Michèle Sidler

Coronary optical frequency domain imaging (OFDI) for in vivo evaluation of stent healing: comparison with light and electron microscopy

Aims Coronary late stent thrombosis, a rare but devastating complication, remains an important concern in particular with the increasing use of drug-eluting stents. Notably, pathological studies have indicated that the proportion of uncovered coronary stent struts represents the best morphometric predictor of late stent thrombosis. Intracoronary optical frequency domain imaging (OFDI), a novel second-generation optical coherence tomography (OCT)-derived imaging method, may allow rapid imaging for the detection of coronary stent strut coverage with a markedly higher precision when compared with intravascular ultrasound, due to a microscopic resolution (axial ∼10–20 µm), and at a substantially increased speed of image acquisition when compared with first-generation time-domain OCT. However, a histological validation of coronary OFDI for the evaluation of stent strut coverage in vivo is urgently needed. Hence, the present study was designed to evaluate the capacity of coronary OFDI by electron (SEM) and light microscopy (LM) analysis to detect and evaluate stent strut coverage in a porcine model. Methods and results Twenty stents were implanted into 10 pigs and coronary OFDI was performed after 1, 3, 10, 14, and 28 days. Neointimal thickness as detected by OFDI correlated closely with neointimal thickness as measured by LM (r = 0.90, P < 0.01). The comparison of stent strut coverage as detected by OFDI and SEM analysis revealed an excellent agreement (r = 0.96, P < 0.01). In particular, stents completely covered by OFDI analysis were also completely covered by SEM analysis. All incompletely covered stents by OFDI were also incompletely covered by SEM. Analyses of fibrin-covered stent struts suggested that these may rarely be detected as uncovered stent struts by OFDI. Importantly, optical density measurements revealed a significant difference between fibrin- and neointima-covered coronary stent struts [0.395 (0.35–0.43) vs. 0.53 (0.47–0.57); P < 0.001], suggesting that differences in optical density provide information on the type of stent strut coverage. The sensitivity and specificity for detection of fibrin vs. neointimal coverage was evaluated using receiver-operating characteristic analysis. Conclusion The present study demonstrates that OFDI is a highly promising tool for accurate evaluation of coronary stent strut coverage, as supported by a high agreement between OFDI and light and electron microscopic analysis. Furthermore, our data indicate that optical density measurements can provide additional information with respect to the type of stent strut coverage, i.e. fibrin vs. neointimal coverage. Therefore, coronary OFDI analysis will provide important information on the biocompatibility of coronary stents.

Biocompatibility and Bone Formation of Flexible, Cotton Wool-like PLGA/Calcium Phosphate Nanocomposites in Sheep

Background: The purpose of this preliminary study was to assess the in vivo performance of synthetic, cotton wool-like nanocomposites consisting of a biodegradable poly(lactide-co-glycolide) fibrous matrix and containing either calcium phosphate nanoparticles (PLGA/CaP 60:40) or silver doped CaP nanoparticles (PLGA/Ag-CaP 60:40). Besides its extraordinary in vitro bioactivity the latter biomaterial (0.4 wt% total silver concentration) provides additional antimicrobial properties for treating bone defects exposed to microorganisms. Materials and Methods: Both flexible artificial bone substitutes were implanted into totally 16 epiphyseal and metaphyseal drill hole defects of long bone in sheep and followed for 8 weeks. Histological and histomorphological analyses were conducted to evaluate the biocompatibility and bone formation applying a score system. The influence of silver on the in vivo performance was further investigated. Results: Semi-quantitative evaluation of histology sections showed for both implant materials an excellent biocompatibility and bone healing with no resorption in the adjacent bone. No signs of inflammation were detectable, either macroscopically or microscopically, as was evident in 5 µm plastic sections by the minimal amount of inflammatory cells. The fibrous biomaterials enabled bone formation directly in the centre of the former defect. The area fraction of new bone formation as determined histomorphometrically after 8 weeks implantation was very similar with 20.5 ± 11.2 % and 22.5 ± 9.2 % for PLGA/CaP and PLGA/Ag-CaP, respectively. Conclusions: The cotton wool-like bone substitute material is easily applicable, biocompatible and might be beneficial in minimal invasive surgery for treating bone defects.

