Mechanical signals regulate blood vessel development in vivo, and have been demonstrated to regulate signal transduction of endothelial cell (EC) and smooth muscle cell (SMC) phenotype in vitro. However, it is unclear how the complex process of angiogenesis, which involves multiple cell types and growth factors that act in a spatiotemporally regulated manner, is triggered by a mechanical input. Here, we describe a mechanism for modulating vascular cells during sequential stages of an in vitro model of early angiogenesis by applying cyclic tensile strain. Cyclic strain of human umbilical vein (HUV)ECs up-regulated the secretion of angiopoietin (Ang)-2 and PDGF-betabeta, and enhanced endothelial migration and sprout formation, whereas effects were eliminated with shRNA knockdown of endogenous Ang-2. Applying strain to colonies of HUVEC, cocultured on the same micropatterned substrate with nonstrained human aortic (HA)SMCs, led to a directed migration of the HASMC toward migrating HUVECs, with diminished recruitment when PDGF receptors were neutralized. These results demonstrate that a singular mechanical cue (cyclic tensile strain) can trigger a cascade of autocrine and paracrine signaling events between ECs and SMCs critical to the angiogenic process.
Cancer vaccines typically depend on cumbersome and expensive manipulation of cells in the laboratory, and subsequent cell transplantation leads to poor lymph-node homing and limited efficacy. We propose that materials mimicking key aspects of bacterial infection may instead be used to directly control immune-cell trafficking and activation in the body. It is demonstrated that polymers can be designed to first release a cytokine to recruit and house host dendritic cells, and subsequently present cancer antigens and danger signals to activate the resident dendritic cells and markedly enhance their homing to lymph nodes. Specific and protective anti-tumour immunity was generated with these materials, as 90% survival was achieved in animals that otherwise die from cancer within 25 days. These materials show promise as cancer vaccines, and more broadly suggest that polymers may be designed to program and control the trafficking of a variety of cell types in the body.
Promoting angiogenesis via delivery of vascular endothelial growth factor (VEGF) and other angiogenic factors is both a potential therapy for cardiovascular diseases and a critical aspect for tissue regeneration. The recent demonstration that VEGF signaling is modulated by the Notch signaling pathway, however, suggests that inhibiting Notch signaling may enhance regional neovascularization, by altering the responsiveness of local endothelial cells to angiogenic stimuli. We tested this possibility with in vitro assays using human endothelial cells, as well as in a rodent hindlimb ischemia model. Treatment of cultured human endothelial cells with DAPT, a gamma secretase inhibitor, increased cell migration and sprout formation in response to VEGF stimulation with a biphasic dependence on DAPT concentration. Further, delivery of an appropriate combination of DAPT and VEGF from an injectable alginate hydrogel system into ischemic hindlimbs led to a faster recovery of blood flow than VEGF or DAPT alone; perfusion levels reached 80% of the normal level by week 4 with combined DAPT and VEGF delivery. Direct intramuscular or intraperitoneal injection of DAPT did not result in the same level of improvement, suggesting that appropriate presentation of DAPT (gel delivery) is important for its activity. DAPT delivery from the hydrogels also did not lead to any adverse side effects, in contrast to systemic introduction of DAPT. Altogether, these results suggest a new approach to promote angiogenesis by controlling Notch signaling, and may provide new options to treat patients with diseases that diminish angiogenic responsiveness.
