The Development Of Novel Hybridized Hyaluronic Acid Biomaterials For Applications In Tissue Engineering And Controlled Drug Delivery

This thesis is focused on the development of novel hybridized biomaterials for applications in tissue engineering and controlled drug delivery. Two types of materials used in my research are biopolymers and nanomaterials. Hyaluronic acid (HA), a natural polymer commonly found in extracellular matrix (ECM), was the primary polymer used in my research due to biocompatibility, biodegradability and the ease of manipulation to offer a wide range of physical and mechanical properties. Meanwhile, the nanomaterials, including Tobacco mosaic virus (TMV), gold nanorods and graphene, provide attractive properties, such as a nanoscale topography, biochemistry, high surface area, and special chemical and electrical properties. These properties could promote cell response, and allow the nanomaterials to react with a myriad of biological small molecules. Hence, in this thesis, we aimed to integrate these two types of materials to create a new type of biomaterials, and provide ideal properties that can leverage the tissue regeneration and targeted drug delivery for various treatment applications. Previously, our research group has found the intriguing effect of TMV on bone differentiation of mesenchymal stem cells (MSCs). We demonstrated that TMV could promote MSC osteogenesis via upregulating bone morphogenetic protein-2 (BMP-2) gene expression, a common gene used to enhance cartilage differentiation. This discovery combined with the clinical demand for cartilage tissue repair, which is limited by the minimum self-healing capacity of cartilage, inspired us to design a hybrid TMV scaffold in a simple injectable form, using thiol-ene “click” chemistry, to promote the MSC differentiation to cartilage. We demonstrated that cysteine-inserted TMV mutants (TMV1cys), containing thiol functional groups, could successfully crosslink to methacrylated hyaluronic acid (MeHA) polymers by thiol–ene “click” chemistry and form hydrogels under physiological conditions. The resulting hydrogels promoted in vitro chondrogenesis of MSCs by upregulating BMP-2 and enhancing collagen accumulation. In addition, incorporation of RGD-inserted TMV mutants (TMV-RGD1) in the HA hydrogels further promoted the in vitro chondrogenesis of BMSCs. Meanwhile, incorporation of gold nanorods, which provide similar size and shape as TMV, HA hydrogels showed no impact on the in vitro chondrogenesis. These results implied that the influences of nanoscale topography and biochemistry provided by TMV and TMV-RGD play critical roles in directing encapsulated MSC chondrogenesis. To better mediate new cartilage tissue formation, the physical and mechanical properties of the HA hydrogels were further optimized by varying the structures of thiol-tailored crosslinker molecules using dithiothreitol (DTT), 4-arm polyethylene glycol (PEG), and a multi-arm polyamidoamine (PAMAM) dendrimer. Chondrogenesis and osteogenesis of MSCs were highly enhanced in 4-arm PEG-crosslinked HA hydrogels, as measured by chondrogenic markers, glycosaminoglycan (GAG) and collagen accumulation, and osteogenic markers, alkaline phosphatase activity, and calcium deposition. It implied that the differentiation performance of MSCs directly correlated to the mechanical stiffness, permeability, pore size, porosity and chemistry of crosslinkers. The 4-arm PEG-crosslinked HA hydrogels seemingly mimicked the architecture of real cartilage and bone closer than other hydrogels. Aside from the application in tissue engineering, we developed a graphene oxide (GO)-hybridized HA-based hydrogel for perivascular drug delivery. The nanoscale GO was used as a novel nanocarrier for controlled drug delivery, owing to its high loading capacity of drugs resulting from the aromatic structure. HA serves as a biodegradable macroscale polymeric scaffold, making the prepared GO nanocarriers localized and stable in different microenvironments. The nanocarrier was firstly synthesized by attaching Senexin A (SNX), a kinase inhibitor and a possible anti-tumor drug, to GO via strong π–π interaction, followed by the in situ encapsulation of GO-SNX with HA-based hydrogel. The results of in vitro testing indicate high loading of SNX onto GO, and subsequent slow release of SNX within the therapeutic window. The slow release of SNX closed correlates to the loading-ratio of GO to SNX. With the in vitro results, we have demonstrated that the SNX loaded-GO hybridized HA hydrogel could be successfully attached to the decellularized scaffolds and form hydrogels under physiological condition. The hybridized materials provided a good biocompatibility and no impact on the proliferation and migration of vessel smooth muscle cells (VSMCs). More importantly, it could inhibit the dedifferentiation of VSMCs in the same manner as the SNX treatment

