are the SEM images of cell adhesion at 500 and 3500 times of the experimental group (the same as Example 3). The red arrows point to the MC3T3-E1 cell clusters.

[0184] It can be seen from the results that MC3T3-E1 cells have good cell adhesion in both commercial bone powder and injectable pearl / gelatin / bovine bone powder composite bone graft materials. The white granular substances on the surface of the commercial bone graft material are MC3T3-E1 cells or cell clusters, and the black shadow areas on the surface of the injectable pearl / gelatin / bovine bone powder composite bone graft material are the adhered MC3T3-E1 cell clusters.

[0185] The adhesion of cells on the material surface, as the first step of the reaction between cells and materials, will affect the subsequent growth and proliferation of cells in the material. The organic substances of pearl powder in the injectable pearl / gelatin / bovine bone powder composite bone graft material can effectively promote cell adhesion. Gelatin, as a thickening agent, can mediate cell adhesion. The pore structure of the injectable pearl / gelatin / bovine bone powder composite bone graft material can increase the surface area of the material and also improve the adhesion of cells on the material surface.

[0186] Cell proliferation assay:

[0187] Use CCK-8 reagent to detect the number of cells after co-culturing in the material for 1 day, 3 days, 5 days, and 7 days, and analyze the cell proliferation situation: Seed the cells at a density of 5×10 3 cells / cm 2Inoculate at a density into a 48-well cell culture plate containing each group of materials. At the above time points, regularly extract the culture medium in the 48-well cell culture plate, then add 150 μL of complete cell culture medium and 15 μL of CCK-8 reagent into the wells. The blank control group only adds 150 μL of complete cell culture medium and 15 μL of CCK-8 reagent. After placing the 48-well cell culture plate with the added CCK-8 reagent in the cell incubator for 1 hour, take out 100 μL of the stained cell supernatant from each well in the 48-well cell culture plate and transfer it to a 96-well cell culture plate. Use a microplate reader to measure the optical density OD value of the cell supernatant in the 96-well cell culture plate at 450 nm. The value obtained after standardizing with the blank control OD value represents the number of cells in the material at this time point.

[0188] The results are as Figure 12 shown:

[0189] Figure 12 It is a schematic diagram of the proliferation of cells cultured in each group of materials for 1, 3, 5, and 7 days. In the figure, A represents the OD value after standardization, and B represents the proliferation curve of cells in each group of materials within 7 days. Figure 12 In A, yellow represents the blank group, blue represents the control group (commercially available products), and gray represents the experimental group (the same as Example 3); Figure 12 In B, the dark purple square mark represents the blank group, the blue-violet triangle mark represents the control group (commercially available products), and the blue diamond mark represents the experimental group (the same as Example 3).

[0190] It can be seen from the results that the OD values and cell proliferation conditions detected at different time points (1, 3, 5, 7 days) after co-culturing MC3T3-E1 cells in the blank group, the commercial bone graft material control group, and the injectable pearl / gelatin / bovine bone meal composite bone graft material experimental group, Figure 12 A represents the OD value after standardization, Figure 12 B represents the growth conditions of cells in each group of materials. The results of the two figures show that: as time increases, the number of cells co-cultured in each group of materials has increased. The number of cells in the injectable pearl / gelatin / bovine bone meal composite bone graft material on the 3rd day is 5.59 ± 2.22 times that on the 1st day, less than the number of cells in the blank group (P < 0.05), and greater than the number of cells in the control group (P < 0.05). As the co-culture time of cells increases, the number of cells in the injectable pearl / gelatin / bovine bone meal composite bone graft material on the 7th day proliferates to 30.15 ± 3.26 times that on the 1st day, and the number of cells in it is greater than that in the blank group and the control group (P < 0.05), which may be due to the pearl water-soluble matrix and gelatin organic substances contained in the pearl / gelatin / bovine bone meal composite bone graft material promoting cell proliferation.

[0191] Alkaline phosphatase relative activity level determination test:

[0192] The protein concentration of cells co-cultured with materials for 5 days and 10 days was detected using a BCA protein concentration assay kit, and the relative activity level of ALP expressed by cells after co-culturing with materials in each group for 5 days and 10 days was detected using an alkaline phosphatase activity assay kit. The experimental steps are as follows:

[0193] (1) Cells were seeded into 48-well cell culture plates containing materials in each group at a density of 5×10 3 cells / cm 2 .

[0194] (2) At two time points of 5 days and 10 days, the cell culture medium in the 48-well cell culture plates was aspirated, and the residual culture medium in the cell culture plates was washed with PBS solution. 200 μL of Ripa cell lysate was added to each well to lyse the cells on the material surface as the test group.

[0195] (3) According to the instructions of the BCA protein concentration assay kit: Eight groups of protein standard solutions with concentrations of 0, 0.025, 0.050, 0.100, 0.200, 0.300, 0.400, and 0.500 mg / mL were prepared.

