Pedro Pohl

Effects of secreted factors in culture medium of annulus fibrosus cells on microvascular endothelial cells: elucidating the possible pathomechanisms of matrix degradation and nerve in-growth in disc degeneration

OBJECTIVE To test whether the interaction between annulus fibrosus cells (AFCs) and endothelial cells (ECs) disrupts matrix homeostasis and stimulates production of innervation mediators. METHODS Human microvascular ECs were cultured in the conditioned media of AF cell culture derived from degenerated human surgical specimen. Matrix-metalloproteinases (MMPs) and platelet-derived growth factor (PDGF) of ECs of this culture were analyzed by qRT-PCR, Western, and immunofluorescence. Vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), and nerve growth factor (NGF) in the media of this cell culture were assayed by ELISA. To determine the effects of ECs on AFCs, qRT-PCR was performed to determine mRNA levels of collagen I, II and aggrecan in AFCs cultured in EC conditioned media. RESULTS Compared to ECs cultured in naïve media, ECs exposed to AFC conditioned media expressed higher mRNA and protein levels of key biomarkers of invasive EC phenotype, MMP-2 (2×), MMP-13 (4×), and PDGF-B (1.5-2×), and NGF (24.9 ± 15.2 pg/mL vs 0 in naïve media). Treatment of AF cells with EC culture conditioned media decreased collagen type II expression two fold. Considerable quantities of pro-angiogenic factors IL-8 (396.7 ± 302.0 pg/mL) and VEGF (756.2 ± 375.9 pg/mL) were also detected in the conditioned media of untreated AF cell culture. DISCUSSION AFCs from degenerated discs secreted factors which stimulated EC production of factors known to induce matrix degradation, angiogenesis, and innervation. IL-8 and VEGF maybe the secreted factors from AFCs which mediate a pro-angiogenic stimulus often implicated in the development of disc degeneration.

Bone tissue repair in patients with open diaphyseal tibial fracture treated with biplanar external fixation or reamed locked intramedullary nailing

INTRODUCTION Open tibial fractures are usually caused by high-energy trauma. There is no consensus about the best treatment for these fractures. Biomechanical studies show that fixing on two planes approaches the rigidity of the bone, whereas the use of interlocking intramedullary nailing is widely used and reported to produce better therapeutic results in fracture healing. OBJECTIVE To compare bone tissue repair in patients with open diaphyseal tibial fracture treated with biplanar external fixation or reamed locked intramedullary nailing. METHOD Prospective randomised study with 68 patients undergoing two types of surgical treatment: biplanar external fixation or reamed locked intramedullary nailing. Consolidation, complications (infection, malunion and non-union) and quality of life using the SF-36 Health Survey were assessed 12 months after surgery. RESULTS Consolidation occurred in 84.6% of patients who underwent reamed intramedullary nailing, and in 90.3% of patients who were treated with biplanar external fixation. In the intramedullary nailing group, there were two cases of non-union, three cases of malunion and two cases of infection. In the patients treated with biplanar fixation, there were three cases of non-union, five cases of malunion and no cases of infection. There were no statistically significant differences between the treatment groups for these results. Patient quality of life was statistically equal for both methods. CONCLUSION Treatment with biplanar external fixation was associated with statistically similar results compared with intramedullary locking.

Catabolic effects of endothelial cell-derived microparticles on disc cells: Implications in intervertebral disc neovascularization and degeneration.

Neovascularization of intervertebral discs, a phenomenon considered pathological since normal discs are primarily avascular structures, occurs most frequently in annulus fibrosus (AF) of degenerated discs. Endothelial cells (ECs) are involved in this process, but the mechanism of the interaction between AF and endothelial cells is unclear. In this study, we evaluated the effects on matrix catabolic activity of AF cells by the extracellular endothelial microparticles (EMPs) and soluble protein factors (SUP fraction) produced from ECs. Passage 1 human AF cells grown in monolayer cultures were treated for 72 h with 250 µg of EMPs or SUP fraction isolated from culture of the microvascular endothelial cell line, HEMC‐I. Live‐cell imaging revealed uptake of EMPs by AF cells. RT‐PCR analysis demonstrated increased mRNA expression of MMP‐1 (50.3‐fold), MMP‐3 (4.5‐fold) and MMP‐13 (5.5‐fold) in AF cell cultures treated with EMPs compared to untreated control. Western analysis also demonstrated increased MMP protein expression in EMP‐treated AF cells. AF cells treated with the SUP fraction also exhibited a dramatic increase in MMP mRNA and protein expression. Increased MMP expression is primarily due to EMP or SUP stimulation of AF cells since EMPs or SUP fraction alone contained negligible amount of MMPs. Interestingly, MMP activity was elevated in AF cell cultures treated with EMPs but not with SUP. This study revealed enhanced matrix catabolism as a molecular consequence of action of ECs on AF cells via EMPs, which might be expected during neo‐angiogenesis of degenerating disc.

