Hydroxyapatite biomaterials derived from aquatic organisms and related methods

Abstract

This invention relates to the preparation of a non-cytotoxic biomaterial comprising hydroxyapatite obtained from bone of aquatic organisms and promoting bone formation, the biomaterial comprising the obtained hydroxyapatite, β-tricalcium phosphate, calcium oxide, magnesium oxide, and / or amorphous biomaterial. The invention also generally relates to compositions comprising said biomaterial and further comprising collagen, and to the use of said compositions in the preparation of products for promoting bone formation.

Description

Technical Field

[0001] This invention generally relates to aquatic-derived biomaterials comprising hydroxyapatite or derivatives thereof, and related methods. For example, this invention relates to a method for obtaining biomaterials based on collagenized hydroxyapatite (HAPCS) from waste generated in the salmon industry. Background Technology

[0002] Bone regeneration technology faces significant challenges within the industry, prompting the ongoing development of biomaterials to facilitate this biological phenomenon. Particularly effective biomaterials in this regard are hydroxyapatite-based biomaterials, which have wide applications in regenerative medicine, particularly in bone regeneration and tissue engineering. Currently available solutions for generating hydroxyapatite biomaterials typically derive from human, bovine, equine, swine, and synthetic sources.

[0003] However, ethical, religious, or cultural considerations have led many countries worldwide (such as Kosher and Halal communities) to exclude the use of human, swine, and bovine grafts. Furthermore, diseases such as mad cow disease (BSE) also make the use of bovine biomaterials taboo. While synthetic derivatives do not have the aforementioned ethical and cultural considerations, their lower clinical efficacy makes them less desirable options. Against this backdrop, there is a need to find effective, non-cytotoxic, and cost-effective alternative sources of natural biomaterials for osteogenesis.

[0004] Document IN202031021533 describes a hydroxyapatite biomaterial derived from the bones of the rose bearded fish (Puntius conchonius), poly(lactic-co-glycolic acid) copolymer (PLGA), and poly(methyl methacrylate) (PMMA), as well as a method for obtaining hydroxyapatite from the bones of P. conchonius. While this biomaterial offers an alternative to conventional sources, it requires combination with additional polymers, which complicates the process and increases costs. Furthermore, the resulting biomaterial exhibits a compressive strength below 12 MPa, indicating a drawback due to its brittleness.

[0005] Currently, there are no readily available, low-cost, high-mechanical-strength, non-cytotoxic, and highly effective alternatives to hydroxyapatite-based biomaterials that are highly effective in promoting osteogenesis. Therefore, there is a need for improved compositions, materials, and methods. Summary of the Invention

[0006] This invention generally relates to hydroxyapatite biomaterials derived from aquatic organisms and related methods.

[0007] In one aspect, biological material is provided. In some embodiments, the biological material is biological material derived from aquatic organisms. In some embodiments, the biological material derived from aquatic organisms comprises a first composition and a second composition, wherein the first composition is present in the biological material in an amount greater than or equal to 10% by weight and less than or equal to 100% by weight relative to the total weight of the biological material, and the second composition is present in the biological material in an amount greater than or equal to 0.1% by weight and less than or equal to 90% by weight relative to the total weight of the biological material, wherein the first composition comprises greater than or equal to 50% by weight and less than or equal to 90% by weight of aquatic organism-derived hydroxyapatite or its derivatives (optionally substituted), and greater than or equal to 5% by weight and The second composition comprises less than or equal to 40% by weight of amorphous biological material, wherein the amorphous biological material comprises: greater than or equal to 10% by weight and less than 20% by weight of β-tricalcium phosphate, greater than or equal to 0.1% by weight and less than or equal to 5% by weight of calcium oxide, greater than or equal to 0.1% by weight and less than or equal to 20% by weight of magnesium oxide relative to the total weight of the first composition, and the second composition comprises one or more of collagen and / or its derivatives, synthetic collagen and / or its derivatives, gelatin and / or its derivatives, fibrin and / or its derivatives, and fibrin and / or its derivatives.

[0008] In another aspect, a method for producing biomaterials of aquatic origin is provided. In some embodiments, the method includes removing non-bone material, including: incubating the aquatic-origin bone at a temperature greater than or equal to 700°C and less than or equal to 120°C for greater than or equal to 20 minutes to less than or equal to 4 hours to remove non-bone material; deproteinizing the aquatic-origin bone, including incubating the aquatic-origin bone in sodium hydroxide at a temperature greater than or equal to 15°C and less than or equal to 25°C for greater than or equal to 30 minutes to less than or equal to 4 hours to deproteinize the bone; and defatting the aquatic-origin bone, including incubating the aquatic-origin bone in acetone at a temperature greater than or equal to 15°C and less than or equal to 30°C for greater than or equal to 4 hours. The bone is degreased for 8 hours or less; the bone of aquatic origin is dried at a temperature of 50°C or higher and 70°C or lower for 6 hours or less and 48 hours; the dried bone is ground; the ground bone is heated to a temperature of 800°C or higher and 1200°C or lower and held for 1 hour or more and 3 hours or less to produce aquatic biological material; and a second composition is added to the aquatic biological material, the second composition comprising one or more of collagen and its derivatives, actin and its derivatives, gelatin and its derivatives, pore-forming additives, and bone morphogenetic proteins.

[0009] Other advantages and novel features of the invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying drawings. In the event of any conflicting and / or inconsistent disclosures between this specification and other documents incorporated by reference, this specification shall prevail. Attached Figure Description

[0010] Non-limiting embodiments of the invention will be described by way of example and with reference to the accompanying drawings, which are schematic and not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown is generally represented by a single number. For clarity, not every component in each figure is labeled, and not every component of each embodiment of the invention is shown where illustration would enable those skilled in the art to understand the invention without the need for diagrams. In the figures:

[0011] Figure 1 An exemplary method for producing biomaterials of aquatic origin is shown according to a set of embodiments.

[0012] Figure 2ARaman spectra of exemplary hydroxyapatite biomaterials obtained during the deproteinization stage optimization according to a set of embodiments are shown. Curve 1 corresponds to the use of the variable NaOH 2 M and 20 °C, curve 2 corresponds to the use of the variable NaOH 2 M and 50 °C, and curve 3 corresponds to the use of the variable NaOH 2.5 M and 20 °C.

[0013] Figure 2B Raman spectra of exemplary hydroxyapatite biomaterials obtained during the degreasing stage optimization according to a set of embodiments are shown. Curve 1 corresponds to the use of acetone variable for 6 hours, curve 2 corresponds to the use of acetone variable for 12 hours, curve 3 corresponds to the use of hexane variable for 6 hours, and curve 4 corresponds to the use of hexane variable for 12 hours.

[0014] Figure 3 Raman spectra of exemplary hydroxyapatite biomaterials obtained during optimization in the calcination stage according to a set of embodiments are shown. Curve 1 corresponds to using the variable 800 °C and 6 hours, curve 2 corresponds to using the variable 800 °C and 4 hours, and curve 3 corresponds to using the variable 800 °C and 2 hours.

[0015] Figure 4 Raman spectra of exemplary hydroxyapatite biomaterials obtained during optimization in the calcination stage according to a set of embodiments are shown. Curve 4 corresponds to using the variable 900 °C and 6 hours, curve 5 corresponds to using the variable 900 °C and 4 hours, and curve 6 corresponds to using the variable 900 °C and 2 hours.

[0016] Figure 5 Raman spectra of exemplary hydroxyapatite biomaterials obtained during optimization in the calcination stage according to a set of embodiments are shown. Curve 7 corresponds to using the variable 1000 °C and 6 hours, curve 8 corresponds to using the variable 1000 °C and 4 hours, and curve 9 corresponds to using the variable 1000 °C and 2 hours.

[0017] Figure 6 Raman spectra of an exemplary hydroxyapatite biomaterial obtained by exemplary embodiment 1 according to a set of embodiments are shown.

[0018] Figure 7 Raman spectra of an exemplary hydroxyapatite biomaterial obtained by exemplary embodiment 2 according to a set of embodiments are shown.

[0019] Figure 8 Raman spectra of an exemplary hydroxyapatite biomaterial obtained by exemplary embodiment 3 according to a set of embodiments are shown.

[0020] Figure 9An X-ray diffraction pattern of an exemplary hydroxyapatite biomaterial obtained by exemplary embodiment 1 according to a set of embodiments is shown.

[0021] Figure 10 An X-ray diffraction pattern of an exemplary hydroxyapatite biomaterial obtained by exemplary embodiment 2 according to a set of embodiments is shown.

[0022] Figure 11 An X-ray diffraction pattern of an exemplary hydroxyapatite biomaterial obtained by exemplary embodiment 3 according to a set of embodiments is shown.

[0023] Figure 12 The changes in weight as a function of temperature are shown according to a set of embodiments following thermogravimetric analysis of an exemplary hydroxyapatite biomaterial obtained by exemplary embodiment 1.

[0024] Figure 13 The following is illustrated, according to a set of embodiments, the weight change as a function of temperature after thermogravimetric analysis of an exemplary hydroxyapatite biomaterial obtained by exemplary embodiment 2.

[0025] Figure 14 The changes in weight as a function of temperature are shown according to a set of embodiments, following thermogravimetric analysis of an exemplary hydroxyapatite biomaterial obtained by exemplary embodiment 3.

[0026] Figure 15 Scanning electron microscope images of exemplary hydroxyapatite biomaterials obtained by exemplary embodiment 1 according to a set of embodiments are shown at magnifications of 220x (left) and 4600x (right).

[0027] Figure 16 Scanning electron microscope images of exemplary hydroxyapatite biomaterials obtained by exemplary embodiment 2 according to a set of embodiments are shown at magnifications of 1000x (left) and 2900x (right).

[0028] Figure 17 Scanning electron microscope images of exemplary hydroxyapatite biomaterials obtained by exemplary embodiment 3 according to a set of embodiments are shown at magnifications of 1000x (left) and 2500x (right).

[0029] Figure 18 Compression strength results are shown, illustrating force-distance curves of hydroxyapatite biomaterials obtained through exemplary schemes 1, 2, and 3 according to a set of embodiments.

[0030] Figure 19The results of cell viability and proliferation obtained from MTT assays according to one set of embodiments are shown. Results are shown after 24 hours of cell culture in the presence of commercially available human hydroxyapatite biomaterial, commercially available bovine hydroxyapatite biomaterial, salmon-derived hydroxyapatite biomaterial from each of three selected exemplary embodiments, and a negative control.

[0031] Figure 20 The results of cell viability and proliferation obtained from MTT assays according to one set of embodiments are shown. Results are shown after culturing cells for 48 hours in the presence of commercially available human hydroxyapatite biomaterial, commercially available bovine hydroxyapatite biomaterial, salmon-derived hydroxyapatite biomaterial from each of three selected exemplary embodiments, and a negative control.

