Combined Host-Pathogen Fate Mapping to Investigate Lung Macrophages in Viral Infection.

Macrophage identity, as defined by epigenetic, transcriptional, proteomic, and functional programs, is greatly impacted by cues originating from the microenvironment. As a consequence, immunophenotyping based on surface marker expression is established and reliable in homeostatic conditions, whereas environmental challenges, in particular infections, severely hamper the determination of identity states. This has become more evident with recent discoveries that macrophage-inherent plasticity may go beyond limits of lineage-defining immunophenotypes. Therefore, transgenic fate mapping tools, such as the phage-derived loxP-cre-system, are essential for the analysis of macrophage adaptation in the tissue under extreme environmental conditions, for example, upon encounter with pathogens. In this chapter, we describe an advanced application of the loxP-cre-system during infection. Here, the host encodes a cell type-specific cre-recombinase, while the pathogen harbors a STOP-floxed fluorescent reporter gene. As an instructive example for the versatility of the system, we demonstrate that alveolar macrophages are predominantly targeted after respiratory tract infection with mouse cytomegalovirus (MCMV). Combined host-pathogen fate mapping not only enables to distinguish between infected and non-infected (bystander) macrophages but also spurs exploration of phenotypic adaptation and tracing of cellular localization in the context of MCMV infection. Moreover, we provide a gating strategy for resolving the diversity of pulmonary immune cell populations.

A non-death function of the mitochondrial apoptosis apparatus in immunity.

Apoptosis is a frequent form of programmed cell death, but the apoptotic signaling pathway can also be engaged at a low level, in the absence of cell death. We here report that such sub-lethal engagement of mitochondrial apoptosis signaling causes the secretion of cytokines from human epithelial cells in a process controlled by the Bcl-2 family of proteins. We further show that sub-lethal signaling of the mitochondrial apoptosis pathway is initiated by infections with all tested viral, bacterial, and protozoan pathogens and causes damage to the genomic DNA. Epithelial cells infected with these pathogens secreted cytokines, and this cytokine secretion upon microbial infection was substantially reduced if mitochondrial sub-lethal apoptosis signaling was blocked. In the absence of mitochondrial pro-apoptotic signaling, the ability of epithelial cells to restrict intracellular bacterial growth was impaired. Triggering of the mitochondrial apoptosis apparatus thus not only causes apoptosis but also has an independent role in immune defense.

Single versus double row suture anchor fixation for greater tuberosity fractures - a biomechanical study.

BACKGROUND: Fractures of the humeral greater tuberosity (GT) are a frequent injury progressively treated with arthroscopic suture anchor repair. Yet, no biomechanical study has been performed comparing fixation strength of arthroscopic single- (SR) vs. double row (DR) fixation. METHODS: Standardized fractures of the greater tuberosity were created in 12 fresh frozen proximal humeri. After random assignation to the SR or DR group the fixed humeri were tested applying cyclic loading to the supraspinatus and infraspinatus tendon. Load to failure and fragment displacement were assessed by means of an electrodynamic material testing machine using an optical tracking system. RESULTS: Load to failure values were higher in the DR group (649 N; ±176) than in the SR group (490 N; ±145) however without statistical significance (p = .12). In greater tuberosity displacement of 3-5 mm surgical treatment is recommended. The fixing constructs in this study did not reach displacement landmarks of 3 or 5 mm before construct failure as shown in previous studies. Thus the applied traction force (N) at 1 mm displacement was analyzed. In the SR group the load at 1 mm displacement was 277 N; ±46 compared to 260 N; ±62 in the DR group (p = .65). CONCLUSION: The results suggest that both techniques are viable options for refixation of greater tuberosity fractures. LEVEL OF EVIDENCE: Laboratory study.

Comparison of 4 Methods for Dynamization of Locking Plates: Differences in the Amount and Type of Fracture Motion.