Traumatic L5-S1 spondylolisthesis in a 15-year-old: a case report.

Traumatic spondylolisthesis is a rare injury resulting from complex trauma and high-energy mechanisms. We present a case report of traumatic spondylolisthesis at the L5–S1 disc space of a patient who was buried after a wall fell on his back. In the physical examination, bilaterally decreased muscle strength was observed. Examination images indicated a 90% slip at L5–S1. Surgical treatment was provided with a posterior and anterior approach using pedicle fixation and an anterior cage. After 4 months, there was significant recovery of muscle strength in the lower limbs.

Adamts5 Deficiency Protects Mice From Chronic Tobacco Smoking-induced Intervertebral Disc Degeneration

Study Design. ADAMTS5-deficient and wild type (WT) mice were chronically exposed to tobacco smoke to investigate effects on intervertebral disc degeneration (IDD). Objective. The aim of this study was to demonstrate a role for ADAMTS5 in mediating tobacco smoking-induced IDD. Summary of Background Data. We previously demonstrated that chronic tobacco smoking causes IDD in mice because, in part, of proteolytic destruction of disc aggrecan. However, it was unknown which matrix proteinase(s) drive these detrimental effects. Methods. Three-month-old WT (C57BL/6) and ADAMTS5−/− mice were chronically exposed to tobacco smoke (four cigarettes/day, 5 day/week for 6 months). ADAMTS-mediated cleavage of disc aggrecan was analyzed by Western blot. Disc total glycosaminoglycan (GAG) content was assessed by dimethyl methylene blue assay and safranin O/fast green histology. Vertebral osteoporosity was measured by microcomputed tomography. Human nucleus pulposus (hNP) cell cultures were also exposed directly to tobacco smoke extract (TSE), a condensate containing the water-soluble compounds inhaled by smokers, to measure ADAMTS5 expression and ADAMTS-mediated cleavage of aggrecan. Activation of nuclear factor (NF)-&kgr;B, a family of transcription factors essential for modulating the cellular response to stress, was measured by immunofluorescence assay. Results. Genetic depletion of ADAMTS5 prevented vertebral bone loss, substantially reduced loss of disc GAG content, and completely obviated ADAMTS-mediated proteolysis of disc aggrecan within its interglobular domain (IGD) in mice following exposure to tobacco smoke. hNP cell cultures exposed to TSE also resulted in upregulation of ADAMTS5 protein expression and a concomitant increase in ADAMTS-mediated cleavage within aggrecan IGD. Activation of NF-&kgr;B, known to be required for ADAMTS5 gene expression, was observed in both TSE-treated hNP cell cultures and disc tissue of tobacco smoke-exposed mice. Conclusion. The findings demonstrate that ADAMTS5 is the primary aggrecanase mediating smoking-induced disc aggrecanolysis and IDD. Mouse models of chronic tobacco smoking are important and useful for probing the mechanisms of disc aggrecan catabolism and IDD. Level of Evidence: N/A

NSAID use in intervertebral disc degeneration: what are the effects on matrix homeostasis in vivo?