[0032] Figure 21 The images shown are 10x images of mesenchymal cells cultured for 21 days in the presence of exemplary hydroxyapatite biomaterial from Exemplary Scheme 1 (Image 1), exemplary hydroxyapatite biomaterial from Exemplary Scheme 2 (Image 2), exemplary hydroxyapatite biomaterial from Exemplary Scheme 3 (Image 3), human allogeneic grafts (Image 4), bovine xenografts (Image 5), a control without hydroxyapatite biomaterials (Image 6), and undifferentiated cells (Image 7), according to a set of embodiments.

[0033] Figure 22 The particle size distribution of exemplary hydroxyapatite biomaterials obtained by exemplary embodiments 1, 2 and 3 using a grinding time of 2 seconds is shown according to a set of embodiments.

[0034] Figure 23 The particle size distribution of exemplary hydroxyapatite biomaterials obtained by exemplary embodiments 1, 2 and 3 using a grinding time of 6 seconds is shown according to a set of embodiments.

[0035] Figure 24 The particle size distribution of exemplary hydroxyapatite biomaterials obtained by exemplary embodiments 1, 2 and 3 using a grinding time of 10 seconds is shown according to a set of embodiments.

[0036] Figure 25 Polarized light microscope images of materials obtained using a 2-second grinding time are shown according to one set of embodiments. The upper left image corresponds to exemplary embodiment 1, the upper right image corresponds to exemplary embodiment 2, and the lower image corresponds to exemplary embodiment 3.

[0037] Figure 26Polarized light microscope images of materials obtained using a 10-second grinding time are shown according to one set of embodiments. The upper left image corresponds to exemplary embodiment 1, the upper right image corresponds to exemplary embodiment 2, and the lower image corresponds to exemplary embodiment 3. Detailed Implementation

[0038] This invention generally relates to aquatic organism-derived biomaterials comprising hydroxyapatite or derivatives thereof, and related methods. In some embodiments, the aquatic organism-derived biomaterial comprises a mixture of a first composition and a second composition, the first composition comprising aquatic organism-derived hydroxyapatite or derivatives thereof, and the second composition comprising collagen (or other scaffold proteins). Advantageously, the aquatic organism-derived biomaterial may comprise primarily aquatic organism-derived hydroxyapatite and / or collagen (or other scaffold proteins). For example, in some embodiments, this invention relates to a method for obtaining collagenized hydroxyapatite (HAPCS)-based biomaterials from waste generated by fisheries (e.g., the salmon industry). In some embodiments, the biomaterial may advantageously be substantially free of synthetic polymers.

[0039] In some embodiments, the present invention generally relates to obtaining biomaterials containing hydroxyapatite from bone of aquatic organisms (e.g., salmon bone), which are low-cost, non-cytotoxic, and / or promote osteogenic processes, and thus serve as an effective alternative to biomaterials derived from humans or other natural sources. In some embodiments, the present invention generally relates to a composition comprising said hydroxyapatite from bone of aquatic organisms, and additionally comprising collagen and / or other scaffold proteins (actin, gelatin, fibroin, etc.). The biomaterials described herein can be used in a variety of applications, including medical applications for treating patients.

[0040] "Object" means any animal, such as a mammal (e.g., a human). Non-limiting examples of objects include humans, non-human primates, cattle, horses, pigs, sheep, goats, dogs, cats, or rodents (e.g., mice, rats, hamsters), birds, fish, or guinea pigs. Generally, this invention relates to use with humans. In some embodiments, the object may exhibit health benefits, for example, after administration of aquatic biologically derived biomaterials. For example, the aquatic biologically derived biomaterials described herein can be used as xenografts (e.g., xenografts comprising aquatic biologically derived biomaterials containing a first composition and a second composition). The term "xenograft" generally refers to a type of graft performed between two different species. The process involves transferring tissue, organs, or cells from one organism (the donor) to another organism of a different species (the recipient). A common example of xenografts is the use of pig tissue to repair human heart valves. This technique is used in medical research and clinical medicine to study diseases, test treatments, and, in some cases, directly treat a patient's condition.

[0041] In some embodiments, the aquatic biologically derived biomaterials described herein may be applied to a subject (e.g., for wound healing, tissue regeneration, etc.). In some embodiments, the biomaterials are applied surgically (e.g., by implantation). In some embodiments, the biomaterials are applied endoscopically. In some embodiments, the biomaterials are applied topically (e.g., on the skin and / or mucous membranes). Other routes of application are also possible (e.g., orally, via the ureter, via the rectum, etc.).

[0042] In some embodiments, the aquatic-derived biomaterial comprises a first composition (e.g., hydroxyapatite or a derivative thereof of an aquatic origin) and a second composition (e.g., a protein scaffold, such as collagen). The term "aquatic-derived" as used herein generally refers to material derived from aquatic organisms. For example, aquatic-derived bone as used herein generally refers to bone found in aquatic species. Non-limiting examples of aquatic biological sources of the biological materials described herein include fish (e.g., salmon, tuna, anchovies, angelfish, catfish, cod, sole, pike, red-spotted salmon, herring, shark, killifish, pufferfish, flatfish, trout, eel, stingray, pufferfish, flounder, remora, sturgeon, tigerfish, grouper, etc.), marine mammals (e.g., cetaceans (e.g., whales, dolphins, porpoises), pinnipeds (e.g., seals, sea lions, walruses), manatees (e.g., manatees, dugongs), sea otters, etc.), crustaceans (e.g., shrimp, prawns, crabs, lobsters, crayfish, krill, barnacles, etc.), marine reptiles (e.g., iguanas, snakes, turtles, crocodiles), marine life (e.g., algae (e.g., seaweed, green algae, red algae), lichens, corals, sponges), etc.).

[0043] In some embodiments, the aquatic-derived biological material does not contain hydroxyapatite of non-aquatic origin. For example, the aquatic-derived biological material does not contain hydroxyapatite derived from cattle, humans, horses, chickens, and / or pigs. In some embodiments, the second composition does not contain scaffold material of non-aquatic origin. For example, in some embodiments, the scaffold material is not derived from cattle, humans, horses, chickens, and / or pigs. In some embodiments, the first composition is not derived from cattle, humans, horses, chickens, synthetic or pig sources, and the scaffold material is derived from cattle, humans, horses, chickens, synthetic and / or pig sources.

[0044] In some embodiments, the first composition comprises hydroxyapatite and / or its derivatives from an aquatic organism. In some embodiments, the hydroxyapatite or its derivatives from an aquatic organism are derived from the bone of a fish (e.g., salmon). For example, in some embodiments, the bone (from which hydroxyapatite is obtained) is obtained from a freshwater fish. In some embodiments, the bone (from which hydroxyapatite is obtained) is obtained from a saltwater fish. In some embodiments, the first composition and the second composition are derived from different organisms. In some embodiments, the first composition and the second composition are derived from the same organism. For example, in an illustrative embodiment, the first composition comprises hydroxyapatite and / or its derivatives derived from salmon bone, and the second composition comprises collagen derived from salmon skin. Those skilled in the art will understand, based on the teachings of this specification, that other aquatic sources of the first composition and / or the second composition are also possible.

[0045] The aquatic biological materials described herein can advantageously provide biomaterials that can be used for xenografts without involving ethical, religious, or cultural considerations (e.g., in many countries worldwide, such as kosher and halal communities, the use of human, pig, and bovine grafts is prohibited), while maintaining clinical efficacy and / or cost-effectiveness.

[0046] In some embodiments, the first composition may be present in the aquatic biological material in any suitable amount. For example, in some embodiments, the first composition is present in the aquatic biological material in an amount greater than or equal to 10% by weight, greater than or equal to 20% by weight, greater than or equal to 30% by weight, greater than or equal to 40% by weight, greater than or equal to 50% by weight, greater than or equal to 60% by weight, greater than or equal to 70% by weight, greater than or equal to 80% by weight, greater than or equal to 90% by weight, greater than or equal to 95% by weight, or greater than or equal to 99.9% by weight, relative to the total weight of the aquatic biological material. In some embodiments, the first composition is present in the aquatic biological material in an amount less than 100% by weight, less than or equal to 99.9% by weight, less than or equal to 95% by weight, less than or equal to 90% by weight, less than or equal to 80% by weight, less than or equal to 70% by weight, less than or equal to 60% by weight, less than or equal to 50% by weight, less than or equal to 40% by weight, less than or equal to 30% by weight, or less than or equal to 20% by weight, relative to the total weight of the aquatic biological material. Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 10% by weight and less than 100% by weight). Other ranges are also possible.

[0047] In some embodiments, as described herein, the first composition comprises hydroxyapatite or a derivative thereof of aquatic origin (e.g., calcium-deficient hydroxyapatite, doped hydroxyapatite, and / or substituted hydroxyapatite). For example, in some embodiments, at least a portion of the hydroxyapatite may be ionically substituted (e.g., wherein the crystal structure of the hydroxyapatite incorporates one or more cations (e.g., Sr...). 2+ Mg 2+ Zn 2+ Na + K + Li + Ag + Fe 2+ Mn 2+ Cu 2+ ) and / or anions (e.g., CO32-) 2− SiO4 4− SeO3 2− SeO4 2− F − Cl − ,Br −(etc.) Replacement). Although the following and most of the description herein generally refers to hydroxyapatite of aquatic origin, those skilled in the art will understand from the teachings of this description that, unless otherwise stated to the contrary, such portions of the description generally also apply to derivatives of hydroxyapatite of aquatic origin and / or substituted hydroxyapatite of aquatic origin (e.g., in amounts present in the first composition).

[0048] Hydroxyapatite of aquatic origin may be present in the first composition in any suitable amount. In some embodiments, hydroxyapatite of aquatic origin is present in the first composition in an amount greater than or equal to 50% by weight, greater than or equal to 54% by weight, greater than or equal to 60% by weight, greater than or equal to 65% by weight, greater than or equal to 70% by weight, greater than or equal to 80% by weight, greater than or equal to 85% by weight, greater than or equal to 90% by weight, greater than or equal to 95% by weight, or greater than or equal to 98% by weight, relative to the total weight of the first composition. In some embodiments, hydroxyapatite is present in the first composition in an amount less than or equal to 100% by weight, less than or equal to 98% by weight, less than or equal to 95% by weight, 90% by weight, less than or equal to 85% by weight, less than or equal to 80% by weight, less than or equal to 75% by weight, less than or equal to 70% by weight, less than or equal to 65% by weight, less than or equal to 60% by weight, or less than or equal to 55% by weight, relative to the total weight of the first composition. Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 50% by weight and less than or equal to 90% by weight, greater than or equal to 54% by weight and less than or equal to 65% by weight, greater than or equal to 50% by weight and less than or equal to 100% by weight). Other ranges are also possible.

