Investigating post-traumatic syringomyelia and local fluid osmoregulation via a rat model.

BACKGROUND: Syringomyelia (SM) is characterized by the development of fluid-filled cavities, referred to as syrinxes, within the spinal cord tissue. The molecular etiology of SM post-spinal cord injury (SCI) is not well understood and only invasive surgical based treatments are available to treat SM clinically. This study builds upon our previous omics studies and in vitro cellular investigations to further understand local fluid osmoregulation in post-traumatic SM (PTSM) to highlight important pathways for future molecular interventions. METHODS: A rat PTSM model consisting of a laminectomy at the C7 to T1 level followed by a parenchymal injection of 2 µL quisqualic acid (QA) and an injection of 5 µL kaolin in the subarachnoid space was utilized 6 weeks after initial surgery, parenchymal fluid and cerebrospinal fluid (CSF) were collected, and the osmolality of fluids were analyzed. Immunohistochemistry (IHC), metabolomics analysis using LC-MS, and mass spectrometry-based imaging (MSI) were performed on injured and laminectomy-only control spinal cords. RESULTS: We demonstrated that the osmolality of the local parenchymal fluid encompassing syrinxes was higher compared to control spinal cords after laminectomy, indicating a local osmotic imbalance due to SM injury. Moreover, we also found that parenchymal fluid is more hypertonic than CSF, indicating establishment of a local osmotic gradient in the PTSM injured spinal cord (syrinx site) forcing fluid into the spinal cord parenchyma to form and/or expand syrinxes. IHC results demonstrated upregulation of betaine, ions, water channels/transporters, and enzymes (BGT1, AQP1, AQP4, CHDH) at the syrinx site as compared to caudal and rostral sites to the injury, implying extensive local osmoregulation activities at the syrinx site. Further, metabolomics analysis corroborated alterations in osmolality at the syrinx site by upregulation of small molecule osmolytes including betaine, carnitine, glycerophosphocholine, arginine, creatine, guanidinoacetate, and spermidine. CONCLUSIONS: In summary, PTSM results in local osmotic disturbance that propagates at 6 weeks following initial injury. This coincides with and may contribute to syrinx formation/expansion.

Comparison of Engineered Liver 3D Models and the Role of Oxygenation for Patient-Derived Tumor Cells and Immortalized Cell Lines Cocultured with Tumor Stroma in the Detection of Hepatotoxins.

In metabolically active tumors, responses of cells to drugs are heavily influenced by oxygen availability via the surrounding vasculature alongside the extracellular matrix signaling. The objective of this study is to investigate hepatotoxicity by replicating critical features of hepatocellular carcinoma (HCC). This includes replicating 3D structures, metabolic activities, and tumor-specific markers. The internal environment of spheroids comprised of cancerous human patient-derived hepatocytes using microparticles is modulated to enhance the oxygenation state and recreate cell-extracellular matrix interactions. Furthermore, the role of hepatic stellate cells in maintaining hepatocyte survival and function is explored and hepatocytes from two cellular sources (immortalized and patient-derived) to create four formulations with and without microparticles are utilized. To investigate drug-induced changes in metabolism and apoptosis in liver cells, coculture spheroids with and without microparticles are exposed to three hepatotoxic drugs. The use of microparticles increases levels of apoptotic markers in both liver models under drug treatments. This coincides with reduced levels of anti-apoptotic proteins and increased levels of pro-apoptotic proteins. Moreover, cells from different origins undergo apoptosis through distinct apoptotic pathways in response to identical drugs. This 3D microphysiological system offers a viable tool for liver cancer research to investigate mechanisms of apoptosis under different microenvironmental conditions.

Fabrication of oxygen-carrying microparticles functionalized with liver ECM-proteins to improve phenotypic three-dimensional in vitro liver assembly, function, and responses.

