Natural functionally-graded composites in hard-to-soft tissue (bone- tendon) junctions

Composite materials are often functionally engineered to imbue desired mechanical properties in materials for structural applications. Nature has long engaged in such composite engineering of biological organisms, which has evolved in both flora and fauna in response to specific mechanical demands. Incorporation of phenolic compounds (like lignin) in stiffening cell assemblies in plant basts, or of silica in plant leaves to resist chomping insect incursions, are good examples in the plant world. Skeletal bone in vertebrates is the classic example in the animal kingdom, a composite of flexible fibrous polymerized organic protein and platy-crystalline inorganic mineral that results in a mechanically strong, hard, tough tissue. The musculo-skeletal system of vertebrates in fact comprises a variety of both hard and soft tissue types (bone, cartilage, tendon, ligament), generative cell types (osteoblasts, chondrocytes, tenocytes, fibroblasts, all of which can derive from multipotent mesenchymal stem cell precursors), and fibrous connective-tissue proteins (chiefly collagen, types I and II) that are susceptible to varying degrees of mineralization. In the case of bone, mineralization is extensive and forms a bi-continuous composite of mineral (chiefly partially-carbonated hydroxyapatite [Ca10(PO4,CO3)6(OH)2] and precursors) and collagen (a triple a-helix polypeptide) that self-assembles into protein fibrils (mostly type I collagen). Bone continually remodels itself and also re-forms as a consequence of injury or around implanted prostheses (such as knee and hip prostheses). High-resolution analytical TEM reveals [1] a mineralization mechanism which entails initial creation, at the mitochondria of bone-forming cells (osteoblasts), of pre-packaged vesicles that fill with a calcium-phosphate hydrogel and thereafter migrate through the cell wall. The vesicle contents subsequently crystallize [2] in the extra-cellular space with the dissolution of the vesicle containment wall, shortly before self-assembling collagen is expressed from the osteoblasts, providing a “just-in-time” ready source of Ca and P for mineralization of collagen fibrils with close to (though not identical with) the Ca/P ratio of hydroxyapatite found in the mature bone composite. The critical connective junctions between different tissue types in the musculo-skeletal system (bone, cartilage, tendon, muscle, ligament) involve several hard-tissue/soft-tissue interfaces, characterized by gradients in mineralization, cell type, cell morphology, and collagen self-assembly modes. For example, standard procedure for re-attachment of ruptured tendons—by surgically re-locating the tendon proximally to bone—re-establishes the important bone-tendon junction (enthesis) in a period of about one year. The process proceeds through growth, contiguous to the (fully mineralized) bone surface, of a partially-mineralized fibrocartilage layer (comprising collagen, expressed by chondrocyte cells, that self-assembles into principally Type II and Type X collagens). TEM [3] of ovine models shows that mineralization of this cartilaginous layer appears to occur via the identical mechanism established [1,2] for bone mineralization but initiated instead by chondrocyte cells. SEM [3] reveals that the cell-type in the remaining unmineralized cartilage portion gradually morphs into tenocytes, which form more elastic tendon fibers comprising, again, mostly Type I collagen (but also Types III, IV, V and IX self-assembly motifs). The resulting hard-tissue/soft-tissue enthesis junction is thus seen [3] to be a multiply graded interface involving three different cell types, several different collagen self-assembly motifs, and the functional gradation of a composite material paradigm spanning fully-hard tissue (bone) to fully-soft tissue (tendon). [1] S. Boonrungsiman, E. Gentleman, R. Carzaniga, N.D. Evans, D.W. McComb, A. E. Porter and M.M. Stevens, PNAS 109 (2012) 141. [2] V. Benezra, L. W. Hobbs and M. Spector, Biomaterials 23 (2001) 725; A. E. Porter, L. W. Hobbs, V. Benezra and M. Spector, Biomaterials 23 (2001) 921. [3] L. W. Hobbs, H. Wang, W. M. Reese, B. M. Tomerline, T. Y. C. Lim, A. E. Porter, M. Walton and M. J. Cotton, Microscopy & Microanalysis 19 (2013) 182

Small scale fracture of bone to understand the effect of fibrillar organization on toughness

