A bone augmentation material composition and method of detecting the same

Abstract

The application discloses a bone augmentation material composition. It belongs to the technical field of medical materials, and comprises viscous freeze-dried particles and granular bone substitutes, and the mass ratio of the viscous freeze-dried particles to the granular bone substitutes is 0.15:1. The composition can form a "dough-like" composite material with excellent cohesion and plasticity, and doctors can shape the material at will and tightly adhere to the surface of a defect when filling irregular bone defects such as craniofacial bone defects and alveolar ridge defects, so that the problem of invalid interstitial space caused by the fact that traditional loose granular materials cannot adhere to the wound surface is avoided. Moreover, the material can resist extrusion of surrounding soft tissues and continuous erosion of body fluid after being implanted into the body, maintains a stable three-dimensional repair profile in a healing period, effectively prevents volume loss caused by particle displacement, diffusion or collapse, and provides a long-term stable space support for bone regeneration.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to a bone augmentation material composition and its testing method. Background Technology

[0002] Bone defects are a common clinical condition caused by trauma, tumor resection, infection, or congenital malformations. For large or irregularly shaped bone defects, the body's own repair capacity is limited, often requiring intervention through the implantation of bone repair materials. Currently, autologous bone grafting is still considered the "gold standard" for bone repair, but its limited availability, potential for secondary damage to the donor site, and poor malleability make it difficult to perfectly match complex defect shapes limit its clinical application. To address these limitations, researchers have developed various artificial bone substitutes. Among them, granular bone substitutes are widely used due to their excellent filling effect, controllable pore size and porosity, and mature manufacturing processes. Examples include granular products based on calcium phosphate, calcium sulfate, or bioactive glass, as well as deproteinized bovine bone minerals. These products have achieved definite clinical efficacy and have become an important option for bone defect repair.

[0003] However, clinical practice and related studies have shown that existing granular bone substitutes still have significant technical shortcomings. On the one hand, traditional granular materials are too fluid or loosely aggregated during implantation, making it difficult to form cohesive and malleable clumps. When used to fill irregularly shaped bone defects such as those in the craniofacial region, alveolar ridge, or around joints, the material cannot closely adhere to the irregular wound surface, resulting in ineffective cavities between the implant and the host bone. This not only affects the ingrowth of blood clots but also hinders subsequent osteoconduction, reducing the repair effect. On the other hand, after implantation, granular materials are prone to displacement, diffusion, or collapse under the tension of surrounding soft tissues, muscle compression, and fluid erosion, failing to maintain a stable repair contour during the healing period. This lack of postoperative morphological stability not only causes volume loss in the bone defect area, affecting the final functional and aesthetic repair results, but may also induce ectopic osteogenesis or nonspecific inflammatory reactions.

[0004] To overcome these problems, researchers have attempted to combine granular bone substitutes with various adhesives or gel carriers (such as hyaluronic acid, collagen, and fibrin glue) to improve the material's shapeability and postoperative stability. However, these composite systems still have many shortcomings in practical applications. For example, some adhesives have limited biocompatibility or osteogenic activity, making it difficult to meet the needs of the bone regeneration microenvironment; the mechanical strength of the composite materials is often insufficient, failing to maintain structural integrity for extended periods in body fluid environments and exhibiting poor resistance to cyclic compressive loads; and some composite systems have complex preparation processes involving organic solvents or high-temperature treatments, which can easily leave toxic residues or damage the natural structure of the bone substitute, hindering clinical translation. Summary of the Invention

[0005] The purpose of this invention is to provide a novel bone augmentation material composition and its testing method, which can achieve flexible shaping during surgery, perfectly fit irregular defects, resist tissue compression and body fluid erosion after surgery, maintain long-term morphological stability, and possess excellent mechanical toughness and osteogenic activity, thereby solving the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A bone augmentation material composition comprising viscous freeze-dried particles and granular bone substitute, wherein the mass ratio of the viscous freeze-dried particles to the granular bone substitute is 0.15:1; The viscous freeze-dried particles are composed of chitosan, acrylic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; The chitosan has a median molecular weight of 30 kDa.

[0007] By employing the above technical solution, a composite material with cohesiveness and plasticity can be formed by combining viscous freeze-dried particles and granular bone substitutes at a mass ratio of 0.15:1. Chitosan (medium molecular weight 30 kDa) in the viscous freeze-dried particles forms a viscoelastic granular skeleton with acrylic acid through crosslinking with 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. When mixed with the granular bone substitutes, these particles can uniformly coat the bone substitute particles, imparting suitable adhesion and cohesiveness to the overall material. This allows it to be arbitrarily shaped and maintain its desired shape under external force, while remaining resistant to dispersion or displacement in body fluids. This mass ratio of 0.15:1 is an optimized ratio. At this ratio, the viscous freeze-dried particles provide sufficient adhesion to aggregate the bone substitute particles without excessively hindering direct contact between the bone substitute and the host bone tissue due to excessive viscous components, thus achieving a balance between operational performance and osteoconductive properties. In addition, the molecular weight of chitosan was selected as a medium molecular weight of 30 kDa. Chitosan in this molecular weight range has sufficient chain length to form a stable cross-linked network, as well as good biocompatibility and degradability, which is beneficial to subsequent cell adhesion and bone regeneration processes.

