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WO2024197316A1 - Composition de croissance osseuse - Google Patents

Composition de croissance osseuse Download PDF

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Publication number
WO2024197316A1
WO2024197316A1 PCT/US2024/021377 US2024021377W WO2024197316A1 WO 2024197316 A1 WO2024197316 A1 WO 2024197316A1 US 2024021377 W US2024021377 W US 2024021377W WO 2024197316 A1 WO2024197316 A1 WO 2024197316A1
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WO
WIPO (PCT)
Prior art keywords
bone
bone growth
growth composition
composition
collagen
Prior art date
Application number
PCT/US2024/021377
Other languages
English (en)
Inventor
Anna Gosiewska
Rajarajeswari SIVALENKA
Brandon MIRABILE
Robert POULIOT
Joseph Gleason
Shamshad AZIMI
Stephen A. BRIGIDO
Robert J. Hariri
Desiree LONG
Maumita BHATTACHARJEE
Nicolas MANN
Lukasz PRZEK
Original Assignee
Celularity Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Celularity Inc. filed Critical Celularity Inc.
Publication of WO2024197316A1 publication Critical patent/WO2024197316A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3633—Extracellular matrix [ECM]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/30—Inorganic materials
    • A61L27/32—Phosphorus-containing materials, e.g. apatite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3645—Connective tissue
    • A61L27/365—Bones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3821—Bone-forming cells, e.g. osteoblasts, osteocytes, osteoprogenitor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3847—Bones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
    • A61L2300/112—Phosphorus-containing compounds, e.g. phosphates, phosphonates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/258—Genetic materials, DNA, RNA, genes, vectors, e.g. plasmids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • This Invention disclosure relates generally to the field of bone growth compositions, methods of making of such compositions, and methods for treating bone defects in a subject using such compositions.
  • Disclosed herein are heretofore unknown bone growth compositions for use as a bone graft, bone implant or bone filler having applications In treating a subject having a bone defect in a subject.
  • Bone is a composite material comprised of collagen, ceils, a form of calcium hydroxyapatite crystals and small quantities of other proteins and organic molecules.
  • the chemistry and physical nature of this composite affords it unique properties of high strength, rigidity, and an ability to adapt to changing loads in the body.
  • injuries to bone it is necessary to find a way to fill voids or gaps, and to encourage the repair and regeneration of the bone tissue.
  • Bone injury due to trauma or diseases requires surgical intervention to replace the diseased area of bone or temporarily fill voids in the bone structure that are to be replaced by the ingrowth of a new bone tissue.
  • Products which consist of or contain hydroxyapatite (HA) (Ca5(PO4)3OH) and/or the crystalline form ⁇ of tricalcium phosphate (Ca3(PO4)2) have been historically known and used for the preparation of scaffolds useful for the bone tissue regeneration.
  • HA is therefore used in a mixture with a second mineral component, the crystalline form ⁇ of tricalcium phosphate (Ca3(PO4)2), commonly known as ⁇ -trlcalcium phosphate or by its abbreviation ⁇ TCP.
  • ⁇ - TCP has a faster resorption than HA, and therefore promotes the regrowth of the bone tissue.
  • the product OPTEMX by Exacted! (Gainesville, Fla., USA) consists of porous HA/ ⁇ - TCP granular composition only and has limited osteoconductive properties when admixed with autologous bone marrow aspirate or autologous blood.
  • the product MASTERGRAFT Putty by Medtronic, having osteoconductive properties, is formed by the same granules as the product MASTERGRAFT mentioned above, evenly distributed in a bovine collagen matrix.
  • the product Integra MOZAIK by Integra Life Sciences Corporation (Plainsboro, NJ., USA), with osteoconductive properties, consists of a mixture containing approximately 80% ⁇ -TCP and 20% crosslinked bovine collagen.
  • VITOSS Scaffold Foam Bone Graft Material is composed of approximately 80% ⁇ -TCP and 20% bovine crosslinked collagen.
  • the product CQLLAGRAFT Bone Graft Matrix by Zimmer is commercialized in the form of strips made of HA/ ⁇ -TCP in a crosslinked bovine collagen matrix.
  • the product HEALOS by DePuy consists of a matrix of bovine cross-linked collagen fibers coated with HA. This product is combined with autologous bone marrow aspirate or autologous blood.
  • a bone restorative composite material that consists of a resorbable polymer that can be collagen, a range of meso, micro and macro porosity to allow for the inclusion of fluid and to assist in bone ingrowth, as well as the inclusion of calcium phosphate particles.
  • the inventions further utilize a specific oxidation-reduction reaction of calcium and phosphorous containing salts to precipitate calcium phosphate within the collagen structure. These devices typically require very precise control of the chemistry to obtain the desired results of the precipitation of the calcium phosphate materials and appear to be limited to calcium based osteoconductive materials.
  • Patent application WO 2011/064724 Al describes mono-, bi- or multi-layer biomimetic materials for use in orthopedic surgery, and the process for their production.
  • the basic material described in this application is a bicomponent material made of the natural polymer bovine collagen added with chitosan (a polymer obtained by basic deacetylation of chitin, the natural component of exoskeleton of crustaceans).
  • the mono-layer biomimetic materials of this application are made of collagen-chitosan only.
  • This bicomponent material contains between 30 and 90% (preferably 50-80%) by weight of collagen, the remainder being chitosan, The role of chitosan is said to be for favoring the fibration of the bicomponent material,
  • bovine collagen-based materials have a long clinical history in a number of medical devices hypersensitivity reactions and immunological responses remains a recognized limitation.
  • the processing steps involved in producing any collagen based medical device can have a significant influence on the in vivo response in terms of tissue infiltration and local inflammatory response and degradation of the collagen material (Chvapif M 1977; Weadock et al., 1983; Bailey A3 2000).
  • the present disclosure is based upon the discovery that, surprisingly and unexpectedly, a novel bone growth composition of the instant disclosure readily has applications for treating a bone defect in subject that exhibits an efficacious result when used to treat a bone defect in a subject, does not suffer from the shortcomings of heretofore known compositions for use in such treatments.
  • the present disclosure extends to a bone growth composition that comprises decellularized placenta derived extracellular matrix substantially free of residual material that comprises a natural biocompatible substrate, and source of Ca 2+ and PCM 3- ions, such as a mineral source.
  • Examples of such bone defects that can be treated with a bone growth composition of the instant disclosure include but certainly are not limited to an injury from trauma, a congenital or acquired bone defect or anomaly, resection of bone due to surgery, a sequalae of bone infection; as a result of a facture, e.g, a comminuted fracture, accompanied by bone mass loss, to name only a few.
