Evaluation of Safety and Efficacy of Skin Cell Sheets in the Treatment of Chronic Wounds: Clinical Case Study

Chronic wounds, for example diabetic wounds or foot ulcers, are defined as wounds that fail be healed by themselves in a timely manner, leading to bacterial infections and severe complications. Immediate treatment of chronic wounds is needed to prevent further amputations. Chronic wound healing is always delayed due to circulatory problems. The presence of capillaries inside the wound is crucial because the regeneration of tissue commonly requires blood flow to carry oxygen and nutrients, as well as to remove wastes. Therefore, increasing the blood vessel density at the wound site can promote wound healing.

Recently, many cell-based therapies have been used as alternative treatments to accelerate wound healing and to treat skin defects after debridement or necrosis removal. For example, the suspension of skin cells is mostly utilized due to its immediately accessible application. However, the limitations of the single-cell suspension are the uncontrolled location of the cells and the unpredictable number of viable cells because the proteins or factors involved in the cell survival process are digested. Due to the drawbacks of the single-cell suspension, tissue engineering with scaffolds is proposed. Although using a biomaterial scaffold can eliminate these issues, it has an effect on the body’s immune response, leading to graft rejection. Most scaffolds are synthetic or made from other species-derived materials that can stimulate an allergic systemic reaction, leading to incomplete healing and graft failure. To overcome this problem, the cell sheet engineering method is suggested.

Technology using temperature-responsive polymers to construct cell sheets has attracted attention for decades due to its benefits including the remaining of the necessary proteins, and the lack of chemical or animal-derived material reagents. The surface of a culture dish is coated with poly(N-isopropylacrylamide) or PNIAM, which can reverse the process of cell adhesion to and detachment from the dish by controlling the hydrophobicity of the surface. The characteristics of the surface are controlled by temperature. The cells are able to attach and proliferate on the polymer-grafted surface at 37°C because the polymer chains are hydrophobic and allow the cells to grow on the culture surface. In contrast, when the temperature is reduced below its lower critical solution temperature (LCST) of PNIAM, these polymer chains change to a hydrophilic phase. Thus, confluent cells can be harvested as single, continuous cell sheets with intact cell-cell  junctions.

Currently, temperature-responsive culture surfaces are commercially available under the trade name UpCell^TM. Unfortunately, this cultureware is more expensive than regular tissue culture dishes because coating PNIAM onto culture dish surfaces requires special and costly equipment. To address this problem, our research group has developed in-house temperature-responsive polymer culture dishes by grafting poly(N-isopropylacrylamide)-co-acrylamide or PNIAM-co-AM onto the culture surfaces via UV polymerization. These surfaces have been shown to possess the thermoreversibility property, enabling the construction of cell sheets.

With cell sheet technology, we successfully constructed human keratinocyte-fibroblast cell sheets from temperature-responsive PNIAM-co-AM grafted surfaces for the treatment of diabetic wounds. Both keratinocytes and fibroblasts were detached as intact layers from the PNIAM-co-AM grafted plates with high cell viability and the reattachment and proliferation capacities. More importantly, keratinocyte-fibroblast cell sheets could release significant amounts of essential cytokines and growth factors which could help promote wound healing. The safety and efficacy of the human keratinocyte-fibroblast sheet for the treatment of full thickness skin defects were evaluated using a xenogeneic model of type II diabetic rats (Zucker diabetic fatty or ZDF rats). The bilayered keratinocyte-fibroblast sheets were transplanted to the wounds, and the wound area as well as the tissue structure underneath the skin were observed on days 7, 14, 21 and 28 after transplantation. During the experiment, no death or severe inflammation was observed in any of the experimental animals, indicating that the cell sheets were considered safe. The re-epithelialization rate of the wounds transplanted with the skin cell sheets was faster than that of the control without cell sheet transplantation. The skin cell sheets helped close the open wounds within 14 days, while without the cell sheets, the wounds could be completely healed by 28 days in diabetic rats. The histological staining of the sectioned wounds with the cell sheets revealed that the inflammation phase of the wound was conformed to normal wound repair. Together, the transplantation of the bilayered keratinocyte-fibroblast cell sheets to diabetic wounds has been shown to be safe and to accelerate wound healing in diabetic wounds.  

To further demonstrate the potential use of human keratinocyte-fibroblast cell sheets in treating chronic wounds, we propose to evaluate the safety and efficacy of the skin cell sheets in human clinical cases. This project is a collaboration between KMUTT researchers and physicians from the Faculty of Medicine Siriraj Hospital, Mahidol University. KMUTT researchers will be responsible for the construction and validation of skin cell sheets according to Good Tissue Practice (GTP) standards, while the Siriraj research team will be in charge of the clinical case study. The outcome of this project will be the standard operating procedure (SOP) of skin cell sheet construction for clinical studies and a manuscript published in a peer-reviewed journal. In addition, the results from this project can be used to apply for ethical approval for the therapeutic assessment of skin cell sheets in a phase I/II human clinical study. We hope that the skin cell sheets from this study can be an alternative approach in treating chronic wounds and potentially become a commercial product in the future.