Search

Polyelectrolytes are known to greatly affect calcium phosphate (CaP) mineralization. The reaction kinetics as well as the CaP phase, morphology and aggregation state depend on the relative concentrations of the polyelectrolyte and the inorganic ions in a complex, nonlinear manner. This study examines the structural evolution and kinetics of polyaspartic acid (pAsp) directed CaP mineralization at high concentrations of polyelectrolytes, calcium, and total phosphate (19-30 mg/mL pAsp, 50-100 mM Ca 2+ , Ca/P = 2). Using a novel combination of characterization techniques including cryogenic transmission electron microscopy (cryo-TEM), spectrophotometry, X-ray total scattering pair distribution function analysis, and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), it was determined that the CaP mineralization occurred over four transition steps. The steps include the formation of aggregates of pAsp stabilized CaP spherical nanoparticles (sNP), crystallization of sNP, oriented attachment of the sNP into nanorods, and further crystallization of the nanorods. The intermediate aggregate sizes and the reaction kinetics were found to be highly polymer concentration dependent while the sizes of the particles were not concentration dependent. This study demonstrates the complex role of pAsp in controlling the mechanism as well as the kinetics of CaP mineralization.

Bone is an important material to study due to its exceptional mechanical properties and relevance with respect to hard tissue regeneration and repair. A significant effort has been directed toward understanding the bone formation process and the production of synthetic bone mimicking materials. Here, the formation and structural evolution of calcium phosphate (CaP) was investigated in the presence of relatively high concentrations of calcium, phosphate, and polyaspartic acid (pAsp) using dynamic light scattering (DLS) and cryo-transmission electron microscopy (cryo-TEM). The incipient CaP aggregates were comprised of spherical nanoparticles (diameter ≈ 3-4 nm); they became preferentially aligned over time and eventually transformed into nanorods. The nanorods remained stable in suspension with no signs of further aggregation for at least four months. Detailed cryo-TEM suggested that the CaP nanorods formed through an oriented attachment mechanism. These results show that the reaction concentration greatly influences the mechanism and final properties of CaP. Mechanistic insights gained from this study will facilitate better design and fabrication of bioinspired materials.

Insight into live microbial biofilm microstructure and mechanical properties and their interactions with the underlying substrate can lead to the development of new remedial strategies and/or materials. Here we report mechanical properties of dental pathogenic Streptococcus mutans biofilms, grown on a polystyrene-coated plate of a shear rheometer in physiologically relevant conditions, precisely controlled in a custom built bioreactor. In situ measurements demonstrated the importance of microstructure and composition of extracellular polymeric substances on the biofilm modulus. The biofilms behave like a weak gel with storage moduli higher than loss moduli. The simple but robust experimental technique presented here can easily be extended to other biofilm-material systems.

Acidic proteins are critical to biomineral formation, although their precise mechanistic function remains poorly understood. A number of recent studies have suggested a nonclassical mineralization model that emphasizes the importance of the formation of polymer-stabilized mineral clusters or particles; however, it has been difficult to characterize the precursors experimentally due to their transient nature. Here, we successfully captured stepwise evolution of transient CaP clusters in mineralizing solutions and studied the roles of functional polymers with laser light scattering (LLS) to determine how these polymers influence the stability of nanoclusters. We found that the polymer structure can alter CaP aggregation mechanisms, whereas the polymer concentration strongly influences the rate of CaP aggregation. Our results indicate that the ability of acidic biomolecules to control the formation of relatively stable nanoclusters in the early stages may be critical for intrafibrillar mineralization. More importantly, LLS provided information about the size and the structural evolution of CaP aggregates, which will help define the process of controlled biomineralization.

