| After puberty or after middle age, the aging, relaxation and atrophy of the surface tissue of human body causing wrinkles. During the skin aging process, dermal fibroblasts become flat gradually, the ability of collagen synthesis decrease, the collagen continously reduce. Elastic fibers in the dermis denature, losing the elasticity, the elastic fabric of leather breed dash forward layer degrade,even disappear. The reduction of the dermal collagen synthesis and the denature of elastic fibers are the primary cause of wrinkles, relaxation and sagging skin.In recent years, with the booming cosmetic surgery, as well as the promotion of beauty standards and awareness, injection of cosmetology with its irreplaceable advantages, such as minimal invasion, remarkable enhancement, quick recovery, make the application of skin filler increasing significantly. The types of dermal fillers can be grouped into three categories:biological original fillers, synthetic filler, and the dynamic cells graft. The common clinical fillers(hyaluronic acid, collagen, etc) have their own shortcomings, such as to a limited maintain time, the occurrence of viral infection, triggering an immune response, and they can not fundamentally solve the problem of facial aging, and can not maintain a long-term effect.Since1982, the FDA approved bovine collagen can be applied to injectable aesthetic, the application of dermal fillers are increasingly common, the species has also increased the overall grouped into three categories:biological sources, fillers, synthetic fillerand dynamic cell transplantation (1). The most frequently used fillers including bovine and human collagen, hyaluronic acid, PMMA, etc. Although these fillers can quickly achieve significant improvement, but they have their own shortcomings, such as to maintain the limited time available, the occurrence of viral infection to trigger an immune response (2-4). Speaking from a deeper level, they are just fillers, only temporarily achieve the purpose of improving the appearance and does not stimulate the synthesis of collagen for skin (5), while the loss of collagen is the fundamental cause of aging, loose skin(6), these fillers are not fundamentally solve the problem of facial aging, can not maintain long-term efficacy.As we all know, the collagen is synthetized and sectioned by dermal fibroblasts, it is the main component of the extracellular matrix of skin tissue. As growing older, the number of fibroblasts in the skin is gradually reduce, the total content of collagen loss at the rate of1%per year (7,8), resulting in thinning of the skin, loss of elasticity, and ultimately the formation of wrinkles. Therefore, the increase in the number of dermal fibroblasts, to stimulate the fibroblasts secrete collagen are a fundamental solution to facial aging.In recent years, the dynamic cell transplantation is becoming the focus of facial rejuvenation. Somatic cell grafts in facial rejuvenation, not only to avoid a variety of side effects of other filler, and the survival of the cells seems to be able to play their respective role, and to radically improve aging skin. Currently there are two expected autologous cells can reach long-term wrinkle effect:autologous dermal fibroblasts (Dermal Fibroblast, DF) and adipose tissue-derived stem cells (adipose-derived stem cells, ASCs).Since1995, the in vitro cultured fibroblasts (Isolagen) had as an active, dynamic protein repair system is used to improve the aging of the skin and subcutaneous tissue defects (9). In isolagen therapy, autologous skin specimens were obtained, in vitro dermal fibroblasts, to be a certain cell of magnitude (1×107), to prepare of cell suspension graft of autologous cells and then injecting to fill the face and neck wrinkles, nasolabial or a small area of pitting scars. Since then, scholars from various countries on the cell survival after injection side effects, effects maintain time made the relevant research and made some progress. Clinical research and animal experiments show that fibroblast injection can achieve the desired lifting effect, and to maintain relatively a long time (9-14), and researches prove that it can’t result in inflammation or immune rejection, studies have shown that there is no tumor risk (10). This may be that the fibroblast cell survival, can produce a large number of normal collagen and slow the rate of loss of autologous skin collagen in order to achieve the effect of wrinkle.Because the richment of adipose tissue, the simple extraction methods, fat is becoming the source of the autologous cell graft in recent years (15). In the subcutaneous adipose tissue stromal vascular fragments containing mesenchymal stem cells, adipose tissue-derived stem cells (ASCs) with multilineage differentiation potential (16-18), and ASC can secrete a variety of cytokines. The clinical studies and animal experiments showed that autologous ASCs can promote the cure of bone trauma(19-21) or to promote the defects skin and soft tissue trauma(22-25), studies have shown that ASCs and their medium (ASCs-CM), have the ability of anti-wrinkle, skin whitening, antioxidant effect (26-29). There are also studies have shown that after autologous fat transplantation the skin was significantly improved compared with previous (30). ASCs has not yet directly used as dermal fillers like fibroblast in clinical cure of wrinkle, but it’s expected to be applied to facial skin rejuvenation treatment (26). In recent years, the platelet-rich plasma (PRP), because the concentration of autologous growth factor, has also become an autologous dermal filler. PRP is an extract of the anticoagulant whole blood, the final extract contain the most of the platelets in whole blood by two-step centrifugation. After intradermal injection of PRP, platelets are activated and release large amounts of growth factor to stimulate the fibroblast to proliferate and secrete collagen to imrove the wrinkle. PRP has been used to improve facial wrinkles or fill the nasolabial fold in clinical.As autologous dermal filler, the