Design, development, and prototyping of a bike chain & gear mechanism cleaning product

Aim of this project is to design, develop, and prototype a new Chain & Gear Mechanism Guard for cleaning bicycles. Sentini Marine managers “Gareth Bransby and Jon Lee have contacted to the University of Huddersfield, Enterprise and Innovation Centre to find a design solution to their initial ideas to find a low cost manufacturing method for their bike chain cleaning product. They were introduced by the Product Design team after the initial meeting. Dr Ertu Unver met the Sentini team initially and agreed to carry out design and development work. Academic staff Robert Silkstone and David Swann also joined the team. A Santander Voucher was awarded to fund this research and development

T-Lymphocytes Enable Osteoblast Maturation via IL-17F during the Early Phase of Fracture Repair

While it is well known that the presence of lymphocytes and cytokines are important for fracture healing, the exact role of the various cytokines expressed by cells of the immune system on osteoblast biology remains unclear. To study the role of inflammatory cytokines in fracture repair, we studied tibial bone healing in wild-type and Rag1−/− mice. Histological analysis, µCT stereology, biomechanical testing, calcein staining and quantitative RNA gene expression studies were performed on healing tibial fractures. These data provide support for Rag1−/− mice as a model of impaired fracture healing compared to wild-type. Moreover, the pro-inflammatory cytokine, IL-17F, was found to be a key mediator in the cellular response of the immune system in osteogenesis. In vitro studies showed that IL-17F alone stimulated osteoblast maturation. We propose a model in which the Th17 subset of T-lymphocytes produces IL-17F to stimulate bone healing. This is a pivotal link in advancing our current understanding of the molecular and cellular basis of fracture healing, which in turn may aid in optimizing fracture management and in the treatment of impaired bone healing

Age Related Tissue Fibrosis During Fracture Repair Is Mediated by Wnt/β-catenin Signaling

The regenerative potential of tissue injury declines with age. Recently, a significant role for Wnt/β-catenin signaling has been shown in tissue specific stem cell aging, leading to increased tissue fibrosis. Wnt/β-catenin signaling regulates the differentiation of multipotent mesenchymal stem cells into osteoblasts during fracture repair. We investigated the potential role of dysregulated Wnt/β-catenin signaling in delayed fracture union and tissue fibrosis in the elderly. Old mice displayed increased total β-catenin protein levels at 4 and 7 days post-fracture and tissue fibrosis at 14 and 21 days post-fracture compared to young mice. Furthermore, treatment with a pharmalogical agent decreased total β-catenin protein levels in the fracture callus at 4 days post-fracture and prevented tissue fibrosis at 21 days post-fracture. Our data suggests that dysregulated Wnt/β-catenin signaling in the elderly contributes to delayed fracture repair and tissue fibrosis and offers a potential therapeutic strategy to improve fracture outcome in the elderly.MAS

Design, development, and prototyping of a bike chain & gear mechanism cleaning product.

Aim of this project is to design, develop, and prototype a new Chain & Gear Mechanism Guard for cleaning bicycles. Sentini Marine managers “Gareth Bransby and Jon Lee have contacted to the University of Huddersfield, Enterprise and Innovation Centre to find a design solution to their initial ideas to find a low cost manufacturing method for their bike chain cleaning product. They were introduced by the Product Design team after the initial meeting. Dr Ertu Unver met the Sentini team initially and agreed to carry out design and development work. Academic staff Robert Silkstone and David Swann also joined the team. A Santander Voucher was awarded to fund this research and development

Osteogenesis is reduced in <i>Rag1</i>

<p><i>⁻</i>^{<b><i>/</i></b><i>−</i>}<b>mice during fracture repair.</b> (<b>A</b>) <i>In vitro</i> cultures of primary mesenchymal stromal cells differentiated to osteoblast colony-forming units (CFU) showed lower gene expression levels of bone markers Col1, Col2 and BSP and osteocalcin in <i>Rag1^−/−</i> mice compared to WT. *p<0.05, **p<0.01 (n = 10). (<b>B</b>) Alizarin red staining of osteoblast CFU showed significant decrease in mineralization at 20 days of culture in <i>Rag1^−/−</i> compared to WT. CFU-osteoblast was quantified by direct counting of all stained nodules positive to Alizarin Red using light microscopy. (<b>C</b>) Digital fluorescent microscopy images of calcein green administered mice 2 and 9 days prior to harvest at 21 and 28 days post fracture exhibited a smaller distance measured between mineralization fronts and hence, less bone formation in <i>Rag1^−/−</i> compared to WT mice (p<0.05). Unfractured limbs showed no significant differences in bone formation.</p

IL-17F promotes osteoblast maturation.

