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.

Nr4a1 enhances Wnt4 transcription to promote mesenchymal stem cell osteogenesis and alleviates inflammation-inhibited bone regeneration.

Intense inflammatory response impairs bone marrow mesenchymal stem cell (BMSC)-mediated bone regeneration, with transforming growth factor (TGF)-ß1 being the most highly expressed cytokine. However, how to find effective and safe means to improve bone formation impaired by excessive TGF-ß1 remains unclear. In this study, we found that the expression of orphan nuclear receptor Nr4a1, an endogenous repressor of TGF-ß1, was suppressed directly by TGF-ß1-induced Smad3 and indirectly by Hdac4, respectively. Importantly, Nr4a1 overexpression promoted BMSC osteogenesis and reversed TGF-ß1-mediated osteogenic inhibition and pro-fibrotic effects. Transcriptomic and histologic analyses confirmed that upregulation of Nr4a1 increased the transcription of Wnt family member 4 (Wnt4) and activated Wnt pathway. Mechanistically, Nr4a1 bound to the promoter of Wnt4 and regulated its expression, thereby enhancing the osteogenic capacity of BMSCs. Moreover, treatment with Nr4a1 gene therapy or Nr4a1 agonist Csn-B could promote ectopic bone formation, defect repair, and fracture healing. Finally, we demonstrated the correlation of NR4A1 with osteogenesis and the activation of the WNT4/ß-catenin pathway in human BMSCs and fracture samples. Taken together, these findings uncover the critical role of Nr4a1 in bone formation and alleviation of inflammation-induced bone regeneration disorders, and suggest that Nr4a1 has the potential to be a therapeutic target for accelerating bone healing.

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.

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.

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.

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.

Macrophage-mediated PDGF Activation Correlates With Regenerative Outcomes Following Musculoskeletal Trauma.

OBJECTIVE: Our objective was to identify macrophage subpopulations and gene signatures associated with regenerative or fibrotic healing across different musculoskeletal injury types. BACKGROUND: Subpopulations of macrophages are hypothesized to fine tune the immune response after damage, promoting either normal regenerative, or aberrant fibrotic healing. METHODS: Mouse single-cell RNA sequencing data before and after injury were assembled from models of musculoskeletal injury, including regenerative and fibrotic mouse volumetric muscle loss (VML), regenerative digit tip amputation, and fibrotic heterotopic ossification. R packages Harmony , MacSpectrum , and Seurat were used for data integration, analysis, and visualizations. RESULTS: There was a substantial overlap between macrophages from the regenerative VML (2 mm injury) and regenerative bone models, as well as a separate overlap between the fibrotic VML (3 mm injury) and fibrotic bone (heterotopic ossification) models. We identified 2 fibrotic-like (FL 1 and FL 2) along with 3 regenerative-like (RL 1, RL 2, and RL 3) subpopulations of macrophages, each of which was transcriptionally distinct. We found that regenerative and fibrotic conditions had similar compositions of proinflammatory and anti-inflammatory macrophages, suggesting that macrophage polarization state did not correlate with healing outcomes. Receptor/ligand analysis of macrophage-to-mesenchymal progenitor cell crosstalk showed enhanced transforming growth factor ß in fibrotic conditions and enhanced platelet-derived growth factor signaling in regenerative conditions. CONCLUSION: Characterization of macrophage subtypes could be used to predict fibrotic responses following injury and provide a therapeutic target to tune the healing microenvironment towards more regenerative conditions.

Discoidin domain receptor 2 regulates aberrant mesenchymal lineage cell fate and matrix organization.

Extracellular matrix (ECM) interactions regulate both the cell transcriptome and proteome, thereby determining cell fate. Traumatic heterotopic ossification (HO) is a disorder characterized by aberrant mesenchymal lineage (MLin) cell differentiation, forming bone within soft tissues of the musculoskeletal system following traumatic injury. Recent work has shown that HO is influenced by ECM-MLin cell receptor signaling, but how ECM binding affects cellular outcomes remains unclear. Using time course transcriptomic and proteomic analyses, we identified discoidin domain receptor 2 (DDR2), a cell surface receptor for fibrillar collagen, as a key MLin cell regulator in HO formation. Inhibition of DDR2 signaling, through either constitutive or conditional Ddr2 deletion or pharmaceutical inhibition, reduced HO formation in mice. Mechanistically, DDR2 perturbation alters focal adhesion orientation and subsequent matrix organization, modulating Focal Adhesion Kinase (FAK) and Yes1 Associated Transcriptional Regulator and WW Domain Containing Transcription Regulator 1 (YAP/TAZ)-mediated MLin cell signaling. Hence, ECM-DDR2 interactions are critical in driving HO and could serve as a previously unknown therapeutic target for treating this disease process.

