Identifying fibrogenic cells following salivary gland obstructive injury.

Fibrosis results from excess extracellular matrix accumulation, which alters normal tissue architecture and impedes function. In the salivary gland, fibrosis can be induced by irradiation treatment for cancer therapy, Sjögren's Disease, and other causes; however, it is unclear which stromal cells and signals participate in injury responses and disease progression. As hedgehog signaling has been implicated in fibrosis of the salivary gland and other organs, we examined contributions of the hedgehog effector, Gli1, to fibrotic responses in salivary glands. To experimentally induce a fibrotic response in female murine submandibular salivary glands, we performed ductal ligation surgery. We detected a progressive fibrotic response where both extracellular matrix accumulation and actively remodeled collagen significantly increased at 14 days post-ligation. Macrophages, which participate in extracellular matrix remodeling, and Gli1+ and PDGFRα+ stromal cells, which may deposit extracellular matrix, both increased with injury. Using single-cell RNA-sequencing, Gli1 + cells were not found in discrete clusters at embryonic day 16 but were found in clusters expressing the stromal genes Pdgfra and/or Pdgfrb. In adult mice, Gli1+ cells were similarly heterogeneous but more cells co-expressed PDGFRα and PDGFRß. Using Gli1-CreERT2; ROSA26tdTomato lineage-tracing mice, we found that Gli1-derived cells expand with ductal ligation injury. Although some of the Gli1 lineage-traced tdTomato+ cells expressed vimentin and PDGFRß following injury, there was no increase in the classic myofibroblast marker, smooth muscle alpha-actin. Additionally, there was little change in extracellular matrix area, remodeled collagen area, PDGFRα, PDGFRß, endothelial cells, neurons, or macrophages in Gli1 null salivary glands following injury when compared with controls, suggesting that Gli1 signaling and Gli1+ cells have only a minor contribution to mechanical injury-induced fibrotic changes in the salivary gland. We used scRNA-seq to examine cell populations that expand with ligation and/or showed increased expression of matrisome genes. Some Pdgfra + /Pdgfrb + stromal cell subpopulations expanded in response to ligation, with two stromal cell subpopulations showing increased expression of Col1a1 and a greater diversity of matrisome genes, consistent with these cells being fibrogenic. However, only a few cells in these subpopulations expressed Gli1, consistent with a minor contribution of these cells to extracellular matrix production. Defining the signaling pathways driving fibrotic responses in stromal cell sub-types could reveal future therapeutic targets.

Identifying Fibrogenic Cells Following Salivary Gland Obstructive Injury.

Fibrosis results from excess extracellular matrix accumulation, which alters normal tissue architecture and impedes function. In the salivary gland, fibrosis can be induced by irradiation treatment for cancer therapy, Sjögren's Disease, and other causes; however, it is unclear which stromal cells and signals participate in injury responses and disease progression. As hedgehog signaling has been implicated in fibrosis of the salivary gland and other organs, we examined contributions of the hedgehog effector, Gli1, to fibrotic responses in salivary glands. To experimentally induce a fibrotic response in female murine submandibular salivary glands, we performed ductal ligation surgery. We detected a progressive fibrotic response where both extracellular matrix accumulation and actively remodeled collagen trended upwards at 7 days and significantly increased at 14 days post- ligation. Macrophages, which participate in extracellular matrix remodeling, Gli1 + and PDGFRα + stromal cells, which may deposit extracellular matrix, both increased with injury. Using single-cell RNA-sequencing, we found that a majority of Gli1 + cells at embryonic day 16 also express Pdgfra and/or Pdgfrb. However, in adult mice, only a small subset of Gli1 + cells express PDGFRα and/or PDGFRß at the protein level. Using lineage-tracing mice, we found that Gli1-derived cells expand with ductal ligation injury. Although some of the Gli1 lineage-traced tdTomato + cells expressed vimentin and PDGFRß following injury, there was no increase in the classic myofibroblast marker, smooth muscle alpha-actin. Additionally, there was little change in extracellular matrix area, remodeled collagen area, PDGFRα, PDGFRß, endothelial cells, neurons, or macrophages in Gli1 null salivary glands following injury when compared with controls, suggesting that Gli1 signaling and Gli1 + cells have only a minor contribution to mechanical injury-induced fibrotic changes in the salivary gland. We used scRNA-seq to examine cell populations that expand with ligation and/or showed increased expression of matrisome genes. Pdgfra + /Pdgfrb + stromal cell subpopulations both expanded in response to ligation, showed increased expression and a greater diversity of matrisome genes expressed, consistent with these cells being fibrogenic. Defining the signaling pathways driving fibrotic responses in stromal cell sub-types could reveal future therapeutic targets.

