The Comparative Research Of Large Volume Engineered Adipose Tissue Fabricating Factors

Background and Objection:In reconstructive and plastic surgery, many treatments aim to restore soft-tissue defects resulting from the post-operative, congenital, or post-traumatic loss of the subcutaneous fat layer. In2011nearly five million reconstructive procedures were reported by The American Society of Plastic Surgeons, of which approximately78%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.The currently preferred treatments use pedicled flaps consisting of subcutaneous fat tissue with microsurgically integrated blood vessel supply.While showing better outcome in terms of reconstruction of the treated tissue defect, considerable donor site morbidity such as hypertrophic scar formation, constricted motility or asymmetry of the affected area is a major drawback of this procedure. In addition, the surgical procedure is complicated and therefore cost-intensive. Specific-shape, define-volume, transplantable soft tissue flap is always the "ultimate dream" of each plastic surgeon. Tissue engineering provides new perspectives for the clinically applicable tissue substitutes. The first study in which cell-containing constructs were successfullyimplanted in order to engineer adipose tissue was conducted by Patrick et al. 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.Several general challenges have to be faced in achieving this ambitious aim:①the 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.Effective ways to enhance in vivo vascularization in engineered tissues include controlled release of angiogenic factors (e.g., bFGF and vascular endothelial growth factor) over a long period and co-transplantation of endothelial cells.However, there is still the unfulfilled need to generate adipose tissue constructs of considerable size that is meaningful for a clinical application.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 vascularization.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. Does mature adipose tissue can be induced by advanced angiogenesis merely? Local microenvironment inside and around the adipose tissue affect the histological structure and maturity? How can we promote the interaction between vascular and adipocyte?At present study, we use tissue engineering chamber technology and prefabrication to enhance angiogenesis in early stage, observe that histology of new tissue. We created new chamber and observed some serial specimen to indicate influence of local microenvironment on new tissue. At last we insert a0.6ml fat flap with vascular pedicle into a protected Chamber space and it growed into3.03ml adipose tissue flap after4weeks.Methods and materials1、Large new tissue formation by prefabricating well-vascularization bed.Tissue engineering chamber technology and prefabrication were used to produce a well-vascularization bed before adding labeled adipose-special stem cells. New tissue volume measurement, HE staining and immunofluorescence were tested to investigate the influence of advanced angiogenesis on large new tissue formation.2、Local microenvironment induce neoadipogenesis.Simple adipogenic microenvironment can not induce mature adipose tissue. We created new chamber to investigate internal environment in chamber to discuss the induction of local environment on new engineered tissue by histology, cell source, environment dynamic change.3、The strategy of fabricating large volume engineered adipose tissue0.6ml fat flap with vascular pedicle was inserted into a sealed chamber space without the addition of growth factors, seed cells, or scaffold, to induce neoadipogenesis by co-exist and mutual effect of angiogenesis and adipogenic environment.The preliminary induced mechanism was discussesed by histology and RT-PCR.4、Statistical analysis.Results were expressed as the mean standard error. SPSS13.0software 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 of0.05was considered to represent a statistical significance.Results:1、Tissue engineering chamber and a vascular pedicel can induced new tissue more than1ml,which has mature vessels. Add exogenous adipose-special stem cells can induce1.1ml new tissue, but organization structure is fibrous connective tissue mainly with a mount of new vessels, no obvious adipose tissue was found.2、Adipose tissue explants in the chamber can only induce fibrous tissue without any vessels formation. We Used a new chamber model to investigate the chamber environment without any interference. After two months a little mature adipose tissue raised above the fat pad.Cell number in interstitial fluid reached peak at day15and stabilized between day45to60.VEGF and MCP-1expression reduced with time and stabilized at last.CD34and perilipin double positive cells found around vessels indicated mesenchymal stem cells differentiated to mature adipocytes.3、3.03ml new tissue was induced in chamber. Haematoxylin staining indicated the adipose tissue remolding process:a great quantity adipocytes similar with normal adipocytes appeared on the contemporary self-assembling fibrinogen matrix gel but with no vessel invasion within7days, microvessel gradually grew into new fat area near capsular region accompanying thick fibre bundle at two weeks around, Vasculature network got richer and maturity accompanied with extensive adipose lobular structure formation in later grow period,no distinct interface was found between with the newly generated tissue and original fat areaDiscussionsThe currently preferred treatments use pedicled flaps consisting of subcutaneous fat tissue with microsurgically integrated blood vessel supply. While considerable donor site morbidity such as hypertrophic scar formation, constricted motility or asymmetry of the affected area is a major drawback of this procedure. Traditional tissue engineering can not fulfill the large volume mature adipose tissue formation because of the unharmonious development between angiogenesis and adipogenesis, delayed blood supply at early stage is bottleneck especially. Tissue engineering chamber technology provides new perspectives for the availability of large volume applicable tissue substitutes.The chamber can induced vascular net maturation quicker through grow factors secreted by tissue injury; enrich the grow and cytokines locally; and the hollow chamber offers the new tissue physics support.But since the particularity of adipose tissue, which elements determine the volume and histology feature is still uncertain. The expansion of adipose tissue needs abundant vessel net and local adipogenic microenvironment, and more important the harmonious development between angiogenesis and adipogenesis.Arteriovenous bundle in chamber can enhance angiogenesis in early stage, but adding exogenous seed cells can but only induce fibrous connective tissue with a mount of new vessels, no obvious adipose tissue was found. It is obviously that the even the adipose-special stem cells can not differentiate spontaneously without adipogenic microenvironment.Though adding exogenous adipose explants can play the adipogenesis induction factor but the new tissue was only fibrous connective tissue without vessels formation due to blood supply deficiency. Through a new chamber model we found the interstitial fluid in chamber at early stage contain a mount of grow factors and cells.These cells came from circulation and may act as the stem cells which can proliferate and differentiate in the fat pad.But the new raised adipose tissue was so small and confined its further grow by the thick capsule.A fat flap with arteriovenous bundle pedicle can provide angiogenesis and adipogenesis factor simultaneously. At last3.03ml new tissue was induced in this chamber. Haematoxylin staining indicated the adipose tissue remolding process:a great quantity adipocytes similar with normal adipocytes appeared on the contemporary self-assembling fibrinogen matrix gel but with no vessel invasion within7days, microvessel gradually grew into new fat area near capsular region accompanying thick fibre bundle at two weeks around, Vasculature network got richer and maturity accompanied with extensive adipose lobular structure formation in later grow period,no distinct interface was found between with the newly generated tissue and original fat areaThis strategy of fabricating large volume engineered adipose tissue provide a safe, steady and effective approach for generating a functional, long-term, stable tissue capable of restoring the natural and aesthetic function of the soft tissue.Conclusion1. Without inductive microenvironment only enhancing angiogenesis can not induce adipogenesis spontaneously.2. Adding exogenous adipose explants can only generate fibrous connective tissue without vessels. A large transplantable adipose tissue cannot be induced quickly without angiogenesis and adipogenesis environment in chamber.3. A new large mature adipose tissue can be induced in chamber with inserting a fat flap with arteriovenous bundle pedicle into a sealed chamber without any seed cells or scaffold,and adipogenesis came before angiogenesis at early stage in chamber.