Biology subjects

Kelly, D. J.

Publications and source records attributed to Kelly, D. J..

14 recordsLinked to original sources

bioRxiv2026

Integrating vascular and hypertrophic cartilage microtissues to fabricatescaled-up grafts for endochondral bone tissue engineering

ABSTRACTThe repair of large bone defects remains a major clinical challenge, in part due to inadequate vascularization and poor integration of graft materials. Tissue engineering strategies that recapitulate the developmental process of endochondral ossification, whereby a cartilage template remodels into bone, have shown significant potential in pre-clinical models of large bone defect healing. However, successfully scaling these approaches to clinically relevant sizes will require the development of strategies to support the rapid vascularization of the graft following implantation in vivo. Here, mechanically reinforced templates were first fabricated by integrating hypertrophic cartilage microtissues derived from human mesenchymal stem/stromal cells (MSCs) within an osteoconductive 3D-printed polycaprolactone (PCL) framework coated with nano-hydroxyapatite (nanoHA). In vitro the cartilage microtissues fused and generated an extracellular matrix rich in sulphated glycosaminoglycans and collagen. To prevascularize these constructs, vascular microtissues derived from a co-culture of endothelial cells and MSCs were incorporated into a central channel within the construct, which generated a microvascular network within the graft in vitro. Following subcutaneous implantation, hypertrophic cartilage templates with ( vascular-channel group) and without ( empty-channel group) this central vascularized channel supported endochondral bone formation. Quantitative microCT and histological analyses revealed significantly greater remaining bone in the empty-channel group, whereas the vascular-channel group supported enhanced vascularization and remodeling of the graft in vivo. These findings support the continued development and testing of a modular biofabrication strategy that combine self-organizing hypertrophic cartilage and vascular microtissues with osteoconductive 3D-printed architectures to generate scalable, prevascularised hypertrophic cartilage templates for endochondral bone repair. Key-words: spheroids, microtissues, hypertrophic cartilage, vascularization, endochondral ossification, bone tissue engineering.

bioengineering↗

bioRxiv2025

Bioassembly of region-specific fibrocartilage microtissues to engineer zonally defined meniscal grafts

Meniscal injuries are common orthopaedic problems which can impair knee function and lead to the development of osteoarthritis. While recent advances in tissue engineering have enabled the fabrication of meniscus-like grafts, these constructs do not fully replicate the zonal structure and composition of the native meniscus. In this study, we used fibrocartilage microtissues generated from meniscus progenitor cells (MPCs) as biological building blocks to biofabricate zonally defined meniscal grafts. MPCs isolated from the inner (iMPC) and outer (oMPC) regions of caprine menisci were used to engineer region-specific meniscal microtissues in a medium-high throughput manner. Both iMPC and oMPC derived microtissues were rich in glycosaminoglycans (GAGs) and collagen, with iMPC microtissues staining more intensely for type II collagen. These microtissues were assembled into two different physically confining moulds (cylindrical and ring-shaped), where they rapidly fused and generated a fibrocartilaginous graft over six weeks of culture. Both iMPC and oMPC assembled microtissues were rich in sGAG and type I collagen, however only the iMPC assembled microtissues stained strongly for type II collagen. We then explored the impact of the catabolic enzyme chondroitinase-ABC (cABC) on the composition and structural organization of the meniscal grafts. This temporal enzymatic treatment increased collagen fiber thickness without altering tissue phenotype. Finally, iMPC and oMPC microtissues were spatially assembled to biofabricate a scaled-up and zonally defined meniscal graft. This graft was phenotypically similar to the native meniscus, with all regions rich in collagen type I and an inner core rich in collagen type II and sGAG. These findings support the use of MPC derived microtissues as biological building blocks for the engineering of zonally defined meniscal grafts. Table of ContentO_LIZonally defined meniscus grafts were biofabricated using inner and outer meniscus progenitor cells microtissues. C_LIO_LIRegion specific microtissues showed phenotypes similar to the native meniscus and successfully fused into cohesive constructs rich in sGAG and collagen. C_LIO_LIcABC treatment modulated collagen fiber formation and organization in assembled grafts. C_LIO_LIThe assembled grafts maintained shape fidelity and may offer a promising strategy for meniscal repair. C_LI O_FIG O_LINKSMALLFIG WIDTH=132 HEIGHT=200 SRC="FIGDIR/small/650568v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@1354a5forg.highwire.dtl.DTLVardef@1a5acd3org.highwire.dtl.DTLVardef@1f558aborg.highwire.dtl.DTLVardef@90666e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Biofabrication of zonally defined meniscal grafts using meniscus progenitor cell (MPC)-derived microtissues. (A) Meniscus progenitor cells (MPCs) derived from the inner (iMPC), and outer (oMPC) regions of the menisci were aggregated into microtissues by cellular self-assembly process using a high-throughput agarose microwell system developed in-house. (B) The iMPC and oMPC microtissues were then separately fused in cylindrical and ring-shaped moulds to form region-specific assembled microtissues. (C) To enhance structural organization, chondroitinase ABC (cABC) enzymatic treatment was applied to the ring-shaped assembled microtissues. (D) Finally, the matured iMPC and oMPC assembled microtissues were assembled to create a scaled-up and zonally defined meniscal graft. C_FIG