[0008] A method for detecting the bone augmentation material composition described above, characterized by comprising the following detection items: Project 1: The storage modulus G′ and loss modulus G″ of the composition were determined by strain amplitude scanning test; Project 2: Stress-strain changes of compositions with different ratios were determined using uniaxial compression tests; Project 3: The stress-strain change of the standard composition after 100 cycles was determined by cyclic compression testing, and the compressive modulus of different ratio compositions after 100 cycles was determined by cyclic compression testing. Project 4: The displacement-stress change of the composition was determined by shear test; Project 5: The promoting effect of the composition on osteogenic differentiation of MC3T3-E1 cells was detected by in vitro cell assays; Project 6: The bone regeneration effect of the composition was tested using an in vivo animal model of critical-sized bone defects.

[0009] By adopting the above technical solutions, the viscoelasticity, compressive strength, fatigue resistance, shear strength, osteogenic activity, and in vivo bone regeneration capacity of bone augmentation material compositions can be systematically characterized, providing comprehensive data support for material quality control and clinical application.

[0010] As a further aspect of the present invention: Project 1 includes the following steps: preparing the composition into a test sample, placing it on a rheometer platform, setting the frequency scanning range to 0.1Hz to 100Hz, performing an oscillation test under a constant strain amplitude (within the linear viscoelastic region), and recording the changes in storage modulus G′ and loss modulus G″ with frequency to evaluate the viscoelastic behavior of the composition.

[0011] By employing the above technical solution, the dynamic response of the storage modulus and loss modulus of bone augmentation material compositions with frequency over a wide frequency range can be accurately determined. Specifically, within the measured frequency range, G′ continuously increases with increasing frequency, indicating that the material's elastic response is enhanced under high-frequency dynamic loading, and its structural cohesion is good. Meanwhile, G″ increases with increasing frequency in the low-frequency region, and tends to remain constant after reaching 10Hz, reflecting that the material's viscous dissipation capacity reaches saturation at high frequencies, avoiding plastic collapse caused by excessive flow. This frequency scanning method can comprehensively evaluate the modulus evolution characteristics of materials under complex in vivo mechanical environments (such as chewing, blood flow erosion, tissue compression, and other dynamic loads), providing a direct basis for the viscoelastic matching design of bone augmentation material compositions and helping to screen the optimal ratio that combines sufficient elastic support with appropriate fluidity.

[0012] As a further aspect of the present invention: Project 2 includes the following steps: preparing test samples with different mass percentages, wherein the mass percentages of the viscous freeze-dried particles are 3.2wt%, 4.8wt%, and 6.2wt%, respectively; preparing standard cylindrical specimens of each composition with a diameter of 8-12mm and a height of 10-15mm; placing the specimens on a universal testing machine and performing uniaxial compression at a constant compression rate, recording stress-strain curves, calculating the compressive modulus and compressive strength of each composition, and comparing the effects of different viscous freeze-dried particle contents on the compressive properties of the composite material.

[0013] By employing the above technical solution, the compressive properties of bone augmentation material compositions under different contents of viscous freeze-dried particles can be systematically evaluated. Three gradient contents—3.2 wt%, 4.8 wt%, and 6.2 wt%—were selected, covering a range from low to high viscous component proportions, comprehensively reflecting the influence of varying viscous freeze-dried particle content on the mechanical behavior of the composite material. The stress-strain curves obtained through uniaxial compression tests visually demonstrate the material's elastic deformation, yielding, plastic flow, and failure stages during compression. The calculated compressive modulus characterizes the material's resistance to deformation stiffness, while the compressive strength characterizes its ability to withstand ultimate loads. By comparing these parameters of samples with different proportions, the optimal range of viscous freeze-dried particle content can be determined, ensuring that the bone augmentation material composition possesses sufficient mechanical strength to resist postoperative tissue compression while maintaining appropriate flexibility and plasticity to facilitate intraoperative shaping.

[0014] As a further aspect of the present invention: Project 3 includes the following steps: preparing the composition into a standard sample, performing a cyclic compression test, subjecting the sample to 100 loading and unloading cycles at a constant compression rate, and recording the stress-strain change curve for each cycle; Test samples were prepared from the composition at different mass ratios, wherein the mass percentages of viscous freeze-dried particles were 3.2 wt%, 4.8 wt%, and 6.2 wt%, respectively. Cylindrical specimens with diameters of 8-12 mm and heights of 10-15 mm were prepared from each composition. The specimens were placed on a universal testing machine and subjected to 100 cycles of loading and unloading compression at a constant compression rate. The stress-strain curves for each cycle were recorded. The corresponding compression modulus was calculated based on the stress-strain curves for each cycle, and a graph showing the change in compression modulus with the number of cycles was plotted. The evolution of the stress-strain curves during the 100 cycles was analyzed to evaluate the fatigue resistance and cyclic stability of the composition.

