Within milliseconds after biomaterials come in contact with a biological fluid such as blood, proteins begin to adhere to the surface through a process known as protein adsorption. Protein adsorption is initially strongly influenced by protein diffusion, but protein affinity for the surface becomes critically important and, over time, higher-affinity proteins can be replaced by lower-affinity proteins in a dynamic process. By the time cells arrive, the material surface has already been coated in a monolayer of proteins; hence, the host cells do not "see" the material but "see" instead a dynamic layer of proteins. Multiple parameters influence protein adsorption to a substrate surface including the chemical and physical properties of both the protein and the material surface, as well as the presence of other proteins on the surface.Many methods have been developed in the last several decades to study protein adsorption to biomaterial surfaces. These new techniques provide information about the type and conformation of adsorbed proteins from multicomponent solutions such as blood serum. Nanomaterials as well as functional group immobilization and novel, stimuli-sensitive polymer surfaces have provided new alternatives for the study and modulation of protein adsorption, with insight into the mechanisms underlying protein adsorption and subsequent cell adhesion. However, a molecular-level understanding of all aspects of protein adsorption is still incomplete. The future of this field, however, is bright as new technologies offer great promise for further elucidation of protein adsorption. Abbreviations AFM

The purpose of this study was to evaluate the biocompatibility and the efficacy in wound healing of a gelatin-based interpenetrating polymer network (IPN) containing poly(ethylene glycol) (PEG)-ylated RGD and soluble KGF-1 (RGD-IPN+KGF). IPNs were applied to full-thickness wounds on a rat model. Wound healing was assessed through histological grading of the host response and percent area contraction at 2 days, 1 week, 2 weeks, and 3 weeks. A control IPN containing unmodified gelatin (unmod-IPN) and a conventional clinical bandage were applied to similar wounds and also evaluated. During the first week of healing, the unmod-IPN and conventional dressing wound showed a greater amount of contraction than that of RGD-IPN+KGF. However, by 3 weeks the extent of wound contraction was comparable between treatments. The RGD-IPN+KGF treated wound demonstrated lower macrophage and fibroblast densities at 3 weeks as compared to unmod-IPN treated wounds. RGD-IPN+KGF acted as a tissue scaffold while preventing the entry of foreign bodies, advantages not seen with the conventional dressing. The extent of cellularity and extracellular matrix organization was higher for wounds healed with RGD-IPN+KGF than those healed with unmod-IPN. These results indicate that both soluble and immobilized bioactive factors can be incorporated into our IPN platform to enhance the rate and the quality of dermal wound healing.

Monocytes/Macrophages have long been recognized as key players in inflammation and wound healing and are often employed in vitro to gain an understanding of the inflammatory response to biomaterials. Previous work has demonstrated a drastic decrease in primary monocyte adherent density on biomaterial surfaces coupled with a change in monocyte behavior over time. However, the mechanism responsible for this decrease was unclear. In this study, we explored active detachment and cellular death as possible regulating factors. Specifically, extracellular TNF-α and ROS production were analyzed as potential endogenous stimulators of cell death. MMPs, but not calpains, were found to play a key role in active monocyte detachment. Monocyte death was found to peak at 24hr and occur by both apoptosis and necrosis as opposed to polymorphonuclear leukocyte death which mainly occurred through apoptosis. Finally, TNF-α and ROS production were not found to have a causal relationship with monocyte death on TCPS or PEG surfaces. The occurrence of primary monocyte apoptosis/necrosis as well as active detachment from a material surface has implications not only in in vitro study, but also in the translation of the in vitro inflammatory response of these cells to in vivo applications.

Ligands presented on biomaterials are a common method to facilitate and control the host response. In a gelatin and polyethylene glycol diacrylate (PEGdA) based semi-interpenetrating network (sIPN), the effects of extracellular matrix (ECM)-derived peptide amount on monocyte adhesion and subsequent protein and mRNA expression were examined. Peptide amount on the sIPN surface was controlled by varying the wt% ratio of the peptide-PEG grafted gelatin to PEGdA. We hypothesized that increasing bioactive peptide amount would modulate human blood derived monocyte adhesion, cytokine expression and gene regulation. Monocyte adhesion, release of gelatin degrading proteases matrix metalloprotease-2 (MMP-2), matrix metalloprotease-9 (MMP-9) and pro-inflammatory protein interleukin-1β (IL-1β), and mRNA expression of these proteins were evaluated. We found RGD-PEG grafted sIPNs with higher surface RGD concentrations showed increased adherent density. MMP-2 and IL-1β protein release was also influenced by the ligand concentration, as initial increase in protein concentration was observed at higher ligand concentrations. MMP-9 protein showed an initial increase that subsided then increased. A decreased IL-1β protein and mRNA expression was observed over time but MMP-2 mRNA was not detected at any time though MMP-2 protein concentrations showed an initial burst. Hence, monocyte behavior was modulated by surface ligand identity in tandem with ligand concentration.

Characterization of the degradation mechanisms and resulting products of biodegradable materials is critical in understanding the behavior of the material including solute transport and biological response. Previous mathematical analyses of a semi-interpenetrating network (sIPN) containing both labile gelatin and a stable cross-linked poly(ethylene glycol) (PEG) network found that diffusion-based models alone were unable to explain the release kinetics of solutes from the system. In this study, degradation of the sIPN and its effect on solute release and swelling kinetics were investigated. The kinetics of the primary mode of degradation, gelatin dissolution, was dependent on temperature, preparation methods, PEGdA and gelatin concentration, and the weight ratio between the gelatin and PEG. The gelatin dissolution rate positively correlated with both matrix swelling and the release kinetics of high-molecular-weight model compound, FITC-dextran. Coupled with previous in vitro studies, the kinetics of sIPN degradation provided insights into the time-dependent changes in cellular response including adhesion and protein expression. These results provide a facile guide in material formulation to control the delivery of high-molecular-weight compounds with concomitant modulation of cellular behavior.