[0049] Aquatic biologically derived biomaterials may have a desired ratio of hydroxyapatite to collagen (or other scaffolds) present in the biomaterial. For example, in some embodiments, the ratio of hydroxyapatite or its derivatives to collagen may be greater than or equal to 70:30, greater than or equal to 80:20, or greater than or equal to 90:10 (e.g., less than or equal to 95:5, less than or equal to 90:10, less than or equal to 80:20, less than or equal to 70:30).

[0050] In some embodiments, the first composition comprises one or more additional components selected from the following: amorphous biomaterials, β-tricalcium phosphate, calcium oxide, magnesium oxide, and β-rhenanite.

[0051] In some embodiments, the first composition comprises amorphous biological material (e.g., amorphous calcium phosphate, non-crystalline calcium, non-crystalline phosphorus, etc.) in an amount relative to the total weight of the first composition of greater than or equal to 5% by weight, greater than or equal to 10% by weight, greater than or equal to 15% by weight, greater than or equal to 19% by weight, greater than or equal to 20% by weight, greater than or equal to 25% by weight, greater than or equal to 30% by weight, greater than or equal to 32% by weight, or greater than or equal to 35% by weight. In some embodiments, the first composition comprises amorphous biological material in an amount relative to the total weight of the first composition of less than or equal to 40% by weight, less than or equal to 35% by weight, less than or equal to 32% by weight, less than or equal to 30% by weight, less than or equal to 25% by weight, less than or equal to 20% by weight, less than or equal to 19% by weight, less than or equal to 15% by weight, or less than or equal to 10% by weight. Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 5% by weight and less than or equal to 40% by weight, greater than or equal to 5% by weight and less than or equal to 25% by weight, greater than or equal to 19% by weight and less than or equal to 32% by weight). Other ranges are also possible.

[0052] In some embodiments, the first composition comprises β-tricalcium phosphate in an amount relative to the total weight of the first composition of greater than or equal to 10% by weight, greater than or equal to 12% by weight, greater than or equal to 14% by weight, greater than or equal to 16% by weight, or greater than or equal to 18% by weight. In some embodiments, the first composition comprises β-tricalcium phosphate in an amount relative to the total weight of the first composition of less than or equal to 20% by weight, less than or equal to 18% by weight, less than or equal to 16% by weight, less than or equal to 14% by weight, or less than or equal to 12% by weight. Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 10% by weight and less than or equal to 20% by weight, greater than or equal to 12% by weight and less than or equal to 16% by weight). Other ranges are also possible.

[0053] In some embodiments, the first composition comprises calcium oxide, if present, in an amount relative to the total weight of the first composition of greater than or equal to 0.1 wt%, greater than or equal to 0.5 wt%, greater than or equal to 1 wt%, greater than or equal to 1.5 wt%, greater than or equal to 2 wt%, greater than or equal to 2.5 wt%, greater than or equal to 3 wt%, greater than or equal to 3.5 wt%, or greater than or equal to 4 wt%. In some embodiments, the first composition comprises calcium oxide in an amount relative to the total weight of the first composition of less than or equal to 5 wt%, less than or equal to 4 wt%, less than or equal to 3.5 wt%, less than or equal to 3 wt%, less than or equal to 2.5 wt%, less than or equal to 2 wt%, less than or equal to 1.5 wt%, less than or equal to 1 wt%, or less than or equal to 0.5 wt%. Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 0.1 wt% and less than or equal to 5 wt%, greater than or equal to 1 wt% and less than or equal to 2 wt%). Other ranges are also possible.

[0054] In some embodiments, the first composition comprises magnesium oxide, if present, in an amount relative to the total weight of the first composition of greater than or equal to 0.1 wt%, greater than or equal to 0.5 wt%, greater than or equal to 1 wt%, greater than or equal to 1.5 wt%, greater than or equal to 2 wt%, greater than or equal to 2.5 wt%, greater than or equal to 3 wt%, greater than or equal to 3.5 wt%, greater than or equal to 4 wt%, greater than or equal to 5 wt%, greater than or equal to 10 wt%, or greater than or equal to 15 wt%. In some embodiments, the first composition comprises magnesium oxide, in an amount relative to the total weight of the first composition of less than or equal to 20 wt%, less than or equal to 15 wt%, less than or equal to 10 wt%, less than or equal to 5 wt%, less than or equal to 4 wt%, less than or equal to 3.5 wt%, less than or equal to 3 wt%, less than or equal to 2.5 wt%, less than or equal to 2 wt%, less than or equal to 1.5 wt%, less than or equal to 1 wt%, or less than or equal to 0.5 wt%. Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 0.1% by weight and less than or equal to 5% by weight, greater than or equal to 1% by weight and less than or equal to 2% by weight, greater than or equal to 0.1% by weight and less than or equal to 20% by weight). Other ranges are also possible.

[0055] In some embodiments, the first composition comprises β-Rainerite, if present, in an amount relative to the total weight of the first composition of greater than or equal to 0.1 wt%, greater than or equal to 0.5 wt%, greater than or equal to 1 wt%, greater than or equal to 1.5 wt%, greater than or equal to 2 wt%, greater than or equal to 2.5 wt%, greater than or equal to 3 wt%, greater than or equal to 3.5 wt%, greater than or equal to 4 wt%, greater than or equal to 5 wt%, greater than or equal to 10 wt%, or greater than or equal to 15 wt%. In some embodiments, the first composition comprises β-Rainerite, in an amount relative to the total weight of the first composition of less than or equal to 20 wt%, less than or equal to 15 wt%, less than or equal to 10 wt%, less than or equal to 5 wt%, less than or equal to 4 wt%, less than or equal to 3.5 wt%, less than or equal to 3 wt%, less than or equal to 2.5 wt%, less than or equal to 2 wt%, less than or equal to 1.5 wt%, less than or equal to 1 wt%, or less than or equal to 0.5 wt%. Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 0.1% by weight and less than or equal to 5% by weight, greater than or equal to 1% by weight and less than or equal to 2% by weight, greater than or equal to 0.1% by weight and less than or equal to 20% by weight). Other ranges are also possible.

[0056] In some embodiments, the first composition has one or more desired properties, including, for example, density, thermal stability, Ca / P ratio, maximum compression force, cell viability, cell proliferation, osteoinductiveness, porosity, absorption and / or moldability.

[0057] For example, in some embodiments, the first composition has a density similar to that of pure hydroxyapatite. In some embodiments, the density of the first composition is greater than or equal to 2 g / cm³. 3 ≥2.2 g / cm 3 ≥2.4 g / cm 3 ≥2.6 g / cm 3 ≥2.8 g / cm 3 ≥3 g / cm 3 ≥3.1g / cm 3 ≥3.15 g / cm 3 ≥3.2 g / cm 3 ≥3.25 g / cm 3 ≥3.3 g / cm 3 ≥3.4 g / cm 3 ≥3.5 g / cm3 ≥3.6 g / cm 3 ≥3.8 g / cm 3 or greater than or equal to 4 g / cm 3 In some embodiments, the density of the first composition is less than or equal to 4.2 g / cm³. 3 Less than or equal to 4 g / cm 3 Less than or equal to 3.8 g / cm 3 Less than or equal to 3.6 g / cm 3 Less than or equal to 3.5 g / cm 3 Less than or equal to 3.4 g / cm³ 3 Less than or equal to 3.3 g / cm 3 Less than or equal to 3.2 g / cm³ 3 Less than or equal to 3.25 g / cm 3 Less than or equal to 3.2 g / cm³ 3 Less than or equal to 3.15 g / cm 3 Less than or equal to 3.1 g / cm 3 Less than or equal to 3 g / cm 3 Less than or equal to 2.8 g / cm 3 Less than or equal to 2.6 g / cm 3 Less than or equal to 2.4 g / cm³ 3 Or less than or equal to 2.2 g / cm³ 3 Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 2 g / cm³). 3 And less than or equal to 4.2 g / cm 3 ≥3 g / cm 3 And less than or equal to 3.4 g / cm³ 3 Other ranges are also possible. Density, as described herein, can be determined using pycnometry.

[0058] In some embodiments, the first composition may be thermally stable within a desired temperature range. For example, in some embodiments, the biomaterial is thermally stable (e.g., exhibiting no significant loss of mass) at temperatures greater than or equal to 0°C, greater than or equal to 10°C, greater than or equal to 25°C, greater than or equal to 50°C, greater than or equal to 100°C, greater than or equal to 150°C, greater than or equal to 200°C, greater than or equal to 300°C, greater than or equal to 400°C, greater than or equal to 500°C, greater than or equal to 600°C, greater than or equal to 700°C, greater than or equal to 800°C, or greater than or equal to 900°C. In some embodiments, the first composition is thermally stable at temperatures less than or equal to 1000°C, less than or equal to 900°C, less than or equal to 800°C, less than or equal to 700°C, less than or equal to 600°C, less than or equal to 500°C, less than or equal to 400°C, less than or equal to 300°C, less than or equal to 200°C, less than or equal to 150°C, less than or equal to 100°C, less than or equal to 50°C, less than or equal to 25°C, or less than or equal to 10°C. Combinations of the above-mentioned ranges are also possible (e.g., greater than or equal to 0°C and less than or equal to 1000°C, greater than or equal to 50°C and less than or equal to 800°C). That is, the first composition can be thermally stable over a relatively wide temperature range. Other ranges are also possible. Thermal stability can be determined using thermogravimetric analysis.

[0059] In some embodiments, the first composition has a desired calcium-to-phosphorus ratio (Ca / P) (e.g., a stoichiometric ratio similar to that of hydroxyapatite). In some embodiments, the Ca / P ratio of the first composition is greater than or equal to 1.5, greater than or equal to 1.55, greater than or equal to 1.6, greater than or equal to 1.65, greater than or equal to 1.7, or greater than or equal to 1.75. In some embodiments, the Ca / P ratio of the first composition is less than or equal to 1.8, less than or equal to 1.75, less than or equal to 1.7, less than or equal to 1.65, less than or equal to 1.6, or less than or equal to 1.55. Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 1.5 and less than or equal to 1.8, greater than or equal to 1.6 and less than or equal to 1.75). Other ranges are also possible. The Ca / P ratio as described herein can be measured using scanning electron microscopy combined with energy-dispersive X-ray spectroscopy (SEM-EDX).

[0060] In some embodiments, the first composition has the desired mechanical properties. For example, in some embodiments, the maximum compressive force of the first composition is greater than or equal to 250 N, greater than or equal to 275 N, greater than or equal to 300 N, greater than or equal to 325 N, greater than or equal to 350 N, greater than or equal to 375 N, greater than or equal to 400 N, or greater than or equal to 500 N. In some embodiments, the maximum compressive force of the first composition is less than or equal to 600 N, less than or equal to 500 N, less than or equal to 400 N, less than or equal to 375 N, less than or equal to 350 N, less than or equal to 325 N, or less than or equal to 300 N. Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 250 N and less than or equal to 600 N). Other ranges are also possible. The maximum compressive force, as used herein, can be measured using a TA.XT plus texture analyzer to determine the compression resistance.

