Cells resident to precision templated 40-µm pore scaffolds generate small extracellular vesicles that affect CD4+ T cell phenotypes through regulatory TLR4 signaling.

Precision porous templated scaffolds (PTS) are a hydrogel construct of uniformly sized interconnected spherical pores that induce a pro-healing response (reducing the foreign body reaction, FBR) exclusively when the pores are 30-40µm in diameter. Our previous work demonstrated the necessity of Tregs in the maintenance of PTS pore size specific differences in CD4+ T cell phenotype. Work here characterizes the role of Tregs in the responses to implanted 40µm and 100µm PTS using WT and FoxP3+ cell (Treg) depleted mice. Proteomic analyses indicate that integrin signaling, monocytes/macrophages, cytoskeletal remodeling, inflammatory cues, and vesicule endocytosis may participate in Treg activation and the CD4+ T cell equilibrium modulated by PTS resident cell-derived small extracellular vesicles (sEVs). The role of MyD88-dependent and MyD88-independent TLR4 activation in PTS cell-derived sEV-to-T cell signaling is quantified by treating WT, TLR4ko, and MyD88ko splenic T cells with PTS cell-derived sEVs. STAT3 and mTOR are identified as mechanisms for further study for pore-size dependent PTS cell-derived sEV-to-T cell signaling. STATEMENT OF SIGNIFICANCE: Unique cell populations colonizing only within 40µm pore size PTS generate sEVs that resolve inflammation by modifying CD4+ T cell phenotypes through TLR4 signaling.

Epidermal and dermal integration into sphere-templated porous poly(2-hydroxyethyl methacrylate) implants in mice.

Percutaneous medical devices remain susceptible to infection and failure. We hypothesize that healing of the skin into the percutaneous device will provide a seal, preventing bacterial attachment, biofilm formation, and subsequent device failure. Porous poly(2-hydroxyethyl methacrylate) [poly(HEMA)] with sphere-templated pores (40 microm) and interconnecting throats (16 microm) were implanted in normal C57BL/6 mice for 7, 14, and 28 days. Poly(HEMA) was either untreated, keeping the surface nonadhesive for cells and proteins, or modified with carbonyldiimidazole (CDI) or CDI reacted with laminin 332 to enhance adhesion. No clinical signs of infection were observed. Epidermal and dermal response within the poly(HEMA) pores was evaluated using light and transmission electron microscopy. Cells (keratinocytes, fibroblasts, endothelial cells, inflammatory cells) and basement membrane proteins (laminin 332, beta4 integrin, type VII collagen) could be demonstrated within the poly(HEMA) pores of all implants. Blood vessels and dermal collagen bundles were evident in all of the 14- and 28-day implants. Fibrous capsule formation and permigration were not observed. Sphere-templated polymers with 40 microm pores demonstrate an ability to recapitulate key elements of both the dermal and the epidermal layers of skin. Our morphological findings indicate that the implant model can be used to study the effects of biomaterial pore size, pore interconnect (throat) size, and surface treatments on cutaneous biointegration. Further, this model may be used for bacterial challenge studies.

A mouse model to evaluate the interface between skin and a percutaneous device.

Percutaneous medical devices are integral in the management and treatment of disease. The space created between the skin and the device becomes a haven for bacterial invasion and biofilm formation and results in infection. We hypothesize that sealing this space via integration of the skin into the device will create a barrier against bacterial invasion. The purpose of this study was to develop an animal model in which the interaction between skin and biomaterials can be evaluated. Porous poly(2-hydroxyethyl methacrylate) [poly(HEMA)] rods were implanted for 7 days in the dorsal skin of C57 BL/6 mice. The porous poly(HEMA) rods were surface-modified with carbonyldiimidazole (CDI) or CDI plus laminin 5; unmodified rods served as control. Implant sites were sealed with 2-octyl cyanoacrylate; corn pads and adhesive dressings were tested for stabilization of implants. All rods remained intact for the duration of the study. There was histological evidence of both epidermal and dermal integration into all poly(HEMA) rods regardless of treatment. This in vivo model permits examination of the implant/skin interface and will be useful for future studies designed to facilitate skin cell attachment where percutaneous devices penetrate the skin.

Novel biophysical techniques for investigating long-term cell adhesion dynamics on biomaterial surfaces.

