**3.3. Flexural strength**

tested, the composition with the most desired properties was an equimolar mixture of TTCP

**Figure 2.** Scanning electron micrograph of CPC added with 5% wt. HNTs showing aggregation of HNTs. Red

arrows = chitosan lactate flakes. Red star = calcium phosphate particles. White star = HNTs concentrations.

Different concentrations of HNTs were added to this composition. These compositions were then tested for compression and flexural strengths. The addition of HNTs has an observable and significant change in the compressive strengths of CPCs (**Figure 3A**). Peak compressive strength was seen in groups that were added with 5% wt. HNTs. Peak compression pressure of 16.48 ± 0.95 MPa was observed for CPCs that were added with 5% wt. HNTs. CPCs that were not added with HNTs had a compressive strength of 11.65 ± 2.24 MPa. Compressive strength of CPCs is increased by 41.47% upon the addition of 5% of HNTs. At concentrations of 10 and 15% wt. HNTs, compressive strength of CPCs was recorded as 14.43 ± 1.64 MPa and 13.54 ± 1.67 MPa. Minimum of five samples in each group were tested and statistical analysis was performed on the data sets. One way ANOVA was done to analyze the statistical significance among the four groups. Results suggest that there is a significant difference in the means of all four groups at α = 0.05. Post hoc analysis was done to identify the group with the highest mean compressive strength. Results from Tukey's HSD suggest that groups contain-

Addition of nanoparticles in the matrix of CPCs improves the compression strength up to certain concentration and beyond this concentration no significant improvement was observed. The optimal concentration for this composition was observed to be at 5% wt. The particle size of TTCP contributes an important role in determining the compressive strength of CPCs. Smaller particles attribute for increased strength compared to larger particles. As shown in **Figure 3B**, at a particle size of 1.9 ± 0.31 μm, the compressive strength of CPCs was 23.49 ± 1.36 MPa. Larger particles with a diameter of 4.1 ± 1.15 μm had a peak compression strength of 8.25 ± 2.56 MPa. Particles with an average diameter of 2.4 ± 0.42 μm had a compression strength

and DCPA in the solid phase with chitosan lactate as the liquid phase.

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

ing 5% wt. HNTs have the highest mean compressive strength.

Three-point bending test was performed to evaluate the flexural strength of CPCs (**Figure 4A**). Flexural strength of control group containing just DCPA, TTCP, and chitosan lactate was recorded as 13.86 ± 2.45 MPa. Addition of HNTs at 5% wt. improved the strength to 17.12 ± 2.49 MPa. This increase was not statistically significant at α = 0.05. CPCs containing 10 and 15% wt. HNTs had flexural strengths of 15.38 ± 0.26 MPa and 17.41 ± 1.45 MPa, respectively. This trend of flexural strengths is graphically represented in **Figure 4B**. The standard deviation error bars in **Figures 3** and **4** may be attributed to air gaps formed while preparing the bone cement and loading them into the molds. Presence of air pockets can reduce the compression and flexural strength of cements. This may be avoided by using a vacuum chamber to mix and prepare the specimens.

The inability to provide significant resistance to applied mechanical loads has limited CPCs to non-load-bearing, mostly craniofacial applications [37–39]. Their toughness and fatigue resistance are much less than those of cortical bone. The incorporation of a polymer during the CPC liquid phase increases ductility, allowing for a higher deformation before breaking [39, 40]. Moreover, polymer fiber reinforcement has been extensively explored as a strategy to increase the toughness and strength of cements [41].

**Figure 4.** (A) Flexural test method. (B) Flexural strength of CPCs with varying amount of HNTs. Error bars = standard deviation.

**3.5. Dexamethasone elution profile**

2− and ▬OH groups, respectively).

HA (CO3

did not have contributed to this release.

**3.6. Cell proliferation**

Drug elution was studied using 50 mg of DEX loaded HNTs (**Figure 6A**). It can be observed that there is an initial burst release from HNTs during the first 10 h of release and from then onwards it was sustained. This 50 mg elution form of HNTs is above the desired micro to nanomolar range to stimulate the differentiation of MSCs towards osteoblastic lineage. DEX release from scaffolds was also studied using scaffolds with only 5% of HNTs loaded with DEX. It can be observed that drug release was slow and the concentration of drug released from scaffolds is much lower compared to HNTs. **Figure 6B** shows the release of DEX from scaffolds. The drug release may be even more extended during scaffolds degradation exposing more HNTs to extracellular fluids (*in vivo*). Drug release from CPC scaffolds was less compared to HNTs. This may be attributed to the surface release and doped HNTs in the matrix

