Tuning the Physical Properties of Collagen/Hyaluronan Hydrogels to favor Mesenchymal Stem Cells Differentiation into NP Cells: A Step forwards Intervertebral Disc Regeneration


Helary, Christophe (Sorbonne Universite - CNRS)


"Back pain is often associated with intervertebral disc (IVD) degeneration. Beside surgery, novel treatments relying on stem cell injection have been tested. Unfortunately, the outcomes are disappointing because of cell leakage and incomplete differentiation. Nowadays, a consensus exists on the necessity to encapsulate stem cells within a hydrogel to maintain them in situ and favor their differentiation. As cell behavior depends on biochemical and physical environment, a biomimetic hydrogel would promote IVD regeneration. Nucleus Pulposus is a highly hydrated tissue working as hydraulic shock absorber. Glycosaminoglycans give a high degree of hydration whereas collagen II gives resistance and allows for cell adhesion. With the aim of developing novel biomimetic hydrogels, collagen/hyaluronic acid composites were developed to mimic the structure and the mechanical properties of Nucleus Pulposus. For this purpose, we first studied the impact of the HA content on physical properties. Then, the potential of the different formulations to differentiate mesenchymal stem cells (MSCs) into NP cells was analyzed in detail.

HA functionalized with tyramine groups (HA-Tyr) was mixed with collagen and gelled using Horse Radish Peroxidase and H2O2 at pH 7.4. With a constant collagen concentration (0.4%), the HA-Tyr content was increased up to 2 % to create a platform of Col/HA hydrogels with different properties. The hydrogel structure, the mechanical properties and the degree of hydration were analyzed. Mesenchymal stem cells were encapsulated within the different hydrogel types and cultivated over 28 days. The impact of MSCs on hydrogel stability, metabolic activity and cell morphology were analyzed. Last, the gene expression of Aggrecan, Collagen I and II was quantified by real time PCR.

The physico-chemical study showed the impact of the HA-Tyr content on the hydrogel physical properties. At low HA-Tyr content (less than 0.4 %), the composite behavior was driven by collagen. Hydrogels exhibited a fibrillary network and were characterized by low mechanical properties. From 0.8% HA-Tyr, the mechanical properties and the hydration degree increased to reach those of NP (5kPa) when 2% HA-Tyr was added. Below 0.4% HA-Tyr, encapsulated cells contracted hydrogels after one week in culture. From 0.8%, hydrogels, MSCs did not contract hydrogels and their mechanical properties were stable over the time course of the experiment. With a high HA-Tyr content, cells did not proliferate, suggesting their commitment towards differentiation. At low content, MSCs spread and adopt a fibroblast like morphology. On the opposite, cells encapsulated within hydrogels at high HA-Tyr content were more rounded and resemble NP cells. The gene expression quantification showed that MSCs orientated towards a NP cell phenotype. When 2% HA-Tyr was used, cells highly expressed NP cells markers, i.e Aggrecan and Collagen II, and weakly expressed Collagen I. In contrast, cells encapsulated in hydrogels with a low HA-Tyr content weakly expressed these NP cell markers.

Taken together, these results show that Collagen/Hyaluronic Acid Composite Hydrogels with a high HA content (2%) mimic the physical properties of the Nucleus Pulposus and promote the differentiation of MSCs into NP cells. Hence, these hydrogels could be useful for IVD regeneration"

Presentation materials

There are no materials yet.