Demineralized bone matrix fibers, methods of making and using same

Abstract

An apparatus for mixing bone material with a liquid is provided. The apparatus includes a chamber having a proximal end and a distal end and bone material disposed within the chamber, the bone material comprising DBM particles of milled and lyophilized demineralized bone fibers; and a plunger having at least a portion slidably disposed within the proximal end of the chamber and configured to dispense the bone material mixed with the liquid from the distal end of the chamber when the plunger is in an extended position.

Description

Background Technology

[0001] It is estimated that more than 500,000 bone graft surgeries are performed annually in the United States, costing over $2.5 billion. These numbers are projected to double by 2020. Both natural bone and bone substitutes are used as bone materials in bone grafts. Natural bone can be autologous or allogeneic grafts. Bone substitutes include natural or synthetic materials such as collagen, silicone, acrylic resins, calcium phosphate, and calcium sulfate.

[0002] Bone grafting employs at least three methods to help repair defects. The first is osteogenic formation, where new bone is formed within the graft due to the presence of osteoblasts called osteoprogenitor cells. The second is osteoinduction, in which molecules within the graft (e.g., bone morphogenetic proteins and other growth factors) convert progenitor cells into osteoblasts. The third is osteoconduction, a physical effect where a matrix, typically containing graft material, acts as a scaffold upon which bone and cells in the recipient can form. This scaffold promotes the migration, proliferation, and differentiation of osteoblasts to achieve bone regeneration.

[0003] Demineralized bone matrix (DBM) is a type of bone material that has been shown to induce and / or guide bone formation. Therefore, it is desirable to implant and maintain demineralized bone matrix at sites where bone growth is required.

[0004] Demineralized bone matrix based on bone fibers for implantation exhibits improved mechanical properties, including adhesion, fiber length, fiber diameter or width, fiber aspect ratio, or a combination of multiple variables.

[0005] Sometimes, DBM fibers exhibit inconsistent processing characteristics when hydrated in closed systems such as chambers or syringes. In cases of uneven hydration, DBM fibers may be improperly hydrated, leading to blockages in the syringe or chamber during the mixing and dispensing of the DBM fibers.

[0006] Therefore, there is a need for a bone material containing DBM fibers that allows the DBM fibers to be uniformly hydrated in a syringe or chamber before delivery. The most advantageous components would be DBM fibers comprising a combination of long and short fiber sizes to enhance hydration, devices for mixing and delivering the DBM fibers, and methods for preparing the DBM fibers. Summary of the Invention

[0007] DBM fibers comprising a combination of long and short fiber sizes that enhance hydration, apparatus for mixing and conveying DBM fibers, and methods for preparing DBM fibers are provided.

[0008] In some embodiments, a method for preparing demineralized bone matrix particles is provided. The method includes providing a plurality of long bone fibers with a diameter of about 0.5 mm to about 8.0 mm; mixing the plurality of long bone fibers with a plurality of short bone fibers with a diameter of about 0.5 mm to about 0.05 mm to form a mixture of long and short bone fibers; demineralizing the mixture of long and short bone fibers to form a demineralized bone matrix slurry; and drying the demineralized bone matrix slurry to form demineralized bone matrix particles.

[0009] In some embodiments, a bone material is provided comprising a mixture of a plurality of long bone fibers with a diameter of about 0.5 mm to about 8.0 mm and a plurality of short bone fibers with a diameter of about 0.5 mm to about 0.05 mm.

[0010] Further features and advantages of the various embodiments will be set forth in part in the description which follows, and will be apparent in part from the description, or may be learned by practice of the various embodiments. The objects and other advantages of the various embodiments will be realized and obtained by means of the elements and combinations specifically pointed out in the description and the appended claims. Attached Figure Description

[0011] Other aspects, features, benefits, and advantages of the embodiments will become apparent in part from the following description, the appended claims, and the accompanying drawings.

[0012] Figure 1A An embodiment of a method for manufacturing DBM granules from macrobone fibers is shown. The macrobone fibers are screened, selected, and sorted into long and short fibers. The long and short fibers are mixed to form a DBM slurry. The DBM slurry is added to a mold to form granules and then dried.

[0013] Figure 1B An embodiment of a method for manufacturing DBM granules from macrobone fibers is shown. The macrobone fibers are screened, selected, and sorted into long and short fibers. The long and short fibers are mixed to form a DBM slurry. The DBM slurry is discharged and then added to a mold to form granules, the granules are freeze-dried, and then removed from the mold in granule form.

[0014] Figure 2 One embodiment of long fibers is described. Large bone fibers are ground from a piece of bone and placed in a sieve to obtain long bone fibers of the desired size.

[0015] Figure 3 One implementation scheme for short fibers is described. The remaining large fibers are ground into short fibers.

[0016] Figure 4An implementation scheme for DBM slurry is described. Target long and short fibers are mixed and demineralized to form DBM slurry, which now contains both large and short demineralized fibers.

[0017] Figure 5A An implementation scheme for filling a portion of DBM slurry into a mold is described. The DBM slurry is gently pressed into the mold using a spatula.

[0018] Figure 5B An embodiment for filling DBM slurry into a mold is described. The DBM slurry is gently compacted into the mold using a scraper, wherein the DBM slurry is shaped to fit the mold and has substantially uniform size and particle shape.

[0019] Figure 6A An implementation scheme for filling DBM slurry into a mold is described. The DBM slurry is gently compacted into the mold using rollers and a compression block.

[0020] Figure 6B An implementation scheme for filling a mold with DBM slurry is described. The DBM slurry is gently compacted into the mold using rollers and a compression block to form DBM particles of uniform size and shape.

[0021] Figure 7A An embodiment of the DBM granules after ejection from the mold is described. The DBM granules are dried, and the granules are in flake form.

[0022] Figure 7B One embodiment of DBM particles is described. The DBM particles are knocked out individually and / or in sheet form for drying.

[0023] Figure 8A An embodiment of DBM granules is described. The dried DBM granules are in flake form and are uniform in size and shape.

[0024] Figure 8B An embodiment of DBM particles is described. The dried DBM particles are uniform in size and shape and are individually separated.

[0025] Figure 8C An embodiment of DBM particles is described. The dried DBM particles are in the form of flakes, from which the particles detach.

[0026] Figure 9 An embodiment of a texture analyzer for measuring the processed texture of DBM particles is shown.

[0027] Figure 10 An embodiment of a syringe or chamber loaded with dried DBM fiber particles is shown.

[0028] Figure 11 An embodiment of hydrating dried DBM particles with a liquid is shown. The liquid comprises blood, and the plunger has been moved between a retracted position and an extended position 20 times to hydrate the DBM particles.

[0029] Figure 12 A graph is shown that correlates the hydration of DBM particles with the injection force applied to the DBM particles. The graph also depicts the different results of adding different volumes of liquid to dried DBM particles.

[0030] It should be understood that the accompanying drawings are not drawn to scale. Furthermore, the relationships between objects in the drawings may not be to scale and may actually have the opposite relationship to their dimensions. These drawings are intended to aid in understanding and clarifying the structure of each object shown, and therefore, some features may be enlarged to illustrate the specific characteristics of a structure. Detailed Implementation

[0031] To facilitate understanding of the principles of this disclosure, reference will now be made to certain embodiments, and they will be described using specific language. However, it will be understood that this is not intended to limit the scope of the disclosure, and such changes and further modifications to the illustrated apparatus, as well as such further applications of the principles of the disclosure as described herein, are considered to be what one would normally think of in the art to which this disclosure relates.

[0032] For the purposes of this specification and the appended claims, unless otherwise indicated, all figures expressing the amount of ingredients, percentages or proportions of materials, reaction conditions, and other numerical values ​​used in this specification and the claims shall be understood to be modified by the term "about" in all cases. Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximations that may vary depending on the desired properties sought to be obtained through this application. To a minimum, and without attempting to limit the application of the equivalence principle to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant digits reported and by applying general rounding techniques.

[0033] Although the numerical ranges and parameters described in this disclosure are approximate, the numerical representations are made as precisely as possible. However, any numerical value inherently contains some error that is necessarily caused by the standard deviation found in its corresponding test measurement. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges contained herein. For example, the range "1 to 10" encompasses (and includes) any and all subranges between (and including) the minimum value of 1 and the maximum value of 10, i.e., any and all subranges having a minimum value equal to or greater than 1 and a maximum value equal to or less than 10, such as 5.5 to 10.

[0034] Furthermore, unless otherwise defined or apparent from the context, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0035] Unless explicitly stated or obvious from the context, the following terms are phrases with the following definitions:

[0036] definition

[0037] It should be noted that, unless explicitly and definitively limited to a single indicator, the singular forms “a,” “an,” and “the” used in this specification and the appended claims encompass a plurality of indicators. Thus, for example, reference to “implant” includes one, two, three, or more implants.

[0038] The term "biodegradable" includes the ability of all or part of the carrier and / or implant to degrade over time through enzymatic action, hydrolysis, and / or other similar mechanisms within the body. In various embodiments, "biodegradable" includes the ability of the carrier and / or implant to break down or degrade into non-toxic components within the body after or during the release of the therapeutic agent. "Bioerodibility" means that the carrier and / or implant will erode or degrade over time, at least partially, due to contact with substances or fluids seen in surrounding tissues or through cellular action.

[0039] The term “mammal” refers to organisms belonging to the class “mammals” in the taxonomic system, including but not limited to humans, other primates (such as chimpanzees, apes, orangutans, and monkeys), rats, mice, cats, dogs, cattle, horses, etc.

[0040] A "therapeutic effective dose" or "effective dose" means that, upon administration, a drug (e.g., a growth factor) causes a change in biological activity, such as promoting the growth of bone, cartilage, and / or other tissues (e.g., vascular tissue), suppressing inflammation, reducing or alleviating pain, or improving symptoms by suppressing the immune response. Depending on various factors, including the pharmacokinetic properties of the drug, the route of administration, the patient's condition and characteristics (sex, age, weight, health status, physique, etc.), the severity of symptoms, concurrent treatments, the frequency of treatment, and the desired effect, the dose administered to the patient may be in the form of a single dose or multiple doses. In some embodiments, the implant is designed for immediate release. In other embodiments, the implant is designed for sustained release. In other embodiments, the implant includes one or more immediate-release surfaces and one or more sustained-release surfaces.

[0041] When used in conjunction with a disease or condition, the term "treating" or "treatment" refers to the execution of a protocol that may include bone repair surgery, in which bone implants and / or one or more medications are administered to a patient (normal or abnormal human or other mammal) in an effort to alleviate the signs or symptoms or immune response of the disease or condition. Relief may occur before or after the onset of signs or symptoms of the disease or condition. Therefore, treating or treatment includes preventing or preventing a disease or undesirable condition. Furthermore, treating, treatment, preventing, or prevention does not require complete relief of signs or symptoms, does not require a cure, and particularly includes protocols with only marginal effects on the patient.

[0042] As used herein, the term "bone" refers to cortical bone, cancellous bone, or cortical-cancellous bone of autogenous, allogeneic, xenogeneic, or transgenic origin.

[0043] The term "allogeneic graft" refers to a tissue graft obtained from a donor of the same species as the recipient but with a different genetic makeup, as a tissue graft between two people.

[0044] The term "autologous" refers to a body that is derived from or transferred from the same individual, such as an autologous bone marrow graft.

[0045] As used in this article, the term "osteogenic" refers to the ability of a non-osteogenic substance to act as a suitable template or material along which bone can grow.

[0046] As used herein, the term "osteoinducible" refers to the property of being able to recruit cells from the host that have the potential to stimulate new bone formation. Any material that can induce ectopic bone formation in the soft tissues of an animal is considered osteoinducible.

[0047] The term "bone induction" refers to the ability to stimulate the proliferation and differentiation of pluripotent mesenchymal stem cells (MSCs). In intrachondral bone formation, stem cells differentiate into chondroblasts and chondrocytes, forming chondrocyte emulsions (ECMs), which subsequently calcify and remodel into sheet-like bone. In intramembranous bone formation, stem cells directly differentiate into osteoblasts, which form bone through a direct mechanism. Osteogenic growth factors can stimulate bone induction, although some ECM proteins can also drive progenitor cells to develop an osteogenic phenotype.

[0048] The term "osteoconduction" refers to the ability of graft materials to stimulate the attachment, migration, and distribution of blood vessels and osteoblasts. Physical characteristics influencing the osteoconductive activity of grafts include porosity, pore size, and three-dimensional structure. Furthermore, direct biochemical interactions between matrix proteins and cell surface receptors play a major role in the host's response to graft materials.

