Immune cell profiling in the joint following human and murine articular fracture.

OBJECTIVE: Human and in vivo animal research implicates inflammation following articular fracture as contributing to post-traumatic arthritis. However, relevant immune cell subsets present following injury are currently undefined. Immunophenotyping human and murine synovial fluid may help to identify immune cell populations that play key roles in the response to articular fracture. METHODS: Immunophenotyping by polychromatic flow cytometry was performed on human and mouse synovial fluid following articular fracture. Specimens were collected in patients with closed ankle fracture at the time of surgical fixation and from C57BL/6 mice with closed articular knee fracture. Immune cells were collected from injured and uninjured joints in mice via a novel cell isolation method. Whole blood samples were also collected. Immunohistochemistry (IHC) was performed on mouse synovial tissue to assess for macrophages and T cells. RESULTS: Following intra-articular fracture, the prominent human synovial fluid immune cell subset was CD3+ T cells, containing both CD4+ and CD8+ T cells. In mice, infiltration of CD45+ immune cells in synovial fluid of the fractured limb was dominated by CD19+ B cells and CD3+ T cells at 7 days after intra-articular fracture. We also detected adaptive immune cells, including macrophages, NK cells, dendritic cells and monocytes. Macrophage and T cell findings were supported by IHC of murine synovial tissue. CONCLUSIONS: Determining specific cell populations that mediate the immune response is essential to elucidating the chain of events initiated after injury and may be an important step in identifying potential immune signatures predictive of PTA susceptibility or potential therapeutic targets.

Intra-articular depletion of macrophages increases acute synovitis and alters macrophage polarity in the injured mouse knee.

OBJECTIVE: Acute synovial inflammation following joint trauma is associated with posttraumatic arthritis. Synovial macrophages have been implicated in degenerative changes. In this study, we sought to elucidate the role of intra-articular macrophages in the acute inflammatory response to fracture in the mouse knee. METHOD: A closed articular fracture was induced in two models of synovial macrophage depletion: genetically-modified MaFIA mice administered AP20187 to induce programmed macrophage apoptosis, and wild-type C57BL/6 mice administered clodronate liposomes, both via intra-articular injection. Synovial inflammation, bone morphology, and levels of F4/80+ macrophages, NOS2+ M1 macrophages, and CD206+ M2 macrophages were quantified 7 days after fracture using histology and micro-computed tomography. RESULTS: Intra-articular macrophage depletion with joint injury did not reduce acute synovitis or the number of synovial macrophages 7 days after fracture in either macrophage-depleted MaFIA mice or in clodronate-treated C57BL/6 mice. In macrophage-depleted MaFIA mice, macrophage polarity shifted to a dominance of M1 macrophages and a reduction of M2 macrophages in the synovial stroma, indicating a shift in M1/M2 macrophage ratio in the joint following injury. Interestingly, MaFIA mice depleted 2 days prior to fracture demonstrated increased synovitis (P = 0.003), reduced bone mineral density (P = 0.0004), higher levels of M1 macrophages (P = 0.013), and lower levels of M2 macrophages (not statistically significant, P=0.084) compared to control-treated MaFIA mice. CONCLUSION: Our findings indicate that macrophages play a critical immunomodulatory role in the acute inflammatory response surrounding joint injury and suggest that inhibition of macrophage function can have prominent effects on joint inflammation and bone homeostasis after joint trauma.

Sustained intra-articular delivery of IL-1RA from a thermally-responsive elastin-like polypeptide as a therapy for post-traumatic arthritis.

Post-traumatic arthritis (PTA) is a rapidly progressive form of arthritis that develops due to joint injury, including articular fracture. Current treatments are limited to surgical restoration and stabilization of the joint; however, evidence suggests that PTA progression is mediated by the upregulation of pro-inflammatory cytokines, such as interleukin-1 (IL-1) or tumor necrosis factor-α (TNF-α). Although these cytokines provide potential therapeutic targets for PTA, intra-articular injections of anti-cytokine therapies have proven difficult due to rapid clearance from the joint space. In this study, we examined the ability of a cross-linked elastin-like polypeptide (xELP) drug depot to provide sustained intra-articular delivery of IL-1 and TNF-α inhibitors as a beneficial therapy. Mice sustained a closed intra-articular tibial plateau fracture; treatment groups received a single intra-articular injection of drug encapsulated in xELP. Arthritic changes were assessed 4 and 8 weeks after fracture. Inhibition of IL-1 significantly reduced the severity of cartilage degeneration and synovitis. Inhibition of TNF-α alone or with IL-1 led to deleterious effects in bone morphology, articular cartilage degeneration, and synovitis. These findings suggest that IL-1 plays a critical role in the pathogenesis of PTA following articular fracture, and sustained intra-articular cytokine inhibition may provide a therapeutic approach for reducing or preventing joint degeneration following trauma.

