Boosting Tissue Vascularization: Nanofat as a Potential Source of Functional Microvessel Segments

Abstract

Nanofat is increasingly applied in plastic surgery for the improvement of scar quality and skin rejuvenation. However, little is known about the underlying regenerative mechanisms. Therefore, we herein investigated nanofat grafts in a murine dorsal skinfold chamber model. Nanofat generated from subcutaneous, inguinal adipose tissue of green fluorescent protein (GFP)⁺ C57BL/6 male and female donor mice was injected intracutaneously into dorsal skinfold chambers of gender-matched GFP^- wild-type mice. The vascularization and tissue composition of the grafted nanofat were analyzed by means of intravital fluorescence microscopy, histology and immunohistochemistry over an observation period of 14 days. The freshly generated nanofat consisted of small fragments of perilipin⁺ adipocytes surrounded by Sirius red⁺ collagen fibers and still contained intact CD31⁺/GFP⁺ vessel segments. After transplantation into the dorsal skinfold chamber, these vessel segments survived and developed interconnections to the surrounding CD31⁺/GFP^- host microvasculature. Accordingly, the grafted nanofat rapidly vascularized and formed new microvascular networks with a high functional microvessel density on day 14 without marked differences between male and female mice. Even though further research is needed to confirm these findings, the present study suggests that nanofat boosts tissue vascularization. Thus, nanofat may represent a versatile resource for many applications in tissue engineering and regenerative medicine.

Keywords: dorsal skinfold chamber; fat graft; intravital fluorescence microscopy; nanofat; tissue engineering; vascularization.

FIGURE 1

(A) Inguinal subcutaneous adipose tissue (borders marked by broken line) with its inguinal lymph node (borders marked by dotted line) for the generation of nanofat. Scale bar: 5 mm. (B) Luer lock connectors with descending internal diameters of 2.4 mm (top), 1.4 mm (middle) and 1.2 mm (below) used for fat emulsification. Scale bar: 5 mm. (C) Cell filter (500 μm pore size) sandwiched between two Luer lock connector halves to filter the emulsified fat. Scale bar: 5 mm. (D) Experimental protocol of the present study. The implantation of the dorsal skinfold chamber was performed 48 h prior to nanofat injection in male (n = 8) and female (n = 8) mice. Graft vascularization was repeatedly analyzed by means of intravital fluorescence microscopy (IVM) on day 0, 3, 6, 10 and 14 after transplantation. (E) Overview of the dorsal skinfold chamber during nanofat injection with a 27G needle. Scale bar: 2 mm. (F) Higher magnification of the graft injection site within the observation window. Scale bar: 2 mm.

FIGURE 2

(A) Histological and immunohistochemical stainings of freshly isolated nanofat exhibiting remnants of burst adipocytes and cell detritus (HE; arrowheads), Sirius red⁺ collagen fibers (asterisks) and intact perilipin⁺ adipocytes (hashtags). Scale bars: 25 μm. (B) Immunofluorescent CD31/GFP stainings of freshly isolated nanofat containing intact vessel segments (arrowheads) in between and around adipocytes and adipocyte remnants. Cell nuclei are stained with Hoechst 33,342 (blue). Scale bars: 25 µm. (C) Number of CD31⁺ vessels per HPF in freshly generated nanofat from female (n = 5; white bars) and male (n = 5; black bars) donor mice. (D) Stereomicroscopic and intravital fluorescence microscopic (IVM) images of a nanofat graft (borders marked by dotted line) within the dorsal skinfold chamber of a female mouse on day 0, 3, 6, 10 and 14 after transplantation. Scale bars (upper panels): 2 mm; scale bars (lower panels): 200 μm. Means ± SEM.

FIGURE 3

(A) Intravital fluorescence microscopic images of nanofat grafts in a male and a female mouse on day 0 and day 14 after transplantation. On day 0, the grafts consist of individual cells, fluid or oil vacuoles of varying sizes (asterisks) without a distinct tissue architecture. On day 14, the grafts contain newly formed, microvascular networks interspersed with adipocytes and not yet reabsorbed oil vacuoles (hashtags). Blood perfusion is evidenced by the injection of FITC-labeled dextran for contrast enhancement by staining of blood plasma. Scale bars: 50 μm. (B–G) Perfused surface area ((B), %), FMD ((C), cm/cm²), diameter ((D), µm), centerline RBC velocity ((E), µm/s), VQ ((F), pL/s) and shear rate ((G), s⁻¹) of nanofat grafts in female (white bars; n = 8) and male (black bars; n = 8) mice on days 0, 3, 6, 10 and 14 after transplantation, as assessed by intravital fluorescence microscopy and computer-assisted image analysis. Means ± SEM.

FIGURE 4

(A) Intravital fluorescence microscopic images of GFP⁺ arterioles (arrowheads), capillaries (arrowheads) and venules (arrowheads) before (native) and after the injection of FITC-labled dextran for contrast enhancement by staining of blood plasma on day 14 after transplantation of nanofat into a male mouse. Scale bars: 50 μm. (B) Intravital fluorescence microscopic images of GFP⁺ microvessel sprouts (arrows) growing out of a nanofat graft (border marked by broken line) into the surrounding host tissue on day 14 after transplantation. Insert (border marked by white line) showing the vessel sprouts after the injection of FITC-labeled dextran for contrast enhancement by staining of blood plasma. Scale bar: 50 μm. (C) Presence of blood-perfused GFP⁺ microvessels in the analyzed ROI of nanofat grafts in female (white bars; n = 8) and male (black bars; n = 8) mice, as assessed by intravital fluorescence microscopy. Means ± SEM.

FIGURE 5

(A–C) Histological and immunohistochemical stainings of nanofat grafts in the subcutis (borders marked by broken line) in between the panniculus carnosus muscle and the dermis of recipient mice on day 14 after transplantation. The grafts exhibit microvessels ((A), arrowheads), collagen fibers ((B), asterisks) and mature adipocytes ((C), hashtags). Scale bars: 50 μm.

FIGURE 6

(A–C) Immunofluorescent CD31/GFP stainings of nanofat grafts on day 14 after transplantation into recipient mice. The grafts exhibit CD31⁺/GFP⁺ microvessels ((A), arrowheads) in between GPF⁺ adipocytes ((A), asterisks). These microvessels ((B), arrowheads) develop interconnections to the CD31⁺/GFP⁻ microvessels ((B), arrows) of the surrounding host tissue. Individual CD31⁺/GFP⁺ microvessels can also be detected outside of the nanofat grafts ((C), arrowheads). Cell nuclei are stained with Hoechst 33,342 (blue). Scale bars: 10 μm. (D) CD31⁺/GFP⁺ microvessels (%) in nanofat grafts of female (white bars; n = 8) and male (black bars; n = 8) mice. Means ± SEM.