Acrylated Hyaluronic-Acid Based Hydrogel for the Treatment of Craniofacial Volumetric Muscle Loss.

Current treatment options for craniofacial volumetric muscle loss (VML) have disadvantages and cannot fully restore normal function. Bio-inspired semisynthetic acrylated hyaluronic acid (AcHyA) hydrogel, which fills irregularly shaped defects, resembles an extracellular matrix, and induces a minimal inflammatory response, has shown promise in experimental studies of extremity VML. We therefore sought to study AcHyA hydrogel in the treatment of craniofacial VML. For this, we used a novel model of masseter VML in the rat. Following the creation of a 5 mm × 5 mm injury to the superficial masseter and administration of AcHyA to the wound, masseters were explanted between 2 and 16 weeks postoperatively and were analyzed for evidence of muscle regeneration including fibrosis, defect size, and fiber cross-sectional area (FCSA). At 8 and 16 weeks, masseters treated with AcHyA showed significantly less fibrosis than nonrepaired controls and a smaller decrease in defect size. The mean FCSA among fibers near the defect was significantly greater among hydrogel-repaired than control masseters at 8 weeks, 12 weeks, and 16 weeks. These results show that the hydrogel mitigates the fibrotic healing response and wound contracture. Our findings also suggest that hydrogel-based treatments have potential use as a treatment for the regeneration of craniofacial VML and demonstrate a system for evaluating subsequent iterations of materials in VML injuries.

Review: Human stem cell-based 3D in vitro angiogenesis models for preclinical drug screening applications.

Vascular diseases are the underlying pathology in many life-threatening illnesses. Human cellular and molecular mechanisms involved in angiogenesis are complex and difficult to study in current 2D in vitro and in vivo animal models. Engineered 3D in vitro models that incorporate human pluripotent stem cell (hPSC) derived endothelial cells (ECs) and supportive biomaterials within a dynamic microfluidic platform provide a less expensive, more controlled, and reproducible platform to better study angiogenic processes in response to external chemical or physical stimulus. Current studies to develop 3D in vitro angiogenesis models aim to establish single-source systems by incorporating hPSC-ECs into biomimetic extracellular matrices (ECM) and microfluidic devices to create a patient-specific, physiologically relevant platform that facilitates preclinical study of endothelial cell-ECM interactions, vascular disease pathology, and drug treatment pharmacokinetics. This review provides a detailed description of the current methods used for the directed differentiation of human stem cells to endothelial cells and their use in engineered 3D in vitro angiogenesis models that have been developed within the last 10 years.

Functionalised biomaterials as synthetic extracellular matrices to promote vascularisation and healing of diabetic wounds.

Diabetic foot ulcers (DFU) are a type of chronic wound that constitute one of the most serious and debilitating complications associated with diabetes. The lack of clinically efficacious treatments to treat these recalcitrant wounds can lead to amputations for those worst affected. Biomaterial-based approaches offer great hope in this regard, as they provide a template for cell infiltration and tissue repair. However, there is an additional need to treat the underlying pathophysiology of DFUs, in particular insufficient vascularization of the wound which significantly hampers healing. Thus, the addition of pro-angiogenic moieties to biomaterials is a promising strategy to promote the healing of DFUs and other chronic wounds. In this review, we discuss the potential of biomaterials as treatments for DFU and the approaches that can be taken to functionalise these biomaterials such that they promote vascularisation and wound healing in pre-clinical models.

Thou shall not heal: Overcoming the non-healing behaviour of diabetic foot ulcers by engineering the inflammatory microenvironment.

Diabetic foot ulcers (DFUs) are a devastating complication for diabetic patients that have debilitating effects and can ultimately lead to limb amputation. Healthy wounds progress through the phases of healing leading to tissue regeneration and restoration of the barrier function of the skin. In contrast, in diabetic patients dysregulation of these phases leads to chronic, non-healing wounds. In particular, unresolved inflammation in the DFU microenvironment has been identified as a key facet of chronic wounds in hyperglyceamic patients, as DFUs fail to progress beyond the inflammatory phase and towards resolution. Thus, control over and modulation of the inflammatory response is a promising therapeutic avenue for DFU treatment. This review discusses the current state-of-the art regarding control of the inflammatory response in the DFU microenvironment, with a specific focus on the development of biomaterials-based delivery strategies and their cargos to direct tissue regeneration in the DFU microenvironment.

