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In proteins, phosphorylation of amino acid residues confers unique functions, including mineral-binding properties. For example, osteopontin, an abundant phosphoprotein in many biomineralized tissu...

Elastogenesis is a hierarchical process by which cells form functional elastic fibers, providing elasticity and the ability to regulate growth factor bioavailability in tissues, including blood vessels, lung, and skin. This process requires accessory proteins, including fibulin-4 and -5, and latent TGF binding protein (LTBP)-4. Our data demonstrate mechanisms in elastogenesis, focusing on the interaction and functional interdependence between fibulin-4 and LTBP-4L and its impact on matrix deposition and function. We show that LTBP-4L is not secreted in the expected extended structure based on its domain composition, but instead adopts a compact conformation. Interaction with fibulin-4 surprisingly induced a conformational switch from the compact to an elongated LTBP-4L structure. This conversion was only induced by fibulin-4 multimers associated with increased avidity for LTBP-4L; fibulin-4 monomers were inactive. The fibulin-4-induced conformational change caused functional consequences in LTBP-4L in terms of binding to other elastogenic proteins, including fibronectin and fibrillin-1, and of LTBP-4L assembly. A transient exposure of LTBP-4L with fibulin-4 was sufficient to stably induce conformational and functional changes; a stable complex was not required. These data define fibulin-4 as a molecular extracellular chaperone for LTBP-4L. The altered LTBP-4L conformation also promoted elastogenesis, but only in the presence of fibulin-4, which is required to escort tropoelastin onto the extended LTBP-4L molecule. Altogether, this study provides a dual mechanism for fibulin-4 in 1) inducing a stable conformational and functional change in LTBP-4L, and 2) promoting deposition of tropoelastin onto the elongated LTBP-4L.

Since Pasteur first successfully separated right-handed and left-handed tartrate crystals in 1848, the understanding of how homochirality is achieved from enantiomeric mixtures has long been incomplete. Here, we report on a chirality dominance effect where organized, three-dimensional homochiral suprastructures of the biomineral calcium carbonate (vaterite) can be induced from a mixed nonracemic amino acid system. Right-handed (counterclockwise) homochiral vaterite helicoids are induced when the amino acid l-Asp is in the majority, whereas left-handed (clockwise) homochiral morphology is induced when d-Asp is in the majority. Unexpectedly, the Asp that incorporates into the homochiral vaterite helicoids maintains the same enantiomer ratio as that of the initial growth solution, thus showing chirality transfer without chirality amplification. Changes in the degree of chirality of the vaterite helicoids are postulated to result from the extent of majority enantiomer assembly on the mineral surface. These mechanistic insights potentially have major implications for high-level advanced materials synthesis., Induction of complex homochiral architectures by chiral transformation in a mixed enantiomer system has remained largely elusive. Here, the authors report a chirality dominance effect which induces homochiral suprastructures of calcium carbonate by a mixed, heterochiral nonracemic amino acid enantiomer system.

Osteopontin (OPN) belongs to the SIBLING family (Small, Integrin-Binding LIgand N-linked Glycoproteins) of mineral-binding matrix proteins found in bones and teeth. OPN is a well-known inhibitor of matrix mineralization, and enzymatic modification of OPN can affect this inhibitory function. In bone, OPN exists both as a monomer and as a high-molecular-weight polymer - the latter is formed by transglutaminase-mediated crosslinking of glutamine and lysine residues in OPN to create homotypic protein assemblies. OPN can be covalently crosslinked by transglutaminase 2 (TG2) and Factor XIII-A. Polymeric OPN has increased binding to collagen and promotes osteoblast adhesion, but despite these initial observations, its role in mineralization is not clear. In this study, we investigated the effect of polymerized OPN on mineralization using a hydroxyapatite crystal growth assay and mineralizing MC3T3-E1 osteoblast cultures. In the cultures, endogenous polymeric OPN was detected after mineralization occurred. In cell-free conditions, TG2 was used to crosslink bovine OPN into its polymeric form, and atomic force microscopy and dynamic light scattering revealed variably-sized, large branched aggregates ranging across hundreds of nanometers. These OPN polymers inhibited the growth of hydroxyapatite crystals in solution at concentrations similar to monomeric OPN, although the crosslinking slightly reduced its inhibitory potency. When added to MC3T3-E1 osteoblast cultures, this exogenous polymeric OPN essentially did not inhibit mineralization when given during the later mineralization stages of culture; however, cultures treated early and then continuously with polymeric OPN throughout both the matrix assembly and mineral deposition stages showed reduced mineralization. Immunoblotting of protein extracts from these continuously treated cultures revealed exogenous OPN polymers incorporated into mature matrix that had not yet mineralized. These results suggest that in bone, the increased size and branched structure of crosslinked inhibitory polymeric OPN near the mineralization front could hinder it from accessing focal mineralization sites in the dense collagen-rich matrix, suggesting that OPN-crosslinking into polymers may represent a way to fine-tune the inhibitory potency of OPN on bone mineralization.