[0061] In some embodiments, the second composition is present in the aquatic biological material in an amount greater than or equal to 0.1% by weight, greater than or equal to 1% by weight, greater than or equal to 5% by weight, greater than or equal to 10% by weight, greater than or equal to 20% by weight, greater than or equal to 30% by weight, greater than or equal to 40% by weight, greater than or equal to 50% by weight, greater than or equal to 60% by weight, greater than or equal to 70% by weight, or greater than or equal to 80% by weight, relative to the total weight of the aquatic biological material. In some embodiments, the second composition is present in the aquatic biological material in an amount less than or equal to 90% by weight, less than or equal to 85% by weight, less than or equal to 80% by weight, less than or equal to 70% by weight, less than or equal to 60% by weight, less than or equal to 50% by weight, less than or equal to 40% by weight, less than or equal to 30% by weight, less than or equal to 20% by weight, less than or equal to 10% by weight, less than or equal to 5% by weight, less than or equal to 3% by weight, less than or equal to 2% by weight, less than or equal to 1% by weight, or less than or equal to 0.5% by weight, relative to the total weight of the aquatic biological material. Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 0.1% by weight and less than 90% by weight). Other ranges are also possible.

[0062] In some embodiments, the second composition comprises one or more scaffold materials, such as protein scaffolds. Non-limiting examples of suitable scaffold materials include collagen (e.g., one or more of collagen type I, collagen type II, collagen type III, and collagen type IV) and derivatives thereof, synthetic collagen and derivatives thereof, gelatin and derivatives thereof, fibroin and derivatives thereof, fibronectin and derivatives thereof, and hydrocolloids (e.g., guar gum, gum arabic, gellan gum, xanthan gum, agar, pectin, starch, carrageenan, pectin, and / or alginate) and derivatives thereof. In one set of exemplary embodiments, the second composition comprises collagen and / or its derivatives. In one set of exemplary embodiments, the scaffold material is of aquatic origin (e.g., freshwater source, marine source). In some embodiments, the scaffold material is derived from bovine, human, horse, chicken, synthetic, and / or swine sources. In one set of exemplary embodiments, the scaffold material comprises salmon-derived collagen. In another set of exemplary embodiments, the scaffold material comprises bovine-derived collagen.

[0063] In some embodiments, the second composition further comprises pore-forming additives (e.g., pore-forming agents such as supersaturated salt solutions (e.g., containing alkali metals, alkaline earth materials and halides, partially neutralized inorganic acids, neutralized organic acids) and / or oils) and / or bone morphogenetic proteins.

[0064] In some embodiments, a combination of methods including the following steps is used to produce aquatic organism-derived biomaterials: initial incubation of aquatic organism-derived bone, deproteinization of aquatic organism-derived bone, defatting of aquatic organism-derived bone, and decalcification of aquatic organism-derived bone. In some embodiments, one or more additional steps (e.g., drying and / or grinding of aquatic organism-derived bone) may be performed. While the embodiments described herein present the steps in a specific order, other arrangements / sequences of the steps are also possible. In one exemplary method, such as... Figure 1 As shown, aquatic organism-derived bones (e.g., salmon bones) can be initially incubated, deproteinized, defatted, dried, ground, calcined, and scaffolded with added materials to form aquatic organism-derived biomaterials.

[0065] In some embodiments, non-bone material (e.g., muscle, connective tissue, etc.) is removed from bone of aquatic origin. For example, in some embodiments, the bone of aquatic origin is initially incubated for 20 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, or 3.5 hours or more to remove at least a portion of the non-bone material. In some embodiments, the bone of aquatic origin is initially incubated for 4 hours or more, 3.5 hours or more, 3 hours or more, 2.5 hours or more, 2 hours or more, or 1 hour or more. Combinations of the ranges mentioned above are also possible (e.g., 20 minutes or more and 4 hours or more, 1 hour or more and 2 hours or more). Other ranges and durations are also possible.

[0066] In some embodiments, the initial incubation step is performed at a temperature greater than or equal to 70°C, greater than or equal to 80°C, greater than or equal to 90°C, greater than or equal to 100°C, greater than or equal to 110°C, or greater than or equal to 120°C. In some embodiments, the initial incubation step is performed at a temperature less than or equal to 120°C, less than or equal to 110°C, less than or equal to 100°C, less than or equal to 90°C, or less than or equal to 80°C. Combinations of the above-mentioned ranges are also possible (e.g., greater than or equal to 70°C and less than or equal to 120°C). Other ranges and temperatures are also possible.

[0067] In some embodiments, the initial incubation step is carried out in air or an aqueous solution (e.g., water, saline). In one set of exemplary embodiments, the removal of non-bone material includes incubating aquatic organism-derived bone at a temperature greater than or equal to 70°C and less than or equal to 120°C for greater than or equal to 20 minutes and less than or equal to 4 hours to remove non-bone material.

[0068] In some embodiments, the bone of aquatic origin is deproteinized (e.g., after an initial incubation step). In some embodiments, the deproteinization step includes incubating the bone of aquatic origin in an alkaline solution. Non-limiting examples of alkaline solutions include sodium hydroxide, potassium hydroxide, sodium bicarbonate, and magnesium hydroxide. Those skilled in the art can select a suitable alkaline solution based on the teachings of this specification.

[0069] In some embodiments, the deproteinization step is performed for 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, or 3.5 hours or more. In some embodiments, the deproteinization step is performed for 4 hours or more, 3.5 hours or more, 3 hours or more, 2.5 hours or more, 2 hours or more, or 1 hour or more. Combinations of the ranges mentioned above are also possible (e.g., 30 minutes or more and 4 hours or more, 1 hour or more and 2 hours or more). Other ranges and durations are also possible.

[0070] In some embodiments, the deproteinization step is performed at a temperature greater than or equal to 15°C, greater than or equal to 16°C, greater than or equal to 18°C, greater than or equal to 20°C, greater than or equal to 22°C, or greater than or equal to 24°C. In some embodiments, the deproteinization step is performed at a temperature less than or equal to 25°C, less than or equal to 24°C, less than or equal to 22°C, less than or equal to 20°C, less than or equal to 18°C, or less than or equal to 16°C. Combinations of the above-mentioned ranges are also possible (e.g., greater than or equal to 15°C and less than or equal to 25°C). Other ranges and temperatures are also possible.

[0071] In one set of exemplary embodiments, deproteinizing bone from aquatic organisms includes incubating the bone in sodium hydroxide at a temperature greater than or equal to 15°C and less than or equal to 25°C for greater than or equal to 30 minutes and less than or equal to 4 hours, thereby deproteinizing the bone.

[0072] In some embodiments, the bone of aquatic origin is defatted (e.g., after an initial incubation step, after a deproteinization step). In some embodiments, defatting the bone of aquatic origin includes incubating the bone of aquatic origin in a defatting solvent. Non-limiting examples of suitable defatting solvents include acetone, hexane, heptane, methanol, ethanol, isopropanol, butanol, chloroform, diethyl ether, and combinations thereof. In one set of exemplary embodiments, the defatting solvent is acetone, hexane, or a mixture thereof.

[0073] In some embodiments, the defatting step is performed for 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, or 13 hours or more. In some embodiments, the defatting step is performed for 14 hours or more, 13 hours or more, 12 hours or more, 11 hours or more, 10 hours or more, 9 hours or more, 8 hours or more, 7 hours or more, 6 hours or more, or 5 hours or more. Combinations of the above ranges are also possible (e.g., 4 hours or more and 12 hours or more, 6 hours or more and 12 hours or more, 4 hours or more and 8 hours or more). Other ranges and durations are also possible.

[0074] In some embodiments, the degreasing step is performed at a temperature greater than or equal to 15°C, greater than or equal to 16°C, greater than or equal to 18°C, greater than or equal to 20°C, greater than or equal to 22°C, or greater than or equal to 24°C. In some embodiments, the degreasing step is performed at a temperature less than or equal to 25°C, less than or equal to 24°C, less than or equal to 22°C, less than or equal to 20°C, less than or equal to 18°C, or less than or equal to 16°C. Combinations of the above-mentioned ranges are also possible (e.g., greater than or equal to 15°C and less than or equal to 25°C). Other ranges and temperatures are also possible.

[0075] In one set of exemplary embodiments, degreasing bone from aquatic organisms includes incubating the bone in acetone at a temperature greater than or equal to 15°C and less than or equal to 30°C for greater than or equal to 4 hours and less than or equal to 8 hours, thereby degreasing the bone.

[0076] In some embodiments, the bone of aquatic origin is dried (e.g., after a deproteinization and / or defatting step). In some embodiments, a drying step is performed to advantageously remove any residual moisture from the bone of aquatic origin. In some embodiments, the drying step is performed for 4 hours or more, 6 hours or more, 8 hours or more, 10 hours or more, 12 hours or more, 16 hours or more, 20 hours or more, or 24 hours or more. In some embodiments, the drying step is performed for 48 hours or more, 24 hours or more, 20 hours or more, 16 hours or more, 12 hours or more, 10 hours or more, 8 hours or more, or 6 hours or more. Combinations of the ranges mentioned above are also possible (e.g., 4 hours or more and 24 hours or more, 6 hours or more and 48 hours or more). Other ranges and durations are also possible.

[0077] In some embodiments, the drying step is performed at a temperature greater than or equal to 40°C, greater than or equal to 45°C, greater than or equal to 50°C, greater than or equal to 55°C, greater than or equal to 60°C, or greater than or equal to 65°C. In some embodiments, the degreasing step is performed at a temperature less than or equal to 70°C, less than or equal to 65°C, less than or equal to 60°C, less than or equal to 55°C, less than or equal to 50°C, or less than or equal to 45°C. Combinations of the above-mentioned ranges are also possible (e.g., greater than or equal to 40°C and less than or equal to 70°C). Other ranges and temperatures are also possible.

[0078] In one set of exemplary embodiments, the drying step includes drying the aquatic organism-derived bone at a temperature greater than or equal to 50°C and less than or equal to 70°C for greater than or equal to 6 hours and less than or equal to 48 hours.

[0079] In some embodiments, bone of aquatic origin is ground (e.g., pulverized, for example by ball milling, manual or electric grinders, mortars and pestles, etc.) to form particles of bone of aquatic origin. In some embodiments, grinding can be performed at any suitable temperature (e.g., room temperature, drying temperature) for greater than or equal to 1 second, greater than or equal to 2 seconds, greater than or equal to 4 seconds, greater than or equal to 6 seconds, greater than or equal to 8 seconds, or greater than or equal to 10 seconds. In some embodiments, the bone of aquatic origin is ground for less than or equal to 12 seconds, less than or equal to 10 seconds, less than or equal to 8 seconds, less than or equal to 6 seconds, less than or equal to 4 seconds, or less than or equal to 2 seconds. Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 1 second and less than or equal to 12 seconds). Other ranges and durations are also possible.

