Fiber-Reinforced Silk Composite for Enhanced Urokinase Production Using High-Density Perfusion Culture and Bioactive Molecule Supplementation

Abstract

Urokinase plasminogen activator (uPA) has been extensively used as a thrombolytic drug in cases of myocardial infarction, thrombo­embolism, and ischemic brain stroke. Media optimization and high-density perfusion culture are the decisive factors that facilitate enhanced urokinase production in a conditioned medium. In this study, we have aimed for a high-density perfusion culture of HT1080, a human fibrosarcoma cell line, by formulating optimal media for enhanced urokinase productivity. Four scaffold variants were fabricated from silk fibroin and microfibers of Bombyx mori (BM) and Antheraea assamensis (AA) and physico-chemically characterized. Field emission scanning electron microscopy studies revealed a heterogeneous distribution of pores with interconnected networks supporting cell infiltration, attachment, and long-term viability. AA-based fiber-reinforced scaffolds (ASAF) demonstrated superior mechanical strength, integral stability, and increased cell proliferation as compared to pure silk scaffolds. Media formulation was accomplished by limiting serum concentration (2% FBS) and supplementing with 20 μg/mL arginine and 20 ng/mL TGF-β1 to retain the stationary phase of cells and augment the urokinase production. A perfusion bioreactor culture of HT1080-laden scaffolds in the presence of formulated media was performed for improving the production of urokinase, with a maximum activity of 432 U/L. Also, gene expression analysis revealed that the individual silk scaffolds have different effects on regulating the expression of plasminogen activator urokinase and plasminogen activator urokinase receptor. In brief, our results suggest that a perfusion bioreactor culture of HT1080-laden ASAF scaffolds in formulated media promotes an increased urokinase production, such that it can be further used as a novel 3D matrix platform for industrial production of the lifesaving uPA drug.

Mimicking Physiologically Relevant Hepatocyte Zonation Using Immunomodulatory Silk Liver Extracellular Matrix Scaffolds toward a Bioartificial Liver Platform

“…Enhanced swelling ratio observed in BA:dLS (1:1) and BA:dLS (0.5:1.5) might be due to its larger pore size and increased porosity (Figure ). The in vitro degradation rate of scaffolds was investigated using a nonspecific Protease XIV enzyme that mimics native MMPs . The higher degradation rates observed in BA:dLS (1:1) and BA:dLS (0.5:1.5) scaffolds might be attributed to their increased hydrophilicity and access to protease enzyme, as confirmed from swelling ratio results.…”

“…The BM and AA silk fibroin solution was isolated from BM cocoons and AA silk glands, as reported previously. , Briefly, BM cocoons were degummed in boiling sodium carbonate solution (0.02 M), dissolved for 4 h in lithium bromide solution (9.3M, 60 °C), and dialyzed against Milli-Q water for 48 h to obtain a regenerated BM silk fibroin solution. However, extruded AA silk fibroin from silk glands harvested from fifth instar silkworms was dissolved in sodium dodecyl sulfate solution (1% ( w / v )) and dialyzed for 4 h against Milli-Q water (4 °C).…”

“…The degradation pattern of LECM-SF scaffolds was evaluated in vitro using Protease XIV, derived from Saccharomyces griseus (Sigma-Aldrich, USA) over 45 days . The initial dry weight (DW i ) of scaffolds (5 mm diameter ×12 mm height) was noted and incubated in 2 U/mL of Protease XIV solution.…”

“…Initially, the PNRHs at a density of 5 × 10 6 were seeded in BA:dLS (0.5:1.5), BA:dLS (1:1), and BA:dLS (1.5:0.5) scaffolds of 14 mm diameter and 3 mm height and cultured for 2 days under static conditions. Simultaneously, the 3D Biotek perfusion bioreactor set up along with tubing, chambers, and medium reservoirs were sterilized and equilibrated with 20 mL of culture medium, as described previously . The PNRHs cultured on BA:dLS (0.5:1.5), BA:dLS (1:1), and BA:dLS (1.5:0.5) scaffolds were stacked in the bioreactor chamber, and the culture medium circulated at a flow rate of 0.5 mL/min, wherein the inlet is close to BA:dLS (0.5:1.5) and the outlet is from the BA:dLS (1.5:0.5) scaffolds.…”

“…The degradation pattern of LECM-SF scaffolds was evaluated in vitro using Protease XIV, derived from Saccharomyces griseus (Sigma-Aldrich, USA) over 45 days. 23 The initial dry weight (DW i ) of scaffolds (5 mm diameter ×12 mm height) was noted and incubated in 2 U/mL of Protease XIV solution. Every 5 days, the enzyme solution was removed, samples were gently rinsed in Milli-Q water to remove residual enzyme solution, and dry weight was noted (DW t ).…”