Cell adhesion on biomaterial surface is crucial for the regeneration and function of clinically viable cell and tissues. In turn, the cellular phenotypes, following the mechanochemical transduction of adherent cells on biomaterials, are directly correlated to the biophysical responses of cells. However, the lack of an integrated bio-analytical system for probing the cell-substrate interface poses significant obstacles to understanding the behavior of cells on biomaterial surface. We have developed a novel method, based on the principle of confocal reflectance interference contrast microscopy (C-RICM) that has enabled us to study the biomechanical deformation of cells on biomaterial surfaces. In this article, we would like to describe our recent development of the C-RICM system that integrates a confocal fluorescence microscope, phase contrast microscope and GFP expression system. We shall demonstrate the system by determining the adhesion contact kinetics, initial deformation rate, cytoskeleton structures of adherent cells on extracellular matrices (e.g., collagen and fibronectin) and biodegradable polymer (e.g., poly(lactic acid)) during long-term culture. We shall demonstrate that this unique approach could provide valuable biophysical information necessary for designing optimized biomaterial surfaces for cell/tissue regeneration applications.

Ultrasonically controlled release of ciprofloxacin from self-assembled coatings on poly(2-hydroxyethyl methacrylate) hydrogels for Pseudomonas aeruginosa biofilm prevention.

Indwelling prostheses and subcutaneous delivery devices are now routinely and indispensably employed in medical practice. However, these same devices often provide a highly suitable surface for bacterial adhesion and colonization, resulting in the formation of complex, differentiated, and structured communities known as biofilms. The University of Washington Engineered Biomaterials group has developed a novel drug delivery polymer matrix consisting of a poly(2-hydroxyethyl methacrylate) hydrogel coated with ordered methylene chains that form an ultrasound-responsive coating. This system was able to retain the drug ciprofloxacin inside the polymer in the absence of ultrasound but showed significant drug release when low-intensity ultrasound was applied. To assess the potential of this controlled drug delivery system for the targeting of infectious biofilms, we monitored the accumulation of Pseudomonas aeruginosa biofilms grown on hydrogels with and without ciprofloxacin and with and without exposure to ultrasound (a 43-kHz ultrasonic bath for 20 min daily) in an in vitro flow cell study. Biofilm accumulation from confocal images was quantified and statistically compared by using COMSTAT biofilm analysis software. Biofilm accumulation on ciprofloxacin-loaded hydrogels with ultrasound-induced drug delivery was significantly reduced compared to the accumulation of biofilms grown in control experiments. The results of these studies may ultimately facilitate the future development of medical devices sensitive to external ultrasonic impulses and capable of treating or preventing biofilm growth via "on-demand" drug release.

Albumin adsorption on alkanethiols self-assembled monolayers on gold electrodes studied by chronopotentiometry.

Chronopotentiometry was used to study the adsorption of human serum albumin (HSA) to self-assembled monolayers with the following terminal functional groups: CH(3), COOH and OH. Surfaces were characterized by X-ray photoelectron spectroscopy, water contact angle measurements and cyclic voltammetry. HSA coverage of the different SAMs was investigated by chronopotentiometry and the total amount of adsorbed protein was determined using radiolabelled albumin. Both techniques have demonstrated that HSA adsorption to the different SAM-modified electrodes increases in the following order: OH

Relative influence of polymer fiber diameter and surface charge on fibrous capsule thickness and vessel density for single-fiber implants.

Single polypropylene microfibers plasma-coated with polymers of different surface charge [N,N-dimethylaminoethyl methacrylate (NN) (positive charge), methacrylic acid (MA) (negative charge), and hexafluoropropylene (HF) (neutral)] were implanted in the subcutaneous dorsum of Sprague-Dawley rats for 5-week intervals. Thee groups of fiber diameters were used: (I) 1.0 to 5.9 microm; (II) 6.0 to 10.9 microm; and (III) 11.0 to 15.9 microm. Fibrous capsule thickness and blood-vessel density (number of vessels within 100 microm of the fiber) were assessed in tissue sections in the planes of microfiber cross-sections. Results from a multifactorial analysis of variance demonstrated statistically significant main effects (p < 0.05) for microfiber diameter but not for surface-charge coating. The mean differences in capsule thickness among the microfiber diameter groups were: between groups II and I: 5.4 microm; between groups III and I: 10.2 microm; and between groups III and II: 4.7 microm. The mean differences in capsule thickness among surface-charge coatings were: between MA and NN: 0.7 microm; between MA and HF: 1.4 microm; and between NN and HF: 0.7 microm. Many of the 1.0 to 5.9 microm-in-diameter fibers had no capsule and no sign of a foreign-body reaction. For the vessel density analysis, neither microfiber diameter nor surface-charge coating had a statistically significant effect. Thus the geometric feature of microfiber diameter was more important than was surface charge relative to fibrous capsule formation but not relative to local vessel density. This ranking of the relative influence of design features in relation to tissue response provides useful information for prioritization in biomaterial design.

