Cell contact guidance via sensing anisotropy of network mechanical resistance

Despite the ubiquitous importance of cell contact guidance, the signal-inducing contact guidance of mammalian cells in an aligned fibril network has defied elucidation. This is due to multiple interdependent signals that an aligned fibril network presents to cells, including, at least, anisotropy of adhesion, porosity, and mechanical resistance. By forming aligned fibrin gels with the same alignment strength, but cross-linked to different extents, the anisotropic mechanical resistance hypothesis of contact guidance was tested for human dermal fibroblasts. The cross-linking was shown to increase the mechanical resistance anisotropy, without detectable change in network microstructure and without change in cell adhesion to the cross-linked fibrin gel. This methodology thus isolated anisotropic mechanical resistance as a variable for fixed anisotropy of adhesion and porosity. The mechanical resistance anisotropy |Y*|⁻¹ − |X*|⁻¹ increased over fourfold in terms of the Fourier magnitudes of microbead displacement |X*| and |Y*| at the drive frequency with respect to alignment direction Y obtained by optical forces in active microrheology. Cells were found to exhibit stronger contact guidance in the cross-linked gels possessing greater mechanical resistance anisotropy: the cell anisotropy index based on the tensor of cell orientation, which has a range 0 to 1, increased by 18% with the fourfold increase in mechanical resistance anisotropy. We also show that modulation of adhesion via function-blocking antibodies can modulate the guidance response, suggesting a concomitant role of cell adhesion. These results indicate that fibroblasts can exhibit contact guidance in aligned fibril networks by sensing anisotropy of network mechanical resistance.

Contact guidance refers to the tendency of a cell to orient and migrate bidirectionally in response to anisotropic topographical features, such as parallel grooves on a two-dimensional (2D) substratum or aligned fibrils in a three-dimensional (3D) collagen or fibrin gel. Contact guidance has been ascribed importance in a number of physiological processes since Weiss first identified and investigated its role in development (1). More recently, the role of contact guidance in cancer metastasis (2) as well as tissue engineering scaffold design (3) has been described.Contact guidance on 2D substrata like parallel grooves or adhesive ligand stripes has been intensively studied (recent examples include refs. 4–6) because in these experiments, the guidance field can be precisely defined and easily manipulated. However, it is the case of aligned fibrils that has the preponderance of physiological relevance. Contact guidance is also vital to the success of engineered connective tissues that mimic native alignment (and thereby function) by harnessing mechanically constrained fibrin gel compaction by fibroblasts and the associated contact guidance response (7–9). Unfortunately, even for the case of collagen and fibrin gels, not to mention tissues, the guidance field generally cannot be precisely defined or controllably manipulated. This is because of the inherent biophysical complexities of an aligned fibril network, resulting in multiple interdependent and simultaneous signals presented to cells. These signals include anisotropy of adhesion, porosity, and mechanical resistance (a combination of elastic stiffness and viscous friction, in general), at least. In a recent publication on this topic (10), for example, a correlation was suggested between protrusion activity with respect to alignment direction and contact guidance. This study made use of varied gel concentrations (typical for contact guidance studies), a method that confounds easy interpretation because all potential anisotropies are almost certainly altered simultaneously.While there are phenomenological studies reporting the contact guidance response of cells in aligned collagen and fibrin gels (11–14), the signal-inducing contact guidance in aligned fibrils has thus far defied elucidation since Dunn first proposed contact guidance in response to these anisotropies nearly 40 y ago (15), illustrated in Fig. 1. The main reason for the lack of progress in distinguishing between these anisotropies is the intrinsic difficulty in changing just one of them in an aligned fibril network—in Dunn’s terms: chemical anisotropy, mechanical anisotropy, or steric anisotropy—without changing the others. This stands in stark contrast to almost every other form of directed cell migration in which the signal is absolutely clear, for example, a chemotactic factor concentration gradient in chemotaxis, an adhesion gradient in haptotaxis, an electric potential gradient in galvanotaxis, and a stiffness gradient in durotaxis.Open in a separate windowFig. 1.Illustration of Dunn’s hypotheses for the signal-inducing cell contact guidance in aligned fibrils. A cell with four pseudopods is depicted. (A) Chemical (adhesion) anisotropy. Cell-binding sites are indicated by blue dots. (B) Mechanical (stiffness/friction) anisotropy. Fibrils presenting a high local modulus to the pseudopod because of their coalignment are colored red, and fibrils presenting a local low modulus to the pseudopods because of their anti-alignment are colored blue. The colored segments are also thickened for visualization. (C) Steric (porosity) anisotropy. Note this figure is not drawn to scale for clarity; as we (59) and others (e.g., ref. 60) have shown with electron microscopy, a pseudopod is much larger than the typical pore size in the standard in vitro models (collagen and fibrin gels) and interacts with many interconnected fibrils simultaneously, not single fibrils.Despite its ubiquitous and powerful role, the signal contained in an aligned fibril network that induces contact guidance remains unknown. The entire mechanism of contact guidance entails many events beyond signal sensing, for example, mechanotransduction pathways and spatial regulation of cell pseudopods. However, before the entire mechanism of contact guidance in response to aligned fibril networks can be understood, the signal(s) contained in aligned fibrils that induce contact guidance must be identified. Thus, in this study, we isolated anisotropic mechanical resistance as a variable, for fixed anisotropy of adhesion and porosity. If contact guidance is shown to depend on varied anisotropic mechanical resistance, for fixed anisotropy of adhesion and porosity, anisotropic mechanical resistance is established as a sufficient signal (not necessarily the only signal or the dominant signal).By combining key technologies—magnetic alignment of fibrin gels during fibrillogenesis, active microrheology (AMR) for local network mechanical resistance measurement, cell-compatible cross-linking of fibrin via dityrosine formation, and adhesion blocking antibodies—and ascertaining the effects of cross-linking on fibrin network mechanical resistance anisotropy, fibrin network microstructure, and fibroblast adhesion to fibrin, we established isolation of mechanical resistance anisotropy as the sole variable when cross-linking an aligned fibrin gel. In addition to quantifying variation in mechanical resistance anisotropy with cross-linking using AMR, absence of network microstructure changes was concluded from morphometric analysis of 3D reconstructed confocal reflectance images, and absence of changes in cell adhesion to the fibrin gel was concluded from a centrifugation assay. Altered contact guidance response in the cross-linked aligned gels was ascertained from the cell anisotropy index (AI) based on the tensor of cell orientation, defined by the orientation of the long axis for each cell. Finally, the same contact guidance experiment performed with cells pretreated with a combination of antibodies against α_vβ₃ integrin and β₁ integrin, which are shown to substantially inhibit cell adhesion in these gels based on the centrifugation assay, was performed to assess whether adhesion is contributing to the contact guidance response when mechanical resistance anisotropy is an operative signal; the same contact guidance experiment was also performed in the presence of a fibrinolysis inhibitor to assess whether fibrinolysis must be considered to interpret the findings. The data show that fibroblasts exhibit contact guidance in a network of aligned fibrin fibrils via sensing anisotropy of mechanical resistance, modulated by integrin-mediated adhesion.     

