Light-triggered in vivo activation of adhesive peptides regulates cell adhesion, inflammation and vascularization of biomaterials.

Materials engineered to elicit targeted cellular responses in regenerative medicine must display bioligands with precise spatial and temporal control. Although materials with temporally regulated presentation of bioadhesive ligands using external triggers, such as light and electric fields, have recently been realized for cells in culture, the impact of in vivo temporal ligand presentation on cell-material responses is unknown. Here, we present a general strategy to temporally and spatially control the in vivo presentation of bioligands using cell-adhesive peptides with a protecting group that can be easily removed via transdermal light exposure to render the peptide fully active. We demonstrate that non-invasive, transdermal time-regulated activation of cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion, inflammation, fibrous encapsulation, and vascularization of the material. This work shows that triggered in vivo presentation of bioligands can be harnessed to direct tissue reparative responses associated with implanted biomaterials.

Bioartificial matrices for therapeutic vascularization.

Therapeutic vascularization remains a significant challenge in regenerative medicine applications. Whether the goal is to induce vascular growth in ischemic tissue or scale up tissue-engineered constructs, the ability to induce the growth of patent, stable vasculature is a critical obstacle. We engineered polyethylene glycol-based bioartificial hydrogel matrices presenting protease-degradable sites, cell-adhesion motifs, and growth factors to induce the growth of vasculature in vivo. Compared to injection of soluble VEGF, these matrices delivered sustained in vivo levels of VEGF over 2 weeks as the matrix degraded. When implanted subcutaneously in rats, degradable constructs containing VEGF and arginine-glycine-aspartic acid tripeptide induced a significant number of vessels to grow into the implant at 2 weeks with increasing vessel density at 4 weeks. The mechanism of enhanced vascularization is likely cell-demanded release of VEGF, as the hydrogels may degrade substantially within 2 weeks. In a mouse model of hind-limb ischemia, delivery of these matrices resulted in significantly increased rate of reperfusion. These results support the application of engineered bioartificial matrices to promote vascularization for directed regenerative therapies.

Single versus dual microgel species for forming guest-host microporous annealed particle PEG-MAL hydrogel.

Inter-particle secondary crosslinks allow microporous annealed particle (MAP) hydrogels to be formed. Methods to introduce secondary crosslinking networks in MAP hydrogels include particle jamming, annealing with covalent bonds, and reversible noncovalent interactions. Here, we investigate the effect of two different approaches to secondary crosslinking of polyethylene glycol (PEG) microgels via reversible guest-host interactions. We generated a dual-particle MAP-PEG hydrogel using two species of PEG microgels, one functionalized with the guest molecule, adamantane, and the other with the host molecule, ß-cyclodextrin (Inter-MAP-PEG). In a different approach, a mono-particle MAP-PEG hydrogel was generated using one species of microgel functionalized with both guest and host molecules (Intra-MAP-PEG). The Intra-MAP-PEG formed a homogenous distribution due to the single type of microgels used. We then compared the mechanical properties of these two types of MAP-PEG hydrogels and found that Intra-MAP-PEG resulted in significantly softer gels with lower yield stress. We investigated the effect of intra-particle guest-host interactions through titrated weight percentage and the concentration of functional groups added to the hydrogel. We found that there was an ideal concentration of guest-host molecules that enables intra- and inter-particle guest-host interactions with sufficient covalent crosslinking. Based on these studies, Intra-MAP-PEG provides a homogeneous guest-host hydrogel that is shear-thinning with reversible secondary crosslinking.

Guest-host interlinked PEG-MAL granular hydrogels as an engineered cellular microenvironment.

We report the development of a polyethylene glycol (PEG) hydrogel scaffold that provides the advantages of conventional bulk PEG hydrogels for engineering cellular microenvironments and allows for rapid cell migration. PEG microgels were used to assemble a densely packed granular system with an intrinsic interstitium-like negative space. In this material, guest-host molecular interactions provide reversible non-covalent linkages between discrete PEG microgel particles to form a cohesive bulk material. In guest-host chemistry, different guest molecules reversibly and non-covalently interact with their cyclic host molecules. Two species of PEG microgels were made, each with one functional group at the end of the four arm PEG-MAL functionalized using thiol click chemistry. The first was functionalized with the host molecule ß-cyclodextrin, a cyclic oligosaccharide of repeating d-glucose units, and the other functionalized with the guest molecule adamantane. These two species provide a reversible guest-host interaction between microgel particles when mixed, generating an interlinked network with a percolated interstitium. We showed that this granular configuration, unlike conventional bulk PEG hydrogels, enabled the rapid migration of THP-1 monocyte cells. The guest-host microgels also exhibited shear-thinning behavior, providing a unique advantage over current bulk PEG hydrogels.

