Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Summary

The goal of this protocol is to generate a nude rat osteoporosis-related vertebral compression fracture model that can be longitudinally evaluated in vivo using a semiautomated microcomputed tomography-based quantitative structural analysis.

Abstract

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (µCT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent µCT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

Introduction

More than 200 million people worldwide suffer from osteoporosis¹. The underlying pathological decrease in bone mineral density (BMD) and altered bone microarchitecture increase bone fragility and, consequently, the relative risk of fracture². Osteoporosis is so prevalent and detrimental to health that the WHO has defined it a major public health concern. Furthermore, as the world's population is expected to age, osteoporosis is expected to become even more common.

Osteoporotic vertebral compression fractures are the most common fragility fractures, estimated at more than 750,000 a year in ....

Protocol

All animal experiments were performed under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Cedars-Sinai Medical Center (Protocol # 3609). Anesthesia was administered for all imaging and surgical procedures. All animals were housed in accordance with approved IACUC protocols.

NOTE: The experimental design of this protocol is shown in Figure 1. Purchase six-week-old rats with their ovaries surgically removed and feed them an LCD consisting of 0.01% calcium and 0.77% phosphate. After a period of 4 months of an LCD, drill a critical-size vertebral defect in the fourth and fifth lu....

Results

Using this protocol, one can image and quantify the regeneration of n = 8 modeled osteoporotic vertebral defects across different time points. The anatomic match obtained by the registration procedure allows for the analysis of the same VOI at all time points. This results in a highly accurate longitudinal 3D histomorphometric analysis, even when the margins of the original defect are no longer recognizable. We used five time points (day 1, week 2, week 4, week 8, and week 12) as an examp.......

Discussion

Osteoporosis is the most prevalent cause of vertebral compression fractures caused by an increased load on the spine and that result in the collapse of the vertebral body. However, it is practically impossible to generate an injury in a rodent that authentically replicates a similar vertebral collapse. Instead, researchers create a cylindrical void in the center of the vertebral body to mimic OVCFs^16,17,18,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Isoflurane	MWI Animal Health, Pasadena, CA	501017
BetadineSolution	MWI Animal Health, Pasadena, CA	4677
Chlorhexidine Gluconate 2% scrub	MWI Animal Health, Pasadena, CA	510083
Isopropyl Alcohol 70%-quart	MWI Animal Health, Pasadena, CA	501044
Carprofen	MWI Animal Health, Pasadena, CA	26357
Buprenorphine 0.3 mg/mL	MWI Animal Health, Pasadena, CA	56163
Ovariectomized Athymic nude rats	Harlan Laboratories, Indianapolis, IN	Hsd:RH-Foxn1 rnu
Low calcium food	Newco Distributors, Inc., CA	1814948 (5AV8 AIN-93M w/low calcium)
Phosphate Buffered Saline	Life Technologies Corporation	14190250
Dermabond	J AND J ETHICON	DHVM12
Anesthesia machine	Patterson Scientific	TEC 3EX
Slide Top Induction Chambers	Patterson Scientific	78917833
ProStation Heated Workstation	Patterson Scientific	78914731
Surgical drape	HALYARD HEALTH INC	89101
Magnetic fixator retraction system	Fine Science Tools, Inc., CA	18200-50
Dissecting Scissors, 10 cm, Curved, SS	World Precision Instruments, FL	14394
Iris Scissors, 11.5 cm, 45 °Angle, Serrated, Sharp/Sharp	World Precision Instruments, FL	503225
Forceps, no. 5	World Precision Instruments, FL	555048FT
Micro Mosquito Hemostatic Forceps	World Precision Instruments, FL	503360
Sterile cotton gauze	Medtronic, MINNEAPOLIS, MN	9024
Absorption Spears - Mounted/Sterile	Fine Science Tools, CA	18105-01
Syringe, 1 mL	TERUMO TERUMO MED	SS-01T
Needle, 25 gauge	BD MED SYS INJECTION SYS	305127
Laminar flow hood	Baker	SterilGARD e3-Class II Type A2 Biosafety Cabinet
Thermal Cautery Unit	World Precision Instruments, FL	501292
Micro-Drill OmniDrill115/230V	World Precision Instruments, FL	503598
Trephines for Micro Drill, 2 mm diameter	Fine Science Tools, CA	18004-20
3-0 Vicryl undyed 27” SH taper	J AND J ETHICON	1663G
4-0 Ethilon black 18” PC3 conventional cutting	J AND J ETHICON	1954G
Conebeam in vivo microCT (vivaCT 40)	Scanco Medical	vivaCT 40
SCANCO Medical microCT systems software suite	Scanco Medical	vivaCT 40
Analyze software	Biomedical Imaging, Mayo Clinic, Rochester, MN	Analyze 12	Image analysis software
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