[0049] The term "osteogenicity" refers to the ability of graft material to independently generate bone. For a graft to possess direct osteogenic activity, it must contain cellular components that directly induce bone formation. For example, allogeneic grafts seeded with activated MSCs can have the potential to directly induce bone formation without recruiting and activating the host MSC population. Since many osteoconductive allogeneic grafts also possess the ability to bind and deliver bioactive molecules, their osteoinductive potential is significantly enhanced.

[0050] As used herein, the term "bone implant" means any bone-derived implant prepared according to embodiments of this disclosure, and is therefore intended to include expressions such as periosteum or bone grafts.

[0051] The term "patient" refers to a biological system to which treatment can be administered. A biological system may include, for example, an individual cell, a group of cells (e.g., a cell culture), an organ, or a tissue. Additionally, the term "patient" may refer to an animal, including but not limited to humans.

[0052] As used herein, “demineralized” means any material produced by removing mineral material from a tissue (e.g., bone tissue). In some embodiments, the demineralized compositions described herein comprise formulations containing less than 5% by weight, 4% by weight, 3% by weight, 2% by weight, or 1% by weight of calcium. Partially demineralized bone (e.g., formulations having more than 5% by weight of calcium but containing less than 100% of the original starting amount of calcium) is also considered to be within the scope of this disclosure. In some embodiments, partially demineralized bone is a formulation containing more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the original starting amount of calcium. In some embodiments, demineralized bone has less than 95% of its original mineral content. In some embodiments, the demineralized bone has less than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of its original mineral content. Demineralization is intended to encompass expressions such as “substantially demineralized,” “partially demineralized,” and “completely demineralized.” In some embodiments, part or all of the surface of the bone may be demineralized. For example, part or all of the surface of an allogeneic graft may be demineralized to a depth of about 100 to about 5000 micrometers or about 150 micrometers to about 1000 micrometers.In some implementations, part or all of the surface of the allogeneic graft can be demineralized to approximately 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500. 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, ​​3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 38 00, 3850, 3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950 to a depth of approximately 5000 micrometers. If necessary, the outer surface of the intervertebral implant may be masked with an acid-resistant coating or otherwise treated to selectively demineralize the unmasked portion of the outer surface of the intervertebral implant, thereby placing the surface demineralized material at discrete locations on the implant.

[0053] As used herein, the term "demineralized bone matrix" refers to any material produced by removing minerals from bone tissue. In some embodiments, as used herein, the DBM composition comprises formulations containing less than 5% by weight, 4% by weight, 3% by weight, 2% by weight, or 1% by weight of calcium.

[0054] As used herein, the term "surface demineralization" refers to a bone-derived element having at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% by weight of its original inorganic mineral content. As used herein, the expression "partially demineralized" refers to a bone-derived element having about 8 to about 90% by weight of its original inorganic mineral content. In some embodiments, partially demineralized refers to a bone-derived element having about 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 to about 90% by weight of its original inorganic mineral content. As used in this article, the term "completely demineralized" means that the bone contains less than 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of its original mineral content.

[0055] As used herein, the terms “comminuted bone,” “powdered bone,” or “bone meal” refer to bone particles with a wide range of average particle sizes, ranging from fine powder to coarse particles or even larger fragments.

[0056] Allogeneic grafts may contain bone fibers. Fibers comprise bone elements with an average length-to-thickness ratio or aspect ratio of about 50:1 to about 1000:1. In their overall appearance, fibrous bone elements can be described as elongated bone fibers, lines, strips, or sheets. Typically, when producing sheets, their edges tend to curl toward each other. The appearance of fibrous bone elements can be substantially linear, or they can be coiled like a spring. In some embodiments, the elongated bone fibers have irregular shapes, including, for example, linear, serpentine, or curved shapes. The elongated bone fibers are preferably demineralized, but some of the original mineral content may be retained when required by a particular embodiment. Fibers loosen when wet because they are porous, and become more entangled as they dry, forming DBM particles as the fibers connect with each other. In some embodiments, they remain sticky even when the fibers are wet.

[0057] As used herein, “non-fibrous” means an element whose average width is substantially less than the average thickness of a fibrous bone element or whose aspect ratio is less than about 50:1 to about 1000:1. For example, allogeneic bone fibers will have a fibrous shape, while non-fibrous materials will not have a fibrous shape but will have shapes such as triangular prisms, spheres, cubes, cylinders, squares, triangles, granules, powders, and other regular or irregular shapes.

[0058] As used herein, “compressed bone fibers” refers to bone fibers formed by applying pressure to bone raw material. The size of the bone used as starting material or raw material can range from relatively small bone fragments to sizes identifiable by their anatomical origin. The bone can be substantially completely demineralized, superficially demineralized, partially demineralized, or non-demineralized. Generally, the median length of a fragment or portion of whole bone raw material can range from about 1 to about 400 mm, about 5 to about 100 mm, the median thickness from about 0.5 to about 20 mm, or about 2 to about 10 mm, and the median width from about 1 to about 20 mm or about 2 to about 10 mm. Forming bone fibers by compression results in longer, more elongated bone fibers than other methods of producing slender bone fibers, thus preserving more of the natural collagen structure. Bone fibers can be produced using a cylinder mill.

[0059] As used in this article, "high porosity" refers to a pore structure that is conducive to cell inward growth and has the ability to promote cell adhesion, proliferation and differentiation.

[0060] As used in this article, “absorbable” means that a material exhibits chemical dissolution when placed in the body of a mammal.

[0061] As used herein, "bioactive agent" or "bioactive compound" refers to a compound or entity that alters, inhibits, activates, or otherwise affects a biological or chemical event. For example, bioactive agents may include, but are not limited to, osteogenic or chondrogenic proteins or peptides, anti-AIDS substances, anticancer substances, antibiotics, immunosuppressants, antiviral substances, enzyme inhibitors, hormones, neurotoxins, opioids, hypnotics, antihistamines, lubricants, sedatives, anticonvulsants, muscle relaxants and anti-Parkinson's substances, anticonvulsants and muscle contractile agents (including channel blockers), miotics and anticholinergics, antiglaucoma compounds, antiparasitic and / or antiprotozoal compounds, modulators of cell-extracellular matrix interactions (including cell growth inhibitors and antiadhesion molecules), vasodilators, inhibitors of DNA, RNA or protein synthesis, antihypertensive drugs, analgesics, antipyretics, steroidal and nonsteroidal anti-inflammatory agents, anti-angiogenic factors, angiogenic factors, antisecretory factors, anticoagulants and / or antithrombotic agents, local anesthetics, ophthalmic drops, prostaglandins, antidepressants, antipsychotic substances, antiemetics, and imaging agents. In some embodiments, the bioactive agent is a drug. In some implementations, the bioactive agent is a growth factor, cytokine, extracellular matrix molecule or fragment or derivative thereof, such as a cell attachment sequence, like RGD.

[0062] The term "flowable" includes compositions that can be applied in an injectable state via a syringe and / or cannula. The composition is flowable when it has a fluid consistency and its viscosity is lower than that of a composition in putty or paste form. Flowable compositions include liquids or fluids (e.g., solutions, suspensions, etc.) or semi-solid compositions (e.g., gels) that are easy to manipulate and can be brushed, sprayed, dripped, injected, shaped, and / or molded at or near a target tissue site. "Flowable" ranges from compositions with low viscosity or aqueous consistency to compositions with high viscosity, such as paste materials. In various embodiments, the flowability of the composition allows it to conform to irregularities, fissures, cracks, and / or voids in bone defect sites (e.g., bone cavities). For example, in various embodiments, the composition can be used to fill one or more voids in an osteolytic lesion.

[0063] The term "injectable" refers to the mode of administration of the composition. The composition can be administered in a variety of ways, such as via syringe and / or cannula. For example, the composition can be administered parenterally, such as via anterior lumbar intervertebral body administration for fusion, or via posterior lumbar intervertebral body administration for fusion, or via transforaminal lumbar vertebral body administration for fusion, other intraspinal injections, or other local administrations.

[0064] The terms “hydrate,” “hydration,” “hydratable,” “hydrating,” or “hydrated” refer to the addition of a certain amount of fluid to bone material to increase the amount of water content in the composition, thereby making it flowable or injectable.

[0065] The term "dehydrated" or "dehydrated" refers to a composition containing a small amount of residual moisture or containing no moisture and may be in the form of a dry composition. Based on the total weight of the composition, a dehydrated composition may have a moisture content of about 0 to about 10%. In some embodiments, a fluid may be added to the composition to hydrate it during dehydration. Dehydrated compositions include lyophilized or freeze-dried compositions.

[0066] The term "bone marrow aspirate" or "BMA" refers to the collection of bone marrow fluid from a patient using a syringe and needle. Bone marrow aspirate is a fluid containing a heterogeneous mixture of stem cells and progenitor cells, platelets, and leukocytes. Bone marrow aspirate can be collected from various sources within the body, including but not limited to the iliac crest.

[0067] Reference will now be made to certain embodiments of this disclosure. This disclosure is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of this disclosure as defined by the appended claims.

[0068] The following headings are not intended to limit this disclosure in any way; embodiments under any heading may be used in conjunction with embodiments under any other heading.

[0069] It will be apparent to those skilled in the art that various modifications and changes can be made to the various embodiments described herein without departing from the spirit or scope of the teachings herein. Therefore, it is contemplated that other modifications and changes to the various embodiments cover the various embodiments within the scope of the teachings of this invention.

[0070] Methods for preparing demineralized bone fibers

[0071] DBM fibers comprising a combination of long and short fiber sizes for enhanced hydration, apparatus for mixing and conveying DBM fibers, and method for preparing DBM fibers are provided.

[0072] During the grinding process, cortical bone fibers are typically formed into various shapes and sizes. A method is provided for preparing demineralized bone matrix particles, which can be tailored to specific sizes to achieve optimal rehydration and ejection properties for use in injectable compositions to repair bone. Figure 1A As shown, in one embodiment, a method is provided for preparing demineralized bone particles of a size sufficient to pass through a chamber or syringe cannula. The method includes screening for 22 large bone fibers that are longer than long bone fibers having a target diameter of approximately 4.0 mm. In some embodiments, the large bone fibers can be screened after grinding and crushing to select those fibers of the desired size. In some embodiments, the bone fibers can be screened using 0.5 mm sieves, 1 mm micrometer sieves, 2 mm sieves, 3 mm sieves, 4 mm sieves, 5 mm sieves, 6 mm sieves, 7 mm sieves, and / or 8 mm sieves.

[0073] Following screening, in some embodiments, a plurality of long bone fibers 126 are provided having a diameter of about 0.5 mm to about 8.0 mm, about 0.5 mm to about 1 mm, about 1 mm to about 2 mm, about 2 mm to about 3 mm, about 3 mm to about 4 mm, about 4 mm to about 5 mm, about 5 mm to about 6 mm, about 6 mm to about 7 mm, or about 6 mm to about 8 mm. In some embodiments, the diameter of the long fibers ranges from about 0.5 mm to about 4 mm. Figure 2 An embodiment of long bone fibers is shown in the figure.

[0074] As is well known, the most widely used method for describing bone fiber size distribution is the D-value. D10, D50, and D90 are commonly used to represent the midpoint and range of bone fiber size for a given sample. Specifically, the bone fiber size distribution D50, also known as the median length or median of the bone fiber size distribution, is the value for bone fiber lengths below 50% in the cumulative distribution; D10 is the value for 10% of samples below the bone fiber size; and D90 is the value for 90% of samples below the bone fiber size. In some embodiments, the D50 values ​​for long bone fibers described in this disclosure vary between about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and about 0.9 mm.

[0075] In some embodiments, the method includes grinding the remaining 24 selected large bone fibers. The ground bone fibers are then ground into multiple short bone fibers. In some embodiments, multiple short bone fibers 128 are produced with a diameter of about 0.5 mm to about 0.05 mm. In some embodiments, short bone fibers are provided ranging from about 0.5 mm to about 0.4 mm, about 0.4 mm to about 0.3 mm, about 0.3 mm to about 0.2 mm, about 0.2 mm to about 0.1 mm, about 0.1 mm to about 0.09 mm, about 0.09 mm to about 0.08 mm, about 0.08 mm to about 0.07 mm, 0.07 mm to 0.06 mm, or about 0.06 mm to about 0.05 mm. In some embodiments, the short bone fibers range from about 0.5 mm to about 0.106 mm. In some embodiments, the D50 value of the short bone fibers described in this disclosure varies between about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08 and about 0.09 mm.

[0076] Figure 3 An embodiment of short bone fibers is illustrated. In some embodiments, the method includes providing long bone fibers 26 and providing short bone fibers 28. In some embodiments, the method further includes mixing a plurality of long bone fibers with a plurality of short bone fibers to form a mixture, and demineralizing the mixture to form a demineralized bone matrix (DBM) slurry 30.

