Melatonin promotes survival of nonvascularized fat grafts and enhances the viability and migration of human adipose-derived stem cells via down-regulation of acute inflammatory cytokines.

Nonvascularized fat grafting is a valuable technique for soft tissue reconstruction but poor survival of fat in the host environment remains a problem. A process known as cell-assisted transfer is used to enhance fat graft retention by adding stromal vascular fraction, an adipose-derived stem cell (ASC) rich content to lipoaspirate. We have recently shown that the use of melatonin, a reactive oxygen species scavenger, protects human ASCs from hydrogen peroxide-induced oxidative stress and cell death in vitro but its role as a pharmacological adjunct in clinical fat grafting has not been studied. Herein, the effect of melatonin was examined on human ASCs in vitro using survival and functional assays including the MTT assay, CellTox Green assay, monolayer scratch assay as well as a human cytokine chemoluminescence, and tumour necrosis factor-α assay. Further, the effect of melatonin-treated fat grafts was tested in vivo with a murine model. Haematoxylin and eosin staining, perilipin and CD31 immunostaining were performed with morphometric analysis of adipose tissue. The results demonstrate that, in vitro, the addition of melatonin to ASCs significantly improved their cell-viability, promoted cell migration and preserved membrane integrity as compared to controls. In addition, it induced a potent anti-inflammatory response by downregulating acute inflammatory cytokines particularly tumour necrosis factor-α. For the first time, it is demonstrated in vivo that melatonin enhances fat graft volume retention by reducing inflammation and increasing the percentage of adipose volume within fat grafts with comparable volumes to that of cell-assisted lipotransfer. Based on these novel findings, melatonin may be a useful pharmacological adjunct in clinical fat grafting.

An adipogenic gel for surgical reconstruction of the subcutaneous fat layer in a rat model.

'Off-the-shelf' tissue-engineered skin alternatives for epidermal and dermal skin layers are available; however, no such alternative for the subdermal fat layer exists. Without this well-vascularized layer, skin graft take is variable and grafts may have reduced mobility, contracture and contour defects. In this study a novel adipose-derived acellular matrix (Adipogel) was investigated for its properties to generate subdermal fat in a rat model. In a dorsal thoracic site, a 1 × 1 cm Adipogel implant was inserted within a subdermal fat layer defect. In a dorsal lumbar site, an Adipogel implant was inserted in a subfascial pocket. Contralateral control defects remained empty. At 8 weeks wound/implant sites were evaluated histologically, immunohistochemically and morphometrically. Identifiable thoracic Adipogel implants lost volume in vivo over 8 weeks. Neovascularization and adipogenesis were evident within implants and adipocyte percentage volume was 33.07 ± 6.55% (mean ± SEM). A comparison of entire cross-sections of thoracic wounds demonstrated a significant increase in total wound fat in Adipogel-implanted wounds (37.19 ± 4.48%, mean ± SEM) compared to control (16.53 ± 4.60%; p = 0.0092), indicating that some Adipogel had been completely converted to normal fat. At the lumbar site, Adipogel also lost volume, appearing flattened, although fat generation and angiogenesis occurred. At both sites macrophage infiltration was mild, whilst many infiltrating cells were PDGFRß-positive mesenchymal cells. Adipogel is adipogenic and angiogenic and is a promising candidate for subcutaneous fat regeneration; it has the potential to be a valuable adjunct to wound-healing therapy and reconstructive surgery practice. Copyright © 2015 John Wiley & Sons, Ltd.

A Streamlined Method for the Preparation of Growth Factor-enriched Thermosensitive Hydrogels from Soft Tissue.

Hydrogels are an ideal medium for the expansion of cells in three dimensions. The ability to induce cell expansion and differentiation in a controlled manner is a key goal in tissue engineering. Here we describe a detailed method for producing hydrogels from soft tissues with an emphasis on adipose tissue. In this method, soluble, extractable proteins are recovered from the tissue and stored while the remaining insoluble tissue is processed and solubilised. Once the tissue has been sufficiently solubilised, the extracted proteins are added. The resulting product is a thermosensitive hydrogel with proteins representative of the native tissue. This method addresses common issues encountered when working with some biomaterials, such as high lipid content, DNA contamination, and finding an appropriate sterilisation method. Although the focus of this article is on adipose tissue, using this method we have produced hydrogels from other soft tissues including muscle, liver, and cardiac tissue.

Preparation of an adipogenic hydrogel from subcutaneous adipose tissue.

The ability to generate controlled amounts of adipose tissue would greatly ease the burden on hospitals for reconstructive surgery. We have previously shown that a tissue engineering chamber containing a vascular pedicle was capable of forming new fat; however, further refinements are required to enhance fat formation. The development and maintenance of engineered adipose tissue requires a suitable source of growth factors and a suitable scaffold. A hydrogel derived from adipose tissue may fulfil this need. Subcutaneous fat was processed into a thermosensitive hydrogel we refer to as adipose-derived matrix (ADM). Protein analysis revealed high levels of basement membrane proteins, collagens and detectable levels of growth factors. Adipose-derived stem cells exposed to this hydrogel differentiated into adipocytes with >90% efficiency and in vivo testing in rats showed significant signs of adipogenesis after 8 weeks. ADM's adipogenic properties combined with its simple gelation, relatively long shelf life and its tolerance to multiple freeze-thaw cycles, makes it a promising candidate for adipose engineering applications.

N-linked keratan sulfate in the aggrecan interglobular domain potentiates aggrecanase activity.

Keratan sulfate is thought to influence the cleavage of aggrecan by metalloenzymes. We have therefore produced a recombinant substrate, substituted with keratan sulfate, suitable for the study of aggrecanolysis in vitro. Recombinant human G1-G2 was produced in primary bovine keratocytes using a vaccinia virus expression system. Following purification and digestion with specific hydrolases, fluorophore-assisted carbohydrate electrophoresis was used to confirm the presence of the monosulfated Gal-GlcNAc6S and GlcNAc6s-Gal disaccharides and the disulfated Gal6S-GlcNAc6S disaccharides of keratan sulfate. Negligible amounts of fucose or sialic acid were detected, and the level of unsulfated disaccharides was minimal. Treatment with keratanases reduced the size of the recombinant G1-G2 by approximately 5 kDa on SDS-PAGE. Treatment with N-glycosidase F also reduced the size of G1-G2 by approximately 5 kDa and substantially reduced G1-G2 immunoreactivity with monoclonal antibody 5-D-4, indicating that keratan sulfate on the recombinant protein is N-linked. Cleavage of G1-G2 by aggrecanase was markedly reduced when keratan sulfate chains were removed by treatment with keratanase, keratanase II, endo-beta-galactosidase, or N-glycosidase F. These results indicate that modification of oligosaccharides in the aggrecan interglobular domain with keratan sulfate, most likely at asparagine residue 368, potentiates aggrecanase activity in this part of the core protein.