In-vitro osteogenesis is affected by composition and stiffness of collagen-GAG scaffold.

Collagen-glycosaminoglycan (GAG) scaffolds were initially designed for use in skin regeneration and have since been utilised in cartilage, nerve and muscle tissue engineering. The FDA-approved formulation contains a ratio of 0.5% collagen to 0.044% GAG. However, the composition and stiffness of the collagen-GAG scaffold has never been optimised to sustain a particular cell phenotype or designed with tissue-specific applications in mind. This study sought to investigate the effect of varying the composition and stiffness of the scaffold on the material andstructural properties, in addition to cell behaviour, with a view to developing an optimised collagen-GAG scaffold for use in bone tissueengineering. Scaffolds were fabricated to contain varying amounts of collagen and GAG independently of each other and crosslinked using two dehydrothermal crosslinking techniques ~ 105°C for 24 hours and 150 °C for 48 hours. The effects of composition and scaffold stiffness on early stage attachment (up to 7 days) and for osteogenesis and mineralisation (up to 28 and 42 days) were investigated. All scaffolds were found to support cell attachment, infiltration, osteogenesis and mineralisation. However, the scaffold that contained the highest amount of collagen (1% Collagen) had consistently superior mechanical properties with high porosity, larger pore sizes, increased permeability and the highest compressive modulus as well as elevatedlevels of cellular metabolic activity, retention, ECM and mineral deposition. All scaffold groups also experienced cell-mediated contractionwith some capsular formation around the scaffold borders by 28 days which, in 42 day culture, resulted in necrotic regions in the centre ofsome constructs. A scaffold containing the highest amount of GAG investigated (0.088% GAG) also demonstrated enhanced osteoblast activity and was the only scaffold not to develop capsular formation during culture. Crosslinking scaffolds at the higher temperature of 150 °C for 48 hours resulted in a 3-fold increase in stiffness across all scaffold groups and an increase in osteoblast attachment and proliferation. Increasing the stiffness of the scaffold also reduced the cell-mediated contraction, thereby allowing scaffolds to retain their porous architecture, without capsular formation or development of necrotic regions in longer term culture. By combining the optimal composition and stiffness (l % Collagen, dehydrothermal crosslinking at 150 °C for 48 hours), osteoblast attachment, proliferation, osteogenesis and mineralisation were all improved. Interestingly, the original scaffold formulation devised for skin regeneration had the lowest cell attachment, proliferation, osteogenesis and mineralisation during culture and experienced the most cell-mediated contraction thereby compromising scaffold architecture. In conclusion, while all scaffold compositions supported osteogenesis and mineral deposition, it was the scaffold containing 1% Collagen (0.044%GAG) and crosslinked at 150 °C for 48 hours that consistently had elevated cell numbers and metabolic activity, ECM and mineral deposition and superior mechanical properties for in-vitro osteogenesis. This scaffold can now be applied within the field of bone tissue engineering to enhance and augment natural bone healing responses.