AbstractWithin the clinical field of human wound healing and chemotherapy induced alopecia (CIA), novel medical devices and technologies have emerged in recent decades with the aim of improving patient outcome. However, there is much to improve in either the efficacy of such therapeutic strategies, or its cost-effectiveness. For example, caring for patients with clinical wounds in the skin such as surgical, leg ulcers, burns and diabetic wounds pose substantial practical and financial problems to healthcare systems worldwide, and the gold standard of treating such wounds (human skin grafting and transplantation), poses various disadvantages. Likewise, CIA causes highly distressing side effects in patients, and while scalp cooling has been shown to be a highly effective method in preventing chemotherapy induced hair loss in human patients, it is still yet, unable to reach a 100% success rate of hair preservation in patients. Therefore, either development of novel alternative therapeutic strategies, or additional improvements of current strategies, are of clinical need. This project had two primary objectives, both in wound healing and prevention of CIA, with the employment of human keratinocyte in vitro culture systems.
The first project has developed a methodology for the creation of an Autologous cell suspension (ACS), which involves enzymatic disaggregation of the skin, and a novel filtration and separation technique using strainers with no specialist equipment or requirement for heating. An additional advantage of the methodology is the ability to perform it as a 30 minute, a 90 minute, and an overnight isolation. Results have shown that the ACS methodology yielded approximately 5 x 105 cells per cm2 of skin, with the overnight method obtaining the highest cell number. ACS consisted of keratinocytes, fibroblasts, and melanocytes. Flow cytometry has shown that the isolated keratinocytes expressed two distinct populations of low and high CD71 expression combined with α6 integrin positivity, indicative of stem cells. Cultures of ACS in vitro demonstrated a variety of secreted growth factors and cytokines, such as epidermal growth factor (EGF), Fibroblast growth factor (FGF), and Interleukin-1 alpha. In addition, damage-associated molecular patterns (DAMPs) such as Hsp90alpha and HMGB1 were also detected extracellularly from cultured ACS. Collected ACS “conditioned medium”, containing such secretion factors, appeared to have attenuated TGFβinduced fibroblast to myofibroblast differentiation, as well as promoting keratinocyte proliferation. These results provided preliminary evidence to suggest that applying ACS from this methodology onto a wound could not only enhance the reepithelialisation process through tissue “take”, but also enhance the intrinsic mechanisms of wound healing.
The second project investigated the hypothesis in which the current strategy in protection against CIA via scalp cooling could be further enhanced through pharmacological intervention. This investigation was carried out by employing primary human keratinocyte cell lines, as well as an immortalised cell line adapted to low calcium conditions (HaCaTa) in vitro. As ROS have been shown to play a role in CIA induced cytotoxicity, the addition of antioxidants was tested in combination with scalp cooling at optimal and sub-optimal temperatures. The results presented here showed that the addition of antioxidants (NAC and Resveratrol) to scalp cooling displayed further protection against CIA compared to scalp cooling alone. Interestingly, there was a diversity in the degree of enhancing scalp cooling between the type of antioxidant and anticancer agent. Nevertheless, these findings provided evidence which supports a promising clinical approach in improving scalp cooling devices without jeopardising the anticancer effects of chemotherapy.
Overall, using in vitro keratinocyte models are an effective practice in generating evidence to either develop or investigate the efficacy of various medical devices. This is exhibited here, by using such models to develop a novel clinical methodology aimed to clinically enhance wound healing, as well as supporting a novel combinatorial approach which may enhance the efficacy of scalp cooling technologies against CIA.
|Date of Award||2023|
|Supervisor||Nik Georgopoulos (Main Supervisor)|