**3. Results and discussion**

## **3.1. SEM analysis**

SEM was used to analyze the nature of the surface topography of CPCs. Surfaces in all formulations had granules of small and large size (**Figure 1**) No definitive difference could be observed visibility in these images between CPS with and without HNTS. Chitosan lactate was flakes or scales that were observed in thin layers around the particles of DCPA and TTCP (**Figures 1** and **2**). Granular particles of DCPA and TTCP are seen in a matrix of chitosan lactate. HNTs were also observed in few images distributed as clumps and as fine particles. As seen in **Figure 2**, aggregations of HNTs were observed distributed within the matrix of CPCs filling up the voids between the calcium and lactate particles. Cell viability and cell proliferation are largely dependent on the substrate surface, and cellular response to surface roughness is different depending on the cell type [32, 33]. Rough surfaces are preferred by many cell types for attachment and further functionality [33, 34]. Osteoblasts grown on surfaces with microtopography were stimulated towards differentiation as compared with a lack of specific bone marker expression when grown on smooth surfaces [35, 36]. SEM images of CPCs clearly indicate that all CPC formulations possess very rough surfaces and provide a conducive environment for cell attachment (**Figure 1**). This observation was confirmed by the cellular response of OBs and MSCs on the CPC surfaces.

**3.2. Compression strength**

Specimens made for the compression test were analyzed for maximum compressive strength and peak load taken before they were fractured. Of the various combinations of calcium salts and setting liquid salts tested, only those samples that had anti-washout property and good compressive strength were chosen. Most of the samples had decent compressive strength when dry, but upon placing them in an aqueous environment, they became soft and collapsed. Specimens made with TTCP and DCPA using chitosan as a setting liquid had good mechanical properties. As chitosan is soluble only in acidic solutions, aqueous solution of chitosan in acidic water was used. This resulted in elution of acid into SBF while testing for their anti-washout property. The pH of SBF was maintained at 7.40, but when these samples were immersed in SBF, the pH of the solution increased as high as pH 2.0. Hence, in-spite-of having good mechanical properties, these samples were not selected as it would create hostile microenvironment for cells. To prevent cytocompatibility issues, chitosan lactate was, accordingly, selected as the setting liquid as it was soluble in neutral water. When all samples were

**Figure 1.** SEM images of CPCs with and without HNTs. (A) CPCs with 0% w/w HNTs (1200× magnification); (B) CPCs with 5% w/w HNTs (1200× magnification); (C) CPCs with 10% w/w HNTs (1500× magnification); and (D) CPCs with 15% w/w HNTs (1500× magnification). (A) and (B) scale bar = 40 microns; (C) and (D) scale bar = 30 microns. Red arrow = chitosan lactate.

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**Figure 1.** SEM images of CPCs with and without HNTs. (A) CPCs with 0% w/w HNTs (1200× magnification); (B) CPCs with 5% w/w HNTs (1200× magnification); (C) CPCs with 10% w/w HNTs (1500× magnification); and (D) CPCs with 15% w/w HNTs (1500× magnification). (A) and (B) scale bar = 40 microns; (C) and (D) scale bar = 30 microns. Red arrow = chitosan lactate.

### **3.2. Compression strength**

for light to penetrate, microscopic imaging and quantification were difficult to accomplish. To quantify staining, de-staining was used and measured for staining the dye concentration

Alcian blue is used to stain sulfate glycosaminoglycans and proteoglycans in the extracellular matrix. The same procedure, as indicated above, was followed using Alcian blue solution as the dying reagent and destained samples were collected. These samples were later analyzed

During *in vitro* bone formation, AP activity is greatly enhanced and hence used as a marker to evaluate the differentiation of MSCs towards osteoblastic lineage or maintenance of the osteoblast phenotype. It is easily detected using BCIP/NBT as a substrate, which stains cells blue-violet when AP is present. Alcohol fixed plates were washed with a buffer and carefully aspirated without disrupting the monolayer of cells. Enough BCIP/ NBT substrate solution was added to just cover the monolayer and left in the dark for 5–10 min. It was then washed and destained using 7% glacial acetic acid for 5 min to quantify the AP activity. The solution was aspirated and analyzed using a NanoDrop 2000c