Relationship between diabetic retinopathy and diabetic peripheral neuropathy - Neurodegenerative and microvascular changes

Purpose:To determine the relationship between diabetic retinopathy (DR) and diabetic peripheral neuropathy (DPN), and their associated risk factors.Methods:We conducted a cross-sectional analysis on 500 patients who attended the Endocrinology department at a quaternary health care center in Kerala between November 2017 and April 2018. Patients above the age of 30 years with type 2 diabetes mellitus (DM) were included. They underwent a detailed medical history, dilated fundus examination for DR, assessment and grading of DPN, and blood investigations. Among these, 49 randomly selected patients without DR had peripapillary retinal nerve fiber layer (RNFL) and ganglion cell inner plexiform layer (GCIPL) assessed by optical coherence tomogram. RNFL and GCIPL changes in different grades of neuropathy were evaluated.Results:Out of 500 patients, 303 (60.6%) were males and 197 (39.4%) were females. Prevalence of DR was 48% and DPN 71.8%. Risk factors for the development of DR included duration of DM >15 years, HbA1c (glycated hemoglobin) greater than 6.5%, serum creatinine more than 1.5 mg/dl, and the presence of DPN. There was a statistically significant association between DR and DPN. There was significant thinning of GCIPL in patients with moderate to severe neuropathy without DR.Conclusion:There is a significant association between DR and DPN and their severities. There are early changes in inner retinal layers of diabetic patients without microvascular changes of DR. These neurodegenerative changes parallel DPN in the course of DM. Our study stresses the importance of multidisciplinary approach in the management of diabetes and its complications.     

Efficacy of Tranexamic Acid in Reducing Blood Loss in Lower Segment Cesearean Section: A Randomised Controlled Study

ObjectivesTo determine the efficacy of tranexamic acid in decreasing blood loss in elective/emergency LSCS.Materials and MethodsA prospective randomised case control study was done in 200 pregnant women undergoing elective/emergency LSCS in the Department of Obstetrics and Gynaecology, at a tertiary care teaching hospital in Mysuru, from December 2018 to September 2019. Women in the age group of 18–35 years were included in the study. Those with anaemia (Hb < 10 gm%), hypertension in pregnancy, bleeding diathesis, GDM on insulin, polyhydramnios, oligohydramnios, cardiac and chronic liver disorders were excluded from the study. Two hundred women undergoing emergency/elective LSCS were divided into case (group 1) or control (group 2) groups using a computer-generated random number table. Tranexamic acid (10 mg/Kg) was given in 100 ml Normal Saline 10 mins prior to skin incision to women in the first group, along with routine care (10 Units of Oxytocin IM soon after extraction of the baby). Routine care, as per institutional protocol, was followed in the second group. The primary outcome was to estimate the intraoperative blood loss. Blood loss was measured by weighing pads, mops, drapes before and after surgery and blood in the suction container after surgery. Two separate suction catheters and containers were used, in order to minimise mixing of blood and amniotic fluid. Total blood loss was calculated as the difference in the weight of the pads, mops and drapes before and after surgery and the sum of the amount of blood in suction container. The difference between the pre-operative and post-operative haemoglobin and haematocrit was compared. The pre-operative, intra-operative and post-operative hemodynamics were also compared.ResultsStatistical analysis was done using MS Excel and R-3.5.1 software. Unpaired and paired t test were used. In our study, there was a significant decrease in intraoperative bleeding in women receiving tranexamic acid. Women in the control group had a significant fall in the postoperative hemoglobin when compared to women who received tranexamic acid. Also, women who received tranexamic acid did not develop any significant hemodynamic changes during or immediately after the surgery.ConclusionTranexamic acid can be safely used as a prophylactic agent to reduce bleeding during elective and emergency LSCS.     