Injectable pH and Thermo-Responsive Hydrogel Scaffold with Enhanced Osteogenic Differentiation of Preosteoblasts for Bone Regeneration

Bone fractures are common in the geriatric population and pose a great economic burden worldwide. While traditional methods for repairing bone defects have primarily been autografts, there are several drawbacks limiting its use. Bone graft substitutes have been used as alternative strategies to improve bone healing. However, there remain several impediments to achieving the desired healing outcomes. Injectable hydrogels have become attractive scaffold materials for bone regeneration, given their high performance in filling irregularly sized bone defects and their ability to encapsulate cells and bioactive molecules and mimic the native ECM of bone. We investigated the use of an injectable chitosan-based hydrogel scaffold to promote the differentiation of preosteoblasts in vitro. The hydrogels were characterized by evaluating cell homogeneity, cell viability, rheological and mechanical properties, and differentiation ability of preosteoblasts in hydrogel scaffolds. Cell-laden hydrogel scaffolds exhibited shear thinning behavior and the ability to maintain shape fidelity after injection. The CNC-CS hydrogels exhibited higher mechanical strength and significantly upregulated the osteogenic activity and differentiation of preosteoblasts, as shown by ALP activity assays and histological analysis of hydrogel scaffolds. These results suggest that this injectable hydrogel is suitable for cell survival, can promote osteogenic differentiation of preosteoblasts, and structurally support new bone growth

Effects of Drug Physicochemical Properties on In-Situ Forming Implant Polymer Degradation and Drug Release Kinetics

In-situ forming implants (ISFIs) represent a simple, tunable, and biodegradable polymer-based platform for long-acting drug delivery. However, drugs with different physicochemical properties and physical states in the polymer-solvent system exhibit different drug release kinetics. Although a few limited studies have been performed attempting to elucidate these effects, a large, systematic study has not been performed until now. The purpose of this study was to characterize the in vitro drug release of 12 different small molecule drugs with differing logP and pKa values from ISFIs. Drug release was compared with polymer degradation as measured by lactic acid (LA) release and change in poly(DL-lactide-co-glycolide) (PLGA) molecular weight (MW) measured by size exclusion chromatography with multi-angle laser light scattering (SEC-MALS). Drug physical state and morphology were also measured using differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). Together, these results demonstrated that hydrophilic drugs have higher burst release at 24 h (22.8–68.4%) and complete drug release within 60 days, while hydrophobic drugs have lower burst release at 24 h (1.8–18.9%) and can sustain drug release over 60–285 days. Overall, drug logP and drug physical state in the polymer–solvent system are the most important factors when predicting the drug release rate in an ISFI for small-molecule drugs. Hydrophilic drugs exhibit high initial burst and less sustained release due to their miscibility with the aqueous phase, while hydrophobic drugs have lower initial burst and more sustained release due to their affinity for the hydrophobic PLGA. Additionally, while hydrophilic drugs seem to accelerate the degradation of PLGA, hydrophobic drugs on the other hand seem to slow down the PLGA degradation process compared with placebo ISFIs. Furthermore, drugs that were in a crystalline state within the ISFI drugs exhibited more sustained release compared with amorphous drugs

Nanotechnology in peripheral nerve repair and reconstruction

The recent progress in biomaterials science and development of tubular conduits (TCs) still fails in solving the current challenges in the treatment of peripheral nerve injuries (PNIs), in particular when disease-related and long-gap defects need to be addressed. Nanotechnology-based therapies that seemed unreachable in the past are now being considered for the repair and reconstruction of PNIs, having the power to deliver bioactive molecules in a controlled manner, to tune cellular behavior, and ultimately guide tissue regeneration in an effective manner. It also offers opportunities in the imaging field, with a degree of precision never achieved before, which is useful for diagnosis, surgery and in the patientâ s follow-up. Nanotechnology approaches applied in PNI regeneration and theranostics, emphasizing the ones that are moving from the lab bench to the clinics, are herein overviewed.The authors acknowledge the Portuguese Foundation for Science and Technology (FCT) for the financial support provided to Joaquim M. Oliveira (IF/01285/2015) and Joana Silva-Correia (IF/00115/2015) under the program “Investigador FCT”.info:eu-repo/semantics/publishedVersio

