Bone growth components

Abstract

A bone growth composition to be applied as a bone implant, bone graft material, or bone graft piece, and a method for treating a subject having a bone defect with the bone composition described herein are provided.

Description

Technical Field

[0001] The disclosure of the present invention generally relates to the field of bone growth compositions, methods of making such compositions, and methods of treating bone defects in a subject using such compositions. Disclosed herein is a bone growth composition heretofore unknown for use as a bone graft, bone implant, or bone filler for treating a subject having a bone defect in a subject.

Background Art

[0002] Bone is a composite material composed of collagen, cells, a form of calcium hydroxyapatite crystals, and small amounts of other proteins and organic molecules. The chemical and physical properties of this composite provide unique properties such as high strength, rigidity, and the ability to adapt to varying loads in the body. However, when bone is damaged, there is a need to find ways to fill voids or gaps and promote the repair and regeneration of bone tissue.

[0003] Bone damage due to trauma or disease requires surgical intervention to replace the affected part of the bone or to temporarily fill the voids in the bone structure that will be replaced by the internal growth of new bone tissue.

[0004] There are several examples of orthopedic-related surgeries where the insertion of a bone filler into a damaged limb bone, pelvis or spine typically functions by providing a bioabsorbable substitute in which bone tissue grows during the healing process. In craniofacial surgery, implants are used to reconstruct damaged facial tissues. In dentistry, implants are mainly used to create stable bone in the upper or lower jaw for the embedding of metal elements to which a prosthesis will then be attached. In cosmetic surgery, implants are mainly used to reconstruct defects including congenital defects.

[0005] Products consisting of or containing hydroxyapatite (HA) (Ca5(PO4)3OH) and / or tricalcium phosphate (Ca3(PO4)2) in crystalline form β have been historically known and used in the preparation of scaffolds useful for bone tissue regeneration.

[0006] Therefore, to produce a scaffold for bone regrowth, HA is used in a mixture with a second mineral component, commonly known as β-tricalcium phosphate or by its abbreviation β-TCP, which is crystalline form β of tricalcium phosphate (Ca3(PO4)2). β-TCP is absorbed faster than HA and therefore promotes bone tissue regrowth.

[0007] Various biocompatible and biodegradable materials consisting of HA / β-TCP are known for use in orthopedic surgery.

[0008] Product OPTEMX by Exactech (Gainesville, Florida, USA) consists solely of a porous HA / β-TCP granular composition, and its osteoconductivity is limited when mixed with autologous bone marrow fluid or autologous blood.

[0009] MASTERGRAFT, a product from Medtronic (Minneapolis, Minnesota, USA), is available only in granular form as an HA / β-TCP composition and possesses osteoconductivity.

[0010] However, these materials are granular in form, limiting their moldability and flexibility. Therefore, they are not very suitable for use in the lumen or surface of irregularly shaped bone.

[0011] Despite the beneficial performance characteristics of HA / β-TCP and other osteoconductive materials, granular morphology remains a challenge from a clinical use perspective. Unless combined with a treatment agent to retain the material in its designated position, ceramic granules tend to migrate from the surgical site (known as "spitting" from the defect). Historically, polymer materials or animal-derived collagen biomaterials have been used in combination with osteoconductive granules to address this problem. Polymer / animal-derived collagen and ceramic constructs create a three-dimensional structure that retains the granules and creates further porosity between them. Numerous devices with similar designs and performance characteristics have been developed. Several products consisting of HA and / or β-TCP in a bovine collagen matrix are known on the market.

[0012] MASTERGRAFT Putty, a product using Medtronic which possesses osteoconductivity, is formed from the same granules as the aforementioned MASTERGRAFT product, uniformly dispersed in a bovine collagen matrix.

[0013] Integra MOZAIK, a bone-conductive product from Integra Life Sciences Corporation (Plainsboro, New Jersey, USA), consists of a mixture containing approximately 80% β-TCP and 20% cross-linked bovine collagen.

[0014] The product VITOSS Scaffold Foam Bone Graft Material consists of approximately 80% β-TCP and 20% bovine cross-linked collagen.

[0015] The product COLLAGRAFT Bone Graft Matrix by Zimmer (Warthau, Indiana, USA) is commercialized in the form of strips made from HA / β-TCP in a cross-linked bovine collagen matrix.

[0016] HEALOS, a product from DePuy (Warthau, Indiana, USA), consists of a matrix of HA-coated bovine cross-linked collagen fibers. This product is combined with autologous bone marrow aspirate or autologous blood.

[0017] U.S. Patents 6,764,517 and 6,902,584 describe three-dimensional mineralized collagen complexes produced by creating a collagen slurry, freezing and freeze-drying the mixture, and then exposing it to a calcium and phosphate solution to form a porous mineralized matrix.

[0018] These patents describe adding soluble collagen in a further step and freeze-drying the mixture to form a porous composite. The invention further describes the ability to enhance physical stability using various crosslinking agents, thereby improving implant absorption time and shape retention. While this technology can offer improvements over previous technologies, the manufacturing process consists of many different steps that become costly and time-consuming.

[0019] Further improvements to these common methods for producing collagen-calcium phosphate composite materials can be found in U.S. Patents 7,156,880 and 7,166,133. These inventions describe the manufacture of implants comprising an osteoconductive matrix containing a blend of both insoluble and soluble collagen, in which at least a portion of the implant is porous. Furthermore, these structures may contain osteoinductive molecules and biodegradable synthetic polymers. The inventions also describe the incorporation of ceramic materials, such as calcium phosphate, calcium sulfate, or hydroxyapatite, in the form of individual particles rather than the formation of compounds by salt precipitation.

[0020] More recent technologies, such as those found in U.S. Patents 7,531,004 and 7,534,451, describe bone repair composite materials comprising an absorbable polymer, which may be collagen, a series of meso, micro, and macroporous structures that allow for fluid encapsulation and aid in bone growth, and the encapsulation of calcium phosphate particles. The invention further utilizes specific redox reactions of calcium and phosphorus-containing salts to precipitate calcium phosphate within the collagen structure. These devices typically require very precise control of chemical reactions to obtain the desired result of precipitation of calcium phosphate material and appear to be limited to calcium-based osteoconductive materials.

[0021] Patent application WO 2011 / 064724 A1 describes single-layer, double-layer, or multi-layer biomimetic materials for use in orthopedic surgery, and processes for producing them. The basic material described in this application is a two-component material made from natural polymeric bovine collagen to which chitosan (a polymer obtained by basic deacetylation of chitin, a natural component of the exoskeleton of crustaceans) has been added. The single-layer biomimetic material of this application is made of collagen-chitosan only. This two-component material contains 30-90% by weight (preferably 50-80%) collagen, with the remainder being chitosan. The role of chitosan is said to be to promote fibrillation of the two-component material.

[0022] While bovine collagen-based materials have a long clinical history in many medical devices, hypersensitivity reactions and immune responses remain recognized limitations. Processing steps involved in the production of any collagen-based medical device can significantly impact the in vivo response in terms of tissue infiltration, local inflammatory responses, and degradation of the collagen material (Chvapil M 1977; Weadock et al., 1983; Bailey AJ 2000). [Overview of the Initiative] [Problems that the invention aims to solve]

[0023] What is needed is a bone growth composition that is not plagued by the problems and limitations of conventional materials and that is applied as a bone graft, bone filler, or bone implant.

[0024] The citation of any reference in this specification should not be construed as an admission that such reference is available as prior art to this disclosure.

Means for Solving the Problem

[0025] The present disclosure is surprisingly and unexpectedly based on the discovery that the novel bone growth compositions of the present disclosure are readily applicable to the treatment of a subject's bone defect, exhibit effective results when used in the treatment of a subject's bone defect, and are not plagued by the drawbacks of known compositions used for such treatment. Broadly speaking, the present disclosure extends to bone growth compositions that include a natural biocompatible matrix, a decellularized placenta-derived extracellular matrix substantially free of residual material, and a source of Ca 2+ and PO4 3- ions, such as a mineral source. Examples of such bone defects that can be treated with the bone growth compositions of the present disclosure include, but are not necessarily limited to, injuries from trauma, congenital or acquired bone defects or abnormalities, surgical bone resection, sequelae of bone infections (such as fractures associated with bone loss, e.g., as a result of comminuted fractures). Further, the form of the bone growth composition of the present disclosure can vary depending on the bone defect in which the composition is being used for treatment. Thus, the bone growth composition of the present invention can be a bone implant, a bone graft, or a bone filler. Further, the bone growth compositions disclosed herein can be porous, osteoconductive, or both porous and osteoconductive.

