Andreas Seekamp

Cytokines and adhesion molecules in elective and accidental trauma-related ischemia/reperfusion.

BACKGROUND The major pathophysiologic role of cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and IL-6, as well as of the (soluble) adhesion molecules ICAM-1 and E-selectin, has been identified using different experimental models of ischemia/reperfusion injury. Moreover, in intensive care management, evaluation of these agents as diagnostic or prognostic tools is of great interest in ischemia/reperfusion injury caused by surgical or accidental trauma. For this reason, inflammatory mediators including those mentioned above were investigated in three different groups of surgical patients. METHODS The first group (A, n = 13) comprised patients undergoing elective limb surgery without a tourniquet. The second group (B, n = 36) included patients subjected to limb surgery with a tourniquet. The third group (C, n = 30) was composed of accidental trauma patients who were retrospectively divided into those with and without multiple organ dysfunction (+MOD and -MOD, respectively) as defined by the Denver Score. Serial blood samples were taken during a 5-day (elective surgery) or 14-day (accidental trauma) observation period for monitoring of cytokines and soluble adhesion molecules. The clinical course and the degree of MOD were recorded daily. RESULTS Only when a tourniquet was applied for a mean time of 105 minutes did elective limb surgery result in significantly increased serum levels of IL-6, IL,-1ra, and IL-10 but not TNF-RII. Yet, the increase in cytokine levels was not sufficient to cause an enhanced shedding of adhesion molecules, and both soluble ICAM-1 and soluble E-selectin remained unchanged in groups A and B throughout the 5-day observation period. In patients with multiple injuries (group C), all parameters increased early after trauma up to 10- to 20-fold in comparison with the elective limb surgery patients in groups A and B. When the accidental trauma patients were divided according to the Denver Score for +MOD (n = 8, mean Injury Severity Score = 33.8) and -MOD (n = 22, mean Injury Severity Score = 31.2), a clear difference became evident in serum IL-6 and IL-1ra levels within the first 4 days and in serum IL-10 levels for the first 2 days after trauma, with cytokine levels being significantly higher in the +MOD patients 3 to 4 days before the onset of MOD. Although highly elevated, TNF-RII levels did not differentiate between +MOD and -MOD at any time. The increase in serum cytokine levels was associated with a remarkable expression and shedding of ICAM-1 and E-selectin made obvious by significantly increased soluble serum ICAM-1 levels in +MOD patients compared with the -MOD group between days 3 and 5 after trauma and increased soluble serum E-selectin levels between days 2 and 4 after trauma. CONCLUSION The release of cytokines and soluble adhesion molecules into the circulation correlates well with the degree of trauma (elective surgery vs. accidental multiple trauma), depending on the extent of the associated ischemia/reperfusion injury. Both groups of mediators are also clearly related to the development of MOD in patients with multiple injuries with generalized ischemia/reperfusion injury caused by hemorrhagic shock. They may be predictive of patients at risk for MOD when measured early in the posttraumatic period.

Survival trends and predictors of mortality in severe pelvic trauma: Estimates from the German Pelvic Trauma Registry Initiative

STUDY OBJECTIVE To determine longitudinal trends in mortality, and the contribution of specific injury characteristics and treatment modalities to the risk of a fatal outcome after severe and complex pelvic trauma. METHODS We studied 5048 patients with pelvic ring fractures enrolled in the German Pelvic Trauma Registry Initiative between 1991 and 1993, 1998 and 2000, and 2004 and 2006. Complete datasets were available for 5014 cases, including 508 complex injuries, defined as unstable fractures with severe peri-pelvic soft tissue and organ laceration. Multivariable mixed-effects logistic regression analysis was employed to evaluate the impact of demographic, injury- and treatment-associated variables on all-cause in-hospital mortality. RESULTS All-cause in-hospital mortality declined from 8% (39/466) in 1991 to 5% (33/638) in 2006. Controlling for age, Injury Severity Score, pelvic vessel injury, the need for emergency laparotomy, and application of a pelvic clamp, the odds ratio (OR) per annum was 0.94 (95% confidence interval [CI] 0.91-0.96). However, the risk of death did not decrease significantly in patients with complex injuries (OR 0.98, 95% CI 0.93-1.03). Raw mortality associated with this type of injury was 18% (95% CI 9-32%) in 2006. CONCLUSION In contrast to an overall decline in trauma mortality, complex pelvic ring injuries remain associated with a significant risk of death. Awareness of this potentially life-threatening condition should be increased amongst trauma care professionals, and early management protocols need to be implemented to improve the survival prognosis.

Intraarticular injection of platelet-rich plasma reduces inflammation in a pig model of rheumatoid arthritis of the knee joint.

