**2.1. Materials**

**1. Introduction**

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

inside the cement [9].

bone formation at the bone-implant interface [21].

Polymethylmethacrylate (PMMA) is widely commercially used bone cement [1, 2]. The most common method of application is the dry mixing of drugs along with the bone cement and administering it into the body [3]. While long considering the 'gold' standard in local antibiotic therapy, it has many disadvantages. A radiolucent fibrous tissue is often observed at the bone/cement interface due to the release of toxic methylmethacrylate (MMA) monomers which damages surrounding tissue [3, 4]. These cements also exhibit a high exothermic setting temperature. Temperatures ranging from 70–120°C have been reported during setting of the PMMA bone cement during implantation [2, 5]. In an experiment conducted by Stańczyk and Rietbergen on bovine cancellous bone, the temperature exposure of 70°C by a fraction (10%) of the bone at the bone/cement interface was recorded [6]. The addition of antibiotics results in reduced mechanical properties in the cement [7]. Furthermore, the release of the antibiotic is short lived and results in less than maximal antibiotic release [7, 8]. Finally, PMMA cements lack elasticity and have dense structure which does not allow bone growth

Brown and Chow were the first to propose the use of calcium phosphate cement (CPC) in bone repair [10]. CPC was approved in 1996 by the Food and Drug Administration (FDA) for repairing craniofacial defects [11]. CPCs have many advantages over PMMA cement. Low shrinkage, durable, dense or porous (depending on the site of injury), and formability (ability to fill cavities of complex configurations) are additional positive qualities of CPCs [12–14]. Due to their similarity to apatite minerals in natural bone, calcium phosphate (CaP) bioceramics, such as hydroxyapatite (HA), are osteoconductive and osteoinductive [15–17]. CPCs implants provide an ideal environment for colonization by osteoblasts to form a functional interface [18, 19]. The end product is easily resorbed by osteoclast cells, leading to new natural

All CPCs are formulated by mixing a solid and a liquid component. The solid component consists of two or more calcium phosphate salts. The solid phase usually consists of a basic and an acidic salt, which reacts together in an aqueous medium and precipitates HA as a final product [19, 20]. The liquids used can be either water, alginates, chitosan, or sodium phosphates [20–22]. To obtain maximum biological use, these components are mixed in predetermined proportions that will lead to the formation of HA. Resorbability of the CPCs completely depends on its end product [22]. The physicochemical reactions that occur during mixing are complex, and cement setting time depends on factors such as solid-liquid composition, liquid-to-powder ratio, and particle size of the powder. Setting conditions also influence the mechanical properties of the cement [23]. However, due to its brittleness, CPCs are restricted to the reconstruction of non-loading bearing bone [24, 25]. Recently, absorbable fibers [23] and chitosan [26] were used to improve the load-bearing capability of CPC [27, 28]. Chitosan and its derivatives are natural biopolymers that are biocompatible, biodegradable, and osteoconductive [29]. CPCs can also be modified through additives (i.e., silicon, strontium, and zinc) and delivered as paste, putty or in an injectable form and set *in situ* to provide intimate adaptation to complex-shaped defects [23, 24]. Polymer materials added as an organic phase to the CPCs have been shown to improve the biological response, physicochemical, and mechanical prop-

erties, such as injectability, cohesion, resorption, and toughness [27, 28, 30].

Calcium phosphate dibasic anhydrous (HCaO4 P, DCPA), β-tri calcium phosphate (Ca<sup>3</sup> O8 P2 , β-TCP), calcium phosphate monobasic monohydrate (H<sup>4</sup> CaO8 P2 H2 0, MCPM), chitosan oligosaccharide lactate ((C12H24N2 O9 ) n ), chitosan (low molecular weight), dexamethasone, gentamicin, neomycin, calcium L-lactate (C6 H10CaO6 × H2 O), and HNTs (H4 Al2 O9 Si2 2H2 O) were purchased from Sigma-Aldrich, St. Louis, MO. Tetra calcium phosphate (Ca4 O4 P, TTCP) was ordered from CaP Biomaterials, E. Troy, WI. Cupric chloride (CuCl2 ) and calcium carbonate (CaCO3 ) were delivered from Nasco, Fort Atkinson, WI. Sodium phosphate dibasic (Na2 HPO4 ) was purchased from Fisher 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.)

*2.2.2. Compression strength testing*

composition of CPCs after setting.

*2.2.3. Flexural strength testing*

**2.3. FTIR analysis**

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

Calcium Phosphate/Clay Nanotube Bone Cement with Enhanced Mechanical Properties and…

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Samples of dimensions 65 mm × 10 mm × 4 mm were prepared using paraffin wax in accordance with the ASTM F417-78 standards. Cement samples were put in these molds and used for flexural testing. The same compression machine was used at a cross-head speed of 1 mm/min. Three samples for each concentration of HNTs were used and an average was calculated.

Potassium bromide (KBr) pellet method was used for FTIR spectroscopy. Thin discs of KBr were prepared by placing a small amount of KBr into the die set and were subjected to a pressure of 8 tons. This pressure caused the KBr to re-crystallize to form thin transparent discs. These discs were used to obtain the background. A small amount of test sample powder was

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.

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,

added to KBr and thin discs were prepared by a similar procedure.

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

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