An in vitro Study to Assess the Potential of a Unique Micro porous Algal Derived Cap Bone Void Filler in Comparison with Clinically-Used Bone Void Fillers

Macroporosity(>100µm) in bone void fillers is a known prerequisite for tissue regeneration, but recent literature has highlighted the added benefit of microporosity(0.5 - 10µm). The aim of this study was to compare the in vitro performances of a novel interconnective microporous hydroxyapatite (HA) derived from red algae to four clinically available macroporous calcium phosphate (CaP) bone void fillers. The use of algae as a starting material for this novel void filler overcomes the issue of sustainability, which overshadows continued use of scleractinian coral in the production of some commercially available materials, namely Pro-OsteonTM and Bio-Coral®. This study investigated the physicochemical properties of each bone voidfiller material using x-ray diffraction, fourier transform infrared spectroscopy, inductive coupled plasma, and nitrogen gas absorption and mercury porosimetry. Biochemical analysis, XTT, picogreen and alkaline phosphatase assays were used to evaluate the biological performances of the five materials. Results showed that algal HA is non-toxic to human foetal osteoblast (hFOB) cells and supports cell proliferation and differentiation. The preliminary in vitro testing of microporous algal-HA suggests that it is comparable to the four clinically approved macroporous bone void fillers tested. The results demonstrate that microporous algal HA has good potential for use in vivo and in new tissue engineered strategies for hard tissue repair.

MicroRNA-199b modulates vascular cell fate during iPS cell differentiation by targeting the notch ligand jagged1 and enhancing VEGF signaling

Aims: Recent ability to derive endothelial cells (ECs) from induced pluripotent stem (iPS) cells holds a great therapeutic potential for personalised medicine and stem cell therapy. We aimed that better understanding of the complex molecular signals that are evoked during iPS cell differentiation towards ECs may allow specific targeting of their activities to enhance cell differentiation and promote tissue regeneration.

Methods and Results: In this study we have generated mouse iPS cells from fibroblasts using established protocol. When iPS cells were cultivated on type IV mouse collagen-coated dishes in differentiation medium, cell differentiation toward vascular lineages were observed. To study the molecular mechanisms of iPS cell differentiation, we found that miR-199b is involved in EC differentiation. A step-wise increase in expression of miR-199 was detected during EC differentiation. Notably, miR-199b targeted the Notch ligand JAG1, resulting in VEGF transcriptional activation and secretion through the transcription factor STAT3. Upon shRNA-mediated knockdown of the Notch ligand JAG1, the regulatory effect of miR-199b was ablated and there was robust induction of STAT3 and VEGF during EC differentiation. Knockdown of JAG1 also inhibited miR-199b-mediated inhibition of iPS cell differentiation towards SMCs. Using the in vitro tube formation assay and implanted Matrigel plugs, in vivo, miR-199b also regulated VEGF expression and angiogenesis.

Conclusions: This study indicates a novel role for miR-199b as a regulator of the phenotypic switch during vascular cell differentiation derived from iPS cells by regulating critical signaling angiogenic responses.

Thermo-Mechanical Properties of Poly ε-Caprolactone/Poly L-Lactic Acid Blends: Addition of Nalidixic Acid and Polyethylene Glycol Additives Journal of the Mechanical Behavior of Biomedical Materials

The search for ideal biomaterials is still on-going for tissue regeneration. In this study, blends of Poly ε-caprolactone (PCL) with Poly l-lactic acid (PLLA), Nalidixic Acid (NA) and Polyethylene glycol (PEG) were prepared. Mechanical and thermal properties of the blends were investigated by tensile and flexural analysis, DSC, TGA, WXRD, MFI, BET, SEM and hot stage optical microscopy. Results showed that the loading of PLLA caused a significant decrease in tensile strength and almost total eradication of the elongation at break of PCL matrix, especially after PEG and NA addition. Increased stiffness was also noted with additional NA, PEG and PLLA, resulting in an increase in the flexural modulus of the blends.
Isothermal degradation indicated that bulk PCL, PLLA and the blends were thermally stable at 200°C for the duration of 2h making extrusion of the blends at this temperature viable. Morphological study showed that increasing the PLLA content and addition of the very low viscosity PEG and powder NA decreased the Melt Flow Indexer and increased the viscosity.
At the higher temperature the PLLA begins to soften and eventually melts allowing for increased flow and, coupling this with, the natural increase in MFI caused by temperature is enhanced further. The PEG and NA addition increased dramatically the pore volume which is important for cell growth and flow transport of nutrients and metabolic waste.