[0026] Numerous naturally occurring biocompatible substrates are applied to the bone growth compositions of this disclosure, and these substrates include, but are not limited to, collagen, elastin, tropoelastin, elastin-like proteins, or any combination thereof of these biocompatible substrates. In certain embodiments of this disclosure, the naturally occurring biocompatible substrate includes a combination of collagen and elastin, and such amounts of collagen in a decellularized placental extracellular matrix substantially free of residues include at least about 60% by weight of collagen and about 20% by weight or less of elastin. The type of collagen applied to the bone growth compositions of this disclosure may vary. In particular, the collagen may be type I collagen, type III collagen, type V collagen, type VI collagen, type VII collagen, or any combination thereof. Type I collagen is the most abundant type of collagen in bone tissue, accounting for about 90% of the total collagen content in bone. Type I collagen provides structural support and strength to bone and also functions as a scaffold for cell adhesion, proliferation, and differentiation during the bone healing process. Type III collagen is less abundant in bone compared to type I collagen, but it still plays a role in bone regeneration. Type III collagen is often found in association with type I collagen and contributes to the formation of the collagen matrix. Type III collagen is involved in the initial stages of tissue repair and remodeling. Collagen types I and III are most suitable for bone regeneration due to their abundance in bone tissue, their contribution to structural integrity, and their role in supporting cellular processes essential for bone healing and regeneration. There are also minor types of collagen. Type V collagen is a trace component of the extracellular matrix but is still present in bone tissue. Type V collagen is often found in association with type I collagen fibrils and is thought to play a regulatory role in collagen fibril formation and organization. While type V collagen may not be as dominant in bone tissue as types I and III, it may potentially influence the mechanical properties and organization of the extracellular matrix, thereby indirectly affecting the bone regeneration process. Type VI collagen is found in bone and forms distinct microfibrilary structures within the ECM, contributing to tissue organization and integrity.Type VII collagen is also a minor collagen that may play a role in the adhesion of osteocytes to the underlying matrix or basement membrane during bone development and repair processes.

[0027] Synthetic biocompatible matrices are also applied to the bone growth compositions of the present disclosure. Examples of such synthetic biocompatible matrices include, but are not limited to, polylactic acid (PLA), poly(lactic-co-glycolic acid) PLGA, biodegradable polyester amide polymers, hydrogels, polyether ketones, or any combination of these synthetic biocompatible matrices.

[0028] Furthermore, in the bone growth compositions of the present disclosure, the sources of Ca 2+ and PO4 3- ions may be different. For example, the mineral sources of these ions may be minerals such as Ca(H2PO4)2, CaHPO4, Ca3(PO4)2 (β-TCP), Ca5(PO4)3(OH), or any combination of these minerals. In certain embodiments, the mineral source is in particulate form, e.g., particulate minerals. Furthermore, the size of the particles of the mineral source and the range of the size of the particles applied to the bone growth compositions of the present disclosure may be different. For example, the range of the particulate size of the mineral source particles in the bone growth compositions of the present disclosure may range from about 250 μm to about 3000 μm, about 250 μm to about 2000 μm, about 250 μm to about 1000 μm, or about 1000 μm to about 2000 μm.

[0029] The weight-based mass ratio of the mineral source to the acellular placental-derived extracellular matrix substantially free of residual substances in the bone growth compositions of the present disclosure may be different. For example, this mass ratio may be about 5% to about 95%, about 10% to about 90%, about 15% to about 85%, about 20% to about 80%, about 15% to about 85%, or about 30% to about 70% (ECM: mineral source).

[0030] Furthermore, the bone growth compositions of this disclosure may further include biomaterials, such as cells, conditioned cell media, cell secretomes, plasma, genetic material, such as nucleic acid molecules (e.g., siRNA and mRNA) and nucleotides, exosomes, bone marrow aspirate, platelet plasma, growth factors, or any combination of such biomaterials. Examples of cells to be applied herein include, but are not limited to, mesenchymal stem cells, adipose tissue-derived stem cells, osteoblasts, and placental-derived cells. In certain embodiments, the conditioned medium and exosomes are produced by the cells discussed above.

[0031] The disclosure further extends to a method for treating a bone defect in a subject, which includes administering an effective amount of the bone growth composition of the disclosure to the subject at the time of the bone defect.

[0032] Also provided herein are the bone growth compositions of this disclosure, and kits including packaging containers specially designed to allow for various product sizes and the addition of rehydration solutions.

[0033] Depending on the circumstances, the kits of this disclosure may further include biomaterials, such as cells, conditioned cell media, cell secretomes, plasma, genetic material, such as nucleic acid molecules (e.g., siRNA and mRNA) and nucleotides, exosomes, bone marrow aspirate, platelet plasma, growth factors, or any combination of such biomaterials. Examples of cells to be applied herein include, but are not limited to, mesenchymal stem cells, adipose tissue-derived stem cells, osteoblasts, and placental-derived cells. In certain embodiments, the conditioned media and exosomes are produced by the cells discussed above. The biomaterials may be housed in containers included in the kit, or the packaging container may be designed to provide a sealed chamber for housing the biomaterials.

[0034] These and other aspects of the present disclosure will be better understood by referring to the following drawings and embodiments for carrying out the invention. [Brief explanation of the drawing]

