Decellularized peripheral nerve supports Schwann cell transplants and axon growth following spinal cord injury
Introduction
Following an insult to the mammalian spinal cord, a complex cascade of reactions contributes to an aggravated expansion of the initial injury, including the formation of fluid-filled cavities that partly contribute to the regenerative failure and cause extreme functional deficits [1]. Cell transplantation strategies following spinal cord injury (SCI) rely on reducing cavitation following damage, re-bridging the injured tissue and creating more favorable conditions for axonal regeneration [2]. Among the prime candidates for repair are Schwann cells (SCs), the myelinating glia of the peripheral nervous system. SCs can be obtained from SCI patient nerve, purified and expanded in culture, and autologously transplanted into the lesioned spinal cord [3]. SCs have been extensively studied and found to be neuroprotective, reduce cavitation, promote axon regeneration and myelination, and modestly improve hindlimb movements in rat SCI models [[4], [5], [6]]. These findings contributed to FDA-approved clinical trials evaluating the safety and efficacy of autologous SC therapy in sub-acute and chronic SCI subjects (www.clinicaltrials.gov NCT01739023; NCT02354625) [[7], [8], [9]].
After transplantation, however, SC survival rates are considerably low which limits the extent of their therapeutic action. Previous studies have shown that after a contusion injury in rats, only approximately 20% of the SCs survived when transplanted in culture medium [[10], [11], [12]]. The harsh injury environment, where oxidative stress, inflammation and immune responses occur, undoubtedly contributes to these low survival levels. In addition, the SC detachment from extracellular matrix (ECM) in the culture dish before transplantation, contributes to significant cell death through an apoptotic process designated anoikis [13]. The vehicle in which SCs are transplanted is thus very important and affects the treatment efficacy. Patel et al. [14] compared different ECM-derived in situ gelling formulations with culture medium as vehicles for SC implantation into a SCI site. Transplanting SCs in these matrices, particularly Matrigel, enhanced SC survival, axon growth, re-vascularization and functional recovery compared to a SC suspension. Matrigel is not clinically relevant, however, as it is derived from a mouse tumor and its composition varies from batch to batch [15]. A supportive and clinically acceptable matrix is needed for human SC implantation.
We hypothesize that peripheral nerve (PN) tissue, the natural environment of SCs, could provide an excellent support scaffold for SC transplantation after SCI. Decellularization technologies allow the removal of cellular components from source tissues that can elicit an immune response, while preserving the ECM components, thus providing attractive biomaterials for tissue engineering [16]. Implantation of decellularized biomaterials does not require immunosuppression, and the chemical intrinsic cues for cell function and guidance are maintained [17]. Nagao et al. [18] have developed an optimized acellular PN graft that has been shown to elicit similar functional recovery to an autograft in a rat sciatic nerve injury model. A similar human-derived scaffold is now FDA-approved for use in patients with PN discontinuities. An injectable formulation of acellular PN, recently established, mimics the composition of native nerve ECM, gels at physiological temperature and possesses mechanical properties similar to rat neural tissue [19]. We here investigate the potential of injectable PN (iPN) matrix to support transplanted SC survival and axon growth following SCI. Accordingly, we transplanted SCs in iPN and compared this to Matrigel, in a rat model of thoracic contusion. We evaluated the subacute immune response, graft volume, axon ingrowth and SC myelination, and locomotor function 8 weeks post-transplantation. SCs transplanted in iPN promoted twice as many axons and equivalent locomotor recovery, compared to Matrigel, raising exciting possibilities that can directly translate to future SCI clinical trial strategies.
Section snippets
Injectable acellular peripheral nerve matrix preparation
Decellularization of rat sciatic nerve was conducted according to methods previously developed in the Schmidt laboratory [20]. Briefly, sciatic nerves were aseptically harvested from Sprague Dawley rats and the epineurium was removed. The nerves were then stored in 1× phosphate buffered saline (PBS) at −20 °C until processing. Nerves were washed with double distilled water and detergent, with buffer washes (100 mM sodium/50 mM phosphate buffer) performed between detergent washes. The detergent
Quantification of SC viability in iPN and Matrigel in vitro
In vitro data demonstrated that the iPN scaffold was not cytotoxic to the seeded SCs. SCs were able to grow abundantly in both iPN and Matrigel, and in both conditions they were evenly distributed. SCs seeded in Matrigel exhibited a bipolar morphology (Fig. 2A), whereas in iPN displayed a rounded phenotype (Fig. 2B). Despite the unusual morphological appearance, SCs cultured in iPN maintained robust viability that was significantly higher (90.0 ± 1.7%) than the cells cultured in Matrigel
Discussion
The current study demonstrates that iPN is an appropriate injectable biomaterial to support transplanted SC survival and axon growth into a SCI site. Moreover, injection of iPN into a lesion is immunologically tolerated and supports as much locomotor recovery as Matrigel, making this matrix an attractive possibility for future translational studies. SC transplantation in Matrigel has been compared to other matrices as well as with SC suspension and shown superior SC survival, axon growth and
Conclusions
Matrices to support SC grafts have been recently explored and shown to improve SC transplantation efficacy after SCI. Nonetheless, no clinically relevant ECM matrix that could be used in human subjects has been reported to date. Our results with iPN revealed this material to be highly supportive for SC survival and myelination, axon growth, and locomotor recovery. Additionally, implantation of this matrix does not aggravate immune responses in the lesion site. As both SC transplantation in SCI
Disclosures
The authors declare no conflict of interest.
Author contributions
SRC,YL, RAW, CES and MBB designed the experiments; SRC, YL, MWM and RCC performed the experimental studies and acquired data; SRC, MWM and RAW analyzed data and performed statistical analysis; SRC prepared the manuscript; CES and MBB edited and reviewed the manuscript.
Acknowledgements
This work was supported by the National Institutes of Health [NIH NS 09923 (MBB)], The Miami Project to Cure Paralysis (MPCP) at the University of Miami, The Buoniconti Fund, the Craig Neilsen Foundation [222456 (CES)] and Conquer Paralysis Now (SRC). The authors would like to acknowledge: Yelena Pressman for preparing SCs for transplantation; the MPCP Virus Core for the virus production; the Animal Facility Core at the MPCP for assisting in animal care; Margaret Bates and Vania Almeida for TEM
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