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Sung Wan Kim's initial efforts as an independent investigator were focused on improving the understanding of the early events in blood/material interactions with the goal to develop blood compatible materials for application in medical devices and prostheses. These initial efforts were centered around blood protein adsorption on biomaterials and related mechanisms of thrombus formation (thrombosis). Ultimately, Sung Wan's efforts were expanded to studies of the non-thrombogenic nature of heparinized biomaterials, prostaglandin biomaterials, and block copolymer systems. These studies were supported by two NIH grants for 22 and 19 years, respectively, and a NIH Career Development Award. Moreover, these studies resulted in over 140 peer-reviewed publications and training of many students and postdoctoral scientists. The intent of this paper is to identify key concepts, papers, and contributions by Sung Wan and his colleagues that fall within the four aforementioned research categories. In this context, many of Sung Wan's early efforts contributed directly to Utah's biomaterials efforts and the Total Artificial Heart program at the time, while providing the foundation for the productive international Triangle Collaboration as well as his following work in polymer-controlled drug releasing systems., (Copyright © 2020. Published by Elsevier B.V.)

Exploiting the inflammatory response on biomaterials research and development requires an in-depth understanding of the cellular and molecular mechanisms involved in the events comprising the tissue response continuum. Examples of how the biomaterial surface chemistry may modulate the foreign body reaction to biomaterials. The utilization of different surface chemistries on biomaterials may provide biological design criteria for the appropriate use and function of biomaterials when used in medical devices and prostheses.

Introduction: Biologic matrices used in abdominal wall reconstruction are purported to undergo remodeling into connective tissue resembling native collagen. Key steps in that process include inflammatory response at the mesh/tissue interface, cellular penetration, and neovascularization of the matrix, followed by fibroblast proliferation and collagen deposition. We aimed to examine the concept of biologic mesh remodeling/regeneration in a series of explanted porcine biologic meshes., Materials and Methods: A cohort of patients who underwent removal of porcine biologic mesh was identified in a prospective database. Mesh/tissue samples were analyzed using standard hematoxylin/eosin and Masson's trichrome staining. Main outcome measures included: inflammatory response at the mesh/tissue interface, foreign body reaction (FBR), cellular penetration, neovascularization, and new collagen deposition. All evaluations were performed by a blinded senior pathologist using established grading scales., Results: A total of 14 cases with implant time ranging from 4 to 33 months were identified and analyzed. All meshes were placed as intraperitoneal underlay. There were 7 non-cross-linked and 7 cross-linked grafts. Cross-linked grafts were associated with mild FBR and moderate fibrous capsule formation. Similarly, non-cross-linked grafts had mild-to-moderate FBR and encapsulation. Furthermore, non-cross-linked grafts were associated with no neovascularization and minimal peripheral mesh neocellularization. Cross-linked grafts demonstrated neither neovascularization nor neocellularization. Although no grafts were associated with any quantifiable new collagen deposition within the porcine biologic matrix, minimal biodegradation/remodeling was observed at the periphery of the non-cross-linked grafts only., Conclusion: The biologic behavior of porcine meshes is predicated on their ability to undergo mesh remodeling with resorption and new collagen deposition. In the largest series of human biologic explants, we detected no evidence of xenograft remodeling, especially in the cross-linked group. Although underlay mesh placement and other patient factors may have contributed to our findings, the concept of porcine biologic mesh regeneration does not seem to be prevalent in the clinical setting., (Copyright © 2014 Mosby, Inc. All rights reserved.)

In previous studies that explored the influence of cytokines on foreign body giant cell (FBGC) formation, we focused on interleukin (IL)-4 and IL-13, each of which was discovered to induce macrophage fusion leading to FBGC formation in vitro. Two correlative in vivo studies also confirmed that IL-4 plays a role in FBGC formation on implanted biomaterials, but that T lymphocytes are not the source of IL-4 or other cytokines that support this process. The present study focused on identification of the cellular source of macrophage fusion-inducing cytokines, including natural killer (NK) or NKT lymphocytes and mast cells using mouse models genetically deficient in each of these cell types, as well as IL-4 receptor alpha(IL-4Rα)-deficient and severe combined immunodeficient (SCID) mice. Polyetherurethane (PEU) and polyethylene terephthalate (PET) polymers were subcutaneously implanted and retrieved after 14, 21, or 28 days. FBGC formation was evaluated using quantitative and qualitative data from retrieved polymer surfaces. Both types of data indicate that, compared to normal control mice, neither NK or NKT lymphocytes nor mast cells are required for FBGC formation. Furthermore, FBGC formation on biomaterials can proceed in IL-4Rα-deficient and in SCID mice. Similar conclusions were made regarding FBGC formation on both PEU and PET biomaterials. These data suggest that other sources of IL-4/IL-13 and/or additional macrophage fusion-inducing cytokines can mediate FBGC formation on implanted biomaterials, or that, in the absence of normal primary pathways, FBGC formation is nevertheless supported by redundant innate mechanisms., (Copyright © 2014 Wiley Periodicals, Inc.)

Background: Despite relatively sparse data regarding their outcomes in the setting of infection, biologic grafts have gained rapid acceptance by the surgical community for complex hernia repair. These materials are heterogeneous in their procurement and processing techniques, which may ultimately have an impact in their ability to withstand infection. The objective of this study is to evaluate the impact of varying levels of contamination on biologic graft performance in a chronic ventral hernia animal model., Methods: Four commonly applied biologic grafts were used in the repair of a chronic ventral hernia rat model (n = 218). Each material was repaired in the setting of 1 of 4 surgical wound classifications (clean, clean contaminated, contaminated, dirty infected) with Staphylococcus aureus as our inoculum agent. After a 30-day survival, repairs underwent quantitative cultures, histological, and biomechanical testing., Results: Marked differences were observed in biologic graft bacterial burden, biomechanical and histological responses at 30 days. Persistent bacterial burden varied among the biologic grafts and increased with increasing wound contamination (P < .05). Delays in wound healing were observed in the contaminated and dirty infected setting (P < .05). Increasing infection weakened the biomechanical strength of repairs (P < .05)., Conclusion: The degree of bacterial contamination at the time of repair affected the rates of bacterial clearance, wound-healing ability, and subsequent repair strength. Material source and processing techniques might alter graft durability, biocompatibility, and ability to clear bacteria in a contaminated field. Clinical trials are warranted in contaminated settings., (Copyright © 2013 Mosby, Inc. All rights reserved.)

