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The demand for materials for bone tissue engineering (BTE) to treat bone disorders has an upward trend, especially in ageing populations. The main aim of the work reported in this thesis was to develop injectable hydrogels comprising the nanoclay Laponite, graphene and phosphonate moieties that would provide biocompatible scaffolds with osteoconductive properties and mechanical properties tailored for use in BTE. The subordinate aim was to produce modified graphene-family nanomaterials (GFNs) that would provide functional groups by which to improve aqueous dispersibility of graphene, tether biomolecules, and accelerate osteogenic mineralisation. Edge-specific functionalisation based upon electrophilic aromatic substitution and radical polymerisation in the presence of graphene were selected to produce functionalised graphene materials without introducing new defects into the graphene. Functionalisation was confirmed using FTIR, Raman, and XPS, which indicated a presence of functional groups and a low degree of defect in graphene structures, as well as demonstrating the change in aqueous dispersibility of graphene sheets. Phosphonate-modified graphene analogues were incorporated into two-dimensional polymer layer-by-layer (LbL) constructs for use in BTE. The biocompatibility and ability to support bone formation of GFN-containing LbL constructs were tested on human osteosarcoma Saos-2 cell line and primary human osteoblasts (HOBs). All GFN-containing LbL constructs supported cell adhesion, proliferation, and osteogenic mineralisation of Saos-2. On the other hand, cell attachment and cell activities of HOBs were highly dependent on protein adsorption, surface chemistry, and topography of constructs. Only HOBs seeded on GO LbL substrate exhibited cell adhesion, proliferation, and mineralisation. Consequently, GO was selected to incorporate into Laponite-based gels. Injectable Laponite gels containing GO and poly(vinylphosphonic acid-co-acrylic acid) (PVPA-co-AA) were fabricated. The addition of PVPA-co-AA increased mechanical properties of Laponite gels whilst GO did not, likely due to a low concentration. Osteogenic mineralisation of Saos-2 within Laponite-based gels were determined to investigate a potential of Laponite-based scaffolds for use in BTE. Positive staining of alizarin red S staining was detected at 3 weeks post-seeding in all samples cultured in non-induction growth medium, suggesting the osteoinductive property of Laponite nanoclay. The addition of GO and PVPA-co-AA did not enhance or accelerate mineralisation of Saos-2 within nanoclay gel scaffolds. All Laponite-based gels maintained structural integrity up to 10 days. In summary, Laponite-based gels offer a potential scaffold to serve as osteogenic microenvironments with cytocompatibility and biodegradability. However, Laponite gels lacked the effective porosity to allow cell migration.

Ventricular arrhythmias (VA) are the leading cause of sudden death in the United States. The only effective treatment for VA is cardiac defibrillation, where a high-energy shock extinguishes the reentrant circuits that initiate and sustain VA. However, these high-energy shocks exceed the pain threshold. The primary goal of this research is to develop new painless strategies to extinguish reentrant VA. The current treatment requires large energy because the current leads capture the tissue from a single point far from the heterogeneous scarred tissue responsible for the electrical disruptions. We hypothesized that flexible electrodes that can access midmyocardium near the scarred area via the cardiac veins, we could terminate arrhythmias with low-energy shocks. However, there were no pacing electrodes small enough to navigate these tributaries to test this hypothesis. The focus of my dissertation was to develop a conductive hydrogel electrode that could be injected across the affected areas of the myocardium, filling the epicardial coronary veins and their mid-myocardial tributaries. When connected to a standard pacing lead, these hydrogels can then act as flexible electrodes that directly capture the previously inaccessible mid-myocardial tissue. Using in vitro and ex vivo models, we developed a hydrogel formulation with adequate viscosity and kinetics to form and retain the hydrogel in the cardiac veins to enable this approach. We tuned the hydrogel composition and combined new macromers chemistries with small molecules to obtain a biostable and biocompatible gel that matches the myocardium modulus. The conductivity is conferred by the ions on the hydrogel matrix and is above the target myocardium values. We demonstrated that the conductivity and network properties were retained after in vivo implantation and successful in vivo deployment demonstrated that the hydrogel electrode reaches cardiac veins and tributaries more deeply than current technologies allow. Feasibility of this approach was then confirmed with successful capture and pacing in a large animal model. This is the first report of an injectable electrode used to successfully pace the midmyocardium and mimic physiologic conduction. In vivo cardiac mapping studies showed fast and uniform capture along the hydrogel before and after myocardium ablation, as well as increased capture compared to traditional point pacing. As such, this injectable hydrogel electrode provides a novel way to improve current defibrillation strategies and opens opportunities for new therapeutic approaches. By capturing a larger area and deeper into the midmyocardium, this technology enhances new ways to study tissue activation that were not possible with current pacing leads.

