Virtual Healthcare Simulation: Implementation, Distractions, and Preferences.

INTRODUCTION: Social distancing guidelines related to COVID-19 resulted in many simulation centers temporarily closing or adopting virtual simulation-based education (SBE). This mixed methods study aimed to evaluate our center's readiness to implement virtual SBE, the preferred method of delivery (virtual vs. nonvirtual), and any reported distractions. METHODS: Educators and simulation operations specialists (SOSs) used by our simulation center completed a survey focused on our center's implementation readiness for virtual SBE at 3 time points over a 3-week period. Three virtual simulation-based styles were developed: observer, vignette, and hybrid. All styles combined the use of Zoom and LearningSpace. Upon the completion of each session, learners, facilitators, and SOSs completed a survey focused on the preferred method of simulation delivery (virtual vs. nonvirtual) as well as any reported distractions during sessions. RESULTS: While some important lessons were learned, simulation team survey scores suggested an overall agreement in the center's preparedness during the 3-week implementation period. Most learners, facilitators, and SOSs preferred a nonvirtual delivery due to the "hands-on" component. Learners participating in the vignette style, however, significantly preferred virtual SBE due to "learning environment comfort" such as reduced anxiety, "better discussion," and "convenience." Reported distractions focused on "challenges with technology," "interruptions at home," "program logistics," and the "remote atmosphere." CONCLUSIONS: Most learners, facilitators, and SOSs preferred nonvirtual SBE; however, virtual SBE may prove beneficial for learners participating in the vignette style or particularly those experiencing anxiety. Future distractions may be mitigated for the simulation team and learners with proper preparedness.

Influence of Socioeconomic Bias on Emergency Medicine Resident Decision Making and Patient Care.

INTRODUCTION: Physician bias impacts clinical decision making, resulting in disparities in patient care. Most existing studies focus on sex and racial bias. This study aimed to investigate disparities in physician decision making among patients of varying socioeconomic status (SES). METHODS: Emergency medicine residents (n = 31) participated in 3 consecutive scenarios of similar disease acuity but with standardized patients of varying SES. Following the scenarios, residents met with a standardized participant acting as an attending physician for a handoff to recount their decision-making processes and care recommendations. Blinded raters evaluated clinical performance using an objective assessment tool. We assessed associations between patient SES and resident-ordered imaging, ordered medication, patient-perceived empathy, and clinical performance. We used qualitative analyses to study residents' decision-making processes. RESULTS: Quantitative analyses revealed no significant relationship between SES and resident-ordered imaging, ordered medications, patient-perceived empathy, and clinical performance. Qualitative analyses revealed 3 themes regarding clinical decision making: (1) overt diagnostic focus, (2) discharge planning, and (3) risk and exposure. CONCLUSIONS: Although quantitative analyses showed that SES did not affect clinical behavior within simulated scenarios, qualitative analyses uncovered 3 themes believed important to physician decision-making processes. Overt diagnostic focus may have resulted from the study environment in addition to organizational factors, policies, and training. Discharge planning, which was not explicitly studied, was often tailored to SES with emphasis placed on risks for patients of low SES. Further research is needed to uncover the nuances of bias, SES, and physician decision making throughout the patient care continuum and within various clinical environments.

Mucin 1, a signal transduction membrane-bound mucin, is present in human disc tissue and is downregulated in vitro by exposure to IL-1ß or TNF-α.

BACKGROUND: Back pain and disc degeneration have a growing socioeconomic healthcare impact. Mucin 1 (MUC1) is a transmembrane glycoprotein whose extracellular and intracellular domains participate in cellular signaling. Little is currently known about the presence or role of MUC1 in human disc degeneration. METHODS: In this IRB-approved research study, 29 human disc specimens were analyzed for MUC1 immunohistochemical localization and gene expression, and annulus fibrosus (annulus) cells were also isolated and cultured in 3D. Microarray analysis assessed expression levels of MUC1 in healthy and degenerated disc tissue and in cells exposed to proinflammatory cytokines (IL-1ß or TNF-α). RESULTS: MUC1 was shown to be present in annulus cells at the protein level using immunochemistry, and its expression was significantly upregulated in annulus tissue from more degenerated grade V discs compared to healthier grade I-II discs (p = 0.02). A significant positive correlation was present between the percentage of MUC1-positive cells and disc grade (p = 0.009). MUC1 expression in annulus cells cultured in 3D was also analyzed following exposure to IL-1ß or TNF-α; exposure produced significant MUC1 downregulation (p = 0.0006). CONCLUSIONS: Here we present the first data for the constitutive presence of MUC1 in the human disc, and its altered expression during disc degeneration. MUC1 may have an important role in disc aging and degeneration by acting as a regulator in the hypoxic environment, helping disc cells to survive under hypoxic conditions by stabilization and by activation of HIF-1α as previously recognized in pancreatic cancer cells.