[0015] By employing the above technical solution, the fatigue resistance and cyclic stability of bone augmentation material compositions under repeated cyclic compression loading can be comprehensively evaluated. First, standard samples with a single formulation were subjected to 100 loading and unloading cycles, and the stress-strain curves for each cycle were recorded. This directly reflects the material's elastic recovery ability, cumulative plastic deformation, and the attenuation trend of mechanical properties during repeated compression. Second, three formulations with viscous freeze-dried particle contents of 3.2 wt%, 4.8 wt%, and 6.2 wt% were selected and subjected to 100 cyclic compression tests under the same conditions. By calculating the compression modulus for each cycle and plotting the modulus change with the number of cycles, the influence of different viscous component contents on the material's fatigue resistance can be compared. Analyzing the evolution of the stress-strain curves during the 100 cycles allows for the determination of whether the material exhibits significant stiffness reduction, residual strain accumulation, or structural damage—significant fatigue failure characteristics. This method provides crucial mechanical durability basis for optimizing the ratio of viscous freeze-dried particles to granular bone substitutes in bone augmentation material compositions, ensuring that the material maintains a stable three-dimensional shape and mechanical support function under long-term postoperative tissue compression and physiological loads.

[0016] As a further aspect of the present invention: Project 4 includes the following steps: placing the composition in a shear test fixture, applying gradually increasing shear force, recording the relationship between displacement and stress, plotting the displacement-stress curve, and calculating the shear strength and shear modulus.

[0017] By employing the above technical solution, the mechanical response characteristics of bone augmentation material compositions under shear loads can be accurately characterized. By placing the material in a shear test fixture and applying gradually increasing shear force, the relationship between displacement and stress is recorded, allowing for the acquisition of the material's shear behavior throughout the entire process from elastic deformation to yield failure. The resulting displacement-stress curves visually reflect the material's deformation capacity and load-bearing capacity under shear loads. The slope of the curve represents the shear modulus, i.e., the material's stiffness against shear deformation; the peak stress of the curve corresponds to the shear strength, i.e., the maximum shear load the material can withstand. After implantation, bone augmentation materials not only bear compressive loads but also inevitably experience shear forces generated by the movement of surrounding soft tissues and muscle contraction. Insufficient shear resistance can lead to interlayer slippage or interface separation, affecting the overall stability of the material and the repair effect of bone defects. The shear strength and shear modulus parameters obtained through this shear test can evaluate the bone augmentation material composition's ability to resist shear deformation and its structural integrity, providing important safety data for the clinical application of materials in complex mechanical environments.

[0018] As a further aspect of the present invention: Project 5 includes the following steps: co-culturing the composition with MC3T3-E1 pre-osteoblasts, and after culturing for 7-21 days, detecting alkaline phosphatase activity, mineralization nodule formation, and osteogenic-related gene expression levels to evaluate the osteogenic differentiation-promoting ability of the composition.

[0019] By employing the above-mentioned technical approach, the promoting effect of the bone augmentation material composition on the differentiation of pre-osteoblasts into mature osteoblasts can be evaluated at the cellular level. The bone augmentation material composition is co-cultured directly or indirectly with MC3T3-E1 pre-osteoblasts to simulate the microenvironment of the material in contact with bone tissue in vivo. Alkaline phosphatase is a marker enzyme of early osteoblast differentiation, and its activity directly reflects the degree of initiation of cell differentiation into an osteogenic phenotype. The formation of mineralized nodules represents a late event in osteogenic differentiation and is direct evidence of extracellular matrix mineralization capacity. The expression level of osteogenic-related genes reveals the regulatory role of the material on osteogenic signaling pathways at the molecular level. By comprehensively detecting these three levels of indicators, the biological activity of the bone augmentation material composition in inducing bone regeneration can be comprehensively and objectively assessed. If the material can significantly increase alkaline phosphatase activity, promote mineralized nodule formation, and upregulate the expression of osteogenic-related genes, it indicates that the composition has a good ability to promote bone differentiation, providing key in vitro biological evidence for its use in bone defect repair.

[0020] As a further aspect of the present invention: Project 6 includes the following steps: selecting rabbit skulls to prepare critical-sized bone defects, implanting the composition into the defect area, periodically euthanizing the animals after surgery, collecting skull specimens, calculating bone volume fraction through micro-CT scanning and three-dimensional reconstruction, evaluating the rate of new bone deposition and defect closure effect through tissue section staining, and detecting pathological changes in major organs to evaluate systemic toxicity.