[0196] (4) 20 μL of each concentration of protein standard solution and cell lysate at each time point were taken into a 96-well cell culture plate, 180 μL of BCA working solution was added, and after incubation in a 37°C cell incubator for 30 minutes, the absorbance at 562 nm was measured using a microplate reader. A protein standard curve was plotted based on the absorbance of each concentration of protein standard solution, and the cell protein concentration in the materials at each time point was calculated based on the absorbance of the cell lysate in combination with the protein standard curve.

[0197] (5) According to the instructions of the alkaline phosphatase activity assay kit: 0.5 mmol / mL phenol standard application solution was used as the standard control group, the cell lysate at each time point was used as the experimental group, and the buffer solution was used as the blank control group.

[0198] (6) 50 μL of phenol standard application solution, cell lysate, and buffer solution were taken into a 96-well cell culture plate respectively, 150 μL of chromogenic substrate solution was added to each well, and the reaction was carried out in a 37°C shaking incubator for 2 h. After the reaction, the absorbance at a wavelength of 405 nm was measured using a microplate reader. According to the following enzyme activity definition formula, the relative activity level of cell alkaline phosphatase in each group of materials was calculated, where A is the measured absorbance, C 标准 is the concentration of phenol standard application solution, and C 样品 is the cell protein concentration in the material sample measured in step (4):

[0199]

[0200] The results are as Figure 13, Figure 14 as shown:

[0201] Figure 13 is the standard protein concentration curve obtained by measurement;

[0202] Figure 14 is the schematic diagram of the protein concentration and relative ALP activity levels measured and calculated after the cells were co-cultured with each group of materials for 5 and 10 days. In the figure, A represents the protein concentration of each group of materials, B represents the relative ALP activity level, ** indicates P < 0.01, and # indicates P > 0.05. Figure 14 In A, blue represents the blank group, brown represents the control group, and gray represents the experimental group; Figure 14 In B, black represents the blank group, dark blue represents the control group, and light blue represents the experimental group.

[0203] The standard protein concentration curve ( Figure 13 ) was obtained by measurement to calculate the protein concentrations of each group of materials at 5 and 10 days ( Figure 14 A), and the OD values of each group of materials were detected by an ALP reagent detection kit to calculate the relative ALP activity levels of each group of materials ( Figure 14 B). The results showed that: with the increase of time, the relative ALP activity levels of each group of materials all increased. The relative ALP activity level of the experimental group increased from 1.23 ± 0.04 to 1.79 ± 0.04, which was higher than that of the control group and the blank group (P < 0.01). As an important marker for osteoblast differentiation, the cell ALP activity level can reflect the osteogenic differentiation ability of the cells on the material surface.

[0204] The reason why the injectable pearl / gelatin / bovine bone meal composite bone graft material exhibits good osteogenic differentiation ability may be that the water-soluble matrix in the pearl improves the cell osteogenic differentiation level. In this experiment, the pearl and bovine bone meal were compounded by low-temperature hydration to retain the activity of the water-soluble organisms in the pearl, thus enabling the injectable pearl / gelatin / bovine bone meal composite bone graft material to exhibit certain osteogenic differentiation ability.

[0205] In summary, it was observed that the injectable pearl / gelatin / bovine bone meal composite bone graft material (same as Example 3) had a cell adhesion ability similar to that of the commercial bone graft material on its surface and could adhere a large number of MC3T3-E1 cells; the number of proliferating cells in the composite bone graft material was higher than that of the commercial bone graft material and the blank group (P < 0.05), and the number of MC3T3-E1 cells after 7 days was 30.15 ± 3.26 times that on the first day (P < 0.01); the relative ALP activity level expressed by MC3T3-E1 cells on the injectable material increased from 1.23 ± 0.04 to 1.79 ± 0.04, which was significantly higher than that of the commercial bone graft material and the blank group (P < 0.01), indicating that the injectable pearl / gelatin / bovine bone meal composite bone graft material has good in vitro biocompatibility and osteogenic ability.

[0206] Experimental Example 5

[0207] Viscosity analysis of the composite slurry:

[0208] The injectable bone graft material is a viscous fluid that can flow under the pressure of a syringe. In the above Experimental Examples 1-4, the sample materials used were all prepared with a solid-liquid ratio of 0.35 mL / g, and the composite slurry had good fluidity and adhesiveness.

[0209] In this experiment, pure bovine bone powder slurry (bovine bone powder + citric acid solution) and pearl / bovine bone powder composite slurry (pearl / bovine bone powder and citric acid solution) were used, as Figure 15 shown.

[0210] Figure 15 Figure 1 shows the schematic diagrams of the morphologies of the two slurries. In the figure, A is the pure bovine bone powder slurry and B is the pearl / bovine bone powder composite slurry.

