**5. Expression of myogenic protein and IGF-1 cell signalling pathway analysis**

The ability of cells to correctly respond to their microenvironment is the basis of tissue repair and development [21, 22]. Here, myogenic protein expression on GO sheet and electrospun GO-PCL scaffolds and cell signalling pathway (insulin-like growth factor-1 (IGF-1)) analysis have been investigated. Better myogenic protein expression was observed on GO-based polymer composite representing superior myogenic differential potential. It was also observed that late myogenic protein, myosin heavy chain (MHC) related to the formation of myotubes, expressed more on GO-PCL composite scaffolds compare to GO sheet and control (tissue culture plate). Myogenic gene (desmin, MyoD and MHC) expression was also found to be better on GO based polymer composite substrate. In addition, IGF-1 pathway, important for skeletal muscle cells growth and maturation [23, 24], was also studied with various intermediate proteins (IRS-1/PI(3)K/Akt/MyoD) expression. Inhibition of Akt was investigated to examine the variation of the expression of myogenic protein MyoD, involved in skeletal muscle differentiation [24, 25]. The expression of MyoD was observed to reduce along with the inhibition of Akt that indicated effectiveness of IGF-1 pathway for skeletal muscle cells differentiation and maturation.

#### **5.1. Expression of myogenic proteins**

**Figure 22.** Immunostaining of desmin, MyoD and MHC (myosin heavy chain) on control (a–c) and GO-PLGA electrospun composite mesh (d–i). Corresponding FESEM micrographs (j–l) of these samples were shown for better demonstration. It is revealed that though GO-PLGA showed better myoblast differentiation compared to that on GO sheet. GO-PCL exhibit superior myoblast differentiation and myotube formation compared to the GO-PLGA meshes [20].

238 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

**5. Expression of myogenic protein and IGF-1 cell signalling pathway**

The ability of cells to correctly respond to their microenvironment is the basis of tissue repair and development [21, 22]. Here, myogenic protein expression on GO sheet and electrospun GO-PCL scaffolds and cell signalling pathway (insulin-like growth factor-1 (IGF-1)) analysis have been investigated. Better myogenic protein expression was observed on GO-based polymer composite representing superior myogenic differential potential. It was also observed that late myogenic protein, myosin heavy chain (MHC) related to the formation of myotubes, expressed more on GO-PCL composite scaffolds compare to GO sheet and control (tissue culture plate). Myogenic gene (desmin, MyoD and MHC) expression was also found to be better on GO based polymer composite substrate. In addition, IGF-1 pathway, important for skeletal muscle cells growth and maturation [23, 24], was also studied with various intermediate proteins (IRS-1/PI(3)K/Akt/MyoD) expression. Inhibition of Akt was investigated to examine the variation of the expression of myogenic protein MyoD, involved in skeletal muscle differentiation [24, 25]. The expression of MyoD was observed to reduce along with the inhibition of Akt that indicated effectiveness of IGF-1 pathway for skeletal muscle cells

**analysis**

differentiation and maturation.

Expressions of myogenic proteins such as desmin, MyoD and MHC were assessed on GO-PCL meshes, GO sheet and control (tissue culture plate) using Western blot analysis. **Figure 25** showed expressions of desmin and MyoD on these substrates after 5 days of culture. Both of these proteins expressed well on GO-based substrates. The electroconducting GO-PCL composite mesh showed the highest expression due to better interconnectivity that enhanced cellular growth and hence their protein expressions.

**Figure 23.** Expression of myogenic protein desmin and MyoD (left) and expression of fold change on GO-PCL mesh, GO sheet and control (tissue culture plate) (right) for the corresponding proteins. \* indicates significant difference compare to control (*p* < 0.05).

From the present observation (**Figure 23**), the expression levels of desmin and MyoD proteins were found to be enhanced on grapheme-based scaffolds, compared with to that of control as follows: 2.4-fold on GO, 4-fold on GO-PCL for desmin and 1.8-fold on GO, 3.1-fold for GO-PCL for MyoD. Data reported as fold change from control (means ± SE). \*indicates significant differences (*n* = 5; *p* < 0.05).

**Figure 24.** Expression of myogenic protein (MHC) (left) and expression of fold change on control, GO and GO-PCL substrates (right). \* indicates significant difference from control (*p* < 0.05).