Acute joint pathology and synovial inflammation is associated with increased intra-articular fracture severity in the mouse knee.

OBJECTIVE: Post-traumatic arthritis is a frequent cause of disability and occurs most commonly and predictably after articular fracture. The objective of this investigation was to examine the effect of fracture severity on acute joint pathology in a novel murine model of intra-articular fracture. DESIGN: Low and high energy articular fractures (n=25 per group) of the tibial plateau were created in adult male C57BL/6 mice. The acute effect of articular fracture severity on synovial inflammation, bone morphology, liberated fracture area, cartilage pathology, chondrocyte viability, and systemic cytokines and biomarkers levels was assessed at 0, 1, 3, 5, and 7 days post-fracture. RESULTS: Increasing intra-articular fracture severity was associated with greater acute pathology in the synovium and bone compared to control limbs, including increased global synovitis and reduced periarticular bone density and thickness. Applied fracture energy was significantly correlated with degree of liberated cortical bone surface area, indicating greater comminution. Serum concentrations of hyaluronic acid (HA) were significantly increased 1 day post-fracture. While articular fracture significantly reduced chondrocyte viability, there was no relationship between fracture severity and chondrocyte viability, cartilage degeneration, or systemic levels of cytokines and biomarkers. CONCLUSIONS: This study demonstrates that articular fracture is associated with a loss of chondrocyte viability and increased levels of systemic biomarkers, and that increased intra-articular fracture severity is associated with increased acute joint pathology in a variety of joint tissues, including synovial inflammation, cortical comminution, and bone morphology. Further characterization of the early events following articular fracture could aid in the treatment of post-traumatic arthritis.

Efficacy of commercial mosquito traps in capturing phlebotomine sand flies (Diptera: Psychodidae) in Egypt.

Four types of commercial mosquito control traps, the Mosquito Magnet Pro (MMP), the Sentinel 360 (S360), the BG-Sentinel (BGS), and the Mega-Catch Ultra (MCU), were compared with a standard Centers for Disease Control and Prevention (CDC) light trap for efficacy in collecting phlebotomine sand flies (Diptera: Psychodidae) in a small farming village in the Nile River Valley 10 km north of Aswan, Egypt. Each trap was baited with either carbon dioxide (CO2) from combustion of butane gas (MMP), dry ice (CDC and BGS traps), light (MCU and S360), or dry ice and light (CDC). Traps were rotated through five sites in a5 x 5 Latin square design, repeated four times during the height of the sand fly season (June, August, and September 2007) at a site where 94% of sand flies in past collections were Phlebotomus papatasi (Scopoli). A total of 6,440 sand flies was collected, of which 6,037 (93.7%) were P. papatasi. Of the CO2-baited traps, the BGS trap collected twice as many P. papatasi as the MMP and CDC light traps, and at least three times more P. papatasi than the light-only MCU and S360 traps (P < 0.05). Mean numbers (+/- SE) of P. papatasi captured per trap night were as follows: BGS 142.1 (+/- 45.8) > MMP 56.8 (+/- 9.0) > CDC 52.3 (+/- 6.1) > MCU 38.2 (+/- 6.4) > S360 12.6 (+/- 1.8). Results indicate that several types of commercial traps are suitable substitutes for the CDC light trap in sand fly surveillance programs.

Novel synovial fluid recovery method allows for quantification of a marker of arthritis in mice.

