Biomechanical Remodeling of Aortic Valve Interstitial Cells During Calcified Lesion Formation In Vitro.

Healthy aortic heart valves are essential to the regulation of unidirectional blood flow. Calcific aortic valve disease (CAVD) is an actively progressive disease that involves the disorganization of valve cells and accumulation of calcium deposits on the aortic valve leaflets. CAVD involves disruption of cell environment homeostasis that prior cell culture models have found difficult to portray and model. As it is still poorly understood how tissue stiffening associates with lesion formation, here, we implement a novel 3D culture platform to characterize the relationship between mechanical stress and tissue remodeling and analyze how the application of pro-osteogenic stimulation dysregulates the native ability of valve cells to organize its matrix. Through a temporal study of macroscopic remodeling, we determine that aortic valve interstitial neo-tissues undergo varying stiffness and mechanical stress, demonstrate greater myofibroblastic gene expression, and show greater remodeling activity in the outer surface of the neo-tissue in a banding pattern when cultured in osteogenic growth medium. In human aortic valve interstitial cells cultured in osteogenic growth medium, we observed an increase in stress but significant decreases in myofibroblastic gene expression with the addition of growth factors. In summary, we are able to see the interplay of biochemical and biomechanical stimuli in valvular remodeling by using our platform to model dynamic stiffening of valve interstitial neo-tissues under different biochemical conditions.

Uncovering transcriptional dark matter via gene annotation independent single-cell RNA sequencing analysis.

Conventional scRNA-seq expression analyses rely on the availability of a high quality genome annotation. Yet, as we show here with scRNA-seq experiments and analyses spanning human, mouse, chicken, mole rat, lemur and sea urchin, genome annotations are often incomplete, in particular for organisms that are not routinely studied. To overcome this hurdle, we created a scRNA-seq analysis routine that recovers biologically relevant transcriptional activity beyond the scope of the best available genome annotation by performing scRNA-seq analysis on any region in the genome for which transcriptional products are detected. Our tool generates a single-cell expression matrix for all transcriptionally active regions (TARs), performs single-cell TAR expression analysis to identify biologically significant TARs, and then annotates TARs using gene homology analysis. This procedure uses single-cell expression analyses as a filter to direct annotation efforts to biologically significant transcripts and thereby uncovers biology to which scRNA-seq would otherwise be in the dark.

Spatiotemporal single-cell RNA sequencing of developing chicken hearts identifies interplay between cellular differentiation and morphogenesis.

Single-cell RNA sequencing is a powerful tool to study developmental biology but does not preserve spatial information about tissue morphology and cellular interactions. Here, we combine single-cell and spatial transcriptomics with algorithms for data integration to study the development of the chicken heart from the early to late four-chambered heart stage. We create a census of the diverse cellular lineages in developing hearts, their spatial organization, and their interactions during development. Spatial mapping of differentiation transitions in cardiac lineages defines transcriptional differences between epithelial and mesenchymal cells within the epicardial lineage. Using spatially resolved expression analysis, we identify anatomically restricted expression programs, including expression of genes implicated in congenital heart disease. Last, we discover a persistent enrichment of the small, secreted peptide, thymosin beta-4, throughout coronary vascular development. Overall, our study identifies an intricate interplay between cellular differentiation and morphogenesis.

Does the presence of the fibronectin-aggrecan complex predict outcomes from lumbar discectomy for disc herniation?

BACKGROUND CONTEXT: Protein biomarkers associated with lumbar disc disease have been studied as diagnostic indicators and therapeutic targets. Recently, a cartilage degradation product, the fibronectin-aggrecan complex (FAC) identified in the epidural space, has been shown to predict response to lumbar epidural steroid injection in patients with radiculopathy from herniated nucleus pulposus (HNP). PURPOSE: Determine the ability of FAC to predict response to microdiscectomy for patients with radiculopathy due to lumbar disc herniation STUDY DESIGN/SETTING: Single-center prospective consecutive cohort study. PATIENT SAMPLE: Patients with radiculopathy from HNP with concordant symptoms to MRI who underwent microdiscectomy. OUTCOMES MEASURES: Oswestry disability index (ODI) and visual analog scores (VAS) were noted at baseline and at 3-month follow-up. Primary outcome of clinical improvement was defined as patients with both a decrease in VAS of at least 3 points and ODI >20 points. METHODS: Intraoperative sampling was done via lavage of the excised fragment by ELISA for presence of FAC. Funding for the ELISA was provided by Cytonics, Inc. RESULTS: Seventy-five patients had full complement of data and were included in this analysis. At 3-month follow-up, 57 (76%) patents were "better." There was a statistically significant association of the presence of FAC and clinical improvement (p=.017) with an 85% positive predictive value. Receiver-operating-characteristic (ROC) curve plotting association of FAC and clinical improvement demonstrates an area under the curve (AUC) of 0.66±0.08 (p=.037). Subset analysis of those with weakness on physical examination (n=48) plotting the association of FAC and improvement shows AUC on ROC of 0.81±0.067 (p=.002). CONCLUSIONS: Patients who are "FAC+" are more likely to demonstrate clinical improvement following microdiscectomy. The data suggest that the inflammatory milieu plays a significant role regarding improvement in patients undergoing discectomy for radiculopathy in lumbar HNP, even in those with preoperative weakness. The FAC represents a potential target for treatment in HNP.