Comparison of the Utilization of Tranexamic Acid and Tourniquet Use in Total Knee Arthroplasty: A Retrospective Case Series.

INTRODUCTION: Total knee arthroplasty (TKA) carries a high risk for significant blood loss due to bone cuts and extensive soft tissue involvement in the knee region. The use of tranexamic acid (TXA) or a tourniquet are two methods commonly employed to prevent significant blood loss and avoid the need for blood transfusion. TXA has been shown to reduce both intraoperative and postoperative bleeding as well as the probability that a patient will require a blood transfusion. The purpose of this study is to compare the efficacy of TXA and tourniquet use, both alone and in combination, in reducing blood loss during TKA. METHODS: Data for this retrospective cohort study were obtained by searching records of patients who underwent TKA at a tertiary care center from January 2019 to October 2020. Data from 526 subjects were available. A chart review was conducted to determine if the patient received TXA only, tourniquet only, or both TXA and tourniquet during the TKA procedure. Primary outcomes for this study including procedure length in minutes, estimated blood loss in cubic centimeters, and total infusion pressor (phenylephrine) administered intraoperatively in milligrams were recorded for the study. Data were summarized using means and standard errors. Statistical methods used for analysis include one-way ANOVA, probability plots, the Shapiro-Wilk test for normality, the Kruskal-Wallis test, and Tukey's test. RESULTS: Data were available for 526 subjects. 122 subjects received tourniquet only (Tourniquet group), 104 received intravenous (IV) TXA only, 264 received both tourniquet and IV TXA (Tourniquet + TXA), and 36 received neither tourniquet nor TXA (None). The groups did not significantly differ in procedure length (p = 0.140) or infusion pressor total (p > 0.20). The groups did significantly differ in estimated blood loss (p < 0.001). Subjects who did not receive either TXA or tourniquet had significantly more blood loss than the Tourniquet and Tourniquet + TXA groups. Similarly, the TXA group had significantly more blood loss than both the Tourniquet and Tourniquet + TXA groups. CONCLUSION: This study supports the conclusion that the use of a tourniquet is superior to the use of TXA in reducing intraoperative blood loss during TKA. All groups that underwent TKA using a tourniquet, either alone or in combination with TXA, exhibited significantly lower levels of blood loss compared to the control (no intervention) group.

Extra-short humeral heads reduce glenohumeral joint overstuffing compared with short heads in anatomic total shoulder arthroplasty.

BACKGROUND: Rotator cuff tears and glenoid loosening remain the two most common causes for revision after anatomic total shoulder arthroplasty. Oversizing of the humeral head leads to increased contact force across the glenohumeral joint and is hypothesized to contribute to clinical and radiographic failure. The purpose of this study is to compare the rate of radiographic overstuffing between standard short humeral heads and newer extra-short heads with decreased lateral offset. METHODS: Fifty-five consecutive anatomic total shoulder arthroplasties performed using extra-short humeral heads were retrospectively reviewed and compared with age- and sex-matched controls receiving standard short heads. A total of 110 postoperative radiographs were analyzed using the Iannotti's perfect circle method to compare the prosthesis' center of rotation (COR) with the native humeral head COR. A difference in the COR of >3.0 mm was considered malpositioned. Malpositioning medially was considered overstuffed, and malpositioning laterally was considered understuffed. The direction of displacement of malpositioned prostheses was categorized using a quadrant system. Furthermore, we used a novel method to evaluate medial and superior overstuffing by measuring the displacement between the anatomic and prosthetic head positions along perpendicular axes. RESULTS: Using the Iannotti's perfect circle method, 56% of heads were malpositioned. Overstuffing occurred more frequently with short heads compared with extra-short heads (47% vs. 4%, P < .001). Conversely, understuffing occurred more frequently with extra-short heads (47% vs. 15%, P = .001). Malpositioned extra-short heads were most frequently placed in the inferomedial quadrant (93% vs. 24%, P < .001), whereas malpositioned short heads were most commonly placed in the superomedial quadrant (56% vs. 7%, P < .001). Our novel measurement method demonstrated that extra-short heads reduced medial overstuffing (2.8 ± 2.8 mm vs. 0.3 ± 2.0 mm, P < .001). Both extra-short and short heads had similar rates of superior malpositioning (1.6 ± 2.2 mm vs. 1.4 ± 1.5 mm, P = .683). CONCLUSION: Routine use of extra-short humeral head sizes reduces the rate of medial glenohumeral joint overstuffing but not superior malpositioning. This is hypothesized to improve clinical outcomes, but future studies are needed to assess the relationship between improved humeral head fit and clinical outcomes.