Human annulus progenitor cells: Analyses of this viable endogenous cell population.

Back pain and intervertebral disc degeneration have growing socioeconomic/health care impacts. Increasing research efforts address use of stem and progenitor cell-based replacement therapies to repopulate and regenerate the disc. Data presented here on the innate human annulus progenitor cells: (i) assessed osteogenic, chondrogenic and adipogenic potentials of cultured human annulus cells; and (ii) defined progenitor-cell related gene expression patterns. Verification of the presence of progenitor cells within primary human disc tissue also used immunohistochemical identification of cell surface markers and microarray analyses. Differentiation analysis in cell cultures demonstrated a viable progenitor cell pool within Thompson grades III-IV discs. Osteogenesis was present in 8 out of 11 cultures (73%), chondrogenesis in 8 of 11 (73%), and adipogenesis in 6 of 6 (100%). Immunolocalization was positive for CD29, CD44, CD105, and CD14 (mean values 80.2%, 81.5%, 85.1%, and 88.6%, respectively); localization of CD45 and CD34 was negative in disc tissue. Compared to controls, surgical discs showed significantly downregulated genes with recognized progenitor cell functions: TCF7L2 (2.7 fold), BMI1 (3.8 fold), FGF receptor 2 (2 fold), PAFAH1B1 (2.3 fold), and GSTP1 (9 fold). Compared to healthier grade I/II discs, grade III/IV discs showed significantly upregulated XRCC5 (3.6 fold), TCF7L2 (6 fold), GSTP1 (3.7 fold), and BMI1 (3 fold). Additional significant cell marker analyses showed expression of platelet-derived growth factor receptor alpha, CD90, CD73, and STRO-1. Statement of Clinical Significance: Findings provide the first identification of progenitor cells in annulus specimens from older, more degenerate discs (in contrast to earlier studies of healthier discs or nondegenerative specimens from teenagers). Findings also increase knowledge on progenitor cells present in the disc and suggest their value in potential future utilization for regeneration and disc cell therapy. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1351-1360, 2016.

Human annulus signaling cues for nerve outgrowth: In vitro studies.

The relationship between neurotrophins produced by human annulus cells, such as neurotrophin-4 (NT4) and brain-derived neurotrophic factor (BDNF) which function in neurite survival and outgrowth, and nerve ingrowth into the disc remains poorly understood. In this work, we tested F11 neurite growth during exposure to control media, media with added nerve growth factor (NGF), conditioned media (CM) harvested from previous human annulus culture, or co-culture with annulus cells. Co-culture of F11 cells with annulus cells significantly increased media levels of amphiregulin, BDNF, glial-derived neurotrophic factor, and vascular endothelial growth factor compared to levels from in culture of F11 cells alone (p ≤ 0.04). Cell-based assays of neurite growth revealed that BDNF levels present in CM bore a significant (p = 0.01) positive relationship to neurite length and accounted for 38.5% of the change in neurite length. NT4 levels produced during co-culture with annulus cells bore a significant (p = 0.04) positive relationship to neurite length and accounted for 40.9% of the change in length. Statement of clinical significance: In vitro findings point to a potential role of annulus cells related to nerve ingrowth in vivo, and may have relevance in the outer annulus (where cell numbers are high) or in regions where nerves penetrate into annular tears or fissures. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1456-1465, 2016.

Autophagy in the Degenerating Human Intervertebral Disc: In Vivo Molecular and Morphological Evidence, and Induction of Autophagy in Cultured Annulus Cells Exposed to Proinflammatory Cytokines-Implications for Disc Degeneration.

