Dose-response of human follicles during laser-based hair removal: Ex vivo photoepilation model with classification system embracing morphological and histological features.

OBJECTIVES: Photoepilation is a commonly used technology in home-use devices (HUDs) and in professional systems to remove unwanted body hair using pulses of laser or intense pulsed light (IPL). Albeit HUDs and professional systems operate at different fluences and treatment regimes, both demonstrate high hair reduction. The underlying mechanisms, however, remain unknown partly due to high divergence of the existing literature data. The objective of this study was to develop an ex vivo photoepilation model with a set of criteria evaluating response to light pulses; and to investigate dose-response behavior of hair follicles (HFs) subjected to a range of fluences. METHODS: After ex vivo treatment (single pulse, 810 nm, 1.7-26.4 J/cm2 , 4-64 ms pulse) human anagen HFs were isolated and maintained in culture for 7-10 days. Response to light was evaluated based on gross-morphology and histological examination (H&E and TUNEL stainings). RESULTS: HFs treated ex vivo demonstrated a dose-dependent response to light with five distinct classes defined by macroscopic and microscopic criteria. Fluences below 13.2 J/cm2 provoked catagen-like transition, higher fluences resulted in coagulation in HF compartments. CONCLUSION: Observed changes in the HF organ culture model were reflected by clinical efficacy. The developed photoepilation model provides an easy and fast method to predict clinical efficacy and permanency of light-based hair removal devices. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

The potential of prolonged tissue culture to reduce stress generation and retraction in engineered heart valve tissues.

In tissue-engineered (TE) heart valves, cell-mediated processes cause tissue compaction during culture and leaflet retraction at time of implantation. We have quantified and correlated stress generation, compaction, retraction, and tissue quality during a prolonged culture period of 8 weeks. Polyglycolic acid/poly-4-hydroxybutyrate strips were seeded with vascular-derived cells and cultured for 4-8 weeks. Compaction in width, generated force, and stress was measured during culture. Retraction in length, generated force, and stress was measured after release of constraints at weeks 4, 6, and 8. Further, the amount of DNA, glycosaminoglycans (GAGs), collagen, and collagen cross-links was assessed. During culture, compaction and force generation increased to, respectively, 63.9% ± 0.8% and 43.7 ± 4.3 mN at week 4, after which they remained stable. Stress generation reached 27.7 ± 3.2 kPa at week 4, after which it decreased to ∼8.5 kPa. At release of constraints, tissue retraction was 44.0% ± 3.7% at week 4 and decreased to 29.2% ± 2.8% and 26.1% ± 2.2% at, respectively, 6 and 8 weeks. Generated force (8-16 mN) was lower at week 6 than at weeks 4 and 8. Generated stress decreased from 11.8 ± 0.9 kPa at week 4 to 1.4 ± 0.3 and 2.4 ± 0.4 kPa at, respectively, weeks 6 and 8. The amount of GAGs increased at weeks 6 and 8 compared to week 4 and correlated to the reduced stress and retraction. In summary, prolonged culture resulted in decreased stress generation and retraction, likely as a result of the increased amount of GAGs. These results demonstrate the potential of prolonged tissue culture in developing functional, nonretracting, TE heart valves.

Passive and active contributions to generated force and retraction in heart valve tissue engineering.

In tissue engineered heart valves, cell-mediated stress development during culture results in leaflet retraction at time of implantation. This tissue retraction is partly active due to traction forces exerted by the cells and partly passive due to release of residual stress in the extracellular matrix and the cells. Within this study, we unraveled the passive and active contributions of cells and matrix to generated force and retraction in engineered heart valve tissues. Tissue engineered rectangular strips, fabricated from PGA/P4HB scaffolds and seeded with human myofibroblasts, were cultured for 4 weeks, after which the cellular contribution was changed at different levels. Elimination of the active cellular traction forces was achieved with Cytochalasin D and inhibition of the Rho-associated kinase pathway. Both active and passive cellular contributions were eliminated by lysation and/or decellularization of the tissue. Maximum cell activity was reached by increasing the fetal bovine serum concentration to 50%. The generated force decreased ~20% after elimination of the active cellular component, ~25% when the passive cellular component was removed as well and remained unaffected by increased serum concentrations. Passive retraction accounted for ~60% of total retraction, of which ~15% was residual stress in the matrix and ~45% was passive cell retraction. Cell traction forces accounted for the remainder ~40% of the retraction. Full activation of the cells increased retraction by ~45%. These results illustrate the importance of the cells in the process of tissue retraction, not only actively retracting the tissue, but also in a passive manner to a large extent.

Low oxygen concentrations impair tissue development in tissue-engineered cardiovascular constructs.