[0035] [Figure 1] This figure shows the SDA-PAGE analysis of the decellularized placenta-derived extracellular matrix of the bone growth composition of the present disclosure, in which the biocompatible substrate contains collagen. MW refers to molecular weight, kDa refers to kilodaltons, COL I refers to type I collagen, COL III refers to type III collagen, COL IV refers to type IV collagen, COL V refers to type V collagen, and COL VI refers to type VI collagen. [Figure 2] This figure shows several forms of the freeze-dried bone growth composition of the present disclosure, which has a cubic structure and contains a particulate mineral source. The size of the mineral source particles ranges from approximately 250 μm to approximately 2000 μm, distributed throughout the bone growth composition, and the ratios of decellularized placental extracellular matrix and β-TCP, which are substantially free of residual material, include 10 / 90%, 15 / 85%, and 20 / 80%. [Figure 3] This figure shows the morphology of a cylindrical bone growth composition of the present disclosure, having a particulate mineral source having a particle size range of approximately 250 to approximately 500 μm distributed throughout the bone growth composition of the present disclosure. [Figure 4] This figure shows a top view and a side view of a cylindrical bone growth composition of the present disclosure, having a particulate mineral source having a particle size range of approximately 1000 to approximately 220 μm distributed throughout the bone growth composition of the present disclosure. [Figure 5] This figure shows a strip-structured bone growth composition of the present disclosure containing approximately 95% by weight of a particulate mineral source having a particle size range of approximately 2050 to 1000 μm, and approximately 5% by weight of decellularized placental extracellular matrix substantially free of residual substances. [Figure 6] This figure shows crosslinking of decellularized placenta-derived extracellular matrix substantially free of residual substances by heat dehydration treatment of the bone growth composition of the present disclosure. [Figure 7]Figures 7A, B, and C show the moldability and plasticity of the bone growth composition of this disclosure, in which the mass ratio of the mineral source to the decellularized placental extracellular matrix substantially free of residues is approximately 20%:approximately 80%. Sterile water was added to the bone growth composition disclosed herein, which has a mass ratio of approximately 20:80% of the decellularized placental extracellular matrix substantially free of residues to β-TCP (Figure 7A). The rehydrated implant was molded into the desired ball shape as shown in Figures 7B and 7C. [Figure 8] Figures 8A, 8B, and 8C show the bone growth composition of the present disclosure, in which the mass ratio of decellularized placental extracellular matrix rehydrated with blood and substantially free of residual substances to β-TCP is approximately 20:approximately 80%. [Figure 9] Figures 9A, 9B, 9C, and 9D show the rehydration and handling of the bone growth composition of this disclosure, having a mass ratio of approximately 20:80% of decellularized placental extracellular matrix to β-TCP that is substantially free of residual material, compared to β-TCP alone. Figure 8A shows that the bone growth composition described herein remains intact after rehydration in saline for 15 minutes, with the β-TCP particles retained therein, and Figure 8B shows the rehydrated bone growth composition of Figure 8A molded into the desired shape. Figures 9C and 9D show β-TCP alone after immersion in saline for 15 minutes. The well after transferring the β-TCP shows residual β-TCP, and Figure 9D shows that the rehydrated β-TCP alone cannot be molded. [Figure 10]Figures 10A and 10B show morphological analyses of the bone growth composition of the present disclosure, having a mass ratio of approximately 20:80% of decellularized placental-derived extracellular matrix to β-TCP, which is substantially free of residual material, compared to β-TCP alone. The particle size range of β-TCP is approximately 250 to 2000 μm (left) and approximately 1000 to 2000 μm (right). Figure 10 shows radiopaque β-TCP particles (white) and porous ECM (black surrounding the β-TCP particles). Microcomputed tomography (micro-CT) of the β-TCP granules shows that the β-TCP granules are uniformly surrounded by the porous placental structure ECM described herein. [Figure 11] This figure shows the XRD spectrum and overlay analysis of the bone growth composition of this disclosure, in which the mass ratio of decellularized placenta-derived extracellular matrix, substantially free of residual substances, to β-TCP is approximately 20:approximately 80%. The correct spectrum was observed to show β-TCP, the β-TCP component was fully characterized, and no further phases or impurities were detected. [Figure 12A] Figure 12 shows the FTIR spectrum and stability analysis of placental-derived ECM, a naturally biocompatible material of the bone growth composition of this disclosure, which is substantially free of residual substances. Figure 12(A) shows collagen standards and decellularized ECM. [Figure 12B] Figure 12(B) shows the stability profile of the bone growth composition of this disclosure over 115 days at ambient temperature. [Figure 12C] Figure 12(C) shows the FTIR profile of the bone growth composition of this disclosure. [Figure 12D] Figure 12D shows the FTIR spectrum of the bone growth composition of this disclosure at 40°C for 10 days. [Figure 13]Figure 13A shows a double tray prototype with a fixation device, Figure 13B shows a double tray prototype with 10cc of the bone growth composition of the present disclosure and a fixation device, and Figure 12C shows a double tray prototype with 5cc of the bone growth composition of the present disclosure and a fixation device, and Figure 12D shows a double tray prototype with 2.5cc of the bone growth composition of the present disclosure and a fixation device. [Figure 14] This figure shows the bone growth composition of the present disclosure after mixing with β-TCP dispersed therein. [Figure 15] This figure shows the bone growth composition of this disclosure alone at 12 weeks. [Figure 16] This figure shows the autologous graft at 12 weeks. [Figure 17] This figure shows the results for experimental group 1 (FUSE 1) at week 12. [Figure 18] This figure shows the results for experimental group 2 (FUSE 2) at week 12. [Figure 19] This figure shows the application of the bone growth composition of this disclosure in the form of bone marrow aspirate generation and bone marrow aspirate and bone implants. [Figure 20] This figure shows the rehydration and implantation of the bone growth composition of this disclosure in the form of a bone implant for a critical-size bone defect. [Figure 21] This figure shows micro-CT images of the bone growth composition of the present disclosure, which has β-TCP having a particle size range of approximately 250 to 500 μm, in the form of a critical-size bone implant at 3 and 6 weeks. [Figure 22] These are micro-CT images of the bone growth composition of this disclosure, which has β-TCP having a particle size range of approximately 1000 to 2000 μm, showing the morphology of bone implants in critical-size bone defects at 3 and 6 weeks. [Figure 23]This figure shows the histological images of the bone growth composition of this disclosure, which contains β-TCP having a particle size range of approximately 1000 to 2000 μm, in the morphology of bone implants in critical bone size defects at 3 and 6 weeks. [Figure 24] This figure shows a histological image of the bone growth composition of this disclosure, which has β-TCP having a particle size range of approximately 1000 to 2000 μm, in the morphology of a bone implant in a critical-size bone defect. [Figure 25] This figure shows the bone growth composition of the present disclosure in the form of a bone implant, having a 20:80% mass ratio of decellularized placental extracellular matrix and β-TCP, which is substantially free of residual material, rehydrated with bone marrow aspirate and filled into a syringe for implantation / placement into a rabbit bone defect in a posterolateral fixation model. [Figure 26] This figure shows the 8-week histological results of the bone growth composition of the present disclosure, having a substantially residue-free decellularized placental extracellular matrix and β-TCP mass ratio of 20:80%, rehydrated with bone marrow aspirate in a rabbit bone defect in a posterolaterally fixed rabbit model. New bone formation and neovascularization are indicated by arrows. In the figure, "TP" refers to tricalcium phosphate, and in the figure, "ECM" refers to a substantially residue-free decellularized placental extracellular matrix. [Figure 27] This figure shows the 8-week histological results of the bone growth composition of the present disclosure, having a mass ratio of 20:80% of decellularized placental-derived extracellular matrix rehydrated in bone marrow and substantially free of residual material, to β-TCP. In the figure, "TP" refers to tricalcium phosphate, and "ECM" refers to decellularized placental-derived extracellular matrix substantially free of residual material. New bone formation and neovascularization are provided by arrows. [Figure 28] This is a diagram illustrating the use of the bone growth composition of this disclosure as a tooth graft. [Figure 29] This figure illustrates the use of a 3D printed mold equipped with a rod that creates channels within the bone growth composition of the present disclosure and controls the channels therein to induce new intravascular growth. [Figure 30]This figure shows the growth of human mesenchymal stem cells in the bone growth composition described herein in implant form, compared to a commercially available product (control product) containing 20% ​​human bovine collagen and 80% beta-TCP. [Modes for carrying out the invention]

[0036] This disclosure is based on the surprising and unexpected discovery that a decellularized placental extracellular matrix, substantially free of residual substances (e.g., DNA, cells, cellular debris, growth factors, exosomes, and cytokines), including naturally biocompatible materials, as well as a bone growth composition containing Ca3- and PO4 ion mineral sources, is readily applicable to the treatment of bone defects in question.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. However, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of this disclosure.

[0038] All numerical specifications, such as volume, mass, and particle count, are approximations that may vary by (+) or (-) 1.0 or 0.1 as needed. While not always explicitly stated, it should be understood that all numerical specifications are preceded by the term "approximately."

[0039] Numerous terms and phrases are used throughout this specification and the claims and are defined below.

[0040] "About" and "approximately" are interchangeable and mean plus or minus a percentage (e.g., ±5%) of the numerical value, parameter, or feature thus modified, and are understood to be appropriate by those skilled in the art in the scientific context in which the terms are used.

[0041] As used herein, the singular forms "a," "an," and "the" include the plural form unless the context specifically indicates otherwise.

[0042] As used herein, "β-TCP" means Ca 2+ and PO4 3- Beta-tricalcium phosphate (Ca3(PO4)2) is a biocompatible ceramic material that serves as an ionic mineral source and is applied to orthopedic and dental uses, including bone void filling and bone regeneration.

[0043] As used herein, the terms “comprising,” “comprises,” and “comprise” are intended to mean that the compositions, preparations, and methods disclosed herein include the listed elements, but do not exclude other elements.

[0044] As used herein, the term “critical size bone defect” refers to a bone defect created in an animal that cannot heal spontaneously within a reasonable timeframe without intervention. The critical size varies depending on factors such as the location of the defect and the specific characteristics of the bone tissue involved. Critical size bone defects typically exceed the body’s natural regenerative capacity and require surgical intervention or the use of tissue engineering constructs or biomaterials to promote healing. In clinical practice, clearly defining the critical size of a bone defect is essential for determining an appropriate treatment strategy. Surgeons often assess factors such as the size, location, and stability of the defect, as well as the condition of the surrounding soft tissue, to determine whether surgical intervention is necessary and what type of intervention would be most effective. A variety of techniques and materials, including bone grafts (autografts, allografts, or xenografts), synthetic bone substitutes, growth factors, and tissue engineering approaches, can be used to address critical size bone defects.

[0045] The terms “delivery” and “administration” are used interchangeably herein and mean and encompass the delivery of a “pharmaceutical composition,” “biological agent,” “active agent formulation,” or “biologically acceptable substance” to a treatment site, such as damaged tissue, by any method suitable for delivering the functional agent, formulation, or pharmaceutical composition to the treatment site. Non-limiting examples of delivery methods include direct injection at the treatment site, transdermal delivery, and topical application.

[0046] As used herein, the terms “extracellular matrix” and “ECM” are used interchangeably and mean and encompass collagen-rich material found between cells of mammalian tissues, and any material derived therefrom, such as decellularized ECM. In certain embodiments, the ECM material is derived from human placenta.

[0047] As used herein, the terms “freeze-dried” and “dehydrated” are used interchangeably and refer to a state from which water has been removed, rather than a method of removing water as a means of preservation.

[0048] As used herein, the term “placenta” refers to the disc-shaped tissue that connects the mother’s uterus to the umbilical cord and is ultimately responsible for the delivery of nutrients and oxygen to the fetus. The placenta is composed of three membranes: (1) the amnion, which is a single layer of ectoderm epithelium that completely encloses the embryo; (2) the chorionic membrane, which surrounds the amniotic sac and contains the chorionic villi and trophoblast; and (3) the decidua, which is the maternal endometrium. The umbilical cord is a conduit between the placenta and the mother’s circulatory system and is therefore not part of the placenta.

