*2.2.2. Compression strength testing*

from Nasco, Fort Atkinson, WI. Sodium phosphate dibasic (Na2

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

**2.2. Sample preparation**

performed.

*2.2.1. Sample preparation for SEM*

before analyzing using SEM.

**Group CPC Composition**

**Table 1.** Control and experimental groups use in osteoconductive studies.

Group 1 CPC powder without HNTs Group 2 CPC powder with 5% wt. HNTs Group 3 CPC powder with 10% wt. HNTs Group 4 CPC powder with 15% wt. HNTs

HPO4

Scientific Company, Waltham, MA. Human osteoblast cells (C-12760) and osteoblast growth medium were purchased from PromoCell, Heidelberg, Germany. Mouse pre-osteoblast cell lines MC3T3-E1 (ATCC® CRL-2593™) and mouse bone marrow stromal cells (MSCs) CRL-12424 (ATCC® CRL-12424™) were obtained from ATCC (Manassas, VA.). Cell culture and lab plastics were obtained from MidScientific, St. Louis, Mo. Alpha minimal essential medium (α-MEM) and Dulbecco's DMEM were obtained from GIBCO Invitrogen, Grand Island, NY. Fetal bovine serum and penicillin-streptomycin were purchased from Phenix Research Products (Candler, NC). TryplE, an animal free trypsin substitute, and Trypan blue were obtained from GIBCO Invitrogen, (Grand Island, NY). Cell Titer Blue assay was purchased from Promega (Sunnyvale, CA). The Picrosirius red staining kit was obtained from Polysciences, Inc. (Warrington, PA.)

Different types of calcium phosphate salts were mixed in different proportions using various chemicals as part of the liquid phase to identify the best composition with respect to their mechanical properties (**Table 1**). All samples were mixed using mortar and pestle under ambient conditions (room temperature and atmospheric pressure). The powder phase was mixed thoroughly until all the salts were uniformly dispersed. To this, the liquid phase was added in small quantities and triturated until the mixture became a thick, moldable paste.

The most suitable formulation was achieved when tetracalcium phosphate (TTCP provided by CaP Biomaterials) and dicalcium phosphate (DCPA provided by Sigma-Aldrich, St. Louis, MO.) were used as the solid phase and chitosan lactate (CL) was used as the setting liquid. An equimolar ratio of TTCP and DCPA was mixed and to this 10% w/v solution of CL was added. This mixture was thoroughly mixed using mortar and pestle to form a soft mass. The soft mass could then be set in different mold shapes depending on the assay or test being

Scanning electron microscopy (SEM) was used to analyze the surface of CPCs. Specimens fractured during compression test were taken for SEM imaging. S4800 Field Emission SEM, HITACHI was used for our research. Gold nanolayer (about 3 nm) was laid over specimens

) was purchased from Fisher

Compression test was performed on cylindrical shape specimens (12 mm length and 6 mm diameter). Compression test was performed on dry specimens and on specimens placed in simulated biological fluid (SBF) for 24 h before testing. Testing was done using an ADMET tensile tester with a load speed of 1 mm/min. FTIR spectroscopy was done to analyze the composition of CPCs after setting.