Chirality is ubiquitous in biology, including in biomineralization, where it is found in many hardened structures of invertebrate marine and terrestrial organisms (for example, spiralling gastropod shells). Here we show that chiral, hierarchically organized architectures for calcium carbonate (vaterite) can be controlled simply by adding chiral acidic amino acids (Asp and Glu). Chiral, vaterite toroidal suprastructure having a ‘right-handed' (counterclockwise) spiralling morphology is induced by L-enantiomers of Asp and Glu, whereas ‘left-handed' (clockwise) morphology is induced by D-enantiomers, and sequentially switching between amino-acid enantiomers causes a switch in chirality. Nanoparticle tilting after binding of chiral amino acids is proposed as a chiral growth mechanism, where a ‘mother' subunit nanoparticle spawns a slightly tilted, consequential ‘daughter' nanoparticle, which by amplification over various length scales creates oriented mineral platelets and chiral vaterite suprastructures. These findings suggest a molecular mechanism for how biomineralization-related enantiomers might exert hierarchical control to form extended chiral suprastructures., Chiral structures are formed in numerous processes including biomineralization of calcium carbonate. Here, the authors demonstrate that the chiral, hierarchically-organized architecture of the calcium carbonate mineral, vaterite, can be controlled simply by the addition of chiral acidic amino acids.

Mammalian otoconia of the inner ear vestibular apparatus are calcium carbonate-containing mineralized structures critical for maintaining balance and detecting linear acceleration. The mineral phase of otoconia is calcite, which coherently diffracts X-rays much like a single-crystal. Otoconia contain osteopontin (OPN), a mineral-binding protein influencing mineralization processes in bones, teeth and avian eggshells, for example, and in pathologic mineral deposits. Here we describe mineral nanostructure and the distribution of OPN in mouse otoconia. Scanning electron microscopy and atomic force microscopy of intact and cleaved mouse otoconia revealed an internal nanostructure (~50 nm). Transmission electron microscopy and electron tomography of focused ion beam-prepared sections of otoconia confirmed this mineral nanostructure, and identified even smaller (~10 nm) nanograin dimensions. X-ray diffraction of mature otoconia (8-day-old mice) showed crystallite size in a similar range (73 nm and smaller). Raman and X-ray absorption spectroscopy – both methods being sensitive to the detection of crystalline and amorphous forms in the sample – showed no evidence of amorphous calcium carbonate in these mature otoconia. Scanning and transmission electron microscopy combined with colloidal-gold immunolabeling for OPN revealed that this protein was located at the surface of the otoconia, correlating with a site where surface nanostructure was observed. OPN addition to calcite growing in vitro produced similar surface nanostructure. These findings provide details on the composition and nanostructure of mammalian otoconia, and suggest that while OPN may influence surface rounding and surface nanostructure in otoconia, other incorporated proteins (also possibly including OPN) likely participate in creating internal nanostructure.

