Rapid Prototypable Biomimetic Peristalsis Bioreactor Capable of Concurrent Shear and Multi-axial Strain

Peristalsis is a nuanced mechanical stimulus comprised of multi-axial strain (radial and axial strain) and shear stress. Forces associated with peristalsis regulate diverse biological functions including digestion, reproductive function, and urine dynamics. Given the central role peristalsis plays in physiology and pathophysiology, we were motivated to design a bioreactor capable of holistically mimicking peristalsis. We engineered a novel rotating screw-drive based design combined with a peristaltic pump, in order to deliver multiaxial strain and concurrent shear stress to a biocompatible polydimethylsiloxane (PDMS) membrane “wall”. Radial indentation and rotation of the screw drive against the wall demonstrated multi-axial strain evaluated via finite element modeling. Experimental measurements of strain using piezoelectric strain resistors were in close alignment of model-predicted values (15.9 ± 4.2% vs. 15.2% predicted). Modeling of shear stress on the ‘wall’ indicated a uniform velocity profile and a moderate shear stress of 0.4 Pa. Human mesenchymal stem cells (hMSCs) seeded on the PDMS ‘wall’ and stimulated with peristalsis demonstrated dramatic changes in actin filament alignment, proliferation, and nuclear morphology compared to static controls, perfusion or strain, indicating that hMSCs sensed and responded to peristalsis uniquely. Lastly, significant differences were observed in gene expression patterns of Calponin, Caldesmon, Smooth Muscle Actin, and Transgelin, corroborating the propensity of hMSCs toward myogenic differentiation in response to peristalsis. Collectively, our data suggests that the peristalsis bioreactor is capable of generating concurrent multi-axial strain and shear stress on a ‘wall’. hMSCs experience peristalsis differently than perfusion or strain, resulting in changes in proliferation, actin fiber organization, smooth muscle actin expression, and genetic markers of differentiation. The peristalsis bioreactor device has broad utility in the study of development and disease in several organ systems.

Alpha Smooth Muscle Actin (αSMA) Immunohistochemistry Use in the Differentiation of Pancreatic Cancer from Chronic Pancreatitis

Aim: Fibrosis is observed both in pancreatic cancer (PDAC) and chronic pancreatitis (CP). The main cells involved in fibrosis are pancreatic stellate cells (PSCs), which activate alpha smooth muscle actin (αSMA), which is considered to be the best-known fibrosis marker. The aim of the study was to evaluate the expression of the αSMA in patients with PDAC and CP as the possible differentiation marker. Methods: We enrolled 114 patients undergoing pancreatic resection: 83 with PDAC and 31 with CP. Normal fragments of resected specimen from 21 patients represented the control tissue. The immunoexpressions of αSMA were detected in tissue specimens with immunohistochemistry (Abcam antibodies, GB). Results: Mean cytoplasmatic expression of αSMA protein in PDAC stromal cells was significantly higher compared to CP: 2.42 ± 0.37 vs 1.95 ± 0.45 (p < 0.01) and control group 0.61 ± 0.45 (p < 0.01). Strong immunoexpression of the αSMA protein was found in the vast majority (80.7%) of patients with PDAC, in about half (58%) of patients with CP, and not at all in healthy tissue. The expression of αSMA of different intensity was found in all patients with PDAC and CP, while in healthy tissue was minimal or absent. In PDAC patients, αSMA expression was significantly higher in tumors of diameter higher than 3 cm compared to smaller ones (p = 0.017). Conclusions: Presented findings confirm the significant role of fibrosis in both PDAC and CP; however, they do not confirm the role of αSMA as a marker of differentiation.

