**4. Tissue engineering applications**

As mentioned in the introduction section, natural and synthetic fibers from different sources have been widely used in many areas for hundreds of years, and tissue engineering area happens to be one of them. TE maintains an alternative way to the restoration and regeneration of the injured tissues. TE is an interdisciplinary field that requires knowledge of biological, chemical, and engineering sciences toward the objective of tissue regeneration using cells, factors, and biomaterials alone or in combination with each other. With the light of this brief information, widely used TE applications of nanofibers are discussed below.

## **4.1 Muscoskeletal tissue engineering applications**

### *4.1.1 Bone tissue engineering (BTE)*

Bone tissue is mainly composed of organic bone matrix, which mostly includes collagen fibers (95% of these fibers are collagen type I) and inorganic compounds such as hydroxyapatite crystals [63]. There is a global need for bone grafts because of the high incidence of bone defects, which are caused by bone tumors, infections, and bone loss by traumas. Main treatment approaches for these injuries are autografts, allografts, or xenografts. But there are some challenges to these approaches such as inflammation, scarring, infection, immunological graft rejection, hematomas, high-cost procedures, etc. [64, 65] At this point, bone tissue engineering (BTE) approaches present an alternative treatment way for these injuries. BTE field aims to form materials that can outperform bone autografts and allografts. The ultimate goal is to manufacture a scaffold that can be implanted to the defect area and then remodeled by patient's own cells. The key is to fulfill the role of the extracellular matrix (ECM) in the defect area. The design of the scaffolds for BTE is also modeled by the structure and function of healthy bone tissue, which is crucial to its function, for example, highly porous trabecular bone or highly dense cortical bone, which surrounds the trabecular bone. But still, regardless of recent advancements in TE and RM, reconstruction of critical-size bone injuries is still challenging [66].

For bone tissue regeneration, wide range of biomaterials can be used to mimic the function, structure, and composition of bone ECM with proper osteogenic activity. First studies for stimulating bone regeneration were done by ES of widely used polymers such as polycaprolactone (PCL), polylactic acid (PLA), gelatin, silk, and chitosan. The common feature of all these polymers was biodegradability because if the scaffolds are not biodegradable, a second surgery is necessary to remove the scaffold, which can result in infection, patient discomfort, or additional costs. According to the study of Cai et al., a 3D PCL/PLA scaffold was produced, and its bone regeneration efficiency was investigated in a rabbit tibia bone defect model by using human embryonic stem cell–derived mesenchymal stem cells (hESC-MSCs) [67]. They reported that the attachment of the hESC-MSCs to the 3D scaffold was successful due to the differentiation of the cells from round-like shape to a spindle-like form. Additionally, the histology and radiography studies resulted in 3D bone tissue formation after 6 weeks. Another study conducted by Nedjari, et al. is based on the development of a novel 3D honeycomb-shaped scaffold made by electrospun hybrid nanofibers, which includes poly(l-lactide-ℇ-caprolactone) and bone ECM protein fibrinogen (FBG) (**Figure 15**) [68].

Results of this study indicate that PLCL/FBG scaffolds support osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs). Besides ES, melt ES writing is a promising method to design scaffolds with controlled structure. Abdalhay et al. manufactured PCL/HAp composite 3D

*Nanofibers: Production, Characterization, and Tissue Engineering Applications DOI: http://dx.doi.org/10.5772/intechopen.102787*

#### **Figure 15.**

*SEM images of random (A, D), aligned (B, E) and honeycomb (C, F) PLGL-FBG nanofibers. Bottom row represents the immunofluorescent imaging of FBG within the fibers (red) on random (G), aligned (H), and honeycomb (I)-shaped scaffolds (the arrows indicate the higher accumulation of FBG at the walls of the honeycomb shapes.). Relative expression of alkaline phosphatase gene (J) and RunX2 gene (K) of ADMS cultured for 21 days in osteogenic medium on different shaped scaffolds. (Figure was reproduced with the permission of Nedjari et al. [68]).*

scaffolds with high porosity (96–98%) by using melt ES writing method [69]. According to the results, infiltration and proliferation of seeded osteoblasts were achieved, which supports high interconnectivity and porosity of the PCL/ HAp scaffolds. Velioglu et al. fabricated 3D-printed PLA scaffolds with different pore sizes for trabecular bone repair and regeneration. Their findings showed that the resemblance between 3D-printed scaffolds and native trabecular bone in terms of pore size, porosity, and mechanical properties of the scaffolds, the 3D-printed PLA scaffolds printed in this study can be considered as candidates for bone substitutes in bone repair [70]. In 2019, Lukasova et al. produced 3D and 2D nanofibrous scaffolds by using centrifugal ES and needleless ES methods, respectively. Cyclone device was used as a spinneret for centrifugal ES, and the spin guidance was sideways. Needleless ES on the other hand was performed by using Nanospider® technology. Scaffolds were then tested for metabolic activity, cell differentiation, and proliferation by using hMSCs. Scaffolds obtained with centrifugal ES showed higher cell proliferation due to their 3D, porous, and interconnective architecture [71].

