Multiscale relationships between fibronectin structure and functional properties

Cell behavior is tightly coupled to the properties of the extracellular matrix (ECM) to which they attach. Fibronectin (Fn) forms a supermolecular, fibrillar component of the ECM that is prominent during development, wound healing and the progression of numerous diseases. This indicates that Fn has an important function in controlling cell behavior during dynamic events in vivo. The multiscale architecture of Fn molecules assembled into these fibers determines the ligand density of cell adhesion sites on the surface of the Fn fiber, Fn fiber porosity for cell signaling molecules such as growth factors, the mechanical stiffness of the Fn matrix and the adhesivity of Fn for its numerous soluble ligands. These parameters are altered by mechanical strain applied to the ECM. Recent efforts have attempted to link the molecular properties of Fn with bulk properties of Fn matrix fibers. Studies of isolated Fn fibers have helped to characterize the fiber’s material properties and, in combination with models of Fn molecular behavior in the fibers, have begun to provide insights into the Fn molecular arrangement and intermolecular adhesions within the fibers. A review of these studies allows the development of an understanding of the mechanobiological functions of Fn.     

Interfacial shear strength in abalone nacre

The shear strength of the interface between tiles of aragonite in the nacre of red abalone Haliotis rufescens was investigated through mechanical tensile and shear tests. Dog-bone shaped samples were used to determine the tensile strength of nacre when loaded parallel to the plane of growth; the mean strength was 65 MPa. Shear tests were conducted on a special fixture with a shear gap of 200 μm, approximately 100 μm narrower than the spacing between mesolayers. The shear strength is found to be 36.9±15.8 MPa with an average maximum shear strain of 0.3. Assuming the majority of failure occurs through tile pull-out and not through tile fracture, the tensile strength can be converted into a shear strength of 50.9 MPa. Three mechanisms of failure at the tile interfaces are discussed: fracture of mineral bridges, toughening due to friction created through nanoasperities, and toughening due to organic glue. An additional mechanism is fracture through individual tiles.     

Effects of organic matrix proteins on the interfacial structure at the bone-biocompatible nacre interface in vitro

The biocompatibility and potential osteoinductivity of nacre have favored its use as a bone-grafting material. The present study is to investigate the interfacial structure at the bone-nacre interface resulting from organic matrix proteins, which emphasizes the mechanism of bone-bonding ability and biocompatibility of the shell tissues such as nacre and biogenic calcite. To understand the interfacial reaction, the zeta potential measurements, provide for a unique method to quantify the actual state of the interface in situ, were used for synthetic and biogenic calcium carbonate suspensions with respect to pH and the organic matrix as an additive. The zeta potentials and surface charge density show that the organic matrix proteins are main charge regulators, resulting in the stabilized tissue properties as compared with synthetic crystals. Also, in forming calcium carbonate crystals with the additives, the conformation of organic matrix has an important role in the understanding of the newly formed interfacial structure. The result provides the primary role of the organic matrix proteins in controlling the formation of interfacial structure and biocompatibility with bone as well as the stability of biogenic tissues. And it gives a new insight into the usefulness of zeta potential measurement to describe the in vivo interaction between the bone and implants.     

The in vitro osteoclastic degradation of nacre

Osteoclast activity was studied on nacre, the mother of pearl (MOP) in order to assess the plasticity of bone resorbing cells and their capacity to adapt to a biomineralized material with a different organic and mineral composition from that of its natural substrate, bone. Pure MOP, a natural biomineralized CaCO(3) material, was obtained from Pinctada oyster shell. When implanted in the living system, nacre has proven to be a sustainable bone grafting material although a limited surface degradation process. Osteoclast stem cells and mature osteoclasts were cultured on MOP substrate and osteoclast precursor cells were shown to differentiate into osteoclasts capable of resorbing nacre substrate. However, analysis of the organization of the cytoskeleton showed that both a sealing zone and a podosome structure were observed on the nacre substrate. Moreover, MOP resorption efficiency was consistently found to be lower than that of bone and appeared to be a limited process.     

