In our present research, we successfully synthesized Qu/BSA NPs with appropriate morphology and release capabilities. Subsequently, these NPs were integrated with an acellular scaffold derived from the spinal cord. This scaffold, containing NPs capable of secreting Qu, was then employed in the treatment of unilateral SCI in a rat model. In our study, we conducted a unilateral hemi-section of the spinal cord on animal models, creating a longitudinal cut that extended 3 mm in length. The purpose of this model was to simulate SCI and prepare a model that could be utilized for scaffold-based interventions. We observed a significant decrease in motor function following the injury. Furthermore, we examined the levels of inflammatory factors, NLRP3, ASC, and Casp-1, at the site of injury and observed a notable increase. This suggests that the injury triggered an inflammatory response, which is consistent with the pathophysiology of SCI. One significant contributor to the disability associated with SCI is the occurrence of secondary injuries, which encompass a range of detrimental events such as inflammation, neuronal cell apoptosis, mitochondrial dysfunction, and oxidative stress (WeiHeng Wang et al., 2017a). Studies have demonstrated that the NLRP3 inflammasome is activated in the injured spinal cord, leading to the release of IL-1β and IL-18, which contribute to the amplification of the inflammatory response and subsequent tissue damage (Mohammed et al., 2020; Yin et al., 2022). In addition, we also investigated the cellular changes at the site of injury. We found an increase in the presence of nestin-positive cells, indicating neural stem/progenitor cell activation. This finding aligns with previous studies that have reported an upregulation of nestin-positive cells following SCI (Mao et al., 2016; Cawsey et al., 2015). Moreover, we observed an increase in GFAP-positive cells at the site of injury. GFAP is a marker for astrocyte activation, and its upregulation is a common response to SCI (Abbaszadeh et al., 2023; Mandwie et al., 2022). A potential scientific strategy for enhancing functional recovery after SCI involves the prevention of adverse secondary outcomes by inhibiting inflammatory responses and regulating the differentiation of neural cells.
In this study, we used an acellular scaffold combined with Qu-encapsulated NPs, as a potential strategy for inhibiting inflammatory responses and enhancing tissue regeneration in SCI rats. This approach aimed to minimize inflammation-induced damage and create a favorable environment for neural tissue regeneration and repair. The first step of study involved the preparation of Qu/BSA NPs. Our result revealed that the resultant NPs exhibited desirable characteristics, including a spherical morphology, an average size of approximately 203 nm, and a negative ZP (− 38 ± 0.1). The EE was found to be 96%, indicating effective loading of Qu into the NPs. The NPs also demonstrated an enhanced dissolution rate of Qu, suggesting improved drug delivery and controlled release. These findings highlight the potential benefits of these NPs, including improved drug delivery and controlled release of Qu, which could enhance its therapeutic efficacy in various applications. BSA has emerged as a promising tool in the field of drug delivery. BSA possesses several characteristics that make it suitable for encapsulating active components and targeting central nervous system (CNS) diseases (Zaman et al., 2018). First, BSA is well-tolerated by the body and can be broken down naturally over time. This is crucial for drug delivery systems as it minimizes the risk of adverse effects (Coelho et al., 2010). Additionally, BSA has a high binding capacity, allowing it to encapsulate a wide range of active components. This versatility opens up possibilities for delivering various therapeutic agents, such as small molecules, proteins, peptides, and nucleic acids, to the CNS (Karimi et al., 2016). Once the BSA-based drug delivery system reaches the CNS, it can release the encapsulated active components in a controlled manner. This enables sustained drug release, improving the therapeutic efficacy and reducing the frequency of administration (Zaman et al., 2018).
Next, an acellular scaffold was prepared from spinal cord tissue. The decellularization process successfully removed all cellular components while preserving the tissue structure. DAPI and H&E staining confirmed the absence of cellular components and the preservation of tissue structure within the scaffold. Immunofluorescence staining further confirmed the presence of important ECM components, such as LM, FN, and Col IV, which play crucial roles in nerve regeneration environment, including their proliferation, migration, and adhesion (Yu et al., 2023). The DNA content of the scaffold was significantly reduced after decellularization, indicating the successful removal of cellular remnants. This reduction in DNA content is important for biocompatibility and functionality, as it minimizes the risk of immune response and adverse reactions. The reduced DNA content during decellularization process also creates an ideal microenvironment for cell seeding and tissue regeneration (Neishabouri et al., 2022). Previous studies have also investigated the preparation of acellular spinal cord using similar methods. For instance, a study conducted by Arslan et al. in 2019 aimed to develop a novel method for creating a 3D biomatrix from bovine spinal cord for nerve regeneration purposes. The researchers utilized enzymatic methods, buffer, and detergent for the process of acellularization and the decellularization process using the 3D-dCBS scaffold was successful in removing cellular components while preserving the ECM. The results obtained from this study aligned with your own research findings, demonstrating the effectiveness of the detergent method for acellularization (Arslan et al., 2019). Based on these results, the acellular scaffold derived from spinal cord tissue exhibited desirable characteristics.
Finally, we combined the prepared NPs with the ASCS and introduced a substance called Qu, which has the potential to create a favorable environment for tissue regeneration. The obtained SEM images showed the presence of NPs on the scaffold, indicating that the integration of the NPs with the acellular scaffold was successful. These NPs were designed to release Qu, which could have specific properties that promote tissue regeneration. Overall, the successful integration of NPs with the acellular scaffold, as confirmed by SEM imaging, suggests that we created a material that has the potential to provide a suitable environment for tissue regeneration.
