[Augmentation with antibiotic-impregnated spacers in sepsis revision surgery]. / Augmentation mit antibiotikaimprägnierten Spacern in der septischen Revisionschirurgie.

BACKGROUND: The development of antibiotic-impregnated polymethyl methacrylate (PMMA) spacers is based on clinical experience and the use of antibiotic-loaded PMMA beads in septic bone surgery as well as antibiotic-loaded bone cement in arthroplasty beginning in the 1970s. MATERIAL AND METHODS: In the meantime hand-formed and prefabricated spacers are implanted in cases of sepsis to achieve high local antibiotic concentrations and bactericidal effects to eradicate the infection. Preformed spacers with gentamicin are commercially available and furthermore, clindamycin-loaded PMMA bone cement can also be used. In principle, all thermostable antibiotics can be mixed with PMMA cement. SIGNIFICANCE: Spacers permit bridging of bone defects originating from trauma or septic bone segment resection. After joint resection spacers allow a certain degree of articulation and inhibit shortening of the extremity which has a positive effect on the soft tissue covering and its perfusion. CONCLUSION: The functional outcome after secondary arthroplasty is better if a spacer has been implanted compared to long-term immobilization without spacers. Nevertheless, spacers can also cause serious complications, such as dislocations and fractures. Antibiotic-loaded spacers have therefore widened the therapeutic options in sepsis surgery.

Early interleukin 12 production by macrophages in response to mycobacterial infection depends on interferon gamma and tumor necrosis factor alpha.

Interleukin 12 (IL-12) produced by macrophages immediately after infection is considered essential for activation of a protective immune response against intracellular pathogens. In the murine Mycobacterium bovis Bacillus Calmette-Guérin (BCG) model we assessed whether early IL-12 production by macrophages depends on other cytokines. In vitro, murine bone marrow-derived macrophages produced IL-12 after infection with viable M. bovis BCG or stimulation with LPS, however, priming with recombinant interferon gamma (rIFN-gamma) was necessary. In addition, IL-12 production by these macrophages was blocked by specific anti-tumor necrosis factor alpha (TNF-alpha) antiserum. Macrophages from gene deletion mutant mice lacking either the IFN-gamma receptor or the TNF receptor 1 (p55) failed to produce IL-12 in vitro after stimulation with rIFN-gamma and mycobacterial infection. In vivo, IL-12 production was induced in spleens of immunocompetent mice early during M. bovis BCG infection but not in those of mutant mice lacking the receptors for IFN-gamma or TNF. Our results show that IL-12 production by macrophages in response to mycobacterial infection depends on IFN-gamma and TNF. Hence, IL-12 is not the first cytokine produced in mycobacterial infections.

[Retrospective Analysis of Diabetics with Regard to Treatment Duration and Costs]. / Retrospektive Analyse von Diabetikern im Hinblick auf Behandlungsdauer und Behandlungskosten in einem überregionalen Traumazentrum.

Background: The increasing incidence of diabetes mellitus is also reflected in the patient population of a trauma and orthopaedic centre. Diabetics also exhibit more comorbidities than non-diabetics. In addition to surgical problems in these patients, hospitalisation is often accompanied by complications, which can prolong treatment and increase costs. The aim of this retrospective study is to analyse hospitalisation of diabetics compared to non-diabetics, as well as differences in treatment costs, depending on associated age and comorbidities. Patients/Material and Methods: 17,185 patients were treated at a transregional trauma and orthopaedic centre and were included in this retrospective analysis between 2012 and 2015. Comorbidities and hospitalisation of diabetics and non-diabetics were recorded. All costs charged by DRG were evaluated to calculate the cost per day and per patient, on the basis of the specific case rate. In this calculation, patient-related case rates were divided by the average residence time and the means of the calculated daily rates were calculated. Inclusion criteria were treatment within the various departments and a minimum hospitalisation of one day. Statistical analysis was performed with the SPSS program (version 22.0, SPSS Inc., Chicago, USA). Results: In comparison to non-diabetics (ND), diabetics (D) exhibited significantly more comorbidities, including: obesity, arterial hypertension, coronary heart disease, myocardial infarction (in the history), peripheral arterial disease, chronic kidney disease and hyperlipidaemia. Pneumonia in hospital was considerably commoner in diabetics (2.45 % [D] vs. 1.02 % [ND], p < 0.001). Time in hospital was significantly longer in diabetics (endoprosthetics 13.52 days [D] vs. 12.54 days [ND], p < 0.001; septic surgery 18.62 days [D] vs. 16.31 days [ND], p = 0.007; traumatology 9.82 days [D] vs. 7.07 days [ND], p < 0.001). For patients aged under 60 years, time in hospital was significantly longer for diabetics than for non-diabetics (9.98 days [D] vs. 6.43 days [ND] p < 0.001). Because of the longer time in hospital, treatment costs were higher by € 1,932,929.42 during the investigated time period. Conclusion: Because of their comorbidities, diabetics need to be categorised at an early stage as high-risk patients in traumatological and orthopaedic departments. Hospitalisation and the associated increased treatment costs, as well as postoperative complications, could be minimised in patients with diabetes by implementing an interdisciplinary treatment concept.