Plasma polymerized N-isopropylacrylamide: synthesis and characterization of a smart thermally responsive coating.

A lower critical solution temperature (LCST) in an aqueous environment has been observed with poly(N-isopropylacrylamide) (pNIPAM) deposited onto solid surfaces from a plasma glow discharge of NIPAM vapor. The synthesis and spectroscopic data (ESCA, FTIR) for the plasma polymerized NIPAM (ppNIPAM) shows a remarkable retention of the monomer structure. The phase transition at 29 degrees C was measured by a novel AFM method. The phase transition was surprising because of the expectation that the plasma environment would destroy the specific NIPAM structure associated with the thermal responsiveness. The phase change of ppNIPAM is also responsible for the changes in the level of the meniscus when coated capillaries are placed in warm and cold water. Plasma polymerization of NIPAM represents a one-step method to fabricate thermally responsive coatings on real-world biomaterials without the need for specially prepared substrates and functionalized polymers.

Self-assembled molecular structures as ultrasonically-responsive barrier membranes for pulsatile drug delivery.

Noninvasive ultrasound has been shown to increase the release rate on demand from drug delivery systems; however, such systems generally suffer from background drug leaching. To address this issue, a drug-containing polymeric monolith coated with a novel ultrasound-responsive coating was developed. A self-assembled molecular structure coating based on relatively impermeable, ordered methylene chains forms an ultrasound-activated on-off switch in controlling drug release on demand, while keeping the drug inside the polymer carrier in the absence of ultrasound. The orderly structure and molecular orientation of these C12 n-alkyl methylene chains on polymeric surfaces resemble self-assembled monolayers on gold. Their preparation and characterization have been published recently (Kwok et al. [Biomacromolecules 2000;1(1):139-148]). Ultrasound release studies showed that a copolymer of 2-hydroxyethyl methacrylate and ethylene glycol dimethacrylate (MW 400) coated with such an ultrasound-responsive membrane maintained sufficient insulin for multiple insulin delivery, compared with a substantial burst release during the first 2 h from uncoated samples. With appropriate surface coating coverage, the background leach rate can be precisely controlled. The biological activity of the insulin releasate was tested by assessing its ability to regulate [C14]-deoxyglucose uptake in 3T3-L1 adipocyte cells in a controlled cell culture environment. Uptake triggered by released insulin was comparable to that of the positive insulin control. The data demonstrate that the released insulin remains active even after the insulin had been exposed to matrix synthesis and the methylene chain coating process.

Replacing and renewing: synthetic materials, biomimetics, and tissue engineering in implant dentistry.

Hundreds of thousands of implantations are performed each year in dental clinical practice. Dental implants are a small fraction of the total number of synthetic materials implanted into the human body in all fields of medicine. Basically, these millions of implants going into humans function adequately. But longevity and complications still are significant issues and provide opportunities for the creation of improved devices. This manuscript briefly reviews the history of dental implant devices and the concepts surrounding the word "biocompatibility." It then contrasts the foreign body reaction with normal healing. Finally, the article describes how ideas gleaned from the study of normal wound healing can be applied to improved dental implants. In a concluding section, three scenarios for dental implants twenty years from now are envisioned.

Inhibition of monocyte adhesion and fibrinogen adsorption on glow discharge plasma deposited tetraethylene glycol dimethyl ether.

Monocytes and macrophages play important roles in host responses to implanted biomedical devices. Monocyte and macrophage interactions with biomaterial surfaces are thought to be mediated by adsorbed adhesive proteins such as fibrinogen and fibronectin. Non-fouling surfaces that minimize protein adsorption may therefore minimize monocyte adhesion, activation, and the foreign body response. Radio-frequency glow discharge plasma deposition (RF-GDPD) of tetraethylene glycol dimethyl ether (tetraglyme) was used to produce polyethylene oxide (PEO)-like coatings on a fluorinated ethylene-propylene (FEP) surface. Electron spectroscopy for chemical analysis (ESCA) and static time of flight secondary ion mass spectrometry (ToF-SIMS) were used to characterize the surface chemistry of tetraglyme coating. Fibrinogen adsorption to the tetraglyme surface was measured with 125I-labeled fibrinogen and ToF-SIMS. Adsorption of fibrinogen to plasma deposited tetraglyme was less than 10 ng cm(-2), a 20-fold decrease compared to untreated FEP or tissue culture polystyrene (TCPS). Monocyte adhesion to plasma deposited tetraglyme was significantly lower than adhesion to FEP or TCPS. In addition, when the surfaces were preadsorbed with fibrinogen, fibronectin, or blood plasma, monocyte adhesion to plasma deposited tetraglyme after 2 h or 1 day was much lower than adhesion to FEP. RF-GDPD tetraglyme coating provides a promising approach to make non-fouling biomaterials that can inhibit non-specific material-host interactions and reduce the foreign body response.

