Hormonal regulation of hyaluronate synthesis in a human dermal equivalent

Hormonal regulation of hyaluronate synthesis in a human dermal equivalent
Deshpande, Madhura S (Author)
Pouyani, Tara (Advisor)
Loring, Ralph H. (Committee member)
Campbell, Robert B. (Committee member)
Boston (Mass.) : Northeastern University
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Type of resource:
Masters theses
Digital origin:
born digital
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.
Subjects and keywords:
health sciences
hormonal regulation
hyaluronate synthesis
neonatal human dermal fibroblasts
T3 thyroid hormone
Medical sciences
Extracellular matrix
Hyaluronic acid
Pharmacy and Pharmaceutical Sciences
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Copyright 2010, Madhura S. Deshpande.