Angiogenic patterning by STEEL, an endothelial-enriched long noncoding RNA.

Endothelial cell (EC)-enriched protein coding genes, such as endothelial nitric oxide synthase (eNOS), define quintessential EC-specific physiologic functions. It is not clear whether long noncoding RNAs (lncRNAs) also define cardiovascular cell type-specific phenotypes, especially in the vascular endothelium. Here, we report the existence of a set of EC-enriched lncRNAs and define a role for spliced-transcript endothelial-enriched lncRNA (STEEL) in angiogenic potential, macrovascular/microvascular identity, and shear stress responsiveness. STEEL is expressed from the terminus of the HOXD locus and is transcribed antisense to HOXD transcription factors. STEEL RNA increases the number and integrity of de novo perfused microvessels in an in vivo model and augments angiogenesis in vitro. The STEEL RNA is polyadenylated, nuclear enriched, and has microvascular predominance. Functionally, STEEL regulates a number of genes in diverse ECs. Of interest, STEEL up-regulates both eNOS and the transcription factor Kruppel-like factor 2 (KLF2), and is subject to feedback inhibition by both eNOS and shear-augmented KLF2. Mechanistically, STEEL up-regulation of eNOS and KLF2 is transcriptionally mediated, in part, via interaction of chromatin-associated STEEL with the poly-ADP ribosylase, PARP1. For instance, STEEL recruits PARP1 to the KLF2 promoter. This work identifies a role for EC-enriched lncRNAs in the phenotypic adaptation of ECs to both body position and hemodynamic forces and establishes a newer role for lncRNAs in the transcriptional regulation of EC identity.

Modular tissue engineering for the vascularization of subcutaneously transplanted pancreatic islets.

The transplantation of pancreatic islets, following the Edmonton Protocol, is a promising treatment for type I diabetics. However, the need for multiple donors to achieve insulin independence reflects the large loss of islets that occurs when islets are infused into the portal vein. Finding a less hostile transplantation site that is both minimally invasive and able to support a large transplant volume is necessary to advance this approach. Although the s.c. site satisfies both these criteria, the site is poorly vascularized, precluding its utility. To address this problem, we demonstrate that modular tissue engineering results in an s.c. vascularized bed that enables the transplantation of pancreatic islets. In streptozotocin-induced diabetic SCID/beige mice, the injection of 750 rat islet equivalents embedded in endothelialized collagen modules was sufficient to restore and maintain normoglycemia for 21 days; the same number of free islets was unable to affect glucose levels. Furthermore, using CLARITY, we showed that embedded islets became revascularized and integrated with the host's vasculature, a feature not seen in other s.c. STUDIES: Collagen-embedded islets drove a small (albeit not significant) shift toward a proangiogenic CD206+MHCII-(M2-like) macrophage response, which was a feature of module-associated vascularization. While these results open the potential for using s.c. islet delivery as a treatment option for type I diabetes, the more immediate benefit may be for the exploration of revascularized islet biology.

Unbiased phosphoproteomic method identifies the initial effects of a methacrylic acid copolymer on macrophages.

An unbiased phosphoproteomic method was used to identify biomaterial-associated changes in the phosphorylation patterns of macrophage-like cells. The phosphorylation differences between differentiated THP1 (dTHP1) cells treated for 10, 20, or 30 min with a vascular regenerative methacrylic acid (MAA) copolymer or a control methyl methacrylate (MM) copolymer were determined by MS. There were 1,470 peptides (corresponding to 729 proteins) that were differentially phosphorylated in dTHP1 cells treated with the two materials with a greater cellular response to MAA treatment. In addition to identifying pathways (such as integrin signaling and cytoskeletal arrangement) that are well known to change with cell-material interaction, previously unidentified pathways, such as apoptosis and mRNA splicing, were also discovered.

Biodegradable scaffold with built-in vasculature for organ-on-a-chip engineering and direct surgical anastomosis.