BACKGROUND CONTEXT Non-steroidal anti-inflammatory drugs (NSAIDs) are a widely used treatment for low back pain (LBP). Literature on NSAID use in articular cartilage has shown detrimental effects; however, minimal data exist to detail the effects of NSAIDs in intervertebral disc degeneration (IDD). As IDD is a major cause of LBP, we explored the effects of indomethacin, a commonly used NSAID, on disc matrix homeostasis in an animal model of IDD. PURPOSE This study aimed to determine the effects of oral indomethacin administration on IDD in an in vivo rabbit model. This study hypothesized that indomethacin use would accelerate the progression of IDD based upon serial imaging and tissue outcomes. STUDY DESIGN/SETTING This was a laboratory-based, controlled, in vivo evaluation of the effects of oral indomethacin administration on rabbit intervertebral discs. METHODS Six skeletally mature New Zealand white rabbits were divided into two groups: disc puncture alone to induce IDD (Puncture group) and disc puncture plus indomethacin (Punc+Ind group). The Punc+Ind group received daily administration of 6mg/kg oral indomethacin. Serial magnetic resonance imaging (MRI) was obtained at 0, 4, 8, and 12 weeks. The MRI index and the nucleus pulposus (NP) area were calculated. Discs were harvested at 12 weeks for determination of disc glycosaminoglycan (GAG) content, relative gene expression measured by real-time polymerase chain reaction, and histologic analyses. RESULTS The MRI index and the NP area of punctured discs in the Punc+Ind group demonstrated no worsening of degeneration compared with the Puncture group. Histologic analysis was consistent with less severe disc degeneration in the Punc+Ind group. Minimal differences in gene expression of matrix genes were observed between Puncture and Punc+Ind groups. The GAG content was higher in animals receiving indomethacin in both annulus fibrosus and NP at adjacent uninjured discs. CONCLUSIONS Oral indomethacin administration did not result in acceleration of IDD in an in vivo rabbit model. Future research is needed to ascertain long-term effects of indomethacin and other NSAIDs on disc matrix homeostasis.

Histological analysis of the repair of dural lesions with silicone mesh in rats subjected to experimental lesions

ABSTRACT Objective To evaluate inflammatory reaction, fibrosis and neovascularization in dural repairs in Wistar rats using four techniques: simple suture, bovine collagen membrane, silicon mesh and silicon mesh with suture. Methods Thirty Wistar rats were randomized in five groups: the first was the control group, submitted to dural tear only. The others underwent durotomy and simple suture, bovine collagen membrane, silicon mesh and silicon mesh with suture. Animals were euthanized and the spine was submitted to histological evaluation with a score system (ranging from zero to 3) for inflammation, neovascularization and fibrosis. Results Fibrosis was significantly different between simple suture and silicon mesh (p=0.005) and between simple suture and mesh with suture (p=0.015), showing that fibrosis is more intense when a foreign body is used in the repair. Bovine membrane was significantly different from mesh plus suture (p=0.011) regarding vascularization. Inflammation was significantly different between simple suture and bovine collagen membrane. Conclusion Silicon mesh, compared to other commercial products available, is a possible alternative for dural repair. More studies are necessary to confirm these findings.

Expressão de fatores da matriz durante o processo de neovascularização do disco intervertebral

OBJETIVO: Analizar el efecto de los productos de proteina de las celulas endoteliales (CEs) en el metabolismo celular del anillo fibroso (AF) en sistema in vitro de cultivo controlado.METODOS: Las celulas del AF humano se ampliaron en monocapa y se las trato con las proteinas obtenidas a partir de los medios de cultivo de la linea de celulas HMEC-1 (Human Microvascular Endothelial Cells) (125µg/ml). Despues de 72h de tratamiento, se aislo el ARN de las celulas de AF para el analisis de la expresion genica y se recogio el medio de cultivo para el analisis de expresion de la proteina.RESULTADOS: El analisis de qRT-PCR demostro una mayor expresion genica de las metaloproteinasas de matriz (MMP) en las celulas tratadas con productos de proteina de AF en las celulas endoteliales, en comparacion con el grupo de control de celulas AF: MMP-1 243,10 veces (p < 0,05), MMP-2 1,37 veces (p < 0,05), MMP-3 39,83 veces (p < 0,05) y MMP-13 5,70 veces (p < 0,05). En contraste, los inhibidores tisulares de las metaloproteinasas (TIMP), presentaron supresion de la expresion del gen TIMP-2 (0,55 veces) (p < 0,05) y TIMP-3 (0,60 veces) (p < 0,05) en los grupos expuestos. La expresion genica de agrecano (0,83 veces) (p < 0,05), importante componente de la matriz extracelular, tambien se redujo. La deteccion de MMP-1 y de MMP-3 fue realizada y se confirmaron los resultados de la PCR mediante la tecnica Western Blot.CONCLUSIONES: En el presente estudio se observo que las proteinas producidas por las CEs indujeron la expresion de MMP y suprimieron la expresion del TIMP y de agrecano en celulas primarias del disco intervertebral humano, con el objetivo de desarrollar posibles tratamientos para la degeneracion del disco intervertebral y el dolor discogenico asociado.