After 11 days of culture on the same substrates, expression of MHC (a late myogenic marker) was assessed using Western blot analysis (**Figure 24**). Expression of MHC, related to formation of myotubes was found to be better on GO and GO-PCL meshes compared to control substrate. Here, better expression of this protein in GO-PCL mesh also indicates better myotube formation on this substrate as described earlier (**Figure 23**).

The expression levels of MHC protein were found to enhance on GO-PCL composite and GO sheet compared with that of control: 1.6-fold on GO and 2.5-fold on GO-PCL mesh. Data were reported as fold change from control (means ± SE).

**Figure 25.** Expression of myogenic genes (desmin, MyoD and MHC) in myoblasts grown on electrospun GO-PCL mesh, GO sheet and control (tissue culture plate) substrates. \* indicates significant difference from control (*p* < 0.05).

#### **5.2. Myogenic gene expression**

Real-time RT-PCR was used to analyse the expression level of MyoD, desmin and MHC on GO sheet, GO-PCL scaffolds and control. It was found, similar to protein expression, that these myogenic genes expressed better on GO-PCL composite and GO sheet.

Highest expression was found on myoblast cells grown on GO-PCL meshes. Quantitative RT-PCR analysis was performed after differentiation for 5 days for desmin and MyoD and 11 days for MHC (as MHC express late). As shown in **Figure 25**, the expression levels of all of the tested genes were enhanced on graphene derivative and GO-polymer composites, compared with that of the control as 1.60-fold on GO and 1.98-fold on GO-PCL for desmin; 1.48-fold on GO and 1.88-fold on GO-PCL for MyoD; 1.86-fold on GO and 2.33-fold on GO-PCL for MHC. \* denotes a significant difference compared to control substrate.

Significant up-regulation of myogenic gene expression indicated that the GO-based substrates enhanced both early and late stages of myogenic differentiation, subsequently stimulating the formation of myotubes. In addition, higher expression of myogenic proteins and genes on GO- PCL substrates again confirmed its superiority perhaps due to its higher conductivity and dielectric constant associated with GO surface charge.

#### **5.3. Cell signalling pathway analysis**

After 11 days of culture on the same substrates, expression of MHC (a late myogenic marker) was assessed using Western blot analysis (**Figure 24**). Expression of MHC, related to formation of myotubes was found to be better on GO and GO-PCL meshes compared to control substrate. Here, better expression of this protein in GO-PCL mesh also indicates better myotube forma-

The expression levels of MHC protein were found to enhance on GO-PCL composite and GO sheet compared with that of control: 1.6-fold on GO and 2.5-fold on GO-PCL mesh. Data were

**Figure 25.** Expression of myogenic genes (desmin, MyoD and MHC) in myoblasts grown on electrospun GO-PCL mesh, GO sheet and control (tissue culture plate) substrates. \* indicates significant difference from control (*p* < 0.05).

Real-time RT-PCR was used to analyse the expression level of MyoD, desmin and MHC on GO sheet, GO-PCL scaffolds and control. It was found, similar to protein expression, that these

Highest expression was found on myoblast cells grown on GO-PCL meshes. Quantitative RT-PCR analysis was performed after differentiation for 5 days for desmin and MyoD and 11 days for MHC (as MHC express late). As shown in **Figure 25**, the expression levels of all of the tested genes were enhanced on graphene derivative and GO-polymer composites, compared with that of the control as 1.60-fold on GO and 1.98-fold on GO-PCL for desmin; 1.48-fold on GO and 1.88-fold on GO-PCL for MyoD; 1.86-fold on GO and 2.33-fold on GO-PCL for MHC. \*

Significant up-regulation of myogenic gene expression indicated that the GO-based substrates enhanced both early and late stages of myogenic differentiation, subsequently stimulating the formation of myotubes. In addition, higher expression of myogenic proteins and genes on GO-

myogenic genes expressed better on GO-PCL composite and GO sheet.

denotes a significant difference compared to control substrate.

tion on this substrate as described earlier (**Figure 23**).

240 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

reported as fold change from control (means ± SE).

**5.2. Myogenic gene expression**

#### *5.3.1. Insulin-like growth factor 1 (IGF-1) pathway study*

IGF-1 is one of the most important pathways that have been studied extensively in the field of muscle growth [23, 24]. Researchers studied IGF-1/IRS-1/PI(3)K/Akt/MyoD pathway and concluded its importance for skeletal muscle differentiation and maturation [23–27]. In the present study, it was attempted to verify this pathway using the novel graphene based composite scaffolds (**Figure 26**). IGF-1 is one of the primary mediators of the effects of growth hormone (GH) in body that stimulates growth via growth promoting effects on almost every cell, especially muscle cells present in the body. IGF-1 is also capable of regulating cellular growth and development. In addition, IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy, very much important for proper development of muscle tissue [24, 28]. So, skeletal muscle healing is associated with IGF-1.