OBJECTIVE: We evaluated three methodologies--a calcium sodium alginate compound (CSAC), polyacrylate beads (PABs), and Whatman paper recovery (WPR)--for the ability to recover synovial fluid (SF) from mouse knees in a manner that facilitated biochemical marker analysis. METHODS: Pilot testing of each of these recovery vehicles was conducted using small volumes of waste human SF. CSAC emerged as the method of choice, and was used to recover and quantify SF from the knees of C57BL/6 mice (n=12), six of which were given left knee articular fractures. SF concentrations of cartilage oligomeric matrix protein (COMP) were measured by enzyme-linked immunosorbent assay. RESULTS: The mean concentration ratio [(COMP(left knee))/(COMP(right knee))] was higher in the mice subjected to articular fracture when compared to the non-fracture mice (P=0.026). The mean total COMP ratio (taking into account the quantitative recovery of SF) best discriminated between fracture and non-fracture knees (P=0.004). CONCLUSIONS: Our results provide the first direct evidence of accelerated joint tissue turnover in a mouse model responding to acute joint injury. These data strongly suggest that mouse SF recovery is feasible and that biomarker analysis of collected SF samples can augment traditional histological analyses in mouse models of arthritis.

Viability and apoptosis of human chondrocytes in osteochondral fragments following joint trauma.

Post-traumatic arthritis is a frequent consequence of articular fracture. The mechanisms leading to its development after such injuries have not been clearly delineated. A potential contributing factor is decreased viability of the articular chondrocytes. The object of this study was to characterise the regional variation in the viability of chondrocytes following joint trauma. A total of 29 osteochondral fragments from traumatic injuries to joints that could not be used in articular reconstruction were analysed for cell viability using the fluorescence live/dead assay and for apoptosis employing the TUNEL assay, and compared with cadaver control fragments. Chondrocyte death and apoptosis were significantly greater along the edge of the fracture and in the superficial zone of the osteochondral fragments. The middle and deep zones demonstrated significantly higher viability of the chondrocytes. These findings indicate the presence of both necrotic and apoptotic chondrocytes after joint injury and may provide further insight into the role of chondrocyte death in post-traumatic arthritis.

Assessment of backside wear from the analysis of 55 retrieved tibial inserts.

Fifty-five retrieved tibial inserts with four different locking mechanisms were evaluated for evidence for polyethylene wear between the inferior surface of the tibial insert and metal tray. This type of wear will be referred to as backside wear. Backside wear was assessed by evaluating manufacturer's stamped markings on the inferior polyethylene surface. Because these markings are embossed into the polyethylene surface, they were used as indicators of backside wear. Decreases in the depths of markings indicated that backside wear was clearly evident, regardless of design, in 24 (44%) of the inserts. In eight of these 24 inserts, the manufacturer's stamped markings were removed completely. The amount of polyethylene wear was as a high as 591 mg from the inferior surface. This corresponds to a polyethylene wear rate from the backside of the tibial insert of greater than 100 mg per year, which is two to four times higher than wear rates associated with total hip replacements. The current work provides direct evidence of backside wear in all four tibial insert designs. Backside wear of tibial inserts can be a significant contributor to polyethylene wear in total knee arthroplasty. Close attention should be given to the fixation of tibial inserts to metal trays by manufacturers and surgeons.

Catastrophic failure of a conforming type of total knee replacement: a case report.

A case study is presented to illustrate the concept of femoral component failure secondary to polyethylene wear in a 67 year old man, 13 years after he had conforming type total knee replacements. This case illustrates the theory that this observed problem may be a leading cause of failure in conforming and nonconforming total knee replacements in the future.

Nonconsolidated polyethylene particles and oxidation in Charnley acetabular cups.

Nonconsolidated particles of ultra high molecular weight polyethylene are believed to be defects that adversely can affect the wear performance of total joint prostheses. The present study was done to determine the number, size, and distribution of these particles and to determine if their presence correlated with wear performance, as well as with other clinical and implant parameters. Forty retrieved and 7 new, never-implanted acetabular components were examined using light microscopy on thin cross sections. Particles were found in 92% of retrieved components and in all the new components. Particles in the retrieved components were either randomly distributed (32 components) or banded (with particles localized in regions approximately 1 mm below the outer surface of the component). No correlations were found between the number or area of particles and the wear performance or any of the clinical or implant variables. The presence of particles in the new implants was found to correlate with the length of time since the components had been radiation sterilized. For retrieved components, the density (and, therefore, the level of oxidative degradation) was high in the areas of banded particles. For new components, the density was higher the longer the time since sterilization. Nonconsolidated polyethylene particles are prevalent in total replacements but their source and cause are unknown. The results of this study show that they do not appear to affect or correlate with the length of implantation of acetabular cups. However, they still may be expected to adversely affect performance in cases where large numbers of particles are banded together near articulating surfaces of high stress environments such as found in the knee.