A Biomimetic, Bilayered Antimicrobial Collagen-Based Scaffold for Enhanced Healing of Complex Wound Conditions.

Chronic, nonhealing wounds in the form of diabetic foot ulcers (DFUs) are a major complication for diabetic patients. The inability of a DFU to heal appropriately leads to an open wound with a high risk of infection. Current standards of care fail to fully address either the underlying defective wound repair mechanism or the risk of microbial infection. Thus, it is clear that novel approaches are needed. One such approach is the use of multifunctional biomaterials as platforms to direct and promote wound healing. In this study, a biomimetic, bilayered antimicrobial collagen-based scaffold was developed to deal with the etiology of DFUs. An epidermal, antimicrobial collagen/chitosan film for the prevention of wound infection was combined with a dermal collagen-glycosaminoglycan scaffold, which serves to support angiogenesis in the wound environment and ultimately accelerate wound healing. Biophysical and biological characterization identified an 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide cross-linked bilayered scaffold to have the highest structural stability with similar mechanical properties to products on the market, exhibiting a similar structure to native skin, successfully inhibiting the growth and infiltration of Staphylococcus aureus and supporting the proliferation of epidermal cells on its surface. This bilayered scaffold also demonstrated the ability to support the proliferation of key cell types involved in vascularization, namely, induced pluripotent stem cell derived endothelial cells and supporting stromal cells, with early signs of organization of these cells into vascular structures, showing great promise for the promotion of angiogenesis. Taken together, the results indicate that the bilayered scaffold is an excellent candidate for enhancement of diabetic wound healing by preventing wound infection and supporting angiogenesis.

Stem cell-based vascularization of microphysiological systems.

Microphysiological systems (MPSs) (i.e., tissue or organ chips) exploit microfluidics and 3D cell culture to mimic tissue and organ-level physiology. The advent of human induced pluripotent stem cell (hiPSC) technology has accelerated the use of MPSs to study human disease in a range of organ systems. However, in the reduction of system complexity, the intricacies of vasculature are an often-overlooked aspect of MPS design. The growing library of pluripotent stem cell-derived endothelial cell and perivascular cell protocols have great potential to improve the physiological relevance of vasculature within MPS, specifically for in vitro disease modeling. Three strategic categories of vascular MPS are outlined: self-assembled, interface focused, and 3D biofabricated. This review discusses key features and development of the native vasculature, linking that to how hiPSC-derived vascular cells have been generated, the state of the art in vascular MPSs, and opportunities arising from interdisciplinary thinking.

Semisynthetic Hyaluronic Acid-Based Hydrogel Promotes Recovery of the Injured Tibialis Anterior Skeletal Muscle Form and Function.

Volumetric muscle loss (VML) injuries are characterized by a degree of tissue loss that exceeds the endogenous regenerative capacity of muscle, resulting in permanent structural and functional deficits. Such injuries are a consequence of trauma, as well as a host of congenital and acquired diseases and disorders. Despite significant preclinical research with diverse biomaterials, as well as early clinical studies with implantation of decellularized extracellular matrices, there are still significant barriers to more complete restoration of muscle form and function following repair of VML injuries. In fact, identification of novel biomaterials with more advantageous regenerative profiles is a critical limitation to the development of improved therapeutics. As a first step in this direction, we evaluated a novel semisynthetic hyaluronic acid-based (HyA) hydrogel that embodies material features more favorable for robust muscle regeneration. This HyA-based hydrogel is composed of an acrylate-modified HyA (AcHyA) macromer, an AcHyA macromer conjugated with the bsp-RGD(15) peptide sequence to enhance cell adhesion, a high-molecular-weight heparin to sequester growth factors, and a matrix metalloproteinase-cleavable cross-linker to allow for cell-dependent remodeling. In a well-established, clinically relevant rat tibialis anterior VML injury model, we report observations of robust functional recovery, accompanied by volume reconstitution, muscle regeneration, and native-like vascularization following implantation of the HyA-based hydrogel at the site of injury. These findings have important implications for the development and clinical application of the improved biomaterials that will be required for stable and complete functional recovery from diverse VML injuries.