In the in vivo phase of our study, we employed two treatment approaches, namely B/BSA/ASCS and Qu/BSA/ASCS, to assess their effectiveness in promoting functional recovery in a rat model of spinal cord injury (SCI). Our results revealed that the utilization of B/BSA/ASCS led to a successful enhancement of functional recovery by increasing the presence of nestin and GFAP positive cells at the site of injury, without involvement of the NLRP3 inflammasome. The presence of Nestin and GFAP proteins is indicative of neural cell regeneration and signifies the potential for tissue repair and recovery. The application of acellular scaffolds in SCI treatment has garnered considerable attention in recent years. These scaffolds are three-dimensional structures that lack cells but retain essential extracellular matrix (ECM) components. They serve as a supportive framework for facilitating cell attachment, migration, and tissue regeneration. One notable study by Liu et al. (2013) demonstrated the development of an acellular spinal cord scaffold through a combination of decellularization techniques and physical treatments. Neural stem cells were then seeded onto the acellular scaffold and transplanted into a rat model of SCI. The outcomes of this investigation exhibited improved functional recovery, enhanced axonal regeneration, and reduced glial scar formation in the treated animals compared to the control group (Jia Liu et al., 2013). Additionally, Wang et al. (2017) conducted a study exploring the effects of an ASCS seeded with mesenchymal stem cells. The researchers observed a reduction in the migration of inflammatory cells and a significant decrease in apoptosis when implementing the aforementioned scaffold (Yu‑Hai Wang et al., 2017b). These examples underscore the increasing attention and promising outcomes associated with the utilization of acellular scaffolds in SCI treatment. By providing a conducive microenvironment for cell attachment and tissue regeneration, these scaffolds possess the potential to promote functional recovery and augment neural repair subsequent to SCI.
Our study aimed at enhancing the efficiency of ASCS, we integrated Qu-encapsulated NPs with ASCS to create an optimal environment for tissue regeneration and facilitate functional recovery in rats with SCI. The treatment involving Qu/BSA/ASCS demonstrated significant improvements in functional recovery within the animal models. An important finding of our study was the effective reduction of NLRP3 inflammasome activity at the injury site through the implementation of the Qu/BSA/ASCS scaffold. Additionally, the Qu/BSA/ASCS scaffold played a crucial role in establishing a conducive environment for tissue regeneration by regulating the proportion of nestin and GFAP-positive cells (astrocytes) at the injury site. Notably, compared to the B/BSA/ASCS group, the Qu/BSA/ASCS scaffold exhibited additional advantages. The results indicated that the B/BSA/ASCS and Qu/BSA/ASCS treatments reduced the presence of GFAP-positive astrocytes, potentially limiting reactive gliosis. Furthermore, both treatment groups exhibited an increase in nestin-positive cells, suggesting their potential for neuroregeneration with superiority of Qu/BSA/ASCS. To the best of our knowledge, our study represents the first report on the utilization of Qu-encapsulated nanoparticles in combination with an acellular scaffold for the treatment of SCI in a rat model. This novel approach holds promise in enhancing the therapeutic outcomes in SCI treatment by providing a tailored and efficient platform for tissue regeneration and functional recovery.
Qu has been the subject of numerous studies due to its potential neuroprotective and anti-inflammatory effects, particularly in the context of SCI. It exerts its neuroprotective effects through multiple mechanisms, including antioxidant activity, anti-inflammatory effects, and the modulation of signaling pathways involved in cell survival and regeneration. Qu has been found to possess potent neuroprotective and anti-inflammatory and properties, which can help mitigate the detrimental effects of inflammation on the injured spinal cord. Fan et al. (2019) reported that Qu administration led to improved functional recovery in SCI rats. Furthermore, the researchers observed a significant reduction in myelin loss and axon loss. The study also revealed that Qu had the ability to suppress the polarization of macrophages/microglia into the M1 phenotype. This effect was achieved through the inhibition of the STAT1/NF-κB signaling pathways, indicating the anti-inflammatory and neuroprotective properties of Qu (Fan et al., 2019). Furthermore, Wang et al. (2018) reported that the Qu resulted in improved locomotor function and electrophysiological recovery in the SCI rats. Additionally, Qu up-regulated the expression of brain-derived neurotrophic factor (BDNF), a molecule known for its neuroprotective properties. At the same time, Qu reduced the expression of phosphorylated JNK2 (p-JNK2) and phosphorylated STAT3 (p-STAT3), which are associated with inflammation and cell death (Yeyang Wang et al., 2018). One important aspect of its mechanism of action involves the modulation of the NLRP3 inflammasome. Jiang et al. (2016) demonstrated that Qu could modulate the activity of the NLRP3 inflammasome via downregulation of NLRP3, ASC and active-caspase-1 and reduced the levels of IL-1β, IL-18 and TNF-α. This inhibition of the NLRP3 inflammasome by Qu may contribute to its anti-inflammatory effects and neuroprotective properties in SCI (Jiang et al., 2016). The NLRP3 inflammasome is a key component of the inflammatory response, and its inhibition can have a positive impact on tissue healing and regeneration (Ding et al., 2022).