The Impact of Defect Size on Bone Healing in Critical-Size Bone Defects Investigated on a Rat Femur Defect Model Comparing Two Treatment Methods.

Critical-size bone defects up to 25 cm can be treated successfully using the induced membrane technique established by Masquelet. To shorten this procedure, human acellular dermis (HAD) has had success in replacing this membrane in rat models. The aim of this study was to compare bone healing for smaller and larger defects using an induced membrane and HAD in a rat model. Using our established femoral defect model in rats, the animals were placed into four groups and defects of 5 mm or 10 mm size were set, either filling them with autologous spongiosa and surrounding the defect with HAD or waiting for the induced membrane to form around a cement spacer and filling this cavity in a second operation with a cancellous bone graft. Healing was assessed eight weeks after the operation using µ-CT, histological staining, and an assessment of the progress of bone formation using an established bone healing score. The α-smooth muscle actin used as a signal of blood vessel formation was stained and counted. The 5 mm defects showed significantly better bone union and a higher bone healing score than the 10 mm defects. HAD being used for the smaller defects resulted in a significantly higher bone healing score even than for the induced membrane and significantly higher blood vessel formation, corroborating the good results achieved by using HAD in previous studies. In comparison, same-sized groups showed significant differences in bone healing as well as blood vessel formation, suggesting that 5 mm defects are large enough to show different results in healing depending on treatment; therefore, 5 mm is a viable size for further studies on bone healing.

Pretreatment of Mesenchymal Stem Cells with Electrical Stimulation as a Strategy to Improve Bone Tissue Engineering Outcomes.

Electrical stimulation (EStim), whether used alone or in combination with bone tissue engineering (BTE) approaches, has been shown to promote bone healing. In our previous in vitro studies, mesenchymal stem cells (MSCs) were exposed to EStim and a sustained, long-lasting increase in osteogenic activity was observed. Based on these findings, we hypothesized that pretreating MSC with EStim, in 2D or 3D cultures, before using them to treat large bone defects would improve BTE treatments. Critical size femur defects were created in 120 Sprague-Dawley rats and treated with scaffold granules seeded with MSCs that were pre-exposed or not (control group) to EStim 1 h/day for 7 days in 2D (MSCs alone) or 3D culture (MSCs + scaffolds). Bone healing was assessed at 1, 4, and 8 weeks post-surgery. In all groups, the percentage of new bone increased, while fibrous tissue and CD68+ cell count decreased over time. However, these and other healing features, like mineral density, bending stiffness, the amount of new bone and cartilage, and the gene expression of osteogenic markers, did not significantly differ between groups. Based on these findings, it appears that the bone healing environment could counteract the long-term, pro-osteogenic effects of EStim seen in our in vitro studies. Thus, EStim seems to be more effective when administered directly and continuously at the defect site during bone healing, as indicated by our previous studies.

One Stage Masquelets Technique: Evaluation of Different Forms of Membrane Filling with and without Bone Marrow Mononuclear Cells (BMC) in Large Femoral Bone Defects in Rats.

The classic two-stage masquelet technique is an effective procedure for the treatment of large bone defects. Our group recently showed that one surgery could be saved by using a decellularized dermis membrane (DCD, Epiflex, DIZG). In addition, studies with bone substitute materials for defect filling show that it also appears possible to dispense with the removal of syngeneic cancellous bone (SCB), which is fraught with complications. The focus of this work was to clarify whether the SCB can be replaced by the granular demineralized bone matrix (g-DBM) or fibrous demineralized bone matrix (f-DBM) demineralized bone matrix and whether the colonization of the DCD and/or the DBM defect filling with bone marrow mononuclear cells (BMC) can lead to improved bone healing. In 100 Sprague Dawley rats, a critical femoral bone defect 5 mm in length was stabilized with a plate and then encased in DCD. Subsequently, the defect was filled with SCB (control), g-DBM, or f-DBM, with or without BMC. After 8 weeks, the femurs were harvested and subjected to histological, radiological, and biomechanical analysis. The analyses showed the incipient bony bridging of the defect zone in both groups for g-DBM and f-DBM. Stability and bone formation were not affected compared to the control group. The addition of BMCs showed no further improvement in bone healing. In conclusion, DBM offers a new perspective on defect filling; however, the addition of BMC did not lead to better results.

