**2.6. Histochemical staining**

deoxyribonucleosides, sodium bicarbonate, and supplemented with 2 mM L-glutamine, 1% antibiotics (100 U/mL penicillin and 100 U/mL streptomycin), and 10% fetal bovine serum

Mouse bone marrow stromal cells (MSCs) CRL-12424 (ATCC® CRL-12424™) were used for evaluating the osteoinductive potential CPC/HNT composites (details are provided below). MSCs were maintained in Dulbecco's Modified Eagles Medium (DMEM) with 10% FBS and 1% antibiotics (100 U/mL penicillin and 100 U/mL streptomycin (complete DMEM)). Cells

Human osteoblast (HOB) cells were used to assess potential cytotoxicity issues. HOBs were cultured in osteoblast growth media according to the manufacturer's directions. Cells from

To check the cytocompatibility of the formulated scaffolds, XTT was performed on human osteoblast (HOB) cells C-12760 purchased from PromoCell, Heidelberg, Germany. XTT assay kit (X4751) with 1% PMS was ordered from Sigma-Aldrich. CPC discs of 5 mm × 1 mm dimensions were molded using different concentrations of HNTs (5, 10, and 15%). Control discs

XTT or 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2*H*-tetrazolium-5-carboxanilide is a sensitive and reliable quantitative assay. It is a colorless or slightly yellow color tetrazolium dye which gets converted into bright orange color formazan on reduction. When this dye is used in cell cultures, the dehydrogenase enzymes produced from mitochondrial cycle of actively respiring cells reduces the dye to orange color formazan. This assay is greatly improved by adding an electron accepting agent such as PMS (*N*-methyl dibenzopyrazine methyl sulfate). Unlike MTT, this assay does not require to go through laborious procedures such as solubilization prior to quantization, making this process quick and easy. Osteoblast cells, upon reaching to 90% confluency, were trypsinized and resuspended into culture media. In a 96 well plate, 100 μL of cell suspension was added in each well and incubated for 24 h. Activated XTT solution (20 μL) was then added to each well and incubated for 2 h and optical densities

without any HNTs and blank cell lines were also cultured for comparative studies.

were measured using a Phenix LT-4000 absorbance microplate reader at 450 nm.

For seeding into CPC/DEX (+/−)/HNTs, OBs and MSCs were detached from the flasks by adding TryplE, an animal free trypsin substitute, and mixed with HBSS. Cells were then centrifuged and the cell pellets resuspended in either complete α-MEM or DEM. Setting liquid remained the same for all the formulations except for changes in solid components as

For cell-culture techniques, disc-shaped specimens 10 × 2 mm were made using steel molds. The liquid phase used in this process was prepared from sterile deionized water. After setting, the samples were removed from the molds, dipped in alcohol, and dried under a class II hood.

*2.5.2. Assessment of CPC/HNT osteoconductive and osteoinductive potential*

(FBS) (complete α-MEM). Cells from passage 3 to 4 were used.

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

from passage 3 to 4 were used.

passage 2 to 3 were used.

*2.5.1. Cell proliferation*

described below.

As the CPC scaffolds were opaque, bright field microscopy or confocal microscopy images could not be taken. Accordingly, an indirect method of estimating the ECM deposition and amount was used. Collagen content was estimated using Picrosirius red (PS) staining and acidic mucopolysaccharides were estimated using Alcian blue (AB). To quantify staining, stained CP discs were destained measured for staining the dye concentration using UV-visible spectrophotometry. Indirect estimation was done using the destaining method. Glacial acetic acid 7% solution was used as destaining solution. After staining the hydrogel films, destaining solution was added to remove the dye that was fixed on the films. Destaining solution was then collected and measured for the concentration of staining the dye using UV spectrophotometry.

#### *2.6.1. Picrosirius red staining*

Picrosirius red is a specific collagen fiber stain and also is capable of detecting thin fibers. Media was removed from the wells and a wash was given with Dulbecco's phosphate buffered saline (DPBS) before fixing the cells using 95% ethanol. These fixed cells were stained with Picrosirius red for quantifying the amount of collagen secreted. The staining kit had three different solutions—A, B (Picrosirius stain), and C (hydrochloric acid—HCl). Solution A was added to the cells and removed after 5 min and solution B was added and was removed after an hour. A wash was given with solution C to remove excess staining. Glacial acetic acid 7% aqueous solution was used as de-staining solution. As the CPC scaffolds were too thick


**Table 2.** Control and experimental groups used in osteoinductive studies.

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 using UV-visible spectrophotometry.