We report the fabrication of a scaffold (hereafter referred to as AngioChip) that supports the assembly of parenchymal cells on a mechanically tunable matrix surrounding a perfusable, branched, three-dimensional microchannel network coated with endothelial cells. The design of AngioChip decouples the material choices for the engineered vessel network and for cell seeding in the parenchyma, enabling extensive remodelling while maintaining an open-vessel lumen. The incorporation of nanopores and micro-holes in the vessel walls enhances permeability, and permits intercellular crosstalk and extravasation of monocytes and endothelial cells on biomolecular stimulation. We also show that vascularized hepatic tissues and cardiac tissues engineered by using AngioChips process clinically relevant drugs delivered through the vasculature, and that millimetre-thick cardiac tissues can be engineered in a scalable manner. Moreover, we demonstrate that AngioChip cardiac tissues implanted with direct surgical anastomosis to the femoral vessels of rat hindlimbs establish immediate blood perfusion.

Effect of Cell Density of a Methacrylic Acid-Based Hydrogel Implant on Embedded Islet Function and Viability.

Subcutaneous delivery of islets in a methacrylic acid-based hydrogel may offer a functional cure for type 1 diabetes. Here we show in mice that the hydrogel is able to provide sufficient vasculature to support islet function and viability, when islets are used at a low islet volume fraction (i.e., cell density). The Krogh cylinder model was used to mathematically estimate the effect of implant volume, for a fixed islet dose (600 islet equivalents [IEQ]), on the minimum vessel density required to maintain sufficient pO2 within the graft. Modeling suggested that 200 µL implants would have low enough islet densities and enough vessels to have islets remain viable, but that 50 µL implants would not; this was confirmed experimentally through measurement of glucose level in streptozotocin-induced diabetic severe combined immunodeficiency disease (SCID/bg) mice, comparing 200 and 50 µL implants, both with 600 IEQ. Vessel densities were ∼20-30 vessels/mm2 independent of implant volume and vessels were sufficient to increase subcutaneous oxygen tension, as measured with microcapsules containing oxygen sensitive material (a platinum [Pt] porphyrin); both these results were determined without cells. These results are useful in thinking about the scale-up of this system to humans: to maintain a low islet density (∼0.5%), many more islets will require attention to the subcutaneous implant configuration to satisfy the oxygen needs of the cells.

Neutrophil depletion for early allogeneic islet survival in a methacrylic acid (MAA) copolymer-induced, vascularized subcutaneous space.

Islet transplantation is a promising treatment for type I diabetes (T1D). Despite the high loss of islets during transplantation, current islet transplant protocols continue to rely on portal vein infusion and intrahepatic engraftment. Because of the risk of portal vein thrombosis and the loss of islets to instant blood mediated inflammatory reaction (IBMIR), other transplantation sites like the subcutaneous space have been pursued for its large transplant volume, accessibility, and amenability for retrieval. To overcome the minimal vasculature of the subcutaneous space, prevascularization approaches or vascularizing biomaterials have been used to subcutaneously deliver islets into diabetic mice to return them to normoglycemia. Previous vascularization methods have relied on a 4 to 6 week prevascularization timeframe. Here we show that a vascularizing MAA-coated silicone tube can generate sufficient vasculature in 2 to 3 weeks to support a therapeutic dose of islets in mice. In order to fully harness the potential of this prevascularized site, we characterize the unique, subcutaneous immune response to allogeneic islets in the first 7 days following transplantation, a critical stage in successful engraftment. We identify neutrophils as a specific cellular target, a previously overlooked cell in the context of subcutaneous allogeneic islet transplantation. By perioperatively depleting neutrophils, we show that neutrophils are a key, innate immune cell target for successful early engraftment of allogeneic islets in a prevascularized subcutaneous site.

Aspects of the alternative host response to methacrylic acid containing biomaterials.

