Histological analysis of fat grafting with platelet-rich plasma for diabetic foot ulcers-A randomised controlled trial.

Diabetic foot ulcers are often unresponsive to conventional therapy and are a leading cause of amputation. Animal studies have shown stem cells and growth factors can accelerate wound healing. Adipose-derived stem cells are found in fat grafts and mixing them with platelet-rich plasma (PRP) may improve graft survival. This study aimed to establish the histological changes when diabetic foot ulcers are treated with fat grafts and PRP. A three-armed RCT was undertaken of 18 diabetic foot ulcer patients: fat grafting; fat grafting with PRP; and routine podiatry care. Biopsies were obtained at week 0, 1, and 4, and underwent quantitative histology/immunohistochemistry (H&E, CD31, and Ki67). Treatment with fat and PRP increased mean microvessel density at 1 week to 1645 (SD 96) microvessels/mm2 (+32%-45% to other arms, P = .035). PRP appeared to increase vascularity surrounding fat grafts, and histology suggested PRP may enhance fat graft survival. There was no clinical difference between arms. This study demonstrates PRP with fat grafts increased neovascularisation and graft survival in diabetic foot ulcers. The histology was not, however, correlated with wound healing time. Future studies should consider using apoptosis markers and fluorescent labelling to ascertain if enhanced fat graft survival is due to proliferation or reduced apoptosis. Trial registration NCT03085550.

Fat grafting and platelet-rich plasma for the treatment of diabetic foot ulcers: A feasibility-randomised controlled trial.

Chronic, nonhealing diabetic foot ulcers (DFU) are increasing in prevalence and are often unresponsive to conventional therapy. Adipose tissue, containing adipose-derived stem cells, and platelet rich plasma (PRP) are regenerative therapies rich in growth factors which may provide a solution to chronic wound healing. This study aimed to assess the feasibility of conducting a definitive randomised controlled trial (RCT) to investigate the efficacy of these therapies for the treatment of DFU. This was a single centre, feasibility, three-arm, parallel group RCT. Eligible DFU patients were randomised on a 1:1:1 basis to three intervention arms: control (podiatry); fat grafting; fat grafting with PRP. The intervention was delivered once and patients were followed-up for 12 weeks. The primary objective was to assess measures of trial feasibility. Clinical outcomes and health-related quality of life (HRQoL) were also evaluated. Three hundred and thirty four patients were screened and 32 patients (9.6%) were deemed eligible with 18 enrolled in the trial (6 per arm) over 17 months. All participants completed the trial with no withdrawals or crossover. Participant engagement was high with most HRQoL questionnaires returned and only 4.8% follow-up appointments missed. There were five adverse events (AEs) related to the trial with no serious AEs. Five (28%) of the wounds healed. There was no difference between any of the groups in terms of clinical outcomes. This feasibility study demonstrated that a multi-centre RCT is safe and feasible with excellent patient engagement. We have highlighted crucial information regarding methodology and recruitment, which will guide future trial design. Registration number: NCT03085550 clinicaltrials.gov. Registered 01/03/2017.

Differential Regulation of Human Bone Marrow Mesenchymal Stromal Cell Chondrogenesis by Hypoxia Inducible Factor-1α Hydroxylase Inhibitors.

