Experimental Research Of Constructing Tissue-engineered Fat Tissue With Dedifferentiated Adipocyte

Background and ObjectiveIn 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.In recent years, the adipose derived stem cells (ASCs) from liposuction have become a hot spot. But ASCs is a mixed populatione residing within adult adipose tissue.Only about 40% of them can differentiate into adipocytes.So it can not satisfy the development of adipose tissue engineering,too.It is still a hot spot of finding a better kind of cells which is isolated easy, highly expandable in culture and highly differentiatable to adipocytes.Adipose tissue is heterogeneous. It contains fully mature adipocytes, vascular endothelial cells, fibroblasts, histiocytes and stem cells et al. Large lipid accumulations make mature adipocytes naturally buoyant and therefore difficult to culture. Little research has been conducted on the nature of them.They have also been considered to be in the terminal stage of differentiation and lacking proliferative activity.Recently,studies suggested that mature adipocytes could be induced to undergo lipolysis,reinitiate proliferative mechanisms and begin to proliferate in vitro. We have termed this physiological transition, adipocyte dedifferentiation.We think that this may prove to be an emerging area of cell physiology and may amend present thinking about tissue renewal capabilities.However, study regarding the cell biology of the dedifferentiated adipocytes (DA) are still scarce.Preliminary study has proved that dedifferentiated adipocytes could differentiate toward the multiple lineages such as adipocyte, chondrocyte, and osteoblast, and had a stronger adipogenic capacity than ASCs. Therefore, DA has a probability to become a kind of more excellent seed cells source for fatty tissue engineering.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.We investigage the gene levels during the induction of adipogenic and osteogenetic differentiation in vitro about the DA which were harvested from adult human.At the same time, to assess the possibility of constructing adipose tissue via the attachment of DA to type I collagen and injectable fibrin glue scaffold. on the other hand, AD.EGFP was used for labeling, the effect of this kind of fluorescent dye on DA is also need to be detected.METHODS1.DA, ASCs Isolation, cultivation and real time PCR analysisMature adipocytes and ASCs were harvested from human fat aspirates via liposuction. Mature adipocytes were cultured and induced to DA by ceiling adherent culture method. Making cells in vitro to adipogenic, osteogenic differentiation. Oil Red O staining and Alizarin red staining staining to identify the success of differentiation. Real time PCR analysis the mRNA levels of PPARγ,C/EBPα,C/EBPβ,Leptin,LPL RUNX2,SOX9. 2.Transfection and marker of EGFP in DAThe DA was infected with adenovirus expressing the EGFP gene by different multiplicity of infection (MOI=0,10,20,30,50,100). The expression of EGFP was detected by fluorscent microsecope and inverted microsecope. The cytotoxicity of adenovirus to DA was evaluated by cell proliferation,cell vitality and cell differentiation.The adipogenic defferentiational ability of DA after labeled was detected by oil red O staining. MTT colorimetric analysis is used to detect of the rate of cell proliferation.3. Biocompatibility of DA attached with typeⅠcollagen sponge,fibrin glue scaffold in vitroDA after GFP labelling attached to the typeⅠcollagen sponge scaffold or fibrin glue scaffold to form compounds respectively, then contrast phase microscope was used to observe cells growth, adhesion or expansion. Whereafter, we observed the cells disposition and proliferation under scanning electron microscope (SEM) in vitro. We determine the adhesion rates of DA marked by GFP. MTT colorimetric analysis is used to detect of the rate of cell proliferation. The adipogenic defferentiational ability of DA was detected by oil red O staining attached with typeⅠcollagen sponge or fibrin glue scaffold.4,Construction of tissue engineered adipose tissue in vivoCollagen typeⅠand fibrin glue mixed with adipogenic differentiated DA or ASCs which is marked with GFP respectively. 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 HE staining are carried out to identify the newly constructive tissues. The capillary density of transplanted the engineered adipose tissue was detected by blood vessel counting.RESULTS1. DA growed appearance as fibroblasts-like, but it has strong tendency for high proliferation and multiple differentiation. With the effect of adipogenic and osteogenic differentiation medium, it is proved to be able to differentiate into mature adipocyte and bone cells. Adipogenic differentiation of ASCs was assessed by Oil Red O staining after 2 weeks and osteogenic induction After 2 weeks, cells were positively stained by alizarin red.Before induction,the gene levels of PPARγ,C/EBPβin DA were higher than its in ASCs,but the gene levels of RUNX2. SOX9 in DA were lower than its in ASCs.After adipogenic differentiation,the expression of PPARγ,C/EBPα,C/EBPβ,Leptin,LPL in DA were always higher than ASCs,and there was a bigger rate of increase in DA. After osteogenic differentiation, the expression of RUNX2 in ASCs was always higher than DA.2. DA were successfully infected by adenovirus. EGFP was initially expressed in DA of all transfected groups 24h following infected by Ad.EGFP. The level increased with the incease of MOI and reached peak on 5d. Transfection efficiency of the Expression were 0%(0); 11.3%(10); 28.5%(20); 43.7%(30); 95.8%(50); 96.3%(100), respectively. During the period of the culture, cell proliferation, cell vitality and cell adipogenic potentiality in the transfected and untransfected groups had no difference.3. DA attached to typeⅠcollagen sponge scaffold or fibrin glue scaffold and normally grew. The adhesion rates different from cells mixing with different scaffolds. The adhesion rates is 77.67%±3.33% when we injected DA into the interior of typeⅠcollagen sponge scaffold (n=6), and the adhesion rates is 91.83%±2.79% when we mixed DA with fibrin glue scaffold (n=6). There was significant difference between the two groups (t=7.996, P<0.001).On the