Biomaterial-centered infections are initiated by adhesion of bacteria to an implant, followed by colonization and mature biofilm formation. Staphylococcus epidermidis is commonly identified as the cause of these device-centered infections. This study used an in vitro model to evaluate temporal changes in the expression of genes-icaADBC, agrBDCA, aap, and atle-that have been identified to play a role in the pathogenesis of S. epidermidis infections. Real-time reverse transcription-polymerase chain reaction was used to determine changes in gene expression from S epidermidis biofilm grown on polyurethanes (Elasthane 80A, hydrophobic) modified with polyethylene oxide (Elasthane 80A-6PEO, hydrophilic) and fluorocarbon (Elasthane 80A-6F, hydrophobic). In vitro expression of the ica locus, which is involved in initial adhesion and intracellular aggregation, increased up to 100-fold from 2 to 48 h, whereas gene expression for autolysin AtlE decreased slightly from 2 to 12 h, followed by a 10-fold increase by 48 h. Upregulation of the aap gene associated with bacterial accumulation and the agr quorum-sensing system was observed during biofilm formation over 48 h. In addition, no correlation was observed between S. epidermidis gene expression and biomaterial surface chemistry. This study used an in vitro model to demonstrate that enhanced expression of the atle, aap, agr, and ica genes plays an important role in initial foreign body colonization and potentially in the establishment of a device-associated infection., (Copyright © 2012 Wiley Periodicals, Inc.)

The aim of current study was to evaluate the effect of nano-apatitic particles (nAp) incorporation on the degradation characteristics and biocompatibility of poly(lactide-co-glycolide) (PLGA)-based nanofibrous scaffolds. Composite PLGA/poly(ɛ-caprolactone) (PCL) blended (w/w = 3/1) polymeric electrospun scaffolds with 0-30 wt% of nAp incorporation (n0-n30) were prepared. The obtained scaffolds were firstly evaluated by morphological, physical and chemical characterization, followed by an in vitro degradation study. Further, n0 and n30 in both virgin and 3-week pre-degraded status were subcutaneously implanted in rats, either directly or in stainless steel mesh cages, to evaluate in vivo tissue response. The results showed that the incorporation of nAp yields an nAp amount-dependent buffering effect on pH-levels during degradation and delayed polymer degradation based on molecular weight analysis. Regarding biocompatibility, nAp incorporation significantly improved the tissue response during a 4-week subcutaneous implantation, showing less infiltration of inflammatory cells (monocyte/macrophages) as well as less foreign body giant cells (FBGCs) formation surrounding the scaffolds. Similar cytokine expression (gene and protein level) was observed for all groups of implanted scaffolds, although marginal differences were found for TNF-α and TGF-β at gene level as well as GRO-KC at protein level after 1 week of implantation. The results of the current study indicate that hybridization of the weak alkaline salt nAp is effective to control the in vivo adverse tissue reaction of PLGA materials, which is beneficial for optimizing final clinical application of different PLGA-based biomedical devices., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The monocyte-derived macrophage is recognized as a critical determinant in biocompatibility, but its appearance in the chronic inflammatory phase is accompanied by the presence of lymphocytes, which have been much less studied in this regard. Here, we first present an overview of the physiologic continuum comprising host reactions to the surgical implantation of biomaterial. Secondly, we describe our collective research efforts, which indicate that lymphocytes are additional and key cellular determinants of biocompatible outcome. Thus, bioengineering advances will require that lymphocyte responses be regarded as integral components of innate inflammatory and immune/immunotoxic cell interactions at sites of biomaterial implantation.

An efficient bacterial synthesis method to harvest cadmium telluride (CdTe) quantum dots (QDs) with tunable fluorescence emission using Escherichia coli is demonstrated. Ultraviolet-visible, photoluminescence, X-ray diffraction and transmission electron microscopy analysis confirmed the superior size-tunable optical properties, with fluorescence emission from 488 to 551 nm, and the good crystallinity of the as synthesized QDs. A surface protein capping layer was confirmed by hydrodynamic size, zeta potential and Fourier transform infrared spectroscopy measurements, which could maintain the viability (92.9%) of cells in an environment with a QD concentration as high as 2 microM. After functionalization with folic acid the QDs were used to image cultured cervical cancer cells in vitro. Investigations of bacterial growth and morphology and the biosynthesis of CdTe QDs in Luria-Bertani medium containing E. coli-secreted proteins showed that extracellular synthesis directly relied on the E. coli-secreted proteins, and a mechanism for protein-assisted biosynthesis of QDs is proposed. This work provides an economical approach to fabricate highly fluorescent biocompatible CdTe QDs via an environmentally friendly production process. The biosynthesized QDs may have great potential in broad bio-imaging and bio-labeling applications., (2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Polyester polyurethanes incorporating polyhedral oligosilsesquioxane (POSS) as the crystalline hard block were evaluated for biocompatibility and degradation over 24 weeks in vivo. In vitro studies were also used to predict the onset of mass loss. The molecular weight of each sample was found to decrease quickly over an 8 week period and then became constant due to the nondegrading POSS hard block. Kinetic analysis of the initial molecular weight change indicated that the degradation rate was dependent on the soft block composition. Crystallinity, melting temperature, and heat of fusion of the polyurethanes were found to increase during degradation as the amorphous polyester soft segments were hydrolyzed. The histological analysis of each polymer demonstrated rapid resolution of the acute and chronic inflammatory responses and the development of expected, normal foreign body reaction, consisting of adherent macrophages and foreign body giant cells on the surface of the polymers, and fibrous capsule formation around the polymer. No acute and/or chronic inflammation was seen after 3 weeks, indicating that the polymers in film form and biodegraded form, that is, particles, were biocompatible and did not elicit inflammatory responses expected for toxic or nonbiocompatible materials., ((c) 2010 Wiley Periodicals, Inc.)

Previous in vitro studies in our laboratory have shown that lymphocytes can influence macrophage adhesion and fusion on biomaterial surfaces. However, few studies have evaluated how material adherent macrophages can influence lymphocyte behavior, specifically T cells. In this study, we cultured human peripheral blood mononuclear cells from healthy donors on three synthetic nonbiodegradable biomedical polymers: elasthane 80A (PEU), silicone rubber (SR), or polyethylene terephthalate (PET) and tissue culture polystyrene (TCPS). Upregulation of T cell surface activation markers (CD69 and CD25), lymphocyte proliferation, and interleukin-2 (IL-2) and interferon-gamma (IFNgamma) concentrations were evaluated by flow cytometry, carboxy-fluorescein diacetate, succinimydyl ester (CFSE) incorporation, and multiplex cytokine immunoassay, respectively, to assess T cell activation. Following 3 and 7 days of culture, CD4+ helper T cells from cultures of any of the material groups did not express the activation markers CD69 and CD25 and lymphocyte proliferation was not present. IL-2 and IFNgamma levels were produced, but dependent on donor. These data indicate that T cells are not activated in response to clinically relevant synthetic biomaterials. The data also suggest that lymphocyte subsets exclusive of T cells are the source of the lymphokines, IL-2 and IFN-gamma, in certain donors.