Oxygen and extracellular matrix (ECM)-derived biopolymers play vital roles in regulating many cellular functions in both the healthy and diseased liver. This study highlights the significance of synergistically tuning the internal microenvironment of three-dimensional (3D) cell aggregates composed of hepatocyte-like cells from the HepG2 human hepatocellular carcinoma cell line and hepatic stellate cells (HSCs) from the LX-2 cell line to enhance oxygen availability and phenotypic ECM ligand presentation for promoting the native metabolic functions of the human liver. First, fluorinated (PFC) chitosan microparticles (MPs) were generated with a microfluidic chip, then their oxygen transport properties were studied using a custom ruthenium-based oxygen sensing approach. Next, to allow for integrin engagements the surfaces of these MPs were functionalized using liver ECM proteins including fibronectin, laminin-111, laminin-511, and laminin-521, then they were used to assemble composite spheriods along with HepG2 cells and HSCs. After in vitro culture, liver-specific functions and cell adhesion patterns were compared between groups and cells showed enhanced liver phenotypic responses to laminin-511 and 521 as evidenced via enhanced E-cadherin and vinculin expression, as well as albumin and urea secretion. Furthermore, hepatocytes and HSCs exhibited more pronounced phenotypic arrangements when cocultured with laminin-511 and 521 modified MPs providing clear evidence that specific ECM proteins have distinctive roles in the phenotypic regulation of liver cells in engineering 3D spheroids. This study advances efforts to create more physiologically relevant organ models allowing for well-defined conditions and phenotypic cell signaling which together improve the relevance of 3D spheroid and organoid models.

Osmotic Contribution of Synthesized Betaine by Choline Dehydrogenase Using In Vivo and In Vitro Models of Post-traumatic Syringomyelia.

Introduction: Syringomyelia (SM) is a debilitating spinal cord disorder in which a cyst, or syrinx, forms in the spinal cord parenchyma due to congenital and acquired causes. Over time syrinxes expand and elongate, which leads to compressing the neural tissues and a mild to severe range of symptoms. In prior omics studies, significant upregulation of betaine and its synthesis enzyme choline dehydrogenase (CHDH) were reported during syrinx formation/expansion in SM injured spinal cords, but the role of betaine regulation in SM etiology remains unclear. Considering betaine's known osmoprotectant role in biological systems, along with antioxidant and methyl donor activities, this study aimed to better understand osmotic contributions of synthesized betaine by CHDH in response to SM injuries in the spinal cord. Methods: A post-traumatic SM (PTSM) rat model and in vitro cellular models using rat astrocytes and HepG2 liver cells were utilized to investigate the role of betaine synthesis by CHDH. Additionally, the osmotic contributions of betaine were evaluated using a combination of experimental as well as simulation approaches. Results: In the PTSM injured spinal cord CHDH expression was observed in cells surrounding syrinxes. We next found that rat astrocytes and HepG2 cells were capable of synthesizing betaine via CHDH under osmotic stress in vitro to maintain osmoregulation. Finally, our experimental and simulation approaches showed that betaine was capable of directly increasing meaningful osmotic pressure. Conclusions: The findings from this study demonstrate new evidence that CHDH activity in the spinal cord provides locally synthesized betaine for osmoregulation in SM pathophysiology. Supplementary Information: The online version of this article contains supplementary material available 10.1007/s12195-022-00749-5.

Modulatory Contribution of Oxygenating Hydrogels and Polyhexamethylene Biguanide on the Antimicrobial Potency of Neutrophil-like Cells.

