In vitro models of normal mammary epithelium have correlated increased extracellular matrix (ECM) stiffness with malignant phenotypes. However, the role of increased stiffness in this transformation remains unclear because of difficulties in controlling ECM stiffness, composition and architecture independently. Here we demonstrate that interpenetrating networks of reconstituted basement membrane matrix and alginate can be used to modulate ECM stiffness independently of composition and architecture. We find that, in normal mammary epithelial cells, increasing ECM stiffness alone induces malignant phenotypes but that the effect is completely abrogated when accompanied by an increase in basement-membrane ligands. We also find that the combination of stiffness and composition is sensed through β4 integrin, Rac1, and the PI3K pathway, and suggest a mechanism in which an increase in ECM stiffness, without an increase in basement membrane ligands, prevents normal α6β4 integrin clustering into hemidesmosomes.
The innate cellular and molecular components required to mediate effective vaccination against weak tumor-associated antigens remain unclear. In this study, we used polymeric cancer vaccines incorporating different classes of adjuvants to induce tumor protection, to identify dendritic cell (DC) subsets and cytokines critical to this efficacy. Three-dimensional, porous polymer matrices loaded with tumor lysates and presenting distinct combinations of granulocyte macrophage colony-stimulating factor (GM-CSF) and various Toll-like receptor (TLR) agonists affected 70% to 90% prophylactic tumor protection in B16-F10 melanoma models. In aggressive, therapeutic B16 models, the vaccine systems incorporating GM-CSF in combination with P(I:C) or CpG-ODN induced the complete regression of solid tumors (≤40 mm(2)), resulting in 33% long-term survival. Regression analysis revealed that the numbers of vaccine-resident CD8(+) DCs, plasmacytoid DCs (pDC), along with local interleukin (IL)-12, and granulocyte colony-stimulating factor (G-CSF) concentrations correlated strongly to vaccine efficacy regardless of adjuvant type. Furthermore, vaccine studies in Batf3(-/-) mice revealed that CD8(+) DCs are required to affect tumor protection, as vaccines in these mice were deficient in cytotoxic T lymphocytes priming and IL-12 induction in comparison with wild-type. These studies broadly demonstrate that three-dimensional polymeric vaccines provide a potent platform for prophylactic and therapeutic protection, and can be used as a tool to identify critical components of a desired immune response. Specifically, these results suggest that CD8(+) DCs, pDCs, IL-12, and G-CSF play important roles in priming effective antitumor responses with these vaccines.
The development of the vertebral column starts with the formation of a linear array of mesenchymal condensations, forming the blueprint for the eventual alternating pattern of bone and cartilage. Despite growing insight into the molecular mechanisms of morphogenesis, the impact of the physical aspects of the environment is not well understood. We hypothesized that geometric boundary conditions may play a pivotal role in the linear patterning of condensations, as neighbouring tissues provide physical constraints to the cell population. To study the process of condensation and the patterning thereof under tightly controlled geometric constraints, we developed a novel in vitro model that combines micropatterning with the established micromass assay. The spacing and alignment of condensations changed with the width of the cell adhesive patterns, a phenomenon that could not be explained by cell availability alone. Moreover, the extent of chondrogenic commitment was increased on substrates with tighter geometric constraints. When the in vivo pattern of condensations was investigated in the developing vertebral column of chicken embryos, the measurements closely fit into the quantitative relation between geometric constraints and inter-condensation distance found in vitro. Together, these findings suggest a potential role of geometric constraints in skeletal patterning in a cellular process of self-organization.
Tissue reinnervation following trauma, disease, or transplantation often presents a significant challenge. Here, we show that the delivery of vascular endothelial growth factor (VEGF) from alginate hydrogels ameliorates loss of skeletal muscle innervation after ischemic injury by promoting both maintenance and regrowth of damaged axons in mice. Nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF) mediated VEGF-induced axonal regeneration, and the expression of both is induced by VEGF presentation. Using both in vitro and in vivo modeling approaches, we demonstrate that the activity of NGF and GDNF regulates VEGF-driven angiogenesis, controlling endothelial cell sprouting and blood vessel maturation. Altogether, these studies produce evidence of new mechanisms of VEGF action, further broaden the understanding of the roles of NGF and GDNF in angiogenesis and axonal regeneration, and suggest approaches to improve axonal and ischemic tissue repair therapies.
