WO2015009256A1 - Greffe osseuse à partir de poudre de coquille d'œuf d'autruche et son procédé de préparation - Google Patents

Greffe osseuse à partir de poudre de coquille d'œuf d'autruche et son procédé de préparation Download PDF

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WO2015009256A1
WO2015009256A1 PCT/TR2014/000232 TR2014000232W WO2015009256A1 WO 2015009256 A1 WO2015009256 A1 WO 2015009256A1 TR 2014000232 W TR2014000232 W TR 2014000232W WO 2015009256 A1 WO2015009256 A1 WO 2015009256A1
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eggshell
particles
until
prepared
temperature levels
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PCT/TR2014/000232
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Durmus ERCAN
Celik ILHAMI
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Ercan Durmus
Ilhami Celik
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Publication of WO2015009256A1 publication Critical patent/WO2015009256A1/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
    • A61L27/50—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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/0005—Ingredients of undetermined constitution or reaction products thereof
    • 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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • A61L24/108—Specific proteins or polypeptides not covered by groups A61L24/102 - A61L24/106
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • 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
    • 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
    • A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • 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

  • the invent is related to preparation method of bone a graft material in powder, particle and sponge form from ostrich eggshell powder and eggshell membranes to use in the treatment of damage of both compact and spongy bones, and bone loses arisen from different reasons, to meet bone loses, to conjunct fracture ends.
  • the prepared material contains biologically active, anti bacterial substances protecting from infection in addition to the substances those strengthening and accelerating bone healing process in the settled defect site.
  • the product contains bovine gelatine and/or chicken eggshell membrane gelatine as matrices and binders or bovine gelatine-carboxyl methylcellulose mixture in addition to 0.5-1.5 mm ostrich eggshell particles. Powder and particle forms of the product can be applied to the defect site by mixing blood, bone marrow or platelet-enriched blood plasma and with antibiotics.
  • the defect region may require filling with different bone graft materials.
  • Such a bone graft material should meet the exposed forces, gain the required size and forms, facilitate osseous tissue development (with osteoconductive effect) or induce bone production (with osteoinductive effect).
  • An ideal bone graft material should have osteoinductive and/or osteoconductive capacity and it should stay in the defect site for a sufficient time, then gradually be resorbed and replaced by a newly formed functional bone tissue of the recipient at a reliable period.
  • graft materials are produced from raw materials with very different origin and sources.
  • Autogenous bone graft which is freshly obtained from the patient's own bone tissues, is the gold standard in orthopaedics, dentistry and maxillofacial surgery since it obtains perfect and fast osteointegration.
  • Patient's own bone sample has both osteoinductive and osteoconductive features, due to its rich osteoprogenitor cell content.
  • autogenously bone graft has a limited application in many of the patients because of the need for more than one surgical operation, bleeding, infection risk, pain in donor site and harvesting the limited amount of the bone.
  • Allografts, Xenografts and synthetic ceramics have been developed to meet the insufficiencies of the autografts.
  • An allograt is the graft material, which is transplanted to a recipient from a different individual of the same species.
  • the most widely used bone allografts are freeze-dried bone allografts (FDBA) and demineralised freeze- dried bone allografts (DFDBA). Although both materials are used under strictly controlled conditions, they have the risk of contaminate infectious agents and are obtained in difficulty and relatively expensive.
  • xsenografts Another graft type used in the treatment of tissue lost is xsenografts.
  • a xsenograft is a graft that obtained from an individual of a different species from the host.
  • Today, corals and bone grafts with bovine origin have largely been used in humans.
  • grafts with the porcine origin have also been developed.
  • the use of bovine origin xsenografts with osteoconductive features has not been widespread, due to transmission risk of bovine spongiform encephalopathy (BSE).
  • BSE bovine spongiform encephalopathy
  • graft materials are biocompatible, inorganic and synthetic.
  • the synthetic graft materials which are available on the market, are dived in two groups, as ceramics and polymers.
  • a synthetic bone graft material should have the features given below:
  • ⁇ should be micro porous, support regenerating bone tissue and physiological fixation
  • ⁇ should have high pressure resistance.
  • Human bone contains high levels calcium and phosphates.
  • Calcium (Ca) and magnesium phosphates, calcium carbonate and their crystal forms such as, hydroxyl apatite (HA) and calcite also form graft materials.
  • di- and tri-calcium phosphate ceramics in the form of the hydroxyl apatite (HA) have a close chemical and structural resemblance with mineralized matrix of the bone, they are largely used as bone graft.
  • a high rate (85%) of bone calcium content gives hardness of the bone tissue.
  • Hydroxyl apatite (HA) may have natural or synthetic origins. Although the synthetic HA is not expensive, it has not the same chemical composition as the teeth. Since the risk of transmission of the infectious diseases such as, Creutzfeldt- Jakob, bovine spongiform encephalopathy
  • HA hydroxyl apatite
  • This protein (human bone morphogenetic protein-2, rhBMP-2), which some problems have been observed in the use of it in experimental studies, have been produced with different techniques, recently. Despite that, there are many bone graft materials on the market and they are produced with advanced technology, clinical results with these materials are not satisfactory yet. Moreover, the mentioned materials are relatively expensive. Therefore, intensive scientific studies have been carried out in order to develop graft materials substituting the natural bone.
  • One of the candidate sources is avian eggshells. Because of the limited mechanical resistance of the chicken eggshell, it is not able to meet the necessary forces in some defect sites. Ostrich eggshell, in addition to its potential use in orbital bone defects as onlay implant because that it can be used as large pieces due to its suitable contour.
  • It can also be used as a bone graft in the powder and particulate forms and also pulp, sponge and injectable forms prepared by mixing with binders and the materials having matrix features. Processing and sterilization of the graft material prepared from ostrich eggshell by preserving bioactive features are easy, and its contour is suitable for orbital bone defect- treatment when used as block.
  • Eggshell which is one of the biomaterials resembling with natural bone, may be preferred because that it has better biodegradation capability and can be provided at lower cost than the synthetic biomaterials. Furthermore, the eggshell is very similar to the structure of coral and contains strontium (Sr) in addition to fluorine (F), it has good potential for use as a bone graft.
