Nanostructured Cellulose-Gellan-Xyloglucan-Lysozyme Dressing Seeded with Mesenchymal Stem Cells for Deep Second-Degree Burn Treatment

Abstract

Purpose: In deep burns, wound contraction and hypertrophic scar formation can generate functional derangement and debilitation of the affected part. In order to improve the quality of healing in deep second-degree burns, we developed a new treatment in a preclinical model using nanostructured membranes seeded with mesenchymal stem cells (MSCs).

Methods: Membranes were obtained by reconstitution of bacterial cellulose (reconstituted membrane [RM]) and produced by a dry-cast process, then RM was incorporated with 10% tamarind xyloglucan plus gellan gum 1:1 and 10% lysozyme (RMGT-LZ) and with 10% gellan gum and 10% lysozyme (RMG-LZ). Membrane hydrophobic/hydrophilic characteristics were investigated by static/dynamic contact-angle measurements. They were cultivated with MSCs, and cell adhesion, proliferation, and migration capacity was analyzed with MTT assays. Morphological and topographic characteristics were analyzed by scanning electron microscopy. MSC patterns in flow cytometry and differentiation into adipocytes and osteocytes were checked. In vivo assays used RMG-LZ and RMGT-LZ (with and without MSCs) in Rattus norvegicus rats submitted to burn protocol, and histological sections and collagen deposits were analyzed and immunocytochemistry assay performed.

Results: In vitro results demonstrated carboxyl and amine groups made the membranes moderately hydrophobic and xyloglucan inclusion decreased wettability, favoring MSC adhesion, proliferation, and differentiation. In vivo, we obtained 40% and 60% reduction in acute/chronic inflammatory infiltrates, 96% decrease in injury area, increased vascular proliferation and collagen deposition, and complete epithelialization after 30 days. MSCs were detected in burned tissue, confirming they had homed and proliferated in vivo.

Conclusion: Nanostructured cellulose-gellan-xyloglucan-lysozyme dressings, especially when seeded with MSCs, improved deep second-degree burn regeneration.

Keywords: cell-based therapy; hydrocolloid dressing; skin; transplantation.

Figure 1

Biparametric graphs divided into four quadrants of results obtained by flow cytometry. Comparison between cell immunostainers is indicated in each graph.

Figure 2

Microscopy of isolated MSCs in culture flasks, 100×. (A) MSCs differentiated in adipocytes; (B) MSCs differentiated in osteocytes; (C) MSCs in standard cultivation medium; (D) MSCs differentiated in adipocytes on RMGT–LZ; (E) MSCs differentiated in osteocytes on RMGT–LZ; (F) MSCs in standard cultivation medium on RMGT–LZ.

Figure 3

Static (A) and hysteretic (B) contact-angle results of DMEM solution on membranes (*p≤0.05, ***p≤0.001).

Figure 4

Results of adhesion (A) and proliferation (B) of mesenchymal stem cells seeded on membranes (*p≤0.05, **p≤0.01, ***p≤0.001).

Figure 5

Scanning electron microscopy of membranes seeded with MSCs and comparison of MSC morphology on the membranes at different magnifications. In the images in the second column, the arrows point to the MSCs. In the images in the third column, the values of the width of some fibers are indicated, allowing the comparison of the mesh of each membrane and its possible relationship with cell interaction.

Figure 6

Acute inflammation analysis on days 5 (A) and 30 (B) and chronic inflammation analysis on days 5 (C) and 30 (D) (*p≤0.05, **p≤0.01).

Figure 7

Vascular proliferation results on day 5 (A) and epithelialization results on day 30 (B) (*p≤0.05). Photographs comparing the healing obtained after debridement and treatment. Photos of the control group on days 5 (C) and 30 (D); other photo of RMGT–LZ seeded with MSCs on days 5 (E) and 30 (F). The dotted circle indicates the scar area.

Figure 8

Histological sections of the burns of all groups (100×, bar 100 µm), allowing comparison of the healing achieved by all groups 30 days after the burn and treatment. RMG–LZ with cells on days 5 (A) and 30 (B), RMG–LZ without cells on days 5 (C) and 30 (D), RMGT–LZ with cells on days 5 (E) and 30 (F), RMGT–LZ without cells on days 5 (G) and 30 (H), MC with cells on days 5 (I) and 30 (J), MC without cells on days 5 (K) and 30 (L), control on days 5 (M) and 30 (N).

Figure 9

Microscopy of the collagen areas (400×, bar 50 µm) of the control group on days 5 (A) and 30 (B) and of MC with cells on days 5 (C) and 30 (D).

Figure 10

Microscopy of the injured area after 15 days of healing, showing MSCs that have migrated from RMGT–LZ to the wound. These MSCs express BrdU (green fluorescence) and have the nuclei marked by DAPI (blue fluorescence). 100×. Yellow circles indicate these cells.