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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.

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.

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.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.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.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.These findings will help guide platelet-rich plasma preparation methods where therapeutic growth factors are used.Therapeutic, V.

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.

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.

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.

Post-mastectomy radiation therapy (PMRT) is known to increase the complication rate and implant loss in implant-based breast reconstruction. The purpose of this study was to systematically review the literature regarding the outcome of PMRT delivered to the permanent/definitive implant.Systematic review and meta-analysis of studies involving immediate implant-based reconstruction and PMRT when delivered to the permanent implant.Seven studies included 2921 patients (520 PMRT, 2401 control). PMRT was associated with significant increase in capsular contracture (7 studies, 2529 patients, 494 PMRT, 2035 control, OR 10.21, 95% CI 3.74 to 27.89, p 0.00001). In addition, PMRT was associated with a significant increase in revisional surgery (7 studies, 2921 patients, 520 PMRT, 2401 control, OR 2.18, 95% CI 1.33 to 3.57, p = 0.002) and reconstructive failure (6 studies, 2814 patients, 496 PMRT, 2318 control, OR 2.52, 95% CI 1.48 to 4.29, p+0.0007). Moreover, it was associated with a significant reduction in patient satisfaction (4 studies, 468 patients, 138 PMRT, 294 control, OR 0.29, 95% CI 0.15 to 0.57, p = 0.0003) and cosmetic outcome (4 studies, 1317 patients, 238 PMRT, 1009 control, OR 28, 95% CI. 0.11 to 0.67, p = 0.005).This meta-analysis demonstrates that within the first 5 years, post implant-based reconstruction for those patients who receive PMRT, the rates of adverse events are increased, and there is a significant reduction in patient satisfaction and cosmetic outcome.

Silicate ions released from bioactive glasses and ceramics have been reported to stimulate osteogenic cell functions. Here, we evaluated osteoblast-like cell reactions to silicate ions released from two different types of materials, 45S5 bioactive glass (BG) and siloxane-doped vaterite (SiV), to investigate the influence of the ionic structure of silicate ions on osteoblast-like cell properties. BG and SiV powders were prepared by using melt-quenching and carbonation methods, respectively. Aminopropyltriethoxysilane was used as a siloxane source of SiV. MC3T3-E1 and SaOS-2 cells were cultured in media containing dissolved BG or SiV ions (10–50 ppm of Si). Cell proliferation (metabolic activity), differentiation (alkaline phosphatase activity) and mineralisation (Ca deposition) were examined. 29Si NMR spectra demonstrated that Q0,1 species and T0–3 species were released from BG and SiV, respectively. Proliferation and mineralisation of the two types of cells were influenced by silicate ions released from BG and SiV in a concentration-dependent manner. In particular, there were significant differences (P < 0.05) in the degree of proliferation and Ca deposition levels in SaOS-2 cells treated with dissolved BG and SiV ions. Furthermore, Ca deposition in SaOS-2 cells was influenced by both the presence of silicate ions and the duration of exposure of cells to them. The structure of silicate ions influenced the proliferation and mineralisation of SaOS-2 cells incubated for different time periods in culture media containing different Si concentrations. Understanding the effect of Si on bone cell behaviour will enable a design-led approach to further BG optimisation.

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.

IntroductionOxygen 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 SpO2of 96% or above. Small pilot studies have demonstrated that permissive hypoxaemia (aiming for a lower SpO2than 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 analysisTargeted 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 SpO2target of either 88%–92% or ≥96% while intubated with an endotracheal tube. Clinical teams can adjust FIO2and 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 disseminationThis 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 numberNCT03287466; Pre-results.

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.

This is a protocol for a Cochrane Review (Intervention). The objectives are as follows: To assess the benefits and harms of antioxidant use in the perioperative period in adults who undergo non‐cardiac surgery.

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-β3-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-β 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.

