Adipose Tissue-Derived Microvascular Fragments Improve Vascularization, Lymphangiogenesis, and Integration of Dermal Skin Substitutes

Abstract

Full-thickness skin defects can be covered with dermal skin substitutes in combination with split-thickness skin grafts. However, slow vascularization of the matrices bears the risk of wound infection and extends the length of hospitalization. To overcome these problems, we describe a promising vascularization strategy. Green fluorescent protein adipose tissue-derived microvascular fragments (ad-MVF) were isolated from epididymal fat pads of C57BL/6-Tg(CAG-EGFP)1Osb/J mice. ad-MVF were seeded on collagen-glycosaminoglycan matrices, which were implanted into full-thickness skin defects in the dorsal skinfold chamber of wild-type C57BL/6 mice. Nonseeded matrices served as controls. Vascularization, lymphangiogenesis, and integration of the implants were studied by using intravital fluorescence microscopy, histology, and immunohistochemistry over 14 days. ad-MVF rapidly reassembled into microvascular networks within the implants, which developed interconnections to the host microvasculature. Accordingly, vascularization of the implants was markedly accelerated, as indicated by a significantly higher microvessel density when compared with controls. Moreover, dense lymphatic networks originating from the green fluorescent protein ad-MVF developed within the implants. This was associated with an improved implant integration. Hence, seeding ad-MVF on collagen-glycosaminoglycan matrices represents a potential strategy to reduce morbidity and hospitalization of patients undergoing the treatment of full-thickness skin defects.

Potentials and limitations of Integra® flowable wound matrix seeded with adipose tissue-derived microvascular fragments

“…As previously described in detail, ad-MVF were isolated from epididymal fat pads of GFP + donor mice (Frueh et al, 2017). Briefly, the bilateral epididymal fat pads were removed, transferred into 10 % Dulbecco's modified eagle medium (DMEM; 100 U/mL penicillin, 0.1 mg/mL streptomycin; Biochrom, Berlin, Germany) and washed thrice with phosphate-buffered saline (PBS).…”

“…To quantify the collagen content of implanted MWD and FWM, additional sections were stained with sirius red. Using a BX60 microscope (Olympus, Hamburg, Germany) and the imaging software cellSens Dimension 1.11 (Olympus), the collagen content was assessed in relation to normal skin in 4 ROIs per sample (Frueh et al, 2017 (Olympus) to assess the distribution and the distance of individual ad-MVF within the samples. For this purpose, the number of CD31 + microvessels was determined in 20 ROIs per implant (equivalent to the entire implant area per section), to calculate the coefficient of variation (cv; standard deviation/mean) of the spatial fragment distribution.…”

“…Recently, we could demonstrate that the seeding of dermal skin substitutes with ad-MVF, which already represent fully functional vessel segments, may be even more effective (Frueh et al, 2017). Accordingly, they only need to develop interconnections with each other and the surrounding host microvasculature to get rapidly reperfused within a few days .…”

“…The kinetics of this biological incorporation process crucially determines the risk of wound infection, which is elevated as long as the physiological skin barrier function is not fully re-established at the defect site (Ruszczak, 2003). Recently, we demonstrated that the seeding of Integra ® matrix wound dressing (MWD) with adipose tissue-derived microvascular fragments (ad-MVF) markedly improves its epithelialisation, vascularisation and incorporation into the host tissue (Frueh et al, 2017). MWD is a clinically well-established dermal skin substitute, consisting of cross-linked bovine tendon collagen and shark glycosaminoglycan (Burke et al, 1981;Reid et al, 2007;Graham et al, 2013;Rowan et al, 2015).…”

“…MWD is a clinically well-established dermal skin substitute, consisting of cross-linked bovine tendon collagen and shark glycosaminoglycan (Burke et al, 1981;Reid et al, 2007;Graham et al, 2013;Rowan et al, 2015). Of interest, the seeding of the matrix with ad-MVF was mainly restricted to its surface, because larger ad-MVF could not penetrate its small pores (Frueh et al, 2017). Accordingly, in the present study, we hypothesised that the seeding of ad-MVF on a dermal matrix allowing a more homogeneous distribution may result in a faster vascularisation and incorporation, when compared to MWD.…”