Surface characterization of hydroxyapatite and related calcium phosphates by XPS and TOF-SIMS.

The surfaces of six biologically interesting calcium phosphate (CaP) phases (hydroxyapatite, dibasic calcium phosphate dihydrate, dibasic calcium phosphate, monobasic calcium phosphate, beta-tribasic calcium phosphate, octacalcium phosphate) have been examined by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). The intensity of an O(1s) shake-up satellite correlates with the phosphate oxygen content. Together with the Ca/P and O/Ca XPS peak ratios, this feature helps provide identification of the CaP phase(s) present in the surface of unknown samples and establish their mole fractions, as proven with a bone sample. Contributions from carbonate impurities can be quantified using its C(1s) peak at 279.9 eV and subtracted from the O(1s) line shape to aid identification. Principal component analysis (PCA) has been applied successfully to analyze TOF-SIMS spectra of these six CaP phases. Multivariate analysis can help differentiate these CaP phases using the first two PCs, which are dominated by the relative intensities of only a few key ions: PO3-, O-, Ca+, CaOH+, PO2-, and OH-.

Characterization of combinatorially designed polyarylates by time-of-flight secondary ion mass spectrometry.

A series of 16 polyarylates, with well-controlled and systematically varying chemistry, has been characterized by time-of-flight secondary ion mass spectrometry (TOF-SIMS). The polymers are structurally identical except for the incremental additions of C2H4 units to the backbone and sidechain. From the spectra, peaks characteristic of all polyarylates are identified. Furthermore, evaluation of the spectra and identification of unique signals allow classification of the polyarylates according to sidechain and backbone chemistry.

Quantitative interrogation of micropatterned biomolecules by surface force microscopy.

Synthetic biomaterials are widely used in medical implants with success in improving and extending quality of life. However, these materials were not originally designed to interact with cells through specific signaling pathways. As a result, the interaction with the body is mediated through passive adsorption of a disorganized protein monolayer. Next generation biomaterials have been proposed to be active in modifying the biological response of the host through the incorporation of specific biorecognition moieties. An important tool in the development of these novel active biomaterials is the scanning force microscope (SFM). The SFM allows for interrogation of bioactive biomaterials in mapping or spectroscopic modes. In this work, micropatterned protein surfaces were prepared using biomolecules implicated in wound healing. The surfaces were imaged via SFM and the specific binding forces between surface associated biomolecules and antibody functionalized tips were quantified.

Plasma-deposited membranes for controlled release of antibiotic to prevent bacterial adhesion and biofilm formation.

Bacterial infection on implanted medical devices is a significant clinical problem caused by the adhesion of bacteria to the biomaterial surface followed by biofilm formation and recruitment of other cells lines such as blood platelets, leading to potential thrombosis and thromboembolisms. To minimize biofilm formation and potential device-based infections, a polyurethane (Biospan) matrix was developed to release, in a controlled manner, an antibiotic (ciprofloxacin) locally at the implant interface. One material set consisted of the polyetherurethane (PEU) base matrix radiofrequency glow discharge plasma deposited with triethylene glycol dimethyl ether (triglyme); the other set had an additional coating of poly(butyl methyacrylate) (pBMA). Triglyme served as a nonfouling coating, whereas the pBMA served as a controlled porosity release membrane. The pBMA-coated PEU contained and released ciprofloxacin in a controlled manner. The efficacy of the modified PEU polymers against Pseudomonas aeruginosa suspensions was evaluated under flow conditions in a parallel plate flow cell. Bacterial adhesion and colonization, if any, to the test polymers were examined by direct microscopic image analysis and corroborated with destructive sampling, followed by direct cell counting. The rate of initial bacterial cell adhesion to triglyme-coated PEU was 0. 77%, and to the pBMA-coated PEU releasing ciprofloxacin was 6% of the observed adhesion rates for the control PEU. However, the rate of adherent cell accumulation due to cell growth and replication was approximately the same for the triglyme-coated PEU and the PEU controls, but was zero for the pBMA-coated PEU releasing ciprofloxacin.

Blood compatibility--a perspective.