Editor's Choice - Aortic Re-operation After Replacement of the Proximal Aorta: A Systematic Review and Meta-Analysis.

OBJECTIVE/BACKGROUND: The aim was to estimate risk of aortic re-operation, and re-operative morbidity and mortality, following replacement of the proximal aorta for aneurysm or dissection. METHODS: A meta-analysis was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement and the Meta-Analysis of Observational Studies in Epidemiology guidelines. A comprehensive literature review was performed to identify all articles reporting aortic re-operation after proximal aortic replacement. The proximal aorta was defined as extending to the origin of the brachiocephalic trunk. The incidence rate for aortic re-operation (IRAR) was calculated, and stratified based on presence/absence of connective tissue disorders, as well as initial surgical indication. Pooled in hospital mortality and post-operative complication rates were estimated. RESULTS: In total, 7821 patients who underwent proximal aortic replacement from 47 studies were included: 8.3% (n = 649) had Marfan syndrome (MS). During a weighted mean follow up of 4.7 ± 0.3 years, 11.5% (n = 903) underwent aortic re-operation. Mean weighted time between initial surgery and re-operation was 5.2 ± 0.2 years. IRAR was 2.4% per person-year (PPY) (confidence interval [CI] 2.1-2.8%). Patients with MFS had a threefold higher IRAR (6.0% PPY, CI 4.1-8.8%) than did patients without a connective tissue disorders (2.3% PPY, CI 1.9-2.7%; p < .001). IRAR was 2.5% PPY (CI 2.1-3.0%) after operation for dissection and 1.3% PPY (CI 0.9-2.0%) after operation for aneurysm (p = .004 for subgroup differences). IRAR proximal and distal to the left subclavian artery was 1.2% PPY (CI 1.0-1.5%) and 1.3% PPY (CI 1.1-1.6%), respectively. The pooled in hospital mortality and complication rates after re-operation were 14.31% (CI 11.28-17.99%) and 18.08% (CI 10.54-29.25%), respectively. On meta-regression, initial operation for dissection was the only significant predictor of aortic re-operation (beta = .030, p = .001). CONCLUSION: Aortic re-operation occurs at a mean rate of 2.4% per person-year in the five years after proximal aortic replacement and is strongly associated with initial operation for dissection.

Totally endoscopic coronary artery bypass surgery: A meta-analysis of the current evidence.

BACKGROUND: Totally endoscopic coronary artery bypass (TECAB) has emerged as an alternative to other minimally invasive techniques. However, limited TECAB results are available to date. The purpose of this systematic review is to examine the existing literature to give an objective estimate of the outcomes of TECAB using a meta-analytical approach. METHODS: A comprehensive online review was performed in Ovid MEDLINE®, Ovid EMBASE and The Cochrane Library from 2000 to July 2017. Eligible studies included single arm TECAB studies as well as comparative studies (TECAB vs minimally invasive direct coronary artery bypass (MIDCAB)). Pooled event rates and odds ratios (ORs) for operative mortality, perioperative myocardial infarction (MI), perioperative stroke, graft patency and repeat revascularization were estimated. Single arm and pairwise comparisons were performed. RESULTS: Seventeen single arm TECAB articles (3721 patients, weighted mean follow-up 3.3years) were included. The pooled event rate was 0.80% (95%CI: 0.60-1.2%) for operative mortality, 2.28% (95%CI: 1.7-3%) for perioperative MI, 1.50% (95%CI: 1.1-2.0%) for perioperative stroke, 2.99% (95%CI: 1.6-5.4%) for repeat revascularization and 94.8% (95%CI: 89.3-97.5%) for early graft patency (weighted mean follow-up 10.1months). On pairwise meta-analysis 376 patients (263 TECAB and 113 MIDCAB) were included. No difference in operative mortality (OR=0.25, 95%CI: 0.02-2.83), perioperative MI (OR=3.09, 95%CI: 0.37-26.12) or perioperative stroke (OR=1.33, 95%CI: 0.17-10.26) was found between the two techniques. CONCLUSIONS: TECAB has an acceptably low operative risk and a good early patency rate. The incidence of perioperative MI requires further investigation. The dearth of data comparing TECAB to open approaches compels the need for future comparative trials.

