Poly(ethylene) glycol is commonly used to stabilize gold nanoparticles (GNPs). In this study, we evaluated the ability of cysteine-functionalized alginate-derived polymers to both provide colloidal stability to GNPs and avoid recognition and sequestration by the body's defense system. These polymers contain multiple reactive chemical groups (hydroxyl and carboxyl groups) that could allow for ready functionalization with, for example, cell-targeting ligands and therapeutic drugs. We report here that alginate-coupled GNPs demonstrate enhanced stability in comparison with bare citrate-coated GNPs and a similar lack of interaction with proteins in vitro and long in vivo circulation as PEG-coated GNPs.
Porous biomaterials have been widely used as scaffolds in tissue engineering and cell-based therapies. The release of biological agents from conventional porous scaffolds is typically governed by molecular diffusion, material degradation, and cell migration, which do not allow for dynamic external regulation. We present a new active porous scaffold that can be remotely controlled by a magnetic field to deliver various biological agents on demand. The active porous scaffold, in the form of a macroporous ferrogel, gives a large deformation and volume change of over 70% under a moderate magnetic field. The deformation and volume variation allows a new mechanism to trigger and enhance the release of various drugs including mitoxantrone, plasmid DNA, and a chemokine from the scaffold. The porous scaffold can also act as a depot of various cells, whose release can be controlled by external magnetic fields.
The treatment of challenging fractures and large osseous defects presents a formidable problem for orthopaedic surgeons. Tissue engineering/regenerative medicine approaches seek to solve this problem by delivering osteogenic signals within scaffolding biomaterials. In this study, we introduce a hybrid growth factor delivery system that consists of an electrospun nanofiber mesh tube for guiding bone regeneration combined with peptide-modified alginate hydrogel injected inside the tube for sustained growth factor release. We tested the ability of this system to deliver recombinant bone morphogenetic protein-2 (rhBMP-2) for the repair of critically-sized segmental bone defects in a rat model. Longitudinal μ-CT analysis and torsional testing provided quantitative assessment of bone regeneration. Our results indicate that the hybrid delivery system resulted in consistent bony bridging of the challenging bone defects. However, in the absence of rhBMP-2, the use of nanofiber mesh tube and alginate did not result in substantial bone formation. Perforations in the nanofiber mesh accelerated the rhBMP-2 mediated bone repair, and resulted in functional restoration of the regenerated bone. μ-CT based angiography indicated that perforations did not significantly affect the revascularization of defects, suggesting that some other interaction with the tissue surrounding the defect such as improved infiltration of osteoprogenitor cells contributed to the observed differences in repair. Overall, our results indicate that the hybrid alginate/nanofiber mesh system is a promising growth factor delivery strategy for the repair of challenging bone injuries.
PURPOSE: The prognosis for glioma patients is poor, and development of new treatments is critical. Previously, we engineered polymer-based vaccines that control GM-CSF, CpG-oligonucleotide, and tumor-lysate presentation to regulate immune cell trafficking and activation, which promoted potent immune responses against peripheral tumors. Here, we extend the use of this system to glioma.
METHODS: Rats were challenged with an intracranial injection of glioma cells followed (1 week) by administration of the polymeric vaccine (containing GM-CSF, CpG, and tumor-lysate) in the tumor bed. Control rats were treated with blank matrices, matrices with GM-CSF and CpG, or intra-tumoral bolus injections of GM-CSF, CpG, and tumor lysate. Rats were monitored for survival and tested for neurological function.
RESULTS: Survival studies confirmed a benefit of the polymeric vaccine as 90% of vaccinated rats survived for > 100 days. Control rats exhibited minimal benefit. Motor tests revealed that vaccination protected against the loss of forelimb use produced by glioma growth. Histological analysis quantitatively confirmed a robust and rapid reduction in tumor size. Long-term immunity was confirmed when 67% of survivors also survived a second glioma challenge.
CONCLUSIONS: These studies extend previous reports regarding this approach to tumor therapy and justify further development for glioma treatment.
PURPOSE: There is evolving evidence that intense exercise may place a disproportionate load on the right ventricle (RV) when compared with the left ventricle (LV) of the heart. Using a novel method of estimating end-systolic wall stress (ES-σ), we compared the RV and LV during exercise and assessed whether this influenced chronic ventricular remodeling in athletes.
METHODS: For this study, 39 endurance athletes (EA) and 14 nonathletes (NA) underwent resting cardiac magnetic resonance (CMR), maximal oxygen uptake (VO2), and exercise echocardiography studies. LV and RV end-systolic wall stress (ES-σ) were calculated using the Laplace relation (ES-σ = Pr/(2h)). Ventricular size and wall thickness were determined by CMR; invasive and Doppler echo estimates were used to measure systemic and pulmonary ventricular pressures, respectively; and stroke volume was quantified by Doppler echocardiography and used to calculate changes in ventricular geometry during exercise.
