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 has limited intrinsic repair capacity due to lack of vasculature and sparse chondrocytes, prompting interest in tissue engineering approaches that integrate cells, scaffolds, and bioactive factors to restore function. Mesenchymal stem cells (MSCs) from Wharton’s Jelly (hWJ-MSCs) exhibit high proliferation and multipotency, making them attractive for cartilage repair. Bioactive supplements such as platelet-rich plasma (PRP) and L-ascorbic acid (LAA) can enhance MSC proliferation and chondrogenic differentiation. Silk fibroin (SF) from Bombyx mori is biocompatible with favorable mechanics but lacks specific cell-adhesive motifs (e.g., RGD), potentially limiting cell attachment. Spider silk spidroin (SS) from Argiope appensa offers excellent mechanical properties and may provide adhesion motifs. This study investigates whether incorporating SS into SF scaffolds improves scaffold properties and hWJ-MSC attachment, proliferation, and chondrogenesis, and compares PRP versus LAA supplementation for inducing chondrogenic differentiation.

Prior studies show SF is a promising scaffold for cartilage and bone due to biocompatibility, biodegradability, and mechanical strength, but cell adhesion can be weak because fibroin lacks RGD motifs recognized by integrins. Non-mulberry silks (e.g., Antheraea mylitta) and engineered spider silk variants containing RGD improve cell adhesion and support osteo/chondrogenesis. Scaffold pore size and interconnectivity critically influence nutrient transport and chondrogenesis, with larger pores (~370–500 µm) favoring MSC chondrogenesis. PRP provides growth factors (e.g., TGF-β, PDGF, EGF) enhancing MSC proliferation and chondrogenesis, with ~10% PRP often optimal. LAA supports proliferation and collagen biosynthesis but high doses may reduce proliferation; moderate doses (around 50 µg/mL) are generally beneficial. These findings motivate blending SS with SF to introduce adhesion motifs and optimize scaffold architecture, while testing PRP and LAA as chondrogenic inducers.

Scaffold fabrication: Porous silk scaffolds were produced via modified salt leaching. SF fibers (Bombyx mori) were degummed in 0.05 wt/v% NaHCO3 for 1 h; SS dragline silk (Argiope appensa) was collected and used without degumming. Total silk (0.06 g) was dissolved in 500 µL of 8 wt% CaCl2-formic acid, poured into molds, mixed with 2.5 g NaCl particles (~500 µm) to template pores, dried overnight, soaked in 70% ethanol (30 min), and leached in water for 3 days. Scaffolds were cut to 5×5 mm. Compositions: SF100, SF95+SS5, SF90+SS10, SF85+SS15, SF80+SS20. Scaffold characterization: FTIR (1000–4000 cm−1) on unprocessed fibers (wet/dry) and processed scaffolds; contact angle (10 µL water, imaged at 10 s) and water uptake after 24 h immersion; SEM for pore morphology and cell distribution; compressive testing on dry cylindrical scaffolds (13 mm diameter, 6 mm thickness) to ~60% strain at 0.1 mm/min using a universal testing machine to obtain stress–strain behavior. Cell isolation and characterization: hWJ-MSCs were isolated from human umbilical cords (ethics-approved) via explant method, expanded to passage 5. Multipotency was confirmed by induction in adipogenic, osteogenic, and chondrogenic media for 21 days and staining (Oil Red O, Alizarin Red, Alcian Blue). Flow cytometry used an MSC marker kit (CD90 FITC, CD105 PerCP, CD73 APC; negative cocktail CD34, CD11b, CD19, CD45, HLA-DR). Cell-scaffold interactions and proliferation: hWJ-MSCs were seeded on SF100, SF90+SS10, and SF85+SS15 and cultured in growth medium. Viability/proliferation was assessed by MTT at days 1, 3, 5, 7, and 14 (n=3). Optimization of PRP and LAA concentrations for proliferation used 2×10^4 cells/well on 96-well plates with PRP 5%, 10%, 20% (v/v) or LAA 25, 50, 100, 200 µg/mL, with MTT at identical time points (n=3). Cell attachment ICC: Integrin β1 immunocytochemistry on cells grown on SF100 and SF90+SS10 at 6, 24, and 48 h; fixation with serial methanol-DMEM, permeabilization (0.05% Tween-20), blocking (3% BSA), primary anti-integrin β1, Alexa Fluor 647 secondary, DAPI nuclear stain; confocal imaging. RGD ICC was performed similarly using anti-RGD primary and Alexa Fluor 488 secondary at 6 and 48 h. Chondrogenic differentiation assays: 5×10^5 cells were seeded on scaffolds; cultures maintained up to 21 days in media supplemented with either PRP 10% (v/v), LAA 50 µg/mL, or control (10% FBS). GAG accumulation quantified by Alcian Blue staining, dye elution, and absorbance at ~605 nm (reported at 650 nm in figure caption); Collagen type II ICC performed at days 7 and 21 using anti-collagen II with Alexa Fluor 488 secondary and DAPI; confocal imaging at three random fields. Statistical analysis used two-way ANOVA with Tukey’s post-hoc; data are mean of three independent experiments.

