| Background and Objection:In plastic and cosmetic surgery,the most common problems were soft tissue defect repair caused by tumor resection,congenital malformation and trauma,and soft tissue augmentation for cosmetic requirements.In 2011 nearly five million reconstructive procedures were reported by The American Society of Plastic Surgeons, of which approximately 78% of them were due to tumor removal. Treating soft-tissue defects is not just a matter of "filling in the gap" but also generating a functional, long-term, stable tissue capable of interacting with adjacent tissues to restore the natural and aesthetic function of the soft tissue.To solve these problems, dermal fat grafts, collagen injections, synthetic materials,and free adipose tissue grafts are usually used for reconstruction of soft tissue defects. All these have disadvantages and limitations. Synthetic materials can cause foreign body reactions and infections. Adipose tissue can provide support for capacity and contour favored, by clinicians. But it was limited in clinical because of as high as 30～50% absorption rate of fat tissue graft at present. Pedicle fat grafts because of its immediate reconstruction of blood circulation with microsurgical technique, the volume reduction after transplantation is low, these approach can achieve the best results of fat graft. However, limited donor area, harvested a lot of fat easily lead to secondary deformity, which limit the pedicle fat flap in clinical application.For these unsatisfictions, tissue engineering provides new perspectives for the clinically applicable tissue substitutes. In adipose tissue engineering cell, scaffolds, and micro environment are the three main factors. Many researchers have focused on investigating seed cells, biomaterials, and the microenvironment. However, no reports have described large-volume engineered adipose tissue, and its clinical applications.Hofer et al reported the "tissue engineering chamber technology’, that a protected chamber space can provide a safe, stable environment for a small tissue flap on a vascular pedicle. This model broadened our insight into large volume engineering adipose tissue formation because it seemed to solve the key vascularizationIn 2007, Dolderer et al implanted mouse inguinal subcutaneous abdominal fat tissue flap with vascular pedicle in the small chambers, and found a significant growth of adipose tissue. Histomorphometric analysis revealed that increased tissue was mature adipose tissue with normal blood vessel, forming "adipose tissue engineering chamber". In 2011 Morrison et al applied this method to the large animal, the maximum was to 56.5ml, and histologicalstudy found their tissue constituents; fibrous connective tissue, adipose tissue, blood vessels, and PLGA, and fibrous connective tissue volume and the amount of fat tissue was the same. Treating soft-tissue defects is not just a matter of "filling in the gap" but also generating a functional, long-term, stable tissue capable of interacting with adjacent tissues to restore the natural and aesthetic function of the soft tissue.Several general challenges have to be faced in achieving this ambitious aim: ① the need for a rapid and sufficient vascularization,which may be one of the main challenges for engineering tissue equivalents; ②the need for a biodegradable, preferably with the degradation rate to match the development kinetics of the engineered adipose tissue;③the need for a grow factors gradient and microenvironment and, last but not least,④the need for a mechanically stable space to facilitate the generation of volume-stable adipose tissue constructs.But when seeking the optimal strategy to engineer fat tissue, it is indispensable to be aware of the unique properties of adipose tissue, its natural development and its biological function. Except a well-defined and dense capillary network,90% space of the tissue is composed by adipocytes marked by high consumption of oxygen. Meanwhile adipose tissue is a main endocrinal organ, a number of the adipokines promote angiogenesis and adipogenesis. Jertta-RiinaSarkanen et al extracted the factors from mature adipose tissue without affecting the cell viability which contained grow factors and cytokines of mature adipose tissue. These factors are known to have an inductive role on adipogenesis and/or angiogenesis in vitro. We investigated whether the application of additional factor into the chamber promoted the key problem of early vascularization to improve the constitutes. The change of local microenvironment inside and around the adipose tissue whether influence the final new constructs of organizational forms and maturity?How can we induce purified adipose tissue in the tissue engineeering chaber model?Around the scientific problem, this experimnet first extracted the factors from mature adipose tissue