Targeted designed variants of alpha-2-macroglobulin (A2M) attenuate cartilage degeneration in a rat model of osteoarthritis induced by anterior cruciate ligament transection.

BACKGROUND: The study was performed to evaluate whether targeted alpha-2-macroglobulin (A2M) variants have a similar or enhanced function at wild-type (wt)-A2M to attenuate cartilage degeneration in vivo. METHODS: In and ex-vivo experiment, bovine cartilage explants (BCE) were incubated with TNF-α and IL-1ß with or without wt-A2M or A2M variants. Cartilage catabolism was measured in culture supernatant by sulfated glycosaminoglycan (sGAG). In an in-vivo experiment, 2-month-old male Wistar rats (n = 77) were randomly divided into seven groups and treated with different doses of A2M or its variants by intra-articular injection at 24 hours and day 14 after anterior cruciate ligament transection (ACLT), receiving (1) ACLT/PBS; (2) ACLT/wt-A2M (0.153 mg); (3) ACLT/CYT-108 A2M (0.153 mg); (4) ACLT/CYT-108 A2M (0.077 mg); (5) ACLT/CYT-98 A2M (0.153 mg); (6) ACLT/CYT-98 A2M (0.077 mg); or (7) sham/PBS. The joints and synovial lavage were collected 8 weeks after surgery. Fluorescence molecular tomography was used to monitor inflammation in vivo using probes ProSense and MMPSense at 24 hours, and weeks 2, 4, and 6 after surgery. The cartilage damage was quantified using Osteoarthritis Research Society International score and matrix metalloproteinase (MMP)-3, -13, collagen (Col) X, Col 2, Runx2, and aggrecan (Acan) were detected by immunohistochemical analysis (IHC), ELISA, and RT-PCR. RESULTS: A2M variants inhibited catabolism in the BCE model by up to 200% compared with wt-A2M. ProSense and MMPSense were dramatically increased in all groups after surgery. Supplemental A2M or its variants reduced ProSense and MMPSense compared with the PBS treatment. Less cartilage damage, lower MMP-13 and Col 2 degraded product, and stronger Col 2 synthesis were detected in animals treated with A2M or its variants compared with PBS-treated animals. A2M and its variants enhanced Col 2 and Acan synthesis, and suppressed MMP-3, MMP-13, Runx2, and Col X production. A2M-108 variant demonstrated less cartilage damage compared with wt-A2M and A2M-98 variant. CONCLUSION: The targeted variants of A2M have a chondroprotective effect similar to wt-A2M. However, A2M-108 variant has enhanced function to attenuate cartilage degeneration compared with wt-A2M.

Ribosomal PCR assay of excised intervertebral discs from patients undergoing single-level primary lumbar microdiscectomy.

PURPOSE: To determine the presence of infectious microorganisms in the herniated discs of immunocompetent patients, using methodology that we hoped would be of higher sensitivity and specificity than has been reported in the past. Recent studies have demonstrated a significant rate of positive cultures for low virulent organisms in excised HNP samples (range 19-53%). These studies have served as the theoretical basis for a pilot trial, and then, a well done prospective randomized trial that demonstrated that systemic treatment with antibiotics may yield lasting improvements in a subset of patients with axial back pain. Whether the reported positive cultures in discectomy specimens represent true positives is as yet not proven, and critically important if underlying the basis of therapeutic approaches for chronic low back pain. METHODS: This consecutive case series from a single academic center included 44 patients with radiculopathy and MRI findings of lumbar HNP. Patients elected for lumbar microdiscectomy after failure of conservative management. All patients received primary surgery at a single spinal level in the absence of immune compromise. Excised disc material was analyzed with a real-time PCR assay targeting the 16S ribosomal RNA gene followed by amplicon sequencing. No concurrent cultures were performed. Inclusion criteria were as follows: sensory or motor symptoms in a single lumbar nerve distribution; positive physical examination findings including positive straight leg raise test, distributional weakness, and/or a diminished deep tendon reflexes; and magnetic resonance imaging of the lumbar spine positive for HNP in a distribution correlating with the radicular complaint. RESULTS: The PCR assay for the 16S rRNA sequence was negative in all 44 patients (100%). 95% CI 0-8%. CONCLUSIONS: Based on the data presented here, there does not appear to be a significant underlying rate of bacterial disc infection in immunocompetent patients presenting with radiculopathy from disc herniation.

