Single cell multi-omics characterise discrete human tendon cells populations that persist in vitro and on fibrous scaffolds.

Chronic tendinopathy represents a growing healthcare burden in the ageing global population. Curative therapies remain elusive as the mechanisms that underlie chronic inflammation in tendon disease remain unclear. Identifying and isolating key pathogenic and reparative cells is essential in developing precision therapies and implantable materials for improved tendon healing. Multiple discrete human tendon cell populations have been previously described ex vivo. To determine if these populations persist in vitro, healthy human hamstring tenocytes were cultured for 8 d on either tissue culture plastic or aligned electrospun fibres of absorbable polydioxanone. Novel single-cell surface proteomics combined with unbiased single-cell transcriptomics (CITE-Seq) was used to identify discrete tenocyte populations. 6 cell populations were found, 4 of which shared key gene expression determinants with ex vivo human cell clusters: PTX3_PAPPA, POSTN_SCX, DCN_LUM and ITGA7_NES. Surface proteomics found that PTX3_PAPPA cells were CD10+CD26+CD54+. ITGA7_NES cells were CD146+ and POSTN_SCX cells were CD90+CD95+CD10+. Culture on the aligned electrospun fibres favoured 3 cell subtypes (DCN_LUM, POSTN_SCX and PTX3_ PAPPA), promoting high expression of tendon-matrix-associated genes and upregulating gene sets enriched for TNF-a and IL-6/STAT3 signalling. Discrete human tendon cell subpopulations persisted in in vitro culture and could be recognised by specific gene and surface-protein signatures. Aligned polydioxanone fibres promoted the survival of 3 clusters, including pro-inflammatory PTX3-expressing CD10+CD26+CD54+ cells found in chronic tendon disease. These results improved the understanding of preferred culture conditions for different tenocyte subpopulations and informed the development of in vitro models of tendon disease.

A surprisingly poor correlation between in vitro and in vivo testing of biomaterials for bone regeneration: results of a multicentre analysis.

New regenerative materials and approaches need to be assessed through reliable and comparable methods for rapid translation to the clinic. There is a considerable need for proven in vitro assays that are able to reduce the burden on animal testing, by allowing assessment of biomaterial utility predictive of the results currently obtained through in vivo studies. The purpose of this multicentre review was to investigate the correlation between existing in vitro results with in vivo outcomes observed for a range of biomaterials. Members from the European consortium BioDesign, comprising 8 universities in a European multicentre study, provided data from 36 in vivo studies and 47 in vitro assays testing 93 different biomaterials. The outcomes of the in vitro and in vivo experiments were scored according to commonly recognised measures of success relevant to each experiment. The correlation of in vitro with in vivo scores for each assay alone and in combination was assessed. A surprisingly poor correlation between in vitro and in vivo assessments of biomaterials was revealed indicating a clear need for further development of relevant in vitro assays. There was no significant overall correlation between in vitro and in vivo outcome. The mean in vitro scores revealed a trend of covariance to in vivo score with 58 %. The inadequacies of the current in vitro assessments highlighted here further stress the need for the development of novel approaches to in vitro biomaterial testing and validated pre-clinical pipelines.

Mechanism of drug-induced gingival overgrowth revisited: a unifying hypothesis.

Drug-induced gingival overgrowth (DIGO) is a disfiguring side effect of anti-convulsants, calcineurin inhibitors, and calcium channel blocking agents. A unifying hypothesis has been constructed which begins with cation flux inhibition induced by all three of these drug categories. Decreased cation influx of folic acid active transport within gingival fibroblasts leads to decreased cellular folate uptake, which in turn leads to changes in matrix metalloproteinases metabolism and the failure to activate collagenase. Decreased availability of activated collagenase results in decreased degradation of accumulated connective tissue which presents as DIGO. Studies supporting this hypothesis are discussed.

Conjugatable water-soluble Pt(II) and Pd(II) porphyrin complexes: novel nano- and molecular probes for optical oxygen tension measurement in tissue engineering.

Measurement of oxygen tension in compressed collagen sheets was performed using matrix-embedded optical oxygen sensors based on platinum(II) and palladium(II) porphyrins supported on polyacrylamide nanoparticles. Bespoke, fully water-soluble, mono-functionalised Pt(II) and Pd(II) porphyrin complexes designed for conjugation under mild conditions were obtained using microwave-assisted metallation. The new sensors display a linear response (1/τ vs. O2) to varying oxygen tension over a biologically relevant range (7.0 × 10(-4) to 2.7 × 10(-1) mM) in aqueous solutions; a behaviour that is maintained following conjugation to polyacrylamide nanoparticles, and following embedding of the nanosensors in compressed collagen sheets, paving the way to innovative approaches for real-time resolution of oxygen gradients throughout 3D matrices useful for tissue regeneration.

