A novel antibiotic class targeting the lipopolysaccharide transporter.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.

Hydrochlorothiazide and Prevention of Kidney-Stone Recurrence.

BACKGROUND: Nephrolithiasis is one of the most common conditions affecting the kidney and is characterized by a high risk of recurrence. Thiazide diuretic agents are widely used for prevention of the recurrence of kidney stones, but data regarding the efficacy of such agents as compared with placebo are limited. Furthermore, dose-response data are also limited. METHODS: In this double-blind trial, we randomly assigned patients with recurrent calcium-containing kidney stones to receive hydrochlorothiazide at a dose of 12.5 mg, 25 mg, or 50 mg once daily or placebo once daily. The main objective was to investigate the dose-response effect for the primary end point, a composite of symptomatic or radiologic recurrence of kidney stones. Radiologic recurrence was defined as the appearance of new stones on imaging or the enlargement of preexisting stones that had been observed on the baseline image. Safety was also assessed. RESULTS: In all, 416 patients underwent randomization and were followed for a median of 2.9 years. A primary end-point event occurred in 60 of 102 patients (59%) in the placebo group, in 62 of 105 patients (59%) in the 12.5-mg hydrochlorothiazide group (rate ratio vs. placebo, 1.33; 95% confidence interval [CI], 0.92 to 1.93), in 61 of 108 patients (56%) in the 25-mg group (rate ratio, 1.24; 95% CI, 0.86 to 1.79), and in 49 of 101 patients (49%) in the 50-mg group (rate ratio, 0.92; 95% CI, 0.63 to 1.36). There was no relation between the hydrochlorothiazide dose and the occurrence of a primary end-point event (P = 0.66). Hypokalemia, gout, new-onset diabetes mellitus, skin allergy, and a plasma creatinine level exceeding 150% of the baseline level were more common among patients who received hydrochlorothiazide than among those who received placebo. CONCLUSIONS: Among patients with recurrent kidney stones, the incidence of recurrence did not appear to differ substantially among patients receiving hydrochlorothiazide once daily at a dose of 12.5 mg, 25 mg, or 50 mg or placebo once daily. (Funded by the Swiss National Science Foundation and Inselspital; NOSTONE ClinicalTrials.gov number, NCT03057431.).

Utility of the SmartPilot® View advisory screen to improve anaesthetic drug titration and postoperative outcomes in clinical practice: a two-centre prospective observational trial.

BACKGROUND: The advisory system SmartPilot® View (Drägerwerk AG, Lübeck, Germany) provides real-time, demographically adjusted pharmacodynamic information throughout anaesthesia, including time course of effect-site concentrations of administered drugs and a measure of potency of the combined drug effect termed the "'Noxious Stimulation Response Index' (NSRI). This dual-centre, prospective, observational study assesses whether the availability of SmartPilot® View alters the behaviour of anaesthetic drug titration of anaesthetists and improves the Anaesthesia Quality Score (AQS; percentage of time spent with MAP 60-80 mm Hg and Bispectral Index [BIS] 40-60 [blinded]). METHODS: We recruited 493 patients scheduled for elective surgery in two university centres. A control group (CONTROL; n=170) was enrolled to observe drug titration in current practice. Thereafter, an intervention group was enrolled, for which SmartPilot® View was made available to optimise drug titration (SPV; n=188). The AQS, haemodynamic and hypnotic effects, recovery times, pain scores, and other parameters were compared between groups. RESULTS: There were 358 patients eligible for analysis. Anaesthesia quality score was similar between CONTROL and SPV (median AQS [Q1-Q3]) 25.3% [7.4-41.5%] and 22.2% [8.0-44.4%], respectively; P=0.898). Compared with CONTROL, SPV patients had less severe hypotension and hypertension, less BIS <40, faster tracheal extubation, and lower early postoperative pain scores. CONCLUSIONS: Adding SmartPilot® View information did not affect average drug titration behaviour. However, small improvements in control of MAP and BIS and early recovery suggest improved titration for some patients without increasing the risk of overdosing or underdosing. CLINICAL TRIAL REGISTRATION: NCT01467167.

Spatio-Temporal Bone Remodeling after Hematopoietic Stem Cell Transplantation.

