Poly‐L‐Lactic acid increases collagen gene expression and synthesis in cultured dermal fibroblast (Hs68) through the TGF‐β/Smad pathway

Poly‐L‐Lactic Acid (PLLA) is a synthetic polymer which possesses biocompatible and biodegradable properties, and is widely used in the clinical filler material. This study focuses on the potential role of PLLA on the collagen production of dermal fibroblasts and its mechanism.


| INTRODUC TI ON
Economic growth and increased longevity have given rise to the pursuit of beauty and the active search for new bioactive substances that can fight age and promote health. 1 Skin aging is mainly characterized by structural and functional changes put into the epidermis and dermis. 2 Collagen, a major component of the extracellular matrix (ECM), is synthesized by dermal fibroblasts to maintain the skin's strength and elasticity. 3 However, UV rays, skin inflammation, intracellular metabolites, and aging can inhibit collagen synthesis, leading to skin aging occurrences such as wrinkle formation, sagging, and laxity. 4 While the current anti-aging process cannot be permanently improved, injectable fillers are a simple, fast-acting, less-invasive, and fast-recovery cosmetic procedure to combat skin aging. 5 As demand increases, safe and effective facial fillers are being updated, such as biodegradable calcium hydroxyapatite, polycaprolactone, hyaluronic acid, and non-biodegradable poly-meth methacrylate silicone, polyacrylamide, and autologous fat grafting. 6,7 Poly-L-Lactic Acid (PLLA) is a biocompatible, biodegradable, absorbable, immunologically inert, malleable, and easy-to-process polymeric synthetic polymer derived from plants that have been used in previous studies of synthetic suture materials. It was approved by the food and Drug Administration (FDA) in 2004 for the treatment of facial fat atrophy following immunodeficiency virus infection and in 2009 for nasolabial folds and other dermal wrinkles. 8 The volumeexpanding of PLLA post-injection is caused by the thinner. As the thinner is absorbed, the PLLA particles slowly degrade, promoting the formation of fibrous tissue and new collagen. Therefore, besides facial fillers, PLLA is also a good material for acne scarring and trunk plasticity in clinical practice. 9 However, the potential mechanisms by which PLLA regulates dermal collagen production and expression are worth exploring.
Various cytokines have been shown to regulate collagen gene expression in fibroblasts. Among them, transforming frown factor (TGF)β is a major regulator of ECM synthesis. TGFβ regulates fibroblast growth, apoptosis, and collagen synthesis by inducing the phosphorylation of smad2 and smad3 10 and inhibits matrixdegrading enzymes. 11 Therefore, the present study attempted to explore whether the mechanism of PLLA-induced collagen synthesis is related to the TGFβ/Smad signaling pathway.

| Cell culture
The human dermal fibroblast cell line (Hs60) was obtained from the American Typical Culture Collection (ATCC). Cells were maintained in DMEM culture containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin in an incubator containing 5% CO 2 at 37°C. For this study, the generation of cells was between 5 and 10th generations. PLLA, purchased from Sculptra®, is injected into sterile water in configurations and final concentrations of 0.1, 0.5, and 1 mg/ml, respectively. SB431542, a specific inhibitor of the TGFβ receptor, was configured according to the manufacturer's instructions. According to the results of previous studies 12,13 and preliminary experiments, 10 μM SB431542 was applied to cells and 2 h incubation was complished. Ploidy changes in gene expression were calculated by the 2 −ΔΔCT formula relative to the internal reference GAPDH. The relevant primer sequences used for RT-qPCR were shown in Table 1. RT-qPCR was performed with the following thermos cycling conditions: an initial 1 cycle at 95°C for 10 min, 40 cycles at 95°C for 15 s, 60°C for the 20 s, and 72°C for elongation for 60 s.

| Western blot
Hs68 cells were spiked with protein lysate containing 1% protease inhibitor and lysed on a shaker at 4°C for 5 min. The precipitation was removed by centrifugation, and the concentration was de- Results were subsequently visualized using chemiluminescence detection reagents. Optical density measurements were performed by NIH Image J to analyze the scanned membranes.

| Procollagen production
A Procollagen Type I C-peptide (PIP) enzyme immunoassay (EIA) assay kit was applied to analyze the levels of procollagen. Hs60 was inoculated into 6-well plates and incubated for 24 h in an incubator with 0.1, 0.5, and 1 mg/ml PLLA after 24 h. The supernatant of each well was collected and assayed according to the PIP EIA commercial assay kit (Takara Bio Inc.).

| Statistical analysis
The results are illustrated as mean ± standard error (SD). Each experiment contained 3 biological replicates per condition and was repeated more than three times. Statistical differences were measured using ANOVA analysis followed Tukey test in GraphPad prism 6.0. A p value of <0.05 was considered statistically significant.

