Thrombospondin 2 (TSP2)-null mice, generated by disruption of the Thbs2 gene, display a variety of connective tissue abnormalities, including fragile skin and the presence of abnormally large ...collagen fibrils with irregular contours in skin and tendon. In this study we demonstrate that TSP2-null skin fibroblasts show a defect in attachment to a number of matrix proteins, and a reduction in cell spreading. To investigate the molecular mechanisms responsible for these abnormal cell-matrix interactions, we compared the levels of matrix metalloproteinases (MMPs) in wild-type and mutant fibroblasts. Isolation and analysis of gelatinases from conditioned media by gelatin-agarose affinity chromatography and gelatinolytic assays demonstrated that TSP2-null fibroblasts produce a 2-fold increase in gelatinase A (MMP2) compared with wild-type cells. The adhesive defect was corrected by treatment of TSP2-null fibroblasts with soluble TSP2, with the MMP inhibitors BB94 and tissue inhibitor of metalloproteinase-2, and with a neutralizing antibody to MMP2. Moreover, stable transfection of TSP2-null fibroblasts with mouse TSP2 cDNA corrected both the adhesive defect and the altered expression of MMP2. Finally, MMP2 was shown to interact with TSP2 in a direct-binding plate assay. We conclude that TSP2 plays an important role in cell-matrix interactions, and that a deficiency in the protein results in increased levels of MMP2 that contribute to the adhesive defect in TSP2-null fibroblasts and could play a role in the complex phenotype of TSP2-null mice.
Abstract Transplantation of endothelial cells (EC) for therapeutic vascularization is a promising approach in tissue engineering but has yet to be proven effective in clinical trials. This cell-based ...therapy is hindered by significant apoptosis of EC upon transplantation as well as poor recruitment of host mural cells to stabilize nascent vessels. Here, we address these deficiencies by augmenting endothelial cell transplantation with dual delivery of vascular endothelial growth factor (VEGF) – to improve survival of transplanted EC – and monocyte chemotactic protein-1 (MCP-1) – to induce mural cell recruitment. We produced alginate microparticles that deliver VEGF and MCP-1 with distinct release kinetics and that can be integrated into a collagen/fibronectin (protein) gel construct for delivery of EC. Combined delivery of VEGF and MCP-1 increased functional vessel formation from transplanted EC and also led to a higher number of smooth muscle cell-invested vessels than did EC therapy alone. Despite the well-known role of MCP-1 in inflammation, these beneficial effects were accomplished without a long-term increase in monocyte/macrophage recruitment or a shift to a pro-inflammatory (M1) macrophage phenotype. Overall, these data suggest a potential benefit of combined delivery of MCP-1 and VEGF from EC-containing hydrogels as a strategy for therapeutic vascularization.
Multinucleated giant cells, formed by fusion of macrophages, are a hallmark of granulomatous inflammation. With a genetic approach, we show that signaling through the adaptor protein DAP12 (DNAX ...activating protein of 12 kD), its associated receptor triggering receptor expressed by myeloid cells 2 (TREM-2), and the downstream protein tyrosine kinase Syk is required for the cytokine-induced formation of giant cells and that overexpression of DAP12 potentiates macrophage fusion. We also present evidence that DAP12 is a general macrophage fusion regulator and is involved in modulating the expression of several macrophage-associated genes, including those encoding known mediators of macrophage fusion, such as DC-STAMP and Cadherin 1. Thus, DAP12 is involved in programming of macrophages through the regulation of gene and protein expression to induce a fusion-competent state.
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.
