| [Background]In recent years following the development of rehabilitation and reconstruction surgery, artificial substitutes and autologous transplantation have become important methods for treating soft tissue defects. Although with promising prospects and encouraging results, there's limits such as short of autologous tissue, lack of blood supply and possibility of donor site morbidity, which greatly restrict the application of these new techniques. Moreover, other methods including autologous dermis transplantation, autologous fat transplantation and collagen injection, filled with artificial material et al. However each method carries considerable disadvantages: for example, synthetic materials invariably result in foreign body reactions, and free adipose tissue grafts shrink to an unpredictable extent. Althogh kinds of methods were used for the repair of soft tissue deformities, No methods was proved to be ideal. Emerging tissue engineering strategies represent an innovative potential solution to many clinical challenges.Tissue engineering involves there items: (1) seed cells, whose growth is needed to be under strict control and high proliferation is alsoessential. (2) three-dimensional scaffold structure, which is needed to be degradable and to maintain stable volume and shape. Moreover, good compatibility with seed cells is also indispensabile; (3) appropriate micro-environment, which can provide adequate blood supply and nutrition, ensure cell proliferation and functions.Previous studies have identified a putative stem cell population within adipose stromal compartment. This cell population, termed adipose-derived stromal cells (ASCs), can be isolated from adipose tissue. ASCs can differentiate toward the multiple lineages like adipocyte, Cartilage cells, muscle cells and cardiomyocyte. ASCs are easier to obtain, carry a relatively lower donor site morbidity, and are available in large numbers of stem cells at harvest. Thus, the use of adult ASCs as the seed cell of to engineer adipose tissue seems to be more inspiring.In general, Scaffolds include synthetic materials and natural materials. Several popular scaffolds like polylactic acid (PLA). polyglycolic acid (PGA), or both poly lactic-co-glycolic acid (PLGA), PTFE, Hydroxyapatite, polyvinyl alcohol, alginate etal were used to perform multiple tissue engineering. Adipose tissue engineering is a new field so only a few scaffolds were tried including the porous biodegradable materials like PLGA, hyaluronic acid, ePTFE. But no consensus was made of which materials is most ideal for adipose tissue engineering. Considering the lack of study on this field, further researches on it may be of great importance.[purpose]To induce the adipogenic differentiation in vitro by ASCs harvested from human being and to assess the possibility of constructing adipose tissue via the attachment of ASCs to typeâ… collagen scaffold and injectable fibrin glue scaffold, on the other hand, DiI was used for labeling, the effect of this kind of fluorescent dye on ASCs is also need to be detected.[Material and Methods]1. ASCs Isolation and identificationUsing the lipid coming from liposuction surgery and using enzyme digestion method for primary culture and observe changes in cell morphology and function of cells. The cells are used for experiments after the generation three. Flow cytometry was used to detect the molecular expressionwhich is associated with the surface of the stem cells such as CD29,CD34, CD44; making cells in vitro to adipogenic, osteogenic differentiation. Oil Red O staining and Alizarin red staining to identify the success of differentiation.2. Changes of biological properties of ASCs which is labeled with DiI fluorescent marker in vitroASCs was labelled with Fluorescent dyes DiI labeled in vitro, then we use inverted microscope and fluorescence microscopy to observe changes of cell growth and cellular fluorescence intensity. Then we compare the difference of Adipogenic capability before and after DiI labeling. XTT colorimetric analysis is used to detect of the rate of cell proliferation; To detected cytotoxicity of fluorescent dyes, cellular lactation of dehydrogenase (LDH) was detected.3. Construction of tissue-engineered adipose tissue using solid typeâ… collagen as scaffold1. Detection of ASCs-Scaffold in vitro biocompatibilityASC_S inoculate to a scaffold to form complexes; Discrepancy microscopy and laser scanning confocal microscope are usedto observe cell growth; XTT colorimetric analysis is used to detect of the rate of cell proliferation; To detected cytotoxicity of fluorescent dyes, cellular lactation of dehydrogenase (LDH) was detected.Adhesion rate changes and SEM observation of ASCs are detected before and after marked by DiI.2. Construction of tissue engineered adipose tissue in vivoCollagen typeâ… and dipogenic differentiated ASC_S which is marked with DiI and are mixed cultured in vitro for three days.The mixed materials are implated subcutaneously under the back of nude mice, at the same time,blank scaffold is used for control, implants are taken out for examination after 12 weeks. Weights of newly constructed tissue are calculated. In order to detect the origin of the newly constructed tissue, fluorescent observation is performed. Histological observation and Oil Red O staining are carried out to identify the newly constructive tissues.4. Construction of tissue-engineered adipose