Effect of cellular fatty acid composition on the phospholipase A2 activity of bone marrow-derived macrophages, and their ability to induce lucigenin-dependent chemiluminescence.

Mouse bone marrow macrophages were obtained by cultivation in serum-free medium. Addition of specific fatty acids to the medium leads to macrophage populations which differ in their fatty acid composition. The fatty acid composition of the cellular membranes directly modulates functional abilities of the macrophages such as the generation of superoxide anion and phospholipase A2 activity in response to phorbol ester and zymosan. Both capacities were lowest in macrophages cultured serum-free without lipids. Incorporation of unsaturated fatty acids into macrophage phospholipids leads to an increase of O2- production as measured by lucigenin-dependent chemiluminescence and to an increased phospholipase A2 activity after challenge with phorbol ester or zymosan.

Effect of human platelet supernatant on proliferation and matrix synthesis of human articular chondrocytes in monolayer and three-dimensional alginate cultures.

Articular cartilage is rich in collagen type II fibres and proteoglycans and is characterized by low cell density. Chondrocytes have specific nutritional requirements and therefore cannot be expanded in vitro without the risk of generating fibroblastoid cells expressing type I collagen. Therefore, various growth conditions were tested for cartilage tissue engineering. Human platelets are a rich source of many growth factors including transforming growth factor-beta and platelet-derived growth factor. To investigate the effect of human platelet supernatant (hPS) on chondrocyte proliferation and differentiation, human articular biopsies obtained from three healthy donors. Chondrocytes were isolated and expanded separately in monolayer cultures and seeded in alginate beads in the presence and absence of hPS of 1% or 10% v/v concentration. Transcript levels of genes encoding chondrogenic factors were determined by quantitative reverse transcriptase-polymerase chain reaction. The deposition of types I and II collagen as well as proteoglycan was detected by indirect immunocytochemistry. Addition of hPS activated chondrocyte proliferation in monolayer cultures but induced a dedifferentiation of chondrocytes towards a fibroblast-like phenotype. The expression levels of mRNAs encoding type II collagen, aggrecan and bone morphogenetic protein-2 were reduced in all samples tested. Seeding chondrocytes in alginate beads in the presence of hPS generated a cell population capable of type II collagen expression, even though hPS induced considerable type I collagen expression as well. Differences (1% vs. 10% group, 1% vs. control, 10% vs. control) in the quantitative gene expression of types I and II collagen or of aggrecan were statistically significant (p<0.001). We conclude that addition of hPS may accelerate chondrocyte expansion but can lead to their dedifferentiation.

Activation of natural killer cells by heat-killed Listeria monocytogenes requires additional signals from lymphoid cells.

Regulatory and protective functions have been attributed to murine natural killer (NK) cells in a number of infectious diseases including listeriosis. We have developed an in vitro model to study parameters underlying the activation of naive NK cells using heat-killed Listeria monocytogenes (HKL) as stimulator. Independent from expression of the cell surface marker NK1.1, NK cells lysed YAC-1 cells after in vitro stimulation with HKL or HKL + Interleukin (IL)-2, but not medium or IL-2 alone. In contrast, NK cells from severely immunocompromised SCID or RAG-1-/-mutant mice failed to respond to HKL alone, but required exogenous IL-2. Using single-gene-disruption mutant mice, we show that NK-cell activation can be supported by either T-cell receptor (TCR) alpha beta cells, TCR- gamma delta cells. MHC class I or MHC class II gene products. We conclude from these data that recognition of listerial components alone is insufficient for activation of naive NK cells, and that additional costimulatory signals are necessary. These can be provided by various lymphoid cells and appear to be cytokines.

