**3.8. Histochemical staining of DEX-doped HNT/CPC discs**

As with the HNT/CPC discs, an indirect method of estimating ECM production was employed using the same staining methods and destaining protocols.

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**Figure 10.** Comparison of absorbance values for samples collected at different concentrations of HNTs at day 7 and day 14 for ALPase activity.

#### *3.8.1. Picrosirius red staining (PSR)*

**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

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

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

Preservation of the osteoblastic phenotype in OB cells was assayed using ALPase activity. It was observed that all CPC groups had a strong ALPase activity showing the preservation of the osteoblast phenotype (**Figure 10**). From the graph, it can be inferred that the cells had enhanced ALPase activity on day 14 compared to day 7. A pronounced increase in ALPase activity was seen in CPC discs without HNTs and CPCs with 10% wt. HNTs. There was, however, no statistical difference among groups on a given day of assay. All histochemical staining results suggest that CPC surfaces are conducive for osteoblast attachment, proliferation, and synthesis of an organic matrix that was subsequently mineralized. Osteogenesis and cell

As with the HNT/CPC discs, an indirect method of estimating ECM production was employed

viability are the critical requirements for bone tissue regeneration.

**3.8. Histochemical staining of DEX-doped HNT/CPC discs**

using the same staining methods and destaining protocols.

HNT concentrations at day 7 and day 14.

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

on given day.

bone tissue regeneration.

*3.7.3. Alkaline phosphatase activity (ALP)*

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

#### *3.8.2. Alcian blue staining*

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 ECM synthesis as revealed by histochemical staining.

**Figure 11.** Picrosirius red staining. Collagen synthesis was consistent and steady across all study groups.

alkaline phosphatase assay are shown in **Figure 13**. Differentiation was more pronounced on Group 2 scaffolds that contained empty HNTs. Scaffolds of Groups 3 and 4, containing DEX loaded HNTs did not induce osteoblastic differentiation in the BSCs. Cell culture wells containing Group 2 scaffolds were deeply stained in purple while the rest other wells were stained slightly or none. Group 2 scaffolds' alkaline phosphatase activity was significantly higher on day 14 compared to other groups. Inability of Group 3 and 4 scaffolds to induce differentiation may be attributed to very high concentrations of DEX released. The optimum concentration of DEX needed to induce the osteogenic differentiation is 10–7 [49–51]. In the CPC discs doped with DEX, DEX may have been eluted in excess and thus had an inhibitory effect. The ability of HNTs alone (no DEX) to induce osteogenic differentiation was previously reported using montmorillonite-doped biopolymers [52]. It may be inferred from this study and the study by Ambre et al. that many aluminosilicate clays may be inherently osteoinductive [53].

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**Figure 13.** Alkaline phosphatase activity assay for osteoinductive scaffolds.

We conclude that CPCs fabricated using TTCP and DCPA as the solid phase and 10% chitosan lactate solution as the setting liquid and doped with HNTs have an osteoconductive nature as evidenced by a production of an abundant ECM synthesis and alkaline phosphatase activity. Surface roughness was increased with HNT addition which favors cell attachment, proliferation, and ECM synthesis. Halloysite increased the compressive strength of CPCs. The composition of CPCs after setting was analyzed using FTIR which reveals that the addition of HNTs increased HA production by acting as seeding agents for the precipitation of

**4. Conclusions and future work**

**Figure 12.** Alcian blue staining for osteoinductive scaffolds. A different trend was observed in terms of acidic mucopolysaccharide synthesis for all four groups.

#### *3.8.3. Alkaline phosphatase activity*

According to the hypothesis, scaffold Groups 3 and 4 would induce differentiation of BSCs towards osteoblastic lineage as they contained different concentrations of DEX- doped HNTs. Hence, more alkaline phosphatase activity was predicted in these groups. Staining results of Calcium Phosphate/Clay Nanotube Bone Cement with Enhanced Mechanical Properties and… http://dx.doi.org/10.5772/intechopen.74341 141

**Figure 13.** Alkaline phosphatase activity assay for osteoinductive scaffolds.

alkaline phosphatase assay are shown in **Figure 13**. Differentiation was more pronounced on Group 2 scaffolds that contained empty HNTs. Scaffolds of Groups 3 and 4, containing DEX loaded HNTs did not induce osteoblastic differentiation in the BSCs. Cell culture wells containing Group 2 scaffolds were deeply stained in purple while the rest other wells were stained slightly or none. Group 2 scaffolds' alkaline phosphatase activity was significantly higher on day 14 compared to other groups. Inability of Group 3 and 4 scaffolds to induce differentiation may be attributed to very high concentrations of DEX released. The optimum concentration of DEX needed to induce the osteogenic differentiation is 10–7 [49–51]. In the CPC discs doped with DEX, DEX may have been eluted in excess and thus had an inhibitory effect.

The ability of HNTs alone (no DEX) to induce osteogenic differentiation was previously reported using montmorillonite-doped biopolymers [52]. It may be inferred from this study and the study by Ambre et al. that many aluminosilicate clays may be inherently osteoinductive [53].