Hyaluronic Acid Macromer Molecular Weight Dictates the Biophysical Properties and in Vitro Cellular Response to Semisynthetic Hydrogels.

In situ-forming hydrogels present a promising approach for minimally invasive cell transplantation and tissue regeneration. Among prospective materials, hyaluronic acid (HyA) has displayed great potential, owing to its inherent biocompatibility, biodegradation, and ease of chemical modification. However, current studies in the literature use a broad range of HyA macromer molecular weights (MWs) from <100 kDa to 1 MDa with no consensus regarding an optimal MW for a specific application. We investigated the effects of different HyA macromer MWs on key biophysical properties of semisynthetic hydrogels, such as viscosity, gelation time, shear storage modulus, molecular diffusion, and degradation. Using higher-MW HyA macromers leads to quicker gelation times and stiffer, more stable hydrogels with smaller mesh sizes. Assessment of the potential for HyA hydrogels to support network formation by encapsulated vascular cells derived from human-induced pluripotent stem cells reveals key differences between HyA hydrogels dependent on macromer MW. These effects must be considered holistically to address the multifaceted, nonmonotonic nature of HyA MW on hydrogel behavior. Our study identified an intermediate HyA macromer MW of 500 kDa as providing optimal conditions for a readily injectable, in situ-forming hydrogel with appropriate biophysical properties to promote vascular cell spreading and sustain vascular network formation in vitro.

Matrix-assisted cell transplantation for tissue vascularization.

Cell therapy offers much promise for the treatment of ischemic diseases by augmenting tissue vasculogenesis. Matrix-assisted cell transplantation (MACT) has been proposed as a solution to enhance cell survival and integration with host tissue following transplantation. By designing semi synthetic matrices (sECM) with the correct physical and biochemical signals, encapsulated cells are directed towards a more angiogenic phenotype. In this review, we describe the choice of cells suitable for pro-angiogenic therapies, the properties that should be considered when designing sECM for transplantation and their relative importance. Pre-clinical models where MACT has been successfully applied to promote angiogenesis are reviewed to show the great potential of this strategy to treat ischemic conditions.

A combined hiPSC-derived endothelial cell and in vitro microfluidic platform for assessing biomaterial-based angiogenesis.

Human induced pluripotent stem cell (hiPSC) derived angiogenesis models present a unique opportunity for patient-specific platforms to study the complex process of angiogenesis and the endothelial cell response to biomaterial and biophysical changes in a defined microenvironment. We present a refined method for differentiating hiPSCs into a CD31 + endothelial cell population (hiPSC-ECs) using a single basal medium from pluripotency to the final stage of differentiation. This protocol produces endothelial cells that are functionally competent in assays following purification. Subsequently, an in vitro angiogenesis model was developed by encapsulating the hiPSC-ECs into a tunable, growth factor sequestering hyaluronic acid (HyA) matrix where they formed stable, capillary-like networks that responded to environmental stimuli. Perfusion of the networks was demonstrated using fluorescent beads in a microfluidic device designed to study angiogenesis. The combination of hiPSC-ECs, bioinspired hydrogel, and the microfluidic platform creates a unique testbed for rapidly assessing the performance of angiogenic biomaterials.

TGF-ß1/CD105 signaling controls vascular network formation within growth factor sequestering hyaluronic acid hydrogels.

Cell-based strategies for the treatment of ischemic diseases are at the forefront of tissue engineering and regenerative medicine. Cell therapies purportedly can play a key role in the neovascularization of ischemic tissue; however, low survival and poor cell engraftment with the host vasculature following implantation limits their potential to treat ischemic diseases. To overcome these limitations, we previously developed a growth factor sequestering hyaluronic acid (HyA)-based hydrogel that enhanced transplanted mouse cardiosphere-derived cell survival and formation of vasculature that anastomosed with host vessels. In this work, we examined the mechanism by which HyA hydrogels presenting transforming growth factor beta-1 (TGF-ß1) promoted proliferation of more clinically relevant human cardiosphere-derived cells (hCDC), and their formation of vascular-like networks in vitro. We observed hCDC proliferation and enhanced formation of vascular-like networks occurred in the presence of TGF-ß1. Furthermore, production of nitric oxide (NO), VEGF, and a host of angiogenic factors were increased in the presence of TGF-ß1. This response was dependent on the co-activity of CD105 (Endoglin) with the TGF-ßR2 receptor, demonstrating its role in the process of angiogenic differentiation and vascular organization of hCDC. These results demonstrated that hCDC form vascular-like networks in vitro, and that the induction of vascular networks by hCDC within growth factor sequestering HyA hydrogels was mediated by TGF-ß1/CD105 signaling.