Multidomain matrix-associated zinc extracellular proteases ADAMTS and ADAMTS-like proteins have important biological activities in cells and tissues. Beyond their traditional role in procollagen and von Willebrand factor processing and proteoglycan cleavage, ADAMTS/ADAMTSL likely participate in or at least have some role in ECM assembly as some of these proteins bind ECM proteins including fibrillins, fibronectin, and LTBPs. In this chapter, we present four biophysical techniques largely used for the characterization, multimerization, and interaction of proteins: surface plasmon resonance spectroscopy, dynamic light scattering, atomic force microscopy, and circular dichroism spectroscopy.

ObjectiveThe plasma protein fetuin-A mediates the formation of protein-mineral colloids known as calciprotein particles (CPP) – rapid clearance of these CPP by the reticuloendothelial system prevents errant mineral precipitation and therefore ectopic mineralization (calcification). The mutant mouse strain D2,Ahsg-/- combines fetuin-A deficiency with the mineralization-prone DBA/2 genetic background, having a particularly severe compound phenotype of microvascular and soft tissue mineralization. Here we studied mechanisms leading to soft tissue mineralization, organ damage and premature aging in these mice.Approach and ResultsWe analyzed mice longitudinally by echocardiography, X-ray-computed tomography, analytical electron microscopy, histology, mass spectrometry proteomics, and genome-wide microarray-based expression analyses of D2 wildtype and Ahsg-/- mice.Fetuin-A deficient mice had calcified lesions in myocardium, lung, brown adipose tissue, reproductive organs, spleen, pancreas, kidney and the skin, associated with reduced growth, cardiac output and premature aging. Importantly, early stage calcified lesions presented in the lumen of the microvasculature suggesting precipitation of mineral containing complexes from the fluid phase of blood. Genome-wide expression analysis of calcified lesions and surrounding (not calcified) tissue, together with morphological observations, indicated that the ectopic mineralization was not associated with osteochondrogenic cell differentiation, but rather with thrombosis and fibrosis.ConclusionsCollectively, these results demonstrate that pathological mineralization can start by intravascular mineral deposition causing microvasculopathy, which impacts on growth, organ function and survival. Our study underscores the importance of fetuin-A and related systemic regulators of mineralized matrix metabolism to prevent cardiovascular disease, especially in dysregulated mineral homeostasis.

Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone.

The extent to which vascular calcification is reversible and the possible mechanisms are unclear. To address this, calcified aortas from uremic mice were transplanted orthotopically into normal mice, and the calcium content, histology, and minerals of the allografts were compared with the nontransplanted donor aorta. Calcium content decreased immediately after transplantation but remained constant thereafter, with 68% ± 12% remaining after 34 weeks. X-ray diffraction showed the presence of apatite in both donor aortas and allografts. Osteoclasts were absent in the allografts and there was no expression of the macrophage marker CD11b, the osteoclast marker tartrate-resistant acid phosphatase, or carbonic anhydrase II. The initial loss of calcium was less in heavily calcified aortas and was associated with an increase in the Ca/P ratio from 1.49 to 1.63, consistent with a loss of nonapatitic calcium. The results indicate that vascular calcification persists after reversal of uremia, because of a lack of active resorption of apatite. This failure to resorb established calcifications may contribute to the severity of vascular calcification and suggests that therapy should be aimed at prevention.

Mineral dissolution events are part of the life history of many protein-rich biomineralized tissues and structures. To assess under dissolution conditions the specificity of interactions between calcite and phosphate moieties in proteins, the ( 1 0 4 ) surface of calcite single crystals were etched with aqueous solutions containing osteopontin (OPN) protein and examined by atomic force microscopy. Etch pits were compared to those modified by constituent acidic peptides of OPN as well as by single relevant amino acids. Exposure to phosphorylated OPN and phosphorylated acidic serine- and aspartate-rich motif (ASARM) peptides of OPN produced oval or nearly circular etch pits (rather than the rhombic shape when etched in water alone) in the ( 1 0 4 ) calcite surface, with a steep acute [ 0 1 0 ] edge, indicating preferential step-specific binding. A carboxyl-group-rich polyaspartic acid peptide, and the nonphosphorylated ASARM peptide, produced pits predominantly elongated parallel to [ 4 2 1 ]. Etch-pit ste...

