Chondrogenic differentiation of Wharton's Jelly mesenchymal stem cells on silk spidroin-fibroin mix scaffold supplemented with L-ascorbic acid and platelet rich plasma

Articular cartilage, lacking vascular, nervous, and lymphatic systems, has limited regenerative capacity. Tissue engineering offers a potential solution, requiring cells, biomaterials, and bioactive factors. Human Wharton's Jelly-derived mesenchymal stem cells (hWJ-MSCs) are a promising cell source due to their high proliferation rate and plasticity, differentiating into chondrocytes and producing hyaluronic acid, glycosaminoglycans, and collagen type II. Platelet-rich plasma (PRP) and L-ascorbic acid (LAA) are bioactive factors known to enhance chondrogenic differentiation. PRP contains growth factors crucial for cell proliferation and chondrogenesis, while LAA is a cofactor in collagen biosynthesis. Silk fibroin (SF) from *Bombyx mori* is biocompatible and biodegradable, but cells attach weakly due to the lack of specific cell-recognition sequences. This study explored the use of a silk spidroin (SS) from *Argiope appensa* and fibroin mix scaffold to improve hWJ-MSC attachment and differentiation, induced by PRP or LAA.

Extensive research highlights the challenges in articular cartilage repair due to its avascular nature. Tissue engineering approaches, utilizing MSCs from various sources, including bone marrow, adipose tissue, and Wharton's Jelly (WJ), show promise. hWJ-MSCs are particularly attractive due to their ease of access and superior proliferation and differentiation potential compared to other sources. The role of bioactive factors like PRP and LAA in promoting chondrogenesis is well-established. PRP, rich in growth factors, stimulates cell proliferation and differentiation, while LAA is essential for collagen synthesis, a major component of the extracellular matrix (ECM). Silk fibroin, a well-studied biomaterial, offers biocompatibility and biodegradability, but its low cell adhesion has limited its application. The incorporation of spidroin, known for its superior mechanical properties, may address this limitation. This study builds upon existing literature by investigating the synergistic effect of a novel silk spidroin-fibroin scaffold and chondrogenic inducers on hWJ-MSC differentiation.

hWJ-MSCs were isolated from umbilical cord Wharton's Jelly using the explant method and characterized by flow cytometry for MSC markers (CD73, CD90, CD105) and multipotency assays. Silk fibroin was extracted from *Bombyx mori* cocoons and silk spidroin from *Argiope appensa* dragline silk. Porous scaffolds with varying compositions (100% SF, 95% SF + 5% SS, 90% SF + 10% SS, 85% SF + 15% SS, 80% SF + 20% SS) were fabricated using a salt leaching method. Scaffold characterization included FTIR spectroscopy, contact angle and water uptake measurements, SEM analysis for pore size and interconnectivity, and compressive strength testing. hWJ-MSCs were seeded on scaffolds and their proliferation was assessed by MTT assay. Optimal PRP (5%, 10%, 20%) and LAA (25 µg/ml, 50 µg/ml, 100 µg/ml, 200 µg/ml) concentrations were determined using MTT assays. Chondrogenic differentiation was induced with PRP or LAA for 7 and 21 days. GAG accumulation was quantified using Alcian Blue staining, and collagen type II expression was evaluated by immunocytochemistry. Cell attachment was analyzed by immunocytochemistry of integrin β1 and RGD sequence. Statistical analysis was performed using two-way ANOVA and Tukey's multiple comparison test.

FTIR analysis showed that the incorporation of spidroin did not alter the amide bonds or secondary structure of the silk scaffolds. Scaffolds containing 10% SS exhibited higher compressive strength and better pore interconnectivity than 100% SF scaffolds. SEM analysis revealed that cells on scaffolds with spidroin had a more flattened morphology compared to the rounded morphology observed on the 100% SF scaffolds. MTT assays indicated that the 90% SF + 10% SS scaffold was optimal for cell proliferation. 10% PRP supplementation was optimal for cell proliferation, while 50 µg/ml LAA showed the best results. Alcian Blue staining and collagen type II immunocytochemistry showed that the 90% SF + 10% SS scaffold supplemented with 10% PRP supported the most significant chondrogenesis after 21 days. Immunocytochemistry of integrin β1 and RGD sequence suggested that the spidroin silk might contain RGD sequences, facilitating better cell attachment.

The findings demonstrate that incorporating silk spidroin into silk fibroin scaffolds significantly enhances the efficacy of cartilage tissue engineering. The improved compressive strength and pore interconnectivity of the spidroin-fibroin mix scaffold provide a more suitable environment for cell growth and differentiation. The presence of potential RGD sequences in the spidroin seems to play a key role in enhancing cell attachment and spreading, leading to increased cell viability and chondrogenic differentiation. The superior performance of PRP over LAA in inducing chondrogenesis in this study aligns with previous research highlighting the potent chondrogenic effects of PRP's growth factors. The results support the development of bio-based materials for cartilage tissue engineering applications.

This study successfully demonstrated that a silk spidroin-fibroin mix scaffold, particularly with 10% spidroin, coupled with 10% PRP supplementation, significantly enhances hWJ-MSC chondrogenic differentiation. The improved mechanical properties, enhanced cell attachment (potentially via RGD sequences), and superior chondrogenic induction make this a promising approach for cartilage tissue engineering. Future research could focus on optimizing scaffold architecture, investigating the specific RGD sequence presence in *A. appensa* spidroin, and conducting in vivo studies to validate these findings.

The study was limited to in vitro experiments. The presence of RGD sequences in *A. appensa* spidroin was inferred from confocal microscopy, and further analysis is needed for confirmation. The long-term biodegradation and biocompatibility of the spidroin-fibroin scaffold in vivo require further investigation.