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Impaired wound healing is a major complication of diabetes, and despite the associated risks, treatment strategies for diabetic wounds remain limited. This is due, in part, to an incomplete understanding of the underlying pathological mechanisms, including the effects of hyperglycemia on components of the extracellular matrix (ECM). In the current study, we explored whether the expression of thrombospondin 2 (TSP2), a matricellular protein with a demonstrated role in response to injury, was associated with delayed healing in diabetes. First, we found that TSP2 expression was elevated in diabetic mice and skin from patients with diabetes. Then, to determine the contribution of TSP2 to impaired healing in diabetes, we developed a novel diabetic TSP2-deficient model. Though the TSP2-deficient mice developed obesity and hyperglycemia comparable with diabetic control mice, they exhibited significantly improved healing, characterized by accelerated reepithelialization and increased granulation tissue formation, fibroblast migration, and blood vessel maturation. We further found that hyperglycemia increased TSP2 expression in fibroblasts, the major cellular source of TSP2 in wounds. Mechanistically, high glucose increased activation of the hexosamine pathway and nuclear factor-κB signaling to elevate TSP2 expression. Our studies demonstrate that hyperglycemia-induced TSP2 expression contributes to impaired healing in diabetes.

Combined targeting of both intra-articular infection and inflammation achieves greater chondroprotection in septic arthritis., Bacterial infections involving joints and vital organs represent a challenging clinical problem because of the two concurrent therapeutic goals of bacterial eradication and tissue preservation. In the case of septic arthritis, permanent destruction of articular cartilage by intense host inflammation is commonly seen even after successful treatment of bacterial infection. Here, we provide scientific evidence of a novel treatment modality that can protect articular cartilage and enhanced eradication of causative bacteria in septic arthritis. Locally delivered cell-penetrating antibiotics such as rifampicin effectively eradicate intracellular reservoirs of methicillin-resistant Staphylococcus aureus within joint cells. Furthermore, mitigation of intra-articular inflammation by targeting the NLRP3 (nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3) inflammasome protects articular cartilage from damage in a murine model of knee septic arthritis. Together, concurrent mitigation of intra-articular inflammation and local adjuvant targeting of intracellular bacteria represents a promising new therapeutic strategy for septic arthritis.

Human-induced pluripotent stem cell-derived-vascular smooth muscle cells (hiPSC-VSMC) have been shown to promote angiogenesis and wound healing. However, there is a paucity of research on how the extracellular matrix (ECM) microenvironment may impact the hiPSC-VSMC’s function. In this study, our objective was to understand the effect of specific ECM ligand-integrin interaction on hiPSC- VSMC’s paracrine secretion, cell proliferation, and morphology. We here showed a precise modulation of hiPSC-VSMC in a fibronectin functionalized fibrillar collagen scaffold by targeting their integrin β3. The secretion of proangiogenic growth factor, basic fibroblast growth factor (bFGF) was found to be fibronectin dependent via αvβ3 integrin interactions. Also, our data indicate the possible role of a positive feedback loop between integrin β3, bFGF, and matrix metalloproteinase-2 in regulating hiPSC- VSMC’s morphology and cell proliferation. Finally, the secretome with improved proangiogenic activity shows potential for future regenerative applications.

Objective The glucocorticoid receptor (GR) is a member of the nuclear receptor family that controls key biological processes in the cardiovascular system and has recently been shown to modulate Wnt signaling in endothelial cells. Wnt/β-catenin signaling has been demonstrated to be crucial in the process of angiogenesis. In the current study, we studied whether GR could regulate angiogenesis via the Wnt/β-catenin pathway. Approach and Resultsa Key components of the Wnt/β-catenin pathway were evaluated using quantitative PCR and Western blot in the presence or absence of GR. Enhanced angiogenesis was found in GR deficiency in vitro and confirmed with cell viability assays, proliferation assays and tube formation assays. Consistent with these in vitro findings, endothelial cell-specific GR loss GR in vivo promoted angiogenesis in both a hind limb ischemia model and sponge implantation assay. Results were further verified in a novel mouse model lacking endothelial LRP5/6, a key receptor in canonical Wnt signaling, and showed substantially suppressed angiogenesis using these same in vitro and in vivo assays. To further investigate the mechanism of GR regulation of Wnt signaling, autophagy flux was investigated in endothelial cells by visualizing auto phagolysosomes as well as by assessing P62 degradation and LC3B conversion. Results indicated that potentiated autophagy flux participated in GR-Wnt regulation. Conclusions Lack of endothelial GR triggers autophagy flux, leads to activation of Wnt/β-catenin signaling and promotes angiogenesis. There may also be a synergistic interaction between autophagy and Wnt/β-catenin signaling.

Human induced pluripotent stem cell-derived vascular smooth muscle cells (hiPSC-VSMCs) with proangiogenic properties have huge therapeutic potential. While hiPSC-VSMCs have already been utilized for wound healing using a biomimetic collagen scaffold, an in situ forming hydrogel mimicking the native environment of skin offers the promise of hiPSC-VSMC mediated repair and regeneration. Herein, the impact of a collagen type-I-hyaluronic acid (HA) in situ hydrogel cross-linked using a PEG-based cross-linker on hiPSC-VSMCs viability and proangiogenic paracrine secretion was investigated. Our study demonstrated increases in cell viability, maintenance of phenotype and proangiogenic growth factor secretion, and proangiogenic activity in response to the conditioned medium. The optimally cross-linked and functionalized collagen type-I/HA hydrogel system developed in this study shows promise as an in situ hiPSC-VSMC carrier system for wound regeneration. This article is protected by copyright. All rights reserved.

Impaired healing leading to the formation of ulcerated wounds is a critical concern in patients with diabetes. Abnormalities in extracellular matrix (ECM) production and remodeling contribute to tissue dysfunction and delayed healing. Specifically, diabetes-induced changes in the expression and/or activity of structural proteins, ECM-modifying enzymes, proteoglycans, and matricellular proteins have been reported. In this review, we provide a summary of the key ECM molecules and associated changes in skin and diabetic wounds. Such information should allow for new insights in the understanding of impaired wound healing and lead to the development of ECM-based therapeutic strategies., Highlights • Wound healing is largely impaired in diabetes due, in part, to dysfunction and dysregulation of extracellular matrix. • This review gives a brief summary of the alterations of key ECM molecules in diabetes. • We also try to elucidate how hyperglycemia impacts ECM and anticipate how these changes contribute to the pathological state of diabetic wounds.