other hand, The ratio of adipogenic differentiation is 63.0%±3.0% when the adipogenic-differentiated DA were injected into the interior of typeⅠcollagen sponge scaffold. The ratio of adipogenic differentiation is 63.2%±3.3% when the adipogenic-differentiated DA were mixed with fibrin glue scaffold. There has no significant difference between groups (t=0.091, P=0.929). There has no influence on the morphous, growth, proliferation,adipogenic potentiality when DA attached scaffolds.4.12 weeks after transplantation, the newly formed tissue formation were observed macroscopically in the operative area of group DA- typeⅠcollagen sponge, ASCs- typeⅠcollagen sponge, DA-fibrin glue, ASCs- fibrin glue. But there has no neogenetic tissue were observed in the operative area of the group blank control. The mean humid weight of the newly formed tissue are 0.0728±0.0184g,0.0732±0.0198g,0.1109±0.0020g and 0.1080±0.0028g respectively(n=10). HE stain show that the newly formed tissue are mature adipose tissue. the positive display of immunofluorescence staining make it sure that the tissue was construted with transplanted cells-scaffolds. There was no significant difference between the group DA- typeⅠcollagen sponge and ASCs- typeⅠcollagen sponge, group DA-fibrin glue and ASCs- fibrin glue (all value of P>0.05). The scaffolds were degraded thoroughly after 12 weeks at the new formed tissue. Histological evaluation of neogenetic adipose tissue showed that capillary density, an index of neovascularization, increased markedly in Group ASCs- typeⅠcollagen sponge and Group ASCs- fibrin glue. A statistically significant increase in capillary density was observed after 12 weeks in group DA- typeⅠcollagen sponge and ASCs- typeⅠcollagen sponge, group DA-fibrin glue and ASCs- fibrin glue:11.22±2.53 capillaries in group DA- typeⅠcollagen sponge versus 15.22±2.39 capillaries in the group ASCs- typeⅠcollagen sponge,10.72±2.42 capillaries in group DA-fibrin glue versus 15.00±2.54 capillaries in the group ASCs- fibrin glue (all value of P<0.05). The fibrosis of the newly formed tissue was 24.90%±0.49% of DA- typeⅠcollagen; 25.10%±0.73% of ASCs- typeⅠcollagen sponge; 25.00%±0.75% of DA-fibrin glue; 24.70%±0.72% of ASCs- fibrin glue。There was no statistically significant difference between every groups. (all value of P>0.05).DA is also proved to be CD31 positive expression in vivo, and to take part in angiopoiesis.DISCUSSIONSSeed cell is the core issues of constructing engineering adipose tissue. In experiment, Human mature adipocytes was confirmed that it could dedifferentiate to DA. These cells have strong proliferate ability,multiple differentiation,high adipogenic differentiation.In this research,we purified mature adipocytes,and induced adipocytes dedifferentiation successfully.DA can be easily obtained using a relatively noninvasive method, be readily expanded in culture, be high adipogenic differentiation.DA offer a potentially unlimited source of cells for tissue engineering.Another of the major challenges facing the emerging field of regenerative medicine is finding a reliable source of scaffold for tissue repair and regeneration. A lot of scaffolds were applied to tissue engineering, but there were much disadvantage. In the experiment, DA were successfully cultured in typeⅠcollagen sponge and fibrin glue. Though fibrin glue has better adhension with cells than typeⅠcollagen sponge,the two scaffolds represent good compatibility and adhension with cells without little toxicity..The typeⅠcollagen sponge and fibrin glue are positived about application of engineering adipose tissue.At the same time, one of the major challenges facing the emerging field of repair mechanism of engineering adipose tissue is finding a better method to label seed cells. It is important for us to find a better method to label seed cells. In the experiment, transduction of EGFP gene to DA by adenovirus is safe and high efficient, and the target protein can be expressed in cell in vitro.EGFP transfection is a better method to gene therapy and label seed cells.In our research, while attached to collagenⅠscaffold or fibrin glue,and transplanted under the skin of nude mouse, DA can successfully form newly adipose tissue at a fairly satisfactory volume as well as ASCs. These results indicate that DA can be used as the cell source for future adipose tissue engineering,it can regenerate adipose tissue with three dimension construction and fuction.Conclusion1. We successfully induced and cultured DA from subcutaneous fat of adult human. And then we established a set of convenient methods about isolation, culture and identification for DA. The cells have great potentiality of proliferation and adipogenic,osteogenic and chondrogenic differentiation in vitro,high adipogenic differentiated ability especially. DA should be used for pluripotent seed cells harvested from adipose tissue. It is ideal in many aspects:they can be easily and effectively harvested, handled and multiplied non-invasively and abundantly;their pluripotency and proliferative efficiency are not less than those of adipose-derived MSCs. So DA has great prospect of clinical application.2. Transduction of EGFP gene to DA by adenovirus is safe and high efficient, and the target protein can be expressed in cell in vitro.EGFP transfection is a better method to gene therapy and label DA. EGFP has no obvious cytotoxicity and influence on the growth, proliferation and differentiation to the cells.3. DA can attach to typeⅠcollagen sponge and fibrin glue scaffold, normally grew and proliferated intro. There has a high adhesion rates when DA was mixed with scaffolds. There has no influence on the morphous, growth, proliferation and differentiation and no cytotoxicity when DA attached scaffolds in vitro. So it has excellent biocompatibility between DA with typeⅠcollagen sponge or fibrin glue scaffold.4,DA mixed with typeⅠcollagen sponge or fibrin glue scaffold can constructe adipose tissue in vivo. DA is also proved to take part in angiogenesis in vivo.Our study implied that DA may be a new seed cell source for tissue engineering to repare soft tissue defect.It also provide a better research model for studing adipocytes'growth and metabolism in vivo.