Evaluation of the Behaviour of Wrinkles Fibroblasts and Normal Aged Fibroblasts in the Presence of Poly-L-Lactic Acid

Carol Courderot-Masuyer, Sophie Robin, Hélène Tauzin, Philippe Humbert

Bioexigence SARL, Espace Lafayette, Besan?on, France.
INSERM UMR1098, SFR FED 4234 IBCT, Besancon, France.

DOI: 10.4236/jcdsa.2012.21006                

Abstract

Background: Wrinkles are characterized by changes in the organization and structure of the dermis. Human wrinkle fibroblasts (WF) have a different functional behaviour in comparison with normal-aged fibroblasts (NF). Decreases in migration capacities and collagen I synthesis are observed. Mitochondrial function is impaired with an increase in lactate production during aging. Sculptra^? (poly-L-lactic acid: PLLA), a biodegradable synthetic polymer, is used for subcutaneous volume restoration. Thus we decided to investigate different fibroblast functions when placed in contact with PLLA. Objectives: The potential of PLLA to compensate for the reduction of metabolic activity, to restore the migration capacity of WF and to inhibit the lactate production, was investigated and compared to NF. Methods: Two different skin samples were used from each of the three women’s facelift (one inside a face wrinkle and one from normal aged skin). Collagen I, lactate productions and proliferation capacities were investigated on monolayer cultures. Migration properties were evaluated using three-dimensional collagen lattices. Results: PLLA increased collagen I synthesis, restored migration capacities and tended to decrease lactate production in WF, whereas PPLA stimulated proliferation in NF and tended to improve the migration of NF. Conclusion: These results suggested that PLLA from Sculptra^? acted as a stimulus for collagen production in WF and that it is suitable for correcting skin depressions, such as wrinkles.

Keywords

Wrinkles; Poly-L-Lactic Acid; Collagen; Fibroblast

1. Introduction

Injectable Poly-L-lactic acid (PLLA) was introduced into Europe in 1999 as New-fill^®, receiving European Union approval to increase the volume of depressed areas, such as skin creases, wrinkles, folds and scars. In February of 2004, this indication was extended to include largevolume corrections of lipoatrophy, and in the same year it received U.S. Food and Drug Administration approval for the restoration and/or correction of the signs of facial lipoatrophy in people with human immunodeficiency virus [1]. Its cosmetic use was approved by the FDA in 2009 [2] and relies upon volumetric expansions as well as the formation of new collagen and elastic fibers. Thus, the gradual increase in dermal thickness in patients receiving PLLA injections is thought to be due to the induction of a local tissue reaction such as acute inflammatory reaction. This effect is characterized by a foreign body reaction that includes an initial increase in fibro blast activity and a subsequent progressive increase in collagen fiber deposition over the course of weeks to months [3,4]. Previous studies have demonstrated a particular behavior of WF different from that of NF. These fibroblasts populations were obtained after punch biopsies performed inside a wrinkle present on discarded tissues from a facelift and also on surrounding normal aged skin. After a comparison of these two populations of fibroblasts, a significant decrease in collagen I production and a significant impairment of migratory capacities were observed in WF compared to NF from the same patient [5]. Reduced synthesis of collagen I is partly responsible for the aging of the skin because it confers resistance to stresses and traction. The reduction in collagen I synthesis was also demonstrated by a lower in vitro production of type I procollagen by dermal fibroblasts isolated from skin of old versus young individuals. There is a failure to replace damaged collagen I with newly synthesized material [6]. To our knowledge, no study was performed in order to evaluate the effect of PLLA on the behavior of specific WF. During aging, an alteration of mitochondrial respiration is often associated with increased lactates production [7]. Indeed it was observed that senescent skin fibroblasts displayed higher rates of utilization of glucose and amino acids and produced more pyruvate and lactate, compared with young skin fibroblasts [8]. We therefore decided to investigate the effects of PLLA on the production of lactates in WF compared to NF. In the present study, the effects of PLLA (0.1% Sculptra^®) on proliferation, migratory capacities, collagen I and lactates production of WF compared to those of NF from the same patients were investigated.

2. Materials and Methods

2.1. Products

Vials of dry powder of Sculptra^® (poly-L-Lacid acid, Sanofi aventis, France) were used in the present study. 5 ml of sterile water were added to the dry powder of Sculptra^® and 0.1% of this solution was added to culture medium of fibroblasts.

2.2. Fibroblast Cultures

Punch biopsies were performed inside a wrinkle and also on surrounding normal aged skin of patients after surgical procedure. Wrinkles fibroblasts (WF) and normal aged fibroblasts (NF) were obtained from discarded tissues from three healthy female patients who had given their written consents (aged 55, 49 and 50 years old). Three fibroblast lines of WF and NF were obtained: L1 (55 years), L2 (49 years) and L3 (50 years). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal calf serum (FCS) and 40 mg/ml of gentamicin and 2 mg/ml of fungizone (DMEMc). They were grown for several weeks in plastic flasks in a moist atmosphere of 5% carbon dioxin and 95% air at 37˚C. Confluent monolayers were propagated by trypsination (trypsin-EDTA 1X solution, Gibco) and replated at 1:2 dilutions. Subcultures of fibroblasts over 10 were not used.