Cell and tissue stiffness is an important biomechanical signalling parameter for dynamic biological processes; responsive polymeric materials conferring responsive functionality are therefore appealing for in vivo implants. We have developed thermoresponsive poly(urea-urethane) nanohybrid scaffolds with 'stiffness memory' through a versatile 3D printing-guided thermally induced phase separation (3D-TIPS) technique. 3D-TIPS, a combination of 3D printing with phase separation, allows uniform phase-separation and phase transition of the polymer solution at a large interface of network within the printed sacrificial preform, leading to the creation of full-scale scaffolds with bespoke anatomical complex geometry. A wide range of hyperelastic mechanical properties of the soft elastomer scaffolds with interconnected pores at multi-scale, controlled porosity and crystallinity have been manufactured, not previously achievable via direct printing techniques or phase-separation alone. Semi-crystalline polymeric reverse self-assembly to a ground-stated quasi-random nanophase structure, throughout a hierarchical structure of internal pores, contributes to gradual stiffness relaxation during in vitro cell culture with minimal changes to shape. This 'stiffness memory' provides initial mechanical support to surrounding tissues before gradually softening to a better mechanical match, raising hopes for personalized and biologically responsive soft tissue implants which promote human fibroblast cells growth as model and potential scaffold tissue integration. STATEMENT OF SIGNIFICANCE: Biological processes are dynamic in nature, however current medical implants are often stronger and stiffer than the surrounding tissue, with little adaptability in response to biological and physical stimuli. This work has contributed to the development of a range of thermoresponsive nanohybrid elastomer scaffolds, with tuneable stiffness and hierarchically interconnected porous structure, manufactured by a versatile indirect 3D printing technique. For the first time, stiffness memory of the scaffold was observed to be driven by phase transition and a reverse self-assembly from a semicrystalline phase to a quasi-random nanostructured rubber phase. Early insight into cell response during the stiffness relaxation of the scaffolds in vitro holds promise for personalized biologically responsive soft implants.

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.

Current commercially available nerve conduits fail to support nerve regeneration gaps larger than 30 mm in length due to the simple intra-luminal design of these conduits which are unable to biomimic the native neural environment. There is, therefore, a major clinical demand for new smart biomaterials, which can stimulate neuronal cell proliferation and migration, and facilitate nerve regeneration across these critical sized defects. In this study, we aimed to investigate Schwann cell (SC) behaviour seeded on the bioabsorbable version of the nanocomposite material, POSS modified poly (caprolactone) urea urethane (PCL), functionalised with arginine-glycine-aspartic acid (RGD) peptide. Successful synthesis of RGD peptide as well as the chemical structure of POSS-PCL nanocomposite film was investigated by Fourier transform infrared spectroscopy. Cell viability assay and morphological assessment were performed to investigate the cytocompatibility of the fabricated constructs. Successful immobilisation of RGD peptide onto the nanocomposite surface was confirmed by water contact angle, Brilliant Blue (BB) staining and thin layer chromatography. Both POSS-PCL and RGD-POSS-PCL nanocomposite scaffolds supported SC attachment, proliferation and morphological differentiation, important aspects for peripheral nerve regeneration. However, a significant increase in SC process length and morphological differentiation towards maturation was observed on the cells grown on RGD-POSS-PCL film. RGD-POSS-PCL nanocomposite demonstrated a significant improvement in SCs spreading and its integrin-dependent process outgrowth (P

Current biomaterials for auricular replacement are associated with high rates of infection and extrusion. The development of new auricular biomaterials that mimic the mechanical properties of native tissue and promote desirable cellular interactions may prevent implant failure. A porous 3D nanocomposite scaffold (NS) based on POSS-PCU (polyhedral oligomeric silsesquioxane nanocage into polycarbonate based urea-urethane) is developed with an elastic modulus similar to native ear. In vitro biological interactions on this NS reveal greater protein adsorption, increased fibroblast adhesion, proliferation, and collagen production compared with Medpor (the current synthetic auricular implant). In vivo, the POSS-PCU with larger pores (NS2; 150-250 μm) have greater tissue ingrowth (≈5.8× and ≈1.4 × increase) than the POSS-PCU with smaller pores (NS1; 100-50 μm) and when compared to Medpor (>100 μm). The NS2 with the larger pores demonstrates a reduced fibrotic encapsulation compared with NS1 and Medpor (≈4.1× and ≈1.6×, respectively; P < 0.05). Porosity also influences the amount of neovascularization within the implants, with no blood vessel observed in NS1 (12 weeks postimplantation). The lack of chronic inflammatory response for all materials may indicate that the elastic modulus and pore size of the implant scaffold could be important design considerations for influencing fibrotic responses to auricular and other soft tissue implants.