The transcriptional profile induced by hypoxia plays important roles in the chondrogenic differentiation of marrow stromal/stem cells (MSC) and is mediated by the hypoxia inducible factor (HIF) complex. However, various compounds can also stabilize HIF's oxygen-responsive element, HIF-1α, at normoxia and mimic many hypoxia-induced cellular responses. Such compounds may prove efficacious in cartilage tissue engineering, where microenvironmental cues may mediate functional tissue formation. Here, we investigated three HIF-stabilizing compounds, which each have distinct mechanisms of action, to understand how they differentially influenced the chondrogenesis of human bone marrow-derived MSC (hBM-MSC) in vitro. hBM-MSCs were chondrogenically-induced in transforming growth factor-ß3-containing media in the presence of HIF-stabilizing compounds. HIF-1α stabilization was assessed by HIF-1α immunofluorescence staining, expression of HIF target and articular chondrocyte specific genes by quantitative polymerase chain reaction, and cartilage-like extracellular matrix production by immunofluorescence and histochemical staining. We demonstrate that all three compounds induced similar levels of HIF-1α nuclear localization. However, while the 2-oxoglutarate analog dimethyloxalylglycine (DMOG) promoted upregulation of a selection of HIF target genes, desferrioxamine (DFX) and cobalt chloride (CoCl2 ), compounds that chelate or compete with divalent iron (Fe2+ ), respectively, did not. Moreover, DMOG induced a more chondrogenic transcriptional profile, which was abolished by Acriflavine, an inhibitor of HIF-1α-HIF-ß binding, while the chondrogenic effects of DFX and CoCl2 were more limited. Together, these data suggest that HIF-1α function during hBM-MSC chondrogenesis may be regulated by mechanisms with a greater dependence on 2-oxoglutarate than Fe2+ availability. These results may have important implications for understanding cartilage disease and developing targeted therapies for cartilage repair. Stem Cells 2018;36:1380-1392.

The use of fat grafting and platelet-rich plasma for wound healing: A review of the current evidence.

Fat grafting is becoming a common procedure in regenerative medicine because of its high content of growth factors and adipose derived stem cells (ADSCs) and the ease of harvest, safety, and low cost. The high concentration of ADSCs found in fat has the potential to differentiate into a wide range of wound-healing cells including fibroblasts and keratinocytes as well as demonstrating proangiogenic qualities. This suggests that fat could play an important role in wound healing. However retention rates of fat grafts are highly variable due in part to inconsistent vascularisation of the transplanted fat. Furthermore, conditions such as diabetes, which have a high prevalence of chronic wounds, reduce the potency and regenerative potential of ADSCs. Platelet-rich plasma (PRP) is an autologous blood product rich in growth factors, cell adhesion molecules, and cytokines. It has been hypothesised that PRP may have a positive effect on the survival and retention of fat grafts because of improved proliferation and differentiations of ADSCs, reduced inflammation, and improved vascularisation. There is also increasing interest in a possible synergistic effect that PRP may have on the healing potential of fat, although the evidence for this is very limited. In this review, we evaluate the evidence in both in vitro and animal studies on the mechanistic relationship between fat and PRP and how this translates to a benefit in wound healing. We also discuss future directions for both research and clinical practice on how to enhance the regenerative potential of the combination of PRP and fat.

Design and development of nanocomposite scaffolds for auricular reconstruction.

Auricular reconstruction using sculpted autologous costal cartilage is effective, but complex and time consuming and may incur donor site sequelae and morbidity. Conventional synthetic alternatives are associated with infection and extrusion in up to about 15% of cases. We present a novel POSS-PCU nanocomposite auricular scaffold, which aims to reduce extrusion rates by mimicking the elastic modulus of human ears and by encouraging desirable cellular interactions. The fabrication, physicochemical properties (including nanoscale topography) and cellular interactions of these scaffolds were compared to Medpor®, the current synthetic standard. Our scaffold had a more similar elastic modulus (5.73 ± 0.17MPa) to ear cartilage (5.02 ± 0.17MPa) compared with Medpor®, which was much stiffer (140.9 ± 0.04MPa). POSS-PCU supported fibroblast ingrowth and proliferation; significantly higher collagen production was also produced by cells on the POSS-PCU than those on Medpor®. This porous POSS-PCU nanocomposite scaffold is therefore a promising alternative biomaterial for auricular surgical reconstruction. FROM THE CLINICAL EDITOR: In this paper, a novel POSS-PCU nanocomposite auricular scaffold is described to reduce extrusion rates by having a much closer elastic modulus of human ears than the currently available synthetic standard. Enabling desirable cellular interactions may lead to the successful clinical application of these novel scaffolds.

Transplantation of human fetal blood stem cells in the osteogenesis imperfecta mouse leads to improvement in multiscale tissue properties.