Methacrylic acid (MAA)-based biomaterials promote a vascularized host response without the addition of exogenous factors such as cells or growth factors. We presume that materials containing MAA favor an alternative foreign body response, rather than the conventional fibrotic response. Here, we characterize selected aspects of the response to two different forms of MAA-a coating, which can be used to prevascularize the subcutaneous tissue for subsequent therapeutic cell delivery or an injectable hydrogel, which can be used to vascularize and deliver cells simultaneously. We show that the MAA-coating quickly vascularized the subcutaneous space compared to an uncoated silicone tube, and after 14 days of prevascularization, the tissue surrounding the MAA-coated tube presented fewer immune cells than the uncoated control. We also compared the host response to a MAA-PEG (polyethylene glycol) hydrogel at day 1, with pancreatic islets in immune-compromised SCID/bg mice and immune-competent Balb/c mice. The Balb/c mouse presented a more inflammatory response with increased IFN-γ production as compared to the SCID/bg. Together with previously published data, this work contributes to a further understanding of tissue responses to a biomaterial in different forms as used for cell delivery.

Immunomodulation by subcutaneously injected methacrylic acid-based hydrogels and tolerogenic dendritic cells in a mouse model of autoimmune diabetes.

Type 1 diabetes is an autoimmune disease associated with the destruction of insulin-producing ß cells. Immunotherapies are being developed to mitigate autoimmune diabetes. One promising option is the delivery of tolerogenic dendritic cells (DCs) primed with specific ß-cell-associated autoantigens. These DCs can combat autoreactive cells and promote expansion of ß-cell-specific regulatory immune cells, including Tregs. Tolerogenic DCs are typically injected systemically (or near target lymph nodes) in suspension, precluding control over the microenvironment surrounding tolerogenic DC interactions with the host. In this study we show that degradable, synthetic methacrylic acid (MAA)-based hydrogels are an inherently immunomodulating delivery vehicle that enhances tolerogenic DC therapy in the context of autoimmune diabetes. MAA hydrogels were found to affect the local recruitment and activation state of macrophages, DCs, T cells and other cells. Delivering tolerogenic DCs in the MAA hydrogel improved the local host response (e.g., fewer cytotoxic T cells) and enhanced peripheral Treg expansion. Non obese diabetic (NOD) mice treated with tolerogenic DCs subcutaneously injected in MAA hydrogels showed a delay in onset of autoimmune diabetes compared to control vehicles. Our findings further demonstrate the usefulness of MAA-based hydrogels as platforms for regenerative medicine in the context of type 1 diabetes.

Hearts by design.

Scalable biofabrication of heart helical tissue pattern augments pumping function.

Degradable methacrylic acid-based synthetic hydrogel for subcutaneous islet transplantation.

Islet transplantation is a promising regenerative therapy that would reduce the dependence of type 1 diabetic patients on insulin injections. However, islet transplantation is not yet widely available, in part because there is no ideal transplant site. The subcutaneous space has been highlighted as a promising transplant site, but it does not have the vasculature required to support an islet graft. In this study we demonstrate that islets engraft in the subcutaneous space when injected in an inherently vascularizing, degradable methacrylic acid-polyethylene glycol (MAA-PEG) hydrogel; no vascularizing cells or growth factors were required. In streptozotocin-induced diabetic mice, injection of 600 rodent islet equivalents in MAA-PEG hydrogels was sufficient to reverse diabetes for 70 days; a PEG gel without MAA had no benefit. MAA-PEG hydrogel scaffolds degraded over the course of a week and were replaced by a host-derived, vascularized, innervated matrix that supported subcutaneous islets. The survival of islet grafts through the inflammatory events of subcutaneous transplantation, hydrogel degradation, and islet revascularization underscore the benefits of the MAA biomaterial. Our findings establish the MAA-PEG hydrogel as a platform for subcutaneous islet transplantation.

Methacrylic acid-based biomaterials promote peripheral innervation in the subcutaneous space of mice.