  • a bone growth composition of the instant disclosure can vary depending upon the bone defect it is being used to treat.
  • a bone growth composition of the instant invention can be a bone implant, a bone graft, or a bone filler.
  • a bone growth composition as disclosed herein can be porous, osteoconductive, or porous and osteoconductive [0002]
  • Numerous natural biocompatible substrates have applications in a bone growth composition of the instant disclosure, including but not limited to a collagen, elastin, tropoelastin, an elastin-like protein, or any combination these biocompatible substrates.
  • the natural biocompatible substrate comprises a combination of a coiiagen and elastin, such the amount of collagen of the decellularlzed placenta derived extracellular matrix substantially free of residual material comprises at least about 60% by weight collagen, and no greater than about 20% by weight elastin.
  • the type of collagen having applications in a bone growth composition of the instant disclosure can vary.
  • the collagen can be Type I collagen, Type III collagen, Type V collagen, Type VI collagen, Type VII collagen, or any combination of these collagens.
  • Type I Collagen is the most abundant collagen type in bone tissue, constituting about 90% of the total collagen content in bone. Type I collagen provides structural support and strength to bones; it also serves as a scaffold for cell attachment, proliferation, and differentiation during the bone healing process.
  • Type III Collagen although less abundant in bone compared to type I coiiagen, type III coiiagen plays a role in bone regeneration. It is often found in association with type I collagen, contributing to the formation of the coiiagen matrix. Type III coiiagen is involved in the early stages of tissue repair and remodeling.
  • Collagen types I and III are the most suitable for bone regeneration due to their abundance in bone tissue and their roles in providing structural integrity and supporting cellular processes essential for bone healing and regeneration.
  • Type V collagen is a minor component of extracellular matrix but is still present in bone tissue. It is often found in association with type I collagen fibrils and is believed to play a regulatory role tn coiiagen fibril logenesis and organization. While type V collagen may not be as predominant as types I and III In bone tissue, it could potentially influence the mechanical properties and organization of the extracellular matrix, thereby indirectly impacting bone regeneration processes.
  • Type VI Coiiagen is found in bone; it forms distinct microfibrillar structures within the ECM and contributes to tissue organization and integrity.
  • Type VII collagen is also a minor collagen that may play a role in the attachment of bone cells to the underlying matrix or basement membrane during bone development and repair processes.
  • Synthetic biocompatible substrates also have applications in a bone growth composition of the instant disclosure.
  • synthetic biocompatible substrates includes a polylactic acid (PLA), a poly (lactic-co-glycolic acid) PUGA, a biodegradable polyester amide polymer, a hydrogel, a polyether ketone, or any combination of these synthetic biocompatible substrates, to name only a few.
  • PHA polylactic acid
  • PUGA poly (lactic-co-glycolic acid) PUGA
  • a biodegradable polyester amide polymer a hydrogel
  • a polyether ketone a polyether ketone
  • the source of Ca 2+ and PO4 3- ions can vary.
  • the mineral source for these ions can be minerals such Ca(H2PO4)2, CaHP04, Ca3(PO4)2 ( ⁇ -TCP), Ca5(PO4)3(OH), or any combination of these minerals.
  • the mineral source is in particle form, e.g., a particulate mineral.
  • the size of the particles of the mineral source, and the range of the size of the particles having applications in a bone growth composition of the instant disclosure can vary.
  • the range of the particle size of mineral source particles in a bone growth composition of the instant disclosure can range from about 250 ⁇ m to about 3000 ⁇ m, about 250 ⁇ m to about 2000 ⁇ m, about 250 ⁇ m to about 1000 ⁇ m* or about 1000 ⁇ m to about 2000 ⁇ m.
  • the mass ratio by weight of the mineral source to the decellularized placenta derived extracellular matrix substantially free of residual material in a bone growth composition of the instant disclosure can vary.
  • this mass ratio can 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%, (ECMimineral source).
  • a bone growth composition of the instant disclosure can further comprise a biological material, such as, for example, cells, conditioned cell medium, cell secretome, plasma, genetic material, e.g. nucleic acid molecules (e.g., siRNA and mRNA) and nucleotides, exosomes, bone marrow aspirate, platelet plasma, growth factors or any combination of such biological material.
  • a biological material such as, for example, cells, conditioned cell medium, cell secretome, plasma, genetic material, e.g. nucleic acid molecules (e.g., siRNA and mRNA) and nucleotides, exosomes, bone marrow aspirate, platelet plasma, growth factors or any combination of such biological material.
  • examples of cells having applications herein include but certainly are not limited to mesenchymal stem cells, adipose tissue derived stem cells, osteoblasts, placental derived cells.
  • conditioned medium and exosomes are produced by the cells discussed above.
  • the Instant disclosure further extends to a method for treating a bone defect in a subject, which comprises administering to the subject at the point of the bone defect an effective amount of a bone growth composition of the instant disclosure.
  • kits that comprises a bone growth composition of the instant disclosure and packaging container specifically designed to allow for different product sizes and the addition of rehydration fluids.
  • a kit of the Instant disclosure can further comprise biological material, such as, for example cells, conditioned cell medium, cell secretome, plasma, genetic material, e.g. nucleic acid molecules (e.g., siRNA and mRNA) and nucleotides, exosomes, bone marrow aspirate, platelet plasma, growth factors or any combination of such biological material.
  • biological material such as, for example cells, conditioned cell medium, cell secretome, plasma, genetic material, e.g. nucleic acid molecules (e.g., siRNA and mRNA) and nucleotides, exosomes, bone marrow aspirate, platelet plasma, growth factors or any combination of such biological material.
  • cells having applications herein include but certainly are not limited to mesenchymal stem cells, adipose tissue derived stem cells, osteoblasts, placental derived cells.
  • conditioned medium and exosomes are produced by the cells discussed above.
  • the biological material can be housed in a container that is included in the
  • FIG. 1 is an SDA-PAGE analysis decellularized placenta derived extracellular matrix of a bone growth composition of the instant disclosure, wherein the biocompatible substrate comprises collagen, wherein 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,
  • FIG, 2 illustrates several forms of a lyophilized bone growth composition of the instant disclosure in cuboidal configurations with the mineral source in particulate form, wherein the size of the particles of the mineral source range from about 250 ⁇ m to about 2000 ⁇ m distributed throughout the bone growth composition and the ratios of decellularized placenta derived extracellular matrix substantially free of residual material and ⁇ -TCP includes 10/90%, 15/85% and 20/80%,
  • FIG, 3 illustrates a form of a bone growth composition of the instant disclosure in cylindrical form having particulate mineral source with a particle size range of about 250- about 500 ⁇ m distributed throughout the bone growth composition of the instant disclosure
  • FIG, 4 are top and size views of a form of a bone growth composition of the instant disclosure in cylindrical form having particulate mineral source with particle size range of about 1000-about 220 ⁇ m distributed throughout the bone growth composition of the instant disclosure.
