Bone graft composition with controlled release of osteogenesis and angiogenesis promoting factor and method for producing the same

Abstract

The present invention relates to a porous bone graft scaffold, a method for manufacturing the same, and an injectable bone graft composition. A porous bone graft scaffold according to one aspect can induce a powerful synergistic effect of bone regeneration by effectively combining bone formation and angiogenesis growth factors with strontium-doped bone minerals through a polydopamine coating. Furthermore, an optimal porous microstructure secured by a controlled process can promote efficient cell penetration and control the release of growth factors to maximize the regenerative effect. Moreover, an injectable formulation according to one aspect can simultaneously exhibit excellent ease of injection and shape stability at the implantation site.

Description

Technology Field

[0001] The present invention relates to a porous bone graft support, a method for manufacturing the same, an injectable bone graft composition, and the like. Background Technology

[0003] The number of patients with bone defects is continuously increasing due to the entry into an aging society, various traumas, and bone diseases. Autologous bone grafting, the standard method for treating such defects, has limitations regarding additional damage to the donor site and the amount of bone that can be harvested. Furthermore, allogeneic or xenogeneic bone grafts also pose potential risks of immune rejection or disease transmission, thus necessitating the development of synthetic bone graft materials to replace them.

[0004] Accordingly, porous scaffolds based on demineralized bone matrix (DBM) or beta-tricalcium phosphate (β-TCP) have been studied; however, when loaded with growth factors such as bone morphogenetic proteins (BMPs), they were rapidly released early in the implantation process, resulting in low bioavailability and difficulty in sustaining effects over the long term. Furthermore, there were technical limitations in implementing an optimal porous microstructure for effective cell penetration into the scaffold.

[0005] Successful bone regeneration requires the organic co-occurrence of osteoogenesis by osteoblasts and rapid angiogenesis to supply oxygen and nutrients to the graft site. However, many existing bone graft materials focus solely on promoting osteoinduction and have been insufficient in maximizing the synergistic effects of the two processes by simultaneously considering angiogenesis.

[0006] Meanwhile, while injectable putty formulations are preferred for irregular bone defects or minimally invasive surgeries, existing compositions present a trade-off where ensuring ease of injection results in insufficient shape stability, whereas enhancing shape stability leads to excessively high injectability, making them difficult to use clinically. Therefore, there is an urgent need for the development of new bone graft materials that can comprehensively resolve these issues.

[0007] delete Prior art literature

[65535] Korean Published Patent Application KR 10-2024-0174819 A (December 17, 2024) The problem to be solved

[0008] One aspect provides a porous bone graft scaffold comprising demineralized bone matrix (DBM) fibers having an average length of 1 mm to 10 mm; a poloxamer matrix; bone morphogenetic protein and vascular endothelial growth factor; bone mineral particles; and ascorbic acid or a pharmaceutically acceptable salt thereof.

[0009] Another aspect provides a method for manufacturing the above-mentioned porous bone graft scaffold.

[0010] Another aspect provides an injectable bone graft composition comprising: demineralized bone matrix (DBM) powder having an average particle size of 100 µm to 800 µm; a heat-sensitive poloxamer hydrogel carrier; a bone morphogenetic protein and vascular endothelial growth factor; bone minerals; and ascorbic acid or a pharmaceutically acceptable salt thereof. means of solving the problem

[0012] One aspect provides a porous bone graft scaffold comprising demineralized bone matrix (DBM) fibers having an average length of 1 mm to 10 mm; a poloxamer matrix; bone morphogenetic protein and vascular endothelial growth factor; bone mineral particles; and ascorbic acid or a pharmaceutically acceptable salt thereof.

[0014] In this specification, the term “Demineralized Bone Matrix (DBM)” refers to a material obtained by removing inorganic components from allogeneic or xenogeneic bone, leaving behind an organic matrix such as collagen and bone-forming proteins. It acts as a key component providing osteoinduction capabilities and forms the structural basis of porous scaffolds.

[0015] Specifically, the fiber form used in the porous support facilitates the formation of an interconnected pore structure, thereby providing a pathway for cell penetration, and the powder form used in the injectable composition can contribute to ensuring flowability within the syringe and enhancing defect filling.