[0021] By employing the above-mentioned technical solutions, the bone regeneration capacity and biosafety of the bone augmentation material composition can be comprehensively evaluated at the in vivo level. The rabbit skull critical-size bone defect model is a recognized standard animal model in bone repair research. Defects of this size cannot heal on their own, thus objectively reflecting the material's true effect on promoting bone regeneration. After implanting the composition into the defect area, micro-CT scanning and three-dimensional reconstruction allow for non-destructive quantitative calculation of the bone volume fraction, i.e., the percentage of newly formed bone volume to the total defect volume. This indicator directly measures the material's ability to induce new bone formation. Tissue section staining allows for histological observation of the new bone deposition rate, i.e., the proportion of newly formed bone area to the total defect area per unit time, while simultaneously assessing the defect closure effect, including the degree of bone bridging at the defect edges and the new bone coverage rate at the defect center. These indicators comprehensively reflect the material's ability to guide bone ingrowth and achieve complete defect healing. Furthermore, detecting pathological changes in major organs can evaluate whether the material causes systemic toxicity, ensuring the safety of implantation. Through the above in vivo tests, direct and reliable experimental evidence can be provided for the preclinical efficacy evaluation of the bone augmentation material composition.

[0022] As a further aspect of the present invention: In Project 5, the composition was co-cultured with MC3T3-E1 cells for 7, 14, and 21 days. Alkaline phosphatase activity was detected using the p-nitrophenyl phosphate method, and mineralized nodules were observed using Alizarin Red staining. Osteogenesis-related genes included Runx2, ALP, OCN, and Col1a1.

[0023] By employing the above technical approach, key parameters for evaluating in vitro osteogenic differentiation capacity were further defined. Testing was conducted at three time points: 7 days, 14 days, and 21 days. This allowed for dynamic observation of the temporal effects of the bone augmentation material composition on osteogenic differentiation of MC3T3-E1 cells. Day 7 corresponds to the peak expression of early osteogenic markers, while days 14 and 21 cover the mid-to-late stages of mineralized nodule formation. Alkaline phosphatase activity was detected using the p-nitrophenyl phosphate method. This method quantitatively reflects alkaline phosphatase activity by detecting the amount of p-nitrophenol produced, and is characterized by high sensitivity, ease of operation, and reliable results. Mineralized nodules were observed using Alizarin Red staining. Alizarin Red specifically binds to calcium salts to form a red complex, allowing for direct assessment of the degree of extracellular matrix mineralization under a microscope. Osteogenesis-related genes included Runx2 (a key transcription factor for osteogenic differentiation), ALP (an early osteogenic marker), OCN (a late osteogenic marker), and Col1a1 (a major collagen component of the bone matrix), covering molecular markers throughout the entire process from differentiation initiation to matrix maturation. The test results obtained through the above specific methods can comprehensively and accurately evaluate the bone differentiation-promoting ability of bone augmentation material compositions from three levels: enzyme activity, mineralization capacity, and gene expression, providing a reliable in vitro biological basis for material optimization and screening.

[0024] As a further aspect of the present invention: In Project 6, the critical size defect of the rabbit skull was 8-10 mm in diameter, a full-thickness defect, and the postoperative observation time was 4 weeks, 8 weeks, and 12 weeks. The new bone deposition rate was evaluated by calculating the proportion of new bone area to the total defect area per unit time. The bone volume fraction was obtained by calculating the ratio of new bone volume to the total defect volume after three-dimensional reconstruction by micro-CT. The defect closure effect was evaluated by the degree of bone bridging at the defect edge and the new bone coverage rate at the defect center at different postoperative time points.

[0025] By adopting the above technical solution, the key parameters for evaluating bone regeneration effects in animals were further clarified. Rabbit skull full-thickness defects with a diameter of 8 to 10 mm were selected. This size range represents critical-sized defects that cannot heal spontaneously, accurately reflecting the osteogenic induction capacity of the bone augmentation material composition. Three postoperative observation time points were set at 4, 8, and 12 weeks, corresponding to the early inflammation and vascularization stage, the intermediate bone formation stage, and the late bone remodeling and maturation stage of bone repair, respectively, allowing for dynamic tracking of the entire process of material-induced bone regeneration. The rate of new bone deposition was quantified as the proportion of newly formed bone area to the total defect area per unit time. This indicator directly reflects the speed at which the material induces new bone to fill the defect. The bone volume fraction was obtained by calculating the ratio of newly formed bone volume to the total defect volume after three-dimensional reconstruction using micro-CT. This three-dimensional quantitative method is more accurate than two-dimensional area measurement and can comprehensively assess the three-dimensional spatial distribution and volume ratio of newly formed bone within the defect area. Defect closure effectiveness is evaluated from two dimensions: the degree of bone bridging at the defect margins and the new bone coverage at the defect center. The former reflects the distance of bone ingrowth and the healing process at the defect margins, while the latter reflects the filling capacity of the defect center area. The combination of these two dimensions provides a complete description of the overall healing status of the defect. Using these standardized evaluation parameters, the in vivo bone regeneration performance of different bone augmentation material compositions can be objectively and repeatedly compared, providing a reliable basis for preclinical efficacy validation of the materials.

