Integra dermal regeneration template single layer without silicone sheet

A 4-mm biopsy punch (kaiEurope GmbH, Solingen, Germany) was used to cut identical
12.5 mm² samples out of a 1.3 mm-thick Integra^® Dermal
Regeneration Template Single Layer without silicone sheet (Integra Life
Sciences, Ratingen, Germany). The matrices were then placed on a 500 µm cell
strainer and 10 µL 0.9% NaCl containing ~20,000 MVF and ~200,000 single cells
were transferred onto the matrices with a 20 µL pipette (Eppendorf,
Wesseling-Berzdorf, Germany) (Figure 1(b)). This pipette was additionally used to induce negative
pressure from underneath the CGAG matrices to ensure a sufficient seeding depth
of MVF.^{10 (link)} The MVF-seeded matrices were then either directly processed for further
histological and immunohistochemical analyses or implanted into full-thickness
skin defects for in vivo analyses.

To generate standardized collagen–glycosaminoglycan matrices for the experiments, 12.5-mm² samples were identically cut out of a 1.3 mm thick Integra^® Dermal Regeneration Template Single Layer without silicone sheet (Integra Life Sciences, Ratingen, Germany) with a 4-mm biopsy punch (kai Europe GmbH, Solingen, Germany) and stored in PBS until their use. Before the seeding, 10 µL PBS was carefully sucked out of the matrices by means of a pipette (Eppendorf, Wesseling-Berzdorf, Germany). They were then placed on a 500-µm cell strainer, and 10 µL 0.9% NaCl containing ~10,000 MVF and ~200,000 single cells were transferred onto the matrices (Figure 1(b)).^{9 (link)} In addition, a 10-µL pipette was used to induce negative pressure from underneath the matrices. In previous studies, we have already shown that this procedure results in a sufficient entrapment of MVFs within the superficial layers of the matrices, which is necessary to guarantee a rapid reassembly of individual MVFs into new microvascular networks.^{10 (link),19 (link)}