Abstract The development of neotissue in tissue engineered vascular grafts remains poorly understood. Advances in mouse genetic models have been highly informative in the study of vascular biology, ...but have been inaccessible to vascular tissue engineers due to technical limitations on the use of mouse recipients. To this end, we have developed a method for constructing sub-1 mm internal diameter (ID) biodegradable scaffolds utilizing a dual cylinder chamber molding system and a hybrid polyester sealant scaled for use in a mouse model. Scaffolds constructed from either polyglycolic acid or poly- l -lactic acid nonwoven felts demonstrated sufficient porosity, biomechanical profile, and biocompatibility to function as vascular grafts. The scaffolds implanted as either inferior vena cava or aortic interposition grafts in SCID/bg mice demonstrated excellent patency without evidence of thromboembolic complications or aneurysm formation. A foreign body immune response was observed with marked macrophage infiltration and giant cell formation by post-operative week 3. Organized vascular neotissue, consisting of endothelialization, medial generation, and collagen deposition, was evident within the internal lumen of the scaffolds by post-operative week 6. These results present the ability to create sub-1 mm ID biodegradable tubular scaffolds that are functional as vascular grafts, and provide an experimental approach for the study of vascular tissue engineering using mouse models.
Bulk metallic glasses (BMGs) are a class of amorphous metals that exhibit high strength, ductility paired with wear and corrosion resistance. These properties suggest that they could serve as an ...alternative to conventional metallic implants that suffer wear and failure. In the present study, we investigated Platinum (Pt)-BMG biocompatibility in bone applications. Specifically, we investigated osteoclast formation on flat and nanopatterned Pt
Cu
Ni
P
(atomic percent) as well as titanium (control). Specifically, receptor activator of NF-κB (RANK) ligand-induced murine bone marrow derived mononuclear cell fusion was measured on multiple nanopatterns and was found to be reduced on nanorods (80 and 200 nm in diameter) and was associated with reduced tartrate-resistant acid phosphatase (TRAP) and matrix metalloproteinase (MMP9) expression. Evaluation of mesenchymal stem cell (MSC) to osteoblast differentiation on nanopatterned Pt-BMG showed significant reduction in comparison to flat, suggesting that further exploration of nanopatterns is required to have simultaneous induction of osteoblasts and inhibition of osteoclasts.
vivo studies were also pursued to evaluate the biocompatibility of Pt-BMG in comparison to titanium. Rods of each material were implanted in the femurs of mice and evaluated by x-ray, mechanical testing, micro-computed tomography (micro-CT), and histological analysis. Overall, Pt-BMG showed similar biocompatibility with titanium suggesting that it has the potential to improve outcomes by further processing at the nanoscale.
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.
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.
In the adult, angiogenesis is mostly restricted to the ovarian cycle, injured tissues, and organ growth. ...endothelial cells are considered stable and almost quiescent showing limited turnover in ...the adult vasculature. The research team that has long-standing experience in the study of the intracellular calcium machinery in different experimental models suggests a novel Ca2+-dependent mechanism activated by angiopoietins that controls important angiogenic processes, including cell migration and the formation of capillary-like structures in vitro.
Macrophages that are recruited to the site of implanted biomaterials undergo fusion to form surface-damaging foreign body giant cells. Exposure of peripheral blood monocytes to interleukin-4 can ...recapitulate the fusion process in vitro . In this study, we used interleukin-4 to induce multinucleation of murine bone marrow-derived macrophages and observed changes in cell shape, including elongation and lamellipodia formation, before fusion. Because cytoskeletal rearrangements are regulated by small GTPases, we examined the effects of inhibitors of Rho kinase (Y-32885) and Rac activation (NSC23766) on fusion. Y-32885 did not prevent cytoskeletal changes or fusion but limited the extent of multinucleation. NSC23766, on the other hand, inhibited lamellipodia formation and fusion in a dose-dependent manner. In addition, we found that in control cells, these changes were preceded by Rac1 activation. However, NSC23766 did not block the uptake of polystyrene microspheres. Likewise, short interfering RNA knockdown of Rac1 limited fusion without limiting phagocytosis. Thus, phagocytosis and fusion can be partially decoupled based on their susceptibility to NSC23766. Furthermore, poly(ethylene-co-vinyl acetate) scaffolds containing NSC23766 attenuated foreign body giant cell formation in vivo . These observations suggest that targeting Rac1 activation could protect biomaterials without compromising the ability of macrophages to perform beneficial phagocytic functions at implantation sites.
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