A specialized metabolic pathway partitions citrate in hydroxyapatite to impact mineralization of bones and teeth.

Citrate is a critical metabolic substrate and key regulator of energy metabolism in mammalian cells. It has been known for decades that the skeleton contains most (>85%) of the body's citrate, but the question of why and how this metabolite should be partitioned in bone has received singularly little attention. Here, we show that osteoblasts use a specialized metabolic pathway to regulate uptake, endogenous production, and the deposition of citrate into bone. Osteoblasts express high levels of the membranous Na+-dependent citrate transporter solute carrier family 13 member 5 (Slc13a5) gene. Inhibition or genetic disruption of Slc13a5 reduced osteogenic citrate uptake and disrupted mineral nodule formation. Bones from mice lacking Slc13a5 globally, or selectively in osteoblasts, showed equivalent reductions in cortical thickness, with similarly compromised mechanical strength. Surprisingly, citrate content in mineral from Slc13a5-/- osteoblasts was increased fourfold relative to controls, suggesting the engagement of compensatory mechanisms to augment endogenous citrate production. Indeed, through the coordinated functioning of the apical membrane citrate transporter SLC13A5 and a mitochondrial zinc transporter protein (ZIP1; encoded by Slc39a1), a mediator of citrate efflux from the tricarboxylic acid cycle, SLC13A5 mediates citrate entry from blood and its activity exerts homeostatic control of cytoplasmic citrate. Intriguingly, Slc13a5-deficient mice also exhibited defective tooth enamel and dentin formation, a clinical feature, which we show is recapitulated in primary teeth from children with SLC13A5 mutations. Together, our results reveal the components of an osteoblast metabolic pathway, which affects bone strength by regulating citrate deposition into mineral hydroxyapatite.

Single-cell mapping of regenerative and fibrotic healing responses after musculoskeletal injury.

After injury, a cascade of events repairs the damaged tissue, including expansion and differentiation of the progenitor pool and redeposition of matrix. To guide future wound regeneration strategies, we compared single-cell sequencing of regenerative (third phalangeal element [P3]) and fibrotic (second phalangeal element [P2]) digit tip amputation (DTA) models as well as traumatic heterotopic ossification (HO; aberrant). Analyses point to a common initial response to injury, including expansion of progenitors, redeposition of matrix, and activation of transforming growth factor ß (TGF-ß) and WNT pathways. Surprisingly, fibrotic P2 DTA showed greater transcriptional similarity to HO than to regenerative P3 DTA, suggesting that gene expression more strongly correlates with healing outcome than with injury type or cell origin. Differential analysis and immunostaining revealed altered activation of inflammatory pathways, such as the complement pathway, in the progenitor cells. These data suggests that common pathways are activated in response to damage but are fine tuned within each injury. Modulating these pathways may shift the balance toward regenerative outcomes.

NELL1 Regulates the Matrisome to Promote Osteosarcoma Progression.

Sarcomas produce an abnormal extracellular matrix (ECM), which in turn provides instructive cues for cell growth and invasion. Neural EGF like-like molecule 1 (NELL1) is a secreted glycoprotein characterized by its nonneoplastic osteoinductive effects, yet it is highly expressed in skeletal sarcomas. Here, we show that genetic deletion of NELL1 markedly reduces invasive behavior across human osteosarcoma (OS) cell lines. NELL1 deletion resulted in reduced OS disease progression, inhibiting metastasis and improving survival in a xenograft mouse model. These observations were recapitulated with Nell1 conditional knockout in mouse models of p53/Rb-driven sarcomagenesis, which reduced tumor frequency and extended tumor-free survival. Transcriptomic and phosphoproteomic analyses demonstrated that NELL1 loss skews the expression of matricellular proteins associated with reduced FAK signaling. Culturing NELL1 knockout sarcoma cells on wild-type OS-enriched matricellular proteins reversed the phenotypic and signaling changes induced by NELL1 deficiency. In sarcoma patients, high expression of NELL1 correlated with decreased overall survival. These findings in mouse and human models suggest that NELL1 expression alters the sarcoma ECM, thereby modulating cellular invasive potential and prognosis. Disruption of NELL1 signaling may represent a novel therapeutic approach to short-circuit sarcoma disease progression. SIGNIFICANCE: NELL1 modulates the sarcoma matrisome to promote tumor growth, invasion, and metastasis, identifying the matrix-associated protein as an orchestrator of cell-ECM interactions in sarcomagenesis and disease progression.