  • Sr strontium
  • F fluorine
  • Eggshell consists of a thin proteinous cuticle film layer, which is located at outside, a middle mineralized layer of calcium carbonate settled in the middle, which is in the form of calcite crystals and two shell membranes settled innermost.
  • a spongy (palisade) layer which consists of vertically located calcite crystals and mamillary layer are distinguished.
  • Inorganic salts contain as 98.4% ratio calcium carbonate, 0.8% magnesium carbonate and 0.8% tricalcium phosphate.
  • Eggshell contains 64% protein, 1 ,7% water and 0,03% lipids.
  • Eggshell is a composite of various minerals, especially calcium carbonate, water-soluble and water insoluble matrix proteins.
  • Calcium carbonate (CaC03) in the form of calcite is the basis of the mineral content.
  • Calcite is the most stable form of calcium carbonate (CaC03) in the nature.
  • Calcified eggshell is formed in shell gland of the avian species. Mineral matrix of the eggshell constitutes more than 97% of the shell content, and eggshell calcification is the fastest and the most amount calcium deposited mineralization process with its 0,33 g/hour calcium deposition rate.
  • the rest of inorganic portion of the eggshell is composed of small amounts of phosphorus (P), magnesium (mg), sodium (Na), potassium (K), zinc (Zn), manganese (Mn), iron (Fe) and copper (Cu).
  • the eggshell contains relatively high amount of strontium (Sr) as well.
  • Lead (Pb), aluminium (Al), cadmium (CD), mercury (Hg), Vanadium (V), boron (B), iron (Fe), zinc (Zn), magnesium (mg), nitrogen (N), fluorine (F), selenium (Se), copper (Cu), and chromium (Cr) levels are below the measurement limits.
  • Fluorine (F), selenium (Se), copper (Cu), chromium (Cr), and strontium (Sr) levels are closely correlated to the levels of the feed, and this is an advantage of producing specific eggshell which is rich in desired elements.
  • the dry matter of the fraction obtained by decalcification of eggshell is composed of approximately % 24,11% % calcium, 0.04% phosphate, 0,23% magnesium.
  • Strontium (Sr) has an anabolic effect on osseous tissues and strengthens enamel layer of the teeth. Although the effective doses have not not been fully understood, when taken 170 mg per day, strontium (Sr) increases bone density at 10-day period. When taken appropriate amount of strontium (Sr), it increases bending resistance of the bone by binding onto hydroxyl apatite crystal.
  • Eggshell has a relatively high (380 mg/g) strontium (Sr) content. The other elements found in the eggshell such as, fluorine (F), copper (Cu), and selenium (SE) have fully positive effects on the bone metabolism.
  • ovocleidin-17 facilitates aggregation of calcite crystals and changes crystal morphology at a concentration of 500 pg/ml. Ovotransferrin reduces crystal size and causes to crystal elongation at 500 pg/ml concentration.
  • Another eggshell protein, ovocalyxin-32 locates in exterior regions of palisade layer, in other words in vertically and cuticle layers of the eggshell. Therefore, it is thought to be associated with the termination of the mineralization.
  • Ovocalyxin-36 plays a significant role in the control of eggshell formation.
  • Ovocleidin-116 locates in palisade layer and plays primary role in the control of mineralization.
  • Osteopontin one of the matrix proteins, is a phosphorylated glycoprotein and abundant in both kidneys and the bone tissue. Because that osteopontin (OPN) increases adhesion of osteoblasts to the bone matrix, in addition to binding to hydroxyl apatite, it has significant roles in the bone formation and remodelling. Osteopontin (OPN) iocates in core regions of proteinous eggshell fibers. Eggshell osteopontin (OPN) ceases calcite crystal growth in vitro. Clusterin, is another eggshell matrix protein, which the presence of it has been shown in 2003, Iocates in mamillary and palisade layers. This molecule possibly plays as a chaperone, which are the molecules being synthesized under stress conditions and stabilizes proteins by preventing their precipitation.
  • Glucosaminoglycans one of the most important components of ground substance of the connective tissue is also abundant in the organic matrix of the eggshell, and they have known to have many physiological functions including electrolyte control and water retention events.
  • Glucosaminoglycans are anionic polysaccharides, which are formed by polymerization of disaccharide molecules, constituted of one hexosamine and one uronic acid molecule. Most of the glucosaminoglycans (GAGs) bind to core protein molecules in order to form proteoglycans.
  • GAGs are the GAGs having the most amount of chondroitin sulphate- dermatan sulphate copolymers. GAGs of the chicken eggshell encompass equal amounts of Hyaluronic acid and galactosaminoglycans. Chondroitin sulphate and dermatan sulphate are dominant in galactosaminoglycans. Keratan sulphate plays significant role at nucleation stage of crystal formation. Ovoglycan, a chain of dermatan sulphate, is a polyanionic and acidic glucosaminoglycan (GAG). It indicates high calcium affinity and regulates crystal growth.
  • Glucosaminoglycans have a wide use in pharmaceutical and food industries.
  • hyaluronic acid is used as a moisturizer in cosmetics and for treatment of joint inflammation (arthritis).
  • Eggshell also contains small amount of transforming growth factor-1 (TGF-1 ), calcitonin, and progesterone.
  • TGF-1 transforming growth factor-1
  • calcitonin calcitonin
  • progesterone progesterone
  • Lysozyme a 14.4 kDa 129-amino-acid enzyme is a bacterial cell wall-degrading enzyme, exerts its activity via catalysing hydrolysis of the 1 ,4- beta bonds between N-acetylmuramic acid and N-acetyl-D-glucosamine residues, and also the beta bonds between N-asetil-D-glukozamin residues in kitodextrins.
  • lysozyme has essential roles in the mineralization events during eggshell formation due to it plays some roles in the determination of calcite crystal morphology. In both egg albumen and eggshell, large amount of lysozyme is found.
  • Lysozyme has a wide application potential due to its antibacterial properties against to bacteria causing periodontitis and oral mucosal infections, in oral care products, such as toothpastes and mouthwashes. Oral and topical application of lysozyme has been found to be effective in the prevention of herpes simplex virus and against HIV. Pseudomonas aureginosa, Bacillus cereus and Staphylococcus aureus are inhibited in the microenvironment containing 100 pg/ ml soluble eggshell matrix protein. On the other hand, Escherichia coli and Salmonella enteritidis are weakly inhibited for up to 4 hours. Matrix protein shows its effect via destroying bacterial cell wall.