[0077] exist Figure 4 One embodiment of the demineralized bone matrix slurry is shown as 130; the demineralized bone matrix slurry is gently filled or compacted 32 into a mold 150 to form particles 134, for example, as... Figure 5A , 5B As shown in 6A and 6B; remove 34DBM particles from the mold, for example, as... Figure 7A and 7B As shown; and freeze-drying in sheet form or as individual granules 36, for example, as Figure 8A , 8BAs shown in 8C.

[0078] Figure 1B Another embodiment of the method for manufacturing demineralized bone particles with a size sufficient to pass through a chamber or syringe is provided. Figure 1B The method shown involves screening out 22 large bone fibers, which are longer than long bone fibers with a target diameter of approximately 4.0 mm. In some embodiments, the large bone fibers can be screened after grinding and crushing to select those fibers with the desired size. In some embodiments, the bone fibers can be screened through 0.5 mm sieves, 1 mm micron sieves, 2 mm sieves, 3 mm sieves, 4 mm sieves, 5 mm sieves, 6 mm sieves, 7 mm sieves, and / or 8 mm sieves. The remaining large bone fibers screened out can be ground into 24 short bone fibers with a diameter of 0.5-0.1 mm. In some embodiments, short bone fibers are provided, ranging from about 0.5 mm to about 0.4 mm, about 0.4 mm to about 0.3 mm, about 0.3 mm to about 0.2 mm, about 0.2 mm to about 0.1 mm, about 0.1 mm to about 0.09 mm, about 0.09 mm to about 0.08 mm, about 0.08 mm to about 0.07 mm, 0.07 mm to 0.06 mm, or about 0.06 mm to about 0.05 mm. In some embodiments, the short bone fibers range from about 0.5 mm to about 0.106 mm. In some embodiments, the D50 value of the short bone fibers described in this disclosure varies between about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08 and about 0.09 mm.

[0079] Figure 1B The method shown includes providing long bone fibers 26 and short bone fibers 28. In some embodiments, the method further includes mixing a plurality of long bone fibers with a plurality of short bone fibers to form a mixture, and demineralizing the mixture to form a demineralized bone matrix (DBM) slurry 30. In some aspects, the method includes discharging the DBM slurry 31, subsequently adding the slurry to a mold 32 to form granules, freeze-drying the granules 33, and then removing the granules from the mold 35.

[0080] In some embodiments, the mixture of long and short bone fibers is demineralized in a slurry. In one embodiment, the slurry is made from long and short fibers, as discussed herein, which have been ground to a suitable particle size. For example, water or saline solutions can be used to form the slurry. In some embodiments, when preparing a slurry containing DBM bone fibers, it may be necessary to select a solution that does not cause or prevents the DBM bone fibers from swelling, as uneven swelling can alter the characteristics of the DBM bone fiber mixture. For example, ethanol solutions can be used to produce a slurry containing DBM bone fibers. Other types of solutions, such as hypertonic saline solutions or sugar solutions, may also be advantageous in achieving this result.

[0081] In one demineralization process, mixed long and short bone fibers undergo an acid demineralization step, followed by a degreasing / sterilization step. The bone fibers are immersed in acid to achieve demineralization. Acids that can be used in this step include inorganic acids, such as hydrochloric acid; and organic acids, such as formic acid, acetic acid, peracetic acid, citric acid, propionic acid, etc. The depth to which the demineralized material penetrates the bone surface can be controlled by adjusting the treatment time, the temperature of the demineralized solution, the concentration of the demineralized solution, and the intensity of agitation during the treatment. Therefore, in various embodiments, DBM bone fibers can be completely demineralized, partially demineralized, or surface-demineralized. Suitable demineralization techniques are described in U.S. Serial No. 15 / 906,788, filed February 27, 2018, and published as U.S. Patent Publication No. 20180185548, assigned to Warsaw Orthopedic, Inc., Warsaw, IN, USA. The entire contents of this publication are incorporated herein by reference.

[0082] Rinse demineralized bone fibers with sterile water and / or a buffer solution to remove residual acid and thus raise the pH. A suitable degreasing / disinfecting solution is an aqueous ethanol solution, as ethanol is a good solvent for lipids and water is a good hydrophilic carrier, allowing the solution to penetrate deeper into the bone particles. The aqueous ethanol solution also disinfects bone by killing nutrient microorganisms and viruses. Typically, at least about 10 to 40% by weight of water (i.e., about 60 to 90% by weight of the degreasing agent, such as ethanol) should be present in the degreasing and disinfecting solution to achieve optimal lipid removal and disinfection over a given time period. A suitable concentration range for the degreasing solution is about 60 to 85% by weight of ethanol or about 70% by weight of ethanol. In some embodiments, a suitable concentration range of the degreasing solution is about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 to about 85% by weight of ethanol.

[0083] In some embodiments, the demineralized bone matrix slurry is added to a mold and dried inside or outside the mold. In some embodiments, the demineralized bone matrix slurry is compacted into the mold using a pressure of about 4 to about 65 Newtons. In some embodiments, the DBM slurry is manually compacted using tools such as a scraper 152. Figure 5A As shown. In some embodiments, the DBM slurry is filled into the mold via roller 154 and passed through... Figure 6A and Figure 6A The compression block 156 in the middle is compacted. In some embodiments, no compression is applied after the DBM slurry is filled into the mold, and the demineralized bone matrix slurry is discharged through a screen until excess fluid is discharged from the slurry.

[0084] In some embodiments, a DBM slurry is added to a mold to form demineralized bone matrix particles, since the mold has a particle shape. The particles are removed from the mold and freeze-dried. In some embodiments, the particles are freeze-dried and then removed from the mold. In some embodiments, the method further includes rehydrating the freeze-dried demineralized bone matrix particles with a physiologically acceptable liquid to form an injectable demineralized bone matrix. In some embodiments, the liquid includes bone marrow aspirate, saline, sterile water, blood for injection, phosphate-buffered saline, dextran, Ringer's lactate solution, or combinations thereof.

[0085] In some embodiments, the liquid used for hydrating DBM particles may include sterile water, saline, phosphate-buffered saline (PBS), hyaluronic acid, cellulose ethers (e.g., carboxymethyl cellulose), water, collagen, gelatin, autoclaved bone meal, osteoconductive carriers, whole blood, blood fractions, concentrated bone marrow aspirate, and mixtures thereof. Non-limiting examples of blood components include serum, plasma, platelet-rich plasma, concentrated platelet-rich plasma, anemic platelet plasma, and concentrated anemic platelet plasma.

[0086] In some embodiments, the fluid-to-particle ratio may be from about 0.5:1 v / v to about 2:1 v / v. In some embodiments, the fluid-to-particle ratio may be from about 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1:1.1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1 to about 2:1 v / v. In some embodiments, the particles may be hydrated with bone marrow aspirate at a 1:1 v / v ratio.

[0087] For example, Figure 11The fluid is blood, and the blood is added to the syringe to mix with dried DBM particles. In some embodiments, the blood volume includes about 3 mL to about 9 mL. In some embodiments, the fluid volume includes about 6 cc to about 12 cc, for example... Figure 12 As shown. In some embodiments, the injection force for injectable demineralized bone matrix is ​​about 10 lbs, about 33 lbs, about 15 lbs to about 33 lbs, or about 24 lbs to about 27 lbs. In some embodiments, the hydrated DBM particles in the syringe (e.g., as shown) are tested by injection force. Figure 11 Processing characteristics (as shown) Figure 12 (As shown). In some embodiments, the injection force decreases as the hydration volume ratio in the syringe increases. In some embodiments, the graft volume comprises DMB particles and liquid. In some embodiments, the injection force increases as the volume of hydrated DBM particles increases.

[0088] In some embodiments, based on the total weight of bone fibers or based on the total weight of demineralized bone matrix particles, the demineralized bone matrix particles comprise about 0 wt.% to about 33 wt.%, about 0.1 wt.% to about 33 wt.%, about 0.5 wt.% to about 33 wt.%, about 1 wt.% to about 33 wt.%, about 5 wt.% to about 33 wt.%, about 10 wt.% to about 33 wt.%, about 15 wt.% to about 33 wt.%, about 20 wt.% to about 33 wt.%, about 25 wt.% to about 33 wt.%, or about 30 wt.% to about 33 wt.%, and about 66 wt.% to about 99.9 wt.%, about 70 wt.% to about 99.9 wt.%, about 75 wt.% to about 99.9 wt.%, or about 80 wt.% to about 99.9 wt.%, respectively, of short demineralized bone fibers. In some embodiments, compaction includes manually applying pressure to the mixture using a trowel or roller and a compression block. In some embodiments, no compression is applied. In some embodiments, the ratio of long demineralized bone fibers to short demineralized bone fibers is about 100:0 to about 90:10, about 80:20, about 70:30, about 66:33, or about 60:40, respectively. In some embodiments, the dried DBM particles are aseptically packaged into syringes. In some embodiments, the diameter of the dried DBM particles ranges from about 7 mm to about 10 mm, about 7 mm to about 8 mm, about 8 mm to about 9 mm, about 9 mm to about 10 mm, about 7 mm to about 10 mm, or about 8 mm to about 10 mm. In some embodiments, the height of the dried DBM particles ranges from 2 mm to about 4 mm, about 2 mm to about 3 mm, or about 3 mm to about 4 mm.

[0089] In some embodiments, a bone material is provided comprising a mixture of a plurality of long bone fibers with a diameter of about 0.5 mm to about 8.0 mm and a plurality of short bone fibers with a diameter of about 0.5 mm to about 0.05 mm. In some embodiments, the bone material is demineralized and molded into lyophilized demineralized bone matrix in granular form. In some embodiments, the bone material comprises demineralized bone powder with a diameter of about 0.106 mm and less. In some embodiments, the bone material comprises DBM particles. In some embodiments, the DBM particles have substantially similar dimensions, about 1 mm to about 15 mm, about 1 mm to about 3 mm, about 3 mm to about 6 mm, about 6 mm to about 10 mm, and about 10 mm to about 15 mm. In some embodiments, each DBM particle has a microporosity and the diameter of each micropore is about 0.01 to about 10 micrometers. In some embodiments, the diameter of each micropore of the DBM particle is about 0.1 to about 10 micrometers or about 1 to about 10 micrometers. In some embodiments, the diameter of each micropore can be from about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9 to about 10 micrometers. In some embodiments, DBM particles have a microporosity percentage of from about 10% to about 100%, or from about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% to about 100%. In some embodiments, DBM particles have a substantially similar size, shape, and porosity.

[0090] In some embodiments, the method includes hydrating the bone material with a liquid to an injectable viscosity of about 50 Pa-s to about 3000 Pa-s. In some embodiments, the demineralized bone powder is about 15 wt.% to about 33 wt.% based on the total weight of the demineralized bone matrix particles.

[0091] In some embodiments, the demineralized bone matrix particles comprise about 0.01 wt.% to about 33 wt.% of short demineralized bone fibers and about 80 wt.% to about 99.99 wt.% of long demineralized bone fibers, based on the total weight of bone fibers or based on the total weight of demineralized bone matrix particles.

[0092] Figure 9 An embodiment of a texture analyzer is illustrated. In some embodiments, the texture analyzer measures processing characteristics, including the processed texture. In some embodiments, DBM particles are placed in the texture analyzer to test their processed texture. In some embodiments, the material consistency of the DBM particles is measured by injection force. In some embodiments, both the processed texture and the material consistency of the DBM particles are tested.

[0093] Figure 10 One embodiment is shown where dried DBM fiber particles 134 are loaded into syringe 136. In some embodiments, the DBM particles are aseptically packaged into a first chamber constituting a first syringe. Liquid 138 disposed in a second chamber constituting a second syringe is added to the first syringe, as... Figure 11 As shown. Figure 11 An embodiment is also shown in which the plunger in the first syringe has moved or pumped 20 times between the retracted and extended positions. In some embodiments, the first chamber may receive 5, 10, 15, 20, or 30 pumps after liquid is added. Figure 12 The hydration of DBM particles was correlated with the injection force applied to the DBM particles.

[0094] Bone materials

[0095] DBM compositions and methods that allow for bone formation, bone induction, and / or bone conduction are provided. DBM compositions, devices, and methods that allow for bone formation, bone induction, and / or bone conduction are also provided. In some embodiments, the provided DBM compositions, devices, and methods are made from binder-free bone materials. DBM compositions, devices, and methods that facilitate the hydration of demineralized bone matrix are also provided.