Peripheral nerve innervation is essential for regulating tissue repair and regeneration. MAA-based biomaterials have been previously shown to promote angiogenesis. Here we show a new role for MAA-based biomaterials in promoting terminal axon nerve growth. Our results demonstrate that MAA-based biomaterials promote peripheral nerve growth in an Igf-1 and Shh dependent manner. The resulting nerves increased the sensitivity of treated mice paws to nociception. iDISCO clearing showed that MAA increased the presence of peripheral nerve structures in whole explants. MAA was also able to increase the expression of key neuronal markers and growth factors in a peripheral neuropathy model, the diabetic db/db mouse, suggesting that MAA-based biomaterials may be relevant to treatment of peripheral neuropathy. Moreover, in a peripheral neuropathy model, MAA was able to up-regulate the expression of growth factors for an extended duration suggesting MAA may prevent degeneration through an effect on factors that promote survival. As all tissues are innervated, MAA-based biomaterials could have broad applications in the promoting regeneration and preventing degeneration of peripheral nerves.

Endothelial cell behaviour within a microfluidic mimic of the flow channels of a modular tissue engineered construct.

To study the effect of disturbed flow patterns on endothelial cells, the channels found within a modular tissue engineering construct were reproduced in a microfluidic chip and lined with endothelial cells whose resulting phenotype under flow was assessed using confocal microscopy. Modular tissue engineered constructs formed by the random packing of sub-millimetre, cylindrically shaped, endothelial cell-covered modules into a larger container creates interconnected channels that permit the flow of fluids such as blood. Due to the random packing, the flow path is tortuous and has the potential to create disturbed flow, resulting in an activated endothelium. At an average shear stress of 2.8 dyn cm⁻², endothelial cells within channels of varying geometries showed higher amounts of activation, as evidenced by an increase in ICAM-1 and VCAM-1 levels with respect to static controls. VE-cadherin expression also increased, however, it appeared discontinuous around the perimeter of the cells. An increase in flow (15.6 dyn cm⁻²) was sufficient to reduce ICAM-1 and VCAM-1 expression to a level below that of static controls for many disturbed flow-prone channels that contained branches, curves, expansions and contractions. VE-cadherin expression was also reduced and became discontinuous in all channels, possibly due to paracrine signaling. Other than showing a mild correlation to VE-cadherin, which may be linked through a cAMP-initiated pathway, KLF2 was found to be largely independent of shear stress for this system. To gauge the adhesiveness of the endothelium to leukocytes, THP-1 cells were introduced into flow-conditioned channels and their attachment measured. Relative to static controls, THP-1 adhesion was reduced in straight and bifurcating channels. However, even in the presence of flow, areas where multiple channels converged were found to be the most prone to THP-1 attachment. The microfluidic system enabled a full analysis of the effect of the tortuous flow expected in a modular construct on endothelial cell phenotype.

Promoting endogenous repair of skeletal muscle using regenerative biomaterials.

Skeletal muscles normally have a remarkable ability to repair themselves; however, large muscle injuries and several myopathies diminish this ability leading to permanent loss of function. No clinical therapy yet exists that reliably restores muscle integrity and function following severe injury. Consequently, numerous tissue engineering techniques, both acellular and with cells, are being investigated to enhance muscle regeneration. Biomaterials are an essential part of these techniques as they can present physical and biochemical signals that augment the repair process. Successful tissue engineering strategies require regenerative biomaterials that either actively promote endogenous muscle repair or create an environment supportive of regeneration. This review will discuss several acellular biomaterial strategies for skeletal muscle regeneration with a focus on those under investigation in vivo. This includes materials that release bioactive molecules, biomimetic materials and immunomodulatory materials.

Methacrylic acid-based hydrogels enhance skeletal muscle regeneration after volumetric muscle loss in mice.