The control of cell behavior to increase biocompatibility of implantable medical devices can be improved by protein coatings. Plasma fibronectin (FN) is a circulating soluble protein capable of assembling into insoluble filaments, fibrils and networks which promote various cellular processes and tissue integrity. FN fibrillogenesis is initiated by a conformational change, which has been proposed to involve charge-mediated opening of its structure. In this study, we have used a bare gold surface polarized electrochemically in the double-layer region to modulate its charge from highly positive to highly negative. The negatively charged surface promoted molecular extension and assembly of FN into beaded filaments and creation of a stable protein coating as examined by atomic force microscopy and electrochemical differential capacitance measurements. Gold surfaces with open and filamentous FN showed significantly improved endothelial cell adhesion while allowing formation of cell–cell contacts. Such surfaces may be used to promote rapid endothelialisation of cardiovascular stents.

Extracellular short fibulins, fibulin-3, -4, and -5, are components of the elastic fiber/microfibril system and are implicated in the formation and homeostasis of elastic tissues. In this study, we report new structural and functional properties of the short fibulins. Full-length human short fibulins were recombinantly expressed in human embryonic kidney cells and purified by immobilized metal ion affinity chromatography. All three fibulins showed various levels of degradation after the purification procedure. N-terminal sequencing revealed that all three fibulins are highly susceptible to proteolysis within the N-terminal linker region of the first calcium-binding epidermal growth factor domain. Proteolytic susceptibility of the linker correlated with its length. Exposure of these fibulins to matrix metalloproteinase (MMP)-1, -2, -3, -7, -9, and -12 resulted in similar proteolytic fragments with MMP-7 and -12 being the most potent proteases. Fibulin-3 proteolysis was almost completely inhibited in cell culture by the addition of 25 μm doxycycline (a broad spectrum MMP inhibitor). Reducible fibulin-4 dimerization and multimerization were consistently observed by SDS-PAGE, Western blotting, and mass spectrometry. Atomic force microscopy identified monomers, dimers, and multimers in purified fibulin-4 preparations with sizes of ∼10-15, ∼20-25, and ∼30-50 nm, respectively. All short fibulins strongly adhered to human fibroblasts and smooth muscle cells. Although only fibulin-5 has an RGD integrin binding site, all short fibulins adhere at a similar level to the respective cells. Solid phase binding assays detected strong calcium-dependent binding of the short fibulins to immobilized heparin, suggesting that these fibulins may bind cell surface-located heparan sulfate.

While advances in biomineralization have been made in recent years, unanswered questions persist on bone- and tooth-cell differentiation, on outside-in signaling from the extracellular matrix, and on the link between protein expression and mineral deposition. In the present study, we validate the use of a bioengineered three-dimensional (3D) dense collagen hydrogel scaffold as a cell-culture model to explore these questions. Dental pulp progenitor/stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into an extracellular matrix-like collagen gel whose fibrillar density was increased through plastic compression. SHED viability, morphology, and metabolic activity, as well as scaffold mineralization, were investigated over 24 days in culture. Additionally, measurements of alkaline phosphatase enzymatic activity, together with immunoblotting for mineralized tissue cell markers ALPL (tissue-non-specific alkaline phosphatase), DMP1 (dentin matrix protein 1), and OPN (osteopontin), demonstrated osteo/odontogenic cell differentiation in the dense collagen scaffolds coincident with mineralization. Analyses of the mineral phase by electron microscopy, including electron diffraction and energy-dispersive x-ray spectroscopy, combined with Fourier-transform infrared spectroscopy and biochemical analyses, were consistent with the formation of apatitic mineral that was frequently aligned along collagen fibrils. In conclusion, use of a 3D dense collagen scaffold promoted SHED osteo/odontogenic cell differentiation and mineralization.