STUDY DESIGN: Autophagy-related gene expression and ultrastructural features of autophagy were studied in human discs. OBJECTIVE: To obtain molecular/morphological data on autophagy in human disc degeneration and cultured human annulus cells exposed to proinflammatory cytokines. SUMMARY OF BACKGROUND DATA: Autophagy is an important process by which cytoplasm and organelles are degraded; this adaptive response to sublethal stresses (such as nutrient deprivation present in disc degeneration) supplies needed metabolites. Little is known about autophagic processes during disc degeneration. METHODS: Human disc specimens were obtained after institutional review board approval. Annulus mRNA was analyzed to determine autophagy-related gene expression levels. Immunolocalization and ultrastructural studies for p62, ATG3, ATG4B, ATG4C, ATG7, L3A, ULK-2, and beclin were conducted. In vitro experiments used IL-1ß- or TNF-α-treated human annulus cells to test for autophagy-related gene expression. RESULTS: More degenerated versus healthier discs showed significantly greater upregulation of well-recognized autophagy-related genes (P ≤ 0.028): beclin 1 (upregulated 1.6-fold); ATG8 (LC3) (upregulated 2.0-fold); ATG12 (upregulated 4.0-fold); presenilin 1 (upregulated 1.6-fold); cathepsin B (upregulated 4.5-fold). p62 was localized, and ultrastructure showed autophagic vacuolization and autophagosomes with complex, redundant whorls of membrane-derived material. In vitro, proinflammatory cytokines significantly upregulated autophagy-related genes (P ≤ 0.04): DRAM1 (6.24-fold); p62 (4.98-fold); PIM-2 oncogene, a positive regulator of autophagy (3-fold); WIPI49 (linked to starvation-induced autophagy) (upregulated 2.3-fold). CONCLUSION: Data provide initial molecular and morphological evidence for the presence of autophagy in the degenerating human annulus. In vivo gene analyses showed greater autophagy-related gene expression in more degenerated than healthier discs. In vitro data suggested a mechanism implicating a role of TNF-α and IL-1ß in disc autophagy. Findings suggest the importance of future work to investigate the relationship of autophagy to apoptosis, cell death, cell senescence, and mitochondrial dysfunction in the aging and degenerating disc. LEVEL OF EVIDENCE: N/A.

Mitochondrial Membrane Potential and Nuclear and Gene Expression Changes During Human Disc Cell Apoptosis: In Vitro and In Vivo Annulus Findings.

STUDY DESIGN: A study using cultured human annulus cells and human annular tissue. OBJECTIVE: To further explore and define mitochondrial mechanisms related to disc cell apoptosis in vitro and in vivo. SUMMARY OF BACKGROUND DATA: Mitochondrial-dependent intrinsic signaling pathways are a well-recognized component of apoptosis (programmed cell death). Disc cell apoptosis is important because it is a major mechanism by which cell numbers decrease during disc degeneration. Our objective was to further explore and define mitochondrial mechanisms related to disc cell apoptosis. METHODS: High-content screening techniques were used to study nuclear morphology and mitochondrial membrane potentials in cultured annulus cells. Gene expression in annulus tissue was studied with microarray analysis. RESULTS: Cultured cells showed significantly increased nuclear size (an indicator of apoptosis) with increasing Thompson grade (P < 0.00001 by analysis of variance). A significant negative correlation for mitochondrial potential (which results from the difference in electrical potential generated by the electrochemical gradient across the inner membrane of the mitochondrion) versus Thompson grade was identified in cultured human annulus cells in control conditions (r = 0.356, P < 0.0001). When exposed to the K ionophore valinomycin at sublethal levels to induce apoptosis, a significant reduction in mitochondrial potential was identified versus nontreated cells. Gene expression patterns in more degenerated Thompson grade III, IV, and V discs versus healthier grade I and II discs showed significant upregulation of a number of genes with well-recognized apoptosis roles in mitochondrial potential decline (ITM2B, beta-2-microglobulin, and cathepsin B, DAP, GAS1, and PDCD5) and TNF-α associations (cathepsin B, RAC1, and PPT1). CONCLUSION: Data presented here show the in vivo expression of apoptosis-related genes associated with the loss of mitochondrial membrane integrity and decreased mitochondrial membrane potential with increasing Thompson scores. These data, which mimic our novel, direct cell-based in vitro findings, stress the importance of mitochondrial changes related to apoptosis and TNF-α during human disc degeneration. LEVEL OF EVIDENCE: N/A.