The Induced Membrane Technique-The Filling Matters: Evaluation of Different Forms of Membrane Filling with and without Bone Marrow Mononuclear Cells (BMC) in Large Femoral Bone Defects in Rats.

The Masquelet technique is used to treat large bone defects; it is a two-stage procedure based on an induced membrane. To improve the induced membrane process, demineralized bone matrix in granular (GDBM) and fibrous form (f-DBM) was tested with and without bone marrow mononuclear cells (BMC) as filling of the membrane against the gold standard filling with syngeneic cancellous bone (SCB). A total of 65 male Sprague-Dawley rats obtained a 5 mm femoral defect. These defects were treated with the induced membrane technique and filled with SCB, GDBM, or f-DBM, with or without BMC. After a healing period of eight weeks, the femurs were harvested and submitted for histological, radiological, and biomechanical analyses. The fracture load in the defect zone was lower compared to SCB in all groups. However, histological analysis showed comparable new bone formation, bone mineral density, and cartilage proportions and vascularization. The results suggest that f-DBM in combination with BMC and the induced membrane technique cannot reproduce the very good results of this material in large, non-membrane coated bone defects, nevertheless it supports the maturation of new bone tissue locally. It can be concluded that BMC should be applied in lower doses and inflammatory cells should be removed from the cell preparation before implantation.

Impact of scaffold granule size use in Masquelet technique on periosteal reaction: a study in rat femur critical size bone defect model.

PURPOSE: The Masquelet technique for the treatment of large bone defects is a two-stage procedure based on an induced membrane. Compared to mature periosteum, the induced membrane differs significantly. However, both play a crucial role in bone regeneration. As part of a histological and radiological post-evaluation of an earlier project, we analyzed the influence of the granule size of the bone void filler Herafill® on development of periosteum regrowth in a critical size defect. METHODS: We compared three different sizes of Herafill® granules (Heraeus Medical GmbH, Wehrheim) in vivo in a rat femoral critical size defect (10 mm) treated with the induced membrane technique. After 8 weeks healing time, femurs were harvested and taken for histological and radiological analysis. RESULTS: A significantly increased regrowth of periosteum into the defect was found when small granules were used. Large granules showed significantly increased occurrence of bone capping. Small granules lead to significant increase in callus formation in the vicinity to the membrane. CONCLUSION: The size of Herafill® granules has significant impact on the development of periosteal-like structures around the defect using Masquelet's induced membrane technique. Small granules show significantly increased regrowth of periosteum and improved bone formation adjacent to the induced membrane.

Fibrous Demineralized Bone Matrix (DBM) Improves Bone Marrow Mononuclear Cell (BMC)-Supported Bone Healing in Large Femoral Bone Defects in Rats.

Regeneration of large bone defects is a major objective in trauma surgery. Bone marrow mononuclear cell (BMC)-supported bone healing was shown to be efficient after immobilization on a scaffold. We hypothesized that fibrous demineralized bone matrix (DBM) in various forms with BMCs is superior to granular DBM. A total of 65 male SD rats were assigned to five treatment groups: syngenic cancellous bone (SCB), fibrous demineralized bone matrix (f-DBM), fibrous demineralized bone matrix densely packed (f-DBM 120%), DBM granules (GDBM) and DBM granules 5% calcium phosphate (GDBM5%Ca2+). BMCs from donor rats were combined with different scaffolds and placed into 5 mm femoral bone defects. After 8 weeks, bone mineral density (BMD), biomechanical stability and histology were assessed. Similar biomechanical properties of f-DBM and SCB defects were observed. Similar bone and cartilage formation was found in all groups, but a significantly bigger residual defect size was found in GDBM. High bone healing scores were found in f-DBM (25) and SCB (25). The application of DBM in fiber form combined with the application of BMCs shows promising results comparable to the gold standard, syngenic cancellous bone. Denser packing of fibers or higher amount of calcium phosphate has no positive effect.

Longitudinal Testing for Respiratory and Gastrointestinal Shedding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Day Care Centers in Hesse, Germany.