Genetic and environmental factors may lead to abnormal growth of the orofacial skeleton, affecting the overall structure of the face. In this study, we investigated the craniofacial abnormalities in a mouse model for Keutel syndrome, a rare genetic disease caused by loss-of-function mutations in the matrix Gla protein (MGP) gene. Keutel syndrome patients show diffuse ectopic calcification of cartilaginous tissues and impaired midface development. Our comparative cephalometric analyses of micro-computed tomography images revealed a severe midface hypoplasia in Mgp−/− mice. In vivo reporter studies demonstrated that the Mgp promoter is highly active at the cranial sutures, cranial base synchondroses, and nasal septum. Interestingly, the cranial sutures of the mutant mice showed normal anatomical features. Although we observed a mild increase in mineralization of the spheno-occipital synchondrosis, it did not reduce the relative length of the cranial base in comparison with total skull length. Contrary to this, we found the nasal septum to be abnormally mineralized and shortened in Mgp−/− mice. Transgenic restoration of Mgp expression in chondrocytes fully corrected the craniofacial anomalies caused by MGP deficiency, suggesting a local role for MGP in the developing nasal septum. Although there was no up-regulation of markers for hypertrophic chondrocytes, a TUNEL assay showed a marked increase in apoptotic chondrocytes in the calcified nasal septum. Transmission electron microscopy confirmed unusual mineral deposits in the septal extracellular matrix of the mutant mice. Of note, the systemic reduction of the inorganic phosphate level was sufficient to prevent abnormal mineralization of the nasal septum in Mgp−/−;Hyp compound mutants. Our work provides evidence that modulation of local and systemic factors regulating extracellular matrix mineralization can be possible therapeutic strategies to prevent ectopic cartilage calcification and some forms of congenital craniofacial anomalies in humans.

Energetic photons (UV, VUV radiation and soft X rays), and particles (ions, electrons, metastable excited atoms) from inert gas plasmas possess sufficient energy to induce multiple bond scission reactions in the macromolecular chains of most polymers. As a result, volatile products of varying molecular weights escape from the polymers' surfaces; they can be captured and identified by mass spectrometry (MS). We report an MS-based study of five commercial polymers, polyethylene (PE), polypropylene (PP), polystyrene (PS), poly(ethylene terephthalate) (PET) and polyamide that were exposed to inert gas (He, Ar) plasmas and to near-monochromatic (λ = 121.6 nm) VUV irradiation. For the cases of PE and PP, quantitative analyses of the most abundant reaction products (H2, C1–C7 alkanes) are compared among the various polymer/excitation-source cases, and with regard to literature data for γ irradiation.

Mineralized tissues such as teeth and bones consist primarily of highly organized apatitic calcium-phosphate crystallites within a complex organic matrix. The dimensions and organization of these apatite crystallites at the nanoscale level determine in part the physical properties of mineralized tissues. After death, geological processes such as diagenesis and dolomitization can alter the crystallographic properties of mineralized tissues through cycles of dissolution and re-precipitation occurring in highly saline environments. Inspired by these natural exchange phenomena, we investigated the effect of hypersalinity on tooth enamel. We discovered that magnesium ions reacted with human tooth enamel through a process of dissolution and re-precipitation, reducing enamel crystal size at the surface of the tooth. This change in crystallographic structure made the teeth harder and whiter. Salt-water rinses have been used for centuries to ameliorate oral infections; however, our discovery suggests that this ancient practice could have additional unexpected benefits. Statement of Significance Here we describe an approach inspired by natural geological processes to modify the properties of a biomineral – human tooth enamel. In this study we showed that treatment of human tooth enamel with solutions saturated with magnesium induced changes in the nanocrystals at the outer surface of the protective enamel layer. As a consequence, the physical properties of the tooth were modified; tooth microhardness increased and the color shade became whiter, thus suggesting that this method could be used as a clinical treatment to improve dental mechanical properties and esthetics. Such an approach is simple and straightforward, and could also be used to develop new strategies to synthesize and modify biominerals for biomedical and industrial applications.