Growth requirements of murine bone marrow macrophages in serum-free cell culture.

A serum-free medium for mass production of pure murine bone marrow-derived macrophages in liquid culture was developed. Iscove's modified Dulbecco's medium (IMDM) was used as a basal nutrient medium supplemented with 2-mercaptoethanol, transferrin and a source of colony-stimulating factor (CSF). Addition of exogenous lipids or bovine serum albumin was not required. The macrophages can be kept viable for at least eight days in the serum-free culture medium, and they are able to incorporate tritiated thymidine. The tritiated thymidine incorporation can be enhanced by retinoic acid, phorbol myristate acetate (PMA), colony-stimulating factor (CSF) and transferrin. Prostaglandin E1, prostaglandin E2 and dexamethasone inhibited tritiated thymidine incorporation. The serum-free culture of bone marrow-derived macrophages has some important implications. First, signal molecules can be defined which enhance or inhibit the proliferative capacity of bone marrow-derived macrophages. Second, the fatty acid composition of membrane phospholipids can be altered to study the relationship between macrophage function and membrane lipid composition.

Functional comparison of bone marrow-derived macrophages obtained by cultivation in serum-free or serum-supplemented medium.

Bone marrow-derived macrophages obtained by cultivation in a serum-free or in a serum-supplemented medium were compared in terms of the activation of the respiratory burst and the activation of tumor cytotoxicity. Serum-free-cultured macrophages responded to interferon-gamma (IFN-gamma) and to lipopolysaccharide (LPS) by an enhancement of the respiratory burst. Macrophages obtained in a serum-supplemented medium are characterized by a diminished capacity to release O2-. These cells did not respond to IFN-gamma unless the stimulation was performed in a serum-containing medium. In terms of activation of tumor cell cytotoxicity, serum-supplemented macrophage cultures seem to be primed by unknown serum constituents because they only need one signal (IFN-gamma or LPS) to become fully cytotoxic. Serum-free cultivated macrophages can be rendered cytotoxic only after exposure to combinations of IFN-gamma and LPS.

Role of cytokines in tuberculosis.

Mycobacterium tuberculosis and Mycobacterium bovis are facultative intracellular pathogens which preferentially utilize the macrophage as their host cell. Acquired resistance against mycobacteria depends on T cells which activate antimicrobial macrophage functions via the release of cytokines. The data summarized below suggest an important role for interferon-gamma (IFN-gamma) as well as the B cell-stimulatory factors interleukin-4 (IL-4) and IL-6 in the induction of tuberculostatic macrophage functions. Growth inhibition of mycobacteria by cytokine-stimulated macrophages is mediated by reactive nitrogen intermediates (RNI) derived from L-arginine. Tumor necrosis factor-alpha (TNF-alpha) and IL-10 act as autocrine regulators in the induction of the enzyme NO-synthase. Both cytokines are produced by macrophages stimulated with IFN-gamma and infected with M. bovis. While TNF-alpha mediates activation of the NO-synthase and production of RNI, IL-10 suppresses this enzyme activity. The outcome of mycobacterial infection is probably regulated by a complex network between stimulatory and inhibitory cytokines.

Effect of fetal calf serum on cytokine release by bone marrow-derived macrophages during infection with intracellular bacteria.

Bone marrow-derived macrophages (BMM) comprise a population of quiescent cells which can be activated by defined signals. Here, we directly compare the release of chemokines and monokines by BMM raised either in serum-supplemented or in serum-free medium in response to Listeria monocytogenes EGD or Mycobacterium bovis BCG infection. We focused on this issue because there have been several controversial reports on the production of cytokines by BMM due to different in vitro culture conditions. Culture in serum-supplemented medium primed BMM for release of monocyte chemoattractant protein (MCP)-1, interleukin (IL)-6, and IL-12, but had no effect on macrophage inflammatory protein (MIP)-1alpha and tumor necrosis factor (TNF)-alpha production in response to L. monocytogenes infection. After challenge infection with M. bovis, BMM raised and stimulated in serum-supplemented medium secreted higher levels of MCP-1, MIP-1alpha, IL-6, and TNF-alpha but not of IL-12 as compared to BMM cultured and infected in a serum-free medium. The effects of serum could be partially mimicked by interferon-gamma. Because the serum components responsible for BMM priming are undefined, BMM cultured under serum-free conditions provide an appropriate cell population for defining macrophage activating signals.