Homoepitaxial meso- and microscale crystal co-orientation and organic matrix network structure in Mytilus edulis nacre and calcite

New developments in high-resolution, low accelaration voltage electron backscatter diffraction (EBSD) enable us to resolve and quantify the co-orientation of nanocrystals constituting biological carbonate crystals with a scan step resolution of 125 nm. This allows the investigation of internal structures in carbonate tablets and tower biocrystals in the nacre of mollusc shells, and it provides details on the calcite–aragonite polymorph interface in bivalves. Within the aragonite tablets of Mytilus edulis nacre we find a mesoscale crystallographic mosaic structure with a misorientation distribution of 2° full width at half maximum. Selective etching techniques with critical point drying reveal an organic matrix network inside the nacre tablets. The size scales of the visible aragonite tablet subunits and nanoparticles correspond to those of the open pore system in the organic matrix network. We further observe by EBSD that crystal co-orientation spans over tablet boundaries and forms composite crystal units of up to 20 stacked co-oriented tablets (tower crystals). Statistical evaluation of the misorientation data gives a probability distribution of grain boundary misorientations with two maxima: a dominant peak for very-small-angle grain boundaries and a small maximum near 64°, the latter corresponding to {1 1 0} twinning orientations. However, the related twin boundaries are typically the membrane-lined {0 0 1} flat faces of the tablets and not {1 1 0} twin walls within tablets. We attribute this specific pattern of misorientation distribution to growth by particle accretion and subsequent semicoherent homoepitaxial crystallization. The semicoherent crystallization percolates between the tablets through mineral bridges and across matrix membranes surrounding the tablets. In the “prismatic” calcite layer crystallographic co-orientation of the prisms reaches over more than 50 micrometers.     

Investigations of voids in the aragonite platelets of nacre

We studied the structure of the aragonite platelets of Haliotis laevigata nacre, using conventional transmission electron microscopy, Z-contrast, electron tomography, energy-dispersive X-ray analysis and electron energy-loss spectroscopy. We observed faceted voids several nanometers wide within the aragonite platelets. The electron tomography investigations showed that the voids are distributed more or less randomly in the studied specimen and allowed an estimation of the order of magnitude of the width and the volumetric content of the voids. Further investigations of these voids revealed that they contain an increased amount of carbon, which suggests the existence of organic material within the voids.     

The growth of nacre in the abalone shell

The process of mineral formation following periods of growth interruption (growth bands) is described. Flat pearl implantation as well as a new trepanning method are used to observe the transitory phases of calcium carbonate which nucleate and grow during this process. An initial random nucleation of the aragonite polymorph is observed followed by a transition towards spherulitic growth. During this transition the animal forms the structure of the shell through both mechanical and chemical actions. About 6 weeks after implantation a steady-state growth of aragonite tiles begins after shorter and more irregular tiles cover the outer surface of the spherulites. The growth rate of aragonitic spherulite during this transition period was calculated to be approximately 0.5 microm per day. An organic scaffolding is observed during the steady-state growth of tiled aragonite. Observations of mineral growth following the deposition of these membranes confirm the presence of mineral bridges originating from subsurface tiles and extending through the organic matrix, confirming the growth model proposed by Sch?ffer et al. [Sch?ffer TE, Ionescu-Zanetti C, Proksch R, Fritz M, Walters, DA, Almqvist N, et al. Does abalone nacre form by heteroepitaxial nucleation or by growth through mineral bridges? Chem Mater 1997;9:1731-40]. Field emission scanning electron microscopy of fractured deproteinated nacre shows the presence of mineral bridges existing between individual layers of tiles. Transmission electron microscopy provides further evidence of mineral bridges.     

Failure mode transition in nacre and bone-like materials

Mineralized biological materials such as nacre or bone achieve remarkable combinations of stiffness and toughness by way of staggered arrangements of stiff components (nanoscale or microscale fibers or tablets) bonded by softer materials. Under applied stress these components slide on one another, generating inelastic deformations and toughness on the macroscale. This mechanism is prominent in nacre, a remarkable material which is now serving as a model for biomimetic materials. In order to better identify which type of nacre should serve as a biomimetic model, the toughness of nacre from four different mollusk species was determined in this study. Nacre from the pearl oyster was found to be toughest, and for the first time remarkable deformation and fracture patterns were observed using in situ optical and atomic force microscopy. Under stress, stair-like deformation bands deformed at an angle to the loading direction, forming a dense, tree-like network. This marks a clear difference from the now well-documented “columnar” failure mode, in which deformation bands are perpendicular to the loading direction. Analytical and numerical models reveal the conditions for the transition between the columnar and stair failure modes, namely large or random overlap between inclusions and local shear stress generated by inhomogeneities in the material. “Stair” failure promotes spreading of non-linear deformation and energy dissipation, which translates into a greater toughness overall. A similar mechanism may also occur in bone, which has a microstructure which is in many ways similar to sheet nacre.     

Interface between bone and nacre implants in sheep.