Neutrophils are a first line of host defense against infection and utilize a series of oxygen-dependent processes to eliminate pathogens. Research suggests that oxygen availability can improve anti-infective mechanisms by promoting the formation of reactive oxygen species. Also, oxygen can synergistically upregulate the antibacterial properties of certain antibiotics against bacteria by altering their metabolism and causing an increase in the antibiotic uptake of bacteria. Therefore, understanding the effects of oxygen availability, as provided via a biomaterial treatment alone or along with potent antibacterial agents, on neutrophil functions can lead us to the development of new anti-inflammatory and anti-infective approaches. However, the study of neutrophil functions in vitro is often limited by their short life span and nonreproducibility, which suggests the need for cell line-based models as a substitute for primary neutrophils. Here, we took advantage of the differentiated human leukemia-60 cell line (HL-60), as an in vitro neutrophil model, to test the effects of local oxygen and antibacterial delivery by fluorinated methacrylamide chitosan (MACF) hydrogels incorporated with polyhexamethylene biguanide (PHMB) antibacterial agent. Considering the natural modes of neutrophil actions to combat bacteria, we studied the impact of our dual functioning oxygenating-antibacterial platforms on neutrophil phagocytosis and antibacterial properties as well as the formation of neutrophil extracellular traps (NETs) and reactive oxygen species (ROS). Our results demonstrated that supplemental oxygen and antibacterial delivery from MACF-PHMB hydrogel platforms upregulated neutrophil antibacterial properties and ROS production. NET formation by neutrophils upon treatment with MACF and PHMB varied when chemical and biological stimuli were used. Overall, this study presents a model to study immune responses in vitro and lays the foundation for future studies to investigate if similar responses also occur in vivo.

Investigating Mechanisms of Subcutaneous Preconditioning Incubation for Neural Stem Cell Embedded Hydrogels.

Stem cells are a vital component of regenerative medicine therapies, however, only a fraction of stem cells delivered to the central nervous system following injury survive the inflammatory environment. Previously, we showed that subcutaneous preconditioning of neural stem cell (NSC) embedded hydrogels for 28 days improved spinal cord injury (SCI) functional outcomes over controls. Here, we investigated the mechanism of subcutaneous preconditioning of NSC-embedded hydrogels, with and without the known neurogenic cue, interferon gamma (IFN-γ), for 3, 14, or 28 days to refine and identify subcutaneous preconditioning conditions by measurement of neurogenic markers and cytokines. Studying the preconditioning mechanism, we found that subcutaneous foreign body response (FBR) associated cytokines infiltrated the scaffold in groups with and without NSCs, with time point effects. A pro-inflammatory environment with upregulated interleukin (IL)-6, IL-10, macrophage inflammatory protein (MIP)-1, MIP-2, IFN-γ-inducible protein 10 (IP-10), tumor necrosis factor-α (TNF-α), and IL-12p70 was observed on day 3. By 14 and 28 days, there was an increase in pro-regenerative cytokines (IL-13, IL-4) along with pro-inflammatory markers IL-1ß, IP-10, and RANTES (regulated on activation, normal T cell expressed, and secreted) potentially part of the mechanism that had an increased functional outcome in SCI. Coinciding with changes in cytokines, the macrophage population increased over time from 3 to 28 days, whereas neutrophils peaked at 3 days with a significant decrease at later time points. Expression of the neuronal marker ßIII tubulin in differentiating NSCs was supported at 3 days in the presence of soluble and immobilized IFN-γ and at 14 days by immobilized IFN-γ only, but it was greatly attenuated in all conditions at 28 days, partially because of dilution via host cell infiltration. We conclude that subcutaneously incubating NSC seeded scaffolds for 3 or 14 days could act as host specific preconditioning through exposure to FBR while retaining ßIII tubulin expression of NSCs to further improve the SCI functional outcome observed with 28 day subcutaneous incubation.

Therapeutic Targeting of Stromal-Tumor HGF-MET Signaling in an Organotypic Triple-Negative Breast Tumor Model.