Biological systems are exquisitely sensitive to the location and timing of physiologic cues and drugs. This spatiotemporal sensitivity presents opportunities for developing new therapeutic approaches. Polymer-based delivery systems are used extensively for attaining localized, sustained release of bioactive molecules. However, these devices typically are designed to achieve a constant rate of release. We hypothesized that it would be possible to create digital drug release, which could be accelerated and then switched back off, on demand, by applying ultrasound to disrupt ionically cross-linked hydrogels. We demonstrated that ultrasound does not permanently damage these materials but enables nearly digital release of small molecules, proteins, and condensed oligonucleotides. Parallel in vitro studies demonstrated that the concept of applying temporally short, high-dose "bursts" of drug exposure could be applied to enhance the toxicity of mitoxantrone toward breast cancer cells. We thus used the hydrogel system in vivo to treat xenograft tumors with mitoxantrone, and found that daily ultrasound-stimulated drug release substantially reduced tumor growth compared with sustained drug release alone. This approach of digital drug release likely will be applicable to a broad variety of polymers and bioactive molecules, and is a potentially useful tool for studying how the timing of factor delivery controls cell fate in vivo.
Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. The unusually long emission lifetime (5-13 μs) of photoluminescent porous silicon nanoparticles can allow the time-gated imaging of tissues in vivo, completely eliminating shorter-lived (<10 ns) emission signals from organic chromophores or tissue autofluorescence. Here using a conventional animal imaging system not optimized for such long-lived excited states, we demonstrate improvement of signal to background contrast ratio by >50-fold in vitro and by >20-fold in vivo when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes is discussed.
Mesenchymal stem cell (MSC)-based transplantation is a promising therapeutic approach for bone regeneration and repair. In the realm of therapeutic bone regeneration, the defect or injured tissues are frequently inflamed with an abnormal expression of inflammatory mediators. Growing evidence suggests that proinflammatory cytokines inhibit osteogenic differentiation and bone formation. Thus, for successful MSC-mediated repair, it is important to overcome the inflammation-mediated inhibition of tissue regeneration. In this study, using genetic and chemical approaches, we found that proinflammatory cytokines TNF and IL-17 stimulated IκB kinase (IKK)-NF-κB and impaired osteogenic differentiation of MSCs. In contrast, the inhibition of IKK-NF-κB significantly enhanced MSC-mediated bone formation. Mechanistically, we found that IKK-NF-κB activation promoted β-catenin ubiquitination and degradation through induction of Smurf1 and Smurf2. To translate our basic findings to potential clinic applications, we showed that the IKK small molecule inhibitor, IKKVI, enhanced osteogenic differentiation of MSCs. More importantly, the delivery of IKKVI promoted MSC-mediated craniofacial bone regeneration and repair in vivo. Considering the well established role of NF-κB in inflammation and infection, our results suggest that targeting IKK-NF-κB may have dual benefits in enhancing bone regeneration and repair and inhibiting inflammation, and this concept may also have applicability in many other tissue regeneration situations.
Alginate is a polysaccharide that can be crosslinked by divalent cations, such as calcium ions, to form a gel. Chemical modification is typically used to improve its cell adhesive properties for tissue engineering applications. In this study, alginates were modified with peptides containing RGD (arginine-glycine-aspartic acid) or PHSRN (proline-histidine-serine-arginine-asparagine) sequences from fibronectin to study possible additive and synergistic effects on adherent cells. Alginates modified with each peptide were mixed at different ratios to form gels containing various concentrations and spacing between the RGD and PHSRN sequences. When normal human osteoblasts (NHOsts) were cultured on or in the gels, the ratio of RGD to PHSRN was found to influence cell behaviors, especially differentiation. NHOsts cultured on gels composed of RGD- and PHSRN-modified alginates showed enhanced differentiation when the gels contained >33 % RGD-alginate, suggesting the relative distribution of the peptides and the presentation to cells are important parameters in this regulation. NHOsts cultured in gels containing both RGD- and PHSRN-alginates also demonstrated a similar enhancement tendency of calcium deposition that was dependent on the peptide ratio in the gel. However, calcium deposition was greater when cells were cultured in the gels, as compared to on the gels. These results suggest that modifying this biomaterial to more closely mimic the chemistry of natural cell adhesive proteins, (e.g., fibronectin) may be useful in developing scaffolds for bone tissue engineering and provide three-dimensional cell culture systems which more closely mimic the environment of the human body.
