Decellularized Porcine Peripheral Nerve Based Injectable Hydrogel as a Schwann Cell Carrier for Injured Spinal Cord Regeneration

Introduction: In the United States alone, ~300,000 patients live with traumatic spinal cord injury (SCI). SCI can result from contusion, compression, laceration, or stretch of spinal cord tissue due to traumatic insult, which leads to permanent loss of function and sensation throughout the body. Presently, there is no effective treatment that results in functional recovery after SCI. In human clinical trials, cell-based treatments for SCI are yet to provide reproducible evidence for clinical efficacy. As noted from preclinical studies, the primary reason for limited cell efficacy is due to poor cell viability and short residence at the SCI site. To address these issues, we developed an injectable hydrogel using porcine peripheral nerves to increase Schwann cell efficacy by maintaining higher cellular viability during injection. Porcine peripheral nerves (sciatic and peroneal) were decellularized by chemical decellularization using a sodium deoxycholate and DNase (SDD) method previously developed by our group. We further studied the effect of lipid remnants in decellularized nerves on the gelation kinetics of the hydrogel. To overcome the effect of lipids remnants on hydrogel gelation kinetics and mechanical strength, we delipidated decellularized nerves using dichloromethane and ethanol solvent. The resultant hydrogels exhibited injectability, and mechanical properties mimicking native CNS tissue. Furthermore, Schwann cells encapsulated within the injectable hydrogel formulations maintained higher cellular viability when passed through a 25 gauge needle. Also, the hydrogel protected Schwann cells against astrocytes-mediated inflammatory milieu. Overall, the developed hydrogel has the potential to be used clinically as a delivery vehicle for Schwann cells.

Materials and

Methods: Porcine peripheral nerves (sciatic and peroneal) were decellularized by chemical decellularization using a sodium deoxycholate and DNase (SDD) method previously developed by our group. The efficiency of decellularization was assessed using immunostaining for DAPI (nuclei presence), and ECM retention (Collagen-I, Collagen-IV, and laminin). The residual DNA was quantified using the Quanti-it DNA quantification kit and compared to the fresh native nerve. The decellularized nerves were delipidated using dichloromethane and ethanol solvent and then digested using pepsin enzyme to form injectable hydrogel formulations. The lipids in the digested nerve solution were quantified using a sulfo-vanillin reagent. The digested solution was neutralized using 10x PBS and 1N NaoH and different concentration of Genipin (0-10mM) was added as a crosslinker to enhance mechanical properties. The mechanical stiffness of the hydrogels was estimated using a frequency sweep, and the injectability was analyzed using the shear-thinning test. The gelation kinetics of the hydrogel solution in the presence/absence of genipin was assessed using time sweep rheology. Schwann cells were encapsulated within the injectable hydrogel formulations and passed through a 25-gauge needle, cell viability was assessed using live/dead staining. To mimic in vitro inflammatory milieu, rat astrocytes were inflamed using 5 micrograms of lipopolysaccharide and co-cultured with Schwann cells within both genipin crosslinked sciatic and peroneal hydrogels. The viability of cells was analyzed using live/dead stains. The decellularized nerves without delipidation are referred to as "decell only", while the decellularized nerves delipidated with dichloromethane and methanol solvent are referred to as "decell+delipid".

Results, Conclusions, and Discussions: Various decellularized porcine tissues have been clinically translated into the market. Therefore, we want to develop an injectable hydrogel formulation that can be clinically translated for cell delivery. The SDD method effectively removes cells, as evidenced by the absence of DAPI stain, and retains the extracellular matrix (Collagen-I, Collagen-IV, and laminin) in decellularized tissues (Figure 1A). DNA content in decellularized nerves was approximately 23 ng/ml, compatible with current decellularization guidelines of < 50 ng dsDNA/mg of tissue (Figure 1B). Treating decellularized nerves with dichloromethane and ethanol solvent significantly reduced lipid remnants, as determined by sulfo-vanillin reagent (Figure 2 A and B). Using rheological studies, we found that the delipidation of decellularized porcine peripheral nerves using dichloromethane and ethanol solvent improves gelation kinetics and mechanical strength of hydrogels as compared to decellularized nerves without delipidation (Figure 2 C-F). Additionally, all injectable formulations could undergo gelation at 37 C (Figure 2 G-H). The delipidated and decellularized hydrogels using both sciatic and peroneal crosslinked using genipin showed an increase in mechanical strength with an increase in genipin concentration, however, 1 mM genipin crosslinking mimicked the mechanical strength of CNS tissue ( 500-2000 Pascals) (Figure 3 A and B). Also, sciatic and peroneal delipidated and decellularized hydrogels underwent complete gelation within 15 minutes of incubation at 37 C (Figure 3 C-D). The hydrogels were found to have shear thinning properties desirable for injectable formulations (Figure 3 E-F). Schwann cells maintained higher viability during injection using both decellularized and delipid sciatic and peroneal hydrogels compared to saline controls. Using in vitro co-culture experiments, we also found that the genipin-crosslinked hydrogels protected Schwann cells from astrocyte-mediated inflammation and cells had higher viability than tissue culture plate cells control (Figure 4). Overall, injectable hydrogels developed using delipidated and decellularized porcine peripheral nerves are a potential clinically relevant solution to deliver Schwann cells, and possibly other therapeutic cells, at the SCI site by maintaining higher cellular viability and increasing therapeutic efficacy for SCI treatment.

Acknowledgements (Optional): The authors would like to acknowledge the funding support received by NIH (P0225954) and Pruitt chair funding awarded to PI Christine E. Schmidt