Characterizing Nonuniform Hydrogel Elastic Moduli Using Autofluorescence

Abstract

Hydrogel biomaterials show promise as implantable cell delivery vehicles that enhance tissue regeneration and the natural healing process. The design of these materials requires that they mimic the natural environment to retain native cell function. Biological tissues often have spatially varying stiffness, allowing them to have a variety of functions within the body. However, this makes them challenging to mimic mechanically with a synthetic scaffold. To enable these complex designs, characterization techniques that measure nonuniform mechanical properties are required, but these methods are limited. Bulk rheological measurements average the stiffness of the sample, microrheological methods cannot characterize high moduli materials (despite being able to resolve spatial variability), and atomic force microscopy measurements can be a function of the selected tip geometry and measurement procedure. We present a new method for determining the stiffness of nonuniform hydrogels. Our technique measures the hydrogel’s autofluorescent brightness, which is related to its degree of cross-linking, and relates this brightness to elastic modulus. We use a well-established 3D cell encapsulation platform. This photopolymerized polymer–peptide hydrogel is composed of poly­(ethylene glycol)–norbornene and a matrix metalloproteinase (MMP)-degradable peptide. We first develop a relationship between hydrogel elastic modulus and brightness, which are systematically varied by controlling UV light exposure during photopolymerization. We then relate elastic modulus and autofluorescent brightness at each exposure time. This relationship enables images of hydrogels that measure brightness to be converted into stiffnesses. To demonstrate the technique, we fabricate hydrogels with nonuniform stiffness profiles: (1) step changes and (2) smooth gradients in elastic moduli. These are made by controlling UV light exposure spatially with a photomask. We then characterize these gels with the new technique. This work provides an alternative characterization method for hydrogels with spatially nonuniform stiffnesses. To effectively design materials for cell encapsulation, they must be characterized so that their properties are finely tuned to match native tissue. This will improve the effectiveness of these scaffolds as cell delivery vehicles and in promoting tissue regeneration.

Measuring human mesenchymal stem cell remodeling in hydrogels with a step-change in elastic modulus

“…After noting the location of the change in UV light exposure, the sample chamber is placed under a light emitting diode (LED) UV lamp (M365LP1-C1, Thor Labs Inc.) which is collimated to a 10 mm diameter beam using a custom-made 3D printed collimator attachment. 36 This beam size matches the size of the PDMS ring in the sample chamber. Prior to exposing the hydrogel precursor, UV light intensity is set to 1.5 mW cm −2 using a UV radiometer and LED driver (LEDD1B, Thor Labs Inc.).…”

“…9 The interface is a 500 μm wide section where the elastic modulus, G ′, changes rapidly (≈1000 Pa mm −1 ) between the two halves of different stiffness. 36 Since the stiffness profile of our hydrogel consists of two relatively uniform halves ( G ′ ≈ 900 and 300 Pa) joined by a region of rapid increase in G ′, cells far from the interface are unaffected by the change in stiffness and behave similar to cells encapsulated in uniform hydrogels. Due to this, we characterize cell-mediated remodeling and degradation around cells that are in the soft or stiff region far from the interface and cells that are in the interface.…”

“…Details of this set-up are provided in our previously published work. 36 To identify the exact location of the interface, which is the location where light starts to be attenuated by the photomask, we use an inverted microscope (Zeiss Observer Z1, Carl Zeiss AG) and a 10Â objective (EC Plan-Neofluar, N.A. 0.3).…”

“…Our previous work verified that this set of UV exposure times for this hydrogel composition produces one half which is E900 Pa and the other half is E300 Pa, with a rapid change in elastic modulus of 600 Pa over an interface that is approximately 500 mm thick. 36 Representative data of the stiffness profile from 3 separate gels formed using this procedure is shown in the ESI † in Fig. S1.…”

“…8,10,12,15,34,35 We recently showed that this hydrogel can be fabricated with a step-change in mechanical stiffness by varying UV light exposure. 36 This work focuses on how individual cells remodel and degrade material in response to rapid changes in their microenvironment. While previous work has characterized motility and morphology of hMSCs in non-uniform hydrogels, most of these studies are done in 2-dimensions with cells seeded on top of hydrogels.…”