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.
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.