- Scaffold chemistry and wettability: FTIR showed that incorporating SS did not alter amide bands or silk secondary structure; salt leaching slightly broadened the ~3300 cm−1 hydrogen-bond region. SF100 contact angle was ~57.6° (consistent with prior work); adding SS increased contact angle (reduced hydrophilicity). Water uptake capacities were broadly similar across groups, with 20% SS showing reduced uptake relative to others. - Microarchitecture: All groups formed macropores in the 400–570 µm range (matching NaCl template). Interconnected pores were present in all but SF95+SS5; SF80+SS20 showed pore formation issues. SF90+SS10 exhibited better pore interconnectivity than SF100. - Mechanics: Compressive strength improved with modest SS addition: SF95+SS5 ~0.0372 MPa and SF90+SS10 ~0.0304 MPa; SF100 and SF80+SS20 were lowest. Despite SF95+SS5 having the highest strength, SF90+SS10 was selected for optimal pore morphology/interconnectivity. - hWJ-MSC characterization: Cells displayed fibroblast-like morphology, tri-lineage differentiation (Oil Red O, Alcian Blue, Alizarin Red), and MSC markers CD73 100%, CD90 100%, CD105 95.3%, and Lin(−) 0.5%. - Cell morphology and attachment: SEM after 48 h showed cells on SF100 remained rounded/aggregated, whereas SS-containing scaffolds supported elongated, well-spread cells. Integrin β1 ICC intensity was higher and more widely distributed on SF90+SS10 than SF100. RGD ICC revealed punctate green signals on SS-containing scaffolds, suggesting presence of RGD-like motifs that may mediate integrin β1 engagement. - Proliferation on scaffolds: MTT showed an initial decrease (days 1–5) followed by increase (days 7–14), consistent with adaptation/infiltration. SS-containing scaffolds outperformed SF100 by day 14. SF90+SS10 yielded significantly higher growth than SF85+SS15 (p<0.005), indicating 10% SS was optimal among blends tested. - PRP and LAA dose optimization: PRP 10% supported the highest viability up to day 14, outperforming 5% and 20% PRP. For LAA, 50 µg/mL sustained the highest viability over 14 days (p<0.05), whereas 100–200 µg/mL led to reduced viability after day 5. - Chondrogenic outcomes: GAG accumulation (Alcian Blue) was greater with PRP 10% than LAA 50 µg/mL or control, and higher on SF90+SS10 than SF100. Collagen II ICC: by day 7, PRP-treated groups produced collagen II (LAA less evident); by day 21, the highest collagen II was observed in SF90+SS10 with PRP 10%. Overall, SF90+SS10 plus PRP 10% most effectively promoted hWJ-MSC chondrogenesis.

Incorporating 10% spidroin into fibroin scaffolds improved pore interconnectivity and compressive strength while enhancing early cell attachment via integrin β1, likely due to RGD-like motifs in spidroin. These enhancements translated into superior hWJ-MSC proliferation and chondrogenic differentiation compared to pure fibroin. The optimal PRP concentration (10%) provided a rich milieu of growth factors (notably TGF-β1) that activate Smad2/3–Sox9 signaling, thereby increasing GAG and collagen II production. LAA at 50 µg/mL supported proliferation but was less effective than PRP at inducing robust chondrogenesis within 21 days. The data collectively indicate that both scaffold composition (SF90+SS10) and bioactive supplementation (PRP 10%) synergize to drive hWJ-MSC chondrogenesis, addressing the study’s goal of enhancing cartilage tissue engineering constructs. Improved cell attachment and survival are critical early steps that likely underpin the observed gains in matrix synthesis and chondrogenic marker expression.

A silk scaffold blending 10% spidroin with 90% fibroin provides improved pore interconnectivity, compressive strength, and cell-adhesive properties, leading to enhanced hWJ-MSC proliferation and chondrogenic differentiation. Supplementation with 10% PRP outperformed LAA (50 µg/mL) in promoting GAG accumulation and collagen II production, with the SF90+SS10 + PRP 10% condition yielding the strongest chondrogenic outcomes. These findings support the use of SF–SS composite scaffolds with PRP as a promising bio-based strategy for cartilage tissue engineering. Future work should quantify and confirm spidroin RGD content, optimize SS ratios for balanced mechanics and permeability, assess long-term matrix maturation, and validate efficacy and safety in vivo.

- The presence of RGD motifs in Argiope appensa spidroin was inferred from ICC signals but not definitively identified/quantified biochemically. - Mechanical properties, while improved with SS addition, remain below native cartilage compressive modulus; only compressive strength was reported for dry scaffolds. - In vitro study with relatively short culture duration (up to 21 days) and small sample size (n=3) limits generalizability. - PRP composition can vary donor-to-donor; standardization and detailed characterization of PRP were not reported. - Only hWJ-MSCs were tested; responses may differ with other clinically relevant cell sources; no in vivo validation.