Fibrillins form multifunctional microfibrils in most connective tissues. Deficiencies in fibrillin assembly can result in fibrillinopathies, such as Marfan syndrome. We demonstrate the presence of heparin/heparan sulfate binding sites in fibrillin-2 and -3. Multimerization of all three fibrillins drastically increased the apparent affinity of their interaction with heparin/heparan sulfate. Surprisingly, contrary to other reports heparin/heparan sulfate strongly inhibited homo- and heterotypic N-to-C-terminal fibrillin interactions. These data suggest that heparin/heparan sulfate controls the formation of microfibrils at the bead interaction stage.

Fibronectin (FN) is an extracellular glycoprotein with critical roles in many fundamental biological processes. A hallmark of FN function is its characteristic assembly into filaments and fibers to form an insoluble matrix which functions as a scaffolding onto which cells attach, migrate, and deposit other matrix constituents. In this study, we have investigated the effects of differently charged and functionalized surfaces on FN conformations using atomic force microscopy. We demonstrate that a negatively charged polysulfonated surface promotes the formation of highly periodic, micrometer-long FN filaments having a "bead-on-a-string" structure with a bead periodicity of about 60 nm. Beaded filaments were observed when FN was adsorbed to polysulfonate surface in water; higher ionic strength allowed formation of filamentous structures but altered the regularity of the beads. FN did not form filaments when adsorbed onto the polysulfonate surface in the presence of soluble polysulfonates emphasizing the role of negatively charged, solid-phase elements on FN assembly. This charge-driven assembly likely derives from the negative surface promoting extension and opening of the protein, and we suggest a model where this assembly pattern is further stabilized by known self-assembly regions. Our results give insight into how FN fibrillogenesis might be promoted in vivo at cell surfaces by the negatively charged and sulfonated environment created by cell-surface, transmembrane proteoglycans.

Microfibrils are essential elements in elastic and nonelastic tissues contributing to homeostasis and growth factor regulation. Fibrillins form the core of these multicomponent assemblies. Various human genetic disorders, the fibrillinopathies, arise from mutations in fibrillins and are frequently associated with aberrant microfibril assembly. These disorders include Marfan syndrome, Weill–Marchesani syndrome, Beals syndrome, and others. Although homotypic and heterotypic fibrillin self-interactions are considered to provide critical initial steps, the detailed mechanisms for microfibril assembly are unknown. We show here that the C-terminal recombinant half of fibrillin-1 assembles into disulfide-bonded multimeric globular structures with peripheral arms and a dense core. These globules are similar to the beaded structures observed in microfibrils isolated from tissues. Only these C-terminal fibrillin-1 multimers interacted strongly with the fibrillin-1 N terminus, whereas the monomers showed very little self-interaction activity. The multimers strongly inhibited microfibril formation in cell culture, providing evidence that these recombinant assemblies can also interact with endogenous fibrillin-1. The C-terminal self-interaction site was fine-mapped to the last three calcium-binding EGF domains in fibrillin-1. These results suggest a new mechanism for microfibril formation where fibrillin-1 first oligomerizes via its C terminus before the partially or fully assembled bead-like structures can further interact with other beads via the fibrillin-1 N termini.