#### *4.1.2 Tendon/ligament tissue engineering*

Tendon/ligament injuries, which are caused by tears, ruptures, traumas, and inflammation, result in severe pain and are generally seen in physically active young patients. Natural healing of these tissues is challenging due to their poor healing capacity and scar tissue formation, which then result in poor mechanical properties. Standard treatment approaches for these injuries are grafts or artificial prosthesis. Autografts are considered "gold standard" because of their lack of immune response, but they are limited by donor site availability and morbidity. Allografts hold the same concerns as in BTE, which are rejection, risk of disease transmission, and high re-rupture rates caused by mismatches between the donor and the recipient. To overcome these challenges, TE approaches are widely used for tendon/ ligament tissue repair [72].

These soft tissues are mainly composed of dense and aligned collagen fibers, so the mechanic load of tendons and ligaments is restricted to one direction. As a result, scaffolds composed of aligned nanofibers are highly promising for tendon/ ligament tissue repair studies because they can mimic the anisotropic nature of the native tissues. In the light of these information, a novel, multilayer scaffold was proposed by Yang et al., which was composed of fibrous PCL and methacrylated gelatin produced by dual ES [73]. The scaffold was formed by five sheets, which were cross-linked. The scaffold was then reinforced with gelatin layer bearing the stem cells, which were treated with TGF-β3 for 7 days to stimulate differentiation. Results showed an increase of tendon markers tenascin-C and scleraxis, which implies the scaffolds were porous enough for the diffusion of bioactive molecules. Another study conducted by Perikamana et al. reported that immobilization of plateletderived growth factor (PDGF) in a gradient scaffold, which is also composed of aligned nanofibers, enhanced the expression of tenomodulin compared with a nonmodified nanofiber scaffold [74]. Rinoldi et al., fabricated a bead-on-string fibrous

#### **Figure 16.**

*The cell proliferation rates from DNA assays (a) and SEM images (b) of tenocytes cultured on different coreshell nanofiber scaffolds. The relative mRNA expression of type-I collagen (c), type III collagen (d), tenascin-C (e), and biglycan (f) by tenocytes after cultured on different core-shell nanofiber scaffolds for 7, 14, and 21 days. (Figures are reproduced with the permission from Chen et al. [76]).*

#### *Nanofibers: Production, Characterization, and Tissue Engineering Applications DOI: http://dx.doi.org/10.5772/intechopen.102787*

scaffold and incorporated with silica particles to enhance the biological activities and modify the properties of the scaffolds such as wettability, degradation rate, etc. The results imply that their bead-to-string fibrous scaffold is a significant candidate for guided tissue regeneration [75]. In a recent study, Chen et al. proposed a three types of core-shell nanofibrous scaffolds [76]. For one group, HA (hyaluronic acid) is the core and PCL (random) is the shell while the other groups are HA/PRP (platelet-rich plasma) core–PCL shell (Random<sup>+</sup> ) and HA/PRP core–PCL shell (Align+ ) (**Figure 16**). Tenocytes were used in in vitro studies, and the cells in Align+ showed the highest cell proliferation rate while Random+ is also significantly higher than Random study group. According to the expression studies, by day 14, Random+ and Align+ showed significant downregulation of collagen III gene expression when shift of collagen III to collagen I occurs during the tendon maturation.

It is safer to study tendon/ligament tissue regeneration compared with tendon/ligament-bone interface regeneration, which is also called the enthesis. Regeneration enthesis is exceptionally challenging due to its complex and gradient structure. The enthesis possesses location-dependent changes such as gradients, in terms of composition of ingredients and structural properties.

Still, there are many studies and research groups trying to fabricate scaffolds to repair tissue-tissue interfaces by incorporating bone-like biomaterials such as hydroxyapatite, hyaluronan, etc. (**Figure 17**) [74, 77]. Yet still, it remains a challenge in the field, which requires much more time and effort.

#### **Figure 17.**

*Energy-dispersive spectroscopy (EDS) spectrum of polycaprolactone (PCL)-only and PCL-nanohydroxyapatite (nHA) meshes at different flow rates corresponding to different nHA concentrations (a–c). Representative scanning electron microscopy (SEM) micrographs taken from (d) polycaprolactone (PCL) rich and (e) nano-hydroxyapatite (nHA)-rich surfaces of the spatially graded meshes. White arrows in E indicate nHA particulates embedded into nanofibers. (Figure was reproduced with the permission from Bayrak et al. [77]).*

#### *4.1.3 Cartilage tissue engineering*

Other than tendons and ligaments, cartilage tissue is another class of connective tissues that presents elastic behavior and protects the end of bones at joints. Nose, ears, knees, and many other parts of the body contain cartilage tissue. The main ECM components of dense cartilage tissue are collagen and proteoglycans, which are produced by a low number of chondrocytes. After an articular cartilage injury such as rupture, trauma, aging, etc., remodeling and regeneration of the native tissue are challenging due to the low availability of chondrocytes and the complex structure of the tissue. Current approaches are mostly grafts, decellularized structures, microfracture, etc.; however, these approaches pose significant risk to the patient such as inflammation, rejection, implant loosening, or failure.

