Electrospun Scaffold Inflammatory Reaction

Immunohistochemical images for staining of CD68-positive total monocytes and macrophages for Poly(L-lactide-co-D/L-lactide) electrospun mesh at day 14[Schnabelrauch M et al. International Journal of Polymer Science, vol. 2014, Article ID 439784, 12 pages, 2014. doi.org/10.1155/2014/439784. This work is licensed under a Creative Commons Attribution 3.0 Unported License.]

For electrospun nanofibrous scaffold to be successful in regenerating the tissue, it is important to understand the foreign body reaction upon implantation. This reaction is a nonspecific protein adsorption, immune and inflammatory cells response to a foreign object. In the first stage, disrupted vasculature during implantation exposes the medical device to blood and its components. Proteins present in the blood adhere on the surface of the medical device depending on factors such as surface topography, hydrophobicity and surface charge [Wilson et al. 2005]. A provisional matrix consisting of fibrin network produced through the hydrolysis of fibrinogen by thrombin would form over the injury site within minutes to hours following implantation [Anderson 2001]. This causes a cascade of biochemical signalling to attract neutrophils over the next 24 to 48 h (Zeller 1983) which is subsequently taken over by emigration of monocytes and fibroblasts[Wilson et al. 2005; Anderson 2001; Anderson et al. 2008]. The monocytes subsequently mature into macrophages and these may be present for weeks to months depending on the implant. In a normal inflammatory reaction, phagocytes would engulf and digest the foreign body. However, as the biomaterials are generally much larger than the phagocytes, frustrated phagocytosis may occur where enzymes are released to degrade the material [Zeller 1983]. Simultaneously, proliferation of fibroblasts and vascular endothelial cells at the implant site lead to the formation of granulation tissue as early as 3 to 5 days following implantation of the biomaterial [Anderson 2001]. Fibroblast is active in synthesizing collagen and proteoglycans while organization of endothelial cells facilitates angiogenesis. Extensive production of collagen around the implant leads to the formation of a fibrous capsule which isolates the biomaterial from the surrounding tissues [Wick et al. 2010]. Differentiation of fibroblast-like cells to myoblasts and their proliferation would later cause capsule shrinkage around the implant resulting in instances such as breast implant dysfunction [Zeller 1983].

Since foreign body reaction is a non-specific immune response to introduction of any implanted material, nanofibrous scaffold will also undergo similar scrutiny by the body's defence. Smith et al (2007) conducted an in vitro study to investigate the immune response of cells to electrospun membrane of different materials. The antibody production of sheep splenocytes was quantified to determine their in vitro hemolytic antibody-forming cell response when exposed to the electrospun membranes. Materials investigated include non-electrospun ePTFE and electrospun membrane of nylon, polydioxanone (PDO), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), and a 50:50 (v:v) blend of PDO and polycaprolactone (PCL). The results showed that electrospun PDO, PGA, PLA, PDO-PCL are immunosuppressive in the Mishell-Dutton assay (lower antibody production compared to cell cultured in media) while e-PTFE and nylon are not. A comprehensive in vitro study of the immune response to electrospun polydioxanone, elastin and their blends involving macrophage biochemical response, T-cell activity, Mishell-Dutton assay and others, demonstrated immunosuppression activity [Smith et al 2009].

The degradation characteristic of a material has a significant effect on the in vivo inflammatory reaction. Implantation of PGA and PGA-LA nanofibrous scaffold in the rat vastus lateralis muscle for 7 days showed evidence of fibrotic encapsulation at the interface of the implants and the surrounding tissue and the presence of foreign body giant cells. Gelatin nanofibrous scaffold also showed poor result with evidence of lymphocytes and fibrotic encapsulation. In the same study, PLA nanofibrous scaffold showed less pronounced fibrotic zone and appear to support cell infiltration better while collagen showed the best result with good integration and vascular infiltration after 7 days [Telemeco et al. 2005]. Another study also showed that PGA induces fibrotic encapsulation with presence of foreign body giant cells [Boland et al. 2004]. Presence of foreign body giant cells on PGA and PGA-LA scaffold is likely to be due to its faster degradation rate and subsequent release hydrolytic fragments which will evoke a greater immune response from the body [Telemeco et al. 2005]. Subcutaneous in vivo study of P-DL-LA-co-PGA electrospun microfibers have shown that the fiber diameter initially increases significantly especially with higher PGA content which probably due to water uptake. Therefore, although the initial diameter of this polymeric fiber may be in the nano-scale, subsequent fluctuation in the fiber diameter may negate its advantage as a biomimetic scaffold. With P-DL-LGA (50:50), crystallization of oligomeric fractions of degraded material formed large crystalline balls at the implant site which were free of cells. Multi-nucleated foreign giant cells were also observed on the implant [Blackwood et al. 2008]. Therefore, this material is unlikely to be suitable as implants. For gelatin, rapid breakdown and release of protein fragments may also evoke a strong immune reaction [Telemeco et al. 2005]. In general, inflammatory reaction of slower degrading synthetic polymers were less pronounced than faster degrading counterpart [Ishii et al. 2009;Telemeco et al. 2005]. However, thin to moderate capsule is likely to surround the biodegradable nanofibrous scaffold [Bölgen et al. 2007] if the degradation rate is too slow and cells and blood vessels are unable to infiltrate into it.

