Search

Abstract 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.

Abstract Improved treatment strategies for sarcoma rely on clarification of the molecular mediators of disease progression. Recently, we reported that the secreted glycoprotein NELL-1 modulates osteosarcoma (OS) disease progression in part via altering the sarcomatous extracellular matrix (ECM) and cell-ECM interactions. Of known NELL-1 interactor proteins, Contactin-associated protein-like 4 (Cntnap4) encodes a member of the neurexin superfamily of transmembrane molecules best known for its presynaptic functions in the central nervous system. Here, CRISPR/Cas9 gene deletion of CNTNAP4 reduced OS tumor growth, sarcoma-associated angiogenesis, and pulmonary metastases. CNTNAP4 knockout (KO) in OS tumor cells largely phenocopied the effects of NELL-1 KO, including reductions in sarcoma cell attachment, migration, and invasion. Further, CNTNAP4 KO cells were found to be unresponsive to the effects of NELL-1 treatment. Transcriptomic analysis combined with protein phospho-array demonstrated notable reductions in the MAPK/ERK signaling cascade with CNTNAP4 deletion, and the ERK1/2 agonist isoproterenol restored cell functions among CNTNAP4 KO tumor cells. Finally, human primary cells and tissues in combination with sequencing datasets confirmed the significance of CNTNAP4 signaling in human sarcomas. In summary, our findings demonstrate the biological importance of NELL-1/CNTNAP4 signaling axis in disease progression of human sarcomas and suggest that targeting the NELL-1/CNTNAP4 signaling pathway represents a strategy with potential therapeutic benefit in sarcoma patients.

Abstract 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.

Abstract Progenitor cells from adipose tissue are able to induce bone repair; however, inconsistent or unreliable efficacy has been reported across preclinical and clinical studies. Soluble inhibitory factors, such as the secreted Wnt signaling antagonists Dickkopf‐1 (DKK1), are expressed to variable degrees in human adipose‐derived stem cells (ASCs), and may represent a targetable “molecular brake” on ASC mediated bone repair. Here, anti‐DKK1 neutralizing antibodies were observed to increase the osteogenic differentiation of human ASCs in vitro, accompanied by increased canonical Wnt signaling. Human ASCs were next engrafted into a femoral segmental bone defect in NOD‐Scid mice, with animals subsequently treated with systemic anti‐DKK1 or isotype control during the repair process. Human ASCs alone induced significant but modest bone repair. However, systemic anti‐DKK1 induced an increase in human ASC engraftment and survival, an increase in vascular ingrowth, and ultimately improved bone repair outcomes. In summary, anti‐DKK1 can be used as a method to augment cell‐mediated bone regeneration, and could be particularly valuable in the contexts of impaired bone healing such as osteoporotic bone repair.

Heterotopic ossification (HO) is the formation of ectopic bone that is primarily genetically driven (fibrodysplasia ossificans progressiva [FOP]) or acquired in the setting of trauma (tHO). HO has undergone intense investigation, especially over the last 50 years, as awareness has increased around improving clinical technologies and incidence, such as with ongoing wartime conflicts. Current treatments for tHO and FOP remain prophylactic and include NSAIDs and glucocorticoids, respectively, whereas other proposed therapeutic modalities exhibit prohibitive risk profiles. Contemporary studies have elucidated mechanisms behind tHO and FOP and have described new distinct niches independent of inflammation that regulate ectopic bone formation. These investigations have propagated a paradigm shift in the approach to treatment and management of a historically difficult surgical problem, with ongoing clinical trials and promising new targets.