**4.** *In-vitro* **cell culture study**

#### **4.1. Isolation and culture of mononuclear cells from UCB**

The mononuclear stem cells (MNCs) were isolated from UCB (**Figure 8a**). Morphology of MNCs was initially round shaped (after 48 h of culture) shown in **Figure 10b** via phase contrast microscope.

**Figure 8.** (a) Mononuclear cells (MNCs) layer (middle) and red blood cells (RBCs) precipitated at the bottom in a 50 ml culture tube. (b) Isolation of MNCs being cultured in a cell culture plate.

**Figure 9.** Gradual morphological changes of umbilical cord blood derived mesenchymal stem cells (MSCs). After fifth passaging fibroblast like morphology (d) of the cultured cells were observed under phase contrast microscope. Scale bar: 50 μm.

Myoblast Differentiation of Umbilical Cord Blood Derived Stem Cells on Biocompatible Composites Scaffold Meshes http://dx.doi.org/10.5772/65032 229

**Figure 10.** Morphology of MSCs analysed using phase contrast microscope (a) and (b) cytoskeleton staining of actin filaments along with nuclei counterstained with DAPI (fluorescence image).

#### **4.2. Isolation of UCB-derived MNCs**

Similar conductivity and dielectric constant behaviour (shown in **Figure 7**) was also observed in case of GOnPs-PLGA meshes soaked in PBS solution. In this case, enhancement of conductivity is little lower than those of GOnPs-PCL scaffold meshes indicating little lower biocom-

The mononuclear stem cells (MNCs) were isolated from UCB (**Figure 8a**). Morphology of MNCs was initially round shaped (after 48 h of culture) shown in **Figure 10b** via phase contrast

**Figure 8.** (a) Mononuclear cells (MNCs) layer (middle) and red blood cells (RBCs) precipitated at the bottom in a 50 ml

**Figure 9.** Gradual morphological changes of umbilical cord blood derived mesenchymal stem cells (MSCs). After fifth passaging fibroblast like morphology (d) of the cultured cells were observed under phase contrast microscope. Scale

patibility of the GOnPs-PLGA composite.

**4.1. Isolation and culture of mononuclear cells from UCB**

228 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

culture tube. (b) Isolation of MNCs being cultured in a cell culture plate.

**4.** *In-vitro* **cell culture study**

microscope.

bar: 50 μm.

UCB samples were diluted with DMEM media in the ratio of 4:1 before use. The mononuclear cells (MNCs) were isolated [20] using the Ficoll Hypaque (Histopaque-1077; Sigma, MO, USA) density gradient centrifugation (with swinging-bucket rotor) at 450 g for 30 min. The erythrocytes were lyzed by incubating with 1% lysis buffer for 10 min and spinning at 200 g for 10 min. The pellet thus obtained was washed twice with phosphate-buffer saline (PBS) by centrifuging at 200 g for 10 min. Finally, the pellet obtained were culture in DMEM, 10% foetal bovine serum, 1% antibiotic with addition of 10 ng/ml basic fibroblast growth factor (bFGF). Initial media was changed after one day to remove the RBC and cell derbies after that media change after 3-day interval. Cells were then cultured for up to fifth passage.

#### **4.3. Characterization of UCB-derived MSCs by flow cytometry**

Cells were analysed by fluorescence-activated cell sorting (FACS) method. In brief, cells were trypsinized and washed with fluorescence activated cell sorting (BDFACS) buffer. After centrifugation at 200 g for 5 min at 4°C, cells were suspended in PBS at a concentration of 1 × 105 cells/ml and repeatedly washed to remove phenol red contained in the media. The cells were finally suspended in 100 μl of FACS buffer. Cells were then labelled with CD90-FITC, CD73-PE, CD105-APC, CD45-PE, CD34-FITC and HLA DR-APC monoclonal antibodies at 4°C for 30 min in the dark. The labelled cells were then analysed by flow cytometry (BDFACS Fortessa II) with at least 10,000 cells being acquired and analysed.

#### **4.4. Morphological characterization of CB-hMSCs**

The morphological variation in the cultured cells was studied by phase contrast microscopic images (**Figure 9**). After initial culture, cells were round/spherical shaped during the initial days of culture and became elongated as spindle fibroblastic shape gradually (**Figure 9a–d**) after fifth passage.