[0080] In some embodiments, bone of aquatic origin is ground (e.g., after a drying step) to produce multiple particles. Advantageously, the duration of the grinding step can produce multiple particles having a particle size similar to that of commercially available allogeneic and / or xenografts. In some embodiments, the multiple particles advantageously have a particle size sufficient to maintain the desired porosity of the particulate material.

[0081] In some embodiments, at least a portion of the plurality of particles has a particle size greater than or equal to 250 micrometers and less than or equal to 1000 micrometers. For example, in some embodiments, the average particle size of the ground aquatic-derived bone is greater than or equal to 100 micrometers, greater than or equal to 250 micrometers, greater than or equal to 500 micrometers, greater than or equal to 750 micrometers, or greater than or equal to 1000 micrometers. In some embodiments, the average particle size is less than or equal to 2000 micrometers, less than or equal to 1000 micrometers, less than or equal to 750 micrometers, less than or equal to 500 micrometers, or less than or equal to 250 micrometers. Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 100 micrometers and less than or equal to 2000 micrometers, greater than or equal to 250 micrometers and less than or equal to 1000 micrometers). Other ranges and sizes are also possible. Those skilled in the art will understand that while the average particle size may be within the range described above (e.g., greater than or equal to 100 micrometers and less than or equal to 2000 micrometers), the plurality of particles may include individual particles with particle sizes less than 100 micrometers and / or greater than 2000 micrometers.

[0082] In some embodiments, the bone of aquatic origin (e.g., after a deproteinization step, a defatting step, a drying step, and / or a grinding step) is calcined. For example, in some embodiments, the bone of aquatic origin (e.g., bone particles of aquatic origin) is calcined to produce a first composition (e.g., a first composition containing hydroxyapatite as described herein). In some embodiments, the calcination step includes heating the (grinded) bone of aquatic origin to a temperature greater than or equal to 600°C, greater than or equal to 650°C, greater than or equal to 700°C, greater than or equal to 800°C, greater than or equal to 900°C, greater than or equal to 1000°C, or greater than or equal to 1100°C. In some embodiments, the calcination step includes heating to a temperature less than or equal to 1200°C, less than or equal to 1100°C, less than or equal to 1000°C, less than or equal to 900°C, less than or equal to 800°C, less than or equal to 700°C, or less than or equal to 650°C. Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 600°C and less than or equal to 1200°C, greater than or equal to 800°C and less than or equal to 1200°C). Other ranges and temperatures are also possible.

[0083] In some embodiments, the calcination step includes heating (ground) aquatic-derived bone (e.g., aquatic-derived bone particles) for greater than or equal to 1 hour, greater than or equal to 1.5 hours, greater than or equal to 2 hours, greater than or equal to 2.5 hours, greater than or equal to 3 hours, greater than or equal to 4 hours, greater than or equal to 5 hours, greater than or equal to 6 hours, or greater than or equal to 8 hours. In some embodiments, the calcination step includes heating (ground) aquatic-derived bone (e.g., aquatic-derived bone particles) for less than or equal to 10 hours, less than or equal to 8 hours, less than or equal to 6 hours, less than or equal to 5 hours, less than or equal to 4 hours, less than or equal to 3 hours, less than or equal to 2.5 hours, less than or equal to 2 hours, or less than or equal to 1.5 hours. Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 1 hour and less than or equal to 10 hours). Other ranges and durations are also possible.

[0084] In some embodiments, after forming the first composition, the first and second compositions are mixed together (e.g., in the weight ratios described above) to form aquatic organism-derived biomaterial. In some embodiments, the first and second compositions are combined such that the ratio of hydroxyapatite present in the aquatic organism-derived biomaterial to the second composition (e.g., collagen or a derivative thereof) is greater than or equal to 70:30, greater than or equal to 75:25, greater than or equal to 80:20, greater than or equal to 85:15, or greater than or equal to 90:10. In some embodiments, the ratio of hydroxyapatite to the second composition (e.g., collagen or a derivative thereof) is less than or equal to 95:5, less than or equal to 90:10, less than or equal to 85:15, less than or equal to 80:20, or less than or equal to 75:25. Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 70:30 and less than or equal to 90:10). Other ranges are also possible. In one set of exemplary embodiments, the ratio of hydroxyapatite to collagen (or its derivatives) in the biomaterial is greater than or equal to 70:30 and less than or equal to 90:10.

[0085] In a set of exemplary embodiments, the method of forming the first composition includes one or more of the following steps:

[0086] 1. Incubate salmon bones in water at 110°C for 1 to 2 hours. This step corresponds to removing substances other than bones, such as muscle, connective tissue, or other components.

[0087] 2. The salmon bones are incubated in a NaOH solution with a concentration of 0.1 M to 0.5 M, preferably 0.2 M to 0.3 M, and more preferably 0.25 M, at a temperature of 15°C to 30°C, preferably 18°C ​​to 23°C, and more preferably 20°C, for 1 to 3 hours, preferably 2 hours. This step corresponds to the deproteinization of the bone.

[0088] 3. Incubate salmon bones in acetone at 20°C to 30°C, preferably 22°C to 28°C, and more preferably 25°C, for 5 to 7 hours, preferably 5.5 to 6.5 hours, and more preferably 6 hours. This step corresponds to defatting the bones.

[0089] 4. Dry the salmon bones at 50°C to 70°C, preferably 55°C to 65°C, and more preferably 60°C for more than 6 hours.

[0090] 5. Grind the bone and calcine it at a temperature of 900°C to 1100°C, preferably 950°C to 1050°C and more preferably 1000°C for 1 to 3 hours, preferably 1.5 to 2.5 hours and more preferably 2 hours.

[0091] 6. Obtain biomaterials containing hydroxyapatite.

[0092] 7. In one set of exemplary embodiments, the method further includes a final step in which 10% to 90% (w / w) of collagen is added to the final composition to obtain the composition of the present invention.

[0093] In some implementations, the method includes the additional step of adding platelet-rich fibrin to aquatic organism-derived biomaterial containing hydroxyapatite.

[0094] In one set of exemplary embodiments, the low-cost, non-cytotoxic, and osteogenic biomaterial containing hydroxyapatite obtained using the above method comprises 54% to 65% hydroxyapatite, 12% to 16% β-tricalcium phosphate, 1% to 2% calcium oxide, 0% to 2% magnesium oxide, and 19% to 32% amorphous biomaterial. The density of the exemplary biomaterial is 3.2 g / cm³. 3 Up to 3.3 g / cm 3 It is thermally stable between 50°C and 800°C. As mentioned above and in this article, other compositions and ranges are also possible.

[0095] In a set of exemplary embodiments of the present invention, the biological material derived from aquatic organisms is in solid, semi-solid, or liquid form, such as solid blocks, gels, pastes, etc.

[0096] A set of exemplary embodiments relates to the use of aquatic biologically derived biomaterials comprising a first composition containing hydroxyapatite derived from salmon bone and a second composition containing collagen in the preparation of products for promoting bone formation.

[0097] In one set of exemplary embodiments, compositions comprising biomaterials containing bone derived from aquatic organisms further comprise collagen, which may be type I, type II, type III, and type IV collagen. Not wishing to be bound by theory, in some embodiments, the addition of collagen (or other scaffold materials as described herein) advantageously enhances the biomaterial's ability to promote bone formation. Collagen may be present in the biomaterial in any suitable amount, such as the amount described in the context of the second composition above.

[0098] Collagen is an important protein found in the extracellular matrix of bone tissue and is crucial for bone structure and strength. Without being bound by theory, introducing collagen into biomaterials can improve the integration of the biomaterial with surrounding bone tissue and promote new bone formation. Furthermore, collagen can provide a favorable environment for osteocyte adhesion and proliferation, which can accelerate the healing process of bone injuries.

[0099] In another exemplary embodiment, the composition comprising hydroxyapatite biomaterial derived from bone of aquatic origin further comprises platelet-rich fibrin. Platelet-rich fibrin (PRF) is an autologous platelet concentrate obtained from a patient's own blood. PRF is rich in growth factors and other bioactive proteins that can promote tissue regeneration. In some cases, incorporating PRF into the biomaterial can improve the biomaterial's ability to promote bone formation, its biocompatibility, and the healing of bone injuries.

[0100] In some embodiments, the aquatic organism-derived biomaterial further comprises platelet-rich fibrin, if present, in an amount relative to the total weight of the biomaterial of greater than or equal to 0.1 wt%, greater than or equal to 0.5 wt%, greater than or equal to 1 wt%, greater than or equal to 1.5 wt%, greater than or equal to 2 wt%, greater than or equal to 2.5 wt%, greater than or equal to 3 wt%, greater than or equal to 3.5 wt%, or greater than or equal to 4 wt%. In some embodiments, the aquatic organism-derived biomaterial comprises platelet-rich fibrin, in an amount relative to the total weight of the biomaterial of less than or equal to 5 wt%, less than or equal to 4 wt%, less than or equal to 3.5 wt%, less than or equal to 3 wt%, less than or equal to 2.5 wt%, less than or equal to 2 wt%, less than or equal to 1.5 wt%, less than or equal to 1 wt%, or less than or equal to 0.5 wt%. Combinations of the ranges mentioned above are also possible (e.g., greater than or equal to 0.1 wt% and less than or equal to 5 wt%, greater than or equal to 1 wt% and less than or equal to 2 wt%). Other ranges are also possible.

[0101] In another embodiment of the invention, the composition comprising hydroxyapatite biomaterial is in solid, semi-solid, or liquid form.

[0102] Another object of the present invention is the use of compositions comprising biomaterials containing hydroxyapatite derived from salmon bone in products for promoting bone formation and for treating bone injuries.

[0103] All technical and scientific terms used to describe this invention have the same meaning as understood by one of ordinary skill in the art. However, to more clearly define the scope of this invention, the following definitions of terms used herein are provided. The term “bone injury” generally refers to various conditions and diseases affecting the structural and functional integrity of bone and bone tissue, including dental injuries. These may include, but are not limited to, fractures, osteoporosis, stress injuries, periodontal disease, dental abscesses, peri-implantitis, osteomyelitis, bone tumors, bone metastases, etc. The term “osteogenesis” generally refers to the biological processes encompassing the formation and development of new bone within the body. It is crucial during individual growth stages, fracture healing, and ongoing bone remodeling, a process that enables the maintenance and renewal of bone throughout a person's life. Osteogenesis can have two types: intramembranous osteoogenesis and endochondrial osteoogenesis. In intramembranous osteoogenesis, bone is formed directly from condensed mesenchymal cells that differentiate into osteoblasts, which then generate bone. This type of osteoogenesis is the primary bone formation process in the face and skull. In the other hand, endochondrial osteoogenesis involves the formation of bone from a cartilage template, which is gradually replaced by bone tissue. This is a major process in the formation of long bones in the body (such as the arm or leg). In the dental context, osteogenesis is a key process in many dental and maxillofacial interventions. For example, in dental implant surgery, titanium implants are inserted into the mandible or maxilla, and efforts are made to promote osteogenesis around the implant to achieve strong and lasting integration, a phenomenon known as osseointegration.

