Hydrostatic Pressure Differentially Regulates Outer and Inner Annulus Fibrosus Cell Matrix Production in 3D Scaffolds

Mechanical stimulation may be used to enhance the development of engineered constructs for the replacement of load bearing tissues, such as the intervertebral disc. This study examined the effects of dynamic hydrostatic pressure (HP) on outer and inner annulus (OA, IA) fibrosus cells seeded on fibrous poly(glycolic acid)-poly(L-lactic acid) scaffolds. Constructs were pressurized (5 MPa, 0.5 Hz) for four hours/day from day 3 to day 14 of culture and analyzed using ELISAs and immunohistochemistry (IHC) to assess extracellular matrix (ECM) production. Both cell types were viable, with OA cells exhibiting more infiltration into the scaffold, which was enhanced by HP. ELISA analyses revealed that HP had no effect on type I collagen production while a significant increase in type II collagen (COL II) was measured in pressurized OA constructs compared to day 14 unloaded controls. Both OA and IA dynamically loaded scaffolds exhibited more uniform COL II elaboration as shown by IHC analyses, which was most pronounced in OA-seeded scaffolds. Overall, HP resulted in enhanced ECM elaboration and organization by OA-seeded constructs, while IA-seeded scaffolds were less responsive. As such, hydrostatic pressurization may be beneficial in annulus fibrosus tissue engineering when applied in concert with an appropriate cell source and scaffold material

Induction of osteoblast differentiation markers in human dermal fibroblasts: potential application to bone tissue engineering.

Tissue engineered constructs have the potential to be used as replacements for current bone graft technologies. One component necessary for bone tissue engineering is a readily available, osteogenic cell source. Human dermal fibroblasts may have the potential to differentiate along an osteoblastic lineage, making them a candidate for use in bone tissue engineering applications. The objective of this study was to validate the ability of dermal fibroblasts to express gene and protein markers of osteoblastic differentiation and to explore their potential, in combination with biomaterial scaffolds and signaling factors, for use in bone tissue engineering

Carboxymethylcellulose hydrogels support central nervous system-derived tumor-cell chemotactic migration: comparison with conventional extracellular matrix macromolecules

The local microenvironment plays an important role in maintaining the dynamics of the extracellular matrix and the cell-extracellular matrix relationship. The extracellular matrix is a complex network of macromolecules with distinct mechanical and biochemical characteristics. Disruptions in extracellular matrix homeostasis are associated with the onset of cancer. The extracellular matrix becomes highly disorganized, and the cell-matrix relationship changes, resulting in altered cell-signaling processes and metastasis. Medulloblastoma is one of the most common malignant pediatric brain tumors in the United States. In order to gain a better understanding of the interplay between cell-extracellular matrix interactions and cell-migratory responses in tumors, eight different matrix macromolecule formulations were investigated using a medulloblastoma-derived cell line: poly-D-lysine, matrigel, laminin, collagen 1, fibronectin, a 10% blend of laminin-collagen 1, a 20% blend of laminin-collagen 1, and a cellulose-derived hydrogel, carboxymethylcellulose. Over time, the average changes in cell morphology were quantified in 2D and 3D, as was migration in the presence and absence of the chemoattractant, epidermal growth factor. Data revealed that carboxymethylcellulose allowed for a cell-extracellular matrix relationship typically believed to be present in tumors, with cells exhibiting a rounded, amoeboid morphology consistent with chemotactic migration, while the other matrices promoted an elongated cell shape as well as both haptotactic and chemotactic motile processes. Therefore, carboxymethylcellulose hydrogels may serve as effective platforms for investigating central nervous system-derived tumor-cell migration in response to soluble factor

Composite biomaterial repair strategy to restore biomechanical function and reduce herniation risk in an ex vivo large animal model of intervertebral disc herniation with varying injury severity

