Scanning electron microscopy analysis

The surface topography and microstructure are important parameters in designing scaffolds for tissue engineering applications. The scaffold morphology significantly influences the

in-vitro and in-vivo behavior of the composite scaffolds. Microstructural analysis was done

on all composite scaffolds with varying concentrations of nHA, CNTs, GNPs and AC to analyze whether the homogeneous dispersion of different reinforcements in the polymer matrix has been achieved.

The FESEM micrographs of different PVA-nHA composite scaffolds are shown in Fig. 4.2. Agglomeration of nHA in the polymer matrix might affect composite properties whereas, homogeneous dispersion of nHA aids in the improvement of physical, mechanical and biological properties of the composite. The SEM micrographs have revealed the distribution of nHA in PVA matrix, which plays a major role in surface roughness of these composites as the concentration of nHA has been increased. The micrograph of PVA scaffold without nHA (Fig. 4.2 (a)) shows the smooth surface owing to the bio-inert nature of PVA. The micrographs show that on the addition of nHA in PVA matrix, the surface roughness was found to increase. The PHA 1 and PHA 2 samples have shown a homogeneous dispersion of uniformly sized clusters of nHA particles (Fig. 4.2 (b-c)). With further increase in the concentration of nHA particles, the roughness was found to increase as in PHA 3 (Fig. 4.2 (d)). The microstructural analysis also reveals that the agglomeration of nHA in PVA matrix increase with the increase in the concentration of nHA. The PHA 3 sample shows some regions of agglomerated nHA particles; with 3% w/v of nHA, whereas, PHA 4 and PHA 5 samples (Fig. 4.2 (e-f)) showed more agglomerated clusters. The formation of agglomerated nHA clusters might attribute to the charged inorganic ions that were not evenly distributed in PVA matrix and thereby getting agglomerated clusters under the effect of van der Waals force and Brownian motion. Brownian motion causes the collision of the nHA particles with each other and the van der Waals forces attract these particles leading to agglomeration [40]. So from the SEM micrographs, it was observed that with the increase in the concentration of nHA above a certain amount (3% w/v), more agglomeration was evident, which could affect the mechanical and biological properties of composites.

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Fig. 4.2 FESEM micrographs of (a) PVA, (b) PHA 1, (c) PHA 2, (d) PHA 3, (e) PHA 4 and (f) PHA 5.

The cross-sectional surfaces of PVA-CNTs nanocomposite scaffolds with varying CNTs concentrations were analyzed using FESEM (Fig. 4.3). The micrographs have revealed the rough, interconnected and porous structure of the PVA-CNTs scaffolds that allow cells to attach, migrate and grow into the scaffold interior. Both micro (<50 µm) and macro (>50 µm) pores were observed in the nanocomposite scaffolds that increase the surface roughness. The pore size was found to decrease slightly with the addition of CNTs in PVA matrix. The pore size obtained in both, PCN 0.5 and PCN 1 (Fig. 4.3 (b-c)) were large enough to fulfill the requirements for osteoblast cell proliferation. However, sample PCN 1.5 (Fig. 4.3 (d)) was observed to have slightly smaller pores. Sample PCN 1 and PCN 1.5 (Fig. 4.3 (e-f)) were also observed at higher magnification to analyze the CNTs dispersion. Sample PCN 1 showed a uniform distribution of CNTs whereas, with further increase in concentration, CNTs started agglomerating. PCN 1.5 showed the presence of CNTs agglomerates in the PVA matrix. These agglomerates may diminish the mechanical and biological properties of the PCN 1.5.

A representative cross-sectional FESEM micrographs of PVA-GNPs composite scaffolds are shown in Fig. 4.4 (a-f). The FESEM micrographs revealed the porous network structure of all the scaffolds. Both, macro and micro sized pores were observed in all the samples which support the cell adhesion, growth and nutrients supply inside the scaffolds [146].

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Fig. 4.3 FESEM micrographs of (a) PCN 0, (b) PCN 0.5, (c) PCN 1 and (d) PCN 1.5. Surfaces of (e) PCN 1 and (f) PCN 1.5 observed at higher magnification. (Arrows show the agglomeration in PCN 1.5.)

Lee et al. have reported that microporous scaffolds encouraged the MG-63 cell adhesion and proliferation, whereas increasing micropore size resulted in improved cell differentiation [147]. With the addition of GNPs in PVA matrix variation in pore architecture was obtained. Pore walls of PGN 0 (Fig. 4.4 (a)) scaffold were thin which led to crumbling of pores while addition of GNPs in PVA provided stability of pore structure by thick pore walls and prevented the pores from collapse, therefore, larger pores were observed in composite scaffolds. The large pore size and stable pore architecture play a significant role in providing mechanical interlocking with surrounding tissue [148]. The FESEM micrographs of PGN 1 and PGN 1.5 scaffolds at higher magnification have also been shown in Fig. 4.4 (e-f). The GNPs were homogeneously dispersed throughout in PGN 1 sample (Fig. 4.4 (e)). The homogeneous dispersion of GNPs is beneficial to enhance the mechanical properties of the scaffold. However, with the further increase of GNPs in PGN 1.5 (Fig. 4.4 (f)), the GNPs started to agglomerate due to van der Waals force [56]. These agglomerates in PGN 1.5 may result in the deterioration of the mechanical and biological properties.

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Fig. 4.4 FESEM micrographs of (a) PGN 0, (b) PGN 0.5, (c) PGN 1 and (d) PGN 1.5. Scaffolds (e) PGN 1 and (f) PGN 1.5 at higher magnification. (Arrows represent the well dispersed GNPs and asterisks represent the agglomeration of GNPs.)

The FESEM micrographs of the cross-sectional surface of PVA-AC composite scaffolds with varying AC concentrations are shown in Fig. 4.5. From these micrographs, it is evident that AC is homogenously dispersed, and there is no trace of agglomeration of AC in PVA matrix. The well dispersed AC was also observed in high magnification micrograph of PC 2.5 (Fig. 4.5 (h)). The cross-sectional morphology of all the composite scaffolds showed macro and micro pores which are an important requirement for influencing cell attachment and growth. Researchers have reported that the pore size plays an important role in encouraging cell attachment and differentiation on a surface roughened biomaterial that develops mechanical interlocking between the implant and surrounding tissues [149, 150]. The cross-section micrographs of PVA (Fig. 4.5 (a) revealed undefined pore shapes. Due to the flexible character of PVA polymer, shrinkage and collapse of the pore structure were observed in PC 0 scaffold. However, comparatively larger and homogenous pore architecture was obtained for PC composite (Fig. 4.5 (b-f)) scaffolds. The AC present in PVA matrix enhanced the stability of the structure and prevented the pores from collapse. The porosity obtained in the PC scaffolds fulfilled the pore size requirements for osteoblast cell to penetrate into the scaffolds. The micrographs of samples have shown the presence

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of micropores that are essential for regulating cell migration and nutrient supply to the cells, thereby permitting cell growth inside the scaffold. The interconnected porosity observed in the PC 2.5 sample has been shown in Fig. 4.5 (g). Thus, the developed scaffolds have the required morphology and porosity which may help in osteoblast growth and proliferation.

Fig. 4.5 FESEM micrographs of (a) PC 0, (b) PC 0.5, (c) PC 1, (d) PC 1.5, (e) PC 2, (f) PC 2.5, (g) interconnected pores in PC 2.5 and (h) higher magnification micrograph of PC 2.5 scaffolds.