EP1337266A1 - Mineralized collagen-polysaccharide matrix for bone and cartilage repair - Google Patents
Mineralized collagen-polysaccharide matrix for bone and cartilage repairInfo
- Publication number
- EP1337266A1 EP1337266A1 EP01979938A EP01979938A EP1337266A1 EP 1337266 A1 EP1337266 A1 EP 1337266A1 EP 01979938 A EP01979938 A EP 01979938A EP 01979938 A EP01979938 A EP 01979938A EP 1337266 A1 EP1337266 A1 EP 1337266A1
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- Prior art keywords
- matrix
- polysaccharide
- collagen
- bone
- growth
- Prior art date
- Legal status (The legal status 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 status listed.)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
- C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
- C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/36—Blood coagulation or fibrinolysis factors
- A61K38/363—Fibrinogen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
- A61K38/4833—Thrombin (3.4.21.5)
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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
- A61L27/14—Macromolecular materials
- A61L27/26—Mixtures of macromolecular compounds
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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
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
- A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
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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
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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
- 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
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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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
Definitions
- the present invention is directed to crosslinked mineralized collagen- polysaccharide matrices for the therapeutic repair of tissue, such as, bone, cartilage and soft tissue; methods of producing such matrices; and methods of using the matrices to repair tissue.
- tissue such as, bone, cartilage and soft tissue
- the present invention provides a crosslinked mineralized collagen- polysaccharide matrix that is administered by implantation or injection alone or in combination with other therapeutics, such as growth factors, for tissue repair.
- Collagens and glycosaminoglycans are two classes of biomaterials suited for use in bone and cartilage regeneration.
- Collagen based matrices have been used in bone grafting.
- Type I collagen has good cell adhesive properties, in particular- for bone forming osteoblast cells.
- Collagen has the capacity to serve both as an active or inert scaffold material for growth.
- Bone is characteristically composed of type I collagen fibrils intimately associated in an orderly manner with calcium phosphate crystals. Minor constituents include an array of macromolecules as well as a series of small molecules associated mainly with the mineral phase.
- Bone crystals are among the smallest biologically formed crystals known and, in fact, crystallographers would intuitively not expect crystals just a few unit cells thick to be stable at all. Therefore, the collagen bone structure has unique characteristics as to its formation, components, and properties.
- Hyaluronic acid is a natural component of the cartilage extracellular matrix, and it is readily sterilized, is biodegradable and can be produced in a wide range of consistencies and formats. It is generally biocompatable and its resorption characteristics can be controlled by the manipulation of monomers to polymer forms, most commonly through the esterification of the carboxylic groups of the glucuronic acid residues.
- Biological glue comprising fibrin has a long history as a tissue adhesive medical device and is believed to be commercially available in Europe (United States patent No. 5,260,420, issued November 9, 1993).
- One obstacle that limits its application is the short turn over and residence time which ranges from a few days to a few weeks depending on the site of implantation.
- the incorporation of collagen fibers into fibrin glue has been reported (Sierra et al., 1993, Trans. Soc. Biomater., vol. 16:257 and United States Patent No. 5,290,552).
- longer coagulation times are required for the collagen/fibrin compositions compared to fibrin alone.
- the present invention provides crosslinked mineralized collagen-polysaccharide matrices, methods for preparing such matrices, and methods of using the matrices by implantation or injection in the repair of tissue, such as, bone, cartilage and other soft connective tissue.
- the mineralized collagen may be formed from purified, native, modified or recombinant collagen of any type.
- the type of polysaccharides which can be used include hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan, heparan sulfate, dextran, dextran sulfate, alginate, and other long chain polysaccharides.
- the polysaccharide is hyaluronic acid.
- a crosslinked mineralized collagen-polysaccharide matrix of the present invention may be used alone to conduct the growth of tissue; or in combination with growth factor.
- Growth factors which can be used with a matrix of the present invention include, but are not limited to, members of the TGF- ⁇ superfamily, including TGF- ⁇ 1,2 and 3, the bone morphogenetic proteins (BMP's), the growth differentiation factors(GDF's), and ADMP-1; members of the fibroblast growth factor family, including acidic and basic fibroblast growth factor (FGF-1 and -2); members of the hedgehog family of proteins, including indian, sonic and desert hedgehog; members of the insulin-like growth factor (IGF) family, including IGF-I and -II; members of the platelet-derived growth factor (PDGF) family, including PDGF-AB, PDGF-BB and PDGF-AA; members of the interleukin (IL) family, including IL-1 thru -6; and members of the colony-stimulating factor (CSF) family, including CSF-1, G-CSF, and GM-CSF.
