| Background and ObjectionIt has been hundreds years of history for using autologous fat transplantation to repair tissue defect and dysplasia. Advantages of autologous fat include rich sources,easy and simple operation, safe and without rejection, natural appearance and tactility, etc. As the most ideal soft tissue filling material, asutologous fat has been wildly used for almost all parts of the soft tissue depression and defection, especially in the field of cosmetic surgeries recently. With the invention of liposuction technology, comes the new technique of autologous fat transplantation----Particle fat injections transplantation. With improvements of the technique, indications of particle fat injections transplantation is also expanding, such as facial deformity, sunken scars, augmentation, post-traumatic defection and vocal cord paralysis, Which representative is breast augmentation and facial rejuvenation.Advent of particle fat injections transplantation technology makes a large number of autologous fat graft possibly, bring a hope to the plastic surgeon for soft tissue filling. Unfortunately, the low survival rate and complications after transplantation of particle fat such as calcification, necrosis, tuberous sclerosis, liquefied cyst and other restrictions, limit the development of particle fat injections transplantation in clinical. Therefore, how to reduce the absorption rate of the particle far after transplantation to improve retention rate, so as to control the effect, is a major problem of plastic surgery clinical application and theoretical study. Over the past decade, many scholars researched from fat of different areas selection, methods of fat acquisition, ways of fat purification and manners of fat injection; from cytokine therapy, gene therapy, cell therapy and other aspects to improve transplanted fat survived. All studies done with have achieved certain results in promoting angiogenesis graft, to prevent or mitigate fibrosis, improve survival, but for saving rate distinction resulting from the different implantation sites is still lacking.Through in-depth research to understand the physical structure of adipose tissue, we find out that adipose tissue have a rich blood supply, each cell has an exclusive blood supply that connect with the trunk to ensure enough nutrient, forms a structure like a bunch of grapes. It will make certain damage to adipose cell during the process of particle fat transplantation, such as acquisition, collection and transplantation. The serious damage of capillaries within adipose tissue was happened so that only a small part of the residual of the blood vessel segments. At the same time, the fat cells also exist some injury. Finally, adipose cells can only get nutrient supply from recipient site. Early stage after fat transplantation, there is not a good blood supply with the recipient site, the nutrition of adipose cell can only supply by the penetration of interstitial fluid. However, interstitial fluid penetration would penetrate only about 150-200 microns. Therefore, necrosis of adipose cells beyond the penetration region due to ischemia and hypoxia, which affects the final long-term retention rate of fat transplantation. Thus, early timely revascularization of grafted particle fat, form a good blood supply is the key to cell survival. Some previous experiments from our team found that 1、Histology source is mainly from host during later stage of fat transplantation.2、Vessel fragments derived from particle fat transplantation can’t participated in the reconstruction of blood supply by anastomosis with vessels of host, and the endothelial cells almost completely died at two weeks after transplantation, the reconstruction of blood supply of the adipose tissue block after transplantation is mainly derived from the angiogenesis of the host.Therefore, vascular conditions by recipient site play an important role in the process of grafts survival.Fat transplantation area involves different tissues anatomy and blood supply conditions. Fat grafting effects in different anatomical level, such as subcutaneous, intramuscular, animal experiments and clinical studies has long been reported. As early as Guerrerosantos in 1996, used 120 rats for fat transplantation to hip, the hindlimb muscle and subcutaneous of forehead,compared effects 3-12 months, intramuscular fat graft was successful. Karacaoglu experiments used rabbit for fat volume after transplantation, compared subcutaneous recipient site with muscles recipient site above and beneath, the masseter saving rate were about 82% six months after, he thought by selecting recipient area can increase fat graft survival. But Rieck,by detecting different anatomical levels of the survival rate of transplanted fat (survival rate:subcutaneous 30%, intramuscular 6%), discovered that intramuscular injection does not increase survival rate. The reason may be:Although muscles have a abundant blood supply, but the space between muscles is limited, tension and pressure when muscle contracts will squeeze transplanted fat, but reduce the blood supply.Clinically, when grafted in the face, the fat was recommended to be placed in the subcutaneous or muscular layer. As for the breasts, Yoshimura et al. considered the spaces anterior and posterior the mammary gland as potential sites for successful autologous fat grafting, based on the rich blood supply and the simplicity for breast shaping, and Coleman et al. pointed out that the fat graft could also survive well within the pectoralis major. However, the glandular tissue was demonstrated by Illouz in 2009 to be in favor of fat grafting, due to its volume and great perfusion. Illouz also argued that the