Faculty Bibliography

Hyaluronan (HA) fragments have been proposed to elicit defensive or pro-inflammatory responses in many cell types. For articular chondrocytes in an inflammatory environment, studies have failed to reach consensus on the endogenous production or effects of added HA fragments. The present study was undertaken to resolve this discrepancy. Cultured primary human articular chondrocytes were exposed to the inflammatory cytokine IL-1β, and then tested for changes in HA content/size in conditioned medium, and for the expression of genes important in HA binding/signaling or metabolism, and in other catabolic/anabolic responses. Changes in gene expression caused by enzymatic degradation of endogenous HA, or addition of exogenous HA fragments, were examined. IL-1β increased the mRNA levels for HA synthases HAS2/HAS3 and for the HA-binding proteins CD44 and TSG-6. mRNA levels for TLR4 and RHAMM were very low and were little affected by IL-1β. mRNA levels for catabolic markers were increased, while type II collagen (α1(II)) and aggrecan were decreased. HA concentration in the conditioned medium was increased, but the HA was not degraded. Treatment with recombinant hyaluronidase or addition of low endotoxin HA fragments did not elicit pro-inflammatory responses. Our findings showed that HA fragments were not produced by IL-1β-stimulated human articular chondrocytes in the absence of other sources of reactive oxygen or nitrogen species, and that exogenous HA fragments from oligosaccharides up to about 40 kDa in molecular mass were not pro-inflammatory agents for human articular chondrocytes, probably due to low expression of TLR4 and RHAMM in these cells.

BACKGROUND:While tranexamic acid (TXA) has been well shown to reduce blood loss after joint replacement surgery, little is known regarding its effectiveness in obese patients. The aim of this study was to evaluate the effect of TXA changes in hematocrit and hemoglobin levels as well as incidence of packed red blood cell (pRBC) transfusions in obese patients undergoing total joint arthroplasty (TJA). MATERIAL AND METHODS/METHODS:Between January 2014 and May 2015, 420 consecutive primary joint replacements were performed by two surgeons at our institution. One-hundred-fifty-seven patients (total hip arthroplasty [THA]=29; total knee arthroplasty [TKA]=128) were obese with a body mass index (BMI) greater than or equal to 30 kg/m2. Medical records were reviewed and identified that TXA was utilized in 85 (54.1%) arthroplasties (study group) and was compared to a consecutive series of 72 (45.9%) TJAs (control group). TXA was given intravenously (IV) in two doses: (1) one gram prior to incision and (2) one gram at the time of femoral preparation in THA or prior to cementation in TKA. Changes in hemoglobin and hematocrit levels, number of pRBC transfusions, and occurrence of thrombolytic events were recorded. RESULTS:The changes in hematocrit (7.2% vs. 8.1%) and hemoglobin levels (3.0 g/dl vs. 3.3 g/dl) were less in the group that received TXA than the control group, albeit not significantly (p=0.100 and p=0.278, respectively). Within the control group, 26 (36.1%) patients required a pRBC transfusion with a mean of 2.0 units per patient (range:1-5); whereas, only eight (9.4%) patients with TXA required a mean of 1.6 units per patient (range: 1-2). The use of TXA significantly reduced the incidence of pRBC transfusions, especially in TKA (p<0.001). Sub-analyses revealed that transfusion rates were even more significantly reduced by TXA in obesity type II and III. Two pulmonary emboli were reported in the group that did not receive TXA; whereas, no thrombolytic events were reported in the group that did receive TXA. CONCLUSION/CONCLUSIONS:Utilization of TXA significantly reduced the rate of pRBC transfusions in obese patients.