The morphology of cells was also analysed using cytoskeleton staining of actin filaments (**Figure 10**). Cells (stained with FITC-phalloidin) were observed under a Zeiss Axivert 40 CFL fluorescence microscope.

#### **4.5. Immunophenotypic characterization of CB-hMSCs**

The immunophenotypic characterizations are shown (**Figure 11**) to be positive for CD90 (99.2%), CD73(98.5%), CD105 (98%) and negligible for hematopoietic markers like CD45 (1.5%), CD45 (0.5%) and HLA-DR (1.0%) indicating mesenchymal stem cells phenotype. Similar behaviour was also exhibited by the GO-PLGA meshes [20].

**Figure 11.** The immunophenotypic analysis was found to be positive for CD90 (99.2%), CD73 (98.5%), CD105 (98%) and negative for CD45 (1.5%), CD34 (0.5%) and HLA-DR (1.0%) indicating mesenchymal stem cells phenotype.

#### **4.6. Cell metabolic activity**

WST-8 assay was used to study the metabolic activity of the MSCs on the prepared scaffolds on various time intervals. The cellular viability of metabolic activity of the cultured MSCs on

**Figure 12.** WST-8 assay of CB-hMSCs grown on GO sheet, GO-PCL and control (tissue culture plate) substrates after 3, 7 and 11 days of culture. Superior cellular metabolic activity has been observed on GO-PCL-based composite meshes. Results presented as the means ± SD. \* indicates significant difference (*n* = 5; *p* < 0.05). Metabolic activity was increased with time with the scaffolds showing the trend GO-PCL > GO > control substrate.

the prepared GO-based scaffold was further evaluated quantitatively by WST-8 assay as shown in **Figure 12**.

#### **4.7. Cell proliferation assay (via DNA quantification)**

**4.5. Immunophenotypic characterization of CB-hMSCs**

230 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

**4.6. Cell metabolic activity**

Similar behaviour was also exhibited by the GO-PLGA meshes [20].

The immunophenotypic characterizations are shown (**Figure 11**) to be positive for CD90 (99.2%), CD73(98.5%), CD105 (98%) and negligible for hematopoietic markers like CD45 (1.5%), CD45 (0.5%) and HLA-DR (1.0%) indicating mesenchymal stem cells phenotype.

**Figure 11.** The immunophenotypic analysis was found to be positive for CD90 (99.2%), CD73 (98.5%), CD105 (98%) and negative for CD45 (1.5%), CD34 (0.5%) and HLA-DR (1.0%) indicating mesenchymal stem cells phenotype.

WST-8 assay was used to study the metabolic activity of the MSCs on the prepared scaffolds on various time intervals. The cellular viability of metabolic activity of the cultured MSCs on

**Figure 12.** WST-8 assay of CB-hMSCs grown on GO sheet, GO-PCL and control (tissue culture plate) substrates after 3, 7 and 11 days of culture. Superior cellular metabolic activity has been observed on GO-PCL-based composite meshes. Results presented as the means ± SD. \* indicates significant difference (*n* = 5; *p* < 0.05). Metabolic activity was increased

with time with the scaffolds showing the trend GO-PCL > GO > control substrate.

The proliferation of MSCs on the prepared scaffolds was evaluated by DNA quantification assay. **Figure 13** shows an increasing rate in DNA content of MSCs with time as observed in different GO-based substrates prepared for investigation.

**Figure 13.** Cell proliferation represented in terms of DNA quantification on GO sheet, GO-PCL mesh and control (tissue culture plate) substrates. An increased trend in DNA content is observed on all the GO-based matrixes. Results represented as mean ± SD, \* indicates significant difference (*n* = 5; *p* < 0.05).Proliferation of MSCs were increased with time with the scaffolds showing the trend GO-PCL > GO > control substrate.

The corresponding DNA contents of hMSCs cultured on tissue culture plate (TCP) was taken as control, GO sheet and GO-PCL composite meshes on 3–11 days of culture are ~50 to ~285, ~58 to ~375 and ~65 to ~430 ng/ml, respectively.