Back pain commonly arises from intervertebral disc (IVD) damage including annulus fibrosus (AF) defects and nucleus pulposus (NP) loss. Poor IVD healing motivates developing tissue engineering repair strategies. This study evaluated a composite injectable IVD biomaterial repair strategy using carboxymethylcellulose-methylcellulose (CMC-MC) and genipincrosslinked fibrin (FibGen) that mimic NP and AF properties, respectively. Bovine ex vivo caudal IVDs were evaluated in cyclic compression-tension, torsion, and compression-to-failure tests to determine IVD biomechanical properties, height loss, and herniation risk following experimentally-induced severe herniation injury and discectomy (4 mm biopsy defect with 20% NP removed). FibGen with and without CMC-MC had failure strength similar to discectomy injury suggesting no increased risk compared to surgical procedures, yet no biomaterials improved axial or torsional biomechanical properties suggesting they were incapable of adequately restoring AF tension. FibGen had the largest failure strength and was further evaluated in additional discectomy injury models with varying AF defect types (2 mm biopsy, 4 mm cruciate, 4 mm biopsy) and NP removal volume (0%, 20%). All simulated discectomy defects significantly compromised failure strength and biomechanical properties. The 0% NP removal group had mean values of axial biomechanical properties closer to intact levels than defects with 20% NP removed but they were not statistically different and 0% NP removal also decreased failure strength. FibGen with and without CMC-MC failed at super-physiological stress levels above simulated discectomy suggesting repair with these tissue engineered biomaterials may perform better than discectomy alone, although restored biomechanical function may require additional healing with the potential application of these biomaterials as sealants and cell/drug delivery carriers

Climatic changes and social transformations in the Near East and North Africa during the ‘long’ 4th millennium BC: A comparative study of environmental and archaeological evidence

This paper explores the possible links between rapid climate change (RCC) and social change in the Near East and surrounding regions (Anatolia, central Syria, southern Israel, Mesopotamia, Cyprus and eastern and central Sahara) during the ‘long’ 4th millennium (∼4500–3000) BC. Twenty terrestrial and 20 marine climate proxies are used to identify long-term trends in humidity involving transitions from humid to arid conditions and vice versa. The frequency distribution of episodes of relative aridity across these records is calculated for the period 6300–2000 BC, so that the results may be interpreted in the context of the established arid episodes associated with RCC around 6200 and 2200 BC (the 8.2 and 4.2 kyr events). We identify two distinct episodes of heightened aridity in the early-mid 4th, and late 4th millennium BC. These episodes cluster strongly at 3600–3700 and 3100–3300 BC. There is also evidence of localised aridity spikes in the 5th and 6th millennia BC. These results are used as context for the interpretation of regional and local archaeological records with a particular focus on case studies from western Syria, the middle Euphrates, southern Israel and Cyprus. Interpretation of the records involves the construction of plausible narratives of human–climate interaction informed by concepts of adaptation and resilience from the literature on contemporary (i.e. 21st century) climate change and adaptation. The results are presented alongside well-documented examples of climatically-influenced societal change in the central and eastern Sahara, where detailed geomorphological studies of ancient environments have been undertaken in tandem with archaeological research. While the narratives for the Near East and Eastern Mediterranean remain somewhat speculative, the use of resilience and adaptation frameworks allows for a more nuanced treatment of human–climate interactions and recognises the diversity and context-specificity of human responses to climatic and environmental change. Our results demonstrate that there is a need for more local environmental data to be collected ‘at source’ during archaeological excavations

Substance P Induces Rapid and Transient Membrane Blebbing in U373MG Cells in a p21-Activated Kinase-Dependent Manner

