Cartilage and subchondral bone synovial tissue interactions

Cartilage and subchondral bone/synovial tissue interactions are well-recognized features of joint failure and osteoarthritis[17](seeFig. 1A, B). The intimate mechanical and biological interactions between these tissues are likely to alter the structural organization and function of either tissue. However, the mechanisms underlying these processes have not yet been fully understood. In this report, we investigated if joint c1v1 c2v2 calculator including CHs and BCs are able to establish intercellular connections and to communicate through GJ channels.
An H/E staining technique was used to study the cartilage and subchondral bone at the conversion area between both tissues in samples from healthy donors and patients with OA (Fig. 1A). The images showed structural changes and loss of normal merge between bone and cartilage in the joint of patients with OA including duplication of the tidemark (Fig. 1A). The biomechanical coupling between subchondral bone and cartilage affects cartilage and bone remodelling[22]. However, it remains unclear whether direct communication exists between cells. Vascular invasion of bone morrow tissue into subchondral bone plate is often observed in cartilage from patients and contributes to cartilage degradation.Fig. 1B exemplifies the concept of OA or RA as diseases of the whole joint that comprises pathologic cellular and structural changes in synovium, bone, ligaments and supporting musculature. OA includes cartilage degradation, osteocyte formation, subchondral sclerosis and synovial hyperplasia. CHs, SCs and BCs were isolated from tissues from joint donors (joint replacement) and cells were kept in monolayer culture. To test if CHs are able to physically interact and electrically couple to BCs and SCs, and to determine the extent of gap junction mediated coupling, we performed experiments on cell pairs to record junctional currents by dual whole-cell voltage clamp.Fig. 1C shows the examples of current recordings obtained from heterologous pair of BC and CH cells, and the homologous pair of BCs. Starting from a holding potential of 0 mV, bipolar pulses of 2 s in duration were administered to establish transjunctional voltage gradients (V) of identical amplitude and either polarity. Vwas then altered from +10 mV to +110 mV using increments of 20 mV. The associated junctional currents (I) increased proportional with Vand showed a voltage- and time- dependent deactivation. The junctional currents (I) obtained from CHs and BCs pairs (Fig. 1C) and CHs and SCs (data not shown) exhibited voltage dependent behaviour similar for GJs that exhibit a dominant pattern for channels formed by Cx43 (Fig. 1E). In heterologous cell co-culture to distinguish CHs from BCs one population of the cells were fluorescently labelled (lower panelFig. 1C). The recordings indicate that functional gap junction channels form between BCs and CHs cells (Fig. 1C). The junctional conductance data from different pairs of CHs (13.4 ± 2.1 nS, n = 11), BCs (13.7 ± 5.6 nS, n = 5) and BC-CHs (12.3 ± 2.0 nS, n = 15) are summarized inFig. 1D. Similar gap junction conductances were observed for all three groups of cell pairs investigated with no statistical difference between groups (P = 0.930). Western-blot analysis against Cx43 along with tubulin loading control shown inFig. 1E confirmed the high levels of Cx43 protein in all three types of primary cells. In order to investigate the metabolic coupling among these cells, and to investigate the exchange of amino acids, peptides and proteins, we used a previously established method represented[9]inFig. 213C) between receivers (CHs) and donors (BCs or SCs) (Fig. 2B). Controls (unlabelled BCs or SCs) were plated 1 mm below the membrane to prevent the establishment of direct cell contacts and GJ intercellular communication (GJIC) (Fig. 2A). For these assays, the LC-MS analysis did not detect the transfer of free L-arginine (13C-15N), because a limited amount of free-labelled arginine used during the labelling protocol in order to avoid arginine-to-proline conversion.