FLASH: Fluorescently LAbelled Sensitive Hydrogel to monitor bioscaffolds degradation during neocartilage generation
Graphical abstract
Introduction
Articular cartilage injuries pose a significant clinical challenge in orthopaedics [1]. Emerging tissue engineering approaches aim to regenerate cartilage tissue using a combination of scaffolds made of biomaterials, along with autologous or allogeneic cells [2,3].
Hydrogels in particular provide a biocompatible, biodegradable and highly hydrated 3D structure, analogous to cartilaginous extracellular matrix (ECM) [4]. A central tenet of this strategy is that the implant material is gradually removed (either through chemical or cell-mediated degradation) and replaced by new ECM deposited by the cells [5]. Efforts to improve the design of such hydrogel bioscaffolds have been accompanied by the persistent challenge of characterising this complex degradation process; namely, to simultaneously identify the breakdown of hydrogels by cellular and acellular processes, while monitoring the deposition of distinct new matrix [6,7].
The use of injectable hydrogels supports a favoured clinical paradigm where the hydrogel degradation and repair occurs in situ, within the defect site [8,9]. Photocrosslinkable hydrogels are well suited to this strategy owing to the control over the crosslinking reaction and kinetics, which has been shown to be suited to an in situ approach [[10], [11], [12]]. Such materials allow for tuning of physical and chemical properties such as porosity, stiffness and degradation [13,14]. However, the interrelatedness of all of these properties presents a challenge towards scaffold optimisation, especially under the influence of cells in a long-term in vitro culture or in vivo environment. On the one hand, cells entrapped in dense, non-degradable gels produce minimal extracellular matrix, which is confined to the space surrounding the cellular membrane, thus impairing a physiological development of new tissue. In degradable gels, on the other hand, the network density decreases with time while the mesh size increases, allowing for further matrix deposition and organization [15,16]. Studies have shown that secretion of specific matrix metalloproteinases (MMPs) by stem cells correlates with their lineage commitment, an example being MMP13 for chondrogenesis [17]. Furthermore, it has been shown that the stiffness of enzymatically degradable Hyaluronic Acid hydrogels increases when its degradation is paired with ECM deposition from stem cells encapsulated within the hydrogel [7]. Recently, Lee et al. demonstrated how the cross-linking density, that regulates both degradation and stiffness, plays a decisive role in directing articular or hypertrophic human Mesenchymal Stem Cells chondrogenesis in polyethylene glycol acrylates (PEG) and oxidized methacrylate alginate (OMA) hydrogels [18].
In all cases, considering the complexity of the biophysical dynamics, characterising the physical behaviour of a material under biological contribution is critical for the clinical translation of cartilage regeneration strategies based on injection of stem-cell laden hydrogels. However, the conventional methods to evaluate the correlation between stem cell activity and hydrogel remodelling are invasive and do not allow for easy manipulation of the samples. Techniques used to quantify biomaterial degradation in vitro are generally destructive and based on mass loss assessment [19,20]. Those techniques are time consuming and do not allow a simultaneous, standardized, and efficient analysis of the same samples, thus making challenging the design of high throughput systems to efficiently screen cartilage regeneration strategies.
Here we propose the application of a Fluorescently LAbelled Sensitive Hydrogel (FLASH) that can be used as sensor to monitor the degradation of a bioscaffold during the neocartilage generation. Gelatine Methacryloyl (GelMA) was used as a model of naturally derived and photo-crosslinkable injectable hydrogel widely used in cartilage regeneration [21,22]. GelMA was covalently bound to a FITC fluorophore and used to label the hydrogel that we then defined as FLASH. As a model to validate the tool, we used different concentrations of FLASH GelMA to generate bioscaffolds in the presence or absence of human adipose-derived stem cells (hADSCs).