Osteogenesis imperfecta (OI or brittle bone disease) is a disorder of connective tissues caused by mutations in the collagen genes. We previously showed that intrauterine transplantation of human blood fetal stem/stromal cells in OI mice (oim) resulted in a significant reduction of bone fracture. This work examines the cellular mechanisms and mechanical bone modifications underlying these therapeutic effects, particularly examining the direct effects of donor collagen expression on bone material properties. In this study, we found an 84% reduction in femoral fractures in transplanted oim mice. Fetal blood stem/stromal cells engrafted in bones, differentiated into mature osteoblasts, expressed osteocalcin, and produced COL1a2 protein, which is absent in oim mice. The presence of normal collagen decreased hydroxyproline content in bones, altered the apatite crystal structure, increased the bone matrix stiffness, and reduced bone brittleness. In conclusion, expression of normal collagen from mature osteoblast of donor origin significantly decreased bone brittleness by improving the mechanical integrity of the bone at the molecular, tissue, and whole bone levels.

The effect of Si species released from bioactive glasses on cell behaviour: A quantitative review.

Despite over 50 years of silicate bioactive glass (SBG) research, commercial success, and 6000+ published articles, there remains a lack of understanding of how soluble silicate (Si) species released from SBGs influences cellular responses. Using a systematic approach, this article quantitatively compares the in vitro responses of cells to SBG dissolution products reported in the literature and determines if there is a Si concentration ([Si]) dependent effect on cell behaviour. Cell behavioural responses to SBGs [Si] in dissolution products included metabolic activity (reported in 52 % of articles), cell number (24 %), protein production (22 %), gene expression (22 %) and biomineralization (24 %). There was a difference in the [Si] reported to cause increased (desirable) cellular responses (median = 30.2 ppm) compared to the [Si] reported to cause decreased (undesirable) cellular responses (median = 52.0 ppm) (P ≤ 0.001). The frequency of undesirable outcomes increased with increasing [Si], with ∼3 times more negative outcomes reported above 52 ppm. We also investigated the effect of [Si] on specific cellular outcomes (e.g., metabolic activity, angiogenesis, osteogenesis), if cell type/species influenced these responses and the impact of other ions (Ca, P, Na) within the SBG dissolution media on cell behaviour. This review has, for the first time, quantitatively compared the cellular responses to SBGs from the literature, providing a quantitative overview of SBG in vitro practices and presents evidence of a range of [Si] where desirable cellular responses may be more likely (30-52 ppm). This review also demonstrates the need for greater standardisation of in vitro methodological approaches and recommends some minimum reporting standards. STATEMENT OF SIGNIFICANCE: This systematic review investigates the relationship between the concentration of Si released from Si-bioactive glasses (SBG) and in vitro cellular responses. Si releasing materials continue to be of considerable scientific, commercial, and medical interest (with 1500+ articles published in the last 3 years) but there is considerable variation in the reported biologically effective Si concentrations and on the importance of Si on cell behaviour. Despite the variation in methodological approaches, this article demonstrated statistical commonalities in the Si concentrations that cause desirable and undesirable cellular behaviours, suggesting a window where positive cellular outcomes are more likely. This review also provides a quantitative analysis of in vitro practices within the bioactive glass field and highlights the need for greater standardisation.

An Evaluation of the Effect of Activation Methods on the Release of Growth Factors from Platelet-Rich Plasma.