- TGF- ⁇ superfamily including TGF- ⁇ 1,2 and 3, the bone morphogenetic
- the method of making a mineralized collagen-polysaccharide matrix of the present invention comprises the steps of oxidizing an exogenous polysaccharide to form a modified exogenous polysaccharide having aldehyde groups, and reacting the modified exogenous polysaccharide with mineralized collagen under conditions such that the aldehyde groups covalently react with mineralized collagen to form a crosslinked matrix.
- the method may further comprise the step of adding a growth factor to the matrix.
- a growth factor can be added before or after the step of reacting the modified polysaccharide with the mineralized collagen.
- the mineralized collagen is prepared from dispersed or solubilized collagen according to the method of U.S. Patent 5,231,169.
- the present invention provides methods of using a crosslinked mineralized collagen-polysaccharide matrix to conduct the growth of tissue by administering the matrix at the sites of desired tissue repair.
- the matrix in combination with a growth factor may be administered by implantation or injection to induce the growth of tissue at sites of desired repair.
- a matrix further comprising fibrin may be administered to anchor the matrix into desired sites, such as, tissue defect sites.
- the prepared matrices may be implanted at a site of desired tissue growth.
- the polysaccharide and mineralized collagen starting materials may also be separately injected into the site of desired tissue growth, along with any growth factor, and the like. Upin mixing at the site, the matrix will form in situ, conforming to the shape of the site.
- repair is defined as growth of new tissue.
- the new tissue may or may not be phenotypically or genotypically identical to the original lost tissue.
- regeneration of tissue means that the new tissue grown is identical to the lost tissue.
- Tissue repair can also be the result of replacing lost tissue with non-identical tissues, e.g., for example, the replacement of hyaline articular cartilage
- the basic cellular properties involved in repair include adhesion, proliferation, migration and differentiation.
- conduction it is meant that the host tissue, e.g.,bone, cartilage or soft tissue grows by extension of existing tissue onto or into the crosslinked collagen-polysaccharide matrix.
- repair cells move onto and into the matrix to synthesize and remodel new tissue identical to the surrounding host tissue.
- induction it is meant that the growth and differentiation of progenitor repair cells is stimulated. These progenitor cells go on to synthesize and remodel new tissue to be continuous with the surrounding host tissue.
- tissue defect can be the result of a congenital condition, trauma, surgery, cancer or other disease.
- an exogenous polysaccharide refers to a free polysaccharide.
- the ratios of the mineralized collagen to polysaccharide can be varied to change both the physical and biological properties of the matrix. A higher proportion of mineralized collagen will result in a more porous sponge-like matrix. A higher proportion of polysaccharide will result in a more gel-like matrix.
- the method of preparing a matrix of the present invention comprises the steps of opening sugar rings on an exogenous polysaccharide and oxidizing terminal hydroxyl groups to aldehydes using, for example, sodium or potassium periodate as a selective oxidizing agent.
- the amount of aldehyde groups produced in this manner can be stoichiometrically controlled. Typically, from about 1% to 50% of the rings can be opened in this manner. More preferably about 1% to 5% of the rings are opened to form the aldehyde groups.
- These aldehyde groups can form covalent crosslinks with the collagen at amine sites on the collagen peptide chains.
- the aldehyde groups are formed in situ without the addition of a separate cross-linking compound, the intermolecular distance between the backbone of the polysaccharide chain and the collagen fibrils which is crosslinked to it is believed to be less than the corresponding distance using a crosslinking compound. Accordingly, the polysaccharide and collagen backbones are relatively closely bound, which produces an advantageous structure for the purpose of providing a matrix that supports, conducts or induces the growth of bone, cartilage or soft connective tissue.
- the starting material for producing the collagen may be purified, native collagen, modified or recombinant collagen of any type.
- a preferred collagen for bone growth is Type I collagen
- a preferred collagen for cartilage growth is Type II collagen.