The growth of tissues and organs is regulated by orchestrated biological signals such as enzymes and growth factors (GFs). The ability to deliver biologics and small molecule drugs in response to these signals holds great promise for tissue repair and regeneration. This dissertation focuses on two aspects, enhancing angiogenesis and preventing infection, as therapeutic and prophylactic means to promoting wound healing. Pro-angiogenic factors regulate the formation of new blood vessels and the renewal of vasculature after wounding, which is crucial for ischemic tissue repair. The mode of GF delivery has become increasingly important, as the narrow therapeutic window and threshold of GF dosage for normal angiogenesis is more and more clear. However the majority of delivery vehicles nowadays rely on hydrolysable scaffolds and thin films of protein-containing polymers for constitutive GF release, which cannot be programmed to respond to biological signals. Here, a delivery platform based on enantiomeric protein nanocapsules is demonstrated, which enables controlled delivery of multiple proteins in response to wound proteases. Exemplified by stroke and diabetic wound healing in mice, sequential delivery of vascular endothelial GF (VEGF) and platelet-derived GF greatly enhances tissue revascularization and vessel maturation. Furthermore, the molecular mechanism for sustained released VEGF in enhancing angiogenesis was studied and revealed differential receptor activation and downstream signaling dynamics.Meanwhile, delivery of hydrophobic drugs to diseased tissue (e.g. antibiotics to an infected wound) is a common therapeutic approach, although little has been done to combine hydrophobic drug release with a scaffold that could provide structural support and additional signals to promote tissue healing. Motivated as such, an injectable amphiphilic synthetic hydrogel is introduced that can effectively solubilize hydrophobic drugs while serving as a scaffold for tissue healing. This self-assembled hydrogel containing solubilized antiobiotics can also be coated as thin films on orthopaedic implants for anti-infectious arthroplasty operations.

We hypothesized that VEGF-containing hydrogels that gelify in situ following injection into a traumatized spinal cord, could stimulate spinal cord regeneration. Injectable hydrogels composed of 0.5% MVG alginate, supplemented or not with fibrinogen, were used. The addition of fibrinogen to alginate had no effect on cell proliferation in vitro but supported neurite growth ex vivo. When injected into a rat spinal cord in a hemisection model, alginate supplemented with fibrinogen was well tolerated. The release of VEGF that was incorporated into the hydrogel was influenced by the VEGF formulation (encapsulated in microspheres or in nanoparticles or in solution (free)). A combination of free VEGF and VEGF-loaded nanoparticles was mixed with alginate:fibrinogen and injected into the lesion of the spinal cord. Four weeks post-injection injection, angiogenesis and neurite growth were increased compared to hydrogel alone. The local delivery of VEGF by injectable alginate:fibrinogen-based hydrogel induced some plasticity in the injured spinal cord involving fiber growth into the lesion site.

We hypothesized that local delivery of GDNF in spinal cord lesion via an injectable alginate hydrogel gelifying in situ would support spinal cord plasticity and functional recovery. The GDNF release from the hydrogel was slowed by GDNF encapsulation in microspheres compared to non-formulated GDNF (free GDNF). When injected in a rat spinal cord hemisection model, more neurofilaments were observed in the lesion when the rats were treated with free GDNF-loaded hydrogels. More growing neurites were detected in the tissues surrounding the lesion when the animals were treated with GDNF microsphere-loaded hydrogels. Intense GFAP (astrocytes), low III tubulin (neural cells) and RECA-1 (endothelial cells) stainings were observed for non-treated lesions while GDNF-treated spinal cords presented less GFAP staining and more endothelial and nerve fiber infiltration in the lesion site. The animals treated with free GDNF-loaded hydrogel presented superior functional recovery compared with the animals treated with the GDNF microsphere-loaded hydrogels and non-treated animals.

Myocardial infarction is caused by blockage of coronary artery. The depletion of nutrient and oxygen supply immediately damages cardiomyocytes, leading to the loss of heart function. The following remodeling results in the formation of fibrous scar tissue at the infarcted site. The stiff and non-contractile scar tissue cannot restore the lost heart function. As a result, left ventricle (LV) dilation was gradually developed which causes thinning of heart wall. It further impairs the heart function, leading to a congestive heart failure.The key to the success therapy for myocardial infarction contains: 1) re-establishing the blood flow to the infarcted area; and 2) regenerating new cardiomyocytes to restore the lost heart functions. Current therapy for MI includes coronary artery by-pass grafting (CABG) or percutaneous coronary intervention (PCI), which focus more on point 1 and cannot have the lost muscle regenerated.Stem cell therapy has been proposed as a promising approach to the future of myocardial infarction therapy. The delivered stem cell is capable to differentiate into new cardiomyocytes to regenerate the damaged heart muscle. However, direct delivery of stem cells into myocardium suffers from low cell differentiation due to the mismatched environment after formation of the scar tissue. The fist half of this dissertation mainly focus on the design of an optimal hydrogel micro-environment to promote stem cell cardiac differentiation. Three families of hydrogels are developed with well controlled matrix moduli and water contents. The differentiation of mesenchymal stem cells (MSCs) and cardiosphere derived cells (CDCs) in those hydrogel matrix was investigated. It was found hydrogel of 40 kPa modulus with 40% water content is optimal for stem cell cardiac differentiation in vitro and in vivo. Furthermore, injection of 40 kPa hydrogel in vivo by a mouse MI model showed it attenuated LV dilation, improved heart function and reduced heart muscle damage after 28 days of injection. Targeting the minimally invasive surgery strategy, a family of dual pH- and thermo-sensitive hydrogels was developed to be used as a promising drug/cell carrier for catheter based PCI delivery therapy.In the real MI situation, stem cells also suffers from low oxygen and nutrient ischemic local environment and high concentration of cytotoxic substances, which are demonstrated to be main issues tor low long-term engraftment when directly stem cell injection. The second half of this work is to develop hydrogel based stem cell delivery system which can protect cells from initial cytotoxic environment, support cell survival and promote stem cell cardiac differentiation under low nutrient and low oxygen conditions. Anti-oxidant enzyme superoxide dismutase is introduced to protect cells from cytotoxic reactive oxygen species attack. Growth factors like bFGF and IGF1 are loaded or conjugated to hydrogel matrix to support the delivered stem cell survival and differentiation under the low oxygen, low nutrient environment. Furthermore, to prolong the survival and differentiation of stem cells under low oxygen ischemic condition, an oxygen releasing hydrogel system was introduced.