Naturally Engineered Maturation of Cardiomyocytes.

Ischemic heart disease remains one of the most prominent causes of mortalities worldwide with heart transplantation being the gold-standard treatment option. However, due to the major limitations associated with heart transplants, such as an inadequate supply and heart rejection, there remains a significant clinical need for a viable cardiac regenerative therapy to restore native myocardial function. Over the course of the previous several decades, researchers have made prominent advances in the field of cardiac regeneration with the creation of in vitro human pluripotent stem cell-derived cardiomyocyte tissue engineered constructs. However, these engineered constructs exhibit a functionally immature, disorganized, fetal-like phenotype that is not equivalent physiologically to native adult cardiac tissue. Due to this major limitation, many recent studies have investigated approaches to improve pluripotent stem cell-derived cardiomyocyte maturation to close this large functionality gap between engineered and native cardiac tissue. This review integrates the natural developmental mechanisms of cardiomyocyte structural and functional maturation. The variety of ways researchers have attempted to improve cardiomyocyte maturation in vitro by mimicking natural development, known as natural engineering, is readily discussed. The main focus of this review involves the synergistic role of electrical and mechanical stimulation, extracellular matrix interactions, and non-cardiomyocyte interactions in facilitating cardiomyocyte maturation. Overall, even with these current natural engineering approaches, pluripotent stem cell-derived cardiomyocytes within three-dimensional engineered heart tissue still remain mostly within the early to late fetal stages of cardiomyocyte maturity. Therefore, although the end goal is to achieve adult phenotypic maturity, more emphasis must be placed on elucidating how the in vivo fetal microenvironment drives cardiomyocyte maturation. This information can then be utilized to develop natural engineering approaches that can emulate this fetal microenvironment and thus make prominent progress in pluripotent stem cell-derived maturity toward a more clinically relevant model for cardiac regeneration.

α2-Macroglobulin: Autologous Protease Inhibition Technology.

α2-Macroglobulin (A2M) is a plasma glycoprotein best known for its ability to inhibit a broad spectrum of serine, threonine, and metalloproteases as well as inflammatory cytokines by a unique bait-and-trap method. A2M has emerged as a unique potential treatment of cartilage-based pathology and inflammatory arthritides. This article describes the unique method by which A2M not only inhibits the associated inflammatory cascade but also disrupts the catabolic process of cartilage degeneration. Autologous concentrated A2M from plasma is currently in use to successfully treat various painful arthritides. Future directions will focus on recombinant variants that enhance its anti-inflammatory and disease-modifying potential.

Cytokines as a predictor of clinical response following hip arthroscopy: minimum 2-year follow-up.

Hip arthroscopy in patients with osteoarthritis has been shown to have suboptimal outcomes. Elevated cytokine concentrations in hip synovial fluid have previously been shown to be associated with cartilage pathology. The purpose of this study was to determine whether a relationship exists between hip synovial fluid cytokine concentration and clinical outcomes at a minimum of 2 years following hip arthroscopy. Seventeen patients without radiographic evidence of osteoarthritis had synovial fluid aspirated at time of portal establishment during hip arthroscopy. Analytes included fibronectin-aggrecan complex as well as a multiplex cytokine array. Patients completed the modified Harris Hip Score, Western Ontario and McMaster Universities Arthritis Index and the International Hip Outcomes Tool pre-operatively and at a minimum of 2 years following surgery. Pre and post-operative scores were compared with a paired t-test, and the association between cytokine values and clinical outcome scores was performed with Pearson's correlation coefficient with an alpha value of 0.05 set as significant. Sixteen of seventeen patients completed 2-year follow-up questionnaires (94%). There was a significant increase in pre-operative to post-operative score for each clinical outcome measure. No statistically significant correlation was seen between any of the intra-operative cytokine values and either the 2-year follow-up scores or the change from pre-operative to final follow-up outcome values. No statistically significant associations were seen between hip synovial fluid cytokine concentrations and 2-year follow-up clinical outcome assessment scores for those undergoing hip arthroscopy.

Can We Improve Workflows in the OR? A Comparison of Quality Perceptions and Preoperative Efficiency across Institutions in Spine Surgery.