- the collagen may be crosslinked or non-cross-linked, but it is preferred that the collagen be non-crosslinked to provide more accessibility to side groups for crosslinking to the polysaccharide aldehyde groups. If Type I collagen is used for tissue repair where it is desired to mask the inherent cell adhesion sites, such as cartilage repair, the adhesion sites can be masked by the use of non cell-adhesive polysaccharides to support the increased cell-to-cell interaction and adhesion.
- the collagen to be mineralized will normally be dispersed or solubilized collagen where solubilization is achieved by dispersing the collagen source in a medium at an elevated pH, using at least about pH 8, more usually about pH 11-12, and generally less than about 1 N.
- a medium at an elevated pH using at least about pH 8, more usually about pH 11-12, and generally less than about 1 N.
- sodium hydroxide is employed, although other hydroxides may find use, such as other alkali metal hydroxides or ammonium hydroxide.
- the concentration of collagen will generally be in the range of about 1 to 10 weight percent, more usually from about 1 to 5 weight percent.
- the collagen medium will generally be at a concentration of the base in the range of about 0.0001 to 0J N.
- the pH is generally maintained during the course of the reaction in the range of about 10-13, preferably about 12.
- the phosphate and calcium are added as solutions, generally at a concentration in the range of about 0. 010.2 M, preferably about 0.025-0.075 M.
- the volume of the solutions added to the collagen medium will generally increase the collagen medium volume by at least 10 percent, usually at least 25 percent and not more than about 400 percent, generally in the range of about 50 to 150 percent.
- the collagen solution will generally not be diluted by more than four-fold.
- the rate of addition is relatively slow, generally requiring at least about one hour and not more than about 72 hours, generally being in the range of about 2 to 18 hours, more usually in the range of about 4 to 16 hours.
- the rate of addition will generally be in the range of 50 to 150 ml per hour.
- the addition of the reagents can be provided in a stoichiometric ratio, although stoichiometry is not required, variations from stoichiometry of up to about 50 percent, preferably not more than about 25 percent are preferred. Thus, where the stoichiometry of addition is not maintained, one of the components may be exhausted, while addition of the other components continue.
- the weight ratio of the collagen to calcium phosphate mineral will generally be in the range of about 8:2 to 1:1, more usually about 7:3.
- agitation e.g., stirring
- stirring will normally be continued, usually at least about 1 h, more usually about 2 h and agitation may continue even more.
- the amount of continued agitation is not critical to the preparation of the product.
- the mineralized collagen may be treated in a variety of ways.
- the product may be washed repeatedly to remove any unbound minerals or other components of the medium, as well as provide a more neutral pH. Washing may be readily accomplished with water, saline, or the like.
- the mineralized collagen may be further treated in a variety of ways.
- the subject compositions may be cross-linked using a variety of cross-linking agents, such as formaldehyde, glutaraldehyde, chromium salts, di-isocyanates or the like.
- the polyaldehyde polysaccharide and mineralized collagen are separately injectable materials which can react when contacted to form the matrix in situ at the site of desired tissue growth.
- the advantages are that no operative implantation procedures are necessary and the flowable starting materials conform to the shape of the site before the reaction is complete. The result is a matrix that conforms in shape to the site without the need for cutting and shaping of a preformed solid matrix.
- polysaccharides which may be utilized include hyaluronic acid, chondroitin sulfate, dermatan, dextran sulfate, alginate, and other long chain polysaccharides.
- the polysaccharide will have an average molecular weight of about 1,000 to 10,000,000 DA.
- the reagents for opening sugar rings on the exogenous polysaccharide may be any selective oxidizing agent which oxidizes a terminal hydroxyl group to an aldehyde, such as potassium or sodium periodate.
- Other reagents include specific sugar oxidases.
- the preferred polysaccharide is hyaluronic acid.
- the relative proportion of polysaccharide to mineralized collagen will impart various physical and biological characteristics to the matrix.
- the proportion of polysaccharide to mineralized collagen may be characterized on a molar ratio basis or on a weight ratio basis.
- the ratio by weight of mineralized collagen to polysaccharide is from 99:1 to about 1:99. This represents an approximate molar ratio of 99.9:0.1 to 1:9, respectively, assuming an average molecular weight of 1,000,000 daltons for hyaluronic acid and 100,000 daltons for the collagen (based on non-mineralized weight).
