Modification and transplantation of anisotropic capillary gels for directed axonal regeneration in the injured spinal cord
Final Report Abstract
Following a traumatic spinal cord injury (SCI), dramatic loss of tissue integrity prohibits axonal regeneration and target reinnervation. For successful functional recovery to occur, axonal growth must be initiated, guided and sustained across a newly formed lesion cavity and its surrounding scar tissue by introducing a permissive physical substrate to support target reinnervation with proper synapse formation. We were previously able to uniquely combine the use of alginate-based anisotropic capillary hydrogels (ACH) with Schwann cell (SC) seeding/transplantation and viral brainderived neurotrophic factor (BDNF) delivery to successfully generate long-distance axonal growth into the ACH and re-entry into the host spinal cord. However, in this funding period we did not observe functional recovery with this treatment greater than controls in motor recovery (BBB score) in a complete low thoracic spinal lesion. Immature cortical astrocytes explored to overcome some possible limitations in the extent of regrowth in the SC/ACH +BDNF model, showed nearly similar functional recovery when seeded in the ACH and injected caudally. Further immature spinal and cortical astrocyte-seeded ACHs with caudal host transplantation in a cervical hemisection were found to benefit axonal regrowth and vascularization, however immature astrocyte-seeded ACH and transplanted surrounding (rostral and caudal) led to the greatest growth (axonal and vascular) into and through the ACH, indicating better host integration leads to greater growth. Without further neurotrophic support we did not observe host re-entry with immature astrocytes. Having previously focused on biological factors to improve ACH integration and the neuronal regenerative capacity, we now have modulated the inherent mechanical characteristics of the ACH - namely its viscoelasticity. We have developed stable ACH with differing viscoelasticities approaching that of the spinal cord. Four groups of ACH could be realized with elastic moduli of ~1 kPa, 2-3 kPa, ~10 kPa and ~20 kPa. The concentration of alginate, the concentration of the diisocyanate crosslinking compound, the crosslinking conditions and the cationic species in the upstream of gel formation were identified as key factors in order to tune the mechanical and morphological requirements of ACH. Two different methods for the determination of the mechanical properties have been applied, i.e. oscillating rheology with a plate diameter of 4 cm to determine the viscoelasticity on a macroscopic level and an atomic force microscope with an indenter of 20 µm in diameter to determine the viscoelasticity on a microscopic level. The results obtained by both methods matched nicely in their order of magnitude with a greater variability found for the microindentation measurements which clearly is a result of the anisotropic structure of the ACH. Differences between both methods were found in the stability testing during incubation in physiological buffer solution, where weakening of the structure was more prone for the macroscopic than for the microscopic mechanical properties. Next, these four ACH groups (~1kPa, 2-3 kPa, ~10kPa and ~20kPa) have been implanted in a rat cervical hemisection model to examine the effect of stiffness on host integration and axonal re-growth. Indeed, softer ACHs provided greater vascularization and axonal re-growth than stiffer ACHs. This was coupled with decreased inflammation, glial scar formation and chondroitin sulfate proteoglycan (CSPG) production. Currently we are examining the mechanical properties of the ACH influencing the host and vice versa with atomic force microscopy in an ex vivo model.
Publications
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Alginate-based anisotropic capillary hydrogels as biomaterial scaffolds for oriented axon regeneration. 6th International Symposium Interface Biology of Implants, Warnemünde, 8-10 May 2019
R. Müller, M. Nützl, M. Brunner, A. Blesch, T. Schackel, R. Puttagunta, N. Weidner
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Peptides and astroglia improve the regenerative capacity of alginate gels in the injured spinal cord, Tissue Engineering A, 2019, 25, 522-537
T. Schackel, M. Günther, S. Liu, M. Brunner, B. Sandner, R. Puttagunta, R. Müller, N. Weidner, A. Blesch
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Impact of stiffness on implant integration and axonal regrowth in rat spinal cord injury, EMBO | EMBL Symposium Mechanobiology in development and disease, Heidelberg, 15-18 May 2022
Y. Zheng, T. Schackel, M. Nützl, R. Müller, N. Weidner, R. Puttagunta
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Mechanical properties and chemical stability of anisotropic alginate-based capillary hydrogels, Journal of the Mechanical Behavior of Biomedical Materials, 2022, 134, 105397
M. Nützl, M. Schrottenbaum, T. Müller, R. Müller