To repair the damaged articular cartilage, Tuli et al., prepared nanofibrous PCL scaffolds by ES method. The fetal bovine chondrocytes (FBCs) were seeded onto these scaffolds and examined in terms of their ability to maintain chondrocytes in a functional state. According to their results, PCL scaffold seeded with FBC is able to preserve the chondrocyte phenotype by expressing cartilage-specific ECM components such as aggrecan and collagen [78]. In another study, electrospun gelatin/ PLA nanofibers were fabricated, and one group was modified by cross-linking with hyaluronic acid to examine the ability to repair cartilage damage. These scaffolds were then subjected to an in vivo study on rabbits using an articular cartilage injury model. Results of the in vivo studies demonstrated that the hyaluronic-acid-modified scaffolds could increase the repair of cartilage along with their super-absorbent properties and cytocompatibility (**Figure 18**) [79].

Another research group fabricated a scaffold by using coaxial ES with poly (L-lactide-co-caprolactone) and collagen as the shell and kartogenin solution as the

#### **Figure 18.**

*Macroscopic images (a, d, and g) of the cartilage joints from three groups at 12 weeks after surgery. Histological analysis of cartilage defect area from three groups at 12 weeks after surgery, stained with safranin O-fast green (b, e, and h) and H&E (c, f, and i). Arrows and dotted lines indicated the defect sites. (OC: Original cartilage tissue. RC: Repaired cartilage tissue.) (Figure was reproduced with the permission from Chen et al. [79]).*

*Nanofibers: Production, Characterization, and Tissue Engineering Applications DOI: http://dx.doi.org/10.5772/intechopen.102787*

core. Kartogenin's release behavior was monitored over 2 months, and it is shown that the proliferation and chondrogenic differentiation of rabbit bone-marrowderived MSCs are increased due to the chondrogenesis inducement properties of kartogenin [80]. Furthermore, incorporation of cartilage-derived ECM into nanofibrous scaffolds is another novel way for stimulation of chondrogenic bioactivity [81].

### **4.2 Skin tissue engineering**

The skin is the largest organ in mammals and acts as a physical barrier between the human body and the external environment, which means it is directly exposed to harmful microbial, thermal, mechanical, and chemical damage. Skin tissue, mainly composed of epidermis, dermis, and subcutaneous layer, suffers from integral skin loss with every injury, which can cause functional imbalance in case of large full-thickness skin defects or loss of large skin areas. Skin loss can occur for many reasons, such as disorders, burns, and chronic wounds. For years, autografts and allografts have been used to treat burns or other skin defects, yet the inability of damaged skin tissue to fully heal has opened up the field of tissue engineering for repair broadly to resolve skin-related defects. The basic prerequisite for a material

#### **Figure 19.**

*In situ deposition of electrospun zein/PEO and zein/PEO/TEO fibrous meshes onto wounds of Kunming mice: (a) no treatment, (b) zein/PEO (control group), and (c) zein/PEO/TEO (study group); gross observation of wounds healing at 0, 3, 7, and 11 days after injury for no treatment (a1*−*4), zein/PEO (b1*−*4), and zein/PEO/ TEO (c1*−*4), respectively. (Figure is reproduced with the permission from Liu et al. [87]).*

to qualify as a biomaterial is biocompatibility, which is the ability of a material to perform with an appropriate host response [82].

Nanofibrous scaffolds with high porosity can enable cell respiration, infiltration, and absorb exudates. Natural polymers such as chitosan, collagen, and elastin are widely used biomaterials for wound dressing according to their biocompatible and biodegradable properties [83]. Ghosal et al. extracted the silk sericin protein (SS) and blended it with PCL, fabricating a scaffold by using emulsion ES method to examine the effect of the silk sericin protein in the scaffold morphology and proliferation of human primary skin fibroblasts. Results showed an increase in proliferation of the cells on PCL/SS scaffolds [84]. Nanoparticles due to their antioxidant and antibacterial properties are also widely used in this field. Augustine et al. incorporated cerium oxide (CeO2) nanoparticles into electrospun poly (3-hydroxybutyrate-co-3-hydroxyvalerate) scaffolds and analyzed the wound healing properties. The results showed increased cell proliferation, angiogenesis, and wound healing [85]. Chantre et al., prepared a scaffold by centrifugal ES composed of hyaluronic acid to repair cutaneous tissue. In vitro test showed that due to the high porosity, the infiltration of seeded dermal fibroblasts was successful, and scaffolds present biocompatible and bioactive properties. In vivo studies supported their research as well by the acceleration of the tissue formation, neovascularization, and re-epithelialization [86]. Recently, portable electrospinning devices have been widely used to understand in situ deposition of fibers for wound coverage. This technology allows fibrous scaffolds to form directly on the wound site in a matter of minutes.

