Cell morphology as a design parameter in the bioengineering of cell-biomaterial surface interactions.

Control of cell-surface interaction is necessary for biomaterial applications such as cell sheets, intelligent cell culture surfaces, or functional coatings. In this paper, we propose the emergent property of cell morphology as a design parameter in the bioengineering of cell-biomaterial surface interactions. Cell morphology measured through various parameters can indicate ideal candidates for these various applications thus reducing the time taken for the screening and development process. The hypothesis of this study is that there is an optimal cell morphology range for enhanced cell proliferation and migration on the surface of biomaterials. To test the hypothesis, primary porcine dermal fibroblasts (PDF, 3 biological replicates) were cultured on ten different surfaces comprising components of the natural extracellular matrix of tissues. Results suggested an optimal morphology with a cell aspect ratio (CAR) between 0.2 and 0.4 for both increased cell proliferation and migration. If the CAR was below 0.2 (very elongated cell), cell proliferation was increased whilst migration was reduced. A CAR of 0.4+ (rounded cell) favoured cell migration over proliferation. The screening process, when it comes to biomaterials is a long, repetitive, arduous but necessary event. This study highlights the beneficial use of testing the cell morphology on prospective prototypes, eliminating those that do not support an optimal cell shape. We believe that the research presented in this paper is important as we can help address this screening inefficiency through the use of the emergent property of cell morphology. Future work involves automating CAR quantification for high throughput screening of prototypes.

Three dimensional porous scaffolds derived from collagen, elastin and fibrin proteins orchestrate adipose tissue regeneration.

Current gold standard to treat soft tissue injuries caused by trauma and pathological condition are autografts and off the shelf fillers, but they have inherent weaknesses like donor site morbidity, immuno-compatibility and graft failure. To overcome these limitations, tissue-engineered polymers are seeded with stem cells to improve the potential to restore tissue function. However, their interaction with native tissue is poorly understood so far. To study these interactions and improve outcomes, we have fabricated scaffolds from natural polymers (collagen, fibrin and elastin) by custom-designed processes and their material properties such as surface morphology, swelling, wettability and chemical cross-linking ability were characterised. By using 3D scaffolds, we comprehensive assessed survival, proliferation and phenotype of adipose-derived stem cells in vitro. In vivo, scaffolds were seeded with adipose-derived stem cells and implanted in a rodent model, with X-ray microtomography, histology and immunohistochemistry as read-outs. Collagen-based materials showed higher cell adhesion and proliferation in vitro as well as higher adipogenic properties in vivo. In contrast, fibrin demonstrated poor cellular and adipogenesis properties but higher angiogenesis. Elastin formed the most porous scaffold, with cells displaying a non-aggregated morphology in vitro while in vivo elastin was the most degraded scaffold. These findings of how polymers present in the natural polymers mimicking ECM and seeded with stem cells affect adipogenesis in vitro and in vivo can open avenues to design 3D grafts for soft tissue repair.

Chronic Leg Ulcers: Are Tissue Engineering and Biomaterials Science the Solution?

Chronic leg ulcers (CLUs) are full thickness wounds that usually occur between the ankle and knee, fail to heal after 3 months of standard treatment, or are not entirely healed at 12 months. CLUs present a considerable burden on patients, subjecting them to severe pain and distress, while healthcare systems suffer immense costs and loss of resources. The poor healing outcome of the standard treatment of CLUs generates an urgent clinical need to find effective solutions for these wounds. Tissue Engineering and Biomaterials Science offer exciting prospects for the treatment of CLUs, using a broad range of skin substitutes or scaffolds, and dressings. In this review, we summarize and discuss the various types of scaffolds used clinically in the treatment of CLUs. Their structure and therapeutic effects are described, and for each scaffold type representative examples are discussed, supported by clinical trials. Silver dressings are also reviewed due to their reported benefits in the healing of leg ulcers, as well as recent studies on new dermal scaffolds, reporting on clinical results where available. We conclude by arguing there is a further need for tissue-engineered products specifically designed and bioengineered to treat these wounds and we propose a series of properties that a biomaterial for CLUs should possess, with the intention of focusing efforts on finding an effective treatment.

In vitro modelling of disease-induced changes in the diabetic wound fibroblast.

OBJECTIVE: Fibroblasts have been shown to play an increasingly important role within diabetic wounds. While several in vitro models of diabetic wound fibroblasts have been reported, none replicate the natural progression of the disease over time, recapitulating the acquisition of the diseased phenotype. Therefore, this study aimed to establish an in vitro model of the diabetic wound fibroblast through sustained exposure of healthy dermal fibroblasts to hyperglycaemia. METHOD: Primary human fibroblasts were isolated from discarded healthy skin tissue and were either exposed to normoglycaemic (control 5.5mM glucose) media or hyperglycaemic (25mM glucose) media for four weeks. Quantitative polymerase chain reaction was performed to measure the expression of inflammatory cytokines and chemokines. RESULTS: In the hyperglycaemia model, stromal cell-derived factor (SDF)-1 expression remained consistently downregulated across all four weeks (p<0.01), while monocyte chemoattractant protein (MCP)-1 (p<0.001), interleukin (IL)-8 (p=0.847) and chemokine (C-X-C motif) ligand 1 (CXCL1) (p=0.872) were initially downregulated at one week followed by subsequent upregulation between 2-4 weeks. CONCLUSION: This hyperglycaemia model may serve as a useful tool to characterise pathological changes in the diabetic wound fibroblast and help identify candidate therapeutic targets, such as SDF-1, that may reverse the pathology.