Although "chaperone molecules" rich in negatively charged residues (i.e., glutamic and aspartic acid) are known to play important roles in the biomineralization process, the precise mechanism by which type I collagen acquires intrafibrillar mineral via these chaperone molecules remains unknown. This study demonstrates a mechanism of cooperative nucleation in which three key components (collagen, chaperone molecules, and Ca(2+) and PO(4)(3-)) interact simultaneously. The mineralization of collagen under conditions in which collagen was exposed to pAsp, Ca(2+), and PO(4)(3-) simultaneously or pretreated with the chaperone molecule (in this case, poly(aspartic acid)) before any exposure to the mineralizing solution was compared to deduce the mineralization mechanism. Depending on the exact conditions, intrafibrillar mineral formation could be reduced or even eliminated through pretreatment with the chaperone molecule. Through the use of a fluorescently tagged polymer, it was determined that the adsorption of the chaperone molecule to the collagen surface retarded further adsorption of subsequent molecules, explaining the reduced mineralization rate in pretreated samples. This finding is significant because it indicates that chaperone molecules must interact simultaneously with the ions in solution and collagen for biomimetic mineralization to occur and that the rate of mineralization is highly dependent upon the interaction of collagen with its environment., (© 2011 American Chemical Society)

Characterization of materials developed for medical usage frequently includes studies in which the materials are inoculated with bacteria in order to assess bacterial colonization and biofilm formation. Observed differences in bacterial growth are typically considered to be due to the material or the incubation conditions. To our knowledge, the method used to prepare the materials has generally not been considered with regard to its influence on bacterial colonization. The objective of this study was to determine the effects that various preparation methods exert on bacterial colonization of polymer disks. Polymer disks of the same dimethacrylate composition were photopolymerized: (1) between untreated glass slides, (2) between polyester release film, (3) between glass slides treated with an alkyl silane, (4) between glass slides treated with a perfluorinated silane, or (5) with one free surface in an argon-purged chamber. Surface chemistry was quantified using X-ray photoelectron spectroscopy, hydrophobicity was assessed by water contact angle, and topography was characterized using atomic force microscopy. The disks were inoculated with Streptococcus mutans for 4 h, fixed, and visualized using confocal laser scanning microscopy. Differences among all groups were found with regard to surface chemistry, hydrophobicity, topography, and bacteria morphology, density, and coverage, indicating that the method of sample preparation strongly affects both the surface properties and the initial bacterial colonization. Polymerization on untreated slides was selected as the preferred method of preparation due to minimal material transfer to the polymer and consistent, reproducible bacterial colonization.

We are interested in developing elastin-mimetic hybrid polymers (EMHPs) that capture the multiblock molecular architecture of tropoelastin as well as the remarkable elasticity of mature elastin. In this study, multiblock EMHPs containing flexible synthetic segments based on poly(ethylene glycol) (PEG) alternating with alanine-rich, lysine-containing peptides were synthesized by step-growth polymerization using α,ω-azido-PEG and alkyne-terminated AKA(3)KA (K = lysine, A = alanine) peptide, employing orthogonal click chemistry. The resulting EMHPs contain an estimated three to five repeats of PEG and AKA(3)KA and have an average molecular weight of 34 kDa. While the peptide alone exhibited α-helical structures at high pH, the fractional helicity for EMHPs was reduced. Covalent cross-linking of EMHPs with hexamethylene diisocyanate (HMDI) through the lysine residue in the peptide domain afforded an elastomeric hydrogel (xEMHP) with a compressive modulus of 0.12 MPa when hydrated. The mechanical properties of xEMHP are comparable to a commercial polyurethane elastomer (Tecoflex SG80A) under the same conditions. In vitro toxicity studies showed that while the soluble EMHPs inhibited the growth of primary porcine vocal fold fibroblasts (PVFFs) at concentrations ≥0.2 mg/mL, the cross-linked hybrid elastomers did not leach out any toxic reagents and allowed PVFFs to grow and proliferate normally. The hybrid and modular approach provides a new strategy for developing elastomeric scaffolds for tissue engineering.

A systematic investigation of tissue engineering scaffolds prepared by salt leaching of a photopolymerized dimethacrylate was performed to determine how the scaffold structure (porosity, pore size, etc.) can be controlled and also to determine how the scaffold structure and the mechanical properties are related. Two series of scaffolds were prepared with (1) the same polymer-to-salt ratio but different salt sizes (ranging from average size of 100 to 390 microm) and (2) the same salt size but different polymer-to-salt ratios (ranging from salt mass of 70 to 90%). These scaffolds were examined to determine how the fabrication parameters affected the scaffold morphometric parameters and corresponding mechanical properties. Combined techniques of X-ray microcomputed tomography (microCT), mercury porosimetry, and gravimetric analysis were used to determine the scaffold parameters, such as porosity, pore size, and strut thickness and their size distributions, and pore interconnectivity. Scaffolds with porosities ranging from 57% to 92% (by volume) with interconnected structures could be fabricated using the current technique. The porosity and strut thickness were subsequently related to the mechanical response of the scaffolds, both of which contribute to the compression modulus of the scaffold. The current study shows that the structure and properties of the scaffold could be tailored by the size and the amount of porogen used in the fabrication of the scaffold.

