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.

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.

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.

Retrograde ejaculation after anterior lumbar interbody fusion using rhBMP-2: a cohort controlled study.

BACKGROUND CONTEXT: The commercially available growth factor recombinant bone morphogenic protein-2 (rhBMP-2) used in spinal fusion has been associated with numerous adverse reactions, including inflammatory reactions in soft tissue, heterotopic bone formation, radiculitis, osteolysis, and cage or graft subsidence. The original Food and Drug Administration Summary of anterior lumbar interbody fusion (ALIF) reported 12 retrograde ejaculation (RE) events (8%) in the rhBMP-2 groups compared with (1.4%) in the control group. It had been debated whether this finding was related to rhBMP-2 use. PURPOSE: To compare the incidence of RE after ALIF in patients with and without rhBMP-2 use. STUDY DESIGN: Retrospective analysis of prospectively gathered outcomes data on consecutive subjects having ALIF with and without rhBMP-2 use. PATIENT SAMPLE: Male patients with lumbar spondylosis or spondylolisthesis having ALIF of the lowest one or two lumbar levels with and without rhBMP-2. OUTCOME MEASURE: Report of RE as a new finding after ALIF. METHODS: From the comprehensive outcome database at a high-volume university practice, male subjects having ALIF for one- (L5/S1) or two-level (L4/L5, L5/S1) lumbar fusion were identified. Retrograde ejaculation events were recorded and comparative incidence compared. RESULTS: The two groups were comparable for age and additional procedures performed. There were 69 L5/S1 ALIFs performed with rhBMP-2 and 174 ALIFs performed without rhBMP-2 during the study period. Of those, 24 and 64 were two-level ALIFs performed with and without rhBMP-2, respectively. There were five RE events (7.2%) reported in the rhBMP-2 group and 1 (0.6%) in the control group. Comparing single-level L5/S1 ALIF, there was a 6.7% and 0% rate of RE in the rhBMP-2 versus control groups, respectively. At 1 year after surgery, three of six affected subjects reported resolution of the RE. CONCLUSION: This study confirms previous reports of a higher rate of RE in ALIF procedures using rhBMP-2. This may be an important consideration in subjects concerned with sterility after surgery.

Are persistently symptomatic vertebral compression fractures associated with abnormal inflammatory profiles? A prospective study.

STUDY DESIGN: A case-control study with prospectively collected samples for laboratory analysis in a series of patients with spinal fragility fractures and a series of patients without fracture who underwent fusion for LBP. OBJECTIVE: Was an exploratory data analysis for candidate cytokine biomarkers present in the fracture milieu of patients with persistent back pain associated with vertebral compression fracture. SUMMARY OF BACKGROUND DATA: Lumbar and thoracic compression fractures are common. Little is known about the presence of inflammatory mediators within fractured vertebra in the clinical setting. METHODS: Thirty patients diagnosed with a single thoracic or lumbar compression fracture were treated with single level vertebroplasty. At the time of intervention, needle aspiration was carried out at the fractured level. A multiplexed bead assay was used to assess the presence of 27 different cytokines and inflammatory mediators. A control group consisted of needle aspiration samples of 30 lumbar vertebra from 13 patients with chronic pain but no fracture undergoing open instrumented fusion. RESULTS: Thirty patients with 30 fractures consisted of 23 female and 7 male with a mean age of 77.5 years (SD 13.6; range 42 to 97) and a mean of 3.9 weeks of pain (SD 3.1; range 1 to 12). The highest levels of inflammatory mediators were (in order): IL-1 receptor antagonist, PDGF, RANTES, IP-10, IL-8, and eotaxin. These mediators were present at concentrations>200 pg/mL. Compared with controls with chronic pain, significant differences were present for 4 mediators: TNF, MIP-1b, IL-9, and IL-12. The panel of these 4 markers was 93.3% specific and 66.7% sensitive for fracture compared with the control group. CONCLUSIONS: Inflammatory mediators are present in needle aspirates of symptomatic vertebral compression fractures. Some of these mediators show different levels than in patients with chronic pain but no fracture. LEVEL OF EVIDENCE: Diagnostic level of evidence II.