BACKGROUND: Activation of platelets in platelet-rich plasma may improve growth factor release, thus enhancing regenerative properties. The authors investigated whether different methods of platelet-rich plasma activation affected growth factor release kinetics over time. METHODS: Platelet-rich plasma from 20 healthy volunteers was processed by six different methods: (1) control (nonactivated); (2) activation with calcium chloride; (3) activation with calcium chloride and ethanol; (4) activation with calcium chloride and ethanol at 4°C; (5) activation with calcium chloride and ethanol with vitamin C; (6) activation with calcium chloride and ethanol with vitamin C at 4°C. Concentration of secreted vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and insulin-like growth factor over 24 hours was measured by immunoassay. RESULTS: Calcium chloride-activated platelet-rich plasma produced significantly more insulin-like growth factor at 1 hour compared to cold and vitamin C platelet-rich plasma, and calcium chloride plus ethanol produced significantly more at 24 hours compared to vitamin C platelet-rich plasma. The addition of vitamin C reduced release of PDGF over time. Activation with calcium chloride and ethanol with or without cold temperature produced a gradual PDGF release as opposed to calcium chloride alone, which caused higher PDGF within 4 hours. There were no significant differences between groups for VEGF, although calcium chloride and cooled platelet-rich plasma approached significance for producing more than vitamin C platelet-rich plasma. CONCLUSIONS: Activation of platelet-rich plasma does not significantly improve growth factor secretion, which is made worse by the addition of vitamin C, a platelet inhibitor. Ethanol does not negatively impact growth factor production and may offer a more gradual release. CLINICAL RELEVANCE STATEMENT: These findings will help guide platelet-rich plasma preparation methods where therapeutic growth factors are used. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Hypoxia mimetics restore bone biomineralisation in hyperglycaemic environments.

Diabetic patients have an increased risk of fracture and an increased occurrence of impaired fracture healing. Diabetic and hyperglycaemic conditions have been shown to impair the cellular response to hypoxia, via an inhibited hypoxia inducible factor (HIF)-1α pathway. We investigated, using an in vitro hyperglycaemia bone tissue engineering model (and a multidisciplinary bone characterisation approach), the differing effects of glucose levels, hypoxia and chemicals known to stabilise HIF-1α (CoCl2 and DMOG) on bone formation. Hypoxia (1% O2) inhibited bone nodule formation and resulted in discrete biomineralisation as opposed to the mineralised extracellular collagen fibres found in normoxia (20% O2). Unlike hypoxia, the use of hypoxia mimetics did not prevent nodule formation in normal glucose level. Hyperglycaemic conditions (25 mM and 50 mM glucose) inhibited biomineralisation. Interestingly, both hypoxia mimetics (CoCl2 and DMOG) partly restored hyperglycaemia inhibited bone nodule formation. These results highlight the difference in osteoblast responses between hypoxia mimetics and actual hypoxia and suggests a role of HIF-1α stabilisation in bone biomineralisation that extends that of promoting neovascularisation, or other system effects associated with hypoxia and bone regeneration in vivo. This study demonstrates that targeting the HIF pathway may represent a promising strategy for bone regeneration in diabetic patients.

Fat grafting and platelet-rich plasma in wound healing: a review of histology from animal studies.

Stem cells could form the basis of a novel, autologous treatment for chronic wounds like diabetic foot ulcers. Fat grafts contain adipose-derived stem cells (ADSC) but low survival of cells within the grafts is a major limitation. Platelet-rich plasma (PRP) may increase graft survival. This review examines the histology from animal studies on fat grafting, ADSC and PRP in wound healing. A literature review of major electronic databases was undertaken, and narrative synthesis performed. Data from 30 animal studies were included. ADSC increase angiogenesis over 14 days and often clinically accelerated wound healing. ADSC had a greater effect in animals with impaired wound healing (e.g. diabetes). Activated PRP increased viability of fat grafts. Despite the high number of studies, the quality is variable which weakens the evidence. It does suggest there is a benefit of ADSC, particularly in impaired wound healing. High-quality evidence in humans is required, to establish its clinical usefulness.

Bioengineering the ameloblastoma tumour to study its effect on bone nodule formation.