  • FIG, 5 is a bone growth composition of the instant disclosure in a strip configuration containing about 95% by weight mineral source in particulate form with the range of the size of the particles of about 2050- about 1000 ⁇ m and about 5% by weight a decellularized placenta derived extracellular matrix substantially free of residual material
  • FIG, 6 illustrates crosslinking of decellularized placenta derived extracellular matrix substantially free of residual material with thermal dehydration treatment of a bone growth composition of the instant disclosure.
  • FIG. 7A, B, and C illustrate the moldability and plasticity of a bone growth composition of the instant disclosure in which the mass ratio of mineral source to decellularized placenta derived extracellular matrix substantially free of residual materials about 20%: about 80%.
  • Sterile water was added to a bone growth composition as disclosed herein with a mass ratio of decellularized placenta derived extracellular matrix substantially free of residual material to ⁇ -TCP is about 20:80% (FIG. 7A).
  • the rehydrated implant was molded into a desired ball shape as depicted in FIGs. 7B and 7C
  • FIGs. 8A, 8B and 8C illustrate a bone growth composition of the instant disclosure in which the mass ratio of decellularized placenta derived extracellular matrix substantially free of residual material to ⁇ -TCP is about 20: about 80%, rehydrated with blood,
  • FIGs. 9A, 9B, 9C, and 9D illustrate rehydration and handling of a bone growth composition of the instant disclosure having a mass ratio of decellularized placenta derived extracellular matrix substantially free of residual material to ⁇ -TCP is about 20:80% as compared to ⁇ -TCP alone.
  • FIG. SA shows a bone growth composition as described herein rehydrating in saline for 15 minutes and remained intact with beta-TCP particles retained therein
  • FIG. 8B shows the rehydrated of FIG. 8A bone growth composition molded into a desired shape.
  • FIGs 9C and 90 shows beta-TCP alone soaked in saline for 15 minutes. The well after transfer of the beta-TCP shows residual beta-TCP and FIG. 9D shows an inability to mold rehydrated beta-TCP alone.
  • FIGs. 10A and 10B illustrate the morphological analysis of a bone growth composition of the instant disclosure having a mass ratio of decellularized placenta derived extracellular matrix substantially free of residual material to ⁇ -TCP is about 20: about 80% as compared to ⁇ -TCP alone, wherein the range of the particle size of the ⁇ -TCP is (left) about 250 to about 2000 ⁇ m and (right) about 1000- about 2000 ⁇ m.
  • FIG. 10 shows radio-opaque beta-TCP particles (white) and porous ECM (black surrounding the beta-TCP particles).
  • Micro Computed Tomography (Micro-CT) of the beta-TCP granules show they were uniformly surrounded by porous placental structure ECM as described herein.
  • FIG. 11 illustrates XRD spectrum and overlay analysis of a bone growth composition of the instant disclosure in which the mass ratio of decellularized placenta derived extracellular matrix substantially free of residual material to ⁇ -TCP is about 20:about 80%.
  • the correct spectra were observed to be indicative of ⁇ -TCP, and the ⁇ -TCP components are fully characterized, and no additional phases or impurities were detected.
  • FIG. 12 illustrates FTIR spectrum and stability analysis for a placenta derived ECM substantially free of residual material that is the natural biocompatible material of a bone growth composition of the instant disclosure.
  • FIG. 12(A) illustrates collagen standards and decellularized ECM.
  • FIG. 12(B) illustrates the stability profile of a bone growth composition of the instant disclosure over 115 days at ambient temperature.
  • FIG. 12(C) is the FTIR profile for a bone growth composition of the instant disclosure.
  • FIG.12D is the FTIR spectra showing stability of a bone growth composition of the instant disclosure at 40° C for 10 days.
  • FIG. 13 shows a bone growth composition of the instant disclosure in the form of bone implants in packaging trays accommodating 0 cc, 4 cc and 2,5 cc and providing a system for adding an appropriate rehydration agent in a clinical setting (kits).
  • FIG. 13A is a double tray prototype with retainer-
  • FIG 13B shows a double tray prototype with a 10 cc bone growth composition of the instant disclosure
  • FIG. 12C shows a double tray prototype with 5 cc bone growth composition of the instant disclosure and retainer
  • FIG. 12 D shows a double tray prototype with a 2.5 cc bone growth composition of the instant disclosure with retainer.
  • FIG. 14 illustrates a bone growth composition of the instant disclosure post admixed with ⁇ -TCP dispersed therein.
  • FIG. 15 Illustrates a bone growth composition of the instant disclosure alone at 12 weeks.
  • FIG. 16 illustrates the autograft at 12 weeks.
  • FIG. 17 illustrates Test Group 1 (FUSE 1) at 12 weeks.
  • FIG. 18 illustrates Test Group 2 (FUSE 2) at 12 weeks.
  • FIG, 19 illustrates bone marrow aspirate generation and application of bone marrow aspirate and a bone growth composition of the instant disclosure in the form of a bone implant.
  • FIG. 20 Illustrates rehydration and placement of a bone growth composition of the instant disclosure in the form of a bone implant In a critical size bone defect.
  • FIG. 21 illustrates a micro*CT image of a bone growth composition of the instant disclosure in the form of a bone implant in a critical size at 3 and 6 weeks with ⁇ -TCP having a particle size range of about 250 to about 500 ⁇ m.
  • FIG. 22 is a micro-CT Image of a bone growth composition of the instant disclosure in the form of a bone Implant in a critical size bone defect at 3 and 6 weeks with ⁇ -TCP having a particle size range of about 1000 to about 2000 ⁇ m.
  • FIG. 23 Illustrates histology of a bone growth composition of the instant disclosure in the form of a bone implant in a critical bone size defect at 3 and 6 weeks with ⁇ -TCP having a particle size range of about 1000 to about 2000 ⁇ m.
  • FIG. 24 illustrates histology of a bone growth composition of the instant disclosure in the form of a bone implant in a critical size bone defect with ⁇ -TCP having a particle size range of about 1000 to about 2000 ⁇ m.
