Multifunctional polyampholyte hydrogels with fouling resistance and protein conjugation capacity.

Materials that are resistant to nonspecific protein adsorption are critical in the biomedical community. Specifically, nonfouling implantable biomaterials are necessary to reduce the undesirable, but natural foreign body response. The focus of this investigation is to demonstrate that polyampholyte hydrogels prepared with equimolar quantities of positively charged [2-(acryloyloxy)ethyl] trimethylammonium chloride (TMA) and negatively charged 2-carboxyethyl acrylate (CAA) monomers are a viable solution to this problem. TMA/CAA hydrogels were prepared and their physical and chemical properties were characterized. The fouling resistance of the TMA/CAA hydrogels were assessed at varying cross-linker densities using enzyme-linked immunosorbant assays (ELISAs). The results clearly demonstrate that TMA/CAA hydrogels are resistant to nonspecific protein adsorption. A unique advantage of the fouling resistant TMA/CAA system is that bioactive proteins can be covalently attached to these materials using standard conjugation chemistry. This was demonstrated in this study through a combination of ELISA investigations and short-term cell adhesion assays. The multifunctional properties of the TMA/CAA polyampholyte hydrogels shown in this work clearly demonstrate the potential for these materials for use as tissue regeneration scaffolds for many biomedical applications.

Mineralization induction effects of osteopontin, bone sialoprotein, and dentin phosphoprotein on a biomimetic collagen substrate.

Native bone tissue is composed of a matrix of collagen, noncollagenous proteins, and calcium phosphate minerals, which are primarily hydroxyapatite. The SIBLING (small integrin-binding ligand, N-linked glycoprotein) family of proteins is the primary noncollagenous protein group found in mineralized tissues. In this work, the mineralization induction capabilities of three of the SIBLING members, bone sialoprotein (BSP), osteopontin (OPN), and the calcium-binding subdomain of dentin sialophosphoprotein, dentin phosphoprotein (DPP), are directly compared on a biomimetic collagen substrate. A self-assembled, loosely aligned collagen fibril substrate was prepared, and then (125) I-radiolabeled adsorption isotherms were developed for BSP, OPN, and DPP. The results showed that BSP exhibited the highest binding capacity for collagen at lower concentrations, followed by DPP and OPN. However, at the highest concentrations, all three proteins had similar adsorption levels. The adsorption isotherms were then used to identify conditions that resulted in identical amounts of adsorbed protein. These substrates were prepared and placed in simulated body fluid for 5, 10, and 24 h at 37°C. The resulting mineral morphology was assessed by atomic force microscopy, and the composition was determined using photochemical assays. Mineralization was seen in the presence of all the proteins. However, DPP was seen to be the only protein that formed individual mineral nodules similar to those seen in developing bone. This suggests that DPP plays a significant role in the biomineralization process and that the incorporation of DPP into tissue engineering constructs may facilitate the induction of biomimetic mineral formation.

Tailoring the protein adsorption properties of whispering gallery mode optical biosensors.

Label-free biosensor technologies have the potential to revolutionize environmental monitoring, medical diagnostics, and food safety evaluation processes due to their unique combinations of high-sensitivity signal transducers and high-specificity recognition elements. This enables their ability to perform real-time detection of deleterious compounds at extremely low concentrations. However, to further improve the biosensors' performance in complex environments, such as wastewater, blood, and urine, it is necessary to minimize nonspecific binding, which in turn will increase their specificity, and decrease the rate of false positives. In the present work, we illustrate the potential of combining emerging high-sensitivity optical signal transducers, such as whispering gallery mode (WGM) microcavities, with covalently bound poly(ethylene glycol) (PEG) coatings of varying thickness, as an effective treatment for the prevention of nonspecific protein adsorption onto the biosensor surface. We monitor the sensitivity of the coated biosensor, and investigate the effect of PEG chain length on minimizing nonspecific adsorption via protein adsorption studies. Experimental results confirm not only that PEG-functionalization reduces nonspecific protein adsorption to the surface of the sensor by as much as a factor of 4 compared to an initialized control surface, but also that chain length significantly impacts the nonfouling character of the microcavity surface. Surprisingly, it is the short chain PEG surfaces that experience the best improvement in specificity, unlike many other systems where longer PEG chains are preferred. The combination of WGM microcavities with PEG coatings tuned specifically to the device will significantly improve the overall performance of biosensor platforms, and enable their wider application in complex, real-world monitoring scenarios.

Adhesion of MC3T3-E1 cells bound to dentin phosphoprotein specifically bound to collagen type I.

Dentin sialophosphoprotein (DSPP) is a member of the SIBLING (small integrin binding N-linked glycoprotein) family of proteins commonly found in mineralized tissues. Dentin phosphoprotein (DPP) is a naturally occurring subdomain of DSPP that contains the cell binding RGD sequence. Previously, the orientation and conformation of other SIBLING family members specifically bound to collagen I have been investigated with respect to their cell adhesion properties. In this study, the orientation of DPP under similar circumstances is examined, and the results are discussed relative to the previous investigations. Radiolabeled adsorption isotherms were developed for DPP adsorbing to both tissue culture polystyrene (TCPS) and collagen coated TCPS. Then, a MC3T3-E1 cell adhesion assay was performed on TCPS and collagen coated TCPS in the presence of identical amounts of adsorbed DPP. It was discovered that there was a significant difference in the number of bound cells on the TCPS and collagen coated TCPS, with a preference for TCPS. Furthermore, a cell inhibition assay was conducted to confirm that the cell binding that occurred was due to specific integrin interactions with the RGD sequence of DPP. These results suggest that the orientation of DPP, rather than its conformation, dictates the accessibility of the cell binding RGD domains of DPP and that the RGD sequence in DPP is less accessible when DPP is specifically bound to collagen. The results obtained in this study are in stark contrast to previous studies with related SIBLING proteins, and suggest that DPP does not play a key role in cell binding to the collagen matrix of developing bone.