Vaccines are largely ineffective for patients with established cancer, as advanced disease requires potent and sustained activation of CD8(+) cytotoxic T lymphocytes (CTLs) to kill tumor cells and clear the disease. Recent studies have found that subsets of dendritic cells (DCs) specialize in antigen cross-presentation and in the production of cytokines, which regulate both CTLs and T regulatory (Treg) cells that shut down effector T cell responses. Here, we addressed the hypothesis that coordinated regulation of a DC network, and plasmacytoid DCs (pDCs) and CD8(+) DCs in particular, could enhance host immunity in mice. We used functionalized biomaterials incorporating various combinations of an inflammatory cytokine, immune danger signal, and tumor lysates to control the activation and localization of host DC populations in situ. The numbers of pDCs and CD8(+) DCs, and the endogenous production of interleukin-12, all correlated strongly with the magnitude of protective antitumor immunity and the generation of potent CD8(+) CTLs. Vaccination by this method maintained local and systemic CTL responses for extended periods while inhibiting FoxP3 Treg activity during antigen clearance, resulting in complete regression of distant and established melanoma tumors. The efficacy of this vaccine as a monotherapy against large invasive tumors may be a result of the local activity of pDCs and CD8(+) DCs induced by persistent danger and antigen signaling at the vaccine site. These results indicate that a critical pattern of DC subsets correlates with the evolution of therapeutic antitumor responses and provide a template for future vaccine design.
Biomaterials, traditionally defined as materials used in medical devices, have been used since antiquity, but recently their degree of sophistication has increased significantly. Biomaterials made today are routinely information rich and incorporate biologically active components derived from nature. In the future, biomaterials will assume an even greater role in medicine and will find use in a wide variety of non-medical applications through biologically inspired design and incorporation of dynamic behaviour.
Growth factors play a crucial role in information transfer between cells and their microenvironment in tissue engineering and regeneration. They initiate their action by binding to specific receptors on the surface of target cells and the chemical identity, concentration, duration, and context of these growth factors contain information that dictates cell fate. Hence, the importance of exogenous delivery of these molecules in tissue engineering is unsurprising, considering their importance for tissue regeneration. However, the short half-lives of growth factors, their relatively large size, slow tissue penetration, and their potential toxicity at high systemic levels, suggest that conventional routes of administration are unlikely to be effective. In this review, we provide an overview of the design criteria for growth factor delivery vehicles with respect to the growth factor itself and the microenvironment for delivery. We discuss various methodologies that could be adopted to achieve this localized delivery, and strategies using polymers as delivery vehicles in particular.
BACKGROUND: There is evidence that ultra-endurance exercise causes myocardial injury. The extent and duration of these changes remains unresolved. Recent reports have speculated that structural adaptations to exercise, particularly of the right ventricle, may predispose to tachyarrhythmias and sudden cardiac death.
OBJECTIVE: To quantify the extent and duration of post-exercise cardiac injury with particular attention to right ventricular (RV) dysfunction.
METHODS: 27 athletes (20 male, 7 female) were tested 1 week before, immediately after and 1 week after an ultra-endurance triathlon. Tests included cardiac troponin I (cTnI), B-type natriuretic peptide (BNP) and comprehensive echocardiographic assessment.
RESULTS: 26 athletes completed the race and testing procedures. Post-race, cTnI was raised in 15 athletes (58%) and the mean value for the entire cohort increased (0.17 vs 0.49 microg/l, p<0.01). BNP rose in every athlete and the mean increased significantly (12.2 vs 42.5 ng/l, p<0.001). Left ventricular ejection fraction (LVEF) was unchanged (60.4% vs 57.5%, p = 0.09), but integrated systolic strain decreased (16.9% vs 15.1%, p<0.01). New regional wall motion abnormalities developed in seven athletes (27%) and LVEF was reduced in this subgroup (57.8% vs 45.9%, p<0.001). RV function was reduced in the entire cohort with decreases in fractional area change (0.47 vs 0.39, p<0.01) and tricuspid annular plane systolic excursion (21.8 vs 19.1 mm, p<0.01). At follow-up, all variables returned to baseline except in one athlete where RV dysfunction persisted.
CONCLUSION: Myocardial damage occurs during intense ultra-endurance exercise and, in particular, there is a significant reduction in RV function. Almost all abnormalities resolve within 1 week.