Cardiovascular tissue engineering has shown considerable progress, but in vitro tissue conditioning to stimulate the development of a functional extracellular matrix still needs improvement. We investigated the environmental factor oxygen concentration for its potential to increase the amount of collagen and collagen cross-links, and therefore improve tissue quality. Cardiovascular tissue engineered (TE) constructs, made of rapidly degrading PGA/P4HB scaffold seeded with human vascular-derived cells, were cultured at 7%, 4%, 2%, 0.5% O(2) for 4 weeks and compared to control cultures at 21% O(2). Tissue properties were evaluated by measuring the extracellular matrix production and mechanical behavior. The culture environment was monitored closely and the oxygen gradient throughout the constructs was simulated with a theoretical model. TE constructs cultured at 21%, 7% and 4% O(2) showed dense and homogeneous tissue formation with comparable strength, stiffness, collagen and collagen cross-link content. At 2% O(2), collagen content and stiffness decreased, whereas at 0.5% O(2), hardly any tissue was formed. Overall, tissue properties deteriorated at the lowest oxygen concentrations, opposing our hypothesis that was based on previous culture at low oxygen concentrations. Further research will focus on establishing the balance between applied oxygen conditions (concentration and exposure time) and optimal tissue outcome.

An in vitro model system to quantify stress generation, compaction, and retraction in engineered heart valve tissue.

Autologous heart valve tissue engineering relies on extracellular matrix production by cells seeded into a degrading scaffold material. The cells naturally exert traction forces to their surroundings, and due to an imbalance between scaffold, tissue, and these traction forces, stress is generated within the tissue. This stress results in compaction during culture and retraction of the leaflets at release of constraints, causing shape loss of the heart valve leaflets. In the present study, an in vitro model system has been developed to quantify stress generation, compaction, and retraction during culture and after release of constraints. Tissue-engineered (TE) constructs based on polyglycolic acid/poly-4-hydroxybutyrate scaffolds seeded with human vascular-derived cells were cultured for 4 weeks. Compaction in width was measured during culture, stress generation was measured during culture and after release of constraints at week 4, and contraction was measured after release of constraints at week 4. Both compaction and stress generation started after 2 weeks of culture and continued up to week 4. TE constructs compacted up to half of their original width and reached an internal stress of 6-8 kPa at week 4, which resulted in a retraction of 36%. The model system has provided a useful tool to unravel and optimize the balance between the different aspects of TE constructs to develop functional TE leaflets.

Controlling matrix formation and cross-linking by hypoxia in cardiovascular tissue engineering.

In vivo functionality of cardiovascular tissue engineered constructs requires in vitro control of tissue development to obtain a well developed extracellular matrix (ECM). We hypothesize that ECM formation and maturation is stimulated by culturing at low oxygen concentrations. Gene expression levels of monolayers of human vascular-derived myofibroblasts, exposed to 7, 4, 2, 1, and 0.5% O(2) (n = 9 per group) for 24 h, were measured for vascular endothelial growth factor (VEGF), procollagen α1(I) and α1(III), elastin, and cross-link enzymes lysyl oxidase (LOX) and lysyl hydroxylase 2 (LH2). After 4 days of exposure to 7, 2, and 0.5% O(2) (n = 3 per group), protein synthesis was evaluated. All analyses were compared with control cultures at 21% O(2). Human myofibroblasts turned to hypoxia-driven gene expression, indicated by VEGF expression, at oxygen concentrations of 4% and lower. Gene expression levels of procollagen α1(I) and α1(III) increased to 138 ± 26 and 143 ± 19%, respectively, for all oxygen concentrations below 4%. At 2% O(2), LH2 and LOX gene expression levels were higher than control cultures (340 ± 53 and 136 ± 29%, respectively), and these levels increased even further with decreasing oxygen concentrations (611 ± 176 and 228 ± 45%, respectively, at 0.5% O(2)). Elastin gene expression levels remained unaffected. Collagen synthesis and LH2 protein levels increased at oxygen concentrations of 2% and lower. Oxygen concentrations below 4% induce enhanced ECM production by human myofibroblasts. Implementation of these results in cardiovascular tissue engineering approaches enables in vitro control of tissue development.

Hypoxia induces near-native mechanical properties in engineered heart valve tissue.

BACKGROUND: Previous attempts in heart valve tissue engineering (TE) failed to produce autologous valve replacements with native-like mechanical behavior to allow for systemic pressure applications. Because hypoxia and insulin are known to promote protein synthesis by adaptive cellular responses, a physiologically relevant oxygen tension and insulin supplements were applied to the growing heart valve tissues to enhance their mechanical properties. METHODS AND RESULTS: Scaffolds of rapid-degrading polyglycolic acid meshes coated with poly-4-hydroxybutyrate were seeded with human saphenous vein myofibroblasts. The tissue-engineered constructs were cultured under normal oxygen tension (normoxia) or hypoxia (7% O(2)) and incubated with or without insulin. Glycosaminoglycan production in the constructs approached that of native values under the influence of hypoxia and under the influence of insulin. Both insulin and hypoxia were associated with enhanced matrix production and improved mechanical properties; however, a synergistic effect was not observed. Although the amount of collagen and cross-links in the engineered tissues was still lower than that in native adult human aortic valves, constructs cultured under hypoxic conditions reached native human aortic valve levels of tissue strength and stiffness after 4 weeks of culturing. CONCLUSIONS: These results indicate that oxygen tension may be a key parameter for the achievement of sufficient tissue quality and mechanical integrity in tissue-engineered heart valves. Engineered tissues of such strength, based on rapid-degrading polymers, have not been achieved to date. These findings bring the potential use of tissue-engineered heart valves for systemic applications a step closer and represent an important improvement in heart valve tissue engineering.