  • FIG. 25 illustrates a bone growth composition of the instant disclosure in the form of a bone implant with a mass ratio of decellularized placenta derived extracellular matrix substantially free of residual material to ⁇ -TCP of 20:30%, rehydrated with bone marrow aspirate and loaded into a syringe for implantation/placement in a rabbit bone defect In a posterolateral fusion model.
  • FIG. 26 illustrates 8-week histology results of a bone growth composition of the instant disclosure with a mass ratio of decellularized placenta derived extracellular matrix substantially free of residual material to ⁇ - TCP is 20:80% rehydrated with bone marrow aspirate in a rabbit bone defect in the posterolateral fusion rabbit model. New bone formation as well as neo vasculature is donated with arrows.
  • TP in the figure denotes tricalcium phosphate
  • ECM in the figure denotes decellularized placenta derived extracellular matrix substantially free of residual material.
  • FIG. 27 iilustratesB-week histology results of a bone growth composition of the instant disclosure having a mass ratio of decellularized placenta derived extracellular matrix substantially free of residual material to ⁇ -TCP is 20:80% rehydrated with bone marrow.
  • TP in the figure denotes tricalcium phosphate
  • ECM in the figure denotes decellularized placenta derived extracellular matrix substantially free of residual material.
  • New bone formation as well as neo vasculature is donated with arrows.
  • FIG, 28 is an illustration of the use of a bone growth composition of the instant disclosure as a dental graft.
  • FIG. 29 is an illustration of the use of a 3D printed mold with rods for creating channels in a bone growth composition of the instant disclosure with controlled channels therein to guide new blood vessel ingrowth.
  • FIG. 30 illustrates human Mesenchymal Stem ceils grown on bone growth composition as described herein in the form of an implant as compared to a commercial product (Control Product) containing 20% human bovine collagen and 80% beta TCP.
  • a bone growth composition comprising a decellularized placenta derived extracellular matrix substantially free of residual materials, such as DMA, cells, cell debris, growth factors, exosomes and cytokines comprising a natural biocompatible material, and a mineral source of Ca3- and PO4 ions readily has applications in treating a bone defect in a subject.
  • ⁇ -TCP is a mineral source for Ca 2+ and PCM 3- ions and is Beta- tricalcium phosphate (Ca3(PO4)2), a biocompatible ceramic material having applications in orthopedic and dental applications, including bone void filling and bone regeneration.
  • critical size bone defect refers to a bone defect created in an animal that is unable to 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-engineered constructs or biomaterials to facilitate healing.
  • defining the critical size of a bone defect is essential for determining the appropriate treatment strategy. Surgeons often assess factors such as the size, location, and stability of the defect, as well as the surrounding soft tissue condition, to determine whether surgical intervention is necessary and what type of intervention would be most effective.
  • Various techniques and materials may be employed to address critical size bone defects, including bone grafts (autografts, allografts, or xenografts), synthetic bone substitutes, growth factors, and tissue engineering approaches
  • delivery and “administration” are used interchangeably herein and mean and include providing a "pharmaceutical composition” or “biologically active agent” or “active agent formulation” or a biologically acceptable material to a treatment site, e.g., damaged tissue, through any method appropriate to deliver the functional agent or formulation or pharmaceutical composition to the treatment site.
  • delivery methods include direct injection, percutaneous delivery and topical application at the treatment site.
  • extracellular matrix and “ECM” are used interchangeably herein and mean and include a collagen-rich substance that is found in between cells in mammalian tissue, and any material processed therefrom, e.g. decellularized ECM.
  • ECM material is derived from human placenta
  • lyophilized and “dehydrated” are used interchangeably, and refer to the state, but not the method of having water removed as a means of preservation.
  • placenta refers to a disc of tissue that connects a mother’s uterus to the umbilical cord and is ultimately responsible for delivering nutrients and oxygen to a fetus.
  • Three layers of membranes make up the placenta: (1) the amnion, which is a single layer of ectodermal epithelium completely enclosing the embryo; (2) the chorion which surrounds the amniotic sac and includes the villi and trophoblast; and (3) the decidua of the maternal endometrium.
  • the umbilical cord is the conduit between the placenta and the mother's circulatory system, and consequently, is riot a part of the placenta .
  • hydrate refers to the addition of a liquid, e.g., a diluent, to a dehydrated material in order suspend the material in a solution and to allow for injection through an adequately sized needle,
  • a liquid e.g., a diluent
  • the phrase "substantially free of residual material” refers to an extracellular matrix that does not Contain a quantifiable amount of residual material, e.g., (1) cells, (2)cell debris, (3) nucleic acid molecules, (4) nucleotides, (5) growth factors, (6) cytokines, (7) chemokines, (8) hormones, or (9) any combination of (l)-(8).
  • an effective amount means that the amount of a "pharmaceutical composition” and/or “biologically active agent” and/or “active agent formulation” such as a bone growth composition as disclosed herein, that is administered at a sufficient quantity to ameliorate one or more causes, symptoms, or sequelae of a disease or condition, such as, for example, a bone defect. Such amelioration only requires a reduction or alteration, not necessarily elimination, of the cause, symptom, or sequelae of a disease or disorder. An effective amount will depend on several factors including severity and course of the disease or condition, previous therapy, the patient's health status, age, weight, and response to the drugs. It is considered well within the skill of the art for one to determine therapeutically effective amounts by routine experimentation (including, but not limited to, a dose escalation clinical trial).
  • exosome refers to naturally existing nanoparticles that are secreted endogenously by many types of in vitro cell cultures and in vivo cells, and are commonly termend in vivo in body fluids, such as blood, urine and malignant ascites. Exosomes are cup-like multivesicular oodies (MVBs) varying in size between 30-100 nm. MVBs are specialized endosomes in the endocytosis pathway of cells and are formed by inward budding and scission of vesicles from the limiting membranes into the endosomal lumen.
  • MVBs cup-like multivesicular oodies
  • MVBs transmembrane and peripheral membrane proteins are absorbed into the vesicle membrane, and at the same time, cytosolic components are also embedded in the vesicles. As this process progresses, the MVBs ultimately fuse with the cellular membrane, triggering the release of the exosomes from the cells.
  • extracellular matrix and “ECM” are used interchangeably and mean and include a collagen-rich substance that is found in between cells in mammalian tissue, and any : material processed therefrom, e.g., decellularized ECM.
  • ECM material is derived frorn human placenta.
  • placenta derived extracellular matrix is derived from the placenta.
  • hydrogel refers to a water-insoluble, three-dimensional network of polymer chains capable of holding large amounts of water.
  • lyophilized and “dehydrated” are used interchangeably, and refer to the state, but not the method of having water removed as a means of preservation.