BACKGROUND: Decreasing the stiffness of locked plating constructs can promote natural fracture healing by controlled dynamization of the fracture. This biomechanical study compared the effect of 4 different stiffness reduction methods on interfragmentary motion by measuring axial motion and shear motion at the fracture site. METHODS: Distal femur locking plates were applied to bridge a metadiaphyseal fracture in femur surrogates. A locked construct with a short-bridge span served as the nondynamized control group (LOCKED). Four different methods for stiffness reduction were evaluated: replacing diaphyseal locking screws with nonlocked screws (NONLOCKED); bridge dynamization (BRIDGE) with 2 empty screw holes proximal to the fracture; screw dynamization with far cortical locking (FCL) screws; and plate dynamization with active locking plates (ACTIVE). Construct stiffness, axial motion, and shear motion at the fracture site were measured to characterize each dynamization methods. RESULTS: Compared with LOCKED control constructs, NONLOCKED constructs had a similar stiffness (P = 0.08), axial motion (P = 0.07), and shear motion (P = 0.97). BRIDGE constructs reduced stiffness by 45% compared with LOCKED constructs (P < 0.001), but interfragmentary motion was dominated by shear. Compared with LOCKED constructs, FCL and ACTIVE constructs reduced stiffness by 62% (P < 0.001) and 75% (P < 0.001), respectively, and significantly increased axial motion, but not shear motion. CONCLUSIONS: In a surrogate model of a distal femur fracture, replacing locked with nonlocked diaphyseal screws does not significantly decrease construct stiffness and does not enhance interfragmentary motion. A longer bridge span primarily increases shear motion, not axial motion. The use of FCL screws or active plating delivers axial dynamization without introducing shear motion.

Dynamic Stabilization of Simple Fractures With Active Plates Delivers Stronger Healing Than Conventional Compression Plating.

OBJECTIVES: Active plates dynamize a fracture by elastic suspension of screw holes within the plate. We hypothesized that dynamic stabilization with active plates delivers stronger healing relative to standard compression plating. METHODS: Twelve sheep were randomized to receive either a standard compression plate (CP) or an active plate (ACTIVE) for stabilization of an anatomically reduced tibial osteotomy. In the CP group, absolute stabilization was pursued by interfragmentary compression with 6 cortical screws. In the ACTIVE group, dynamic stabilization after bony apposition was achieved with 6 elastically suspended locking screws. Fracture healing was analyzed weekly on radiographs. After sacrifice 9 weeks postsurgery, the torsional strength of healed tibiae and contralateral tibiae was measured. Finally, computed tomography was used to assess fracture patterns and healing modes. RESULTS: Healing in both groups included periosteal callus formation. ACTIVE specimens had almost 6 times more callus area by week 9 (P < 0.001) than CP specimens. ACTIVE specimens recovered on average 64% of their native strength by week 9, and were over twice as strong as CP specimens, which recovered 24% of their native strength (P = 0.008). Microcomputed tomography demonstrated that compression plating induced a combination of primary bone healing and gap healing. Active plating consistently stimulated biological bone healing by periosteal callus formation. CONCLUSIONS: Compared with compression plating, dynamic stabilization of simple fractures with active plates delivers significantly stronger healing.

Load distribution between cephalic screws in a dual lag screw trochanteric nail.

BACKGROUND: It has been observed clinically that the Z-effect is a potential cause of failure of an intramedullary nail with two cephalic screws. It describes the migration behavior of the cephalic screws in the femoral head. The primary objective was to examine different cephalic screw configurations and test the load distribution between them as a function of their relative placement and their relative movement in the nail. It has been hypothesized that different cephalic screw positions may have an influence on the stress in the implant and bone and therefore on implant failures, such as the Z-effect. METHODS: To quantify the load distribution of a dual cephalic screw intramedullary femoral nail (Citieffe, Calderara di Reno, BO, Italy), a finite element model of the femur, focusing on the loading of the cephalic screws, was prepared. Four different screw lengths (90-105 mm) were examined. The investigation considered the stresses and strains in the bone and implant as well as the relative movement of the screws. RESULTS: If the inferior cephalic screw had a shorter length, then the superior one and the femoral nail had to bear higher loads. In that case, the "equivalent von Mises stress" increased up to 10 % at the superior cephalic screw and up to 5 % at the femoral nail. The analysis of the relative movement showed that sliding of the inferior cephalic screw occurred in the nail. The total movement ranged from 0.47 to 0.73 mm for the different screw configurations. CONCLUSIONS: The stresses were distributed more equally between the two cephalic screws in the bone and the implant if a longer inferior screw was used. The stresses in the bone and implant were reduced with a longer inferior cephalic screw. Therefore, a configuration using a longer inferior cephalic screw is preferable for trochanteric fracture fixation with a dual cephalic screw intramedullary device.

