*5.3. Growth Factor*

Growth factors are naturally occurring endogenous signaling molecules (i.e., proteins or steroid hormones) which are efficient for inciting cellular development, differentiation, regeneration and proliferation by binding to cellular receptors. Recently, marked progress has been made in preparing several types of ultrathin electrospun nanofiber scaffolds to successfully load and deliver several growth factors (Figure 6a) as well as regenerative medicines, owing to the unique characteristics of nanofibers such as large surface area, porous formation, high loading capacity, easy access, and cost-effectiveness [133]. Notably, the large surface area to volume ratio of electrospun nanofibers facilitates cell adhesion, loading, storage and release of growth factors [134]. Those parameters are required for guiding cellular behaviors and transmitting signals that regulate proliferation, differentiation, metabolism, and apoptosis of cells, including extracellular matrix deposition in tissue engineering.

The quick inactivation and very short biological half-life of growth factors are the most important factors hindering their effectual delivery. In addition, it has been noted that the growth factors' carrier should have slow and sequential releasing properties. The establishment of growth factors into nanofiber scaffolds play very crucial roles in repairing and regenerating damaged tissues by mimicking signal transduction from cell to cell, or from cell to its extracellular matrix [135]. Either electrospinning procedures alone (for example, bending, emulsion, and coaxial electrospinning) or in combination with other traditional methods such as hydrogel (electrospinning) are ranked at the top among all nanofiber fabrication techniques for their controlled and sustained release properties. On the other hand, surface modified nanofibers promote high loading efficiency, protection of incorporated growth factors and the binding of growth factors to cell receptors. Those surface modified nanofibers exhibit biochemical and morphological uniformity to natural tissues.

Recently, the polysaccharide matrix is being used as a superior nanofiber material to fabricate a growth factor carrier owing to their low toxicity, biocompatibility, and extracellular matrix mimicking capability. However, introducing the sulfation state to the alginate scaffolds ensures the appointed binding and loading of growth factor into the polymer backbone, as well as maintenance of its control release properties. For instance, Mohammadi S, et al. fabricated electrospun scaffolds by blending alginate sulfate and polyvinyl alcohol to load and deliver transforming growth factor-beta1 (TGF-β1) for tissue engineering applications. For the first time, this research revealed that more successful binding, encapsulation and sustain release of heparin-like proteins from electrospun scaffolds could be achieved by the addition of an affinity site to the alginate [134]. A part of mammalian polysaccharides—glycosaminoglycans (heparin-like substance)—would be another example of an excellent nanofiber matrix because of its efficiency in conjugating with GFs by successfully interacting with its (GFs) anionic sulfated backbones and mimicking a pathway similar to ECM.

**Figure 6.** Schematic of the synthesis of growth factor loaded electrospun nanofiber with the controlled release properties (**a**), and VEGF (vascular endothelial growth factor) release profile with respect to time from PCL nanofibers (Reproduced from ref no [123] with permission from ACS Publications, 2018) (**b**).

Usually, the traditional nanofiber scaffolds-based GFs delivery methods such as dip coating and covalent binding are associated with low entrapment efficiency and primary burst release properties. Currently, researchers are looking for new, innovative and stable strategies by focusing on controlled and sustained release properties. To control the release properties of encapsulated GFs, the usage of gelatin particles in nanofiber fabrication is becoming popular. Generally, the in vitro degradation time of gelatin particles is about 240 h, so researchers tend to use gelatin particle-mediated nanofiber scaffolds as a promising GF nanocarrier. The basic advantages of using gelatin particles are their slow degradation and controlled release properties in a biological environment because of

introducing cross-linking treatment during fabrication. An example would be the work of Jiang and co-workers who prepared PCL fibrous scaffolds containing vascular endothelial growth factor (VEGF)-incorporated gelatin particles which were able to exhibit both diffusion and degradation mediated release for a long period of time (Figure 6b), differentiate mesenchymal stem cells from endothelial cells, sustain the durability of the tubular composition, and form new blood vessels among endothelial cells [136]. The integration of various sustained release characteristics within nanofiber scaffolds protects the encapsulated GFs from biological degradation, generates cellular signal transduction as well as repairs and regenerates damage.