- the molar ratio may vary depending on the actual molecular weight of the polysaccharide and collagen used. In a preferred embodiment disclosed herein, the ratio by weight of collagen to polysaccharide is from 9:1 to about 1:9.
- the ratios of the mineralized collagen to polysaccharide can be varied to change both the physical and biological properties of the matrix. Biologically, a higher proportion of Type I collagen will more closely mimic the composition and architecture of bone, whereas a higher proportion of Type II collagen will more closely mimic the composition of cartilage. Bone forming cells will interact with specific cell adhesion sites on collagen and will divide, migrate and differentiate to form new bone.
- polysaccharide preferably hyaluronic acid
- increasing the proportion of polysaccharide will more closely mimic a natural cartilage matrix.
- a higher proportion of polysaccharide will mask some specific cell adhesive sites on collagen and will favor other cell-cell interactions and aggregation important in the development of cartilage tissue.
- Growth factors which can be used with a matrix of the present invention include, but are not limited to, members of the TGF- ⁇ superfamily, including TGF- ⁇ 1,2 and 3, the bone morphogenetic proteins (BMP's), the growth differentiation factors(GDF's), and ADMP-1; members of the fibroblast growth factor family, including acidic and basic fibroblast growth factor (FGF-1 and -2); members of the hedgehog family of proteins, including indian, sonic and desert hedgehog; members of the insulin- like growth factor (IGF) family, including IGF-I and -II; members of the platelet-derived growth factor (PDGF) family, including PDGF-AB, PDGF-BB and PDGF-AA; members of the interleukin (IL) family, including IL-1 thru -6; and members of the colony-stimulating factor (CSF) family, including CSF-1, G-CSF, and GM-CSF.
- TGF- ⁇ superfamily including TGF- ⁇ 1,2 and 3, the bone morphogenetic
- Growth factor preparations are obtained either commercially or isolated and purified from tissue or from recombinant sources. Growth factors can be loaded into the collagen/HA/fibrin matrices across a wide dose range (fentogram to millgram range). Factors such as cost, safety and the desired growth factor release profile will dictate the amount of growth factor that is loaded onto the matrix.
- Thrombin acts as a catalyst for fibrinogen to provide fibrin.
- the fibrinogen and thrombin are added individually to a reaction mixture containing oxidized exogenous polysaccharide and mineralized collagen.
- the concentration of fibrinogen used in forming the matrix is preferably 10 mg/ml or greater.
- the thrombin is added to the fibrinogen in a concentration of from about 0.01 H units to about 100 MH units/ml and preferably from about 0J - 2.0 NIH units/ml.
- the thrombin is commercially available from a variety of sources. Fibrinogen may be derived from autologous patient plasma or from commercial sources.
- the matrices according to the present invention may be formed into any shape by lyophilization, or wet-laying and air drying in molds of the desired shape.
- the wet-laid material having a high proportion polysaccharides may also be formed into viscous gels for injection or direct application into a fracture or joint. As described above, the starting materials are also injectable and may be separately injected to mix at the site of desired tissue growth to react without formation of undesired side products.
- the usefulness of the matrices according to the present invention can be shown by both in vitro and in vivo tests.
- in vitro tests primary fetal rat calvarial cells, harvested by a series of collagenase digestions, according to the method of Wong and Cohn (PNAS USA 72:3167-3171, 1975), or primary rat epiphyseal cartilage Thyberg and Moskalewski, (Cell Tissue Res. 204:77-94, 1979) or rabbit articular chondrocytes, harvested by the method of Blein-Sella O. et al., (Methods Mol. Biol..43:169-175, 1995), are seeded into the matrices and cultured under conventional conditions for 1-4 weeks. Cultures are then processed and evaluated histologically.
- polysaccharide mineralized collagen matrices according to the invention have comparable growth factor binding ability to cross-linked mineralized collagen but much better osteoconductivity. Furthermore, while the polysaccharide-mineralized collagen matrices have comparable osteoconductivity to polysaccharide-nonmineralized collagen, they have slower growth factor release kinetics, which is an advantage for growth factor retention within the matrix.