Physical topography of the scaffold such as surface roughness, pore size and fiber diameter also has a significant influence on the foreign body response. Comparison of separate in vitro studies have suggested that film with rough surfaces in the nanometer scale (Ra of 150 nm to 4500 nm) tend to led to a decrease or stagnation in fibroblast proliferation [Vance et al. 2004;Prasad et al. 2010;Kunzler et al. 2007] while smoother surface (Ra < 200 nm) favours increased proliferation [Prasad et al. 2010; Kunzler et al. 2007;Mao et al. 2009] although the surface roughness does not seem to affect the level of initial cell adhesion. In vivo studies have also demonstrated thinner fibrous capsule formation when the surface roughness is greater than 500 Ra [Kim et al. 2006]. As compared to a film, electrospun nanofibers scaffold will have a greater surface roughness. Cao et al (2009) showed that with electrospun polycaprolactone scaffold with fiber diameter of 300 to 500 nm, the fibrous capsule thickness in a Sprague Dawley rat subcutaneous model was less than 8 µm compared to more than 37 µm on film. However, examination of the severity of inflammatory reaction by macrophages and foreign body giant cell activity was found to be the highest in random nanofiber mesh and film having the least at 4 weeks.

The immune response on the scaffold is due to biochemical cue expressed by the cells in response to its presence. The level of fibrous capsule formation and scaffold isolation is partly dependent on the immune response. Saino et al (2011) examine macrophage activation and secretion of proinflammatory cytokines and chemokines on aligned and random PLLA nanofibers (about 600 nm) and microfibers (about 1.5 µm) and film in an in vitro study at 24 h and 7 days. Their results suggest that nanofibers evoke the least secretion of pro-inflammatory molecules with film registering the highest number of foreign body giant cell. This is in agreement with a study by Sanders et al (2000) which compares macrophage density in tissue implanted with single polypropylene microfibers ranging from 6.5 µm to 26.7 µm and showed less fibrous capsule thickness and macrophage density for small fibers. However, a separate study by Cai et al (2010) showed the highest number of monocytes and macrophages on random polycaprolactone nanofibers (fiber diameter of 313 nm) compared to film and align nanofibers (fiber diameter of 506 nm). Evidence from an in vitro study of growth factors secretion by macrophages cultured on electrospun polydioxanone (PDO) of different diameters also suggested potential lower fibroblast activity for smaller diameter fibers. Garg et al (2009) showed that while vascular endothelial growth factor (VEGF) secretion was largely similar across all diameters over a 28 days period, acidic fibroblast growth factor (aFGF) and transforming growth factor beta-1 (TGF-β1) were generally lower across all time points for the smallest diameter fibers compared to larger diameter fibers. Addition of elastin into PDO was also found to reduce the aFGF secretion from day 7 to day 28. While most evidences seem to favor smaller diameters fiber for evoking less inflammatory response, more studies need to be carried out to determine whether there is a lower diameter limit.

In the last few decades, there are many research on seeding scaffold with cells prior to implantation such that integration and recovery can be accelerated. The presence of cells in the scaffold will certainly influence the host body reaction to it as the host tissues interacts with the introduced cells. 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) significantly reduces the number of polymorphonuclear neutrophil (PMN) infiltrate compared to pure SF electrospun scaffold. Macrophage, leukocyte and T cell were also found to be significantly lower in SF seeded with MSCs.Therefore, the presence of seeded stem cells have been shown to reduce immune infiltrates.