Implanted biomaterials elicit a series of distinct immune and repair-like responses that are collectively known as the foreign body reaction (FBR). These include processes involving innate immune inflammatory cells and wound repair cells that contribute to the encapsulation of biomaterials with a dense collagenous and largely avascular capsule. Numerous studies have shown that the early phase is dominated by macrophages that fuse to form foreign body giant cells that are considered a hallmark of the FBR. With the advent of more precise cell characterization techniques, specific macrophage subsets have been identified and linked to more or less favorable outcomes. Moreover, studies comparing synthetic- and natural-based polymer biomaterials have allowed the identification of macrophage subtypes that distinguish between fibrotic and regenerative responses. More recently, cells associated with adaptive immunity have been shown to participate in the FBR to synthetic polymers. This suggests the existence of cross-talk between innate and adaptive immune cells that depends on the nature of the implants. However, the exact participation of adaptive immune cells, such as T and B cells, remains unclear. In fact, contradictory studies suggest either the independence or dependence of the FBR on these cells. Here, we review the evidence for the involvement of adaptive immunity in the FBR to synthetic polymers with a focus on cellular and molecular components. In addition, we examine the possibility that such biomaterials induce specific antibody responses resulting in the engagement of adaptive immune cells.

Polarization of macrophages by chemical, topographical and mechanical cues presents a robust strategy for designing immunomodulatory biomaterials. Here, we studied the ability of nanopatterned bulk metallic glasses (BMGs), a new class of metallic biomaterials, to modulate murine macrophage polarization. Cytokine/chemokine analysis of IL-4 or IFNγ/LPS-stimulated macrophages showed that the secretion of TNF-α, IL-1α, IL-12, CCL-2 and CXCL1 was significantly reduced after 24-hour culture on BMGs with 55 nm nanorod arrays (BMG-55). Additionally, under these conditions, macrophages increased phagocytic potential and exhibited decreased cell area with multiple actin protrusions. These in vitro findings suggest that nanopatterning can modulate biochemical cues such as IFNγ/LPS. In vivo evaluation of the subcutaneous host response at 2 weeks demonstrated that the ratio of Arg-1 to iNOS increased in macrophages adjacent to BMG-55 implants, suggesting modulation of polarization. In addition, macrophage fusion and fibrous capsule thickness decreased and the number and size of blood vessels increased, which is consistent with changes in macrophage responses. Our study demonstrates that nanopatterning of BMG implants is a promising technique to selectively polarize macrophages to modulate the immune response, and also presents an effective tool to study mechanisms of macrophage polarization and function. Statement of Significance Implanted biomaterials elicit a complex series of tissue and cellular responses, termed the foreign body response (FBR), that can be influenced by the polarization state of macrophages. Surface topography can influence polarization, which is broadly characterized as either inflammatory or repair-like. The latter has been linked to improved outcomes of the FBR. However, the impact of topography on macrophage polarization is not fully understood, in part, due to a lack of high moduli biomaterials that can be reproducibly processed at the nanoscale. Here, we studied macrophage interactions with nanopatterned bulk metallic glasses (BMGs), a class of metallic alloys with amorphous microstructure and formability like polymers. We show that nanopatterned BMGs modulate macrophage polarization and transiently induce less fibrotic and more angiogenic responses. Overall, we demonstrate nanopatterning of BMG implants as a technique to polarize macrophages and modulate the FBR.

Objective— The importance of PI3K/Akt signaling in the vasculature has been demonstrated in several models, as global loss of Akt1 results in impaired postnatal ischemia- and VEGF-induced angiogenesis. The ubiquitous expression of Akt1, however, raises the possibility of cell-type–dependent Akt1-driven actions, thereby necessitating tissue-specific characterization. Approach and Results— Herein, we used an inducible, endothelial-specific Akt1-deleted adult mouse model (Akt1iECKO) to characterize the endothelial cell autonomous functions of Akt1 in the vascular system. Endothelial-targeted ablation of Akt1 reduces eNOS (endothelial nitric oxide synthase) phosphorylation and promotes both increased vascular contractility in isolated vessels and elevated diastolic blood pressures throughout the diurnal cycle in vivo. Furthermore, Akt1iECKO mice subject to the hindlimb ischemia model display impaired blood flow and decreased arteriogenesis. Conclusions— Endothelial Akt1 signaling is necessary for ischemic resolution post-injury and likely reflects the consequence of NO insufficiency critical for vascular repair.

Impaired wound healing is a major complication of diabetes, and can lead to development of chronic foot ulcers in a significant number of patients. Despite the danger posed by poor healing, very few specific therapies exist, leaving patients at risk of hospitalization, amputation, and further decline in overall health. Recent Advances: Redox signaling is a key regulator of wound healing, especially through its influence on the extracellular matrix (ECM). Normal redox signaling is disrupted in diabetes leading to several pathological mechanisms that alter the balance between reactive oxygen species (ROS) generation and scavenging. Importantly, pathological oxidative stress can alter ECM structure and function.There is limited understanding of the specific role of altered redox signaling in the diabetic wound, although there is evidence that ROS are involved in the underlying pathology.Preclinical studies of antioxidant-based therapies for diabetic wound healing have yielded promising results. Redox-based therapeutics constitute a novel approach for the treatment of wounds in diabetes patients that deserve further investigation. Antioxid. Redox Signal. 27, 823-838.

Collagen fibrillogenesis and crosslinking have long been implicated in extracellular matrix (ECM)-dependent processes such as fibrosis and scarring. However, the extent to which matricellular proteins influence ECM protein production and fibrillar collagen crosslinking has yet to be determined. Here we show that thrombospondin 2 (TSP2), an anti-angiogenic matricellular protein, is an important modulator of ECM homeostasis. Specifically, through a fractionated quantitative proteomics approach, we show that loss of TSP2 leads to a unique ECM phenotype characterized by a significant decrease in fibrillar collagen, matricellular, and structural ECM protein production in the skin of TSP2 KO mice. Additionally, TSP2 KO skin displays decreased lysyl oxidase (LOX), which manifests as an increase in fibrillar collagen solubility and decreased levels of LOX-mediated fibrillar collagen crosslinking. We show that these changes are indirectly mediated by miR-29, a major regulator of ECM proteins and LOX, as miR-29 expression is increased in the TSP2 KO. Altogether, these findings indicate that TSP2 contributes to ECM production and assembly by regulating miR-29 and LOX.