Genomic classification of betanodavirus by molecular phylogenetic analysis of the coat protein gene

The classification of betanodavirus into four species was reviewed including newer and well-characterised isolates. Six major clusters were identified, four of which were similar to the classic species. Two single isolate clusters were worth consideration as new species.     

Burn‐McKeown syndrome with biallelic promoter type 2 deletion in TXNL4A in two siblings

Burn‐McKeown syndrome (BMKS) (MIM# 608572) is a rare condition caused by biallelic variants in TXNL4A. BMKS is characterized by craniofacial dysmorphism, choanal atresia, and normal intellect in affected individuals. BMKS has overlapping clinical features with Treacher Collins syndrome. Till date, 15 families have been described with BMKS. Homozygosity or compound heterozygosity of promoter deletions and null variants in TXNL4A are known to cause most cases of BMKS. We describe the first Indian family with two siblings with BMKS and promoter type 2 deletion in homozygous state.     

Circulating adiponectin and resistin levels in relation to metabolic factors, inflammatory markers, and vascular reactivity in diabetic patients and subjects at risk for diabetes

OBJECTIVE: Adiponectin and resistin, two recently discovered adipocyte-secreted hormones, may link obesity with insulin resistance and/or metabolic and cardiovascular risk factors. We performed a cross-sectional study to investigate the association of adiponectin and resistin with inflammatory markers, hyperlipidemia, and vascular reactivity and an interventional study to investigate whether atorvastatin mediates its beneficial effects by altering adiponectin or resistin levels. RESEARCH DESIGN AND METHODS: Associations among vascular reactivity, inflammatory markers, resistin, and adiponectin were assessed cross-sectionally using fasting blood samples obtained from 77 subjects who had diabetes or were at high risk to develop diabetes. The effect of atorvastatin on adiponectin and resistin levels was investigated in a 12-week-long randomized, double-blind, placebo-controlled study. RESULTS: In the cross-sectional study, we confirm prior positive correlations of adiponectin with HDL and negative correlations with BMI, triglycerides, C-reactive protein (CRP), and plasma activator inhibitor (PAI)-1 and report a negative correlation with tissue plasminogen activator. The positive association with HDL and the negative association with PAI-1 remained significant after adjusting for sex and BMI. We also confirm prior findings of a negative correlation of resistin with HDL and report for the first time a positive correlation with CRP. All of these associations remained significant after adjusting for sex and BMI. No associations of adiponectin or resistin with any aspects of vascular reactivity were detected. In the interventional study, atorvastatin decreased lipid and CRP levels, but adiponectin and resistin were not specifically altered. CONCLUSIONS: We conclude that adiponectin is significantly associated with inflammatory markers, in part, through an underlying association with obesity, whereas resistin's associations with inflammatory markers appear to be independent of BMI. Lipid profile and inflammatory marker changes produced by atorvastatin cannot be attributed to changes of either adiponectin or resistin.     

A Study on Dentigerous Cystic Changes with Radiographically Normal Impacted Mandibular Third Molars

Background

Dentigerous cyst develops in the follicular tissue surrounding the impacted lower third molar. A study was carried out to know the incidence of Association of Dentigerous cyst with radiographically normal impacted lower third molars and to draw the attention of the Oral Surgeons towards the prophylactic removal of impacted third molars.

Methods

A prospective study was done on 30 patients with impacted lower third molars which were indicated for extraction. The follicle tissue surrounding the impacted tooth was subjected for histopathologic investigations. Only those teeth with a radiographic finding of pericoronal space of less than 2.5 mm were considered. Two Oral Pathologists reviewed the slides for any changes suggestive of cystic pathology.

Results

Pathologic changes suggestive of Dentigerous cyst was found in 7 of the 30 follicular tissue sent for histopathologic testing. It was found to be statistically significant (P < 0.001).

Conclusion

This study shows statistically high incidence of Dentigerous cyst association with radiographically normal impacted lower third molar teeth. Hence the Oral and Maxillofacial surgeons should consider histopathologic evaluation and radiographic diagnosis in the management of impacted lower third molars. Prophylactic extractions of normal impacted lower third molars should be considered as a treatment option.