Multivalent conjugates of basic fibroblast growth factor enhance in vitro proliferation and migration of endothelial cells.

Growth factors hold great promise for regenerative therapies. However, their clinical use has been halted by poor efficacy and rapid clearance from tissue, necessitating the delivery of extremely high doses to achieve clinical effectiveness which has raised safety concerns. Thus, strategies to either enhance growth factor activity at low doses or to increase their residence time within target tissues are necessary for clinical success. In this study, we generated multivalent conjugates (MVCs) of basic fibroblast growth factor (bFGF), a key growth factor involved in angiogenesis and wound healing, to hyaluronic acid (HyA) polymer chains. Multivalent bFGF conjugates (mvbFGF) were fabricated with minimal non-specific interaction observed between bFGF and the HyA chain. The hydrodynamic radii of mvbFGF ranged from ∼50 to ∼75 nm for conjugation ratios of bFGF to HyA chains at low (10 : 1) and high (30 : 1) feed ratios, respectively. The mvbFGF demonstrated enhanced bioactivity compared to unconjugated bFGF in assays of cell proliferation and migration, processes critical to angiogenesis and tissue regeneration. The 30 : 1 mvbFGF outperformed the 10 : 1 conjugate, which could be due to either FGF receptor clustering or interference with receptor mediated internalization and signal deactivation. This study simultaneously investigated the role of both protein to polymer ratio and multivalent conjugate size on their bioactivity, and determined that increasing the protein-to-polymer ratio and conjugate size resulted in greater cell bioactivity.

Engineered systems for therapeutic angiogenesis.

Ischemic disease caused by insufficient blood supply leads to a lack of oxygen and nutrients and a build-up of waste products in the affected tissue. Therapeutic angiogenesis, as a means to enhance perfusion of tissues with an inadequate blood supply, holds great promise for the treatment of ischemic disease. A wide range of factors that play a key role in physiological angiogenesis have been identified and trialed as pro-angiogenic agents. However, as yet pro-angiogenic treatments have failed to be translated clinically, owing to both lack of efficacy and safety concerns regarding the use of doses considerably larger than is typical present under physiological conditions. Thus, there is a clear need for the design and development of systems to overcome these hurdles and allow for the translation of safe and efficacious treatments to induce angiogenesis. In this regard, much progress has been made in the development of biomaterials as delivery systems for angiogenic factors to control the delivery and release of angiogenic therapies to induce vascularization. Thus, we review progress towards the development of translatable biomaterial-based systems to deliver angiogenic therapies, and point towards burgeoning advances in the field.

Drug-eluting biodegradable ureteral stent: New approach for urothelial tumors of upper urinary tract cancer.

Upper urinary tract urothelial carcinoma (UTUC) accounts for 5-10% of urothelial carcinomas and is a disease that has not been widely studied as carcinoma of the bladder. To avoid the problems of conventional therapies, such as the need for frequent drug instillation due to poor drug retention, we developed a biodegradable ureteral stent (BUS) impregnated by supercritical fluid CO2 (scCO2) with the most commonly used anti-cancer drugs, namely paclitaxel, epirubicin, doxorubicin, and gemcitabine. The release kinetics of anti-cancer therapeutics from drug-eluting stents was measured in artificial urine solution (AUS). The in vitro release showed a faster release in the first 72h for the four anti-cancer drugs, after this time a plateau was achieved and finally the stent degraded after 9days. Regarding the amount of impregnated drugs by scCO2, gemcitabine showed the highest amount of loading (19.57µg drug/mg polymer: 2% loaded), while the lowest amount was obtained for paclitaxel (0.067µg drug/mg polymer: 0.01% loaded). A cancer cell line (T24) was exposed to graded concentrations (0.01-2000ng/ml) of each drugs for 4 and 72h to determine the sensitivities of the cells to each drug (IC50). The direct and indirect contact study of the anti-cancer biodegradable ureteral stents with the T24 and HUVEC cell lines confirmed the anti-tumoral effect of the BUS impregnated with the four anti-cancer drugs tested, reducing around 75% of the viability of the T24 cell line after 72h and demonstrating minimal cytotoxic effect on HUVECs.

