Christoph Nau

Establishment and characterization of the Masquelet induced membrane technique in a rat femur critical‐sized defect model

The Masquelet induced membrane technique for reconstructing large diaphyseal defects has been shown to be a promising clinical treatment, yet relatively little is known about the cellular, histological and biochemical make‐up of these membranes and how they produce this positive clinical outcome. We compared cellular make‐up, histological changes and growth factor expression in membranes induced around femur bone defects and in subcutaneous pockets at 2, 4 and 6 weeks after induction, and to the periosteum. We found that membranes formed around bone defects were similar to those formed in subcutaneous pockets; however, both were significantly different from periosteum with regard to structural characteristics, location of blood vessels and overall thickness. Membranes induced at the femur defect (at 2 weeks) and in periosteum contain mesenchymal stem cells (MSCs; STRO‐1+) which were not found in membranes induced subcutaneously. BMP‐2, TGFβ and VEGF were significantly elevated in membranes induced around femur defects in comparison to subcutaneously induced membranes, whereas SDF‐1 was not detectable in membranes induced at either site. We found that osteogenic and neovascular activity had mostly subsided by 6 weeks in membranes formed at both sites. It was conclude that cellular composition and growth factor content in induced membranes depends on the location where the membrane is induced and differs from periosteum. Osteogenic and neovascular activity in the membranes is maximal between 2 and 4 weeks and subsides after 6. Based on this, better and quicker bone healing might be achieved if the PMMA cement were replaced with a bone graft earlier in the Masquelet technique. Copyright

High calcium bioglass enhances differentiation and survival of endothelial progenitor cells, inducing early vascularization in critical size bone defects.

Early vascularization is a prerequisite for successful bone healing and endothelial progenitor cells (EPC), seeded on appropriate biomaterials, can improve vascularization. The type of biomaterial influences EPC function with bioglass evoking a vascularizing response. In this study the influence of a composite biomaterial based on polylactic acid (PLA) and either 20 or 40% bioglass, BG20 and BG40, respectively, on the differentiation and survival of EPCs in vitro was investigated. Subsequently, the effect of the composite material on early vascularization in a rat calvarial critical size defect model with or without EPCs was evaluated. Human EPCs were cultured with β-TCP, PLA, BG20 or BG40, and seeding efficacy, cell viability, cell morphology and apoptosis were analysed in vitro. BG40 released the most calcium, and improved endothelial differentiation and vitality best. This effect was mimicked by adding an equivalent amount of calcium to the medium and was diminished in the presence of the calcium chelator, EGTA. To analyze the effect of BG40 and EPCs in vivo, a 6-mm diameter critical size calvarial defect was created in rats (n = 12). Controls (n = 6) received BG40 and the treatment group (n = 6) received BG40 seeded with 5×105 rat EPCs. Vascularization after 1 week was significantly improved when EPCs were seeded onto BG40, compared to implanting BG40 alone. This indicates that Ca2+ release improves EPC differentiation and is useful for enhanced early vascularization in critical size bone defects.

Alteration of Masquelet's induced membrane characteristics by different kinds of antibiotic enriched bone cement in a critical size defect model in the rat's femur.

The Masquelet technique for the treatment of large bone defects consists of a 2-stage procedure. In the first stage, a polymethylmethacrylate (PMMA) cement spacer is inserted into the bony defect of a rats femur and over a period of 2-4 weeks a membrane forms that encapsulates the defect/spacer. In a second operation the membrane is opened, the PMMA spacer is removed and the resulting cavity is filled with autologous bone. Different kinds of bone cements are available, with or without supplemental antibiotics. Both might influence the development and the characteristics of the induced membrane which might affect the bone healing response. Hence, this comparative study was performed to elucidate the effect of different bone cements with or without supplemental antibiotics on the development of an induced membrane in a critical size femur defect model in rats. A total of 72 male SD rats received a 10mm critical size defect of the femur which was stabilised by a plate osteosynthesis and filled with either Palacos+Gentamycin, Copal Gentamycin+Vancomycin, Copal+Gentamycin+Clindamycin or Copal Spacem. The induced membranes were analysed after two, four and six weeks (wks) after insertion of the cement spacers (n=6/group). Paraffin embedded histological sections of the membrane were microscopically analysed for membrane thickness, elastic fibres, vascularisation and proliferation by an independent observer blinded to the group setup. The thickness of the induced membrane increased significantly from 2 wks (553 μm) to 6 wks (774 μm) in group Palacos+Gentamycin whereas membrane thickness decreased significantly in groups Copal+Gentamycin+Clindamycin (682-329 μm) and Copal Spacem (916 μm to 371 μm). The comparison between the groups revealed significantly increased membrane thickness in group Palacos+Gentamycin and Copal Gentamycin+Vancomycin in comparison to group Copal+Gentamycin+Clindamycin six weeks after induction. However, the fraction of elastic fibres was significantly increased in groups Copal+Gentamycin+Clindamycin (71%, 80%) and Copal Spacem (82%, 81%) after 2 and 4 weeks in comparison to the groups Palacos+Gentamycin (56%, 57%) and Copal Gentamycin+Vancomycin (63%, 69%). Those differences however were partly diminished after 6 wks. The ratio of immature (vWF+) to more mature (CD31+) blood vessels increased significantly in groups Palacos+Gentamycin and Copal Gentamycin+Vancomycin whereas no significant alterations were noted in groups Copal+Gentamycin+Clindamycin and Copal Spacem. For the first time we demonstrated that thickness and proportion of elastic fibres in induced membranes were influenced by the type of cement and the kind of supplemental antibiotics being used. Whether these alterations of the induced membrane have an effect on bone healing remains to be proven in future studies.

