Vegfc-expressing cells form heterotopic bone after musculoskeletal injury.

Heterotopic ossification (HO) is a challenging condition that occurs after musculoskeletal injury and is characterized by the formation of bone in non-skeletal tissues. While the effect of HO on blood vessels is well established, little is known about its impact on lymphatic vessels. Here, we use a mouse model of traumatic HO to investigate the relationship between HO and lymphatic vessels. We show that injury triggers lymphangiogenesis at the injury site, which is associated with elevated vascular endothelial growth factor C (VEGF-C) levels. Through single-cell transcriptomic analyses, we identify mesenchymal progenitor cells and tenocytes as sources of Vegfc. We demonstrate by lineage tracing that Vegfc-expressing cells undergo osteochondral differentiation and contribute to the formation of HO. Last, we show that Vegfc haploinsufficiency results in a nearly 50% reduction in lymphangiogenesis and HO formation. These findings shed light on the complex mechanisms underlying HO formation and its impact on lymphatic vessels.

The HIF-1α/PLOD2 axis integrates extracellular matrix organization and cell metabolism leading to aberrant musculoskeletal repair.

While hypoxic signaling has been shown to play a role in many cellular processes, its role in metabolism-linked extracellular matrix (ECM) organization and downstream processes of cell fate after musculoskeletal injury remains to be determined. Heterotopic ossification (HO) is a debilitating condition where abnormal bone formation occurs within extra-skeletal tissues. Hypoxia and hypoxia-inducible factor 1α (HIF-1α) activation have been shown to promote HO. However, the underlying molecular mechanisms by which the HIF-1α pathway in mesenchymal progenitor cells (MPCs) contributes to pathologic bone formation remain to be elucidated. Here, we used a proven mouse injury-induced HO model to investigate the role of HIF-1α on aberrant cell fate. Using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics analyses of the HO site, we found that collagen ECM organization is the most highly up-regulated biological process in MPCs. Zeugopod mesenchymal cell-specific deletion of Hif1α (Hoxa11-CreERT2; Hif1afl/fl) significantly mitigated HO in vivo. ScRNA-seq analysis of these Hoxa11-CreERT2; Hif1afl/fl mice identified the PLOD2/LOX pathway for collagen cross-linking as downstream of the HIF-1α regulation of HO. Importantly, our scRNA-seq data and mechanistic studies further uncovered that glucose metabolism in MPCs is most highly impacted by HIF-1α deletion. From a translational aspect, a pan-LOX inhibitor significantly decreased HO. A newly screened compound revealed that the inhibition of PLOD2 activity in MPCs significantly decreased osteogenic differentiation and glycolytic metabolism. This suggests that the HIF-1α/PLOD2/LOX axis linked to metabolism regulates HO-forming MPC fate. These results suggest that the HIF-1α/PLOD2/LOX pathway represents a promising strategy to mitigate HO formation.

Intersections of Fibrodysplasia Ossificans Progressiva and Traumatic Heterotopic Ossification.

Heterotopic ossification (HO) is a debilitating pathology where ectopic bone develops in areas of soft tissue. HO can develop as a consequence of traumatic insult or as a result of dysregulated osteogenic signaling, as in the case of the orphan disease fibrodysplasia ossificans progressiva (FOP). Traumatic HO (tHO) formation is mediated by the complex interplay of signaling between progenitor, inflammatory, and nerve cells, among others, making it a challenging process to understand. Research into the pathogenesis of genetically mediated HO (gHO) in FOP has established a pathway involving uninhibited activin-like kinase 2 receptor (ALK2) signaling that leads to downstream osteogenesis. Current methods of diagnosis and treatment lag behind pre-mature HO detection and progressive HO accumulation, resulting in irreversible decreases in range of motion and chronic pain for patients. As such, it is necessary to draw on advancements made in the study of tHO and gHO to better diagnose, comprehend, prevent, and treat both.

The Experience of Medical Scribing: No Disparities Identified.