- the chondroconductive capability of the matrices of the present invention can be determined by successful support of adhesion, migration, proliferation and differentiation of primary rat bone marrow and stromal cells as well as retinoic acid-treated primary rat or rabbit chondrocytes or human mesenchyme stem cells.
- Bone marrow and bone marrow stromal cells closely approximate the early chondroprogenitor cells found in the subchondral bone marrow of full-thickness defects. Bone marrow are harvested from the long bones of 2-3 week-old inbred Lewis rats and added directly to a matrix and cultured for 2 weeks under standard conditions. The adherent stromal cell population that grows out of these cultures are passaged and frozen for use. Cells from up to six passages are used for culturing or seeding on the matrix.
- Retinoic acid-treated chondrocytes represent the latter stages of chondrogenesis. Retinoic acid treatment of primary is performed prior to culturing or seeding the cells on a candidate matrix (Dietz, U. et al., 1993, J. Cell Biol. 52(l):57-68).
- in vitro studies of the early and late stage chondrocytes are merged to allow stromal cells to condition the matrices and then to replace them with more mature chondrocytes.
- evolution of the matrices during the early phases of chondrogenesis may be tested for effects on the later stages of the process.
- Cell adhesion and proliferation on the matrix are monitored using an MTS assay that can measure cell number and viability based on mitochondrial activity.
- Stromal cells or chondrocytes are cultured on matrices for 6-18 hrs. in the presence or absence of serum for adhesion analysis and for 1-2 weeks for proliferation assessment.
- matrices are coated or fitted onto porous Trans-well membrane culture inserts (Corning). Stromal cells are seeded on top of the matrices in the upper chamber of the Trans-well and a chemoattractant (growth factor, PDGF) placed in the bottom chamber. After 12-18 hrs of culture the cells that have migrated through the matrix to the bottom side of the Trans- well membrane are quantitated by the MTS assay. Matrices are removed from the upper chamber and processed histologically to assess degree of infiltration.
- PDGF chemoattractant
- the analysis of differentiation markers relevant to chondrogenesis and osteogenesis are evaluated at both the protein and transcriptional level.
- the specific markers that may be analyzed include: 1) Type II collagen and IIA, IIB isoforms; 2) Aggrecan proteoglycan; 3) Type IX, X and XI collagen; 4) Type I collagen; 5) Cartilage matrix protein (CMP); 6) Cart-1 transcription factor; 7) Fibronectin (EDA, EDB isoforms); 8) Decorin proteoglycan; 9) Link protein; 10) NG-2 proteoglycan; 11) Biglycan proteoglycan; 12) Alkaline phosphatase. Differentiation may be measured by Northern/PCR analysis, Western blotting or by metabolic cell labeling.
- RNA are isolated by standard procedures from stromal cells or chondrocytes that have been cultured on composite matrices. Time course tests may be used to determine optimal culture periods that range from 1 to 6 weeks depending on the cell type.
- the isolated RNA is analyzed by Northern gel and hybridization techniques with specific cDNA or PCR amplified probes. Northern analysis is quantified by densitometric scanning of autoradiographs and normalization to housekeeping gene signals (G3PDH). Northern analysis may be supplemented with quantitative PCR analysis using primers generated from the published cDNA sequences of the genes to be analyzed.
- solubilized protein lysates are isolated from cells cultured on composite matrices by standard techniques (Spiro R.C., et al., 1991, J. Cell. Biol., 115:1463-1473). After the lysis of cells the matrices are extracted in stronger denaturants (8 M urea, GnHCL) to remove and examine matrix-bound or incorporated proteins. Protein samples are analyzed by standard Western blotting techniques using specific polyclonal or monoclonal antibodies.
- cells cultured on a composite matrix are metabolically radiolabeled with SO , S-methionine or H/ C-labeled amino acids by standard techniques (Spiro et al., supra).
- Solubilized cellular and matrix-associated proteins are quantitatively immunoprecipitated with antibodies specific for the protein of interest and analyzed by SDS-PAGE (Spiro et al., supra). Quantitation of results are performed by densitometric scanning of autoradiographs and signals will be normalized to either cell equivalents or to a house-keeping protein such as actin.
- a matrix of the present invention to support chondrogeneic differentiation in vivo may be tested in an inbred rat soft tissue implant model.