INTRODUCTION: Cost containment and surgical inefficiencies are major concerns for hospitals in this era of declining resources. The primary aim of this investigation was to understand subjective perceptions of perioperative spine surgical quality across three practice settings and to identify potential factors contributing to these perceptions. Subsequently, we objectively evaluated factors that influence the duration of time in which the patient is in the operating room (OR) prior to the surgical incision and assessed the influence of fluoroscopy technician expertise on radiation dose and imaging efficiency. METHODS: One hundred and eight medical device representatives with at least 1 year of OR experience were surveyed at a national conference. Three distinct healthcare facilities were identified: university, small volume, and large volume private hospitals. Respondents rated facilities on a five-point scale for staff quality; size and consistency of surgical teams; and overall likelihood of recommending the facility. Separately, 140 posterior lumbar procedures from two institutions were retrospectively reviewed. Two time periods were quantified for each surgical case: patient arrival in the OR to induction of anesthesia (T1) and induction to surgical incision (T2). T1 and T2 were compared between university and large private hospital settings using t tests and multivariate analysis. For 44 separate lumbar spine surgical procedures, practice setting, patient BMI, number of vertebral levels requiring imaging, number of localizing fluoroscopy images taken, total fluoroscopy time, total radiation dose, fluoroscopy machine, and whether the fluoroscopist could correctly state his or her role, which was to obtain a lateral lumbar localizing image, were recorded. T-tests were used to compare cases in which the fluoroscopist could and could not correctly state the task. RESULTS: Survey ratings for surgeons were not significantly different across university, large private, and small private hospitals. Fewer circulating nurses were rated as excellent or good in university versus private hospitals (p < 0.001). Small volume private hospital surgical teams were more likely to have worked together before than university teams (p < 0.05), and university teams were larger (p < 0.05). Respondents were more likely to recommend a university or large private hospital for complex instrumentation cases (p < 0.001). On objective measures, university patients were older, less obese, and had higher mean ASA scores (2.5 versus 2.2, p < 0.001). Compared to the university setting, private hospital cases had significantly shorter Time 1 (8 versus 37 min, p < 0.001) and Time 2 (23 versus 30 min, p < 0.001), even after adjusting for ASA score, BMI, and age. Cases in which the fluoroscopist knew the imaging purpose were associated with significantly fewer images (mean 1.8 versus 3.4 images, p < 0.0001) and shorter total exposure times (2.3 versus 4.0 sec, p < 0.001). Operations performed in the university setting were associated with significantly more images (2.7 versus 1.8 images, p < 0.001), longer total exposure times (3.2 versus 2.3 sec, p = 0.0027), and total radiation dose (27.8 versus 53.3 rad, p < 0.001) when compared with those performed in the private setting. The university practice setting was associated with significantly more images (2.7 versus 1.8 images, p < 0.001), longer total exposure times (3.2 versus 2.3 sec, p = 0.003), and total radiation dose (27.8 versus 53.3 rad, p < 0.001) when compared with non-university settings. CONCLUSION: Large private and university hospitals had higher surgeon ratings. The university setting was associated with larger and less consistent surgical teams and lower nurse ratings. Surgical staff awareness of the procedure and attention to preoperative tasks specific to the procedure reduced pre-operative time spent in the OR as well as fluoroscopy radiation. These data suggest that nurses and support staff make substantial contributions to overall quality of care, and that leadership and interpersonal coordination are especially important within large teams at teaching hospitals.

Fibronectin-aggrecan complex as a marker for cartilage degradation in non-arthritic hips.

PURPOSE: To report hip synovial fluid cytokine concentrations in hips with and without radiographic arthritis. METHODS: Patients with no arthritis (Tonnis grade 0) and patients with Tonnis grade 2 or greater hip osteoarthritis (OA) were identified from patients undergoing either hip arthroscopy or arthroplasty. Synovial fluid was collected at the time of portal establishment for those undergoing hip arthroscopy and prior to arthrotomy for the arthroplasty group. Analytes included fibronectin-aggrecan complex (FAC) as well as a standard 12 cytokine array. Variables recorded were Tonnis grade, centre-edge angle of Wiberg, as well as labrum and cartilage pathology for the hip arthroscopy cohort. A priori power analysis was conducted, and a Mann-Whitney U test and regression analyses were used with an alpha value of 0.05 set as significant. RESULTS: Thirty-four patients were included (17 arthroplasty, 17 arthroscopy). FAC was the only analyte to show a significant difference between those with and without OA (p < 0.001). FAC had significantly higher concentration in those without radiographic evidence of OA undergoing microfracture versus those not receiving microfracture (p < 0.05). CONCLUSION: There was a significantly higher FAC concentration in patients without radiographic OA. Additionally, those undergoing microfracture had increased levels of FAC. As FAC is a cartilage breakdown product, no significant amounts may be present in those with OA. In contrast, those undergoing microfracture have focal area(s) of cartilage breakdown. These data suggest that FAC may be useful in predicting cartilage pathology in those patients with hip pain but without radiographic evidence of arthritis.

Does a fibronectin and aggrecan complex play a role in painful vertebral disks?