[0049] As used herein, the term “hydrate” refers to the addition of a liquid, such as a diluent, to a dehydrated material in order to suspend it in a solution so that it can be injected through a needle of appropriate size.

[0050] As used herein, the phrase “substantially free of residues” means extracellular matrix that does not contain quantifiable amounts of residues, such as (1) cells, (2) cellular debris, (3) nucleic acid molecules, (4) nucleotides, (5) growth factors, (6) cytokines, (7) chemokines, (8) hormones, or (9) any combination of (1) to (8).

[0051] Where used herein, the term “effective dose” means an amount of a “pharmaceutical composition” and / or “biological activator” and / or “activator formulation,” such as a bone growth composition disclosed herein, administered in an amount sufficient to improve one or more causes, symptoms, or sequelae of a disease or condition, such as bone loss. Such improvement requires only a reduction or alteration of the cause, symptoms, or sequelae of the disease or disorder, and does not necessarily require their complete disappearance. The effective dose is determined by several factors, including the severity and course of the disease or condition, previous treatments, the patient’s health status, age, weight, and response to the drug. Determining the therapeutic effective dose by routine experiments (including, but not limited to, dose-escalation clinical trials) is considered well within the art of the art.

[0052] As used herein, the term “exosome” refers to naturally occurring nanoparticles that are endogenously secreted by many types of in vitro cell cultures and in vivo cells and are commonly found in vivo in bodily fluids, such as blood, urine, and malignant ascites. Exosomes are cup-shaped polyvesicles (MVBs) of varying sizes, ranging from 30 to 100 nm. MVBs are specialized endosomes in the cellular endocytosis pathway, formed by the budding and cleavage of vesicles from the boundary membrane into the endosomal lumen. During MVB formation, transmembrane and peripheral membrane proteins are absorbed into the vesicle membrane, while cytoplasmic components are simultaneously incorporated into the vesicle. As this process progresses, the MVB eventually fuses with the cell membrane, inducing the release of exosomes from the cell.

[0053] As used herein, the terms “extracellular matrix” and “ECM” are used interchangeably and mean and encompass collagen-rich material found between cells of mammalian tissues, and any material derived therefrom, such as decellularized ECM. In certain embodiments, the ECM material is derived from human placenta. Naturally, placental extracellular matrix is ​​derived from the placenta.

[0054] As used herein, the term "hydrogel" refers to a polymer chain with a water-insoluble, three-dimensional network structure that can hold a large amount of water.

[0055] As used herein, the terms “freeze-dried” and “dehydrated” are used interchangeably and refer to a state from which water has been removed, rather than a method of removing water as a means of preservation.

[0056] As used herein, the term “osteoconductive” refers to a material or substance that supports and promotes bone growth. In the context of medical devices or implants, osteoconductivity describes the ability of a material to function as a scaffold or framework for new bone formation. Osteoconductive materials promote the attachment, proliferation, and differentiation of osteocytes, and facilitate the integration of implants with surrounding bone tissue. This property is critical to the success of bone grafts, implants, and other orthopedic or dental devices designed to repair or replace bone tissue. Materials, such as certain ceramics, calcium phosphate, and bioactive glass, are often considered osteoconductive due to their ability to stimulate bone regeneration.

[0057] As used herein, the term "pharmaceutical composition" refers to a composition comprising at least one ophthalmic agent, excipient, carrier, etc. The pharmaceutical composition, rather than the ophthalmic agent alone, is administered to the patient.

[0058] As used herein, the terms “particle” and “granule” are interchangeable and refer to very small parts, pieces, fragments, or quantities.

[0059] As used herein, the term “porous” refers to a material or structure characterized by the presence of interconnected voids or pores within the material. These voids allow substances, such as fluids or gases, to pass through or diffuse into the material. The porosity of a material can result in properties, such as increased surface area, permeability, or absorption, which may be advantageous for a variety of applications, including filtration, insulation, catalysis, or controlled release of substances. The degree of porosity can range from fine porosity, which contains very small interconnected voids, to coarse porosity, which contains larger voids. “Osteoconductive” refers to a material or substance that supports and promotes bone growth.

[0060] As used herein, the terms “porous and osteoconductive” refer to materials or structures that possess both porous and osteoconductive properties. Such materials are not only permeable, allowing the passage or diffusion of substances, but also promote bone growth and integration with surrounding bone tissue. This combination of properties is particularly advantageous for medical devices or implants intended for orthopedic or dental applications. The porous nature of the material provides a framework for internal cell growth and angiogenesis, while its osteoconductivity stimulates bone formation and promotes osseointegration between the implant and surrounding tissue. This dual functionality increases the effectiveness and long-term success of implants in supporting bone regeneration and structural integrity.

[0061] As used herein, the term “placenta” refers to the disc-shaped tissue that connects the mother’s uterus to the umbilical cord and is ultimately responsible for the delivery of nutrients and oxygen to the fetus. The placenta consists of three layers of membranes: (1) the amnion, which is a single layer of ectoderm epithelium that completely encloses the embryo; (2) the chorionic membrane, which surrounds the amniotic sac and contains the chorionic and trophoblast membranes; and (3) the decidua, which is the maternal endometrium. For the purposes of this disclosure, the umbilical cord is not considered part of the placenta.

[0062] As used herein, the term “placental-derived” in relation to biomaterials indicates that the source of the biomaterial is the placenta, which is typically obtained from animal (e.g., pig or sheep) or human placental tissue. Examples of placental-derived biomaterials to be applied herein are decellularized placental-derived extracellular matrix and placental-derived exosomes. Placental-derived biomaterials are proposed to be immunoprivileged, so as to be proposed to rapidly re-establish healing signals and limit xenobiotic reactions when presented physically and biochemically to the “natural” administration site of a subject. A specific example of decellularized placental-derived extracellular matrix to be applied to the pharmaceutical compositions of this disclosure is decellularized dehydrated human amniotic membrane (DDHAM).

[0063] As used herein, “therapeutic agent” means a compound or substance in a pharmaceutical composition that is biologically active and produces the effect of the pharmaceutical composition.

[0064] The bone growth compositions of this disclosure may be hydrated for point-of-care administration or stored at low temperatures, for example, about 2°C to about 8°C. Examples of hydration solutions for the bone growth compositions of this disclosure may vary. Examples of such liquids include sterile sodium chloride solution, for example about 0.9% sodium chloride (hereinafter referred to as "saline"), blood, plasma, bone marrow, etc. The effective amount of the bone growth composition may vary depending on the extent of the bone defect and whether the composition is used as a bone graft, bone implant, or bone graft material.

[0065] In certain embodiments, the bone growth composition of the present disclosure is administered directly to a target site (e.g., a bone defect to be treated).

[0066] The mass ratio of the mineral source to the biocompatible material, such as a substantially residue-free decellularized placental extracellular matrix, in the bone growth compositions described herein may vary. Examples of such ratios applied herein include 5:95%, 10:90%, 15:85%, 20:80%, 25:75%, or 30:80%. In certain embodiments, the biocompatible material comprises collagen and elastin, with the amount of collagen exceeding about 60% by weight of the bone growth composition described herein and the amount of elastin being less than about 20% by weight of the bone growth composition.

[0067] Depending on the circumstances, the bone growth compositions of this disclosure may include pharmaceutically acceptable diluents, excipients, or carriers. Furthermore, the bone growth compositions of this disclosure may further include drugs or pharmaceuticals, carriers, or adjuncts for addressing potential infections, pain, or adjuncts for preventing or limiting the development of blood clots (anticoagulants).

[0068] This disclosure can be better understood by referring to the following non-limiting embodiments provided as examples of this disclosure. The following embodiments are presented to better illustrate preferred embodiments of this disclosure; however, they should not be construed as limiting the broad scope of this disclosure. [Examples]

[0069] [Example 1] Procurement of placenta for producing the bone growth composition described herein. Placental starting material is sourced using established methods designed to reduce the risk of further contamination and prevent improper labeling and / or tracking errors. Tissue is sourced from accredited hospitals, and collection is performed by authorized medical professionals. Tissue collection kits used include sufficient containers and barcode labels for the biomaterials, along with appropriate documentation and instructions for umbilical cord collection. Furthermore, placental tissue is screened for the presence of transfusion-transmitting viruses in accordance with FDA Guidance for Industry: Eligibility Determination for Donors of Human Cells, Tissues and Cellular and Tissue-Based Products (HCT / Ps) - August 2007.

