Low temperature, or cryogenic, electrospinning has been previously reported to be an effective technique for inducing cell infiltration into synthetic electrospun scaffolds12, 17–20
. The current report demonstrates the application of this technique for the first time to the natural polymer, silk. Cell distribution throughout the entire volume of the electrospun silk scaffold was achieved only using the cryogenic electrospinning technique, whereas conventional electrospun silk did not exhibit the same structural properties and no cell infiltration was observed. Fibroblast infiltration throughout the electrospun silk scaffolds was only observed in cryogenically electrospun scaffolds. The three dimensional cell culture conditions also allowed for variation in cell type distribution with the invasive HN12 cells infiltrating the entire volume of the scaffolds, whereas the immortalized keratinocytes formed multi-cell layers on the surface of the scaffolds at the air-liquid interface (). Poor fibroblast attachment and limited keratinocyte infiltration were observed in co-culture experiments with conventional electrospun silk scaffolds, with no formation of distinct cell layers (). Co-culture systems of cryogenic electrospun silk scaffolds seeded with fibroblasts and NOKSI cells exhibited two distinct layers: a deeper fibroblast layer and a more superficial epithelial layer more reminiscent of dermal and epidermal structures, respectively (). Immunofluorescent staining for keratin revealed low, evenly distributed keratin expression in the epithelial cells, confirming their lineage (). The NOKSI cells showed high expression of the proliferation marker Ki67 () even after three weeks in air-liquid interface co-culture, but extremely low or undetectable levels of the terminal differentiation marker involucrin (). Thus, NOKSI cells may show blocked terminal differentiation. In order to investigate further whether cryogenic scaffolds support epithelial differentiation, we conducted similar co-culture experiments with the hTERT-immortalized HFK-398 keratinocytes which have previously been shown to undergo terminal differentiation in organotypic culture23
. Similar to NOKSI cells, HFK-398 cells were immunoreactive with a pan-keratin antibody (). However, after three weeks of air-liquid interface culture, Ki67 expression in HFK-398 cells was minimal and involucrin expression was prominent, features that are consistent with epithelial differentiation (). Therefore, our data suggest that terminal differentiation of epithelial cells is possible on cryogenic electrospun silk scaffolds, and this system may be useful for in vitro
mucosal modeling. In addition, co-culture systems of cryogenic scaffolds seeded with fibroblasts and lymph node metastasis-derived HN12 cells resulted in the tumor cells occupying and sharing the entire scaffold volume with the fibroblasts, raising the possibility that the silk scaffolds may also show utility for in vitro
investigation of tumor cell invasion. Furthermore, all cells that we have tested have remained viable for at least three weeks, with higher viability observed on cryogenic scaffolds, possibly due at least in part to increased cell-cell and cell-matrix interactions in three dimensions compared to two dimensions29
Fluorescent labeling of cells was used to facilitate identification of cell types in co-culture experiments. Silk auto-fluorescence in the red region of the spectrum makes it difficult to identify mCherry labeled cells, therefore DAPI was also used to counterstain cell nuclei. Electrospun silk exhibited no auto-fluorescence in the ultra-violet or blue regions of the spectrum. Although silk also fluoresces in the green region, the intensity of the YFP signal was much greater than that of the scaffold, allowing a clear distinction of YFP labeled cells. A major advantage of using fluorescently labeled cells is the ability to differentiate between the cell types in co-culture systems. A disadvantage of using fluorescently labeled cell lines is the possible variation in protein expression between labeled and original cells lines. However, no notable differences in cell proliferation, attachment or viability were observed between labeled and corresponding unlabeled cell lines (unpublished observations); therefore, in these studies the labeled cells seeded on scaffolds are predicted to behave in a similar fashion to unlabeled cell lines.
We found that maintaining relative humidity above 30% was a critical requirement for creating electrospun silk scaffolds capable of inducing cell infiltration, a similar result to that found with other cryogenic electrospun scaffolds12, 17, 18
. Cryogenic electrospun silk scaffolds that were manufactured at lower humidity levels did not support cell infiltration and did not appear to differ appreciably from conventional electrospun scaffolds, as judged by SEM analysis (unpublished observations). The cryogenic electrospun silk fibers were measured to have narrower widths then conventional electrospun silk fibers. This observation might be attributable to a possible change in the electric field during the electrospinning process due to ice accumulation on the mandrel, as well as diameter change of the collecting mandrel. Another critical element of cryogenic electrospinning of silk is maintaining the scaffold frozen until the sample is lyophilized. Any melting of the ice crystals dissolves the electrospun silk, which does not become insoluble in aqueous solution until after dehydration and ethanol treatment.
A common technique to increase porosity of electrospun scaffolds is to introduce a sacrificial polymer into the electrospinning process. The sacrificial polymer, such as poly(ethylene oxide), is mixed in the electrospinning solution with the polymer of interest, such as silk. The solution of both polymers is electrospun conventionally and then the sacrificial polymer is dissolved to leave a scaffold composed only of the desired material14
. Although this procedure does result in increased void volume, minimal cell infiltration has been demonstrated using this approach15
. Thus, cryogenic electrospinning of silk would appear to be advantageous. The porosity increase between cryogenic and conventional electrospinning remains consistent from polymer to polymer as demonstrated by previous studies and confirmed by this study18
. Another recent method for increasing porosity of electrospun scaffolds is the cotton-ball electrospinning method16
. Cell infiltration does appear to increase with this method compared to conventional electrospinning. However, these studies were carried out over a 7-day period 16
, whereas experiments in the present study show high cell viability after 21 days in culture.
In the present study, we performed uniaxial tensile testing to characterize the cryogenic electrospun silk further, and found a 3.5-fold decrease in strength of the scaffolds compared to conventional electrospun silk. Although mechanical strength did decrease with increased porosity, the decrease for silk was considerably less than the 40-fold reduction in tensile strength reported for cryogenically electrospun poly(D,L- lactide) when compared to conventional electrospun poly(D,L-lactide) 12
Cellular infiltration appears to be comparable between the cryogenic electrospun silk and poly(D,L-lactide).
In this work, we have demonstrated the potential of cryogenic electrospinning as an improvement over conventional electrospinning for true three-dimensional cell culture on a natural polymer. To our knowledge, we report the first electrospun silk scaffold with enhanced cell infiltration and cell viability as a result of increased void volume throughout the scaffolds, which has been a limitation of previous studies21
. We have successfully demonstrated epithelial cell viability in co-culture with fibroblasts, as well as terminal differentiation of keratinocyte cultures, and suggest that this new scaffold system can, potentially, be used for many applications ranging from in vitro
three-dimensional cell culture for tissue modeling as well as in vivo
applications for tissue regeneration and reconstruction.