Biostimulating fillers and induction of inflammatory pathways: A preclinical investigation of macrophage response to calcium hydroxylapatite and poly-L lactic acid.

INTRODUCTION: Initial macrophage response to biostimulatory substances is key in determining the subsequent behavior of fibroblasts and the organization of newly synthesized collagen. Though histological studies suggest that calcium hydroxylapatite (CaHA) filler initiates a regenerative healing response with collagen and elastin deposition similar to natural, healthy tissue rather than an inflammatory response with fibrosis, the relative activity of macrophages stimulated by CaHA, as well as how this activity compares to that induced by other biostimulatory fillers, has not been explored. The aim of the study is to characterize the in vitro macrophage response to two biostimulory fillers, CaHA and PLLA (poly-L lactic acid), and to evaluate their inflammatory potential. METHODS: Primary human macrophages were incubated with two dilutions (1:50 and 1:100) of commercially available CaHA or PLLA. After 24 h incubation, an inflammation array was used to screen for the expression of 40 cytokines, released by macrophages. ELISA was used to confirm array results. RESULTS: Four cytokines were significantly upregulated in M1 macrophages incubated with PLLA compared to both unstimulated controls and CaHA: CCL1 (p < 0.001), TNFRII (p < 0.01), MIP-1α (p < 0.05), and IL-8 (p < 0.001). In M2 macrophages, MIP-1α (p < 0.01) and MIP-1ß (p < 0.01) were significantly upregulated by PLLA compared to CaHA and unstimulated controls. CONCLUSION: Together, these findings indicate that the CaHA mode of action is a non-inflammatory response while PLLA initiates expression of several cytokines known to play a role in inflammation. Our study supports the concept that these two "biostimulatory" fillers follow distinct pathways and should be considered individually with regard to mechanism of action.

L-Cystine-Containing Hair-Growth Formulation Supports Protection, Viability, and Proliferation of Keratinocytes.

PURPOSE: Clinical studies have confirmed that the hair-growth-promoting effect of approved oral drug combinations is beneficial for the treatment of diffuse telogen effluvium, which is characterized by the excessive loss of telogen club hairs. Since data elucidating the mode of action of such combinations are limited, our study focused on the identification of cellular processes potentially supporting the treatment of hair loss. MATERIALS AND METHODS: A minimal growth culture system (MGM) was used to mimic in vitro the reduced activity of human hair follicular keratinocytes (HHFKs). The effect of four core compounds (L-cystine, thiamine, calcium D-pantothenate, and folic acid) of a marketed oral combination (Panto[vi]gar®), which are approved for the treatment of diffuse hair loss, was examined by comparing HHFKs cultured either with or without the compounds. After determining their impact on metabolic activity and proliferation, we conducted a comparative whole-genome gene expression study with subsequent functional grouping of differentially expressed genes to identify cellular processes influenced by the tested compounds. RESULTS: The four core compounds of an oral hair-growth formulation enhanced proliferation and metabolic activity of HHFKs compared to HHFKs cultivated in MGM only. Functional grouping of differentially expressed genes confirmed the regulation of cell cycle-/proliferation-associated genes (cdk1, HJURP) and revealed regulation of cell death- and oxidative stress-associated gene groups. A supportive effect of the compounds on cell viability was demonstrated by lower sensitivity to solar-simulated UV-radiation and increased protection against oxidative stress. We established a central role for L-cystine, as changes in the expression of the anti-oxidative gene hmox1 were L-cystine-dependent. However, to reach a maximal stimulating effect on proliferation, the combination of all four compounds was necessary. CONCLUSION: The tested compound combination had positive effects on metabolic activity, cell viability, and proliferation of keratinocytes. Furthermore, this study suggested that L-cystine primarily contributes to the observed protection against endogenous oxidative stress.

Cystine-thiamin-containing hair-growth formulation modulates the response to UV radiation in an in vitro model for growth-limiting conditions of human keratinocytes.

BACKGROUND: Ultraviolet radiation (UVR) is known to be harmful to normal human epidermal keratinocytes (NHEKs) of the epidermal skin layer, as well as to hair-follicle-associated keratinocytes. An oral formulation containing l-cystine, thiamin, calcium d-pantothenate, medicinal yeast, keratin and p-aminobenzoic acid (Panto[vi]gar®) has demonstrated clinical efficacy for the treatment of diffuse telogen effluvium; however, its mode of action at the cellular level, and in particular whether protective mechanisms are involved, has yet to be elucidated. OBJECTIVES: To assess the capacity of ingredients of this oral formulation, both separately and in combination, to modulate the effects of UVR in growth-limited NHEKs in vitro. METHODS: NHEKs were incubated in keratinocyte basal medium, keratinocyte basal medium lacking cystine, thiamin, calcium d-pantothenate, folic acid and biotine (minimal growth medium [MGM]) or MGM plus test compound. Test compounds comprised the following four ingredients related to the oral formulation: l-cystine, thiamin, calcium d-pantothenate and folic acid (a proposed metabolite of p-aminobenzoic acid), and a combination of these (Panto[vi]gar®-in vitro correlate; P-IC). The effect of different doses of these compounds on the metabolic activity and proliferation of NHEKs was tested, as well as their influence on the impact of UV light on NHEKs assessed by monitoring metabolic activity, cell number and apoptosis induction. RESULTS: Compared with basal medium, MGM reduced the proliferation of NHEKs in a time-dependent manner. Reduced proliferation is a characteristic of the multifactorial and complex phenotype associated with diffuse hair loss. l-cystine (50 µM) increased metabolic activity and proliferation 3-fold versus MGM (p < 0.05). Thiamin also had a significant effect (p < 0.05) on proliferation and metabolic activity of NHEKs, but calcium d-pantothenate and folic acid did not when tested individually in this in vitro model. In the presence of P-IC, metabolic activity increased 4-fold and proliferation 3-fold compared with MGM alone (p < 0.05 for both). Following UV irradiation, cells in MGM showed a 72% reduction in metabolic activity, while P-IC-treated cells showed only a 12-18% reduction. The observed prevention of the UV-induced reduction in metabolic activity was not simply due to filtering UVR by the P-IC components, as P-IC-mediated reduction of this effect persisted even when P-IC was washed out during UV irradiation. CONCLUSION: This study demonstrated that l-cystine and thiamin are essential for proliferation of epidermal keratinocytes and suggests a novel, UV-protective potential of formulations combining l-cystine and thiamin in growth-limited inter-follicular NHEKs in vitro.