  • Transferrins are also found in the eggshell. Among them, ovotransferrins, when present at 1 mg/ml and higher concentrations, have a negative effect on the bacteria since they bind free iron in the medium. Ovotransferrin, which settled close to the eggshell pores, exerts its antibacterial activity there. Ovocalyxin-32 (OCX-32) is another antibacterial substance, which locates in cuticle and external layer of the eggshell. It has bacteriostatic activity on B. subtilis and inhibits carboxypeptidase activity. In the eggshell matrix, ovocalyxin-36 (OCX-36) is found.
  • This protein has similarities with Plunck protein family members serving as defensing proteins of the eggshell, and with lipopolysaccharide-binding proteins serving as bacterial permeability increasing substances.
  • Potential anti-microbial proteins such as histones and avian beta-defensins, which are found in acid soiubie-fraction of eggsheii matrix protein fraction, have been shown to be effective against to P. aeruginosa, Bacillus cereus and Staph, aureus.
  • eggshell membranes There are two eggshell membranes, which one of them is inner membrane surrounding albumen and the second one is outer eggshell membrane locating underneath the calcified eggshell.
  • Organic matter constitutes 70%, inorganic matter constitutes 10% and water constitutes 20% of the membranes.
  • the eggshell membranes contain 16% nitrogen, 2% saccharide, 1.35-1.4% lipid.
  • the ratio of neutral lipids to complex lipids is 86:14. 63% of complex lipids is sphingomyelin and 12% is phosphatidylcholine.
  • a water-insoluble protein web forms the membranes.
  • Membrane proteins which forms a fibrous web enables a semi-permeable feature to the membranes.
  • the eggshell membrane provides a large adhesion surface for stromal cells to attach.
  • cystine there is significant amount of cystine in the eggshell membranes. Since the membranes contain 5-hydroxylisine, they include significant amount of collagen. Thus, fibrous elements of both outer and inner membranes are mainly composed of Tip-I, Tip-V ve Tip-X collagens. In addition to desmosin and isodesmosin, a non-elastin protein is found as a cross-linker in the eggshell membranes.
  • hyaluronic acid ratio in eggshell membrane and its enzymatic hydrolysates were found to be between 0.1-2%. With ELISA, on the basis of wet weight, the ratio was found as 5-10% (practically between 1 -5%). In the membranes, 2-5% hexosamine (w/w), 0.3-3% chondroitin (w/w), 5-30% collagen (w/w) are found. Collagen ratio was calculated by regarding hydroxy proline amino acid content as 4.5% and on the basis of 35% wet weight. The most abundant collagen is Type-I, less amounts of Type-V and Type-X collagens are also found. Type X -is known as tissue mineralization-controlling collagen. Glucosamine ratio is 10%, chondroitin ratio was determined to be 9% on the basis of wet weight.
  • the eggshell membranes contain high amounts of arginine, glutamic acid, methionine, histidine, cystine and proline amino acids. In addition, hydroxyproline, hydroxylysine and desmosine amino acids are very abundant. Cystine is a sulphur-containing amino acid that is very necessary for healthy skin, hair, bone and connective tissue.
  • cystine In the structure of alpha-keratin protein, which is abundant in the nail, hair, skin and oral mucosa, high amount of cystine is found. Because that cysteine is involved in the formation of collagen containing tissue, it improves skin's elasticity. Cysteine supplementation improves the healing of burns and other wound types, increases the flexibility of the joints with arthritis.
  • the eggshell membranes also contain bacteriolytic enzymes, such as N-acetyl glucosaminidase. Certain components of the eggshell membranes decrease thermal resistance of Gram-positive and Gram- negative pathogenic bacteria (Salmonella enteritis, Escherichia coli 0157: H7, Listeria monocytogenes and Staphylococcus aureus).
  • the eggshell membranes also contain acidic glycosaminoglycans (GAGs). Of these, dermatan sulphate and chondroitin sulphate are the most important members. Sulphated glycoproteins are found in the eggshell membranes. Glycoproteins contain hexosamines, hexos and fucose.
  • Hyaluronic acid is in abundance in the eggshell membranes.
  • Other components of the eggshell membrane are ovotransferrin, desmosin, isodesmosin, lysyl oxidase and lysozyme.
  • Contents of the eggshell membrane such as GAGs, chondroitin sulphate, hydrolyzed or natural form of collagen, sodium hyaluronate, ascorbate chelate of manganese and L- malic acid-containing oral and injectable preparations have long been used in the treatment and prevention of connective tissue and skin diseases.
  • Compositions containing such materials accelerate the synthesis activity of chondrocytes and wound healing, are effective in maintaining tissue health.
  • Collagen containing preparations aiming to accelerate wound healing and preparations containing glycosaminoglycan (GAG) suspensions have been used.
  • GAG glycosaminoglycan
  • EC 1 .4.3.13 amine oxidase form of lysyl oxidase enzyme
  • LTQ Lysine Tyrosyl Quinone Cofactor
  • This enzyme has significant role in embryonic development and repair of the connective tissues.
  • connective tissue damage occurs in the gum in addition to the other body regions.
  • chicken eggshell membrane as a biomaterial source is possibility of production of limited membrane area.
  • the total area of the chicken egg membrane is about 40-50 cm 2 , the only area can be obtained for clinical use is about 20 cm 2 .
  • This condition reveals that ostrich eggshell membrane would be more efficient for medical purposes. Due to larger useful surface area of the ostrich eggshell membrane and high mechanical resistance arising from its more thickness, ostrich eggshell membrane may be more suitable to use as wound-dressing material.
  • Ostrich eggshell membrane is also mainly consisted of collagen fibres, such as chicken eggshell membrane.
  • Outer layer of the ostrich eggshell membrane is a meshwork, which is comprised of a number of fibrous protein layers, wherein each layer is arranged perpendicular directions.