[0104] Furthermore, in orthognathic surgery aimed at correcting jaw deformities, incisions are made in the bone, which is then fixed in its new position. During recovery, osteogenics is the process by which the repositioned bone segments heal and fuse. Finally, in guided bone regeneration surgery used to treat bone loss due to periodontal disease or to prepare the foundation for future dental implants, biomaterials and techniques that stimulate osteogenics are used to replace lost bone tissue. The term "hydroxyapatite" or "HAPS" refers to a calcium biomineral, specifically a form of calcium phosphate with the chemical formula Ca5(PO4)3(OH). Hydroxyapatite is a major mineral component of human bone and teeth, forming approximately 70% of bone mass and up to 90% of tooth enamel. It is recognized for its excellent biocompatibility and bioactivity, making it an ideal biomaterial for orthopedic, dental, and regenerative medicine applications. In microcrystalline form, hydroxyapatite provides hardness and structural stiffness in bone and teeth. Furthermore, due to its chemical composition being similar to bone, it is frequently used in implant and bone-filling biomaterials because it promotes new bone formation and integration with surrounding tissues. The term "osteoinduction" refers to the biological process involving the stimulation of undifferentiated progenitor cells to differentiate into osteocytes or osteoblasts. This process occurs primarily through biochemical signaling from bone growth factors and is crucial in the regeneration and reconstruction of bone tissue. For example, osteoinduction plays a vital role in the success of bone grafting, where the transplanted biomaterial induces the formation of new bone at the graft site. The potential to differentiate into bone tissue is important and explains osteogenesis and osteoinduction, because if mesenchymal cells have the ability to differentiate into osteoblasts (osteoblasts are bone tissue cells that specialize in producing a bone matrix composed primarily of Ca and P and formed from hydroxyapatite crystals), they will also exhibit the ability to produce these structures.

[0105] Osteoprogenitor cells present in the patient can potentially proliferate and differentiate into osteoblasts in the presence of a graft; therefore, based on the above, in some embodiments, the biomaterial may have osteogenic and osteoinductive activity because it may be associated with bone formation in addition to stimulating and activating mesenchymal stem cells in the host tissue, respectively.

[0106] The term "allogeneic graft" refers to a specific type of transplant where tissue is transferred from one individual to another individual, a twin of the same species but not genetically identical. Allogeneic grafts are common in medical practice and are used in a variety of surgical procedures, such as organ transplantation, treatment of skin tissue from severe burns, or orthopedic surgeries using allogeneic bone grafts. Because the donor and recipient are not genetically identical, the risk of immune rejection must be considered, and therefore, immunosuppressive drugs are often used to prevent this problem.

[0107] Example

[0108] The following examples are intended to illustrate certain embodiments described herein (including certain aspects of the invention), but do not represent the full scope of the invention.

[0109] Example 1 - Optimization of Deproteinization Variables

[0110] In this embodiment, the variables of the production method of the present invention were optimized, with a focus on the deproteinization stage. A series of experiments were conducted to test different values ​​of alkaline solution concentration and temperature during this stage.

[0111] step:

[0112] 1. Initially, salmon bones are suspended in water at 110°C for 2 hours to remove most of the material other than bones, such as muscle and connective tissue.

[0113] 2. Then, the deproteinization stage is carried out, in which the bone is treated with NaOH solution of different concentrations (2 M or 0.25 M) and temperatures (20°C or 50°C) for 2 hours.

[0114] 3. After deproteinization, the bone was defatted with acetone at room temperature for 24 hours.

[0115] 4. Then, dry the bones at 60°C overnight to remove any residual moisture.

[0116] 5. Finally, the dried bone is ground in preparation for the final heating (i.e., calcination) stage, in which it is converted into hydroxyapatite biomaterial by a process at 650°C for 5 hours.

[0117] Results: Yield results based on deproteinization stage variables are shown in Table 1. The combination of NaOH 2 M and 20°C showed the most efficient yield, at 12%.

[0118] In addition, Raman spectroscopy measurements were performed on the biomaterials produced under each combination of conditions. Figure 2A Four vibrational modes belonging to the PO4 group of apatite were observed, among which the characteristic peak of hydroxyapatite was the most prominent.

[0119] Table 1: Yield results based on variables in the deproteinization stage

[0120] NaOH 2 M and 20℃ 12.0 NaOH 2 M and 50℃ 6.0 NaOH 0.25 M and 20℃ 7.8 NaOH 0.25 M and 50℃ -

[0121] Conclusion: Based on the results, the treatment conditions of 2.5 M NaOH and 50 °C were discarded because they did not show sufficient yield. The combination of 2 M NaOH and 20 °C was identified as the most effective for deproteinizing salmon bones. This example illustrates the process of optimizing variables in the deproteinization stage, which is crucial for obtaining high-quality and high-yield hydroxyapatite biomaterials.

[0122] Example 2 - Optimization of defatting variables

[0123] The variables in the production method of this invention, particularly the solvent and time in the degreasing stage, were determined through a series of experiments testing different values ​​for each variable.

[0124] step:

[0125] 1. First, suspend the salmon bones in water at 110°C for 2 hours.

[0126] 2. Subsequently, a deproteinization stage was performed, in which the bone was treated with a 2 M NaOH solution. This process was carried out at a constant temperature of 20°C for 2 hours.

[0127] 3. After deproteinization, the bone is defatted with acetone or hexane at 25°C for 6 or 12 hours.

[0128] 4. Then, dry the bones at 60°C overnight to remove any residual moisture.

[0129] 5. Finally, the dried bone is ground in preparation for the final calcination stage, in which it is converted into hydroxyapatite biomaterial by a process at 650°C for 5 hours.

[0130] Results: Yield results based on defatting stage variables are shown in Table 2. The combination of acetone and 6 hours showed the most efficient yield, at 17.8%.

[0131] Table 2: Yield results based on defatting stage variables

[0132] Acetone and 6 hours 17.8 Acetone and 12 hours 17.7 Hexane and 6 hours 16.0 Hexane and 12 hours 17.0

[0133] In addition, Raman spectroscopy measurements of the biomaterials produced under each combination of conditions were performed using the XploRA™ PLUS instrument (Horiba Scientific, France), such as... Figure 2B As shown. Although no significant time-related effects were observed, the intensity peak at 910 cm⁻¹ was more pronounced for the experiment using hexane, and the intensity of the hydroxyapatite peak was lower compared to the sample using acetone.

[0134] Example 3 - Optimization of Calcination Variables

[0135] The variables in the production method of this invention, particularly the temperature and time during the calcination stage, were determined through a series of experiments testing different values ​​for each variable.

[0136] step:

[0137] 1. First, suspend the salmon bones in water at 110°C for 2 hours.

[0138] 2. Subsequently, a deproteinization stage was performed, in which the bone was treated in a 2 M NaOH solution. This process was carried out at a constant temperature of 20°C for 2 hours.

[0139] 3. After deproteinization, the bone is defatted with acetone at 25°C for 6 hours.

[0140] 4. Then, dry the bones at 60°C overnight to remove any residual moisture.

[0141] 5. Finally, the dried bone is ground in preparation for the final calcination stage, in which it is converted into hydroxyapatite biomaterial by a process at 800°C, 900°C or 1000°C for 2, 4 or 6 hours.

[0142] The yield results based on the calcination stage variables are shown in Table 3. The combination of 800℃ and 2 hours showed the most efficient yield, with a yield of 19.0%.

[0143] Table 3: Yield results based on variables in the calcination stage

[0144] 800℃ and 6 hours 18.9 800℃ and 4 hours 18.9 800℃ and 2 hours 19.0 900℃ and 6 hours 18.0 900℃ and 4 hours 18.0 900℃ and 2 hours 18.8 1000℃ and 6 hours 18.0 1000℃ and 4 hours 18.0 1000℃ and 2 hours 18.0

[0145] In addition, Raman spectroscopy measurements of the biomaterials produced under each combination of conditions were performed using the XploRA™ PLUS instrument (Horiba Scientific, France), such as... Figure 3 , 4 As shown in Figure 5

[0146] In all samples, the characteristic maximum intensity of hydroxyapatite was identified at the previously reported locations. The time variable did not appear to affect the formation of hydroxyapatite, as the spectra at a given temperature did not show significant differences, and all exhibited characteristic peaks of the hydroxyapatite structure. However, temperature did affect the purity of hydroxyapatite, with clearer spectra observed at higher temperatures.

[0147] Example 4 - Selected Scheme

[0148] The three complete schemes were evaluated as follows:

[0149] • Option 1: Deproteinize at 20℃ with 0.25 M for 2 hours / defatt with acetone for 6 hours / calcine at 1000℃ for 2 hours.

[0150] • Option 2: Deproteinize at 20°C with 0.25 M for 2 hours / defatt with acetone for 6 hours / calcine at 900°C for 2 hours.

[0151] • Option 3: Deproteinize at 20°C with 0.25 M for 2 hours / defatt with acetone for 6 hours / calcine at 800°C for 2 hours.

[0152] The results shown in Table 4 were obtained from the above schemes, in which the biomaterials obtained from the three test schemes were selected as having comparable quality.

[0153] Table 4: Results of three complete schemes for obtaining hydroxyapatite biomaterials.

[0154] 1 19.0 64.1 80.49 2 21.0 60.21 78.45 3 18.9 54.33 68.42

[0155] Raman spectroscopy measurements of the biomaterials produced in each protocol were then performed using an XploRA™ PLUS instrument (Horiba Scientific, France), such as... Figure 6 , 7 As shown in Figure 8.

[0156] Four vibrational modes belonging to the phosphate group (PO4) of apatite were observed in each spectrum. Hydroxyapatite from Scheme 1 corresponded to a sharper peak, while the biomaterials from Schemes 2 and 3 exhibited both apatite structural peaks and a noisy peak. The peak appearing in the 1070 cm⁻¹ region corresponded to the carbonate group peak, which overlapped with the phosphate group ν3. The presence of carbonate groups in bone graft tissue is crucial because it is described as a factor mimicking the extracellular matrix, thereby improving bone integration, biocompatibility, and reabsorption properties.

[0157] Subsequently, X-ray diffraction (XRD) measurements were performed on the biomaterials produced from each of the three schemes using a powder X-ray diffractometer (Bruker, Germany). The resulting diffraction patterns can be seen on... Figure 9 , 10 And 11, and convert them into the results shown in Table 5.

[0158] Table 5: Diffraction patterns of biomaterials obtained from the three schemes.