Engineered microenvironments and microdevices for modeling the pathophysiology of type 1 diabetes.

The pathophysiology of type 1 diabetes is a complex process involving tightly controlled microenvironments, a number of highly specific immune cell - islet cell interactions, and the eventual breaking of immune tolerance leading to beta cell death. Modeling this process can provide researchers with powerful insights into how and when to best provide treatment, but has proven difficult to accurately model due to its complex nature and differences between animal models and humans. Much progress has been made in determining the genetic, molecular, and cellular mechanisms of type 1 diabetes, yet translating that knowledge to clinical treatments remains challenging. Thus, there exists a capabilities gap between understanding the disease pathophysiology and engineering effective clinical treatment strategies. Biomimetic modeling of human type 1 diabetes is a valuable tool to study and manipulate islet function and can be employed to address immunological aspects of type 1 diabetes. This article will review recent advances in this field, and will suggest ways to synergize systems to model and observe the pathophysiology of autoimmune diabetes with bioengineered therapeutic strategies.

Vaccine nanocarriers: Coupling intracellular pathways and cellular biodistribution to control CD4 vs CD8 T cell responses.

Nanoparticle delivery systems are known to enhance the immune response to soluble antigens (Ags) and are thus a promising tool for the development of new vaccines. Our laboratory has engineered two different nanoparticulate systems in which Ag is either encapsulated within the core of polymersomes (PSs) or decorated onto the surface of nanoparticles (NPs). Previous studies showed that PSs are better at enhancing CD4 T cells and antibody titers, while NPs preferentially augment cytotoxic CD8 T cells. Herein, we demonstrate that the differential activation of T cell immunity reflects differences in the modes of intracellular trafficking and distinct biodistribution of the Ag in lymphoid organs, which are both driven by the properties of each nanocarrier. Furthermore, we found that Ags within PSs promoted better CD4 T cell activation and induced a higher frequency of CD4 T follicular helper (Tfh) cells. These differences correlated with changes in the frequency of germinal center B cells and plasma cell formation, which reflects the previously observed antibody titers. Our results show that PSs are a promising vector for the delivery of Ags for B cell vaccine development. This study demonstrates that nanocarrier design has a large impact on the quality of the induced adaptive immune response.

Primary Human and Rat ß-Cells Release the Intracellular Autoantigens GAD65, IA-2, and Proinsulin in Exosomes Together With Cytokine-Induced Enhancers of Immunity.

The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular ß-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by ß-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D.

Crystalline Oligo(ethylene sulfide) Domains Define Highly Stable Supramolecular Block Copolymer Assemblies.

With proper control over copolymer design and solvation conditions, self-assembled materials display impressive morphological variety that encompasses nanoscale colloids as well as bulk three-dimensional architectures. Here we take advantage of both hydrophobicity and crystallinity to mediate supramolecular self-assembly of spherical micellar, linear fibrillar, or hydrogel structures by a family of highly asymmetric poly(ethylene glycol)-b-oligo(ethylene sulfide) (PEG-OES) copolymers. Assembly structural polymorphism was achieved with modification of PEG-OES topology (linear versus multiarm) and with precise, monomer-by-monomer control of OES length. Notably, all three morphologies were accessed utilizing OES oligomers with degrees of polymerization as short as three. These exceptionally small assembly forming blocks represent the first application of ethylene sulfide oligomers in supramolecular materials. While the assemblies demonstrated robust aqueous stability over time, oxidation by hydrogen peroxide progressively converted ethylene sulfide residues to increasingly hydrophilic and amorphous sulfoxides and sulfones, causing morphological changes and permanent disassembly. We utilized complementary microscopic and spectroscopic techniques to confirm this chemical stimulus-responsive behavior in self-assembled PEG-OES colloidal dispersions and physical gels. In addition to inherent stimulus-responsive behavior, fibrillar assemblies demonstrated biologically relevant molecular delivery, as confirmed by the dose-dependent activation of murine bone marrow-derived dendritic cells following fibril-mediated delivery of the immunological adjuvant monophosphoryl lipid A. In physical gels composed of either linear or multiarm PEG-OES precursors, rheologic analysis also identified mechanical stimulus-responsive shear thinning behavior. Thanks to the facile preparation, user-defined morphology, aqueous stability, carrier functionality, and stimuli-responsive behaviors of PEG-OES supramolecular assemblies, our findings support a future role for these materials as injectable or implantable biomaterials.