High-mobility group box-1 gene, a potent proinflammatory mediators, is upregulated in more degenerated human discs in vivo and its receptor upregulated by TNF-α exposure in vitro.

Mechanisms which control and enhance proinflammatory cytokine expression during human disc degeneration are still poorly understood. The high-mobility group box-1 gene (HMGB1) produces a protein which can itself act as a cytokine, or can function as a potent proinflammatory mediator. Little is known about expression of HMGB1 in the human disc. Since proinflammatory cytokines increase significantly during human disc degeneration, in this work we hypothesized that HMGB1 may show upregulation with advancing stages of degeneration, and upregulation in cells exposed to TNF-α. Immunohistochemistry was performed to confirm the presence of HMGB1 in the human disc, and human annulus cells were cultured and challenged with 10(3)pM TNF-α for 14days in 3D culture. Cells with positive HMGB1 immunolocalization were abundant in the outer annulus. Molecular analysis of cultured cells showed an 8-fold significant increase in HMGB1 expression in more degenerated Thompson grade V discs compared to healthier grade I/II discs (p=0.033). Human disc tissue was assessed in molecular studies. Herniated specimens showed a 6.3-fold significantly greater expression level than that seen in control specimens (p=0.001). In culture experiments, expression of the receptor to HMGB1, toll-like receptor 2, showed a 24-fold upregulation in vitro in cells exposed to TNF-α vs. controls (p=0.0003).

Proinflammatory cytokines modulate the chemokine CCL2 (MCP-1) in human annulus cells in vitro: CCL2 expression and production.

Chemokines are important secondary inflammatory mediators released in response to stimuli which act as second-order cytokines with specialized functions in inflammation. The role of many of these specialized mediators is as yet poorly understood in the human intervertebral disc. Here we investigated CCL2 (chemokine (C-C motif) ligand 2, also known as monocyte chemotactic protein-1 (MCP-1)) in a study of its immunolocalization in disc tissue, and then hypothesized that exposure of cultured human annulus cells to proinflammatory cytokines might alter CCL2 gene expression and CCL2 production. CLL2 was localized to many disc cells in both herniated and non-herniated tissue specimens. Molecular analyses showed that cells exposed to IL-1ß showed a 5.5 fold upregulation in CCL2 gene expression vs. controls, p=0.017. Cells exposed to TNF-α showed a 7.7 fold upregulation vs. controls, p=0.005. Cultured cells (grades II-V) showed increased MCP-1 production in IL1-ß-treated cells vs. controls (p=0.016), with no significant difference in production in TNF-α-treated cells. Local production of CCL2 in vivo and vitro suggests that annulus cells may be primary effector cells (as well as target cells), with the ability to mediate physiological immune-related processes during disc degeneration by both autocrine and paracrine signaling.

A novel catechol-O-methyltransferase variant associated with human disc degeneration.

BACKGROUND: Disc degeneration and its associated low back pain are a major health care concern causing disability with a prominent role in this country's medical, social and economic structure. Low back pain is devastating and influences the quality of life for millions. Low back pain lifetime prevalence approximates 80% with an estimated direct cost burden of $86 billion per year. Back pain patients incur higher costs, greater health care utilization, and greater work loss than patients without back pain. METHODS: Research was performed following approval of our Institutional Review Board. DNA was isolated, processed and amplified using routine techniques. Amplified DNA was hybridized to Affymetrix Genome-Wide Human SNP Arrays. Quality control and genotyping analysis were performed using Affymetrix Genotyping Console. The Birdseed v2 algorithm was used for genotyping analysis. 2589 SNPs were selected a priori to enter statistical analysis using lotistic regression in SAS. RESULTS: Our objective was to search for novel single nucleotide polymorphisms (SNPs) associated with disc degeneration. Four SNPs were found to have a significant relationship to disc degeneration; three are novel. Rs165656, a new SNP found to be associated with disc degeneration, was in catechol-O-methyltransferase (COMT), a gene with well-recognized pain involvement, especially in female subjects (p=0.01). Analysis confirmed the previously association between COMT SNP rs4633 and disc degeneration. We also report two novel disc degeneration-related SNPs (rs2095019 and rs470859) located in intergenic regions upstream to thrombospondin 2. CONCLUSIONS: Findings contribute to the challenging field of disc degeneration and pain, and are important in light of the high clinical relevance of low back pain and the need for improved understanding of its fundamental basis.