The higher patency rates of cardiovascular implants, including vascular bypass grafts, stents, and heart valves are related to their ability to inhibit thrombosis, intimal hyperplasia, and calcification. In native tissue, the endothelium plays a major role in inhibiting these processes. Various bioengineering research strategies thereby aspire to induce endothelialization of graft surfaces either prior to implantation or by accelerating in situ graft endothelialization. This article reviews potential bioresponsive molecular components that can be incorporated into (and/or released from) biomaterial surfaces to obtain accelerated in situ endothelialization of vascular grafts. These molecules could promote in situ endothelialization by the mobilization of endothelial progenitor cells (EPC) from the bone marrow, encouraging cell-specific adhesion (endothelial cells (EC) and/or EPC) to the graft and, once attached, by controlling the proliferation and differentiation of these cells. EC and EPC interactions with the extracellular matrix continue to be a principal source of inspiration for material biofunctionalization, and therefore, the latest developments in understanding these interactions will be discussed.

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.

Understanding the biochemical and biophysical properties of live cells is fundamental for unravelling the secrets of many diseases and developing new therapies. Raman micro-spectroscopy is a powerful non-invasive technique that allows in vitro studies of individual living cells or groups of cells without the use of any labels or contrast enhancing chemicals. We describe the use of various multivariate statistical methods, such as Principal Component Analysis (PCA), Linear Discriminant Analysis (LDA) and Classical Least Square (CLS) fitting, to extract biochemical information related to various cellular events. Such methods are required because of the high complexity of the Raman spectra obtained from living cells. PCA and LDA are used to discriminate between healthy and tumor cells. A leave-one-out cross-validation method indicated high prediction accuracy (95%) in identification of tumorogenic bone cells. The CLS fitting method using commercially available biopolymers makes it possible to monitor biochemical changes during the differentiation of embryonic stem cells and foetal bone cells. The results suggest that in both cases differentiated cells are characterised by lower concentrations of RNA compared to undifferentiated cells. These studies suggest that Raman micro-spectroscopy could become an invaluable tool for in vitro cellular biochemistry studies.

A new type of ceramic-polymer biomaterial having excellent apatite-forming ability in simulated body fluid (SBF) was prepared by hot-pressing a mixture consisting of poly(-L-lactic acid)(PLA) and calcium carbonate (vaterite). The composite containing 30% vaterite showed no brittle fracture behavior and comparably high bending strength of ~50 MPa. Apatite layer of several micrometers in thickness formed on its surface after soaking in SBF at 37°C only for 1 day. Osteoclast culture tests showed that the composite coated with apatite has excellent bioresorbability. After incubation of human osteoblasts, mineralized bone nodule formation was seen on the surface.

The Classical Least Square (CLS) fitting method was used to analyze the Raman spectra of living cells with the aim of identification of new phenotype-specific spectral markers for osteoblasts. The following chemicals were used for the CLS model: DNA, RNA, serum albumin, chymotrypsin and phosphatidyl choline. In this study we analyzed primary mature osteoblasts as well as two other cell types used as potential sources of osteoblasts: embryonic stem cells and fetal bone cells. The results obtained suggest that the Raman spectra of the cell types can be well approximated with a linear combination of the Raman spectra of the biopolymers used in the CLS model. The relative concentrations of the CLS components varied significantly between cell types, indicating that this analytical method could be used for phenotypic identification of osteoblasts.

Oxygen tension is a known regulator of mesenchymal stem cell (MSC) plasticity, differentiation, proliferation, and recruitment to sites of injury. Materials capable of affecting the MSC oxygen-sensing pathway, independently of the environmental oxygen pressure, are therefore of immense interest to the tissue engineering (TE) and regenerative medicine community. In this study, we describe the evaluation of the effect of hypoxia inducible factor (HIF)-stabilizing bioactive glasses (BGs) on human MSCs. The dissolution products from these hypoxia-mimicking BGs stabilized HIF-1α in a concentration-dependent manner, altered cell proliferation and metabolism, and upregulated a number of genes involved in the hypoxic response (HIF1A, HIF2A, and VHL), MSC survival (SAG and BCL2), extracellular matrix remodeling (MMP1), and angiogenesis (VEGF and PDGF). These HIF-stabilizing materials can therefore be used to improve MSC survival and enhance regeneration in a number of TE strategies.