[0096] Compositions and methods for hydrating bone materials with liquids are provided, the bone material comprising ground and demineralized bone fiber DBM particles, the demineralized fiber DBM particles having no binder disposed within or on the DBM particles. In some embodiments, the bone material is freeze-dried. In some embodiments, the demineralized bone fibers are cylindrically ground and have a ribbon shape and increased surface area. In some embodiments, the ground and freeze-dried demineralized bone fiber DBM particles are cylindrically ground fibers having a ribbon shape, increased surface area, and coiled portions. In some embodiments, the ground and freeze-dried demineralized bone fiber DBM particles comprise autologous bone grafts or allogeneic bone grafts. In some embodiments, the diameter of the bone fibers is from about 100 μm to about 2 mm. In some embodiments, the length of the bone fibers is from about 0.5 mm to about 50 mm. In some embodiments, the average length of the bone fibers is from about 0.5 cm to about 10 cm. In some embodiments, the aspect ratio of the fibers is about 50:1 to about 1000:1, about 50:1 to about 950:1, about 50:1 to about 750:1, about 50:1 to about 500:1, about 50:1 to about 250:1, about 50:1 to about 100:1, about 10:1 to about 50:1, or about 5:1 to about 10:1. In some embodiments, the liquid used for fiber hydration includes blood, water, saline, or combinations thereof. In some embodiments, the liquid used for fiber hydration is mixed with DBM particles of milled and demineralized bone fibers, which are then freeze-dried without a binder to form moldable freeze-dried demineralized bone fibers.

[0097] In some embodiments, the bone fibers have a band-like shape and an increased surface area of ​​approximately 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%, 18.5%, and 19% compared to bone fragments or bone powder. 0%, 19.5%, 20.0%, 20.5%, 21.0%, 21.5%, 22.0%, 22.5%, 23.0%, 23.5%, 24.0%, 24.5%, 25.0%, 25.5%, 26.0%, 26.5%, 27.0%, 27.5%, 28.0%, 28.5%, 29.0%, 29.5%, 30.0%, 30.5%, 31.0% 31.5%, 32.0%, 32.5%, 33.0%, 33.5%, 34.0%, 34.5%, 35.0%, 35.5%, 36.0%, 36.5%, 37.0%, 37.5%, 38.0%, 38.5%, 39.0%, 39.5%, 40.0%, 40.5%, 41.0%, 41.5%, 42.0%, 42.5%, 43.0%, 43.5%, 44.0%, 44.5%, 45.0%, 45.5%, 46.0%, 46.5%, 47.0%, 47.5%, 48.0%, 48.5%, 49.0%, 49.5%, 50.0%, 55.0%, 60.0%, 65.0%, 70.0%, 75.0%, 80.0%, 85.0%, 90.0%, 95.0% to approximately 100.0%.

[0098] In some embodiments, an apparatus for mixing bone material with a liquid is provided. The apparatus includes a chamber having a proximal and a distal end, and bone material disposed within the chamber comprising DBM particles of ground and lyophilized demineralized bone fibers; and a plunger having at least a portion slidably disposed within the proximal end of the chamber and configured to dispense the liquid-mixed bone material from the distal end of the chamber when the plunger is in an extended position. In some embodiments, the chamber comprises a syringe barrel. In some embodiments, the DBM particles of ground and lyophilized demineralized bone fibers do not contain a binder. In some embodiments, the DBM particles of ground and lyophilized demineralized bone fibers comprise cylindrically ground fibers with coiled portions. In some embodiments, the DBM particles of ground and lyophilized demineralized bone fibers comprise autologous or allogeneic bone grafts. In some embodiments, the diameter of the bone fibers is from about 100 μm to about 2 mm. In some embodiments, the length of the bone fibers is from about 0.5 mm to about 50 mm. In some embodiments, the average length of the bone fibers is from about 0.5 cm to about 10 cm. In some embodiments, the aspect ratio of the fibers is from about 50:1 to about 1000:1, from about 50:1 to about 950:1, from about 50:1 to about 750:1, from about 50:1 to about 500:1, from about 50:1 to about 250:1, from about 50:1 to about 100:1, from about 10:1 to about 50:1, or from about 5:1 to about 10:1. In some embodiments, the liquid used for fiber hydration includes blood, water, saline, or combinations thereof. In some embodiments, the liquid is mixed with lyophilized demineralized bone fibers to form moldable lyophilized demineralized bone fibers. In some embodiments, the liquid is mixed with the lyophilized demineralized bone fibers using negative pressure generated by a plunger in a chamber. In some embodiments, the distal end of the chamber includes a removable cap.

[0099] In some embodiments, a method for hydrating bone material with a liquid is provided. The method includes mixing a liquid with bone material comprising ground and lyophilized demineralized bone fiber (DBM) particles in a device having a first chamber having a proximal and a distal end, the bone material being disposed within the first chamber; a first plunger having at least a portion slidably disposed within the proximal end of the first chamber; a second chamber having a proximal and a distal end, the liquid being disposed within the second chamber, the liquid being configured to hydrate the ground and lyophilized demineralized bone fiber (DBM) particles; a second plunger having at least a portion slidably disposed within the proximal end of the second chamber; and a connector fluidly connecting the distal end of the first chamber to the distal end of the second chamber, wherein the second plunger, when moved to an extended position, causes liquid flow to hydrate the ground and lyophilized demineralized bone fiber (DBM) particles in the first chamber.

[0100] Compositions and methods for use with bone materials are provided, the bone material comprising cylindrically ground and demineralized bone fiber DBM particles, the cylindrically ground and demineralized bone fiber DBM particles having no binder disposed within or on the DBM particles. In some embodiments, the bone material comprises cortical bone, cancellous bone, cortical-cancellous bone, or mixtures thereof. In some embodiments, the bone material is obtained from autologous bone, allogeneic bone, xenogeneic bone, or mixtures thereof. In some embodiments, the DBM particles are freeze-dried and shaped. In some embodiments, the freeze-dried DBM particles are cubic, square, triangular, rectangular, circular, disc-shaped, or cylindrical. In some embodiments, the freeze-dried DBM particles are disc-shaped, and the disc has a reservoir configured to contact a liquid. In some embodiments, the freeze-dried DBM particles are cylindrical. In some embodiments, the DBM particles have a plurality of longitudinally extending channels through the center of the cylindrical bone material to allow fluid hydration of the bone material. In some embodiments, the DBM particles have a plurality of longitudinally extending channels through the exterior of the cylindrical bone material to allow fluid hydration of the bone material. In some implementations, the cylindrical bone material also includes multiple longitudinally extending channels through the exterior of the bone material to allow fluid hydration of the bone material.

[0101] Compositions and methods for implantable bone grafts are provided, the bone grafts comprising fibers obtained from allogeneic bone, the fibers comprising hook-shaped portions configured to interlock with each other to form DBM particles, wherein the composition does not contain an adhesive.

[0102] Typically, when bone is processed into granules or fibers, it is electrostatically charged and not coherent or adhesive. Processed bone is usually contained within an external structure (i.e., a pouch or covering) or mixed with a carrier or adhesive to provide a bonding structure. This external structure or carrier must be removed from the patient's body during implantation, which can affect the graft's osteoinductive potential.

[0103] In some embodiments, bone fiber DBM particles without additional carriers comprise bone treated in a way that provides cohesive forces between fibers without additional inclusions or binders. The bone diaphysis is milled to produce crimped bone fibers, which are then demineralized and freeze-dried. The fiber shape changes during the drying process, resulting in physical entanglement and surface-to-surface interactions between adjacent fibers. The entanglement / interaction of the fibers determines the adhesiveness of the final product. Therefore, this disclosure provides a fibrous bone material with dimensions and shapes that provide increased surface area and the ability to mechanically interlock with each other to form implantable DBM particles.

[0104] The compositions disclosed herein are used in effective bone graft products. Bone graft materials are reabsorbed / remodeled and replaced by host bone during the healing process. In some embodiments, the bone materials disclosed herein contain additional additives, such as synthetic ceramics and / or biodegradable polymers, which produce high concentrations of calcium, phosphate, and silicon ions, as discussed herein, acting as nests for re-bone formation. Because biodegradable polymers degrade faster than ceramics, more and more bone-inducing DBM particles are exposed. Slower-absorbed ceramics can serve as a solid surface for stem cells and osteoblasts to attach to new bone and begin laying new bone.

[0105] The DBM particles of this invention possess excellent flexibility and compressive strength. They also exhibit osteoinductive activity and retain demineralized bone matrix. These properties make them an excellent alternative to bone grafts because they do not rupture, crack, or deform upon implantation.

[0106] The implantable composition may be a combination of bone matrix fibers from allogeneic bone and fibers from allogeneic bone material. The fibers of the allogeneic bone material comprise non-fibrous demineralized bone matrix particles embedded within or dispersed on the fibers of the allogeneic bone material. The ratio of fibers from the allogeneic demineralized bone matrix to fibers from the allogeneic material ranges from about 20:80 to about 70:30. In one embodiment, the ratio of fibers from the allogeneic material to fibers from the allogeneic material ranges from about 40:60 to about 60:40. In one embodiment, the ratio of fibers from the allogeneic demineralized bone matrix to fibers from the allogeneic material is about 50:50.

[0107] In some embodiments, the demineralized bone material comprises non-fibrous particles. In some embodiments, the particles are powders, microspheres, sponges, pastes, gels, and / or granules. In one embodiment, the particles are powders.

[0108] In some embodiments, the demineralized bone material fibers comprise about 1 to about 70 micrometers or about 125 to about 250 micrometers. In some embodiments, the demineralized bone material fibers include approximately 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248 and / or 250 micrometers. In some embodiments, the bone fibers comprise a length of about 100 micrometers to about 2 millimeters. In some embodiments, the length of the bone fibers is about 0.5 cm to about 10 cm, about 1 cm to about 8 cm, about 3 cm to about 5 cm, about 0.5 mm to about 50 mm, about 1.0 mm to about 25 mm, or about 5 mm to about 10 mm. The fibers comprise a diameter of about 100 micrometers to about 4 millimeters.

[0109] The fibers are milled in a manner that provides increased surface area in a compact shape and size. In some embodiments, the fibers include crimped shapes such that the diameter of the crimped fibers is between about 50 micrometers and about 3 millimeters, and the diameter of the fibers in a flattened configuration is between about 125 micrometers and about 5 millimeters. In some embodiments, the fibers include crimped shapes such that the diameter of the crimped fibers is between about 100 micrometers and about 1 millimeter, and the diameter of the fibers in a flattened configuration is between about 250 micrometers and about 2 millimeters.

[0110] In various embodiments, the aspect ratio of the fiber length to width may be about 50:1 to about 1000:1, about 50:1 to about 950:1, about 50:1 to about 750:1, about 50:1 to about 500:1, about 50:1 to about 250:1, about 50:1 to about 100:1, about 10:1 to about 50:1, or about 5:1 to about 10:1. In other embodiments, the aspect ratio of the fiber length to width is about 4:1, 17:1, or 23:1.

[0111] The composition has very low immunogenicity and good biocompatibility to fill bone voids.

[0112] Typically, favorable osteoinductive DBM materials can be prepared by decalcification of cortical and / or cancellous bone fibers, usually by acid extraction. The fibers can be milled, for example, using a milling machine. The acid extraction process can be performed to retain collagen, non-collagenous proteins, and growth factors together in the solid fibers. Methods for preparing bioactive demineralized bone are described, for example, in U.S. Patent Nos. 5,073,373; 5,484,601; and 5,284,655. DBM products are also commercially available, including, for example, from sources such as Regeneration Technologies, Inc. (Alachua, Florida) and The American Red Cross (Arlington, Virginia). Similar techniques can be used to prepare osteoconductive bone fibers, which have been modified or supplemented to remove or inactivate (e.g., by crosslinking or otherwise denaturing) the osteoinductive components in the bone matrix. The osteoinductive and / or osteoconductive DBM materials used in this disclosure may be derived from human donor tissue, particularly in relation to implantable devices intended for use in human subjects.

[0113] Regarding the fiber content of DBM particles based on dry weight, bone fiber material may constitute about 5% to about 100% by weight, about 20% to about 80% by weight, or about 25% to about 75% by weight of the composition.

[0114] In some embodiments, the average length-to-thickness ratio, or aspect ratio, of the xenograft bone fibers is from about 50:1 to about 1000:1. In overall appearance, the bone fibers can be in the form of bands, lines, narrow strips, or sheets. The elongated bone fibers can appear substantially linear, or they can be coiled into a spring-like shape. In some embodiments, the bone fibers have linear and coiled portions. In some embodiments, the bone fibers have irregular shapes, including, for example, linear, serpentine, or curved shapes. In some embodiments, the fibers can be curled at the edges to have a substantially semi-circular cross-section. In some embodiments, the fibers can be fully or partially helical, coiled, or spiral. The elongated bone fibers can be demineralized, but some of the original mineral content can be retained when required by a particular embodiment. Bone graft fibers may also include mineralized bone material.