Histological and biomechanical analysis of porous additive manufactured implants made by direct metal laser sintering: a pilot study in sheep.

OBJECTIVES It was the aim of this study to analyze osseointegrative properties of porous additive manufactured titanium implants made by direct metal laser sintering in a sheep model after an implantation period of 2 and 8 weeks. MATERIAL AND METHODS Three different types of implants were placed in the pelvis of six sheep. In each sheep were placed three standard machined (M), three sandblasted and etched (SE), and three porous additive manufactured (AM) implants. Of these three implants (one per type) were examined histologically and six implants were tested biomechanically. Additionally a semiquantitative histomorphometrical and qualitative fluorescent microscopic analysis were performed. RESULTS After 2 and 8 weeks bone-to-implant-contact (BIC) values of the AM surface (2w: 20.49% ± 5.18%; 8w: 43.91% ± 9.69%) revealed no statistical significant differences in comparison to the M (2w: 20.33% ± 11.50%; 8w: 25.33% ± 4.61%) and SE (2w: 43.67 ± 12.22%; 8w: 53.33 ± 8.96%) surfaces. AM surface showed the highest increase of the BIC between the two observation time points. Considering the same implantation period histomorphometry and fluorescent labelling disclosed no significant differences in the bone surrounding the three implants groups. In contrast Removal-torque-test showed a significant improve in fixation strength (P ≤ 0.001) for the AM (1891.82 ± 308, 44 Nmm) surface after eight weeks in comparison to the M (198.93±88,04 Nmm) and SE (730.08 ± 151,89 Nmm) surfaces. CONCLUSION All three surfaces (M, SE, and AM) showed sound osseointegration. AM implants may offer a possible treatment option in clinics for patients with compromised bone situations.

The dynamisation of locking plate osteosynthesis by means of dynamic locking screws (DLS)—An experimental study in sheep

In this in vivo study a new generation of locking screws was tested. The design of the dynamic locking screw (DLS) enables the dynamisation of the cortex underneath the plate (cis-cortex) and, therefore, allows almost parallel interfragmentary closure of the fracture gap. A 45° angle osteotomy was performed unilaterally on the tibia of 37 sheep. Groups of 12 sheep were formed and in each group a different osteotomy gap (0, 1 and 3mm) was fixed using a locking compression plate (LCP) in combination with the DLS. The healing process was monitored radiographically every 3 weeks for 6, respectively 12 weeks. After this time the sheep were sacrificed, the bones harvested and the implants removed. The isolated bones were evaluated in the micro-computed tomography unit, tested biomechanically and evaluated histologically. The best results of interfragmentary movement (IFM) were shown in the 0mm configuration. The bones of this group demonstrated histomorphometrically the most distinct callus formation on the cis-cortex and the highest torsional stiffness relative to the untreated limb at 12 weeks after surgery. This animal study showed that IFM stimulated the synthesis of new bone matrix, especially underneath the plate and thus, could solve a current limitation in normal human bone healing. The DLS will be a valuable addition to the locking screw technology and improve fracture healing.

In vitro fabrication of autologous living tissue‐engineered vascular grafts based on prenatally harvested ovine amniotic fluid‐derived stem cells

