*Article* **Infrapatellar Fat Pad Gene Expression and Protein Production in Patients with and without Osteoarthritis**

**Elisa Belluzzi 1,**†**, Veronica Macchi 2,3,**†**, Chiara Giulia Fontanella 4,5, Emanuele Luigi Carniel 5,6, Eleonora Olivotto 7, Giuseppe Filardo 8, Gloria Sarasin 2, Andrea Porzionato 2,3, Marnie Granzotto 9, Assunta Pozzuoli 1, Antonio Berizzi 10, Manuela Scioni 11, Ra**ff**aele De Caro 2,3, Pietro Ruggieri 10, Roberto Vettor 9, Roberta Ramonda 12, Marco Rossato 9,\* and Marta Favero 12,13**


Received: 24 July 2020; Accepted: 19 August 2020; Published: 21 August 2020

**Abstract:** Osteoarthritis (OA) is one of the most common joint disorders. Evidence suggests that the infrapatellar fat pad (IFP) is directly involved in OA pathology. However, a comparison between OA versus non-OA IFP is still missing. Thus, the aim of this study was to compare IFP molecular, adipocytes and extracellular matrix characteristics of patients affected by OA, and patients undergoing anterior cruciate ligament (ACL) reconstruction. We hypothesized that not only inflammation but also changes in adipocytes and extracellular matrix (ECM) composition might be involved in OA pathogenesis. Fifty-three patients were enrolled. IFP biopsies were obtained, evaluating: (a) lymphocytic infiltration and vascularization; (b) adipocytes area and number; (c) adipo-cytokines and extracellular matrix gene expression levels; (d) IL-6 and VEGF protein production; (e) collagen fibers distribution. OA IFP was more inflamed and vascularized compared to ACL IFP. OA IFP adipocytes were larger and numerically lower (1.3-fold) than ACL IFP adipocytes. An increase of gene expression of typical white adipose tissue genes was observed in OA compared to ACL

IFP. Collagen-types distribution was different in the OA IFP group compared to controls, possibly explaining the change of the biomechanical characteristics found in OA IFP. Statistical linear models revealed that the adipocyte area correlated with BMI in the OA group. In conclusion, inflammation and fibrotic changes of OA IFP could represent novel therapeutic targets to counteract OA.

**Keywords:** adipocyte; infrapatellar fat pad; osteoarthritis; anterior cruciate ligament; inflammation

#### **1. Introduction**

Osteoarthritis (OA) is one of the most frequent forms of arthritis and an important cause of pain and disability in elderly people [1]. The most affected joint is the knee, with a worldwide estimated radiographic prevalence of 3.8% [2].

Nowadays, OA is considered a whole joint disease involving cartilage, meniscus, synovial membrane, and infrapatellar fat pad (IFP) [3–6]. It is well-known that OA is characterized by synovial inflammation, determined by synoviocytes, which secrete pro-inflammatory cytokines that induce chondrocytes to produce degradative enzymes of the extracellular matrix (ECM) and inhibit both tissue repair and regeneration [7]. Actually, there is no cure for this pathology and the OA management relies on symptomatic interventions. Total joint replacement represents the only treatment available for end-stage OA. However, physical activity and nutrition supplements are considered as nonpharmacological and preventive treatment for OA and sarcopenia [7–9].

Recently, attention has focused on the role of the IFP in OA pathophysiology [3,10,11]. It has been shown that IFP produces pro-inflammatory mediators inducing synovial inflammation and seems to act as an anatomo-functional unit with synovial membrane contributing to OA onset and progression [3,10–14].

Moreover, during the last years, research has focused on the study of IFP-derived stem cells for regenerative medicine [15]. Recently, we showed that OA-IFP stem cells seem to be primed by the pathological environment and to exert incomplete protective activity from OA inflammation [16].

A decrease of IFP volume and an increase of hypointense signal at the magnetic resonance imaging (hallmarks of fibrosis) were described in OA patients compared to controls [17]. It has been reported that IFP signal intensity alterations were associated with the incidence of radiographic OA [18] and that IFP hypointense signals were associated with increased knee cartilage defects and bone marrow lesions [19]. Moreover, IFP undergoes biomechanical changes in OA, showing a nonorganized distribution of the stresses within the interlobular septa affecting the mechanical (and possibly functional) response of the adipose lobules [20]. Usually, gene expression of OA IFP is compared with that of subcutaneous adipose tissue of the same subject, although these depots are very different. In this regard, the comparison of different IFP conditions could allow us to better understand and quantify the specific disease-related changes of the IFP.

The aim of the present study was to compare the histological, morphometric, and molecular characteristics of IFP of patients undergoing total knee replacement (TKR) for end-stage OA with those of patients undergoing anterior cruciate ligament reconstruction (ACLR) after traumatic rupture. We hypothesized that not only inflammation but also changes in adipocytes and extracellular matrix (ECM) composition might be involved in OA pathogenesis.

### **2. Results**

#### *2.1. Demographic and Clinical Characteristics of Patients*

Twenty-eight patients undergoing ACLR for traumatic rupture and twenty-five patients undergoing TKR for end-stage OA were enrolled. Patients' characteristics are summarized in Table S2, Supplementary Materials. The time between the injury and the surgery of patients with ACLR was at least 6 months (median 8 months; interquartile range (IQR), 14.5–6). Males were 75% in the ACL group and 28% in the OA group (*p* = 0.001). Moreover, ACL subjects were statistically younger (median age 31; IQR, 42–22) compared to OA patients (median age 68; IQR, 75–62) (*p* < 0.0001). The BMI of the ACL group (median BMI 23.04; IQR, 25.26–20.57) was statistically lower than that of OA patients (median 29.52; IQR, 32.25–25.95) (*p* < 0.0001).
