Potential Applications of Snail Mucus in Cartilage Repair and Fabrication of Porous 3D Scaffolds

Abstract

Articular cartilage damage is a major challenge in orthopedics. This medical condition is exacerbated by the cartilage's inability to self-repair due to the non-vascularization of the tissue and many other factors. Several treatment strategies for cartilage repair have been explored, including autologous chondrocyte implantation, microfracture, osteochondral plug transplantation, pridie perforation, spongilization and debridement. However, these strategies require complex surgery and have limited success in fully regenerating functional cartilage. In this regard, tissue engineering approaches are promising alternatives to the existing approaches for cartilage tissue repair and regeneration. The success of this approach heavily depends on three major factors, usually referred to as the triad of tissue engineering, which includes appropriate cells of interest, scaffolds, and bioactive factors. The cartilage tissue mainly contains chondrocyte cells, which are the cells of interest explored for cartilage tissue engineering applications. On the other hand, agarose is one of the biopolymers explored for cartilage regeneration due to its unique thermal and mechanical properties, biocompatibility, biodegradability and ease of fabrication. However, this biopolymer lacks cell adhesion property, a major requirement for tissue engineering scaffolds. Therefore, using agarose for tissue engineering applications requires modification of the polymer to achieve the desired properties that will enhance cell attachment and proliferation on the scaffolds. Snail mucus is a complex viscoelastic bioactive substance containing 90 – 97% water by weight, a mixture of glycosaminoglycans (GAGs), proteoglycan, enzymes, metal ions, copper, and antimicrobial peptides, collagen, glycolic acid, allantoin, and elastin. The GAGs reduce cartilage deterioration and inflammation, while other molecules exhibit wound healing, anti-inflammatory, and antibacterial properties, making the gastropod mucus a very rich and suitable material for biomedical applications. Despite these facts, the application of snail mucus in cartilage regeneration has not been explored. Therefore, this thesis reports the application of Achatina fulica (A. fulica) mucus, a novel and cheaper substance, as a therapeutic repair agent in cartilage damage and its use to enhance the bioactivity and cell adhesion properties of bioinert agarose scaffolds. Firstly, the use of A. fulica mucus as a novel potential therapeutic repair agent for osteoarthritis and cartilage tissue repair in vitro was explored. The snail mucus was isolated, sterilized, and characterized using FTIR, XPS, Rheology, and LC-MS/MS. The GAGs, sugar, phenol, and protein contents were estimated using standard assays. The LC-MS/MS detected 6-gingerol and some other small molecules in the snail mucus for the first time. The effects of the sterilized mucus were studied on human chondrocytes using the C28/I2 cells as a model for the in vitro assays. The MTT assay indicates that mucus extracted from the pedal of A. fulica is biocompatible with the cells up to a concentration of 50 µg/mL. The mucus promoted cell migration and proliferation and completely closed the scratch wound within 72 h compared to the control, as indicated in the in vitro scratch assay. In addition, the snail mucus reduced apoptosis significantly (p < 0.05) in the treated cells by 74.5% and preserved the cells’ cytoskeletal integrity, attributed mainly to GAGs and 6-gingerol present in the SMu. Having established the therapeutic effects of the snail mucus and its biocompatibility on the human chondrocytes, C28/I2 cells, the biomaterial was then explored for designing and fabricating porous 3D scaffolds that can be explored for cartilage tissue engineering. Next, porous 3D blend scaffolds containing agarose polymer (AG) and sterilized snail mucus (SMu) were prepared by the freeze-drying method. The scaffolds were characterized by FTIR, FESEM, DMA and other parameters such as open porosity, pore size, swelling capacity, and biodegradability. The scaffolds' bioactivity and cell adhesion effects were determined on the C28/I2 cells. FTIR showed that SMu was successfully incorporated into the scaffolds, and the FESEM result revealed the microporous morphology of the scaffolds with an average pore size of 245 µm. The compression test showed that SMu significantly (p < 0.05) increased the mechanical strength of the composite scaffolds by more than 80% compared to the pristine AG scaffold. The degradation study indicated that SMu enhanced the scaffolds' degradation properties. The MTT assay (24 h and 48 h) and FACS established the biocompatibility of all the scaffolds, while the hemolysis assay with porcine RBCs confirmed the hemocompatibility of the scaffolds having hemolysis levels below 10%. At the same time, CLSM revealed that the cells presented spheroidal morphology with filopodia and lamellipodia protrusions of the actin filaments, confirming cell attachment on the AGSMu blend scaffolds compared to AG scaffolds where no cytoskeletal protrusions were observed for the days 1, 3 and 7 study. Finally, the scaffold’s properties were further enhanced, especially to maintain the structural integrity of the scaffolds and reduce the degradation rate long enough to allow cell proliferation and extracellular matrix (ECM) deposition. In order to achieve this, reduced graphene oxide (rGO) was used as a filler to prepare AG-rGO-SMu composite scaffolds. Scaffolds containing agarose with and without rGO and SMu were designed and fabricated via freeze-drying. The scaffolds were also characterized by FTIR, FESEM, DMA and other parameters such as open porosity, pore size, swelling capacity, and biodegradability. The bioactivity of the scaffolds for cell adhesion properties was also investigated on the C28/I2 cells. The FTIR data showed that SMu and rGO were successfully incorporated into the composite scaffolds and significantly increased the mechanical properties of the composite scaffolds, AG-rGO (0.69 MPa), AG-SMu (0.73 MPa), AG-rGO-SMu-0.5 (0.98 MPa), AG-rGO-SMu-1 (1.09 MPa), AG-rGO-SMu-2 (1.22 MPa) compared to the AG scaffold (0.43 MPa) as revealed by the compressive mechanical analysis results. Incorporating the duo, rGO and SMu, also improved the porosity of the scaffolds from 58% in AG to 74% in AG-rGO-SMu-2 but synergistically reduced the pore sizes based on rGO content in the composite scaffolds. The swelling capacity reduced as the rGO content in the composite scaffolds increased. In addition, rGO significantly reduced the enzymatic and hydrolytic degradation of the composite scaffolds from 40% in AG to 15% in AG-rGO-SMu-2 after 28 days, indicating that the composite scaffolds degraded slowly and could maintain structural integrity long enough to support the deposition of cellular ECM which is required for cartilage regeneration. The MTT assay results showed that all the scaffolds are biocompatible with the C28/I2 cells, as cell viability on all the scaffolds was about 100% and higher for the 24 and 48 h studies. Likewise, all the composite scaffolds incubated with the porcine RBCs demonstrated a high level of hemocompatibility as hemolysis was less than 5%, which is a significant improvement from the less than 10% and 20% hemolysis levels recorded from the AGSMu scaffolds. Furthermore, the CLSM micrographs showed that only the scaffolds containing rGO, SMu or both showed spheroids with filopodia and lamellipodia protrusions of the actin filaments, while pristine AG scaffolds presented cells with only spheroidal morphologies without any observable cytoskeletal protrusions. Therefore, it can be concluded that SMu is a suitable therapeutic repair agent for cartilage repair, and it improved the bioactivity of agarose polymer by enhancing the attachment of C28/I2 cells on the modified AGSMu blend scaffolds. In addition, rGO acted synergistically with SMu to further modify the agarose polymer to enhance its mechanical and cell adhesion properties and, more importantly, reduce the degradation rate of the AG-rGO-SMu composite scaffolds. The scaffolds can, therefore, be explored for cartilage tissue engineering.