Basic and Clinical Evidence of an Alternative Method to Produce Vivo Nanofat

Abstract

Background: Fat grafting technologies are popularly used in plastic and reconstructive surgery. Due to its size limitation, it is hard to directly inject untreated fat tissue into the dermal layer. Nanofat, which was introduced by Tonnard, solves this problem by mechanically emulsifying fat tissue. However, the viability of the cells was greatly destroyed. In this study, we reported a new method by "gently" digesting the fat tissue to produce viable adipocytes, progenitors, and stromal stem cells using collagenase I digestion and centrifugation. This was named "Vivo nanofat".

Methods: Human liposuction aspirates were obtained from five healthy female donors with mean age of 28.7 ± 5.6 years. Colony-forming assay, flow cytometry analysis, and adipogenic and osteogenic induction of the adherent cells from the Vivo nanofat were used to characterize the adipose mesenchymal stem cells (MSCs). To investigate in vivo survival, we respectively injected Vivo nanofat and nanofat subcutaneously to the back of 8-week-old male BALB/c nude mice. Samples were harvested 2 days, 2 weeks, and 4 weeks postinjection for measurement, hematoxylin and eosin staining, and immunostaining.

Results: Our results showed that the Vivo nanofat contained a large number of colony-forming cells. These cells expressed MSC markers and had multi-differentiative potential. In vivo transplantation showed that the Vivo nanofat had lower resorption ratio than that of nanofat. The size of the transplanted nanofat was obviously smaller than that of Vivo nanofat 4 weeks postinjection (0.50 ± 0.17 cm vs. 0.81 ± 0.07 cm, t = -5783, P = 0.01).

Conclusion: Vivo nanofat may serve as a cell fraction injectable through a fine needle; this could be used for cosmetic applications.

Keywords: Adipose Tissue; Cell Therapy; Mesenchymal Stromal Cells; Rejuvenation.

Figure 1

Characters of Vivo nanofat. (a) Macroscopy of the fresh isolated fat tissue. (b) Softly digested “golden sand-like” Vivo nanofat. (c) Microscopy of the fresh fat tissue (left) and Vivo nanofat (right). Bar = 500 μm. (d) Bar graph of trypan blue stain positive cells (n = 4, t = 7.626. *P = 0.005).

Figure 2

Vivo nanofat containing more multipotential colony-forming stromal cells. (a) Colony forming assay: the Vivo nanofat has more colony number formed (n = 3, t = −4.918, *P = 0.039). (b and e) Flow cytometry analysis showed passage 4 stromal cells in the Vivo nanofat (b) and nanofat (e) expressed the positive markers CD90, CD44, CD105, and CD73 of mesenchymal stem cells while did not express the negative markers such as CD45, CD34 CD11b, CD19, and HLA-DR. (c) Adipogenic induction. Oil red staining showed the adipocyte after induction (Bar = 10 μm). (d) Alizarin red staining showed the calcium node after induction.

Figure 3

Less resorption in the Vivo nanofat transplantation. (a) Size changes of nanofat and Vivo nanofat after transplantation (n = 4 in each group, t = −5.783, *P = 0.01). (b) Hematoxylin and eosin staining showed that the fibrosis changes of the nanofat after transplantation and more adipocyte survival in the implant (Bar = 200 μm). (c) Perilipin immunofluorescences staining showed that more viable adipocyte in the Vivo nanofat graft (Bar = 200 μm).

Figure 4

Vivo nanofat correction of the neck wrinkles. (a) Fat tissue after liposuctions. (b) Filtration of the “soft” digested Vivo nanofat. (c and d) Injection of the Vivo nanofat into the dermal and subdermal. (e) Six-month follow-up after Vivo nanofat treatment.