# US 10,478,525 B2 · Process for demineralization of bone matrix with preservation of natural growth factors

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- Number: US 10,478,525 B2
- Document ID: US10478525B2
- Status: Granted
- Kind: B2
- Application number: 14/812,777
- Filed: 2015-07-29
- Published: 2015-11-19
- Granted: 2019-11-19
- Earliest claimed: 2007-06-01
- Assignees: Bacterin International, Inc.
- Applicants: Bacterin International, Inc.
- Inventors: Nancy J. Shelby; Steven M. Scott; Benjamin P. Luchsinger; Gregory A. Juda; Kelly R. Kirker; Darrel L. Holmes; Jesus Hernandez
- Web page: https://patentgrep.com/patent/US10478525B2

## CPC

- A61L 27/3608: {Bone, e.g. demineralised bone matrix [DBM], bone powder}
- A61F 2/4644: {Preparation of bone graft, bone plugs or bone dowels, e.g. grinding or milling bone material (bone material A61F2/28; disintegrating or milling devices B02C)}
- A61L 27/227: {Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24}
- A61L 27/28: Materials for coating prostheses
- A61L 27/3683: {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}
- A61L 27/3691: {characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation}
- A61L 27/38: {containing added animal cells (organs or tissue containing native cells A61L27/36)}
- A61L 27/3804: {characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells}
- A61L 27/3834: {Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells}
- A61L 27/54: Biologically active materials, e.g. therapeutic substances {(A61L27/227 takes precedence)}
- A61L 27/56: Porous materials, {e.g. foams or sponges}
- A61P 19/00: Drugs for skeletal disorders
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 5/0062: {General methods for three-dimensional culture}
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- A61L 2300/414: Growth factors
- A61L 2420/00: Materials or methods for coatings medical devices
- A61L 2430/02: for reconstruction of bones; weight-bearing implants
- A61L 2430/06: for cartilage reconstruction, e.g. meniscus
- C12N 2503/02: Drug screening
- C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
- C12N 2537/00: Supports and/or coatings for cell culture characterised by physical or chemical treatment
- 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 (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)
- A61F: FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS (dental prosthetics A61C)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A demineralized bone matrix is produced by a process in which a bone body is placed in a first processing solution comprising an acid to demineralize the bone body. The bone body is periodically removed from the first solution at specific time intervals to perform at least one test, such as a compression test, on a mechanical property of the bone body. When the test yields a desired result, the bone body is exposed to a second processing solution that is less acidic than the first, thus minimizing the exposure of the bone body to the harsh acidic conditions of the demineralization phase of the process.

## Claims

### Claim 1

A method for producing a three dimensional matrix, comprising:
seeding a demineralized bone matrix with cells to form the three dimensional matrix, wherein the demineralized bone matrix was produced by a method comprising:
placing a bone body in a first processing solution comprising an acid to demineralize the bone body, wherein the demineralized bone body comprises bone fragments with at least one dimension greater than 2 mm;
periodically removing the bone body from the first processing solution to perform at least one test on a mechanical property of the bone body; and
exposing the bone body to a second processing solution when the test yields a desired result to produce a demineralized bone matrix, wherein the demineralized bone matrix comprises less than 2% residual calcium, wherein the test comprises a compression test on the bone body, wherein the compression test comprises applying a compressive force in a range from about 10 g-force/cm2 to about 4000 g-force/cm2 until the bone body uniformly compresses to less than about 60 percent of its original shape; or wherein the test comprises a bending test, wherein the bending test comprises applying a bending force in a range from about 10 g-force/cm2 to about 4000 g-force/cm2.

### Claim 2 (depends on 1)

The method of claim 1, further comprising culturing the cells in the three dimensional matrix.

### Claim 3 (depends on 2)

The method of claim 2, wherein the step of culturing the cells is conducted to establish cell attachment.

### Claim 4 (depends on 1)

The method of claim 1, further comprising contacting the three dimensional matrix with factors for cellular differentiation and proliferation of the cells.

### Claim 5 (depends on 4)

The method of claim 4, further comprising studying new tissue growth, cell differentiation, proliferation, and function in the three dimensional matrix.

### Claim 6 (depends on 1)

The method of claim 1, wherein the cells are selected from the group consisting of osteoprogenitor cells, skeletal muscle stem cells, cartilage progenitor cells, differentiated osteoblasts, osteoclasts, chondrocytes, adipose stem cells, bone marrow stem cells, mesenchymal stem cells, adult stem cells, embryonic stem cells, and combinations thereof.

### Claim 7 (depends on 1)

The method of claim 1, wherein the cells are selected from the group consisting of autogenic cells of a patient, allogeneic cells of the patient, xenogenic cells of the patient, and combinations thereof.

### Claim 8 (depends on 1)

The method of claim 1, further comprising mixing the cells with a biocompatible carrier selected from the group consisting of fibrin, gelatin, cellulose, synthetic extracellular matrices, synthetic polymers, natural polymers, and combinations thereof to form a cell-containing carrier.

### Claim 9 (depends on 8)

The method of claim 8, wherein the step of seeding comprises injecting the three dimensional matrix with the cell-containing carrier.

### Claim 10 (depends on 1)

The method of claim 1, wherein the demineralized bone matrix is a sponge-like bone graft matrix.

### Claim 11 (depends on 1)

The method of claim 1, further comprising coating the three dimensional matrix with bone marrow aspirate or stem cells.

### Claim 12 (depends on 1)

The method of claim 1, wherein the demineralized bone matrix comprises less than 2% of magnesium or phosphate.

### Claim 13 (depends on 1)

The method of claim 1, wherein the demineralized bone matrix is compressible to 5 to 60% of an original shape.

### Claim 14 (depends on 1)

The method of claim 1, wherein the first processing solution has a pH between about 0 and about 0.5.

### Claim 15 (depends on 1)

The method of claim 1, wherein the second processing solution has a pH greater than about 4.

### Claim 16 (depends on 1)

The method of claim 1, wherein the test comprises the bending test, and wherein the bending force does not cause the cortical bone body to fracture.

### Claim 17 (depends on 1)

The method of claim 1, wherein the matrix is compressible to 5 to 60 percent of an original shape of the matrix when between 10 and 1000 grams-force/cm2 of force is applied to the matrix and wherein the matrix is a sponge-like bone graft matrix.

### Claim 18 (depends on 1)

The method of claim 1, further comprising adding an additive to the three dimensional matrix, wherein the additive is selected from the group consisting of a bioactive agent, a chemotherapeutic agent, a chondrocyte, a trophic growth enhancing agent, and combinations thereof.

### Claim 19 (depends on 1)

The method of claim 1, wherein the demineralized bone matrix has two or more of the characteristics selected from the group consisting of:
a. having greater than about 5000 pg/g of demineralized bone of bone morphogenetic protein-2 (BMP-2);
b. having greater than about 600 pg/g of demineralized bone of bone morphogenetic protein-4 (BMP-4)
c. having greater than about 12000 pg/g of demineralized bone of bone morphogenetic protein-7 (BMP-7); and
d. having greater than about 60 pg/g of demineralized bone of insulin-like growth factor-1 (IGF-1) per gram.

### Claim 20 (depends on 1)

The method of claim 1, wherein the demineralized bone matrix is not subjected to hydrogen peroxide.