OBJECTIVE: To determine the presence of a fibronectin-aggrecan complex (FAC) in the disk space of persons with chronic low back pain as relates to provocative diskography. DESIGN: A single-center prospective consecutive case series. SETTING: A single private practice setting. PATIENTS: Thirty-seven patients with symptomatic degenerative disk disease of the cervical, thoracic, or lumbar spine undergoing provocative diskography to identify a source of pain. METHODS: Diskographic lavage for analysis was simultaneously performed at each disk level injected during diskography. MAIN OUTCOME MEASURES: Visual analog scale (VAS) pain scores, Pfirrmann magnetic resonance imaging grade, and biochemical analysis of disk material were statistically analyzed. RESULTS: A total of 105 levels in 37 patients had a complete set of data (mean age 43.2 ± 11.9 years; 15 male/22 female). The FAC was present in 43 of 108 levels and in at least one level in 25 of 37 patients. The Pfirrmann magnetic resonance imaging grade did not differ between complex-positive and negative levels (P = .125), nor did the intraoperative VAS (IO-VAS) score for pain by level (P = .206). A significant but loose correlation was found between Pfirrmann grade and IO-VAS (R(2) = 0.4, P < .001), but no significant correlation was found between VAS or IO-VAS and complex concentration (R(2) = 0.08, P = .11 and R(2) = 0.003, P = .5). CONCLUSIONS: The FAC was identified in some painful disks by diskography. There was no significant correlation between the Pfirrmann grade or pre/intraoperative pain scores during diskography and complex concentrations within the disk measured by disk lavage.

Cytokine expression in the epidural space: a model of noncompressive disc herniation-induced inflammation.

STUDY DESIGN: Animal study. OBJECTIVE: Development of an animal model for the study of biochemical changes that occur in the epidural space after intervertebral disc herniation. SUMMARY OF BACKGROUND DATA: Although strong evidence for an inflammatory component exists, the biochemical processes underlying pain after disc herniation remain unknown. METHODS: Epidural lavage was performed in 48 rats after L5 dorsal root ganglion exposure at baseline and 3, 6, or 24 hours after placement of autologous nucleus pulposus (NP) (N = 15), saline (N = 15), or NP + an interferon-γ antibody (anti-IFN-γ; N = 18) directly onto the dorsal root ganglion. Multiplex assays quantifying interleukin (IL)-1α, IL-1ß, IL-2, IL-4, IL-6, IL-10, tumor necrosis factor α (TNF-α), IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were performed. NP (N = 7) was also analyzed for these cytokines by placing NP into saline and measuring the relative concentration. RESULTS: Cytokines measured low at baseline (0-100 pg/mL) in all groups. Compared with saline, NP application caused IL-6 elevation, peaking at T = 3 hours, that was prevented by anti-IFN-γ. NP induced elevation of TNF-α, peaking at T = 24 hours and was prevented by anti-IFN-γ. IFN-γ was elevated after NP at T = 3 hours and T = 24 hours. IL-1α was similar after saline versus NP. The concentrations of IL-1ß and IL-10 were elevated at T = 3 hours, 6 hours, and 24 hours in all groups without between-groups difference. The level of IL-4 peaked at T = 3 hours in the NP group and was different than saline and NP + anti-IFN-γ groups, but the time effect was insignificant. There was no change for GM-CSF. The concentration of cytokines measured in normal NP was less than 2 pg/mL for all cytokines except TNF-α. CONCLUSION: In this model of acute noncompressive disc herniation, NP caused the elevation of epidural IL-6, TNF-α, and IFN-γ--all attenuated by IFN-γ blockade. IL-1ß and IL-10 were both significantly elevated by saline alone and their response was not prevented by IFN-γ blockade. This model may prove useful for the study of the biochemical processes by which NP induces inflammation-induced nerve root irritation and radiculopathic pain.

The effect of local anaesthetics on synoviocytes: a possible indirect mechanism of chondrolysis.