Fracture toughness is a critical component of bone quality and derives from the hierarchical arrangement of collagen and mineral from the molecular level to the whole bone level. Molecular defects, disease, and age affect bone toughness, yet there is currently no treatment to address deficits in toughness. Toughening mechanisms occur at every length scale, making it difficult to isolate the influence of specific components. Most experimental studies on the fracture behaviour of bone use milled samples of bone or whole bones. Toughness deficits can be identified but may be caused by a multitude of parameters across length-scales, making it difficult to develop targeted therapies. Herein, we measure the toughness of bone in micropillars where porosity and heterogeneities are minimized, allowing us to determine the role of fibril anisotropy on fracture toughness. Double cantilever beam micromechanical tests were conducted in a scanning electron microscope on 4x6x15 mm pillars of mouse bone femorae produced in the longitudinal and transverse orientations. Subsequent transmission electron microscopy of the fractured pillars revealed a role of the local organization of the mineralized collagen fibrils in influencing crack propagation. We demonstrate that fibril orientation is a critical factor in deflection during crack propagation, significantly contributing to fracture toughness

Identifying Spatial Variability in Greenland’s Outlet Glacier Response to Ocean Heat

Although the Greenland ice sheet is losing mass as a whole, patterns of change on both local and regional scales are complex. Spatial statistics reveal large spatial variability of dynamic thinning rates of Greenland’s marine-terminating glaciers between 2003 and 2009; only 18% of glacier thinning rates co-vary with neighboring glaciers. Most spatially-correlated thinning rates are clusters of stable glaciers in the Thule, Scoresby Sund, and Southwest regions. Conversely, where spatial-autocorrelation is low, individual glaciers are more strongly controlled by local, glacier-scale features than by regional influences. We investigate possible sources of local control of oceanic forcing by combining grounding line depths and ocean model output to estimate mean ocean heat content adjacent to 74 glaciers. Linear regression models indicate stronger correlation of dynamic thinning rates with ocean heat content compared to those with grounding line depths alone. The correlation between ocean heat and dynamic thinning is robust for all of Greenland except glaciers in the West, and strongest in the Southeast (R2 ∼ 0.81 ± 0.15, ρ = 0.009), implying that glaciers with deeper grounded termini here are most sensitive to changes in ocean forcing. In the Northwest, accounting for shallow sills in the regressions improves the correlation of water depth with glacial thinning, highlighting the need for comprehensive knowledge of fjord geometry

Tissue-specific calibration of extracellular matrix material properties by transforming growth factor-beta and Runx2 in bone is required for hearing

Publisher version: http://www.nature.com/embor/journal/v11/n10/full/embor2010135.htmlDA - 20100917 IS - 1469-3178 (Electronic) IS - 1469-221X (Linking) LA - ENG PT - JOURNAL ARTICLEDA - 20100917 IS - 1469-3178 (Electronic) IS - 1469-221X (Linking) LA - ENG PT - JOURNAL ARTICLEDA - 20100917 IS - 1469-3178 (Electronic) IS - 1469-221X (Linking) LA - ENG PT - JOURNAL ARTICLEPhysical cues, such as extracellular matrix stiffness, direct cell differentiation and support tissue-specific function. Perturbation of these cues underlies diverse pathologies, including osteoarthritis, cardiovascular disease and cancer. However, the molecular mechanisms that establish tissue-specific material properties and link them to healthy tissue function are unknown. We show that Runx2, a key lineage-specific transcription factor, regulates the material properties of bone matrix through the same transforming growth factor-beta (TGFbeta)-responsive pathway that controls osteoblast differentiation. Deregulated TGFbeta or Runx2 function compromises the distinctly hard cochlear bone matrix and causes hearing loss, as seen in human cleidocranial dysplasia. In Runx2(+/-) mice, inhibition of TGFbeta signalling rescues both the material properties of the defective matrix, and hearing. This study elucidates the unknown cause of hearing loss in cleidocranial dysplasia, and demonstrates that a molecular pathway controlling cell differentiation also defines material properties of extracellular matrix. Furthermore, our results suggest that the careful regulation of these properties is essential for healthy tissue functio

