Analyzing the Effect of Blood-Induced Arthropathies on ACL Repair Capacity

Introduction: Anterior cruciate ligament (ACL) injury is typically accompanied by joint bleeding, but the individual contributions of whole blood
components in the joint are not fully understood. Hemarthrosis, or articular bleeding, has been shown to induce joint inflammation and has been implicated in the degeneration of various joint tissues, such as cartilage and synovium. Additionally, iron homeostasis is vital to joint health as excess iron content can lead to oxidative stress damage which is closely associated with osteoarthritis and hemophilic arthropathy. Previous work has shown that acute blood exposure increases pro-inflammatory cytokine production and cellular damage. Initial research has also shown that TGF𝛽-1 is typically elevated in injury and plays an important role in fibrotic scarring during the wound healing process.5 Here, we analyze the contributions of whole blood, intact red blood cells (RBCs), and lysed RBCs on the ACL in the context of biochemical content, oxidative stress, and various cell death pathways including ferroptosis, a specific type of cell death characterized by iron dependency and oxidative stress. We employed the use of a 3D collagen gel contraction assay as a measure of wound healing capacity. Using the contraction assay, this study will assess the impact of acute short-term blood exposure on the contractile phenotype of ACL fibroblasts (ACLF) as a measure of cell function and viability. We hypothesized that human ACLF seeded collagen constructs exposed to lysed RBCs and by extension, iron, will exhibit lower rates of contraction and increased forms of cell death and inflammation.

Materials and

Methods: Whole human blood was separated via the Ficoll-Paque separation technique. Healthy human ACL was obtained from the Musculoskeletal Transplant Foundation. ACL fibroblasts were isolated via enzymatic digestion and cultured with various blood concentrations for 4 days. Previous dose response experiments have shown that 20% v/v is the minimum blood concentration to induce significant cell toxicity. Therefore, isolated intact RBC, lysed blood, and whole blood were added to CM at 20% v/v. To study collagen contraction, human ACLF were cultured at 3520 cells/cm2 until confluency in 10% DMEM supplemented with 5 ng/ml FGF-2. To cast collagen gel, 5X DMEM, rat tail type I collagen, 1N NaOH, and cell suspension were combined until homogeneously mixed. After solid gels formed at 37°C for 60 minutes in 24 well plates, 2 mL of experimental blood treatment group was added to each well. Wells were treated with CM (control), CM + TGF𝛽1 (1 ng/ml), and corresponding blood experimental groups (20% v/v). Images were collected at day 0, 1, 2, 3, 4 following gel preparation. Using FIJI, percent contraction was measured over time. At day 4, collagen constructs were assessed for biochemical content (DNA and collagen). ACL engineered constructs were prepared using 35 mm agarose-coated plates with pinned brushite anchors. After polymerization of master mix containing cells, fibrinogen, and thrombin, tensile testing was performed until failure. Biochemical content and gene expression were analyzed with ANOVA on GraphPad Prism 9 with a Tukey post-hoc for multiple comparisons, and significance was determined at p < 0.05.

Results, Conclusions, and Discussions: In the current study using human cells, we partially accept our hypothesis, with ACLF seeded collagen constructs exposed to lysed RBCs exhibiting more cell death and inflammation while not exhibiting lower rates of contraction compared to intact RBCs. Greater cell death in the lysed blood treatment group may indicate that blood lysing significantly induces ACLF cytotoxicity and potentially lowers their rate of cellular proliferation. After 4 days, the whole blood showed the least amount of contraction which could be due to a significant increase in fibrosis and DNA content, and may suggest that blood exposure may lead to induction of ferroptosis and inhibited contractility which can delay wound healing. Collagen production was significantly increased in the whole blood treatment group in comparison to all treatment groups. This potentially means that other blood constituents in whole blood aside from RBCs induce significant fibrosis within the wound healing response. The lysed blood treatment group did not exhibit as much collagen production which could reflect the lack of secretory factors by RBCs and other blood constituents. Blood treatment did significantly induce inflammation through upregulation of IL6. Furthermore, upregulation of COL1, COL2, and COL3 suggest presence of RBCs and blood constituents increases general collagen production, indicative of fibrosis. Additionally, these experiments highlight that different blood constituents have varied effects on cell function and viability. In addition to ferroptosis, various other forms of cell death could be at play that are elicited by different blood components.

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