To characterize the effects of adherent macrophages and biomaterial surface chemistries on lymphocyte adhesion and activation, lymphocytes were co-cultured with monocytes alone and together, directly and separated by a porous membrane transwell on hydrophobic, hydrophilic/neutral, hydrophilic/anionic, and hydrophilic/cationic biomaterial surfaces. Surface adherent cells were quantitatively analyzed after 3 days utilizing immunofluorescence and phase contrast imaging. After periods of 3, 7, and 10 days, secreted interferon-gamma (IFN-gamma) was quantified by ELISA. Limited direct biomaterial-adherent lymphocytes were identified regardless of the presence of macrophages or foreign body giant cells (FBGC). The majority of adherent lymphocytes, which were T cells (>95%) rather than natural killer cells, predominantly interacted with adherent macrophages and FBGCs; greater than 90% were interacting on surfaces with higher levels of adherent macrophages and FBGCs and greater than 55% were interacting on surfaces with lower levels of macrophages and FBGCs. The hydrophilic/anionic surface promoted higher levels of macrophage- and FBGC-adherent lymphocytes but was nonselective for lymphocyte subtype interactions. The hydrophilic/neutral surface was selective for CD4+ T lymphocyte interactions while the hydrophobic surface was selective for CD8+ T lymphocyte interactions. IFN-gamma was produced in direct and indirect co-cultures but not in lymphocyte- and monocyte-only cultures suggesting that lymphocytes are activated via macrophage-derived cytokines rather than direct biomaterial contact. Direct lymphocyte interactions with adherent macrophages/FBGCs enhanced IFN-gamma production relative to indirect co-cultures. These results suggest that lymphocytes prefer interactions with adherent macrophages and FBGCs, resulting in lymphocyte activation, and these interactions can be influenced by biomaterial surface chemistries.

To further elucidate the foreign body reaction, investigation of cytokines at biomaterial implant sites was carried out using a multiplex immunoassay and ELISA. Macrophage activation cytokines (IL-1beta, IL-6, and TNFalpha), cytokines important for macrophage fusion (IL-4 and IL-13), antiinflammatory cytokines (IL-10 and TGFbeta), chemokines (GRO/KC, MCP-1), and the T-cell activation cytokine IL-2 were quantified at biomaterial implant sites. Empty cages (controls) or cages containing synthetic biomedical polymer (Elasthane 80A (PEU), silicone rubber (SR), or polyethylene terephthalate (PET)) were implanted subcutaneously in Sprague-Dawley rats for 4, 7, or 14 days, and cytokines in exudate supernatants and macrophage surface adhesion and fusion were quantified. The presence of a polymer implant did not affect the levels of IL-1beta, TGFbeta, and MCP-1 in comparison to the control group. IL-2 was not virtually detected in any of the samples. Although the levels of IL-4, IL-13, IL-10, and GRO/KC were affected by polymer implantation, but not dependent on a specific polymer, IL-6 and TNFalpha were significantly greater in those animals implanted with PEU and SR, materials that do not promote fusion. The results indicate that differential material-dependent cytokine profiles are produced by surface adherent macrophages and foreign body giant cells in vivo., (Copyright 2008 Wiley Periodicals, Inc.)

The role of lymphocytes in the biological response to synthetic polymers is poorly understood despite the transient appearance of lymphocytes at the biomaterial implant site. To investigate cytokines, chemokines, and extracellular matrix (ECM) proteins produced by lymphocytes and macrophages in response to biomaterial surfaces, human peripheral blood monocytes and lymphocytes were co-cultured on polyethylene terephthalate (PET)-based material surfaces displaying distinct hydrophobic, hydrophilic/neutral, hydrophilic/anionic, and hydrophilic/cationic chemistries. Antibody array screening showed the majority of detected proteins are inflammatory mediators that guide the early inflammatory phases of wound healing. Proteomic ELISA quantification and adherent cell analysis were performed after 3, 7, and 10 days of culture. IL-2 and IFN-gamma were not detected in any co-cultures suggesting lack of lymphocyte activation. The hydrophilic/neutral surfaces increased IL-8 relative to the hydrophobic PET surface (p < 0.05). The hydrophilic/anionic surfaces promoted increased TNF-alpha over hydrophobic and cationic surfaces and increased MIP-1beta compared to hydrophobic surfaces (p < 0.05). Since enhanced macrophage fusion was observed on hydrophilic/anionic surfaces, the production of these cytokines likely plays an important role in the fusion process. The hydrophilic/cationic surface promoted IL-10 production and increased matrix metalloproteinase (MMP)-9/tissue inhibitor of MMP (TIMP) relative to hydrophilic/neutral and anionic surfaces (p < 0.05). These results suggest hydrophilic/neutral and anionic surfaces promote pro-inflammatory responses and reduced degradation of the ECM, whereas the hydrophilic/cationic surfaces induce an anti-inflammatory response and greater MMP-9/TIMP with an enhanced potential for ECM breakdown. The study also underscores the usefulness of protein arrays in assessing the role of soluble mediators in the inflammatory response to biomaterials., ((c) 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2008.)

Novel self-assembled monolayers (SAMs) designed to present homogenous surface chemistries were utilized to further investigate the material surface chemistry dependent macrophage and foreign-body giant cell (FBGC) behaviors, including macrophage adhesion, fusion, and apoptosis. Contact angle analysis revealed instabilities in the --CH(3) and --COOH terminated SAM surfaces upon incubation in serum-free media (SFM) at 37 degrees C or under dry, room temperature conditions. Further analysis indicated that the --CH(3) terminated SAM surface degraded rapidly within 2 h and loss of sufficient SAM units to be comparable to the gold (Au) control surface, within 24 h of incubation in SFM at 37 degrees C. After 5 days of incubation in SFM at 37 degrees C, the contact angles for the --COOH terminated SAM surfaces increased markedly. AFM analysis confirmed the desorption of --CH(3) terminated SAM molecules from the surface with increased roughness and marked appearance of peaks and valleys within 2 h. A decrease in the thickness of the --COOH terminated SAM surface also suggests molecular desorption over time. No significant changes in contact angle or AFM analyses were observed on the --OH terminated SAM surfaces. Cellular adhesion decreased more rapidly on the Au control and --CH(3) terminated SAM surfaces in comparison to the other surfaces. However by day 10, cellular adhesion, fusion, and apoptosis were comparable on all SAM surfaces and the Au control. These studies suggest that SAM surfaces may not be suitable for long-term studies where material dependent properties are investigated., ((c) 2008 Wiley Periodicals, Inc.)

Synthetic biomaterials are considered to be nonimmunogenic. Therefore, the role that adaptive immunity may play in the host response to implanted synthetic biomaterials has not been extensively studied. Cardinal features of adaptive immunity include specificity and T cell responses, which are greater and more effective with upregulation of activation receptors upon rechallenge. We compared the primary and secondary in vivo host response to three synthetic biomaterials: Elasthane 80A, silicone rubber, and polyethylene terephthalate using a cage implant model in Sprague Dawley rats. The synthetic biomedical polymers were subcutaneously implanted in cages for 14 days. Following explantation of the cages and a 2 week healing period, rats were implanted with cages containing the biomedical polymers for an additional 2 weeks. The cellular exudates within the cages were analyzed 4, 7, and 14 days post primary and secondary implantation by flow cytometry for the following cell types: T cells (inclusive of CD8(+), CD4(+), and CD4(+)/CD25(+) subsets), B cells, granulocytes, and macrophages. At day 14 following secondary implantation, there was an increase in T cells, granulocytes, and macrophages in the exudates when compared with primary implantation for all groups inclusive of the empty cage control. However, CD4(+)/CD8(+) ratios, the percentage of CD4(+)CD25(+) T cells, and the macrophage surface adhesion/fusion did not vary significantly upon secondary implantation. Despite a quantitative increase in T cells following secondary biomaterial exposure, T cell subset distribution did not change, indicating nonspecific recruitment rather than an adaptive immune response., (Copyright 2007 Wiley Periodicals, Inc.)

