2.2 FACTORES HUMANOS:
2.4.2 Repaso mayor y tiempo entre repasos mayores (Overhaul y TBO)
Hydrogels, which are defined as hydrated 3D polymer networks (Figure 1.1),1-3 are characterised by their high water content and porosity.4, 5 As a result, they have a wide range of applications, from contact lenses to food additives.6-8 Specifically, hydrogels are most commonly used as soft contact lenses, first introduced by Wichterle and Lím in 1954.9 This application clearly highlights the advantageous properties of hydrogels (e.g. oxygen permeability and mechanical strength) and their relevance to clinical applications.10-12
The formation of a hydrogel occurs when the viscosity of a polyfunctional system dramatically increases, defined as the “gel point” or sol-gel transition point.13 Gelation can occur through many different routes and mechanisms (e.g. chain or step growth polymerisations, intramolecular forces, crosslinking reactions) depending on the precursors and conditions used.14 There are many different theories which can be applied to predict the gelation of a polymeric system. The most popular theory of gelation, through
Figure 1.1. a) Schematic of a hydrated polymer network, adapted from ref. 1, b) The components of a
hydrogel adapted from ref. 3.
Crosslinks Solvent molecules Polymer chains
Polymer Water Hydrogel
3 the crosslinking of polymers via a step-growth polymerisation, is the Flory-Stockmayer theory.15-18 The theory predicts the point of gelation for a system of high functionality (i.e. more than 2 functional groups per molecule), producing a polymer network with infinite molecular weight. The theory builds on the Carothers equation, which is limited to branched stoichiometrically balanced systems, to calculate the probability of finding a crosslinked point during gelation. The Flory-Stockmayer equation can be used to calculate the theoretical critical number of crosslinks needed to form a gel (Equation 1.1). Hence, the gelation point is reached when a critical number of intermolecular linkages has been exceeded. This theory predicts the gel point of a system assuming; 1) all the functional groups are equally reactive, 2) the reaction occurs in an A-B fashion and 3) there are no intramolecular forces.
𝑝𝑐 =
1
√(𝑓𝐴 − 1)(𝑓𝐵− 1)/𝑟
Equation 1.1. Flory-Stockmayer equation to calculate the theoretical critical number of crosslinks needed
to form a gel, 𝑝𝑐, where 𝑓𝐴 is the functionality of polymer A, 𝑓𝐵 is the functionality polymer B, and r is the ratio between the total number of A and B groups.
Hydrogels can swell to thousands of times their dry weight, dictated through the crosslinking structure and charge density of the hydrated polymer network. Their swelling profile is defined by important parameters; 1) polymer volume fraction, the amount of water that can be absorbed and retained in the structure, 2) mesh size, the correlation distance between two adjacent crosslink points and 3) the effective molecular weight of the polymer chain between crosslink points. The Flory-Rehner theory can be used to analyse the structure of hydrogels through the combination of thermodynamic and elasticity theories. For non-ionic hydrogels it states that a crosslinked polymer gel that is immersed in a fluid and allowed to reach equilibrium with its surroundings is subject to
4 two opposing forces: the thermodynamic force of mixing and the retractive force of the crosslinked polymer chains.18, 19 At equilibrium these forces are equal and can be defined in terms of Gibbs free energy (Equation 1.2).
∆𝐺𝑡𝑜𝑡𝑎𝑙 = ∆𝐺𝑒𝑙𝑎𝑠𝑡𝑖𝑐 + ∆𝐺𝑚𝑖𝑥𝑖𝑛𝑔
Equation 1.2. Gibbs free energy equation for the forces on a polymer network immersed in a fluid.
∆𝐺𝑒𝑙𝑎𝑠𝑡𝑖𝑐= the elastic forces of the polymer network, ∆𝐺𝑚𝑖𝑥𝑖𝑛𝑔= the spontaneous mixing of the fluid molecules within the polymer chains, ∆𝐺𝑡𝑜𝑡𝑎𝑙= total Gibbs free energy change of a polymer network immersed in a fluid.