Amniotic fluid cells (AFCs) have been proposed as a valuable source for tissue engineering and regenerative medicine. However, before clinical implementation, rigorous evaluation of this cell source in clinically relevant animal models accepted by regulatory authorities is indispensable. Today, the ovine model represents one of the most accepted preclinical animal models, in particular for cardiovascular applications. Here, we investigate the isolation and use of autologous ovine AFCs as cell source for cardiovascular tissue engineering applications. Fetal fluids were aspirated in vivo from pregnant ewes (n = 9) and from explanted uteri post mortem at different gestational ages (n = 91). Amniotic non‐allantoic fluid nature was evaluated biochemically and in vivo samples were compared with post mortem reference samples. Isolated cells revealed an immunohistochemical phenotype similar to ovine bone marrow‐derived mesenchymal stem cells (MSCs) and showed expression of stem cell factors described for embryonic stem cells, such as NANOG and STAT‐3. Isolated ovine amniotic fluid‐derived MSCs were screened for numeric chromosomal aberrations and successfully differentiated into several mesodermal phenotypes. Myofibroblastic ovine AFC lineages were then successfully used for the in vitro fabrication of small‐ and large‐diameter tissue‐engineered vascular grafts (n = 10) and cardiovascular patches (n = 34), laying the foundation for the use of this relevant pre‐clinical in vivo assessment model for future amniotic fluid cell‐based therapeutic applications. Copyright

The influence of different osteosynthesis configurations with locking compression plates (LCP) on stability and fracture healing after an oblique 45° angle osteotomy

BACKGROUND Locking compression plates are used in various configurations with lack of detailed information on consequent bone healing. STUDY DESIGN In this in vivo study in sheep 5 different applications of locking compression plate (LCP) were tested using a 45° oblique osteotomy simulating simple fracture pattern. 60 Swiss Alpine sheep where assigned to 5 different groups with 12 sheep each (Group 1: interfragmentary lag screw and an LCP fixed with standard cortex screws as neutralisation plate; Group 2: interfragmentary lag screw and LCP with locking head screws; Group 3: compression plate technique (hybrid construct); Group 4: internal fixator without fracture gap; Group 5: internal fixator with 3mm gap at the osteotomy site). One half of each group (6 sheep) was monitored for 6 weeks, and the other half (6 sheep) where followed for 12 weeks. METHODS X-rays at 3, 6, 9 and 12 weeks were performed to monitor the healing process. After sacrifice operated tibiae were tested biomechanically for nondestructive torsion and compared to the tibia of the healthy opposite side. After testing specimens were processed for microradiography, histology, histomorphometry and assessment of calcium deposition by fluorescence microscopy. RESULTS In all groups bone healing occurred without complications. Stiffness in biomechanical testing showed a tendency for higher values in G2 but results were not statistically significant. Values for G5 were significantly lower after 6 weeks, but after 12 weeks values had improved to comparable results. For all groups, except G3, stiffness values improved between 6 and 12 weeks. Histomorphometrical data demonstrate endosteal callus to be more marked in G2 at 6 weeks. DISCUSSION AND CONCLUSION All five configurations resulted in undisturbed bone healing and are considered safe for clinical application.

Inorganic pyrophosphatase in uncultivable hemotrophic mycoplasmas: identification and properties of the enzyme from Mycoplasma suis

BackgroundMycoplasma suis belongs to a group of highly specialized hemotrophic bacteria that attach to the surface of host erythrocytes. Hemotrophic mycoplasmas are uncultivable and the genomes are not sequenced so far. Therefore, there is a need for the clarification of essential metabolic pathways which could be crucial barriers for the establishment of an in vitro cultivation system for these veterinary significant bacteria.Inorganic pyrophosphatases (PPase) are important enzymes that catalyze the hydrolysis of inorganic pyrophosphate PPi to inorganic phosphate Pi. PPases are essential and ubiquitous metal-dependent enzymes providing a thermodynamic pull for many biosynthetic reactions. Here, we describe the identification, recombinant production and characterization of the soluble (s)PPase of Mycoplasma suis.ResultsScreening of genomic M. suis libraries was used to identify a gene encoding the M. suis inorganic pyrophosphatase (sPPase). The M. suis sPPase consists of 164 amino acids with a molecular mass of 20 kDa. The highest identity of 63.7% was found to the M. penetrans sPPase. The typical 13 active site residues as well as the cation binding signature could be also identified in the M. suis sPPase. The activity of the M. suis enzyme was strongly dependent on Mg2+ and significantly lower in the presence of Mn2+ and Zn2+. Addition of Ca2+ and EDTA inhibited the M. suis sPPase activity. These characteristics confirmed the affiliation of the M. suis PPase to family I soluble PPases. The highest activity was determined at pH 9.0. In M. suis the sPPase builds tetramers of 80 kDa which were detected by convalescent sera from experimentally M. suis infected pigs.ConclusionThe identification and characterization of the sPPase of M. suis is an additional step towards the clarification of the metabolism of hemotrophic mycoplasmas and, thus, important for the establishment of an in vitro cultivation system. As an antigenic and conserved protein the M. suis sPPase could in future be further analyzed as a diagnostic antigen.