[0016] A porous bone graft scaffold according to one aspect may comprise 30 to 60 parts by weight of the demineralized bone matrix (DBM) fibers per 100 parts by weight of the dry weight of the scaffold.

[0017] If the above content is less than 30 parts by weight, it is difficult to expect a sufficient osteoinduction effect at the implantation site, and because the solid content is relatively low compared to the poloxamer substrate, the pore structure may easily collapse during the freeze-drying process or the mechanical strength of the final scaffold may be significantly reduced, which may impair the ability to maintain space at the implantation site. On the other hand, if the above content exceeds 60 parts by weight, fibers may aggregate during the preparation of the aqueous slurry, causing the viscosity of the slurry to rise sharply and the uniformity to decrease. This may induce a non-uniform pore structure that hinders cell penetration, and the final scaffold may be too hard and brittle, making it difficult to trim and use to fit the shape of the bone defect.

[0018] In this specification, the term “poloxamer” may be a temperature-sensitive polymer that exists in a liquid state at low temperatures to enable uniform mixing with other components, and forms a gel at temperatures similar to body temperature to act as a carrier that provides shape stability at the implantation site.

[0019] A porous bone graft scaffold according to one aspect may comprise 30 to 60 parts by weight of the poloxamer substrate per 100 parts by weight of the dry weight of the scaffold.

[0020] If the above content is less than 30 parts by weight, the binding force to solid components is insufficient, which reduces the shape stability of the scaffold, and as a result, loss of solid components or particles may occur during transportation or implantation. On the other hand, if the above content exceeds 60 parts by weight, the relative proportion of bone regeneration effective components, such as DBM fibers or bone mineral particles, decreases, which may reduce the bone regeneration promoting effect per unit volume, and due to the hydrophobic nature of poloxamer, it may hinder the penetration of body fluids, nutrients, and cells into the scaffold during the initial implantation, thereby acting as a factor that inhibits rapid bone regeneration.

[0021] In this specification, the term “bone morphogenetic protein” refers to a growth factor that promotes the differentiation of mesenchymal stem cells into osteoblasts, and “vascular endothelial growth factor” may refer to a growth factor that induces the formation of new blood vessels.

[0022] In a composition according to one aspect, the combination of the bone morphogenetic protein and the vascular endothelial growth factor induces the rapid establishment of a vascular network that supplies oxygen and nutrients essential for the bone formation process, thereby exhibiting a synergistic effect that promotes significantly faster and more robust bone regeneration than when using a single growth factor.

[0023] A porous bone graft scaffold according to one aspect may comprise a total of 0.05 to 1.5 parts by weight of the bone morphogenetic protein and vascular endothelial growth factor per 100 parts by weight of the dry weight of the scaffold.

[0024] If the above content is less than 0.05 parts by weight, it may not reach the minimal effective concentration required to effectively attract and differentiate mesenchymal stem cells or endothelial cells at the transplant site, and this relies solely on the osteoconductive / osteoinductive effects of DBM or bone mineral particles, making it difficult to express the potent and rapid bone regeneration synergistic effect intended by the present invention. On the other hand, if the above content exceeds 1.5 parts by weight, local overdose of a potent growth factor, such as rhBMP-2, may cause side effects such as ectopic bone formation, inflammatory response, or excessive stimulation of early osteoclast activity, and may interfere with the harmonious bone remodeling process.

[0025] In this specification, the term “bone mineral particles” may refer to a material that acts as an osteoconductive scaffold providing a physical surface on which new bone can be deposited, which can be gradually absorbed in vivo and replaced by new bone tissue.

[0026] A porous bone graft scaffold according to one aspect may comprise 5 to 20 parts by weight of the bone mineral particles per 100 parts by weight of the dry weight of the scaffold.

[0027] If the above content is less than 5 parts by weight, there is insufficient effective surface area for osteoblasts to attach and proliferate, so bone formation induced by DBM may not proceed efficiently and may be delayed. On the other hand, if the above content exceeds 20 parts by weight, it may physically interfere with the osteoinductive properties of DBM fibers and the biological activity of growth factors, reduce the porosity of the entire scaffold, or impede the interconnectivity between pores, thereby hindering cell penetration and causing central necrosis.

