Heat Preconditioning of Nanofat Does Not Improve Its Vascularization Properties

Abstract

Heat preconditioning has been shown to promote nutritive perfusion and tissue survival in autologous fat grafting as well as in flap and breast surgery. However, its impact on the vascularization properties of nanofat has not been investigated so far. Therefore, we exposed nanofat from donor mice to a temperature of 43 °C for 1 h and assessed the effects of this heat stress on cell viability and the expression of heat shock proteins (HSPs) and angiogenesis-related factors. Moreover, dermal substitutes seeded with heat-preconditioned and non-preconditioned control nanofat were implanted into dorsal skinfold chambers of recipient mice to study their vascularization and tissue integration in vivo by means of repeated intravital fluorescence microscopy, histology and immunohistochemistry. Heat preconditioning upregulated the expression of HSPs in nanofat without affecting cell viability. Moreover, it resulted in the downregulation of many pro-angiogenic factors and the increased expression of anti-angiogenic factors, indicating a shift towards an anti-angiogenic phenotype. Accordingly, implanted dermal substitutes seeded with heat-preconditioned nanofat exhibited a reduced vascularization and were not better integrated into the host tissue when compared to controls. These findings indicate that heat preconditioning cannot be recommended for enhancing the vascularization capacity of nanofat.

Keywords: Integra®; angiogenesis; dermal substitutes; heat preconditioning; inflammation; nanofat; survival; vascularization.

Figure 1

In vitro characterization of control and heat-preconditioned nanofat. (A) Fraction (%) of vital, apoptotic, necroptotic and necrotic cells in non-preconditioned nanofat (control; white bars, n = 4) and heat-preconditioned nanofat (heat; black bars, n = 4). Mean ± SEM. (B,C) Immunohistochemical assessment of Casp-3⁺ apoptotic cells ((B), arrows) and their quantitative analysis (C) in non-preconditioned nanofat (control; white bar, n = 4) and heat-preconditioned nanofat (heat; black bar, n = 4). Mean ± SEM. (D) mRNA expression (% of control) of HSP-27, HSP-70, HSP-90 and HSP-32/HO-1 in non-preconditioned nanofat (control; white bars, n = 4) and heat-preconditioned nanofat (heat; black bars, n = 4). Mean ± SEM; * p < 0.05 vs. control. (E,F) Immunohistochemical assessment of HO-1⁺ cells ((E), arrows) and their quantitative analysis (F) in non-preconditioned nanofat (control; white bar, n = 4) and heat-preconditioned nanofat (heat; black bar, n = 4). Mean ± SEM; * p < 0.05 vs. control.

Figure 2

In vivo microscopy of nanofat-seeded implants. (A,B) Representative intravital fluorescence microscopic images of dermal substitutes seeded with non-preconditioned (control, (A)) and heat-preconditioned nanofat (heat, (B)) (implant border = closed line; border of non-vascularized implant area = broken line). (C–F) Perfused ROIs (%) (C,D) and functional microvessel density (cm/cm²) (E,F) in the border (C,E) and center (D,F) of dermal substitutes seeded with non-preconditioned (control; white bars, n = 8) and heat-preconditioned nanofat (heat; black bars, n = 8). Mean ± SEM.

Figure 3

Interaction of leukocytes with the microvascular endothelium in response to nanofat-seeded implants. (A) Representative intravital fluorescence microscopic images of a venule next to an implanted dermal substitute seeded with non-preconditioned nanofat (blue light epi-illumination, contrast enhancement by 5% fluorescein isothiocyanate (FITC)-labeled dextran (left panel); green light epi-illumination, in situ staining of leukocytes with 0.1% rhodamine 6G (right panel); arrows = leukocytes). (B,C) Rolling leukocytes (min⁻¹) (B) and adherent leukocytes (mm⁻²) (C) within venules next to dermal substitutes seeded with non-preconditioned (control; white bars, n = 8) and heat-preconditioned nanofat (heat; black bars, n = 8). Mean ± SEM.