Ameloblastoma is a benign, epithelial cancer of the jawbone, which causes bone resorption and disfigurement to patients affected. The interaction of ameloblastoma with its tumour stroma drives invasion and progression. We used stiff collagen matrices to engineer active bone forming stroma, to probe the interaction of ameloblastoma with its native tumour bone microenvironment. This bone-stroma was assessed by nano-CT, transmission electron microscopy (TEM), Raman spectroscopy and gene analysis. Furthermore, we investigated gene correlation between bone forming 3D bone stroma and ameloblastoma introduced 3D bone stroma. Ameloblastoma cells increased expression of MMP-2 and -9 and RANK temporally in 3D compared to 2D. Our 3D biomimetic model formed bone nodules of an average surface area of 0.1 mm2 and average height of 92.37 [Formula: see text] 7.96 µm over 21 days. We demonstrate a woven bone phenotype with distinct mineral and matrix components and increased expression of bone formation genes in our engineered bone. Introducing ameloblastoma to the bone stroma, completely inhibited bone formation, in a spatially specific manner. Multivariate gene analysis showed that ameloblastoma cells downregulate bone formation genes such as RUNX2. Through the development of a comprehensive bone stroma, we show that an ameloblastoma tumour mass prevents osteoblasts from forming new bone nodules and severely restricted the growth of existing bone nodules. We have identified potential pathways for this inhibition. More critically, we present novel findings on the interaction of stromal osteoblasts with ameloblastoma.

Clinical relevance assessment of animal preclinical research (RAA) tool: development and explanation.

BACKGROUND: Only a small proportion of preclinical research (research performed in animal models prior to clinical trials in humans) translates into clinical benefit in humans. Possible reasons for the lack of translation of the results observed in preclinical research into human clinical benefit include the design, conduct, and reporting of preclinical studies. There is currently no formal domain-based assessment of the clinical relevance of preclinical research. To address this issue, we have developed a tool for the assessment of the clinical relevance of preclinical studies, with the intention of assessing the likelihood that therapeutic preclinical findings can be translated into improvement in the management of human diseases. METHODS: We searched the EQUATOR network for guidelines that describe the design, conduct, and reporting of preclinical research. We searched the references of these guidelines to identify further relevant publications and developed a set of domains and signalling questions. We then conducted a modified Delphi-consensus to refine and develop the tool. The Delphi panel members included specialists in evidence-based (preclinical) medicine specialists, methodologists, preclinical animal researchers, a veterinarian, and clinical researchers. A total of 20 Delphi-panel members completed the first round and 17 members from five countries completed all three rounds. RESULTS: This tool has eight domains (construct validity, external validity, risk of bias, experimental design and data analysis plan, reproducibility and replicability of methods and results in the same model, research integrity, and research transparency) and a total of 28 signalling questions and provides a framework for researchers, journal editors, grant funders, and regulatory authorities to assess the potential clinical relevance of preclinical animal research. CONCLUSION: We have developed a tool to assess the clinical relevance of preclinical studies. This tool is currently being piloted.

Comparative materials differences revealed in engineered bone as a function of cell-specific differentiation.

An important aim of regenerative medicine is to restore tissue function with implantable, laboratory-grown constructs that contain tissue-specific cells that replicate the function of their counterparts in the healthy native tissue. It remains unclear, however, whether cells used in bone regeneration applications produce a material that mimics the structural and compositional complexity of native bone. By applying multivariate analysis techniques to micro-Raman spectra of mineralized nodules formed in vitro, we reveal cell-source-dependent differences in interactions between multiple bone-like mineral environments. Although osteoblasts and adult stem cells exhibited bone-specific biological activities and created a material with many of the hallmarks of native bone, the 'bone nodules' formed from embryonic stem cells were an order of magnitude less stiff, and lacked the distinctive nanolevel architecture and complex biomolecular and mineral composition noted in the native tissue. Understanding the biological mechanisms of bone formation in vitro that contribute to cell-source-specific materials differences may facilitate the development of clinically successful engineered bone.

Hypoxia Inducible Factor-1α in Osteochondral Tissue Engineering.