Cortistatin is endogenous to the human intervertebral disc and exerts in vitro mitogenic effects on annulus cells and a downregulatory effect on TNF-α expression.

BACKGROUND CONTEXT: Cortistatin (CST) is a recently discovered cyclic neuropeptide with biologic anti-inflammatory properties relevant to disc degeneration. PURPOSE: To test whether CST is present in the disc tissue, whether its expression is influenced by tumor necrosis factor-α (TNF-α), and whether it influences cell proliferation. STUDY DESIGN: Institutional review board-approved study using immunohistochemistry on human disc tissue, in vitro annulus cultures to determine the effect of CST on cell proliferation, and the effect of TNF-α on CST gene expression. PATIENT SAMPLE: Discs from 12 subjects used for immunohistochemistry, four annulus specimens used for cell culture with proinflammatory cytokines, and 11 used for cell proliferation analyses. OUTCOME MEASURES: Immunohistochemical localization of CST, gene expression of CST, and cell proliferation analyses. METHODS: Immunohistochemistry localized CST in disc tissue. Microarray analysis measured CST gene expression. Human annulus cells were exposed to CST for proliferation tests or cultured for the effect of TNF-α on CST expression. Standard statistical analyses were performed. RESULTS: Immunohistochemistry identified CST in outer annulus, inner annulus, and nucleus tissue. Annulus cells exposed to TNF-α revealed significantly lower CST expression (p=.013). Exposure to CST significantly increased proliferation. Quantitative real-time polymerase chain reaction also confirmed expression of CST in vitro. CONCLUSIONS: Data provide the first evidence that CST is present in the human disc. Addition of CST significantly increased cell proliferation. Cortistatin expression was significantly downregulated by TNF-α exposure in vitro. Findings suggest possible in vivo reduction of the anti-inflammatory actions of CST because of elevated proinflammatory cytokines during degenerating disc.

Growth and differentiation factor-5 (GDF-5) in the human intervertebral annulus cells and its modulation by IL-1ß and TNF-α in vitro.

Growth and differentiation factor-5 (GDF-5) is a member of the TGF-ß superfamily which regulates cell division and differentiation. GDF-5 attracted high interest because of its role in skeletal development, especially in cartilaginous sites. Little is known, however, about the role of GFD-5 in disc cell biology. The present work demonstrated the immunohistologic presence of GDF-5 in human outer and inner annulus tissue. Microarray analysis of annulus cells showed significant upregulation of GDF-5 expression in herniated vs. non-herniated lumbar discs (2.14-fold change, p=0.021). In vitro three-dimensional culture studies challenged human annulus cells with IL-1ß and TNF-α, two proinflammatory cytokines known to be elevated in the human degenerating disc. Exposure resulted in significant downregulation of GDF-5 during both TNF-α exposure (5.83-fold change, p=0.044) and IL-1ß exposure (3.38-fold change, p=0.015). In vitro findings suggest that the degenerating disc milieu, with high proinflammatory cytokine levels, may limit expression of GDF-5, resulting in limited regenerative capacity of the intact disc.

Production and expression of RANTES (CCL5) by human disc cells and modulation by IL-1-ß and TNF-α in 3D culture.