  • osteoconductive refers to a material or substance that supports and promotes bone growth.
  • osteoconductivity describes the ability of a material to serve as a scaffold or framework for new bone formation.
  • Osteoconductive materials encourage the attachment, proliferation, and differentiation of bone ceils, facilitating the integration of the implant With surrounding bone tissue. This property is crucial for 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 phosphates, and bioactive glasses are often considered osteoconductive due to their ability to stimulate bone regeneration.
  • composition refers to a composition that comprises at least one ophthalmic therapeutic agent, excipient, a carrier, etc. Pharmaceutical compositions are administered to a patient rather than the ophthalmic therapeutic agent alone.
  • particle and “granule” are used interchangeably and refer to a minute portion, piece, fragment, or amount.
  • porous refers to a material or structure characterized by the presence of interconnected voids or pores within its substance. These voids allow for the passage or diffusion of substances, such as fluids or gases, through the material.
  • the porous nature of a material provides properties such as increased surface area, permeability, or absorbency, which may be advantageous for various applications including filtration, insulation, catalysis, or controlled release of substances.
  • the extent of porosity can vary, ranging from fine porosity with very small interconnected voids to coarse porosity with larger voids.
  • Osteoconductive refers to a material or substance that supports and promotes bone growth.
  • porous and osteoconductive refers to a material or structure that possesses both porous characteristics and osteoconductive properties. Such a material is not only permeable, allowing for the passage or diffusion of substances, but also promotes bone growth and integration with surrounding bone tissue. This combination of traits is particularly advantageous for medical devices or implants intended for orthopedic or dental applications.
  • the porous nature of the material provides a framework for cellular ingrowth and vascularization, white its osteoconductive properties stimulate bone formation and facilitate the osseointegration of the implant with the surrounding tissue. This dual functionality enhances the effectiveness and long-term success of the implant in supporting bone regeneration and structural integrity.
  • placenta refers to a disc of tissue that connects a mother's uterus to the umbilical cord and is ultimately responsible for delivering nutrients and oxygen to a fetus.
  • Three layers of membranes make up the placenta: (1) the amnion, which is a single layer of ectodermal epithelium completely enclosing the embryo; (2) the chorion which surrounds the amniotic sac and includes the villi and trophoblast; and (3) the decidua of the maternal endometrium.
  • umbilical cord is not considered part of a placenta.
  • placenta derived with respect to a biomaterial indicates that the source of the biomaterial is the placenta, which is typically obtained from animals (such as pigs or sheep) or from human placental tissue.
  • placenta derived biomaterials having applications herein are a decellularized placenta derived extracellular matrix and placenta derived exosomes.
  • Placental derived biomaterials been proposed to be immune-privileged such that, when presented physically and biochemically to a "native" administration site on a subject, they are proposed to rapidly re-establish healing cues and limit foreign body reactions.
  • a particular example of a decellularized placenta derived extracellular matrix having applications in a pharmaceutical composition of the instant disclosure is a decellularized, dehydrated human amniotic membrane (DDHAM).
  • therapeutic agent refers to -a compound or substance in a pharmaceutical composition that is biologically active and produces the effects of the pharmaceutical composition
  • a bone growth composition of the present disclosure may be hydrated for administration at the point of care or may be stored at a cold temperature, such as from about 2® C to about 8° G.
  • a hydration fluid for a bone growth composition of the instant disclosure can vary.
  • examples of such fluids include sterile sodium chloride solution, for example about 0.9% sodium chloride (hereinafter referred to as "saline"), blood, plasma, bone marrow, etc.
  • An effective amount of a bone growth composition can vary depending upon magnitude of the bone defect, whether the composition is being used as a bone graft, a bone implant, or a bone filler
  • a bone growth composition of the instant disclosure is administered directly to the target site (e.g., the bone defect to be treated.).
  • the mass ratio of the mineral source to biocompatible material can vary. -Examples of such ratios having applications herein include 5:95%, 10:90%, 15:85%, 20:80%, 25:75%, or 30:80%.
  • the biocompatible material comprises a collagen and an elastin, wherein the amount collogen is more than about 60% by weight of a bone growth composition as described herein, and elastin is less than about 20% by weight of a bone growth composition.
  • a bone growth composition of the instant disclosure can comprise pharmaceutically acceptable diluent(s), excipient(s), or carrier(s). Furthermore, a bone growth composition of the instant disclosure can further comprise a medicinal or pharmaceutical agent, carrier, or adjuvants to address possible infection, pain, prevent or limit development of blood clots (an anti -coagulant), etc.
  • the placental starting material is sourced by established methods designed to reduce the risk of additional contamination and prevent mislabeling and/or tracking errors.
  • the tissue originates from accredited hospitals, with collections performed by licensed health care professionals.
  • the tissue collection kit utilized includes sufficient containers and barcoded labels for biomaterials along with appropriate documentation and instructions for collecting the umbilical cord.
  • the placental tissue is screened for the presence of transfusion transmissible viruses in accordance with the FDA Guidance for Industry: Eligibility Determination for Donors of Human Cells, Tissues and Cellular and Tissue-Based Products (HCT/Ps) - August 2007.
  • Making a bone growth composition as disclosed herein begins with the procurement of placental tissue, obtained from a normal, full-term delivery and thus is sourced from whole human placentas.
  • the frozen placental tissue is released from quarantine for processing and is thawed at 4 - 8®C for up to 96 hours.
  • the placental tissue is cut into small segments and homogenized using an Omni mixer homogenizer.
  • the homogenization step serves to generate small tissue particulates, with increased surface area, allowing for more effective separation and removal of cells and cellular debris from the placental tissue.
  • the homogenized tissue from a single placenta is transferred into a sterile processing container with sodium chloride (NaCI) solution.
  • NaCI sodium chloride
  • the tissue is washed repeatedly with sterile NaCI by shaking on an orbital shaker whereby the NaQ solution is exchanged by centrifugation to setle tissue particulate, followed by removal of the supernatant, and replacement with fresh sterile NaCI solution.
  • the placental tissue is incubated at controlled room temperature (20 to 25 O C) for up to 24 hours while shaking in sterile NaCI solution, followed by centrifugation to remove supernatant and washed with sterile water.
  • the exposure of the placental tissue to a high concentration of NaCI, followed by hypotonic water constitutes an "osmotic shock" to the tissue.
  • the osmotic shock serves to remove blood, blood components, cells, and cell debris from the tissue.
  • the placental tissue is subjected to a second osmotic shock before a nucleic acid removal step.