[0028] In a porous bone graft scaffold according to one aspect, the bone morphogenetic protein may be rhBMP-2 or rhBMP-7, the vascular endothelial growth factor may be rhVEGF, and the bone mineral may be β-TCP.

[0029] In one experimental example, the example containing all of rhBMP-2, rhVEGF, and β-TCP showed statistically the highest ALP activity and calcium deposition of hMSC compared to the comparative example that did not. Additionally, in the analysis of tube formation of HUVEC, it was confirmed that it exhibited the longest total tube length and the largest number of branching points, demonstrating the most superior angiogenic ability. From this, it was confirmed that the composite composition of the present invention organically links bone induction and angiogenesis to have a significantly superior bone regeneration promoting effect.

[0030] A porous bone graft scaffold according to one aspect may have a total porosity of 70% to 95% and an average pore size of 200 μm to 400 μm.

[0031] If the total porosity and pore size are below the above range, physical movement and internal penetration of cells are inhibited, which may result in minimal bone formation in the center of the scaffold; conversely, if they exceed the above range, the initial mechanical properties of the scaffold deteriorate and the surface area for cell attachment decreases, making it difficult to induce effective bone regeneration.

[0032] In one experimental example, SEM analysis results showed that the porous bone graft scaffold of the present invention exhibited a uniform and interconnected pore structure with an average size of about 300 μm, and the porosity was measured to be about 85%. In the evaluation of cell penetration ability, it was confirmed that the number of cells passing through the porous bone graft scaffold of the present invention was overwhelmingly higher than that of the comparative example. This implies that the total porosity of 70% to 95% and the pore size in the range of 200-400 μm specified in the present invention provide the most suitable environment for cells to physically move and penetrate.

[0033] The above bone morphogenetic protein and vascular endothelial growth factor may be characterized by being included in a weight ratio of 5:1 to 15:1, and specifically, may be included in a weight ratio of 10:1.

[0034] If the above weight ratio is less than 5:1, bone formation may be delayed or fibrous tissue may be formed due to excessive angiogenesis, and if it exceeds 15:1, osteoblasts induced by insufficient blood supply may necrose, preventing effective bone formation.

[0035] In one experimental example, it was confirmed that the porous bone graft scaffold of the present invention (10:1 ratio) exhibited the highest ALP activity and excellent tube-forming ability, thereby promoting bone differentiation and angiogenesis in a balanced manner. On the other hand, in the comparative example with an excessively high VEGF ratio, tube formation was active but ALP activity was lower than that of Example 1, and in the comparative example with a very low VEGF ratio, tube formation was poor, confirming that long-term bone formation would be difficult without vascular support. From this, it was confirmed that the specific weight ratio range of the present invention is the optimal condition for maximizing the synergistic effect of angiogenesis and bone formation.

[0036] In a porous bone graft scaffold according to one aspect, the demineralized bone matrix fibers and bone mineral particles may be characterized by having polydopamine coated on their surfaces, and the bone morphogenetic protein and vascular endothelial growth factor immobilized on the polydopamine coating.

[0037] This plays a role in maximizing bioavailability by preventing the rapid release and loss of growth factors during the early stages of transplantation and controlling their continuous release over a long period.

[0038] In one experimental example, the porous bone graft scaffold of the present invention with a polydopamine coating showed a cumulative release rate of less than about 30% within 24 hours, which was significantly lower than that of a comparative example without a polydopamine coating. From this, it was confirmed that the polydopamine coating and fixation prevent the loss of growth factors in the early stages and control their release effectively over a long period.

[0039] In addition, the above β-TCP may be characterized by being doped with strontium.

[0040] Strontium exhibits a dual effect of promoting the proliferation of osteoblasts and inhibiting the activity of osteoclasts, and can more powerfully promote bone regeneration through synergistic action with bone morphogenetic proteins.

