Vascular endothelial growth factor (VEGF) is a potent peptide with well-documented pro-angiogenic effects. Recently, it has also become clear that exogenous administration of VEGF is neuroprotective in animal models of central nervous system diseases. In the present study, VEGF was incorporated into a sustained release hydrogel delivery system to examine its potential benefits in a rat model of Huntington's disease (HD). The VEGF-containing hydrogel was stereotaxically injected into the striatum of adult rats. Three days later, quinolinic acid (QA; 225 nmol) was injected into the ipsilateral striatum to produce neuronal loss and behavioral deficits that mimic those observed in HD. Two weeks after surgery, animals were tested for motor function using the placement and cylinder tests. Control animals received either QA alone or QA plus empty hydrogel implants. Behavioral testing confirmed that the QA lesion resulted in significant deficits in the ability of the control animals to use their contralateral forelimb. In contrast, the performance of those animals receiving VEGF was significantly improved relative to controls with only modest motor impairments observed. Stereological counts of NeuN-positive neurons throughout the striatum demonstrated that VEGF implants significantly protected against the loss of striatal neurons induced by QA. These data are the first to demonstrate that VEGF can be used to protect striatal neurons from excitotoxic damage in a rat model of HD.
Label-free nanosensors can detect disease markers to provide point-of-care diagnosis that is low-cost, rapid, specific and sensitive. However, detecting these biomarkers in physiological fluid samples is difficult because of problems such as biofouling and non-specific binding, and the resulting need to use purified buffers greatly reduces the clinical relevance of these sensors. Here, we overcome this limitation by using distinct components within the sensor to perform purification and detection. A microfluidic purification chip simultaneously captures multiple biomarkers from blood samples and releases them, after washing, into purified buffer for sensing by a silicon nanoribbon detector. This two-stage approach isolates the detector from the complex environment of whole blood, and reduces its minimum required sensitivity by effectively pre-concentrating the biomarkers. We show specific and quantitative detection of two model cancer antigens from a 10 microl sample of whole blood in less than 20 min. This study marks the first use of label-free nanosensors with physiological solutions, positioning this technology for rapid translation to clinical settings.
Pulmonary transit of agitated contrast (PTAC) occurs to variable extents during exercise. We tested the hypothesis that the onset of PTAC signifies flow through larger-caliber vessels, resulting in improved pulmonary vascular reserve during exercise. Forty athletes and fifteen nonathletes performed maximal exercise with continuous echocardiographic Doppler measures [cardiac output (CO), pulmonary artery systolic pressure (PASP), and myocardial velocities] and invasive blood pressure (BP). Arterial gases and B-type natriuretic peptide (BNP) were measured at baseline and peak exercise. Pulmonary vascular resistance (PVR) was determined as the regression of PASP/CO and was compared according to athletic and PTAC status. At peak exercise, athletes had greater CO (16.0 ± 2.9 vs. 12.4 ± 3.2 l/min, P < 0.001) and higher PASP (60.8 ± 12.6 vs. 47.0 ± 6.5 mmHg, P < 0.001), but PVR was similar to nonathletes (P = 0.71). High PTAC (defined by contrast filling of the left ventricle) occurred in a similar proportion of athletes and nonathletes (18/40 vs. 10/15, P = 0.35) and was associated with higher peak-exercise CO (16.1 ± 3.4 vs. 13.9 ± 2.9 l/min, P = 0.010), lower PASP (52.3 ± 9.8 vs. 62.6 ± 13.7 mmHg, P = 0.003), and 37% lower PVR (P < 0.0001) relative to low PTAC. Right ventricular (RV) myocardial velocities increased more and BNP increased less in high vs. low PTAC subjects. On multivariate analysis, maximal oxygen consumption (VO(2max)) (P = 0.009) and maximal exercise output (P = 0.049) were greater in high PTAC subjects. An exercise-induced decrease in arterial oxygen saturation (98.0 ± 0.4 vs. 96.7 ± 1.4%, P < 0.0001) was not influenced by PTAC status (P = 0.96). Increased PTAC during exercise is a marker of pulmonary vascular reserve reflected by greater flow, reduced PVR, and enhanced RV function.