Expression of matrix factors in the process of neovascularization of intervertebral disc

OBJETIVO: Analizar el efecto de los productos de proteina de las celulas endoteliales (CEs) en el metabolismo celular del anillo fibroso (AF) en sistema in vitro de cultivo controlado.METODOS: Las celulas del AF humano se ampliaron en monocapa y se las trato con las proteinas obtenidas a partir de los medios de cultivo de la linea de celulas HMEC-1 (Human Microvascular Endothelial Cells) (125µg/ml). Despues de 72h de tratamiento, se aislo el ARN de las celulas de AF para el analisis de la expresion genica y se recogio el medio de cultivo para el analisis de expresion de la proteina.RESULTADOS: El analisis de qRT-PCR demostro una mayor expresion genica de las metaloproteinasas de matriz (MMP) en las celulas tratadas con productos de proteina de AF en las celulas endoteliales, en comparacion con el grupo de control de celulas AF: MMP-1 243,10 veces (p < 0,05), MMP-2 1,37 veces (p < 0,05), MMP-3 39,83 veces (p < 0,05) y MMP-13 5,70 veces (p < 0,05). En contraste, los inhibidores tisulares de las metaloproteinasas (TIMP), presentaron supresion de la expresion del gen TIMP-2 (0,55 veces) (p < 0,05) y TIMP-3 (0,60 veces) (p < 0,05) en los grupos expuestos. La expresion genica de agrecano (0,83 veces) (p < 0,05), importante componente de la matriz extracelular, tambien se redujo. La deteccion de MMP-1 y de MMP-3 fue realizada y se confirmaron los resultados de la PCR mediante la tecnica Western Blot.CONCLUSIONES: En el presente estudio se observo que las proteinas producidas por las CEs indujeron la expresion de MMP y suprimieron la expresion del TIMP y de agrecano en celulas primarias del disco intervertebral humano, con el objetivo de desarrollar posibles tratamientos para la degeneracion del disco intervertebral y el dolor discogenico asociado.

Expresión de factores de la matriz en el proceso de neovascularización del disco intervertebral

OBJETIVO: Analizar el efecto de los productos de proteina de las celulas endoteliales (CEs) en el metabolismo celular del anillo fibroso (AF) en sistema in vitro de cultivo controlado.METODOS: Las celulas del AF humano se ampliaron en monocapa y se las trato con las proteinas obtenidas a partir de los medios de cultivo de la linea de celulas HMEC-1 (Human Microvascular Endothelial Cells) (125µg/ml). Despues de 72h de tratamiento, se aislo el ARN de las celulas de AF para el analisis de la expresion genica y se recogio el medio de cultivo para el analisis de expresion de la proteina.RESULTADOS: El analisis de qRT-PCR demostro una mayor expresion genica de las metaloproteinasas de matriz (MMP) en las celulas tratadas con productos de proteina de AF en las celulas endoteliales, en comparacion con el grupo de control de celulas AF: MMP-1 243,10 veces (p < 0,05), MMP-2 1,37 veces (p < 0,05), MMP-3 39,83 veces (p < 0,05) y MMP-13 5,70 veces (p < 0,05). En contraste, los inhibidores tisulares de las metaloproteinasas (TIMP), presentaron supresion de la expresion del gen TIMP-2 (0,55 veces) (p < 0,05) y TIMP-3 (0,60 veces) (p < 0,05) en los grupos expuestos. La expresion genica de agrecano (0,83 veces) (p < 0,05), importante componente de la matriz extracelular, tambien se redujo. La deteccion de MMP-1 y de MMP-3 fue realizada y se confirmaron los resultados de la PCR mediante la tecnica Western Blot.CONCLUSIONES: En el presente estudio se observo que las proteinas producidas por las CEs indujeron la expresion de MMP y suprimieron la expresion del TIMP y de agrecano en celulas primarias del disco intervertebral humano, con el objetivo de desarrollar posibles tratamientos para la degeneracion del disco intervertebral y el dolor discogenico asociado.