[0041] In one experimental example, the porous bone graft scaffold of the present invention containing strontium showed results in a statistically greater increase of about 40% in ALP activity and final calcium deposition compared to the comparative example, and from this, it was confirmed that strontium doping technology can significantly promote bone differentiation and regeneration through a strong synergistic effect with growth factors, going beyond simply acting as a bone mineral.

[0042] According to one aspect, a porous bone graft scaffold may comprise 0.1 to 2.0 parts by weight of the ascorbic acid or its pharmaceutically acceptable salt per 100 parts by weight of the dry weight of the scaffold.

[0043] If the above content is less than 0.1 parts by weight, the effect of promoting collagen synthesis in osteoblasts or acting as an antioxidant is negligible, which may inhibit the formation of bone matrix in the early stages of transplantation and make cells vulnerable to oxidative stress. On the other hand, if the above content exceeds 2.0 parts by weight, it may excessively lower the local pH of the transplant site, thereby inducing an inflammatory response in surrounding tissues, impair the stability of the poloxamer matrix, or cause denaturation of immobilized growth factors (rhBMP-2, rhVEGF), which may reduce biological activity.

[0045] Another aspect is to provide a method for manufacturing the above-mentioned porous bone graft scaffold.

[0046] Specifically, a method for manufacturing a porous bone graft scaffold according to one aspect may comprise: (a) forming a polydopamine coating layer on the surface of demineralized bone matrix (DBM) fibers and bone mineral particles, and immobilizing bone morphogenetic protein and vascular endothelial growth factor on the coating layer; (b) preparing an aqueous slurry by mixing the solid components of step (a) with a poloxamer hydrogel carrier and ascorbic acid; (c) cooling the slurry to a temperature of -10°C to -40°C at a controlled cooling rate of -0.5°C / min to -5°C / min; (d) an annealing step of maintaining at -10°C to -2°C for 1 to 4 hours; (e) forming a porous scaffold by sublimating a frozen solvent under vacuum; and (f) sterilizing the porous scaffold with gamma rays.

[0048] In step (a) above, the polydopamine coating is a surface modification technology with excellent biocompatibility that forms a strong adhesive layer on the surface of DBM fibers and bone mineral particles, thereby providing a foundation for the stable immobilization of growth factors thereafter. This step plays a key role in controlling the initial over-release of growth factors and inducing their gradual release over a long period at the implantation site, thereby ensuring the sustainability of the therapeutic effect.

[0049] In step (b) above, the preparation of the aqueous slurry is a process that determines the uniformity of the final porous support, and by uniformly dispersing solid components with immobilized growth factors within the poloxamer hydrogel carrier, mechanical properties and biological activity can be consistently expressed throughout the entire support to be prepared.

[0050] In step (c) above, the cooling rate is a key variable determining the final pore structure. If the cooling rate is too fast, exceeding -5°C / min, small and non-uniform ice crystals may form, leading to micropores unsuitable for cell penetration. Conversely, if the cooling rate is too slow, below -0.5°C / min, excessively large pores may form, which can reduce the mechanical strength of the support.

[0051] In step (d) above, the annealing process recrystallizes the microcrystals of the frozen solvent to induce the formation of larger, more uniform, and highly interconnected pore structures. If this step is omitted or time is insufficient, closed pores with poor connectivity between pores may be formed, which can hinder internal penetration of cells.

[0052] In one experimental example, the porous bone graft scaffold of the present invention exhibited a uniform and interconnected pore structure with an average size of approximately 300 μm, and the porosity was measured to be approximately 85%. In the evaluation of cell penetration ability, it was confirmed that the number of cells passing through the porous bone graft scaffold of the present invention was overwhelmingly higher compared to the comparative example, which was manufactured by rapid cooling and had non-uniform and fine pores or was in a non-porous form.

[0053] From this, it was confirmed that the manufacturing process specified in the present invention, in particular the controlled cooling and annealing steps, plays a decisive role in forming an optimal microstructure for efficient internal penetration of cells.

[0054] In step (e) above, a vacuum may be applied to the frozen slurry to directly sublimate the solvent in the form of ice crystals into a gaseous state. Since this freeze-drying process does not go through a liquid state, it is possible to form an interconnected porous network by emptying the space occupied by the ice crystals while maintaining the arrangement structure of the solid components within the slurry.