Long-chained polymers in alginate hydrogels can form networks by either ionic or covalent crosslinks. This paper shows that the type of crosslinks can markedly affect the stress-relaxation behavior of the gels. In gels with only ionic crosslinks, stress relaxes mainly through breaking and subsequent reforming of the ionic crosslinks, and the time scale of the relaxation is independent of the size of the sample. By contrast, in gels with only covalent crosslinks, stress relaxes mainly through migration of water, and the relaxation slows down as the size of the sample increases. Implications of these observations are discussed.
PURPOSE: The aim was to investigate that a bio-degradable alginate and poly lactide-co-glycolide (PLG) system capable of delivering growth factors sequentially would be superior to single growth factor delivery in promoting neovascularization and improving perfusion.
METHODS: Three groups of apoE null mice underwent unilateral hindlimb ischemia surgery and received ischemic limb intramuscular injections of alginate (Blank), alginate containing VEGF(165) (VEGF), or alginate containing VEGF(165) combined with PLG microspheres containing PDGF-BB (VEGF/PDGF). Vascularity in the ischemic hindlimb was assessed by morphologic and immunohistochemical end-points, while changes in blood flow were assessed by Laser Doppler Perfusion Index. Muscle VEGF and PDGF content was assessed at multiple time points.
RESULTS: In the VEGF/PDGF group, local tissue VEGF and PDGF levels peaked at week 2 and 4, respectively, with detectable PDGF levels at week 6. At week 6, mean vessel mean diameter was significantly greater in the VEGF/PDGF group compared to the VEGF or Blank groups with evidence of well-formed smooth muscle-lined arterioles.
CONCLUSIONS: Sequential delivery of VEGF and PDGF using an injectable, biodegradable platform resulted in stable and sustained improvements in perfusion. This sustained, control-released, injectable alginate polymer system is a promising approach for multiple growth factor delivery in clinical application.
This paper describes a simple reversible hydrogel patterning method for 3D cell culture. Alginate gel is formed in select regions of a microfluidic device through light-triggered release of caged calcium. In the pre-gelled alginate solution, calcium is chelated by DM-nitrophen (DM-n) to prevent cross-linking of alginate. After sufficient UV exposure the caged calcium is released from DM-n causing alginate to cross-link. The effect of using different concentrations of calcium and chelating agents as well as the duration of UV exposure is described. Since the cross-linking is based on calcium concentration, the cross-linked alginate can easily be dissolved by EDTA. We also demonstrate application of this capability to patterned microscale 3D co-culture using endothelial cells and osteoblastic cells in a microchannel.
Therapeutic angiogenesis relies on the delivery of angiogenic factors capable of reversing tissue ischemia. Polymeric materials that can provide spatial and temporal over vascular endothelial growth factor (VEGF) presentation provide clear benefit, but the influence of VEGF dose, temporal, and spatial presentation on the resultant angiogenic process are largely unknown. The influence of the temporal profile of VEGF concentration, dose, and the impact of VEGF spatial distribution on angiogenesis in in vitro models of angiogenesis and ischemic murine limbs was analyzed in this study. Importantly, a profile consisting of a high VEGF concentration initially, followed by a decreasing concentration over time was found to yield optimal angiogenic sprouting. A total VEGF dose 0.1 microg/g, when delivered with kinetics found to be optimal in vitro, provided a favorable therapeutic dose in murine hindlimb ischemia model, and distributing this VEGF dose in two spatial locations induces a higher level of vascularization and perfusion than a single location. These findings suggest that material systems capable of controlling and regulating the temporal and spatial presentation of VEGF maybe useful to achieve a robust and potent therapeutic angiogenic effect in vivo.