Polyethylenimine mediated recovery of SARS-CoV-2 and total viral RNA: Impact of aqueous conditions on behaviour and recovery.

Wastewater-based epidemiology (WBE) is an emerging, practical surveillance tool for monitoring community levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, SC2). However, a paucity of data exists regarding SARS-CoV-2 and viral biomarker behaviour in aqueous and wastewater environments. Therefore, there is a pressing need to develop efficient and robust methods that both improve method sensitivity and reduce time and cost. We present a novel method for SARS-CoV-2, Human Coronavirus 229E (229E), and Pepper Mild Mottle Virus (PMMoV) recovery utilizing surface charge-based attraction via the branched cationic polymer, polyethylenimine (PEI). Initially, dose-optimization experiments demonstrated that low concentrations of PEI (0.001% w/v) proved most effective at flocculating suspended viruses and viral material, including additional unbound SC2 viral fragments and/or RNA from raw wastewater. A design-of-experiments (DOE) approach was used to optimize virus and/or viral material aggregation behaviour and recovery across varying aqueous conditions, revealing pH as a major influence on recoverability in this system, combinatorially due to both a reduction in viral material surface charge and increased protonation of PEI-bound amine groups. Overall, this method has shown great promise in significantly improving quantitative viral recovery, providing a straightforward and effective augmentation to standard centrifugation techniques.

Engineering stable topography in dense bio-mimetic 3D collagen scaffolds.

Topographic features are well known to influence cell behaviour and can provide a powerful tool for engineering complex, functional tissues. This study aimed to investigate the mechanisms of formation of a stable micro-topography on plastic compressed (PC) collagen gels. The uni-directional fluid flow that accompanies PC of collagen gels creates a fluid leaving surface (FLS) and a non-fluid leaving surface (non-FLS). Here we tested the hypothesis that the resulting anisotropy in collagen density and stiffness between FLS and non-FLS would influence the fidelity and stability of micro-grooves patterned on these surfaces. A pattern template of parallel-aligned glass fibres was introduced to the FLS or non-FLS either at the start of the compression or halfway through, when a dense FLS had already formed. Results showed that both early and late patterning of the FLS generated grooves that had depth (25 ±7 µm and 19 ±8 µm, respectively) and width (55 ±11 µm and 50 ±12 µm, respectively) which matched the glass fibre diameter (50 µm). In contrast, early and late patterning of the non-FLS gave much wider (151 ±50 µm and 89 ±14 µm, respectively) and shallower (10 ±2.7 µm and 13 ±3.5 µm, respectively) grooves than expected. The depth to width ratio of the grooves generated on the FLS remained unaltered under static culture conditions over 2 weeks, indicating that grooves were stable under long term active cell-mediated matrix remodelling. These results indicate that the FLS, characterised by a higher matrix collagen density and stiffness than the non-FLS, provides the most favourable mechanical surface for precise engineering of a stable micro-topography in 3D collagen hydrogel scaffolds.

Clickable decellularized extracellular matrix as a new tool for building hybrid-hydrogels to model chronic fibrotic diseases in vitro.

Fibrotic disorders account for over one third of mortalities worldwide. Despite great efforts to study the cellular and molecular processes underlying fibrosis, there are currently few effective therapies. Dual-stage polymerization reactions are an innovative tool for recreating heterogeneous increases in extracellular matrix (ECM) modulus, a hallmark of fibrotic diseases in vivo. Here, we present a clickable decellularized ECM (dECM) crosslinker incorporated into a dynamically responsive poly(ethylene glycol)-α-methacrylate (PEGαMA) hybrid-hydrogel to recreate ECM remodeling in vitro. An off-stoichiometry thiol-ene Michael addition between PEGαMA (8-arm, 10 kg mol-1) and the clickable dECM resulted in hydrogels with an elastic modulus of E = 3.6 ± 0.24 kPa, approximating healthy lung tissue (1-5 kPa). Next, residual αMA groups were reacted via a photo-initiated homopolymerization to increase modulus values to fibrotic levels (E = 13.4 ± 0.82 kPa) in situ. Hydrogels with increased elastic moduli, mimicking fibrotic ECM, induced a significant increase in the expression of myofibroblast transgenes. The proportion of primary fibroblasts from dual-reporter mouse lungs expressing collagen 1a1 and alpha-smooth muscle actin increased by approximately 60% when cultured on stiff and dynamically stiffened hybrid-hydrogels compared to soft. Likewise, fibroblasts expressed significantly increased levels of the collagen 1a1 transgene on stiff regions of spatially patterned hybrid-hydrogels compared to the soft areas. Collectively, these results indicate that hybrid-hydrogels are a new tool that can be implemented to spatiotemporally induce a phenotypic transition in primary murine fibroblasts in vitro.

