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WO2003020840A1 - Methodes de purification de gelatine d'os insoluble - Google Patents

Methodes de purification de gelatine d'os insoluble Download PDF

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Publication number
WO2003020840A1
WO2003020840A1 PCT/IB2002/003856 IB0203856W WO03020840A1 WO 2003020840 A1 WO2003020840 A1 WO 2003020840A1 IB 0203856 W IB0203856 W IB 0203856W WO 03020840 A1 WO03020840 A1 WO 03020840A1
Authority
WO
WIPO (PCT)
Prior art keywords
bone
gelatin
insoluble
powder
tissue
Prior art date
Application number
PCT/IB2002/003856
Other languages
English (en)
Other versions
WO2003020840A9 (fr
Inventor
Ying Fan
Ming Hao Zheng
David Wood
Original Assignee
Perth Bone And Tissue Bank, 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 Perth Bone And Tissue Bank, Inc. filed Critical Perth Bone And Tissue Bank, Inc.
Priority to PCT/IB2002/003856 priority Critical patent/WO2003020840A1/fr
Publication of WO2003020840A1 publication Critical patent/WO2003020840A1/fr
Publication of WO2003020840A9 publication Critical patent/WO2003020840A9/fr

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Classifications

    • 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
    • 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
    • A61L27/222—Gelatin
    • 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
    • A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H1/00—Macromolecular products derived from proteins
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09H—PREPARATION OF GLUE OR GELATINE
    • C09H3/00—Isolation of glue or gelatine from raw materials, e.g. by extracting, by heating
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09H—PREPARATION OF GLUE OR GELATINE
    • C09H7/00—Preparation of water-insoluble gelatine
    • 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 present invention relates to methods for isolation and purification of Insoluble Bone Gelatin (ISBG) and the application of ISBG produced by the present invention, for example, for impaction bone grafting, nonunion fracture and dental conditions.
  • ISBG Insoluble Bone Gelatin
  • bone is the most frequently transplanted tissue in humans. With an aging population and with an increasing number of younger and more active patients undergoing, for example, hip replacement, revision surgery is often required. Currently, fresh frozen donor bone is the most effective graft material available for difficult clinical situations. Although bone transplantation has been used as the standard approach for reconstruction following excision of diseased bone, some problems remain unsolved. Among these, non-union fracture and loosening are major problems responsible for the failure of operations (especially for more extensive operations or after multiple operations), due to poor incorporation of allografts into host bone. Bone formation in adult humans is a complex and closely regulated process.
  • All bone is remodeled by the coordinated actions of bone resorbing (osteoclasts) and bone forming (osteob lasts) cells that are under the regulatory control of local cytokines generated in the environment of the remodeling cells.
  • These local factors are comprised of peptides and nonpeptides. These factors are often incorporated into non-collagenous protein of bone matrix, .released in an active form when bone is remodeled. Complex interactions between these factors and their target cells are responsible for the normal delicate balance between bone resorption and bone formation.
  • the specific cellular components of bone only account for a minor portion of tissue weight.
  • the major component of bone is matrix, which accounts for 90-95% of the tissue weight.
  • Bone matrix includes mineral phases and protein phases, which are portioned 60-65% and 40- 35%), respectively, of non-cellular bone weight.
  • the non-collagenous proteins are heterogeneous in origin and some appear to be produced by bone cells while others are incorporated from or are concentrated from serum.
  • the non-collagenous proteins are laid down into bone matrix by binding to the mineral phase, collagen or other matrix proteins.
  • There are two fractions of non-collagenous bone proteins soluble and insoluble non-collagenous bone proteins.
  • the fraction of insoluble non- collagenous bone proteins consist of bone growth factors - the growth factor 'cocktail', which is responsible for regulating bone formation. In the 1970's, M.
  • Urist established procedures to isolate soluble and insoluble bone morphogenetic proteins from animal bones (for example, as described in "Bone morphogenesis in implants of insoluble bone gelatin.” Urist MR, Iwata H, Ceccoth PL, Doriman RL, Boyd SD, McDowell RM and Chien C. Proc Natl Acad Sci USA 1973:351 1-351 5, hereby incorporated by reference in its entirety (the Urist 1973 article).
  • the method taught in Urist begins with crushed bone, and does not have a pre- ashing step. A chloroform-methanol extraction is performed to remove blood, cells, and other debris from the bone. The method also includes a treatment step with lithium chloride, and treatment with HC1, CaCl 2 , and EDTA at 2°C. The duration of the process is about 3.5 days, and the resulting ISBG contains BMP-2 only.
  • Bone morphogenetic activities were observed in implants of insoluble bone gelatin in several animal models described by Urist and other researchers including the present inventors. It has now been well accepted that bone gelatin can induce bone formation and control bone morphogenetic reaction.
  • Gie et al. was first to describe the use of impaction bone allografting in revision surgery (for example, as described in "Contained morselized allograft in revision total hip arthroplasty.” Gie GA, Linder L, Ling RS, Simon JP, Slooff TJ, Timperley AJ Orthop Clin North Am 1993 24:717-725, hereby incorporated by reference in its entirety) and this technique has been used widely since then. Even with the improvement of techniques and materials used in this field, loosening still remains a big problem.
  • the present invention discloses methods for isolating and purifying insoluble bone gelatin (ISBG).
  • ISBG insoluble bone gelatin
  • the present invention teaches isolation and purification of ISBG and its clinical application, for example, in impaction bone grafts and non-union fracture healing.
  • the invention involves methods for preparing bone powder and, isolating and purifying ISBG, and the resulting purified ISBG and use of it in, for example, impaction bone grafts and non-union fracture healing.
  • an insoluble bone gelatin including about 10 percent growth factor is also included in the present invention.
  • the growth factors are BMP-2, FGF, TGF-beta, and IGF, or any combination thereof.
  • the present technique for purification of ISBG differs from the prior art in terms of preparation of bone powder (for example, as described below), the pre-treatment procedure, temperature and the chemicals used.
  • a method for isolating bone gelatin was established by Urist (for example, in U.S. Patent No. 4,294,753, hereby incorporated by reference in its entirety).
  • a novel ISBG containing one or more of BMP-2, FGF, TGF-beta, IGF, and IGF binding protein, or any combination thereof is produced by milling bone powder to up to about 1.0 millimeter particles and pre-washing the bone powder with saline at 35-55°C, preferably 40-45°C for 5 minutes.
  • the milled bone powder is treated with HCl, CaCl 2 , and EDTA at 4°C, as described in detail below, and the entire procedure takes approximately 48 hours. No chloroform or methanol extraction process is used, and no lithium chloride solution is used in the process for obtaining the novel ISBG of this particular embodiment.
  • the ISBG produced is useful in preparing impaction bone grafts.
  • the present invention includes the steps of screening and testing human bones to determine suitability for human transplantation, preparing bone powder (preferably having a particle size up to 1.0 millimeter, more preferably 0.5-1.0 millimeter), isolating and purifying insoluble bone gelatin from the bone powder, and using the purified insoluble bone gelatin, for example, for impaction bone grafting, non-union fracture, and dental use.
  • bone powder prepared according to the present invention was demineralized using an acid such as hydrochloric acid or acetic acid, then treated with a neutralizing salt such as calcium chloride or calcium phosphate, and then treated with a stabilizer such as ethylene diamine tetraacetic acid (EDTA). The resulting gelatin was then treated with sterilized water.
  • Protocol 1 is according to the Urist 1973 article.
  • Protocols 2 and 3 are according to the present invention.
  • Bone powders without any pre-preparation step were subjected to the following procedure for ISBG extraction.
  • Step 1 chloroform and methanol (1 : 1 ratio) for 4 hours at 25°C; Step 2 0.6 N HCl for 24 hours at 4°C; Step 3 2.0 M CaCl 2 for 24 hours at 4°;C Step 4 0.5 M EDTA for 24 hours at 4°;C Step 5 8.0 M LiCl for 4 hours at 4°C; and Step 6 sterilized H O for 4 hours at 55°C
  • Protocol 2 Bone powders prepared by the method described below were subjected to the following procedure for ISBG extraction. • Step 1 0.6 N HCl up to 24 hours at 4°C;
  • Step 2 2.0 M CaCl 2 up to 24 hours at 4°C; Step 3 0.5 M EDTA up to 24 hours at 4°C; Step 4 8.0 M LiCl for 4 hours at 4°C; and Step 5 sterilized H 2 O for 4 hours at 55°C.
  • Protocol 3 Bone powders prepared by the method described below were subjected to the following procedure for ISBG extraction. Step 1 0.6 N HCl up to 24 hours at 4°C; Step 2 2.0 M CaCl 2 up to 24 hours at 4°C;
  • Step 3 0.5 M EDTA for 4 hours at 4°C; and Step 4 sterilized H 2 O for 4 hours at 55°C.
  • the present invention provides very promising results to meet clinical requirements, such as osteoinductive activities and mechanical stability for impaction bone grafts. Indeed, there was no significant difference between protocols 2 & 3 as to the degree of new bone formation produced.
  • An embodiment as described in Protocol 3 is preferable from an economic standpoint as this protocol includes fewer steps and chemicals and provides a desirable product. No adverse effects were observed using this protocol.
  • the extraction period was reduced from 3.5 days to about 2 days, and produced an ISBG material having potent osteoinductive activity as evidenced in rat models in which bone formation was observed after the implantation of ISBG. Mechanical stability in impaction bone grafts was also observed, and is thought to be related to the osteoinductivity of the ISBG.
  • an mjectable ISBG material can be generated by using the ISBG produced according to the present invention with any acceptable pharmaceutical carriers. In one embodiment, 1% alginate gel with distilled water can be used as a pharmaceutical carrier.
  • the ISBG isolated and purified according to the present invention has preferred biological properties, such as osteoinductivity, and mechanical stability. It will provide exceptional benefits for example, for impaction bone graft, non-union fracture and dental implantation.
  • the results provided herein regarding the present invention indicate that the process of bone repair can be improved by introducing insoluble bone gelatin (ISBG) into a site of the operation, for example, for impaction bone grafting.
  • ISBG insoluble bone gelatin
  • the donors of human bones were screened and tested to determine suitability for human transplant according to international standards (e.g., American Association of Tissue Banks, European Association of Tissue Banks, Therapeutic Goods Administration of Australia).
  • Bone powder with a particle size of up to 1.0 millimeter was produced with a bone mill. Bone powder was collected in a sterile container and rinsed thoroughly with sterile normal saline at 50°C to remove as much as possible blood, fat and bone marrow. Important aspects of using a fine particle size of the bone powder include enabling the success of further purification procedure for ISBG and the ability to produce an mjectable form of ISBG.
  • Step l 0.6 N HCl up to 24 hr; Step 2 2.0 M CaCl 2 up to 24 hr; and
  • Step 3 0.5 M EDTA for 4 hr.
  • the ISBG materials were then maintained below -70°C to be used for further assessments such as osteoinductivity and mechanical stability.
  • Rat models were used to assess the biological activities of each ISBG material produced according to the present invention.
  • the so called attribute of osteoinductivity was assessed by introducing ISBG produced according to the present invention into two rat models.
  • ISBG was implanted into the tibialis anterior (TA) muscle or under skin of anterior abdominal wall of the animals.
  • TA tibialis anterior
  • the skin over the TA muscle was cut to expose the muscle and the TA muscle was then cut longitudinally halfway through the muscle.
  • ISBG prepared according to the present invention was then implanted into the site and the muscle closed by suturing the cut muscle with silk sutures. The skin was then sutured using silk thread and the animal was left to recover.
  • the skin over the anterior abdominal wall was opened and the ISBG produced according to the present invention was implanted into the pocket.
  • the skin was then closed by suturing the cut skin with silk thread and the animal left to recover.
  • a control group was produced using the same procedures, expect bone powder was implanted rather than ISBG produced according to the present invention.
  • the ISBG material produced by the present invention has potent osteoinductivity and mechanical stability so that we indicate that it can be used in impaction bone grafting.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dermatology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • Botany (AREA)
  • Zoology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Urology & Nephrology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