  • Ostrich eggs have an average of 1.5 kg in weight and 16 cmX12 cm in size. Mechanical resistance of the mineralized eggshelPis about 55 kg/cm 2 . Among the avian eggs, mechanical properties of the ostrich eggshell are excellent because of its perfect crystalline structure. Calcite in different iayers of the eggsheil has a different microstructure. This mechanical feature of the eggshell is imparted by biopolymers, which plays significant roles during eggshell formation. This biological process is quite similar to the processes observed in the teeth and bone formation.
  • Inorganic matrix of the ostrich eggshell is consisted of 96-97% is calcium carbonate, which is mainly in calcite form, 1.9% of calcium phosphate and 0.7% of of tricalcium phosphate.
  • Chicken eggshell calcite is also used as a raw material in the synthesis of hydroxilapatite (HA) due to its hydroxyl apatite content.
  • the organic matrix is around 4% and mainly consists of glycoproteins and proteoglycans.
  • HA hydroxilapatite
  • 520 different proteins have been identified. A significant number of these are involved in crystal nucleation, crystal growth and in the control of crystal formation. The most abundant of them are ovocleidin-17, ovocleidin-116 and ovocalyxin-36.
  • Type-X collagen which controls crystal nucleation and growth is also found.
  • Carbon (C)/calcium (Ca) ratio of the ostrich eggshell is 0.6 in the crystal layer and 2 times more in the cone layer.
  • the magnesium ratio in the crystal layer is 3%, whereas the ratio is 1 % in the cone layer.
  • Avian eggshell is formed in acellular environment of the avian uterus, in which calcium (Ca) and bicarbonate ions are at supersaturated conditions and rich in the proteins, which their concentrations depends on the stage of eggshell formation.
  • the absorption rate of the bone grafts is also quite important.
  • the position of the graft in the defect site, such as top cover material (onlay), the intermediate region (interpositional) or perimplant grafts requires different absorption rates. While in the onlay graft, minimal resorption rate is expected, whereas a rapid resorption is expected in the interpositional and periimplant grafts.
  • the resorption rate of bone grafts prepared from the ostrich eggshell varies depending on particle size and the nature of the matrix material.
  • biodegradability of the graft material plays an important role in addition to the micro porosity of the surface structure in healing. Particle surface of the ostrich eggshell is suitable for bone healing and has sufficient surface micro porosity.
  • Type-I collagen which can be used for medical purposes.
  • Type I collagen grafts when used alone, have some drawbacks, such as unstability of 3-dimensional structure over time depending on swelling, reduction of mechanical resistance and elasticity, risk of the antigenic response, the tissue irritation depending on cross linking treatment, weak patient tolerance, different dissolution kinetics and lack of effectiveness in the infected field.
  • Gelatine does not occur naturally in the free form, it is a hydrocolloid harvested by partial hydrolysis resulting from thermal denaturation or degradation with acid or alkali treatment of Type-I collagen, which is mainly prepared from animal skin and demineralised bone.
  • Type A gelatine is obtained by acid treatment of pork skins
  • Type B gelatine is obtained by alkaline treatment of bovine bone.
  • Gelatine is a material that its structural, physical, chemical, immunological properties are well known; it is also biodegradable, biocompatible, non-toxic, supporting cell proliferation; cross linked films, sheet, net, fibre, sponge and the bead forms can be prepared and it is non-immunogenic due to it does not contain aromatic rings in the molecular structure.
  • bovine-derived gelatine So in the medicine, it has a wide usage as different products. Use of the gelatine with porcine origin for medicinal purposes is limited because of religious reasons. Although, the use of bovine-derived gelatine might have BSA (bovine spongiform encephalopathy) transmission risk, acid demineralization decreases the risk
  • alkaline treatment reduces 100 times and a combined effect reduces 1000 times the risk.
  • Reducing effect of utra high temperature (UHT) treatment has been calculated as 100, probably 1000 times. Washing, filtration, ion exchange and other chemical applications reduce the BSE (bovine encephalopathy spongiform) risk of about 100 times.
  • BSE bovine encephalopathy spongiform
  • Carboxymethylcellulose is one of the water-soluble polymers and has the most common use nowadays.
  • Carboxymethylcellulose is a white or whitish powder, when dissolved in water forms a transparent and odourless viscous liquid. Cross bonds occur between the carboxymethylcellulose molecules themselves when dissolved in the water, to form a 3-dimensional network. Because that retention of significant amount of water occurs in this spongy and porous structure, carboxymethylcellulose is widely used in many fields including the food and drug industry.
  • Carboxymethylcellulose is widely been used for thickening as food additive and numbered as E 466. Because that carboxymethylcellulose is produced from natural sources, it has no side effect.
  • Carboxymethylcellulose is soluble in either hot or cold water, insoluble in organic solvents and compliant with water/alcohol systems.
  • carboxymethyl cellulose Functional properties depend on the hydroxyl groups of cellulose molecule. While the natural pH of carboxymethylcellulose is 8.25, more preferably, commercial carboxymethylcellulose products are produced at pH 7-7.5 to provide better dissolution. As the pH drops, the solubility of the product is reduced and insoluble in water at pH values below
  • Carboxymethylcellulose can be used as phase-emulsion stabilizer and suspending agent due to its rapid dissolution in cold water, controllable viscosity and thickener effect.
  • carboxymethylcellulose in addition to the thickening, water absorbing, suspension forming and the thin film-forming effects of carboxymethylcellulose; its good chemical stability, being physiologically inert and biocompatible, odourless, tasteless and non-toxic features makes carboxymethylcellulose a suitable substance to use in the preparation of bone graft materials as a binder.
  • carboxymethylcellulose is a polymeric binder and diluent, it facilitates the clinical use of the other graft materials.
  • Carboxymethylcellulose was reported to have a very positive effect in graft compatibility.
  • Carboxymethylcellulose is also very functional in polymer stabilization and controlled drug delivery systems.
  • carboxymethylcellulose creates 3-dimensional network, it may provide a support to the cells similar to the extracellular matrix at the early stages of healing.
  • the sodium carboxymethylcellulose which is more abundant chemical form found on the market, has perfect capacity of water absorbing, gel forming and biodegradation.
  • Chicken uterine fluid contains many bioactive substances, which are also found in the eggshell, those are starting and controlling crystal nucleation and crystal growth during the eggsheii construction.
