*2.2. Micro-CT Imaging Confirmed Decreased Cartilage and Bone Destruction in Arthritic Nkx2-3 Knock-Out Mice*

Next, we wanted to visualize the radiological changes induced by GIA in Nkx2-3−/<sup>−</sup> and control BALB/c mice. RA causes not only cartilage destruction but the adjacent bone is always affected as a result of the inflammation-induced osteoclast activity. Typical changes associated with RA are bone loss, osteophyte formation and, in the latter stages of the disease, ankylosis. So, we performed a micro-CT analysis of arthritic Nkx2-3−/<sup>−</sup> and control BALB/c mice. As shown by the pseudocolor enhanced micro-CT images wild-type mice demonstrated marked osteophyte formation and bone surface irregularity predominantly in the tarsal and metatarsal region (Figure 2A,C,E). In comparison, Nkx2-3−/<sup>−</sup> animals showed less severe detrimental bone structural damage secondary to the autoimmune arthritis manifested by a more limited surface porosity and periarticular inflammatory osteoporosis (Figure 2A,C,E).

We also analyzed the micro-CT scans for some quantitative markers of the bone microarchitecture, e.g., number (Po.N), volume (Po.V) and surface of bone pores (Po.S), and the bone surface/Total Volume (BS/TV) values. In arthritic Nkx2-3 KO mice, the slightly decreased Po.N together with the increased Po.V and Po.S values could be due to decreased reactive bone formation, whereas the slightly decreased BS/TV value might indicate less osteophyte formation, all of which correlate with the milder arthritis (Figure A1).

**Figure 1.** The comparison of the clinical parameters of recombinant human G1 (rhG1)-induced arthritis (GIA) in Nkx2-3−/<sup>−</sup> and control BALB/c mice. Female Nkx2-3−/<sup>−</sup> (*n* = 40) and control BALB/c (*n* = 27) mice were immunized with rhG1 and dimethyl-dioctadecyl-ammonium (DDA) adjuvant intraperitoneally three times every third week. The severity score (**A**) and incidence (**B**) of the induced arthritis is shown on the diagrams. Black arrows show the time of the third immunization (Day 42). Severity of the disease was determined every second day with the help of a scoring system ranging from 1 to 4, based on the swelling, redness and ankylosis of the joints of the paws. Clinical scores are visualized as mean ± standard error of mean (SEM). The thickness of the limbs (**C**) were measured with a digital caliper two weeks after the third immunization. The diagrams show the thickness values of the wrist (**C**/**a**), legs (**C**/**b**) and ankles (**C**/**c**) as mean ± SEM. Statistically significant differences (∗ *p* < 0.05) are indicated.