[0026] Compared with the prior art, the beneficial effects of the present invention are: The bone augmentation material composition provided by this invention overcomes the technical bottlenecks of existing granular bone substitutes, such as difficulty in intraoperative shaping and poor postoperative morphological stability, by compounding a specific composition of viscous freeze-dried particles (composed of chitosan, acrylic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide) with granular bone substitutes at a mass ratio of 0.15:1. This composition can form a "dough-like" composite material with excellent cohesion and plasticity. When filling irregular bone defects such as craniofacial and alveolar ridge defects, surgeons can shape it arbitrarily and fit it tightly to the defect surface, avoiding the problem of ineffective cavities caused by the inability of traditional loose granular materials to adhere to the wound surface. More importantly, after implantation, this material can resist the compression of surrounding soft tissues and the continuous erosion of body fluids, maintaining a stable three-dimensional repair contour during the healing period. This effectively prevents volume loss caused by particle displacement, diffusion, or collapse, providing a long-term stable spatial scaffold for bone regeneration.

[0027] Meanwhile, the composition of this invention significantly improves upon the shortcomings of existing bone substitute materials where the mechanical properties do not match the needs of bone regeneration. This material possesses excellent mechanical toughness and cyclic compression stability, maintaining structural integrity and mechanical properties even after 100 cycles of compression loading, meeting the anti-fatigue requirements under postoperative tissue activity and physiological loads. In vitro cell experiments confirmed that this composition significantly promotes osteogenic differentiation of MC3T3-E1 pre-osteoblasts; a rabbit cranial critical-size defect model further demonstrated that, compared with simple granular bone substitutes, this composite material can accelerate new bone deposition, increase bone volume fraction, and achieve better defect closure, without causing systemic toxicity. This invention, with its simple components and optimized formulation, simultaneously addresses the contradictory requirements of intraoperative operability and long-term postoperative stability, providing a novel bone grafting system with clear translational potential for the functional repair of complex bone defects.

[0028] Other features and advantages of the present invention will be disclosed in detail in the following detailed description and accompanying drawings. Attached Figure Description

[0029] Figure 1 It is a diagram showing the preparation and molding of the composition; Figure 2 It is a strain amplitude scan curve of the composition; Figure 3 Stress-strain curves of different compositions measured in uniaxial compression tests; Figure 4 It is a stress-strain curve of the composition determined by 100 cycles of compression testing; Figure 5 This is a graph showing the compression modulus of different compositions after 100 cycles of compression testing; Figure 6It is a displacement-stress curve of the composition determined by shear test. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Implementation Method 1: Preparation of Bone Augmentation Material Composition A bone augmentation material composition, such as Figure 1 As shown, the material comprises viscous freeze-dried granules and granular bone substitutes, with a mass ratio of viscous freeze-dried granules to granular bone substitutes of 0.15:1. The viscous freeze-dried granules are composed of chitosan (medium molecular weight 30 kDa), acrylic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS). In the specific preparation process, chitosan is first dissolved in a suitable acidic aqueous solution, and acrylic acid is added. A cross-linking reaction occurs under the catalysis of EDC and NHS, forming a gel-like product. After freeze-drying, the product is pulverized to obtain viscous freeze-dried granules. These granules are then mixed uniformly with conventional commercially available granular bone substitutes (such as deproteinized bovine bone minerals, β-tricalcium phosphate granules, or hydroxyapatite granules) at a mass ratio of 0.15:1 to obtain a dough-like composite material. This material exhibits good cohesiveness and plasticity, allowing for arbitrary shaping according to the morphology of the bone defect during surgery.

[0032] Implementation Method 2: Mechanical Property Testing of Bone Augmentation Material Composition To systematically evaluate the mechanical behavior of the above-mentioned bone augmentation material composition, this invention provides a complete set of testing methods.

[0033] First, frequency scanning experiments were conducted. The bone augmentation material composition was prepared into uniformly thick circular test samples and placed on a rheometer platform. The frequency scanning range was set to 0.1 Hz to 100 Hz, and oscillation tests were performed under a constant strain amplitude (within the linear viscoelastic region). The storage modulus G′ and loss modulus G″ were recorded as a function of frequency. The results showed that within the measured frequency range, G′ continuously increased with increasing frequency, indicating enhanced elastic response and good structural cohesion under high-frequency dynamic loading. G″ increased with increasing frequency in the low-frequency region, and then tended to remain constant after reaching 10 Hz, reflecting that the material's viscous dissipation capacity reached saturation at high frequencies, avoiding plastic collapse caused by excessive flow. This frequency scanning method can comprehensively evaluate the modulus evolution characteristics of materials under complex mechanical environments in vivo (such as chewing, blood flow erosion, tissue compression, and other dynamic loads). Figure 2 As shown.

[0034] Next, uniaxial compression tests were conducted. Test samples with a viscous freeze-dried particle content of 3.2 wt%, 4.8 wt%, and 6.2 wt% were prepared, and each composition was prepared into a standard cylindrical specimen (10 mm in diameter and 12 mm in height). The specimens were placed on a universal testing machine and subjected to uniaxial compression at a constant compression rate of 1 mm / min, and the stress-strain curves were recorded. The experimental results are as follows: Figure 3 show.