Regenerative efforts typically focus on the delivery of single factors, but it is likely that multiple factors regulating distinct aspects of the regenerative process (e.g., vascularization and stem cell activation) can be used in parallel to affect regeneration of functional tissues. This possibility was addressed in the context of ischemic muscle injury, which typically leads to necrosis and loss of tissue and function. The role of sustained delivery, via injectable gel, of a combination of VEGF to promote angiogenesis and insulin-like growth factor-1 (IGF1) to directly promote muscle regeneration and the return of muscle function in ischemic rodent hindlimbs was investigated. Sustained VEGF delivery alone led to neoangiogenesis in ischemic limbs, with complete return of tissue perfusion to normal levels by 3 weeks, as well as protection from hypoxia and tissue necrosis, leading to an improvement in muscle contractility. Sustained IGF1 delivery alone was found to enhance muscle fiber regeneration and protected cells from apoptosis. However, the combined delivery of VEGF and IGF1 led to parallel angiogenesis, reinnervation, and myogenesis; as satellite cell activation and proliferation was stimulated, cells were protected from apoptosis, the inflammatory response was muted, and highly functional muscle tissue was formed. In contrast, bolus delivery of factors did not have any benefit in terms of neoangiogenesis and perfusion and had minimal effect on muscle regeneration. These results support the utility of simultaneously targeting distinct aspects of the regenerative process.
Stem cells sense and respond to the mechanical properties of the extracellular matrix. However, both the extent to which extracellular-matrix mechanics affect stem-cell fate in three-dimensional microenvironments and the underlying biophysical mechanisms are unclear. We demonstrate that the commitment of mesenchymal stem-cell populations changes in response to the rigidity of three-dimensional microenvironments, with osteogenesis occurring predominantly at 11-30 kPa. In contrast to previous two-dimensional work, however, cell fate was not correlated with morphology. Instead, matrix stiffness regulated integrin binding as well as reorganization of adhesion ligands on the nanoscale, both of which were traction dependent and correlated with osteogenic commitment of mesenchymal stem-cell populations. These findings suggest that cells interpret changes in the physical properties of adhesion substrates as changes in adhesion-ligand presentation, and that cells themselves can be harnessed as tools to mechanically process materials into structures that feed back to manipulate their fate.
Diabetes can diminish the responsiveness to angiogenic factors (e.g., VEGF) important for wound healing and the treatment of ischemic diseases, and this study investigated the hypothesis that this effect can be reversed by altering Notch signaling. Aortic endothelial cells (ECs) isolated from diabetic mice demonstrated reduced sprouting capability in vitro, but adding a Notch inhibitor (DAPT) led to cell-density and VEGF-dose dependent enhancement of proliferation, migration and sprouting, in both 2-D and 3-D cultures, as compared to VEGF alone. The in vivo effects of VEGF and DAPT were tested in the ischemic hind limbs of diabetic mice. Combining VEGF and DAPT delivery resulted in increased blood vessel density (∼150%) and improved tissue perfusion (∼160%), as compared to VEGF alone. To examine if DAPT would interfere with vessel maturation, DAPT was also delivered with a combination of VEGF and platelet derived growth factor (PDGF). DAPT and PDGF did not interfere with the effects of the other, and highly functional and mature networks of vessels could be formed with appropriate delivery. In summary, modulating Notch signaling enhances neovascularization and perfusion recovery in diabetic mice suffering from ischemia, suggesting this approach could have utility for human diabetics.
Lyophilization of polycation/pDNA complexes provides stable, long-term storage of complexes prior to clinical use but also reduces gene delivery efficiency. We examined whether polycation structure mediates effects of lyophilization on gene expression. Linear and branched PEI of the same molecular weight were used as a model system. Interestingly, pDNA/linear PEI complexes led to much smaller effects on gene expression following lyophilization compared with branched PEI complexes. The effect of polycation structure correlated with changes in dissociation ability of pDNA/PEI complexes. These results will be useful for developing new gene delivery vehicles.
