Comparison of the adsorption kinetics and surface arrangement of "as received" and purified bovine submaxillary gland mucin (BSM) on hydrophilic surfaces.

The effect of bovine serum albumin (BSA) as impurity in a commercial bovine submaxillary gland mucin preparation (BSM; Sigma M3895) on the adsorption of BSM to hydrophilic surfaces (mica and silica) has been studied in terms of adsorption kinetics, amount and structure of the formed adlayer. The Surface Force Apparatus (SFA) was used to gain information about the extended and compressed structure of adsorbed "as received" BSM, purified BSM, BSA extracted from the "as received" BSM and mixtures of the latter purified proteins. The adsorbed amount was estimated using a combination of X-ray Photoelectron Spectroscopy (XPS), Enzyme-Linked Immuno Sorbent Assay (ELISA), Enzyme-Linked Lectin Assay (ELLA), Dual Polarization Interferometry (DPI) and Quartz Crystal Microbalance (QCM-D) measurements. Under the used conditions, purified BSM showed very low affinity for silica and only small amounts were found to adsorb on mica. Initially, the BSM molecules adopted an extended conformation on the mica surface with tails extending into the bulk phase. These tails were irreversibly compressed into a very thin (10A) layer upon applying a high load. "As received" BSM formed considerably thicker compressed layers (35A); however, the extended layer structure was qualitatively the same. When mixtures of purified BSM and BSA were coadsorbed on mica, a 9wt-% albumin content gave a comparable layer thickness as the "as received" BSM and from XPS data we draw the conclusion that the albumin content in the layer adsorbed from "as received" BSM was approximately 5wt-%. Adsorption from an equal amount of BSM and BSA revealed that even though the amount of BSM is scarce in the mixed layer, the few BSM molecules have a drastic effect on the adsorbed thickness and structure. Clearly, this study shows the importance of characterizing the mucin used since differences in purity give rise to different adsorption behaviours in terms of both adsorbed amount and layer structure.

Surface analysis of pure and complex mucin coatings on a real-type substrate using individual and combined mBCA, ELLA, and ELISA.

In the past, we introduced the idea of using mucin coatings to improve biomaterials performance. Here, we evaluate non-radioactive methods for the analysis of pure and human host protein-containing (complex) mucin coatings on a real-type substrate (Thermanox). A common protein quantification assay (mBCA) was combined with mass-calibrated, enzyme-amplified assays based on lectin (ELLA) and antibody (ELISA) recognition, to determine the total and specific amounts of surface-associated proteins. Model studies showed the mBCA assay to be of limited use at low mass loads, and steric effects to influence the ELLA at high surface layer densities. Non-specific responses due to substrate interaction were low for the ELLA and ELISAs. Cross-reactions were observed during ELLA analysis of analytes sharing high degree of O-glycosylation. Combined mBCA-ELLA-ELISA analysis suggested that mucin desorption was low upon protein addition and that low concentrations of ELISA-determined protein for the complex coatings could be explained in terms of low accessibility of proteins to the bulk environment. Specifically, a methodology is presented for the determination of the fraction of surface-exposed, presumed bioactive proteins in a complex mucin coating. Finally, X-ray photoelectron spectroscopy and infrared reflectance spectroscopy combined with multivariate data analysis were proven useful in the evaluation of mucin-based coatings.

Interactions between human neutrophils and mucin-coated surfaces.

Recently, we showed microscopically that bovine (BSM), porcine (PGM) and human (MG1) mucin coatings could suppress the adhesion of neutrophils to a polyethylene terephthalate-based model biomaterial (Thermanox). Here, using the release of reactive oxygen species (ROS) as a marker of material-induced neutrophil activation, the strong surface-passivating effects of these mucin coatings were corroborated. Under optimal adsorption conditions, all mucin species performed equally well, thus indicating a high degree of functional homology between the mucins. Cell adhesion and morphology correlated well with the release of ROS. Quartz crystal microbalance (QCM-D) analysis linked low neutrophil activation to efficient mucin surface-shielding. Interestingly, the shielding power appeared equal for thick expanded and thin compact mucin coatings. Combined mucin-serum coatings were found to be highly surface-passivating. Particularly, since our data suggested partly synergistic mucin-serum action, we highlight the possibility that pre-adsorbed mucins could provide favorable support for adsorbing host components.

Potential use of mucins as biomaterial coatings. II. Mucin coatings affect the conformation and neutrophil-activating properties of adsorbed host proteins--toward a mucosal mimic.