Introduction: The chronic failure to significantly increase the number of underrepresented minorities (URM) in medicine requires that we look for new mechanisms for channelling URM students through pre-medical education and into medical school. One potential mechanism is medical scribing, which involves a person helping a physician engage in real-time documentation in the electronic medical record. Methods: As a precursor to evaluating this mechanism, this survey pilot study explored individuals' experiences working as a medical scribe to look for any differences related to URM status. Of 248 scribes, 159 (64% response rate) completed an online survey. The survey was comprised of 11 items: demographics (4 items), role and length of time spent as a scribe (2 items), and experience working as a scribe (5 items). Results: The vast majority (>80%) of participants reported that working as a medical scribe gave them useful insight into being a clinician, provided valuable mentoring, and reinforced their commitment to pursue a career in medicine. The experiences reported by scribes who identified as URM did not differ from those reported by their majority counterparts. Discussion: It remains to be seen whether medical scribing can serve as an effective pipeline for URM individuals to matriculate into medical school. But the present findings suggest that the experience of working as a medical scribe is a positive one for URM.

Seraph 100 Microbind Affinity Blood Filter Does Not Clear Antibiotics: An Analysis of Antibiotic Concentration Data from PURIFY-OBS.

INTRODUCTION: Novel hemoperfusion systems are emerging for the treatment of sepsis. These devices can directly remove pathogens, pathogen-associated molecular patterns, cytokines, and other inflammatory markers from circulation. However, significant safety concerns such as potential antibiotic clearance need to be addressed prior to these devices being used in large clinical studies. METHODS: Prospective, observational study of 34 participants undergoing treatment with the Seraph 100® Microbind Affinity Blood Filter (Seraph 100) device at 6 participating sites in the USA. Patients were included for analysis if they had a record of receiving an antibiotic concurrent with Seraph 100 treatment. Patients were excluded if there was missing information for blood flow rate. Blood samples were drawn pre- and post-filter at 1 h and 4 h after treatment initiation. These average pre- and post-filter time-concentration observations were then used to estimate antibiotic clearance in L/h (CLSeraph) due to the Seraph 100 device. RESULTS: Of the 34 participants in the study, 17 met inclusion and exclusion criteria for the antibiotic analysis. Data were obtained for 7 antibiotics (azithromycin, cefazolin, cefepime, ceftriaxone, linezolid, piperacillin, and vancomycin) and one beta-lactamase inhibitor. Mean CLSeraph for the antibiotics investigated ranged from -0.57 to 0.47 L/h. No antibiotic had a CLSeraph statistically significant from 0. DISCUSSION/CONCLUSION: The Seraph 100 did not significantly clear any measured antibiotic in clinical samples. These data give further evidence to suggest that these therapies may be safely administered to critically ill patients and will not impact concentrations of administered antibiotics.

What Counts as a Surrogate Decision?

When patients lose decision-making capacity, others must make surrogate decisions on their behalf. What counts as a surrogate decision might seem self-evident. But as clinician-researchers in the field of advance care planning, we have found that it is not always so clear-cut. In this paper, we describe how and why this is a matter of concern, a novel approach for assessing whether a surrogate decision occurred, and findings from this assessment.

Assessing a Structured Mental Fitness Program for Academic Acute Care Surgeons: A Pilot Study.