[0070] [Example 2] Production of bone growth compositions according to the disclosure The preparation of the bone growth compositions disclosed herein begins with the procurement of placental tissue obtained from a normal full-term birth, and is therefore procured from the entire human placenta. The frozen placental tissue is released from isolation for processing and thawed at 4–8°C for up to 96 hours. The placental tissue is cut into small fragments and homogenized using an Omni Mixer homogenizer. The homogenization process helps to generate small tissue microparticles with increased surface area, allowing for more effective separation and removal of cells and cellular debris from the placental tissue. The homogenized tissue derived from a single placenta is transferred to a sterile processing container containing a sodium chloride (NaCl) solution. The tissue is shaken in an orbital shaker and repeatedly washed with sterile NaCl, the NaCl solution is replaced by centrifugation to precipitate the tissue microparticles, the supernatant is removed and replaced with a fresh sterile NaCl solution. The placental tissue is incubated at controlled room temperature (20–25°C) for up to 24 hours with shaking in a sterile NaCl solution, the supernatant is removed by centrifugation and washed with sterile water. Exposure of placental tissue to high concentrations of NaCl, followed by hypotonic water, results in an "osmotic shock" to the tissue. Osmotic shock helps remove blood, blood components, cells, and cellular debris from the tissue. Placental tissue is subjected to a second osmotic shock before the nucleic acid removal process.

[0071] After washing with water, remove the supernatant from the sterile container and replace it with a solution containing BENZONASE. Incubate in an orbital shaker at room temperature for up to 24 hours to aid in the degradation of all forms of nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)).

[0072] After thoroughly rinsing with sterile water to remove BENZONASE, the tissue is subjected to surfactant washing with sodium deoxycholate (DOC) solution for up to 24 hours, followed by a further washing with freshly replaced DOC solution for up to 72 hours. DOC is a mild, water-soluble bile acid-derived ionic surfactant used to remove residues such as cells, cell membranes, and other cellular debris. After surfactant washing is complete, the tissue is washed with sterile water to remove debris and residual DOC.

[0073] To concentrate collagen, a placental structural protein, the placental ECM is treated with a sodium hydroxide (NaOH) solution to denature and remove all soluble extracellular matrix elements, non-crosslinked proteins, i.e., cytokines / chemokines, growth factors, and hormones. Subsequently, the ECM is treated with hydrochloric acid (HCl) to neutralize the NaOH and remove the aforementioned impurities. The NaOH and subsequent HCl incubation are used as a "concentration step." Titration of this step allows for careful selection of a matrix composition containing a concentrated collagen content and a titrated, desirable level of elastin content of less than 20%. This concentration process and subsequent thorough washing with water further facilitate the removal of solubilized product residues, such as cells, cell debris, nucleic acids, growth factors, chemokines / cytokines, hormones, and processing reagents, such as salts, DOC, and BENZONASE.

[0074] The resulting placental structural protein concentrate is tested for collagen and elastin content. The methods used are well-established in the field of biochemical testing; collagen is tested using a hydroxyproline-based colorimetric assay, and elastin is tested using a dye-5,10,15,20-tetraphenyl-21H,23H-porphinetetra-sulfonate-based colorimetric assay.

[0075] After confirming the collagen and elastin ratios, transfer the placental decellularized structure protein concentrate composition to a sterile mold, freeze at -80°C, and freeze-dry. Seale the freeze-dried material and store at room temperature until ready for use in formulations with β-TCP granules in various ratios, including the examples discussed below.

[0076] [Example 3] Determination of the composition of the bone growth composition of this disclosure When the obtained placental structural protein concentrate was tested for collagen and elastin levels, it was found that the 100% composition contained a concentration of over 60% collagen, elastin was optimally titrated to less than 20%, and residual water content was titrated to less than 20%.

[0077] [Table 1]

[0078] To investigate the collagen types in the placental structural protein concentrate, collagen types I, III, IV, V, and VI were analyzed by molecular weight using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and compared to their respective human placental collagen standards according to ASTM 2122-192. The results showed that alpha-1 type I collagen and alpha-1 type III collagen moved at the same rate on the SDS-PAGE gel, and the gel band patterns overlapped, confirming that the prominent bands in the placental structural protein concentrate correspond to collagen types I and III. Collagen type IV was below the assay limit of detection (LOD). Collagen types V and VI could not be detected in the placental structural protein concentrate due to the absence of their characteristic bands. Only protein bands corresponding to collagen types I and III were observed. These collagen types account for the majority of the collagen content in the placental structural protein concentrate. Figure 1 shows the results of SDS-PAGE analysis of the growth composition of this disclosure, shown as ECM compared to collagen standards.

[0079] [Example 4] Determination of the mass ratio of mineral sources to decellularized placental extracellular matrix that is substantially free of residual substances. The bone growth composition of this disclosure, developed with the desired ratio of collagen to elastin as confirmed by biochemical assay, is then transferred to a sterile mold, frozen at -80°C, lyophilized, and stored at room temperature. The lyophilized matrix is ​​formulated into β-TCP granules, with a size ranging from approximately 250 micrometers to approximately 3000 micrometers, in various mass ratios of β-TCP and biocompatible material, such as decellularized placental extracellular matrix substantially free of residues, including but not limited to 5 / 95%, 10 / 90%, 15 / 85%, 20 / 80%, 25 / 75%, and 30 / 70%.

[0080] To give just a few examples, we calculated the dry weight of decellularized placental extracellular matrix, substantially free of residual substances, relative to the required ratio of mineral components and β-TCP to form ratios of 5 / 95%, 10 / 90%, 15 / 85%, 20 / 80%, 25 / 75%, and 30 / 70%.

[0081] Sterile water was added to the decellularized placental matrix, transferred to a sterile plastic container, and homogenized using a probe. The placental ECM mixture was combined with β-TCP granules in the desired mass ratio and dispensed into sterile molds of the desired shape (cylindrical, cubic, etc.). The molds were frozen and dehydrated by freeze-drying.

[0082] Figure 2 shows several forms of cubic lyophilized biocompatible graft material, in which β-TCP granules or particles have a size ranging from about 250 to about 2000 micrometers and are distributed throughout the bone growth composition of this disclosure, with matrix / β-TCP ratios of 10 / 90%, 15 / 85%, and 20 / 80%. Figure 3 shows one form of cylindrical lyophilized biocompatible graft material, having small-sized mineral granules having a size ranging from about 250 to about 1000 micrometers and being distributed throughout the bone growth composition of this disclosure. Figure 4 shows another form of cylindrical bone growth composition described herein (top view and side view), having particle sizes ranging from about 1000 to 2000 micrometers and being distributed throughout the bone growth composition.

[0083] Figure 5 shows another form of the bone growth composition described herein in strip structure, containing 95% of a mineral source in particulate form with particle sizes ranging from approximately 250 to 1000 micrometers, and 5% of a biocompatible material, such as placental extracellular matrix substantially free of residues.

[0084] [Example 5] Crosslinking of decellularized placenta-derived extracellular matrix As discussed above, the extracellular matrix enriched with placental structural proteins, developed with the desired ratio of collagen to elastin and confirmed by biochemical assay, is then transferred to a sterile mold, frozen at -80°C, lyophilized, and stored at room temperature. The lyophilized matrix is ​​formulated into β-TCP granules ranging in size from approximately 250 micrometers to approximately 3000 micrometers, in various mass ratios containing, but not limited to, 5 / 95%, 10 / 90%, 15 / 85%, 20 / 80%, 25 / 75%, and 30 / 70% placental matrix / β-TCP.

[0085] The dry weight of the extracellular matrix enriched with placental structural proteins was calculated for the required ratios of mineral components and β-TCP to form the ratios of 5 / 95%, 10 / 90%, 15 / 85%, 20 / 80%, 25 / 75%, and 30 / 70%.

[0086] Sterile water was added to the decellularized placental matrix, transferred to a sterile plastic container, and homogenized using a probe. The placental ECM mixture was combined with β-TCP granules in the desired mass ratio and dispensed into sterile molds of the desired shape (cylindrical, cubic, etc.). The molds were frozen and dehydrated by freeze-drying.

[0087] [Example 5] Crosslinking of the bone growth composition of this disclosure in the form of a bone implant Crosslinking tests were conducted to examine various methods for stabilizing the structure and handling properties of the bone growth composition disclosed herein. The crosslinking methods tested included a) chemical crosslinking using carbodiimide and glutaraldehyde chemical reactions, which require lengthy washing procedures to remove chemicals, such as crosslinking agents and catalysts; and b) physical crosslinking using heat treatment, one of the earliest published physical methods used for collagen crosslinking (Yannas IV, Tobolsky A V. Nature. 1967). As a result of heat dehydration, collagen crosslinks were formed in the absence of catalysts, excipients, or crosslinking agents. No further chemicals, excipients, or crosslinking agents were present during the heat dehydration crosslinking process. Figure 6 shows one embodiment of excellent crosslinking of the bone growth composition disclosed herein by heat dehydration treatment.