Nanomaterials (NMs) have revolutionized multiple aspects of medicine by enabling novel sensing, diagnostic, and therapeutic approaches. Advancements in processing and fabrication have also allowed significant expansion in the applications of the major classes of NMs based on polymer, metal/metal oxide, carbon, liposome, or multi-scale macro-nano bulk materials. Concomitantly, concerns regarding the nanotoxicity and overall biocompatibility of NMs have been raised. These involve putative negative effects on both patients and those subjected to occupational exposure during manufacturing. In this review, we describe the current state of testing of NMs including those that are in clinical use, in clinical trials, or under development. We also discuss the cellular and molecular interactions that dictate their toxicity and biocompatibility. Specifically, we focus on the reciprocal interactions between NMs and host proteins, lipids, and sugars and how these induce responses in immune and other cell types leading to topical and/or systemic effects.

Objective— It is widely accepted that the presence of a glycosaminoglycan-rich glycocalyx is essential for endothelialized vasculature health; in fact, a damaged or impaired glycocalyx has been demonstrated in many vascular diseases. Currently, there are no methods that characterize glycocalyx functionality, thus limiting investigators’ ability to assess the role of the glycocalyx in vascular health. Approach and Results— We have developed novel, easy-to-use, in vitro assays that directly quantify live endothelialized surface’s functional heparin weights and their anticoagulant capacity to inactivate Factor Xa and thrombin. Using our assays, we characterized 2 commonly used vascular models: native rat aorta and cultured human umbilical vein endothelial cell monolayer. We determined heparin contents to be ≈10 000 ng/cm 2 on the native aorta and ≈10-fold lower on cultured human umbilical vein endothelial cells. Interestingly, human umbilical vein endothelial cells demonstrated a 5-fold lower anticoagulation capacity in inactivating both Factor Xa and thrombin relative to native aortas. We verified the validity and accuracy of the novel assays developed in this work using liquid chromatography–mass spectrometry analysis. Conclusions— Our assays are of high relevance in the vascular community because they can be used to establish the antithrombogenic capacity of many different types of surfaces such as vascular grafts and transplants. This work will also advance the capacity for glycocalyx-targeting therapeutics development to treat damaged vasculatures.

Decellularized tissue scaffolds are commonly used in the clinic because they can be used as substitutes for more traditional biomaterials, while imparting additional physiological effects. Nevertheless, reports of complications associated with their use are widespread and poorly understood. This study probes possible causes of these complications by examining cell viability and apoptosis in response to eluents from decellularized dermis. Using multiple sources of decellularized dermis, this study shows that typical decellularized scaffolds (prepared with commonly used laboratory techniques, as well as purchased from commercial sources) contain soluble components that are cytotoxic and that these components can be removed by extensive washes in cell culture media. In addition, this study demonstrates that these observed in vitro phenotypes correlate with increased apoptosis and acute inflammation when implanted subcutaneously in mice.

Background: Vascular smooth muscle cells (SMCs) synthesize extracellular matrix (ECM) that contributes to tissue remodeling after revascularization interventions. The cytokine transforming growth factor β (TGF-β) is induced on tissue injury and regulates tissue remodeling and wound healing, but dysregulated signaling results in excess ECM deposition and fibrosis. The LIM (Lin11, Isl-1 & Mec-3) domain protein LIM domain only 7 (LMO7) is a TGF-β1 target gene in hepatoma cells, but its role in vascular physiology and fibrosis is unknown. Methods: We use carotid ligation and femoral artery denudation models in mice with global or inducible smooth muscle–specific deletion of LMO7, and knockout, knockdown, overexpression, and mutagenesis approaches in mouse and human SMC, and human arteriovenous fistula and cardiac allograft vasculopathy samples to assess the role of LMO7 in neointima and fibrosis. Results: We demonstrate that LMO7 is induced postinjury and by TGF-β in SMC in vitro. Global or SMC-specific LMO7 deletion enhanced neointimal formation, TGF-β signaling, ECM deposition, and proliferation in vascular injury models. LMO7 loss of function in human and mouse SMC enhanced ECM protein expression at baseline and after TGF-β treatment. TGF-β neutralization or receptor antagonism prevented the exacerbated neointimal formation and ECM synthesis conferred by loss of LMO7. Notably, loss of LMO7 coordinately amplified TGF-β signaling by inducing expression of Tgfb1 mRNA, TGF-β protein, αv and β3 integrins that promote activation of latent TGF-β, and downstream effectors SMAD3 phosphorylation and connective tissue growth factor. Mechanistically, the LMO7 LIM domain interacts with activator protein 1 transcription factor subunits c-FOS and c-JUN and promotes their ubiquitination and degradation, disrupting activator protein 1–dependent TGF-β autoinduction. Importantly, preliminary studies suggest that LMO7 is upregulated in human intimal hyperplastic arteriovenous fistula and cardiac allograft vasculopathy samples, and inversely correlates with SMAD3 phosphorylation in cardiac allograft vasculopathy. Conclusions: LMO7 is induced by TGF-β and serves to limit vascular fibrotic responses through negative feedback regulation of the TGF-β pathway. This mechanism has important implications for intimal hyperplasia, wound healing, and fibrotic diseases.

Hydrogels composed of solubilized decellularized extracellular matrix (ECM) are attractive materials because they combine the complexity of native ECM with injectability and ease of use. Nevertheless, these materials are typically only tunable by altering the concentration, which alters the ligand landscape, or by incorporating synthetic components, which can result in an unfavorable host response. Herein, we demonstrate the fabrication of genetically tunable ECM-derived materials, by utilizing wild type (WT) and (thrombospondin-2 knockout) TSP-2 KO decellularized skins to prepare hydrogels. The resulting materials exhibited distinct mechanical properties characterized by rheology and different concentrations of collagens when characterized by quantitative proteomics. Mixtures of the gels achieved intermediate effects between the WT and the KO, permitting tunability of the gel properties. In vivo, the hydrogels exhibited tunable cell invasion with a correlation between the content of TSP-2 KO hydrogel and the extent of cell invasion. Additionally, TSP-2 KO hydrogels significantly improved diabetic wound healing at 10 and 21 days. Furthermore, hydrogels derived from genetically engineered in vitro cell-derived matrix mimicked the trends observed for tissue-derived matrix, providing a platform for faster screening of novel manipulations and easier clinical translation. Overall, we demonstrate that genetic engineering approaches impart tunability to ECM-based hydrogels and can result in materials capable of enhanced regeneration.