We have investigated the interface between bone and chronic implants of nacre in sheep. There was no foreign body reaction over the period of 10 months and the implants were not broken down. Light microscopy indicated activity within an osteoprogenitor cellular layer lining the implant, resulting in a complete sequence of new bone formation. Nacre appeared to bind directly to newly formed bone without any intervening fibrous tissue. Scanning electron microscopy and energy dispersive photon X-microanalysis showed calcium and phosphate ions lining the nacre within the osteoprogenitor tissue. These studies show a dynamic activity of the bone/nacre interface, leading to continuity between the nacre and the bone.     

Structural and mechanical properties of the organic matrix layers of nacre

The type of nanostructure referred to in biomineralization as a mineral bridge has been directly observed and measured in the organic matrix layers of nacre by transmission electron microscopy and scanning electron microscopy. Statistical analysis provides the geometric characteristics and a distribution law of the mineral bridges in the organic matrix layers. Experiments reveal that the nanostructures significantly influences the mechanical properties of the organic matrix layers. In addition, the mechanical analysis illustrates the effects of the nanostructures on the behaviors of the organic matrix layers, and the analytical results explain the corresponding experimental phenomena fairly well. The present study shows that the mineral bridges play a key role in the mechanical performances of the organic matrix layers of nacre. The results obtained provide a guide to the interfacial design of synthetic materials.     

Dimensional analysis and parametric studies for designing artificial nacre

Nacre, the iridescent material found in Abalone shells, exhibits remarkable strength and toughness despite its composition of over 95% brittle ceramic. Its hierarchical structure over multiple length scales gives rise to its increase in toughness despite its material composition. In this work we develop a computational model of composites incorporating key morphological features of nacre's microstructure. By conducting a parametric analysis we are able to determine an optimal geometry that increases energy dissipation over 70 times. We discuss the contribution of varying ceramic strengths and size effect to see how this affects the overall performance of the composite. We then compare our simulations to experiments performed on a material possessing the same microstructure investigated computationally. For both simulations and experiments we show that our optimal geometry corresponds to that of natural nacre indicating the importance of specifically incorporating nacre's key morphological and constituent features. This combination of simulations and experiments gives great insight to the delicate interplay between material parameters and microstructure showing that if we optimally combine all aspects, we can develop novel synthetic materials with superior performance.     

珍珠层人工骨修复兔桡骨节段性缺损的放射学评价

目的:用珍珠层/聚乳酸人工骨(N/P)复合同种异体成骨细胞(osteoblastsOBs)修复兔桡骨节段性骨缺损,并从放射学角度评价成骨效果。方法:手术制作18只新西兰大白兔15mm双侧桡骨缺损模型;体外培养同种异体成骨细胞,分别种植到珍珠层/聚乳酸人工骨和聚乳酸(PLA)材料上。以复合OBs的珍珠层/聚乳酸人工骨为实验组(ON),以复合OBs的聚乳酸(OP)和未复合OBs的珍珠层/聚乳酸人工骨(N)作对照组,移植修复兔桡骨缺损。分别于植入后4、8、12周取材,经大体观察、X线、放射学评分和X线阻射密度值来评价修复兔桡骨节段性骨缺损的效果并进行统计学分析。结果:复合OBs的N/P人工骨的试验组放射学评分和X线阻射密度值均高于对照组,统计学差异有显著性。在新骨形成的同时,珍珠层粉未完全降解。结论:珍珠层人工骨能较好地修复兔桡骨骨缺损,在复合成骨细胞后修复效果更佳,是一种很有前途的骨组织工程材料。     

心电图的多尺度熵分析

我们使用Costa等人提出的算法,研究了心电图(ECG)的多尺度熵(MSE)的特性。发现健康人的样本熵要高于冠心病人和心梗病人且健康人的复杂度最高。而冠心病人的样本熵(SampEn)要高于心梗病人,但是已很接近心梗病人。说明冠心病人和心梗病人的复杂度明显低于健康人,而冠心病人很容易导致心梗发作,从而引起生命危险。     

Multiscale imaging of bone microdamage

Abstract

Bone is a structural and hierarchical composite that exhibits remarkable ability to sustain complex mechanical loading and resist fracture. Bone quality encompasses various attributes of bone matrix from the quality of its material components (type-I collagen, mineral and non-collagenous matrix proteins) and cancellous microarchitecture, to the nature and extent of bone microdamage. Microdamage, produced during loading, manifests in multiple forms across the scales of hierarchy in bone and functions to dissipate energy and avert fracture. Microdamage formation is a key determinant of bone quality, and through a range of biological and physical mechanisms, accumulates with age and disease. Accumulated microdamage in bone decreases bone strength and increases bone’s propensity to fracture. Thus, a thorough assessment of microdamage, across the hierarchical levels of bone, is crucial to better understand bone quality and bone fracture. This review article details multiple imaging modalities that have been used to study and characterize microdamage; from bulk staining techniques originally developed by Harold Frost to assess linear microcracks, to atomic force microscopy, a modality that revealed mechanistic insights into the formation diffuse damage at the ultrastructural level in bone. New automated techniques using imaging modalities, such as microcomputed tomography are also presented for a comprehensive overview.     