bioengineering↗

bioRxiv2024

A new class of binding protein-dependent solute transporter exemplified by the TAXI-GltS system from Bordetella pertussis

Tripartite ATP-dependent periplasmic (TRAP) transporters are widespread in prokaryotes, but absent in eukaryotes, and facilitate the uptake of a wide variety of substrates. TRAP transporters are composed of a substrate binding protein (SBP) and two unequally sized membrane components that exist as either separate proteins or are fused into a single polypeptide. Almost all TRAP SBPs exist as monomeric globular proteins that bind substrate and present it to the membrane component. Here, we describe the discovery and characterisation of a novel TRAP SBP from the TAXI subfamily with a previously unidentified architecture. BP0403 from the human pathogen Bordetella pertussis is a predicted lipoprotein composed of 3 distinct domains; an /{beta} globular domain with a unique fold, a long helical domain and a C-terminal TAXI SBP domain. Expression and purification of the full-length protein reveals that it forms a stable dimer. Structural modelling of the dimer interface and molecular weight analysis using size exclusion chromatography of the individual domains reveals that the interdomain helical region is solely responsible for dimerization. Differential scanning fluorimetry (DSF) and intrinsic tyrosine fluorescence reveal that BP0403 binds L-glutamate with nanomolar affinity. Unexpectedly, analysis of the genome context of BP0403 reveals the complete absence of characteristic genes for TRAP membrane components but co-localisation and translational coupling with gltS, encoding a Na+/glutamate symporter. In other bacteria, we identified fused BP0403-GltS homologues, strongly suggesting that this constitutes a completely novel SBP-dependent secondary active transporter. Structural comparisons suggest GltS operates by an elevator-type mechanism, like TRAP transporters; the association of an SBP with this class of secondary transporter is an emerging theme.

biochemistry↗

bioRxiv2022

Temporal enzymatic treatment to enhance the remodelling of multiple cartilage microtissues into a structurally organised tissue