Transforming growth factor-ß (TGF-ß) signaling plays an important role in regulating crucial biological processes such as cell proliferation, differentiation, apoptosis, and extracellular matrix remodeling. Many of these processes are also an integral part of amelogenesis. In order to delineate a precise role of TGF-ß signaling during amelogenesis, we developed a transgenic mouse line that harbors bovine amelogenin promoter-driven Cre recombinase, and bred this line with TGF-ß receptor II floxed mice to generate ameloblast-specific TGF-ß receptor II conditional knockout (cKO) mice. Histological analysis of the teeth at postnatal day 7 (P7) showed altered enamel matrix composition in the cKO mice as compared to the floxed mice that had enamel similar to the wild-type mice. The µCT and SEM analyses revealed decreased mineral content in the cKO enamel concomitant with increased attrition and thinner enamel crystallites. Although the mRNA levels remained unaltered, immunostaining revealed increased amelogenin, ameloblastin, and enamelin localization in the cKO enamel at the maturation stage. Interestingly, KLK4 mRNA levels were significantly reduced in the cKO teeth along with a slight increase in MMP-20 levels, suggesting that normal enamel maturation is regulated by TGF-ß signaling through the expression of KLK4. Thus, our study indicates that TGF-ß signaling plays an important role in ameloblast functions and enamel maturation.
It has been observed that microbubbles may pass through the pulmonary circulation of dogs and humans during exercise. In humans, this phenomenon has been associated with lower pulmonary artery pressures, enhanced right ventricular function and greater exercise capacity. In the exercising Thoroughbred horse, extraordinarily high cardiac outputs exert significant pulmonary vascular stresses. The aim of this study was to determine, using contrast echocardiography, whether Thoroughbred horses performing strenuous exercise developed pulmonary transit of agitated contrast microbubbles (PTAC). At rest, agitated contrast was observed in the right ventricle, but not in the left ventricle. However, post-exercise microbubbles were observed in the left ventricle, confirming the occurrence of PTAC with exercise but not at rest. Further investigation is warranted to investigate whether this phenomenon may be associated with superior physiology and performance measures as has been implicated in other species.
BACKGROUND: Vascularization underlies the success of guided bone regeneration (GBR) procedures. This study evaluates the regenerative potential of GBR in combination with vascular endothelial growth factor (VEGF) delivery via an injectable hydrogel system.
METHODS: Critical-sized defects were created in rat calvariae, and GBR procedures were performed with a collagen membrane alone (control), or plus bolus delivery of VEGF, or plus application of VEGF-releasing hydrogels (VEGF-Alg). Four and 8 weeks after treatment, defect sites were evaluated with microcomputed tomographic and histomorphometric analyses for blood vessel and bone formation.
RESULTS: At 4 weeks, relative to the control condition, the bolus addition of VEGF did not affect blood vessel density within the defect site, yet the application of VEGF-Alg significantly (P <0.05) increased blood vessel density. Although there was no difference in bone regeneration at 4 weeks, at 8 weeks there was a significant (P <0.05) increase in bone regeneration in the VEGF-Alg-treated defects.
CONCLUSIONS: These data demonstrate that the application of VEGF-Alg enhanced early angiogenesis, whereas at a later time point, it enhanced bone regeneration. Controlled delivery approaches of angiogenic growth factors used adjunctively with GBR may be a promising strategy for enhancing outcomes of GBR.
Immunotherapy is a promising approach for treating cancer. However, there are limitations inherent to current approaches which may be addressed by integrating them with biomaterial-based strategies. Material platforms have been fabricated to interact with immune cells through spatially controlled and temporally controlled delivery of immune modulators and to promote immune cell crosstalk. Particle vaccines have been developed to specifically target and deliver agents to organs, cells and subcellular compartments. These strategies have been shown to generate antigen-specific CTL responses and, in some cases, tumor regression. Therefore, collaboration between immunology and materials engineering is likely to result in the creation of strong vaccines to combat cancer in the future.