Characterization of Bone Marrow Mononuclear Cells on Biomaterials for Bone Tissue Engineering In Vitro

Bone marrow mononuclear cells (BMCs) are suitable for bone tissue engineering. Comparative data regarding the needs of BMC for the adhesion on biomaterials and biocompatibility to various biomaterials are lacking to a large extent. Therefore, we evaluated whether a surface coating would enhance BMC adhesion and analyze the biocompatibility of three different kinds of biomaterials. BMCs were purified from human bone marrow aspirate samples. Beta tricalcium phosphate (β-TCP, without coating or coated with fibronectin or human plasma), demineralized bone matrix (DBM), and bovine cancellous bone (BS) were assessed. Seeding efficacy on β-TCP was 95% regardless of the surface coating. BMC demonstrated a significantly increased initial adhesion on DBM and β-TCP compared to BS. On day 14, metabolic activity was significantly increased in BMC seeded on DBM in comparison to BMC seeded on BS. Likewise increased VEGF-synthesis was observed on day 2 in BMC seeded on DBM when compared to BMC seeded on BS. The seeding efficacy of BMC on uncoated biomaterials is generally high although there are differences between these biomaterials. Beta-TCP and DBM were similar and both superior to BS, suggesting either as suitable materials for spatial restriction of BMC used for regenerative medicine purposes in vivo.

Effect of the harvest procedure and tissue site on the osteogenic function of and gene expression in human mesenchymal stem cells

Evidence has indicated that mesenchymal stem cells (MSCs) harvested with the Reamer/Irrigator/Aspirator (RIA) procedure exhibited an improved osteogenic differentiation capability compared with MSCs obtained by bone marrow aspiration from the iliac crest. In the present study, we hypothesized that the harvest procedure indeed influences the osteogenic activity of human MSCs more than the tissue site itself. Concentration [by colony forming unit-fibroblast (CFU-F) assay], calcification (by von Kossa staining), collagen deposition, gene expression and the gene methylation of the bone morphogenetic protein (BMP)-2 pathway [BMP2, SMAD5 and runt-related transcription factor 2 (RUNX2)], the Wnt pathway [WNT3, dickkopf-1 (DKK1), low-density lipoprotein receptor-related protein 5 (LRP5) and β-catenin] and osteogenic genes [alkaline phosphatase (ALP), collagen, type I, alpha 1 (COL1A) and osteocalcin] were analyzed in the MSCs isolated intraoperatively from the iliac crest with a spoon (n=14), from the femur with a spoon (n=7), from the femur with the RIA procedure (n=13) and from the iliac crest by fine-needle aspiration (n=8, controls). A Bonferroni-Holm corrected p-value <0.05 indicated a statistically significant difference. The concentration of CFU-F in the MSCs was increased in the RIA debris in comparison with that in the iliac crest aspirates (trend) and the femur (spoon, significant). Calcium deposition was highest in the femur-derived MSCs (by RIA) and was significantly increased in comparison with that in the iliac crest-derived MSCs (spoon, aspirate). The gene expression of BMP2, SMAD5, RUNX2, osteocalcin, and COL1A was significantly increased in the femur-derived MSCs (spoon) and the iliac crest aspirate derived-MSCs in comparison with that in the femur-derived MSCs (by RIA). There was no significant diversity between the samples obtained using a spoon (from the femur or iliac crest). Calcium deposition and osteogenic gene expression decreased significantly with the increasing passage number in all the samples. The methylation of genes did not correlate with their respective gene expression and inconsistent differences were observed between the groups. Herein, we provide evidence that the harvest procedure is a critical factor in the osteogenesis of MSCs in vitro. The MSCs isolated from the femur and iliac crest using a spoon exhibit no significant differences. The altered gene expression and function of the femur-derived MSCs (by RIA) may be due to the harsh isolation procedure. The variable differentiation ability of the MSCs, which depends on the harvest site and the harvest technique, as well as the rapid loss of the osteogenic differentiation capacity with the increasing culture duration should be taken into consideration when using MSCs as a potential therapeutic application for bone tissue engineering.