The tumor microenvironment (TME) promotes proliferation, drug resistance, and invasiveness of cancer cells. Therapeutic targeting of the TME is an attractive strategy to improve outcomes for patients, particularly in aggressive cancers such as triple-negative breast cancer (TNBC) that have a rich stroma and limited targeted therapies. However, lack of preclinical human tumor models for mechanistic understanding of tumor-stromal interactions has been an impediment to identify effective treatments against the TME. To address this need, we developed a three-dimensional organotypic tumor model to study interactions of patient-derived cancer-associated fibroblasts (CAF) with TNBC cells and explore potential therapy targets. We found that CAFs predominantly secreted hepatocyte growth factor (HGF) and activated MET receptor tyrosine kinase in TNBC cells. This tumor-stromal interaction promoted invasiveness, epithelial-to-mesenchymal transition, and activities of multiple oncogenic pathways in TNBC cells. Importantly, we established that TNBC cells become resistant to monotherapy and demonstrated a design-driven approach to select drug combinations that effectively inhibit prometastatic functions of TNBC cells. Our study also showed that HGF from lung fibroblasts promotes colony formation by TNBC cells, suggesting that blocking HGF-MET signaling potentially could target both primary TNBC tumorigenesis and lung metastasis. Overall, we established the utility of our organotypic tumor model to identify and therapeutically target specific mechanisms of tumor-stromal interactions in TNBC toward the goal of developing targeted therapies against the TME. IMPLICATIONS: Leveraging a state-of-the-art organotypic tumor model, we demonstrated that CAFs-mediated HGF-MET signaling drive tumorigenic activities in TNBC and presents a therapeutic target.

Generation of Oxygenating Fluorinated Methacrylamide Chitosan Microparticles to Increase Cell Survival and Function in Large Liver Spheroids.

Despite advances in the development of complex culture technologies, the utility, survival, and function of large 3D cell aggregates, or spheroids, are impeded by mass transport limitations. The incorporation of engineered microparticles into these cell aggregates offers a promising approach to increase spheroid integrity through the creation of extracellular spaces to improve mass transport. In this study, we describe the formation of uniform oxygenating fluorinated methacrylamide chitosan (MACF) microparticles via a T-shaped microfluidic device, which when incorporated into spheroids increased extracellular spacing and enhanced oxygen transport via perfluorocarbon substitutions. The addition of MACF microparticles into large liver cell spheroids supported the formation of stable and large spheroids (>500 µm in diameter) made of a heterogeneous population of immortalized human hepatoma (HepG2) and hepatic stellate cells (HSCs) (4 HepG2/1 HSC), especially at a 150:1 ratio of cells to microparticles. Further, as confirmed by the albumin, urea, and CYP3A4 secretion amounts into the culture media, biological functionality was maintained over 10 days due to the incorporation of MACF microparticles as compared to controls without microparticles. Importantly, we demonstrated the utility of fluorinated microparticles in reducing the number of hypoxic cells within the core regions of spheroids, while also promoting the diffusion of other small molecules in and out of these 3D in vitro models.

Detection of locomotion deficit in a post-traumatic syringomyelia rat model using automated gait analysis technique.

Syringomyelia (SM) is a spinal cord disorder in which a cyst (syrinx) filled with fluid forms in the spinal cord post-injury/disease, in patients syrinx symptoms include loss of pain and temperature sensation or locomotion deficit. Currently, there are no small animal models and connected tools to help study the functional impacts of SM. The objective of this study was to determine the detectability of subtle locomotion deficits due to syrinx formation/expansion in post-traumatic syringomyelia (PTSM) rat model using the recently reported method of Gait Analysis Instrumentation, and Technology Optimized for Rodents (GAITOR) with Automated Gait Analysis Through Hues and Areas (AGATHA) technique. First videos of the rats were collected while walking in an arena (using GAITOR) followed by extracting meaningful locomotion information from collected videos using AGATHA protocol. PTSM injured rats demonstrated detectable locomotion deficits in terms of duty factor imbalance, paw placement accuracy, step contact width, stride length, and phase dispersion parameters compared to uninjured rats due to SM. We concluded that this technique could detect mild and subtle locomotion deficits associated with PTSM injury, which also in future work could be used further to monitor locomotion responses after different treatment strategies for SM.

Osmoregulatory Role of Betaine and Betaine/γ-Aminobutyric Acid Transporter 1 in Post-Traumatic Syringomyelia.