Attenuation of Expression of Splenocytes Pro-Inflammatory Cytokines and Lung Alpha-Smooth Muscle Actin By Human Amniotic Mesenchymal Stem Cells-Conditioned Medium in Asthmatic Mice

Background Asthma is a chronic respiratory illness characterized by lung tissue remodeling, T helper cell imbalance, and the generation of inflammatory factors. The Human amniotic mesenchymal stem cells-conditioned medium (hAM-MSC-CM) contains various immunomodulatory components and has been utilized in certain studies as a source for anti-inflammatory factors. We investigated the impacts of CM on splenocytes pro-inflammatory cytokines and alpha-smooth muscle actin (α-SMA) expression in Balb/c mice with Ovalbumin (OVA)-induced asthma.Methods and results Forty mice were separated into four groups of ten: control (challenged and sensitized with normal saline solution), asthma (sensitized on days 1, 8, and 14 and challenged daily with OVA from days 21 to 28), OVA+CM (asthmatic mice treated with CM on days 29 and 30), and OVA+DMEM (DMEM-treated asthmatic mice on days 29 and 30). The spleen and lung tissues were removed 48 hours after the final challenge, and the expression of the inflammatory factors in the splenocyte culture supernatant was determined by ELISA, while the α-SMA expression in the lung was determined using western blotting. α-SMA protein expression was significantly greater in lung tissue. Also, inflammatory agents were significantly higher in the splenocytes supernatant in the DMEM and asthma groups compared to the control group. CM therapy has been shown to inhibit the production of the α-SMA protein and inflammatory cytokines. Conclusions Results showed that CM Treatment was able to decrease the α-SMA expression in lung and splenocytes pro-inflammatory cytokines.

Periductal Stromal Tumor of the Breast: One Institution's Review of 6 Tumors Over a 22 Year Period With Immunohistochemical Analysis

Introduction. Periductal stromal tumor (PST) of the breast is a rare fibroepithelial neoplasm with controversial pathogenesis. Methods. A retrospective search of our Pathology database from 2000 to 2021 identified 6 PST, all evaluated according to the Armed Forces Institute of Pathology (AFIP) criteria. Immunohistochemistry for CD10, CD34, CD117, GATA3, p63, SOX10, ER, PR, HER2, smooth muscle actin (SMA), beta-catenin, and myogenin was performed as well. Results. All 6 patients were female and age ranged from 29 to 55 years (mean 40 years). Tumor size ranged from 2.9 to 5.9 cm (mean 3.0 cm). Data showed absence of leaf-like architecture (0/6), at least moderate hypercellularity (6/6), lack of a circumscribed border (5/6), coalescing nodules with intermixed adipose tissue (4/6), at least moderate stromal atypia (4/6), and an elevated mitotic activity ≥3mitotic figures/10 HPF (6/6). The stromal cells were positive for CD10 (4/4), CD34 (4/4), CD117 (3/4), and SMA (3/4), and negative for GATA3 (0/6), p63 (0/6), SOX10 (0/6), ER (0/4), PR (0/4), HER2 (0/4), nuclear beta-catenin (0/5), and myogenin (0/4). No patient had a PST recurrence or metastasis (average follow-up of 91 months). Conclusion. We confirm that PST shares morphologic and immunophenotypic similarities with phyllodes tumor (PT). However, PST can be reliably differentiated from PT using the AFIP criteria. Additionally, PST's immunoprofile of positive CD117 and CD34 stromal expression alongside the negative GATA3, p63, and SOX10 reactivity can aid the pathologist in excluding metaplastic carcinoma. All 6 of our PST behaved as benign neoplasms akin to benign PT.