Nature frequently utilizes opposing factors to create a stable activator gradient to robustly control pattern formation. This study employs a biomimicry approach, by delivery of both angiogenic and antiangiogenic factors from spatially restricted zones of a synthetic polymer to achieve temporally stable and spatially restricted angiogenic zones in vivo. The simultaneous release of the two spatially separated agents leads to a spatially sharp angiogenic region that is sustained over 3 wk. Further, the contradictory action of the two agents leads to a stable level of proangiogenic stimulation in this region, in spite of significant variations in the individual release rates over time. The resulting spatially restrictive and temporally sustained profiles of active signaling allow the creation of a spatially heterogeneous and functional vasculature.
Improvement of long-term drug release and design of mechanically more stable encapsulation devices are still major challenges in the field of cell encapsulation. This may be in part due to the weak in vivo stability of calcium-alginate beads and to the use of inactive biomaterials and inert scaffolds that do not mimic the physiological situation of the normal cell milieu. We hypothesized that designing biomimetic cell-hydrogel capsules might promote the in vivo long-term functionality of the enclosed drug-secreting cells and improve the mechanical stability of the capsules. Biomimetic capsules were fabricated by coupling the adhesion peptide arginine glycine aspartic acid (RGD) to alginate polymer chains and by using an alginate-mixture providing a bimodal molecular weight distribution. The biomimetic capsules provide cell adhesion for the enclosed cells, potentially also leading to mechanical stabilization of the cell-polymer system. Strikingly, the novel cell-hydrogel system significantly prolonged the in vivo long-term functionality and drug release, providing a sustained erythropoietin delivery during 300 days without immunosuppressive protocols. Additionally, controlling the cell-dose within the biomimetic capsules enables a controlled in vitro and in vivo drug delivery. Biomimetic cell-hydrogel capsules provide a unique microenvironment for the in vivo long-term de novo delivery of drugs from immobilized cells.
Current efforts in surface functionalization have not produced a robust technique capable of creating specific two-dimensional microscale geometrical arrays composed of multiple proteins. Such a capability is desirable for engineering substrates in sensing and cell patterning applications where at least two different protein functionalities in a specific configuration are required. Here we introduce a new approach for the creation of arrays of microscale geometries. We demonstrate our approach with a biomimetic structure inspired by the immunological synapse, a cell-cell interfacial structure characterized by two concentric rings of proteins: an outer adhesion protein structure and an inner recognition ligand core. The power of the technique lies in its ability to pattern any protein in any defined geometry as well as to create arrays in parallel.
Stem cell fate is influenced by a number of factors and interactions that require robust control for safe and effective regeneration of functional tissue. Coordinated interactions with soluble factors, other cells, and extracellular matrices define a local biochemical and mechanical niche with complex and dynamic regulation that stem cells sense. Decellularized tissue matrices and synthetic polymer niches are being used in the clinic, and they are also beginning to clarify fundamental aspects of how stem cells contribute to homeostasis and repair, for example, at sites of fibrosis. Multifaceted technologies are increasingly required to produce and interrogate cells ex vivo, to build predictive models, and, ultimately, to enhance stem cell integration in vivo for therapeutic benefit.
Injectable delivery vehicles in tissue engineering are often required for successful tissue formation in a minimally invasive manner. Shear-reversibly crosslinked hydrogels, which can recover gel structures from shear-induced breakdown, can be useful as an injectable, because gels can flow as a liquid when injected but re-gel once placed in the body. In this study, injectable and shear-reversible alginate hydrogels were prepared by combination crosslinking using cell-crosslinking and ionic crosslinking techniques. The addition of a small quantity of calcium ions decreased the number of cells that were required to form cell-crosslinked hydrogels without changing the shear reversibility of the system. The physical properties and gelation behavior of the gels were dependent on the concentration of both the cells and the calcium ions. We found that gels crosslinked by combination crosslinking methods were effective to engineer cartilage tissues in vivo. Using both ionic and cell-crosslinking methods to control the gelation behavior may allow the design of novel injectable systems that can be used to deliver cells and other therapeutics for minimally invasive therapy, including tissue engineering.
