Degradable Microchannel Nerve Guidance Scaffolds for Central and Peripheral Nerve Repair - From Soft to Rigid.

Traumatic nerve injury is generally permanent and debilitating. There is no available therapy primarily owing to the lack of spontaneous axon growth in the adult human central nervous system. In this doctoral work, an interventional technology was investigated to promote and guide axons through nerve gaps to provide nerve repair. Previously, agarose hydrogel microchannel scaffolds linearly guided axons through lesion gaps of spinal cords in rats. However, these scaffolds were non-degradable. In this work, first the efficacy of degradable hydrogels such as alginate, chitosan and poly(ethylene glycol) (PEGDA) as nerve guidance scaffolds was studied. All the hydrogels, however, were concluded unstable in vivo and provided limited axon growth. To fabricate scaffolds effective for nerve repair poly caprolactone (PCL) with slow degradation rate (reported over 8 months) was selected and investigated. In addition, to increase the open volume of scaffolds, a novel scaffold architecture and fabrication process were introduced in which, both the channels open space and the interstitial space between the channels could be utilized for axon growth. A salt-leaching process was developed to optimize PCL properties such as porosity, stiffness and cell adhesion. The scaffold design entailed the fabrication of PCL tubes and their assembly inside a PCL outer tube resulting scaffolds with >60% open volume (a 3-fold improvement to state-of-the-art microchannel scaffolds). When implanted in transected spinal cords in rats, linear axon growth inside and between the channels was observed. The PCL scaffolds, with 3 orders of magnitude higher stiffness than the nerve tissue, provided the highest axon integration and growth in close proximity to the scaffold walls when compared to soft hydrogels. This observation is contradictory to the general belief that an implant with stiffness more closely matching the tissue is more effective. Indeed, this doctoral work is the first study that suggests axon/implant integration is enhanced in vivo when the substrate stiffness is orders of magnitude higher than the host tissue. This technology was translated to poly lactic-co-glycolic acid (PLGA), for a higher degradation rate, and was fabricated to clinically-relevant dimensions. Overall, this dissertation introduces a promising microchannel scaffold for its translation to human nerve repair.PhDMacromolecular Science and EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/133319/1/sdena_1.pd

Characterizing the degradation of alginate hydrogel for use in multilumen scaffolds for spinal cord repair

Alginate was studied as a degradable nerve guidance scaffold material in vitro and in vivo. In vitro degradation rates were determined using rheology to measure the change in shear modulus vs time. The shear modulus decreased from 155 kPa to 5 kPa within 2 days; however, alginate samples maintained their superficial geometry for over 28 days. The degradation behavior was supported by materials characterization data showing alginate consisted of high internal surface area (400 m2/g), which likely facilitated the release of cross‐linking cations resulting in the rapid decrease in shear modulus. To assess the degradation rate in vivo, multilumen scaffolds were fabricated using a fiber templating technique. The scaffolds were implanted in a 2‐mm‐long T3 full transection rodent spinal cord lesion model for 14 days. Although there was some evidence of axon guidance, in general, alginate scaffolds degraded before axons could grow over the 2‐mm‐long lesion. Enabling alginate‐based scaffolds for nerve repair will likely require approaches to slow its degradation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 611–619, 2016.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137597/1/jbma35600.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137597/2/jbma35600_am.pd

Peripheral nerve growth within a hydrogel microchannel scaffold supported by a kink‐resistant conduit

Nerve repair in several mm‐long nerve gaps often requires an interventional technology. Microchannel scaffolds have proven effective for bridging nerve gaps and guiding axons in the peripheral nervous system (PNS). Nonetheless, fabricating microchannel scaffolds at this length scale remains a challenge and/or is time consuming and cumbersome. In this work, a simple computer‐aided microdrilling technique was used to fabricate 10 mm‐long agarose scaffolds consisting of 300 µm‐microchannels and 85 µm‐thick walls in less than an hour. The agarose scaffolds alone, however, did not exhibit adequate stiffness and integrity to withstand the mechanical stresses during implantation and suturing. To provide mechanical support and enable suturing, poly caprolactone (PCL) conduits were fabricated and agarose scaffolds were placed inside. A modified salt‐leaching technique was developed to introduce interconnected porosity in PCL conduits to allow for tuning of the mechanical properties such as elastic modulus and strain to failure. It was shown that the PCL conduits were effective in stabilizing the agarose scaffolds in 10 mm‐long sciatic nerve gaps of rats for at least 8 weeks. Robust axon ingress and Schwann cell penetration were observed within the microchannel scaffolds without using growth factors. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3392–3399, 2017.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/139110/1/jbma36186_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/139110/2/jbma36186.pd

Hierarchically Ordered Porous and High-Volume Polycaprolactone Microchannel Scaffolds Enhanced Axon Growth in Transected Spinal Cords.

