4.1.4. Decellularized Matrix and Demineralized Bone

Decellularized cell-derived matrices (dCDMs) could further provide a way to mimic natural tissues. It has been reported that aligned dCDMs could contribute to producing a more homogeneous environment, which resulted in a uniform response of cells to the biophysical environment, displaying a highly homogeneous phenotype and can undergo differentiation if properly stimulated [165]. Substrates displaying linear topographic patterns were obtained by replica molding, using PDMS, while flat PDMS substrates were produced by using a polystyrene dish as a control.

Cell-derived matrices (CDMs) were attained by cultivating MC3T3-E1 pre-osteoblasts in the presence of ascorbic acid for two weeks on linear or flat surfaces. MC3T3-E1 cultivated on nanopatterned substrates produced an aligned fibrillar matrix, whose microarchitectural features remained intact after the decellularization process. Atomic force microscope measurements performed on bare dCDMs revealed very low Young's moduli in the range of (0.01–0.1 kPa) that was increased by genipin crosslinking to reach (i.e., 0.1–1.5 kPa). These matrices were further seeded with murine MSCs and cultivated in the presence of either osteogenic or adipogenic media for two weeks. Both the aligned and random dCDMs promoted murine MSC adhesion and proliferation in their pristine state, while maintaining high levels of stemness markers, with a more homogeneous population of undifferentiated cells, were seen on aligned dCDMs. On the pristine dCDMs, MSCs promptly underwent adipogenic differentiation when stimulated with induction media, while they were minimized in the presence of osteogenic medium, due to very low stiffness. On the contrary, MSCs responded consistently on stiff dCDMs, displaying a significant adipogenic and osteogenic differentiation potential [165].

In another study, 3D demineralized bone matrices with the same 3D microstructure (porosity and pore size), but with various compressive moduli (high: 66.06 ± 27.83 MPa, medium: 26.90 ± 13.16 MPa, and low: 0.67 ± 0.14 MPa), were fabricated by controlling the decalcification duration (1 h, 12 h, and 5 d, respectively). Low-stiffness scaffolds promoted BMMSCs' osteogenic differentiation. Subcutaneous implantation in a rat model further revealed efficient improvement of cells' infiltration and deposition of collagen fibers, in addition to upregulated positive osteocalcin and osteopontin expression, as well as angiogenesis upon utilizing the low-stiffness scaffolds. Further implantation in a femoral condylar defect rabbit model supported the previous findings and revealed that stromal-cell-derived factor-1α/CXC chemokine receptor (SDF-1α/CXCR4) signaling pathway was essential for the stiffness-mediated stem/progenitor recruitment and osteogenic differentiation during bone repair [166].