One of the biggest challenges in regenerative medicine is promoting sufficient vascularisation of tissue-engineered constructs. One approach to overcome this challenge is to target the cellular hypoxia inducible factor (HIF-1α) pathway, which responds to low oxygen concentration (hypoxia) and results in the activation of numerous pro-angiogenic genes including vascular endothelial growth factor (VEGF). Cobalt ions are known to mimic hypoxia by artificially stabilising the HIF-1α transcription factor. Here, resorbable bioactive glass particles (38 μm and 100 μm) with cobalt ions incorporated into the glass network were used to create bioactive glass/collagen–glycosaminoglycan scaffolds optimised for bone tissue engineering. Inclusion of the bioactive glass improved the compressive modulus of the resulting composite scaffolds while maintaining high degrees of porosity (>97%). Moreover, in vitro analysis demonstrated that the incorporation of cobalt bioactive glass with a mean particle size of 100 μm significantly enhanced the production and expression of VEGF in endothelial cells, and cobalt bioactive glass/collagen–glycosaminoglycan scaffold conditioned media also promoted enhanced tubule formation. Furthermore, our results prove the ability of these scaffolds to support osteoblast cell proliferation and osteogenesis in all bioactive glass/collagen–glycosaminoglycan scaffolds irrespective of the particle size. In summary, we have developed a hypoxia-mimicking tissue-engineered scaffold with pro-angiogenic and pro-osteogenic capabilities that may encourage bone tissue regeneration and overcome the problem of inadequate vascularisation of grafts commonly seen in the field of tissue engineering.

Achieving complete functional recovery from peripheral nerve (PN) damage remains a major challenge for plastic surgeons. Nerve autografting is the current “gold standard” technique to bridge large diameter nerves with gap size greater than 30 mm. Advancements in the field of biomaterial science and tissue engineering have led to the development of synthetic nerve conduits. These conduits overcome the major limitations associated with autografting technique including donor site morbidity and lack of suitable graft material. The available Food and Drug Administration (FDA) and Conformite Europeenne (CE)-approved nerve conduits for PN repair are either natural based (collagen and decellularized allograft) or synthetic (polyester)-based conduits. These commercially available nerve conduits have shown promising results on repairing short defects. However, so far, no ideal biomaterial and design has been found to be a perfect substitution to nerve autografting for bridging large gaps (>30 mm). This chapter briefly reviews currently available surgical options for restoring nerve functionality. Furthermore, it will highlight the promising hopes held by tissue engineering to develop the next generation of nerve conduits. The ultimate goal is to determine potential alternatives for nerve regeneration which are simply applicable for clinical cases.

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 ± 0.17 MPa) to ear cartilage (5.02 ± 0.17 MPa) compared with Medpor®, which was much stiffer (140.9 ± 0.04 MPa). 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.

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 cellular response to hypoxia (low oxygen pressure) is vital for skeletal tissue development and regeneration. Numerous processes, including progenitor cell recruitment, differentiation and angiogenesis, are activated via the hypoxia pathway. Novel materials-based strategies designed to activate the hypoxia pathway are therefore of great interest for orthopaedic tissue engineering. Resorbable bioactive glasses (BGs) were developed to activate the hypoxia pathway by the controlled release of cobalt ions (at physiological relevant concentrations) whilst controlling BG apatite-forming ability. Two series of soda-lime–phosphosilicate glasses were synthesised with increasing concentrations of cobalt. Compositions were calculated to maintain constant network connectivity (2.13) by considering that cobalt is taking part in the network in the first series, and is acting as a network modifier in the second series. Mg2+ and Zn2+ were added to one of the Co2+-containing glasses to inhibit HCA formation. The presence of HCA formation is undesirable for the use of BG in soft tissues e.g. cartilage. Cobalt was present in both the silicate and phosphate phases of the BG. In addition, evidence was found that it plays a dual role in the silicate phase, entering the network as well as disrupting it as a network modifying oxide. Consistent with this dual role, the presence of cobalt in the BG was shown to decrease ion release. HCA formation was delayed with cobalt addition as well as incorporation of Mg2+ and Zn2+ into the BGs. Importantly, cobalt release was found to be proportional to cobalt content of the BGs enabling the controlled delivery of cobalt in therapeutically active doses.