Model tissue engineering scaffolds based on photocurable resin mixtures with sodium chloride have been prepared for optical imaging studies of cell attachment. A photoactivated ethoxylated bisphenol A dimethacrylate was mixed with sieved sodium chloride (NaCl) crystals and photocured to form a cross-linked composite. Upon soaking in water, the NaCl dissolved to leave a porous scaffold with desirable optical properties, mechanical integrity, and controlled porosity. Scaffolds were prepared with salt crystals that had been sieved to average diameters of 390, 300, 200, and 100 microm, yielding porosities of approximately 75 vol %. Scanning electron microscopy and X-ray microcomputed tomography confirmed that the pore size distribution of the scaffolds could be controlled using this photocuring technique. Compression tests showed that for scaffolds with 84% (by mass fraction) salt, the larger pore size scaffolds were more rigid, while the smaller pore size scaffolds were softer and more readily compressible. The prepared scaffolds were seeded with osteoblasts, cultured between 3 and 18 d, and examined using confocal microscopy. Because the cross-linked polymer in the scaffolds is an amorphous glass, it was possible to optically image cells that were over 400 microm beneath the surface of the sample.

Blends of cross-linked poly(ethylene glycol) dimethacrylate (PEGDMA) and poly(d,l-lactide) (PLA) were prepared by mixing photoactive PEGDMA (molecular mass: 875 g/mol) and PLA, and subsequently photopolymerizing the mixture with visible light. The effects of PLA molecular mass and mass fraction on the rheological properties of the PEGDMA/PLA mixtures, and on the degree of methacrylate vinyl conversion (DC), as well as blend miscibility, microstructure, mechanical properties, in vitro swelling behavior, and cell responses were studied. PLA-2K (molecular mass: 2096 g/mol) and PLA-63K (molecular mass: 63 000 g/mol) formed miscible and partially miscible blends with cross-linked PEGDMA, respectively. The addition of the PLA-2K did not affect the immediate or post-cure (>24 h) DC of the PEGDMA upon photopolymerization. However, the addition of PLA-63K decreased the immediate DC of the PEGDMA, which can be increased through extending the curing time or post-curing period. Compared to the cross-linked neat PEGDMA and PLA-2K/PEGDMA blends, PLA-63K/PEGDMA blends were significantly stronger, stiffer, and tougher. Both types of blends and the cross-linked PEGDMA swelled when soaked in a phosphate buffered saline (PBS) solution. The attachment and spreading of MCT3-E1 cells increased with increasing PLA-63K content in the blends. The facile and rapid formation of PEGDMA/PLA blends by photopolymerization represents a simple and efficient approach to a class of biomaterials with a broad spectrum of properties.

Facile synthesis and detailed characterization of photopolymerizable and biocompatible poly(ethylene glycol) dimethacrylates (PEGDM) and poly(ethylene glycol) urethane-dimethacrylates (PEGUDM) are described. Poly(ethylene glycol)s of various molecular masses (M(n) = 1000 to 8000 g/mol) were reacted with methacrylic anhydride or with 2-isocyanatoethyl methacrylate to form PEGDMs and PEGUDMs, respectively. PEGDMs were also prepared by a microwave-assisted route to achieve fast reaction conversions under solvent free conditions. Combined analyses of (1)H NMR and MALDI-TOF MS confirmed the formation of prepolymers of high purity and narrow mass distribution (PD < 1.02). Aqueous solutions of the PEGDMs and PEGUDMs (10% and 20% by mass fraction) were photopolymerized to yield hydrogels. Bovine chondrocytes, seeded in the hydrogels, were used to assess the biocompatibility. Preliminary rheology and uniaxial compression measurements showed varied mechanical response, and biocompatibility studies showed that cells are completely viable in both types of hydrogels after two weeks.