Crosslinking is crucial to enhance the physicochemical properties of collagen-based edible scaffolds for cell-cultured meat (CCM). This study aims to systematically investigate the precise and controllable enhancement of cross-linking agents proanthocyanidins (PA), sodium tripolyphosphate (TPP) and different cross-linking means (PA/TPP/PA+TPP) on the performance of edible CCM scaffolds. Bovine bone collagen (BBC)/chitosan (CS) self-crosslinked fibrillar gels (B/C) were first prepared at the optimal stoichiometric ratio of 4∶1 (w/w). Afterward, BBC/CS scaffolds, including 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) were prepared by immersing B/C 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, and degree of crystallinity), physicochemical properties (porosity, thermal stability, crosslinking degree, swelling/water retention ratio, dissolution degree, enzymatic resistance capacity, and mechanical strength), and biocompatibility (seeding efficiency and cell viability) of the samples before (B/C) and after crosslinking (B/C/P, B/C/T, and B/C/PT). Results from Fourier Transform Infrared Spectroscopy (FTIR) and X-ray Diffraction (XRD) showed that the crosslinking improved the molecular structure of the BBC/CS scaffolds without affecting the triple-helical structure integrity of BBC. The BBC/CS scaffolds exhibited a higher degree of molecular order and crystallinity, as well as more and tighter hydrogen bonding interactions, compared with B/C. Scanning electron microscopy (SEM) revealed that the B/C/P (98.3%/136 μm), B/C/PT (96.5%/175 μm), and B/C/T (94.8%/203 μm) shared higher porosity but smaller pore sizes, compared with the BBC/CS fibrillar gels (93.2%/265 μm). Differential scanning calorimetry (DSC) results showed that the crosslinking improved the thermal stability of the BBC/CS scaffolds within a smaller temperature range (10~100 ℃) rather than a larger temperature range (30~500 ℃), compared with B/C. The thermal stability of crosslinked scaffolds was ranked as follows: B/C/P (T_m 66.8 ℃, ΔH 13.52 J/g), B/C/PT (T_m 62.2 ℃, ΔH 10.26 J/g), and B/C/T (T_m 60.6℃, ΔH 8.71 J/g), compared with the B/C (T_m 55.6 ℃, ΔH 5.26 J/g) and BBC (T_m 51.3 ℃, ΔH 3.11 J/g). The cross-linked BBC/CS scaffolds exhibited a lower dissolution ratio (B/C/P: 17.3%, B/C/PT: 31.6%, and B/C/T: 35.9%), while 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: 23.3%, degradability), mechanical strength (B/C/P: 12.5 kPa, B/C/PT: 11.9 kPa, B/C/T: 10.2 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 with the B/C (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 shared superior physicochemical properties, compared with B/C. The B/C/P showed better performance than B/C/T and B/C/PT. The potential crosslinking mechanisms of PA and TPP predominantly relied on the extensive hydrogen bonding between the abundant hydroxyl groups of PA and BBC, as well as the ionic interactions between the phosphate ions of TPP and the amino groups of CS. The three different crosslinking means (PA, TPP, PA+TPP) can accurately improve the performance of BBC/CS scaffolds with different costs and degrees based on their different mechanisms. Therefore, in the production of different edible CCM scaffolds, appropriate crosslinking methods can be comprehensively considered based on the specific performance requirements, production costs, and crosslinking efficiency to achieve precise control. The study provide reference for the construction of edible CCM scaffolds with excellent properties and controllable cost based on PA and TPP cross-linking.

Constructing an edible extracellular matrix (ECM) scaffold with good thermal stability, self-assembly properties and biocompatibility is crucial for manufacturing structured cell cultured meat products. In this study, carboxymethyl chitosan (CMCS) was introduced into the bovine bone collagen (BBC) system. Using ultraviolet (UV) absorption, infrared(IR) and fluorescence spectroscopy, it was found that the interaction between BBC and CMCS was enhanced with the increase in CMCS concentration, without affecting the triple-helical structure. The results of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) showed that the introduction of CMCS reinforced the thermal stability of BBC. Turbidity test, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) confirmed an increase in the degree of fibrillogenesis and aggregation behavior and changes in self-assembly rate; a looser and twisted three-dimensional structure with a larger fibril diameter and a wider diameter distribution was observed after the introduction of CMCS. However, the introduction of CMCS did not significantly affect the formation and length of D-periodicity (a characteristic alternating light/dark transverse stripe structure formed during the self-assembly process of collagen fibers) in BBC, and there was no significant difference in the cell compatibility of the system before and after the introduction of CMCS. The BBC-CMCS interaction might be dominated more by electrostatic forces than covalent interactions and hydrogen bonds with the increase in CMCS concentration. These results indicated that the introduction of CMCS improved the thermal stability and self-assembly properties of BBC without affecting its biocompatibility or triple-helical integrity. This study provides a reference for the development of excellent edible collagen-based ECM as a biomimetic scaffold for cultured meat and for the deep processing and high-value utilization of livestock and poultry bone by-products.