Spatial transcriptomics reveals metabolic changes underly age-dependent declines in digit regeneration.

De novo limb regeneration after amputation is restricted in mammals to the distal digit tip. Central to this regenerative process is the blastema, a heterogeneous population of lineage-restricted, dedifferentiated cells that ultimately orchestrates regeneration of the amputated bone and surrounding soft tissue. To investigate skeletal regeneration, we made use of spatial transcriptomics to characterize the transcriptional profile specifically within the blastema. Using this technique, we generated a gene signature with high specificity for the blastema in both our spatial data, as well as other previously published single-cell RNA-sequencing transcriptomic studies. To elucidate potential mechanisms distinguishing regenerative from non-regenerative healing, we applied spatial transcriptomics to an aging model. Consistent with other forms of repair, our digit amputation mouse model showed a significant impairment in regeneration in aged mice. Contrasting young and aged mice, spatial analysis revealed a metabolic shift in aged blastema associated with an increased bioenergetic requirement. This enhanced metabolic turnover was associated with increased hypoxia and angiogenic signaling, leading to excessive vascularization and altered regenerated bone architecture in aged mice. Administration of the metabolite oxaloacetate decreased the oxygen consumption rate of the aged blastema and increased WNT signaling, leading to enhanced in vivo bone regeneration. Thus, targeting cell metabolism may be a promising strategy to mitigate aging-induced declines in tissue regeneration.

Neuron-to-vessel signaling is a required feature of aberrant stem cell commitment after soft tissue trauma.

The functional interdependence of nerves and blood vessels is a well-established concept during tissue morphogenesis, yet the role of neurovascular coupling in proper and aberrant tissue repair is an emerging field of interest. Here, we sought to define the regulatory relationship of peripheral nerves on vasculature in a severe extremity trauma model in mice, which results in aberrant cell fate and heterotopic ossification (HO). First, a high spatial degree of neurovascular congruency was observed to exist within extremity injury associated heterotopic ossification. Vascular and perivascular cells demonstrate characteristic responses to injury, as assessed by single cell RNA sequencing. This vascular response to injury was blunted in neurectomized mice, including a decrease in endothelial proliferation and type H vessel formation, and a downregulation of key transcriptional networks associated with angiogenesis. Independent mechanisms to chemically or genetically inhibit axonal ingrowth led to similar deficits in HO site angiogenesis, a reduction in type H vessels, and heterotopic bone formation. Finally, a combination of single cell transcriptomic approaches within the dorsal root ganglia identified key neural-derived angiogenic paracrine factors that may mediate neuron-to-vascular signaling in HO. These data provide further understanding of nerve-to-vessel crosstalk in traumatized soft tissues, which may reflect a key determinant of mesenchymal progenitor cell fate after injury.

NGF-p75 signaling coordinates skeletal cell migration during bone repair.

Bone regeneration following injury is initiated by inflammatory signals and occurs in association with infiltration by sensory nerve fibers. Together, these events are believed to coordinate angiogenesis and tissue reprogramming, but the mechanism of coupling immune signals to reinnervation and osteogenesis is unknown. Here, we found that nerve growth factor (NGF) is expressed following cranial bone injury and signals via p75 in resident mesenchymal osteogenic precursors to affect their migration into the damaged tissue. Mice lacking Ngf in myeloid cells demonstrated reduced migration of osteogenic precursors to the injury site with consequently delayed bone healing. These features were phenocopied by mice lacking p75 in Pdgfra+ osteoblast precursors. Single-cell transcriptomics identified mesenchymal subpopulations with potential roles in cell migration and immune response, altered in the context of p75 deletion. Together, these results identify the role of p75 signaling pathway in coordinating skeletal cell migration during early bone repair.