PURPOSE: The purpose of this study was to modify the method of platelet-rich plasma (PRP) preparation for obtaining optimal angiogenic potential and accelerate bone healing. Also, the potential synergistic effect of a suboptimal concentration of bone morphogenic protein-2 (BMP-2) and modified PRP (mPRP) on bone healing was evaluated in vivo.
MATERIALS AND METHODS: The angiogenic factor-enriched PRP, which included peripheral blood mononuclear cells (mostly lymphocytes and monocytes, excluding polymorphonuclear leukocytes [PMNs], was achieved by lowering concentrations of thrombin and CaCl2, after pre-activation with shear stress using a table-top vortex machine and collagen. In vitro, endothelial cell migration activity in the mPRP group was compared to conventional PRP preparation using a modified Boyden chamber assay. In an animal study, PGA scaffold, PGA scaffold + mPRP, PGA scaffold + mPRP + rhBMP-2, and PGA scaffold + rhBMP-2 were applied to critical-sized calvarial defects in 28 nude rats. At 2 weeks, periosteal blood flow was measured using laser Doppler perfusion imaging, and bone formation was evaluated at 8 weeks by histology, dual energy x-ray absorptiometry, and micro-computed tomography.
RESULTS: mPRP induced faster migration of cord blood-derived outgrowth endothelial-like cells. In vivo, the group with mPRP with a low dose of rhBMP-2 showed significantly increased numbers of blood vessels at 2 weeks and notable synergistic effect on bone healing at 8 weeks as evaluated with histology, bone mineral density and bone mineral content, and muCT.
CONCLUSION: The mPRP used in this study improved vascular perfusion around the defect and resulted in enhanced bone healing. Also, combining mPRP with a suboptimal dosage of rhBMP-2 improved bone formation and enhanced bone density.
Specific interactions between cells and cell-interactive polymers in solution were investigated by the fluorescence resonance energy transfer (FRET) technique and rheological measurements. The green fluorescence emission was dramatically reduced when rhodamine-stained cells were mixed with a fluorescein-labeled RGD-alginate solution, compared with those mixed with no RGD-containing alginate solution, which indicated an occurrence of FRET and existence of specific interactions between the cells and the polymers in solution. Rheological measurements also confirmed the formation of ordered structures of cell/polymer mixtures, caused by specific cell-polymer interactions. The FRET method was able to provide a useful means of investigating cell-polymer interactions, both in a qualitative and quantitative manner, and this approach to monitoring and controlling specific interactions between cells and polymers could be useful in the design and tailoring of polymeric carriers for cells, as well as for biological drugs, especially for tissue engineering applications.
Cues from the material to which a cell is adherent (e.g., adhesion ligand presentation, substrate elastic modulus) clearly influence the phenotype of differentiated cells. However, it is currently unclear if stem cells respond similarly to these cues. This study examined how the overall density and nanoscale organization of a model cell adhesion ligand (arginine-glycine-aspartic acid [RGD] containing peptide) presented from hydrogels of varying stiffness regulated the proliferation of a clonally derived stem cell line (D1 cells) and preosteoblasts (MC3T3-E1). While the growth rate of MC3T3-E1 preosteoblasts was responsive to nanoscale RGD ligand organization and substrate stiffness, the D1 stem cells were less sensitive to these cues in their uncommitted state. However, once the D1 cells were differentiated towards the osteoblast lineage, they became more responsive to these signals. These results demonstrate that the cell response to material cues is dependent on the stage of cell commitment or differentiation, and these findings will likely impact the design of biomaterials for tissue regeneration.
Several high-resolution imaging techniques such as FESEM, TEM and AFM are compared with respect to their application on alginate hydrogels, a widely used polysaccharide biomaterial. A new AFM method applicable to RGD peptides covalently conjugated to alginate hydrogels is described. High-resolution images of RGD adhesion ligand distribution were obtained by labeling biotinylated RGD peptides with streptavidin-labeled gold nanoparticles. This method may broadly provide a useful tool for sECM characterization and design for tissue regeneration strategies.
