A Novel Protocol to Generate Decellularized Bovine Spinal Cord Extracellular Matrix-based Scaffolds (3D-dCBS)

Background

Recently, nerve injuries have become a widespread issue that affects an approximate number of 20 million people in the United States alone and remain a significant burden on society (Du et al., 2018). According to data from the World Health Organization (2013), 250,000 to 500,000 people suffer a spinal cord injury due to traffic injuries, falls, and violence. When investigated the National Spinal Cord Injury Database, it is seen that spinal cord injury (SCI) caused by falls has dramatically increased. For instance, while that kind of injuries had occurred 17% in the 1970s, the value reached 31% during 2010-2013. In addition to this, the rate of fall-related SCI is indicated as 75% among persons 76 years of age and over (Chen et al., 2015). Following the SCI, mechanical stress and secondary injuries cause axonal degeneration. Following the SCI, mechanical stress and secondary injuries cause axonal degeneration. Although there are a few treatment approaches for axonal regeneration, it is still impossible to completely cure the damaged axons (Ban et al., 2017). Central nervous system (CNS) axons, unfortunately, have limited regeneration capacity after injury in contrast to axons of the peripheral nervous system (PNS). Therefore, the physiological response is considerably different in the repair of CNS and PNS. Because the macrophages assigned to repair of CNS injury cannot entry to the damaged zone due to blood-spine barrier, and thus tissue repair takes place slightly below of the desired level (Schmidt and Leach, 2003). Many strategies have been developed to provide axonal reinnervation and direct axonal growth, but recent approaches aim at preventing secondary injury, regeneration, and replacement of damaged spinal cord tissues.

Surgical operations and pharmacological methods have been preferred to minimize secondary damage, only treatment for SCI patients. However, there is currently no treatment available to repair nerve tissue function completely (Cristante et al., 2012; Varma et al., 2013; Schmidt and Leach, 2003). Cell-based regenerative therapy and tissue engineering strategies have a promising potential for recovering severe SCI (Assunção-Silva et al., 2015). More recently, neural tissue engineering has focused on natural or synthetic biopolymers that are combined to create a tissue engineering product (Boni et al., 2018). Natural and synthetic derived materials have some advantages and disadvantages, including mechanical, chemical, and biological features (O’ Brien, 2011). The most important properties of natural biopolymers such as gelatin, elastin, fibrinogen, silk, and collagen are a biocompatible, biodegradable and inherent structural similarity to mimic natural ECM. Despite these unique advantages, they have weak mechanical strength and inconsistency, which limit to develop ideal scaffolds for regenerative medicine applications (Haraguchi et al., 2012; Ulery et al., 2011; Sekula and Zuba‐Surma, 2018).

On the other hand, synthetic biopolymers such as poly (ε-caprolactone), poly (lactic acid) and their copolymers, poly (p-dioxanone), copolymers of trimethylene carbonate and glycolide are easily fabricated on a large scale by controlling their degradation rates (Gunatillake and Adhikari, 2003; Abbasian et al., 2019). However, various disadvantages such as toxic by-products resulting from biodegradation of the synthetic polymers restrict their applications in the field of tissue engineering (Tabata, 2009; Arslan et al., 2017). Due to limitations of synthetic and/or natural biopolymers and shortage of donors, acellular biologic scaffolds have become a new option for the treatment of missing or damaged tissues (Yu et al., 2016). Decellularization technique is a promising technology for tissue engineering and regenerative medicine applications because the technique aims at removing host cells from ultrastructure of native ECM by protecting unique ECM molecules (Kelleher and Vacanti, 2010). Many different methods including chemical, enzymatic, physical and/or their combinations, are used for the decellularization of native ECM. Preservation of structural integrity and mechanical properties of ECM in the decellularization process, which allows removing cellular components such as cells, cell debris, chromosome fragments, and xenogeneic antigens, is a crucial fact for achieving effective healing of the tissues (Crapo et al., 2011; Patnaik et al., 2014). Furthermore, acellular biologic scaffolds regulate the homeostasis and regeneration of tissues and organs by mimicking the ECM that acts as a niche for the cells (Tapias and Ott, 2014; Dzobo et al., 2018).

The ultimate goal of the proposed decellularization protocol is to effectively remove the nuclear contents (i.e., dsDNA) of native tissues and organs while maintaining the critical structural, biochemical, and biomechanical cues present in the ECM. This study has demonstrated a novel decellularization protocol, which enables an optimized strategy to fabricate a 3D bioscaffold for neural tissue engineering applications (Arslan et al., 2019). By using 1% sodium dodecyl sulfate (SDS), 50 mM Trizma^® hydrochloride and 5 mM EDTA, we have executed the decellularization process at room temperature (RT) for 72 h. The results showed that the amounts of dsDNA in the native BSC and 3D-dCBS were found to be 520.76 ± 18.11 and 28.80 ± 0.20 ng/mg dry weight, respectively (n = 3; P < 0.001; ANOVA). The proposed method removed approximately 94.47% of nuclear material from the native BSC. It has been reported that the amount of dsDNA should be < 50 ng per mg dry weight and < 200 bp residual of the DNA fragment for the optimal decellularization (Pati et al., 2014).

Consequently, the main benefit of the presented method is constructing a 3D biomatrix from decellularized BSC with desired geometry. We believe that the recellularization of 3D-dCBS with neural stem and/or progenitor cells could have a positive effect on the supporting of deformed nerve tissue. In conclusion, we believe that the proposed approach to decellularization of BSC will open new horizons for the experts working in the field of neural tissue engineering.