Immunologic characteristics of nerves within osteoarthritic marrow regions of human femoral heads
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Osteoarthritis (OA) is the most common degenerative joint disease, affecting primarily the hip, knee, and hand (Ip 2005). OA of the knee and hip is the 11th highest contributor to global disability (Cross et al. 2014). Hip OA is diagnosed using radiographs, computed tomography (CT), and/or magnetic resonance imaging (MRI). Hip OA affects the whole hip joint, causing pain and reduced range of motion (Altman et al. 1991). The cause of pain in OA is not well understood, but it may be attributed to abnormal growth of blood vessels and nerves in the subchondral bone of the femoral head (Kumar et al. 2013). Quantifying blood vessels and nerves and correlating their presence with diagnostic techniques such as MRI will establish a relationship between femoral head degeneration and pain levels and current diagnostic signs. The aims of this study were: (1) to develop a reproducible histological technique to identify blood vessels and nerves in bone sections; and (2) to apply this protocol to identify blood vessel and nerve characteristics within osteoarthritic femoral heads. Femoral heads were retrieved from 8 OA patients (age range: 40-76; 5 female and 3 male) undergoing total hip replacement surgery. Each sample was evaluated for the presence of subchondral bone cysts using micro-computed tomography (µCT), then regions containing cysts were isolated and dissected for histological processing. Different fixation times in paraformaldehyde (PFA) were tested in three samples to assess the effect of fixation time on binding of the primary antibody to the target. Samples were stained with hematoxylin and eosin to evaluate overall tissue morphology, safarin-O and fast green to visualize the integrity of articular cartilage, anti-CD31 to identify vascular endothelium, and anti-PGP 9.5 to identify peripheral nerve fibers. A protocol was successfully developed to identify CD31-positive blood vessels and PGP 9.5-positive nerves in osteoarthritic femoral heads. The immunohistochemistry protocols for staining with anti-CD31 and anti-PGP 9.5 were optimized for maximum intensity of target staining, minimal background staining, and minimal artefactual tissue folding. Fixation time in PFA was not found to have an effect on quality of staining with anti-CD31. Blood vessels were found in all eight of the samples collected, and peripheral nerves were found in five of the samples. Special attention was paid to regions with fibrous subchondral bone cysts, because these are the most likely type to undergo neurovascular invasion. Out of the five samples with fibrous subchondral bone cysts, blood vessels were identified in all five cyst regions, and peripheral nerves were identified in three cyst regions. Using the protocols developed in this study, blood vessels and nerves were found in osteoarthritic femoral heads. In future, a larger sample set will be used to correlate the nature of blood vessel density and nerves that are found in bone marrow lesions identified on MRI scans obtained before surgery and are associated with bone cysts. This is an important step towards identifying more effective treatments for OA that address the specific underlying causes and the development of non-treatable pain.
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