Syringomyelia (SM) is primarily characterized by the formation of a fluid-filled cyst that forms in the parenchyma of the spinal cord following injury or other pathology. Recent omics studies in animal models have identified dysregulation of solute carriers, channels, transporters, and small molecules associated with osmolyte regulation during syrinx formation/expansion in the spinal cord. However, their connections to syringomyelia etiology are poorly understood. In this study, the biological functions of the potent osmolyte betaine and its associated solute carrier betaine/γ-aminobutyric acid (GABA) transporter 1 (BGT1) were studied in SM. First, a rat post-traumatic SM model was used to demonstrate that the BGT1 was primarily expressed in astrocytes in the vicinity of syrinxes. In an in vitro system, we found that astrocytes uptake betaine through BGT1 to regulate cell size under hypertonic conditions. Treatment with BGT1 inhibitors, especially NNC 05-2090, demonstrated midhigh micromolar range potency in vitro that reversed the osmoprotective effects of betaine. Finally, the specificity of these BGT1 inhibitors in the CNS was demonstrated in vivo, suggesting feasibility for targeting betaine transport in SM. In summary, these data provide an enhanced understanding of the role of betaine and its associated solute carrier BGT1 in cell osmoregulation and implicates the active role of betaine and BGT1 in syringomyelia progression.

Micro-computed tomography utility for estimation of intraparenchymal spinal cord cystic lesions in small animals.

Precise assessment of spinal cord cystic lesions is crucial to formulate effective therapeutic strategies, yet histological assessment of the lesion remains the primary method despite numerous studies showing inconsistent results regarding estimation of lesion size via histology. On the other hand, despite numerous advances in micro-computed tomography (micro-CT) imaging and analysis that have allowed precise measurements of lesion size, there is not enough published data on its application to estimate intraspinal lesion size in laboratory animal models. This work attempts to show that micro-CT can be valuable for spinal cord injury research by demonstrating accurate estimation of syrinx size and compares between micro-CT and traditional histological analysis. We used a post-traumatic syringomyelia rat model to compare micro-CT analysis to conventional histological analysis. The study showed that micro-CT can detect lesions within the spinal cord very similar to histology. Importantly, micro-CT appears to provide more accurate estimates of the lesions with more measures (e.g., surface area), can detect compounds within the cord, and can be done with the tissue of interest (spinal cord) intact. In summary, the experimental work presented here provides one of the first investigations of the use of micro-CT for estimating the size of intraparenchymal cysts and detecting materials within the spinal cord. All animal procedures were approved by the University of Akron Institutional Animal Care and Use Committee (IACUC) (protocol # LRE 16-05-09 approved on May 14, 2016).

Softening of the chronic hemi-section spinal cord injury scar parallels dysregulation of cellular and extracellular matrix content.

Regeneration following spinal cord injury (SCI) is challenging in part due to the modified tissue composition and organization of the resulting glial and fibrotic scar regions. Inhibitory cell types and biochemical cues present in the scar have received attention as therapeutic targets to promote regeneration. However, altered Young's modulus of the scar as a readout for potential impeding factors for regeneration are not as well-defined, especially in vivo. Although the decreased Young's modulus of surrounding tissue at acute stages post-injury is known, the causation and outcomes at chronic time points remain largely understudied and controversial, which motivates this work. This study assessed the glial and fibrotic scar tissue's Young's modulus and composition (scar morphometry, cell identity, extracellular matrix (ECM) makeup) that contribute to the tissue's stiffness. The spatial Young's modulus of a chronic (~18-wks, post-injury) hemi-section, including the glial and fibrotic regions, were significantly less than naïve tissue (~200 Pa; p < 0.0001). The chronic scar contained cystic cavities dispersed in areas of dense nuclei packing. Abundant CNS cell types such as astrocytes, oligodendrocytes, and neurons were dysregulated in the scar, while epithelial markers such as vimentin were upregulated. The key ECM components in the CNS, namely sulfated proteoglycans (sPGs), were significantly downregulated following injury with concomitant upregulation of unsulfated glycosaminoglycans (GAGs) and hyaluronic acid (HA), likely altering the foundational ECM network that contributes to tissue stiffness. Our results reveal the Young's modulus of the chronic SCI scar as well as quantification of contributing elastic components that can provide a foundation for future study into their role in tissue repair and regeneration.

