Generation of Connective Tissue-Free Microvascular Fragment Isolates from Subcutaneous Fat Tissue of Obese Mice

Abstract

Background: Microvascular fragment (MVF) isolates are generated by short-term enzymatic digestion of adipose tissue and contain numerous vessel segments for the vascularization of tissue defects. Recent findings indicate that the functionality of these isolates is determined by the quality of the fat source. Therefore, we compared MVF isolates from subcutaneous adipose tissue of obese and lean mice.

Methods: MVF isolates were generated from subcutaneous adipose tissue of donor mice, which received a high fat or control diet for 12 weeks. The isolates were analyzed in vitro and in vivo.

Results: Feeding of mice with a high fat diet induced obesity with adipocyte hypertrophy, resulting in a significantly lower collagen fraction and microvessel density within the subcutaneous fat depots when compared to lean controls. Accordingly, MVF isolates from obese mice also contained a reduced number of MVF per mL adipose tissue. However, these MVF tended to be longer and, in contrast to MVF from lean mice, were not contaminated with collagen fibers. Hence, they could be freely seeded onto collagen-glycosaminoglycan scaffolds, whereas MVF from lean controls were trapped in between large amounts of collagen fibers that clogged the pores of the scaffolds. In line with these results, scaffolds seeded with MVF isolates from obese mice exhibited a significantly improved in vivo vascularization after implantation into full-thickness skin defects.

Conclusion: Subcutaneous adipose tissue from obese mice facilitates the generation of connective tissue-free MVF isolates. Translated to clinical conditions, these findings suggest that particularly obese patients may benefit from MVF-based vascularization strategies.

Keywords: Microvascular fragments; Obesity; Subcutaneous fat tissue; Tissue engineering; Vascularization.

Fig. 1

Histomorphology of subcutaneous adipose tissue from lean and obese mice. A, B HE-stained sections of the subcutaneous adipose tissue from a lean (A) and an obese (B) mouse. Scale bars: 30 µm. C, D Adipocyte diameter (C, given in µm) and adipocyte density (D, given in mm⁻²) within the subcutaneous adipose tissue from lean (white bars, n = 5) and obese (black bars, n = 5) mice. Means ± SEM. *p < 0.05 versus lean. E, F Sirius red-stained sections under polarized light, displaying larger blood vessels (arrows) with surrounding type I collagen within the subcutaneous adipose tissue from a lean (E) and an obese (F) mouse Scale bars: 40 µm. G Collagen fraction (given in % of adipose tissue) within the subcutaneous adipose tissue from lean (white bar, n = 5) and obese (black bar, n = 5) mice. Means ± SEM. *p < 0.05 vs. lean. H, I Immunohistochemical detection of CD31⁺ microvessels (arrows) between adipocytes (asterisks) within the subcutaneous adipose tissue from a lean (H) and an obese (I) mouse. Cell nuclei were stained with Hoechst 33342. Scale bars: 15 µm. J Microvessel density (given in mm⁻²) within the subcutaneous adipose tissue from lean (white bar, n = 5) and obese (black bar, n = 5) mice. Means ± SEM. *p < 0.05 versus lean

Fig. 2

Characterization of MVF isolates. A Number of MVF (given per mL adipose tissue) isolated from the subcutaneous adipose tissue of lean (white bar, n = 10) and obese (black bar, n = 10) mice. Means ± SEM. *p < 0.05 versus lean. B, C Fluorescence microscopic images of PI-stained MVF isolated from the subcutaneous adipose tissue of a lean (B) and an obese (C) mouse (arrows = dead PI⁺ cells). Cell nuclei were stained with Hoechst 33342. Scale bars: 45 μm. D PI⁺ cells (given in % of all counted cells) within MVF isolated from the subcutaneous adipose tissue of lean (white bar, n = 5) and obese (black bar, n = 5) mice. Means ± SEM. E Length distribution (given in %) of MVF isolated from the subcutaneous adipose tissue of lean (white bars, n = 5) and obese (black bars, n = 5) mice. Means ± SEM. *p < 0.05 versus lean. F, G Light microscopic images of MVF isolates from the subcutaneous adipose tissue of a lean (F) and an obese (G) mouse (arrows = MVF; arrowheads = connective tissue fibers). Scale bars: 90 µm