#### **4.8. MSCs seeding, attachment and spreading**

After seeding of MSCs (by static seeding method with ~2×104 cells/ml), attachment and spreading of these cells was evaluated by scanning electron microscopic (SEM) micrographs on increasing time interval (**Figure 14**). Cellular attachment and spreading rate also indicates their compatibility to the scaffold environment.

**Figure 14.** Attachment and spreading of CB-hMSCs on GO-PCL composite scaffolds on days 3 (a), 5 (b) and 7 (c) of culture. The MSCs are well visualized from the micrographs.

#### **4.9. Differentiation potential of CB-hMSCs on GO-PCL composite meshes**

#### *4.9.1. Myoblast differentiation potential*

After confirming viability and proliferation of cord blood derived mesenchymal stem cells (CB-hMSCs or simply MSCs) onto the novel GO-PCL composite scaffolds, MSCs were further allowed for myoblast differentiation on these substrates. Along with differentiation of MSCs, elongated bipolar morphology of myoblasts have been observed as seen from FESEM micrographs (**Figure 15a** and **b**).

**Figure 15.** Morphology of CB-MSCs (a) changes towards bipolar structure (b), same as myoblasts, on GO-PCL electrospun composite scaffold that indicates differentiation of MSCs to myoblast cells (morphology wise).

#### *4.9.2. Myoblast viability and proliferation*

#### *4.9.2.1. Cell viability and proliferation assay*

The vastly used methyl thiazolyl diphenyl-tetrazolium bromide (MTT) assay which is a typical nontoxicity assay may not correctly predict the toxicity of GO because of the mild reaction of MTT salt with GO resulting in an incorrect positive signal. Therefore, we used, alternatively, a water soluble tetrazolium salt (WST-8) assay. Cell viability and proliferation on GO/PCL composite meshes, thin GO sheet and controls were measured by water-soluble tetrazolium salt (WST-8) assay after 3, 7and 11 days of cell seeding in 96-well culture plate. Ten microlitre of cell proliferation reagent (WST-8) was added into each well containing sample with 100 μl of culture medium and incubated for 4 h at 37°C. Absorbance (OD) of the solution was then measured at 450 nm by a microplate reader (Varioskan Flash, Thermo Scientific). The cells seeded on collagen scaffolds were evaluated as control. WST-8 was reduced by dehydrogenase activities of living cells that give rise yellow-colour formazan dye. The amount of formazan dye generated (by the activities of dehydrogenases) was directly proportional to the number of living cells.

## *4.9.2.2. Evaluation of myotubes formation*

**Figure 14.** Attachment and spreading of CB-hMSCs on GO-PCL composite scaffolds on days 3 (a), 5 (b) and 7 (c) of

After confirming viability and proliferation of cord blood derived mesenchymal stem cells (CB-hMSCs or simply MSCs) onto the novel GO-PCL composite scaffolds, MSCs were further allowed for myoblast differentiation on these substrates. Along with differentiation of MSCs, elongated bipolar morphology of myoblasts have been observed as seen from FESEM micro-

**Figure 15.** Morphology of CB-MSCs (a) changes towards bipolar structure (b), same as myoblasts, on GO-PCL electro-

The vastly used methyl thiazolyl diphenyl-tetrazolium bromide (MTT) assay which is a typical nontoxicity assay may not correctly predict the toxicity of GO because of the mild reaction of

spun composite scaffold that indicates differentiation of MSCs to myoblast cells (morphology wise).

**4.9. Differentiation potential of CB-hMSCs on GO-PCL composite meshes**

culture. The MSCs are well visualized from the micrographs.

232 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

*4.9.1. Myoblast differentiation potential*

*4.9.2. Myoblast viability and proliferation*

*4.9.2.1. Cell viability and proliferation assay*

graphs (**Figure 15a** and **b**).