application of DF, ASCs and PRP in facial minimally invasive cosmetic is becoming wide. But which kind of autologous dermal filler can effect the dermis mostly,rechieve the best filling effect, the experiments with rabbit for the model will do an initial comparative study.Materials and methods1. Reagents and equipmentDMEM/F12(1:1), FBS, PBS, trypsin from Hyclone; eosin, methylene blue dye, Sirius red-picric acid dye, oil red O dye, alizarin red dye. Alcian blue dye, Dil dye, DMSO solution, type I collagenase were purchased from Sigma; rabbit type I collagen antibodies were purchased from Abcam company; sodium citrate solution were purchased from Shanghai Jiahe biotechnology Co., Ltd.; centrifuge tubes, petri dishes, straws, filters were purchased from Corning; coverslip, slide, organizational scissors, tissue forceps, syringes, filters, fluorescence microscopy, polarized light microscope, centrifuge, Sysmex kx-21blood count analyzer.2. The isolation and cultivation of fibroblasts, adipose tissue-derived stem cellAll the8New Zealand white rabbits in this experiment are bought from the Southern Medical University Experimental Animal Center. After anesthesia, the groin skin and adipose tissue of rabbits were isolated and the fibroblst,ASCs were cultured,after the amount of cells achieve1×107, prepare cell suspension,and then these autologous skin were injected into rabbits dermis.3. PRP extractionWhole blood taken from the rabbit ear vein were approximately5ml with3.8%sodium citrate for anticoagulant, at400×g centrifugal force,the blood were centrifugated by10min, the whole blood was divided into three layers:the top as the PPP, the middle layer as the "buffy coat", the lower as red blood cell. Extracting the PPP and buffy-coat layer with a straw carefully, then moving them into another centrifuge tube, at800×g centrifugal force,the extraction were centrifuged again by10min,then there were visible platelet deposition at the bottom, discarding the upper excess of PPP, the remaining serum and platelet mixing is the PRP, approximately0.5ml.4Cell marking and IdentificationRabbit dermal fibroblasts (Dermal Fibroblast, DF) cells were prepared for cell crawling piece,applying immune HRP staining techniques to identify dermal fibroblasts and the type I collagen protein synthesis is detected. ASCs were staining with oil red O for dectecting lip forming, with alizarin red for bone forming, with Alcian blue line for cartilage forming. In vitro cultured fibroblasts and ASCs were labeled with Dil dye.5. The animal experimentsThe cultured cells were prepared into a concentration of1×107cells/ml of cell suspension with an equal volume of PRP inhaled1ml syringe alternate.0.5ml of PBS as a control group. Six New Zealand white rabbits back shaving each cell of each rabbit were injected with2points, each point of injection0.1ml; Similarly, PRP, and PBS each rabbit injected with2points, each point of injection0.1ml. Injection level of the deep dermis, showing significantly uplift the skin to mark the injection points Violet. Two weeks, the points were repeated injections once. In addition, the another two rabbits were injected with Dil labeled DF and ASCs, cell concentration and injection volume were the same as the experimental group.after two weeks each place were repeated injected.6. Histological examinationEach injection site were drawn after four weeks, the sample is divided into four groups:the DF group (fibroblast injection group), group ASCs (adipose tissue-derived stem cells injection group), the PRP group (platelet rich plasma injection group), PBS group (PBS injection group). Each organization block sections were eosin-methylene blue, sirius red-picric acid, and immunohistochemical staining. Observe and compare the thickness of the skin samples, the proportion of collagen Ⅰ and Ⅲ collagen in the skin of each sample, the collagen protein expression levels. At the same time to take the type I collagen in the skin samples, using the Western blot technique (Western blot) to detect the difference of whether the expression of type I collagen protein in the sample. Dil labeled cells around the injection site, subjects, organizations block by rapid frozen section, and then observed under fluorescence microscope responsely, to identify the survival cells.7. Statistical analysisAll the data were analyzed with the SPSS13.0for statistical analysis, the rabbit dermal thickness results and the analysis of Sirius red staining were statistically analyzed using ONE WAY-ANNOVA, P<0.05was considered statistically significant differences.Results1.P3fibroblast cultured in vitro can still systhenzied type I collagen.ASCs cultured in vitro can differentiated into fat cells, osteoblasts, and cartilage cell,which can be used for injection.2.DiI labeled fibroblasts and ASCs were injected into the dermis of the back of two rabbits, after four weeks, under a fluorescence microscope to observing the material, there are still large areas of scattered red fluorescence, to prove that the cells which were injected into the dermis, can survive.3.The collagen fibers of skin tissue in experimental groups is slightly disordered compared with control group.Under microscope there can be found that spindle blue staining nuclei in the dermis, in DF group the nuclei was significantly more than other groups. Skin thickness of the DF group is the thickest, followed by ASCs group,(P<0.05), the skin thickness of PRP group and the PBS group were no significant difference,(P>0.05).4.Type Ⅰ collagen protein content in each group were no significant statistical difference (P>0.05), but in DF group and ASCs group, the type Ⅲ collagen protein content were higher than the other two groups (P<0.05), between the DF group and ASCs group and between the PRP group and PBS group there were no significant differences(P>0.05).Western Blot test results show the expression levels of type Ⅰ collagen protein in the PBS groups was lightly higher, but there were no significant difference in other groups. |
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