Ingo Flesch

Crucial Role of Vitamin D in the Musculoskeletal System

Vitamin D is well known to exert multiple functions in bone biology, autoimmune diseases, cell growth, inflammation or neuromuscular and other immune functions. It is a fat-soluble vitamin present in many foods. It can be endogenously produced by ultraviolet rays from sunlight when the skin is exposed to initiate vitamin D synthesis. However, since vitamin D is biologically inert when obtained from sun exposure or diet, it must first be activated in human beings before functioning. The kidney and the liver play here a crucial role by hydroxylation of vitamin D to 25-hydroxyvitamin D in the liver and to 1,25-dihydroxyvitamin D in the kidney. In the past decades, it has been proven that vitamin D deficiency is involved in many diseases. Due to vitamin D’s central role in the musculoskeletal system and consequently the strong negative impact on bone health in cases of vitamin D deficiency, our aim was to underline its importance in bone physiology by summarizing recent findings on the correlation of vitamin D status and rickets, osteomalacia, osteopenia, primary and secondary osteoporosis as well as sarcopenia and musculoskeletal pain. While these diseases all positively correlate with a vitamin D deficiency, there is a great controversy regarding the appropriate vitamin D supplementation as both positive and negative effects on bone mineral density, musculoskeletal pain and incidence of falls are reported.

Elution kinetics, antimicrobial efficacy, and degradation and microvasculature of a new gentamicin-loaded collagen fleece

Management of bone and soft tissue infections generally includes surgical procedures as well as attendant treatment and prevention with gentamicin-loaded fleeces. Conventional gentamicin-containing collagen fleeces currently in use are strongly acidic and exhibit limited biocompatibility thereby adversely affecting wound healing. To improve the antibiotic delivery system, a new phosphate-buffered, gentamicin-loaded fleece with pH-neutral properties has been developed (Jason G). This study aimed at comparing the elution kinetics of gentamicin release and the antimicrobial efficacy of conventional fleeces with the newly developed fleece in vitro. In addition, degradation and microvasculature of implanted fleeces were examined in a rat model and assessed using histology, as well as detection of ED-1 and PECAM-expression using immunohistochemistry. We show that the phosphate-buffered fleeces have reduced release (p < 0.05) of the integrated gentamicin. However, all of the fleeces tested had a significant antimicrobial effect on the growth of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa strains (p < 0.01). Among the fleeces tested, the new Jason G fleece had the weakest but nevertheless sufficient antimicrobial effectiveness. Evaluation of the antibiotic effect in the prevention of an infection showed no differences between the applied fleeces. Following surgical implantation of fleece in the backs of Wistar rats we observed, on day 5 after implantation, an increase in cell infiltration and microvascularization with the phosphate-buffered fleece as compared with conventional fleeces, which show necrotic cells on their surface. Unlike the acidic fleeces, on day 15 after implantation the pH-neutral fleece was resorbed widely. Here, we show that the new, pH-neutral, gentamicin-containing fleece Jason G exhibits good overall antimicrobial effectiveness against both gram-positive and gram-negative bacteria in vitro with improved degradation properties and microvasculature formation in vivo.

New blood vessel formation and expression of VEGF receptors after implantation of platelet growth factor-enriched biodegradable nanocrystalline hydroxyapatite.

Vascular endothelial growth factor (VEGF) plays a key role for the interaction of osteoblasts and endothelial cells and, therefore, is an important factor for the osteointegration of bone substitutes. The aim of the current work was to study the effects of platelet growth factors (PLF) on new blood vessel formation and VEGF-receptors expression pattern in bone defects filled with nanocrystalline hydroxyapatite (HA) paste in miniature-pigs. Conventional histology, RT-PCR for VEGF and receptors mRNA, Western blot analysis, immunohistochemical staining and quantitative assessment of newly formed vessels was performed. HA enriched with platelet growth factor (HA/PLF+) led to an up-regulation of VEGF-R1 synthesis, a slightly enhanced number of newly formed vessels with higher sprouting activity compared with HA without PLF (HA/PLF−) filling defects. These observation are most likely attributable to a stimulating effect of TGF-β from the platelet factor on VEGF expression in osteoblasts.

