*3.7.2. Alcian blue staining*

samples without HNTs. CPCs with 10% HNTs had the highest absorbance value. Finally, at day 14, all wells containing CPC scaffolds showed higher absorbance values than control wells and it was the largest in scaffolds with 15% HNTs. The results further suggest that HNT/CPC composites are cytocompatible and enhance the osteoblast viability of the osteoblast cells. In

**Figure 6.** (A) Dexamethasone elution from loaded HNTs. (B) Dexamethasone release from CPC scaffolds loaded with

As all CPC scaffolds were opaque, bright field microscopy or confocal microscopy micrographs were unable to be taken. An indirect method of estimating ECM production was

**Figure 7.** Cell viability assessed in human osteoblast culture on different CPC-HNT scaffolds (mean ± SD; n = 6).

addition, further increase in viability (cell growth) was seen with HNT addition.

**3.7. Histochemical staining of HNT-doped CPC discs**

136 Current Topics in the Utilization of Clay in Industrial and Medical Applications

DEX-doped HNTs.

Alcian blue staining was used to determine the acidic mucopolysaccharides synthesis. **Figure 9** is graphical representation of Alcian blue staining for all scaffolds over the testing period. Higher absorbance indicates higher acidic mucopolysaccharides synthesis. On day 7, acidic mucopolysaccharides synthesis was almost same across all CPC scaffold groups. Day 14 staining showed an improvement in mucopolysaccharide synthesis with an increase in the HNT concentration. CPC scaffolds with 15% HNTs showed the highest level of Alcian

**Figure 8.** Destained well-plates showing red color, due to Picrosirius red, staining on and around osteoblast seeded CPC scaffolds indicating the presence of collagen. A = 7 days in culture and B = 14 days in culture. (C) Absorbance values for different HNT concentrations at day 7 and 14.

**Figure 9.** Destained well-plates showing blue color, due to Alcian blue dye, on and around CPC scaffolds indicating the presence of acid mucosubstances. A = 7 days in culture and B = 14 days in culture. (C) Absorbance values for different HNT concentrations at day 7 and day 14.

blue staining indicating the higher acidic mucopolysaccharides synthesis. However, there was no significant difference in the amount of collagen synthesized among all the groups on given day.

*3.8.1. Picrosirius red staining (PSR)*

14 for ALPase activity.

MSC Group #2 (CPCs with 10% HNTs).

the ECM synthesis as revealed by histochemical staining.

*3.8.2. Alcian blue staining*

MSC collagen production was observed across all the test groups and throughout the length of the experimental period (**Figure 11**). Collagen synthesis remained almost same for any given group on all the days except for Group 3. By day 7 and 14, these scaffolds were stained equally compared to the rest other scaffold groups. By the end of day 14, all the scaffolds were equally stained with PSR stain referring to similar amount of collagen production by cells on all the scaffolds. However, no significant difference in the amount of collagen is synthesized among all the groups on given day. In comparing OB and MSC collagen synthesis on CPC/ HNT discs, the pattern observed was similar for Group 1 (CPCs no HNTs) and OB Group #3/

**Figure 10.** Comparison of absorbance values for samples collected at different concentrations of HNTs at day 7 and day

Calcium Phosphate/Clay Nanotube Bone Cement with Enhanced Mechanical Properties and…

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Each group of scaffolds followed a different trend for each day of experiment in terms of acidic mucopolysaccharide synthesis (**Figure 12**). Group 1 scaffolds had an increase in staining for day 3 and slight decrease on the following days 7 and 14. Staining on Group 2 scaffolds had deep staining on day 7, while on rest other days, similar amount of staining was observed. Group 3 scaffolds showed a similar staining for all the test days, in contrast, Group 4 scaffolds were deeply stained on day 14 compared to other days (**Figure 12**). Results from Picrosirius red and Alcian blue stains suggest cytocompatibility of these scaffolds with bone marrow stromal cells. The presence of dexamethasone does not appear to enhance or decrease

The Picrosirius red and Alcian blue staining results suggest that CPC surfaces are conducive for osteogenesis and cell viability. Production of ECM molecules is a critical factor during bone tissue regeneration.