| PLLA enhances the synthesis of type 1 collagen
To elucidate the effect of PLLA on collagen synthesis, the PIP EIA assay kit was first employed to determine the level of procollagen products in PLLA-treated dermal fibroblasts Hs60. As demonstrated in Figure 1A, PLLA typically increased procollagen production in a concentration-dependent manner (p < 0.05), and the mRNA levels of COL1A1 and COL1A2, which encode both strands of collagen, also showed a PLLA dose-dependent increase (p < 0.001, Figure 1B,C).
Finally, COL1A1 protein levels were upregulated significantly in a dose-dependent manner with the increase of PLLA concentration, which was confirmed by Western blot (p < 0.001, Figure 1D).

| PLLA regulates the levels of elastin, MMP-1, TIMP-1, and TIMP-2
Together with collagen, elastin forms the ECM produced by dermal fibroblasts, providing elasticity and flexibility to the skin. 14 As

| PLLA enhanced the activity of the TGFβ/ Smad signaling pathway
The mechanism of PLLA in regulating type I collagen in dermal fibroblasts was subsequently explored. TGFβ/Smad signaling pathway was identified to be critical in dermal collagen synthesis. RT-qPCR confirmed that TGFβ mRNA levels were significantly elevated with the increase of PLLA dose (p < 0.001, Figure 3A). What's more, Western blot revealed that TGFβ protein was also gradually enhanced, and p-Smad2 and p-Smad3 proteins were also increased in a dose manner (p < 0.001, Figure 3B).

| TGFβ inhibitor reverses the promotion of collagen synthesis by PLLA in dermal fibroblasts
SB431542 was added to PLLA-treated Hs60 as an inhibitor of TGFβ.

| DISCUSS ION
The appearance of skin and face is considered an essential factor in happiness and health, and as a result, aesthetic procedures have proliferated worldwide. 15,16 They are less invasive, have the fastest recovery, and have less scarring to achieve the best results. 15 PLLA is a synthetic polymer obtained from 100% natural sources such as corn starch and sugar cane 17 and is biocompatible, biodegradable, and immunologically inert. 18 As PLLA particles degrade, they gradually regain volume by stimulating collagen synthesis, and thus, PLLA is widely used as a dermal filler to improve appearance and repair imperfections, as well as in various areas of medicine. 19,20 The Hs68 cell line has the typical characteristics of primary dermal fibroblasts and is the most widely used dermal fibroblast cell line in the study of dermal physiology, which can regulate various components of the cell matrix including collagen. 4,21 Therefore, we explored the regulation of collagen synthesis in dermal cells by PLLA in Hs68 cells and focused on its potential molecular mechanisms.
In our study, we first examined the effect of PLLA on collagen synthesis, and the PIP EIA assay confirmed that procollagen synthesis in dermal fibroblasts gradually increased with increasing PLLA concentration. It is known that type I collagen is the most common type of collagen in the human body, consisting of a type I collagen α 1 chain and a type I collagen α 2 chains, encoded by the COL1A1 and COL1A2 genes. 22 Here, we confirmed that the mRNA levels of COL1A1 and COL1A2 gradually increased with increasing PLLA concentration. The protein level of COL1A1 also increased gradually. Our findings are consistent with previous studies 23 that found PLLA promotes collagen synthesis.
In our study, elastin levels were also found to increase gradually with increasing PLLA concentration. Previous studies have confirmed that elastin, a type of dermal ECM cross-linked fiber, together with collagen, forms the thicker connective tissue of the dermis, proving the mechanical framework and elasticity needed for the skin. 24,25 Therefore, cosmetic skin materials should mainly be biomaterials based on collagen and elastin. In our study, it was also confirmed that the PLLA gradient inhibited MMP-1, but significantly   33 Pyropia yezoensis as a marine alga promotes collagen synthesis by activating the TGFβ/Smad signaling pathway in dermal fibroblasts. 34 Based on the above study background, we attempted to investigate whether the TGFβ/ Smad signaling pathway is involved in the mechanism by which PLLA promotes collagen gene expression and synthesis in dermal fibroblasts. It was found that with the increase of PLLA, the protein and mRNA levels of TGFβ were significantly decreased, and the protein levels of p-Smad2 and p-Smad3 were also significantly reduced, but this reduction was significantly reversed by TGFβ receptor-specific inhibitors SB431542. Finally, PLLA-induced elevation of COL1A1, COL1A2, TIMP-1, TIMP-2, and reduction of MMP-1 were significantly reversed by SB431542. This study does have several limitations that needed to be considered when interpreting the findings. For example, the lack of animal models and clinical data are potential limitations of this study, which is also the direction of our further research. Additionally, we confirmed the mRNA levels of MMP but did not use a more visual gel enzyme profiling assay to confirm its activity is another limitation of this study.
Collectively, our study provides evidence that PLLA stimulates collagen expression and synthesis in dermal fibroblasts through activation of the TGFβ/Smad signaling pathway. In conclusion, our study lays the foundation for the clinical application of PLLA.

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.