Bioactive glasses and electrospun composites that release cobalt to stimulate the HIF pathway for wound healing applications.

BACKGROUND: Bioactive glasses are traditionally associated with bonding to bone through a hydroxycarbonate apatite (HCA) surface layer but the release of active ions is more important for bone regeneration. They are now being used to deliver ions for soft tissue applications, particularly wound healing. Cobalt is known to simulate hypoxia and provoke angiogenesis. The aim here was to develop new bioactive glass compositions designed to be scaffold materials to locally deliver pro-angiogenic cobalt ions, at a controlled rate, without forming an HCA layer, for wound healing applications. METHODS: New melt-derived bioactive glass compositions were designed that had the same network connectivity (mean number of bridging covalent bonds between silica tetrahedra), and therefore similar biodegradation rate, as the original 45S5 Bioglass. The amount of magnesium and cobalt in the glass was varied, with the aim of reducing or removing calcium and phosphate from the compositions. Electrospun poly(ε-caprolactone)/bioactive glass composites were also produced. Glasses were tested for ion release in dissolution studies and their influence on Hypoxia-Inducible Factor 1-alpha (HIF-1α) and expression of Vascular Endothelial Growth Factor (VEGF) from fibroblast cells was investigated. RESULTS: Dissolution tests showed the silica rich layer differed depending on the amount of MgO in the glass, which influenced the delivery of cobalt. The electrospun composites delivered a more sustained ion release relative to glass particles alone. Exposing fibroblasts to conditioned media from these composites did not cause a detrimental effect on metabolic activity but glasses containing cobalt did stabilise HIF-1α and provoked a significantly higher expression of VEGF (not seen in Co-free controls). CONCLUSIONS: The composite fibres containing new bioactive glass compositions delivered cobalt ions at a sustained rate, which could be mediated by the magnesium content of the glass. The dissolution products stabilised HIF-1α and provoked a significantly higher expression of VEGF, suggesting the composites activated the HIF pathway to stimulate angiogenesis.

Decellularised scaffolds: just a framework? Current knowledge and future directions.

The use of decellularised matrices as scaffolds offers the advantage of great similarity with the tissue to be replaced. Moreover, decellularised tissues and organs can be repopulated with the patient's own cells to produce bespoke therapies. Great progress has been made in research and development of decellularised scaffolds, and more recently, these materials are being used in exciting new areas like hydrogels and bioinks. However, much effort is still needed towards preserving the original extracellular matrix composition, especially its minor components, assessing its functionality and scaling up for large tissues and organs. Emphasis should also be placed on developing new decellularisation methods and establishing minimal criteria for assessing the success of the decellularisation process. The aim of this review is to critically review the existing literature on decellularised scaffolds, especially on the preparation of these matrices, and point out areas for improvement, finishing with alternative uses of decellularised scaffolds other than tissue and organ reconstruction. Such uses include three-dimensional ex vivo platforms for idiopathic diseases and cancer modelling.

Cobalt from metal-on-metal hip replacements may be the clinically relevant active agent responsible for periprosthetic tissue reactions.

Some types of metal-on-metal (MOM) hip replacements have unacceptably high rates of failure, such as the Ultima TPS MOM hip, with 13.8% failure at 5 years. This has been attributed to an inflammatory reaction following the release of cobalt (Co) and chromium (Cr) from the bearing surfaces and modular junctions. There is in vitro evidence that Co is more important than Cr in the inflammatory process, but there are no reported human tissue studies of the analysis of implant-derived metals.

Simple isolation method for the bulk isolation of wear particles from metal on metal bearing surfaces generated in a hip simulator test.

Isolation and characterization of metal-on-metal (MoM) wear particles from simulator lubricants is essential to understand wear behaviour, ion release and associated corrosive activity related to the wear particles. Substantial challenges remain to establish a simple, precise and repeatable protocol for the isolation and analysis of wear particles due to their extremely small size, their tendency to agglomerate and degrade. In this paper, we describe a simple and efficient method for the bulk isolation and characterisation of wear particles from MoM bearings. Freeze drying was used to remove the large volume of water from the serum lubricant, enzymes used to digest the proteins and ultracentrifugation to finally isolate and purify the particles. The present study involved a total of eight steps for the isolation process and a wear particle extraction efficiency of 45% was achieved.