INTRODUCTION: There is a well-established positive correlation between improved physician wellness and patient care outcomes. Mental fitness is a component of wellness that is understudied in academic medicine. We piloted a structured mental fitness Positive Intelligence (PQ) training program for academic surgeons, hypothesizing this would be associated with improvements in PQ scores, wellness, sleep, and trainee evaluations. METHODS: This is a single-institution, prospective, mixed-methods pilot study. All active Burn/Trauma/Acute & Critical Care Surgical faculty and fellows in our division were offered the PQ program and the option to participate in this research study. The 6-wk program consists of daily exercises on a smartphone application, weekly readings, and small-group meetings with a trained mindfulness coach. Study outcomes included changes in pretraining versus post-training PQ scores, sleep hygiene, wellness, and teaching scores. A Net Promoter Score was calculated to measure user overall experience (range -100 to 100; positive scores being supportive). For secondary analysis, participants were stratified into high versus low user groups by "muscle" scores, which were calculated by program use over time. A postintervention focus group was also held to evaluate perceptions of wellness and experience with the PQ program. RESULTS: Data were analyzed for 15 participants who provided consent. The participants were primarily White (73.3%), Assistant Professors (66.7%) with Surgical Critical Care fellowship training (86.7%), and a slight female predominance (53.3%). Comparison of scores pretraining versus post-training demonstrated statistically significant increases in PQ (59 versus 65, P = 0.004), but no significant differences for sleep (24.0 versus 29.0, P = 0.33) or well-being (89.0 versus 94.0, P = 0.10). Additionally, there was no significant difference in teaching evaluations for both residents (9.1 versus 9.3, P = 0.33) and medical students (8.3 versus 8.5, P = 0.77). High versus low user groups were defined by the median muscle score (166 [Interquartile range 95.5-298.5]). High users demonstrated a statistically higher proportion of ongoing usage (75% versus 14%, P < 0.05). The final Net Promoter Score score was 25, which demonstrates program support within this group. Focus group content analysis established eight major categories: current approaches to wellness, preknowledge, reasons for participation, expected gains, program strengths, suggestions for improvement, recommendations for approaches, and sustainability. CONCLUSIONS: Our pilot study highlighted certain benefits of a structured mental fitness program for academic acute care surgeons. Our mixed-methods data demonstrate significant improvement in PQ scores, ongoing usage in high user participants, as well as interpersonal benefits such as improved connectedness and creation of a shared language within participants. Future work should evaluate this program on a higher-powered scale, with a focus on intentionality in wellness efforts, increased exposure to mental fitness, and recruitment of trainees and other health-care providers, as well as identifying the potential implications for patient outcomes.

Inhibition of focal adhesion kinase 2 results in a macrophage polarization shift to M2 which attenuates local and systemic inflammation and reduces heterotopic ossification after polysystem extremity trauma.

Introduction: Heterotopic ossification (HO) is a complex pathology often observed in combat injured casualties who have sustained severe, high energy polytraumatic extremity injuries. Once HO has developed, prophylactic therapies are limited outside of surgical excision. Tourniquet-induced ischemia injury (IR) exacerbates trauma-mediated musculoskeletal tissue injury, inflammation, osteogenic progenitor cell development and HO formation. Others have shown that focal adhesion kinase-2 (FAK2) plays a key role in regulating early inflammatory signaling events. Therefore, we hypothesized that targeting FAK2 prophylactically would mitigate extremity trauma induced IR inflammation and HO formation. Methods: We tested whether the continuous infusion of a FAK2 inhibitor (Defactinib, PF-573228; 6.94 µg/kg/min for 14 days) can mitigate ectopic bone formation (HO) using an established blast-related extremity injury model involving femoral fracture, quadriceps crush injury, three hours of tourniquet-induced limb ischemia, and hindlimb amputation through the fracture site. Tissue inflammation, infiltrating cells, osteogenic progenitor cell content were assessed at POD-7. Micro-computed tomography imaging was used to quantify mature HO at POD-56. Results: In comparison to vehicle control-treated rats, FAK2 administration resulted in no marked wound healing complications or weight loss. FAK2 treatment decreased HO by 43%. At POD-7, marked reductions in tissue proinflammatory gene expression and assayable osteogenic progenitor cells were measured, albeit no significant changes in expression patterns of angiogenic, chondrogenic and osteogenic genes. At the same timepoint, injured tissue from FAK-treated rats had fewer infiltrating cells. Additionally, gene expression analyses of tissue infiltrating cells resulted in a more measurable shift from an M1 inflammatory to an M2 anti-inflammatory macrophage phenotype in the FAK2 inhibitor-treated group. Discussion: Our findings suggest that FAK2 inhibition may be a novel strategy to dampen trauma-induced inflammation and attenuate HO in patients at high risk as a consequence of severe musculoskeletal polytrauma.