Factors circulating in the blood of type 2 diabetes mellitus patients affect osteoblast maturation - Description of a novel in vitro model

Type 2 diabetes mellitus (T2DM) is one of the most frequent metabolic disorders in industrialized countries. Among other complications, T2DM patients have an increased fracture risk and delayed fracture healing. We have demonstrated that supraphysiological glucose and insulin levels inhibit primary human osteoblasts׳ maturation. We aimed at developing a more physiologically relevant in vitro model to analyze T2DM-mediated osteoblast changes. Therefore, SCP-1-immortalized pre-osteoblasts were differentiated with T2DM or control (non-obese and obese) sera. Between both control groups, no significant changes were observed. Proliferation was significantly increased (1.69-fold), while AP activity and matrix mineralization was significantly reduced in the T2DM group. Expression levels of osteogenic marker genes and transcription factors were altered, e.g. down-regulation of RUNX2 and SP-7 or up-regulation of STAT1, in the T2DM group. Active TGF-β levels were significantly increased (1.46-fold) in T2DM patients׳ sera. SCP-1 cells treated with these sera showed significantly increased TGF-β signaling (2.47-fold). Signaling inhibition effectively restored osteoblast maturation in the T2DM group. Summarizing our data, SCP-1 cells differentiated in the presence of T2DM patients׳ serum exhibit reduced osteoblast function. Thus, this model has a high physiological impact, as it can identify circulating factors in T2DM patients׳ blood that may affect bone function, e.g. TGF-β.

Effect of metallo-β-lactamase production and multidrug resistance on clinical outcomes in patients with Pseudomonas aeruginosa bloodstream infection: a retrospective cohort study

BackgroundBlood stream infections (BSI) with Pseudomonas aeruginosa lead to poor clinical outcomes. The worldwide emergence and spread of metallo-β-lactamase (MBL) producing, often multidrug-resistant organisms may further aggravate this problem. Our study aimed to investigate the effect of MBL-producing P. aeruginosa (MBL-PA) and various other resistance phenotypes on clinical outcomes.MethodsA retrospective cohort study was conducted in three German hospitals. Medical files from 2006 until 2012 were studied, and a number of 113 patients with P. aeruginosa BSI were included. The presence of VIM, IMP and NDM genes was detected using molecular techniques. Genetic relatedness was assessed through multilocus sequence typing (MLST). The effect of resistance patterns or MBL production on clinical outcomes was investigated by using multivariate Cox regression models.ResultsIn-hospital mortality was significantly higher in patients with MBL-PA and multidrug-resistant P. aeruginosa. However, neither BSI with MBL-PA nor BSI with various resistance phenotypes of P. aeruginosa were independently associated with mortality or length of hospital stay. In multivariate models, the SAPS II score (HR 1.046), appropriate definitive treatment (HR range 0.25-0.26), and cardiovascular disease (HR range 0.44-0.46) were independent predictors of mortality. Concomitant infections were associated with an excess length of stay (HR < 1).ConclusionsMedication with appropriate antimicrobial agents at any time during the course of infection remains the key for improving clinical outcomes in patients with P. aeruginosa BSI and should be combined with a strict implementation of routine infection control measures.

BMP9 a possible alternative drug for the recently withdrawn BMP7? New perspectives for (re-)implementation by personalized medicine.