  • the transmission risk of zoonotic diseases is relatively high for example mad cow disease (bovine spongiform encephalopathy , BSE) and other prion diseases of the bovine origin and HIV infection transmission risk of the human origin.
  • BSE bovine spongiform encephalopathy
  • Eggshell, eggshell membranes and their products do not contain any animal tissues and genetic material. Eggshell membrane is the unique biological material free of DNA and its antigenicity is very low. Therefore, the possibility of allergic reactions is poor.
  • the eggshell powder and eggshell membrane or their products can be used in safe as a bone graft.
  • Eggshell and eggshell membrane which also known to posses antibacterial properties, are relatively easy to supply, has a high potential for use in the healthcare.
  • the treatment of eggshell and eggshell membranes with 2.5% glutaraldehyde eliminates the risk of transmission of viral agents.
  • the organic matrix of the eggshell membrane contains antibacterial substances in addition to bioactive molecules initiating calcium mineralization and controlling the form and size of the calcite crystals.
  • the eggshell membrane proteins alone or in combination with shell particles are suitable in the preparation of bone graft materials.
  • Graft material prepared from the ostrich eggshell can be used alone in the block, particle and powder form, or in combination with other matrix and binder substances.
  • Ostrich eggshell is a readily available, safe osteoconductive material and has many positive effects on the bone healing. DETAILED DESCRIPTION OF THE INVENTION
  • the invent is related to preparation and bone graft material itself in the powder, particulate, pulp and sponge forms, which are prepared by separately processing ostrich eggshell and eggshell membranes, coating eggshell particles with water-soluble eggshell membrane proteins, recombinant bone morphogenetic proteins, carboxymethylcellulose, gelatine and chicken uterine fluid, and also preparation of bone graft material in the form of powder, particulate, memep and sponge by addition of excipients and using the coated-eggshell particles.
  • the prepared material strengths and accelerates bone defect healing, provides porosity in the placed defect site, contains bioactive substances of biological origin and includes antibacterial agents protecting from infection.
  • the product contains ostrich eggshell particles sized 500- 1500 ⁇ , gelatine with bovine or chicken origin as binder and bovine gelatine or carboxymethylcellulose as matrix. Powder or particle forms can be applied to the defect site by mixing blood, bone marrow or platelet enriched blood plasma of the recipient.
  • Preparation of the bone graft comprises of the following steps:
  • Eggshell particles are ground into a powder with a suitable mill, and the powder is passed through a sieve with 1.5 mm pore size. The residue on the sieve is separated and not used. The fraction passing through the sieve with 1.5 mm pore size is passed through again with a sieve with 0.5 mm pore size. The residue on the 0.5 mm mesh-sieve (the particles between ⁇ 0.5-1.5 mm size) is used in graft preparation.
  • the membrane fragments are decalcified in 10% EDTA aqueous solution (w/v, tetra sodium salt, pH 7.2) containing 0.02% sodium azide as bacterial protectant, for 12 hours. Then, the fragments are rinsed 3 times in distilled water, by rinsing 2 hours in each, and dried under vacuum up to 50°C temperature levels. Dried membrane fragments are ground into a powder and ⁇ 5 ⁇ particles are separated by passing through a suitable sieve. The separated powder is kept in a freezer until use.
  • 10% EDTA aqueous solution w/v, tetra sodium salt, pH 7.2
  • sodium azide sodium azide
  • Eggshell membrane powder prepared as in the item 3 is added at 3% ratio (w/v) into 1 ,25 N aqueous 3-mercapto propionic acid solution (v/v), which is prepared in 100 ml of 10% acetic acid at room temperature (22°C). Temperature of the mixture is increased to a temperature up to 50°C, and by agitating on a shaker, the powder is completely dissolved and subsequently cooled to room temperature. Insoluble fractions are precipitated by high-speed centrifugation. Supernatant is recovered and the pH adjusted to 5 with 5M NaOH. The precipitate is filtered, washed with absolute methanol and dried.
  • Eggshell membrane powder prepared as in the item 3 is added into 3 parts 1.5 N NaOH and 1 part absolute ethanol (v/v) mixture at %25 ratio (w/v) and shaken for sufficient time at appropriate temperature. Upon completion of dissolution, liquid fraction is evaporated. The residue is washed with absolute methanol and dried. The material is stored in the freezer until use.
  • NaOH sodium hydroxide
  • Eggshell membrane powder prepared as in the item 3 is added into 100 ml of performic acid which is prepared by mixing 10 ml of hydrogen peroxide (H2O2) and 90 ml of formic acid (v/v) and the mixture is treated for 24 hours at 25°C.
  • the material, which is filtered through glass filter, is washed distilled water and treated with 3200 U/mg pepsin prepared by dissolving in 1 % (v/v) 0.5 M acetic acid solution, at 25°C for 48 hours by agitating every 2 hours. Then, enzymatic activity is blocked by adding 0.2 mg/100 ml a pepstatin.
  • Water-soluble protein solution is prepared by dissolving eggshell membrane protein prepared as in the item 4.2 (by treating with NaOH- absolute ethanol) in 10% acetic acid. The solution is stored in the freezer until use.
  • Preparation of water-soluble membrane protein solution with the membrane protein obtained by perform ic acid treatment Water- soluble protein solution is prepared by dissolving eggshell membrane protein prepared as in the item 4.3 method (treating with performic acid) in 10% acetic acid. The solution is stored in the freezer until use.
  • Preparation of carboxy methylcellulose solution 10% carboxymethylcellulose solution is prepared by heating distilled water up to 50°C temperatures with a magnetic stirrer, meanwhile by slow addition of pharmacological quality carboxymethylcellulose. The solution is stored in the freezer until use.
  • Preparation of water-soluble membrane protein and carboxy methylcellulose mixture is prepared by dissolving eggshell membrane protein prepared as in the item 4.3 method (treating with performic acid) in 10% acetic acid. The solution is stored in the freezer until use.
