4.1.2. Collagen

Collagen is considered to be the most abundant protein in mammals [144]. Being the main ECM protein, collagen, together with soluble factors, may act as a niche for MSCs osteogenesis and bone mineralization [145]. Mechanical properties of collagen fibers vary depending upon their location in different tissues. Thus, the cells can sense local fibrillar microenvironments with different physical cues. Collagen gels were engineered to attain varying fiber stiffness (from 1.1 to 9.3 kPa), while maintaining bulk stiffness below 200 Pa, by changing the polymerization temperature to 4 ◦C (Col-4), 21 ◦C (Col-21), and 37 ◦C (Col-37) without changing the density of the collagen. A polymerization temperature of 4 ◦C led to shorter, thicker, and stiffer collagen fibers (Col-4), with limited fiber recruitment and force transmission and fewer focal adhesions. Cells grown on Col-4 showed much slower spreading as compared to Col-37 with similar bulk stiffness but with more flexible and longer fibers that can be easily remodeled. Human MSCs cultured on Col-4 revealed a much lower ratio of osteogenic differentiation (21.1%) compared to that on Col-37 with (34.1%) ALP positive reactivity [146].

Matrix stiffness possesses a high impact on cellular bioactivity regardless of the presence or absence of growth factors. This was proved by culturing porcine adipose-derived stem cells (ADSCs) on sequentially integrated benzophenone photo-immobilization and carbodiimide, crosslinking collagen–glycosaminoglycan, with stiffness ranging from 2.85 to 5 MPa, in the presence or absence of covalently immobilized platelet-derived growth factor (PDGF-BB) and BMP-2 [147].

In the presence of osteogenic culture media, human MSCs coated with three bilayers of collagen/alginate nanofilm with relatively high stiffness 24 and 53 MPa revealed an augmented osteogenic differentiation efficiency with a significant increase in ALP by activating transcriptional co-activators with the PDZ binding motif through extracellular signal-related kinase and p38-MAPK signaling [148].

Nanoparticulate mineralized collagen–glycosaminoglycan scaffolds chemically crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide had a higher range of elastic moduli 3.90 ± 0.36 kPa as compared to non-crosslinked materials. Cultured human MSCs on crosslinked substrates showed higher expression of osteogenic genes and proteins compared to non-crosslinked versions. This was maintained via the mechanotransduction mediators YAP/TAZ and the Wnt signaling pathway [149].

MSCs seeded within a 3D collagen gel with an elastic modulus of ~108 Pa stimulated by vibrations of nanoscale amplitude in a vibrational bioreactor showed increased expression of Runx2, collagen I, ALP, osteopontin, osteocalcin, and BMP-2. This indicated that the viscoelastic properties of the collagen gel allowed the transfer of high-frequency vibrations to the cells seeded in 3D [150].

In the absence of any differentiation supplementation, MSCs grew on stiffer (1.5 kPa) dehydrothermal and 1-ethyl-3-3-dimethyl aminopropyl carbodiimide (EDC) crosslinked collagen–glycosaminoglycan scaffolds showed the greatest level of Runx2 expression, while substrates with lower stiffness (0.5 kPa) resulted in significant elevation of SOX9 expression, indicating that MSCs are directed towards a chondrogenic lineage [151].

Additionally, 3D scaffolds with the highest proportion of collagen in collagen and hydroxyapatite mixture coated on the decellularized cancellous bone with various stiffness (13.00 ± 5.55 kPa, 13.87 ± 1.51 kPa, and 37.7 ± 19.6 kPa) exhibited the highest stiffness that, in turn, promoted higher expressions of osteopontin and osteocalcin [152].