BACKGROUND: With the pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ongoing in Europe in June 2020, day care centers were reopened in the state of Hesse, Germany, after the lockdown. The role young children play in the dynamics of the transmission was unknown. METHODS: We conducted a longitudinal study over 12 weeks and 2 days (18 June 2020-10 September 2020) to screen attendees and staff from day care centers in the state of Hesse, Germany, for both respiratory and gastrointestinal shedding of SARS-CoV-2. A total of 859 children (age range, 3 months-8 years) and 376 staff members from 50 day care centers, which were chosen representatively from throughout the state, participated in the study. Parents were asked to collect both a buccal mucosa and an anal swab from their children once a week. Staff were asked to self-administer the swabs. Reverse transcriptas polymerase chain reaction for SARS-CoV-2 was performed in a multiple-swab pooling protocol. RESULTS: A total of 7366 buccal mucosa swabs and 5907 anal swabs were analyzed. No respiratory or gastrointestinal shedding of SARS-CoV-2 was detected in any of the children. Shedding of SARS-CoV-2 was detected in 2 staff members from distinct day care centers. One was asymptomatic at the time of testing, and one was symptomatic and did not attend the facility on that day. CONCLUSION: Detection of either respiratory or gastrointestinal shedding of SARS-CoV-2 RNA in children and staff members attending day care centers was rare in the context of limited community activity and with infection prevention measures in the facilities in place.

First Human Leucocyte Antigen (HLA) Response and Safety Evaluation of Fibrous Demineralized Bone Matrix in a Critical Size Femoral Defect Model of the Sprague-Dawley Rat.

Treatment of large bone defects is one of the great challenges in contemporary orthopedic and traumatic surgery. Grafts are necessary to support bone healing. A well-established allograft is demineralized bone matrix (DBM) prepared from donated human bone tissue. In this study, a fibrous demineralized bone matrix (f-DBM) with a high surface-to-volume ratio has been analyzed for toxicity and immunogenicity. f-DBM was transplanted to a 5-mm, plate-stabilized, femoral critical-size-bone-defect in Sprague-Dawley (SD)-rats. Healthy animals were used as controls. After two months histology, hematological analyses, immunogenicity as well as serum biochemistry were performed. Evaluation of free radical release and hematological and biochemical analyses showed no significant differences between the control group and recipients of f-DBM. Histologically, there was no evidence of damage to liver and kidney and good bone healing was observed in the f-DBM group. Reactivity against human HLA class I and class II antigens was detected with mostly low fluorescence values both in the serum of untreated and treated animals, reflecting rather a background reaction. Taken together, these results provide evidence for no systemic toxicity and the first proof of no basic immunogenic reaction to bone allograft and no sensitization of the recipient.

Introduction of a New Surgical Method to Improve Bone Healing in a Large Bone Defect by Replacement of the Induced Membrane by a Human Decellularized Dermis Repopulated with Bone Marrow Mononuclear Cells in Rat.

The Masquelet technique for the treatment of large bone defects is a two-stage procedure based on an induced membrane. We eliminate the first surgical step by using a decellularized dermal skin graft (Epiflex®) populated with bone marrow mononuclear cells (BMC), as a replacement for the induced membrane. The aim of this study was to demonstrate the feasibility of this technology and provide evidence of equivalent bone healing in comparison to the induced membrane-technique. Therefore, 112 male Sprague-Dawley rats were allocated in six groups and received a 10 mm femoral defect. Defects were treated with either the induced membrane or decellularized dermis, with or without the addition of BMC. Defects were then filled with a scaffold (ß-TCP), with or without BMC. After a healing time of eight weeks, femurs were taken for histological, radiological and biomechanical analysis. Defects treated with Epiflex® showed increased mineralization and bone formation predominantly in the transplanted dermis surrounding the defect. No significant decrease of biomechanical properties was found. Vascularization of the defect could be enhanced by addition of BMC. Considering the dramatic reduction of a patient's burden by the reduced surgical stress and shortened time of treatment, this technique could have a great impact on clinical practice.

Determination of the effective dose of bone marrow mononuclear cell therapy for bone healing in vivo.

