MTT test for evaluation of dermal viability on reticular dermis.

<p>Results of the vitality index assessment showed the absence of viable cells in the HADM after glycerolization and decellularization processes.</p

Evaluation scores of biomechanical parameters of reticular dermis grafts of increasing thickness treated for 5 weeks with NaOH or DMEM, assessed by two surgeons (S1 and S2).

<p>Evaluation scores of biomechanical parameters of reticular dermis grafts of increasing thickness treated for 5 weeks with NaOH or DMEM, assessed by two surgeons (S1 and S2).</p

Antibodies used for immunohistochemical reactions.

<p>Antibodies used for immunohistochemical reactions.</p

Histochemical reactions showing histological features of human acellular reticular dermal matrices at different incubation times (20X magnification).

<p>In comparison with control (T0) samples (<b>A</b>: hematoxylin-eosin; <b>D</b>: trichrome staining), after 5 weeks of treatment (T5), specimens showed the presence of stromal shrinkage and tissue fragmentation, edema and focal condensations with spongy patterns; these denaturation artifacts were rare in samples treated with DMEM (<b>B</b>: hematoxylin-eosin; <b>E</b>: trichrome staining) and more evident in those treated with NaOH (<b>C</b>: hematoxylin-eosin; <b>F</b>: trichrome staining). Elastic fibers, stained in black using Elastic Von Gieson histochemical reaction, showed no significant alterations in terms of length, diameter and mean number comparing T0 samples (<b>G</b>) with T5 specimens treated with DMEM (<b>H</b>) or NaOH (<b>I</b>).</p

Quantity of DNA in 1 mg of tissue obtained from RD matrices at different incubation times using different decellularization protocols.

<p>The amount of DNA is reported on the y-axis (ng/mg) and the time of incubation is reported on the x-axis (from T1 to T3), using different colors referring to different decellularization methods (blue for DMEM and red for NaOH).</p

Immunohistochemical reactions showing histological features of the cellular component of reticular dermal matrices at different incubation times (20X magnification).

<p>In control samples (T0), the CD31 immunohistochemical reaction showed the presence of intact vessels (<b>A</b>; endothelial cells are stained in brown), while immunostaining was focal and weak after 5 weeks (T5) of treatment with DMEM (<b>B</b>) and completely absent in NaOH treated samples (<b>C</b>). Immunohistochemistry also showed that at T0 both macrophages (<b>D</b>, showing CD68 positive cells) and lymphocytes (<b>G</b>, showing CD45/CLA positive cells) were present, concentrated in perivascular spaces and near the residual lower portion of hair follicles, while only non-specific focal and weak staining was present after 5 weeks (T5) of treatment with DMEM (<b>E</b>, <b>H</b>); reactions were completely negative in NaOH-treated samples (<b>F</b>, <b>I</b>).</p

Mann-Whitney test.

<p>The amount of residual DNA from decellularized RD matrices was not significantly different using DMEM and NaOH.</p