In Vivo and In Vitro Evaluation of a Novel Hyaluronic Acid–Laminin Hydrogel as Luminal Filler and Carrier System for Genetically Engineered Schwann Cells in Critical Gap Length Tubular Peripheral Nerve Graft in Rats

Abstract

In the current study we investigated the suitability of a novel hyaluronic acid–laminin hydrogel (HAL) as luminal filler and carrier system for co-transplanted cells within a composite chitosan-based nerve graft (CNG) in a rat critical nerve defect model. The HAL was meant to improve the performance of our artificial nerve guides by giving additional structural and molecular support to regrowing axons. We filled hollow CNGs or two-chambered nerve guides with an inserted longitudinal chitosan film (CNG[F]s), with cell-free HAL or cell-free HA or additionally suspended either naïve Schwann cells (SCs) or fibroblast growth factor 2-overexpressing Schwann cells (FGF2-SCs) within the gels. We subjected female Lewis rats to immediate 15 mm sciatic nerve gap reconstruction and comprehensively compared axonal and functional regeneration parameters with the gold standard autologous nerve graft (ANG) repair. Motor recovery was surveyed by means of electrodiagnostic measurements at 60, 90, and 120 days post-reconstruction. Upon explantation after 120 days, lower limb target muscles were harvested for calculation of muscle-weight ratios. Semi-thin cross-sections of nerve segments distal to the grafts were evaluated histomorphometrically. After 120 days of recovery, only ANG treatment led to full motor recovery. Surprisingly, regeneration outcomes revealed no regeneration-supportive effect of HAL alone and even an impairment of peripheral nerve regeneration when combined with SCs and FGF2-SCs. Furthermore, complementary in vitro studies, conducted to elucidate the reason for this unexpected negative result, revealed that SCs and FGF2-SCs suspended within the hydrogel relatively downregulated gene expression of regeneration-supporting neurotrophic factors. In conclusion, cell-free HAL in its current formulation did not qualify for optimizing regeneration outcome through CNG[F]s. In addition, we demonstrate that our HAL, when used as a carrier system for co-transplanted SCs, changed their gene expression profile and deteriorated the pro-regenerative milieu within the nerve guides.

Modified Hyaluronic Acid-Laminin-Hydrogel as Luminal Filler for Clinically Approved Hollow Nerve Guides in a Rat Critical Defect Size Model

“…For ethical and animal welfare reasons (reduction of animal numbers used), we have not again investigated in the current study, experimental groups we have already been investigating before in the same model (15 mm rat sciatic nerve gap, animals from the same breeder, strain, sex, and matched age). However, for a more comprehensive evaluation of the new data derived from the current study, we want to report specific previously published data again [ 30 , 31 ]. These data relate to the use of empty chitosan-based nerve guides (CNG) in Stößel et al 2018 [ 31 ], and to the use of chitosan-based nerve guides filled with hyaluronic acid (HA) or with the non-modified HAL both in a concentration of 0.2% in Dietzmeyer et al 2020 [ 30 ].…”

“…In our previous work, we have already evaluated different hydrogel formulations as luminal fillers for either collagen-based NeuraGen ® Nerve Guides [ 27 , 28 ] or chitosan-based Reaxon ® Nerve Guides [ 29 , 30 ]. Recently we evaluated a 0.2% hyaluronic acid-laminin-hydrogel (HAL) as luminal filler of chitosan-based nerve guides in the acute repair of 15 mm critical length sciatic nerve defects in the adult rat [ 30 ]. Our results indicated that the previous formula of the HAL was not yet optimal for robust support of axonal regeneration in the immediate repair approach.…”

“…Over a period of 120 days, we compared four experimental groups with hydrogel filled nerve guides to one group in which we applied the gold standard, reversed autograft repair (ANG), and one group in which we used the empty collagen-based graft. We further compared our new data with our own previously published data on the use of empty chitosan-based nerve guides in the same model [ 30 , 31 ]. Also in comparison to our previous work evaluating HAL [ 30 ], we performed additional gene expression profiling in vitro in Schwann cells cultured within M-HAL.…”

“…We further compared our new data with our own previously published data on the use of empty chitosan-based nerve guides in the same model [ 30 , 31 ]. Also in comparison to our previous work evaluating HAL [ 30 ], we performed additional gene expression profiling in vitro in Schwann cells cultured within M-HAL.…”

Natural Biomaterials as Instructive Engineered Microenvironments That Direct Cellular Function in Peripheral Nerve Tissue Engineering

“…The effect of including growth factors such as basic fibroblast growth factor (FGF-2) in hydrogels has a positive effect on neuron growth ( Sakiyama-Elbert and Hubbell, 2000 ; Al-Zer et al, 2015 ; Fujimaki et al, 2017 ). Indeed, although Dietzmeyer et al (2020) saw an impairment of regeneration when Schwann cells modified to overexpress FGF-2 were present in their hyaluronic acid and laminin conduit versus the acellular conduits, it was found the inclusion of the Schwann cells had instead induced a downregulation of neurotrophic factor expression compared to that of the in vitro 2D culture. Sakiyama-Elbert and Hubbell (2000) additionally modelled the release of FGF-2 from fibrin hydrogels and found its inclusion would improve neurite extension as long as the immobilised heparin it was bound to was in 500-molar excess relative to the FGF-2.…”