[0115] Bone fibers are elongated and coiled to increase the surface area of ​​the strips. The coiled fibers may include abrasion portions along the edges to facilitate interaction with other bone fibers. In some embodiments, the coiled fibers are ground to have hook-shaped portions along the fiber edges, which are configured to engage with other fibers. The hook-shaped portions may engage other hook-shaped portions, abrasion portions, straightened portions, or coiled portions of other fibers. The hook-shaped and abrasion portions of the fibers, along with the coiled shape, provide entanglement between the fibers, allowing the fibers to form DBM particles without the need for a carrier or binder.

[0116] Bone fiber size and shape can be produced in a variety of ways, such as by barrel milling. One such embodiment of a suitable barrel mill is the Osteobiologic Milling Machine, as described in U.S. Patent Publication No. 2012 / 0160945, assigned to Warsaw Orthopaedic Corporation and incorporated herein by reference in its entirety. However, it is contemplated that bone fibers may be milled alternatively using pliers, cutters, rollers, rotary files, or reciprocating blade mills.

[0117] In some embodiments, the bone material may be combined with non-bone material additives after demineralization and / or lyophilization and before implantation. For example, the bone material may be combined with a biodegradable polymer. Biodegradable polymers exhibit solubility when placed in a mammalian body and may be hydrophilic (e.g., collagen, hyaluronic acid, polyethylene glycol). According to this disclosure, synthetic polymers are suitable because they are biocompatible and can be obtained in a range of copolymer ratios to control their degradation.

[0118] In some embodiments, hydrophobic polymers (e.g., poly(lactide-co-glycolic acid), polyanhydride) may be used. Alternatively, a combination of hydrophilic and hydrophobic polymers may be used in the bone graft compositions of this disclosure.

[0119] Exemplary materials may include biopolymers and synthetic polymers, such as human skin, human hair, bone, collagen, fat, thin cross-linked sheets containing fibers and / or fibers and fragments, polyethylene glycol (PEG), chitosan, alginate sheets, cellulose sheets, hyaluronic acid sheets, and copolymer blends of poly(lactide-co-glycolic acid) PLGA.

[0120] In some embodiments, the particles disclosed herein may also include other biocompatible and bioabsorbable substances. These materials may include, for example, natural polymers such as proteins and peptides, glycosaminoglycans, proteoglycans, elastin, hyaluronic acid, dermatan sulfate, gelatin, or mixtures or complexes thereof. Synthetic polymers may also be incorporated into bone graft complexes. These include, for example, biodegradable synthetic polymers such as polylactic acid, polyglycolic acid lactide, polylactic acid-polyglycolic acid copolymer (“PLGA”), polycaprolactone (“PCL”), poly(dioxane), poly(trimethylene carbonate) copolymer, polygluconate, poly(propylene fumarate), poly(ethylene terephthalate), poly(butylene terephthalate), polyethylene glycol, polycaprolactone copolymer, polyhydroxybutyrate, polyhydroxyvalerate, tyrosine-derived polycarbonate, and any random or (multi)block copolymers, such as binary copolymers, ternary copolymers, and quaternary copolymers, which may be polymerized from monomers associated with the previously listed homopolymers and copolymers.

[0121] The molecular weight of the biocorrosive polymer can be from about 1,000 to about 30,000 Daltons (Da). In various embodiments, the molecular weight of the polymer can be from about 2,000 to about 10,000 Da. In some embodiments, the molecular weight of the polymer can be from about 2,000 to 4,000 Da or from about 3,000 to 4,000 Da. In some embodiments, the molecular weight of the bio-erodible polymer can be 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, or about 30,000 Da.

[0122] In some embodiments, the bio-erosible polymer is collagen. Collagen exhibits excellent tissue compatibility without antibody formation or transplant rejection. Any suitable collagen raw material can be used, including known collagen raw materials, or those disclosed in U.S. Patent Application Serial No. 12 / 030,181, filed February 12, 2008 (the entire contents of which are incorporated herein by reference). Various collagen raw materials can be used alone or in combination with other materials.

[0123] The insoluble collagen raw materials used in this disclosure can be derived from natural tissue sources (e.g., xenogeneic, allogeneic, or autologous relative to the recipient or other patient) or recombinantly prepared. Collagen can be subdivided into several different types based on its amino acid sequence, carbohydrate content, and the presence or absence of disulfide crosslinks. Type I and Type III collagen are the two most common collagen subtypes and are used in this disclosure. Type I collagen is found in skin, tendons, and bone, while Type III collagen is primarily found in skin. The collagen used in the compositions of this disclosure can be obtained from skin, bone, tendons, or cartilage and purified using methods well known in the art and industry. Alternatively, collagen can be purchased from commercial sources.

[0124] Collagen can be telopeptide collagen and / or telopeptide collagen. Further, one or both of non-fibrous and fibrous collagen can be used. Non-fibrous collagen is collagen that has been dissolved and not reconstituted into its natural fibrillary form.

[0125] Suitable collagen products are commercially available, including, for example, those from Kensey Nash (Eston, PA), which produces a fibrous collagen called Semed F from bovine hide. Bovine hide-derived collagen raw materials are also produced by Integra Life Sciences Holdings, Inc. (Princeborough, NJ). Naturally derived or recombinant human collagen raw materials are also suitable for this disclosure. For example, recombinant human collagen products are available from Fibrogen, Inc. (San Francisco, CA).

[0126] In some embodiments, the fibers can be combined with a synthetic ceramic that effectively provides a scaffold for bone growth and is fully bioresorbable and biocompatible. The synthetic ceramic should provide locally high concentrations of calcium, phosphate, and silicon ions as a focal point for new bone formation. Using such a resorbable ceramic offers numerous advantages over alternative conventional materials. For example, it eliminates the need for post-treatment surgery for removal and degrades within the body into a biocompatible, bioresorbable product.

[0127] In some embodiments, the synthetic ceramics disclosed herein may be selected from one or more materials comprising calcium phosphate ceramics or silicon ceramics. Bioglasses such as calcium silicate-based bioglasses, calcium silicate phosphate, tricalcium phosphate (TCP), biphasic calcium phosphate, calcium sulfate, hydroxyapatite, coral hydroxyapatite, silicon carbide, silicon nitride (Si3N4), and biocompatible ceramics may be used. In some embodiments, the ceramic is tricalcium phosphate or biphasic calcium phosphate and silicon ceramics. In some embodiments, the ceramic is tricalcium phosphate.

[0128] In some embodiments, the ceramic is a combination of calcium phosphate ceramic and silicon ceramic. In some embodiments, the calcium phosphate ceramic is reabsorbable biphasic calcium phosphate (BCP) or reabsorbable tricalcium phosphate (TCP), most preferably reabsorbable TCP.

[0129] The biphasic calcium phosphate can have a tricalcium phosphate:hydroxyapatite weight ratio of about 50:50 to about 95:5, about 70:30 to about 95:5, about 80:20 to about 90:10, or about 85:15. The mineral material can be granular particles with an average particle size of about 0.2 to 5.0 mm, about 0.4 to 3.0 mm, or about 0.4 to 2.0 mm.

[0130] The ceramics disclosed herein can also be oxide ceramics, such as alumina (Al2O3) or zirconium oxide (ZrO2), or composite combinations of oxides and non-oxides, such as silicon nitride.

[0131] In some embodiments, an adhesive may be added to the DBM particles of the DBM fibers before implantation, after the DBM particles have formed. However, in some embodiments, the DBM particles of the DBM fibers do not contain an adhesive and remain together without the use of an adhesive. Examples of suitable binders that may be optionally included after DBM particle formation include, but are not limited to: (i) polyhydroxy compounds, such as acyclic polyols, non-reducing sugars, sugar alcohols, sugar acids, monosaccharides, disaccharides, water-soluble or water-dispersible oligosaccharides, polysaccharides, and known derivatives of the above substances. Specific polyhydroxy compounds include 1,2-propanediol, glycerol, 1,4-butanediol, trimethylolethane, trimethylolpropane, erythritol, pentaerythritol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, propylene glycol, and dipropylene glycol; polyoxyethylene-polyoxypropylene copolymers, such as those known and commercially available under the trade names Pluronic and Emkalyx; polyoxyethylene-polyoxypropylene block copolymers, such as those known and commercially available under the trade name Poloxamer; and alkylphenol hydroxyl polyoxyethylene, for example, those known under the trade name... Triton's known and commercially available types of polyoxyalkylene glycols, such as polyethylene glycol, xylitol, sorbitol, mannitol, galactitol, arabinose, xylose, ribose, arabinitol, inositol, fructose, galactose, glucose, mannose, sorbitol, sucrose, maltose, lactose, maltitol, lactitol, stachyose, maltopentose, cyclomaltohexaose, carrageenan, agar, dextran, alginate, guar gum, tragacanth gum, locust bean gum, gum arabic, xanthan gum, amylose, and any mixtures thereof.

[0132] The carrier or binder may also include hydrogels, such as hyaluronic acid, dextran, polyoxyethylene, and Prönkel block copolymers of polypropylene. Suitable polyhydroxy compounds include compounds such as acyclic polyols, non-reducing sugars, sugar alcohols, sugar acids, monosaccharides, disaccharides, water-soluble or water-dispersible oligosaccharides, polysaccharides, and known derivatives of the above substances. Exemplary carriers include glyceryl monolaurate dissolved in glycerol or mixtures of glycerol and propylene glycol in a weight ratio of 4:1 to 1:4. Curable materials can be used, and they can be set in situ or prior to implantation. Optionally, xenogeneic bone meal carriers can also be treated with proteases such as trypsin. Xenogeneic carriers can be treated with one or more fibrillation modifiers to increase intraparticle invasion volume (porosity) and surface area. Useful agents include solvents such as dichloromethane, trichloroacetic acid, acetonitrile, and acids such as trifluoroacetic acid and hydrogen fluoride. The choice of carrier may depend on the desired properties of the composition. In some embodiments, lubricants such as water, glycerol, or polyethylene glycol may be added.

[0133] In some embodiments, the fiber-containing composition may also contain other beneficial substances, including, for example, preservatives, solubilizers, suspending agents, viscosity enhancers, ionic strength and gravimetric osmolarity modifiers, and / or other excipients. Suitable buffers may also be used, including but not limited to alkaline earth metal carbonates, phosphates, bicarbonates, citrates, borates, acetates, succinates, etc. Exemplary specific buffers include, for example, sodium phosphate, sodium citrate, sodium borate, sodium acetate, sodium bicarbonate, sodium carbonate, and sodium tromethamine (TRIS).

[0134] In some embodiments, the bone fiber DBM particles may be mixed with a porogen material, which is subsequently removed during manufacturing to increase the porosity of the dried DBM particles. Suitable porogen materials can be made from any biocompatible, biodegradable substance that can form particles and is able to retain at least substantially its shape during implant manufacturing, but is subsequently removed, degraded, or dissolved upon contact with aqueous solutions or other liquids. In some embodiments, the porogen can be inorganic or organic; for example, they can be made from gelatin, organic polymers (e.g., polyvinyl alcohol), polyurethane, polyorthoesters, PLA, PGA and PLGA copolymers, sugars, calcium salts, sodium chloride, calcium phosphate, or mixtures thereof. The porogen particles can be from about 100 to about 500 micrometers in size.

[0135] In one embodiment, all porogen particles of a given morphology may have at least one average axial, transverse, or lateral dimension of about 100 to about 500 micrometers. In some embodiments, all porogen particles used may independently have at least one axial, transverse, or lateral dimension of about 100 to about 500 micrometers. In some embodiments, all porogen particles used may collectively have at least one average axial, transverse, or lateral dimension of about 100 to about 500 micrometers. In some embodiments, at least one size of the porogen particles may be about 100 micrometers or larger, or about 120 micrometers or larger, or about 140 micrometers or larger. In some embodiments, at least one size of the porogen particles may be about 500 micrometers or smaller, about 425 micrometers or smaller, about 350 micrometers or smaller, about 300 micrometers or smaller, or about 250 micrometers or smaller. In some embodiments, the porogen particles may have at least one size of about 120 to about 400 micrometers.

[0136] In some implementations, DBM fiber particles may contain single or multiple size-controlled fibers of varying concentrations to influence the consistency of the DBM particles and the treatment of the hydrated material.

[0137] In some cases, fibers may mix with microparticles in DBM particles, affecting the consistency of DBM particles and the handling of hydrated materials.

[0138] In some cases, multiple DBM particles can be packaged together to improve the hydration and / or treatment of DBM particles before and after hydration.

[0139] In some cases, DBM particles can be hydrated with polar or nonpolar solutions and / or salt solutions prior to drying to enhance subsequent material rehydration.

