EuropeanW Wei et Cellsal. and Materials Vol. 30 2015 (pages 303-314) DOI: 10.22203/eCM.v030a21 Anti-chondrogenic effect ofISSN IPFP 1473-2262 on MSCs THE INFRAPATELLAR FAT PAD FROM DISEASED JOINTS INHIBITS CHONDROGENESIS OF MESENCHYMAL STEM CELLS W. Wei1, R. Rudjito1, N. Fahy2, J.A.N. Verhaar1, S. Clockaerts1, Y.M. Bastiaansen-Jenniskens1,§ and G.J.V.M. van Osch1,3,§,* 1Department of Orthopaedics, Erasmus MC University Medical Center, Rotterdam, The Netherlands 2Regenerative Medicine Institute, National University of Ireland, Galway, Ireland 3Department of Otorhinolaryngology, Erasmus MC University Medical Center, Rotterdam, The Netherlands §Both authors contributed equally Abstract Introduction Cartilage repair by bone marrow derived mesenchymal Articular cartilage has limited self-healing capabilities stem cells (MSCs) can be influenced by inflammation and surgical intervention using bone marrow stimulation in the knee. Next to synovium, the infrapatellar fat pad techniques, such as the microfracture procedure, can be (IPFP) has been described as a source for inflammatory used to activate mesenchymal stem cells (MSCs) from factors. Here, we investigated whether factors secreted the underlying bone marrow to repair the defect (Shapiro by the IPFP affect chondrogenesis of MSCs and whether et al., 1993; Steadman et al., 2003). However, instead of this is influenced by different joint pathologies or obesity. a normal hyaline cartilage matrix, a fibrocartilaginous Furthermore, we examined the role of IPFP resident cartilage matrix fills up the defect (Frisbie et al., 2003; macrophages. First, we made conditioned medium from Kaul et al., 2012; Shapiro et al., 1993). An explanation IPFP obtained from osteoarthritic joints, IPFP from why hyaline cartilage production is inhibited could be traumatically injured joints during anterior cruciate that the cartilage defect is located in a post-traumatic ligament reconstruction, and subcutaneous adipose inflammatory environment (Irie et al., 2003). In this tissue. Additionally, we made conditioned medium of post-traumatic inflammatory environment, circulating macrophages isolated from osteoarthritic IPFP and of inflammatory factors stimulate matrix degradation and polarised monocytes from peripheral blood. We evaluated inhibit chondrogenic differentiation (Heldens et al., 2012). the effect of different types of conditioned medium on Besides surgical intervention, research is currently MSC chondrogenesis. Conditioned medium from IPFP focused on MSC-based tissue-engineered cartilage repair decreased collagen 2 and aggrecan gene expression as strategies (Hunziker et al., 2015). In these strategies, well as thionin and collagen type 2 staining. This anti- repair tissues are engineered in-vitro using a combination chondrogenic effect was the same for conditioned medium of MSCs, scaffolds and growth factors and subsequently from IPFP of osteoarthritic and traumatically injured joints. implanted into the cartilage defect. When implanted, the Furthermore, IPFP from obese (Body Mass Index >30) MSCs in these pre-engineered constructs will be exposed to donors did not inhibit chondrogenesis more than that of the same post-traumatic inflammatory environment as the lean (Body Mass Index <25) donors. Finally, conditioned MSCs after the microfracture procedure. An inflammatory medium from macrophages isolated from IPFP decreased environment can inhibit hyaline cartilage matrix production the expression of hyaline cartilage genes, as did peripheral (Wehling et al., 2009). blood monocytes stimulated with pro-inflammatory Another factor that could influence MSC chondrogenic cytokines. The IPFP and the resident pro-inflammatory differentiation is obesity. The clinical results after the macrophages could therefore be targets for therapies to microfracture procedure are reported to be worse in patients improve MSC-based cartilage repair. with obesity (Mithoefer et al., 2005). Obesity is known to cause systemic metabolic and inflammatory changes, with Keywords: Cartilage repair, mesenchymal stem cells, increased circulating inflammatory factors (Lumeng and infrapatellar fat pad, osteoarthritis, inflammation, obesity. Saltiel, 2011). Inflammatory factors can be produced by different tissues of the knee joint, the most well described being the synovium and cartilage. The infrapatellar fat pad (IPFP) *Address for correspondence: is an adipose tissue located extra-synovially and intra- Gerjo JVM van Osch, PhD capsularly. It is richly vascularised, innervated and known Department of Orthopaedics and Otorhinolaryngology, to also secrete many adipokines and cytokines (Clockaerts Ee1655 et al., 2012; Clockaerts et al., 2010). Adipose tissue in Erasmus MC University Medical Centre obese patients has