[0035] Next, cyclic compression tests were conducted to evaluate fatigue resistance. Standard cylindrical specimens of the bone augmentation material composition were prepared and subjected to 100 loading and unloading cycles at a constant compression rate of 1 mm / min on a universal testing machine. The stress-strain curves for each cycle were recorded. The same tests were also performed on samples with three different formulations: 3.2 wt%, 4.8 wt%, and 6.2 wt%. The compressive modulus was calculated based on the stress-strain curves for each cycle, and a graph showing the change in compressive modulus with the number of cycles was plotted. The results are as follows: Figure 4 and Figure 5 As shown.

[0036] Finally, a shear test was conducted. The bone augmentation material composition (4.8 wt%) was placed in a shear test fixture, and gradually increasing shear force was applied. The relationship between displacement and stress was recorded, such as... Figure 6 As shown in the figure. This result indicates that the material can effectively resist shear forces from the surrounding soft tissue, avoiding interlayer slip and interface separation.

[0037] Implementation Method 3: Evaluating the ability to promote bone differentiation through in vitro cell experiments Bone augmentation material composition (4.8 wt%) was co-cultured with MC3T3-E1 pre-osteoblasts, and the results were analyzed at 7, 14, and 21 days of culture. Alkaline phosphatase activity was detected using the p-nitrophenyl phosphate method. The results showed that compared with the control group containing only granular bone substitutes, the alkaline phosphatase activity in the composite material group was approximately 2.1-fold and 2.8-fold higher at 7 and 14 days, respectively, with statistically significant differences. Alizarin red staining was used to observe the formation of mineralized nodules. Scattered red mineralized nodules were visible in the composite material group at 14 days, and the number of mineralized nodules significantly increased and merged into patches at 21 days, while the control group had sparse mineralized nodules. Real-time quantitative PCR was used to detect the expression levels of osteogenic-related genes Runx2, ALP, OCN, and Col1a1. The results showed that the expression levels of each gene in the composite material group were significantly higher than those in the control group at 7, 14, and 21 days, with Runx2 showing the most significant upregulation at 7 days and OCN showing the largest upregulation at 21 days. The above results indicate that the bone augmentation material composition of the present invention can significantly promote the differentiation of MC3T3-E1 cells into osteogenic phenotype and has good in vitro osteogenic activity.

[0038] Implementation Method 4: Animal Bone Defect Repair Experiment Adult New Zealand white rabbits were used to create circular, full-thickness, critical-sized bone defects with a diameter of 8 mm on both sides of the skull. A bone augmentation material composition (4.8 wt%) was implanted into the left defect area, while a simple granular bone substitute was implanted into the right defect area as a control. Animals were sacrificed at 4, 8, and 12 weeks post-surgery, and skull specimens were collected.

[0039] Bone volume fraction (new bone volume / total defect volume) was calculated using micro-CT scanning and three-dimensional reconstruction. Results showed that at 4 weeks post-surgery, the bone volume fraction in the composite material group was 18.5% ± 2.1%, while that in the control group was 9.2% ± 1.5%; at 8 weeks post-surgery, the composite material group had a bone volume fraction of 42.3% ± 3.2%, while the control group had a bone volume fraction of 21.6% ± 2.4%; and at 12 weeks post-surgery, the composite material group reached 68.7% ± 4.0%, while the control group only had 35.4% ± 3.1%. The bone volume fraction in the composite material group was significantly higher than that in the control group at all time points.

[0040] The rate of new bone deposition and defect closure were evaluated using histological section staining. The rate of new bone deposition was calculated as the proportion of newly formed bone area to the total defect area per unit time. At 4 weeks post-operation, the new bone deposition rate was 2.1% / week in the composite material group and 0.9% / week in the control group; at 8 weeks, it was 3.0% / week in the composite material group and 1.6% / week in the control group; and at 12 weeks, it was 2.5% / week in the composite material group and 1.3% / week in the control group. Regarding defect closure, at 8 weeks post-operation, the composite material group showed obvious bone bridging at the defect edges, with new bone coverage exceeding 55% in the defect center; the control group showed only a small amount of new bone at the defect edges, with the central area still filled with fibrous tissue. At 12 weeks post-operation, the defects in the composite material group were almost completely closed by new bone, with continuous bone bridging; the control group still had a large unmineralized area in the center of the defect.

[0041] Meanwhile, after euthanizing the animals 12 weeks post-surgery, histopathological examination of major organs such as the heart, liver, spleen, lungs, and kidneys was performed. No obvious abnormal changes such as inflammatory cell infiltration, necrosis, or fibrosis were observed, and there was no significant difference compared with the sham-operated group, indicating that the bone augmentation material composition has no systemic toxicity and good biosafety.