Figure 4

Tissue incorporation of nanofat-seeded implants. (A,B) HE-stained sections of implanted dermal substitutes seeded with non-preconditioned (control, (A)) and heat-preconditioned nanofat (heat, (B)) (implant border = closed line; border zone = broken line; ROIs in the border and center zones of the implants shown in higher magnification in (C,D) = blue and red frame; panniculus carnosus muscle = arrows; granulation tissue = asterisks). (C,D) Higher magnification of blue and red frames in (A) and (B) in the border (C) and center (D) zones of the implants.

Figure 5

Collagen content of nanofat-seeded implants. (A,C) Immunohistochemical assessment of Col I (A) and III (C) in the border and center zones of dermal substitutes seeded with non-preconditioned (control) and heat-preconditioned nanofat (heat). (B,D) Total Col I (B) and Col III (D) ratio (implant/skin) in the border and center zones of dermal substitutes seeded with non-preconditioned (control; white bars, n = 8) and heat-preconditioned nanofat (heat; black bars, n = 8). Mean ± SEM.

Figure 6

(A) Immunohistochemical assessment of CD31⁺ microvessels in the border zones (arrowheads) and the center (arrows) of dermal substitutes seeded with non-preconditioned (control) and heat-preconditioned nanofat (implant border = closed line; border zone = dotted line). (B) Microvessel density (mm⁻²) of dermal substitutes seeded with non-preconditioned (control; white bars, n = 8) and heat-preconditioned nanofat (heat; black bars, n = 8). Mean ± SEM. * p < 0.05 vs. control. (C) Immunohistochemical assessment of CD31⁺/GFP⁻ (arrowheads) and CD31⁺/GFP⁺ (arrows) microvessels within dermal substitutes seeded with non-preconditioned (control) and heat-preconditioned nanofat (heat). (D) CD31⁺/GFP⁺ microvessels (%) in the border and center zones of dermal substitutes seeded with non-preconditioned (control; white bars, n = 8) and heat-preconditioned nanofat (heat; black bars, n = 8). Mean ± SEM. * p < 0.05 vs. control. (E) Immunohistochemical assessment of LYVE-1⁺ lymph vessels in the border zones (arrowheads) of dermal substitutes seeded with non-preconditioned (control) and heat-preconditioned nanofat (heat) (implant border = closed line; border zone = dotted line). (F) Lymph vessel density (mm⁻²) of dermal substitutes seeded with non-preconditioned (control; white bars, n = 8) and heat-preconditioned nanofat (heat; black bars, n = 8). Mean ± SEM. (G) Immunohistochemical assessment of LYVE-1⁺/GFP⁻ (arrowheads) and LYVE-1⁺/GFP⁺ (arrows) lymph vessels in a dermal substitute seeded with non-preconditioned nanofat (control). (H) LYVE-1⁺/GFP⁺ microvessels (%) in the border and center zones of dermal substitutes seeded with non-preconditioned (control; white bars, n = 1–5) and heat-preconditioned nanofat (heat; black bars, n = 0–5). Mean ± SEM.

Figure 7

Immune cell infiltration of nanofat-seeded implants. (A,C,E) Immunohistochemical assessment of CD68⁺ macrophages ((A), arrows), MPO⁺ granulocytes ((C), arrows) and CD3⁺ lymphocytes ((E), arrows) in the border and center zones of dermal substitutes seeded with non-preconditioned (control) and heat-preconditioned nanofat (heat). (B,D,F) CD68⁺ macrophages (mm⁻²) (B), MPO⁺ granulocytes (mm⁻²) (D) and CD3⁺ lymphocytes (mm⁻²) (F) in the border and center zones of dermal substitutes seeded with non-preconditioned (control; white bars, n = 8) and heat-preconditioned nanofat (heat; black bars, n = 8). Mean ± SEM. * p < 0.05 vs. control.