and its characteristics. We applied the ATE into the adipose tissue engineering chamber model, then observed what happened in the chamber by means of HE, immunohistochemistry, ELISA and PCR analysis.Methods and materials1, Extracting the adipose tissue extracts and related detectionUseing the back adipose tissue of rabbits adipose,after the isolation, culture of 24 hours, and centrifugation, the supernatant was extracted, which was detected by ELISA. The adipose tissue extract and extraction (ATE) was added to adipose derived stem cells to observe the effect on adipogenesis,and added to the vascular endothelial cells and fibroblasts to observe its effect on angiogenesis.2, Changes of the constructs after adipose tissue ectract injecting into the adipose tissue engineering chamber.The rabbit adipose tissue engineering chamber model was constructed. ATE was injected into the chamber after lweek the model construction. At different time points, we extracted the liquid of the chamber to observe the changes of the construction ande the cytokines and the growth factors.3-, Statistical analysis.Results were expressed as the mean standard error. SPSS 13.0 software was used for data analysis. The data was statistically analyzed by using a Two-way ANOVA. Differences of the different time points using analysis of covariance. A p-level of 0.05 was considered to represent a statistical significance.Results:1,ATE was added into the vascular endothelial cell and fibroblast and blood vessel formation experiment showed that vascular endothelial cells formed a continuous network structure in the ATE group. ATE joined the ASCs culture mediun, oil O staining showed that ASCs in non adipogenic conditions appeared lipid droplets in ATE group.2.The early constructure of chamber was jelly. At late capsule formation was visible and touched tough. After injection of ATE, the experimental group new tissue volume was significantly increased compared with the control group.3,In the beginning stage, the control group and the experimental group have a large number of new small blood vessels appeared in theinterstitial region. The density of capillaries was higher in experimental group than in control group. Many small adipocytes (immature adipocytes) were observed around newly formed capillaries in control group and experimental group. More small adipocytes were found in connective tissue and large vessels were also present in experimental group. There were fewer small adipocytes and more connective tissue in control group. Adipogenesis was in advance and vascularization gradual mature over time. At 7 weeks post-injection, its structure was normal and regular. Newly formed adipose tissue had a better morphology and structure and the capsule was thinner in experimental group than in control group.4.CD31 immunohistochemistry found that after post-injection ATE angiogenesis was increased remarkably and after post-injection lweek the number of capillaries was the highest. The experimental group was more than the control group.DiscussionThe pattern that engineering room model induced the rebirth of fat can be treated as a unique process of adipose tie wound healing. Separated from situ, the fat flap produced the pedicle island fat flap, then the continuity of adipose tissue was broken, the normal vascular function was impaired. As a consequence, the body initiates the nonspecific wound healing which was mainly reflected as three independent and overlapping periods:inflammation, proliferation and tissue remodeling.Vascular endothelial growth factor(VEGF) originally called vascular permeability factor, was considered as a powerful stimulus of the endothelial cell’s proliferation and migration; Fibroblast growth factor 2 (FGF-2) was important in wound healing and angiogenesis. As a factor of angiogenic cytokines, FGF-2 promoted endothelial cell proliferation and formed the capillary, and also stimulated the proliferation and migration of mesenchymal cells. John A. Rophael and others blend VEGF, platelet growth factor (PDGF-BB), FGF-2, and then joined them into Matrigel small room which contains the pure vascular pedicle. It was discovered that the growth factor started to work in early period. Meanwhile, the infiltrating cells which included macrophages, pericyte, and all kinds of preadipocytes increasing in the fibrous tissue. Macrophages and pericyte were associated with angiogenesis and fat formation. The research also found that the addition of angiogenesis factor can promoted wound healing. The reason was that the early new blood vessel provided local tissue oxygen and nutrients, and it was also the main method of stem cell chemotactic migration. Thus, the earlier established the blood supply, the better the tissue regenerated. ATE contained growth factors and cytokines of mature adipose tissue, such as VEGF, bFGF, interleukin 