For immunostaining analysis, human skeletal myoblast cells (hSkMCs) grown after 5 days of culture on different substrates (i.e. collagen and glass controls, GO sheets and GO-PCL meshes) were analysed for the expression of myogenin, an early myogenic differentiation marker. Briefly, to detect myogenin, cells were fixed and incubated with primary antibody (1:100) at 4°C overnight and after washed with PBS, again incubated with secondary antibody DyLight 488-conjugated goat anti-mouse IgG (1:100) at RT for 1 h. before viewing. On 11 days of culture, cells were analysed for further expression of muscle specific antigens such as myosin heavy chain (MHC) and dystrophin. Cells were fixed with 4% paraformaldehyde, permeabilized with

**Figure 16.** Viability and proliferation of myoblasts observed bytetrazolium salt (WST-8) assay. Superior viability of cells has been found on GO-PCL composite meshes compare to GO sheet and other controls (collagen and tissue culture plate) indicating better myoblast differentiation potential and hence biocompatibility. Results presented as the means ± standard deviation). \* indicates significant difference (*n* = 5; *p* < 0.05).

0.1% Triton X-100, and then incubated in goat polyclonal anti-MHC (1:100) and rabbit polyclonal anti-dystrophin (1:100) as primary antibodies for 1 h. Next, after washing with PBS, a FITC conjugate rabbit anti-goat secondary antibody (1:500) was used to detect MHC, while Texas Red conjugated goat anti-rabbit secondary antibody (1:150) was also employed to detect dystrophin. Both MHC and Dystrophin are myotubes-specific markers. The samples stained without primary antibody served as negative controls. Nuclei were counterstained with 4ʹ,6 diamidino-2-phenylindole (DAPI). Substrates with cells were then mounted for fluorescence microscopic studies using a Zeiss Axivert 40 CFL fluorescence microscope.

**Figure 16** shows viability and proliferation of myoblast cells on GO sheets, GO-PCL mesh and controls. Cell viability (from WST-8 assay analysis) was found to increase significantly for GO sheets and GO-PCL meshes compared to the control surfaces (\**p* < 0.05).

This was ascribed to be due to the better myogenic differential potential contributed by the favourable physicochemical properties of graphene oxide. This result implied that GO sheets and GO-PCL meshes were cytocompatible and supported cell viability that increased the biocompatibility of GO-PCL composite scaffolds.

**Figure 17.** Myoblast cells viability and proliferation observed by tetrazolium salt (WST-8) assay. Results presented as mean ± standard deviation. \* indicates significant difference (*n* = 5, *p* < 0.05). Viability was found to increase with time on the samples showing the trend of GO-PLGA > GO > control substrate (tissue culture plate).

The viability of myoblast cells on GO-PLGA composite scaffolds were also analysed by WST-8 assay. **Figure 17** showed the viability of GO-PLGA mesh along with control and GO sheet (for comparison). Cell viability was found to increase significantly on GO sheets and GO-PLGA meshes compared to that on the control surfaces (\**p* < 0.05). This result implies that GO-PLGA mesh is cytocompatible and supported myoblast proliferation as in the case of GO-PCL composite meshes. It is thus seen that electrospun GO-polymer meshes with low GO concentration (not toxic to human cells) provided favourable circumstance for the growth and proliferation of myoblast cells.

#### *4.9.3. Immunophenotypic characterization of myoblast cells*

Flow cytometric analysis of cells adhered on thin GO sheets and GO-PCL meshes was performed to confirm the positive expression of myogenic markers CD56 and desmin indicating skeletal muscle cell phenotype (**Figure 18**).

**Figure 18.** FACS analysis of trypsinized hSkMCs from GO-PCL meshes and GO sheets after 7 days of culture. Cells were highly expressed for skeletal muscle markers CD56 and desmin indicating myoblast cells phenotype.

#### *4.9.4. Aspect ratio analysis*

0.1% Triton X-100, and then incubated in goat polyclonal anti-MHC (1:100) and rabbit polyclonal anti-dystrophin (1:100) as primary antibodies for 1 h. Next, after washing with PBS, a FITC conjugate rabbit anti-goat secondary antibody (1:500) was used to detect MHC, while Texas Red conjugated goat anti-rabbit secondary antibody (1:150) was also employed to detect dystrophin. Both MHC and Dystrophin are myotubes-specific markers. The samples stained without primary antibody served as negative controls. Nuclei were counterstained with 4ʹ,6 diamidino-2-phenylindole (DAPI). Substrates with cells were then mounted for fluorescence