INTRODUCTION: Cell-based therapy by bone marrow mononuclear cells (BMC) in a large-sized bone defect has already shown improved vascularization and new bone formation. First clinical trials are already being conducted. BMC were isolated from bone marrow aspirate and given back to patients in combination with a scaffold within some hours. However, the optimal concentration of BMC has not yet been determined for bone healing. With this study, we want to determine the optimal dosage of the BMC in the bone defect to support bone healing. MATERIAL AND METHODS: Scaffolds with increasing BMC concentrations were inserted into a 5 mm femoral defect, cell concentrations of 2 × 106 BMC/mL, 1 × 107 BMC/mL and 2 × 107 BMC/mL were used. Based on the initial cell number used to colonize the scaffolds, the groups are designated 1 × 106, 5 × 106 and 1 × 107 group. Bone healing was assessed biomechanically, radiologically (µCT), and histologically after 8 weeks healing time. RESULTS: Improved bone healing parameters were noted in the 1 × 106 and 5 × 106 BMC groups. A significantly higher BMD was observed in the 1 × 106 BMC group compared to the other groups. Histologically, a significantly increased bone growth in the defect area was observed in group 5 × 106 BMC. This finding could be supported radiologically. CONCLUSION: It was shown that the effective dose of BMC for bone defect healing ranges from 2 × 106 BMC/mL to 1 × 107 BMC/mL. This concentration range seems to be the therapeutic window for BMC-supported therapy of large bone defects. However, further studies are necessary to clarify the exact BMC-dose dependent mechanisms of bone defect healing and to determine the therapeutically effective range more precisely.

From two stages to one: acceleration of the induced membrane (Masquelet) technique using human acellular dermis for the treatment of non-infectious large bone defects.

INTRODUCTION: The induced membrane technique for the treatment of large bone defects is a two-step procedure. In the first operation, a foreign body membrane is induced around a spacer, then, in the second step, several weeks or months later, the spacer is removed and the Membrane pocket is filled with autologous bone material. Induction of a functional biological membrane might be avoided by initially using a biological membrane. In this study, the effect of a human acellular dermis (hADM, Epiflex, DIZG gGmbH) was evaluated for the treatment of a large (5 mm), plate-stabilised femoral bone defect. MATERIAL AND METHODS: In an established rat model, hADM was compared to the two-stage induced membrane technique and a bone defect without membrane cover. Syngeneous spongiosa from donor animals was used for defect filling in all groups. The group size in each case was n = 5, the induction time of the membrane was 3-4 weeks and the healing time after filling of the defect was 8 weeks. RESULTS: The ultimate loads were increased to levels comparable with native bone in both membrane groups (hADM: 63.2% ± 29.6% of the reference bone, p < 0.05 vs. no membrane, induced membrane: 52.1% ± 25.8% of the reference bone, p < 0.05 vs. no membrane) and were significantly higher than the control group without membrane (21.5%). The membrane groups were radiologically and histologically almost completely bridged by new bone formation, in contrast to the control Group where no closed osseous bridging could be observed. CONCLUSION: The use of the human acellular dermis leads to equivalent healing results in comparison to the two-stage induced membrane technique. This could lead to a shortened therapy duration of large bone defects.

Size matters: Effect of granule size of the bone graft substitute (Herafill®) on bone healing using Masquelet's induced membrane in a critical size defect model in the rat's femur.

The Masquelet technique for the treatment of large bone defects is a two-stage procedure based on an induced membrane. The size of a scaffold is reported to be a critical factor for bone healing response. We therefore aimed to investigate the influence of the granule size of a bone graft substitute on bone marrow derived mononuclear cells (BMC) supported bone healing in combination with the induced membrane. We compared three different sizes of Herafill® granules (Heraeus Medical GmbH, Wehrheim) with or without BMC in vivo in a rat femoral critical size defect. A 10 mm defect was made in 126 rats and a membrane induced by a PMMA-spacer. After 3 weeks, the spacer was taken out and membrane filled with different granule sizes. After 8 weeks femurs were taken for radiological, biomechanical, histological, and immunohistochemical analysis. Further, whole blood of the rat was incubated with granules and expression of 29 peptide mediators was assessed. Smallest granules showed significantly improved bone healing compared to larger granules, which however did not lead to an increased biomechanical stability in the defect zone. Small granules lead to an increased accumulation of macrophages in situ which could be assigned to the inflammatory subtype M1 by majority. Increased release of chemotactic respectively proangiogenic active factors in vitro compared to syngenic bone and beta-TCP was observed. Granule size of the bone graft substitute Herafill® has significant impact on bone healing of a critical size defect in combination with Masquelet's technique in terms of bone formation and inflammatory potential.