TrkA-mediated sensory innervation of injured mouse tendon supports tendon sheath progenitor cell expansion and tendon repair.

Peripheral neurons terminate at the surface of tendons partly to relay nociceptive pain signals; however, the role of peripheral nerves in tendon injury and repair remains unclear. Here, we show that after Achilles tendon injury in mice, there is new nerve growth near tendon cells that express nerve growth factor (NGF). Conditional deletion of the Ngf gene in either myeloid or mesenchymal mouse cells limited both innervation and tendon repair. Similarly, inhibition of the NGF receptor tropomyosin receptor kinase A (TrkA) abrogated tendon healing in mouse tendon injury. Sural nerve transection blocked the postinjury increase in tendon sensory innervation and the expansion of tendon sheath progenitor cells (TSPCs) expressing tubulin polymerization promoting protein family member 3. Single cell and spatial transcriptomics revealed that disruption of sensory innervation resulted in dysregulated inflammatory signaling and transforming growth factor-ß (TGFß) signaling in injured mouse tendon. Culture of mouse TSPCs with conditioned medium from dorsal root ganglia neuron further supported a role for neuronal mediators and TGFß signaling in TSPC proliferation. Transcriptomic and histologic analyses of injured human tendon biopsy samples supported a role for innervation and TGFß signaling in human tendon regeneration. Last, treating mice after tendon injury systemically with a small-molecule partial agonist of TrkA increased neurovascular response, TGFß signaling, TSPC expansion, and tendon tissue repair. Although further studies should investigate the potential effects of denervation on mechanical loading of tendon, our results suggest that peripheral innervation is critical for the regenerative response after acute tendon injury.

Spatial transcriptomic interrogation of the murine bone marrow signaling landscape.

Self-renewal and differentiation of skeletal stem and progenitor cells (SSPCs) are tightly regulated processes, with SSPC dysregulation leading to progressive bone disease. While the application of single-cell RNA sequencing (scRNAseq) to the bone field has led to major advancements in our understanding of SSPC heterogeneity, stem cells are tightly regulated by their neighboring cells which comprise the bone marrow niche. However, unbiased interrogation of these cells at the transcriptional level within their native niche environment has been challenging. Here, we combined spatial transcriptomics and scRNAseq using a predictive modeling pipeline derived from multiple deconvolution packages in adult mouse femurs to provide an endogenous, in vivo context of SSPCs within the niche. This combined approach localized SSPC subtypes to specific regions of the bone and identified cellular components and signaling networks utilized within the niche. Furthermore, the use of spatial transcriptomics allowed us to identify spatially restricted activation of metabolic and major morphogenetic signaling gradients derived from the vasculature and bone surfaces that establish microdomains within the marrow cavity. Overall, we demonstrate, for the first time, the feasibility of applying spatial transcriptomics to fully mineralized tissue and present a combined spatial and single-cell transcriptomic approach to define the cellular components of the stem cell niche, identify cell‒cell communication, and ultimately gain a comprehensive understanding of local and global SSPC regulatory networks within calcified tissue.

Identifying what works for whom: Implementation outcomes following iLookOut, a child abuse identification and referral training program.

