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

开发一种负载丝心蛋白的生物活性前脂肪细胞生物杂交体，用于增强软组织缺陷。

• 批准号: 241346278
• 负责人: Dr. Henning Hanken
• 金额: --
• 依托单位: Institut für Textilmaschinen und Textile Hochleistungswerkstofftechnik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/241346278?language=en
• 关键词: Development; bioactive; preadipocyte; loaded; fibroin

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.

这项建议起源于2010年在乌尔姆举行的DFG Nachwuchsakademie Zahnmedizin，代表了Nachwuchsakademie项目的第二阶段。面部区域皮下脂肪组织缺损伴隔离层向深层结构的缺失，如果不使用合适的软组织移植物，则无法充分重建。如今，通常使用通过吸脂获得的自体脂肪组织，即使它具有已知的缺点：脂肪细胞被完全分化或被去除技术损坏，因此易于缺血性损伤。移植物会随着时间的推移而收缩和硬化，甚至完全消失。新血管形成和营养供应是移植脂肪细胞存活的关键因素。拟议的项目旨在通过利用由丝心蛋白组成的新型支架材料开发这些常规技术的替代品，该丝心蛋白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物的血管化。使用能够进行细胞分裂的脂肪组织前体细胞（前脂肪细胞）对于使用分化的脂肪细胞是有利的，这主要是由于它们对局部缺血具有更高的耐受性。计划构建专门设计的由丝素蛋白组成的生物活性三维支架，将其与自体前脂肪细胞一起接种，并检查其作为移植物的适用性。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。为了评价丝心蛋白作为前脂肪细胞的载体材料，将具有不同三维结构和具有不同VEGF和FGF-2呈递密度的支架材料与人前脂肪细胞接种。随后，通过在转录水平（真实的时间PCR）以及在蛋白质水平（ELISA）上测定脂肪形成分化的特异性标志物（PPAR-gamma、Glut 4、FABP 4、ACS、GPDH）的表达水平来监测脂肪形成分化21天。将在功能性酶和代谢试验（GPDH活性、脂解和葡萄糖摄取试验）以及免疫组织学（PPAR-gamma、Glut 4、FABP 4、ACS、GDPH、vWF、CD 31）和形态学（油红-O染色）中验证成功分化。如果分化的脂肪组织移植成功产生，在后续项目的移植体内评价计划。