Histopathology, matrix metalloproteinase-2 and alpha smooth muscle actin expression in Tenon's capsule of eyes with congenital glaucoma

Purpose To evaluate the ultrastructural features of collagen fibrils, matrix metalloproteinase (MMP) and alpha-smooth muscle actin (α-SMA) expression in the Tenon's capsule of buphthalmic eyes. Methods A prospective comparative case series study was conducted on 35 buphthalmic eyes vs 25 control eyes. Children with congenital glaucoma (CG) who underwent a combined trabeculectomy-trabeculotomy procedure with mitomycin C (CTTM); the Tenon's capsule was obtained during the surgical procedure. The control group included children with strabismus, the Tenon's capsule was obtained in the course of strabismus surgery. Both H&E and Masson's trichrome staining were done. The Metalloprotenease-2 (MMP-2), alpha smooth muscle actin (α-SMA) immunohistochemical staining was performed. Results Mean collagen percentage area by Masson trichrome stain in Tenons capsule was significantly higher in buphthalmic eyes (54.76% ± 0.32 vs 33.71% ± 1.4; P < 0.001). The percentage area of αSMA expression in Tenons capsule was significantly higher in buphthalmic eyes (4.93% ± 0.7 vs 2.00% ± 0.5; P < 0.001). However, MMP2 expression in Tenons capsule of the buphthalmic group was significantly lower than that of the control group (12.88% ± 2.95 vs 27.91% ± 0.2 respectively) P = 0.02. Conclusions Tenon capsule of buphthalmic eyes have their own histopathological features and properties making them more liable for fibrosis with high rate of failure following antiglaucoma surgeries. Such detailed information has not been published before which may aid in the identification of new antifibrotic therapies in management of glaucoma.

A deep learning approach to identify and segment alpha-smooth muscle actin stress fiber positive cells

Cardiac fibrosis is a pathological process characterized by excessive tissue deposition, matrix remodeling, and tissue stiffening, which eventually leads to organ failure. On a cellular level, the development of fibrosis is associated with the activation of cardiac fibroblasts into myofibroblasts, a highly contractile and secretory phenotype. Myofibroblasts are commonly identified in vitro by the de novo assembly of alpha-smooth muscle actin stress fibers; however, there are few methods to automate stress fiber identification, which can lead to subjectivity and tedium in the process. To address this limitation, we present a computer vision model to classify and segment cells containing alpha-smooth muscle actin stress fibers into 2 classes (α-SMA SF+ and α-SMA SF-), with a high degree of accuracy (cell accuracy: 77%, F1 score 0.79). The model combines standard image processing methods with deep learning techniques to achieve semantic segmentation of the different cell phenotypes. We apply this model to cardiac fibroblasts cultured on hyaluronic acid-based hydrogels of various moduli to induce alpha-smooth muscle actin stress fiber formation. The model successfully predicts the same trends in stress fiber identification as obtained with a manual analysis. Taken together, this work demonstrates a process to automate stress fiber identification in in vitro fibrotic models, thereby increasing reproducibility in fibroblast phenotypic characterization.

Dynamic Stromal Changes in Myelofibrosis Patients Pre/Post JAK Inhibition Is Revealed in Clinically Archived Bone Marrow Biopsies By Smooth Muscle Actin (SMA)-CD34 Dual Immunohistochemistry