[0140] One or more bioactive ingredients can be added to the resulting composition (e.g., lyophilized bone fibers). These active ingredients may or may not be related to the bone repair capacity of the composition. Suitable active ingredients include hemostatic agents, bone morphogenetic proteins (BMPs), genes, growth differentiation factors (GDFs) or other non-collagenous proteins such as TGF-β, PDGF, osteopontin, osteonectin, cytokines, etc.

[0141] In one embodiment, the composition may comprise at least one BMP, which is a class of proteins believed to have osteoinductive or growth-promoting activity in endogenous bone tissue, or to function as a procollagen precursor. Known members of the BMP family include, but are not limited to, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, BMP-16, BMP-17, BMP-18, and their polynucleotides or polypeptides, as well as mature polypeptides or polynucleotides encoding them.

[0142] BMPs used as bone inducers comprise one or more of the following: BMP-1; BMP-2; BMP-3; BMP-4; BMP-5; BMP-6; BMP-7; BMP-8; BMP-9; BMP-10; BMP-11; BMP-12; BMP-13; BMP-15; BMP-16; BMP-17; or BMP-18; and any combination of one or more of these BMPs, comprising full-length BMPs or fragments thereof, or combinations thereof, or as polypeptides or polynucleotides encoding polypeptide fragments of all said BMPs. Isolated BMP bone inducers can be administered as polynucleotides, polypeptides, full-length proteins, or combinations thereof.

[0143] In another embodiment, the particles may include one or more growth differentiation factors (“GDFs”) disposed within a compartment or on or within the DBM particles. Known GDFs include, but are not limited to, GDF-1, GDF-2, GDF-3, GDF-7, GDF-10, GDF-11, and GDF-15. For example, GDFs that can be used as isolated bone inducers include, but are not limited to, the following GDFs: GDF-1 polynucleotides or polypeptides corresponding to GenBank accession numbers M62302, AAA58501, and AAB94786, and the mature GDF-1 polypeptides or polynucleotides encoding them; GDF-2 polynucleotides or polypeptides corresponding to GenBank accession numbers BC069643, BC074921, Q9UK05, AAH69643, or AAH74921, and the mature GDF-2 polypeptides or polynucleotides encoding them. GDF-3 polynucleotides or peptides corresponding to GenBank accession numbers AF263538, BCO30959, AAF91389, AAQ89234, or Q9NR23, and the mature GDF-3 peptides or polynucleotides encoding them. GDF-7 polynucleotides or peptides corresponding to GenBank accession numbers AB158468, AF522369, AAP97720, or Q7Z4P5, and the mature GDF-7 peptides or polynucleotides encoding them. GDF-10 polynucleotides or peptides corresponding to GenBank accession numbers BC028237 or AAH28237, and the mature GDF-10 peptides or polynucleotides encoding them.

[0144] GDF-11 polynucleotides or polypeptides corresponding to GenBank accession numbers AF100907, NP005802, or 095390, and the mature GDF-11 polypeptides or polynucleotides encoding them. GDF-15 polynucleotides or polypeptides corresponding to GenBank accession numbers BC008962, BC000529, AAH00529, or NP004855, and the mature GDF-15 polypeptides or polynucleotides encoding them.

[0145] In some embodiments, the implantable composition contains other bioactive agents that can be delivered together with the materials disclosed herein. In some embodiments, the bioactive agent is a pharmaceutical. These bioactive agents can include, for example, antimicrobial agents, antibiotics, antimyobacterial agents, antifungal agents, antiviral agents, antitumor agents, antitumor agents, agents that affect immune responses, blood calcium regulators, agents that can be used to regulate glucose, anticoagulants, antithrombotic agents, antilipidemic drugs, cardiac drugs, thyroid hormones and antithyroid drugs, adrenaline, antihypertensive agents, cholinergic drugs, anticholinergic drugs, anticonvulsants, antiulcer drugs, skeletal and smooth muscle relaxants, prostaglandins, general inhibitors of allergic reactions, antihistamines, local anesthetics, analgesics, narcotic antagonists, antitussives, sedative-hypnotics, anticonvulsants, antipsychotics, anxiolytics, antidepressants, anorexia nervosa, nonsteroidal anti-inflammatory drugs, steroidal anti-inflammatory drugs, antioxidants, vasoactive agents, osteoactive agents, osteogenic factors, antiarthritis drugs, and diagnostic agents.

[0146] A more complete list of bioactive agents and specific medicines applicable to this disclosure can be found in "The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals" edited by Susan Budavari et al.; and United States Pharmacopeia / National Formulary XXXVII / XXXII, Rockville, MD, 2013, each of which is incorporated herein by reference.

[0147] Bioactive agents can also be provided by incorporation into implantable compositions. Bioactive agents such as those described herein can be uniformly or locally incorporated into implant materials by simple mixing or other methods. Furthermore, they can be incorporated alone or together with another carrier form or medium such as microspheres or another particulate formulation. Suitable techniques for forming microparticles are well known in the art and can be used to encapsulate or package bioactive agents, after which the microparticles can be dispersed within the bone graft complex during or after its preparation.

[0148] It should be understood that the amount of additive used will vary depending on the type of additive, the specific activity of the particular additive formulation used, and the intended use of the composition. Users can easily determine the desired amount.

[0149] Any of a variety of medically and / or surgically useful substances can be incorporated into or combined with allogeneic graft bone material before, during, or after the preparation of the implantable composition. Thus, for example, when using non-allogeneic graft bone material, one or more such substances can be introduced into bone fibers, for example, by soaking or immersing these bone fibers in a solution or dispersion of the desired substance.

[0150] In some implementations, the DBM particles of the fiber can be freeze-dried together with one or more growth factors (e.g., BMP, GDF, etc.) and drugs, so that they can be released from the DBM particles in a sustained release manner.

[0151] Bone fiber shape

[0152] Bone fibers can be obtained from autologous, allogeneic, xenogeneic, or transgenic cortical, cancellous, or corticocancellous bone. Such bone can be barrel-milled to obtain bone fibers of the desired size and diameter. A suitable barrel mill for obtaining fibers of the desired size and diameter is described in U.S. Patent Serial No. 13 / 333,279, entitled “Osteobiological Milling Machine,” filed December 21, 2011, published as U.S. Publication No. 20120160945. The entire disclosure is incorporated herein by reference, specifically as follows: Figure 2 The grinding apparatus described in U.S. Publication No. 20120160945 comprises a cutter housing and a feed chute, a rotary cutter at least partially housed within the cutter housing and communicating with the feed chute, and a feed piston removably positioned within the feed chute for holding the workpiece against the rotary cutter. The feed chute and the feed piston can be selectively positioned at one of several angular positions relative to the rotary cutter. In this way, the force exerted on the workpiece by the feed piston is a function of the weight of the feed piston and the angular position of the feed piston relative to the rotary cutter. These types of bone grinders and methods of use result in workpiece utilization rates as high as approximately one percent (approximately 100%). That is, the bone grinder described in U.S. Publication No. 20120160945 utilizes most of the bone placed in the machine and can utilize up to one percent. After grinding the bone to the desired fiber size and shape, the resulting bone fibers can subsequently be demineralized.

[0153] In some implementations, fibers are ground from bone using any suitable equipment, such as a barrel mill. The fibers are ground into a coiled shape including abrasive portions and / or hook-like portions to facilitate mechanical interlocking of the fibers. For example, grinding bone material produces fibers and bone particles separated from the fibers. The shape of the allograft can be customized to suit its location. For example, it can be in the shape of a small piece, plug, pin, nail, cylinder, block, wedge, ring, plate, etc.

[0154] In one embodiment, the method includes placing allogeneic bone graft fibers into a mold before demineralization and / or freeze-drying. The fibers are then demineralized, sterilized, and / or freeze-dried to produce shaped DBM fiber particles. The fibers can be placed into a mold and then demineralized and / or freeze-dried to form a desired shape, or the fibers can be demineralized and / or freeze-dried and then shaped into a desired shape by compression molding or stamping. As discussed herein, the demineralization and freeze-drying steps alter the shape of the fibers to promote entanglement and mechanical interlocking. Therefore, in some embodiments, the fibers are shaped into DBM particles by undergoing demineralization and / or freeze-drying in a molding cavity (not shown). The fibers form such DBM particles without the use of binders or carriers.

[0155] In some embodiments, fibers are placed in a mold and shaped to form DBM particles with a predetermined shape and size range, as required by the medical procedure. In some embodiments, allogeneic grafts can be manufactured by injection molding, compression molding, compression molding, slip casting, laser cutting, waterjet processing, sand casting, shell mold casting, degenerative scaffold casting, plaster mold casting, vacuum casting, permanent mold casting, hollow casting, pressure casting, die casting, centrifugal casting, extrusion casting, rolling, forging, die forging, extrusion, shearing, spinning, or combinations thereof.

[0156] Fibers can be molded into disc-shaped DBM granules, the granules having reservoirs that promote hydration. DBM granules may have a uniform or variable thickness on their surface to facilitate packaging and / or hydration. The reservoir includes recessed areas on the surface of the DBM granule for retaining liquid during hydration. The reservoir is circular. However, in other embodiments, the reservoir may include a variable cross-sectional shape, such as polygonal, elliptical, or irregular. In some embodiments, the fibers may be molded into a conical, plug-shaped, cubic, or cylindrical shape. Fibers can be molded into a conical or plug-shaped form to form DBM granules. The DBM granule includes a first end having a first diameter and a second end having a second diameter. In some embodiments, the first diameter is larger than the second diameter. Fibers can be molded into a cubic shape to form DBM granules. In other embodiments, DBM granules may include other prismatic configurations similar to DBM granules. For example, DBM granules may be rectangular, pyramidal, triangular, pentagonal, or other polygonal or irregular prismatic shapes.

[0157] Demineralization

[0158] After bone is obtained from the donor and ground into fibers, it is processed using methods well known in the art, such as cleaning, sterilization, and degreasing. The whole bone can then be demineralized, or, if necessary, the bone can be sliced ​​before demineralization. The whole bone or one or more slices thereof are then demineralized to reduce the inorganic content to a low level, for example, containing less than about 10% by weight, preferably less than about 5% by weight, and more preferably less than about 1% by weight of residual calcium.

[0159] DBM can be prepared by any suitable method. In one embodiment, DBM is prepared by acid extraction of minerals from bone. It contains a collagen matrix of bone and acid-insoluble proteins, including bone morphogenetic proteins (BMPs) and other growth factors. It can be formulated as granules, gels, sponge materials, putty, or pastes, and can be freeze-dried for storage. Sterilization procedures used to prevent the spread of disease may reduce the activity of beneficial growth factors in DBM. DBM provides an initial osteoconductive matrix and exhibits a degree of osteoinductive potential, inducing the infiltration and differentiation of osteoprogenitor cells from surrounding tissues. As noted, in embodiments of bone particles derived from cortical long bones, the osteoinductive potential of the bone particles during demineralization can vary based on the origin of the bone particles, whether from the periosteal layer, intermediate layer, or endosteal layer.

[0160] DBM formulations have been used in orthopedic medicine for many years to promote bone formation. For example, DBMs have been found to repair fractures, fuse vertebrae, perform joint replacement surgery, and treat bone destruction caused by underlying diseases such as rheumatoid arthritis. DBMs are believed to promote bone formation in vivo through osteoconduction and osteoinduction processes. The osteoinductive effect of implanted DBM compositions is thought to be caused by the presence of active growth factors present on a separated collagen-based matrix. These factors include members of the TGF-β, IGF, and BMP protein families. Specific examples of osteoinductive factors include TGF-β, IGF-1, IGF-2, BMP-2, BMP-7, parathyroid hormone (PTH), and angiogenesis factors. Other osteoinductive factors, such as osteocalcin and osteopontin, may also be present in DBM formulations. Other unnamed or undiscovered osteoinductive factors may also be present in DBMs.

[0161] In one demineralization process, a mixture of long and short bone fibers undergoes an acid demineralization step, followed by a degreasing / sterilization step, during which bone fiber-derived bone microparticles (DBMs) can be formed. The bone is immersed in acid to achieve demineralization. Acids that can be used in this step include inorganic acids, such as hydrochloric acid; and organic acids, such as formic acid, acetic acid, peracetic acid, citric acid, propionic acid, etc. The depth to which the demineralized material penetrates the bone surface can be controlled by adjusting the treatment time, the temperature of the demineralized solution, the concentration of the demineralized solution, and the intensity of agitation during treatment. Therefore, in various embodiments, DBM can be completely demineralized, partially demineralized, or surface-demineralized.

