Smith MK, Mooney DJ. Hypoxia leads to necrotic hepatocyte death. J Biomed Mater Res A. 2007;80 (3) :520-9.Abstract
Hepatocyte transplantation is being investigated as a therapy for liver disease; however, its success has been limited by rapid death of the cells following transplantation. This study was dedicated to elucidating the mode of death responsible for loss of transplanted hepatocytes in order to guide future strategies for promoting their survival. Using a tissue engineering model, it was found that the environment within polymer scaffolds containing transplanted cells was hypoxic after 5 days in vivo, with (90 +/- 3)% of hepatocytes existing at pO(2) < 10 mmHg. The primary mode of hepatocyte death in response to hypoxic conditions of 0 or 2 vol % oxygen was then determined in vitro. Several assays for features of apoptosis and necrosis demonstrated that hepatocytes cultured in an anoxic environment died via necrosis, while culture at 2% oxygen inhibited proliferation. These results suggest it will not be possible to prevent hepatocyte death by interfering with the apoptotic process, and hypoxic conditions in the transplants must instead be addressed. The finding that the environment within cell transplantation scaffolds is hypoxic is likely applicable to many cell-based therapies, and a similar analysis of the primary mode of death for other cell types in response to hypoxia may be valuable in guiding future strategies for their transplantation.
Riddle KW, Kong H-J, Leach KJ, Fischbach C, Cheung C, Anseth KS, Mooney DJ. Modifying the proliferative state of target cells to control DNA expression and identifying cell types transfected in vivo. Mol Ther. 2007;15 (2) :361-8.Abstract
Although the majority of current gene transfer techniques have focused on increasing the ability of the DNA to enter the cell, it is possible that changing the proliferative and migratory state of cells will influence the cells ability to take up and express plasmid DNA. This study was designed to test the hypothesis that growth factors (basic fibroblast growth factor (bFGF) and hepatocyte growth factor/scatter factor (HGF/SF)) used to alter the proliferative and migratory state of cells can alter plasmid DNA uptake and expression. In vitro studies indicate that enhancing cell proliferation with growth factor exposure enhances plasmid DNA uptake and expression. Furthermore, dual localized delivery of bFGF and plasmid DNA in vivo increases the expression, 3-6 times over control, as compared to plasmid delivery alone. Dual delivery of a factor promoting cell proliferation and a plasmid led to a further increase in the expression of the plasmid encoding bone morphogenetic protein-2 in a rat cranial defect by specific cell populations. The results of these studies suggest that increasing the proliferative state of target cell populations can enhance non-viral gene transfer.
Kong H, Hsiong S, Mooney DJ. Nanoscale cell adhesion ligand presentation regulates nonviral gene delivery and expression. Nano Lett. 2007;7 (1) :161-6.Abstract
It is hypothesized that the nanoscale organization of cell adhesion ligands in a synthetic ECM regulates nonviral gene delivery. This hypothesis was examined with pre-osteoblasts cultured on substrates which present varied density and spacing of synthetic adhesion ligands. The levels of gene transfer and expression were increased with the density of adhesion ligands, but decreased with the spacing of ligands, due to changes in the cell growth rate. This study provides a material-based control point on the nanometer scale for nonviral gene based therapies.
Fischbach C, Mooney DJ. Polymers for pro- and anti-angiogenic therapy. Biomaterials. 2007;28 (12) :2069-76.Abstract
Dysregulated growth factor signaling is traditionally targeted via bolus injections of therapeutic molecules, but this approach may not recreate necessary qualitative and quantitative aspects of biologic growth factor delivery systems. Polymeric delivery systems may, instead, mimic certain sequestration and binding characteristics of the extracellular matrix and lead to the provision of therapeutic molecules at therapeutically efficient local concentrations [V], in the form of spatial gradients (d[V]/dx) and temporal gradients (d[V]/dt), and in combination with other morphogenetic cues. Both physicochemical and biological attributes dictate their design, and they may be fabricated from synthetic and natural polymers. General concepts for manipulating growth factor signaling with these systems are discussed in the context of angiogenesis with vascular endothelial growth factor (VEGF), and these strategies may be broadly adapted to a multitude of other morphogens and growth factors.
