Injectable Chitosan hydrogels with high Mechanical Properties for IVD Regeneration

Adoungotchodo, Atma-Luseck G* (École de technologie supérieure(ÉTS); LBeV, Centre de recherche du CHUM (CRCHUM) , Montréal, Canada)
Alinejad, Yasaman* (École de technologie supérieure(ÉTS); LBeV, Centre de recherche du CHUM (CRCHUM) , Montréal, Canada)
Grant, Michael P (Lady Davis Institute for Medical Research, SMBD-Jewish General Hospital, McGill University)
Epure, Laura M (Lady Davis Institute for Medical Research, SMBD-Jewish General Hospital, McGill University)
Mwale, Fackson** (Lady Davis Institute for Medical Research, SMBD-Jewish General Hospital, McGill University)
Lerouge, Sophie** (École de technologie supérieure(ÉTS); LBeV, Centre de recherche du CHUM (CRCHUM) , Montréal, Canada)
*co-first authors, **co-corresponding authors ()


Low back pain is one of the most prevalent debilitating diseases that affects approximately 1 in 50 Canadians. Although its etiology is often unclear, it is believed that intervertebral disc (IVD) degeneration, typified by alterations in composition and structure of the extra cellular matrix (ECM), plays a major role. Current treatments (surgical disc excision and vertebral fusion) offer short-term pain relief in many instances. But they are quite invasive and alter spinal mechanics leading to subsequent adjacent-level disc degeneration and elevated early failure rates (20-40% at 5 years). Minimally invasive approaches are therefore needed. Chitosan (CH) thermosensitive hydrogels are particularly attractive as injectable scaffolds for IVD repair since CH cationic nature makes it tissue-adhesive and facilitates the entrapment of the highly anionic glycoaminoglycans (GAGs). Yet mechanical properties and cytocompatibility of such hydrogels are poor. Our team has developed new CH thermosensitive hydrogels which present greatly enhanced mechanical properties and cytocompatibility and thus offer a possible solution to these challenges(1). These gels are in liquid state at physiological pH at room temperature and can therefore easily be mixed with cell suspension or bioactive agents and be injected through small needles, which is necessary to avoid disruption of the annulus fibrosus (AF). They gel around 37°C and rapidly form a strong cohesive structure after in vivo injection. They have a physiological pH and isoosmolarity and lead to excellent survival of encapsulated cells(1). We hypothesized that these newly developed CH hydrogels can be used for IVD regeneration by providing an injectable and cytocompatible environment with enough mechanical resistance to deliver cells and bioactive materials to the IVD. The specific aims of the present work were to optimize gel composition for IVD repair and establish the proof of concept using nucleus pulposus (NP) cells.

Materials and Methods

CH hydrogels were prepared by mixing an acidic chitosan solution (2% w/v) with different formulations of gelling agent (GA) (either BGP0.4M, SHC0.075M-BGP0.1M, SHC0.075M-PB0.02M or SHC0.075M-PB0.04M). All give a solution at room temperature to which the cell suspension can be added and gelation occurs at 37°C. The gelation speed was assessed by following rheological properties within 1h at 37°C (strain 5% and 1Hz). Mechanical properties were characterized and compared with human NP tissues. Elastic properties of the hydrogels were studied by evaluating the secant modulus in unconfined compression. Equilibrium modulus was also measured, using an incremental stress-relaxation test 24h after gelation in unconfined compression (5% strain at 5%/s followed by 5min relaxation, five steps). Complex shear modulus was evaluated by Anton Paar rheometer (parallel plate, strain 1%, 1Hz) after 72h gelation at 37°C. The pH and osmolality of the hydrogels were measured after 24h of gelation at 37°C, as a first assessment of gel cytocompatibility. Bovine NP cells were then entrapped in hydrogels (up to 14 days) and their viability was evaluated using LIVE/DEAD assay followed by confocal imaging, while cell metabolic activity was estimated by AlamarBlue assay. The sulfated glycosaminoglycan content was monitored using the 1,9-dimethylmethylene blue (DMMB) assay. Finally, the injectability of the hydrogels was tested by injecting the toluidine blue stained hydrogels with a 25G needle in explanted human discs.


Hydrogels synthesized using BGP0.4 were hypertonic (>800 mOsmol/L) while others showed pH and osmolarity values close to physiological range (300-430 mOsmol/L, pH~7). 85% cell survival was observed after 14 days in SHC0.075PB0.02 hydrogels (Figure 1 A and B). The cells entrapped in SHC0.075PB0.02 hydrogels also showed the highest metabolic activity after 14 days (Figure 1C). Interestingly, this hydrogel also exhibited the highest GAG retention (2-10 folds more than the other formulations tested) (Figure 1D). Unconfined compression confirmed drastically enhanced mechanical properties compared to conventional CH-BGP hydrogels (secant Young modulus of 105 kPa for SHC0.075PB0.02 versus 3-6 kPa for BGP0.04). More importantly, SHC0.075PB0.02 and SHC0.075BGP0.1 hydrogels exhibited mechanical properties very similar to NP tissue. For instance, equilibrium modulus was 5.20.6 KPa for SHC0.075PB0.02 and 8.0±0.8 KPa for SHC0.075BGP0.1 compared to 5.3±2.6 KPa for human NP tissue(2). Complex shear modulus of both hydrogels was around 12 KPa, comparable to that of human NP (7-21KPa)(3). Rheological properties and gelation time (G’ = G’’   after less than 15 s at 37°C, and rapid increase of G’ )  of these hydrogels also appear to be adapted to this application. Injectability through a 25G syringe, filling of nuclear clefts and good retention in human degenerated discs was demonstrated for SHC0.075PB0.02 hydrogel (Figure 2).

Discussion and Conclusion

SHC0.075PB0.02 appears to be a particularly promising injectable scaffold for IVD repair by providing suitable structural environment for cell survival, ECM production and retention and mechanical properties very similar to that of NP tissue. In the next steps, the benefit of adding a bioactive factor with growth factor-like properties to the hydrogels will be evaluated.


Funding by the Canada Research Chair program and Canadian Institutes of Health Research (CIHR); YA also acknowledges FRQS scholarship; AA acknowledges CRSNG and FQRNT scholarship.


1. Ceccaldi C, Assaad E, Hui E, Buccionyte M, Adoungotchodo A, Lerouge S. Optimization of Injectable Thermosensitive Scaffolds with Enhanced Mechanical Properties for Cell Therapy. Macromolecular Bioscience. 2017:1600435-n/a.

2. Cloyd JM, Malhotra NR, Weng L, Chen W, Mauck RL, Elliott DM. Material properties in unconfined compression of human nucleus pulposus, injectable hyaluronic acid-based hydrogels and tissue engineering scaffolds. Eur Spine J. 2007;16(11):1892-8.

3. Iatridis JC, Weidenbaum M, Setton LA, Mow VC. Is the Nucleus Pulposus a Solid or a Fluid? Mechanical Behaviors of the Nucleus Pulposus of the Human Intervertebral Disc. Spine. 1996;21(10):1174-84.

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