- Rat bone marrow or stromal cells described above are seeded onto matrices at high density, cultured overnight in MEM medium containing 10% FBS serum and antibiotics, then transferred into Millipore diffusion chambers and implanted intraperitoneally or subcutaneously into 8 week-old recipients. Chambers are harvested after 3 weeks and evaluated histologically for cartilage formation.
- a transplantation model in outbred rats is used to evaluate the ability of the composite matrices to maintain the cartilage phenotype in vivo.
- Rib costal cartilage chondrocytes are seeded onto matrices at high density and cultured overnight in Ham's F- 12 containing 1% rat serum and antibiotics.
- the seeded matrices are then implanted into posterior tibial muscle pouches created by blunt dissection in 8 week-old male Sprague- Dawley rats. Explants are taken at 14 and 28 days and evaluated histologically for matrix compatibility, cartilage growth, and maintenance of the differentiated phenotype based on staining for aggrecan and type II collagen.
- a matrix of the present invention to interact with extracellular matrix proteins (proteoglycans, proteins and growth factors) found in the surrounding serum, tissue fluid, or in the secretion products of chondroprogenitor cells correlate with the chondroconductive potential of a matrix.
- extracellular matrix proteins proteoglycans, proteins and growth factors
- the interaction of the matrices of the present invention with extracellular matrix proteins may be measured by means known to those of skill in the art such as, Western blotting, affinity co- electrophoresis techniques and binding characteristics.
- the matrix is incubated in culture media containing increasing amounts of serum (various species and sources). After washing, bound proteins are eluted by boiling in SDS-PAGE sample buffer and unsolubilized matrix will be removed by centrifugation. SDS-PAGE analysis is used to initially document the binding pattern of the matrices. Western blotting is then performed to identify specifically bound components such as fibronectin and vitronectin.
- Affinity coelectrophoresis is used to analyze proteoglycan binding to a matrix of the present invention.
- 35 SO 4 -labeled or iodinated proteoglycan (aggrecan) isolated from bovine and rat (or other sources) is loaded into ACE gels (Lee, M.K. et al., 1991, 88:2768-2772) containing composite matrices or collagen scaffolds alone.
- the binding affinity of aggrecan for collagen scaffolds plus and minus hyaluronic acid or dextran sulfate are taken as a measure of the ability of composite matrices to organize a cartilage matrix.
- the shift in expression from Type I to Type II collagen and the splicing of the Type II collagen transcript from the Type II A to the Type IIB isoform are measured by means known to those of skill in the art to determine differentiation down a chondrogenic pathway.
- the expression of the cartilage-associated proteoglycan, aggrecan (Schmid, T.M., et al., 1985, J. Cell Biol. 100:598-605 and Kuettner K.E. 1992, Clin. Biochem. 25:155-163) and a cartilage homeoprotein transcription factor (Cart-1) appear to be markers for cells committed to the chrondrocytic lineage.
- the matrices are evaluated for the capabilities for supporting osseous healing in a rat cranial defect model by implantation into a 5 mm by 3 mm defect created in the parietal bone of 6 weeks old male Sprague-Dawley rats.
- the defects are evaluated at 28 days by radiographic and histologic analysis.
- the in vivo model for cartilage repair is a full-thickness articular cartilage defect in the rabbit (Amiel et al., 1985, j. Bone Joint Surg. 67A:911). Defects measuring approximately 3.7 mm in diameter and 5 mm deep defect are created in the center of the medial femoral condyles of adult male New Zealand white rabbits. The defects are then either filled with matrix or left unfilled as controls. The defects are evaluated morphologically and histologically at 6 and 12 weeks.
- the matrices of the present invention may be used for the treatment of bone and/or cartilage defects associated with surgical resection, such as spinal fusions; trauma; disease; infection; cancer or genetic defects.
- the matrices according to the present invention may be administered through implantation, direct application or injection depending on the intended application of the matrix, the physical properties of the matrix and the ratio by weight of mineralized collagen to polysaccharide in the matrix.
- the matrix is provided having a higher proportion of mineralized collagen compared to polysaccharide, is in a sponge-like form and is surgically implanted at a site where growth of new bone tissue is desired, such as in spinal fusions.
- the matrix further comprises a growth factor, such as BMP-2.