PURPOSE: While the effect of local anaesthetics on chondrocyte viability is widely documented, the effect of these medications on synoviocytes is largely unknown. The purpose of this study was to understand the effect of 0.5 % bupivacaine and 0.5 % bupivacaine with epinephrine on synoviocyte viability, cytokine and growth factor release, and breakdown product formation. METHODS: Rabbit fibroblast-like synoviocyte (Type B) cultures were perfused with 0.5 % bupivacaine or 0.5 % bupivacaine with epinephrine (1:200,000) for 24 h. Cell viability was evaluated using a two-colour fluorescence assay. The supernatant was analysed using multiplex inflammatory and matrix metalloproteinase assays. RESULTS: Synoviocytes treated for 24 h with 0.5 % bupivacaine with epinephrine demonstrated a significant decrease in viability (31.3 ± 19.4 % cell death) when compared with synoviocytes cultured in control media (3.8 ± 1.3 % cell death, p = 0.000) and those cultured in 0.5 % bupivacaine alone (12.6 ± 11.1 % cell death, p = 0.003). No significant decrease in cell viability was observed in synoviocytes treated with 0.5 % bupivacaine compared to those in control media (12.6 ± 11.1 % vs 3.8 ± 1.3 % cell death, p = 0.194). Significantly greater amounts of MMP-1 (47.0 ± 9.2 pg/ml) and MMP-3 (250.0 ± 68.8 pg/ml) were observed in 0.5 % bupivacaine cultures compared with controls (14.3 ± 14.3, p = 0.023 and 72.0 ± 84.9, p = 0.045, respectively). CONCLUSIONS: 0.5 % bupivacaine with epinephrine caused a significant increase in cell death of the synoviocytes, while 0.5 % bupivacaine alone produced cell injury and a significant release of matrix metalloproteinases, which may also lead to indirect injury of the surrounding chondrocytes. These results may help explain the onset of chondrolysis observed in patients who have been treated with intra-articular local anaesthetics.

Platelet-rich plasma increases matrix metalloproteinases in cultures of human synovial fibroblasts.

BACKGROUND: The effect of platelet-rich plasma on chondrocytes has been studied in cell and tissue culture. Less attention has been given to the effect of platelet-rich plasma on nonchondrocytic cell lineages within synovial joints, such as fibroblast-like synoviocytes, which produce cytokines and matrix metalloproteinases (MMPs) that mediate cartilage catabolism. The purpose of the present study was to determine the effect of platelet-rich plasma on cytokines and proteases produced by fibroblast-like synoviocytes. METHODS: Platelet-rich plasma and platelet-poor plasma from harvested autologous blood were prepared with a commercially available system. Fibroblast-like synoviocytes were treated with platelet-rich plasma, platelet-poor plasma, recombinant PDGFßß (platelet-derived growth factor ßß), or phosphate-buffered saline solution and incubated at 37°C for forty-eight hours. The concentrations of IL-1ß (interleukin-1ß), IL-1RA (IL-1 receptor antagonist), IL-6, IFN-γ (interferon-γ), IP-10 (interferon gamma-induced protein 10), MCP-1 (monocyte chemotactic protein-1), MIP-1ß (macrophage inflammatory protein-1ß), PDGFßß, RANTES, TNF-α (tumor necrosis factor-α), VEGF (vascular endothelial growth factor), MMP-1, MMP-3, and MMP-9 in the culture medium were determined by multiplex immunoassay. RESULTS: Platelet-rich plasma cultured in medium contained multiple catabolic mediators in substantial concentrations, including MMP-9 (15.8 ± 2.3 ng/mL) and MMP-1 (2.5 ± 0.8 ng/mL), as well as proinflammatory mediators IL-1ß, IL-6, IFN-γ, IP-10, MCP-1, MIP-1ß, RANTES, and TNF-α in concentrations between 20 pg/mL and 20 ng/mL. Platelet-poor plasma contained significantly lower concentrations of these compounds. Platelet-rich plasma was used to treat human fibroblast-like synoviocytes, and the resulting concentrations of mediators were corrected for the concentrations in the platelet-rich plasma alone. Compared with untreated fibroblast-like synoviocytes, synoviocytes treated with platelet-rich plasma exhibited significantly greater levels of MMP-1 (363 ± 94.0 ng/mL, p = 0.018) and MMP-3 (278 ± 90.0 ng/mL, p = 0.018). In contrast, platelet-poor plasma had little effect on mediators secreted by the synoviocytes. PDGFßß-treated fibroblast-like synoviocytes exhibited a broad proinflammatory cytokine response at four and forty-eight hours. CONCLUSIONS: Platelet-rich plasma was shown to contain a mixture of anabolic and catabolic mediators. Synoviocytes treated with platelet-rich plasma responded with substantial MMP secretion, which may increase cartilage catabolism. Synoviocytes responded to PDGF with a substantial proinflammatory response.

Correlation of intra-articular ankle pathology with cytokine biomarkers and matrix degradation products.