The foreign body reaction composed of macrophages and foreign body giant cells is the end-stage response of the inflammatory and wound healing responses following implantation of a medical device, prosthesis, or biomaterial. A brief, focused overview of events leading to the foreign body reaction is presented. The major focus of this review is on factors that modulate the interaction of macrophages and foreign body giant cells on synthetic surfaces where the chemical, physical, and morphological characteristics of the synthetic surface are considered to play a role in modulating cellular events. These events in the foreign body reaction include protein adsorption, monocyte/macrophage adhesion, macrophage fusion to form foreign body giant cells, consequences of the foreign body response on biomaterials, and cross-talk between macrophages/foreign body giant cells and inflammatory/wound healing cells. Biomaterial surface properties play an important role in modulating the foreign body reaction in the first two to four weeks following implantation of a medical device, even though the foreign body reaction at the tissue/material interface is present for the in vivo lifetime of the medical device. An understanding of the foreign body reaction is important as the foreign body reaction may impact the biocompatibility (safety) of the medical device, prosthesis, or implanted biomaterial and may significantly impact short- and long-term tissue responses with tissue-engineered constructs containing proteins, cells, and other biological components for use in tissue engineering and regenerative medicine. Our perspective has been on the inflammatory and wound healing response to implanted materials, devices, and tissue-engineered constructs. The incorporation of biological components of allogeneic or xenogeneic origin as well as stem cells into tissue-engineered or regenerative approaches opens up a myriad of other challenges. An in depth understanding of how the immune system interacts with these cells and how biomaterials or tissue-engineered constructs influence these interactions may prove pivotal to the safety, biocompatibility, and function of the device or system under consideration.

Matrix metalloproteinases (MMPs) can degrade structural components within the extracellular matrix and at the cellular surface producing changes in cellular behavior (i.e., adhesion and migration) and subsequent pathological responses (i.e., the foreign body reaction and wound healing). We continue to study the foreign body reaction that occurs following biomaterial implantation by investigating secretory responses of biomaterial-adherent macrophages and foreign body giant cells (FBGCs) as directed by material surface chemistry and further this research by determining whether secreted MMPs play a role in macrophage adhesion and fusion. We have identified numerous MMPs and their tissue inhibitors (TIMPs) in in vitro cell-culture supernatants using antibody arrays and quantified select MMP/TIMPs with ELISAs. MMP-9 concentrations were significantly greater than both TIMP-1 and TIMP-2 on all materials. The ratios of MMP-9/TIMP-1 and MMP-9/TIMP-2 increased with time because of an increase in MMP-9 concentrations over time, while the TIMP concentrations remained constant. Total MMP-9 concentrations in the supernatants were comparable on all materials at each timepoint, while TIMP-1 and TIMP-2 concentrations tended to be greater on hydrophilic/anionic surfaces. Analysis of the MMP/TIMP quantities produced per cell revealed that the hydrophilic/neutral surfaces, which inhibited macrophage adhesion, activated the adherent macrophages/FBGCs to produce a greater quantity of MMP-9, TIMP-1, and TIMP-2 per cell. Pharmacological inhibition of MMP-1,-8,-13, and -18 reduced macrophage fusion without affecting adhesion, while inhibitors of MMP-2,-3,-9, and -12 did not affect adhesion or fusion. These findings demonstrate that material surface chemistry does modulate macrophage/FBGC-derived MMP/TIMP secretion and implicates MMP involvement in macrophage fusion., ((c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2008.)

To better understand the relationship between macrophage/foreign body giant cell adhesion and activation on surface-modified biomaterials, quantitative assessment of adherent cell density (cells per mm(2)) and cytokine production (pgs per mL) were determined by ELISA. Further analysis to identify cellular activation was carried out by normalizing the cytokine concentration data to provide a measure of cellular activation. This method of analysis demonstrated that hydrophobic surfaces provided statistically significantly greater adherent cell densities than hydrophilic/neutral surfaces. However, when cell activation parameters were determined by normalization to the adherent cell density, the hydrophilic/neutral surfaces demonstrated statistically significantly greater levels of activation and production of IL-10, IL-1beta, IL-6, IL-8, and MIP-1beta. With increasing time, production of the anti-inflammatory cytokine IL-10 increased, whereas IL-1beta, IL-6, and IL-8 decreased and MIP-1beta was relatively constant over the culture time period. This observed dichotomy or disparity between adhesion and activation may be related to surface-induced adherent cell apoptosis. Further evaluation of macrophage activation on biomaterial surfaces indicated that an apparent phenotypic switch in macrophage phenotype occurred over the course of the in vitro culture. Analysis of cytokine/chemokine profiles with surface-modified biomaterials revealed similarities between the classically activated macrophages and the biomaterial-adherent macrophages early (day 3) in culture, while at later timepoints the biomaterial-adherent macrophages produced profiles similar to alternatively activated macrophages. Classically activated macrophages are those commonly activated by lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma) and alternatively activated macrophages are those activated by IL-4/IL-13 or IL-10. Surface modification of biomaterials offer an opportunity to control cellular activation and cytokine profiles in the phenotypic switch, and may provide a means by which macrophages can be induced to regulate particular secretory proteins that direct inflammation, the foreign body reaction, wound healing, and ultimately biocompatibility.

Implantation of biomaterial devices results in the well-known foreign body reaction consisting of monocytes, macrophages, and foreign body giant cells (FBGCs) at the material/tissue interface. We continue to address the hypothesis that material surface chemistry modulates the phenotypic expression of these cells. Utilizing our human monocyte culture system, we have used surface-modified polymers displaying hydrophobic, hydrophilic, and/or ionic chemistries to determine the cytokines/chemokines released from biomaterial-adherent macrophages/FBGCs. This study broadens our approach by using proteomic analysis to identify important factors expressed by these cells and further quantifies these molecules with ELISAs. Proteomic profiles changed over time suggesting that the adherent macrophages underwent a phenotypic switch. Macrophage/FBGC-derived proinflammatory cytokines, IL-1beta and IL-6, decreased with time, while the anti-inflammatory cytokine, IL-10, gradually increased with time. Resolution of the inflammatory response was also demonstrated by a decrease in chemoattractant IL-8 and MIP-1beta production with time. Material-dependent macrophage/FBGC activation was analyzed using cytokine/chemokine production and cellular adhesion. Monocyte/macrophage adhesion was similar on all surfaces, except for the hydrophilic/neutral surfaces that showed a significant decrease in cellular density and minimal FBGC formation. Normalizing the ELISA data based on the adherent cell population provided cytokine/chemokine concentrations produced per cell. This analysis showed that although there were fewer cells on the hydrophilic/neutral surface, these adherent cells were further activated to produce significantly greater amounts of each cytokine/chemokine tested than the other surfaces. This study clearly presents evidence that material surface chemistry can differentially affect monocyte/macrophage/FBGC adhesion and cytokine/chemokine profiles derived from activated macrophages/FBGCs adherent to biomaterial surfaces., (Copyright 2007 Wiley Periodicals, Inc.)