retromammary space was less ideal, because its blood supply is relatively poor. Despite of the controversy, there is currently no solid evidence showing which layer is superior in the survival of fat graft. In order to identify the ideal recipient site, to assess the outcomes with more objectivity, and to improve the long-term outcome of fat grafting, further studies are needed to investigate the difference of the preserving rate among these sites.Based on the above question, Fat harvested from inguinal fat pads of GFP mice was grafted into the subcutaneous and intramuscular planes and the fat pads of C57 mice. Specimens collected after 1,4,8,12, and 16 weeks were stained with hematoxylin and eosin to determine angiogenesis and fibrosis in the grafts. The survival rate of donor adipose tissue was determined by measuring GFP expression. To determine the optimal recipient layer for fat transplantation.Methods and materialsC57BL/6 and GFP C57BL/6 mice. Fat was harvested from the inguinal fat pads of GFP mice and gently dissected into very small pieces, similar to the size of aspirated fat tissue used for clinical fat injection in humans. GFP mouse fat grafts were placed under the dorsal dermis of C57 mice using a 16-gauge cannula, or were injected into the biceps femoris muscle or the inguinal region fat pads of C57 mice using a 16-gauge needle For each experimental condition,0.1 ml of prepared adipose tissue was deposited per location. a、Particles fat of GFP mice transplant to C57 mouse’s dorsal dermis. b、Particles fat of GFP mice transplant to C57 mouse’s biceps femoris muscle. c、Particles fat of GFP mice transplant to C57 mouse’s inguinal region fat pads.1、wet weight measuredThe weight of 0.1 ml prepared adipose tissue served as the wet weight baseline for fat grafts. Fat grafts were harvested from each of seven mice in each group 1,4,8, 12, and 16 weeks after fat transplantation. Grafts from one side were carefully separated from surrounding tissue, and their wet weight measured (n=7). The retention of wet weight was calculated as the ratio of graft wet weight to baseline wet weight.2、Histological examination, capillary density measuring and connective tissue quantificationGrafts from all three groups were fixed in 4% paraformaldehyde, dehydrated, and paraffin-embedded for hematoxylin and eosin (H&E) staining. Tissue blocks were serially sectioned (5μm sections) along the longitudinal axis and assessed under an Olympus BX51 microscope and photographed using an Olympus DP71 digital camera. Neovascularization was assessed by counting the capillaries in 20 fields of each H&E slide (40x magnification). Counting was performed by two blinded investigators.3、GFP expressionGFP expression in frozen sections was examined signal by fluorescence microscopy (Olympus, Japan). Images at 100x magnification were merged into a panoramic image for each graft, and the percentage of total area positive for GFP was measured using ImageJ software.4、Statistical analysis.The data are expressed as means±SEM and analyzed by repeated measures analysis of variance. If the differences were statistically significant, the three groups were compared at one time point using an independent Student’s t-test, and each group was compared at all time points by one-way analysis of variance (ANOVA). A P value < 0.05 was considered statistically significant.Results:1、Capsules were present in all three groups, with no obvious differences in gross appearance after 1 week. The graft volume of the fat pad group was slightly bigger than that of the two other groups at 4 weeks, whereas the intramuscular group had the most vessels surrounding the face of the grafts. Graft volume at 16 weeks was highest in the fat pad group, with these grafts having the most normal appearance.The retention of wet weight was calculated as the ratio of graft wet weight to baseline wet weight. This ratio decreased dramatically in all three groups after fat transplantation, especially at 1-8 weeks, but leveled off at 8-16 weeks. Interestingly, the retention ratio was highest in the fat pad group (P<0.05) and was significantly higher in the subcutaneous than in the intramuscular group at 8-16 weeks (P<0.05).2、The most necrotic areas and disorganized structures were observed in the subcutaneous group, while the least necrotic areas and most intact structure were observed in the intramuscular group. At 4 weeks,Vascularized connective tissue and small size adipocytes were present in all grafts at the same time, but were lowest in the subcutaneous group. At 16 weeks, the graft structure in the fat pad and intramuscular groups was very similar to that of normal adipose tissue. The intramuscular group showed no obvious fibrosis or oil droplets and had the thinnest capsules. Moreover, the diameter of mature adipocytes was significantly smaller in the intramuscular than in the two other groups.Quantification of blood vessels indicated that capillary density increased similarly and significantly in all three groups at 1-4 weeks, but slightly decreased during 4-16 weeks. From 4 to 16 weeks, capillary density was significantly higher in the intramuscular than in the other two groups, but there were no statistically significant differences between the subcutaneous and fat pad groups. The change in fibrosis in the three groups was similar to their change in capillary density. The area of connective tissue was higher in the intramuscular group than in the other two groups at 4,8, and 12 weeks.3、This One week after transplantation, most of the grafted area remained GFP+, with only the tissue located in the central zone being GFP-. Over time, the GFP-area expanded from the central zone to the outer layer of