Efficiency assessment of irrigation as an alternative method for improving the regenerative potential of non-healing wounds

Abstract

The application of mesenchymal stem/stromal cells (MSC) in regenerative medicine offers hope for the effective treatment of incurable or difficult-to-heal diseases. However, it requires the development of unified protocols for both safe and efficient cell acquisition and clinical usage. The therapeutic effect of fat grafts (containing stem cells) in non-healing wounds has been discussed in previous studies, although the application requires local or general anaesthesia. The treatment of MSC derived from adipose tissue (ASC) could be a less invasive method, and efficient delivery could lead to more favourable outcomes, which should encourage clinicians to use such therapeutic approaches more frequently. Therefore, the aim of this study was to optimise the methods of ASC isolation, culture and administration while maintaining their high survival, proliferation and colonisation potential. The ASC were isolated by an enzymatic method and were characterised according to International Society for Cellular Therapy and International Federation for Adipose Therapeutics and Science guidelines. To assess the opportunity to obtain a sufficient number of cells for transplantation, long-term cell cultures in two oxygen concentrations (5% vs. 21%) were conducted. For these cultures, the population doubling time, the cumulative time for cell population doublings and the rate of cell senescence were estimated. In a developed and pre-defined protocol, ASC can be efficiently cultured at physiological oxygen concentrations (5%), which leads to faster proliferation and slower cell senescence. Subsequently, to select the optimal and minimally invasive methods of ASC transplantation, direct cell application with an irrigator or with skin dressings was analysed. Our results confirmed that both the presented methods of cell application allow for the safe delivery of isolated ASC into wounds without losing their vitality. Cells propagated in the described conditions and applied in non-invasive cell application (with an irrigation system and dressings) to treat chronic wounds can be a potential alternative or supplement to more invasive clinical approaches.

Keywords: cell therapy; irrigation; mesenchymal stem/stromal cells; non-healing wounds.

FIGURE 1

Scheme of the experiments carried out. The first step of our study was to optimise the method of ASC isolation for easy implementation in the clinical procedure. Additionally, the aim of our study was to optimise cell culture conditions (based on the analysis of ASC with the application of microscopic and cytometric techniques as well as microplate tests) and methods for cell delivery into wounds while maintaining their high survival, proliferation and colonisation potential

FIGURE 2

Characterisation of the ASC. (A) Flow cytometry analysis showed typical and relatively high expression of specific mesenchymal markers (CD73, CD90 and CD105). Similarly, less than 1% of the ASC expressed negative markers (CD34, CD11b, CD19, CD45 or HLA‐DR). (B) Immunocytochemical analysis of the ASC. Scale bar: 50 μM. (C) Multilineage mesodermal differentiation potential of the ASC. A positive differentiation result was noted for the ASC differentiated into bone, adipose and cartilage lineages after oil red O (adipogenesis), Alcian blue (chondrogenesis) and Alizarin red S (osteogenesis) staining, respectively. (D) ASC vasculogenic potential analysis. ASC labelled with CMFDA formed a network, and pictures were taken at two time points: 2 h (left image) and 4 h (right image) after the ASC were seeded on Matrigel. Scale bar: 100 μM

FIGURE 3

Proliferation and senescence analysis of ASC cultured under 5% and 21% O₂ conditions. (A) Morphological and SA‐β‐gal activity changes in ASC during long‐term culture. Photos magnified 100×. Scale bar: 200 μm. (B) Growth curve of the ASC. ASC cultured with 5% O₂ proliferated faster. The results showed significantly higher cell numbers per cm² for ASC in the 1st passage and a stable growth phase from the 8th to the 15th passages. (C) ASC population doubling time analysis. The population doubling time was shorter at all passages for the cells cultured with 5% oxygen and significantly shorter at the 1st, 2nd, 4th, 6th—13th, 15th, 17th, and 18th passages. (D) Cumulative population doubling. The CPD was relatively higher for cells cultured with 5% oxygen in the 6th passage and later. (E) One‐week ASC growth analysis. ASC presented a three‐phase growth rate, which was significantly higher for the cells cultured with 5% oxygen. The results are presented as the mean ± SD of 4–6 experiments; *𝑝 < 0.05, **𝑝 < 0.01; and ***𝑝 < 0.001. Blue points—parameters of the cells cultured with 5% oxygen; green points—parameters of the cells cultured with 21% oxygen

FIGURE 4

Characterisation of the ASC and fibroblast coculture. (A) ASC and fibroblast integration analysis. Good integration, possible niche sharing and a common monolayer of ASC and fibroblasts were visualised, with vimentin and fibronectin labelling shown in red, ASC shown by the green CMFDA tracer, and the cell nuclei stained blue with Hoechst stain. Scale bar: 50 μm. (B) The effect of ASC on fibroblast migration—scratch assay. Photos show the migratory properties of cocultured ASC and fibroblasts at three different time points—0, 24 and 48 h at a magnification of 100×. The graph shows the migratory properties of fibroblasts compared with fibroblasts cocultured with ASC. Fibroblasts cocultured with ASC presented a faster overgrowth rate, especially after 48 h. The results are presented as the mean ± SD of at least 4 experiments; ***𝑝 < 0.001

FIGURE 5

Optimization of the methods for ASC wound application. (A) ASC cocultured with hydrocolloid and paraffin dressings. Photos show the CMFDA tracer‐labelled ASC in coculture with hydrocolloid and paraffin patches and live/dead staining with live cells labelled with calcein (green) and dead cells labelled with the ethidium homodimer (red). High survival (±77% and ±82%) of these cells 24 h after seeding on hydrocolloid and paraffin patch surfaces was proven. (B) Analysis of the vasculogenic potential of ASC seeded on gelatin patches. ASC formed a network, and pictures were taken at three time points: 0 h (the first image presents ASC cocultured with a gelatin sponge before application on Matrigel), 2 h (second image) and 4 h (third image) after seeding of the ASC on Matrigel. Scale bar: 100 μM. (C) Irrigator application to cover extensive wounds. Comparable ASC survival was confirmed for concentrated and distributed streams: ±83% and ±82.5%. The rate of fluid delivery (slow—1st mode, intermediate—3rd mode and fast—5th mode) did not influence ASC survival during wound application. Suspending ASCs in Ringer's solution resulted in greater cell viability than suspending ASCs in sodium chloride (0.9% NaCl) in the three different speed modes of liquid delivery. Scale bar: 100 μM

FIGURE 6

Clinical ASC application to an extensive wound. (A and B) A 70‐year‐old patient with a seven‐year history of arteriosclerosis with a chronic wound on the lower leg with tibial bone exposure after vascular angioplasties in both legs and failed attempts with conservative and surgical wound treatments. (C) Two weeks after cortical bone layer removal, ASC were administered with the irrigator and a wound were covered with split thickness skin graft. Total wound closure was achieved in 4 weeks. (D and E) Sixty‐month follow‐up; no signs of wound recurrence

FIGURE 7

Clinical ASC application to a deep, penetrating wound. (A and B) A deep and penetrating wound after complicated fracture of the proximal tibia and fibula in a 60‐year‐old obese man. Two applications of ASC with dressing were performed. (C) At the 36‐month follow‐up, no sign of wound recurrence was observed