Most infections due to implanted cardiovascular biomaterials are initiated by bacterial adhesion of Staphylococcus epidermidis, followed by colonization and biofilm formation on the surface of the implant. This study examined the role of serum proteins and material surface chemistry in the formation of S. epidermidis biofilm on polyurethanes (Elasthane 80A, hydrophobic) modified with polyethylene oxide (Elasthane 80A-6PEO, hydrophilic) and fluorocarbon (Elasthane 80A-6F, hydrophobic). Initial adhesion, aggregation, biofilm thickness, viability, and slime formation of S. epidermidis strain, RP62A in phosphate buffered saline (PBS), tryptic soy broth (TBS), and 20% pooled human serum was quantified. In the presence of adsorbed serum proteins, initial bacterial adhesion was suppressed significantly to <2% relative to adhesion in TSB or PBS. However, adhesion, aggregation, and proliferation increased dramatically in the 12-24 h period on Elasthane 80A and Elasthane 80A-6F, which resulted in an extensive network of biofilm. A contrasting trend was observed on the hydrophilic Elasthane 80A-6PEO surface, with minimal bacterial adhesion, which decreased steadily over 24 h. In the presence of serum proteins, an increasingly thick ( approximately 20 mum) biofilm formed on the hydrophobic surfaces over 48 h whereas the formation of a mature biofilm on the hydrophilic surface was impeded with few viable bacteria present over 48 h. Furthermore, slime was detected during the initial phase of bacterial adhesion at 2 h and increased over time with the formation of biofilm. These results have shown that while initial S. epidermidis adhesion is suppressed in the presence of adsorbed proteins, inter-bacterial adhesion possibly aided by slime production leads to the formation of a robust mature biofilm. Also, biomaterial surface chemistry affected biofilm formation and, most notably, polyethylene oxide significantly inhibited S. epidermidis biofilm formation over 48 h in vitro.

Infection due to implanted cardiovascular biomaterials is a serious complication initiated by bacterial adhesion to the surface of the implant. The release of reactive oxygen species by neutrophils, particularly superoxide anion, is a well-known bactericidal mechanism. Additionally, nitric oxide (NO) has also been identified as an important cytotoxic mediator in acute and chronic inflammatory responses with enhanced NO production by upregulation of inducible nitric oxide synthase (iNOS). The interaction of NO and superoxide anion will result in the formation of peroxynitrite (OONO-), a potent cytotoxic oxidant. In this study, we have shown that biomaterial-induced neutrophil activation does not cause upregulation of iNOS and activation of iNOS-mediated pathways. However, NO and O2- production does occur over time upon adhesion to a biomaterial and is modulated by biomaterial surface chemistry. With no stimulus, the polyethylene oxide-modified polyurethane induced greater neutrophil activation than did the control as indicated by the increased production of NO and O2- over time. Adherent-stimulated neutrophils generally produced lower amounts of NO over time in comparison with unstimulated cells. Furthermore, there is no evidence of peroxynitrite activity in unstimulated neutrophils adherent to the Elasthane 80A. However, upon stimulation with adherent Staphylococcus epidermidis, peroxynitrite formation did occur. Our results suggest that bactericidal mechanisms in neutrophils involving NO generation (NOS pathway) are further compromised than O2- producing pathways (NADPH oxidase) upon exposure to biomaterials, resulting in a diminished microbial killing capacity, which can increase the probability of device-centered infections., (Copyright 2006 Wiley Periodicals, Inc.)

The purpose of this communication is to present the author's perspectives on the future of biomedical materials that were presented at the Larry L. Hench Retirement Symposium held at Imperial College, London, in late September 2005. The author has taken a broad view of the future of biomedical materials and has presented key ideas, concepts, and perspectives necessary for the future research and development of biomedical polymers and their future role as an enabling technology for the continuing progress of tissue engineering, regenerative medicine, prostheses, and medical devices. This communication, based on the oral presentation, is meant to be provocative and generate discussion. In addition, it is targeted for students and young scientists who will play an ever-increasing role in the future of biomedical materials.

The formation of biofilm, a structured community of bacteria enclosed in slime, is a significant virulence factor in medical-device-centered infection. The development of cardiovascular device infection can be separated into two phases: initial bacterial adhesion and aggregation, followed by proliferation and production of slime. It is possible to modulate the adhesion and biofilm formation of Staphylococcus epidermidis, a commensal skin bacterium commonly found on infected medical devices, through biomaterial surface chemistry. This study examines bacterial adhesion and biofilm formation on surface-modified polyethylene terephthalate (PET), including surfaces with varying hydrophilic, hydrophobic, and ionic character. Bacterial adhesion and biofilm formation were observed over 48 hours in phosphate-buffered saline (PBS) and 20% pooled human serum. The hydrophilic surface (PAAm) had significantly less nonspecific adhesion of bacteria than that in the control (PET) and other surfaces, when cultured in PBS (P < 0.0001). Charged surfaces, both anionic and cationic, had increased adhesion and aggregation of bacteria in comparison with the control (PET) in the presence of serum proteins over 24 hours (P < 0.0001). Bacteria cultured in serum on the charged surfaces did not have significantly different amounts of biofilm formation compared with that of the control (PET) surface after 48 hours. This study showed that biomaterial surface chemistry characteristics impact initial adhesion and aggregation of S. epidermidis on biomaterials., (2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006.)

This study examined the effect of cholesterol esterase (CE) on the degradation of commercial poly(ether urethane) (PEU) and poly(carbonate urethane) (PCU). Unstrained PEU and PCU films were incubated in 400 U/mL CE solution or a buffer control for 36 days. The study used a concentration of cholesterol esterase that was considerably higher than the estimated physiological level in order to accelerate degradation. However, characterization of treated polyurethane films with SEM, attenuated total reflectance Fourier transform infrared (ATR-FTIR) and GPC analysis revealed only a small loss in surface soft segment content. Comparison with implanted PEU and PCU films led to the conclusion that any effect of enzymatic hydrolysis was confined to the immediate surface, and the magnitude of the effect was too small to contribute significantly to in vivo degradation. The study confirmed that oxidation, rather than enzymatic hydrolysis, is the primary mechanism responsible for the observed biodegradation of PEU and PCU. The oxidative H(2)O(2)/CoCl(2) treatment continues to accurately predict the long-term biostability of polyurethanes.

