Biodegradable poly (lactic acid-co-glycolic acid) scaffolds as carriers for genetically-modified fibroblasts

Abstract

Recent advances in gene delivery into cells allow improved therapeutic effects in gene therapy trials. To increase the bioavailability of applied cells, it is of great interest that transfected cells remain at the application site and systemic spread is minimized. In this study, we tested clinically used biodegradable poly(lactic acid-co-glycolic acid) (PLGA) scaffolds (Vicryl & Ethisorb) as transient carriers for genetically modified cells. To this aim, we used human fibroblasts and examined attachment and proliferation of untransfected cells on the scaffolds in vitro, as well as the mechanical properties of the scaffolds at four time points (1, 3, 6 and 9 days) of cultivation. Furthermore, the adherence of cells transfected with green fluorescent protein (GFP) and vascular endothelial growth factor (VEGF165) and also VEGF165 protein secretion were investigated. Our results show that human fibroblasts adhere on both types of PLGA scaffolds. However, proliferation and transgene expression capacity were higher on Ethisorb scaffolds most probably due to a different architecture of the scaffold. Additionally, cultivation of the cells on the scaffolds did not alter their biomechanical properties. The results of this investigation could be potentially exploited in therapeutic regiments with areal delivery of transiently transfected cells and may open the way for a variety of applications of cell-based gene therapy, tissue engineering and regenerative medicine.

Fig 1. Visualisation of Hs27 cell growth on Vicryl and Ethisorb scaffolds.

During the co-cultivation period of 9 days, Hs27 fibroblast cells on Vicryl (A) and Ethisorb (B) scaffolds were investigated at four different time points by means of light microscopy with 100-fold magnification. In the upper row of each panel, the scaffolds containing no cells (control) are presented. The fibroblasts were visualized with Giemsa staining and can be seen in the images of the bottom row of each panel as small blue dots.

Fig 2. Comparison of proliferation activity of Hs27 cells grown on Vicryl and Ethisorb scaffolds.

Hs27 cells were seeded on Vicryl and Ethisorb scaffolds and cultivated for a period of 9 days. At day (d) 1, 3, 6 and 9 the proliferation activity was measured by means of colorimetric WST assay. The results of one experiment are presented as means of absorbance at 450 nm. The error bars represent SEM of five technical replicates (n = 5; two-way ANOVA comparing proliferation activity of cells between Vicryl and Ethisorb for each time point: ****p<0.00005).

Fig 3. Analysis of scaffold fibers and Hs27 cell morphology by scanning electron microscopy.

Hs27 fibroblasts are grown on Vicryl and Ethisorb scaffolds and visualized at day 1, day 3 day6 and day 9 in culture by means of scanning electron microscopy (Scale bars in the upon two rows in A and B are 100μm, in the lowest row are 25μm)

Fig 4. Determination of mechanical properties for Vicryl and Ethisorb scaffolds with and without seeded cells.

The mechanical properties of Vicryl (A) and Ethisorb (B) scaffolds were measured by using a uniaxial test system. Meshes were clamped along their long side with an initial length L₀ of 20 mm. Maximum force values for the two types of scaffolds (both with and without Hs27 cells) were measured in a single experiment at day (d) 1, 3, 6 and 9 and presented graphically (C). All error bars attached to the mean values represent the SEM of five technical replicates (n = 5; two-way ANOVA comparing maximum force values of Vicryl and Ethisorb scaffolds for each time point: ****p<0.00005).

Fig 5. Visualisation of GFP-expressing Hs27 cells adhered on Vicryl and Ethisorb scaffolds.

5 x 10⁶ Hs27 cells were electroporated with 20 μg GFP plasmid by using Nucleofector transfection apparatus. After a short period in culture, transfected cells were transferred on scaffolds. The expression of GPF protein in Hs27 fibroblast grown on Vicryl (A) and Ethisorb (B) scaffolds was recorded at day 1, 3, 6 and 9 by a fluorescent microscope with 100-fold magnification. In the upper row of each panel, the scaffolds containing no cells are depicted.

Fig 6. Comparison of VEGF165 protein secretion from Hs27 cells grown on Vicryl and Ethisorb scaffolds.

Human fibroblasts were transfected with 20 μg VEGF165 plasmid DNA and co-cultured with Vicryl or Ethisorb scaffolds. After 1, 3, 6 and 9 days (d) the supernatant was collected and the level of VEGF165 protein was determined by the enzyme-linked immunosorbent assay. Data shown are mean ± SEM from three experiments with three technical replicates (n = 3; two-way ANOVA comparing VEGF165 concentration of cells grown on Vicryl and Ethisorb scaffolds for each time point: **p<0.005, ****p<0.00005).