Scaffold-free tissue engineering strategies aim to recapitulate key aspects of normal developmental processes as a means of generating highly biomimetic grafts. Cartilage and fibrocartilaginous tissues have successfully been engineered by bringing together large numbers of cells, cellular aggregates or microtissues and allowing them to self-assemble or self-organize into a functional graft. Despite the promise of such approaches, considerable challenges still remain, such as engineering scaled-up tissues with predefined geometries, ensuring robust fusion between adjacent cellular aggregates or microtissues, and directing the (re)modelling of such biological building blocks into a unified scaled-up graft with hierarchical matrix organisation mimetic of the native tissue. In this study, we first demonstrate the benefits of engineering cartilage via the fusion of multiple cartilage microtissues compared to conventional scaffold-free approaches where (millions of) individual cells are allowed to aggregate and generate a cartilaginous graft. Key advantages include the engineering of a tissue with a richer extracellular matrix, a more hyaline-like cartilage phenotype and a final graft which better matched the intended geometry. A major drawback associated with this approach is that individual microtissues did not completely (re)model and remnants of their initial architectures where still evident throughout the macrotissue. In an attempt to address this limitation, the enzyme chondroitinase ABC (cABC) was employed to accelerate structural (re)modelling of the engineered tissue. Temporal enzymatic treatment supported robust fusion between adjacent microtissues, enhanced microtissue (re)modelling and supported the development of a more biomimetic tissue with a zonally organised collagen architecture. Additionally, we observed that cABC treatment modulated matrix composition (rebalancing the collagen:glycosaminoglycans ratio), tissue phenotype, and to a lesser extent, tissue mechanics. Ultimately, this work demonstrates that microtissue self-organisation is an effective method for engineering scaled-up cartilage grafts with a pre-defined geometry and near-native levels of ECM accumulation. Importantly we have demonstrated that key limitations associated with tissue engineering using multiple cellular aggregates, microtissues or organoids can be alleviated by temporal enzymatic treatment during graft development.

bioengineering↗

bioRxiv2022

Engineering High-Quality Cartilage Microtissues using Hydrocortisone Functionalised Microwells

Engineering clinically-relevant musculoskeletal tissues at a human scale is a considerable challenge. Developmentally-inspired scaffold-free approaches for engineering cartilage tissues have shown great promise in recent years, enabling the generation of highly biomimetic tissues. Despite the relative success of these approaches, the absence of a supporting scaffold or hydrogel creates challenges in the development of large scale tissues. Combining numerous scaled-down tissue units (herein termed microtissues) into a larger macrotissue represents a promising strategy to address this challenge. The overall success of such approaches, however, relies on the development of strategies to support the robust and consistent chondrogenic differentiation of clinically relevant cell sources such as mesenchymal stem/stromal cells (MSCs) within microwell arrays to biofabricate numerous microtissues rich in cartilage-specific extracellular matrix components. In this paper, we first describe a simple method to manufacture cartilage microtissues at various scales using novel microwell array stamps. This system allows the rapid and reliable generation of cartilage microtissues, and can be used as a platform to study microtissue phenotype and development. Based on the unexpected discovery that Endothelial Growth Medium (EGM) enhanced MSC aggregation and chondrogenic capacity within the microwell arrays, this work also sought to identify soluble factors within the media capable of supporting robust differentiation using heterogeneous MSC populations. Hydrocortisone was found to be the key factor within EGM that enhanced the chondrogenic capacity of MSCs within these microwell arrays. This strategy represents a promising means of generating large numbers of high-quality, scaffold-free cartilage microtissues for diverse biofabrication applications.

bioengineering↗

bioRxiv2021

Integrating melt-electrowriting and inkjet bioprinting for engineering structurally organized articular cartilage

Successful cartilage engineering requires the generation of biological grafts mimicking the structure, composition and mechanical behaviour of the native tissue. Here melt-electrowriting (MEW) was used to produce arrays of polymeric structures whose function was to orient the growth of cellular aggregates spontaneously generated within these structures, and to provide tensile reinforcement to the resulting tissues. Inkjeting was used to deposit defined numbers of cells into MEW structures, which self-assembled into an organized array of spheroids within hours, ultimately generating a hybrid tissue that was hyaline-like in composition. Structurally, the engineered cartilage mimicked the histotypical organization observed in skeletally immature synovial joints. This biofabrication framework was then used to generate scaled-up (50mm x 50mm) cartilage implants containing over 3,500 cellular aggregates in under 15 minutes. After 8 weeks in culture, a 50-fold increase in the compressive properties of these MEW reinforced tissues were observed, while the tensile properties were still dominated by the polymer network, resulting in a composite construct demonstrating tension-compression nonlinearity mimetic of the native tissue. Helium ion microscopy further demonstrated the development of an arcading collagen network within the engineered tissue. This hybrid bioprinting strategy provides a versatile and scalable approach to engineer cartilage biomimetic grafts for biological joint resurfacing.

