Angiogenesis in Electrospun Scaffold

Muscle tissues implanted with (A) scaffolds with aligned fiber orientation loaded with bFGF showing tubular constructs from CD31+ cells, (B) non bFGF containing groups showing numerous pockets of CD31+ cells without vessel formation, and (C) non-ligated/untreated control with small numbers of CD31+ cells. [Montero et al 2014 Open Journal of Regenerative Medicine 2014; 3: 1. This work is licensed under a Creative Commons Attribution International License (CC BY).]

Long term survival and integration of an implant is dependent on the presence of vasculature within it. Some researchers have argued that since nutrients and metabolic exchanges are known to be limited to less than 200 µm (Tannock 1972), implants that are larger than this is doomed for failure even with cells inside as they would die anyway without sufficient supplements. Having adequate vascularisation shortly after implanting a reasonable thick scaffold is vital for successful clinical application. Since the rate of blood vessel growth is about half a millimeter or less per day [Clark 1939], it is a challenge to ensure adequate angiogenesis for cell survival.

A requirement for vascularization in a scaffold is that the pore size needs to be sufficiently large. This is to allow migration of endothelial cells and to form tubules inside scaffold. Study by Leong et al (2009) used low temperature electrospinning to construct a scaffolds with pore sizes ranging from 900 µm² to 5000 µm². Such large pore size allows cell infiltration and blood vessels containing intraluminal red blood cells were observed in the 400 µm-thick scaffold at day 56. In contrast, cells showed poor infiltration into electrospun scaffold constructed using conventional electrospinning with no evidence of blood vessels. Poor vascularization of conventional electrospun membrane was also demonstrated by Cheng et al (2013) where an implanted nonwoven plasma treated poly(L-lactide) membrane into the subcutaneous abdominal cavity of Sprague-Dawley rats showed no CD31 positive cells within the membrane depth after 14 days. Instead, monocytes and other cells were found through the membrane thickness. Alternate post processing techniques such as laser cutting to introduce macro pores into the electrospun membrane has been used to promote cellular infiltration and vascular ingrowth. Joshi et al (2013) created a laser perforated scaffold made from electrospun membrane sheet with pore sizes of 80 µm, 160 µm and 300 µm and rolled into the form of a cylinder. The perforation was designed to ensure overlapping of the pores after rolling. This subsequently implanted in the peritoneal cavity of Lewis Rats after wrapping in omentum. They found that only scaffold with 300 µm exhibits full cell penetration after two weeks and significant vascular ingrowth to a distance of 850 µm. This shows that scaffold with sufficiently large pore size is required for adequate vascularization.

Formation of vascular tubules requires the endothelial cells to come together and align in a given direction. Thus, scaffold-directed endothelial cell alignment may be the first step towards encouraging angiogenesis. Studies by Montero et al (2014) and Brown (2012) have shown that aligned fibers were able to guide endothelial cells growth and their formation to functional vessels while random fibers results in scattered endothelial cells. Brown (2012) further 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.

It is unclear whether the presence of nanofibers will encourage endothelial cell to form capillaries. Santos et al (2008) compared the density of tubules formation within a collagen gel by migrating endothelial cells from microfiber scaffold and nanofiber-microfiber scaffold under the influence of growth factors. Their results showed significantly more tubule formation from the nanofiber-microfiber scaffold. It was noted that endothelial cells on the nanofibers have a stretched morphology while cells on the microfiber are confluent and flat on the microfiber surface. Stretched cells on the nanofibers were said to be more responsive to angiogenic growth factors thus organizing themselves to form tubules unlike the less responsive cells on the microfibers. However, the tubule formation may be due to more endothelial cells coming into contact with the collagen gel as they were found in the spaces between the microfibers due to the presence of inter-fiber nanofibers. Endothelial cells generally do not self organize to form tubules on a flat surface and on nanofibers membrane. However, there are some studies that reported signs of endothelial cells organization into tubules on electrospun fibers scaffold. Al Rez et al (2017) tested the potential use of electrospun poly(ε-caprolactone)/chitosan (PCL/Chitosan) nanofibrous scaffold in vascular tissue engineering. After 12 days of culturing Primary Human Vascular Endothelial cells on PCL/Chitosan nanofibrous scaffold, some endothelial cells were observed to form vessel like structures. However, these vessels were isolated and it remains to be seen whether it will form long and viable vessels given a longer culture duration.

To encourage angiogenesis for Implants, the scaffold may be populated with cells before implantation. Millán-Rivero et al (2019) showed in an hairless SKH1 mice full thickness wound model, that silk fibroin (SF) electrospun scaffold seeded with mesenchymal stem cells (MSCs) exhibited higher vascular surface area. Without MSCs, the vascular surface area on silk fibroin scaffold dropped off on week 3 and 4. The vascular surface area was significantly higher if MSCs were injected at the periphery of the silk fibroin scaffold. The group with MSCs seeded on silk fibroin scaffolds and injected at the periphery has the highest vascular surface area at week 3 and 4.

Incorporation of biochemical cues onto scaffolds may be able to encourage and accelerate angiogenesis. However, when used with oriented electrospun scaffold, the combination has been shown to increase the rate of angiogenesis with directed vessel formation. Growth factors have been added to electrospun scaffold through simple blending with the spinning solution prior to electrospinning, using core-shell fiber [Joung et al 2011] and growth factor encapsulated particles on fibers [Devolder 2013]. Montero et al (2014) examined the angiogenic potential of electrospun aligned and randomly oriented gelatin B nanofiber with and without growth factor (bFGF) added in a murine critical limb ischemic model. The results showed low level of reperfusion in scaffolds with bFGF and the highest level of reperfusion for aligned scaffolds loaded with bFGF. Aligned scaffolds with bFGF showed long straight vessels parallel to the nanofiber alignment while isolated vessel structures were found on random scaffolds demonstrating the benefit of fiber alignment in directing blood vessel formation and encouraging tube formation. No difference in the blood vessel diameters were observed for all the groups. Devolder (2013) electrosprayed particles containing vascular endothelial cell growth factor (VEGF) onto electrospun fibers (diameter of 1.5 µm) and showed that the presence of fibers increases the number of blood vessel formation in a Chorioallantoic Membrane (CAM) model. Particles containing VEGF but without electrospun fibers were unable to stimulate greater number of blood vessel formation compared to control CAMs. Using electrospun fibers coated with VEGF-free particles resulted in randomly oriented blood vessels with small diameter. Randomly aligned electrospun fibers coated with VEGF-loaded particles also resulted in smaller size and irregularly spaced blood vessels. It is only with VEGF-loaded particles on aligned fibers where large number of blood vessels was formed with smaller spacings.