The goal of this work was to design nerve guidance scaffolds with a unique architecture to maximize the open volume available for nerve growth. Polycaprolactone (PCL) was selected as the scaffold material based on its biocompatibility and month-long degradation. Yet, dense PCL does not exhibit suitable properties such as porosity, stiffness, strength, and cell adhesion to function as an effective nerve guidance scaffold. To address these shortcomings, PCL was processed using a modified salt-leaching technique to create uniquely controlled interconnected porosity. By controlling porosity, we demonstrated that the elastic modulus could be controlled between 2.09 and 182.1 MPa. In addition, introducing porosity and/or coating with fibronectin enhanced the PCL cell attachment properties. To produce PCL scaffolds with maximized open volume, porous PCL microtubes were fabricated and translated into scaffolds with 60 volume percent open volume. The scaffolds were tested in transected rat spinal cords. Linear axon growth within both the microtubes as well as the interstitial space between the tubes was observed, demonstrating that the entire open volume of the scaffold was available for nerve growth. Overall, a novel scaffold architecture and fabrication technique are presented. The scaffolds exhibit significantly higher volume than state-of-the-art scaffolds for promising spinal cord nerve repair

Functional Skeletal Muscle Regeneration with Thermally Drawn Porous Fibers and Reprogrammed Muscle Progenitors for Volumetric Muscle Injury

Skeletal muscle has an inherent capacity for spontaneous regeneration. However, recovery after severe injuries such as volumetric muscle loss (VML) is limited. There is therefore a need to develop interventions to induce functional skeletal muscle restoration. One suggested approach includes tissue-engineered muscle constructs. Tissue-engineering treatments have so far been impeded by the lack of reliable cell sources and the challenges in engineering of suitable tissue scaffolds. To address these challenges, muscle extracellular matrix (MEM) and induced skeletal myogenic progenitor cells (iMPCs) are integrated within thermally drawn fiber based microchannel scaffolds. The microchannel fibers decorated with MEM enhance differentiation and maturation of iMPCs. Furthermore, engraftment of these bioengineered hybrid muscle constructs induce de novo muscle regeneration accompanied with microvessel and neuromuscular junction formation in a VML mouse model, ultimately leading to functional recovery of muscle activity.11Nsciescopu

<i>In Vivo</i> Microcomputed Tomography of Nanocrystal-Doped Tissue Engineered Scaffolds

Tissue engineered scaffolds (TES) hold promise for improving the outcome of cell-based therapeutic strategies for a variety of biomedical scenarios, including musculoskeletal injuries, soft tissue repair, and spinal cord injury. Key to TES research and development, and clinical use, is the ability to longitudinally monitor TES location, orientation, integrity, and microstructure following implantation. Here, we describe a strategy for using microcomputed tomography (microCT) to visualize TES following implantation into mice. TES were doped with highly radiopaque gadolinium oxide nanocrystals and were implanted into the hind limbs of mice. Mice underwent serial microCT over 23 weeks. TES were clearly visible over the entire time course. Alginate scaffolds underwent a 20% volume reduction over the first 6 weeks, stabilizing over the next 17 weeks. Agarose scaffold volumes were unchanged. TES attenuation was also unchanged over the entire time course, indicating a lack of nanocrystal dissolution or leakage. Histology at the implant site showed the presence of very mild inflammation, typical for a mild foreign body reaction. Blood work indicated marked elevation in liver enzymes, and hematology measured significant reduction in white blood cell counts. While extrapolation of the X-ray induced effects on hematopoiesis in these mice to humans is not straightforward, clearly this is an area for careful monitoring. Taken together, these data lend strong support that doping TES with radiopaque nanocrystals and performing microCT imaging, represents a possible strategy for enabling serial <i>in vivo</i> monitoring of TES