# US 10,865,811 B2 · Engineered tissue substitute system

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- Number: US 10,865,811 B2
- Document ID: US10865811B2
- Status: Granted
- Kind: B2
- Application number: 15/547,320
- Filed: 2016-02-03
- Published: 2018-02-01
- Granted: 2020-12-15
- Earliest claimed: 2015-02-06
- Assignees: The University of British Columbia
- Applicants: The University of British Columbia
- Inventors: Aziz Ghahary; Ryan Hartwell
- Web page: https://patentgrep.com/patent/US10865811B2

## CPC

- F15B 1/165: {in the form of a bladder}
- A61L 27/26: Mixtures of macromolecular compounds
- A61L 27/48: with macromolecular fillers
- A61L 27/52: Hydrogels or hydrocolloids
- A61L 27/60: Materials for use in artificial skin
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- E21B 33/064: specially adapted for underwater well heads
- F15B 1/24: with rigid separating means, e.g. pistons
- A61L 2300/64: Animal cells
- A61L 2400/06: Flowable or injectable implant compositions
- C12N 2533/54: Collagen; Gelatin
- C12N 2533/70: Polysaccharides
- C12N 2537/10: Cross-linking
- F15B 2201/205: using gas
- F15B 2201/31: having rigid separating means, e.g. pistons
- F15B 2201/32: having multiple separating means, e.g. with an auxiliary piston sliding within a main piston, multiple membranes or combinations thereof
- F15B 2201/41: Liquid ports
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- F15B: SYSTEMS ACTING BY MEANS OF FLUIDS IN GENERAL; FLUID-PRESSURE ACTUATORS, e.g. SERVOMOTORS; DETAILS OF FLUID-PRESSURE SYSTEMS, NOT OTHERWISE PROVIDED FOR
- 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)
- 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)
- E21B: EARTH OR ROCK DRILLING; OBTAINING OIL, GAS, WATER, SOLUBLE OR MELTABLE MATERIALS OR A SLURRY OF MINERALS FROM WELLS
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Compositions and methods of preparation and use are provided for an engineered tissue substitute system comprising collagen, glycosaminoglycan and hydrogel in a cross-linked matrix. The compositions may be further lyophilized and reconstituted with a physiological fluid prior to use in methods, such as in the treatment of wounds, tissue engineering and cell transplantation.

## Claims

### Claim 1

A lyophilized powder composition, the composition comprising:
(a) collagen, wherein the collagen is at a concentration of between 3-10 mg/ml;
(b) glycosaminoglycan, wherein the ratio of the glycosaminoglycan to collagen is a weight ratio from about 4:1 to about 8:1;
(c) a biocompatible small molecule cross-linker of collagen and glycosaminoglycan;
(d) a hydrogel, wherein the hydrogel is 0.01%-1.0% w/vol of the final composition; and
(e) a biocompatible, small-molecule, hydrogel cross-linker.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the biocompatible small molecule cross-linker of collagen and glycosaminoglycan is selected from one or more of the following: glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC), EDC (1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide):NHS (N-hydroxysuccinimide), EDC (1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide):sulfo-NHS (N-hydroxysulfoxuccinimide), hexamethylene diisocyanate and Genipin.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the hydrogel is formed from hydrogel polymers, and wherein the hydrogel polymers are selected from one or more of: polyvinyl alcohol (PVA); polyvinyl acetate (PVacetate); thiolated polyvinyl alcohol; polyvinyl alcohol block polymers containing polyethylene glycol (PVA-PEG); polyvinylpyrrolidone (PVP); and a copolymer thereof of any two or more of the preceding polymers.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the hydrogel is 0.01-0.5% w/vol of the final composition.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the composition further comprises a solvent.

### Claim 6 (depends on 5)

The composition of claim 5, wherein the solvent is cell media, water, or a physiological fluid.

### Claim 7 (depends on 6)

The composition of claim 6, wherein the physiological fluid is blood, serum or plasma.

### Claim 8 (depends on 1)

The composition of claim 1, wherein fibrillogenesis begins within 13-16 minutes of solvent addition.

### Claim 9 (depends on 1)

The composition of claim 1, wherein gelation of the powder occurs:
between 25°-40° C.;
between 25°-37° C.;
between 30°-37° C.; or
at 37° C.

### Claim 10 (depends on 1)

A wound treatment method, the method comprising reconstituting the powdered composition of claim 1 in a solvent and administration of the reconstituted composition to a patient in need thereof.

### Claim 11 (depends on 1)

A method of preparing a composition of claim 1, the method comprising:
(a) mixing collagen with glycosaminoglycan, wherein the ratio of the glycosaminoglycan to collagen is a weight ratio from about 4:1 to about 8:1;
(b) cross-linking of collagen and glycosaminoglycan;
(c) adding a hydrogel to the cross-linked collagen and glycosaminoglycan, wherein the hydrogel is 0.3%-1.0% w/vol of the final composition; and
(d) cross-linking the hydrogel.

### Claim 12 (depends on 11)

The method of claim 11, wherein the cross-linking of collagen and glycosaminoglycan is by dehydrothermal treatment (DHT), ultraviolet irradiation (UV) or enzymatic crosslinking.

### Claim 13 (depends on 11)

The method of claim 11, wherein the cross-linking of collagen and glycosaminoglycan is by small molecule cross-linker of collagen and glycosaminoglycan.

### Claim 14 (depends on 13)

The method of claim 13, wherein the cross-linker of collagen and glycosaminoglycan is selected from one or more of the following: glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC), EDC (1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide):NHS (N-hydroxysuccinimide), EDC (1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide):sulfo-NHS (N-hydroxysulfoxuccinimide), hexamethylene diisocyanate and Genipin.

### Claim 15 (depends on 14)

The method of claim 14, wherein the method further comprises adding dextran.

### Claim 16 (depends on 11)

The method of claim 11, wherein the hydrogel comprises one or more of: polyvinyl alcohol (PVA); polyvinyl acetate (PVacetate); thiolated polyvinyl alcohol; polyvinyl alcohol block polymers containing polyethylene glycol (PVA-PEG); polyvinylpyrrolidone (PVP); and a copolymer thereof of any two or more of the preceding polymers.

### Claim 17 (depends on 16)

The method of claim 16, wherein the hydrogel is a PVA hydrogel.

### Claim 18 (depends on 17)

The method of claim 17, wherein the PVA hydrogel is 0.4-0.6% w/vol of the final composition.

### Claim 19 (depends on 17)

The method of claim 17, wherein the cross-linker of the hydrogel is:
borate; or
sodium borate decahydrate.

### Claim 20 (depends on 11)

The method of claim 11, wherein the cross-linker is:
0.01%-0.0001% w/vol of the final composition; or
0.01-0.1% w/vol of the final composition.

### Claim 21 (depends on 11)

The method of claim 11, wherein the method further comprises lyophilizing of the composition.

### Claim 22 (depends on 21)

The method of claim 21, wherein the method further comprises powdering the lyophilized composition.

### Claim 23 (depends on 22)

The method of claim 22, wherein the method further comprises reconstituting the powder in a solvent.

### Claim 24 (depends on 23)

The method of claim 23, wherein the solvent is a physiological fluid or water.

### Claim 25 (depends on 24)

The method of claim 24, wherein the physiological fluid is blood, serum or plasma.