The natural history of BCR-ABL1 negative myeloproliferative neoplasms (MPNs) is progression towards an overt myelofibrotic (MF) phase with variable risk to develop secondary acute myeloid leukemia. Current treatments include Janus kinase inhibitors (JAKi) which can temporarily alleviate MF-related symptoms but are non-curative and most patients eventually progress to a more advanced stage. Given the negative prognostic impact of bone marrow fibrosis in MPNs and generally poor outcome post JAKi failure, it would be important to identify in situ biomarkers that address the initiation, perpetuation and early reversal of the fibrotic reaction. The current clinical standard for bone marrow fibrosis assessment involves reticulin/trichrome stains that detect relatively static extracellular matrix products rather than the fibrosis driving cells directly. To address this, we have developed a smooth muscle actin stromal-vascular (SMA-CD34) dual immunohistochemical (IHC) technique amenable to morphologic scoring and complemented with a CellProfiler image analysis pipeline. SMA was prioritized over other validated stromal IHC markers given work by others in experimental models demonstrating SMA+ myofibroblasts to be the differentiated output of critical fibrosis inducing Gli1+ 'driver' mesenchymal stem/progenitor cells in MPN. Herein, we demonstrate the feasibility of our translational approach using a clinically annotated cohort of MF patients from the Princess Margaret Cancer Centre MPN Registry. After selecting for high quality (&gt;1.0 cm) paired pre and post JAKi biopsies amenable to image and transcriptome-based analysis, the pilot cohort was comprised of 13 cases with 38% high-risk, 54% intermediate-2 and 8% intermediate-1 risk by DIPSS. Driver mutations were JAK2 V617F (77%), CALR (15%) and other (8%). JAKi therapies included ruxolitinib (31%) + pelabresib (23%), momelotinib (15%), itacitinib (15%) and pacritinib (8%). The SMA-CD34 stromal assessment at baseline revealed distinct interstitial myofibroblast patterns and vascular perturbations not captured by conventional clinical hematopathology assessment (e.g. SMA+ dilated sinusoids). A SMA-CD34 scoring system was developed using a 4-point scale representing normal (0 pts), increased vascularity (1 pt), focal interstitial SMA (2 pts), multifocal interstitial SMA (3 pts) and diffuse SMA (4 pts). Scoring was then performed by blinded hematopathologists. A trend towards JAK2 mutated MF cases demonstrating higher SMA grade at baseline was noted. Interestingly, variable trajectories in SMA scores emerged following treatment with JAKi. Specifically, SMA signals had increased in 15%, decreased in 46% and were stable in 38% post-JAKi when using a morphologic SMA grading scheme. When compared to reticulin fibrosis, the severity of SMA signals had diverged in 1/3 of the cases (e.g. SMA grade decreased, reticulin grade stable). To further complement the SMA-CD34 morphologic grading, a CellProfiler image analysis pipeline was developed yielding a non-vessel associated normalized SMA area metric as a supervised correlate of the clinical SMA scoring system (R 2 = 0.68). Additional supervised and unsupervised bioinformatic approaches for clustering of relevant SMA-CD34 features including an algorithm that informs SMA spatial patterns with respect to niche elements such as arterioles (CD34+SMA+), sinusoids (CD34+) and adipocytes is in development. Lastly, Nanostring Fibrosis V2 panel was employed on a subset that met RNA concentration and quality metrics. Exploratory interpretation showed significant differentially expressed genes in pre vs. post JAKi specimens related to lipid metabolism such as ADIPOR1, SCD, ELOVL6 as well as the chemokine CXCL16. This may suggest a link between fatty acid metabolism and inflammatory differentiation along the SMA-vascular axis in the bone marrow modulated by JAKi treatment. SMA-CD34 IHC stratifies MF bone marrow biopsies differentially from standard WHO reticulin/trichome grading providing a practical formalin-fixed paraffin embedded (FFPE) tissue-based biomarker for assessing fibrosis related bone marrow niche elements from archived clinical samples. While our pilot numbers precluded statistical evaluation by JAKi-type, clinical response and NGS mutational profile at this time, further studies are underway to validate the SMA-CD34 signature on a larger MF cohort. Figure 1 Figure 1. Disclosures Gupta: Sierra Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS-Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy; Incyte: Honoraria, Research Funding; Constellation Pharma: Consultancy, Honoraria; Pfizer: Consultancy.

Titanium Substratum Roughness as a Determinant of Human Gingival Fibroblast Fibronectin and α-Smooth Muscle Actin Expression