  • the supernatant is removed from the sterile processing container after the completion of the water wash and replaced with a solution containing BENZONASE and is incubated up to 24 hours on an orbital shaker at room temperature to aid in degradation of all forms of nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the tissue is subjected to detergent washes with sodium deoxycholate (DOC) solution for up to 24 hours followed by freshly exchanged DOC solution for up to an additional 72 hours.
  • DOC sodium deoxycholate
  • DOC is a water soluble, bile-acid derived, mild ionic detergent used to remove residuals such as cells, cell membranes, and other cell debris. Upon completion of the detergent washes, the tissue is washed with sterile water to remove debris and residual DOC.
  • the placental ECM is treated with sodium hydroxide (NaOH) solution to denature and remove any soluble extracellular matrix elements, non-crosslinked proteins, i.e., cytokines/chemokines, growth factors, and hormones. Thereafter, the ECM is subjected to hydrochloric acid (HCI) treatment to neutralize the NaOH and to remove the above-mentioned impurities.
  • HCI hydrochloric acid
  • the incubation of NaOH followed by HCI is utilized as an "enrichment step". Titration of this step allows for careful selection of the matrix composition containing enriched collagen content and titrated desirable levels of elastin content of less than 20%.
  • solubilized product residuals such as cells, cellular debris, nucleic acids, growth factors, chemokines/cytokines, hormones, and process reagents, such as ; salt, DOC, and BENZONASE.
  • the resultant placental structural protein enriched composition is tested for collagen and elastin content.
  • the methods used are well established in the art for biochemical testing and are based on hydroxyproline- based colorimetric assay for collagen and a dye- 5,10, 15, 20-tetraphenyl- 21H,23H-porphine tetra-sulfonate based colorimetric assay for elastin.
  • the placental decell ularized structural protein enriched composition is transferred into a sterile mold, frozen at -80° C, and lyophilized.
  • the lyophilized material is sealed and stored at room temperature until it is used in formulation with ⁇ -TCP granules at different ratios, including examples discussed below.
  • FIG.l shows results of SDS-PAGE analysis of growth composition of the instant disclosure denoted as ECM in com parison to col lagen sta ndards .
  • the lyophilized matrix is formulated with ⁇ -TCP granules of sizes including, but not limited to about 250 micrometers to about 3000 micrometers at different mass ratios, including but not limited to 5/95%, 10/90%; 15/85%, 20/80%, 25/75%, and 30/70% ⁇ -TCP to the biocompatible material, e.g., decellularized placenta derived extracellular matrix substantially free of residual material.
  • the dry weight of the decellularized placenta derived extracellular matrix substantially free of residual material was calculated for the required ratio of the mineral component, ⁇ -TCP, to form a ratio of 5/95%, 10/90%, 15/85%, 20/80%, 25/75%, and 30/70%, to name only a few
  • FIG. 2 illustrates several forms of the lyophilized biocompatible graft material in cuboidal configurations with granules or particles of ⁇ -TCP having a size ranging from about 250- about 2000 micrometers distributed throughout the bone growth composition of the instant disclosure, and matrix/ ⁇ -TCP ratios including 10/90%, 15/85%, and 20/80%.
  • FIG. 3 illustrates one form of the lyophilized biocompatible graft material in a cylindrical form having small size mineral granules having a size ranging from about 250- about 1000 micrometers distributed throughout a bone growth composition of the instant disclosure.
  • FIG. 4 illustrates another form of a bone growth composition as described herein in a cylindrical form having particle size ranging from about 1000-2000 micrometers, distributed throughout the bone growth composition, top and side view.
  • FIG. 5 illustrates another form of a bone growth composition described herein in a strip configuration containing 95% of mineral source in particle form with the size of the particles ranging from about 250-1000 micrometers and 5% of biocompatible material, e.g., placenta derived extracellular matrix substantially free of residual material.
  • biocompatible material e.g., placenta derived extracellular matrix substantially free of residual material.
  • Placental structural protein enriched extracellular matrix that is developed with desired ratio of collagen to elastin as discussed above and confirmed by biochemical assays, is then transferred into a sterile mold, frozen at -80° C, lyophilized arid stored at room temperature.
  • the lyophilized matrix is formulated with ⁇ -TCP granules of sizes ranging from about 250 micrometers to about 3000 micrometers at different mass ratios, including but not limited to 5/95%, 10/90%; 15/85%, 20/80%, 25/75%, and 30/70% placental Matrix / ⁇ -TCP.
  • crosslinking studies were performed testing different methods for stabilizing the Structure and handling properties of a bone growth composition of the instant disclosure.
  • the crosslinking methods tested included a) chemical crosslinking, using carbodiimides and glutaraldehyde chemistry, which require lengthy rinsing procedures to remove chemicals, such as crosslinkers, catalysts, etc., b) a physical crosslinking method, using thermal treatment, which is one of the earliest published physical techniques utilized for Collagen cross-linking (Yannas I V, Tobolsky A V. Nature. 1967).
  • thermal treatment which is one of the earliest published physical techniques utilized for Collagen cross-linking (Yannas I V, Tobolsky A V. Nature. 1967).
  • thermal dehydration collagen cross-links were formed in the absence of any catalysts, excipients or crosslinkers.
  • FIG. 6 illustrates one embodiment of superior crosslinking a bone growth composition as disclosed herein with thermal dehydration treatment.
  • FIG. 8 illustrates the bone growth composition of the instant disclosure in the form of an implant at 20:80% ⁇ -TCP rehydrated with blood.
  • FIG. 9 illustrates rehydration and handling of the bone Implant at 20:80% ⁇ -TCP as compared to g-TCP alone,
  • FIG. 10 illustrates the morphological analysis of a bone growth composition of the instant disclosure in the form of a bone implant, at 20:80% ⁇ -TCP with ⁇ -TCP particle size ranges of about 250 to about 2000 micrometers (Left) and about 1000 to about 2000 micrometers (Right), SEM analysis was used to perform morphological analysis of the bone implant at 20:80% ⁇ -TCP.
  • the organic to inorganic ratio and inorganic phase composition of a bone growth composition as described herein in the form of a bone implant was tested.
  • Acquiring the organic and inorganic weight composition involves heating/sintering the samples to a temperature that will decompose (ash) the organic components, leaving behind the inorganic granules.
  • the heat treatment/sintering will simultaneously prepare the inorganic granules, which are left over after sintering, to be analyzed for phase composition using x- ray diffraction (XRD).
  • Ash testing was performed to establish a specification for the weight percentage of organic material to inorganic components.
