Daily Suction Provided By External Volume Expansion Inducing Regeneration Of Grafted Fat In A Murine Model

BackgroundFat grafting is currently considered the standard procedure for soft-tissue defection. Because of its major advantages:a wide range of sources, easy to obtain, low immunogenicity, etc., fat grafting has been more than 100 years of history. Although fat grafting has obvious effect, it always associated with induration, cyst, calcification and unpredictable long-term graft retention, commonly varying between 20% and 70%, which remains a substantial challenge for both surgeons and patients. The unprediction of the final volume of fat retention limit its clinical application.With the aim of overcoming these limitations, researchers have recently devoted much effort toward optimizing the procedure. Nonsurgical breast enlargement using an external soft-tissue expansion system was first reported in 2000 by Khouri et al. More recently, Khouri and colleagues have successfully developed a more attractive approach, Brava-assisted autologous fat grafting, for esthetic augmentation and breast reconstruction in patients after lumpectomies without requiring surgical intervention. Using Brava, the mean volume of the autologous fat tissue grafted was 346 ml per breast, while the post-operative mean volume was 266 ml per breast, which indicated that graft retention was higher. Growing number of clinical trials showed that using external soft-tissue expansion system assisted fat grafting was a safe, effective and economial technology, which brought hope to solve the problem of low fat retention rate after transplantation.The application principle of Brava is an external soft-tissue expansion system to provide negative attraction. Some animal studies have indicated that application of external volume expansion could increase cellular proliferation of the recipient site, promote the formation of new vessles and survival of adipocytes, stressing the importance of autologous fat grafting with pre-expansion. Finally, the retention rate of grafts increased. So far, evidence from both animal and clinical trials showed that external soft-tissue expansion system assisted fat grafting can significantly improve the retention rate of grafts. At present, more in-depth researches on mechanism of pre-expansion of recipient site before the fat grafting, while, few studies on the mechanism of external soft-tissue expansion system after the fat grafting. Then, what is the effect on the regeneration of adipose tissue by suction? A large number of in vitro experiments indicated that mechanical action like tension can change the ability of cell proliferation and differentiation. But one problem is whether suction could influence the proliferation and differentiation of cells in order to reconstruct vascular system and promote adipogenesis in vivo? There is still a lack of compelling experimental evidence. There are several problems to be solved:(a) How to establish a stable and effective suction-assisted fat grafting model? (b) Suction assisted fat grafting compared with fat grafting alone, in the transplantation process, what’s the changes in the origins and biological behaviors of regeneration-related cells (e.g., endothelial cells, macrophages, adipose precursor cells) in free fat after transplantation? (c) What’s the source and the morphological changes of new adipocytes in the final grafts under suction? (d) Analysis on the molecular level, what consequences would the suction have for the expression of regeneration related transcription factor?To address these questions, in this study, we fabricated a modified device to mimic external soft-tissue expansion system used in clinic that provides a three-dimensional array of mechanical force (i.e., vacuum distraction force). The device was tested in two animal models:a suction model (based on Heit et al.’s protocol) and a fat exchange transplantation model (based on Dong et al.’s protocol). To observe morphological changes of grafted fat assisted with suction or fat transplantation alone using Magnetic Resonance technology. At each time point, the weight of mice, mass measurement of grafted fat, analysis of HE staining, immunohistochemistry and western blot technology were used to evaluate the effect of fat grafting between two groups. The aim of this experiment is to explore the mechanism of regeneration of grafted fat induced by daily suction. The study results provide important experimental basis for the clinical application of fat grafting, and their contribution will be of great significance.Objective1、Comparative analysis of suction assisted fat grafting and pure fat grafting. To explore the dynamic change of regeneration-related cells.2、To explore the origins and morphological changes of new adipocytes in grafted fat under suction.3、Comparative analysis of the expression of regeneration-related protein (PPARy).4、To explore the potential mechanisms of fat regeneration induced by suction after fat transplantation.Methods1. Animal ModelsThe source of animal in the experiment:Eight-week-old male C57BL/6 mice (B6 mice) and C57BL/6-Tg (CAG-EGFP) mice (GFP mice) were respectively obtained from Southern Medical University, Guangzhou, P, R. China and Model Animal Research Center of Nanjing University, Nanjing, P, R. China.1.1 Fat transplantation modelThe mice were anesthetized with intraperitoneal pentobarbital (50 mg/kg). Inguinal hair on the back of 8 weeks old C57BL/6 male mice was cut short, in which to unhair the required parts by depilatory creams with tampon. The inguinal skin of C57/BL6 mice was incised and the subcutaneous inguinal fat pad (150-200 mg) was harvested and gently dissected into small pieces, similar to the size of aspirated fat tissue used in clinics. Then, the GFP-free fat tissue obtained was injected into the subcutaneous tissue in the backs of GFP mice. This procedure was performed in 55 GFP mice, which were evenly divided into two groups