Osteoinductive effect of soluble transforming growth factor beta receptor 3 on human osteoblast lineage.

The development of bone requires carefully choregraphed signaling to bone progenitors to form bone. Our group recently described the requirement of transforming growth factor beta receptor 3 (TGFßR3), a receptor involved in TGFß pathway signaling, during osteoblast lineage commitment in mice. The TGFß pathway is known to play multiple osteo-inductive and osteo-inhibitory roles during osteoblast development and TGFßR3 human mutations are associated with reduced bone mineral density, making TGFßR3 a unique target for bone inductive therapy. In this article, we demonstrated increased mineralization of human pediatric bone-derived osteoblast-like cells (HBO) when treated with soluble TGFßR3 (sR3) using Alizarin Red staining. Osteogenic commitment of HBO cells was demonstrated by induction of osteogenic genes RUNX2, osteocalcin, osteopontin, and osterix. Evaluation of the canonical TGFß pathway signaling demonstrated that sR3 was able to induce bone formation in HBO cells, mainly through activation of noncanonical targets of TGFß pathway signaling including AKT, ERK, and p38 MAP kinases. Inhibition of these osteogenic noncanonical pathways in the HBO cells also inhibited mineralization, suggesting they are each required. Although no induction of SMAD1, 5, and 9 was observed, there was the activation of SMAD2 and 3 suggesting that sR3 is primarily signaling via the noncanonical pathways during osteogenic induction of the HBO. Our results highlight the important role of TGFßR3 in osteoblast induction of mineralization in human bone cells through noncanonical targets of TGFß signaling. Future studies will focus on the ability of sR3 to induce bone regeneration in vivo using animal models.

Local delivery of FTY720 induces neutrophil activation through chemokine signaling in an oronasal fistula model.

PURPOSE: Cleft palate repair surgeries lack a regenerative reconstructive option and, in many cases, develop complications including oronasal fistula (ONF). Our group has developed a novel murine phenocopy of ONF to study the oral cavity wound healing program. Using this model, our team previously identified that delivery of FTY720 on a nanofiber scaffold had a unique immunomodulatory effect directing macrophages and monocytes into a pro-regenerative state during ONF healing. Here, the objective of this study was to determine the effects of local biomaterial-based FTY720 delivery in the ONF model on the early bulk gene expression and neutrophil phenotypic response within the regenerating tissue. METHODS: Using a mouse model of ONF formation, a palate defect was created and was treated with FTY720 nanofiber scaffolds or (blank) vehicle control nanofibers. At 1 and 3 days post-implantation, ONF oral mucosal tissue from the defect region was collected for RNA sequencing analysis or flow cytometry. For the RNA-seq expression profiling, intracellular pathways were assessed using the KEGG Pathway database and Gene Ontology (GO) Terms enrichment interactive graph. To assess the effects of FTY720 on different neutrophil subpopulations, flow cytometry data was analyzed using pseudotime analysis based on Spanning-tree Progression Analysis of Density-normalized Events (SPADE). RESULTS: RNA sequencing analysis of palate mucosa injured tissue identified 669 genes that were differentially expressed (DE) during the first 3 days of ONF wound healing after local delivery of FTY720, including multiple genes in the sphingolipid signaling pathway. Evaluation of the DE genes at the KEGG Pathway database also identified the inflammatory immune response pathways (chemokine signaling, cytokine-cytokine receptor interaction, and leukocyte transendothelial migration), and the Gene Ontology enrichment analysis identified neutrophil chemotaxis and migration terms. SPADE dendrograms of CD11b+Ly6G+ neutrophils at both day 1 and day 3 post-injury showed significantly distinct subpopulations of neutrophils in oral mucosal defect tissue from the FTY720 scaffold treatment group compared to the vehicle control group (blank). Increased expression of CD88 and Vav1, among other genes, were found and staining of the ONF area demonstrated increased VAV1 staining in FTY720-treated healing oral mucosa. CONCLUSION: Treatment of oral mucosal defects using FTY720 scaffolds is a promising new immunotherapy to improve healing outcomes and reducing ONF formation during cleft palate surgical repair. Local delivery of FTY720 nanofiber scaffolds during ONF healing significantly shifted early gene transcription associated with immune cell recruitment and modulation of the immune microenvironment results in distinct neutrophil subpopulations in the oral mucosal defect tissue that provides a critical shift toward pro-regenerative immune signaling. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40883-021-00208-z.