Volumetric muscle loss (VML) impairs the regenerative ability of skeletal muscle resulting in scar tissue formation and loss of function. Current treatments are of limited efficacy as they do not fully restore function, i.e., force generation. Regenerative biomaterials, such as those containing methacrylic-acid (MAA), are proposed as a novel approach to enhancing muscle regeneration without added cells, growth factors or drugs. Here, the regenerative effects of two hydrogels were investigated: MAA-poly(ethylene glycol) (MAA-PEG) and MAA-collagen. These hydrogels were used to treat VML injuries in murine tibialis anterior muscles. The MAA-collagen hydrogel significantly increased regenerating muscle fiber size and muscle force production. While both hydrogels increased vascularization, only the MAA-collagen hydrogel increased apparent muscle innervation. The MAA-collagen hydrogel also significantly reduced a pro-inflammatory macrophage (MHCII+CD206-) population. Furthermore, the hydrogels had distinct gene expression profiles indicating that their regenerative abilities were carrier dependent. Overall, this study suggests MAA-collagen as a cell-free and drug-free approach to enhancing skeletal muscle regeneration after traumatic injury.

A scalable device-less biomaterial approach for subcutaneous islet transplantation.

The subcutaneous space has been shown to be a suitable site for islet transplantation, however an abundance of islets is required to achieve normoglycemia, often requiring multiple donors. The loss of islets is due to the hypoxic conditions islets experience during revascularization, resulting in apoptosis. Therefore, to reduce the therapeutic dosage required to achieve normoglycemia, pre-vascularization of the subcutaneous space has been pursued. In this study, we highlight a biomaterial-based approach using a methacrylic acid copolymer coating to generate a robust pre-vascularized subcutaneous cavity for islet transplantation. We also devised a simple, but not-trivial, procedure for filling the cavity with an islet suspension in collagen. We show that the pre-vascularized site can support a marginal mass of islets to rapidly return streptozotocin-induced diabetic SCID/bg mice to normoglycemia. Furthermore, immunocompetent Sprague Daley rats remained normoglycemia for up to 70 days until they experienced graft destabilization as they outgrew their implants. This work highlights methacrylic acid-based biomaterials as a suitable pre-vascularization strategy for the subcutaneous space that is scalable and doesn't require exogenous cells or growth factors.

Poly-Methacrylic Acid Cross-Linked with Collagen Accelerates Diabetic Wound Closure.

Impaired blood vessel formation limits the healing of diabetic ulcers and leaves patients at high risk for amputation. Nonbiologic vascular regenerative materials made of methacrylic acid (MAA) copolymer, such as MAA-co-methyl methacrylate beads, have shown to enhance wound healing in a diabetic animal model, but their lack of biodegradability precludes their clinical implementation. Here, a new MAA-based gel was created by cross-linking polyMAA with collagen using carbodiimide chemistry. Using this gel on full-thickness wounds in diabetic db/db mice augmented vascularization of the wound bed, resulting in a faster closure compared to untreated or collagen-only treated wounds. After 21 days, almost all the wounds were closed and re-epithelialized in the polyMAA-collagen group compared to that in the other groups in which most wounds remained open. Histological and fluorescent gel tracking data suggested that the gel resorbed during the phase of tissue remodeling, likely because of the action of macrophages that colonized the gel. We expect the addition of the polyMAA to commercially available collagen-based dressing to be a good candidate to treat diabetic ulcers.

Methacrylic acid copolymer coating of polypropylene mesh chamber improves subcutaneous islet engraftment.

Subcutaneous devices can be used to house therapeutic cells such as pancreatic islets so that the cells can be retrieved. However, a high number of cells may be required to reverse diabetes, since a portion of the graft can be lost after transplantation due to ischemia and therefore the right device design is important. Increasing the vascularity of the subcutaneous space prior to cell transplantation is a strategic goal for cell transplantation, as it promotes islet survival, glucose-sensing and insulin secretion. In this study, a porous cell transplantation device was coated with 40% methacrylic acid-co-isodecyl acrylate (MAA-co-IDA), a biomaterial which promotes a vascular response without additional biologics. Three weeks after device implantation, the vessel density surrounding the device was double that of an uncoated device. The vasculature was mature and connected to the host bloodstream, as demonstrated by perfusion studies and histology. The tissue response to coated devices demonstrated lower levels of inflammation, measured by reduced gene expression of i-NOS and IL1ß, and increased expression of IL4. Syngeneic islets (300 islet equivalents) transplanted into the prevascularized coated device were able to return diabetic animals to normoglycemia for up to 11 weeks and resolve a glucose bolus similarly to non-diabetic mice by 3 weeks post-transplantation. We expect that the vessels and microenvironment resulting from the device coating are permissive to islet survival and thus enabled islets to reverse diabetes.