[0088] [Example 7] Blood rehydration of the bone growth composition of the present disclosure having a 20:80% mass ratio of decellularized placenta-derived ECM substantially free of residual substances and a mineral source. Blood was added to the molded bone growth composition described herein in a 20:80% ratio of β-TCP and a biocompatible material (e.g., decellularized placenta-derived ECM substantially free of residual material in the form of implants as described above). The rehydrated implant was molded into the desired ball shape as shown in Figure 7, enabling excellent positioning and implantation within the defect. When the implant was cut in half after rehydration, it was shown that the blood had completely penetrated the implant to the center and hydrated it. Figure 8 shows the bone growth composition of this disclosure in the form of an implant at 20:80% β-TCP, rehydrated with blood.

[0089] [Example 8] Rehydration and handling of the bone growth composition of the present disclosure When a placental structural protein-enriched bone implant with β-TCP in a 20 / 80 ratio, such as the bone growth composition disclosed herein, was rehydrated with saline for 15 minutes, the implant remained intact and the β-TCP granules were retained within the implant. The rehydrated implant was removed from the well and molded into a ball, mimicking a clinical procedure. Unexpectedly, the implant exhibited excellent integrity, moldability, and handling without any apparent separation of β-TCP. However, such excellent integrity, moldability, and handling were not observed when β-TCP alone was rehydrated and molded, as evidenced by the multiple granules remaining in the well. Figure 9 shows the rehydration and handling of bone implants with 20:80% β-TCP compared to β-TCP alone.

[0090] [Example 9] Morphological analysis of bone growth compositions of the present disclosure of bone implant morphology Figure 10 shows the morphological analysis of the bone growth composition of the present disclosure in 20:80% β-TCP having a β-TCP particle size range of approximately 250 to 2000 micrometers (left) and approximately 1000 to 2000 micrometers (right). Morphological analysis of the bone implant in 20:80% β-TCP was performed using SEM analysis.

[0091] [Example 10] Ratio analysis of bone growth composition of bone implant morphology in this disclosure The organic-to-inorganic ratio and inorganic phase composition of the bone growth compositions described herein in the form of bone implants were tested. Obtaining the organic and inorganic weight composition requires heating / sintering the sample to a temperature that decomposes (ashes) the organic components, leaving inorganic granules. The heat treatment / sintering simultaneously prepares the inorganic granules (remaining after sintering) which are analyzed for phase composition using X-ray diffraction (XRD). Ash content testing was performed to establish the specifications for the weight percentage of organic material to inorganic components. Before the sample undergoes XRD analysis to obtain phase purity and phase weight composition, the sample was sintered at 100°C ± 25°C to increase and normalize the microcrystal size compared to the XRD standard used for XRD calibration. Increasing the microcrystal size is necessary to better define peaks during XRD scanning and to provide a flat baseline. The required sintering of the sample occurs during the heat treatment for ash content testing.

[0092] Sintered granules obtained as a result of ash content testing were ground using a mortar and pestle made of synthetic sapphire and analyzed according to established test procedures. The ground samples were scanned at a rate of 1.20 degrees per minute at 20–60° using a Rigaku Miniflex 11 X-ray diffractometer according to established procedures. Calcium phosphate patterns from the International Center for Diffraction Data / Joint Committee on Powder Diffraction Standards (ICDD / JCPDS) library, stored in JADE 9 analysis software, were compared with the sample patterns. All matching calcium phosphate patterns were reported as crystalline phase impurities. The sample diffraction patterns were provided by superimposing the patterns of each phase from the pattern library.

[0093] Phase purity quantification: Calibration curves were created for various calcium phosphate phases (hydroxyapatite / P-tricalcium phosphate / pyrophosphate). These calibration curves were used to quantify the impurity phases detected in the test samples. The detection limit for hydroxyapatite and P-tricalcium phosphate is 1%, and pyrophosphate has a detection limit of 4%, although it may be visible down to 0.5%. Figure 11 shows the XRD spectra and overlay analyses of the bone growth compositions described herein in the form of bone implants in 20:80% β-TCP.

[0094] [Example 11] FTIR analysis of bone growth composition of the present disclosure of bone implant morphology Fourier transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode was used to characterize placental structural protein-enriched biocomposite bone implants, e.g., the bone growth compositions disclosed herein in the form of bone implants, and control samples. A minimum test sample size of 10 mm × 10 mm was used for analysis. Five batches were included as part of this feasibility study. Five FTIR spectra were randomly acquired from each test sample. A minimum of 32 scans were collected per spectrum at a spectral resolution of 4 cm - 1. Data processing was performed at two levels using OMNIC software: 1) identify collagen characteristic peaks; compare and contrast characteristic peaks from the product and control groups for potential changes due to additives or degradation; and 2) perform further spectral processing to establish range and standard deviation. Level 2 analysis included: performing curve fitting of selected regions to access comparative percentages of structural proteins in the product; and 3) establishing range and standard deviation of selected peaks or indices (peak ratios). Figure 12 shows the FTIR spectra and stability analysis of placental decellularized ECM and PSPE-BI.

[0095] [Example 12] Placement of the bone growth composition of this disclosure in the form of a bone implant in packaging designed for shipment and clinical rehydration. The freeze-dried bone implants are placed in an inner plastic tray sealed with an aluminum cover using a medical sealing device. The sealed inner tray is then placed inside an outer tray sealed with aluminum foil, as shown in Figure 13. The double-sealed trays are labeled and prepared for sterilization. Figure 13 shows the bone growth composition of this disclosure in the form of bone implants in packaging trays containing 10 cc, 5 cc, and 2.5 cc, providing a system for adding appropriate rehydrating agents in a clinical setting.

[0096] [Example 13] Bone growth compositions formed with various mass ratios of ECM and mineral sources. Placental structural protein-enriched matrix, such as the decellularized placental extracellular matrix containing collagen and elastin, substantially free of residual substances as discussed above, was mixed with β-TCP in various ratios, including 5 / 95%, 10 / 90%, 15 / 85%, 20 / 80%, 25 / 75%, 30 / 70%, 40 / 60%, and various β-TCP particle sizes including about 250 to about 3000 micrometers. The mixture was then homogenized to completely disperse the β-TCP within the ECM to form the bone growth composition described herein, containing dispersed β-TCP granules. Figure 14 shows the bone growth composition after homogenization with the dispersed β-TCP therein. The method for dispersing a mineral source within the bone growth composition of this disclosure provides an unexpected method for generating a finely pulverized, small-sized mineral source in particulate form.

[0097] [Example 14] In vivo studies of the bone growth composition of this disclosure in ECM vs. β-TCP ratios of 15 / 85 and 30 / 70% Pilot evaluation study of the bone growth composition disclosed herein in the form of bone graft material implanted in critical-size defects in the long bones of New Zealand white rabbits: Implants were placed in 6 mm (mm) femoral condyle defects drilled to a depth of 10-12 mm. Implants #1 = 15% ECM: 85% β-TCP and implant #2 = 30% ECM: 70% β-TCP were rehydrated with saline or BMA (bone marrow aspirate). Implants #1 and #2 were compared to autologous grafts and tricalcium phosphate (β-TCP) alone at 0, 42, and 84 days. BMA was obtained from the iliac crest and combined with the test material. New bone growth was evaluated by microcomputed tomography (micro-CT) analysis at 0, 6, and 12 weeks post-implantation, and by histological evaluation at 6 and 12 weeks post-implantation. The local tissue response, inflammation, and potential for intra-bone growth after implantation of the test material were evaluated compared to β-TCP and autografts. Figure 15 shows the bone growth composition alone at 12 weeks. Figure 16 shows the autograft at 12 weeks. Figure 17 shows test group 1 (FUSE 1) at 12 weeks. Figure 18 shows group 2 (FUSE 2) at 12 weeks.