**Figure 16** shows viability and proliferation of myoblast cells on GO sheets, GO-PCL mesh and controls. Cell viability (from WST-8 assay analysis) was found to increase significantly for GO

This was ascribed to be due to the better myogenic differential potential contributed by the favourable physicochemical properties of graphene oxide. This result implied that GO sheets and GO-PCL meshes were cytocompatible and supported cell viability that increased the

**Figure 17.** Myoblast cells viability and proliferation observed by tetrazolium salt (WST-8) assay. Results presented as mean ± standard deviation. \* indicates significant difference (*n* = 5, *p* < 0.05). Viability was found to increase with time

The viability of myoblast cells on GO-PLGA composite scaffolds were also analysed by WST-8 assay. **Figure 17** showed the viability of GO-PLGA mesh along with control and GO sheet (for comparison). Cell viability was found to increase significantly on GO sheets and GO-PLGA meshes compared to that on the control surfaces (\**p* < 0.05). This result implies that GO-PLGA mesh is cytocompatible and supported myoblast proliferation as in the case of GO-PCL composite meshes. It is thus seen that electrospun GO-polymer meshes with low

on the samples showing the trend of GO-PLGA > GO > control substrate (tissue culture plate).

microscopic studies using a Zeiss Axivert 40 CFL fluorescence microscope.

234 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

sheets and GO-PCL meshes compared to the control surfaces (\**p* < 0.05).

biocompatibility of GO-PCL composite scaffolds.

After 3 days of culture, the aspect ratios measured on GO-PCL meshes, GO sheets and controls were found to be ~6.6, ~5.4 and (~4.7 for collagen mesh and ~4.3 for tissue culture plate (TCP)), respectively (**Figure 19**).

**Figure 19.** Analysis of cytoskeleton development of hSkMCs grown on (a) control (tissue culture plate (TCP)), (b) collagen mesh, (c) GO sheets, (d) GO-PCL meshes. (e) Cell aspect ratio quantification from (a) to (d) after 3 days of culture. Increased aspect ratio indicates better elongation of these cells on GO-based substrates.

#### *4.9.5. Formation of myotubes*

The myoblast cells were bipolar at the initial stage (**Figure 20a** and **b**) and they were fused together on extended time interval (day 11) under proper condition and formed myotubes that were further clarified by FESEM micrograph (**Figure 20c**) as well as immunostaining assay (**Figure 20d–l**). These figures showed improved myoblasts cell proliferation, differentiation and formation of myotubes onto GO-PCL meshes compared to GO-sheet and control.

**Figure 20.** FESEM micrographs of GO-PCL electrospun scaffolds representing cells attachment as well as spreading at increasing time interval (day 3 (a)–day 7 (b)) and also formation of myotubes at extended time of differentiation (day 11 (c)). For better comparison, immunostaining (with desmin-FITC and MHC-FITC conjugated) images of the corresponding FESEM images have also been shown alongside (d–l) with GO sheet and control (tissue culture plate (TCP)) substrate [6, 19].

#### *4.9.6. Immunohistochemical characterization*

This process has widely been used for the detection of cells specific antigens (proteins, e.g. desmin, MyoD, Myosin Heavy Chain, Dystrophin, etc.) to verify the presence of specific cells/ tissues. Immunohistochemical analysis (**Figure 21a–l**), along with FESEM analysis of the corresponding samples (**Figure 21m–p**), confirmed differentiation of CB-hMSCs to myoblasts via early expression of myogenin-positive nuclei on controls, GO sheet and GO-PCL mesh (**Figure 21a–d**).

Moreover, muscle-specific antigens like myosin heavy chain (MHC) shown in **Figure 22e–h** and dystrophin (**Figure 21i–l**) were expressed more intensely on GO-PCL meshes compared to those on thin GO sheets or control substrates. Myotubes formed on GO sheets and GO-PCL meshes were found to be more aligned compared to those on the control substrates. Quantitative analysis of the percentage of myogenin-positive nuclei showed in **Figure 21** that myogenin expression increased more on thin GO sheets and GO-PCL meshes compared to that on control substrates (collagen mesh and tissue culture plate), which also indicated a better differentiation potential of the GO-based substrates. Importantly, GO-PCL meshes showed the highest percentage of myogenin positive nuclei (~19%) (**Figure 21**). Significantly higher expression of early myogenic marker (myogenin) indicated superior myogenic differentiation potential of GO-PCL electrospun meshes compared to GO sheets and controls.

