(i) Fluorescence microscopy
The fluorescent images were taken to observe the viability of MSCs cultured on the developed scaffolds. Cells are found to increase in number with culture time (as denoted by green fluorescence) from 3rd day [Figure 5.46 (a, c)] to 14th day [Figure 5.46 (b, d)]. The morphology of the cells is spherical in shape till day 3 and found to spread throughout the scaffold matrix at later time points. The cell density of dead cells (as denoted by red fluorescence) is also seen on 3rd day and the number is found to decrease with time. Number
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of dead cells is very few on both the scaffolds indicating excellent viability of seeded hMSCs. More number of viable cells is seen upto 14th day indicating excellent biocompatibility of developed scaffolds. Furthermore, it is also evident from the figures that, the cell proliferation is higher on GN cross-linked scaffold than TPP cross-linked scaffold
Figure 5.46 Fluorescent images of hMSCs on CS/nano β-TCP/GN and CS/nano β- TCP/TPP composite scaffolds upto 14 days of culture. Scaffolds are stained with calcein (green, which transport into cytoplasm of live cells) and ethedium homodimer (red, binds to DNA of dead cells). Number of viable cells is found to increase on both kinds of scaffolds from day 3 to 14. However, more number of viable cells on GN cross-linked CS/nano β- TCP [d] scaffold is seen as compared to TPP cross-linked CS/nano β-TCP [b] scaffold on day 14 indicating increased proliferation on GN cross-linked scaffold
(ii) DNA quantification assay
The proliferation of hMSCs seeded on the developed scaffolds was further assessed by DNA quantification assay. An increased DNA content is observed with both GN and TPP cross- linked scaffolds with culture time as shown in Figure 5.47. However, the DNA content of hMSCs on GN cross-linked scaffold is slightly higher (452 ± 22 ng/ml) as compared to TPP cross-linked scaffold (376 ± 13 ng/ml) upto 21 days of culture which represents the enhanced
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proliferation rate on GN cross-linked scaffold, (p>0.05). Similar trend in increased DNA content of osteoblasts was observed earlier on TPP cross-linked CS fibrous mats and TCP incorporated PCL 3D porous scaffolds as reported [16, 244].
Figure 5.47: Cell proliferation in terms of DNA quantification on CS/nano β-TCP/GN and CS/nano β-TCP/TPP cross-linked composite scaffolds upto 21 days of incubation. Cell number (DNA content) increases with incubation time. The highest proliferation is observed with GN cross-linked composite scaffolds. Each point represents the mean ± SD (n= 3) * and ** denotes significant differences between DNA content with other scaffolds at p > 0.05 and p < 0.05
5.3.13 Cytoskeletal organization
Confocal images of hMSCs cultured on CS/nano β-TCP/GN scaffold upto 14 days were analyzed for cytoskeletal organization as depicted in Figure 5.48 (i). On day 7, less intensity of blue color in the figure denotes minimal growth of cytoskeleton [Figure 5.48 (i-a)]. Whereas, on day 14, images show prominent green color confirming the extensive development of cytoskeleton as a result of increased cell proliferation as shown in [Figure 5.48 (i-b)]. 3-dimensional confocal laser scanning microscopic images (Z-stacks) depict cell penetration in GN cross-linked composite scaffold matrix upto ~ 50 µm, from the surface of
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scaffolds as depicted in Figure 5.48 (ii). Similar pattern of cell infiltration and uniform distribution of fibroblasts were observed earlier by Steward et al on fibronectin fibrils [245].
Figure 5.48 (i): Confocal laser scanning microscopic images of hMSCs cultured on
CS/nano β-TCP/GN composite scaffolds on day 7 and 14. Images depict the development of cytoskeleton with culture time resulting in an extensive cytoskeletal development indicating increased cell proliferation and cell-cell interaction on day 14. Nuclei of the cells stained with Hoechst 33342 (blue) and actin filaments with phalloidin (green). Bar indicates 300µm
Figure 5.48 (ii): 3D confocal laser scanning images (Z-stacks) of hMSCs after 14 days of culture on CS/nano β-TCP/GN composite scaffolds (c, d) on day 14. Z-stacks rotated in 3D space (d) to represent internalization of seeded cells in the porous structure of CS/nano β- TCP/GN composite scaffolds. Nuclei of the cells stained with Hoechst 33342 (blue) and actin filaments with phalloidin (green)
112 5.3.14 Osteogenic differentiation potential (i) Alkaline Phosphatase assay
Endogenous alkaline phosphatase activity of hMSCs seeded on CS/nano β-TCP/GN and CS/nano β-TCP/TPP scaffolds were measured by ALP assay. ALP activity of hMSCs seeded on scaffolds is bimodal with respect to incubation time, which increased from day 7 to 14 and decreased from day 14 to 21 (Figure 5.49), consistent pattern of ALP activity by MSCs undergoing osteogenesis [221, 246]. Figure 5.49 demonstrates an enhanced ALP activity (p < 0.05) from day 7 to 14 and then a decline in ALP activity is evident for GN and TPP cross- linked scaffolds. The ALPase activity of GN and TPP cross-linked scaffolds at 7th day is 3.51 ± 0.04 and 3.26 ± 0.036 IU/μg where as at 14th day; the activity is 6.09 ± 0.06 and 5.51 ± 0.07 IU/μg respectively. This increase in ALPase activity is thus an evident of osteoblast differentiation. The cells cultured on both types of scaffolds show ALPase activity of 5.01 ± 0.02 and 5.14 ± 0.07 IU/μg respectively on 21st day.
Figure 5.49:Alkaline Phosphatase activity of CS/nano β-TCP/GN and CS/nano β-TCP/TPP composite scaffolds. Each point represents the mean ± SD (n= 3) * and ** denotes significant differences between groups at p > 0.05 and p < 0.05 respectively. Significant increase in ALP is seen on both scaffolds from day 7 to day 14. Highest ALP activity is evident on GN cross-linked scaffolds
113 (ii) Total calcium content
Figure 5.50 depicts the calcium content of hMSCs seeded scaffolds until 21 days of culture. The calcium content is found to be increased in logarithmic pattern with time indicating an increased degree of mineralization with both GN and TPP cross-linked scaffolds. However, GN cross-linked scaffold has expressed higher calcium content (2054 mg/mg DNA) than with TPP cross-linked scaffold (1860 mg/mg DNA) as observed for 21 days of incubation, (p < 0.05). Higher mineralization activity of differentiated MSCs represents the superior osteogenic potential of GN cross-linked scaffold compared to TPP cross-linked scaffold. The result is in good agreement with published data where fibers were reinforced into hydrogels [201].
Figure 5.50: Total calcium content of CS/nano β-TCP/GN and CS/nano β-TCP/TPP cross- linked CS/β-TCP composite scaffolds. The calcium content increased with culture time. Highest calcium content is obtained with GN cross-linked scaffolds representing its higher mineralization activity depicting superior osteogenic potentiality. Each point represents the mean ± SD (n= 3) ** and *** denotes significant differences between groups at p > 0.05 and p < 0.05 respectively