Bioactive glasses (BG) which contain strontium have the potential to combine the known bone regenerative properties of BG with the anabolic and anti-catabolic effects of strontium cations. Here we created a BG series (SiO(2)-P(2)O(5)-Na(2)O-CaO) in which 0-100% of the calcium was substituted by strontium and tested their effects on osteoblasts and osteoclasts in vitro. We show that ions released from strontium-substituted BG enhance metabolic activity in osteoblasts. They also inhibit osteoclast activity by both reducing tartrate resistant acid phosphatase activity and inhibiting resorption of calcium phosphate films in a dose-dependent manner. Additionally, osteoblasts cultured in contact with BG show increased proliferation and alkaline phosphatase activity with increasing strontium substitution, while osteoclasts adopt typical resorption morphologies. These results suggest that similarly to the osteoporosis drug strontium ranelate, strontium-substituted BG may promote an anabolic effect on osteoblasts and an anti-catabolic effect on osteoclasts. These effects, when combined with the advantages of BG such as controlled ion release and delivery versatility, may make strontium-substituted BG an effective biomaterial choice for a range of bone regeneration therapies.

Raman spectroscopy can offer a non-invasive, information-rich biochemical “snapshot” of living human cells, tissues or material-cell tissue constructs rapidly(seconds–minutes), without the need of labels or contrast enhancers. This chapter details the exciting potential and challenges associated with the use of Raman spectroscopy in tissue engineering (TE). The use of Raman spectroscopy in three interlinked areas of TE will be considered: (1) the characterisation of the various scaffolds and “smart” materials, (2) the biochemical analysis of cellular behaviour important in TE (e.g. differentiation) and (3) the use of Raman spectroscopy for the analysis of tissue/extracellular matrix (ECM) formation in vitro or possibly in vivo.

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.

Engineering the surface of materials to promote cell adhesion and direct cellular behaviour is vital for the development of materials capable of restoring, replacing and/or enhancing tissue function. Cells are inherently sensitive to physical, biochemical and chemical stimuli from their surroundings. In vivo, the local cell environment or “niche” provides specific environmental cues that determine cell-specific recruitment, migration, proliferation, differentiation and the production of the numerous proteins needed for hierarchical tissue organisation. However, when cells are cultured in vitro or when materials are implanted into the body, cells encounter very different, unfamiliar surfaces and environments (Figs. 35.1 and 35.2). This chapter will introduce various approaches to modifying these unfamiliar material surfaces to promote desirable cell responses.

The remarkable intrinsic properties of carbon nanotubes, including their high mechanical strength, electrical conductivity, and nanoscale 3D architecture, create promising opportunities for the use of nanotube composites in a number of fields, particularly for composites in which conventional fillers cannot be accommodated. In the current study, 3D polyurethane (PU) nanocomposite foams were developed, and their potential biomedical applications were investigated. Multiwalled carbon nanotubes (CNTs) were synthesized by chemical vapor deposition and, following suitable chemical modification, uniformly distributed within the walls of PU foams produced by direct reaction. Although the loading fraction was too low to observe significant mechanical effects, CNT incorporation improved the wettability of the nanocomposite surfaces in a concentration-dependent manner, supporting the claim that the nanotubes are active at the pore surface. Studies of bone cell interactions with the nanocomposite foams revealed that increasing CNT loading fraction did not cause osteoblast cytotoxicity nor have any detrimental effects on osteoblast differentiation or mineralization. The application of "fixed" or embedded CNTs in nondegradable scaffolds is likely advantageous over "loose" or unattached CNTs from a toxicological point of view.

Here, we report on a rapid, noninvasive biophotonics system using Raman spectroscopy to detect real-time biochemical changes in foetal osteoblasts (FOBs) following exposure to 45S5 Bioglass® (BG)-conditioned media. Bio-Raman spectroscopy, combined with multivariate statistical analysis techniques (principal component analysis and least squares analysis), was able to noninvasively identify biochemical differences in FOBs cultured for different time periods and between FOBs exposed/or not to BG-conditioned media. Gene and protein expression studies were also performed for known markers of osteoblastic differentiation, namely, alkaline phosphatase, bone sialoprotein, and collagen type I. Quantitative RT-PCR confirmed upregulation of genes associated with osteoblast differentiation after exposure to BG-conditioned media. These results suggest that Raman spectroscopy can noninvasively detect biochemical changes in FOBs associated with differentiation. This technique could have important applications in the field of regenerative medicine by enabling rapid characterization of cell or organoid behavior on novel bioactive scaffolds without damage to either cell or biomaterial. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res 2008

