Alopecia is a clinical condition caused by excessive hair loss which may result in baldness, the causes of which still remain elusive. Conditioned media (CM) from stem cells shows promise in regenerative medicine. Our aim was to evaluate the potential CM of dental pulp stem cells obtained from human deciduous teeth (SHED-CM) to stimulate hair growth under in vitro and in vivo conditions. SHED and hair follicle stem cells (HFSCs) (n = 3) were cultured in media combinations; i) STK2, ii) DMEM-KO+10% FBS, iii) STK2+2% FBS and profiled for the presence of positive hair growth-regulatory paracrine factors; SDF-1, HGF, VEGF-A, PDGF-BB and negative hair growth-regulatory paracrine factors; IL-1α, IL-1β, TGF-β, bFGF, TNF-α, and BDNF. The potential of CM from both cell sources to stimulate hair growth was evaluated based on the paracrine profile and measured dynamics of hair growth under in vitro conditions. The administration of CM media to telogen-staged synchronized 7-week old C3H/HeN female mice was carried out to study the potential of the CM to stimulate hair growth in vivo. SHED and HFSCs cultured in STK2 based media showed a shorter population doubling time, higher viability and better maintenance of MSC characteristics in comparison to cells cultured in DMEM-KO media. STK2 based CM contained only two negative hair growth-regulatory factors; TNF-α, IL-1 while DMEM-KO CM contained all negative hair growth-regulatory factors. The in vitro study confirmed that treatment with STK2 based media CM from passage 3 SHED and HFSCs resulted in a significantly higher number of anagen-staged hair follicles (p<0.05) and a significantly lower number of telogen-staged hair follicles (p<0.05). Administration of SHED-CM to C3H/HeN mice resulted in a significantly faster stimulation of hair growth in comparison to HFSC-CM (p<0.05), while the duration taken for complete hair coverage was similar for both CM sources. Thus, SHED-CM carries the potential to stimulate hair growth which can be used as a treatment tool for alopecia.
Dutasteride, used for treating benign prostate hyperplasia (BPH), promotes hair growth. To enhance delivery to the hair follicles and reduce systemic effects, in this study dutasteride has been formulated for topical application, in a nanostructured lipid carrier (NLC) coated with chitosan oligomer-stearic acid (CSO-SA). CSO-SA has been successfully synthesized, as confirmed using1H NMR and FTIR. Formulation of dutasteride-loaded nanostructured lipid carriers (DST-NLCs) was optimized using a 23full factorial design. This formulation was coated with different concentrations of stearic acid-chitosan solution. Coating DST-NLCs with 5% SA-CSO increased mean size from 187.6±7.0nm to 220.1±11.9nm, and modified surface charge, with zeta potentials being -18.3±0.9mV and +25.8±1.1mV for uncoated and coated DST-NLCs respectively. Transmission electron microscopy showed all formulations comprised approximately spherical particles. DST-NLCs, coated and uncoated with CSO-SA, exhibited particle size stability over 60days, when stored at 4-8°C. However, NLCs coated with CSO (without conjugation) showed aggregation when stored at 4-8°C after 30days. The measured particle size for all formulations stored at 25°C suggested aggregation, which was greatest for DST-NLCs coated with 10% CSO-SA and 5% CSO. All nanoparticle formulations exhibited rapid release in an in vitro release study, with uncoated NLCs exhibiting the fastest release rate. Using a Franz diffusion cell, no dutasteride permeated through pig ear skin after 48h, such that it was not detected in the receptor chamber for all samples. The amount of dutasteride in the skin was significantly different (p<0.05) for DST-NLCs (6.09±1.09μg/cm2) without coating and those coated with 5% CSO-SA (2.82±0.40μg/cm2), 10% CSO-SA (2.70±0.35μg/cm2) and CSO (2.11±0.64μg/cm2). There was a significant difference (p<0.05) in the cytotoxicity (IC50) between dutasteride alone and in the nanoparticles. DST-NLCs coated and uncoated with CSO-SA increased the maximum non-toxic concentration by 20-fold compared to dutasteride alone. These studies indicate that a stearic acid-chitosan conjugate was successfully prepared, and modified the surface charge of DST-NLCs from negative to positive. These stable, less cytotoxic, positively-charged dutasteride-loaded nanostructured lipid carriers, with stearic acid-chitosan oligomer conjugate, are appropriate for topical delivery and have potential for promotion of hair growth.