bioengineering↗

bioRxiv2021

Cross-species evolutionary rewiring in the enteric bacterium Campylobacter

Significance StatementGenetic exchange among bacteria shapes the microbial world. From the acquisition of antimicrobial resistance genes to fundamental questions about the nature of bacterial species, this powerful evolutionary force has preoccupied scientists for decades. However, the mixing of genes between species rests on a paradox. On one hand, promoting adaptation by conferring novel functionality, on the other potentially introducing disharmonious gene combinations (negative epistasis) that will be selected against. Taking an interdisciplinary approach to analyse natural populations of the enteric bacteria Campylobacter, an ideal example of long-range admixture, we demonstrate that genes can independently transfer across species boundaries and rejoin in epistasis in a recipient genome. This challenges conventional ideas and highlights the possibility of single step evolution by saltation. AbstractRecombination of short DNA fragments via horizontal gene transfer (HGT) can both introduce beneficial alleles and create genomic disharmony through negative epistasis. For non-core (accessory) genes, the negative epistatic cost is likely to be minimal because the incoming genes have not co-evolved with the recipient genome. By contrast, for the core genome, interspecific recombination is expected to be rare because disruptive allelic replacement is likely to introduce negative epistasis. Why then is homologous recombination common in the core of bacterial genomes? To understand this enigma we take advantage of an exceptional model system, the common enteric pathogens Campylobacter jejuni and Campylobacter coli, that are known for very high magnitude interspecies gene flow in the core genome. As expected, HGT does indeed disrupt co-adapted allele pairings (negative epistasis). However, multiple HGT events enable recovery of the genomes co-adaption between introgressing alleles, even in core metabolism genes (e.g., formate dehydrogenase). These findings demonstrate that, even for complex traits, genetic coalitions can be decoupled, transferred and independently reinstated in a new genetic background - facilitating transition between fitness peaks. In this example, the two-step recombinational process is associated with C. coli that are adapted to the agricultural niche.

microbiology↗

bioRxiv2020

Badger territoriality maintained despite disturbance of major road construction

Road ecology has traditionally focused on the impact of in-situ and functional roads on wildlife. However, road construction also poses a major, yet understudied, threat and any implications for key aspects of animal behaviour are unknown. There are also concerns that environmental disturbances, including major roadworks, can disrupt badger territoriality, promoting the spread of tuberculosis to cattle. To address these knowledge gaps, the ranging behaviour of a medium-density Irish badger population was monitored, using GPS-tracking collars, before, during and after a major road realignment project that bisected the study area. We estimated badgers home range sizes, nightly distances travelled and the distance and frequency of extra-territorial excursions during each phase of the study and quantified any changes to these parameters. We show that roadworks had a very limited effect on ranging behaviour. A small increase in nightly distance during the roadworks did not translate into an increase in home range size, nor an increase in the distance or frequency of extra-territorial excursions during the roadworks. In addition, suitable mitigation measures to prevent badger deaths appeared to ensure that normal patterns of ranging behaviour continued once the new road was in place. Our analysis supports the view that road construction did not cause badgers to change their ranging behaviour in ways likely to increase the spread of tuberculosis.

ecology↗

bioRxiv2020

Effects of chondrogenic priming duration on mechanoregulation of engineered cartilage anlagen.