[0055] In step (f) above, gamma sterilization is a final process for using the manufactured porous scaffold for medical purposes. Gamma rays have high penetrating power and penetrate into the complex internal structure of the scaffold to effectively kill all microorganisms, thereby ensuring the sterility of the product and preventing the risk of infection during clinical application.

[0057] Another aspect provides an injectable bone graft composition comprising: demineralized bone matrix (DBM) powder having an average particle size of 100 µm to 800 µm; a heat-sensitive poloxamer hydrogel carrier; bone morphogenetic protein and vascular endothelial growth factor; bone mineral; and ascorbic acid or a pharmaceutically acceptable salt thereof.

[0059] In the above-described injectable bone graft composition, the demineralized bone matrix may be included in an amount of 25 to 45 parts by weight relative to 100 parts by weight of the total composition, and the poloxamer hydrogel carrier may be included in an amount of 40 to 60 parts by weight.

[0060] In addition, the above composition may be characterized by exhibiting a storage modulus of 500 Pa to 2,500 Pa at 25°C and 1 Hz.

[0061] In one experimental example, it was confirmed that an example satisfying the optimal DBM:poloxamer ratio (35:55) and storage modulus range (500-2500 Pa) of the present invention showed a high retention rate of over 90%, indicating excellent shape stability in a surgical environment, while simultaneously exhibiting a clinically acceptable injection force of 45.7 N, thereby ensuring ease of injection. From this, it was proven that the injectable composition of the present invention exhibits the effect of optimally satisfying both shape stability and ease of injection simultaneously within a specific composition ratio and a specific storage modulus range expressed therefrom. Effects of the invention

[0063] A porous bone graft scaffold according to one aspect can induce a powerful synergistic effect of bone regeneration by effectively combining bone formation and angiogenesis growth factors with strontium-doped bone minerals through a polydopamine coating, and the optimal porous microstructure secured by a controlled process can maximize the regenerative effect by promoting efficient cell penetration and controlling the release of growth factors. Furthermore, an injectable formulation according to one aspect can simultaneously exhibit excellent ease of injection and shape stability at the graft site. Brief explanation of the drawing

[0065] Figure 1 briefly shows a flowchart of a method for manufacturing a bone-guided bone graft composition according to one aspect. Figure 2 shows an example of a bone-guided bone graft composition according to one aspect. Figure 3 shows an example of an injectable bone graft composition according to one aspect. Figure 4 shows an example of a bone-guided bone graft composition according to one aspect. Specific details for implementing the invention

[0066] The following examples will be explained in more detail. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0068] (1) Example 1

[0069] First, strontium (Sr)-doped β particles were prepared. DBM fibers and the strontium-β-TCP particles were dispersed in a Tris buffer solution at pH 8.5, and then dopamine hydrochloride was added and stirred for 6 hours to form a polydopamine (PDA) layer on the surface. After washing and drying the PDA-coated DBM fibers and strontium-β-TCP particles, they were immersed in a solution containing rhBMP-2 and rhVEGF (10:1 weight ratio) to immobilize the growth factors onto the PDA layer.

[0070] Based on 100 parts by weight of the dry weight of the final support, a uniform aqueous slurry was prepared by dispersing 45 parts by weight of DBM fibers with the growth factor immobilized and 10 parts by weight of strontium-β-TCP particles in 44 parts by weight of Poloxamer 407 carrier (substrate), 1 part by weight of ascorbic acid, and distilled water. Subsequently, the prepared aqueous slurry was cooled to a temperature of -20°C at a controlled cooling rate of -1.0°C / min and subjected to an annealing step of maintaining at -5°C for 2 hours. Then, the frozen solvent was sublimated under vacuum to form a porous support, and finally, gamma ray sterilization was performed.

[0072] (2) Example 2

[0073] 35 parts by weight of DBM powder (average particle size 400 μm), 55 parts by weight of Poloxamer 407 carrier, and the remaining components (rhBMP-2, rhVEGF, β-ascorbic acid, etc.) were uniformly mixed using a planetary stirrer. As a result, the storage modulus measured at 25°C and 1 Hz ( G ') manufactured an injectable putty with a strength of approximately 1500 Pa.