been shown to have an inflammatory Wytemaweg 80 phenotype with abundant immune cell infiltration and 3015 CN, Rotterdam, The Netherlands secretion of inflammatory cytokines (Grant and Dixit, Telephone Number: + 31-10-7043661 2015). Furthermore, joint trauma and joint degradation can FAX Number: + 31-10-7044690 both increase inflammation in the IPFP and the secretion of E-mail: [email protected] inflammatory factors (Bastiaansen-Jenniskens et al., 2012; www.ecmjournal.org 303 W Wei et al. Anti-chondrogenic effect of IPFP on MSCs Clockaerts et al., 2012; Gandhi et al., 2011; Gierman et Modified Eagle Medium with Glutamax (DMEM-HG; al., 2013). Gibco) supplemented with 1 % insulin-transferrin- The IPFP contains immune cells such as macrophages selenium (ITS+; BD Biosciences, Erembodegem, Belgium) (Bastiaansen-Jenniskens et al., 2012; Klein-Wieringa et al., for 24 h at 37 °C. Afterwards, the medium was harvested, 2011). Macrophages play an important role in inflammation centrifuged at 250 ×g for 8 min and the supernatant was and wound healing. Macrophages can be categorised into stored at −80 °C for culture experiments. Unconditioned pro-inflammatory macrophages and anti-inflammatory medium was generated by incubation of DMEM-HG or wound healing macrophages (Mosser and Edwards, supplemented with ITS+ for 24 h at 37 °C, centrifuged at 2008; Murray et al., 2014). Inflammation or obesity often 250 ×g for 8 min and stored at −80 °C. results in increased numbers of inflammatory macrophages. Previously, we have shown that only pro-inflammatory Isolation of macrophages from adipose tissue and and not anti-inflammatory macrophages inhibit MSC preparation of IPFP macrophage CM chondrogenesis in vitro (Fahy et al., 2014). To isolate macrophages from the stromal vascular Since the IPFP from end-stage OA patients is a source fraction of adipose tissue, six OA IPFP samples (age 66.6 of inflammatory and catabolic cytokines (Clockaerts et (54.1-80.2), BMI 28.9 (21.9-33.9)) were used. Adipose al., 2012), it is to be expected that IPFP secreted factors tissue samples were cut into pieces of around 9 mm2 and influence other joint tissues. Against our expectation that incubated overnight at 37 °C with 1 mg/mL collagenase the factors released from IPFP would stimulate tissue B (Roche, Penzberg, Germany) in DMEM-HG with 10 % degradation, our previous studies demonstrated that FCS. After centrifugation, the supernatant containing the factors released from IPFP from end-stage osteoarthritis floating adipocytes and fat was removed, and the cell pellet (OA) patients inhibit catabolic mediators in cartilage was re-suspended and filtered through a 100 µm filter (Bastiaansen-Jenniskens et al., 2012) and stimulate fibrotic followed by a 40 µm filter. The resulting cell suspension processes in synovial fibroblasts (Bastiaansen-Jenniskens was layered on top of 15 mL Ficoll (Ficoll-Paque™ PLUS, et al., 2013). This indicates that the factors released by GE Healthcare, Little Chalfont, UK) and separated by the IPFP not only have a pro-inflammatory effect on joint density gradient following centrifugation at 1,000 ×g for environment and but that this effect could be different in 15 min. The interphase was removed and washed with each type of joint tissue. phosphate buffered saline (PBS; Gibco) containing 2 % Considering the IPFP’s potential to produce factors foetal calf serum (FCS) followed by incubation with CD45- that influence the joint environment, and the knowledge PE (BD Biosciences) for 1 h and incubation in 20 µL of that the joint environment influences cartilage repair, anti-mouse-IgG magnetic beads (Miltenyi Biotec, Bergisch we hypothesised that factors secreted by the IPFP affect Gladbach, Germany) per 10,000,000 cells for 30 min in MSC-based cartilage repair. In this study, we investigated the dark at 4 °C. CD45 positive cells were separated by whether and how factors secreted by the IPFP affect the magnetic activated cell sorting (MACS, MACS Separation chondrogenesis of MSCs in vitro and whether this could be Columns LS and MidiMACS™ Separator, Miltenyi influenced by joint pathology or obesity. Furthermore, we Biotec) according to manufacturer’s instructions. Flow evaluated whether macrophages present in the IPFP could cytometric analysis was performed to confirm a >90 % be responsible for the effect seen on MSC chondrogenesis. CD45+ population purity. The CD45 reduced fraction contained <10 % CD45+ cells. Isolated CD45+ cells were seeded at a cell density of 100,000 cells/cm2 in 24 wells Materials and Methods plates and cultured overnight at 37 °C in DMEM-HG supplemented with 10 % FCS. Cells were then carefully Preparation of adipose tissue conditioned medium washed twice with phosphate-buffered saline (PBS) to (CM) remove non-attached