[0211] It can be seen that the slurry formed by adding bovine bone powder to the citric acid solution is a fluid with low viscosity: perhaps the HA in the bovine bone powder with small particle size is not easily soluble in the liquid, and the Ca in the HA 2+ and the citrate radical are not easily crosslinked and entangled, resulting in low viscosity of the bovine bone powder slurry, not being easily shaped arbitrarily, and unable to maintain a stable height at the transplantation site. The slurry formed by adding pearl / bovine bone powder to the citric acid solution has a relatively high viscosity. Pearl / bovine bone powder is more easily soluble in the solution than pure bovine bone powder, and the citrate radical can form more crosslinking bonds with the Ca in the pearl / bovine bone powder, 2+ thus making the pearl / bovine bone powder composite slurry have a relatively high viscosity, being able to maintain a stable shape at the implantation site, being able to be solidified in situ, meeting the clinical requirements. Therefore, the present application selects the pearl / bovine bone powder composite slurry for key research.

[0212] In summary, the embodiments of the present application provide an injectable composite bone graft material and its preparation method. It uses bovine bone powder as the base material for injectable modification. The prepared injectable composite bone graft material has obvious improvements in osteogenic efficiency, spatial stability, surgical operation convenience, and cost compared with commercial bone powder, and its physical and chemical properties and biological properties meet clinical needs, having more excellent cell compatibility, cell adhesion, and cell osteogenic induction ability. It has low cost and excellent performance, and has great application potential in the maxillary sinus floor elevation surgery.

[0213] The embodiments described above are some embodiments of the present application, rather than all embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application claimed, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present application.

Claims

1. An injectable composite bone graft material, characterized in that: The method comprises the following components: a mixed powder and a composite solution; Wherein, the raw materials of the mixed powder include cattle bone powder and pearl powder; The raw materials of the composite solution include gelatin solution and citric acid solution.

2. The injectable composite bone graft material according to claim 1, characterized in that: In the mixed powder, the mass ratio of pearl powder is 25-40%; In the composite solution, the volume ratio of the gelatin solution is 15-35%.

3. The injectable composite bone graft material according to claim 1, characterized in that: In the mixed powder, the cow bone powder is prepared by calcining and grinding cow femur, and the pearl powder is pharmaceutical grade pearl powder.

4. The injectable composite bone graft material according to claim 1, characterized in that: In the composite solution, the gelatin solution used is prepared from dry gelatin and has a concentration of 11.11%; The concentration of the citric acid solution used was 25%.

5. The injectable composite bone graft material according to claim 1, characterized in that: Among the raw material components, the solid-to-liquid ratio of the mixed powder to the composite solution is 0.35 mL / g.

6. A method for preparing an injectable composite bone graft material according to any one of claims 1 to 5, characterized in that: It includes the following steps: Preparation of cattle bone powder: Take cattle bones and remove impurities such as surface muscle, periosteum, fat, etc., boil them in water, soak them in sodium hydroxide solution and hydrogen peroxide solution respectively, wash them, calcine them, grind them and sieve them to obtain cattle bone powder for later use; Preparation of mixed powder: Mix the prepared cow bone powder and pearl powder in proportion to obtain a mixed powder for later use; Preparation of gelatin solution: weigh dry gelatin and add UP water to make the dry gelatin swell to a gel state, then perform constant temperature treatment to prepare a gelatin solution for later use; Preparation of composite solution: Mix the prepared gelatin solution with the citric acid solution in proportion to obtain a composite solution for later use; Product material preparation: The prepared mixed powder is mixed with the composite solution in proportion to obtain a composite slurry, and the composite slurry is then injected into a mold and then cured at a constant temperature to obtain the product injectable composite bone transplant material.

7. The method for preparing the injectable composite bone graft material according to claim 6, characterized in that: In the step of preparing the bovine bone powder, the concentration of the sodium hydroxide solution used is 1 mol / L, and the concentration of the hydrogen peroxide used is 30%.

8. The method for preparing the injectable composite bone graft material according to claim 6, characterized in that: In the step of preparing the cattle bone powder, calcination is carried out in a muffle furnace at 850° C., and then grinding is carried out in a planetary ball mill at a rate of 50 r / min, and the cattle bone powder is obtained after sieving.

9. The method for preparing the injectable composite bone graft material according to claim 6, characterized in that: In the gelatin solution preparation step, the gelatin solution is prepared by subjecting the solution to a constant temperature treatment in a 37° C. electric thermostat.

10. The method for preparing the injectable composite bone graft material according to claim 6, characterized in that: In the product material preparation step, the product is cured at a constant temperature in an electric constant temperature blast drying oven at a temperature of 37° C. and a humidity of 100% to obtain an injectable composite bone transplant material.

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