[0075] (3) Comparative example

[0076] In the case of Comparative Examples 1 to 11, specific components or conditions were changed in the composition or manufacturing method of the above examples as described in each experimental example below, and the compositions of each example and comparative example are shown in Table 1 and Table 2 below.

[0077] [Table 1]

[0078]

[0080] [Table 2]

[0081]

[0083] Experimental Example 1. Evaluation of synergistic effects on bone formation and angiogenesis based on composite composition

[0084] We intended to verify whether the combination of bone morphogenetic protein (rhBMP-2), vascular endothelial growth factor (rhVEGF), and bone mineral (β) in the bone graft composition of one embodiment exhibits a superior synergistic effect in the ability to induce bone differentiation and angiogenesis compared to when each component is used alone or only partially combined.

[0085] Specifically, a co-culture system using human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) was established to simulate the interaction between osteoogenesis and angiogenesis. HUVECs were cultured in the lower chamber of a Transwell culture plate, while hMSCs were cultured in the upper chamber (0.4 µm pore size insert). Sculptural pieces of each group were placed in the upper chamber along with the hMSCs to allow eluted growth factors and ions to affect both cell types.

[0086] For the evaluation of osteogenic differentiation potential, hMSCs from the upper chamber were collected on days 7 and 14 of culture, and alkaline phosphatase (ALP) activity was measured. ALP is an early indicator of osteoblast differentiation and was quantified through a colorimetric reaction using a p-nitrophenyl phosphate (pNPP) substrate. On day 21 of culture, the degree of calcium deposition (mineralization) in the extracellular matrix was evaluated using Alizarin Red S staining, and the stained pigment was extracted with cetylpyridinium chloride and quantitatively analyzed by measuring the absorbance.

[0087] For the evaluation of angiogenesis, HUVECs from the lower chamber were collected on day 7 of culture, and a tube formation assay was performed on Matrigel. After 6 hours of culture, the formed tube-like structures were observed using a phase-contrast microscope, and the total tube length and number of junctions were quantified using ImageJ software.

[0088] [Table 3]

[0089]

[0091] As a result, Example 1 showed statistically significantly higher ALP activity and calcium deposition of hMSCs compared to Comparative Examples 1, 2, and 3. At the same time, the analysis of HUVEC tube formation also demonstrated the longest total tube length and the highest number of branching points, proving superior angiogenic ability. In particular, it showed significantly higher osteodifferentiation ability even compared to Comparative Example 3, which excluded rhVEGF, implying that an interaction occurred in which HUVECs activated by rhVEGF secreted factors that promote osteodifferentiation of hMSCs. From this, it indicates that the composite composition of the present invention organically links osteoinduction and angiogenesis to possess a remarkably superior bone regeneration-promoting effect (Table 3).

[0093] Experimental Example 2. Evaluation of bone formation and angiogenesis ability according to the optimal weight ratio of growth factors

[0094] The same co-culture system of hMSC and HUVEC as in Experimental Example 1 was used. After treating the scaffolds of each group in the upper chamber, the ALP activity of hMSC was measured on day 14 of culture, and the osteodifferentiation ability and angiogenic ability were simultaneously evaluated by performing a tube formation analysis using HUVEC.

[0095] [Table 4]

[0096]

[0098] As a result, in the Example 1 (10:1 ratio) group, ALP activity was the highest and tube formation ability was also excellent, confirming that bone differentiation and angiogenesis were promoted in a balanced manner. On the other hand, in Comparative Example 4, where the VEGF ratio was excessively high, tube formation was active, but ALP activity was actually lower than that of Example 1. In Comparative Example 5, where the VEGF ratio was too low, ALP activity was higher than that of Comparative Example 4, but tube formation was poor, confirming that long-term bone formation would be difficult without vascular support. From this, it was confirmed that the specific weight ratio range of the present invention is the optimal condition for maximizing the synergistic effect of angiogenesis and bone formation (Table 4).