Silva EA, Mooney DJ. Spatiotemporal control of vascular endothelial growth factor delivery from injectable hydrogels enhances angiogenesis. J Thromb Haemost. 2007;5 (3) :590-8.Abstract
Therapeutic angiogenesis with vascular endothelial growth factor (VEGF) delivery may provide a new approach for the treatment of ischemic diseases, but current strategies to deliver VEGF rely on either bolus delivery or systemic administration, resulting in limited clinical utility, because of the short half-life of VEGF in vivo and its resultant low and transient levels at sites of ischemia. We hypothesize that an injectable hydrogel system can be utilized to provide temporal control and appropriate spatial biodistribution of VEGF in ischemic hindlimbs. A sustained local delivery of relatively low amounts of bioactive VEGF (3 mug) with this system led to physiologic levels of bioactive VEGF in ischemic murine (ApoE(-/-)) hindlimbs for 15 days after injection of the gel, as contrasted with complete VEGF deprivation after 72 h with bolus injection. The gel delivery system resulted in significantly greater angiogenesis in these limbs as compared to bolus (266 vs. 161 blood vessels mm(-2)). Laser Doppler perfusion imaging showed return of tissue perfusion to normal levels by day 28 with the gel system, whereas normal levels of perfusion were never achieved with saline delivery of VEGF or in control mice. The system described in this article could represent an attractive new generation of therapeutic delivery vehicle for treatment of cardiovascular diseases, as it combines long-term in vivo therapeutic benefit (localized bioactive VEGF for 1-2 weeks) with minimally invasive delivery.
Chen RR, Silva EA, Yuen WW, Mooney DJ. Spatio-temporal VEGF and PDGF delivery patterns blood vessel formation and maturation. Pharm Res. 2007;24 (2) :258-64.Abstract
PURPOSE: Biological mechanisms of tissue regeneration are often complex, involving the tightly coordinated spatial and temporal presentation of multiple factors. We investigated whether spatially compartmentalized and sequential delivery of factors can be used to pattern new blood vessel formation. MATERIALS AND METHODS: A porous bi-layered poly(lactide-co-glycolide) (PLG) scaffold system was used to locally present vascular endothelial growth factor (VEGF) alone in one spatial region, and sequentially deliver VEGF and platelet-derived growth factor (PDGF) in an adjacent region. Scaffolds were implanted in severely ischemic hindlimbs of SCID mice for 2 and 6 weeks, and new vessel formation was quantified within the scaffolds. RESULTS: In the compartment delivering a high dose of VEGF alone, a high density of small, immature blood vessels was observed at 2 weeks. Sequential delivery of VEGF and PDGF led to a slightly lower blood vessel density, but vessel size and maturity were significantly enhanced. Results were similar at 6 weeks, with continued remodeling of vessels in the VEGF and PDGF layer towards increased size and maturation. CONCLUSIONS: Spatially localizing and temporally controlling growth factor presentation for angiogenesis can create spatially organized tissues.
Comisar WA, Kazmers NH, Mooney DJ, Linderman JJ. Engineering RGD nanopatterned hydrogels to control preosteoblast behavior: a combined computational and experimental approach. Biomaterials. 2007;28 (30) :4409-17.Abstract
The adhesion ligand arginine-glycine-aspartic acid (RGD) has been coupled to various materials to be used as tissue culture matrices or cell transplantation vehicles, and recent studies indicate that nanopatterning RGD into high-density islands alters key cell behaviors. Previous studies have failed, however, to conclusively decouple the effects of RGD bulk density and individual pattern parameters (i.e. RGDs/island and island distribution) on these altered cell responses. Using a nanopatterned RGD-coupled alginate hydrogel matrix, this work combines computational, statistical and experimental approaches to elucidate the effects of RGD patterns on four key cell responses. This study shows that in MC3T3 preosteoblasts focal adhesion kinase (FAK) Y397 phosphorylation, cell spreading, and osteogenic differentiation can be controlled by RGD nanopatterning, with the distribution of islands throughout the hydrogel (i.e. how closely spaced the islands are) being the most significant pattern parameter. More closely spaced islands favor FAK Y397 phosphorylation and cell spreading, while more widely spaced islands favor differentiation. Proliferation, in contrast, is primarily a function of RGD bulk density. Nanopatterning of cell adhesion ligands has tremendous potential as a simple tool to gain significant control over multiple cell behaviors in engineered extracellular matrix (ECM).