Engineered VEGF-releasing PEG-MAL hydrogel for pancreatic islet vascularization.

Biofunctionalized polyethylene glycol maleimide (PEG-MAL) hydrogels were engineered as a platform to deliver pancreatic islets to the small bowel mesentery and promote graft vascularization. VEGF, a potent stimulator of angiogenesis, was incorporated into the hydrogel to be released in an on-demand manner through enzymatic degradation. PEG-MAL hydrogel enabled extended in vivo release of VEGF. Isolated rat islets encapsulated in PEG-MAL hydrogels remained viable in culture and secreted insulin. Islets encapsulated in PEG-MAL matrix and transplanted to the small bowel mesentery of healthy rats grafted to the host tissue and revascularized by 4 weeks. Addition of VEGF release to the PEG-MAL matrix greatly augmented the vascularization response. These results establish PEG-MAL engineered matrices as a vascular-inductive cell delivery vehicle and warrant their further investigation as islet transplantation vehicles in diabetic animal models.

Vasculogenic bio-synthetic hydrogel for enhancement of pancreatic islet engraftment and function in type 1 diabetes.

Type 1 diabetes (T1DM) affects one in every 400 children and adolescents in the US. Due to the limitations of exogenous insulin therapy and whole pancreas transplantation, pancreatic islet transplantation has emerged as a promising therapy for T1DM. However, this therapy is severely limited by donor islet availability and poor islet engraftment and function. We engineered an injectable bio-synthetic, polyethylene glycol-maleimide hydrogel to enhance vascularization and engraftment of transplanted pancreatic islets in a mouse model of T1DM. Controlled presentation of VEGF-A and cell-adhesive peptides within this engineered material significantly improved the vascularization and function of islets delivered to the small bowel mesentery, a metabolically relevant site for insulin release. Diabetic mice receiving islets transplanted in proteolytically degradable hydrogels incorporating VEGF-A exhibited complete reversal of diabetic hyperglycemia with a 40% reduction in the number of islets required. Furthermore, hydrogel-delivered islets significantly improved weight gain, regulation of a glucose challenge, and intra-islet vascularization and engraftment compared to the clinical standard of islet infusion through the hepatic portal vein. This study establishes a simple biomaterial strategy for islet transplantation to promote enhanced islet engraftment and function.

Dual delivery of hepatocyte and vascular endothelial growth factors via a protease-degradable hydrogel improves cardiac function in rats.

Acute myocardial infarction (MI) caused by ischemia and reperfusion (IR) is the most common cause of cardiac dysfunction due to local cell death and a temporally regulated inflammatory response. Current therapeutics are limited by delivery vehicles that do not address spatial and temporal aspects of healing. The aim of this study was to engineer biotherapeutic delivery materials to harness endogenous cell repair to enhance myocardial repair and function. We have previously engineered poly(ethylene glycol) (PEG)-based hydrogels to present cell adhesive motifs and deliver VEGF to promote vascularization in vivo. In the current study, bioactive hydrogels with a protease-degradable crosslinker were loaded with hepatocyte and vascular endothelial growth factors (HGF and VEGF, respectively) and delivered to the infarcted myocardium of rats. Release of both growth factors was accelerated in the presence of collagenase due to hydrogel degradation. When delivered to the border zones following ischemia-reperfusion injury, there was no acute effect on cardiac function as measured by echocardiography. Over time there was a significant increase in angiogenesis, stem cell recruitment, and a decrease in fibrosis in the dual growth factor delivery group that was significant compared with single growth factor therapy. This led to an improvement in chronic function as measured by both invasive hemodynamics and echocardiography. These data demonstrate that dual growth factor release of HGF and VEGF from a bioactive hydrogel has the capacity to significantly improve cardiac remodeling and function following IR injury.

Update on therapeutic vascularization strategies.

The ability to exploit angiogenesis and vascularization as a therapeutic strategy will be of enormous benefit to a wide range of medical and tissue-engineering applications. Angiogenic growth factor and cell-based therapies have thus far failed to produce a robust healing response in clinical trials for a variety of ischemic diseases, while engineered tissue substitutes are still size-limited by a lack of vascularization. The purpose of this review is to investigate current research advances in therapeutic vascularization strategies applied to ischemic disease states, tissue engineering and regenerative medicine. Recent advances are discussed that focus on better regulation of growth factor delivery and attempts to better mimic natural processes by delivering combinations of multiple growth factors, cells and bioactive materials in the right spatial and temporal setting. Some unconventional approaches and novel therapeutic targets that hold significant potential are also discussed.