Influence of fiber diameter on cells

Figure 1. Hela cells on glass coverslip showing preferential proliferation and adhesion on nanofibers instead of exposed smooth surface.

Electrospinning is able to fabricate scaffolds comprising of fibers from less than 100 nm to a few microns. Given that most collagen fibers in native extracellular matrix (ECM) are in the nanometer dimension, it is important to understand the influence of fiber diameter on cell behaviour. This will allow investigators to tailor fiber diameter according to the desired cell response. More common and established fiber manufacturing techniques are able to produce fibers with diameter from 5 µm onwards and these techniques may be used instead of electrospinning if smaller fiber diameter does not give a significant benefit in terms of cell response. Tuin et al (2016) showed that electrospun fibers with diameter of 1.1 µm demonstrate no significant advantage for human adipose derived stem cells (hASC) in terms of attachment, proliferation, and both adipogenic and osteogenic differentiation of hASC compared to scaffolds produced by melt blown, spunbond and carded with fiber diameters ranging from 5 µm to 27 µm. However, numerous studies have suggested that the benefit of smaller fiber diameter is only obvious when it is below 1 µm.

Fiber diameter and its surface architecture have a significant influence on fibroblast adhesion. Using electrospun polycaprolactone fibers, Chen et al (2009) found that 3T3 fibroblast attachment on 428 nm nanofibrous scaffold was significantly better than fibers with larger diameter up to 1.6 µm from 2 hrs to 24 hrs. Beaded fibers generally fare worst although it have the smallest diameter of 117 nm. Cell proliferation was also the best on 428 nm fiber across all time point up to 7 days. Beaded fibers have the least cell proliferation. On smooth fibers, cell proliferation was the least at fiber diameter 900 nm to 1050 nm. Surprisingly, for fiber diameter 1.6 µm, 3T3 fibroblast proliferation is comparable to that on 428 nm fiber scaffold for day 3 and 5.

Chondrocytes were also positively influenced by fibers with diameter less than 500 nm. Noriega et al (2012) compared the response of bovine articular chondrocytes cultured on electrospun chitosan fibers with diameters of 300 nm and 1 µm and freeze dried chitosan scaffolds. Chondrocytes cultured on scaffold with mean fiber diameters of 300 nm showed a 2-fold higher ratio of collagen II/collagen I compared to freeze dried chitosan scaffold. Both electrospun scaffolds with 1 µm and 300 nm diameter fibers showed more aggrecan expression freeze dried chitosan scaffold. Their studies also suggested that up-regulation of the expression of collagen II and aggrecan in the electrospun scaffold with 300 nm diameter fibers was probably due to up-regulation in mDia1 and Sox-5 compared to ROCK-1 and Sox-9.

Osteoblastic differentiation has also been observed to be influenced by fiber diameter. Using electrospun gelatin fibers with diameters of 110 nm and 600 nm, Sisson et al (2010) found that MG63, a human osteoblast-like cell line, showed significantly higher levels of Alkaline Phosphatase (ALP) when cultured on the smaller diameter fibers at day 3 and 7. ALP level started to decrease from day 7 to day 14 for both scaffolds and the ALP activity was statistically similar for both fiber diameters scaffolds. ALP is an early differentiation marker for osteoblastic cells and this positively demonstrated the influence of fiber diameter on early cell differentiation.

Cell alignment due to fiber orientation is also dependent on the fiber diameter and cell type. For adult human dermal fibroblasts, Liu et al (2009) found that a minimum diameter of 0.97 µm is required for cell orientation to occur. At lower fiber diameter, the aspect ratio of the cell is comparable to film. On the contrary, for endothelial cells, aligned fiber diameter over 1.2 µm seems to have less influence on cell orientation. Using electrospun aligned polycaprolactone (PCL)/collagen fibers with different fiber diameters (100 nm, 300 nm and 1200 nm), Whited and Rylander (2014) found that influence on fiber orientation on primary human umbilical vein endothelial cells (HUVEC) orientation was weakest for 1200 nm fibers. There was no significant difference in fiber directed cell orientation for diameter of 100 nm and 300 nm. Brown (2012) studied the effect of aligned fiber diameter on the quantity of in vivo blood vessel formation in rat spinal cord injury. Using polydioxanone aligned fibers of average diameter 1 µm and 2 µm, it was found that the smaller fiber diameter yield more blood vessels.

Another favourable characteristic of fibrous scaffold with smaller fiber diameter for use as vascular graft is in its blood activation. Milleret et al (2012) found that scaffold composed of fibers less than 1 µm triggered very low coagulation with negligible platelet adhesion. However, larger fiber diameter (2- 3 µm and 5 µm) triggered higher thrombin formation and platelet adhesion. Han et al (2019) investigated the effect of electrospun polycaprolactone (PCL) diameter on smooth muscle vascular cell (SMVC) infiltration and proliferation in In vitro tests and macrophage activation in a rat subcutaneous model. The tested fiber diameters range from 0.5 µm to 10 µm. Their results showed that smaller fiber diameter encourages greater cell proliferation with fiber diameters less than 1 µm showing fastest proliferation and fiber diameters of 7 and 10 µm, the slowest. However, for cell Infiltration, fiber diameters of 5, 7 and 10 µm showed the best cell Infiltration. In terms of maintaining the SMVC contractile phenotype, there is a general reduction in the contractile phenotype as the fiber diameters increase. Fiber diameters less than 1 µm have the most number of cells maintaining the contractile phenotype at 10 days. In the In vivo studies larger fiber diameters increases the number of activated macrophages. The macrophages secrete angiogenic factors that stimulate neovascularization which may help to recruit sufficient my fibroblasts to populate the scaffold.

Fiber diameter has a significant effect on the differentiation and proliferation of rat adult neural stem/progenitor cell (rNSC). Using laminin-coated electrospun Polyethersulfone (PES) of different fiber diameters (283 nm, 749 nm and 1452 nm), Christopherson et al (2009) showed a 40% increase in oligodendrocyte differentiation on the smallest diameter fiber in a randomly oriented scaffold when rNSC was cultured in differentiation condition compared to tissue culture polystyrene surface (TCPS). At a larger fiber diameter of 749 nm, the differentiation was reduced to just 20%. On 283 nm diameter fibers, the cell stretched multi-directionally under the influence of the underlying fibers. However, on larger diameter fibers, the cell extended along the single fiber. Larger diameter fibers also reduced migration, spreading and proliferation of rNSCs in the presence of FGF-2 and serum free medium. Christopherson et al (2009) suggested that under differentiation medium, rNSCs preferentially differentiate into oligodendrocytes on smaller fiber diameter substrate while on larger fiber diameter (749 nm and 1452 nm) substrate, rNSCs preferentially differentiate into neuronal lineage.

In the differentiation of mesenchymal stem cells (MSC) to ligament phenotype, Bashur et al (2009) found that fiber diameter is a key parameter driving its differentiation. Expression of collagen I/αI, decorin and tenomodulin decreases with increasing fiber diameter (from 0.28 µm to 0.82 µm).

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Content [Biomedical (General)]