The interaction of hematopoietic cells and the bone microenvironment to maintain bone homeostasis is increasingly appreciated. We hypothesized that the transfer of allogeneic T lymphocytes has extensive effects on bone biology and investigated trabecular and cortical bone structures, the osteoblast reconstitution, and the bone vasculature in experimental hematopoietic stem cell transplantations (HSCT). Allogeneic or syngeneic hematopoietic stem cells (HSC) and allogeneic T lymphocytes were isolated and transferred in a murine model. After 20, 40, and 60 days, bone structures were visualized using microCT and histology. Immune cells were monitored using flow cytometry and bone vessels, bone cells and immune cells were fluorescently stained and visualized. Remodeling of the bone substance, the bone vasculature and bone cell subsets were found to occur as early as day +20 after allogeneic HSCT (including allogeneic T lymphocytes) but not after syngeneic HSCT. We discovered that allogeneic HSCT (including allogeneic T lymphocytes) results in a transient increase of trabecular bone number and bone vessel density. This was paralleled by a cortical thinning as well as disruptive osteoblast lining and loss of B lymphocytes. In summary, our data demonstrate that the adoptive transfer of allogeneic HSCs and allogeneic T lymphocytes can induce profound structural and spatial changes of bone tissue homeostasis as well as bone marrow cell composition, underlining the importance of the adaptive immune system for maintaining a balanced bone biology.

Poikilothermic Animals as a Previously Unrecognized Source of Fecal Indicator Bacteria in a Backwater Ecosystem of a Large River.

Quantitative information regarding the presence of Escherichia coli, intestinal enterococci, and Clostridium perfringens in poikilotherms is notably scarce. Therefore, this study was designed to allow a systematic comparison of the occurrence of these standard fecal indicator bacteria (SFIB) in the excreta of wild homeothermic (ruminants, boars, carnivores, and birds) and poikilothermic (earthworms, gastropods, frogs, and fish) animals inhabiting an alluvial backwater area in eastern Austria. With the exception of earthworms, the average concentrations of E. coli and enterococci in the excreta of poikilotherms were equal to or only slightly lower than those observed in homeothermic excreta and were 1 to 4 orders of magnitude higher than the levels observed in the ambient soils and sediments. Enterococci reached extraordinarily high concentrations in gastropods. Additional estimates of the daily excreted SFIB (E. coli and enterococcus) loads (DESL) further supported the importance of poikilotherms as potential pollution sources. The newly established DESL metric also allowed comparison to the standing stock of SFIB in the sediment and soil of the investigated area. In agreement with its biological characteristics, the highest concentrations of C. perfringens were observed in carnivores. In conclusion, the long-standing hypothesis that only humans and homeothermic animals are primary sources of SFIB is challenged by the results of this study. It may be necessary to extend the fecal indicator concept by additionally considering poikilotherms as potential important primary habitats of SFIB. Further studies in other geographical areas are needed to evaluate the general significance of our results. We hypothesize that the importance of poikilotherms as sources of SFIB is strongly correlated with the ambient temperature and would therefore be of increased significance in subtropical and tropical habitats and water resources.IMPORTANCE The current fecal indicator concept is based on the assumption that the standard fecal indicator bacteria (SFIB) Escherichia coli, intestinal enterococci, and Clostridium perfringens multiply significantly only in the guts of humans and other homeothermic animals and can therefore indicate fecal pollution and the potential presence of pathogens from those groups. The findings of the present study showed that SFIB can also occur in high concentrations in poikilothermic animals (i.e., animals with body temperatures that vary with the ambient environmental temperature, such as fish, frogs, and snails) in an alluvial backwater area in a temperate region, indicating that a reconsideration of this long-standing indicator paradigm is needed. This study suggests that poikilotherms must be considered to be potential primary sources of SFIB in future studies.

Clinical and Research Approaches to Treat Non-union Fracture.

