# Effect of Blood on Synovial Joint Tissues: Potential Role of Ferroptosis
## 4 In Vitro Models  
Much about blood effects on synovial joint tissues has been gleaned from tissue culture studies of 2D and 3D cultures of isolated cells or tissue explants with applied physiologic loading and modern bioengineering and biology tools. These devised systems permit a systematic approach for a myriad of experimental conditions, including blood concentration and duration of application, and provide opportunities for mechanistic studies. Isolating the contribution of synovium-dependent and independent blood effects on joint tissues that share synovial fluid, as well as identifying anti-ferrotopsis agents may help to define the role of bleeding in joint damage [68; 69]. Current research has not investigated blood injury of the cartilage, bone, meniscus, and ligament in co-culture with synovium and/or under physiologic loading conditions in culture. There also are no established models for recurrent microbleeds in 2D in vitro models. To gain mechanistic insights into the role of individual blood constituents, whole blood, RBCs and lysed RBCs (such as occurs via phagocytosis by macrophages), WBCs (buffy coat), and platelets will need to be further studied [45; 47; 48; 70]. In culture experiments, the concentration of blood stays constant, whereas, in situ, the synovial joint concentration decreases due to blood clearance. The majority of published studies of blood and joint tissues have been undertaken in a similar manner, which has also led to the development of a robust culture protocol for studying blood-induced injury of human cartilage to characterize blood-induced cell death, particularly ferrotopsis, and testing the efficacy of anti-ferrotopsis agents on preserving synovial joint health (Table 1) [71]. The contribution of ferrotopsis to the co-culture of the cartilage, bone, meniscus, and ligament with synovium and applied loading will provide new mechanistic insights as well as potential therapeutic targets to arrest hemorrhosis. Establishing in vitro model systems is necessary to capture critical aspects of the inflammatory joint environment that may drive bleeding-induced synovial arthropathy. \begin{table}
\begin{tabular}{|p{113.8 pt}|p{113.8 pt} p{113.8 pt}|} \hline
**Reference** & **Experimental Design** & **Research Findings** \\ \hline \multirow{8}{*}{[PERSON] et al., 1976 [55]} & \multirow{8}{*}{Biochemical and biomechanical analyses of mongrel dogs subject to continuous hemarthrosis [55]} & Significant decrease in GAG content observed as early as 4 weeks. Total collagen content was not significantly affected until after 12 and 16 weeks. Experimental cartilage lost significant amount of mechanical integrity as a result of continuous hemarthrosis [55]. \\ \cline{2-3}  & \multirow{8}{*}{[PERSON] et al., 1999 [52]} & Co-culture of human cartilage tissue with RBC or mononuclear cells (MNC) + RBC followed by a 12-day recovery period [52] \\ \cline{2-3}  & \multirow{8}{*}{[PERSON] et al., 2003 [9]} & Co-culture of human cartilage tissue with whole blood, mononuclear cells + RBC, or caspase inhibitors followed by a 12-day recovery period [9] \\ \cline{2-3}  & \multirow{8}{*}{[PERSON] et al., 2003 [61]} & Co-culture of human cartilage tissue with RBC, mononuclear cells + RBC, or caspase inhibitors followed by a 12-day recovery period [9] \\ \cline{2-3}  & \multirow{8}{*}{[PERSON] et al., 2003 [61]} & Co-culture of human cartilage tissue with RBC, mononuclear cells + RBC, CD14+ cells + RBC, or lysed RBC with IL-1\(\beta\) followed by a 12-day recovery period [61] \\ \cline{2-3}  & \multirow{8}{*}{[PERSON] et al., 2003 [61]} & \multirow{8}{*}{[PERSON] et al., 2003 [61]} & Lysed RBC + IL-1\(\beta\) produced by monocytes inhibited proteoglycan synthesis and increased production of hydrogen peroxide. Hemoglobin-derived iron + hydrogen peroxide resulted in formation of hydroxyl radicals that lead to further chondrocyte damage. DMSO and hydroxyl radicals can restore cartilage proteoglycan synthesis [61].