Liver cirrhosis but not alcohol abuse is associated with impaired outcome in trauma patients – A retrospective, multicentre study

INTRODUCTION Liver cirrhosis has been shown to be associated with impaired outcome in patients who underwent elective surgery. We therefore investigated the impact of alcohol abuse and subsequent liver cirrhosis on outcome in multiple trauma patients. MATERIALS AND METHODS Using the multi-centre population-based Trauma Registry of the German Society for Trauma Surgery, we retrospectively compared outcome in patients (ISS ≥ 9, ≥ 18) with pre-existing alcohol abuse and liver cirrhosis with healthy trauma victims in univariate and matched-pair analysis. Means were compared using Students t-test and analysis of variance (ANOVA) and categorical variables using χ(2) (p<0.05=significant). RESULTS Overall 13,527 patients met the inclusion criteria and were, thus, analyzed. 713 (5.3%) patients had a documented alcohol abuse and 91 (0.7%) suffered from liver cirrhosis. Patients abusing alcohol and suffering from cirrhosis differed from controls regarding injury pattern, age and outcome. More specific, liver cirrhotic patients showed significantly higher in-hospital mortality than predicted (35% vs. predicted 19%) and increased single- and multi-organ failure rates. While alcohol abuse increased organ failure rates as well this did not affect in-hospital mortality. CONCLUSIONS Patients suffering from liver cirrhosis presented impaired outcome after multiple injuries. Pre-existing condition such as cirrhosis should be implemented in trauma scores to assess the individual mortality risk profile.

Effects of positive blood alcohol concentration on outcome and systemic interleukin-6 in major trauma patients

BACKGROUND The influence of alcohol on the outcome after major trauma remains controversial. In several recent studies, alcohol has been associated with neuroprotective effects in head injuries, while others reported negative or no effects on survival and/or the in-hospital stay in major trauma patients (TP). The purpose of this study was to examine the relationship of alcohol with injury characteristics and outcome as well as to analyze possible anti-inflammatory properties in major TP. PATIENTS/METHODS 184 severely injured TP with an Injury Severity Score (ISS) ≥16 were successively enrolled. All patients had measured blood alcohol concentration (BAC). Patients were grouped according to their positive BAC (>0.5‰, BAC) vs. <0.5‰ alcohol (no BAC) upon arrival at the emergency department (ED). Injury characteristics, physiologic parameters and outcome with respect to organ or multiple organ failure (MOF), SIRS, sepsis, pneumonia, ARDS or mortality were assessed. Systemic levels of interleukin (IL)-6 at ED were determined. RESULTS Forty-nine TP had positive BAC without chronic alcohol abuse history and 135 patients had BAC levels below 0.5‰. Overall injury severity and age were comparable in both groups. No BAC TP received significantly higher numbers of packed red blood cells and fresh frozen plasma (transfused within the initial 24h or in total) compared to BAC TP. Organ failure, MOF, SIRS, sepsis, pneumonia, ARDS and the in-hospital mortality were not different between both groups. Trauma patients with positive BAC had significantly decreased leukocyte numbers and systemic IL-6 levels compared to no BAC group. There was a significant positive correlation between leukocyte counts and IL-6 as well as BAC and leukocytes. BAC levels did not correlate with IL-6. CONCLUSIONS Positive BAC is associated with reduced leukocyte numbers and lowered systemic IL-6 levels at admittance indicating immune-suppressive effects of alcohol in major trauma patients.

Treatment of Large Bone Defects with a Vascularized Periosteal Flap in Combination with Biodegradable Scaffold Seeded with Bone Marrow-Derived Mononuclear Cells: An Experimental Study in Rats.