Extracellular matrix (ECM) derived components are emerging sources for the engineering of biomaterials that are capable of inducing desirable cell-specific responses. This review explores the use of biomaterials derived from naturally occurring ECM proteins and their derivatives in approaches that aim to regulate cell function. Biomaterials addressed are grouped into six categories: purified single ECM proteins, combinations of purified ECM proteins, cell-derived ECM, tissue-derived ECM, diseased and modified ECM, and ECM-polymer coupled biomaterials. Purified ECM proteins serve as a material coating for enhanced cell adhesion and biocompatibility. Cell-derived and tissue-derived ECM, generated by cell isolation and decellularization technologies, can capture the native state of the ECM environment and guide cell migration and alignment patterns as well as stem cell differentiation. We focus primarily on recent advances in the fields of soft tissue, cardiac, and dermal repair, and explore the utilization of ECM proteins as biomaterials to engineer cell responses.

Mesenchymal stem cell (MSC) differentiation is regulated by surface modification including texturing, which is applied to materials to enhance tissue integration. Here, we used Pt57.5Cu14.7Ni5.3P22.5 bulk metallic glass (Pt-BMG) with nanopatterned surfaces achieved by thermoplastic forming to influence differentiation of human MSCs. Pt-BMGs are a unique class of amorphous metals with high strength, elasticity, corrosion resistance, and an unusual plastic-like processability. It was found that flat and nanopattened Pt-BMGs induced osteogenic and adipogenic differentiation, respectively. In addition, osteogenic differentiation on flat BMG exceeded that observed on medical grade titanium and was associated with increased formation of focal adhesions and YAP nuclear localization. In contrast, cells on nanopatterned BMGs exhibited rounded morphology, formed less focal adhesions and had mostly cytoplasmic YAP. These changes were preserved on nanopatterns made of nanorods with increased stiffness due to shorter aspect ratios, suggesting that MSC differentiation was primarily influenced by topography. These observations indicate that both elemental composition and nanotopography can modulate biochemical cues and influence MSCs. Moreover, the processability and highly tunable nature of Pt-BMGs enables the creation of a wide range of surface topographies that can be reproducibly and systematically studied, leading to the development of implants capable of engineering MSC functions.

Decellularized biologic scaffolds are gaining popularity over synthetic biomaterials as naturally derived materials capable of promoting improved healing. Nevertheless, the most widely used biologic material – acellular dermal matrix (ADM) – exhibits slow repopulation and remodeling, which prevents integration. Additionally, engineering control of these materials is limited because they require a natural source for their production. In the current report, we demonstrate the feasibility of using genetically engineered animals to create decellularized biologic scaffolds with favorable extracellular matrix (ECM) properties. Specifically, we utilized skin from thrombospondin (TSP)-2 KO mice to derive various decellularized products. Scanning electron microscopy and mechanical testing showed that TSP-2 KO ADM exhibited an altered structure and a reduction in elastic modulus and ultimate tensile strength , respectively. When a powdered form of KO ADM was implanted subcutaneously, it was able to promote enhanced vascularization over WT. Additionally, when implanted subcutaneously, intact slabs of KO ADM were populated by higher number of host cells when compared to WT. In vitro studies confirmed the promigratory properties of KO ADM. Specifically, degradation products released by pepsin digestion of KO ADM induced greater cell migration than WT. Moreover, cell-derived ECM from TSP-2 null fibroblasts was more permissive to fibroblast migration. Finally, ADMs were implanted in a diabetic wound model to examine their ability to accelerate wound healing. KO ADM exhibited enhanced remodeling and vascular maturation, indicative of efficient integration. Overall, we demonstrate that genetic manipulation enables engineered ECM-based materials with increased regenerative potential.

We investigated the ability of cryopreserved human amniotic membrane (hAM) scaffold sealed with an underwater adhesive, bio-inspired by marine sandcastle worms to promote healing of iatrogenic fetal membrane defects in a pregnant swine model.Twelve Yucatan miniature pigs underwent laparotomy under general anesthesia at 70 days gestation (term = 114 days). The gestational sacs were assigned to uninstrumented (n = 24) and instrumented with 12 Fr trocar, which was further randomized into four different arms-no hAM patch, (n = 22), hAM patch secured with suture (n = 16), hAM patch with no suture (n = 14), and hAM patch secured with adhesive (n = 9). The animals were euthanized 20 days after the procedure. Gross and histological examination of the entry site was performed for fetal membrane healing.There were no differences in fetal survival, amniotic fluid levels, or dye-leakage from the amniotic cavity between the groups. The fetal membranes spontaneously healed in instrumented sacs without hAM patches. In sacs with hAM patches secured with sutures, the patch was incorporated into the swine fetal membranes. In sacs with hAM patches without sutures, 100% of the patches were displaced from the defect site, whereas in sacs with hAM patches secured with adhesive 55% of the patches remained in place and showed complete healing (p = 0.04).In contrast to humans, swine fetal membranes heal spontaneously after an iatrogenic injury and thus not an adequate model. hAM patches became incorporated into the defect site by cellular ingrowth from the fetal membranes. The bioinspired adhesive adhered the hAM patches within the defect site.

Poly(ethylene glycol) (PEG) hydrogels with their highly tunable properties are promising implantable materials, but as with all non-biological materials, they elicit a foreign body response (FBR). Recent studies, however, have shown that incorporating the oligopeptide RGD into PEG hydrogels reduces the FBR. To better understand the mechanisms involved and the role of RGD in mediating the FBR, PEG, PEG-RGD and PEG-RDG hydrogels were investigated. After a 28-day subcutaneous implantation in mice, a thinner and less dense fibrous capsule formed around PEG-RGD hydrogels, while PEG and PEG-RDG hydrogels exhibited stronger, but similar FBRs. Protein adsorption to the hydrogels, which is considered the first step in the FBR, was also characterized. In vitro experiments confirmed that serum proteins adsorbed to PEG-based hydrogels and were necessary to promote macrophage adhesion to PEG and PEG-RDG, but not PEG-RGD hydrogels. Proteins adsorbed to the hydrogels in vivo were identified using liquid chromatography-tandem mass spectrometry. The majority (245) of the total proteins (≥300) that were identified was present on all hydrogels with many proteins being associated with wounding and acute inflammation. These findings suggest that the FBR to PEG hydrogels may be mediated by the presence of inflammatory-related proteins adsorbed to the surface, but that macrophages appear to sense the underlying chemistry, which for RGD improves the FBR.