For example, Liu et al. fabricated electrospun zein/poly (ethylene oxide) nanofibrous scaffolds modified with thyme essential oil (TEO) by using portable handheld ES device directly onto partial thickness wounds on mice dermal tissue defect (**Figure 19**). It is found that electrospun nanofibers improved the wound healing process within 11 days [87].

#### **4.3 Cardiovascular tissue engineering**

Cardiovascular diseases such as coronary artery, cardiomyopathy, hypertension, valve disorders, heart failure, etc., are the leading cause of death globally, and the incidence rates are drastically increasing day by day. Common approach is vascular graft transplantation, but it has some limitations such as lack of organ donors, mismatches, preexisting vascular diseases. These limitations cause a need for more stable, flexible grafts with low toxicity and immunity. Since cardiac tissue ECM causes cardiomyocytes to form into fiber-like cell bundles and these bundles elongate and align themselves, a polymeric scaffold that could mimic this specific feature of cardiac tissue could be a potential candidate for cardiovascular tissue engineering.

To stimulate myocardial regeneration, a 3D PCL-based scaffold with hexagonal structure was fabricated using melt electrowriting method. The aim of the study was to create functional cardiac patches, human induced pluripotent stem cellderived cardiomyocytes (iPSC-CM) were seeded onto these scaffolds. Results of the in vitro studies showed increased cell alignment, cardiac-maturation-related markers, and sarcomere content. Furthermore, in vivo studies, which are conducted on a contracting porcine heart with a minimally invasive approach, showed that the scaffolds express successful biaxial deformation and also supported high tensile stress [88]. Recently, many studies focus on the development of conductive nanoporous scaffolds for cardiovascular tissue engineering approaches. Bertuoli et al. developed an electrospun conducting and biocompatible uniaxial and core-shell fibers having PLA, PEG, and polyaniline (PAni) for cardiac tissue engineering (**Figure 20**) [89]. They produced PLA, PLA/PAni, and PLA/PEG/PAni fibers and

*Nanofibers: Production, Characterization, and Tissue Engineering Applications DOI: http://dx.doi.org/10.5772/intechopen.102787*

#### **Figure 20.**

*Biocompatibility of PLA/PAni uniaxial fibers expressed as relative viability of normal rat fibroblasts (NRK) and osteosarcoma (MG-63) culture cells onto the fibrous mats after (a) 24 h (cell adhesion) and (b) 96 h (cell proliferation). Biocompatibility of (c, d) PLA/PEG/PAni uniaxial and (e, f) PLA/PEG//PLA/PAni core-shell fibers expressed as relative viability of NRK and MG-63 cells onto the fibrous mats after (c, e) 24 h (cell adhesion) and (d, f) 96 h (cell proliferation). (g) Electrical conductivity of PLA, PLA/PAni, PLA/ PEG/PAni, PLA//PLA/PAni, and PLA/PEG//PLA/PAni fibrous mats. (Figures were reproduced with the permission from Bertuoli et al. [89]).*

core-shell PLA/PLA/PAni and PLA/PEG//PLA/PAni fibers successfully via uniaxial and coaxial electrospinning, respectively. The proposed PLA/PAni-5% uniaxial and PLA//PLA/PAni coaxial fibers offer very good adhesion for cardiac cells, also being able to modulate cell shape and orientation, something important for the characteristic anisotropy of the cardiac tissue.

Liang et al. fabricated a conductive nanofibrous scaffold by encapsulating polypyrole (PPY), which is a conductive polymer, in silk fibroin electrospun fibers. Neonatal rat cardiomyocytes (NRCM) and iPSC-CM cells were used to evaluate cardiomyocyte contraction studies. Results showed that both cell lines attached and proliferated onto these scaffolds successfully. Contraction study indicated that scaffolds with different amount of PPY exhibit contraction behavior starting from day 5 [90]. Another group developed a lab-on-a-chip system integrated with PLLA and PU nanofiber mats for cardiovascular diseases. The aim of the study was to create a model of hypoxic myocardial tissue. The microfluidic system allows simultaneously conducting cell cultures under different circumstances. Cardiac cell lines were used for this study, and results showed that cell viability was high, and cells were positioned parallelly on the scaffolds. The hypoxia study indicates that the amount of ATP molecules decreased during biochemical simulation [91].