Characterization of cellulose production in Escherichia coli Nissle 1917 and its biological consequences.

Bacterial species of the Enterobacteriaceae family produce cellulose and curli fimbriae as extracellular matrix components, and their synthesis is positively regulated by the transcriptional activator CsgD. In this group of bacteria, cellulose biosynthesis is commonly regulated by CsgD via the GGDEF domain protein AdrA, a diguanylate cyclase that produces cyclic-diguanylic acid (c-di-GMP), an allosteric activator of cellulose synthase. In the probiotic Escherichia coli strain Nissle 1917 and its recent clonal isolates, CsgD activates the production of curli fimbriae at 28 degrees C, but neither CsgD nor AdrA is required for the c-di-GMP-dependent biosynthesis of cellulose at 28 degrees C and 37 degrees C. In these strains, the GGDEF domain protein YedQ, a diguanylate cyclase that activates cellulose biosynthesis in certain E. coli strains, is not required for cellulose biosynthesis and it has in fact evolved into a novel protein. Cellulose production in Nissle 1917 is required for adhesion of bacteria to the gastrointestinal epithelial cell line HT-29, to the mouse epithelium in vivo, and for enhanced cytokine production. The role of cellulose in this strain is in contrast to the role of cellulose in the commensal strain E. coli TOB1. Consequently, the role of cellulose in bacterial-host interaction is dependent on the E. coli strain background.

Evidence for an intramembrane component associated with a cellulose microfibril-synthesizing complex in higher plants.

Freeze-fracture of rapidly frozen, untreated plant cells reveals terminal complexes on E-fracture faces and intramembrane particle rosettes on P-fracture faces. Terminal complexes and rosettes are associated with the ends of individual microfibril impressions on the plasma membrane. In addition, terminal complexes and rosettes are associated with the impressions of new orientations of microfibrils. These structures are sparse within pit fields where few microfibril impressions are observed, but are abundant over adjacent impressions of microfibrils. It is proposed that intramembrane rosettes function in association with terminal complexes to synthesize microfibrils. The presence of a cellulosic microfibril system in Zea mays root segments is confirmed by degradation experiments with Trichoderma cellulase.

Alteration of in vivo cellulose ribbon assembly by carboxymethylcellulose and other cellulose derivatives.

In vivo cellulose ribbon assembly by the Gram-negative bacterium Acetobacter xylinum can be altered by incubation in carboxymethylcellulose (CMC), a negatively charged water-soluble cellulose derivative, and also by incubation in a variety of neutral, water-soluble cellulose derivatives. In the presence of all of these substituted celluloses, normal fasciation of microfibril bundles to form the typical twisting ribbon is prevented. Alteration of ribbon assembly is most extensive in the presence of CMC, which often induces synthesis of separate, intertwining bundles of microfibrils. Freeze-etch preparations of the bacterial outer membrane suggest that particles that are thought to be associated with cellulose synthesis or extrusion may be specifically organized to mediate synthesis of microfibril bundles. These data support the previous hypothesis that the cellulose ribbon of A. xylinum is formed by a hierarchical, cell-directed, self-assembly process. The relationship of these results to the regulation of cellulose microfibril size and wall extensibility in plant cell walls is discussed.

Calcofluor white ST Alters the in vivo assembly of cellulose microfibrils.

The fluorescent brightener, Calcofluor White ST, prevents the in vivo assembly of crystalline cellulose microfibrils and ribbons by Acetobacter xylinum. In the presence of more than 0.01 percent Calcofluor, Acetobacter continues to synthesize high-molecular-weight beta-1,4 glucans. X-ray crystallography shows that the altered product exhibits no detectable crystallinity in the wet state, but upon drying it changes into crystalline cellulose I. Calcofluor alters cellulose crystallization by hydrogen bonding with glucan chains. Synthesis of this altered product is reversible and can be monitored with fluorescence and electron microscopy. Use of Calcofluor has made it possible to separate the processes of polymerization and crystallization leading to the biogenesis of cellulose microfibrils, and has suggested that crystallization occurs by a cell-directed. self-assembly process in Acetobacter xylinum.

Human granulocyte collagenase.

