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This study describes a screening platform for a guided in situ vascular tissue engineering approach. Polymer rods were developed that upon 3 weeks of subcutaneous implantation evoke a controlled inflammatory response culminating in encapsulation by a tube-shaped autologous fibrocellular tissue capsule, which can form a basis for a tissue-engineered blood vessel. Rods of co-polymer were produced using different ratios of poly(ethylene oxide terephthalate) and poly(butylene terephthalate) to create a range of physicochemical properties. In addition, a set of different physical, chemical, and biological surface modifications were tested on their ability to actively steer this tissue capsule formation using a rat model as testing platform. Tissue capsules were mainly composed of circumferentially aligned collagen and myofibroblasts. Different implant material resulted in distinct differences in tissue capsule formation. Compared to its unmodified counterparts, all surface modifications resulted in increased wall thickness, collagen, and myofibroblasts. Oxygen plasma-treated rods resulted in loose tissue arrangement, collagen, and collagen/TGF-β-coated rods yielded thick, collagen-rich, densely packed tissue capsules, though with a random distribution of myofibroblasts. In contrast, chloroform-etched rods provided homogenous densely packed tissue capsules, completely populated by myofibroblasts. In conclusion, by varying the implant's surface characteristics, tissue capsule composition, cell distribution, and tissue arrangement could be tailored, enabling controlled guidance of the tissue response for in vivo vascular tissue engineering.

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.)

The effect of polymorphonuclear leukocytes (PMNs) on the subsequent chronic phase macrophage-mediated foreign body reaction has not been previously investigated. Furthermore, while monocyte/macrophage-produced cytokines such as GM-CSF, G-CSF, or IL-1beta have been shown to increase PMN survival in vitro, few studies have examined the impact of directly cocultured monocytes/macrophages on PMN viability. To this end, we used our established in vitro system of interleukin (IL)-4-induced monocyte-derived macrophage fusion to examine the role of PMNs in the subsequent foreign body reaction. Monocytes were directly cultured with PMNs for 3 days before the addition of IL-4 to induce monocyte-derived macrophage fusion to facilitate foreign body giant cell (FBGC) formation by days 7 and 10 of culture. Optical microscopy was used to quantitatively determine adherent monocyte density, percent macrophage fusion, and FBGC density. A colorimetric MTT assay was used to assess PMN viability for direct cocultures of monocytes/macrophages and PMNs. Our results strongly suggest that the presence of PMNs inhibit IL-4-induced macrophage fusion and FBGC formation. Additionally, our findings demonstrate that cocultures containing PMNs and monocytes/macrophages increases PMN survival with respect to PMN-only cultures in vitro., ((c) 2010 Wiley Periodicals, Inc.)

Topographical cues play an important role in influencing cellular behavior and are considered as significant parameters to be controlled in tissue engineering applications. This work investigated the biocompatibility with regard to scaffold architecture and topographical effect of nanofibrous scaffolds on the in vivo and in vitro foreign body reaction. Random and aligned polycaprolactone (PCL) nanofibers were fabricated by electrospinning technique, with diameters of 313 +/- 5 nm and 506 +/- 24 nm, respectively. Primary monocytes isolated from five human donors were cultured on PCL nanofibers, PCL film, and RGD-coated glass in vitro and cell density and morphology was evaluated at time points of day 0 (2 h), day 3, day 7, and day 10. The in vivo study was carried out by implanting PCL nanofibers and film scaffolds subcutaneously in rats to test the biocompatibility and host response at time points of week 1, week 2, and week 4. The in vitro studies revealed that the initial monocyte adhesion on the aligned fiber scaffold was significantly less (p < 0.001) when compared to the random fiber scaffold. The in vivo study showed that the thicknesses of fibrous capsule on fibrous scaffolds were 7.55 +/- 0.54 microm for aligned fibers and 4.13 +/- 0.31 microm for random fibers, which were significantly thinner than that of film implants 37.7 +/- 0.25 microm (p < 0.001). Additionally, cell infiltration was observed in aligned fibrous scaffolds both in vitro and in vivo, while on random fibers and films, distinct fibrous capsule boundaries were found on the surfaces. These results indicate that aligned electrospun nanofibers may serve as a promising scaffold for tissue engineering by minimizing host response, enhancing tissue-scaffold integration, and eliciting a thinner fibrous capsule.