**Figure 5.** FTIR spectra of HA and CPCs with and with HNTs. Peaks at 1450 cm−1 and 3100–3700 cm−1 are characteristic of

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The viability of HOBs on the surface of the CPC scaffolds was analyzed at days 3, 7, and 14 (**Figure 7**). The absorbance values of the XTT assay reflects the mitochondrial activity of the live cells. The absorbance values across the 14-day period indicate that all samples showed no cytotoxic effects. The mean values of absorbance also varied strongly with the HNT concentration. On day 3, a 20.35% increase in absorbance values was observed for control CPC scaffolds when compared to wells containing only cells. On addition of 5% HNTs to the CPC scaffolds, 28.3% significant increase in the absorbance value was noticed. For 10 and 15% HNT absorbance was 9.8 and 12.8% larger, respectively. But, when compared with control CPC scaffold (no HNTs added), 8.7 and 6.1% decrease in the absorbance values for 10 and 15% CPC-HNT scaffolds were seen. By day 7, absorbance was larger for CPC scaffolds than for control wells. Wells containing 5% HNT samples showed 26.7% larger absorbance compared to CPC

One explanation for a portion of the gain in material strength could be HNT addition to the CPC cement. HNTs have been widely used as a nanofiller for the modification of nanocomposite materials [24, 25, 27]. HNTs have been used to increase surface area and impart high surface reactivity, improving mechanical and overall strength (see Zhang et al. for an excellent review [42]). To achieve the increase in mechanical strength, thermal stability, and toughness HNTs has been combined into a variety of polymers. Examples of polymer studied modification by HNTs include: poly(butylene succinate) [44], polyamide [12, 45], styrene-butadiene [46], epoxy [47], poly(methyl-methacrylate) [48], and chitosan [43]. In our case, we speculate that HNTs distribution the CPC cement fills voids resulting in the increase of mechanical strength by preventing crack propagation. HNTs have been suggested as mechanism nanoparticle deflect or absorb a propagating crack, thus preventing the formation of weak points and improving strengths. However, when the HNT concentration exceeds a certain value, the CPC deformability decreased sharply. This may be because of the pattern of HNT dispersion [47]. The interfacial binding between HNT/chitosan composites was proposed as resulting from hydrogen bonding and electrostatic interactions and a uniform dispersion results in a uniform interfacialbinding matrix and favorable to force conduction [43]. In contrast, too many HNTs and HNT clustering may inhibit the dispersion state and create interfacial gaps, which are easy to break.

## **3.4. FTIR analysis**

The final setting product of apatite cements was hypothesized to be hydroxyapatite. To evaluate the end-product formation, FTIR analysis was used. A comparison of IR spectra of CPC control, CPCs (no HNTs added), CPCs with 5% HNTs, and hydroxyapatite is shown in **Figure 5**. Band of peaks seen at wavenumber 900–1200 cm−1 are due to PO + 4 groups present in all the test samples. Peaks at 1450 cm−1 are characteristic of the CO3 2− group found in HA. Peaks at 3100–3700 cm−1 are due to the ▬OH groups of hydroxyapatite. Comparing peaks for the ▬OH group, we observe that CPCs with HNTs have stronger peak than control CPCs. This observation can be attributed to the presence of HNTs that acted as seeding/nucleating agents facilitating the precipitation of hydroxyapatite crystals.

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**Figure 5.** FTIR spectra of HA and CPCs with and with HNTs. Peaks at 1450 cm−1 and 3100–3700 cm−1 are characteristic of HA (CO3 2− and ▬OH groups, respectively).

#### **3.5. Dexamethasone elution profile**

One explanation for a portion of the gain in material strength could be HNT addition to the CPC cement. HNTs have been widely used as a nanofiller for the modification of nanocomposite materials [24, 25, 27]. HNTs have been used to increase surface area and impart high surface reactivity, improving mechanical and overall strength (see Zhang et al. for an excellent review [42]). To achieve the increase in mechanical strength, thermal stability, and toughness HNTs has been combined into a variety of polymers. Examples of polymer studied modification by HNTs include: poly(butylene succinate) [44], polyamide [12, 45], styrene-butadiene [46], epoxy [47], poly(methyl-methacrylate) [48], and chitosan [43]. In our case, we speculate that HNTs distribution the CPC cement fills voids resulting in the increase of mechanical strength by preventing crack propagation. HNTs have been suggested as mechanism nanoparticle deflect or absorb a propagating crack, thus preventing the formation of weak points and improving strengths. However, when the HNT concentration exceeds a certain value, the CPC deformability decreased sharply. This may be because of the pattern of HNT dispersion [47]. The interfacial binding between HNT/chitosan composites was proposed as resulting from hydrogen bonding and electrostatic interactions and a uniform dispersion results in a uniform interfacialbinding matrix and favorable to force conduction [43]. In contrast, too many HNTs and HNT clustering may inhibit the dispersion state and create interfacial gaps, which are easy to break.