Identification of a novel fibronectin-aggrecan complex in the synovial fluid of knees with painful meniscal injury.

BACKGROUND: Molecular biomarkers associated with knee pain may be useful as diagnostic modalities, prognostic indicators, and surrogate end points for therapeutic trials. The present study describes a novel complex of fibronectin and aggrecan that is present in the affected knee of patients with pain and meniscal abnormality. METHODS: The present prospective study included thirty patients with knee pain, mechanical symptoms, and magnetic resonance imaging findings that were positive for a meniscal tear who chose arthroscopic partial meniscectomy after unsuccessful nonoperative management. Synovial fluid was aspirated at the time of surgery and was assayed for the fibronectin-aggrecan complex with use of a heterogeneous enzyme-linked immunosorbent assay (ELISA). The results were compared with knee aspirates from ten asymptomatic volunteers with no pain who underwent magnetic resonance imaging of the knee. RESULTS: The mean optical density (and standard deviation) of the fibronectin-aggrecan complex was significantly greater in synovial fluid from knees undergoing arthroscopic surgery as compared with fluid from asymptomatic controls (13.29 ± 8.48 compared with 0.03 ± 0.09; p < 0.001). The mean age in the study group was significantly greater than in control group (46.0 ± 12.6 compared with 38.5 ± 6.0 years; p = 0.02), but controlling for age did not affect the results. Post hoc, an optical density cutoff value of 0.3 distinguished the study group from the control group with 100% accuracy. CONCLUSIONS: A novel fibronectin-aggrecan complex is present in the synovial fluid of painful knees with meniscal abnormality. The fibronectin-aggrecan complex may prove to be useful as a clinical biomarker or therapeutic target. Further research is warranted to correlate functional outcome after surgery with the fibronectin-aggrecan complex and other cartilage biomarkers.

Outcome of lumbar epidural steroid injection is predicted by assay of a complex of fibronectin and aggrecan from epidural lavage.

STUDY DESIGN: A single-center, prospective, consecutive case series of patients undergoing epidural lavage before the treatment of radiculopathy due to lumbar disc herniation. OBJECTIVE: To determine whether a novel complex of fibronectin and aggrecan predicts clinical response to epidural steroid injection (ESI) for the indication of radiculopathy from lumbar herniated nucleus pulposus (HNP). SUMMARY OF BACKGROUND DATA: ESI for lumbar radiculopathy due to HNP is widely used despite variable effectiveness for this indication. With increased attention aimed at cost containment, it would be beneficial to identify those in whom ESI may be helpful. There are currently no accurate diagnostic tests to predict response to ESI in back pain and sciatica syndromes. We have previously investigated biomarkers of disc degeneration associated with radiculopathy. METHODS: We embarked to determine whether a molecular complex of fibronectin and aggrecan predicts clinical response to ESI for the indication of radiculopathy from HNP. This prospective study was conducted at a single center and included 26 patients with radiculopathic pain and magnetic resonance imaging positive for HNP, who elected ESI. Epidural lavage with physiologic saline was performed immediately before ESI. The lavage fluid was assayed for the fibronectin-aggrecan complex (FAC) by using a heterogeneous sandwich enzyme-linked immunosorbent assay. The results were compared with the interval improvement in the physical component summary (PCS) score of the Medical Outcomes Study Short Form-36 instrument (SF-36) after injection compared with baseline. RESULTS: The mean improvement from baseline PCS in patients with the FAC was 22.9 (SD, 12.4) and without the complex was 0.64 (SD, 3.97; P < 0.001). Differences in total SF-36 improvement were also highly significant (P < 0.001). The presence of the FAC predicts a clinically significant increase in PCS after lumbar ESI by receiver-operating-characteristic analysis (area under the curve = 0.97; P < 0.001). There was no significant difference in age (P = 0.25), sex (P = 0.84), laterality (P = 0.06), lumbar spinal level (P = 0.75), or payer type (worker's compensation vs. private insurance; P = 0.90) between groups with and without the marker. CONCLUSION: A molecular complex of fibronectin and aggrecan predicts response to lumbar ESI for radiculopathy with HNP. The biomarker is accurate, objective, and not affected by demographic or psychosocial variables in this series.