Damage to osteochondral (OC) tissues can lead to pain, loss of motility, and progress to osteoarthritis. Tissue engineering approaches offer the possibility of replacing damaged tissues and restoring joint function; however, replicating the spatial and functional heterogeneity of native OC tissue remains a pressing challenge. Chondrocytes in healthy cartilage exist in relatively low-oxygen conditions, while osteoblasts in the underlying bone experience higher oxygen pressures. Such oxygen gradients also exist in the limb bud, where they influence OC tissue development. The cellular response to these spatial variations in oxygen pressure, which is mediated by the hypoxia inducible factor (HIF) pathway, plays a central role in regulating osteo- and chondrogenesis by directing progenitor cell differentiation and promoting and maintaining appropriate extracellular matrix production. Understanding the role of the HIF pathway in OC tissue development may enable new approaches to engineer OC tissue. In this review, we discuss strategies to spatially and temporarily regulate the HIF pathway in progenitor cells to create functional OC tissue for regenerative therapies. Impact statement Strategies to engineer osteochondral (OC) tissue are limited by the complex and varying microenvironmental conditions in native bone and cartilage. Indeed, native cartilage experiences low-oxygen conditions, while the underlying bone is relatively normoxic. The cellular response to these low-oxygen conditions, which is mediated through the hypoxia inducible factor (HIF) pathway, is known to promote and maintain the chondrocyte phenotype. By using tissue engineering scaffolds to spatially and temporally harness the HIF pathway, it may be possible to improve OC tissue engineering strategies for the regeneration of damaged cartilage and its underlying subchondral bone.

Electrospinning 3D bioactive glasses for wound healing.

An electrospinning technique was used to produce three-dimensional (3D) bioactive glass fibrous scaffolds, in the SiO2-CaO sol-gel system, for wound healing applications. Previously, it was thought that 3D cotton wool-like structures could only be produced from sol-gel when the sol contained calcium nitrate, implying that the Ca2+ and its electronic charge had a significant effect on the structure produced. Here, fibres with a 3D appearance were also electrospun from compositions containing only silica. A polymer binding agent was added to inorganic sol-gel solutions, enabling electrospinning prior to bioactive glass network formation and the polymer was removed by calcination. While the addition of Ca2+ contributes to the 3D morphology, here we show that other factors, such as relative humidity, play an important role in producing the 3D cotton-wool-like macrostructure of the fibres. A human dermal fibroblast cell line (CD-18CO) was exposed to dissolution products of the samples. Cell proliferation and metabolic activity tests were carried out and a VEGF ELISA showed a significant increase in VEGF production in cells exposed to the bioactive glass samples compared to control in DMEM. A novel SiO2-CaO nanofibrous scaffold was created that showed tailorable physical and dissolution properties, the control and composition of these release products are important for directing desirable wound healing interactions.

Impact of post mastectomy radiotherapy on the silicone breast implant.

Implant based reconstruction accounts for over half of breast reconstruction performed in the UK. Patients with implant based breast reconstructions undergoing post mastectomy radiotherapy are at increased risk of capsular contracture and reconstructive failure. This study sought to determine the effect of treatment dose radiotherapy on the bulk mechanical, surface chemical properties of silicone implants as well as their cellular response. Silicone breast implant shells were submitted to treatment dose radiotherapy, 2.67 Gy (one daily fraction) and 40.05 Gy (15 fractions) using non-irradiated shells as controls. Bulk mechanical and surface chemical properties of the shells were evaluated using tensile and tear testing, attenuated total reflectance - fourier transform infrared spectroscopy (ATR-FTIR), water contact angle measurements. HDFa cells were seeded on the shells and Alamar Blue assay was performed to study cell metabolic activity. Cell morphology was evaluated using phalloidin and DAPI staining. There was no significant difference in tensile, tear strength and Young's modulus however there was reduction in maximum elongation following irradiation. Irradiation of the shells did not significant alter spectroscopy measurements nor wettability of the shells. Cell metabolism was not significantly affected by irradiation. Further analysis is warranted of the micromechanical properties to fully elucidate the effect of irradiation on the breast implant which could explain the increased rate of capsular contracture and reconstructive failure in patients undergoing post-mastectomy radiotherapy.

Mechanical and surface chemical analysis of retrieved breast implants from a single centre.