SEM was used to analyze the nature of the surface topography of CPCs. Surfaces in all formulations had granules of small and large size (**Figure 1**) No definitive difference could be observed visibility in these images between CPS with and without HNTS. Chitosan lactate was flakes or scales that were observed in thin layers around the particles of DCPA and TTCP (**Figures 1** and **2**). Granular particles of DCPA and TTCP are seen in a matrix of chitosan lactate. HNTs were also observed in few images distributed as clumps and as fine particles. As seen in **Figure 2**, aggregations of HNTs were observed distributed within the matrix of CPCs filling up the voids between the calcium and lactate particles. Cell viability and cell proliferation are largely dependent on the substrate surface, and cellular response to surface roughness is different depending on the cell type [32, 33]. Rough surfaces are preferred by many cell types for attachment and further functionality [33, 34]. Osteoblasts grown on surfaces with microtopography were stimulated towards differentiation as compared with a lack of specific bone marker expression when grown on smooth surfaces [35, 36]. SEM images of CPCs clearly indicate that all CPC formulations possess very rough surfaces and provide a conducive environment for cell attachment (**Figure 1**). This observation was confirmed by the

using a Thermo Scientific NanoDrop 2000c spectrophotometer.

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

using UV-visible spectrophotometry.

*2.6.3. Alkaline phosphatase (AP) assay*

*2.6.2. Alcian blue staining*

spectrophotometer.

**3.1. SEM analysis**

**3. Results and discussion**

cellular response of OBs and MSCs on the CPC surfaces.

Specimens made for the compression test were analyzed for maximum compressive strength and peak load taken before they were fractured. Of the various combinations of calcium salts and setting liquid salts tested, only those samples that had anti-washout property and good compressive strength were chosen. Most of the samples had decent compressive strength when dry, but upon placing them in an aqueous environment, they became soft and collapsed. Specimens made with TTCP and DCPA using chitosan as a setting liquid had good mechanical properties. As chitosan is soluble only in acidic solutions, aqueous solution of chitosan in acidic water was used. This resulted in elution of acid into SBF while testing for their anti-washout property. The pH of SBF was maintained at 7.40, but when these samples were immersed in SBF, the pH of the solution increased as high as pH 2.0. Hence, in-spite-of having good mechanical properties, these samples were not selected as it would create hostile microenvironment for cells. To prevent cytocompatibility issues, chitosan lactate was, accordingly, selected as the setting liquid as it was soluble in neutral water. When all samples were

**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.

of 17.59 ± 1.13 MPa. It can be inferred that smaller particles may be arranged in a compact manner resulting in smaller voids between the particles and thus resulting in greater compression strengths of CPCs. This improvement in compression strength by the addition of HNTs can be useful in applications where primary forces acting on cements are compression. Bone cements used in vertebral augmentation are primarily subjected to compressive forces and the CPC/

**Figure 3.** (A) Compressive strength of CPCs with varying amount of HNTs. (B) Compression strength as a function of

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Much research was directed towards improvement of CPCs by introducing additives in solid and liquid phases. Improvements were observed in mechanical strengths, setting times, bioactivity, and degradability. Addition of gelatin and chitosan resulted in increased setting time, improved cell adhesion and ECM formation, and improved compressive strengths. Addition of other polymers including collagen, alginate, hyaluronate, cellulose, and synthetic polymers had various effects depending on the CPC composition and the amount of polymer [37, 38].

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

HNTs composite may be of use in this surgical procedure.

particle size of TTCP. Error bars = standard deviation.

increase the toughness and strength of cements [41].

**3.3. Flexural strength**

tested, the composition with the most desired properties was an equimolar mixture of TTCP and DCPA in the solid phase with chitosan lactate as the liquid phase.

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

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

**Figure 3.** (A) Compressive strength of CPCs with varying amount of HNTs. (B) Compression strength as a function of particle size of TTCP. Error bars = standard deviation.

of 17.59 ± 1.13 MPa. It can be inferred that smaller particles may be arranged in a compact manner resulting in smaller voids between the particles and thus resulting in greater compression strengths of CPCs. This improvement in compression strength by the addition of HNTs can be useful in applications where primary forces acting on cements are compression. Bone cements used in vertebral augmentation are primarily subjected to compressive forces and the CPC/ HNTs composite may be of use in this surgical procedure.

Much research was directed towards improvement of CPCs by introducing additives in solid and liquid phases. Improvements were observed in mechanical strengths, setting times, bioactivity, and degradability. Addition of gelatin and chitosan resulted in increased setting time, improved cell adhesion and ECM formation, and improved compressive strengths. Addition of other polymers including collagen, alginate, hyaluronate, cellulose, and synthetic polymers had various effects depending on the CPC composition and the amount of polymer [37, 38].