Although hydrogels now see widespread use in a host of applications, low fracture toughness and brittleness have limited their more broad use. As a recently described interpenetrating network (IPN) of alginate and polyacrylamide demonstrated a fracture toughness of ≈ 9000 J/m(2), we sought to explore the biocompatibility and maintenance of mechanical properties of these hydrogels in cell culture and in vivo conditions. These hydrogels can sustain a compressive strain of over 90% with minimal loss of Young's Modulus as well as minimal swelling for up to 50 days of soaking in culture conditions. Mouse mesenchymal stem cells exposed to the IPN gel-conditioned media maintain high viability, and although cells exposed to conditioned media demonstrate slight reductions in proliferation and metabolic activity (WST assay), these effects are abrogated in a dose-dependent manner. Implantation of these IPN hydrogels into subcutaneous tissue of rats for 8 weeks led to mild fibrotic encapsulation and minimal inflammatory response. These results suggest the further exploration of extremely tough alginate/PAAM IPN hydrogels as biomaterials.
In general, alginate hydrogels are considered to be biologically inert and are commonly used for biomedical purposes that require minimum inflammation. However, Ca(2+), which is commonly used to crosslink alginate, is a critical second messenger in immune cell signaling, and little has been done to understand its effect on immune cell fate when delivered as a component of alginate gels. We found that dendritic cells (DCs) encapsulated in Ca(2+)-crosslinked alginate (calcium alginate) secreted at least fivefold more of the inflammatory cytokine IL-1β when compared to DCs encapsulated in agarose and collagen gels, as well as DCs plated on tissue-culture polystyrene (TCPS). Plating cells on TCPS with the alginate polymer could not reproduce these results, whereas culturing DCs on TCPS with increasing concentrations of Ca(2+) increased IL-1β, MHC class II and CD86 expression in a dose-dependent manner. In agreement with these findings, calcium alginate gels induced greater maturation of encapsulated DCs compared to barium alginate gels. When injected subcutaneously in mice, calcium alginate gels significantly upregulated IL-1β secretion from surrounding tissue relative to barium alginate gels, and similarly, the inflammatory effects of LPS were enhanced when it was delivered from calcium alginate gels rather than barium alginate gels. These results confirm that the Ca(2+) used to crosslink alginate gels can be immunostimulatory and suggest that it is important to take into account Ca(2+)'s bioactive effects on all exposed cells (both immune and non-immune) when using calcium alginate gels for biomedical purposes. This work may strongly impact the way people use alginate gels in the future as well as provide insights into past work utilizing alginate gels.
During embryonic development, morphogenetic processes give rise to a variety of shapes and patterns that lead to functional tissues and organs. While the impact of chemical signals on these processes is widely studied, the role of physical cues is less understood. The aim of this study was to test the hypothesis that the interplay of cell mediated contraction and mechanical boundary conditions alone can result in spatially regulated differentiation in simple 3D constructs. An experimental model consisting of a 3D cell-gel construct and a finite element (FE) model were used to study the effect of cellular traction exerted by mesenchymal stem cells (MSCs) on an initially homogeneous matrix under inhomogeneous boundary conditions. A robust shape change is observed due to contraction under time-varying mechanical boundary conditions, which is explained by the finite element model. Furthermore, distinct local differences in osteogenic differentiation are observed, with a spatial pattern independent of osteogenic factors in the culture medium. Regions that are predicted to have experienced relatively high shear stress at any time during contraction correlate with the regions of distinct osteogenesis. Taken together, these results support the underlying hypothesis that cellular contractility and mechanical boundary conditions alone can result in spatially regulated differentiation. These results will have important implications for tissue engineering and regeneration.
Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.
Therapeutic stimulation of angiogenesis to re-establish blood flow in ischemic tissues offers great promise as a treatment for patients suffering from cardiovascular disease or trauma. Since angiogenesis is a complex, multi-step process, different signals may need to be delivered at appropriate times in order to promote a robust and mature vasculature. The effects of temporally regulated presentation of pro-angiogenic and pro-maturation factors were investigated in vitro and in vivo in this study. Pro-angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2) cooperatively promoted endothelial sprouting and pericyte detachment in a three-dimensional in vitro EC-pericyte co-culture model. Pro-maturation factors platelet-derived growth factor B (PDGF) and angiopoietin 1 (Ang1) inhibited the early stages of VEGF- and Ang2-mediated angiogenesis if present simultaneously with VEGF and Ang2, but promoted these behaviors if added subsequently to the pro-angiogenesis factors. VEGF and Ang2 were also found to additively enhance microvessel density in a subcutaneous model of blood vessel formation, while simultaneously administered PDGF/Ang1 inhibited microvessel formation. However, a temporally controlled scaffold that released PDGF and Ang1 at a delay relative to VEGF/Ang2 promoted both vessel maturation and vascular remodeling without inhibiting sprouting angiogenesis. Our results demonstrate the importance of temporal control over signaling in promoting vascular growth, vessel maturation and vascular remodeling. Delivering multiple growth factors in combination and sequence could aid in creating tissue engineered constructs and therapies aimed at promoting healing after acute wounds and in chronic conditions such as diabetic ulcers and peripheral artery disease.
