Ralph H. Loring, Robert B. Campbell
Date of Award
Master of Science
Department or Academic Unit
Bouve College of Health Sciences. Department of Pharmaceutical Sciences
health sciences, hormonal regulation, hyaluronate synthesis, neonatal human dermal fibroblasts, T3 thyroid hormone
Medical sciences, Extracellular matrix, Hyaluronic acid, Hydrocortisone, Triiodothyronine
Pharmacy and Pharmaceutical Sciences
It has been previously reported that an organized extracellular matrix can be produced in vitro under serum free conditions from neonatal human dermal fibroblasts cultured without using a three dimensional artificial scaffold. These constructs resemble the human dermis and produce large amounts of hyaluronic acid (hyaluronate, HA). According to previous studies documented in the literature, hyaluronate synthesis and regulation in human dermal fibroblasts is hormonally controlled. We have studied the effects of the hormones Hydrocortisone and Triiodothyronine (T3) on HA and extracellular matrix synthesis, as these hormones are present in the base culture media used to prepare these constructs. We have also studied the effects of an exogenous hormone Retinoic acid, which was not present in the construct base media. The constructs were prepared in polycarbonate Transwell® inserts by culturing human neonatal dermal fibroblasts in serum free media. In the normal constructs, Hydrocortisone and T3 were used at physiological concentrations of 0.4 ug/ mL and 20 pM respectively. The concentrations of Hydrocortisone were varied in the base media from 12 ug/ mL to 400 ug/ mL and then combined with 0.2 nM T3. Retinoic acid concentrations were varied from 250 nM to 10 uM. The constructs were harvested after 21 days and then analyzed for Cell Count, Quantification of Hyaluronic acid, Quantification of Collagen, Transmission Electron Microscopy, Histology and Glycosaminoglycan disaccharide analysis. Lower concentrations of hydrocortisone enhance HA synthesis while higher concentrations inhibit HA synthesis without marked inhibition of collagen synthesis and cell replication. T3 specifically inhibits HA synthesis. Hydrocortisone and T3 in conjunction with each other show higher inhibition of HA synthesis than they do independently. Retinoic acid inhibits HA synthesis but the inhibition is non-specific. Total RNA was also isolated from the constructs for gene array analysis in order to study the regulation of genes in HA and extracellular matrix synthesis due to hormonal treatment. Implantation of these constructs in vivo may shed more light on the potential role of HA in promoting scarless wound healing and regenerative process.
Madhura S. Deshpande
Deshpande, Madhura S, "Hormonal regulation of hyaluronate synthesis in a human dermal equivalent" (2010). Pharmaceutical Science Master's Theses. Paper 16. http://hdl.handle.net/2047/d20000797
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