(I-346) It Will All Get Better with Time: Exploring the Role of Circadian Rhythms in Skin Tissue Engineering

Introduction:: The United States has more than 1.25 million people with burns and 6.5 million people with chronic ulcers per year. Even though several tissue engineering products have successfully transitioned into the clinic, many wounds still fail to heal. One aspect of physiology that tissue engineers have ignored to date is circadian rhythms which are the body’s natural 24-hour cycles that coordinate the body’s behavior, physiology, and metabolism. In the skin, when these rhythms are disrupted through mutation of the circadian genes, it impairs wound healing. This suggests that the influence of circadian rhythms should be considered in skin tissue engineering. Additionally, the pharmaceutical industry has increasingly appreciated the importance of time-of-day of dosing for drugs and that these should be aligned with circadian rhythms to increase the drug’s efficacy. Yet, there are few tests for locally-delivered drugs, and no examples in tissue engineering. Here, we induced circadian rhythms in in vitro wound healing assays and delivered biologics at select times to these circadian-established models, to detect whether their presence will increase the rates of wound closure.

Materials and Methods:: Circadian rhythms were induced in in vitro models using established techniques. Cells and tissues were synchronized through dexamethasone stimulation (100 nM, 30mins), or through temperature pulsing (42°C, 30 mins); or by entrainment (12hr/12hr cycles of 36°C/38.5°C). Several wound healing models were then used. In 2D, proliferation, and wound scratch assays of fibroblasts and keratinocytes were tested to compare ‘wound closure’ rates in circadian synchronized vs. unsynchronized cells at 18hrs after scratching with a 200mL pipette tip. By synchronizing the cells 12hrs out of phase, we also compare the effects of day vs. night time wounding. In 3D, a re-epithelialization assay was used. Briefly, human skin equivalents (HSE) are formed by first seeding fibroblasts in Type I collagen and grown in epidermal growth media for 7 days. Keratinocytes are seeded on the surface of the collagen matrix and raised to an air liquid interface on day 4, followed by epithelial differentiation in cornification media for 1 week. To study re-epithelialization in synchronized and control tissues, these HSE are wounded with a 8mm biopsy punch, placed on a fibroblast-seeded support collagen gel and re-epithelialization at 48hrs measured via histology. In both the 2D and 3D models, fibroblast growth factor (FGF, 10ng/ml) was given either during the ‘day’ phase or the ‘night’ phase to see the effect of circadian growth factor delivery on the cells.


Results, Conclusions, and Discussions:: In 2D models of wound healing scratch assays, circadian synchronized fibroblasts (dexamethasone and temperature pulsing) resulted in the higher wound closure percentages when compared with unsynchronized controls (see figure 1). In our 3D re-epithelialization model, circadian synchronized (dexamethasone) and entrained (temperature cycling) also resulted in higher percentages of wound closure. Finally, when fibroblast growth factor was delivered to synchronized and entrained conditions in 2D and 3D wound healing models, the percentage area closed was higher than the controls. While some effects of phase were observed (i.e., day vs. night), they were more subtle than the differences for circadian vs. non-circadian. While there are few prior studies in this space, this data corresponds with previous work that showed decreased wound healing in vivo when circadian genes were knocked out. Our in vitro data to date suggests the importance of circadian rhythms in wound healing. Currently, we are establishing in vivo models to corroborate these studies and to understand the interplay between wound tissue engineering and circadian rhythms.


Acknowledgements (Optional): :

References (Optional): : References

[1] Cañedo-Dorantes, Luis, and Mara Cañedo-Ayala. “Skin Acute Wound Healing: A Comprehensive Review.” International Journal of Inflammation, vol. 2019, 2019, pp. 1–15., doi:10.1155/2019/3706315.

[2] Singer, Adam J., and Richard A.F. Clark. “Cutaneous Wound Healing.” New England Journal of Medicine, vol. 341, no. 10, 1999, pp. 738–746., https://doi.org/10.1056/nejm199909023411006.

[3] Sherratt, Michael J., et al. “Circadian Rhythms in Skin and Other Elastic Tissues.” Matrix Biology, vol. 84, 2019, pp. 97–110., doi:10.1016/j.matbio.2019.08.004.

[4] Schibler, Ueli, et al. “Clock-Talk: Interactions between Central and Peripheral Circadian Oscillators in Mammals.” Cold Spring Harbor Symposia on Quantitative Biology, vol. 80, 2015, pp. 223–232., doi:10.1101/sqb.2015.80.027490.

[5] Hoyle, Nathaniel P., et al. “Circadian Actin Dynamics Drive Rhythmic Fibroblast Mobilization during Wound Healing.” Science Translational Medicine, vol. 9, no. 415, 2017, doi:10.1126/scitranslmed.aal2774.

[6] A. Sunil, S., et al. “Chronotherapeutic Drug Delivery Systems - an Approach to Circadian Rhythms Diseases.” Current Drug Delivery, vol. 8, no. 6, 2011, pp. 622–633., doi:10.2174/156720111797635559.

[7] Sulli, Gabriele, et al. “Training the Circadian Clock, Clocking the Drugs, and Drugging the Clock to Prevent, Manage, and Treat Chronic Diseases.” Trends in Pharmacological Sciences, vol. 39, no. 9, 2018, pp. 812–827., doi:10.1016/j.tips.2018.07.003.