This perspective on blood- materials interactions is intended to introduce the set of papers stemming from the symposium, "Devices and Diagnostics in Contact with Blood: Issues in Blood Compatibility at the Close of the 20th Century," organized on August 4-6, 1999 at the University of Washington by the University of Washington Engineered Biomaterials (UWEB) Engineering Research Center. This article outlines some of the history of blood contacting materials, overviews the work that has originated at the University of Washington over the past 28 years, speculates on the origins of the controversies on blood compatibility and considers the issues that should be addressed in future studies.

Surface modification of polymers with self-assembled molecular structures: multitechnique surface characterization.

A simple, one-step procedure for generating ordered, crystalline methylene chains on polymeric surfaces via urethane linkages was developed. The reaction of dodecyl isocyanate with surface hydroxyl functional groups, catalyzed by dibutyltin dilaurate, formed a predominantly all-trans, crystalline structure on a cross-linked poly(2-hydroxyethyl methacrylate) (pHEMA) substrate. Allophanate side-branching reactions were not observed. Both X-ray photoelectron spectrocopy and time-of-flight secondary ion mass spectrometry show that the surface reaction reached saturation after 30 min at 60 degrees C. Unpolarized Fourier transform infrared-attenuated total reflection showed that, after 30 min, the stretching frequencies, vCH2,asym and vCH2,sym, decreased and approached 2920 and 2850 cm-1, indicative of a crystalline phase. The distance between two hydroxyl groups is roughly 4 A. A tilt angle of 33.5 degrees +/- 2.4 degrees was estimated by dichoric ratios measured in polarized ATR according to the two-phase and Harrick thin film approximations. The findings reported here are significant in that the possibilities for using structures similar to self-assembled monolayers (SAMs) are expanded beyond the rigid gold and silicon surfaces used through most of the literature. Thus, SAMs, biomimetics for ordered lipid cell wall structures, can be applied to real-world biomedical polymers to modify biological interactions. The terminal groups of the SAM-like structure can be further functionalized with biomolecules or antibodies to develop surface-based diagnostics, biosensors, or biomaterials.

Template recognition of protein-imprinted polymer surfaces.

Synthetic materials capable of specifically recognizing proteins are important in separations, biosensors, and biomaterials. In this study, polysaccharide-like surfaces with tailored protein-binding nanocavities were prepared by a novel templating approach based on radiofrequency plasma deposition of thin films. The template-imprinted proteins included albumin, immunoglobulin, fibrinogen, lysozyme, ribonuclease A, alpha-lactalbumin, and glutamine synthetase. Transmission electron microscopy showed that nanometer-sized "pits" in the shape of imprinted protein were formed on the surfaces of template-imprinted polymer films. Electron spectroscopy for chemical analysis and time-of-flight secondary ion mass spectrometry indicated the saccharide covering of imprint surfaces and the removal of template proteins. (125)I-labeled protein adsorption from single solutions showed a similar amount of protein was adsorbed to its own imprint as to the imprint of another protein. However, more protein remained on the former surface than on the latter following elution with the detergents Tween 20 or sodium dodecyl sulfate. Competitive adsorption of a binary protein mixture showed a highly preferential adsorption of template protein to the corresponding imprint. This template recognition diminished as the number of protein-imprinted pits decreased. Structurally unstable proteins such as alpha-lactalbumin exhibited weaker template recognition that "robust" proteins such as lysozyme. The hypothesis that protein recognition is due to complementarity between the protein and its imprinted nanopit was supported by protein turnover experiments that showed template protein adsorbed to its own imprint was less readily displaced by a nontemplate protein.

Design of infection-resistant antibiotic-releasing polymers: I. Fabrication and formulation.

Biomaterials-related infections are often observed with prosthetic implants and in many cases result in the failure of the devices. To design a biomedically useful polymer that is intrinsically infection-resistant, we have developed a ciprofloxacin-loaded polyurethane (PU) matrix that releases antibiotic locally at the implant surface, thereby minimizing bacterial accumulation. We report here the methods of fabrication and formulation for making such antibiotic-loaded devices, as well as evidence of their bactericidal properties. Specifically, various pore-forming agents and drug loadings were examined. An optimum formulation consisting of BIOSPAN PU, poly(ethylene glycol) and ciprofloxacin offered the longest effective period of sustained release (5 days). The bactericidal efficacy of the released ciprofloxacin against Pseudomonas aeruginosa (PA) was four times that of the control PU without antibiotics. This bactericidal efficiency was due to an increase in the PA detachment from the surface. These observations suggested that the released ciprofloxacin was biologically active in preventing the bacteria from permanently adhering to the substratum, and thus decreasing the possibility of biofilm-related infection.