Mice
All animal experiments were performed under IACUC protocols approved by the Thomas Jefferson University. Mice heterozygous for the progressive ankylosis allele (ank) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA; C3FeB6 A/Aw−J-Ankank/J, stock number 000200). Obtained Hets were used to breed Ankank/ank, heterozygous and WT littermates. 4-, 13- and 20-week-old mice were used for µCT studies whereas all remaining analyses were carried out using 13-week-old mice.
Histological Analysis
Dissected spines were fixed in 4% PFA in PBS for 48 hours, decalcified in 20% EDTA and embedded in paraffin. Spines used for calcified sections were fixed similarly, treated with 30% sucrose, OCT embedded and snap-frozen. 7 µm mid-coronal sections were cut from decalcified L3-S1 levels and 10 µm mid-coronal sections were cut from calcified cervical to L1/2 levels. Safranin-O/Fast Green/hematoxylin staining was performed for assessing histology or Picrosirius Red staining was done to visualize the collagen content. Alizarin Red staining was used to detect calcium. Staining was visualized using a Axio Imager 2 microscope (Carl Zeiss, Jena, Germany) using 5×/0.15 N-Achroplan or 20×/0.5 EC Plan-Neofluar objectives (Carl Zeiss) or a polarizing microscope (Eclipse LV100 POL, Nikon, Tokyo, Japan) using 10×/0.25 Pol/WD 7.0 objective, Digital Sight DS-Fi2 camera, and NIS Elements Viewer software. To evaluate degeneration, mid-coronal sections from L3-S1 levels per mouse were scored using a modified Thompson Grading scale and a scoring system described by Tessier et al.42 by at least 3 blinded observers17,43. For Picrosirius Red staining, under polarized light, stained collagen bundles appear either green, yellow, or red pixels that correlate to fiber thickness: green (thin), yellow (intermediate), and red (thick). Color threshold levels remained constant.
Immunohistochemistry and analysis
De-paraffinized sections following antigen retrieval were blocked in 5–10% normal serum in PBS-T and incubated with antibodies against COL I (1:100, Millipore, Burlington, MA, USA, ABT123), COL II (1:400, Fitzgerald, North Acton, MA, USA 70R-CR008), COMP (1:200, Abcam ab231977), COL X (1:500, Abcam, Cambridge, UK, ab58632), ACAN (1:50, Millipore AB1031); CS (1:300, Abcam ab11570); CA3 (1:150, Santa Cruz Biotechnology, Dallas, TX, USA), MMP13 (1:200, Abcam ab39012), Ki67 (1:100, Abcam ab15580), OCN (1:200, Abcam ab93876), CD31 (1:400, Abcam ab124432), CD68 (1:500, Abcam ab125212), and IHH (1:120, Abcam ab39634). For CD14 (1:1000, Abcam ab182032), CTPK (1:2000, Abcam, ab37259), and ARGxx (1:200, Abcam, ab3773) staining, MOM kit (Vector laboratories, Newark, CA, USA, BMK-2202) was used for blocking and primary antibody incubation. Tissue sections were washed and incubated with Alexa Fluor-594-conjugated secondary antibody (Jackson ImmunoResearch Lab, Inc., West Grove, PA, USA, 1:500–700). The sections were mounted with ProLong® Gold or Diamond Antifade Mountant with DAPI (Fisher Scientific, Waltham, MA USA, P36934; P36966) and visualized and acquired the images with Axio Imager 2 microscope using 5×/0.15 N-Achroplan or 20×/0.5 EC Plan-Neofluar objectives (Carl Zeiss). Percent positive staining area and cell number quantification was performed using the ImageJ/FIJI software. Images were set the thresholds to subtract the background, transformed into binary, and then staining area and cell number were calculated using the analyze particles function.
TUNEL assay
TUNEL staining was performed using “In situ cell death detection” Kit (Roche Diagnostic, Zug, Switzerland). Briefly, sections were de-paraffinized and permeabilized using Proteinase K (20 µg/mL) for 15 min in room temperature and TUNEL assay was carried out per the manufacturer's protocol. Sections were washed and mounted with ProLong® Gold or Diamond Antifade Mountant with DAPI and visualized with Axio Imager 2 microscope.
TNAP and TRAP staining
Calcified sections were prepared and stained according to the previous report44. Briefly, specimens attaching to the cryotape were glued on the glass slides with chitosan adhesive solution, and dried overnight in 4℃. As for TNAP staining, slides were incubated for 10 minutes in 100mM Tris-HCl buffer (AP Buffer) (pH 8-8.5) mixed with Vector Blue (Vector laboratories, SK-5300) at room temperature following the instruction of the manufacturer. Subsequently, the slides were rinsed in 1X PBS for 5 minutes 3 times and mounted with DAPI and visualized under Cy5 channel. For TRAP staining, ELF97 (ThermoFisher Scientific, E6588) was diluted 1:75 in TRAP buffer (9.2 g of sodium acetate anhydrous and 11.4 g of sodium tartrate dibasic dihydrate dissolved in water; total volume 1000ml; pH4.2). TRAP buffer was applied to the slides for 15 minutes in room temperature. Subsequently ELF97 dissolved TRAP buffer was applied to the slides for minutes in room temperature. The slides were rinsed in 1X PBS for 5 minutes 3 times and mounted with DAPI and visualized under GFP channel.