Raman micro-spectroscopy combined with multivariate analysis was employed to monitor real-time biochemical changes induced in living cells in vitro following exposure to a pharmaceutical. The cancer drug etoposide (topoisomerase II inhibitor) was used to induce double-strand DNA breaks in human type II pneumocyte-like cells (A549 cell-line). Raman spectra of A549 cells exposed to 100 microM etoposide were collected and classical least squares (CLS) analysis used to determine the relative concentrations of the main cellular components. It was found that the concentrations of DNA and RNA significantly (P < 0.05) decreased, whilst the concentration of lipids significantly (P < 0.05) increased with increasing etoposide exposure time as compared to control untreated A549 cells. The concentration of DNA decreased by 27.5 and 87.0% after 24 and 48 h exposure to etoposide respectively. Principal components analysis (PCA) successfully discriminated between treated and untreated cells, with the main variance between treatment groups attributed to changes in DNA and lipid. DNA fragmentation was confirmed by Western blot analysis of apoptosis regulator protein p53 and cell metabolic activity determined by MTT assay. The over-expression of p53 protein in the etoposide treated cells indicated a significant level of DNA fragmentation and apoptosis. MTT tests confirmed that cellular metabolic activity decreased following exposure to etoposide by 29.4 and 61.2% after 24 and 48 h, respectively. Raman micro-spectroscopy may find applications in the toxicology screening of other drugs, chemicals and new biomaterials, with a range of cell types.

The lymphatic system plays an essential physiological role in homeostasis, interstitial fluid composition, and immunity while impaired lymphatic function has been implicated in a number of pathological conditions, including arthritis and delayed wound healing. This study investigated lymphatic capillary growth and lymphangiogenesis pathways in the bone-implant interface from patients with aseptically loosened prosthetic joints. The newly developed lymphatic specific marker, podoplanin, has enabled the first demonstration of lymphatic capillaries in peri-prosthetic tissues (60% of cases contained podoplanin positive vessels). The pro-lymphangiogenic factor (VEGF-C) and its receptor VEGFR-3 showed high level of expression in these tissues, (often in areas of high levels of wear debris). However despite the upregulation of the lymphangiogenesis pathway by a VEGF-C/VEGFR-3-mediated mechanism, there were relatively few podoplanin positive lymphatic vessels in the bone-implant interface (3.4% of total vessels). This may have important pathological consequences in terms of perpetuating inflammation and edema by inhibiting the removal of macromolecules, cells, and interstitial fluid. The identification of lymphatic vessels with internalized polyethylene wear particles provides evidence of this route of wear debris transportation to distal sites. This paper highlights the importance of lymphatic vessels in the maintenance of local and distal inflammatory responses to prosthetic wear particles.

Publisher Summary This chapter presents that many of the advancements in understanding cell biology have arisen from cell isolation and in vitro studies. However, caution must be extended when comparing in vitro models to the multi-factorial and multi-cellular in vivo environment. Cells invariably behave differently in vivo due to the presence of other cell types, numerous cell signaling factors, and the extracellular matrix and physiological differences in terms of mechanical stress, blood flow, and 3-D growth. Cell type also has to be carefully selected for in vitro experiments to relate to the in vivo environment. For example, due to the ease of isolation, the vast majority of cell culture studies on endothelial cells have been performed on endothelial cells isolated from macro-vessels (large vessels), in human foreskin. However, it has now been shown that endothelial cell phenotypic expression and behavior alters both between the type of vessel (blood, vein or lymphatic vessel), size of vessel (capillary, arteriole or artery) and also the location of the vessels (skin, brain, etc). Whether these differences in endothelial cell behavior are induced by factors produced in the local environment and/or due to different endothelial cell lineages is uncertain, but clearly the selection of endothelial cell-type has important consequences for the success of tissue-engineered tissues and biomaterial research.

Bioactive glasses have been shown to regulate gene expression in both hard and soft tissue repair. New resorbable bioactive glass constructs are now being developed that can influence gene expression in the local environment by manipulating material properties such as the surface chemistry, topography and the release of dissolution ions. The success of these scaffolds, however, may depend upon a greater understanding of the bioactive glass stimulated gene expression pathways. This will allow the construction of tissue specific scaffolds with tailored surface chemistry, topography and ion release rates. This paper summarises the advances made in understanding gene expression in response to bioactive glasses and discusses the future steps required for further insights into these molecular mechanisms.