  • the samples Prior to the samples undergoing XRD analysis to acquire the phase purity and phase weight composition, the samples were sintered at I 100°C ⁇ 25°C in order to increase and normalize the crystallite size relative to the XRD standards used to calibrate the XRD. Increasing the crystallite size is necessary to better define the peaks and provide a fiat baseline during the XRD scan. The required sintering of the samples takes place during the heat treatment for ash testing.
  • the sintered granules resultant to ash testing were pulverized in a mortar and pestle composed of synthetic sapphire and analyzed according to established testing procedure.
  • the pulverized sample(s) were scanned at a rate of 1.20 degrees per minute from 20- 60° using a Rigaku Miniflex 11 X-ray diffractometer following the established procedure.
  • Calcium phosphate patterns from the International Center for Diffraction Data/ Joint Committee on Powder Diffraction Standards (ICDD/JCPDS) library that are stored in the JADE 9 analysis software were compared to the sample pattern. All matching calcium phosphate patterns will be reported as crystalline phase impurities.
  • the sample diffraction patterns were provided with superimposed patterns of each phase from the pattern library,
  • Phase Purity Quantification Calibration curves for various calcium phosphate phases (Hydroxyapatite/P-Tricalcium Phosphate/ pyrophosphate) have been prepared. 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%, while pyrophosphate has a detection limit of 4% but can be visible as low as 0.5%.
  • FIG. 11 illustrates XRD spectrum and overlay analysis of bone growth composition as described herein in the form of a bone implant at 20:80% ⁇ -TCP.
  • FTIR Fourier-transform infrared spectroscopy
  • ATR Attenuated Total Reflectance
  • FIG. 12 illustrates FTIR spectrum and stability analysis of Placental decellularized ECM and PSPE-BI.
  • FIG. 13 shows bone growth compositions of the instant disclosure in the form of bone implants in packaging trays accommodating 10cc, 5ccc and 2,5 cc and providing a system for adding an appropriate rehydration agent In a clinical setting.
  • Placental structural protein enriched matrix e.g., decellularized placenta derived extracellular matrix substantially free of residual material and comprising collagen and elastin, as discussed above, was mixed with ⁇ -TCP at various ratios, 5/95%, 10/90%, 15/85%, 20/80%, 25/75%, 30/70%, 40/60% and various ⁇ -TCP particle sizes including about 250 to about 3000 micrometers and homogenized to fully disperse the ⁇ -TCP within the ECM to form a bone growth composition as described herein with dispersed ⁇ -TCP granules, FIG, 14 illustrates the bone growth composition post homogenization with ⁇ -TCP dispersed therein.
  • the method of dispersing the mineral resource within a bone growth composition of the instant disclosure provides an unexpected way of generating micronized small size of the mineral source in particle form.
  • Example 14 In Vivo Testing of a Bone Growth Composition of the instant Disclosure at Ratios 15/85 and 30/70% ECM to ⁇ -TCP.
  • FIG. 15 illustrates the bone growth composition alone at 12 weeks
  • FIG. 16 illustrates the autograft at 12 weeks
  • FIG. 17 illustrates Test group 1 (FUSE 1) at 12 weeks.
  • FIG, 18 Illustrates group 2 (FUSE2) at 12 weeks.
  • Bone growth composition in the form of two implants with one having ⁇ -TCP particles ranging in size from about 250 to about 2000 micrometers and the other having ⁇ -TCP particles ranging in size from about 1000 to about 2000 micrometers were placed within the bone defect.
  • the bone growth composition in the form of a bone implant was further combined with a biological material, e.g., Bone Marrow Aspirate (BMA) and was tested at 3 and 6 weeks. As part of the procedure.. BMA was obtained from each tibia and combined with test articles.
  • BMA Bone Marrow Aspirate
  • FIG. 19 shows BMA generation and application of bone marrow aspirate the bone growth composition implant
  • FIG. 20 Rehydration and placement of the bone growth composition implant in a critical size bone defect.
  • FIG. 21 micro-CT imaging of the bone growth composition implant in a critical size defect at 3 and 6 weeks with ⁇ -TCP particles ranging in size from 250 to 2000 micrometers.
  • FIG. 22 micro-CT imaging of the bone growth composition implant in a critical size defect at 3 and 6 weeks - ⁇ -TCP particle size range of about 1000 to about 2000 micrometers.
  • FIG. 23 Histology of a bone growth composition of the instant disclosure in the form of a bone implant in a critical size defect at 3 and 6 weeks - ⁇ -TCP particle size range is 1000-2000 micrometers
  • FIG. 24 Histology of a bone growth composition of the instant disclosure in the form of a bone implant in a critical size defect at 3 and 6 weeks - ⁇ -TCP particle size range is 1000-2000 micrometers.
  • Bilateral critical distal femur (cancellous region) defects (6mm x 10mm) will be created in skeletally mature New Zealand White rabbits. Treatment of both defects in a single animal will consist of no treatment (empty defect), a bone growth composition as disclosed herein in the form of a bone implant,, or Comparator Scaffold Foam Material, New bone growth will be evaluated at both 6- and 12-weeks post implantation into the defect. Bone growth will be measured by micro- CT and polymethylmethacrylate (PMMA) histology and histomorphometry. The Day 3 timepoint will provide a baseline of micro-CT and histomorphometry. The 6-week time allows for an assessment of the initial biologic responses to the device. The final time point (12-weeks) should be of a sufficient duration to demonstrate bone healing and the effects of any residual device material. Paraffin histology sections will also be used for ISO 10993-6 evaluation from the timepoints referenced above.
  • FIG. 25 illustrates the bone growth composition in the form of a bone implant at 20:80% ⁇ -TCP rehydrated with bone marrow aspirate and loaded into a syringe for implantation/placement in a rabbit defect in a posterolateral fusion model.
  • FIG. 27 illustrates 8-week histology results of the bone growth composition implant at 20:80% ⁇ -TCP rehydrated with bone - marrow aspirate in a rabbit defect in the posterolateral fusion model.
  • a line of osteoblast cells migrating into the defect and neovascularization are denoted by arrows.
  • FIG. 27 illustrates 8-week histology results of the bone growth composition implant at 20:80% ⁇ - TCP rehydrated with bone marrow aspirate in a rabbit defect in the posterolateral fusion model.
  • a line of osteoblast cells migrating into the defect and neovascularization are denoted by arrows.
  • a bilateral canine humeral defect implant study is undertaken to evaluate bone growth composition as described herein in the form of a bone graft (the bone graft material) that is in direct contact with bone tissue. Bone remodeling, new bone formation, and implant resorption is evaluated at periodic time intervals.
  • the bone graft material is supplied in a form that results in a flexible graft upon wetting.