A collagenase, operative at neutral and alkaline pH, has been extracted from the granule fraction of human granulocytic leukocytes. It digests reconstituted collagen fibrils and reduces the viscosity of collagen solutions. Cleavage of collagen in solution with purified enzyme produces the discrete products characteristic of other animal collagenases.

Cellulosic wall component produced by the golgi apparatus of Pleurochrysis scherffelii.

The Golgi apparatus of a marine chrysophycean alga Pleurochrysis scherffelii Pringsheim produces wall fragments (circular-to-ellipsoidal "scales") which are released to the periphery by an exocytotic process involving the fusion of cisternae and the plasma membrane. The cellulosic component of the scales is a complex network of fibrils (10 to 25 angstroms in diameter) that resist treatment with strong alkali. Untreated washed scales yield galactose, ribose, arabinose, and traces of glucose; alkali-purified scales yield much more glucose. The fibrillar scale constituent shows a positive iodine dichroism of the intact wall, a positive zinc chloride-iodine reaction, breakage sites characteristic of highly crystalline cellulose, and solubility in Schweizer's reagent.

Collagen stiffness regulates cellular contraction and matrix remodeling gene expression.

Cell-level mechanical and 3D spatial cues are essential to the organization and architecture of new tissues that form during growth, repair or in bioreactors. Fibroblast-seeded 3D collagen constructs have been used as bioartifical extracellular matrix (ECM) providing a 3D environment to embedded resident cells. As cells attach to scaffold fibrils, they generate quantifiable contractile forces which depend on cell type, cell attachment, cell density, growth factors, and matrix stiffness. The aim of this study was to quantify the cytomechanical and molecular responses of human dermal (HDF) and neonatal foreskin fibroblasts (HNFF) seeded in constructs of increased stiffness. We also tested the effect of blocking early attachment using serum starvation on these outputs. Constructs were placed under uniaxial strains of 0-10% to increase scaffold stiffness, prior to gel contraction, and force generation was monitored using a tensional culture force monitor (t-CFM). Increased matrix stiffness reduced generation of quantifiable cellular force (up to 70%) over 24 h in both cell types and delayed the onset of measurable contraction (upto sevenfold). The delay of measurable force generation was cell lineage dependent but not FCS dependent. Gene expression of MMP-2, TIMP-2, and collagen type III expression in HDFs were significantly upregulated in constructs of increased stiffness. HNFFs did not show any significant changes in these gene expressions indicating a lineage specific response.

Cellulose synthases and related enzymes.

The discovery of a large number of genes encoding cellulose synthases and related glycosyltransferases in plants has led to a renewed interest in the biosynthesis of cell-wall polysaccharides. A number of approaches, including virus-induced gene silencing have proven useful in the functional analysis of these genes. X-ray analysis of the structures of a few glycosyltransferases has led to the identification and confirmation of the role of conserved residues within this group of enzymes. Analysis of related enzymes has provided useful information on the possible domain organization of cellulose synthases and the requirement for at least two separate glycosyltransferase activities in the processive synthesis of sugar chains.

Microbial cellulose--the natural power to heal wounds.

Microbial cellulose (MC) synthesized in abundance by Acetobacter xylinum shows vast potential as a novel wound healing system. The high mechanical strength and remarkable physical properties result from the unique nanostructure of the never-dried membrane. This article attempts to briefly summarize the recent developments and applications of MC in the emerging field of novel wound dressings and skin substitutes. It considers the properties of the synthesized material, its clinical performance, as well as progress in the commercialization of MC for wound care products. Efficient and inexpensive fermentation techniques, not presently available, will be necessary to produce large quantities of the polymer.

Complex dependence of substrate stiffness and serum concentration on cell-force generation.

Collagen is a widely used biomaterial in tissue engineering. Mechanical stimulation of cell-seeded collagen constructs and its effects on cell orientation, intracellular signaling, and molecular responses have been reported. Our aim was to study the transfer of applied mechanical load to resident cells in 3D collagen constructs. Stainless steel markers were embedded in constructs as reporters of micromovement and uniaxial (0-15%) strain was applied. Cell-seeded collagen constructs were also subjected to (0-15%) uniaxial strain and material responses recorded. The viscoelastic properties of collagen resulted in comparatively small movement of the marker bars relative to gel deformation. Cell seeding density of 1 million/mL had no significant effect on the viscoelastic properties of collagen for the range of strain tested. Our findings indicate that viscoelastic properties of collagen result in minimal force transfer of applied loads as recorded by movement of stainless steel markers. At higher strain rates as collagen got stiffer the movement decreased. These findings indicate that as cell-seeded collagen constructs mature in a bioreactor and become stiffer due to ECM production/deposition, mechanical stimulation will have to be tailored over time to account for increased stiffness of constructs in vitro to elicit predictable and consistent cellular responses.