Chemokines act as important secondary inflammatory mediators which are released by cells in response to a variety of stimuli. Chemokines bind to cell surface receptors and act as second-order cytokines with specialized functions in inflammation. The role of RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted) (also called CCL5 (chemokine (C-C motif) ligand 5)) has received little attention to date in disc tissue. Microarray analyses of lumbar disc annulus tissue revealed that RANTES expression was significantly upregulated in more degenerated Thompson grades IV and V discs compared to expression levels in grades I, II and III discs (p=0.032). Immunolocalization confirmed the presence of RANTES in the annulus and nucleus of the disc, and localized the RANTES receptors CCR1, CCR3 and CCR5 to cells in the disc. In vitro studies with IL-1-ß and TNF-α challenges, both proinflammatory cytokines resulted in elevated levels of RANTES in conditioned media (p<0.01); TNF-α exposure, however, produced significantly greater levels than did IL-1alpha (p<0.0001), suggesting a differential regulation by TNF-α. Local production of RANTES in vivo by annulus and nucleus cells, and in vitro induction of RANTES by proinflammatory cytokines suggest that disc cells are primary effector cells as well as target cells, and thus can mediate physiological immune-related processes during disc degeneration by both autocrine and paracrine signaling.

Human annulus cells regulate PAPP-A and IGFBP-4 expression, and thereby insulin-like growth factor bioavailability, in response to proinflammatory cytokine exposure in vitro.

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloproteinase which cleaves IGF binding protein (BP)-4 in the extracellular matrix, making IGF available to nearby cells. We have shown that PAPP-A is present in the human intervertebral disc, and is significantly upregulated in more degenerated discs where increased proinflammatory cytokine levels are present. We hypothesized that increased proinflammatory cytokines present in the degenerating disc might be related to PAPP-A expression. Experiments exposed human annulus cells to IL-1-ß or TNF-α to test this hypothesis. Treated cells showed significantly increased PAPP-A in conditioned media versus controls (p < 0.001). PAPP-A production following exposure to IL-1ß was significantly greater in cells derived from more degenerated versus healthier discs (p = 0.05). PAPP-A gene expression (microarray analysis) was significantly upregulated in IL-1ß- or TNF-α-exposed cells (p = 0.01-0.004). Quantitative RT-PCR confirmed significant upregulation of IGFBP-4 in IL-1ß- or TNF-α-exposed cells. Data have potential relevance to future cell-based biologic therapies for disc degeneration.

Genomewide molecular and biologic characterization of biomembrane formation adjacent to a methacrylate spacer in the rat femoral segmental defect model.

OBJECTIVES: This study focuses upon the morphologic and molecular features of the layer of cells, termed the "biomembrane," which forms around methacrylate spacers in bone segmental defects. The objective of this research was to assess the biomembrane formed in a novel rodent femoral segmental defect model at 4, 8, and 16 weeks with histologic and molecular studies. METHODS: Following Institutional Animal Care and Use Committee approval, a segmental defect was created in the rat femur and stabilized with the AO LockingRatNail and analyzed at 4, 8, and 16 weeks postsurgery using digital radiologic imaging, morphological and immunohistochemical studies, and genomewide gene expression studies employing microarray analysis. RESULTS: The biomembrane formed around the methacrylate spacer was rich in vasculature, which showed vascular endothelial growth factor immunolocalization. The biomembrane supported development of foci of bone and cartilage within it. Bone morphogenetic protein 2 immunolocalization and gene expression were positive within developing osseous and chondrocyte foci. Microarray analysis showed significant expression of key genes related to bone and cartilage formation and angiogenesis. CONCLUSIONS: This rat bone model was effective in creation of the biomembrane. Bone and cartilage foci were formed within the vascularized biomembrane with associated expression of genes critical for bone and cartilage development/formation and vascularization. The polymethyl methacrylate-induced biomembrane offers an exciting potential solution for segmental defects; the biomembrane, may act as a receptive bed and also serve as a source for mesenchymal stem cells, which could be recruited/directed for the healing process.

Cell-based tissue engineering augments tendon-to-bone healing in a rat supraspinatus model.

Rotator cuff pathology causes substantial pain/disability and health care costs. Cell-based tissue engineering offers promise for improved outcomes in tendon to bone healing. Cells from the tendon-bone interface were used here to amplify surgical defect healing in a rat model. Cells from tendon-to-bone interface of the rotator cuff were seeded in sponges and implanted into critical rotator cuff defects: Group I, control; II, surgical defect only; III, suture-repaired defect; IV, surgical defect, repair with sponge only; V, surgical defect, repair with sponge with cells. Three, 6-, and 12-week results were assessed for histologic features. At 3 weeks, histologic indices in Group V were significantly increased versus other treatment groups. Group V (12 weeks) showed significantly improved collagen organization versus other treatment groups; there was no difference in collagen organization in Group I versus V. In summary, increased cellularity, inflammation, vascularity, and collagen organization were present at 3 weeks; increased collagen organization at 12 weeks in Group V provides evidence for improved healing with cells. Data further support the utility of tendon-bone interface cells in rotator cuff healing.

