Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening

Summary

This manuscript describes a murine calvarial osteolysis model by exposure to CoCrMo particles, which constitutes an ideal animal model for assessing the interactions between wear particles and various cells in aseptic loosening.

Abstract

Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long follow-ups necessary for detection and sporadic occurrence, it is challenging to assess the pathogenesis ofparticle-induced osteolysis in clinical cases. Hence, optimal animal models are required for further studies. The murine model of calvarial osteolysis established by exposure to CoCrMo particles is an effective and valid tool for assessing the interactions between particles and various cells in aseptic loosening. In this model, CoCrMo particles were first obtained by high-vacuum three-electrode direct current and resuspended in phosphate-buffered saline at a concentration of 50 mg/mL. Then, 50 µL of the resulting suspension was applied to the middle of the murine calvaria after separation of the cranial periosteum by sharp dissection. After two weeks, the mice were sacrificed, and calvaria specimens were harvested; qualitative and quantitative evaluations were performed by hematoxylin and eosin staining and micro computed tomography. The strengths of this model include procedure simplicity, quantitative evaluation of bone loss, rapidity of osteolysis development, potential use transgenic or knockout models, and a relatively low cost. However, this model cannot to be used to assess the mechanical force and chronic effects of particles in aseptic loosening. Murine calvarial osteolysis model generated by exposure to CoCrMo particles is an ideal tool for assessing the interactions between wear particles and various cells, e.g., macrophages, fibroblasts, osteoblasts and osteoclasts, in aseptic loosening.

Introduction

Aseptic loosening is the most common cause of total hip arthroplasty (THA) and total knee arthroplasty (TKA) failure, which requires revision surgery¹. However, the underlying mechanism remains unclear². A long follow-up is required to detect particle-induced osteolysis, whose occurrence is rare; therefore, it is challenging to explore its pathogenesis in clinical cases. Hence, further studies focusing on complex cellular and tissue mechanisms require both in vivo experiments in wear particle-induced osteolysis models and in vitro assays in cells related to bone homeostasis³. A valid animal model is important in revealing the effects of wear particles on bone loss, providing evidence for further cellular assays.

A murine calvarial osteolysis model constructed by exposure to CoCrMo particles is an effective and valid method for assessing the interactions between particles and various cells in aseptic loosening. In this model, CoCrMo particles cause calvarial osteolysis by inducing inflammatory cytokines in macrophages, activating osteoclasts, inhibiting osteoblast proliferation, and promoting osteoblast apoptosis.

It only takes two weeks to establish this model. Osteolysis can be visualized and quantified by hematoxylin and eosin (H&E) staining and micro computed tomography (micro-CT)². In addition, this model has a relatively low cost, and transgenic and knockout mouse models can be used to screen a large number of compounds at various doses³.

The procedure to establish and evaluate this model is simple. First, CoCrMo particles were obtained by high-vacuum three-electrode direct current and resuspended in phosphate-buffered saline (PBS) at a concentration of 50 mg/mL. Then, 50 µL of the resulting suspension was applied to the middle of the murine calvaria after separation of the cranial periosteum by sharp dissection. The mice were sacrificed after two weeks, and calvaria samples were harvested; qualitative and quantitative analyses were performed by H&E staining andmicro-CT.

A murine calvarial osteolysis model constructed by exposure to CoCrMo particles is an ideal tool for assessing the interactions between CoCrMo particles and various cells, such as macrophages, fibroblasts, osteoblasts, and osteoclasts, in aseptic loosening.

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing University.

1. CoCrMo Particle Preparation

1. Obtain CoCrMo particles by using a fabricated high-vacuum three-electrode direct current⁴. Place CoCrMo alloy in the instrument under 10^-3 Pa vacuum, 0.04 MPa argon and hydrogen 3:2 (v/v), and 650 A cathode current.
2. Measure the diameters of CoCrMo particles.
   1. Add 1 mg of CoCrMo particles into 1.5 mL of anhydrous ethanol.
   2. Resuspend CoCrMo particles in anhydrous ethanol by ultrasonic shaking at 28 kHz and 600 W for 5 min.
   3. Apply one drop (about 20 µL) of the resulting suspension on the objective table of a transmission electron microscope (TEM). Capture series of TEM photos at 200 kV acceleration voltage and 0.24 nm resolution.
   4. Use the provided software to calculate the mean diameter and particle size distribution in TEM micrographs.
3. Decontaminate endotoxins
   1. Autoclave 50 g of particles for 15 min at 121 °C and 15 psi.
   2. Detect endotoxins by a quantitative Limulus Amebocyte Lysate (LAL) Assay (<0.25% EU/mL was considered to indicate endotoxin absence)⁵.
4. Resuspend the particles in phosphate-buffered saline (PBS) at a concentration of 50 mg/mL as stock solution⁶.

2. Construction of the Calvarial Osteolysis Model

1. Anesthetize 6 week old C57BL/J6 mice (six mice per group) withpentobarbital (50 mg/kg). Use the pinch test to assess the level of anesthesia. Prevent drying of eyes with normal saline.
2. Place mice in the prone position. Remove the fur on the cranium with a shaver and disinfect the skin using medical cotton balls containing 75% ethanol.
3. For point localization, identify two points, including the midpoints between the two eyes and ears, respectively. Then, determine the line between the two above points, and incise the skin along the above line with scissors (Figure 1A).
4. Remove the cranial periosteum from the calvaria with a scalpel (Figure 1B)⁶.
5. Suture skin at both ends with simple interrupted suture.
6. Make a suture line through the middle of the incision without knotting. Hold the two ends of the suture line.
7. Embed 50 µL of CoCrMo particle suspension (50 mg/mL, in PBS) in the middle of the calvarias (Figure 1C)².
8. Knot the last stitch within simple interrupted suture (Figure 1D).
9. Maintain mice for another 2 weeks.