Implantation of biomaterials elicits a foreign body response characterized by fusion of macrophages to form foreign body giant cells and fibrotic encapsulation. Studies of the macrophage polarization involved in this response have suggested that alternative (M2) activation is associated with more favorable outcomes. Here we investigated this process in vivo by implanting mixed cellulose ester filters or polydimethylsiloxane disks in the peritoneal cavity of wild-type (WT) and monocyte chemoattractant protein-1 (MCP-1) knockout mice. We analyzed classical (M1) and alternative (M2) gene expression via quantitative polymerase chain reaction, immunohistochemistry and enzyme-linked immunosorbent assay in both non-adherent cells isolated by lavage and implant-adherent cells. Our results show that macrophages undergo unique activation that displays features of both M1 and M2 polarization including induction of tumor necrosis factor α (TNF), which induces the expression and nuclear translocation of p50 and RelA determined by immunofluorescence and Western blot. Both processes were compromised in fusion-deficient MCP-1 KO macrophages in vitro and in vivo. Furthermore, inclusion of BAY 11-7028, an inhibitor of NFκB activation, reduced nuclear translocation of RelA and fusion in WT macrophages. Our studies suggest that peritoneal implants elicit a unique macrophage polarization phenotype leading to induction of TNF and activation of the NFκB pathway.

Thrombospondin-2 (TSP2) is a potent inhibitor of angiogenesis whose expression is dynamically regulated following injury. In the present study, it is shown that HIF-1α represses TSP2 transcription. Specifically, in vitro studies demonstrate that the prolyl hydroxylase inhibitor DMOG or hypoxia decrease TSP2 expression in fibroblasts. This effect is shown to be via a transcriptional mechanism as hypoxia does not alter TSP2 mRNA stability and this effect requires the TSP2 promoter. In addition, the documented repressive effect of nitric oxide (NO) on TSP2 is shown to be non-canonical and involves stabilization of hypoxia inducible factor-1a (HIF-1α). The regulation of TSP2 by hypoxia is supported by the in vivo observation that TSP2 has spatiotemporal expression distinct from regions of hypoxia in gastrocnemius muscle following murine hindlimb ischemia (HLI). A role for TSP2 regulation by HIF-1α is supported by the dysregulation of TSP2 expression in SM22α-cre HIF-1α KO mice following HLI. Indeed, there is a reduction in blood flow recovery in the SM22a-cre HIF-1α KO mice compared to littermate controls following HLI surgery, associated with impaired recovery and increased TSP2 levels. Moreover, SM22α-cre HIF-1α KO smooth muscle cells mice have increased TSP2 mRNA levels that persist in hypoxia. These findings identify a novel, ischemia-induced pro-angiogenic mechanism involving the transcriptional repression of TSP2 by HIF-1α.

In order to bypass the limitation of bulk metallic glasses fabrication, we synthesized thin film metallic glasses to study the corrosion characteristics of a wide atomic% composition range, Mg(35.9-63%)Ca(4.1-21%)Zn(17.9-58.3%), in simulated body fluid. We highlight a clear relationship between Zn content and corrosion current such that Zn-medium metallic glasses exhibit minimum corrosion. In addition, we found higher Zn content leads to a poor in vitro cell viability. These results showcase the benefit of evaluating a larger alloy compositional space to probe the limits of corrosion resistance and prescreen for biocompatible applications.

Each year, hundreds of thousands coronary bypass procedures are performed in the US, yet there currently exists no off-the-shelf alternative to autologous vessel transplant. In the present study, we investigated the use of mouse thrombospondin-2 knockout (TSP2 KO) cells, which secrete a non-thrombogenic and pro-migratory extracellular matrix (TSP2 KO ECM), to modify small diameter vascular grafts. To accomplish this, we first optimized the incorporation of TSP2 KO ECM on decellularized rat aortas. Because MMP levels are known to be elevated in TSP2 KO cell culture, it was necessary to probe the effect of the modification process on the graft's mechanical properties. However, no differences were found in suture retention, Young's modulus, or ultimate tensile strength between modified and unmodified grafts. Platelet studies were then performed to determine the time point at which the TSP2 KO ECM sufficiently reduced thrombogenicity. Finally, grafts modified by either TSP2 KO or WT cells or unmodified grafts, were implanted in an abdominal aortic interposition model in rats. After 4 weeks, grafts with incorporated TSP2 KO ECM showed improved endothelial and mural cell recruitment, and a decreased failure rate compared to control grafts. Therefore, our studies show that TSP2 KO ECM could enable the production of off-the-shelf vascular grafts while promoting reconstruction of native vessels.

Aim: Inflammatory myeloid lineage cells mediate neotissue formation in tissue-engineered vascular grafts, but the molecular mechanism is not completely understood. We examined the role of vasculogenic PDGF-B in tissue-engineered vascular graft neotissue development. Materials & methods: Myeloid cell-specific PDGF-B knockout mice (PDGF-KO) were generated using bone marrow transplantation, and scaffolds were implanted as inferior vena cava interposition grafts in either PDGF-KO or wild-type mice. Results: After 2 weeks, grafts from PDGF-KO mice had more remaining scaffold polymer and less intimal neotissue development. Increased macrophage apoptosis, decreased smooth muscle cell proliferation and decreased collagen content was also observed. Conclusion: Myeloid cell-derived PDGF contributes to vascular neotissue formation by regulating macrophage apoptosis, smooth muscle cell proliferation and extracellular matrix deposition.

Pharmacological modulation of responses to injury is complicated by the need to deliver multiple drugs with spatiotemporal resolution. Here, a novel controlled delivery system containing three separate compartments with each releasing its contents over different timescales is fabricated. Core-shell electrospun fibers create two of the compartments in the system, while electrosprayed spheres create the third. Utility is demonstrated by targeting the foreign body response to implants because it is a dynamic process resulting in implant failure. Sequential delivery of a drug targeting nuclear factor-κB (NF-κB) and an antifibrotic is characterized in in vitro experiments. Specifically, macrophage fusion and p65 nuclear translocation in the presence of releasate or with macrophages cultured on the surfaces of the constructs are evaluated. In addition, releasate from pirfenidone scaffolds is shown to reduce transforming growth factor-β (TGF-β)-induced pSMAD3 nuclear localization in fibroblasts. In vivo, drug eluting constructs successfully mitigate macrophage fusion at one week and fibrotic encapsulation in a dose-dependent manner at four weeks, demonstrating effective release of both drugs over different timescales. Future studies can employ this system to improve and prolong implant lifetimes, or load it with other drugs to modulate other dynamic processes.