Promotion of rhBMP2 and rhBMP7 for the routine use to support fracture healing has been hampered by high costs, safety concerns and reasonable failure rates, imposing restrictions in its clinical use. Since there is little debate regarding its treatment potential, there is rising need for a better understanding of the mode of action of these BMPs to overcome its drawbacks and promote more efficacious treatment strategies for bone regeneration. Recently, BMP9, owing to its improved osteogenic potential, is gaining attention as a promising therapeutic alternative. Our study aimed at identifying specific gene expression patterns which may predict and explain individual responses to rhBMP7 and rhBMP9 treatments. Therefore, we investigated the effect of rhBMP7 and rhBMP9 on primary human osteoblasts from 110 donors and corresponding THP-1-derived osteoclasts. This was further compared with each other and our reported data on rhBMP2 response. Based on the individual donor response, we found three donor groups profiting from rhBMP treatment either directly via stimulation of osteoblast function or viability and/or indirectly via inhibition of osteoclasts. The response on rhBMP7 treatment correlated with expression levels of the genes BAMBI, SOST, Noggin, Smad4 and RANKL, while the response of rhBMP9 correlated to the expression levels of Alk6, Endoglin, Smurf1, Smurf2, SOST and RANKL in these donors. Noteworthy, rhBMP9 treatment showed significantly increased osteogenic activity (AP activity and Smad nuclear translocation) when compared to the two clinically used rhBMPs. Based on patient’s respective expression profiles, clinical application of rhBMP9 either solely or in combination with rhBMP2 and/or rhBMP7 can become a promising new approach to fit the patient’s needs to promote fracture healing.

Time to positivity as prognostic tool in patients with Pseudomonas aeruginosa bloodstream infection

OBJECTIVES The time to positivity (TTP), measured as the time span between the start of incubation and the alert signal from the blood culture device, has been described as useful tool of prognosis in patients suffering from blood stream infection with Staphylococcus aureus, Escherichia coli and Klebsiella pneumonia. The present study investigates the relationship between TTP and in-hospital mortality in patients with monomicrobial Pseudomonas aeruginosa blood stream infection (PA-BSI). METHODS From 2006 until 2012 a retrospective cohort study was undertaken in 3 hospitals in the region surrounding Tübingen, Germany. Seventy-four patients with monomicrobial PA-BSI were studied. TTP and clinical parameters were determined and analyzed by receiver operating characteristic (ROC) analysis and Cox regression. RESULTS The in-hospital mortality of our clinical cohort was 33.78%. In multivariate Cox regression, a TTP ≤ 18 h proved to be independently associated with mortality (HR 3.83, P = 0.012) along with SAPS II score (HR 1.04, P = 0.006), cardiac disease (HR 0.33, P = 0.008) and appropriate definitive antimicrobial treatment (HR 0.21, P = 0.013). CONCLUSIONS TTP is an easy-to-measure laboratory tool for prognosis in patients with monomicrobial PA-BSI, providing useful information in addition to clinical parameters.

Malnutrition – An underestimated factor in the inpatient treatment of traumatology and orthopedic patients: A prospective evaluation of 1055 patients

INTRODUCTION Suboptimal nutritional status is often observed among hospitalized patients across all medical specialties. The objective of the present study was to (1) analyze the prevalence of malnutrition in hospitalized orthopedic and trauma patients and (2) to evaluate the relationship between malnutrition and selected clinical outcomes. MATERIALS AND METHODS The prospective field study was conducted between 06/2014 and 06/2015 in a German level I trauma center (Department of Traumatology, Septic Trauma Surgery and Arthroplasty) with a total number of 1055 patients. At hospital admission, patients were checked for malnutrition using the validated Nutritional Risk Screening (NRS). Patients at risk for malnutrition were defined as NRS≥3. Quality of life (SF-36) was assessed to evaluate the physical and mental health status prior to hospitalization. Clinical outcomes under consideration included 1) rate of adverse events, 2) length of hospitalization, and 3) mobilization after operative and conservative treatment. Patients were included independently of surgical intervention or age. RESULTS 22.3% (235) of our patients were at risk for malnutrition (NRS≥3) while a regular nutritional status (NRS<3) was diagnosed in 77.7% (819). The highest prevalence of malnutrition was found in Septic Surgery with 31.0% (106), followed by Traumatology with 19.2% (100) and Arthroplasty with 15.1% (29). Higher prevalence of malnutrition was observed among patients with typical fractures of the elderly, such as lumbar spine and pelvis (47.4%), proximal femur (36.4%) and proximal humeral (26.7%) fractures. Furthermore, patients at risk for malnutrition showed prolonged hospitalization (13.7±11.1 vs. 18.2±11.7days), delayed postoperative mobilization (2.2±2.9 vs. 4.0±4.9days) and delayed mobilization after conservative treatment (1.1±2.7 vs. 1.8±1.9days). A statistically significant correlation of NRS with each parameter (Spearmans rank correlation, p<0.05) was observed. The incidence of adverse events in patients at risk for malnutrition was statistically significantly higher compared to that of patients with a regular nutritional status (37.2% vs. 21.1%, p<0.001). CONCLUSIONS Malnutrition is widespread regarding hospitalized patients in the field of orthopedic and trauma surgery and results in suboptimal clinical outcome. It should be considered as an important factor that significantly contributes to delayed recovery. Especially elderly trauma patients and patients suffering from postoperative infections should be monitored carefully during hospitalization.

