Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Summary

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Abstract

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

Introduction

Articular cartilage (AC) is a load bearing tissue that covers and protects bones in synovial joints, providing smooth joint articulation. Tissue homeostasis is dependent on the viability of chondrocytes, the sole cell type residing in AC. However, exposure of cartilage to extreme forces due to trauma (e.g., falls, vehicle accident or sports injuries) or due to post-traumatic joint instability can induce chondrocyte death, leading to irreversible breakdown of the joint (osteoarthritis)¹. Furthermore, in osteochondral grafting procedures that aim to repair local defects in damaged cartilage, graft insertion-associated mechanical trauma r....

Protocol

All animal work was approved by the University of Rochester Committee on Animal Resources.

1. Solutions

1. Prepare Hank's Balanced Salt Solution (1X HBSS) containing calcium, magnesium and no phenol red. Adjust the pH to 7.4 by adding small amounts of HCl or NaOH.
2. Adjust the osmolarity to 303 mOsm by adding NaCl or deionized water. Use the buffer during the dissections, specimen preparation and mechanical testing.

2. Dissections of the Distal Femur and Proximal Humerus with Fully Intact Articular Cartilage

1. Euthanize the mouse according to institutional guidelines using a ....

Results

Six different applied loading protocols (static loading: 0.1 N, 0.5 N and 1 N for 5 min; and impact loading: 1 mJ, 2 mJ and 4 mJ) reproducibly induced quantifiable localized areas of cell injury in femoral and humeral cartilage obtained from 8-10-week-old BALB/c mice (Figure 2). Importantly, the spatial extent of chondrocyte injury on the articular surface was measured quickly and easily in ImageJ. Representative results demonstrate that the mechanical vulner.......

Discussion

The methods described above were successfully employed to visualize viable and injured/dead in situ articular chondrocytes from mouse joints after prescribed mechanical loads or impacts. In particular, we were able to analyze the mechanical vulnerability of chondrocytes within fully intact articular cartilage from two different synovial joints: the knee joint (distal femurs) and shoulder (humeri). Our representative results show that the spatial extent of cell injury on the articular surface depends sensitively .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Calcein, AM	Invitrogen by Thermo Fisher Scientific	C3100MP	20x50mg , Eugene, OR, USA
Propidium Iodide	Invitrogen by Thermo Fisher Scientific	P3566	1 mg/mL solution in water, 10mL, Eugene, OR, USA
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	276855	1L DMSO, anhydrous, ≥99.9%, St. Louis, MO, USA
HBSS (calcium, magnesium, no phenol red)	Gibco by Thermo Fisher Scientific	14025-092	1X, 500mL, Grand Island, NY, USA
Feather surgical blade (#11)	VWR	102097-822	Hatfield, PA, USA
Vapor pressure osmometer, VAPRO	ELITechGroup	Model 5520	Puteaux, France
pH meter	Beckman	Model Phi 32	Brea, CA, USA
Eppendorf thermomixer	Eppendorf AG	Model 5350	Hamburg, Germany
Motorized inverted research microscope	Olypmus	Model IX-81	Center Valley, PA, USA
Wooden applicator	Puritan Medical Products Company, LLC	807	6"x100, Guilford, ME, USA
1.5 Glass coverslips	Warner Instruments, LLC	64-1696	#1.5, 0.17mm thick, 40mm diameter, Hamden, CT, USA