Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Summary

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Abstract

Lipotransfer is a vital tool in the surgeon’s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.¹ However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. ^1-3

To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 µl of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.

Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Introduction

Soft tissue defects arise from a variety of causes including trauma, tumor resection, aging, and congenital anomaly. They can be debilitating for patients, and represent one of the most common, yet challenging problems for reconstructive surgeons. Many methods exist for addressing soft tissue deficiencies, such as local and free flaps, collagen injections, and synthetic fillers.^4-8 However, since its first documented use by Neuber in 1893¹, autologous fat transfer remains the gold standard for the repair of soft tissue deficits, as it is ready available, easy and safe to harvest, and naturally compatible.^1,2

Protocol

NOTE: Experimental protocols and patient consent forms for obtaining fat were reviewed and approved by the Stanford University Institutional Review Board (Protocol #2188). All animal procedures were approved by the Stanford Administrative Panel on Laboratory Animal Care (APLAC) under Protocol #9999. All experiments were conducted with strict adherence to animal safety and humane care guidelines.

1. Fat Harvesting

1. Using the Coleman procedure^17-19, obtain human adipose tissue from the abdomen, flank, and/or thigh regions of healthy female patients undergoing elective liposuction.
2. To process the lipoaspirate fo....

Results

Fat grafts progressively decreased in volume over the course of study, resulting in 62.2% average survival by Week 8. (Figure 4A)²⁴At the completion of the Week 8 scan, each fat graft was extracted in a single piece. A Wilcoxan rank sum test was used to compare the difference between volume measurements of fat grafts obtained by either micro-CT or calculated from physical mass. No significant difference was found between these two methods (two-sided p-value = 0.9362). (Figure .......

Discussion

Up until this point, most researchers have relied on non-imaging modalities to quantify the long-term survival of fat grafts, but these methods require the sacrifice of the study animal and yield only a single measurement. ^3,10-12 Our study represents an improved analysis method that permits objective, real-time quantification of fat graft survival in a mouse model.

Critical in this process is ensuring that sufficiently immunocompromised mice are used for the study, as this prevents.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SAL lipoaspirate
Centrifuge	Beckman Coulter, Inc., Pasadena, CA
50 ml conical tubes	BD Biosciences, San Jose, CA
CD-1 nude mice (Crl:CD1-Foxn1nu)	Charles River Laboratories, Inc., Wilmington, MA
Isoflurane	Henry Schein, Dublin, OH
2.5% Betadine	Purdue Pharma, L.P., Stamford, CT
70% Ethanol solution	Gold Shield, Hayward, CA
1cc luer-lock syringe	BD Biosciences, San Jose, CA
14 gauge cannula	Shippert Medical, Centennial, CO
Forceps	Fine Science Tools, Heidelberg, Germany
Tenotomy scissors	Fine Science Tools, Heidelberg, Germany
6-0 nylon suture	Ethicon, Blue Ash, OH
Phosphate buffered saline	Gibco, Carlsbad, CA
micro-CT scanner	Siemens Healthcare, Pleasanton, CA
Phantom	TriFoil Imaging, Northridge, CA
Imaging analysis software	IRW, Siemens Healthcare, Pleasanton, CA
Scale	Mettler-Toledo International, Inc., Columbus, OH