PURPOSE OF REVIEW: Impaired healing outcomes or even non-unions after bone injury are still a highly relevant problem in the daily clinical life. Especially within an aging population, the occurrence of bone fractures increases and thus novel treatment approaches to overcome compromised bone regeneration are needed. RECENT FINDINGS: The gold standard to treat delayed or non-healing bone injuries is still the use of autologous bone grafts to foster regeneration. Besides its successful treatment outcome, it also has disadvantages: a second surgery is needed in order to harvest the bone material and the material is highly limited. Looking into the recent literature, a multitude of different research approaches were already conducted to identify new possible strategies to treat impaired bone regeneration: application of mesenchymal stromal cells, platelet lysates, growth factors, interference in the immune system, or bone formation stimulation by ultrasound. This review gives an overview of the treatment approaches actually performed in the clinic as well as at the bench in the context of compromised bone healing. It clearly highlights the complexity of the nature of non-healing bone fractures as well as patient-dependent factors influencing the healing process.

Influence of postmenopausal state, solar radiation, drugs and comorbidities on serum calcium and phosphate in 13,000 hospital admissions.

AIMS: Our goal was to determine the relative contributions of demographic variables, drugs, comorbidities, and weather conditions on serum calcium (Ca) and phosphate (Pi) in patients admitted to a tertiary referral center. METHODS: For 12,667 consecutive patients admitted to the Kantonsspital St. Gallen, drug history on admission, age, sex, body weight, ICD-10 diagnoses, and laboratory data were extracted from electronic medical records. Weather parameters prior to hospital admission were also integrated in a regression analysis. RESULTS: Serum Ca was normally distributed with a median (interquartile range) of 2.3 (2.2/2.4) mmol/L. In contrast Pi showed a right tailed distribution of 1.0 (0.9/1.2) mmol/L. Ca was increased in postmenopausal women. Solar radiation prior to admission was associated both with higher Ca and higher Pi. Lower blood pressure was associated with lower Ca and higher Pi. In addition Ca increased by 0.017 mmol/L per g/L increase of albumin (p < 0.0001). CONCLUSIONS: Serum Ca and Pi at hospital admission are highly dependent on patient characteristics, drugs, and comorbidities. In particular, we found higher Ca in postmenopausal women. The commonly applied albumin correction formula of Payne (0.025 mmol/L Ca per g/L albumin) may overestimate the effect of albumin; we propose using 0.017 mmol/L Ca per g/L albumin or measurement of free (ionized) Ca.

Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum.

The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged organs, including limbs, brain, heart, tail, and others. Studies on axolotl shed light on cellular and molecular pathways ruling progenitor activation and tissue restoration after injury. This knowledge can be applied to facilitate the healing of regeneration-incompetent injuries, such as bone non-union. In the current protocol, the femur osteotomy stabilization using an internal plate fixation system is described. The procedure was adapted for use in aquatic animals (axolotl, Ambystoma mexicanum). ≥20 cm snout-to-tail tip axolotls with fully ossified, mouse-size comparable femurs were used, and special attention was paid to the plate positioning and fixation, as well as to the postoperative care. This surgical technique allows for standardized and stabilized bone fixation and could be useful for direct comparison to axolotl limb regeneration and analogous studies of bone healing across amphibians and mammals.

Caloric restriction reduces trabecular bone loss during aging and improves bone marrow adipocyte endocrine function in male mice.

Introduction: Caloric restriction (CR) is a nutritional intervention that increases life expectancy while lowering the risk for cardio-metabolic disease. Its effects on bone health, however, remain controversial. For instance, CR has been linked to increased accumulation of bone marrow adipose tissue (BMAT) in long bones, a process thought to elicit detrimental effects on bone. Qualitative differences have been reported in BMAT in relation to its specific anatomical localization, subdividing it into physiological and potentially pathological BMAT. We here examine the local impact of CR on bone composition, microstructure and its endocrine profile in the context of aging. Methods: Young and aged male C57Bl6J mice were subjected to CR for 8 weeks and were compared to age-matched littermates with free food access. We assessed bone microstructure and BMAT by micro-CT, bone fatty acid and transcriptomic profiles, and bone healing. Results: CR increased tibial BMAT accumulation and adipogenic gene expression. CR also resulted in elevated fatty acid desaturation in the proximal and mid-shaft regions of the tibia, thus more closely resembling the biochemical lipid profile of the distally located, physiological BMAT. In aged mice, CR attenuated trabecular bone loss, suggesting that CR may revert some aspects of age-related bone dysfunction. Cortical bone, however, was decreased in young mice on CR and remained reduced in aged mice, irrespective of dietary intervention. No negative effects of CR on bone regeneration were evident in either young or aged mice. Discussion: Our findings indicate that the timing of CR is critical and may exert detrimental effects on bone biology if administered during a phase of active skeletal growth. Conversely, CR exerts positive effects on trabecular bone structure in the context of aging, which occurs despite substantial accumulation of BMAT. These data suggest that the endocrine profile of BMAT, rather than its fatty acid composition, contributes to healthy bone maintenance in aged mice.

