2.4.1. Inflammation and Vascularization

In order to compare cytokines and chemokines gene expression of ACL IFP to OA IFP, qRT-PCR was performed in eight samples for each group (Figures S1 and S2 Supplementary Materials).

The median age and BMI were statistically different between the two subgroups as well as in the whole population (*p* < 0.0001 and *p* = 0.010, respectively).

A statistical difference was observed between the two subgroups in *IL-6* and *VEGF* gene expression levels (*p* = 0.0039 and *p* = 0.0183, respectively) (Figure 2a–d), while no differences were observed in *MCP-1*/*CCL-2* and *TNF-*α expression (Figure S1, Supplementary Materials). A decrease in *TGF-*β gene expression levels was determined in OA IFP compared to ACL IFP (*p* = 0.0368) (Figure S1, Supplementary Materials). On the basis of these results, IL-6 and VEGF proteins were evaluated by IHC or HE in a group of patients confirming the difference detected by gene expression between ACL (*n* = 21) and OA IFP (*n* = 14) (Figure 2b,c,e,f) (Table S3, Supplementary Materials).

**Figure 2.** Evaluation of inflammation and vascularization in IFP of ACL and end-stage OA patients. *IL-6* gene expression revealed an increase of *IL-6* expression in end-stage OA IFP (*n* = 8) compared to ACL IFP (*n* = 8) (**a**). Immunohistochemistry of IL-6 end-stage OF IFP showed a marked positivity (highlighted by the arrows) (**c**) compared to ACL IFP (**b**). *VEGF* gene expression levels were increased in end-stage OA (*n* = 8) compared to ACL IFP (*n* = 8) (**d**). Hematoxylin–eosin image showed an increased in end-stage OA IFP (highlighted by the arrows) (**f**) compared to ACL IFP (**e**). Scale bar b = 50 μm; c = 23.8 μm; e,f = 37.5 μm. IL = interleukin; ACL = anterior cruciate ligament; IFP = infrapatellar fat pad; OA = osteoarthritis; VEGF = vascular endothelial grow factor.

#### 2.4.2. Adipokines

Gene expression analysis of different adipokines was performed comparing ACL and OA IFP samples (Figure 3). There was an increase of white adipose tissue typical genes (*adiponectin*, *leptin,* and *FAB4*) in end-stage OA compared to ACL IFP (*p* = 0.0167, *p* = 0.0122, and *p* = 0.0056, respectively), while no differences were observed regarding *PPAR-*γ (*p* = 0.1590). The expression of *GPMNB* was evaluated, showing a decrease in end-stage OA compared to ACL IFP (*p* = 0.0007). On the contrary, an increase of *ITH5* gene expression was shown in end-stage OA compared to ACL IFP (*p* = 0.0277). No differences were evident in *SERPIN2* gene expression between the two groups (Figure S1, Supplementary Materials).

**Figure 3.** Adipokines evaluation in IFP ACL (*n* = 8) and end-stage OA patients (*n* = 8). (**a**) *Adiponectin* gene expression levels were increased in end-stage OA compared to ACL IFP. (**b**) *Leptin* gene expression levels were increased in end-stage OA compared to ACL IFP. (**c**) *PPAR-*γ gene expression levels were increased in end-stage OA compared to ACL IFP. (**d**) *FAB4* gene expression levels were increased in end-stage OA compared to ACL IFP. (**e**)*ITH5* gene expression was higher in end-stage OA IFP. (**f**) *GPNMB* gene expression was higher in ACL IFP. IFP = infrapatellar fat pad; ACL = anterior cruciate ligament; OA = osteoarthritis; PPAR-γ = peroxisome proliferative activated receptor gamma; FABP4 = fatty acid-binding protein 4; ITH5 = inter-α-trypsin inhibitor heavy chain 5; GPNMB = transmembrane glycoprotein NMB.