BACKGROUND: Articular cartilage degeneration is mediated by inflammatory cytokines and fragments of structural matrix proteins. Few studies have examined the role of these biomarkers in intra-articular pathology of the ankle. METHODS: Four groups of patients with increasing ankle pathology were enrolled. Group 1 included controls with no pain who underwent unrelated forefoot surgery. Group 2 included patients undergoing arthroscopy with intraoperative mild chondrosis. Group 3 included patients undergoing arthroscopy with moderate/severe chondrosis, osteochondral lesions, impingement, or loose bodies. Group 4 included positive controls with severe arthrosis undergoing ankle arthrodesis/arthroplasty. Ankle fluid was obtained by intra-articular aspiration and was assayed for IL-6, IFN-γ, MCP, MIP-1ß, and fibronectin-aggrecan complex (FAC), a matrix-degradation marker. There were 36 patients total, 21 males and 15 females with a mean age 45 (±16; range 18 to 76) years and a mean VAS for pain of 4.7 (±3.5; range 0 to 9). In groups 1 through 4, there were 11, 6, 15 and 4 patients respectively. RESULTS: The mean values of MCP-1 were 49.8 (±8.0) for minimal pathology and 133.9 (±33.0) for substantial pathology (pg/ml). The mean values of the FAC were 2.83 (±1.16) for minimal pathology and 9.62 (±2.23) for substantial pathology (optical density at 450 nm). The groups differed significantly in age, preoperative VAS, FAC, IL-6, and MCP-1 (p<0.05). CONCLUSION: There are differences in FAC and MCP-1 with increasing grades of severity of intra-articular pathology. CLINICAL RELEVANCE: These tests may play a role in determining the necessity for arthroscopy or intra-articular procedures in equivocal candidates.

Vaginal myomectomy is a safe and feasible procedure: a retrospective study of 46 cases.

AIM: The aim of this study was to establish the feasibility and safety of vaginal myomectomy via posterior colpotomy in a series of consecutive procedures performed by one surgeon. MATERIAL AND METHODS: We conducted a retrospective study in a tertiary care university hospital, involving 46 patients with symptomatic myomas and uteruses smaller than 16 gestational weeks and with no signs of pelvic disease. After a presurgical study, the patients underwent vaginal myomectomy. Characteristics of patients, position and size of myomas, operative data, intraoperative and postoperative complications, and length of hospital stay were recorded. RESULTS: Forty-four women underwent vaginal myomectomy and conversion to laparotomy was required in two cases (4.3%). Two patients suffered from infertility and one of these achieved pregnancy after the procedure. The median size of myomas was 50 mm (range 16-81). In two cases a culdoscopy was performed with a flexible fiberoptic gastroscope to better evaluate size and localization of myomas. Thirty-two patients underwent vaginal myomectomy under general anesthesia and 12 under locoregional anesthesia. The median vaginal operating time was 70 min (range 30-120). The estimated hemoglobin loss was 0.70 g/dL (range 0.40-3.35 g/dL). No severe intraoperative complications occurred. The median duration of hospital stay was 1 day (range 1-6). CONCLUSIONS: Vaginal myomectomy is a safe and feasible surgical procedure if performed by a well-trained, experienced surgeon.

Is the fibronectin-aggrecan complex present in cervical disk disease?

OBJECTIVE: To investigate the presence of inflammatory cytokines and the fibronectin-aggrecan complex (FAC) in persons undergoing surgical treatment for cervical radiculopathy caused by disk herniation. DESIGN: Single-center, prospective, consecutive case series. SETTING: A single large academic institution. PATIENTS: A total of 11 patients with radiculopathic pain and magnetic resonance imaging findings positive for disk herniation elected to undergo single-level cervical diskectomy. METHODS OR INTERVENTIONS: Lavage was performed by needle injection and aspiration upon entering the disk space for fluoroscopic localization before diskectomy. MAIN OUTCOME MEASUREMENTS: The lavage fluid was assayed for pH and the FAC, as well as for the cytokines interleukin-6 (IL-6), interferon-γ, monocyte chemotactic protein (MCP), and macrophage inhibitory protein-1ß. RESULTS: The subjects were 7 women and 4 men with a mean age of 50.6 years (SE 9.7; range, 36-70 years). The mean concentrations (SE; range) in picograms per milliliter were 7.9 (4.4; 0-44) for IL-6, 25.3 (15.5; 0-159) for interferon-γ, 16.1 (11.9; 0-121) for MCP, and 6.1 (2.8; 0-29) for macrophage inhibitory protein-1ß. The optical density of the FAC at 450 nm was 0.151 (0.036; 0.1-0.32), and the pH was 6.68 (0.1; 6.10-7.15). Statistically significant correlations were found between MCP and FAC (P = .036) and between FAC and pH (P = .008). CONCLUSIONS: Biochemical analysis of injured cervical intervertebral disks reveals the presence of inflammatory markers such as MCP, fragments of structural matrix proteins such as FAC, and a correlation with pH. Further evaluation of the FAC as a potential diagnostic biomarker or therapeutic target is warranted in the cervical spine.