  • Preparation of carboxy methylcellulose solution 10% carboxymethylcellulose solution is prepared by heating distilled water up to 50°C temperatures with a magnetic stirrer, meanwhile by slow addition of pharmacological quality carboxymethylcellulose. The solution is
  • Gelatine solution is prepared by heating distilled water up to 50°C with a magnetic stirrer, meanwhile by slow addition 10 g of bovine origin gelatine and dissolved.
  • Preparation of the chicken uterine fluid The uterus of the chicken is exposed by midline incision of the abdominal wall, 6-19 hours after 50 pg prostaglandin F20: injection. After removal of the egg in the oviduct, a plastic tube is inserted into the vaginal outlet and uterine fluid is discharged completely by massaging uterus with hand. Through a longitunal incision of the uterine wall, the exposed uterine mucosa is scraped. Scraping is homogenized with a mechanical tissue homogenizer in Tris HCI buffer solution (0.0625 M, pH 6.8.
  • the homogenate is passed through a membrane filter (0,2 pm), and the filtrate is diluted at a 1 :1 ratio with proteinase inhibitor (benzamidine HCI, 2.5 mM ; ⁇ - amino - 2 - caproic acid, 50 mM N - etil malei- imide, 0.5 mM phenyl methyl sulfonyl fluoride (PMSF), 1.0 mM) in Tris HCI buffer ( 0.0625 M, pH 6.8) and quenched in liquid nitrogen, the frozen content is stored in the freezer until use.
  • proteinase inhibitor benzamidine HCI, 2.5 mM ; ⁇ - amino - 2 - caproic acid, 50 mM N - etil malei- imide, 0.5 mM phenyl methyl sulfonyl fluoride (PMSF), 1.0 mM
  • Tris HCI buffer 0.0625 M, pH 6.8
  • Preparation of bone graft in the powder form by absorbing eggshell membrane protein with 3-mercaptopropionic acid treatment prepared as in the item 4.1 To a definite volume of membrane protein solution (prepared with 3-mercaptopropionic acid treatment) prepared as in the item 4.1 , eggshell material prepared as in the item 3 is added until the particles completely getting wet. The material is dried under vacuum up to 50°C temperature levels, sterilized with ethylene oxide and stored in the freezer until use.
  • Preparation of bone graft in the powder form by absorbing eggshell membrane protein prepared with performic acid treatment as in the item 4.3 To a definite volume of membrane protein solution (prepared with performic acid treatment treatment) prepared as in the item 4.3, eggshell material prepared as in the item 3 is added until the particles completely getting wet. The material is dried under vacuum up to 50°C temperature levels, sterilized with ethylene oxide and stored in the freezer until use.
  • Preparation of bone graft in the powder form by coating eggshell membrane protein and carboxymethylcellulose mixture
  • Preparation of bone graft in the powder form by absorbing eggshell membrane protein prepared with 3-mercaptopropionic acid treatment and carboxymethylcellulose mixture To a definite volume of membrane protein solution (prepared with 3-mercaptopropionic acid treatment) prepared as in the item 7.1 , eggshell material prepared as in the item 3 is added until the particles completely getting wet. The material is dried under vacuum up to 50°C temperature levels, sterilized with ethylene oxide and stored in the freezer until use.
  • Preparation of bone graft in the powder form by absorbing eggshell membrane protein prepared with performic acid treatment and carboxymethylcellulose mixture To a definite volume of membrane protein solution (prepared with performic acid treatment) prepared as in the item 7.3, eggshell material prepared as in the item 3 is added until the particles completely getting wet. The material is dried under vacuum up to 50°C temperature levels, sterilized with ethylene oxide and stored in the freezer until use.
  • Preparation of bone graft in the powder form by absorbing eggshell membrane protein prepared with 3-mercaptopropionic acid treatment and gelatine mixture To a definite volume of membrane protein solution (prepared with 3-mercaptopropionic acid treatment) prepared as in the item 9.1 , eggshell material prepared as in the item 3 is added until the particles completely getting wet. The material is dried under vacuum up to 50°C temperature levels, sterilized with ethylene oxide and stored in the freezer until use.
  • Preparation of bone graft in the powder form by absorbing eggshell membrane protein prepared with NaOH-absolute ethanol treatment and gelatine mixture To a definite volume of membrane protein solution (prepared with NaOH-absolute ethanol treatment) prepared as in the item 9.2, eggshell material prepared as in the item 3 is added until the particles completely getting wet. The material is dried under vacuum up to 50°C temperature levels, sterilized with ethylene oxide and stored in the freezer until use.
  • Preparation of bone graft in the powder form by coating chicken uterine fluid Eggshell particles prepared as in the item 3 are coated by continuous agitation with uterine fluid under vacuum and sterile conditions by drying up to 40°C temperature levels. The material is sterilized with ethylene oxide and stored in the freezer until use.
  • Preparation of bone graft in the pulp form by absorbing eggshell membrane protein prepared with 3-mercaptopropionic acid treatment as in the item 4.1 To a definite volume of membrane protein solution (prepared with 3-mercaptopropionic acid treatment) prepared as in the item 4.1 , eggshell material prepared as in the item 3 is added until the particles completely getting wet. The particles are dried under vacuum, at 50°C with continuous agitation overnight. To this material, gelatine solution at 60°C temperature levels and glycerine are added by continuous agitation. The material is sterilized with ethylene oxide and stored in the freezer until use.
  • eggshell material prepared as in the item 3 is added until the particles completely getting wet.
  • the particles are dried under vacuum, at 50°C with continuous agitation overnight.
  • gelatine solution at 60°C temperature levels and glycerine are added by continuous agitation.
  • the material is sterilized with ethylene oxide and stored in the freezer until use.
  • Preparation of bone graft in the pulp form by absorbing eggshell membrane protein prepared with performic acid treatment as in the item 4.3 To a definite volume of membrane protein solution (prepared with performic acid treatment) prepared as in the item 4.3, the eggshell material prepared as in the item 3 is added until the particles completely getting wet. The particles are dried under vacuum, at 50°C with continuous agitation overnight. To this material, gelatine solution at 60°C temperature levels and glycerine are added by continuous agitation. The material is sterilized with ethylene oxide and stored in the freezer until use. 16. Preparation of bone graft in the pulp form by absorbing eggshell membrane protein and carboxymethylcellulose mixture:
  • Preparation of bone graft in the pulp form by absorbing eggshell membrane protein prepared with 3-mercaptopropionic acid treatment and carboxymethylcellulose mixture To a definite volume of membrane protein (prepared with 3-mercaptopropionic acid treatment) and carboxymethylcellulose mixture prepared as in the item 7.1 , the eggshell material prepared as in the item 3 is added until the particles completely getting wet. The particles are dried under vacuum, at 50°C with continuous agitation overnight. To this material, gelatine solution at 60°C temperature levels and glycerine are added by continuous agitation.