First mid-term results after cancellous allograft vitalized with autologous bone marrow for infected femoral non-union.

SummaryBackgroundSurgical treatment of infected femoral non-union is challenging. Only few reports exist including autologous bone grafting (ABG) from the iliac crest promoting union. Vitalized allogeneic bone grafting (VABG) is an alternative promoting osseous healing and reconstructing bone defects. VABG contains allogeneic cancellous bone, impregnated with autologous bone marrow puncture harvested from the iliac crest. Yet, no systematic trial exists summarizing the results of septic femoral non-union using VABG analyzing the infection eradication rate, rate of osseous integration with union, and osseous remodeling.MethodsIn this prospective non-randomized cohort study, 18 patients treated by nailing or plating for femur fractures that subsequently developed a septic non-union were included. The surgical intervention included a standardized protocol by eradicating infection first, followed by implantation VABG to promote osseous union. Main outcome measurements were radiographic union and clinical parameters.ResultsMean follow-up was 5.9 years (range: 2–8 years). Infection eradication was achieved for all patients, while union was achieved in 15 out of 18 cases (83.3 %). Mean time for union took 16.9 weeks (range: 12–24). Radiographic analysis proved osseous remodeling and full integration of VABG within 12 months for 15 patients. No infection recurrence occurred at final follow-up.ConclusionsVABG demonstrated a high union rate without donor site morbidity as the main advantage over ABG. Sufficient osseous integration within 3 months and remodeling within 12 months are promising aspects, as no late fatigue fractures occurred. However, further trials are necessary due to the limitations of this study.

Multi-omics approach identifies novel pathogen-derived prognostic biomarkers in patients with Pseudomonas aeruginosa bloodstream infection

Pseudomonas aeruginosa is a human pathogen that causes health-care associated blood stream infections (BSI). Although P. aeruginosa BSI are associated with high mortality rates, the clinical relevance of pathogen-derived prognostic biomarker to identify patients at risk for unfavorable outcome remains largely unexplored. We found novel pathogen-derived prognostic biomarker candidates by applying a multi-omics approach on a multicenter sepsis patient cohort. Multi-level Cox regression was used to investigate the relation between patient characteristics and pathogen features (2298 accessory genes, 1078 core protein levels, 107 parsimony-informative variations in reported virulence factors) with 30-day mortality. Our analysis revealed that presence of the helP gene encoding a putative DEAD-box helicase was independently associated with a fatal outcome (hazard ratio 2.01, p = 0.05). helP is located within a region related to the pathogenicity island PAPI-1 in close proximity to a pil gene cluster, which has been associated with horizontal gene transfer. Besides helP, elevated protein levels of the bacterial flagellum protein FliL (hazard ratio 3.44, p < 0.001) and of a bacterioferritin-like protein (hazard ratio 1.74, p = 0.003) increased the risk of death, while high protein levels of a putative aminotransferase were associated with an improved outcome (hazard ratio 0.12, p < 0.001). The prognostic potential of biomarker candidates and clinical factors was confirmed with different machine learning approaches using training and hold-out datasets. The helP genotype appeared the most attractive biomarker for clinical risk stratification due to its relevant predictive power and ease of detection.