Different groups were used for each experiment:

• NF: normal aged fibroblasts (n = 6)

• NF + PLLA (0.1% Sculptra^®) (n = 6)

• WF: wrinkle fibroblasts (n = 6)

• WF + PLLA (0.1% Sculptra^®) (n = 6)

2.3. Fibroblasts Proliferation

Fibroblast proliferation in monolayers was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye-reduction assay after the action of PLLA for 24 hours, 48 hours and 72 hours. All cultures were set up in 10 wells. Fibroblasts were placed in 96-well microliter plates at a cell density of 5 × 10³ cells per well. After 24 hours of incubation, PLLA was added to the culture medium of WF or NF. The fibroblasts were seeded with PLLA, one for 24 hours, one for 48 hours and the final one for 72 hours. Then MTT was added. The extraction buffer was added after 4 hours. Absorbance of each well was assessed using a Multiscan Ex (Thermo, France) equipped with a 550 nm filter.

2.4. Migratory Capacity

NF and WF were embedded in three-dimensional hydrated collagen using a modified version of the technique developed by Bell et al. [9]. Collagen lattices were made by mixing: six volumes of 1.76 X concentrated medium, three volumes of rat tail type I collagen solution (2 mg/ml) and one volume of fibroblast suspension (8·10⁵ cells/ml). This mixture was poured into plastic Petri dishes (60 mm diameter) (n = 10). The gel polymerized in less than 10 minutes at 37˚C. After one hour, culture medium was added and changed every 48 hours. To measure the retracted lattice diameter, the culture dishes were placed on a transparent metric scale. The measurement of the lattice diameter was taken each day for 10 days.

2.5. Collagen I Synthesis

Fibroblasts were seeded in 12-well plates at 0.04 × 10⁶ cells per well. The plates were incubated at 37˚C with and without the presence of PLLA in DMEMc with 1% foetal calf serum for 7, 21, 30 and 42 days respectively (n = 6). The culture medium was changed twice a week. After this period, the supernatant was placed in an antiprotease solution (10% v/v, Calbiochem, France). The samples were stored at –80˚C until the analysis by Elisa method was performed. Collagen I was evaluated using murine anticollagen I monoclonal antibody (5). The antimurine second antibody was peroxidase conjugated (Sigma-Aldrich, France). Color was developed using a TMB substrate. The absorbance was read by a spectrophotometer at 620 nm (Multiscan, Thermo, France). The number of cells was counted and the amount of collagen I was expressed as µg of collagen I/million of cells. Different groups were used at T = 0, 7, 21, 30 and 42 days of fibroblast cultures.

2.6. Lactate Production

Fibroblasts were seeded in 12-wells plates at 0.04 × 10⁶ cells per well. The plates were incubated at 37˚C both with and without the presence of PLLA for 7, 21, 30 and 42 days of fibroblast cultures (n = 6). The culture medium was changed twice a week. After the incubation period, the supernatant was placed in an antiprotease solution (10%, v/v, Calbiochem, France). The samples were stored at –20˚C until the analysis. The dosage of lactates was performed by the biochemistry department of St Jacques hospital (Besançon, France) using Dimension^Ò system. The oxidation of lactate in pyruvate with reduction of NAD is obtained with rabbit lacticodeshydrogenase. One molecule of NAD is transformed into one molecule of NADH for each molecule of lactate. Absorbance of NADH is proportional to the concentration of lactate and measured by spectrophotometry (340 nm). The number of cells is counted and the amount of lactates is expressed as µmoles of lactates/million of cells.

2.7. Statistical Analysis

The results shown were expressed as the mean of the obtained results with the 3 cell lines (L1, L2 and L3).

The groups with PLLA and those without PLLA were compared by using one way (for cell proliferation) or two ways (for the other experiments) variance analysis followed if necessary by Fisher test. Values were considered significantly different when p < 0.05.

3. Results

3.1. Cell Proliferation

The viability of NF and WF of the 3 women was higher than 80% in the presence of PLLA (0.1% of Sculptra^®) providing the chosen dose for the following experiments. PLLA induced a cell proliferation in NF after 72 hours of culture (Figure 1).

3.2. Migration Capacity

The migration of fibroblasts was evaluated through their capacity to retract free floating collagen lattices. The retraction of collagen lattices obtained with normal aged fibroblasts depended on the time (a decrease of 68% from the initial diameter was observed at the end of 10 days). A significant difference was observed between the retraction of collagen lattices obtained with NF and WF. The cell migration was slower with WF than NF. The presence of PLLA tended to improve the retraction of collagen lattices from NF (the difference was significant for cell lines L1 and L2) suggesting a beneficial effect of PLLA on migratory capacities of NF (Figure 2). PLLA restored the migration capacities of WF to the same level as basal values of NF.

3.3. Collagen I Production

Figure 3 shows the basal amount of collagen I in NF and WF groups (D0). The production of collagen I was significantly lower in WF than in NF. PLLA increased the mean production of collagen I of WF after 42 days of culture but did not modify the collagen I production of NF (Figure 4).

Conflicts of Interest

The authors declare no conflicts of interest.

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