Acquisition of a Unique Mesenchymal Precursor-like Blastema State Underlies Successful Adult Mammalian Digit Tip Regeneration.

Here, we investigate the origin and nature of blastema cells that regenerate the adult murine digit tip. We show that Pdgfra-expressing mesenchymal cells in uninjured digits establish the regenerative blastema and are essential for regeneration. Single-cell profiling shows that the mesenchymal blastema cells are distinct from both uninjured digit and embryonic limb or digit Pdgfra-positive cells. This unique blastema state is environmentally determined; dermal fibroblasts transplanted into the regenerative, but not non-regenerative, digit express blastema-state genes and contribute to bone regeneration. Moreover, lineage tracing with single-cell profiling indicates that endogenous osteoblasts or osteocytes acquire a blastema mesenchymal transcriptional state and contribute to both dermis and bone regeneration. Thus, mammalian digit tip regeneration occurs via a distinct adult mechanism where the regenerative environment promotes acquisition of a blastema state that enables cells from tissues such as bone to contribute to the regeneration of other mesenchymal tissues such as the dermis.

Endothelialized collagen based pseudo-islets enables tuneable subcutaneous diabetes therapy.

Pancreatic islets are fragile cell clusters and many isolated islets are not suitable for transplantation. Furthermore, following transplantation, islets will experience a state of hypoxia and poor nutrient diffusion before revascularization, which is detrimental to islet survival; this is affected by islet size and health. Here we engineered tuneable size-controlled pseudo-islets created by dispersing de-aggregated islets in an endothelialized collagen scaffold. This supported subcutaneous engraftment, which returned streptozotocin-induced diabetic mice to normoglycemia. Whole-implant imaging after tissue clearing demonstrated pseudo-islets regenerated their vascular architecture and insulin-secreting ß-cells were within 5 µm of a perfusable vessel - a feature unique to this approach. By using an endothelialized collagen scaffold, this work highlights a novel "bottom-up" approach to islet engineering that provides control over the size and composition of the constructs, while enabling the critical ability to revascularize and engraft when transplanted into the clinically useful subcutaneous space.

Injectable and degradable methacrylic acid hydrogel alters macrophage response in skeletal muscle.

After severe trauma, skeletal muscle cannot repair itself leading to scar tissue formation and functional impairment. A novel approach to overcome this issue is to alter the fibrotic response in muscle using regenerative biomaterials, such as those containing methacrylic acid (MAA). In the skin, MAA-based materials have been shown to promote wound healing and new vessel formation, through endogenous mechanisms, including macrophage polarization; however, MAA has yet to be studied outside the skin. To study the innate immune response to MAA in skeletal muscle, MAA-poly(ethylene glycol) (MAA-PEG) hydrogels were synthesized with degradation rates of either 2 (fast-degrading) or 7 days (slow-degrading). When injected into the tibialis anterior muscle of mice, both slow- and fast-degrading MAA hydrogels increased the expression of Il-10, Tnfα and M2 macrophage markers (Fizz1 and Arg for slow-and fast-degrading, respectively). Moreover, the slow degrading MAA hydrogel decreased the number of pro-inflammatory MHCII+ macrophages. An unbiased t-distributed stochastic neighbor embedding (tSNE) analysis suggested the involvement of other immune cells beyond just macrophages in the effect of MAA on skeletal muscle. Overall, this study shows that MAA hydrogels bias macrophages towards a pro-regenerative phenotype.