6 (IL-6), insulin-like growth factor 1 (IGF 1), leptin, adiponectin, and so on. Some growth factors and cytokines, can also stimulate angiogenesis and the required capillary network for adipose tissue expansion formation. We injected ATE into the closet, and found that the obvious stroma in the early time, while the effusion of tissue fluid formed the gel-like fibrinogen matrix. Meanwhile, a significant angiogenesis appeared, and many immature fat cells have been produced around the vessel which showed that mesenchymal stem cells migrated to chamber and start to induced differentiation in the local microenvironment.Along with the development, the new blood vessels formed a complete network with body’s natural blood. As a consequence, the inflammatory gradually relieved, and the tissue repair entered into the breeding period. At this stage of our experimental group, we have observed that a large number of new blood vessels form. In the early formation of fibrinogen matrix, a large amount of island fat cell differented and prolifered, and they gradually formed the new fat lobule. The fat precursor cells and fat cells in different differentiation stages attached to the blood vessel and formed the basis of fat rebirth. At this stage, the experimental group formed the island new fat cells early. On the one hand, in inflammation period, the angiogenic factors of ATE induced more vascularnetwork informed quickly in matrix, which resulted in more mesenchymal stem cells through the circulation into the chamber induced adipogenesis; on the other hand, in the proliferative phase probably because of adipogenesis factors of ATE, such as leptin, insulin-like growth factor, can promote the migration of mesenchymal stem and induced to differentiate into adipocyte. As the maturity of the building organization went into the reshaping period, the blood impassability occured during the process of angiogenesis which is accompanied by th e reshaping of the blood vessels. This explained that why the control group and experi mental group vascular density decreased in the two weeks after the injection of ATE. Adipose tissue structure gradually formed new normal lobular structure of adipose tis sue.The actual volume of new adipose tissue was less than the volume of engineering chamber, and the outer was a layer of densefibrous capsule. Our previous experiments have found that using silica gel Chambers and the back fat with vascular pedicle flapof New Zealand would induce new tissue which was smaller than the silicone chamber. Through the present experiment that injecting the adipose tissue extract into the engineering indoor of the rabbit’s back, we found that the early injection of ATE in the engineering indoor construct vascularization appeared in advance, and more abundant. In the early time, the ATE promoted the vascularization, made fat organization in advance, and accelerated the mature speed of the fat structure. The building material’s outer surface without the ATE’s injection was packaged of a dense fiber layer, and this layer probably was the main reason of limiting the further growth of new tissue.the main reason.Through experiment, we found that the injection of the ATE changed the capsular reaction, and reduced the capsular contracture of the late new suppression, and induced the regeneration of the mass of adipose tissue..Through the continuous observation of engineering indoor exudate in our experiment, we found that the fat project indoor exudate contains a large number of growth factors. After injecting ATE, the concentration of growth factorin the exudate increases, and this tissue exudate can provide better vascularization and adipogenesis, and provide a better environment to support the growth of adipose tissue.Adipose tissue engineering room has an important application prospect because of its’rich source of fat tissue and the small area damage. Engineering room not only provides the required space required for the indoor growth, but also builds a relatively independent space to facilitate the experimental intervention.The local application of adipose tissue extracts accelerate the growth of the interior organization, and won’t affect the whole body organization; this perspective may be research focus of the future use of tissue engineering room for tissue engineering.Conclusion1、The rich growth factors and fat factors in ATE can improve the environment of the blood vessels in the engineering indoor, early induce a large network of blood vessels mature, establish and improve blood supply, and promote the adipose tissue renewal.2、Meanwhile, the rich growth factors and fat factors in ATE can remove an obstacle which is the further increase of new tissue volume through improving the local vascularization to reduce capsular response. |
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