ROCK inhibitor increases proacinar cells in adult salivary gland organoids.

Salisphere-derived adult epithelial cells have been used to improve saliva production of irradiated mouse salivary glands. Importantly, optimization of the cellular composition of salispheres could improve their regenerative capabilities. The Rho Kinase (ROCK) inhibitor, Y27632, has been used to increase the proliferation and reduce apoptosis of progenitor cells grown in vitro. In this study, we investigated whether Y27632 could be used to improve expansion of adult submandibular salivary epithelial progenitor cells or to affect their differentiation potential in different media contexts. Application of Y27632 in medium used previously to grow salispheres promoted expansion of Kit+ and Mist1+ cells, while in simple serum-containing medium Y27632 increased the number of cells that expressed the K5 basal progenitor marker. Salispheres derived from Mist1CreERT2; R26TdTomato mice grown in salisphere media with Y27632 included Mist1-derived cells. When these salispheres were incorporated into 3D organoids, inclusion of Y27632 in the salisphere stage increased the contribution of Mist1-derived cells expressing the proacinar/acinar marker, Aquaporin 5 (AQP5), in response to FGF2-dependent mesenchymal signals. Optimization of the cellular composition of salispheres and organoids can be used to improve the application of adult salivary progenitor cells in regenerative medicine strategies.

RARα and RARγ reciprocally control K5+ progenitor cell expansion in developing salivary glands.

Understanding the mechanisms of controlled expansion and differentiation of basal progenitor cell populations during organogenesis is essential for developing targeted regenerative therapies. Since the cytokeratin 5-positive (K5+) basal epithelial cell population in the salivary gland is regulated by retinoic acid signaling, we interrogated how isoform-specific retinoic acid receptor (RAR) signaling impacts the K5+ cell population during salivary gland organogenesis to identify RAR isoform-specific mechanisms that could be exploited in future regenerative therapies. In this study, we utilized RAR isoform-specific inhibitors and agonists with murine submandibular salivary gland organ explants. We determined that RARα and RARγ have opposing effects on K5+ cell cycle progression and cell distribution. RARα negatively regulates K5+ cells in both whole organ explants and in isolated epithelial rudiments. In contrast, RARγ is necessary but not sufficient to positively maintain K5+ cells, as agonism of RARγ alone failed to significantly expand the population. Although retinoids are known to stimulate differentiation, K5 levels were not inversely correlated with differentiated ductal cytokeratins. Instead, RARα agonism and RARγ inhibition, corresponding with reduced K5, resulted in premature lumenization, as marked by prominin-1. With lineage tracing, we demonstrated that K5+ cells have the capacity to become prominin-1+ cells. We conclude that RARα and RARγ reciprocally control K5+ progenitor cells endogenously in the developing submandibular salivary epithelium, in a cell cycle-dependent manner, controlling lumenization independently of keratinizing differentiation. Based on these data, isoform-specific targeting RARα may be more effective than pan-RAR inhibitors for regenerative therapies that seek to expand the K5+ progenitor cell pool. SUMMARY STATEMENT: RARα and RARγ reciprocally control K5+ progenitor cell proliferation and distribution in the developing submandibular salivary epithelium in a cell cycle-dependent manner while regulating lumenization independently of keratinizing differentiation.