Biocompatible and biodegradable polymeric nanoparticles and biomimetic hydrogel have gained more attention for encapsulating various types of growth factors and confirming their controlled and prolonged releasing [133]. VEGF and bone morphogenetic protein 2 (BMP2) were incorporated and injected to improve the angiogenesis and regeneration of bone in rabbits [133]. Another preclinical study suggested that VEGF-loaded and hMSC (human bone marrow stroma cells)-seeded PDLA nanofiber scaffolds are able to regenerate blood vessels and enhance bone growth [137]. Interestingly, to achieve dual actions from two separate GFs at a time, core/shell nanofibers or emulsion electrospinning methods are widely used in tissue engineering applications. Liu et al. prepared bicomponent scaffolds with the emulsion electrospinning method for delivering two GFs at the same time in nerve tissue engineering. In this study, the nerve growth factor was encapsulated in poly(D,L-lactic acid) (PDLLA) nanofiber scaffolds whereas the glial cell line-derived neurotrophic factor was incorporated in poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds [138]. This dual factor core/shell electrospun nanofiber scaffold not only revealed its sustained release and degradation-dependent weight loss properties over 42 days but also ensured the high entrapment efficiency and the intact bioactivity of GFs. Apart from degradation-based controlled release behavior of electrospun scaffolds, providing the affinity sites and/or covalent binding of heparin to the surface of nanofibers is also established. The covalent binding of heparin-like proteoglycans on the surface of the nanofibers preserves the biological integrity of GFs and promotes the sustained release properties of the scaffolds. The aminated PLGA was immobilized with VEGF in the presence of heparin via EDC/NHS coupling to facilitate the sustained release properties of the encapsulated growth factor [139]. The presence of hydroxyl (–OH) and carboxylic acid (–COOH) groups on the surface of the heparin simplifies its modification with both GFs and nanofiber scaffolds. Heparin also increased the binding capacity of VEGF to VEGF receptors on the cell surface.

The time period of sustained release of GFs from its carrier should be proportional to the time period of GFs capability to preserve the nanofiber scaffolds; therefore, this important factor is considered during the design of nanofiber-mediated GFs delivery. Employing several electrospinning techniques, the preservation of GFs' bioactivity is achieved for the proliferation and migration of specific cells. The heterogeneous electrospun matrix made of biocompatible poly(L-lactide-co-caprolactone) and poly(2-ethyl-2-oxazoline) was able to protect the bioactivity of dual angiogenic basic fibroblast growth factor and vascular endothelial growth factor during and after their physical blending. It is also capable of mimicking the topography and chemical cues of native cardiac tissue structures [140]. The structural integrity and bioactivity of encapsulated GFs remained the same over the total period of sustained release in comparison with the pure GFs. In another study, for the first time, Naskar et al. reported the anteriority of using natural polymer silk protein fibroin reinforced synthetic carbon nanofiber scaffolds for preserving bioactivity and controlling the sustained release kinetics of BMP-2 and TGF-β1 GFs over a prolonged period of 50 days [141]. The primary attachment of GFs and the regeneration, excess proliferation, and differentiation of both osteoblastic cells and MSC proved the structural integrity of encapsulated GFs in the nanofiber scaffolds. Moreover, a few linear polysaccharides (i.e., chitosan) are very popular for encapsulating GFs (transforming growth factor beta-1). The major challenge associated with the polysaccharide-based GFs delivery system is their short biological half-life. Hence, they are further composited with nanofiber scaffolds in order to improve the long term presence of GFs in stem cells through maintaining their

sustained release from electrospun scaffolds [142]. Previously reported data proved the efficiency of electrospinning methods to load and achieve the sustained release properties of two different GFs at the same time in tissue engineering applications, but now it is also possible to accomplish the distinct release kinetics and spatiotemporal delivery of two different GFs from the same scaffolds with various layered configurations by applying several electrospinning techniques. The combination of emulsion, sequential, high speed and double-sourced double power electrospinning were employed to fabricate tri and tetra layered nanofiber scaffolds using poly(lactic-co-glycolic acid) and poly(D,L-lactic acid) [135]. This glial cell line-derived growth factor and nerve growth factor incorporated multilayer electrospun nanofibers was able to maintain various release actions of these GFs due to aligned fiber coating in each layer with a controlled thickness. The cumulative release studies of encapsulated-GFs indicated distinct release properties from different sites of tri-layered and tetra-layered nanofiber scaffolds over a period of 42 days.