Bone development and repair occur by endochondral ossification of a cartilage anlage, or template. Endochondral ossification is regulated by mechanical cues. Recently, we found that in vivo mechanical loading promoted regeneration of large bone defects through endochondral ossification, in a manner dependent on the timing of load initiation. Here, we have developed an in vitro model of the cartilage anlage to test whether the chondrogenic differentiation state alters the response to dynamic mechanical compression. We cultured human bone marrow stromal cells (hMSCs) at high cell density in fibrin hydrogels under chondrogenic priming conditions for periods of 0, 2, 4, or 6 weeks prior to two weeks of dynamic mechanical loading. Samples were evaluated by biomechanical testing, biochemical analysis of collagen and glycosaminoglycan (GAG) deposition, gene expression analysis, and immunohistological analysis, in comparison to time-matched controls cultured under static conditions. We found that dynamic loading increased the mechanical stiffness of engineered anlagen in a manner dependent on the duration of chondrogenic priming prior to load initiation. For chondrogenic priming times of 2 weeks or greater, dynamic loading enhanced the expression of type II collagen and aggrecan, although no significant changes in overall levels of matrix deposition was observed. For priming periods less than 4 weeks, dynamic loading generally supressed markers of hypertrophy and osteogenesis, although this was not observed if the priming period was extended to 6 weeks, where loading instead enhanced the expression of type X collagen. Taken together, these data demonstrate that the duration of chondrogenic priming regulates the endochondral response to dynamic mechanical compression in vitro, which may contribute to the effects of mechanical loading on endochondral bone development, repair, and regeneration in vivo.

bioengineering↗

bioRxiv2020

Marcksl1 modulates endothelial cell mechanoresponse to haemodynamic forces to control blood vessel shape and size

The formation of vascular tubes is driven by extensive changes in endothelial cell (EC) shape. Here, we have identified a novel role of the actin-binding protein, Marcksl1, in modulating the mechanical properties of EC cortex to regulate cell shape and vessel structure during angiogenesis. Increasing and depleting Marcksl1 expression level in vivo resulted in an increase and decrease, respectively, in EC size and the diameter of microvessels. Furthermore, endothelial overexpression of Marcksl1 induced ectopic blebbing on both apical and basal membranes, during and after lumen formation, that is suppressed by reduced blood flow. High resolution imaging revealed that Marcksl1 promotes the formation of linear actin bundles and decreases actin density at the EC cortex. Our findings demonstrate that a balanced network of linear and branched actin at the EC cortex is essential in conferring cortical integrity to resist the deforming forces of blood flow to regulate vessel structure.

cell biology↗

bioRxiv2019

The cryo-EM structure of the bacterial flagellum cap complex suggests a molecular mechanism for filament elongation.

The bacterial flagellum is a remarkable molecular motor, present at the surface of many bacteria, whose primary function is to allow motility through the rotation of a long filament protruding from the bacterial cell. A cap complex, consisting of an oligomeric assembly of the protein FliD, is localized at the tip of the flagellum, and is essential for filament assembly, as well as adherence to surfaces in some bacteria. However, the structure of the intact cap complex, and the molecular basis for its interaction with the filament, remains elusive. Here we report the cryo-EM structure of the Campylobacter jejuni cap complex. This structure reveals that FliD is pentameric, with the N-terminal region of the protomer forming an unexpected extensive set of contacts across several subunits, that contribute to FliD oligomerization. We also demonstrate that the native C. jejuni flagellum filament is 11-stranded and propose a molecular model for the filament-cap interaction.

microbiology↗

bioRxiv2019

Domestication of Campylobacter jejuni NCTC 11168

Reference and type strains of well-known bacteria have been a cornerstone of microbiology research for decades. The sharing of well-characterised isolates among laboratories has parallelised research efforts and enhanced the reproducibility of experiments, leading to a wealth of knowledge about trait variation in different species and the underlying genetics. Campylobacter jejuni strain NCTC 11168, deposited at the National Collection of Type Cultures in 1977, has been adopted widely as a reference strain by researchers worldwide and was the first Campylobacter for which the complete genome was published (in 2000). In this study, we collected 23 C. jejuni NCTC 11168 reference isolates from laboratories across the UK and compared variation in simple laboratory phenotypes with genetic variation in sequenced genomes. Putatively identical isolates identified previously to have aberrant phenotypes varied by up to 281 SNPs (in 15 genes) compared to the most recent reference strain. Isolates also display considerable phenotype variation in motility, morphology, growth at 37{degrees}C, invasion of chicken and human cell lines and susceptibility to ampicillin. This study provides evidence of ongoing evolutionary change among C. jejuni isolates as they are cultured in different laboratories and highlights the need for careful consideration of genetic variation within laboratory reference strains.\n\nImpact statementIn this paper, we comment on the changing role of laboratory reference strains. While the model organism allows basic comparison within and among laboratories, it is important to remember the effect even small differences in isolate genomes can have on the validity and reproducibility of experimental work. We quantify differences in 23 reference Campylobacter genomes and compare them with observable differences in common laboratory phenotypes.\n\nData summaryShort read data are archived on the NCBI SRA associated with BioProject accession PRJNA517467 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA517467).\n\nAll assembled genomes are also available on FigShare (doi: 10.6084/m9.figshare.7849268). Phylogeny visualised on microreact: https://microreact.org/project/NCTC11168.\n\nRepositoriesShort read data are archived on the NCBI SRA repository, associated with BioProject accession PRJNA517467 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA517467; Table S1).\n\nThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2019