Airway injury complicating excision of thyroglossal duct cysts.

OBJECTIVES: We report on four cases of thyroglossal duct cyst (TGDC) excision using the Sistrunk procedure (resection of the mid-portion of the hyoid bone in continuity with a thyroglossal duct cyst tract) in which the airway was significantly injured. The patterns of injury, their treatment and outcomes as well as preventative measures are detailed. METHODS: Retrospective analysis of four patients referred to a tertiary medical center after sustaining injury to the cricothyroid membrane and/or thyroid cartilages while undergoing a Sistrunk excision of a TGDC. RESULTS: Three patients were repaired after a delay; one patient was immediately repaired. All four patients required application of cartilage grafts, and all ultimately required tracheotomy. Decannulation was achieved in the four patients after an average of 4.5 months, and none suffered from aspiration. Voice outcomes were poor in 3/4. CONCLUSIONS: The Sistrunk procedure has been advocated for TGDC excision, citing a low recurrence rate. However, if the thyroid cartilage is mistaken for the hyoid bone, significant airway injury occurs. Urgent laryngotracheoplasty is indicated, but poor voice outcomes are anticipated. SIGNIFICANCE: Surgeons employing the Sistrunk procedure to excise TGDC must remain oriented to midline cervical anatomy, particularly as the hyoid my override the thyroid notch in young children, placing the larynx at risk for significant injury.

Deglycosylated products of endogenous digoxin-like immunoreactive factor in mammalian tissue.

Digoxin-like immunoreactive factor (DLIF) from adrenal cortex is an endogenous molecule with structural features remarkably similar to those of digoxin, a plant-derived cardiac glycoside (Shaikh, I. M., Lau, B. W. C., Siegfried, B. A., and Valdes, R., Jr. (1991) J. Biol. Chem. 266, 13672-13678). Two characteristic structural and functional features of digoxin are a lactone ring and three digitoxose sugars attached to a steroid nucleus. Digoxin is known to undergo deglycosylation during metabolism in humans. We now demonstrate the existence of several naturally occurring deglycosylated components of DLIF in human serum. The components are identified as DLIF-genin, DLIF-mono, and DLIF-bis, corresponding to the aglycone, and the aglycone with one and two sugars, respectively. Similar components are produced by acid-induced deglycosylation of DLIF isolated from bovine adrenal cortex. The elution pattern and sequence of DLIF-deglycosylation was identical to that of digoxin suggesting identical sugar stoichiometry. However, analysis of these newly discovered congeners by reverse-phase chromatography, spectrophotometry, antibody reactivity, and kinetics of deglycosylation, demonstrates that subtle structural and physical differences do exist when compared to digoxin. DLIF was chromatographically distinct from digoxin, and interestingly, the mobility of the DLIF-genin was shifted toward increased polarity relative to digoxigenin. DLIF and DLIF-bis, -mono, and -genin congeners have absorbance maxima at 216 nm, whereas digoxin and its congeners absorb at 220 nm. Reaction with specific antibodies directed at the lactone portion of these molecules shows DLIF and its deglycosylated congeners to be 10(3)-fold less reactive than digoxin. Kinetics of sugar removal suggests that DLIF is 8-fold more susceptible to deglycosylation than is digoxin. Two less polar DLIF components produced from the DLIF-genin have lambdamax at 196 nm and are 4-fold less immunoreactive than DLIF. Our data suggest that subtle structural differences exist between DLIF and digoxin at or near the lactone ring as well as in the nature of the sugars. The presence of deglycosylated congeners of DLIF in human serum, including the less polar components, suggests in vivo deglycosylation of these factors. This is the first demonstration of the existence of naturally occurring deglycosylated derivatives of DLIF and establishes the likelihood of active metabolism of DLIF in mammals.