Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

Summary

This protocol establishes a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats and measuring the subsequent pain behavior and histopathological changes.

Abstract

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.

Introduction

Articular cartilage is a highly differentiated and dense tissue consisting of chondrocytes and extracellular matrix¹. The surface layer of articular cartilage is a form of hyaline cartilage, which has a smooth surface, low friction, good strength and elasticity, and excellent mechanical stress tolerance². The extracellular matrix comprises collagen proteoglycan and water, and type II collagen is the main structural component of the collagen, as it accounts for about 90% of the total collagen³. As no blood vessels or nerves exist in cartilage tissue, it lacks the ability to self-repair after injury....

Protocol

The animal experiments were approved by the Medical Standards and Ethics Committee of Zhejiang University of Traditional Chinese Medicine, which conforms to the China legislation on the use and care of laboratory animals. In the present study, 6 week old male Sprague-Dawley (SD) rats weighing 150-180 g were used. The animals were obtained from a commercial source (see the Table of Materials).

1. Establishment of a full-thickness cartilage defects model in rats

Discussion

This study describes an animal model for mimicking clinical cartilage defects by drilling holes in the femoral trochlear groove of rats (Supplementary Figure 1). After cartilage injury, the excitability or responsiveness of peripheral nociceptors is enhanced, which can result in a decrease in the pain threshold and the enhancement of responsiveness to stimulation¹⁸. In preclinical studies, the modeling of cartilage defects in different species of animals has .......

Materials

Name	Company	Catalog Number	Comments
3, 3 '-diaminobenzidine	Hangzhou Zhengbo Biotechnology Co., Ltd.	ZLI-9019	The dye for IHC staining
Anti-Collagen III antibody	Novus	NB600-594	Primary antibody for IHC
Anti-Collagen II antibody	Abcam (UK)	34712	Primary antibody for IHC
Anti-Collagen I antibody	Novus	NB600-408	Primary antibody for IHC
Bouin solution	Shanghai Yuanye Technology Co., Ltd.	R20381	The dye for Masson staining
Celestite blue	Shanghai Yuanye Technology Co., Ltd.	R20381	The dye for Masson staining
Corncob paddings	Xiaohe Technology Co., Ltd		Bedding for animal
Eosin	Sigma-Aldrich	861006	The dye for HE staining
Fast Green FCF	Sigma-Aldrich	F7252	The dye for SO staining
Goat anti-mouse antibody	ZSGQ-BIO (Beijing, China)	PV-9002	Secondary antibody for IHC
Goat anti-rabbit antibody	ZSGQ-BIO (Beijing, China)	PV-9001	Secondary antibody for IHC
Hematoxylin	Sigma-Aldrich	H3163	The dye for HE staining
Masson	Shanghai Yuanye Technology Co., Ltd.	R20381	The dye for Masson staining
Microdrill	Rwd Life Science Co., Ltd	78001	Equipment for surgery
MMP13	Cell Signaling Technology, Inc. (Danvers, MA, USA)	69926	Primary antibody for IHC
Modular tissue embedding center	Thermo Fisher Scientific (USA)	EC 350	Produce paraffin blocks
Neutral resin	Hangzhou Zhengbo Biotechnology Co., Ltd.	ZLI-9555	Seal for IHC
Nonabsorbable suture	Hangzhou Huawei Medical Supplies Co.,Ltd.	4-0	Equipment for surgery
Pentobarbital sodium	Hangzhou Zhengbo Biotechnology Co., Ltd.	WBBTN5G	Anesthetized animal
phosphomolybdic acid	Shanghai Yuanye Technology Co., Ltd.	R20381	The dye for Masson staining
Ponceau fuchsin	Shanghai Yuanye Technology Co., Ltd.	R20381	The dye for Masson staining
Rotary and Sliding Microtomes	Thermo Fisher Scientific (USA)	HM325	Precise paraffin sections
Safranin-O	Sigma-Aldrich	S2255	The dye for SO staining
Scalpel blade	Shanghai Lianhui Medical Supplies Co., Ltd.	11	Equipment for surgery
Sodium citrate solution (20x)	Hangzhou Haoke Biotechnology Co., Ltd.	HK1222	Antigen retrieval for IHC
Sprague Dawley (SD) rats	Shanghai Slake Experimental Animal Co., Ltd.	SD	Experimental animal
Tissue-Tek VIP 5 Jr	Sakura (Japan)		Vacuum Infiltration Processor
Toluidine Blue	Sigma-Aldrich	89640	The dye for TB staining
Von Frey filament	UGO Basile (Italy)	37450-275	Equipment for MWT assay
Wire mesh platform	Shanghai Yuyan Instruments Co.,Ltd.		Equipment for MWT assay