# US 11,998,660 B2 · Method for making a porous scaffold suitable for use in repair of osseous, chondral, or osteochondral defects in a mammal

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- Number: US 11,998,660 B2
- Document ID: US11998660B2
- Status: Granted
- Kind: B2
- Application number: 16/795,770
- Filed: 2020-02-20
- Published: 2020-06-18
- Granted: 2024-06-04
- Earliest claimed: 2014-08-15
- Assignees: The Provost, Fellows, Scholars and Other Members of Board of Trinity College Dublin
- Applicants: The Provost, Fellows, Scholars and Other Members of Board of Trinity College Dublin
- Inventors: Daniel John Kelly; Grainne Cunniffe; Henrique Almeida; Rajalakshmanan Eswaramoorthy; Conor Buckley; Pedro Jose Diaz Payno; David Browe
- Web page: https://patentgrep.com/patent/US11998660B2

## CPC

- A61L 27/3633: {Extracellular matrix [ECM]}
- A61L 27/3612: {Cartilage, synovial fluid}
- A61L 27/365: {Bones}
- A61L 27/3654: {Cartilage, e.g. meniscus}
- 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 27/3695: {characterised by the function or physical properties of the final product, where no specific conditions are defined to achieve this (A61L27/3687, A61L27/3691 take precedence)}
- A61L 27/3817: {Cartilage-forming cells, e.g. pre-chondrocytes}
- A61L 27/3821: {Bone-forming cells, e.g. osteoblasts, osteocytes, osteoprogenitor cells}
- A61L 27/3847: {Bones}
- A61L 27/3852: {Cartilage, e.g. meniscus}
- A61L 27/44: having a macromolecular matrix
- A61L 27/52: Hydrogels or hydrocolloids
- A61L 27/54: Biologically active materials, e.g. therapeutic substances {(A61L27/227 takes precedence)}
- A61L 27/56: Porous materials, {e.g. foams or sponges}
- A61L 2300/414: Growth factors
- A61L 2300/64: Animal cells
- A61L 2430/02: for reconstruction of bones; weight-bearing implants
- A61L 2430/06: for cartilage reconstruction, e.g. meniscus
- A61L 2430/40: Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
- 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)

## Abstract

A method for making a porous devitalised scaffold suitable for use in repair of osseous, chondral, or osteochondral defects in a mammal comprises the steps of providing micronized extracellular matrix (ECM) tissue, mixing the micronized extracellular matrix with a liquid to provide a slurry, and freeze-drying the slurry to provide the porous scaffold. A porous scaffold suitable for use in repair of osseous, chondral, or osteochondral defects in a mammal and comprising a porous freeze-dried matrix formed from micronised decellularised extracellular matrix tissue is also described.

## Claims

### Claim 1

A method for making a porous scaffold suitable for use in repair of osseous, chondral, or osteochondral defects in a mammal, the method comprising the steps of:
A) providing a gel comprising solubilized extracellular matrix (ECM) tissue;
B) chemically crosslinking the extracellular matrix (ECM) tissue utilizing glyoxal as the chemically crosslinking agent;
C) freeze-drying the gel to provide the porous scaffold;
D) dehydrothermally (DHT) crosslinking the scaffold to produce a first layer; and
E) repeating steps A-D to make a second layer, wherein the first layer is attached to the second layer;
wherein the extracellular matrix is treated to reduce the GAG content to less than 90% of the GAG content of untreated ECM, and wherein the ECM tissue in at least one layer is cartilage ECM.

### Claim 2 (depends on 1)

The method according to claim 1, in which the ECM is solubilized by enzymatic digestion.

### Claim 3 (depends on 1)

The method according to claim 1, in which the ECM is micronized prior to solubilization.

### Claim 4 (depends on 3)

The method according to claim 3, in which the micronized cartilage extracellular matrix tissue has a mean particle size of 10-200 microns.

### Claim 5 (depends on 1)

The method according to claim 1, in which the cartilage ECM is hyaline cartilage ECM or growth plate ECM.

### Claim 6 (depends on 3)

The method according to claim 3, in which the cartilage ECM is decellularised before or after micronizing.

### Claim 7 (depends on 1)

The method according to claim 1, in which the method of the invention includes an additional step of seeding the scaffold with a biological material selected from the group consisting of: cells and a biological growth factor.

### Claim 8 (depends on 7)

The method according to claim 7, in which the cells are selected from the group consisting of: stem cells, chondrocytes, mesenchymal cells and osteoblasts; and/or
in which the biological growth factor is selected from the group consisting of: one or more of the TGF-β superfamily or cannabinoids.

### Claim 9 (depends on 1)

The method according to claim 1, in which the method further comprises a step of attaching the first layer to the second layer to form the multilayer scaffold, in which the first layer comprises hyaline cartilage ECM and the second layer comprises growth plate ECM.

### Claim 10 (depends on 1)

A multilayer scaffold suitable for repair of osteochondral defects in a mammal and having a first layer comprising a porous scaffold according to claim 1 in which the cartilage ECM is hyaline cartilage ECM and a second layer comprising a porous scaffold according to claim 1 in which the cartilage ECM is growth plate ECM, in which the first layer is attached to the second layer.

### Claim 11 (depends on 1)

The method according to claim 1, in which the cartilage ECM is broken up and treated with a basic solution to reduce the GAG content to less than 90% of the GAG content of untreated ECM.

### Claim 12 (depends on 11)

The method according to claim 11, wherein the basic solution comprises a sodium hydroxide solution.

### Claim 13 (depends on 1)

The method of claim 1, wherein the extracellular matrix is treated to reduce the GAG content to 5% or less of the GAG content of untreated ECM.

### Claim 14 (depends on 1)

The method according to claim 1, wherein the gel comprises solubilized ECM at a concentration range of 1 mg/ml to 200 mg/ml in an aqueous solution cross-linked with the glyoxal at a concentration range of 1 mM to 50 mM.

### Claim 15 (depends on 14)

The method according to claim 14, wherein the gel comprises solubilized ECM at a concentration of 20 mg/ml.

### Claim 16 (depends on 14)

The method according to claim 14, wherein the gel comprises solubilized ECM at a concentration of 10 mg/ml.

### Claim 17 (depends on 14)

The method according to claim 14, wherein the solubilized ECM is cross-linked with the glyoxal at a concentration of 10 mM.

### Claim 18 (depends on 1)

The method according to claim 1, in which the cartilage ECM is articular cartilage ECM.

### Claim 19

A method for making a porous scaffold suitable for use in repair of osseous, chondral, or osteochondral defects in a mammal, the method comprising the steps of:
A) providing a gel comprising a least a first layer of solubilized extracellular matrix (ECM) tissue and a second layer of solubilized ECM tissue;
B) chemically crosslinking the extracellular matrix (ECM) tissue utilizing glyoxal as the chemically crosslinking agent;
C) freeze-drying the gel to provide the porous scaffold;
D) dehydrothermally (DHT) crosslinking the scaffold;
wherein the extracellular matrix is treated to reduce the GAG content to less than 90% of the GAG content of untreated ECM, and wherein the ECM tissue in at least one layer is cartilage ECM.