[0100] Experimental Example 3. Evaluation of Microstructure and Cell Penetration Ability of Porous Supports According to Manufacturing Process

[0101] For microstructure analysis, freeze-dried supports from each group were cut, and cross-sections were captured using a scanning electron microscope (SEM). From the acquired images, the average pore size was calculated by measuring the diameters of over 100 pores using ImageJ software. Additionally, the total porosity of the supports was measured using the liquid displacement method. The volume of pores inside the supports was calculated by impregnating the supports with a liquid of known density (e.g., ethanol) and measuring the change in weight.

[0102] For the evaluation of cell penetration ability, the scaffolds (2 mm thick) of each group were placed on the upper chamber insert (8 µm pore size) of a Transwell culture plate. hMSCs were seeded on the upper part of the scaffold, and the lower chamber was filled with medium containing 10% FBS as a chemoattractant. After 48 hours of incubation, the inserts were detached, and cells that had passed through the scaffold and migrated to the underside of the insert membrane were stained with crystal violet. The stained cells were observed under a microscope, and the number of cells was counted and quantified in at least five random regions.

[0103] [Table 5]

[0104]

[0106] As a result of SEM analysis, Example 1 showed a uniform and interconnected pore structure with an average size of about 300 μm, and the porosity was measured to be about 85%. On the other hand, Comparative Example 6 showed small and non-uniform pores with an average size of less than 100 μm, and Comparative Example 7 had a high density and hardly any pore structure was observed.

[0107] In addition, in the evaluation of cell penetration ability, the number of cells passing through the Example 1 group was overwhelmingly higher than that of Comparative Examples 6 and 7, which means that the pore size in the range of 200-400 μm of the present invention provides the most suitable environment for cells to physically move and penetrate. On the other hand, it was confirmed that the small pores of Comparative Example 6 and the non-porous structure of Comparative Example 7 acted as physical barriers to cell migration, significantly inhibiting penetration (Table 5).

[0109] Experimental Example 4. Evaluation of Effects of Polydopamine Coating and Strontium Doping

[0110] We intended to confirm the effects of polydopamine coating and strontium doping, which are technical features of porous scaffolds according to one aspect, on controlling the release of growth factors and promoting bone formation, respectively, and conducted experiments on Example 1 and Comparative Examples 8 to 10 as shown in Table 6 below.

[0111] [Table 6]

[0112]

[0114] First, to confirm the effect of polydopamine coating on controlling growth factor release, the supports of each group were immersed in phosphate-buffered saline (PBS) and cultured at 37°C, and eluates were collected at predetermined time intervals (1 hour, 24 hours, 7 days, 14 days, 21 days). The concentration of rhBMP-2 in the collected eluates was measured by enzyme-linked immunosorbent assay (ELISA), and the cumulative release rate (%) was calculated.

[0115] The synergistic effect of bone formation by strontium doping was investigated using the same co-culture system of human mesenchymal stem cells (hMSC) and human umbilical vein endothelial cells (HUVEC) as in Experimental Example 1. On day 14 of culture, ALP activity, an early indicator of bone differentiation in hMSCs, was measured, and on day 21 of culture, the final degree of calcium deposition (mineralization) was quantitatively analyzed through Alizarin Red S staining.

[0116] [Table 7]

[0117]

[0119] As a result, Example 1 and Comparative Example 9, which had a polydopamine coating, showed a cumulative release rate of less than about 30% within 24 hours, which was significantly lower than the approximately 75% of Comparative Examples 8 and 10, which did not have a polydopamine coating. From this, it was confirmed that polydopamine coating and immobilization prevent the growth factor from being lost in the early stages and control its release to ensure it is effectively released over a long period.

[0120] In addition, Example 1 containing strontium showed results in a statistically greater increase in ALP activity and final calcium deposition of about 40% compared to Comparative Example 9, confirming that strontium doping technology can significantly promote bone differentiation and regeneration through a strong synergistic effect with growth factors, going beyond simply acting as a bone mineral (Table 7).