Multiscale Models of Cell Signaling

Computational models of signal transduction face challenges of scale below the resolution of a single cell. Here, we organize these challenges around three key interfaces for multiscale models of cell signaling: molecules to pathways, pathways to networks, and networks to outcomes. Each interface requires its own set of computational approaches and systems-level data, and no single approach or dataset can effectively bridge all three interfaces. This suggests that realistic "whole-cell" models of signaling will need to agglomerate different model types that span critical intracellular scales. Future multiscale models will be valuable for understanding the impact of signaling mutations or population variants that lead to cellular diseases such as cancer.     

Bone reactions to nacre injected percutaneously into the vertebrae of sheep

We have studied the osteogenic effects of nacre (mother of pearl) placed in experimental cavities prepared in the lumbar vertebrae of sheep. Some of cavities were filled with nacre, some with PMMA, and some were left empty. The vertebrae were removed 1, 8, 12 weeks after surgery, and assessed histologically and morphometrically. The nacre particles in the bone cavity and the surrounding intertrabecular spaces gradually dissolved beginning at 8 weeks after surgery. There were layers of newly formed bone, both woven and lamellar, in various stages of maturation in contact with or adjacent to the dissolving nacre. Quantitative assessment of the activation of bone formation adjacent to the cavities filled with nacre indicated significant activation of bone formation, which continued until week 12. There was also increased mineralization of the host bone at this time. There was no new bone formation in the empty cavities, or in those filled with PMMA. PMMA also caused necrosis of surrounding bone cells with a change in bone architecture and significant reductions in bone formation and mineralization. This study demonstrates that nacre stimulates bone-forming cells in vertebrae and appears to result in new bone formation.     

Self-organisation of nacre in the shells of Pterioida (Bivalvia: Mollusca)

Changes in the morphology and orientation of crystals have been tracked across the nacreous layer in pterioid bivalves, by SEM and X-ray diffraction. Early crystals nucleated on the organic membrane usually covering the outer prismatic layer. They formed polycrystalline aggregates of various shapes, first at prism boundaries and progressively invading the prisms in a centripetal fashion. Their c-axes were perpendicular to the substrate, but the a- and b-axis were variously arranged. As they grew, nacreous crystals became individualized and acquired a common crystallographic orientation also in the a-b plane (with the b-axis in the direction of shell growth). Initial lamellae had well delineated growth fronts, which later changed to diffuse. The increasing orientation was explained by a competition model between nacreous plates within the growing lamellae, which was particularly effective in lamellae with delineated fronts. This competition between adjacent crystals could not take place in gastropod nacre because, contrary to the stepped mode of growth of bivalve nacre, vertical stacking predominated. Our model is an alternative to the heteroepitaxial model where the organic matrix supposedly acted as an orienting template, but is nevertheless compatible with the mineral bridge hypothesis that implied epitaxial growth of stacking nacreous tablets.     

Multiscale Models of Breast Cancer Progression

Breast cancer initiation, invasion and metastasis span multiple length and time scales. Molecular events at short length scales lead to an initial tumorigenic population, which left unchecked by immune action, acts at increasingly longer length scales until eventually the cancer cells escape from the primary tumor site. This series of events is highly complex, involving multiple cell types interacting with (and shaping) the microenvironment. Multiscale mathematical models have emerged as a powerful tool to quantitatively integrate the convective-diffusion-reaction processes occurring on the systemic scale, with the molecular signaling processes occurring on the cellular and subcellular scales. In this study, we reviewed the current state of the art in cancer modeling across multiple length scales, with an emphasis on the integration of intracellular signal transduction models with pro-tumorigenic chemical and mechanical microenvironmental cues. First, we reviewed the underlying biomolecular origin of breast cancer, with a special emphasis on angiogenesis. Then, we summarized the development of tissue engineering platforms which could provide high-fidelity ex vivo experimental models to identify and validate multiscale simulations. Lastly, we reviewed top-down and bottom-up multiscale strategies that integrate subcellular networks with the microenvironment. We present models of a variety of cancers, in addition to breast cancer specific models. Taken together, we expect as the sophistication of the simulations increase, that multiscale modeling and bottom-up agent-based models in particular will become an increasingly important platform technology for basic scientific discovery, as well as the identification and validation of potentially novel therapeutic targets.