sFlt Multivalent Conjugates Inhibit Angiogenesis and Improve Half-Life In Vivo.

Current anti-VEGF drugs for patients with diabetic retinopathy suffer from short residence time in the vitreous of the eye. In order to maintain biologically effective doses of drug for inhibiting retinal neovascularization, patients are required to receive regular monthly injections of drug, which often results in low patient compliance and progression of the disease. To improve the intravitreal residence time of anti-VEGF drugs, we have synthesized multivalent bioconjugates of an anti-VEGF protein, soluble fms-like tyrosine kinase-1 (sFlt) that is covalently grafted to chains of hyaluronic acid (HyA), conjugates that are termed mvsFlt. Using a mouse corneal angiogenesis assay, we demonstrate that covalent conjugation to HyA chains does not decrease the bioactivity of sFlt and that mvsFlt is equivalent to sFlt at inhibiting corneal angiogenesis. In a rat vitreous model, we observed that mvsFlt had significantly increased intravitreal residence time compared to the unconjugated sFlt after 2 days. The calculated intravitreal half-lives for sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, we show that mvsFlt is more effective than the unconjugated form at inhibiting retinal neovascularization in an oxygen-induced retinopathy model, an effect that is most likely due to the longer half-life of mvsFlt in the vitreous. Taken together, our results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF and this conjugation significantly improves drug half-life. These in vivo results suggest that our strategy of multivalent conjugation could substantially improve upon drug half-life, and thus the efficacy of currently available drugs that are used in diseases such as diabetic retinopathy, thereby improving patient quality of life.

Matrix metalloproteinase-13 mediated degradation of hyaluronic acid-based matrices orchestrates stem cell engraftment through vascular integration.

A critical design parameter for the function of synthetic extracellular matrices is to synchronize the gradual cell-mediated degradation of the matrix with the endogenous secretion of natural extracellular matrix (ECM) (e.g., creeping substitution). In hyaluronic acid (HyA)-based hydrogel matrices, we have investigated the effects of peptide crosslinkers with different matrix metalloproteinases (MMP) sensitivities on network degradation and neovascularization in vivo. The HyA hydrogel matrices consisted of cell adhesive peptides, heparin for both the presentation of exogenous and sequestration of endogenously synthesized growth factors, and MMP cleavable peptide linkages (i.e., QPQGLAK, GPLGMHGK, and GPLGLSLGK). Sca1(+)/CD45(-)/CD34(+)/CD44(+) cardiac progenitor cells (CPCs) cultured in the matrices with the slowly degradable QPQGLAK hydrogels supported the highest production of MMP-2, MMP-9, MMP-13, VEGF165, and a range of angiogenesis related proteins. Hydrogels with QPQGLAK crosslinks supported prolonged retention of these proteins via heparin within the matrix, stimulating rapid vascular development, and anastomosis with the host vasculature when implanted in the murine hindlimb.

Co-transfection of decorin and interleukin-10 modulates pro-fibrotic extracellular matrix gene expression in human tenocyte culture.

Extracellular matrix synthesis and remodelling are driven by increased activity of transforming growth factor beta 1 (TGF-ß1). In tendon tissue repair, increased activity of TGF-ß1 leads to progressive fibrosis. Decorin (DCN) and interleukin 10 (IL-10) antagonise pathological collagen synthesis by exerting a neutralising effect via downregulation of TGF-ß1. Herein, we report that the delivery of DCN and IL-10 transgenes from a collagen hydrogel system supresses the constitutive expression of TGF-ß1 and a range of pro-fibrotic extracellular matrix genes.

Variability in Endogenous Perfusion Recovery of Immunocompromised Mouse Models of Limb Ischemia.

