Cell Sheets from Adipose Tissue MSC Induce Healing of Pressure Ulcer and Prevent Fibrosis via Trigger Effects on Granulation Tissue Growth and Vascularization

Abstract

We report a comparative study of multipotent mesenchymal stromal cells (MSC) delivered by injection, MSC-based cell sheets (CS) or MSC secretome to induce healing of cutaneous pressure ulcer in C57Bl/6 mice. We found that transplantation of CS from adipose-derived MSC resulted in reduction of fibrosis and recovery of skin structure with its appendages (hair and cutaneous glands). Despite short retention of CS on ulcer surface (3–7 days) it induced profound changes in granulation tissue (GT) structure, increasing its thickness and altering vascularization pattern with reduced blood vessel density and increased maturation of blood vessels. Comparable effects on GT vascularization were induced by MSC secretome, yet this treatment has failed to induce repair of skin with its appendages we observed in the CS group. Study of secretome components produced by MSC in monolayer or sheets revealed that CS produce more factors involved in pericyte chemotaxis and blood vessel maturation (PDGF-BB, HGF, G-CSF) but not sprouting inducer (VEGF165). Analysis of transcriptome using RNA sequencing and Gene Ontology mapping found in CS upregulation of proteins responsible for collagen binding and GT maturation as well as fatty acid metabolism enzymes known to be negative regulators of blood vessel sprouting. At the same time, downregulated transcripts were enriched by factors activating capillary growth, suggesting that in MSC sheets paracrine activity may shift towards matrix remodeling and maturation of vasculature, but not activation of blood vessel sprouting. We proposed a putative paracrine trigger mechanism potentially rendering an impact on GT vascularization and remodeling. Our results suggest that within sheets, MSC may change their functional state and spectrum of soluble factors that influence tissue repair and induce more effective skin healing inclining towards regeneration and reduced scarring.

Measurement of the Adipose Stem Cells Cell Sheets Transmittance

“…In general, the cell sheet could be digested with an enzyme, and the number of cells per cell sheet could be counted with the trypan blue methodology. However, ASCCS are very difficult to digest with dispase, trypsin and another enzyme, and it was observed in other laboratories [30]. We decided to isolate the genomic DNA from the entire cell sheets and to measure the content of the double strand DNA (dsDNA).…”

“…This device could address the needs in the increasing cell sheet technology field and could help inexperienced people in the field of cell sheet to determine when a cell sheet is ready for harvesting, in well-defined cell culture conditions (culture media, type of cells, initial seeding, etc.). Moreover, additional studies will be necessary to study the transmittance of different type of cell sheets, that can be engineered with different sources of cells: cardiac, corneal, dental, epithelial [30,35,[69][70][71].…”

“…Because of the difficulty to dissociate the cells from the multilayer cell sheet (data not published and [30]) to obtain single cells, the number of cells was estimated by isolating total genomic DNA from entire cell sheets and compared to the quantity of genomic DNA from a determined number of isolated ASC. Genomic DNA, from isolated ASC and multilayer cell sheets, was isolated with the Wizard Genomic DNA purification Kit (Promega, Madison, WI, USA), following the manufacturer protocol.…”

Systematic Review: Adipose-Derived Mesenchymal Stem Cells, Platelet-Rich Plasma and Biomaterials as New Regenerative Strategies in Chronic Skin Wounds and Soft Tissue Defects

“…Full-thickness skin defects caused by severe trauma, extensive burns, diabetes, and other reasons are common clinical disorders; their healing is a dynamic and multi-stage process that includes inflammation, angiogenesis, matrix deposition, and cell recruitment [63,64]. Additionally, there are multiple kinds of cells involved, including keratinocytes, endothelial cells, fibroblasts, inflammatory cells, and so forth.…”

Cell Sheets Restore Secretory Function in Wounded Mouse Submandibular Glands

“…These results indicate that cell sheets promote rapid wound healing, consistent with previous findings with other organs. Specifically, a wounded skin mouse model treated with mesenchymal stem cell sheets formed organized blood vessels and exhibited increased expression of angiogenic factors, thus leading to tissue healing at twenty-one days post-surgery [ 63 ]. Likewise, mesenchymal stem cell sheets were shown to improve kidney function by reducing fibrosis and microvasculature damage in a renal ischemia-reperfusion injury rat model fourteen days post-surgery [ 64 ].…”