MicroRNA-199a induces differentiation of induced pluripotent stem cells into endothelial cells by targeting sirtuin 1

The ability to reprogram induced pluripotent stem (iPS) cells from somatic cells may facilitate significant advances in regenerative medicine. MicroRNAs (miRNAs) are involved in a number of core biological processes, including cardiogenesis, hematopoietic lineage differentiation and oncogenesis. An improved understanding of the complex molecular signals that are required for the differentiation of iPS cells into endothelial cells (ECs) may allow specific targeting of their activity in order to enhance cell differentiation and promote tissue regeneration. The present study reports that miR‑199a is involved in EC differentiation from iPS cells. Augmented expression of miR‑199a was detected during EC differentiation, and reached higher levels during the later stages of this process. Furthermore, miR‑199a inhibited the differentiation of iPS cells into smooth muscle cells. Notably, sirtuin 1 was identified as a target of miR‑199a . Finally, the ability of miR‑199a to induce angiogenesis was evaluated in vitro, using Matrigel plugs assays. This may indicate a novel function for miR‑199a as a regulator of the phenotypic switch during vascular cell differentiation. The present study provides support to the notion that with an understanding of the molecular mechanisms underlying vascular cell differentiation, stem cell regenerative therapy may ultimately be developed as an effective treatment for cardiovascular disease.

ADP-Ribose-1"-Monophosphatase: a Conserved Coronavirus Enzyme That Is Dispensable for Viral Replication in Tissue Culture

Replication of the ~30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.

Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration

Distinct cell populations with regenerative capacity have been reported to contribute to myofibres after skeletal muscle injury, including non-satellite cells as well as myogenic satellite cells. However, the relative contribution of these distinct cell types to skeletal muscle repair and homeostasis and the identity of adult muscle stem cells remain unknown. We generated a model for the conditional depletion of satellite cells by expressing a human diphtheria toxin receptor under control of the murine Pax7 locus. Intramuscular injection of diphtheria toxin during muscle homeostasis, or combined with muscle injury caused by myotoxins or exercise, led to a marked loss of muscle tissue and failure to regenerate skeletal muscle. Moreover, the muscle tissue became infiltrated by inflammatory cells and adipocytes. This localised loss of satellite cells was not compensated for endogenously by other cell types, but muscle regeneration was rescued after transplantation of adult Pax7(+) satellite cells alone. These findings indicate that other cell types with regenerative potential depend on the presence of the satellite cell population, and these observations have important implications for myopathic conditions and stem cell-based therapeutic approaches.

Therapeutic revascularisation of ischaemic tissue. The opportunities and challenges for therapy using vascular stem/ progenitor cells

Ischaemia-related diseases such as peripheral artery disease and coronary heart disease constitute a major issue in medicine as they affect millions of individuals each year and represent a considerable economic burden to healthcare systems. If the underlying ischaemia is not sufficiently resolved it can lead to tissue damage, with subsequent cell death. Treating such diseases remains difficult and several strategies have been used to stimulate the growth of blood vessels and promote regeneration of ischaemic tissues, such as the use of recombinant proteins and gene therapy. Although these approaches remain promising, they have limitations and results from clinical trials using these methods have had limited success. Recently, there has been growing interest in the therapeutic potential of using a cell-based approach to treat vasodegenerative disorders. In vascular medicine, various stem cells and adult progenitors have been highlighted as having a vasoreparative role in ischaemic tissues. This review will examine the clinical potential of several stem and progenitor cells that may be utilised to regenerate defunct or damaged vasculature and restore blood flow to the ischaemic tissue. In particular, we focus on the therapeutic potential of endothelial progenitor cells as an exciting new option for the treatment of ischaemic diseases. © 2012 BioMed Central Ltd

Physical activity and the rejuvenation of Connswater (PARC study): protocol for a natural experiment investigating the impact of urban regeneration on public health

Background: There is a dearth of evidence regarding the impact of urban regeneration projects on public health, particularly the nature and degree to which urban regeneration impacts upon health-related behaviour change. Natural experiment methodology enables comprehensive large-scale evaluations of such interventions. The Connswater Community Greenway in Belfast is a major urban regeneration project involving the development of a 9 km linear park, including the provision of new cycle paths and walkways. In addition to the environmental improvements, this complex intervention involves a number of programmes to promote physical activity in the regenerated area. The project affords a unique opportunity to investigate the public health impact of urban regeneration.

