**2.4. Loading of the HNTs with dexamethasone (DEX)**

HNTs were loaded using the vacuum loading technique. Saturated solution of Dex was made by dissolving 200 mg of Dex in 50 mL of ethanolic water (50%v/v). To this solution, 9 g of HNTs were added and subjected to vacuum cycles. The suspension of HNTs in Dex was kept in a vacuum for 20 min and at atmospheric pressure for 20 min, this was repeated thrice. This suspension was left under the vacuum overnight and centrifuged. The supernatant was discarded and HNTs were washed twice with deionized water to remove drug adsorbed on the HNT surface. Washed HNTs were dried in a vacuum and these were used in further experiments.

Drug release from HNTs was measured for a period of 96 h. Accurately measured quantity of 50 mg of Dex loaded HNTs was taken in a centrifuge tube and 1 mL of simulated biological fluid (SBF) was added and placed in a shaker. After specific time intervals, the tube was centrifuged to precipitate HNTs and the supernatant was collected. Equal volume of SBF was replaced every time supernatant was collected to maintain sink conditions. Collected supernatant was observed using a UV-visible spectrophotometer (NanoDrop) at a wavelength of 240 nm.

#### **2.5. Cell culture, proliferation, and differentiation**

Mouse pre-osteoblast cell lines MC3T3-E1 (ATCC® CRL-2593™) were obtained from ATCC (Manassas, VA.). Mouse pre-osteoblast (OB) was used for evaluating the osteoconductive potential CPC/HNT composites (details are provided below). OBs were maintained in a minimum essential medium, Eagle's α-modification (α-MEM), containing ribonucleosides, 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 (FBS) (complete α-MEM). Cells from passage 3 to 4 were used.

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.

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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.

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

Group 3 CPC powder with 5% wt. DEX-doped HNTs and 5% wt. plain HNTs

**2.6. Histochemical staining**

*2.6.1. Picrosirius red staining*

**Group CPC composition**

Group 1 CPC powder without HNTs Group 2 CPC powder with 10% wt. HNTs

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

Group 4 CPC powder with 10% wt. DEX-doped HNTs

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 from passage 3 to 4 were used.

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 passage 2 to 3 were used.
