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.

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.