Dynamic Stabilization with Active Locking Plates Delivers Faster, Stronger, and More Symmetric Fracture-Healing.

BACKGROUND: Axial dynamization of fractures can promote healing, and overly stiff fixation can suppress healing. A novel technology, termed active plating, provides controlled axial dynamization by the elastic suspension of locking holes within the plate. This prospective, controlled animal study evaluated the effect of active plates on fracture-healing in an established ovine osteotomy model. We hypothesized that symmetric axial dynamization with active plates stimulates circumferential callus and delivers faster and stronger healing relative to standard locking plates. METHODS: Twelve sheep were randomly assigned to receive a standard locking plate or an active locking plate for stabilization of a 3-mm tibial osteotomy gap. The only difference between plates was that locking holes of active plates were elastically suspended, allowing up to 1.5 mm of axial motion at the fracture. Fracture-healing was analyzed weekly on radiographs. After sacrifice at nine weeks postoperatively, callus volume and distribution were assessed by computed tomography. Finally, to determine their strength, healed tibiae and contralateral tibiae were tested in torsion until failure. RESULTS: At each follow-up, the active locking plate group had more callus (p < 0.001) than the standard locking plate group. At postoperative week 6, all active locking plate group specimens had bridging callus at the three visible cortices. In standard locking plate group specimens, only 50% of these cortices had bridged. Computed tomography demonstrated that all active locking plate group specimens and one of the six standard locking plate group specimens had developed circumferential callus. Torsion tests after plate removal demonstrated that active locking plate group specimens recovered 81% of their native strength and were 399% stronger than standard locking plate group specimens (p < 0.001), which had recovered only 17% of their native strength. All active locking plate group specimens failed by spiral fracture outside the callus zone, but standard locking plate group specimens fractured through the osteotomy gap. CONCLUSIONS: Symmetric axial dynamization with active locking plates stimulates circumferential callus and yields faster and stronger healing than standard locking plates. CLINICAL RELEVANCE: The stimulatory effect of controlled motion on fracture-healing by active locking plates has the potential to reduce healing complications and to shorten the time to return to function.

Effect of Coracoid Drilling for Acromioclavicular Joint Reconstruction Techniques on Coracoid Fracture Risk: A Biomechanical Study.

PURPOSE: To biomechanically compare the stability of the coracoid process after an anatomic double-tunnel technique using two 4-mm drill holes or a single-tunnel technique using one 4-mm or one 2.4-mm drill hole. METHODS: For biomechanical testing, 18 fresh-frozen cadaveric scapulae were used and randomly assigned to one of the following groups: two 4-mm drill holes (group 1), one 4-mm drill hole (group 2), or one 2.4-mm drill hole (group 3). After standardized coracoid drilling, load was applied to the conjoined tendons at a rate of 120 mm/min and ultimate failure load, along with the failure mode, was recorded. RESULTS: There was no significant difference between groups regarding load to failure. Mean load to failure in group 1 was 392 N; group 2, 459 N; and group 3, 506 N. The corresponding P values were .55, .74, and .20 for group 1 versus group 2, group 2 versus group 3, and group 1 versus group 3, respectively. However, the failure mode for the group with one 4-mm drill hole and the group with two 4-mm drill holes was coracoid fracture, whereas the group with one 2.4-mm drill hole showed 5 tears of the conjoined tendons and only 1 coracoid fracture (P = .015). CONCLUSIONS: Although there was no significant difference regarding load-to-failure testing between groups, the failure mechanism analysis showed that one 2.4-mm drill hole led to less destabilization of the coracoid than one or two 4-mm drill holes. CLINICAL RELEVANCE: Techniques with small, 2.4-mm drill holes might decrease the risk of severe iatrogenic fracture complications.