Gold nanorod reshaping in vitro and in vivo using a continuous wave laser

Funding for this project was provided by ERC grant 242991 (D. Elson), and by Cancer Research UK via the CRUK Cancer Imaging Centre at the Institute of Cancer Research (ICR) to J. Bamber. We acknowledge an ERC starting grant (project number 257182) to A. Porter, and BRC funding (project number P46143) to A. Porter, D. Elson and P. Ruenraroengsak. We acknowledge NHS funding to the NIHR Biomedical Research Centre at The Royal Marsden (J. Bamber) and at Imperial College London, as well as support provided by the Cancer Research UK Imperial Centre.Gold nanorods (GNRs) are increasingly being investigated for cancer theranostics as they possess features which lend themselves in equal measures as contrast agents and catalysts for photothermal therapy. Their optical absorption spectral peak wavelength is determined by their size and shape. Photothermal therapy using GNRs is typically established using near infrared light as this allows sufficient penetration into the tumour matrix. Continuous wave (CW) lasers are the most commonly applied source of near infrared irradiation on GNRs for tumour photothermal therapy. It is perceived that large tumours may require fractionated or prolonged irradiation. However the true efficacy of repeated or protracted CW irradiation on tumour sites using the original sample of GNRs remains unclear. In this study spectroscopy and transmission electron microscopy are used to demonstrate that GNRs reshape both in vitro and in vivo after CW irradiation, which reduces their absorption efficiency. These changes were sustained throughout and beyond the initial period of irradiation, resulting from a spectral blue-shift and a considerable diminution in the absorption peak of GNRs. Solid subcutaneous tumours in immunodeficient BALB/c mice were subjected to GNRs and analysed with electron microscopy pre- and post-CW laser irradiation. This phenomenon of thermally induced GNR reshaping can occur at relatively low bulk temperatures, well below the bulk melting point of gold. Photoacoustic monitoring of GNR reshaping is also evaluated as a potential clinical aid to determine GNR absorption and reshaping during photothermal therapy. Aggregation of particles was coincidentally observed following CW irradiation, which would further diminish the subsequent optical absorption capacity of irradiated GNRs. It is thus established that sequential or prolonged applications of CW laser will not confer any additional photothermal effect on tumours due to significant attenuations in the peak optical absorption properties of GNRs following primary laser irradiation.Publisher PDFPeer reviewe

High resolution and dynamic imaging of biopersistence and bioreactivity of extra and intracellular MWNTs exposed to microglial cells

Multi-walled carbon nanotubes (MWNTs) are increasingly being developed both as neuro-therapeutic drug delivery systems to the brain and as neural scaffolds to drive tissue regeneration across lesion sites. MWNTs with different degrees of acid oxidation may have different bioreactivities and propensities to aggregate in the extracellular environment, and both individualised and aggregated MWNTs may be expected to be found in the brain. Before practical application, it is vital to understand how both aggregates and individual MWNTs will interact with local phagocytic immune cells, the microglia, and ultimately to determine their biopersistence in the brain. The processing of extra- and intracellular MWNTs (both pristine and when acid oxidised) by microglia was characterised across multiple length scales by correlating a range of dynamic, quantitative and multi-scale techniques, including: UV-vis spectroscopy, light microscopy, focussed ion beam scanning electron microscopy and transmission electron microscopy. Dynamic, live cell imaging revealed the ability of microglia to break apart and internalise micron-sized extracellular agglomerates of acid oxidised MWNTs, but not pristine MWNTs. The total amount of MWNTs internalised by, or strongly bound to, microglia was quantified as a function of time. Neither the significant uptake of oxidised MWNTs, nor the incomplete uptake of pristine MWNTs affected microglial viability, pro-inflammatory cytokine release or nitric oxide production. However, after 24 h exposure to pristine MWNTs, a significant increase in the production of reactive oxygen species was observed. Small aggregates and individualised oxidised MWNTs were present in the cytoplasm and vesicles, including within multilaminar bodies, after 72 h. Some evidence of morphological damage to oxidised MWNT structure was observed including highly disordered graphitic structures, suggesting possible biodegradation. This work demonstrates the utility of dynamic, quantitative and multi-scale techniques in understanding the different cellular processing routes of functionalised nanomaterials. This correlative approach has wide implications for assessing the biopersistence of MWNT aggregates elsewhere in the body, in particular their interaction with macrophages in the lung

Reply to Comment on Conopeptide-Functionalized Nanoparticles Selectively Antagonize Extrasynaptic N-Methyl-d-aspartate Receptors and Protect Hippocampal Neurons from Excitotoxicity In Vitro

In this manuscript, we provide precise answers to the concerns expressed by Molokanova et al. in their comment. In our reply, we highlight that there is indeed substantial agreement between our study and the one reported in Nano Letters by the Molokanova’s group.1 We believe this is a very important aspect because it proves the validity of the chosen approach, i.e. PEGylated AuNPs carrying NMDAR antagonists and with an overall dimension large enough to prevent their diffusion into the synapse can exclusively antagonize extrasynaptic NMDAR-mediated currents and are thereby neuroprotective