Chen RR, Snow JK, Palmer JP, Lin AS, Duvall CL, Guldberg RE, Mooney DJ. Host immune competence and local ischemia affects the functionality of engineered vasculature. Microcirculation. 2007;14 (2) :77-88.Abstract
OBJECTIVE: Localized and sustained delivery of vascular endothelial growth factor (VEGF) is a promising approach to overcome the limited efficacy of bolus delivery. The authors examined the effects of host immune competence and local ischemia on the functionality of new vessel networks formed with this approach. METHODS: Vessel structure and perfusion resulting from implantation of porous 85:15 poly(lactide-co-glycolide) scaffolds releasing VEGF165 were measured in both subcutaneous tissue and ischemic hindlimbs of immune competent C57BL/6 and immune deficient SCID mice. RESULTS: Sustained VEGF delivery resulted in a similar approximately 100% increase in vessel density within scaffolds in both implant sites, and both animal models. However, the resulting perfusion within scaffolds implanted in subcutaneous tissue increased modestly versus control (18-35%), while perfusion increased 52-110% above control when VEGF-releasing scaffolds were placed in ischemic hindlimbs of C57BL/6 or SCID mice. VEGF delivery improved perfusion in the entire ischemic limb (55 +/- 18% of the normal value by week 6; 138% increase over control) in SCID mice. Although C57BL/6 mice demonstrated spontaneous recovery from ischemia, VEGF delivery accelerated recovery as compared to control. CONCLUSIONS: Localized and sustained VEGF delivery can create functional vasculature that amplifies recovery of tissue ischemia. However, increases in local and regional perfusion were highly dependent on the implantation site and the animal model.
Chen RR, Silva EA, Yuen WW, Brock AA, Fischbach C, Lin AS, Guldberg RE, Mooney DJ. Integrated approach to designing growth factor delivery systems. FASEB J. 2007;21 (14) :3896-903.Abstract
Growth factors have been widely used in strategies to regenerate and repair diseased tissues, but current therapies that go directly from bench to bedside have had limited clinical success. We hypothesize that engineering successful therapies with recombinant proteins will often require specific quantitative information of the spatiotemporal role of the factors and the development of sophisticated delivery approaches that provide appropriate tissue exposures. This hypothesis was tested in the context of therapeutic angiogenesis. An in vitro model of angiogenesis was adapted to quantify the role of the concentration/gradient of vascular endothelial growth factor [VEGF(165)] on microvascular endothelial cells, and a delivery system was then designed, based on a mathematical model, to provide the desired profile in ischemic mice hindlimbs. This system significantly enhanced blood vessel formation, and perfusion and recovery from severe ischemia. This general approach may be broadly applicable to growth factor therapies.