Human embryonic and induced pluripotent stem cells (hESC/hiPSC) are promising cell sources for the derivation of large numbers of specific cell types for tissue engineering and cell therapy applications. We have describe a directed differentiation protocol that generates fibroblasts from both hESC and hiPSC (EDK/iPDK) that support the repair and regeneration of epithelial tissue in engineered, 3D skin equivalents. In the current study, we analyzed the secretory profiles of EDK and iPDK cells to investigate the production of factors that activate and promote angiogenesis. Analysis of in vitro secretion profiles from EDK and iPDK cells demonstrated the elevated secretion of pro-angiogenic soluble mediators, including VEGF, HGF, IL-8, PDGF-AA, and Ang-1, that stimulated endothelial cell sprouting in a 3D model of angiogenesis in vitro. Phenotypic analysis of EDK and iPDK cells during the course of differentiation from hESCs and iPSCs revealed that both cell types progressively acquired pericyte lineage markers NG2, PDGFRβ, CD105, and CD73 and demonstrated transient induction of pericyte progenitor markers CD31, CD34, and Flk1/VEGFR2. Furthermore, when co-cultured with endothelial cells in 3D fibrin-based constructs, EDK and iPDK cells promoted self-assembly of vascular networks and vascular basement membrane deposition. Finally, transplantation of EDK cells into mice with hindlimb ischemia significantly reduced tissue necrosis and improved blood perfusion, demonstrating the potential of these cells to stimulate angiogenic responses in vivo. These findings demonstrate that stable populations of pericyte-like angiogenic cells can be generated with high efficiency from hESC and hiPSC using a directed differentiation approach. This provides new cell sources and opportunities for vascular tissue engineering and for the development of novel strategies in regenerative medicine.
Macroscale drug delivery (MDD) devices are engineered to exert spatiotemporal control over the presentation of a wide range of bioactive agents, including small molecules, proteins and cells. In contrast to systemically delivered drugs, MDD systems act as a depot of drug localized to the treatment site, which can increase drug effectiveness while reducing side effects and confer protection to labile drugs. In this Review, we highlight the key advantages of MDD systems, describe their mechanisms of spatiotemporal control and provide guidelines for the selection of carrier materials. We also discuss the combination of MDD technologies with classic medical devices to create multifunctional MDD devices that improve integration with host tissue, and the use of MDD technology in tissue-engineering strategies to direct cell behaviour. As our ever-expanding knowledge of human biology and disease provides new therapeutic targets that require precise control over their application, the importance of MDD devices in medicine is expected to increase.
During infection, inflammatory cytokines mobilize and activate dendritic cells (DCs), which are essential for efficacious T cell priming and immune responses that clear the infection. Here we designed macroporous poly(lactide-co-glycolide) (PLG) matrices to release the inflammatory cytokines GM-CSF, Flt3L and CCL20, in order to mimic infection-induced DC recruitment. We then tested the ability of these infection mimics to function as cancer vaccines via induction of specific, anti-tumor T cell responses. All vaccine systems tested were able to confer specific anti-tumor T cell responses and longterm survival in a therapeutic, B16-F10 melanoma model. However, GM-CSF and Flt3L vaccines resulted in similar survival rates, and outperformed CCL20 loaded scaffolds, even though they had differential effects on DC recruitment and generation. GM-CSF signaling was identified as the most potent chemotactic factor for conventional DCs and significantly enhanced surface expression of MHC(II) and CD86(+), which are utilized for priming T cell immunity. In contrast, Flt3L vaccines led to greater numbers of plasmacytoid DCs (pDCs), correlating with increased levels of T cell priming cytokines that amplify T cell responses. These results demonstrate that 3D polymer matrices modified to present inflammatory cytokines may be utilized to effectively mobilize and activate different DC subsets in vivo for immunotherapy.
Congrats to David and team on their recent publication in Nature Communications! Here, they utilized antigen presenting cell-mimetic scaffolds to tune CAR T-cell product functionality by controlling the precise level of stimulation during T-cell activation to accommodate individual differences in the donor cells. Check out the publication here: Enhancing CAR-T cell functionality in a patient-specific manner