Distance fields on unstructured grids: Stable interpolation, assumed gradients, collision detection and gap function.

This article presents a novel approach to collision detection based on distance fields. A novel interpolation ensures stability of the distances in the vicinity of complex geometries. An assumed gradient formulation is introduced leading to a [Formula: see text]-continuous distance function. The gap function is re-expressed allowing penalty and Lagrange multiplier formulations. The article introduces a node-to-element integration for first order elements, but also discusses signed distances, partial updates, intermediate surfaces, mortar methods and higher order elements. The algorithm is fast, simple and robust for complex geometries and self contact. The computed tractions conserve linear and angular momentum even in infeasible contact. Numerical examples illustrate the new algorithm in three dimensions.

Asynchronous variational integration using continuous assumed gradient elements.

Asynchronous variational integration (AVI) is a tool which improves the numerical efficiency of explicit time stepping schemes when applied to finite element meshes with local spatial refinement. This is achieved by associating an individual time step length to each spatial domain. Furthermore, long-term stability is ensured by its variational structure. This article presents AVI in the context of finite elements based on a weakened weak form (W2) Liu (2009) [1], exemplified by continuous assumed gradient elements Wolff and Bucher (2011) [2]. The article presents the main ideas of the modified AVI, gives implementation notes and a recipe for estimating the critical time step.

The benefits of adipocyte metabolism in bone health and regeneration.

Patients suffering from musculoskeletal diseases must cope with a diminished quality of life and an increased burden on medical expenses. The interaction of immune cells and mesenchymal stromal cells during bone regeneration is one of the key requirements for the restoration of skeletal integrity. While stromal cells of the osteo-chondral lineage support bone regeneration, an excessive accumulation of cells of the adipogenic lineage is thought to promote low-grade inflammation and impair bone regeneration. Increasing evidence indicates that pro-inflammatory signaling from adipocytes is responsible for various chronic musculoskeletal diseases. This review aims to summarize the features of bone marrow adipocytes by phenotype, function, secretory features, metabolic properties and their impact on bone formation. In detail, the master regulator of adipogenesis and prominent diabetes drug target, peroxisome proliferator-activated receptor γ (PPARG), will be debated as a potential therapeutic approach to enhance bone regeneration. We will explore the possibilities of using clinically established PPARG agonists, the thiazolidinediones (TZDs), as a treatment strategy to guide the induction of a pro-regenerative, metabolically active bone marrow adipose tissue. The impact of this PPARG induced bone marrow adipose tissue type on providing the necessary metabolites to sustain osteogenic-as well as beneficial immune cells during bone fracture healing will be highlighted.

COMMBINI: an experimentally-informed COmputational Model of Macrophage dynamics in the Bone INjury Immunoresponse.

Bone fracture healing is a well-orchestrated but complex process that involves numerous regulations at different scales. This complexity becomes particularly evident during the inflammatory stage, as immune cells invade the healing region and trigger a cascade of signals to promote a favorable regenerative environment. Thus, the emergence of criticalities during this stage might hinder the rest of the process. Therefore, the investigation of the many interactions that regulate the inflammation has a primary importance on the exploration of the overall healing progression. In this context, an in silico model named COMMBINI (COmputational Model of Macrophage dynamics in the Bone INjury Immunoresponse) has been developed to investigate the mechano-biological interactions during the early inflammatory stage at the tissue, cellular and molecular levels. An agent-based model is employed to simulate the behavior of immune cells, inflammatory cytokines and fracture debris as well as their reciprocal multiscale biological interactions during the development of the early inflammation (up to 5 days post-injury). The strength of the computational approach is the capacity of the in silico model to simulate the overall healing process by taking into account the numerous hidden events that contribute to its success. To calibrate the model, we present an in silico immunofluorescence method that enables a direct comparison at the cellular level between the model output and experimental immunofluorescent images. The combination of sensitivity analysis and a Genetic Algorithm allows dynamic cooperation between these techniques, enabling faster identification of the most accurate parameter values, reducing the disparity between computer simulation and histological data. The sensitivity analysis showed a higher sensibility of the computer model to the macrophage recruitment ratio during the early inflammation and to proliferation in the late stage. Furthermore, the Genetic Algorithm highlighted an underestimation of macrophage proliferation by in vitro experiments. Further experiments were conducted using another externally fixated murine model, providing an independent validation dataset. The validated COMMBINI platform serves as a novel tool to deepen the understanding of the intricacies of the early bone regeneration phases. COMMBINI aims to contribute to designing novel treatment strategies in both the biological and mechanical domains.