Subcutaneous priming of protein-functionalized chitosan scaffolds improves function following spinal cord injury.

Strategies using neural stem cells (NSCs) to aid regeneration following spinal cord injury (SCI) show much promise, but challenges remain regarding implementation and efficacy. In this work, we explored the use of an NSC-seeded scaffold consisting of covalently immobilized interferon-γ and rat NSCs within a hydrogel matrix (methacrylamide chitosan). We placed the scaffolds within the subcutaneous environment of rats, allowing them to incubate for 4 weeks in order to prime them for regeneration prior to being transplanted into a right lateral hemisection SCI model in the same animal. We found that subcutaneous priming reduced the lineage commitment of encapsulated NSCs, as observed by increased nestin expression and decreased NeuN expression. When combined with intracellular σ peptide administration (which reduces inhibition from the glial scar), subcutaneous maturation improved functional outcomes, which were assessed by BBB score and quantitative gait parameters (fore and hind limb duty factor imbalance, right and left paw placement accuracy). Although we did not observe any direct reconnection of the transplanted cells with the host tissue, we did observe neurofilament fibers extending from the host tissue into the scaffold. Importantly, the mechanism for improved functional outcomes is likely an increase in trophic support from subcutaneously maturing the scaffold, which is enhanced by the administration of ISP.

Automated Gait Analysis Detects Improvements after Intracellular σ Peptide Administration in a Rat Hemisection Model of Spinal Cord Injury.

A promising treatment strategy for spinal cord injury (SCI) is to reduce inhibition from chondroitin sulfate proteoglycans (CSPGs). For example, administering intracellular σ peptide (ISP) can improve the ability of axons to cross inhibitory CSPGs and improve function in rodent models of SCI. To translate such treatments into the clinic, we need robust and sensitive methods for studying rodent models. In this study, we applied a newly developed suite of quantitative gait analysis tools: gait analysis instrumentation and technology optimized for rodents (GAITOR), which consists of an arena and open-source software (AGATHA: automated gait analysis through hues and areas). We showed that GAITOR can be used to detect subtle functional improvements (measured by hindlimb duty factor imbalance) in rats following ISP administration in a T10 hemisection injury model. We demonstrated that SCI-specific parameters (right paw placement accuracy and phase dispersion) can be easily added to GAITOR to track recovery. We confirmed the gait observations via retrograde tracer uptake. We concluded that GAITOR is a powerful tool for measuring recovery after moderate/mild SCI, and could be used to replace expensive/inflexible commercially-available gait analysis techniques.

Biomaterial strategies for limiting the impact of secondary events following spinal cord injury.

The nature of traumatic spinal cord injury (SCI) often involves limited recovery and long-term quality of life complications. The initial injury sets off a variety of secondary cascades, which result in an expanded lesion area. Ultimately, the native tissue fails to regenerate. As treatments are developed in the laboratory, the management of this secondary cascade is an important first step in achieving recovery of normal function. Current literature identifies four broad targets for intervention: inflammation, oxidative stress, disruption of the blood-spinal cord barrier, and formation of an inhibitory glial scar. Because of the complex and interconnected nature of these events, strategies that combine multiple therapies together show much promise. Specifically, approaches that rely on biomaterials to perform a variety of functions are generating intense research interest. In this review, we examine each target and discuss how biomaterials are currently used to address them. Overall, we show that there are an impressive amount of biomaterials and combinatorial treatments which show good promise for slowing secondary events and improving outcomes. If more emphasis is placed on growing our understanding of how materials can manage secondary events, treatments for SCI can be designed in an increasingly rational manner, ultimately improving their potential for translation to the clinic.