Mycolactone inhibits monocyte cytokine production by a posttranscriptional mechanism.

The virulence and immunosuppressive activity of Mycobacterium ulcerans is attributed to mycolactone, a macrolide toxin synthesized by the bacteria. We have explored the consequence and mechanism of mycolactone pretreatment of primary human monocytes activated by a wide range of TLR ligands. The production of cytokines (TNF, IL-1beta, IL-6, IL-10, and IFN-gamma-inducible protein-10), chemokines (IL-8), and intracellular effector molecules (exemplified by cyclooxygenase-2) was found to be powerfully and dose dependently inhibited by mycolactone, irrespective of the stimulating ligand. However, mycolactone had no effect on the activation of signaling pathways that are known to be important in inducing these genes, including the MAPK and NF-kappaB pathways. Unexpectedly, LPS-dependent transcription of TNF, IL-6, and cyclooxygenase-2 mRNA was found not to be inhibited, implying that mycolactone has a novel mechanism of action and must function posttranscriptionally. We propose that mycolactone mediates its effects by inhibiting the translation of a specific subset of proteins in primary human monocytes. This mechanism is distinct from rapamycin, another naturally occurring immunosuppressive lactone. The current findings also suggest that monocyte-derived cytokine transcript and protein levels may not correlate in Buruli ulcer lesions, and urge caution in the interpretation of RT-PCR data obtained from patient biopsy samples.

Rac mediates TNF-induced cytokine production via modulation of NF-kappaB.

TNF is a key factor in a variety of inflammatory diseases. Here we report that TNF induced pro-inflammatory cytokine synthesis of IL-6 and IL-8 is mediated by the Rho GTPase Rac. TNF induces p42/p44, p54 and p38 MAPK kinase; these kinases have been implicated in control of cytokine synthesis. However, over-expression of a dominant negative form of Rac strongly inhibited TNF-induced p42/44 MAPK kinase activation, but had little effect upon JNK and no effect upon p38 MAPK activity. Another key signalling pathway controlling cytokine expression is NF-kappaB. When analyzing TNF-induced NF-kappaB activity via luciferase-reporter assays or via EMSA, we were able to show that the dominant negative version of Rac could completely abrogate TNF-induced NF-kappaB activity. In addition, we also observed that inhibition of the ERK pathway led to a reduction in TNF-induced NF-kappaB transcriptional activity; this was accompanied by an ablation of TNF-induced p65 phosphorylation at serine 276. This would suggest that TNF-induced activation of Rac, lies upstream of NF-kappaB activation, and that the inhibition of this pathway results in inhibition of cytokine production.

A late, prolonged activation of the phosphatidylinositol 3-kinase pathway is required for T cell proliferation.

Activation of the phosphatidylinositol-3 kinase (PI 3-K) pathway is associated with the proliferation of many cell types, including T lymphocytes. However, recent studies in cell lines stably expressing deletion mutants of IL-2R that fail to activate PI 3-K have questioned the requirement for this pathway in cell cycle regulation. In this study with IL-2 and IL-7, we show in primary T cells that, unlike IL-2, IL-7 fails to induce the early activation of PI 3-K seen within minutes and normally associated with cytokine signaling. However, kinetic experiments showed that both of these T cell growth factors induce a distinct and sustained phase of PI 3-K activity several hours after stimulation. This delayed activation correlates with cell cycle induction and from studies using inhibitors of PI 3-K signaling, we show that this later phase, unlike the early activation within minutes, is required for cell cycle induction. The data presented here will have major implications for our understanding of the mechanism of T cell proliferation as well as the regulation of PI 3-K activity.

IL-4 regulation of p38 MAPK signalling is dependent on cell type.

p38 MAPK was originally characterized as a stress-induced kinase, along with JNK. Subsequently, p38 MAPK was found to be activated by stimuli other than cellular stress, such as growth factors and mitogens, like interleukin (IL)-2, IL-7 and IL-3. A notable exception was IL-4, as studies in mast cells showed no activation of p38 MAPK by this cytokine. In this study we show that the regulation of p38 MAPK is cell type dependent. Like other cytokines that signal through the gamma (gamma)(c), IL-4 can activate p38 MAPK in the CT6 T-cell line and BA/F3 pro-B-cells. However, IL-4 was unable to activate p38 MAPK in the murine macrophage cell line, RAW 264.7 and, indeed, prolonged exposure of cells to IL-4 results in suppression of LPS-induced MAPK activation. This result correlates with the well defined inhibitory effect of IL-4 on tumour necrosis factor alpha (TNFalpha) production. In contrast, studies in primary human monocytes showed that prolonged exposure to IL-4 resulted in enhanced activation of LPS-stimulated p38 MAPK; this correlated with an enhanced TNFalpha production. These data highlight the complexity of IL-4 signalling mechanisms, the diversity that can exist in the regulation of a given signalling pathway by a given cytokine and, furthermore, indicate the problems that can arise from extrapolation between different cell systems.