2.2.2. Bone Tissue Engineering Applications

Bone is a strong rigid organ that plays an essential role in our body. It protects our vital interior organs, movements, manufactures white blood cells and red blood cells, and also stores minerals [98]. Bone extracellular matrix mainly consists of organic and inorganic components, such as collagen and hydroxyapatite (HAp). Incorporation of these components results in suitable scaffolds for bone tissue engineering applications. The architecture of the electrospun scaffolds, including microstructure, porosity and surface properties, plays an essential role in successful bone regeneration [99]. Electrospun fibers should offer better mechanical properties to support the structure, and provide space for osteochondral adhesion, proliferation, and differentiation. Hence, the development of an ideal scaffold for tissue regeneration could be achieved by using a porous ceramic material, lamellar material and a fiber matrix material for imrpoved biological and physical properties. Subramanian Uma Maheshwari developed a scaffold comprised of a polymer–ceramic combination in a PCL/PVA bilayer scaffold blended with HAp NPs [100]. (PVA-PCL)-HAp has an improved porosity of around 64%, as well as hydrophilicity of around 141%. Also, MTT assay studies with MG-63 osteoblast cells had better cell adhesion and proliferation, which indicates promise for application in tissue regeneration. However, the incorporation of growth factors (GFs) or drug to the scaffold is also crucial for enhancing the regrowth of broken bones. Many GFs, including bone morphogenetic protein-2 (BMP-2) and VEGF, have been added to electrospun scaffolds in order to achieve long-lasting sustained release of GFs to mimic the natural healing process. For instance, co-axial electrospun of collagen-PCL incorporating BMP-2 and dexamethasone (DEX) have shown a more controlled release of GFs, thereby encouraging the osteogenic expression of human mesenchymal stromal cells (hMSCs) [101]. In this design, the shell layer was loaded with DEX, and the core incorporated BMP-2. Dual drug release was exhibited, in which DEX showed a fast release. However, BMP-2 demonstrated a sustained release over 22 days. This scaffold provides an efficient healing process, as well as osteogeneration.

On the other hand, incorporation of stem cells into the biomaterials is also a novel approach for tissue regeneration of the cells. For instance, Abbas Shafie has studied, in vitro and in vivo, cartilage tissue regeneration from rabbit bone marrow mesenchymal stem cells (BM-MSC) seeded on the electrospun scaffold of PVA/PCL nanofibers [102]. In vitro, the MTT assay showed that the scaffolds supported the chondrogenic differentiation of MSC. In vivo, the scaffold with and without MSC loading was implanted in rabbit full-thickness cartilage defects. To study cartilage regeneration, histological and semi-quantitative grading was executed. The results showed that scaffold seeded with MSC enhanced the healing process in comparison to non-seeded scaffolds. These results indicate that PVA/PCL scaffold seeded with MSC is suitable for grafts for articular cartilage repair.

Recently, the effect of polyacrylonitrile/MoS2 nanofibers on the growth behavior of bone marrow mesenchymal stem cells (BMSCs) was discussed by Wu et al. [103]. The nanofibers were realized to enhance the contact of BMSCs with each other, to enhance cellular behavior, and also to provide positive promotion of regulation of cellular proliferation [103]. In addition, for guided spinal fusion, an injectable and thermosensitive hydrogel made of collagen/n-HA/BMP-2@PCEC/PECE enclosed in poly(D,L-lactide) (PDLLA) nanofibrous membranes was made by Qu et al. [104]. This system restricted the escape factor in order to maintain osteogenesis in the desired position [104].

#### 2.2.3. Skeletal Muscle Regeneration

Skeletal muscle makes up around 40% of the human body. Skeletal muscle is made of various fibers, with diameters ranging from 10 to 80 μm [105]. These fibers are unidirectional and produce an enormous amount of force during contraction [106]. If a muscle cell gets injured or wounded, it will not be possible to contract, and satellite cells are switched on in order to perform their muscle cell regeneration activities. However, this healing process can create scar tissue and block muscle function [106]. Many efforts have been made to study the initial steps of muscle regeneration, such as autologous muscle transplant, satellite cells, exogenousmyogenic cells, and myoblasts, but these methods have met with limited success [107]. Therefore, long-term denervation and severe injuries can lead to the loss of skeletal muscle function.

Muscle tissue engineering materials require better contraction ability and mechanical properties [108]. Muscle cell adhesion and proliferation have been studied using both mechanical properties and electric stimulus in cell culture. Mckeon-Fischer K D prepared co-axial electrospun fibers with a PCL core and a modified outser shell layer comprising multiwalled carbon nanotubes (MWCNT) and a blend of (83/17, 60/40, 50/50, and 40/60) poly(acrylic acid/poly(vinyl alcohol) (PAA/PVA) [109]. All four components were electrically conductive, although the scaffold was not actuated when an electric field was applied. The best results occurred at 20 V. MTA assay in soleus and vastus lateralis (VL) muscles extracted from rats showed that 0%, 0.14% and 0.7% concentrations of MWCNT in the scaffold were non-toxic for cells over a four-week period. Based on the different percentages of blend solutions, 40/60 PAA/PVA in the outer layer illustrated a higher number cells than other scaffolds. The scaffold has tensile properties that are higher than those of skeletal muscle. Further modification of these scaffolds for contraction, rather than bending, could lead to promising scaffolds for artificial muscle applications.

#### 2.2.4. Nerve Tissue Engineering

Electro-conducting polymers such as polypyrrole (PPy), polyaniline (PANI), polythiophene (PT), poly(3,4-ethylene dioxythiophene) (PEDOT)) show attractive electrical and optical phenomena. Thus, they have been researched in the past few decades for various applications such as microelectronics, actuators and polymer batteries [110]. Electro-conducting polymers that possess the advantages of biocompatibility and good conductivity can be applied as biosensors and tissue engineering scaffolds [111]. Electrospun electro-conductive polymers are an excellent tool for electrically stimulating neurons and for nerve tissue engineering, as well as for application in neural prostheses for therapeutical function [88,89,112]. Schmidt et al. first studied PC12 cells using the polypyrrole (PPy) electroconductive polymer, recognizing the growth of PC12 cells on the PPy thin film, they enhanced the neurite outgrowth from the cells; these results suggest significant application of these type of

scaffolds for nerve tissue regeneration [113]. Many studies have proposed the improvement of the electro-conducting polymer for nerve tissue regeneration applications by adding cell adhesive [114], neurotrophins [115], and topographical features [116]. Jae Y Lee prepared electrospun nanofibers coated with the conductive polymer PPy for nerve tissue engineering applications [117]. PPy-PLGA showed improved growth of rat pheochromocytoma 12 PC12 cells and hippocampal neurons compared to non-coated PLGA as a control. This suggests that PPy-PLGA could be used for nerve tissue engineering applications. Simultaneously, electrical stimulus studies on the scaffold indicated that a stimulus of 10 mV/cm improved the neurites such that they were 40–50% longer, as well as exhibiting 40–90% greater neuron formation, compared to the same scaffolds with no stimulus. Moreover, aligned scaffolds show greater neurite elongation and formation than randomly oriented PPy-PLGA fiber scaffolds. The good results for electric stimulus suggest that biocompatible polymers prepared by electrospinning have significant advantages in biomedical applications such as nerve tissue engineering.