INTRODUCTION: Breast implants are associated with complications such as capsular contracture, implant rupture and leakage often necessitating further corrective surgery. Re-operation rates have been reported to occur in up to 15.4% of primary augmentation patients and up to 27% in primary reconstructions patients within the first three years (Cunningham, 2007). The aim of this study was to examine the mechanical and surface chemical properties as well as the fibroblast response of retrieved breast implants in our unit to determine the in vivo changes which occur over time. METHODS: Ethical approval was obtained. 47 implants were retrieved. Implantation time ranged from 1 month to 388 months (Mean 106.1 months). Tensile strength, elongation, Young's modulus and tear strength properties were measured using Instron 5565 tensiometer on anterior and posterior aspects of the implant. Attenuated total reflectance-fourier transform infra-red spectroscopy (ATR-FTIR), wettability and scanning electron microscopy (SEM) analysis was performed on the shell surfaces. Bicinchoninic acid assay was performed to determine shell protein content. The fibroblast response was determined by seeding HDFa cells on the retrieved implants and cell metabolism measured using Alamar Blue™ assay. RESULTS: Mechanical properties fall with increasing duration of implantation. There were no significant changes in ATR-FTIR spectra between ruptured and intact implants nor significant changes in wettability in implants grouped into 5 year categories. SEM imaging reveals surface degradation changes with increasing duration of implantation. CONCLUSIONS: With increasing duration of implantation, mechanical properties of the breast implants fall. However this was not associated with surface chemical changes as determined by ATR-FTIR and wettability nor protein content of the shells. Thus the reduction in mechanical properties is associated with breast implant failure but further research is required to elucidate the mechanisms.

Protocol for a feasibility randomised controlled trial of targeted oxygen therapy in mechanically ventilated critically ill patients.

INTRODUCTION: Oxygen is the most commonly administered drug to mechanically ventilated critically ill adults, yet little is known about the optimum oxygen saturation (SpO2) target for these patients; the current standard of care is an SpO2 of 96% or above. Small pilot studies have demonstrated that permissive hypoxaemia (aiming for a lower SpO2 than normal by using a lower fractional inspired oxygen concentration (FIO2)) can be achieved in the critically ill and appears to be safe. This approach has not been evaluated in a National Health Service setting. It is possible that permissive hypoxaemia may be beneficial to critically ill patients thus it requires robust evaluation. METHODS AND ANALYSIS: Targeted OXygen therapY in Critical illness (TOXYC) is a feasibility randomised controlled trial (RCT) to evaluate whether recruiting patients to a study of permissive hypoxaemia is possible in the UK. It will also investigate biological mechanisms that may underlie the links between oxygenation and patient outcomes. Mechanically ventilated patients with respiratory failure will be recruited from critical care units at two sites and randomised (1:1 ratio) to an SpO2 target of either 88%-92% or ≥96% while intubated with an endotracheal tube. Clinical teams can adjust FIO2 and ventilator settings as they wish to achieve these targets. Clinical information will be collected before, during and after the intervention and blood samples taken to measure markers of systemic oxidative stress. The primary outcome of this study is feasibility, which will be assessed by recruitment rate, protocol adherence and withdrawal rates. Secondary outcomes will include a comparison of standard critical care outcome measures between the two intervention groups, and the measurement of biomarkers of systemic oxidative stress. The results will be used to calculate a sample size, likely number of sites and overall length of time required for a subsequent large multicentre RCT. ETHICS AND DISSEMINATION: This study was approved by the London - Harrow Research Ethics Committee on 2 November 2017 (REC Reference 17/LO/1334) and received HRA approval on 13 November 2017. Results from this study will be disseminated in peer-reviewed journals, at medical and scientific meetings, in the NIHR Journals Library and patient information websites. TRIAL REGISTRATION NUMBER: NCT03287466; Pre-results.

Hypoxia impacts human MSC response to substrate stiffness during chondrogenic differentiation.