Introduction: iLookOut, a web-based child abuse training for early childcare professionals (ECPs), has been shown to improve knowledge and attitudes related to correctly identifying and reporting suspected cases of child abuse. The overarching goal of the present study is to examine "what works for whom" for iLookOut in order to identify strategies for optimizing learner outcomes. Methods: This prospective study enrolled 12,705 ECPs who completed iLookOut (November 2014-December 2018). We used structural equation models to test whether learner demographic and professional characteristics were differentially associated with implementation outcomes (i.e., acceptability and appropriateness) and whether these mediated subsequent indicators of training effectiveness (i.e., gains in knowledge). Results: Consistent with previous research, individuals with lower baseline knowledge scores showed greater knowledge gains (ß = -.57; p < .001). Greater knowledge gains were seen for learners who reported higher acceptability (ß = .08; p < .001) or appropriateness (ß = .14; p < .001). Implementation outcomes strongly associated with knowledge gains included acceptability for female learners and appropriateness for learners who had not completed high school or had >15 years of experience in childcare settings. Where mediation was found, for the majority of groups, appropriateness emerged as the driving mediator. Conclusion: Implementation outcomes emerged as important drivers of knowledge change for most groups. The iLookOut Core Training's use of a multimedia learning environment, video-based storylines, and game-based techniques were endorsed by learners and correlated with increases in knowledge. Future work should explore why aspects of the iLookOut training are rated as less acceptable or appropriate by some groups and what changes would improve efficacy for low performing learners.

Proteome-wide abundance profiling of yeast deletion strains for GET pathway members using sample multiplexing.

The GET pathway is associated with post-translational delivery of tail-anchored (TA) proteins to the endoplasmic reticulum (ER) in yeast, as well as other eukaryotes. Moreover, dysfunction of the GET pathway has been associated with various pathological conditions (i.e., neurodegenerative disorders, cardiovascular ailments, and protein misfolding diseases). In this study, we used yeast deletion strains of Get complex members (specifically, Get1, Get2, Get3, Get4, and Get5) coupled with sample multiplexing-based quantitative mass spectrometry to profile protein abundance on a proteome-wide scale across the five individual deletion strains. Our dataset consists of over 4500 proteins, which corresponds to >75% of the yeast proteome. The data reveal several dozen proteins that are differentially abundant in one or more deletion strains, some of which are membrane-associated, yet the abundance of many TA proteins remained unchanged. This study provides valuable insights into the roles of these Get genes, and the potential for alternative pathways which help maintain cellular function despite the disruption of the GET pathway.

Itaconate-producing neutrophils regulate local and systemic inflammation following trauma.

Modulation of the immune response to initiate and halt the inflammatory process occurs both at the site of injury as well as systemically. Due to the evolving role of cellular metabolism in regulating cell fate and function, tendon injuries that undergo normal and aberrant repair were evaluated by metabolic profiling to determine its impact on healing outcomes. Metabolomics revealed an increasing abundance of the immunomodulatory metabolite itaconate within the injury site. Subsequent single-cell RNA-Seq and molecular and metabolomic validation identified a highly mature neutrophil subtype, not macrophages, as the primary producers of itaconate following trauma. These mature itaconate-producing neutrophils were highly inflammatory, producing cytokines that promote local injury fibrosis before cycling back to the bone marrow. In the bone marrow, itaconate was shown to alter hematopoiesis, skewing progenitor cells down myeloid lineages, thereby regulating systemic inflammation. Therapeutically, exogenous itaconate was found to reduce injury-site inflammation, promoting tenogenic differentiation and impairing aberrant vascularization with disease-ameliorating effects. These results present an intriguing role for cycling neutrophils as a sensor of inflammation induced by injury - potentially regulating immune cell production in the bone marrow through delivery of endogenously produced itaconate - and demonstrate a therapeutic potential for exogenous itaconate following tendon injury.

Cas9-mediated knockout of Ndrg2 enhances the regenerative potential of dendritic cells for wound healing.

Chronic wounds impose a significant healthcare burden to a broad patient population. Cell-based therapies, while having shown benefits for the treatment of chronic wounds, have not yet achieved widespread adoption into clinical practice. We developed a CRISPR/Cas9 approach to precisely edit murine dendritic cells to enhance their therapeutic potential for healing chronic wounds. Using single-cell RNA sequencing of tolerogenic dendritic cells, we identified N-myc downregulated gene 2 (Ndrg2), which marks a specific population of dendritic cell progenitors, as a promising target for CRISPR knockout. Ndrg2-knockout alters the transcriptomic profile of dendritic cells and preserves an immature cell state with a strong pro-angiogenic and regenerative capacity. We then incorporated our CRISPR-based cell engineering within a therapeutic hydrogel for in vivo cell delivery and developed an effective translational approach for dendritic cell-based immunotherapy that accelerated healing of full-thickness wounds in both non-diabetic and diabetic mouse models. These findings could open the door to future clinical trials using safe gene editing in dendritic cells for treating various types of chronic wounds.

