Stem cell migration drives lung repair in living mice.

Tissue repair requires a highly coordinated cellular response to injury. In the lung, alveolar type 2 cells (AT2s) act as stem cells to replenish both themselves and alveolar type 1 cells (AT1s); however, the complex orchestration of stem cell activity after injury is poorly understood. Here, we establish longitudinal imaging of AT2s in murine intact tissues ex vivo and in vivo in order to track their dynamic behavior over time. We discover that a large fraction of AT2s become motile following injury and provide direct evidence for their migration between alveolar units. High-resolution morphokinetic mapping of AT2s further uncovers the emergence of distinct motile phenotypes. Inhibition of AT2 migration via genetic depletion of ArpC3 leads to impaired regeneration of AT2s and AT1s in vivo. Together, our results establish a requirement for stem cell migration between alveolar units and identify properties of stem cell motility at high cellular resolution.

Primary tumor associated macrophages activate programs of invasion and dormancy in disseminating tumor cells.

Metastases are initiated by disseminated tumor cells (DTCs) that colonize distant organs. Growing evidence suggests that the microenvironment of the primary tumor primes DTCs for dormant or proliferative fates. However, the manner in which this occurs remains poorly understood. Here, using the Window for High-Resolution Intravital Imaging of the Lung (WHRIL), we study the live lung longitudinally and follow the fate of individual DTCs that spontaneously disseminate from orthotopic breast tumors. We find that spontaneously DTCs have increased levels of retention, increased speed of extravasation, and greater survival after extravasation, compared to experimentally metastasized tumor cells. Detailed analysis reveals that a subset of macrophages within the primary tumor induces a pro-dissemination and pro-dormancy DTC phenotype. Our work provides insight into how specific primary tumor microenvironments prime a subpopulation of cells for expression of proteins associated with dissemination and dormancy.

A Permanent Window for Investigating Cancer Metastasis to the Lung.

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Sport activity levels following ankle fusion.

PURPOSE: Ankle arthrodesis (AA) is frequently employed in the treatment of end-stage ankle arthritis, which is common following trauma and athletic injuries. While AA remains a popular therapeutic option, little data exists about activity and sporting capacity following AA. The objective of this research was to determine functional outcomes and sporting activity levels in patients following Ankle Arthrodesis. METHODS: Validated questionnaires were emailed to 35 patients with a history of AA at an average follow-up of 52 months. Functional outcomes were assessed using the Foot and Ankle Disability Index (FADI), as well as the associated FADI-Sport. Responses were compared to those from a control population of RESULTS: 24 patients scheduled for AA, at an average pre-operative visit of three months. Activity levels were assessed prior to injury as well as pre- and post-operatively using the Tegner activity level scale. Average Tegner scores of the 35 surgical patients decreased from 3.82 ± 0.38 before their injury, to 1.15 ± 0.19 immediately pre-op, with recovery to 2.67 ± 0.26 following fusion. Average post arthrodesis FADI and FADI-sport scores in our patients were 76.5 ± 3.19% and 33.8 ± 23.06%. For the pre-operative control population, corresponding scores were 47.41 ± 2.61% and 22.24 ± 1.03%. Following AA, we found that patients improved upon their pre-op Tegner score, although they did not return to their pre-injury level of sporting participation. Additionally, patients reported that they had no current dysfunction with their lower leg. CONCLUSION: Patients undergoing Ankle Arthrodesis should expect improved lower leg function, though will not likely return to their pre-injury level of activity.

Development of a Small Animal Ankle Arthrodesis Model.

BACKGROUND: Our understanding of the biology of ankle arthrodesis is based largely on work in spine and long bone animal models. However, the local soft tissue and vascular anatomy of the foot and ankle is different from that of the spine. Accordingly, the objective of this study was to develop a small animal ankle arthrodesis model. METHODS: A total of 12 Lewis rats successfully underwent ankle arthrodesis with stabilization consisting of a single Kirschner wire across the prepared tibiotalar joint. Based on high nonunion rates with this initial procedure, a modification was made consisting of a second pin crossing the joint. A total of 6 rats underwent the second procedure. Radiographs were taken postoperatively and in 2-week intervals up to 10 weeks. Micro computed tomography (µCT) and histological analysis was conducted at 10 weeks to assess the fusion mass. Osseous bridging of greater than 50% across the tibiotalar joint was deemed a successful fusion. RESULTS: µCT analysis determined that 11 of the 12 rats in the single-pin cohort developed nonunions (8.3% fusion rate). In the dual-pin cohort, all 6 animals successfully fused (100% fusion rate). Histological analysis supported the radiographic imaging conclusions. CONCLUSION: While the initial procedure had a high nonunion rate, enhancing the stability of the fixation greatly increased the union rate. CLINICAL RELEVANCE: The present work demonstrates the first reliable small animal ankle arthrodesis model. We believe that this model can be used in the development of novel therapies aimed at decreasing complications and increasing fusion rates.

Impact of Vancomycin Treatment on Human Mesenchymal Stromal Cells During Osteogenic Differentiation.

BACKGROUND: Vancomycin is frequently applied locally to the operative site during foot and ankle procedures to help prevent infection. Although the efficacy of locally applied vancomycin has been demonstrated in spine surgery, there is no consensus on dosing and indication within foot and ankle surgery. Osteogenic differentiation of human mesenchymal stromal cells (hMSCs) is key to healing of both fractures and arthrodesis. The purpose of this research was to determine the impact of vancomycin on human hMSCs during the process of osteogenic differentiation. METHODS: hMSCs were cultured in osteogenic differentiation media to promote osteogenic differentiation. Cells were treated with vancomycin at differing concentrations of 0, 50, 500, and 5000 µg/mL. Viability and cell growth were assessed via LIVE/DEAD viability/cytotoxicity kit (Invitrogen, Waltham, MA) after 1, 3, and 7 days of vancomycin treatment. Differentiation and mineralization was assessed via alizarin red staining after 21 days of treatment. Mean cell viability, cell number, and mineralization were compared between treatment groups using 1-way analysis of variance and the Tukey-Kramer method for post hoc pairwise comparisons. RESULTS: At the highest concentrations of vancomycin, there was a significant reduction in cell viability and proliferation after 3 days compared with all other treatment groups. Mineralization was also significantly decreased with higher doses of vancomycin. CONCLUSION: At high concentrations, vancomycin may impair hMSC viability and osteogenic differentiation. CLINICAL RELEVANCE: Surgeons should exercise caution and consider the limited soft tissue envelope when applying vancomycin locally during foot and ankle surgery, especially during arthrodesis procedures.

Exercise-Induced Acute Bilateral Upper-Arm Compartment Syndrome.

We present a rare case of acute exercise-induced bilateral upper-arm compartment syndrome in a patient who, after a year-long hiatus from exercise, subjected his upper-extremities to the stress of over 100 pushups. The patient presented with severe pain of the bilateral biceps and triceps and complaints of dark urine. Decompressive fasciotomy was performed followed by an intensive care unit (ICU) stay for associated myoglobinuria secondary to rhabdomyolysis. The patient suffered no long-term sequelae as a result of his conditions and recovered full function of the bilateral upper-extremities. Albeit rare, acute exercise-induced compartment syndrome should be considered as a diagnosis following unaccustomed bouts of exercise.