The genome of the Gulf pipefish enables understanding of evolutionary innovations.

BACKGROUND: Evolutionary origins of derived morphologies ultimately stem from changes in protein structure, gene regulation, and gene content. A well-assembled, annotated reference genome is a central resource for pursuing these molecular phenomena underlying phenotypic evolution. We explored the genome of the Gulf pipefish (Syngnathus scovelli), which belongs to family Syngnathidae (pipefishes, seahorses, and seadragons). These fishes have dramatically derived bodies and a remarkable novelty among vertebrates, the male brood pouch. RESULTS: We produce a reference genome, condensed into chromosomes, for the Gulf pipefish. Gene losses and other changes have occurred in pipefish hox and dlx clusters and in the tbx and pitx gene families, candidate mechanisms for the evolution of syngnathid traits, including an elongated axis and the loss of ribs, pelvic fins, and teeth. We measure gene expression changes in pregnant versus non-pregnant brood pouch tissue and characterize the genomic organization of duplicated metalloprotease genes (patristacins) recruited into the function of this novel structure. Phylogenetic inference using ultraconserved sequences provides an alternative hypothesis for the relationship between orders Syngnathiformes and Scombriformes. Comparisons of chromosome structure among percomorphs show that chromosome number in a pipefish ancestor became reduced via chromosomal fusions. CONCLUSIONS: The collected findings from this first syngnathid reference genome open a window into the genomic underpinnings of highly derived morphologies, demonstrating that de novo production of high quality and useful reference genomes is within reach of even small research groups.

Comparison of four purified extracellular 1,4-beta-D-glucan cellobiohydrolase enzymes from Trichoderma viride.

Four electrophoretically distinct 1,4-beta-D-glucan cellobiohydrolase enzymes (exo-cellobiohydrolase, EC 3.2.1.91) from Trichoderma viride have been purified to homogeneity. Three enzymes (A, B, and C) were from a commercial T. viride preparation whereas the other (D) was from T. viride QM 9123 grown on cellulose in submerged culture. The enzymes were similar with respect to ultraviolet light absorption, amino acid and amino sugar composition, heat stability, molecular weight, specific activity, and carboxyterminal residues, indicating very nearly identical polypeptide portions. The enzymes also exhibited immunological cross-reactivity. The enzymes differed most in the content and composition of covalently bound neutral carbohydrate.

Aggregation of ampholine on heparin and other acidic polysaccharides in isoelectric focusing.

The mechanism of complexation of pI range 3.5--5 Ampholine to heparin in isoelectric focusing has been explored by the dye-binding technique at different pH values in solution. There is no significant interaction between heparin and Ampholine at pH 6.7. Weak, or selective, binding occurs at pH 5.1, and very strong interaction at pH 3.5. In the latter system, the Ampholine components appear to behave as polycations due to their ordered sequence of positive charges, each two methylene groups apart, which favors a strong binding to polyanions. In addition, there appear to be variable stoichiometries for the strong binding between heparin and Ampholine, depending on their relative amounts. It is proposed that at a low ratio of heparin to Ampholine (Ampholine excess), aggregation is perpendicular to the heparin chain, with the end ammonium charge of each Ampholine molecule neutralizing one negative charge along the heparin molecule; at higher ratios (heparin excess), the bound Ampholine segment is aligned parallel to the heparin molecule, so that on the average one Ampholine component neutralizes approx. three negative charges. The banding of heparin in isoelectric focusing in the pH range 3.0--4.5 can be explained by aggregation of the various components on heparin in amounts dependent upon the net charge on the Ampholine species at the given pH, and upon the changing stoichiometries as a function of the variation in ratio of heparin to Ampholine along the pH gradient. Binding of Ampholine to polygalacturonate was also demonstrated in excess Ampholine in a pH range dependent on the degree of protonation of the carboxyl groups of this acidic polysaccharide as well as on the net positive charge of the Ampholine. The aggregation seen at pH 4.2--4.5 led to the prediction and subsequent demonstration that polygalacturonate would also exhibit binding upon isoelectric focusing. This supports the hypothesis that aggregation of Ampholine on polyanions having sufficient charge density is a general phenomenon which can lead to spurious banding of certain polymers at appropriate pH ranges in isoelectric focusing. On the basis of their behavior in isoelectric focusing at pH 3.0--4.5, strength of aggregation of the polyanions studied appears to be heparin A = heparin B greather than polyglutamate greater than carboxyl-reduced heparin B greater than polygalacturonic acid.