Matsumoto T, Yung YC, Fischbach C, Kong H, Nakaoka R, Mooney DJ. Mechanical strain regulates endothelial cell patterning in vitro. Tissue Eng. 2007;13 (1) :207-17.Abstract
Blood vessels of the vertebrate circulatory system typically exhibit tissue-specific patterning. However, the cues that guide the development of these patterns remain unclear. We investigated the effect of cyclic uniaxial strain on vascular endothelial cell dynamics and sprout formation in vitro in two-dimensional (2D) and three-dimensional (3D) culture systems under the influence of growth factors. Cells preferentially aligned and moved in the direction perpendicular to the major strain axis in monolayer culture, and mechanical strain also regulated the spatial location of cell proliferation in 2D cell culture. Cells in 3D cell culture could be induced to form sprouts by exposure to appropriate growth factor combinations (vascular endothelial growth factor and hepatocyte growth factor), and the strain direction regulated the directionality of this process. Moreover, cyclic uniaxial strain inhibited branching of the structures formed by endothelial cells and increased their thickness. Taken together, these data support the importance of external mechanical stimulation in the regulation of endothelial cell migration, proliferation, and differentiation into primitive vessels.
Kong H, Mooney DJ. Microenvironmental regulation of biomacromolecular therapies. Nat Rev Drug Discov. 2007;6 (6) :455-63.Abstract
There is currently great interest in molecular therapies to treat various diseases, and this has prompted extensive efforts to achieve target-specific and controlled delivery of bioactive macromolecules (for example, proteins, antibodies, DNA and small interfering RNA) through the design of smart drug carriers. By contrast, the influence of the microenvironment in which the target cell resides and the effect it might have on the success of biomacromolecular therapies has been under-appreciated. The extracellular matrix (ECM) component of the cellular niche may be particularly important, as many diseases and injury disrupt the normal ECM architecture, the cell adhesion to ECM, and the subsequent cellular activities. This Review will discuss the importance of the ECM and the ECM-cell interactions on the cell response to bioactive macromolecules, and suggest how this information could lead to new criteria for the design of novel drug delivery systems.
Kong H, Kim C, Huebsch N, Weitz D, Mooney DJ. Noninvasive probing of the spatial organization of polymer chains in hydrogels using fluorescence resonance energy transfer (FRET). J Am Chem Soc. 2007;129 (15) :4518-9.
Dong Z, Neiva KG, Jin T, Zhang Z, Hall DE, Mooney DJ, Polverini PJ, Nör JE. Quantification of human angiogenesis in immunodeficient mice using a photon counting-based method. Biotechniques. 2007;43 (1) :73-7.Abstract
Testing new antiangiogenic drugs for cancer treatment requires the use of animal models, since stromal cells and extracellular matrices mediate signals to endothelial cells that cannot be fully reproduced in vitro. Most methods used for analysis of antiangiogenic drugs in vivo utilized histologic examination of tissue specimens, which often requires large sample sizes to obtain reliable quantitative data. Furthermore, these assays rely on the analysis of murine vasculature that may not be correlated with the responses of human endothelial cells. Here, we engineered human blood vessels in immunodeficient mice with human endothelial cells expressing luciferase, demonstrated that these cells line functional blood vessels, and quantified angiogenesis over time using a photon counting-based method. In a proof-of-principle experiment with PTK/ZK, a small molecule inhibitor of vascular endothelial growth factor (VEGF) tyrosine kinase receptors, a strong correlation was observed between the decrease in bioluminescence (9.12-fold) in treated mice and the actual decrease in microvessel density (9.16-fold) measured after retrieval of the scaffolds and immunohistochemical staining of endothelial cells. The method described here allows for quantitative and noninvasive investigation into the effects of anti-cancer drugs on human angiogenesis in a murine host.