[0098] [Example 15] In vivo testing of the bone growth composition of this disclosure in critical size bone defects in long bones of New Zealand white rabbits - β-TCP of different particle sizes A 6 mm femoral condyle defect was drilled to a depth of 10–12 mm. Two bone growth compositions in the form of implants (one with β-TCP particles ranging in size from approximately 250–2000 micrometers, and the other with β-TCP particles ranging in size from approximately 1000–2000 micrometers) were placed in the bone defect. The bone growth compositions in the form of bone implants were further combined with biomaterials, such as bone marrow aspirate (BMA), and tested at 3 and 6 weeks. As part of the procedure, BMA was obtained from each tibia and combined with the test material. Micro-CT and histological analysis at 3 and 6 weeks post-implantation were performed to evaluate local tissue response, inflammation, and intra-bone growth after implantation. Figure 19 shows BMA generation and application of bone marrow aspirate and bone growth composition implants. Figure 20 shows rehydration and placement of bone growth composition implants in a critical-size bone defect. Figure 21, Micro-CT imaging of bone growth composition implants in critical-size defects at 3 and 6 weeks, with β-TCP particle sizes ranging from 250 to 2000 micrometers. Figure 22, Micro-CT imaging of bone growth composition implants in critical-size defects at 3 and 6 weeks - β-TCP particle size range of approximately 1000 to 2000 micrometers. Figure 23, Histological image of the bone growth composition of this disclosure showing the morphology of the bone implant in critical-size defects at 3 and 6 weeks - β-TCP particle size range of 1000 to 2000 micrometers. Figure 24, Histological image of the bone growth composition of this disclosure showing the morphology of the bone implant in critical-size defects at 3 and 6 weeks - β-TCP particle size range of 1000 to 2000 micrometers.

[0099] [Example 16] In vivo study of bone growth compositions of the present disclosure in the form of bone implants in critical-size bone defects in long bones of New Zealand white rabbits. Bilateral severe distal femoral (spongiform region) defects (6 mm × 10 mm) are created in New Zealand white rabbits with mature skeletons. Treatment of both defects in one animal consists of no treatment (empty defect), bone growth compositions disclosed herein in the form of bone implants, or comparative scaffold foam material. New bone growth is evaluated at both 6 and 12 weeks after implantation into the defect. Bone growth is measured by micro-CT and polymethyl methacrylate (PMMA) histological imaging and histological morphometry. Day 3 provides a baseline for micro-CT and histological morphometry. Week 6 allows for evaluation of the initial biological response to the device. The final time point (12 weeks) should be sufficient to demonstrate bone healing and the effects of any residual device material. Paraffin tissue sections are also used for ISO 10993-6 evaluation from the time points mentioned above.

[0100] [Example 17] In vivo study of bone growth composition of the present disclosure regarding the morphology of bone implants in a rabbit posterolateral fixation model. An 8-week posterolateral fixation study was conducted in skeletal mature New Zealand white rabbits (n=15). Each rabbit underwent sterile bilateral surgery to resect the transverse processes (TPs) of the fourth and fifth lumbar vertebrae (L4-L5). Bone marrow aspirate was obtained from the iliac crest and combined with the bone growth composition disclosed herein in the form of a bone implant. Approximately 2.5-3 cc of the test material mixture was spread in the paraspinal bed where the resected L4-L5 TPs were implanted. X-ray examinations were performed immediately before surgery to identify the L4-L5 surgical site, postoperatively to verify implant placement, and at the end of the study (excised specimens) to evaluate fusion. On day 0, surgery was performed at L4-L5, and bone marrow aspirate from the iliac crest was combined with the bone growth composition disclosed herein in the form of a bone implant and implanted at L4-L5. At 8 weeks (excised specimens) at the end of the study, the fusion of the test material was evaluated by X-ray, micro-CT, and histological imaging, and compared with the β-TCP fusion rate of previous autologous grafts and negative controls. Figure 25 shows the bone growth composition in the form of a bone implant with 20:80% β-TCP, rehydrated with bone marrow aspirate and filled into a syringe for implantation / placement in the rabbit defect in a posterolateral fixation model. Figure 27 shows the histological results at 8 weeks for a bone growth composition implant with 20:80% β-TCP rehydrated with bone marrow aspirate in a rabbit defect in a posterolateral fixation model. Rows of osteoblasts that have migrated to the defect and angiogenesis are indicated by arrows. Figure 27 shows the histological results at 8 weeks for a bone growth composition implant with 20:80% β-TCP rehydrated with bone marrow aspirate in a rabbit defect in a posterolateral fixation model. Rows of osteoblasts that have migrated to the defect and angiogenesis are indicated by arrows.

[0101] [Example 18] In vivo testing of the morphology of bone implants in a canine model of the bone growth composition disclosed herein. To evaluate the bone growth compositions described herein in the form of bone grafts (bone graft materials) that come into direct contact with bone tissue, a bilateral canine humeral defect implant test is performed. Bone remodeling, new bone formation, and implant resorption are evaluated periodically. The bone graft material is supplied in a form that yields a flexible graft when moistened. A bovine collagen containing a bone space filler, such as VITROSS Foam Bone Implant, is used as a control.

[0102] The test material or control material is implanted into bilateral drill defects surgically created in the proximal humerus cancellous bone of 18 dogs. According to a randomization schedule established before the start of the study, the bone graft material is implanted into the drill defect of one humerus, and the control material is implanted into the drill defect of the opposite humerus in each animal. The drill defects are approximately 10 mm in diameter and 25 mm deep. Lateral and dorsopalmar images are obtained immediately postoperatively, and all animals are subsequently radiographed at each sacrifice time. After a predetermined exposure period (3, 6, 12, 24, or 52 weeks), the animals are sacrificed and the implantation sites are exposed. The implant sites are observed visually, retrieved, wrapped in gauze sponge soaked in saline, and frozen at approximately -20°C for further analysis.

[0103] Analysis of the recovered implant sites includes mechanical tests to evaluate intra-bone growth and remodeling at the defect site. Tissue implant sites are also examined using standard histological methods well known in the art. The degree of healing and the nature of the tissue contained within the defect are characterized by histopathological and histomorphometric evaluations. Defects treated with the bone growth compositions described herein exhibit bone healing that is equal to or better than that observed in defects treated with control graft materials, or a degree of healing equal to or better than that observed in defects treated with control graft materials.

[0104] [Example 19] Clinical application of bone growth compositions of the present disclosure in the form of bone implants for critical size defects Several surgical approaches are described for the surgical treatment of late osteochondrosis dissecans (OCD) of the knee and ankle that has not been successfully managed with non-surgical methods. The bone growth compositions described herein in the form of bone implants may be the optimal surgical option. All surgeries are performed using retrograde drilling for OCD in the talar dome and femoral condyle. Approximately 10 patients are treated with retrograde drilling or treffin drilling under arthroscopic and fluoroscopic guidance to prevent damage to the cartilage surface. The subchondral space is filled with a mixture of 20-40 mg bone growth composition bone implants rehydrated with either saline, blood, or small fragments of bone graft. The mixture is packed using a drill guide and positioned with a Steinman pin. Thus, only the subchondral defect is filled, while the area around the drill hole remains empty. Evaluation is obtained by clinical evaluation, radiography, and magnetic resonance imaging. The follow-up period will be an average of 24 months to a maximum of 48 months. In all cases, bone density is expected to increase in the subchondral region of bone growth composition and bone implant grafts, and mechanical indentation of the joint surface can be prevented. Favorable clinical outcomes (range of motion, pain) and radiographic results (bone healing) are expected after this new treatment method.

[0105] [Example 20] Bone growth compositions disclosed herein using microparticle-sized βTCP for dental bone growth The bone growth compositions disclosed herein, prepared from finer-grained calcium phosphate, can function as dental implants (Figure 28) and are evaluated in a rat cranial critical-size defect dental implant model to demonstrate osteoconductivity, bone formation, and test remodeling. The bone growth compositions are used to fill a 5.0 mm diameter cranial defect in rats. The defects are evaluated by histological and histomorphological analysis of demineralized tissue sections stained with hematoxylin and eosin at 6 and 10 weeks postoperatively, respectively. Figure 28 shows the use of the bone growth compositions of this disclosure in the morphology of a tooth graft.

[0106] [Example 21] Bone growth composition in the form of a bone implant as a scaffold for delivering exosomes (disclosure of this disclosure) To fill large critical-size bone defects and achieve desired therapeutic outcomes, the bone growth composition of this disclosure, in the form of a bone implant, is integrated with exosomes. Exosomes isolated from placental cells or placental tissue are packed into the bone implant by physical absorption in the operating room, and the exosome-containing implant is placed in a critical-size bone defect in a rat tibial critical defect using the bone implant as a carrier. Bone regeneration is quantified using microcomputed tomography and histological examination. Combined with the induction of early migration and mineral deposition by preosteoblasts, exosome treatment demonstrates that the positive effect on bone mineralization by local delivery of exosomes packed into a delayed-absorption bone implant may offer a potential alternative to autologous grafts for bone reconstruction.