A case of late epidural hematoma in a patient on clopidogrel therapy postoperatively: when is it safe to resume antiplatelet agents?

BACKGROUND CONTEXT: The use of antiplatelet agents after coronary artery stent placement is currently recommended to prevent coronary stent obstruction. These patients may have concurrent disabling spinal stenosis and require spinal decompression. Resuming antiplatelet agents as soon as possible after spinal surgery is recommended. PURPOSE: To describe a unique case of late postoperative epidural hematoma occurring with the use of clopidogrel. STUDY DESIGN: A case report and review of the literature. METHODS: The hospital chart, history, physical examination, and imaging of a single patient were reviewed. RESULTS: A 59-year-old man underwent spinal decompression and fusion for neurogenic claudication with lumbar spinal stenosis and spondylolisthesis while managed on clopidogrel for prevention of thrombosis after cardiac stent placement. He developed a symptomatic epidural hematoma 12 days postoperatively, well outside the usual time frame for this complication. The patient was closely monitored, and lumbar radiculopathy resolved over the ensuing days. CONCLUSION: After spinal surgery and resumption of antiplatelet therapy, the physician needs to maintain vigilance in observing patients for late postoperative complications such as epidural hematoma, which could have catastrophic consequences if not recognized in a timely manner.

Accelerated degeneration after failed cervical and lumbar nucleoplasty.

STUDY DESIGN: Observational cohort study. SUMMARY OF BACKGROUND DATA: Studies evaluating the treatment of presumed discogenic spine pain using nucleoplasty have reported variable success rates. It has been suggested that these procedures lower the intradiscal pressure, reduce disk protrusion, improve disk hydration, and restore disk height. It is proposed that such structural changes in treated disks correspond to the clinical improvement in patients. Radiographic and clinical evidence showing the efficacy of nucleoplasty remains inadequate. OBJECTIVE: To document the comparative changes in magnetic resonance imaging (MRI) the appearance of disk morphology as reflected by Pfirrmann classification scores before and after the nucleoplasty treatment in patients with continued symptoms. METHODS: Twenty-eight consecutive patients with persistent symptoms after nucleoplasty within 1 year of treatment were evaluated. Prenucleoplasty and postnucleoplasty MRIs were evaluated and Pfirrmann scores of the symptomatic level were determined. RESULTS: In all the treated patients, comparison between the prenucleoplasty and the postnucleoplasty MRI of the targeted disks failed to show increased signal hydration, disk space height improvement, or shrinkage of the preoperative disk bulge at a mean time of 6 months after the procedure. Of the 17 cervical levels treated in 12 patients, 5 seemed to show progressive degeneration at treated levels (42% of the patients). Of the 17 lumbar procedures in 16 patients, 4 seemed to show progressive degeneration (25% of the patients) and 1 developed a new spondylolisthesis (6.3%). Thus, 32% of the patients in our cohort showed progressive degeneration at the treated level. The median Pfirrmann score in both prenucleoplasty and postnucleoplasty was 2, and the mean Pfirrmann classification prenucleoplasty and postnucleoplasty was 1.8 and 2.1, respectively (P<0.05, 2-tailed t test). CONCLUSION: This study failed to detect any morphologic improvement of disk abnormalities by MRI evaluation in patients with persistent pain, who then underwent nucleoplasty. Thirty-two percent showed progressive degeneration in less than 1 year after nucleoplasty, a rate greater than expected by natural progression during the interval of examination.