Mice lacking thrombospondin-2 (TSP2) represent an animal model of impaired collagen fibrillogenesis. Collagen constitutes ~1/3 of the wall of the normal murine descending thoracic aorta (DTA) and is thought to confer mechanical strength at high pressures. Microstructural analysis of the DTA from TSP2-null mice revealed irregular and disorganized collagen fibrils in the adventitia and at the interface between the media and adventitia. Yet, biaxial mechanical tests performed under physiologic loading conditions showed that most mechanical metrics, including stress and stiffness, were not different between mutant and control DTAs at 20- and 40-weeks of age, thus suggesting that the absence of TSP2 is well compensated under normal conditions. A detailed bilayered analysis of the wall mechanics predicted, however, that the adventitia of TSP2-null DTAs fails to engage at high pressures, which could render the media vulnerable to mechanical damage. Failure tests confirmed that the pressure at which the DTA ruptures is significantly lower in 20-week-old TSP2-null mice compared to age-matched controls (640±37 vs. 1120±45 mmHg). Moreover, half of the 20-week-old and all 40-week-old mutant DTAs failed by delamination, not rupture. This delamination occurred at the interface between the media and the adventitia, with separation planes often observed at ~45 degrees with respect to the circumferential/axial directions. Combined with the observed microstructural anomalies, our theoretical-experimental biomechanical results suggest that TSP2-null DTAs are more susceptible to material failure when exposed to high pressures and this vulnerability may result from a reduced resistance to shear loading at the medial/adventitial border.

The PI3K/Akt pathway is necessary for several key endothelial cell (EC) functions, including cell growth, migration, survival, and vascular tone. However, existing literature supports the idea that Akt can be either pro- or antiangiogenic, possibly due to compensation by multiple isoforms in the EC when a single isoform is deleted. Thus, biochemical, genetic, and proteomic studies were conducted to examine isoform-substrate specificity for Akt1 vs. Akt2. In vitro, Akt1 preferentially phosphorylates endothelial nitric oxide synthase (eNOS) and promotes NO release, whereas nonphysiological overexpression of Akt2 can bypass the loss of Akt1. Conditional deletion of Akt1 in the EC, in the absence or presence of Akt2, retards retinal angiogenesis, implying that Akt1 exerts a nonredundant function during physiological angiogenesis. Finally, proteomic analysis of Akt substrates isolated from Akt1- or Akt2-deficient ECs documents that phosphorylation of multiple Akt substrates regulating angiogenic signaling is reduced in Akt1-deficient, but not Akt2-deficient, ECs, including eNOS and Forkhead box proteins. Therefore, Akt1 promotes angiogenesis largely due to phosphorylation and regulation of important downstream effectors that promote aspects of angiogenic signaling.

Angiopoietin-2 (Ang2) is an extracellular protein and one of the principal ligands of Tie2 receptor that is involved in the regulation of vascular integrity, quiescence, and inflammation. The mode of secretion of Ang2 has never been established, however. Here, we provide evidence that Ang2 is secreted from endothelial cells via exosomes and that this process is inhibited by the PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling pathway, whereas it is positively regulated by the syndecan-4/syntenin pathway. Vascular defects in Akt1 null mice arise, in part, because of excessive Ang2 secretion and can be rescued by the syndecan-4 knock-out that reduces extracellular Ang2 levels. This novel mechanism connects three critical signaling pathways: angiopoietin/Tie2, PI3K/Akt/eNOS, and syndecan/syntenin, which play important roles in vascular growth and stabilization.

We developed a multi-functional construct capable of controlled delivery of bioactive substances that can improve wound repair by supporting the intrinsic ability of the skin to heal. We synthesized electrospun scaffolds—composed of a blend of the degradable polymers poly( l -lactide) (PLA) or polycaprolactone (PCL)—that produce highly efficient non-viral in vivo gene delivery to cells in the wound bed, provide a protective barrier during early wound healing, and support cell migration and growth. This multi-functional material was tested for its influence on wound healing: scaffolds were loaded with plasmids encoding keratinocyte growth factor (KGF) and applied to full-thickness wounds in mice. Compared to scaffolds with control plasmids, animals receiving the KGF plasmid-loaded scaffold produced significant enhancements in wound healing, which was quantified by improvements in the rate of wound re-epithelialization, keratinocyte proliferation, and granulation response. Further, we quantified the expression level of endogenous and plasmid-derived KGF in wound samples: qRT-PCR on wound sections revealed a correlation between the levels of plasmid-derived protein expression and histological analysis of wound healing, revealing an inverse relationship between the expression level of exogenous KGF and the size of the unhealed epithelial layer in wounds. Our findings suggest that engineered nanofiber PLA/PCL scaffolds are capable of highly efficient controlled DNA delivery and are promising materials for treatment of cutaneous wounds.

Poly(ethylene glycol) (PEG)-based hydrogels are promising in situ cell carriers for tissue engineering. However, their success in vivo will in part depend upon the foreign body reaction (FBR). This study tests the hypothesis that the FBR affects cells encapsulated within PEG hydrogels, and in turn influences the severity of the FBR. Fibroblasts were encapsulated within PEG hydrogels containing RGD to support cell attachment. Macrophages were seeded on top of cell-laden hydrogels to mimic in vivo macrophage interrogation and treated with lipopolysaccharide to induce an inflammatory phenotype. The presence of activated macrophages reduced fibroblast gene expression for extracellular matrix molecules and remodeling, but stimulated VEGF and IL-1β gene expression. Fibroblasts impacted macrophage phenotype leading to increased iNOS, IL-1β and TNF-α expressions. Syngeneic cell-laden and acellular hydrogels were also implanted subcutaneously into C57bl/6 mice for 2, 7 and 28 days. Encapsulated fibroblasts secreted collagen type I during the first week, but tissue deposition and cellularity decreased by 28 days. The presence of encapsulated fibroblasts led to greater acute inflammation, but did not influence the fibrotic response. In summary, this work emphasizes the importance of the host response in tissue engineering, and the potentially deleterious impact it may have on cell-laden synthetic scaffolds.

Cell-cell fusion is fundamental to a multitude of biological processes ranging from cell differentiation and embryogenesis to cancer metastasis and biomaterial-tissue interactions. Fusogenic cells are exposed to biochemical and biophysical factors, which could potentially alter cell behavior. While biochemical inducers of fusion such as cytokines and kinases have been identified, little is known about the biophysical regulation of cell-cell fusion. Here, we designed experiments to examine cell-cell fusion using bulk metallic glass (BMG) nanorod arrays with varying biophysical cues, i.e. nanotopography and stiffness. Through independent variation of stiffness and topography, we found that nanotopography constitutes the primary biophysical cue that can override biochemical signals to attenuate fusion. Specifically, nanotopography restricts cytoskeletal remodeling-associated signaling, which leads to reduced fusion. This finding expands our fundamental understanding of the nanoscale biophysical regulation of cell fusion and can be exploited in biomaterials design to induce desirable biomaterial-tissue interactions.