BACKGROUND:/UNASSIGNED:Changes in the joint microenvironment after an injury to the articular surface of the knee have been implicated in the pathogenesis of osteoarthritis. While prior studies focused on changes in this microenvironment after anterior cruciate ligament ruptures, few have explored the biomarker changes that occur in the setting of meniscal injuries. PURPOSE:/UNASSIGNED:To determine whether meniscal injury results in significant alterations to synovial fluid biomarker concentrations as compared with noninjured contralateral knees. Additionally, to explore the relationship between synovial fluid biomarkers and the degree of cartilage injury seen in these patients. STUDY DESIGN:/UNASSIGNED:Cross-sectional study; Level of evidence, 3. METHODS:/UNASSIGNED:Patients undergoing surgery for unilateral meniscal injury were prospectively enrolled from October 2011 to December 2016, forming a cohort that had synovial fluid samples collected from both the injured knee and the contralateral uninjured knee at the time of meniscal surgery. Synovial fluid samples were collected just before incision, and the concentrations of 10 biomarkers of interest were determined with a multiplex magnetic bead immunoassay. The concentrations of synovial fluid biomarkers from the operative and contralateral knees were compared. Additionally, the synovial fluid biomarker concentrations of operative knees from patients with associated high-grade cartilage lesions were compared with those with low-grade lesions. RESULTS:/UNASSIGNED:The current analysis included synovial fluid samples from 82 knees (41 operative and 41 contralateral) from 41 patients undergoing arthroscopic surgery to treat a symptomatic meniscal injury. The mean ± SD age of patients was 49.86 ± 11.75 years. There were significantly greater concentrations of 4 of the 5 proinflammatory biomarkers (IL-6, MCP-1, MIP-1β, and MMP-3) in symptomatic knees as compared with asymptomatic knees when controlling for the duration of symptoms, body mass index, age, and the random effects of by-patient variability. In the injured knees, associated high-grade cartilage lesions were predictive of elevated MCP-1, MIP-1β, and VEGF levels. Low synovial fluid concentration of TIMP-1 or a greater ratio of MMP-3 to TIMP-1 was associated with the presence of synovitis. Increasing age was found to be an independent predictor of increased IL-6, MCP-1, and VEGF concentrations in the setting of symptomatic meniscal injury. CONCLUSION:/UNASSIGNED:The authors identified 4 proinflammatory synovial fluid biomarkers whose concentrations were significantly different after meniscal injury as compared with uninjured contralateral knees. Furthermore, they describe the effects of associated cartilage damage, synovitis, and patient age on biomarker concentrations.

Osteoarthritis (OA) results from degenerative and abnormal function of joints, with localized biochemistry playing a critical role in its onset and progression. As high levels of all- trans retinoic acid (ATRA) in synovial fluid have been identified as a contributive factor to OA, the synthesis of de novo antagonists for retinoic acid receptors (RARs) has been exploited to interrupt the mechanism of ATRA action. BMS493, a pan-RAR inverse agonist, has been reported as an effective inhibitor of ATRA signaling pathway; however, it is unstable and rapidly degrades under physiological conditions. We employed an engineered cartilage oligomeric matrix protein coiled-coil (C_cc^S) protein for the encapsulation, protection, and delivery of BMS493. In this study, we determine the binding affinity of C_cc^S to BMS493 and the stimulator, ATRA, via competitive binding assay, in which ATRA exhibits approximately 5-fold superior association with C_cc^S than BMS493. Interrogation of the structure of C_cc^S indicates that ATRA causes about 10% loss in helicity, while BMS493 did not impact the structure. Furthermore, C_cc^S self-assembles into nanofibers when bound to BMS493 or ATRA as expected, displaying 11-15 nm in diameter. Treatment of human articular chondrocytes in vitro reveals that C_cc^S·BMS493 demonstrates a marked improvement in efficacy in reducing the mRNA levels of matrix metalloproteinase-13 (MMP-13), one of the main proteases responsible for the degradation of the extracellular cartilage matrix compared to BMS493 alone in the presence of ATRA, interleukin-1 beta (IL-1β), or IL-1 β together with ATRA. These results support the feasibility of utilizing coiled-coil proteins as drug delivery vehicles for compounds of relatively limited bioavailability for the potential treatment of OA.

A method for specific quantification of hyaluronan (HA) concentration using AlphaScreen® (Amplified Luminescent Proximity Homogeneous Assay) technology is described. Two types of hydrogel-coated and chromophore-loaded latex nanobeads are employed. The proximity of the beads in solution is detected by excitation of the donor bead leading to the production of singlet oxygen, and chemiluminescence from the acceptor bead upon exposure to singlet oxygen. In the HA assay, the donor bead is modified with streptavidin, and binds biotin-labeled HA. The acceptor bead is modified with Ni(II), and is used to bind a specific recombinant HA-binding protein (such as HABP; aggrecan G1-IGD-G2) with a His-tag. Competitive inhibition of the HA-HABP interaction by free unlabeled HA in solution is used for quantification. The assay is specific for HA, and not dependent on HA molecular mass above the decasaccharide. HA can be quantified over a concentration range of approximately 30-1600 ng/mL using 2.5 μL of sample, for a detectable mass range of approximately 0.08-4 ng HA. This sensitivity of the AlphaScreen assay is greater than existing ELISA-like methods, due to the small volume requirements. HA can be detected in biological fluids using the AlphaScreen assay, after removal of bound proteins from HA and dilution or removal of other interfering proteins and lipids.