[0107] [Example 22] Bone growth composition disclosed in the form of a bone implant as a scaffold for delivering biomaterials To fill large critical-size bone defects and obtain desired therapeutic outcomes, the bone growth compositions described herein in the form of bone implants are seeded with cells known to stimulate bone tissue formation (including, but not limited to, mesenchymal stem cells, adipose tissue-derived stem cells, osteoblasts, and placental-derived cells). The cells are seeded into the bone implant, where they settle and produce cellular byproducts within the implant. Implants containing cells and cellular byproducts are placed in critical-size bone defects in a rat tibia model. Bone regeneration is quantified using microcomputed tomography and histological examination. Combined with the induction of early migration and mineral deposition by preosteoblasts, treatments based on cells and cellular byproducts demonstrate a positive effect on bone mineralization by local delivery of cells or cellular byproducts filled into slow-resorbing bone implants. Cellular byproducts include, but are not limited to, cell-conditioned media and cellular secretome contents. Such applications may offer a potential alternative to autologous grafts for bone reconstruction.

[0108] [Example 23] Bone growth compositions disclosed herein in the form of bone implants as scaffolds for delivering biomaterials To fill large critical-size bone defects and obtain desired therapeutic outcomes, the bone growth compositions described herein in the form of bone implants are combined with biomaterials (including, but not limited to, growth factors known to stimulate bone growth, e.g., platelet-derived growth factor BB (PDGF-BB), bone morphogenesis protein 2 (BMP-2)). The biomaterials are filled into bone implants, and the resulting bioactive material-containing implants are placed in critical-size bone defects in a rat tibia model. Bone regeneration is quantified using microcomputed tomography and histological examination. Combined with the induction of early migration and mineral deposition by preosteoblasts, the bioactive treatment demonstrates a positive effect on bone mineralization through local delivery of cells filled into slow-resorbing bone implants. This may offer a potential alternative to autologous grafts for bone reconstruction.

[0109] [Example 24] Bone growth compositions disclosed herein in the form of bone implants having controlled channels that induce new intravascular growth. Scaffolds with controlled pore structures were produced using a 3D printed mold based on a computer-aided design (CAD) model in which rods were uniformly arranged within the mold. After shaping, these rods created horizontal channels within the implant. These channels would induce new vascular endovascular growth into the bone tissue. Figure 29 shows the use of a 3D printed mold with rods that create channels within the bone growth composition described herein in the morphology of a bone implant and control the channels to induce new vascular endovascular growth into the bone tissue. Rods of different spacings and configurations are utilized to enable more effective vascular endovascular growth. Figure 29 shows the use of a 3D printed mold with rods that create channels within the bone growth composition described herein in the morphology of a bone implant and control the channels to induce new vascular endovascular growth. The dimensions of these horizontal projections are optimized for implant integrity and handling. Pores ranging in size from 20 to 1,500 μm were used. Initial tests demonstrated that the minimum pore size for significant bone growth was 75–100 μm, with an optimal range of 100–270 μm.

[0110] In addition to the use of molds to modify the layout of the bone growth compositions of this disclosure, and due to the beneficial properties of the bone growth compositions described herein, such as their plasticity and moldability, the compositions can be readily used as materials that can be easily modified to easily alter the shape of the bone growth compositions of this disclosure when applied as three-dimensional printing materials. Thus, for example, to produce bone implants or grafts for extremely precise bone defects, the bone defect may optionally be mapped using a laser, and the information may be sent to a three-dimensional printer, allowing for the printing of a perfectly fitting bone graft or implant from the bone growth compositions described herein.

[0111] [Example 25] Bone growth compositions disclosed herein in the form of bone implants that support mesenchymal stem cell (MSC) attachment and growth. Human mesenchymal stem cells were seeded in the bone growth composition of the present disclosure in the form of a bone implant, compared to a commercially available product containing 20% ​​human bovine collagen and 80% β-TCP. Figure 30 shows human mesenchymal stem cells grown in the bone growth composition of the present disclosure in the form of a bone implant, compared to a commercially available product (control product) containing 20% ​​bovine collagen and 80% β-TCP, such as VITOSS bone implant.

[0112] This disclosure should not be limited in scope by the specific embodiments described herein. In fact, various modifications of this disclosure in addition to those described herein will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the accompanying claims.

Claims

1. (a) Decellularized placental extracellular matrix that is substantially free of residual substances and contains naturally biocompatible substrates; and (b) Ca 2- and PO 4 3- Ion mineral source A bone growth composition containing the following:

2. The bone growth composition according to claim 1, wherein the natural biocompatible substrate comprises (a) collagen, (b) elastin, (c) tropoelastin, (d) elastin-like protein, or (e) any combination of (a) to (d).

3. The bone growth composition according to claim 1 or 2, wherein the natural biocompatible substrate comprises collagen and elastin.

4. The bone growth composition according to any one of claims 2 to 3, wherein the collagen of the decellularized placenta-derived extracellular matrix, which is substantially free of residual substances, comprises at least about 60% by weight of collagen and about 20% by weight or less of elastin.

5. The bone growth composition according to any one of claims 1 to 4, further comprising a synthetic biocompatible substrate.

6. The bone growth composition according to claim 5, wherein the synthetic biocompatible substrate comprises (a) PLA, (b) PLGA, (c) biodegradable polyesteramide polymer, (d) hydrogel, (e) polyether ketone, or (f) any combination of (a) to (e).

7. A bone growth composition according to any one of claims 1 to 6, wherein the mass ratio of the mineral source to the decellularized placenta-derived extracellular matrix substantially free of residual substances in the bone growth composition includes (a) about 5% to about 95%, (b) about 10% to about 90%, (c) about 15% to about 85%, (d) about 20% to about 80%, (e) about 15% to about 85%, or (f) about 30% to about 70%.

8. The bone growth composition according to any one of claims 1 to 7, wherein the mineral source is in the form of particles ranging in size from (a) about 250 μm to about 3000 μm, (b) about 250 μm to about 2000 μm, (c) about 250 μm to about 1000 μm, or (d) about 1000 μm to about 2000 μm.

9. Ca 2+ and PO 4 3- The mineral source of ions is (a) Ca(H2PO4)2, (b) CaHPO4, (c) Ca3(PO4)2, (d) Ca5(PO4)3(OH), or (e) any combination of (a) to (d). A bone growth composition according to any one of claims 1 to 8, comprising:

10. A bone growth composition according to any one of claims 1 to 9, further comprising a biological material.

11. The bone growth composition according to claim 10, wherein the biological material comprises cells, a conditioned cell culture medium, a cell secretome, plasma, genetic material, exosomes, bone marrow aspirate, platelet plasma, growth factors, or any combination thereof.

12. (a) The genetic material includes nucleic acids, nucleotides, or combinations thereof, (b) The cells include mesenchymal stem cells, adipose tissue-derived stem cells, osteoblasts, placental-derived cells, or any combination thereof. (c) Exosomes are produced by the cells in (b), (d) The conditioned medium is produced by the cells of (b), The bone growth composition according to claim 11.

13. The bone growth composition according to any one of claims 1 to 12, wherein the bone growth composition is (a) porous, (b) osteoconductive, or (c) porous and osteoconductive.

14. The bone growth composition according to any one of claims 1 to 13, wherein the bone composition is a bone graft, a bone implant, or a bone graft material.

15. A method for treating a bone defect in a subject, comprising administering an effective amount of the bone growth composition described in any one of claims 1 to 14 to the subject at the time of the bone defect.

16. The method according to claim 15, wherein the bone defect includes (a) injury from trauma, (b) congenital or acquired bone defect or abnormality, (c) surgical bone resection, (d) sequelae of bone infection, and (e) comminuted fracture with bone loss.

17. (a) a bone growth composition according to any one of claims 1 to 14, and (b) Packaging containers specially designed to accommodate various product sizes and the addition of rehydration solution. A kit that includes this.

18. The kit according to claim 17, further comprising a container for storing biological material.

19. The kit according to claim 17, wherein the packaging container is designed to provide a sealed chamber for containing biological material.

20. The kit according to any one of claims 18 or 19, wherein the biological material comprises cells, a conditioned cell medium, a cell secretome, plasma, genetic material, exosomes, bone marrow aspirate, platelet plasma, growth factors, or any combination thereof.

21. (a) The genetic material includes nucleic acids, nucleotides, or combinations thereof, (b) The cells include mesenchymal stem cells, adipose tissue-derived stem cells, osteoblasts, placental-derived cells, or any combination thereof. (c) Exosomes are produced by the cells in (b), (d) The conditioned medium is produced by the cells of (b), The kit according to claim 20.

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