  • the bovine collagen containing bone void filler such as VITROSS Foam Bone Implant, wiil be used as a control,
  • the test or control materials will be implanted into bilateral drill defects surgically created in the cancellous bone of the proximal humerus of X8 canines.
  • Bone graft material graft will be implanted into the drill defect of one humerus, and the control article will be implanted into the drill defect in the opposite humerus of each animal in accordance with a randomization schedule generated prior to study start
  • the drill defects will be approximately 10 mm In diameter and approximately 25 mm in depth.
  • a lateral and dorse- palmar view will be obtained immediately post-operatively and all animals wiil be subsequently radiographed at each sacrifice time point.
  • the animals After the predetermined exposure period (3, 6, 12, 24; or 52 weeks), the animals will be sacrificed, and the implantation sites exposed. The implant sites will be grossly observed, harvested, wrapped in a saline soaked gauze sponge and frozen at approximately -20° C. for further analysis.
  • Analysis of the harvested implant sites will include mechanical testing to assess the bony ingrowth and remodeling of the defect site.
  • the tissue implant sites will also be examined using standard histology techniques well-known in the art.
  • the extent of healing and nature of tissue contained within the defect will be characterized by the histopathological and histomorphometry evaluation. Defects treated with bone growth composition as described herein will show bone healing with the rate or extent of healing in defects treated with graft material equal to or better than that observed in defects treated with the control graft material.
  • a bone growth composition as described herein in the form of a bone implant may be an optimal surgical option. All surgeries will be performed using retrograde drilling procedures for OCD in the talar dome and the femoral condyles. Approximately 10 patients will be treated by retrograde drilling or trephine drilling under arthroscopic and fluoroscopic control preventing injury to the cartilage surface. The subchondral cavity will be filled with a mixture of 20-40 mg bone growth composition bone implant either rehydrated with saline, blood, or morselized bone graft.
  • Example 20 A bone Growth Composition as Disclosed herein with Fine Particle Size pTCP for
  • a bone growth composition as disclosed herein prepared from finer particle size Calcium phosphate can serve as dental implants (FIG. 28) and will be evaluated in rat calvarial critical size- defect dental implant model to demonstrate osteoconductivity, bone formation and to study remodeling.
  • the bone growth composition will be used to fill a 5.0 mm- diameter calvarial defect in rats.
  • the defects will be evaluated by histological and histomorphological analyses of decalcified histological sections with hematoxylin and eosin staining at 6 and 10 weeks, respectively, after surgery.
  • FIG. 28 illustrates use a Bone Growth Composition of the instant disclosure in the form of a dental graft.
  • a Bone Growth Composition of the Instant Disclosure in the form of a Bone Implant as a Scaffold for Delivery of Exosomes
  • a bone growth composition of the instant disclosure in the form of a bone implant will be integrated with exosomes. Exosomes isolated from placental cells or placental tissue will be loaded into the Bone Implant by physical absorption at the operating theater and the exosome-containing implant will be positioned within the critical size bone defect in rat tibia critical defects using a Bone Implant as the carrier. Bone regeneration will be quantified using micro-computed tomography and histology.
  • exosome treatment will demonstrate a positive effect on bone mineralization by providing a local delivery of exosomes loaded onto a slowly resorbing Bone Implant can provide a potential alternate to autografts for bone reconstruction.
  • a bone growth composition as described herein in the form of a bone implant will be seeded with cells known to stimulate bone tissue formation including but not limited to Mesenchymal Stem Cells, Adipose Tissue Derived Stem Celis, Osteoblasts, Placental derived Celis. Cells will be seeded onto the Bone Implant and allowed to colonize it generating cell byproducts within the Implant. The cell and cel i byproducts-contalning implant will be positioned within the critical size bone defect in a rat tibia model Bone regeneration will be quantified using micro-computed tomography and histology.
  • cell- and cell-byproducts - based treatment will demonstrate a positive effect on bone mineralization by providing local delivery of cells or ceil byproduct - loaded onto a slowly resorbing Bone Implant.
  • Cell byproducts include but are not limited to cell conditioned media, cell secretome content. Such application can provide a potential alternative to autografts for bone reconstruction.
  • a bone growth composition as described herein in the form of a bone implant will be combined with biological material, including but not limited to growth factors known to stimulate bone growth, such as Platelet Derived Growth Factor BB (PDGF-BB), Bone Morphogenetic Protein 2 (BMP-2).
  • biological material including but not limited to growth factors known to stimulate bone growth, such as Platelet Derived Growth Factor BB (PDGF-BB), Bone Morphogenetic Protein 2 (BMP-2).
  • PDGF-BB Platelet Derived Growth Factor BB
  • BMP-2 Bone Morphogenetic Protein 2
  • bioactive- treatment will demonstrate a positive effect on bone mineralization by providing local delivery of cells loaded onto a slowly resorbing Bone Implant, which can provide a potential alternate to autografts for bone reconstruction.
  • FIG. 29 illustrates use of 3D printed mold with rods for creating channels a bone growth composition as described herein in the form of a bone implant with controlled channels to guide new blood vessel ingrowth into the bone tissue. Rods of different spacing and configuration are utilized allowing for more effective blood vessel ingrowth.
  • FIG. 29 illustrates use of 3D printed mold with rods for creating channels in bone growth composition as described herein in the form of a bone implant with controlled channels to guide new blood vessel ingrowth.
  • the bone defect can be mapped using lasers, and that information fed into a three dimension printer, which can print an exact fitting bone graft or implant from a bone growth composition as described herein.
  • a Bone Growth Composition as Disclosed Herein in the Form of a Bone Implant that Supports Mesenchymal Stem Cell (MSC) Attachment and Growth.
  • MSC Mesenchymal Stem Cell
  • FIG. 30 illustrates human Mesenchymal Stem cells grown on bone growth composition of the Instant disclosure in the form of a bone implant as compareci to a commercial product (Control Product) containing 20% bovine collagen and 80% ⁇ -TCP, e.g. VITOSS Bone implant.

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Abstract

L'invention concerne des compositions de croissance osseuse qui ont des applications en tant qu'implants osseux, charges osseuses ou greffes osseuses, une méthode de traitement d'un sujet présentant un défaut osseux avec une composition osseuse telle que décrite dans la description.
PCT/US2024/021377 2023-03-23 2024-03-25 Composition de croissance osseuse WO2024197316A1 (fr)

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US7156880B2 (en) 2002-06-13 2007-01-02 Kensey Nash Corporation Devices and methods for treating defects in the tissue of a living being
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