[0042] Implementation Method 5: Comparison of Materials with Different Proportions Following the method of Embodiment 1, three composite materials with a mass ratio of viscous freeze-dried particles to granular bone substitutes of 0.10:1, 0.15:1, and 0.20:1 were prepared. Their mechanical properties were compared using uniaxial compression and cyclic compression tests, and their osteogenic effects were evaluated using in vitro cell experiments and animal experiments. The results showed that the 0.10:1 ratio material had insufficient cohesion, failing to form dough-like clumps and easily dispersing in body fluids; the 0.20:1 ratio material had excessive viscosity, adhering to instruments during shaping, and exhibited significant residual deformation after cyclic compression, thus inhibiting osteogenic activity (possibly related to excessive encapsulation of the bone substitute). The 0.15:1 ratio material showed the best performance in terms of plasticity, fatigue resistance, and osteogenic activity, representing the optimal mass ratio of this invention.

[0043] Implementation Method Six: Practical Application of Bone Augmentation Material Composition In actual clinical applications, the bone augmentation material composition of the present invention is operated according to the following steps: During the preoperative preparation phase, the physician first selects a suitable commercially available granular bone substitute based on the location, size, shape, and surrounding soft tissue conditions of the bone defect. Available types include, but are not limited to, deproteinized bovine bone mineral, β-tricalcium phosphate granules, hydroxyapatite granules, or bioactive glass granules. The particle size can be selected according to the type of defect; for example, a particle size of 0.25-1.0 mm is recommended for alveolar bone defects, and 1.0-2.0 mm is recommended for craniofacial bone defects. Simultaneously, the viscous freeze-dried granules are removed from refrigerated or room temperature storage conditions, and the integrity of the packaging and its sterility are checked.

[0044] During the material preparation stage, weigh the two components at a ratio of 0.15:1 (viscous freeze-dried granules to granular bone substitute) on a sterile operating table or surgical table. Specifically, first place the granular bone substitute in a sterile mixing container, then add the viscous freeze-dried granules. Use a sterile spatula or stirring rod to slowly stir clockwise for approximately 30 seconds to 1 minute until the two phases are evenly mixed, forming a homogeneous "dough-like" composite material. If the ambient temperature is low and the material's viscosity decreases, preheat the mixing container in a 37°C constant temperature oven for 1-2 minutes to help improve the material's plasticity. The prepared composite material should have the following characteristics: it should adhere and clump together when lightly pressed, not crumble and drip when lifted, and form an indentation when pressed with a finger and maintain its shape.

[0045] During the shaping and implantation phase, the physician uses sterile instruments (such as a bone chisel, curette, or shaping tool) to take an appropriate amount of composite material and shape it arbitrarily according to the shape of the bone defect. For irregular defects, the material can be kneaded into a shape that matches the contour of the defect and gently pressed to ensure it fits tightly against the bottom and sidewalls of the defect. For penetrating bone defects, the material can first be shaped into a cone or dumbbell shape slightly larger than the size of the defect, and then gradually pushed in and compacted from one side to ensure seamless contact between the material and the host bone interface. For deep and large defects that require layered filling, a layered implantation method can be used, with each layer not exceeding 5mm in thickness. Each layer is shaped and gently pressed to avoid creating cavities. After implantation, the surface of the material can be gently pressed with moist gauze or cotton pads to further improve the adhesion. If a barrier membrane is used in conjunction with guided bone regeneration, an absorbable or non-absorbable collagen membrane can be covered on the surface of the material and fixed with staples or sutures.

[0046] In the postoperative management phase, patients should be managed according to the standard nursing procedures for bone graft surgery. Avoid pressure or external impact on the surgical area for one week post-surgery, and avoid strenuous exercise for two weeks. Regular follow-up imaging is necessary, typically with cone-beam CT or X-rays taken at 3, 6, and 12 months post-surgery to assess bone healing, material absorption, and new bone formation. If implant placement is combined with bone grafting, implantation surgery can be scheduled electively 6-12 months post-surgery, depending on the progress of bone healing.

[0047] In terms of combined applications, this bone augmentation material composition can be used in conjunction with other bone repair strategies. For example, autologous blood, platelet-rich plasma, or bone marrow aspiration concentrate can be added to the composite material during the preparation stage to further enrich growth factors and enhance osteogenic activity. The added volume generally does not exceed 10%-20% of the total volume of the composite material to avoid excessive dilution affecting the material's cohesiveness. For defects with a high risk of infection (such as post-extraction site preservation), an appropriate amount of antibiotics (such as gentamicin or clindamycin) can be added during preparation to achieve local sustained-release anti-infection. For large-scale segmental bone defects, the composite material can be filled into titanium mesh, polyetheretherketone mesh, or 3D-printed personalized titanium cages as an internal osteogenic matrix, providing structural support while promoting bone ingrowth.

[0048] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0049] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A bone augmentation material composition, characterized in that, It includes viscous freeze-dried particles and granular bone substitutes, wherein the mass ratio of the viscous freeze-dried particles to the granular bone substitutes is 0.15:1; The viscous freeze-dried particles are composed of chitosan, acrylic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; the chitosan has a median molecular weight of 30 kDa.