Cardiac quadruple-fusion imaging: A brief report on a novel integrated multimodality approach for in vivo visualization of transplanted stem cells

a Cardiovascular Center, Cardiac Imaging, University Hospital Zurich, Raemistrasse 100, 8091 Zurich, Switzerland b Cardiovascular Center, Cardiology, University Hospital Zurich, Raemistrasse 100, 8091 Zurich, Switzerland c Veterinary Anaesthesia Services—International, Winterthur, Switzerland d Musculoskeletal Research Unit (MRSU), Vetsuisse Faculty, University of Zurich, Switzerland e Leibniz Research Laboratories for Biotechnology and Artificial Organs, Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

Comparison of two dental implant surface modifications on implants with same macrodesign: an experimental study in the pelvic sheep model

OBJECTIVES The aim of this study was to compare two different surfaces of one uniform macro-implant design in order to focus exclusively on the osseointegration properties after 2, 4 and 8 weeks and to discuss the animal model chosen. MATERIAL AND METHODS In six mature sheep, n = 36 implants with a highly crystalline and phosphate-enriched anodized titanium oxide surface (TiU) and n = 36 implants with a hydrophilic, sandblasted, large grit and acid-etched surface (SLA) were placed in the pelvic bone. TiU implants were custom-made to match the SLA implant design. The implant stability and bone-to-implant contact (BIC) were assessed by resonance frequency (ISQ), backscatter scanning electron microscopy (B-SEM), light microscopy (LM), micro-CT and intravital fluorochrome staining. Biomechanical removal torque testing was performed. RESULTS Overall, no statistically significant differences in BIC total (trabecular + cortical) between TiU and SLA were found via LM and B-SEM. BIC values (B-SEM; LM) in both groups revealed a steady rise in trabecular bone attachment to the implant surface after 2, 4 and 8 weeks. In the 2- to 4-week time interval in the TiU group (P = 0.005) as well as in the SLA group (P = 0.01), a statistically significant increase in BIC trabecular could be observed via LM. B-SEM values confirmed the statistically significant increase for TiU (P = 0.001). In both groups, BIC trabecular values after 8 weeks were significantly higher (P ≤ 0.05) than after 2 weeks (B-SEM; LM). Biomechanical data confirmed the histological data. CONCLUSION The two surfaces proved comparable osseointegration in this sheep model.

A Novel Multi-Phosphonate Surface Treatment of Titanium Dental Implants: A Study in Sheep

The aim of the present study was to evaluate a new multi-phosphonate surface treatment (SurfLink®) in an unloaded sheep model. Treated implants were compared to control implants in terms of bone to implant contact (BIC), bone formation, and biomechanical stability. The study used two types of implants (rough or machined surface finish) each with either the multi-phosphonate Wet or Dry treatment or no treatment (control) for a total of six groups. Animals were sacrificed after 2, 8, and 52 weeks. No adverse events were observed at any time point. At two weeks, removal torque showed significantly higher values for the multi-phosphonate treated rough surface (+32% and +29%, Dry and Wet, respectively) compared to rough control. At 52 weeks, a significantly higher removal torque was observed for the multi-phosphonate treated machined surfaces (+37% and 23%, Dry and Wet, respectively). The multi-phosphonate treated groups showed a positive tendency for higher BIC with time and increased new-old bone ratio at eight weeks. SEM images revealed greater amounts of organic materials on the multi-phosphonate treated compared to control implants, with the bone fracture (from the torque test) appearing within the bone rather than at the bone to implant interface as it occurred for control implants.