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

这项建议起源于2010年在乌勒姆举行的DFG Nachwuchsakademie Zahnmedizin，代表着Nachwuchsakademie计划的第二阶段。如果不使用适当的软组织移植，面部皮下脂肪组织的缺陷将失去隔离层到更深的铺设结构，无法得到充分的重建。目前，通过吸脂获得的自体脂肪组织仍被广泛使用，尽管它有一些已知的缺点：脂肪细胞被充分分化或被去除技术破坏，因此容易发生缺血性损伤。随着时间的推移，移植的器官会缩小、变硬，甚至完全消失。新生血管和营养供应是移植脂肪细胞存活的关键因素。这项拟议的项目旨在开发一种替代这些传统技术的方法，利用一种由丝素蛋白组成的新型支架材料，该材料呈现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)。这些生长因子促进脂肪细胞的分化和移植血管的形成。脂肪组织前体细胞(前脂肪细胞)的使用具有细胞分裂的能力，有利于分化的脂肪细胞的使用，主要是因为它们对缺血的耐受性更高。该公司计划构建由丝素蛋白组成的特殊设计的生物活性三维支架，这些支架将与自体前脂肪细胞一起种植，并检查其作为移植的适用性。丝素是一种丝蛋白，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。为了评价丝素蛋白作为前脂肪细胞载体材料的作用，将不同三维结构、不同表达密度的血管内皮细胞生长因子和成纤维细胞生长因子2的支架材料接种于人前脂肪细胞。随后，通过检测成脂分化特异性标志物(PPAR-γ、GLUT4、FABP4、ACS、GPDH)在转录水平(实时定量聚合酶链式反应)和蛋白质水平(ELISA)上的表达水平，对成脂分化进行21天的监测。成功的分化将在功能酶和代谢试验(GPDH活性、脂解和葡萄糖摄取试验)以及免疫组织学(PPAR-γ、GLUT4、FABP4、ACS、GDPH、vWF、CD31)和形态(油红O染色)中得到验证。如果分化的脂肪组织移植成功，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.