Human preadipocytes seeded on freeze-dried collagen scaffolds investigated in vitro and in vivo

Abstract

Currently, there is no adequate implant material for the correction of soft tissue defects such as after extensive deep burns, after tumor resection and in hereditary and congenital defects (e.g. Romberg's disease, Poland syndrome). The autologous transplantation of mature adipose tissue has poor results. In this study human preadipocytes of young adults were isolated and cultured. 10(6) preadipocytes were seeded onto collagen sponges with uniform 40 microm pore size and regular lamellar structure and implanted into immunodeficient mice. Collagen sponges without preadipocytes were used in the controls. Macroscopical impression, weight, thickness, histology, immunohistochemistry (scaffold structure, cellularity, penetration depth of the seeded cells) and ultrastructure were assessed after 24 h in vitro and after explantation at 3 and 8 weeks. Preadipocytes penetrated the scaffolds 24 h after seeding at a depth of 299+/-55 microm before implantation. Macroscopically after 3 and 8 weeks in vivo layers of adipose tissue accompanied by new vessels were found on all preadipocyte/collagen grafts. The control grafts appeared unchanged without vessel ingrowth. There was a significant weight loss of all grafts between 24 h in vitro and 3 weeks in vivo (p < 0.05), whereas there was only a slight weight reduction from week 3 to 8. The thickness decreased in the first 3 weeks (p < 0.05) in all grafts. The preadipocyte/collagen grafts were thinner but had a higher weight than the controls at this point in time. The histology showed adipose tissue and a rich vascularisation adherent to the scaffolds under a capsule. The control sponges contained only few cells and a capsule but no adipose tissue. Human-vimentin positive cells were found in all preadipocyte/collagen grafts but not in the controls, penetrating 1188+/-498 microm (3 weeks) and 1433+/-685 microm (8 weeks). Ultrastructural analysis showed complete in vivo differentiation of viable adipocytes in the sponge seeded with preadipocytes. Formation of extracellular matrix was more pronounced in the preadipocyte/collagen grafts. The transplantation of isolated and cultured preadipocytes within a standardised collagen matrix resulted in well-vascularised adipose-like tissue. It is assumed that a pore size greater than 40 microm is required, as preadipocytes enlarge during differentiation due to incorporation of lipids.