Macrophages and hepatocytic cells as chemokine producers in murine listeriosis.

The major target organ of systemic infection with the intracellular bacterium Listeria monocytogenes is the liver, to where inflammatory leukocytes are rapidly recruited. We determined by reverse transcriptase polymerase chain reaction the early chemokine response in the liver after systemic infection of mice with listeriae, and in parallel compared chemokine release from macrophages and hepatocytic cells in vitro. Murine bone marrow-derived macrophages (BMM) grown in fetal calf serum-supplemented medium were used as macrophages and the TIB75 cell line as hepatocytic cells. Within 1-3 hours, gene expression of monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1 alpha, MIP-2, KC, and interferon-gamma inducible protein-10 (IP-10) was upregulated in liver tissue of infected mice. BMM infected in vitro with L. monocytogenes showed a generalized chemokine response, and readily released MCP-1, MIP-1 alpha, MIP-2, and KC, as measured by enzyme-linked immunosorbent assay. In contrast, the chemokine response of hepatocytic cells was more restricted, and infection induced MCP-1 and KC, but not MIP-2 and MIP-1 alpha. Interferon gamma enhanced chemokine release from hepatocytic cells, but unexpectedly had either no or a negative effect on chemokine secretion by BMM cultured in serum-supplemented medium. Listeriolysin (Hly)-negative avirulent listeriae as well as listeriae killed by heat or gentamycin initiated a similar chemokine response in BMM and hepatocytic cells as did wild-type L. monocytogenes. Stimulation of hepatocytic cells with the monokines, tumor necrosis factor alpha and interleukin (IL-)1 alpha, but not IL-6, augmented liberation of chemokines. Together, our data demonstrate an early hepatic chemokine response to L. monocytogenes in murine listeriosis. Probably, not only macrophages but also parenchymal cells participate in chemokine production.

Therapeutic effect of argon plasma coagulation on small malignant gastrointestinal tumors.

In endoscopy, argon plasma coagulation (APC) is a new principle of non-contact electrocoagulation and has proved to be a sufficient tool for palliative endoscopic treatment of gastrointestinal neoplasms, predominantly of the oesophagus and colorectum. In a study of 67 patients suffering from histologically confirmed and endosonographic T1-staged tumours of the gastrointestinum, 10 patients were selected for endoscopic APC treatment because of the impossibility of surgical therapy. Although the application was primarily of a palliative nature, in 9 of 10 cases of minor neoplasms, no further tumour could be detected in biopsies during the observation period (9.45 +/- 2.8 months). One patient was not cured locally. In none of the patients was any serious complication noticed during the outpatient follow-up. The effective results and lack of severe complications suggest this technique as an alternative therapy in selected patients with smaller gastrointestinal tumours.

Effects of platelet growth factors on human mesenchymal stem cells and human endothelial cells in vitro.

The aim of the present in vitro study has been to investigate the effects of a enriched platelet derived growth factors on proliferation and migration of human endothelial and mesenchymal stem cells and on osteogenic differentiation of stem cells. Platelet rich plasma has been produced, yielding a four time higher number of thrombocytes than normal plasma. Degranulation of platelets has been performed by means of calcium and thrombin. Plasma has served as a control, whereas plasma in combination with calcium and thrombin was used to distinguish the difference between calcium and/or thrombin mediated effects and growth factor induced effects on the cells. The observed enhanced proliferation and migration of endothelial cells towards the platelet derived growth factors was driven by the plasma component of these preparations. However PDGF solely stimulated the migration and proliferation of mesenchymal stem cells. The increased osteogenic differentiation of growth factor treated mesenchymal stem cells was mostly driven by the high level of calcium used for the platelets degranulation. In summery, the different components of platelet derived growth factors work together to influence human endothelial and mesenchymal stem cells. This is of special clinically interest regarding the stimulation of bone healing in orthopaedic and traumatic surgery.

[Measuring microcirculation after replantation and revascularization]. / Mikrozirkulationsmessungen nach Replantation und Revaskularisation.

