## 3 Results
### PTFH Group Displayed a Higher Expression of Fibrotic Markers than NTFH Specimens  
The control group showed low protein expression for type I collagen in cartilage, whereas we noticed a high percentage of this marker in the underlying subchondral bone (Figure 2a,b). Fibrosis reactions in terms of the presence of type I collagen were more robust in the cartilage of the PTFH group compared to the NTFH group (Figure 2a,b). This latter group showed higher type I collagen expression compared to the control (\(p<0.05\)) and NTFH groups (\(p<0.05\)). As for autotaxin, the two ON groups did not show any difference; however, the PTFH group reported a higher amount of this marker compared to the control group (\(p<0.05\)). The NTFH group showed mild protein expression for both collagen type I and autotaxin, especially in the articular cartilage. Figure 1: (a) Safranin-O/Fast Green staining of cartilage and subchondral bone from healthy controls, and two groups of patients with non-traumatic femoral head (NTFH) ON (\(n=5\)), and post-traumatic femoral head (PTFH) ON (\(n=6\)). Red: proteoglycans; green: collagen. Scale bar: 50 \(\upmu\)m. IG isogenic groups: CL cell clusters; L: empty lacunae; FIS fibrillation processes; HC hypertrophic chondrocytes; T tears; Bo bone tissue; BM bone marrow; AT adipose tissue; BV blood vessels; FIB fibrous tissue; OC osteoclasts; E erythrocytes. (b) Graphical representation of ICRS and Ficat scores of the osteochondral unit of control, NTFH and PTFH groups. Data are reported in a scatter plot graph with mean \(\pm\) standard deviation (SD). \({}^{*}\)\(p<0.05\): NTFH versus PTFH; \({}^{***}\)\(p<0.001\): PTFH versus the control group. We did not find any difference for caspase-3 between the NTFH and PTFH groups. Both specimens displayed higher cell positivity for this marker when compared to the control group (Figure 3a,b). Regarding CB-2, a molecule involved in bone remodelling and pain responses, the control group displayed moderate expression, especially near bone marrow spaces, and at a lesser extent in the osteocytes. The NTFH group revealed higher protein expression for CB-2 in chondrocytes than in osteocytes and bone marrow precursors within the subchondral bone. The PTFH group displayed a similar behaviour (Figure 3a,b). To assess the breakdown of the extracellular matrix, we analysed ADAMTS-5 and MMP-13 as catabolic markers. All cartilage and bone specimens showed low levels of ADAMTS-5, with no difference between the two ON groups (Figure 4a,b). As for MMP-13, the PTFH group displayed a higher cell positivity in the middle and deep layers of articular cartilage when compared to the control and NTFH groups (\(p<0.05\)) (Figure 4a,b). Figure 2: (**a**) Immunostaining for collagen type I and autotaxin of control, NTFH and PTFH groups. Scale bar: 50 \(\upmu\)m. Black arrows: positive areas for markers. (**b**) Graphical representation of quantitative measurements for collagen type I and autotaxin in control, NTFH and PTFH groups. Data are reported in a scatter plot graph with mean \(\pm\) SD. Collagen type I: * \(p<0.05\): Control versus PTFH group; * \(p<0.05\) NTFH versus PTNH group. Autotaxin: * \(p<0.05\): Control versus PTFH group. Figure 4: _Cont._  
Figure 3: (**a**) Immunostaining for caspase-3 and CB-2 of the control, NTFH and PTFH groups. Scale bar = 50 \(\upmu\)m. Black arrows show positive cells. (**b**) Graphical representations of the percentage of positivity for caspase 3 and CB-2. Data are expressed in a scatter plot graph with mean \(\pm\) standard deviation (SD). Caspase 3: * \(p<0.05\): Control versus NTFH group; * \(p<0.05\): NTFH versus PTFH group. We analysed NGF and the substance P (SP) nociceptive fibre pattern to test the pain response. Both NTFH and PTFH groups showed higher immunostaining for NGF in the cartilage rather than in the underlying subchondral bone (\(p<0.05\)) (Figure 5a,b). Both ON groups displayed an increased SP positivity near cell clones of the articular cartilage and hypercellular and fibrotic areas of the subchondral bone (Figure 5a,b).