[0122] Experimental Example 5. Comprehensive Evaluation of Physical Properties and Performance of Injectable Composition

[0123] We aimed to comprehensively evaluate the shape stability (wash resistance) and injectability, which are key requirements for injectable compositions, and to determine how they relate to the compositional ratio and the resulting rheological properties (storage modulus).

[0124] First, to measure rheological properties, the rheological properties of each group were measured at 25°C using a rheometer equipped with parallel plate geometry. First, the linear viscoelastic region (LVER) was determined through an amplitude sweep. Subsequently, a frequency sweep (0.1–100 rad / s) was performed at constant strain values ​​within the LVER to determine the storage modulus at 1 Hz (6.28 rad / s). G I obtained the value of ')

[0125] Wash resistance was evaluated by washing 1 cc of each group's putty in phosphate-buffered saline (PBS) at 37°C at a flow rate of 50 mL / min for 1 minute and calculating the mass percentage (residual rate, %) of the putty remaining in the mold, and injectability was evaluated by measuring the maximum force (injection force, N) required to extrude each group's putty with a 5cc syringe (18G needle) at a speed of 10 mm / min using a universal testing machine (UTM), and 50 N or less was determined to be a clinically usable standard.

[0126] [Table 8]

[0127]

[0129] As a result, Example 2, which satisfies the DBM:poloxamer ratio (35:55) and storage modulus range (500-2500 Pa) of the present invention, showed a high retention rate of over 90%, indicating excellent shape stability in a surgical environment, while simultaneously exhibiting a clinically acceptable injection force of 45.7 N, thereby ensuring ease of injection.

[0130] On the other hand, Comparative Examples 11 and 12, in which the ratio of DBM to poloxamer deviated from the optimal range, had a retention rate of less than 60% and lacked shape stability; therefore, the evaluation of secondary properties such as storage modulus and injection force was excluded for experimental purposes. In addition, Comparative Example 13, which had an excessively low storage modulus, was judged to be easy to inject (8.9 N) but had very poor shape stability, while Comparative Example 14, which had an excessively high storage modulus, had excellent shape stability but had an injection force exceeding 100 N, making it unsuitable for clinical use.

[0131] From this, it was confirmed that the injectable composition of the present invention exhibits the effect of optimally satisfying shape stability and ease of injection simultaneously within a specific composition ratio and a specific storage modulus range expressed therefrom (Table 8).

[0133] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 A porous bone graft scaffold comprising demineralized bone matrix (DBM) fibers having an average length of 1 mm to 10 mm; a poloxamer matrix; a bone morphogenetic protein and a vascular endothelial growth factor; bone mineral particles; and ascorbic acid or a pharmaceutically acceptable salt thereof, wherein the total porosity is 70% to 95% and the average pore size is 200 μm to 400 μm, wherein the bone morphogenetic protein and the vascular endothelial growth factor are included in a weight ratio of 10:1, the bone morphogenetic protein is rhBMP-2, the vascular endothelial growth factor is rhVEGF, the bone mineral is β-TCP, and the β-TCP is doped with strontium. Claim 2 delete Claim 3 A porous bone graft support according to claim 1, wherein the demineralized bone matrix fibers and bone mineral particles are coated with polydopamine on their surface, and the bone morphogenetic protein and vascular endothelial growth factor are immobilized on the polydopamine coating. Claim 4 delete Claim 5 ◈Claim 5 was abandoned upon payment of the registration fee.◈ (a) forming a polydopamine coating layer on the surface of demineralized bone matrix (DBM) fibers and strontium-doped β-TCP particles, and immobilizing a bone morphogenetic protein rhBMP-2 and a vascular endothelial growth factor rhVEGF mixed in a weight ratio of 10:1 on the coating layer; (b) preparing an aqueous slurry by mixing the solid components of step (a) with a poloxamer hydrogel carrier and ascorbic acid; (c) cooling the slurry to a temperature of -10°C to -40°C at a controlled cooling rate of -0.5°C / min to -5°C / min; (d) an annealing step of maintaining at -10°C to -2°C for 1 to 4 hours; (e) forming a porous support by sublimating the frozen solvent under vacuum; A method for manufacturing a porous bone graft scaffold, characterized by including the step of (f) sterilizing the porous scaffold with gamma rays.

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