RESULTS: In EA, compared with NA, resting CMR measures showed greater RV than LV remodeling. The ratios RV ESV/LV ESV (1.40 ± 0.23 vs 1.26 ± 0.12, P = 0.007) and RV mass/LV mass (0.29 ± 0.04 vs 0.25 ± 0.03, P = 0.012) were greater in EA than in NA. RVES-σ was lower at rest than LVES-σ (143 ± 44 vs 252 ± 49 kdyn · cm, P < 0.001) but increased more with strenuous exercise (125% vs 14%, P < 0.001), resulting in similar peak exercise ES-σ (321 ± 106 vs 286 ± 77 kdyn · cm, P = 0.058). Peak exercise RVES-σ was greater in EA than in NA (340 ± 107 vs 266 ± 82 kdyn · cm, P = 0.028), whereas RVES-σ at matched absolute workloads did not differ (P = 0.79).
CONCLUSIONS: Exercise induces a relative increase in RVES-σ which exceeds LVES-σ. In athletes, greater RV enlargement and greater wall thickening may be a product of this disproportionate load excess.
We previously engineered a macroporous, polymer-based vaccine that initially produces GM-CSF gradients to recruit local dendritic cells and subsequently presents CpG oligonucleotides, and tumor lysate to cell infiltrates to induce immune cell activation and immunity against tumor cells in peripheral tumor models. Here, we demonstrate that this system eradicates established intracranial glioma following implantation into brain tissue, whereas implantation in resection cavities obviates vaccine efficacy. Rats bearing seven-day old, intracranial glioma tumors were treated with PLG vaccines implanted into the tumor bed, resulting in retention of contralateral forelimb function (day 17) that is compromised by tumor formation in control animals, and 90% long-term survival (>100 days). Similar benefits were observed in animals receiving tumor resection plus vaccine implants into the adjacent parenchyma, but direct implantation of PLG vaccines into the resection cavity conferred no benefit. This dissociation of efficacy was likely related to GM-CSF distribution, as implantation of PLG vaccines within brain tissue produced significant GM-CSF gradients for prolonged periods, which was not detected after implantation in resection cavities. These studies demonstrate that PLG vaccine efficacy is correlated to GM-CSF gradient formation, which requires direct implantation into brain tissue, and justify further exploration of this approach for glioma treatment.
Osteogenic growth factors that promote endogenous repair mechanisms hold considerable potential for repairing challenging bone defects. The local delivery of one such growth factor, bone morphogenetic protein (BMP), has been successfully translated to clinical practice for spinal fusion and bone fractures. However, improvements are needed in the spatial and temporal control of BMP delivery to avoid the currently used supraphysiologic doses and the concomitant adverse effects. We have recently introduced a hybrid protein delivery system comprised of two parts: a perforated nanofibrous mesh that spatially confines the defect region and a functionalized alginate hydrogel that provides temporal growth factor release kinetics. Using this unique spatiotemporal delivery system, we previously demonstrated BMP-mediated functional restoration of challenging 8mm femoral defects in a rat model. In this study, we compared the efficacy of the hybrid system in repairing segmental bone defects to that of the current clinical standard, collagen sponge, at the same dose of recombinant human BMP-2. In addition, we investigated the specific role of the nanofibrous mesh tube on bone regeneration. Our results indicate that the hybrid delivery system significantly increased bone regeneration and improved biomechanical function compared to collagen sponge delivery. Furthermore, we observed that presence of the nanofiber mesh tube was essential to promote maximal mineralized matrix synthesis, prevent extra-anatomical mineralization, and guide an integrated pattern of bone formation. Together, these results suggest that spatiotemporal strategies for osteogenic protein delivery may enhance clinical outcomes by improving localized protein retention.
Oral Diseases (2011) 17, 241-251 The rapid advancement in basic biology knowledge, especially in the stem cell field, has created new opportunities to develop biomaterials capable of orchestrating the behavior of transplanted and host cells. Based on our current understanding of cellular differentiation, a conceptual framework for the use of materials to program cells in situ is presented, namely a domino vs a switchboard model, to highlight the use of single vs multiple cues in a controlled manner to modulate biological processes. Further, specific design principles of material systems to present soluble and insoluble cues that are capable of recruiting, programming and deploying host cells for various applications are presented. The evolution of biomaterials from simple inert substances used to fill defects, to the recent development of sophisticated material systems capable of programming cells in situ is providing a platform to translate our understanding of basic biological mechanisms to clinical care.
