Collagen fiber network architecture regulates astrocyte activation in bioengineered fibrotic spinal cord milieu

Introduction:: Spinal cord injury (SCI) is characterized by traumatic damage to the central nervous system that results in hindered axonal regeneration and functional recovery[1]. The microenvironment of the lesion site is characterized with scar-forming reactive astrocytes and fluid-filled lesion cavity that later is filled with glial and fibrotic scars. These scars are neuro-inhibitory, yet there currently exist no treatment strategies that aim at normalizing the lesion microenvironment. Furthermore, while glial scarring on the periphery of the lesions has been widely studied, including the widespread research on chondroitinase ABC (ChABC) to cleave glial scar, fibrotic scar forming at the center of the lesion remains largely elusive[2]. Using tissue-engineered in vitro models of spinal cord lesions allows mechanistic investigations into fibrotic scarring, including astrocyte activation and subsequent changes in the lesion milieu. To this end, we sought to understand the role of scar-mimetic extracellular matrix (ECM) environment on astrocyte activation and its implications in functional recovery after SCI. Our data show that astrocytes are responsive to collagen fiber architecture, where pro-fibrotic “cold-cast” collagen fibers promote astrocyte activation as demonstrated by changes in phenotype, ECM deposition, cytokine secretion, and invasion of pro-regenerative cells such as endothelial cells and neuronal cells.


Materials and Methods:: Composite hydrogels consisting of spinal cord (SC) gel to collagen I gel were fabricated by decellularization and acidic extraction of rat tail tendons, respectively. The 3D cultures were created by embedding normal human astrocytes (NHA) into the composite hydrogels with varying volumetric ratios of SC matrix and collagen I. Microarchitecture was manipulated using previously established temperature-controlled casting methods, with “cold-casting” mimicking a fibrotic environment and “warm-casting” mimicking healthy ECM[3]. Confocal reflectance imaging was used to image the fiber network produced by the different casting methods, while immunofluorescence was performed to visualize markers of reactive astrocytes and nascent ECM deposition. Nuclear counterstaining was done with DAPI. Pro-angiogenic factor secretion by astrocytes in 3D cultures was probed using Luminex. Therapeutic implications of normalizing fibrotic activation of astrocytes were assessed by endothelial sprouting, rat dorsal root ganglia (DRG) neurite infiltration, and neuroblast invasion.


Results, Conclusions, and Discussions:: Confocal reflectance images showed successful manipulation of ECM fiber thickness in cold- versus warm-cast composite hydrogels. Astrocytes embedded in cold-cast composite gels had elevated levels of activation markers glial fibrillary acidic protein (GFAP), alpha smooth muscle actin (α-SMA) and N-cadherin. Astrocytes also deposited significantly greater amounts of collagen. Previous studies have shown that collagen I is highly expressed in the spinal cord during the scar forming phase and induces astrocytic scar formation via the integrin β1-N-Cadherin pathway[4]. Our results indicate that astrocyte interaction with collagen I is mediated by not only its presence but also its fiber architecture. As such, pharmacological inhibitors targeting ECM-integrin interactions and mechanosensing pathways reversed astrocyte activation with the effect of the inhibitors being more significant in cold-cast compared to warm-cast controls. DRG neurite and neuroblast invasion was observed significantly more in warm-cast gels, demonstrating neuro-inhibitory effect of cold-cast gels. Interestingly, astrocytes embedded in varying ratios of the composite gels secreted different amounts of pro-angiogenic factors, with most heightened secretion observed in collagen-rich, cold-cast composite gels. This aligns with other studies of fibrotic environment where activated tissue-resident cells increase pro-angiogenic factor secretion[3, 5, 6]. Accordingly, endothelial sprouting was most greatly observed in the same collagen-rich, cold-cast gels in the presence of astrocytes. Overall, this study demonstrates the need to account for ECM organization in studying astrocyte behavior after spinal cord injury to not only better understand the lesion environment but also to devise therapeutic strategies to effectively normalize the injury site.


Acknowledgements (Optional): : This work was funded by the National Institutes of Health (R15NS121884 and P20GM139768) awarded to YS and the Student Undergraduate Research Fellowship (SURF) from the Arkansas Department of Higher Education awarded to DG.


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