Complex Spatio-Temporal Interplay of Distinct Immune and Bone Cell Subsets during Bone Fracture Healing.

BACKGROUND: The healing of a bone injury is a highly complex process involving a multitude of different tissue and cell types, including immune cells, which play a major role in the initiation and progression of bone regeneration. METHODS: We histologically analyzed the spatio-temporal occurrence of cells of the innate immune system (macrophages), the adaptive immune system (B and T lymphocytes), and bone cells (osteoblasts and osteoclasts) in the fracture area of a femoral osteotomy over the healing time. This study was performed in a bone osteotomy gap mouse model. We also investigated two key challenges of successful bone regeneration: hypoxia and revascularization. RESULTS: Macrophages were present in and around the fracture gap throughout the entire healing period. The switch from initially pro-inflammatory M1 macrophages to the anti-inflammatory M2 phenotype coincided with the revascularization as well as the appearance of osteoblasts in the fracture area. This indicates that M2 macrophages are necessary for the restoration of vessels and that they also play an orchestrating role in osteoblastogenesis during bone healing. The presence of adaptive immune cells throughout the healing process emphasizes their essential role for regenerative processes that exceeds a mere pathogen defense. B and T cells co-localize consistently with bone cells throughout the healing process, consolidating their crucial role in guiding bone formation. These histological data provide, for the first time, comprehensive information about the complex interrelationships of the cellular network during the entire bone healing process in one standardized set up. With this, an overall picture of the spatio-temporal interplay of cellular key players in a bone healing scenario has been created. CONCLUSIONS: A spatio-temporal distribution of immune cells, bone cells, and factors driving bone healing at time points that are decisive for this process-especially during the initial steps of inflammation and revascularization, as well as the soft and hard callus phases-has been visualized. The results show that the bone healing cascade does not consist of five distinct, consecutive phases but is a rather complex interrelated and continuous process of events, especially at the onset of healing.

A buprenorphine depot formulation provides effective sustained post-surgical analgesia for 72 h in mouse femoral fracture models.

Adequate pain management is essential for ethical and scientific reasons in animal experiments and should completely cover the period of expected pain without the need for frequent re-application. However, current depot formulations of Buprenorphine are only available in the USA and have limited duration of action. Recently, a new microparticulate Buprenorphine formulation (BUP-Depot) for sustained release has been developed as a potential future alternative to standard formulations available in Europe. Pharmacokinetics indicate a possible effectiveness for about 72 h. Here, we investigated whether the administration of the BUP-Depot ensures continuous and sufficient analgesia in two mouse fracture models (femoral osteotomy) and could, therefore, serve as a potent alternative to the application of Tramadol via the drinking water. Both protocols were examined for analgesic effectiveness, side effects on experimental readout, and effects on fracture healing outcomes in male and female C57BL/6N mice. The BUP-Depot provided effective analgesia for 72 h, comparable to the effectiveness of Tramadol in the drinking water. Fracture healing outcome was not different between analgesic regimes. The availability of a Buprenorphine depot formulation for rodents in Europe would be a beneficial addition for extended pain relief in mice, thereby increasing animal welfare.

Odd skipped-related 1 controls the pro-regenerative response of fibro-adipogenic progenitors.