Improvement of Bone Healing by Neutralization of microRNA-335-5p, but not by Neutralization of microRNA-92A in Bone Marrow Mononuclear Cells Transplanted into a Large Femur Defect of the Rat.

Transplanted bone marrow mononuclear cells (BMC) support the healing of large bone defects. Neutralization of microRNA (MiR) that negatively affects key processes of the reparative response in BMC might help to further improve the beneficial effect of transplanted BMC in bone healing. Hence, the aim of this study was to evaluate if the neutralization of MiR-92A (vascularization) and MiR-335-5p (osteogenic differentiation) in BMC using specific antiMiRs leads to a further improvement of the BMC-supported therapy of large bone defects. BMC transiently transfected with antiMiR- 92A, antiMiR-335, antiMiR-92A, and antiMiR-355 or control antiMiR were seeded on ß-TCP (beta-tricalcium phosphate) and placed in a femoral large bone defect (5 mm) in Sprague-Dawley rats. Ultimate load as well as osseous integration of the ß-TCP-scaffolds were significantly improved in the antiMiR-335 group compared to the control group after 8 weeks, whereas neutralization of antiMiR-92A lead to an improvement of early vascularization after 1 week, but not to enhanced bone healing after 8 weeks. We demonstrated that the targeted inhibition of MiRs in transplanted BMC is a new approach that enhances BMC-supported bone healing.

Influence of the induced membrane filled with syngeneic bone and regenerative cells on bone healing in a critical size defect model of the rat's femur.

INTRODUCTION: The induced membrane 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 rat's 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. Since little effort has been made to replace the need for autologous bone this study was performed to elucidate the influence of different stem cells and the membrane itself on bone healing in a critical size femur defect model in rats. Especially the question should be addressed whether the use of stem cells seeded on a ß-TCP scaffold is equivalent to syngeneic bone as defect filling in combination with the induced membrane technique. MATERIALS AND METHODS: A total of 96 male Sprague-Dawley (SD) rats received a 10 mm critical size defect of the femur, which was stabilized by a plate osteosynthesis and filled with PMMA cement. In a second step the spacer was extracted and the defects were filled with syngeneic bone, ß-TCP with MSC + EPC or BM-MNC. In order to elucidate the influence of the induced membrane on bone defect healing the induced membrane was removed in half of the operated femurs. The defect area was analysed 8 weeks later for bone formation (osteocalcin staining), bone mineral density (BMD) and bone strength (3-point bending test). RESULTS: New bone formation, bone mineral density and bone stiffness increased significantly, if the membrane was kept. The transplantation of biologically active material (syngeneic bone, stem cells on b-TCP) into the bone defect mostly led to a further increase of bone healing. Syngeneic bone had the greatest impact on bone healing however defects treated with stem cells were oftentimes comparable. CONCLUSION: For the first time we demonstrated the effect of the induced membrane itself and different stem cells on critical size defect healing. This could be a promising approach to reduce the need for autologous bone transplantation with its' limited availability and donor site morbidity.

Oxidative stress-driven pulmonary inflammation and fibrosis in a mouse model of human ataxia-telangiectasia.

Lung failure is responsible for significant morbidity and is a frequent cause of death in ataxia-telangiectasia (A-T). Disturbance in the redox balance of alveolar epithelial cells must be considered as a causal factor for respiratory disease in A-T. To investigate bronchoalveolar sensitivity to reactive oxygen species (ROS) and ROS-induced DNA damage, we used bleomycin (BLM) to induce experimental inflammation and fibrotic changes in the Atm-deficient mouse model. BLM or saline was administered by oropharyngeal instillation into the lung of Atm-deficient mice and wild-type mice. Mice underwent pulmonary function testing at days 0, 9, and 28, and bronchoalveolar lavage (BAL) was analysed for cell distribution and cytokines. Lung tissue was analysed by histochemistry. BLM administration resulted in a tremendous increase in lung inflammation and fibrotic changes in the lung tissue of Atm-deficient mice and was accompanied by irreversible deterioration of lung function. ATM (ataxia telangiectasia mutated) deficiency resulted in reduced cell viability, a delay in the resolution of γH2AX expression and a significant increase in intracellular ROS in pulmonary epithelial cells after BLM treatment. This was confirmed in the human epithelial cell line A549 treated with the ATM-kinase inhibitor KU55933. Our results demonstrate high bronchoalveolar sensitivity to ROS and ROS-induced DNA damage in the Atm-deficient mouse model and support the hypothesis that ATM plays a pivotal role in the control of oxidative stress-driven lung inflammation and fibrosis.