First, we validated the FLASH sensitivity by correlating the fluorescence loss to the weight loss of the FLASH scaffold, using a controlled degradation test incorporating collagenase. Then, we evaluated the ability of FLASH to monitor the degradation rates of FLASH bioscaffolds bearing different network density undergoing chondrogenesis in vitro. Chondrogenesis was assessed by evaluating the expression of chondrogenic markers, the accumulation of extracellular matrix and hyaline like cartilage, changes in mechanical properties, and second harmonic generation microscopy imaging. The capability of FLASH to represent the regenerative output was further validated using an ex vivo (human) and in vivo (animal) critical chondral defect model. Finally, we explored the capability of FLASH to act as a proxy measurement for cartilage regeneration through correlative analysis with the other biophysical features evaluated in the study.
Section snippets
Materials
Gelatin-methacryloyl (GelMA) was synthesized and provided by TRICEP (Wollongong, NWS, Australia). The same GelMA was conjugated to the amine group with the isothiocyanate group of the fluorescein isothiocyanate isomer I (FITC) molecule, resulting in a thiourea derivative, which is stable under physiological pH condition. To label GelMA, 0.4 g GelMA was dissolved in 10 mL phosphate buffered saline (PBS). 4 mg of FITC (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.4 mL dimethyl sulfoxide
FLASH allows a contactless monitoring of scaffold degradation rate under enzymatic digestion
Our primary hypothesis is that FLASH can be used as a sensor to monitor the degradation of a bioscaffold during neocartilage generation. To generate FLASH, GelMA was covalently bound to a FITC fluorophore and used to label the hydrogel. FITC was conjugated between the amine group of GelMa and the isothiocyanate group of the fluorescent molecule, resulting in a thiourea derivative, which is stable under standard cell culturing condition. After the labelling procedure, the unbound FITC was
Discussion
Tissue engineering raises hope in the field of orthopaedics, particularly with respect to restoring articular cartilage function [37]. Tissue engineered products have the potential to replace deficient or injured tissue that results from trauma, infection, or chronic diseases [38]. Although there is significant research interest in tissue engineering and critical advances have been made in the last decade, very few have had success in the clinical marketplace.
This lack of translation can be in
Conclusions
FLASH is a sensitive tool to monitor photocrosslinkable hydrogels in tissue engineered constructs. Regardless of the experimental models used, the extracellular matrix production within a hydrogel correlates with the fluorescence loss profiles, which in turn depicts the degradation rate of the bioscaffold. The usage of FLASH can be implemented with a scalable system for sample maintenance and fluorescence recording to produce an analytical real time monitoring system, suitable for a contactless
Credit author statement
Carmine Onofrillo, Serena Duchi conceptualize, designed, and performed the study, executed the data analyses, wrote the original draft, and prepared the figures. Sam Francis designed and performed the in vivo rabbit experiments, executed the surgeries and the post operation analyses together with Carmine Onofrillo and Serena Duchi, critically revised the manuscript. Cathal D. O'Connell gave intellectual input throughout the project, critically revised the data analyses, and edited the
Ethical statement
St. Vincent Hospital Ethics Committee [HREC/16/SVHM/186] approved use of all human samples and procedures (isolation of hADSCs from human infrapatellar fat pad and osteochondral plug generation) in this study and all the experiments were performed in accordance with relevant guidelines and regulations.
This study was approved by the Animal Ethics Committee [AEC/002/19-r1] and the Experimental Medical and Surgical Unit (EMSU) of St. Vincent's Hospital, Melbourne, Australia. The animal study was
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by (1) Melbourne medical School – Early and Mid-Career Seed Grant Scheme, (2) The Foundation for Surgery Senior Lecturer Fellowship (Royal Australasian College of Surgeons IMIS N 164037), (3) The Australian Research Council Centre of Excellence Scheme (Project Number CE 140100012), (4) Aikenhead Centre for Medical Discovery (ACMD) Research Endowment Fund (90264), (5) AVANT doctor in training research grant and (6) the Victorian Medical Research Acceleration Fund.
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These authors contributed equally to this work.