The identification and production of recombinant morphogens and growth factors that play key roles in tissue regeneration have generated much enthusiasm and numerous clinical trials, but the results of many of these trials have been largely disappointing. Interestingly, the trials that have shown benefit all contain a common denominator, the presence of a material carrier, suggesting strongly that spatio-temporal control over the location and bioactivity of factors after introduction into the body is crucial to achieve tangible therapeutic effect. Sophisticated materials systems that regulate the biological presentation of growth factors represent an attractive new generation of therapeutic agents for the treatment of a wide variety of diseases. This review provides an overview of growth factor delivery in tissue engineering. Certain fundamental issues and design strategies relevant to the material carriers that are being actively pursued to address specific technical objectives are discussed. Recent progress highlights the importance of materials science and engineering in growth factor delivery approaches to regenerative medicine.
Our understanding of immunological regulation has progressed tremendously alongside the development of materials science, and at their intersection emerges the possibility to employ immunologically active biomaterials for cancer immunotherapy. Strong and sustained anticancer, immune responses are required to clear large tumor burdens in patients, but current approaches for immunotherapy are formulated as products for delivery in bolus, which may be indiscriminate and/or shortlived. Multifunctional biomaterial particles are now being developed to target and sustain antigen and adjuvant delivery to dendritic cells in vivo, and these have the potential to direct and prolong antigen-specific T cell responses. Three-dimensional immune cell niches are also being developed to regulate the recruitment, activation and deployment of immune cells in situ to promote potent antitumor responses. Recent studies demonstrate that materials with immune targeting and stimulatory capabilities can enhance the magnitude and duration of immune responses to cancer antigens, and preclinical results utilizing material-based immunotherapy in tumor models show a strong therapeutic benefit, justifying translation to and future testing in the clinic.
Chemotaxis plays a critical role in tissue development and wound repair, and is widely studied using ex vivo model systems in applications such as immunotherapy. However, typical chemotactic models employ 2D systems that are less physiologically relevant or use end-point assays, that reveal little about the stepwise dynamics of the migration process. To overcome these limitations, we developed a new model system using microfabrication techniques, sustained drug delivery approaches, and theoretical modeling of chemotactic agent diffusion. This model system allows us to study the effects of 3D architecture and chemotactic agent gradient on immune cell migration in real time. We find that dendritic cell migration is characterized by a strong interplay between matrix architecture and chemotactic gradients, and migration is also influenced dramatically by the cell activation state. Our results indicate that Lipopolysaccharide-activated dendritic cells studied in a traditional transwell system actually exhibit anomalous migration behavior. Such a 3D ex vivo system lends itself for analyzing cell migratory behavior in response to single or multiple competitive cues and could prove useful in vaccine development.
Integrin receptors bind to adhesion ligand (e.g. arginine-glycine-aspartic acid or RGD containing peptides) on extracellular matrix and organize into high-density complexes which mediate many cell behaviors. Biomaterials with RGD nanopatterned into multivalent "islands" (∼30-70 nm diameter) have been shown to alter cell responses, although the length scale of pattern features is orders of magnitude smaller than adhesion complexes. In this work, we employ together for the first time an extensive data set on osteoblast responses as a function of ligand nanopatterns, a computational model of integrin binding to ligand nanopatterns, and new measures of integrin organization on the cell surface. We quantify, at multiple length scales, integrin organization generated in silico as a function of RGD nanopattern parameters. We develop a correlative model relating these measures of in silico integrin organization and in vitro MC3T3 preosteoblast cell responses as functions of the same RGD nanopatterns: cell spreading correlates with the number of bound integrins, focal adhesion kinase (FAK) phosphorylation correlates with small, homogeneously distributed clusters of integrins, and osteogenic differentiation correlates with large, heterogeneously distributed integrin clusters. These findings highlight the significance of engineering biomaterials at the nanolevel and suggest new approaches to understanding the mechanisms linking integrin organization to cell responses.
Cancer vaccines are typically formulated for bolus injection and often produce short-lived immunostimulation resulting in poor temporal control over immune cell activation and weak oncolytic activity. One means of overcoming these limitations utilizes immunologically active biomaterial constructs. We previously reported that antigen-laden, macroporous PLG scaffolds induce potent dendritic cell (DC) and cytotoxic T-lymphocyte (CTL) responses via the controlled signaling of inflammatory cytokines, antigen and toll-like receptor agonists. In this study, we describe the kinetics of these responses and illustrate their fundamental relationship to potent tumor rejection when implanted subcutaneously in a mouse B16 model of melanoma. By explanting scaffolds from mice at times ranging from 1-7 d, a seamless relationship was observed between the production of controlled CTL responses, tumor growth and long-term survival in both prophylactic and therapeutic models. Scaffolds must be implanted for > 7 d to augment CTL responses via the prolonged presentation of tumor antigen, and the benefits included a notable regression of established tumors. Host DC infiltration into the porous material persisted for 12 days (peaking at day 5 ~1.4 x 10(6) cells), and a sharp attenuation in DC numbers coincided with peak CD8(+) CTL infiltration at day 12 (~8 x 10(5) cells). Importantly, these PLG systems enhanced DC numbers in the draining lymph node, resulting in increased CD(+) CTL subsets at days 10-16 of vaccination. These results indicate that material systems can finely control innate and adaptive immune cell responses to kill typically untreatable melanoma tumors and provide critical kinetic data for the design of vaccine carriers.