[0162] Rinse demineralized bone with sterile water and / or a buffer solution to remove residual acid, thereby raising the pH. A suitable degreasing / disinfecting solution is an aqueous ethanol solution, as ethanol is a good solvent for lipids and water is a good hydrophilic carrier, allowing the solution to penetrate deeper into the bone particles. The aqueous ethanol solution also disinfects bone by killing nutrient microorganisms and viruses. Typically, at least about 10 to 40% by weight of water (i.e., about 60 to 90% by weight of the degreasing agent, such as ethanol) should be present in the degreasing and disinfecting solution to achieve optimal lipid removal and disinfection over a given time period. A suitable concentration range for the degreasing solution is about 60 to 85% by weight of ethanol or about 70% by weight of ethanol.

[0163] In some embodiments, demineralized bone may be further processed to affect bone properties. For example, DBM may be treated to disrupt its collagen structure. Such processing may include collagenase treatment, heat treatment, mechanical treatment, etc. For further processing options, see U.S. Provisional Patent Applications 60 / 944,408, 60 / 944,417, and 60 / 957,614, which are incorporated herein by reference.

[0164] freeze-dried

[0165] Bone fibers can be freeze-dried in a mold to obtain the desired shape or freeze-dried outside a mold, where they can be shaped (e.g., molding, stamping, cutting, etc.). For example, a vial containing bone and preservative is initially frozen to -76°C, followed by placing the bone and preservative in a vacuum of less than 100 mTorr while maintaining the temperature at -35°C or below. The endpoint of the freeze-drying process is a determination of approximately 5% residual moisture. Once the bone is freeze-dried, it is stored in a sealed vacuum vial for later reconfiguration and use.

[0166] In some embodiments, the demineralization and freeze-drying steps alter the shape of the fibers to promote entanglement and mechanical interlocking. Therefore, in some embodiments, the fibers are shaped into DBM granules by undergoing demineralization and / or freeze-drying in a molding cavity (not shown). These DBM granules are formed without the use of binders or carriers.

[0167] To facilitate the on-site preparation and / or use of the compositions described herein, demineralized fibrous bone elements and non-fibrous bone elements (preferably in lyophilized or freeze-dried form) and fluid carriers (the latter containing one or more optional components, such as those identified above) can be stored under sterile conditions in separate packages or containers and thoroughly mixed together upon use for immediate application to the bone defect site using any suitable method (e.g., spatula, forceps, syringe, tamping device, etc.). Alternatively, the implant compositions can be prepared in advance and stored under sterile conditions until required for use. When the implant compositions are prepared in advance, they are preferably lyophilized before packaging and storage. In some embodiments, the compositions described herein can be combined with autologous bone marrow aspirate, autologous bone graft, a formulation of selected autologous transplant cells, or autologous transplant cells containing genes encoding bone-promoting effects before placement at the defect site. In various embodiments, the implant compositions are already mixed, packaged, and ready for use in suitable containers, such as syringes, resealable non-toxic vials, mesh bags, or pouches, or provided as kits that can be prepared under the guidance of a surgeon when needed.

[0168] Hydration

[0169] DBM particles, made from a combination of short and long fibers, are hydrophilic and have the desired porosity, allowing for easy hydration and synthesis of injectable compositions.

[0170] In some embodiments, DBM particles are hydrated with a liquid comprising bone marrow aspirate, saline, sterile water, injectable blood, phosphate-buffered saline, dextran, Ringer's lactate solution, or combinations thereof. Once hydrated, the hydrated DBM particles have a flowable consistency for injection into bone voids determined by a physician. The fibers in the hydrated DBM bone material retain their cohesiveness and mechanical interactions, thus eliminating the need for adhesives or carriers during in-situ placement. In some embodiments, DBM particles can be hydrated into a putty or paste form and applied to bone voids.

[0171] In some embodiments, the bone repair composition is hydrated with a physiologically acceptable liquid and a biocompatible carrier. Non-limiting examples of physiologically acceptable liquids include saline, phosphate-buffered saline (PBS), hyaluronic acid, cellulose ethers (e.g., carboxymethyl cellulose), collagen, gelatin, autoclaved bone powder, osteoconductive carriers, whole blood, blood components, bone marrow aspirate, concentrated bone marrow aspirate, and mixtures thereof. Non-limiting examples of blood components include serum, plasma, platelet-rich plasma, concentrated platelet-rich plasma, anemic platelet plasma, and concentrated anemic platelet plasma. After hydration, the bone repair composition becomes a putty or paste that can be molded into a predetermined shape or applied to a bone defect and manipulated in a manner that promotes healing to fit the bone defect. For example, the composition can be hydrated with approximately 2 ml of saline blood per 2.5 g of the combined DBM fibers.

[0172] In some embodiments, the bone material can be expressed as approximately 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%, 18.5%, 19.0%, 19.5%, 20.0%, 20.5%, 21.0%, 21.5%, 2 2.0%, 22.5%, 23.0%, 23.5%, 24.0%, 24.5%, 25.0%, 25.5%, 26.0%, 26.5%, 27.0%, 27.5%, 28.0%, 28.5%, 29.0%, 29.5%, 30.0%, 30.5%, 31.0%, 31.5%, 32.0%, 32.5%, 33.0%, 33.5%, 34.0%, 34.5%, 35.0%, 35 0.5%, 36.0%, 36.5%, 37.0%, 37.5%, 38.0%, 38.5%, 39.0%, 39.5%, 40.0%, 40.5%, 41.0%, 41.5%, 42.0%, 42.5%, 43.0%, 43.5%, 44.0%, 44.5%, 45.0%, 45.5%, 46.0%, 46.5%, 47.0%, 47.5%, 48.0%, 48.5%, 49 Hydrate 0.0%, 49.5%, 50.0%, 50.5%, 51.0%, 51.5%, 52.0%, 52.5%, 53.0%, 53.5%, 54.0%, 54.5%, 55.0%, 55.5%, 56.0%, 56.5%, 57.0%, 57.5%, 58.0%, 58.5%, 59.0%, 59.5%, 60% to about 99.9% w / v, w / w and / or v / v fluids to the desired consistency.

[0173] mixing device

[0174] In various embodiments, an apparatus for mixing bone material with a liquid is provided. The apparatus includes a first syringe comprising a first chamber having a proximal end and a distal end. The first chamber includes a syringe barrel. Bone material is disposed within the chamber. The bone material comprises ground and freeze-dried demineralized bone fiber (DBM) particles. The first syringe includes a plunger having at least a portion slidably disposed within the proximal end of the chamber and configured to dispense bone material when the plunger is in an extended position, such as when mixed with liquid from the distal end of the chamber. Figure 10-11 middle.

[0175] In some embodiments, the device includes a second syringe comprising a second chamber having a proximal end and a distal end. The second chamber includes a syringe barrel. A liquid is disposed within the second chamber. The liquid is configured to hydrate milled and lyophilized demineralized bone fiber (DBM) particles. In some embodiments, the liquid includes blood, water, saline, or a combination thereof. The second syringe includes a second plunger having at least a portion slidably disposed within the proximal end of the second chamber.

[0176] In some embodiments, the device includes a connector that fluidly connects the distal end of a first chamber to the distal end of a second chamber via a dispensing channel and a hydration channel. The dispensing channel is connected to the distal end of the first chamber, while the hydration channel is connected to the distal end of the second chamber.

[0177] The second plunger moving to the extended position causes a negative pressure (e.g., a vacuum) to be generated in the first chamber and sets up liquid flow in the second syringe.

[0178] The second plunger moves to the retracted position, forcing gas into the second chamber and mixing with the liquid. The air and liquid are displaced, creating pressure in the second chamber, causing the second plunger to move to the extended position, thus allowing fluid to enter the connector's hydration and dispensing channels and into the first chamber to replace the vacuum space. The negative pressure created by the plunger in the first chamber mixes the liquid with the lyophilized demineralized bone fibers. The hydration fluid can be 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, or more. This hydrates the DBM particles of ground and lyophilized demineralized bone fibers in the first chamber, as it is porous. In some embodiments, the DBM particles of ground and lyophilized demineralized bone fibers are hydrated within approximately 60 seconds. In some embodiments, the DBM particles of ground and lyophilized demineralized bone fibers are hydrated within more than 60 seconds. In some embodiments, the ground and freeze-dried demineralized bone fiber DBM particles are hydrated within approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. Suitable apparatus for mixing the bone material of this application is described in U.S. Patent Application Serial No. 15 / 912,038, filed March 5, 2018, now U.S. Patent No. 9,913,676, assigned to Warsaw Plastic Surgery, Inc., Warsaw, Indiana, USA. The entire disclosure is incorporated herein by reference.

[0179] The first chamber is detachable from the connector to dispense hydrated DBM particles of ground and lyophilized demineralized bone fibers, which can be molded onto the surgical site. When disassembling the first chamber, the cap attached to the distal end of the chamber is removed, and the hydrated DBM particles of ground and lyophilized demineralized bone fibers are ejected from the first chamber, as shown below. Figure 10-11 As shown.

[0180] In some embodiments, the ground and freeze-dried demineralized bone fiber DBM particles do not contain a binder. In some embodiments, the ground and freeze-dried demineralized bone fiber DBM particles comprise cylindrically ground fibers with coiled portions. In some embodiments, the ground and freeze-dried demineralized bone fiber DBM particles comprise autologous or allogeneic bone grafts. In some embodiments, the diameter of the bone fibers is from about 100 μm to about 2 mm.

[0181] In various embodiments, the length of the bone fibers is from about 0.5 mm to about 50 mm. In some embodiments, the average length of the bone fibers is from about 0.5 cm to about 10 cm.

[0182] In some embodiments, the aspect ratio of the fibers may be about 50:1 to about 1000:1, about 50:1 to about 950:1, about 50:1 to about 750:1, about 50:1 to about 500:1, about 50:1 to about 250:1, about 50:1 to about 100:1, about 10:1 to about 50:1, or about 5:1 to about 10:1.

[0183] In various embodiments, a method for hydrating bone material with a liquid is provided. The method includes mixing a liquid with bone material comprising ground and lyophilized demineralized bone fiber (DBM) particles in an apparatus. The apparatus includes a first chamber having a proximal end and a distal end, and the bone material is disposed within the first chamber. A first plunger is provided having at least a portion slidably disposed within the proximal end of the first chamber. The apparatus includes a second chamber having a proximal end and a distal end. A liquid is disposed within the second chamber. The liquid is configured to hydrate the ground and lyophilized demineralized bone fiber (DBM) particles. A second plunger is provided having at least a portion slidably disposed within the proximal end of the second chamber. The apparatus includes a connector fluidly coupling the distal end of the first chamber to the distal end of the second chamber, wherein movement of the second plunger to an extended position causes liquid flow to hydrate the ground and lyophilized demineralized bone fiber (DBM) particles in the first chamber.

[0184] The first chamber is disassembled from the connector to dispense moldable, ground and freeze-dried hydrated DBM particles of demineralized bone fibers.

[0185] In some embodiments, the mixing device includes a first syringe and a second syringe. The first syringe includes bone material comprising DBM particles of ground and freeze-dried demineralized bone fibers. The second syringe includes a liquid, such as blood, saline, sterile water, dextrose, or combinations thereof, or other physiological fluids (e.g., moldable putty, paste, gel, etc.) that can be used to hydrate the bone material to give it a desired consistency. The syringes are connected via a connector. The first and second syringes can be threaded into the connector; suitable threads may be in a leur lock fitting, or a friction or slip-fit ​​fitting may be present so that the syringes can engage the connector and provide a seal against fluid leakage. In some embodiments, the threads extend radially at discrete locations within the connector and are configured to engage the reciprocating threads of the first and / or second syringes to provide an airtight seal.

[0186] In some embodiments, the device can be integral or a single piece. In some embodiments, the first and second plungers may have handles to allow for easier mixing between the second and first chambers. The plungers may slide longitudinally once or multiple times within their respective chambers to allow the bone material to mix and hydrate between the first and second chambers, thereby providing the required hydration of the bone material.

[0187] In some embodiments, a second syringe containing fluid may be docked to a first syringe containing bone material (e.g., bone graft material to be hydrated). Fluid from the second syringe is introduced into the first syringe containing the bone graft material, and the bone material is hydrated. The second syringe, which no longer contains fluid, is removed. The first syringe, containing both bone material and fluid, is capped. The plunger of the first syringe containing both fluid and bone material is moved multiple times to pressurize the device and force fluid into the graft to achieve the desired hydration. The cap is then removed, and the hydrated bone material is removed.

[0188] In some embodiments, the device includes one or more index marks disposed on a first syringe, a second syringe, and / or a connector to visually indicate the alignment of the syringe with the connector and to ensure proper alignment of the chambers and channels for mixing bone material and to prevent leakage of liquid and bone material from the device.