- the matrix further comprises fibrin to facilitate anchoring of the matrix into the desired site.
- the starting polyaldehyde polysaccharide and mineralized collagen are separately injected into the site of the desired tissue growth along with any desired growth factors. The materials react in situ to form the matrix at the desired site.
- the matrix has a higher proportion of polysaccharide compared to mineralized collagen, is formed into a viscous gel and is either directly applied or injected into a site where growth of new bone tissue is desired, such as in filling bone defects, fracture repair and grafting periodontal defects.
- the matrix is provided with a higher proportion of polysaccharide, is formed into a viscous gel and is injected directly or delivered through an arthoscopic procedure into a site where growth of cartilage tissue is desired, such as in injury induced cartilage damage or disease-induced cartilage damage such as in, osteoarthritis or rheumatoid arthritis.
- the amount of matrix to be administered to conduct growth of bone or cartilage tissue depends upon the extent of the bone or cartilage defect to be treated. As will also be understood by those of skill in the art, the cost, safety, and desired growth factor release profile will dictate the type and amount of growth factor that is loaded onto the matrix.
- Example 1 Implantable Matrices of Amine-linked Mineralized Collagen and Polysaccharides
- the raw materials, mineralized Type I collagen and polysaccharide-polyaldehyde were prepared by the methods disclosed in U.S. Patent No. 5,231,169 and U.S. Patent No. 5,866,165, respectively.
- Mineralized semed F collagen (63 mg/ml) was mixed with a hyaluronate-polyaldehayde solution (7 mg/ml, 5% repeating units were oxidized; pH 7.5- 9.0) at the equal volume ratio in the container of a heavy duty blender.
- Sodium cyanoborohydride NaCNBH 3 5.0 M in 1.0 M NaOH was added to the mixture to the final concentration of 10 mM.
- the mixture was then blended 3 times at low speed for 10 seconds.
- the reaction was continued carrying on by pouring the slurry into a heavy-wall bottle incorporated with a tight-fitting polypropylene screw cap.
- the bottle was rotated at the speed of 100 rotes/min. at ambient temperature in dark for 24 hr.
- the slurry was then poured into a mold and lyophilized. This formed a matrix, which was washed with D.I. water to removed NaCNBH 3 and re-lyophilized. This procedure was followed to make a series of matrices from mCOL with other oxidized polysaccharides.
- the surface property, structures and biological activity of the matrices were controlled by altering the ratio of mCOL to the polysaccharides, the type of polysaccharides, the density of aldehyde groups generated on the polysaccharides, the density of matrix, as well as the process of lyophilization.
- Example 2 Implantable Matrices of Imine-linked Mineralized Collagen and Polysaccharides The matrices were prepared by the procedure as described in Example 1, except that no NaCNBH 3 was used.
- the gel-matrices were prepared using a FibrinJetTM surgical sealant delivery system. The typical procedure in detail is described in following:
- the mCOL fibers with the diameter of about 100 ⁇ m and fibrinogen (mCOL, 42 mg/ml; fibrinogen, 21 mg/ml; pH 7.5) were loaded in one syringe, an equal volume of activated hyaluronate solution (7 mg/ml, pH 7.5) containing thrombin (1-5 U/ml) was placed in another syringe. Both of the syringes were mounted onto the FibrinJetTM surgical sealant delivery system connected with a 18 G needle. Gel-matrix was formed in less than three minutes at the exit site of the needle upon pushing the two parts of composition to flow through the syringes simultaneously.
- the above procedure was followed to make a series of injectable gel-matrices from mCOL, fibrinogen, and other oxidized polysaccharides.
- the surface property, the porous structures and biological activity of the gel-matrices were controlled by altering the ratio of mCOL and polysaccharides to fibrinogen, the type of polysaccharides, the density of aldehyde groups, the density of the final matrix and the concentration of thrombin.
- the gelation time and the gel hardness were controlled by amount of thrombin added.
- Example 4 Injectable Gel-Matrices with Plasma The gel-matrices were prepared by the method described in Example 3, except that blood plasma was used instead of fibrinogen. The blood plasma was prepared from citrate (10 w%) added whole blood, which was centrifuged at 3,000 rpm for 15 minutes.