Metallic alloys are normally composed of multiple constituent elements in order to achieve integration of a plurality of properties required in technological applications. However, conventional alloy development paradigm, by sequential trial-and-error approach, requires completely unrelated strategies to optimize compositions out of a vast phase space, making alloy development time consuming and labor intensive. Here, we challenge the conventional paradigm by proposing a combinatorial strategy that enables parallel screening of a multitude of alloys. Utilizing a typical metallic glass forming alloy system Zr-Cu-Al-Ag as an example, we demonstrate how glass formation and antibacterial activity, two unrelated properties, can be simultaneously characterized and the optimal composition can be efficiently identified. We found that in the Zr-Cu-Al-Ag alloy system fully glassy phase can be obtained in a wide compositional range by co-sputtering and antibacterial activity is strongly dependent on alloy compositions. Our results indicate that antibacterial activity is sensitive to Cu and Ag while essentially remains unchanged within a wide range of Zr and Al. The proposed strategy not only facilitates development of high-performing alloys, but also provides a tool to unveil the composition dependence of properties in a highly parallel fashion, which helps the development of new materials by design.

Interactions between collagenous extracellular matrices and von Willebrand factor (VWF) are critical for hemostasis and thrombosis. In the present study, we investigated the contribution of an extracellular matrix (ECM) abnormality to the bleeding diathesis in thrombospondin-2 (TSP2) knockout (KO) mice. First, we performed adoptive bone marrow transplantation and observed that introduction of wild-type (WT) marrow into lethally irradiated TSP2 KO mice did not rescue the bleeding diathesis. However, platelets in transplanted mice displayed an inherent aggregation defect, which complicated interpretation. Second, we performed interposition of arterial segments denuded of endothelium. Denuded TSP2 KO arteries grafted into WT mice remained patent in vivo. In contrast, WT grafts underwent thrombosis and were completely occluded within 24 to 48 hours. The nonthrombogenic property of the TSP2 KO ECM was confirmed in vitro by exposing platelets to TSP2 KO dermal fibroblast (DF)-derived ECM. To further probe the effect of TSP2 deficiency, ECM production and deposition by WT and TSP2 KO DFs was analyzed via polymerase chain reaction, immunofluorescence, and scanning electron microscopy and showed similar patterns. In addition, atomic force microscopy (AFM) analysis of WT and TSP2 KO ECM did not reveal differences in stiffness. In contrast, reduced VWF accumulation on TSP2 KO ECM was observed when matrices were subjected to plasma under physiological flow. AFM utilizing VWF-coated 2-μm beads confirmed the weak binding to TSP2 KO ECM, providing a mechanistic explanation for the lack of thrombus formation. Therefore, our studies show that ECM assembly is critical for interaction of collagen with VWF and subsequent thrombogenic responses.

Poly(ω-pentadecalactone-co-p-dioxanone) [poly(PDL-co-DO)] copolyesters are copolymers of an isodimorphic system, which remain semicrystalline over the whole range of compositions. Here, we evaluated enzymatically synthesized poly(PDL-co-DO) copolymers as new materials for biomedical applications. In vivo experiments using mice, showed that the copolyesters are well tolerated, with tissue responses that are comparable to poly(p-dioxanone). In addition, the copolymers were found to degrade hydrolytically at controlled rates over a period of several months under physiological conditions. The poly(PDL-co-DO) copolymers with up to 69 mol% DO units were successfully transformed to free-standing nanoparticles that are capable of encapsulating an anticancer drug, doxorubicin, or a polynucleotide, siRNA. Drug- or siRNA-loaded nanoparticles exhibited controlled and continuous release of agent over many weeks. In addition, siLUC-encapsulated poly(PDL-co-DO) nanoparticles were active in inhibiting luciferase gene expression in LUC-RKO cells. Because of substantial differences in structure and hydrophobicity between PDL and DO units, poly(PDL-co-DO) biodegradation rate and physical properties can be tuned over a wide range depending on the copolymer composition. Our results demonstrate that the semicrystalline and biodegradable poly(PDL-co-DO) copolyesters are promising biomaterials to serve as drug carriers, as well as potential raw materials for constructing bioabsorbable sutures and other medical devices.

Homotypic cell fusion occurs in several cell types including macrophages in the formation of foreign body giant cells. Previously, monocyte chemoattractant protein-1 (MCP-1) was demonstrated to be required for foreign body giant cell formation in the foreign body response. The present study investigated the fusion defect in MCP-1-null macrophages by implanting biomaterials intraperitoneally in wild-type and MCP-1-null mice and monitoring the macrophage response at 12 hours to 4 weeks. MCP-1-null mice exhibited reduced accumulation and fusion of macrophages on implants, which was associated with attenuation of the foreign body response. Consistent with previous in vitro findings, the level of matrix metalloproteinase-9 (MMP-9) was reduced in MCP-1-null macrophages adherent to implants. In contrast, CCR2 expression was unaffected. In vitro studies revealed reduced tumor necrosis factor-α (TNF-α) production and abnormal subcellular redistribution of E-cadherin and β-catenin during fusion in MCP-1-null macrophages. Exogenous TNF-α caused an increase in the production of MMP-9 and rescued the fusion defect. Addition of GM6001 (MMP inhibitor) or NSC23766 (Rac1 inhibitor) indicated two distinct induction pathways, one for E-cadherin/β-catenin and one for MCP-1, TNF-α, and MMP-9. Considered together, these observations demonstrate that induction of E-cadherin/β-catenin is not sufficient for fusion in the absence of MCP-1 or the downstream mediators TNF-α and MMP-9. Moreover, attenuation of the foreign body response in intraperitoneal implants in MCP-1-null mice demonstrates that the process depends on tissue-specific factors.

Poly(ethylene glycol) (PEG) hydrogels hold great promise as in vivo cell carriers for tissue engineering. To ensure appropriate performance of these materials when implanted, the host response must be well understood. The objectives for this study were to characterize the temporal evolution of the foreign body reaction (FBR) to acellular PEG-based hydrogels prepared from PEG diacrylate precursors when implanted subcutaneously in immunocompentent c57bl/6 mice by (immuno)histochemical analysis and gene expression. Compared with a normal FBR elicited by silicone (SIL), PEG hydrogels without or with a cell adhesion ligand RGD elicited a strong early inflammatory response evidenced by a thick band of macrophages as early as day 2, persisting through two weeks, and by increased interleukin-1β expression. PEG-only hydrogels showed a slower, but more sustained progression of inflammation over PEG-RGD. Temporal changes in gene expression were observed in response to PEG-based materials and in general exhibited, elevated expression of inflammatory and wound healing genes in the tissues surrounding the implants, while the expression patterns were more stable in response to SIL. While a stabilized FBR was achieved with SIL and to a lesser degree with PEG-RGD, the PEG-only hydrogels had not yet stabilized after 4 weeks. In summary, PEG-only hydrogels elicit a strong early inflammatory reaction, which persists throughout the course of the implantation even as a collagenous capsule begins to form. However, the incorporation of RGD tethers partially attenuates this response within 2 weeks leading to an improved FBR to PEG-based hydrogels.

