Utilization of glycosaminoglycans to improve biological activities of biomaterials

Abstract

Glycosaminoglycans (GAGs) are present abundantly in the extracellular matrix (ECM) of higher organisms. GAGs play an important role on many cell surfaces in connective tissues and ECM. They displayed a variety of biological functions and modulated many biological processes through their interactions with a wide range of proteins in ECM. Therefore, in an attempt to capture these biological functions, GAGs have been incorporated into a range of biomaterials either in 2D or in 3D systems for tissue engineering, protein/drug delivery, and regenerative medicine applications. Herein, in this thesis, GAGs (heparin and hyaluronic acid) are utilized for improving the bioactivities of the biomaterials through surface modification and physical incorporation. A general introduction of GAGs in terms of their structure, their role in the biomedical application as well as the way of incorporation into biomaterials for bioactivities improvement were first reviewed in chapter 1. In chapter 2, heparin possesses specific binding affinities and controlled release capability to bone morphogenetic protein-2 (BMP-2) was coated on the polyetheretherketone (PEEK) to improve its performance in terms of osteogenesis. PEEK is a biocompatible synthetic thermoplastic polymer that has gained increased interest as an alternative material in orthopedic and dental applications recently mainly due to its closely matched elastic modulus with natural bone. However, the bio-inertness of PEEK which results in poor osseointegration has limited its potential applications. Delivery of bone morphogenetic protein-2 (BMP-2) in a controlled manner has emerged as a potential approach for osseointegration improvement through osteogenic differentiation of osteoblast progenitors or stem cells. Therefore, in this study, surface modification of PEEK with heparin was carried out via a combination of ozone and UV treatment. Heparin was successfully grafted on PEEK through the thiol-ol reaction. BMP-2 loading efficiency on PEEK was enhanced and controlled release of it was achieved in the presence of heparin on it compared to pristine PEEK. Surprisingly, bioactivity enhancement was observed on hydrophilic heparin-grafted PEEK itself in terms of proliferation rate as well as osteogenic differentiation of MG 63 compared to pristine PEEK. However, the synergistic effect of BMP-2 on PEEK especially in osteogenic differentiation of MG 63 became significant only on heparin-grafted PEEK. Overall, we demonstrated a relatively safe method where no harsh chemical reagent or organic solvent is involved in the process of heparin grafting onto PEEK. The BMP-2 loaded heparin-grafted PEEK could be served as a potential platform for osseointegration improvement. In chapter 3, hyaluronic acid (HA), the most abundant glycosaminoglycans in native ECM of stem cells was physically incorporated into cellulose nanofiber (CNF) microbeads to improve the bioactivities of human adipose-derived stem cells (hADSCs). Cell microencapsulation is a process to entrap viable and functional cells within a biocompatible and semi-permeable matrix that aims to provide a favorable or closely mimic microenvironment to the cells. Cellulose nanofiber (CNF), a low-cost and resources sustainable cellulose-derived natural polymer has been widely studied as the matrix for 3D stem cell culture in the form of a bulk hydrogel. Therefore, in chapter 3, the formation of CNF microbeads by ion crosslinking for the 3D culture of human adipose-derived stem cells (hADSCs) was first demonstrated followed by HA incorporation. Since HA can affect most of the stem cell activities, such as cell proliferation, signaling, adhesion, and differentiation through the interaction with stem cell surface receptors, hence, it is expected that the bioactivities of encapsulated cells in CNF-HA microbeads could be enhanced in the presence of HA. All the CNF-HA microbeads showed no significant differences from CNF microbeads in terms of rheological property. Moreover, hADSCs encapsulated in HA incorporated CNF microbeads (CNF-HA) showed the enhancement in cell proliferation, endogenous growth factor secretion as well as stemness and differentiation ability preservation compared to CNF microbeads. The results were noticeable at the highest molecular weight (MW) and concentration of HA (700kDa and 0.2%) incorporated CNF microbeads. All of our results demonstrate that the CNF microbeads which are tunable with HA could serve as a promising matrix for hADSCs 3D culture. In chapter 4, heparin possesses anti-inflammatory potential was physically absorbed on tempo-oxidized CNF (CNF) film together with chitosan by layer-by-layer technique to reduce the pro-inflammatory property of bare CNF film. The demand for biomaterials and medical devices has rapidly increased. However, foreign body reaction is still one of the big challenges for successful implantation. The foreign body response is an immune-mediated reaction to implanted materials that leads to the rejection of implanted devices. Therefore, the implanted materials with anti-inflammatory and anti-fibrotic properties are needed to achieve successful implantation. Cellulose nanofiber (CNF) is considered a biocompatible nanomaterial that has been widely studied in various biomedical applications. However, there is still no certainty that CNF-based biomaterials possess either pro-inflammatory or anti-inflammatory. CNF is a relatively low risk for human health, as well as the environment and it is represented as a low-cost and sustainable biomaterial to be widely used in biomedical applications compared to the other types of biopolymer materials. Hence, it is worth reducing the inflammatory properties of CNF-based biomaterials as this could provide more beneficial effects for their various biomedical applications. Herein, a simple and cost-effective layer-by-layer method was utilized to deposit chitosan-heparin (Chi/Hep) multilayers film on CNF film to reduce its pro-inflammatory property. This process could be carried out without any modification of CNF film due to its intrinsic negatively charged property. By heparin deposition, the macrophage's attachment, as well as the pro-inflammatory cytokine secretion were significantly decreased compared to the only CNF and chitosan deposited CNF films. The positive effects of heparin deposition were significant compared to CNF films as the (Chi/Hep) bilayers increased from 1 to 3 layers. Overall, the present study demonstrated the potential of Chi/Hep multilayer films to improve the anti-inflammatory property of CNF film. In summary, this thesis described the use of heparin and hyaluronic acid in improving the bioactivities of the biomaterials where they are incorporated on or in.