OBJECTIVE Treatment options for rheumatoid arthritis range from symptomatic approaches to modern molecular interventions such as inhibition of inflammatory mediators. Inhibition of inflammation by platelet-rich plasma (PRP) has been proposed as a treatment for tendinitis and osteoarthritis. The present study was undertaken to investigate the effect of PRP on antigen-induced arthritis (AIA) of the knee joint in a large animal model. METHODS Six-month-old pigs (n = 10) were systemically immunized by bovine serum albumin (BSA) injection, and arthritis was induced by intraarticular BSA injection. PRP was injected into the knee joints of 5 of the animals after 2 weeks. An additional 5 animals received no systemic immunization (controls). Signs of arthritis were documented by plain histologic analysis, Safranin O staining, and immunohistochemistry analysis for type II collagen (CII), interleukin-6 (IL-6), and vascular endothelial growth factor (VEGF). Interleukin-1β (IL-1β), IL-6, tumor necrosis factor α (TNFα), VEGF, and insulin-like growth factor 1 (IGF-1) protein content was measured by Luminex assay. RESULTS In the pigs with AIA, plain histologic analysis revealed severe arthritic changes in the synovium. Safranin O and CII staining showed decreased proteoglycan and CII content in cartilage. Immunohistochemistry analysis revealed increased levels of IL-6 and VEGF in synovium and cartilage, and protein concentrations of IL-6, VEGF, IL-1β, and IGF-1 in synovium and cartilage were elevated as well; in addition, TNFα protein was increased in cartilage. Treatment with PRP led to attenuation of these arthritic changes in the synovium and cartilage. CONCLUSION We have described a porcine model of AIA. Experiments using this model demonstrated that PRP can attenuate arthritic changes as assessed histologically and based on protein synthesis of typical inflammatory mediators in the synovial membrane and cartilage.

Platelet-released growth factors can accelerate tenocyte proliferation and activate the anti-oxidant response element

Little is know about the pathophysiology of acute and degenerative tendon injuries. Although most lesions are uncomplicated, treatment is long and unsatisfactory in a considerable number of cases. Besides the common growth factors that were shown to be relevant for tendon integrity more recently protection against oxidative stress was shown to promote tendon healing. To improve tendon regeneration, many have advocated the use of platelet-rich plasma (PRP), a thrombocyte concentrate that can serve as an autologous source of growth factors. In this study, we investigated the effect of platelet-released growth factors (PRGF) on tenocytes. Tenocytes were isolated from the Achilles tendon of postnatal rats. Tenocyte cell cultures were stimulated with PRGF. We used a CyQuant assay and WST assay to analyse tendon cell growth and viability in different concentrations of PRGF. Migration and proliferation of cells grown in PRGF were assessed by a scratch test. A dual-luciferase assay was used to demonstrate the activation of the anti-oxidant response element (ARE) in tenocytes. A positive effect of PRGF could be shown on tendon cell growth and migratory capacity. PRGF activated the Nrf2–ARE pathway in a dose-dependent manner. Here, we provide evidence of a biological effect of PRGF on tenocytes by the promotion of tenocyte growth and activation of the Nrf2–ARE pathway. This is a novel aspect of the action of platelet concentrates on tendon growth.

Osteoblasts participate in the innate immunity of the bone by producing human beta defensin-3

Gram-positive bacterial bone infections are an important cause of morbidity particularly in immunocompromised patients. Antimicrobial peptides (AP) are effectors of the innate immune system and directly kill microorganisms in the first hours after microbial infection. The aim of the present investigation was to study the expression and regulation of gram-positive specialized human β-defensin-3 (HBD-3) in bone. Samples of healthy and osteomyelitic human bone were assessed for the expression of HBD-3. Using primary and immortalized osteoblasts (SAOS-2 cells), release and regulation of HBD-3 was evaluated after exposure to Staphylococcus aureus supernatant and/or corticosteroids using PCR, immunohistochemistry, Western blot and ELISA. To determine the role of toll-like-receptors-2 and -4 (TLR-2/-4), shRNA was used to downregulate TLRs. An osteomyelitis mouse model was created performed to investigate the release of murine β-defensins using immunohistochemistry and RT-PCR. Cultured osteoblasts and human bone produce HBD-3 under standard conditions. The release increases within hours of bacterial supernatant exposure in cultured osteoblasts. This observation was not made in chronically infected bone samples. The shRNA-technology revealed the necessity of TLR-2 and -4 in HBD-3 induction in osteoblasts. Blocking protein synthesis with cycloheximide showed that the rapid release of HBD-3 is not dependent on a translational de novo synthesis and is not affected by glucocorticoids. The murine osteomyelitis model confirmed the in vivo release uptake of mouse β-defensins-4 (MBD-4) in bone. This report shows the bacterial induction of HBD-3 via TLR-2 and -4 in osteoblasts and suggests a central role of antimicrobial peptides in the prevention of bacterial bone infection. The rapid and effective induction of HBD-3 in osteoblasts incubated with conditioned media from bacteria is more likely a result of a rapid secretion of preformed HBD-3 by osteoblasts rather than a result of enhanced biosynthesis. The increased incidence of gram-positive bacterial bone infection in patients with regular intake of glucocorticoids does not seem to be caused by a deranged HBD-3 release in osteoblasts.

