# US 10,865,256 B2 · Method for preparing cross-linked hyaluronic acid-based cell scaffold material

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- Number: US 10,865,256 B2
- Document ID: US10865256B2
- Status: Granted
- Kind: B2
- Application number: 16/289,666
- Filed: 2019-03-01
- Published: 2019-09-05
- Granted: 2020-12-15
- Earliest claimed: 2018-03-01
- Assignees: Hangzhou Singclean Medical Products Co., Ltd
- Applicants: Hangzhou Singclean Medical Products Co., Ltd.
- Inventors: Jin Zeng; Weiqing Sun
- Web page: https://patentgrep.com/patent/US10865256B2

## CPC

- C08B 37/0072: {Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates}
- A61L 27/20: Polysaccharides
- 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 27/60: Materials for use in artificial skin
- C08L 5/08: Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
- A61L 2300/416: Anti-neoplastic or anti-proliferative or anti-restenosis or anti-angiogenic agents, e.g. paclitaxel, sirolimus
- A61L 2300/602: Type of release, e.g. controlled, sustained, slow
- A61L 2400/18: Modification of implant surfaces in order to improve biocompatibility, cell growth, fixation of biomolecules, e.g. plasma treatment
- A61L 2430/06: for cartilage reconstruction, e.g. meniscus
- C08B: POLYSACCHARIDES; DERIVATIVES THEREOF (polysaccharides containing less than six saccharide radicals attached to each other by glycosidic linkages C07H; fermentation or enzyme-using processes C12P19/00; sugar industry C13; production of cellulose D21)
- 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)
- C08L: COMPOSITIONS OF MACROMOLECULAR COMPOUNDS (compositions based on polymerisable monomers C08F, C08G; artificial filaments or fibres D01F; textile treating compositions D06)

## Abstract

Disclosed is a method for preparing cross-linked hyaluronic acid-based cell scaffold material. The hyaluronic acid-based cell scaffold is obtained by subjecting a hyaluronic acid and a disulfide cross-linking agent to an amidation reaction, followed by dialysis-freeze drying. The cell scaffold has abundant pores, good mechanical strength which ensures that the scaffold does not rupture in transplantation, and good biocompatibility. The method is advantageous in that the raw material is easy to obtain, the reaction condition is moderate, and the process is simple. Cross-linked networks of the prepared hydrogel contain disulfide bonds, which can quickly split into single chains at the presence of small molecular glutathione. The hyaluronic acid-based cell scaffold has flexibly controllable mechanical property, disaggregation ability, and swelling property, and therefore has wide applications in facilitating cartilage injury repair, skin repair, cell culture, etc.

## Claims

### Claim 1

A method for preparing a cross-linked hyaluronic acid-based cell scaffold material, comprising the steps of:
adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide (NHS) in sequence to an aqueous solution of hyaluronic acid having a pH value of 5.0-6.0, to form a mixture;
stirring the mixture at room temperature to obtain a uniform mixture;
adding an aqueous solution of cystamine dihydrochloride to the uniform mixture to form a reaction system;
adjusting the reaction system to have a pH value of 5.0-6.0 for reaction;
placing the reaction system after reaction in a phosphate buffer for dialysis to form a dialyzed product; and
freeze-drying the dialyzed product to obtain the cross-linked hyaluronic acid-based cell scaffold,
wherein the step of freeze-drying comprises: at a first pre-freezing phase, keeping the dialyzed product at a temperature ranging from −60° C. for 2-5 hours; at a second sublimating phase, keeping the dialyzed product at a temperature ranging from −40° C. to −25° C. for 4-8 hours and keeping at a temperature ranging from −20° C. to 0° C. for 2-10 hours; and at a third vacuum drying phase, keeping the dialyzed product at a temperature ranging from 10° C. to 30° C. for 3-6 hours.

### Claim 2 (depends on 1)

The method according to claim 1, wherein a molar ratio of —COOH in the hyaluronic acid to —NH2 in the cross-linking agent is in a range of 1:1-1:0.1, and a molar ratio of —COOH in the hyaluronic acid to EDC and NHS is in a range of 1:0.1-0.6:0.1-1.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the reaction is carried out at a temperature in a range of 0-10° C.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the reaction is carried out for 24-120 hours.

### Claim 5 (depends on 1)

The method according to claim 1, wherein an aqueous alkali or an acid solution is used for adjusting the pH value.

### Claim 6 (depends on 1)

The method according to claim 1, wherein hyaluronic acid based cell scaffold is cut into small pieces having a weight in a range of 0.2-0.5 g for conducting the dialysis.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the phosphate buffer contains 0.1-0.2 M phosphate and has a pH value of 7.0-7.4.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the dialysis is conducted for 24-72 hours.

### Claim 9 (depends on 3)

The method according to claim 3, wherein the reaction is carried out at a temperature in a range of 1-4° C.

### Claim 10 (depends on 4)

The method according to claim 4, wherein the reaction is carried out for 72-96 hours.

### Claim 11 (depends on 5)

The method according to claim 5, wherein the aqueous alkali is 0.1 M sodium hydroxide solution, and the acid solution is 0.1 M HCl.

### Claim 12 (depends on 8)

The method according to claim 8, wherein the dialysis is conducted for 48-72 hours.