**Figure 4.** (A) Flexural test method. (B) Flexural strength of CPCs with varying amount of HNTs. Error bars = standard

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

The final setting product of apatite cements was hypothesized to be hydroxyapatite. To evaluate the end-product formation, FTIR analysis was used. A comparison of IR spectra of CPC control, CPCs (no HNTs added), CPCs with 5% HNTs, and hydroxyapatite is shown in **Figure 5**. Band of peaks seen at wavenumber 900–1200 cm−1 are due to PO + 4 groups present in all

at 3100–3700 cm−1 are due to the ▬OH groups of hydroxyapatite. Comparing peaks for the ▬OH group, we observe that CPCs with HNTs have stronger peak than control CPCs. This observation can be attributed to the presence of HNTs that acted as seeding/nucleating agents

2− group found in HA. Peaks

the test samples. Peaks at 1450 cm−1 are characteristic of the CO3

facilitating the precipitation of hydroxyapatite crystals.

**3.4. FTIR analysis**

deviation.

Drug elution was studied using 50 mg of DEX loaded HNTs (**Figure 6A**). It can be observed that there is an initial burst release from HNTs during the first 10 h of release and from then onwards it was sustained. This 50 mg elution form of HNTs is above the desired micro to nanomolar range to stimulate the differentiation of MSCs towards osteoblastic lineage. DEX release from scaffolds was also studied using scaffolds with only 5% of HNTs loaded with DEX. It can be observed that drug release was slow and the concentration of drug released from scaffolds is much lower compared to HNTs. **Figure 6B** shows the release of DEX from scaffolds. The drug release may be even more extended during scaffolds degradation exposing more HNTs to extracellular fluids (*in vivo*). Drug release from CPC scaffolds was less compared to HNTs. This may be attributed to the surface release and doped HNTs in the matrix did not have contributed to this release.

#### **3.6. Cell proliferation**

The viability of HOBs on the surface of the CPC scaffolds was analyzed at days 3, 7, and 14 (**Figure 7**). The absorbance values of the XTT assay reflects the mitochondrial activity of the live cells. The absorbance values across the 14-day period indicate that all samples showed no cytotoxic effects. The mean values of absorbance also varied strongly with the HNT concentration. On day 3, a 20.35% increase in absorbance values was observed for control CPC scaffolds when compared to wells containing only cells. On addition of 5% HNTs to the CPC scaffolds, 28.3% significant increase in the absorbance value was noticed. For 10 and 15% HNT absorbance was 9.8 and 12.8% larger, respectively. But, when compared with control CPC scaffold (no HNTs added), 8.7 and 6.1% decrease in the absorbance values for 10 and 15% CPC-HNT scaffolds were seen. By day 7, absorbance was larger for CPC scaffolds than for control wells. Wells containing 5% HNT samples showed 26.7% larger absorbance compared to CPC

employed using a staining and destaining method. Collagen content was estimated using the Picrosirius red stain, the presence of acidic mucopolysaccharides using an Alcian blue stain, and alkaline phosphatase activity was examined to confirm the osteoblastic phenotype of cells

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Graphical representation of PSR stain absorbance is shown in **Figure 8**. Higher absorbance indicates greater collagen content secreted by osteoblasts on the scaffolds. All test groups supported collagen production and throughout the length of the experimental period. On average, a higher amount of collagen synthesis was observed on day 14 stain as compared to day 7. However, there was no significant difference in the amount of collagen synthesized

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

cultured of HNT/CPC discs.

*3.7.1. Picrosirius red staining (PSR)*

among all the groups on given day.

different HNT concentrations at day 7 and 14.

*3.7.2. Alcian blue staining*

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

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

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

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).

employed using a staining and destaining method. Collagen content was estimated using the Picrosirius red stain, the presence of acidic mucopolysaccharides using an Alcian blue stain, and alkaline phosphatase activity was examined to confirm the osteoblastic phenotype of cells cultured of HNT/CPC discs.