[0189] In some implementations, once the syringe containing the mixed bone material and liquid is removed from the connector, the needle can be connected to the syringe and the mixed bone material can be dispensed.

[0190] Treatment

[0191] Exemplary bone repair sites that can be treated with the implantable compositions disclosed herein include those caused by injury, defects resulting during surgery, infection, malignancy, or developmental malformation. The composite bone graft compositions can be used in a variety of orthopedic, periodontal, neurosurgical, and oral and maxillofacial surgeries, including but not limited to repairing simple and complex fractures and non-fusion; external and internal fixation; joint reconstruction, such as arthroplasty; general arthroplasty; hip cupping; femoral and humeral head replacement; femoral head resurfacing and total arthroplasty; spinal repair, including spinal fusion and internal fixation; tumor surgery, e.g., lack of filling; discectomy; laminectomy; resection of spinal cord tumors; anterior cervical and thoracic spine surgery; repair of spinal injuries; treatment of scoliosis, kyphosis, and lordosis; intermaxillary fixation of fractures; genioplasty; temporomandibular joint replacement; alveolar ridge enhancement and reconstruction; inlay bone implants; implant placement and modification; sinus floor elevation; cosmetic enhancement; and so on. Specific bones that can be repaired or replaced by the composite bone graft composition or implants containing said composition include the ethmoid bone; frontal bone; nasal bone; occipital bone; parietal bone; temporal bone; mandible; maxilla; zygomatic bone; cervical vertebrae; thoracic vertebrae; lumbar vertebrae; sacrum; ribs; sternum; clavicle; scapula; humerus; radius; ulna; carpal bones; metacarpals; phalanges; ilium; ischium; pubis; femur; tibia; fibula; patella; calcaneus; tarsus and metatarsals.

[0192] According to certain aspects of this disclosure, the bone graft compositions of this disclosure can be used as bone gap fillers, or can be incorporated into, on, or around load-bearing implants such as spinal implants, hip implants (e.g., in or around the implant stem, and / or posterior to the acetabular cup), and knee implants (e.g., in or around the stem). In some embodiments, the implantable compositions of this disclosure can be incorporated into, on, or around load-bearing spinal implant devices having a compressive strength of at least about 10,000 N, such as fusion cages, PEEK implants, pins, or other devices, which may have pockets, chambers, or other cavities for receiving the osteoinductive composition, and are used in spinal fusion procedures such as interbody fusion. One exemplary use of this is in combination with a load-bearing interbody spacer to achieve interbody fusion. In these applications, the implantable compositions can be placed in and / or around the spacer to facilitate fusion.

[0193] Methods for preparing DBM are well known in the art, such as those described in U.S. Patent Nos. 5,314,476, 5,507,813, 5,073,373, and 5,405,390, each of which is incorporated herein by reference. Methods for preparing ceramic powders of calcium phosphate and / or hydroxyapatite are described in, for example, U.S. Patent Nos. 4,202,055 and 4,713,076, each of which is incorporated herein by reference.

[0194] In some embodiments, the method includes obtaining fibers by scraping, grinding, or pressing sheets or blocks under aseptic conditions. The shape of the fibers can be optimized to induce new bone formation and processing properties through the fiber network.

[0195] In another aspect, this disclosure provides a method for accelerating bone formation at an implantable tissue regeneration scaffold. In yet another aspect, this disclosure provides a method for regenerating bone in a patient in need, comprising implanting an implantable composition into the patient.

[0196] In another aspect, this disclosure provides a method for treating bone defects caused by injury, disease, trauma, or surgery using an implantable composition comprising a combination of demineralized bone matrix fibers obtained from allogeneic bone and bone fibers of allogeneic bone graft material, wherein the fibers of the allogeneic bone graft material comprise non-fibrous demineralized bone particles embedded within or disposed on the fibers of the allogeneic bone graft material.

[0197] Reagent test kit

[0198] This application also provides a medical kit for preparing the implantable compositions of this disclosure for treating patients, the kit comprising at least a delivery system containing bone material as described above and packaging for aseptically encapsulating the medical implant. Such kits may include a dry material containing the solid components of the composition and an aqueous medium or other biocompatible wetting liquid for combining with the dry material to form a wetting material, or may include a formulated wetting implant material in a suitable container, such as a syringe or vial (e.g., terminally sterilized), and / or another article, such as a weight-bearing implant (e.g., a spinal pad), and / or a transfer device, such as a syringe, and / or a therapeutic substance, such as an osteogenic substance, such as BMP. In one specific form, such a medical kit may include a dry material, such as microparticles or desiccated bodies, lyophilized BMP (e.g., rhBMP-2), and an aqueous medium for reconstituted BMP to prepare an aqueous formulation, which can then be added to the dry material during the preparation of the composite bone graft composition of this disclosure.

[0199] DBM particles can possess functional properties. Alternatively, other materials with functional properties can be incorporated into the DBM particles. Functional properties may include radiation impermeability, bacterial absorbability, source of released materials, viscosity, etc. Such properties may be imparted substantially throughout the DBM particles or only at certain locations or portions of the DBM particles.

[0200] Suitable radiopaque materials include, for example, ceramics, mineralized bone, ceramic / calcium phosphate / calcium sulfate, metal particles, fibers, and iodinated polymers. Polymer materials can be used to form DBM particles and made radiopaque by iodination. Other techniques can also be used to increase the radiopaque linearity of the polymer by incorporating biocompatible metals or metal salts into it. Suitable bactericidal materials may include, for example, trace metal elements. In some embodiments, trace metal elements may also promote bone growth.

[0201] Functional materials, such as radiopaque markers, may be provided at one or more locations on the DBM particle, or substantially throughout the entire DBM particle. Thus, for example, in a cylindrical DBM particle, radiopaque markers may be provided at the tip of the cylindrical DBM particle. Such markers facilitate placement of the DBM particle. Radiopaque materials may be incorporated into the DBM particle and / or substances for delivery via the DBM particle. Furthermore, radiopaque materials may be provided only at certain locations on the DBM particle, such that visualization of those locations provides an indication of the DBM particle's orientation within the body.

[0202] The implantable compositions of the present invention can be used alone as bone graft materials, as scaffolds for bone tissue engineering for the repair, enhancement and replacement of bone tissue, or as carriers of growth factors or genes.

[0203] These and other aspects of this application will be further understood upon consideration of the following embodiments, which are intended to illustrate certain specific implementations of this application but are not intended to limit the scope of this application as defined by the claims.

[0204] Example 1

[0205] Preparation of injectable demineralized bone matrix particles

[0206] A method for forming DBM particles is provided. Large cortical bone is provided. The cortical bone is immersed in a special denaturing alcohol (SDA). The cortical bone is then ground into small bone flakes, which are then screened and barrel-milled to produce large bone fibers of various sizes. The large bone fibers are screened to separate long fibers with a target diameter between approximately 4 mm and approximately 0.5 mm. The remaining large bone fibers are further barrel-milled to produce short bone fibers with a diameter range between 0.5 mm and 4 mm. The long fibers have a diameter range between 0.5 mm and 4 mm, and the short fibers have a diameter range between 0.05 mm and 0.5 mm. The long and short bone fibers are mixed at a predetermined ratio. The mixture is demineralized to form a DBM slurry. The slurry is gently compacted into a mold to form DBM particles. The DBM particles are then freeze-dried. The processing characteristics of different combinations of long and short fibers are tested by measuring the injection force, as shown in Table 1 below.

[0207] Table 1.

[0208] Target (long) fibers (wt.%) Short fibers (wt.%) Injection force (pounds) 80 20 24 90 10 26 100 0 33

[0209] The above represents the ratio / percentage of target (long) fibers and short fibers that have established good handling and ejection capabilities. DBM particles containing 80% long bone fibers and 20% short bone fibers establish optimal handling and ejection capabilities, which is ideal for injectable DBM products.

[0210] Example 2

[0211] Preparation of injectable hydrated demineralized bone matrix particles

[0212] A method similar to Embodiment 1 described above is provided. DBM particles are set, for example, as... Figure 10 The mixing device shown includes a chamber and a plunger. The plunger moves back and forth between an extended position and a retracted position. The plunger is in the extended position when its distal end approaches the distal end of the chamber. The plunger is in the retracted position when its distal end moves away from the distal end of the chamber. Twenty repetitive movements of the plunger are used to hydrate the DBM particles. The hydrated DBM particles are aseptically packaged in a syringe for delivery. The material consistency of the hydrated DBM particles is tested by measuring the maximum injection force applied to deliver the hydrated DBM particles, as shown in Table 2 below.

[0213] Table 2.

[0214]

[0215] As mentioned above, the injection force decreases with increasing hydration volume ratio in the syringe, while increasing with increasing DBM particle weight.

[0216] Example 3

[0217] Testing of the treated texture of hydrated demineralized bone matrix particles

[0218] A method similar to that in Example 2 is provided. For example, as... Figure 9 The texture analyzer shown is used to qualitatively determine the treated texture of DBM particles, as shown in Table 3 below, where a treatment score of 1 is unacceptable and 5 is excellent.

[0219] Table 3.

[0220]

[0221] Processed texture quality decreased with increasing short fiber percentage, but lower short fiber percentages increased waste from donor bone. These samples were hydrated to 100% hydration, which was determined to reduce processed texture to an unacceptable level. Further texture quality testing determined the optimal range for processed texture to be 50–75% hydration.

[0222] Example 3

[0223] Testing of the treatment properties of hydrated demineralized bone matrix particles

[0224] A method similar to that of Example 3 was provided. Furthermore, as shown in Table 4 below, different percentages of short fibers were tested for addition to DBM granule formulations for injection, force measurement, and qualitative treatment of texture.

[0225] Table 4.

[0226]

[0227] As shown in Table 4, formulations with 33% short bone fibers were below acceptable levels prior to texture assessment, while formulations with 100% target long bone fiber length were still injected at 50 psi. A combination of 10% short fibers and 90% long fibers yielded the optimal treatment grade.

[0228] It should be understood that the foregoing relates to exemplary embodiments of this disclosure and may be modified without departing from the spirit and scope of this disclosure as set forth in the appended claims.

Claims

1. A method for preparing demineralized bone matrix particles, the method comprising: After grinding and crushing, the bone fibers are passed through an 8mm sieve to screen out large bone fibers; After screening, multiple long bone fibers are provided, the long bone fibers having a diameter of 1 mm to 8.0 mm; The large bone fibers are ground and screened to provide a plurality of short bone fibers with a diameter of 0.5 mm to 0.05 mm; The plurality of long bone fibers are mixed with the plurality of short bone fibers to form a mixture of long bone fibers and short bone fibers; The mixture of long and short bone fibers is demineralized to form a demineralized bone matrix slurry; and the demineralized bone matrix slurry is dried to form demineralized bone matrix particles, wherein the demineralized bone matrix particles comprise 0 wt.% to 33 wt.% of short demineralized bone fibers and 80 wt.% to 100 wt.% of long demineralized bone fibers, based on the total weight of the bone fibers or based on the total weight of the demineralized bone matrix particles.

2. The method of claim 1, wherein the demineralized bone matrix slurry is added to a mold and dried in the mold.

3. The method of claim 1, wherein the demineralized bone matrix slurry is compacted into a mold using a pressure of 4 to 65 Newtons.

4. The method of claim 1, wherein the slurry is added to a mold to form the demineralized bone matrix particles and the particles are removed from the mold and freeze-dried.

5. The method of claim 4, further comprising rehydrating the freeze-dried demineralized bone matrix particles with a physiologically acceptable liquid to form an injectable demineralized bone matrix.

6. The method of claim 5, wherein the liquid comprises bone marrow aspirate, saline, sterile water, blood for injection, phosphate-buffered saline, dextran, Ringer's lactate solution, or a combination thereof.

7. The method of claim 6, wherein the blood volume comprises 3 mL to 9 mL.

8. The method of claim 5, wherein the injection force of the injectable demineralized bone matrix is ​​15 to 33 pounds or 24 to 27 pounds.

9. The method of claim 1, wherein, based on the total weight of the bone fibers or based on the total weight of the demineralized bone matrix particles, the demineralized bone matrix particles comprise 0.1 wt.% to 33 wt.% of short demineralized bone fibers and 80 wt.% to 99.9 wt.% of long demineralized bone fibers.

10. The method of claim 3, wherein the compaction comprises (i) manually applying pressure to the mixture using a trowel or roller and a compression block; or (ii) without applying pressure, allowing the demineralized bone matrix slurry to pass through a sieve until excess fluid is discharged from the slurry.

11. A bone material made by the method according to any one of claims 1-10, wherein the bone material comprises a mixture of a plurality of long bone fibers with a diameter of 1 mm to 8.0 mm and a plurality of short bone fibers with a diameter of 0.5 mm to 0.05 mm.

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