**Figure 26.** A schematic representation of IGF-1 cell signalling pathway that is involved in skeletal muscle differentiation and maturation.

#### *5.3.2. Protein expression for IGF pathway*

To study IGF pathway, expression of IRS-1/PI(3)K/Akt/MyoD proteins is important. Initially, the expression of IRS-1/PI(3)K/Akt/MyoD proteins on GO, GO-PCL and control (tissue culture plate) substrates (**Figure 27**) was analysed. All of these proteins expressed better on GO and GO-PCL, while the expression was highest for GO-PCL. This indicates that the GO-PCL substrate is the most suitable candidate for myoblast differentiation of CB-hMSCs.

To evaluate IGF-1 pathway, Akt is the most important protein that regulates skeletal muscle differentiation and hypertrophy. It was also reported that IGF-1 dependent Akt-phosphoralization increased along with higher rate of myogenic differentiation. Properties of MyoD, responsible for skeletal muscle differentiation, are controlled by Akt and blockage of Akt prevents formation of skeletal muscle transcriptosome [24–26]. The present investigation demonstrates that the investigated pathway proteins expressed better on GO-PCL composite meshes than those on GO sheet or control (tissue culture plate). Up-regulation of such pathway proteins on GO-PCL further emphasize the IGF-1 pathway which is related to skeletal muscle development. Thus, it reveals the importance GO-PCL composite meshes for skeletal muscle tissue engineering applications.

**Figure 27.** Expression of PI(3)k, Akt, pAkt, IRS-1 (a) and expression of fold change for these corresponding signalling proteins (b) on control (tissue culture plate), GO sheet and GO-PCL meshes. \* indicates significant difference (*p* < 0.05).

#### *5.3.3. Effect of Akt inhibition on MyoD expression*

As mentioned above, Akt is a major component that regulates IGF-1 pathway. Inhibition of Akt deters IGF-I-stimulated nuclear translocation of Akt and also suppresses the growth of various cells. Akt is a vital component of the cell survival pathway as it functions by resisting apoptosis via improving communication between cells that are damaged [26, 27]. Inhibition of Akt results in much higher rate of apoptosis and a decrease in IGF-1 cell signalling. Inhibition of Akt was done using 10-DEBC hydrochloride, a selective and precise cell permeable Akt phosphorylation inhibitor that shows no activity at PDK1, SGK1 or PI 3-kinase [27, 28]. In the present experiment, the expression of MyoD, a vital myogenic protein that helps in myogenic differentiation has been studied by pre- and post-Akt inhibition. The results showed (**Figure 28**) that after inhibition of Akt, the expression of MyoD intensity decreased rapidly. Akt inhibitor inhibits IGF-I-stimulated nuclear translocation of Akt that results in much reduction of MyoD protein assessed by Western blotting. This finding indicates the importance of IGF-1/ IRS-1/PI(3)K/Akt/MyoD cell signalling pathway for the system of present investigation.

**Figure 28.** Expression of MyoD (a) and the corresponding relative intensity (b) indicating pre- and post-inhibition of Akt. \* indicates significant difference from post Akt inhibition (*p* < 0.05).

The expressions of myogenic proteins on graphene oxide-based substrates (GO sheet and GO-PCL scaffold) have been assessed. It has been demonstrated that myogenic proteins expressed better on the GO-PCL composite meshes than GO sheet and the control (tissue culture plate). This highlighted the superiority of grapheme-based substrates for myoblast differentiation. Favourable physicochemical and biological properties established these scaffolds as potential platforms for myogenic differentiation and maturation for tissue engineering and other biomedical applications. In addition, we have also shown preliminary study of IGF-1 pathway that is important for skeletal muscle differentiation and maturation as well. This pathway proteins (IRS-1/PI(3)K/Akt/MyoD) also expressed better on graphene based substrates, particularly on GO-PCL scaffolds. Moreover, selective inhibition of Akt has reduced the expression level of MyoD, a myogenic protein important for skeletal muscle differentiation.

Hence, it was noticed that lower expression of MyoD along with Akt inhibition indicated the importance of the IGF-1 cell signalling pathway observed from this preliminary investigation. Further elaborate study seems to be more interesting.
