INTRODUCTION
Poly-l-lactic acid (PLLA-SCA) is a synthetic polymer used as an injectable to restore volume and stimulate collagen formation.1 It was initially approved by the US Food and Drug Administration (FDA) in 2004 under the name Sculptra (Galderma) for the treatment of HIV-associated lipoatrophy.2 Later in 2009 it was approved as Sculptra Aesthetic for the correction of shallow to deep nasolabial folds and other facial wrinkles in immunocompetent patients.3 In 2023, the FDA has approved Sculptra for the correction of cheek wrinkles.4 PLLA-SCA has a biostimulatory effect by activating resident fibroblasts to produce collagen.3,5 Animal experiments revealed that after injection, PLLA-SCA induces a response through phagocytosis by tissue macrophages and then slowly converts into lactic acid monomers, which are metabolized into carbon dioxide or incorporated into glucose while stimulating the production of new collagen type-I fibers in the skin.1,6 However, the underlying molecular mechanisms are not yet known in detail. Since our previous study aimed to better understand the molecular effects of HA-based fillers with and without subsequent additional fractional laser co-treatment,7 we now focused on gaining molecular insights into the stimulatory effects of PLLA-SCA injections on collagen I production in novel human 3D skin models comprising macrophages.
MATERIALS AND METHODS
In this in vitro study, the PLLA-SCA filler Sculptra was injected into previously described human full-thickness 3D skin models,8 in which macrophages were incorporated. Macrophages were isolated from peripheral blood mononuclear cells (PBMCs) by plastic adherence as published before9 and added to the models on day 2 of culture. Sculptra is composed of 150 mg of PLLA-SCA microparticles with a median particle size of approximately 50 micrometers suspended in sodium carboxymethylcellulose (NaCMC).10 After one single injection of 100 microliters of Sculptra, models were harvested after 5 and 14 days for histological and gene expression analyses. Untreated models were used as negative controls. Experiments were performed three times independently with three different cell donors.
Statistical analysis was performed using the Mann-Whitney U test. Values of P<0.05 were considered statistically significant.
For monolayer experiments, primary dermal fibroblasts were stimulated with human recombinant CXCL6 (50 ng/ml) for 24 hours.
Statistical analysis was performed using the Mann-Whitney U test. Values of P<0.05 were considered statistically significant.
RESULTS
Histological analysis revealed a significantly increased epidermal thickness in our macrophage-containing 3D skin models at days 5 and 14 after PLLA-SCA injection compared to untreated controls (Figure 1A and B). Using immunohistochemical staining, we could prove that our models contained CD163-positive macrophages (Figure 1C).
To test whether CXCL6, which was upregulated in our microarray analysis, could be a potential stimulator of collagen I in the human skin, we stimulated primary dermal fibroblast monolayers with a human recombinant CXCL6 protein. An ELISA assay revealed an upregulation of collagen I in dermal fibroblasts after CXCL6 stimulation for 24 hours (Figure 3C).
On the molecular level, microarray analyses showed an upregulation of integrins (ITGA6), laminins (LAMA3, LAMC2), and desmogleins (DSG2) at day 14 after PLLA-SCA injection into the models, compared to untreated controls (Figure 2). Furthermore, we found an upregulation of cytokines (TGFB2, IL1B) and chemokines (CXCL6).
Focusing on the biostimulatory effects of PLLA-SCA on collagen production, we performed an immunofluorescence analysis of collagen I expression (Figure 3A). Quantitative fluorescence measurements revealed a significant upregulation of collagen I at day 14 after PLLA-SCA injection (Figure 3B).
To test whether CXCL6, which was upregulated in our microarray analysis, could be a potential stimulator of collagen I in the human skin, we stimulated primary dermal fibroblast monolayers with a human recombinant CXCL6 protein. An ELISA assay revealed an upregulation of collagen I in dermal fibroblasts after CXCL6 stimulation for 24 hours (Figure 3C).
