Modified Agarose Hydrogel Scaffold for Neural Stem Cell Transplantation in Spinal Cord Injuries
Authors:   , Spicer Maleah , Allen Fred D. , Zhong Yinghui 
Published: October 2023

Spinal cord injury (SCI) is a debilitating neurological condition that results from direct trauma to the tissue and causes a loss of motor, sensory, and autonomic functions caudal to or below the site of injury. Globally, SCI has an incidence rate of 250,000 and 500,000 cases with an annual economic impact of $4 billion USD. In the United States, there are between 11,000 and 20,000 new cases of SCI each year. SCIs result from direct trauma to the tissue and cause a partial or complete loss of motor, sensory, and autonomic functions caudal to or below the site of injury. The most frequent SCI are contusion injuries where the lesion site expands over time due to a wave of secondary degeneration resulting in a cystic cavity surrounded by a thin rim of tissue. The secondary injury leads to further neuronal loss, demyelination, and axon transection caused by mechanisms such as bleeding, swelling, and elevated reactive oxygen species (ROS). Since neurons have limited regenerative capacity, the transected axons cannot regenerate in the hostile microenvironment after a SCI. This results in SCI patients having a lifelong disability that requires continuous care. Neural stem/progenitor cells (NSPCs) transplantation is a promising therapeutic strategy to replace lost neurons and rebuild the damaged neural circuitry in SCI. As a result of the large lesion cavity after SCI, a biomaterial scaffold composed of an agarose hydrogel with laminin incorporated is needed as a supporting matrix to promote attachment and survival of the transplanted NSPCs. Agarose is a biocompatible polysaccharide with thermo-reversible properties commonly used as a biomaterial for neural tissue engineering. However, agarose lacks cell adhesion motifs and cannot encourage neuron growth. The incorporation of a cell binding domain can encourage cell attachment and growth. Laminin is a key extracellular matrix regulator of cell adhesion, differentiation, proliferation, and migration in addition to maintaining blood-brain barrier integrity. To develop agarose hydrogels with the ability to have thiol-functionalized laminin attached, it is modified with divinyl sulfone (DVS) via a Michael Addition to the primary hydroxyl groups. This modification allows for a thiolated bioactive molecule, thiolated-laminin, to be attached to the DVS groups on the modified agarose. There are three types of agaroses modified with DVS: SeaPrep (SP), SeaPlaque (SPQ), and SeaKem (SK). The stability, swelling and degradation degree, mechanical properties, and cytocompatibility of the DVS agarose (DVS-Ag) hydrogel at various concentrations were conducted. A seven-day stability study revealed the most stable concentration (w/v) was 1.2% for DVS-SP, 1.0% for DVS-SPQ, and 0.75% for DVS-SK. A seven-day degradation study further demonstrated that the 1.0% DVS-SPQ and 0.75% DVS-SK had less than 30% degradation over the course of 7 days. Additionally, a four-day swelling study showed that 1.0% DVS-SPQ had minimal swelling while 0.75% DVS-SK experienced some degradation prior to swelling. For determining the mechanical properties of 1.0% DVS-SPQ and 0.75% DVS-SK, the hydrogels were compressed to find an elastic modulus of 0.0116 kPa and 0.0107 kPa, respectively. Additionally, the compressive modulus was not statistically significant between 0.75% DVS-SK and 1.0% DVS-SPQ. The modification of agarose with DVS, disorganized the double helical structure and subsequently impacted the stability, degradation, swelling, and compressive modulus. When assessing the amount of thiolated molecules in comparison to non-thiolated molecules released, it confirmed the successful modification of agarose with DVS. Furthermore, it identified that a lower dosage of thiolated-molecules at 100 [mu]g/mL allowed for more thiolated-molecules to adhere to the DVS groups. Moreover, the cytotoxic analysis study indicated that the DVS modification combined with the conjugation of thiolated-laminin, to some degree, was successful in promoting Nph viability and proliferation for 0.75% DVS-SK and 1.0% DVS-SPQ. Overall, 1.0% DVS-SPQ seemed the most promising in promoting the survival of NSPCs as it promoted Nph survival in addition it had lower cycling in swelling degree and degradation. While in some measures this approach was successful, this modification is promising in exploring different thiolated bioactive molecules conjugated to the DVS groups to create a biomaterial scaffold that promotes the survival of NSPCs.