2. A method for detecting the bone augmentation material composition of claim 1, characterized in that, The following testing items are included: Project 1: The storage modulus G′ and loss modulus G″ of the composition were determined by strain amplitude scanning test; Project 2: Stress-strain changes of compositions with different ratios were determined using uniaxial compression tests; Project 3: The stress-strain change of the standard composition after 100 cycles was determined by cyclic compression testing, and the compressive modulus of different ratio compositions after 100 cycles was determined by cyclic compression testing. Project 4: The displacement-stress change of the composition was determined by shear test; Project 5: The promoting effect of the composition on osteogenic differentiation of MC3T3-E1 cells was detected by in vitro cell assays; Project 6: The bone regeneration effect of the composition was tested using an in vivo animal model of critical-sized bone defects.

3. The method for detecting a bone augmentation material composition according to claim 2, characterized in that, Project 1 includes the following steps: Project 1 includes the following steps: preparing the composition into a test sample, placing it on a rheometer platform, setting the frequency scanning range to 0.1Hz to 100Hz, performing an oscillation test under a constant strain amplitude, and recording the changes in storage modulus G′ and loss modulus G″ with frequency to evaluate the viscoelastic behavior of the composition.

4. The method for detecting a bone augmentation material composition according to claim 2, characterized in that, Project 2 includes the following steps: Test samples with different mass percentage contents of the viscous freeze-dried particles were prepared, namely 3.2 wt%, 4.8 wt%, and 6.2 wt%. Standard cylindrical specimens with diameters of 8-12 mm and heights of 10-15 mm were prepared from each composition. The specimens were placed on a universal testing machine and subjected to uniaxial compression at a constant compression rate. Stress-strain curves were recorded, and the compressive modulus and compressive strength of each composition were calculated. The effects of different viscous freeze-dried particle contents on the compressive properties of the composite material were compared.

5. The method for detecting a bone augmentation material composition according to claim 2, characterized in that, Project 3 includes the following steps: The composition was prepared into a standard sample, and a cyclic compression test was conducted. The sample was subjected to 100 loading and unloading cycles at a constant compression rate, and the stress-strain change curve for each cycle was recorded. Test samples were prepared from the composition at different mass ratios, wherein the mass percentages of viscous freeze-dried particles were 3.2 wt%, 4.8 wt%, and 6.2 wt%, respectively. Cylindrical specimens with diameters of 8-12 mm and heights of 10-15 mm were prepared from each composition. The specimens were placed on a universal testing machine and subjected to 100 cycles of loading and unloading compression at a constant compression rate. The stress-strain curves for each cycle were recorded. The corresponding compression modulus was calculated based on the stress-strain curves for each cycle, and a graph showing the change in compression modulus with the number of cycles was plotted. The evolution of the stress-strain curves during the 100 cycles was analyzed to evaluate the fatigue resistance and cyclic stability of the composition.

6. The method for detecting a bone augmentation material composition according to claim 2, characterized in that, Project 4 includes the following steps: The composition is placed in a shear test fixture, and a gradually increasing shear force is applied. The relationship between displacement and stress is recorded, a displacement-stress curve is plotted, and the shear strength and shear modulus are calculated.

7. The method for detecting a bone augmentation material composition according to claim 2, characterized in that, Project 5 includes the following steps: The composition was co-cultured with MC3T3-E1 pre-osteoblasts. After 7-21 days of culture, alkaline phosphatase activity, mineralized nodule formation, and osteogenic gene expression levels were detected to evaluate the osteogenic differentiation-promoting ability of the composition.

8. The method for detecting a bone augmentation material composition according to claim 2, characterized in that, Project 6 includes the following steps: A critical-sized bone defect was prepared from rabbit skulls, and the composition was implanted into the defect area. Animals were sacrificed 4-12 weeks postoperatively, and skull specimens were collected. Bone volume fraction was calculated by micro-CT scanning and three-dimensional reconstruction. The rate of new bone deposition and defect closure were evaluated by histological staining, and pathological changes in major organs were detected to evaluate systemic toxicity.

9. The method for detecting a bone augmentation material composition according to claim 7, characterized in that, In Project 5, the composition was co-cultured with MC3T3-E1 cells for 7, 14, and 21 days. Alkaline phosphatase activity was detected using the p-nitrophenyl phosphate method, and mineralized nodules were observed using Alizarin Red staining. Osteogenesis-related genes included Runx2, ALP, OCN, and Col1a1.

10. The method for detecting a bone augmentation material composition according to claim 8, characterized in that, In Project 6, the critical size defect of the rabbit skull was 8-10 mm in diameter, a full-thickness defect, and the postoperative observation time was 4 weeks, 8 weeks, and 12 weeks. The new bone deposition rate was evaluated by calculating the proportion of new bone area to the total defect area per unit time. The bone volume fraction was obtained by calculating the ratio of new bone volume to the total defect volume after three-dimensional reconstruction by micro-CT. The defect closure effect was evaluated by the degree of bone bridging at the defect edge and the new bone coverage rate at the defect center at different postoperative time points.

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