Injectable pH Thermo-Responsive Hydrogel Scaffold for Tumoricidal Neural Stem Cell Therapy for Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults and despite recent advances in treatment modalities, GBM remains incurable. Injectable hydrogel scaffolds are a versatile delivery system that can improve delivery of drug and cell therapeutics for GBM. In this report, we investigated an injectable nanocellulose/chitosan-based hydrogel scaffold for neural stem cell encapsulation and delivery. Hydrogels were prepared using thermogelling beta-glycerophosphate (BGP) and hydroxyethyl cellulose (HEC), chitosan (CS), and cellulose nanocrystals (CNCs). We evaluated the impact of neural stem cells on hydrogel gelation kinetics, microstructures, and degradation. Furthermore, we investigated the biomaterial effects on cell viability and functionality. We demonstrated that the incorporation of cells at densities of 1, 5 and 10 million does not significantly impact rheological and physical properties CS scaffolds. However, addition of CNCs significantly prolonged hydrogel degradation when cells were seeded at 5 and 10 million per 1 mL hydrogel. In vitro cell studies demonstrated high cell viability, release of TRAIL at therapeutic concentrations, and effective tumor cell killing within 72 h. The ability of these hydrogel scaffolds to support stem cell encapsulation and viability and maintain stem cell functionality makes them an attractive cell delivery system for local treatment of post-surgical cancers

Injectable pH and Thermo-Responsive Hydrogel Scaffold with Enhanced Osteogenic Differentiation of Preosteoblasts for Bone Regeneration

Bone fractures are common in the geriatric population and pose a great economic burden worldwide. While traditional methods for repairing bone defects have primarily been autografts, there are several drawbacks limiting its use. Bone graft substitutes have been used as alternative strategies to improve bone healing. However, there remain several impediments to achieving the desired healing outcomes. Injectable hydrogels have become attractive scaffold materials for bone regeneration, given their high performance in filling irregularly sized bone defects and their ability to encapsulate cells and bioactive molecules and mimic the native ECM of bone. We investigated the use of an injectable chitosan-based hydrogel scaffold to promote the differentiation of preosteoblasts in vitro. The hydrogels were characterized by evaluating cell homogeneity, cell viability, rheological and mechanical properties, and differentiation ability of preosteoblasts in hydrogel scaffolds. Cell-laden hydrogel scaffolds exhibited shear thinning behavior and the ability to maintain shape fidelity after injection. The CNC-CS hydrogels exhibited higher mechanical strength and significantly upregulated the osteogenic activity and differentiation of preosteoblasts, as shown by ALP activity assays and histological analysis of hydrogel scaffolds. These results suggest that this injectable hydrogel is suitable for cell survival, can promote osteogenic differentiation of preosteoblasts, and structurally support new bone growth

Effects of Drug Physicochemical Properties on In-Situ Forming Implant Polymer Degradation and Drug Release Kinetics

In-situ forming implants (ISFIs) represent a simple, tunable, and biodegradable polymer-based platform for long-acting drug delivery. However, drugs with different physicochemical properties and physical states in the polymer-solvent system exhibit different drug release kinetics. Although a few limited studies have been performed attempting to elucidate these effects, a large, systematic study has not been performed until now. The purpose of this study was to characterize the in vitro drug release of 12 different small molecule drugs with differing logP and pKa values from ISFIs. Drug release was compared with polymer degradation as measured by lactic acid (LA) release and change in poly(DL-lactide-co-glycolide) (PLGA) molecular weight (MW) measured by size exclusion chromatography with multi-angle laser light scattering (SEC-MALS). Drug physical state and morphology were also measured using differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). Together, these results demonstrated that hydrophilic drugs have higher burst release at 24 h (22.8–68.4%) and complete drug release within 60 days, while hydrophobic drugs have lower burst release at 24 h (1.8–18.9%) and can sustain drug release over 60–285 days. Overall, drug logP and drug physical state in the polymer–solvent system are the most important factors when predicting the drug release rate in an ISFI for small-molecule drugs. Hydrophilic drugs exhibit high initial burst and less sustained release due to their miscibility with the aqueous phase, while hydrophobic drugs have lower initial burst and more sustained release due to their affinity for the hydrophobic PLGA. Additionally, while hydrophilic drugs seem to accelerate the degradation of PLGA, hydrophobic drugs on the other hand seem to slow down the PLGA degradation process compared with placebo ISFIs. Furthermore, drugs that were in a crystalline state within the ISFI drugs exhibited more sustained release compared with amorphous drugs