Many cell populations, derived from both adult tissues and embryonic stem cells, show promise for the treatment of a variety of diseases. Although the major effort in stem cell therapies in the past has been identifying potentially therapeutic cells, it is now clear that developing systems to deliver these cells and promote their efficient engraftment will provide an equally challenging task. More sophisticated pretransplantation manipulations and material carriers may dramatically improve the survival, engraftment, and fate control of transplanted stem cells and their ultimate clinical utility.
PURPOSE: To attain the effective local and sustained delivery of plasmid DNA (pDNA) encoding for a growth factor.
METHODS: We hypothesized that controlling the degradation rate of biomaterials encapsulating pDNA via concurrent physical dissociation of the cross-linked structure and hydrolytic chain breakage of polymers would allow one to significantly broaden the range of pDNA release rate. This hypothesis was examined using ionically cross-linked polysaccharide hydrogels which were previously designed to rapidly degrade via engineering of ionic cross-linking junction and partial oxidation of polysaccharide chains.
RESULTS: The hydrogel degradation rates were varied over the broad range, and pDNA release correlated with the gel degradation rate. Degradable hydrogels were used for the local and sustained delivery of a pDNA encoding for vascular endothelial growth factor (VEGF) in the ischemic hindlimbs of mice, and local pDNA release significantly improved the recovery of blood perfusion as compared with a bolus injection of VEGFencoding pDNA.
CONCLUSION: This strategy to control the hydrogel degradation rate may be useful in regulating the delivery of a broad array of macromolecular drugs, and subsequently improve their therapeutic efficacy.
Our group has previously created a functional neointestine that is capable of restoring absorptive function. However, the endogenous level of vascular endothelial growth factor (VEGF) is markedly reduced in the construct compared to native bowel. Therefore, we wanted to locally deliver VEGF in a sustained fashion to upregulate angiogenesis in the neointestine. Rat recombinant VEGF was encapsulated in poly(lactide-co-glycolide) microspheres by a double emulsion method. Release kinetics and bioactivity were determined in vitro. Tissue-engineered intestine was generated by seeding donor neonatal rat intestinal organoid units onto a biodegradable polyglycolic acid scaffold along with VEGF-containing or empty microspheres, and wrapped in the omentum of recipient rats. After 4 weeks, the neointestinal cysts were analyzed for morphometry, VEGF levels, epithelial proliferation, and capillary density. Sustained release of biologically active VEGF was confirmed by in vitro studies. Intestinal constructs with VEGF microspheres were significantly larger than those containing empty microspheres. Tissue VEGF levels were significantly higher in neointestine loaded with encapsulated VEGF compared to those without growth factor. Epithelial cellular proliferation and capillary density were significantly increased in the VEGF-containing neointestinal constructs compared to empty constructs. Tissue-engineered intestine responds to sustained delivery of VEGF by upregulating microvasculature and epithelial proliferation.
Cell-interactive polymers have been widely used as synthetic extracellular matrices to regulate cell function and promote tissue regeneration. However, there is a lack of quantitative understanding of the cell-material interface. In this study, integrin-adhesion ligand bond formation of preosteoblasts and D1 stem cells with RGD presenting alginate matrices were examined using FRET and flow cytometry. Bond number increased with adhesion ligand density but did not change with RGD island spacing for both cell types. Integrin expression varied with cell type and substrate in 2D culture, but the integrin expression profiles of both cell types were similar when cultured in 3D RGD presenting substrates and distinct from 2D culture. In summary, combining a FRET technique to quantify bond formation with flow cytometry to elucidate integrin expression can define specific cell-material interactions for a given material system and may be useful for informing biomaterial design strategies for cell-based therapies.