PDGFRα reporter activity identifies periosteal progenitor cells critical for bone formation and fracture repair.

The outer coverings of the skeleton, which is also known as the periosteum, are arranged in concentric layers and act as a reservoir for tissue-specific bone progenitors. The cellular heterogeneity within this tissue depot is being increasingly recognized. Here, inducible PDGFRα reporter animals were found to mark a population of cells within the periosteum that act as a stem cell reservoir for periosteal appositional bone formation and fracture repair. During these processes, PDGFRα reporter+ progenitors give rise to Nestin+ periosteal cells before becoming osteoblasts and osteocytes. The diphtheria toxin-mediated ablation of PDGFRα reporter+ cells led to deficits in cortical bone formation during homeostasis and a diminutive hard callus during fracture repair. After ossicle transplantation, both mouse PDGFRα reporter+ periosteal cells and human Pdgfrα+ periosteal progenitors expand, ossify, and recruit marrow to a greater extent than their counterpart periosteal cells, whereas PDGFRα reporter- periosteal cells exhibit a predisposition to chondrogenesis in vitro. Total RNA sequencing identified enrichment of the secreted factors Fermt3 and Ptpn6 within PDGFRα reporter+ periosteal cells, which partly underlie the osteoblastogenic features of this cell population.

Multimodal Targeted Nanoparticle-Based Delivery System for Pancreatic Tumor Imaging in Cellular and Animal Models.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC), which ranks forth on the cancer-related death statistics still is both a diagnostic and a therapeutic challenge. Adenocarcinoma of the exocrine human pancreas originates in most instances from malignant transformation of ductal epithelial cells, alternatively by Acinar-Ductal Metaplasia (ADM). RA-96 antibody targets to a mucin M1, according to the more recent nomenclature MUC5AC, an extracellular matrix component excreted by PDAC cells. In this study, we tested the usability of multimodal nanoparticle carrying covalently coupled RA-96 Fab fragments for pancreatic tumor imaging. METHODS: In order to make and evaluate a novel, better targeting, theranostic nanoparticle, iron nanoparticles and the optical dye indocyanin green (ICG) were encapsulated into the cationic sphingomyelin (SM) consisting liposomes. RA-96 Fab fragment was conjugated to the liposomal surface of the nanoparticle to increase tumor homing ability. ICG and iron nanoparticle-encapsulated liposomes were studied in vitro with cells and (i) their visibility in magnetic resonance imaging (MRI), (ii) optical, (iii) Magnetic particle spectroscopy (MPS) and (iv) photoacoustic settings was tested in vitro and also in in vivo models. The targeting ability and MRI and photoacoustic visibility of the RA-96-nanoparticles were first tested in vitro cell models where cell binding and internalization were studied. In in vivo experiments liposomal nanoparticles were injected into the tail vain using an orthotopic pancreatic tumor xenograft model and subcutaneous pancreatic cancer cell xenografts bearing mice to determine in vivo targeting abilities of RA-96-conjugated liposomes Results: Multimodal liposomes could be detected by MRI, MPS and by photoacoustic imaging in addition to optical imaging showing a wide range of imaging utility. The fluorescent imaging of ICG in pancreatic tumor cells Panc89 and Capan-2 revealed an increased association of ICG-encapsulated liposomes carrying RA-96 Fab fragments in vitro compared to the control liposomes without covalently linked RA-96. Fluorescent molecular tomography (FMT) studies showed increased accumulation of the RA96-targeted nanoparticles in the tumor area compared to non-targeted controls in vivo. Similar accumulation in the tumor sites could be seen with liposomal ferric particles in MRI. Fluorescent tumor signal was confirmed by using an intraoperative fluorescent imaging system, which showed fluorescent labeling of pancreatic tumors. CONCLUSION: These results suggest that RA-96-targeted liposomes encapsulating ICG and iron nanoparticles can be used to image pancreatic tumors with a variety of optical and magnetic imaging techniques. Additionally, they might be a suitable drug delivery tool to improve treatment of PDAC patients.