the graft, with the GFP+area located at the edge of the graft at 4 weeks. At 16 weeks, almost the entire graft was GFP-, with only a slight area located at the edge of the graft being GFP+. However, the GFP+area was significantly larger in the intramuscular than in the other two groups. Semiquantitative results indicated that the GFP+area decreased in all three groups from 1 to 8 weeks, but decreased slightly at 8-16 weeks. The GFP+area was larger in the intramuscular than in the other two groups, beginning 4 weeks after transplantation (P<0.05). At all time points, however, there were no statistically significant differences between the subcutaneous and fat pad groups.DiscussionsThese findings indicate that the retention rates of injected fat were greatest for fat pad injections than for subcutaneous injections and were greater for the latter than for intramuscular injections. In addition, graft angiogenesis and the amount of vascularized connective tissue soon after fat transplantation were highest for intramuscularly injected than for subcutaneous and fat pad injected grafts. Furthermore, the survival rate of donor adipose tissue was highest for intramuscularly injected than for subcutaneous and fat pad injected group grafts.The mechanism of graft retention was recently shown to conform to a three zones theory, consisting of a surviving area (the outer layer), a necrotic area (the central layer), and a regenerating area (the layer between the outer and central layers). Long-term graft retention depends on two vital factors:the survival of the graft and regeneration by the host. To survive, fat grafts require tissue perfusion and nutrient supply soon after transplantation. The vascular nature of muscle increases the survival of intramuscularly injected fat grafts. In addition, a comparison of submuscular, supramuscular, and subcutaneous fat injections found that survival was greatest for supramuscular injections. Evaluation of graft angiogenesis and tissue regeneration found that intramuscular injections resulted in better revascularization and more vascularized connective tissue, with a larger GFP+graft survival area.Interestingly, graft retention rate was highest in the fat pad group, whereas the retention rate of grafts was lowest in the intramuscular group, which had the best vascular environment. Two possible reasons may explain this apparent discrepancy. First, the survival area of fat grafts is very small, having little influence on long-term graft retention rates. Adipose tissue has a very low tolerance to ischemia, with the viable zone restricted to the peripheral area, located<300μm from the edge of the fat graft. Muscular tissue with better vascularization promotes the survival of fat grafts, similar to our finding that GFP+area was greatest in the intramuscular group. However, the long-term graft retention rate is determined by later adipose regeneration. Tissue-resident or host original stem/progenitor cells (such as ASCs) play an important role in adipose regeneration after fat transplantation. Moreover, the niche environment determines the fate and activity of ASCs. An appropriate ASCs niche is therefore crucial for adipose regeneration after fat transplantation, explaining in part why intramuscular injections showed poor graft retention rate even with a better blood supply.Second, the differentiation of stem/progenitor cells depends on the microenvironment. By appropriately manipulating the chemical and mechanical environments, the stem/progenitor cells can become dysregulated and progress preferentially towards an adipocyte lineage. Intramuscular injections provide sufficient blood supply for fat grafts, but do not provide an appropriate adipogenic microenvironment. Moreover, the mechanical force induced by muscle movement may inhibit the adipogenic differentiation of stem/progenitor cells. Injection of free fat plus botulinum toxin A was found to significantly reduce the level of fat graft resorption. Various cells in adipose tissue, especially ASCs, are mechanosensitive and mechanoresponsive. Static stretching or low tension has been found to promote adipogenesis, whereas dynamic mechanical forces such as cyclic stretching or vibration and static compression inhibit adipogenesis. Not only can mechanical force influence differentiation, but also substrate stiffness can direct the specific differentiation of mesenchymal stem cells. This may explain why the diameter of mature adipocyte was significantly smaller in the intramuscular than in the two other groups at 16 weeks after fat transplantation. Our findings suggest that graft retention rate in response to intramuscular injection is associated with muscle movement, with more movement correlated with lower retention rate.The graft retention rate was higher for the fat pad than for the subcutaneous group, although the capillary density of these two groups did not differ significantly. The difference in graft retention rate may be due to differences in chemical and mechanical microenvironments, which play a key role in adipose regeneration.Conclusion1、Fat was retained longer in fat pads than in subcutaneous layers of recipient mice and longer in subcutaneous than in intramuscular layers. Fat pad injection showed the highest graft retention rate, suggesting that areas of fat are the optimal recipient areas for fat transplantation.2、Angiogenesis and vascularized connective tissue were greater in intramuscular than in subcutaneous grafts or fat pads. Neovascularization, however, was similar in fat pads and subcutaneous grafts.3、Survival rate was higher for intramuscularly injected than subcutaneously and fat pad injected adipose tissue. |
PDF Full Text Request