Bioactive, patterned micro- and nanoscale surfaces that can be spatially engineered for three-dimensional ligand presentation and sustained release of signaling molecules represent a critical advance for the development of next-generation diagnostic and therapeutic devices. Lithography is ideally suited to patterning such surfaces due to its precise, easily scalable, high-throughput nature; however, to date polymers patterned by these techniques have not demonstrated the capacity for sustained release of bioactive agents. We demonstrate here a class of lithographically-defined, electropolymerized polymers with monodisperse micro- and nanopatterned features capable of sustained release of bioactive drugs and proteins. We show that precise control can be achieved over the loading capacity and release rates of encapsulated agents and illustrate this aspect using a fabricated surface releasing a model antigen (ovalbumin) and a cytokine (interleukin-2) for induction of a specific immune response. We further demonstrate the ability of this technique to enable three-dimensional control over cellular encapsulation. The efficacy of the described approach is buttressed by its simplicity, versatility, and reproducibility, rendering it ideally suited for biomaterials engineering.
Three-dimensional culture alters cancer cell signaling; however, the underlying mechanisms and importance of these changes on tumor vascularization remain unclear. A hydrogel system was used to examine the role of the transition from 2D to 3D culture, with and without integrin engagement, on cancer cell angiogenic capability. Three-dimensional culture recreated tumor microenvironmental cues and led to enhanced interleukin 8 (IL-8) secretion that depended on integrin engagement with adhesion peptides coupled to the polymer. In contrast, vascular endothelial growth factor (VEGF) secretion was unaffected by 3D culture with or without substrate adhesion. IL-8 diffused greater distances and was present in higher concentrations in the systemic circulation, relative to VEGF. Implantation of a polymeric IL-8 delivery system into GFP bone marrow-transplanted mice revealed that localized IL-8 up-regulation was critical to both the local and systemic control of tumor vascularization in vivo. In summary, 3D integrin engagement within tumor microenvironments regulates cancer cell angiogenic signaling, and controlled local and systemic blockade of both IL-8 and VEGF signaling may improve antiangiogenic therapies.
The development of a multi-sample strain device and elastomeric culture wells designed to systematically assess strain effects on cell cultures is presented in this report. This device enables one to precisely conduct experimental analyses in sterile conditions while delivering cyclic uniaxial tensile strain. The input to the computer interface allows one to alter variables of frequency, duration, and amplitude of strain. The influence of strain on the migration of human umbilical vein endothelial cell (HUVEC) cultured on 2D polydimethylsiloxane (PDMS) surfaces was examined to verify the utility of this system.
The role of morphogens in bone regeneration has been widely studied, whereas the effect of matrix cues, particularly on stem cell differentiation, are less well understood. In this work, we investigated the effects of arginine-glycine-aspartate (RGD) ligand conformation (linear vs cyclic RGD) on primary human bone marrow stromal cell (hBMSC) and D1 stem cell osteogenic differentiation in three-dimensional (3D) culture and compared their response with that of committed MC3T3-E1 preosteoblasts to determine whether the stage of cell differentiation altered the response to the adhesion ligands. Linear RGD densities that promoted osteogenic differentiation of committed cells (MC3T3-E1 preosteoblasts) did not induce differentiation of hBMSCs or D1 stem cells, although matrices presenting the cyclic form of this adhesion ligand enhanced osteoprogenitor differentiation in 3D culture. This may be due to enhanced integrin-ligand binding. These studies indicate that biomaterial design parameters optimized for differentiated cell types may not directly translate to stem cell populations, because less-committed cells may require more instruction than differentiated cells. It is likely that design of synthetic extracellular matrices tailored to promote stem cell differentiation may enhance bone regeneration by transplanted cells.