La présente invention concerne des méthodes destinées à purifier de la gélatine d'os insoluble ainsi que les utilisations de cette gélatine d'os insoluble. Une technique d'isolement de gélatine d'os insoluble à partir du tissu osseux consiste à broyer le tissu osseux en poudre d'os, à nettoyer la poudre d'os avec une solution saline, à déminéraliser le tissu osseux, à mettre la poudre d'os en contact avec un sel neutre, puis à mettre ladite poudre d'os en contact avec un stabilisant. La présente invention concerne également une gélatine d'os insoluble comprenant un facteur de croissance en concentration d'environ 10 %. Cette gélatine d'os insoluble est utile, par exemple, dans la préparation de greffes osseuses par impaction.
PCT/IB2002/003856 2002-09-06 2002-09-06 Methodes de purification de gelatine d'os insoluble WO2003020840A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008134814A1 (fr) * 2007-05-04 2008-11-13 Perth Bone & Tissue Bank Procédé et matériau à libération contrôlée pour traiter l'inflammation
EP2349365A4 (fr) * 2008-11-04 2013-01-23 Perth Bone & Tissue Bank Matériau de support pour des cellules ostéogéniques

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4619989A (en) * 1981-05-05 1986-10-28 The Regents Of The University Of Cal. Bone morphogenetic protein composition
US6180606B1 (en) * 1994-09-28 2001-01-30 Gensci Orthobiologics, Inc. Compositions with enhanced osteogenic potential, methods for making the same and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4619989A (en) * 1981-05-05 1986-10-28 The Regents Of The University Of Cal. Bone morphogenetic protein composition
US6180606B1 (en) * 1994-09-28 2001-01-30 Gensci Orthobiologics, Inc. Compositions with enhanced osteogenic potential, methods for making the same and uses thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008134814A1 (fr) * 2007-05-04 2008-11-13 Perth Bone & Tissue Bank Procédé et matériau à libération contrôlée pour traiter l'inflammation
AU2008247320B2 (en) * 2007-05-04 2013-08-29 Perth Bone & Tissue Bank A method for treating inflammation and controlled-release material capable of providing same
EP2349365A4 (fr) * 2008-11-04 2013-01-23 Perth Bone & Tissue Bank Matériau de support pour des cellules ostéogéniques

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Publication number Publication date
WO2003020840A9 (fr) 2003-10-30

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