One goal of tissue engineering is to replace lost or compromised tissue function, and an approach to this is to control the interplay between materials (scaffolds), cells and growth factors to create environments that promote the regeneration of functional tissues and organs. An increased understanding of the chemical signals that direct cell differentiation, migration and proliferation, advances in scaffold design and peptide engineering that allow this signaling to be recapitulated and the development of new materials, such as DNA-based and stimuli-sensitive polymers, have recently given engineers enhanced control over the chemical properties of a material and cell fate. Additionally, the immune system, which is often overlooked, has been shown to play a beneficial role in tissue repair, and future endeavors in material design will potentially expand to include immunomodulation.
Cell-based therapies are attractive for revascularizing and regenerating tissues and organs, but clinical trials of endothelial progenitor cell transplantation have not resulted in consistent benefit. We propose a different approach in which a material delivery system is used to create a depot of vascular progenitor cells in vivo that exit over time to repopulate the damaged tissue and participate in regeneration of a vascular network. Microenvironmental conditions sufficient to maintain the viability and outward migration of outgrowth endothelial cells (OECs) have been delineated, and a material incorporating these signals improved engraftment of transplanted cells in ischemic murine hindlimb musculature, and increased blood vessel densities from 260 to 670 vessels per mm(2), compared with direct cell injection. Further, material deployment dramatically improved the efficacy of these cells in salvaging ischemic murine limbs, whereas bolus OEC delivery was ineffective in preventing toe necrosis and foot loss. Finally, material deployment of a combination of OECs with another cell population commonly isolated from peripheral or cord blood, endothelial progenitor cells (EPCs) returned perfusion to normal levels in 40 days, and prevented toe and foot necrosis. Direct injection of an EPC/OEC combination was minimally effective in improving limb perfusion, and untreated limbs underwent autoamputation in 3 days. These results demonstrate that vascular progenitor cell utility is highly dependent on the mode of delivery, and suggest that one can create new vascular beds for a variety of applications with this material-controlled deployment of cells.
Many functions of the extracellular matrix can be mimicked by small peptide fragments (e.g., arginine-glycine-aspartic acid (RGD) sequence) of the entire molecule, but the presentation of the peptides is critical to their effects on cells. It is likely that some effects of peptide presentation from biomaterials simply relate to the number of bonds formed between cell receptors and the adhesion ligands, but a lack of tools to quantify bond number limits direct investigation of this assumption. The impact of different ligand presentations (density, affinity, and nanoscale distribution) on the proliferation of C2C12 and human primary myoblasts was first examined in this study. Increasing the ligand density or binding affinity led to a similar enhancement in proliferation of C2C12 cells and human primary myoblasts. The nanoscale distribution of clustered RGD ligands also influenced C2C12 cells and human primary myoblast proliferation, but in an opposing manner. A theological technique and a FRET technique were then utilized to quantify the number of receptor-ligand interactions as a function of peptide presentation. Higher numbers of bonds were formed when the RGD density and affinity were increased, as measured with both techniques, and bond number correlated with cell growth rates. However, the influence of the nanoscale peptide distribution did not appear to be solely a function of bond number. Altogether, these findings provide significant insight to the role of peptide presentation in the regulation of cell proliferation, and the approaches developed in this work may have significant utility in probing how adhesion regulates a variety of other cellular functions and aid in developing design criterion for cell-interactive materials.
Current techniques to educate dendritic cells (DCs) ex vivo for immunotherapy are plagued by inefficient protocols and DC modifications are often transient and lost upon transplantation. This study investigated the role of sustained presentation of GM-CSF and PEI condensed pDNA (PEI-DNA) on gene transfer and long-term gene expression. Appropriate GM-CSF signaling during DC transfection promoted PEI-DNA uptake, although high cytokine concentrations induced intercellular DNA degradation, indicating the need for controlled presentation. Poly(lactide-co-glycolide) scaffolds that continuously stimulated DCs with both GM-CSF and PEI-DNA led to a 20-fold increase in gene expression, and high levels of expression persisted for a period of 10 days, in vitro. These results encourage the exploitation of biomaterials and GM-CSF to develop novel delivery vectors for genetically modified DCs or to genetically program host DCs in situ for vaccination and the treatment of autoimmunity.