Materials and Methods

4. SYNTHETIC CALCIUM POLYPHOSPHATE AS AN ALTERNATIVE TO ILIAC CREST BONE

4.3 Materials and Methods

4.3.1 Ca-polyP nanoparticle synthesis and classification 4.3.1.1 Fabricating Ca-polyP nanoparticles

Ca-polyP nanoparticles were fabricated following the previously published method by Müller, W.E.G., et al ¹⁹. Longer chain-length sodium polyphosphate (Na-polyP) salt, provided as a gift (RegeneTiss, Inc.; Nagano, Japan), was dissolved in deionized water (20 g L^-1), and aqueous sodium hydroxide (NaOH, 1M) was added to the Na-polyP solution until the pH was approximately 10. Calcium chloride dihydrate (CaCl2, Sigma-Aldrich; St. Louis, MO) was dissolved in distilled water (112 g L^-1) and added dropwise to the Na-polyP solution (1 mL/min). The 1M NaOH solution was added throughout to maintain a pH close to 10. Once all of the CaCl2 solution was added to the Na-polyP solution, the suspension was stirred for an additional 4 h while using the 1M NaOH solution to maintain a pH close to 10. After 4 h, the particles were washed twice with ethanol by centrifuging the suspended particles, aspirating the supernatant, and resuspending the particles in ethanol. After the second wash, the particles were dried in a 60 ^oC oven.

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4.3.1.2 Quantifying the average chain-length of Na-polyP by NMR

The average chain-length of the Na-polyP salt was quantified by Phosphorus-31 nuclear magnetic resonance spectroscopy (³¹P-NMR) ²⁴. 100 mg Na-polyP was dissolved in 550 µL deionized water and 50 µL D2O (Sigma-Aldrich; St. Louis, MO), and the NMR analysis was conducted at 500 MHz on a DRX-500 FT-NMR spectrometer (Bruker; Billerica, MA). The spectrum peaks of the internal and external phosphates were integrated in TopSpin (Bruker; Billerica, MA), and the average chain-length was calculated using Equation 4.1.

𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝐶ℎ𝑎𝑖𝑛 𝐿𝑒𝑛𝑔𝑡ℎ = 2∗𝑃𝑒𝑎𝑘 𝐴𝑟𝑒𝑎𝐼𝑛𝑡𝑒𝑟𝑛𝑎𝑙 𝑃ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒𝑠

𝑃𝑒𝑎𝑘 𝐴𝑟𝑒𝑎𝐸𝑥𝑡𝑒𝑟𝑛𝑎𝑙 𝑃ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒𝑠 (4.1)

4.3.1.3 Quantifying the average particle size of the Ca-polyP nanoparticles by DLS and SEM

The average particle size of the Ca-polyP nanoparticles was quantified by digital light scattering (DLS) and by scanning electron microscopy (SEM). For DLS analysis, the Ca-polyP nanoparticles were suspended in deionized water at a concentration of 5 mg mL^-1. After sonication for less than 1 minute and dilution to 0.1 mg mL

-1, the average particle size was measured using a Zetasizer (Malvern Panalytical Ltd; Malvern, Worcestershire, England). For SEM imaging, the Ca-polyP nanoparticles were dispersed across an SEM stub with carbon tape and gold sputter-coated with a 108 Auto Sputter Coater (Ted Pella Inc.; Redding, CA). The nanoparticles were imaged using a MERLIN SEM with a GEMINI II column (Carl Zeiss Inc.; Thornwood, NY), and the average particle size was quantified using ImageJ (NIH, https://imagej.nih.gov/ij/). The diameters of at least 50 particles were measured in each of 15 SEM images for a total of 868 measurements.

4.3.1.4 Characterizing the chemical composition of the Ca-polyP nanoparticles by FTIR

The chemical composition of the Ca-polyP nanoparticles was characterized by Fourier transform infrared spectroscopy (FTIR). 2 mg Ca-polyP was pressed into a 200 mg KBr pellet, and the pellet was scanned using a Tensor 27 FTIR (Bruker; Billerica, MA).

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4.3.1.4 Characterizing the crystallinity of the Ca-polyP nanoparticles by XRD

The crystallinity of the Ca-polyP nanoparticles was analyzed by x-ray diffraction (XRD). Ca-polyP nanoparticles were evenly dispersed across a glass slide and were scanned from 10^o to 80^o (2-theta) using a SmartLab diffractometer with Cu Kα X-ray source (Rigaku; Tokyo, Japan).

4.3.2 Harvesting and implanting ICBG

All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Vanderbilt University Medical Center. Male C57BL/6J mice were purchased from Jackson Laboratory and housed at Vanderbilt University in a 12-h light/dark cycle with food and water provided ad libitum. At approximately 8-9 weeks of age, a cohort of mice were sacrificed by CO2 inhalation, and ICBG was harvested. The harvested ICBG was standardized by volume.

Immediately after harvesting the ICBG, posterolateral lumbar surgeries were performed on separate, yet genetically identical, male mice. Following adequate anesthesia and analgesic, ICBG, Ca-polyP nanoparticles, or saline (sham), was transplanted into the posterolateral gutters of age-matched mice. The internal fascia and skin were closed with absorbable and nylon sutures, respectively, and the mice were transferred to their respective cages and monitored until they regained normal ambulation. For the first 3 d after surgery, analgesic was administered every 12 h to minimize pain.

4.3.3. Quantifying bone formation and lumbar fusion by μCT

The mice were sacrificed 42 d post-surgery, and their lumbar spines were scanned at an isotropic voxel size of 20 μm (55 kVp, 145 μA, 232 ms) using a μCT 40 (Scanco Medical AG; Wangen-Brüttisellen, Switzerland). To quantify fusion, the μCT scans were converted to DICOM image stacks and imported into RadiAnt (Medixant;

Poznań, Poland). The image stacks were visually inspected, serially, in all three planes (sagittal, coronal, and axial planes), and the number of fused vertebrae were counted. Fusion had to be seen in at least two planes to be considered. Volume renderings of the posterior lumbar spine, between the L3 and L5 vertebrae, were also generated using RadiAnt.

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Next, to quantify the amount of new bone and evaluate its quality, all of the newly formed bone was contoured using Scanco’s evaluation module (Supp. Fig. 4.1). For simplicity, any remaining ICBG was included.

The calcified tissue was segmented from the soft tissue, using a global threshold of 150 per mille of the X-ray attenuation coefficient (1/1000) and no Gaussian noise filter, and the total bone volume (BV), bone volume fraction (BV/TV), trabecular spacing (Tb.Sp.), and trabecular thickness (Tb.Th.) were evaluated using a built-in evaluation script. To compare the bone morphometric properties of the new bone to those of native host bone, a 200 μm thick region of trabecular bone, in the trabecular arch of each vertebra of interest (three vertebrae), was contoured and segmented using the same global parameters.

4.3.4 Statistical analysis

Spinal fusion, bone formation, and bone volume were assessed by Kruskal–Wallis one-way analysis of variance (ANOVA). Tb.Th. and Tb.Sp. were assessed by Welch and Brown-Forsythe ANOVA. The statistical analyses were conducted in Prism V9 (GraphPad; San Diego, California), and all of the averaged results are presented as mean ± standard deviation.