at random:those treated with or without suction. All GFP mice were killed, and the grafted fat was harvested after 0 (n= 5),1,2,4,8, and 12 weeks (n= 5 at each time point per group).1.2 Suction modelThe suction device is composed of three parts:vacuum pump, connecting device, animal fixing device. The dome-shaped devices with a diameter of 1.5 cm and an internal volume of 1.5 ml were obtained by 3D-printing of a photosensitive resin material, known to be non-toxic to mice. Animal fixed device is a cylindrical device, the size of which slightly larger than that of mice. At the top of this device is a rectangular window with the size of 5* 2cm for connecting dome-shaped device. The fat grafts in the backs of mice were connected to the suction pump (low-vacuum suction unit DYX-1A, SMAF Corp., Shanghai, P.R. China) at a pressure of-3 kPa (about-23 mmHg) through a rubber hose. This pressure setting was chosen, as this is the standard rage used with Brava in clinical settings. Mice in the experimental group were treated with suction for 10 hours/day at a fixed time for 4 weeks. After cessation of suction, the mice were observed continuously until week 12. Grafted samples were fixed in 4% paraformaldehyde for 24 h, and then dehydrated and paraffin-embedded for histological examination.2、Comparative observation of morphological changes of fat tissue using Magnetic Resonance Imaging (MRI)MRI scans of the treated area were performed on mice in the suction group by using a scanner (Philips Achieva 3.0T). The mice were anesthetized with intraperitoneal pentobarbital injection (50 mg/kg), and kept warm during the procedure. Thin-section T1- and T2-weight images (WI) of the treated area were obtained.3、Gross and Histological ObservationDigital images were captured to display the macroscopic results at each time point. Samples, sectioned serially at 5 μm, were stained with hematoxylin and eosin according to standard protocols. Results were acquired with an Olympus BX51 microscope and photographed using an Olympus DP71 digital camera (Olympus Crop., Tokyo, Japan). Neovascularization was assessed by counting the number of blood vessels in 10 fields per slide (40 magnification). All data were collected and analyzed by two independent, blinded observers.4、Observe the changes of regeneration-related cells in the graft using immunohistochemistry technologyAfter preparing 5-μm-thick sections of samples, we performed immunostaining with the following primary antibodies:chicken anti-GFP (dilution 1:200; Abcam, Cambridge, UK), rat anti-mouse CD34 (dilution 1:300; Abcam), guinea pig anti-mouse perilipin (dilution 1:400; Progen, Heidelberg, Germany), rat anti-mouse F4/80 (dilution 1:25; Abcam). For double-fluorescence staining, the following secondary antibodies were applied as appropriate:Alexa Fluor 488-conjugated goat anti-chicken immunoglobulin Y (dilution 1:200; Thermo Fisher, Australasia), Alexa Fluor 555-conjugated donkey anti-rat immunoglobulin G (dilution 1:200; Abcam), Alexa Fluor 647-conjugated goat anti-guinea pig immunoglobulin G (dilution 1:200; Abcam). Nuclei and new capillaries were stained with DAPI (dilution 1:200; Sigma, St. Louis, MO, USA) and Lectin594 (dilution 1:200; Invitrogen, Carlsbad, CA, USA), respectively. The number of new capillaries (Lectin+) was counted in at least three field images for each sample.5、Detect the expression of regeneration-related protein (PPARy) in grafted fat using the Western blot technique.In order to detect adipogenesis of the fat grafts at the molecular level, we performed the western blot technique for a semi-quantitative analysis of the expression of peroxisome proliferator-activated receptor (PPAR), a transcription factor that induces adipogenesis. The primary antibody was rabbit anti-mouse PPAR (dilution 1:500; Abcam), and goat anti-rabbit immunoglobulin G (dilution 1:8000, Sangon Biotech Co. Ltd, P.R. China) was used as the secondary antibody. In addition, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (dilution 1:5000, Bioworld Technology, Co., Ltd., P.R. China) was used as the internal reference.6、Statistical AnalysisAll data were analyzed with IBM SPSS 20.0 software. Because the initial weight of the mice could not be controlled, factorial analysis of covariance was performed to identify the difference between different groups and times using body weight as a covariate. The body weights of the mice and the normalized sample weight are presented with the original and modified mean±S.E, whereas the other data are expressed as the mean±SD. Furthermore, an independent t-test was performed to compare data between the two groups at any given point. A value of p < 0.05 was considered significant.Results1. MRI results showed that the graft confirmed as fat tissue and good fixation under suction surrounded with substantial jelly exudate.2. The total retention of grafted fat under suction was higher than that of controls at weeks 1,4,8, and 12. At 12 weeks after transplantation. The grafts’normalized sample weight in two groups were 0.060 ± 0.001,0.001 ± 0.046, respectively.3. Blood supply increased from new host-derived capillaries or macrophage infiltration under suction.4. CD34+cells, key regeneration cells, showed increased migration from the host into the grafts under suction.5. At week 12, nearly half of the mature adipocytes regenerated in the grafts in the experimental group was derived from the host.6. PPARy expression of the experimental group was significantly higher than that of controls at days 14 and 28 during adipogenesis.Conclusions1. This study observed the grafted fat between two groups at tissue, cell and molecular level. The mechanical effect of suction favors the regeneration of grafted fat and improves retention by promoting the migration of regeneration-related cells, the formation of new blood vessels and the differentiation of adipocytes. Thus, more mature fat tissue with a well-organized structure was formed under daily suction.