Development of an bioactive preadipocyte loaded fibroin biohybrid for the augmentation of soft tissue defects.

该提案起源于2010年乌尔姆DFG Nachwuchsakademie Zahnmedizin，代表了Nachwuchsakademie计划的第二阶段。面部皮下脂肪组织缺损，若缺乏隔离层，则需采用合适的软组织移植，才能充分重建。目前，通过吸脂获得的自体脂肪组织是常用的，尽管它有已知的缺点：脂肪细胞被完全分化或被移除技术破坏，因此容易发生缺血性损伤。随着时间的推移，移植物会萎缩、变硬，甚至完全消失。新生血管的形成和营养的供应是移植脂肪细胞存活的关键因素。该项目旨在开发一种替代这些传统技术的新支架材料，该材料由含有生长因子血管内皮生长因子（VEGF）和成纤维细胞生长因子2 （FGF-2）的纤维蛋白组成。这些生长因子促进脂肪细胞的分化和移植血管的形成。使用具有细胞分裂能力的脂肪组织前体细胞（preadipocytes）有利于使用已分化的脂肪细胞，这主要是因为它们对缺血的耐受性更高。计划构建由纤维蛋白组成的专门设计的、具有生物活性的三维支架，并将其植入自体前脂肪细胞，并检查其移植的适用性。丝素蛋白是一种经过充分研究和验证的生物材料，不会引起异物反应或伤口愈合障碍。为了评估纤维蛋白作为前脂肪细胞的载体材料，我们将具有不同三维结构、不同VEGF和FGF-2呈现密度的支架材料植入人前脂肪细胞。随后，通过在转录水平（real - time PCR）和蛋白水平（ELISA）上检测成脂分化特异性标志物（ppar - γ、Glut4、FABP4、ACS、GPDH）的表达水平，监测成脂分化21天。成功分化将通过功能性酶和代谢试验（GPDH活性、脂肪分解和葡萄糖摄取试验）、免疫组织学（ppar - γ、Glut4、FABP4、ACS、GDPH、vWF、CD31）和形态学（油-红- o染色）进行验证。如果分化脂肪组织移植成功生成，则计划在后续项目中对移植进行体内评估。

This proposal originated at the DFG Nachwuchsakademie Zahnmedizin 2010 in Ulm and represents the second stage of the Nachwuchsakademie program.Defects of subcutaneous adipose tissue in the facial area with a loss of the isolating layer to the deeper laying structures cannot be reconstructed adequately without the use of suitable soft tissue transplants. Nowadays, autologous adipose tissue obtained by liposuction is commonly used, even though it has known shortcomings: the adipose cells are fully differentiated or damaged by the removal technique and are therefore prone to ischemic damage. The transplants shrink and harden or are even lost completely over time. Neovascularisation and nutritive supply are crucial factors for the survival of the transplanted adipose cells. The proposed project aims to develop an alternative to these conventional techniques by utilizing a novel scaffold material consisting of fibroin that presents the growth factors vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF-2). These growth factors promote the differentiation of adipocytes and vascularization of the transplant. The use of adipose tissue precursor cells (preadipocytes), which are capable of cell division, is advantageous to the use of differentiated adipocytes, mainly due to their higher tolerance for ischaemia. It is planned to construct specifically designed, bioactive three-dimensional scaffolds consisting of fibroin, that will be seeded with autologous preadipocytes and examined regarding their applicability as transplants. Fibroin, a silk protein, is a well-studied and validated biomaterial, that does not cause foreign body reactions or wound healing disorders. For the evaluation of fibroin as a carrier material for preadipocytes, scaffold materials in different three-dimensional structures and with different presentation densities of VEGF and FGF-2 are seeded with human preadipocytes. Subsequently, the adipogenic differentiation is monitored for 21 days by determining the expression levels of specific markers for adipogenic differentiation (PPAR-gamma, Glut4, FABP4, ACS, GPDH) on the transcriptional level (real time PCR) as well as on the protein level (ELISA). Successful differentiation will be verified in functional enzymatic and metabolic assays (GPDH activity, lipolysis and glucose-uptake assay) as well as immunohistologically (PPAR-gamma, Glut4, FABP4, ACS, GDPH, vWF, CD31) and morphologically (Oil-Red-O-staining). If differentiated adipose tissue transplants are successfully generated, an in vivo evaluation of the transplants in a follow-up project is planned.