Fibronectin (FN) is a ubiquitously expressed cell adhesion protein capable of assembling into large, extended fibrillar networks as part of an extracellular matrix (ECM) that regulates cell behavior. FN is a substrate for certain members of the transglutaminase family of protein-crosslinking enzymes-enzymes which can modify the ability of FN to support cell adhesion. In this study, we have analyzed the thermo-chemical stability of plasma FN in its noncrosslinked form, and after crosslinking by transglutaminase 2 (TG2), using dynamic light scattering. We report that FN is found in a generally globular (8.7 nm hydrodynamic radius), dimerized form in aqueous solutions, but unfolds into a linear arrangement at high ionic (1 M NaCl) and chaotropic (5 M urea) environments. FN conformation remained stable after multiple heating and cooling cycles ranging from 4 to 60 degrees C. Crosslinking of FN with TG2 formed large, multimeric complexes having high chemical stability in aqueous, high ionic and chaotropic environments, demonstrating that this covalent modification stabilizes FN. Given recent data that substrate (e.g. ECM) rigidity profoundly affects cell differentiation and behavior, we further studied how TG2 crosslinking affects the molecular rigidity of FN by obtaining atomic force microscopy nanoindentation measurements from untreated and crosslinked FN samples embedded in acrylamide gels. We demonstrate that TG2-mediated crosslinking of FN significantly increases Young's modulus (of elasticity), an observation of increased rigidity having important implications with respect to the biological role of ECM protein-crosslinking in cell signaling and guiding cell differentiation.

Calcium phosphate has been used for over 30 years to deliver genetic material to mammalian cells. This vector has proven advantages over other transfection species such as viruses and dendrimers in terms of superior biocompatibility and reduced immune response. However, clinical application of calcium phosphate based transfection techniques is hampered by poor understanding of the key factors underlying its action. Despite widespread in vitro use, little attention has been given to the physico-chemical characteristics of the calcium phosphate particles mediating transfection. In this study parameters were optimised to produce calcium phosphate nanoparticles onto which plasmid DNA (pDNA) was adsorbed that were more effective than a commercial dendrimer vector in delivering pDNA to an osteoblastic cell line and compared favourably in a fibroblastic cell line without the need for special culture conditions such as cell cycle synchronization or glycerol shock treatment. Addition of the pDNA after nanoparticle synthesis allowed for characterisation of particle morphology, size, surface charge and composition. We found that the key parameters for effective calcium phosphate nanoparticle transfection were an optimal concentration of calcium and chloride ions and a nanosized non-agglomerated precipitate.

Fibronectin (FN) is a cell adhesion protein that binds integrins in a process also involving the protein-crosslinking enzyme transglutaminase 2 (TG2) as a co-receptor. The cell-adhesive property of TG2 has been linked to a complex formation with FN and to its ability to crosslink and polymerize FN on the cell surface. We tested here the effects of extracellular FN, before and after in vitro crosslinking and polymerization by TG2, on MC3T3-E1 osteoblast adhesion. We show that TG2-mediated crosslinking creates large, compacted chain-like protein clusters that include both TG2 and FN molecules as analyzed by Western blotting and atomic force microscopy. Crosslinking of FN significantly promotes osteoblast adhesion as measured by crystal violet staining, and enhances beta(1)-integrin clustering on the cell surface as visualized by immunofluorescence microscopy. We hypothesize that TG2-mediated crosslinking enhances the cell-adhesive properties of FN by increasing the molecular rigidity of FN in the extracellular matrix.

Little is known of the effect of material surfaces on stem cell differentiation. The present study has addressed the hypothesis that the interaction of mesenchymal stem cells (MSCs) with material surfaces modified by glow discharge plasma is a major regulator of osteogenic differentiation. We found that biaxially oriented polypropylene (BOPP) plasma treated in ammonia significantly reduced up-regulation of expression of osteogenic marker genes, such as alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OC). In contrast, ALP expression was up-regulated when cultured on treated Nylon-6 polyamide (Ny-t) but was substantially reduced when cultured on its pristine counterpart (Ny-p) on day 3. On day 7, ALP expression was down-regulated with MSCs cultured on Ny-t although its expression level was up again on day 14. BSP was expressed weakly on day 3, but was up-regulated when cultured on Ny-t and Ny-p. Its expression reached its maximum on day 14 when cultured on a polystyrene control, while it was cyclically up-regulated on Ny-t. Similarly, there was a slight increase in OC expression when MSCs were cultured on Ny-t and Ny-p on day 3, when compared to control. Thus, the nature of the surface can directly influence MSCs differentiation, ultimately affecting the quality of new tissue formation with BOPP-t suppressing osteogenic differentiation.