[0159] 1 64.10 15.40 0.99 - 19.51 2 60.21 14.33 1.91 2.00 21.55 3 54.33 12.62 1.33 0.14 31.58

[0160] Example 5 - Thermogravimetric Analysis (TGA)

[0161] Thermogravimetric analysis (TGA) was performed on the biomaterials generated from schemes 1, 2, and 3 using a TGA Q50 V20.10 Build 36 instrument (Thermal Analysis, USA), with temperatures varying between 50°C and 800°C. Results for each scheme are available in [link to relevant documentation]. Figure 12 , 13 And 14. For the hydroxyapatite of the selected schemes, there was no evidence of significant mass loss. However, for both Schemes 1 and 2, slight variations were observed in the temperature range of 500°C to 600°C, which could indicate the combustion of trace organic matter remaining in the biomaterial. This confirms that the produced hydroxyapatite is thermally stable.

[0162] Example 6 - Scanning electron microscopy combined with energy-dispersive X-ray spectroscopy (SEM-EDX)

[0163] Biomaterials generated from schemes 1, 2, and 3 were sent for SEM-EDX microscopy using a Phenom Pro X instrument (Thermo Fisher Scientific, USA). The resulting images are available on [website address missing]. Figure 15 , 16 And 17. In addition, the local elemental composition extracted from EDX analysis is shown in Table 6.

[0164] Table 6: Local elemental composition of biomaterials obtained from each scheme.

[0165] Ca 7.64 18.20 18.87 P 4.68 10.46 10.84 Ca / P 1.63 1.74 1.74

[0166] The Ca / P ratio of the obtained hydroxyapatite is close to the stoichiometric value of hydroxyapatite, 1.67. In addition to the elements listed in Table 5, the amounts of C, O, Mg, Na, N, Cu, Al, and Si are also shown.

[0167] Example 7 - Gas Specific Gravity Bottle Determination Method

[0168] The biomaterials obtained through three selected protocols were analyzed using a gas pyrometer via an AccuPyc II 1340 series instrument (Micromeritics, USA). The measured densities are shown in Table 7.

[0169] Table 7: Density of biomaterials obtained from each extraction scheme.

[0170] 1 3.24 ± 1.10 2 3.29 ± 0.01 3 3.37 ± 0.01

[0171] The obtained value is close to the theoretical density of pure hydroxyapatite, 3.156 g / cm³. 3 .

[0172] Example 8 - Texture Analysis

[0173] The compressive resistance of the samples obtained from each scheme was analyzed using a TA.XT Plus texture analyzer (Stable Micro Systems, UK), and the results were as follows: Figure 18 The slope is shown. Based on the obtained data, each scheme was considered for 6 repetitions. Measurements were taken using the compressive force at a maximum of 2 mm. The slope was calculated and the maximum force was determined considering data from 0.5 to 1.6 mm compression, and the results are shown in Table 8.

[0174] Table 8: Maximum compressive force of biomaterials obtained from each extraction scheme.

[0175] 1 320.60 2 326.26 3 339.55

[0176] These results indicate that, with lower density (from the material in Scheme 1) and therefore higher porosity, the mechanical resistance is lower than that of other materials. Therefore, not wanting to be bound by theory, in some cases, the higher temperatures during the calcination process of hydroxyapatite production may interfere with the resulting structure of the final biomaterial, affecting its compressive resistance by exhibiting higher porosity.

[0177] Example 9 - Cell Viability and Proliferation

[0178] To evaluate the viability and proliferation of different cell types in the presence of hydroxyapatite biomaterials obtained from three selected protocols, gingival mesenchymal cells were cultured for 48 hours in Dulbecco's Modified Eagle Medium (DMEM) in the presence of human commercial hydroxyapatite biomaterials, bovine commercial hydroxyapatite biomaterials, biomaterials from each of the three selected protocols, and a negative control. Viability and proliferation were then measured by measuring metabolic activity using a 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results at 24 and 48 hours of culture are available in [the table / data missing]. Figure 19 and 20 .

[0179] Under all test conditions, the number of live cells was similar, except for the hydroxyapatite biomaterial of Protocol 1 (HAPSP1), which showed a significant difference at 24 hours compared with the control and the hydroxyapatite biomaterial of Protocol 3 (HAPS P3) (p < 0.01). Then, in Figure 20 In the study, changes in cell viability of HAPS at 48 hours were observed. The number of cells treated with HAPS P3 did not increase compared to the 24-hour experiment, while the viability of cells under other conditions did increase, reaching similar cell numbers.

[0180] Although HAPS P3 showed significant differences compared to controls and other hydroxyapatite at 48 hours, the number of viable cells remained acceptable, suggesting that hydroxyapatite derived from salmon bones, which are considered waste from the salmon industry, has value in the field of bone repair.

[0181] Example 10 - Cell differentiation of mesenchymal cells into osteoblasts

[0182] Mesenchymal cells were cultured for 21 days to differentiate, and then stained with Alizarin Red in differentiation medium (DMEM supplemented with ascorbic acid, sodium phosphate, and dexamethasone) at 37°C. Differentiation was performed under various conditions, particularly in the presence of hydroxyapatite biomaterials from three selected protocols: HAPS P1, HAPS P2, HAPS P3, human allogeneic grafts, and bovine xenografts; in the absence of biomaterials; and without differentiation. Results after 21 days were available. Figure 21 The arrows indicate calcium deposits present in different cultures. These deposits, observed through deeper staining, are a clear indication of mineralization produced by osteoblasts (which specialize in producing bone matrix).

[0183] Cell differentiation assays enabled the determination that the three HAPS production pathways induce mesenchymal cells to differentiate into osteoblasts, as confirmed by the presence of calcium deposition. HAPS P1 and P2 showed significant mineralization, while P3 showed lower mineralization, similar to that produced by bovine xenografts and significantly higher than that produced by human allogeneic grafts and cells differentiating in the absence of biomaterials.

[0184] These results indicate that the biomaterials produced by the three protocols are osteoinducible and that mesenchymal cells differentiate into osteoblasts in their presence.

[0185] Example 11 - Grinding Time

[0186] Particle sizes were measured for each of the three selected schemes using grinding times of 2, 6, and 10 seconds. The size distribution results are available in... Figure 22 , 23 And 24. The particle size distribution of commercially available allogeneic and xenografts ranges from 250 to 1000 μm. Based on this, a size distribution closest to this spectrum was obtained by milling for 2 seconds.

[0187] Subsequently, the porosity of the biomaterials obtained at grinding times of 2 seconds and 10 seconds was measured using polarized light microscopy. These results are available in... Figure 25 and 26 .

[0188] These results conclude that shorter grinding times generally result in larger particle sizes while preserving the porosity of the biomaterial, while longer grinding times lead to smaller particle sizes and a loss of porosity. Therefore, based on these results, grinding for 2 seconds provides a better microstructure, making it the preferred condition for producing hydroxyapatite biomaterials from the selected scheme.

[0189] Example 12 - Combination of hydroxyapatite biomaterials with leukocyte- and platelet-rich fibrin (L-PRF) and its clinical application evaluation

[0190] The behavior of hydroxyapatite biomaterials in combination with leukocyte-rich and platelet fibrin (L-PRF) was investigated, and the results are shown in Table 9, where each attribute was evaluated based on its qualitative properties and assigned a maximum score of 5.

[0191] Surgical procedures It is easy to deliver to the surgical site and can be shaped as needed. It's easy to operate. It's easy to shape small portions as needed, and it's simple to use. It is easy to shape in small portions as needed and is easy to operate. hydrophobic Biomaterials absorb liquids. Biomaterials absorb liquids. Biomaterials absorb liquids. Gathering Agglutinates within 5 minutes. Brittle materials, aggregated in small portions. The material aggregates but is easily dispersed. Consistency It is hard and can withstand pressure. Soft material. It is a hard material that can withstand a small amount of pressure. Total score obtained 20 15 17

[0192] The biomaterial obtained using scheme 1 (higher calcination temperature) achieved full marks in the evaluation in combination with L-PRF. No material loss occurred during the operation using this hydroxyapatite biomaterial. Biomaterials from schemes P1 and P2 showed excessive, but incomplete, L-PRF absorption.

[0193] Furthermore, P1 and P2 did not exhibit complete aggregation, resulting in the loss of biomaterial particles. Nevertheless, the biomaterials from P2 and P3 achieved acceptable scores in the evaluation.

[0194] While some embodiments of the invention have been described and illustrated herein, those skilled in the art will readily conceive of a variety of other means and / or structures for performing the said functions and / or obtaining the said results and / or one or more advantages described herein, and each such variation and / or modification is considered to be within the scope of the invention. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and constructions described herein are intended to be exemplary, and actual parameters, dimensions, materials, and / or constructions will depend on one or more specific applications using the teachings of this invention. Those skilled in the art will recognize, or be able to determine, many equivalents of the specific embodiments of the invention described herein using only conventional experiments. Therefore, it should be understood that the foregoing embodiments are presented by way of example only, and that the invention may be practiced in ways other than those specifically described and claimed within the scope of the appended claims and their equivalents. The invention relates to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the invention if such features, systems, articles, materials, kits, and / or methods are not contradictory.

[0195] Unless otherwise expressly stated, nouns not limited by quantifiers as used herein in the specification and claims shall be understood to mean "at least one / a."

[0196] As used herein in the specification and claims, the phrase “and / or” should be understood to mean “any one or both” of the elements so connected, that is, elements that exist together in some cases and separately in others. In addition to the elements explicitly identified by the “and / or” clause, other elements may optionally exist, whether related to or unrelated to those explicitly identified elements, unless explicitly indicated otherwise. Thus, as a non-limiting example, when used in conjunction with open-ended language such as “comprising / including,” a reference to “A and / or B” may in one embodiment refer to A, excluding B (optionally including elements other than B); in another embodiment, refer to B, excluding A (optionally including elements other than A); in yet another embodiment, refer to both A and B (optionally including other elements); and so on.

[0197] As used herein in the specification and claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted as inclusive, i.e., including multiple elements or at least one of the elements in the list, but also including more than one of them, and optionally including additional unlisted items. Only terms that explicitly indicate the opposite, such as “only one of” or “exactly one of”, or when used in a claim, “consisting of”, will refer to including multiple elements or exactly one of the elements in the list. Generally, the term “or” as used herein should only be interpreted as indicating an exclusive alternative (i.e., “one of or the other but not both”) when preceding an exclusive term (e.g., “any one,” “one of,” “only one of,” or “exactly one of”). When used in a claim, “consisting substantially of” should have its usual meaning as used in the field of patent law.

[0198] As used herein in the specification and claims, the phrase "at least one" for a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element expressly listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those expressly identified in the list of elements referred to by the phrase "at least one," whether related to or unrelated to those expressly identified elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B," or equivalently, "at least one of A and / or B") in one embodiment may refer to at least one (optionally including more than one) A, excluding B (and optionally including elements other than B); in another embodiment, it refers to at least one (optionally including more than one) B, excluding A (and optionally including elements other than A); in yet another embodiment, it refers to at least one (optionally including more than one) A and at least one (optionally including more than one) B (and optionally including other elements); and so on.

