Introduction: Adipose tissue transplantation is commonly used in reconstructive surgery[1]. However, even vascularized fat flaps undergo considerable resorption after transplantation[2]. Adipocytes can only survive near the surface of the transplant (less than 300 µm in depth). Adipocytes that locate in deeper areas start to die on the first day of transplantation due to ischemia[3]. Oxygen deficiency is one of the factors that jeopardizes viability of many cells after transplantation[4]. We have developed a self-oxygenating scaffold consisting of metal peroxides and hydrogel. In this work, we aimed to examine adipose tissue viability in anoxia with and without our oxygen generating scaffold.
Materials and Methods: Oxygen release scaffolds were made from calcium peroxide (CaO2) and calcium cross-linked sodium alginate.Subcutaneous adipose tissue was collected from freshly sacrificed rats and kept on ice during processing. Then the tissue was cut into 3×3 mm pieces and mixed with sodium alginate with or without calcium peroxide suspension. The mixtures were cross-linked by calcium ions to form cylinders 7 mm in diameter and 10 mm in length. The encapsulated adipose tissue was cultured under anoxia (95% N2, 5% CO2, 37 ) for up to seven days. The viability of cells in adipose tissue was inspected by a live/dead assay under a fluorescent microscope. The cultured tissue were also fixed and embedded in paraffin. H&E staining and perilipin staining were performed to examine the tissue structure and live cells, respectively.
The scaffolds containing adipose tissue with and without oxygen release material will be further implanted subcutaneously into a rat model. After two and four weeks, the transplants will be retrieved. The transplants will be weighed before and after transplantation to determine the weight changes of the transplanted tissue. The volume of the adipose tissue after transplantation will be assessed by immersion[5]. The size of adipocytes in the transplants will be measured. Cell apoptosis will be examined using a caspase 3/7 assay. In addition, the retrieved transplants will be fixed and embedded in paraffin and histological characterizations will be performed as described previously.
Results and Discussion: According to the live/dead assay results (Fig. 1a), adipose tissue preserved with oxygen release material under anoxia maintained high cell viability and scarce red nuclei from dead cells were observed. However, adipose tissue preserved under anoxia without oxygen release material (Fig. 1b) exhibited low cell viability. Our in vitro culture results show that adipose tissue can be preserved by oxygen delivery.
Figure 1. Fluorescent images of live/dead assay stained adipose tissue after seven days culture in the absence of oxygen. Live cells were stained by green color. The nuclei of dead cells were stained into red and the nuclei of all the cells blue. Adipose tissue preserved with the oxygen release materials in the scaffold displayed very good viability (a), but adipose tissue preserved under anoxia without oxygen release materials (b) showed poor viability.
Conclusion: We successfully preserved adipose tissue by oxygen delivery using a self-oxygenating scaffold for up to seven days under anoxia. Oxygen delivery can be a promising method to maintain cell viability in adipose tissue following transplantation.
The Natural Sciences and Engineering Research Council of Canada; China Scholarship Council
References:
[1] Casadei A, Epis R, Ferroni L, Tocco I, Gardin C, Bressan E, et al. Adipose tissue regeneration: a state of the art. BioMed Research International. 2012;2012.
[2] Oashi K, Furukawa H, Akita S, Nakashima M, Matsuda K, Oyama A, et al. Vascularised fat flaps lose 44% of their weight 24 weeks after transplantation. Journal of Plastic, Reconstructive & Aesthetic Surgery. 2012;65:1403-9.
[3] Eto H, Kato H, Suga H, Aoi N, Doi K, Kuno S, et al. The fate of adipocytes after nonvascularized fat grafting: evidence of early death and replacement of adipocytes. Plastic and reconstructive surgery. 2012;129:1081-92.
[4] Suga H, Eto H, Aoi N, Kato H, Araki J, Doi K, et al. Adipose tissue remodeling under ischemia: death of adipocytes and activation of stem/progenitor cells. Plastic and reconstructive surgery. 2010;126:1911-23.
[5] Dolderer JH, Abberton KM, Thompson EW, Slavin JL, Stevens GW, Penington AJ, et al. Spontaneous large volume adipose tissue generation from a vascularized pedicled fat flap inside a chamber space. Tissue Engineering. 2007;13:673-81.