Wound healing is a complex process initiated by the formation of fibrin fibers and endothelialization. Normally, this process is triggered in a wound by thrombin cleavage of fibrinopeptides on fibrinogen molecules, which allows them to self spontaneously-assemble into large fibers that provide the support structure of the clot and promote healing. We have found that the fibrous structures can also form without thrombin on most polymer or metal surfaces, including those commonly used for stents. We show that the relatively hydrophobic E and D regions of the fibrinogen molecule are adsorbed on these surfaces, exposing the αC domains, which in turn results in the formation of large fiber structures that promote endothelial cell adhesion. We show that the entire process can be suppressed when stents or other substrates are coated with polymers that are functionalized to bind the αC domains, leading to the development of potentially nonthrombogenic implant materials.

We have investigated the effects of moderate static magnetic fields (SMFs) on murine MC3T3-E1 osteoblasts, and found that they enhance proliferations and promote differentiation. The increase in proliferation rates in response to SMFs was greater in cultures grown on partially sulfonated polytstyrene (SPS, degree of sulfonation: 33%) than in cultures grown on tissue culture plastic. We have previously shown that when the degree of sulfonation exceeded a critical value (12%) [1], spontaneous fibrillogenesis occured which allowed for direct observation of the ECM fibrillar organization under the influence of external fields. We found that the ECM produced in cultures grown on the SPS in the presence of the SMFs assembled into a lattice with larger dimensions than the ECM of the cultures grown in the absence of SMFs. During the early stages of the biomineralization process (day 7), the SMF exposed cultures also templated mineral deposition more rapidly than the control cultures. The rapid response is attributed to orientation of diamagnetic ECM proteins already present in the serum, which could then initiate further cellular signaling. SMFs also influenced late stage osteoblast differentiation as measured by the increased rate of osteocalcin secretion and gene expression beginning 15 days after SFM exposure. This correlated with a large increase in mineral deposition, and in cell modulus. GIXD and EDXS analysis confirmed early deposition of crystalline hydroxyapatite. Previous studies on the effects of moderate SMF had focused on cellular gene and protein expression, but did not consider the organization of the ECM fibers. Our ability to form these fibers has allowed us explore this additional effect and highlight its significance in the initiation of the biomineralization process.

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The effects of the components of extracellular matrix on the bone formation and the kinetics of crystal growth of calcium phosphate have remained unknown. In this paper, we reported a method to investigate the role of Type I collagen and the interactions with other ECM proteins such as fibronectin and elastin during biomimic mineralization process in vitro. The early stage of mineralization was characterized by scanning probe microscopy (SPM) and shear modulation force microscopy (SMFM). The late stage of mineralization was investigated by synchrotron grazing incident x-ray diffraction (GIXD). The results demonstrate the cooperative interaction between type I collagen and noncollagenous proteins such as fibronectin or elastin could be essential for the biomineralization.

The extracellular matrix (ECM) is composed of mixed protein fibers whose precise composition affects biomineralization. New methods are needed to probe the interactions of these proteins with calcium phosphate mineral and with each other. Here we follow calcium phosphate mineralization on protein fibers self-assembled in vitro from solutions of fibronectin, elastin and their mixture. We probe the surface morphology and mechanical properties of the protein fibers during the early stages. The development of mineral crystals on the protein matrices is also investigated. In physiological mineralization solution, the elastic modulus of the fibers in the fibronectin-elastin mixture increases to a greater extent than that of the fibers from either pure protein. In the presence of fibronectin, longer exposure in the mineral solution leads to the formation of amorphous calcium phosphate particles templated along the self-assembled fibers, while elastin fibers only collect calcium without any mineral observed during early stage. TEM images confirm that small needle-shape crystals are confined inside elastin fibers which suppress the release of mineral outside the fibers during late stage, while hydroxyapatite crystals form when fibronectin is present. These results demonstrate complementary actions of the two ECM proteins fibronectin and elastin to collect cations and template mineral, respectively.

Understanding how biomineralization occurs in the extracellular matrix (ECM) of bone cells is crucial to the understanding of bone formation and the development of a successfully engineered bone tissue scaffold. It is still unclear how ECM mechanical properties affect protein-mineral interactions in early stages of bone mineralization. We investigated the longitudinal mineralization properties of MC3T3-E1 cells and the elastic modulus of their ECM using shear modulation force microscopy, synchrotron grazing incidence X-ray diffraction (GIXD), scanning electron microscopy, energy dispersive X-ray spectroscopy, and confocal laser scanning microscopy (CLSM). The elastic modulus of the ECM fibers underwent significant changes for the mineralizing cells, which were not observed in the nonmineralizing cells. On substrates conducive to ECM network production, the elastic modulus of mineralizing cells increased at time points corresponding to mineral production, whereas that of the nonmineralizing cells did not vary over time. The presence of hydroxyapatite in mineralizing cells and the absence thereof in the nonmineralizing ones were confirmed by GIXD, and CLSM showed that a restructuring of actin occurred only for mineral-producing cells. These results show that the correct and complete development of the ECM network is required for osteoblasts to mineralize. This in turn requires a suitably prepared synthetic substrate for bone development to succeed in vitro.

Extracellular matrix (ECM) proteins play an essential role during biomineralization in bone and engineered tissues. In a previous study ([1]), we showed that calcite preferentially nucleated on pure elastin fibers. However, the actual cellular ECM fibers are composed of a combination of proteins, primarily collagen, fibronectin and some elastin. Here we follow the calcium carbonate- and calcium phosphate- mineralization process in vitro when these ECM proteins are combined and determine the differences between these proteins in the biomineralization process. The surface morphology and mechanical properties of the protein fibers during the early stages were probed by atomic force microscopy (AFM) and shear modulation force microscopy (SMFM). The nucleation of the mineral crystals on the protein matrices was investigated by scanning electron microscopy (SEM). Preliminary data showed that the moduli of all protein fibers increased at the early stages, with collagen having the largest increase in supersaturated calcium bicarbonate solution. In metastable calcium phosphate solutions the modulus of the mixed elastin-fibronectin fibres increased to a greater extent than the moduli of the fibers composed of the single proteins. Longer exposure in the mineral solutions led to the formation of crystals templated along the self-assembled fiber structures.