In continuation of our recent fractionation and characterization study on mucins of bovine salivary (BSM), porcine gastric (PGM), and human salivary (MG1) origin, this study evaluates the effect of mucin precoating on the conformation and neutrophil-activating properties of host proteins adsorbed to a polyethylene terephthalate-based model biomaterial. Microscopy combined with assays for the neutrophil releases of reactive oxygen species and human neutrophil lipocalin showed that mucin precoating greatly reduced the strong immune-response normally induced by adsorbed immunoglobulin G (IgG) and secretory immunoglobulin A (sIgA), respectively. A similar finding was made for the proinflammatory fibrinogen. Although the total uptakes of these proteins depended on the mucin surface concentration, a detailed composite analysis suggested the fraction of surface-exposed protein to be a stronger determinant of coating performance. The unexpectedly low neutrophil activation showed by composites containing near-monolayer concentrations of exposed IgG and sIgA, respectively, suggested that these act synergistically with mucin on the surface. In support of this hypothesis, quartz crystal microbalance with dissipation monitoring measurements revealed that a preadsorbed BSM layer stabilizes IgG through complexation on a polymeric model surface. Our findings link well to the complex in vivo situation and suggest that functional mucosal mimics can be created in situ for improved biomaterials performance.

Potential use of mucins as biomaterial coatings. I. Fractionation, characterization, and model adsorption of bovine, porcine, and human mucins.

Previously, we presented evidence that mucins have potential as biomaterial coatings. Here, we reveal substantial batch-to-batch variations for a frequently used commercial bovine salivary mucin preparation (BSM) and stress the importance of standardizing mucins intended for comparative purposes. "Mild" fractionation strategies, aiming at preserving natural mucin functions, were used to prepare two more defined BSM fractions as well as three mucin fractions from porcine gastric (PGM) and human salivary (MG1) sources. While the BSM and PGM were highly purified and mainly adopted random coil conformations in solution, the MG1 contained mucin-bound components (1.6 wt% albumin) and appeared compact. Average molar masses and root-mean-square radii for the predominant BSM, PGM, and MG1 species spanned 0.8-4.2 MDa and 46-86 nm, respectively. An ellipsometric evaluation, using hydrophilic and hydrophobic silica, showed the mucin adsorption to be slow and related to mucin charge, size, conformation, and compositional complexity. The mass uptakes on hydrophobic silica averaged 2.6, 2.6, and 5.0 mg/m(2), for BSM, PGM, and MG1, respectively. Finally, we find that stable mucin coatings can be formed on polymers of different wettability. The reported mucin preparations serve as platforms for a series of studies on the biocompatibility of mucin coatings.

Adsorption and viscoelastic properties of fractionated mucin (BSM) and bovine serum albumin (BSA) studied with quartz crystal microbalance (QCM-D).

The adsorption profile and viscoelastic properties of bovine submaxillary gland mucin (BSM) and bovine serum albumin (BSA), extracted from a commercial mucin preparation, adsorbing to polystyrene surfaces has been studied using quartz crystal microbalance with dissipation monitoring (QCM-D). A significant difference in the adsorption properties of the different proteins was detected; with the BSA adsorbing in a flat rigid layer whilst the mucin adsorbed in a diffuse, highly viscoelastic layer. Subsequent addition of BSA to the preadsorbed mucin layer resulted in stiffening of the protein layer which was attributed to complexation of the mucin by BSA. In contrast, a preadsorbed layer of BSA prevented mucin adsorption altogether. Combined mixtures of mucin and BSA in well defined ratios revealed intermediate properties between the two separate protein species which varied systematically with the protein ratios. The results shed light on the synergistic effects of complexation of lower molecular weight biomolecular species with mucin. The possibility to selectively control protein uptake and tailor the physical properties of the adsorbed layer makes mucin an attractive option for application in biomaterial coatings.

Mucin coatings suppress neutrophil adhesion to a polymeric model biomaterial.

Following our recent study on the fractionation, characterization, and model adsorption of mucins derived from bovine salivary glands (BSM), porcine stomach scrapings (PGM), and human whole saliva (MG1), we here present a microscopic evaluation of the interactions between mucin-coated substrates and human neutrophils. Our results show that surface-coating with BSM, PGM, and MG1 can be effectively used to suppress the adhesion of neutrophils to a polymeric model biomaterial (Thermanox). Neutrophil morphologies found on Thermanox substrates coated with mucins resemble those observed for nonactivated neutrophils found in circulation. Notably, low neutrophil adhesion can be obtained at a significantly lower coating concentration (0.125 mg/mL) for the compositionally complex MG1 mucin than for the relatively pure BSM and PGM mucins (1 mg/mL). Furthermore, since coating at a low BSM and PGM concentration (0.25 mg/mL) results in higher cell counts and more spread cells than in the high-concentration case, we suggest that dense mucin surface packing is critical for good coating performance. In conclusion, the present study demonstrates how mucins from three different sources, of different compositional and structural status, efficiently can be used to suppress neutrophil adhesion and activation. This finding makes them potent candidates for use as biomaterial coatings.