*2.5.1. Cell proliferation*

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

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

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

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 described below.

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.

Two different scaffold types were made for culturing cells (**Tables 1** and **2**). One set of scaffolds were made to evaluate the osteoconductive nature of CPCs while the other set was used to assess its osteoinductive potential. Osteoconductive scaffolds contained CPC powder with varied amounts of HNTs (**Table 1**). Four groups were prepared containing 0, 5, 10, and 15% wt. of HNTs to CPC powders. These HNTs were not loaded with DEX or any other growth factors. Mouse OBs were used for seeding on these scaffolds to evaluate the osteoconductive property of fabricated CPC. To evaluate the osteoinductive potential of our formulation with DEX-doped HNTs, four set of scaffolds were prepared (**Table 2**). Scaffolds in both groups were tested for cell viability, proliferation, ECM production, and alkaline phosphatase activity over a 14-day period. Standard histochemical staining techniques were used. We predicted that MSCs would differentiate into osteoblasts after the exposure to DEX-doped HNT/CPC composites.
