Performance enhancement of edible cell culture meat scaffolds crosslinked by procyanidins and sodium tripolyphosphate

Crosslinking is crucial for enhancing the physicochemical properties of collagen-based edible scaffolds for cell cultured meat (CCM). To systematically investigate the precise enhancement effect of the crosslinking agents proanthocyanidins (PA) and sodium tripolyphosphate (TPP) on the physicochemical performance of edible CCM scaffolds, bovine bone collagen (BBC)/chitosan (CS) self-crosslinked fibrillar gels were first prepared at the optimal stoichiometric ratio of 4:1 (w/w). Afterwards, BBC/CS scaffolds were immersed in crosslinking agents to obtain PA-crosslinked BBC/CS scaffolds (B/C/P), TPP-crosslinked BBC/CS scaffolds (B/C/T), and PA & TPP-crosslinked BBC/CS scaffolds (B/C/PT). These were prepared by immersing in PA for 2 h, TPP for 2 h, and a combination of PA for 1 h followed by TPP for 1 h, respectively. Subsequently, a comparative assessment was conducted on the molecular structure (hydrogen bonding, degree of molecular order, triple-helical structure integrity, degree of crystallinity), physicochemical properties (porosity, thermal stability, crosslinking degree, swelling/water retention ratio, dissolution degree, enzymatic resistance capacity, mechanical strength), and biocompatibility (seeding efficiency and cell viability) of the samples before (BBC/CS fibrillar gels) and after crosslinking (BBC/CS scaffolds, including B/C/P, B/C/T, and B/C/PT). Results from Fourier Transform Infrared Spectroscopy (FTIR) and X-ray Diffraction (XRD) showed that crosslinking improved the molecular structure of the scaffolds without affecting the triple-helical structure integrity of BBC in BBC/CS scaffolds. The BBC/CS scaffolds exhibited a higher degree of molecular order and crystallinity, as well as more and tighter hydrogen bonding interactions, compared to BBC/CS fibrillar gels. Scanning electron microscopy revealed that B/C/P (98.3%/136 μm), B/C/PT (96.5%/175 μm), and B/C/T (94.8%/203 μm) had higher porosity but smaller pore sizes compared to BBC/CS fibrillar gels (93.2%/265 μm). Differential scanning calorimetry results showed that crosslinking improved the thermal stability of the BBC/CS scaffolds within a smaller temperature range (10～100 ℃) but did not significantly enhance thermal stability over a larger temperature range (30～500 ℃) compared to BBC/CS fibrillar gels. The thermal stability of the crosslinked scaffolds was as follows: B/C/P (Tm 66.8 ℃, ΔH 13.52 J/g), B/C/PT (Tm 62.2 ℃, ΔH 10.26 J/g), and B/C/T (Tm 60.6 ℃, ΔH 8.71 J/g), compared to BBC/CS fibrillar gels (Tm 55.6 ℃, ΔH 5.26 J/g) and BBC (Tm 51.3 ℃, ΔH 3.11 J/g). The cross-linked BBC/CS scaffolds exhibit a lower dissolution ratio (B/C/P: 17.3%, B/C/PT: 31.6%, B/C/T: 35.9%) but a higher degree of crosslinking (B/C/P: 46.53%, B/C/PT: 38.96%, B/C/T: 30.61%), swelling/water retention ratio (B/C/P: 68/7.0 times, B/C/PT: 45/5.3 times, B/C/T: 37/4.4 times, weight after moisture absorption equilibrium to initial dry weight), enzymatic resistance capacity (B/C/P: 11.1%, B/C/PT: 20.6%, B/C/T: 23.2%), mechanical strength (B/C/P: 12.5 kPa, B/C/PT: 10.2 kPa, B/C/T: 11.9 kPa), and biocompatibility (B/C/P: 86.37%/ 118.33%, B/C/PT: 79.33%/ 115.68%, B/C/T: 63.52%/ 112.82%, seeding efficiency and cell viability, respectively) compared to BBC/CS fibrillar gels (dissolution ratio 55.6%; swelling/water retention ratio 25/2.9 times; enzyme resistance capacity 35.2%; mechanical strength 5.6 kPa; seeding efficiency 56.59%/cell viability 100%). Generally, BBC/CS scaffolds exhibit superior physicochemical properties compared to BBC/CS fibrillar gels, and B/C/P shows better performance improvement than B/C/T and B/C/PT. The potential crosslinking mechanisms of PA and TPP may predominantly rely on extensive hydrogen bonding between the abundant hydroxyl groups of PA and BBC, as well as ionic interactions between the phosphate ions of TPP and the amino groups of CS. The crosslinking of PA and TPP can improve the physicochemical performance of BBC/CS scaffolds to varying degrees based on their different molecular mechanisms. Therefore, in the production of edible CCM scaffolds, precise control can be achieved by comprehensively considering crosslinking methods such as PA, TPP, or PA+TPP based on factors such as CCM scaffold performance requirements and production cost control. This study provides a reference for the application and development of edible CCM scaffolds with excellent properties based on PA and TPP crosslinking.