Tppp3+ synovial/tendon sheath progenitor cells contribute to heterotopic bone after trauma.

Heterotopic ossification (HO) is a pathological process resulting in aberrant bone formation and often involves synovial lined tissues. During this process, mesenchymal progenitor cells undergo endochondral ossification. Nonetheless, the specific cell phenotypes and mechanisms driving this process are not well understood, in part due to the high degree of heterogeneity of the progenitor cells involved. Here, using a combination of lineage tracing and single-cell RNA sequencing (scRNA-seq), we investigated the extent to which synovial/tendon sheath progenitor cells contribute to heterotopic bone formation. For this purpose, Tppp3 (tubulin polymerization-promoting protein family member 3)-inducible reporter mice were used in combination with either Scx (Scleraxis) or Pdgfra (platelet derived growth factor receptor alpha) reporter mice. Both tendon injury- and arthroplasty-induced mouse experimental HO models were utilized. ScRNA-seq of tendon-associated traumatic HO suggested that Tppp3 is an early progenitor cell marker for either tendon or osteochondral cells. Upon HO induction, Tppp3 reporter+ cells expanded in number and partially contributed to cartilage and bone formation in either tendon- or joint-associated HO. In double reporter animals, both Pdgfra+Tppp3+ and Pdgfra+Tppp3- progenitor cells gave rise to HO-associated cartilage. Finally, analysis of human samples showed a substantial population of TPPP3-expressing cells overlapping with osteogenic markers in areas of heterotopic bone. Overall, these data demonstrate that synovial/tendon sheath progenitor cells undergo aberrant osteochondral differentiation and contribute to HO after trauma.

Neutrophil and NETosis Modulation in Traumatic Heterotopic Ossification.

OBJECTIVE: To characterize the role of neutrophil extracellular traps (NETs) in heterotopic ossification (HO) formation and progression and to use mechanical and pharmacological methods to decrease NETosis and mitigate HO formation. BACKGROUND: Traumatic HO is the aberrant osteochondral differentiation of mesenchymal progenitor cells after traumatic injury, burns, or surgery. While the innate immune response has been shown to be necessary for HO formation, the specific immune cell phenotype and function remain unknown. Neutrophils, one of the earliest immune cells to respond after HO-inducing injuries, can extrude DNA, forming highly inflammatory NETs. We hypothesized that neutrophils and NETs would be diagnostic biomarkers and therapeutic targets for the detection and mitigation of HO. METHODS: C57BL6J mice underwent burn/tenotomy (a well-established mouse model of HO) or a non-HO-forming sham injury. These mice were either (1) ambulated ad libitum, (2) ambulated ad libitum with daily intraperitoneal hydroxychloroquine, ODN-2088 (both known to affect NETosis pathways), or control injections, or (3) had the injured hind limb immobilized. Single-cell analysis was performed to analyze neutrophils, NETosis, and downstream signaling after the HO-forming injury. Immunofluorescence microscopy was used to visualize NETosis at the HO site and neutrophils were identified using flow cytometry. Serum and cell lysates from HO sites were analyzed using enzyme-linked immunosorbent assay for myeloperoxidase-DNA and ELA2-DNA complexes to identify NETosis. Micro-computerized tomography was performed on all groups to analyze the HO volume. RESULTS: Molecular and transcriptional analyses revealed the presence of NETs within the HO injury site, which peaked in the early phases after injury. These NETs were highly restricted to the HO site, with gene signatures derived from both in vitro NET induction and clinical neutrophil characterizations showing a high degree of NET "priming" at the site of injury, but not in neutrophils in the blood or bone marrow. Cell-cell communication analyses revealed that this localized NET formation coincided with high levels of toll-like receptor signaling specific to neutrophils at the injury site. Reducing the overall neutrophil abundance within the injury site, either pharmacologically through treatment with hydroxychloroquine, the toll-like receptor 9 inhibitor OPN-2088, or mechanical treatment with limb offloading, results in the mitigation of HO formation. CONCLUSIONS: These data provide a further understanding of the ability of neutrophils to form NETs at the injury site, clarify the role of neutrophils in HO, and identify potential diagnostic and therapeutic targets for HO mitigation.