Candida albicans biofilms form on indwelling medical devices (e.g., denture acrylic or intravenous catheters) and are associated with both oral and invasive candidiasis. Here, we determined whether surface modifications of polyetherurethane (Elasthane 80A [E80A]), polycarbonateurethane, and poly(ethyleneterephthalate) (PET) can influence fungal biofilm formation. Polyurethanes were modified by adding 6% polyethylene oxide (6PEO), 6% fluorocarbon, or silicone, while the PET surface was modified to generate hydrophilic, hydrophobic, cationic, or anionic surfaces. Formation of biofilm was quantified by determining metabolic activity and total biomass (dry weight), while its architecture was analyzed by confocal scanning laser microscopy (CSLM). The metabolic activity of biofilm formed by C. albicans on 6PEO-E80A was significantly reduced (by 78%) compared to that of biofilm formed on the nonmodified E80A (optical densities of 0.054 +/- 0.020 and 0.24 +/- 0.10, respectively; P = 0.037). The total biomass of Candida biofilm formed on 6PEO-E80A was 74% lower than that on the nonmodified E80A surface (0.46 +/- 0.15 versus 1.76 +/- 0.32 mg, respectively; P = 0.003). Fungal cells were easily detached from the 6PEO-E80A surface, and we were unable to detect C. albicans biofilm on this surface by CSLM. All other surface modifications allowed formation of C. albicans biofilm, with some differences in thearchitecture. Correlation between contact angle and biofilm formation was observed for polyetherurethane substrates (r = 0.88) but not for PET biomaterials (r = -0.40). This study illustrates that surface modification is a viable approach for identifying surfaces that have antibiofilm characteristics. Investigations into the clinical utility of the identified surfaces are warranted.

We developed a novel bactericidal surface based on a catechin-loaded surface-erodible polymer. (-)-Epigallocatechin-3-gallate (EGCg), which is the main constituent of tea catechins, showed a dose-dependent inhibitory effect on Escherichia coli biofilm formation and a dose-dependent enhanced destructive effect on biofilm. EGCg-immobilized surfaces were prepared by photopolymerization of liquid biodegradable polyesters. The releasing rate was enhanced with an increase in surface-erosion rate of photocured polymers. Polymers with high releasing capacity dose-dependently reduced biofilm formation on the surfaces. The confocal laser scanning microscopic and scanning electron microscopic observations revealed that EGCg induced biofilm-destructing activities, which include bacterial membrane damage, degradation of exopolysaccharides, and detachment of colonized cells. From these results, potential advantages of the clinical use of catechin-loaded polymer-coated implants or catheters are discussed in terms of a reduced occurrence of biomaterial-centered infections without substantial toxicity or adverse effects., ((c) 2005 Wiley Periodicals, Inc.)

To determine the effect of biomaterial surface chemistry on leukocyte interaction and activity at the material/tissue interface, human peripheral blood monocytes and lymphocytes were cultured on a series of poly(ethylene terephthalate) (PET)-based biomaterials. Both monocytes and lymphocytes were isolated from whole human blood and separated by a nonadherent density centrifugation method before being plated on PET disks, surface modified by photograft copolymerization to yield hydrophobic, hydrophilic, anionic, and cationic surface properties. Monocytes and lymphocytes were cultured separately, to elicit baseline levels of activity, in direct coculture, to promote direct cell surface interactions, or in an indirect coculture system with both cell types separated by a -0.02-microm Transwell apparatus, to promote indirect paracrine interactions. Monocyte adhesion, macrophage fusion, and lymphocyte proliferation were measured on days 3, 7, 10, and 14 of culture. Results demonstrated that the presence of monocytes increased the activity of cocultured lymphocytes at the biomaterial/tissue interface, while the corresponding presence of lymphocytes increased the activation and fusion of indirectly cocultured monocytes. Biomaterial surface chemistry was also found to have a significant effect on monocyte adhesion and activation, and lymphocyte activity. Hydrophilic surfaces significantly inhibited both initial and longterm monocyte adhesion, and inhibited lymphocyte proliferation at longer time points. Anionic and cationic surfaces both exhibited mild inhibition of monocyte adhesion at prolonged time points and increased levels of macrophage fusion, while cationic surfaces decreased levels of lymphocyte proliferation and inhibited monocyte activity. These results elucidate the complex role of juxtacrine and paracrine interactions between monocytes and lymphocytes in the foreign body response, as well as promote the consideration of hydrophilic surfaces in future designs of implantable biomedical devices and prostheses.

In this study, a fatty acid urethane derivative of dehydroepiandrosterone (DHEA) was synthesized and evaluated as a polyurethane additive to increase long-term biostability. The modification was hypothesized to reduce the water solubility of the DHEA and physically anchor the additive in the polyurethane during implantation. Polyurethane film weight loss in water as a function of time was studied to determine the polymer retention of the modified DHEA. The polyurethane film with unmodified DHEA had significant weight loss in the first day (10%) that was previously correlated to rapid leaching of the additive. The polyurethane film with modified DHEA had significantly less weight loss at all time points indicating improved polymer retention. The effect of the modified DHEA additive on the biostability of a poly(ether urethane urea) was examined after 5 weeks of subcutaneous implantation in Sprague-Dawley rats. Optical micrographs and infrared analysis of the specimens indicated that the modified DHEA bloomed to the surface of the film forming a crystalline surface layer approximately 10-15 microns thick. After explantation, this surface layer was intact without measurable differences in surface chemistry as monitored by attenuated total reflectance-Fourier transform infrared spectroscopy. There was no evidence of degradation of the polyurethane underneath the modified DHEA surface layer as compared with the polyurethane control. We have concluded that the modified DHEA self-assembled into a protective surface coating that inhibited degradation of the polyurethane. The roughness of the modified DHEA surface layer prevented adherent cell analysis to determine if the additive retained the ability to down-regulate macrophage activity. Subsequent studies will investigate the ability of surface-modifying additives to modulate cellular respiratory bursts in addition to the formation of an impermeable barrier. This bimodal approach to improving biostability holds great promise in the field of polyurethane biomaterials., (Copyright (c) 2005 Wiley Periodicals, Inc.)