U373MG astrocytoma cells endogenously express the full-length neurokinin 1 receptor (NK1R). Substance P (SP), the natural ligand for NK1R, triggers rapid and transient membrane blebbing and we report that these morphological changes have different dynamics and intracellular signaling as compared to the changes that we have previously described in HEK293-NK1R cells. In both cell lines, the SP-induced morphological changes are Gq-independent, and they require the Rho, Rho-associated coiled-coil kinase (ROCK) signaling pathway. Using confocal microscopy we have demonstrated that tubulin is phosphorylated subsequent to cell stimulation with SP and that tubulin accumulates inside the blebs. Colchicine, a tubulin polymerization inhibitor, blocked SP-induced blebbing in U373MG but not in HEK293-NK1R cells. Although p21-activated kinase (PAK) is expressed in both cell lines, SP induced rapid phosphorylation of PAK in U373MG, but failed to phosphorylate PAK in HEK293-NK1R cells. The cell-permeable Rho inhibitor C3 transferase inhibited SP-induced PAK phosphorylation, but the ROCK inhibitor Y27632 had no effect on PAK phosphorylation, suggesting that Rho activates PAK in a ROCK-independent manner. Our study demonstrates that SP triggers rapid changes in cell morphology mediated by distinct intracellular signaling mechanisms in U373MG versus HEK293-NK1R cells

A novel formulation of inhaled sodium cromoglicate (PA101) in idiopathic pulmonary fibrosis and chronic cough: a randomised, double-blind, proof-of-concept, phase 2 trial

Background Cough can be a debilitating symptom of idiopathic pulmonary fibrosis (IPF) and is difficult to treat. PA101 is a novel formulation of sodium cromoglicate delivered via a high-efficiency eFlow nebuliser that achieves significantly higher drug deposition in the lung compared with the existing formulations. We aimed to test the efficacy and safety of inhaled PA101 in patients with IPF and chronic cough and, to explore the antitussive mechanism of PA101, patients with chronic idiopathic cough (CIC) were also studied. Methods This pilot, proof-of-concept study consisted of a randomised, double-blind, placebo-controlled trial in patients with IPF and chronic cough and a parallel study of similar design in patients with CIC. Participants with IPF and chronic cough recruited from seven centres in the UK and the Netherlands were randomly assigned (1:1, using a computer-generated randomisation schedule) by site staff to receive PA101 (40 mg) or matching placebo three times a day via oral inhalation for 2 weeks, followed by a 2 week washout, and then crossed over to the other arm. Study participants, investigators, study staff, and the sponsor were masked to group assignment until all participants had completed the study. The primary efficacy endpoint was change from baseline in objective daytime cough frequency (from 24 h acoustic recording, Leicester Cough Monitor). The primary efficacy analysis included all participants who received at least one dose of study drug and had at least one post-baseline efficacy measurement. Safety analysis included all those who took at least one dose of study drug. In the second cohort, participants with CIC were randomly assigned in a study across four centres with similar design and endpoints. The study was registered with ClinicalTrials.gov (NCT02412020) and the EU Clinical Trials Register (EudraCT Number 2014-004025-40) and both cohorts are closed to new participants. Findings Between Feb 13, 2015, and Feb 2, 2016, 24 participants with IPF were randomly assigned to treatment groups. 28 participants with CIC were enrolled during the same period and 27 received study treatment. In patients with IPF, PA101 reduced daytime cough frequency by 31·1% at day 14 compared with placebo; daytime cough frequency decreased from a mean 55 (SD 55) coughs per h at baseline to 39 (29) coughs per h at day 14 following treatment with PA101, versus 51 (37) coughs per h at baseline to 52 (40) cough per h following placebo treatment (ratio of least-squares [LS] means 0·67, 95% CI 0·48–0·94, p=0·0241). By contrast, no treatment benefit for PA101 was observed in the CIC cohort; mean reduction of daytime cough frequency at day 14 for PA101 adjusted for placebo was 6·2% (ratio of LS means 1·27, 0·78–2·06, p=0·31). PA101 was well tolerated in both cohorts. The incidence of adverse events was similar between PA101 and placebo treatments, most adverse events were mild in severity, and no severe adverse events or serious adverse events were reported. Interpretation This study suggests that the mechanism of cough in IPF might be disease specific. Inhaled PA101 could be a treatment option for chronic cough in patients with IPF and warrants further investigation

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification