(B-70) Impact of the use of stabilized ascorbic acid in the production of engineered stromal tissue for regenerative medicine

Introduction:: Ascorbic acid or vitamin C is a critical building block of collagen, and its deficiency is associated with impaired extracellular matrix deposition. Thus, sodium ascorbate is often added to growth media in tissue engineering applications to stimulate the production and maturation of an endogeneous extracellular matrix by fibroblasts. For instance, production of various tissues by the self-assembly technique relies on the ability of stromal cells to produce their own extracellular matrix support scaffold. The use of sodium ascorbate is therefore crucial for this technique. However, sodium ascorbate is unstable in aqueous solution even when stored at 4°C, meaning that fresh solutions have to be made at every medium change. This poses a challenge for the production of engineered tissues as it increases cost and manipulation time. A more stable form of sodium ascorbate, 2-Phospho-L-Ascorbate (2PAA), allows the formation of connective tissue-like structures in fibroblast cultures. It remains unclear if 2PAA constitutes an adequate substitute to sodium ascorbate for tissues production. In this context, we evaluated the physico-chemical properties of sodium ascorbate or 2PAA solutions and how 2PAA impact the properties of dermis, bladder and vaginal tissues reconstructed using the self-assembly technique.

Materials and Methods:: Sodium ascorbate (AA) or 2-phospho-L-ascorbate (2PAA) solutions were reconstituted at 10 mg/ml in DMEM without phenol red. Compound stability was assessed for a working dilution (50µg/ml) under storage conditions (4°C or -80°C) as well as under culture conditions (37°C humidified 5% CO2 incubator). Solution samples were taken at different time points and measurements were performed at room temperature on a UV–vis scanning spectrophotometer in the 230–820 nm wavelength range, with a slot of 2 nm, and an integration time of 0.2 s. Engineered dermis, bladder and vaginal tissues were produced using 3 populations of primary fibroblasts from each respective tissue. Cells were seeded (500,000 cells/well in 6-well plate) with a paper anchor and weight. Cells were cultured in 10% FBS DMEM supplemented with 50µg/ml freshly made AA or 2PAA. After 4 weeks, 3 stromal sheets were stacked and allowed to fuse for 4 days. Mechanical characterization was performed under uniaxial tensile stretch on an Instron Electropuls® E1000 (10N load cell and 0.2 mm/sec load rate). Tissue test samples were obtained with a dog bone-shaped biopsy punch. Stroma structure and organization were assessed on histological cryosection stained with Masson’s trichrome stain (collagen organization and tissue thickness), Picrosirius Red (type I collagen content, polarized microscopy) or picogreen (cell number). Enzymatic activity of matrix metalloproteinases (MMPs) remodeling enzymes secreted during tissue production was assessed using the Sensolyte fluorogenic substrate kit. Finally, a price analysis was performed.

Results, Conclusions, and Discussions:: We first tested the stability of both AA and 2PAA under different storage conditions. Direct UV-Vis spectrophotometry showed a maximum of absorption around 260 nm for both freshly prepared AA and 2PAA solutions. After 15 to 30 days of storage at 4°C, the AA solution underwent yellowing along with a shift of the maximum absorption peak, indicating degradation. To a lower extent, this trend was also observed when the AA solution was stored at -80°C. In contrast, no changes in color or spectra were observed for the 2PAA solution. Experiments performed over 72h at 37°C under culture condition showed even faster change in the color and spectrum of AA while 2PAA remained stable (Fig 1a).

We then tested if tissues produced using 2PAA were equivalent to those obtained with AA. Notably, tissues produced with 2PAA had comparable maximum thicknesses and equivalent mechanical properties compared to those produced with AA, including the measured maximal rupture force (Fig. 1b). At the molecular level, measurement of matrix metalloproteinases activity also revealed no significant difference between AA and 2PAA (Fig. 1c). Furthermore, collagen content and organization were found to be similar. Nevertheless, a higher rate of delamination of the stromal sheets was observed for tissues produced with AA compared with those produced with 2PAA. This suggests that the use of 2PAA may yield decreased rate of faulty tissues.

A price comparison study also revealed that 2PAA ($0.51 /cm2) might be particularly advantageous compared to freshly made AA ($4.79/cm2). This difference in cost is mainly due to the more frequent use of relatively expensive 0.22µm filters as AA and 2PAA cost are relatively insignificant overall. While not yet factored in, the difference in failure rate and decreased hands-on time is likely to further improve the cost effectiveness of using 2PAA.

Overall, our results demonstrate the possibility of substituting sodium ascorbate with 2-Phospho-L-Ascorbate for tissue engineering by self-assembly approach. This protocol would significantly reduce the costs associated with tissue production while lowering required hands-on time. These are key factors for any scaling activity.

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