Local Delivery of Hepatocyte Growth Factor Reduces Glial and Fibrotic Scar Formation After Spinal Cord Injury Zhicheng Wang1 Mengxi Yang1, Jia Nong1, Robert B. Shultz1, Yao Zhao1, Shaoping Hou2, and Yinghui Zhong1* 1School of Biomedical Engineering, Science and Health Systems, Drexel University 2Department of Neurobiology and Anatomy, Drexel University College of Medicine

Statement of Purpose: Traumatic spinal cord injury Methods: Dextran sulfate (DS), chitosan (CH), and HGF (SCI) is followed by secondary injury processes that can solutions were mixed to form DS-CH complexes lead to scar-encapsulated cavity many times larger than encapsulating HGF. After centrifugation to remove the initial lesion, resulting in deleterious functional loss. supernatant, the complexes were loaded into 20 µL The scar tissue, which has been attributed to gliosis, is agarose hydrogel. 250–300 g Sprague Dawley rats were known to inhibit axon regeneration. Chondroitin sulfate unilaterally contused at C5 using the Infinite Horizon proteoglycans (CSPGs) are the major inhibitory Spinal Impactor at a force of 200 kdyn. The hydrogel molecules in glial scar. Therefore, numerous therapeutic encapsulating HGF administered into the intrathecal space strategies have been developed to resolve astrogliosis and at the injury site. Another 100 µl of blank agarose CSPGs. Recently, fibrotic scar, formed by perivascular hydrogel was applied epidurally to protect the injury site. fibroblasts, was reported to be present after contusive SCI, the most clinically relevant injury model. Fibrosis Results: Figure 1 shows stable and sustained release of has been associated with increased production of collagen HGF from agarose hydrogel loaded with DS-CH-HGF type I, III and IV in kidney, liver, and type I and III in complexes. The release lasted for at least 23 days. lung. In the injured spinal cord, increased deposition of collagen type I and IV has been reported. Collagen type I interacts with astrocytes to induce astrocytic scar formation after SCI, while collagen IV deposits do not inhibit axon regrowth. On the other hand, no previous studies have examined collagen type III production after SCI. We for the first time found that collagen III production was greatly increased after SCI. It mostly co- localizes with fibroblasts, but not with astrocytes or CSPGs. Moreover, reduced neurofilament (axon) staining was correlated with regions with positive collagen III or Figure 1. HGF release from agarose hydrogel loaded CS56 (CSPGs) staining, suggesting that both collagen III with DS-CH-HGF complexes. and CSPGs are inhibitory for axon growth. Hepatocyte growth factor (HGF) has been shown to A B promote a normal wound healing process by promoting tissue regeneration and inhibiting fibrotic scar/collagen deposition (type I and III) in various tissues outside of the central nervous system (CNS). Moreover, it has been shown to inhibit astrocytic scar formation and depositions of CSPGs after SCI. In this study, HGF was delivered to hemisection spinal cord lesions via HGF overexpressing mesenchymal stem cells (HGF-MSCs). However, survival of transplanted MSCs after contusive SCI remains an issue (survival was detected in only 2 out of 6 rats at day 14). Another study used gene delivery, however, the Figure 2. Quantification of (A) CS56 for CSPGs and vector encoding HGF was injected 3 days before SCI to (B) collagen type III staining within a 2 mm spinal allow sufficient time for gene expression, which is not cord segment spanning 1 mm rostral and caudal to clinically feasible. HGF is a positively charged growth lesion epicenter. *P < 0.05 compared to blank gel. factor. Utilizing this property, we developed polysaccharide-based complex self-assembled through Conclusions: We developed a biomaterial-based drug electrostatic interactions for sustained delivery of HGF. delivery system capable of stable and sustained release of Further, we encapsulated HGF-loaded complexes in HGF. Local delivery of HGF significantly reduced CSPG agarose hydrogel, which was applied intrathecally on top and collagen type III production. Moreover, we also of the injured spinal cord, for safe local delivery of HGF observed significantly reduction of GFAP (reactive without causing additional damage to the injured spinal astrocytes) and PDGFR-β staining (fibroblasts) (data not cord. Using a clinically relevant rat unilateral cervical shown), suggesting that local delivery of HGF can contusion injury model, we investigated the efficacy of effectively inhibit both astrocytic and fibrotic scar local delivery of HGF in reducing fibrotic and astrocytic formation, which may facilitate nerve regeneration after scar formation after SCI. SCI.