[8] Jacob, Haritha, et al. “Therapeutics on the Clock: Circadian Medicine in the Treatment of Chronic Inflammatory Diseases.” Biochemical Pharmacology, vol. 182, 2020, p. 114254., doi:10.1016/j.bcp.2020.114254.

[9] Sen, Chandan K. “Human Wound and Its Burden: Updated 2020 Compendium of Estimates.” Advances in Wound Care, vol. 10, no. 5, 2021, pp. 281–292., doi:10.1089/wound.2021.0026.

[10] Chaudhari, Atul, et al. “Future Prospects for Scaffolding Methods and Biomaterials in Skin Tissue Engineering: A Review.” International Journal of Molecular Sciences, vol. 17, no. 12, 2016, p. 1974., https://doi.org/10.3390/ijms17121974.

[11] Luo, Beibei, et al. “Circadian Rhythms Affect Bone Reconstruction by Regulating Bone Energy Metabolism.” Journal of Translational Medicine, vol. 19, no. 1, 2021, doi:10.1186/s12967-021-03068-x.

[12] Dickmeis, Thomas. “Glucocorticoids and the Circadian Clock.” Journal of Endocrinology, vol. 200, no. 1, 2008, pp. 3–22., doi:10.1677/joe-08-0415.

[13] Shimba, Akihiro, and Koichi Ikuta. “Glucocorticoids Regulate Circadian Rhythm of Innate and Adaptive Immunity.” Frontiers in Immunology, vol. 11, 2020, doi:10.3389/fimmu.2020.02143.

[14] Aitzetmüller, Matthias M., et al. “Challenges and Opportunities in Drug Delivery for Wound Healing.” Chronic Wounds, Wound Dressings and Wound Healing, 2018, pp. 37–49., doi:10.1007/15695_2017_91.

[15] Chan, B. P., and K. W. Leong. “Scaffolding in Tissue Engineering: General Approaches and Tissue-Specific Considerations.” European Spine Journal, vol. 17, no. S4, 2008, pp. 467–479., doi:10.1007/s00586-008-0745-3.

[16] Brown, Steven A, et al. “Rhythms of Mammalian Body Temperature Can Sustain Peripheral Circadian Clocks.” Current Biology, vol. 12, no. 18, 2002, pp. 1574–1583., doi:10.1016/s0960-9822(02)01145-4.

[17] Tsuchiya, Yoshiki, et al. “Temperature Compensation and Temperature Resetting of Circadian Rhythms in Mammalian Cultured Fibroblasts.” Genes to Cells, vol. 8, no. 8, 2003, pp. 713–720., doi:10.1046/j.1365-2443.2003.00669.x.

[18] Tamaru, Teruya, et al. “Synchronization of Circadian per2 Rhythms and HSF1-BMAL1:Clock Interaction in Mouse Fibroblasts after Short-Term Heat Shock Pulse.” PLoS ONE, vol. 6, no. 9, 2011, doi:10.1371/journal.pone.0024521.

[19] Boelsma, Esther, et al. “Reconstruction of a Human Skin Equivalent Using a Spontaneously Transformed Keratinocyte Cell Line (HaCaT).” Journal of Investigative Dermatology, vol. 112, no. 4, 1999, pp. 489–498., doi:10.1046/j.1523-1747.1999.00545.x.

[20] Szymański, Łukasz, et al. “A Simple Method for the Production of Human Skin Equivalent in 3D, Multi-Cell Culture.” International Journal of Molecular Sciences, vol. 21, no. 13, 2020, p. 4644., doi:10.3390/ijms21134644.

[21] Gangatirkar, Pradnya, et al. “Establishment of 3D Organotypic Cultures Using Human Neonatal Epidermal Cells.” Nature Protocols, vol. 2, no. 1, 2007, pp. 178–186., doi:10.1038/nprot.2006.448.

[22] Phillips, Derrick J., et al. “Environmental Disruption of the Circadian Clock Leads to Altered Sleep and Immune Responses in Mouse.” Brain, Behavior, and Immunity, vol. 47, 2015, pp. 14–23., https://doi.org/10.1016/j.bbi.2014.12.008.

[23] Beta, Rafailia A., et al. “Core Clock Regulators in Dexamethasone-Treated HEK 293T Cells at 4 h Intervals.” BMC Research Notes, vol. 15, no. 1, 2022, doi:10.1186/s13104-021-05871-7.

[24] El Ghalbzouri, Abdoelwaheb, et al. “Fibroblasts Facilitate Re-Epithelialization in Wounded Human Skin Equivalents.” Laboratory Investigation, vol. 84, no. 1, 2003, pp. 102–112., doi:10.1038/labinvest.3700014.

[25] Bruguerolle, Bernard, et al. “Temporal Variations in Transcutaneous Passage of Drugs: The Example of Lidocaine in Children and in Rats.” Chronobiology International, vol. 8, no. 4, 1991, pp. 277–282., https://doi.org/10.3109/07420529109063932.

[26] Reinberg, A.E., et al. “Circadian Dosing Time Dependency in the Forearm Skin Penetration of Methyl and Hexyl Nicotinate.” Life Sciences, vol. 57, no. 16, 1995, pp. 1507–1513., https://doi.org/10.1016/0024-3205(95)02123-z.