Comparison of three different types of scaffolds preseeded with human bone marrow mononuclear cells on the bone healing in a femoral critical size defect model of the athymic rat.

Large bone defects often pose major difficulties in orthopaedic surgery. The application of long-term cultured stem cells combined with a scaffold lead to a significant improvement of bone healing in recent experiments but is strongly restricted by European Union law. Bone marrow mononuclear cells (BMC), however, can be isolated and transplanted within a few hours and have been proven effective in experimental models of bone healing. The effectivity of the BMC-supported therapy might be influenced by the type of scaffold. Hence, we compared three different scaffolds serving as a carrier for BMC in a rat femoral critical size defect with regard to the osteogenic activity in the defect zone. Human demineralized bone matrix (DBM), bovine cancellous bone hydroxyapatite ceramic (BS), or ß-tricalcium phosphate (ß-TCP) were seeded with human BMC and hereafter implanted into critically sized bone defects of male athymic nude rats. Autologous bone served as a control. Gene activity was measured after 1 week, and bone formation was analysed histologically and radiologically after 8 weeks. Generally, regenerative gene expression (BMP2, RUNX2, VEGF, SDF-1, and RANKL) as well as bony bridging and callus formation was observed to be most pronounced in defects filled with autologous bone, followed in descending order by DBM, ß-TCP, and BS. Although DBM was superior in most aspects of bone regeneration analysed in comparison to ß-TCP and BS, the level of autologous bone could not be attained.

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 rat's 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.

Pre- or post-treatment with ethanol and ethyl pyruvate results in distinct anti-inflammatory responses of human lung epithelial cells triggered by interleukin-6.

Increased local and systemic levels of interleukin (IL)-6 are associated with inflammatory processes, including neutrophil infiltration of the alveolar space, resulting in lung injury. Our previous study demonstrated the beneficial anti-inflammatory effects of acute exposure to ethanol (EtOH) in an acute in vivo model of inflammation. However, due to its side-effects, EtOH is not used clinically. In the present study, the effects of EtOH and ethyl pyruvate (EtP) as an alternative anti-inflammatory drug prior to and following application of an IL-6 stimulus on cultured A549 lung epithelial cells were compared, and it was hypothesized that treatment with EtOH and EtP reduces the inflammatory potential of the A549 cells. Time- and dose-dependent release of IL-8 from the A549 cells was observed following stimulation with IL-6. The release of IL-8 from the A549 cells was assessed following treatment with EtP (2.5-10 mM), sodium pyruvate (NaP; 10 mM) or EtOH (85-170 mM) for 1, 24 or 72 h, prior to and following IL-6 stimulation. The adhesion capacities of neutrophils to the treated A549 cells, and the expression levels of cluster of differentiation (CD)54 by the epithelial cells were measured. Treatment of the A549 cells with either EtOH or EtP significantly reduced the IL-6-induced release of IL-8. This effect was observed in the pre- and post-stimulatory conditions, which is of therapeutic importance. Similar data was revealed regarding the IL-6-induced neutrophil adhesion to the treated A549 cells, in which pre- and post-treatment with EtOH or EtP decreased the adhesion capacity, however, the results were dependent on the duration of incubation. Incubation durations of 1 and 24 h decreased the adhesion rates of neutrophils to the stimulated A549 cells, however, the reduction was only significant at 72 h post-treatment. The expression of CD54 was reduced only following treatment for 24 h with either EtOH or EtP, prior to IL-6 stimulation. Therefore, EtOH and EtP reduced the inflammatory response of lung epithelial cells, and the potential of EtP to mimic EtOH was observed in the pre- and post-treatment conditions.

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 (ß-TCP, without coating or coated with fibronectin or human plasma), demineralized bone matrix (DBM), and bovine cancellous bone (BS) were assessed. Seeding efficacy on ß-TCP was 95% regardless of the surface coating. BMC demonstrated a significantly increased initial adhesion on DBM and ß-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.