Evaluation of Cross-Linking Methods for Electrospun Gelatin on Cell Growth and Viability

Abstract

The creation of a tissue engineering scaffold via electrospinning that has minimal toxicity and uses a solvent system composed of solvents with low toxicity and different cross-linking agents was investigated. First, a solvent system of acetic acid/ethyl acetate/water (50:30:20) with gelatin as a solute was evaluated. The optimum system for electrospinning a scaffold with the desired properties resulted from a gelatin concentration of 10 wt %. Several different methods were used to cross-link the electrospun gelatin fibers, including vapor-phase glutaraldehyde, aqueous phase genipin, and glyceraldehyde, as well as reactive oxygen species from a plasma cleaner. Because glutaraldehyde at high concentrations has been shown to be toxic, we explored other cross-linking methods. Using reactive oxygen species from a plasma cleaner is an easy alternative; however, the degradation reaction dominated the cross-linking reaction and the scaffolds degraded after only a few hours in aqueous medium at 37 °C. Glyceraldehyde and genipin were established as good options for cross-linking agents because of the low toxicity of these cross-linkers and the resistance to dissolution of the cross-linked fibers in cell culture medium at 37 °C. MG63 osteoblastic cells were grown on each of the cross-linked scaffolds. A proliferation assay showed that the cells proliferated as well or better on the cross-linked scaffolds than on traditional two-dimensional polystyrene culture plates.

Cytocompatible cross-linking of electrospun zein fibers for the development of water-stable tissue engineering scaffolds

“…Cell attachment on electrospun PLA fibers was 1.8-fold of that on PLA films, while cell attachment on the cross-linked electrospun zein fibers was up to 3-fold that on cross-linked zein films. The high surface to volume ratio and large number of pores in the electrospun structures could enhance the adhesion of cells and proteins in the culture medium [6,10]. A preference for cell attachment to electrospun fibers over films was also observed by Xua et al [37].…”

“…It has been reported that increasing scaffold stiffness promotes cell attachment and growth [36]. In addition, the cross-linked electrospun zein fibers had improved water stability and retained their high surface to volume ratio and the large volume of interconnected pores, which facilitated cell attachment [6,10]. Figure 3 also shows that cell attachment to electrospun scaffolds cross-linked with SHP (SHPXL) was lower than that to the fibers cross-linked without SHP (XL-L, XL-M, and XL-H).…”

“…The results of the MTS and genomic DNA quantification assays showed that the final numbers of cells on the crosslinked zein fibers (SHPXL, XL-L, XL-M, and XL-H) and zein films (ZFXL) after 24 h were higher than the cell numbers on uncross-linked zein fibers (UXL) and films (ZFUL), due to the enhanced stiffness of the scaffolds [36], and that they retained interconnected pores [6,10).…”

“…Electrospun structures closely mimic the three-dimensional (3D) fibrous network found in the natural extracellular matrix (ECM) [6][7][8][9]. Electrospun materials also have a high surface to volume ratio that facilitates cell attachment and drug sorption [6,10] and contain a large volume of interconnected pores, which assist in the transportation of oxygen, nutrients and the metabolic waste of cells and cell migration and communication [8]. Therefore, protein-based electrospun fibers have been developed in order to combine the advantages of protein materials and electrospun structures for biomedical applications [11-17J. However, the electrospun protein fibers developed so far have poor stability in aqueous environments, which restricts their biomedical applications [16].…”

“…Therefore, chemical modifications such as cross-linking have been used as alternatives to polymer blending. However, most cross-linking agents used to crosslink electrospun protein fibers are toxic [21][22][23][24][25][26][27][28][29], expensive [10] and/or inefficient in providing the desired improvement in water stability of the fibers [10,16].…”