This study used an in vitro environment that simulated the microenvironment at the adherent cell-material interface to reproduce and accelerate the biodegradation of poly(ether urethane) (PEU) and poly(carbonate urethane) (PCU). Polyurethane films were treated in vitro for 24 days in 20% hydrogen peroxide/0.1 M cobalt chloride solution at 37 degrees C. Characterization with ATR-FTIR and SEM showed soft segment and hard segment degradation consistent with the chemical changes observed after long-term in vivo treatment. Overall, the PCU underwent less degradation and the degraded surface layer was much thinner than PEU. Nevertheless, the results supported a common oxidation mechanism for biodegradation of these polymers. The observed in vitro degradation was inhibited by adding an antioxidant to the polyurethane film. Our findings further support the use of the in vitro H(2)O(2)/CoCl(2) system in evaluating the biostability of polyurethanes under accelerated conditions., (Copyright 2004 Wiley Periodicals, Inc. J Biomed Mater Res 70A: 245-255, 2004)

Compromised neutrophil function in the presence of an implanted biomaterial may represent an important mechanism that allows for the development of implant-associated infections. Here, human neutrophil mobility has been investigated on a polyurethane (ChronoFlex AR), a hydrophobic surface consisting of an octadecyltrichlorosilane (OTS) self-assembled monolayer, and a glass reference material. Neutrophil mobility was quantified, based on cell movement speed and persistence time obtained from time-lapse optical microscopy, while neutrophil cytoskeletal structures and morphology were visualized using confocal microscopy and atomic force microscopy. Our results show that material surface properties affect neutrophil-surface interactions, as reflected by morphological changes, and the mobility of neutrophils stimulated by N-formylmethionyl-leucyl-phenylalanine (fMLP). In the absence of adsorbed plasma proteins, the mobility of stimulated neutrophils increased with increasing material hydrophobicity from glass, to polyurethane, to OTS. The opposite trend was observed in the presence of adsorbed plasma proteins, such that neutrophil mobility increased with decreasing material hydrophobicity. Analysis of the results showed that the mobility of fMLP-stimulated neutrophils cells was inversely related to the extent of cell spreading on the materials., (Copyright 2004 Wiley Periodicals, Inc.)

Several strategies have been used to increase the biostability of medical-grade polyurethanes while maintaining biocompatibility and mechanical properties. One approach is to chemically modify or replace the susceptible soft segment. Currently, poly(carbonate urethanes) (PCUs) are being evaluated as a replacement of poly(ether urethanes) (PEUs) in medical devices because of the increased oxidative stability of the polycarbonate soft segment. Preliminary in vivo and in vitro studies have reported improved biostability of PCUs over PEUs. Although several studies have reported evidence of in vitro degradation of these new polyurethanes, there has been no evidence of significant in vivo degradation that validates a degradation mechanism. In this study, the effect of soft segment chemistry on the phase morphology, mechanical properties, and in vivo response of commercial-grade PEU and PCU elastomers was examined. Results from dynamic mechanical testing and infrared spectroscopy suggested that the phase separation was better in PCU as compared with PEU. In addition, the higher modulus and reduced ultimate elongation of PCU was attributed to the reduced flexibility of the polycarbonate soft segment. Following material characterization, the in vivo biostability and biocompatibility of PEU and PCU were studied using a subcutaneous cage implant protocol. The results from the cage implant study and cell culture experiments indicated that monocytes adhere, differentiate, and fuse to form foreign body giant cells on both polyurethanes. It is now generally accepted that the reactive oxygen species released by these adherent macrophages and foreign body giant cells initiate PEU biodegradation. Attenuated total reflectance-Fourier transform infrared analysis of explanted samples provided evidence of chain scission and crosslinking in both polyurethanes. This indicated that the PCU was also susceptible to biodegradation by agents released from adherent cells. These results reinforce the need to evaluate and understand the biodegradation mechanisms of PCUs., (Copyright 2004 Wiley Periodicals, Inc. J Biomed Mater Res 69A: 407-416, 2004)

The effect of soft-segment chemistry on biostability of polyurethane elastomers was studied with a diaphragm-type film specimen under conditions of static and dynamic loading. During testing, the films were exposed to an H(2)O(2)/CoCl(2) solution, which simulated the oxidative component of the in vivo environment. Films treated for up to 24 days were evaluated by IR spectroscopy and by optical and scanning electron microscopy. Biostability of a poly(ether urethane) (PEU), which is known to undergo oxidative degradation, was compared with biostability of a poly(carbonate urethane) (PCU), which is thought to be more resistant to oxidation than PEU. Materials similar to PEU and PCU, in which the polyether or polycarbonate soft segment was partially replaced with poly(dimethylsiloxane) (PDMS), were also tested with the expectation that PDMS would improve soft-segment biostability. Oxidative degradation of the polyether soft segment of PEU was manifest chemically as chain scission and cross-linking and physically as surface pitting. Biaxial fatigue accelerated chemical degradation of PEU and eventually caused brittle stress cracking. In comparison, the polycarbonate soft segment was more stable to oxidation; there was minimal chemical or physical degradation of PCU, even in biaxial fatigue. Partial substitution of the polyether soft segment with PDMS enhanced oxidative stability of PEU. Although both strategies for modifying soft-segment chemistry improved the resistance to oxidative degradation, the outstanding mechanical properties of PEU were compromised to some extent., (Copyright 2004 Wiley Periodicals, Inc. J Biomed Mater Res 68A: 668-683, 2004)

Adherent macrophages and foreign body giant cells (FBGCs) are known to release degradative molecules that can be detrimental to the long-term biostability of polyurethanes. The modification of polyurethanes using surface modifying endgroups (SMEs) and/or the incorporation of silicone into the polyurethane soft segments may alter macrophage adhesion, fusion and apoptosis resulting in improved long-term biostability. An in vitro study of macrophage adhesion, fusion and apoptosis was performed on polyurethanes modified with fluorocarbon SMEs, polyethylene oxide (PEO) SMEs, or poly(dimethylsiloxane) (PDMS) co-soft segment and SMEs. The fluorocarbon SME and PEO SME modifications were shown to have no effect on macrophage adhesion and activity, while silicone modification had varied effects. Macrophages were capable of adapting to the surface and adhering in a similar manner to the silicone-modified and unmodified polyurethanes. In the absence of IL-4, macrophage fusion was comparable on the modified and unmodified polyurethanes, while macrophage apoptosis was promoted on the silicone modified surfaces. In contrast, when exposed to IL-4, a cytokine known to induce FBGC formation, silicone modification resulted in more macrophage fusion to form foreign body giant cells. In conclusion, fluorocarbon SME and PEO SME modification does not affect macrophage adhesion, fusion and apoptosis, while silicone modification is capable of mediating macrophage fusion and apoptosis. Silicone modification may be utilized to direct the fate of adherent macrophages towards FBGC formation or cell death through apoptosis.

The biocompatibility and biodegradation rate of component materials are critical when designing a drug-delivery device. The degradation products and rate of degradation may play important roles in determining the local cellular response to the implanted material. In this study, we investigated the biocompatibility and relative biodegradation rates of PLA, PGA and two poly(lactic-co-glycolic acid) (PLGA) polymers of 50:50 mol ratio, thin-film component materials of a drug-delivery microchip developed in our laboratory. The in vivo biocompatibility and both in vivo and in vitro degradation of these materials were characterized using several techniques. Total leukocyte concentration measurements showed normal acute and chronic inflammatory responses to the PGA and low-molecular-weight PLGA that resolved by 21 days, while the normal inflammatory responses to the PLA and high-molecular-weight PLGA were resolved but at slower rates up to 21 days. These results were paralleled by thickness measurements of fibrous capsules surrounding the implants, which showed greater maturation of the capsules for the more rapidly degrading materials after 21 days, but less mature capsules of sustained thicknesses for the PLA and high-molecular-weight PLGA up to 49 days. Gel-permeation chromatography of residual polymer samples confirmed classification of the materials as rapidly or slowly degrading. These materials showed thinner fibrous capsules than have been reported for other materials by our laboratory and have suitable biocompatibility and biodegradation rates for an implantable drug-delivery device.