Boontheekul T, Hill EE, Kong H-J, Mooney DJ. Regulating myoblast phenotype through controlled gel stiffness and degradation. Tissue Eng. 2007;13 (7) :1431-42.Abstract
Mechanical stiffness and degradability are important material parameters in tissue engineering. The aim of this study was to address the hypothesis that these variables regulate the function of myoblasts cultured in 2-D and 3-D microenvironments. Development of cell-interactive alginate gels with tunable degradation rates and mechanical stiffness was established by a combination of partial oxidation and bimodal molecular weight distribution. Higher gel mechanical properties (13 to 45 kPa) increased myoblast adhesion, proliferation, and differentiation in a 2-D cell culture model. Primary mouse myoblasts were more highly responsive to this cue than the C2C12 myoblast cell line. Myoblasts were then encapsulated in gels varying in degradation rate to simultaneously investigate the effect of degradation and subsequent reduction of mechanical properties on cells in a 3-D environment. C2C12 cells in more rapidly degrading gels exhibited lower proliferation, as they exited the cell cycle to differentiate, compared to those in nondegradable gels. In contrast, mouse primary myoblasts illustrated significantly higher proliferation in degradable gels than in nondegradable gels, and exhibited minimal differentiation in either type of gel. Altogether, these studies suggest that a critical balance between material degradation rate and mechanical properties may be required to regulate formation of engineered skeletal muscle tissue, and that results obtained with the C2C12 cell line may not be predictive of the response of primary myoblasts to environmental cues. The principles delineated in these studies may be useful to tailor smart biomaterials that can be applied to many other polymeric systems and tissue types.
Zhong Y, Zhang L, Ding AG, Shenderova A, Zhu G, Pei P, Chen RR, Mallery SR, Mooney DJ, Schwendeman SP. Rescue of SCID murine ischemic hindlimbs with pH-modified rhbFGF/poly(DL-lactic-co-glycolic acid) implants. J Control Release. 2007;122 (3) :331-7.Abstract
Site-specific controlled release of biologically active angiogenic growth factors such as recombinant human basic fibroblast growth factor (rhbFGF) is a promising approach to improve collateral circulation in patients suffering from ischemic heart disease or peripheral vascular disease. Previously, we demonstrated stabilization of rhbFGF encapsulated in injectable poly(DL-lactic-co-glycolic acid) (PLGA) millicylindrical implants upon co-incorporation of Mg(OH)2 to raise the microclimate pH in the polymer. The purpose of this study was to compare stabilized (S; +Mg(OH)2+other stabilizers), partially stabilized (PS; -Mg(OH)2+other stabilizers), unstabilized (US; no stabilizers), and blank (B) PLGA-encapsulated rhFGF formulations to promote angiogenesis in SCID mice. Following 4 weeks subcutaneous implantation at a 0.1 microg dose in healthy animals, the S group exhibited significantly higher blood vessel density (62+/-17 vessels/mm2) compared with PS, US, and B groups (11+/-2*, 17+/-7*, and 3+/-1** respectively) (* p<0.05; ** p<0.01). Furthermore, the S group developed a thicker granulation layer at the tissue/implant interface relative to the other groups (39+/-7 vs 25+/-2**, 21+/-1***, and 12+/-1 microm*** respectively) (*** p<0.001). After 6 weeks implantation in mice with ischemic hindlimbs, the S group implants also markedly augmented both limb reperfusion (87+/-14%) and limb survival (5/5), whereas ischemic limbs did not recover in PS, US and B groups. Stabilized rhbFGF incorporated in pH modified PLGA millicylinders effectively promotes site-directed in vivo angiogenesis and also enables preservation of ischemic hindlimb function.
Fischbach C, Chen R, Matsumoto T, Schmelzle T, Brugge JS, Polverini PJ, Mooney DJ. Engineering tumors with 3D scaffolds. Nat Methods. 2007;4 (10) :855-60.Abstract
Microenvironmental conditions control tumorigenesis and biomimetic culture systems that allow for in vitro and in vivo tumor modeling may greatly aid studies of cancer cells' dependency on these conditions. We engineered three-dimensional (3D) human tumor models using carcinoma cells in polymeric scaffolds that recreated microenvironmental characteristics representative of tumors in vivo. Strikingly, the angiogenic characteristics of tumor cells were dramatically altered upon 3D culture within this system, and corresponded much more closely to tumors formed in vivo. Cells in this model were also less sensitive to chemotherapy and yielded tumors with enhanced malignant potential. We assessed the broad relevance of these findings with 3D culture of other tumor cell lines in this same model, comparison with standard 3D Matrigel culture and in vivo experiments. This new biomimetic model may provide a broadly applicable 3D culture system to study the effect of microenvironmental conditions on tumor malignancy in vitro and in vivo.