Hemiarthroplasty of the shoulder after four-part fracture of the humeral head: a long-term analysis of 34 cases.

OBJECTIVE To assess the treatment outcomes of patients with four-part fracture of the humeral head after primary and secondary hemiarthroplasty. PATIENTS Retrospective long-term analysis of 46 patients from 1996 to 2002 of patients with 47 four-part fractures of humeral head. Patients with malignant disease were excluded. INTERVENTION Aequalis (Tornier, Burscheid, Germany). MAIN OUTCOME MEASUREMENTS Absolute and relative constant scores at 5-year follow-up examination without age or sex normalization, radiographic parameters of calcification, dislocation of tuberosities, prosthetic loosening, and dislocation of joint. RESULTS Eighteen patients treated by primary and 16 patients treated by secondary arthroplasty were assessed clinically and radiologically after a mean follow-up of 64 (60-96) months. The absolute Constant scores at follow-up were 54.9 to 48.5 points, respectively. The relative scores were 61.4% and 57.3%, respectively. Dislocation of tuberosities with severe loss of function was found in five cases treated by primary arthroplasty (13.5%) and in 12 treated by secondary arthroplasty (75.0%). CONCLUSIONS The majority of patients in both groups was free of pain or suffered minor pain as determined by the Constant score. Safe fixation of the tuberosities is a prerequisite for functional exercises and is better achieved in primary arthroplasty. A computed tomography scan before operative therapy aids in making the decision between open reduction and internal fixation or hemiarthroplasty.

Platelets display potent antimicrobial activity and release human beta-defensin 2

Platelet-rich plasma (PRP) is a potent agent that improves soft tissue and bone healing. By the release of growth factors and cytokines, PRP is believed to locally boost physiologic healing processes. Recently, antimicrobial activity of PRP has been demonstrated against S. aureus strains. Major scientific effort is being put into the understanding and prevention of infections i.e. by delivery of antimicrobial substances. In previous studies we showed the ideal antibacterial activity-profile of the human beta-defensin 2 (hBD-2) for orthopaedic infections and therefore hypothesized that hBD-2 may be the effector of antimicrobial platelet action. Platelet concentrates were produced from human platelet phresis obtained from a hospital blood bank. They were screened by immunohistochemistry, Western Blot and ELISA for the human beta defensin-2. In vitro susceptibility to PRP was investigated by a standard disc diffusion test with or without pre-incubation of PRP with anti-hBD-2 antibody. SPSS statistical software was used for statistical analysis. PRP contains hBD-2 470 pg/109 platelets or 1786 pg/ml, respectively, (ELISA), which was confirmed by immunohistochemistry and Western Blot. In antimicrobial testing, PRP demonstrates effective inhibition of E. coli, B. megaterium, P. aeruginosa, E. faecalis and P. mirabilis. With this study we confirm the previously reported antimicrobial action of platelet concentrates i.e. PRP. In opposition to previously reported effects against gram positive bacteria our study focuses on gram negative and less common gram positive bacteria that do frequently cause clinical complications. We provide a possible molecular mechanism at least for E. coli and P. mirabilis for this effect by the detection of an antimicrobial peptide (hBD-2). This study may advocate the clinical use of PRP by highlighting a new aspect of platelet action.

Thrombocytes are effectors of the innate immune system releasing human beta defensin-3.

BACKGROUND Thrombocyte concentrate i.e. platelet-rich plasma (PRP) has become a popular adjunct for many surgical procedures. It is believed to improve bone and soft tissue healing. Recently antimicrobial effects of the autologous preparation were reported by several groups. In this study we investigated the antimicrobial effect of PRP against gram-negative microbes which frequently cause severe complications in orthopaedic trauma surgery. METHODS Platelet-rich plasma was produced from liquid preserved thrombocyte concentrates. ELISA, Western blot and immunohistochemistry were preformed to investigate the release and content of platelet concentrates. A radial diffusion assay was used to detect antimicrobial effects of PRP. RESULTS We detected the human beta defensin-3 in bactericidal concentrations in platelet preparations by ELISA, Western blot and immunohistochemistry. In antimicrobial testing we demonstrated effective inhibition of Escherichia coli (ATCC 11303), Bacterium megaterium (ATCC 14581), Klebsiella pneumoniae (ATCC 13883), Enterococcus faecalis (ATCC 29212) and Proteus mirabilis (ATCC21100). CONCLUSION With this study we demonstrate antimicrobial action of a popular adjunct for orthopaedic and trauma surgery against gram-positive and gram-negative bacteria. We have identified a possible mechanism of action via the secretion of HBD-3 as a first line defence in contaminated wounds and in elective application of PRP. This finding supports a broader spectrum of clinical indications for an autologous platelet preparation.