Spatial transcriptomics reveals a role for sensory nerves in preserving cranial suture patency through modulation of BMP/TGF-ß signaling.

The patterning and ossification of the mammalian skeleton requires the coordinated actions of both intrinsic bone morphogens and extrinsic neurovascular signals, which function in a temporal and spatial fashion to control mesenchymal progenitor cell (MPC) fate. Here, we show the genetic inhibition of tropomyosin receptor kinase A (TrkA) sensory nerve innervation of the developing cranium results in premature calvarial suture closure, associated with a decrease in suture MPC proliferation and increased mineralization. In vitro, axons from peripheral afferent neurons derived from dorsal root ganglions (DRGs) of wild-type mice induce MPC proliferation in a spatially restricted manner via a soluble factor when cocultured in microfluidic chambers. Comparative spatial transcriptomic analysis of the cranial sutures in vivo confirmed a positive association between sensory axons and proliferative MPCs. SpatialTime analysis across the developing suture revealed regional-specific alterations in bone morphogenetic protein (BMP) and TGF-ß signaling pathway transcripts in response to TrkA inhibition. RNA sequencing of DRG cell bodies, following direct, axonal coculture with MPCs, confirmed the alterations in BMP/TGF-ß signaling pathway transcripts. Among these, the BMP inhibitor follistatin-like 1 (FSTL1) replicated key features of the neural-to-bone influence, including mitogenic and anti-osteogenic effects via the inhibition of BMP/TGF-ß signaling. Taken together, our results demonstrate that sensory nerve-derived signals, including FSTL1, function to coordinate cranial bone patterning by regulating MPC proliferation and differentiation in the suture mesenchyme.

NGF-TrkA signaling dictates neural ingrowth and aberrant osteochondral differentiation after soft tissue trauma.

Pain is a central feature of soft tissue trauma, which under certain contexts, results in aberrant osteochondral differentiation of tissue-specific stem cells. Here, the role of sensory nerve fibers in this abnormal cell fate decision is investigated using a severe extremity injury model in mice. Soft tissue trauma results in NGF (Nerve growth factor) expression, particularly within perivascular cell types. Consequently, NGF-responsive axonal invasion occurs which precedes osteocartilaginous differentiation. Surgical denervation impedes axonal ingrowth, with significant delays in cartilage and bone formation. Likewise, either deletion of Ngf or two complementary methods to inhibit its receptor TrkA (Tropomyosin receptor kinase A) lead to similar delays in axonal invasion and osteochondral differentiation. Mechanistically, single-cell sequencing suggests a shift from TGFß to FGF signaling activation among pre-chondrogenic cells after denervation. Finally, analysis of human pathologic specimens and databases confirms the relevance of NGF-TrkA signaling in human disease. In sum, NGF-mediated TrkA-expressing axonal ingrowth drives abnormal osteochondral differentiation after soft tissue trauma. NGF-TrkA signaling inhibition may have dual therapeutic use in soft tissue trauma, both as an analgesic and negative regulator of aberrant stem cell differentiation.

Genomic variants within chromosome 14q32.32 regulate bone mass through MARK3 signaling in osteoblasts.

Bone mineral density (BMD) is a highly heritable predictor of osteoporotic fracture. GWAS have identified hundreds of loci influencing BMD, but few have been functionally analyzed. In this study, we show that SNPs within a BMD locus on chromosome 14q32.32 alter splicing and expression of PAR-1a/microtubule affinity regulating kinase 3 (MARK3), a conserved serine/threonine kinase known to regulate bioenergetics, cell division, and polarity. Mice lacking Mark3 either globally or selectively in osteoblasts have increased bone mass at maturity. RNA profiling from Mark3-deficient osteoblasts suggested changes in the expression of components of the Notch signaling pathway. Mark3-deficient osteoblasts exhibited greater matrix mineralization compared with controls that was accompanied by reduced Jag1/Hes1 expression and diminished downstream JNK signaling. Overexpression of Jag1 in Mark3-deficient osteoblasts both in vitro and in vivo normalized mineralization capacity and bone mass, respectively. Together, these findings reveal a mechanism whereby genetically regulated alterations in Mark3 expression perturb cell signaling in osteoblasts to influence bone mass.