  • the material is sterilized with ethylene oxide and stored in the freezer until use.
  • Preparation of bone graft in the pulp form by absorbing eggshell membrane protein prepared with NaOH-absolute ethanol treatment and carboxymethylcellulose mixture To a definite volume of membrane protein (prepared with NaOH-absolute ethanol treatment) and carboxymethylcellulose mixture prepared as in the item 7.2, the eggshell material prepared as in the item 3 is added until the particles completely getting wet. The particles are dried under vacuum, at 50°C with continuous agitation overnight. To this material, gelatine solution at 60°C temperature levels and glycerine are added by continuous agitation. The material is sterilized with ethylene oxide and stored in the freezer until use.
  • Preparation of bone graft in the pulp form by absorbing eggshell membrane protein prepared with performic acid treatment and carboxymethylcellulose mixture To a definite volume of membrane protein (prepared with performic acid treatment) and carboxymethylcellulose mixture prepared as in the item 7.3, the eggshell material prepared as in the item 3 is added until the particles completely getting wet. The particles are dried under vacuum, at 50°C with continuous agitation overnight. To this material, gelatine solution at 60°C temperature levels and glycerine are added by continuous agitation. The material is sterilized with ethylene oxide and stored in the freezer until use. Preparation of bone graft in the pulp form by absorbing eggshell membrane protein and gelatine mixture:
  • Preparation of bone graft in the pulp form by absorbing eggshell membrane protein prepared with 3-mercaptopropionic acid treatment and gelatine mixture To a definite volume of membrane protein (prepared with 3-mercaptopropionic acid treatment) and gelatine mixture prepared as in the item 9.1 , the eggshell material prepared as in the item 3 is added until the particles completely getting wet. The particles are dried under vacuum, at 50°C with continuous agitation overnight. To this material, gelatine solution at 60°C temperature levels and glycerine are added by continuous agitation. The material is sterilized with ethylene oxide and stored in the freezer until use.
  • Preparation of bone graft in the pulp form by absorbing eggshell membrane protein prepared with NaOH-absolute ethanol treatment and gelatine mixture To a definite volume of membrane protein (prepared with NaOH-absolute ethanol treatment) and gelatine mixture prepared as in the item 9.2, the eggshell material prepared as in the item 3 is added until the particles completely getting wet. The particles are dried under vacuum, at 50°C with continuous agitation overnight. To this material, gelatine solution at 60°C temperature levels and glycerine are added by continuous agitation. The material is sterilized with ethylene oxide and stored in the freezer until use.
  • Preparation of bone graft in the pulp form by absorbing eggshell membrane protein prepared with performic acid treatment and gelatine mixture To a definite volume of membrane protein (prepared with performic acid treatment) and gelatine mixture prepared as in the item 9.3, the eggshell material prepared as in the item 3 is added until the particles completely getting wet. The particles are dried under vacuum, at 50°C with continuous agitation overnight. To this material, gelatine solution at 60°C temperature levels and glycerine are added by continuous agitation. The material is sterilized with ethylene oxide and stored in the freezer until use.
  • Preparation of bone graft in the pulp form by coating eggshell particles with uterine fluid Eggshell particles prepared as in the item 3 are coated by continuous agitation with uterine fluid prepared as in the item 10, under vacuum and sterile conditions by drying up to 40°C temperature levels. To this material, gelatine solution at 60°C temperature levels and glycerine are added by continuous agitation. The material is sterilized with ethylene oxide and stored in the freezer until use.
  • the eggshell material prepared as in the item 3 is added until the particles completely getting wet.
  • the particles are dried under vacuum, at 50°C with continuous agitation overnight.
  • gelatine solution at 60°C temperature levels and glycerine are added by continuous agitation.
  • the prepared material is poured into molds of different depths and sizes before solidification, waited to harden, sterilized with ethylene oxide and stored in the freezer until use. .
  • Preparation of bone graft in sponge form by absorbing membrane protein solution prepared with NaOH-absolute ethanol treatment as in the item 4.2 To a definite volume of membrane protein (prepared with NaOH-absolute ethanol treatment) prepared as in the item 4.2, the eggshell material prepared as in the item 3 is added, until the particles completely getting wet. The particles are dried under vacuum, at 50°C with continuous agitation overnight. To this material, gelatine solution at 60°C temperature levels and glycerine are added by continuous agitation. The prepared material is poured into molds of different depths and sizes before solidification, get hardened, sterilized with ethylene oxide and stored in the freezer until use. .
  • Preparation of bone graft in the sponge form by absorbing membrane protein solution prepared with 3-mercaptopropionic acid treatment and carboxymethylcellulose mixture to the eggshell particles To a definite volume of membrane protein (prepared with 3- mercaptopropionic acid treatment) prepared as in the item 7.1 and carboxymethylcellulose mixture, the eggshell material prepared as in the item 3 is added until the particles completely getting wet. The particles are dried under vacuum, at 50°C with continuous agitation overnight. To this material, 10% carboxymethylcellulose solution at 60°C temperature levels and glycerine are added by continuous agitation. The prepared material is poured into molds of different depths and sizes before solidification, waited to harden, sterilized with ethylene oxide and stored in the freezer until use. 0.2. Preparation of bone graft in the sponge form by absorbing membrane protein solution, which is prepared with NaOH and absolute ethanol treatment and carboxymethylcellulose mixture to the eggshell particles: To a definite volume of membrane protein solution, which is prepared with NaOH and absolute ethanol treatment and carb
  • the eggshell material prepared as in the item 3 is added, until the particles completely getting wet.
  • the particles are dried under vacuum, at 50°C with continuous agitation overnight.
  • 10% carboxymethylcellulose solution at 60°C temperature levels and glycerine are added by continuous agitation.