Fig. 3

Surface morphology of MVF-seeded CGAG scaffolds. A–F Scanning electron microscopic images of CGAG scaffolds directly after their seeding with MVF isolates from the subcutaneous tissue of a lean (A–C) and an obese (D–F) MVF mouse. B and C as well as E and F display higher magnifications of white frames in A and B as well as D and E (arrows = MVF; asterisks = scaffold pores). Scale bars: A, D = 790 µm; B, E = 170 µm; C, F = 45 µm

Fig. 4

Vascularization of implanted MVF-seeded CGAG scaffolds. A–D Intravital fluorescence microscopy in blue light epi-illumination (contrast enhancement with 5% FITC-labeled dextran) of CGAG scaffolds (borders marked by broken lines in A and B) seeded with MVF isolates from the subcutaneous adipose tissue of a lean (A, C) and an obese (B, D) mouse on day 14 after implantation into full-thickness skin defects within the dorsal skinfold chamber of recipient mice (arrows = blood-perfused microvessels, asterisks = non-perfused implant areas). Scale bars: A, B = 280 µm; C, D = 60 µm. E, F Perfused scaffold areas (E, given in %) and functional microvessel density (F, given in cm/cm²) of CGAG scaffolds seeded with MVF isolates from the subcutaneous adipose tissue of lean (white circles, n = 10) and obese (black circles, n = 10) mice on day (d) 0, 3, 6, 10 and 14 after implantation into full-thickness skin defects within the dorsal skinfold chamber of recipient mice. Means ± SEM. *p < 0.05 versus lean

Fig. 5

Histomorphology of implanted MVF-seeded CGAG scaffolds. A, B HE-stained sections of CGAG scaffolds (borders marked by broken lines) seeded with MVF isolates from the subcutaneous adipose tissue of a lean (A) and an obese (B) mouse on day 14 after implantation into full-thickness skin defects within the dorsal skinfold chamber of recipient mice. Scale bars: 230 µm. C–E Sirius red-stained sections under polarized light of normal skin (C) as well as CGAG scaffolds seeded with MVF isolates from the subcutaneous adipose tissue of a lean (D) and an obese (E) mouse (arrows = collagen fibers). Scale bars: 12 µm. F Total collagen ratio (given as implant/skin) of CGAG scaffolds seeded with MVF isolates from the subcutaneous adipose tissue of lean (white bar, n = 10) and obese (black bar, n = 10) mice on day 14 after implantation into full-thickness skin defects within the dorsal skinfold chamber of recipient mice. Means ± SEM. G, H Immunohistochemical detection of CD31⁺ microvessels (arrows) within CGAG scaffolds seeded with MVF isolates from the subcutaneous adipose tissue of a lean (G) and an obese (H) mouse. Cell nuclei were stained with Hoechst 33342. Scale bars: 30 µm. I Microvessel density (given in mm⁻²) of CGAG scaffolds seeded with MVF isolates from the subcutaneous adipose tissue of lean (white bar, n = 10) and obese (black bar, n = 10) mice on day 14 after implantation into full-thickness skin defects within the dorsal skinfold chamber of recipient mice. Means ± SEM. *p < 0.05 versus lean. J–L Immunohistochemical detection of a CD31⁺/GFP⁺ microvessel (arrowhead) and a CD31⁺/GFP⁻ microvessel (arrow) within a CGAG scaffold seeded with an MVF isolate from the subcutaneous adipose tissue of a lean mouse. Cell nuclei were stained with Hoechst 33342. Scale bars: 20 µm. M CD31⁺/GFP⁺ microvessels (given in %) within CGAG scaffolds seeded with MVF isolates from the subcutaneous adipose tissue of lean (white bar, n = 10) and obese (black bar, n = 10) mice on day 14 after implantation into full-thickness skin defects within the dorsal skinfold chamber of recipient mice. Means ± SEM