∆𝐺𝑚𝑖𝑥𝑖𝑛𝑔is typically expressed as the polymer-solvent interaction parament (
𝜒
1) and can be used to calculate the molecular weight between the polymer chains (Equation 1.3).201 𝑀̅𝐶
=
2 𝑀̅𝑛−
ῡ 𝑉1(ln(1−𝑣2)+𝑣2+ 𝜒1𝑣2 2 𝑣21/3−𝑣22Equation 1.3. Equation to determine the molecular weight between two adjacent crosslinks of a non-ionic
hydrogel (𝑀̅𝐶). 𝑀̅𝑛 = the number-average molecular weight of the un-crosslinked hydrogel 𝑉1= the molar volume of the solvent (18 cm3 mol-1 for water), 𝑣
2 = the polymer volume fraction in the equilibrium swollen hydrogel, ῡ = the specific volume of the polymer and 𝜒1 = the polymer-solvent interaction parameter.
Once calculate the molecular weight between two adjacent crosslinks can be used to determine the mesh size (§) of the hydrogels (Equation 1.4).21 This parameter is important for many drug delivery applications and the flow of molecules in and out of the hydrogel structure. It also defines a hydrogel as macroporous, microporous or nonporous.
§ =
𝑣
2−1/3(𝑟̅02)1/2Equation 1.4. Equation to calculate the mesh size of a hydrogel. 𝑣2 = the polymer volume fraction in the
equilibrium swollen hydrogel, (𝑟̅02)1/2 = the root-mean-square end-to-end distance of the polymer chain in the unperturbed state calculated from average bond length for the polymer, the characteristic ratio of the polymer (Cn) and the molecular weight of the repeating unit (𝑀𝑟).
The ability to use these parameters to tailor the molecular structure of hydrogels results in mechanical properties with responsive and diffusive properties ideal for many biological and medical applications.22 Hence the popularity of hydrogel materials has
5 become very apparent in recent years, especially in the tissue engineering field. Cells are influenced by their surrounding environment, made up of extracellular matrix (ECM) proteins, soluble bioactive factors and neighbouring cells.23 These components influence how cells grow, divide and differentiate, in response to stimuli and most importantly, how they interact through signalling with the ECM. Hence, hydrogels with highly tuneable properties can simulate in vivo conditions, thus creating an environment that mimics the native ECM.
The importance of accurately mimicking in vivo settings was highlighted by Discher and co workers24-26 through the response stem cells showed to different substrate stiffnesses. On a 2D culture configuration, cell morphology differed depending on the environment the cells were sensing (i.e. soft substrates resulted in fat tissue cells, while stiff substrates resulted in cartilage growth). This observation has redefined the area of tissue engineering for the culture of cells in vitro.27-29 It has created a drive to form hydrogels, which mimic the features of the native ECM, enabling cell culture in vitro but capturing the behaviour and characteristics of cells in vivo.6, 30-37 However to recreate in
vivo environments, hydrogels require; 1) adequate porosity for the flow of nutrients in and
out of the matrix, 2) degradability to allow cell proliferation and communication38, 39 and 3) the correct signalling factors to enable cell growth.3, 31, 40-43 An additional factor to address, is the ability to encapsulate cells during the crosslinking reaction to allow for a good distribution of cells within the hydrogel matrix allowing cells to sense a true representation of an in vivo setting.44 Hence, it is crucial that the crosslinking chemistry is biocompatible and does not influence the viability or growth of encapsulated cells.45-47
6 In turn the chemistry behind hydrogels underpins their final properties and enables them to be highly versatile and synthetically flexible.22, 48-50
Exploitation of this phenomena could enable further understanding into disease progression. For example by modelling the change in stiffness of tissue during the growth of a cancerous tumour or by a myocardial infarction.51 Synthetic hydrogel materials have also been used to capture different cell processes (e.g. differentiation of stem cells or the reorganisation of the microenvironment).52 These applications highlight the importance these scaffolds can have in recreating in vivo settings in vitro with the ability to encapsulate a range of different cell cultures. In this respect, hydrogel synthesis benefits from a range of chemical reactions (e.g. supramolecular interactions, intramolecular forces and click reactions).53-55 As a result, hydrogels can be prepared with tailored specifications that meet the requirements for a range of different biological environments. Additionally, the ability to control the chemistry behind these hydrated polymer networks expands the library of dynamic stimuli-responsive materials.56
This chapter will focus on the choice of crosslinking chemistry used to prepare hydrogels for biomedical applications (i.e. ECM mimics or injectable robust scaffolds). It aims to highlight the use of alkyne functional groups, to synthesise dynamic hydrogels which can encapsulate cells without impacting their viability.
7