Eighty-four patients were evaluated following replantation or revascularization. Extremity function in daily and professional life was altogether satisfactory. Microcirculation investigations in eight patients included thermography, laser-doppler-flowmetry and photoplethysmography. The rewarming period and postocclusive hyperemia after standardized cooling of the upper extremity were determined. In areas of hypesthesia, the rewarming period was clearly delayed. In addition, reactive hyperemia after occlusion of circulation was significantly reduced.

[Plaster filling in surgical treatment of enchondroma--a justified therapeutic procedure?]. / Gipsplombenauffütterung bei der operativen Behandlung des Enchondroms--Ein berechtigtes Therapieverfahren?

Enchondroma are the most frequent tumors of the hand. These benign tumours are characterized by slow growth, the lack of clinical symptoms and by accidental discovery. The surgical treatment of enchondroma includes resection of the tumor tissue from the bone matrix, and subsequent filling of the defect with autologous or homologous spongiosa or with sterile plaster of Paris. We showed in a period from 1982 to 1989 that there is no difference with respect to the clinical outcome between our patients receiving autologous spongiosa (n = 25) or plaster filling (n = 35). Owing to its simplicity and lack of additional surgery at the iliac crest, we prefer the method of plaster filling. Animal studies performed by our group have demonstrated that implanted plaster is transformed to spongiosa within four to ten weeks without adverse effects.

[Stimulation of wound healing with thrombocyte growth factors in treatment of chronic unhealed wounds]. / Stimulation der Wundheilung mit thrombozytären Wachstumsfaktoren bei der Behandlung chronisch nicht-heilender Wunden.

The management of chronic wounds remains a challenge for various medical and surgical specialties. General strategies include local wound care, surgery, skin grafting, debridement, and vascular reconstruction. However, many wounds remain resistant to conventional therapies. Because platelet factors are important in the normal mechanism of wound healing, we report on the topical application of platelet-derived growth factor. Our initial results suggest that the topical application may stimulate and promote healing of chronic wounds.

[Legal aspects in early defibrillation by trained lay respondents]. / Rechtliche Aspekte bei der Frühdefibrillation durch geschulte Laienhelfer.

Ventricular fibrillation is the main reason for cardiac arrest. The probability for survival decreases by 10% every minute, therefore immediate resuscitation is necessary. Cardiopulmonary Resuscitation (CPR) by trained first responders is already established, when a doctor is not available. Today automated external defibrillators (AED) are available to first responders for an effective therapy of ventricular fibrillation. Thanks to the high reliability of today's automated external defibrillators they can be used by trained first responders without any legal reservations. If a defibrillator is available, a trained first responder is obliged to use it in an emergency.

[Definition of the Diagnosis Osteomyelitis-Osteomyelitis Diagnosis Score (ODS)]. / Zur Definition der Diagnose Osteomyelitis - Osteomyelitis-Diagnose-Score (ODS).

AIM: The disease "osteomyelitis" is characterised by different symptoms and parameters. Decisive roles in the development of the disease are played by the causative bacteria, the route of infection and the individual defense mechanisms of the host. The diagnosis is based on different symptoms and findings from the clinical history, clinical symptoms, laboratory results, diagnostic imaging, microbiological and histopathological analyses. While different osteomyelitis classifications have been published, there is to the best of our knowledge no score that gives information how sure the diagnosis "osteomyelitis" is in general. METHOD: For any scientific study of a disease a valid definition is essential. We have developed a special osteomyelitis diagnosis score for the reliable classification of clinical, laboratory and technical findings. The score is based on five diagnostic procedures: 1) clinical history and risk factors, 2) clinical examination and laboratory results, 3) diagnostic imaging (ultrasound, radiology, CT, MRI, nuclear medicine and hybrid methods), 4) microbiology, and 5) histopathology. RESULTS: Each diagnostic procedure is related to many individual findings, which are weighted by a score system, in order to achieve a relevant value for each assessment. If the sum of the five diagnostic criteria is 18 or more points, the diagnosis of osteomyelitis can be viewed as "safe" (diagnosis class A). Between 8-17 points the diagnosis is "probable" (diagnosis class B). Less than 8 points means that the diagnosis is "possible, but unlikely" (class C diagnosis). Since each parameter can score six points at a maximum, a reliable diagnosis can only be achieved if at least 3 parameters are scored with 6 points. CONCLUSION: The osteomyelitis diagnosis score should help to avoid the false description of a clinical presentation as "osteomyelitis". A safe diagnosis is essential for the aetiology, treatment and outcome studies of osteomyelitis.