Current strategies to limit macrophage adhesion, fusion and fibrous capsule formation in the foreign body response have focused on modulating material surface properties. We hypothesize that topography close to biological scale, in the micron and nanometric range, provides a passive approach without bioactive agents to modulate macrophage behavior. In our study, topography-induced changes in macrophage behavior was examined using parallel gratings (250 nm-2 mum line width) imprinted on poly(epsilon-caprolactone) (PCL), poly(lactic acid) (PLA) and poly(dimethyl siloxane) (PDMS). RAW 264.7 cell adhesion and elongation occurred maximally on 500 nm gratings compared to planar controls over 48 h. TNF-alpha and VEGF secretion levels by RAW 264.7 cells showed greatest sensitivity to topographical effects, with reduced levels observed on larger grating sizes at 48 h. In vivo studies at 21 days showed reduced macrophage adhesion density and degree of high cell fusion on 2 mum gratings compared to planar controls. It was concluded that topography affects macrophage behavior in the foreign body response on all polymer surfaces examined. Topography-induced changes, independent of surface chemistry, did not reveal distinctive patterns but do affect cell morphology and cytokine secretion in vitro, and cell adhesion in vivo particularly on larger size topography compared to planar controls., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The role(s) of T lymphocytes in the foreign body response has not been thoroughly elucidated. Lymphocytes are known to augment macrophage adhesion and fusion in vitro. Furthermore, T lymphocytes are a possible source of the cytokines, IL-4 and IL-13, which induce macrophage fusion. In this study, we used BALB/c mice and BALB/c (nu/nu) nude mice to investigate foreign body giant cell (FBGC) formation in a T-cell-deficient setting. Mice were implanted with Elasthane 80A (PEU), silicone rubber (SR), or poly(ethylene terephthalate) (PET) for 7, 14, or 21 days using the cage implant system. Exudate cells and IL-4 and IL-13 levels in exudate supernatants were analyzed by flow cytometry and a multiplex immunoassay, respectively, at Days 7, 14, and 21. Macrophage adhesion and fusion on material surfaces were analyzed using optical microscopy. T-cell-deficient mice had lower total leukocyte concentrations at the biomaterial implant site at all time points. Adherent cell density was comparable between normal and T-cell-deficient mice except in the PEU group at Day 21. However, percent fusion, average nuclei per FBGC, and FBGC morphology were comparable between normal and T-cell-deficient mice. IL-4 was not detected in any sample, but IL-13 levels were also comparable between normal and T-cell-deficient mice indicating Th2-polarized T-cells are not the sole source of this cytokine. We have shown that there are pathways that do not require thymus-matured T lymphocytes, which lead to a normal foreign body response to biomaterials in a murine model.

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 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.

This study compared the effect of an antioxidant on the in vivo biodegradation of a poly(carbonate urethane) (PCU) and a poly(ether urethane) (PEU). Unstrained PEU and PCU films with and without Santowhite were implanted subcutaneously into 3-month-old Sprague-Dawley rats for 3, 6, and 12 months. Characterization of unstabilized PEU and PCU with ATR-FTIR and SEM showed soft-segment and hard-segment degradation consistent with previous studies. In particular, evidence of chain scission and crosslinking of the surface was present in the ATR-FTIR spectra of explanted specimens. Addition of 2.2 wt % antioxidant inhibited the in vivo degradation of both PCU and PEU. Although the antioxidant probably improved polyurethane biostability by decreasing the susceptibility of the polymer to degradation, modulation of the cellular response to prevent the release of degradative agents was also possible. To differentiate the effects, the foreign-body response was investigated with the use of a standard cage implant protocol. Polyurethane films were implanted in wire mesh cages subcutaneously in rats for 4, 7, and 21 days. There were no statistical differences among materials in the inflammatory exudate cell counts, adherent cell densities, or percent fusion of macrophages into foreign-body giant cells (FBGCs). Therefore, it was concluded that the antioxidant inhibited degradation by capturing oxygen radicals that would otherwise cause polyurethane chain scission and crosslinking., ((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.

The host foreign body response ensues immediately following implantation of medical devices and prostheses. We have previously identified the role of macrophages in adhering to biomaterial surfaces and guiding the foreign body response while fusing into foreign body giant cells (FBGCs) and concentrating degradative and phagocytic activities. Despite their early and transient presence around implanted biomaterials, few studies have focused on the role of lymphocytes in the foreign body response and biocompatibility. To address this, an in vitro human lymphocyte/macrophage coculture system has been developed. Using this system, it has been shown that when lymphocytes are present during the initial adhesion of monocytes, the rate of monocyte adhesion and fusion is significantly increased (1,500 cells/mm2 and 60%, respectively) when compared to either no lymphocytes present (500 cells/mm2 adhesion and 0% fusion). Although lymphocytes adhered to the tissue culture polystyrene surface, 90% of the lymphocytes were associated with adherent macrophages. However, these cell-cell direct interactions were not necessary to influence macrophage adhesion or fusion as separating the two cell types by a Transwell insert still resulted in significantly increased levels of macrophage adhesion (p < 0.05 when compared to macrophage only cultures). Conversely, the presence of macrophages in Transwell experiments increased lymphocyte proliferation rates at all time points tested. These studies begin to detail the interactions between lymphocytes and macrophages in the absence of known antigen that appropriately relates to the scenarios experienced upon implantation of biomedical devices and the initiation of the foreign body response., ((c) 2005 Wiley Periodicals, Inc.)

The in vivo biocompatibility and biofouling of gold electrodes for a microelectromechanical systems drug delivery device were investigated in a rodent model. The role of the applied voltage and gold electrolysis products in modulating the inflammatory response (biocompatibility), and the temporal adhesion of cellular populations onto macroscopic gold film electrodes (biofouling) were analyzed in reference to two controls, devices to which voltage was not applied (uncorroded) or voltage was applied to inert platinum electrodes (electrical controls). Voltammetry was applied to the gold surfaces once (day 4, 7, 14, 21, 28, 35, 42, or 49), while voltage of identical magnitude was applied to the electrical controls. An inflammatory response characterized by a rapid decrease of leukocyte concentration to control levels was observed 48 h following voltage application with no significant cell concentration difference (p > 0.05) between the corroded devices and electrical controls. The histological evaluation of the direct implant fibrous capsule showed comparable thickness of voltage applied and control specimens. The gold corrosion peak current showed no significant difference (p > 0.05) among peak values at all time points. It was concluded that gold electrode corrosion was biocompatible and its electrochemical performance was not hindered by fibrous capsule formation.

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.