Gut microbiome features associated with Clostridium difficile colonization in puppies

In people, colonization with Clostridium difficile, the leading cause of antibiotic-associated diarrhea, has been shown to be associated with distinct gut microbial features, including reduced bacterial community diversity and depletion of key taxa. In dogs, the gut microbiome features that define C. difficile colonization are less well understood. We sought to define the gut microbiome features associated with C. difficile colonization in puppies, a population where the prevalence of C. difficile has been shown to be elevated, and to define the effect of puppy age and litter upon these features and C. difficile risk. We collected fecal samples from weaned (n=27) and unweaned (n=74) puppies from 13 litters and analyzed the effects of colonization status, age and litter on microbial diversity using linear mixed effects models.\n\nColonization with C. difficile was significantly associated with younger age, and colonized puppies had significantly decreased bacterial community diversity and differentially abundant taxa compared to non-colonized puppies, even when adjusting for age. C. difficile colonization remained associated with decreased bacterial community diversity, but the association did not reach statistical significance in a mixed effects model incorporating litter as a random effect.\n\nEven though litter explained a greater proportion (67%) of the variability in microbial diversity than colonization status, we nevertheless observed heterogeneity in gut microbial community diversity and colonization status within more than half of the litters, suggesting that the gut microbiome contributes to colonization resistance against C. difficile. The colonization of puppies with C. difficile has important implications for the potential zoonotic transfer of this organism to people. The identified associations point to mechanisms by which C. difficile colonization may be reduced.

epidemiology↗

bioRxiv2017

Recapitulating bone development for tissue regeneration through engineered mesenchymal condensations and mechanical cues

Large bone defects cannot heal without intervention and have high complication rates even with the best treatments available. In contrast, bone fractures naturally healing with high success rates by recapitulating the process of bone development through endochondral ossification.1 Endochondral tissue engineering may represent a promising paradigm, but large bone defects are unable to naturally form a callus. We engineered mesenchymal condensations featuring local morphogen presentation (TGF-{beta}1) to mimic the cellular organization and lineage progression of the early limb bud. As mechanical forces are 2,3 critical for proper endochondral ossification during bone morphogenesis2,3 and fracture healing, we hypothesized that mechanical cues would be important for endochondral regeneration.4,5 Here, using fixation plates that modulate ambulatory load transfer through dynamic tuning of axial compliance, we found that in vivo mechanical loading was necessary to restore bone function to large bone defects through endochondral ossification. Endochondral regeneration produced zonal cartilage and primary spongiosa mimetic of the native growth plate. Live human chondrocytes contributed to endochondral regeneration in vivo, while cell devitalization prior to condensation transplantation abrogated bone formation. Mechanical loading induced regeneration comparable to high-dose BMP-2 delivery, but without heterotopic bone formation and with order-of-magnitude greater mechanosensitivity.6-8 In vitro, mechanical loading promoted chondrogenesis, and upregulated pericellular collagen 6 deposition and angiogenic gene expression. Consistently, in vivo mechanical loading regulated cartilage formation and neovascular invasion dependent on load timing. Together, this study represents the first demonstration of the effects of mechanical loading on transplanted cell-mediated bone defect regeneration, and provides a new template for recapitulating developmental programs for tissue engineering.

bioengineering↗