3. Evaluation of Calvarial Osteolysis Model by Micro-CT Scanning

1. Sacrifice the mice with carbon dioxide. Decapitate the mice in the horizontal plane. Remove the brain tissue inside, and the skin and fur outside. Harvest the calvarias for further experiments.
2. Gently clear up all the soft tissue on the calvaria with tweezers. Fix the cleared calvarias in 4% paraformaldehyde at 4 °C for 24 h. Immerse the calvarias in PBS 24 h before micro-CT scanning.
3. Analyze the mice calvarias by high-resolution micro-CT at an isometric resolution of 18 µm and X-ray energy settings of 45 kV and 550 mA.
4. Conduct three-dimensional reconstruction of micro-CT data with the software.
5. Qualitative and quantitative analysis.
   1. First, select the square region around the midline suture as the region of interest.
   2. Secondly, measure bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation/spacing (Tb.Sp), and percentage of total porosity with the provided software for micro-CT.
   3. Thirdly, compare the three groups for various measurements by one-way ANOVA. For post hoc analysis of variance, apply the Bonferroni method².

4. Evaluation of Calvarial Osteolysis Model by H&E Staining

1. Decalcify calvaria samples in 15% ethylene diamine tetraacetic acid (EDTA)-PBS at 4 °C. Change the decalcification solution every day for 3 weeks.
2. Embed the decalcified samples in paraffin for a 2 cm x 1 cm x 1 cm cube, and slice them into 2 µm sections in the area of particle deposition.
3. Stain the sections with hematoxylin and eosin as previously described⁷.
4. Capture micrographs of the overall pathomorphism by light microscopy.

Results

The in-house produced nanoscale CoCrMo particles were around 50 nm (standard error of 3.56) in diameter, as quantified by TEM (Figure 2). After exposure of mouse calvarias to CoCrMo particles, the animals (n=6 per group) were maintained for another two weeks. Within the two weeks, the calvarial incision was completely healed, and the suture may fall. Any local infection or nonunion may affect bone loss assessment. After mouse sacrifice, calvaria samples were ...

Discussion

There are two main methods for wear particle-induced osteolysis in mice: the air-pouch model and the calvarial osteolysis model. In the air-pouch model, a subcutaneously generated air-pouch is first established, followed by wear particle introduction and implantation into the bone tissue⁸. The pouch wall mimics the periosteum in aseptic loosening. However, bone implantation is nonvascular with no biological activity, which makes it difficult to assess direct interactions between particles and the ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CoCrMo alloy from prosthesis	Waldemar Link GmbH & Co	GEMINI MK II	Raw material to obtain CoCrMo nanoparticles
Fabricated high-vacuum three-electrode direct current	College of Materials Science & Engineering , Nanjing University of Technology		Self designed machine
6 week old male C57BL/6J mice	Model animal research center of Nanjing University	N000013
100% Ethanol	Nanjing Reagent	C0691514023	Solvent of CoCrMo nanoparticles for transmission electron microscope scanning
1.5 ml Microcentrifuge tubes	Taizhou Weierkang Medical Supplies co., LTD	W603
Microanalytical balance	Shenzhen Qun long Instrument Equipment Co,. LTD	EX125DZH
Ultrasonic shaker	Shanghai Yuhao scientific instrument co., LTD	YH-200DH	To suspend CoCrMo nanoparticles
Transmission Electron Microscope	FEI	Tecnai G20
SimplePCI software	Compix Inc.	6.6 version	To calculate the mean diameter and particle size distribution.
High-handed sterilization pan	QIULONGYIQI	KYQL-100DS	To decontaminate endotoxin
Limulus Amebocyte Lysate (LAL) Assay	Charles River	R13025	To detect endotoxin
15 ml Microcentrifuge tubes	Taizhou Suyi Medical	B122
Phosphate-buffered saline	Boster Biological Technology	AR0030	Solvent of CoCrMo nanoparticles stock solution
Pentobarbital Sodium	Sigma	P3761	To anesthetize mice
Normal saline	SACKLER	SR8572EP-15	To prevent drying of mice eyes
75% Ethanol	Nanjing Reagent	C0691560275	Disinfection
Medical cotton ball	Shuitao	1278298933	Disinfection
Shaver	Kemei	KM-3018	To shave the fur
Scissor	RWD LIFE SCIENCE	S12005-10	To incise skin
Suture	RWD LIFE SCIENCE	F34001-01	To suture skin
Needle holder	RWD LIFE SCIENCE	F33001-01	To suture skin
Needle	RWD LIFE SCIENCE	R14003-12	To suture skin
Vessel forceps	RWD LIFE SCIENCE	F22003-09	To suture skin
Scalpel	RWD LIFE SCIENCE	S31010-01	To harvest calvaria
Tweezers	RWD LIFE SCIENCE	F12006-10	To harvest calvaria
100 µL pipettes	Eppendorf	3120000240	To embed particles suspension in the calvatias
100 µL pipette tips	AXYGEN	T-200-Y	To embed particles suspension in the calvatias
5 ml Microtubes	Taizhou Weierkang Medical Supplies co., LTD	W621
4% Paraformaldehyde	Servicebio	G1101	Fixation
Micro Computed Tomography	SkyScan	SkyScan1176
Ethylene Diamine Tetraacetic Acid	Servicebio	G1105	Decalcification
Paraffin	Servicebio	#0001
Paraffin slicing machine	Leica	RM2125RTS
Glass slide	Servicebio	G6004
Cover glass	Servicebio	200
HE staining kit	Servicebio	#1-5	HE staining
Light microscope	Nikon	E200