Chapter 7 Biomimetic Mineralization and Shear Modulation Force Microscopy of Self-Assembled Protein Fibers Elaine Dimasi, Search for more papers by this authorSeo-Young Kwak, Search for more papers by this authorNadine Pernodet, Search for more papers by this authorXiaolan Ba, Search for more papers by this authorYizhi Meng, Search for more papers by this authorVladimir Zeitsev, Search for more papers by this authorKarthikeyan Subburaman, Search for more papers by this authorMiriam Rafailovich, Search for more papers by this author Elaine Dimasi, Search for more papers by this authorSeo-Young Kwak, Search for more papers by this authorNadine Pernodet, Search for more papers by this authorXiaolan Ba, Search for more papers by this authorYizhi Meng, Search for more papers by this authorVladimir Zeitsev, Search for more papers by this authorKarthikeyan Subburaman, Search for more papers by this authorMiriam Rafailovich, Search for more papers by this author Book Editor(s):Prof. Dr. Edmund Bäuerlein, Max-Planck-Institute for Biochemistry, Department of Membrane Biochemistry, Am Klopferspitz 18 A, 82152 Planegg, GermanySearch for more papers by this author First published: 25 May 2007 https://doi.org/10.1002/9783527619443.ch31Citations: 1 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Summary Biological mineralization relies upon proteins which preferentially nucleate minerals and control their growth. This process is often referred to as "templating", but the term is inclusive of a variety of mineral-organic interactions demonstrated in diverse model systems. In this chapter, details are presented of studies designed to differentiate between structured and unstructured proteins, which can be assembled together on submicron length scales and probed simultaneously at early stages of biomimetic mineralization. The approach utilizes extracellular matrix proteins, which self-assemble into fiber networks when induced onto negatively charged sulfonated polystyrene surfaces. A novel technique, based on atomic force microscopy is introduced; this is used to measure the elastic modulus of both structured and disorganized protein, prior to and during calcium carbonate mineralization. Mineral-induced thickening and stiffening of the protein fibers occurs during the early stages of mineralization, well before discrete mineral crystals are large enough to image. Calcium carbonate stiffens the protein fibers selectively, without affecting the regions of disorganized protein between them. Secondary ion mass spectroscopy reveals calcium to be concentrated along protein fibers. In this unique model system, organized versus unstructured proteins can be assembled only nanometers apart and probed in identical environments, demonstrating a mineralization process which requires the structural organization imposed by fibrillogenesis of the extracellular matrix. Citing Literature Handbook of Biomineralization: Biological Aspects and Structure Formation RelatedInformation

Understanding how biomineralization occurs in the extracellular matrix (ECM) of bone cells is crucial to the development of a successfully engineered bone tissue scaffold, and to date there has not been a well-established method for the quantitative examination of bone mineralization in situ. We investigated the mechanical properties of MC3T3-E1 osteoblast-like cells in vitro using shear modulation force microscopy (SMFM). The elastic modulus of the mineralizing cells increased at time points corresponding to mineral production, whereas that of the non-mineralizing cells did not vary significantly over time.

Understanding how biomineralization occurs in the extracellular matrix (ECM) of bone cells is crucial to the development of a successfully engineered bone tissue scaffold, and to date there has not been a well-established method for the quantitative examination of bone mineralization in situ. We investigated the mechanical properties of MC3T3-E1 osteoblast-like cells and the crystalline properties of their biomineralized ECM in vitro using shear modulation force microscopy (SMFM), confocal laser scanning microscopy (CLSM), synchrotron X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR). The elastic modulus of the mineralizing cells increased at time points corresponding to mineral production, whereas that of the non-mineralizing cells did not vary significantly over time. CLSM showed a restructuring of the F-actin fiber network of mineralizing cells with time, which indicates remodeling activities in the cytoskeleton and was not seen in the non-mineralizing cells. Both XRD and FTIR showed that the mineralizing subclone produced hydroxyapatite in situ and that the non-mineralizing subclone was in fact weakly biomineralizing.

Biological mineralization of tissues in living organisms relies on proteins that preferentially nucleate minerals and control their growth. This process is often referred to as “templating,” but this term has become generic, denoting various proposed mineral–organic interactions including both chemical and structural affinities. Here, we present an approach using self-assembled networks of elastin and fibronectin fibers, similar to the extracellular matrix. When induced onto negatively charged sulfonated polystyrene surfaces, these proteins form fiber networks of ≈10-μm spacing, leaving open regions of disorganized protein between them. We introduce an atomic force microscopy-based technique to measure the elastic modulus of both structured and disorganized protein before and during calcium carbonate mineralization. Mineral-induced thickening and stiffening of the protein fibers during early stages of mineralization is clearly demonstrated, well before discrete mineral crystals are large enough to image by atomic force microscopy. Calcium carbonate stiffens the protein fibers selectively without affecting the regions between them, emphasizing interactions between the mineral and the organized protein fibers. Late-stage observations by optical microscopy and secondary ion mass spectroscopy reveal that Ca is concentrated along the protein fibers and that crystals form preferentially on the fiber crossings. We demonstrate that organized versus unstructured proteins can be assembled mere nanometers apart and probed in identical environments, where mineralization is proved to require the structural organization imposed by fibrillogenesis of the extracellular matrix.