Methods/Design: The evaluation framework was informed by the socio-ecological model and guided by the RE-AIM Framework. Key components include: (1) a quasi-experimental before-and-after survey of the Greenway population (repeated cross-sectional design), in tandem with data from a parallel Northern Ireland-wide survey for comparison; (2) an assessment of changes in the local built environment and of walkability using geographic information systems; (3) semi-structured interviews with a purposive sample of survey respondents, and a range of community stakeholders, before and after the regeneration project; and (4) a cost-effectiveness analysis. The primary outcome is change in proportion of individuals identified as being regularly physically active, according to the current UK recommendations. The RE-AIM Framework will be used to make an overall assessment of the impact of the Greenway on the physical activity behaviour of local residents.

Discussion: The Connswater Community Greenway provides a significant opportunity to achieve long-term, population level behaviour change. We argue that urban regeneration may be conceptualised meaningfully as a complex intervention comprising multiple components with the potential, individually and interactively, to affect the behaviour of a diverse population. The development and implementation of our comprehensive evaluation framework reflects this complexity and illuminates an approach to the empirical, rigorous evaluation of urban regeneration. More specifically, this study will add to the much needed evidence-base about the impact of urban regeneration on public health as well as having important implications for the development of natural experiment methodology.

Marine organisms for bone repair and regeneration

Bioprospecting has led to increased interest in potential applications for marine organisms and their by-products. As a rich source of mineralising porous organisms, our seas and oceans could provide new directions for bone tissue engineering particularly in the supply of biomimetic templates that may enhance in vivo and ex vivo bone formation. In this chapter we examine the history of marine organism use in this field; exploring how these organisms could be utilised, given the problems of sustainability, and reviewing the current evidence to support their use for bone repair and regeneration.

Modelling responses to spatially fractionated radiation fields using preclinical image-guided radiotherapy

OBJECTIVES: Radiotherapy is planned to achieve the optimal physical dose distribution to the target tumour volume whilst minimising dose to the surrounding normal tissue. Recent in vitro experimental evidence has demonstrated an important role for intercellular communication in radiobiological responses following non-uniform exposures. This study aimed to model the impact of these effects in the context of techniques involving highly modulated radiation fields or spatially fractionated treatments such as GRID therapy.

METHODS: Using the small animal radiotherapy research platform (SARRP) as a key enabling technology to deliver precision imaged-guided radiotherapy, it is possible to achieve spatially modulated dose distributions that model typical clinical scenarios. In this work, we planned uniform and spatially fractionated dose distributions using multiple isocentres with beam sizes of 0.5 - 5 mm to obtain 50% volume coverage in a subcutaneous murine tumour model, and applied a model of cellular response that incorporates intercellular communication to assess the potential impact of signalling effects with different ranges.

RESULTS: Models of GRID treatment plans which incorporate intercellular signalling showed increased cell killing within the low dose region. This results in an increase in the Equivalent Uniform Dose (EUD) for GRID exposures compared to standard models, with some GRID exposures being predicted to be more effective than uniform delivery of the same physical dose.

CONCLUSIONS: This study demonstrates the potential impact of radiation induced signalling on tumour cell response for spatially fractionated therapies and identifies key experiments to validate this model and quantify these effects in vivo.

ADVANCES IN KNOWLEDGE: This study highlights the unique opportunities now possible using advanced preclinical techniques to develop a foundation for biophysical optimisation in radiotherapy treatment planning.

Fiducial Marker Guided Prostate Radiotherapy: A Review

Image guided radiotherapy (IGRT) is an essential tool in the accurate delivery of modern radiotherapy techniques. Prostate radiotherapy positioned using skin marks or bony anatomy may be adequate for delivering a relatively homogenous whole pelvic radiotherapy dose but these are not reliable when using reduced margins, dose escalation or hypo-fractionated stereotactic radiotherapy. Fiducial markers (FMs) for prostate IGRT have been in use since the 1990's. They require surgical implantation and provide a surrogate for the position of the prostate gland. A variety of FMs are available and they can be used in a number of ways. This review aims to establish the evidence for using prostate FMs in terms of feasibility, implantation procedures, types of FMs used, FM migration, imaging modalities used and the clinical impact of FMs. A search strategy was defined and a literature search was carried out in Medline. Inclusion and exclusion criteria were applied which resulted in 50 papers being included in this review. The evidence demonstrates that FMs provide a more accurate surrogate for the position of the prostate than either external skin marks or bony anatomy. A combination of FM alignment and soft tissue analysis is currently the most effective and widely available approach to ensuring accuracy in prostate IGRT. FM implantation is safe and well tolerated. FM migration is possible but minimal. Standardisation of all techniques and procedures in relation to the use of prostate FMs is required. Finally a clinical trial investigating a non-surgical alternative to prostate FMS is introduced.