[0199] In the claims and the foregoing description, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” etc., shall be understood as open-ended, meaning including but not limited to. Only the transitional phrases “consisting of” and “consisting substantially of” shall be closed or semi-closed transitional phrases, respectively, as described in Section 2111.03 of the U.S. Patent Examination Procedure Manual.

[0200] Unless otherwise defined or indicated, any term used herein relating to the shape, orientation, alignment and / or geometric relationship of, for example, one or more articles, structures, forces, fields, flows, directions / trajectories and / or their sub-components and / or combinations thereof and / or any other tangible or intangible element not listed above that is suitable for characterization by such terms, shall be understood not to require absolute conformity to the mathematical definition of such terms, but rather to indicate conformity to the mathematical definition of such terms to the extent possible for the subject matter so characterized, as understood by a person skilled in the art most closely associated with such subject matter. Examples of such terms relating to shape, orientation, and / or geometric relationships include, but are not limited to, terms describing the following: shape – such as circle, square, gomboc, ring / ring, rectangle / rectangle, triangle / triangle, cylinder / cylindrical, ellipse / ellipse, (n) polygon / (n) polygon, etc.; angular orientation – such as perpendicular, orthogonal, parallel, longitudinal, transverse, collinear, etc.; profile and / or trajectory – such as planar / planar, coplanar, hemispherical, quasi-hemispherical, line / linear, hyperbolic, parabolic, flat, curved, straight, arc, sine, tangent / tangent, etc.; direction – such as north, south, east, west, etc.; surface and / or bulk material properties and / or spatial / temporal resolution and / or distribution – such as smooth, reflective, transparent, clear, opaque, rigid, impermeable, homogeneous, inert, non-wetting, insoluble, stable, invariant, constant, homogeneous, etc.; and many other terms that are obvious to those skilled in the art. As an example, a manufactured article described herein as a "square" does not require that the article have perfectly planar or linear faces or sides that intersect at exactly 90-degree angles (in fact, such an article can only exist as a mathematical abstraction). Rather, the shape of the article should be interpreted as approximating a mathematically defined "square" to the extent that the manufacturing technology generally makes and has achieved it, as understood or specifically described by those skilled in the art. As another example, two or more manufactured articles described herein as "aligned" do not require that such articles have perfectly aligned faces or sides (in fact, such articles can only exist as a mathematical abstraction). Rather, the arrangement of such articles should be interpreted as approximating a mathematically defined "alignment" to the extent that the manufacturing technology generally makes and has achieved it, as understood or specifically described by those skilled in the art.

Claims

1. Biological materials derived from aquatic organisms, including: The first composition is present in the biological material in an amount greater than or equal to 10% by weight and less than or equal to 100% by weight relative to the total weight of the biological material; The second composition is present in the biological material in an amount greater than or equal to 0.1% by weight and less than or equal to 90% by weight relative to the total weight of the biological material; The first composition comprises: 50% or more and 90% or less by weight of hydroxyapatite or its derivatives from aquatic organisms, which may optionally be substituted; Greater than or equal to 5% by weight and less than or equal to 40% by weight of amorphous biomaterials, wherein the amorphous biomaterials comprise: The total weight of β-tricalcium phosphate in the first composition is greater than or equal to 10% by weight and less than 20% by weight. Calcium oxide with a content greater than or equal to 0.1% by weight and less than or equal to 5% by weight, Magnesium oxide greater than or equal to 0.1% by weight and less than or equal to 20% by weight; and The second composition comprises one or more of collagen and / or its derivatives, synthetic collagen and / or its derivatives, gelatin and / or its derivatives, fibrin and / or its derivatives, and fibrin and / or its derivatives.

2. The aquatic biologically derived biomaterial according to claim 1, wherein the aquatic biologically derived biomaterial is substantially free of synthetic polymers.

3. A biological material derived from an aquatic organism according to any one of the preceding claims, wherein the second composition comprises a pore-forming additive and / or bone morphogenetic protein.

4. The aquatic organism-derived biomaterial according to any one of the preceding claims, wherein the collagen, actin and / or gelatin, or derivatives thereof, are each derived from an aquatic organism.

5. A biological material derived from an aquatic organism according to any one of the preceding claims, wherein platelet-rich fibrin is present in the biological material in an amount greater than or equal to 0.1% by weight and less than or equal to 5% by weight relative to the total weight of the biological material.

6. The aquatic biological material according to any one of the preceding claims, wherein the ratio of hydroxyapatite to the second composition is greater than or equal to 70:30 and less than or equal to 90:

10.

7. A method for producing biological material of aquatic origin, the method comprising: Removal of non-bone material includes incubating bone of aquatic origin at a temperature of 700°C or higher and 120°C or lower for 20 minutes to 4 hours to remove non-bone material. Deproteinizing the bone from the aquatic organism includes incubating the bone in sodium hydroxide at a temperature greater than or equal to 15°C and less than or equal to 25°C for greater than or equal to 30 minutes to less than or equal to 4 hours, thereby deproteinizing the bone. Degreasing the bone from the aquatic organism includes incubating the bone in acetone at a temperature greater than or equal to 15°C and less than or equal to 30°C for greater than or equal to 4 hours and less than or equal to 8 hours, thereby degreasing the bone. The bone derived from the aquatic organism was dried at a temperature greater than or equal to 50°C and less than or equal to 70°C for greater than or equal to 6 hours and less than or equal to 48 hours. The dried bone is ground. Grinded bone is heated to a temperature greater than or equal to 800°C and less than or equal to 1200°C and held for a period of 1 hour and less than or equal to 3 hours to produce biomaterials derived from aquatic organisms. as well as A second composition is added to the aquatic biological material, the second composition comprising one or more of collagen and its derivatives, actin and its derivatives, gelatin and its derivatives, pore-forming additives, and bone morphogenetic proteins.

8. The article or method according to any one of the preceding claims, wherein the aquatic biological material is derived from one or more of fish, marine mammals, crustaceans, marine reptiles, and marine organisms.

9. The article or method according to any one of the preceding claims, wherein the fish is selected from salmon, tuna, anchovy, angelfish, catfish, cod, sole, pike, red-spotted salmon, herring, shark, killifish, pufferfish, flatfish, trout, eel, stingray, pufferfish, flounder, remora, sturgeon, tigerfish and grouper.

10. The article or method according to any one of the preceding claims, wherein the aquatic biological material is free of hydroxyapatite from non-aquatic biological sources.

11. The article or method according to any one of the preceding claims, wherein the first composition comprises hydroxyapatite and / or derivatives thereof of aquatic origin.

12. The article or method according to any one of the preceding claims, wherein at least a portion of the first composition is derived from salmon bone.

13. The article or method according to any one of the preceding claims, wherein the aquatic organism-derived hydroxyapatite is present in the first composition in an amount greater than or equal to 54% by weight and less than or equal to 65% by weight.

14. The article or method according to any one of the preceding claims, wherein the ratio of hydroxyapatite or a derivative thereof to collagen is less than or equal to 95:

5.

15. The article or method according to any one of the preceding claims, wherein the first composition further comprises β-Rainerite, and if present, the amount of β-Rainerite is greater than or equal to 0.1% by weight and less than or equal to 20% by weight relative to the total weight of the first composition.

16. The article or method according to any one of the preceding claims, wherein the density of the first composition is greater than or equal to 2 g / cm³. 3 And less than or equal to 4.2 g / cm 3 The specific gravity was determined using the gas specific gravity bottle method.

17. The article or method according to any one of the preceding claims, wherein the first composition is thermally stable at a temperature greater than or equal to 0°C and less than or equal to 1000°C, as determined by thermogravimetric analysis.

18. The article or method according to any one of the preceding claims, wherein the calcium-to-phosphorus ratio (Ca / P) of the first composition is greater than or equal to 1.5 and less than or equal to 1.8, as determined using scanning electron microscopy combined with energy-dispersive X-ray spectroscopy (SEM-EDX).

19. The article or method according to any one of the preceding claims, wherein the maximum compressive force of the first composition is greater than or equal to 250 N and less than or equal to 600 N, as measured using a TA.XT plus texture analyzer.

20. The article or method according to any one of the preceding claims, wherein the second composition comprises one or more of collagen type I, collagen type II, collagen type III, collagen type IV and / or derivatives thereof.

21. The article or method according to any one of the preceding claims, wherein the second composition comprises bovine collagen.

22. The article or method according to any one of the preceding claims, wherein the pore-forming additive comprises a supersaturated salt solution, the supersaturated salt solution comprising alkali metals, alkaline earth materials and halides, partially neutralized inorganic acids and / or neutralized organic acids.

23. The article or method according to any one of the preceding claims, wherein the first incubation step is carried out in air or an aqueous solution.

24. The article or method according to any one of the preceding claims, wherein the deproteinization step comprises incubating the aquatic organism-derived bone in an alkaline solution.

25. The article or method according to any one of the preceding claims, wherein defatting the aquatic organism-derived bone comprises incubating the aquatic organism-derived bone in a defatting solvent.

26. The article or method according to any one of the preceding claims, wherein the bone of the aquatic organism source is ground to form particles of the bone of the aquatic organism source.

27. The article or method according to any one of the preceding claims, wherein the grinding is performed for a time greater than or equal to 1 second and less than or equal to 12 seconds.

28. The article or method according to any one of the preceding claims, wherein grinding the bone derived from the aquatic organism produces a plurality of particles.

29. The article or method according to any one of the preceding claims, wherein at least a portion of the plurality of particles has a particle size greater than or equal to 250 micrometers and less than or equal to 1000 micrometers.

30. The article or method according to any one of the preceding claims, wherein the first composition and the second composition are combined such that the ratio of the hydroxyapatite to the second composition in the aquatic biological material is greater than or equal to 70:30 and less than or equal to 90:

10.

31. A non-cytotoxic biomaterial composition for promoting bone formation, comprising hydroxyapatite, characterized in that... The composition comprises 54% to 65% (w / w) hydroxyapatite derived from salmon bone, 12% to 16% (w / w) tricalcium β-phosphate, 1% to 2% (w / w) calcium oxide, 0% to 2% (w / w) magnesium oxide, and 19% to 32% (w / w) amorphous biological material; wherein the composition further comprises collagen at a concentration of 10% to 90% (w / w) of the final composition.

32. The composition according to claim 31, characterized in that... The density of the biomaterial is 3.1 g / cm³ to 3.4 g / cm³. 3 .

33. The composition according to claim 31, characterized in that... The biomaterial is thermally stable between 50°C and 800°C.

34. The composition according to claim 31, characterized in that... The collagen is selected from type 1 collagen, type 2 collagen, type 3 collagen and type 4 collagen.

35. The composition according to any one of claims 31 to 34, characterized in that... It also contains platelet-rich fibrin.

36. The composition according to any one of claims 31 to 35, characterized in that... It exists in solid, semi-solid, or liquid form.

37. Use of the composition according to any one of claims 31 to 36, characterized in that... It is used to prepare products that can be used to promote bone formation.

Images