Genetic models for lineage tracing in musculoskeletal development, injury, and healing.

Musculoskeletal development and later post-natal homeostasis are highly dynamic processes, marked by rapid structural and functional changes across very short periods of time. Adult anatomy and physiology are derived from pre-existing cellular and biochemical states. Consequently, these early developmental states guide and predict the future of the system as a whole. Tools have been developed to mark, trace, and follow specific cells and their progeny either from one developmental state to the next or between circumstances of health and disease. There are now many such technologies alongside a library of molecular markers which may be utilized in conjunction to allow for precise development of unique cell 'lineages'. In this review, we first describe the development of the musculoskeletal system beginning as an embryonic germ layer and at each of the key developmental stages that follow. We then discuss these structures in the context of adult tissues during homeostasis, injury, and repair. Special focus is given in each of these sections to the key genes involved which may serve as markers of lineage or later in post-natal tissues. We then finish with a technical assessment of lineage tracing and the techniques and technologies currently used to mark cells, tissues, and structures within the musculoskeletal system.

In vivo screening identifies SPP2, a secreted factor that negatively regulates liver regeneration.

BACKGROUND AND AIMS: The liver is remarkably regenerative and can completely recover even when 80% of its mass is surgically removed. Identification of secreted factors that regulate liver growth would help us understand how organ size and regeneration are controlled but also provide candidate targets to promote regeneration or impair cancer growth. APPROACH AND RESULTS: To enrich for secreted factors that regulate growth control, we induced massive liver overgrowth with either YAP or MYC . Differentially expressed secreted factors were identified in these livers using transcriptomic analysis. To rank candidates by functionality, we performed in vivo CRISPR screening using the Fah knockout model of tyrosinemia. We identified secreted phosphoprotein-2 (SPP2) as a secreted factor that negatively regulates regeneration. Spp2 -deficient mice showed increased survival after acetaminophen poisoning and reduced fibrosis after repeated carbon tetrachloride injections. We examined the impact of SPP2 on bone morphogenetic protein signaling in liver cells and found that SPP2 antagonized bone morphogenetic protein signaling in vitro and in vivo. We also identified cell-surface receptors that interact with SPP2 using a proximity biotinylation assay coupled with mass spectrometry. We showed that SPP2's interactions with integrin family members are in part responsible for some of the regeneration phenotypes. CONCLUSIONS: Using an in vivo CRISPR screening system, we identified SPP2 as a secreted factor that negatively regulates liver regeneration. This study provides ways to identify, validate, and characterize secreted factors in vivo.

Tourniquet-induced ischemia creates increased risk of organ dysfunction and mortality following delayed limb amputation.

Tourniquets are critical for the control of traumatic extremity hemorrhage. In this study, we sought to determine, in a rodent blast-related extremity amputation model, the impact of prolonged tourniquet application and delayed limb amputation on survival, systemic inflammation, and remote end organ injury. Adult male Sprague Dawley rats were subjected to blast overpressure (120±7 kPa) and orthopedic extremity injury consisting femur fracture, one-minute soft tissue crush injury (20 psi), ± 180 min of tourniquet-induced hindlimb ischemia followed by delayed (60 min of reperfusion) hindlimb amputation (dHLA). All animals in the non-tourniquet group survived whereas 7/21 (33%) of the animals in the tourniquet group died within the first 72 h with no deaths observed between 72 and 168 h post-injury. Tourniquet induced ischemia-reperfusion injury (tIRI) likewise resulted in a more robust systemic inflammation (cytokines and chemokines) and concomitant remote pulmonary, renal, and hepatic dysfunction (BUN, CR, ALT. AST, IRI/inflammation-mediated genes). These results indicate prolonged tourniquet application and dHLA increases risk of complications from tIRI, leading to greater risk of local and systemic complications including organ dysfunction or death. We thus need enhanced strategies to mitigate the systemic effects of tIRI, particularly in the military prolonged field care (PFC) setting. Furthermore, future work is needed to extend the window within which tourniquet deflation to assess limb viability remains feasible, as well as new, limb-specific or systemic point of care tests to better assess the risks of tourniquet deflation with limb preservation in order to optimize patient care and save both limb and life.