Genome-wide analysis of pain-, nerve- and neurotrophin -related gene expression in the degenerating human annulus.

BACKGROUND: In spite of its high clinical relevance, the relationship between disc degeneration and low back pain is still not well understood. Recent studies have shown that genome-wide gene expression studies utilizing ontology searches provide an efficient and valuable methodology for identification of clinically relevant genes. Here we use this approach in analysis of pain-, nerve-, and neurotrophin-related gene expression patterns in specimens of human disc tissue. Control, non-herniated clinical, and herniated clinical specimens of human annulus tissue were studied following Institutional Review Board approval. RESULTS: Analyses were performed on more generated (Thompson grade IV and V) discs vs. less degenerated discs (grades I-III), on surgically operated discs vs. control discs, and on herniated vs. control discs. Analyses of more degenerated vs. less degenerated discs identified significant upregulation of well-recognized pain-related genes (bradykinin receptor B1, calcitonin gene-related peptide and catechol-0-methyltransferase). Nerve growth factor was significantly upregulated in surgical vs. control and in herniated vs. control discs. All three analyses also found significant changes in numerous proinflammatory cytokine- and chemokine-related genes. Nerve, neurotrophin and pain-ontology searches identified many matrix, signaling and functional genes which have known importance in the disc. Immunohistochemistry was utilized to confirm the presence of calcitonin gene-related peptide, catechol-0-methyltransferase and bradykinin receptor B1 at the protein level in the human annulus. CONCLUSIONS: Findings point to the utility of microarray analyses in identification of pain-, neurotrophin and nerve-related genes in the disc, and point to the importance of future work exploring functional interactions between nerve and disc cells in vitro and in vivo. Nerve, pain and neurotrophin ontology searches identified numerous changes in proinflammatory cytokines and chemokines which also have significant relevance to disc biology. Since the degenerating human disc is primarily an avascular tissue site into which disc cells have contributed high levels of proinflammatory cytokines, these substances are not cleared from the tissue and remain there over time. We hypothesize that as nerves grow into the human annulus, they encounter a proinflammatory cytokine-rich milieu which may sensitize nociceptors and exacerbate pain production.

Deleterious effects of discography radiocontrast solution on human annulus cell in vitro: changes in cell viability, proliferation, and apoptosis in exposed cells.

BACKGROUND CONTEXT: Carragee et al. have recently shown that modern discography injections are associated with subsequent acceleration of disc degeneration, herniation, and loss of disc height. Although needle puncture and pressurization are known trauma events that can create disc degeneration in animal models, another likely culprit in clinical discography-associated degeneration is a direct effect of the contrast agent itself on disc cells. PURPOSE: To test the hypothesis that discography contrast solution would have a deleterious effect on human annulus cells in vitro. STUDY DESIGN: An in vitro study using cultured human annulus cells to assay cell death, cell proliferation, and apoptosis. PATIENT SAMPLE: Annulus cells from eight surgical disc specimens were evaluated (two Thompson Grade III discs and six Grade IV discs) for cell death and proliferation, and an additional five cultures were tested for apoptosis. OUTCOME MEASURES: The proportion of dead and live cells, cell proliferation, and the proportion of apoptotic cells in control and experimental groups. METHODS: After internal review board approval, experimental design used two sets of controls: untreated cells under our normal culture conditions (control) and a set with added glucose to adjust the osmolality to match respective Isovue radiocontrast solution treatments (glucose controls) using a freezing point osmometer. Treated cells received Isovue 200 (iopamidol, Isovue-M 200; Bracco Diagnostics, Inc., Princeton, NJ, USA) at 12.5, 25, 50, or 100 mg/mL. Twenty thousand cells/well were seeded in triplicate in 24 well plates, control or test media added, and incubated for 24 hours. At termination, dead cells were identified with trypan blue staining and percentage dead cells determined. Cells were also tested to determine the percentage of apoptotic cells after 50 or 100 mg/mL Isovue exposures. Proliferation assays used standard plate reader methods. Statistical analysis used repeated measures analysis of variance with SAS software (version 9.2; SAS Institute, Inc., Cary, NC, USA). RESULTS: Analysis of cell death showed a significant increase in the percentage of dead cells with increasing Isovue concentrations compared with control cells (p=.018-.0008). Cell proliferation analyses showed significantly reduced division in Isovue-treated cells (p=.004), and apoptosis assays revealed a significantly higher proportion of apoptotic cells in cells exposed to 50 and 100 mg/mL Isovue (p=.016 and .0003, respectively). CONCLUSIONS: Discography is used extensively in the evaluation of low back pain. Because the lifetime prevalence of disc degeneration and low back pain is high (80% in the general population), many patients may undergo this procedure. Data presented here show that cells exposed in vitro to a radiocontrast agent with adjustments for osmolality have significantly reduced proliferation, increased cell death, and increased programmed cell death (apoptosis). In light of the well-recognized age- and degeneration-related decrease in disc cell numbers, it is possible that radiocontrast exposure may be contributing significantly to disc cell loss with subsequent progression of disc degeneration. Findings presented here provide a plausible cell-based explanation for the previously reported disc degeneration in patients receiving discography contrast solutions.

