Kerstin Kontradowitz

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.

Nlrp1 inflammasome is downregulated in trauma patients

After a major trauma, IL-1β-producing capacity of monocytes is reduced. Generation of IL-1β is important for appropriate immune response after trauma and requires not only synthesis and transcription of inflammasome components but also their activation. Altered IL-1β-processing due to deregulated NLRP inflammasomes assembly is associated with several inflammatory diseases. However, the precise role of NLRP1 inflammasome in monocytes after trauma is unknown. Here, we investigated if NLRP1 inflammasome components are responsible for depressed monocyte function after trauma. We found in ex vivo in vitro assays that LPS-stimulation of CD14+-isolated monocytes from healthy volunteers (HV) results in remarkably higher capacity of the IL-1β-release compared to trauma patients (TP). During the 10-day time course, this monocyte depression was highest immediately after admission. Inflammasome activation correlating with this inflammatory response was demonstrated by enhanced protein production of cleaved IL-1β and caspase-1. Furthermore, we found that the gene expression of IL-1β, caspase-1, and ASC was comparable in TP and HV after LPS-stimulation during the 10-day course, while NLRP1 was markedly reduced in TP. We demonstrated that transfected monocytes from TP, which expressed the lacking components, were recovered in their LPS-induced IL-1β-release and that lacking of NLRP1 is responsible for the suppressed monocyte activity after trauma. The restoration of NLRP1 inflammasome suggests new mechanistic target for the recovery of dysbalanced immune reaction after trauma.Key messageSuppression in monocyte function occurs early after a major trauma or surgery.Reduced gene expression abrogates NLRP1 inflammasome assembly after trauma.Limited availability of inflammasome components may cause reduced host defense.Restoring NLRP1 in immune-suppressed monocytes recovers NLPR1 activity after trauma.Recovered inflammasome activity may improve the immune response to PAMPs/DAMPs.

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.

Decreased Inflammatory Responses of Human Lung Epithelial Cells after Ethanol Exposure Are Mimicked by Ethyl Pyruvate

Background and Purpose. Leukocyte migration into alveolar space plays a critical role in pulmonary inflammation resulting in lung injury. Acute ethanol (EtOH) exposure exerts anti-inflammatory effects. The clinical use of EtOH is critical due to its side effects. Here, we compared effects of EtOH and ethyl pyruvate (EtP) on neutrophil adhesion and activation of cultured alveolar epithelial cells (A549). Experimental Approach. Time course and dose-dependent release of interleukin- (IL-) 6 and IL-8 from A549 were measured after pretreatment of A549 with EtP (2.5–10 mM), sodium pyruvate (NaP, 10 mM), or EtOH (85–170 mM), and subsequent lipopolysaccharide or IL-1beta stimulation. Neutrophil adhesion to pretreated and stimulated A549 monolayers and CD54 surface expression were determined. Key Results. Treating A549 with EtOH or EtP reduced substantially the cytokine-induced release of IL-8 and IL-6. EtOH and EtP (but not NaP) reduced the adhesion of neutrophils to monolayers in a dose- and time-dependent fashion. CD54 expression on A549 decreased after EtOH or EtP treatment before IL-1beta stimulation. Conclusions and Implications. EtP reduces secretory and adhesive potential of lung epithelial cells under inflammatory conditions. These findings suggest EtP as a potential treatment alternative that mimics the anti-inflammatory effects of EtOH in early inflammatory response in lungs.

Impaired Surface Expression of HLA-DR, TLR2, TLR4, and TLR9 in Ex Vivo-In Vitro Stimulated Monocytes from Severely Injured Trauma Patients

Objective. Trauma patients (TP) frequently develop an imbalanced immune response that often causes infectious postinjury complications. Monocytes show a diminished capability of both producing proinflammatory cytokines and antigen presentation after trauma. TLR2, TLR4, and TLR9 recognize pathogens and subsequently activate monocytes. While there are conflictive data about TLR2 and TLR4 expression after trauma, no studies about the expression of TLR2, TLR4, TLR9, and HLA-DR on monocytes from TP after their secondary ex vivo-in vitro “hit” have been reported. Methods/Results. Ex vivo-in vitro lipopolysaccharide- (LPS-) stimulated blood from TP showed diminished interleukin- (IL-) 1β-release in TP for five postinjury days compared to healthy volunteers (HV). The recovery was observed at day 5. In parallel, monocytes from TP showed an impaired capability of TLR2, TLR4, and TLR9 expression after secondary stimulation compared to HV, while the measurement of unstimulated samples showed significant reduction of TLR4 and TLR9 at ED. Furthermore, HLA-DR decreased after trauma and was even more profound by stimulation of monocytes. Ratio of monocytes to leukocytes was significantly increased at days 6 and 7 after trauma compared to HV. Conclusion. Impaired expression of TLRs and HLA-DR in acute inflammatory conditions may be responsible for the well-described monocyte paralysis after severe trauma.

Cultivation of EPC and co-cultivation with MSC on β-TCP granules in vitro is feasible without fibronectin coating but influenced by scaffolds’ design

IntroductionMeanwhile, the osteoconductive properties of frequently used synthetic bone grafts can be improved by the use of osteoinductive cells and growth factors. Nevertheless, the cultivation of endothelial progenitor cells (EPC) seems to be difficult and requires a pre-conditioning of the scaffolds with fibronectin. Additionally, the influence of the scaffolds’ design on cell cultivation is not fully elucidated.MethodsAs scaffold, a commercially available β-tricalcium phosphate was used. 5 × 105 EPC, or 5 × 105 MSC or a combination of each 2.5 × 105 cells was seeded onto the granules. We investigated seeding efficiency, cell morphology, cell metabolism, adherence, apoptosis and gene expression of EPC and MSC in this in vitro study on days 2, 6 and 10.ResultsTotal number of adherent cells was higher on the β-TCP without fibronectin coating. The number of cells in all approaches significantly declined when a solid β-TCP was used. Metabolic activity of MSC was comparable throughout the scaffolds and increased until day 10. Additionally, the amount of supernatants VEGF was higher for MSC than for EPC.DiscussionOur results demonstrate that a coating of the scaffold for successful cultivation of EPC in vitro is not necessary. Furthermore, our study showed that structural differences of the scaffolds significantly influenced cell adherence and metabolic activity. Thereby, the influence on EPC seems to be higher than on MSC.

Corrigendum to "Characterization of Bone Marrow Mononuclear Cells on Biomaterials for Bone Tissue Engineering In Vitro".

In the paper titled “Characterization of Bone Marrow Mononuclear Cells on Biomaterials for Bone Tissue Engineering In Vitro,” [1] there was an error in the Acknowledgments. The changes made to the Acknowledgments are as follows. The AO-Research Foundation and the respective Grant no. S-11-64-N were added since the AO provided financial support to this study. Therefore the Acknowledgments was changed as follows: “This work was partially founded by the German Institute for Cell and Tissue Replacement gGmbH (DIZG), Berlin, the LOEWE Center for Cell and Gene Therapy Frankfurt funded by Hessian Ministry of Higher Education, Research and the Arts (funding reference no. III L 4-518/17.004 (2010)), and the AO-Research Fund, Davos, Switzerland (funding reference no. S-11-64-N).”