<p>(<b>A</b>) Treatment of MC3T3-E1 pre-osteoblast cell line cultures directly with pro-inflammatory cytokine IL-17F showed increased bone marker gene expression of Col1, Col2, BSP and osteocalcin, whereas, anti-inflammatory cytokine TGFβ treatment inhibited osteoblast maturation and showed significant decreases in expression of the entire panel of bone markers analyzed. (<b>B</b>) Left: Treatment of <i>Rag1^−/−</i> mice primary mesenchymal stromal cell cultures directly with pro-inflammatory cytokine IL-17F showed increased bone marker gene expression of Col1, Col2, and Runx2. Right: In contrast, treatment with anti-inflammatory cytokine TGFβ inhibited WT mice primary mesenchymal stromal cell maturation and showed significant decreases in expression of all the bone markers analyzed. (*p<0.05 and **p<0.01).</p

Proposed mechanistic scheme of T-cell mediated osteoblast differentiation and maturation.

<p>Upstream IL-6 increase early post fracture promotes naïve CD4+ T-cells to Th17 cells stimulating pre-osteoblast cell differentiation via IL-17F. Concomitantly, the Treg pathway is suppressed, decreasing TGFβ and IL-10 and inhibiting pre-osteoblast differentiation. The effect of IL-6 also has a direct role in the later stages of osteoblast differentiation in fracture healing.</p

Healing of the fracture callus in <i>Rag1</i>

<p><i>⁻</i>^{<b><i>/</i></b><i>−</i>}<b>mice is delayed.</b> (<b>A</b>) Safranin O staining of WT and <i>Rag1^−/−</i> mice 28 days post fracture showed persistence of a cartilage template with more proteoglycan staining (black arrows) and less bridging bone across the fracture gap in <i>Rag1^−/−</i> compared to WT mice. (<b>B</b>) µCT analysis confirmed the presence of a wider, lower density callus in <i>Rag1^−/−</i> mice tibias at 28 days. (<b>C</b>) This was reflected by increased total callus volume (TV) measurements (p = 0.002) and decreased tissue mineral density (TMD) at 28 days in the <i>Rag1^−/−</i> compared to WT mice (p = 0.004). Torsional rigidity (TR) was significantly higher in <i>Rag1^−/−</i> mice compared to WT (p = 0.002). (<b>D</b>) Mechanical assessment of the samples using torsional mechanical testing showed a significantly higher ultimate torque and torsional stiffness in the <i>Rag1^−/−</i> mice at 28 days compared to WT (p = 0.002).</p

Cytokine expression during the early phase of fracture repair.

<p>(<b>A</b>) WT and <i>Rag1^−/−</i> mice serums drawn 2 days post-fracture show elevated IL-6 and G-CSF levels compared to unfractured baseline mice. However, WT mice were found to have twice the increase in cytokine levels of IL-6 and G-CSF compared to <i>Rag1^−/−</i> mice during this early phase of fracture healing (p<0.05). (<b>B</b>) Fracture callus RNA expression analysis of cytokine levels at a similar early time point post fracture, shows a greater than 2 fold up-regulation of pro-inflammatory cytokines IL-6, IL-17F and IL-23 in WT mice compared to <i>Rag1^−/−</i> and baseline mice (p<0.05). Conversely, expression of anti-inflammatory cytokine IL-10 is up-regulated in <i>Rag1^−/−</i> and TGFβ is down-regulated in WT mice. (<b>C</b>) Immunohistochemistry analysis during the early phase of fracture healing at 3 days post fracture confirmed IL-17F positive staining (black arrows) in WT mice similar to the distribution of CD3 staining shown previously with no positive IL-17F staining in <i>Rag1^−/−</i> mice (200X).</p

T-cell presence and increased bone marker expression are demonstrated in early fracture repair.

<p>(<b>A</b>) Although no cartilage or new bone formation is expected at 3 days post tibial fracture, low magnification (20X) Safranin O staining of WT mice at this time point helps to illustrate the region of interest (inset). (<b>B</b>) Higher magnification images corroborate the histology in this region and (<b>C</b>) positive CD3 staining of T-cells localizing to the endosteal fracture hematoma in contiguous sections (200X). Immunohistochemistry analysis confirms presence of T-cell infiltration (CD3 – black arrows) in WT mice. (<b>D</b>) In a similar region, no positive staining was seen in <i>Rag1^−/−</i> mice (200X). (<b>E</b>) T-cell presence is shown to correlate with gene expression of mature bone markers detected on quantitative RT-PCR normalized to GAPDH. Expression of Col1 (p<0.01), Col2 (p<0.05), BSP (p<0.01), BMP2 (p<0.01) and Runx2 (p<0.05) were increased in WT compared to <i>Rag1^−/−</i> mice at 3 days post fracture.</p