Osteogenic and chondrogenic potential of biomembrane cells from the PMMA-segmental defect rat model.

A layer of cells (the "biomembrane") has been identified in large segmental defects between bone and surgically placed methacrylate spacers or antibiotic-impregnated cement beads. We hypothesize that this contains a pluripotent stem cell population with potential valuable applications in orthopedic tissue engineering. Objectives using biomembranes harvested from rat segmental defects were to: (1) Culture biomembrane cells in specialized media to direct progenitor cells along bone or cartilage cell differentiation lineages; (2) evaluate harvested biomembranes for mesenchymal stem cell markers, and (3) define relevant gene expression patterns in harvested biomembranes using microarray analysis. Culture in osteogenic media produced mineralized nodules; culture in chondrogenic media produced masses containing chondroitin sulfate/sulfated proteoglycans. Molecular analysis of biomembrane cells versus control periosteum showed significant upregulation of key genes functioning in mesenchymal stem cell differentiation, development, maintenance, and proliferation. Results identified significant upregulation of WNT receptor signaling pathway genes and significant upregulation of BMP signaling pathway genes. Findings confirm that the biomembrane has a pluripotent stem cell population. The ability to heal large bone defects is clinically challenging, and novel tissue engineering uses of the biomembrane hold great promise in treating non-unions, open fractures with large bone loss and/or infections, and defects associated with tumor resection.

Matrix metalloproteinase-26, a novel MMP, is constitutively expressed in the human intervertebral disc in vivo and in vitro.

Matrix metalloproteinase (MMP) regulation and expression is important in the aging/degenerating human intervertebral disc. MMP-26 (also known as matrilysin-2 or endometase) is a newly discovered MMP which degrades type IV collagen, fibronectin, fibrinogen, vitronectin, denatured collagen types I-IV, insulin-like growth factor binding protein 1, and activated pro-MMP-9. Our objective here was to determine if it is present in human disc tissue and cultured disc cells. Immunohistochemistry and microarray gene expression analyses were used to evaluate the presence of MMP-26 in human disc tissue from healthy and degenerated discs. Immunohistochemistry was also applied to human annulus cells cultured in a collagen sponge. Cellular and matrix localization of MMP-26 was identified in the outer and inner annulus and in the nucleus pulposus. Fewer cells showed localization in the inner vs. outer annulus, and localization was sparse in the nucleus. During in vitro culture of annulus cells, MMP-26 was also expressed. Molecular analyses showed significant downregulation of expression of MMP-26 (p=0.03), and significant 9.8-fold upregulation of TGF-beta (p=0.01) in more degenerated discs vs. healthier discs. Findings document the first identification of MMP-26 in the disc at the molecular and protein levels. Results point to the potentially important role of MMP-26 in matrix modulation during disc health and degeneration.