development of hyaluronic acid derivatives for applications in biomedical engineering
Dalila Petta is a PhD Student in the research group Biomaterials Science and Technology. Her supervisor is professor Dirk Grijpma from the Faculty of Science and Technology.
Hyaluronic acid (HA) is a non-sulphated glycosaminoglycan. Ubiquitous in the human body, this natural polymer is widely used in the biomedical research thanks to its unique chemical, physical and biological properties [1-3]. Over forty years of use in clinics makes it one of the most successfully naturally-derived polymers in the medical field. The versatility of the HA processing and its unique biological interaction with cells, make it an important building block for the development of new biofunctional materials. HA biomedical applications are related to its physicochemical and biological properties. The first biomedical applications of HA have been as aid in eye surgery [4] and as viscosupplement in osteoarthritis [5]. Not surprising, both applications are connected with its viscoelastic properties, which can be modulated with concentration, molecular weight or chemical modification for creating semi-synthetic derivatives giving physical or covalent gels [6, 7].
The HA chemical groups available for modifications are carboxyl, hydroxyl and N-acetyl group. Chemical alterations are numerous and enable the synthesis of a wide range of HA derivatives targeting applications in the field of tissue engineering and regenerative medicine [8-10]. It is possible to create HA derivatives retaining the cyto- and bio-compatibility of the pristine HA, while having modified mechanics, degradation and interactions with biologics such as cells and proteins. Still, HA derivatives synthesis methods that employ more simple and controlled chemistries, with less toxic by-products to ensure biological biocompatibility of the conjugation and further chemical functionalization are required. This is especially true for the delivery of active biological agents and cells through advanced fabrication technologies.
One of the most widespread chemical modification on the HA carboxyl group is the amide formation by carbodiimide chemistry [11]. This is usually performed in presence of 1-ethyl-3-[3-(dimethylamino)-propyl]-carbodiimide (EDC) and N-hydroxylsuccinamide (NHS). This carbodiimide conjugation presents the advantage of being performed in water with no significant cleavage of the HA chains, however the reaction requires high quantities of reagents and it is strongly pH-dependent [10]. An alternative for the activation of the carboxyl groups is the use of the triazine-chemistry. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) has shown to promote the grafting of amino groups on HA showing higher efficiency and better control compared to the carbodiimide-mediated reaction [12, 13].
The simplicity of the DMTMM chemistry prompted the interest in investigating a range of HA derivatives for applications in drug delivery, tissue engineering and biofabrication. In this thesis, I have researched on two physical gels, namely a thermoresponsive HA derivatives and short-alkyl derivatives, and a covalent gel (tyramine-modified HA). Specifically, the thermoresponsive hydrogel, where the HA carboxyl group was functionalized with poly(N-isopropylacrylamide), was combined with beta tricalcium phosphate particles. The non- covalent network of the thermoresponsive HA is exploited to obtain a better cohesion of the formulation together with a modulation of drugs delivery for bone tissue engineering. A second physical gel network was obtained grafting short-alkyl moieties to the HA. Here, a low degree of modification with small molecules such as propylamine and butylamine was able to drastically change the viscoelastic properties of HA, indicating profound modification of its structure. This same chemistry was employed to develop a new bioink for 3D printing, where the crosslinking density of a covalent tyramine-modified gel was optimized and controlled with a double crosslinking mechanism.
Aim and outline of the thesis
The aim of this thesis was to introduce a series of new HA derivatives for biomedical applications. These derivatives embrace a range of gelation mechanisms and applications as illustrated in the following outline:
In Chapter 2 an introduction to HA and its chemical, physical and biological properties are provided. The chemical modifications on the main functional groups and the possible crosslinking strategies for hydrogel creation are described. Finally, the main applications of HA in the field of musculoskeletal repair and regeneration are reported.
In Chapter 3 DMTMM is employed for grafting propylamine and butylamine to HA. The impact on the HA viscoelastic properties after the conjugation of these short-alkyl moieties is particularly relevant at low degree of substitution and opens a variety of possibilities in applications such as drug delivery and 3D printing. Additionally, a parametric study on the DMTMM reaction conditions shows the reproducibility of this chemistry and the accurate control over a wider range of degrees of substitutions compared to the traditional carbodiimide modification [14].
Chapter 4 describes the preparation of a thermoresponsive poly(N-isopropylacrylamide) HA derivative (HApN) combined with beta-tricalcium phosphate particles (βTCP) as bone substitute and drug delivery system. The asset of this novel composite is the improved cohesion at body temperature that the HApN gives, thus giving the possibility to address bone defects. A range of composite formulations is tested as an injectable or putty formulation: the handling properties and the injectability of the composite are analysed as function of the particle size and the HA molecular weight and concentration. The most promising formulations are tested as drug delivery system for the recombinant human bone morphogenetic protein-2 (rhBMP-2) and dexamethasone (DEX) as models of hydrophilic and small hydrophobic drugs, respectively [15].
In Chapter 5 a simple and versatile HA derivative is introduced as an effective biofunctional ink for extrusion-based 3D printing. HA is modified with tyramine functional groups via DMTMM chemistry. A double-crosslinking mechanism consisting in an enzymatic crosslinking that allows good extrudability followed by a visible-light crosslinking is implemented to ensure the stability of the 3D printed constructs. The ink is still available after printing for a functionalization with cell-adhesive motives for improving the construct-cell interaction.
Chapter 6 explores the possibility to 3D print viable cells encapsulated in a tyramine-modified HA and to obtain 3D constructs directly on cartilage tissue. The viscoelastic properties of the cell-laden ink are investigated targeting low shear stress for high cell viability and good extrudability. The influence of the photoinitiator and visible light-photocrosslinking on the cell viability is assessed on three different cell types and the printing of the cartilage-adhesive bioink on a piece of cartilage tissue is shown.
Chapter 7 focuses on the future perspectives for the HA derivatives in the biomedical field and the need of developing a new generation of derivatives due to the constant expansion of new technologies.
