Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Summary

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

Abstract

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

Introduction

Cartilage tissue engineering is a rapidly developing field that seeks to regenerate damaged or diseased cartilage tissue¹. One key challenge in this field is the development of biomimetic scaffolds that can support the growth and differentiation of chondrocytes, the cells responsible for producing cartilage². The ECM of cartilage tissue plays a critical role in regulating the behavior of chondrocytes. DC-ECM is an effective scaffold for tissue engineering applications³.

A number of techniques have been developed to produce DC-ECM from cartilage tissue, including chemica....

Protocol

This study was approved by the Ethics Committee of Tongde Hospital of Zhejiang Province.

1. Preparation of the DC-ECM hydrogel

NOTE: In this study, the cartilage was obtained from the knee joints of 12 month old Bama miniature pigs, avoiding the collection of bone tissue.

1. Take the collected cartilage, and block and chop it into 1-2 mm³ pieces with a scalpel.
2. Place 20 g of the minced cartilage in a 50 mL centrifuge tube, and add 20 mL of hypotonic Tris-HCl buffer (10 mM Tris-HCl, pH 8.0) to submerge the tissue completely. Place the centrifuge tube in a −80 &#....

Results

To prepare a better DC-ECM cartilage hydrogel, we studied and reviewed the previous literature and compared the various decellularization protocols in terms of the decellularization ratio, immunogenicity, and mechanical functionality⁹.

On this basis, we prepared the DC-ECM cartilage hydrogel and explored the effect of a radially oriented extractive matrix/mesenchymal stem cell exosome bio-ink in treating osteochondral defects. The results showed that the DC-ECM cartilag.......

Discussion

This protocol provides a systematic approach for the preparation of decellularized cartilage extracellular matrix hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical, chemical, and enzymatic disruption to remove cellular material while preserving the structure and composition of the ECM. The protocol's critical steps include adjusting the decellularization time and methods and ensuring complete decellularization.

Compared to other ex.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 M Tris-HCl, pH7.6	Beyotime	ST776-100 mL
1 M Tris-HCl, pH8.0	Beyotime	ST780-500 mL
-80 °C Freezer	Eppendorf	F440340034
Deoxyribonuclease	Aladdin	D128600-80KU
DNEasy Blood &Tissue Kit	Qiagen	No. 69506
GAG colorimetric quantitative detection kit	Shanghai Haling	HL19236.2
HCP-2 dryer	Hitachi	N/A
Nanodrop8000	Thermo Fisher	N/A	Spectrophotometer
PBS (10x)	Gibco	70011044
Ribonuclease	Aladdin	R341325-100 mg
Sigma500	ZIESS	N/A	Scanning electron microscope
Spectra S	Thermo Fisher	N/A	Transmission electron microscope
Stainless steel sieve	SHXB-Z-1	Shanghai Xinbu
Triton X-100	Beyotime	P0096-500 mL
Trypsin	Gibco	15050065
Ultraviolet lamp	Omnicure 2000	N/A
Vitamin B2	Gibco	R4500-5G
Vortex mixer	Shanghai Qiasen	78HW-1