Tissue RNA isolation
The AF and NP tissue were separately dissected from caudal discs of 13-week-old WT and Ank mutants (each n = 5) under microscope (Zeiss, Stemi 503) and pooled the tissue from single animal to serve as an individual sample. The tissue was immediately placed in RNAlater® Reagent (Invitrogen, Carlsbad, CA, USA). NP and AF tissues were homogenized with a Pellet Pestle Motor (Sigma Aldrich, St. Louis, MO, USA, Z359971), and RNA was extracted from the lysates using an RNeasy® Mini kit (Qiagen, Venlo, Netherlands).
RNA microarray analysis and gene set enrichment analysis
Total RNA with RIN around 5 was used for the analysis. Fragmented biotin-labelled cDNA was synthesized using the GeneChip WT Plus kit (Thermo Fisher). Gene chips (Mouse Clariom S) were hybridized with biotin-labelled cDNA in 100µL of hybridization cocktail. Arrays were
washed and stained with GeneChip hybridization wash and stain kit and scanned on an Affymetrix Gene Chip Scanner 3000 7G, using Command Console Software. CHP files were
generated by sst-rma normalization from Affymetrix CEL file using the Transcriptome Analysis Console (TAC) v4.0.2 (Affymetrix, Santa Clara, CA, USA). Only protein-coding genes were included in the analyses. The experimental group was compared to the control group in the TAC, including all probe sets where at least 50% of the samples had a DABG (detected above background) p < 0.05. Inclusion cutoffs were set to a 1.75-fold change and p-value < 0.05. Significantly differentially up- and down-regulated genes from the AF and NP compartments were analyzed using Enrichr25–27, GO database annotation, statistical test with p-value < 0.05. Analyses and visualizations were done in Affymetrix Transcriptome array console 4.0 software.
Micro-CT analysis
Micro-CT scanning (Bruker, Billerica, MA, USA, SkyScan 1275) was performed on fixed spines using parameters: 50 kV (voltage), 200 µA (current) at 15 µm resolution. Occupation ratios of the disc mineralization were compared between WT and mutant discs. Disc height and vertebral height were measured at three different points equidistant from the center of the bone on the sagittal plane and disc height index (DHI) was calculated. The three-dimensional data sets were assessed for cortical bone analysis, and two-dimensional assessments computed cortical cross-sectional thickness (Cort. Cs. Th), cortical bone area (Cort. B. Ar), cortical bone volume (Cort. BV), and cortical bone mass density (Cort. BMD). For trabecular bone analysis, trabecular number (Tb. N.), thickness (Tb. Th), trabecular separation (Tb. Sp), trabecular BV/TV (Tb. BV/TV), and trabecular bone mass density (Tb. BMD) were computed. For bony EP analysis, EP bone volume (EP BV), EP BV/TV, EP total porosity, and EP BMD were computed.
FTIR imaging spectroscopy
Spectral imaging data in the mid-IR region, 4,000–800 cm− 1 at 8 cm− 1 spectral resolution and 25 µm spatial resolution was acquired from 10-µm-thick calcified T10/11 and T12/L1 disc sections from 13-week-old WT and Ank mutant mice (n = 3/group) using Spotlight 400 FT-IR
Imaging system (Perkin Elmer, Waltham, MA, USA). The spectral images were analyzed using ISys Chemical Imaging Analysis software (v. 5.0.0.14; Malvern Panalytical Ltd, Malvern, UK). Briefly, spectral subtraction was initially carried out to remove bands from the cryotape used for sample preparation, the spectra were smoothed (9-pt) and baseline-corrected, then single-wavenumber images reflecting the distribution of specific tissue components were acquired at 1660 cm− 1 (protein, collagen), 960 cm− 1 (apatite mineral, phosphate), and 870 cm− 1 (apatite mineral, carbonate). Overlays of mineral and protein images were carried out using Fiji (ImageJ 2.1.0/1.53c) as previously described45. For quantification of mineral content, the mineralized regions were masked using the ISys, and the 2nd derivative of the spectra were obtained as a mathematically objective approach to assess peak intensity46. The intensity ratio of the phosphate-to-amide I peak (960/1660 ratio) was calculated to inform on the relative amount of mineral-to-protein present in the tissues. The main phosphate peak at 1030 cm-1 was not used due to the influence of the cryotape at that spectral region. To assess the variation in mineral content across the MIA-MOA interface, the Image Topology tool on the ISys was used in regions of interest on phosphate-to-amide I 2nd derivative peak ratio images.
Statistical analysis
Statistical analysis was performed depending on the results of D’Agostino and Pearson test and Shapiro-Wilk test to check the normality of data distribution using Prism 9.2.0 (GraphPad, La Jolla, CA, USA) with data presented as mean ± standard deviation (SD). The differences between two groups were analyzed by t test or Mann-Whitney test. The differences between three groups were analyzed by ANOVA or Kruskal-Wallis test followed by Dunnett’s T3 multiple comparisons test or Dunn's multiple comparison test. p ≤ 0.05 was considered a statistically significant difference.
Data availability
RNA microarray data are deposited in the GEO database (GSE206997).