Skeletal muscle regeneration requires the coordinated interplay of diverse tissue-resident- and infiltrating cells. Fibro-adipogenic progenitors (FAPs) are an interstitial cell population that provides a beneficial microenvironment for muscle stem cells (MuSCs) during muscle regeneration. Here we show that the transcription factor Osr1 is essential for FAPs to communicate with MuSCs and infiltrating macrophages, thus coordinating muscle regeneration. Conditional inactivation of Osr1 impaired muscle regeneration with reduced myofiber growth and formation of excessive fibrotic tissue with reduced stiffness. Osr1-deficient FAPs acquired a fibrogenic identity with altered matrix secretion and cytokine expression resulting in impaired MuSC viability, expansion and differentiation. Immune cell profiling suggested a novel role for Osr1-FAPs in macrophage polarization. In vitro analysis suggested that increased TGFß signaling and altered matrix deposition by Osr1-deficient FAPs actively suppressed regenerative myogenesis. In conclusion, we show that Osr1 is central to FAP function orchestrating key regenerative events such as inflammation, matrix secretion and myogenesis.

Local immune cell contributions to fracture healing in aged individuals - A novel role for interleukin 22.

With increasing age, the risk of bone fractures increases while regenerative capacity decreases. This variation in healing potential appears to be linked to adaptive immunity, but the underlying mechanism is still unknown. This study sheds light on immunoaging/inflammaging, which impacts regenerative processes in aging individuals. In an aged preclinical model system, different levels of immunoaging were analyzed to identify key factors that connect immunoaged/inflammaged conditions with bone formation after long bone fracture. Immunological facets, progenitor cells, the microbiome, and confounders were monitored locally at the injury site and systemically in relation to healing outcomes in 12-month-old mice with distinct individual levels of immunoaging. Bone tissue formation during healing was delayed in the immunoaged group and could be associated with significant changes in cytokine levels. A prolonged and amplified pro-inflammatory reaction was caused by upregulated immune cell activation markers, increased chemokine receptor availability and a lack of inhibitory signaling. In immunoaged mice, interleukin-22 was identified as a core cell signaling protein that played a central role in delayed healing. Therapeutic neutralization of IL-22 reversed this specific immunoaging-related disturbed healing. Immunoaging was found to be an influencing factor of decreased regenerative capacity in aged individuals. Furthermore, a novel therapeutic strategy of neutralizing IL-22 may successfully rejuvenate healing in individuals with advanced immune experiences.

The multifaceted roles of macrophages in bone regeneration: A story of polarization, activation and time.

To present knowledge, macrophages are found in all tissues of the human body. They are a cell population with high plasticity which come with a multitude of functions which appear to be adapted to the respective tissue niche and micro-environment in which they reside. Bone harbors multiple macrophage subpopulations, with the osteoclasts as classical representative of a bone resorbing cells and osteomacs as a bone tissue resident macrophage first described by the expression of F4/80. Both subtypes are found throughout all phases in bone healing. In vivo data on bone regeneration have demonstrated their essential role in initiating the healing cascade (inflammatory phase) but also of the later phases of healing (e.g. endochondral and intramembranous bone formation). To participate in such diverse processes macrophages have to be highly plastic in their functionality. Thus, the widely used M1/M2 paradigm to distinguish macrophage subpopulations may not mirror the comprehensive role of the dynamics of macrophage plasticity. From a clinical perspective it is especially relevant to distinguish what drives macrophages in impaired healing scenarios, implant loosening or infections, where their specific role of a misbalanced inflammatory setting is so far only partially known. With this review we aim at illustrating current knowledge and gaps of knowledge on macrophage plasticity and function during the cascades of regeneration and reconstitution of bone tissue. We propose aspects of the known biological mechanisms of macrophages and their specific subsets that might serve as targets to control their function in impaired healing and eventually support a scar-free regeneration. STATEMENT OF SIGNIFICANCE: Macrophages are essential for successful regeneration. In scar-free healing such as in bone, a complete failure of healing was shown if macrophages were depleted; the M1/M2 switch appears to be key to the progression from pro-inflammation to regeneration. However, experimental data illustrate that the classical M1/M2 paradigm does not completely mirror the complexity of observed macrophage functions during bone healing and thus demands a broader perspective. Within this review we discuss the high degree of plasticity of macrophages and the relevant contribution of the different and more specific M2 subtypes (M2a-M2f) during (bone) regeneration. It summarizes the versatile roles of macrophages in skeletal regeneration and thereby highlights potential target points for immunomodulatory approaches to enable or even foster bone repair.