The most appropriate surface treatment to enhance gingival connective tissue formation on the abutment of dental implants remains undefined, with healing associated with a scar-like response. We have previously shown that topographies with an arithmetic average of the absolute profile height deviations (Ra) = 4.0 induces an anti-fibrotic phenotype in human gingival fibroblasts (HGFs) by causing nascent adhesion formation. With bacterial colonization considerations, we hypothesized that a lower Ra could be identified that would alter adhesion stability and promote a matrix remodeling phenotype. Focal adhesions (FAs) area decreased with increasing roughness, although no differences in cell attachment or proliferation were observed. Alpha smooth muscle actin (α-SMA) protein levels were significantly reduced on Ra = 3.0 and 4.0 vs. 0.1 (p < 0.05), with incorporation of α-SMA into stress fibers most prominent on Ra = 0.1. Fibronectin protein levels were reduced on 3.0 and 4.0 vs. 0.1 (p < 0.05), and Ra = 1.5 and deeper significantly altered fibronectin deposition. Addition of exogenous TGF-β3 increased HGF adhesion size on 0.1 surfaces, but not on any other topography. We conclude that Ra = 1.5 is sufficient to reduce adhesion size and inhibit α-SMA incorporation into stress fibers in HGFs, but 3.0 is required in the presence of exogenous TGF-β3. Our findings have implications for inhibiting fibrotic tissue formation surrounding percutaneous devices such as dental implants.

The Anti-Fibrotic Effect of Cold Atmospheric Plasma on Localized Scleroderma In Vitro and In Vivo

Cold Atmospheric Plasma (CAP) has shown promising results in the treatment of various skin diseases. The therapeutic effect of CAP on localized scleroderma (LS), however, has not yet been evaluated. We investigated the effects of CAP on LS by comparing human normal fibroblasts (hNF), human TGF-β-activated fibroblasts (hAF), and human localized scleroderma-derived fibroblasts (hLSF) after direct CAP treatment, co-cultured with plasma-treated human epidermal keratinocytes (hEK) and with an experimental murine model of scleroderma. In hAF and hLSF, 2 min CAP treatment with the MicroPlaSterβ® plasma torch did not affect pro-fibrotic gene expression of alpha smooth muscle actin, fibroblast activating protein, and collagen type I, however, it promoted re-expression of matrix metalloproteinase 1. Functionally, CAP treatment reduced cell migration and stress fiber formation in hAF and hLSF. The relevance of CAP treatment was confirmed in an in vivo model of bleomycin-induced dermal fibrosis. In this model, CAP-treated mice showed significantly reduced dermal thickness and collagen deposition as well as a decrease in both alpha smooth muscle actin-positive myofibroblasts and CD68-positive macrophages in the affected skin in comparison to untreated fibrotic tissue. In conclusion, this study provides the first evidence for the successful use of CAP for treating LS and may be the basis for clinical trials including patients with LS.

Fibrin with Laminin-Nidogen Reduces Fibrosis and Improves Soft Palate Regeneration Following Palatal Injury

This study aimed to analyze the effects of fibrin constructs enhanced with laminin-nidogen, implanted in the wounded rat soft palate. Fibrin constructs with and without laminin-nidogen were implanted in 1 mm excisional wounds in the soft palate of 9-week-old rats and compared with the wounded soft palate without implantation. Collagen deposition and myofiber formation were analyzed at days 3, 7, 28 and 56 after wounding by histochemistry. In addition, immune staining was performed for a-smooth muscle actin (a-SMA), myosin heavy chain (MyHC) and paired homeobox protein 7 (Pax7). At day 56, collagen areas were smaller in both implant groups (31.25 ± 7.73% fibrin only and 21.11 ± 6.06% fibrin with laminin-nidogen)) compared to the empty wounds (38.25 ± 8.89%, p < 0.05). Moreover, the collagen area in the fibrin with laminin-nidogen group was smaller than in the fibrin only group (p ˂ 0.05). The areas of myofiber formation in the fibrin only group (31.77 ± 10.81%) and fibrin with laminin-nidogen group (43.13 ± 10.39%) were larger than in the empty wounds (28.10 ± 11.68%, p ˂ 0.05). Fibrin-based constructs with laminin-nidogen reduce fibrosis and improve muscle regeneration in the wounded soft palate. This is a promising strategy to enhance cleft soft palate repair and other severe muscle injuries.