INTRODUCTION The surgical treatment of large bone defects continues to pose a major challenge in modern trauma and orthopedic surgery. In this study we test the effectiveness of a tissue engineering approach, using three-dimensional (3D) β-tricalcium phosphate (β-TCP) scaffolding plus bone marrow-derived mononuclear cells (BM-MNCs), combined with a vascularized periosteal flap, in a rat femur critical size defect model. METHODS Eighty rats were randomly allocated into four equal groups. Under general anesthesia, critical size defects were created on their femurs and were treated with (1) Vascularized periosteal flap alone, (2) Vascularized periosteal flap+β-TCP scaffolding, (3) Vascularized periosteal flap+β-TCP scaffolding+ligated vascular pedicle, and (4) Vascularized periosteal flap+β-TCP scaffolding+BM-MNCs. After 4 and 8 weeks animals were euthanized and the bone defects were harvested for analysis of new bone formation, vascularization, and strength using histology, immunohistology, micro-CT, and biomechanical testing, respectively. RESULTS Group 1: (P. flap) Increase in new bone formation and vascularization. Group 2: (P. flap+scaffold) Increase in new bone formation and vascularization. Group 3: (P. flap+scaffold+ligated vascular pedicle) No new bone formation and no vascularization. Group 4: (P. flap+scaffold+BM-MNCs) A significant (p < 0.05) increase was seen in new bone formation, vascularization, and strength in bones treated with flaps, scaffold, and BM-MNCs, when compared with the other treatment groups. CONCLUSION Combining a vascularized periosteal flap with tissue engineering approach (β-TCP scaffolding and BM-MNC) results in significantly improved bone healing in our rat femur critical size bone defect model.

Evaluation of a dual-room sliding gantry CT concept for workflow optimisation in polytrauma and regular in- and outpatient management.

OBJECTIVES To reveal the impact on workflow from introducing a dual-room sliding gantry CT to the trauma room for polytrauma and regularly scheduled in- outpatients with regard to efficiency and degree of capacity utilisation. MATERIALS AND METHODS Time analysis was performed for 30 polytrauma patients each in 2 different trauma room settings, the new trauma room comprising a sliding gantry CT, the old one a stationary single-room CT. Complete trauma room and diagnostic workup times were manually measured and compared for both groups. In a third scenario, the number of CT scans performed with one single sliding gantry CT and the two-room concept was compared to the number of CT scans performed on two separate regular CT units in a 5 days clinical routine sample. RESULTS Patients demographics and type of CT examinations were comparable for all patient groups. The median time from patient arrival in the trauma room until beginning of CT scanning was 6 min shorter for the sliding gantry CT group (21 vs.15 min). Sliding gantry CT embedded in a two-room solution achieved 252 CT scans in 5 working days, compared to 250 CT scans on two separate regular CT units with the same man power. CONCLUSIONS Sliding gantry CT in the trauma room allows for significant time saving in the diagnostic workup of polytrauma patients and faster resumption of the regular in- outpatients CT schedule is possible. With the same man power, the dual-room solution is able to generate the same throughput as two separate CT units.

Time dependency and topography of hepatic nuclear factor κB activation after hemorrhagic shock and resuscitation in mice.

The leading causes of death in people aged 1 to 44 years are unintentional injuries with associated hemorrhagic shock. Hemorrhagic shock followed by resuscitation (H/R) activates the nuclear factor &kgr;B (NF-&kgr;B) pathway. To further address the association between liver damage and NF-&kgr;B activation, we analyzed the H/R-induced activation of NF-&kgr;B using cis-NF-&kgr;Begfp reporter gene mice. In these mice, the expression of green fluorescent protein (GFP) is linked to the activation of NF-&kgr;B, and therefore tracing of GFP colocalizes NF-&kgr;B activation. Mice were hemorrhaged to a mean arterial blood pressure of 30mmHg for 90 min, followed by resuscitation. Six, 14, or 24 h after resuscitation, mice were killed. Compared with sham-operated mice, H/R led to a profound hepatic and cellular damage as measured by aspartate aminotransferase, creatine kinase, and lactate dehydrogenase levels, which was accompanied by an elevation in interleukin 6 levels and hepatic leukocyte infiltration. Interleukin 10 levels in plasma were elevated 6 h after H/R. Using serial liver sections, we found an association between necrotic areas, oxidative stress, and enhanced GFP–positive cells. Furthermore, enhanced GFP–positive cells surrounded areas of necrotic liver tissue, predominantly in a penumbra-like–shape pericentrally. These results elucidate spatial relationship between oxidative stress, liver necrosis, and NF-&kgr;B activation, using an in vivo approach and therefore might help to further analyze mechanisms of NF-&kgr;B activation after resuscitated blood loss. ABBREVIATIONS ALT—alanine transaminase eGFP—enhanced green fluorescent protein H/R—hemorrhagic shock and reperfusion KC—Kupffer cells NF-&kgr;B—nuclear factor &kgr;B light-chain enhancer of activated B cells TLR—Toll-like receptor