TiO2 doped chitosan/hydroxyapatite/halloysite nanotube membranes with enhanced mechanical properties and osteoblast-like cell response for application in bone tissue engineering

The current therapeutic strategies for healing bone defects commonly suffer from the occurrence of bacterial contamination on the graft, resulting in nonunion in the segmental bone defects and the requirement for secondary surgery to remove or sterilize the primary graft. A membrane with enhanced anti-bacterial efficacy, mechanical strength and osteoconductivity would represent an improvement in the therapeutic strategy for guided bone regeneration. The present study aims to optimize the content of halloysite nanotubes (HNTs) and TiO2 in the polymer matrix of chitosan (CTS) with a constant amount of nano-hydroxyapatite (5%) with the objective of mimicking the mechanical and biological microenvironment of the natural bone extracellular matrix with enhanced anti-bacterial efficacy. HNTs are a low-cost alternative to MWNCTs for enhancing the mechanical properties and anti-bacterial efficacy of the composite. From the first stage of the study, it was concluded that the membranes possessed enhanced mechanical properties and optimum biological properties at 7.5% (w/w) loading of HNTs in the composite. In the second stage of this investigation, we studied the effect of the addition of TiO2 nanoparticles (NPs) and TiO2 nanotubes (NTs) in small amounts to the CTS/n-HAP/HNT nanocomposite at 7.5% HNT loading, with an aim to augment the anti-bacterial efficacy and osteoconductivity of this mechanically strong membrane. The study revealed a significant enhancement in the anti-bacterial efficacy, osteoblast-like MG-63 cell proliferation and ALP expression with the addition of TiO2 NTs. The CHH-TiT membrane successfully inhibited the S. aureus and E. coli growth within 16 hours and simultaneously assisted the enhanced proliferation of osteoblast-like cells on its surface. The study supports the potential exploitation of CHH-TiT (7.5% HNT & 0.2% TiO2 NT) membranes as a template for guided bone tissue regeneration.

At first, the dry weights (W 0 ) of CHH I-III, CHH-TiP, CHH-TiT and CH were measured. Thereafter the samples were immersed in distilled water for up to 24 hrs. The samples were then displaced periodically from the water and gently blotted with filter paper to remove water adsorbed at the surface. The samples were then weighed again (W t ), after 1, 2, 3, 4, 6, 12 and 24 h of first immersing them into the water.

SI 1.2 Hemolytic assay
Following approval from the Institute human ethics committee of IIT Roorkee and with due consent, blood was collected from 3 adult individuals. The lysis of the RBC leads to the release of hemoglobin and subsequent centrifugation separates other cell debris and the intact cells. The amount of hemoglobin in the supernatant depends on the number of cells lysed by the respective sample solutions of CHH I-III, CHH-TiP, CHH-TiT and CH. The freshly collected human blood sample was centrifuged at 3000 rpm and 4°C, to separate the blood into erythrocytes (RBC), plasma and Buffy Coat. The RBC's were then collected at the bottom of the centrifuge tube and were then subsequently washed with phosphate buffer solutions (PBS) at pH 7.4 and thereafter re-suspended in the same buffer. Then, 50 μl of each sample solution was taken in a centrifuge tube, that was then subsequently diluted with an additional 900 μl of PBS and finally, 50 μl of RBC sample was added to the above tube. The tubes were then incubated in dark for 10 minutes at 4°C and were then subsequently centrifuged. The optical density (OD) of the supernatant following the centrifugation was measured at 540 nm using a spectrophotometer. The Positive control sample (50 μl RBC + 950 μl H 2 O) and Negative control sample (50 μl RBC + 950 μl PBS) were also prepared to compare their OD values with the OD of the supernatant of the respective sample solutions.

SI 1.3 MTT assay
The culture was maintained using Alpha Minimum Essential Medium (α-MEM) (Sigma Aldrich). The medium was replaced at regular intervals. To carry out the MTT assay, 1 mg of each sample was maintained in polystyrene 96-well sterilized plates. 5X103 cells were seeded on each scaffold (experimental) and wells without scaffolds (experimental control) in 200 μL of the medium. The cells were incubated at 37°C for 24 h in a humidified incubator with 5% CO 2 . Subsequently, 20 μL of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Aldrich) dissolved in 1X PBS (5mg/mL) was added to each well (experimental variables and experimental control) followed by a 4 h incubation at 37°C. Thereafter, the MTT containing medium was replaced from each well with 200 μL of dimethyl sulfoxide (DMSO), to dissolve the formazan crystals. Finally, the absorbance of the solution was recorded at 570 nm using a Fluostar Optima plate reader (Germany).

SI 1.4 Antibacterial efficacy study
Both the microbes were procured from NCCS, Pune. In outline, E. coli was inoculated in Luria-Bertani broth (LB) and S. aureus was inoculated in Nutrient Broth (NB), for the exponential growth of the microbes. 0.03 mg of each film was added into 30 mL of bacterial suspension and subsequently incubated at 37°C in a vibrator at 180 rpm for 18 h. 300 microliter of suspension aliquots were taken out from each suspension at time intervals of 1, 2, 4, 6, 18 h to measure their optical density (OD) at 600 nm. An increase in OD signifies bacterial growth in the suspension. To examine the bacterial structure of E. coli and S. aureus before and after treating them with CHH-TiT film we drop-fixed the respective suspensions on a square glass film, which were subsequently taken for imaging on a Field Emission Scanning Electron Microscope (FE-SEM Quanta 200 FEG).

SI 1.5 Alkaline Phosphatase (ALP) activity test
The MG-63 cells were seeded on the films for 3 or 7 days in a humidified incubator at 37°C with 5% CO 2 . The cells were then lysed using 500.0 μL of lysis solution (0.2% TritonX-100 (w/v)). 50 μL of supernatant from lysis was incubated along with 100 μL of pNPP substrate solution for ELISA (Sigma-Aldrich) at 25°C. Then the absorbance reading of the wells were taken using a microplate reader at 405 nm after ageing for 30 minutes. ALP activity of the cells cultured without any films served as the control. The ALP activity values were normalized by the amount of protein present in the well. The protein was quantified using Bicinchoninic acid (BCA) protein assay kit (Sigma-Aldrich). Briefly, 50 μL of cell lysate supernatant was added to 200 μL of the BCA reagent, followed by incubation at 37°C for 30 min. The optical absorbance was measured for each well at 562 nm using a microplate reader to quantify the protein. Finally, the Protein content and ALP activity were evaluated using the standard curve derived from the serial dilutions of the standard reagents.