A method utilizing expansion of a diaphragm-type film specimen was developed to study in vitro biodegradation of poly(etherurethane urea) (PEUU) under conditions of dynamic loading (fatigue). A finite element model was used to describe the strain state, which ranged from uniaxial at the edges of the film to balanced biaxial tensile strain at the center. During testing, the film was exposed to a H(2)O(2)/CoCl(2) solution, which simulated in vivo oxidative biodegradation of PEUU. The extent of chemical degradation was determined by infrared analysis. Physical damage of the film surface was characterized by optical microscopy and scanning electron microscopy. Dynamic loading did not affect the rate of degradation relative to unstressed and constant stress (creep) controls in regions of the film that experienced primarily uniaxial fatigue; however, degradation was accelerated in regions that experienced balanced biaxial or almost balanced biaxial fatigue. It was concluded that the combination of dynamic loading and biaxial tensile strain accelerated oxidative degradation in this system. Chemical degradation produced a brittle surface layer that was marked by numerous pits and dimples. Physical damage of the surface in the form of cracking occurred only in fatigue experiments. Cracking was not observed in unstressed or creep tests. Cracks initiated at the dimples produced by chemical degradation, and propagated in a direction that was determined by the strain state., (Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 65A: 524-535, 2003)

The biocompatibility and biofouling of the microfabrication materials for a MEMS drug delivery device have been evaluated. The in vivo inflammatory and wound healing response of MEMS drug delivery component materials, metallic gold, silicon nitride, silicon dioxide, silicon, and SU-8(TM) photoresist, were evaluated using the cage implant system. Materials, placed into stainless-steel cages, were implanted subcutaneously in a rodent model. Exudates within the cage were sampled at 4, 7, 14, and 21 days, representative of the stages of the inflammatory response, and leukocyte concentrations (leukocytes/microl) were measured. Overall, the inflammatory responses elicited by these materials were not significantly different than those for the empty cage controls over the duration of the study. The material surface cell density (macrophages or foreign body giant cells, FBGCs), an indicator of in vivo biofouling, was determined by scanning electron microscopy of materials explanted at 4, 7, 14, and 21 days. The adherent cellular density of gold, silicon nitride, silicon dioxide, and SU-8(TM) were comparable and statistically less (p<0.05) than silicon. These analyses identified the MEMS component materials, gold, silicon nitride, silicon dioxide, SU-8(TM), and silicon as biocompatible, with gold, silicon nitride, silicon dioxide, and SU-8(TM) showing reduced biofouling.

An in vivo mouse cage implant system was used to determine whether leukocyte cytokine mRNA responses to implanted biomaterials were dependent on surface chemistry. Surfaces displaying various chemistries (hydrophobic, hydrophilic, anionic, and cationic) were placed into stainless steel cages and implanted subcutaneously. Semiquantitative RT-PCR analyses revealed that hydrophilic surfaces showed a decreased expression of proinflammatory cytokines, IL-6 and IL-8, and pro-wound healing cytokines, IL-10 and TGF-beta by adherent cells, and mRNA levels of the proinflammatory cytokine, IL-1beta, and the pro-wound healing cytokine IL-13 were decreased in surrounding, exudate cells. Cytokine responses by adherent and exudate cells to hydrophobic, anionic and cationic surfaces were similar and indicative of a strong inflammatory response at the earliest time point followed by a wound healing response at later time points. However, no differences in the types or levels of exudate cells for any of the surfaces or the empty cage at each of the respective time points were observed, indicating their respective biocompatibility. These studies identify hydrophilic surface chemistries as having significant effects on leukocyte cytokine responses in vivo by decreasing the expression of inflammatory and wound healing cytokines by inflammatory cells adherent to the biomaterial as well as present in the surrounding exudate., (Copyright 2002 Wiley Periodicals, Inc. J Biomed Mater Res 64A: 320-329, 2003)

Within the complex environment of an implanted cardiovascular device comprised of dynamic flow and foreign materials, phagocytic neutrophils may be ineffective in combating infection due to cellular responses to shear stress. This may be explained, in part, by our recent reports of apoptosis of biomaterial-adherent leukocytes induced through exposure to shear stress. Here we utilize a rotating disk system to generate physiologically relevant shear stress levels (0-18 dynes/cm(2)) at the surface of a polyetherurethane urea (PEUU) and investigate neutrophil intracellular pathways involved in shear-induced apoptosis. In situ detection of activated caspases, the enzymatic mediators of the apoptosis cascade, showed qualitatively that these proteases participate in shear-induced apoptosis and are activated in a shear-dependent manner. The involvement of caspase 3 was confirmed through immunoprecipitation and immunoblotting of extracted neutrophil proteins. Comparative studies with neutrophils adherent under static conditions demonstrated time-dependent activation of caspases in TNF-alpha/cycloheximide-induced apoptosis, for which caspase-3 also was implicated. These findings are the first steps toward elucidation of the mechanisms behind the inappropriate induction of apoptosis by adhesion to biomaterials, which may contribute to the development and persistence of device-related infections., (Copyright 2002 Wiley Periodicals, Inc.)

Biocompatibility of implanted materials is determined by the host foreign-body response, which is comprised of cellular (adherent monocytes and macrophages) and soluble (secreted cytokines) components. Modulating the presence, activity or both of adherent macrophages may increase or decrease the biocompatibility of implants because these cells remain adherent to the implant surface and fuse to form foreign-body giant cells (FBGCs), leading to failure of the implant. An attractive mechanism of eliminating these cells is through the induction of apoptosis; therefore ways of inducing or inhibiting apoptosis of biomaterial-adherent inflammatory cells are being investigated. We hypothesized that interleukin-4 (IL-4) promotes macrophage survival by inhibiting tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. We found that TNF-alpha induces apoptosis in a time- and dose-dependent manner, whereas IL-4 inhibits TNF-alpha-induced and spontaneous apoptosis of biomaterial-adherent macrophages. Blocking experiments and evaluation of shedding of soluble TNF receptor type I (TNF-RI) demonstrated that endogenous TNF-alpha production is responsible for spontaneous apoptosis of biomaterial-adherent cells and that IL-4 inhibits this apoptosis by increasing levels of shedding of soluble TNF-RI. These findings suggest that TNF-alpha and IL-4 play key roles in determining the fate of biomaterial-adherent cells and that fusion of macrophages into FBGCs is a mechanism for promoting inflammatory-cell survival on implanted materials.