Many cell types of therapeutic interest, including myoblasts, exhibit reduced engraftment if cultured prior to transplantation. This study investigated whether polymeric scaffolds that direct cultured myoblasts to migrate outwards and repopulate the host damaged tissue, in concert with release of angiogenic factors designed to enhance revascularizaton of the regenerating tissue, would enhance the efficacy of this cell therapy and lead to functional muscle regeneration. This was investigated in the context of a severe injury to skeletal muscle tissue involving both myotoxin-mediated direct damage and induction of regional ischemia. Local and sustained release of VEGF and IGF-1 from macroporous scaffolds used to transplant and disperse cultured myogenic cells significantly enhanced their engraftment, limited fibrosis, and accelerated the regenerative process. This resulted in increased muscle mass and, improved contractile function. These results demonstrate the importance of finely controlling the microenvironment of transplanted cells in the treatment of severe muscle damage.
Targeting of nanoparticles to ischemic tissues was studied in a murine ischemic hindlimb model. Intravenously injected fluorescent nanoparticles allowed ischemia-targeted imaging of ischemic muscles due to increased permeability of blood vessels in hypoxic tissues. Targeting efficiency correlated with blood perfusion after induction of ischemia and was enhanced in early stages of ischemia (<7 days). Therapeutic delivery of vascular endothelial growth factor (VEGF) was achieved by VEGF-conjugated nanoparticles and resulted in a 1.7-fold increase in blood perfusion, as compared to control mice. This work supports the application of nanoparticles as imaging and therapeutic modalities for ischemia treatment.
Many cell-based therapies aim to transplant functional cells to revascularize damaged tissues and ischemic areas. However, conventional cell therapy is not optimally efficient: massive cell death, damage, and non-localization of cells both spatially and temporally all likely contribute to poor tissue functionality. An alginate cell depot system has been proposed as an alternative means to deliver outgrowth endothelial cells (OECs) in a spatiotemporally controllable manner while protecting them in the early stages of tissue re-integration. Here OECs exiting the alginate scaffold were measured for viability, functionality, and migration speed and characterized for cytokine and surface marker profiles. OECs were highly viable in the alginate and were depleted from the scaffold via migration at a speed of 21 ± 6 μm/h following release. Prolonged interaction with the alginate scaffold microenvironment did not detrimentally change OECs; they retained high functionality, displayed a similar angiogenic cytokine profile as control OECs, and did not have significantly altered surface markers. These results suggest that alginate-OEC interactions do not adversely affect these cells, validating control of cellular migration as a means to control the cell delivery profile from the material system, and supporting usage of the alginate scaffold as an efficient cell delivery vehicle.
Therapeutic cancer vaccines are emerging as novel and potent approaches to treat cancer. These vaccines enhance the body's immune response to cancerous cells, and dendritic cells (DCs), an initiator of adaptive immunity, are a key cell type targeted by these strategies. Current DC-based cancer vaccines are based on ex vivo manipulation of the cells following their isolation from the patient, followed by reintroduction to the patient, but this approach has many limitations in practical cancer treatment. However, recent progress in materials science has allowed the design and fabrication of physically and chemically functionalized materials platforms that can specifically target DCs in the body. These materials, through their in vivo modulation of DCs, have tremendous potentials as new cancer therapies. Nanoparticles, which are several orders of magnitude smaller than DCs, can efficiently deliver antigen and danger signals to these cells through passive or active targeting. Three-dimensional biomaterials, with sizes several orders of magnitude larger than DCs, create microenvironments that allow the effective recruitment and programming of these cells, and can be used as local depots of nanoparticles targeting resident DCs. Both material strategies have shown potential in promoting antigen-specific T cell responses of magnitudes relevant to treating cancer.
Advances in tissue engineering have traditionally led to the design of scaffold- or matrix-based culture systems that better reflect the biological, physical and biochemical environment of the natural extracellular matrix. Although their clinical applications in regenerative medicine tend to receive most of the attention, it is obvious that other areas of biomedical research could be well served by the powerful tools that have already been developed in tissue engineering. In this article, we review the recent literature to demonstrate how tissue engineering platforms can enhance in vitro and in vivo models of tumorigenesis and thus hold great promise to contribute to future cancer research.