Linking clinical vascular dark circles reduction to laboratory in vitro modelization can be achieved using vascularized 3D Bioprinted skin models.
Podium 8
Presented by: Colin McGuckin
INTRODUCTION: Translational cosmetics testing is the theory of making laboratory in vitro testing relate better to in vivo testing and user efficacy. Often, however, it is just a theory, with many laboratory tests hardly transferable, nor reliable in delivering true efficacy data. This is particularly a problem with cosmetics ingredients. Skin angiogenesis is one area of much difficulty for this. Over and under vascularization can have debilitating effects on sufferers. Such conditions, including Rosacea and Periorbital Edema, while not life-threatening can lead to personal embarrassment, mental health issues and depression. Solutions to gently treat the condition without changing the normal pathobiology of the skin are not easy and hampered by the existence of good in vitro models, particularly those which can prove layer penetration from the epidermis to the vascular bed of the skin. Existing lab models include co-cultures of endothelial cells, keratinocytes and fibroblasts, but this does not well represent the structure and barrier performance of the skin.
For this reason, we have developed a 3D bioprinted layered full thickness skin model with a vascularized bed within the dermis, as a screening and efficacy tool. At the same time, we have mirrored this promising in vitro system with live donor clinical testing to evaluate cross-over efficacy on the angiogenic contribution known in the etiology of vascular dark circles.
METHODS: Clinically, a double-blind randomized study enrolled 18 women, aged 18 to 35 years old and presenting persistent dark circles. Participants applied a test formulation active at 1% or a placebo, twice daily for 28 days. Clinical efficacy was evaluated by hyperspectral imaging with a SpectraCam system (Newtone, France), which investigated the spectral reflectance of the skin and producing a qualitative map of the underlying blood vessels structures. On the in vitro side, epidermal keratinocytes, dermal fibroblasts and Human Dermal Microvascular Endothelial Cells (HDMEC), were expanded from juvenile skin donations <30 mo following ethical consent. Cells at early passage 2 or 3 were selected for optimal growth and mixed with a bioink (CELLINK, Sweden), into which adhesion proteins were added and cartridged into a CELLINK pneumatic 3D bioprinting system, after modelization with Sketchup and SLIC3R slicing software (first described at IFSCC2020, Henry Maso award 2022). Resulting full thickness models had a vascularization bed created in the lower quadrant of the dermal structure, which was confocal microscopy imaged with anti-CD31 fluorescent labelling. Challenge of the individual cell types, with the same active tested clinically, and additionally on the 3D model was performed. Additionally Anti-angiogenic Endostatin (3 µg/mL) and proangiogenic VEGF (5 ng/mL) controls were made.
RESULTS: Reduction or inhibition of pro-angiogenic factors resulted in the sequential reduction of the vascular bed over 14 days, whilst continual supplementation maintains the model and cellular interconnections, whilst the clinical study similarly demonstrated after 28 days of use the reduction of vascular network in dark circle area thanks to the active ingredient.
DISCUSSION AND CONCLUSIONS: Modelization in the laboratory is an expensive part of ingredients development, but an essential step for safety and efficacy, but is often a step that does not work. Our models allow now a better translation between the lab and clinical testing to evaluate deep bed effects at the stratified skin levels of both epidermis and dermis, which should help to create safer more effective cosmetics of the future.
For this reason, we have developed a 3D bioprinted layered full thickness skin model with a vascularized bed within the dermis, as a screening and efficacy tool. At the same time, we have mirrored this promising in vitro system with live donor clinical testing to evaluate cross-over efficacy on the angiogenic contribution known in the etiology of vascular dark circles.
METHODS: Clinically, a double-blind randomized study enrolled 18 women, aged 18 to 35 years old and presenting persistent dark circles. Participants applied a test formulation active at 1% or a placebo, twice daily for 28 days. Clinical efficacy was evaluated by hyperspectral imaging with a SpectraCam system (Newtone, France), which investigated the spectral reflectance of the skin and producing a qualitative map of the underlying blood vessels structures. On the in vitro side, epidermal keratinocytes, dermal fibroblasts and Human Dermal Microvascular Endothelial Cells (HDMEC), were expanded from juvenile skin donations <30 mo following ethical consent. Cells at early passage 2 or 3 were selected for optimal growth and mixed with a bioink (CELLINK, Sweden), into which adhesion proteins were added and cartridged into a CELLINK pneumatic 3D bioprinting system, after modelization with Sketchup and SLIC3R slicing software (first described at IFSCC2020, Henry Maso award 2022). Resulting full thickness models had a vascularization bed created in the lower quadrant of the dermal structure, which was confocal microscopy imaged with anti-CD31 fluorescent labelling. Challenge of the individual cell types, with the same active tested clinically, and additionally on the 3D model was performed. Additionally Anti-angiogenic Endostatin (3 µg/mL) and proangiogenic VEGF (5 ng/mL) controls were made.
RESULTS: Reduction or inhibition of pro-angiogenic factors resulted in the sequential reduction of the vascular bed over 14 days, whilst continual supplementation maintains the model and cellular interconnections, whilst the clinical study similarly demonstrated after 28 days of use the reduction of vascular network in dark circle area thanks to the active ingredient.
DISCUSSION AND CONCLUSIONS: Modelization in the laboratory is an expensive part of ingredients development, but an essential step for safety and efficacy, but is often a step that does not work. Our models allow now a better translation between the lab and clinical testing to evaluate deep bed effects at the stratified skin levels of both epidermis and dermis, which should help to create safer more effective cosmetics of the future.