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.
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.
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
Despite these advantages, autologous fat grafts suffer from unpredictable and variable survival, with retention rates ranging anywhere from 10-80% over time.1-3,9 In order to account for this expected loss of volume and symmetry, surgeons must often overcorrect when filling soft tissue defects, or perform multiple follow-up procedures.
Poorly vascularized graft beds are partly to blame for this tissue resorption. Additionally, the lack of a benchmark analysis method to compare graft survival may also contribute to the inconsistency in reported results. A precise method for measuring graft volume would reduce measurement error when evaluating retention rates. This in turn would help researchers more accurately identify the causative factors that affect graft survival. Although many laboratory animal models have facilitated both quantitative and qualitative assessment of human fat graft survival, most are based on histological and biochemical means and require sacrificing the study animal to yield a single measurement.3,10-12 Little has been reported on the use of imaging techniques to enumerate fat graft volume retention in vivo.
A handful of clinical studies have shown more effective measurement techniques using imaging. Magnetic Resonance Imaging (MRI) was employed by Hörl et al. to measure fat graft survival13, and CT was utilized by Har-Shai et al. and Fontdevila et al. in their analyses of volume retention after grafting in patients who suffered from HIV.14,15 Employing three-dimensional (3D) imaging software, Meier et al. measured volume retention in humans after autologous fat grafting by comparing images from the preoperative and postoperative period.16
Yet, a standardized method employing imaging to measure fat graft survival is lacking in basic science research. A high resolution imaging approach for assessing the volumes of fat grafts would allow not only for accurate and reproducible volume measurements, but also for repeated measurements allowing visualization of the evolution of fat graft survival in a real time fashion.
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
2. Fat Grafting
3. Micro-CT
4. Micro-CT Analysis
5. Fat Harvest
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 4B)
With 5 centiGy per scan and five scan time points, each mouse received no more than a total of 25 centiGy over the course of the study. Consistent with this, none of the mice displayed any gross evidence of cutaneous radiation burns.
Figure 1. (A) A 14 G cannula on a 1 ml syringe, positioned in the midline and at the rostral-most aspect of the pocket prior to beginning fat injection. (B) Nude mouse upon completed fat grafting, with a single nylon suture used to bring wound edges together. The pocket has been filled but is not tense.
Figure 2. (A) The reconstructed images initially displayed in axial, coronal, and sagittal views. (B) Using the axial view as a guide to navigate to the left most aspect of the graft on sagittal view. Setting a threshold for pixel intensity so that all voxels selected within the threshold range will represent adipose tissue, thus allow for the demarcation of the fat graft volume. (C) ROI’s defined on sagittal view starting at the left most aspect of the graft and continued every fifth slice moving to the other end of the graft. (D) All selected voxels from 2D ROI’s interpolated into a single 3D ROI. (E) A three-dimensional surface was created using cubic-spline interpolation to visualize the total fat graft volumes.
Figure 3. (A) Fat graft before explantation, with dorsal patch of skin removed. (B) Fat graft after explantation.
Figure 4. (A) Micro-CT volumetric analysis demonstrated gradual loss of fat graft volume over eight weeks. (B) Final fat graft volumes, as measured by micro-CT, corresponded closely to volumes calculated from the masses of explanted fat grafts. The average density of human fat (0.9 g/ml) was used as a conversion rate.
Table 1. Calculated Micro-CT Volume vs. Actual Measured Fat Volume24
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 the graft rejection that would occur if mice with intact immune systems are used. Preserving fat integrity is critical during the harvest, processing, and placement phases of grafting. In accordance with traditionally accepted standards, fat for grafting should be obtained by suction assisted liposuction (SAL). During placement, fat should be injected at a steady flow rate no faster than 0.5 ml/sec. A 14 gauge cannula is preferred for grafting in a mouse, but larger diameter cannulas may be used without any injury to the fat. Smaller cannulas and needles—especially those narrower than 16 gauge—are discouraged during placement as they may cause the fat to breakdown due to increased shear stress. Although we describe our preferred technique for processing above, any combination of sedimentation, centrifugation, and/or filtration may be used as long as the oil and blood layers are adequately separated out from the fat prior to grafting.
Fat grafts should be at least 200 µl in size to minimize variance in results due to the inconsistent nature of fat grafting. Larger grafts up to 400 µl in size may be used, but above this volume, impaired vascular supply and excessive skin tension may result in fat and skin necrosis. Ultimately, maximum fat graft size will be determined by surface area and volume of the pocket. To increase the volume of a graft that can be safely delivered, the pocket can be expanded by more extensive dissection. However, this may place fat beyond the boundaries of the top of the skull, which will make the contrast between the graft and surrounding tissue less clear. Hence, subsequent voxel selection will become more difficult.
If initial skin incision is small enough, a suture may not be needed as long as fat remains contained and is not seen leaking out of the pocket. If a suture is placed, care must be taken not to tie the first knot too tightly, otherwise skin breakdown may occur. A non-absorbable monofilament suture such as nylon is preferred, as it limits the inflammatory reaction and is less likely to harbor infection. Re-epithelialization of the incision will occur within 24 to 48 hr post-operatively, and the suture may be removed at this time. Absorbable and braided sutures should not be used. Skin should always be handled with the least force necessary, and the surgeon should take care not to crush the skin while holding up wound edges.
Depending on the investigators’ imaging analysis software, the exact relationship between pixel intensity and the density of tissue may vary. Investigators should choose pixel intensity thresholds to obtain a maximum and minimum range that most accurately distinguishes the fat graft from the surrounding tissue. The same maximum and minimum threshold values should be used during all volume analyses to maintain consistency.
There are multiple methods to select graft volume once pixel intensity threshold values have been set. Although we find painting with a brush tool in the sagittal view best in our hands, other voxel selection methods to create an ROI can be used such as drawing with the spline tool or painting in the axial view. It is preferable if a single person performs all volume analyses as consistently as possible in order to reduce measurement error.
The non-invasive nature of this method and the real-time visualization of graft evolution offer significant advantages over traditional techniques. However, this technique is limited in its ability to identify the viability and health of remnant grafts. Furthermore, it cannot demonstrate relative revascularization of grafts. Though changes in appearance and graft density may hint at fat necrosis, infection, cyst formation, or liquefaction, it is difficult to draw exact conclusions from micro-CT alone.
We hope this technique will serve as a foundation upon which future studies can be conducted to better understand the causative factors in fat graft survival and loss. Variations on this theme may elucidate the role that stem cells, growth factors, cytokines, genes, and cell surface markers play in the ultimate preservation of fat graft volume. With this improved tool to test contrasting hypotheses, we look forward to a better understanding of fat transfer that transforms a capricious technique for treating soft tissue deficits into a more predictable one.
The authors have nothing to disclose.
This study was supported by the Oak Foundation, the Hagey Laboratory for Pediatric Regenerative Medicine, and the National Institute of Health, Grants NIHR21DE019274, NIHR01DE019434, NIHR01DE021683, and NIHU01HL099776 to M.T.L. D.C.W. was supported by the ACS Franklin H. Martin Faculty Research Fellowship, the Hagey Laboratory for Pediatric Regenerative Medicine, and the Stanford University Child Health Research Institute Faculty Scholar Award. Micro-CT was conducted at the Stanford Center for Innovation in In Vivo Imaging.
Reagents and Materials | Manufacturer |
SAL lipoaspirate | N/A |
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 |