Sustained changes in blood flow modulate the size of conduit arteries through structural alterations of the vessel wall that are dependent on the transient accumulation and activation of perivascular macrophages. The leukocytic infiltrate appears to be confined to the adventitia, is responsible for medial remodeling, and resolves once hemodynamic stresses have normalized without obvious intimal changes. We report that inward remodeling of the mouse common carotid artery after ligation of the ipsilateral external carotid artery is dependent on the chemokine receptor CXCR3. Wild-type myeloid cells restored flow-mediated vascular remodeling in CXCR3-deficient recipients, adventitia-infiltrating macrophages of Gr1low resident phenotype expressed CXCR3, the perivascular accumulation of macrophages was dependent on CXCR3 signaling, and the CXCR3 ligand IP-10 was sufficient to recruit monocytes to the adventitia. CXCR3 also contributed to selective features of macrophage activation required for extracellular matrix turnover, such as production of the transglutaminase factor XIII A subunit. Human adventitial macrophages displaying a CD14+/CD16+ resident phenotype, but not circulating monocytes, expressed CXCR3, and such cells were more frequent at sites of disturbed flow. Our observations reveal a CXCR3-dependent accumulation and activation of perivascular macrophages as a necessary step in homeostatic arterial remodeling triggered by hemodynamic stress in mice and possibly in humans as well.

Thrombospondin (TSP) 1 and TSP2 have been implicated in the regulation of several processes during tissue repair. Due to their matricellular nature, these proteins are thought to modulate cell-matrix interactions through a variety of mechanisms specific to the spatio-temporal context of their expression. Most notably, TSP1 and TSP2 appear to play distinct, non-overlapping roles in the healing of skin wounds. In contrast, both proteins have been implicated as regulators of ischemia-induced angiogenesis. Moreover, TSP2 has been shown to be a critical regulator of angiogenesis in the foreign body response (FBR). In this review, we discuss the role of TSPs in tissue repair and examine the mechanistic data regarding the ability of the thrombospondins to modulate cell-matrix interactions in this context.

Blood vessel formation during ischemia and wound healing requires coordination of the inflammatory response with genes that regulate blood vessel assembly. Here we show that the reticulon family member 4B, aka Nogo-B, is upregulated in response to ischemia and is necessary for blood flow recovery secondary to ischemia and wound healing. Mice lacking Nogo-B exhibit reduced arteriogenesis and angiogenesis that are linked to a decrease in macrophage infiltration and inflammatory gene expression in vivo. Bone marrow-derived macrophages isolated from Nogo knock-out mice have reduced spreading and chemotaxis due to impaired Rac activation. Bone marrow reconstitution experiments show that Nogo in myeloid cells is necessary to promote macrophage homing and functional recovery after limb ischemia. Thus, endogenous Nogo coordinates macrophage-mediated inflammation with arteriogenesis, wound healing, and blood flow control.

The selection criteria for biomaterials include the material’s properties and biocompatibility, and the ability to fabricate the desired shapes. Bulk metallic glasses (BMGs) are relative newcomers in the field of biomaterials but they exhibit an excellent combination of properties and processing capabilities desired for versatile implant applications. To further evaluate the suitability of BMGs for biomedical applications, we analyzed the biological responses they elicited in vitro and in vivo. The BMGs promoted cell adhesion and growth in vitro and induced improved foreign body responses in vivo suggesting their potential use as biomaterials. Because of the BMGs’ flexible chemistry, atomic structure, and surface topography, they offer a unique opportunity to fabricate complex implants and devices with a desirable biological response from a material with superior properties over currently used metallic biomaterials.

Despite many successful applications of microelectrode arrays (MEAs), typical two-dimensional in-vitro cultures do not project the full scale of the cell growth environment in the three-dimensional (3D) in-vivo setting. This study aims to on-line monitor in-vitro cell growth in a 3D matrix on the surface-modified MEAs with a dynamic perfusion culture system. A 3D matrix consisting of poly(ethylene glycol) hydrogel supplemented with poly-D-lysine was subsequently synthesized in situ on the self-assembled monolayer modified MEAs. FTIR spectrum analysis revealed a peak at 2100 cm(-1) due to the degradation of the structure of the 3D matrix. After 2 wks, microscopic examination revealed that the non-degraded area was around 1500 microm(2) and provided enough space for cell growth. Fluorescence microscopy revealed that the degraded 3D matrix was non-cytotoxic allowing the growth of NIH3T3 fibroblasts and cortical neurons in vitro. Time-course changes of total impedance including resistance and reactance were recorded for 8 days to evaluate the cell growth in the 3D matrix on the MEA. A consistent trend reflecting changes of reactance and total impedance was observed. These in-vitro assays demonstrate that our 3D matrix can construct a biomimetic system for cell growth and analysis of cell surface interactions.

Matrix metalloproteinases (MMPs) are enzymes with specificity towards extracellular matrix (ECM) components. MMPs, especially MMP-9, have been shown to degrade components of the basal lamina and disrupt the blood-brain barrier (BBB) and thus, contribute to neuroinflammation. In the present study we examined the role of MMP-9 in the foreign body response in the brain. Millipore filters of mixed cellulose ester were implanted into the brain cortex of wild type and MMP-9-null mice for a period of 2 d to 8 wks and the response was analyzed by histology and immunohistochemistry. We observed enhanced and prolonged neuroinflammation in MMP-9-null mice, evidenced by persistence of neutrophils, macrophages/microglia, and reactive astrocytes up to 8 wks post-implantation. In addition, blood vessel density around implants was increased in MMP-9-null mice and detection of mouse serum albumin (MSA) indicated that vessels were leaky. Immunohistochemical and western blot analyses indicated that this defect was associated with the absence of tight junction proteins zonula occludens-1 (ZO-1) and ZO-2 from vessels in proximity to implants. Analysis of brain sections and brain protein extracts revealed that the levels of the pro-inflammatory cytokine interleukin-1beta (IL-1beta), which is a substrate for MMP-9, were significantly higher in MMP-9-null mice at 8wks post-implantation. Collectively, our studies suggest that increased levels of IL-1beta and the delayed repair of BBB are associated with prolongation of the FBR in MMP-9-null mice.