Schockraummanagement bei Verletzungen des Beckens

ZusammenfassungFragestellung.Beckenverletzungen können eine vitale Bedrohung darstellen, was sofortiges operatives Handeln erfordert. Eine Literaturübersicht soll hierzu aktuelle Empfehlungen gegenüberstellen.Methodik.Klinische Studien wurden über systematische Literatursuchen (Medline, Cochrane und Handsuchen) gesichtet und nach Klassifikation und Evidenzgüte (nach Oxforder Schema) zusammengetragen.Ergebnisse.Aus der Literaturübersicht lassen sich weitestgehend übereinstimmende Empfehlungen bezüglich der Dringlichkeit der operativen Versorgung und der Kreislaufstabilisierung bei komplexen Beckenverletzungen ableiten. In Diskussion ist die Art der Notfallstabilisierung (Fixateur externe oder Beckenzwinge), Art und Umfang der Röntgendiagnostik (Nativ und/oder nur CT) und die Art der Blutstillung (Tamponade oder Embolisation).Schlussfolgerungen.Das Schockraummanagement bei Beckenverletzungen beinhaltet in erster Linie die Therapie einer vitalen Bedrohung, die von einer komplexen Beckenverletzung ausgehen kann. Hierzu stehen verschiedene Methoden zur Auswahl, einheitlich ist das Prinzip der parallelen Kreislaufstabilisierung und mechanischen Stabilisierung des Beckenrings.AbstractObjective.Injuries to the pelvis may occur as a life threatening situation which then requires immediate surgical treatment. A review of the literature represents the range of current recommendations.Methods.Clinical trials were systematically collected (Medline, Cochrane and hand searches) reviewed and classified into evidence levels (1 to 5 according to the Oxford system).Results.According to the literature there is a consent that immediate resuscitation and surgical intervention is essential in complex pelvic fractures. In contrast the way of emergency stabilization of the pelvis (fixateur externe or c-clamp) is still under discussion as well as the radiological diagnostic (x-ray or CT) and the way of bleeding control (tamponade or embolization).Conclusion.Emergency management of pelvic fractures means treatment of a life threatening injury in first place. Although there are different methods that can be used, they all follow the same principle of resuscitation and mechanical stabilization of the pelvis in parallel.

Shock trauma room management of pelvic injuries. A systematic review of the literature

ZusammenfassungFragestellung.Beckenverletzungen können eine vitale Bedrohung darstellen, was sofortiges operatives Handeln erfordert. Eine Literaturübersicht soll hierzu aktuelle Empfehlungen gegenüberstellen.Methodik.Klinische Studien wurden über systematische Literatursuchen (Medline, Cochrane und Handsuchen) gesichtet und nach Klassifikation und Evidenzgüte (nach Oxforder Schema) zusammengetragen.Ergebnisse.Aus der Literaturübersicht lassen sich weitestgehend übereinstimmende Empfehlungen bezüglich der Dringlichkeit der operativen Versorgung und der Kreislaufstabilisierung bei komplexen Beckenverletzungen ableiten. In Diskussion ist die Art der Notfallstabilisierung (Fixateur externe oder Beckenzwinge), Art und Umfang der Röntgendiagnostik (Nativ und/oder nur CT) und die Art der Blutstillung (Tamponade oder Embolisation).Schlussfolgerungen.Das Schockraummanagement bei Beckenverletzungen beinhaltet in erster Linie die Therapie einer vitalen Bedrohung, die von einer komplexen Beckenverletzung ausgehen kann. Hierzu stehen verschiedene Methoden zur Auswahl, einheitlich ist das Prinzip der parallelen Kreislaufstabilisierung und mechanischen Stabilisierung des Beckenrings.AbstractObjective.Injuries to the pelvis may occur as a life threatening situation which then requires immediate surgical treatment. A review of the literature represents the range of current recommendations.Methods.Clinical trials were systematically collected (Medline, Cochrane and hand searches) reviewed and classified into evidence levels (1 to 5 according to the Oxford system).Results.According to the literature there is a consent that immediate resuscitation and surgical intervention is essential in complex pelvic fractures. In contrast the way of emergency stabilization of the pelvis (fixateur externe or c-clamp) is still under discussion as well as the radiological diagnostic (x-ray or CT) and the way of bleeding control (tamponade or embolization).Conclusion.Emergency management of pelvic fractures means treatment of a life threatening injury in first place. Although there are different methods that can be used, they all follow the same principle of resuscitation and mechanical stabilization of the pelvis in parallel.