Despite advances in thromboprophylaxis, the relationship between preexisting deep venous thromboses (DVTs) and postoperative thromboembolic complications is not fully established. The aim of this study was to assess the utility of selective and nonselective preoperative lower extremity venous Doppler screening protocols as tools in reducing the incidence of thromboembolic events (DVT/pulmonary embolism [PE]) after total joint arthroplasty. In the 2-year period from August 2013 to August 2015, a total of 455 consecutive elective primary total joint arthroplasties were identified from the authors' database. During the first year, a selective preoperative Doppler ultrasound screening protocol (only patients with a history of DVT/PE) was used for 182 patients, 31 of whom had preoperative ultrasound scans. The following year, a nonselective screening protocol was used for 273 consecutive patients, all of whom had preoperative Doppler ultrasound scans. All patients were followed for a minimum of 3 months postoperatively for postoperative Doppler ultrasound scans, emergency department visits, and readmissions related to DVT/PE. Preoperatively, there was no difference between the selective and nonselective cohorts regarding preoperative DVTs (3 and 1, respectively; P=.307); all patients were known to have prior DVT. Postoperative thromboembolic events were found in 4 (2.6%) of the patients in the selective cohort and 2 (0.7%) of the patients in the nonselective cohort (P=.196). Use of a nonselective preoperative Doppler ultrasound screening protocol did not improve the identification of preoperative DVTs or reduce postoperative thromboembolic complications. [Orthopedics. 201x; xx(x):xx-xx.].

The purpose of this study was to examine the influence of topical vancomycin on cell migration and survival of tissue healing cells. Human osteoblasts, myoblasts and fibroblasts were exposed to vancomycin at concentrations of 1, 3, 6, or 12 mg/cm² for either a 1-h or 48-h (continuous) duration. Continuous exposure to all vancomycin concentrations significantly reduced cell survival (<22% cells survived) and migration in osteoblasts and myoblasts (P < 0.001). 1-h vancomycin exposure reduced osteoblast and myoblast survival and migration only at 12 mg/cm² (P < 0.001). Further in vivo studies are warranted to optimize the dosage of intrawound vancomycin.

Objective Focal chondral defects alter joint mechanics and cause pain and debilitation. Microfracture is a surgical technique used to treat such defects. This technique involves penetration of subchondral bone to release progenitor cells and growth factors from the marrow to promote cartilage regeneration. Often this results in fibrocartilage formation rather than structured hyaline cartilage. Some reports have suggested use of growth hormone (GH) with microfracture to augment cartilage regeneration. Our objective was to test whether intra-articular (IA) GH in conjunction with microfracture, improves cartilage repair in a rabbit chondral defect model. We hypothesized that GH would exhibit a dose-dependent improvement in regeneration. Design Sixteen New Zealand white rabbits received bilateral femoral chondral defects and standardized microfracture repair. One group of animals ( n = 8) received low-dose GH by IA injection in the left knee, and the other group ( n = 8) received high-dose GH in the same manner. All animals received IA injection of saline in the contralateral knee as control. Serum assays, macroscopic grading, and histological analyses were used to assess any improvements in cartilage repair. Results Peripheral serum GH was not elevated postoperatively ( P = 0.21). There was no improvement in macroscopic grading scores among either of the GH dosages ( P = 0.83). Scoring of safranin-O-stained sections showed no improvement in cartilage regeneration and some evidence of increased bone formation in the GH-treated knees. Conclusions Treatment with either low- or high-dose IA GH does not appear to enhance short-term repair in a rabbit chondral defect model.

Annexin A6 (AnxA6) is expressed in articular chondrocytes at levels higher than in other mesenchymal cell types. However, the role of AnxA6 in articular chondrocytes is not known. Here we show that complete lack of AnxA6 functions resulted in increased ß-catenin activation in Wnt3a-treated murine articular chondrocytes, whereas AnxA6 expressing articular chondrocytes showed decreased ß-catenin activation. High expression of AnxA6 in human articular chondrocytes showed the highest inhibition of Wnt/ß-catenin signaling. Inhibition of Wnt/ß-catenin signaling activity by AnxA6 together with cytosolic Ca2+ was achieved by interfering with the plasma membrane association of the Wnt signaling complex. AnxA6 also affected the cross-talk between Wnt/ß-catenin signaling and NF-κB signaling by decreasing ß-catenin activity and increasing NF-κB activity in Wnt3a-, interleukin-1beta (IL-1ß)-, and combined Wnt3a/IL-1ß-treated cells. Wnt3a treatment increased the mRNA levels of catabolic markers (cyclooxygenase-2, interleukin-6, inducible nitric oxide synthase) to a much lesser degree than IL-1ß treatment in human articular chondrocytes, and decreased the mRNA levels of matrix metalloproteinase-13 (MMP-13) and articular cartilage markers (aggrecan, type II collagen). Furthermore, Wnt3a decreased the mRNA levels of catabolic markers and MMP-13 in IL-1ß-treated human articular chondrocytes. High expression of AnxA6 resulted in decreased mRNA levels of catabolic markers, and increased MMP-13 and articular cartilage marker mRNA levels in Wnt3a-treated human articular chondrocytes, whereas leading to increased mRNA levels of catabolic markers and MMP-13 in human articular chondrocytes treated with IL-1ß, or combined Wnt3a and IL-1ß. Our findings define a novel role for AnxA6 in articular chondrocytes via its modulation of Wnt/ß-catenin and NF-κB signaling activities and the cross-talk between these two signaling pathways.