Sands WR, Mooney DJ. Polymers to direct cell fate by controlling the microenvironment. Curr Opin Biotechnol. 2007;18 (5) :448-53.Abstract
Enhanced understanding of the signals within the microenvironment that regulate cell fate has led to the development of increasingly sophisticated polymeric biomaterials for tissue engineering and regenerative medicine applications. This advancement is exemplified by biomaterials with precisely controlled scaffold architecture that regulate the spatio-temporal release of growth factors and morphogens, and respond dynamically to microenvironmental cues. Further understanding of the biology, qualitatively and quantitatively, of cells within their microenvironments and at the tissue-material interface will expand the design space of future biomaterials.
Cao L, Mooney DJ. Spatiotemporal control over growth factor signaling for therapeutic neovascularization. Adv Drug Deliv Rev. 2007;59 (13) :1340-50.Abstract
Many of the qualitative roles of growth factors involved in neovascularization have been delineated, but it is unclear yet from an engineering perspective how to use these factors as therapies. We propose that an approach that integrates quantitative spatiotemporal measurements of growth factor signaling using 3-D in vitro and in vivo models, mathematic modeling of factor tissue distribution, and new delivery technologies may provide an opportunity to engineer neovascularization on demand.
Nakaoka R, Hsiong SX, Mooney DJ. Regulation of chondrocyte differentiation level via co-culture with osteoblasts. Tissue Eng. 2006;12 (9) :2425-33.Abstract
The close apposition of osteoblasts and chondrocytes in bone and their interaction during bone development and regeneration suggest that they may each regulate the other's growth and differentiation. In these studies, osteoblasts and chondrocytes were co-cultured in vitro, with both direct and indirect contact. Proliferation of the co-cultured chondrocytes was enhanced using soluble factors produced from the osteoblasts, and the differentiation level of the osteoblasts influenced the differentiation level of the chondrocytes. In addition, the chondrocytes regulated differentiation of the co-cultured osteoblasts using soluble factors and direct contact. These data support the possibility of direct, reciprocal instructive interactions between chondrocytes and osteoblasts in a variety of normal processes and further suggest that it may be necessary to account for this signaling in the regeneration of complex tissues comprising cartilage and mineralized tissue.
Zhao B-Q, Wang S, Kim H-Y, Storrie H, Rosen BR, Mooney DJ, Wang X, Lo EH. Role of matrix metalloproteinases in delayed cortical responses after stroke. Nat Med. 2006;12 (4) :441-5.Abstract
Matrix metalloproteinases (MMPs) are zinc-endopeptidases with multifactorial actions in central nervous system (CNS) physiology and pathology. Accumulating data suggest that MMPs have a deleterious role in stroke. By degrading neurovascular matrix, MMPs promote injury of the blood-brain barrier, edema and hemorrhage. By disrupting cell-matrix signaling and homeostasis, MMPs trigger brain cell death. Hence, there is a movement toward the development of MMP inhibitors for acute stroke therapy. But MMPs may have a different role during delayed phases after stroke. Because MMPs modulate brain matrix, they may mediate beneficial plasticity and remodeling during stroke recovery. Here, we show that MMPs participate in delayed cortical responses after focal cerebral ischemia in rats. MMP-9 is upregulated in peri-infarct cortex at 7-14 days after stroke and is colocalized with markers of neurovascular remodeling. Treatment with MMP inhibitors at 7 days after stroke suppresses neurovascular remodeling, increases ischemic brain injury and impairs functional recovery at 14 days. MMP processing of bioavailable VEGF may be involved because inhibition of MMPs reduces endogenous VEGF signals, whereas additional treatment with exogenous VEGF prevents MMP inhibitor-induced worsening of infarction. These data suggest that, contrary to MMP inhibitor therapies for acute stroke, strategies that modulate MMPs may be needed for promoting stroke recovery.