Tissue engineering strategies often aim to direct tissue formation by mimicking conditions progenitor cells experience within native tissues. For example, to create cartilage in vitro, researchers often aim to replicate the biochemical and mechanical milieu cells experience during cartilage formation in the developing limb bud. This includes stimulating progenitors with TGF-ß1/3, culturing under hypoxic conditions, and regulating mechanosensory pathways using biomaterials that control substrate stiffness and/or cell shape. However, as progenitors differentiate down the chondrogenic lineage, the pathways that regulate their responses to mechanotransduction, hypoxia and TGF-ß may not act independently, but rather also impact one another, influencing overall cell response. Here, to better understand hypoxia's influence on mechanoregulatory-mediated chondrogenesis, we cultured human marrow stromal/mesenchymal stem cells (hMSC) on soft (0.167 kPa) or stiff (49.6 kPa) polyacrylamide hydrogels in chondrogenic medium containing TGF-ß3. We then compared cell morphology, phosphorylated myosin light chain 2 staining, and chondrogenic gene expression under normoxic and hypoxic conditions, in the presence and absence of pharmacological inhibition of cytoskeletal tension. We show that on soft compared to stiff substrates, hypoxia prompts hMSC to adopt more spread morphologies, assemble in compact mesenchymal condensation-like colonies, and upregulate NCAM expression, and that inhibition of cytoskeletal tension negates hypoxia-mediated upregulation of molecular markers of chondrogenesis, including COL2A1 and SOX9. Taken together, our findings support a role for hypoxia in regulating hMSC morphology, cytoskeletal tension and chondrogenesis, and that hypoxia's effects are modulated, at least in part, by mechanosensitive pathways. Our insights into how hypoxia impacts mechanoregulation of chondrogenesis in hMSC may improve strategies to develop tissue engineered cartilage. STATEMENT OF SIGNIFICANCE: Cartilage tissue engineering strategies often aim to drive progenitor cell differentiation by replicating the local environment of the native tissue, including by regulating oxygen concentration and mechanical stiffness. However, the pathways that regulate cellular responses to mechanotransduction and hypoxia may not act independently, but rather also impact one another. Here, we show that on soft, but not stiff surfaces, hypoxia impacts human MSC (hMSC) morphology and colony formation, and inhibition of cytoskeletal tension negates the hypoxia-mediated upregulation of molecular markers of chondrogenesis. These observations suggest that hypoxia's effects during hMSC chondrogenesis are modulated, at least in part, by mechanosensitive pathways, and may impact strategies to develop scaffolds for cartilage tissue engineering, as hypoxia's chondrogenic effects may be enhanced on soft materials.

Non-invasive analysis of cell cycle dynamics in single living cells with Raman micro-spectroscopy.

Raman micro-spectroscopy is a laser-based technique which enables rapid and non-invasive biochemical analysis of cells and tissues without the need for labels, markers or stains. Previous characterization of the mammalian cell cycle using Raman micro-spectroscopy involved the analysis of suspensions of viable cells and individual fixed and/or dried cells. Cell suspensions do not provide cell-specific information, and fixing/drying can introduce artefacts which distort Raman spectra, potentially obscuring both qualitative and quantitative analytical results. In this article, we present Raman spectral characterization of biochemical changes related to cell cycle dynamics within single living cells in vitro. Raman spectra of human osteosarcoma cells synchronized in G(0)/G(1), S, and G(2)/M phases of the cell cycle were obtained and multivariate statistics applied to analyze the changes in cell spectra as a function of cell cycle phase. Principal components analysis identified spectral differences between cells in different phases, indicating a decrease in relative cellular lipid contribution to Raman spectral signatures from G(0)/G(1) to G(2)/M, with a concurrent relative increase in signal from nucleic acids and proteins. Supervised linear discriminant analysis of spectra was used to classify cells according to cell cycle phase, and exhibited 97% discrimination between G(0)/G(1)-phase cells and G(2)/M-phase cells. The non-invasive analysis of live cell cycle dynamics with Raman micro-spectroscopy demonstrates the potential of this approach to monitoring biochemical cellular reactions and processes in live cells in the absence of fixatives or labels.