- Example 5 Injectable Gel-Matrices with Plasma The gel-matrices were prepared by the method described in Example 4, except that 50 mM calcium chloride solution was used instead of thrombin.
- Example 6 Injectable Gel-Matrices with Whole Blood The gel-matrices were prepared by the method described in Example 3, except that whole blood was used instead of blood plasma.
- Matrices prepared from mCOL and active polysaccharides as prepared in Examples 1 and 2 were used. Pre-dried matrices were cut to cubes with the size of 5 x 4 x 2 mm. The water uptake of the cubes was measured and found to be 85 ⁇ 5 ⁇ per piece, Growth and differentiation factor-5 (GDF-5, 0.588 mg/ml, 20 mM acetic acid) was drop- wise added to the matrix specimens at 85 ⁇ l for each piece. The GDF-5 loaded matrix specimens were allowed to stand at ambient temperature in hood for 5 minutes, then froze at -78 °C, and lyophilized.
- GDF-5 Growth and differentiation factor-5
- the above procedure was followed to load a series of growth factors with various concentrations, such as bone morphogenic proteins, transferin growth factor- ⁇ , and insulin-like growth factors to matrices prepared from mCOL with other oxidized polysaccharides.
- the above procedure was followed to load DNA, hormones, and cytokines to matrices prepared from mCOL with other oxidized polysaccharides.
- Example 9 Comparative Sustained Release of GDF-5 from Matrices Implantable mCOL/HA matrices (5 x 4 x 2 mm) with pre-loaded GDF-5 at the ratio of 50 ⁇ g/piece were prepared as described in Example 7, using radio-lebeled GDF-5 as a tracer. Glutaraldehyde cross-linked mCOL and hyaluronate polyaldehyde cross- linked COL (COL/HA) were also loaded with GDF-5 at the same ratio and served as controls. The release kinetics of GDF-5 from these matrices was investigated.
- Example 10 Osteoconductivity of Implanted Matrices Matrices comprising 9 parts of mCOL and 1 part of HA polyaldehyde (5% repeat units oxidized) were prepared as described in Example 2. Specimens of the matrix with the size of 5 mm x 3 mm x 2 mm were sterilized with ethanol and implanted into the defects created in parietal bone of 6 week old male Spregue-Dawley rats. The defects were evaluated at 28 days by radiographic and histologic analysis, and the results summarized in Table 1.
- Example 11 In Vitro Growth of Cells and Expression of Bone Phenotype
- FRCs fetal rat calvarial cells
- FRCs fetal rat calvarial cells
- FIG. 11 This example illustrates that the mCOL/HA matrix supports the growth of fetal rat calvarial cells (FRCs) and demonstrates the expression of bone phenotype in vitro.
- FRCs were prepared from a 19 day old fetus and expanded, seeded into the matrix made by the method described in Example 2 comprising 90% mCOL and 10% HA (5% repeat units were oxidized) and cultured under standard conditions for 4 weeks. Cultures were then evaluated for cell growth and the express of alkaline phosphatase activity (ALP). Results showed that RFCs seeded on the matrix grew continually, and the cell number was increased by 9 fold at day 28, compared to day 1.
- ALP alkaline phosphatase activity
Abstract
Description
Claims
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US703438 | 1996-09-16 | ||
US70343800A | 2000-10-31 | 2000-10-31 | |
PCT/US2001/042477 WO2002036147A1 (en) | 2000-10-31 | 2001-10-05 | Mineralized collagen-polysaccharide matrix for bone and cartilage repair |
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EP1337266A1 true EP1337266A1 (en) | 2003-08-27 |
EP1337266A4 EP1337266A4 (en) | 2006-11-02 |
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EP01979938A Withdrawn EP1337266A4 (en) | 2000-10-31 | 2001-10-05 | Mineralized collagen-polysaccharide matrix for bone and cartilage repair |
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EP (1) | EP1337266A4 (en) |
JP (1) | JP2004512145A (en) |
AU (2) | AU2002211850B2 (en) |
CA (1) | CA2427047A1 (en) |
NZ (1) | NZ525435A (en) |
WO (1) | WO2002036147A1 (en) |
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EP1337266A4 (en) | 2006-11-02 |
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AU2002211850B2 (en) | 2006-07-13 |
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