Immunocompromised hind limb ischemia (HLI) murine models are essential for preclinical evaluation of human cell-based therapy or biomaterial-based interventions. These models are used to generate proof of principle that the approach is effective and also regulatory preclinical data required for translation to the clinic. However, surgical variations in creation of HLI models reported in the literature introduce variability in the pathological manifestation of the model, in consequence affecting therapeutic endpoints. This study aims to compare the extent of vascular regeneration in HLI-induced immunocompromised murine models to obtain a stable and more reproducible injury model for testing. Athymic and Balb/C nude mice underwent HLI surgery with single and double ligation of femoral artery (FA). The recovery from surgery was observed over a period of 2 weeks with respect to ischemia reperfusion using laser Doppler and clinical signs of necrosis and ambulatory impairment. Double ligation of the FA results in a more severe response to ischemia in Balb/C with endogenous perfusion recovery up to 50% ± 10% compared with 75% ± 20% in athymic nude mice. Single iliac artery (IA) and FA lead to creation of mild ischemia compared with femoral artery-vein (FAV) pair ligation in Balb/C. Microcirculatory parameters indicate significantly lower capillary numbers (26 ± 3/mm(2)) and functional capillary density (203 ± 5 cm/cm(2)) in the FAV group. In this study, we demonstrate a reproducible, arterial double ligation in an immunocompromised Balb/C nude mouse model that exhibits characteristic pathological signs of ischemia with impaired endogenous recovery.

Modulation of inflammation and angiogenesis and changes in ECM GAG-activity via dual delivery of nucleic acids.

Tissue-engineered organs and implants hold promise for the replacement of damaged and diseased organs. However, the foreign body response (FBR) is a major obstacle that compromises the function of tissue-engineered constructs, typically causing them to fail. Two components of FBR are an inflammatory response and a lack of vascularization. To overcome these limitations, a collagen system was developed to release interleukin-6 (IL-6) siRNA and endothelial nitric oxide synthase (eNOS) pDNA in a staggered manner. Hollow collagen microspheres were assembled into a collagen sphere-in-hydrogel system that displayed a staggered release profile in vitro. This system was assessed in vivo in a subcutaneous rat model. The doses of IL-6 siRNA and eNOS pDNA were first individually optimized for their ability to reduce the volume fraction of inflammatory cells (7 days) and increase the length density of blood vessels (14 days), respectively. The identified optimal doses were combined, and the ability of the system to decrease the volume fraction of inflammatory cells and increase the length density of blood vessels was confirmed at both 7 and 14 days. Analysis of the tissue using Raman microspectroscopy revealed that in addition to changes in inflammation and angiogenesis, there were also changes in the extracellular matrix (ECM) at seven days. While changes in sulfated glycosaminoglycan (sGAG) content of the ECM were not detected, changes in the binding of sGAG of the ECM to growth factors were observed. Two growth factors tested, VEGF165 and bFGF showed increased binding to sGAG extracted from eNOS pDNA-treated samples at seven days, increasing the angiogenic potential of the ECM. Thus, we observe that changes in the tissue in terms of the balance of inflammation and angiogenesis as well changes in the activity of sGAG of the ECM occurs following dual delivery of nucleic acids from the collagen sphere-in-hydrogel system.

Biomaterial-mediated modification of the local inflammatory environment.

Inflammation plays a major role in the rejection of biomaterial implants. In addition, despite playing an important role in the early stages of wound healing, dysregulated inflammation has a negative impact on the wound healing processes. Thus, strategies to modulate excessive inflammation are needed. Through the use of biomaterials to control the release of anti-inflammatory therapeutics, increased control over inflammation is possible in a range of pathological conditions. However, the choice of biomaterial (natural or synthetic), and the form it takes (solid, hydrogel, or micro/nanoparticle) is dependent on both the cause and tissue location of inflammation. These considerations also influence the nature of the anti-inflammatory therapeutic that is incorporated into the biomaterial to be delivered. In this report, the range of biomaterials and anti-inflammatory therapeutics that have been combined will be discussed, as well as the functional benefit observed. Furthermore, we point toward future strategies in the field that will bring more efficacious anti-inflammatory therapeutics closer to realization.