tissue using injectable fibrin glue as scaffold1. The observation of biocompatibility of Scaffold fibrin glue in vitroA (fibrinogen). B (prothrombin) comprising fibrin glue, ASC_S mixed with A, A and B into double-pumped syringe separately. mixed the two liquid through a mixture tube to form cell-gel complex in vitro.Discrepancy microscopy and laser scanning confocal microscope are used to observe cell growth. XTT colorimetric analysis is used to detect of the rate of cell proliferation; To detected cytotoxicity of fluorescent dyes, cellular lactation of dehydrogenase (LDH) was detected.Adhesion rate changes and SEM observation of ASCs are detected before and after marked by DiI.2. Construction of tissue engineered adipose tissue in vivoA Solution is mixed with adipogenic differentiated ASC_S marked with DiI, mixe A with B into double-pumped syringe. The mixed materials are injected subcutaneously under the back of nude mice, at the same time, blank scaffold is used for control on the contrary side, implants are taken out for examination after 12 weeks. Weights of newly constructed tissue are calculated. In order to detect the origin of the newly constructed tissue, fluorescent observation is performed. Histological observation and Oil Red O staining are carried out to identify the newly constructive tissues.[Results]1. The original cultured ASCs looks like tofibroblasts, but it has strong tendency for high proliferation and multiple differentiation. With the effect of adiposic and osteogenic differentiation medium, it is proved to be able to differentiate into mature adipocyte and bone cells. The differentiation can be proved by Oil Red O, Alizarin red positive staining respectively. These kind of cell (ASCs) is also proved to be CD29, CD34 and CD44 positive expression which are one of the main proof for stem cells.2. Almost all ASCs is labelled with DiI, a kind of fluorescent dye, It is proved not to destroy cells and interfere with cell proliferation. No changes of fluorescence is detected during the first week cultured in vitro, But as it was passaged to the third generation cells, intercellualr fluorescence weakens, After six passage, fluorescence was decreased to less than 30%.3. Both collagen and fibrin glue represent good compatibility and adhesion with ASCS without little toxicity; newly constructed tissue are found in both experiments. The average weight of newly constructed tissue is about 0.019g mixed with collagen scaffold and 0.028g mixed with fibrin glue respecitively. Results of HE staining and Oil Red O staining testify that the newly constructed tissue is mature adipose tissue, the positive fluorescence staining make it sure that the tissue was construted with implated human ASCs. No new tissue was found in the control side.[Discussion]The reconstruction of soft tissue defects remains a challenge in plastic and reconstructive surgery. In patients suffering from soft tissue defects, free adipose tissue grafts transplantation has been popularly applied clinically. But it is reported that there will be 40-60% volume loss during conventional grafting procedures with autologous fat tissue transplantation.Emerging tissue engineering strategies represent an innovative potential solution to many clinical challenges. Perhaps sufficient supply of autologous soft tissue can be obtained by this technique.Adult mesenchymal stem cells have been proven to be a well-established source for multiple mesenchymal tissue-forming cell lineages, including adipocytes. Fat tissues have been reported to contain such multipotent cells. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Previous studies have identified a putative stem cell population within the adipose stromal compartment. This cell population is termed adipose-derived stromal cells (ASCs).ASCs are easier to obtain, carry a relatively lower donor site morbidity, and are available in large numbers of stem cells at harvest. Thus, the use of adult ASCs to engineer adipose tissue implants seems to be more inspiring.Adipose derived stem cells can successfully differentiated into mature adipocyte exhibiting a adipose-like morphology and expressing of intracytoplasmic lipid droplet. While attached to collagen I scaffold and transplanted under the skin of nude mouse, new formed adipose tissue is found at a fairly satisfactory volume. These results indicate that only adipo-ASCs can be used as the cell source for future adipose tissue engineering.In plastic and reconstructive surgery, minimal post-operation scars are ideal. The conventional sheet- or sponge-type scaffolds leave scars when transplanted via incision. We successflully use fibrin glue as scaffolds, which is injectable and also provide appropriate rigidity in our last part. The newly formed tissue is more inspiring. Thus, injectable fibrin glue scaffold is more valuable in vivo adipose tissue formation.[Conclusion]Adipose derived stem cells can be successfully obtained from the human liposuction extacat with enzymatic digestion. While attached to both solid collagen I scaffold and injectable fibrin glue scaffold, and then transplanted under the skin of nude mouse, new formed adipose tissue is found at a fairly satisfactory volume. Injectable fibrin glue scaffold represents inspring advantages like scarless, minimal invasive and plastic, which shows more value in vivo adipose tissue formation.
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