  • the prepared material is poured into molds of different depths and sizes before solidification, waited to harden, sterilized with ethylene oxide and stored in the freezer until use. 20.3.
  • Preparation of bone graft in the sponge form by absorbing membrane protein solution, which is prepared with performic acid treatment and carboxy methylcellulose mixture to the eggshell particles To a definite volume of membrane protein (prepared with performic acid treatment) prepared as in the item 7.3 and carboxymethylcellulose mixture, the eggshell material prepared as in the item 3 is added, until the particles completely getting wet. The particles are dried under vacuum, at 50°C with continuous agitation overnight. To this material, 10% carboxymethylcellulose solution at 60°C temperature levels and glycerine are added by continuous agitation. The prepared material is poured into molds of different depths and sizes before solidification, waited to harden, sterilized with ethylene oxide and stored in the freezer until use.
  • Preparation of bone graft in sponge form by absorbing eggshell membrane protein and gelatine To a definite volume of membrane protein (prepared with performic acid treatment) prepared as in the item 7.3 and carboxymethylcellulose mixture, the eggshell material prepared as in the item 3 is added, until the particles completely getting wet
  • Preparation of bone graft in sponge form by absorbing membrane protein solution prepared with 3-mercaptopropionic acid treatment and gelatine mixture to the eggshell particles To a definite volume of membrane protein (prepared with 3-mercaptopropionic acid treatment) prepared as in the item 9.1 and gelatine mixture, the eggshell material prepared as in the item 3 is added, until the particles completely getting wet. The particles are dried under vacuum, at 50°C with continuous agitation overnight. To this material, 10% gelatine solution at 60°C temperature levels and glycerine are added by continuous agitation. The prepared material is poured into molds of different depths and sizes before solidification, waited to harden, sterilized with ethylene oxide and stored in the freezer until use.
  • Preparation bone graft in sponge form by coating eggshell particles with uterine fluid The eggshell particles prepared as in the item 3 are coated by continuous agitation and drying, up to 40°C temperature levels at under vacuum and sterile conditions with chicken uterine fluid prepared as in the the item 10. To this material, 10% gelatine and glycerine are added until the material gaining sponge consistency, at 60°C. The material is poured into molds of different depths and sizes before solidify and then, sterilized with ethylene oxide and stored in freezer until use.
  • the prepared bone graft forms described as processing steps given above can be used for treatment of the compact or cancellous bone tissue damage and in the bone losses with different origin and in orthopaedic problems, dentistry and veterinary medicine to meet the losses, induction of bone modelling, conjunction of fracture ends.
  • the prepared materials might find a widespread use in in different areas of medicine, dentistry and veterinary medicine since they are cheaper and have not any risk of zoonotic disease transmission.

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Abstract

L'invention se rapporte à la préparation de matériaux de greffe osseuse sous forme pulvérulente, particulaire et spongieuse à partir de coquille d'œuf et de membranes de coquille d'œuf d'autruche afin de corriger des pertes osseuses survenant pour différentes raisons, pour la régénération à la fois de l'os compact et de l'os spongieux, afin de traiter des fractures et de joindre des extrémités fracturées.
PCT/TR2014/000232 2013-07-17 2014-07-16 Greffe osseuse à partir de poudre de coquille d'œuf d'autruche et son procédé de préparation WO2015009256A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016207355A1 (fr) * 2015-06-24 2016-12-29 Biovotec As Échafaudages produits par ingénierie tissulaire comprenant de la membrane de coquille d'œuf sous forme particulaire
US10932952B2 (en) 2015-11-11 2021-03-02 Biovotec As Dry biocompatible disintegratable films for delivering particulate egg shell membrane to a wound
CN115382877A (zh) * 2022-08-15 2022-11-25 武汉新华扬生物股份有限公司 一种分离蛋壳与蛋壳膜的方法
US11992508B2 (en) 2014-10-28 2024-05-28 Biovotec As Micronized eggshell membrane particles and the use thereof to promote the healing of wounds

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2746652A1 (fr) * 1996-03-28 1997-10-03 Dupoirieux Laurent Pierre Utilisation de la coquille d'oeuf comme implant osseux et son mode de fabrication
WO2012036645A2 (fr) * 2010-09-15 2012-03-22 Ercan Durmus Procédé de préparation d'un gel buccal pour hygiène bucco-dentaire contenant de la poudre de coquilles d'œufs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2746652A1 (fr) * 1996-03-28 1997-10-03 Dupoirieux Laurent Pierre Utilisation de la coquille d'oeuf comme implant osseux et son mode de fabrication
WO2012036645A2 (fr) * 2010-09-15 2012-03-22 Ercan Durmus Procédé de préparation d'un gel buccal pour hygiène bucco-dentaire contenant de la poudre de coquilles d'œufs

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11992508B2 (en) 2014-10-28 2024-05-28 Biovotec As Micronized eggshell membrane particles and the use thereof to promote the healing of wounds
WO2016207355A1 (fr) * 2015-06-24 2016-12-29 Biovotec As Échafaudages produits par ingénierie tissulaire comprenant de la membrane de coquille d'œuf sous forme particulaire
CN108136073A (zh) * 2015-06-24 2018-06-08 生物科技有限公司 包括颗粒蛋壳膜的组织工程化支架
US20200030492A1 (en) * 2015-06-24 2020-01-30 Biovotec As Tissue engineering scaffolds comprising particulate egg shell membrane
CN108136073B (zh) * 2015-06-24 2021-02-02 生物科技有限公司 包括颗粒蛋壳膜的组织工程化支架
AU2016282918B2 (en) * 2015-06-24 2021-04-22 Biovotec As Tissue engineering scaffolds comprising particulate egg shell membrane
US11045578B2 (en) 2015-06-24 2021-06-29 Biovotec As Tissue engineering scaffolds comprising particulate egg shell membrane
US10932952B2 (en) 2015-11-11 2021-03-02 Biovotec As Dry biocompatible disintegratable films for delivering particulate egg shell membrane to a wound
CN115382877A (zh) * 2022-08-15 2022-11-25 武汉新华扬生物股份有限公司 一种分离蛋壳与蛋壳膜的方法

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