Control of craniofacial development by the collagen receptor, discoidin domain receptor 2.

Development of the craniofacial skeleton requires interactions between progenitor cells and the collagen-rich extracellular matrix (ECM). The mediators of these interactions are not well-defined. Mutations in the discoidin domain receptor 2 gene (DDR2), which encodes a non-integrin collagen receptor, are associated with human craniofacial abnormalities, such as midface hypoplasia and open fontanels. However, the exact role of this gene in craniofacial morphogenesis is not known. As will be shown, Ddr2-deficient mice exhibit defects in craniofacial bones including impaired calvarial growth and frontal suture formation, cranial base hypoplasia due to aberrant chondrogenesis and delayed ossification at growth plate synchondroses. These defects were associated with abnormal collagen fibril organization, chondrocyte proliferation and polarization. As established by localization and lineage-tracing studies, Ddr2 is expressed in progenitor cell-enriched craniofacial regions including sutures and synchondrosis resting zone cartilage, overlapping with GLI1 + cells, and contributing to chondrogenic and osteogenic lineages during skull growth. Tissue-specific knockouts further established the requirement for Ddr2 in GLI +skeletal progenitors and chondrocytes. These studies establish a cellular basis for regulation of craniofacial morphogenesis by this understudied collagen receptor and suggest that DDR2 is necessary for proper collagen organization, chondrocyte proliferation, and orientation.

We each have unique facial features that are key to our identities. These features are inherited, but the mechanisms are poorly understood. People with the genetic disease spondylo-meta-epiphyseal dysplasia, or SMED, have characteristic facial and skull abnormalities including a flattened face and shortened skull. SMED is associated with mutations that inactivate the gene encoding a protein called discoidin domain receptor 2 (DDR2), which is a receptor for collagen. Collagen is the major structural protein in the human body, supporting the structure of cells and tissues. It also controls cell behaviors including growth, migration and differentiation, and it helps form tissues such as cartilage or bone. At least some of the effects of collagen on cells depend on its interaction with DDR2. Since the facial and skull abnormalities in mice with mutations that stop DDR2 from working correctly resemble those of SMED patients, these mice can be used to understand the cellular basis for this disease, as well as the role of DDR2 in the embryonic development of the face and skull. Therefore, Mohamed et al. set out to understand how loss of DDR2 causes the characteristic facial and skull defects associated with SMED. Mohamed et al. used mice that had been genetically modified so that DDR2 could be inactivated in skeletal progenitor cells, cartilage cells and bone cells (osteoblasts). Examining these mice, they found that the shortened skulls and flat face characteristic of mice lacking DDR2 are due to bones at the skull base failing to elongate correctly due to defects in the growth centers that depend on cartilage. Mohamed et al. also discovered that the cells that normally produce DDR2 are the progenitors of cartilage and bone-forming cells, which partly explains why lacking this protein leads to issues in growth of these tissues. In addition to shedding light on the causes of SMED, Mohamed et al.'s results also provide general insights into the mechanisms controlling the formation of facial and skull bones that depend on interactions between cells and collagen. This information may help explain how other abnormalities in the face and skull emerge, and provide a basis for how the shape of the skull has changed during human evolution. In the future, it may be possible to manipulate the activity of DDR2 to correct skull defects.