*4.9.5. Formation of myotubes*

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substrate [6, 19].

(**Figure 21a–d**).

*4.9.6. Immunohistochemical characterization*

The myoblast cells were bipolar at the initial stage (**Figure 20a** and **b**) and they were fused together on extended time interval (day 11) under proper condition and formed myotubes that were further clarified by FESEM micrograph (**Figure 20c**) as well as immunostaining assay (**Figure 20d–l**). These figures showed improved myoblasts cell proliferation, differentiation

**Figure 20.** FESEM micrographs of GO-PCL electrospun scaffolds representing cells attachment as well as spreading at increasing time interval (day 3 (a)–day 7 (b)) and also formation of myotubes at extended time of differentiation (day 11 (c)). For better comparison, immunostaining (with desmin-FITC and MHC-FITC conjugated) images of the corresponding FESEM images have also been shown alongside (d–l) with GO sheet and control (tissue culture plate (TCP))

This process has widely been used for the detection of cells specific antigens (proteins, e.g. desmin, MyoD, Myosin Heavy Chain, Dystrophin, etc.) to verify the presence of specific cells/ tissues. Immunohistochemical analysis (**Figure 21a–l**), along with FESEM analysis of the corresponding samples (**Figure 21m–p**), confirmed differentiation of CB-hMSCs to myoblasts via early expression of myogenin-positive nuclei on controls, GO sheet and GO-PCL mesh

Moreover, muscle-specific antigens like myosin heavy chain (MHC) shown in **Figure 22e–h** and dystrophin (**Figure 21i–l**) were expressed more intensely on GO-PCL meshes compared to those on thin GO sheets or control substrates. Myotubes formed on GO sheets and GO-PCL meshes were found to be more aligned compared to those on the control substrates. Quantitative analysis of the percentage of myogenin-positive nuclei showed in **Figure 21** that myogenin expression increased more on thin GO sheets and GO-PCL meshes compared to

and formation of myotubes onto GO-PCL meshes compared to GO-sheet and control.

**Figure 21.** Expression of the early myogenic differentiation marker myogenin-positive nuclei (green) on controls (a and b), GO sheets (c) and GO-PCL meshes (d). Immunostaining of MHC (green), respectively, on controls (collagen and tissue culture plate) (e and f), GO sheets (g) and GO-PCL meshes (h) and dystrophin (red) similarly on controls (i and j), GO sheets (k) and GO-PCL meshes (l). Nuclei were counterstained with DAPI.FESEM micrographs (m–p) of the corresponding samples were also shown for better demonstration (q). Quantitative analysis of percentage myogenin-positive nuclei (cells cultured in differentiation medium for 5 days before staining). \* represents significant difference (*p* < 0.05) compare to collagen mesh and tissue culture plate (TCP) taken as controls [6].

Similar to the GO-PCL composite meshes, immunohistochemical study has also been performed with myoblasts grown on electrospun GO-PLGA meshes. The experimental results confirmed the differentiation of CB-hMSCs into skeletal myoblasts by the expressions of desmin and MyoD, and formation of myotubes by the expression of MHC on GO-PLGA composite meshes and control (tissue culture plate) (**Figure 22**). Immunostaining of desmin and MyoD after 3–7 days of culture expressed almost similar on both control and GO-PLGA substrates. But, formation of MHC on GO-PLGA mesh was much better compare to control.

Formation of myotubes was also more aligned, similar to natural orientation. This indicates better myotubes formation hence better myogenic maturation potential of GO-PLGA mesh. But, GO-PCL composite meshes showed even superior myoblast proliferation as well as differentiation potential compare to GO-PLGA meshes. This might be due to the fact that PLGA released acidic byproducts upon its degradation that hamper cellular interaction. Moreover, conductivity of GO-PCL was also higher than that made GO-PLGA mesh that also makes GO-PCL more suitable for electro-responsive skeletal muscle tissue regeneration.

**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].