Fluorinated Methacrylamide Chitosan Hydrogels Enhance Cellular Wound Healing Processes.

Low availability of oxygen can lead to stalled wound healing processes and chronic wounds. To address local hypoxia and to better understand direct cellular benefits, a perfluorocarbon conjugated chitosan (MACF) hydrogel that delivers oxygen was created and applied for the first time to in vitro cultures of human dermal fibroblasts and human epidermal keratinocytes under both normoxic (21% O2) and hypoxic (1% O2) environments. Results revealed that local application of MACF provided 233.8 ± 9.9 mmHg oxygen partial pressure at 2 h and maintained equilibrium oxygen levels that were approximately 17 mmHg partial pressure greater than untreated controls. Cell culture experiments showed that MACF oxygenating gels improved cellular functions involved in wound healing such as cell metabolism, total DNA synthesis and cell migration under hypoxia in both fibroblasts and keratinocytes. Adenosine triphosphate (ATP) quantification also revealed that MACF treatments improved cellular ATP levels significantly over controls under both normoxia and hypoxia (p < 0.005). In total, these studies provide new data to indicate that supplying local oxygen via MACF hydrogels under hypoxic environments improves key wound healing cellular functions.

A Hydrogel Bridge Incorporating Immobilized Growth Factors and Neural Stem/Progenitor Cells to Treat Spinal Cord Injury.

Spinal cord injury (SCI) causes permanent, often complete disruption of central nervous system (CNS) function below the damaged region, leaving patients without the ability to regenerate lost tissue. To engineer new CNS tissue, a unique spinal cord bridge is created to deliver stem cells and guide their organization and development with site-specifically immobilized growth factors. In this study, this bridge is tested, consisting of adult neural stem/progenitor cells contained within a methacrylamide chitosan (MAC) hydrogel and protected by a chitosan conduit. Interferon-γ (IFN-γ) and platelet-derived growth factor-AA (PDGF-AA) are recombinantly produced and tagged with an N-terminal biotin. They are immobilized to streptavidin-functionalized MAC to induce either neuronal or oligodendrocytic lineages, respectively. These bridges are tested in a rat hemisection model of SCI between T8 and T9. After eight weeks treatments including chitosan conduits result in a significant reduction in lesion area and macrophage infiltration around the lesion site (p < 0.0001). Importantly, neither immobilized IFN-γ nor PDGF-AA increased macrophage infiltration. Retrograde tracing demonstrates improved neuronal regeneration through the use of immobilized growth factors. Immunohistochemistry staining demonstrates that immobilized growth factors are effective in differentiating encapsulated cells into their anticipated lineages within the hydrogel, while qualitatively reducing glial fibrillary acid protein expression.

Functional immobilization of interferon-gamma induces neuronal differentiation of neural stem cells.

Stem cell transplantation provides significant promise to regenerative strategies after injury in the central nervous system. Neural stem/progenitor cells (NSPCs) have been studied in terms of their regenerative capacity and their ability to differentiate into neurons when exposed to various soluble factors. In this study, interferon-gamma (IFN-gamma) was compared with brain-derived neurotrophic factor (BDNF) and erythropoietin and was shown to be the best single growth factor for inducing neuronal differentiation from adult rat brain-derived NSPCs. Next, IFN-gamma was surface immobilized to a methacrylamide chitosan (MAC) scaffold that was specifically designed to match the modulus of brain tissue and neuronal differentiation of NSPCs was examined in vitro by immunohistochemistry. Bioactive IFN-gamma was successfully immobilized and quantified by ELISA. Both soluble and immobilized IFN-gamma on MAC surfaces showed dose dependent neuronal differentiation with soluble saturation occurring at 100 ng/mL and the most effective immobilized IFN-gamma dose at 37.5 ng/cm(2), where significantly more neurons resulted compared with controls including soluble IFN-gamma.